Differential expression of GABAA/benzodiazepine receptor beta 1, beta 2, and beta 3 subunit mRNAs in the developing mouse cerebellum. Gamma aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian cerebellum. Cerebellar granule, Purkinje, and deep nuclear neurons are known to receive GABAergic afferents. Since GABA exerts its inhibitory effects via GABA receptors, it is of interest to determine the temporal relationship between the formation of GABAergic synapses and the expression of genes coding for the GABA receptor. In a previous study, we have examined the developmental expression of binding sites for [3H]muscimol, which binds with high affinity to the beta subunits of the GABAA/benzodiazepine (GABAA/BZ) receptor. In the present study, [35S]cRNA probes were used to examine the appearance and distribution of GABAA/BZ beta 1, beta 2, and beta 3 subunit mRNAs in the developing C57BL/6 mouse cerebellum by in situ hybridization. In the adult cerebellum, the distribution of the three subunit mRNAs was clearly different, despite considerable overlap, and their temporal expression differed throughout postnatal development. The beta 1 hybridization signal appeared within the cerebellar cortex during the second postnatal week as a discrete band at the interface of the molecular and granule cell layers. Grains were distributed diffusely over small densely staining cells surrounding the Purkinje cells; relatively few grains were visible over Purkinje cell bodies themselves. This distribution may reflect an association with Bergmann glia or basket cells. The beta 2 and beta 3 hybridization signals were present considerably earlier than that of the beta 1 mRNA. The beta 2 signal was present at birth in the molecular/Purkinje cell layer; as development progressed, the signal became increasingly intense over both granule and Purkinje cells. At birth, the beta 3 subunit mRNA was present in the external germinal and molecular layers, later becoming largely localized within the granule cell layer. Dense beta 2 and beta 3 cRNA probe labeling was present over the adult granule cell layer. Moderate levels of beta 2 signal were seen over Purkinje cell bodies; considerably less labeling was observed with the beta 3 probe. The adult distribution of beta 2 and beta 3 cRNA probes showed good spatial correspondence with the known GABAA receptor beta subunit markers, [3H]-muscimol and the mAb 62-3G1 antibody, each being present within the granule cell layer. Our results indicate that the temporal expression of GABAA/BZ receptor beta subunit messages within a given cell type may be independently regulated, and that acquisition of the beta 2 and beta 3 mRNAs occurs before these cells become integrated into mature synaptic circuits.


Direct and indirect "cortico"-rubral and rubro-cerebellar cortical projections in the pigeon. In birds the red nucleus is the most rostral cell group in the brain having projections to all levels of the spinal cord (Cabot et al., Prog. Brain Res., 57:79-108, 1982), but its sources of afferents are incompletely known. In order to determine these, a series retrograde and anterograde tracing experiments was carried out, largely with cholera toxin B-chain conjugated to horseradish peroxidase. The results show that a sparse and diffuse projection to the red nucleus arises from deep regions of the hyperstriatum accessorium (HA) of the anterior Wulst, and that a much more dense projection arises from the caudal part of the nucleus principalis precommissuralis and the medial part of the medial spiriform nucleus (SpMm). These last two sources were themselves shown to receive a substantial projection from HA of the anterior Wulst. The red nucleus was also shown to project upon the cerebellar cortex of lobule VI, and SpM upon the cerebellar cortex of lobules VI through IX (Karten and Finger, Brain Res., 102:335-338, 1976; Clarke, J. Comp. Neurol., 174:535-552, 1977). Double retrograde labelling experiments with fluorescein and rhodamine labelled latex microspheres injected into the cerebellar cortex and spinal cord showed that the rubrocerebellar cortical neurons are a different population from, although intermixed with, the rubrospinal neurons.


[Cancer of the testis after Hodgkin's disease. Apropos of 2 cases]. We report about two cases of cancer of the testis occurring 2 and 7 years after Hodgkin's disease treated with combined chemotherapy and radiation therapy. From an etiopathogenic point of view, this association may, of course, be accidental, but it may also have an iatrogenic origin or be caused by contiguous carcinogenesis, or even by an immune deficiency. In addition to orchidectomy, the therapeutic history of these patients requires an adaptation of the treatment of their tumors of the testes.


Immunohistochemical detection of desmosomes in oral squamous cell carcinomas: correlation with differentiation, mode of invasion, and metastatic potential. An expression of desmosomal glycoprotein 1 (DG 1) was immunohistochemically examined in 77 biopsies and 21 metastatic cervical lymph nodes of oral squamous cell carcinomas (SCC). In the primary tumors the DG 1 expression was significantly reduced at the invasive site of poorly differentiated and highly invasive tumors. In cases of metastases in cervical lymph nodes, the DG 1 staining at the invasive site of the primary tumor was significantly less than that of nonmetastatic cases. The DG 1 expression in the metastatic lymph nodes was as weak as that in the primary tumor. Thus, we suggest that immunohistochemical investigation of DG 1 expression in oral SCC is valuable in predicting tumor behavior.


Measurement of proliferating cell nuclear antigen (PCNA) and its clinical application in oral cancers. The PCNA score was measured in oral squamous cell carcinoma (SCC), and its relationship to other cell proliferation markers, Ki-67 score, S-phase fraction (SPF), and AgNORs counts was investigated. The PCNA score ranged from 0.4% to 43.5% with an average value of 22.8%, the Ki-67 score ranged from 4.9% to 40% with an average of 24.1%, and the SPF ranged from 0.4% to 32.5% with an average of 12.4%, while AgNORs counts ranged from 2.53/nucleus to 7.03/nucleus with an average of 4.74/nucleus. These four parameters were closely interrelated. There was a significant difference in PCNA score between malignant and nonmalignant lesions, suggesting a difference in growth activity. The mean PCNA score decreased significantly from 20.0% to 8.0% after cancer chemotherapy. The response of cancer cells to anticancer agents may be estimated by consecutive measurement of PCNA, since the PCNA score dropped after treatment in cases showing a favorable prognosis.


The technical presentation.

The technical presentation. A technical presentation, like any other presentation, requires organization so the audience can follow the speaker's core message smoothly and logically. Remember to incorporate these "tips" to make your technical presentation successful: Keep it short and simple (KISS) Using fewer visual aids is better Use large, bold and colorful, and unclutered visual aids Communicate with simplistic language Use vocal variety Use gestures Know your audience.


The human antibody V region repertoire to the type B capsular polysaccharide of Haemophilus influenzae. The V region repertoire of the human antibody response to the type b capsular polysaccharide of Haemophilus influenzae (Hib-PS) is being defined at the molecular level using antibodies purified from serum of immunized adults. The VH of this response is restricted to the VHIII subgroup while the VL can be divided into two categories. The most common VL, expressed in > 90% of adults and usually constituting the majority of a subjects anti-Hib-PS antibody response, is restricted to the product of a single V kappa II gene known as A2 that probably lacks somatic mutations. The product of the A2 gene is invariably joined to one of several J kappa products by an inserted arginine at the V kappa-J kappa junction. In contrast to the restricted nature of the dominant VL clonotype, the second category of VL constitutes a heterogeneous group of at least seven different VL gene products that often contain somatic mutations and generally exhibit crossreactivity with a related polysaccharide from E. coli. Elucidation of anti-Hib-PS V regions at the molecular level will permit examination of structure-function relationships among these clinically important antibodies and should make the V region repertoire to Hib-PS a useful model for studying human V gene responses.


Role of K+ channels in the modulation of cholinergic neural responses in guinea-pig and human airways. 1. Several agonists modulate cholinergic neurotransmission in airways raising the possibility that there may be a common inhibitory mechanism, such as the activation of a common K+ channel in the nerve ending. To test this hypothesis, we examined whether blockers of K+ channels are able to depress the prejunctional inhibitory modulation of cholinergic contractile responses by various agonists in guinea-pig and human airways in vitro. 2. Electrical field stimulation (40 V, 0.5 ms) was applied to guinea-pig (0.5 Hz) or human (1 Hz) tracheal strips every 4 min to elicit cholinergic neural responses. The effects of the K+ channel blockers, charybdotoxin (ChTX, 10 nM), apamin (100 nM) and glibenclamide (1 microM), on the prejunctional inhibition of cholinergic contraction evoked by neuropeptide Y (NPY, 100 nM), an alpha 2-agonist, clonidine (10 nM), a mu-opioid agonist, [D-Ala2, NMePhe4, Gly-ol5]-enkephalin (DAMGO, 100 nM), and a KATP channel opener, lemakalim (300 nM) were tested in guinea-pigs. In human tissues, the effect of ChTX (10 nM) on the mu-opioid (DAMGO, 300 nM)-induced inhibition of cholinergic nerves was examined. 3. In guinea-pigs, ChTX (10 nM) significantly reversed the prejunctional inhibition of cholinergic contraction by NPY (84.2 +/- 16.2%), clonidine (71.9 +/- 22.4%), DAMGO (67.3 +/- 13.1%) and lemakalim (20.9 +/- 9.4%) (n = 5, P < 0.05, respectively), while apamin (100 nM) had no effect. Glibenclamide (10 microM) reduced only the lemakalim-induced inhibitory modulation. ChTX (10 nM) itself potentiated cholinergic contraction (24.6 +/- 9.4%, n = 5, P < 0.05) without affecting exogenously applied acetylcholine dose-response curves. Pretreatment with ChTX (10 nM) significantly reduced the inhibitory modulation of cholinergic nerves by NPY, clonidine and DAMGO, but not by lemakalim. 4. In human tissues, ChTX significantly reduced DAMGO-induced prejunctional inhibition of cholinergic contraction (13.6 +/- 8.5% with and 46.5 +/- 5.5% without ChTX, respectively; n = 5, P < 0.05). 5. These results may support a hypothesis that the activation of ChTX-sensitive K+ channels is involved in the inhibitory modulation of cholinergic neuro-transmission by agonists acting on presynaptic receptors in guinea-pig and human airways.


Calcium homeostasis in the outer segments of retinal rods from the tiger salamander. 1. The processes regulating intracellular calcium in the outer segments of salamander rods have been investigated. The main preparation used was the isolated rod loaded with the Ca(2+)-sensitive photoprotein aequorin, from which outer segment membrane current and free [Ca2+]i could be recorded simultaneously. Two other preparations were also used: outer segment membrane current was recorded from intact, isolated rods using a suction pipette, and from detached outer segments using a whole-cell pipette. 2. Measurements of free intracellular [Ca2+] in Ringer solution were obtained from two aequorin-loaded rods. Mean [Ca2+]i in darkness was 0.41 microM, and after a bright flash [Ca2+]i fell to below detectable levels ( < 0.3 microM). No release of intracellular Ca2+ by a bright flash of light could be detected ( < 0.2 microM). 3. Application of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) caused an increase in the size of the light-sensitive current and a rise in [Ca2+]i, but application of IBMX either when the light-sensitive channels had been closed by a bright light or in the absence of external Ca2+ caused no detectable rise in [Ca2+]i. It is concluded that IBMX increases [Ca2+]i by opening light-sensitive channels, and does not release Ca2+ from stores within the outer segment. 4. Removal of external Na+ caused a rise in [Ca2+]i to around 2 microM and completely suppressed the light-sensitive current. 5. The Na(+)-Ca2+, K+ exchange current in aequorin-loaded rods was activated in first-order manner by internal free calcium, with a mean Michaelis constant, KCa, of 1.6 microM. 6. The KCa of the Na(+)-Ca2+, K+ exchange was increased by elevating internal [Na+]. 7. The Michaelis relation between [Ca2+]i and the activity of the Na(+)-Ca2+, K+ exchange was used to calculate the change in [Ca2+]i occurring during the response to a bright light. In aequorin-loaded rods in Ringer solution the mean change in free [Ca2+]i after a bright flash was 0.34 microM. In these rods 10% of the dark current was carried by Ca2+. 8. Most of the calcium entering the outer segment was taken up rapidly and reversibly by buffer systems. The time constant of equilibration between free and rapidly bound Ca2+ was less than 20 ms. No slow component of calcium uptake was detected. 9. Two components of calcium buffering could be distinguished in the outer segments of aequorin-loaded rods.(ABSTRACT TRUNCATED AT 400 WORDS)


Glutamate receptor channels in isolated patches from CA1 and CA3 pyramidal cells of rat hippocampal slices. 1. Currents activated by glutamate receptor (GluR) agonists were recorded from outside-out patches isolated from the soma of visually identified pyramidal neurones of the CA3 and CA1 region of rat hippocampal slices. alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), L-glutamate (L-Glu), and kainate (KA) were delivered either by bath application through perfusion of the recording chamber or by rapid application via a piezo-driven two-barrelled fast application system. 2. Bath application of each of the three agonists activated inward currents in all patches (n = 134) at holding potentials of -50 or -60 mV. The current amplitude increased in size between 3 to 30 microM-AMPA and 100 microM to 1 mM-KA. With this slow mode of bath application, the responses showed no apparent desensitization even at saturating concentrations of AMPA (30 microM) and KA (1 mM). 3. The ratio of currents activated by 30 microM-AMPA and 300 microM-KA showed a characteristic difference between CA3 and CA1 neurones. The ratio was 0.242 +/- 0.028 (mean +/- S.E.M., n = 16) for CA3 cell patches and 0.097 +/- 0.012 (n = 8) for CA1 cell patches indicating that GluRs in the two cell populations are different. 4. The steady-state current-voltage relations (I-Vs) for AMPA- and KA-activated currents showed pronounced outward rectification for both cell types (when the main cations are Na+ in the bath and Cs+ in the pipette solution). The current reversed close to 0 mV and the ratio of chord conductances 80 mV on either side of the reversal potential was 2.66 for KA-activated currents in CA3 cell patches and 2.60 in CA1 cell patches. AMPA-activated currents showed a time-dependent increase after steps to positive membrane potentials and a decrease after steps to negative voltages, indicating that a gating process is responsible for outward rectification of the steady-state I-V. 5. The permeability (P) of GluR channels was high for Na+ as compared to Cs+ for both cell types (PNa/PCs = 0.88 and 0.84). The permeability was low for N-methyl-D-glucamine+ (PNMG/PCs < or = 0.03) and Ca2+ (PCa/PCs < or = 0.05). 6. The current noise level increased during application of AMPA or KA. Apparent single-channel conductances obtained from fluctuation analysis were higher for AMPA than for KA, but similar for both cell types. In CA3 cell patches, AMPA activated channels with an apparent chord conductance of 7.2 pS, KA of 3.0 pS conductance.(ABSTRACT TRUNCATED AT 400 WORDS)


Dependence of electrical slow waves of canine colonic smooth muscle on calcium gradient. 1. The ionic dependence of the upstroke and plateau components of slow waves of canine colonic circular muscles was studied. 2. Reduced extracellular Ca2+ caused a decrease in the amplitude of the upstroke and plateau components, a decrease in the depolarization velocity, and a decrease in frequency. The reduction in the upstroke phase per 10-fold reduction in external Ca2+ was close to the value predicted by the Nernst relationship, suggesting that the membrane permeability to Ca2+ increases steeply during this phase. 3. Nifedipine (10(-9)-10(-6)) reduced the plateau component, but concentrations of 10(-6) M did not abolish the upstroke component. The data suggest that a nifedipine-resistant component of Ca2+ current may be involved in the upstroke. 4. Inorganic Ca2+ channel blockers (Mn2+ and Ni2+) blocked spontaneous slow waves at concentrations of 1.0 mM or less. 5. The upstroke component was more sensitive to Ni2+ than to Mn2+; a concentration of 0.040 mM-Ni2+ caused more than a 50% reduction in upstroke velocity. Ni2+ also reduced the plateau phase of slow waves. 6. The results suggest that the upstroke and plateau components of slow waves are dependent upon activation of voltage-dependent Ca2+ currents. The current responsible for the upstroke is partially resistant to dihydropyridines (at least at 10(-6) M). The current responsible for the plateau component is nifedipine-sensitive.


Upstroke component of electrical slow waves in canine colonic smooth muscle due to nifedipine-resistant calcium current. 1. Electrical slow waves of gastrointestinal smooth muscles are not abolished by organic Ca2+ channel blocking drugs, such as nifedipine or D600. These compounds reduce the amplitude and duration of the plateau phase, but the upstroke phase of slow waves persists. 2. Voltage clamp experiments were performed on isolated circular muscle cells from the canine proximal colon to characterize the dihydropyridine-resistant component of inward current. Inward currents were measured at 25 and 35 degrees C. The higher temperature increased the amplitudes of the transient and sustained phases of the inward current. The voltage dependence of activation and inactivation of the inward current was not significantly changed at 35 vs. 25 degrees C. 3. At 35 degrees C the transient phase of the inward current was reduced but not blocked by nifedipine (10(-6) M). The sustained phase was blocked by nifedipine. 4. The block by nifedipine was voltage dependent, increasing with depolarization. At voltages reached during the upstroke depolarization about 35% of the inward current persisted in the presence of nifedipine (10(-6) M). This may be sufficient inward current to sustain the upstroke depolarization in intact muscles. 5. Nifedipine caused a 20 mV negative shift in the voltage dependence of inactivation suggesting that dihydropyridines may preferentially bind to Ca2+ channels in an inactivated state. 6. Ni2+ (< 100 microM) significantly decreased the transient phase of inward current. A combination of Ni2+ (40 microM) and nifedipine (10(-6) M) blocked all of the inward current at 35 degrees C. Combination of nifedipine (10(-6) M) and Ni2+ (40 microM) blocked slow waves in intact muscles. 7. Bay K 8644 (10(-6) M) increased the amplitude of the transient and sustained components of inward current. On a percentage basis the increase in the sustained component was greater than the increase in the transient component with test potentials in the range of -50 to -20 mV. This may explain why Bay K 8644 preferentially increases the plateau component of slow waves vs. the upstroke component. 8. The findings of this study suggest that the nifedipine resistance of the upstroke depolarization could be due to the voltage dependence of the block of Ca2+ channels by dihydropyridines. Thus a single class of voltage-dependent Ca2+ channels could be responsible for the upstroke and plateau phases of slow waves.


A patch clamp study of the nicotinic acetylcholine receptor of bovine adrenomedullary chromaffin cells in culture. 1. Acetylcholine-induced currents recorded from bovine adrenal medullary chromaffin cells maintained in culture were studied during pressure or ionophoretic applications of ACh, using the 'whole-cell' and 'outside-out' configurations of the patch clamp technique. In standard salines, ACh evoked whole-cell currents of -38 pA to -1 nA at -60 mV, which had a reversal potential (EACh) of -7.1 +/- 0.6 mV. The ACh current-voltage relationship was characteristically linear at negative holding potentials and biphasic at positive holding potentials, displaying a region of almost zero slope conductance between 0 and +40 mV followed by a region of positive slope conductance at more positive potentials. 2. Relative permeation to cations was examined. Substitution of external Na+ by sucrose resulted in a -42 mV shift of EACh for a 10-fold reduction in [Na+]o. Using isotonic substitutions, the permeability ratios (relative to Na+) for monovalent cations were determined to be 1.32 +/- 0.02 for Cs+ (n = 11), 1.03 +/- 0.02 for Li+ (n = 8) and 0.18 +/- 0.02 for Tris+ (n = 7). Elevated external Ca2+ salines were found to shift EACh to more positive potentials, especially in the presence of low external Na+. 3. The nicotinic agonists nicotine, tetramethylammonium and lobeline evoked inward currents in bovine chromaffin cells. In contrast, decamethonium and the muscarinic agonist, methacholine, had no effect. 4. The nicotinic antagonists mecamylamine, trimetaphan, (+)-tubocurarine and hexamethonium caused dose-dependent reductions in the amplitude of ACh-evoked inward currents. The estimated IC50's were 0.25, 0.33, 0.63 and 2.2 microM respectively, for cells voltage clamped at -60 mV. High concentrations (> 2 microM) of the muscarinic antagonist, atropine, also produced a dose-dependent reduction in the amplitude of ACh-induced currents. 5. Inhibition by trimetaphan was voltage independent. With the other drugs the antagonism was voltage sensitive, increasing with membrane hyperpolarization. The voltage sensitivity was most marked for hexamethonium. Neither hexamethonium nor mecamylamine were found to depress ACh-evoked outward currents at concentrations which severely depressed inward currents. In addition to its antagonist actions, (+)-tubocurarine activated unitary currents in these cells and on isolated membrane patches. 6. The results indicate that nicotinic ion channels of bovine chromaffin cells have a similar ionic selectivity to monovalent cations, but that Ca2+ ions permeate the channels to a greater degree than at the motor endplate. The ACh current-voltage relationship resembles that described for other types of 'neuronal' nicotinic receptors.(ABSTRACT TRUNCATED AT 400 WORDS)


The gp120 glycoprotein of HIV-1 binds to sulfatide and to the myelin associated glycoprotein. We investigated the binding of the gp120 glycoprotein of the human immunodeficiency virus (HIV-1) to neural glycolipids and glycoproteins by ELISA. The gp120 protein bound to sulfatide (GalS), a sulfated glycolipid autoantigen implicated in sensory neuritis, and to the myelin associated glycoprotein (MAG), an autoantigen in demyelinating neuropathy. Binding of gp120 to MAG was inhibited by the HNK-1 antibody, which recognizes a sulfated glucuronic acid epitope, suggesting that the interaction involves carbohydrate determinants. Sulfatide and MAG are potential receptors for gp120 in peripheral nerve and may have a role in the neuropathy associated with HIV-1 infection.


Specific binding of cytotactin to sulfated glycolipids. The binding of the glial glycoprotein, cytotactin, to a variety of purified glycolipids was examined. Clear-cut evidence was found for binding of radiolabeled cytotactin to sulfatides purified from bovine brain, but the molecule did not bind to gangliosides or cerebrosides. The sulfatide binding was sensitive to pH and ionic strength and was dependent on the presence of divalent cations. Binding was inhibited by purified unlabeled cytotactin, by polyclonal antibodies to cytotactin, and by several monosaccharides and polysaccharides. It was not inhibited by fibronectin, a chondroitin sulfate proteoglycan, or the HNK-1 monoclonal antibody, all of which are known to bind to cytotactin. These findings raise the possibilities that sulfated glycolipids may function as cellular receptors for cytotactin and that binding by sulfatides may modulate the varied effects of cytotactin on cellular processes.


L2/HNK-1 carbohydrate and protein-protein interactions mediate the homophilic binding of the neural adhesion molecule P0. The neural adhesion molecule P0, the most abundant glycoprotein in peripheral myelin of mammals, is a member of the immunoglobulin superfamily and expresses the L2/HNK-1 and L3 oligosaccharides at a single N-glycosylation site. It acts in both homophilic and heterophilic binding mechanisms. To investigate the molecular requirements for homophilic interaction, we have used P0 from human sciatic nerve and the extracellular domain of P0 expressed in bacteria to determine binding of P0 to P0 in solid phase and bead aggregation assays. The binding of P0 to P0 could be partially inhibited in both assays by antibodies to the L2/HNK-1 epitope and by the L2/HNK-1 carbohydrate, but not by L3 antibodies or other carbohydrates. Inhibition of binding was also seen with polyclonal antibodies reacting with the protein backbone of P0. These observations indicate that both carbohydrate and protein structures are involved in the binding of P0 to P0 and that P0 acts as a presenter of and a receptor for a functionally important carbohydrate.


[Detection of glycosylated protein of renal tissues using NBT reaction in STZ-induced diabetic rats]. We are carried out to determine the detection of glycosylated protein of renal tissues using nitroblue tetrazolium (NBT) reaction in streptozotocin (STZ)-induced diabetic rats. The levels of glomerular non-enzymatic glycosylation were measured by thiobarbituric acid (TBA) method. There was no significant difference in the intensity of NBT in renal tissues between the 4-week diabetic rats and the same age control rats. The intensity of NBT in the glomerular mesangial areas and capillary walls was marked in 12-week diabetic rats. The staining of NBT was also observed in the tubular epithelial cells in 4- and 12-week diabetic rats. The levels of glomerular non-enzymatic glycosylated protein in 4- or 12-week diabetic rats were significantly greater than those in the same age control rats. It appears that the non-enzymatic glycosylation of renal tissues increased in the early stage of diabetic nephropathy, i.e. 4- or 12-week diabetic rats. It is concluded that the non-enzymatic glycosylation may play an important role in the initiation of renal injuries in diabetic nephropathy.


Molecular cloning of human endothelin (ET) receptors ETA and ETB. Complementary DNAs coding for two different endothelin (ET) receptors were isolated from a human lung cDNA library. DNA sequencing revealed that they corresponded to either the ET-1-selective receptor subtype originally identified in cows (ETA) or the nonselective subtype first found in rats (ETB). An open reading frame coding for a 427 (ETA) or a 442 (ETB) amino acid-long protein, with a transmembrane topology also found in other G protein-coupled receptors, was determined. Whereas the amino acid identity is about 90% between the characterized members of a given receptor subtype, with the best conserved parts being the hydrophobic transmembrane domains, less than 60% identity was found between the human A and B subtypes. Southern blot analysis of human genomic DNA revealed the existence of multiple hybridizing bands when using probes derived from the full-length ETA or ETB cDNAs. However, when shorter fragments were selected as probes, only one band remained detectable, which suggests the existence of a single-copy gene for each subtype of ET receptor.


Bradykinin-induced, N omega-nitro-L-arginine-insensitive endothelium-dependent relaxation of porcine coronary arteries is not mediated by bioassayable relaxing substances. The effect of the arginine analogue, N omega-nitro-L-arginine (L-NNA) was studied on bradykinin-induced relaxation in porcine coronary arteries. In the presence of indomethacin (3 x 10(-6) M) and captopril (10(-6) M), treatment with L-NNA (10(-4) M) had no effect on the bradykinin-induced (10(-10)-10(-7) M) relaxations in strips contracted with U-46619. In contrast to the findings in organ chamber experiments, bradykinin-induced release of endothelium-derived relaxing factor(s) (EDRFs) was abolished after 45 min of treatment of perfused porcine coronary artery segments with L-NNA (10(-4) M) in a superfusion bioassay system. These results show that, in addition to the release of nitric oxide, endothelium-dependent relaxation of porcine coronary arteries to bradykinin involves an alternative mechanism(s), which accounts for the relaxation in the presence of L-NNA. Since the release of a relaxing mediator could not be detected from L-NNA-treated porcine coronary artery segments under bioassay conditions, it is postulated that either no diffusible factor(s) is involved in the L-NNA-insensitive endothelium-dependent relaxation, or it is mediated by an extremely labile endothelium-derived substance(s).


N omega-nitro-L-arginine blocks the second phase but not the first phase of the endothelium-dependent relaxations induced by substance P in isolated rings of pig carotid artery. Endothelium-dependent relaxations can be evoked by a variety of stimuli, among them substance P (SP), which is found in sensory nerve fibers supplying the adventitia-media junction of most muscular arteries. This study determined the role of endothelium-derived nitric oxide as a mediator of endothelium-dependent relaxations to SP in isolated rings of the pig carotid artery suspended in organ chambers for isometric tension recording. SP (10(-12)-10(-7) M) caused concentration-dependent relaxations of arteries precontracted with norepinephrine (10(-7) M). The relaxations were characterized by a partially transient relaxation (phase 1) and a sustained relaxation of the artery (phase 2). The inhibitor of nitric oxide formation, N omega-nitro-L-arginine (L-NNA) methyl ester caused a gradual increase in tension, the phase I response at 3 x 10(-10) to 3 x 10(-7) M SP was shifted to the right, but the maximal relaxation was comparable in the presence of L-NNA. However, the sustained relaxation after addition of substance P (phase II) was lost and tension in the presence of L-NNA returned to a level above that induced by L-NNA and norepinephrine (10(-9) M). These results suggest that the endothelium-dependent relaxations to SP, particularly the prolonged relaxation (phase II), are due to de novo synthesis of nitric oxide and hence fully abolished by a specific inhibitor.


Biphasic response to substance P in canine basilar arteries. The responses to intraluminally applied substance P (SP) were examined in isolated and perfused canine basilar arteries using the stainless-steel cannula inserting method. In control vessels with intact endothelium, this peptide induced a monophasic dilation at lower doses, and a biphasic response, i.e., an initial dilation followed by a secondary constriction at higher doses. After extraluminal treatment with oxyhemoglobin, the dilation was attenuated and the constriction was augmented. After endothelial removal with intraluminal saponin, the dilation was reduced and the constriction was enhanced significantly. This potentiated constriction was significantly depressed by indomethacin (a cyclooxygenase inhibitor), OKY-046 (a thromboxane synthetase inhibitor), and nimodipine (a calcium antagonist), but not by AA-861 (a lipoxygenase inhibitor). These results suggest that SP has two distinct effects (an endothelium-dependent dilation and a direct constriction) and that the potentiated constriction in the absence of endothelium may be related to the action of thromboxane A2, linked with calcium influx into the smooth muscle cells of cerebral arteries. This mechanism may be implicated in cerebral vasospasm after subarachnoid hemorrhage.


In vitro biological profile of a highly potent novel endothelin (ET) antagonist BQ-123 selective for the ETA receptor. The novel endothelin (ET) receptor antagonists BE-18257A and BE-18257B were isolated from the fermentation products of Streptomyces misakiensis. The above-mentioned compounds inhibited [125I]ET-1 binding to ETA receptors (selective for ET-1) on porcine aortic vascular smooth muscle cells (VSMCs) with IC50 values of 1.4 and 0.47 microM, respectively. [125I]ET-1 binding to ETB receptors (nonselective to ET isopeptides) in cerebellar membranes was not inhibited by either of these compounds even at 100 microM. The synthesized analogue BQ-123 induced extremely potent inhibition of [125I]ET-1 binding to ETA receptors (IC50 of 7.3 nM), but it barely inhibited [125I]ET-1 binding to ETB receptors (IC50 of 18 microM) and binding of various other peptides to their receptors. BQ-123 shifted the concentration-response curve for ET-1 toward the right in porcine isolated coronary arteries, indicative of competitive antagonism for the ETA receptor. However, there was a small amount of BQ-123-insensitive vasoconstriction that paralleled the incomplete inhibition of [125I]ET-1 binding in the membrane of the vascular smooth muscle layer. These data suggest that the artery contracts via both ETA and ETB receptors and that BQ-123 selectively inhibits ETA-mediated contraction. Furthermore, BQ-123 revealed large tissue and species differences in the distribution of ETA receptors. Thus, the potent ETA antagonist BQ-123 should be useful in clarifying the (patho)physiological roles of ETA receptors.


Endothelium-dependent contraction produced by acetylcholine and relaxation produced by histamine in monkey basilar arteries. The present experiments were carried out to investigate the endothelium dependence of the responses to acetylcholine (ACh), arachidonic acid, and histamine in monkey basilar arteries. ACh and arachidonic acid caused endothelium-dependent contraction (EC) in both monkey and canine basilar arteries. The endothelium-derived contracting factor (EDCF) was probably thromboxane A2 (TxA2), as the EDC was attenuated by a cyclooxygenase inhibitor, TxA2 synthetase inhibitors, and TxA2 antagonists. On the other hand, histamine caused endothelium-dependent relaxation (EDR) in monkey and EDC in canine basilar arteries. The EDR in monkey basilar arteries was attenuated by a nitric oxide synthase inhibitor. The EDR and EDC were antagonized by tripelennamine but not by cimetidine, indicating that they are mediated by H1-receptors. From these results, we suggest that in the monkey basilar artery, either there are two types of endothelium (an EDCF type for ACh and arachidonic acid and an EDRF type for histamine) or there is a single type of endothelium with two types of signalling processes (one for EDC and one for EDR).


Different mechanisms of hypoxic relaxation in canine coronary arteries and rat abdominal aortas. Hypoxia causes complex changes in vascular tone of isolated blood vessels. This study was performed in rings with and without endothelium of rat abdominal aortas and canine coronary arteries suspended in organ chambers for isometric tension recording. In both aortic and coronary rings with endothelium precontracted with a half-maximal concentration of phenylephrine or prostaglandin F2 alpha, respectively, hypoxia induced transient relaxations (20 +/- 2 and 15 +/- 3%, respectively); removal of the endothelium prevented the response in aortas, but not coronary arteries. The transient hypoxic relaxation was followed in both preparations by endothelium-dependent contractions (EDCs). Hypoxic relaxations were prevented by indomethacin (10 microM) in canine arteries, but not in rat aortas. The inhibitor of nitric oxide (NO) synthase, N omega-nitro-L-arginine (30 microM), inhibited hypoxic relaxations in intact rat aortas, but left those in coronary arteries unchanged. Similar results were obtained with methylene blue and LY 83583. In preparations without endothelium, sodium nitroprusside (30 nM) elicited a reappearance of hypoxic relaxations in the rat but not the dog coronary artery. Thus, hypoxic relaxation is mediated by a prostaglandin in the dog coronary artery, but by endothelium-derived NO in the rat aorta. As the response was dependent on the level of contraction, this suggests that the release or action of NO decreases with increasing tone.


Calcium activation of hyperpolarization response to acetylcholine in coronary endothelial cells. Hyperpolarization response to acetylcholine (ACh) in endothelial cells was studied by intracellular recording of an intact endothelium preparation of guinea pig coronary artery. The hyperpolarization requires the presence of extracellular Ca2+ and the response was progressively blocked by MnCl2 or by removal of extracellular Ca2+. The intracellular Ca2+ antagonist, 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester hydrochloride (TMB-8, 10 microM), was also effective in abolishing the hyperpolarization. Cyclopiazonic acid, an inhibitor of the sarcoplasmic reticulum Ca2+ pump, potentiated the hyperpolarization to ACh but inhibited caffeine-induced hyperpolarization. These findings suggest that ACh-induced hyperpolarization is mediated by an increase in cytosolic Ca2+ due to influx of extracellular Ca2+ and release of Ca2+ from an internal IP3-sensitive store. The rise in cytosolic Ca2+ level was modulated by a caffeine-sensitive pool that is inhibited by cyclopiazonic acid.


Endothelial modulation of DNA synthesis in isolated arteries of the rat. The endothelium can modulate the supply of growth factors to the underlying smooth muscle. In vitro experiments suggest that it may also influence the responsiveness of arterial smooth muscle to mitogens. In these experiments, we measured DNA synthesis in segments of carotid and renal arteries that were isolated from Wistar-Kyoto (WKY) rats and exposed to serum. Nuclear incorporation of the thymidine analogue, 5-bromo-2'-deoxyuridine (BrdUrd), was visualized by immunocytochemistry and the percentage of labeled nuclei (BrdUrd L%) was determined in the tunica media. In both types of artery isolated from 6- and 20-week-old WKY rats, mechanical removal of endothelium increased the BrdUrd L% in the tunica media. In carotid arteries of 20-week-old WKY rats, gentle denudation increased the incorporation of [3H]thymidine but not [14C]leucine. In denuded renal arteries of adult WKY rats, exogenous prostaglandin E2, iloprost, and transforming growth factor-beta (TGF-beta) reduced media labeling, which was not affected by Na nitroprusside. In renal arteries with endothelium, methylene blue and indomethacin did not affect medial DNA synthesis. These findings demonstrate that in arteries of young and adult rats, the endothelium reduces stimulated DNA synthesis. It is unlikely that basal production of nitric oxide or prostaglandins is involved herein. Endothelial inhibition of DNA synthesis but not protein synthesis in arteries indicates that the endothelium may influence the extent of arterial smooth muscle hypertrophy and hyperplasia.


Vascular smooth muscle proliferation in hypertensive transgenic rats. In vascular smooth muscle cell (VSMC) cultures from Sprague-Dawley (SD) and hypertensive transgenic rats for the mouse renin gene Ren-2 (TGR), the DNA synthesis, which was analyzed by the uptake of [3H]thymidine, was higher in TGR than SD VSMCs (2.5- to 8-fold, mean of 5.6-fold) under basal conditions. DNA synthesis was increased by fetal calf serum (10%) in SD cells more than in TGR VSMCs, and was decreased by heparin (400 micrograms/ml) and by phorbol-12,13-dibutyrate (10(-7) M) in TGR VSMCs to a higher degree than in SD cells. Neither endothelin (10(-7) M), angiotensinogen (10(-8) M), the renin inhibitor CGP 29,287 (10(-4) M), angiotensin I (10(-7) M), captopril (10(-5) M), angiotensin II (10(-7) M), nor saralasin (10(-6) M) modified DNA synthesis in either type of VSMCs. Sodium nitroprusside (10(-4) and 10(-3) M) increased DNA synthesis in both kinds of VSMCs but in TGR cultures it became toxic at 10(-3) M. 8-Bromocyclic GMP (10(-7) to 10(-5) M) reduced DNA synthesis in SD cells more than in TGR VSMCs. These results suggest that (a) cellular mechanisms of proliferation appear to be more activated in TGR VSMCs, likely involving a protein kinase C-dependent pathway but not the renin-angiotensin system, and (b) in both type of cells, sodium nitroprusside possesses proliferative properties whereas 8-bromocyclic GMP has antiproliferative properties.


The role of nitric oxide in endotoxic shock: effects of NG-monomethyl-L-arginine. The role of nitric oxide (NO) in the changes in blood pressure and plasma levels of nitrate and nitrite (NOx) and alanine aminotransferase (ALT) were determined over a 5-h period in anesthesized rats after intravenous administration of S. typhosa endotoxin (LPS, 4 mg/kg). Rats treated with LPS showed a sustained fall in blood pressure accompanied by an increase in plasma NOx and ALT. Forty percent of these rats died during the experiment. There was no change in blood pressure in rats treated with dexamethasone (1 mg/kg) 1 h before and 2 h after LPS and the increase in NOx and ALT was significantly inhibited. None of the rats in this group died. Administration of 10 mg/kg of NG-monomethyl-L-arginine (L-NMMA) prevented the fall in blood pressure and partially prevented the increase in NOx and ALT. None of the animals in this group died. In contrast, 300 mg/kg of L-NMMA caused an initial increase in blood pressure followed by a rapid fall and enhanced the increase in ALT while abolishing the elevation of NOx. All of these animals died before the end of the experiment. However, when rats treated with high doses of L-NMMA were given a continuous infusion of S-nitroso-N-acetylpenicillamine (SNAP, 300 micrograms/kg/h), the blood pressure was maintained at control levels and no mortality was observed.(ABSTRACT TRUNCATED AT 250 WORDS)


Endothelium-derived kinins account for the immediate response of endothelial cells to bacterial lipopolysaccharide. We have recently shown that cultured endothelial cells produce kinins that can stimulate endothelial nitric oxide (NO) production in an autocrine manner. Because both the kallikrein-kinin system and the L-arginine/NO pathway have been implicated in the pathogenesis of septic shock, we investigated the possible involvement of endothelium-derived kinins in the response of cultured endothelial cells to bacterial lipopolysaccharide (LPS). In primary cultures of human umbilical vein and porcine aortic endothelial cells, LPS (0.3 to 3 micrograms/ml) induced significant concentration-dependent increases in cyclic GMP and 6-keto-PGF1 alpha, both of which were abolished in the presence of the selective bradykinin B2-receptor antagonist HOE 140 (0.1 microM). These LPS-induced increases in cyclic GMP and 6-keto-PGF1 alpha were short lived, being maximal after 5 min but were not apparent after 60 min. In parallel with these effects, LPS (30 micrograms/ml) induced a distinct, HOE 140-sensitive increase in the intracellular calcium concentration of human endothelial cells loaded with indo-1. In summary, these data suggest that the release of endothelium-derived kinin and subsequent stimulation of endothelial cells, followed by the enhanced production of NO and prostacyclin (PGI2), are implicated in the immediate hypotension induced by LPS in vivo.


Characterization of a microsomal calcium-dependent nitric oxide synthase in activated J774.2 monocyte/macrophages. The nitric oxide (NO) synthase activity present in murine J774.2 monocyte/macrophages was characterized in terms of its intracellular localization, substrate specificity, and Ca2+ dependency. Traces of constitutive NO synthase activity were found in the microsomal fraction from noninduced J774.2 cells, whereas no NO synthase activity was detected in the cytosol. After 24 h in the presence of bacterial lipopolysaccharide and mouse interferon, NO synthase activity was substantially increased in both fractions with 51-60% of the total activity present in the cytosol. These activities, however, were clearly different, for the microsomal enzyme was Ca2+ dependent, whereas the cytosolic NO synthase was not. Moreover, NG-hydroxy-L-arginine (L-HOArg), L-homo-arginine, and several L-arginine (L-Arg)-containing dipeptides could replace L-Arg as substrates for the Ca(2+)-independent NO synthase, whereas the Ca(2+)-dependent enzyme accepted only L-Arg, L-HOArg, or L-Arg-L-Arg as substrates. Thus, a microsomal Ca(2+)-dependent NO synthase is induced in J774.2 monocyte/macrophages with a substrate specificity different from the inducible Ca(2+)-independent NO synthase as well as the constitutive NO synthase in, for example, endothelial cells. Irrespective of their intracellular localization, therefore, at least three isoforms of NO synthase exist, all of which can accommodate substrates different from L-Arg in size, charge, and hydrophobicity.


The inducible nitric oxide synthase is impaired by thrombin in vascular smooth muscle cells. The present study investigated whether or not thrombin may affect the induction of nitric oxide (NO) synthase caused by interleukin-1 beta in cultured smooth muscle cells (SMCs) from the rat aorta. The release of nitrite, an oxidation product of NO, from interleukin-1 beta-activated SMCs was inhibited by thrombin. The inhibitory effect of thrombin was prevented by hirudin, a thrombin inhibitor, and required the presence of thrombin during the induction period. Under bioassay conditions, the perfusate from interleukin-1 beta-activated SMCs relaxed endothelium-denuded rat aortic rings precontracted with phenylephrine. The perfusate from untreated SMCs or cells exposed to thrombin alone or to interleukin-1 beta in combination with thrombin relaxed only minimally the detector blood vessel. These observations demonstrate that thrombin inhibits the production of NO by the inducible NO synthase in cultured vascular SMCs.


Lipoteichoic acid: a new inducer of nitric oxide synthase. Inducible nitric oxide (NO) synthase in vascular smooth muscle cells (SMCs) appears to play a major role for the diminished responsiveness to vasoconstrictors observed in endotoxemia. However, cardiovascular dysfunctions associated with septic shock are also observed in the absence of endotoxin (LPS). Similar hemodynamic changes are produced either by a gram-negative bacteria (Escherichia coli) or by a gram-positive bacteria (Staphylococcus aureus), a microorganism without LPS, suggesting a common pathway leading to cardiovascular abnormalities. In the present study, we describe the induction of NO synthase in vascular SMCs by lipoteichoic acid (LTA), a component of the membrane of gram-positive bacteria. In cultured vascular SMCs, a 24-h incubation with LTA produced an increase in intracellular cyclic GMP. This effect was inhibited by methylene blue (MB), an inhibitor of guanylate cyclase. Incubation with a specific inhibitor of L-arginine, i.e., NG-nitro-L-arginine methyl ester (L-NAME), or depletion of L-arginine attenuated the LTA-induced cGMP production. A 5-h incubation of endothelium-free rings of rat aorta in the presence of LTA induced a loss of tonicity to the contractile response of phenylephrine. The contractions were restored by MB and by L-NAME. The effect of L-NAME was reversed by L-arginine. These results show that LTA, like LPS, expresses NO synthase in vascular SMCs.


Exogenous nitric oxide stress on endothelial cells and macrophages. Bovine endothelial cells (ECs, P1) and lipopolysaccharide/gamma-interferon-induced mouse macrophages (MMs) were incubated in the presence of SIN-1 and C 3754 (1 microM to 1 mM), sydnonimine metabolites of the antianginal predrugs molsidomine and pirsidomine, respectively up to 48 h. No change of the endogenous nitric oxide output from MMs and A23187- or adenosine triphosphate-stimulated ECs was found by means of the methemoglobin method. Data indicate that downregulation of the nitric oxide (NO) synthase is not obvious within the intact cells under exogenous NO stress supplied by high concentrations of the spontaneous NO donors. Cytosolic MM NO synthase extracts, however, revealed reduction in the enzymic [3H]arginine turnover to [3H]citrulline by SIN-1, but not by C 3786, the pharmacologically active metabolite of pirsidomine.


Modulation of arterial endothelin-1 receptors following chronic increases in blood flow. Experiments were designed to determine whether or not increased contractions to endothelin-1 (ET-1) in arteries chronically exposed to increased blood flow were due to a change in the subtype, number, or affinity of ET receptors on the smooth muscle. A fistula was created between one femoral artery and vein in dogs; contralateral blood vessels were operated as shams. After 6 weeks, the femoral arteries were removed. In both fistula- and sham-operated arteries suspended for the measurement of isometric force in organ chambers, the contractile potency was ET-1 > ET-2 > ET-3. Specific binding of [125I]ET-1 as a function of membrane protein was significantly greater in fistula compared to sham-operated arteries (Bmax = 184.8 +/- 35.9 and 117.4 +/- 25.6 fmol/mg, n = 6, respectively). Affinity of binding was the same in each group of arteries. These results suggest that ETA receptors mediate contraction on canine femoral arterial smooth muscle. These receptors may be upregulated when the artery is exposed chronically to increased blood flow.


Balloon injury induces nitric oxide synthase activity in rat carotid arteries. Experiments were performed to investigate whether or not balloon injury induces nitric oxide (NO) synthase activity in the arterial wall removed. Contractions to phenylephrine were compared in left carotid arteries of the rat, previously injured by balloon catheterization and collected 24 h after the procedure, and in control right carotid arteries in which the endothelium was removed acutely. Phenylephrine evoked concentration-dependent contractions in balloon-injured and control carotid arteries without endothelium. However, in the balloon-injured carotid arteries, the concentration-contraction curves to phenylephrine were shifted significantly to the right compared to those observed in control carotid arteries without endothelium. In balloon-injured arteries, nitro-L-arginine restored full contractions, whereas superoxide dismutase potentiated the hyporesponsiveness to phenylephrine. The depressed contractions were associated with a concomitant increase in the basal level of guanosine-3',5'-cyclic monophosphate; this production was abolished by nitro-L-arginine. These observations indicate that in vivo endothelial injury of the rat carotid arteries induces the production of nitric oxide from L-arginine in the arterial wall.


Involvement of nitric oxide in tumor cell adhesion to cytokine-activated endothelial cells. The aim of the present experiments was to test the possible involvement of nitric oxide (NO) in cytokine-induced enhancement of tumor cell (TC) adhesion to endothelial cells (ECs). Exposure of EA hyb 926 cells to TNF (500 U/ml) plus IFN (100 U/ml) for 24 h significantly enhanced their adhesivity for the 51Cr-labeled GLC1 (small cell lung carcinoma) TCs. Conversely, exposure of TCs to cytokines did not result in an increased adhesion of these cells to ECs. TC-stimulated adhesion to EA hyb 926 was abrogated by the glucocorticoid dexamethasone (Dex, 10(-7) M), the NO synthase inhibitors N omega-nitro-L-arginine methyl ester (L-NAME, 10(-5) M) and NG-monomethyl-L-arginine (L-NMMA, 10(-5) M) and the protein synthesis inhibitor cycloheximide (Cex, 10(-6) M). Furthermore, GLC1-stimulated adhesion to EA hyb 926 was reversed following removal of L-arginine from the medium or pretreatment with the guanylate cyclase inhibitor methylene blue. TC-stimulated adhesion was also prevented when TCs were pretreated with the monoclonal antibody CD15 directed against the endothelial-leukocyte adhesion molecule (ELAM-1) ligand or following exposure of ECs to anti-ELAM-1 monoclonal antibody. Although suppressing TC-stimulated adhesion, L-NMMA failed to modify significantly cytokine-induced ELAM-1 expression in EA hyb 926. These results (a) provide evidence for the NO-inducible pathway contributing to cytokine-induced enhancement of tumor cell adhesion to the vascular endothelium and (b) demonstrate the involvement of the ELAM-1/CD15 adhesion system in tumor cell-stimulated adhesion to ECs.


Control of renal hemodynamics during intrarenal and systemic blockade of nitric oxide synthesis in conscious dogs. Systemic blockade of nitric oxide (NO) synthesis results in marked increases in the renal vascular resistance (RVR) and decreases in the glomerular filtration rate (GFR) and renal plasma flow (RPF). The renal hemodynamic effect of systemic NO blockade is most likely due to an integration of direct and indirect renal actions. The quantitative importance of local intrarenal blockade of NO synthesis on renal hemodynamics has not been fully elucidated. Therefore, the purpose of this study was to compare the renal hemodynamic effects of intrarenal vs. systemic NO synthesis blockade on renal hemodynamics. Nitro-L-arginine methyl ester (L-NAME) was infused intrarenally at a rate of 3 micrograms/kg/min for 180 min in conscious chronically instrumented dogs (n = 7). Intrarenal infusion of L-NAME for 180 min resulted in a 12% decrease in RPF, 14% increase in RVR, and no effect on mean arterial pressure (MAP) or GFR. In contrast, infusion of L-NAME intravenously at a rate of 10 micrograms/kg/min for 180 min increased the RVR by 124% and decreased the RPF by 55% and GFR by 45%. The MAP and heart rate both increased in response to intravenous administration of L-NAME. The results of this study suggest that the renal hemodynamic effects of systemic administration of L-NAME may, in large part, be secondary to extrarenal effects of NO synthesis blockade, possibly via activation of the sympathetic nervous system.


Endothelium-derived relaxing factor modulates renal interstitial cyclic GMP. The arterial vasodilator activity of endothelium-derived relaxing factor (EDRF) is mediated by activation of the soluble form of guanylate cyclase, causing increased levels of guanosine-3',5'-cyclic monophosphate (cGMP). Because of its extreme lability, the actions of EDRF are local. The ability to monitor changes in renal interstitial fluid cGMP would be of great advantage in clarification of local mechanisms controlling renal function. Utilizing a renal interstitial microdialysis technique, we investigated changes in renal interstitial and urinary cGMP in response to right intrarenal arterial administration of the EDRF inhibitor, NG-monomethyl-L-arginine (L-NMMA), in anesthetized dogs (n = 5) in metabolic balance at a sodium intake of 40 mEq/day. Urine was collected directly from the right and left ureter. L-NMMA at 20-60 micrograms/kg/min significantly decreased right renal interstitial and right urinary cGMP levels (p < 0.01) without changing left renal interstitial and urinary cGMP levels (p < 0.01). L-NMMA at 100 micrograms/kg/min decreased both right and left renal interstitial and urinary cGMP levels (p < 0.01). These data demonstrate the ability to monitor renal interstitial cGMP in vivo. There was a dose-dependent decrease in renal interstitial and urinary cGMP in response to intrarenal EDRF inhibition. Additionally, they suggest that EDRF acts as a renal paracrine substance through the modulation of renal interstitial cGMP.


Collateral perfusion: the role of endothelium-derived relaxing factor and effects of vasodilators. An in vitro rabbit ear model has been used to investigate the role of endothelium-derived relaxing factor (EDRF) in collateral perfusion after acute arterial occlusion and also the effects of vasodilators. Collateral perfusion of an arterial segment isolated between occlusions was assessed by x-ray microangiography and was found to develop in a time-dependent manner. Inhibition of EDRF synthesis with NG-nitro-L-arginine methyl ester (L-NAME) greatly impaired collateral perfusion, indicating that the development of collateral perfusion was dependent on EDRF activity. This inhibitory effect was reversed by an excess of L-arginine. Further studies using vasodilators with different modes of action indicated that BRL 38227 (the active enantiomer of cromakalim, a potassium channel activator) substantially enhanced collateral perfusion, sodium nitroprusside had early beneficial effects, and verapamil had no effect.


Functional hyperemia of skeletal muscle: role of endothelium. The role of the endothelium and endothelium-derived vasoactive factors in the development of skeletal muscle functional hyperemia was studied. Experiments were performed in anesthetized dogs. Functional hyperemia of isometrically contracted gastrocnemius muscle was produced by direct electric stimulation. Five series of experiments were conducted to investigate the effect of (a) de-endothelialization of gastrocnemius vessels, (b) inhibition of prostanoids biosynthesis by indomethacin, (c) inhibition by gossipol of lipoxygenase, which mediates biosynthesis of endothelium-derived relaxing factor (EDRF), (d) inhibition of cGMP formation by methylene blue, and (e) stimulation of EDRF biosynthesis of L-arginine. The gastrocnemius muscle work was accompanied by an increase in blood flow by 6.8 +/- 0.6 ml/min/N (8 Hz) and by 2.5 +/- 0.2 ml/min/N) (40 Hz). Chemical deendothelialization of gastrocnemius vessels decreased functional hyperemia, and blood flow increased by only 1.2 +/- 0.3 ml/min/N (8 Hz) and by 0.5 +/- 0.1 ml/min/N (40 Hz). Cyclooxygenase inhibition by indomethacin had no significant effect on functional hyperemia, whereas methylene blue and gossipol inhibited functional hyperemia by 4.2- and 6.6-fold, respectively, and L-arginine significantly elevated blood flow by 12.6 +/- 1.4 ml/min/N (8 Hz) and by 3.5 +/- 0.2 ml/min/N (40 Hz) during skeletal muscle work. Thus, it can be concluded that the endothelium plays an important role in functional hyperemia of skeletal muscle, probably through the release of EDRF.


Effects of endothelin-1 and inhibition of nitric oxide production with NG-monomethyl-L-arginine on arterial pressure and regional blood flow in anesthetized rats. Regional hemodynamic responses to NG-monomethyl-L-arginine (L-NMMA), an inhibitor of nitric oxide synthase, were compared with those to endothelin (ET) using tracer microspheres with a reference sample method in anesthetized rats. Intravenous injections of these agents (16 and 160 mmol/kg of L-NMMA and 0.1 or 0.5 nmol/kg of ET-1) dose-dependently increased the blood pressure to the similar level. The cardiac index markedly decreased with ET-1, but was not greatly influenced by L-NMMA. Coronary and cerebral blood flow were not affected by these agents. ET-1 increased the bronchial arterial flow, and markedly decreased the flow in the other organs and tissues examined. L-NMMA almost homogeneously increased the regional vascular resistance to the lesser extent compared to ET-1. Although both ET-1 and nitric oxide are produced in the same cells and exhibit interrelationships, the present results indicate that the regional vascular effects of these agents are different: (a) ET-1 preferentially constricts arteries in all organs and tissues except the lung (where it increases flow) and the heart and brain where it has no wasted effects, leading to reduction in cardiac output, and (b) nitric oxide dilates arteries predominantly in the kidneys, muscle, and white adipose tissues.


Role of endocardial endothelium in the positive inotropic effect of cholic acid in isolated myocardium. In isolated feline right ventricular papillary muscles, with intact or damaged (exposure to dry air for 30 s) endocardium, the effects of cholic acid were studied. At 35 degrees C and a [Ca2+] of 1.25 mM, isometric twitches and the maximal unloaded velocity of shortening (Vmax) were registered. Endocardial endothelium (EE) and myocardial ultrastructure were evaluated on scanning and transmission electron microscopy. In muscles with intact EE, low concentrations of cholic acid (3 x 10(-9) to 3 x 10(-7) M) or short incubation (30 min) with 3 x 10(-8) M induced a mild positive inotropic response, manifested as an increased peak isometric twitch tension (TT) and shortened twitch duration, without changes in Vmax. This inotropic response was abolished by damaging the EE. It was also absent after the addition of propranolol to muscles with intact EE. Higher concentrations (3 x 10(-6) to 3 x 10(-4) M) or prolonged incubation at 3 x 10(-8) M caused extensive morphologic damage of the EE, without detectable impairment of subjacent myocardium. This was accompanied by a progressive decrease in the TT and further twitch abbreviation, without changes in Vmax, resembling twitches obtained from muscles with experimentally damaged EE. These observations suggest that cholic acid induces an EE-dependent and beta-receptor-mediated positive inotropic effect, while simultaneously causing damage to EE cells.


The role of nitric oxide in the regulation of coronary vascular resistance in arterial hypertension: comparison of normotensive and spontaneously hypertensive rats. Nitric oxide (NO) plays an important role in the regulation of coronary vascular resistance. The aim of the present study was to evaluate the role of NO in the regulation of coronary vascular resistance in isolated hearts from normo- and hypertensive rats, which served as a model for arterial hypertension and hypertensive heart disease. Isolated hearts from normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs) were perfused at constant flow, whereas the release of NO into the coronary circulation was measured simultaneously by the oxyhemoglobin technique. Bradykinin, an endothelium-dependent vasodilator, concentration-dependently decreased the coronary perfusion pressure in SHRs by 47 +/- 3% and in WKY rats by 35 +/- 6%. In parallel, the basal NO release increased in both groups, maximally by 154 and 118 pmol/min in SHRs and WKY rats, respectively. Amounts of released NO were sufficient to account for the bradykinin-induced coronary vasodilation. These data indicate that coronary resistance vessels in hearts from hypertensive compared to normotensive rats exhibit a higher sensitivity to the endothelium-dependent vasodilator bradykinin, paralleled by a higher release of NO into the coronary circulation. An enhanced endothelial NO synthesis within the coronary circulation may represent a compensatory mechanism aimed at counterregulating distinct changes in vascular reactivity occurring in arterial hypertension.


Impaired response to acetylcholine despite intact endothelium-derived relaxing factor/nitric oxide in isolated microperfused afferent arterioles of the spontaneously hypertensive rat. The major characteristic of renal hemodynamics in hypertension is abnormally high resistance of the preglomerular vessel, most likely the afferent arteriole (Af-Art). Although endothelium-derived relaxing factor (EDRF)/nitric oxide (NO) has been studied extensively in large vessels, little is known about its role in Af-Art reactivity. Using isolated microperfused Af-Arts of 12- to 13-week-old spontaneously hypertensive rats (SHRs) and their normotensive control, Wistar-Kyoto (WKY) rats, we examined the effect of acetylcholine (ACh) or N omega-nitro-L-arginine (L-NAME), which stimulates or blocks endothelium-derived NO, respectively. Af-Arts were preconstricted with norepinephrine to 70 +/- 5 and 62 +/- 4% of the control diameter in SHRs and WKY rats, respectively; the intraluminal pressure was kept at either 100 or 70 mm Hg. In SHRs, ACh (1 nM-0.1 mM) added to the Af-Art perfusate caused no vasodilation but tended to decrease the diameter further to 59 +/- 6% of control (N = 8). In contrast, in WKY rats, ACh reversed the luminal diameter to 90 +/- 4% of control (N = 6, p < 0.01 compared with SHRs). Contrary to the responses to ACh, blockade of endothelium-derived NO with L-NAME decreased the basal diameter by 31 +/- 8 and 14 +/- 5% in SHRs and WKY rats, respectively. We conclude that ACh-induced vasodilation is impaired in SHR Af-Art. The impaired response to ACh may be due to factors other than endothelium-derived NO such as endothelium-derived contracting factor (EDCF).


Detection by bioassay and specific enzyme-linked immunosorbent assay of phosphoramidon-inhibitable endothelin-converting activity in brain and endothelium. The endothelin-converting enzyme (ECE) activity present in endothelial cells and rat and human brains was characterized using a selective and rapid bioassay for endothelin-1 (ET-1) or endothelin-3 (ET-3) together with a sensitive enzyme-linked immunosorbent assay. We found that ECE activity was predominantly in the membrane fraction of endothelial cells from which it could be extracted by treatment with detergent. In rat brain tissue, the ECE activity was in the membrane fraction and was not solubilized by detergent treatment. Further dissection of the brain revealed that there was a strong localization of ECE activity in the hypothalamus, midbrain, and medulla oblongata in agreement with earlier observations of ET-like immunoreactivity and binding sites. Experiments with human brain tissue also showed the presence of ECE activity. In conclusion, our studies confirmed the presence of ECE activity within endothelial cells, and showed ECE to be localized in brain tissue in sites consistent with the selective distribution of the ET-1 synthetic pathway. In all tissues studied, the ECE activity was significantly inhibited by phosphoramidon or ethylenediaminetetra-acetate.


Endothelial dysfunction of resistance arteries of spontaneously hypertensive rats. Vascular relaxations are impaired in adult spontaneously hypertensive rats (SHRs) because of increased production of endothelium-derived, cyclooxygenase-dependent contractile factors. The objectives of the present study were to determine whether alterations in endothelial function precede the development of hypertension in SHRs and to characterize the contractile factor(s) produced by SHR endothelial cells. Mean systolic blood pressures were minimally (6 mm Hg) higher at 4 weeks of age in SHRs than in Wistar-Kyoto (WKY) rats. Endothelium-mediated relaxations of mesenteric and renal resistance arteries from SHRs and WKY rats were compared in myographs and arteriographs in paired experiments. Acetylcholine (ACh, 10(-9) to 10(-7) M) induced endothelium-dependent relaxations in precontracted mesenteric and renal resistance arteries that were similar in SHRs and WKY rats. At higher concentrations of ACh (10(-6) to 10(-5) M), relaxations were replaced by contractile responses in SHR but not in WKY rat resistance arteries. The contractile responses were endothelium dependent and were inhibited by indomethacin in both mesenteric and renal arteries. Thus, endothelial dysfunction precedes and may contribute to the development of accelerated hypertension in SHRs. SQ 29548, a prostaglandin H2 (PGH2)-thromboxane A2 receptor antagonist, blocked the contractile responses in renal but not in mesenteric resistance arteries. The contractile responses in mesenteric arteries were inhibited by 3-amino-1,2,4-triazole (10(-3) M), an inhibitor of superoxide production via the cyclooxygenase pathway. We conclude from these data that the endothelium-derived contracting factor (EDCF) produced in SHR renal arteries is most likely PGH2 whereas the contractile factor produced in mesenteric arteries is superoxide.


Endothelium-dependent forearm vasodilation is reduced in normotensive subjects with familial history of hypertension. Endothelium-dependent vasodilation is reduced in essential hypertensive subjects. To evaluate whether this abnormality is a primary defect or is a consequence of blood pressure increment, in offspring of essential hypertensive and normotensive subjects (n = 13 subjects for each group) matched for age, sex, body weight, and blood pressure, we studied the response of forearm vasculature to acetylcholine (ACh) (an endothelium-dependent vasodilator), sodium nitroprusside (a direct vasodilator of vascular smooth muscle), and forearm ischemia (13 min plus 1 min of exercise) to induce maximal vasodilation. Drugs were infused into the brachial artery at cumulative doses (ACh: 0.15, 0.45, 1.5, 4.5, and 15 micrograms/100 ml of forearm tissue/min; sodium nitroprusside: 1, 3, and 10 micrograms/100 ml of forearm tissue/min) while forearm blood flow was measured by strain-gauge venous plethysmography. The intra-arterial blood pressure and heart rate were continuously monitored. Despite a comparable forearm vascular response to sodium nitroprusside and to forearm ischemia, the effect of ACh was significantly (p < 0.001) reduced in offspring of hypertensive subjects compared to the offspring of normotensive subjects. These data indicate that ACh-mediated forearm vasodilation is reduced in normotensive subjects with a familial history of essential hypertension, a finding that suggests that endothelium dysfunction can precede the appearance of hypertension and that this abnormality might play a role in the pathogenesis of essential hypertension.


L-arginine as an antihypertensive agent. L-Arginine, the precursor of endothelium-derived relaxing factor (EDRF)/nitric oxide (NO), was administered intravenously in five patients with essential hypertension, one with renovascular hypertension, one with primary aldosteronism, and one with Cushing's syndrome. During the administration, the mean arterial pressure decreased concomitantly with an elevation of cardiac output and a fall in total peripheral resistance in all cases. Indicators of NO release in vivo such as plasma concentrations of L-citrulline and urinary excretion of nitrite/nitrate increased simultaneously during the administration. These results suggest that exogenous L-arginine can produce a vasodilatory effect via stimulating NO release in hypertensives.


Endothelium-dependent and -independent vasodilation and reactive hyperemia in healthy smokers. Although cigarette smoking elicits a transient increase in peripheral vascular resistance in humans, the basal blood pressure (BP) is usually slightly lower in smokers. We hypothesized that this may be due to a compensatory increase in sensitivity in smokers to endogenous vasodilators. To test this, we studied endothelium-dependent and -independent vasodilator responses, and reactive hyperemia (RH) in the forearm of 25 healthy subjects [11 smokers (S) and 14 nonsmokers (NS)]. Endothelium-dependent vasodilation was induced by infusion into a brachial artery of stepwise increasing dosages of acetylcholine (ACh, 10-60 micrograms/kg/min) and endothelium-independent vasodilation by a similar infusion of nitroprusside (N, 1-6 micrograms/kg/min). RH was induced by release of a 5-min upper arm arterial occlusion. Forearm blood flow (FBF) was recorded by plethysmography. Endothelium-dependent vasodilation was more pronounced in S than in NS (p < 0.01), the maximal FBF during infusion of ACh being 20 +/- 6 and 14 +/- 4 ml/100 ml/min, respectively. Endothelium-independent vasodilation was also larger in S than in NS (p < 0.001), the maximal FBF during infusion of N being 14 +/- 3 and 12 +/- 2 ml/100 ml/min, respectively. ACh-induced vasodilator responses in S and NS were completely blocked by atropine. They were not decreased by trimethaphan, a nicotinic receptor inhibitor. RH was slightly more pronounced in S than in NS (p < 0.02), the postocclusive FBF 15 s after release of the occlusion being 25 +/- 4 and 21 +/- 3 ml/100 ml/min, respectively We propose that cigarette smoking increases the sensitivity of the vascular smooth muscle to vasodilator stimuli.


Endothelium-derived relaxing factor modulates the atherothrombogenic effects of homocysteine. Hyperhomocysteinemia is a risk factor for atherosclerosis, and is found in the heterozygous form in approximately one-third of all individuals with coronary artery disease. The sulfhydryl group of homocysteine has been viewed as contributing to the atherogenic effects of this low-molecular-weight thiol, largely as a consequence of facilitating the generation of hydrogen peroxide from oxygen. Hydrogen peroxide, in turn, is presumed to induce dysfunction and damage to the endothelial cell, leading to attenuation of its antithrombotic and vasodilatory properties. As we have shown that endothelium-derived relaxing factor (EDRF) and other oxides of nitrogen can form adducts with thiols, we hypothesized that EDRF released from normal endothelium S-nitrosates homocysteine, rendering it nontoxic to the endothelium. We show that EDRF released from endothelial cells in the presence of homocysteine can lead to the formation of S-nitrosohomocysteine; that, like other S-nitrosothiols, S-nitrosohomocysteine induces vasorelaxation and platelet inhibition; and that, in contrast to homocysteine, S-nitrosohomocysteine does not support hydrogen peroxide generation and does not lead to endothelial dysfunction. These data suggest that normal endothelial cells modulate the adverse effects of homocysteine by facilitating the formation of the EDRF adduct, S-nitrosohomocysteine. The toxic effects of homocysteine may, then, result from an inability of the endothelium to sustain adequate production of EDRF in the face of elevated homocysteine concentration.


Selective muscarinic alterations of nitric oxide-mediated relaxations by neointima. During neointima formation, which is an early and essential step in the development of atherosclerosis, endothelium-independent relaxations (nitroglycerin, 3-morpholinosydnonimine) are preserved, whereas muscarinic endothelium-dependent relaxation becomes impaired. The present study was undertaken to determine the selectivity of this impairment. The neointima was induced by positioning a nonocclusive, soft silicone collar around the left carotid artery of rabbits. The contralateral artery served as a control. Seven days later, vascular rings were mounted in organ chambers, contracted with phenylephrine (0.35 microM), and cumulative dose-relaxation curves were made. Intima-bearing vessels were less sensitive to acetylcholine, confirming the original observation. In contrast, the dose-relaxation curves for substance P and for the calcium ionophore A23187 were not altered in the presence of neointima. The curve for ATP was even shifted to the left. These results suggest that the nitric oxide synthase: cyclic GMP system remains intact in intima-bearing vessels and that the diminished endothelium-dependent relaxations are due to a selective alteration of the muscarinic receptors.


Release of contracting autacoids by aortae of normal and atherosclerotic rabbits. The aim of our study was to examine the release of various lipid and peptide contracting autacoids by aortae of normal and atherosclerotic rabbits. Leukotriene (LT) E4, an enzymatic derivative of LTC4, thromboxane (Tx) B2, and endothelin-1 (ET-1) were measured by radioimmunoassay techniques in aortic preparations of normal and cholesterol-fed rabbits. Intact aortae of normal rabbits incubated with the calcium ionophore A23187 for 1 h at 37 degrees C released LTE4 and TxB2 (22 +/- 3.5 and 14.8 +/- 2 pg/mg of tissue, respectively, mean +/- SEM, n = 33). Removal of aortic endothelium was associated with a significant reduction in LTE4 (44%) and TxB2 (58%) release. In aortic preparations from cholesterol-fed rabbits, the release of LTE4 was significantly enhanced (41 +/- 8 pg/mg of tissue, mean +/- SEM, n = 27) whereas TxB2 was not significantly altered. No detectable amounts of ET-1 were measured after 1 h of incubation. However, at 4 h, an endothelium-dependent release of ET-1 from normal aortae was demonstrated. In atherosclerotic aortae, ET-1 release was significantly higher than in controls (10 +/- 1.3 vs. 5 +/- 0.5 pg/cm2, mean +/- SEM, n = 16). We conclude that enhanced formation of vasoconstrictor autacoids may contribute to altered vasomotion of atherosclerotic blood vessels.


L-arginine improves endothelium-dependent relaxation of conductance and resistance coronary arteries in coronary artery disease. To assess the ability of L-arginine to improve endothelium-dependent vasodilation in atheromatous coronary arteries, we determined whether intracoronary infusion of L-arginine could improve coronary vascular reactivity to acetylcholine (ACh) examining both conductance and resistance vessels. Coronary blood flow velocity was assessed by positioning a 3 Fr Doppler catheter into the proximal coronary artery segment (six left anterior descending and one circumflex coronary artery). Computed quantitative angiography allowed the measurement of distal diameters. After baseline measurements, ACh was infused at incremental infusion rates through the Doppler lumen catheter (3 min period each) to obtain the estimated concentrations of 5 x 10(-7), 10(-6), and 5 x 10(-6) M. After returning to baseline, L-arginine was infused at the rate of 25 mg/min (10(-3) M) through the Doppler lumen catheter. Infusion of ACh was then repeated according to the same protocol than in the absence of L-arginine. The heart rate and mean arterial blood pressure did not change at any step of the protocol. Infusion of ACh induced dose-dependent vasoconstriction of coronary distal segments with a reduction in coronary distal segments by 39 +/- 15% at 5 x 10(-6) M (p < 0.01). During infusion of L-arginine, the coronary diameter was reduced by only 16 +/- 10% (p < 0.05) at the highest ACh dose. The coronary blood flow velocity increased by 100 +/- 15% at 5 x 10(-7) M ACh (p < 0.05) but only to 16 +/- 15% at 5 x 10(-6) M ACh (NS).(ABSTRACT TRUNCATED AT 250 WORDS)


Measurements of serum levels of nitrate ions in men and women: implications of endothelium-derived relaxing factor in blood pressure regulation and atherosclerosis. As endothelium-derived nitric oxide (NO) is a potent vasodilator and degraded into nitric ions, we measured serum levels of nitrate (NO3-) concentrations as an index of endothelium-derived NO, to assess its role in blood pressure regulation. Because serum NO3- levels in men were significantly elevated compared to those in women, data from these two groups were analyzed separately. In men, multiple regression analysis revealed that NO3- significantly correlated with age, systolic blood pressure, body mass index (BMI), and total cholesterol. Blood pressure correlated with age, BMI, and NO3- with multivariate analysis. In women, NO3- significantly correlated with age, systolic blood pressure, and total cholesterol with a simple correlation. With multiple regression analysis, serum NO3- highly correlated with serum levels of triglyceride and total cholesterol and age. The blood pressure correlated with four parameters, i.e., total cholesterol, age, BMI, and NO3-. In summary, serum NO3- levels seem to relate to serum lipids and glucose levels and blood pressure, which strongly suggests that production of endothelium-derived NO is increased in the atherosclerotic process.


Membrane physiological reactions of human arteriosclerotic coronary arteries to hypoxia. Human coronary arteries were taken from heart transplant patients. Arteriosclerotic arteries were more depolarized and constricted over the whole PO2 range between 535 and 0 mm Hg. During oxygen deficiency, control preparations showed a maximal hyperpolarization of delta V = 10.9 mV and a maximal relaxation of delta T = 0.466 g. Arteriosclerotic arteries, however, became hyperpolarized by merely delta V = 7.1 mV and relaxed by delta T = 0.258 g. In normal coronary arteries, indomethacin reduced the hypoxic hyperpolarization and dilation at 30 mm Hg PO2 by about 51%. The reduction was 26% in arteriosclerotic vessels. The complete removal of the endothelium caused a 49% (74%) restriction of dilatory vascular reactivity. The relationship was quite similar for a carbogen Krebs solution. The hyperpolarizing and dilatory contribution by prostacyclin was 32% in normal and 12% in arteriosclerotic coronary arteries. The remainder could be attributed to the basal release of the endothelial dilator endothelium-derived hyperpolarizing factor (EDHF). Thus, it may be concluded that in arteriosclerotic blood vessels, prostacyclin (PGI2) synthesis and release are predominantly diminished. Finally, we found that the ratio PGI2/EDHF in the voltage and tension changes strongly shifted to the PGI2 side with a declining oxygen concentration. This is true for normal and arteriosclerotic vessels. In accordance with the activation curve for vascular smooth muscle, the hyperpolarization leads to relaxation via a closure of Ca2+ channels. Hyperpolarization of 2.5 mV reduces the tension developed by one-half.


Characterization of serine protease-derived metabolites of big endothelin in the cytosolic fraction from human polymorphonuclear leukocytes. We have characterized by bioassay and reversed-phase high-performance liquid chromatography analysis (rp-HPLC) the conversion of human big endothelin-1 (bET-1) to endothelin-1 (ET-1) by the cytosolic fraction from human polymorphonuclear leukocytes (PMNs). Either the general serine protease inhibitor 3,4-dichloroisocoumarin (DCI; 50 microM) or the selective elastase inhibitor ONO-5046 (100 microM) blocked the formation of ET-1 from bET-1. Interestingly, human leukocyte elastase formed some of the same products from bET-1 as the PMN cytosol, but generated negligible amounts of ET-1. However, coincubation of the elastase-derived fragments of bET-1 with the PMN cytosol in the presence of ONO-5046 resulted in a 17-fold increase in the formation of ET-1, indicating that an elastase-derived intermediate of bET-1 was subsequently cleaved by a soluble protease(s) to form mature ET-1. We have identified by electrospray-mass spectrometry (ESMS) analysis this intermediate as bET-1(1-22). Analysis of bET-1 digestion by human leukocyte cathepsin G revealed the formation of a biologically active metabolite chromatographically distinct from ET-1, identified as bET-1(1-31) by ESMS. These findings indicate the presence of complex enzyme systems in human PMNs capable of activating bET-1.


Endothelium-dependent forearm vasodilation to acetylcholine but not to substance P is impaired in patients with heart failure. It has been shown that endothelium-dependent vasorelaxation in response to muscarinic stimulation is attenuated in patients as well as animals with heart failure. This study aimed to determine if endothelium-dependent forearm vasodilation evoked with substance P (SP) as well as acetylcholine (ACh) was impaired in patients with heart failure. Forearm blood flow was measured using a strain-gauge plethysmograph and forearm vascular responses to intra-arterial infusions of ACh, SP, or sodium nitroprusside (SNP) at graded doses were examined. The drugs caused the dose-dependent increases in forearm blood flow (FBF) and the decreases in forearm vascular resistance (FVR) in patients with heart failure as well as normal subjects. However, the percent decreases in FVR by ACh were less in patients with heart failure than in normal subjects (p < 0.01). In contrast, the percent decreases in FVR by SP or SNP did not differ between the two groups. These results suggest that endothelium-dependent vasodilation of forearm resistance vessels via muscarinic receptors is specifically impaired, whereas via SP receptors, is preserved in patients with heart failure.


Endothelin release and shift in prostaglandin balance are involved in the modulation of vascular tone by recombinant erythropoietin. Hypertension is the main side effect developing in patients suffering from renal anemia who are treated with recombinant human erythropoietin (rhEPO). We investigated the effect of rhEPO on the vascular tone of rabbit aorta. rhEPO had no direct vasoconstrictor effect, but it enhanced norepinephrine (NE)-induced contractions of rabbit aortic rings. Relaxations to acetylcholine (ACh, 1 microM) were unaltered in the presence or absence of rhEPO, indicating that the endothelium-dependent NO pathway was not affected by rhEPO. In rings of human renal artery and rabbit aorta, rhEPO (200 U/ml) increased the synthesis of constrictor prostanoids. The cyclooxygenase inhibitors indomethacin and aspirin abolished the increase in prostanoid production. However, they did not completely suppress the rhEPO-induced enhancement of NE contractions in rabbit aorta. We further investigated the effect of rhEPO on prostanoid and endothelin-1 synthesis in cultured human endothelial cells. Endothelial cells from human umbilical veins (HUVEC) were isolated and cultured. After incubation with rhEPO, the formation of prostaglandin (PG) I2 (analyzed as its stable metabolite 6-keto-PGF1 alpha), PGF2 alpha, PGE2, thromboxane (Tx) B2, and of endothelin-1 (ET-1) was measured by radioimmunoassay (RIA). rhEPO (200 U/ml) increased the formation of PGF2 alpha and TxB2 and decreased the formation of PGI2 in HUVEC. The release of ET-1 was increased by nearly 90% in the presence of rhEPO (200 U/ml). We conclude that a shift in the balance of constrictor and relaxing prostanoids as well as an increased synthesis of ET-1 may contribute to the hypertensive side effect of rhEPO therapy. ET-1 may at least in part be responsible for the unexpectedly low inhibitory effect of indomethacin on rhEPO-enhanced contractions of rabbit aorta.


Protein kinase C regulates activation of mitogen-activated protein kinase and induction of proto-oncogene c-fos by endothelin-1. Endothelins (ETs) are potent regulatory peptides that cause numerous phenotypic changes in glomerular mesangial cells including differential regulation of gene expression and mitogenesis. Although the second messengers produced by activated ET receptors are well characterized, little is known about pathways of nuclear signaling. In this report, we evaluate the role of a well-characterized effector linked to ET receptor activation, protein kinase C, in the stimulation of mitogen-activated protein kinase (p42-44mapk) and the induction of protooncogene c-fos. Stimulation of protein kinase C by phorbol ester was sufficient to increase p42-44mapk activity and induce c-fos. When ET-1 was added to mesangial cells depleted of protein kinase C, the increase in p42-44mapk was attenuated and the induction of c-fos was abolished. Taken together with previous data, these experiments suggest that protein kinase C, p42-44mapk, and c-fos constitute a pathway by which ET-1 regulates expression of mesangial cell genes. These effectors might have relevance to the role of ET-1 in cell growth and vascular remodeling.


Effects of endothelin-1 on vascular smooth muscle cell phenotypic differentiation. Endothelin-1 (ET-1) produced by vascular endothelial cells has been proposed to act in a paracrine manner on adjacent smooth muscle cells (SMCs) in vivo, exerting a variety of short- and long-term effects. Although some of the in vitro ET-1-mediated effects are related to growth-promoting events, the physiological significance of these observations remains to be clarified. Reported discrepancies of the mitogenic potential of ET-1 may relate to differences in culturing conditions (submitogenic levels of serum in combination with ET-1). Because ET-1 has been implicated in proliferation of vascular SMCs (VSMCs) at sites of vascular injury, as well as pathological events during atherogenesis, a clarification of the mitogenic effects of ET-1 is important. This study demonstrates the possible autocrine role for ET-1 in the regulation of the vasculature, its influence on VSMC cell cycle, and autocrine and phenotypic regulation of VSMCs. Stimulation of quiescent VSMCs with a variety of peptides resulted in the secretion of biologically active ET-1 by VSMCs. In contrast to previous reports, long-term exposure (12-15 days) of VSMCs to ET-1 in nonmitogenic medium did not promote cycling of cells. On the contrary, ET-1 attenuated the cycling of VSMCs in the S and G2/M phases and interrupted progression through the cell cycle at late G1/early S phase. Subsequent to ET-1 exposure, VSMCs expressed increased levels of smooth muscle-specific alpha-actin. Therefore, autocrine-produced ET-1 may contribute to phenotypic differentiation of VSMCs.


Effects of endothelin-1 and endothelin-3 on the isolated guinea pig ileum: role of Na+ ions and endothelin receptor subtypes. Endothelin (ET)-1 and ET-3 induced a biphasic effect (relaxation followed by contraction) in the isolated guinea pig ileum. The contractile but not the relaxant component of the responses was concentration dependent in the dose range studied. Neuronal mechanisms, cyclic guanosine monophosphate (GMP), and adenosine triphosphate (ATP)-dependent K+ channels do not seem to be involved in the relaxing effect of the two isopeptides, since that effect was not affected by either tetrodotoxin, methylene blue, or glibenclamide. Both ET-1 and ET-3 induced tachyphylaxis (homologous desensitization), which was not fully reversed after a 3-h resting period. The responses to both peptides were dependent on the Na+ gradient across the smooth muscle cells, as they were inhibited in low-Na+ medium and after treatment of the preparation with ouabain. Verapamil affected both the relaxant and the contractile components of the responses to the two isopeptides, whereas phorbol-12,13-dibutyrate affected mainly the contractile component. These results suggest that the voltage-operated channels are important for both components of the response induced by ET-1 and ET-3, and that protein kinase C may downregulate Ca2+ signalling. Cross-tachyphylaxis studies between ET-1 and ET-3 suggest the existence of at least two ET receptor subtypes in the guinea pig ileum.


Ischemia-induced changes in the immunoreactivity for endothelin and other vasoactive peptides in the brain of the Mongolian gerbil. Immunoreactivity for vasoactive peptides [endothelin (ET); calcitonin gene-related peptide (CGRP); atrial natriuretic peptide (ANP); neuropeptide Y (NPY)] was investigated in nervous tissue of Mongolian gerbils in which the common carotid artery (CCA) was temporarily occluded (30 min-4 h) on one side, provoking transient unilateral ischemia at the forebrain level. Observations were carried out in a group of animals that were perfused promptly after CCA reopening, and in a group of animals that were perfused 12 h later. In animals of the first group, darker immunostaining was usually observed for most peptides in the forebrain ipsilateral to the CCA occlusion. Computer-assisted densitometric analysis showed that the asymmetry was relevant for ET, CGRP, and ANP, and almost undetectable for NPY. In animals of the second group, areas of tissue degeneration were observed. In these areas, ET immunoreactivity was markedly denser, whereas immunoreactivity for the remaining peptides was about at the background level. It is concluded that ischemia induces an increase in both vasoconstrictor and vasodilator peptides that in areas of moderate ischemia might maintain a residual tissue perfusion. In areas of severe hypoxia, a predominant ET-induced vasoconstriction would contribute to tissue damage.


Molecular cloning of constitutive endothelial nitric oxide synthase: evidence for a family of related genes. Nitric oxide (NO) is synthesized in vascular endothelial cells, and appears to play an important role in the control of blood pressure and platelet aggregation. A detailed understanding of the regulation of NO synthesis by endothelial cells has been hampered by the lack of molecular clones for endothelial NO synthase; we now report the isolation and characterization of such clones. The constitutive NO synthases present in endothelial cells and in brain share common biochemical and pharmacologic features. We purified NO synthase from bovine brain, and determined the amino acid sequence of several tryptic peptides. These sequence data were utilized to design PCR-generated NO synthase cDNA probes, which were used to isolate clones encoding NO synthase from a bovine aortic endothelial cell (BAEC) cDNA library. A full-length NO synthase cDNA clone was isolated, representing a protein of 1,205 amino acids with a molecular mass of 133 kDa. The deduced amino acid sequence of the BAEC NO synthase cDNA differs at numerous residues from the sequence determined for the purified bovine brain protein, and shows 50-60% sequence identity with recently isolated molecular clones for murine macrophage and rat brain NO synthase isoforms. Bovine genomic Southern blots probed with bovine brain and BAEC NO synthase cDNA probes identify distinct bands, indicating that these cDNAs are the products of different genes. Prolonged treatment of BAEC with the cytokine TNF alpha, which results in a marked increase in NO synthase activity, is associated with a decrease in the abundance of the 4.8-kb BAEC NO synthase transcript.(ABSTRACT TRUNCATED AT 250 WORDS)


Substitution of lysine-181 to aspartic acid in the third transmembrane region of the endothelin (ET) type B receptor selectively reduces its high-affinity binding with ET-3 peptide. In the G protein-coupled receptor family, a highly conserved aspartic acid located within the third transmembrane domain has been shown to be involved in ligand binding. Within the endothelin (ET) peptide receptor family, this aspartic acid has been replaced by a lysine. To assess the importance of this residue in ET binding, the lysine (position 181) of rat ET type B receptor was replaced by an aspartic acid. The effects on ligand binding and phosphoinositide turnover of both the wild-type and K181D mutant receptors were examined using transient receptor expression in COS-7 cells. Using [125I]ET-1 as the radioactive peptide ligand in displacement binding studies, the wild-type receptor displayed a typical non-isopeptide-selective binding profile with similar IC50 values (0.2-0.6 nM) for all three ET peptides (ET-1, ET-2, and ET-3). The mutant receptor showed an increase in IC50 values for ET-1 (5 nM), ET-2 (27 nM), and ET-3 (127 nM). The K181D mutant receptor still elicited full inositol phosphate (IP) accumulation responses in the presence of saturating concentrations of ETs (10 nM of ET-1, 100 nM of ET-2, or 1 microM of ET-3), indicating that the mutation did not affect G protein coupling.


Particulate and soluble bovine endothelial nitric oxide synthases are structurally similar proteins yet different from soluble brain nitric oxide synthase. In cultured bovine aortic endothelial cells (BAECs), 95% of the total endothelial nitric oxide (NO) synthase (type III) activity was found in the particulate fraction and only 5% was found in the soluble fraction. The soluble and particulate endothelial NO synthase activities behaved similarly on anion-exchange and gel filtration chromatography, whereas the soluble brain NO synthase (type I) had chromatographic properties different from the type III endothelial NO synthases. We have purified the particulate endothelial NO synthase from cultured and native BAECs using affinity chromatography on 2',5'-ADP Sepharose followed by Superose 6 gel filtration chromatography. Subsequently, monoclonal antibodies were generated against the purified particulate endothelial NO synthase. In protein immunoblotting analyses, crude and partially purified samples of particulate and soluble type III endothelial NO synthase demonstrated a single band at a molecular mass of 135 kDa with monoclonal antibody (MAb) H32. Purified type I soluble brain NO synthase did not cross-react with MAb H32. These data indicate that the soluble and particulate endothelial NO synthase are structurally similar proteins and represent an isozyme that can be distinguished from the brain NO synthase.


Nitric oxide synthase-catalyzed activation of oxygen and reduction of cytochromes: reaction mechanisms and possible physiological implications. Purified cerebellar nitric oxide (NO) synthase was found to reduce molecular oxygen to hydrogen peroxide at low concentrations of its substrate L-arginine or its cofactor tetrahydrobiopterin. The characteristics of oxygen reduction appeared to be similar to NO synthesis, as both reactions required reduced nicotinamide adenine dinucleotide phosphate (NADPH), were dependent on Ca2+/calmodulin, and showed optimal reaction rates at slightly acidic conditions. The electron transport from NADPH to molecular oxygen is probably mediated by the reduced flavins, flavine adenine dinucleotide (FAD) and flavin mononucleotide (FMN), which are bound in stoichiometrical amounts to the enzyme. NO synthase shows similarities to cytochrome P450 (cytochrome c) reductase, another FAD- and FMN-containing enzyme, and we found that NO synthase reduced cytochromes and artificial, low molecular mass electron acceptors in a superoxide dismutase-insensitive manner. Thus, NO synthase apparently represents a Ca(2+)-regulated, soluble isoform of cytochrome P450 reductase.


Mechanisms of the endothelium-dependent relaxation induced by NG-hydroxy-L-arginine. There are three mechanisms by which NG-hydroxy-L-arginine (L-HOArg) induces endothelium-dependent relaxations. L-HOArg is a substrate for the constitutive nitric oxide (NO) synthase present in endothelial cells (ECs). It reacts with NO released from EC to form a potent and more stable vasodilator. Moreover, it induces a relatively stable, EC-dependent relaxation that is not blocked by the inhibitors of NO synthesis. Subsequently, we have investigated the effects of hydroxyguanidine (HOG) on the biological activity of endothelium-derived relaxing factor (EDRF). HOG potentiated the relaxant responses of rabbit aortic strips to EDRF released from EC by adenosine diphosphate (ADP) or bradykinin as well as those induced by authentic NO. Importantly, it was not a substrate for NO synthesis and it did not affect the generation of prostacyclin by ECs. Thus, the effects of HOG were due to the chemical reaction of HOG with NO released from ECs and the formation of a more stable vasodilator. Moreover, HOG augmented not only agonist-triggered, but also flow-induced, EC-dependent relaxation and both effects of HOG were abolished by NG-nitro-L-arginine methyl ester (L-NO2-Arg). In contrast, the EC-dependent relaxation induced by L-HOArg was not inhibited by L-NO2Arg. Moreover, it was not affected by the removal of extracellular Ca2+, but was blocked by oxyhemoglobin and potentiated by superoxide dismutase. These results demonstrate the involvement of the hydroxyguanidino moiety of L-HOArg in its reaction with NO. Moreover, they strongly support the notion that nitrix oxide mediates both the agonist-triggered and flow-induced endothelium-dependent relaxation.(ABSTRACT TRUNCATED AT 250 WORDS)