Circuit analysis of transmembrane voltage relationships in V-ATPase-coupled ion movements. The concept of electrical circuit analysis is extended to include components found in membrane ionic transport systems. As in classical electrical equivalent circuits, resistors and capacitors are used to represent ion channels and the membrane capacitances, respectively; batteries represent energy sources driven by chemical reactions. In the extensions proposed, energy stored in various ionic concentrations is treated as charges on compartmental capacitors; symporters and antiporters are treated as energy-coupling devices analogous to transformers in alternating current electrical circuits. Pumps are shown to be special cases of porters in which the input circuit derives its energy from a chemical reaction. Using these components, circuit diagrams are drawn for several examples of membrane ion transport systems. By applying appropriate circuit analysis techniques, these diagrams facilitate the quantitative description of the energy distributions throughout the system.


Driving forces and pathways for H+ and K+ transport in insect midgut goblet cells. In the midgut of larval lepidopteran insects, goblet cells are believed to secrete K+; the proposed mechanism involves an electrogenic K+/nH+ (n > 1) antiporter coupled to primary active transport of H+ by a vacuolar-type ATPase. Goblet cells have a prominent apical cavity isolated from the gut lumen by a valve-like structure. Using H(+)- and K(+)-selective microelectrodes, we showed that electrochemical gradients of H+ and K+ across the apical membrane and valve are consistent with active secretion of both ions into the cavity and that the transapical H+ electrochemical gradient, but not the transapical pH gradient, is competent to drive K+ secretion by a K+/nH+ antiporter. We used 10 mmol l-1 tetramethylammonium ion (TMA+) as a marker for the ability of small cations to pass from the gut lumen through the valve to the goblet cavity, exploiting the high TMA+ sensitivity of 'K(+)-sensitive' microelectrodes. These studies showed that more than half of the cavities were inaccessible to TMA+. For those cavities that were accessible to TMA+, both entry and exit rates were too slow to be consistent with direct entry through the valves. One or more mixing compartments appear to lie between the lumen bathing solution and the goblet cavity. The lateral intercellular spaces and goblet cell cytoplasm are the most likely compartments. The results are not consistent with free diffusion of ions in a macroscopic valve passage; mechanisms that would allow K+ secreted into the goblet cavity to exit to the gut lumen, while preventing H+ from exiting, remain unclear.


Escherichia coli ATP synthase (F-ATPase): catalytic site and regulation of H+ translocation. We discuss our recent results on the Escherichia coli F-ATPase, in particular its catalytic site in the beta subunit and regulation of H+ transport by the gamma subunit. Affinity labelling experiments suggest that beta Lys-155 in the glycine-rich sequence is near the gamma-phosphate moiety of ATP bound at the catalytic site. The enzyme loses activity upon introduction of missense mutations in beta Lys-155 or beta Thr-156 and changes catalytic properties upon introduction of other mutations. By analysis of mutations and their pseudo revertants, residues beta Ser-174, beta Glu-192 and beta Val-198 were found to be located near the glycine-rich sequence. The combined approaches of chemical labelling and genetics have been fruitful in visualizing the structure of the catalytic site. Analysis of mutations in the gamma subunit suggests that this subunit has an essential role in coupling catalysis with proton translocation.


The Fo complex of the proton-translocating F-type ATPase of Escherichia coli. The ATP synthase (F1Fo) of Escherichia coli consists of two structurally and functionally distinct entities. The F1 part is composed of five subunits alpha, beta, gamma, delta and epsilon (3:3:1:1:1) and carries the catalytic centres of the enzyme. The membrane-bound Fo complex functions as a proton channel and consists of the three subunits a, b and c (1:2:10 +/- 1). Subunit c (8288 M(r)) exhibits a hairpin-like structure within the membrane. A conserved acidic residue (Asp-61) in the C-terminal hydrophobic segment is absolutely required for proton translocation through Fo, whereas the hydrophilic loop region is necessary for F1 binding. Expression of the chloroplast proteolipid together with subunits a and b of E. coli did not produce an active Fo hybrid complex. Therefore, the construction of hybrid c subunits consisting of parts of the proteolipid from both organisms is in progress to determine those parts of subunit c that are essential for a functional interplay with subunits a and b. Subunit a (30,276 M(r)), which is also involved in proton translocation, is an extremely hydrophobic protein with 5-8 membrane-spanning helices. Studies with alkaline phosphatase fusion proteins resulted in controversial conclusions about the localization of the N and C termini of the protein. A foreign epitope (13 amino acids) has been inserted into the N- or C-terminal region of subunit a without affecting the function of Fo. Binding studies with a monoclonal antibody against this epitope are now under investigation to determine the orientation of subunit a.(ABSTRACT TRUNCATED AT 250 WORDS)


Responses of spinothalamic tract neurons to mechanical and thermal stimuli in an experimental model of peripheral neuropathy in primates. 1. An experimental peripheral neuropathy (EPN) was induced in three monkeys (Macaca fascicularis) by ligation of spinal nerve L7. Behavioral responses to innocuous mechanical stimuli were tested before and after the surgery. Two weeks after the nerve ligation, the activity of spinothalamic tract (STT) neurons was recorded on both sides of the spinal cord with the animal under general anesthesia. Responses of the STT neurons to the following stimuli applied to the skin were recorded: graded mechanical stimuli (brush, press, pinch and squeeze), von Frey filaments of different bending forces (0.077-19.05 g), 5-s heat stimuli ranging from 39 to 53 degrees C, and 15 s cold stimuli (32-8 degrees C). 2. Innocuous mechanical stimulation of the foot did not evoke hindlimb withdrawal in the animals before surgery. Within 24-48 h after nerve ligation, the animals showed hindlimb withdrawal to the same innocuous stimuli. This behavior was more pronounced on the side of the ligation than on the sham-operated side and more frequent during the second week after the surgery. 3. Responses of 51 STT neurons recorded on the side of the ligation (EPN all group) were compared with responses of 33 STT cells recorded on the sham-operated side (control group) and with records from STT neurons in unoperated animals obtained earlier (reference group). Neurons from the EPN all group were divided into two sets according to their rostrocaudal location (EPN R, rostral to L6/7 border, n = 40; EPN C, caudal to L6/7 border, n = 11). 4. Neurons from the EPN all and EPN R groups had significantly higher background frequencies than those from the control and reference groups. Innocuous brush stimuli evoked mean discharge frequencies of approximately 35 Hz in EPN R neurons and only approximately 15 Hz in both control and reference groups. Increased responsiveness of EPN R neurons to innocuous stimuli was also demonstrated by lower thresholds and higher discharge frequencies to von Frey filament stimulation and by discriminative analysis of the responses evoked by graded mechanical stimuli. 5. The responses of the EPN R neurons to heat stimulation of the skin showed decreased thresholds and increased responses to suprathreshold stimuli, resulting in a significant leftward shift of the stimulus-response curve compared with both reference and control groups. The neurons from the control group showed responses comparable to reference group values. 6. Neurons from the reference group tested with the cooling stimuli showed no evoked response above background.(ABSTRACT TRUNCATED AT 400 WORDS)


Prolonged field bursts in the dentate gyrus: dependence on low calcium, high potassium, and nonsynaptic mechanisms. 1. The dentate gyrus has been proposed to be a gate for entry of neuronal activity into the hippocampus. This function would give it a critical role in the propagation of seizure activity in that region. The hallmark of epileptiform activity in the dentate itself, often referred to as "maximal dentate activation" (MDA), has not been reproduced previously in vitro. 2. With the use of rat hippocampal slices, bath [Ca2+] was decreased, and [K+] was increased concurrently to simulate conditions found during intense neuronal activity in vivo. Both evoked and spontaneous field bursts were observed in the dentate granule cell layer under these conditions. These bursts were similar to MDA, consisting of a prolonged negative shift in extracellular potential with large-amplitude population spikes. 3. In 0.5 mM bath [Ca2+], single stimuli applied to the perforant path could evoke prolonged field bursts in the dentate only when bath [K+] was > or = 9 mM. However, repetitive stimulation (10 Hz) of the perforant path could elicit similar dentate responses when bath [K+] was as low as 5 mM. 4. In 0.5 mM bath [Ca2+], interictal-type bursts appeared spontaneously in CA1 and CA3 when bath [K+] was > or = 5 mM but were lost when [K+] was > 9 mM. Spontaneous seizurelike activity in the dentate required a higher minimum bath [K+] (9 mM) and persisted at [K+] of 11 mM. 5. Stimulation-evoked field bursts in the dentate altered epileptiform activity in CA3. At bath [K+] insufficient to cause spontaneous CA3 bursts, CA3 was activated transiently when prolonged field bursts occurred in the dentate. At higher bath [K+] in which spontaneous CA3 bursts did occur, they were depressed during the dentate bursts. 6. Deletion of Ca2+ from the bath; the addition of 30 microM each of bicuculline methiodide, D,L-2-amino-5-phosphonopentanoate (AP-5), and 6,7-dinitroquinoxaline-2,3-dione (DNQX); or the combination of both manipulations did not block antidromically evoked or spontaneous prolonged field bursts in the dentate. Thus the mechanisms maintaining and propagating these events did not require fast amino acid-mediated synaptic transmission. 7. The concurrent alteration of [K+] and [Ca2+] required to produce prolonged field bursts in the dentate underscores the positive feedback relationship between neuronal excitation and extracellular ionic concentrations, whereas the ability of synaptic stimulation to trigger nonsynaptic seizurelike events such as these prolonged field bursts may be relevant to the transition from interictal to ictal activity in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)


Roles of Ca2+ influx through ATP-activated channels in catecholamine release from pheochromocytoma PC12 cells. 1. Extracellular ATP evokes catecholamine release concomitant with depolarization in pheochromocytoma PC12 cells. Roles of Ca2+ influx through ATP-activated channels during the catecholamine release were investigated. 2. Norepinephrine or dopamine release induced by > or = 100-microM concentrations of ATP was insensitive to 300 microM Cd2+, whereas the release induced by increasing extracellular KCl (50-150 mM) was completely blocked by this concentration of Cd2+. 3. ATP (100 microM) increased the intracellular free Ca2+ concentration measured with fura-2. The increase was not affected by 300 microM Cd2+ or 100 microM nicardipine, suggesting that Ca2+ influx through ATP-activated channels but not through voltage-gated Ca2+ channels contributes to the ATP-evoked catecholamine release. 4. Inward currents permeating through voltage-gated Ca2+ channels were measured using the whole-cell voltage clamp. In the presence of 10 microM ATP, a concentration that induces an ATP-activated channel-mediated current equivalent to that induced by 100 microM ATP during the depolarization in "non-voltage clamped" cells, the Ca2+ current activated by a voltage step to +10 mV was reduced. The reduction in the Ca2+ channel-mediated current was not observed when the extracellular Ca2+ was replaced with Ba2+. 5. The ATP (100 microM)-evoked dopamine release was inhibited by 300 microM Cd2+ when measured with extracellular Ba2+ instead of Ca2+. This effect of Ba2+ may not be related to K+ channel-blocking activity, because the ATP-evoked dopamine release obtained with 5 mM tetraethylammonium (TEA) was not inhibited by Cd2+.(ABSTRACT TRUNCATED AT 250 WORDS)


Cholinergic transmission at the first synapse of the circuit mediating the crayfish lateral giant escape reaction. 1. The chemical synapses between mechanoreceptor neurons and first-order interneurons in the lateral giant (LG) neuron escape circuit of the crayfish have plastic properties, some of which are believed to be the basis for behavioral habituation and sensitization. In this investigation pharmacological experiments were conducted to assess the role of cholinergic synaptic transmission in this pathway. 2. Arterial perfusion of the cholinergic agonist carbachol produced increased activity of many abdominal nerve cord units, including an identified first-order interneuron (interneuron A) in the LG circuit. A general increase in activity of interneurons in this circuit in the presence of certain cholinergic agonists was inferred from an increase in the frequency of occurrence of spontaneous excitatory postsynaptic potentials (EPSPs) recorded in the LG. 3. Cholinergic antagonists reduced the amplitude of spontaneous and evoked sensory neuron-to-interneuron A EPSPs and decreased the disynaptic (via 1st-order interneurons) component of evoked EPSPs in the LG. These effects indicate that postsynaptic cholinergic receptors are utilized in mechanosensory synaptic transmission to the first-order interneurons of this circuit. The relative potencies of the blockers tested (mecamylamine > picrotoxin >>> curare > atropine) suggest that the receptors on the interneurons belong to a previously characterized class of crustacean cholinergic receptors that resemble the ganglionic nicotinic subtype of vertebrates. 4. Nicotinic agonists (carbachol, tetramethylammonium hydroxide, 1,1-dimethyl-4-phenyl-piperazium iodide) produced depolarizing (decreased input resistance) responses on the LG neuron itself. These responses persisted during blockade of chemical transmission by cobalt. The presence of cholinergic receptors on the LG, a cell in which all known inputs mediating sensory excitation are electrical, is discussed. 5. Application of muscarinic agonists (pilocarpine, oxotremorine) resulted in a long-lasting reduction of the evoked sensory neuron-to-interneuron A EPSP and the disynaptic component of the evoked EPSP in the LG. No effects on the membrane potential or input resistance of the interneurons were detected. It is proposed that presynaptic receptors with a muscarinic profile are present on mechanosensory neurons and that these receptors mediate a reduction of transmitter release.


Effects of membrane voltage on receptive field properties of lateral geniculate neurons in the cat: contributions of the low-threshold Ca2+ conductance. 1. Thalamic relay cells, including those of the lateral geniculate nucleus, display a low-threshold spike (LT spike), which is a large depolarization due to an increased Ca2+ conductance. Typically riding the crest of each LT spike is a burst of from two to seven action potentials, which we refer to as the LT burst. The LT spike is voltage dependent, because if the cell's resting membrane potential is more depolarized than roughly -60 mV, the LT spike is inactivated, but if more hyperpolarized, the spike is deinactivated and can be activated by a depolarization, such as from an afferent excitatory postsynaptic potential (EPSP). Thalamic relay cells thus display two response modes: a relay or tonic mode, when the cell is depolarized and LT spikes are inactivated, leading to tonic firing of action potentials; and a burst mode, when the cell is hyperpolarized and tends to respond with LT spikes and their associated bursts of action potentials. 2. We were interested in the contribution of the LT spike on the transmission of visually evoked signals through geniculate relay cells to visual cortex. We recorded intracellularly from geniculate cells in an anesthetized, paralyzed, in vivo cat preparation to study the effects of membrane voltage, and thus the presence or absence of LT spikes, on responses to drifting sine-wave gratings. We monitored the visually evoked responses of 14 geniculate neurons (6 X, 7 Y, and 1 unclassified) at different membrane potentials at which LT spikes were inactivated or deinactivated. 3. Changing membrane voltage during visual stimulation switched the response mode of every cell between the relay and burst modes. In the burst mode, LT spikes occurred in phase with the visual stimulus and not at rhythmic intervals uncorrelated to visual stimuli. To any given stimulus cycle, the cell responded usually with an LT burst or a tonic response, and rarely was more than one LT burst evoked by a stimulus cycle. Occasionally a single cycle evoked both an LT burst and tonic response, but always the LT burst occurred first. 4. The spatial tuning characteristics of the cells did not differ dramatically as a function of membrane potential, because the tuning of the LT bursts was quite similar to that of the tonic response component. Although we did not obtain complete temporal tuning properties, we did note that hyperpolarized cells responded reliably with LT bursts at several temporal frequencies. 5. A consistent difference was seen between the LT burst and tonic response components in terms of response linearity.(ABSTRACT TRUNCATED AT 400 WORDS)


A model of NMDA receptor-mediated activity in dendrites of hippocampal CA1 pyramidal neurons. 1. The role of synaptic activation of NMDA (N-methyl-D-aspartate) receptor-mediated conductances on CA1 hippocampal pyramidal cells in short-term excitability changes was studied with the use of a computational model. Model parameters were based on experimental recordings from dendrites and somata and previous hippocampal simulations. Representation of CA1 neurons included NMDA and non-NMDA excitatory dendritic synapses, dendritic and somatic inhibition, five intrinsic membrane conductances, and provision for activity-dependent intracellular and extracellular ion concentration changes. 2. The model simulated somatic and dendritic potentials recorded experimentally. The characteristic CA1 spike afterdepolarization was a consequence of the longitudinal spread of dendritic charge, reactivation of slow Ca(2+)-dependent K+ conductances, slow synaptic processes (NMDA-dependent depolarizing and gamma-aminobutyric acid-mediated hyperpolarizing currents) and was sensitive to extracellular potassium accumulation. Calcium currents were found to be less important in generating the spike afterdepolarization. 3. Repetitive activity was influenced by the cumulative activation of the NMDA-mediated synaptic conductances, the frequency-dependent depression of inhibitory synaptic responses, and a shift in the potassium reversal potential. NMDA receptor activation produced a transient potentiation of the excitatory postsynaptic potential (EPSP). The frequency dependence of EPSP potentiation was similar to the experimental data, reaching a maximal value near 10 Hz. 4. Although the present model did not have compartments for dendritic spines, Ca2+ accumulation was simulated in a restricted space near the intracellular surface of the dendritic membrane. The simulations demonstrated that the Ca2+ component of the NMDA-operated synaptic current can be a significant factor in increasing the Ca2+ concentration at submembrane regions, even in the absence of Ca2+ spikes. 5. Elevation of the extracellular K+ concentration enhanced the dendritic synaptic response during repetitive activity and led to an increase in intracellular Ca2+ levels. This increase in dendritic excitability was partly mediated by NMDA receptor-mediated conductances. 6. Blockade of Ca(2+)-sensitive K+ conductances in the dendrites increased the size of EPSPs leading to a facilitation of dendritic and somatic spike activity and increased [Ca2+]i. NMDA receptor-mediated conductances appeared as an amplifying component in this mechanism, activated by the relatively depolarized membrane potential. 7. The results suggest that dendritic NMDA receptors, by virtue of their voltage-dependency, can interact with a number of voltage-sensitive conductances to increase the dendritic excitatory response during periods of repetitive synaptic activation. These findings support experimental results that implicate NMDA receptor-mediated conductances in the short-term response plasticity of the CA1 hippocampal pyramidal neuron.


Specific involvement of Ca(2+)-calmodulin kinase II in cholinergic modulation of neuronal responsiveness. 1. Muscarinic agonists when applied in the hippocampus at low concentrations suppress intrinsic controls on neuronal excitability through the block of Ca(2+)-activated K conductance(s), gK (Ca), underlying the adaptation of firing and slow afterhyperpolarization (sAHP) in CA1 and CA3 neurons. Carbachol, for example, is effective at 0.1-0.3 microM suggesting activation of a relatively high-affinity receptor. 2. We have examined the mechanism of this action by using a new, highly specific, peptide inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaMKII) as well as other kinase inhibitors and show that the muscarinic block of gK (Ca) relies on CaMKII activation in both CA1 and CA3 neurons. Thus phosphorylation of these channels or of an intermediary protein causes the channels to remain closed in the presence of Ca2+ and depolarization. 3. The very similar electrophysiological effects of serotonergic and glutamatergic agonists are mediated either through other kinases or by entirely different processes. 4. Block of intrinsic phosphatase activity by okadaic acid also reduced adaptation and sAHP, and muscarinic agonists had no further effect on these quantities. 5. The removal of presynaptic cholinergic inputs to the hippocampus in animals has a deleterious effect on the performance of tasks requiring spatial memory and is also implicated as a cause of cognitive disorders in humans. By increasing Ca2+ accumulation during electrical activity and promoting CaMKII activity, muscarinic input provides parallel reinforcing pathways for the induction of long-term potentiation, an important cellular memory mechanism. This suggests a possible link between behavioral and cellular approaches to the analysis of learning and memory.


Building a bridge between neurobiology and mental illness. GABA (gamma amino butyric acid) is the most abundant and important inhibitory transmitter in mammalian CNS. It counterbalances the glutamate mediated neuronal excitation. Abnormalities of the interaction of these two transmitters might change the mechanisms of neuronal group selection that according to Edelman [Neural Darwinism. Basic Books, New York] play a role in mediating several brain functions including cognition processes. Indeed imbalances in GABAergic functions were shown to elicit psychoses. They can be obtained by administration of drugs that affect synthesis, metabolism and uptake of GABA and thereby cause a persistent stimulation of GABAA receptors or perhaps by genetic abnormalities in DNA transcription, pre-mRNA splicing, mRNA translation and posttranslation modifications of GABAA receptor subunits. The complexities in the regulation of GABAA receptor subunit structure, synthesis, assembly and the brain location of specific mRNA encoding for these subunits are investigated with in situ mRNA hybridization specific for subunits of GABAA receptors. The role of the variability resulting from the complexities in the regulation of GABAA receptor allosteric modulation by drugs and putative endogenous allosteric modulators of GABA action at GABAA receptors is discussed. This discussion gives relevance to the possibility that genetic abnormalities in the expression of proteins participating in GABAergic function are to be considered as a possible target of the genetic defects operative in psychoses. In line with this thinking, it is suggested that partial allosteric modulators (partial agonists) of GABAA receptors and the phosphothioate or methylphosphonate analogs antisense to specific mRNA oligonucleotides that mediate the expression of genetic information concerning GABAA and glutamate receptor subunits may become valuable tools in psychiatric research. Perhaps in the future these studies might generate new ideas useful in the therapy of genetically determined psychiatric illness.


Pituitary-adrenal axis responsiveness to ovine corticotropin releasing hormone in patients with rheumatoid arthritis treated with low dose prednisone. Ovine corticotropin-releasing hormone (oCRH) stimulation tests were performed in 8 female patients with active rheumatoid arthritis treated chronically with daily low dose prednisone and 16 age matched female controls. Patients were tested on the day of treatment, 12 h after their last prednisone dose, and after withholding prednisone for 36 h. Basal levels of plasma ACTH and to a lesser extent plasma cortisol levels were elevated before each test, and significant increases in ACTH and cortisol were induced with oCRH. The dose response relationship between total ACTH and total cortisol was shifted to the right, suggesting that the patient group had mildly deficient adrenocortical responsiveness compensated for by elevated basal evening ACTH concentrations. It is not known whether the neuroendocrine abnormalities demonstrated are due to an intrinsic preexisting abnormality, active disease, drug therapy or all these factors.


Plasma thrombomodulin and alpha 2-plasmin inhibitor-plasmin complex are elevated in active systemic lupus erythematosus. Plasma levels of thrombomodulin and alpha 2-plasmin inhibitor-plasmin complex were measured by ELISA in patients with rheumatic diseases. Thrombomodulin levels in patients with active systemic lupus erythematosus (SLE) were significantly higher than those in patients with inactive SLE or in healthy controls. This suggests that thrombomodulin, normally a component of vascular endothelial cell membrane, is easily released to plasma in patients with active SLE. High titers of the thrombomodulin level and the correlated alpha 2-plasmin inhibitor-plasmin complex elevations imply vascular injury, and consequently, excessive fibrinolytic processes in active SLE.


Effect of prolyl 4-hydroxylase inhibitor on fibroblast collagen production in vitro: an approach to the treatment of systemic sclerosis. Prolyl 4-hydroxylase (PH), which catalyzes the conversion of prolyl residues to 4-hydroxyproline, plays a central role in the synthesis and secretion of collagen. We demonstrated that fibroblasts from patients with systemic sclerosis produced significantly more PH than those from healthy subjects. To regulate excessive collagen production by scleroderma fibroblasts, we examined the effect of a PH inhibitor on collagen production by scleroderma fibroblasts. Fibrostatin-C, a PH inhibitor, significantly decreased the amount of procollagen (type I) production by scleroderma fibroblasts (p < 0.01). We suggest that this PH inhibitor might be a valid antifibrotic agent useful for the treatment of patients with scleroderma.


Factors affecting chloride conductance in apical membrane vesicles from human placenta. Apical membrane vesicles from human term placenta were isolated using a magnesium precipitation technique, and the purity of the vesicles was assessed morphologically using scanning and transmission electron microscopy, and biochemically, using marker enzymes. The vesicles were found to be morphologically intact and significantly enriched in enzymes associated with apical membranes. 36Cl- uptake into these vesicles was studied in the presence of an outwardly directed Cl- gradient. This uptake was found to be time dependent, with an initial rapid uptake tending to peak between 10 and 20 min and thereafter decline. Uptake was found to be voltage dependent since 5 microM valinomycin caused a decrease in uptake. The effects of N-phenylanthranilic acid (NPA) and 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS) and bumetanide on the initial rate of Cl- were examined in the presence and absence of 5 microM valinomycin. NPA and DIDS inhibited isotope uptake strongly with IC50 values of 0.83 +/- 0.35 microM and 3.43 +/- 0.37 microM, respectively, in the absence of valinomycin. Although valinomycin reduced 36Cl- uptake by about 80% when added before the isotope, DIDS reduced the uptake which remained in a concentration-dependent fashion with an IC50 of 5.6 +/- 2.1 microM. Under these conditions, NPA was without effect at concentrations below 100 microM. Bumetanide was without effect at the concentrations used in the absence of valinomycin. However, following valinomycin pretreatment, bumetanide reduced 36Cl- uptake significantly at 100 microM concentration. Vesicle diameter, as assessed by flow cytometry, did not change under the conditions employed. The effects of some fatty acids were also investigated. Arachidonic acid and linoleic acid inhibited Cl- uptake with IC50 values of 37.6 +/- 14.9 microM and 4.59 +/- 0.51 microM, respectively. Arachidonyl alcohol and elaidic acid were found to be without effect. These studies show that human placental brush border membrane vesicles possess a chloride conductance channel, the activity of which can be measured in the presence of an outwardly directed Cl- gradient and this channel is sensitive to Cl- channel inhibitors, especially N-phenylanthranilic acid, and can be inhibited by unsaturated fatty acids such as arachidonic acid and linoleic acid.


Sonographic evaluation of pregnancies with maternal varicella infection. We reviewed 37 cases of mothers who had varicella-zoster virus (VZV) infection during early pregnancy, who subsequently underwent fetal sonography in the 13th to 37th week of gestation. Of the five infants later shown to have congenital varicella infection at postnatal examination (1 case) or autopsy (4 cases), all showed ultrasonographic abnormalities suggesting infection, and all died by 4 months of age. None of the 32 unaffected infants demonstrated in utero ultrasonographic abnormalities. No statements about relative risk of being affected by the virus can be made from this data.


[Direct in vivo gene introduction into rat kidney]. We established a simple and efficient method in vivo gene transfer using Sendai virus (HVJ) and liposomes. Plasmid DNA and high mobility group 1 (MHG1) protein were co-encapsulated in liposomes and co-introduced into target cells by HVJ-mediated membrane fusion. pAct-SVT DNA, as a reporter gene, was introduced into the kidney of intact rats through a cannula in the renal artery, and SV 40 large T antigen was detected by enzyme immunohistochemically in glomerular cells (mesangial cells and/or capillary cells) 4 days after it's introduction. Thereafter expression of SV 40 large T antigen gene decreased and was not detected on day 10. This gene delivery system could be useful in research and development of gene therapy of renal disease.


[Vasopressin (ADH)].

[Vasopressin (ADH)]. Vasopressin is thought to play an important role, not only in the metabolism of water and electrolytes, but also in the regulation of renal hemodynamics. This year, great progress has been achieved in molecular biology of vasopressin receptors. First, the cloning of a complementary DNA, encoding the rat liver V1a arginine vasopressin receptor, was reported. The liver cDNA encodes a protein with seven putative transmembrane domains, which binds arginine vasopressin and related compounds with affinities similar to the native rat V1a receptor. The messenger RNA, corresponding to the cDNA, is distributed in rat tissues, known to contain V1a receptors. Second, the cloning of a complementary DNA encoding the rat kidney V2 arginine vasopressin receptor was also successful. The kidney cDNA encodes a protein with a transmembrane topography characteristic of G protein-coupled receptors. The receptor messenger RNA is detected only in the kidney. Last year, an orally active and specific vasopressin V1 receptor antagonist, OPC-21268 was first reported. The i.v. or p.o. administration of OPC-21268 dose-dependently inhibited vasopressin-induced vasoconstriction, while that induced by angiotensin II was not affected. OPC-21268 may have clinical potentials in certain hypertensive cardiovascular disorders. In addition, an orally active and specific vasopressin V2 receptor antagonist, OPC-31260 was also reported. Oral administration of OPC-31260 inhibited antidiuretic action of arginine vasopressin. OPC-31260 is thought to be useful in the treatment of certain disorders, such as the syndrome of inappropriate secretion of ADH (SIADH).


[Renal kallikrein-kinin system].

[Renal kallikrein-kinin system]. The renal kallikrein-kinin system seems to participate in the regulation of blood pressure, control of sodium and water excretion, renal vascular resistance and renin release. By using ultrastructural immunocytochemistry, in situ hybridization or polymerase chain reaction experiments with the appropriate cDNA probes, kallikrein and kininogen were observed to exist and be synthesized in different types of cells of the nephron. Recently, new finding of a kinin receptor has been reported, since specific kinin receptor antagonists have become available. The localization of components of kallikrein-kinin system in the kidneys and its relation to renal function is discussed.


[Molecular biology of regulation of renal function--structure, function and distribution of the receptor--insulin, glucagon]. Insulin and glucagon are known to regulate both metabolic and transport functions in the nephron, directly. Insulin receptor is a heterotetrameric glycoprotein, consisting of two alpha-subunits and two beta-subunits linked by disulfide bonds. Binding of insulin to its receptor in the proximal basolateral membranes results in phosphorylation of beta-subunit which is considered to be necessary to subsequent activation of receptor tyrosine kinase. Insulin receptors are also present in the luminal membranes of the proximal tubules. Proximal tubules are major sites of insulin and glucagon degradation. Insulin and glucagon regulate mineral transport mainly at the thick ascending limb. Insulin and glucagon regulate gluconeogenesis in the kidney.


[Parathyroid hormone and calcitonin].

[Parathyroid hormone and calcitonin]. Parathyroid hormone (PTH) is essential for the physiologic maintenance of mineral homeostasis. PTH regulates the mineral transport in bone and kidney and through its secondary actions on mineral transport in intestine (mediated by 1.25 (OH)2D). Calcitonin, in many ways, acts as a physiologic antagonist to PTH. Recently the techniques of molecular biology have been applied to the study of these hormones and more precise mechanism of action of these hormones has been elucidated. Last year both PTH receptor and calcitonin receptor were cloned. This review briefly summarizes new informations about their biosynthesis, secretion, metabolism, action, and structures of their receptors.


[Metabolism and action mechanisms of eicosanoids and platelet-activating factor in the kidney]. Recent advances in molecular biological studies on the metabolism and action mechanisms of lipid mediators: arachidonic acid metabolites (eicosanoids) and platelet-activating factor (PAF), are reviewed, in relation to their roles in the kidney. For the last decade, almost all the enzymes involved in three major arachidonic acid metabolic pathways; cyclooxygenase, lipoxygenase and epoxygenase pathways, have been purified and almost all of their cDNA have been cloned. Recently, cDNAs encoding three receptors for lipid mediators; PAF receptor, thromboxane A2/prostaglandin (PG) H2 receptor and a subtype of PGE2 receptor; EP3, were cloned. These advances have facilitated our knowledges of the physiological, pathophysiological roles of these lipid mediators in the kidney.


[Vitamin D3].

[Vitamin D3]. With the application of biochemical technique came the discovery that vitamin D is metabolized sequentially to 25-hydroxyvitamin D in liver and then to 1.25-dihydroxyvitamin D (1.25D), the hormonal form of vitamin D, in kidney. The latter process is strictly controlled with the demand of calcium and phosphorus. The produced 1.25D binds its specific receptor probably located in the nuclei. With the application of modern tools of molecular biology came the discovery that vitamin D receptor (VDR), which belongs to steroid receptor family, acquires increased affinity for DNA when it binds to 1.25D and then VDR complexes bind to DNA with its two zinc-finger projections. VDR complexes recognize vitamin D responsive element on DNA, two tandemly repeated hexanucleotide sequences separated by three base pairs. Furthermore, to interact with the VDRE, VDR complexes require nuclear accessory factor, which might be retinoid X receptor (RXR). As above, molecular biology helps us to extend our knowledge in the field of vitamin D.


[Molecular biology of membrane transport proteins of renal tubules]. Molecular biology techniques are now opening a new era in renal epithelial transport physiology and biology. Two things emerge as a result of molecular biology studies. 1) Transporters are usually composed of several isoforms. Expressions of each isoforms are different in timing and localization, thus, they play different physiological roles. 2) Comparison of sequence homology allows speculation of superfamily and root of transporter gene. Based on this idea it is not surprising that one transporter shows three functions; channel, enzyme, and carrier. In this review current understanding of kidney transport protein by molecular biology is summarized. Topics are: GLUT, Na/glucose cotransporter, Na/H exchanger, Cl/HCO3 exchanger, amino acids transporter, water channel, CFTR, MDR.


[Analyses of Na, K-ATPase subunit genes]. Na, K-ATPase is the enzyme responsible for the maintenance of the electrochemical gradients of Na+ and K+ across the plasma membrane. The enzyme consists of a heterodimer of an alpha and a beta subunits. Na, K-ATPase belongs to a multigene family. Three alpha subunit (alpha 1 alpha 2 alpha 3) and three beta subunit (beta 1 beta 2 beta 3) isoforms have been described so far. The gene expression of each subunit is differentially regulated in various tissues and developmental stages. To clarify the molecular mechanisms of the regulation of Na, K-ATPase gene expression and of the coordinated regulation between the alpha and beta subunit genes, extensive analyses of the genes encoding alpha and beta subunit isoforms have been performed. The rapid progress of the study in this field is reviewed.


[Molecular biology of cytochrome P450 in kidney]. Cytochrome P450-catalyzed monooxygenation system is widespread in nature and many isoforms have been found in almost all the tissues of vertebrates. In the kidney, several isoforms of cytochrome P450 families, CYP1, 2, 3, 4, 24, 27 have been characterized by biological, immunochemical and molecular biological techniques. These cytochrome P450 isoforms are localized in the endoplasmic reticulum or mitochondria of kidney cortex and functional in the monooxygenation of a number of substrates, such as steroids, fatty acids, xenobiotics, and vitamin D. Particularly, hydroxylation of vitamin D at positions of 1 and 24 is known to be one of the most important functions of kidney specific P450 isoforms.


[A case of an aged patient suspected of craniopharyngioma with a chief symptom of eunuchoidism accompanied with panhypopituitarism]. A 78-year-old man with developmental disturbance of the genital organs and eunuchoidism was reported. He also had a high pitched voice, thickness of the lower lip and kyphosis of the thorax. He seemed to be fretful, but his intelligence was normal. Neurological tests revealed bilateral hemianopsia and decreased tendon reflexes. A plain skull radiograph clearly showed an egg shaped calcified mass extending upward from the sella turcica which resembled a ballooning shape. Brain CTs showed a high density round mass which expanded the sella turcica and raised the floor of the third ventricle. The inner part of the tumor showed irregular high density. T1-weighted MR imaging revealed an iso signal intensity, and T2 showed low signal intensity in the mass. These findings strongly supported the diagnosis of calcificated craniopharyngioma. Endocrinological study showed panhypopituitarism caused by the tumor compressing the pituitary gland and the hypothalamus. The main reasons why there were no apparent symptoms of hypopituitarism were because the receptors were up-regulated and secondarily because the thyroid and the adrenal cortical functions decreased while struggling to maintain balance with each other. There was also a possibility that these symptoms might have been masked by normal aging. Benign monoclonal hypergammopathy was also indicated, although we could not find a clear correlation between this finding and others.


Hemodynamic responses during acute normovolemic hemodilution in anesthetized dogs. Acute normovolemic hemodilution was induced by progressive replacement of blood by dextran (molecular weight 150,000) in anesthetized artificially ventilated dogs. Experiments were performed on dogs with intact autonomic innervation, dogs with beta-adrenergic blockade, dogs with cholinergic blockade, dogs with bilateral vagotomy, and dogs with combined bilateral vagotomy plus beta-adrenergic blockade. Hemodilution induced an increase in cardiac-output in all the groups. However, in dogs with low control heart rate (dogs with intact autonomic innervation, and dogs with beta-adrenergic blockade), cardiac-output increase was almost wholly due to an increase in heart rate. Whereas, in dogs with high control heart rate (dogs with cholinergic blockade, dogs with bilateral vagotomy, and dogs with combined bilateral vagotomy plus beta-adrenergic blockade), the increase in cardiac-output was almost wholly due to an increase in the stroke-volume. The increase in heart rate in dogs with intact autonomic innervation was not significantly different from the heart rate increase in dogs with beta-adrenergic blockade. In dogs with low control heart rate (beta-receptor blockade), hemodilution induced tachycardia, which was not significantly different from the response induced in intact dogs. This shows that the cardioacceleration was primarily mediated through the efferent vagus nerves, and the efferent sympathetic nerves did not make significant contribution in the reflex.


In vivo pharmacologic profile of ONO-1078: a potent, selective and orally active peptide leukotriene (LT) antagonist. We investigated the in vivo antagonistic activity of ONO-1078 against peptide leukotrienes (LTs) in guinea pigs. ONO-1078, when administered p.o. (0.3-3 mg/kg), caused a dose-dependent reduction of LTC4-, LTD4- and LTE4-induced bronchoconstriction, LTD4-induced airway microvascular leakage and LTD4-induced increase in cutaneous vascular permeability. When administered intravenously, ONO-1078 (3-30 micrograms/kg) inhibited these responses approximately 200-600 fold more potently than FPL55712. When guinea pigs were treated with indomethacin to examine the antagonism of ONO-1078 on the direct action against peptide LTs, intravenous (3-30 micrograms/kg) and oral (0.3-3 mg/kg) administration of ONO-1078 also inhibited LTC4- and LTD4-induced bronchoconstriction, and its activity was approximately 300-500 fold more potent than that of FPL55712. ONO-1078 (10 mg/kg, i.v.) had no inhibitory effect on bronchoconstrictions induced by histamine, acetylcholine, serotonin, arachidonic acid, LTB4, prostaglandin (PG) F2 alpha, PGD2, 9 alpha, 11 beta-PGF2, a stable thromboxane A2 mimetic agent and platelet activating factor. Furthermore, oral administration of ONO-1078 (1-10 mg/kg) inhibited slow-reacting substance of anaphylaxis mediated bronchoconstriction induced by antigen in a dose-dependent manner. These results indicate that ONO-1078 is an extremely potent, selective and orally active peptide LT antagonist and that oral administration of ONO-1078 antagonizes not only exogenously administered peptide LTs but also endogenous peptide LTs.


Protective effect of ulinastatin against liver injury caused by ischemia-reperfusion in rats. The effects of ulinastatin (ULN), a human urinary protease inhibitor, on liver injury caused by ischemia-reperfusion were studied in rats. In the liver ischemia-reperfusion model, ULN suppressed the elevation of serum transaminase levels and tissue lipid peroxide levels in the liver. ULN did not exhibit a radical-trapping action on the superoxide and hydroxyl radicals as measured by electron spin resonance (ESR). ULN suppressed formylmethionyl-leucyl-phenylalanine (FMLP) and phorbol myristate acetate (PMA)-induced superoxide production from polymorphonuclear leukocytes (PMNs) as measured by the cytochrome c assay. ULN did not inhibit either xanthine oxidase (XO) activity or the conversion of xanthine dehydrogenase (XDH) to XO during the ischemic period. ULN also strongly protected against the hypotonic hemolysis of rat erythrocytes. These results suggest that ULN's membrane stabilizing action and suppressive effect against PMNs superoxide production might be attributed to its suppressive effect on the liver's lipid peroxidation caused by ischemia-reperfusion.


Calcium is essential for ATP-induced steroidogenesis in bovine adrenocortical fasciculata cells. The effect of extracellular ATP on steroidogenesis in primary cultured bovine adrenocortical fasciculata cells was investigated. I observed that in the presence of extracellular Ca2+, ATP caused a dose-dependent elevation of intracellular Ca2+ ([Ca2+]i) and induced steroidogenesis concentration- and time-dependently. However, in the absence of extracellular Ca2+, ATP had no effect on steroidogenesis. In the presence of extracellular Ca2+, calmodulin inhibitors inhibited the ATP-induced steroidogenesis, but dihydropyridine calcium channel blockers did not. Furthermore, ATP did not cause an elevation of cyclic AMP in bovine adrenocortical fasciculata cells even if extracellular Ca2+ existed. These results suggest that extracellular ATP might have an influence on bovine adrenocortical cells via the purinoceptor (P2Y) in connection with calcium mobilization, open the non-selective calcium channel and induce steroidogenesis by means of an elevation of [Ca2+]i via the calcium-calmodulin system.


Possible mechanisms of action of the antispasmodic agent tiropramide in the isolated detrusor from rats. The effects of tiropramide hydrochloride on Ca(2+)-induced contraction, cytoplasmic free Ca2+ levels and tissue cyclic AMP concentrations were investigated to elucidate the mechanisms of its antispasmodic action in the isolated detrusor from rats. Tiropramide inhibited the Ca2+ (3 mM)-induced contractions of the isolated urinary bladder depolarized in a Ca(2+)-free medium, and the IC50 value was 3.3 x 10(-6) M. When tiropramide was added during the sustained phase of the K+ (60 mM)-contracture, IC50 values of tiropramide for the contraction and the increased fluorescence were 1.9 x 10(-5) M and 16.4 x 10(-5) M, respectively. On the other hand, the IC50 values for the K(+)-induced contraction and fluorescence after pretreatment of the isolated urinary bladder with tiropramide were 2.1 x 10(-5) M and 2.6 x 10(-5) M, respectively. Tissue cyclic AMP levels at 1 min after addition of 10(-5) M tiropramide were significantly increased. Papaverine, IBMX or forskolin potentiated the inhibitory effect of tiropramide on carbachol-induced contraction and its cyclic AMP-elevating effect. However, a good correlation between the degrees of potentiation of the inhibitory effect and the increase in cyclic AMP levels was not observed. The present results suggest that the smooth muscle relaxant activity of tiropramide in the isolated detrusor from rats may be intimately associated with predominant inhibition of Ca2+ influx and, to a lesser extent, an increase in intracellular cyclic AMP levels.


Melibiose transport system in Lactobacillus plantarum. Lactobacillus plantarum ATCC 8014 grew on melibiose at 30 C, but not at 37 C, although it grew on galactose or lactose at either temperature. ATCC 8014 grown on lactose at 30 or 37 C accumulated melibiose slowly, suggesting that melibiose may partly be transported by a lactose transport system. A lactose-negative mutant, NTG 21, derived from ATCC 8014 was isolated. The mutant was totally deficient in lactose transport, but retained normal melibiose transport activity. In NTG 21, the melibiose transport activity was induced by melibiose at 30 C, but not at 37 C. The transport activity itself was found to be stable for at least 3 hr at 37 C, suggesting that the induction process in the cytoplasm rather than the inducer entrance is temperature-sensitive in the organism. The organism also failed to form alpha-galactosidase at 37 C when grown on melibiose. The enzyme synthesis, however, was induced by galactose in NTG 21 (and also by lactose in ATCC 8014) even at 37 C, indicating that the induction of the enzyme is essentially not temperature-sensitive. In NTG 21, melibiose transport system and alpha-galactosidase were induced by galactose, melibiose and o-nitrophenyl-alpha-D-galactopyranoside when the strain was grown at 30 C. Raffinose induced melibiose transport system only a little, while it was a good inducer for alpha-galactosidase. Inhibition studies revealed that galactose may be a weak substrate of the melibiose transport system; no inhibition was demonstrated with lactose and raffinose.


Detection of herpes simplex and varicella-zoster virus DNA by field-inversion gel electrophoresis from clinical materials. A simple method using field-inversion gel electrophoresis (FIGE) was applied to detect herpes simplex virus (HSV) and varicella-zoster virus (VZV) genomes in clinical specimens. The whole genomes of these viruses could be detected in small vesicle tissues by the FIGE method regardless of their clinical stages of skin lesions. And the sensitivity of the FIGE method was equivalent to that of an immunofluorescent (IF) method. These data indicated usefulness of the FIGE method to detect the whole genomes of HSV and VZV in clinical specimens.


A rationale for NMDA receptor antagonist therapy of the neuroleptic malignant syndrome. The triad of rigidity, fever, and elevation of serum creatine phosphokinase (CPK) levels, labeled 'neuroleptic malignant syndrome' (NMS), is a dangerous complication of neuroleptic drug treatment. Amantadine was introduced for the pharmacological management of NMS because of its beneficial effects in Parkinson's disease which were attributed to direct or indirect dopaminomimetic properties of amantadine. While the dopaminomimetic effects of amantadine are weak under experimental conditions, recent studies have confirmed that amantadine is an antagonist at the N-methyl-D-aspartate (NMDA) type of glutamate receptor. Two lines of evidence suggest that amantadine or other NMDA receptor antagonists could be effective drugs for the reversal of NMS symptoms. First, glutamate antagonists restore the balance between glutamatergic and dopaminergic systems when dopaminergic transmission has been antagonized by neuroleptic drugs. Second, by virtue of their effects against rigor and spasticity, NMDA antagonists may reduce increased muscle tone and prevent rhabdomyolysis. In conclusion, NMS may be considered an iatrogenic excitatory aminoacid syndrome which is amenable to NMDA receptor antagonist therapy.


An efficient method for detecting connectivity in neural ensembles. Modern technology is allowing researchers to collect data from neural ensembles with a large number of units, and the analysis of interaction between these units can be very time consuming. Estimation of pairwise connectivity is the most common method of determining the neural 'network' but usually necessitates the production of numerous histograms for each pair considered. We present a method which will indicate which pairs in a network represent potential connections and thereby simplify the postexperimental analysis. The technique uses cross-interval information to create an n x n matrix which represents all possible connections in an n neuron ensemble and can be calculated recursively on-line. The performance of this technique is analyzed with respect to data size and strength of the connections. It is compared to 2 similar techniques that are also presented here, one in which perfect knowledge of the timing of the excitation is known, and one in which the timing can be bounded.


Paradoxical decrease in myf-5 messenger RNA levels during induction of myogenic differentiation by insulin-like growth factors. Having previously demonstrated that the insulin-like growth factors (IGFs) induce expression of the myogenin gene, we have now extended our investigation of the induction of myogenesis by the IGFs to a second member of the MyoD family, myf-5. This is the only myogenesis gene other than myogenin expressed early in the differentiation of L6 myoblasts, so its regulation was of particular interest because of our observations on myogenin. In contrast to myogenin, myf-5 mRNA was detectable in proliferating myoblasts, but the steady state levels of myf-5 mRNA fell strikingly for 48 h after the cells were switched to low serum medium containing IGF-II in both murine cell lines and myoblasts cultured from human muscle. In spite of this decrease, translation of myf-5 mRNA appeared essential during the early stages of stimulation of myogenesis by the IGFs; an antisense oligodeoxynucleotide complementary to the first five codons of myf-5 blocked the increase in myogenin mRNA and inhibited morphological (cell fusion) and biochemical (creatine kinase elevation) aspects of myogenesis. We conclude that expression of myf-5 is essential for the initial induction of myogenin by the IGFs, but that subsequent elevation of myogenin expression is independent of myf-5, possibly resulting from autoinduction of the myogenin gene. The functional significance of the dramatic decrease in myf-5 mRNA levels during differentiation is not obvious.


Identification of positive and negative placenta-specific basal elements and a cyclic adenosine 3',5'-monophosphate response element in the human gene for P450scc. The chronic regulation of steroiodgenesis is mediated principally by transcriptional regulation of the genes encoding the various steroidogenic enzymes. The cholesterol side-chain cleavage enzyme, P450scc, is rate limiting and hormonally regulated in a tissue-specific fashion. Human placental steroidogenesis is regulated by LH and hCG through increased intracellular cAMP, and forskolin and 8-bromo-cAMP increase the abundance of human P450scc mRNA in human JEG-3 choriocarcinoma cells. We transfected JEG-3 cells with 24 promoter/reporter constructions to examine the tissue-specific and hormonally induced transcription of the human P450scc gene in these cells. A reporter construction containing only bases -79 to +49 of the human P450scc gene was expressed in JEG-3 cells. This basal expression was increased by four elements, especially by a powerful element between -152 to -142. Adding DNA sequences to -177 suppressed the basal expression seen with the -152 construction, indicating that a repressor element lies between -177 and -152. Thus, basal expression of the human P450scc gene in JEG-3 cells is mediated by the interplay of several separate cis-acting DNA elements. Forskolin induction was conferred by sequences between -108 and -89. The mechanism for cAMP induction appears to be direct, as this induction is rapid and is not blocked by inhibiting protein synthesis with cycloheximide. Gel mobility shift experiments identified six specific DNA-protein complexes. Five of these complexes correlate closely with the basal transcription activities identified by the reporter assays. The powerful basal element, the repressor element, and the cAMP element differ from those identified by similar experiments in mouse adrenal Y1 cells, suggesting that the human P450scc gene is regulated by the tissue-specific use of different regulatory elements.


Cyclic adenosine 3',5'-monophosphate activation of the rat prolactin promoter is restricted to the pituitary-specific cell type. Pituitary lactotroph cell function and PRL gene expression are highly regulated by the cAMP-protein kinase-A (PKA) pathway. To further our understanding of the molecular mechanisms by which cAMP/PKA regulates rat (r) PRL promoter activity and to determine whether cAMP regulation is cell type specific, we 1) transected intact (-425), internal and 5'-deletion, and site-specific mutants of the rPRL promoter ligated to the firefly luciferase reporter gene into both pituitary and nonpituitary cell lines; and 2) assessed the role of the cAMP-cAMP response element-binding protein (CREB) pathway in GH4 rat pituitary cells. The data show that deleting the rPRL promoter from -425 to -116 did not abolish cAMP regulation, implying that proximal elements, such as the basal transcription element (-112/-80) or the pituitary-specific footprint (FP) I (-67/-45), mediate the cAMP response. However, nucleotide changes within FP I or FP II (-130/-120) did not alter the rPRL promoter response to 1 microM forskolin (FSK), despite the 77% and 26% reductions in basal rPRL promoter activity caused by these mutations, respectively. Furthermore, internal deletion of either the basal transcription element of FP I element also failed to affect cAMP regulation of the rPRL promoter, again despite the 90% and 93% reductions in basal promoter activity by these deletions, respectively. Since these internal deletion constructs otherwise contain rPRL promoter sequences from -425 to +73, including the up-stream pituitary-specific FPs III and IV, the data suggest that any one of these cell-specific elements is capable of imparting cAMP regulation to the proximal rPRL promoter. To directly test the implication that the cAMP response of the rPRL promoter is restricted to the pituitary-specific cell type, we took advantage of a 5'-deletion mutant truncated at position -116 and a FP II site-specific mutant, since constructs containing these rPRL promoters are active in nonpituitary cells. Despite the 6.6- and 18.5-fold stimulations over wild-type rPRL promoter activity in nonpituitary cells, respectively, these mutations remained completely unresponsive to FSK treatment. To document that the cAMP-CREB pathway was functional in GC/GH4 rat pituitary cells, CREB was affinity purified from GC rat pituitary cells, and DNase-I protection studies showed that it does not bind to the proximal rPRL promoter. Also, the human glycoprotein alpha-subunit promoter was induced 10-fold by FSK in GH4 rat pituitary cells.(ABSTRACT TRUNCATED AT 400 WORDS)


[Specific DNA cleavage by an analog of netropsin containing a copper(II) chelating peptide Gly-Gly-His]. Experimental data are reported on DNA-cleaving activity of the synthetic netropsin analogs consisting of the two N-propylpyrrole carboxamide units linked covalently through two or three glycine residues to a copper-chelating tripeptide glycyl-glycyl-L-histidine. Incubation of DNA restriction fragment and netropsin analog in the presence of ascorbate, hydrogen peroxide and Cu2+ ions resulted in selective cleavage of the DNA at or near the preferred sites for binding of netropsin analog. A similar cleavage pattern is observed after X-ray irradiation of DNA complexes with netropsin analogs tethered with Cu2+ ions. The cleavage patterns are found to be dependent on the length of the connecting chain between the histidine-containing tripeptide and netropsin analog. The netropsin analog containing three glycine residues in the connecting chain, but not the analog with a shorter linker chain, can generate an intense cleavage of one of the two polynucleotide chains at a position corresponding to the presumed binding site for the dimeric ligand species. More than 50% of the total DNA can be cleaved at this position after X-ray irradiation. From analysis of the nucleotide sequences surrounding the preferred cleavage site on several DNA fragments we found that the consensus is 5'-TTTTNCA*AAA-3', where N is an arbitrary nucleotide. The Cu(2+)-mediated cleavage of DNA occurs at the second adenine (indicated by an asterisk) from the 5'-end of the sequence. The greatest cleavage activity is observed when the molar ratio of Cu2+ to the netropsin analog is equal to 0.5. Evidently, the Cu(2+)-ligated and unligated oligopeptide species interacts with each other to form a heterodimer bound to DNA at the cleavage site. To test the validity of this model we have studied the binding of unligated netropsin analog and netropsin analog complexed with Cu2+ ion to a self-complementary oligonucleotide 5'-GCGTTTTGCAAAACGC-3'. It is found that binding of Cu(2+)-ligated netropsin analog to the DNA oligomer preincubated with unligated form of the oligopeptide is a cooperative process for which interactions between the two bound ligands are responsible. The cooperativity parameter is estimated to be on the order of factor 6. Finally, a model is proposed in which a heterodimer stabilized by interligand beta-sheet binds in the minor DNA groove.


Switching agonistic, antagonistic, and mixed transcriptional responses to 11 beta-substituted progestins by mutation of the progesterone receptor. The study of transcription activation by a series of RU486-related 11 beta-substituted progestins revealed three types of ligands: agonists, antagonists, and a novel type of compounds that exerted a mixed activity. These ligands conferred to the human progesterone receptor (hPR) only weak activation properties despite high affinity binding and, hence, acted as agonists and, at the same time, as partial antagonists of pure agonists. When the same series of ligands was tested with mutant PRs, transcriptional activation/inactivation profiles were different from those seen with the wild-type PR, since several steroids initially classified as antagonists switched to mixed responses. One compound that acted as an antagonist for the hPR was an agonist for a mutated PR in which 15 amino acids of the hormone-binding domain were replaced by the corresponding divergent residues of the chicken homolog. In analyzing a series of steroids with wild-type and mutant PRs, we observed that a phenyl group (or a phenyl derivative) in the 11 beta position of RU486-related steroids generates antagonism with hPR, but has to be bound in a critical position in the hormone-binding domain to exert its antagonistic activity. Apparently, a deviation from this positioning by mutations in the hormone-binding domain can generate mixed or even agonistic activities.


[Kinetics of formation of 8-oxy-2'-deoxyguanosine-5'-monophosphate under the effect of heat: determination of rate constants and activation energy]. Rate constants of 8-oxy-dGMP (8-hydroxy-dGMP) formation upon incubating dGMP in H2O solutions at different temperatures were determined with differential UV-spectroscopy. Extrapolation of rate constant values obtained at elevated temperatures to 37 degrees C gives k = 5.8 x 10(-10) s-1.M-1. The activation energy for the process was estimated to be 24 kcal/mole. In D2O solutions essential lowering of the activation energy (13 kcal/mole) and rising of rate constant (k = 3.7 x 10(-9) s-1.M-1 at 37 degrees C) were observed. The strong influence of D2O on the process points to the possible participation of singlet oxygen in a heat-induced formation of 8-oxy-dGMP. The obtained values of rate constants and activation energy induced by heat show that of all types of DNA damages currently known such as single strand scission, depurination, cytosine deamination and oxidation of guanyl residues to the 8-oxo-derivatives- the last process seems to be the strongest damage of DNA resulting in such biological consequences as mutagenesis, carcinogenesis and aging.


Activating transcription factor-2 DNA-binding activity is stimulated by phosphorylation catalyzed by p42 and p54 microtubule-associated protein kinases. Recent studies have detailed the ability of activating transcription factor-2 (ATF-2) to mediate adenoviral E1a stimulation of gene expression; however, an endogenous regulator for the transcriptional activity of this protein has not been described. To characterize the regulation of ATF-2 activity, we have expressed full-length and truncated peptides corresponding to various regions of the ATF-2 protein in bacteria and the baculovirus insect cell system. Bacterially expressed truncated (350-505) but not full-length ATF-2, was able to bind a consensus cAMP response element-containing oligonucleotide, suggesting the N-terminal moiety may serve as a negative regulator of DNA-binding activity. In contrast, the full-length ATF-2 protein expressed in Spodoptera frugiperda (Sf9) cells using a recombinant baculovirus was fully competent to bind DNA. Protein phosphatase 2A reversed the DNA-binding activity by dephosphorylating the ATF-2 polypeptide. Microtubule-associated protein kinase catalyzed the phosphorylation and stimulated the DNA-binding activity of bacterially expressed full-length ATF-2. Phosphopeptide mapping of phosphorylated ATF-2 proteins identified a single peptide in the N-terminal moiety of ATF-2 phosphorylated by p42 or p54 microtubule-associated protein kinase. Therefore, we propose that phosphorylation of this regulatory site is sufficient to induce an allosteric structural change in the ATF-2 protein, which allows dimerization and subsequent DNA binding.


Somatostatin receptors, an expanding gene family: cloning and functional characterization of human SSTR3, a protein coupled to adenylyl cyclase. We previously reported the cloning of two distinct somatostatin receptor (SSTR) subtypes, SSTR1 and SSTR2. Although both SSTR1 and SSTR2 bound somatostatin specifically and with high affinity, neither was coupled to adenylyl cyclase, a major cellular effector of somatostatin's actions. Here we report the cloning and functional characterization of a third member of the SSTR family. Human SSTR3 is a protein of 418 amino acids and has 45% and 46% identity with human SSTR1 and SSTR2, respectively. RNA blotting studies showed that SSTR3 mRNA could be readily detected in brain and pancreatic islets. The pharmacological properties of human SSTR3 were characterized by transiently expressing the human SSTR3 gene in COS-1 cells. Membranes from cells expressing human SSTR3 bound the somatostatin agonist [125I]CGP 23996 specifically and with high affinity, with a rank order of potency of somatostatin-28 = CGP 23996 > somatostatin-14 > SMS-201-995. Studies using cells transiently coexpressing the human dopamine D1 receptor and human SSTR3 showed that somatostatin was able to inhibit dopamine-stimulated cAMP formation in a dose-dependent manner, indicating that SSTR3 was functionally coupled to adenylyl cyclase. These results indicate that the diverse biological effects of somatostatin are mediated by a family of receptor with distinct, but overlapping, tissue distributions, unique pharmacological properties, and potentially different functions.


Cloning of a novel alpha 1-subunit of the voltage-dependent calcium channel from the beta-cell. To study the molecular regulation of voltage-dependent Ca2+ channels (VDCCs) in the beta-cell, we have cloned a cDNA for the alpha 1-subunit from a hamster insulin-secreting cell line (HIT-T15). The cDNA (HCa3a) encodes a 1610-amino acid protein with four repeating membrane domains and an overall structure characteristic of other alpha 1-subunits. Although the cDNA shows a high degree of sequence homology (97%) with a rat brain alpha 1-subunit (RB alpha 1), the C-terminal 15 amino acids of HCa3a share no similarity with any cloned alpha 1 protein. High stringency Northern blot analysis revealed a single transcript of approximately 8.6 kilobases in HIT cells and hamster pancreas. A similarly sized species was detected in hamster brain, heart, and skeletal muscle. Using polymerase chain reaction and a primer set unique to HCa3a, this alpha 1 isoform was found to be expressed in islet cell lines derived from rat, mouse, and hamster. The HIT cell alpha 1-subunit is also expressed in discrete regions of the rat central nervous system, including the cortex, cerebellum, hypothalamus, and brain stem. The expression of two alpha 1 isoforms (HCa3a and cardiac) in the HIT cell underscores the possible complexity of VDCCs in the regulation of beta-cell signal transduction. With its widespread tissue distribution, HCa3a does not conform to the current classification system used for L-type VDCCs; this suggests that an alternative system of classification is required.


Parathyroid hormone induces transcription of collagenase in rat osteoblastic cells by a mechanism using cyclic adenosine 3',5'-monophosphate and requiring protein synthesis. Collagenase is synthesized and secreted by rat osteoblastic cells in response to PTH. We have previously demonstrated that this effect involves a substantial increase in collagenase mRNA via transcription. Northern blots and nuclear run-on assays were performed to further investigate the induction of collagenase by PTH in the rat osteoblastic cell line UMR 106-01. Detectable amounts of collagenase mRNA were not apparent until 2 h of PTH treatment, showed the greatest abundance at 4 h, and declined to approximately 30% of maximum by 8 h. The changes in the rate of transcription of the collagenase gene in response to PTH paralleled and preceded the changes in the steady state mRNA levels. After an initial lag period of about 1 h, collagenase transcription rates increased from very low levels to a maximal response at 2 h, returning to about 50% of maximum by 10 h. The increased transcriptional rate of the collagenase gene was found to be dependent on the concentration of PTH, with a half-maximal response at approximately 7 x 10(-10) M rat PTH-(1-34) and a maximal effect with a dose of 10(-8) M. The PTH-mediated induction of collagenase transcriptional activity was completely abolished by cycloheximide, while transcription of the beta-actin gene was unaffected by the translation inhibitor. These data suggest that a protein factor(s) is required for PTH-mediated transcriptional induction of collagenase. Since PTH increases intracellular levels of several potential second messengers, agents that mimic these substances were employed to determine which signal transduction pathway is predominant in the PTH-mediated stimulation of collagenase transcription.(ABSTRACT TRUNCATED AT 250 WORDS)


Regulation of prodynorphin gene expression in the ovary: distal DNA regulatory elements confer gonadotropin regulation of promoter activity. Examination of the regulation of prodynorphin (pro-DYN) promoter activity is limited by the absence of a good cell model. The discovery of pro-DYN mRNA and derived peptides in the reproductive tract led us to examine the cellular localization and hormonal regulation of ovarian pro-DYN expression and to evaluate normal granulosa cells as a model for studying pro-DYN gene regulation. Ovarian pro-DYN mRNA levels were significantly elevated in PMSG-primed immature rats 12-24 h after receiving an ovulatory dose of hCG. Levels peaked 2 days after hCG, remained elevated throughout the ensuing pseudopregnancy, and rose again at the end of pseudopregnancy. In situ hybridization localized pro-DYN expression predominantly to granulosa and luteal cells. Transfection of primary cultures of granulosa cells revealed that the activity of the rat pro-DYN promoter [-1858 to 133 base pairs (bp)] was increased 18- to 19-fold by hCG and human FSH treatments and 7-fold by cAMP analog treatment. Deletion analysis identified a 358 bp fragment as the primary hormone-responsive sequence (-1858 to -1500 bp; containing three potential cAMP-responsive elements); its deletion resulted in severely reduced FSH responsiveness, and its ligation to hormone-unresponsive basal promoter sequences completely restored FSH responsiveness. This is an unusually distal position for cAMP-responsive elements compared to other cAMP-regulated genes. These data demonstrate specific expression of pro-DYN in granulosa and luteal cells, which is under sensitive gonadotropin regulation, and identify a distal hormone-responsive sequence within the promoter.


Dialysis of lactotropes with antisense oligonucleotides assigns guanine nucleotide binding protein subtypes to their channel effectors. This article describes a new approach for determining the role of endogenous guanine nucleotide binding (G) protein subunits in signal transduction. Sequential patch-clamping was applied to BSA gradient-enriched cultured lactotropes from lactating rats, first to dialyze antisense oligodeoxyribonucleotides (AS) directed against G alpha protein mRNAs and 48 h later to record ion-current responses to the PRL release inhibitor, dopamine. The effectiveness and specificity of action of six types of AS were determined by their effects on the in vitro translation of alpha o, alpha i1, alpha i2, alpha i3, and alpha s. The specificity of AS could be enhanced by replacing guanine by cytosine bases within the center core of AS and by maximizing the number of mismatches against nontargeted mRNAs within the extremities of AS. A total of 59 out of 240 cells could be investigated using the sequential patch clamp procedure in the absence of antibiotics. The typical decrease of the voltage-activated calcium current in response to 10 nM dopamine was diminished or abolished by AS, in correlation with the inhibition of in vitro translation of the alpha o subunit. The typical increase of the voltage-activated potassium current in response to dopamine was abolished by AS directed against alpha i3 but not alpha o mRNA. Control experiments showed that culture conditions or loss of receptor affinity for dopamine were not responsible for the loss of response. The results suggest that dopamine D2 receptors are linked via alpha o to calcium channels and via alpha i3 to potassium channels.(ABSTRACT TRUNCATED AT 250 WORDS)


Neurotransmitter and drug receptor genes. Studies of the two neurotransmitter receptor genes described here illustrate the rich possibilities that this area of molecular neurobiology holds for drug abuse research. Since many of the 100 to 150 different neurotransmitters could have multiple receptor genes, as many as 1,000 different genes or 1 percent of the human genome might encode neurotransmitter receptors. Clearly, as the other chapters in this monograph also indicate, the field is ripe with possibilities of discovery with implications for drug action.


[The role of HBV and HCV viruses in the pathogenesis of hepatic lesions in alcoholics]. Due to the high prevalence in the population of both viral hepatitis and alcoholism it is not uncommon to find patients with chronic hepatitis in whom both these stigmata are simultaneously present. In such cases, the risk of evolution to cirrhosis is thought to be greater. In this study was evaluated 104 alcoholic patients (50 of whom had hepatic biopsy), with or without antibodies (ab) to hepatitis C virus (HCV). In addition, we retrospectively examined 183 hepatic biopsies of alcoholic patients with or without hepatitis B virus (HBV markers). At the end of the study the following conclusions were drawn: (i) Contact with HBV is unlikely to effect either the histologic picture or the course of alcoholic liver disease. (ii) The incidence of ab to HCV is higher in more advanced stages of the disease. (iii) As regards biopsies of alcoholic patients, sometimes it was impossible to establish a relationship between piece-meal necrosis or portitis and viral markers of hepatitis; therefore, other factors may be synergic with alcohol (ie nutritional, immunologic, genetic factors, concomitant diseases, etc.). For each of these factors, the relative risk of cirrhotic evolution could be assessed by a multicenter survey with case-control design.


[The role of colposcopy in the diagnosis of HPV infection]. Human papillomavirus (HPV) has been implicated as an important etiologic factor in cervical carcinoma. This study evaluates the efficacy of colposcopy in the detection of cervical lesions with koilocytosis features. Colposcopy, cervical smears and biopsy specimens from 217 women seen in our department between January-December 1991 were examined. The colposcopy examination detected 77.5% of the viral presence the histological diagnosis detected 85% of HPV, but the cytologic smears showed only 52% of infection. The data suggests that colposcopy is a good examination to evidence koilocytosis infection.


Renal replacement therapy for end-stage renal failure before 2 years of age. This report concerns 296 children (67% males and 33% females) from 24 countries who started renal replacement therapy (RRT) for end-stage renal failure between 1969 and 1988. Children under 2 years of age represented 3.6%, 4.4%, and 8.9% of all children under 15 years of age who started RRT in 1978-1982, 1983-1985, and 1986-1988 respectively. During the first 2 years of life, the most frequent causes of end-stage renal failure were renal hypoplasia and dysplasia (24%), and haemolytic-uraemic syndrome (17%). During 1986-1988 the initial therapy for ESRF was continuous ambulatory peritoneal dialysis (CAPD) in 60%, haemodialysis 25%, intermittent peritoneal dialysis 8%, and 7% were transplanted without prior dialysis. Between 1978 and 1988, 139 of these children were grafted; 53 received a graft (39 cadaveric, 10 living donor, 4 donor uncertain) below, and 86 (71 cadaveric, 14 living donor, 1 donor uncertain) above 2 years of age. One-year graft survival was 54% in the 53 children grafted below 2 years of age and 65% in the 86 grafted above 2 years of age. Only two of the 24 living donor grafts were lost during the first year after grafting. These results compare favourably with the 67% 1-year graft survival of all 278 children aged 2 to less than 6 years at grafting in 1978-1988 on the Registry's file. The 3-year survival of all children aged less than 2 years at start of RRT was 65% in 1978-1982 and rose to 78% in 1986-1988. Twenty-three percent of all deaths were caused by infections.(ABSTRACT TRUNCATED AT 250 WORDS)


Angiotensin-converting enzyme inhibitor renography in the diagnosis of renovascular hypertension. Studies before and after angioplasty. Renography with [99mTc] diethylenetriaminepenta-acetate (DTPA) was performed in 26 patients with renal artery stenosis (RAS), unilateral in 15 and bilateral in 11, and in 16 patients with essential hypertension with a normal renal angiogram. Nine of the patients with unilateral RAS were restudied after a successful percutaneous transluminal renal angioplasty (PTRA), i.e. complete removal of the stenosis and a normalization of the blood pressure without antihypertensive treatment. Single-kidney [99mTc]-DTPA clearance and parenchymal mean transit time (MTT) were determined at each examination. All patients were studied on two different days using the same procedure except that captopril 25 mg was given orally before renography at the second examination. In unilateral RAS captopril reduced single-kidney [99mTc]-DTPA clearance significantly on the affected side (-42.7%, median) but not on the unaffected side (-3.2%). In bilateral RAS single-kidney [99mTc]-DTPA clearance was reduced to the greatest extent on the most affected side (-43.0%) compared with the least affected side (-17.2%). In essential hypertension no significant changes were recorded on any side (-1.5% for both). After PTRA, single-kidney [99mTc]-DTPA clearance was not significantly changed by captopril either on the previously affected side (4.3%) or on the unaffected side. MTT was significantly prolonged after captopril on the affected side in unilateral RAS and on the most affected side in bilateral RAS, whereas no significant changes were found on the unaffected side in unilateral RAS, on the least affected side in bilateral RAS, or on any side in essential hypertension.(ABSTRACT TRUNCATED AT 250 WORDS)


Differential effect of L-nitroarginine methyl ester on urinary cGMP and kidney growth in the conscious rat following uninephrectomy. The role of cGMP in compensatory renal growth (CRG) is uncertain, since inconsistent changes in renal cGMP have been reported following uninephrectomy (UNx) in the rat. The aim of this study was to reassess the change in cGMP following UNx in the conscious rat by sequential measurements of cGMP excretion and to determine the contribution of nitric oxide, an activator of guanylate cyclase, using L-NAME an inhibitor of nitric oxide synthase. In the conscious rat a sustained increase in the urinary excretion of cGMP was produced in the 7-day period following UNx. L-NAME (20 mg/kg per day) prevented the increase in cGMP excretion following UNx, but not compensatory renal growth. Total kidney DNA, however, was reduced by L-NAME. These observations dissociate the increase in cGMP after UNx from the process of renal hypertrophy.


Renal venous erythropoietin concentrations in hypertensive patients with unilateral renal disease. In 24 hypertensive patients with unilateral renal disease, the erythropoietin (Epo) concentration ratio in the renal veins was compared with the renin ratio. Seven patients showed moderately elevated peripheral Epo values. Epo and plasma renin activity were significantly positively correlated both in peripheral and renal veins. This suggests that the reduction of renal blood flow was a common, but not unique, stimulating factor of Epo and renin secretion. Epo ratio appeared insensitive since it was greater than 1.5 in only 30% of patients with a renin ratio > 2. Our results indicate that the Epo concentration ratio in renal veins cannot be proposed as a substitute for the currently used renin ratio.


Are antiphospholipid antibodies clinically relevant in dialysis patients? A cohort of 60 stable dialysis patients (56 haemodialysis, 4 continuous ambulatory peritoneal dialysis) was followed for 1 year to determine the relationship between anticardiolipin (aCL) antibodies and lupus anticoagulant (LA), and clinical events. The outcome measures were death, arterial, venous, and fistula thromboses, and fistula repairs. At baseline 15% had antibodies (5/60 IgG aCL, 3/60 LA, and 1/60 had both); 15 patients had a history of arterial thrombosis (1 patient was aCL positive), seven venous thrombosis (1 patient LA positive), 10 fistula thromboses (1 aCL positive); and 21 had a history of fistula repairs (4 aCL positive). Renal diagnosis, age (66.78 versus 59.67 years) and duration of dialysis (38.11 versus 45.25 months) were similar in patients with and without aCL antibodies or LA. Only the sex ratio showed a female predominance in the aCL- or LA-positive patients compared to the negative patients (3M:6F versus 36M:15F), but this was not significant (P = 0.07). After 1 year there were 10 deaths (1 LA positive), 12 thrombotic events in eight patients (none aCL or LA positive), and nine fistula repairs in seven patients (1 aCL-positive patient). We are unable to show higher rates of death, thrombotic event, or fistula repair in dialysis patients with aCL antibodies or LA followed up for 1 year in one centre. The clinical importance of antiphospholipid antibodies in dialysis patients is uncertain.


Haemodynamic changes and physical performance at comparative levels of haemoglobin after long-term treatment with recombinant erythropoietin. Physical performance and haemodynamic parameters at rest and with exercise were compared in a prospective, cross-over fashion in 12 anaemic haemodialysis patients (Hb 6.4 +/- 0.5, mean +/- SEM) at two levels of haemoglobin (Hb 9 and 12 g/dl) before and after long-term treatment with recombinant human erythropoietin (rHuEpo). Patients were divided into two groups and measurements made prior to treatment, upon reaching, and after 4 months at the first target Hb (9 g/dl group A, 12 g/dl group B), and after 4 months at the alternative target Hb. Tests included an exercise radionuclide ventriculogram, Doppler echocardiogram, and respiratory function exercise test. Compared to pretreatment, there was a significant reduction in resting pulse rate (P < 0.001), and in pulse rate (P < 0.001) and arterial lactate (P < 0.01) concentrations at specified levels of exercise. Work capacity improved 60% (P < 0.001), and left ventricular mass fell by 26% (P < 0.001). Although cardiac output (CO) during and after exercise was reduced (P < 0.05), resting CO, cardiac index, stroke volume and ejection fraction (rest and exercise) were not significantly altered. There appeared little benefit in having the higher target Hb: no significant difference could be found between target levels for almost any measure. In addition, despite marked improvement from pretreatment levels, performance parameters were still below those of non-uraemic age-matched controls. These results demonstrate the beneficial but incomplete effect of rHuEpo on resting and exercise-related factors, and suggest that most improvement is achieved with modest increments in haemoglobin.


Effect of 1,25 (OH)2D3 treatment on glucose intolerance in uraemia. The aim of this study was to evaluate the effect of calcitriol treatment on glucose intolerance in uraemia. Thirty one patients on haemodialysis who had never been treated with vitamin D or related drugs, and 12 healthy control subjects with normal renal functions were studied. Uraemic patients were randomly divided into two groups; 16 patients were treated with oral calcitriol (0.5 micrograms/day) for 8 weeks, and 15 uraemic patients and 12 healthy subjects were given a placebo. In all these cases, before and 8 weeks after treatment, baseline serum glucose, insulin, calcium, parathormone (PTH), and 1,25 (OH)2D3 were measured. After an oral load of 75 g glucose, blood glucose and insulin were determined at 30, 60, 90, and 120 min. The same measurements were repeated after 8 weeks. HbA1c and fructosamine were also measured at 0 and 8 weeks. Baseline serum insulin was significantly elevated after calcitriol treatment (7.81 versus 11.63 microIU/ml) there was also a significant increase in insulin following calcitriol treatment at 30, 60, 90, and 120 min. On the other hand, glycosylated haemoglobin (HbA1c) and fructosamine decreased after calcitriol treatment (HbA1c 7.09% versus 5.22% P < 0.01 and fructosamine 2.92 versus 2.50 mmol/l P < 0.01). Blood glucose significantly decreased after calcitriol treatment at 0, 30, 60, 90, and 120 min. In the other two groups there were no significant changes in any parameters. These results seem to confirm that vitamin D influences pancreatic beta (beta) cell secretion and suggest that calcitriol may improve glucose intolerance in uraemic haemodialysis patients.(ABSTRACT TRUNCATED AT 250 WORDS)


Encephalopathy in patients on continuous ambulatory peritoneal dialysis and patients on chronic haemodialysis. A group of 121 patients, 22 with a preterminal chronic renal insufficiency (PCRI), 74 on chronic haemodialysis (CHD), and 25 on continuous ambulatory peritoneal dialysis (CAPD), was evaluated by means of neurophysiological and neuropsychological studies to detect signs of central nervous system dysfunction. CHD patients were studied the day before dialysis treatment. In each patient the neurophysiological and neuropsychological studies were performed on the same day. The same overall result emerged from the neurophysiological and neuropsychological studies: all three patient groups showed significant deviations from the values obtained from a healthy reference group, whereas no differences were found between the three patient groups. Biochemical variables (a.o. PTH, Al, PO4) showed inconsistent or only minor correlations with the encephalopathic parameters. Apparently traditional biochemical variables are not a reliable measure to safeguard renal patients from neurotoxic damage. With respect to central nervous system dysfunction CAPD appears to be as 'safe' as CHD.


Low-calcium dialysis fluid and oral calcium carbonate in CAPD. A method of controlling hyperphosphataemia whilst minimizing aluminium exposure and hypercalcaemia. Oral calcium carbonate is an effective phosphate binder in dialysis patients. Its use minimizes aluminium intake, and by maintaining a high-normal serum ionized calcium, suppresses serum parathyroid hormone levels. However, the dose required to control hyperphosphataemia may cause hypercalcaemia. We performed prospective studies in 50 previously undialysed patients starting CAPD (28 study group, 22 control group). Calcium carbonate was the only phosphate binder used in the study group which utilized a low calcium PD fluid (calcium 1.25 mmol/l), whilst the control group used standard PD solution (calcium 1.75 mmol/l) with calcium carbonate plus aluminium hydroxide phosphate binders as clinically indicated. The study group was able to take larger doses of oral calcium carbonate with no increase in episodes of hypercalcaemia compared to the control group. There were no instances of hypocalcaemia in any patient using the low-calcium dialysis fluid. Phosphate control was better in the study group, despite the additional use of aluminium-containing phosphate binders by some patients in the control group. Serum aluminium levels in the study group were maintained at < 11.5 mumol/l, but increased significantly in the control group from 3 months onward. Mean serum parathyroid hormone in the study group declined significantly from baseline values over the first 6 months, and remained at the lower level. Bone histology showed a tendency towards improvement over the 12 months, in terms of osteoclast numbers and activity. We conclude that using dialysis fluid with a reduced calcium concentration in compliant, well-monitored patients is safe.(ABSTRACT TRUNCATED AT 250 WORDS)


Double-blind, placebo-controlled trial of human lymphoblastoid interferon prophylaxis of cytomegalovirus infection in renal transplant recipients. We have conducted a double-blind, placebo-controlled trial of human lymphoblastoid interferon prophylaxis of cytomegalovirus (CMV) infection in 74 renal transplant recipients. Interferon (3 x 10(6) units was given thrice weekly for the first 6 weeks, then twice weekly for a further 8 weeks. During the period of interferon therapy, the incidence of CMV excretion was lower in the interferon group (28% versus 50%, P = 0.065), mainly due to a significant reduction of CMV reactivation (9% versus 56%, P = 0.02). However, for the whole study period (including the follow-up period after interferon therapy), there was no difference in the incidence of CMV excretion (44% versus 53%). The onset of CMV excretion was delayed (8.2 +/- 0.8 to 20.9 +/- 5.5 weeks, P = 0.04). The duration of CMV excretion was also reduced (11.1 +/- 3.1 to 29.4 +/- 5.7 weeks, P = 0.008). The number of positive CMV isolates from urine and saliva was significantly less in the interferon group. There was no difference in the site of CMV excretion. Of the patients in the treatment group who excreted CMV, 43% developed disease as compared to 63% in the control group (difference not significant). There was also no significant difference in the severity of the CMV infection between the two groups. Benefit appears to be restricted to seropositive recipients of seronegative kidneys. The interferon regime used in this study was well tolerated, with mild fever being the only reported side-effect. No patient had to stop therapy because of toxicity. The incidence of rejection and graft loss was not different between the two groups.


1 alpha-Hydroxycholecalciferol adsorption to peritoneal dialysis bags: influence of time, glucose concentration, temperature, and albumin. The present study examines the adsorptive phenomenon of 1 alpha-hydroxycholecalciferol (1 alpha-OHD3) to peritoneal dialysis bags. One minute after injection of 1 alpha-OHD3 into the dialysis bags, a significant adsorption to the bags was observed in experiments where albumin was not added. A higher temperature (37 degrees versus 23 degrees C) in bags containing 13.6 mg glucose/ml resulted in less adsorption (P < 0.05). Adding albumin to bags at 37 degrees C with a glucose concentration of 13.6 mg/ml, reduced the adsorptive phenomenon significantly (P < 0.01). Bags tested at 37 degrees C with a glucose concentration of 38.6 mg/ml resulted in a significantly higher 1 alpha-OHD3 adsorption than bags containing 13.6 mg glucose/ml (P < 0.01). It is concluded that adsorption of 1 alpha-OHD3 to dialysis bags is affected by time, albumin, temperature and glucose concentrations. Substantial amounts of 1 alpha-OHD3 is retained by the peritoneal dialysis system when albumin is not added.


Serum lipoprotein (a) in men with proteinuria due to idiopathic membranous nephropathy. Patients with heavy proteinuria have an increased incidence of venous thrombosis and coronary heart disease (CHD). They also exhibit perturbations in lipoprotein metabolism. Lipoprotein (a) (Lp(a)) predisposes to CHD; it may also promote intravascular thrombosis since it contains apolipoprotein (a), which could act as a competitive inhibitor of plasminogen activation. We have measured the concentration of serum Lp(a) in 10 men with proteinuria due to idiopathic membranous nephropathy (IMN), in eight men with a similar diagnosis but who were in remission, and in 103 healthy men. Serum Lp(a) levels were significantly elevated in the men with active IMN, having a median value of 31.3 (range 3.2-75.0) mg/dl, whereas they were 8.4 (3.4-31.5) mg/dl in the patients in remission, which was similar to the value of 11.3 (< 0.8-87.4) mg/dl found in the healthy controls. Lp(a) is unique in containing an apolipoprotein which is a giant mutant of plasminogen and it is thus possible that the high circulating levels of Lp(a) contribute to the vascular morbidity associated with proteinuria by promoting thrombosis or atherogenesis or both.


Secondary membranous glomerulonephritis. Membranous glomerulonephritis of defined aetiology (secondary MG) is a common finding, particularly in children and older adults. Secondary MG can be readily detected by a combination of clinical, serological and morphological analysis in a majority of instances. Recognition of secondary MG has significant prognostic and therapeutic implications.


Membranous nephropathy and its treatment. Membranous nephropathy is a histological appearance which indicates a particular immunopathogenesis, but may have many basic aetiologies and is probably not a single disease. It is predominantly an appearance seen in middle-aged and elderly individuals, and in general has a slow progression over years or even decades towards remission or renal impairment in almost equal proportions. An aggressive search for associated disease is worthwhile, and one should wait to see what the evolution of proteinuria and renal function may be. If a progressive course becomes evident, then a trial of treatment with corticosteroids is worthwhile, but if this is ineffective, a more aggressive approach involving the use of alkylating agents may be justified. Cyclosporin does not appear to have a major effect in the majority of patients, and intravenous gammaglobulin is under evaluation at the moment with some encouraging results. The best treatment regime remains to be determined.


Remissions and relapses in idiopathic membranous nephropathy. To assess the significance of remissions and relapses in idiopathic nephrotic syndrome the outcome of 169 patients was reviewed of them III had either a complete or partial remission. The probability of obtaining a complete remission within 5 years was 44% for the whole group, being significantly higher (P = 0.015) for those patients who had been treated with steroids plus cytotoxic drugs. Of the 74 patients who had complete remission only two showed renal function deterioration over time. Relapse of non-nephrotic proteinuria occurred in 22 patients. Of these, 12 again entered complete remission spontaneously, which persisted in nine until the last observation. Relapse of nephrotic syndrome occurred in 42 of III patients. Six patients showed a moderate renal function deterioration. Of 27 untreated patients, 17 had again a partial or complete remission that persisted in 13 until the last observation. All the 15 patients submitted to treatment again entered remission, which persisted in nine of 11 patients given prednisolone and chlorambucil and only in one of four given steroids alone. In conclusion, (a) the development of complete remission is a good marker of an excellent renal outcome, (b) patients with relapse of non-nephrotic proteinuria usually carry a good prognosis, (c) about half of patients with relapse of nephrotic syndrome show a spontaneous remission, and (d) the chances of a stable remission may be increased by treatment with steroids and cytotoxic agents.


Antiproteinuric drugs in patients with idiopathic membranous glomerulopathy. We studied the effects of symptomatic, antiproteinuric treatment with NSAID's (n = 28) and ACE-inhibitors (n = 14) in patients with proteinuria due to idiopathic membranous glomerulopathy (MGP). These two treatment groups were compared with a group of patients who did not receive antiproteinuric medication (n = 14). Urinary protein loss was effectively lowered by NSAID and ACE inhibitor therapy from 9.5 +/- 1.0 to 4.5 +/- 0.5 g/day (mean +/- SEM) and from 9.8 +/- 1.4 to 3.9 +/- 0.7 g/day respectively, whereas the control group showed a slight fall in proteinuria from 6.9 +/- 0.8 to 5.5 +/- 0.8 g/day. As a result of this treatment hypoalbuminaemia and hypercholesterolaemia improved significantly: serum albumin rose in the NSAID group from 25.4 +/- 1.2 to 29.0 +/- 1.0, and in the ACEi group from 29.9 +/- 1.8 to 32.7 +/- 1.2 g/l (control group from 27.4 +/- 1.6 to 27.8 +/- 1.6 g/l, while cholesterol was lowered in the NSAID group from 8.5 +/- 0.5 to 7.5 +/- 0.4 and in the ACEi group from 8.7 +/- 0.5 to 7.6 +/- 0.4 mmol/l (control group from 9.7 +/- 1.1 to 8.5 +/- 1.0 mmol/l). The antiproteinuric effect of both drugs was well maintained during an 18-month follow-up. Progression towards end-stage renal failure was observed especially in patients with impaired renal function at entry. Remission of proteinuria occurred particularly in patients with lower baseline values of proteinuria, irrespective of the treatment modality.(ABSTRACT TRUNCATED AT 250 WORDS)