Antiflammins suppress the A23187- and arachidonic acid-dependent chloride secretion in rabbit distal colonic mucosa. Antiflammins are synthetic peptides with sequence homology to proteins inhibitory for phospholipase A2. The effects of antiflammins on chloride secretion induced by the calcium ionophore A23187, arachidonic acid (AA) and prostaglandin E2 (PGE2) were investigated in distal rabbit colonic mucosa mounted in Ussing-type chambers. 100 nM AF-2 (antiflammin 2, peptide HDMNKVLDL) fully prevented the increase in Isc, net chloride secretion, tissue PGE2 and second messenger cAMP induced by 5 microM A23187. AF-1 (antiflammin 1, peptide MQMKKVLDS) also inhibited, although to a lesser extent, the A23187-mediated increase in Isc. AF-2 inhibition began at doses of 100 nM (delta Isc -0.20 +/- 0.08, microEq h-1 cm-2, mean +/- S.E.M., N = 3) and was maximal at doses comprised between 200 and 250 nM (delta Isc -0.51 +/- 0.12, microEq h-1 cm-2, mean +/- S.E.M., N = 3). AF-2 also inhibited the increase in Isc and chloride secretion induced by the incubation with 10 microM AA and bradykinin but it was ineffective when tissues were stimulated with 10 microM PGE2. 100 nM AF-2 brought about an inhibition of the increase in AA-mediated increases in PGs and cAMP comparable to that of 10 microM of the cyclooxygenase inhibitor indomethacin. In vitro assay of colonic cyclooxygenase activity showed that 100 nM AF-2 and AF-1 inhibited cyclooxygenase activity (73 and approximately 30%, respectively), but they did not modify the in vitro activity of porcine phospholipase A2.(ABSTRACT TRUNCATED AT 250 WORDS)


Characterization of the rate-dependent effects of ethmozine on conduction, in vivo, and on the sodium current, in vitro, in the newborn heart. We evaluated the effects of ethmozine on resting conduction intervals, myocardial refractory periods and His-Purkinje and intraventricular conduction in vivo in 11 neonatal dogs aged 7 to 16 days. Ethmozine produced significant prolongation in resting sinus cycle length (P < .05), in the atrioventricular nodal (P < .001) and His-Purkinje conduction time (P < .01) intervals and in the QRS duration (P < .01). Ventricular, but not atrial refractory periods were significantly prolonged (P < .05). Rate-dependent changes in His-Purkinje and intraventricular conduction were demonstrated after ethmozine by direct pacing of the bundle of His and right ventricular pacing. The development of steady-state conduction delay at a paced cycle length of 200 msec was characterized by a time constant (tau on) of 23.5 beats. The time constant of diastolic recovery (tau off) from rate-dependent conduction delay, determined during His bundle extrastimulation, was 171 msec. Ethmozine was highly proarrhythmic. A total of 7 arrhythmias were induced in 6 out of 12 neonates after administration of ethmozine. We also characterized ethmozine block of cardiac sodium channels in isolated neonatal canine ventricular myocytes using the whole cell variation of the patch clamp technique. Ethmozine (1.3-40 microM) produced a use-dependent block of cardiac Na channels that was dependent upon both drug concentration and pulse duration. Drug binding to inactivated channel states accounted for the observed use-dependent block. The time constant of recovery from use-dependent block ranged between 10 and 30 sec at 16 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)


Blocking effects of V1 (OPC-21268) and V2 (OPC-31260) antagonists on the negative inotropic response to vasopressin in isolated dog heart preparations. We investigated the effects of V1 IOPC-21268, 1-(1-[4-(3-acetylaminopropoxy)benzoyl]-4-piperidyl)-3,4-dihydro-2( 1H)- quinolinone) and V2 (OPC-31260, 5-dimethylamino-1[4-(2-methylbensoylamino)benzoyl]-2,3,4,5-tetrahy dro-1H- benzazepine) vasopressin receptor antagonists on the negative inotropic response to arginine vasopressin (AVP) in the isolated perfused heart preparations of the dog. AVP (7.5-750 pmol) decreased the atrial and ventricular contractile force when the preparation was perfused with constant pressure or constant flow. AVP induced a small increase in sinus rate. Desmopressin, a selective vasopressin V2 agonist, did not change the sinus rate and atrial or ventricular contractile force. OPC-21268 (0.01-3 mumol) and OPC-31260 (0.01-1 mumol) induced a small negative inotropic effect. Both OPC-21268 and OPC-31260 inhibited the negative inotropic response to AVP in a dose-dependent manner. The doses of 50% inhibition (ID50) for OPC-21268 and OPC-31260 on the inotropic effect were 0.30 +/- 0.16 mumol and 0.084 +/- 0.034 mumol, respectively. Neither OPC-21268 nor OPC-31260 affected the acetylcholine-, adenosine- or norepinephrine-induced inotropic and chronotropic effects. It has been reported that the concentration of OPC-21268 that displaced 50% of specific AVP binding is 0.4 microM for V1 receptors and > 100 microM for V2 receptors and the concentration of OPC-31260 is 0.01 microM for V2 receptors and 1 microM for V1 receptors. We, therefore, suggest that AVP directly causes negative inotropic effects mediated at least in part by V1 receptors in the dog heart.


Effects of acute administration of alcohol on in vivo binding of [3H]Ro 15-1788 to mouse brain. Mice received i.p. injections of either saline or drug before i.p. administration of [3H]Ro 15-1788 (flumazenil), a selective benzodiazepine (BZD) receptor antagonist. Subjects were decapitated and six brain regions (cortex, cerebellum, striatum, hippocampus, hypothalamus and brain stem) were analyzed for the levels of tritium present in each tissue. Benzyl, ethyl and methyl alcohol enhanced in vivo binding of [3H]Ro 15-1788 in a dose-dependent manner in all brain regions studied with mean ED50 values of 2.4, 34.8 and 38.4% volume/kg body weight, respectively. Lorazepam (18 mg/kg) completely blocked effects of benzyl, ethyl and methyl alcohol to increase [3H]Ro 15-1788 binding. These data indicate that the alcohol-induced increases in [3H]Ro 15-1788 binding occurred at the level of the BZD receptor. In addition, these alcohols did not alter the in vivo binding of [3H]diprenorphine, an opioid receptor ligand, further indicating the selective effect of alcohol on BZD receptors. Administration of lorazepam or alprazolam produced a dose-dependent decrease in the binding of [3H]Ro 15-1788 to brain tissue after administration of either saline or 30% volume/kg ethanol. However, low doses of alprazolam increased binding of [3H]Ro 15-1788 in the absence of ethanol. Together, these results suggest that ethanol produces an increase in the number of BZD binding sites in vivo. These alcohol-induced alterations in BZD receptor binding may mediate, in part, the anxiolytic or sedative properties of ethanol.


Binding and functional selectivity of himbacine for cloned and neuronal muscarinic receptors. The binding potencies for the putative M2-selective antagonist himbacine were determined in radioligand binding and in functional response assays in neuronal tissue and Chinese hamster ovary cells containing transfected muscarinic receptors. Himbacine was shown to bind to all five cloned muscarinic receptor subtypes in the order of potencies: hM2 = hM4 > hM3 > hM1 > hM5 (Kd values were 4, 7, 59, 83 and 296 nM, respectively). Himbacine was shown to bind to M2 receptors in rat heart and brain stem with Kd values of 6.9 and 4.6 nM, respectively. In rat brain tissues with complex mixtures of muscarinic receptors, and using the radioligand [3H] +/- -5,11-dihydro-11-([(2-(2-[(dipropylamino)methyl]-1- peperidinyl)ethyl)amino]carbonyl)-6H-pyrido(2,3-b)(1,4)benzodiazep ine-6-one to demarcate M2 and M4 receptors, himbacine was shown to bind to 80% of cortical or striatal receptors with Kd values of 4.5 and 3.8 nM, respectively, consistent with the involvement of M2 and/or M4 receptors in both these brain regions. Himbacine was a potent blocker of oxotremorine-M-mediated cyclic AMP inhibition in rat striatum (4.4 nM) and in N1E-115 neuroblastoma cells (10.6 nM), responses mediated by M4 receptors. Himbacine also reversed oxotremorine-M-mediated inhibition of evoked acetylcholine release from hippocampal tissue with a Kd value of 8.6 nM, a value consistent with the involvement of M2 or M4 receptors. At the cortical postsynaptic muscarinic receptors involved with phosphoinositide turnover (putative M1 and M3 receptors), himbacine was 21-fold less potent. Himbacine appears to be a potent muscarinic antagonist that displays selectivity for M2 or M4 receptors, as compared to M1 or M3 receptors.(ABSTRACT TRUNCATED AT 250 WORDS)


Intermediate efficacy mu opioids: examination of their morphine-like stimulus effects and response rate-decreasing effects in morphine-tolerant rats. The present study examined the effects of morphine, the intermediate efficacy mu opioids (-)-pentazocine, (-)-metazocine, proxorphan, levallorphan, (-)-NANMY (N-allylnormetazocine) and (-)-cyclazocine, and the mu antagonist naloxone 1) in rats responding under a FR (fixed-ratio) 30 schedule before, during and after a chronic morphine regimen, and 2) in rats trained to discriminate 10.0 [10-MS (morphine sulfate)] or 3.0 mg/kg (3-MS) of morphine from saline. Under the FR30 schedule, chronic administration of morphine produced tolerance to morphine's rate-decreasing effects and conferred cross-tolerance to (-)-metazocine, proxorphan and (-)-pentazocine. However, the effects of these intermediate efficacy mu opioids could be differentiated from those of morphine on the basis of their 1) shallow dose-effect curves, and 2) large differences in the degree to which tolerance developed in individual rats. In the drug discrimination procedure, (-)-metazocine, proxorphan and (-)-pentazocine produced high levels of substitution for the 3-MS stimulus and intermediate levels for the 10-MS stimulus. In contrast to the pattern of substitution observed with morphine in the 10-MS group, the effects of these drugs were characterized by 1) shallow dose-effect curves, 2) large individual differences in the lowest dose of each drug that substituted completely for the 10-MS stimulus and 3) the failure to obtain complete substitution for the 10-MS stimulus in all of the rats tested. The behavioral profile obtained with the intermediate efficacy mu opioids (-)-NANM, levallorphan and (-)-cyclazocine was indicative of opioids with intrinsic efficacy lower than that of (-)-pentazocine, (-)-metazocine and proxorphan. Under the FR30 schedule, chronic administration of morphine produced an enhanced sensitivity to the rate-decreasing effects of (-)-NANM and levallorphan but not (-)-cyclazocine. In the drug discrimination procedure, (-)-NANM, levallorphan and (-)-cyclazocine produced high levels of substitution for the 3-MS stimulus and low levels for the 10-MS stimulus. Like naloxone, these drugs produced a dose-related attenuation of the 10-MS stimulus. The results of the present study suggest that the relative order of intrinsic efficacy among the opioids tested is: morphine > (-)-metazocine = (-)-pentazocine = proxorphan > (-)-cyclazocine = levallorphan = (-)-NANM > naloxone.


Potentiation of cyclic adenosine monophosphate production by thrombin in the human erythroleukemia cell line, HEL. The human erythroleukemia cell line (HEL) has been used as a model system for studying signal transduction processes as they might relate to platelet/megakaryocyte function. We were interested in examining the role of thrombin in the regulation of adenylyl cyclase in this cell line. As opposed to its predominantly inhibitory effects on cyclic AMP production in platelets or in membranes from HEL cells, our initial experiments in intact HEL cells revealed that thrombin markedly potentiated the cyclic AMP response to prostaglandin E1 (2.9 +/- 0.2-fold), prostacyclin (1.9 +/- 0.2-fold) and carbacyclin (2.5 +/- 0.5-fold), measured either by radioimmunoassay or by the [3H]adenine preloading procedure. Thrombin, although ineffective alone, also potentiated cyclic AMP production stimulated by vasoactive intestinal peptide (1.6 +/- 0.2-fold), cholera toxin (3.0 +/- 0.6-fold) and AIF4- (2.3 +/- 0.6-fold), but not by forskolin (0.9 +/- 0.1-fold). The thrombin effect 1) produced an increase in the efficacy of the prostaglandins with no change in potency; 2) was long-lived; 3) required the proteolytic activity of thrombin; 4) was insensitive to pertussis toxin; and 5) was at least partially mimicked by trypsin, extracellular ATP and UTP, platelet activating factor and activators of protein kinase C. Down-regulation of protein kinase C or pre-exposure to the protein kinase inhibitor staurosporine blocked the potentiating effect. Together, these results suggest that in HEL cells, the mechanism of thrombin potentiation of cyclic AMP production may involve alterations in the interaction between stimulatory guanine nucleotide binding protein and the catalytic subunit of adenylyl cyclase, possibly involving protein kinase C-mediated phosphorylation.


Characterization of the inhibitory effect of adrenocorticotropin/melanocyte-stimulating hormone-like peptides on the binding of dopamine receptor ligands to the dopamine D2 receptor in vitro. Adrenocorticotropin (ACTH)-(1-24) decreased the binding of the dopamine D2 agonist [3H]N-n-propylnorapomorphine [3H](NPA) to the dopamine D2 receptor in rat striatal membranes in vitro. The association and dissociation of [3H]NPA to the dopamine D2 receptor was inhibited by ACTH-(1-24), suggesting an apparent competitive interaction between ACTH-(1-24) and the binding of [3H]NPA. ACTH-(1-24) was able to inhibit the binding of the dopamine D2 receptor antagonist [3H]spiperone to the dopamine D2 receptor, both in the high- and the low-affinity state. These observations suggest a G-protein-independent mechanism of action. The inhibitory effect of ACTH-(1-24) and ACTH-(7-16)-NH2 was diminished after the addition of polylysine chains, presumably via a blockade of the attachment sites for ACTH-(1-24) on the dopamine D2 receptor. The effect of ACTH-(1-24) on membrane fluidity and on the inhibition of the binding of [3H]NPA to the dopamine D2 receptor appeared to be unrelated because lowering the incubation temperature from 25 degrees C to 4 degrees C, which causes a strong decrease of membrane fluidity, did not diminish the effect of ACTH-(1-24) on the binding of [3H]NPA to the dopamine D2 receptor. Furthermore, in both young and old rats, whose membranes are reported to differ in lipid composition and membrane fluidity, ACTH-(1-24) inhibited the binding of [3H]NPA to the dopamine D2 receptor to nearly the same extent.(ABSTRACT TRUNCATED AT 250 WORDS)


Antinociceptive mechanisms of [D-Arg2]-dermorphin tripeptide analogs. These studies examined the antinociceptive effects and mechanisms of opioid activity of synthetic dermorphin tripeptide analogs containing D-Arg as the second amino acid, H-Tyr-D-Arg-Phe-NHCH3 and H-Tyr-D-Arg-Phe-N(CH3)2. Both tripeptides, administered i.c.v., i.t. and s.c. in mice, produced a potent and long-lasting antinociceptive activity as compared with morphine. In the guinea pig isolated ileum (GPI) assay, the IC50 value of both peptides was lower than that of morphine on the electrically induced contractions of the GPI. In radioligand binding studies with rat brain membrane synaptosomes, both tripeptides bound with high affinity to [3H]DAMGO-labeled mu-type sites, whereas they bound with no or negligible affinity to [3H]DPDPE-labeled delta sites and [3H]U-69593-labeled kappa sites. In the enzymatic degradation using high-performance liquid chromatography, both tripeptides showed good enzymatic stability after 25 hr of incubation with solubilized enzymes of mouse brain or spinal cord, in contrast to a rapid degradation of Met-enkephalin. The in vitro and in vivo pharmacological profile of [D-Arg2]-dermorphin tripeptide analogs demonstrates that they are potent and selective agonists at the mu opioid receptor. A high resistance of the tripeptides to enkephalin-degrading enzymes may largely contribute to their prolonged opioid activity.


A second cysteinyl leukotriene receptor in human lung. Leukotrienes (LT) are potent spasmogenic agents in human isolated bronchial and pulmonary venous muscle preparations. Treatment of human isolated pulmonary veins with the L-serine borate complex (45 mM; 30 min) did not alter the LTC4 pD2 values in these preparations. The cysteinyl LT antagonists, ICI 198615, MK 571 and SKF 104353, significantly shifted to the right the LT concentration-effect curves in airways with pKB values against LTC4 of 8.4 for ICI 198615, 8.6 for MK 571 and 8.0 for SKF 104353. Similar results were found against LTD4. In contrast, these antagonists did not inhibit the LTC4 and LTD4 contractions in human pulmonary veins. LTE4 was a partial agonist on the human pulmonary veins and blocked the contractions with a pKp value of 6.3 against LTD4 and 6.6 against LTC4. An LT analog, BAY u9773, also blocked the LT contractions in bronchial and venous muscle preparations with pKp values against LTD4 and LTC4 of 6.5 and 6.7, respectively. These data provide pharmacological evidence for a second cysteinyl LT receptor in the human lung. One LT receptor (LT-1) is stimulated by all cysteinyl LT, found on airways and inhibited by the LT-1 antagonists, and a second receptor (LT-2) can also be stimulated by all cysteinyl LT and is found on pulmonary veins, resistant to LT-1 antagonists but blocked by LTE4 and the dual LT-1/LT-2 antagonist BAY u9773.


Dexamethasone prevents epileptiform activity induced by morphine in in vivo and in vitro experiments. The inhibitory effects exerted by dexamethasone on the epileptiform activity induced by morphine were investigated in two different experimental models with two different animal species. In the first series of experiments, dexamethasone administered i.v. in rabbits 30 min before i.c.v. administration of morphine completely prevented both epileptiform and background EEG as well as behavioral alterations induced by morphine. Cycloheximide (a protein synthesis inhibitor) pretreatment reversed the antagonistic effect induced by dexamethasone on the behavioral and EEG alterations induced by morphine. In the second series of experiments, the effects exerted by dexamethasone were investigated on morphine-induced CA1 epileptiform bursting on rat hippocampal slices in vitro. Dexamethasone pretreatment 10 to 60 min before morphine strongly prevented the morphine effects in a concentration- and time-dependent manner. Sixty min of dexamethasone pretreatment also prevented the epileptiform bursting induced by the selective mu opiate receptor agonist DAMGO, whereas it did not significantly affect the increase of the CA1 population spike amplitude due to the selective delta opiate receptor agonist DPDPE. The addition of cycloheximide to the slice-perfusing medium containing dexamethasone prevented the inhibitory effects of the drug toward the morphine and DAMGO-induced CA1 epileptiform bursting. Our results indicate that dexamethasone induces an inhibition on the epileptiform activity induced by morphine and DAMGO. The time lag (30-60 min) which is necessary for revealing the inhibitory influence of dexamethasone on opiate epileptiform activity induced both in vivo and in vitro, and the inhibitory effect exerted by cycloheximide on dexamethasone activity strongly support the hypothesis of a genomic corticosteroid effect within the central nervous system.


Differential actions of corticotropin releasing factor on basolateral and central amygdaloid neurones, in vitro. Intracellular current- and voltage-clamp recordings were obtained from basolateral (BLA) and central (ACe) neurones of the rat amygdala in vitro. The effect of superfusion of rat corticotropin releasing factor (rCRF) was examined on the ACe and BLA, nuclei with a high density of CRF-immunoreactive cell bodies and CRF receptors, respectively. rCRF (12.5-250 nM) had no effect on either resting membrane potential or input resistance in BLA neurones. In contrast, at low nanomolar concentrations rCRF (10-50 nM) caused hyperpolarisation and decreased membrane input resistance in the majority of ACe neurones. The equilibrium potential for this effect was -84 mV in the presence of tetrodotoxin (0.5 microM), suggesting that this response may be mediated by a postsynaptic increase in K+ conductance. Furthermore, rCRF consistently reduced the amplitude of, and the current mediating, the slow after hyperpolarisation following evoked action potential firing in both nuclei. This effect was concentration-dependent with an EC50 of 40 nM in BLA neurones and was insensitive to tetrodotoxin and noradrenergic antagonists. In addition, the putative CRF antagonist alpha-helical CRF9-41 (5 microM) blocked the effect of rCRF and showed partial agonist action on slow after hyperpolarisations. In BLA neurones, rCRF (50 nM) prolonged the Ca(2+)-spike evoked in the presence of tetrodotoxin and tetraethylammonium (2 mM), suggesting that the blockade of the slow after hyperpolarisation occurs subsequent to Ca2+ entry. These results suggest that rCRF has a differential effect on the membrane properties of BLA and ACe neurones possibly by activation of a heterogeneous population of CRF receptors.


Characterization of antisera to the naloxone-insensitive receptor for beta-endorphin on U937 cells generated by using the complementary peptide strategy. Antisera to the naloxone-insensitive receptor for beta-endorphin expressed on the U937 cell line were generated by using the complementary peptide strategy. A nanopeptide complementary to a C-terminal fragment of human beta-endorphin was synthesized as predicted by reading the beta-endorphin antisense mRNA 3' to 5'. By using enzyme-linked immunosorbent assay, rabbit antisera specific for the peptide complementary to beta-endorphin (C'-peptide) were characterized. With the exception of C'-peptide, preabsorption of the antisera with human-beta-endorphin1-31 or 10 unrelated peptides of 5 to 21 amino acids failed to reduce the enzyme-linked immunosorbent assay titer. Sucrose gradient separation was also used to show that the antisera failed to recognize beta-[125I]endorphin. Immunoglobulin to C'-peptide (10-800 micrograms/tube) inhibited the binding of beta-[125I]endorphin (1-2 nM) to intact U937 cells in a dose-dependent manner, whereas control immunoglobulin was ineffective. Moreover, immunoglobulin to C'-peptide failed to reduce [3H]naloxone binding to rat brain membrane. After binding and cross-linking of beta-[125I]endorphin to U937 cell membrane in the presence of beta-endorphin (2.5 x 10(-5) M), control immunoglobulin or anti-C'-peptide immunoglobulin, sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that anti-C'-peptide immunoglobulin inhibited binding to 44 and 59 kDa bands. Nonspecific antibody was completely ineffective, whereas beta-endorphin completely inhibited binding to the 44 kDa and partially to the 59 kDa band. Western blot analysis of U937 cell membrane showed bands at 64, 58 and 56 kDa. In summary, antibodies selective for C'-peptide displaced beta-endorphin from the naloxone-insensitive receptor on U937 cells.(ABSTRACT TRUNCATED AT 250 WORDS)


Comparative in vivo and in vitro regional selectivity of central omega (benzodiazepine) site ligands in inhibiting [3H]flumazenil binding in the rat central nervous system. The in vivo selectivity for central omega (benzodiazepine) modulatory site subtypes of ligands from several chemical classes has been evaluated by measuring the displacement of the in vivo binding of [3H]flumazenil to several rat central nervous system structures differentially enriched in omega 1 and omega 2 sites. This labeling was prevented in a dose-related manner by the i.p. administration, 30 min before the radioligand, of several benzodiazepine derivatives, the cyclopyrrolone derivatives suriclone and zopiclone, the triazolopyridazine derivative CL 218,872 and the imidazopyridine derivative zolpidem. Most of the benzodiazepine derivatives studied displayed in vivo some selectivity for omega 2-enriched structures. In contrast, oxoquazepam and CL 218,872 were 2- to 3-fold more potent at preventing [3H]flumazenil binding in omega 1-enriched (cerebellum) than in omega 2-enriched structures. Maximal inhibitions by zolpidem of in vivo [3H]flumazenil binding [cerebellum (100%) > cerebral cortex (79%) > or = striatum (74%) > hippocampus (52%) > spinal cord (37%)] were related to the relative omega 1/omega 2 distribution ratio in each structure. These differences did not result from an uneven distribution of this compound in the central nervous system. Quantitative autoradiographic studies performed on 30 central nervous system regions showed a strong correlation between the in vitro and in vivo regional selectivity of zolpidem. For all the drugs studied there was a significant global correlation between their potency at inhibiting [3H]flumazenil binding in vitro and in vivo either in the cerebellum (P < .001) or in the spinal cord (P < .01) and between the in vitro and in vivo cerebellum/spinal cord selectivity. The differential in vivo selectivity of zolpidem may account for the reported hypnoselective profile of this imidazopyridine in the rodent.


The location of the receptors involved in the human diuretic response to drinking an isotonic electrolyte solution. 1. This study aimed to shed light on the receptors involved in the diuretic response to drinking isotonic fluids in man by employing a polyethylene glycol-based bowel lavage solution (Golytely) which is reported to cause no net movement of fluid across the gut. 2. Drinking Golytely resulted in a transient hypotonic diuresis. Mean urine flow rose from control values of 0.9 ml min-1 to 10.1 ml min-1 70 min after the start of drinking. The increase in urine output was accompanied by a fall in urine osmolality from control values of 879 mosM kg-1 to 105 mosM kg-1. The diuresis is similar to that produced by ingestion of an equal volume of an absorbable electrolyte-based solution (Tyrode). 3. Neither solution produced changes in plasma osmolality or electrolytes, but Golytely provoked a 6.8% contraction of plasma volume, whereas drinking Tyrode resulted in plasma expansion. Copious diarrhoea was experienced by all subjects who drank Golytely solution and by none on drinking Tyrode solution. 4. The infusion of Golytely into the stomach resulted in a hypotonic diuresis similar in magnitude to that elicited by drinking. Drinking with simultaneous aspiration of gastric contents ('sham-drinking') did not produce a significant diuresis. 5. Plasma arginine vasopressin (AVP) levels did not fall following the drinking of Golytely. The assay used was sufficiently sensitive to measure changes of 0.6 pg AVP (ml plasma)-1. 6. The findings show that signals from the oropharynx do not mediate the diuretic response to drinking Golytely in man and that a mechanism other than inhibition of AVP release appears to be involved. The receptors mediating this response may lie in the stomach and/or small bowel since Golytely is not absorbed, as evidenced by the contraction of plasma volume and diarrhoea.


Voltage-dependent currents in isolated single Merkel cells of rats. 1. Merkel cells were dissociated enzymatically from the footpad epidermis of 10- to 20-day-old rats pretreated with fluorescent dye, quinacrine, for purposes of staining. The fluorescent Merkel cells had an elongated or elliptic shape in situ, yet the dissociated ones were round (7-12 microns in diameter). 2. Electrical recordings were performed in the whole-cell configuration using a conventional patch-clamp technique. The mean resting membrane potential of fluorescent Merkel cells was -54.0 mV, the value being greater than the -26.1 mV of non-fluorescent epidermal cells. No voltage-dependent channel was observed in non-fluorescent cells. 3. The Merkel cells had no Na+ spike in an external standard solution, but tetrodotoxin-resistant long-lasting action potentials were evoked by depolarization with injection of constant currents in an external solution containing Ba2+. 4. In Merkel cells under voltage clamp, depolarizing step pulses (800 ms) from a holding potential (VH) of -80 mV elicited predominantly outward K+ currents composed of transient and sustained components: the former was selectively inhibited by 4-aminopyridine (4-AP), while the latter was inhibited by both tetraethylammonium (TEA) and quinacrine. Quinacrine was more effective and selective than TEA in blocking the sustained K+ current but had no effect on the current at the low concentration (10(-7) or 3 x 10(-6) M) used for staining the Merkel cells. 5. The sustained outward K+ current (IKD) was activated at potentials more positive than -20 or -10 mV at a VH of -50 mV, at which potential the transient outward K+ channel was completely inactivated. The potential for half-inactivation in the steady-state inactivation curve for IKD was -33 mV. 6. The transient outward K+ current (IA) was activated at potentials more positive than -50 mV at a VH of -80 mV. The potential for half-inactivation in the steady-state inactivation curve for IA was -64 mV. 7. When the outward K+ currents were blocked by adding both TEA and 4-AP, only a sustained inward Ca2+ current was observed. In an external solution containing 10 mM-Ca2+, ICa was evoked by potentials more positive than -20 mV at a VH of -80 mV, and the maximum inward current appeared around +10 mV. Increases in external Ca2+ concentration ([Ca2+]o) induced a hyperbolic increase in ICa and shifted the current-voltage (I-V) relationship along the voltage axis in a more positive direction. Saturation of ICa occurred at about 25 mM [Ca2+]o.(ABSTRACT TRUNCATED AT 400 WORDS)


Blockade by calcium antagonists of chemical excitation and sensitization of polymodal nociceptors in the cat's cornea. 1. The possibility that a modified Ca2+ channel mediates chemical and thermal excitation of nociceptors was examined in single polymodal nociceptive fibres of the cat's cornea. 2. Ca2+ channel blockers cadmium (2.5 mM) and diltiazem (1 mM), and high external [Ca2+] (40 mM), markedly reduced nociceptive responses to topical acidic solutions (pH 4.5). 3. Decreasing the pH to 6.5 did not cause excitation, but reduced the subsequent response to pH 4.5 buffer. 4. Diltiazem (1 mM), applied after repeated stimulation with stepped heating pulses (35 to 47-49 degrees C in 2 degrees C steps) decreased the impulse responses elicited by heat. The decrease in threshold to thermal stimulation produced by repeated heating was blocked by diltiazem. 5. Mechanical threshold and mechanical responsiveness of corneal polymodal nociceptors was not modified by Ca2+ antagonists or by a high-Ca2+ solution. 6. These results offer indirect evidence that proton-activated Ca2+ channels mediate stimulation of nociceptors by acidic solutions. The same type of ionic channel appears to be involved in the response of nociceptors to heat and in sensitization, but not in their responsiveness to mechanical stimulation. The blockade of nociceptive responses by Ca2+ antagonists opens the possibility of using Ca2+ blockers as selective analgesic drugs.


Role of proton pump of mitochondria-rich cells for active transport of chloride ions in toad skin epithelium. 1. Active Cl- currents were studied in short-circuited toad skin epithelium in which the passive voltage-activated Cl- current is zero. Under visual control double-barrelled microelectrodes were used for impaling principal cells from the serosal side, or for measuring the pH profile in the solution bathing the apical border. 2. The net inward (active) 36Cl- flux of 27 +/- 8 pmol s-1 cm-2 (16) (mean +/- S.E.M (number of observation)) was abolished by 2 mM-CN- (6.3 +/- 3.5 pmol s-1 cm-2 (8)). The active flux was maintained in the absence of active Na+ transport when the latter was eliminated by either 100 microM-mucosal amiloride, replacement of mucosal Na+ with K+, or by 3 mM-serosal ouabain. 3. In Ringer solution buffered by 24 mM-HCO3- -5% CO2 mucosal amiloride reversed the short circuit current (ISC). The outward ISC was maintained when gluconate replaced mucosal Cl-, and it was reversibly reduced in CO2-free 5 mM-Tris-buffered Ringer solution (pH = 7.40) or by the proton pump inhibitor oligomycin. These observations indicate that the source of the outward ISC is an apical proton pump. 4. Amiloride caused principal cells to hyperpolarize from a basolateral membrane potential, Vb, of -73 +/- 3 (22) to -93 +/- 1 mV (26), and superfusion with CO2-free Tris-buffered Ringer solution induced a further hyperpolarization (Vb = -101 +/- 1 mV (26)) which could be blocked by Ba2+. The CO2-sensitive current changes were null at Vb = EK (potassium reversal potential, -106 +/- 2 mV (55)) implying that they are carried by K+ channels in the basolateral membrane. Such a response cannot account for the inhibition of the outward ISC which by default seems to be located to mitochondria-rich (MR) cells. 5. In the absence of mucosal Cl- a pH gradient was built up above MR cells with pH = 7.02 +/- 0.04 (42) and pH increasing to 7.37 +/- 0.02 (10) above principal cells (pH = 7.40 in bulk solution buffered by 0.1 mM-Tris). This observation localizes a proton pump to the apical membrane of MR cells. Using the integrated diffusion equation it was shown that the measured external pH gradient would account within an order of magnitude for measured currents. 6. Standing gradients of protons were eliminated in the presence of mucosal Cl- suggesting that active uptake of Cl- is associated with the exit of base equivalents across the apical membrane of MR cells.(ABSTRACT TRUNCATED AT 400 WORDS)


Calcium gradients and buffers in bovine chromaffin cells. 1. Digital imaging and photometry were used in conjunction with the fluorescent Ca2+ indicator, Fura-2, to examine intracellular Ca2+ signals produced by depolarization of single adrenal chromaffin cells. 2. Depolarization with a patch pipette produced radial gradients of Ca2+ within the cell, with Ca2+ concentration highest in the vicinity of the plasma membrane. These gradients dissipated within a few hundred milliseconds when the voltage-gated Ca2+ channels were closed. 3. Dialysis of Fura-2 into the chromaffin cell caused concentration-dependent changes in the depolarization-induced Ca2+ signal, decreasing its magnitude and slowing its recovery time course. These changes were used to estimate the properties of the endogenous cytoplasmic Ca2+ buffer with which Fura-2 competes for Ca2+. 4. The spatially averaged Fura-2 signal was well described by a model assuming fast competition between Fura-2 and an endogenous buffer on a millisecond time scale. Retrieval of calcium by pumps and slow buffers occurs on a seconds-long time scale. No temporal changes indicative of buffers with intermediate kinetics could be detected. 5. Two independent estimates of the capacity of the fast endogenous Ca2+ buffer suggest that 98-99% of the Ca2+ entering the cell normally is taken up by this buffer. This buffer appears to be immobile, because it does not wash out of the cell during dialysis. It has a low affinity for Ca2+ ions, because it does not saturate with 1 microM-Ca2+ inside the cell. 6. The low capacity, affinity and mobility of the endogenous Ca2+ buffer makes it possible for relatively small amounts of exogenous Ca2+ buffers, such as Fura-2, to exert a significant influence on the characteristics of the Ca2+ concentration signal as measured by fluorescence ratios. On the other hand, even at moderate Fura-2 concentrations (0.4 mM) Fura-2 will dominate over the endogenous buffers. Under these conditions radiometric Ca2+ concentration signals are largely attenuated, but absolute fluorescence changes (at 390 nm) accurately reflect calcium fluxes.


Ionic currents in cultured rat hypothalamic neurones. 1. Dissociated neurones from embryonic rat hypothalamus were grown for several weeks in culture where they formed complex networks. These synaptically coupled networks were capable of generating synchronized bursting activity. Voltage-activated membrane currents were studied in these neurones using a patch clamp in the whole-cell configuration. 2. Outward currents were carried by K+ ions and consisted of an inactivating and a non-inactivating component. These components were similar to the transient K+ current (IA) and the delayed rectifier current (IK) reported in neurones from the postnatal rat hypothalamus. Application of Zn2+ (1 mM) blocked the transient component completely while reducing the non-inactivating component by only approximately 20%. 3. Inward currents were carried by Na+ and Ca2+ ions. Rapidly activating transient Na+ currents were activated at approximately -25 mV. TTX entirely blocked these currents at low concentration (300 nM). Voltage sensitivity of the Na+ conductance was 5.8 mV per e-fold change with half-maximal activation occurring at -8 mV. Na+ current kinetics could be well described by the Hodgkin-Huxley model (m3h). 4. With depolarizing pulses from a holding potential of -80 mV two Ca2+ current components with different ranges of activation were identified. Low voltage-activated (LVA, T-type) Ca2+ currents were activated at approximately -50 mV. High voltage-activated (HVA; also called L- or N-type) Ca2+ currents were observed at membrane potentials more positive to approximately -30 mV. LVA Ca2+ currents were observed in hypothalamic neurones that had developed a network of dendritic processes in the course of several weeks in culture. Activation and inactivation time constants of LVA Ca2+ currents were 15-25 ms and 30-100 ms (-30 to -45 mV). In contrast to HVA Ca2+ currents, no LVA Ca2+ currents were seen in neuronal somata obtained from the network cultures by mechanical dissociation. This suggests that most of the LVA Ca2+ channels are located on the dendritic tree rather than on the soma membrane. 5. HVA Ca2+ currents were maximal between 0 and +10 mV (external [Ca2+] = 5 mM). The time-to-peak was in the range of 1.7-5.4 ms (+30 to -10 mV). Tail currents following repolarization decayed monoexponentially with a time constant of approximately 210 microseconds. During 500 ms depolarizations, 90% of the current inactivated. The time course of inactivation showed two time constants of approximately 40 and approximately 700 ms.(ABSTRACT TRUNCATED AT 400 WORDS)


Ca2+ current in myotome cells of the lancelet (Branchiostoma lanceolatum). 1. By using the whole-cell patch clamp method Ca2+ and Ba2+ currents were measured in the extremely thin twitch muscle cells of the protochordate Branchiostoma lanceolatum whose Ca2+ channels are likely to resemble the evolutionary ancestors of those found in vertebrate skeletal muscle. 2. When using 10 mM-Ca2+ in the artificial external solution and 1 mM-EGTA in the internal solution two kinetically different Ca2+ inward current components could be observed, showing very similar voltage dependence of activation and inactivation. 3. In solutions containing 10 mM-Ba2+ as an external charge carrier the biphasic inward current turned into a single rapidly activated and slowly inactivating current. 4. Inspecting peak currents, the voltage dependence of fractional activation and inactivation was nearly the same in Ca2+ and in Ba2+. 5. A transformation into a single component of the Ca2+ current could also be observed after perfusing the intracellular lumen with 10 mM of either EGTA or BAPTA. In the case of EGTA this transformation required considerably more time. Probably a higher internal concentration of EGTA is necessary since it binds Ca2+ more slowly than BAPTA. 6. Soon after establishing the whole-cell configuration a gradual increase of the second, slow inward current phase was observed relative to the fast component, indicating an enhancement of the slow component by intermediate intracellular buffer concentrations. 7. We conclude that the Branchiostoma myotome cells have only one Ca2+ channel system. The biphasic appearance of the inward current is caused by an unusually rapid inactivation due to Ca2+ ions, which enter the myoplasm during the current and temporarily bind to an inactivation site at the channel. The second phase probably reflects reactivation from the inactivated state upon dissociation of Ca2+ from the binding site.


Two types of neurone in the rat ventral tegmental area and their synaptic inputs. 1. Intracellular recordings were made from 241 ventral tegmental neurones in slices of rat midbrain. Seventy-seven per cent of neurones were hyperpolarized by dopamine (principal cells); 16% were hyperpolarized by opioid peptides (secondary cells). 2. Most principal cells fired spontaneously (1-3 Hz) with a threshold of -53 mV; most secondary cells did not fire spontaneously. Action potentials of principal cells were longer (0.9 ms) than those of secondary cells (0.5 ms). 3. Focal electrical stimulation within the ventral tegmental area evoked a biphasic synaptic potential, depolarization followed by hyperpolarization, with a duration of about 200 ms. Experiments with receptor antagonists showed that the depolarizing component resulted from activation of both N-methyl-D-aspartate (NMDA) and non-NMDA receptors and the hyperpolarizing component resulted from activation of GABAA receptors. 4. A later hyperpolarizing synaptic potential developed after a latency of 50 ms, reached its peak in 250 ms and had a duration of about 1 s. It reversed polarity at -108 mV (external potassium concentration was 2.5 mM), was blocked by phaclofen (30 microM-1 mM) or 2-hydroxysaclofen (100-300 microM). In some cells, a phaclofen-resistant component remained that was increased by cocaine and blocked by sulpiride (1 microM). 5. It is concluded that the ventral tegmental area contains two types of neurone having properties similar to those in the substantia nigra. The cells receive synaptic inputs mediated by excitatory amino acids acting at NMDA and non-NMDA receptors, GABA acting at GABAA and GABAB receptors, and dopamine acting at D2 receptors.


Tetrodotoxin-sensitive calcium-conducting channels in the rat hippocampal CA1 region. 1. Tetrodotoxin (TTX)-sensitive Ca2+ conducting channels which produce a transient inward current were investigated in pyramidal neurones freshly dissociated from the dorsal part of rat hippocampal CA1 region by the use of the suction-pipette technique, which allows for intracellular perfusion under a single-electrode voltage clamp. 2. In all cells superfused with Na(+)- and K(+)-free external solution containing 10 mM-Ca2+ and 10(-5) M-La3+, a transient inward Ca2+ current was evoked by a step depolarization to potentials more positive than about -50 mV from a holding potential (VH) of -100 mV. This current was inhibited by either removing the extracellular Ca2+ or adding TTX (termed as 'TTX-ICa'). 3. Activation and inactivation processes of the TTX-ICa were highly potential dependent at 20-22 degrees C, and the latter was fitted by a double exponential function. The time to peak of the current decreased from 5.0 to 2.3 ms at a test potential change from -50 to 0 mV. The time constants of the current decay decreased from 2.8 to 2.2 ms for fast component (tau if) and from 16.0 to 8.2 ms for slow component (tau is) at a potential change from -35 to -10 mV. 4. The TTX-ICa was activated at threshold potential of about -55 mV and reached full activation at -30 mV. The steady-state inactivation of TTX-ICa could be fitted by a Boltzmann equation with a slope factor of 6.0 mV and a half-inactivation voltage of -72.5 mV. 5. Biphasic recovery (reactivation) from the complete inactivation of TTX-ICa was observed. The time constant of the major component (78.8 to 91.6% of total) of the reactivation was 13.1 ms, and that of the minor one was 120 to 240 ms. Therefore, TTX-ICa remained fairly constant at a train of stimulation up to 3 Hz. However, the inhibition of current amplitude occurred as the repetitive stimulation increased more than 10 Hz, and considerable tonic inhibition occurred with increasing stimulation frequency. 6. When the peak amplitudes in the individual current-voltage (I-V) relationships of TTX-ICa at various extracellular Ca2+ concentrations ([Ca2+]o) were plotted as a function of [Ca2+]o, the current amplitude increased linearly without showing any saturation. 7. The ratio of peak amplitude in the individual I-V relationships of Ca2+, Sr2+ and Ba2+ currents passing through the TTX-sensitive Ca2+ conducting channel was 1:0.33:0.05, although the current kinetics were much the same.(ABSTRACT TRUNCATED AT 400 WORDS)


Time-resolved changes in intracellular calcium following depolarization of rat brain synaptosomes. 1. Changes of cytoplasmic free calcium levels ([Ca2+]i) in isolated rat brain nerve terminals (synaptosomes), previously loaded with the fluorescent intracellular calcium indicator Fura-2, were measured 1-2 ms after depolarization with elevated K+ by stopped-flow fluorescence spectroscopy. 2. In physiological saline (PSS) containing 4 mM-K+, intraterminal Ca2+ was estimated to be in the range 150-250 nM. Depolarization of the nerve terminals with elevated external K+ in the presence of Ca2+ induced a prompt rise in [Ca2+]i, which occurred in two phases. No change in [Ca2+]i was seen when the terminals were depolarized in nominally Ca(2+)-free solutions, and only a small change was seen when the terminals were acutely exposed to Ca2+ in 4 mM-K+. 3. Predepolarization of the nerve terminals with K+ in nominally Ca(2+)-free solutions several seconds before the introduction of Ca2+ greatly decreased the magnitude of the fast phase, whilst leaving the slow phase largely intact. 4. In Na(+)-depleted nerve terminals, the fast phase of K(+)-stimulated Ca2+ uptake was essentially unaltered, but the slow phase of Ca2+ uptake was dramatically reduced. 5. The rapid phase of K(+)-stimulated uptake displayed voltage-dependent inactivation (tau approximately 50 ms at -10 mV), and the rate of inactivation was accelerated with increasing depolarization. In contrast, at constant [K+]o, increasing [Ca2+]o had little or no effect on the rate of inactivation, but did increase the initial rate of Ca2+ uptake. 6. The dihydropyridine calcium channel blockers nifedipine and nitrendipine had little effect on either component of Ca2+ uptake. However, the inorganic Ca2+ channel blockers La3+, Cd2+, and Co2+ were potent blockers of the fast phase of Ca2+ uptake, but blocked the slow phase only at higher concentrations. No consistent effect of the peptide neurotoxin omega-conotoxin was observed on either component of the Ca2+ rise. 7. These studies demonstrate that the dynamics of depolarization-activated intraterminal Ca2+ changes can be studied on a millisecond time scale in isolated nerve terminals. Moreover, our results indicate that two pathways contribute to depolarization-induced [Ca2+]i changes, namely a voltage-activated, inactivating Ca2+ channel, possibly of the N-type, and Na(+)-Ca2+ exchange operating in the 'reverse' mode.


Presynaptic inhibitory action of enkephalin on excitatory transmission in superficial dorsal horn of rat spinal cord. 1. Tight-seal whole-cell recordings were made from marginal neurones visually identified in thin slices of 1- to 2-week-old rat lumbar spinal cord. Excitatory postsynaptic currents (EPSCs), either evoked by extracellular stimulation or those arising spontaneously in tetrodotoxin, i.e. miniature EPSCs (mEPSCs), were recorded after blocking inhibitory synaptic inputs with strychnine and bicuculline. 2. The EPSCs were abolished reversibly by kynurenic acid or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) but not affected by (+)-2-amino-5-phosphonovalerate (APV), suggesting that they were mediated by non-NMDA (N-methyl-D-aspartate) glutamate receptors. Micromolar concentrations of methionine [Met5]enkephalin reversibly reduced the magnitude of evoked EPSCs and the frequency of mEPSCs. 3. The enkephalin action on the mEPSC frequency was blocked by naloxone. A specific agonist of mu-opiate receptor, [D-Ala2,N-Me-Phe4, Gly5]enkephalin-ol (DAGO) suppressed the mEPSC frequency. In contrast, neither a delta-opiate receptor agonist, [D-Pen2, L-Pen5]enkephalin (DPLPE) nor a kappa-opiate receptor agonist, (5 alpha, 7 alpha, 8 beta)-(-)-N-methyl-N-[7-(1-pyrrolidinyl)-1- oxaspiro(4,5)-dec-8-yl]benzeneacetamide (U-69,593) significantly affected the mEPSC frequency. 4. The amplitude of mEPSCs or of currents induced by exogenous L-glutamate, was not affected by [Met5]enkephalin. It is suggested that [Met5]enkephalin presynaptically inhibits glutamatergic EPSCs by activating the mu-opiate receptor. 5. The frequency of mEPSC was reduced by about 50% by replacement of external Ca2+ with Mg2+ or by addition of Cd2+. In Ca(2+)-free-Mg2+ solution, [Met5]enkephalin did not reduce the remaining mEPSCs' frequency any further. 6. It is concluded that the opiates may suppress presynaptic Ca2+ entry, thereby inhibiting synaptic transmission.


Efficacy of a porcelain refinishing system in restoring surface finish after grinding with fine and extra-fine diamond burs. Commercial porcelain refinishing kits are claimed to restore the surface finish on porcelain after adjustments in circumstances that preclude reglazing. This study investigates the efficacy of one such kit in restoring a Vitadur N porcelain surface finish after grinding with fine (30 microns grit-red band) and extra-fine (15 microns grit-yellow band) high-speed diamond burs. The production of the porcelain test samples, the refinishing procedures, and the refinishing times were standardized throughout. Techniques were selected to simulate clinical practice. Randomly selected examples of surfaces created during refinishing were subjected to scanning electron microscopy, using standard kV and tilt angle settings, and to surface profilometry tracings. Roughness average (Ra) readings were recorded for each test specimen for every surface finish. Ra readings were analyzed statistically using a two-way unbalanced model for analysis of variance. Although refinishing after grinding with a 15 microns grit bur produced surfaces significantly smoother than on specimens previously ground with the 30 microns grit burs, the surfaces remained significantly rougher than when originally glazed. It is concluded that, using the type of kit tested, burs of a grade finer than the existing 15 microns grit yellow band types would be appropriate for porcelain adjustments to permit subsequent refinishing to a surface smoothness comparable to the original glaze.


Surface roughness of finished composite resins. This study evaluated the results of polishing composite resin by using the following finishing and polishing techniques: Arkansas stone burs; eight-blade tungsten-carbide burs; diamond burs; aluminum-oxide disks; no polishing; and 12- and 30-blade tungsten-carbide burs. The results were compared to ascertain which technique delivers the smoothest surface. The study was done with 120 class V restorations on extracted human teeth distributed in two groups (I and II) of 60 restorations each. Group I teeth samples were filled with microfilled composite resin and Group II with a hybrid composite resin. Readings were made with a profilometer to evaluate the final polishing. The conclusions from the results of the study were that microfilled composite resins provided a better finish when treated with aluminum-oxide disks. These results were statistically significant (p < 0.001). When hybrid composites were used the control group recorded the lowest values.


Perception of breath components by the tropical bont tick, Amblyomma variegatum Fabricius (Ixodidae). I. CO2-excited and CO2-inhibited receptors. Wall-pore olfactory sensilla located in the capsule of Haller's organ on the tarsus of Amblyomma variegatum ticks bear cells responding to vertebrate breath: one of these sensilla contains a CO2-excited receptor and a second sensillum has a CO2-inhibited receptor. Each of these antagonistic CO2-receptors, which display typical phasic-tonic responses, monitors a different CO2-concentration range. The CO2-inhibited receptor is very sensitive to small concentration changes between 0 and ca. 0.2%, but variations of 0.01% around ambient (ca. 0.04%) induce the strongest frequency modulation of this receptor. An increase of just 0.001-0.002% (10-20 ppm) above a zero CO2-level already inhibits this receptor. By contrast, the CO2-excited receptor is not so sensitive to small CO2 shifts around ambient, but best monitors changes in CO2 concentrations above 0.1%. This receptor is characterized by a steep dose-response curve and a fast inactivation even at high CO2-concentrations (greater than 2%). In a wind-tunnel, Amblyomma variegatum is activated from the resting state and attracted by CO2 concentrations of 0.04 to ca. 1%, which corresponds to the sensitivity range of its CO2-receptors. The task of perceiving the whole concentration range to which this tick is attracted would thus appear to be divided between two receptors, one sensitive to small changes around ambient and the other sensitive to the higher concentrations normally encountered when approaching a vertebrate host.


Inhibitory actions of dopamine on Limulus visceral muscle involve a cyclic AMP-dependent mechanism. 1. The catecholamines dopamine, epinephrine and norepinephrine were detected in alumina extracts of Limulus midgut tissue using high performance liquid chromatography with electrochemical detection. Moderate levels of norepinephrine (28.2 +/- 2.1 ng/g) and dopamine (24.0 +/- 5.2 ng/g) were detected in the midgut, while epinephrine levels (7.4 +/- 0.9 ng/g) were less. Catecholamines were present in all regions along the longitudinal axis of the midgut, and norepinephrine and dopamine levels were highest in posterior regions. 2. Catecholamines decreased muscle tonus and inhibited spontaneous contractions of the Limulus midgut. Dopamine typically decreased spontaneous midgut activity at doses of 10(-8) M or greater, and produced inhibitory actions on all regions of the Limulus midgut. In some preparations epinephrine and norepinephrine elicited a secondary rhythmicity. The actions of dopamine opposed the excitatory effects produced by either proctolin or octopamine. 3. Catecholamines significantly elevated levels of cyclic AMP in Limulus midgut muscle rings. Dopamine (10(-5) M) increased cyclic AMP with a time course consistent with its physiological effects. Forskolin and several methyl xanthines increased Limulus midgut cyclic AMP levels and mimicked the inhibitory effects of dopamine on the isolated midgut preparation. Cyclic nucleotide analogues also produced dopamine-like effects on the isolated midgut preparation. Inhibition of cyclic nucleotide phosphodiesterase prior to addition of dopamine enhanced the effect of this amine to decrease baseline muscle tension. 4. The inhibitory effects of 10(-5) M dopamine on the midgut persisted in solutions of zero sodium and in the presence of tetrodotoxin. Zero calcium solutions gradually reduced spontaneous midgut activity and the effects of dopamine. Calcium channel blockers did not prohibit dopamine-induced relaxation.(ABSTRACT TRUNCATED AT 250 WORDS)


[Granular cell tumors (Abrikossoff's tumors) of the breast. Apropos of 4 cases]. Granular cell tumors are rarely localized in the breast gland because of diagnosis is difficult compared to breast skin lesions. Clinically and radiologically, pseudo-neoplastic aspect can be encountered. Differential diagnosis with an invasive carcinoma can be difficult on frozen section. But final histological analysis easily confirms the diagnosis. The treatment consists in local excision of the tumor. Today we can admit that granular cell tumors are derived from schwann cells.


Androgenic regulation of enzymes involved in DNA synthesis in epididymis of young rats. In the epididymis of young rats, activities of DNA polymerases alpha, beta and gamma and DNA topoisomerase I decreased after castration. DNA polymerase alpha and gamma increased with androgen administration and activity reached 81.3% and 78.0%, respectively, of the activity in the sham-operated group on day 21. Activity of DNA polymerase beta remained at the activity of day 7 during androgen administration and was almost the same as that in the sham-operated group on day 21. DNA topoisomerase I activity showed a slight increase with androgen administration and reached 50.3% of that in the sham-operated group. The activities of these enzymes were not fully restored to those in the sham-operated group. These results indicate that in young rats activities of epididymal DNA polymerase alpha and gamma and DNA topoisomerase I are partially, and that of DNA polymerase beta wholly, dependent on androgens and may provide a means of investigating the regulation of epididymal cell proliferation.


Lack of effect of hormones and inducers of intracellular messengers on plasminogen activator production by bovine embryos in vitro. Several hormones and inducers of intracellular messengers, known to affect plasminogen-activator (PA) production in other systems, were investigated for putative effects on bovine embryos. Day 8 embryos were cultured for 5 days in a humidified atmosphere of 5% CO2 in air at 37 degrees C in media containing different concentrations of progesterone, oestradiol, dexamethasone, retinoic acid, dibutyryl cyclic AMP (dbcAMP) and phorbol myristate acetate (PMA). At intervals of 24 h, the medium was recovered for PA analysis and overall embryonic diameter was measured. While none of the hormones and agents tested affected PA production (P > 0.05), dimethyl sulfoxide, which was used to dissolve PMA, inhibited PA production during the first 72 h of culture (P < 0.05). PA production was affected by duration of culture (P < 0.05). Concentrations of plasminogen activator in the media were low during the first 48 h, had increased after 72 and 96 h in culture, and either remained high or decreased slightly toward the end of the culture period. With the exceptions of dbcAMP and PMA, the hormones tested in this study did not affect embryonic size. Dibutyryl cAMP caused a progressive decrease in embryonic diameter. PMA resulted in embryo death at high concentrations but at lower concentrations it enhanced overall embryonic diameter throughout the time of culture (P < 0.05). These results suggest that cultured bovine embryos produce PA in a fixed, time-dependent manner, independent of exogenous hormonal regulation.


Prevalence of human papillomavirus infection in men. Comparison of the partners of infected and uninfected women. Men were checked for the presence of genital human papillomavirus (HPV)-associated lesions. The evaluation included acetic acid-guided, high-magnification examination of the penis and scrotum employing a colposcope and biopsy confirmation of HPV-associated lesions in the genital area. The men were in a high-risk group if they were sexual partners (for at least six months) of women known to harbor genital condyloma. The men were in a low-risk group if they were sexual partners of women with no known genital condyloma. Seventy-eight of 113 men (69%) in the high-risk group had genital HPV-associated lesions, while 31% did not. Two of the 94 men (2%) in the low-risk group had genital HPV-associated lesions, while 98% did not. The differences were highly statistically significant (P less than .01). The male sexual partners of women with HPV infection had a significantly higher risk of harboring genital HPV infection as compared to partners of uninfected women.


Diagnosis of cauda equina abnormalities by using electromyography, discography, and epidurography in dogs. Electromyography (EMG), L7-S1 discography and epidurography were investigated in 15 dogs with clinical signs of cauda equina dysfunction and in 7 control dogs without such clinical signs. Electromyography of paraspinal and pelvic limb muscles was done in 13 of 15 affected dogs. An L7-S1 discogram followed by an epidurogram was performed in all 22 dogs using 20% iopamidol. Results of discograms, epidurograms, and gross necropsy examinations were normal in six of seven control dogs. The one dog in which these studies were abnormal had a mild L7-S1 disc protrusion that did not result in nerve root compression at necropsy. Electromyographic analysis was 100% accurate in predicting the presence or absence of cauda equina disease. None of the results of discograms were falsely negative. Twelve of 15 discograms in clinically affected dogs indicated dorsal disc protrusion, but 2 of these protrusions were found to be noncompressive at surgery (13% error). Abnormal epidurograms occurred in 9 of 15 clinically affected dogs. There was one false positive and two false negatives (20% error). Electromyography was a sensitive screening technique for the presence of cauda equina disease. Discography may be more sensitive for detection of L7-S1 disc protrusion than epidurography. An abnormal radiographic contrast study of the cauda equina may only be useful when combined with an abnormal EMG.


Inclusion body myositis: analysis of 32 cases. Inclusion body myositis is characterized by an insidious onset, progressive indolent course, and is generally felt to be refractory to standard therapy for myositis. We reviewed the charts of 32 patients with muscle biopsy findings suggestive of inclusion body myositis. The average time from symptom onset to diagnosis was 37 months, but initially 40% were incorrectly diagnosed. Twenty-eight patients (88%) were classified as definite or probable inclusion body myositis and were treated with various combinations of prednisone and immunosuppressive agents. Sixty-eight percent of those treated experienced a decrement in function and muscle strength. Three patients exhibited longterm improvement while 12 patients experienced delayed progression, defined by short term improvement in strength or a stable functional class, All of these patients received therapy, 5 in the form of methotrexate and prednisone. All untreated patients deteriorated clinically. In summary, (1) inclusion body myositis is a clinically distinct entity which is frequently misdiagnosed initially. (2) While clinical improvement with therapy is rare, our observations support recent reports that therapy may be associated with a slower rate of clinical progression. (3) Optimal therapy remains uncertain, but the use of low dose methotrexate and prednisone may warrant further study.


A study of the effects of lisinopril when used in addition to atenolol. A double-blind, parallel group multicentre study was carried out to compare the effects of adding once daily treatment with lisinopril 10 or 20 mg and placebo to the treatment of 100 patients whose blood pressure was inadequately controlled with once daily atenolol 50 mg. Following a two-week run-in period, patients with a lying DBP between 95 mmHg and 115 mmHg were randomised to either lisinopril 10 mg or placebo once daily for four weeks. Blood pressure measurements were made approximately 24 h after the previous dose of study medication. After four weeks' treatment the dose of study medication was doubled for those patients whose lying DBP was greater than or equal to 90 mmHg and a final assessment was made after a further two weeks of treatment. Overall, six weeks' treatment with lisinopril produced a greater fall in lying blood pressures than placebo when added to atenolol therapy. The difference in favour of the additional ACE inhibitor therapy was 7.1 +/- 2.6/5.4 +/- 1.5 mmHg (mean +/- SEM) (P less than 0.01). Standing blood pressures showed similar behaviour in favour of the additional ACE inhibitor treatment (7.6 +/- 2.4/4.7 +/- 1.6 mmHg) (P less than 0.005). Heart rate was not altered significantly by either lisinopril or placebo treatment. The addition of lisinopril to treatment with atenolol produced a slight increase in the reported number of adverse events compared with placebo. The results of this study indicate that the addition of lisinopril 10-20 mg once daily to treatment with a beta-adrenoceptor blocking drug produces a worthwhile decrease in blood pressure in patients not responsive to beta-blocker therapy alone.


Twenty-four hour blood pressure effect of once-daily lisinopril, enalapril, and placebo in patients with mild to moderate hypertension. This multicentre, double-blind, parallel-group, placebo-controlled study compared the antihypertensive effects of equal doses of two long-acting angiotensin converting enzyme (ACE) inhibitors. After a two-week, placebo run-in phase, 110 patients with mild to moderate hypertension were randomised to receive 10 mg lisinopril or enalapril, or placebo for 4 weeks. Office BPs were measured at regular intervals throughout the study. Twenty-four hour ambulatory blood pressure (ABP) was measured at baseline and after the first and final doses of study drug. Serum ACE activity and aldosterone were obtained concomitantly with each ABP monitoring. Office BP differences from placebo reached (P less than 0.05) or approached (P less than 0.10) statistical significance at all observations for the lisinopril group but were not significant for any observation in the enalapril group and approached significance on two occasions. After four weeks of treatment, ABP analysis revealed that the lisinopril and enalapril groups, when compared with placebo, had similar and significant systolic and diastolic AUC reductions (P less than 0.01) from baseline over the 24 h dosing interval. During the second half of the dosing interval, 13-24 h post drug administration, the lisinopril group was significantly different from placebo (systolic BP, P = 0.002; diastolic BP, P = 0.005) while the enalapril group was not. Both drugs were well tolerated. The results indicate that monotherapy with 10 mg of lisinopril is as effective as with 10 mg of enalapril, and that ABP monitoring is useful in more precisely depicting the clinical effect of the known pharmacokinetic properties of these two agents.


Novel positive inotropic agents: synthesis and biological activities of 6-(3-amino-2-hydroxypropoxy)-2(1H)-quinolinone derivatives. A series of 6-(3-amino-2-hydroxypropoxy)-2(1H)-quinolinones has been synthesized and evaluated for positive inotropic activity on the canine heart. Some of these derivatives have a potent activity with none or negative chronotropic effect in isolated, blood-perfused dog heart preparations. They also display a high selectivity for positive inotropic effect over chronotropic and vasodilatory effects in anesthetized dogs. (+/-)-6-[2-Hydroxy-3-[(3-methoxybenzyl)amino]propoxy]-2(1H)-quinolinone (39) and (+/-)-6-[3-(3,4-dimethoxybenzyl)amino]-2-hydroxypropoxy]-2 (1H)-quinolinone (40) were further investigated in conscious dogs. After iv administration, they did not affect heart rate or mean blood pressure at the dose producing a 50% increase in the peak of the first derivative of the left ventricular pressure. The compounds (39, OPC-18750, and 40, OPC-18790) are the most promising agents with desirable biological activities, and now are currently undergoing clinical evaluation.


Comparative molecular field analysis of polyhalogenated dibenzo-p-dioxins, dibenzofurans, and biphenyls. Comparative molecular field analysis (CoMFA) was performed on polyhalogenated dibenzo-p-dioxins, dibenzofurans, and biphenyls for Ah (dioxin) receptor binding and associated enzyme inducing activities determined by others using in vitro assays. Since various members of all three classes of compounds have been shown to produce qualitatively similar toxicities, a separate CoMFA was performed on each class of compounds and combinations of the different classes for each bioactivity which included combining all three classes of molecules in one CoMFA study. For the Ah receptor binding, the CoMFA-derived QSARs for all three classes of compounds and combinations thereof showed strong crossvalidated correlations indicating that they are highly predictive. For enzyme induction, the CoMFA-derived QSARs were highly predictive for the dibenzofurans but were only partially successful for the dioxins. For the biphenyls, the results were clearly unpredictive. The overall results of these CoMFA studies which include both steric and electrostatic considerations are compared and contrasted to other SAR models that have met with some success in making qualitative predictions about the potential for receptor binding and associated toxicity in these classes of compounds. The CoMFA-derived QSAR for the dioxin series of molecules in most cases significantly overestimates the enzyme inducing ability of the ortho-substituted biphenyls. This weak inducing activity of the o-biphenyls is, however, consistent with their relatively low dioxin-like toxicity as measured in other biological systems. Fundamentally different mechanisms may be operating in the expression of dioxin-like toxic responses for the o-biphenyls, and their direct, dioxin-like toxic equivalency perhaps needs to be reconsidered in this light.


Development of a novel series of styrylquinoline compounds as high-affinity leukotriene D4 receptor antagonists: synthetic and structure-activity studies leading to the discovery of (+-)-3-[[[3-[2-(7-chloro-2-quinolinyl)-(E)-ethenyl]phenyl][[3- (dimethylamino)-3-oxopropyl]thio]methyl]thio]propionic acid. Based on LTD4 receptor antagonist activity of 3-(2-quinolinyl-(E)-ethenyl)pyridine (2) found in broad screening, structure-activity studies were carried out which led to the identification of 3-[[[3-[2-(7-chloro-2-quinolinyl)-(E)-ethenyl]phenyl][[3- (dimethylamino)-3-oxopropyl]thio]methyl]thio]propionic acid (1, MK-571) as a potent and orally active LTD4 receptor antagonist. These studies demonstrated that a phenyl ring could replace the pyridine in 2 without loss of activity, that 7-halogen substitution in the quinoline group was optimal for binding, that the (E)-ethenyl linkage was optimal, that binding was enhanced by incorporation of a polar acidic group or groups in the 3-position of the aryl ring, and that two acidic groups could be incorporated via a dithioacetal formed from thiopropionic acid and the corresponding styrylquinoline 3-aldehyde to yield compounds such as 20 (IC50 = 3 nM vs [3H]LTD4 binding to the guinea pig lung membrane). It was found that one of the acidic groups could be transformed into a variety of the amides without loss of potency and that the dimethylamide 1 embodied the optimal properties of intrinsic potency (IC50 = 0.8 nM on guinea pig lung LTD4 receptor) and oral in vivo potency in the guinea pig, hyperreactive rat, and squirrel monkey. The evolution of 2 to 1 involves the increase of > 6000-fold in competition for [3H]LTD4 binding to guinea pig lung membrane and a > 40-fold increase in oral activity as measured by inhibition of antigen-induced dyspnea in hyperreactive rats.


Development of a novel class of cyclic hexapeptide oxytocin antagonists based on a natural product. A new structural class of cyclic hexapeptide oxytocin antagonists derived from Streptomyces silvensis and typified by L-365,209 (cyclo-[L-prolyl1-D-phenylalanyl2-L- isoleucyl3-D-dehydropiperazyl4-L-dehydroperazyl5-D-(N- methyl)phenylalanyl6]) was recently reported. In this paper we further delineate the structure-activity profile for this new class by systematic study of L-365,209 analogs obtained by total synthesis. The optimal combination of cyclic amino acid ring sizes at positions 1, 4, and 5 and the role of the N-alkyl substituent at position 6 was elucidated. The lipophilic amino acids at positions 2 and 3 and the unusual amino acid D-dehydropiperazic acid at position 4 were found to be the most critical residues for obtaining good oxytocin receptor affinity. Analogs containing a basic side chain at the less critical 5- and 6-positions maintained good receptor affinity and also had useful levels of water solubility for intravenous formulation. By combining potency- and solubility-enhancing substitutions, several analogs were identified that have the desired combination of properties in vitro (22, cyclo-[L-prolyl-D-tryptophanyl-L-isoleucyl-D-pipecolyl-L-pipeco lyl-D- histidyl]; 25, cyclo-[L-prolyl-D-2-naphthylalanyl-L-isoleucyl-D-pipecolyl-L -pipecolyl-D- histidyl]; 26, cyclo-[L-prolyl-D-tryptophanyl-L-isoleucyl-D-dehydropiperazyl-L-++ pipecolyl-D-histidyl]; 33, cyclo-[L-prolyl-D-tryptophanyl-L-isoleucyl-D-pipecolyl-L- piperazinylcarboxy-D-(N-methyl)phenylalanyl]; 34, cyclo-[L-prolyl-D-phenylalanyl-L-isoleucyl-D-dehydropiperazyl-L-or nithyl- D-(N-methyl)phenylalanyl]). In general, this class exhibited good selectivity for binding to the oxytocin receptor versus the arginine vasopressin V1a and V2 receptor subtypes, although increased V2 receptor affinity was observed in one case (32, cyclo[L-prolyl-D-2-naphthylalanyl-L-isoleucyl-D-pipecolyl-L- lysyl-D-(N- methyl)phenylalanyl]). Unexpectedly, compound 33 was found to stimulate contractions of the isolated rat uterus via activation of the uterine bradykinin receptor. Compounds 22, 25, 26, 33, and 34 were found to be potent antagonists of oxytocin-stimulated contraction of the rat uterus in vitro and in vivo. Compounds 22 and 25 were additionally characterized as potent antagonists of oxytocin-stimulated uterine contractions in the near-term pregnant rhesus monkey. These studies thus demonstrate the selectivity and efficacy of certain members of this novel class of antagonists and suggest their use as pharmacological tools in further defining the role of oxytocin in both term and preterm labor.


Orally active, nonpeptide oxytocin antagonists. The first nonpeptide antagonists of the neurohypophyseal hormone, oxytocin (OT) are described. Derivatives of the spiroindenepiperidine ring system, these compounds include L-366,509, an orally bioavailable OT antagonist with good in vivo duration. The potential use of these agents for treatment of preterm labor and their significance as new nonpeptide ligands for peptide receptors are discussed.


A new class of high affinity ligands for the neurokinin A NK2 receptor: psi (CH2NR) reduced peptide bond analogues of neurokinin A4-10. Analogues of [Leu10]NKA4-10 were synthesized in which each of the amide bonds was sequentially replaced with the reduced amide psi (CH2NH) bond to determine the effect of this structural modification on the antagonism of NKA binding to the HUB NK2 receptor. [psi (CH2-NH)9,Leu10]NKA4-10 (6) retained significant affinity for the NK2 receptor (IC50 = 115 nM) and showed weak partial stimulation of PI turnover (approximately 10-15% of NKA maximum). 6 behaves as a competitive antagonist of NKA-stimulated PI turnover with a pA2 = 6.7. The secondary amine of the psi (CH2NH) moiety of 6 was converted to a tertiary amine by alkylation. This modification was found to have a small effect upon receptor affinity but did result in attenuation of partial agonist activity. A combination of amino acid substitutions and psi (CH2NH) alkylation yielded [beta Ala8,psi (CH2N(CH2)2CH3)9,Phe10]NKA4-10 (21) which has very high affinity for the HUB NK2 receptor. This compound inhibited [125I]NKA binding with an IC50 = 1 nM which is equal to the receptor affinity of NKA. Compound 21 also shows very weak partial agonism of PI turnover (< or = 5% of NKA maximum) which makes this the most potent member of a new class of NKA ligands: psi(CH2NR)9-NKA4-10 analogues which potently antagonize NKA binding and possess minimal partial agonist activity.


Conformationally restricted deltorphin analogues. Conformationally restricted deltorphin analogues were synthesized either through incorporation of cyclic phenylalanine analogues in position 2 or 3 of the peptide sequence or through various side chain-to-side chain cyclizations. Compounds were tested in mu-, delta-, and kappa-receptor selective binding assays and in the guinea pig ileum (GPI) and mouse vas deferens (MVD) bioassays. Replacement of Phe3 in [D-Ala2]deltorphin I with 2-aminoindan-2-carboxylic acid (Aic) or L- or D-2-aminotetralin-2-carboxylic acid (Atc) resulted in agonist compounds which retained the high delta receptor selectivity of the parent peptide. Substitution of a tetrahydroisoquinoline-3-carboxylic acid (Tic) residue in the 2-position of [D-Ala2]deltorphin I and of [Phe4,Nle6]deltorphin produced a partial delta agonist, H-Tyr-Tic-Phe-Asp-Val-Val-Gly-NH2, and a pure delta antagonist, H-Tyr-Tic-Phe-Phe-Leu-Nle-Asp-NH2, respectively. The latter antagonist displayed high delta selectivity (Ki mu/Ki delta = 502) and was a potent antagonist against selective delta agonists in the MVD assay (Ke congruent to 10 nM). Various [D-Ala2]-deltorphin I analogues cyclized between the side chains of Orn (or Lys) and Asp (or Glu) residues substituted in positions 2 and 4, 4 and 7, and 2 and 7 were essentially nonselective. Comparison with corresponding N-terminal tetrapeptide analogues revealed that the C-terminal tripeptide segment in the deltorphin heptapeptides made a crucial contribution to delta affinity and delta selectivity in the case of the agonist peptides but not in the case of the antagonist.


Predictive binding of beta-carboline inverse agonists and antagonists via the CoMFA/GOLPE approach. The synthesis and affinities of six new 3-substituted beta-carbolines (6-10, 12) for the benzodiazepine receptor (BzR) are described. These analogs were used both to probe the dimensions of the hydrophobic pocket in the benzodiazepine receptor and to test the predictive ability of a previously reported 3D-QSAR regression model. Of the new analogs synthesized, the gamma-branched derivatives (isobutoxy, 7, IC50 = 93 nM; isopentoxy, 9, IC50 = 104 nM) display significantly higher affinity for the BzR than either the beta-branched (sec-butoxy, 6, IC50 = 471 nM; tert-butyl ketone, 12, IC50 = 358 nM) or delta-branched (isopentoxy, 8, IC50 = 535 nM) analogs. An exception to this rule is the gamma-branched 3-benzyloxy derivative 10 (IC50 > 1000 nM) which appears to have a chain length that is too long to be accommodated by the BzR. The standard error of prediction for these six new beta-carbolines using the original regression model is significantly lower than the standard error estimate of the cross validation runs on the training set, hence the predictions made using this model are much better than expected. In order to obtain more credible predictions, a new procedure called GOLPE (generating optimal linear PLS estimates) was used to eliminate irrelevant electrostatic and steric descriptors from the regression equation. A substantial reduction in the standard error estimate resulted. The predictions from this new regression equation were somewhat less accurate than the ones obtained with the original regression equation; however the standard error of prediction and the standard error estimate are in much closer agreement. Finally, to probe the effect that the quality of the steric and electrostatic potentials has on 3D-QSAR analyses, the semiempirical MNDO parallel PRDDOE geometries and Mulliken charges used in the original analyses were replaced with ab initio 3-21G parallel 6-31G* geometries and electrostatic potential fit charges. A modest decrease in the standard error estimate and increase in cross validated R2 resulted.


Effects of alkyl substitutions of xanthine skeleton on bronchodilation. Structure-activity relationships in a series of 1,3,7-trialkyl-xanthine were studied with guinea pigs. Relaxant actions in the tracheal muscle were increased with alkyl chain length at the 1- and 3-positions of the xanthine skeleton, but decreased by alkylation at the 7-position. Positive chronotropic actions in the right atrium were potentiated with 3-alkyl chain length but tended to decrease with 1-alkylation and diminish by 7-substitution. Consequently, while the 1- and 3-substitutions were equally important for the tracheal smooth muscle relaxation, the substitution at the 1-position was more important than the 3-substitution for bronchoselectivity. The 7-alkylation may be significant to cancel heart stimulation. There were good correlations between the smooth muscle relaxant action and the cyclic AMP-PDE inhibitory activity in 3-substituents and the affinity for adenosine (A1) receptors in 1-, 3-, and 7-substituents. This suggests that not only the cyclic AMP-PDE inhibitory activity but also the adenosine antagonistic activity is important in the bronchodilatory effects of alkylxanthines. Among these xanthine derivatives, 1-butyl-3-propylxanthine and its 7-methylated derivative showed high bronchoselectivity in the in vitro and in vivo experiments compared to theophylline and enprofylline and may be new candidates for bronchodilator.


New bronchodilators. 1. 1,5-Substituted 1H-imidazo[4,5-c]quinolin-4(5H)-ones. A series of novel xanthine-based tricyclic heterocycles in 1H-imidazo[4,5-c]quinolin-4(5H)-ones was designed, synthesized, and tested as potential active bronchodilators. Inhibition of the Schulz-Dale (SD) reaction-induced contraction in trachea and inhibition of antigen inhalation-induced bronchospasm in passively sensitized guinea pigs served as primary in vitro and in vivo assays, respectively. Simultaneous measurement of acute lethal toxicity (minimum lethal dose; MLD, po) in mice allowed determination of a safety margin. The bronchodilatory activity of these heterocycles was considerably varied with the nature of substituents at the 5-position. The most active substituents at the 2- and 5-positions and on the aromatic ring were found to be hydrogen, n-butyl, and hydrogen, respectively. There was a bulk tolerance for lipophilic substituents at the 1-position. 5-Butyl-substituted compounds appeared to be less toxic than theophylline on the basis of MLD data. Thus 5-butyl-1-methyl-1H-imidazo[4,5-c]quinolin-4(5H)-one (10) (IC50 value of the SD assay = 0.25 microM, MLD > 300 mg/kg) was selected for further studies. Compound 10 (KF15570) reduced bronchoconstriction produced by antigen (Konzett-Rössler preparation in anesthetized guinea pigs, ED50 = 0.42 mg/kg, iv) more effectively than aminophylline (ethylenediamine salt of theophylline, ED50 = 7.8 mg/kg, iv) but had fewer side effects on the heart and CNS than theophylline. Compound 10 and its derivatives showed weak adenosine antagonism and phosphodiesterase (PDE) inhibition which could not account for their potent bronchodilation. Although their precise mechanism of action remains unclear, this series of novel tricyclic heterocycles represents a new class of bronchodilator.


Delta opioid antagonist activity and binding studies of regioisomeric isothiocyanate derivatives of naltrindole: evidence for delta receptor subtypes. The isothiocyanate group was attached to the 4'-, 5'-, 6'-, or 7'-position of naltrindole in an effort to determine the importance of the position of this electrophilic group on the selectivity for subtypes of delta opioid receptors. All of the ligands were delta-selective when tested against standard agonists in smooth muscle preparations. However, the rank-order delta antagonism of antinociception in mice did not parallel the in vitro pharmacologic data. The 5'-isothiocyanate 2 was the most potent and selective antagonist in vivo, causing a 52-fold increase of the ED50 for [D-Ser2,D-Leu5]enkephalin-Thr6 (DSLET) and no increase for [D-Pen2,D-Pen5]enkephalin (DPDPE). The effect of each of the ligands on the binding of [3H]DSLET and [3H]DPDPE to guinea pig brain membranes clearly differentiated between the binding sites that recognize these radioligands. These studies provide additional evidence for the presence of two subtypes of delta opioid receptors.


Condensation of muscimol or thiomuscimol with aminopyridazines yields GABA-A antagonists. Ten analogs of muscimol and thiomuscimol in which the amino function was delocalized in an amidinic system were prepared by N2 alkylation of 6-aryl-3-aminopyridazines with (chloromethyl)isoxazole or (chloromethyl)isothiazole derivatives. These muscimol and thiomuscimol derivatives show potent binding properties for GABA-A receptors (they displace [3H]GABA and [3H]gabazine) and provoke convulsions after iv injections. They fit well with the model pharmacophore proposed by our group for the GABA-A antagonists and show similar structure-activity profiles to that of the pyridazinyl-GABAs.


Molecular yardsticks. Rigid probes to define the spatial dimensions of the benzodiazepine receptor binding site. A series of rigid planar azadiindoles (8a, 8b, and 8d), benzannelated pyridodiindoles (11a, 11b, and 11d), and indolopyridoimidazoles (11c, 20, and 24) were synthesized from 4-oxo-1,2,3,4-tetrahydro-beta-carboline 5 via the Fischer indole cyclization with the appropriate arylhydrazines. These analogues were employed as probes ("molecular yardsticks") to define the spatial dimensions of the lipophilic regions of the benzodiazepine receptor (BzR) binding cleft. Benzannelated indoles 11a-d and indolopyridoimidazoles 20 and 24 were important in establishing an area of negative interaction (S1, see Figure 6, part b) in the binding cleft common to the interactions of both inverse agonists and agonists. Data from this chemical and computer-assisted analysis of the pharmacophore (see Figure 6) indicates that inverse agonists and agonists bind to the same binding region, but the pharmacophoric descriptors required for the two activities are different, in keeping with previous studies with these planar ligands. However, the hydrogen bond donating site H1 and the lipophilic region L1 in the receptor binding site are common interactions experienced by both series of ligands. The low affinities of both indolo[3,2-c]carbazole (3a) and indolo[3,2-b]isoquinoline (3b) for the BzR are consonant with the requirements of a hydrogen bond acceptor interaction at donor site H1 and a hydrogen bond donor interaction at acceptor site A2 for potent inverse agonist activity in the beta-carboline series. The hydrochloride salts of 1-aza- 8a (IC50 10.6 nM), 2-aza- 8b (IC50 51.5 nM), and 4-azadiindole 8d (IC50 11.2 nM) were found to be much more soluble in water than the corresponding salt of the parent diindole 2. Moreover, aza analogues 8a and 8b were shown to be partial inverse agonists with proconvulsant potencies comparable to that of the parent diindole 2.


Structural modifications of camptothecin and effects on topoisomerase I inhibition. Camptothecin (1), a potent antitumor alkaloid, is known to inhibit topoisomerase I, an enzyme that relaxes supercoiled DNA. Modifications have been made to the B, D, and E rings of this natural product. Specifically, compounds 2-10 either have an ester moiety in place of the E ring lactone, a methyl ester attached to position 14, a saturated (or nonexistent) deaza B ring, or contain a combination of these permutations. We have conducted in vitro assays against the topoisomerase I relaxation reaction which verify the necessity for a lactone in the E ring. Furthermore, steric requirements at position 14 are shown to be crucial for activity, and planarity of the A and B rings of camptothecin is also implicated in the ability of the drug to inhibit topoisomerase I. Speculation on the nature of the drug binding pocket is presented.


The first mechanism-based inactivators for angiotensin-converting enzyme. The first example of mechanism-based inactivation of angiotensin-converting enzyme (ACE) is described for N-[N-(cyanoacetyl)-L-phenylalanyl]-L-phenylalanine (compound 1). It is proposed that an ACE-mediated deprotonation of 1 unmasks a ketenimine intermediate, which traps an active-site nucleophile, and hence irreversibly modifies the enzyme. In competition with the inactivation reaction, ACE also hydrolyzes 1 with a partition ratio of 8300 (i.e., kcat/kinact). Since the corresponding keto analogue, N-[(R)-2-benzyl-5-cyano-4-oxopentanyl]-L-phenylalanine (compound 4), does not inactivate the enzyme, it is suggested that the NH in compound 1 is critical for the proper active-site anchoring of the inhibitor for the inactivation process to take place.


Conformationally constrained tachykinin analogues: potent and highly selective neurokinin NK-2 receptor agonists. The design and synthesis of potent and selective neurokinin NK-2 receptor agonists 12 (GR64349) and 31 are described, together with structure-activity relationships for related analogues. Compound 12 (EC50 = 3.7 nM at NK-2 receptors in the rat colon; selectivity > 1000- and > 300-fold with respect to NK-1 and NK-3 receptors, respectively) was derived by incorporation of a Gly-Leu gamma-lactam conformational constraint into the C-terminal region of the neurokinin A octapeptide analogue [Lys3]-NKA(3-10). Compound 31 (EC50 = 15 nM in rat colon) contains a novel fused-bicyclic constraint at the corresponding site in the substance P hexapeptide analogue [Ava6]-SP(6-11).


L-thymidine is phosphorylated by herpes simplex virus type 1 thymidine kinase and inhibits viral growth. We have demonstrated that herpes simplex 1 (HSV1) thymidine kinase (TK) shows no stereospecificity for D- and L-beta-nucleosides. In vitro, L enantiomers are not recognized by human TK, but function as specific substrates for the viral enzyme in the order: L-thymidine (L-T) >> 2'-deoxy-L-guanosine (L-dG) > 2'-deoxy-L-uridine (L-dU) > 2'-deoxy-L-cytidine (L-dC) > 2'-deoxy- L-adenosine (L-dA). HSV1 TK phosphorylates both thymidine enantiomers to their corresponding monophosphates with identical efficiency and the Ki of L-T (2 microM) is almost identical to the Km for the natural substrate D-T (2.8 microM). The L enantiomer reduces the incorporation of exogenous [3H]T into cellular DNA in HeLa TK-/HSV1 TK+ but not in wild-type HeLa cells, without affecting RNA, protein synthesis, cell growth, and viability. L-T markedly reduces HSV1 multiplication in HeLa cells. Our observations could lead to the development of a novel class of antiviral drugs characterized by low toxicity.


Background calcium permeable channels in glomerulosa cells from adrenal gland. The cell-attached recording mode of the patch-clamp technique was used to study Ca2+ permeable background currents of glomerulosa cells from rat and bovine adrenal gland. With a pipette filled with 110 mM BaCl2 or 90 mM CaCl2, three different types of unitary currents were detected. The B1 channel demonstrates a nonlinear I-V curve. The conductances are 4 and 7 pS at -40 and -70 mV, respectively. The curve of the opening probability vs. membrane potential is bell shaped with its maximum at -70 mV. The B2 channel has a conductance of 6 pS, while the B3 channel shows a nonlinear I-V relationship with conductances close to 17 and 10 pS at HPs of -60 and -20 mV. The three types of currents are insensitive to dihydropyridines. We suggest that these background currents could be responsible for the basal calcium influx and aldosterone secretion previously observed in nonstimulated glomerulosa cells.


Sulfhydryl alkylating agents induce calcium current in skeletal muscle fibers of a crustacean (Atya lanipes). Voltage-clamp experiments using the three-microelectrode voltage clamp technique were performed on ventroabdominal flexor muscles of the crustacean Atya lanipes. Potassium and chloride currents were found to underlie the normal, passive response of the muscle. Blocking potassium currents with tetraethylammonium and replacing chloride ions with methanesulfonate did not unmask an inward current. By treating the muscle with the sulfhydryl-alkylating agent 4-cyclopentene-1,3-dione an inward current was detected. The current induced by the agent is carried by Ca2+, since it is abolished in Ca(2+)-free solutions. The induced Ca2+ current is detected at about -40 mV and reaches a mean maximum value of -78 microA/cm2 at ca. -10 mV. At this potential the time to peak is close to 15 msec. The induced Ca2+ current inactivated with 1-sec prepulses which did not elicit detectable Ca2+ current; the fitted hx curve had a midpoint of -38 mV and a steepness of 5.0 mV. Measurements of isometric tension were performed in small bundles of fibers, and the effects of the sulfhydryl-alkylating agents 4-cyclopentene-1,3-dione and N-ethylmaleimide were investigated. Tetanic tension was enhanced in a strictly Ca(2+)-dependent manner by 4-cyclopentene-1,3-dione. The amplitude of K+ contractures increased after treatment with N-ethylmaleimide. It is concluded that Ca2+ channels are made functional by the sulfhydryl-specific reagents and that the increase in tension is probably mediated by an increase in Ca2+ influx through the chemically induced Ca2+ channels.


pH regulation in tissue-cultured bovine lens epithelial cells. The intracellular pH (pHi) of tissue-cultured bovine lens epithelial cells was measured in small groups of 6 to 10 cells using the trapped fluorescent dye 2',7'-bis-(2-,carboxyethyl)-5 (and 6)carboxyfluorescein (BCECF). When perifused at 35 degrees C with artificial aqueous humour solution (AAH) containing 16 mM HCO3- and 5% CO2, pH 7.25, pH(i) was 7.19 +/- 0.02 (SEM, n = 95). On removing HCO3- and CO2 there was an initial transient alkalinization followed by a fall in pH to a steady value of 6.97 +/- 0.03 (SEM, n = 54). Addition of 0.25 mM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) to AAH containing HCO3- and CO2 led to a rapid and pronounced fall in pH. Exposure to Na(+)-free AAH again led to a marked fall in pH(i), but in this case the addition of DIDS did not produce a further fall. Substitution of the impermeant anion gluconate for Cl- in the presence of HCO3- led to a rise in pHi, while substitution in the absence of HCO3- led to a fall in pHi. The above data indicate a significant role for a sodium-dependent Cl(-)-HCO3- exchange mechanism in the regulation of pHi. Addition of 1 mM amiloride to control AAH in both the presence and absence of HCO3- led to a marked fall in pH(i), indicating that a Na+/H+ exchange mechanism also has a significant role in the regulation of pHi.(ABSTRACT TRUNCATED AT 250 WORDS)


Involvement of different S4 parts in the voltage dependency of Na channel gating. Three synthetic peptides corresponding to parts of S4 of the first repeat of eel electroplax sodium channel were synthesized. The basic peptide was C1+ which corresponds to amino acids 210-223 (eel channel numbering) and two subfractions: an external fraction, C1+ex (amino acid 210-217); and an internal part, C1+in (amino acid 218-221). Peptide C1+ includes four of the charged amino acids of this domain; peptide C1+ex includes three of the charged amino acids and is closer to the external membrane surface (according to channel models) than peptide C1+in which includes the fourth charged amino acid alone. Antibodies generated in rabbits against these peptides were shown to be site specific. Using the whole-cell patch-clamp technique, we found that in rat dorsal root ganglion (DRG) cells, the antibodies against C1+in but not against C1+ex had an effect on the gating parameters. They shifted the Na-channel inactivation curve towards hyperpolarization and decreased the slope of the Na-channel activation curve. These results demonstrate that during the conformational changes associated with channel gating, the fourth charged amino acid of S4 must be accessible to antibodies given to the external solution. Furthermore, they indicate a specific involvement of S4 in the voltage dependency of the gating processes.


Volume-dependent regulation of sodium and potassium fluxes in cultured vascular smooth muscle cells: dependence on medium osmolality and regulation by signalling systems. To identify ion transport systems involved in the maintenance of vascular smooth muscle cell volume the effects of incubation medium osmolality and ion transport inhibitors on the volume and 86Rb and 22Na transport in cultured smooth muscle cells from rat aorta (VSMC) have been studied. A decrease of medium osmolality from 605 to 180 mosm increased intracellular water volume from 0.6 to 1.3 microliters per 10(6) cells. Under isosmotic conditions, cell volume was decreased by ouabain (by 10%, P less than 0.005) but was not influenced by bumetanide, furosemide, EIPA and quinidine. These latter compounds were also ineffective in cell volume regulation under hypotonic buffer conditions. Under hyperosmotic conditions, cell volume was decreased by bumetanide (by approximately 7%, P less than 0.05) and by ethylisopropyl amiloride (by approximately 13%, P less than 0.005). Ouabain-sensitive 86Rb influx was decreased by 30-40% under hypoosmotic conditions. An increase in medium osmolality from 275 to 410 mosm resulted in an approximately eightfold increase in bumetanide-inhibited 86Rb influx and 86Rb efflux. The (ouabain and bumetanide)-insensitive component of 86Rb influx was not dependent on the osmolality of the incubation medium. However (ouabain and bumetanide)-insensitive 86Rb efflux was increased by approximately 1.5-2 fold in VSMC incubated in hypotonic medium. Ethylisopropyl amiloride-inhibited 22Na influx was increased by approximately sixfold following osmotic-shrinkage of VSMC. The data show that both Na+/H+ exchange and Na+/K+/2Cl- cotransport may play a major role in the regulatory volume increase in VSMC. Basal and shrinkage-induced activities of Na+/K+/2Cl- cotransport in VSMC were similarly sensitive to inhibition by either staurosporin, forskolin, R24571 or 2-nitro-4-carboxyphenyl N,N-diphenylcarbomate (NCDC). In contrast basal and shrinkage-induced Na+/K+/2Cl- cotransport were differentially inhibited by NaF (by 30 and 65%, respectively), suggesting an involvement of guanine nucleotide binding proteins in the volume-sensitive activity of this carrier. Neither staurosporin, forskolin, R24571 nor NCDC influenced shrinkage-induced Na+/H+ exchange activity. NaF increased Na+/H+ exchanger activity under both isosmotic and hyperosmotic conditions. These data demonstrate that different intracellular signalling mechanisms are involved in the volume-dependent activation of the Na+/K+/2Cl- cotransporter and the Na+/H+ exchanger.