Showing papers in "Journal of Pharmacology and Experimental Therapeutics in 1988"

Opposite effects of mu and kappa opiate agonists on dopamine release in the nucleus accumbens and in the dorsal caudate of freely moving rats.

Abstract: We studied the effect of opiates acting preferentially on mu receptors, like morphine, methadone and fentanyl (mu agonists) and on kappa receptors, like U50,488, bremazocine and tifluadom (kappa agonists) on the release of dopamine (DA) and of its metabolites, dihydroxyphenylacetic acid and homovanillic acid, from the nucleus accumbens and from the dorsal caudate of freely moving rats using brain dialysis coupled to high-performance liquid chromatography with electrochemical detection. Spontaneous behavior was videotaped and analyzed by estimating the percentage of time spent by the animals in performing certain specific behavioral items. Mu agonists stimulated DA-release and metabolism in the accumbens at lower doses than in the caudate. Maximal stimulation of DA release did not exceed 100% except after high doses of methadone (10 mg/kg) which stimulated DA release in the accumbens by more than 300%, possibly as a result of hypoxia. Stimulation of DA release was associated to stimulation of behavior at low doses and to a biphasic inhibitory-stimulatory syndrome after higher doses of the opiates. Pretreatment with low doses of naloxone (0.1 mg/kg s.c.) or with the irreversible mu antagonist beta-funaltrexamine (10 nmol i.c.v.) increased the ED50 for the stimulation of DA release by the three opiates. In contrast with mu agonists, agonists of kappa receptors like U50,488, bremazocine and tifluadom decreased DA release in the accumbens and in the caudate and reduced motor activity. These effects were antagonized only by rather high doses of naloxone (2.5 mg/kg s.c.) and were not affected by pretreatment with beta-funaltrexamine (10 nmol i.c.v.).(ABSTRACT TRUNCATED AT 250 WORDS)

Steroid modulation of the chloride ionophore in rat brain: structure-activity requirements, regional dependence and mechanism of action.

Abstract: Further in vitro studies of steroids active at the gamma-aminobutyric acidA (GABAA) receptor regulated Cl- channel labeled by [35S]-t-butylbicyclophosphorothionate ([35S]TBPS) reveal additional structural requirements necessary for activity. Evaluation of selected steroids for activity against TBPS-induced convulsions show similar requirements for activity. Interestingly, steroids (e.g., 5 alpha-pregnan-3 alpha, 20 alpha-diol) were identified that have high potency but limited efficacy as modulators of [35S]TBPS binding. These characteristics are reminiscent of the clinically useful benzodiazepines (BZs) such as clonazepam. However, interactions between the prototypical anesthetic-barbiturate, sodium pentobarbital, and steroids active at the Cl- channel suggest that they do not share a common site of action as allosteric modulators of [35S]TBPS and BZ receptor binding. The most potent steroid evaluated, 5 alpha-pregnan-3 alpha-ol-20-one, modulates [35S]TBPS binding at low concentrations (IC50 approximately 17 nM) in a regionally dependent manner. All [35S]TBPS binding sites appear to be functionally coupled to a steroid "modulatory site." Because several of the active steroids are metabolites of progesterone, their ability to inhibit the binding of [3H]promegestrone to the cytosolic progestin receptor in rat uterus was evaluated. Those steroids showing potent activity at the GABAA receptor-Cl- ionophore were inactive at the intracellular progestin receptor. Such specificity coupled with their high potency provide additional support for the hypothesis that some of these steroids may be involved in the homeostatic regulation of brain excitability via the GABAA-BZ receptor complex.

Pharmacology of risperidone (R 64 766), a new antipsychotic with serotonin-S2 and dopamine-D2 antagonistic properties.

Abstract: Comparative studies of the benzisoxazole derivative risperidone (R 64 766) were made with ritanserin, a selective centrally acting serotonin-S2 antagonist and with haloperidol, a selective centrally acting dopamine-D2 antagonist. Risperidone like ritanserin shows activity in all tests related to serotonin-S2 antagonism, but at even lower doses (peripheral S2-antagonism at 0.0011 mg/kg, central S2-antagonism at 0.014 mg/kg). Like haloperidol, risperidone shows activity in all tests related to dopamine-D2 antagonism; activity in rats for both compounds starts at 0.016 mg/kg, but some central nervous system controlled functions, including the induction of catalepsy, are relatively much less affected by risperidone. Qualitatively, risperidone is a mixed serotonin-dopamine antagonist. Quantitatively, its study in dogs reveals potent dopamine-D2 antagonistic activity with excellent p.o. bioavailability and a relatively long duration of action. From the obtained pharmacological data, risperidone could be expected to possess the complementary clinical effects of a ritanserin-like serotonin-S2 and an haloperidol-like dopamine-D2 antagonist. Serotonin-S2 antagonism may improve the quality of sleep, reduce negative and affective symptoms in schizophrenic patients and decrease extrapyramidal symptoms induced by classical neuroleptics. Because risperidone is a dopamine-D2 antagonist, antidelusional, antihallucinatory and antimanic actions are expected. The first clinical studies indicate that two additional therapeutic targets, which are not reached with classical neuroleptics, may be obtained with risperidone in the monotherapy of schizophrenia and related disorders: very important contact and mood-elevating properties and extrapyramidal symptoms-free maintenance therapy.

Nor-binaltorphimine, a highly selective kappa-opioid antagonist in analgesic and receptor binding assays.

Abstract: Previously, we reported on an opioid antagonist, nor-binaltorphimine (nor-BNI), that had high selectivity for kappa opioid receptors in smooth muscle preparations. In this study, nor-BNI administered either s.c. or i.c.v. was shown to antagonize significantly the antinociceptive effects of the kappa opioid agonists, ethylketazocine and U-50,488H at doses that had no effect on the antinociceptive effect of mu agonists, morphine and [D-Ala3, MePhe4, Gly-ol5]enkephalin and the delta agonist, [D-Pen3, D-Pen5]enkephalin. Nor-BNI and U-50,488H were used to demonstrate that kappa opioid receptors in the spinal cord were more important than those located supraspinally for kappa-mediated analgesia. Nor-BNI also possessed high affinity and high selectivity for kappa opioid receptors in the receptor binding assay. However, the comparatively low selectivity of BNI in receptor binding studies did not correlate with the high pharmacologic selectivity for kappa receptors.

Biochemical profile of risperidone, a new antipsychotic.

Abstract: Risperidone was compared to the 5-hydroxytryptamine2 antagonist ritanserin and to the dopamine-D2 antagonist haloperidol. The in vitro receptor binding (neurotransmitter-, peptide- and ion channel binding sites) and neurotransmitter uptake profile were investigated. Risperidone revealed, like ritanserin, a very high binding affinity for 5-hydroxytryptamine2 receptors (Ki = 0.16 and 0.30 nM, respectively) and a slow dissociation (half-time, 31 and 160 min). In accordance, risperidone (IC50 = 0.5 nM) and ritanserin (IC50 = 1.8 nM) potently blocked serotonin-induced 32P-phosphatidic acid formation in human blood platelets. Risperidone showed, like haloperidol, high binding affinity for dopamine-D2 receptors (Ki = 3.13 and 1.55 nM, respectively) and rapid dissociation (half-time, 2.7 and 5.8 min). Risperidone displayed higher binding affinity than ritanserin and haloperidol for alpha-1 adrenergic (Ki = 0.8 nM), histamine-H1 (Ki = 2.23 nM) and alpha-2 adrenergic receptors (Ki = 7.54 nM). In in vitro superfusion experiments, risperidone and haloperidol reversed at nanomolar concentrations the inhibition by LY 171555 (a dopamine-D2 agonist) and by amphetamine of potassium and electrically evoked release of [3H]acetylcholine from striatal slices (postsynaptic dopamine-D2 effects). Both drugs reversed with similar potency the inhibition by LY 171555 of electrically evoked release of [3H]dopamine (a presynaptic dopamine-D2 effect). Risperidone did not affect the activation by amphetamine of [3H]dopamine efflux from rat striatal slices. Risperidone enhanced at nanomolar concentrations the stimulated [3H]norepinephrine efflux from cortical slices and it similarly reversed the inhibition by clonidine, at concentrations corresponding to its binding affinity for alpha-2 adrenergic receptors. The in vitro biochemical properties of risperidone are in agreement with the reported in vivo pharmacological profile, the relation to clinical findings is discussed.

Glycyrrhetinic acid derivatives: a novel class of inhibitors of gap-junctional intercellular communication. Structure-activity relationships.

Abstract: Glycyrrhetinic acid was shown previously to inhibit intercellular gap-junctional communication between human fibroblasts. In the present study 31 derivatives of glycyrrhetinic acid were tested for their ability to inhibit communication. Eight of the compounds inhibited communication with high potency (IC50 less than 3 microM) and showed low toxicity, properties which suggest they may be useful pharmacological probes for studies of gap-junction function.

Amphetamine injection into the ventral mesencephalon sensitizes rats to peripheral amphetamine and cocaine.

Abstract: The daily administration of indirect dopamine agonists, including amphetamine and cocaine, results in a progressive increase in the behavioral stimulant effect of these drugs. Behavioral augmentation also has been shown with opioids such as morphine, and it is known that a stimulant action on dopaminergic perikarya in the ventromedial mesencephalon is critical to the development of behavioral sensitization to morphine. To determine if amphetamine-induced behavioral sensitization might also involve the mesencephalic dopamine neurons, amphetamine was microinjected daily for 2 days into regions of the rat brain containing dopamine cell bodies (A10 and A9 dopamine regions), or dopamine terminals (nucleus accumbens and striatum), and 6 days later amphetamine was given peripherally. It was found that daily amphetamine injection into the A10 or A9 dopamine region, but not into the dopamine terminal fields, significantly potentiated the motor stimulant effect of peripherally administered amphetamine. The behavioral sensitization produced by intracranial injection of amphetamine was found to be dose-dependent. Intra-A10 injection of amphetamine also was found to potentiate the motor stimulant effect of peripheral cocaine. These data indicate that an action by amphetamine in the A10 and A9 dopamine regions may play a critical role in the development of behavioral sensitization.

Effect of 17 beta-estradiol on endothelium-dependent responses in the rabbit.

Abstract: Experiments were designed to determine the effect of 17 beta-estradiol on endothelium-dependent responses of isolated arteries. Ovariectomized female New Zealand rabbits were treated either with 17 beta-estradiol (100 micrograms i.m.) or with solvent for 4 days. After excision, femoral arteries were cut into rings and suspended for isometric tension recording in organ chambers filled with modified Krebs-Ringer bicarbonate solution. Rings from the 17 beta-estradiol-treated rabbits showed an enhanced endothelium-dependent relaxation to acetylcholine (3 x 10(-9)-3 x 10(-8) M) in the absence or presence of indomethacin. Under the same experimental conditions, the endothelium-dependent responses to the calcium ionophore A23187 were unchanged. In the absence of indomethacin, the response to adenosine diphosphate was depressed in rings with endothelium taken from animals treated with with 17 beta-estradiol; in the presence of the inhibitor of cyclooxygenase, the endothelium-dependent responses were comparable in blood vessels from treated and untreated rabbits. This study suggests that endothelium-dependent responses of arteries can be modulated by steroid hormones.

Evidence that mesolimbic dopaminergic activation underlies the locomotor stimulant action of nicotine in rats.

Abstract: L-Nicotine stimulates locomotor activity in rats which have had prior experience of the drug. The present study investigated whether this behavioral effect is related to activation of the mesolimbic dopamine system. In the first experiment, l-nicotine (0.2-0.8 mg/kg s.c.) stimulated locomotor activity and increased dopamine utilization in the olfactory tubercle, as judged by the ratio of the concentration of dihydroxyphenylacetic acid to dopamine. In other experiments, l-nicotine (0.1-0.4 mg/kg) stimulated locomotor activity in a dose-related, stereoselective manner; after pretreatment with the l-aromatic amino acid decarboxylase inhibitor NSD-1015, l-nicotine increased 3,4-dihydroxyphenylalanine/dopamine ratios in olfactory tubercle and nucleus accumbens, suggesting increased dopamine utilization, although absolute concentrations of 3,4-dihydroxyphenylalanine and dopamine were in general not significantly altered. This neurochemical action of l-nicotine was dose-dependent, stereoselective and absent in the caudate-putamen at the doses tested. l-Nicotine did not alter indices of 5-hydroxytryptamine utilization. The locomotor stimulant effect of l-nicotine was abolished by bilateral intra-accumbens microinjection of 6-hydroxydopamine, which depleted markedly mesolimbic terminal areas of dopamine. Thus, in rats which have been chronically treated with l-nicotine, a selective activation of mesolimbic dopamine appears to mediate the locomotor stimulant effect of this drug.

Substance P and vasoactive intestinal peptide degradation by mast cell tryptase and chymase.

Abstract: The peptides substance P (SP) and vasoactive intestinal peptide (VIP) released from peptidergic neurons have potent effects on gland secretion and on smooth muscle tone. Because mast cells release proteases during degranulation, and are located in many of the same tissue microenvironments into which SP and VIP are released, we wished to examine whether mast cell proteases, by cleaving and thus inactivating these peptides, could modulate their effects. We used active site-titrated preparations of the two major neutral proteases of mast cell granules, tryptase and chymase, to determine the sites and rates of cleavage of SP and VIP. The proteases were purified from dog mastocytomas. Tryptase cleaved VIP rapidly at two sites with a kcat/Km of 2.2 X 10(5) sec-1 M-1, but had no effect on SP. Chymase cleaved both SP and VIP at primarily a single site with kcat/Km of 3.9 X 10(4) and 5.4 X 10(4) sec-1 M-1, respectively. Thus, these data show that mast cell proteases degrade SP and VIP. The differences in peptidase activity between tryptase and chymase suggest that the consequences of protease release could vary according to mast cell protease phenotype and location in various tissues and species. Tryptase, by cleaving the bronchodilator VIP but not the bronchoconstrictor SP, might promote bronchial hyper-responsiveness in asthma by decreasing the nonadrenergic neural inhibitory influence mediated by VIP. In skin and other tissues, chymase might interrupt axon reflex-mediated neurogenic inflammation by cleaving SP.

MK-801, a proposed noncompetitive antagonist of excitatory amino acid neurotransmission, produces phencyclidine-like behavioral effects in pigeons, rats and rhesus monkeys.

Abstract: The behavioral effects of MK-801 [(+)-5-methyl-10,11-dihydroxy-5H-dibenzo(a,d)cyclohepten-5,10-imin e], a proposed noncompetitive N-methyl-D-aspartate (NMDA) antagonist, were compared to those of phencyclidine (PCP). In pigeons, MK-801 produced PCP-like catalepsy (i.e., loss of righting without eye closure and without muscle relaxation) and PCP-like discriminative stimulus effects. In rats, MK-801 produced PCP-like behavior (i.e., locomotion, sniffing, swaying and falling). In rhesus monkeys, like PCP, MK-801 produced 1) ketamine-like discriminative stimulus effects, 2) positive reinforcing effects and 3) ketamine-like anesthetic effects (i.e., anesthesia without eye closure and without respiratory depression, but with profuse salivation and with some muscle relaxation). Thus, MK-801 produced PCP-like behavioral effects in each species and with each procedure. MK-801 was 2 to 10 times more potent than PCP, depending on the effect measured and the species tested. Because MK-801 has been shown to have NMDA-antagonist properties, the findings of this study offer further support for the hypothesis that certain behavioral effects of PCP-like drugs may result from a reduction of neurotransmission at excitatory synapses utilizing NMDA-preferring receptors. The behavioral similarities between MK-801 and PCP make it relevant to evaluate PCP-like activity in clinical trials of MK-801.

Alpha-2A and alpha-2B adrenergic receptor subtypes: antagonist binding in tissues and cell lines containing only one subtype.

Abstract: The affinities of 34 adrenergic antagonists for alpha-2 adrenergic receptors were determined from homogenate radioligand binding studies using [3H]yohimbine and [3H]rauwolscine. It has been suggested that alpha-2 adrenergic receptors can be subdivided into alpha-2A and alpha-2B subtypes. Oxymetazoline is selective for alpha-2A receptors, whereas prazosin is alpha-2B selective. Five different tissues were used, each of which has only one of the two subtypes: human platelet (alpha-2A), HT29 cell line (alpha-2A), human cerebral cortex (alpha-2A), neonatal rat lung (alpha-2B), and NG108-15 cell line (alpha-2B). The drug affinities were highly correlated when alpha-2A tissues were compared with alpha-2A tissues (r = 0.97 to 0.98) or when the two alpha-2B tissues were compared (r = 0.99). By contrast, comparison of an alpha-2A tissue with an alpha-2B tissue resulted in poor correlations (r = 0.77 to -0.87). Three new subtype selective drugs were identified among these drugs on the basis of at least a 10-fold greater affinity for one subtype. All three were selective for the alpha-2B subtype: ARC-239 (100-fold selective), chlorpromazine (18-fold selective), and 7-hydroxychlorpromazine (17-fold selective). These studies, by demonstrating distinct pharmacological profiles for the two alpha-2 adrenergic receptor subtypes in several different tissues, further support the existence and definition of these subtypes. The identification of a cell line for each subtype should be useful in the further study of alpha-2 adrenergic receptor subtypes.

Behavioral and neurochemical effects of acute and daily cocaine administration in rats.

Abstract: Daily cocaine injection into rodents produces a progressive increase in the motor stimulant effect of acute cocaine administration. In this study it was found that daily cocaine injection (15 mg/kg i.p. x 3 days) produced an enhanced motor stimulant response to acute cocaine injection. The behavioral augmentation was linear with regards to dose in horizontal activity and behavioral intensity rating, but was biphasic in vertical activity. Augmented vertical, but not horizontal, activity in response to acute cocaine was found to persist for 2 weeks after the last daily injection of cocaine. Acute injection of cocaine was found to significantly decrease the level of dopamine (DA) metabolites in the nucleus accumbens, striatum and A10 DA region. In rats pretreated with daily injections of cocaine (15 mg/kg i.p. x 3 days), an acute challenge of cocaine 14 days after the last daily injection produced a more consistent decrease in DA metabolites in the nucleus accumbens, striatum and prefrontal cortex compared to daily saline-pretreated rats. In contrast, daily cocaine treatment abolished the decrease in DA metabolites produced in the A10 region by an acute cocaine challenge. Acute injection with cocaine was found to significantly depress dopa accumulation in the A10 region, nucleus accumbens and striatum. This effect was abolished in the A10 region in rats pretreated 14 days previously with daily injections of cocaine (7.5, 15.0 or 30 mg/kg i.p. x 3 days), but remained intact in the nucleus accumbens and striatum, except after daily pretreatment with the highest dose of cocaine.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in cytosolic calcium level in vascular smooth muscle strip measured simultaneously with contraction using fluorescent calcium indicator fura 2.

Abstract: In rat aortic strips, muscle contraction was recorded simultaneously with cytosolic Ca++ level, which was indicated by the 500 nm fluorescence of Ca++ indicator, fura 2, due to excitation at either 340 nm (F340) or 380 nm (F380) and the ratio of F340 to F380 (R340/380). On the addition of 72.7 nM K+ or 1 microM norepinephrine, muscle contraction followed the increase in R340/380 (resulted from the increased F340 and the decreased F380). Cytosolic Ca++ concentrations of resting, 72.7 mM K+-stimulated and 1 microM norepinephrine-stimulated aortas were tentatively calculated as 228 +/- 25, 1784 +/- 154 and 1528 +/- 180 nM, respectively. Cumulative addition of K+ or norepinephrine induced concentration-dependent increase in both muscle tension and R340/380. However, norepinephrine induced greater contraction than K+ when both of these stimulants induced similar increase in R340/380. Addition of 10 mM tetraethylammonium and 1 microM Bay k8644 caused rhythmic contractions which followed the rhythmic changes in R340/380. EGTA decreased the muscle contraction and decreased R340/380. In Ca++-free solution, addition of 10 microM norepinephrine or 20 mM caffeine induced transient increase in both muscle tension and R340/380. Tension changes always were preceded by the fluorescent changes. Verapamil (10 microM) decreased both tension development and R340/380 in high K+- and norepinephrine-stimulated tissues. Sodium nitroprusside (1 microM) also decreased both tension and R340/380 in norepinephrine-stimulated tissues, whereas it decreased tension more strongly than R340/380 in high K+-stimulated tissues. These results indicate that vasoconstrictors and vasodilators may modulate smooth muscle contraction by changing the cytosolic Ca++ concentrations and also by changing the sensitivity of contractile elements to Ca++.

Pharmacology and stereoselectivity of structurally novel cannabinoids in mice.

Abstract: The pharmacological effects of three stereoisomeric pairs of structurally novel cannabinoids were tested after i.v. administration in mice for depression of spontaneous activity and the production of hypothermia, antinociception and catalepsy. The (-)-enantiomers were as much as 770 times more potent than delta 9-6a,10a-trans-tetrahydrocannabinol and were 7 to 2000 times more potent than their respective (+)-enantiomers. The order of potency for cannabinoid-induced effects was spontaneous activity greater than antinociception greater than hypothermia greater than or equal to catalepsy. Levonantradol was active between 0.123 to 1.5 mg/kg, whereas dextronantradol, its (+)-enantiomer was inactive. (-)-CP 55,244 and (-)-CP55,940 analogs which lack the dihydropyran ring were 5 to 775 times more potent than delta 9-6a,10a-trans-tetrahydrocannabinol and 30 to 2000 times more potent than their respective (+)-enantiomers. Some separation of effects was demonstrated with (+)-CP 55,243 and (-)-CP 56,667 which were inactive in producing hypothermia and catalepsy but were active in the spontaneous activity and tail-flick procedures. The high degree of enantioselectivity and potency of these nonclassical cannabinoids are indicative of a highly specific mechanism of action such as a receptor.

Acute and chronic opiate-regulation of adenylate cyclase in brain: specific effects in locus coeruleus.

Abstract: Acutely, morphine and D-ala2-D-leu-enkephalin (DADLE) inhibited adenylate cyclase in vitro in locus coeruleus (LC), dorsal raphe, frontal cortex and neostriatum and the inhibition by each agonist was blocked by the opiate-receptor antagonist naloxone. Although morphine was equally efficacious in the four brain regions examined (10-15% inhibition), DADLE inhibited cyclic AMP (cAMP) production to a greater extent in cortex and striatum (20-25% inhibition). Pertussis toxin treatment in vitro significantly reduced DADLE-inhibition of adenylate cyclase in all brain areas, indicating that this opiate response is mediated by a pertussis toxin-sensitive G-protein (i.e., Gi and/or Go). Chronic (in vivo) administration of morphine pellets for 5 days, treatment known to induce opiate tolerance and dependence, increased basal, GTP- and forskolin-stimulated adenylate cyclase in the LC, but not in the other three brain regions studied. DADLE was found to inhibit cAMP production in LC in vitro to the same extent in control and morphine-treated rats, suggesting a lack of opiate receptor tolerance. The morphine-induced increase in adenylate cyclase required chronic exposure to the opiate, as shorter treatment times, namely 2 hr and 1 day, failed to produce this effect. In fact, at 2 hr a small decrease in adenylate cyclase in the LC was observed that did not appear to be due to morphine being retained in the membrane fraction. Taken together, the findings of this study provide support for the view that changes in the cAMP system in the LC play a role in mediating acute opiate action as well as in underlying the development of opiate tolerance, dependence and/or withdrawal.

Ethanol potentiation of GABAergic transmission in cultured spinal cord neurons involves gamma-aminobutyric acidA-gated chloride channels.

Abstract: The interaction of ethanol with gamma-aminobutyric acid (GABA)-mediated 36-Cl-influx and its modulation by various drugs was investigated in C57 mice spinal cord cultured neurons. Ethanol (5-100 mM) potentiated the effect of GABA on 36Cl-influx; whereas at concentrations greater than or equal to 50 mM ethanol activated Cl- channels directly. The effect of ethanol was specific for GABAA receptor-gated Cl- channels, as ethanol did not potentiate glycine-induced 36Cl-influx in the same neurons. Both the enhancing and direct effects of ethanol on 36Cl-influx were blocked by GABA antagonists like bicuculline, picrotoxinin and inverse agonists of the benzodiazepine site like the imidazodiazepine R015-4513 (ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo [1,5 alpha], [1,4]benzodiazepine-3-carboxylate) and N-methyl-beta-carboline-3-carboxamide (FG-7142). Ethanol potentiating effect of GABA-induced 36Cl-influx was also reversed by methyl-6,7-dimethyl-4-ethyl-beta-carboline-3-carboxylate. The effects of the inverse agonists were blocked by the benzodiazepine receptor antagonist R015-1788. Both R015-4513 and FG-7142 reversed direct and GABA potentiating effects of ethanol effect at concentrations lower than those that exhibit inverse agonistic activity in the 36Cl-influx assay in cultured neurons. These results suggest that ethanol facilitation of GABAAergic transmission involves GABA receptor-gated Cl- channels and that this interaction may be responsible for some of the pharmacological effects of ethanol.

Pharmacological evidence that endothelium-derived relaxing factor is nitric oxide: use of pyrogallol and superoxide dismutase to study endothelium-dependent and nitric oxide-elicited vascular smooth muscle relaxation.

Abstract: The principal objective of this study was to elucidate the influence of superoxide anion on both endothelium-dependent arterial relaxation elicited by acetylcholine and endothelium-independent arterial relaxation produced by nitric oxide (NO). Pyrogallol was used to generate superoxide in the oxygenated bathing medium, and superoxide dismutase was used to scavenge superoxide. Pyrogallol caused endothelium-dependent contractions of bovine intrapulmonary arterial and venous smooth muscle after precontraction of muscle by phenylephrine. Acetylcholine- and NO-elicited arterial relaxations were promptly converted to marked contractions upon addition of pyrogallol. Moreover, pyrogallol markedly inhibited the development of arterial relaxant responses to acetylcholine and NO. However, isoproterenol- and glyceryl trinitrate-elicited arterial relaxations were unaffected by pyrogallol. Both pyrogallol and oxyhemoglobin enhanced arterial contractile responsiveness to phenylephrine in an endothelium-dependent manner, whereas indomethacin was without effect. Similarly, both pyrogallol and oxyhemoglobin inhibited acetylcholine- and NO-elicited arterial cyclic GMP accumulation, whereas indomethacin was without effect. Uncontracted arterial rings maintained under tension showed endothelium-dependent contraction and decreased cyclic GMP levels in response to oxyhemoglobin but not pyrogallol. Superoxide dismutase enhanced arterial relaxation and cyclic GMP accumulation in response to both acetylcholine and NO. Using a bioassay superfusion cascade system in which intact perfused artery was the source of endothelium-derived relaxing factor (EDRF) and three endothelium-denuded arterial strips mounted in series served as the detector of EDRF, superfusion of strips with pyrogallol blocked relaxation caused by perfusion of artery with acetylcholine. Superoxide dismutase enhance the relaxations produced by arterial perfusion with acetylcholine and prevented the effects of pyrogallol.

Ifenprodil and SL 82.0715 as cerebral anti-ischemic agents. I. Evidence for efficacy in models of focal cerebral ischemia.

Abstract: Recent studies have strongly implicated the excitatory neurotransmitter glutamate in the cascade of pathological mechanisms that cause neuronal loss after certain types of brain ischemia. The neurotoxic effects of glutamate are mediated, at least in global ischemia, via NMDA receptors. In the present study we have examined the effects of compounds that possess NMDA receptor antagonist properties (ifenprodil, SL 82.0715 [(+/-)-alpha-(4-chlorophenyl)-4-[(4-fluorophenyl)methyl]- 1-piperidineethanol] and 1-[1-(2-thienyl)cyclohexyl]piperidine) on the histological consequences of focal, as opposed to global, cerebral ischemia in both the rat and the cat. Ifenprodil (0.3-3 mg/kg i.v.) administered as a perfusion over 3 hr after occlusion of the feline middle cerebral artery reduced the volume of infarcted tissue (measured 4 days after occlusion) in a dose-related manner. At the highest dose a 42% reduction of infarcted volume was noted, essentially in cortical tissue. In an identical protocol, a derivative of ifenprodil, SL 82.0715, reduced the volume of infarction in a manner comparable to that described for ifenprodil. As SL 82.0715 possesses better p.o. bioavailability, this compound was also evaluated in the rat, again after middle cerebral artery occlusion. First administered 30 min after the induction of ischemia, SL 82.0715 (1 and 10 mg/kg p.o.) reduced infarction volume by 34 and 48%, respectively. The quantitative histology was performed 2 days after middle cerebral artery occlusion. The noncompetitive receptor antagonist, 1-[1-(2-thienyl)cyclohexyl]piperidine, administered (1 mg/kg i.p.) before the induction of focal ischemia, similarly and significantly decreased the final volume of infarction. As both ifenprodil and SL 82.0715 are noncompetitive antagonists of the NMDA receptor, two conclusions may be drawn from the present investigation. First, NMDA antagonism by ifenprodil and its derivative is an effective approach for tissue sparing in animal models of stroke and brain infarction. Second, these pharmacological observations provide evidence for the involvement of excitatory amino-acid induced-neurotoxicity in the evolution and consequences of focal cerebral ischemia.

Release-regulating D-2 dopamine receptors are located on striatal glutamatergic nerve terminals.

Abstract: The effects of dopamine (DA) and other dopaminergic receptor agonists on the depolarization-evoked release of endogenous glutamic acid (GLU) have been studied using synaptosomes prepared from rat corpus striatum and depolarized in superfusion with 15 mM KCl DA and the selective D-2 receptor agonists quinpirole (LY-171555, the levorotatory enantiomer of LY-141865) and pergolide inhibited GLU release in a concentration-dependent way The natural agonist was particularly effective causing 50% inhibition of GLU release at 10 nM In contrast, the selective D-1 receptor agonist SK&F 38393 did not affect the release of GLU The inhibitory effect of DA on the K+-evoked release of GLU was antagonized in a concentration-dependent manner by the selective D-2 receptor antagonist S-sulpiride, but not by the R-enantiomer The data represent a direct demonstration that receptors sensitive to nanomolar concentrations of DA and belonging to the D-2 type are located on GLU axon terminals in the rat corpus striatum where they may modulate the release of GLU from glutamatergic afferents including the cortico-striatal pathway

Buprenorphine: dose-related blockade of opioid challenge effects in opioid dependent humans.

Abstract: This study assessed the blockade of hydromorphone challenge effects (cumulative s.c. doses of 0, 6 and 18 mg) during chronic buprenorphine treatment of opioid dependent subjects. Buprenorphine was administered daily via the sublingual route in ascending doses of 2, 4, 8 and 16 mg. Hydromorphone challenges were conducted after 10 to 14 days of chronic administration of each buprenorphine dose and 24 h after the last buprenorphrine dose. Buprenorphrine itself only produced dose-related effects in respiration rate. While subjects were maintained on 2 mg of buprenorphine, the hydromorphone challenge produced dose-related changes in physiological and self-report measures. As the dose of buprenorphine was increased, hydromorphone effects on self-report measures were attenuated to a greater extent than was noted for pupil diameter. The results of this study confirm previous reports of buprenorphine blockade of opioid agonist effects in humans and extent those findings by demonstrating both a buprenorphine dose-effect function and a dissociation between the potency of buprenorphine's opioid blockade for self-report vs. physiological measures.

CGS 19755, a selective and competitive N-methyl-D-aspartate-type excitatory amino acid receptor antagonist.

Abstract: CGS 19755 (cis-4-phosphonomethyl-2-piperidine carboxylic acid) was found to be a potent, stereospecific inhibitor of N-methyl-D-aspartate (NMDA)-evoked, but not KCl-evoked, [3H] acetylcholine release from slices of the rat striatum. The concentration-response curve to NMDA was shifted to the right by CGS 19755 (pA2 = 5.94), suggesting a competitive interaction with NMDA-type receptors. CGS 19755 inhibited the binding of [3H]-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid to NMDA-type receptors with an IC50 of 50 nM, making it the most potent NMDA-type receptor antagonist reported to date. CGS 19755 failed to interact with 23 other receptor types as assessed by receptor binding, including the quisqualate- and kainate-type excitatory amino acid receptors. In crude P2 fractions, no evidence was obtained to suggest that CGS 19755 is taken up by an active transport system. Furthermore, CGS 19755 failed to affect the uptake of L-[3H]glutamate, or to interact with aconitine-induced inhibition of L-[3H]glutamate uptake, the latter finding suggesting a lack of membrane-stabilizing or local anesthetic properties. CGS 19755 selectively antagonized the excitatory effect of iontophoretically applied NMDA in the red nucleus of the rat without affecting the excitatory effects of quisqualate. CGS 19755 blocked the harmaline-induced increase in cerebellar cyclic GMP levels at a dose of 4 mg/kg i.p. with a duration of action exceeding 2 hr. CGS 19755 inhibited convulsions elicited by maximal electroshock in rat (ED50 = 3.8 mg/kg i.p. 1 hr after administration) and in mouse (ED50 = 2.0 mg/kg i.p. 0.5 hr after administration). Likewise, convulsions elicited by picrotoxin were inhibited by CGS 19755, whereas the compound was relatively weak in protecting against convulsions elicited by pentylenetetrazole or strychnine. CGS 19755 produced retention performance deficits in a dark avoidance task. However, CGS 19755 did not show a unique propensity for learning and memory disruption compared to other anticonvulsants.

Luzindole (N-0774) : a novel melatonin receptor antagonist

Abstract: The pharmacological potencies of 2-substituted N-acetyltryptamines were determined on the presynaptic melatonin receptor site of rabbit retina labeled in vitro with [3H]dopamine. Calcium-dependent release of [3H]dopamine was elicited by electrical stimulation at 3 Hz for 2 min (20 mA, 2 msec). Melatonin (5-OCH3-N-acetyltryptamine) and 6-chloromelatonin were equipotent in inhibiting the calcium-dependent release of [3H]dopamine (IC50 = 40 pM). 2-Substituted N-acetyltryptamines with a methyl (i.e., 6,7-dichloro-2-methylmelatonin, IC50 = 10 pM) or iodine (i.e., 2-iodomelatonin, IC50 = 5 pM) group were more potent than melatonin in inhibiting [3H]dopamine release. I report here the pharmacological properties of the novel N-acetyltryptamine, 2-benzyl-N-acetyltryptamine (N-0774, luzindole) on the presynaptic melatonin receptor of rabbit retina. Luzindole (0.1-10 microM) did not affect the spontaneous outflow of radioactivity or the stimulation-evoked release of [3H]dopamine when added alone. However, luzindole (0.1-10 microM) shifted the concentration effect curve for melatonin to the right in a parallel fashion. The pA2 extrapolated from the Schild plot (slope, 0.91) was 7.7, with a KB = 20 nM. The dissociation constants for luzindole (KB), determined in the presence of 6,7-dichloro-2-methylmelatonin (10 pM-1 nM) or 6-chloromelatonin (10 pM-100 nM) were 16 and 40 nM, respectively. These data suggest that luzindole and the various melatonin agonists are competing for the same presynaptic melatonin receptor site in the rabbit retina.(ABSTRACT TRUNCATED AT 250 WORDS)

LY 83583 interferes with the release of endothelium-derived relaxing factor and inhibits soluble guanylate cyclase.

Abstract: LY 83583 (6-anilino-5,8-quinolinedione) has been reported to lower intracellular cyclic GMP by an unknown mechanism. The objective of the present study was to investigate the effect of LY 83583 on different types of vasorelaxation and to study its mechanism of action. Low concentrations of LY 83583 (less than or equal to 0.1 microM) inhibited endothelium-dependent relaxations of rabbit aortic strips induced by acetylcholine or by the calcium ionophore A23187. Higher concentrations (greater than or equal to 0.3 microM) were required to produce partial inhibition of relaxation to sodium nitroprusside and glyceryl trinitrate. Cyclic AMP-mediated relaxations, induced by isoprenaline or forskolin, were not affected by LY 83583 (10 microM). The site of interference of LY 83583 with endothelium-dependent relaxation was examined with endothelium-derived relaxing factor (EDRF) released from cultured endothelial cells that were grown on microcarrier beads and stimulated by superfusion with ATP or thimerosal. EDRF in the superfusate was detected by endothelium-denuded segments of rabbit femoral artery, which responded with dilation and, simultaneously, by purified soluble guanylate cyclase (GC) in test tubes, which was activated by EDRF. When LY 83583 was added to the glutathione-containing GC-assay or to the superfusate from cultured endothelial cells, it did not affect stimulation of soluble GC by EDRF but it slowly reversed the dilator response of the arterial detector segment. Superfusion of cultured endothelial cells with LY 83583 (1 microM), rapidly and reversibly inhibited EDRF release.(ABSTRACT TRUNCATED AT 250 WORDS)

Chronic benzodiazepine administration. I. Tolerance is associated with benzodiazepine receptor downregulation and decreased gamma-aminobutyric acidA receptor function.

Abstract: Tolerance occurs to a number of the pharmacodynamic effects of benzodiazepines. To assess pharmacokinetic and neurochemical aspects of tolerance, lorazepam (LRZ) was administered chronically to mice via implantable osmotic pumps and rotarod ataxia, plasma and brain LRZ concentrations, benzodiazepine receptor binding in vivo and in vitro, chloride channel binding and muscimol-stimulated chloride uptake were examined in various brain regions over a 14-day period. Behavioral tolerance, indicated by diminished rotarod ataxia, developed at all doses examined (1, 2, 4 and 10 mg/kg/day), with little change occurring before day 4. The greatest decrease in rotarod ataxia occurred between days 4 and 7. Plasma and brain LRZ concentrations were proportional to dose and were constant over time at each dose, indicating that tolerance was not pharmacokinetic. Benzodiazepine receptor binding as determined by the specific uptake of [3H]Ro15-1788 decreased in cortex, hypothalamus and hippocampus primarily between days 4 and 7, with an approximately 50% decrement in each region by day 7. Receptor binding and rotarod ataxia in cortex were highly correlated at each dose. Apparent affinity in vivo at the receptor was unchanged in cortex, indicating that altered ligand uptake was due to decreased receptor number. Similar results were observed in membrane preparations. There was a small, nonsignificant decrease in chloride channel binding at day 7 compared to day 1. Muscimol-stimulated chloride uptake into cortical synaptoneurosomes was decreased at day 7 compared to day 1. Thus, downregulation of benzodiazepine receptor binding and of gamma-aminobutyric acidA receptor function is closely associated with behavioral tolerance to benzodiazepines.

Differential establishment and maintenance of oral ethanol reinforced behavior in Lewis and Fischer 344 inbred rat strains.

Abstract: Oral ethanol self-administration was investigated systematically in two inbred strains of rats, Fischer 344 CDF (F-344)/CRLBR (F344) and Lewis LEW/CRLBR (LEW). For both strains ethanol maintained higher response rates and was consumed in larger volumes than the water vehicle. In addition, blood ethanol levels increased with increases in ethanol concentration. However, LEW rats drank substantially more ethanol than F344 rats. The typical inverted U-shaped function between ethanol concentration and number of deliveries was observed for the LEW rats, whereas for the F344 rats much smaller differences were seen between ethanol and water maintained responding. For the LEW strain, as the fixed-ratio size was increased, the number of responses increased almost in direct proportion to the fixed-ratio size increase, so that at least at the lower fixed-ratio values the rats were obtaining similar numbers of deliveries at different fixed-ratio sizes. However, a decrease in ethanol deliveries and blood ethanol levels was observed at higher fixed-ratio sizes. Similar results were obtained in F344 rats, but the amount of responding was lower and less consistent. LEW rats showed significantly higher response rates, numbers of ethanol deliveries and blood ethanol levels. Ethanol-induced behavioral activation also was observed in LEW rats, but not in F344 rats. These results support the conclusion that ethanol serves as a strong positive reinforcer for LEW rats and as a weak positive reinforcer for F344 rats, and that genotype is a determinant of the degree to which ethanol functions as a reinforcer.

Alterations of central serotonin and dopamine turnover in rats treated with ipsapirone and other 5-hydroxytryptamine1A agonists with potential anxiolytic properties.

Abstract: Measurements of tissue levels of monoamines and their metabolites, and of the rates of 5-hydroxytryptophan and dihydroxy-phenylalanine accumulation after blockade of aromatic amino acid decarboxylase by benserazid indicated that ipsapirone (1-10 mg/kg i.p.) decreased 5-hydroxytryptamine (5-HT) turnover and accelerated dopamine (DA) turnover in various brain regions. The reduced 5-HT turnover probably resulted from the stimulation of 5-HT1A autoreceptors within the anterior raphe nuclei as in vitro tests [( 3H]-8-hydroxy-2-[di-n-propylamino]tetralin binding and adenylate cyclase assays) demonstrated that ipsapirone was a 5-HT1A agonist almost as potent as 8-OH-DPAT, and the same decrease in 5-hydroxytryptophan accumulation could be induced by the i.p. (5 mg/kg) or intraraphe (1 microgram) injection of ipsapirone. Ipsapirone-induced acceleration of DA turnover persisted after the selective degeneration of serotoninergic neurons by intraraphe 5,7-dihydroxytryptamine infusion, and could be reproduced by i.p. administration of other 5-HT1A agonists like buspirone and gepirone, but not 8-OH-DPAT. These results demonstrate that ipsapirone-induced acceleration of DA turnover did not result from the stimulation of 5-HT1A (auto)receptors, but involved additional target(s) of the drug. The possible participation of dopaminergic systems in the "anxiolytic" properties of ipsapirone should deserve further investigations.

Metabolic basis for a drug hypersensitivity: antibodies in sera from patients with halothane hepatitis recognize liver neoantigens that contain the trifluoroacetyl group derived from halothane.

Abstract: Previous studies have demonstrated that antibodies in sera from patients with halothane hepatitis recognize halothane-induced liver microsomal polypeptide neoantigens, and have suggested that these antibodies may play a role in the pathogenesis of the hepatitis. In the present study, the mechanism of neoantigen generation was investigated. Liver microsomes from rats treated in vivo with halothane or deuterated halothane were tested by immunoblotting for reactivity with patients' sera and with an antiserum specific for the covalently bound trifluoroacetyl (TFA) halide metabolite of halothane. Rat liver microsomes incubated aerobically or anaerobically with halothane or deuterated halothane in vitro, +/- NADPH and/or NADH, were also analyzed. The results obtained demonstrate that neoantigen expression involves oxidative halothane metabolism by cytochromes P-450 to TFA halide and covalent binding of the TFA group to the proteins. Incubation of microsomes from halothane-treated rats with 1 M piperidine cleaved the TFA groups from the proteins and abolished antigenicity, confirming this conclusion. Recognition of the neoantigens by the patients' antibodies was inhibited only partially using the hapten derivative N-E-TFA-L-lysine. It appears that the patients' antibodies recognize epitopes consisting of the TFA group plus associated structural features of the protein carriers (100 kDa, 76 kDa, 59 kDa, 57 kDa and 54 kDa), not the TFA hapten alone. To our knowledge, this constitutes the first characterization of drug metabolite-tissue protein neoantigens implicated in a drug hypersensitivity. The approach described may be of general utility for characterization of drug-induced neoantigens associated with other drug hypersensitivities.

Alterations in pulmonary mRNA encoding procollagens, fibronectin and transforming growth factor-beta precede bleomycin-induced pulmonary fibrosis in mice.

Abstract: Female C57Bl/6 mice treated by constant s.c. infusion for 1 week with 100 mg of bleomycin per kg of body weight develop a more pronounced pulmonary fibrosis than BALB/c mice. Within 4 weeks after bleomycin treatment, the pulmonary content of mRNAs encoding fibronectin, alpha 2I procollagen and alpha 1III procollagen was increased. The increases were greater and occurred earlier in C57Bl/6 mice compared to BALB/c mice. Fibronectin mRNA increased 12-fold in C57Bl/6 mice and only 3-fold in BALB/c mice, whereas alpha 1III procollagen mRNA increased 4-fold in C57Bl/6 mice and 2-fold in BALB/c mice. alpha 2I procollagen mRNA was increased only in C57Bl/6 mice (2-fold). The increases were sequential in C57Bl/6 mice: fibronectin mRNA was elevated first, followed by alpha 2I procollagen, then alpha 1III procollagen mRNA. The temporal relationship between these mRNA elevations and extracellular matrix accumulation, and the exaggerated responses in C57Bl/6 mice, suggest that matrix accumulation is a function of the mRNA levels. Transforming growth factor-beta mRNA relative to total polyadenylated RNA was elevated 5-fold in C57Bl/6 mice and depressed 80% in BALB/c mice 1 week after treatment. Early alterations in transforming growth factor-beta mRNA may contribute to murine strain variation in bleomycin-induced pulmonary fibrosis and suggest the involvement of transforming growth factor-beta in this disease.

Pharmacology of the allodynia in rats evoked by high dose intrathecal morphine.

Abstract: Morphine sulfate in doses of 90 to 150 micrograms/3 microliters evoke a prominent behavioral syndrome characterized by 1) periodic bouts of spontaneous agitation during which the rat scratches and bites at the skin of the caudal dermatomes and 2) vigorous agitation, vocalization and coordinated efforts to bite and escape evoked by a light tactile stimulus applied to the flank, suggestive of a pain state (allodynia). The phenomenon is not reversed by naltrexone or is it subject to tolerance. The ordering of activity of an opioid alkaloid related agent in producing this touch-evoked agitation is: noroxymorphone-3-glucuronide, morphine-3-glucuronide, morphine-3-ethereal sulfate, dihydromorphine, noroxymorphone dihydrate, hydromorphone, dihydrocodeine tartrate, morphine sulfate, dihydroisomorphine, morphine-HCl, 6-acetylmorphine, N-normorphine-HCl and (+)-morphine. The following agents were essentially without effect at the highest doses examined: 3,6-diacetylmorphine, N-normeperidine-HCl, nalorphine-HCl, alfentanil, sufentanil, naloxone, naltrexone, methadone, dextrorphan tartrate, meperidine-HCl, oxycodone, levorphanol, oxymorphone, codeine phosphate, thebaine, nalbuphine and naltrexone-3-glucuronide. The observations that the sulfated and conjugated metabolites are 10 to 50 times more potent than their unmetabolized precursor suggest the possibility that, in high concentrations certain phenanthrene opioid alkaloids with a free 3-OH position, an ether bridge and no N-methyl extension will be subject to conjugation and this metabolite will alter the processing of otherwise innocuous tactile stimuli. The fact that the phenomenon appeared at least partially stereospecific may reflect upon the fact that other laboratories have shown that glucuronyl transferase may preferentially convert (-)-morphine to the 3-glucuronide and (+)-morphine to the 6-glucuronide which may be less active.(ABSTRACT TRUNCATED AT 250 WORDS)