Showing papers in "Journal of Pharmacology and Experimental Therapeutics in 1986"

Preferential stimulation of dopamine release in the nucleus accumbens of freely moving rats by ethanol.

Abstract: The effect of the i.p. administration of ethanol on the release of dopamine (DA) and on the output of its main metabolites, dihydroxyphenylacetic acid and homovanillic acid, was estimated in the rat by transcerebral dialysis of two terminal dopaminergic areas, the nucleus accumbens and the dorsal caudate. Low doses of ethanol (0.25-0.5 g/kg i.p.) stimulated DA release specifically in the n. accumbens and elicited pure behavioral stimulation. Higher doses of ethanol (1.0-2.5 g/kg) elicited sedation and hypnosis and stimulated further DA release and dihydroxyphenylacetic acid and homovanillic acid output in the accumbens and, although less, also in the caudate. High doses of ethanol (5 g/kg i.p.) elicited long-lasting hypnosis and sedation and induced a depression followed by stimulation of DA release in the accumbens. DA release in the caudate was stimulated further. Low doses of apomorphine (0.05 mg/kg s.c.) reversed completely the stimulant effect of 0.5 g/kg of ethanol on behavior and on DA release in the accumbens. Moreover, the stimulation of behavior and of DA release in the accumbens elicited by 0.5 g/kg of ethanol were abolished completely by pretreatment with 700 mg/kg of gamma-butyrolactone, an agent which blocks DA firing and DA release. The results indicate that ethanol preferentially stimulates DA transmission in the mesolimbic system probably by activating the firing activity of mesolimbic DA neurons and provide direct evidence that these changes are involved in the motor stimulant effects of ethanol.

Characterization of the 5-hydroxytryptamine1a receptor-mediated inhibition of forskolin-stimulated adenylate cyclase activity in guinea pig and rat hippocampal membranes.

Abstract: The inhibition of forskolin-stimulated adenylate cyclase activity by 5-hydroxytryptamine (5-HT) receptor agonists was measured in guinea pig and rat hippocampal membranes. The results were consistent with the inhibition being mediated by a single, homogeneous population of receptors. In guinea pig hippocampal membranes 8-hydroxy-2-(di-n-propylamino)tetralin, d-lysergic acid diethylamide, 5-HT and buspirone were potent in inhibiting forskolin-stimulated adenylate cyclase activity, with EC50 values of 18, 24, 53 and 146 nM, respectively. Spiperone (Kb = 26 nM) and methiothepin (Kb = 13 nM) were potent competitive antagonists at this receptor whereas ketanserin, a high affinity 5-HT2 receptor ligand, and ICS 205-930, a high affinity peripheral neuronal (M) receptor ligand, were not. In rat hippocampal membranes, 8-hydroxy-2-(di-n-propylamino)tetralin, d-lysergic acid diethylamide, 5-HT and buspirone were potent agonists and exhibited the same rank order of potency as in guinea pig hippocampal membranes. The maximal percentage of inhibition by buspirone was significantly less than the maximal percentage of inhibition by 5-HT in rat membranes, suggesting that it is a partial agonist at this receptor, with an intrinsic activity relative to 5-HT of 0.5. The concentration-response data show that the inhibition of forskolin-stimulated adenylate cyclase activity in guinea pig and rat hippocampal membranes is mediated by a receptor with the characteristics of the 5-HT1A binding site. We propose that the inhibition of adenylate cyclase activity is a functional correlate of this binding site. This response is suitable for measuring activities and affinities of drugs acting at 5-HT1A receptors.

Activation of purified soluble guanylate cyclase by endothelium-derived relaxing factor from intrapulmonary artery and vein: stimulation by acetylcholine, bradykinin and arachidonic acid.

Abstract: The objective of this study was to ascertain whether "endothelium-derived relaxing factor" (EDRF) released from bovine intrapulmonary artery and vein is capable of directly activating soluble guanylate cyclase, thereby accounting for elevated vascular levels of cyclic GMP during EDRF release. Isolated arterial and venous rings, after equilibration and depolarization in bath chambers, were transferred to reaction tubes and incubated with soluble guanylate cyclase that had been purified to homogeneity from bovine lung. Addition of test agents to either bath chambers or enzyme reaction mixtures enabled the determination of their sites of action. Arterial and venous rings caused an endothelium-dependent 2- to 3-fold enzyme activation that was inhibited by methylene blue. Endothelium-dependent enzyme activation in artery but not vein was enhanced several-fold by acetylcholine in an atropine-sensitive manner. Bradykinin, which relaxes both artery and vein when endothelium is intact, activated guanylate cyclase upon addition of endothelium-intact rings to enzyme reaction mixtures. Vasoactive intestinal peptide, which causes endothelium-dependent relaxation of artery but not vein, also activated guanylate cyclase in the presence of endothelium-intact artery but not vein. Arachidonic acid activated the enzyme directly as well as through EDRF release from artery but not vein. Atrial peptides, prostacyclin, isoproterenol and nitroglycerin were inactive. Methylene blue was a powerful inhibitor of EDRF-elicited activation of guanylate cyclase but was without effect when rings were merely pretreated with methylene blue in bath chambers with no further addition to enzyme reaction mixtures. Thus, methylene blue did not interfere with the formation, release or chemical stability of EDRF, but rather inhibited its influence on guanylate cyclase. No agent was found to inhibit EDRF generation or release.(ABSTRACT TRUNCATED AT 250 WORDS)

Depression of contractile responses in rat aorta by spontaneously released endothelium-derived relaxing factor.

Abstract: Removal of endothelial cells on rings of rat aorta increased the sensitivity to the selective alpha-1 adrenoceptor agonist phenylephrine, to the nonselective alpha adrenoceptor agonist norepinephrine and to the selective alpha-2 adrenoceptor agonist clonidine. In the case of the first two, which are strong agonists for the alpha-1 adrenoceptor-mediating contraction, removal of endothelium increased sensitivity 4- and 6-fold at the EC30 level, but produced little or no increase in maximum. In the case of clonidine, a partial agonist for the alpha-1 adrenoceptor, which gave only about 15% of the maximum given by phenylephrine on endothelium-containing rings, removal of the endothelium not only shifted the curve to the left but also increased the maximum to about 50% of that given by phenylephrine. The depression of sensitivity to these agonists in rings with endothelium appeared to be due to the vasodepressor action of endothelium-derived relaxing factor (EDRF), as hemoglobin, a specific blocking agent of EDRF, abolished this depression. It is unlikely that the endothelium-dependent depression was due to stimulation of release of EDRF, because clonidine did not produce endothelium-dependent relaxation in precontracted rings even when its contractile action was blocked by the alpha-1 adrenoceptor antagonist prazosin. Further evidence against alpha adrenoceptor agents stimulating release of EDRF was that neither phenylephrine nor clonidine induced a rise in cyclic GMP in aortic rings, whereas acetylcholine, which does release EDRF, caused a large rise in cyclic GMP content. The possibility that the muscle cells of intact rat aortic rings were under the tonic influence of released EDRF was supported by the finding that, in the absence of any contractile agent, hemoglobin induced a fall in the basal level of cyclic GMP in endothelium-containing rings. Also consistent with EDRF being released spontaneously was the finding that contraction induced by 5-hydroxytryptamine, like that by alpha-adrenergic agonists, was also depressed in endothelium-containing rings of aorta. When the efficacy of phenylephrine as an alpha-1 agonist was reduced to about the initial efficacy of clonidine by irreversible inactivation of a very large fraction of alpha-1 adrenoceptors of the smooth muscle cells by pretreatment with dibenamine, the concentration-contraction curves for phenylephrine for both endothelium-containing rings and for endothelium-denuded rings now became very similar to the corresponding curves obtained for clonidine before receptor inactivation.(ABSTRACT TRUNCATED AT 400 WORDS)

Pharmacological and autoradiographic discrimination of sigma and phencyclidine receptor binding sites in brain with (+)-[3H]SKF 10,047, (+)-[3H]-3-[3-hydroxyphenyl]-N-(1-propyl)piperidine and [3H]-1-[1-(2-thienyl)cyclohexyl]piperidine.

Abstract: The benzomorphan opioid, SKF 10,047, is the prototypical agonist for the sigma receptor. In this study, pharmacological and autoradiographic analyses reveal that (+)-[3H]SKF 10,047 labels two sites in brain: a high affinity site resembling the sigma receptor and a second site, labeled with lower affinity by (+)-[3H] SKF 10,047, similar to the phencyclidine (PCP) receptor. The drug specificity of the high affinity site for (+)-[3H]SKF 10,047 resembles that of the putative sigma receptor labeled with (+)-[3H]-3-[3-hydroxyphenyl]-N-(1-propyl)piperidine [(+)-[3H]-3-PPP], being potently inhibited by (+)-3-PPP, haloperidol and (+/-)-pentazocine, and demonstrating stereoselectivity for the (+)-isomer of SKF 10,047. In contrast, these drugs are weak in inhibiting binding of (+)-[3H]SKF 10,047 to the low affinity site, whereas PCP analogs, such as 1-[1-(2-thienyl)cyclohexyl]piperidine (TCP) and 1-[1-(m-aminophenyl)cyclohexyl]piperidine (m-NH2-PCP), are potent inhibitors. No stereoselectivity for the isomers of SKF 10,047 is noted at the low affinity binding site. Autoradiographic localizations of high affinity (+)-[3H]SKF 10,047 binding sites closely resemble those of (+)-[3H]-3-PPP labeled sites with high levels of binding in the hippocampal pyramidal cell layer, hypothalamus, pontine and cranial nerve nuclei and cerebellum. By contrast, low affinity (+)-[3H]SKF 10,047 sites are most abundant in nonpyramidal layers of the hippocampus, the cerebral cortex and thalamic nuclei, similar to the distribution of [3H]TCP labeled PCP receptors.

Induction of depolarization block in midbrain dopamine neurons by repeated administration of haloperidol: analysis using in vivo intracellular recording.

Abstract: Acute administration of the antipsychotic drug haloperidol causes an increase in the firing rate and in the proportion of spontaneously active dopamine neurons in the substantia nigra. In contrast, repeated administration of haloperidol for 21 days results in a dramatic decrease in the number of dopamine neurons displaying spontaneous electrophysiological activity. In vivo intracellular recordings revealed that these nonfiring dopamine neurons have a membrane potential significantly more depolarized than dopamine neurons in control rats, although their input resistances were equivalent. Neurons displaying this depolarization block could not be activated by depolarizing current injection, but could be made to fire in response to hyperpolarization of the membrane. Furthermore, administration of autoreceptor-selective doses of the direct acting dopamine agonist apomorphine hyperpolarized inactivated dopamine neurons in treated rats, resulting in initiation of spontaneous activity. Thus, repeated administration of neuroleptics appears to cause a feedback excitatory drive to nigral dopamine neurons of sufficient magnitude to depolarize the membrane beyond the spike generating region. As a result, a tonic depolarization blockade of spontaneous spike activity is induced.

Sodium valproate, but not ethosuximide, produces use- and voltage-dependent limitation of high frequency repetitive firing of action potentials of mouse central neurons in cell culture.

Abstract: Effects of the anticonvulsant drugs sodium valproate (NaVP) and ethosuximide (ES) on mouse central (spinal cord and cortical) neurons in primary dissociated cell culture were studied using intracellular recording techniques. Drug effects on two properties of the neurons were assayed: the ability to sustain high frequency repetitive firing (SRF) of sodium-dependent action potentials, a voltage sensitive nonsynaptic membrane property; and the amplitude of responses to iontophoretically applied gamma-aminobutyric acid (GABA), a postsynaptic effect of this inhibitory amino acid neurotransmitter. At concentrations equivalent to the clinically useful therapeutic range in cerebrospinal fluid (6-200 microM), NaVP limited SRF to a few action potentials in both spinal cord and cortical neurons during long (450 msec) depolarizing current pulses. The limitation of SRF was paralleled by use-dependent reduction of maximal rate of rise (Vmax) of the action potentials and prolongation of recovery of Vmax from inactivation. This action was similar to limitation of SRF produced by phenytoin and carbamazepine. The 2-en-metabolite of NaVP, sodium 2-propyl, 2-pentenoate, did not limit SRF at 12 to 120 microM. However, the diphenyl analog of NaVP, sodium diphenylacetate, limited SRF at concentrations between 4.7 to 23.5 microM. ES did not affect SRF at concentrations up to 700 microM. At concentrations of 120 to 1000 microM, including the upper limit of therapeutic range, NaVP did not affect postsynaptic GABA responses in 80% of spinal cord neurons. In the remaining 20%, GABA responses were augmented less than 33%. ES reduced slightly (22%) GABA responses at a high concentration (700 microM). These findings suggest that limitation of SRF may be an important cellular mechanism by which NaVP, but not ES, exerts anticonvulsant efficacy and that neither ES nor NaVP have anticonvulsant action by enhancing postsynaptic GABA action.

Zolpidem, a novel nonbenzodiazepine hypnotic. I. Neuropharmacological and behavioral effects.

Abstract: Zolpidem [N,N,6-trimethyl-2-(4-methylphenyl)imidazo[1,2-a]pyridine-3-acetamide hemitartrate] is reported to be a rapid onset, short duration hypnotic that interacts at the benzodiazepine recognition site. The present report establishes the neuropsychopharmacological profile of zolpidem and compares it with those of benzodiazepine hypnotics. Although in mice the effects of zolpidem are qualitatively similar to those of midazolam, triazolam and flunitrazepam, sedation with zolpidem occurs at doses 10 and 20 times lower than those inducing anticonvulsant and myorelaxant effects, respectively. In contrast, the benzodiazepines studied induce sedation at doses causing myorelaxation and which are 2 to 6 times superior to those antagonizing pentetrazole-induced convulsions. In the rat, zolpidem induces sleep (as indicated behaviorally and electrocorticographically) and displays anticonflict activity in a punished drinking paradigm, as do the benzodiazepines. However, whereas benzodiazepine hypnotics induce EEG sleep patterns in curarized rats at doses similar or inferior to those active in the conflict test (in freely moving animals), the hypnotic effect of zolpidem is seen at doses 10 times lower than those producing an anticonflict effect. Moreover, a qualitative difference between the effects of zolpidem and benzodiazepines is observed in electrocorticographic recordings obtained in curarized rats: electrocorticographic hypersynchronization induced by zolpidem is dominated by the energy increase within the 2 to 4 Hz band whereas the benzodiazepines increase predominantly energy levels within the 12 to 14 Hz band. Studies of the sleep-wakefulness cycle in the rat and the cat revealed that hypnotic doses of zolpidem do not alter the pattern of physiological sleep, although elevated doses of the drug decrease paradoxical sleep and increase slow wave sleep. In rats trained to discriminate chlordiazepoxide, zolpidem fails to generalize with the chlordiazepoxide-associated lever indicating that the compound and benzodiazepines do not share the same discriminative stimulus properties. Nevertheless, the anticonvulsant, hypnotic, myorelaxant and anticonflict effects of zolpidem are antagonized by benzodiazepine receptor antagonist Ro 15-1788 and CGS 8216 indicating an involvement of the benzodiazepine recognition site in the action of this drug. The highly selective sedative effect of zolpidem (as compared to myorelaxant and anticonvulsant effects) suggests that it may possess a specificity for certain subtypes of benzodiazepine receptors.

Carbamazepine and 10,11-epoxycarbamazepine produce use- and voltage-dependent limitation of rapidly firing action potentials of mouse central neurons in cell culture.

Abstract: Effects of the anticonvulsant, carbamazepine (CBZ), on mouse central neurons in cell culture were examined using intracellular recording techniques. Spinal cord and cortical neurons demonstrated sustained repetitive firing (SRF) of action potentials (APs) at high frequency in response to depolarizing current pulses. Hippocampal neurons fired rapidly only in the pressure of the calcium channel blocker verapamil (1 microM). Concentrations of CBZ equivalent to therapeutic anticonvulsant levels in cerebrospinal fluid (greater than 4.2 microM) limited firing to a few action potentials in all three cell types in parallel with use-dependent reduction of AP maximal rate of rise (Vmax). Resting membrane properties and postsynaptic responses to ionophoretically applied gamma-aminobutyric acid and glutamate were unaffected. The CBZ metabolite, 10,11-epoxycarbamazepine, limited SRF at a concentration (4 microM) encountered at therapeutic CBZ levels. Another major CBZ metabolite, the 10,11-diol, limited SRF only at concentrations greater than those encountered with therapeutic serum CBZ levels. In control medium, APs could be evoked at a maximal rate of 450 Hz while in CBZ (10.6 microM) AP frequency was limited to 100 to 200 Hz. Limitation of SRF was voltage-dependent and could be partially reversed, or prevented, by membrane hyperpolarization. In addition, recovery of Vmax of sodium-dependent action potentials from inactivation was slowed, suggesting an effect on voltage-dependent sodium channels. CBZ and phenytoin effects of SRF were similar. We suggest that limitation of sustained high frequency repetitive firing may be a significant anticonvulsant action of carbamazepine. The 10,11-epoxy derivative of CBZ, but not the 10,11-diol, may contribute to anticonvulsant efficacy by the same mechanism.

Airway hyperresponsiveness induced by platelet-activating factor: role of thromboxane generation.

Abstract: The effect of platelet-activating factor (acetyl glyceryl ether phosphorylcholine; PAF), a potent inflammatory mediator, on airway responsiveness was studied. In six dogs airway responsiveness was determined by measuring the provocative concentration of acetylcholine aerosol that increased total pulmonary resistance (RL) by 5 cm H2O X L-1 X s, before and after inhalation of PAF (1 mg). PAF caused a 2.3-fold increase in RL (P less than .001) that lasted approximately 30 min. Airway hyperresponsiveness was maximal at 3 hr (mean 3.7-fold increase, P less than .001), persisted at 6 hr (P less than .005) and disappeared by 24 hr. Inhalation of 0.9% NaCl had no effect on RL or on responsiveness. PAF caused an 8-fold increase in neutrophil recovery in bronchoalveolar lavage fluid at 3 hr. OKY-046, a thromboxane synthetase inhibitor, inhibited PAF-induced bronchoconstriction and hyperresponsiveness but did not alter the increase in neutrophil recovery. We tested the specificity of the effect of OKY-046 on the release of cyclooxygenase products from canine neutrophils in vitro; OKY-046 suppressed PAF-induced generation of thromboxane and caused a small increase in prostaglandin F2 alpha release. The studies suggest that the airway hyperresponsiveness induced by PAF may depend on thromboxane generation.

Role of glutathione in prevention of acetaminophen-induced hepatotoxicity by N-acetyl-L-cysteine in vivo: studies with N-acetyl-D-cysteine in mice.

Abstract: The revelation that many covalent binding estimates are falsely low due to flawed normalization discloses that protection by N-acetyl-L-cysteine against acetaminophen hepatotoxicity is accompanied routinely by a 50 to 80% decline in arylation. Elevated glutathione may be responsible for inhibiting covalent binding but above-normal concentrations have never been demonstrated in vivo after N-acetyl-L-cysteine treatment or separated adequately from other possible hepatoprotective actions including direct reduction of the toxic acetaminophen metabolite by the antidote. This led us to compare the conventional L-isomer of the antidote to its nonphysiologic stereoisomer, N-acetyl-D-cysteine, because the latter should be capable of reducing the toxic metabolite but not of stimulating glutathione biosynthesis. Oral coadministration of N-acetyl-D-cysteine (1200 mg/kg), however, failed in preventing the elevation of serum alanine aminotransferase activity, in decreasing hepatocellular necrosis, in interdicting covalent binding of the toxic metabolite to hepatocellular proteins and in preventing the depletion of liver glutathione caused by 500 mg/kg of acetaminophen. N-acetyl-L-cysteine succeeded in decreasing these measures of acetaminophen hepatotoxicity while driving liver glutathione concentrations 2-3 fold above control values. The L-isomer also increased urinary excretion of glutathione-derived acetaminophen metabolites whereas the D-isomer increased only acetaminophen sulfate excretion and reversed the customary predominance of acetaminophen cysteine over the mercapturic acid conjugate. Liver uptake of N-acetyl-D-cysteine was reflected in organ concentrations 7-fold higher than noted for the L-isomer.(ABSTRACT TRUNCATED AT 250 WORDS)

Decline in beta adrenergic receptor-mediated vascular relaxation with aging in man.

Abstract: Beta adrenergic relaxation of vascular smooth muscle, mediated by cyclic AMP, is blunted with age in a variety of experimental animals. The applicability of these observations to man is uncertain. The dorsal hand vein technique provides an excellent method to examine the direct effects of aging on vascular responsiveness. Thirty-nine healthy male volunteers over the age range of 19 to 79 were studied. No differences in vascular responsiveness to phenylephrine, an alpha adrenergic agonist, were found for either the ED50 (dose producing 50% vasoconstriction) or Emax (maximum vasoconstriction attained). In marked contrast, vascular relaxation induced by isoproterenol, a beta adrenergic agonist, was significantly different in both the ED50 (dose producing 50% of maximum relaxation from a preconstricted state) and Emax (maximum relaxation attained). ED50 +/- S.E.M. for the youngest and oldest deciles were 8.9 +/- 2.3 and 60 +/- 17.0 ng/min, respectively (P less than .05); Emax +/- S.E.M. were 96.7 +/- 3.3 and 37.7 +/- 8.7%, respectively (P less than .001). Nitroglycerin, a smooth muscle relaxant whose effects are not mediated through the cyclic AMP system, was also used to examine the specificity of this blunted response to isoproterenol. Almost complete relaxation was achieved with the infusion of nitroglycerin in the older group. These results suggest that aging is associated with a specific decrease in beta adrenoreceptor-mediated vascular relaxation.

Characterization of the inhibition of excitatory amino acid-induced neurotransmitter release in the rat striatum by phencyclidine-like drugs.

Abstract: In the present study, the authors found that, in Mg++-free buffer, N-methyl-D-aspartate (NMDA) was able to evoke the Ca++-dependent and tetrodotoxin-sensitive release of striatal acetylcholine (ACh), presumably via interaction with receptors on cholinergic interneurons. In Mg++-free buffer containing pargyline, NMDA also evoked a Ca++-dependent and tetrodotoxin-sensitive release of striatal [3H]dopamine (DA). Phencyclidine (PCP) and physiological concentrations of Mg++ (1.2 mM) also inhibited ACh release evoked by L-glutamate, L-aspartate and DL-homocysteate, but not ACh release evoked by the glutamate analogs quisqualate and kainate, suggesting that PCP is selective for the magnesium-sensitive, NMDA-preferring glutamate-aspartate receptor subtype. Comparison of PCP inhibition of NMDA-stimulated ACh and DA release with that produced by the competitive NMDA antagonist 2-amino-5-phosphonovalerate indicates that PCP is probably not altering release by a direct action on the NMDA recognition site. The ability of 2-amino-5-phosphonovalerate, but not PCP, to prevent desensitization of NMDA-induced ACh release is consistent with this interpretation. Binding studies did, however, reveal a reduction in the apparent affinity of the PCP binding site by high concentrations of NMDA. This may suggest an allosteric link between the PCP-sigma receptor and the NMDA-type glutamate-aspartate receptor. The receptors mediating excitatory amino acid-induced DA release were somewhat less selective than those on cholinergic neurons in their sensitivity to both Mg++ and PCP. Structure-activity-relationship studies suggested that the inhibition off ACh and DA release evoked by NMDA involves biding to the PCP-sigma receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization and autoradiographic localization of multiple tachykinin binding sites in gastrointestinal tract and bladder.

Abstract: Binding sites for the [125I]Bolton-Hunter-labeled tachykinins substance K (BHSK), eledoisin (BHE) and substance P (BHSP) were investigated using crude membrane suspensions and autoradiography. In smooth muscle membranes from guinea-pig small intestine and rat duodenum, specific binding of BHSK was saturable and reversible, showing a single class of sites with a KD of 1 to 3 nM and maximum number of specific binding sites of 1 to 2 fmol/mg of wet weight tissue. Pharmacological characterization of this binding revealed a novel receptor site (K) with affinity for substance K greater than kassinin greater than or equal to eledoisin greater than neuromedin K greater than substance P greater than physalaemin. Inhibition of the binding of BHSK in membranes from mouse urinary bladder exhibited a similar K-type pattern. In rat duodenum and mouse bladder membranes, the binding of BHE was inhibited by substance K greater than kassinin greater than eledoisin greater than neuromedin K greater than substance P greater than physalaemin indicating the same receptor site as for BHSK. On the other hand, in rat cerebral cortex membranes BHE binding was inhibited by neuromedin K = kassinin = eledoisin greater than physalaemin greater than substance K greater than substance P indicating a definitive tachykinin E receptor site. The same displacement pattern of BHE binding was also detected in longitudinal muscle membranes from the guinea-pig small intestine. In mouse bladder membranes and in rat and guinea-pig intestine, the binding of BHSP was inhibited by substance P greater than physalaemin greater than substance K greater than or equal to eledoisin = kassinin greater than neuromedin K indicating a definitive tachykinin P receptor site. Autoradiographic binding sites for both BHSK and BHSP were seen in circular muscle of the rat stomach, small intestine and colon and in circular and longitudinal muscle of the guinea-pig small intestine and colon. Binding sites for BHSK, but not for BHSP, were seen in the muscularis mucosae of the gastric fundus and colon of the rat. Binding sites for BHSP, but not for BHSK, were seen in mucosa of guinea-pig colon and were densely clustered over ganglia of the myenteric and submucous plexuses in rat and guinea-pig colon. The guinea-pig intestine probably contains all three types of tachykinin binding sites whereas rat duodenum and mouse bladder contain only K and P sites. Some tissues classified previously as SP-P or SP-E may actually contain P and/or K sites.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of methadone on human cigarette smoking and subjective ratings.

Abstract: In order to study possible interactions between opioids and cigarette smoking, we examined the effects of oral methadone administration on the smoking behavior of five male methadone-maintenance patients. Isolated subjects smoked their regular brand of cigarettes ad libitum in a naturalistic laboratory environment while reading or watching television. Ninety minutes before each daily 2-hr smoking session subjects received either placebo, dextromethorphan (a taste blind) or one of three doses of methadone, 0.5, 1.0 or 2.0 times their regular maintenance dose (40-60 mg). Each subject received each treatment five times, in a mixed order across days. Methadone pretreatment resulted in a dose-related increase in the number of cigarettes smoked per session (from a mean of 2.8 after placebo to 5.6 after the high dose of methadone). The total time spent puffing during the session increased from a mean of 27 sec after placebo to 74 sec after the high dose of methadone. CO levels in expired air (a measure of actual smoke inhalation) showed corresponding dose-related increases. Methadone administration also resulted in dose-related decreases in pupil diameter and increases in subjective ratings of smoking satisfaction and dose-strength. Dextromethorphan had no significant effects on any measure of smoking behavior or subjective response. The results demonstrate that methadone can produce substantial increases in cigarette smoking and may have implications regarding the proposed role of endogenous opioids in the smoking process.

Sustained coronary vasoconstriction provoked by a peptidergic substance released from endothelial cells in culture.

Abstract: Recent reports have shown that cultured endothelial cells release into the culture medium a substance (or substances) that contracts isolated preparations of arterial smooth muscle [endothelial-derived constrictor factor (EDCF)]. To evaluate the coronary and cardiac effects of EDCF, isolated rabbit hearts retroperfused in a nonrecirculating system with Krebs-Henseleit solution were challenged with bolus injections (100-600 microliter) of either serum-free minimum essential medium (vehicle) or aortic endothelial cell supernates concentrated in minimum essential medium (EDCF). EDCF, but not its vehicle, produced dose-dependent coronary vasoconstriction unaccompanied by changes in left ventricular contraction or heart rate. The constrictor responses were remarkably well sustained with little or no decrement in resistance occurring over a 15-min observation period. Nitroprusside inhibited the development of and reversed on-going vasoconstriction evoked by EDCF. Neither meclofenamate nor diethylcarbamazine influenced EDCF-induced pressor responses, thereby negating a role for arachidonic acid metabolites. Coronary vasoconstriction induced by EDCF also was unaffected by blockade of alpha-adrenergic, histaminergic or serotonergic receptors. Incubation of EDCF with trypsin attenuated the pressor effects markedly, suggesting that EDCF may be a peptide. Roles for angiotensin or substance P were ruled out, however, as saralasin failed to influence EDCF-induced constriction and since substance P was inactive in the perfused rabbit heart preparation. We conclude that a substance (or substances), probably a peptide, released from cultured endothelial cells provokes sustained coronary vasoconstriction by an unknown mechanism.

Inhibition of gastrointestinal transit by morphine in rats results primarily from direct drug action on gut opioid sites.

Abstract: The local intestinal component of the constipating action of morphine was assessed through several integrated approaches in an in vivo animal model. The doses of systemically administered morphine reducing to 50% of drug-free controls (ID50) the small intestinal transit of a charcoal meal fed by gavage to overnight fasted rats were 0.04 and 3.8 mg/kg i.p. and p.o. and 0.5 mg/kg either s.c. or i.v., respectively. Transit inhibition with any of these morphine doses occurred within 10 min and was still measurable 20, 30, 45 and 240 min after i.p., i.v., s.c. and p.o. administration, respectively. Morphine given by any of these systemic routes did not reduce significantly transit in rats receiving the putative peripheral antagonist quaternary naloxone (1 mg/kg i.p., 5 min before morphine) that, unlike naloxone, failed to reverse transit inhibition (to about 50% of drug-free controls) induced by 0.08 mg/kg i.c.v. of morphine. Radioassay of thin-layer chromatograms of extracts of central nervous system, plasma, small intestine and small intestinal longitudinal muscle of rats given tritium-labeled morphine and also tested for gastrointestinal transit, showed that morphine concentrations in the longitudinal muscle (with attached myenteric plexus) after i.v., i.p. and s.c. injection fell within a relatively narrow range and were consistent with the appropriate transit scores. Morphine levels in the central nervous system of the same rats were lower than in any other tissue assayed, presented considerable differences depending on administration routes and did not correlate at all with the corresponding intestinal effects. Morphine administered directly into the rat cerebral ventricles effectively inhibits gastrointestinal transit through an opioid-sensitive central nervous system-located action site.(ABSTRACT TRUNCATED AT 250 WORDS)

Kynurenic acid and quinolinic acid act at N-methyl-D-aspartate receptors in the rat hippocampus.

Abstract: Responses evoked by several amino acid excitants, including the tryptophan metabolite quinolinic acid, were recorded intracellularly from CA1 pyramidal neurons in rat hippocampal slices. Quinolinate, N-methyl-D-aspartate (NMDA), ibotenate and (+/-)-cis-1-amino-1,3-dicarboxycyclopentane produced excitations characterized by burst firing of action potentials, tetrodotoxin-resistant spiking and apparent increases in input resistance measured with brief hyperpolarizing current pulses. L-Glutamate, kainate, quisqualate and (+/-)-2'-amino-3-hydroxy-5-methyl-4-isoxazole-3'-propionate depolarized CA1 pyramidal neurons and induced apparent decreases in input resistance. Quinolinate-, NMDA-, and ibotenate-induced focal depolarizations, but not L-glutamate, kainate- or quisqualate-induced responses, were strongly antagonized by specific NMDA receptor antagonists. The tryptophan metabolite kynurenic acid, at concentrations that antagonized focal depolarizations produced by NMDA, ibotenate and the endogenous excitant quinolinate, did not antagonize quisqualate or L-glutamate responses. In addition to its NMDA-type antagonist action, kynurenate blocked kainate-induced focal depolarizations.

Pharmacological studies on stress-induced increase in frontal cortical dopamine metabolism in the rat.

Abstract: The effects of a variety of minor tranquilizers and of benzodiazepine inverse agonists on the stress-induced increase in frontal cortical dopamine metabolism have been studied in the rat. Electric footshock stress increased 3,4-dihydroxyphenylacetic acid (DOPAC) levels in the frontal (but not parietal) cortex and in the nucleus accumbens but not in the striatum or ventral tegmental area. Similar stress-induced alterations of frontal cortical DOPAC levels were observed after DSP4-induced noradrenergic denervation or after adrenalectomy. Other types of stress, e.g. conditioned fear (exposure to an environment paired previously with footshock) or swim stress also provoked an elevation of DOPAC levels in the prefrontal cortex. When administered systemically, the anxiolytic agents meprobamate, CL 218,872, CGS 9896, suriclone and the hypnotic/anxiolytic drugs zolpidem and zopiclone all prevented the electric footshock stress-induced augmentation of cortical DOPAC levels whereas the gamma-aminobutyric acid receptor agonists progabide, muscimol and depamide or the sedative alpha-1 adrenoceptor antagonist prazosin were ineffective. The preventive effect of diazepam and zolpidem on the stress-induced biochemical response was antagonized by the benzodiazepine antagonist CGS 8216 but not by the gamma-aminobutyric acid receptor antagonist bicuculline. In nonstressed rats, systemic administration of the anxiogenic benzodiazepine inverse agonists beta-CCM (methyl-beta-carboline-3-carboxylate) and beta-CCE (ethyl-beta-carboline-3-carboxylate), but not of the benzodiazepine antagonists Ro 15-1788 or CGS 8216, caused an increase in frontal cortical DOPAC similar to that provoked by stress and which was antagonized by zolpidem.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphodiesterase inhibitors induce endothelium-dependent relaxation of rat and rabbit aorta by potentiating the effects of spontaneously released endothelium-derived relaxing factor.

Abstract: The selective cyclic GMP phosphodiesterase inhibitor M&B 22948 and the less selective phosphodiesterase inhibitors papaverine and isobutylmethylxanthine (IBMX) each induced a component of relaxation of rat aortic rings that was endothelium-dependent. The most selective agent at inducing endothelium-dependent relaxation was M&B 22948, which caused little relaxation of endothelium-denuded rings at concentrations that produced almost complete relaxation of endothelium-containing rings. Although endothelium-dependent components of relaxation induced by papaverine and IBMX were clearly present, they were less well separated from the endothelium-independent components of relaxation. In the aorta of the rabbit, M&B 22948 and papaverine were less affective at inducing an endothelium-dependent component of relaxation than in the aorta of the rat, and IBMX produced no discernible endothelium-dependent component. The endothelium-dependent components of relaxation induced by M&B 22948, papaverine and IBMX on rat and rabbit aorta were probably dependent on endothelium-derived relaxing factor (EDRF), because they were associated with concomitant endothelium-dependent rises in cyclic GMP, and these components of relaxation as well as the rises in cyclic GMP were completely blocked by the EDRF-blocking agent hemoglobin. The action of hemoglobin was entirely specific, as none of the endothelium-independent components of relaxation induced by any of the phosphodiesterase inhibitors was affected by this hemoprotein. It is likely that the phosphodiesterase inhibitors induce their endothelium-dependent components of relaxation by inhibiting the hydrolysis of cyclic GMP formed in response to EDRF released spontaneously from endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Human coffee drinking: reinforcing and physical dependence producing effects of caffeine.

Abstract: In a residential research ward coffee drinking was studied in nine volunteer human subjects with histories of heavy coffee drinking. The presence or absence of caffeine in the coffee was manipulated under double-blind conditions by using caffeinated (C) or decaffeinated (D) coffee. When subjects were switched alternately for 10 or more consecutive days between C and D, the daily number of cups consumed tended to be relatively stable. In a different experiment, preference for C vs. D was assessed. After experimenter-scheduled exposures, subjects were given choices between C and D. When subjects were presumably caffeine tolerant/dependent, C was rated as being better liked than D and was reliably preferred to D in choice tests. When subjects were not caffeine tolerant/dependent, C was not reliably preferred to D, nor were there pronounced differences in ratings of liking. Under these conditions, some subjects preferred D to C, citing adverse symptoms (suggesting caffeine toxicity) as reasons for avoiding C. The effects of caffeine withdrawal were studied by abruptly substituting D for C for 10 or more days. This resulted in an orderly withdrawal syndrome, having an onset latency of 19 hr, peaking on days 1 and 2, and decreasing progressively over the next 5 or 6 days. The withdrawal syndrome, which was detected on subject-rated, staff-rated and objective behavioral measures, was characterized by increased headache, sleepiness and laziness and decreased alertness and activeness. The present study demonstrates the reinforcing effects of caffeine in humans and also documents the severity of the caffeine withdrawal syndrome. It is concluded that caffeine has the cardinal features of a prototypic drug of abuse.

Dynorphin A and related peptides administered intrathecally in the rat: a search for putative kappa opiate receptor activity.

Abstract: The intrathecal delivery of dynorphin A (1-17) and (1-13) induces, with rapid onset, a severe motor dysfunction characterized by flaccid extension of the hindlimbs and complete loss of muscle tone. This motor effect does not appear to be mediated via opiate receptors as high doses of naloxone neither block nor reverse motor dysfunction, and it is produced by dynorphin A fragments inactive at opiate receptors. At doses just below those which produce motor dysfunction, dynorphin A has no effect on nociceptive responses in the hot-plate, tail-flick and writhing tests. The selective, kappa opiate agonist, U50488H, produces a significant, dose-dependent inhibition of writhing, which is antagonized by pretreatment with naloxone, but has no effect on hot-plate and tail-flick latency. The kappa agent U50488H does not produce motor dysfunction with doses as high as 300 nmol/rat. It appears that the potent kappa opiate activity exhibited by dynorphin A in vitro may not reflect in vivo effects of dynorphin after intrathecal administration.

Roles of D1 and D2 dopamine receptor subtypes in mediating the methamphetamine-induced changes in monoamine systems.

Abstract: Previous studies have demonstrated that the effects of methamphetamine (METH) on dopaminergic and serotonergic systems are likely related to METH-induced increases in dopamine release. The ability of haloperidol to prevent these effects of METH suggests that dopamine receptor activation is involved in mediating these METH actions. The present studies were undertaken to determine what role the dopamine receptor subtype(s) might have in such METH effects. We found that the D1 antagonist, SCH23390, attenuated or blocked the effects of METH on dopaminergic and serotonergic systems, whereas the D2 antagonist, sulpiride, blocked the effects of METH on only the dopaminergic system. The results of the present studies suggest that METH-induced increases in dopaminergic action on both D1 and D2 receptors are associated with the effects of METH on the dopaminergic system. In contrast, dopamine action on D1, but not on D2, receptors appears to be involved in the effects of METH on the serotonergic systems of the neostriatum and the cerebral cortex.

Identification and characterization of functional angiotensin II receptors on cultured heart myocytes.

Abstract: Cultured neonatal rat myocytes have been investigated as a potential system to study the molecular mechanism of angiotensin II (All) stimulation of heart contractile behavior. Intact cultured cells and the membranes from these cells bind [125I] All in a biphasic manner. The data were analyzed as two classes of binding sites on membrane preparations: Kd1 = 0.65 nM; maximum binding (Bmax1) = 245 fmol/mg of protein and Kd2 = 5.57 nM, Bmax2 = 720 fmol/mg of protein. The receptor on intact cells is specific as All peptide analogs were potent at inhibiting the binding of [125I]All. An All antagonist, [125I]Sar1,Leu8-All, recognized a single class of high affinity receptors on intact cells: Kd = 0.63 nM, Bmax = 318 fmol/mg of protein, or 45,300 sites per cell. Guanine nucleotides regulated the binding of All to membranes by shifting the receptor from a high affinity to a low affinity form without changing Kd2. Conversely, these nucleotides altered the high affinity binding of [125I]Sar1,Leu8-All by increasing Kd without changing Bmax. All was found to stimulate the contractile frequency of spontaneously beating myocytes with maximal effects (a 50% stimulation) observed at 5 nM hormone. The responses were reversible with half-maximal effects observed at doses around 1 nM. The antagonist, Sar1,Ala8-All, could inhibit the chronotropic stimulatory responses. It is concluded that these cultured myocytes express an All receptor system with properties consistent with a true hormone receptor system. Furthermore, studies on the pharmacology of the chronotropic responses of these cells to All demonstrate that these receptors are functional.

Regulation of dopamine synthesis in the medial prefrontal cortex is mediated by release modulating autoreceptors: studies in vivo.

Abstract: Previous studies have suggested that rat mesoprefrontal dopamine (DA) neurons are devoid of synthesis and impulse-modulating autoreceptors. We have examined further the presynaptic regulatory parameters of these neurons in vivo and have developed a model based on the following observations. Prefrontal DA turnover, measured as the rate of DA disappearance after inhibition of tyrosine-3-monooxygenase by alpha-methyltyrosine, is relatively rapid (T1/2 = 15 min) and is suppressed by apomorphine (50 micrograms/kg) pretreatment, suggesting that prefrontal DA release is sensitive to regulation by DA agonists. Prefrontal DOPA accumulation (measured after inhibition of decarboxylase with m-hydroxybenzylamine) is also diminished after administration of DA agonists, such as apomorphine, BHT-920, 3-[4-(4-phenyl)-1,2,3,6-tetrahydropyridyl-1] butyl indole (EMD 23 448) and 3-(3-hydroxyphenyl)-N-n-propylpiperidine[(+)-3-PPP]. However, the apomorphine-induced inhibition of prefrontal, but not striatal, DOPA synthesis is blocked by either cessation of impulse-dependent DA release (after gamma-butyrolactone) or by depletion of intraneuronal DA (after reserpine), suggesting that DA agonists affect prefrontal DA synthesis only in the presence of DA release. Inhibition of impulse flow after administration of gamma-butyrolactone does not increase the rate of prefrontal tyrosine hydroxylation in vivo, suggesting that synaptic DA does not influence medial prefrontal DA synthesis under basal conditions. After treatment with m-hydroxybenzylamine (30 min), prefrontal DA levels are substantially reduced (-70%) and inhibition of prefrontal synthesis by DA agonists is associated with an increase in intraneuronal DA relative to m-hydroxybenzylamine-treated controls, suggesting that agonist-induced synthesis inhibition is a consequence of activation of release-modulating autoreceptors with a subsequent decrease in DA release and increase in end product inhibition of tyrosine hydroxylation. These and other data in the accompanying paper suggest that the nerve terminals of mesoprefrontal DA neurons are unique in their constitution of functional autoreceptors in that they contain only a release-modulating mechanism.

Pharmacological properties of acetorphan, a parenterally active "enkephalinase" inhibitor.

Abstract: Acetorphan, i.e. N-[(R,S)-3-acetylmercapto-2-benzylpropanoyl]-glycine, benzyl ester, is a lipophilic derivative of Thiorphan, a potent inhibitor of "enkephalinase" (EC 3.4.24.11). On purified enkephalinase its inhibitory potency was approximately 1000 fold less than that of Thiorphan but became close to the latter (nanomolar) when it was incubated previously with cerebral membranes. After parenteral administration to mice and rats (1-10 mg/kg) extensive inhibition of cerebral enkephalinase was shown by the depressed enzyme activity in brain membranes from treated animals and the long-lasting potentiation of analgesia elicited by (D-Ala2,Met5)enkephalin (i.c.v.). This suggests that acetorphan easily enters the brain where the active Thiorphan is released. Parenteral acetorphan elicited a series of naloxone-reversible, opioid-like effects, most of which were described previously with intracerebral Thiorphan or other enkephalinase inhibitors. Antinociceptive effects were found in some tests (hot plate jump and phenylbenzoquinone-induced writhing) but not in others (hot plate licking and tail withdrawal). "Antidepressant" effect was found in the "mouse despair" test and antidiarrhoeal effect in the rat castor oil test. Acetorphan also elicited significant increases and decreases in turnover indexes of serotonin and noradrenaline, respectively, in mouse cerebral cortex. In mice chronically treated with acetorphan, the antinociceptive activity of the compound was not modified markedly and no overt withdrawal symptom could be observed after either treatment interruption or administration of naloxone.

5-Hydroxytryptamine2 receptors coupled to phospholipase C in rat aorta: modulation of phosphoinositide turnover by phorbol ester.

Abstract: In rat aorta, 5-hydroxytryptamine (5-HT) stimulated phosphoinositide (PI) turnover and contraction (EC50 = 10 +/- 3 microM); these two responses were highly correlated (r = 0.95; P less than .01). We have characterized the inhibitory potency of a variety of 5-HT-antagonists against the stimulation of PI turnover elicited by 5-HT. Classic 5-HT2 antagonists mianserin, ketanserin, metergoline and pizotifen were found to inhibit this response in the low nanomolar range; amitryptiline and haloperidol were 10- to 20-fold less potent. The alpha-1 receptor antagonist, prazosin, was inactive in micromolar concentrations. The potency of the 5-HT2 antagonists was correlated with their ability to displace [3H] ketanserin binding from rat frontal cortex membranes (r = 0.90; P less than .05). The tumor promoter phorbol dibutyrate was found to inhibit 5-HT-stimulated PI turnover at low nanomolar concentrations whereas the biologically inactive substance 4-alpha-phorbol was ineffective. Pretreatment of rat aorta with phorbol dibutyrate at concentrations that inhibited 5-HT-induced PI turnover also attenuated the aortic contraction induced by 5-HT in the presence of a calcium channel blocker nitrendipine. Our results suggest that phorbol esters may desensitize 5-HT2-receptor-mediated PI turnover and contraction of rat aorta, possibly via an activation of protein kinase C.

[3H]pirenzepine and (-)-[3H]quinuclidinyl benzilate binding to rat cerebral cortical and cardiac muscarinic cholinergic sites. I. Characterization and regulation of agonist binding to putative muscarinic subtypes.

Abstract: The binding and regulation of selected muscarinic agonists to putative subtypes in rat cerebral cortex and heart were studied. Parallel inhibition studies of [3H]pirenzepine ([3H]PZ) and (-)-[3H]quinuclidinylbenzilate [(-)-[3H]QNB]-labeled membranes were done with and without 30 microM guanyl-5'-yl imidodiphosphate [Gpp(NH)p] at 25 degrees C in 10 mM Na-K-phosphate buffer which enhances PZ binding affinity and in modified Krebs-phosphate buffer, which mimics physiological conditions. Classical agonists such as carbachol, oxotremorine and acetylcholine inhibited (-)-[3H]QNB binding to membranes with shallow Hill values (nH less than 1), were better fit to a 2-state model, were Gpp(NH)p-regulated and showed lower affinity in modified Krebs-phosphate buffer than in 10 mM Na-K-phosphate buffer. Some agonists were not significantly better fit to a 2-state model in [3H]PZ-labeled cortical membranes, especially in 10 mM Na-K-phosphate buffer. Whereas putative M1 and M2 binding sites distinguished by PZ possessed multiple agonist affinity states, as judged by carbachol, and agonist binding to [3H]PZ-labeled sites were Gpp(NH)p modulated, the partial agonist pilocarpine and nonclassical agonist McN-A-343 [3-(m-chlorophenylcarbamoyloxy)-2-butynyl trimethylammonium chloride] showed little Gpp(NH)p-induced shift in [3H]PZ-labeled cortical membranes in physiological conditions. Agonist binding to (-)-[3H]QNB-labeled putative M2 cardiac sites was more sensitive to Gpp(NH)p than (-)-[3H]QNB-labeled cortical sites. Carbachol and acetylcholine showed significant selectivity for putative M2 sites.(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo uptake kinetics of aminoglycosides in the kidney cortex of rats.

Abstract: The renal cortical uptake kinetics of four aminoglycosides were studied in vivo. Gentamicin, netilmicin, tobramycin or amikacin were administered to rats by continuous infusion over 6 hr achieving constant serum levels ranging from 0.2 to 100 micrograms/ml. Renal cortical concentrations at the end of the infusion were plotted against the steady-state serum levels. Steady-state elevations of serum gentamicin and netilmicin were associated with nonlinear increases in cortical levels, suggesting saturable uptake. Analysis of the data using Michaelis-Menten kinetics indicates that the apparent Km for gentamicin and netilmicin were 15.01 and 23.84 micrograms/ml and Vmax 149.83 and 178.36 micrograms/g of cortex per hr, respectively. The "initial" rate of uptake (at serum levels below 15 micrograms/ml) was highest for gentamicin. The cortical uptake of tobramycin was linearly related to elevations in serum levels [cortex concentration (conc) = 9.24 + 1.40 serum conc]. The initial rate of tobramycin uptake was considerably lower than that for gentamicin and netilmicin. For amikacin, the initial rate of uptake followed Michaelis Menten kinetics and the second phase of the titration curve was linear. The equation for total amikacin uptake was: cortex conc = 12.98 + 1.71 serum conc. Aminoglycosides exhibit differing kinetics for renal cortical uptake in the rat during constant infusions. These results indicate that more than one mechanism probably mediates the uptake of each aminoglycoside. Depending on which mechanism predominates, the kinetic pattern may be saturable, linear or mixed.

Effects of d-amphetamine and dopamine synthesis inhibitors on dopamine and acetylcholine neurotransmission in the striatum. I. Release in the absence of vesicular transmitter stores.

Abstract: The release of endogenous dopamine (DA) elicited by electrical stimulation and by d-amphetamine (AMPH) from superfused striatal slices of reserpine-pretreated rabbits was examined. Although reserpine pretreatment reduced tissue DA levels by greater than 95%, the basal efflux of DA and the DA metabolite dihydroxyphenylacetic acid (DOPAC) was slightly greater than that observed in untreated slices. DOPAC constituted the large majority of the basal efflux of endogenous compounds. No overflow of endogenous compounds was evoked by electrical stimulation (3 Hz, 3 min) after reserpine pretreatment. Superfusion with alpha-methyl-p-tyrosine (100 microM) abolished the efflux of endogenous DA and DOPAC. AMPH (0.3-10 microM) produced a concentration-dependent increase in the basal efflux of endogenous DA and a concomitant decrease in endogenous DOPAC efflux. The total efflux of endogenous compounds (DA + DOPAC) tended to be decreased by AMPH. No electrically evoked overflow of endogenous compounds was observed in the presence of AMPH. The increase in synaptic DA produced by AMPH was reflected by a concentration-dependent reduction in the electrically evoked overflow of [3H]acetylcholine (ACh). The ability of AMPH to increase DA efflux and inhibit [3H]ACh release was blocked by inhibition of DA synthesis with alpha-methyl-p-tyrosine (100 microM) or by blockade of the DA neuronal uptake carrier with nomifensine (NOM) (10 microM) and was potentiated by inhibition of monoamine oxidase with pargyline (10 microM). NOM also blocked partially the ability of AMPH to reduce endogenous DOPAC efflux. NOM increased the basal efflux of endogenous DA and inhibited electrically evoked [3H]ACh release but these effects were quantitatively much less than those produced by AMPH. NOM had no effect on DOPAC efflux. Pargyline had little effect on endogenous DA efflux or electrically evoked [3H]ACh release but abolished DOPAC efflux and increased tissue DA levels measured at the end of superfusion. When given in combination, NOM and pargyline produced a similar degree of inhibition of [3H]ACh release as AMPH, although the increase in DA efflux produced by this drug combination was less than that produced by AMPH. These results suggest that in the absence of vesicular transmitter stores (reserpine-pretreatment): synthesis provides a continuous supply of DA which is metabolized rapidly within the neuron and is lost as DOPAC; AMPH facilitates the synthesis-dependent efflux of extravesicular DA probably by an accelerated exchange diffusion mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)