Plasma pharmacokinetics and tissue depletion of cyromazine and its metabolite melamine following oral administration in laying chickens.

The study was designed to characterize the plasma pharmacokinetics and tissue depletion profiles (including eggs) of cyromazine (CYR) in chickens following oral administration alone or in combination with melamine (MEL). In order to assess the pharmacokinetic profile of CYR, chickens were administered 1 or 10 mg/kg (single oral doses), whereas residue studies were conducted in chickens fed CYR alone (5 or 10 mg/kg) or CYR (5 mg/kg) and MEL (5 mg/kg) for a period of 14 days. Estimates for the apparent volume of distribution (1.66 L/kg), clearance (7.17 mL/kg/min), and elimination half-life (2.82 h) were derived by noncompartmental analyses. The highest concentration of CYR occurred in liver but fell below detectable limits within 3 days following drug withdrawal from feed. Combined feeding of MEL with CYR did not significantly alter CYR tissue levels. CYR residues were detected only in egg white and were undetectable at the 2nd day postadministration. No MEL was found in eggs unless it had been added to the feed, and when present, it almost exclusively restricted to the egg white. Based upon the results of this initial study of CYR pharmacokinetics and residue depletion, it appears that use of CYR as a feed additive either alone (5 or 10 mg/kg) or in combination with MEL (both agents at 5 mg/kg) does not produce unsafe residue levels in edible products as long as appropriate withdrawal periods are followed for tissues (3 days) and eggs (2 days). However, our results indicate that adoption of a zero-day withdrawal period should be reconsidered in light of these results.

Pharmacokinetics of ceftiofur sodium in equine pregnancy.

Eleven pregnant pony mares (D270-326) were administered ceftiofur sodium intramuscularly at 2.2 mg/kg (n = 6) or 4.4 mg/kg (n = 5), once daily. Plasma was obtained prior to ceftiofur administration and at 0.5, 1, 2, 4, 8, 12, and 24 hr after administration. Eight pony mares were re-enrolled in the study at least 3 days from expected foaling to ensure steady-state concentrations of drug at the time of foaling. Mares were administered ceftiofur sodium (4.4 mg/kg, IM) daily until foaling. Parturition was induced using oxytocin 1 hr after ceftiofur sodium administration. Allantoic and amniotic fluid, plasma, and colostrum samples were collected at time of foaling. Serial foal plasma samples were obtained. Placental tissues were collected. Desfuroylceftiofur acetamide (DCA) concentrations were measured in samples by high-performance liquid chromatography (HPLC). Mean (±SD) peak serum concentrations of DCA were 3.97 ± 0.50 µg/ml (low dose) and 7.45 ± 1.05 µg/ml (high dose). Terminal half-life was significantly (p = .014) shorter after administration of the low dose (2.91 ± 0.59 hr) than after administration of the high dose (4.10 ± 0.72 hr). The mean serum concentration of DCA from mares at time of foaling was 7.96 ± 1.39 µg/ml. The mean DCA concentration in colostrum was 1.39 ± 0.70 µg/ml. DCA concentrations in allantoic fluid, amniotic fluid, placental tissues, and foal plasma were below the limit of quantification (<0.1 µg/ml) and below the minimum inhibitory concentration of ceftiofur against relevant pathogens. These results infer incomplete passage of DCA across fetal membranes after administration of ceftiofur sodium to normal pony mares.

Disposition of ampicillin trihydrate in plasma, uterine tissue, lochial fluid, and milk of postpartum dairy cattle.

The objective of this study was to determine the disposition of ampicillin in plasma, uterine tissue, lochial fluid, and milk of postpartum dairy cattle. Ampicillin trihydrate was administered by intramuscular (i.m.) injection at a dose of 11 mg/kg of body weight every 24 h (n = 6, total of 3 doses) or every 12 h (n = 6, total of 5 doses) for 3 days. Concentrations of ampicillin were measured in plasma, uterine tissue, lochial fluid, and milk using HPLC with ultraviolet absorption. Quantifiable ampicillin concentrations were found in plasma, milk, and lochial fluid of all cattle within 30 min, 4 h, and 4 h of administration of ampicillin trihydrate, respectively. There was no significant effect of dosing interval (every 12 vs. every 24 h) and no significant interactions between dosing interval and sampling site on the pharmacokinetic variable measured or calculated. Median peak ampicillin concentration at steady-state was significantly higher in lochial fluid (5.27 µg/mL after q 24 h dosing) than other body fluids or tissues and significantly higher in plasma (3.11 µg/mL) compared to milk (0.49 µg/mL) or endometrial tissue (1.55 µg/mL). Ampicillin trihydrate administered once daily by the i.m. route at the label dose of 11 mg/kg of body weight achieves therapeutic concentrations in the milk, lochial fluid, and endometrial tissue of healthy postpartum dairy cattle.

Pharmacokinetics and pharmacodynamics of glycopyrrolate following a continuous-rate infusion in the horse.

Glycopyrrolate (GLY) is an antimuscarinic agent that is used in humans and domestic animals primarily to reduce respiratory tract secretions during anesthesia and to reverse intra-operative bradycardia. Although GLY is used routinely in veterinary patients, there is limited information regarding its pharmacokinetic (PK) and pharmacodynamic (PD) properties in domestic animals, and an improved understanding of the plasma concentration-effect relationship in racehorses is warranted. To accomplish this, we characterize the pharmacokinetic-pharmacodynamic (PK-PD) actions of GLY during and after a 2-h constant-rate intravenous infusion (4 µg/kg/h) and evaluate potential PK-PD models for cardiac stimulation in adult horses. Measurements of plasma GLY concentrations, heart and respiration rates, and frequency of bowel movements were performed in six Thoroughbred horses. The time course for GLY disposition in plasma followed a tri-exponential equation characterized by rapid disappearance of GLY from blood followed by a prolonged terminal phase. Physiological monitoring revealed significant (P < 0.01) increases in heart (>70 bpm) and respiratory rates accompanied by a marked and sustained delay in the frequency of bowel movements (1.1 ± 0.2 h [saline group] vs. 6.0 ± 2.0 h [GLY group]). Two of six horses showed signs of colic during the 8-h observation period after the end of the GLY infusion, but were treated and recovered without further complications. The relationship between plasma GLY concentration and heart rate exhibited counterclockwise hysteresis that was adequately described using an effect compartment.

Disposition of desfuroylceftiofur acetamide in serum, placental tissue, fetal fluids, and fetal tissues after administration of ceftiofur crystalline free acid (CCFA) to pony mares with placentitis.

The objective of this study was to determine the pharmacokinetics of CCFA in mares with placentitis and evaluate the disposition of the drug in fetal fluids, fetal membranes, colostrum, and serum of foals. A secondary objective was to obtain pilot data regarding the efficacy of CCFA for improving foal survival in mares with placentitis. Twelve pregnant pony mares were enrolled in the study, inoculated with Streptococcus zooepidemicus, intracervically and assigned to one of three groups: CEFT (n = 3; administered CCFA only; 6.6 mg/kg, i.m., q96h); COMBO (n = 6; administered combination therapy of CCFA, altrenogest, and pentoxifylline); UNTREAT (n = 3, no treatment). Treatment was initiated at the onset of clinical signs. Concentrations of desfuroylceftiofur acetamide (DCA), the acetamide derivative of ceftiofur and desfuroylceftiofur metabolites, were measured using high-performance liquid chromatography. Maximum and minimum serum concentrations of DCA at steady state in treated mares were 2.40±0.40 µg/mL and 1.06±0.29 µg/mL, respectively. Concentration of DCA in colostrum was 1.51±0.60 µg/mL. DCA concentrations in placenta and fetal tissues were very low (median = 0.03 µg/mL) and below the minimum inhibitory concentration of relevant pathogens. DCA was not detected in amniotic fluid or foal serum. Treatment did not appear to improve foal survival (CEFT: 0/3; COMBO: 2/6; UNTREAT: 2/3). Bacteria were recovered from the uterus of most mares postpartum and from blood cultures of most foals regardless of treatment.

Caffeine-induced hyperactivity in the horse: comparisons of drug and metabolite concentrations in blood and cerebrospinal fluid.

The goals of this study were to elucidate the temporal and quantitative relationships between caffeine and its major bioactive metabolites in blood and cerebrospinal fluid (CSF) and to characterize the pharmacokinetic-pharmacodynamic relationship for caffeine-induced changes in spontaneous locomotor activity in the horse. We hypothesized that caffeine and its metabolites distribute efficiently into the CSF to antagonize adenosine A1 and A2a receptors and that spontaneous locomotor activity correlates well with caffeine and/or metabolite concentrations in CSF and blood. A microdialysis system was developed to allow simultaneous monitoring of locomotor activity and collection of CSF and blood samples for pharmacokinetic analysis. CSF concentrations of caffeine and its metabolites were evaluated to determine the percentage of central adenosine receptor blockade by the established standard inhibition curves. Caffeine increased the spontaneous locomotor activity for up to 4 h in a dose-dependent manner. After 3 mg/kg caffeine administration, blood caffeine concentration as well as locomotor activity increased sharply to near peak level while CSF caffeine concentrations exhibited a slow rise to a steady-state 75 min later. High correlation coefficient was found between locomotor activity and caffeine concentrations in blood (R(2 )=0.95) and in CSF (R(2) = 0.93). At 3 mg/kg dosage, theophylline was the only detectable caffeine metabolite in the CSF. The concentrations reached in the CSF were sufficient to partially block central adenosine A1 (14% blockade) and A2a (11% blockade) receptors. There were no statistically significant differences between the pharmacokinetics of caffeine in the blood and CSF. This study provides novel evidence that locomotor stimulation in horses is closely correlated with caffeine concentrations in the blood and CSF and, furthermore, is consistent with blockade of central adenosine receptors.

Continuous monitoring of penicillin G and gentamicin in allantoic fluid of pregnant pony mares by in vivo microdialysis.

REASONS FOR PERFORMING STUDY: Most current treatments for placentitis in mares are empirical with few control studies to evaluate their effectiveness. OBJECTIVE: To monitor drug concentrations in allantoic fluid of pregnant pony mares using in vivo microdialysis and establish if this method would be useful for determining allantoic concentrations of drugs in normal mares and those with placentitis. METHODS: Five late gestational pony mares had microdialysis probes inserted into the allantoic fluid using transabdominal ultrasound-guided allantocentesis. Single injections of penicillin G (22,000 u/kg), gentamicin (6.6 mg/kg bwt) and flunixin meglumine (1 mg/kg bwt) were administered i.v. and dialysate samples collected continuously for 24 h. In a separate study, drug concentrations were monitored in allantoic fluid of 2 mares with experimental placentitis induced by intracervical inoculation with Streptococcus equi ssp. zooepidemicus. Drug concentrations were measured by high performance liquid chromatography (penicillin G, flunixin meglumine) or enzyme-linked immunosorbent assay (gentamicin). RESULTS: Penicillin G and gentamicin achieved average peak concentrations of 9.8+/-2.2 and 8.5+/-3.1 microg/ml, respectively, in allantoic fluid of noninfected mares. Pharmacokinetic comparisons indicate that penicillin G persists much longer in allantoic fluid than blood, whereas gentamicin exhibited similar profiles in the 2 compartments. Flunixin meglumine was not detected in allantoic fluid. In infected mares, penicillin G achieved a similar peak concentration in allantoic fluid (11.2 microg/ml) whereas peak gentamicin concentration (3.9 microg/ml) appeared to be reduced relative to drug concentrations in noninfected mares. CONCLUSIONS: Microdialysis is a useful technique for continuous in vivo monitoring of drugs in equine allantoic fluid. Our results indicate that penicillin G and gentamicin undergo effective placental transfer in pregnant mares and in 2 mares that transplacental drug transfer may be altered selectively if active placental infection is present. POTENTIAL RELEVANCE: Further studies are needed to evaluate the feasibility of using increased dose intervals for penicillin G and an increased dose rate of gentamicin to effectively combat placental infections in mares.

Neurochemical basis of disruption of hippocampal long term potentiation by chronic alcohol exposure.

The aim of this review is to summarize the possible mechanisms underlying the long-term impairment of learning and memory resulting from chronic ethanol treatment (CET) especially that involving decrements in long-term potentiation (LTP) in hippocampus. CET for a 28-week duration affects the rat hippocampal formation in such a way as to decrease the magnitude of LTP; an effect that can last as long as 7 months after ethanol withdrawal. It appears that NMDA receptor number in hippocampus is unchanged after CET whereas the data suggest a more pronounced role for changes in GABAergic and cholinergic synaptic transmission in determining how CET influences the induction of LTP in hippocampus. In particular, changes in presynaptic modulation of neurotransmitter release in hippocampus may be one mechanism by which CET inhibits LTP. Thus, the mechanisms underlying the effect of CET on LTP are a result of changes in a number of neurotransmitter systems in hippocampus (GABAergic and cholinergic) rather than based solely on changes in glutamate transmission.

Effects of phencyclidine on the release and synthesis of newly formed dopamine.

The effect of phencyclidine (PCP) on the release and synthesis of [3H]-dopamine ( [3H]-DA) newly synthesized from [3,5-3H]-L-tyrosine was studied under both basal and potassium-stimulated conditions in slices from the rat striatum. Phencyclidine (3-100 microM) stimulated the release of [3H]-DA during superfusion with low level potassium (4.5 mM) buffer, but had no effect on the release of [3H]-DA elicited by superfusion with buffer containing 40 mM potassium. On the other hand, phencyclidine had no effect (except at 100 microm) on the spontaneous release of [3H]-H2O (formed as a result of the hydroxylation of [3,5-3H]-L-tyrosine), but inhibited the increase in formation of [3H]-H2O normally associated with potassium-induced depolarization. These data are discussed in relation to results obtained with phencyclidine on other in vitro and in vivo models of dopaminergic function.

Pharmacological heterogeneity among calcium channels that subserve acetylcholine release in vertebrate forebrain.

Inhibition of calcium-evoked [3H]ACh release by different classes of calcium channel blockers was compared among calcium-naive synaptosomes from chick, frog and rat forebrain tissues. In all three species, [3H]ACh release was insensitive to nifedipine (0.03-3 microM) but was completely inhibited by cadmium (IC50 range = 0.7-1.7 microM) or cobalt (190-350 microM). In contrast, the peptide omega-conotoxin revealed marked species heterogeneity in that [3H]ACh release was potently blocked in chick and rat synaptosomes (IC50 congruent to 1 nM), whereas frog tissue was notably resistant (IC50 greater than 100 nM). Together, these data provide functional evidence for pharmacological heterogeneity among presynaptic calcium channels that subserve [3H]ACh release.

Irreversible blockade of high-affinity choline uptake in rat brain by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ).

N-Ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), an agent that causes irreversible covalent modification of protein carboxyl residues, has been used previously to produce irreversible occlusion of neurotransmitter receptors as well as other cellular proteins. The present investigation was undertaken to ascertain the mechanism by which EEDQ inhibits stimulus-dependent acetylcholine (ACh) release from rat brain hippocampal synaptosomes. Brief pretreatment with EEDQ (up to 100 microM) eliminated completely calcium-evoked [3H]acetylcholine ([3H]ACh) release and reduced de novo synthesis of transmitter by greater than 90%. Studies revealed that pretreatment with EEDQ in vitro caused a time- and concentration-dependent inhibition of high-affinity [3H]choline uptake (HACU) by synaptosomes. EEDQ-induced inhibition of HACU was not reversed by repeated tissue washing; however, co-incubation with hemicholinium-3, a highly specific and reversible inhibitor of HACU, protected against EEDQ-induced inhibition of HACU, as well as the loss of stimulus-dependent [3H]-ACh release. In vivo administration of EEDQ (20 mg/kg, s.c.) to rats caused marked reductions (46-65%) in synaptosomal HACU as well as the number of membrane binding sites for the muscarinic cholinergic antagonist L-[benzilic-4,4'-3H]quinuclidinyl benzilate ([3H]QNB) in the hippocampus and striatum. Treatment with atropine (100 mg/kg) prevented the reduction in [3H]QNB binding but did not influence EEDQ-induced inhibition of HACU. Taken together, these results indicate that EEDQ causes a direct and irreversible inhibition of high-affinity choline transporters on CNS cholinergic nerve terminals and, therefore, may be a useful investigational tool for characterization of the turnover and regulation of this transporter protein in vivo.

Selective loss of phorbol-12,13-dibutyrate-facilitated L-glutamate transport in forebrain neurons of aged rats.

Phorbol-12,13-dibutyrate (PDBu), an activator of protein kinase C, was used to evaluate potential age-related changes in phosphorylation-dependent facilitation of high-affinity L-glutamate uptake in the rat central nervous system (CNS). Forebrain homogenates from male Fischer-344/brown Norway F1 hybrid rats were separated into glia-enriched (glial plasmalemmal vesicles) and neuron-enriched fractions (synaptosomes) and assayed for sodium-dependent transport of L-[3H]glutamate. Glial fractions from rats aged 5, 25, 31, and 37 months exhibited similar rates of basal L-[3H]glutamate transport and demonstrated no significant age-related differences with respect to the maximal facilitatory effect of PDBu (1-100 microM). In contrast, neuronal fractions exhibited an age-related decline in both indices, with basal L-[3H]glutamate transport decreasing from 710+/-31 to 560+/-40 pmoL/mg protein/90 s for the 5- and 37-month groups, respectively (p < .03) and PDBu having a significantly attenuated effect in aged animals. Together, these results provide support for the hypothesis that aging is associated with a decrease in the number of neuronal L-glutamate transporters as well as a diminished capacity to up-regulate these transporters through a protein kinase C-dependent pathway.

Pharmacological and mechanistic studies of cholecystokinin-facilitated [3H]dopamine efflux from rat nucleus accumbens.

Cholecystokinin (CCK) is co-localized with dopamine (DA) in mesolimbic neurons of the CNS and appears to selectively regulate the output of this system. In an attempt to characterize the nature of CCK interactions with mesolimbic DA-containing nerve terminals, we have investigated CCK regulation of [3H]DA overflow from rat nucleus accumbens slices. CCK-8 produced a saturable and potent (EC50 = 3 nM) facilitation of KCl (35 mM)-evoked [3H]DA efflux from nucleus accumbens, but failed to significantly alter [3H]DA efflux from striatum: The stimulatory action of CCK-8 was unaffected by the muscarinic antagonist atropine, the opiate antagonist naloxone, or the selective ion channel blockers tetrodotoxin and nifedipine. Pharmacological studies revealed that non-sulfated CCK-8 and CCK-4 (up to low micromolar concentrations) did not facilitate [3H]DA efflux from accumbens slices. Furthermore, the effect of CCK-8 was selectively and potently (IC50 = 300 nM) inhibited by the Type A-selective CCK antagonist CR-1409. Taken together, these results indicate that CCK regulates DA efflux from mesolimbic nerve terminals via a direct presynaptic action on receptors which display a pharmacological profile that is similar to Type A CCK receptors in gastrointestinal tissues.

Neurobiochemical evidence for calcium-induced depolarization of EGTA-pretreated hippocampal synaptosomes.

The neurochemical effects of calcium were determined in hippocampal cholinergic synaptosomes which had been prepared and preincubated in calcium-free medium containing 50 microM EGTA. Calcium (and barium) reversibly stimulated [3H]acetylcholine release and produced a long-lasting elevation of high-affinity [3H]choline uptake. Both effects were blocked by omega-conotoxin and substantially reduced by tetrodotoxin. Together, these data indicate that calcium causes membrane depolarization and is associated with opening of voltage-gated sodium channels in EGTA-pretreated synaptosomes.

Effects of bilateral ibotenate-induced lesions of the nucleus basalis magnocellularis upon selective cholinergic biochemical markers in the rat anterior cerebral cortex.

The relationship of choline acetyltransferase (ChAT) activity and high affinity binding of the potent and selective sodium-dependent choline uptake inhibitor [3H]hemicholinium-3 ([3H]HC-3) to high-affinity binding of the muscarinic agonist [3H](+)-cis-methyldioxolane ([3H](+)CD), the putative M1 selective antagonist [3H]pirenzepine ([3H]PZ) and the classical antagonist [3H](-)-quinuclidinyl benzilate ([3H](-)QNB) in homogenates of the rat neocortex was studied. ChAT activity was 42% lower in rats with ibotenate-induced lesions of the nucleus basalis magnocellularis (nbm) when compared to controls, and [3H]HC-3 binding was similarly reduced by 44%. However, equilibrium dissociation constants (Kd values) for [3H]HC-3 (0.8-1.0 nM), [3H](-)QNB (11-24 pM), [3H]PZ (4.0-4.3 nM) and [3H](+)CD (2.1-2.9 nM) were each unchanged. Mean Bmax values (total binding site densities) for [3H](+)CD were significantly altered in both hemispheres of the anterior cerebral cortex, showing a 25% reduction in the number of sites which display the highest affinity conformation for this potent muscarinic agonist. The decreased ChAT activity and [3H]HC-3 binding after nbm lesions were associated with only slight reductions in putative M1 muscarinic site density (14%) and [3H](-)QNB binding site density (13%). Thus, it appears that while [3H]PZ and [3H](-)QNB label predominantly postsynaptic muscarinic binding sites, a significant number of sites labeled by [3H](+)CD may be associated with presynaptic cholinergic nerve terminals. These data suggest that cholinergic input differentially regulates the drug binding sites of anterior cerebral cortical muscarinic receptors, exerting a substantial effect upon the highest affinity conformational state for agonists.

Autoradiographic localization of muscarinic agonist binding sites in the rat central nervous system with (+)-cis-[3H]methyldioxolane.

(+)-cis-[3H]Methyldioxolane ((+)-[3H]CD), a potent muscarinic agonist, was used to label high-affinity agonist states of muscarinic receptors in thin tissue sections of the rat central nervous system. Light microscopic autoradiography of atropine-sensitive (+)-[3H]CD binding sites revealed regions of dense labeling (superior colliculus, inferior colliculus, lateral geniculate body, hypoglossal (XII) nucleus, facial (VII) nucleus, tractus diagonalis) and regions of sparse labeling (hippocampus, dentate gyrus). The inverse regional correlation between high-affinity (+)-[3H]CD states and binding sites for the muscarinic antagonists [3H]pirenzepine (r = -0.79) and (-)-[3H]quinuclidinyl benzilate (r = -0.30) underscores potentially important differences between agonist and antagonist binding to CNS tissue slices.

Quantitative light microscopic autoradiography of [3H]hemicholinium-3 binding sites in the rat central nervous system: a novel biochemical marker for mapping the distribution of cholinergic nerve terminals.

The distribution of specific [3H]hemicholinium-3 ( [3H]HC-3) binding sites sites throughout the rat forebrain was studied by means of quantitative light microscopic autoradiography. Tissue sections were labeled with 2.5 nM [3H]HC-3, apposed to tritium-sensitive film for 2 months and analyzed by computer-assisted densitometry. Regions of intense [3H]HC-3 labeling include the caudate-putamen, nucleus accumbens, olfactory tubercle, amygdala, habenula and the granule cell layer of the dentate gyrus. Little or no specific binding was detected in the corpus callosum, a white matter region. This distribution of specific [3H]HC-3 binding sites is compatible with a selective labeling of central cholinergic nerve terminals.

The effects of cholecystokinin (CCK-8) on dopamine-containing nerve terminals in the caudate nucleus and nucleus accumbens of the anesthetized rat: an in vivo electrochemical study.

The action of cholecystokinin (CCK) on presynaptic function of dopaminergic nerve terminals has been the subject of much debate in the literature. In efforts to resolve some of the reported ambiguities, high speed in vivo electrochemical recordings were carried out in the caudate nucleus and nucleus accumbens of the urethane anesthetized rat, to determine effects of locally applied sulfated (CCK-8S) and unsulfated (CCK-8US) CCK octapeptide. Locally-applied CCK-8S and CCK-8US caused no increase in the baseline electrochemical signals recorded from either brain region. However, locally applied CCK-8S potentiated the potassium-evoked overflow of dopamine (DA) into the extracellular space in both the caudate and nucleus accumbens. In contrast, pressure ejection of CCK-8US produced no significant effects on the potassium-evoked overflow of DA in either structure. These data support a facilitatory effect of CCK-8S on potassium-evoked overflow from DA-containing nerve terminals in the urethane anesthetized rat that is likely mediated through a peripheral type CCK receptor.

Stimulation of synaptosomal tyrosine hydroxylation by phencyclidine in vitro.

Phencyclidine (PCP), a potent psychoactive drug, produces some animal behaviors that are believed to be mediated by dopaminergic and/or cholinergic neurons in the basal ganglia. In this study, we have monitored the effects of PCP in vitro on the synthesis, uptake, and release of dopamine (DA) in rat striatal synaptosomes. Using tyrosine hydroxylation as an index of DA synthesis, we observed a concentration-dependent stimulation of DA synthesis by PCP. The stimulatory effect was antagonized by reserpine (1 micro M) and was observed only when synaptosomes were preincubated under conditions which prevented the spontaneous release of [3H]DA. Two hydroxylated metabolites of PCP were also tested and found to have little effect on tyrosine hydroxylation. Like PCP these metabolites are potent inhibitors of synaptosomal [3H]DA uptake, but they apparently lack PCP's ability to release synaptosomal DA. Taken together, these results support our hypothesis that PCP stimulates synaptosomal DA synthesis by releasing DA from an inhibitory pool.

Regulation of stimulus-dependent hippocampal acetylcholine release by okadaic acid-sensitive phosphoprotein phosphatases.

Isolated nerve endings (synaptosomes) from rat hippocampus were used to characterize the influence by serine/threonine-specific phosphoprotein phosphatase (PP) inhibitors on acetylcholine release. Brief exposure to low concentrations of selective PP inhibitors (okadaic acid and calyculin A) caused a concentration-dependent attenuation of stimulus-dependent (calcium-evoked or potassium-evoked) [3H]acetylcholine ([3H]ACh) release, while having no effect on the rate of basal transmitter efflux. In view of the observed potencies for okadaic acid and calyculin A (pseudo-IC50 values near 3 nM), these data indicate that Type 1 (PP1) or Type 2A (PP2A) enzymes play a permissive role in exocytotic [3H]ACh release. In contrast, the absence of any measurable effect by sodium orthovanadate argues against a similar influence by tyrosine-specific phosphoprotein phosphatases. While the neuronal substrate(s) responsible for PP regulation of [3H]ACh release are unknown, the underlying mechanism clearly differs from that through which muscarinic autoreceptors act since inhibition by okadaic acid and oxotremorine (an autoreceptor agonist) are additive and the former is not blocked by the muscarinic receptor antagonist atropine. Based upon these results, we conclude that dephosphorylation steps catalyzed by okadaic acid-sensitive PP represent an important regulatory mechanism for stimulus-dependent transmitter release in septo-hippocampal cholinergic neurons.