Effects of low extracellular chloride on dopamine release and the dopamine transporter

Removal of chloride (Cl-) from the superfusion medium results in increased spontaneous efflux of dopamine (DA) but not acetylcholine from rabbit striatal slices prelabeled with [3H]DA and [14C]choline. Cl- was substituted to varying degrees with the impermeant anion, isethionate (IS-), or the permeant anion, nitrate (NO3-). The magnitude of low Cl(-)-induced DA efflux was inversely related to the external [Cl-] and was greater with IS- than with NO3-. Analysis of the composition of the 3H efflux in terms of DA and its metabolites revealed an increase in [3H]DA with decreasing Cl- concentration. Reduction of external Ca++ from 1.3 to 0.13 mM increased low Cl(-)-induced DA efflux. In slices depleted of vesicular DA by reserpine pretreatment and subsequently labeled and superfused in the presence of monoamine oxidase and catechol-O-methyltransferase inhibitors, the same inverse relationship between [Cl-] and spontaneous DA efflux was observed. Neuronal DA uptake inhibitors, nomifensine, mazindol, GBR-12909 and cocaine, all increased the rate of low Cl(-)-induced DA efflux in the reserpinized preparation. Cl(-)-induced DA efflux in untreated and reserpinized preparations was not blocked by tetrodotoxin, amiloride, furosemide, picrotoxin or 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid). Low Cl- inhibited initial rates of [3H]DA uptake. At Cl- concentrations producing significantly different efflux rates (0 and 7.4 mM Cl-, IS- and NO3- substitution), DA uptake was inhibited in all cases by greater than 90%. Additionally, the binding of [3H]mazindol, one of the uptake inhibitors, to striatal membranes was unaffected by removal of Cl-. In summary, low Cl- produces a nonexocytotic rapid outward transport of DA. Extracellular Cl- is not required for the binding to transporter sites or for the inhibition of neuronal uptake produced by neuronal uptake inhibitors.     

Muscarinic receptors mediating inhibition of gamma-aminobutyric acid release in rat corpus striatum and their pharmacological characterization

The effects of acetylcholine (ACh) and of cholinergic agonists on the release of tritiated gamma-aminobutyric acid ([3H]GABA) were studied in superfused synaptosomes prepared from rat corpus striatum and prelabeled with the radioactive amino acid. ACh, oxotremorine or (-)-nicotine, all tested at 100 microM had no effect on the spontaneous outflow of [3H]GABA. The depolarization-evoked overflow obtained by exposing the synaptosomes to 9 mM KCl was decreased in a concentration-dependent manner by ACh, oxotremorine, oxotremorine-M or carbachol. The maximal inhibition caused by ACh was 50%. The EC50 (agonist concentration causing half-maximal effect) amounted to 1 microM. Oxotremorine and oxotremorine-M were almost equipotent to ACh, whereas the concentration-response curve of carbachol was slightly (although not significantly) shifted to the right with respect to that of ACh. (-)-Nicotine (100 microM) did not affect the K(+)-evoked [3H]GABA overflow. ACh also inhibited the K(+)-evoked release of endogenous GABA. The inhibitory effect of 10 microM ACh on the release of [3H]GABA evoked by 9 mM KCl was insensitive to the nicotinic antagonist mecamylamine (10 microM) but it was potently blocked by the muscarinic antagonist atropine (IC50 = 5 nM) and weakly antagonized by pirenzepine, dicyclomine and AF-DX 116. The pharmacological profile of this receptor was very similar to that of the muscarinic autoreceptors regulating [3H]ACh release. The extent of [3H]GABA release inhibition caused by ACh did not differ between dorsal and ventral striatum. The inhibitory effect of ACh was much less pronounced in hippocampus and cortex than in the striatum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative dopamine-cholinergic mechanisms in the olfactory tubercle and the striatum: effects of metoclopramide

The olfactory tubercle (OT) is a limbic structure containing high dopamine (DA) and acetylcholine (ACh) concentrations. We performed a comparative study of the DA-ACh interactions, the efficacy of autoreceptor control and the effects of metoclopramide in the OT and the nucleus caudate (striatum). Rabbit brain slices from both regions of the same animal were prelabeled with radioactive DA and/or choline and then superfused. Comparable magnitude of DA and ACh release was evoked by electrical stimulation from both regions. DA release was unaltered, whereas ACh release was inversely related to the stimulation frequency, both in OT and striatum. Apomorphine (APO), a D1-D2 agonist, an LY-171555 (LY), a D2 agonist, inhibited DA and ACh release from OT and striatum with similar EC50 and Emax (maximal percentage of inhibition). However, the maximal degree of inhibition of ACh release achieved with APO, LY or DA in the OT was only one-half that observed in the striatum. In both regions, the inhibitory effects of DA agonists on DA and ACh release were reduced markedly when the number of electrical pulses and/or the frequency of stimulation were increased. l-Sulpiride, a DA D2 antagonist, increased the evoked release of DA and ACh from OT in direct relationship with the frequency of stimulation. In the OT, increases in synaptic DA achieved by administration of amphetamine or by blockade of the neuronal uptake pump with nomifensine inhibited the evoked release of ACh. Again these drug treatments produced only a 40 to 50% inhibition of ACh release. SKF 38393, a D1 agonist, had no effect per se on DA or ACh release in OT slices from control or from reserpine-treated animals (2 mg/kg s.c. for 3 or 7 days). With the exception of one specific dose combination, coadministration of SKF 38393 and LY produced no additive or synergistic effects on DA or ACh release from OT. APO- and LY-induced inhibition of DA and ACh release were antagonized by l-sulpiride. However, 300 nM SCH 23390, a D1 antagonist, reduced APO inhibition of DA and ACh release without affecting the inhibitory action of LY on DA and ACh release. Metoclopramide, "a DA antagonist with poor limbic activity", had a similar affinity for OT (pA2: 7.59) and striatal (pA2: 7.59) DA autoreceptors. Its antidopaminergic efficacy on DA receptors modulating ACh release from OT and striatum was also compared.(ABSTRACT TRUNCATED AT 400 WORDS)     

Frequency-dependent muscarinic receptor modulation of acetylcholine and dopamine release from rabbit striatum

The effects of muscarinic receptor activation on the electrically evoked release of [3H]dopamine (DA) and [14C]acetylcholine (ACh) or [3H]ACh were investigated in rabbit striatal slices. Release was measured in the presence of 10 microM hemicholinium and 1 microM sulpiride to block choline uptake and prevent the effects of released DA on DA receptors modulating release. Stimulation (120 pulses, 20 mA, 2 msec) at 0.3, 3 and 10 Hz produced (3H or 14C) ACh release that sharply declined with increasing stimulation frequency. A flat frequency-release curve was obtained for DA. Oxotremorine (OXO), a direct muscarinic agonist (1-100 microM), produced a concentration-dependent inhibition of ACh release, inversely related to stimulation frequency, at a fixed number of pulses (120). When the number of pulses was modified to produce similar amounts of ACh release (20 pulses at 0.1 Hz, 39 pulses at 0.3 Hz, 120 pulses at 3 Hz and 350 pulses at 10 Hz), much greater inhibition of ACh release by OXO (0.3 and 3 microM) was obtained with lower frequencies and lower number of pulses. Physostigmine, an acetylcholinesterase inhibitor, decreased ACh release with an inverse relationship to stimulation frequency. Atropine (1 microM), a selective muscarinic antagonist, enhanced the release of ACh more at 10 Hz than at 0.3 and 3 Hz and completely antagonized the effects of OXO (10 microM) and physostigmine (1 microM) at all three stimulation frequencies. OXO (3 and 10 microM) enhanced DA release at 3 Hz. Physostigmine (1 microM) and atropine (1 microM) had no effect on DA release.(ABSTRACT TRUNCATED AT 250 WORDS)     

N-methyl-D-aspartate increases acetylcholine release from rat striatum and cortex: its effect is augmented by choline.

We examined the effects of N-methyl-D-aspartate (NMDA), a glutamate agonist, and of glutamate itself, on acetylcholine (ACh) release from superfused rat striatal slices. In a Mg(++)-free medium, NMDA (32-1000 microM) as well as glutamate (1 mM) increased basal ACh release by 35 to 100% (all indicated differences, P less than .05), without altering tissue ACh or choline contents. This augmentation was blocked by Mg++ (1.2 mM) or by MK-801 (10 microM). Electrical stimulation (15 Hz, 75 mA) increased ACh release 9-fold (from 400 to 3660 pmol/mg of protein): this was enhanced (to 4850 pmol/mg of protein) by NMDA (100 microM). ACh levels in stimulated slices fell by 50 or 65% depending on the absence or presence of NMDA. The addition of choline (40 microM) increased ACh release both basally (570 pmol/mg of protein) and with electrical stimulation (6900 pmol/mg of protein). In stimulated slices choline acted synergistically with NMDA, raising ACh release to 10,520 pmol/mg of protein. The presence of choline also blocked the fall in tissue ACh. No treatment affected tissue phospholipid or protein levels. NMDA (32-320 microM) also augmented basal ACh release from cortical but not hippocampal slices. Choline efflux from striatal and cortical (but not hippocampal) slices decreased by 34 to 50% in Mg(++)-free medium. These data indicate that NMDA-like drugs may be useful, particularly in combination with choline, to enhance striatal and cortical cholinergic activity. ACh release from rat hippocampus apparently is not affected by NMDA receptors.     

Effects of amiloride and 5-(N,N-hexamethylene)amiloride on dopaminergic and cholinergic neurotransmission

In order to establish the role of the Na+/H+ exchange transport on neurotransmission, we investigated the effects of amiloride and of 5-(N,N-hexamethylene)amiloride (HMA) on dopamine (DA) and acetylcholine (ACh) release and on receptor-mediated modulation of DA and ACh release. Superfused rabbit striatal slices prelabeled with [3H]DA and [14C]choline were stimulated electrically in the presence and absence of several concentrations of these agents. Amiloride (3-10 microM) and HMA (0.3-10 microM) reduced the basal efflux and the stimulation evoked overflow of total 3H and of [3H]-3,4-dihydroxyphenylacetic acid and inhibited monoamine oxidase activity. The inhibition of stimulation evoked overflow of total 3H was blocked by pretreatment with nomifensine but not by sulpiride. Amiloride had no effect on the basal efflux and the stimulation evoked overflow of ACh or it did modify apomorphine-induced inhibition of DA and ACh release. However, at 3 to 10 microM, HMA enhanced the basal efflux of 3H; this effect was not prevented either by uptake inhibition with nomifensine or by low extracellular calcium. These results suggest that amiloride-sensitive Na+ transport and the amiloride and HMA-sensitive Na+/H+ antiporter play no role on the secretion of DA and ACh, or on the mechanisms by which activation of pre- and postsynaptic DA receptors lead to inhibition of neurotransmitter release. Amiloride- and HMA-induced monoamine oxidase inhibition accounts for the effects of amiloride and HMA on DA efflux and overflow. The guanidine moiety present in the amiloride and HMA molecules is most likely responsible for these effects.     

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

The release of endogenous dopamine (DA) elicited by electrical stimulation and by d-amphetamine (AMPH) from superfused striatal slices of untreated rabbits was examined. AMPH (0.3-10 microM) produced a concentration-dependent increase in basal DA efflux (30-fold increase at 10 microM) and stimulation-evoked (SE) DA overflow (11-fold increase at 10 microM). Although AMPH had little effect on the basal efflux of dihydroxyphenylacetic acid (DOPAC), the drug was an effective inhibitor of the SE overflow of the DA metabolite (66% inhibition at 0.3 microM). AMPH increased significantly the total basal efflux of endogenous compounds (DA + DOPAC) only at high concentrations (3-10 microM) whereas the total SE overflow of total endogenous compounds was decreased at all concentrations of AMPH tested. AMPH inhibited SE [3H]acetylcholine (ACh) release in a concentration-dependent manner (71% inhibition at 10 microM). Inhibition of DA synthesis with alpha-methyl-p-tyrosine (100 microM) or 3-iodotyrosine (100 microM) reduced both the basal efflux and SE overflow of endogenous DA and DOPAC; synthesis inhibition had greater effects on the SE overflow. Neither synthesis inhibitor altered SE [3H]ACh release. alpha-Methyl-p-tyrosine and 3-iodotyrosine reduced the absolute values of the basal efflux and SE overflow of DA elicited by AMPH by approximately 60%; however, the inhibition of SE [3H]ACh release produced by AMPH was attenuated only slightly (approximately 20%). Synthesis inhibitors also reduced tissue DA levels (approximately 30%). These results suggest that: basal efflux of endogenous DA from superfused rabbit striatal slices may derived both from DA newly synthesized in the cytoplasm and from spontaneous leakage of DA from storage vesicles. In addition, synthesis may provide a continuous supply of DA to vesicles that are used for exocytotic DA release during electrical stimulation. However, the depletion of tissue DA produced by synthesis inhibitors as well as other extraneous pharmacological actions of these drugs makes firm conclusions difficult. AMPH increases the synaptic concentration of DA by accelerating the basal efflux as well as the SE overflow of unchanged DA. At concentrations less than 1 microM AMPH has no effect on basal efflux of DA or DOPAC but reduces SE overflow of DOPAC via an unknown mechanism. At higher concentrations (greater than or equal to 1 microM) acceleration of carrier-mediated DA efflux coupled with displacement of DA from vesicular stores, as well as interference with the uptake of exocytotically released DA produces a marked increase in synaptic DA which in turn inhibits SE ACh release.(ABSTRACT TRUNCATED AT 400 WORDS)     

Dissociation between muscarinic receptor-mediated inhibition of adenylate cyclase and autoreceptor inhibition of [3H] acetylcholine release in rat hippocampus

Activation of muscarinic cholinergic receptors (mAChRs) in the central nervous system reduces the catalytic activity of membrane-bound adenylate cyclase and attenuates depolarization-dependent release of acetylcholine (ACh). Inasmuch as reports have indicated that these mAChR-mediated responses exhibit pharmacological profiles similar to the M2 subclass of mAChR, the present studies were undertaken to ascertain whether attenuation of presynaptic adenylate cyclase activity [and concurrent reduction of intraneuronal cyclic AMP (cAMP) levels] underlies mAChR-mediated autoinhibition of electrically evoked ACh release. In [3H]choline-prelabeled rat hippocampal slices, the mAChR agonists oxotremorine (EC50 = 15 microM) and carbachol (EC50 = 80 microM) caused atropine-reversible inhibition of [3H]ACh release up to a maximum of 80% reduction. The rank order of potency for antagonist reversal of this inhibitory action (N-methylatropine = atropine greater than scopolamine much greater than pirenzepine) was generally consistent with an M2 mAChR-mediated response although pirenzepine was ineffective up to 1 mM. Under these assay conditions, forskolin (1-10 microM) and 8-bromo-cAMP (30-300 microM) enhanced electrically evoked [3H]ACh release maximally by 50 to 60%; however, neither agent significantly reversed mAChR agonist-induced inhibition of [3H]ACh release. Additional studies were undertaken to determine the consequences of chemically uncoupling mAChR from their G protein-adenylate cyclase effector system in this tissue. Whereas brief pretreatment with the sulfhydryl alkylating agent N-ethylmaleimide (30 microM) or pertussis toxin (1 microgram/ml) markedly attenuated carbachol inhibition of adenylate cyclase activity in hippocampal tissue, there was no concurrent reduction of carbachol-inhibited [3H] ACh release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kappa receptor regulation of dopamine release from striatum and cortex of rats and guinea pigs

The effects of opioid agonists with selectivity for kappa, mu and delta types of opioid receptors on the K+-stimulated release of [3H]dopamine (DA) from striatum and cortex of rat and guinea pig loaded previously with the monoamine have been studied. The kappa agonist U50488H did not affect base-line release of [3H]DA measured in 5 mM K+, but produced a dose-dependent inhibition of the release of [3H]DA stimulated by 20 mM K+ from slices of striatum in rat and guinea pig, with an IC50 of about 0.5 nM in each case. In contrast, the mu-selective agonist, Tyr-D-Ala-Gly-(Me)Phe-Gly-ol, and the delta-selective agonist, [D-Pen2-D-Pen5]enkephalin, did not inhibit stimulated release from the slice preparations at concentrations up to 1 microM. The inhibitory effects of U50488H were antagonized by naloxone, and the potent and selective kappa antagonist, nor-binaltorphimine (nor-BNI). Similar results were obtained when release of [3H]DA from rat and guinea pig cortex slices was examined. In guinea pig cortex, the maximum inhibition of DA release induced by U50488H was 80% of control-stimulated fractional release. In rat cortex and in striatum of both species the maximum release was about 40% of control fractional release. Thus, in the guinea pig, the mesocortical dopaminergic pathway appears more sensitive to the inhibitory effects of U50488H than the nigrostriatal system. The effects of the opioids on the K+ (12.5 mM)-stimulated release of [3H]DA from guinea pig striatal synaptosomes also were determined.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phorbol esters and D2-dopamine receptors

4-beta-Phorbol-12,13-dibutyrate (PDBu), a powerful activator of protein kinase C (PKC), enhanced dopamine (DA) release evoked by electrical stimulation (1 Hz, 2 min) from the striatum and the prefrontal cortex of the rabbit. However, acetylcholine (ACh) release from the striatum (1 Hz, 2 min), was only enhanced slightly by PDBu. The increase in DA release induced by PDBu was reduced markedly at higher frequencies of stimulation. Sulpiride (10 microM) alone, a D2 DA-receptor antagonist, or combined with nomifensine (3 microM), a neuronal-uptake inhibitor, did not prevent PDBu-induced facilitation of DA release from prefrontal cortex or striatum. The D2 DA agonists (LY-171555, bromocriptine and apomorphine) inhibited in a concentration-dependent manner the stimulation-evoked overflow of DA and ACh from the striatum, and of DA from the prefrontal cortex. Pretreatment with PDBu antagonized the inhibitory effect of the three agonists on DA and ACh release. A reduction both in Emax and IC50 was observed in PDBu-treated slices. Removal of endogenous DA by pretreatment with reserpine and alpha-methyl-p-tyrosine, failed to prevent PDBu-induced antagonism of apomorphine effects on ACh release, indicating that the antagonism of agonist effects was not due to higher synaptic levels of endogenous DA. The inactive enantiomer of PDBu, 4-alpha-12,13-dibutyrate did not enhance DA release and failed to modify the effects of D2 agonists on DA release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacological characterization of endogenous acetylcholine release from primary septal cultures

A detailed investigation of endogenous acetylcholine (ACh) release from primary embryonic septal cultures is described in this study. Applications of veratridine (25 microM) or increasing extracellular concentrations of K(+) (6-100 mM) induced robust increases of endogenous ACh release ( approximately 500-15,000 fmol/well/10 min). Release stimulated with K(+) (25 mM) was sustainable and did not differ significantly over 180 min. ACh release was dependent on extracellular choline and decreased proportionally to choline concentrations (0-10 microM). For example, after 30 min of stimulation with K(+) (25 mM), release in the absence of extracellular choline was approximately 25% of that associated with 10 microM choline. The vesicular transport blocker vesamicol (0-5 microM) almost completely prevented stimulated and basal ACh release at the highest concentration evaluated, which suggests a mostly vesicular mode of release in this model. The M(2)-like muscarinic receptor antagonist AF-DX 384 (0-10 microM) enhanced stimulated ACh release ( approximately 150% at the highest concentration evaluated), whereas the nonspecific muscarinic receptor agonist oxotremorine (0-10 microM) decreased stimulated release (approximately 60% at the highest concentration evaluated), suggesting that functional muscarinic autoreceptors exist in primary embryonic septal cultures. Novel findings concerning ACh release from primary embryonic septal cultures are reported herein, and the demonstration of ACh release gives further credit to the use of these cultures for studying cholinergic system functioning and in relation to physiology and pathology.     

Relationships between choline uptake, acetylcholine synthesis and acetylcholine release in isolated rat atria

Isolated rat atria take up [3H]choline and synthesize [3H]acetylcholine (ACh). The uptake of [3H]choline has a high-affinity component with a Km of approximately 0.2 microM and a Vmax of approximately 6 fmol/min/mg wet wt. This high-affinity component of choline uptake is difficult to measure directly because it represents only a small portion of total [3H]choline uptake. However, the rate of synthesis of [3H] ACh from [3H]choline appears to reflect the activity of the high-affinity choline uptake system. Thus, [3H]ACh synthesis is most efficient at low choline concentrations and is inhibited in the presence of hemicholinium-3 and low NaCl medium. The neuronal localization of the [3H]Ach synthesized from [3H]choline is demonstrated by the finding that [3H]ACh is released from the atria by depolarization with 57 mM K+ medium. The release is Ca++ -dependent and there is a compensatory increase in the synthesis of [3H]ACh after depolarization-induced ACh release. These data suggest that [3H]choline can be specifically incorporated into a releasable pool of [3H]ACh localized in cardiac parasympathetic neurons. The synthesis of [3H]ACh is inhibited by blockade of high-affinity choline uptake and is regulated in response to neuronal activity. The application of these methods will provide a means for directly examining the physiological and pharmacological control of ACh synthesis and release from cardiac parasympathetic neurons.     

Wash-resistantly bound xanomeline inhibits acetylcholine release by persistent activation of presynaptic M(2) and M(4) muscarinic receptors in rat brain

We studied the effects of 3-[3-hexyloxy-1,2,5-thiadiazo-4-yl]-1,2,5,6-tetrahydro-1-methylpyridine (xanomeline) wash-resistant binding on presynaptic muscarinic regulation of electrically evoked [(3)H]acetylcholine (ACh) release from rat brain slices. In both cortical and striatal tissues that possess M(2) and M(4) autoreceptors, respectively, immediate application of 10 microM xanomeline had no effect on evoked [(3)H]ACh release or its inhibition by 10 microM carbachol. In contrast, preincubation with 1, 10, or 100 microM xanomeline for 15 min decreased evoked release of ACh measured after 53 min of washing in xanomeline-free medium in a concentration-dependent manner. The maximal inhibitory effect equaled the immediate effect of the muscarinic full agonist carbachol, and it was completely (at 1 and 10 microM xanomeline) or partially (at 100 microM xanomeline) blocked by 1 microM N-methylscopolamine. Neither presence of N-methylscopolamine during 100 microM xanomeline treatment nor previous irreversible inactivation of the classical receptor binding site using propylbenzylcholine mustard in cortical slices prevented the inhibitory effect of wash-resistantly bound xanomeline. Treatment of cortical slices with xanomeline slightly decreased the number of muscarinic binding sites, and it markedly decreased affinity for N-methylscopolamine. When applied as in acetylcholine release experiments, xanomeline did not impair presynaptic alpha(2)-adrenoceptor-mediated regulation of noradrenaline release. The functional studies in brain tissue reported in this work demonstrate that xanomeline can function as a wash-resistant agonist of native presynaptic muscarinic M(2) and M(4) receptors with both competitive and allosteric components of action.     

Evidence for autoreceptor modulation of endogenous dopamine release from rabbit caudate nucleus in vitro

This study was designed to determine if the release of endogenous dopamine (DA), like [3H]DA, is modulated by inhibitory autoreceptors. A high-performance liquid chromatographic assay was developed which was capable of detecting the basal efflux and electrically evoked overflow of endogenous DA and dihydroxyphenylacetic acid (DOPAC), the primary DA metabolite. In the absence of neuronal uptake inhibitors the stimulation-evoked overflow of endogenous DA was entirely in the form of DOPAC, whereas overflow consisted primarily of DA in the presence of uptake inhibition. The evoked overflow of DA and DOPAC was abolished by reduction of the Ca++ concentration of the superfusion medium from 1.3 to 0.13 mM. The DA receptor antagonist sulpiride (1 microM) increased DOPAC overflow by 41%. Nomifensine (10 microM) increased slightly and cocaine (10 microM) decreased slightly the total overflow of endogenous compounds (DA plus DOPAC). Combination of nomifensine and sulpiride or cocaine and sulpiride increased total overflow of endogenous compounds by 217 and 120%, respectively, as compared to the neuronal uptake inhibitors alone. The DA receptor agonists apomorphine (0.3 microM) and bromocriptine (1 microM) inhibited DOPAC overflow by 92 and 83%, respectively. However, apomorphine and bromocriptine failed to inhibit endogenous DA release in the presence of nomifensine. Sulpiride antagonized the inhibitory effects of both apomorphine and bromocriptine. In experiments in which [3H]DA and endogenous DA overflow were measured simultaneously, radiolabeled DA behaved exactly like the endogenous transmitter.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sustained high release at rapid stimulation rates and reduced functional autoreceptors characterize prefrontal cortex dopamine terminals

The release of dopamine (DA) from mesocortical and nigrostriatal nerve terminal fields, as well as its modulation by auto- and heteroreceptors was investigated. Rabbit brain slices obtained from medial prefrontal cortex (PFC) and nucleus caudate (striatum) were prelabeled with [3H]DA in the presence of 0.3 microM desipramine. Neuronal depolarization was elicited by electrical stimulation. Higher stimulation-evoked overflow of [3H]DA (release) was observed from PFC than from striatal slices. At 0.3 Hz (120 pulses) release from the PFC was 60% higher than from the striatum, and at higher frequencies (10 Hz and 120 or 1200 pulses) the fraction of tissue radioactivity released from the PFC was 550% greater than that released from the striatum. These differences were not eliminated by blockade of autoreceptors with haloperidol, or by inhibition of neuronal uptake with nomifensine. These results suggest that the coupling between neuronal depolarization and DA release is more efficient in the PFC than in the striatum. This may allow the PFC terminals to sustain neurotransmission under continuous fast firing. Selective D2 agonists, as well as nonselective DA agonists, inhibited DA release in a concentration-dependent fashion from the PFC and the striatum. Their effects were blocked by l-sulpiride or haloperidol. SKF 38393, a selective D1 agonist, produced a small facilitation of release from both regions; its effects were blocked by SCH 23390 (a selective D1 antagonist). The latter was ineffective on its own. The maximal degree of inhibition of release produced by apomorphine, bromocriptine and LY-171555 was lower in the PFC than in the striatum; these differences were accentuated greatly at high stimulation rates. When the slices were stimulated at frequencies comparable to the "in vivo" firing rates for each neuronal group, apomorphine and LY-171555 were much weaker in inhibiting DA release from the PFC (10 Hz) than from the striatum (3 Hz). In the striatum, strong modulation of DA release by endogenous DA was observed; whereas little modulation was seen in the PFC. Nomifensine produced larger increases in the stimulation-evoked overflow of DA from PFC and there was no synergistic interaction between nomifensine and haloperidol in this structure. In the striatum, marked facilitation of DA overflow was observed when nomifensine and haloperidol were given together. Furthermore, haloperidol per se facilitated DA release from both brain regions; however, the degree of facilitation was frequency dependent in the striatum, but not in the PFC.(ABSTRACT TRUNCATED AT 400 WORDS)     

gamma-Aminobutyric acid-induced modulation of acetylcholine release from the guinea pig lung

gamma-Aminobutyric acid (GABA) content was measured biochemically and the effect of GABA on the release of [3H]acetylcholine (ACh) was studied in strips of the guinea pig lung preloaded with [3H]choline. GABA contents were highest in the middle sections of the lung, as compared with proximal and distal areas. GABA evoked the release of [3H]ACh from the strips of the lung. The effect of GABA was mimicked by muscimol and antagonized by bicuculline and furosemide. Perfusion with Ca++-free medium and tetrodotoxin, but not nipecotic acid, inhibited the GABA- and muscimol-evoked release of [3H]ACh, thereby indicating that the released ACh was of neuronal origin. Diazepam and pentobarbital potentiated the muscimol-evoked [3H]ACh release. On the other hand, GABA reduced the KCl (40 mM)-evoked release of [3H]ACh in the presence of tetrodotoxin and bicuculline and baclofen mimicked the inhibitory effect of GABA. The effects of GABA and baclofen were not altered by alpha and beta adrenergic antagonists. These findings provide evidence for two types of GABA receptors in the lung of the guinea pig, and these receptors are involved in regulating the release of ACh.     

Effects of microdialyzed oxotremorine, carbachol, epibatidine, and scopolamine on intraspinal release of acetylcholine in the rat

Intrathecally administered cholinergic agonists such as oxotremorine (muscarinic), carbachol (mixed nicotinic and muscarinic agonist), and epibatidine (nicotinic) have all been shown to reduce nociception in behavioral studies. Thus, there is substantial evidence for a role of acetylcholine (ACh) in the control of nociception in the spinal cord, but the mechanisms regulating ACh release are not known. The present study was initiated to establish a rat model to study which mechanisms are involved in the control of ACh release. Spinal microdialysis probes were inserted intraspinally at the C1-C5 spinal level in isoflurane-anesthetized rats. The probes were perfused with Ringer's solution containing 10 microM neostigmine to prevent degradation of ACh. Oxotremorine, carbachol, epibatidine, and scopolamine, dissolved in Ringer's solution, were administered intraspinally via dialysis and 30 microliter/10-min samples of dialysate were collected for HPLC analysis of ACh content. The release of ACh was found to be constant in the control (Ringer's only) situation during the experimental period of 150 min. Oxotremorine (100-1000 microM), carbachol (1 mM), and epibatidine (50-5000 microM) enhanced but scopolamine (50-200 nM) decreased the intraspinal release of ACh. Oxotremorine (ED(50) = 118 microM) and epibatidine (ED(50) = 175 microM) were found to produce a dose-dependent increase of ACh release. Cholinergic agonists caused an increase of intraspinal ACh and the antagonist scopolamine caused a decreased release of ACh. The data do not support an autoreceptor function of either nicotinic or muscarinic receptors in the spinal cord, contrary to what has been observed in the brain.     

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

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)     

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

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)     

Pharmacologic characterization and functional role of muscarinic autoreceptors in the rabbit striatum

The objectives of the present studies were 1) to pharmacologically characterize the muscarinic autoreceptors in the striatum and 2) to examine their role in the regulation of physiologic acetylcholine (ACh) release. Schild plots were generated for atropine and pirenzepine against oxotremorine-induced inhibition of [3H]ACh release. Atropine, a nonselective antagonist, yielded a pA2 of 8.92. The pA2 for pirenzepine, a purported M1-selective antagonist, was 7.14. Both Schild plots had slopes not significantly different from one. Four agonists [oxotremorine, carbachol (CARB), McN-A-343 and pilocarpine] were tested for their effectiveness in inhibiting [3H]ACh release. McN-A-343 and pilocarpine have been reported to be selective for M1 receptors. Oxotremorine and carbachol were effective and potent inhibitors of [3H] ACh release, whereas McN-A-343 and pilocarpine were weak. Although the existence of muscarinic receptor subtypes remains an open question, these data are consistent with the "low" pirenzepine affinity (M2) subtype. Chronic treatments (14 days) with several agents were carried out (in vivo) to assess the role of muscarinic autoreceptors in the regulation of physiologic ACh release. Scatchard analyses of binding studies with [3H]quinuclidinyl benzilate were also performed to assess changes in the muscarinic receptor population in the striatum. Chronic treatment with scopolamine caused a 100% increase in the Bmax for [3H]quinuclidinyl benzilate binding but had no effect on the sensitivity of [3H]ACh release to inhibition by CARB. Fourteen-day treatment with physostigmine (3 mg/kg) produced a decrease in the sensitivity of [3H]ACh release to CARB plus a 42% decrease in Bmax and a 48% decrease in Kd for [3H]quinuclidinyl benzilate binding. Chronic haloperidol treatment caused an increase in the sensitivity of [3H]ACh release to CARB accompanied by a 46% increase in Bmax for 3H quinuclidinyl benzilate binding. These data suggest that muscarinic autoreceptors in the striatum do not regulate physiologic ACh release in the presence of intact acetylcholinesterase and that the interaction of dopaminergic and cholinergic neurons in the striatum may not be simple trans-synaptic inhibition.