Differential Effects of Bromocriptine on Dopamine and Acetylcholine Release Modulatory Receptors

Rabbit neostriatal slices were prelabeled with [3H]dopamine (DA) and [14C]choline and then superfused. The electrical stimulation-evoked release of DA and of acetylcholine (ACh) was abolished by 0.33 microM tetrodotoxin and by low calcium concentrations (0.13 mM). Bromocriptine, a selective D2-DA receptor agonist, inhibited in a concentration-dependent manner the evoked overflow of DA and ACh, without affecting the basal efflux of both transmitters. The effects of bromocriptine were antagonized by sulpiride, a specific antagonist of D2-DA receptors. With stimulation at 0.3 Hz and 120 pulses, bromocriptine was eight times more potent in inhibiting the evoked overflow of DA (IC50: 11 nM) than that of ACh (IC50: 83 nM). Stimulations at 3 Hz and 360 pulses markedly reduced the potency of bromocriptine in inhibiting DA and ACh release, and diminished its selectivity for presynaptic receptors. These results indicate that DA receptors that modulate the release of DA and ACh are of the D2 subtype. The greater potency of bromocriptine at pre- than at postsynaptic sites suggests that these receptors may be different in quantity and/or quality [D2-alpha (presynaptic) versus D2-beta (postsynaptic)]. Finally, marked differences in the potency and efficacy of DA agonist actions on DA and ACh release modulatory receptors are obtained, depending on the parameters of stimulation used.     

Nicotinic Autoreceptors Mediating Enhancement of Acetylcholine Release Become Operative in Conditions of "Impaired" Cholinergic Presynaptic Function

Abstract: The existence in the mammalian CNS of release-inhibiting muscarinic autoreceptors is well established. In contrast, few reports have focused on nicotinic autoreceptors mediating enhancement of acetylcholine (ACh) release. Moreover, it is unclear under what conditions the function of one type of autoreceptor prevails over that of the other. Rat cerebrocortex slices, prelabeled with [³H]choline, were stimulated electrically at 3 or 0.1 Hz. The release of [³H]ACh evoked at both frequencies was inhibited by oxotremorine, a muscarinic receptor agonist, and stimulated by atropine, a muscarinic antagonist. Nicotine, ineffective at 3 Hz, enhanced [³H]ACh release at 0.1 Hz; mecamylamine, a nicotinic antagonist, had no effect at 3 Hz but inhibited [³H]ACh release at 0.1 Hz. The cholinesterase inhibitor neostigmine decreased [³H]ACh release at 3 Hz but not at 0.1 Hz; in the presence of atropine, neostigmine potentiated [³H]ACh release, an effect blocked by mecamylamine. In synaptosomes depolarized with 15 mM KCI, ACh inhibited [³H]ACh release; this inhibition was reversed to an enhancement when the external [Ca²⁺] was lowered. The same occurred when, at 1.2 mM Ca²⁺, external [K⁺] was decreased. Oxotremorine still inhibited [³H]ACh release at 0.1 mM Ca²⁺. When muscarinic receptors were inactivated with atropine, the K⁺ (15 mM)-evoked release of [³H]ACh (at 0.1 mM Ca²⁺) was potently enhanced by ACh acting at nicotinic receptors (EC₅₀? 0.6 µM). In conclusion, synaptic ACh concentration does not seem to determine whether muscarinic or nicotinic autoreceptors are activated. Although muscarinic autoreceptors prevail under normal conditions, nicotinic autoreceptors appear to become responsive to endogenous ACh and to exogenous nicotinic agents under conditions mimicking impairment of ACh release. Our data may explain in part the reported efficacy of cholinesterase inhibitors (and nicotinic agonists) in Alzheimer's disease.     

Dopaminergic Receptors in Bovine Retina and Their Interaction with Thyrotropin-Releasing Hormone

A superfusion technique was employed to study the release of [3H]dopamine from isolated bovine retina. Only K+-stimulated release was observed from both light- and dark-adapted retina; release by other stimuli was from dark-adapted retina only. Light-evoked release of [3H]dopamine from dark-adapted retina was blocked by thyrotropin-releasing hormone (TRH), which has previously been identified as a retinal neuropeptide. TRH itself released small amounts of [3H]dopamine from dark-adapted retina. These results are interpreted as indicating that TRH acts as a modulator of dopaminergic activity in retina through the agency of presynaptic autoreceptors. Evidence of the existence of a feedback inhibition system, probably mediated by dopaminergic autoreceptors, was found by the inclusion of sulpiride, a dopaminergic D2 receptor antagonist in the perfusate, which, in a stereoselective manner, enhanced spontaneous and light-evoked release of [3H]dopamine. On the other hand, dopamine (1 microM) reduced these effects. TRH did not affect the high-affinity uptake system for dopamine in retina; this, then, could not account for the effects on release. Radioligand binding showed a specific, saturable high-affinity binding system for [3H]TRH, with an apparent KD of 2.2 nM and a Bmax of 23 fmol/mg protein in bovine retinal membranes. Displacement experiments showed that specific [3H]TRH binding was displaced in the nanomolar range by spiperone and in the micromolar range by dopamine, whereas L-(--)-sulpiride was virtually inactive in displacing [3H]TRH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Frequency-Dependent Release of Acetylcholine and Dopamine From Rabbit Striatum: Its Modulation by Dopaminergic Receptors

Abstract: The release of [³H]dopamine (DA) and [¹⁴C]acetylcholine (ACh) was monitored from single slices of the rabbit striatum. In all cases, the evoked overflow of ACh showed a higher peak and was of shorter duration than that of ³H products. For ACh, the release per pulse showed a marked decline with increasing frequency of stimulation, whereas flat frequency-release curves were obtained for DA. At 0.1 and 1 Hz the evoked overflows of ACh were 15 and 7 times greater, respectively, than those of DA. Haloperidol (0.03 μM) and sulpiride (1 μM) produced large increases in the evoked overflow of DA and ACh at 3 and 10 Hz; little effect was observed at lower frequencies. These results indicate that the frequency-release curves for DA and ACh are different and that at high frequencies the slope of the curves is modified by activation of pre- and postsynaptic DA receptors. Apomorphine inhibited in a concentration-dependent fashion the evoked overflow of DA and ACh; greater inhibition was obtained at lower frequencies of stimulation. At 0.3 Hz the- DA agonist was two times more potent in inhibiting DA than ACh overflow (IC₅₀: 12.0 ± 2.2 versus 22.0 ± 2.8 nM; p < 0.01). The greater sensitivity of pre-than postsynaptic sites to apomorphine was also seen at higher frequencies (3 Hz). Benztropine (1/μ) reduced the evoked overflow of ACh at 10 Hz, and enhanced that of ³H products at all rates of stimulation (0.3–10 Hz). These results suggest that the release of DA and ACh is regulated by dopaminergic receptors. They also indicate that the effects of DA agonists and antagonists and of uptake inhibitors on DA and ACh release are highly dependent on the frequency of stimulation used.     

GABA Autoreceptors Are Not Coupled to Benzodiazepine Receptors in Rat Cerebral Cortex

Abstract: Depolarization-induced release of [³H] γ -aminobutyric acid ([³H]-GABA) from preloaded slices of rat cerebral cortex was inhibited by muscimol and THIP in a dose-dependent fashion. This inhibition of release was prevented by the GABA antagonists bicuculline and picrotoxin. These results confirm previous reports postulating the existence of GABA autoreceptors on GABAergic terminals. Since benzodiazapines are known to facilitate postsynaptic GABA actions, the effect of flunitrazepam on the inhibition of GABA release mediated through the autoreceptors has been examined. At a concentration of 1 μ m or 10 μ m , flunitrazepam had no effect on the IC₅₀ values for muscimol or THIP in inhibiting stimulated GABA release. It thus seems that GABA autoreceptors are not functionally coupled to benzodiazepine receptors in rat cerebral cortex.     

Regulation of Neuropeptide Y Release by Neuropeptide Y Receptor Ligands and Calcium Channel Antagonists in Hypothalamic Slices

Neuropeptide Y (NPY) is an important regulator of energy balance in mammals through its orexigenic, antithermogenic, and insulin secretagogue actions. We investigated the regulation of endogenous NPY release from rat hypothalamic slices by NPY receptor ligands and calcium channel antagonists. High-potassium stimulation (60 mM) of the slices produced a calcium-dependent threefold increase in NPY release above basal release. The Y2 receptor agonists NPY(13-36) and N-acetyl[Leu28,Leu31]NPY(24-36), the Y4 agonist rat pancreatic polypeptide (rPP), and the Y4/Y5 agonist human pancreatic polypeptide (hPP) significantly reduced both basal and stimulated NPY release. NPY(13-36)-induced reduction of NPY release could be partially prevented in the presence of the weak Y2 antagonist T4-[NPY(33-36)]4, whereas the hPP- and rPP-induced inhibition of release was not affected by the Y5 antagonist CGP71683A or the Y1 antagonist BIBP3226. The selective Y1, Y2, and Y5 antagonists had no effect on either basal or potassium-stimulated release when administered alone. The calcium channel inhibitors omega-conotoxin GVIA (N-type), omega-agatoxin TK (P/Q-type), and omega-conotoxin MVIIC (Q-type) all significantly inhibited potassium-stimulated NPY release, without any effect on basal release, whereas nifedipine had no effect on either basal or stimulated release. Addition of both omega-conotoxin GVIA and omega-agatoxin TK together completely inhibited the potassium-stimulated release. In conclusion, we have demonstrated that NPY release from hypothalamic slices is calcium-dependent, involving N-, P-, and Q-type calcium channels. NPY release is also inhibited by Y2 agonists and rPP/hPP, suggesting that Y2 and Y4 receptors may act as autoreceptors on NPY-containing nerve terminals.     

A Microdialysis Study of the Regulation of Endogenous Noradrenaline Release in the Rat Hippocampus

In vivo microdialysis was used to investigate the regulation of noradrenaline release in rat hippocampus. Idazoxan, an alpha 2-adrenoreceptor antagonist (1-10 mg/kg), increased noradrenaline release in a dose-dependent manner. Inhibition of noradrenaline uptake by desipramine (0.05-20 microM; via the probe) also increased the extracellular content of the transmitter. In the presence of this increased noradrenaline content (desipramine via the probe), the effect of a low dose of idazoxan (1 mg/kg) was potentiated. Local perfusion of idazoxan (1-500 microM) in the hippocampus also increased noradrenaline release but not to the same extent as following systemic administration. In the presence of desipramine, unlike the systemic injection of idazoxan, local perfusion did not potentiate noradrenaline release. The data are consistent with the regulation of extracellular noradrenaline content in the hippocampus by neuronal uptake and to a lesser extent by presynaptic autoreceptors.     

Dynamic Observation of Dopamine Autoreceptor Effects in Rat Striatal Slices

Fast-scan cyclic voltammetry has been used to measure dopamine (DA) synaptic overflow in slices of rat caudate nucleus induced by electrical stimulation with one-, two-, and 50-pulse, 10-Hz trains. Synaptic overflow in this preparation is shown to be the result of the competing effects of release and cellular uptake. Release caused by all pulses was attenuated by the D2 agonist quinpirole (1 microM). The rapid time response of the measurements (100 ms) allows the autoinhibition induced by endogenous, released DA to be resolved in real time. The concentration of DA released during the second pulse of a train was 58% of that released by the first pulse, an effect that is partially blocked by the addition of 2 microM sulpiride, a D2 antagonist, to the perfusion buffer. DA release during the first stimulus pulse is unaffected by 2 microM sulpiride, suggesting that autoreceptors are not normally occupied in this preparation. Release caused by the third pulse was 14% of the first pulse and also could be partially enhanced by 2 microM sulpiride. The duration of the inhibition of release induced by endogenous DA was estimated by varying the interval between one-pulse stimulations until the overflow of DA induced by the second pulse was equal to that on the first; a half-time of approximately 17 s was found. The addition of picrotoxin (100 microM) and glutamate (10 microM) to the perfusion buffer did not affect stimulated release of DA, although the addition of atropine (100 microM) attenuated overflow for all the trains tested.     

Multiple Factors Influencing the In Vitro Release of [Met5]-Enkephalin from Rat Hypothalamic Slices

This study examined several in vivo and in vitro factors which influence the release of [Met5]-enkephalin (Met-ENK) from male rat hypothalamic slices superfused in vitro. Met-ENK release was significantly stimulated by corticotropin-releasing hormone (CRH; 10(-12)-10(-8) M), an effect which was abolished in the presence of the CRH-receptor antagonist, alpha-helical CRF9-41 (10(-6) M). The amount of Met-ENK release diminished with time in experiments in which the slices were continuously exposed to CRH. The opioid receptor antagonist naloxone (10(-6) M) stimulated Met-ENK release, even in the presence of the Na+ -channel blocker tetrodotoxin (10(-6) M), a result indicating presynaptic opioid feedback inhibition of Met-ENK release. The role of gonadal steroids in the control of Met-ENK release in vitro was also examined. It was found that the basal and CRH-induced release of Met-ENK was not changed 1 week after castration. However, a significant increase in the basal release of this peptide was observed 4 weeks after gonadectomy, and the Met-ENK-releasing efficacy of CRH was found to be reduced. The Met-ENK content of hypothalami from 1-week castrates was not significantly changed from control levels, but was significantly reduced in those from 4-week castrates. These long-term effects of castration could be overcome by the subcutaneous implantation of testosterone-containing capsules at the time of castration.     

Distinct Muscarinic Receptor Subtypes Differentially Modulate Acetylcholine Release from Corticocerebral Synaptosomes

The effect of McN-A-343 and oxotremorine on acetylcholine (ACh) release and choline (Ch) transport was studied in corticocerebral synaptosomes of the guinea pig. The synaptosomes were preloaded with [3H]Ch after treatment with the irreversible cholinesterase inhibitor, diisopropyl fluorophosphate, and then tested for their ability to release isotope-labeled ACh and Ch in the presence and absence of these agents. The kinetics of release were determined at the resting state (basal release) and in the presence of 50 mM K+. Under either condition, McN-A-343 enhanced the release of isotope-labeled ACh, whereas oxotremorine inhibited the K(+)-evoked release but had no effect on the basal release. The enhancing effect of McN-A-343 on basal ACh release was fully blocked by the selective M1 muscarinic antagonist, pirenzepine (100 nM). In contrast to its enhancing effect on ACh release, McN-A-343 potently inhibited Ch efflux as well as Ch influx. These effects were not blocked by atropine, a nonselective muscarinic antagonist. Oxotremorine had no effect on Ch transport. Binding studies showed that McN-A-343 was 3.6-fold more potent in displacing radiolabeled quinuclidinyl benzilate from cerebral cortex muscarinic receptors (mostly M1 subtype) than from cerebellar receptors (mostly M2 subtype), whereas oxotremorine was 2.6-fold more potent in the cerebellum. The displacements of radio-labeled pirenzepine and cis-dioxolane confirmed the M1 subtype preference of McN-A-343 and the M2 subtype preference of oxotremorine.(ABSTRACT TRUNCATED AT 250 WORDS)