Modulation of GABA-augmented norepinephrine release in female rat brain slices by opioids and adenosine

GABA_A receptor activation augments electrically-stimulated release of norepinephrine (NE) from rat brain slices. Because this effect is not observed in synaptoneurosomes, GABA probably acts on inhibitory interneurons to disinhibit NE release. To determine whether opioids or adenosine influence GABA-augmented NE release, hypothalamic and cortical slices from female rats were superfused with GABA or vehicle in the presence and absence of 10 M morphine or 100 M adenosine. GABA augments [³H]NE release in the cortex and hypothalamus. Morphine alone has no effect on [³H]NE release, but attenuates GABA augmentation of [³H]NE release in both brain regions. Adenosine alone modestly inhibits [³H]NE release in the cortex, but not in the hypothalamus. Adenosine inhibits GABA-augmented [³H]NE release in both brain regions. The general protein kinase inhibitor H-7, augments [³H]NE release in both brain regions and may have additive effects with GABA in cortical slices. These results implicate opioid and adenosine interneurons and possibly protein kinases in regulating GABAergic influences on NE transmission.     

Effects of neuropeptide Y on alpha 1-and beta-adrenoceptor-stimulated second messenger systems in rat frontal cortex

Neuropeptide Y (NPY) (1 microM) significantly reduced the basal cAMP concentration in slices of rat frontal cortex. However, NPY (10(-9)-10(-6)M) did not alter the isoproterenol-stimulated (10(-9)-10(-5) M) accumulation of cAMP in the frontal cortical slices, showing that Y2 NPY receptors do not modulate the beta-adrenoceptor-stimulated adenylase cyclase activity. NPY (10(-8)-2.5 x 10(-5) M) was also demonstrated to stimulate inositol phosphate accumulation in rat frontal cortex slices in a dose-dependent manner. However, NPY (1 microM) did not potentiate the ability of phenylephrine (5 X 10(-8)-10(-4) M), an alpha 1-adrenoceptor agonist, to stimulate inositol phosphate hydrolysis. The combined effects of phenylephrine and NPY (1 microM) on inositol phosphate hydrolysis were additive, suggesting that the alpha 1-adrenoceptor and NPY Y1 receptor sites are located on different postsynaptic sites in rat frontal cortex. This study demonstrates the existence of both Y2 and Y1 NPY receptors in the rat frontal cortex based on second messenger systems, but there does not appear to be an interaction of NPY with either alpha 1- or beta-adrenoceptors.     

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.     

Inhibitory effects of neuropeptide Y on sympathetic neurotransmission in the rabbit iris-ciliary body

Neuropeptide Y (NPY, 1–300 nM) mediated a concentration-dependent inhibition of field stimulation-evoked [³H]norepinephrine (NE) overflow from the isolated, superfused rabbit iris-ciliary body. At equimolar concentrations (100 nM), the homologous neuropeptide peptide YY (PYY) mimicked the effects of NPY, whereas pancreatic polypeptide (PP) and the C-terminal fragment of NPY_16–36 did not modify [³H]NE release. NPY-induced inhibition of [³H]NE release was unaffected by pretreatment of tissues with atropine (100 nM) plus yohimbine (100 nM) and was nonadditive with the maximal prejunctional effects of carbamycholine or clonidine, indicating that NPY acts independently of prejunctional muscarinic or alpha₂-adrenergic receptor activity to reduce [³H]NE overflow. It is concluded that NPY is a specific, potent modulator of adrenergic neurosecretion in the rabbit iris-ciliary body. These findings confirm the role of NPY as a co-transmitter at ocular sympathetic neuroeffector junctions, either mimicking or augmenting the actions of endogenously released norepinephrine.To whom to address reprint requests.     

Clonidine Inhibition of Norepinephrine Release from Normal and Morphine-Tolerant Guinea Pig Cortical Slices

Endogenous norepinephrine (NE) release in cerebral cortex slices taken from normal and morphine-tolerant guinea pigs was measured by HPLC. In normal slices, a linear relationship was found between electrically evoked NE release and the log of the frequency of stimulation in the range of 1-20 Hz. The efficiency of the alpha 2-mediated autofeedback was tested by adding the alpha 2-agonist clonidine and the alpha 2 agonist idazoxan. NE release was dose-dependently reduced by clonidine (1 nmol/L-1 mumol/L) and increased by idazoxan (10-100 nmol/L). The inhibition by clonidine was significantly greater at 1 Hz than at 3 Hz, whereas the absolute increase in NE release induced by idazoxan was greater at 3 Hz than at 1 Hz. Morphine at 1 mumol/L (a concentration per se ineffective) shifted to the left the clonidine concentrations able to inhibit NE release at 3 and 1 Hz (1-10 nmol/L), but at both frequencies, the opiate reduced the maximal inhibition induced by clonidine at 1 mumol/L. In slices taken from morphine-tolerant guinea pigs (in the presence of morphine at 1 mumol/L), clonidine (1 nmol/L-1 mumol/L) was ineffective at the stimulation rate of 3 Hz, but it was more active than in normal slices at 1 Hz. Such a response pattern suggests a reduced availability of alpha 2 receptors and an increase in their sensitivity to clonidine. However, chronic morphine treatment did not influence the physiological autoinhibition because the increase in NE release elicited by idazoxan (10-100 nmol/L) at 1 and 3 Hz was the same in normal and in "morphine-tolerant" slices.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of N-[3H]Methylcarbamylcholine Binding Sites and Effect of N-Methylcarbamylcholine on Acetylcholine Release in Rat Brain

The present experiments show that N-[3H]-methylcarbamylcholine ([3H]MCC) binds specifically and with high affinity to rat hippocampus, frontal cortex, and striatum. The highest maximal density of binding sites was apparent in frontal cortex and the lowest in hippocampus. [3H]MCC binding was potently inhibited by nicotinic, but not muscarinic, agonists and by the nicotinic antagonist dihydro-beta-erythroidine in all three brain regions studied. The effect of unlabeled MCC on acetylcholine (ACh) release from slices of rat brain was tested. The drug significantly enhanced spontaneous ACh release from slices of hippocampus and frontal cortex, but not from striatal slices. This effect of MCC to increase ACh release from rat hippocampus and frontal cortex was antagonized by the nicotinic antagonists dihydro-beta-erythroidine and d-tubocurarine, but not by alpha-bungarotoxin or by the muscarinic antagonist atropine. The MCC-induced increase in spontaneous ACh release from hippocampal and frontal cortical slices was not affected by tetrodotoxin. The results suggest that MCC might alter cholinergic transmission in rat brain by a direct activation of presynaptic nicotinic receptors on the cholinergic terminals. That this alteration of ACh release is apparent in hippocampus and frontal cortex, but not in striatum, suggests that there may be a regional specificity in the regulation of ACh by nicotinic receptors in rat brain.     

Studies on the Role of α2-Adrenoceptors in the Control of Synaptosomal [3H]5-Hydroxytryptamine Release: Effects of Antidepressant Drugs

The sensitivity of alpha 2-adrenoceptors on 5-hydroxytryptamine (5-HT) nerve endings obtained from rat cerebral cortex was investigated following treatment with the antidepressant drugs desipramine (10 mg/kg/day for 21-28 days) or clorgyline (1 mg/kg/day for 21-28 days). [3H]5-HT (100 nM) was used to load cortical synaptosomes (P2) after experiments with uptake inhibitors confirmed that this concentration of amine ensured exclusive uptake into 5-HT nerve terminals. The sensitivity of K+-stimulated release of [3H]5-HT to alpha 2-adrenoceptor occupancy was assessed in a superfusion system by means of the dose-dependent inhibition of [3H]5-HT release by clonidine. This is blocked by yohimbine (1 microM), which, when administered alone, enhances release, suggesting that endogenous catecholamines released from other synaptosomes act on these alpha 2-heteroreceptors. The effect of addition of citalopram (1 microM) to superfusates suggests that some reuptake of [3H]5-HT occurs during superfusion. Of the tritium released into superfusates during "background" and K+-stimulated release, 17 and 90%, respectively is [3H]5-HT. The attenuation of K+-stimulated release by clonidine is apparently diminished by the chronic clorgyline regimen but not by desipramine. However, clorgyline elevates catecholamine levels, and this might increase endogenous noradrenaline (NA) efflux, which by competition with clonidine could appear to alter alpha 2-adrenoceptor sensitivity. This possibility was investigated by depleting NA with the neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4). These studies showed that the apparent effect of chronic clorgyline on alpha 2-adrenoceptor sensitivity to clonidine was due to competition with increased levels of endogenous NA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential presynaptic effects of hexachlorocyclohexane isomers on noradrenaline release in cerebral cortex.

To investigate presynaptic effects of hexachlorocyclohexane (HCH) isomers, the release of noradrenaline (NA) in brain tissue was analyzed using rat cerebral cortical slices preloaded with [3H]-NA. gamma-HCH (lindane) 50 microM significantly enhanced the [3H]-NA release evoked by 15-25 mM K+. alpha- and beta-HCH (50 microM) did not produce any significant effect on K(+)-evoked [3H]-NA release. delta-HCH (50 microM) induced a significant decrease of the 25 mM K(+)-evoked release of [3H]-NA. The effect of the gamma- and delta-HCH isomers on the presynaptic action of the alpha 2-agonist clonidine and the alpha 2-antagonist yohimbine was also studied. The presynaptic inhibitory effect of clonidine and the stimulatory effect of yohimbine on [3H]-NA release was attenuated by lindane and delta-HCH, respectively. These results are consistent with a presynaptic action of the HCH isomers on noradrenergic release processes.     

Platelet-Activating Factor: Diminished Acetylcholine Release from Rat Brain Slices Is Mediated by a Gi Protein

Abstract: The effect of platelet-activating factor (PAF) on neurotransmitter release from rat brain slices prelabeled with [³H]acetylcholine ([³H]ACh), [³H]norepinephrine ([³H]NE), or [³H]serotonin ([³H]5-HT) was studied. PAF inhibited K⁺ depolarization-induced [³H]ACh release in slices of brain cortex and hippocampus by up to 59% at 10 n M but did not inhibit [³H]ACh release in striatal slices. PAF did not affect 5-HT or NE release from cortical brain slices. The inhibition of K⁺-evoked [³H]ACh release induced by PAF was prevented by pretreating tissues with several structurally different PAF receptor antagonists. The effect of PAF was reversible and was not affected by pretreating brain slices with tetrodotoxin. PAF-induced inhibition of [³H]ACh release was blocked 90 ± 3 and 86 ± 2% by pertussis toxin and by anti-Gαi_1/2 antiserum incorporated into cortical synaptosomes, respectively. The results suggest that PAF inhibits depolarization-induced ACh release in brain slices via a Gαi_1/2 protein-mediated action and that PAF may serve as a neuromodulator of brain cholinergic system.     

α1Adrenoreceptpr-Mediated Increase in Acetylcholine Release in Braip Slices During Morphine Tolerance

Norepinephrine, clonidine, and phenylephrine increased the electrically evoked release of endogenous acetylcholine in cortical slices taken from morphine-tolerant guinea pigs. This effect was alpha 1-adrenoreceptor mediated and was opposite to the alpha 2-adrenoreceptor-mediated inhibition of acetylcholine release, normally elicited by norepinephrine and clonidine. In the presence of prazosin, clonidine recovered its normal inhibitory properties, suggesting that morphine tolerance induced the appearance of an alpha 1-adrenoreceptor-mediated response that overshadowed, but did not cancel, the still present alpha 2-adrenoreceptor inhibitory control. The attempt to prove the presence of alpha-adrenoreceptors on the nerve endings by testing the effect of norepinephrine in synaptosomal preparations (preloaded with [3H]choline and depolarized with KCl and veratridine) was unsuccessful. Therefore the problem of the exact location of this excitatory input remains to be solved. These results confirm previous findings reporting the increase in cortical acetylcholine release induced by the alpha-adrenoreceptor agonists in morphine-tolerant, freely moving guinea pigs and demonstrate that opiate tolerance inverts the direction of the noradrenergic modulation even in the isolated intracortical cholinergic structures.     

Receptor selectivity of Met-enkephalin-Arg6-Phe7, an endogenous opioid peptide, in cerebral cortex of human and rat

This study was undertaken to examine the receptor selectivity of Met-enkephalin-Arg6-Phe7 (MERF) employing radioreceptor binding assays in human cerebral cortex membranes, and to elucidate the responsible receptors that mediate the regulatory action of MERF on high (20 mM) K+-stimulated release of [3H]norepinephrine ([3H]-NE) in rat cortex slices. Specific binding of [3H]MERF was inhibited by DAMGO, Tyr-D-Arg-Phe-Sar(TAPS), bremazocine and ethylketocyclazocine (EKC), but not by U69,593 (U69) and DPDPE. MERF showed high affinity for specific binding sites of [3H]DAMGO. However, MERF had little influence on the specific binding of [3H]DPDPE, [3H]U69 and [3H]diprenorphine ([3H]DIP) in the presence of 1 microM each of DAMGO, DPDPE and U69. In [3H]NE release experiments using rat cortex slices, DAMGO, MERF and EKC, in order of their potency, inhibited K+-stimulated release of [3H]NE. The inhibitory effects of MERF and DAMGO were more sensitive than that of EKC to antagonism by CTAP, nor-binaltorphimine (nor-BNI) and naloxone. These results suggested that MERF possesses high affinity for mu-receptors, but not for delta-, kappa1-, and very low affinity for kappa2-receptors in human cerebral cortex membranes. Also, the inhibitory effect of MERF on the K+-stimulated release of [3H]NE appears to be mediated by mu-receptors in rat cerebral cortex slices.     

Involvement of norepinephrine activity in the regulation of alpha 1 adrenergic receptors in the medial preoptic nucleus of estradiol-treated rats

To establish whether the diurnal decrease in the density of alpha 1 receptors observed in the medial preoptic nucleus (MPN) of estrogen (E2)-treated rats is related to the concomitant diurnal increase in norepinephrine (NE) turnover rates, we quantitated the density of [3H]-Prazosin binding to alpha 1 receptors after blockade of NE turnover with alpha-methyl-paratyrosine (alpha MPT). A series of preliminary studies was performed to rule out an interference of this drug with [3H]-Prazosin binding to alpha 1 adrenergic receptors in vitro and in vivo. Incubation of brain slices with alpha MPT produced a dose-dependent inhibition of [3H]-Prazosin binding to alpha 1 adrenergic receptors with an IC50 of approximately 6 mM. Scatchard analysis demonstrated that alpha MPT exhibited a simple competitive interaction with [3H]-Prazosin binding sites as shown by an increase in the apparent dissociation constant (Kd) of the ligand and no change in the number of alpha 1 receptors (Bmax). In contrast, preincubation of brain slices with alpha MPT and prior in vivo administration of alpha MPT did not affect [3H]-Prazosin binding to alpha 1 adrenergic receptors. Once we established that alpha MPT could be used to suppress NE turnover without interfering with the measurement of alpha 1 receptor densities, we repeatedly injected this drug to ovariectomized (OVX) and E2-implanted rats. The density of alpha 1 adrenergic receptors in MPN was quantitated autoradiographically. Blockade of NE turnover with alpha MPT only partially prevented the reduction in alpha 1 receptor density observed in the E2-treated rats, suggesting that the decrease in the level of [3H]-Prazosin binding sites cannot be completely ascribed to increased NE turnover rates.     

Selective alteration of neurotransmitter release by low oxygen in vitro

The potassium-stimulated release of acetylcholine, norepinephrine, serotonin, glutamate, and 4-aminobutyrate from superfused rat cortical slices was studied during hypoxia. A reduction in oxygen tensions from 603±6 to 22±2 mm Hg selectively altered the calcium-dependent efflux of these neurotransmitters, but did not change their calcium-independent release. The calcium-dependent release of [¹⁴C]acetylcholine decreased (39%), while that of glutamate increased (66%) and 4-aminobutyrate, [³H]norepinephrine, and [³H]serotonin were unaffected. Thus, low oxygen reveals variations in the calcium-dependent release mechanisms of several neurotransmitters. These differences may have important implications for pharmacological intervention of neurotransmitter release.     

Regulation of Noradrenaline Release from Rat Occipital Cortex Tissue Chops by α2-Adrenergic Agonists

Noradrenaline (NA) and the alpha 2-adrenergic agonists clonidine, BHT-920, and UK 14304-18 inhibit potassium-evoked release of [3H]NA from rat occipital cortex tissue chops with similar potencies. NA (10(-5) M) was most effective as up to 85% inhibition could be observed compared with 75%, 55%, and 35% for UK 14304-18, clonidine, and BHT-920, respectively, all at 10(-5) M. Potassium-evoked release was enhanced by both forskolin (10(-5) M) and 1 mM dibutyryl cyclic AMP. Pretreatment of tissue chops with 1 mM dibutyryl cyclic AMP in the presence of 3-isobutyl-1-methylxanthine partially reversed the alpha 2-adrenergic agonist inhibition of NA release. No reversal of inhibition was observed following pretreatment with 10(-5) M forskolin. The effects of clonidine, BHT-920, UK-14308-18, and NA on cyclic AMP formation stimulated by (a) forskolin, (b) isoprenaline, (c) adenosine, (d) potassium, and (e) NA were examined. Only cAMP formation stimulated by NA was inhibited by these alpha 2-adrenergic agonists. These results suggest that only a small fraction of adenylate cyclase in rat occipital cortex is coupled to alpha 2-adrenergic receptors. These results are discussed in relation to recent findings that several alpha 2-adrenergic receptor subtypes occur, not all of which are coupled to the inhibition of adenylate cyclase, and that alpha 2-adrenergic receptors inhibit NA release in rat occipital cortex by a mechanism that does not involve decreasing cyclic AMP levels.     

High affinity dopamine D2 receptor radioligands. 2. [125I]epidepride, a potent and specific radioligand for the characterization of striatal and extrastriatal dopamine D2 receptors.

Epidepride, (S)-N-[(1-ethyl-2-pyrrolidinyl)methyl]-5-iodo-2,3-dimethoxybenzamide+ ++, the iodine analogue of isoremoxipride (FLB 457), was found to be a very potent dopamine D2 receptor antagonist. Optimal in vitro binding required incubation at 25 degrees C for 4 h at pH 7.4 in a buffer containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl2 and 1 mM MgCl2. Scatchard analysis of in vitro binding to striatal, medial frontal cortical, hippocampal and cerebellar membranes revealed a KD of 24 pM in all regions, with Bmax's of 36.7, 1.04, 0.85, and 0.37 pmol/g tissue, respectively. The Hill coefficients ranged from 0.91-1.00 in all four regions. The IC50's for inhibition of [125I]epidepride binding to striatal, medial frontal cortical, and hippocampal membranes for SCH 23390, SKF 83566, serotonin, ketanserin, mianserin, naloxone, QNB, prasozin, clonidine, alprenolol, and norepinephrine ranged from 1 microM to greater than 10 microM. Partial displacement of [125I]epidepride by nanomolar concentrations of clonidine was noted in the frontal cortex and hippocampus, but not in the striatum. Scatchard analysis of epidepride binding to alpha 2 noradrenergic receptors in the frontal cortex and hippocampus revealed an apparent KD of 9 nM. At an epidepride concentration equal to the KD for the D2 receptor, i.e. 25 pM, no striatal alpha 2 binding was seen and only 7% of the specific epidepride binding in the cortex or hippocampus was due to binding at the alpha 2 site. Correlation of inhibition of [3H]spiperone and [125I]epidepride binding to striatal membranes by a variety of D2 ligands revealed a correlation coefficient of 0.99, indicating that epidepride labels a D2 site. In vitro autoradiography revealed high densities of receptor binding in layers V and VI of prefrontal and cingulate cortices as well as in striatum. In vivo rat brain uptake revealed a hippocampal:cerebellar and frontal cortical:cerebellar ratio of 2.2:1 which fell to 1.1:1 following haloperidol pretreatment. These properties suggest that [125I]epidepride is a superior radioligand for the in vitro and in vivo study of striatal and extrastriatal dopamine D2 receptors.     

Multiple binding sites for [3H]clonidine and [3H]WB-4101 in rat brain

Binding of the alpha-adrenergic agonist [3H]clonidine and the alpha-adrenergic antagonist [3H]WB-4101 exhibited multiple binding site characteristics in both rat frontal cortex and cerebellum. Kinetic analysis of the dissociation of both radioligands in rat frontal cortex suggests two high affinity sites for each ligand. Competition of various noradrenergic agonists and antagonists for [3H]WB-4101 binding yielded shallow competition curves, with Hill coefficients ranging from 0.45 to 0.7. This further suggests multiplicity in [3H]WB-4101 binding. In the rat cerebellum, competition of various noradrenergic drugs for [3H]clonidine binding yielded biphasic competition curves. Furthermore Scatchard analysis of [3H]clonidine binding in rat cerebellum showed two high affinity sites with KD = 0.5 nM and 1.9 nM, respectively. Competition of various noradrenergic drugs for [3H]WB-4101 binding in the rat cerebellum yielded biphasic competition curves. Lesioning of the dorsal bundle with 6-hydroxydopamine did not significantly affect the binding of either [3H]clonidine or [3H]WB-4101. These findings for both [3H]clonidine and [3H]WB-4101 binding in rat frontal cortex and cerebellum can be explained by the existence of postsynaptic binding sites for both 3H ligands.     

Release of [3H]Dopamine from Striatal and Cerebral Cortical Slices from Rats with Thioacetamide-Induced Hepatic Encephalopathy: Different Responses to Stimulation by Potassium Ions and Agonists of Ionotropic Glutamate Receptors

The effects of depolarizing stimuli; high (50 mM) potassium ions and the glutamate receptor agonists N-methyl-D-aspartate, kainate and 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) on the release of newly-loaded [³H]dopamine were studied in frontal cortical and striatal slices from control rats and from rats with acute hepatic encephalopathy induced with a hepatotoxin, thioacetamide. Hepatic encephalopathy enhanced the stimulatory effect of potassium ions by 20% in striatal slices and by 34% in frontal cortical slices. In striatal slices the stimulatory effects of N-methyl-D-aspartate and kainate were depressed in hepatic encephalopathy by 46% and 21%, respectively, which may be taken to reflect impaired modulation of striatal dopamine release by glutamate acting at N-methyl-D-aspartate or kainate receptors. In frontal cortical slices, the stimulatory effect of kainate was enhanced by 35% in hepatic encephalopathy but N-methyl-D-aspartate-stimulated release was not affected. The release evoked by 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate was not affected in hepatic encephalopathy in either brain region. Stimulation of dopamine release in the frontal cortex by depolarization or glutamate acting at kainate receptors could inhibit the activity of descending corticostriatal glutamatergic pathways, further impairing regulation of dopamine release by glutamate in the stratum.     

Comparative regulation of potassium and amphetamine induced release of 3H-norepinephrine from rat brain via presynaptic mechanisms.

This study examined the ability of various drugs to modify the potassium (K) or d-amphetamine (d-A) induced release of ³H-norepinephrine ³HNE) from chopped rat cortical tissue. The K induced release of the transmitter, which occurs from reserpine sensitive sites of cortical tissue, was significantly reduced by the beta receptor antagonist propranolol, the alpha receptor agonist clonidine and also by PGE₂. Pretreatment with eicosatetrynoic acid, an inhibitor of prostaglandin synthesis, did not influence the effect of clonidine on ³HNE release; thus this latter effect appears to be independent of enhanced prostaglandin formation. The proposed alpha receptor mediated negative feedback exhibits stereospecificity since addition of exogenous 1-, but not d-, NE decreased release of the transmitter. Blockade of alpha receptors by phentolamine or stimulation of beta receptors by isoproterenol significantly enhanced the K induced release of ³HNE from cortical tissue. By contrast, the d-A induced release of ³HNE which occurs from reserpine-insensitive sites, was reduced by propranolol and clonidine; and was not altered by phentolamine, isoproterenol or PGE₂. These data indicate that the K, but no d-A, induced release of ³HNE from cortical tissue is modified in accordance with postulated presynaptic negative and positive feedback mechanisms.     

Ethanol-Induced Increase in Endogenous Dopamine Release May Involve Endogenous Opiates

The effect of opiate peptides on basal and potassium-stimulated endogenous dopamine (DA) release from striatal slices was studied in vitro. Dual stimulation of the striatal slices gave a reproducible increase in DA release that was calcium dependent. Addition of the delta-opiate receptor agonists Met5-enkephalin, [D-Ala2,D-Leu5]enkephalin (DADLE), and [D-Ser2]Leu-enkephalin-Thr (DSLET), increased the basal DA release without affecting potassium-stimulated release in a dose-dependent manner. The effect of DADLE was antagonized by the addition of naloxone. In contrast, the mu-opioid receptor agonist [D-Ala2,N-MePhe4,Gly-ol5]enkephalin (DAGO) and the epsilon-opioid agonist beta-endorphin inhibited the stimulated DA release without changing the basal release. The inhibitory effect of DAGO on potassium-stimulated release was antagonized by naloxone. The addition of ethanol (75 mM) to the incubation media produced a delayed increase of both the basal and stimulated DA release. There was no change in stimulated DA release when the change in basal release was subtracted, suggesting that ethanol produced a dose-dependent, selective increase in basal DA release. Naloxone and the selective delta-opiate antagonist ICI 174864 inhibited the ethanol-induced increase in basal DA release. Naloxone and ICI 174864 added alone did not alter either basal or stimulated DA release. We therefore suggest that the ethanol-induced increase in basal DA release is an indirect effect involving an endogenous delta-opiate agonist.     

Phosphatidylinositol Metabolism During In Vitro Hypoxia

The effects of in vitro histotoxic hypoxia (0.5 mM KCN) on potassium-stimulated phosphatidylinositol turnover were determined. In rat cortical slices that were prelabeled with [2-3H]inositol, depolarization with 60 mM KCl increased [2-3H]inositol monophosphate and [2-3H]inositol bisphosphate accumulation in a Ca2+-dependent manner. At early times (10 s and 1 min), histotoxic hypoxia enhanced potassium-stimulated [2-3H]inositol monophosphate and inositol bisphosphate accumulation. Under basal conditions, hypoxia did not alter the accumulation of [2-3H]inositol phosphates. These results are consistent with the following hypothesis. The hypoxic-induced increase in cytosolic free calcium that we reported previously may lead to the early stimulation of inositol phosphates formation during hypoxia through activation of phospholipase C. The impairment of inositol phosphates formation during more prolonged hypoxia may be due to negative feedback regulation of the phosphatidylinositol cascade by protein kinase C or to a reduction in ATP levels.