Potassium-evoked Release Research Articles

Nitric oxide inhibits the release of norepinephrine and dopamine from the medial basal hypothalamus of the rat.

Previous research indicates that norepinephrine and dopamine stimulate release of luteinizing hormone (LH)-releasing hormone (LHRH), which then reaches the adenohypophysis via the hypophyseal portal vessels to release LH. Norepinephrine exerts its effect via alpha 1-adrenergic receptors, which stimulate the release of nitric oxide (NO) from nitricoxidergic (NOergic) neurons in the medial basal hypothalamus (MBH). The NO activates guanylate cyclase and cyclooxygenase, thereby inducing release of LHRH into the hypophyseal portal vessels. We tested the hypothesis that these two catecholamines modulate NO release by local feedback. MBH explants were incubated in the presence of sodium nitroprusside (NP), a releaser of NO, and the effect on release of catecholamines was determined. NP inhibited release of norepinephrine. Basal release was increased by incubation of the tissue with the NO scavenger hemoglobin (20 micrograms/ml). Hemoglobin also blocked the inhibitory effect of NP. In the presence of high-potassium (40 mM) medium to depolarize cell membranes, norepinephrine release was increased by a factor of 3, and this was significantly inhibited by NP. Hemoglobin again produced a further increase in norepinephrine release and also blocked the action of NP. When constitutive NO synthase was inhibited by the competitive inhibitor NG-monomethyl-L-arginine (NMMA) at 300 microM, basal release of norepinephrine was increased, as was potassium-evoked release, and this was associated in the latter instance with a decrease in tissue concentration, presumably because synthesis did not keep up with the increased release in the presence of NMMA. The results were very similar with dopamine, except that reduction of potassium-evoked dopamine release by NP was not significant. However, the increase following incubation with hemoglobin was significant, and hemoglobin, when incubated with NP, caused a significant elevation in dopamine release above that with NP alone. In this case, NP increased tissue concentration of dopamine along with inhibiting release, suggesting that synthesis continued, thereby raising the tissue concentration in the face of diminished release. When the tissue was incubated with NP plus hemoglobin, which caused an increase in release above that obtained with NP alone, the tissue concentration decreased significantly compared with that in the absence of hemoglobin, indicating that, with increased release, release exceeded synthesis, causing a fall in tissue concentration. When NO synthase was blocked by NMMA, the release of dopamine, under either basal or potassium-evoked conditions, was increased. Again, in the latter instance the tissue concentration declined significantly, presumably because synthesis did not match release. Therefore, the results were very similar with both catecholamines and indicate that NO acts to suppress release of both amines. Since both catecholamines activate the release of LHRH, the inhibition of their release by NO serves as an ultra-short-loop negative feedback by which NO inhibits the release of the catecholamines, thereby reducing the activation of the NOergic neurons and decreasing the release of LHRH. This may be an important means for terminating the pulses of release of LHRH, which generate the pulsatile release of LH that stimulates gonadal function in both male and female mammals.

High potassium-evoked release of ATP from rabbit pulmonary artery via endogenous noradrenaline

1. 1. High potassium 30 mM and noradrenaline at 10 μM significantly evoked the release of ATP and produced remarkable vasoconstriction in the rabbit pulmonary artery. 2. 2. Phentolamine and prazosin at 0.1 μM inhibited ATP release but not vasoconstriction by 30 mM potassium. 3. 3. 30 mM potassium significantly evoked the release of noradrenaline. 4. 4. There was a significant positive correlation between the amounts of release of total purines, sum of ATP, ADP, AMP and adenosine, and noradrenaline evoked by 30 mM potassium. 5. 5. 30 mM potassium-evoked ATP release was significantly reduced by denudation of endothelium.

Effects of inorganic mercury (Hg2+) on calcium channel currents and catecholamine release from bovine chromaffin cells

The effects of inorganic mercury (Hg2+) on calcium channel currents and the potassium-evoked catecholamine release of bovine chromaffin cells in culture were examined. The effects of cadmium (Cd2+), known to block calcium channels and reduce catecholamine release of chromaffin cells, were studied for comparison. Calcium channel currents were recorded in the whole-cell configuration of the patch-clamp technique. Hg2+ is a potent calcium channel blocker in bovine chromaffin cells. The IC50 value is about 3 microM, the Hill slope 1.46. In a concentration of 100 microM, Hg2+ blocked the currents completely; 100 microM Cd2+ had the same effect. Potassium-evoked catecholamine release from chromaffin cells was measured at different time-points with high-performance-liquid-chromatography (HPLC) under control conditions and in the presence of different Hg2+ concentrations. Low Hg2+ concentrations (0.1 and 1 microM) did not affect the amount of the catecholamines epinephrine (E) and norepinephrine (NE) which was released. Under identical conditions 1 microM Cd2+ also had no effect on release. With 10 microM Hg2+ there was a time-dependent increase in the potassium-evoked catecholamine release (by 27% after 8 min). The E/NE ratio was not altered, suggesting that the release of both hormones was increased similarly. In contrast to this, the release was slightly reduced with 10 microM Cd2+. In the presence of 100 microM Hg2+, there was a reduction of the release during an early phase, followed by an increase. The reduction is most probably due to the fast and effective calcium channel block by Hg2+ in this high concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Different calcium channels mediate transmitter release evoked by transient or sustained depolarization at mammalian sympathetic ganglia.

We have compared the effect of calcium channel blockers on the potassium-evoked release of tritium-labeled acetylcholine and on preganglionic spike-evoked synaptic transmission in the rat superior cervical ganglion. Transmitter release at the nerve terminals is mediated by the influx of calcium through voltage-gated calcium channels. While four types of voltage-gated calcium channels (T, L, N and P) have been identified in neurons, it is not clear which may actually be involved in excitation-secretion coupling. Release of tritiated acetylcholine evoked by sustained depolarization in high (40 mM) extracellular potassium decreased markedly in the absence of calcium or the presence of cadmium. High potassium-evoked release was substantially inhibited by the P-type channel blockers, purified from funnel-web spider toxin, and omega-agatoxin-IVA, and by the N-type channel blocker omega-conotoxin-GVIA, but was unaffected by the L-type channel blocker nitrendipine. In contrast, postganglionic compound action potentials synaptically triggered by preganglionic stimulation were strongly blocked by funnel-web spider toxin and slightly blocked by a high concentration of omega-agatoxin-IVA, but were unaffected by either omega-conotoxin-GVIA, nitrendipine or a low concentration of omega-agatoxin-IVA. Thus, at the superior cervical ganglion, funnel-web spider toxin-sensitive calcium channels play a dominant role in transmitter release evoked by transient, spike-mediated depolarization, but other types of voltage-gated calcium channels in addition to the funnel-web spider toxin-sensitive channel mediate the transmitter release that is evoked by sustained high potassium depolarization.

P-Chloroamphetamine (PCA), 3,4-methylenedioxy-methamphetamine (MDMA) and d-fenfluramine pretreatment attenuates d-fenfluramine-evoked release of 5-HT in vivo.

Previous work has suggested that repeated treatment with substituted amphetamines including PCA, MDMA and d-fenfluramine produces a persistent neurodegeneration which is relatively selective for the fine serotoninergic terminals arising from the dorsal raphe nucleus. The aim of the present study was to investigate whether the acute releasing effect of d-fenfluramine might also be sensitive to lesions produced by PCA, MDMA and d-fenfluramine itself. Basal and 5-HT release evoked by d-fenfluramine or 100 mM KCl was measured by microdialysis in frontal or parietal cortex of rats 2 weeks after they had been treated with a neurodegenerative regime of PCA, MDMA, d-fenfluramine, or vehicle. In frontal cortex of vehicle controls, d-fenfluramine (10 mg/kg IP) and KCl (100 mM via microdialysis probe) evoked an increase in 5-HT of 1740% and 779% of basal, respectively. PCA pretreatment reduced d-fenfluramine-evoked 5-HT release by 90.9% while potassium-evoked release was reduced by only 66.8%. Similar results were obtained in parietal cortex. MDMA (20 mg/kg x 8) and d-fenfluramine (1.25 mg/kg x 8) pretreatment reduced d-fenfluramine-evoked release of 5-HT in frontal cortex by 45.2% and 72.0%, respectively. Overall, the present data are consistent with the hypothesis that the acute release of 5-HT evoked by d-fenfluramine occurs via those terminals destroyed by pretreatment with PCA, MDMA and d-fenfluramine, while KCl evokes release from both PCA-sensitive and PCA-insensitive terminals. The significance of these results for the interpretation of neuroendocrine data from d-fenfluramine challenge tests is discussed.

Enhancement of retinal acetylcholine release by DAMGO: possibly a direct opioid receptor-mediated excitatory effect.

1. An eye-cup preparation in anaesthetized rabbits was used to examine opioid modulation of acetylcholine (ACh) release from cholinergic neurones in the retina. 2. The mu-opioid receptor agonist, [D-Ala2, MePhe4, Gly-ol5]-enkephalin (DAMGO), when applied locally to the retina at concentrations between 1-30 microM significantly increased the light-evoked release of ACh. The effect of DAMGO was completely blocked by the selective mu-receptor antagonist CTOP but the kappa-receptor antagonist nor-binaltorphimine (norBNI) did not affect the action of DAMGO on ACh release indicating that the opioid produced its effect by activation of mu-receptors (the rabbit retina has negligible delta-receptors). 3. Blockade with bicuculline and strychnine of GABAergic and glycinergic inputs to the cholinergic neurones did not affect the action of DAMGO on ACh release. Also DAMGO did not reduce the potassium-evoked release of either GABA or glycine from rat isolated retinas. 4. Exposure of the rabbit retina to a combination of an A1-adenosine receptor antagonist, 8-cyclopentyl-1,3 dipropylxanthine (DPCPX), and adenosine deaminase did not affect the enhancing action of DAMGO on the light-evoked release of ACh. 5. When the retina in the rabbit eye-cup was exposed to kainate, the release of ACh was increased by approximately three times the resting release. In the presence of DAMGO the kainate-evoked release of ACh was enhanced by 44%. 6. These experiments show that activation of mu-opioid receptors by DAMGO increases the release of ACh elicited by physiological stimulation (flickering light). Since we could find no evidence thatDAMGO reduces inhibitory inputs to the cholinergic neurones, it seems that the enhancing action ofDAMGO on the light-evoked release of ACh involves a direct excitatory effect rather than disinhibition.This conclusion is supported by the enhancing action of DAMGO on the kainate-evoked release of ACh because kainate is thought to act directly on the cholinergic neurones.

Nicotine tolerance in chromaffin cell cultures: acute and chronic exposure to smoking-related nicotine doses.

Nicotine tolerance and dependence are key aspects of tobacco addiction; however, the cellular mechanisms underlying these phenomena are poorly understood. Adrenal chromaffin cells release catecholamines upon exposure to nicotine and with repeated exposure this response exhibits nicotine tolerance. Using bovine adrenal chromaffin cells in culture, we have demonstrated acute and chronic nicotine tolerance at doses relevant to that in the blood and tissues of smokers (10(-7) M to 10(-6) M). Chromaffin cells are preexposed to low doses of nicotine for time periods ranging from 10 min to 7 days and then subsequently challenged with a maximally stimulating dose of nicotine (10(-5) M) for 10 min, all at 37 degrees C. Preexposure to nicotine results in a depression of 45Ca uptake and catecholamine release upon subsequent nicotine challenge. Acute tolerance or desensitization of nicotine-stimulated catecholamine release begins to occur in minutes after preexposure to 10(-6) M nicotine at 37 degrees C. The depression of catecholamine release upon preexposure to nicotine is both dose and temperature dependent and is not seen with potassium-evoked release. Chronic exposure to 10(-7) M nicotine for 3 days led to a depression of the secretory response to approximately 70% of control responses. There was a trend toward recovery of full response by days 5 and 7 of 10(-7) M nicotine preexposure. Nearly complete depression of the nicotine-evoked release occurs within the first day of exposure to 10(-6) M nicotine and persists for at least a week of nicotine exposure at 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Neuropeptide-Y potentiates the secretion of vasopressin from the neurointermediate lobe of the rat pituitary gland

Neuropeptide-Y (NPY) is colocalized with vasopressin and oxytocin in magnocellular neurons of the hypothalamo-neurohypophysial system, and a very high density of NPY-binding sites is present within the neurohypophysis. To investigate the possibility that NPY exerts a modulatory role on the release of neurohypophysial hormones, we have studied the actions of NPY on potassium-evoked release of vasopressin and oxytocin from the rat neurointermediate lobe in vitro. NPY dose-dependently potentiated vasopressin release evoked by high extracellular potassium (56 mM), with a maximal enhancement of 223% (10(-7) M NPY). A similar effect was obtained with the Y2-selective agonist NPY-(13-36). In contrast, no effect on the potassium-evoked release of oxytocin was observed at this concentration. In the absence of Ca2+ in the incubation medium, NPY did not potentiate vasopressin secretion, indicating that the effect of NPY on potassium-evoked secretion of neurohypophysial vasopressin is critically dependent on extracellular calcium ions. The number of neurohypophysial NPY-binding sites is drastically down-regulated in animals subjected to chronic osmotic stimulation. In the present study, it was observed that the potentiating effect of NPY on vasopressin secretion was completely abolished in neurointermediate lobes recovered from animals that had been drinking 2% NaCl for 12 days, reflecting the concomitant down-regulation of neurohypophysial NPY-binding sites observed during this state. Finally, it was confirmed that stimulation by high K+ significantly evoked the release of endogenous NPY from neurointermediate lobes of the pituitary gland. The present results provide evidence that NPY selectively and potently enhances evoked vasopressin secretion. Considering the coexistence of the two neuropeptides in magnocellular hypothalamo-neurohypophysial neurons, this action is likely to be part of an autostimulatory feedforward loop. NPY may be an important component in the mechanisms associated with the control of body fluid homeostasis.

Neuropeptide-Y potentiates the secretion of vasopressin from the neurointermediate lobe of the rat pituitary gland.

Neuropeptide-Y (NPY) is colocalized with vasopressin and oxytocin in magnocellular neurons of the hypothalamo-neurohypophysial system, and a very high density of NPY-binding sites is present within the neurohypophysis. To investigate the possibility that NPY exerts a modulatory role on the release of neurohypophysial hormones, we have studied the actions of NPY on potassium-evoked release of vasopressin and oxytocin from the rat neurointermediate lobe in vitro. NPY dose-dependently potentiated vasopressin release evoked by high extracellular potassium (56 mM), with a maximal enhancement of 223% (10(-7) M NPY). A similar effect was obtained with the Y2-selective agonist NPY-(13-36). In contrast, no effect on the potassium-evoked release of oxytocin was observed at this concentration. In the absence of Ca2+ in the incubation medium, NPY did not potentiate vasopressin secretion, indicating that the effect of NPY on potassium-evoked secretion of neurohypophysial vasopressin is critically dependent on extracellular calcium ions. The number of neurohypophysial NPY-binding sites is drastically down-regulated in animals subjected to chronic osmotic stimulation. In the present study, it was observed that the potentiating effect of NPY on vasopressin secretion was completely abolished in neurointermediate lobes recovered from animals that had been drinking 2% NaCl for 12 days, reflecting the concomitant down-regulation of neurohypophysial NPY-binding sites observed during this state. Finally, it was confirmed that stimulation by high K+ significantly evoked the release of endogenous NPY from neurointermediate lobes of the pituitary gland. The present results provide evidence that NPY selectively and potently enhances evoked vasopressin secretion. Considering the coexistence of the two neuropeptides in magnocellular hypothalamo-neurohypophysial neurons, this action is likely to be part of an autostimulatory feedforward loop. NPY may be an important component in the mechanisms associated with the control of body fluid homeostasis.

The ontogeny of apomorphine-induced alterations of neostriatal dopamine release: effects on potassium-evoked release.

The effects of apomorphine (0.05, 0.1, and 1.0 mg/kg, s.c.) on K(+)-evoked dopamine release were studied through the use of in vivo microdialysis in the neostriatum of developing and adult rats. Fifteen-minute samples were collected from urethane-anesthetized rats 5, 10-11, 21-22, 35-36 days of age, and adults, and quantified by high performance liquid chromatography with electrochemical detection. Apomorphine attenuated K(+)-evoked dopamine release in all age groups, suggesting that the dopamine autoreceptor modulating release in the neostriatum is functional by 5 days of age. A dose-response effect of apomorphine was observed in all age groups except at 5 and 10 days of age. Absolute levels of extracellular dopamine were significantly lower at 5 and 10 days of age compared with the other ages, and the effectiveness of a high-K+ artificial cerebrospinal fluid to evoke dopamine release increased with age.

Fentanyl inhibits the release of [3H]noradrenaline from SH-SY5Y human neuroblastoma cells

We have examined the effect of fentanyl on [3H]noradrenaline release in a human neuroblastoma cell preparation, SH-SY5Y. Fentanyl produced a significant, concentration-dependent inhibition of [3H]noradrenaline release with IC50 values of 5.5 x 10(-6) mol litre-1 and 15.5 x 10(-6) mol litre-1 for carbachol- and potassium-evoked release, respectively. The small difference in IC50 between the two evoking stimuli may be explained by the weak binding affinity of fentanyl to muscarinic receptors (Ki = 570 nmol litre-1). The minimum concentrations at which a significant effect was observed were 0.3 x 0.10(-6) mol litre-1 and 10.0 x 10(-6) mol litre-1 for carbachol- and potassium-evoked release, respectively; these values are considerably in excess of the serum concentration of fentanyl required to produce analgesia. Naloxone failed to antagonize the fentanyl inhibition and, furthermore, morphine and an enkephalin had no effect on evoked release, implying a non-opioid receptor mediated effect.

Pharmacological characterization of the release of neuropeptide Y-like immunoreactivity from the rat hypothalamus

Radioimmunoassay for NPY and detection by HPLC-EC were used to detect the concurrent release of norepinephrine and NPY from slices of the rat hypothalamus and to examine the modulation by adrenoceptors of the release of this neuropeptide. Basal and potassium-evoked (56 mM K +) release of both compounds were easily measured, with evoked release occuring in a calcium-dependent manner. The effect of the α 2-adrenoceptor agonist clonidine, the antagonists prazosin ( α 1 selective) and yohimbine ( α 2 selective) and the β-adrenoceptor antagonist propranotol were all shown to modulate the evoked release of NPY. The α 2 agonist clonidine decreased evoked release of NPY, while the α 2 antagonist yohimbine increased the potassium-evoked release. Prazosin decreased both the basal and potassium-evoked release of NPY. Propranolol had the most profound effect on release of NPY, causing a significant decrease in basal release and a large decrease in the potassium-evoked release of NPY from slices of hypothalamus.

Calcium channel antagonist omega-conotoxin binds to intramembrane particles of isolated nerve terminals

Voltage-sensitive calcium channels play a key role in evoked neurotransmitter release and their distribution in presynaptic membranes can be critical for fast signalling at chemical synapses. Using a biotinylated derivative of the neuronal calcium channel antagonist, omega-conotoxin, and a combination of colloidal gold labeling and freeze-fracture techniques, we have labeled calcium channels present at the membrane of nerve terminals isolated from the electric organ of Torpedo marmorata. The biotinylated blocker exerts an inhibitory action on the high potassium-evoked release of adenosine triphosphate as the native toxin does and its interaction with biological membranes is specific as shown in displacement experiments. This study shows that an antagonist specific for voltage-activated calcium channels binds to intramembrane particles in presynaptic membranes, reinforcing the idea that these particles, concentrated at neurotransmitter release sites, effectively represent calcium channels.

Depolarization by K + and glutamate activates different neurotransmitter release mechanisms in gabaergic neurons: Vesicular versus non-vesicular release of GABA

Neurotransmitter release and changes in the concentration of intracellular free calcium ([Ca ++] i) were studied in cultured GABAergic cerebral cortical neurons, from mice, upon depolarization with either an unphysiologicaLly high potassium concentration (55 mM) or the physiological excitatory neurotransmitter glutamate (100 μM). Both depolarizing stimuli exerted prompt increases in the release of preloaded [ 3H]GABA as well as in [Ca ++] i. However, the basic properties of transmitter release and the increase in [Ca ++] i, under a variety of conditions were different during stimulation with K + or glutamate. Potassium-evoked release of [ 3H]GABA consisted of two phases, a rapid, large and transient phase followed by a smaller, more persistent second phase. The rapid phase was inhibited (60%) by nocodazole which reduced the number of vesicles in the neuntes by 80%. This rapid phase of the GABA release was also reduced by organic (verapamil) and inorganic (Co ++) Ca ++ channel blockers but was insensitive to the GABA transport inhibitor SKF 89976A. In contrast, the second phase was less sensitive to nocodazole and Ca ++ channel antagonists but could be inhibited by SKF 89976A. The glutamateinduced [ 3H]GABA release, which was mainly mediated by N-methyl- d-aspartate receptors, consisted of a single, sustained phase. This was insensitive to nocodazole, partly inhibited by verapamil and could be blocked by Co ++ as well as SKF 89976A. The action of Co ++ could be attributed to a block of N-methyl- d-aspartate-associated ion channels. These findings strongly suggest that the majority of the K +-stimulated GABA release is dependent upon vesicles whereas the glutamate induced release is non-vesicular and mediated by a depolarization-dependent reversal of the direction of high-affinity GABA transport. The basic differences in the mode of action of the two depolarizing stimuli were reflected in the properties of the increase in [Ca ++] i elicited by 55 mM K + and 100μM glutamate, respectively. The K +-induced increase in [Ca ++] i was reduced by both verapamil and Ca ++-free media whereas the corresponding glutamate response was only sensitive to Ca ++-free conditions. Exposure of the cells to nocodazole or SKF 89976A had no effect on the ability of K + or glutamate to increase [Ca ++] i. Altogether, the results clearly demonstrate that K +-induced transmitter release from these GABAergic neurons is vesicular in nature whereas that induced by the neurotransmitter glutamate is not.

Effect of inhibitors of eicosanoid metabolism on release of [3H]noradrenaline from the human neuroblastoma, SH-SY5Y.

Nordihydroguaiaretic acid (NDGA; a lipoxygenase inhibitor), LY-270766 (an inhibitor of 5-lipoxygenase), and the diacylglycerol lipase inhibitor RG 80267 completely eliminated potassium-evoked release of [3H]-noradrenaline ([3H]NA) from the human neuroblastoma clone SH-SY5Y with IC50 values of 10, 15, and 30 microM, respectively. In contrast, these inhibitors only partially inhibited carbachol-evoked release and had little effect on the calcium ionophore A23187-evoked release of NA in this cell line. Arachidonic acid partially inhibited potassium- and A23187-evoked release but did not reverse the inhibition of potassium-evoked release observed in the presence of RG 80267. These studies suggest that arachidonic acid (or its lipoxygenase products) are not important intermediates in the regulation of exocytosis in SH-SY5Y. This conclusion is strengthened by our studies in which SH-SY5Y cells were grown in medium supplemented with bovine serum albumin-linoleic acid (50 microM). Under these conditions there was a selective increase in content of membrane polyunsaturated fatty acids of the omega 6 series, including arachidonic acid; however, these changes did not effect potassium-, veratridine-, carbachol-, or calcium ionophore-evoked release of [3H]NA.

Interleukin-2 regulates monoamine and opioid peptide release from the hypothalamus.

The present study determined whether interleukin-2 (IL-2) and IL-2 receptors are present in the adult rat hypothalamus. In addition, we determined whether IL-2 is involved in the regulation of neurotransmitter release from hypothalamic slices. In the hypothalamus, the highest levels of endogenous IL-2 and IL-2 receptors were localized to the median eminence and arcuate nucleus. Application of exogenous IL-2 to hypothalamic slices produced significant decreases of potassium (25 mM)-evoked [3H]noradrenaline release (24%) without significantly affecting the evoked release of [14C]glutamate, [14C]5-hydroxytryptamine, [3H]dopamine or [3H] gamma-amino butyric acid. In addition, IL-2 increased the potassium-evoked release of methionine-enkephalin (by 78%) and beta-endorphin (by 38%) from hypothalamic slices without affecting the release of leucine-enkephalin. In contrast, the spontaneous release of neurotransmitters and neuropeptides was not affected by exogenous IL-2. Overall, the findings of this study suggest that hypothalamic IL-2 is an endogenous neurokine that may be of consequence to the regulation of hypothalamic function.

Glutamate in the inferior colliculus plays a critical role in audiogenic seizure initiation

Alterations of excitant amino acid (EAA) action are implicated in seizure susceptibility in the genetically epilepsy-prone rat (GEPR). The inferior colliculus (IC) is critical for audiogenic seizure (AGS) initiation in the GEPR. The present study observed that bilateral microinjection into the IC of l-canaline, a glutamate synthesis inhibitor, decreased AGS severity in the GEPR and also decreased potassium-evoked release of glutamate from IC slices. Bilateral microinjection of NMDA receptor antagonists, 2-amino-7-phosphonoheptanoate (AP7) or 3-((±)-2-carboxypiperazin-4-yl)-propyl-1-phosphonate (CPP) into IC blocked AGS, and an antagonist at non-NMDA EAA receptors, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), also blocked AGS. NMDA receptor antagonists were 5–200 times more effective than CNQX. Microinjection of a non-competitive NMDA receptor antagonist, dizocilpine (MK-801), into IC had little effect except with very high doses. Microinjection of CPP or AP7 into the IC blocked AGS at considerably lower doses as compared to pontine reticular formation (PRF). However, MK-801 attenuated AGS when microinjected into PRF at doses that were ineffective in IC. Systemically administered CPP blocked AGS and significantly reduced IC neuronal firing in the behaving GEPR, suggesting an important action of systemically administered NMDA receptor antagonists on brainstem auditory nuclei critical to AGS. The present results support a critical role for glutamate acting, in part, through NMDA receptors in IC in initiation of AGS.

Calcium-dependent pro-cholecystokinin V-9-M immunoreactive peptide release from rat brain slices and CCK-secreting rat medullary thyroid carcinoma cells in culture

The release of peptides immunoreactive for a synthetic peptide (V-9-M) contained in the amino-terminal of pro-CCK was examined. The potassium-evoked release of V-9-M immunoreactive peptides from rat cerebral cortical slices in vitro was calcium dependent. Cholecystokinin-secreting rat medullary thyroid carcinoma cells also secreted significant quantities of these peptides. Sephadex column chromatography of the release media from slices and cells showed two V-9-M immunoreactive peptides, one larger and one smaller than V-9-M itself. Previous behavioral studies have suggested that V-9-M has a distinct neuropharmacological profile. These results demonstrate that V-9-M-like peptides are released along with CCK-8 and are consistent with the hypothesis that V-9-M-like peptides may be neurotransmitters or neuromodulators or may be involved in the sorting or transport of CCK-8.

Fiber-optic ammonia sensor for measuring synaptic glutamate and extracellular ammonia.

A fiber-optic ammonia gas sensor designed for neurochemical applications is presented. Parameters evaluated in terms of effect on the steady-state and dynamic response of this sensor include the indicator dye, concentrations of indicator and total ammonia nitrogen in the internal solution, volume of the internal solution, structure of the gas-permeable membrane, and temperature. The final ammonia sensor responds over the concentration range from 7 to 3000 nM with a limit of detection of 7 nM and response times ranging from 2 to 5 min. Glutamate oxidase is immobilized at the tip of this ammonia sensor to provide a glutamate biosensor with a detection limit of 0.1 microM when operated at pH 7.8. In addition, this ammonia sensor is used to measure extracellular ammonia levels in perfused retinal and eye-cup tissue preparations. These measurements indicate a calcium-dependent, potassium-evoked release of ammonia during these depolarization conditions.

Modulation of GABA release by α-amino-3-hydroxy-5-methylisoxazole-4-propionate and N-methyl- d-aspartate receptors in matrix-enriched areas of the rat striatum

Using a new in vitro superfusion device, the release of preloaded [ 3H]GABA was examined in microdiscs of tissues taken from sagittal slices in matrix-enriched areas of the rat striatum. Potassium (9 mM, 15 mM) stimulated the release of [ 3H]GABA in a concentration- and calcium-dependent manner and the veratridine (1 μM)-evoked release of [ 3H]GABA was completely abolished in the presence of tetrodotoxin (1 μM). The selective glutamatergic agonist α-amino-3-hydroxy-5-methylisoxazole-4-propionate (1 mM) enhanced the potassium-evoked release of [ 3H]GABA as well as the basal outflow of [ 3H]GABA. This latter effect was found to be calcium-dependent, partially diminished by tetrodotoxin (1 μM), completely blocked by 6,7-dinitro-quinoxaline-2,3-dione (0.1 mM), which is generally used as an antagonist of α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors, but not affected by ( + )-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK801, 10μM), a specific antagonist of N-methyl- d-aspartate receptors. Similarly, N-methyl- d-aspartate (1 mM) enhanced both the potassium (9mM) and the α-amino-3-hydroxy-5-methylisoxazole-4-propionate (1 mM )-evoked release of [ 3H]GABA but when used alone, due to the presence of magnesium in the superfusion medium, was ineffective on the basal efflux of [ 3H]GABA. A stimulatory effect of N-methyl- d-aspartate (1 mM) on the basal outflow of [ 3H]GABA was observed, however, when magnesium was omitted from the superfusion medium. The stimulatory effect of N-methyl- d-aspartate (1 mM) observed in the presence of α-amino-3-hydroxy-5-methylisoxazole-4-propionate was not potentiated by glycine (1 μM, in the presence of strychnine 1 gmM) and the N-methyl- d-aspartate-evoked response seen in the absence of magnesium was not enhanced by d-serine (1 mM). suggesting that endogenous glycine is already acting on N-methyl- d-aspartate receptors. In fact, in the absence of magnesium, 7-chloro-kynurenate (1 mM) completely abolished the stimulatory effect of N-methyl- d-aspartate on the release of [ 3H]GABA confirming that under our conditions, the glycine site of the N-methyl- d-aspartate receptor is saturated. N-methyl- d-aspartate-evoked responses were all blocked by MK801 (10 μM). Finally, the N-methyl- d-aspartate-evoked response seen in the absence of magnesium was markedly reduced in the presence of tetrodotoxin (1 μM). While potassium (9 mM) markedly stimulated the release of [ 3H]GABA from purified synaptosomes from the rat striatum, neither α-amino-3-hydroxy-5-methylisoxazole-4-propionate alone nor N-methyl- d-aspartate in the presence of α-amino-3-hydroxy-5-methylisoxazole-4-propionate or in the absence of magnesium enhanced the release of [ 3H]GABA. A slight but not significant stimulatory effect was observed with N-methyl- d-aspartateand d-serine using a magnesium-free superfusion medium. Altogether, these results strongly suggest that α-amino-3-hydroxy-5-methylisoxazole-4-propionate and N-methyl- d-aspartate receptors are present on dendrites and/or cell bodies of efferent GABAergic neurons but not on their nerve terminals in matrix-enriched areas of the rat striatum.