Control of ionic currents and fluid secretion by muscarinic agonists in exocrine glands

Stimulation of muscarinic receptors in exocrine glands leads to a complex set of intracellular events involving activation of a GTP-binding protein, production of inositol trisphosphate, and release of Ca²⁺ from the endoplasmic reticulum. One of the consequences of the ensuing rise in Ca²⁺ is the activation of three classes of Ca²⁺-dependent channels that are involved in the fluid secretion response of the glands. In addition, muscarinic stimulation closes the gap junction channels that link adjacent cells. Application of patch-clamp methods has helped to characterize the channels implicated in these processes. Whole-cell recordings using intracellular solutions containing GTP derivatives or inositol trisphosphate are currently used to study the chain of events that take place during muscarinic stimulation.     

N-methyl-D-aspartate receptor-mediated signaling in the supraoptic nucleus involves activation of a nitric oxide-dependent pathway

N-Methyl-D-aspartate (NMDA) microinjection (1 mM, 0.2 μl) into the hypothalamic supraoptic nucleus (SON) stimulated heart rate in urethane-anaesthetized rats. This effect was inhibited by coinjection of a competitive blocker of NMDA receptors, CPP (20 nmol) or by pretreatment with a sympathetic ganglionic blocker, chlorisondamine chloride (5 mg/kg i.p.), but not by prior hypophysectomy. Furthermore, the cardioexcitatory effect of intra-SON NMDA was inhibited by prior intra-SON injection of a competitive blocker of nitric oxide (NO) synthesis, N^G-nitro-L-arginine methyl ester (40 nmol) or a blocker of the soluble guanylate cyclase, Methylene blue (20 nmol), and was mimicked by intra-SON injection of a calcium ionophore, A23187 (10 nmol), which stimulates NO production by raising intracellular free calcium levels. Finally, intra-SON microinjection of a membrane-permeating cGMP analog, 8-bromo-cGMP (20 nmol) stimulated heart rate in urethane-anaesthetized rats. The results point to a functional link between a sympathetically mediated cardiophysiological effect of NMDA receptor stimulation in the SON and activation of the NO/cGMP signal transduction pathway.     

Endothelin stimulates nitric oxide-dependent cyclic GMP formation in rat cerebellar astroglia

Endothelins (ETs) elicit a diversity of cellular responses in cultured astrocytes that suggest an important role of these peptides in glial cell function. Stimulation of astroglial ET receptors induces phosphoinositide (PI) hydrolysis and intracellular calcium mobilization, but little is known about the signalling events that occur downstream of this system. Here we show that in rat cerebellar astroglia in culture ETs produce a receptor-mediated stimulation of cyclic GMP (cGMP) formation that is rapid and totally dependent on nitric oxide synthase (NOS) activity. The effect is blocked by an inhibitor of PI phospholipase C, compound U73122, and by depletion of intracellular calcium stores with thapsygargin. These results indicate that calcium released by inositol trisphosphate is responsible for NOS activation and subsequent cGMP formation.     

Inositol trisphosphate releases intracellularly stored calcium and modulates ion channels in molluscan neurons

Stimulation of the bag cell neurons of Aplysia triggers a long-lasting afterdischarge in these cells. In vivo, such a discharge causes the onset of a sequence of reproductive behaviors. We have found that treatments that trigger discharges in vitro stimulate the hydrolysis of phosphoinositides in the bag cell neurons, as measured by increased incorporation of 3H-inositol into fractions containing membrane lipids and water-soluble inositol phosphates. The electrophysiological effects of inositol trisphosphate, one of the products of phosphoinositide turnover that has been shown to mobilize intracellular calcium in non-neuronal cells, were investigated using isolated bag cell neurons in cell culture. Microinjection of inositol trisphosphate into cultured bag cell neurons caused a transient hyperpolarization of the membrane (approximately 35 sec), together with an increase in conductance. This effect of inositol trisphosphate was abolished by 50 mM tetraethylammonium ions. Inositol trisphosphate also reduced the amplitude of action potentials. Injection of calcium ions directly into bag cell neurons mimicked these responses seen after inositol trisphosphate injection. Using the cell-attached patch-clamp technique in conjunction with inositol trisphosphate microinjection, we observed that inositol trisphosphate evoked increases in the activity of a channel carrying outward current at the resting potential and more positive potentials. The estimated slope conductance of the channel modulated by inositol trisphosphate was approximately 40 pS, and its reversal potential was close to that predicted for potassium ions. The increased opening of this channel in response to inositol trisphosphate injection appeared to result from a transient shift of its voltage-dependence to more negative potentials. In a few cases, inositol trisphosphate injection also elicited an increase in the activity of a channel passing inward current at rest. Direct measurements of changes in intracellular calcium in response to inositol trisphosphate were made using digital imaging of isolated neurons loaded with the fluorescent calcium indicator fura-2. These revealed that injection of inositol trisphosphate significantly elevated intracellular calcium levels, and that this inositol trisphosphate-induced rise in cytosolic calcium was not affected by removal of extracellular calcium. In contrast to the effects of trains of action potentials in calcium-containing media, which produced increases in calcium primarily in neurites, the inositol trisphosphate-induced elevation of calcium appeared more localized to the somata of these neurons.(ABSTRACT TRUNCATED AT 400 WORDS)     

Taurine Release Evoked by NMDA Receptor Activation is Largely Dependent on Calcium Mobilization from Intracellular Stores

It is known that the activation of N-methyl-d -aspartate (NMDA) receptors leads to an increase in extracellular taurine concentration in different brain regions. The mechanism that mediates this effect is not totally understood. In this study, rat hippocampal slices were used to determine the dependence of NMDA-induced taurine release on extracellular calcium and/or on calcium mobilization from intracellular stores. NMDA was administered through a microdialysis probe inserted into the slice, at the level of CA1 stratum radiatum, which was also used to collect amino acids from the extracellular space. Field potentials evoked by stimulation of the Schaffer collaterals and recorded in the stratum pyramidale of CA1 were used as a control of NMDA receptor activation. NMDA induced a marked increase in extracellular taurine levels and a decrease in field potential amplitude, and both effects were suppressed in the presence of MK-801, a blocker of the NMDA receptor-linked channel. Dantrolene, an inhibitor of calcium release from intracellular stores, partially inhibited the extracellular taurine increase, while 2-nitro-4-carboxyphenyl-N,N-diphenyl carbamate (NCDC), an inhibitor of phosphatidylinositol-specific phospholipase C activation, had no effect. Removal of extracellular calcium diminished, but did not abolish, the extracellular taurine increase caused by NMDA. The remaining taurine response was totally suppressed by dantrolene, and also by NCDC. These results demonstrate that the release of taurine induced by NMDA receptor activation is triggered by the increase in cytoplasmic calcium concentration. We suggest that, under physiological conditions, calcium influx provides the signal for NMDA-induced taurine release, which is amplified by calcium-dependent calcium mobilization from intracellular stores. In the absence of extracellular calcium, NMDA is still able to evoke taurine release through a mechanism that implies calcium release from inositol 1,4,5-trisphosphate-sensitive stores.     

Imaging upregulated brain arachidonic acid metabolism in HIV-1 transgenic rats

Human immunodeficiency virus (HIV)-associated infection involves the entry of virus-bearing monocytes into the brain, followed by microglial activation, neuroinflammation, and upregulated arachidonic acid (AA) metabolism. The HIV-1 transgenic (Tg) rat, a noninfectious HIV-1 model, shows neurologic and behavioral abnormalities after 5 months of age. We hypothesized that brain AA metabolism would be elevated in older HIV-1 Tg rats in vivo. Arachidonic acid incorporation from the plasma into the brain of unanesthetized 7-to-9-month-old rats was imaged using quantitative autoradiography, after [1-¹⁴C]AA infusion. Brain phospholipase (PLA₂) activities and eicosanoid concentrations were measured, and enzymes were localized by immunostaining. AA incorporation coefficients k^* and rates J_in, measures of AA metabolism, were significantly higher in 69 of 81 brain regions in HIV-1 Tg than in control rats, as were activities of cytosolic (c)PLA₂-IV, secretory (s)PLA₂, and calcium independent (i)PLA₂-VI, as well as prostaglandin E₂ and leukotriene B₄ concentrations. Immunostaining of somatosensory cortex showed elevated cPLA₂-IV, sPLA₂-IIA, and cyclooxygenase-2 in neurons. Brain AA incorporation and other markers of AA metabolism are upregulated in HIV-1 Tg rats, in which neurologic changes and neuroinflammation have been reported. Positron emission tomography with [1-¹¹C]AA could be used to test whether brain AA metabolism is upregulated in HIV-1-infected patients, in relation to cognitive and behavioral disturbances.     

Inhibition by soman of NMDA-stimulated [H]norepinephrine release from rat cortical slices, studies of non-cholinergic effect

Effects of soman, an irreversible cholinesterase (ChE) inhibitor, on [³H]norepinephrine (NE) release evoked by N-methyl-

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-aspartate (NMDA) were studied in rat brain cortical slices. Soman inhibited NMDA-stimulated [³H]NE release in a concentration-dependent manner. This effect was neither reversed by atropine, an antagonist of the muscarinic receptor, nor by

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-tubocurarine, an antagonist of the nicotinic receptor. Incubation of the slices with NMDA antagonists, AP5, MK-801, ketamine or magnesium, resulted in inhibitory effects on NMDA-stimulated [³H]NE release. Soman significantly shifted the inhibition curves downward and significant interactions between these chemicals and soman were observed. Glycine potentiated the release of [³H]NE stimulated by NMDA, and soman did not alter this effect of glycine. Soman also inhibited the release of [³H]NE evoked by K⁺ in a concentration-dependent manner. NMDA-stimulated [³H]NE release was inhibited by tetrodotoxin (TTX), an antagonist of voltage-dependent sodium channels, and a significant interaction between soman and TTX was observed. The [³H]NE release induced by NMDA was dependent on extracellular calcium concentrations and was inhibited by nifedipine, a selective blocker of the L-type voltage-dependent calcium channels (VDCC), or cadmium, a non-specific blocker of VDCC. However, no significant interaction between the effects of soman and calcium, nifedipine, or cadmium was observed. Taken together, the results suggested that: (1) soman has a direct action at non-cholinergic sites; (2) soman may interfere with some of the regulatory sites of the NMDA receptor–ion channel complex; and (3) the voltage-dependent sodium channel, but not VDCC, may be a site of action for soman.     

N-methyl-d-aspartate stimulates dopamine release through nitric oxide formation in the nucleus accumbens of rats

Intracerebral microdialysis technique was utilized to study the effect ofN^G-nitro-l-arginine, a nitric oxide (NO) synthase inhibitor, onN-methyl-d-aspartate (NMDA)-induced dopamine overflow in the nucleus accumbens of unanesthetized, freely moving rats. Perfusion of 1 and 3 mM NMDA through the microdialysis probe dose-dependently increased the extracellular dopamine level in the nucleus accumbens. Coapplication of 0.5 mMd-(−)-2-amino-5-phosphonovaleric acid (D-AP5), a selective and competitive NMDA receptor antagonist, significantly reduced the dopamine overflow induced by 3 mM NMDA. Perfusion of 0.5 mMN^G-nitro-l-arginine alone did not affect the basal dopamine level, whereas it suppressed the NMDA-evoked dopamine overflow in the nucleus accumbens when concurrently applied with 3 mM NMDA. These results suggest that NO mediates, at least in part, dopamine release resulting from NMDA receptor activation in the nucleus accumbens of rats.     

N-methyl-d-aspartate stimulates dopamine release through nitric oxide formation in the nucleus accumbens of rats

Intracerebral microdialysis technique was utilized to study the effect ofN^G-nitro-l-arginine, a nitric oxide (NO) synthase inhibitor, onN-methyl-d-aspartate (NMDA)-induced dopamine overflow in the nucleus accumbens of unanesthetized, freely moving rats. Perfusion of 1 and 3 mM NMDA through the microdialysis probe dose-dependently increased the extracellular dopamine level in the nucleus accumbens. Coapplication of 0.5 mMd-(−)-2-amino-5-phosphonovaleric acid (D-AP5), a selective and competitive NMDA receptor antagonist, significantly reduced the dopamine overflow induced by 3 mM NMDA. Perfusion of 0.5 mMN^G-nitro-l-arginine alone did not affect the basal dopamine level, whereas it suppressed the NMDA-evoked dopamine overflow in the nucleus accumbens when concurrently applied with 3 mM NMDA. These results suggest that NO mediates, at least in part, dopamine release resulting from NMDA receptor activation in the nucleus accumbens of rats.     

Human immunodeficiency virus type 1 clade B and C gp120 differentially induce neurotoxin arachidonic acid in human astrocytes: implications for neuroAIDS

HIV-1 clades (subtypes) differentially contribute to the neuropathogenesis of HIV-associated dementia (HAD) in neuroAIDS. HIV-1 envelop protein, gp120, plays a major role in neuronal function. It is not well understood how these HIV-1 clades exert these neuropathogenic differences. The N-methyl-d-aspartate (NMDA) receptor-reduced glutamine synthesis could lead to secretion of neurotoxins such as arachidonic acid (AA) which plays a significant role in the neuropathogenic mechanisms in neuroAIDS. We hypothesize that clade B and C gp120 proteins exert differential effects on human primary astrocytes by production of the neurotoxin arachidonic acid. Our results indicate that clade B gp120 significantly downregulated NMDA receptor gene and protein expression, and level of glutamine while increasing expression of prostaglandin E2 (PGE₂) and thromboxane A2 receptor (TBXA₂ R) compared to HIV-1 clade C gp120 protein. Thus, our studies for the first time demonstrate that HIV-1 clade B-gp120 protein appears to induce higher levels of expression of the neuropathogenic molecule cyclooxygenase-2 (COX-2)-mediated arachidonic acid by-products, PGE₂, and TBXA₂ R compared to HIV-1 clade C gp120 protein. These studies suggest that HIV-1 clade B and C gp120 proteins may play a differential role in the neuropathogenesis of HAD in neuroAIDS.     

Differential responses of rat cerebral somatostatinergic and cholinergic cells to glutamate agonists

Reductions in cortical somatostatin (SRIH) and choline acetyltransferase (ChAT) are major biochemical deficits in Alzheimer disease (AD). SRIH and ChAT were measured in fetal rat cerebral neurons after exposure to the glutamate agonistsN-methyl-d-aspartate (NMDA), kainate (KA), and quisqualate (Q). NMDA (96 h incubation) stimulated SRIH release and content in a dose-dependent manner with aB _max of 10^?5 M and EC₅₀ of 2?3×10^?6 M. KA showed a small stimulation in SRIH levels at 10^?5 M, but produced marked inhibition at 10^?4 M. Q decreased both intracellular and secreted SRIH. KA (51–76% of basal) and Q (27–56% of basal) but not NMDA (91–114% of basal) also inhibited the incorporation of [³⁵S]methionine into proteins. In similar experiments 10^?4 M Q (23±9% of basal) and KA (20±3% of basal) but not NMDA (80±16% of basal) reduced ChAT levels in hypothalamic/septal cultures. These inhibitory actions on ChAT activity by KA and Q were reversed by γ-glutamyltaurine (GT) but not by 2-amino-5-phosphonopentanoic acid (AP5). Chronic NMDA exposure partially inhibited muscarinic acetylcholine receptor (mAChR) mediated inositol phospholipid (PI) turnover, whereas it was abolished after KA and Q pretreatment. These findings suggest that in cerebral cell cultures, NMDA has a stimulatory action on somatostatinergic neurons and non-NMDA receptor agonism could play an important role in EAA-mediated neural damage.     

In vivo modulation of nitric oxide concentration dynamics upon glutamatergic neuronal activation in the hippocampus

Nitric oxide (^?NO) is a labile endogenous free radical produced upon glutamatergic neuronal activity in hippocampus by neuronal nitric oxide synthase (nNOS), where it acts as a modulator of both synaptic plasticity and cell death associated with neurodegeneration. The low CNS levels and fast time dynamics of this molecule require the use of rapid analytical methods that can more accurately describe its signaling in vivo. This is critical for understanding how the kinetics of ^?NO‐dependent signaling pathways is translated into physiological or pathological functions. In these studies, we used ^?NO selective microelectrodes coupled with rapid electrochemical recording techniques to characterize for the first time the concentration dynamics of ^?NO endogenously produced in hippocampus in vivo following activation of ionotropic glutamate receptors. Both L ‐glutamate (1–100 mM) and N‐methyl‐D ‐aspartate (NMDA; 0.01–5 mM) produced transient, dose‐dependent increases in extracellular ^?NO concentration. The production of ^?NO in the hippocampus by glutamate was decreased by the nNOS inhibitor 7‐NI. Intraperitoneal administration of the NMDA receptor blocker, MK‐801, and the inhibitor of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoazolepropionic acid (AMPA) receptor, NBQX, applied locally greatly attenuated glutamate‐evoked overflow of ^?NO. Thus, ^?NO overflow elicited by activation of glutamate receptors appeared to result from an integrated activation of ionotropic glutamate receptors, both of the NMDA and AMPA receptors subtypes. Additionally, distinct concentration dynamics was observed in the trisynaptic loop with stronger and longer lasting effects of glutamate activation on ^?NO overflow seen in the CA1 region as compared with the dentate gyrus. Overall, the results provide a quantitative and temporal basis for a better understanding of ^?NO activity in the rat hippocampus. © 2010 Wiley‐Liss, Inc.     

Bed nucleus of the stria terminalis N‐methyl‐D‐aspartate receptors and nitric oxide modulate the baroreflex cardiac component in unanesthetized rats

The bed nucleus of the stria terminalis (BST) plays a tonic role modulating the baroreflex bradycardiac response. In the present study, we verified whether local BST glutamatergic receptors and nitric oxide (NO) system modulate baroreflex bradycardiac responses. Bilateral BST‐ N‐methyl‐D‐aspartate (NMDA) receptor inhibition by treatment with the selective NMDA receptor antagonist LY235959 increased bradycardiac response to mean arterial pressure increases. Treatment with the selective non‐NMDA antagonist NBQX did not affect reflex bradycardia. These results suggest an involvement of local NMDA receptors in the BST‐related tonic inhibitory modulation of baroreflex bradycardiac response. BST treatment with the nonselective NO synthase (NOS) inhibitor L‐NAME or the selective neuronal NOS (nNOS) inhibitor N^ω‐propyl‐L‐arginine increased bradycardiac response, indicating that NO generated by nNOS activation modulates baroreflex. The NO involvement was further reinforced by observation that BST treatment with the NO scavenger carboxy‐PTIO caused an effect similar to that observed after NMDA receptor blockade or treatment with NOS inhibitors. Additionally, it was observed that LY235959 effects on baroreflex bradycardiac response were reverted by BST treatment with the NO‐donor sodium nitroprusside, suggesting an NMDA receptor–NO interaction. Baroreflex bradycardiac responses observed before and after BST treatment with LY235959 or N^ω‐propyl‐L‐arginine were no longer different when animals were pretreated intravenously with the anticholinergic drug homatropine methyl bromide. These results indicate that parasympathetic activation accounts for the effects observed after BST pharmacological manipulation. In conclusion, our data point out that local NMDA and nNOS interaction mediates the tonic inhibitory influence of the BST on the baroreflex bradycardiac response, modulating the parasympathetic cardiac activity. © 2009 Wiley‐Liss, Inc.     

Exposure to gp120 of HIV-1 Induces an Increased Release of Arachidonic Acid in Rat Primary Neuronal Cell Culture Followed by NMDA Receptor-mediated Neurotoxicity

After incubation of highly enriched neurons from rat cerebral cortex with the HIV-1 coat protein gp120 for 18 h, cells showed fragmentation of DNA at internucleosomal linkers followed by NMDA receptor-mediated neurotoxicity. We report that in response to exposure to gp120 cells react with an increased release of arachidonic acid (AA) via activation of phospholipase A₂. This process was not inhibited by NMDA receptor antagonists. To investigate the role of AA on the sensitivity of the NMDA receptor towards its agonist, low concentrations of NMDA were co-administered with AA. This condition enhanced the NMDA-mediated cytotoxicity. Administration of mepacrine reduced cytotoxicity caused by gp120. We conclude that gp120 causes an activation of phospholipase A₂, resulting in the increased release of AA, which may in turn sensitize the NMDA receptor.     

In vivo evidence for functional NMDA receptor blockade by memantine in rat hippocampal neurons

Antagonising the NMDA (N-methyl-d-aspartate) receptor complex is a widely hypothesised therapeutic approach in several neurodegenerative conditions, such as Alzheimer’s disease. Memantine, a moderate affinity uncompetitive NMDA receptor antagonist, has been in clinical use for several years and numerous experimental data support its NMDA receptor blocking effects. It has recently been reported in transfected HEK293T cells that physiological concentrations of Mg²⁺ may impart partial NMDA receptor subtype selectivity and weaken the overall inhibitory actions of memantine in NMDA receptor-mediated cellular events. In the present study, we set out to investigate the effect of intravenously applied memantine on iontophoresed NMDA-evoked firing of hippocampal CA1 neurons using in vivo conditions. Cumulative doses of memantine in the rat (4, 8 and 16 mg/kg i.v.) caused the firing rate to decrease in a dose-dependent manner to 77 ± 7, 58 ± 8 and 34 ± 12% of control, respectively, while saline application had no significant effect. We show that therapeutic doses of memantine are able to antagonize NMDA receptor-mediated activity in the principal cells of the hippocampus in vivo, i.e. in the presence of physiological concentrations of Mg²⁺.     

Mechanism of synchronized Ca oscillations in cortical neurons

Dissociated rat cortical neurons reassociate in vitro to form synaptically connected networks. Removal of Mg²⁺ from the extracellular medium then induces neurons in the network to undergo synchronized oscillations of cytoplasmic calcium. Previous studies have shown that these calcium oscillations involve the activation of NMDA receptors and that the rising phase of each calcium spike is coincident with a brief burst of action potentials (Robinson et al., Jpn. J. Physiol. 43 (Suppl. 1) (1993) S125–130; Robinson et al., J. Neurophysiol. 70 (1993) 1606–1616; Murphy et al., J. Neurosci. 12 (1992) 4834–4845). We have found that these calcium oscillations are dependent on an influx of extracellular calcium but are independent of mobilization of calcium from intracellular stores. The influx of extracellular Ca²⁺ occurs primarily through L-type voltage-gated calcium channels (VGCCs), since diltiazem inhibits calcium oscillations under all conditions. On the other hand, N-, P/Q-, and T-type VGCCs are not required for calcium oscillations, although inhibitors of these channels may act as partial antagonists. In addition to removal of Mg²⁺, oscillations can also be induced by the inhibition of voltage-gated K⁺ channels with 4-aminopyridine (4-AP), a treatment known to increase neurotransmitter release. In the presence of 4-AP, synchronized calcium oscillations become independent of NMDA receptor activation, although they continue to require activation of AMPA/KA receptors. A model for the mechanism of neuronal calcium oscillations and the reason for their synchrony is presented.     

Effects of a nitric oxide synthase inhibitor on NMDA receptor function in organotypic hippocampal cultures

Nitric oxide (NO) has been proposed to trigger long-term potentiation (LTP) at CA3 to CA1 synapses. We previously reported that NO synthesis inhibitors and blockers reduce an electrophysiological index of NMDA receptor activation in acute hippocampal slices. We now show that the NOS inhibitor, N^G-methyl-

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-arginine (MLA), also reversibly prevents LTP induction in organotypic hippocampal slices and significantly reduces a biochemical index of NMDA receptor function. These results further indicate that MLA inhibits LTP induction by interfering with NMDA receptor functions.     

Neurotoxicity evoked by N-methyl-d-aspartate in the organotypic static slice cultures of rat cerebral cortices: effect of GABAA receptor activation

We investigated the neurotoxicity evoked by N-methyl-d-aspartate (NMDA) receptor stimulation in the organotypic static slice cultures of rat cerebral cortices. We also examined whether the γ-aminobutyric acid (GABA)_A receptor agonist muscimol has a protective effect on the NMDA-mediated neurotoxicity in this culture system. NMDA-mediated cytotoxicity was evaluated histologically and quantified by the measurement of lactate dehydrogenase (LDH) release into the culture medium. There was an NMDA-induced, dose-dependent leakage of LDH release and neuronal cell death, which were not attenuated by muscimol treatment. The results suggested that NMDA neurotoxicity is reproduced in the organotypic culture, and that GABA_A receptor activation exerted no protective action against the NMDA cytotoxicity. Received: 25 July 1997 / Revised, accepted: 1 December 1997     

Phospholipase A2 and Somatostatin Release are Activated in Response to N-Methyl-D-Aspartate Receptor Stimulation in Hypothalamic Neurons in Primary Culture

We have recently shown that glutamate primarily induces somatostatin release in hypothalamic neurons through N-methyl-D-aspartate (NMDA)-type receptor sites. Here we report that glutamate and NMDA also stimulate the release of [³H]arachidonic acid in a dose-dependent manner. The NMDA-induced effects (arachidonic acid release and somatostatin secretion) were both inhibited by MK-801, an NMDA receptor-type antagonist, or mepacrine, a phospholipase A₂ inhibitor. In addition, mepacrine was able to inhibit A23187-stimulated arachidonic acid release and somatostatin secretion. p-Bromophenacylbromide, another phospholipase A₂ inhibitor, also blocked NMDA-induced secretion of somatostatin. However, responses to NMDA were unaffected by H7 (inhibitor of protein kinase C), nordihydroguaiaretic acid or indomethacin (inhibitors of lipoxygenase and cyclooxygenase). Melittin, a phospholipase A₂ activator, was found to stimulate both responses, but omission of extracellular Ca²⁺ from the incubation media strongly reduced melittin-induced somatostatin release. Six-h pertussis toxin pretreatment did not significantly reduce the action of NMDA on either of the two parameters studied. High-performance liquid chromatography analysis of [₃H]metabolites released in the medium after NMDA stimulation revealed that [₃H]arachidonic acid was the only detectable metabolite. External addition of arachidonic acid increased the release of somatostatin, whereas E² and F²α prostaglandins had no effect. Our results show a close correlation between arachidonic acid release and somatostatin secretion, the two parameters we investigated.     

Nitric oxide modulates NMDA-induced increases in intracellular Ca2+ in cultured rat forebrain neurons.

We studied the effects of nitric oxide (NO) and the NO-releasing agents sodium nitroprusside (SNP), S-nitroso-N-acetylpenicillamine (SNAP) and isosorbide dinitrate (ISDN) on N-methyl-D-aspartate (NMDA)-induced increases in intracellular Ca2+ ([Ca2+]i), whole-cell patch-clamp currents and on glutamate-stimulated [3H]dizocilpine binding. NO and agents that release NO partially inhibit increases in [Ca2+]i at concentrations between 1 microM and 1 mM. These agents also decrease [Ca2+]i changes produced by kainate and potassium, but to a smaller extent. As the effects of NO are still present following alkylation of the redox modulatory site on the NMDA receptor this action of NO is probably not a consequence of oxidation of the redox site. In contrast to SNP, ISDN does not inhibit NMDA-induced whole cell patch-clamp currents suggesting that NO modulates [Ca2+]i via perturbation of a Ca2+ homeostatic process. Furthermore, SNP may have a direct action on the NMDA receptor complex in addition to the generation of NO. 8-Bromo-cGMP does not mimic the inhibitory effect of NO suggesting that this effect is not the result of NO stimulation of neuronal cGMP production. As the production of NO in neurons is dependent on increases in [Ca2+]i associated with NMDA receptor activation, these data suggest that NO-mediated decreases in [Ca2+]i may represent a novel feedback inhibitory mechanism for NO production in the brain.