Glutamate triggers neurosecretion and apoptosis in bovine chromaffin cells through a mechanism involving NO production by neuronal NO synthase activation.

Previous work from our group stated that nitric oxide (NO), via cytokines, induces apoptosis in chromaffin cells by a mechanism involving iNOS, nNOS, and NF-κB. In this paper the involvement of glutamate as a possible intracellular trigger of neurosecretion and NO-mediated apoptosis has been evaluated. We show that chromaffin cells express different ionotropic and metabotropic glutamate receptors, this exerting different effects on the regulation of basal and glutamate-induced catecholamine secretion, via NO/cGMP. In addition, we studied the effects of endogenously generated NO, both basal and glutamate-stimulated, on apoptosis of chromaffin cells. Our results show that glutamate agonists are able to induce cell death and apoptosis in bovine chromaffin cells, parallel to an increase in NO production. Such effects were reversed by NOS inhibitors and glutamate receptor antagonists. Under basal conditions, iNOS inhibitors did not have any effect on apoptosis, whereas nNOS inhibitors induced apoptosis, indicating a neuroprotective effect of constitutive nNOS-generated NO. In contrast, glutamate-induced apoptosis was strongly reversed by nNOS inhibitors and weakly by iNOS inhibitors, thus indicating nNOS involvement in glutamate-mediated apoptosis. These results were confirmed by the fact that nNOS expression, but not iNOS, is specifically activated by glutamate. Finally, our results suggest the participation of PKG, PKA, PKC, and MAPK pathways in glutamate-mediated nNOS activation in chromaffin cells and point out the involvement of both PKA and PKC signaling pathways in the apoptotic effect of glutamate.

Review: Could neurotransmitters influence neurogenesis and neurorepair after stroke?

Brain ischaemia and reperfusion produce alterations in the microenvironment of the parenchyma, including ATP depletion, ionic homeostasis alterations, inflammation, release of multiple cytokines and abnormal release of neurotransmitters. As a consequence, the induction of proliferation and migration of neural stem cells is redirected towards the peri-infarct region. The success of new neurorestorative treatments for damaged brain implies the need to describe with greater accuracy the mechanisms in charge of regulating adult neurogenesis, under both physiological and pathological conditions. Recent evidence demonstrates that many neurotransmitters, glutamate in particular, control the subventricular zone (SVZ), thus being part of the complex signal network that exerts a remarkable influence on the production of new neurones. Neurotransmitters provide a link between brain activity and SVZ neurogenesis. Therefore, a deeper knowledge of the role of neurotransmitters systems, such as glutamate and its transporters, in adult neurogenesis, may prove a valuable tool to be utilized as a neurorestorative therapy in this pathology.

Plasma membrane and vesicular glutamate transporter expression in chromaffin cells of bovine adrenal medulla.

The study of the functional expression of glutamate signaling molecules in peripheral tissues has received relatively little attention. However, evidence is increasing for a role of glutamate as an extracellular signal mediator in endocrine systems, in addition to having an excitatory amino acid neurotransmitter role in the CNS. Chromaffin cells are good models of catecholaminergic neurons, in which previous work from our group demonstrated the existence of both functional glutamate receptors and specific exocytotic and nonexocytotic glutamate release. In this work, the presence of specific plasma membrane (EAATs) and vesicular glutamate (VGLUTs) transporters has been investigated by using confocal microscopy, flow cytometric analysis, Western blot, and qRT-PCR techniques. We found specific expression of EAAT3, EAAT2, VGLUT1, and VGLUT3 in about 95%, 65%, 55%, and 25%, respectively, of the whole chromaffin cell population. However, chromaffin cells do not express VGLUT2 and have a very low expression of EAAT1. VGLUTs are localized mainly in the membrane fraction, and EAATs share their subcellular location between membrane and cytosolic fractions. Their estimated molecular weights were about 70 kDa for EAAT2, about 65 kDa for EAAT3, about 50 kDa for VGLUT1, and about 60 kDa for VGLUT3. RT-qPCR techniques confirm the expression of these glutamate transporters at the mRNA level and show a different regulation by cytokines and glucocorticoids between VGLUT1 and -3 and EAAT2 and -3 subfamilies. These interesting results support the participation of these glutamate transporters in the process of glutamate release in chromaffin cells and in the regulation of their neurosecretory function in adrenal medulla.

Involvement of NMDA receptor in the modulation of excitatory and inhibitory amino acid neurotransmitters release in cortical neurons.

The purpose of this paper was to examine the function of N-methyl-D-aspartate (NMDA) glutamate receptor in cortical neurons on amino acid neurotransmitters release as well as the fraction of neurons implicated in the response of this receptor. Local stimulation of these cells at different concentrations of NMDA, agonist of this ionotropic glutamate receptor, produced a dose dependent release of aspartate, glutamate, glycine and GABA. These effects were blocked by DAP5, an antagonist of the NMDA receptor. The amino acid Ca(2+) dependent release mediated by the NMDA receptor, is induced by the opening of voltage-dependent Ca(2+) channels that this receptor promotes. Ca(++) movements were explored in single cells loaded with fura-2. When single cells were stimulated with 100 microM NMDA, the calcium recording performed showed that 82% of the cells responded to this agonist increasing the intracellular calcium concentration, although the amplitude of these increments was variable. The results suggest that NMDA-elicited neurotransmitter release from cortical neurons involves Ca(2+)-dependent and Ca(2+)-independent components, as well as neuron depolarisation, and different VDCC subtypes of N, P/Q or L depending of the amino acid neurotransmitter release elicited by this receptor.

Nitric oxide and peroxynitrite induce cellular death in bovine chromaffin cells: evidence for a mixed necrotic and apoptotic mechanism with caspases activation.

Treatment of chromaffin cells with nitric oxide (NO) donors (SNP and SNAP) and peroxynitrite produces a time- and dose-dependent necrotic and apoptotic cell death. Necrotic cell death was characterized by both an increase in lactate dehydrogenase and ATP release and changes in nuclei and cell morphology (as seen with fluorescence microscopy analysis with propidium iodide and Hoechst 33342). Apoptotic cell death was characterized by nuclear fragmentation and presence of apoptotic cell bodies, by a decrease in DNA content, and by an increase in DNA fragmentation. Treatment of chromaffin cells with lipopolysaccharide (LPS) or cytokines (interferon-gamma, tumor necrosis factor-alpha) resulted only in apoptotic cell death. Apoptotic effects of NO-inducing compounds were specifically reversed, depending on the stimuli, by the NO scavenger carboxy-PTIO (CPTio) or by the NOS inhibitors L-NMA and thiocitrulline. NO-induced apoptotic death in chromaffin cells was concomitant to a cell cycle arrest in G0G1 phase and a decrease in the number of chromaffin cells in the G2M and S phases of cell cycle. All NO-producing compounds were able to induce activation of caspase 3 and cytochrome c release, and specific inhibitors of caspase 3 and 9, such as Ac-DEVD-CHO (CPP32) and Ac-Z-LEHD-FMK, respectively, prevented NO-induced apoptosis in chromaffin cells. These results suggest that chromaffin cells could be good models for investigating the molecular basis of degeneration in diseases showing death of catecholaminergic neurons, phenomenon in which NO plays an important role.

Cadmium induces reactive oxygen species generation and lipid peroxidation in cortical neurons in culture.

Cadmium is a toxic agent that it is also an environmental contaminant. Cadmium exposure may be implicated in some humans disorders related to hyperactivity and increased aggressiveness. This study presents data indicating that cadmium induces cellular death in cortical neurons in culture. This death could be mediated by an apoptotic and a necrotic mechanism. The apoptotic death may be mediated by oxidative stress with reactive oxygen species (ROS) formation which could be induced by mitochondrial membrane dysfunction since this cation produces: (a) depletion of mitochondrial membrane potential and (b) diminution of ATP levels with ATP release. Necrotic death could be mediated by lipid peroxidation induced by cadmium through an indirect mechanism (ROS formation). On the other hand, 40% of the cells survive cadmium action. This survival seems to be mediated by the ability of these cells to activate antioxidant defense systems, since cadmium reduced the intracellular glutathione levels and induced catalase and SOD activation in these cells.

Mitochondrial involvement in nitric oxide-induced cellular death in cortical neurons in culture.

Nitric oxide (NO) is an unstable molecule with physiological and pathological properties. In brain, NO acts as a modulator of neurotransmission as well as a protector against neuronal death from several death stimuli. However, beside this protector effect, high NO concentrations produce neuronal death by a mechanism in which the caspase pathway is implicated. In this work, we demonstrate that in cortical neurons the NO toxicity is mediated by mitochondrial dysfunction. SNAP, an NO donor, induces apoptosis in these cells because it 1) increases the p53 and 2) induces cytochrome c release and activation of caspase-9 and caspase-3. SNAP also induces necrosis, through 1) breakdown of the mitochondrial membrane potential, 2) ATP decrease, 3) ROS formation, and 4) LDH and ATP release, indicative of oxidative stress and death by necrosis. To sum up, in cortical neurons, high NO concentrations produced cellular death by both an apoptotic and a necrotic mechanism in which the mitochondria are implicated.

SNAP, a NO donor, induces cortical neuron death by a mechanism in which the caspase pathway is implicated.

In this paper, we present data which demonstrate that, in cortical neurons, SNAP induces loss in cell viability as evaluated by the XTT test. This cell death started at 250 microM SNAP when the treatment was performed in a serum-free medium and at 10 microM when the treatment was given in the presence of serum. This death was mediated, at least in part, by an apoptotic mechanism detected by flow cytometry and DNA fractionation. The highest SNAP concentrations induced a dual behavior on caspase-3 activity. Concentrations of 250 microM in the absence of serum and 10 microM to 300 microM in the presence of serum produced caspase-3 activation. This indicates that NO induces neuronal death by an apoptotic mechanism in which the caspase pathway is implicated. Higher SNAP concentrations (500 microM to 1 mM) diminished the caspase-3 activity to levels similar or even lower than control values. This profile was observed in the absence as well as in the presence of serum in the medium. The caspase-3 inhibition mediated by the highest SNAP concentrations did not imply NO cellular protection since the caspase-3 inhibition mediated by these SNAP concentrations neither correlated with cellular viability nor with cellular apoptosis. The possible mechanism of caspase-3 inhibition at the highest SNAP concentrations used is discussed.

SNAP, a NO donor, induces cellular protection only when cortical neurons are submitted to some aggression process.

Nitric oxide is a versatile molecule, which plays important physiological and pathological roles. Its protective and toxic actions have been already evidenced in several cell types. However, the protective effect in cortical neurons remains elusive. In this work, we demonstrate that the NO-donor SNAP may induce both neuroprotection and neurotoxicity in this sort of cells. The protective effect of NO was evidenced when cortical neurons were exposed to deleterious conditions, such as serum deprivation. Serum deprivation induces apoptotic cortical neuron death through a caspase-dependent mechanism. Under these conditions, SNAP was able to oppose cell death through both caspase-3 inhibition and/or increase of antiapoptotic protein levels (Bcl-2 and Bcl-x(L)). On the other hand, in a normally serum-supplemented medium, high dose of SNAP behaves as a neurotoxic agent, through a mechanism which involves caspase-3 activation.

Nitric oxide donors induce calcium-mobilisation from internal stores but do not stimulate catecholamine secretion by bovine chromaffin cells in resting conditions.

The potential role of nitric oxide (NO) donors and peroxynitrites on both basal catecholamine (CA) secretion and modulation of calcium levels has been investigated in primary cultures of bovine chromaffin cells. NO donors did not modulate catecholamine secretion, while peroxynitrites induced a time dose-dependent increase in basal CA secretion. Two facts may explain the lack of these compounds on basal CA secretion. NO donors induce, on the one hand, an increase in intracellular calcium levels by depletion of internal IP3-stores from endoplasmic reticulum. On the other hand, a small calcium influx through N-type voltage-dependent calcium channels (VDCC), which seem not to be coupled to exocytosis of adrenaline and noradrenaline in chromaffin cells. Both effects, calcium-mobilisation from internal stores and calcium entry through N-type VDCC are mediated by cGMP synthesis. In contrast, peroxynitrites induce an increase in basal CA secretion by both a decrease of intracellular catecholamine content and a toxic effect on cellular membrane. All these results, taken together, could explain contradictory results in the literature on the role of NO on basal catecholamine secretion and on modulation of intracellular calcium in chromaffin cells.

Expression and functional properties of group I metabotropic glutamate receptors in bovine chromaffin cells.

We demonstrate the presence and functional properties of Group I metabotropic glutamate receptors (mGluRs) expressed in chromaffin cells. Immunocytochemical techniques revealed that two mGluR subtypes (mGluR1alpha and mGluR5) are expressed in chromaffin cells, located in both the cytoplasmic membrane and the cytosol surrounding the nucleus. These mGluRs are functionally active on catecholamine (CA) secretion in chromaffin cells because both (1S, 3R)-1-aminocyclopentane-1,3-dicarboxylic acid (t-ACPD) and the specific agonist of Group I mGluRs, (S)-3,5-dihydroxyphenylglycine (DHPG), were able to stimulate the release of CAs (adrenaline and noradrenaline) in a dose-response manner. These effects were specifically reversed by L-(+)-2-amino-3-phosphonopropionic acid (L-AP3), a selective antagonist of the Group I metabotropic glutamate receptors. t-ACPD induced an increase in CA secretion in both the presence and absence of extracellular calcium, the former effect being accompanied by cell membrane depolarization. Noradrenaline (NA) release was higher in the presence of extracellular calcium than in its absence, whereas adrenaline release was of the same order under both conditions. These results indicate that different subtypes of Group I mGluRs are present in noradrenergic and adrenergic cells. Fluorescence imaging techniques in single cells showed different t-ACPD-induced increases in intracellular calcium in different chromaffin cells: in chromaffin cells, 67% expressed functional metabotropic glutamate receptors and with nicotinic receptors, whereas the remaining 33% expressed only nicotinic receptors. In the absence of external calcium, only about 25% of cells responded to t-ACPD-increased intracellular calcium by increasing inositol 1,4,5-trisphosphate (IP(3)) concentration and subsequent calcium mobilization from intracellular stores, whereas the remaining 75% increased intracellular calcium by promoting Ca(2+) influx from the extracellular medium through L- and N- but not P/Q voltage-dependent calcium channels.

Apoptosis and necrosis: two distinct events induced by cadmium in cortical neurons in culture.

(1) Cadmium is an extremely toxic metal commonly found in industrial workplaces, a food contaminant and a major component of cigarette smoke. Cadmium can severely damage several organs, including the brain. In this work, we have studied both the cadmium toxicity on rat cortical neurons in culture and the possible protective effect of serum. (2) Our results indicate that: (1) cadmium is taken up by the neurons in a dose and serum dependent way; (2) cadmium, at concentrations from 1 micro M or 10 micro M (depending on the absence or the presence of serum) up to 100 micro M, decreases the metabolic capacity, which was evaluated by the XTT (tetrazolium salt) test; (3) cadmium induces apoptosis and LDH (lactate dehydrogenase) release in a dose dependent way; (4) in a serum-free medium, the cadmium-induced apoptosis is accompanied by caspase-3 activation; (5) both the caspase-3 activation and the cadmium-induced apoptosis are reversed by N-acethyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO), a selective caspase-3 inhibitor, indicating that the caspase-3 pathway is involved in cadmium-induced apoptosis in cortical neurons; and (6) the cadmium concentrations which produce caspase-3 activation do not modify the intracellular ATP levels; however, higher cadmium concentrations lead to both intracellular ATP depletion and ATP release, but do not increase the caspase-3 activity, indicating that cadmium also produces cellular death by necrosis. (3) These results suggest that cadmium induces either apoptosis or necrosis in rat cortical neurons, depending on the cadmium concentration.

Molecular mechanisms of glutamate release by bovine chromaffin cells in primary culture.

Previous work indicated that glutamate could be involved in the regulation of catecholamine secretion in bovine chromaffin cells. Thus, the question arises on the source of this putative regulatory glutamate. In this work we have examined the possibility that glutamate could be released from chromaffin cells. Data from this study indicate that chromaffin cells are able to release glutamate when they are stimulated by different depolarising agents such as 60 mM KCl, 1 mM 4-aminopyridine or 50 microM veratridine. The amount of glutamate released by these compounds was 0.32 nmol/10(6) cells (9.24% of cellular glutamate content), 0.275 (7.86%) and 0.158 (4.52%) for KCl, 4-AP and veratridine stimulation, respectively. All these catecholamine-secretagogues induced glutamate secretion by two mechanisms: 1) a Ca(2+)-dependent, probably exocytotic, mechanism and 2) a Ca(2+)-independent mechanism mediated by reversion of the electrogenic glutamate transporter. Analysis of Ca(2+)-dependent and independent releases for different compounds carried out by several experimental approaches, indicate that Ca(2+)-dependent release was the predominant mechanism for release induced by 4-aminopyridine (84% of total release) and high KCl (63%) whilst Ca(2+)-independent release was predominant for veratridine (67%). The Ca(2+)-dependent glutamate release evoked by depolarisation of chromaffin cells with high KCl and 4-AP could be split into both a fast and a slow kinetic component, which might correspond to the release of docked and mobilised chromaffin granules, respectively. On the other hand, depolarisation of cells with veratridine result in glutamate release with only the fast kinetic component. In the case of 60 mM KCl-evoked glutamate release, the fast component exhibited a decay time of <1 s and accounted for 0.63 nmol glu/6x10(6) cells (70% of total exocytotic release), whereas the slow component, which exhibited a decay time of 231 s, accounted for the release of 0.27 nmol glu/6x10(6) cells (30% of total exocytotic release). By contrast in the case of 4-aminopyridine the fast component of exocytosis only represents a 19% of total secretion and the slow a 81% with a decay time of 94 s. These data are very similar to those found in neurones and support the possible intracellular origin of glutamate having a role in the regulation of catecholamine secretion from chromaffin cells. In support of this, we have found that glutamate secretion could be evoked by stimulation of the nicotinic cholinergic receptors.

Neuronal nitric oxide synthase modulates basal catecholamine secretion in bovine chromaffin cells.

The role of endogenously produced nitric oxide (NO) in the regulation of basal catecholamine (CA) secretion was studied in chromaffin cells. Treatment of chromaffin cells with nitric oxide synthase (NOS) inhibitors produced a dose-dependent increase in basal catecholamine secretion, which paralleled their ability to inhibit NOS activity. This inhibitory profile was similar to that found in neurons, suggesting the constitutive expression of neuronal NOS (nNOS) in these cells, which was confirmed by Western blot analysis. A study of the kinetics and pharmacology of nNOS activity expressed in chromaffin cells in culture indicated that NOS activity is calcium-dependent, increases with time, and is highly dependent on both intracellular concentrations of L-arginine (K(m) approximately 4 microM, V(max) = 908 +/- 60 pmol/hr x 10(6) cells) and transport of L-arginine into the cells (exhibiting two affinity constants of k(1) = 3.2 +/- 0.3 microM and k(2) = 126 +/- 5.5 microM). The effects of NOS inhibitors on CA secretion were mediated by the L-arginine-NO-cGMP pathway, insofar as exogenous L-arginine was able to partially block the increase in CA secretion evoked by them, and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ), a specific inhibitor of guanylate cyclase, and zaprinast, an inhibitor of the cGMP phosphodiesterase, were able to increase and inhibit, respectively, basal CA secretion in a dose-dependent manner. These results suggest that chromaffin cells exhibit a tonic production of NO by nNOS that keeps the basal CA secretion at low levels, and this could be necessary for maintaining a normotensive state.

Calcium channel types involved in intrinsic amino acid neurotransmitters release evoked by depolarizing agents in cortical neurons.

Although numerous biochemical and electrophysiological studies have already established many of the properties of the putative Ca2+ receptor for exocytosis at the synapse, the molecular mechanism that involves the influx of Ca2+ and the release of neurotransmitters has remained elusive. Several relationships have been established between neurotransmitter release and Ca2+ channel involved, but no work attempting to connect a particular neurotransmitter release, the effector which produces the release and the opening of a Ca2+ channel type has been performed. This work shows, data dealing with this subject. Based on our results, we have reached the following conclusions: (1) Ca2+ channel types P/Q, N and L mediate Ca2+ entry evoked by high KCl and veratridine, and P/Q and N but not L-type Ca2+ channels are involved when the effector is 4-aminopyridine (4-AP); (2) When we compare the relationship between the amino acid release and the Ca2+ channels which are opened by different depolarizing agents, we find that the release of a particular amino acid neurotransmitter not only depends on the opening of the voltage-dependent Ca2+ channel but also on the effector which produces the opening; and (3) the amount of amino acid release evoked by the different depolarizing agents is not correlated with the elevation of intracellular Ca2+ produced by them. From all of these results, we may conclude that calcium concentration in the active zone is not the only important factor in mediating amino acid release.

Nicotinic receptors mediate the release of amino acid neurotransmitters in cultured cortical neurons.

Nicotine stimulation of cortical neurons obtained from gestation day 19 rats provoked a dose-dependent release of aspartate, glutamate, glycine and GABA, indicating a functional role for the nicotinic receptor in this model. This release was exclusively Ca2+-dependent (vesicular release) in the case of aspartate and dual Ca2+-dependent and Ca2+-independent) for glutamate, glycine and GABA. Nicotine also raised the membrane potential and the intracellular calcium concentration. These effects were specific, since they were reversed by hexamethonium, an antagonist of the nicotinic receptor. It was shown that L, N, and P/Q type Ca2+ channels are involved in nicotine-mediated Ca2+ entry into cortical neurons. Evaluation of the effects of nicotine on Ca2+ entry in isolated cells showed that 100% of the cells responded to nicotine, although the intensity of the response was variable: 63% of the neurons showed an increase in intracellular Ca(2+) of 152 +/- 5 grey levels, 25% of 88 +/- 12 grey levels and 12% of 48 +/- 1 grey levels. Tetrodotoxin, which blocks voltage-dependent Na(+) channels, completely reversed nicotine-induced Ca2+ entry into single cells. This suggests that the Ca2+ increment is mediated by opening of Ca2+ channels and not by the nicotinic receptor.

Mechanism by which GABA, through its GABA(A) receptor, modulates glutamate release from rat cortical neurons in culture.

In cortical neurons, the GABA(A) agonist, muscimol, increases: (a) basal glutamate release (with a EC50 of 99 +/- 7 microM); (b) intracellular calcium and (c) membrane potential, all of these in a dose-dependent manner. These muscimol effects were specific since they were reversed by bicuculline, a GABA(A) antagonist. When the action of muscimol was measured at different KCl concentrations, an increase or decrease of the glutamate secretion was observed, depending on the KCl concentration in the medium. At low KCl concentration (5.6 mM of KCl), it depolarized, at 20 mM of KCl it had no effect, but at higher KCl concentrations (30-100 microM of KCl), it produced a hyperpolarization in these cells. The mechanism by which the GABA-Cl(-)-channel permits Cl- fluxes, inward or outward, depending on the membrane potential.

Effect of glutamate receptor agonists on catecholamine secretion in bovine chromaffin cells.

In this study, the effects of glutamate and glutamate receptor agonists in cultured chromaffin cells from bovine adrenal medulla were investigated. It was found that glutamate increases basal catecholamine (CA) secretion in a dose-dependent manner. This effect is mimicked by specific agonists of the four known glutamate receptors N-methyl-D-aspartate (NMDA), quisqualate/(RS)-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainate (KA), and trans-(+)-1-amino-1,3-cyclopentane dicarboxylic acid (t-ACPD), which increased both basal and nicotine-evoked CA secretion. The NMDA receptor antagonist 2-amino-5-phosphonopentanoic acid, 6-cyano-7-nitroquinoxaline-2,3-dione, an antagonist of KA and AMPA receptors, and L-(+)-2-amino-3-phosphonopropionic acid, an antagonist of the t-ACPD receptor, inhibited the stimulatory effect of related glutamate agonists. Hexamethonium, an antagonist of the nicotinic receptor, failed to influence glutamate agonists except for a 15% inhibition of KA. The increase in CA secretion produced by a 100 microM concentration of glutamate agonists was about 20-60% of that obtained with 10 microM of nicotine, an agonist of the physiological stimulatory cholinergic receptor. The increase in CA secretion produced by glutamate was accompanied by both an increase in bisoxonol fluorescence, suggesting membrane depolarization, and by an increase in intracellular Ca2+ concentrations. Results obtained with image analysis on single cells indicated that the percentage of cells which respond to the stimulation of 50 microM of glutamate is 42%. From these results, we conclude that glutamate, through its four known glutamate receptors, can increase both basal and nicotine-evoked CA secretion in chromaffin cells by a process which involves membrane depolarization and an increase in intracellular calcium levels.

Effect of various depolarizing agents on endogenous amino acid neurotransmitter release in rat cortical neurons in culture.

The purpose of this study is to determine whether differences in membrane potential and/or intracellular Ca2+ increments are implicated in a programmed release of amino acid neurotransmitters (aspartate, glutamate, glycine and GABA) in cortical neurons in culture. According to our results, it is possible to assume that difference in membrane potential is not the only signal which starts the amino acid neurotransmitter release, but there are other necessary conditions at the start of this amino acid release. One of these conditions could be the increment in intracellular Ca2+, but our results indicate that, in cortical neurons in culture, the total intracellular Ca2+ increments are not important on release levels, but are the stimulating agent which produces this intracellular Ca2+ increment. From these results we may infer: (1) that in rat cortical neurons there are neurons which contain and release glutamate, aspartate, glycine and GABA, (2) that in cortical neurons the 36.6 +/- 5.8% of the neurons are GABA-ergic, (3) that the membrane potential and the total intracellular calcium are not only responsible for the release of these amino acids but also the depolarizing agent which plays an important role in this release, and (4) that glutamate and aspartate and glutamate and GABA are localized in different vesicular pools or in different cell neurons.

Segregation of nitric oxide synthase expression and calcium response to nitric oxide in adrenergic and noradrenergic bovine chromaffin cells.

Previous work has demonstrated that nitric oxide can be an important intracellular messenger in the regulation of neurosecretion in chromaffin cells. Since standard chromaffin cell cultures are mixed populations of noradrenaline and adrenaline producing cells, it would seem important to understand the functional differences between these individual components. The use of fluorescence imaging techniques for the recording of cytosolic calcium from single chromaffin cells together with the immunoidentification of individual cells with specific antibodies against tyrosine hydroxylase, N-phenyl ethanolamine methyl transferase and nitric oxide synthase, has allowed us to measure single-cell calcium responses in identified adrenergic, noradrenergic and nitrergic chromaffin cells, thus helping us to clarify the differential role of nitric oxide in the function of these chromaffin cell types. 53 +/- 2% of chromaffin cells were able to synthesize nitric oxide (nitric oxidesynthase-positive cells), these cells being mainly noradrenergic (82 +/-2%). Results indicate that nitric oxide donors such as sodium nitroprusside, molsidomine and isosorbide dinitrate evoke [Ca2+]i increases in a 62 +/- 4% of chromaffin cells, the response to nitric oxide donors being between 30 and 50% of that of 20 microM nicotine. Cells responding to nitric oxide donors were mainly adrenergic (68 +/- 5%) although 45 +/- 9% of noradrenergic cells also gave [Ca2+]i increasing responses. The distribution of nitric oxide responding cells between nitric oxide synthase-positive and negative was very similar in the whole population (63 +/- 5 and 60 +/- 7%, respectively), but these differences were more prominent when considering the distribution of nitric oxide response between noradrenergic and adrenergic nitric oxide synthase-positive cells; while 73 6% of adrenergic nitric oxide synthase-positive cells evoke [Ca2+]i increases by nitric oxide stimulation, only 35 +/- 11% of noradrenergic nitric oxide synthase-positive cells respond. Taken together these results seem to indicate that (i) nitric oxide could act within adrenal medulla as both an intracellular and intercellular messenger; and (ii) noradrenergic cells seem to be specialized in nitric oxide synthesis while adrenergic cells with an endocrine function could mainly act as a target of neurosecretory action of this second messenger.