Intracellular, nonreceptor-mediated signaling by adenosine: induction and prevention of neuronal apoptosis.

Inhibitory effect of adenosine on the isolated heart muscle and vascular system were first described in 1929. Since then, numerous reviews have been published on the diverse actions of this nucleoside on a wide variety of cell types. Essentially all effects of adenosine in neurons and non-neuronal cells are mediated by activation of nucleoside membrane receptors coupled to specific intracellular second messenger pathways. This brief review describes two novel actions of adenosine in peripheral sympathetic neurons, which are not mediated by adenosine receptors. First is described how adenosine and related nucleosides are able to induce apoptosis during the initial stages of neuronal growth and development in vitro and in vivo. Second is discussed how adenosine is able to prevent or delay apoptosis in more mature sympathetic neurons subjected to nerve growth factor deprivation in culture. Both the induction and prevention of apoptosis are independent of receptor activation, and totally dependent on the intracellular accumulation and subsequent phosphorylation of adenosine. The physiological significance and mechanisms by which adenosine can induce apoptosis in one situation, and rescue from apoptosis in another, are described in this article.

Embryonic mesenchymal cells share the potential for smooth muscle differentiation: myogenesis is controlled by the cell's shape.

Undifferentiated embryonic mesenchymal cells are round/cuboidal in shape. During development, visceral myogenesis is shortly preceded by mesenchymal cell elongation. To determine the role of the cell's shape on smooth muscle development, undifferentiated embryonic mesenchymal cells from intestine (abundant visceral muscle), lung (some visceral muscle) or kidney (no visceral muscle) were plated under conditions that maintained cell rounding or promoted elongation. Regardless of their fate in vivo, all the cells differentiated into smooth muscle upon elongation as indicated by the expression of smooth muscle-specific proteins and the development of membrane potentials of -60 mV and voltage-dependent Ca2+ currents, characteristic of excitable cells. Smooth muscle differentiation occurred within 24 hours and was independent of cell proliferation. Regardless of their fate in vivo, all the round cells remained negative for smooth muscle markers, had membrane potentials of -30 mV and showed no voltage-activated current. These cells, however, differentiated into smooth muscle upon elongation. The role of the cell's shape in controlling smooth muscle differentiation was not overcome by treatment with retinoic acid, TGF-beta1, PDGF BB or epithelial-conditioned medium (all modulators of smooth muscle differentiation). These studies suggest that the mesenchymal cell shape plays a main role in visceral myogenesis.

Pituitary adenylyl cyclase-activating polypeptide and nerve growth factor use the proteasome to rescue nerve growth factor-deprived sympathetic neurons cultured from chick embryos.

Removal of nerve growth factor (NGF) from sympathetic neurons initiates a neuronal death program and apoptosis. We show that pituitary adenylyl cyclase-activating polypeptide (PACAP) prevents apoptosis in NGF-deprived sympathetic neurons. PACAP (100 nM) added to culture medium at the time of plating failed to support neuronal survival. However, in neurons grown for 2 days with NGF and then deprived of NGF, PACAP prevented cell death for the next 24-48 h. Uptake of [3H]norepinephrine ([3H]NE) was used as an index of survival and decreased >50% in NGF-deprived cultures within 24 h. PACAP (1-100 nM) restored [3H]NE uptake to 92 +/- 8% of that of NGF-supported controls. Depolarization-induced [3H]NE release in neurons rescued by PACAP was the same as that in NGF-supported neurons. PACAP rescue was not mimicked by forskolin or 8-bromo-cyclic AMP and was not blocked by the protein kinase A inhibitor Rp-adenosine 3',5'-cyclic monophosphothioate. Mobilization of phosphatidylinositol by muscarine failed to support NGF-deprived neurons. Thus, PACAP may use novel signaling to promote survival of sympathetic neurons. The apoptosis-associated caspase CPP32 activity increased approximately fourfold during 6 h of NGF withdrawal (145 +/- 40 versus 38 +/- 17 nmol of substrate cleaved/min/mg of protein) and returned to even below the control level in NGF-deprived, PACAP-rescued cultures (14 +/- 7 nmol/min/mg of protein). Readdition of NGF or PACAP to NGF-deprived cultures reversed CPP32 activation, and this was blocked by lactacystin, a potent and specific inhibitor of the 20S proteasome, suggesting that NGF and PACAP target CPP32 for destruction by the proteasome. As PACAP is a preganglionic neurotransmitter in autonomic ganglia, we propose a novel function for this transmitter as an apoptotic rescuer of sympathetic neurons when the supply of NGF is compromised.

Adenosine induces apoptosis by inhibiting mRNA and protein synthesis in chick embryonic sympathetic neurons.

Our previous work has established that adenosine is toxic to chick embryonic sympathetic neurons and kills freshly plated neurons by a process of apoptosis. Although the exact mechanism remains unknown, we found that phosphorylation of adenosine was essential to the toxicity. Using markers for RNA ([3H]uridine) and protein ([35S]methionine) synthesis we demonstrate here that in freshly plated sympathetic neurons adenosine inhibits RNA and protein synthesis by about 50%. The inhibitory effects of adenosine on RNA and protein synthesis, and increased ATP synthesis were blocked by adenosine kinase inhibitor, suggesting that phosphorylated products are responsible for inhibition of RNA and protein synthesis and cell death. Adenosine-induced inhibition of RNA and protein synthesis in neuronal cells provides a new role for adenosine in the regulation of cell function.

Chronic activation of protein kinase C by phorbol ester reduces calcium channel expression in chick sympathetic neurons.

Chronic activation of protein kinase C (PKC) has been implicated in regulation of Ca2+ entry responsible for normal development of transmitter properties in cultured sympathetic neurons. The idea that PKC alters the expression of Ca2+ channels was tested using phorbol 12,13-dibutyrate (PDB) which activates PKC and also supports survival of chick sympathetic neurons in the absence of nerve growth factor (NGF). Whole cell voltage-clamp showed that neurons supported by PDB for 2 days had significantly lower Ca2+ current density (0.243 +/- 0.025 pA/microm2) than those supported by NGF (0.356 +/- 0.033 pA/microm2). [125I]omega-Conotoxin GVIA binding showed that PDB-supported neurons had significantly lower maximum binding (617 +/- 223 fmol/mg protein) compared with those supported by NGF (1099 +/- 192 fmol/mg protein). These results support the conclusion that chronic activation of PKC limits the expression of N-type Ca2+ channels. A reduction in Ca2+ channel number is consistent with, and could account for the mature type Ca2+ handling and transmitter release properties seen in sympathetic neuro-effector preparations, sympathetic neurons co-cultured with their targets, and neurons supported by PDB.

2'-deoxyadenosine induces apoptosis in rat chromaffin cells.

We show here that 2'-deoxyadenosine (2'-dAdo) but not adenosine was toxic to chromaffin cells of 3-4-week-old rat adrenal glands. More than 75% of the cells plated in culture gradually died over a 3-day period in the presence of 100 microM 2'-dAdo plus 3 microM deoxycoformycin (DCF). Morphological observations together with bisbenzimide staining and terminal deoxynucleotidyl transferase-mediated nick and labeling showed membrane blebbing, shrinkage of cell bodies, chromatin condensation, and DNA fragmentation, suggesting apoptosis-like cell death by 2'-dAdo. Lethal effects of 2'-dAdo were potentiated by DCF, a drug that inhibits adenosine deaminase. 2'-dAdo-prompted cell death was not prevented by inhibitors of nucleoside transporter (3 microM dilazep or 1 microM nitrobenzylthioinosine), precursors of pyrimidine nucleotide biosynthesis (300 microM uridine or 100 microM 2'-deoxycytidine), or 5 mM nicotinamide. Cells incubated with 2'-dAdo (100 and 300 microM) showed a three- and ninefold, respectively, increase in content of dATP, a product known to be an inhibitor of ribonucleotide reductase, an enzyme essential for DNA synthesis. Formation of dATP was completely prevented by iodotubercidin (ITu), a drug that inhibits phosphorylation of 2'-dAdo to dATP by nucleoside kinase. It is interesting that nanomolar concentrations of ITu also completely protected chromaffin cells from 2'-dAdo lethality. Our study demonstrates for the first time that mammalian adrenal chromaffin cells undergo apoptotic cell death by a natural nucleoside and suggests that this model could be used to study apoptosis and cell function.

Exocytosis coupled to mobilization of intracellular calcium by muscarine and caffeine in rat chromaffin cells.

We used cultured rat chromaffin cells to test the hypothesis that Ca2+ entry but not release from internal stores is utilized for exocytosis. Two protocols were used to identify internal versus external Ca2+ sources: (a) Ca2+ surrounding single cells was transiently displaced by applying agonist with or without Ca2+ from an ejection pipette. (b) Intracellular stores of Ca2+ were depleted by soaking cells in Ca2+ -free plus 1 mM EGTA solution before transient exposure to agonist plus Ca2+. Exocytosis from individual cells was measured by microelectrochemical detection, and the intracellular Ca2+ concentration ([Ca2+]i) was measured by indo-1 fluorescence. KCl (35 mM) and nicotine (10 microM) caused an immediate increase in [Ca2+]i and secretion in cells with or without internal Ca2+ stores, but only when applied with Ca2+ in the ejection pipette. Caffeine (10 mM) and muscarine (30 microM) evoked exocytosis whether or not Ca2+ was included in the pipette, but neither produced responses in cells depleted of internal Ca2+ stores. Pretreatment with ryanodine (0.1 microM) inhibited caffeine- but not muscarine-stimulated responses. Elevated [Ca2+]i and exocytosis exhibited long latency to onset after stimulation by caffeine (2.9 +/- 0.38 s) or muscarine (2.2 +/- 0.25 s). However, the duration of caffeine-evoked exocytosis (7.1 +/- 0.8 s) was significantly shorter than that evoked by muscarine (33.1 +/- 3.5 s). The duration of caffeine-evoked exocytosis was not affected by changing the application period between 0.5 and 30 s. An approximately 20-s refractory period was found between repeated caffeine-evoked exocytosis bursts even though [Ca2+]i continued to be elevated. However, muscarine or nicotine could evoke exocytosis during the caffeine refractory period. We conclude that muscarine and caffeine mobilize different internal Ca2+ stores and that both are coupled to exocytosis in rat chromaffin cells. The nicotinic component of acetylcholine action depends primarily on influx of external Ca2+. These results and conclusions are consistent with our original observations in the perfused adrenal gland.

Importance of protein kinase C for normal development of transmitter release properties in embryonic chick sympathetic neurons in culture.

The hypothesis that multiple trophic inputs are essential for normal development of transmitter release properties in sympathetic neurons was tested using two supportive agents (excess KCl and phorbol 12,13-dibutyrate which produce marked activation of protein kinase C and also support survival of chick sympathetic neurons in culture) in addition to nerve growth factor, ciliary neurotrophic factor and neurotrophin-3. Basal and electrically evoked (10 pulses at 1 Hz and 10 Hz) release of [(3)H]norepinephrine from neurons supported by nerve growth factor was very high (1.5 to 2% of total [(3)H]norepinephrine content) and relatively insensitive to facilitation by tetraethylammonium as compared to release in neuroeffector organs, and the frequency-release response was negative. In K+-supported neurons, basal [(3)H]norepinephrine release was almost four-fold lower, evoked release was four- to eight-fold lower, the frequency response was flat to positive, and tetraethylammonium increased evoked release up to four-fold. Inclusion of nerve growth factor in culture did not modify the effects of K+ on basal or evoked release, and nerve growth factor plus ciliary neurotrophic factor and/or neurotrophin-3 did not produce the changes observed in K+-supported neurons. Neurons supported by phorbol ester had a low background release, low evoked release, a positive frequency-release response, and 10- to 30-fold facilitation by tetraethylammonium of release evoked by 1 Hz or 1 pulse stimulation. Thus, physiological and pharmacological behavior of transmitter release of sympathetic neurons supported by excess KCl or phorbol ester was very similar to their counterparts growing in the body. Neurons supported by nerve growth factor showed an immediate rise in stimulated [Ca(2)+]i that was three- to five-fold above basal levels with either 1 Hz or 10 Hz stimulation. However, in phorbol supported neurons, [Ca(2)+]i rose gradually to about 1.5 times basal levels during 1 Hz stimulation and increased further with 10Hz stimulation. Tetraethylammonium had little effect on stimulated [Ca(2)+]i in nerve growth factor-supported neurons, but greatly facilitated the stimulated rise in [Ca(2)+]i in phorbol-supporte neurons. The data show that multiple trophic inputs distinct from nerve growth factor, neurotrophin-beta or ciliary neurotrophic factor are required for normal physiological function of sympathetic neurons.

A non-cholinergic transmitter, pituitary adenylate cyclase-activating polypeptide, utilizes a novel mechanism to evoke catecholamine secretion in rat adrenal chromaffin cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is the most potent non-cholinergic neurotransmitter to stimulate catecholamine secretion from rat chromaffin cells; however, the mechanism of action is not clear. We used amperometric detection of exocytosis and indo-1 monitoring of [Ca2+]i to identify PACAP actions in cultured chromaffin cells. PACAP (100 nM) required external Ca2+ to evoke secretion. However, unlike nicotine and KCl which caused immediate and relatively brief secretion, PACAP has a latency of 6.8 +/- 0.96 s to the first secretory response and secretion continued for up to 2 min. PACAP elevation of [Ca2+]i showed similar latency and often remained above base line for several minutes following a brief exposure. ZnCl2 (100 microM) selectively inhibited PACAP-stimulated secretion and [Ca2+]i with little effect on nicotine-evoked responses. Nifedipine (10 microM) had little effect on PACAP-evoked secretion but inhibited nicotine-evoked secretion by more than 80%, while omega-conotoxin (100 nM) failed to affect either agonist. PACAP-stimulated cAMP levels required 5 s to significantly increase, consistent with the latency of exocytotic and Ca2+ responses. Forskolin (10 microM) caused responses similar to PACAP. PACAP-evoked exocytosis was blocked by the protein kinase A inhibitor adenosine 3'5'-cyclic monophosphorothioate Rp-diastereomer (Rp-cAMPS). These data showed that PACAP stimulates exocytosis by a mechanism distinctly different from cholinergic transmitters that appears to involve cAMP-mediated Ca2+ influx. Differences in receptor coupling mechanisms and pharmacology of Ca2+ entry stimulated by cholinergic and peptidergic agonists support the idea that the peptidergic system maintains catecholamine secretion under conditions where the cholinergic system desensitizes or otherwise fails.

Cardiac cells control transmitter release and calcium homeostasis in sympathetic neurons cultured from embryonic chick.

1. The contribution of target cells in controlling the functional properties of sympathetic neurons was investigated using pure neuronal cultures and co-cultures of neurons with their physiological target cells. 2. Chick embryo sympathetic neurons cultured alone exhibited maximal elevation of cytosolic free Ca2+ ([Ca2+]i) and release of tritiated noradrenaline ([3H]NA) when given ten stimulating pulses at 1 Hz but not at 10 Hz, yielding a negative frequency-release response. Stimulation-evoked release was only slightly enhanced by the K+ channel blocker tetraethylammonium (TEA, 10 mM). 3. When sympathetic neurons were co-cultured with cardiac cells of the chick embryo, electrically stimulated transmitter release and neuronal [Ca2+]i were reduced by 3- to 5-fold. Co-cultured neurons had a positive stimulation frequency--[3H]NA release response and 5- to 7-fold facilitation of release by TEA. 4. Voltage-clamped Ca2+ current density was decreased from 0.61 +/- 0.13 pA micron-2 in neurons alone to 0.19 +/- 0.03 pA micron-2 in co-cultured neurons. 5. Neonatal rat superior cervical ganglion (SCG) neurons were also relatively insensitive to TEA when cultured alone, but [3H]NA release was greatly facilitated by TEA when tested in SCG neurons co-cultured with rat neonatal cardiac myocytes. 6. The cardiac cell-induced changes in Ca2+ handling and release properties were produced within 24 h by sympathetic neuroeffector cells, but not by skeletal muscle cells or sensory neurons, and did not occur spontaneously in neurons grown alone for up to 6 days. 7. The frequency and TEA responses of neurons grown with cardiac cells are characteristic of responses seen in sympathetic neuroeffector organs. We conclude that physiological targets play a crucial role in development of normal transmitter-release properties by controlling Ca2+ homeostasis in sympathetic neurons.

Adenosine-induced apoptosis in chick embryonic sympathetic neurons: a new physiological role for adenosine.

1. A newly found action of adenosine in neurons, which may have an important physiological function in the growth and development of the sympathetic nervous system, is described. Adenosine (1-100 microM) inhibited neurite outgrowth within the first 24 h and killed about 80% of sympathetic neurons supported by nerve growth factor over the next 2 days in culture. Neurons supported by excess KCl, forskolin or phorbol 12,13-dibutyrate were equally susceptible to the toxic actions of adenosine. Inosine, guanosine or hypoxanthine (all 100-300 microM) were without effect on neuronal growth and survival. 2. Specific agonists of adenosine A1 and A2 receptors were not neurotoxic, and toxic effects of adenosine were not antagonized by aminophylline. These results rule out involvement of adenosine receptors and the adenylyl cyclase-cAMP signalling system in neurotoxic actions of adenosine. 3. Adenosine toxicity was prevented by inhibitors of the adenosine membrane transporter, suggesting an intracellular site of action of adenosine. 4. Inhibitors of adenosine deaminase dramatically facilitated the toxic action so that physiologically relevant concentrations of adenosine were neurotoxic. 5. Adenosine kinase activity of sympathetic neurons was dose-dependently inhibited by 5'-iodotubercidin (3-100 nM). 5'-Iodotubercidin (100 nM) completely protected neurons against toxicity of adenosine plus adenosine deaminase inhibitors. These results provide convincing evidence that phosphorylation of the nucleoside is an essential requirement for initiation of adenosine toxicity. 6. Sympathetic neurons were successfully rescued from the lethal effects of adenosine deaminase inhibitor plus adenosine by uridine or 2-deoxycytidine, but not by nicotinamide or 2-deoxyguanosine, suggesting that depletion of pyrimidine nucleotides by phosphorylated adenosine compounds and consequent inhibition of DNA synthesis produces neuronal death. 7. DNA fragmentation, assessed by the fluorescent dye bisbenzimide and by the TUNEL (terminal deoxynucleotidyl transferase-mediated nick end labelling) method, indicated that neuronal death induced by adenosine was apoptotic. 8. We conclude that adenosine deaminase and adenosine kinase play an important role in the metabolism of intracellular concentrations of adenosine and thereby regulate the growth and development of sympathetic neurons. Our study highlights, for the first time, the importance of adenosine as a mediator of programmed cell death of neurons supported by nerve growth factor.

Morphological and transmitter release properties are changed when sympathetic neurons are cultured in low Ca2+ culture medium.

The stimulated elevation of [Ca2+]i can either promote neuronal survival or lead to Ca(2+)-mediated neurotoxicity. Similarly, growth cone mobility and neurite outgrowth may be promoted or arrested by elevated [Ca2+]i. We examined survival, development and transmitter release properties of chick sympathetic neurons maintained in culture medium containing varying concentrations of Ca2+. Neurons maintained in medium with no added Ca2+ or as low as 0.1 mM external Ca2+ show a dramatic change in growth and development compared to neurons kept in 1-2 mM Ca(2+)-containing medium. Furthermore, neurons in Ca(2+)-free medium (+ 100 microM EGTA) survived up to 24 h and, following change to 0.1 mM Ca2+, grew neurites and survived for several weeks. Neurons grown in high Ca2+ medium (0.6-2 mM) exhibited thick neurites, aggregated cell bodies, and neurites began to detach after six to eight days in culture. Neurons in low Ca2+ medium (no added Ca2+ to 0.3 mM) grew as single cells with extensive, thin branching neurites, remained firmly attached to the substrate and survived for several weeks. Neurons initially plated in 0.1 mM Ca2+ (or 2 mM Ca2+) medium and switched over to 2 mM (or 0.1 mM) Ca2+ medium after two days acquired the characteristic morphology of high (and low) Ca2+ medium over the next six days, demonstrating the plasticity of effects of external Ca2+. The above characteristic changes in growth of sympathetic neurons in low Ca2+ medium occurred when neurons were supported by 35 mM KCl or 30 nM phorbol 12,13-dibutyrate instead of nerve growth factor. The uptake and retention of tritiated norepinephrine in neurons grown in low or high Ca2+ medium were similar. However, basal release of [3H]norepinephrine in neurons maintained in low Ca2+ medium was one-third of that in neurons kept in high Ca2+ medium. Furthermore, electrically stimulated (10 pulses at 1 or 10 Hz) [3H]norepinephrine release from neurons grown in high Ca2+ had a high fractional release (> 1%) which did not change during six days in culture. On the other hand, fractional release in neurons grown in low Ca2+ medium for six to 10 days decreased about 50% and 75%, respectively, compared to release after two days in culture. The resulting low fractional release (< 0.5%) is characteristic of sympathetic neurons in neuroeffector organs.(ABSTRACT TRUNCATED AT 400 WORDS)

Deoxynucleoside induces neuronal apoptosis independent of neurotrophic factors.

Postmitotic sympathetic neurons are known to undergo a programmed cell death (apoptosis) when they are deprived of nerve growth factor (NGF) or treated with arabinofuranosyl nucleoside antimetabolites. Here we report the existence of a biochemical mechanism for the induction of neuronal death by an endogenous nucleoside in the presence of NGF. In support of such a mechanism we show that 2-deoxyadenosine (dAdo) induces apoptosis in chick embryonic sympathetic neurons supported in culture by NGF, excess K+, phorbol 12,13-dibutyrate, or forskolin. Neuronal death was related to a dramatic increase in the dATP content of sympathetic neurons exposed to dAdo (34.96 +/- 5.98 versus 0.75 +/- 0.16 pmol/micrograms protein in untreated controls, n = 9), implicating dATP in the toxicity. Supportive evidence for a central role of dATP was gained by inhibition of kinases necessary for phosphorylation of dAdo. 5'-Iodotubercidin in nanomolar concentrations completely and dose-dependently inhibited formation of dATP and also protected against toxicity of submillimolar concentrations of dAdo in sympathetic neurons. Although some of these actions of dAdo were remarkably similar to those reported for human lymphoid cells, several were uniquely different. For example, [3H]dAdo was not transported into neurons by the nucleoside transporter, and therefore inhibition of the transporter (dilazep, nitrobenzylthioinosine) did not prevent neurotoxicity by dAdo. Precursors of pyrimidine synthesis (2'-deoxycytidine, uridine) or NAD+ synthesis (nicotinamide) were ineffective in protecting sympathetic neurons against dAdo toxicity. Finally, inhibition of adenosine deaminase by deoxycoformycin or erythro-9-(2-hydroxy-3-nonyl) adenine did not potentiate the toxic effects of dAdo. Our results provide evidence for the first time that neuronal cells are as susceptible to nucleoside lethality as human lymphocytes are, and provide a new model to study the salvage pathway of deoxyribonucleosides in controlling neuronal populations through programmed cell death.

Exocytosis from a single rat chromaffin cell by cholinergic and peptidergic neurotransmitters.

Secretion of catecholamines from chromaffin cells is mediated by cholinergic and peptidergic neurotransmitters. The cholinergic transmitter acetylcholine activates both nicotinic and muscarinic receptors to trigger catecholamine secretion in rat adrenal medulla. Vasoactive intestinal polypeptide (VIP) has been identified as the peptidergic transmitter in rat adrenal medulla and may also be the non-cholinergic transmitter in bovine adrenal. Pituitary adenylate cyclase activating polypeptide (PACAP), a VIP-like secretin peptide, is also found in the adrenal, and is a potent secretagogue. Thus, PACAP may be another peptidergic transmitter at the adrenal synapse. A most intriguing property of rat chromaffin cells is that stimulation of nicotinic, muscarinic, VIP or PACAP receptors are each able to produce robust catecholamine secretion on their own. This raises the question of whether a single chromaffin cell can respond to each of the above agonists or whether the secretion is due to subpopulations of chromaffin cells. This issue was addressed by using electrochemical techniques to monitor exocytosis from individual chromaffin cells in culture. We demonstrate that acetylcholine, nicotine, muscarine, VIP and PACAP are each able to evoke catecholamine secretion from a single chromaffin cell. Some cells only responded to acetylcholine. Furthermore, each agonist produced a distinct pattern of exocytosis. Muscarine-evoked secretion exhibited a latency of 0.5-2 s, but exocytosis persisted up to 30 s following 500 ms stimulation. Nicotine produced an immediate response which usually ended within 10 s. The secretory pattern following acetylcholine appeared to be the sum of the nicotinic and muscarinic patterns, showing both rapid onset and longer duration.(ABSTRACT TRUNCATED AT 250 WORDS)

Tetraethylammonium facilitation of single-pulse mediated action potential, [Ca2+]i and transmitter release in sympathetic neurons.

A single stimulus applied to sympathetic neurons co-cultured with cardiac cells produced a very small increase in the release of tritiated norepinephrine and intracellular free Ca2+ concentration ([Ca2+]i), but evoked a typical neuronal action potential. Treatment with 10 mM tetraethylammonium caused a dramatic increase in the responses to a single stimulus, and [3H]norepinephrine release remained above baseline for as long as 20 s. [Ca2+]i increased in cell bodies and neurites from basal levels of 50 to 100 nM to over 300 nM. [Ca2+]i remained elevated for 18 +/- 1 s and required 6.8 +/- 0.5 s for 50% recovery to basal levels. Action potential duration at 50% repolarization was increased from 4.5 +/- 0.3 ms to 88 +/- 20 ms and resting membrane potential decreased from -55 +/- 2 to -43 +/- 4 mV in the presence of tetraethylammonium. Repetitive firing was not observed after a single stimulus in current clamped neurons before or during tetraethylammonium exposure. These findings show a direct relation between action potential duration, Ca2+ entry and transmitter release in response to a single stimulus.

Massive exocytosis triggered by sodium-calcium exchange in sympathetic neurons is attenuated by co-culture with cardiac cells.

Entry of Ca2+ through voltage-dependent Ca2+ channels is known to be linked to the exocytotic release of transmitter from sympathetic neurons. In this paper we provide evidence that transmitter release can also be stimulated by Ca2+ influx via the Na-Ca exchanger. Furthermore, the release linked to Na-Ca exchange is regulated by cardiac target cells. Cultured sympathetic neurons of the chick embryo incubated in Ca2(+)-Mg(2+)-free Krebs solution for 20 min and then switched to Ca(2+)-containing solution exhibited 15-20-fold increases in [3H]noradrenaline release over the spontaneous release. Electrophysiologic studies showed that neurons were completely depolarized in Ca(2+)-Mg(2+)-free medium. Indo-1 fluorescence revealed a large and sustained increase in intracellular free Ca2+ concentration ([Ca2+]i) after addition of Ca2+ to Ca(2+)-Mg(2+)-free medium. The increased [3H]noradrenaline release and [Ca2+]i were dependent on external Na+ and Ca2+, but were not affected by the Ca2+ channel blockers lanthanum, cadmium, verapamil or omega-conotoxin. A conventional depolarizing stimulus (125 mM K+) produced a 13-fold increase in [3H]noradrenaline release over spontaneous release. However, K(+)-induced release and rise in [Ca2+]i declined rapidly and were sensitive to the Ca2+ channel blockers. When sympathetic neurons were co-cultured with embryonic cardiac cells the release induced by change from Ca(2+)-Mg(2+)-free to Ca(2+)-Krebs solution was dramatically reduced. The change from Ca(2+)-Mg(2+)-free to Ca(2+)-Krebs solution was ineffective in evoking [3H]noradrenaline release from sympathetic neurons in situ using perfused hearts of 15-day-old chick embryos.(ABSTRACT TRUNCATED AT 250 WORDS)

Sites of transmitter release and relation to intracellular Ca2+ in cultured sympathetic neurons.

Fluorescence imaging of indo-1 loaded cells was used to monitor influx and distribution of Ca2+ in cell bodies, neurites and growth cones of sympathetic neurons cultured from embryonic chick. Similar experiments on release of tritiated noradrenaline were performed to assess the relationship between intracellular Ca2+ concentration ([Ca2+]i) and transmitter release. Effects of Ca2+ channel antagonists on electrically stimulated rise in [Ca2+]i were dependent on the neuronal region examined. Cadmium and verapamil blocked Ca2+ entry in cell bodies but were less effective in neurites and growth cones. Nifedipine partially inhibited Ca2+ entry in cell bodies and was less effective in neurites and growth cones. Combination of cadmium and nifedipine blocked [Ca2+]i rise in all neuronal regions. Omega-conotoxin was an effective Ca2+ channel blocker in all regions. Ca2+ channel blockers had effects on [3H]noradrenaline release which paralleled effects on [Ca2+]i in neurites (and growth cones) but not cell bodies. Cadmium, verapamil and nifedipine each caused a partial, reversible block of the evoked release. Combination of cadmium and nifedipine completely blocked evoked [3H]noradrenaline release. Omega-conotoxin caused complete, irreversible block of electrically evoked release. During prolonged depolarization with 125 mM K+ Krebs solution, elevation of [Ca2+]i was maintained in cell bodies but was transient in neurites and growth cones. The amplitude and time course of [3H]noradrenaline release paralleled [Ca2+]i in neurites and growth cones, but not the cell body under the above conditions. A new method is described to study localized uptake and release of [3H]noradrenaline in cell bodies versus neurites of sympathetic neurons. Incubation of these modified cultures with [3H]noradrenaline showed that cell bodies had very low [3H]noradrenaline uptake (0.23 x 10(-6) c.p.m./mg protein), whereas neurites contained approximately 20 times more radioactivity. Depolarization of neurites by excess K+ and field stimulation caused a large increase in the net release of [3H]noradrenaline. The release was unaffected by removal of cell bodies. Neurites remained functionally viable for more than 2 h after separation from their cell bodies. [3H]Noradrenaline release could be evoked repeatedly over this time. [3H]Noradrenaline release from isolated neurites was partially blocked by nifedipine and fully blocked by combination of cadmium and nifedipine or by omega-conotoxin. The uptake and release of [3H]noradrenaline by neurites alone (expressed per mg protein) accounted for the total [3H]noradrenaline in intact cultures with neurites and cell bodies. Therefore, we conclude that neurites (and growth cones) are the prominent sites of uptake, storage and release of sympathetic transmitter.(ABSTRACT TRUNCATED AT 400 WORDS)

Barium-induced exocytosis is due to internal calcium release and block of calcium efflux.

The concentration of cytosolic free Ca2+ ([Ca2+]i) and the release of tritiated norepinephrine ([3H]NE) were monitored during Ba2+ stimulation of sympathetic neurons cultured from chick embryos. Ba2+ (2.5 mM in Ca(2+)-free medium) caused a rise in [Ca2+]i in all regions (cell bodies, neurites, and growth cones) of sympathetic neurons and evoked [3H]NE release in the absence of other stimuli. The increase in [Ca2+]i and release of [3H]NE were sustained for up to 30 min in the presence of Ba2+. When Ba(2+)-stimulated cells were immediately washed in Ca(2+)-free Ba(2+)-free EGTA solution, both the elevated [Ca2+]i and [3H]NE release returned to basal levels, with similar, fast, time courses. Ba2+ also blocked Ca2+ efflux from neurons loaded with 45Ca. We conclude from the parallel effects of Ba2+ on [Ca2+]i and [3H]NE release that Ba2+ stimulates exocytosis by a Ca(2+)-dependent mechanism. The Ba(2+)-induced rise in [Ca2+]i is a result of two separate actions: (i) the release of Ca2+ from intracellular sites and (ii) an effective block of Ca2+ extrusion. The ability of Ba2+ to release Ca2+ in growth cones that are insensitive to caffeine suggests that Ba2+ may displace Ca2+ from binding sites other than endoplasmic reticulum.

Activation of K+ channels by lanthanum contributes to the block of transmitter release in chick and rat sympathetic neurons.

We studied the effects of lanthanum (La3+) on the release of 3H-norepinephrine (3H-NE), intracellular Ca2+ concentration, and voltage clamped Ca2+ and K+ currents in cultured sympathetic neurons. La3+ (0.1 to 10 microM) produced concentration-dependent inhibition of depolarization induced Ca2+ influx and 3H-NE release. La3+ was more potent and more efficacious in blocking 3H-NE release than the Ca(2+)-channel blockers cadmium and verapamil, which never blocked more than 70% of the release. At 3 microM, La3+ produced a complete block of the electrically stimulated rise in intracellular free Ca2+ ([Ca2+]i) in the cell body and the growth cone. The stimulation-evoked release of 3H-NE was also completely blocked by 3 microM La3+. However, 3 microM La3+ produced only a partial block of voltage clamped Ca2+ current (ICa). Following La3+ (10 microM) treatment 3H-NE release could be evoked by high K+ stimulation of neurons which were refractory to electrical stimulation. La3+ (1 microM) increased the hyperpolarization activated, 4-aminopyridine (4-AP) sensitive, transient K+ current (IA) with little effect on the late outward current elicited from depolarized holding potentials. We conclude that the effective block of electrically stimulated 3H-NE release is a result of the unique ability of La3+ to activate a stabilizing, outward K+ current at the same concentration that it blocks inward Ca2+ current.

Dissociation between intracellular Ca2+ and modulation of [3H]noradrenaline release in chick sympathetic neurons.

1. We studied the relation between cyclic AMP, intracellular Ca2+ concentration and release of [3H]noradrenaline ([3H]NA) in sympathetic neurons cultured from chick embryos. 2. Forskolin (10 microM) and vasoactive intestinal polypeptide (VIP, 3 microM) increased cellular levels of cyclic AMP 8- and 3-fold, respectively, either in the absence or presence of electrical stimulation. Electrical stimulation (1 Hz for 10 s) alone had no effect on cyclic AMP levels. 3. Electrically evoked (1 Hz for 10 s) release of [3H]NA was facilitated by 10 microM-forskolin, 3 microM-VIP and by the non-hydrolysable cyclic AMP analogue, 8-bromoadenosine 3': 5'-cyclic monophosphate (8-Br-cyclic AMP). The inactive analogue of forskolin, dideoxyforskolin, had no effect on [3H]NA release. 4. The stimulation-evoked release of [3H]NA was completely inhibited by the neuronal blocking drugs guanethidine (1 microM) and bretylium (3 microM). 5. Whole-cell voltage-clamp studies showed that forskolin and VIP did not facilitate and guanethidine and bretylium did not block voltage-activated Ca2+ currents in the cell bodies of sympathetic neurons. 6. Fluorescence measurements using the Ca(2+)-sensitive dye Indo-1 revealed that forskolin and guanethidine had no effect on the electrically stimulated increase in intracellular Ca2+ concentration recorded from the cell bodies and the growth cones. 7. We conclude that release of [3H]NA can be enhanced or blocked without affecting the increase in intracellular Ca2+ concentration produced by electrical stimulation. Therefore, it is possible that pharmacological agents enhance or depress the release of [3H]NA by acting on steps of exocytosis that are down-stream from Ca2+ mobilization.