Ca(2+)-dependent K(+) current and exocytosis in responses to caffeine and muscarine in voltage-clamped guinea-pig adrenal chromaffin cells.

We characterized changes in membrane currents and the cytosolic Ca(2+) concentration, [Ca(2+)](i), in response to caffeine, and compared them with those in response to muscarine using the perforated patch-clamp technique and fura-2 microfluorimetry in guinea-pig adrenal chromaffin cells. Catecholamine release from single voltage-clamped cells was monitored with amperometry using carbon microelectrodes. Caffeine produced a transient outward current (I(out)) at holding potentials over - 60 mV, increasing in amplitude with increasing the potentials. It also evoked a rapid increase of [Ca(2+)](i) at all potentials examined. The current-voltage relation revealed that the activation of K(+) channels was responsible for the I(out) evoked by caffeine. Both current and [Ca(2+)](i) responses were reversibly abolished by cyclopiazonic acid, an inhibitor of Ca(2+)-pump ATPase. At - 30 mV, the caffeine-induced I(out), but not [Ca(2+)](i), was partly inhibited by either charybdotoxin or apamin. In the majority of cells tested, caffeine induced a larger I(out) but a smaller [Ca(2+)](i) increase than muscarine. Caffeine and muscarine increased catecholamine release from voltage-clamped single cells concomitant with the transient increase of [Ca(2+)](i), and there was a positive correlation between them. These results indicate that caffeine activates Ca(2+)-dependent K(+) channels and catecholamine secretion due to the release of Ca(2+) from internal stores in voltage-clamped adrenal chromaffin cells of the guinea-pig. There seems to be a spatial difference between [Ca(2+)](i) increased by Ca(2+) release from caffeine-sensitive stores and that released from muscarine (inositol 1,4,5-trisphosphate)-sensitive ones.

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.

Primary structure and function of the catecholamine release inhibitory peptide catestatin (chromogranin A(344-364)): identification of amino acid residues crucial for activity.

The novel chromogranin A fragment catestatin (bovine chromogranin A(344-364); RSMRLSFRARGYGFRGPGLQL) is a potent inhibitor of catecholamine release (IC50, approximately 0.2-0.3 microM) by acting as a nicotinic cholinergic antagonist. To define the minimal active region within catestatin, we tested the potencies of synthetic serial three-residue deletion (amino-terminal, carboxyl-terminal, or bidirectional) fragments to inhibit nicotine-stimulated catecholamine secretion from PC12 pheochromocytoma cells. The results revealed that a completely active core sequence of catestatin was constituted by chromogranin A(344-364). Nicotinic cationic signal transduction was affected by catestatin fragments in a manner similar to that for secretion (confirming the functional importance of the amino-terminus). To identify crucial residues within the active core, we tested serial single amino acid truncations or single residue substitutions by alanine on nicotine-induced catecholamine secretion and desensitization. Nicotinic inhibition by the active catestatin core was diminished by even single amino acid deletions. Selective alanine substitution mutagenesis of the active core revealed important roles for Met346, Leu348, Phe350, Arg351, Arg353, Gly354, Tyr355, Phe357, and Arg358 on catecholamine secretion, whereas crucial roles to inhibit desensitization of catecholamine release were noted for Arg344, Met346, Leu348, Ser349, Phe350, Arg353, Gly354, Tyr355, Gly356, and Arg358. We conclude that a small, 15-amino acid core of catestatin (chromogranin A(344-364)) is sufficient to exert the peptide's typical inhibitory effects on nicotinic cholinergic-stimulated catecholamine secretion, signal transduction, and desensitization. These studies refine the biologically active domains of catestatin and suggest that the pharmacophores for inhibition of nicotinic secretion and desensitization may not be identical.

2'-Deoxyadenosine causes cell death in embryonic chicken sympathetic ganglia and brain.

Previous work has shown that nucleosides produce apoptosis in sympathetic ganglion (SG) cells in vitro. The present study examined the effects of nucleosides on the development of the chick embryo in vivo with special attention to the SG and the optic tectum of the central nervous system. In the presence of an adenosine deaminase inhibitor, adenosine and 2'-deoxyadenosine (2'-dAdo) produced different toxicity patterns: both adenosine and 2'-dAdo were toxic to E3 embryos, but only 2'-dAdo was toxic at later stages (E6 1/2, E11). Dosage experiments on E6 1/2 embryos showed that adenosine was less toxic than 2'-dAdo and that 2'-dAdo in sublethal doses was teratogenic. We also examined the effects of 2'-dAdo on embryonic chicken SG and optic tectum in vivo to determine whether sublethal doses of 2'-dAdo produced cell death in these centers on E6 1/2 and 10. In the E6 1/2 SG, 2'-dAdo produced significant neuron loss (83%) and a decrease in SG volume (65%); however, at E10, there was only minor cell loss (7%) and no significant change in SG volume. In the optic tectum at E6 1/2, cell loss was confined mainly to the tectal ventricular zone, but there was little sign of cell loss in this organ at E10. Since cell production is vigorous in the SG and optic tectum at E6 1/2 but relatively low at E10, 2'-dAdo appears to work by stopping cell proliferation. The ineffectiveness of 2'-dAdo at E10 may result from the lethality of 2'-dAdo to the embryo at low concentrations (30 microM) in vivo, well below the apoptosis-inducing concentrations employed in vitro (100-300 microM). These data extend previous findings showing that purine and pyrimidine metabolism plays an important role in development.

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 protects chick embryonic sympathetic neurons from apoptotic death by 2'-deoxyadenosine--importance of ATP in apoptosis.

Our past work on nucleoside toxicity in sympathetic neurons has clearly revealed that adenosine and 2'-deoxyadenosine (dAdo) have different mechanisms of action in inducing apoptotic death. For example, adenosine is toxic to neurons only during early phase of growth whereas dAdo kills even mature neurons. In this study, we hypothesize that dAdo-induced apoptosis is initiated when ATP concentration of sympathetic neurons decreases below a critical level. To prove our hypothesis we used adenosine as a tool to replenish ATP levels of sympathetic neurons. We demonstrate that dAdo toxicity in mature sympathetic neurons was fully prevented by adenosine treatment. Furthermore, we demonstrate that depletion of ATP caused by dAdo was prevented by pretreatment with adenosine. These data suggest that intracellular accumulation of adenosine could play a neuroprotective role in preventing death associated with reduction in neuronal ATP concentration.

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.

2'-Deoxyadenosine selectively kills nonneuronal cells without affecting survival and growth of chick dorsal root ganglion neurons.

Recently, we have demonstrated that adenosine and 2'-deoxyadenosine are toxic to embryonic sympathetic neurons and proposed that purine and pyrimidine metabolism may play a critical role in the growth and development of sympathetic neurons. To extend this hypothesis further, we examined the effects of these nucleosides on two other neuronal populations in the chick embryo, sensory dorsal root ganglion neurons and parasympathetic ciliary ganglion neurons. Now, we show that 2'-deoxyadenosine and adenosine have no visible adverse effect on the viability of either sensory or parasympathetic neurons. Instead, 2'-deoxyadenosine proved to be highly toxic to the nonneuronal cells. The toxic effects of 2'-deoxyadenosine were markedly enhanced by inhibition of adenosine deaminase. In contrast, adenosine was much less toxic to nonneuronal cells than 2'-deoxyadenosine and its effect was not potentiated by inhibition of adenosine deaminase. Priming of pyrimidine pools by exogenous uridine and the specific inhibitor of the nucleoside transporter, nitrobenzylthioinosine, did not protect nonneuronal cells from 2'-deoxyadenosine toxicity. Since phosphorylation of internalized nucleosides was a key step in the initiation of toxicity in sympathetic neurons, adenosine kinase activity was compared in sensory and sympathetic neuronal cultures. The adenosine kinase activity in dorsal root ganglion cultures was only 20% of that in sympathetic ganglion cultures. Furthermore, inhibition of phosphorylation by blocking 2'-deoxyadenosine kinase with iodotubercidin and 5'-amino-5'-deoxyadenosine had no protective effect against 2'-deoxyadenosine toxicity. [3H]-thymidine incorporation was inhibited over 90% by 2'-deoxyadenosine as early as 6 h following its addition and for up to 4 days, suggesting inhibition of proliferation of nonneuronal cells by 2'-deoxyadenosine. The nucleoside was also able to wipe out already well established nonneuronal cells, leaving behind an enriched population of sensory neurons. The selective vulnerability of nonneuronal cells to 2'-deoxyadenosine offers a convenient and effective tool for removing nonneuronal cells from neuronal cultures as well as providing a new model for studying the mechanisms of nucleoside toxicity.

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.

Quantitative analysis of similarities and differences in neurotoxicities caused by adenosine and 2'-deoxyadenosine in sympathetic neurons.

These experiments characterize the nucleoside transport and quantify the neurotoxicity of adenosine and 2'-deoxyadenosine (dAdo) in chick sympathetic neurons. We show that [3H]adenosine transport was sensitive to low temperature, specific inhibitors of nucleoside transport, and an excess concentration of adenosine. However, many of these treatments had a marginal effect on [3H]dAdo transport. Total retention of [3H]dAdo over short and long periods was approximately 10 times less than that of [3H]adenosine. These data suggest that adenosine and dAdo enter sympathetic neurons by different routes. Uptake of (3H]norepinephrine ([3H]NE) decreased in neurons damaged by nucleosides and increased to control levels when neurons were protected by various agents against adenosine or dAdo toxicity. These results indicate that [3H]NE uptake serves as a quantitative index of toxicity by the nucleosides. Using this approach we demonstrate that phosphorylation of both nucleosides is essential for their lethal action. For example, iodotubercidin prevented nucleoside-induced neuronal death, but the effect was much more pronounced in the case of dAdo toxicity (IC50 of 0.83 +/- 0.4 vs. 30 +/- 1.6 nM). Another kinase inhibitor, 5'-amino 5'-deoxyadenosine, was effective in protecting neurons against dAdo but had no effect against adenosine toxicity. These results suggest that specific kinases are associated with the phosphorylation of adenosine and dAdo in sympathetic neurons to produce toxic metabolic products. Finally, neurons were susceptible to dAdo toxicity from the time of plating to 4 weeks in culture but were resistant to adenosine toxicity 8 h after plating. In conclusion, our results highlight major differences in the mechanism of neurotoxicity by adenosine and dAdo and provide insights for identification of biochemical pathways leading to neuronal death.

Age-dependent sensitivity of cultured peripheral sympathetic neurons to 1-methyl-4-phenylpyridinium: role of glutathione.

We demonstrate that 1-methyl-4-phenylpyridinium (MPP+) is toxic to chick peripheral sympathetic neurons maintained in culture in the presence of nerve growth factor (NGF). When MPP+ was added to the culture medium at the time the neurons were plated, cell loss after 3 days in culture was evident at concentrations as low as 3 nM, and near maximal at 1 microM. Toxicity was blocked by brief preincubation with the norepinephrine (NE)-reuptake blocker desipramine (DMI; 10 microM for 30 min). MPP+ blocked the uptake of [3H]NE by sympathetic neurons in a dose-dependent manner with a potency roughly equal to DMI. At concentrations up to 10 microM, MPP+ had no neurotoxic effect on the survival of sensory neurons maintained in the presence of NGF. The sensitivity of sympathetic neurons to the toxic effects of MPP+ diminished gradually with increasing lengths of time in culture. When MPP+ was added to the culture medium 48 h after plating, concentrations up to 100 microM did not cause neuronal death. This increasing resistance of sympathetic neurons to MPP+-induced cell death could not be explained by an increasing capacity for sequestration of MPP+ within synaptic vesicles. The loss of sensitivity with time in culture was, however, accompanied by a threefold increase in the levels of glutathione (GSH). Furthermore, addition of MPP+ (1 microM) to cultures previously maintained for 2 days in the presence of the GSH-synthesis inhibitor L-buthionine-[S,R]-sulfoximine (1 microM) caused the same degree of cell death as when added to freshly plated neurons. These results suggest that the observed toxicity of MPP+ in freshly plated chick sympathetic neurons may involve the formation of free radicals and that GSH plays a role in protecting sympathetic neurons in vivo from the toxicity of MPP+.

Noradrenaline transport and transporter mRNA of rat chromaffin cells are controlled by dexamethasone and nerve growth factor.

1. The biochemical basis for differences in noradrenaline (NA) transporter function between chromaffin cells in the adrenal medulla and those maintained in primary culture was investigated. 2. Intact adrenal medullae of neonatal rats accumulated small amounts of [3H]NA. In contrast, dissociated chromaffin cells placed in culture for 2-6 days accumulated 100-1000 times more [3H]NA. 3. Nerve growth factor (NGF) stimulated, whereas glucocorticoids dose dependently and reversibly inhibited, [3H]NA transport in chromaffin cells maintained in culture up to 6 days. During this period, no change in the morphological or biochemical characteristics of either NGF-treated or -untreated chromaffin cells was evident. 4. A rat NA transporter cDNA clone was isolated for use in the quantification of NA transporter mRNA. Intact adrenal medullae contained 40% less NA transporter mRNA than an equivalent number of chromaffin cells in culture. Furthermore, dexamethasone produced nearly 90% loss and NGF elicited approximately 60% increase in NA transporter mRNA levels in cultured cells. 5. In cultured cells, and possibly in vivo, glucocorticoids inhibit NA transporter function of chromaffin cells at least in part through a decrease in NA transporter mRNA.

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.

Pituitary adenylate cyclase activating polypeptide-immunoreactive sensory neurons innervate rat adrenal medulla.

Rat adrenal chromaffin cells were invested by a dense network of nerve fibers immunoreactive to pituitary adenylate cyclase activating polypeptide-38 (PACAP-IR). Immunohistochemical studies demonstrated the presence of PACAP-IR in nodose and dorsal root ganglion cells, but not in neurons of the intermediolateral cell column and other autonomic nuclei of the thoracic and upper lumbar spinal cord. Somata of the T7 to T12 paravertebral ganglia were PACAP-negative. A few lightly labeled neurons were occasionally noted in the dorsal motor nucleus of the vagus. Injection of the retrograde tracer Fluorogold into the left adrenal medulla 3 days prior to sacrifice resulted in the labeling of a population of neurons in the ipsilateral spinal cord intermediolateral cell column (T1 to L1), ipsilateral and contralateral nodose ganglia and ipsilateral dorsal root ganglia from T7 to T10 inclusive. A small number of lightly labeled somata was occasionally noted in the dorsal motor nucleus of the vagus. Combined retrograde tracing and PACAP immunohistochemistry showed that a population of Fluorogold-containing nodose and dorsal root ganglion cells were also PACAP-positive. Pre-treatment of the rats with capsaicin caused a marked reduction of the PACAP-IR in the adrenal gland as well as in the superficial layers of the dorsal horn and caudal spinal trigeminal nucleus. These findings, in conjunction with the apparent absence of PACAP-IR in spinal sympathetic preganglionic neurons, sympathetic postganglionic neurons, and dorsal motor nucleus of the vagus, raise the possibility that PACAP-IR fibers observed in the adrenal medulla are primarily sensory in origin. As a corollary, catecholamine secretion from chromaffin cells may be modulated by the peptidergic sensory afferents in addition to the cholinergic sympathetic preganglionic nerve fibers.