Distinct distribution of immunoreactive dynorphin and leucine enkephalin in various populations of isolated adrenal cromaffin cells

The distribution of immunoreactive-dynorphin (ir-Dyn) in isolated subpopulations of bovine adrenal chromaffin cells was examined and compared with that of adrenaline (A), noradrenaline (NA) and ir-Leucine-Enkephalin (ir-Leu-Enk). Using a stepwise bovine serum albumin (BSA) gradient, various populations of catecholamine-storing cells were separated and designated as cell layers I, II and III. Cell layer I contained more NA than A; cell layer II contained slightly more A than NA whereas cell layer III was highly enriched in A. The original cell preparation contained 2.9 times more ir-Leu-Enk than ir-Dyn (4.7 and 1.6 pmoles per 10(6) cells, respectively). After separation of the cells on BSA gradient, ir-Dyn was mainly detected in cell layer I (4.0 pmoles/10(6) cells) whereas ir-Leu-Enk was concentrated in cell layer III (8.3 pmoles/10(6) cells). Both peptides were secreted in response to acetylcholine (5 x 10(-5) M), but the amount secreted was in accordance with the cell content in each peptide. After subcellular fractionation of the adrenal medulla, the neuropeptides were found in close association with catecholamines in the secretory granules. These results indicate that bovine adrenal chromaffin cells can be isolated according to their specific content in A, NA and opioid peptides and are consistent with the hypothesis of distinct biosynthetic pathways for Dyn and the Enk.     

Bombesin-like immunoreactivity in bovine adrenal medulla

The presence of immunoreactive (ir)-bombesin in bovine adrenal medulla, isolated adrenal chromaffin cells and subcellular fractions of the adrenal medulla was demonstrated using a specific antibody to the synthetic peptide. High levels of ir-bombesin were detected in acid (HCl) extracts of the adrenal tissue (27 pmol/g) and isolated cells (0.35 pmol per 10(6) cells). Subpopulations of adrenal chromaffin cells were also obtained by centrifugation of the original cell preparation through a stepwise bovine serum albumin gradient (cell layers I, II and III). The highest concentration of ir-bombesin (0.77 pmol/10(6) cells) was found in a cell population (cell layer I) enriched in noradrenaline (adrenaline/noradrenaline ratio of 0.6). At the subcellular level, ir-bombesin was mainly concentrated in the secretory granules (0.61 pmol/mg protein) along with catecholamines (1097 nmol/mg protein), but a relatively high concentration of ir-bombesin (0.26 pmol/mg protein) was also found in the microsomal fraction. Isolation and high performance liquid chromatography (HPLC) analysis of adrenomedullary ir-bombesin revealed the presence of four molecular forms, one of them corresponding to gastrin releasing peptide (GRP), another one (major peak) eluting closely to synthetic neuromedin B and another one coeluting with GRP-(18-27). HPLC analysis of the molecular forms of ir-bombesin in the microsomes and secretory granules indicated that GRP- and neuromedin B-like materials can be generated between the two fractions.     

Presence of the Novel Pituitary Protein „7B2” in Bovine Chromaffin Granules: Possible Co-Release of 7B2 and Catecholamine as Induced by Nicotine

We observed the presence of the novel pituitary protein "7B2" and its release in the bovine adrenal medulla. The 7B2 concentration (mean +/- SEM) in extracts of the bovine adrenal medulla was 952 +/- 155 pg/mg tissue (n = 6). 7B2 was distributed in the chromaffin granule fraction prepared from the bovine adrenal medulla and was released by high K+ and/or nicotine from cultured cells of the bovine adrenal medulla. Co-release of 7B2 with catecholamine induced by nicotine from the cultured bovine chromaffin cells was also observed. In an analysis of the bovine adrenal medulla chromaffin granule fraction on gel permeation chromatography, there was a major peak with an apparent molecular weight of 45,000, whereas a major peak with an apparent molecular weight of 20,000 was found in that on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On reverse-phase HPLC, a major peak with a retention time of 35 min was observed in the bovine chromaffin granule fraction and in the bovine anterior pituitary extract. These findings indicate that 7B2 is a secretory protein in the bovine adrenal medulla. The possibility that 7B2 might be released with catecholamine, possibly in response to stress, warrants investigation.     

Heterogeneous distribution of neurocalcin-immunoreactive nerve terminals in the mouse adrenal medulla

Neurocalcin is a novel calcium-binding protein found in bovine brain tissue. We investigated immunoreactivity for neurocalcin in the mouse adrenal medulla using light and electron microscopy. The immunoreactivity was present in nerve fibers, nerve terminals, and ganglion cells in the adrenal medulla, but chromaffin cells, sustentacular cells, and Schwann cells were negative in reaction. Nerve bundles containing neurocalcin-immunoreactive fibers passed through the adrenal cortex and extended into the medulla. Immunopositive nerve fibers branched off and projected varicose terminals around the chromaffin cells. These varicose terminals contained small and large-cored vesicles and made synapses with the chromaffin cells. We performed paraformaldehyde-induced fluorescence-histochemical studies for catecholamine combined with immunohistochemical studies for neurocalcin. Neurocalcin-immunoreactive nerve terminals were more abundant at noradrenaline (fluorescent) cell-rich regions than at adrenaline (non-fluorescent) cell-rich regions. These results show that neurocalcin-immunoreactive nerves mainly innervate noradrenaline-containing chromaffin cells in the mouse adrenal medulla and that neurocalcin may regulate synaptic function in the nerve terminals. Received: 21 October 1996 / Accepted: 12 February 1997     

Prostaglandin E receptors in bovine adrenal medulla are coupled to adenylate cyclase via Gi and to phosphoinositide metabolism in a pertussis toxin-insensitive manner

Prostaglandin E (PGE) receptor is coupled to a pertussis toxin-insensitive GTP-binding protein in bovine adrenal medulla, but PGE receptor partially purified from bovine adrenal medulla was functionally reconstituted with Gi into phospholipid vesicles (Negishi, M., Ito, S., Yokohama, H., Hayashi, H., Katada, T., Ui, M., and Hayaishi, O. (1988) J. Biol. Chem. 263, 6893-6900). We demonstrate here that PGE2 inhibited forskolin-induced accumulation of cAMP in cultured bovine chromaffin cells. In plasma membranes prepared from bovine adrenal medulla, PGE2 inhibited forskolin-stimulated adenylate cyclase activity in a GTP-dependent manner. This inhibitory action of PGE2 was abolished by treatment of the membrane with pertussis toxin. Reconstitution of the membranes ADP-ribosylated by pertussis toxin with Gi purified from bovine brain restored the potency of PGE2 to inhibit the adenylate cyclase activity. Inhibition of forskolin-induced cAMP accumulation by PGE2 was also abolished by exposure to the toxin in the cells, indicating that PGE receptors are coupled to Gi. In contrast, PGE2 stimulated the formation of inositol phosphates in chromaffin cells, but this effect was not affected by treatment of the cells with pertussis toxin, suggesting that the PGE receptors are coupled to phosphoinositide metabolism via a pertussis toxin-insensitive G-protein. Both the inhibitory action of cAMP accumulation and stimulation of phosphoinositide metabolism were specific for PGE1 and PGE2, and the Scatchard plot analysis of PGE2 binding to the membrane showed a single high-affinity binding site (Kd = 2 nM). In bovine adrenal chromaffin cells PGE2 enhanced catecholamine release in the presence of ouabain by stimulation of phosphoinositide metabolism (Yokohama, H., Tanaka, T., Ito, S., Negishi, M., Hayashi, H., and Hayaishi, O. (1988) J. Biol. Chem. 263, 1119-1122). We further examined the modulation of catecholamine release by PGE2 through its inhibitory coupling to the adenylate cyclase system. Prior exposure of chromaffin cells to forskolin or dibutyryl-cAMP reduced nicotine-stimulated catecholamine release, and PGE2 attenuated forskolin-induced inhibition of catecholamine release stimulated by nicotine, but not dibutyryl-cAMP-induced inhibition. In the absence of evidence that PGE receptor subtypes exist, these results suggest that the PGE receptor is coupled to two signal transduction systems leading to inhibition of cAMP accumulation via Gi and to production of inositol phosphates via a pertussis toxin-insensitive G-protein, both of which may modulate catecholamine release from bovine chromaffin cells.     

Chromostatin receptors control calcium channel activity in adrenal chromaffin cells.

One of the functions of chromogranin A (CGA), the major soluble component of secretory granules in both adrenal medullary chromaffin cells and many other endocrine cell types appears to be that of a prohormone. CGA is the precursor of several peptides including pancreastatin, a 49-residue peptide, and a 20-residue peptide, chromostatin, which have been identified as biologically active peptides. Chromostatin produces a dose-dependent inhibition (ID50 of 5 nM) of the secretagogue-evoked catecholamine secretion from chromaffin cells. Here we report that chromostatin potently inhibits L-type calcium currents recorded with the nystatin-perforated patch technique in cultured chromaffin cells. This inhibitory effect of chromostatin on calcium currents was not observed in experiments using the classical patch-clamp whole-cell approach which induces the leakage of cytoplasmic components. Using 125I-chromostatin, we show that chromostatin exhibits a fully reversible and saturable binding to the plasma membrane of cultured chromaffin cells. Analysis of binding experiments at equilibrium indicates the existence of one class of binding sites with a Bmax of 2.7 pmol/mg of chromaffin cell proteins and an apparent Kd of 6.5 nM. This high affinity is in good correlation with the half-maximal concentration (ID50 5 nM) of chromostatin inhibiting catecholamine secretion from chromaffin cells. Specificity of the chromostatin binding was further assessed by displacement experiments with unlabeled CGA-related or -unrelated peptides. We found an excellent quantitative correlation between the affinities of the various peptides determined by binding assays and their functional potency tested on catecholamine secretion: bovine chromostatin greater than human chromostatin greater than CGA much greater than rat chromostatin, pancreastatin, CAP-14, substance P, and Leu-enkephalin. Cross-linking experiments reveal that chromostatin associates specifically with an 80-kDa plasma membrane protein. These results together with the patch-clamp experiments support the idea that chromaffin cells possess specific chromostatin receptors and that activation of such receptors leads to the inhibition of L-type voltage-sensitive calcium channels through an intracellular second messenger pathway.     

Reduced calcium current density in female versus male mouse adrenal chromaffin cells in situ

Neuroendocrine adrenal medullary chromaffin cells are a main output of the sympathetic nervous system. Acute stress activates the sympatho-adrenal stress reflex, excites adrenal chromaffin cells, and elicits catecholamine secretion into the circulation. Previous studies have demonstrated that stress-evoked serum catecholamine levels are greater in males. We investigated potential mechanistic bases for this gender dimorphism at the level of the adrenal medulla. We utilized in situ single-cell perforated patch voltage clamp to measure basic electrophysiological parameters that affect cell excitability. We found that chromaffin cells from male and female mice exhibit statistically identical depolarization-evoked calcium currents. However, the resting capacitance, an index of cell surface area, was significantly higher in cells from female mice. Thus the current density in female cells was significantly lower. We found that inhibition of protein kinase C, an enzyme shown to regulate both exocytosis and endocytosis, eliminates the cell surface area gender dimorphism. Finally, we performed kinetic simulations of the secretion process and report a predicted elevated secretory capacity in male cells. Thus, regulation of cell size may act to decrease cell excitability in female cells and may in-part represent the mechanistic basis for increased stress-evoked catecholamine secretion described in males.     

Difference in the Effectiveness of Ca2+ to Evoke Catecholamine Secretion Between Adrenaline-and Noradrenaline-Containing Cells of Bovine Adrenal Medulla

Abstract: Differential adrenaline (Ad) and noradrenaline (NA) secretions evoked by secretagogues were investigated using digitonin-permeabilized adrenal chromaffin cells, cultured adrenal chromaffin cells, and perfused adrenal glands of the ox. In digitonin-permeabilized cells, Ca²⁺ (0.8-160 μM) caused a concentration-dependent increase in catecholamine secretion, which was characterized by a predominance of NA over Ad secretion. Acetylcholine (10-1,000 μM), high K⁺ (14-56 μM), and bradykinin (0.1-1,000 μM) all were confirmed to induce the release of more NA than Ad at all concentrations used. There was no apparent difference in the ratios of NA/Ad between Ca²⁺-induced catecholamine secretion from digitonin-permeabilized cells and those induced by secretagogues from cultured cells. Qualitatively the same result was obtained in the secretory responses to acetylcholine and high K⁺ in perfused adrenal glands. These results indicate that the effectiveness of Ca²⁺ for catecholamine secretion is higher in the secretory apparatus of NA cells than in that of Ad cells of the bovine adrenal medulla. This may be one of the reasons why the secretagogues cause a predominance of NA secretion over Ad secretion in the bovine adrenal medulla.     

Effects of substance P on the long-term regulation of tyrosine hydroxylase activity and catecholamine levels in cultured adrenal chromaffin cells

Acetylcholine, released from splanchnic nerve terminals innervating adrenal chromaffin cells, is known to increase synthesis of adrenal tyrosine hydroxylase, the rate-limiting enzyme in catecholamine synthesis. The neuropeptide substance P is also present in the splanchnic nerve innervating the adrenal medulla, and this study examined whether substance P has any long-term effects on tyrosine hydroxylase activity and catecholamine levels in cultures of adult bovine adrenal chromaffin cells. When cultures were incubated for 3 days with substance P and carbachol, a cholinergic agonist, substance P (10(-6) M, and greater) completely inhibited the increase in tyrosine hydroxylase activity normally induced by carbachol. Long-term stimulation with carbachol also depleted endogenous catecholamines from the cells and substance P prevented this carbachol-induced depletion of catecholamine content. Substance P by itself, in the absence of carbachol, had only a slight effect on tyrosine hydroxylase activity. 8-Bromoadenosine 3':5'-cyclic monophosphate, an analogue of adenosine 3':5'-cyclic monophosphate, also increases tyrosine hydroxylase activity in chromaffin cells; however, substance P had no effect on the increase in tyrosine hydroxylase activity induced by this analogue. These results indicate that substance P's effects are relatively specific for the carbachol-induced increased in tyrosine hydroxylase activity and that the primary site of action of substance P is not a site common to the mechanism of tyrosine hydroxylase induction by carbachol and 8-bromoadenosine 3':5'-cyclic monophosphate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adrenomedullin and proadrenomedullin N-terminal 20 peptide (PAMP) in adrenal chromaffin cells.

Adrenomedullin (AM) and proadrenomedullin N-terminal 20 peptide (PAMP) are peptides having multiple physiological functions and are most abundantly expressed in the adrenal medulla. In addition to PAMP, PAMP12, a 12 amino acid peptide with sequence identity to PAMP between amino acids 9-20, has also been shown to be expressed in the adrenal medulla. AM, PAMP and PAMP12 are released along with catecholamines by regulated exocytosis upon stimulation of adrenal chromaffin cells. PAMP and PAMP12 regulate catecholamine release and synthesis by interfering with nicotinic cholinergic receptors in these chromaffin cells. AM may also cause gradual release of catecholamine from these cells. AM, PAMP and PAMP12 are endogenous peptides that modulate chromaffin cell function via different mechanisms.     

Role of TrkB expression in rat adrenal gland during acute immobilization stress

Expression of tyrosine receptor kinase B (TrkB), a receptor for brain‐derived neurotrophic factor (BDNF), is markedly elevated in the adrenal medulla during immobilization stress. Catecholamine release was confirmed in vitro by stimulating chromaffin cells with recombinant BDNF. We investigated the role of TrkB and the localization of BDNF in the adrenal gland during immobilization stress for 60 min. Blood catecholamine levels increased after stimulation with TrkB expressed in the adrenal medulla during 60‐min stress; however, blood catecholamine levels did not increase in adrenalectomized rats. Furthermore, expression of BDNF mRNA and protein was detected in the adrenal medulla during 60‐min stress. Similarly, in rats undergoing sympathetic nerve block with propranolol, BDNF mRNA and protein were detected in the adrenal medulla during 60‐min stress. These results suggest that signal transduction of TrkB in the adrenal medulla evokes catecholamine release. In addition, catecholamine release was evoked by both the hypothalamic–pituitary–adrenal axis and autocrine signaling by BDNF in the adrenal gland. BDNF–TrkB interaction may play a role in a positive feedback loop in the adrenal medulla during immobilization stress.     

Vasoactive intestinal peptide and substance P increase levels of enkephalin-containing peptides in adrenal chromaffin cells

The neuropeptides substance P and vasoactive intestinal peptide (VIP), reported to exist in the splanchnic nerve terminals innervating the adrenal medulla, elevate the levels of enkephalin-containing peptides (ECPs) in cultured bovine adrenal medullary chromaffin cells. Cellular ECP stores were increased over 48 hr by 72 and 46 percent, respectively, following incubation with 5 microM VIP or 10 microM substance P, maximally effective concentrations. The results suggest that VIP and substance P may be trans-synaptic modulators of chromaffin cell ECP stores.     

Characterization of Two Chromaffin Cell Populations Isolated from Bovine Adrenal Medulla

Cultures of chromaffin cells isolated from the bovine adrenal medulla have been extremely useful for investigating secretory mechanisms, but such cultures used up to the present time represent mixed populations of adrenergic and noradrenergic cells. This report describes how, with slight modifications to standard procedures, two separate chromaffin cell populations may be separated from bovine adrenal medullae. These two cell fractions have been characterized by biochemical, immunocytochemical, and morphological techniques as enriched populations of adrenergic or noradrenergic cells, respectively. The adrenergic cell-enriched fraction consists of greater than 90% adrenergic cells, whereas the noradrenergic cell-enriched fraction contains greater than 60% noradrenergic cells. We also demonstrate that these cells may be cultured with their secretory machinery intact: analysis of secreted catecholamines from nicotine- or high K+ concentration-stimulated cells cultured from each fraction confirms that adrenaline is the major catecholamine secreted by one fraction, whereas noradrenaline is mainly secreted by the other.     

Pituitary adenylate cyclase-activating peptide enhances electrical coupling in the mouse adrenal medulla

Neuroendocrine adrenal medullary chromaffin cells receive synaptic excitation through the sympathetic splanchnic nerve to elicit catecholamine release into the circulation. Under basal sympathetic tone, splanchnic-released acetylcholine evokes chromaffin cells to fire action potentials, leading to synchronous phasic catecholamine release. Under elevated splanchnic firing, experienced under the sympathoadrenal stress response, chromaffin cells undergo desensitization to cholinergic excitation. Yet, stress evokes a persistent and elevated adrenal catecholamine release. This sustained stress-evoked release has been shown to depend on splanchnic release of a peptide transmitter, pituitary adenylate cyclase-activating peptide (PACAP). PACAP stimulates catecholamine release through a PKC-dependent pathway that is mechanistically independent of cholinergic excitation. Moreover, it has also been reported that shorter term phospho-regulation of existing gap junction channels acts to increase junctional conductance. In this study, we test if PACAP-mediated excitation upregulates cell-cell electrical coupling to enhance chromaffin cell excitability. We utilize electrophysiological recordings conducted in adrenal tissue slices to measure the effects of PACAP stimulation on cell coupling. We report that PACAP excitation increases electrical coupling and the spread of electrical excitation between adrenal chromaffin cells. Thus PACAP acts not only as a secretagogue but also evokes an electrical remodeling of the medulla, presumably to adapt to the organism's needs during acute sympathetic stress.     

The effects of ketamine, phencyclidine and lidocaine on catecholamine secretion from cultured bovine adrenal chromaffin cells

The ability of ketamine, phencyclidine and analogues to alter catecholamine secretion from cultured bovine adrenal chromaffin cells was investigated. Both ketamine and phencyclidine specifically inhibited nicotinic agonist-induced secretion at concentrations which did not alter secretion induced by elevated K+ depolarization. The inhibition of nicotinic agonist-induced secretion was not overcome by increasing concentrations of nicotinic agonist. The effects of stereoisomer pairs of phencyclidine-like drugs - dexoxadrol, levoxadrol and (+)PCMP, (-)PCMP - did not reveal stereospecificity for the inhibition, in contrast to the stereospecific behavioral effects of the drugs. The local anesthetic lidocaine (0.3 mM) also noncompetitively inhibited nicotinic agonist-induced secretion without inhibiting elevated K+-induced secretion. The data indicate that ketamine and phencyclidine at clinically relevant concentrations specifically inhibit the adrenal chromaffin cell nicotinic receptor at a site similar to or identical with the site of action of local anesthetic. Although the nicotinic receptor inhibition is probably not related to the anesthetic and behavioral effects of ketamine and phencyclidine, it is likely that the centrally mediated increase in sympathetic nervous system activity which is characteristic of these drugs is moderated by the peripheral blocking effects on catecholamine secretion from the adrenal medulla.     

Bovine adrenal chromaffin granules are a site of synthesis of atrial natriuretic factor

We have previously reported the existence of a peptide factor in the adrenal medulla which inhibits aldosterone secretion in cultured bovine zona glomerulosa cells. The acid extracts of chromaffin granules from bovine adrenal medulla were purified by a four step high performance liquid chromatography procedure. Two active fractions exhibited sequence homology with bovine atrial natriuretic factor ANF (Ser99-Tyr126) and its polypeptide precursor (Asn1-Tyr126). The occurrence of both precursor and mature forms of ANF within chromaffin granules indicates the endogenous character of ANF in the adrenal medulla and suggests the potential usefulness of cultured adrenal chromaffin cells for investigating the synthesis, maturation and secretion of atrial peptides.     

Binding of prostaglandin E2 to cultured bovine adrenal chromaffin cells and its effect on catecholamine secretion

We have previously shown that plasma membranes from adrenal medulla possess specific high-affinity binding sites for prostaglandins (PGs) E1 and E2. We have now investigated the binding of PGE2 to intact bovine adrenal chromaffin cells and the effects of prostaglandins on the release of catecholamines from these cells. Adrenal chromaffin cells specifically bound PGE2 with a dissociation constant of 2 nM and a concentration of about 40,000 binding sites per cell. Low concentrations of PGE2 inhibited the nicotine-stimulated release of catecholamines from these cells. The effect of PGE2 was biphasic, the maximal inhibitory effect being observed at a concentration of between 1 and 10 nM. Higher concentrations (1 microM) of PGE2 had minimal inhibitory effects on nicotine-evoked noradrenaline release, but instead had a direct stimulatory effect in the absence of cholinergic agonists. Although the stimulatory effects of high concentrations of PGE2 were reproducibly observed in all cell preparations, only about one-half of the cultures tested responded to the inhibitory effects of this prostaglandin. It is possible that PGE2 plays a modulatory role in the regulation of catecholamine secretion from the adrenal medulla.     

The caffeine-sensitive Ca2+ store in bovine adrenal chromaffin cells; an examination of its role in triggering secretion and Ca2+ homeostasis

The effect of caffeine on catecholamine secretion and intracellular free Ca2+ concentration [( Ca2+]i) in bovine adrenal chromaffin cells was examined using single fura-2-loaded cells and cell populations. In cell populations caffeine elicited a large (approximately 200 nM) transient rise in [Ca2+]i that was independent of external Ca2+. This rise in [Ca2+]i triggered little secretion. Single cell measurements of [Ca2+]i showed that most cells responded with a large (greater than 200 nM) rise in [Ca2+]i, whereas a minority failed to respond. The latter, whose caffeine-sensitive store was empty, buffered a Ca2+ load induced by a depolarizing stimulus more effectively than those whose store was full. The caffeine-sensitive store in bovine chromaffin cells may be involved in Ca2+ homeostasis rather than in triggering exocytosis.     

PACAP regulates immediate catecholamine release from adrenal chromaffin cells in an activity-dependent manner through a protein kinase C-dependent pathway

Adrenal medullary chromaffin cells are a major peripheral output of the sympathetic nervous system. Catecholamine release from these cells is driven by synaptic excitation from the innervating splanchnic nerve. Acetylcholine has long been shown to be the primary transmitter at the splanchnic-chromaffin synapse, acting through ionotropic nicotinic acetylcholine receptors to elicit action potential-dependent secretion from the chromaffin cells. This cholinergic stimulation has been shown to desensitize under sustained stimulation, yet catecholamine release persists under this same condition. Recent evidence supports synaptic chromaffin cell stimulation through alternate transmitters. One candidate is pituitary adenylate cyclase activating peptide (PACAP), a peptide transmitter present in the adrenal medulla shown to have an excitatory effect on chromaffin cell secretion. In this study we utilize native neuronal stimulation of adrenal chromaffin cells in situ and amperometric catecholamine detection to demonstrate that PACAP specifically elicits catecholamine release under elevated splanchnic firing. Further data reveal that the immediate PACAP-evoked stimulation involves a phospholipase C and protein kinase C-dependant pathway to facilitate calcium influx through a Ni²⁺ and mibefradil-sensitive calcium conductance that results in catecholamine release. These data demonstrate that PACAP acts as a primary secretagogue at the sympatho-adrenal synapse under the stress response.