Biochemical characterization and immunocytochemical localization of EM66, a novel peptide derived from secretogranin II, in the rat pituitary and adrenal glands.

Characterization of secretogranin II (SgII) mRNA in various vertebrates has revealed selective conservation of the amino acid sequences of two regions of the protein, i.e., the bioactive peptide secretoneurin and a flanking novel peptide that we named EM66. To help elucidate the possible role of EM66, we examined the occurrence as well as the cellular and subcellular distribution of EM66 in rat pituitary and adrenal glands by using a polyclonal antibody raised against the recombinant human EM66 peptide. High-performance liquid chromatography (HPLC) analysis of rat pituitary and adrenal extracts combined with a radioimmunoassay resolved EM66-immunoreactive material exhibiting the same retention time as recombinant EM66. In the rat pituitary, double-labeling immunohistochemical (IHC) studies showed that EM66 immunoreactivity (IR) was present in gonadotrophs, lactotrophs, thyrotrophs, and melanotrophs, whereas corticotrophs were devoid of labeling. EM66-IR was also observed in nerve endings in the neural lobe. Immunocytochemical staining at the electron microscopic level revealed that EM66-IR is sequestered in the secretory granules within gonadotrophs and lactotrophs. In the adrenal medulla, double IHC labeling showed that EM66-IR occurs exclusively in epinephrine-synthesizing cells. At the ultrastructural level, EM66-IR was seen in chromaffin vesicles of adrenomedullary cells. These results demonstrate that post-translational processing of SgII generates a novel peptide that exhibits a cell-specific distribution in the rat pituitary and adrenal glands where it is stored in secretory granules, supporting the notion that EM66 may play a role in the endocrine system.     

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.     

Temperature effects in the stimulus-secretion process from isolated chromaffin cells

Temperature effects on the stimulus-secretion coupling process was studied by inducing release of catecholamines (CA) from isolated chromaffin cells of the bovine adrenal medulla. Use was made of three different secretagogues: acetylcholine (ACH), high potassium concentration, and the calcium ionophore A23187, at various incubation temperatures. The latter two agents induced a monotonic increase in secretion with rise in temperature, suggesting different regions of the dependence of total release on temperature. The ACH-induced secretion was, however, markedly different and exhibited a maximal release at 30 degrees C. Kinetic experiments using ACH stimulus revealed that this maximum is produced by different temperature dependence in the stages of activation and desensitization. A proposed model for the total release process yields temperature-dependent parameters that can be divided into three regions of initial rates of secretory activity corresponding to the above independent findings using high K+ concentration and the calcium ionophore. The transitions between the various regions indicate possible transitions in the physical properties of the plasma and secretory granule membranes. Elucidation of the interaction between the membranes is of primary importance in the determining mechanism of CA secretion from the isolated adrenal medulla cell.     

Pituitary adenylate cyclase-activating peptide (PACAP) recruits low voltage-activated T-type calcium influx under acute sympathetic stimulation in mouse adrenal chromaffin cells

Low voltage-activated T-type Ca_v3.2 calcium channels are expressed in neurosecretory chromaffin cells of the adrenal medulla. Previous studies have shown that naïve adrenal chromaffin cells express a nominal Ca_v3.2-dependent conductance. However, Ca_v3.2 conductance is up-regulated following chronic hypoxia or long term exposure to cAMP analogs. Thus, although a link between chronic stressors and up-regulation of Ca_v3.2 exists, there are no reports testing the specific role of Ca_v3.2 channels in the acute sympathoadrenal stress response. In this study, we examined the effects of acute sympathetic stress on T-type Ca_v3.2 calcium influx in mouse chromaffin cells in situ. Pituitary adenylate cyclase-activating peptide (PACAP) is an excitatory neuroactive peptide transmitter released by the splanchnic nerve under elevated sympathetic activity to stimulate the adrenal medulla. PACAP stimulation did not evoke action potential firing in chromaffin cells but did cause a persistent subthreshold membrane depolarization that resulted in an immediate and robust Ca²⁺-dependent catecholamine secretion. Moreover, PACAP-evoked secretion was sensitive to block by nickel chloride and was acutely inhibited by protein kinase C blockers. We utilized perforated patch electrophysiological recordings conducted in adrenal tissue slices to investigate the mechanism of PACAP-evoked calcium entry. We provide evidence that stimulation with exogenous PACAP and native neuronal stress stimulation both lead to a protein kinase C-mediated phosphodependent recruitment of a T-type Ca_v3.2 Ca²⁺ influx. This in turn evokes catecholamine release during the acute sympathetic stress response.     

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.     

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.     

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.     

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.     

Pituitary adenylate cyclase activating polypeptide provokes cultured rat chromaffin cells to secrete adrenaline.

Pituitary adenylate cyclase activating polypeptide (PACAP) provoked the rat chromaffin cells to secrete adrenaline. Within 20 min, the amount of adrenaline secreted by PACAP (10(-8) M) was as much as that caused by acetylcholine (10(-4) M). PACAP, but not acetylcholine, induced a long-term (over 120 min) increase in secretion of adrenaline. PACAP also activated adenylate cyclase and elevated cytosolic Ca2+ concentration. Furthermore, we found immunoreactive PACAP and PACAP binding sites in the rat adrenal medulla. These results suggest that PACAP has an important role in stimulating secretion of adrenaline in the adrenal medulla.     

Primary cultures of bovine chromaffin cells synthesize and secrete vasoactive intestinal polypeptide (VIP)

Dispersed cells of the bovine adrenal medulla express immunoreactive vasoactive intestinal polypeptide (VIP) after 24 hours in culture, although VIP could not be detected in extracts of bovine adrenal medulla or cortex. Immunoreactive VIP eluted from a reversed-phrase chromatography column with the same retention time as authentic porcine VIP_1–28. VIP in chromaffin cells in culture appears to be contained in a secretory granule pool, since it, like methionine-enkephalin (met-enk) was released into the medium after exposure of cells to nicotine, carbachol, veratridine and elevated potassium in a dose-dependent manner. Doseresponse curves for VIP and enkephalin release by the above secretagogues were similar but not identical. Enkephalins and VIP may either be contained in separate subpopulations of chromaffin cells or co-stored in the same cells.     

Expression of Trophic Peptides and Their Receptors in Chromaffin Cells and Pheochromocytoma

Pheochromocytomas are catecholamine-producing tumors arising from chromaffin cells of the adrenal medulla or extra-adrenal location. Along with catecholamines, tumoral cells produce and secrete elevated quantities of trophic peptides which are normally released in a regulated manner by the normal adrenal medulla. Among these peptides, the amounts of pituitary adenylate cyclase-activating polypeptide (PACAP), adrenomedullin (AM), and neuropeptide Y (NPY) are particularly high. These peptides can exert endocrine, paracrine or autocrine effects in numerous cell types. In particular, they have been shown to be involved in cell proliferation and survival, catecholamine production and secretion, and angiogenesis. Some of these processes are exacerbated in pheochromocytomas, raising the possibility of the involvement of trophic peptides. Here, we review the expression levels of NPY, PACAP, and AM and theirs receptors in chromaffin cells and pheochromocytomas, and address their possible implication in the adrenal medulla tumorigenesis and malignant development of pheochromocytomas.     

Pituitary Adenylate Cyclase-Activating Polypeptide Causes Ca2+ Release from Ryanodine/Caffeine Stores Through a Novel Pathway Independent of Both Inositol Trisphosphates and Cyclic AMP in Bovine Adrenal Medullary Cells

Abstract: Pituitary adenylate cyclase-activating polypeptide (PACAP) causes both Ca²⁺ release and Ca²⁺ influx in bovine adrenal chromaffin cells. To elucidate the mechanisms of PACAP-induced Ca²⁺ release, we investigated expression of PACAP receptors and measured inositol trisphosphates (IP₃), cyclic AMP, and the intracellular Ca²⁺ concentration in bovine adrenal medullary cells maintained in primary culture. RT-PCR analysis revealed that bovine adrenal medullary cells express the PACAP receptor hop, which is known to couple with both IP₃ and cyclic AMP pathways. The two naturally occurring forms of PACAP, PACAP38 and PACAP27, both increased cyclic AMP and IP₃, and PACAP38 was more potent than PACAP27 in both effects. Despite the effects of PACAP on IP₃ production, the Ca²⁺ release induced by PACAP38 or by PACAP27 was unaffected by cinnarizine, a blocker of IP₃ channels. The potencies of the peptides to cause Ca²⁺ release in the presence of cinnarizine were similar. The Ca²⁺ release induced by PACAP38 or by PACAP27 was strongly inhibited by ryanodine and caffeine. In the presence of ryanodine and caffeine, PACAP38 was more potent than PACAP27. PACAP-induced Ca²⁺ release was unaffected by Rp-adenosine 3′,5′-cyclic monophosphothioate, an inhibitor of protein kinase A. Ca²⁺ release induced by bradykinin and angiotensin II was also inhibited by ryanodine and caffeine, but unaffected by cinnarizine. Although IP₃ production stimulated by PACAP38 or bradykinin was abolished by the phospholipase C inhibitor, U-73122, Ca²⁺ release in response to the peptides was unaffected by U-73122. These results suggest that PACAP induces Ca²⁺ release from ryanodine/caffeine stores through a novel intracellular mechanism independent of both IP₃ and cyclic AMP and that the mechanism may be the common pathway through which peptides release Ca²⁺ in adrenal chromaffin cells.     

Muscarinic release of inositol trisphosphate without mobilization of calcium in bovine adrenal chromaffin cells

Chromaffin cells of bovine adrenal medulla release catecholamines in response to activation of nicotinic ACh receptors which open voltage-sensitive calcium channels. Catecholamine secretion by exocytosis requires an increase in cytosolic free calcium. The cells also possess muscarinic ACh receptors but muscarinic agents do not provoke catecholamine release. Quin-2 studies show that they do not increase cytosolic free Ca2+ concentration, but unlike the nicotinic agents, they cause phosphoinositide hydrolysis. Muscarinic stimulation leads to rapid loss of labelled phosphatidylinositol 4-phosphate and of phosphatidylinositol 4,5-bisphosphate. At the same time there is release of inositol trisphosphate, inositol bisphosphate and inositol phosphate. In a number of other cells inositol trisphosphate may act as a second messenger releasing Ca2+ from storage sites in the endoplasmic reticulum but this is not its function in bovine chromaffin cells.     

PAC1 receptor activation by PACAP-38 mediates Ca2+ release from a cAMP-dependent pool in human fetal adrenal gland chromaffin cells

Previous studies have shown that human fetal adrenal gland from 17- to 20-week-old fetuses expressed pituitary adenylate cyclase-activating polypeptide (PACAP) receptors, which were localized on chromaffin cells. The aim of the present study was to identify PACAP receptor isoforms and to determine whether PACAP can affect intracellular calcium concentration ([Ca(2+)](i)) and catecholamine secretion. Using primary cultures and specific stimulation of chromaffin cells, we demonstrate that PACAP-38 induced an increase in [Ca(2+)](i) that was blocked by PACAP (6-38), was independent of external Ca(2+), and originated from thapsigargin-insensitive internal stores. The PACAP-triggered Ca(2+) increase was not affected by inhibition of PLC beta (preincubation with U-73122) or by pretreatment of cells with Xestospongin C, indicating that the inositol 1,4,5-triphosphate-sensitive stores were not mobilized. However, forskolin (FSK), which raises cytosolic cAMP, induced an increase in Ca(2+) similar to that recorded with PACAP-38. Blockage of PKA by H-89 or (R(p))-cAMPS suppressed both PACAP-38 and FSK calcium responses. The effect of PACAP-38 was also abolished by emptying the caffeine/ryanodine-sensitive Ca(2+) stores. Furthermore, treatment of cells with orthovanadate (100 microm) impaired Ca(2+) reloading of PACAP-sensitive stores indicating that PACAP-38 can mobilize Ca(2+) from secretory vesicles. Moreover, PACAP induced catecholamine secretion by chromaffin cells. It is concluded that PACAP-38, through the PAC(1) receptor, acts as a neurotransmitter in human fetal chromaffin cells inducing catecholamine secretion, through nonclassical, recently described, ryanodine/caffeine-sensitive pools, involving a cAMP- and PKA-dependent phosphorylation mechanism.     

Inhibition of voltage-gated Ca(2+) current by PACAP in rat adrenal chromaffin cells.

It is well established that pituitary adenylate cyclase-activating polypeptide (PACAP) can stimulate catecholamine biosynthesis and secretion in adrenal chromaffin cells. Recent studies from this laboratory demonstrated that PACAP pretreatment inhibits nicotine (NIC)-induced intracellular Ca(2+) transients and catecholamine secretion in porcine adrenal chromaffin cells. Mechanistically, this effect is mediated by protein kinase C (PKC), and based on indirect evidence, is thought to primarily target voltage-gated Ca(2+) channels. The present study used whole-cell patch-clamp analysis to test this possibility more directly in rat chromaffin cells. Consistent with the porcine data, pretreatment with PACAP or with phorbol ester [phorbol myristate acetate (PMA)] significantly suppressed NIC-induced intracellular Ca(2+) transients and catecholamine secretion in rat chromaffin cells. Exposure to PACAP and PMA significantly reduced peak Ca(2+) current in rat cells. The effects of both PACAP and PMA on Ca(2+) current could be blocked by treating cells with the PKC inhibitor staurosporine. Exposure to selective channel blockers demonstrated that rat chromaffin cells contain L-, N- and P/Q-type Ca(2+) channels. PACAP pretreatment significantly reduced Ca(2+) current gated through all three channel subtypes. These data suggest that PACAP can negatively modulate NIC-induced catecholamine secretion in both porcine and rat adrenal chromaffin cells.     

Calelectrin, a Calcium-Dependent Membrane-Binding Protein Associated with Secretory Granules in Torpedo Cholinergic Electromotor Nerve Endings and Rat Adrenal Medulla

Calelectrin, a calcium-dependent membrane-binding protein of subunit molecular weight 32,000 has been isolated from the electric organ of Torpedo, and shown to occur in cholinergic neurones and in bovine adrenal medulla. In this study a monospecific antiserum against the Torpedo protein has been used to study the localization of calelectrin in the rat adrenal gland. The cortex was not stained, whereas in the medulla the cytoplasm of the chromaffin cells was stained in a particulate manner. An identical staining pattern was obtained with an antiserum against the chromaffin granule enzyme dopamine beta-hydroxylase, although the two antisera did not cross-react with the same antigen. The purified protein aggregates bovine chromaffin granule membranes and cholinergic synaptic vesicles and also self aggregates in a calcium-dependent manner. Negative staining results demonstrate that calcium induces a transformation of the purified protein from circular structures 30-80 nm in diameter into a highly aggregated structure. Calelectrin may have a structural or regulatory role in the intracellular organization of secretory cells.     

乙酰胆碱和A23187对离体大鼠肾上腺髓质细胞肾上腺素分泌的作用

通过胆碱能激动剂乙酰胆碱及离子诱导剂A23187（以下简称激动剂）作用于分离的肾上腺髓质细胞,以引起离体细胞的刺激－分泌耦联过程,运用细胞立体形态计量法计算分泌过程中的嗜铬颗粒数目的变化,运用电镜X射线显微分析法测量分泌过程中嗜铬颗粒内钙含量的变化,并运用高 液相色谱分析法测定离体细胞在激动剂作用后的肾上腺素分泌情况,结果发现,分离的肾上腺髓质细胞嗜铬颗粒内钙含量在激动剂作用10min时有明显下降,颗粒数目在激动剂作用过程中呈缓慢下降趋势,而细胞悬液中的肾上腺素含量在激动剂作用20min以后有明显的升高,激动剂作用引起的离体肾上腺髓质的细胞分泌时颗粒内钙含量的下降早于颗粒数目减少或肾上腺素升高,提示颗粒释放的Ca^2 可能是引起细胞分泌的原因之一。     

Uptake of calcium in chromaffin granules of bovine adrenal medulla stimulated in vitro

The calcium content of bovine adrenal medulla perfused in vitro has been shown to increase about 30% in response to extensive acetylcholine stimulation. The calcium accumulated during secretion was mainly associated with the mitochondria and chromaffin granule fractions and to a lesser extent in the microsome fraction. While the calcium taken up by the mitochondria and microsomes was partly or totally removed by treatment with EDTA, the chelating agent had no effect on the granule content of calcium. The uptake of calcium in the mitochondria and microsomes during secretion is consistent with a function of these organelles in regulating the cellular calcium concentration. It is suggested that also the chromaffin granules may act as a “Ca-pump” in the chromaffin cell of the adrenal medulla.     

PAC1hop receptor activation facilitates catecholamine secretion selectively through 2-APB-sensitive Ca2+ channels in PC12 cells

PACAP is a critical regulator of long-term catecholamine secretion from the adrenal medulla in vivo, however the receptor or pathways for Ca²⁺ entry triggering acute and sustained secretion have not been adequately characterized. We have previously cloned the bovine adrenal chromaffin cell PAC1 receptor that contains the molecular determinants required for PACAP-induced Ca²⁺ elevation and is responsible for imparting extracellular Ca²⁺ influx-dependent secretory competence in PC12 cells. Here, we use this cell model to gain mechanistic insights into PAC1hop-dependent Ca²⁺ pathways responsible for catecholamine secretion. PACAP-modulated extracellular Ca²⁺ entry in PC12 cells could be partially blocked with nimodipine, an inhibitor of L-type VGCCs and partially blocked by 2-APB, an inhibitor and modulator of various transient receptor potential (TRP) channels. Despite the co-existence of these two modes of Ca²⁺ entry, sustained catecholamine secretion in PC12 cells was exclusively modulated by 2-APB-sensitive Ca²⁺ channels. While IP3 generation occurred after PACAP exposure, most PACAP-induced Ca²⁺ mobilization involved release from ryanodine-gated cytosolic stores. 2-APB-sensitive Ca²⁺ influx, and subsequent catecholamine secretion was however not functionally related to intracellular Ca²⁺ mobilization and store depletion. The reconstituted PAC1hop-expessing PC12 cell model therefore recapitulates both PACAP-induced Ca²⁺ release from ER stores and extracellular Ca²⁺ entry that restores PACAP-induced secretory competence in neuroendocrine cells. We demonstrate here that although bPAC1hop receptor occupancy induces Ca²⁺ entry through two independent sources, VGCCs and 2-APB-sensitive channels, only the latter contributes importantly to sustained vesicular catecholamine release that is a fundamental characteristic of this neuropeptide system. These results emphasize the importance of establishing functional linkages between Ca²⁺ signaling pathways initiated by pleotrophic signaling molecules such as PACAP, and physiologically important downstream events, such as secretion, triggered by them.