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In C. auratus the adrenal chramaffin tissue is situated around the posterior cardinal veins, in the head kidney. Chromaffin tissue consists of two types of cells containing secretory granules, adrenaline and nor adrenaline cells. The cells produced catecholamine hormones. Adrenaline cell contains electron-lucent granules, whereas nor adrenaline cells possesses electron-dense granules. Cholinergic fibers embedded in the head kidney innervated the chromaffin cell. Two types of secretory structures, synaptic vesicles and secretory granules are found within the presynaptic terminal. Secretory granules discharge their contests, as neuropeptide in non synaptic area of nerve terminal by exocytosis, whereas synaptic vesicles discharge their contents as neurotransmitters at the synaptic thickening (active zone) in the presynaptic terminal by exocytosis.

Initial PACAP-regulated transcriptomes of PACAP-treated cultured chromaffin cells, and the adrenal gland of wild-type versus PACAP-deficient mice, have been assembled using microarray analysis. These were compared to previously acquired PACAP-regulated transcriptome sets from PC12 cells and mouse central nervous system, using the same microarray platform. The Ingenuity Pathways Knowledge Base was then employed to group regulated transcripts into common first and second messenger regulatory clusters. The purpose of our meta-analysis was to identify sets of genes regulated distinctly or in common by the neurotransmitter/neurotrophin PACAP in specific physiological contexts. Results suggest that PACAP participates in both the basal differentiated expression, and the induction upon physiological stimulation, of distinct sets of transcripts in neuronal and endocrine cells. PACAP in both developmental and acute regulatory paradigms acts on target genes also regulated by either TNFalpha or TGFbeta, two first messengers acting on transcription mainly through NFkappaB and Smads, respectively.

The sympathoadrenal (SA) cell lineage is a derivative of the neural crest (NC), which gives rise to sympathetic neurons and neuroendocrine chromaffin cells. Signals that are important for specification of these two types of cells are largely unknown. MASH1 plays an important role for neuronal as well as catecholaminergic differentiation. Mash1 knockout mice display severe deficits in sympathetic ganglia, yet their adrenal medulla has been reported to be largely normal suggesting that MASH1 is essential for neuronal but not for neuroendocrine differentiation. We show now that MASH1 function is necessary for the development of the vast majority of chromaffin cells. Most adrenal medullary cells in Mash1(-/-) mice identified by Phox2b immunoreactivity, lack the catecholaminergic marker tyrosine hydroxylase. Mash1 mutant and wild-type mice have almost identical numbers of Phox2b-positive cells in their adrenal glands at embryonic day (E) 13.5; however, only one-third of the Phox2b-positive adrenal cell population seen in Mash1(+/+) mice is maintained in Mash1(-/-) mice at birth. Similar to Phox2b, cells expressing Phox2a and Hand2 (dHand) clearly outnumber TH-positive cells. Most cells in the adrenal medulla of Mash1(-/-) mice do not contain chromaffin granules, display a very immature, neuroblast-like phenotype, and, unlike wild-type adrenal chromaffin cells, show prolonged expression of neurofilament and Ret comparable with that observed in wild-type sympathetic ganglia. However, few chromaffin cells in Mash1(-/-) mice become PNMT positive and downregulate neurofilament and Ret expression. Together, these findings suggest that the development of chromaffin cells does depend on MASH1 function not only for catecholaminergic differentiation but also for general chromaffin cell differentiation.

1. Gastrointestinal endocrine cells produce biogenic amines which are transported into secretory vesicles by one of two proton-amine exchangers, vesicular monoamine transporters type 1 and 2 (VMAT1 and 2). We report here the presence of VMAT1 in rat gastrin (G) cells and the relevance of VMAT1 function for the modulation of progastrin processing by biogenic and dietary amines. 2. In immunocytochemical studies VMAT1, but not VMAT2, was localized to subpopulations of G cells and enterochromaffin (EC) cells; neither was found in antral D cells. The expression of VMAT1 in antral mucosa was confirmed by Northern blot analysis, which revealed an mRNA band of approximately 3.2 kb, and by Western blot analysis, which revealed a major protein of 55 kDa. 3. In pulse-chase labelling experiments, the conversion of the amidated gastrin G34 to G17 was inhibited by biogenic amine precursors (L-DOPA and 5-hydroxytryptophan). This inhibition was stereospecific and sensitive to reserpine (50 nM), which blocks VMAT1 and VMAT2, but resistant to tetrabenazine, which is a selective inhibitor of VMAT2. 4. Dietary amines such as tyramine and tryptamine also inhibited G34 cleavage. This effect was associated with a loss of the electron-dense core of G cell secretory vesicles. It was not stereospecific or reserpine sensitive, but was correlated with hydrophobicity. 5. Thus rat antral G cells can express VMAT1; transport of biogenic amines into secretory vesicles by VMAT1 is associated with inhibition of G34 cleavage, perhaps by raising intravesicular pH. Dietary amines also modulate cleavage of progastrin-derived peptides, but do so by a VMAT1-independent mechanism; they may act as weak bases that passively permeate secretory vesicle membranes and raise intravesicular pH.

Introduction: The progress made by contemporary neurobiology opens new horizons both for study and for treatment of disorders of the nervous system. At the present time we are in the age of growth factors. These are molecules which affect survival, development and the normal functioning of cell populations., Development: One of the most widely studied growth factors is nervous growth factor (NGF) which is necessary for normal life of various types of neurones, including sensory nerves and nerves derived from the neural crests. Diabetic neuropathy, worldwide a major neurological disorder, is primarily characterized by involvement of the fine fibres for temperature and pain perception and also by a variety of autonomic disorders. The great dependence of sensory and sympathetic nerves on NGF, the quantity of results which show alterations in the levels of neurotrophic factors in diabetic neuropathy (DN), and the encouraging experimental and clinical results of using NGF as a new alternative to treatment with DN, were the basic reasons which led us to do this study., Conclusions: We started by considering neurotrophic factors, especially NGF and its connection with DN disorders and its clinical applications, we made a summary of the main findings in this field to date.

In this study, we investigated the contribution of different subtypes of voltage-dependent Ca2+ channels to changes in cytosolic free Ca2+ ([Ca2+]i) and secretion in noradrenergic and adrenergic bovine chromaffin cells. In single immunocytochemically identified chromaffin cells, [Ca2+]i increased transiently during high K+ depolarization. Furnidipine and BAY K 8644, L-type Ca2+ channel blocker and activator, respectively, affected the [Ca2+]i rise more in noradrenergic than in adrenergic cells. In contrast, the Q-type Ca2+ channel blocker omega-conotoxin MVIIC inhibited the [Ca2+]i rise more in adrenergic cells. omega-Agatoxin IVA (30 nM), which blocks P-type Ca2+ channels, had little effect on the [Ca2+]i signal. The N-type Ca2+ channel blocker omega-conotoxin GVIA similarly inhibited the [Ca2+]i rise in both cell types. The effects of furnidipine, BAY K 8644, and omega-conotoxin MVIIC on K+-evoked norepinephrine and epinephrine release paralleled those effects on [Ca2+]i signals. However, omega-conotoxin GVIA and 30 nM omega-agatoxin IVA did not affect the secretion of either amine. The data suggest that, in the bovine adrenal medulla, the release of epinephrine and norepinephrine are preferentially controlled by Q- and L-type Ca2+ channels, respectively. P- and N-type Ca2+ channels do not seem to control the secretion of either catecholamine.

Background: Although somatostatin is recognized as an inhibitor of neuroendocrine cell secretion, its effect on cell proliferation has not been well defined. Generation of low acid and hypergastrinemia through irreversible H2-receptor blockade (loxtidine) in the African rodent mastomys results in gastric carcinoids (ECLomas) within 4 months. This study was undertaken to evaluate and characterize the precise somatostatin receptor (SSTR) subtype on the mastomys enterochromaffin-like (ECL) cell and to define its role in the regulation of ECL cell secretion and proliferation., Methods: A pure preparation (approximately 90%) of ECL cells was derived by a combination of pronase digestion and density gradient separation. We assessed the effect of somatostatin (10(-15) to 10(-7) mol/L) on gastrin-stimulated ECL cell histamine secretion and DNA synthesis (bromodeoxyuridine uptake). SSTR2 subtype was evaluated by reverse transcription-polymerase chain reaction (RT-PCR) using gene specific primers and mRNA isolated from normal and hypergastrinemia-induced ECLoma. The polymerase chain reaction product was confirmed by Southern analysis, subcloned, and sequenced., Results: Somatostatin inhibited both gastrin-stimulated histamine secretion (IC50, 5 x 10(-13) mol/L) and DNA synthesis (IC50, 10(-10) mol/L). SSTR2 was identified in the mastomys' brain, and both normal and tumor ECL cells and comparison of the brain and ECL cell SSTR2 nucleotide sequences revealed homology of 99%., Conclusions: The SSTR2 is expressed by the mastomys' ECL cell and ECLoma. Receptor activation inhibits both ECL cell secretory and proliferative functions.

Previous morphological and physiological evidence indicates that the adrenal medulla can modulate adrenocortical steroidogenesis, most likely via paracrine or neuronal interactions. To study directly chromaffin-adrenocortical cellular interactions, we previously developed co-cultures of frog (Rana pipiens) adrenal (interrenal) cells. Importantly, chromaffin cells in these co-cultures extend processes that project toward or onto adrenocortical cells, thereby providing the substrate for direct autonomic regulation of adrenocortical function and also mimicking the organization in vivo. To test whether chromaffin cells in our co-cultures affect adrenocortical steroidogenesis, we used veratridine, a sodium ionophore, to depolarize chromaffin cells. Chronic veratridine (50 microM) results in increased corticosterone secretion on days 3 (950%), and 4 (350%). These results indicate that chromaffin cell activation results in the modulation of corticosteroidogenesis.

Many of the molecules involved in biological signaling processes are easily oxidized and have been monitored by electrochemical methods. Temporal response, spatial considerations, and sensitivity of the electrodes must be optimized for the specific biological application. To monitor exocytosis from single cells in culture, constant potential amperometry offers the best temporal resolution, and a low-noise picoammeter improves the detection limits. Smaller electrodes, with 1-micron diameters, provided spatial resolution sufficient to identify the locations of release sites on the surface of single cells. For the study of neurotransmitter release in vivo, larger cylindrical microelectrodes are advantageous because the secreted molecules come from multiple terminals near the electrode, and the greater amounts lead to a larger signal that emerges from the Johnson noise of the current amplifier. With this approach, dopamine release elicited by two electrical stimulus pulses at 10 Hz was detected with fastscan cyclic voltammetry in vivo. Nafion-coated elliptical electrodes have previously been shown to be incapable of detecting such concentration changes without extensive signal averaging. In addition, we demonstrate that high-pass filtering (200 Hz) of cyclic voltammograms recorded at 300 V/s decreases the background current and digitization noise at these microelectrodes, leading to an improved signal. Also, high-pass filtering discriminated against ascorbic acid, DOPAC, and acidic pH changes, three common interferences in vivo.

An increase in extracellular Ca2+ concentration from 0.25 to 10 mM enhanced secretion of norepinephrine and epinephrine induced by a high extracellular K+ concentration (75 mM). The increment in extracellular Ca2+ concentration also increased the observed peak inward Ca2+ current in response to long (10-s) depolarizing pulses from a holding potential of -55 mV to +5 mV, from about -26 to -400 pA. However, the total amount of Ca2+ influx into the cell only increased when the extracellular Ca2+ concentration was raised from 0.25 to 1 mM and then remained constant up to 10 mM extracellular Ca2+. ATP is cosecreted with catecholamines following a depolarizing stimulus. Kinetic studies indicated that ATP secretion had two components with time constants, in the presence of 2.5 mM extracellular Ca2+, of approximately 4 and 41 s, being the fast component of secretion produced by the exocytosis of approximately 220 chromaffin granules. The results suggest that, for a given depolarizing stimulus, the size and rate of release for the fast and slow components of secretion are dependent on extracellular Ca2+ concentration.

Chromaffin cells were isolated from bovine adrenal glands and fractionated into two distinct subpopulations by density gradient centrifugation on Percoll. Cells in the more dense fraction stored epinephrine (E) as their predominant catecholamine (81% of total catecholamines), contained high levels of phenylethanolamine N-methyltransferase (PNMT) activity, and exhibited intense PNMT immunoreactivity. This population of chromaffin cells was termed the E-rich cell population. Cells in the less dense fraction, the norepinephrine (NE)-rich cell population, stored predominantly NE (75% of total catecholamines). Although the NE-rich cells had only 3% as much PNMT activity as did the E-rich cells, 20% of the NE-rich cells were PNMT immunoreactive. This suggested that the PNMT-positive cells in the NE-rich cell cultures contained less PNMT per cell than did E-rich cells and may not be typical adrenergic cells. The regulation of PNMT mRNA levels and PNMT activity in primary cultures of E-rich and NE-rich cells was compared. At the time the cells were isolated, PNMT mRNA levels in NE-rich cells were approximately 20% of those in E-rich cells; within 48 h in culture, PNMT mRNA in both populations declined to almost undetectable levels. Treatment with dexamethasone increased PNMT mRNA levels and PNMT activity in both populations. In E-rich cells, dexamethasone restored PNMT mRNA to the level seen in freshly isolated cells and increased PNMT activity twofold. In NE-rich cells, dexamethasone increased PNMT mRNA to levels twice those found in freshly isolated cells and increased PNMT activity sixfold. Cycloheximide blocked the effects of dexamethasone on PNMT mRNA expression in NE-rich cells but had little effect in E-rich cells. Angiotensin II, forskolin, and phorbol 12,13-dibutyrate elicited large increases in PNMT mRNA levels in E-rich cells but had no effect in NE-rich cells. Our data suggest that PNMT expression is regulated differently in the two chromaffin cell subpopulations.

Vesicular monoamine transport (VMAT) inhibitors, such as reserpine and tetrabenazine, impair vesicular catecholamine storage in chromaffin cells and sympathetic neurons, thereby lowering blood pressure. Here we describe a novel action of VMAT inhibitors-blockade of L-type voltage-gated calcium channels-that may also influence catecholamine release from both PC12 rat pheochromocytoma cells and bovine adrenal chromaffin cells. When given alone, VMAT inhibitors acutely release catecholamines from chromaffin cells in a dose-dependent fashion. However, VMAT inhibitors block catecholamine secretion stimulated by either nicotinic cholinergic agonists or cell membrane depolarization, each of which rely on the opening of L-type channels; the inhibition was more potent after long-term exposure to VMAT inhibitors (IC50 < 100 nmol/L). Reserpine blocked nicotinic-stimulated catecholamine release from neurite-bearing PC12 cells. Reserpine also antagonized catecholamine release triggered by combined membrane depolarization and the dihydropyridine L-type channel agonist Bay K8644, and reserpine blocked cellular uptake of extracellular 45Ca2+ in response to nicotine. Taken together, these results indicate that VMAT inhibitors are also antagonists at L-type voltage-gated calcium channels. Classic L-type channel antagonists (verapamil or nifedipine) also exhibited the reciprocal actions; acutely, they released norepinephrine from chromaffin cells, and chronically, they depleted cellular catecholamine stores, albeit with inferior molar potency to reserpine (IC50 < 1 nmol/L). We conclude that VMAT inhibitors and L-type calcium channel antagonists exert reciprocal inhibitory actions on each other's more classic pharmacological targets. Furthermore, these novel actions are seen at concentrations of these compounds frequently taken to be specific in vitro and likely to occur during antihypertensive treatment in vivo.

The cortical F-actin cytoskeleton represents a negative control for secretion, and it must be locally disassembled to allow chromaffin vesicle exocytosis. Recombinant scinderin (a Ca(2+)-dependent F-actin-severing protein) potentiated Ca(2+)-evoked F-actin disassembly and exocytosis in permeabilized chromaffin cells, an effect blocked by peptides Sc-ABP1 and Sc-ABP2 (with sequences corresponding to two actin-binding sites of scinderin), exogenous gamma-actin, or phosphatidylinositol 4,5-bisphosphate (PIP2). PIP2 effect was blocked by peptide Sc-PIP2BP (with sequence corresponding to a PIP2-binding site of scinderin). Truncated scinderin254-715 (lacking actin-severing domains) did not potentiate exocytosis. Sc-ABP1, Sc-ABP2, and gamma-actin also inhibited exocytosis in the absence of recombinant scinderin, suggesting an inhibition of endogenous scinderin. Results suggest that scinderin-evoked cortical F-actin disassembly is required for secretion and that scinderin is an important component of the exocytotic machinery.

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.

Background & Aims: Peripheral regulation of acid secretion depends mainly on stimulation or inhibition of the three major gastric endocrine cells (enterochromaffin-like, gastrin, and somatostatin). The aim of this paper was to define physiological responses of enterochromaffin-like, gastrin, and somatostatin cells in a mixed endocrine cell population by measuring ligand-selective changes of intracellular calcium ([Ca2+]i) in individual cells., Methods: Endocrine cells were enriched from a rat gastric cell suspension by elutriation, a density-gradient fractionation, and a 48-hour short-term culture. [Ca2+]i responses of individual cells to various ligands such as gastrin/carboxy-terminal cholecystokinin octapeptide and selective cholecystokinin antagonists, carbachol, and gastrin-releasing peptide were monitored using video imaging in a perfusion chamber. Characteristic [Ca2+]i changes distinguished the three cell types, confirmed by immunostaining., Results: All enterochromaffin-like cells respond to cholecystokinin-B receptor stimulation, but only a few respond to carbachol. Gastrin cells respond to both gastrin-releasing peptide and carbachol but not to cholecystokinin-receptor agonists. Somatostatin cells have both stimulatory cholecystokinin-A and cholecystokinin-B receptors and inhibitory muscarinic receptors. All cells have inhibitory somatostatin receptors., Conclusions: Calcium-signaling responses of gastric endocrine cells are distinctive. This allows individual cell types in a mixed population to be characterized and permits an analysis of the hormones and transmitters that act directly on a specific cell type.

KN-62, an inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II), inhibited significantly catecholamine secretion and tyrosine hydroxylase activity stimulated by acetylcholine in cultured bovine adrenal medullary cells. KN-62, however, showed an additional inhibitory effect on acetylcholine-induced 45Ca2+ influx, which is essential for functional responses. Carbachol-stimulated 22Na+ influx, veratridine-induced 22Na+ influx, and 56 mM K(+)-evoked 45Ca2+ influx were also attenuated by KN-62. Inhibitions by KN-62 of these ion influxes were correlated closely with those of catecholamine secretion. KN-04, which is a structural analogue of KN-62 but does not inhibit CaM kinase II activity, elicited inhibitory effects on the three kinds of stimulant-evoked ion influxes with an inhibitory potency similar to KN-62. These results suggest that KN-62 inhibits catecholamine secretion and tyrosine hydroxylase activation due to mainly its ion channel blockade on the plasma membrane rather than the inhibition of CaM kinase II activity in the cells.

In bovine chromaffin cells forskolin, phorbol ester, or high potassium levels induce a rapid increase of c-fos, c-jun, and junB mRNA levels, which precede an induction of proenkephalin gene expression. Preincubation of the cells with cycloheximide inhibited induction of proenkephalin mRNA levels by each of these agents, indicating that newly synthesized transcription factors are involved. Transient transfection of reporter genes showed that the ENKCRE-2 element of the proenkephalin promoter was sufficient for basal and second messenger-induced expression. Gel mobility shift assays revealed that stimulation increased the binding of nuclear proteins to ENKCRE-2 and AP-1 oligonucleotides but not to CRE oligonucleotides. Western analysis showed that the induction of AP-1 binding activity was associated with Fos protein synthesis. Moreover, cotransfection of c-fos, but not of c-jun or CREB, expression plasmids transactivated the expression of the PENKCAT reporter genes. These results suggest that Fos and/or other components of AP-1 transcription factors, rather than CREB or other preexisting proteins, play a specific role in the induction of the proenkephalin gene in bovine chromaffin cells.

The gastric enterochromaffin-like (ECL) cell secretes histamine in response to secretagogues (gastrin, acetylcholine) by calcium signaling-dependent exocytosis of intracellular vacuoles containing the hormone. ECL cells were isolated from rat fundic gastric mucosa by elutriation and density-gradient centrifugation. Currents across the plasma membrane were measured using whole cell patchclamp methods. These cells had a low conductance of 0.5 nS and resting potential of -50 mV. Depolarization activated a K+ current that was blocked by Ba2+. Steady-state current in absence of K+ was due to Cl- because of the magnitude of the reversal potential and the effects of Cl- removal. Stimulation of secretion by gastrin, cholecystokinin octapeptide (CCK-8), and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate activated the Cl- conductance with a time course similar to that of histamine release. Therefore the ECL cell maintains a high resting potential, largely due to K+ currents, and stimulation of secretion activates a Cl- current, perhaps deriving from the membrane of the secretory granule that fuses with the plasma membrane. The depolarization that ensues may activate the K+ current to maintain the membrane potential during exocytosis.

Amperometric detection of exocytosis at single chromaffin cells has shown that the distribution of spike areas, or quantal size, is dependent on the volume and catecholamine concentration of individual secretory vesicles. The present work offers an alternate, simplified model to analyze the current spikes due to single exocytotic events. When the cube root of these spike areas is plotted as a histogram, a Gaussian distribution is obtained for chromaffin cells and also mast, pheochromocytoma, and pancreatic beta-cells. It was found that the relative SD of these distributions is similar to that for the vesicular radii, which also have a Gaussian distribution in all four cell types. In addition, this model was used to evaluate conditions where the quantal size of individual events was altered. When chromaffin cells were maintained in culture for < 6 days, spikes of approximately double the quantal size were obtained on repeated exposure to 60 mM K+. The results suggest a heterogeneous distribution of catecholamine-containing vesicles at later days in culture is responsible for this alteration.

Transmission electron microscopy has been used to morphometrically evaluate exocytosis in bovine adrenal medulla chromaffin cells as the mechanism of catecholamine release. Purified cell suspensions were stimulated with KCl at varying strengths and durations and then conventionally processed for ultrastructural analysis. Quantitation of exocytotic images of dense cored chromaffin granules was a major objective and such images were found in all preparations, attesting to the efficacy of chemical fixation to preserve this event. However, because hundreds of cell profiles had to be screened to find a single granule in the process of release this low frequency precluded any meaningful correlations with estimates of granular involvement based on catecholamine release. Neither KCl molarity nor duration altered this finding nor did these variables significantly affect other parameters linked to exocytotic activity. For example, cell size and numbers of "empty' granules and vesicles remained constant and attempts to label "any' organelle with 30-nm colloidal gold or lanthanum precipitate proved unsuccessful. In short, if exocytosis is responsible for release, it would appear to function without leaving a morphological trace. An alternative hypothesis, therefore, is outlined which better accommodates existing data.

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.

Adrenal chromaffin cells contain at least two subtypes of nicotinic acetylcholine receptors (nAChRs). These studies were designed to identify and characterize the subtype of nAChR mediating adrenal catecholamine release using the monoclonal antibody mAb35, which recognizes the alpha-subunit of muscle nAChRs and cross-reacts with some neuronal nAChRs. Immunocytochemical studies demonstrated that mAb35 interacts with specific sites on cultured chromaffin cells. Pretreatment with mAb35 reduced nAChR-stimulated catecholamine release (IC50 of approximately 10nM). mAb35 had no effects on release stimulated through non-nAChR mechanisms. Unlike agonist-induced nAChR desensitization, the mAb35-induced reduction in nAChR-mediated secretion developed slowly. Although not immediately reversible, nAChR-stimulated release recovered after mAB35 removal. However, unlike recovery from agonist pretreatment, recovery from mAb35 pretreatment was relatively slow and was par tially blocked by vinblastine. Hybridization of adrenal chromaffin RNA with a rat alpha3 cDNA revealed two strong bands and two fainter bands: two higher-molecular-weight bands, 6.9 and 8.5 kb; a strong band of 3.2 kb; and a lower amount of 2.3kb RNA. With recovery of nAChR function after agonist or mAb35 treatment, no significant effects on alpha 3 subunit mRNA levels were seen. In summary, these studies demonstrate the presence of mAb35-nAChRs on adrenal chromaffin cells and provide evidence that these receptors represent the major population that regulates secretory events in adrenal chromaffin cells.

Treatment of cultured bovine adrenal chromaffin cells with 12-O-tetradecanoylphorbol 13-acetate (TPA), an activator of protein kinase C (PKC), decreased [3H]saxitoxin ([3H]STX) binding in a concentration (IC50 = 19 nM)- and time (t1/2 = 4.5 h)-dependent manner. TPA (100 nM for 15 h) lowered the Bmax of [3H]STX binding by 53% without altering the KD value. Phorbol 12,13-dibutyrate (PDBu) also reduced [3H]STX binding, whereas 4 alpha-TPA, an inactive analogue, had no effect. The inhibitory effect of TPA was abolished when H-7 (an inhibitor of PKC), but not H-89 (an inhibitor of cyclic AMP-dependent protein kinase), was included in the culture medium for 1 h before and during TPA treatment. Simultaneous treatment with TPA in combination with either actinomycin D or cycloheximide, an inhibitor of protein synthesis, nullified the effect of TPA. TPA treatment also attenuated veratridine-induced 22Na+ influx but did not alter the affinity of veratridine for Na channels as well as an allosteric potentiation of veratridine-induced 22Na+ influx by brevetoxin. These results suggest that an activation of PKC down-regulates the density of Na channels without altering their pharmacological features; this down-regulation is mediated via the de novo synthesis of an as yet unidentified protein(s), rather than an immediate effect of Na channel phosphorylation.

The time course of the spontaneous current spikes produced by release of the catecholamine contents of individual vesicles was examined in bovine chromaffin cells using carbon filament electrodes. The rate of spontaneous release was enhanced by adding either LaCl3 (0.01-0.5 mM) or BaCl2 (2 mM) to the extracellular solution. A paucity of events of very short duration was evident from the frequency histograms of the rise and the decay times. In the scatterograms of the rise and of the decay times the regression lines are invariably positive (i.e. the longer the duration of the rise times the longer the duration of the decay times). However, the regression lines never go through the origin but intercept the ordinate (the axis of the decay times) at (+/- SD) 16.1 +/- 6.4 ms (n = 11). On the other hand, the regression lines of paired rise and decay times for the time courses of diffusion are both linear and go through the origin. This relationship holds irrespective of whether the diffusion from an instantaneous point source was assumed to occur in an infinite plane or in an infinite volume. Therefore our experimental findings are incompatible with the model(s) assuming that diffusional broadening determines entirely the time course of current spikes. However, they can be explained, although only partially, by the possible slow speed of the electrode. They thus suggest that in chromaffin cells the duration of exocytosis of individual vesicles is much longer than in synapses.

Adrenal chromaffin cells have been utilized for several neural grafting applications, but limitations in allogeneic donor availability and dangers inherent in autografting limit the widespread use of this approach clinically. While xenogeneic donors offer promise as a source for cell transplantation in the central nervous system (CNS), immunologic responses to cellular components of the adrenal medulla have not been well characterized. To further study the host T cell xenogeneic response to chromaffin and passenger cells of the adrenal medulla, an in vitro lymphocyte proliferation assay was used. Lymphocyte proliferation was determined by mixing rat lymphocytes with potential stimulator cell subpopulations of the bovine adrenal medulla: isolated chromaffin cells, isolated endothelial cells, or passenger nonchromaffin cells, which include a mixture of fibroblasts, smooth muscle cells, and endothelial cells. As a positive control, bovine aortic endothelial cells were also used. 3[H]-thymidine incorporation, corresponding to lymphocyte proliferation, was measured. Results indicated that isolated bovine chromaffin cells produce only a mild, statistically insignificant stimulation of rat lymphocytes. In contrast, there was a significant response to passenger nonchromaffin cells of the adrenal medulla, especially endothelial cells. The inclusion of low levels of cyclosporin A in the cultures completely eliminated the mild proliferative response to isolated bovine chromaffin cells, while near toxic levels were necessary to abrogate the response to endothelial cells. Immunocytochemical analysis revealed that routine chromaffin cell isolation procedures result in the inclusion of a small percentage of endothelial cells, which may be responsible for the slight lymphocyte stimulation. The results of this study indicate that isolated chromaffin cells possess low immunogenicity, and suggest that passenger cells in the adrenal medulla, particularly endothelial cells, may be primarily responsible for progressive rejection in CNS grafts. Thus, removal of passenger nonchromaffin cells from xenogeneic donor tissues prior to transplantation may produce a more tolerated graft in rodent models of neural transplantation.

In this report we investigate the isoforms of protein kinase C (PKC) present in cultured adrenal chromaffin cells with respect to their modulation by treatment with phorbol ester and their possible differential involvement in the regulation of responses to histamine and bradykinin. The presence of individual isoforms of PKC was investigated by using eight isoform specific antisera, as a result of which PKC-alpha, epsilon, and zeta were identified. To characterize down-regulation of these enzymes, cells were incubated for 6-48 h with 1 microM phorbol myristate acetate (PMA). PKC-epsilon down-regulated more rapidly than PKC-alpha. At 12 h, PMA pretreatment, for example, PKC-epsilon was maximally down-regulated (23 +/- 4% of controls), whereas PKC-alpha was unchanged. PKC-alpha showed partial down-regulation by 24 h of PMA pretreatment. PKC-zeta did not down-regulate at any of the times tested. Translocation from cytosol to membrane in response to PMA was also more rapid for PKC-epsilon than for PKC-alpha. The accumulation of total 3H-inositol (poly) phosphates in response to bradykinin or histamine was essentially abolished by prior treatment with 10-min PMA treatment (1 microM). However, with 12-h exposure to PMA, the bradykinin response was restored to the level seen with no prior PMA exposure. The histamine response showed no recovery by 12 h of PMA, but showed partial recovery by 24 h of PMA pretreatment. These observations showed that the restoration of the response to bradykinin corresponds to the loss of PKC-epsilon, whereas the restoration of the histamine response corresponds to the loss of PKC-alpha. This picture was confirmed with further studies on cytosolic Ca2+. The results show that chromaffin cells exhibit an unusual pattern of down-regulation of PKC isoforms on prolonged exposure to PMA, and that there is a differential effect of exposure to PMA on the histamine and bradykinin responses, suggesting that different PLC-linked receptors in chromafin cells are differentially regulated by PKC isoforms.

Background: The adrenal medulla is a typical paraganglion, having the same origin as the sympathetic ganglia, and contains at least two types of parenchymal cells: chromaffin cells and supporting cells. We previously reported that the extent of cellular association of chromaffin cells with supporting cells was remarkably higher in noradrenaline (NA)-than in adrenaline (A)-cell regions in the adrenal medullae of the rat and pig., Methods: Cryostat sections of adrenal medullae of nine mammalian species fixed with Zamboni fluid for 24 h were immunostained by ABC methods using antisera to S-100 protein and PNMT., Results: The distribution patterns of A and NA cells in the adrenal medullae were classified into four types. In the chipmunk and rabbit, adrenomedullary chromaffin cells consisted of A cells. S-100-immunoreactive cells were present more frequently in NA- than in A-cell regions in seven species (rat, golden hamster, cat, dog, pig, ox, and horse). These cells sent out cytoplasmic processes and formed a network by immunoreactive elements among NA cells. The cell-association patterns of S-100-positive cells with NA cell were classified into three types. In A-cell regions, only a few S-100-positive cells were seen in most of the species, although the frequency of S-100-labeled cells were exceptionally high in the horse., Conclusions: The close association of supporting cells with NA cells was commonly found in the adrenal medulla in many mammalian species, irrespective of the proportions and distribution patterns of A cells and NA cells. On the other hand, species differences existed in details of the cellular association between supporting cells and NA cells.

The adrenal gland of mammals contains high concentrations of peripheral-type benzodiazepine receptors (PBR) and diazepam-binding inhibitor (DBI), a polypeptide which acts as an endogenous ligand for PBR. The aim of the present study was to investigate the localization of DBI and PBR in the adrenal gland of the frog Rana ridibunda. Reverse transcription followed by polymerase chain reaction with specific primers for the frog DBI cDNA showed the presence of DBI mRNA in frog adrenal gland extracts. The cellular distribution of DBI and PBR was investigated using an antiserum against the octadecaneuropeptide DBI [33-50] (ODN) and antibodies against the 18-kDa isoquinoline binding protein subunit of PBR (IBP), respectively. ODN-like immunoreactivity was found in chromaffin cells and in Stilling s cells, but not in adrenocortical cells. IBP-like immunoreactivity was observed in chromaffin cells, in Stilling s cells and in a small proportion (11%) of steroid-secreting cells. The ODN- and IBP-immunoreactive materials were homogeneously distributed in the cytoplasm of chromaffin cells and concentrated at the periphery of large cytoplasmic vesicles in Stilling s cells. The proportion of ODN-positive Stilling s cells showed marked circannual variations with a maximum in July. Similarly, the proportion of IBP-positive Stilling s cells was 17 times higher in July than in December. These results indicate that, in the frog adrenal gland, DBI-related peptides and PBR are simultaneously expressed in chromaffin cells and Stilling s cells, suggesting that endogenous ligands for PBR may play a physiological role in the control of adrenal cell activity.

Nicotine-induced catecholamine secretion in bovine adrenomedullary chromaffin cells is accompanied by rapid tyrosine phosphorylation of multiple cellular proteins, most notably the mitogen-activated protein kinases (MAPKs). The requirement for activation of tyrosine kinases and MAPKs in chromaffin cell exocytosis was investigated using a panel of tyrosine kinase inhibitors. Genistein and tyrphostin 23, two compounds that inhibit tyrosine kinases by distinct mechanisms, were found to inhibit secretion by > 90% in cells stimulated by nicotine, 55 mM KCl, or the Ca2+ ionophore A23187. Inhibition of secretion induced by all three secretagogues correlated with a block in both protein tyrosine phosphorylation and activation of the MAPKs and their activators (MEKs) in situ. However, neither genistein nor tyrphostin 23 inhibited the activities of the MAPKs or MEKs in vitro. These results indicate that the target(s) of inhibition lie downstream of Ca2+ influx and upstream of MEK activation. This Ca(2+)-activated tyrosine kinase activity could not be accounted for entirely by c-Src or Fyn (two nonreceptor tyrosine kinases that are expressed abundantly in chromaffin cells), because their in vitro kinase activities were not inhibited by tyrphostin 23 and only partially inhibited by genistein. These results demonstrate that an unidentified Ca(2+)-activated tyrosine kinase(s) is required for MAPK activation and exocytosis in chromaffin cells and suggest that MAPK participates in the regulation of secretion.

Pituitary adenylate cyclase-activating polypeptide (PACAP) has been reported to increase intracellular Ca2+ concentrations ([Ca2+]i) and catecholamine release in adrenal chromaffin cells. We measured [Ca2+]i with fura-2 and recorded ion currents and membrane potentials with the whole cell configuration of the patch-clamp technique to elucidate the mechanism of PACAP-induced [Ca2+]i increase in bovine adrenal chromaffin cells. PACAP caused [Ca2+]i to increase due to Ca2+ release and Ca2+ influx, and this was accompanied by membrane depolarization and inward currents. The Ca2+ release was suppressed by ryanodine, an inhibitor of caffeine-sensitive Ca2+ stores, but was unaffected by cinnarizine, an inhibitor of inositol trisphosphate-induced Ca2+ release. Ca2+ influx and inward currents were both inhibited by replacement of extracellular Na+, and Ca2+ influx was inhibited by nicardipine, an L-type Ca2+ channel blocker, or by staurosporine, a protein kinase C (PKC) inhibitor, but was unaffected by a combination of omega- conotoxin-GVIA, omega-agatoxin-IVA, and omega-conotoxin- MVIIC, blockers of N-, P-, and Q-type Ca2+ channels. Moreover, 1-oleoyl-2-acetyl-sn-glycerol, a PKC activator, induced inward currents and Ca2+ influx. These results indicate that PACAP causes both Ca2+ release, mainly from caffeine-sensitive Ca2+ stores, and Ca2+ influx via L-type Ca2+ channels activated by membrane depolarization that depends on PKC-mediated Na+ influx.

Single-vesicle release of catecholamines from chromaffin cells can be detected in real time as current spikes by the electrochemical method of amperometry. About 70% of spikes are preceded by a small "foot," the trickle of transmitter out of the early fusion pore. In addition, 20-50% of foot signals exhibit rapid fluctuations that we interpret as flickering of the fusion pore. There are also "stand-alone" foot signals, which may reflect transient fusions, in which the vesicles do not collapse completely into the plasma membrane. The number and frequency of the foot flickering are affected by intracellular Ca2+ concentration.

LR White and Unicryl are members of the same family of acrylic embedding resins and are very suitable for "on grid" postembedding immunogold labeling. We studied the ultrastructure of LR White- and Unicryl-embedded cultured chromaffin cells and the immunolocalization of three chromaffin cell proteins, the enzymes dopamine-beta-hydroxylase (DbetaH) and tyrosine hydroxylase (TH), and the membrane fusion and Ca2+ channel protein synexin (annexin VII). We report here that Unicryl is preferable to LR White as an embedding medium for electron microscopy when osmium tetroxide fixation is omitted. The basis for this distinction is better ultrastructural preservation and improved immunodetection efficiency.

Dehydroepiandrosterone sulfate (DHEAS) dose-dependently inhibited [3H]norepinephrine (NE) secretion and the corresponding [Ca2+]i rise induced by the nicotinic receptor agonist 1,1-dimethyl-4-phenylpoperazimium (DMPP) in bovine chromaffin cells. DHEAS at 10 microM, the physiological concentration in human serum, significantly inhibited both the release of [3H]NE and the rise of [Ca2+]i induced by DMPP in chromaffin cells. DHEAS also inhibited the [3H]NE release induced by the Na+ channel activator veratridine. However, DHEAS did not affect either the [3H]NE release, or the corresponding [Ca2+]i rise induced by high K+. Moreover, DHEAS suppressed the [Na+]i rise induced by either DMPP or high K+ as monitored by the fluorescence 340/380 ratio of SBFI loaded chromaffin cells. Our results suggest that the inhibitory effects of DHEAS on secretion mainly occur at nicotinic receptors as well as at the voltage-dependent Na+ channels.

A potential role of the intracellular Ca2+ stores in modulating catecholamine release has been investigated in bovine chromaffin cells maintained in tissue culture. Pharmacological depletion of the stores with a combination of caffeine, histamine and thapsigargin in Ca2+-free media resulted in a significantly greater release of catecholamines on re-exposure to Ca2+-containing media compared with that from non-store depleted cells. The increase in catecholamine release was prevented by intracellular BAPTA indicating that the increase was caused by a rise in Ca2+. Measurement of intracellular free Ca2+ concentration with the fluorescent indicator, fura-2, over the same time-course as the catecholamine release experiments showed that upon restoration of external Ca2+ there was an immediate, substantial and maintained increase in cytosolic Ca2+. It is most probable that the increase in catecholamine release was a consequence of an increase in Ca2+ influx triggered by prior depletion of the internal Ca2+ stores. However, the data suggest that capacitative Ca2+ entry is poorly linked to catecholamine release; although Ca2+ entry on restoration of external Ca2+ was immediate and substantial, the increase in catecholamine release, although quantitatively significant, was slowly realised.

Transmitter release from chromaffin cells differs from that in synapses in that it persists for a longer time after Ca2+ entry has stopped. This prolonged secretion is not due to a delay between vesicle fusion and transmitter release, nor to slow detection of released substance: step increases in capacitance due to single vesicle fusion precede the release detected by amperometry by only a few milliseconds. The persistence of secretion after a depolarization is reduced by addition of mobile calcium buffer. This suggests that most of the delay is due to diffusion of Ca2+ between channels and release sites, implying that Ca2+ channels and secretory vesicles are not colocalized in chromaffin cells, in contrast to presynaptic active zones.

1. Otilonium, a clinically useful spasmolytic, behaves as a potent blocker of neuronal nicotinic acetylcholine receptors (AChR) as well as a mild wide-spectrum Ca2+ channel blocker in bovine adrenal chromaffin cells. 2. 45Ca2+ uptake into chromaffin cells stimulated with high K+ (70 mM, 1 min) was blocked by otilonium with an IC50 of 7.6 microM. The drug inhibited the 45Ca2+ uptake stimulated by the nicotinic AChR agonist, dimethylphenylpiperazinium (DMPP) with a 79 fold higher potency (IC50 = 0.096 microM). 3. Whole-cell Ba2+ currents (IBa) through Ca2+ channels of voltage-clamped chromaffin cells were blocked by otilonium with an IC50 of 6.4 microM, very close to that of K(+)-evoked 45Ca2+ uptake. Blockade developed in 10-20 s, almost as a single step and was rapidly and almost fully reversible. 4. Whole-cell nicotinic AChR-mediated currents (250 ms pulses of 100 microM DMPP) applied at 30 s intervals were blocked by otilonium in a concentration-dependent manner, showing an IC50 of 0.36 microM. Blockade was induced in a step-wise manner. Wash out of otilonium allowed a slow recovery of the current, also in discrete steps. 5. In experiments with recordings in the same cells of whole-cell IDMPP, Na+ currents (INa) and Ca2+ currents (ICa), 1 microM otilonium blocked 87% IDMPP, 7% INa and 13% ICa. 6. Otilonium inhibited the K(+)-evoked catecholamine secretory response of superfused bovine chromaffin cells with an IC50 of 10 microM, very close to the IC50 for blockade of K(+)-induced 45Ca2+ uptake and IBa. 7. Otilonium inhibited the secretory responses induced by 10 s pulses of 50 microM DMPP with an IC50 of 7.4 nM. Hexamethonium blocked the DMPP-evoked responses with an IC50 of 29.8 microM, 4,000 fold higher than that of otilonium. 8. In conclusion, otilonium is a potent blocker of nicotinic AChR-mediated responses. The drugs also blocked various subtypes of neuronal voltage-dependent Ca2+ channels at a considerably lower potency. Na+ channels were unaffected by otilonium. This extraordinary potency of otilonium in blocking nicotinic AChR, unrecognised until now, might account in part for its well known spasmolytic effects.

Cytosolic Ca2+ oscillations are physiologically important in a range of excitable and non-excitable cells. The combined techniques of whole-cell patch clamp and photometric measurement of cytosolic Ca2+ has enabled us to identify the components of Ca2+ spiking in rat chromaffin cells. We show that Ca2+ oscillations continue at a fixed membrane potential and that infusion of the InsP3 receptor antagonist, heparin, substantially blocked the cytosolic Ca2+ spikes. However, even in the presence of heparin we observed spikes of membrane potential depolarization due to the repetitive activation of a transient inward cation current. We conclude that Ca2+ oscillations are dependent on Ca2+ release from heparin sensitive Ca2+ stores and possibly on Ca2+ entry associated with the repetitive activation of a transient cation current. The depolarizing action of the cation current would, in turn, recruit voltage-sensitive Ca2+ channels and further Ca2+ entry would augment the cytosolic Ca2+ spikes. Our results demonstrate that Ca2+ oscillations in rat chromaffin cells are due to a complex interplay of Ca2+ entry and Ca2+ release.

The effect of calmodulin on exocytosis in bovine adrenal medullary chromaffin cells was examined by the use of patch-clamp capacitance recording. Calmodulin was dialysed into cells via the patch-pipette and cells stimulated by depolarisation. Following a test stimulation, cells were dialysed with a control or with a calmodulin containing buffer for 10 mins and then were stimulated at 2 min intervals thereafter. The inclusion of calmodulin in the pipette did not increase Ca2+ currents which instead decreased during dialysis. The presence of calmodulin, however, resulted in a 2-fold increase in the initial rate of exocytosis during the 10 min depolarisation step. These results demonstrate the utility of the patch-clamp capacitance technique for the examination of the effect of soluble proteins on exocytosis and in conjunction with previous work on permeabilised chromaffin cells suggest that calmodulin regulates late steps in Ca2+-dependent exocytosis.

The effect of adrenomedullin, a hypotensive peptide, on Ca2+ efflux from cultured bovine adrenal chromaffin cells was examined. Adrenomedullin stimulated the efflux of 45Ca2+ from the cells in a concentration-dependent manner (10(-7)M - 3x10(-6)M). Adrenomedullin did not increase the intracellular free Ca2+ ([Ca2+]i) level and catecholamine secretion. The adrenomedullin-stimulated 45Ca2+ efflux was not inhibited by incubation with Ca2+-free medium, but was inhibited by incubation with Na+-free medium. These results indicate that adrenomedullin stimulates extracellular Na+-dependent 45Ca2+ efflux from cultured bovine adrenal chromaffin cells, probably through its stimulatory effect on membrane Na+/Ca2+ exchange.

The effects of extracellular adenosine 5'-triphosphate (ATP) on voltage-dependent Ca2+ currents were examined using the whole-cell voltage-clamp technique in guinea-pig isolated adrenal chromaffin cells. ATP (500 microM) reversibly suppressed Ca2+ currents in the presence of 5mM Ca2+ in the extracellular solution. The inhibitory effect of ATP on Ca2+ currents tended to increase with increases in the peak amplitude of ATP-evoked current when the intracellular solution contained 0.1 or 1 mM ethylenebis(oxonitrilo)tetraacetate(EGTA). Using the intracellular solution containing 10mM EGTA, on the other hand, the inhibitory effect did not change regardless of the amplitude of current responses to ATP. In the presence of 10 mM Ba2+, ATP (100 microM) reduced Ba2+ currents in a manner similar to Ca2+ currents. This reduction was decreased by dialysis of cells with the internal solution containing guanosine 5'-O-(2-thiodiphosphate) (GDP [beta-S]; 1 mM) or guanosine 5'-O-(3-thiotriphosphate) (GTP [gamma-S]; 100 microM). A depolarizing prepulse to + 100 mV partly relieved ATP-induced reduction of Ba2+ currents. ADP, AMP and adenosine also reduced Ba2+ currents and the effect of adenosine was the most potent. Adenosine (0.5 and 1 mM) significantly inhibited adrenaline secretion induced by nicotine (50 microM). These results suggest that Ca2+ entry through ATP-activated non-selective cation channels results in the inactivation of voltage-dependent Ca2+ channels. In addition, ATP seems to modulate Ca2+ channels via the pathway related to G-protein. Adenine nucleotides and adenosine may play a role in controlling secretory activity in guinea-pig adrenal chromaffin cells.

The purpose of this study was to determine whether the loss of protein kinase C (PKC) from adrenal chromaffin cells affected the enhancement of high K(+)- and forskolin-stimulated tyrosine hydroxylase (tyrosine 3-monooxygenase, EC 1.14.16.2) activity observed in cells treated with insulin-like growth factor-I (IGF-I). Forskolin-stimulated tyrosine hydroxylase activation was not affected by down-regulation of PKC. High K(+)-stimulated tyrosine hydroxylase activity decreased substantially after treating the cells for approximately 18 h with active, but not inactive, phorbol ester (300 nM). After down-regulation of PKC, high K(+)-stimulated tyrosine hydroxylase activity in cells cultured with IGF-I decreased by 61 +/- 5% (n = 14) compared to 36 +/- 8% (n = 14) in cells cultured without IGF-I. These data suggest that PKC is required for the enhancement of high K(+)-stimulated tyrosine hydroxylase activity observed with IGF-I treatment.

The effects of several physiological agonists on the cytosolic Ca2+ concentration ([Ca2+]i) of immunnocytochemically identified single adrenergic and noradrenergic bovine chromaffin cells were compared. No differences were observed in the responses to stimulation by high-K+ solutions with or without BAY K 8644, suggesting that the density and properties of voltage-dependent Ca2+ channels were similar in both cell types. The increase of [Ca2+]i induced by acetylcholine was greater in adrenergic cells, and this was due to differences in the response mediated through nicotinic receptors. The responses to bradykinin and to ATP were slightly greater in noradrenergic cells. Only a small fraction of the cells (18-28%) was responsive to ATP. The responses to angiotensin II and to histamine were much greater in adrenergic than in noradrenergic cells. Histamine was almost a selective stimulator of adrenergic cells. These differences suggest differential distribution of functional membrane receptors in both cell types and may be relevant to understanding the differential contribution of epinephrine- and norepinephrine-secreting cells during stressful conflicts in physiological or pathophysiological situations.

Fast superfusion of electroporated bovine adrenal chromaffin cells with a K+ glutamate-based solution containing 50 nM free Ca2+ and 2 mM adenosine 5'-triphosphate, dipotassium salt (K2ATP), produced a steady-state low catecholamine secretion, measured on-line with an electrochemical detector (about 20 nA). Rapid switching to electroporation solutions containing increasing Ca2+ concentrations ([Ca2+]) produced a rapid increase in the rate and peak secretion, followed by a decline. At intermediate [Ca2+] (3-100 microM), a fast peak and a slow secretory plateau were distinguished. The fast secretory peak identifies a readily releasable catecholamine pool consisting of about 200-400 vesicles per cell. Pretreatment of cells with tyramine (10 microM for 4 min before electroporation) supressed the initial fast secretory peak, leaving intact the slower phase of secretion. With [Ca2+] in the range of 0.1-3 microM, the activation rate of secretion increased from 2.3 to 35.3 nA.s-1, reached a plateau between 3-30 microM and rose again from 100 to 1000 microM [Ca2+] to a maximum of 91.9 nA.s-1. In contrast, total secretion first increased (0.1-1 microM Ca2+), then plateaud (1-100 microM Ca2+) and subsequently decreased (100-1000 microM Ca2+). At 30 and 1000 microM extracellular [Ca2+] or [Ca2+]o, the activation rates of secretion from intact cells depolarised with 70 mM K+ were close to those obtained in electroporated cells. However, secretion peaks were much lower in intact (93 nA at 30 microM Ca2+) than in electroporated cells (385 nA). On the other hand, inactivation of secretion was much faster in intact than in electroporated cells; as a consequence, total secretion in a 5-min period was considerably smaller in intact (10.6 microA.s at 1000 microM Ca2+) than in electroporated cells (42.4 microA.s at 1 microM Ca2+). Separation of the time-courses of changes in intracellular [Ca2+] or [Ca2+]i and secretion in intact chromaffin cells depolarised with 70 mM K+ was demonstrated at different [Ca2+]o. The increase in the rate of catecholamine release was substantially higher than the increase of the average [Ca2+]i. In contrast, the decline of secretion was faster than the decline of the peak [Ca2+]i. The results are compatible with the idea that the peak and the amount of catecholamine released from depolarised intact cells is determined essentially by plasmalemmal factors, rather than by vesicle supply from reserve pools. These plasmalemmal factors limit the supply of Ca2+ by the rates of opening and closing of voltage-dependent Ca2+ channels of the L- and Q-subtypes, which control the local [Ca2+]i near to exocytotic sites.

We have studied osmotically induced catecholamine secretion from bovine adrenal chromaffin cells by combining patch-clamp measurements, electrochemical detection of secretion, and Fura-2 measurements of intracellular free calcium concentration ([Ca2+]i). We find that osmotically induced catecholamine release is exocytotic and calcium dependent. Furthermore, we demonstrate that cell swelling is coupled to such secretion via a volume-activated current, carrying predominantly chloride, which causes a plateau depolarization of the cell membrane potential and thus promotes voltage-activated calcium influx. Therefore, cell volume changes may modulate the secretory activity.

We have investigated the regulation of the morphological phenotype of chromaffin cells cultured from 6-day-old rat adrenal glands. We show that pituitary adenylate cyclase activating polypeptide (PACAP), which is present in and released from nerves innervating chromaffin cells, rapidly induces neuritic growth, affecting 25% of tyrosine hydroxylase-positive chromaffin cells after 3 days at an optimal concentration of about 20 nM. PACAP does not synergistically act with other factors known to promote neurite growth, including nerve growth factor (NGF), basic fibroblast growth factor (bFGF, FGF-2), and ciliary neurotrophic factor (CNTF). The neurite promoting effect of PACAP and FGF-2 is entirely overridden by dexamethasone (2 x 10(-8) M) suggesting that, despite the presence of these promoting factors in the adrenal medulla, glucocorticoids from the adrenal cortex are probably sufficient to prevent the development of neuronal traits in adrenal chromaffin cells.

ATP, an established neurotransmitter, causes elevation of cytosolic Ca2+ and catecholamine secretion when applied to chromaffin cells in the intact adrenal gland. The ATP-induced rise in Ca2+ is due both to release from internal stores and to entry across the plasma membrane. The latter source of Ca2+ causes secretion; the primary role of Ca2+ released from internal stores remains undetermined. In this article, we have studied the nucleotide specificity for activating the two types of Ca2+ increases. The agonist potency order for the increase in fluorescence from fura-2-loaded chromaffin cells due to release of Ca2+ from internal stores is ATP = UTP > ADP > 2-methylthio-ATP, alpha, beta-methylene ATP, identifying the receptor as a P2u purinoceptor. The potency order for secretion is 2-methylthio-ATP > ATP > alpha, beta-methylene ATP, ADP, UTP, placing the receptor in the P2Y subtype. Thus, two distinct receptors are responsible for Ca2+ release and secretion. Agonists were more effective in the absence of extracellular Mg2+, suggesting that ATP uncomplexed with divalent cations binds preferentially to both receptors. The low response of both receptors to ADP distinguishes them from the ATP receptor on these cells that inhibits voltage-dependent Ca2+ current and secretion.

The adrenal medullary chromaffin cell is a commonly used model for the adrenergic neuron. Although much work has been done to study the transport system in the adrenal chromaffin vesicles, relatively little is known about cellular transport, especially with regard to structural features of phenethylamines required for intracellular accumulation. We have now investigated the structural requirements of phenethylamine-related compounds for their accumulation into cultured adrenal chromaffin cells. We find that two types of cellular uptake, previously described only for dopamine, norepinephrine, and epinephrine, are also present for [3H]tyramine. Although two types of accumulation occur, tyramine accumulation occurs mainly via a cocaine-insensitive process, whereas dopamine accumulation occurs predominantly via a cocaine-sensitive process. The accumulation of [14C]-phenethylamine and p-methoxyphenethylamine is not affected by cocaine, suggesting that a ring hydroxyl substituent is necessary for cocaine-sensitive accumulation. The compounds p-hydroxyphenylpropylamine and p-hydroxyphenyl-2-aminoethyl sulfide accumulate in the cell only via a cocaine-insensitive process, indicating that lengthening of the aminoalkyl side chain prevents cocaine-sensitive accumulation. We have performed conformational analyses of this series of compounds to determine whether the conformation of these compounds can be related to the kinetic data. For dopamine, tyramine, phenethylamine, and p-methoxyphenethylamine, two groups of energy-minimized conformers were found. We find that there is an approximately linear relationship between the Km values for these phenethylamines and the differences in minimized energies between the low- and highest energy conformer groups of each compound. A similar correlation was found for p-hydroxyphenyl-2-aminoethyl sulfide. These results are consistent with the hypothesis that these compounds undergo a conformational change from the low-energy conformer to the highest energy conformer before their cocaine-insensitive accumulation.

The effects of the nematocyst venom of the sea anemone Bunodosoma cangicum on depolarization-activated currents were studies in opener crayfish muscle fibers and in cultured bovine chromaffin cells. The venom selectively and reversibly blocked the Ca(2+)-dependent K+ current (IK(Ca)) present in crayfish muscle in a dose-dependent manner without affecting voltage-gated Ca2+ or K+ currents. Furthermore, the venom also reduced IK(Ca) in chromaffin cells, without modifying voltage-gated Na+, Ca2+, or K+ currents. Synaptic transmission in crayfish muscle was also affected by the venom. Repetitive excitatory and inhibitory postsynaptic currents (each associated with a presynaptic action potential) were evoked by each nerve stimulus, suggesting that presynaptic IK(Ca) may control the electrical activity of excitatory and inhibitory presynaptic fibers. We conclude that B. cangicum venom includes a toxin that selectively and reversibly blocks Ca(2+)-dependent K+ currents in crayfish muscle and in bovine chromaffin cells, and modifies excitatory and inhibitory synaptic transmission, probably abolishing a similar conductance at the presynaptic fibers.

The object of these experiments was to investigate whether noradrenaline is the signal neurotransmitter between the sympathetic nervous system and rat thymocytes. Using immunocytochemistry, evidence was obtained that the rat thymus (thymic capsule, subcapsular region and connective tissue septa) is innervated by noradrenergic varicose axons terminals (tyrosine hydroxylase- and dopamine-beta-hydroxylase-immunostained nerve fibres). This innervation is mainly associated with the vasculature and separately from vessels along the thymic tissue septa it branches into the thymic parenchyma. Using electron microscopy, classical synapses between thymocytes and neuronal elements were not observed. The neurochemical study revealed that these nerve terminals are able to take up, store and release noradrenaline upon axonal stimulation in a [Ca2+]o-dependent manner. The release was tetrodotoxin (1 microM)-sensitive, and reserpine pretreatment prevented axonal stimulation to release noradrenaline, indicating vesicular origin of noradrenaline. In addition, it was found that the release of noradrenaline was subjected to negative feedback modulation via presynaptic alpha 2-adrenoreceptors. Using a patch-clamp technique, electrophysiological evidence was obtained showing that noradrenaline inhibits in a concentration-dependent manner outward voltage-dependent potassium (k+) current recorded from isolated thymocytes. Since noradrenergic varicose axon terminals enter the parenchyma thymocytes and the boutons are not in close apposition to their target cells, noradrenaline released from these terminals diffuses away from release site to reach its targets, thymocytes, and to exert its inhibitory effect on voltage-dependent K+ -current. Since K+ channels are believed to be involved in T cell proliferation and differentiation, the modulation of K+ channel gating by noradrenaline released in response to axonal activity suggests that signals from blood-born or locally released hormones and cytokines. In this respect, noradrenaline released from non-synaptic neuronal varicosities and exerting its effect within the radius of diffusion may serve as a chemical link between the sympathetic nervous system and thymocytes and may have physiological and pathological importance in the thymus during stress and inflammatory/immune responses.

The effect of ATP on catecholamine secretion and intracellular Ca2+ concentration ([Ca2+]i) was examined using perfused adrenal glands and dispersed chromaffin cells of the guinea-pig. The application of ATP (2 to 10 mM) for 2 min caused a dose-dependent increase in catecholamine secretion from perfused adrenal glands. ADP, but neither AMP nor adenosine, was also effective in increasing catecholamine secretion, though its potency was much less than that of ATP. 3. The ATP-induced secretory response was observed even under Na(+)-deficient conditions, but was reversibly abolished by removal of extracellular Ca2+. 4. In dispersed chromaffin cells, ATP (0.5 mM) caused increases in catecholamine secretion and [Ca2+]i, both of which were abolished after the removal of extracellular Ca2+. 5. These results indicate that ATP released from adrenal chromaffin cells together with catecholamine may exert a positive feedback influence on the following exocytotic catecholamine secretion. This effect may be accomplished by increasing the entry of extracellular Ca2+, but not by mobilizing intracellular Ca2+ stores in adrenal chromaffin cells of the guinea-pig.