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Background: Neurons in sympathetic ganglia and neuroendocrine cells in the adrenal medulla share not only their embryonic origin from sympathoadrenal precursors in the neural crest but also a range of functional features. These include the capacity for noradrenaline biosynthesis, vesicular storage and regulated release. Yet the regulation of neuronal properties in early neuroendocrine differentiation is a matter of debate and the developmental expression of the vesicle fusion machinery, which includes components found in both neurons and neuroendocrine cells, is not resolved., Results: Analysis of synaptic protein and pan-neuronal marker mRNA expression during mouse development uncovers profound differences between sympathetic neurons and adrenal chromaffin cells, which result in qualitatively similar but quantitatively divergent transcript profiles. In sympathetic neurons embryonic upregulation of synaptic protein mRNA follows early and persistent induction of pan-neuronal marker transcripts. In adrenal chromaffin cells pan-neuronal marker expression occurs only transiently and synaptic protein messages remain at distinctly low levels throughout embryogenesis. Embryonic induction of synaptotagmin I (Syt1) in sympathetic ganglia and postnatal upregulation of synaptotagmin VII (Syt7) in adrenal medulla results in a cell type-specific difference in isoform prevalence. Dicer 1 inactivation in catecholaminergic cells reduces high neuronal synaptic protein mRNA levels but not their neuroendocrine low level expression. Pan-neuronal marker mRNAs are induced in chromaffin cells to yield a more neuron-like transcript pattern, while ultrastructure is not altered., Conclusions: Our study demonstrates that remarkably different gene regulatory programs govern the expression of synaptic proteins in the neuronal and neuroendocrine branch of the sympathoadrenal system. They result in overlapping but quantitatively divergent transcript profiles. Dicer 1-dependent regulation is required to establish high neuronal mRNA levels for synaptic proteins and to maintain repression of neurofilament messages in neuroendocrine cells.

Background: Maturation of enterocytes along the small intestinal crypt-villus axis is associated with significant changes in gene expression profiles. fls485 coding a putative chaperone protein has been recently suggested as a gene involved in this process. The aim of the present study was to analyze fls485 expression in human small intestinal mucosa., Methods: fls485 expression in purified normal or intestinal mucosa affected with celiac disease was investigated with a molecular approach including qRT-PCR, Western blotting, and expression strategies. Molecular data were corroborated with several in situ techniques and usage of newly synthesized mouse monoclonal antibodies., Results: fls485 mRNA expression was preferentially found in enterocytes and chromaffine cells of human intestinal mucosa as well as in several cell lines including Rko, Lovo, and CaCo2 cells. Western blot analysis with our new anti-fls485 antibodies revealed at least two fls485 proteins. In a functional CaCo2 model, an increase in fls485 expression was paralleled by cellular maturation stage. Immunohistochemistry demonstrated fls485 as a cytosolic protein with a slightly increasing expression gradient along the crypt-villus axis which was impaired in celiac disease Marsh IIIa-c., Conclusions: Expression and synthesis of fls485 are found in surface lining epithelia of normal human intestinal mucosa and deriving epithelial cell lines. An interdependence of enterocyte differentiation along the crypt-villus axis and fls485 chaperone activity might be possible.

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.

To date, there are no vasopressin (VP) agonists that exhibit a high affinity and selectivity for the VP V1b receptor with respect to the V1a, V2, and oxytocin receptors. In this study, we describe the synthesis and pharmacological properties of [1-deamino-4-cyclohexylalanine] arginine vasopressin (d[Cha4]AVP). Binding experiments performed on various membrane preparations revealed that d[Cha(4)]AVP exhibits a nanomolar affinity for V1b receptors from various mammalian species (rat, bovine, human). It exhibits high V1b/V1a and V1b/oxytocin selectivity for rat, human, and bovine receptors. Furthermore, it exhibits high V1b/V2 specificity for both bovine and human vasopressin receptors. Functional studies performed on biological models that naturally express V1b receptors indicate that d[Cha4]AVP is an agonist. Like VP, it stimulated basal and corticotropin-releasing factor-stimulated ACTH secretion and basal catecholamine release from rat anterior pituitary and bovine chromaffin cells, respectively. In vivo experiments performed in rat revealed that d[Cha4]AVP was able to stimulate both ACTH and corticosterone secretion and exhibits negligible vasopressor activity. It retains about 30% of the antidiuretic activity of VP. This long-sought selective VP V1b receptor ligand with nanomolar affinity will allow a better understanding of V1b-mediated VP physiological effects and is a promising new tool for V1b receptor structure-function studies.

Ontogenetic changes in neuropeptide Y-like immunoreactivity (NPY-LI) were studied in chromaffin tissue of the cloudy dogfish, Scyliorhinus torazame. In adults and post-hatching juveniles, NPY-LI was demonstrated in chromaffin cells, but not in ganglion cells and supporting cells. Immunoreactive fibers were also found in the axillary body (the major chromaffin tissue) of the adult fish. During the embryonic period, NPY-LI was found at first in chromaffin tissue in the 34-mm stage. In this stage, cells in the periphery of the tissue were positive for NPY. Afterwards, changes were not observed in the topography and relative dominance of labelled cells in the tissue. Transmission electron microscopy of chromaffin tissue of the 26-mm stage showed an early phase of histogenesis in rudimental cell clusters composed of agranular cells and a few granular cells, i.e. pheochromoblasts. In the 43-mm stage, differentiation of the chromaffin tissue enabled ultrastructural classification of adrenalin-producing cells, noradrenalin-producing cells, ganglion cells, supporting cells, and unmyelinated nerve fibers. These results suggest that in the dogfish the appearance of NPY-LI in the developing sympathoadrenal system is related to differentiation of chromaffin cells.

Brain stimulation or activation of certain reflexes can result in differential activation of the two populations of adrenal medullary chromaffin cells: those secreting either epinephrine or norepinephrine, suggesting that they are controlled by different central sympathetic networks. In urethan-chloralose-anesthetized rats, we found that antidromically identified adrenal sympathetic preganglionic neurons (SPNs) were excited by stimulation of the rostral ventrolateral medulla (RVLM) with either a short (mean: 29 ms) or a long (mean: 129 ms) latency. The latter group of adrenal SPNs were remarkably insensitive to baroreceptor reflex activation but strongly activated by the glucopenic agent 2-deoxyglucose (2-DG), indicating their role in regulation of adrenal epinephrine release. In contrast, adrenal SPNs activated by RVLM stimulation at a short latency were completely inhibited by increases in arterial pressure or stimulation of the aortic depressor nerve, were unaffected by 2-DG administration, and are presumed to govern the discharge of adrenal norepinephrine-secreting chromaffin cells. These findings of a functionally distinct preganglionic innervation of epinephrine- and norepinephrine-releasing adrenal chromaffin cells provide a foundation for identifying the different sympathetic networks underlying the differential regulation of epinephrine and norepinephrine secretion from the adrenal medulla in response to physiological challenges and experimental stimuli.

The development of the adrenal gland in the lizard Calotes versicolor was studied histologically and histochemically from the day of oviposition (stage 27) to 60 days after hatching. At stage 27, the adrenocortical cells are found in association with the genital ridge (primordial gonad). The separation of adrenocortical cells from the gonad takes place at stage 31. Organization of adrenocortical cells into cords takes place at stage 34. The catecholamine-secreting chromaffin cells can be seen distinctly on the dorsal region of the adrenal at stage 36, indicating the presence of biologically active catecholamines; the noradrenaline-secreting chromaffin cells appear first at stage 36 and the adrenaline-secreting cells appear later at stage 41. The cortico-medullary ratio of 6:1 during early embryonic development decreases with the increase in age and is 3:1 in posthatching lizards. The histochemical localization of Delta(5)-3beta-hydroxysteroid dehydrogenase (3beta-HSD) and glucose-6-phosphate dehydrogenase in the adrenocortical cells as early as at stage 27 (prior to the gonadal differentiation) indicates the capability of these cells to synthesize steroids. The intensity of the enzyme activity is maximum on the day of hatching and remains more or less the same in the posthatching lizards. The localization of 17beta-HSD enzyme activity observed in the adrenocortical cells at stage 34 is suggestive of their ability to synthesize sex steroids during embryonic life. The intense 3beta-HSD activity on the day of hatching in C. versicolor suggests high production of steroids which may be corticoids. The results of the present work also suggest that the onset of steroid secretion occurs prior to catecholamine secretion during embryogenesis of the adrenal gland in C. versicolor. In addition, there is a significant relationship between ontogenic steroidogenesis of the adrenal gland and sexual differentiation of the gonad., (Copyright 2000 Academic Press.)

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.

The developmental coexpression of galanin-like immunoreactivity with the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH) was studied in the avian embryo sympathoadrenal system using double-labeling immunocytochemistry. Galanin-like immunoreactivity is expressed by various catecholaminergic cell populations, namely sympathoblasts, chromaffin and small intensely fluorescent (SIF) cells, but not by principal neurons of the paravertebral sympathetic ganglia. Both galanin and somatostatin immunoreactivities are coexpressed in the adrenal and sympathetic ganglion primordia by the neural precursors, but the subsequent expression pattern of both peptides differs. Our results support the hypothesis that early sympathoblasts express a large repertoire of neuroactive substances and that the expression of these becomes restricted during further development as the sympathoblasts become principal neurons.

Biological transport of moderately permeant compounds is obscured by diffusion of the compounds back across the membrane, so characterization of the transport of such compounds requires correction for permeability. A relatively simple method for determining kinetic parameters for moderately permeant compounds is presented here. After evaluating a compound's apparent permeability coefficient, its steady-state uptake is measured as a function of concentration. By comparing the concentration dependence of uptake measured both in the presence and in the absence of a complete inhibitor of the transporter, K(m) and Vmax for transport of that substrate may be calculated. When used to analyze transport of tyramine and hydroxyephedrine by the vesicular amine transporter, this method yields results consistent with other methods and with values for analogous impermeant substrates. In bovine adrenal chromaffin vesicles, tyramine and (-)erythro-hydroxyephedrine have apparent permeability coefficients of 4.7 +/- 1.0 x 10(-9) and 1.1 +/- 0.4 x 10(-8) cm/s, respectively. Values for K(m) are 15 +/- 9 and 34 +/- 14 microM and for Vmax are 1.3 +/- 0.2 and 1.4 +/- 0.9 nmol/min.mg of membrane protein, respectively.

In this study, the effects of glutamate and glutamate receptor agonists in cultured chromaffin cells from bovine adrenal medulla were investigated. It was found that glutamate increases basal catecholamine (CA) secretion in a dose-dependent manner. This effect is mimicked by specific agonists of the four known glutamate receptors N-methyl-D-aspartate (NMDA), quisqualate/(RS)-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainate (KA), and trans-(+)-1-amino-1,3-cyclopentane dicarboxylic acid (t-ACPD), which increased both basal and nicotine-evoked CA secretion. The NMDA receptor antagonist 2-amino-5-phosphonopentanoic acid, 6-cyano-7-nitroquinoxaline-2,3-dione, an antagonist of KA and AMPA receptors, and L-(+)-2-amino-3-phosphonopropionic acid, an antagonist of the t-ACPD receptor, inhibited the stimulatory effect of related glutamate agonists. Hexamethonium, an antagonist of the nicotinic receptor, failed to influence glutamate agonists except for a 15% inhibition of KA. The increase in CA secretion produced by a 100 microM concentration of glutamate agonists was about 20-60% of that obtained with 10 microM of nicotine, an agonist of the physiological stimulatory cholinergic receptor. The increase in CA secretion produced by glutamate was accompanied by both an increase in bisoxonol fluorescence, suggesting membrane depolarization, and by an increase in intracellular Ca2+ concentrations. Results obtained with image analysis on single cells indicated that the percentage of cells which respond to the stimulation of 50 microM of glutamate is 42%. From these results, we conclude that glutamate, through its four known glutamate receptors, can increase both basal and nicotine-evoked CA secretion in chromaffin cells by a process which involves membrane depolarization and an increase in intracellular calcium levels.

The characteristics of the binding sites for the Conus magus toxins omega-conotoxin MVIIC and omega-conotoxin MVIID, as well as their effects on K+-evoked 45Ca2+ entry and whole-cell Ba2+ currents (IBa), and K+-evoked catecholamine secretion have been studied in bovine adrenal chromaffin cells. Binding of [125I] omega-conotoxin GVIA to bovine adrenal medullary membranes was displaced by omega-conotoxins GVIA, MVIIC and MVIID with IC50 values of around 0.1, 4 and 100 nM, respectively. The reverse was true for the binding of [125I] omega-conotoxin MVIIC, which was displaced by omega-conotoxins MVIIC, MVIID and GVIA with IC50 values of around 30, 80 and 1.200 nM, respectively. The sites recognized by omega-conotoxins MVIIC and MVIID in bovine brain exhibited higher affinities (IC50 values of around 1 nM). Both omega-conotoxin MVIIC and MVIID blocked IBa by 70-80%; the higher the [Ba2+]o of the extracellular solution the lower the blockade induced by omega-conotoxin MVIIC. This was not the case for omega-conotoxin MVIID; high Ba2+ (10 mM) slowed down the development of blockade but the maximum blockade achieved was similar to that obtained in 2 mM Ba2+. A further difference between the two toxins concerns their reversibility; washout of omega-conotoxin MVIIC did not reverse the blockade of IBa while in the case of omega-conotoxin MVIID a partial, quick recovery of current was produced. This component was irreversibly blocked by omega-conotoxin GVIA, suggesting that it is associated with N-type Ca2+ channels. Blockade of K+-evoked 45Ca2+ entry produced results which paralleled those obtained by measuring IBa. Thus, 1 microM of each of omega-conotoxin GVIA and MVIIA inhibited Ca2+ uptake by 25%, while 1 microM of each of omega-conotoxin MVIIC and MVIID caused a 70% blockade. K+-evoked catecholamine secretory responses were not reduced by omega-conotoxin GVIA (1 microM). In contrast, at 1 microM both omega-conotoxin MVIIC and MVIID reduced the exocytotic response by 70%. These data strengthen the previously established conclusion that Q-type Ca2+ channels that contribute to the regulation of secretion and are sensitive to omega-conotoxins MVIIC and MVIID are present in bovine chromaffin cells. These channels, however, seem to possess binding sites for omega-conotoxins MVIIC and MVIID whose characteristics differ considerably from those described to occur in the brain; they might represent a subset of Q-type Ca2+ channels or an entirely new subtype of voltage-dependent high-threshold Ca2+ channel.

Background & Aims: GATA transcription factors may regulate gene expression in developing tissues, including gut epithelium. In the stomach, their expression has been linked to regulation of proton pump genes. However, GATA consensus sequences also occur in the promoter of the histidine decarboxylase gene, located in enterochrommafin-like cells. The aim of this study was to determine if GATA factors are located in gastric endocrine cells and to examine their expression during development and in response to changes in the gastric luminal environment., Methods: Polymerase chain reaction cloning, Northern blot, and gel shift assays were used to examine GATA expression in gastric endocrine cells; changes in GATA messenger RNA during development and in response to fasting, feeding, and gastric achlorhydria were determined by Northern blot., Results: GATA-6 was expressed strongly in rodent gastric endocrine cell fractions, in a human ECL cell tumor, and in an endocrine cell line (STC-1) derived from gut epithelium; proteins from STC-1 cells bound specifically to GATA consensus sequences in the human histidine decarboxylase promoter. GATA messenger RNA abundance was up-regulated during terminal differentiation of the rat stomach and on feeding after a fast., Conclusions: The GATA-6 transcription factor is expressed in gastric endocrine cells and is a potential regulator of gastric differentiation and of genes involved in the response to feeding.

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.

The adrenal medulla of several species and some human pheochromocytomas contain CRH. The first aim of the present work was to find out whether normal rat adrenal chromaffin cells and the PC12 rat pheochromocytoma cell line produce CRH in vitro and what regulates its production. CRH was measured and characterized in the media of both types of chromaffin cells under basal conditions and after exposure to K+, nicotine, interleukin-1 beta, and nerve growth factor (NGF). The second aim was to examine the biological effect of exogenous CRH (and of its antagonist) on the production of catecholamines from these two types of cells. Our results are as follows: 1) Both types of chromaffin cells contained and secreted comparable amounts of immunoreactive-CRH under basal conditions and after K(+)-induced depolarization, nicotine, and interleukin-1 beta; 2) the physicochemical characteristics of the immunoreactive-CRH in the cells and the media were identical to the putative CRH peptide on both sieve chromatography and RP-HPLC; 3) synthetic CRH induced the production of catecholamines from both cell types in a dose- and time-dependent manner; this effect was abolished by the antagonist, alpha helical CRH; 4) exposure of PC12 cells to NGF (for 1 week) resulted in their neuronal differentiation and the stimulation of their production of CRH by 30 times and of dopamine by 10 times, compared with parallel controls; this effect of NGF was abolished by alpha helical CRH. In conclusion, our data suggest that the production of CRH by PC12 cells represents the preservation of a normal chromaffin cell characteristic rather than a tumor-induced ectopic phenomenon.

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.

alpha-Latrotoxin (alpha-LT), from black widow spider venom, is a potent enhancer of the spontaneous quantal release of neurotransmitter from a variety of nerve terminals and clonal neurosecretory cells. Using electrochemical amperometry and estimation of membrane impedance by phase detection, we present evidence that alpha-LT induces exocytosis of catecholamines from rat adrenal chromaffin cells beginning as rapidly as 30 s after close application of the toxin. This release is largely dependent on adequate levels of extracellular Ca2+ ([Ca2+]o). Lowering [Ca2+]o from 2 mM to

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.

Trichosporin-B-VIa (TS-B-VIa) has a Pro14-kinked helical structure which is considered to be important for the formation of peptaibol-type ion-channels in lipid bilayer membranes. TS-B-VIa and its analog [Aib14]TS-B-VIa with Pro-->Aib substitution at position 14, resulting in a straight helical structure, were tested for ion-channel-forming activity in planar lipid bilayer membranes and for ability to induce catecholamine secretion from cultured bovine adrenal chromaffin cells. Voltage-dependent multi-channel conductance, which is characteristic of TS-B-VIa, was also observed for [Aib14]TS-B-VIa. In single-channel measurements, current fluctuations induced by [Aib14]TS-B-VIa had a shorter life-time and showed fewer substates than those induced by TS-B-VIa. Catecholamine secretion induced by these peptides at low concentrations is completely Ca(2+)-dependent. At high concentrations, TS-B-VIa-induced secretion was partly independent of external Ca2+, but this was not the case for the analog. The differences of behavior can be explained in terms of the differences of hydrophobicity, and magnitude of dipole moment due to the conformational changes around position 14 and the C-terminal domain caused by the Pro-->Aib substitution.

Submembrane [Ca2+]i changes were examined in rat chromaffin cells by monitoring the activity of an endogenous Ca(2+)-dependent protein: the large conductance Ca(2+)-and voltage-activated K+ channel (also known as the BK channel). The Ca2+ and voltage dependence of BK current inactivation and conductance were calibrated first by using defined [Ca2+]i salines. This information was used to examine submembrane [Ca2+]i elevations arising out of Ca2+ influx and muscarine-mediated release of Ca2+ from intracellular stores. During Ca2+ influx, some BK channels are exposed to [Ca2+]i of at least 60 microM. However, the distribution of this [Ca2+]i elevation is highly nonuniform so that the average [Ca2+]i detected when all BK channels are activated is only approximately 10 microM. Intracellular dialysis with 1 mM or higher EGTA spares only the BK channels activated by the highest [Ca2+]i during influx, whereas dialysis with 1 mM or higher BAPTA blocks activation of all BK channels. Submembrane [Ca2+]i elevations fall rapidly after termination of short (5 msec) Ca2+ influx steps but persist above 1 microM for several hundred milliseconds after termination of long (200 msec) influx steps. In contrast to influx, the submembrane [Ca2+]i elevations produced by release of intracellular Ca2+ by muscarinic actetylcholine receptor (mAChR) activation are much more uniform and reach peak levels of 3-5 microM. Our results suggest that during normal action potential activity only 10-20% of BK channels in each chromaffin cell see sufficient [Ca2+]i to be activated.

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.

Adrenal medullary chromaffin cells derive from the neural crest during embryogenesis and differentiate into dedicated secretory cells that release catecholamines in response to acetylcholine in vivo or nicotinic agonists in vitro. Previous studies have indicated that tyrosine kinases participate in early secretagogue-induced events in these cells and are required for exocytosis. Abundant levels of the cytoplasmic tyrosine kinases, c-Src and c-Yes, have been detected in chromaffin cells, thereby implicating them as kinases relevant to these events. However, c-Src has been found to undergo a decrease in activity following secretagogue-stimulation, and c-Yes appears to exist in a constituitively low activity state, suggesting that other tyrosine kinases are involved. Furthermore, other members of the Src family of tyrosine kinases have been implicated as playing roles in secretion in a variety of cell types. Therefore, we sought to determine if other Src family members were present in chromaffin cells, and if so, to examine them for subcellular localization and changes in activity following treatment with nicotinic agonists. To this end, antibodies for Fyn, Lck, Lyn, and Fgr were assembled and used in immunoprecipitation, in vitro autokinase, and Western immunoblotting assays. Of these four kinases, only Fyn was found to be expressed at detectable levels. Differential centrifugation studies revealed that Fyn resides predominantly (> 95%) in the crude plasma membrane fraction and undergoes nicotinic-and carbachol-induced activation. This activation is reduced by the nicotinic antagonist, mecamylamine, is not elicited by muscarine, and is dependent upon the presence of extracellular Ca2+. These results suggest that Fyn is involved in signalling through the nicotinic receptor and may be one of the relevant kinases responsible for at least some of the tyrosine phosphorylations detected after stimulation.

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.

The relationship between cytosolic Ca2+ and catecholamine secretion during stimulus-secretion coupling has been examined at individual chromaffin cells isolated from the cow and rat. Vesicular catecholamine exocytosis was determined via amperometric measurements with carbon fibre microelectrodes and fura-2 was used for simultaneous fluorescent monitoring of cytosolic Ca2+ at the same cell. Individual secretory vesicles in cells from the two species were found to release similar amounts of catecholamine. In addition, the time courses for secretion from individual vesicles was similar with rat and bovine chromaffin cells. The total quantity of catecholamine released and the change in cytosolic Ca2+ were also similar in response to exposure to K+ (60 mM), 1,1-dimethyl-4-phenylpiperazinium (50 microM), and histamine (50 microM), although both responses were more prolonged following 1,1-dimethyl-4-phenylpiperazinium and histamine at bovine cells. Agents that mobilize intracellular Ca(2+)-stores such as methacholine, caffeine and bradykinin resulted in different cytosolic Ca2+ and exocytosis responses at the rat and bovine chromaffin cells. Results indicate a heightened Ca(2+)-store activity or a more filled state in chromaffin cells from the rat. The results of this study clearly show that single-cell techniques can be used to characterize stimulus-secretion coupling. The requirement for lower numbers of cells with these techniques means that chromaffin cells from rodents can be routinely employed. This can be advantageous to minimize biological variability which occurs with organs obtained from slaughter houses and enables the investigation of genetically-altered animals.

Pituitary adenylate cyclase-activating polypeptide (PACAP)i a potent stimulant of catecholamine secretion, increased catecholamine production in cultured porcine adrenal medullary chromaffin cells. PACAP induced dose-and time-dependent increases in mRNAs for the catecholamine synthesizing enzymes, tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH), with maximal 6- and 4-fold increases occurring at 8-16 h, respectively. The half-maximally and maximally effective PACAP concentrations for stimulation of TH and DBH gene expression were 0.5 and 3 nM, respectively. The TH protein level also showed an increase over the unstimulated basal level at 16-24 h in PACAP-stimulate cells. We previously demonstrated that PACAP activates both phospholipase C and adenylate cyclase in adrenal medullary cells. Addition of forskolin alone induced increases in mRNA expression of both TH and DBH. The phosphodiesterase inhibitor 3- isobutyl-1-methylxanthine potentiated the induction of TH and DBH mRNAs by PACAP. Addition of the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) also caused increases in TH and DBH mRNA levels. In protein kinase C-downregulated cells pretreated with PMA for 24 h, the stimulatory effect of PACAP on TH and DBH gene expression was diminished. These results suggest that cAMP and protein kinase C mediate the PACAP-induced TH and DBH gene expression. Removal of extracellular Ca2+ with EGTA enhanced the PACAP-induced increases in both cellular cAMP and mRNA levels of TH and DBH, suggesting that Ca2+ has an inhibitory effect on the induction of TH and DBH mRNAs. In conclusion, the present study indicates that PACAP coordinately upregulates the gene expression of both TH and DBH by activating the cAMP and protein kinase C signaling pathways, leading to simulation of cate-cholamine synthesis, while Ca2+ negatively regulates TH and DBH gene expression in porcine adrenal medullary cells.

1. In this study the secretory response of individual bovine adrenal chromaffin cells was monitored using amperometric carbon-fibre microelectrodes. Cells were stimulated to secrete by exposure to 20-100 microseconds long tonebursts of ultrasound (2-4 x 10(5) Pa; peak pressure at 1 MHz). 2. Three types of secretory responses were observed: an almost instantaneous response, a delayed release of catecholamines, or a series of 'burst-like' secretory bouts. 3. Fura-2 measurements of intracellular Ca2+ concentrations showed that the release of catecholamines was accompanied by an increase in the intracellular Ca2+ concentration. In the absence of extracellular Ca2+, secretory responses were not evoked showing that Ca2+ entry was necessary to elicit catecholamine release.

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.

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.

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.

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.

The seven types (A--G) of botulinum neurotoxin (BoNT) are Zn2+ -dependent endoproteases that potently block neurosecretion. Syntaxin is presently thought to be the sole substrate for BoNT/C1, and synaptosomal-associated protein of Mr = 25 000 (SNAP-25) is selectively proteolyzed by types A and E. In this study, the effects of C1 on Ca2+ -regulated exocytosis of dense core granules from adreno-chromaffin cells were examined together with its underlying molecular action. Intact chromaffin cells were exposed to the toxin, and catecholamine release therefrom was then measured in conjunction with the monitoring of syntaxin cleavage by Western blotting. A good correlation was obtained between degradation of syntaxin 1A/B and reduction in Ca2+- or Ba2+-dependent secretion. However, blotting with antibodies against a C-terminal peptide of SNAP-25 revealed the additional disappearance of immunoreactivity, with the same toxin concentration dependency as syntaxin breakdown. Notably, the cleaved SNAP-25 product was similar in size to that produced by BoNT/A; however, contamination of BoNT/C1 by serotypes A or E was eliminated. Therefore, it is concluded that syntaxin 1A/B and SNAP-25 are cleaved in intact cells poisoned with only C1. Notably, C1 treatment of chromaffin cells abolished Ca2+ -evoked secretion following digitonin permeabilization, compared with partial inhibition by BoNT/A, suggesting the importance of syntaxin for catecholamine release. Unexpectedly, C1 failed to proteolyze a soluble recombinant SNAP-25, even though it served as an efficient substrate for BoNT/A. These interesting observations suggest that C1 can only efficiently cleave SNAP-25 in intact cells, possibly due to the existence therein of a unique conformation and/or the participation of accessory factors.

We used the perforated-patch technique to examine the relationship between Ca2+ entry and exocytosis of large dense-cored vesicles in bovine adrenal chromaffin cells. Exocytosis evoked by single-step depolarizations was monitored by capacitance detection. Ca2+ entry was varied by changing external calcium concentration, stepping to different test potentials, depolarizing for different durations, or applying blockers of specific calcium channel subtypes. Regardless of protocol, the amount of exocytosis was strictly related to the integral of the voltage-clamped calcium current, raised to a power of approximately 1.5. Thus, despite the complexities of transient and nonuniform changes in submembrane calcium concentration produced by voltage-gated calcium entry, the calcium dependence of large dense-cored vesicle fusion under conditions of minimal stimulation is well approximated by a simple transfer function of summed calcium entry.

Clonidine inhibited the uptake of calcium and the overall phosphorylation of tyrosine hydroxylase induced by nicotinic receptor activation in bovine adrenal medullary chromaffin cells in culture. However, clonidine did not inhibit the increase in these parameters that accompanied K+ depolarisation of the cells. There was also no effect of clonidine on the overall phosphorylation of tyrosine hydroxylase when cells were stimulated by muscarine. Nicotinic receptor activation increased the phosphorylation of Ser-19, Ser-31, and Ser-40 on tyrosine hydroxylase, and this was inhibited by clonidine in a concentration-dependent manner. On the other hand, clonidine had no effect on calcium uptake, yet increased the phosphorylation of Ser-19 under basal conditions. Using calcium and calmodulin-stimulated protein kinase II obtained from rat brain clonidine increased the autophosphorylation of the alpha-subunit of the kinase by 37%, and also its activity against an exogenous peptide substrate by 29%. These data are consistent with the hypothesis that clonidine inhibits nicotinic receptor-induced tyrosine hydroxylase phosphorylation by decreasing calcium influx into chromaffin cells, perhaps by an action at the nicotinic receptor. Clonidine also increases the basal phosphorylation of tyrosine hydroxylase at Ser-19, perhaps by directly activating calcium and calmodulin-stimulated protein kinase II.

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.

Tumor necrosis factor (TNF) is gaining increasing importance in clinical medicine. It plays a role in the interaction of the immune system with the hypothalamic-pituitary-adrenal axis. In the present study various morphological methods, including immunohistochemistry, electron microscopy, and in situ hybridization were applied to characterize the localization and distribution of TNF in the human adrenal gland. Double immunostaining revealed an astonishing degree of intermingling of steroid-producing cells and chromaffin cells. Macrophages could be found in all regions of the adrenal gland, but particularly in the transition zone of cortex and medulla. The steroid-producing cells of the inner zone of the cortex express major histocompatibility complex class II molecules. On the ultrastructural level, immune cells, steroid cells, and catecholamine-producing cells were found in direct contact. The combination of immunohistochemistry and in situ hybridization was optimally suited to define the exact cellular source of TNF in the human adrenal. TNF is produced in macrophages, but above all in 17 alpha-hydroxylase-positive cells (steroid-producing cells) in the zona reticularis and medulla. No signal was found in chromaffin cells. TNF may induce major histocompatibility complex class II in human adrenal gland in a paracrine or autocrine manner. It is concluded that TNF may have an important role in normal human adrenal physiology.

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.

In sea-level natives, exposure to hypoxia for a few weeks is characterized by an increased dependence on blood glucose and a decreased reliance on lactate for energy metabolism during exercise. These metabolic adjustments have been attributed to behavioral changes in the sympathoadrenergic and pancreatic systems. The aim of this study was to test the hypothesis of a reduced sympathoadrenergic activation and subsequent metabolic changes when high-altitude natives are acutely exposed to normoxia. Young Andean natives performed incremental exercise to exhaustion during hypoxia (arterial PO2 55.1 +/- 1.1 Torr) or during acute normoxia (arterial PO2 78.7 +/- 1.7 Torr). As a whole, oxygen uptake was increased in normoxia compared with hypoxia during graded exercise. This finding is not related to a decrease in anaerobic metabolism but rather is interpreted as a consequence of a shift in substrate utilization during exercise (increased contribution of fat as assessed by a reduction in the respiratory exchange ratio). These metabolic changes are not accompanied by modifications of glucoregulatory hormones (catecholamines, insulin, and glucagon). In particular, the exercise-induced catecholamine secretion was similar in chronic hypoxia and acute normoxia. As a consequence, blood lactate accumulation during incremental exercise was similar in both conditions. It is concluded that high-altitude natives do not display any sign of a greater sympathoadrenergic activation during chronic hypoxia and that the exercise-induced hormonal changes remained unaffected by acute inhalation of a normoxic gas mixture.

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.

Infusion of the G protein activator, AlF complex, into guinea-pig adrenal chromaffin cells produced an inward non-selective cation current (INS) at -55 mV. Under metabolically suppressed conditions, this current was abolished by removal of ATP from the AlF-containing pipette solution. ADP could substitute for ATP in generation of the current. This supporting action of ADP was eliminated by addition to the AlF solution of the ATP-consuming system (hexokinase and 2-deoxyglucose). A similar generation of INS occurred when AMP and phosphocreatine were simultaneously added to AlF solution, but not when either of the agents was added separately. These results suggest that under conditions of whole-cell current recordings, the machinery for ATP production is preserved and that ADP indirectly supports generation of INS by the AlF complex.

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.

Growth associated protein 43 (GAP43) mRNA was found in scattered groups of chromaffin cells prenatally, and in chromaffin noradrenaline cells from postnatal day 6 (P6). The distinct adult distribution of GAP43 mRNA in chromaffin cells appeared between P10 and P16. High levels of GAP43 mRNA were found in intra-adrenal ganglion neurons also expressing neuropeptide tyrosine (NPY) mRNA (type I ganglion neurons), and at prenatal and early postnatal stages in large medullarly clusters of NPY mRNA containing cells, probably representing type I ganglion neurons precursors. The expression of GAP43 mRNA in noradrenaline-producing chromaffin cells and type I ganglion neurons suggests an important role for GAP43 in these cells both during development and in adult life.

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.

Dopamine agonists or grafts compensate impaired motor functions in aged rats. However, there is no evidence showing whether grafting in adulthood retard aging manifestations. Motor performance of 13-month-old rats was tested on 2 meter-long wooden beams which had a 15 degree inclination and whose thickness varied from 3, 6, 12, 18, to 24 mm. Rats at 14 months were randomly assigned to 3 groups: sham graft (Group 1); intrastriatal graft of chromaffin cells cultured with NGF (Group 2); intrastriatal graft of chromaffin cells (Group 3). Motor performance was tested at monthly intervals up until rats were 26 months old. Two more groups were included: 26-month-old naive rats (Group 4); and 3- to 5-month-old naive rats (Group 5) both evaluated only once. At 26 months, the basal activity of ventral mesencephalic dopaminergic neurons was recorded. Results showed in Group 2 delay of motor detriments seen in aged rats, maintenance of basal firing rates of nigral cells compared to those of younger rats, and greater survival of substantia nigra cells. It is suggested that NGF cultured chromaffin cells produce a delay of motor detriments in aged rats, as a result of inducing survival and firing rates of nigral cells comparable to those seen in young rats.

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.