Acute reversible SERCA blockade facilitates or blocks exocytosis, respectively in mouse or bovine chromaffin cells.

Pre-blockade of the sarco-endoplasmic reticulum (ER) calcium ATPase (SERCA) with irreversible thapsigargin depresses exocytosis in adrenal bovine chromaffin cells (BCCs). Distinct expression of voltage-dependent Ca2+-channel subtypes and of the Ca2+-induced Ca2+ release (CICR) mechanism in BCCs versus mouse chromaffin cells (MCCs) has been described. We present a parallel study on the effects of the acute SERCA blockade with reversible cyclopizonic acid (CPA), to repeated pulsing with acetylcholine (ACh) at short (15 s) and long intervals (60 s) at 37 °C, allowing the monitoring of the initial size of a ready-release vesicle pool (RRP) and its depletion and recovery in subsequent stimuli. We found (i) strong depression of exocytosis upon ACh pulsing at 15-s intervals and slower depression at 60-s intervals in both cell types; (ii) facilitation of exocytosis upon acute SERCA inhibition, with back to depression upon CPA washout in MCCs; (iii) blockade of exocytosis upon acute SERCA inhibition and pronounced rebound of exocytosis upon CPA washout in BCCs; (iv) basal [Ca2+]c elevation upon stimulation with ACh at short intervals (but not at long intervals) in both cell types; and (v) augmentation of basal [Ca2+]c and inhibition of peak [Ca2+]c amplitude upon CPA treatment in both cell types, with milder effects upon stimulation at 60-s intervals. These results are compatible with the view that while in MCCs the uptake of Ca2+ via SERCA contributes to the mitigation of physiological ACh triggered secretion, in BCCs the uptake of Ca2+ into the ER facilitates such responses likely potentiating a Ca2+-induced Ca2+ release mechanism. These drastic differences in the regulation of ACh-triggered secretion at 37 °C may help to understand different patterns of the regulation of exocytosis by the circulation of Ca2+ at a functional ER Ca2+ store.

The receptor tyrosine kinase EPHB6 regulates catecholamine exocytosis in adrenal gland chromaffin cells.

The erythropoietin-producing human hepatocellular receptor EPH receptor B6 (EPHB6) is a receptor tyrosine kinase that has been shown previously to control catecholamine synthesis in the adrenal gland chromaffin cells (AGCCs) in a testosterone-dependent fashion. EPHB6 also has a role in regulating blood pressure, but several facets of this regulation remain unclear. Using amperometry recordings, we now found that catecholamine secretion by AGCCs is compromised in the absence of EPHB6. AGCCs from male knockout (KO) mice displayed reduced cortical F-actin disassembly, accompanied by decreased catecholamine secretion through exocytosis. This phenotype was not observed in AGCCs from female KO mice, suggesting that testosterone, but not estrogen, contributes to this phenotype. Of note, reverse signaling from EPHB6 to ephrin B1 (EFNB1) and a 7-amino acid-long segment in the EFNB1 intracellular tail were essential for the regulation of catecholamine secretion. Further downstream, the Ras homolog family member A (RHOA) and FYN proto-oncogene Src family tyrosine kinase (FYN)-proto-oncogene c-ABL-microtubule-associated monooxygenase calponin and LIM domain containing 1 (MICAL-1) pathways mediated the signaling from EFNB1 to the defective F-actin disassembly. We discuss the implications of EPHB6's effect on catecholamine exocytosis and secretion for blood pressure regulation.

EPHB6 controls catecholamine biosynthesis by up-regulating tyrosine hydroxylase transcription in adrenal gland chromaffin cells.

EPHB6 is a member of the erythropoietin-producing hepatocellular kinase (EPH) family and a receptor tyrosine kinase with a dead kinase domain. It is involved in blood pressure regulation and adrenal gland catecholamine (CAT) secretion, but several facets of EPHB6-mediated CAT regulation are unclear. In this study, using biochemical, quantitative RT-PCR, immunoblotting, and gene microarray assays, we found that EPHB6 up-regulates CAT biosynthesis in adrenal gland chromaffin cells (AGCCs). We observed that epinephrine content is reduced in the AGCCs from male Ephb6-KO mice, caused by decreased expression of tyrosine hydroxylase, the rate-limiting enzyme in CAT biosynthesis. We demonstrate that the signaling pathway from EPHB6 to tyrosine hydroxylase expression in AGCCs involves Rac family small GTPase 1 (RAC1), MAP kinase kinase 7 (MKK7), c-Jun N-terminal kinase (JNK), proto-oncogene c-Jun, activator protein 1 (AP1), and early growth response 1 (EGR1). On the other hand, signaling via extracellular signal-regulated kinase (ERK1/2), p38 mitogen-activated protein kinase, and ELK1, ETS transcription factor (ELK1) was not affected by EPHB6 deletion. We further report that EPHB6's effect on AGCCs was via reverse signaling through ephrin B1 and that EPHB6 acted in concert with the nongenomic effect of testosterone to control CAT biosynthesis. Our findings elucidate the mechanisms by which EPHB6 modulates CAT biosynthesis and identify potential therapeutic targets for diseases, such as hypertension, caused by dysfunctional CAT biosynthesis.

Variations in adrenal gland medulla and dopamine effects induced by the lack of Irs2.

The adrenomedullary chromaffin cells' hormonal pathway has been related to the pathophysiology of diabetes mellitus. In mice, the deletion of insulin receptor substrate type 2 (Irs2) causes peripheral insulin resistance and reduction in ß-cell mass, leading to overt diabetes, with gender differences on adrenergic signaling. To further unravel the relevance of Irs2 on glycemic control, we analyzed in adult Irs2 deficient (Irs2-/-) mice, of both sexes but still normoglycemic, dopamine effects on insulin secretion and glycerol release, as well as their adrenal medulla by an immunohistochemical and morphologic approach. In isolated islets, 10 µM dopamine significantly inhibited insulin release in wild-type (WT) and female Irs2-/- mice; however, male Irs2-/- islets were insensitive to that catecholamine. Similarly, on isolated adipocytes, gender differences were observed between WT and Irs2-/- mice in basal and evoked glycerol release with crescent concentrations of dopamine. By immunohistochemistry, reactivity to tyrosine hydroxylase (TH) in female mice was significantly higher in the adrenal medulla of Irs2-/- compared to WT; although no differences for TH-immunopositivity were observed between the male groups of mice. However, compared to their corresponding WT animals, adrenomedullary chromaffin cells of Irs2-/- mice showed a significant decrease in the cellular and nuclear areas, and even in their percentage of apoptosis. Therefore, our observations suggest that, together with gender differences on dopamine responses in Irs2-/- mice, disturbances in adrenomedullary chromaffin cells could be related to deficiency of Irs2. Accordingly, Irs2 could be necessary for adequate glucose homeostasis and maintenance of the population of the adrenomedullary chromaffin cells.

Hydralazine administration activates sympathetic preganglionic neurons whose activity mobilizes glucose and increases cardiovascular function.

Hypotensive drugs have been used to identify central neurons that mediate compensatory baroreceptor reflex responses. Such drugs also increase blood glucose. Our aim was to identify the neurochemical phenotypes of sympathetic preganglionic neurons (SPN) and adrenal chromaffin cells activated following hydralazine (HDZ; 10mg/kg) administration in rats, and utilize this and SPN target organ destination to ascribe their function as cardiovascular or glucose regulating. Blood glucose was measured and adrenal chromaffin cell activation was assessed using c-Fos immunoreactivity (-ir) and phosphorylation of tyrosine hydroxylase, respectively. The activation and neurochemical phenotype of SPN innervating the adrenal glands and celiac ganglia were determined using the retrograde tracer cholera toxin B subunit, in combination with in situ hybridization and immunohistochemistry. Blood glucose was elevated at multiple time points following HDZ administration but little evidence of chromaffin cell activation was seen suggesting non-adrenal mechanisms contribute to the sustained hyperglycemia. 16±0.1% of T4-T11 SPN contained c-Fos and of these: 24.3±1.4% projected to adrenal glands and 29±5.5% projected to celiac ganglia with the rest innervating other targets. 62.8±1.4% of SPN innervating adrenal glands were activated and 29.9±3.3% expressed PPE mRNA whereas 53.2±8.6% of SPN innervating celiac ganglia were activated and 31.2±8.8% expressed PPE mRNA. CART-ir SPN innervating each target were also activated and did not co-express PPE mRNA. Neurochemical coding reveals that HDZ administration activates both PPE+SPN, whose activity increase glucose mobilization causing hyperglycemia, as well as CART+SPN whose activity drive vasomotor responses mediated by baroreceptor unloading to raise vascular tone and heart rate.

Depressed excitability and ion currents linked to slow exocytotic fusion pore in chromaffin cells of the SOD1(G93A) mouse model of amyotrophic lateral sclerosis.

Altered synaptic transmission with excess glutamate release has been implicated in the loss of motoneurons occurring in amyotrophic lateral sclerosis (ALS). Hyperexcitability or hypoexcitability of motoneurons from mice carrying the ALS mutation SOD1(G93A) (mSOD1) has also been reported. Here we have investigated the excitability, the ion currents, and the kinetics of the exocytotic fusion pore in chromaffin cells from postnatal day 90 to postnatal day 130 mSOD1 mice, when motor deficits are already established. With respect to wild-type (WT), mSOD1 chromaffin cells had a decrease in the following parameters: 95% in spontaneous action potentials, 70% in nicotinic current for acetylcholine (ACh), 35% in Na(+) current, 40% in Ca(2+)-dependent K(+) current, and 53% in voltage-dependent K(+) current. Ca(2+) current was increased by 37%, but the ACh-evoked elevation of cytosolic Ca(2+) was unchanged. Single exocytotic spike events triggered by ACh had the following differences (mSOD1 vs. WT): 36% lower rise rate, 60% higher decay time, 51% higher half-width, 13% lower amplitude, and 61% higher quantal size. The expression of the α3-subtype of nicotinic receptors and proteins of the exocytotic machinery was unchanged in the brain and adrenal medulla of mSOD1, with respect to WT mice. A slower fusion pore opening, expansion, and closure are likely linked to the pronounced reduction in cell excitability and in the ion currents driving action potentials in mSOD1, compared with WT chromaffin cells.

Cardiac sympathetic denervation in 6-OHDA-treated nonhuman primates.

Cardiac sympathetic neurodegeneration and dysautonomia affect patients with sporadic and familial Parkinson's disease (PD) and are currently proposed as prodromal signs of PD. We have recently developed a nonhuman primate model of cardiac dysautonomia by iv 6-hydroxydopamine (6-OHDA). Our in vivo findings included decreased cardiac uptake of a sympathetic radioligand and circulating catecholamines; here we report the postmortem characterization of the model. Ten adult rhesus monkeys (5-17 yrs old) were used in this study. Five animals received 6-OHDA (50 mg/kg i.v.) and five were age-matched controls. Three months post-neurotoxin the animals were euthanized; hearts and adrenal glands were processed for immunohistochemistry. Quantification of immunoreactivity (ir) of stainings was performed by an investigator blind to the treatment group using NIH ImageJ software (for cardiac bundles and adrenals, area above threshold and optical density) and MBF StereoInvestigator (for cardiac fibers, area fraction fractionator probe). Sympathetic cardiac nerve bundle analysis and fiber area density showed a significant reduction in global cardiac tyrosine hydroxylase-ir (TH; catecholaminergic marker) in 6-OHDA animals compared to controls. Quantification of protein gene protein 9.5 (pan-neuronal marker) positive cardiac fibers showed a significant deficit in 6-OHDA monkeys compared to controls and correlated with TH-ir fiber area. Semi-quantitative evaluation of human leukocyte antigen-ir (inflammatory marker) and nitrotyrosine-ir (oxidative stress marker) did not show significant changes 3 months post-neurotoxin. Cardiac nerve bundle α-synuclein-ir (presynaptic protein) was reduced (trend) in 6-OHDA treated monkeys; insoluble proteinase-K resistant α-synuclein (typical of PD pathology) was not observed. In the adrenal medulla, 6-OHDA monkeys had significantly reduced TH-ir and aminoacid decarboxylase-ir. Our results confirm that systemic 6-OHDA dosing to nonhuman primates induces cardiac sympathetic neurodegeneration and loss of catecholaminergic enzymes in the adrenal medulla, and suggests that this model can be used as a platform to evaluate disease-modifying strategies aiming to induce peripheral neuroprotection.

The V-ATPase membrane domain is a sensor of granular pH that controls the exocytotic machinery.

Several studies have suggested that the V0 domain of the vacuolar-type H(+)-adenosine triphosphatase (V-ATPase) is directly implicated in secretory vesicle exocytosis through a role in membrane fusion. We report in this paper that there was a rapid decrease in neurotransmitter release after acute photoinactivation of the V0 a1-I subunit in neuronal pairs. Likewise, inactivation of the V0 a1-I subunit in chromaffin cells resulted in a decreased frequency and prolonged kinetics of amperometric spikes induced by depolarization, with shortening of the fusion pore open time. Dissipation of the granular pH gradient was associated with an inhibition of exocytosis and correlated with the V1-V0 association status in secretory granules. We thus conclude that V0 serves as a sensor of intragranular pH that controls exocytosis and synaptic transmission via the reversible dissociation of V1 at acidic pH. Hence, the V-ATPase membrane domain would allow the exocytotic machinery to discriminate fully loaded and acidified vesicles from vesicles undergoing neurotransmitter reloading.

Phospholipid scramblase-1-induced lipid reorganization regulates compensatory endocytosis in neuroendocrine cells.

Calcium-regulated exocytosis in neuroendocrine cells and neurons is accompanied by the redistribution of phosphatidylserine (PS) to the extracellular space, leading to a disruption of plasma membrane asymmetry. How and why outward translocation of PS occurs during secretion are currently unknown. Immunogold labeling on plasma membrane sheets coupled with hierarchical clustering analysis demonstrate that PS translocation occurs at the vicinity of the secretory granule fusion sites. We found that altering the function of the phospholipid scramblase-1 (PLSCR-1) by expressing a PLSCR-1 calcium-insensitive mutant or by using chromaffin cells from PLSCR-1⁻/⁻ mice prevents outward translocation of PS in cells stimulated for exocytosis. Remarkably, whereas transmitter release was not affected, secretory granule membrane recapture after exocytosis was impaired, indicating that PLSCR-1 is required for compensatory endocytosis but not for exocytosis. Our results provide the first evidence for a role of specific lipid reorganization and calcium-dependent PLSCR-1 activity in neuroendocrine compensatory endocytosis.

Regulation of neuropeptide processing enzymes by catecholamines in endocrine cells.

Treatment of cultured bovine adrenal chromaffin cells with the catecholamine transport blocker reserpine was shown previously to increase enkephalin levels severalfold. To explore the biochemical mechanism of this effect, we examined the effect of reserpine treatment on the activities of three different peptide precursor processing enzymes: carboxypeptidase E (CPE) and the prohormone convertases (PCs) PC1/3 and PC2. Reserpine treatment increased both CPE and PC activity in extracts of cultured chromaffin cells; total protein levels were unaltered for any enzyme. Further analysis showed that the increase in CPE activity was due to an elevated V(max), with no change in the K(m) for substrate hydrolysis or the levels of CPE mRNA. Reserpine activation of endogenous processing enzymes was also observed in extracts prepared from PC12 cells stably expressing PC1/3 or PC2. In vitro experiments using purified enzymes showed that catecholamines inhibited CPE, PC1/3, and PC2, with dopamine quinone the most potent inhibitor (IC(50) values of ∼50-500 µM); dopamine, norepinephrine, and epinephrine exhibited inhibition in the micromolar range. The inhibition of purified CPE with catecholamines was time-dependent and, for dopamine quinone, dilution-independent, suggesting covalent modification of the protein by the catecholamine. Because the catecholamine concentrations found to be inhibitory to PC1/3, PC2, and CPE are well within the physiological range found in chromaffin granules, we conclude that catecholaminergic transmitter systems have the potential to exert considerable dynamic influence over peptidergic transmitter synthesis by altering the activity of peptide processing enzymes.

Vesicular monoamine transporters (VMATs) in adrenal chromaffin cells: stress-triggered induction of VMAT2 and expression in epinephrine synthesizing cells.

Vesicular monoamine transporters (VMATs) mediate transmitter uptake into neurosecretory vesicles. There are two VMAT isoforms, VMAT1 and VMAT2, encoded by separate genes and displaying different cellular distributions and pharmacological properties. We examined the effect of immobilization stress (IMO) on expression of VMATs in the rat adrenal medulla. Under basal conditions, VMAT1 is widely expressed in all adrenal chromaffin cells, while VMAT2 is co-localized with tyrosine hydroxylase (TH) but not phenylethanolamine N-methyltransferase (PNMT), indicating its expression in norepinephrine (NE)-, but not epinephrine (Epi)-synthesizing chromaffin cells. After exposure to IMO, there was no change in levels of VMAT1 mRNA. However, VMAT2 mRNA was elevated after exposure of rats to 2 h IMO once (1× IMO) or daily for 6 days (6× IMO). The changes in VMAT2 mRNA were reflected by increased VMAT2 protein after the repeated IMO. Immunofluorescence revealed an increased number of cells expressing VMAT2 following repeated IMO and its colocalization with PNMT in many chromaffin cells. The findings suggest an adaptive mechanism in chromaffin cells whereby enhanced catecholamine storage capacity facilitates more efficient utilization of the well-characterized heightened catecholamine biosynthesis with repeated IMO stress.

Neuropeptides, growth factors, and cytokines: a cohort of informational molecules whose expression is up-regulated by the stress-associated slow transmitter PACAP in chromaffin cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a co-transmitter with acetylcholine at the adrenomedullary synapse, mediating sustained hormone secretion and regulation of cellular plasticity in response to stress at the level of gene transcription. Here we have extended our investigation of PACAP-regulated neuroendocrine cell-specific genes from PC12 cells to PC12 cells expressing physiological levels of the PAC1hop receptor found on chromaffin cells in vivo. PACAP induces in these PC12_bPAC1hop cells an additional cohort of genes, compared to PC12 cells, enriched in informational molecules including cytokines, neuropeptides, and growth factors. Using two newly developed microarray platforms for expressed bovine transcripts, we further examined PACAP-induced genes in bovine chromaffin cells during a period of exposure (6 h) corresponding to a period of prolonged metabolic or psychogenic stress in vivo during which PACAP is released from the splanchnic nerve onto chromaffin cells. As in PC12_bPAC1hop cells, PACAP induced in bovine chromaffin cells a cohort of genes encoding secretory proteins, identified by tiling for cellular localization using Ingenuity Pathway Analysis, which were highly enriched in informational molecules (secreted proteins acting at extracellular receptors). These included cytokines, growth factors and hormones, as well as converting enzymes, or protease inhibitors modulating converting enzyme function. Several neuropeptide prohormone transcripts not previously shown to be PACAP-regulated in chromaffin cells, such as thyrotropin-releasing hormone, and tachykinin precursor 1, were identified. Identification of this cohort of informational molecule-encoding transcripts suggests a wider, more integrative role for PACAP as a co-transmitter specific to stress transduction in the adrenal medulla.

SDHD-related chromaffin tumours: disease localisation to genetic dysfunction.

BACKGROUND/AIMS: To describe the management of a subject with multiple chromaffin tumours found to have a novel succinate dehydrogenase D (SDHD) mutation. CASE: A 15-year-old boy with marked hypertension was found to have elevated urinary catecholamines and initial imaging thought to represent bilateral adrenal phaeochromocytomas. An adrenal venous catheter was required to clarify a right adrenal phaeochromocytoma and a left abdominal paraganglioma, distinct from the left adrenal gland. Excision of these tumours, with preservation of the left adrenal gland, provided a cure for this subject without the need for lifelong steroid replacement. Genetic analysis revealed a novel SDHD mutation (c. 169 + 1 G>A) which was shown to result in loss of the 5' splice site and exclusion of exon 2 during splicing. This suggests the likely pathogenicity of this mutation. Disease surveillance in this subject and genetic screening of first degree relatives is ongoing. CONCLUSIONS: Genetic testing should be considered in all subjects presenting with a chromaffin tumour. In certain circumstances an adrenal venous sampling catheter for catecholamines may clarify diagnostic uncertainty. The complex management issues raised in the care of these subjects requires the involvement of a multidisciplinary team with the relevant expertise.

Reduction of sympathetic activity via adrenal-targeted GRK2 gene deletion attenuates heart failure progression and improves cardiac function after myocardial infarction.

Chronic heart failure (HF) is characterized by sympathetic overactivity and enhanced circulating catecholamines (CAs), which significantly increase HF morbidity and mortality. We recently reported that adrenal G protein-coupled receptor kinase 2 (GRK2) is up-regulated in chronic HF, leading to enhanced CA release via desensitization/down-regulation of the chromaffin cell alpha(2)-adrenergic receptors that normally inhibit CA secretion. We also showed that adrenal GRK2 inhibition decreases circulating CAs and improves cardiac inotropic reserve and function. Herein, we hypothesized that adrenal-targeted GRK2 gene deletion before the onset of HF might be beneficial by reducing sympathetic activation. To specifically delete GRK2 in the chromaffin cells of the adrenal gland, we crossed PNMTCre mice, expressing Cre recombinase under the chromaffin cell-specific phenylethanolamine N-methyltransferase (PNMT) gene promoter, with floxedGRK2 mice. After confirming a significant ( approximately 50%) reduction of adrenal GRK2 mRNA and protein levels, the PNMT-driven GRK2 knock-out (KO) offspring underwent myocardial infarction (MI) to induce HF. At 4 weeks post-MI, plasma levels of both norepinephrine and epinephrine were reduced in PNMT-driven GRK2 KO, compared with control mice, suggesting markedly reduced post-MI sympathetic activation. This translated in PNMT-driven GRK2 KO mice into improved cardiac function and dimensions as well as amelioration of abnormal cardiac beta-adrenergic receptor signaling at 4 weeks post-MI. Thus, adrenal-targeted GRK2 gene KO decreases circulating CAs, leading to improved cardiac function and beta-adrenergic reserve in post-MI HF. GRK2 inhibition in the adrenal gland might represent a novel sympatholytic strategy that can aid in blocking HF progression.

Effect of urotensin II on PC12 rat pheochromocytoma cells.

Urotensin II (U-II), initially identified as a cyclic peptide from fish urophysis, acts both as a strong vasoconstrictor and vasodilator in the vasculature via its receptor, G-protein coupled receptor 14. In addition, U-II and its receptor are co-expressed in the adrenal medulla, as well as in human pheochromocytomas, suggesting that this peptide may have some function in chromaffin cells. However, the precise role of U-II in these cells is unknown. In the present study, we initially demonstrate that U-II and its receptors mRNA are co-expressed in the rat pheochromocytoma cell line PC12. Moreover, U-II has not effect on tyrosine hydroxylase (TH), the rate-limiting enzyme involved in the biosynthesis of catecholamine, in terms of enzyme activity or at the mRNA level. However, U-II does induce an increase in the phosphorylation of TH specifically at Ser31 without affecting phosphorylation at the two other sites (Ser19 and Ser40). U-II also markedly activates extracellular signal-regulated kinases (ERKs) and p38, but not Jun N-terminal kinase. Blockade of the epidermal growth factor (EGF) receptor by AG1478 significantly reduces activation of ERK, suggesting that EGF receptor transactivation could act upstream of the ERK pathway in PC12 cells. Furthermore, U-II significantly increases dopamine secretion from PC12 cells. Finally, we show that U-II induced significant DNA synthesis in a ERKs and P38 mitogen-activated protein kinase-dependent manner. The results obtained indicate that U-II may exert its effects as a neuromodulator in chromaffin cells.

Electrophysiological and morphological features underlying neurotransmission efficacy at the splanchnic nerve-chromaffin cell synapse of bovine adrenal medulla.

The ability of adrenal chromaffin cells to fast-release catecholamines relies on their capacity to fire action potentials (APs). However, little attention has been paid to the requirements needed to evoke the controlled firing of APs. Few data are available in rodents and none on the bovine chromaffin cell, a model extensively used by researchers. The aim of this work was to clarify this issue. Short puffs of acetylcholine (ACh) were fast perifused to current-clamped chromaffin cells and produced the firing of single APs. Based on the currents generated by such ACh applications and previous literature, current waveforms that efficiently elicited APs at frequencies up to 20 Hz were generated. Complex waveforms were also generated by adding simple waveforms with different delays; these waveforms aimed at modeling the stimulation patterns that a chromaffin cell would conceivably undergo upon strong synaptic stimulation. Cholinergic innervation was assessed using the acetylcholinesterase staining technique on the supposition that the innervation pattern is a determinant of the kind of stimuli chromaffin cells can receive. It is concluded that 1) a reliable method to produce frequency-controlled APs by applying defined current injection waveforms is achieved; 2) the APs thus generated have essentially the same features as those spontaneously emitted by the cell and those elicited by fast-ACh perifusion; 3) the higher frequencies attainable peak at around 30 Hz; and 4) the bovine adrenal medulla shows abundant cholinergic innervation, and chromaffin cells show strong acetylcholinesterase staining, consistent with a tight cholinergic presynaptic control of firing frequency.

Melittin promotes exocytosis in neuroendocrine cells through the activation of phospholipase A2.

Regulated exocytosis requires the formation of trans-SNARE complexes that assemble at the interface between vesicles and the plasma membrane. Recent evidence has also highlighted the importance of lipid dynamic in this process. For instance, small cone-shaped lipids generating membrane curvature of the plasma membrane are synthesized at the exocytotic sites. Among those lipids, phosphatidic acid (PA) synthesized through the activity of phospholipase D (PLD) has been recently shown to be necessary to hormonal release in various cell types as well as in neurotransmitter release. In this paper we examined the possible role of arachidonic acid (AA), a fatty acid that is generated by the activity of phospholipase A2 (PLA2). Melittin a well-known activator of PLA2 was found to concomitantly promote catecholamine and chromogranin A (CGA) release in a calcium-dependent manner and also to increase AA synthesis in chromaffin cells. The effects of melittin on exocytosis and AA synthesis did not involve heterotrimeric G protein activation, but were suppressed by PLA2 inhibitors. Accordingly addition of exogenous PLA2 stimulated AA synthesis and catecholamine release in permeabilized chromaffin cells, whereas provision of exogenous AA directly increased exocytosis. These results suggest that AA produced by PLA2 activation during exocytosis may play an important regulatory role in hormonal and neurotransmitter release. The possibility that CGA-derived peptides released during exocytosis mimic the activity of melittin is discussed.

Mitochondrial Na+/Ca2+-exchanger blocker CGP37157 protects against chromaffin cell death elicited by veratridine.

Mitochondrial calcium (Ca(2+)) dyshomeostasis constitutes a critical step in the metabolic crossroads leading to cell death. Therefore, we have studied here whether 7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one (CGP37157; CGP), a blocker of the mitochondrial Na(+)/Ca(2+)-exchanger (mNCX), protects against veratridine-elicited chromaffin cell death, a model suitable to study cell death associated with Ca(2+) overload. Veratridine produced a concentration-dependent cell death, measured as lactate dehydrogenase released into the medium after a 24-h incubation period. CGP rescued cells from veratridine-elicited death in a concentration-dependent manner; its EC(50) was approximately 10 microM, and 20 to 30 microM caused near 100% cytoprotection. If preincubated for 30 min and washed out for 3 min before adding veratridine, CGP still afforded significant cytoprotection. At 30 microM, CGP blocked the veratridine-elicited free radical production, mitochondrial depolarization, and cytochrome c release. At this concentration, CGP also inhibited the Na(+) and Ca(2+) currents by 50 to 60% and the veratridine-elicited oscillations of cytosolic Ca(2+). This drastic cytoprotective effect of CGP could be explained in part through its regulatory actions on the mNCX.

Phospholipase D in calcium-regulated exocytosis: lessons from chromaffin cells.

Membrane fusion remains one of the less well-understood processes in cell biology. A variety of mechanisms have been proposed to explain how the generation of fusogenic lipids at sites of exocytosis facilitates secretion in mammalian cells. Over the last decade, chromaffin cells have served as an important cellular model to demonstrate a key role for phospholipase D1 (PLD1) generated phosphatidic acid in regulated exocytosis. The current model proposes that phosphatidic acid plays a biophysical role, generating a negative curvature and thus promoting fusion of secretory vesicles with the plasma membrane. Moreover, multiple signaling pathways converging on PLD1 regulation have been unraveled in chromaffin cells, suggesting a complex level of regulation dependant on the physiological context.

Involvement of protein kinase C in the regulation of Na+/Ca2+ exchanger in bovine adrenal chromaffin cells.

1. The Na(+)/Ca(2+) exchanger (NCX) exchanges Na+ and Ca(2+) bidirectionally through the forward mode (Ca(2+) extrusion) or the reverse mode (Ca(2+) influx). The present study was undertaken to clarify the role of protein kinase C (PKC) in the regulation of NCX in bovine adrenal chromaffin cells. The Na(+)-loaded cells were prepared by treatment with 100 micromol/L ouabain and 50 micromol/L veratridine. Incubation of Na(+)-loaded cells with Na(+)-free solution in the presence of the Ca(2+) channel blockers nicardipine (3 micromol/L) and omega-conotoxin MVIIC (0.3 micromol/L) caused Ca(2+) uptake and catecholamine release. 2. The Na(+)-dependent Ca(2+) uptake and catecholamine release were inhibited by 2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline (SEA0400; 1 micromol/L) and 2-[2-[4-(4-nitrobenzyloxy)phenyl]isothiourea (KB-R7943; 10 micromol/L), both NCX inhibitors. These results indicate that the Na(+)-dependent responses are mostly due to activation of the NCX working in the reverse mode. 3. In addition, we examined the effects of PKC inhibitors and an activator on the NCX-mediated Ca(2+) uptake and catecholamine release. Bisindolylmaleimide I (0.3-10 micromol/L) and chelerythrine (3-100 micromol/L), both PKC inhibitors, inhibited NCX-mediated responses. In contrast, phorbol 12,13-dibutyrate (0.1-10 micromol/L), a PKC activator, enhanced the responses. Bisindolylmaleimide I and chelerythrine, at effective concentrations for inhibition of Na(+)-dependent catecholamine release, had a little or no effect on high K(+)-induced catecholamine release in intact cells or on Ca(2+)-induced catecholamine release in beta-escin-permeabilized cells. 4. These results suggest that PKC is involved in the activation of NCX in bovine adrenal chromaffin cells.