Characteristics of cytosolic Ca2+ elevation induced by muscarinic receptor activation in single adrenal chromaffin cells of the guinea pig

In Fura-2 loaded-single guinea pig adrenal chromaffin cells, muscarine, nicotine and KCl all caused an early peak rise in intracellular Ca concentration ([Ca²⁺]_i) followed by a sustained rise. In Ca²⁺-free solution, muscarine, but neither nicotine nor KCl, caused a transient increase in [Ca²⁺]_i, which was partially reduced by preceding application of caffeine or by treatment with ryanodine plus caffeine. In voltage-clamped cells at a holding potential of −60 mV, the muscarine-induced [Ca²⁺]_i, rise, especially its sustained phase, decreased in magnitude. intracellular application of inositol 1,4,5-trisphosphate caused a transient increase in [Ca²⁺]_i and inhibited the following [Ca²⁺]_i response to muscarine without affecting responses to nicotine and a depolarizing pulse. Muscarine evoked membrane depolarization following brief hyperpolarization in most cells tested. There was a significant positive correlation between the amplitude of the depolarization and the magnitude of the sustained rise in [Ca²⁺]_i. Muscarine-induced sustained [Ca²⁺]_i rise was much greater in the current-clamp mode than that in the voltage-clamp mode. The sustained phase of [Ca²⁺]_i rise and Mn²⁺ influx in response to muscarine were suppressed by a voltage-dependent Ca²⁺ channel blocker, methoxyverapamil. These results suggest that stimulation of muscarinic receptors causes not only extracellular Ca²⁺ entry, but also Ca²⁺ mobilization from inositol 1,4,5-trisphosphate-sensitive intracellular stores. Voltage-dependent Ca²⁺ channels may function as one of the Ca²⁺ entry pathways activated by muscarinic receptor in guinea pig adrenal chromaffin cells.     

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

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

Ca2+ influx and clearance at hyperpolarized membrane potentials modulate spontaneous and stimulated exocytosis in neuroendocrine cells

Neuroendocrine adrenal chromaffin cells release neurohormones catecholamines in response to Ca²⁺ entry via voltage-gated Ca²⁺ channels (VGCCs). Adrenal chromaffin cells also express non-voltage-gated channels, which may conduct Ca²⁺ at negative membrane potentials, whose role in regulation of exocytosis is poorly understood. We explored how modulation of Ca²⁺ influx at negative membrane potentials affects basal cytosolic Ca²⁺ concentration ([Ca²⁺]_i) and exocytosis in metabolically intact voltage-clamped bovine adrenal chromaffin cells. We found that in these cells, Ca²⁺ entry at negative membrane potentials is balanced by Ca²⁺ extrusion by the Na⁺/Ca²⁺ exchanger and that this balance can be altered by membrane hyperpolarization or stimulation with an inflammatory hormone bradykinin. Membrane hyperpolarization or application of bradykinin augmented Ca²⁺-carrying current at negative membrane potentials, elevated basal [Ca²⁺]_i, and facilitated synchronous exocytosis evoked by the small amounts of Ca²⁺ injected into the cell via VGCCs (up to 20 pC). Exocytotic responses evoked by the injections of the larger amounts of Ca²⁺ via VGCCs (> 20 pC) were suppressed by preceding hyperpolarization. In the absence of Ca²⁺ entry via VGCCs and Ca²⁺ extrusion via the Na⁺/Ca²⁺ exchanger, membrane hyperpolarization induced a significant elevation in [Ca²⁺]_i and asynchronous exocytosis. Our results indicate that physiological interferences, such as membrane hyperpolarization and/or activation of non-voltage-gated Ca²⁺ channels, modulate basal [Ca²⁺]_i and, consequently, segregation of exocytotic vesicles and their readiness to be released spontaneously and in response to Ca²⁺ entry via VGCCs. These mechanisms may play role in homeostatic plasticity of neuronal and endocrine cells.     

Immunolocalization of synexin (annexin VII) in adrenal chromaffin granules and chromaffin cells: evidence for a dynamic role in the secretory process

Summary Synexin (annexin VII) is a Ca²⁺- and phospholid-binding protein which has been proposed to play a role in Ca²⁺-dependent membrane fusion processes. Using a monoclonal antibody against synexin, Mab 10E7, and immunogold, we carried out a semiquantitative localization study of synexin in bovine adrenal medullary chromaffin granules, and in resting and nicotine-stimulated adrenal chromaffin cells. Isolated chromaffin granules contained very little synexin, whereas chromaffin granules aggregated with synexin (24 g/mg) and Ca²⁺ (1 mM) clearly showed synexin-associated immunogold particles in the vicinity of the granule membrane (1.88 gold particles per granule profile). In isolated, cultured adrenal chromaffin cells, synexin was present in the nucleus (5.5 particles/m²) and in the cytosol (5.3 particles/m²), but mainly around the granule membrane in the granular cell area (11.7 particles/m²). During the active phase of cholinergically stimulated catecholamine secretion, the amount of synexin label was reduced by 33% in the nucleus, by 23% in the cytosol, and by 51% in the granule area. The plasma membrane contained a small amount of synexin, which did not significantly change upon stimulation of the cells. We conclude that synexin is involved in the secretory process in chromaffin cells.     

Muscarinic and nicotinic receptor-mediated Ca2+ dynamics in rat adrenal chromaffin cells during development

To clarify when the cholinergic receptor-mediated secretion mechanism of developing adrenal chromaffin cells is expressed and becomes functional, morphological changes and intracellular calcium dynamics were studied by immunohistochemistry, electron microscopy, and Fura-2 digital image analysis. From embryonic day 14 to 16, adrenal medullary cells were immunoreactive to noradrenaline-synthesizing enzyme (dopamine β-hydroxylase) but not to adrenaline-synthesizing enzyme (phenylethanolamine N-methyltransferase). These cells contained either no granules or just a few granules of high electron density. Exocytotic figures were rarely observed in cells of the control or in cells after carbamylcholine stimulation. Nerve fibers in the adrenal medulla contained either no clear vesicles or very few. Neither methacholine nor nicotine caused a change of intracellular Ca²⁺ in most chromaffin cells. From embryonic day 18 to 20, chromaffin cells were immunoreactive to both dopamine β-hydroxylase and phenylethanolamine N-methyltransferase and they contained relatively numerous secretory granules. Exocytotic figures were often seen in cells after carbamylcholine stimulation. The intra-adrenal nerve fibers contained numerous clear vesicles and a few dense-cored vesicles. Methacholine caused no rise of intracellular Ca²⁺, but nicotine induced a low to relatively high rise in many cells. From postnatal day 2 or 3 to postnatal week 1, numerous cells were immunoreactive to both dopamine β-hydroxylase and phenylethanolamine N-methyltransferase, whereas some cells were reactive to dopamine β-hydroxylase alone. Chromaffin cells were divisible into noradrenaline cells and adrenaline cells based on the ultrastructural features of their granules. Methacholine induced a moderate rise of intracellular Ca²⁺ and nicotine caused a high rise in many chromaffin cells, whereas, in some chromaffin cells, methacholine induced no rise of intracellular Ca²⁺ and nicotine induced a high rise. These results suggest that morphological changes of the developing cells and the intra-adrenal nerve fibers are related to the expression of a cholinergic receptor-mediated secretion mechanism and that this mechanism via a nicotinic receptor-mediated Ca²⁺ signaling pathway precedes the muscarinic receptor-mediated one during development.     

Synexin protein is non-selective in its ability to increase Ca2+-dependent aggregation of biological and artificial membranes

Synexin, a soluble protein which increases the specificity of Ca²⁺ to aggregate isolated bovine chromaffin granules was prepared from bovine adrenal medullary tissue by the method of Creutz, Pazoles and Pollard (J. Biol. Chem. $\text{253}$, 2858–2866, 1978). We also find that synexin increases both the initial rate and final amplitude of Ca²⁺-promoted aggregation of granule membranes. This effect is Ca²⁺-specific. However in contrast to Creutz $\text{et}\text{al}$, we find that synexin also potentiates aggregation of adrenal medulla and liver mitochondria and microsomes as well as phosphatidylserine vesicles. This lack of membrane specificity argues against the suggestion of Creutz $\text{et}\text{al}$ that synexin specifically binds the granule to the plasma membrane prior to exocytosis $\text{in}\text{vivo}$.     

Roles of Nicotinic and Muscarinic Receptors in Calcium Signaling and Transmitter Release

Activation of acetylcholine receptors (AChR) triggers catecholamine release from adrenal chromaffin cells and release of neurotransmitters in neuron-to-neuron and neuromuscular junctions, including those on smooth muscle cells. Calcium ions play the role of the main intracellular messenger, which mediates these processes. In our study, we explored the properties of Ca²⁺ signaling triggered by activation of AChR by analyzing the characteristics of Ca²⁺ transients induced by selective activation of nicotinic (nAChR) and muscarinic (mAChR) cholinoreceptors using Fura-2 fluorescent measurements in experiments on rat chromaffin cells. Two populations of chromaffin cells, which in a different manner responded to AChR stimulation, were classified. We found that the mean frequency of quantum release induced by ACh is considerably higher than that during hyperpotassium cell depolarization. Comparative analysis of single secretory events showed that, in the case of stimulation by ACh, single secretory spikes demonstrate faster kinetic characteristics than those induced by depolarization. Statistical analysis of the integral magnitude (area) of single secretory spikes evoked by both types of stimulation showed no significant difference despite amplitude and kinetic dissimilarities between such secretory events. Mathematical modeling of the dynamics of the exocytotic processes led to the conclusion that the reason for the specific kinetic characteristics of single secretory responses may be different diameters of the secretory pores formed during fusion of secretory vesicles with the plasma membrane.     

The relationship between secretion and intracellular free calcium in bovine adrenal chromaffin cells

The effect of carbamylcholine and the calcium ionophore A23187 on catecholamine release and intracellular free calcium, [Ca²⁺]i, in bovine adrenal chromaffin cells was determined. At 10^–4M carbamylcholine maximal release occurred with an accompanying increase i n [Ca²⁺]i from a basal level of 168 nM to less than 300 nM. An increase in [Ca²⁺]i of a similar magnitude was found following challenge with 40 nM A23187. However, in this case, no catecholamine release occurred. These results suggest that stimulation of secretion from chromaffin cells by carbamylcholine may involve additional triggers which stimulate secretion at low [Ca²⁺]i.     

Expression of voltage-gated calcium channels augments cell susceptibility to membrane disruption by nanosecond pulsed electric field

We compared membrane permeabilization by nanosecond pulsed electric field (nsPEF) in HEK293 cells with and without assembled CaV1.3 L-type voltage-gated calcium channel (VGCC). Individual cells were subjected to one 300-ns pulse at 0 (sham exposure); 1.4; 1.8; or 2.3 kV/cm, and membrane permeabilization was evaluated by measuring whole-cell currents and by optical monitoring of cytosolic Ca²⁺. nsPEF had either no effect (0 and 1.4 kV/cm), or caused a lasting (>80 s) increase in the membrane conductance in about 50% of cells (1.8 kV/cm), or in all cells (2.3 kV/cm). The conductance pathway opened by nsPEF showed strong inward rectification, with maximum conductance increase for the inward current at the most negative membrane potentials. Although these potentials were below the depolarization threshold for VGCC activation, the increase in conductance in cells which expressed VGCC (VGCC+ cells) was about twofold greater than in cells which did not (VGCC− cells). Among VGCC+ cells, the nsPEF-induced increase in membrane conductance showed a positive correlation with the amplitude of VGCC current measured in the same cells prior to nsPEF exposure. These findings demonstrate that the expression of VGCC makes cells more susceptible to membrane permeabilization by nsPEF. Time-lapse imaging of nsPEF-induced Ca²⁺ transients confirmed permeabilization by a single 300-ns pulse at 1.8 or 2.3 kV/cm, but not at 1.4 kV/cm, and the transients were expectedly larger in VGCC+ cells. However, it remains to be established whether larger transients reflected additional Ca²⁺ entry through VGCC, or were a result of more severe electropermeabilization of VGCC+ cells.     

pH modulation of large conductance potassium channel from adrenal chromaffin granules

We report here that large conductance K⁺ selective channel in adrenal chromaffin granules is controlled by pH. We measured electrogenic influx of ⁸⁶Rb⁺ into chromaffin granules prepared from bovine adrenal gland medulla. The ⁸⁶Rb⁺ influx was inhibited by acidic pH. Purified chromaffin granule membranes were also fused with planar lipid bilayer. A potassium channel with conductance of 432±9 pS in symmetric 450 mM KCl was observed after reconstitution into lipid bilayer. The channel activity was unaffected by charybdotoxin, a blocker of the Ca²⁺-activated K⁺ channel of large conductance. It was observed that acidification to pH 6.4 cis side of the membrane lowered the channel open probability and single channel conductance. Whereas only weak influence on the single channel current amplitude and open probability were observed upon lowering of the pH at the trans side. We conclude that a pH-sensitive large conductance potassium channel operates in the chromaffin granule membrane.     

Modulation of biological responses to 2 ns electrical stimuli by field reversal

Nanosecond bipolar pulse cancellation, a recently discovered phenomenon, is modulation of the effects of a unipolar electric pulse exposure by a second pulse of opposite polarity. This attenuation of biological response by reversal of the electric field direction has been reported with pulse durations from 60 ns to 900 ns for a wide range of endpoints, and it is not observed with conventional electroporation pulses of much longer duration (>100 μs) where pulses are additive regardless of polarity. The most plausible proposed mechanisms involve the field-driven migration of ions to and from the membrane interface (accelerated membrane discharge). Here we report 2 ns bipolar pulse cancellation, extending the scale of previously published results down to the time required to construct the permeabilizing lipid electropores observed in molecular simulations. We add new cancellation endpoints, and we describe new bipolar pulse effects that are distinct from cancellation. This new data, which includes transport of cationic and anionic permeability indicators, fluorescence of membrane labels, and patterns of entry into permeabilized cells, is not readily explained by the accelerated discharge mechanism. We suggest that multi-step processes that involve first charged species movement and then responses of cellular homeostasis and repair mechanisms are more likely to explain the broad range of reported results.     

Docking of chromaffin granules—A necessary step in exocytosis?

Putative docking of secretory vesicles comprising recognition of and attachment to future fusion sites in the plasma membrane has been investigated in chromaffin cells of the bovine adrenal medulla and in rat phaeochromocytoma (PC 12) cells. Upon permeabilization with digitonin, secretion can be stimulated in both cell types by indreasing the free Ca²⁺-concentration to M levels. Secretory activity can be elicited up to 1 hr after starting permeabilization and despite the loss of soluble cytoplasmic components indicating a stable attachment of granules to the plasma membrane awaiting the trigger for fusion. Docked granules can be observed in the electron microscope in permeabilized PC 12 cells which contain a large proportion of their granules aligned underneath the plasma membrane. The population of putatively docked granules in chromaffin cells cannot be as readily discerned due to the dispersal of granules throughout the cytoplasm. Further experiments comparing PC 12 and chromaffin cells suggest that active docking but not transport of granules can still be performed by permeabilized cells in the presence of Ca²⁺: a short (2 min) pulse of Ca²⁺ in PC 12 cells leads to the secretion of almost all releasable hormone over a 15 min observation period whereas, in chromaffin cells, with only a small proportion of granules docked, withdrawal of Ca²⁺ leads to an immediate halt in secretion. Transport of chromaffin granules from the Golgi to the plasma membrane docking sites seems to depend on a mechanism sensitive to permeabilization. This is shown by the difference in the amount of hormone released from the two permeabilized cell types, reflecting the contrast in the proportion of granules docked to the plasma membrane in PC 12 or chromaffin cells. Neither docking nor the docked state are influenced by cytochalasine B or colchicine. The permeabilized cell system is a valuable technique for thein vitro study of interaction between secretory vesicles and their target membrane.     

A Sequential Vesicle Pool Model with a Single Release Sensor and a Ca2+-Dependent Priming Catalyst Effectively Explains Ca2+-Dependent Properties of Neurosecretion

Neurotransmitter release depends on the fusion of secretory vesicles with the plasma membrane and the release of their contents. The final fusion step displays higher-order Ca²⁺ dependence, but also upstream steps depend on Ca²⁺. After deletion of the Ca²⁺ sensor for fast release – synaptotagmin-1 – slower Ca²⁺-dependent release components persist. These findings have provoked working models involving parallel releasable vesicle pools (Parallel Pool Models, PPM) driven by alternative Ca²⁺ sensors for release, but no slow release sensor acting on a parallel vesicle pool has been identified. We here propose a Sequential Pool Model (SPM), assuming a novel Ca²⁺-dependent action: a Ca²⁺-dependent catalyst that accelerates both forward and reverse priming reactions. While both models account for fast fusion from the Readily-Releasable Pool (RRP) under control of synaptotagmin-1, the origins of slow release differ. In the SPM the slow release component is attributed to the Ca²⁺-dependent refilling of the RRP from a Non-Releasable upstream Pool (NRP), whereas the PPM attributes slow release to a separate slowly-releasable vesicle pool. Using numerical integration we compared model predictions to data from mouse chromaffin cells. Like the PPM, the SPM explains biphasic release, Ca²⁺-dependence and pool sizes in mouse chromaffin cells. In addition, the SPM accounts for the rapid recovery of the fast component after strong stimulation, where the PPM fails. The SPM also predicts the simultaneous changes in release rate and amplitude seen when mutating the SNARE-complex. Finally, it can account for the loss of fast- and the persistence of slow release in the synaptotagmin-1 knockout by assuming that the RRP is depleted, leading to slow and Ca²⁺-dependent fusion from the NRP. We conclude that the elusive ‘alternative Ca²⁺ sensor’ for slow release might be the upstream priming catalyst, and that a sequential model effectively explains Ca²⁺-dependent properties of secretion without assuming parallel pools or sensors.     

Ca2+ dependence and kinetics of cell membrane repair after electropermeabilization

Electroporation, in particular with nanosecond pulses, is an efficient technique to generate nanometer-size membrane lesions without the use of toxins or other chemicals. The restoration of the membrane integrity takes minutes and is only partially dependent on [Ca²⁺]. We explored the impact of Ca²⁺ on the kinetics of membrane resealing by monitoring the entry of a YO-PRO-1 dye (YP) in BPAE and HEK cells. Ca²⁺ was promptly removed or added after the electric pulse (EP) by a fast-step perfusion. YP entry increased sharply after the EP and gradually slowed down following either a single- or a double-exponential function. In BPAE cells permeabilized by a single 300- or 600-ns EP at 14 kV/cm in a Ca²⁺-free medium, perfusion with 2 mM of external Ca²⁺ advanced the 90% resealing and reduced the dye uptake about twofold. Membrane restoration was accomplished by a combination of fast, Ca²⁺-independent resealing (τ = 13–15 s) and slow, Ca²⁺-dependent processes (τ ~70 s with Ca²⁺ and ~ 110 s or more without it). These time constants did not change when the membrane damage was doubled by increasing EP duration from 300 to 600 ns. However, injury by microsecond-range EP (300 and 600 μs) took longer to recover even when the membrane initially was less damaged, presumably because of the larger size of pores made in the membrane. Full membrane recovery was not prevented by blocking both extra- and intracellular Ca²⁺ (by loading cells with BAPTA or after Ca²⁺ depletion from the reticulum), suggesting the recruitment of unknown Ca²⁺-independent repair mechanisms.     

Neuronal differentiation of PC12 cells abolishes the expression of membrane androgen receptors

Sex steroids affect adrenal chromaffin cell function. In the present work, we have examined the expression and functional significance of membrane androgen receptor sites in normal rat adrenal chromaffin cells and in the PC12 rat pheochromocytoma cell line which can differentiate to either a neuronal or to an epithelial phenotype and expresses membrane estrogen receptor sites. Our data are as follows: (a) no cytosolic androgen receptors were found in both normal chromaffin and PC12 cells; (b) both types of chromaffin cells expressed high affinity membrane testosterone binding sites; (c) activation of these sites increased cytosolic Ca²⁺, decreased catecholamine secretion and induced apoptosis; (d) NGF-induced neuronal differentiation of PC12 cells resulted in the suppression of the number of membrane testosterone sites. In conclusion, our data provide evidence for the existence of specific membrane testosterone receptors on adrenal chromaffin cells via which androgens, (some of them originating in the cortex) modulate their function. Neuronal differentiation of chromaffin cells results in a significant attenuation of these effects, via suppression of the expression of membrane androgen receptors suggesting, that the latter are specific for epithelioid chromaffin cells.     

Nanosecond Electric Pulses: A Novel Stimulus for Triggering Ca<Superscript>2+</Superscript> Influx into Chromaffin Cells Via Voltage-Gated Ca<Superscript>2+</Superscript> Channels

Exposing bovine chromaffin cells to a single 5 ns, high-voltage (5 MV/m) electric pulse stimulates Ca²⁺ entry into the cells via L-type voltage-gated Ca²⁺ channels (VGCC), resulting in the release of catecholamine. In this study, fluorescence imaging was used to monitor nanosecond pulse-induced effects on intracellular Ca²⁺ level ([Ca²⁺]_i) to investigate the contribution of other types of VGCCs expressed in these cells in mediating Ca²⁺ entry. ω-Conotoxin GVIA and ω-agatoxin IVA, antagonists of N-type and P/Q-type VGCCs, respectively, reduced the magnitude of the rise in [Ca²⁺]_i elicited by a 5 ns pulse. ω-conotoxin MVIIC, which blocks N- and P/Q-type VGCCs, had a similar effect. Blocking L-, N-, and P\Q-type channels simultaneously with a cocktail of VGCC inhibitors abolished the pulse-induced [Ca²⁺]_i response of the cells, suggesting Ca²⁺ influx occurs only via VGCCs. Lowering extracellular K⁺ concentration from 5 to 2 mM or pulsing cells in Na⁺-free medium suppressed the pulse-induced rise in [Ca²⁺]_i in the majority of cells. Thus, both membrane potential and Na⁺ entry appear to play a role in the mechanism by which nanoelectropulses evoke Ca²⁺ influx. However, activation of voltage-gated Na⁺ channels (VGSC) is not involved since tetrodotoxin (TTX) failed to block the pulse-induced rise in [Ca²⁺]_i. These findings demonstrate that a single electric pulse of only 5 ns duration serves as a novel stimulus to open multiple types of VGCCs in chromaffin cells in a manner involving Na⁺ transport across the plasma membrane. Whether Na⁺ transport occurs via non-selective cation channels and/or through lipid nanopores remains to be determined.     

Positive and negative controls by protein kinases of sodium-dependent Ca2+ efflux from cultured bovine adrenal chromaffin cells stimulated by lysophosphatidic acid

We previously found that lysophosphatidic acid (LPA), a bioactive phospholipid, induced Na⁺-dependent Ca²⁺ efflux from cultured bovine adrenal chromaffin cells, possibly by activating a Na⁺/Ca²⁺ exchanger. The present study on the structure-activity relationship of its action revealed that 1-acyl type LPAs were stronger stimulants than the corresponding 1-O-alkyl type LPAs having a long alkyl moiety with the same chain length. Lysophosphatidylglycerol, suramin and N-palmitoyl-tyrosine phosphoric acid have all been reported to inhibit the action of LPA in some animal cells and platelets, but only lysophosphatidylglycerol was found to inhibit selectively LPA-induced Ca²⁺ efflux from chromaffin cells. LPA-induced Ca²⁺ extrusion was suggested to be involved in both acceleration of return of intracellular Ca²⁺ in Fura 2-loaded bovine chromaffin cells after addition of carbachol, and inhibition of carbachol-induced catecholamine release when the cells were co-incubated with LPA. The Ca²⁺ efflux from chromaffin cells stimulated by LPA was augmented by their pretreatment with staurosporine or calphostin C, inhibitors of protein kinase C, but reduced by their preincubation with phorbol 12-myristate 13-acetate. Furthermore, the response to LPA was potentiated by sodium vanadate, a protein tyrosine phosphatase inhibitor, but inhibited by genistein, an inhibitor of protein tyrosine kinase. These results suggest that protein kinase C and protein tyrosine kinase are involved negatively and positively, respectively, in the signal transduction triggered by LPA, leading to activation of the Na⁺/Ca²⁺ exchanger.     

CaV1.3 as pacemaker channels in adrenal chromaffin cells: Specific role on exo- and endocytosis?

Voltage-gated L-type calcium channels (LTCCs) are expressed in adrenal chromaffin cells. Besides shaping the action potential (AP), LTCCs are involved in the excitation-secretion coupling controlling catecholamine release and in Ca²⁺-dependent vesicle retrieval. Of the two LTCCs expressed in chromaffin cells (Ca_V1.2 and Ca_V1.3), Ca_V1.3 possesses the prerequisites for pacemaking spontaneously firing cells: low-threshold, steep voltage-dependence of activation and slow inactivation. By using Ca_V1 .3^-/- KO mice and the AP-clamp it has been possible to resolve the time course of Ca_V1.3 pacemaker currents, which is similar to that regulating substantia nigra dopaminergic neurons. In mouse chromaffin cells Ca_V1.3 is coupled to fast-inactivating BK channels within membrane nanodomains and controls AP repolarization. The ability to carry subthreshold Ca²⁺ currents and activate BK channels confers to Ca_V1.3 the unique feature of driving Ca²⁺ loading during long interspike intervals and, possibly, to control the Ca²⁺-dependent exocytosis and endocytosis processes that regulate catecholamine secretion and vesicle recycling.     

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

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

Arachidonic Acid Activates Cation Channels in Bovine Adrenal Chromaffin Cells

Abstract— Microscopic fluorescence analysis of fura-2-loaded bovine adrenal chromaffin cells demonstrates that ～70% of the cells responded to arachidonic acid in increasing the intracellular Ca²⁺ concentration. Because this increase was markedly less in the absence of external Ca²⁺, we examined the effect of arachidonic acid on Ca²⁺ influx electrophysiologically. Bath application of 10 μM arachidonic acid induced a long-lasting inward current when the cell was clamped at -50 mV. Other fatty acids, such as oleic acid, linoleic acid, eicosatrienoic acid, and eicosa-pentaenoic acid, were all ineffective. The current-voltage relationships suggest that arachidonic acid may activate voltage-insensitive channels. Arachidonic acid (2μM) activated a single-channel current in the inside-out patch, even in the presence of inhibitors of cyclooxygenase and lipoxygenase, possibly suggesting that arachidonic acid could activate channels directly. The onset delay of the inward channel current in the outside-out patch configuration (54.02 ± 63.5 s; mean SD) was significantly shorter than that in the inside-out patch one (197.3 ± 177.7 s). Washout of arachidonic acid decreased the probability of channel openings in the outside-out patch but not in the inside-out one. These results suggest that arachidonic acid activates channels reversibly from outside of the plasma membrane. The unitary conductarce for Ca²⁺ of arachidonic acid-activated channel was ～17 pS. The arachidonic acid-activated channel was permeable to Ba²⁺, Ca²⁺, and Na⁺ but not to Cl^?. The opening probability of the arachidonic acid-activated channel did not depend on membrane potential. These results demonstrate that arachidonic acid activates cation-selective, Ca²⁺-permeable channels in bovine adrenal chromaffin cells.