Recapture after exocytosis causes differential retention of protein in granules of bovine chromaffin cells

After exocytosis, chromaffin granules release essentially all their catecholamines in small fractions of a second, but it is unknown how fast they release stored peptides and proteins. Here we compare the exocytic release of fluorescently labelled neuropeptide Y (NPY) and tissue plasminogen activator from single granules. Exocytosis was tracked by measuring the membrane capacitance, and single granules in live cells were imaged by evanescent field microscopy. Neuropeptide Y left most granules in small fractions of a second, while tissue plasminogen activator remained in open granules for minutes. Taking advantage of the dependence on pH of the fluorescence of green fluorescent protein, we used rhythmic external acidification to determine whether and when granules re-sealed. One-third of them re-sealed within 100 s and retained significant levels of tissue plasminogen activator. Re-sealing accounts for only a fraction of the endocytosis monitored in capacitance measurements. When external [Ca²⁺] was raised, even neuropeptide Y remained in open granules until they re-sealed. It is concluded that a significant fraction of chromaffin granules re-seal after exocytosis, and retain those proteins that leave granules slowly. We suggest that granules vary the stoichiometry of release by varying both granule re-sealing and the association of proteins with the granule matrix.     

Calcium signaling and exocytosis in adrenal chromaffin cells

At a given cytosolic domain of a chromaffin cell, the rate and amplitude of the Ca2+ concentration ([Ca2+]c) depends on at least four efficient regulatory systems: 1) plasmalemmal calcium channels, 2) endoplasmic reticulum, 3) mitochondria, and 4) chromaffin vesicles. Different mammalian species express different levels of the L, N, P/Q, and R subtypes of high-voltage-activated calcium channels; in bovine and humans, P/Q channels predominate, whereas in felines and murine species, L-type channels predominate. The calcium channels in chromaffin cells are regulated by G proteins coupled to purinergic and opiate receptors, as well as by voltage and the local changes of [Ca2+]c. Chromaffin cells have been particularly useful in studying calcium channel current autoregulation by materials coreleased with catecholamines, such as ATP and opiates. Depending on the preparation (cultured cells, adrenal slices) and the stimulation pattern (action potentials, depolarizing pulses, high K+, acetylcholine), the role of each calcium channel in controlling catecholamine release can change drastically. Targeted aequorin and confocal microscopy shows that Ca2+ entry through calcium channels can refill the endoplasmic reticulum (ER) to nearly millimolar concentrations, and causes the release of Ca2+ (CICR). Depending on its degree of filling, the ER may act as a sink or source of Ca2+ that modulates catecholamine release. Targeted aequorins with different Ca2+ affinities show that mitochondria undergo surprisingly rapid millimolar Ca2+ transients, upon stimulation of chromaffin cells with ACh, high K+, or caffeine. Physiological stimuli generate [Ca2+]c microdomains in which the local subplasmalemmal [Ca2+]c rises abruptly from 0.1 to approximately 50 microM, triggering CICR, mitochondrial Ca2+ uptake, and exocytosis at nearby secretory active sites. The fact that protonophores abolish mitochondrial Ca2+ uptake, and increase catecholamine release three- to fivefold, support the earlier observation. This increase is probably due to acceleration of vesicle transport from a reserve pool to a ready-release vesicle pool; this transport might be controlled by Ca2+ redistribution to the cytoskeleton, through CICR, and/or mitochondrial Ca2+ release. We propose that chromaffin cells have developed functional triads that are formed by calcium channels, the ER, and the mitochondria and locally control the [Ca2+]c that regulate the early and late steps of exocytosis.     

Rab3A negatively regulates activity-dependent modulation of exocytosis in bovine adrenal chromaffin cells

Members of the Rab family of monomeric GTPases have been implicated in vesicle trafficking, and Rab3A, located on synaptic vesicles in neurones and secretory vesicles in neuroendocrine cells, is likely to be involved in vesicle fusion leading to neurotransmitter release. A hydrolysis-deficient mutant of Rab3A, Rab3AQ81L, has been shown to potently inhibit hormone release. Here we show that the inhibition of hormone release by Rab3AQ81L is activity-dependent. Bovine adrenal chromaffin cells were induced to express Rab3AQ81L and green fluorescent protein by adenoviral gene transfer of a bicistronic construct. Fluorescent cells were stimulated with single depolarizations and trains of depolarizing pulses in whole cell perforated patch clamp recordings, and exocytosis was detected with cell capacitance measurements and carbon fibre amperometry. When single depolarizations were used to evoke exocytosis, cells expressing Rab3AQ81L showed a 50% reduction in response amplitude. When trains of brief depolarizations (10 or 40 ms) were used to evoke exocytosis, responses rapidly declined to zero in cells expressing Rab3AQ81L. Wild-type Rab3A had effects similar to Rab3AQ81L, causing significant inhibition of exocytosis only during repetitive stimulation. Expression of Rab5A did not alter exocytosis evoked by single depolarizations or repetitive stimulation. Applying a long duration depolarization in the middle of a stimulus train revealed that exocytotic efficacy (capacitance increase per amount of calcium influx) was not decreased in Rab3AQ81L-expressing cells. Instead, the activity-dependent increase in exocytotic efficacy observed in control cells did not occur in Rab3AQ81L-expressing cells. Our results suggest that Rab3A in the GTP bound conformation prevents activity-dependent facilitation.     

Nicotinic receptor-mediated intracellular calcium release in cultured bovine adrenal chromaffin cells

When cultured bovine adrenal chromaffin cells were stimulated by a nicotinic agonist, carbamylcholine (0.3 mM) or 1,1-dimethyl-4-phenylpiperazinium (50 microM), in the Ca2+-free medium containing 0.1 mM ethyleneglycoltetraacetic acid, intracellular free Ca2+ concentration ([Ca2+]i) rose from approximately 90 to 149 nM. High K+ (56 mM) and veratridine (50 microM) had no effect on the [Ca2+]i in Ca2+-free medium. The carbamylcholine-evoked rise in [Ca2+]i was blocked by hexamethonium (0.1 mM) but not by atropine (1 microM). Furthermore, the carbamylcholine-evoked rise in [Ca2+]i was inhibited by an intracellular Ca2+ antagonist, 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxy-benzoate hydrochloride (10 microM) but not by a calmodulin antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (20 microM). These results show the existence of intracellular Ca2+ store sites, from which Ca2+ is released upon nicotinic receptor stimulation, in cultured adrenal chromaffin cells.     

Enhancement of asynchronous and train-evoked exocytosis in bovine adrenal chromaffin cells infected with a replication deficient adenovirus

Bovine adrenal chromaffin cells share many characteristics with neurons and are often used as a simple model system to study ion channels and neurotransmitter release. We infected bovine adrenal chromaffin cells with a replication deficient adenovirus that induces expression of the common reporters beta-galactosidase and Green Fluorescent Protein via a bicistronic sequence. In perforated-patch recordings performed 48-h postinfection, peak calcium currents were reduced 32%, primarily due to loss of omega-conotoxin-GVIA-sensitive current. In contrast, sodium currents were increased 17%. Exocytosis, detected as an increase in membrane capacitance immediately after a single step depolarization, was reduced in proportion to the decrease in calcium influx. However, capacitance continued to increase for seconds after the depolarization. The amplitude of this poststimulus drift, or asynchronous exocytosis, was approximately three times that which occurred in a small fraction of control cells. Exocytosis evoked by repetitive stimulation with a train of brief depolarizations was increased 50%. Intracellular calcium levels measured during and after stimulation were lower, not higher, in adenovirus-infected cells. Electroporated cells showed reduced calcium currents but no enhancement of exocytosis. Cells infected with UV-irradiated virus showed reduced calcium currents and enhancement of exocytosis, but the changes were smaller than those caused by intact virus. Our results are consistent with the idea that adenovirus capsid and adenoviral DNA contribute to a Ca2+ influx- and [Ca2+]i-independent enhancement of exocytosis in bovine chromaffin cells.     

Cytoskeletal control of vesicle transport and exocytosis in chromaffin cells

Chromaffin cell exocytosis is a fascinating interplay between secretory vesicles and cellular components. One of these components is the cytoskeleton and its associated regulatory proteins. Transport of chromaffin secretory granules from their site of biosynthesis towards the active site of exocytosis requires both F-actin fine remodelling as well as microtubule trails. At least two molecular motors, myosins II and V, seem to play a crucial role in the control of F-actin dynamics and vectorial vesicle displacement respectively. Vesicle movement experiences spatial restrictions as they approach the cell cortical region, where the F-actin meshwork constitutes a barrier-limiting vesicle access to the plasmalemma. During secretion, cortical F-actin is locally disrupted providing access of vesicles to release sites on the plasmalemma. Removal of the stimulus restores cortical F-actin. Two pathways (Ca2+-scinderin and PKC-MARCKS) control F-actin changes during the secretory cycle . Furthermore, GTPases such as RhoA, that controls F-actin network integrity, and Cdc42 signalling which induces the formation of local actin filaments at active sites, provide additional evidence on the importance of F-actin as a key element in vesicle transport and in the exocytotic machinery of chromaffin cells.     

Extracellular ATP regulates exocytosis by inhibiting multiple Ca2+ channel types in bovine chromaffin cells

Feedback modulation of voltage-dependent Ca2+ channels by ATP is a well documented phenomenon in bovine chromaffin cells. However, its influence in the control of hormone release is at present poorly understood. By using combined patch-clamp and fura-2 fluorescence measurements we provide evidence that the three Ca2+ channel types (L, N and P/Q) expressed in bovine chromaffin cells are inhibited by ATP (30 microM), and that their involvement in the secretory response, as assayed by capacitance measurements, is roughly proportional to their contribution to the whole-cell Ca2+ current (ICa) both in the absence and presence of ATP. ATP did not modify the capacitance increase observed in cells dialyzed with Ca(2+)-EGTA buffers (1.5 microM free Ca2+), thus excluding a direct effect of ATP on the secretory machinery. Voltage predepolarizations or long chemical (2 s, 70 mM KCl) depolarizations attenuate the effect of ATP on exocytosis by partially relieving the inhibition of ICa Likewise, a strong stimulation that depletes the readily releasable pool of vesicles prevents an inhibitory effect of ATP on the secretory response. While these results lend support to the hypothesis of autocrine modulation of exocytosis by endogenously released ATP acting on P2y-purinoceptors to inhibit ICa, feedback regulation of the rate of release will be a complex function of the occupancy of those receptors and of the electrical and secretory activity of the cell.     

Hypophosphorylated neurofilament subunits in the cytoskeletal and soluble fractions of cultured bovine adrenal chromaffin cells

The neurofilament proteins in cultured bovine adrenal chromaffin cells are in a hypophosphorylated state, as determined by the co-migration of the 160,000 and 210,000 molecular weight subunits with in vitro dephosphorylated bovine brain subunits on sodium dodecyl sulfate polyacrylamide gels. In addition, chromaffin cells were not stained by anti-heavy neurofilament subunit that binds only to phosphorylated epitopes. Pulse-labeling with 32Pi in the presence and absence of the protein synthesis inhibitor emetine indicated that some neurofilament protein phosphorylation occurred co-translationally and/or immediately after synthesis of the proteins. Pulse-chase experiments showed that the three neurofilament proteins rapidly attained their maximal phosphorylation levels, as multiple forms of either of the respective subunits were not seen after a one hour chase. We found that Triton X-100-soluble forms of high molecular weight neurofilament and middle molecular weight neurofilament subunits were present in chromaffin cells, and they also co-migrated with standard neurofilament proteins dephosphorylated in vitro. However, there were differences between the phosphopeptide maps of cytoskeleton-associated and soluble middle molecular weight neurofilament subunit, suggesting that the localization of phosphate moieties rather than extent of phosphorylation influences the association of the subunit with neurofilaments. Double immunofluorescence staining of cell cultures with antibody to the 70,000 molecular weight subunit and with anti-vimentin showed that chromaffin cells do not express vimentin.     

Effects of pertussis toxin on the affinity of exocytosis for Ca2+ in bovine adrenal chromaffin cells

Effects of pertussis toxin (islet-activating protein, IAP) on the secretory function of bovine adrenal chromaffin cells in culture were studied. Treatment of chromaffin cells with IAP resulted in an increase in both basal release of catecholamine and evoked-release by either acetylcholine (ACh) or high K+. In the dose-response curve for ACh-evoked release, IAP treatment produced an increase of the maximal response without affecting the half-maximal concentration of ACh. When the cells were permeabilized with digitonin after IAP-pretreatment, Ca2(+)-dependent exocytosis was markedly increased where the affinity of exocytosis for Ca2+ was augmented. These findings suggest that IAP-sensitive GTP-binding protein (or proteins) directory controls the Ca2(+)-triggered process in the machinery of exocytosis by modulating the affinity for Ca2+ of its unknown target.     

Localized L-type calcium channels control exocytosis in cat chromaffin cells

Depolarizing 1-s pulses to 0 mV from a holding potential of −70 mV, induced whole-cell currents through Ca²⁺ channels (I _Ca) in patch-clamped cat adrenal medulla chromaffin cells. The dihydropyridine (DHP) furnidipine (3 μM) reduced the peak current by 47% and the late current by 80%. ω-Conotoxin GVIA (CgTx, 1 μM) reduced the peak I _Ca by 42% and the late I _Ca by 55%. Pulses (10 s duration) with 70 mM K⁺/2.5 mM Ca²⁺ solution (70 K⁺/2.5 Ca²⁺), applied to single fura-2-loaded cat chromaffin cells increased the cytosolic Ca²⁺ concentration ([Ca²⁺]_i from 0.1 to 2.21 μM; this increase was reduced by 43.7% by furnidipine and by 42.5% by CgTx. In the perfused cat adrenal gland, secretion evoked by 10-s pulses of 70 K⁺/2.5 Ca²⁺ was reduced by 25% by CgTx and by 96% by furnidipine. Similar results were obtained when secretion from superfused isolated cat adrenal chromaffin cells was studied and when using a tenfold lower [Ca²⁺]_o. The results are compatible with the existence of DHP-sensitive (L-type) as well as CgTx-sensitive (N-type) voltage-dependent Ca²⁺ channels in cat chromaffin cells. It seems, howevever, that though extracellular Ca²⁺ entry through both channel types leads to similar increments of averaged [Ca²⁺]_i, the control of catecholamine release is dominated only by Ca²⁺ entering through L-type Ca²⁺ channels. This supports the idea of a preferential segregation of L-type Ca²⁺ channels to localized “hot spots” in the plasmalemma of chromaffin cells where exocytosis occurs.     

Loose coupling between calcium channels and sites of exocytosis in chromaffin cells

Calcium microdomains generated by tight clusters of calcium channels regulate fusion of small vesicles at the synaptic terminal and have also been suggested to trigger exocytosis of large dense-core vesicles from neuroendocrine cells. To test this idea, we have compared sites of exocytosis and the spatial distribution of calcium channels in chromaffin cells. Fusion of individual vesicles was visualized using interference reflection microscopy and the submembranous calcium signal was assessed using total internal reflection fluorescence microscopy. Depolarization triggered a burst of exocytosis from up to seven sites in a membrane area of 11 μm², but these sites did not colocalize with calcium microdomains. Instead, calcium influx occurred in large patches (averaging 34 μm²) containing a mixture of P/Q- and N-type channels. About 20% of fusion events occurred outside calcium channel patches. Further, the delay between the onset of stimulation and a burst of exocytosis was prolonged for several seconds by increasing the concentration of the slow calcium chelator EGTA from 1.5 to 5 m m . These results demonstrate that while calcium channels and release sites tend to congregate in specialized regions of the surface membrane, these have dimensions of several micrometres. The dominant calcium signal regulating release in chromaffin cells is generated by the cooperative action of many channels operating over distances of many micrometres rather than discrete clusters of calcium channels generating localized microdomains.     

Presence of a high affinity uptake system for catecholamines in cultured bovine adrenal chromaffin cells

The uptake of catecholamines was investigated in bovine adrenal chromaffin cells cultured for 2 and 7 days. These cells, after their attachment onto the collagen-coated plates, began to develop processes which progressively increased in length with time in culture. Process outgrowth of a few cells was apparent on day 2 in culture. However, by day 7 most of the chromaffin cells possessed processes with a mean length of52.68 ± 1.27μm(n= 202). There was found to exist in both 2-day and 7-day-old cultures an uptake mechanism for (?)noradrenaline which in many aspects simulated the neuronal uptake₁ transport system in that it was saturable, followed Michaelis-Menten kinetics, had a high affinity for (?)noradrenaline with apparentK_m values ranging from 0.35–0.48 μm (n = 4) and 0.46 ? 0.67 μm (n = 4) on day 2 and day 7 respectively. This uptake process was blocked by low concentrations of desipramine (10^?7 M), a specific inhibitor of uptake₁, and like the neuronal uptake mechanism exhibited absolute Na⁺ dependency in which Li⁺ could not adequately replace Na⁺. However, uptake by the chromaffin cells did not show any stereochemical specificity towards the (?) form of noradrenaline nor did it exhibit structural specificity for (?)noradrenaline over (?)adrenaline.Therefore, it appeared that a high affinity accumulating mechanism for noradrenaline was present in cultured chromaffin cells isolated from adult bovine adrenal medulla. The properties of this uptake system did not vary significantly from day 2 to day 7 and thus did not parallel the morphological changes seen in culture.     

Physiological aspects of exocytosis in chromaffin cells of the adrenal medulla

The adrenal medulla is composed principally of groups of adrenergic and noradrenergic chromaffin cells, with minor populations of small intensely fluorescent cells and ganglionic neurones. Different molecular stimuli evoke distinct secretory events in the gland, involving the release of either adrenaline or noradrenaline together with various neuroactive peptides. The nature of the secretory response can be controlled at a central level or regulated locally within the gland. Specific innervation patterns to the different types of chromaffin cell have been implicated in central regulatory mechanisms, while several explanations for regulating secretion locally have been proposed. The differential distribution of various types of receptors between cell phenotypes, such as muscarinic or nicotinic acetylcholine receptors, histamine receptors, angiotensin receptors and different classes of opiate receptors between the two principal chromaffin cell populations could be involved in local control. In addition exocytosis parameters could be modulated differently in adrenergic and noradrenergic cells by phenotype-specific mechanisms, possibly involving molecules like Growth Associated Protein-43, Synaptosomal Associated Protein-25 isoforms or the p11 annexin subunit. The distribution of the various types of calcium channels is also known to vary between chromaffin cell subtypes. This short review examines possible ways in which specific innervation patterns in the adrenal gland could be programmed and discusses exocytosis mechanisms that could differ between chromaffin cell phenotypes. Data reviewed here suggest that the adrenal medulla should no longer be viewed as a homogeneous entity but as consisting of an ensemble of individual cell subpopulations each with a distinct secretory response that could in part reflect its local history.     

Altered exocytosis in chromaffin cells from mouse models of neurodegenerative diseases

Chromaffin cells from the adrenal gland (CCs) have extensively been used to explore the molecular structure and function of the exocytotic machinery, neurotransmitter release and synaptic transmission. The CC is integrated in the sympathoadrenal axis that helps the body maintain homoeostasis during both routine life and in acute stress conditions. This function is exquisitely controlled by the cerebral cortex and the hypothalamus. We propose the hypothesis that damage undergone by the brain during neurodegenerative diseases is also affecting the neurosecretory function of adrenal medullary CCs. In this context, we review here the following themes: (i) How the discharge of catecholamines is centrally and peripherally regulated at the sympathoadrenal axis; (ii) which are the intricacies of the amperometric techniques used to study the quantal release of single‐vesicle exocytotic events; (iii) which are the alterations of the exocytotic fusion pore so far reported, in CCs of mouse models of neurodegenerative diseases; (iv) how some proteins linked to neurodegenerative pathologies affect the kinetics of exocytotic events; (v) finally, we try to integrate available data into a hypothesis to explain how the centrally originated neurodegenerative diseases may alter the kinetics of single‐vesicle exocytotic events in peripheral adrenal medullary CCs.     

Calmodulin increases the initial rate of exocytosis in adrenal chromaffin cells

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

Facilitation of Ca2+-dependent exocytosis by Rac1-GTPase in bovine chromaffin cells

Rho family GTPases are primary mediators of cytoskeletal reorganization, although they have also been reported to regulate cell secretion. Yet, the extent to which Rho family GTPases are activated by secretory stimuli in neural and neuroendocrine cells remains unknown. In bovine adrenal chromaffin cells, we found Rac1, but not Cdc42, to be rapidly and selectively activated by secretory stimuli using an assay selective for the activated GTPases. To examine effects of activated Rac1 on secretion, constitutively active mutants of Rac1 (Rac1-V12, Rac1-L61) were transiently expressed in adrenal chromaffin cells. These mutants facilitated secretory responses elicited from populations of intact and digitonin-permeabilized cells as well as from cells under whole cell patch clamp. A dominant negative Rac1 mutant (Rac1-N17) produced no effect on secretion. Expression of RhoGDI, a negative regulator of Rac1, inhibited secretory responses while overexpression of effectors of Rac1, notably, p21-activated kinase (Pak1) and actin depolymerization factor (ADF) promoted evoked secretion. In addition, expression of effector domain mutants of Rac1-V12 that exhibit reduced activation of the cytoskeletal regulators Pak1 and Partner of Rac1 (POR1) resulted in a loss of Rac1-V12-mediated enhancement of evoked secretion. These findings suggest that Rac1, in part, functions to modulate secretion through actions on the cytoskeleton. Consistent with this hypothesis, the actin modifying drugs phalloidin and jasplakinolide enhanced secretion, while latrunculin-A inhibited secretion and eliminated the secretory effects of Rac1-V12. In summary, Rac1 was activated by secretory stimuli and modulated the secretory pathway downstream of Ca²⁺ influx, partly through regulation of cytoskeletal organization.     

Induction of neurofilament phosphorylation in cultured chromaffin cells

The distribution, structural organization and state of phosphorylation of neurofilaments have been examined in chromaffin cells from adult bovine adrenal medulla cultured under various conditions using a series of monoclonal antibodies directed against phosphorylated and nonphosphorylated epitopes of the 200,000 mol. wt subunit. Nonphosphorylated neurofilament epitopes were detected immunocytochemically to varying extents in chromaffin cells maintained under standard culture conditions for up to 3 weeks. Staining was usually limited to a perinuclear region from which fine filaments sometimes appeared to radiate around the nucleus. In marked contrast, none of the antibodies directed against phosphorylated neurofilament epitopes stained these structures. When cells were cultured under conditions favouring neurite outgrowth, in conditioned medium derived from intermediate lobe cultures, there was a more extensive expression of the nonphosphorylated neurofilament epitopes. In addition, phosphorylation of neurofilaments was induced. The phosphorylated neurofilament epitopes were restricted to the neurite, whereas the nonphosphorylated neurofilament epitopes were localized in both neurite extensions and perikarya. These results demonstrate that conditioned medium from intermediate lobe cells of the hypophysis not only provokes neurite outgrowth from chromaffin cells, but also supports neuronal maturation as demonstrated by the phosphorylation of neurofilaments in neurites.     

Palytoxin: a potent stimulator of catecholamine release from cultured bovine adrenal chromaffin cells.

The effect of palytoxin (PTX), a potent marine toxin, on catecholamine release from cultured bovine adrenal chromaffin cells was examined. PTX at concentrations of over 10(-9) M induced catecholamine release dose-dependently. About 40-50% of the total cellular catecholamine was released during 20-min incubation with 3 x 10(-8) M PTX. PTX-induced catecholamine release was dependent on both extracellular Na+ and Ca2+, and was inhibited by organic and inorganic Ca2+ channel blockers, but not by tetrodotoxin. PTX-induced increase in 45Ca2+ influx into the cells, which was associated with catecholamine release, was also inhibited by these Ca2+ channel blockers. These results indicated that PTX-induced catecholamine release was mediated by activation of Na(+)-dependent, tetrodotoxin (TTX) insensitive voltage-dependent Ca2+ channels.     

Excitatory and inhibitory actions of isoflurane in bovine chromaffin cells

Isoflurane, a halogenated volatile anesthetic, is thought to produce anesthesia by depressing CNS function. Many anesthetics, including isoflurane, are thought to modulate and/or directly activate GABA(A) receptors. Chromaffin cells are known to express functional GABA(A) receptors. We previously showed that activation of the GABA(A) receptors, with specific agonists, leads to cellular excitation resulting from the depolarized anion equilibrium potential. In this study, our goal was to determine whether isoflurane mimicked this response and to explore the functional consequences of this activation. Furthermore, we sought to study the actions of isoflurane on nicotinic acetylcholine receptors (nAChRs) as they mediate the "sympathetic drive" in these cells. For these studies the Ca(2+)-indicator dye fura-2 was used to assay [Ca(2+)](i). Amperometric measurements were used to assay catecholamine release. We show that bovine adrenal chromaffin cells were excited by isoflurane at clinically relevant concentrations. Isoflurane directly activated GABA(A) receptors found in chromaffin cells, which depolarized the cells and elevated [Ca(2+)](i). Application of isoflurane directly to the chromaffin cells elicited catecholamine secretion from these cells. At the same time, isoflurane suppressed activation of nAChRs, which presumably blocks "sympathetic drive" to the chromaffin cells. These latter results may help explain why isoflurane produces the hypotension observed clinically.