Temperature dependence of release of vesicular content in bovine chromaffin cells

Recent studies suggest that the time course of secretion of the vesicular content in bovine chromaffin cells is much slower than in the peripheral or the central nervous system, but the reasons for this marked difference are not known. In this study we try to assess the importance of factors that may influence the time course of release of the vesicular content of bovine chromaffin cells, namely: (1) diffusion of catecholamines in the extracellular solution, (2) dissociation of catecholamines from the matrix of chromogranin A, and (3) the kinetics of opening and closing of the fusion pore. The temperature dependence of the time course and the amplitude of the spontaneous current spikes were examined using the carbon filament recording technique in amperometric mode. The change in amplitude was not statistically significant, but both the rise and the decay times were shortened (from 29 ± 12 to 16 ± 5 ms, and from 87 ± 26 to 57 ± 11 ms respectively) as temperature was raised by 20 °C [from 15 to 35 °C; n = 6; the changes were statistically significant at the level of P = 0.05; their respective temperature coefficients (Q ₁₀) were 1.4 and 1.3]. The areas underneath the spontaneous current spikes, however, were not altered significantly. Neither the relationship between the rise and the decay times nor the frequency of occurrence of the spontaneous current spikes changed consistently as the temperature was raised. However, the frequency histograms could, in all cases, be well described by a monoexponential function. It is concluded that the release of catecholamine content from the individual vesicles in bovine chromaffin cells is probably mostly determined by the dissociation of catecholamines from the matrix of chromogranin A. Received: 10 October 1995/Received after revision and accepted: 29 April 1996     

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.     

Catecholamine release from bovine chromaffin cells: the role of sodium-calcium exchange in ouabain-evoked release

Spontaneous catecholamine (CA) release from bovine chromaffin cells maintained in primary tissue culture has been measured after pre-loading the cells with [3H]noradrenaline. Ouabain inhibited 86Rb+ uptake and increased 3H release in a concentration-dependent manner during a 60 min incubation period. Low external Na+ (5 mM: Li+ substitution) also increased 3H release. Whereas the 3H-releasing action of ouabain was maintained, the Li(+)-evoked release decreased with time. The effects of both ouabain and low Na+ solution on 3H release were completely inhibited by removal of Ca2+ from the external medium even though in Ca2(+)-free solution ouabain further inhibited 86Rb+ uptake into the cells. Readmission of Ca2+ to Na(+)-loaded cells (10-4 M-ouabain in Ca2(+)-free-1 mM-EGTA solution for 60 min) markedly increased the release of 3H. In the additional presence of diphenylhydantoin (DPH, 10-4 M) 3H release was significantly less on Ca2+ readmission. The 3H release from Na(+)-loaded cells was proportional to the concentration of Ca2+ readmitted. The 3H release was further increased from Na(+)-loaded cells in response to Ca2+ readmission when [Na+]o was lowered from 149 to 5 mM (Li+, choline+, Tris+ or sucrose substitution) though Li+ was less effective than the other Na+ substitutes. Potassium removal from the external medium significantly inhibited the 3H release evoked by Ca2+ readmission to Na(+)-loaded cells, even when [Ca2+]o was greater than normal (7.5 mM) or if Ca2+ was readmitted in low [Na+]o solution. Rb+, Cs+ or Li+ could substitute for K+ with the order of potency: Rb+ greater than or equal to K+ greater than Cs+ greater than Li+. A slight increase of external K+ (10.8 mM) potentiated the 3H release from Na(+)-loaded cells on Ca2+ readmission, but a higher concentration of K+ (149.4 mM) had the opposite action. The data is consistent with the hypothesis that ouabain-evoked CA release from bovine chromaffin cells is, in part, a consequence of an internal Na(+)-dependent Ca2+ influx. The evidence also suggests that there is Na(+)-Ca2+ competition at the external arm of the exchanger together with a monovalent cation activation site.     

Time course of release of content of single vesicles in bovine chromaffin cells

The time course of the spontaneous current spikes produced by release of the catecholamine contents of individual vesicles was examined in bovine chromaffin cells using carbon filament electrodes. The rate of spontaneous release was enhanced by adding either LaCl₃ (0.01–0.5 mM) or BaCl₂ (2 mM) to the extracellular solution. A paucity of events of very short duration was evident from the frequency histograms of the rise and the decay times. In the scatterograms of the rise and of the decay times the regression lines are invariably positive (i.e. the longer the duration of the rise times the longer the duration of the decay times). However, the regression lines never go through the origin but intercept the ordinate (the axis of the decay times) at (± SD) 16.1 ± 6.4 ms (n = 11). On the other hand, the regression lines of paired rise and decay times for the time courses of diffusion are both linear and go through the origin. This relationship holds irrespective of whether the diffusion from an instantaneous point source was assumed to occur in an infinite plane or in an infinite volume. Therefore our experimental findings are incompatible with the model(s) assuming that diffusional broadening determines entirely the time course of current spikes. However, they can be explained, although only partially, by the possible slow speed of the electrode. They thus suggest that in chromaffin cells the duration of exocytosis of individual vesicles is much longer than in synapses. Received: 25 April 1995 /Received after revision: 7 August 1995 /Accepted: 19 September 1995     

Change of quantal size and parameters of release with stimulation in bovine chromaffin cells

Changes in quantal size and in the parameters of release were examined in chromaffin cells using amperometric recordings during and following various stimuli that induce secretion. As a general rule, a greater quantal content was associated with a greater quantal size. Following a short depolarizing pulse (0.5-2 s; 100 mV from a holding potential of -80 mV), the mean value of quantal size increased by 54% over several seconds before gradually (over tens of seconds) returning toward the control value, whilst its variability rose by 62%. The changes observed following 30-s applications of high extracellular K+ (50 mM) were more modest. A rapid application of short depolarizing pulses (2 s every 10-20 s; 100 mV from a holding potential of -80 mV) also led, at least initially, to greater quantal content and quantal size. Mean quantal size rose initially by 68%, but decreased subsequently to 31% below pre-stimulation levels. In whole-clamped cells, the frequency of quantal release can rise abruptly, probably owing to a mechanical disturbance that makes the membrane leaky to Ca2+. In such cases, a marked rise in quantal content (>ten-fold) was paralleled by an almost as dramatic (up to ten-fold) rise in quantal size and an important, although less pronounced and slower, rise in its variability (up to four-fold). The return toward control values of mean quantal size occurred over several minutes, whilst its variability decayed more slowly. The release parameters were evaluated directly from the number of events to avoid a large and time-dependent contribution of the amplitude variability of spontaneous amperometric current spikes (minis). In general, the greater probability of release contributed more than the greater size of the immediately available store to the increase in quantal output. In conclusion, quantal size was found to be highly labile. Its change can alter strongly the facilitation and depression of evoked quantal output and probably occurs due to a preferential release of large vesicles that are more efficient barriers to Ca2+ diffusion when Ca2+ rises rapidly following a synchronous opening of several Ca2+ channels. When intracellular Ca2+ levels rise slowly to threshold levels for secretion, as during an asynchronous and generally spontaneous release, vesicles are less effective diffusion barriers and quantal size changes less.     

Adrenal medulla calcium channel population is not conserved in bovine chromaffin cells in culture

During the stress response adrenal medullary chromaffin cells release catecholamines to the bloodstream. Voltage-activated calcium channels present in the cell membrane play a crucial role in this process. Although the electrophysiological and pharmacological properties of chromaffin cell calcium channels have been studied in detail, the molecular composition of these channels has not been defined yet. Another aspect that needs to be explored is the extent to which chromaffin cells in culture reflect the adrenal medulla calcium channel characteristics. In this sense, it has been described that catecholamine release in the intact adrenal gland recruits different calcium channels than those recruited during secretion from cultured chromaffin cells. Additionally, recent electrophysiological studies show that chromaffin cells in culture differ from those located in the intact adrenal medulla in the contribution of several calcium channel types to the whole cell current. However there is not yet any study that compares the population of calcium channels in chromaffin cells with that one present in the adrenal medulla. In order to gain some insight into the roles that calcium channels might play in the adrenal medullary cells we have analyzed the alpha1 subunit mRNA expression profile. We demonstrate that the expression pattern of voltage-dependent calcium channels in cultured bovine chromaffin cells markedly differs from that found in the native adrenal medulla and that glucocorticoids are only partially involved in those differences. Additionally, we show, for the first time, that the cardiac isoform of L-type calcium channel is present in both bovine adrenal medulla and cultured chromaffin cells and that its levels of expression do not vary during culture.     

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.     

Histamine promotes excitability in bovine adrenal chromaffin cells by inhibiting an M-current

The current study has investigated the electrophysiological responses evoked by histamine in bovine adrenal chromaffin cells using perforated-patch techniques. Histamine caused a transient hyperpolarization followed by a sustained depolarization of 7.2 ± 1.4 mV associated with an increase in spontaneous action potential frequency. The hyperpolarization was abolished after depleting intracellular Ca²⁺ stores with thapsigargin (100 n m ), and was reduced by 40 % with apamin (100 n m ). Membrane resistance increased by about 60 % during the histamine-induced depolarization suggesting inhibition of a K⁺ channel. An inward current relaxation, typical of an M-current, was observed in response to negative voltage steps from a holding potential of −30 mV. This current reversed at −81.6 ± 1.8 mV and was abolished by the M-channel inhibitor linopirdine (100 μ m ). During application of histamine, the amplitude of M-currents recorded at a time corresponding with the sustained depolarization was reduced by 40 %. No inward current rectification was observed in the range −150 to −70 mV, and glibenclamide (10 μ m ) had no effect on either resting membrane potential or the response to histamine. The results show that an M-current is present in bovine chromaffin cells and that this current is inhibited during sustained application of histamine, resulting in membrane depolarization and increased discharge of action potentials. These results demonstrate for the first time a possible mechanism coupling histamine receptors to activation of voltage-operated Ca²⁺ channels in these cells.     

Pervanadate inhibits volume-sensitive chloride current in bovine chromaffin cells

 The role of protein tyrosine phosphorylation in the activation of the volume-sensitive Cl^– current in bovine chromaffin cells was investigated by studying the effects of inhibitors of protein tyrosine kinases (PTKs) and phosphatases (PTPs). The whole-cell current was induced by intracellular guanosine-5’-0-(3-thiotriphosphate) (GTP-[γ-S], 100–250 μM), the nonhydrolysable GTP analogue, or by cell inflation through the patch pipette under voltage-clamp conditions. PTK inhibitors tyrphostin B46 (5–50 μM) and genistein (200 μM) did not inhibit the volume-sensitive Cl^– current nor did they induce it in the absence of other stimuli. In contrast, the PTP inhibitor pervanadate (200 μM) applied intracellularly prevented activation of the current. Voltage-activated Na⁺ and Ca²⁺ currents were unaffected by pervanadate. Neither sodium orthovanadate nor hydrogen peroxide alone mimicked the action of pervanadate. Other PTP inhibitors tested, i.e. ammonium molybdate (10–100 μM), phenylarsine oxide (10 μM), and ZnCl₂ (500 μM), as well as the serine/threonine protein phosphatases inhibitor okadaic acid (200 nM) failed to inhibit the volume-sensitive Cl^– current. It is suggested that the inhibitory action of pervanadate indicates the involvement of protein tyrosine phosphorylation in the regulation of volume-sensitive Cl^– channels in bovine chromaffin cells. The possibility of pervanadate acting via a pathway unrelated to protein phosphorylation is also discussed. Received: 27 June / Received after revision: 15 September / Accepted: 16 September     

gamma-Aminobutyric acid elevates cytosolic Ca in bovine chromaffin cells

Measurements of the cytosolic Ca concentration ([Ca]i) with the Ca-sensitive dye, fura-2, showed that in intact, but not in voltage-clamped, bovine chromaffin cells gamma-aminobutyric acid (GABA, 10 microM) elicited a transient increase in [Ca]i. The Ca transient of intact cells was inhibited by bicuculline (20 microM), by removal of extracellular Ca or by treatment with the Ca channel blocker cobalt (2.5 mM), and enhanced by lowering the extracellular Cl. We conclude, that GABA elevates [Ca]i by inducing a GABAA-receptor-linked Cl current which depolarizes the cell membrane sufficiently to activate potential-operated Ca channels and cause Ca entry into the cell.     

The involvement of specific phospholipase C isozymes in catecholamine release from digitonin permeabilized bovine adrenal medullary chromaffin cells

The role of phospholipase C (PLC) in exocytosis has been investigated using digitonin permeabilized, [(3)H]noradrenaline ([(3)H]NA) loaded, bovine adrenal medullary chromaffin cells. The PLC inhibitor U-73122 caused a concentration-dependent suppression of Ca(2+)-evoked [(3)H]NA release but increased basal release (that occurring in the absence of Ca(2+)). Preincubation with antibodies against PLCgamma1 or PLCbeta3 (but not PLCdelta1, delta2, beta1 and beta2) also inhibited [(3)H]NA release evoked by Ca(2+) and increased basal release, indicating that only specific PLC isozymes are involved in these actions. Interestingly, PLCgamma1 (but not PLCbeta3) antibodies inhibited the ability of Ca(2+) to increase PLC activity in these permeabilized cells. These data therefore suggest that PLCgamma1 activity may have a specific role in regulating the exocytotic response from the adrenal chromaffin cell.     

Spatio-temporal regulation of neurotransmitter release by PKC; studies in adrenal chromaffin cells

Activation of protein kinase C (PKC) seems to promote vesicle recruitment to the release-ready state prior to Ca2+ -triggered fusion in chromaffin cells. To understand spatio-temporal regulation of vesicle recruitment by PKC, we studied the effects of a phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), on the vesicle movements in living chromaffin cells by imaging with a fluorescence microscope-cooled CCD system. About 60 approximately 80% of the chromaffin vesicles showed a rapid movement, about 20% showed a moderate movement, and the rest showed slow/no movement in resting and post-stimulation. The vesicles with slow/no movement increased to 40% upon a depolarizing stimulation, and TPA increased this population to about 70%. TPA treatment, in addition, increased the number of visible chromaffin vesicles beneath the plasma membrane, suggesting that the potentiation of vesicle recruitment by PKC involves a substantial increase in the subplasmalemmal distribution of vesicles.     

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.     

Xenotransplantation of microencapsulated bovine chromaffin cells into hemiparkinsonian monkeys

This study examines the effects of xenografts of microencapsulated bovine chromaffin cells (BCCs) on the rotational behavior of hemiparkinsonian monkey recipients. In addition, it determines the content of monoamine neurotransmitters and their major metabolites in the neostriatum in hemiparkinsonian monkeys. The hemiparkinsonian model in monkeys was induced by a unilateral intracarotid injection of methyl-phenyl-tetrahydropyridine (MPTP). Unencapsulated BCCs, BCCs microencapsulated in alginate-polylysine-alginate (ALA) membranes as well as empty microencapsules were grafted into the neostriatum of the hemiparkinsonian monkeys. Following the transplantation the hemiparkinsonian symptoms subsided and the number of rotations induced by apomorphine decreased for up to nine months in the group of recipients grafted with microencapsulated BCCs, while only a temporary improvement (one month) was detected in the recipients of the unencapsulated BCCs. No change was observed in the recipients of empty microencapsules. Dopamine and its metabolites were found considerably depleted in the MPTP-lesioned side versus the unlesioned side of the neostriatum in the hemiparkinsonian monkeys(P<0.05).     

Pertussis-toxin-sensitive inhibition by (-) baclofen of Ca signals in bovine chromaffin cells

Ca signals in bovine adrenal chromaffin cells were studied both in Fura-2/AM-loaded intact cells, and in voltage-clamped cells under whole-cell patch-clamp conditions. The effects of gamma-aminobutyric acid b subtype (GABA_b) receptor activation on K⁺-depolarization-induced signals and on voltage-activated Ca²⁺ currents were investigated. Both GABA (20 M) plus bicuculline (20 M) and (-)baclofen (20–100 M), effectively inhibited the Ca signal in intact cells. The effects caused by baclofen continued to develop during the time interval between two successive stimuli. The restoration of the Ca signal during washout of baclofen was also delayed and continued in some experiments for 10–20 min. The inhibitory effect of baclofen on the Ca signal was eliminated by pre-treatment of the cells with pertussis toxin (PTX, 1g/ml, for 4–6h at 37°C). Baclofen (50 M) inhibited Ca²⁺ current in whole-cell mode by at most 20%. The effect developed quickly and was reversible. Infusion into the cells of a non-hydrolyzable analogue of guanosine 5-triphosphate GTP S (100 M), led to complete inhibition of the Ca²⁺ conductance and of voltage-evoked intracellular Ca ([CA]_i) transients within 2 min. In paired cells intracellularly perfused with GTPS-free solution, the Ca²⁺ current amplitude decreased by only about 30% for 5–6 min. It is concluded that bovine chromaffin cells have functional GABA_b receptors the activation of which, mediated by a PTX-sensitive GTP-binding protein, inhibits the evoked increase in cytosolic free Ca²⁺. The small size of the effect on Ca²⁺ current in whole-cell mode as compared to that on the Ca signal in intact cells may be explained by washout of some regulatory element during cell dialysis, or by a relatively small contribution of the normal voltage-activated Ca²⁺ current to the Ca signal. Alternatively, it might indicate GABA_b effects on mechanisms other than Ca²⁺ channels.     

All nucleoside transporters in bovine chromaffin cells are nitrobenzylthioinosine sensitive

Nitrobenzylthioinosine is an effective inhibitor of adenosine transport in chromaffin cells. When adenosine transport was measured at a 0.15 microM adenosine concentration, in the presence of variable concentrations of nitrobenzylthioinosine, ranging from 10(-14) to 10(-6) M, a half-maximal inhibitory concentration value (IC50) of 1 +/- 0.3 nM was deduced. This compound has the capacity to inhibit the total adenosine transport at 10(-7) M concentration. Nitrobenzylthioinosine acts in a non-competitive manner in blocking adenosine transport, as deduced from a Dixon plot, with a constant inhibition value (K1) of 0.01 +/- 0.003 nM. The results suggest that all nucleoside transporters present in bovine chromaffin cells are sensitive to the nitrobenzylthioinosine inhibition.     

Host-graft relationships of isolated bovine chromaffin cells in rat periaqueductal grey

Summary The transplantation of chromaffin cells from the adrenal medulla into pain modulatory regions of the CNS has previously been shown to reduce pain sensitivity, most likely via local release of neuroactive substances from the transplanted cells. The ready availability of bovine adrenal glands, as well as the high levels of opioid peptides produced by their chromaffin cells, make these glands a potentially valuable donor source for antinociception studies. However, the success of these xenografts depends on their ability to survive and integrate within the host CNS.The aim of the present study was to assess host-graft relationships of bovine chromaffin cells transplanted to the rat CNS. We have found that isolated bovine chromaffin cells survive for at least three months in the rat periaqueductal grey, with no evidence of immunological response following a short-term course of immunosuppressant treatment. In the early stages following transplantation, only minor pathology is found at the injection site, which apparently recovers completely at later stages. The host-graft borders are not well demarcated, in contrast to solid tissue grafts.Neuronal processes of host origin, forming numerous synapses with the transplanted bovine chromaffin cells, are apparent by three weeks following transplantation. Migration also occurs from the graft into the host parenchyma, as evidenced by individual chromaffin cells found near host parenchymal blood vessels. The clusters of chromaffin cells found in the graft itself are generally not very vascular, in contrast to solid tissue grafts. The chromaffin cell clusters are surrounded by blood vessels of the non-fenestrated CNS type at the host-graft border. It is likely that the small size of the graft does not require extensive angiogenesis. The lack of fenestrated peripheral-type endothelial capillaries, normally seen in adrenal medullary tissue grafts, may contribute to the survival of these xenografts in the rat brain.