Román Olivares

Voltage inactivation of Ca2+ entry and secretion associated with N‐ and P/Q‐type but not L‐type Ca2+ channels of bovine chromaffin cells

1 In this study we pose the question of why the bovine adrenal medullary chromaffin cell needs various subtypes (L, N, P, Q) of the neuronal high‐voltage activated Ca2+ channels to control a given physiological function, i.e. the exocytotic release of catecholamines. One plausible hypothesis is that Ca2+ channel subtypes undergo different patterns of inactivation during cell depolarization. 2 The net Ca2+ uptake (measured using 45Ca2+) into hyperpolarized cells (bathed in a nominally Ca2+‐free solution containing 1·2 mM K+) after application of a Ca2+ pulse (5 s exposure to 100 mM K+ and 2 mM Ca2+), amounted to 0·65 ± 0·02 fmol cell−1; in depolarized cells (bathed in nominally Ca2+‐free solution containing 100 mM K+) the net Ca2+ uptake was 0·16 ± 0·01 fmol cell−1. 3 This was paralleled by a dramatic reduction of the increase in the cytosolic Ca2+ concentration, [Ca2+]i, caused by Ca2+ pulses applied to fura‐2‐loaded single cells, from 1181 ± 104 nM in hyperpolarized cells to 115 ± 9 nM in depolarized cells. 4 A similar decrease was observed when studying catecholamine release. Secretion was decreased when K+ concentration was increased from 1·2 to 100 mM; the Ca2+ pulse caused, when comparing the extreme conditions, the secretion of 807 ± 35 nA of catecholamines in hyperpolarized cells and 220 ± 19 nA in depolarized cells. 5 The inactivation by depolarization of Ca2+ entry and secretion occluded the blocking effects of combined ω‐conotoxin GVIA (1 μM) and ω‐agatoxin IVA (2 μM), thus suggesting that depolarization caused a selective inactivation of the N‐ and P/Q‐type Ca2+ channels. 6 This was strengthened by two additional findings: (i) nifedipine (3 μM), an L‐type Ca2+ channel blocker, suppressed the fraction of Ca2+ entry (24 %) and secretion (27 %) left unblocked by depolarization; (ii) FPL64176 (3 μM), an L‐type Ca2+ channel ‘activator’, dramatically enhanced the entry of Ca2+ and the secretory response in depolarized cells. 7 In voltage‐clamped cells, switching the holding potential from ‐80 to ‐40 mV promoted the loss of 80 % of the whole‐cell inward Ca2+ channel current carried by 10 mM Ba2+ (IBa). The residual current was blocked by 80 % upon addition of 3 μM nifedipine and dramatically enhanced by 3 μM FPL64176. 8 Thus, it seems that the N‐ and P/Q‐subtypes of calcium channels are more prone to inactivation at depolarizing voltages than the L‐subtype. We propose that this different inactivation might occur physiologically during different patterns of action potential firing, triggered by endogenously released acetylcholine under various stressful conditions.

Mitochondrial calcium sequestration and protein kinase C cooperate in the regulation of cortical F-actin disassembly and secretion in bovine chromaffin cells

Mitochondria play an important role in the homeostasis of intracellular Ca2+ and regulate its availability for exocytosis. Inhibitors of mitochondria Ca2+ uptake such as protonophore CCCP potentiate the secretory response to a depolarizing pulse of K+. Exposure of cells to agents that directly (cytochalasin D, latrunculin B) or indirectly (PMA) disrupt cortical F‐actin networks also potentiate the secretory response to high K+. The effects of cytochalasin D and CCCP on secretion were additive whereas those of PMA and CCCP were not; this suggests different mechanisms for cytochalasin D and CCCP and a similar mechanism for PMA and CCCP. Mitochondria were the site of action of CCCP, because the potentiation of secretion by CCCP was observed even after depletion of Ca2+ from the endoplasmic reticulum. CCCP induced a small increase in the cytosolic Ca2+ concentration ([Ca2+]c) that was not modified by the protein kinase C (PKC) inhibitor chelerythrine. Both CCCP and PMA induced cortical F‐actin disassembly, an effect abolished by chelerythrine. In addition, rotenone and oligomycin A, two other mitochondrial inhibitors, also evoked cortical F‐actin disassembly and potentiated secretion; again, these effects were blocked by chelerythrine. CCCP also enhanced the phosphorylation of PKC and myristoylated alanine‐rich C kinase substance (MARCKS), and these were also inhibited by chelerythrine. The results suggest that the rapid sequestration of Ca2+ by mitochondria would protect the cell from an enhanced PKC activation and cortical F‐actin disassembly, thereby limiting the magnitude of the secretory response.

Effect of inositol 1,4,5-trisphosphate receptor stimulation on mitochondrial [Ca2+] and secretion in chromaffin cells

Ca(2+) uptake by mitochondria is a potentially important buffering system able to control cytosolic [Ca(2+)]. In chromaffin cells, we have shown previously that stimulation of either Ca(2+) entry or Ca(2+) release via ryanodine receptors triggers large increases in mitochondrial [Ca(2+)] ([Ca(2+)](M)) approaching the millimolar range, whose blockade dramatically enhances catecholamine secretion [Montero, Alonso, Carnicero, Cuchillo-Ibañez, Albillos, Garcia, Carcia-Sancho and Alvarez (2000) Nat. Cell Biol. 2, 57-61]. In the present study, we have studied the effect of stimulation of inositol 1,4,5-trisphosphate (InsP(3)) receptors using histamine. We find that histamine produces a heterogeneous increase in [Ca(2+)](M), reaching peak levels at approx. 1 microM in 70% of the mitochondrial space to several hundred micromolar in 2-3% of mitochondria. Intermediate levels were found in the rest of the mitochondrial space. Single-cell imaging experiments with aequorin showed that the heterogeneity had both an intercellular and a subcellular origin. Those mitochondria responding to histamine with increases in [Ca(2+)](M) much greater than 1 microM (30%) were the same as those that also responded with large increases in [Ca(2+)](M) following stimulation with either high-K(+) medium or caffeine. Blocking mitochondrial Ca(2+) uptake with protonophores or mitochondrial inhibitors also enhanced catecholamine secretion induced by histamine. These results suggest that some InsP(3) receptors tightly co-localize with ryanodine receptors and voltage-dependent Ca(2+) channels in defined subplasmalemmal functional units designed to control secretion induced by different stimuli.

A choline-evoked [Ca2+]c signal causes catecholamine release and hyperpolarization of chromaffin cells

In bovine chromaffin cells fast‐superfused with Krebs‐HEPES solution containing 1–2 mM Ca2+, 5 s pulses of choline (1–10 mM), elicited catecholamine secretory responses that were only ~10% of those evoked by ACh (0.01–0.1 mM). However, in high‐Ca2+ solutions (10–20 mM) the size of the choline secretory responses approached those of ACh. The choline responses (10 mM choline in 20 mM Ca2+, 10Cho/20Ca2+) tended to decline upon repetitive pulsing, whereas those of ACh were well maintained. The confocal [Ca2+]c increases evoked by 10Cho/20Ca2+ were similar to those of ACh. Whereas 10Cho/20Ca2+ caused mostly hyperpolarization of chromaffin cells, 0.1ACh/20 Ca2+ caused first depolarization and then hyperpolarization; in regular solutions (2 mM Ca2+), the hyperpolarizing responses did not show up. In Xenopus oocytes injected with mRNA for bovine α7 nicotinic receptors (nAChRs), 10Cho/20 Ca2+ fully activated an inward current; in oocytes expressing α3β4, however, the inward current elicited by choline amounted to only 4% of the size of α7 current. Our results suggest that choline activates the entry of Ca2+ through α7 nAChRs; this leads to a cytosolic concentration of calcium ([Ca2+]c) rise that causes the activation of nearby Ca2+‐dependent K+ channels and the hyperpolarization of the chromaffin cell. This response, which could be unmasked provided that cells were stimulated with high‐Ca2+ solutions, may be the underlying mechanism through which choline exerts a modulatory effect on the electrical activity of the chromaffin cell and on neurotransmitter release at cholinergic synapses.

Control of secretion by mitochondria depends on the size of the local [Ca2+] after chromaffin cell stimulation

In chromaffin cells, plasma membrane calcium (Ca2+) channels and mitochondria constitute defined functional units controlling the availability of Ca2+ nearby exocytotic sites. We show here that, when L‐/N‐type Ca2+ channels were inhibited with nisoldipine and ω‐conotoxin GVIA, cytosolic [Ca2+] ([Ca2+]c) peaks measured in fura‐4F‐loaded cells were reduced by 36%; however, mitochondrial Ca2+ uptake was unaffected and secretion was potentiated by protonophores as in control cells. By contrast, when non L‐type Ca2+ channels were inhibited with ω‐conotoxin MVIIC, [Ca2+]c peaks induced by high K+ were reduced by 73%, mitochondrial Ca2+ uptake was abolished, and secretion was not modified by protonophores. However, if Ca2+ entered only through L‐type channels activated by FPL64176, high K+ stimulation induced fast mitochondrial Ca2+ uptake and catecholamine secretion was strongly increased and potentiated by protonophores. These results confirm the close association of catecholamine secretion to mitochondrial Ca2+ uptake, and indicate the sharp threshold of local [Ca2+]c (about 5 µm) required for triggering fast mitochondrial Ca2+ uptake that is able to modulate secretion. The entry of Ca2+ through L‐type channels generated local [Ca2+]c increases just below that, inducing little mitochondrial Ca2+ uptake unless FPL64176 was present. By contrast, Ca2+ entry through P/Q‐type channels fully activated mitochondrial Ca2+ uptake. Control of secretion by mitochondria therefore depends critically on the ability of the stimulus to create large local [Ca2+]c microdomains.

Blockade by agmatine of catecholamine release from chromaffin cells is unrelated to imidazoline receptors

The blockade of exocytosis induced by the putative endogenous ligand for imidazoline receptors, agmatine, was studied by using on-line measurement of catecholamine release in bovine adrenal medullary chromaffin cells. Agmatine inhibited the acetylcholine-evoked release of catecholamines in a concentration-dependent manner (IC(50)=366 microM); the K(+)-evoked release of catecholamines was unaffected. Clonidine (100 microM) and moxonidine (100 microM) also inhibited by 75% and 50%, respectively, the acetylcholine-evoked response. In cells voltage-clamped at -80 mV, the intermittent application of acetylcholine pulses elicited whole-cell inward currents (I(ACh)) that were blocked 63% by 1 mM agmatine. The onset of blockade was very fast (tau(on) = 31 ms); the recovery of the current after washout of agmatine also occurred very rapidly (tau(off = 39 ms). Efaroxan (10 microM) did not affect the inhibition of I(ACh) elicited by 1 mM agmatine. I(ACh) was blocked 90% by 100 microM clonidine and 50% by 100 microM moxonidine. The concentration-response curve for acetylcholine to elicit inward currents was shifted to the right in a non-parallel manner by 300 microM agmatine. The blockade of I(ACh) caused by agmatine (100 microM) was similar at various holding potentials, around 50%. When intracellularly applied, agmatine did not block I(ACh). At 1 mM, agmatine blocked I(Na) by 23%, I(Ba) by 14%, I(K(Ca)) by 16%, and I(K(VD)) by 18%. In conclusion, agmatine blocks exocytosis in chromaffin cells by blocking nicotinic acetylcholine receptor currents. In contrast to previous views, these effects seem to be unrelated to imidazoline receptors.

Plasmalemmal sodium-calcium exchanger shapes the calcium and exocytotic signals of chromaffin cells at physiological temperature.

The activity of the plasmalemmal Na(+)/Ca(2+) exchanger (NCX) is highly sensitive to temperature. We took advantage of this fact to explore here the effects of the NCX blocker KB-R7943 (KBR) at 22 and 37°C on the kinetics of Ca(2+) currents (ICa), cytosolic Ca(2+) ([Ca(2+)]c) transients, and catecholamine release from bovine chromaffin cells (BCCs) stimulated with high K(+), caffeine, or histamine. At 22°C, the effects of KBR on those parameters were meager or nil. However, at 37°C whereby the NCX is moving Ca(2+) at a rate fivefold higher than at 22°C, various of the effects of KBR were pronounced, namely: 1) no effects on ICa; 2) reduction of the [Ca(2+)]c transient amplitude and slowing down of its rate of clearance; 3) blockade of the K(+)-elicited quantal release of catecholamine; 4) blockade of burst catecholamine release elicited by K(+); 5) no effect on catecholamine release elicited by short K(+) pulses (1-2 s) and blockade of the responses produced by longer K(+) pulses (3-5 s); and 6) potentiation of secretion elicited by histamine or caffeine. Furthermore, the more selective NCX blocker SEA0400 also potentiated the secretory responses to caffeine. The results suggest that at physiological temperature the NCX substantially contributes to shaping the kinetics of [Ca(2+)]c transients and the exocytotic responses elicited by Ca(2+) entry through Ca(2+) channels as well as by Ca(2+) release from the endoplasmic reticulum.

Desensitized nicotinic receptors that, however, afford cytoprotection in bovine chromaffin cells

Neuronal nicotinic receptors for acetylcholine (nAChRs) are among the ionotropic receptors that suffer the most desensitization upon prolonged exposure to their agonists. This is particularly true for the alpha7 subtype of nAChRs, although alpha3beta4 receptors also suffer quick desensitization. This study was planned to test the hypothesis that even after suffering desensitization, a given nAChR might still afford cell protection against a noxious stimulus. Of the many agonists developed for nAChRs, we selected the poorly desensitizing ligand dimethylphenylpiperazinium (DMPP) (Britt and Brenner, 1997) and the highly desensitizing agent epibatidine (EPB) (Marks et al., 1996). We have measured nAChR currents, catecholamine secretory responses, and changes of [Ca2+]c elicited by stimulation of nAChRs with DMPP or EPB. We have also investigated cytoprotection elicited by DMPP and EPB against the cytotoxic effects of veratridine in bovine chromaffin cells.