Antonio R. Artalejo

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 (ICa) 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 ICa by 42% and the late ICa 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.

A dopaminergic receptor modulates catecholamine release from the cat adrenal gland.

Nicotine evokes the release of catecholamines from perfused cat adrenal glands in a concentration‐dependent manner, the median effective concentration for nicotine being 5 microM. Two 2 min pulses of 5 microM‐nicotine, 40 min apart (S1 and S2) gave net catecholamine outputs of 7.64 and 3.55 micrograms/8 min, respectively. The ratio S2/S1 in control glands was 0.5. Increasing concentrations of apomorphine (1‐10 microM) markedly inhibited catecholamine release during the second nicotine pulse (S2). At 1 microM‐apomorphine, the release during S2 was significantly reduced to 16% of S1; with 10 microM‐apomorphine, the secretory response was reduced further to only 3% of S1, the ratio S2/S1 being 0.03. The presence of haloperidol, sulpiride or picobenzide (each 0.5 microM) during S2, completely reversed the inhibition of catecholamine release produced by apomorphine. Haloperidol itself increased the nicotinic secretory response during S2; so, while the ratio S2/S1 was 0.5 in control conditions, this ratio increased significantly to 0.95 if haloperidol (0.5 microM) was present during S2, suggesting that the presence of this dopaminergic antagonist removed a negative feed‐back mechanism that inhibits nicotine‐evoked catecholamine release. If present during S2, dopamine (1 microM) also markedly inhibited catecholamine release evoked by nicotine; this inhibition was again reversed by 0.5 microM‐haloperidol. Neither the opiate antagonist naloxone nor the alpha‐adrenoceptor blocking agent phentolamine (at concentrations of 0.5‐5 microM) affected the inhibition by apomorphine of the secretory response to nicotine. These data strongly suggest that the cat adrenal medulla chromaffin cell membrane contains a dopaminergic receptor which modulates the catecholamine secretory process triggered by stimulation of the nicotinic cholinoceptor. The fact that dopamine is released in measurable amounts, together with adrenaline and noradrenaline, from perfused cat adrenal glands in response to nicotinic stimulation (V. Ceña, unpublished results), favours a role for this dopaminergic receptor in modulating catecholamine release from the chromaffin cell.

Ca(2+)-activated K+ channels modulate muscarinic secretion in cat chromaffin cells.

1. This study was aimed at testing the hypothesis that Ca(2+)‐dependent K+ channels regulate the release of catecholamines mediated by muscarinic stimulation of cat adrenal chromaffin cells. Two parameters were measured: the secretory response to brief pulses of methacholine (100 microM for 10 s) in intact cat adrenal glands perfused at a high rate with oxygenated Krebs solution; and the changes in cytosolic Ca2+ concentrations, [Ca2+]i, produced by puff applications of methacholine pulses (also 100 microM for 10 s) in isolated single cat adrenal chromaffin cells loaded with Fura‐2. 2. A pulse of methacholine released 805 +/‐ 164 ng of catecholamines (mean of thirty‐two pulses). d‐Tubocurarine (DTC) increased the secretory response in a concentration‐dependent manner. The maximum increase (around 1000 ng catecholamines over control values) was reached at 100 microM‐DTC and the EC50 was around 10 microM. 3. The secretory responses to methacholine alone, or to the combination of methacholine plus DTC, were strongly dependent on the extracellular Ca2+ concentration, [Ca2+]o. Thus Ca2+o removal from the perfusing solution for 5‐10 min abolished catecholamine release. 4. At 0.1 microM, isradipine (an L‐type Ca2+ channel blocker) inhibited by 71% the secretory response to DTC plus methacholine. At 1 microM, Bay K 8644 (an L‐type Ca2+ channel activator) increased 2‐fold the secretory response to DTC plus methacholine (2746 ng of catecholamines). 5. Apamin (1 microM) increased 3.5‐fold the secretory response to methacholine pulses (from 500 to 1800 ng of catecholamines). 6. Methacholine pulses enhanced [Ca2+]i from the resting level of 100 nM to a peak of 1000 nM which quickly declined to basal level. DTC (100 microM) enhanced by 20% the [Ca2+]i peak and substantially prolonged its duration. 7. Apamin (1 microM) increased by 60% the [Ca2+]i peak evoked by methacholine, and delayed the initiation of decline of the [Ca2+]i peak. 8. These results are compatible with the idea that muscarinic stimulation depolarizes the cat adrenal chromaffin cell through an unidentified mechanism. Depolarization is probably counteracted by activation of Ca2+i‐dependent K+ channels. Therefore, inhibition of these channels enhances depolarization and firing of action potentials which activate voltage‐dependent L‐type Ca2+ channels to increase further the Ca2+i signal and the secretory response. Thus Ca2+i‐dependent K+ channels, probably of the small‐conductance type (SK), seem to be involved in the modulation of muscarinic‐evoked catecholamine release responses in cat adrenal chromaffin cells.

ATP from subplasmalemmal mitochondria controls Ca2+-dependent inactivation of CRAC channels.

A sustained Ca2+ entry is the primary signal for T lymphocyte activation after antigen recognition. This Ca2+ entry mainly occurs through store-operated Ca2+ channels responsible for a highly selective Ca2+ current known as ICRAC. Ca2+ ions act as negative feedback regulators of ICRAC, promoting its inactivation. Mitochondria, which act as intracellular Ca2+ buffers, have been proposed to control all stages of CRAC current and, hence, intracellular Ca2+ signaling in several types of non-excitable cells. Using the whole-cell configuration of the patch clamp technique, which allows control of the intracellular environment, we report here that respiring mitochondria located close to CRAC channels can regulate slow Ca2+-dependent inactivation of ICRAC by increasing the Ca2+-buffering capacity beneath the plasma membrane, mainly through the release of ATP.

Characterization of a Dopaminergic Receptor that Modulates Adrenomedullary Catecholamine Release

Abstract: Nicotine evokes the release of catecholamines from bovine adrenal glands perfused with oxygenated Krebs‐bicarbonate solution. Two 2‐min pulses of 5 μM nicotine, at 40‐min intervals (S1 and S2), gave net catecholamine outputs of 45.2 ± 3.6 and 29.1 ± 3.5 μg/8 min, respectively. Apomorphine (1 or 10 μM) markedly inhibited catecholamine release during S2 to 9.1 ± 2.2 and 0.5 μg/8 min, respectively. Haloperidol (0.5 μM) reversed the inhibitory effects of apomorphine. Haloperidol alone enhanced catecholamine release induced by nicotine to 67.9 ± 7.9 μg/8 min. [3H]Spiperone binds to adrenomedullary membranes with a KD of 0.24 nM and a Bmax of 117 fmol/mg of protein. Whereas spiperone and haloperidol potently displaced such binding, 3,4‐dihydroxyphenylethylamine (dopamine) and sulpiride were poorer displacers, and SCH23390, prazosin, phenoxybenzamine, propranolol, BAY‐K‐8644, and nitrendipine did not displace [3H]spiperone bound. These data strongly suggest that, as in the cat, the bovine adrenal medulla chromaffin cell contains a dopaminergic receptor that modulates the catecholamine secretory process triggered by stimulation of the nicotinic cholinoceptor. Such a receptor seems to be of the D2 type and might be involved in a sympatho‐adrenal cooperative mechanism contributing to the maintenance of cardiovascular homeostasis during stressful situations as well as to the pathogenesis of hypertension. If so, selective dopaminergic agonists might prove clinically useful in the treatment of hypertension.

Permeation by zinc of bovine chromaffin cell calcium channels: relevance to secretion

Zn2+ increased the rate of spontaneous release of catecholamines from bovine adrenal glands. This effect was Ca2+ independent; in fact, in the absence of extracellular Ca2+, the secretory effects of Zn2+ were enhanced. At low concentrations (3–10 μM), Zn2+ enhanced the secretory responses to 10-s pulses of 100 μM 1,1-dimethyl-4-phenylpiperazinium (DMPP, a nicotinic receptor agonist) or 100 mM K+. In the presence of DMPP, secretion was increased 47% above controls and in high-K+ solutions, secretion increased 54% above control. These low concentrations of Zn2+ did not facilitate the whole-cell Ca2+ (ICa) or Ba2+ (IBa) currents in patch-clamped chromaffin cells. Higher Zn2+ concentrations inhibited the currents (IC50 values, 346 μM for ICa and 91 μM for IBa) and blocked DMPP- and K+-evoked secretion (IC50 values, 141 and 250 μM, respectively). Zn2+ permeated the Ca2+ channels of bovine chromaffin cells, although at a much slower rate than other divalent cations. Peak currents at 10 mM Ba2+, Ca2+, Sr2+ and Zn2+ were 991, 734, 330 and 7.4 pA, respectively. Zn2+ entry was also evidenced using the fluorescent Ca2+ probe fura-2. This was possible because Zn2+ causes an increase in fura-2 fluorescence at the isosbestic wavelength for Ca2+, i.e. 360 nm. There was a slow resting entry of Zn2+ which was accelerated by stimulation with DMPP or high-K+ solution. The entry of Zn2+ was concentration dependent, slightly antagonized by 1 mM Ca2+ and completely blocked by 5 mM Ni2+. The entry of Ca2+ evoked by depolarization with high-K+ solution was antagonized by Zn2+. We conclude that inhibition by Zn2+ of evoked catecholamine secretion is associated with blockade of Ca2+ entry through Ca2+ channels recruited by DMPP or K+. However, the facilitation of secretion observed at low Zn2+ concentrations, or in the absence of Ca2+, may be exerted at an intracellular site on the secretory machinery. This is plausible because Zn2+ permeates the bovine chromaffin cell Ca2+ channels and in this way gains access to the cytosol. In addition, we have established conditions for measuring Zn2+ transients in fura-2-loaded cells with a very high sensitivity, taking advantage of the high-affinity binding of Zn2+ to fura-2 and the modification of its fluorescence spectrum.

R56865 inhibits catecholamine release from bovine chromaffin cells by blocking calcium channels

1 The effects of R56865 (a new class of cardioprotective agent which prevents Na+ and Ca2+ overload in cardiac myocytes) on catecholamine release, whole‐cell current through Ca2+ channels (IBa) and cytosolic Ca2+ concentrations, [Ca2+]i, have been studied in bovine chromaffin cells. 2 R56865 caused a time‐ and concentration‐dependent blockade of catecholamine release from superfused cells stimulated intermittently with 5 s pulses of 59 mm K+. After 5 min superfusion, a 3 μm concentration inhibited secretion by 20%; the blockade increased gradually with perfusion time, to reach 85% after 40 min. The IC50 to block secretion after 5 min periods of exposure to increasing concentrations of R56865 was around 3.1 μm. The blocking effects of R56865 were reversible after 5–15 min wash out. In high Ca2+ solution (10 mm Ca2+), the degree of blockade of secretion diminished by 20% in comparison with 1 mm Ca2+. 3 In electroporated cells, R56865 (10 μm) did not modify the secretory response induced by the application of 10 μm free Ca2+. 4 R56865 blocked the peak IBa current in a concentration‐ and time‐dependent manner; its IC50 was very similar to that obtained for secretion (3 μm). The compound not only reduced the size of the peak current but also promoted its inactivation; when the effects of R56865 were measured at the most inactivated part of the current, its IC50 was 1 μm. Both the inactivation and the reduction of the peak currents were reversible upon washing out the drug. 5 In fura‐2‐loaded single chromaffin cells the basal [Ca2+]i of around 100 nm was elevated to a peak of 1.5 μm by the application of a 5 s pulse of 59 mm K+. R56865 (10 μm) did not affect the basal [Ca2+]i but blocked by 90% the K+‐evoked increase. This effect was fully reversible after 5–10 min of wash out. 6 The results are compatible with the idea that R56865 blocks Ca2+ entry into K+‐depolarized chromaffin cells by promoting the inactivation of voltage‐dependent Ca2+ channels; this would lead to the limitation of the rise in [Ca2+]i and of the release of catecholamines. The restriction of catecholamine release may favour indirectly the known direct beneficial cardioprotective actions of R56865.

Sodium‐dependent inhibition by PN200‐110 enantiomers of nicotinic adrenal catecholamine release

1 Dimethylphenylpiperazinium (DMPP) or high K concentrations evoke catecholamine release from perfused cat adrenal glands; in both cases the secretory response was significantly enhanced in the absence of Na. Tetrodotoxin did not modify the nicotinic secretory response. 2 The (+)‐ and (−)‐enantiomers of the dihydropyridine Ca channel blocker PN200‐110 show a high degree of stereoselectivity in the inhibition of catecholamine secretion evoked by high K or by DMPP in the presence of Na, the (+)‐enantiomer being 57 and 80 times more potent, respectively, than the (−)‐enantiomer. Both, noradrenaline and adrenaline release were equally depressed by PN200‐110. 3 The IC50 values for (+)‐ and (−)‐PN200‐110 for blockade of the secretory response induced by K or DMPP in the presence of Na are in the same range. In the absence of Na, (−)‐PN200‐110 did not affect DMPP‐evoked secretion; however, the (+)‐enantiomer partially inhibited it. 4 The results suggest that the physiological catecholamine release from chromaffin cells is preceded by Na entry through the nicotinic receptor‐associated ionophore; this causes cell depolarization, opening of voltage‐dependent, dihydropyridine‐sensitive Ca channels and Ca entry into the cell. In the absence of Na, additional Ca influx through an alternative pathway (the nicotinic cholinoceptor ionophore?) might also activate secretion.

Interactions between Ca2+, PCA50941 and Bay K 8644 in bovine chromaffin cells.

We describe here the effects of PCA50941 (a novel 1,4-dihydropyridine derivative) comparatively with Bay K 8644 on various parameters in bovine adrenal chromaffin cells. The binding of [3H](+)-isradipine to bovine adrenal medulla plasma membranes was inhibited similarly by PCA50941 and Bay K 8644 at various [Ca2+]o suggesting a common binding site for both compounds on the dihydropyridine receptor. In voltage-clamped chromaffin cells PCA50941 (1 microM) and Bay K 8644 (1 microM) shifted the I-V relationship of whole-cell Ca2+ currents by about 5-10 mV towards more hyperpolarizing potentials. At -20 mV, PCA50941 enhanced ICa by 195 +/- 16% and Bay K 8644 by 288 +/- 51%. Stimulation of fura 2-loaded chromaffin cell suspensions with 17.7 K+/0.5 Ca2+ increased 3-fold the basal [Ca2+]i. PCA50941 increased further the K(+)-evoked peak to 655 nM, and Bay K 8644 to 1129 nM. In the presence of 5 mM Ca2+, PCA50941 or Bay K 8644 increased the [Ca2+] peaks to 427 and 350 nM, respectively. PCA50941 potentiated the release of catecholamines from perfused bovine adrenal glands evoked by 30 s pulses of 17.7 mM K+ in a manner dependent on the [Ca2+]o. Thus at 1, 2.5, 5 and 10 mM Ca2+, secretion was 2.3-, 3.8-, 5- and 4-fold greater than in control glands. Bay K 8644 enhanced the K(+)-induced response 3- and 9-fold at [Ca2+]o of 0.25 or 0.5 mM, respectively; at higher [Ca2+]o the potentiation was similar to that of PCA50941.(ABSTRACT TRUNCATED AT 250 WORDS)

Two components in the adrenal nicotinic secretory response revealed by cobalt ramps

Prolonged stimulation with nicotine (50 microM) enhanced the secretion of catecholamines from perfused cat adrenal glands. The profile of secretion consisted of a quick activation phase to a peak of 7.68 micrograms/min followed by a second inactivation phase which exhibited a t1/2 of 3.75 min. Sustained stimulation with a solution enriched in K+ (59 mM) also evoked a transient secretory response, with a peak release of 8.62 micrograms/2 min and a t1/2 for inactivation of 4.8 min. Co2+ (10 mM) blocked the nicotinic response by 58% and the K(+)-evoked secretory response by over 96%. In the presence of Co2+ (5 mM), continuous perfusion with nicotine produced a transient but large initial secretory response; the gradual decrease of the extracellular Co2+ concentration, [Co2+]0, as a continuous ramp allowed the development of a second component of secretion which inactivated later on. When the glands were continuously stimulated with 59 mM K+ in the presence of Co2+, the first component of secretion was missing; the second component appeared as [Co2+]0 decreases as a ramp. In similar experiments performed in low-Na+ solution (10 mM Na+), only the first secretion component evoked by nicotine was observed. This finding suggests that the second component of secretion depends on Na+ entry through the nicotinic receptor, on the ensuing cell depolarization and on Ca2+ entry through voltage-dependent Ca2+ channels.(ABSTRACT TRUNCATED AT 250 WORDS)