Almudena Albillos

Chromaffin-cell stimulation triggers fast millimolar mitochondrial Ca2+ transients that modulate secretion.

Activation of calcium-ion (Ca2+) channels on the plasma membrane and on intracellular Ca2+ stores, such as the endoplasmic reticulum, generates local transient increases in the cytosolic Ca2+ concentration that induce Ca2+ uptake by neighbouring mitochondria. Here, by using mitochondrially targeted aequorin proteins with different Ca2+ affinities, we show that half of the chromaffin-cell mitochondria exhibit surprisingly rapid millimolar Ca2+ transients upon stimulation of cells with acetylcholine, caffeine or high concentrations of potassium ions. Our results show a tight functional coupling of voltage-dependent Ca2+ channels on the plasma membrane, ryanodine receptors on the endoplasmic reticulum, and mitochondria. Cell stimulation generates localized Ca2+ transients, with Ca2+ concentrations above 20–40 µM, at these functional units. Protonophores abolish mitochondrial Ca2+ uptake and increase stimulated secretion of catecholamines by three- to fivefold. These results indicate that mitochondria modulate secretion by controlling the availability of Ca2+ for exocytosis.

Q‐ and L‐type Ca2+ channels dominate the control of secretion in bovine chromaffin cells

Potassium‐stimulated catecholamine release from superfused bovine adrenal chromaffin cells (70 mM K+ in the presence of 2 mM Ca2+ for 10 s, applied at 5‐min intervals) was inhibited by the dihydropyridine furnidipine (3 μM) by 50%. ω‐Conotoxin MVIIC (CTx‐MVIIC, 3 μM) also reduced the secretory response by about half. Combined CTx‐MVIIC plus furnidipine blocked 100% catecholamine release. 45Ca2+ uptake and cytosolic Ca2+ concentrations ([Ca2+]i) in K+‐depolarized cells were partially blocked by furnidipine or CTx‐MVIIC, and completely inhibited by both agents. The whole cell current through Ca2+ channels carried by Ba2+ (I Ba) was partially blocked by CTx‐MVIIC. Although ω‐conotoxin GVIA (CTx‐GVIA, 1 μM) and ω‐agatoxin IVA (Aga‐IVA, 0.2 μM) partially inhibited 45Ca2+ entry, I Ba and the increase in [Ca2+]i, the combination of both toxins did not affect the K+‐evoked secretory response. The results are compatible with the presence in bovine chromaffin cells of a Q‐like Ca2+ channel which has a prominent role in controlling exocytosis. They also suggest that Q‐ and L‐type Ca2+ channels, but not N‐ or P‐types are localized near exocytotic active sites in the plasmalemma.

Multiple calcium channel subtypes in isolated rat chromaffin cells

By using the whole-cell configuration of the patch-clamp technique we have investigated the pharmacological properties of Ca2+ channels in short-term cultured rat chromaffin cells. In cells held at a membrane potential of −80 mV, using 10 mM Ba2+ as the charge carrier, only high-voltage-activated (HVA) Ca2+ channels were found. Ba2+ currents (IBa) snowed variable sensitivity to dihydropyridine (DHP) Ca2+ channel agonists and antagonists. Furnidipine, a novel DHP antagonist, reversibly blocked the current amplitude by 22% and 48%, at 1 μM and 10 μM respectively, during short (15–50 ms) depolarizing pulses to 0 mV. The L-type Ca2+ channel agonist Bay K 8644 (1 μM) caused a variable potentiation of HVA currents that could be better appreciated at low rather than at high depolarizing steps. Increase of IBa was accompanied by a 20-mV shift in the activation curves for Ca2+ channels towards more hyperpolarizing potentials. Application of the conus toxin ω-conotoxin GVIA (GVIA; 1 μM) blocked 31% of IBa; blockade was irreversible upon removal of the toxin from the extracellular medium, ω-Agatoxin IVA (IVA; 100 nM) produced a 15% blockade of IBa. ω-Conotoxin MVIIC (MVIIC; 5 μM) produced a 36% blockade of IBa; such blockade seems to be related to both GVIA-sensitive (N-type) and GVIA-resistant Ca2+ channels. The sequential addition of supramaximal concentrations of furnidipine (10 μM), GVIA (1 μM), IVA (100 nM) and MVIIC (3 μM) produced partial inhibition of IBa, which were additive. Our data suggest that the whole cell IBa in rat chromaffin cells exhibits at least four components. About 50% of IBa is carried by L-type Ca2+ channels, 30% by N-type Ca2+channels and 15% by P-type Ca2+ channels. These figures are close to those found in cat chromaffin cells. However, they differ considerably from those found in bovine chromaffin cells where P-like Ca2+channels account for 45% of the current, N-type carry 35% and L-type Ca2+ channels are responsible for only 20–25% of the current. These drastic differences might have profound physiological implications for the relative contribution of each channel subtype to the regulation of catecholamine release in different animal species.

ω‐Agatoxin‐IVA‐sensitive calcium channels in bovine chromaffin cells

A large component of the whole‐cell currents through Ca2+ channels in bovine adrenomedullary chromaffin cells has been shown to be insensitive to both L‐type and N‐type Ca2+ channel Mockers, suggesting the existence of a third type of Ca2+ channel. In the present paper, ω‐agatoxin‐IVA (AgTx), a selective blocker of P‐type Ca2+ channels in mammalian neurons, has been used to investigate the presence of this subtype of Ca2+ channel in bovine chromaffin cells. Barium currents (I Ba) through Ca2+ channels were recorded in whole‐cell patch‐clamped bovine chromaffin cells. I Ba was blocked by AgTx in a dose‐dependent and irreversible manner. At the maximal concentration used (1 μM), AgTx inhibited T Ba by 49.5 ± 3%. Such a blockade was also present when bovine chromaffin cells were pretreated with 10 μM fumidipine, a novel 1,4‐dihydropyridine L‐type channel blocker, and after treatment with 1 μM of the N‐type channel blocker, ω‐conotoxin GVIA (CgTx). A combination of these three types of Ca2+ channel blockers suppressed the macroscopic Ba2+ currents by 88%. We conclude that bovine chromaffin cells, in addition to N‐ and L‐type Ca2+ channels, possess a P‐like component in their whole‐cell currents through the Ca2+ channels.

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.

Re-evaluation of the P/Q Ca2+ channel components of Ba2+ currents in bovine chromaffin cells superfused with solutions containing low and high Ba2+ concentrations

This study was undertaken to reassess the set of voltage-dependent Ca2+ channel subtypes expressed by bovine adrenal chromaffin cells maintained in primary cultures. Previous views on the pharmacology of such channels had to be revised in the light of the novel data which arose from the use in this study of low and high micromolar concentrations of ω-agatoxin IVA, and low (2 mM) and high (10 mM) concentrations of the charge carrier Ba2+. Whole-cell Ba2+ currents (IBa) through Ca2+ channels were elicited in voltage-clamped chromaffin cells, with a holding potential of −80 mV and depolarising pulses to 0 mV. Mean peak IBa was 425 pA in 2 mM Ba2+ (59 cells) and 787 pA in 10 mM Ba2+ (42 cells). In 2 mM Ba2+, ω-conotoxin MVIIC (3 μM) inhibited IBa by 79%; in 10 mM Ba2+, the blockade developed much more slowly and reached only 44%. A low concentration of ω-agatoxin IVA (20 nM) inhibited IBa by 9%; 2 μM inhibited IBa by 60%. This blockade was similar in low and high Ba2+ concentrations. After giving furnidipine (3 μM) and ω-conotoxin GVIA (1 μM), 2 μM ω-agatoxin IVA inhibited the remaining current (about 40–45%); this blockade was independent of the Ba2+ concentration. The current could be fully blocked by the cocktail furnidipine/gw-conotoxin GVIA/high ω-agatoxin IVA, both in low and high Ba2+ concentrations. The large Q-type channel component of IBa is blocked by micromolar concentrations of ω-agatoxin IVA and ω-conotoxin MVIIC. While solutions with a high Ba2+ concentration strongly delayed the development of blockade by ω-conotoxin MVIIC, the blockade by high concentrations of ω-agatoxin IVA was equally effective in solutions with a low or a high Ba2+ concentration. Hence, the use of appropriate Ba2+ and toxin concentrations in this study reveals that P-type Ca2+ channels are poorly expressed in bovine chromaffin cells; in contrast, a robust component of the current depends on Q-type Ca2+ channels. An R-type residual current is not present in these cells.

Opioid inhibition of Ca2+ channel subtypes in bovine chromaffin cells: selectivity of action and voltage-dependence.

Bovine chromaffin cells possess a mixture of high‐voltage‐activated Ca2+ channel subtypes: L‐type, dihydropyridine‐sensitive channels, and N‐, P‐ and Q‐types, ω‐conotoxin MVIIC‐sensitive channels. In these cells, we studied the reversible, naloxone‐antagonized inhibition of Ba2+ currents by the opioid agonist met‐enkephalin (IC50= 272 nM). This inhibition could be resolved into a voltage‐dependent and a voltage‐independent component. The first was revealed by its slow Ba2+ current activation kinetics at 0 mV and by the current facilitation induced by short prepulses to +90 mV. The second was estimated as the residual inhibition persisting after the facilitation protocol. The two inhibitory components varied markedly from cell to cell and each contributed to about half of the total inhibition. Replacement of internal GTP by GDP‐β‐S or cell pretreatment with pertussis toxin completely abolished the voltage‐dependent inhibition by opioids, partially preserving the voltage‐independent component. The opioid‐induced inhibition was not selective for any Ca2+ channel subtype, being not prevented after the addition of specific Ca2+ channel antagonists. However, when separately analyzing the contribution of each channel type to the voltage‐dependent and voltage‐independent modulation, a clear‐cut distinction could be achieved. The voltage‐independent inhibition was effective on all Ca2+ channel subtypes but predominantly on L‐type Ca2+ channels. The voltage‐dependent process was abolished by ω‐conotoxin‐MVIIC, but unaffected by nifedipine, and was thus sharply restricted to non‐L‐type channels (N‐, P‐ and Q‐types). Our data suggest a functionally distinct opioid receptor‐mediated modulation of L‐ and non‐L‐type channels, i.e. of the two channel classes sharing major control of catecholamine secretion from bovine chromaffin cells.

The mechanism of calcium channel facilitation in bovine chromaffin cells

1. This study was planned to clarify the mechanism of Ca2+ channel facilitation by depolarizing prepulses given to voltage‐clamped bovine chromaffin cells. The hypothesis for an autocrine modulation of such channels was tested by studying the effects of a soluble vesicle lysate (SVL) on whole‐cell Ba2+ currents (IBa). 2. SVL was prepared from a bovine adrenal medullary homogenate. The ATP content in this concentrated SVL amounted to 3.18 +/‐ 0.12 mM (n = 4). The concentration of noradrenaline and adrenaline present in the SVL was 11.2 +/‐ 0.97 and 15.2 +/‐ 2 mM, respectively (n = 5). A 1:1000 dilution of SVL in the external solution halved the magnitude of IBa and produced a 7‐fold slowing of its activation kinetics. The blocking effects of SVL were concentration dependent and quickly reversed upon washout. 3. Inhibition and slowing of the kinetics of IBa by SVL could be partially reversed by strong depolarizing prepulses (+90 mV, 45 ms). This reversal of inhibition, called Ca2+ channel facilitation, persisted in the presence of 3 microM nifedipine. 4. Intracellular dialysis of GDP‐beta‐S (0.5 mM) or pretreatment of the cells with pertussis toxin (100 ng ml‐1 for 18‐24 h) prevented the reduction in peak current caused by a 1:100 dilution of SVL; no prepulse facilitation could be observed under these conditions. 5. The receptor blockers naloxone (10 microM) or suramin (100 microM) and PPADS (100 microM) largely antagonized the effects of SVL. Treatment of SVL with alkaline phosphatase or dialysis against a saline buffer to remove low molecular mass materials (< 10 kDa) considerably reduced the activity of SVL. 6. Stopping the flow of the external solution (10 mM Ba2+) gradually reduced the size, and slowed down the activation phase, of the current. Prepulse facilitation of IBa was absent or weak in a superfused cell, but was massive upon flow‐stop conditions in the presence or absence of 3 microM nifedipine. 7. Our experiments suggest that facilitation by prepulses of whole‐cell current through Ca2+ channels is due to the suppression of an autoinhibitory autocrine loop present in bovine chromaffin cells. By acting at least on purinergic and opiate receptors, the exocytotic release of ATP and opiates will cause a tonic inhibition of the current through a G‐protein‐mediated mechanism. Such a mechanism will be removed by strong depolarizing prepulses, and will involve preferentially non‐L‐type channels. In the light of these and other recent results, previously held views on the selective recruitment by prepulses of dihydropyridine‐sensitive Ca2+ channels are not tenable.

SNX482 selectively blocks P/Q Ca2+ channels and delays the inactivation of Na+ channels of chromaffin cells

The effects of the toxin SXN482 on Ca2+ channel currents (ICa), Na+ currents (INa), and K+ currents (IK) have been studied in bovine adrenal medullary chromaffin cells voltage-clamped at -80 mV. Currents were elicited by depolarising pulses to 0-10 mV (ICa and INa) or to +60 mV (IK). SNX482 blocked ICa in a concentration-dependent manner. The inhibition curve exhibited two phases. The first high-affinity phase comprised 28% of the whole-cell current and exhibited an IC50 of 30.2 nM. The second low-affinity phase comprised over 70% of ICa and had an IC50 of 758.6 nM. Blockade was rapid and fully reversible upon washout of the toxin. Occlusion experiments showed additivity of blockade exerted by nifedipine plus SNX482 (0.3 microM) and by omega-conotoxin GVIA plus SNX482. In contrast, blockade exerted by combined omega-agatoxin IVA plus SNX482 (about 50% of the whole cell) did not show additivity. At 0.3 microM and higher concentrations, SNX482 delayed the inactivation of INa. The time constant (tau) for inactivation of INa in control conditions doubled in the presence of 0.5 microM SNX482. At 0.3 microM, SNX482 did not affect IK. Our data demonstrate that: (i) SNX482 selectively blocks P/Q Ca2+ channels at submicromolar concentrations; (ii) the toxin partially blocks Na+ channels; (iii) SNX482 delays the inactivation of Na+ channels. These results reveal novel properties of SNX482 and cast doubts on the claimed selectivity and specificity of the toxin to block the R-type Ca2+ channel.

Greater diversity than previously thought of chromaffin cell Ca2+ channels, derived from mRNA identification studies

Using reverse transcription followed by PCR amplification (RT‐PCR), we have identified multiple messenger RNAs encoding for the neuronal pore‐forming Ca2+ channel subunits α1A (P/Q channel), α1B (N channel), α1D (neuronal/endocrine L channel), α1E (R channel), α1G‐H (T channel) and α1S (skeletal muscle L channel) in bovine chromaffin cells. mRNAs for the auxiliary β2, β3, β4, α2/δ and γ2 subunits were also identified. In agreement with these molecular data, perforated patch‐clamp recordings of whole‐cell Ca2+ currents reveal the existence of functional R‐type Ca2+ channels in these cells that were previously undetected with other techniques. Our results provide a molecular frame for a much wider functional diversity of Ca2+ channels in chromaffin cells than that previously established using pharmacological and electrophysiological approaches.