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 (I_Ba) 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 I _Ba 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 I _Ba 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 I _Ba by 9%; 2 μM inhibited I _Ba 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/ω-conotoxin GVIA/high ω-agatoxin IVA, both in low and high Ba²⁺ concentrations. The large Q-type channel component of I _Ba 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. Received: 22 April 1996 / Received after revision: 11 June 1990 / Accepted: 11 June 1996     

Autocrine/paracrine modulation of calcium channels in bovine chromaffin cells

 Applying 10-s pulses of 10 mM Ba²⁺ to resting or K⁺-depolarized (70 mM) bovine adrenal chromaffin cells superfused with a nominal 0Ca²⁺ solution produced a large catecholamine secretory peak. In contrast, pulses of 10 mM Sr²⁺ or Ca²⁺ did not induce secretion from polarized resting cells, and induced smaller and narrower secretory peaks from depolarized cells; the areas of the secretory peaks from depolarized cells were 1.87, 3.06 and 27.4 nA s, respectively, for Ca²⁺, Sr²⁺ and Ba²⁺. Ca²⁺ channel currents in isolated cells or in cells surrounded by other unpatched cells (cell cluster) were studied with either the continuous-flow or the flow-stop method. When applied to an isolated cell, flow-stop reduced the amplitude of I _Ca by 19%, I _Sr by 31%, and I _Ba by 53%, compared with the current amplitude measured under continuous-flow conditions. This decrease in current amplitude was accompanied by a pronounced slowing down of current activation and could be largely relieved by applying strong depolarizing prepulses (facilitation). Under continuous-flow conditions, 10 μM exogenous ATP reduced (about 50%) I _Ca, I _Sr and I _Ba similarly. On the other hand, the use of Na⁺ as a charge carrier through Ca²⁺ channels, or intracellular dialysis with 1 mM BAPTA prevented the modulation of current by flow-stop. In cell clusters, activating secretion from unpatched cells, by either 10 mM Ba²⁺, 100 μM acetylcholine or 70 mM K⁺, caused a pronounced slowing down of current activation, as well as a decrease of its magnitude in the voltage-clamped cell immersed in the cluster. Such modulation of isolated cells was not observed. These data are compatible with the idea that the secretory activity of adrenal medullary chromaffin cells ”in situ” controls the activity of their Ca²⁺ channels through autocrine/paracrine mechanisms. Received: 29 June 1998 / Received after revision: 20 August 1998 / Accepted: 1 September 1998     

Analogies and differences between ω-conotoxins MVIIC and MVIID: binding sites and functions in bovine chromaffin cells

 The characteristics of the binding sites for the Conus magus toxins ω-conotoxin MVIIC and ω-conotoxin MVIID, as well as their effects on K⁺-evoked ⁴⁵Ca²⁺ entry and whole-cell Ba²⁺ currents (I _Ba), and K⁺-evoked catecholamine secretion have been studied in bovine adrenal chromaffin cells. Binding of [¹²⁵I] ω-conotoxin GVIA to bovine adrenal medullary membranes was displaced by ω-conotoxins GVIA, MVIIC and MVIID with IC₅₀ values of around 0.1, 4 and 100 nM, respectively. The reverse was true for the binding of [¹²⁵I] ω-conotoxin MVIIC, which was displaced by ω-conotoxins MVIIC, MVIID and GVIA with IC₅₀ values of around 30, 80 and 1.200 nM, respectively. The sites recognized by ω-conotoxins MVIIC and MVIID in bovine brain exhibited higher affinities (IC₅₀ values of around 1 nM). Both ω-conotoxin MVIIC and MVIID blocked I _Ba by 70–80%; the higher the [Ba²⁺]_o of the extracellular solution the lower the blockade induced by ω-conotoxin MVIIC. This was not the case for ω-conotoxin MVIID; high Ba²⁺ (10 mM) slowed down the development of blockade but the maximum blockade achieved was similar to that obtained in 2 mM Ba²⁺. A further difference between the two toxins concerns their reversibility; washout of ω-conotoxin MVIIC did not reverse the blockade of I _Ba while in the case of ω-conotoxin MVIID a partial, quick recovery of current was produced. This component was irreversibly blocked by ω-conotoxin GVIA, suggesting that it is associated with N-type Ca²⁺ channels. Blockade of K⁺-evoked ⁴⁵Ca²⁺ entry produced results which paralleled those obtained by measuring I _Ba. Thus, 1 μM of each of ω-conotoxin GVIA and MVIIA inhibited Ca²⁺ uptake by 25%, while 1 μM of each of ω-conotoxin MVIIC and MVIID caused a 70% blockade. K⁺-evoked catecholamine secretory responses were not reduced by ω-conotoxin GVIA (1 μM). In contrast, at 1 μM both ω-conotoxin MVIIC and MVIID reduced the exocytotic response by 70%. These data strengthen the previously established conclusion that Q-type Ca²⁺ channels that contribute to the regulation of secretion and are sensitive to ω-conotoxins MVIIC and MVIID are present in bovine chromaffin cells. These channels, however, seem to possess binding sites for ω-conotoxins MVIIC and MVIID whose characteristics differ considerably from those described to occur in the brain; they might represent a subset of Q-type Ca²⁺ channels or an entirely new subtype of voltage-dependent high-threshold Ca²⁺ channel. Received: 16 April 1997 / Received after revision: 10 July 1997 / Accepted: 23 July 1997     

Separation of calcium channel current components in mouse chromaffin cells superfused with low- and high-barium solutions

 This study was carried out to characterize the set of voltage-dependent Ca²⁺ channel subtypes expressed by mouse adrenal chromaffin cells superfused with solutions containing low (2 mM) or high (10 mM) Ba²⁺ concentrations. Using 50-ms test pulses at 0 mV from a holding potential of –80 mV, averaged peak current in 10 mM Ba²⁺ was around 1 nA, and in 2 mM Ba²⁺ 0.36 nA. When using 2 mM Ba²⁺ as the charge carrier, nifedipine (3 μM) blocked I _Ba by 40–45%. ω-Conotoxin GVIA (1 μM) caused 26% inhibition, while ω-conotoxin MVIIC (3 μM) produced a 48% blockade. At low concentrations (20 nM), ω-agatoxin IVA caused 5–15% of current inhibition, while 2 μM gave rise to a 35–40% blockade. In 10 mM Ba²⁺, the blocking effects of nifedipine (40%) and ω-conotoxin GVIA (25%) were similar to those seen in 2 mM Ba²⁺. In contrast, blockade by ω-conotoxin MVIIC was markedly reduced in 10 mM Ba²⁺ (20–25%) as compared to 10 mM Ba²⁺ (48%). The blocking actions of ω-agatoxin IVA (2 μM) were also slowed down in 10 mM Ba²⁺, though the final blockade was unaffected. In 2 mM Ba²⁺, I _Ba was quickly inhibited by over 94% with combined nifedipine + ω-conotoxin MVIIC + ω-conotoxin GVIA; in 10 mM Ba²⁺, I _Ba was blocked by 70% with this combination. The data suggest that mouse chromaffin cells express L-type (40%) as well as non-L-type (60%) high-threshold voltage-dependent Ca²⁺ channels. The current carried by non-L-type Ca²⁺ channels consists of about 25% N-type and 35% P/Q-type; P-type channels, if anything, are poorly expressed. The data also indicate that the fraction of current blocked by ω-conotoxin MVIIC and ω-agatoxin IVA might considerably change as a function of the Ba²⁺ concentration of the extracellular solution; taking this fact into consideration, it seems that a residual R-type current is not expressed in mouse chromaffin cells. Received: 21 January 1998 / Received after revision and accepted: 20 February 1998     

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.     

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 (I _Ba) 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 M and 10 M respectively, during short (15–50 ms) depolarizing pulses to 0 mV. The L-type Ca²⁺ channel agonist Bay K 8644 (1 M) caused a variable potentiation of HVA currents that could be better appreciated at low rather than at high depolarizing steps. Increase of I _Ba was accompanied by a 20-mV shift in the activation curves for Ca²⁺ channels towards more hyperpolarizing potentials. Application of the conus toxin -conotoxin GVIA (GVIA; 1 M) blocked 31% of I _Ba; blockade was irreversible upon removal of the toxin from the extracellular medium, -Agatoxin IVA (IVA; 100 nM) produced a 15% blockade of I _Ba. -Conotoxin MVIIC (MVIIC; 5 M) produced a 36% blockade of I _Ba; 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 M), GVIA (1 M), IVA (100 nM) and MVIIC (3 M) produced partial inhibition of I _Ba, which were additive. Our data suggest that the whole cell I _Ba in rat chromaffin cells exhibits at least four components. About 50% of I _Ba 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.     

Ca2+ channel inhibition induced by nitric oxide in rat insulinoma RINm5F cells

 The effect of nitric oxide (NO) donors on high-voltage-activated Ca²⁺ channels in insulin-secreting RINm5F cells was investigated using the patch-clamp technique in the whole-cell configuration. Sodium nitroprusside (SNP, 2–400 μM) induced a dose-dependent reduction in Ba²⁺ currents with maximal inhibition of 58%. The IC₅₀ for SNP was 45 μM. A different NO donor, (±)S-nitroso-N-acetylpenicillamine (SNAP, 500 μM), also produced a 50% decrease in current amplitude. When 200 μM SNP was administered together with the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidozoline-1-oxyl-3-oxide (carboxy-PTIO, 300 μM), the Ba²⁺ current inhibition was lowered to 7%. Administration of 500 μM 8-bromoguanosine 3′:5′-cyclic monophosphate sodium salt (8-Br-cGMP) mimicked the effects of SNP, causing a comparable decrease (56%) in peak-current amplitude. When soluble guanylyl cyclase was blocked by 10 μM 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ), the inhibitory effect of 200 μM SNP was reduced from 39% to 15%. The SNP-induced current decrease was 36% of controls after the blockade of L-type Ca²⁺ channels and 30% in the presence of 2.5 μM ω-conotoxin-MVIIC. These data indicate that NO inhibits both L-type and P/Q-type Ca²⁺ channels in RINm5F cells, probably by an increase in the intracellular levels of cGMP. NO may then significantly influence the Ca²⁺-dependent release of hormones from secretory cells as well as that of neurotransmitters from nerve terminals. Received: 14 April 1998 / Received after revision: 14 September 1998 / Accepted: 25 September 1998     

Tyrosine kinase inhibitors suppress N-type and T-type Ca2+ channel currents in NG108–15 cells

 Modulation of Ca²⁺ channel activity by protein kinases constitutes one of the major mechanisms regulating neuronal functions. Here, we explored the possible modulation of neuronal Ca²⁺ channels by protein tyrosine kinases (PTKs). To this end, the effects of PTK inhibitors on whole-cell Ba²⁺ currents (I _Ba) through voltage-gated Ca²⁺ channels were analysed in differentiated NG108–15 neuroblastoma × glioma hybrid cells. Genistein suppressed I _Ba in a concentration-dependent fashion (IC₅₀ = 22 μM). Although daidzein, an analogue of genistein that is devoid of PTK inhibitory activity, also suppressed I _Ba, we estimated that specific PTK inhibition by genistein reduced I _Ba amplitude by 30%. In addition, lavendustin A (20 μM) and herbimycin A (20 μM), two other distinct PTK inhibitors, depressed I _Ba by 22% and 20%, respectively. Genistein suppressed N-type and T-type currents, sparing L-type current, and its effect was independent of G protein activation. The results suggest that the activity of neuronal Ca²⁺ channels can be modulated by PTKs, opening the possibility that some of the functions of PTKs in the nervous system are mediated by Ca²⁺ channel modulation. Received: 21 November 1997 / Received after revision: 12 January 1998 / Accepted: 13 January 1998     

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 M), Zn²⁺ enhanced the secretory responses to 10-s pulses of 100 M 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⁺ (I _Ca) or Ba²⁺ (I _Ba) currents in patch-clamped chromaffin cells. Higher Zn²⁺ concentrations inhibited the currents (IC₅₀ values, 346 M for I _Ca and 91 M for I _Ba) and blocked DMPP- and K⁺-evoked secretion (IC₅₀ values, 141 and 250 M, 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.     

Q-type Ca2+ channels are located closer to secretory sites than L-type channels: functional evidence in chromaffin cells

 This study uses a new strategy to investigate the hypothesis that, of the various Ca²⁺ channels expressed by a neurosecretory cell, a given channel subtype is coupled more tightly to the exocytotic apparatus than others. The approach is based on the prediction that the degree of inhibition of the secretory response by various Ca²⁺ channel blockers will differ at low (0.5 mM) and high (5 mM) extracellular Ca²⁺ concentrations ([Ca²⁺]_o). So, at low [Ca²⁺]_o the K⁺-evoked catecholamine release from superfused bovine chromaffin cells was depressed 60–70% by 2 μM ω-agatoxin IVA (P/Q-type Ca²⁺ channel blockade), by 3 μM ω-conotoxin MVIIC (N/P/Q-type Ca²⁺ channel blockade), or by 3 μM lubeluzole (N/P/Q-type Ca²⁺ channel blockade); in high [Ca²⁺]_o these blockers inhibited the responses by only 20–35%. At 1–3 μM ω-conotoxin GVIA (N-type Ca²⁺ channel blockade) or 3 μM furnidipine (L-type Ca²⁺ channel blockade), secretion was inhibited by 30 and 50%, respectively; such inhibitory effects were similar in low or high [Ca²⁺]o. Combined furnidipine plus ω-conotoxin MVIIC, ω-agatoxin IVA or ω-conotoxin GVIA exhibited additive blocking effects at both Ca²⁺ concentrations. The results suggest that Q-type Ca²⁺ channels are coupled more tightly to exocytotic active sites, as compared to L-type channels. This hypothesis if founded in the fact that external Ca²⁺ that enters the cell through a Ca²⁺ channel located near to chromaffin vesicles will saturate the K⁺ secretory response at both [Ca²⁺]_o, i.e. 0.5 mM and 5 mM. In contrast, Ca²⁺ ions entering through more distant channels will be sequestered by intracellular buffers and, thus, will not saturate the secretory machinery at lower [Ca²⁺]_o. Received: 23 September 1997 / Received after revision: 29 October 1997 / Accepted: 30 October 1997     

Two types of ω-agatoxin IVA-sensitive Ca2+ channels are coupled to adrenaline and noradrenaline release in bovine adrenal chromaffin cells

 To clarify the role of P-type Ca²⁺ channels in catecholamine release from adrenal chromaffin cells we examined the concentration dependence of the effect of ω-agatoxin IVA on the release both of adrenaline and noradrenaline induced by a K⁺-evoked depolarization. ω-Agatoxin IVA caused a biphasic dose-dependent inhibition of secretion with a high-potency component (IC₅₀<1 nM), responsible for 10–15% of catecholamine release evoked by 70 mM K⁺, and a low-potency component that accounted for about 40% of release, with IC₅₀ values of 57 nM and 48 nM for noradrenaline and adrenaline release, respectively. The release of catecholamines from chromaffin cells was also inhibited dose dependently by ω-conotoxin MVIIC with IC₅₀ values of 182 and 218 nM for noradrenaline and adrenaline release, respectively. The effects of 3 nM ω-agatoxin IVA and 3 μM ω-conotoxin MVIIC were additive, indicating that at the concentrations used the toxins were acting at independent sites, presumably, P- and Q-type Ca²⁺ channels. The blockade of Q-type channels inhibited the release of adrenaline (72 ± 4.1%) significantly more than the release of noradrenaline (50 ± 2.7%), suggesting a higher density or a closer coupling of these channels to exocytosis in adrenergic chromaffin cells. The blockade of P-type channels caused a greater inhibition of catecholamine secretion at low levels of K⁺-evoked depolarization and shorter times of stimulation than that observed at higher levels of stimulation. The contribution of Q-type channels to catecholamine secretion did not change significantly with the intensity of stimulation. The data show that two types of ω-agatoxin IVA-sensitive Ca²⁺ channels are coupled to catecholamine release in chromaffin cells, and that the contribution of P-type channels to secretion is larger at low levels of depolarization. Received: 6 March 1997 / Received after revision and accepted: 28 April 1997     

Calcium channel subtypes in porcine adrenal chromaffin cells

 The effects of nifedipine, ω-conotoxin GVIA (ω-CgTx) and ω-agatoxin IVA (ω-AgTx) on Ca²⁺ currents, a 60-mM-K⁺-induced increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) and catecholamine secretion were examined to clarify the subtypes of Ca²⁺ channels in cultured adrenal chromaffin cells from the pig. Nifedipine, ω-CgTx, and ω-AgTx inhibited Ca²⁺ currents in a dose-dependent manner, suggesting the presence of L-, N- and P-type Ca²⁺ channels. The maximal doses of nifedipine (10 μM), ω-CgTx (1 μM), and ω-AgTx (0.1 μM) inhibited Ca²⁺ currents to 85%, 22%, and 94% of control currents, respectively. The inhibitory effects of these three blockers were observed in the same cell, indicating that at least three subtypes of Ca²⁺ channels are present in porcine chromaffin cells. The increase in [Ca²⁺]_i and catecholamine secretion induced by 60 mM K⁺ were inhibited equally by nifedipine (10 μM) and ω-CgTx (1 μM), but not by ω-AgTx (0.1 μM). These results suggest that L-, N- and P-type Ca²⁺ channels are present in porcine adrenal chromaffin cells, and that the major pathways of Ca²⁺ entry evoked by a high concentration of K⁺ are L- and N-type Ca²⁺ channels. Received: 6 September 1996 / Received after revision: 3 February 1997 / Accepted: 18 February 1997     

Ca2+-channel-dependent and -independent inhibition of exocytosis by extracellular ATP in voltage-clamped rat adrenal chromaffin cells

 Membrane currents and capacitance were measured to examine the effects of extracellular ATP on exocytosis in voltage-clamped rat adrenal chromaffin cells. ATP reversibly inhibited Ca²⁺ current (I _Ca) and exocytosis. The dependency of exocytosis on I _Ca evoked by 1-s depolarizations was determined. However, inhibition of exocytosis was 2.6 times larger than that estimated from the reduction of I _Ca, implying the existence of a Ca²⁺-channel-independent pathway. This inhibition did not rely on a further reduction of the intracellular Ca²⁺ concentration spike. ATP reduced the rate of exocytosis induced by clamping the intracellular Ca²⁺ concentration. Pertussis toxin blocked the inhibitory effects of ATP on I _Ca and exocytosis. Although RB-2, a P_2Y antagonist, blocked the inhibitory effect of ATP on I _Ca, RB-2 itself produced large increase or decrease in membrane capacitance. Adenosine inhibited I _Ca via a pertussis-toxin-sensitive pathway but did not significantly inhibit exocytosis. Our data show that extracellular ATP inhibits exocytosis via inhibition of I _Ca by activation of a pertussis-toxin-sensitive G-protein linked to P_2Y receptors. Furthermore, our data strongly suggest that ATP activates another pathway, which is also G-protein dependent and accounts for the majority of the inhibitory effect of ATP on exocytosis. Received: 20 February 1997 / Received after revision: 10 July 1997 / Accepted: 23 July 1997     

Calcium and barium permeation through calcium release-activated calcium (CRAC) channels

A Ca²⁺ current activated by store depletion has been described recently in several cell types and has been termed I _CRAC (for Ca²⁺ release-activated Ca²⁺ current). In this paper, the Ca²⁺ and Ba²⁺ permeability of CRAC channels is investigated in mast cells, rat basophilic leukaemia cells (RBL) and human T-lymphocytes (Jurkat). The selectivity of CRAC channels for Ca²⁺ over monovalent cations is identical in all three cell types and is at least as high as that of voltage-operated Ca²⁺ (VOC) channels in the various tissues tested. The amplitude of Ba²⁺ currents relative to Ca²⁺ currents (I _Ba/I _Ca) through CRAC channels was found to be strongly dependent on the membrane potential and was much smaller in Jurkat cells compared to mast and RBL cells. An anomalous mole-fraction behavior was observed at very negative membrane potentials in all three cell types when using different mixtures of external Ca²⁺ and Ba²⁺. In contrast to VOC channels, the anomalous mole-fraction effect was not observed at potentials positive to–20 mV.     

Calcium channel types contributing to excitatory and inhibitory synaptic transmission between individual hypothalamic neurons

The contribution of L-, N-, P- and Q-type Ca²⁺ channels to excitatory and inhibitory synaptic transmission and to whole-cell Ba²⁺ currents through Ca²⁺ channels (Ba²⁺ currents) was investigated in rat hypothalamic neurons grown in dissociated cell culture. Excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs) were evoked by stimulating individual neurons under whole-cell patch-clamp conditions. The different types of high-voltage-activated (HVA) Ca²⁺ channels were identified using nifedipine, ω-Conus geographus toxin VIA (ω-CTx GVIA), ω-Agelenopsis aperta toxin IVA (ω-Aga IVA), and ω-Conus magus toxin VIIC (ω-CTx MVIIC). N-, but not P- or Q-type Ca²⁺ channels contributed to excitatory as well as inhibitory synaptic transmission together with Ca²⁺ channels resistant to the aforementioned Ca²⁺ channel blockers (resistant Ca²⁺ channels). Reduction of postsynaptic current (PSC) amplitudes by N-type Ca²⁺ channel blockers was significantly stronger for IPSCs than for EPSCs. In most neurons whole-cell Ba²⁺ currents were carried by L-type Ca²⁺ channels and by at least two other Ca²⁺ channel types, one of which is probably of the Q-type and the others are resistant Ca²⁺ channels. These results indicate a different contribution of the various Ca²⁺ channel types to excitatory and inhibitory synaptic transmission and to whole-cell currents in these neurons and suggest different functional roles for the distinct Ca²⁺ channel types. Received: 15 November 1995/Accepted: 30 January 1996     

Calcium channel current facilitation in porcine adrenal chromaffin cells

 The mechanisms of depolarizing-prepulse-induced facilitation of Ca²⁺ channel current were investigated in a study of porcine chromaffin cells. The Ba²⁺ current evoked by a pulse to 0 mV was increased by a strong depolarizing prepulse (conditioning pulse), termed ”facilitation”. This facilitation increased with an increase in either the duration or the voltage of the conditioning pulse, and decreased with an increase in the interpulse interval. For example, the Ba²⁺ current was increased to 1.14 times the control (facilitation ratio) by a 150-ms conditioning pulse to +100 mV followed by a 10-ms interpulse interval. Forskolin, 8-bromo-adenosine 3′,5′-cyclic monophosphate (8-bromo-cAMP) and Rp-adenosine 3′,5′-cyclic monophosphothioate (Rp-cAMPS) did not affect the facilitation of the Ba²⁺ current, suggesting that a cAMP-dependent mechanism is not involved. Intracellular guanosine 5′-O-(3-thiotriphosphate) (GTPγS) decreased the Ba²⁺ current to 0.59 times the control and GDPβS increased it to 1.19. However, neither GTPγS nor guanosine 5′-O-(2-thiodiphosphate) (GDPβS) changed the amplitude of the Ba²⁺ current that was facilitated by the conditioning pulse. Thus, GTPγS increased the facilitation ratio to 2.05 and GDPβS decreased it to 1.05. Furthermore, the facilitation of the Ba²⁺ current was abolished by ω-conotoxin GVIA but not by either ω-agatoxin IVA or nifedipine. These results suggest that, in porcine chromaffin cells, there is a ω-conotoxin GVIA-sensitive N-type Ca²⁺ channel that is under the inhibitory control of a G protein, which can be relieved by a conditioning pulse. Received: 25 September 1997 / Received after revision: 14 November 1997 / Accepted: 16 December 1997     

Ca2+ entry through cardiac L-type Ca2+ channels modulates β-adrenergic stimulation in mouse ventricular myocytes

 β-adrenergic receptor (β-AR) stimulation increases cardiac L-type Ca²⁺ channel (CaCh) currents via cAMP-dependent phosphorylation. We report here that the affinity and maximum response of CaCh to isoproterenol (Iso), in mouse ventricular myocytes were significantly higher when Ba²⁺ was used as the charge carrier (I _Ba) instead of Ca²⁺ (I _Ca). The EC₅₀ and maximum increase of peak currents were 43.7 ± 7.9 nM and 1.8 ± 0.1-fold for I _Ca and 23.3 ± 4.7 nM and 2.4 ± 0.1-fold for I _Ba. When cells were dialyzed with the faster Ca²⁺ chelator, BAPTA, both sensitivity and maximum response of I _Ca to Iso were significantly augmented compared to cells with EGTA (EC₅₀ of 23.1 ± 5.2 nM and maximal increase of 2.2 ± 0.1-fold). Response of I _Ca to forskolin was also significantly increased when cells were dialyzed with BAPTA or when currents were measured in Ba²⁺. In contrast, depletion of the sarcoplasmic reticulum (SR) Ca²⁺ stores by ryanodine did not alter sensitivity of I _Ca to Iso or forskolin. These results suggest that the Ca²⁺ entering through CaCh regulates cAMP-dependent phosphorylation, and such negative feedback may play a significant role in cellular Ca²⁺ homeostasis and contraction in cardiac cells during β-AR stimulation. Received: 10 December 1997 / Received after revision: 19 January 1998 / Accepted: 21 January 1998     

Opioid potentiation of N-type Ca2+ channel currents via pertussis-toxin-sensitive G proteins in NG108-15 cells

Opioids have both inhibitory and stimulatory effects on neurotransmitter release. While the inhibitory effect has been ascribed to presynaptic inhibition of Ca²⁺ channels, the cellular mechanism underlying the stimulatory effect is not clear. In order to address this issue, we analyzed the effects of [d-Ala², d-Leu⁵]-enkephalin (DADLE) on whole-cell Ba²⁺ currents (I _Ba) through voltage-gated Ca²⁺ channels in NG108–15 neuroblastoma × glioma hybrid cells. Application of DADLE inhibited and washout of DADLE transiently potentiated I _Ba. Furthermore, potentiation of I _Ba was elicited even in the presence of DADLE, when inhibition was relieved by a large depolarizing prepulse. DADLE-induced potentiation, as well as inhibition, had both voltage-sensitive and -insensitive components and was abolished by treatment with ICI174864, a δ-opioid antagonist, pertussis toxin (PTX) and ω-conotoxin GVIA. Potentiation developed over @3 min and took 5–20 min to recover, whereas inhibition was complete within 30 s and recovered within 1 min. Although this potentiation should contribute to DADLE-induced desensitization of Ca²⁺ channel inhibition, it was not the sole mechanism for desensitization. We conclude that the δ-opioid receptor exerts a dual action on N-type Ca²⁺ channels via PTX-sensitive G proteins, i.e., rapid inhibition followed by slowly developing potentiation. Received: 31 March 1999 / Received after revision: 27 April 1999 / Accepted: 28 April 1999     

Calcium- and barium-dependent exocytosis from the rat insulinoma cell line RINm5F assayed using membrane capacitance measurements and serotonin release

Electrophysiological measurements of cell capacitance (C _m) and biochemical assays of [³H] serotonin ([³H]5-hydroxytryptamine or [³H]5-HT) release were combined to study the control of secretion in rat insulinoma RINm5F cells. Depolarizing pulses produced C _m changes (ΔC _m), indicative of exocytosis, with the same voltage and Ca²⁺ dependency as the inward Ca²⁺ currents (I _Ca). Ba²⁺ was able to substitute for Ca²⁺ in stimulating exocytosis, but not endocytosis. However, both the relative potency and kinetics of Ca²⁺-versus Ba²⁺-triggered exocytosis differed significantly. 5-HT synthesis and uptake were demonstrated in RINm5F cells. This allowed the use of [³H]5-HT to study hormone release from cell populations. [³H]5-HT was released in a depolarization-, Ca²⁺- and time-dependent manner. Ba²⁺ also substituted for Ca²⁺ in depolarization-induced [³H]5-HT release. Thapsigargin, used to deplete Ca²⁺ stores, had no effects on Ca²⁺-triggered C _m increases, but Ca²⁺-triggered [³H]5-HT release was abolished. Ba²⁺-triggered [³H]5-HT release, however, was only slightly affected by Ca²⁺ store depletion. Ba²⁺ was found to act directly as a secretagogue of [³H]5-HT in intact cells, but not in C _m measurements of voltage-clamped cells, suggesting that cell depolarization is a prerequisite for this action. Received: 18 October 1995/Received after revision and accepted: 9 January 1996     

VIP and secretin augment cardiac L-type calcium channel currents in isolated adult rat ventricular myocytes

Vasoactive intestinal peptide (VIP) is colocalized in parasympathetic nerve terminals in the heart and coreleased from these nerve terminals with the “classical” neurotransmitter acetylcholine (Ach). VIP also exerts a positive inotropic effect on the intact heart and enhances adenylyl cyclase activity in isolated heart membranes. Using the whole-cell patch-clamp technique, we show here that VIP enhances Ca²⁺ and Ba²⁺ currents (I _Ba) through voltage-dependent L-type Ca²⁺ channels in adult rat ventricular myocytes. Neither the kinetics nor the voltage-dependent properties of the currents are affected. The effect of VIP on I _Ba is dose dependent with a half-maximal concentration of approximately 0.4 μM. The onset of the effect of VIP and the recovery phase are slow, suggesting the involvement of an intracellular second messenger. The effect of VIP on I _Ba is antagonized by a peptide analog of the growth hormone releasing factor ([Ac-Tyr¹, D-Phe²]-GRF) which belongs to the same peptide family as VIP. Although VIP and the β-adrenergic receptor agonist isoproterenol (ISO) enhance I _Ba peak amplitudes to approximately the same extent, the effect of VIP is not seen on all cells. Only approximately 50% of the isolated myocytes respond to 5 μM VIP, whereas 95% of the cells respond to ISO. Similar results were obtained using the amphotericin B perforated-patch whole-cell-recording technique, suggesting that the variable response to VIP does not reflect the loss of a pivotal intracellular regulator. The gastrointestinal hormone secretin, a peptide structurally related to VIP, also potentiates I _Ba in adult rat ventricular myocytes, although secretin is substantially more potent than VIP (half-maximal concentration for secretin is about 0.7 nM). Taken together, these results suggest that the VIP- (and secretin-) induced potentiation of I _Ba in adult rat ventricular myocytes is mediated through a non-VIP-preferring class of VIP receptors. Received: 7 December 1995/Received after revision and accepted: 31 May 1996