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     

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     

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     

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.     

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     

Human adrenal chromaffin cell calcium channels: drastic current facilitation in cell clusters, but not in isolated cells

 Human adrenal medullary chromaffin cells were prepared and cultured from a cystic tumoral adrenal gland whose medullary tissue was unaffected. Adrenaline-containing and noradrenaline-containing cells were identified using a confocal fluorescence microscope and antibodies against dopamine beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT). Current/voltage (I/V) curves performed with the voltage-clamped cells bathed in 10 mM Ba²⁺ (holding potential, V _h=–80 mV) revealed the presence of only high-threshold voltage-dependent Ca²⁺ channels; T-type Ca²⁺ channels were not seen. By using supramaximal concentrations of selective Ca²⁺ channel blockers, the whole-cell I _Ba could be fractionated into various subcomponents. Thus, I _Ba had a 25% fraction sensitive to 1 μM nifedipine (L-type channels), 21% sensitive to 1 μM ω-conotoxin GVIA (N-type channels), and 60% sensitive to 2 μM ω-agatoxin IVA (P/Q-type channels). The activation of I _Ba was considerably slowed down, and the peak current was inhibited upon superfusion with 10 μM ATP. The slow activation and peak current blockade were reversed by strong depolarizing pre-pulses to +100 mV (facilitation). A drastic facilitation of I _Ba was also observed in voltage-clamped human chromaffin cell surrounded by other unclamped cells; in contrast, in voltage-clamped cells not immersed in a cell cluster, facilitation was scarce. So, facilitation of Ca²⁺ channels in a voltage-clamped cell seems to depend upon the exocytotic activity of neighbouring unclamped cells, which is markedly increased by Ba²⁺. It is concluded that human adrenal chromaffin cells mostly express P/Q-types of voltage-dependent Ca²⁺ channels (60%). L-Type channels and N-type channels are also expressed, but to a considerably minor extent (around 20% each). This dominance of P/Q-type channels in human chromaffin cells clearly contrasts with the relative proportion of each channel type expressed by chromaffin cells of five other animal species studied previously, where the P/Q-type channels accounted for 5–50%. The results also provide strong support for the hypothesis that Ca²⁺ channels of human chromaffin cells are regulated in an autocrine/paracrine fashion by materials co-secreted with the catecholamines, i.e. ATP and opiates. Received: 1 May 1998 / Received after revision and accepted: 21 May 1998     

P/Q-type calcium channels activate neighboring calcium-dependent potassium channels in mouse motor nerve terminals

 The identity of the voltage-dependent calcium channels (VDCC), which trigger the Ca²⁺-gated K⁺ currents (I _K(Ca)) in mammalian motor nerve terminals, was investigated by means of perineurial recordings. The effects of Ca²⁺ chelators with different binding kinetics on the activation of I _K(Ca) were also examined. The calcium channel blockers of the P/Q family, ω-agatoxin IVA (ω-Aga-IVA) and funnel-web spider toxin (FTX), have been shown to exert a strong blocking effect on I _K(Ca). In contrast, nitrendipine and ω-conotoxin GVIA (ω-CgTx) did not affect the Ca²⁺-activated K⁺ currents. The intracellular action of the fast Ca²⁺ buffers BAPTA and DM-BAPTA prevented the activation of the I _K(Ca), while the slow Ca²⁺ buffer EGTA was ineffective at blocking it. These data indicate that P/Q-type VDCC mediate the Ca²⁺ influx which activates I _K(Ca). The spatial association between Ca²⁺ and Ca²⁺-gated K⁺ channels is discussed, on the basis of the differential effects of the fast and slow Ca²⁺ chelators. Received: 20 December 1996 / Received after revision: 21 March 1997 / Accepted: 2 April 1997     

Cholinergic agonists decrease quantal output at the frog neuromuscular junction by targeting a calcium channel blocked by ω-conotoxin

 Nicotinic cholinergic agonists are known to decrease synchronous evoked quantal output at the frog neuromuscular junction [Van der Kloot 1993, J Physiol (Lond) 468:567–589]. Here we also show that carbachol decreases the frequency of miniature endplate potentials (F _MEPP) in solutions containing elevated levels of K⁺ and Ca²⁺. Carbachol did not decrease F _MEPP in hypertonic solutions or in solutions containing the Ca²⁺ ionophore ionomycin and Ca²⁺. We conclude that the nicotinic agonists decrease Ca²⁺ influx through voltage-gated Ca²⁺ channels. Carbachol did not alter two-pulse facilitation. A blocker of N-type Ca²⁺ channels, ω-conotoxin GVIA, antagonized the nicotinic agonist-induced decrease in evoked quantal output. The effect of carbachol was not altered by ω-conotoxin MVIIC, a blocker of P-type and certain other Ca²⁺channels. The Ca²⁺ channel targeted by the nicotinic agonists appears to be of the N-type. Received: 6 March 1997 / Received after revision: 6 May 1997 / Accepted: 4 June 1997     

Functional expression of rat brain cloned α1E calcium channels in COS-7 cells

 The properties of the rat brain α1E Ca²⁺ channel subunit and its modulation by accessory rat brain α2-δ and β1b subunits were studied by transient transfection in a mammalian cell line in order to attempt to reconcile the debate as to whether α1E forms a low-voltage-activated (LVA) or high-voltage-activated (HVA) Ca²⁺ channel and to examine its pharmacology in detail. α1E alone was capable of forming an ion-conducting pore in COS-7 cells. The properties of heteromultimeric α1E/α2-δ/β1b channels were largely dictated by the presence of the β1b subunit, which increased current density and tended to produce a hyperpolarizing shift in the voltage dependence of activation and inactivation. α1E/α2-δ/β1b channels did not appear to be regulated by Ca²⁺-induced inactivation. α1E was shown to exhibit a unique pharmacological profile. ω-Agatoxin IVA blocked the current in a dose-dependent manner with an IC₅₀ of approximately 50 nM and a maximum inhibition of about 80%, whilst ω-conotoxin MVIIC was without effect. The 1,4-dihydropyridine (DHP) antagonist nicardipine (1 μM) produced an inhibition of 51 ± 7%, whereas the DHP agonist S-(–)BAY K 8644 was without effect. Our findings suggest a re-evaluation of the classification of the α1E Ca²⁺ channel subunit; we propose that rat brain α1E forms a novel Ca²⁺ channel with properties more similar to a subtype of LVA than HVA Ca²⁺ current. Received: 30 August 1996 / Received after revision and accepted: 28 October 1996     

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     

Role of calcium in stretch-induced release and mRNA synthesis of natriuretic peptides in isolated rat atrium

 To investigate the role of Ca²⁺ in stretch-induced synthesis and release of atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) isolated superfused rat atria were stretched by raising intra-atrial pressure. The immunoreactive (ir-) ANP and BNP concentrations were analysed by radioimmunoassay and the corresponding mRNA levels were quantified by Northern blot and dot blot analyses. Stretch-induced ir-ANP release and a rise in BNP mRNA levels increased at high (3.0 mM) compared to low (0.5 mM) extracellular Ca²⁺ concentration ([Ca²⁺]_o). Moreover, the adaptation of stretch-induced ir-ANP release was dependent on [Ca²⁺]_o. Atrial BNP mRNA levels were increased by stretch also in non-paced, electrically silent atria, where voltage-activated Ca²⁺ channels are not activated. The stretch-induced rise in BNP mRNA was blocked by gadolinium (80 μM), but not by the L-type channel blocker diltiazem (3.0 μM). This study indicates that both the stretch-secretion coupling of ir-ANP release and the pressure-stimulated synthesis of BNP mRNA are Ca²⁺-dependent processes. Gadolinium inhibits the stretch-stimulated rise in BNP mRNA levels in contracting and non-contracting atria, which is similar to its ability to block stretch-activated ir-ANP release, suggesting the involvement of Ca²⁺-permeable stretch-activated channels. Received: 29 April 1996 / Received after revision and accepted: 17 June 1996     

Effects of T-type, L-type, N-type, P-type, and Q-type calcium channel blockers on stimulus-induced pre-and postsynaptic calcium fluxes in rat hippocampal slices

The contribution of T-, L-, N-, P-, and Q-type Ca²⁺ channels to pre-and postsynaptic Ca²⁺ entry during stimulus-induced high neuronal activity in area CA1 of rat hippocampal slices was investigated by measuring the effect of specific blockers on stimulus-induced decreases in extracellular Ca²⁺ concentration ([Ca²⁺]₀). [Ca²⁺]₀ was measured with ion-selective electrodes in stratum radiatum (SR) and stratum pyramidale (SP), while Ca²⁺ entry into neurons was induced with stimulus trains (20 Hz for 10 s) alternately delivered to SR and the alveus, respectively. The [Ca²⁺]₀ decreases recorded in SR in response to SR stimulation represented mainly presynaptic Ca²⁺ entry (Ca_pre), while [Ca²⁺]⁰ decreases recorded in SP in response to alvear stimulation were predominantly based on postsynaptic Ca²⁺ entry (Ca_post). Ethosuximide and trimethadione were ineffective m concentrations up to 1 mM. At 10 mM, they reduced Ca_post and, much less, also Ca_pre Nimodipine (25 M) reduced Ca_post and, to a minor extent, Ca_pre. -Agatoxin IVA (0.4–1 M) and -conotoxin MVIIC (1 M) also reduced both Ca_pre and Ca_post, but with a stronger action on Ca_pre. -Conotoxin GVIA (3–8 M) reduced Ca_post without effect on Ca_pre. We conclude that during stimulus-induced, high-frequency neuronal activity Ca_post is carried by P/Q-, N-, and L-type channels and probably a further channel type different from these channels. Ca_pre includes at least P/Q-and possibly L-type channels. N-type channels did not contribute to Ca_pre in our experiments. Since ethosuximide and trimethadione were only effective in high concentrations, their action may be unspecific. Thus, T-type channels do not seem to play a major part in Ca²⁺ entry in this situation.     

Voltage-gated calcium channels may be involved in the regulation of the mechanosensitivity of slowly conducting knee joint afferents in rat

Voltage-gated Ca²⁺ channels play an important role in the central processing of nociceptive information. Recently, it has been shown that L- and N-type voltage-gated Ca²⁺ channels are also present on peptidergic, fine afferent nerve fibers in the knee joint capsule. Therefore, the influence of specific blockers for L-type (verapamil) or N-type (ω-conotoxin GVIA) Ca²⁺ channels on the mechanosensitivity of slowly conducting afferents was tested in the rat knee joint. Topical application of 100 μM verapamil onto the receptive field reduced the mean response to knee joint rotation to 67±8% (SEM, n=12), obtained by outward rotations with a torque of 10 mNm above the mechanical threshold and compared with control movements. In the presence of 50 μM ω-conotoxin GVIA, the mean response decreased to 44±5% (n=12), a reduction that was also observed during rotations of other intensities. Simultaneous application of both substances further reduced the response to 25±11% (n=6). In additional experiments it was shown that L- and N-type voltage-gated Ca²⁺ channels do not influence activity-dependent changes of the mechanical excitability. In conclusion, the data of the present study indicate that voltage-gated Ca²⁺ channels may also be involved in the regulation of the mechanosensitivity of nociceptive nerve fiber endings. Electronic Publication     

Membrane potential modulation of ionomycin-stimulated Ca<Superscript>2+</Superscript> entry via Ca<Superscript>2+</Superscript>/H<Superscript>+</Superscript> exchange and SOC in rat submandibular acinar cells

Ionomycin (IM) at 5 μM mediates the Ca²⁺/H⁺ exchange, while IM at 1 μM activates the store-operated Ca²⁺ entry channels (SOCs). In this study, the effects of depolarization on both pathways were examined in rat submandibular acinar cells by increasing extracellular K⁺ concentration ([K⁺]_o). IM (5 μM, the Ca²⁺/H⁺ exchange) increased the intracellular Ca²⁺ concentration ([Ca²⁺]_i) to an extremely high value at 151 mM [K⁺]_o. However, with increasing [K⁺]_o, the rates of Ca²⁺ entry decreased in a linear relationship. The reversal potential (E _rev) for the Ca²⁺/H⁺ exchange was +93 mV, suggesting that IM (5 μM) exchanges 1 Ca²⁺ for 1 H⁺. Thus, depolarization decreases the Ca²⁺ influx via the Ca²⁺/H⁺ exchange because of its electrogenicity (1 Ca²⁺ for 1 H⁺). On the other hand, IM (1 μM, the SOCs) abolished an increase in [Ca²⁺]_i at 151 mM [K⁺]_o. With increasing [K⁺]_o, the rate of Ca²⁺ entry immediately decreased linearly. The E _rev for the SOC was +3.7 mV, suggesting that the SOCs are nonselective cation channels and less selective for Ca²⁺ over Na⁺ (P _Ca/P _Na = 8.2). Moreover, an increase in extracellular Ca²⁺ concentration (20 mM) enhanced the Ca²⁺ entry via the SOCs at 151 mM [K⁺]_o, suggesting depolarization does not inhibit the SOCs and decreases the driving force for the Ca²⁺ entry. This suggests that membrane potential changes induced by a secretory stimulation finely regulate the [Ca²⁺]_i via the SOCs in rat submandibular acinar cells. In conclusion, IM increases [Ca²⁺]_i via two pathways depending on its concentration, the exchange of 1 Ca²⁺ for 1 H⁺ at 5 μM and the SOCs at 1 μM.     

Blockade of HERG channels expressed in Xenopus oocytes by external H+

 We have investigated the effect of external H⁺ concentration ([H⁺]_o)on the human-ether-a-go-go-related gene (HERG) current (I _HERG), the molecular equivalent of the cardiac delayed rectifier potassium current (I _Kr), expressed in Xenopus oocytes, using the two-microelectrode voltage-clamp technique. When [H⁺]_o was increased, the amplitude of the I _HERG elicited by depolarization decreased, and the rate of current decay on repolarization was accelerated. The activation curve shifted to a more positive potential at lower external pH (pH_o) values (the potential required for half-maximum activation, V _1/2, was: –41.8 mV, –38.0 mV, –33.7 mV, –26.7 mV in pH_o 8.0, 7.0, 6.6, 6.2, respectively). The maximum conductance (g _max) was also affected by [H⁺]_o: a reduction of 7.9%, 14.6%, and 22.8% was effected by decreasing pH_o from 8.0 to 7.0, 6.6, and 6.2, respectively. We then tested whether this pH effect was affected by the external Ca²⁺ concentration, which is also known to block HERG channels. When the extracellular Ca²⁺ concentration was increased from 0.5 mM to 5 mM, the shift in V _1/2 caused by increasing [H⁺]_o was attenuated, suggesting that these two ions compete for the same binding site. On the other hand, the decrease in g _max caused by increasing [H⁺]_o was not significantly affected by changing external Ca²⁺ levels. The results indicate that HERG channels are inhibited by [H⁺]_o by two different mechanisms: voltage-dependent blockade (shift of V _1/2) and the decrease in g _max. With respect to the voltage-dependent blockade, the interaction between H⁺ and Ca²⁺ is competitive, whereas for the decreasing g _max, their interaction is non-competitive. Received: 12 January 1999 / Received after revision: 15 February 1999 / Accepted: 16 February 1999     

Neomycin inhibits K+-induced force and Ca2+ channel current in rat arterial smooth muscle

 The techniques of small vessel isometric myography and patch clamp were used to investigate the action of neomycin on K⁺-induced isometric force and voltage-gated Ca²⁺ channel currents in rat arterial smooth muscle. Neomycin and the dihydropyridine (DHP) Ca²⁺ channel antagonist (–)202–791 concentration-dependently and reversibly inhibited 40 mM K⁺-induced isometric force in rings of rat mesenteric and basilar arteries (IC₅₀ values of 70 μM and 1.2 nM, respectively, n = 10 and 4). Elevation of [Ca²⁺]_o by a factor of 2 significantly reduced the IC₅₀ values for inhibition of K⁺-induced force for both neomycin and (–)202–791 (192 μM and 3.7 nM, respectively, n = 6 and 4), but did not affect the Hill coefficient of the concentration/effect relationships. In patch-clamp experiments using freshly isolated basilar arterial myocytes, the voltage-gated inward current carried by Ba²⁺ was reversibly and concentration-dependently inhibited by neomycin (IC₅₀ 32 μM, n = 3). The concentration/effect curve for inhibition of the inward Ba²⁺ current by neomycin was significantly shifted to the right when [Ba²⁺]_o was raised from 1.8 mM to 10 mM (IC₅₀ 144 μM, n = 8). Our findings suggest that neomycin relaxes high-K⁺-induced force in rat isolated mesenteric and basilar arteries largely by inhibition of voltage-dependent and DHP-sensitive Ca²⁺ channels. Received: 1 August 1996 / Received after revision and accepted: 11 September 1996     

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.     

Small (SKCa) Ca2+-activated K+ channels in cultured rat hippocampal pyramidal neurones

 Small (SK_Ca) Ca²⁺-activated K⁺ channels were identified in membrane patches excised from cultured CA1-CA3 pyramidal neurones of the neonatal rat hippocampus. When recorded in low-K⁺ extracellular solution ([K⁺]_o=2.5 mM), SK_Ca channels had a low conductance (@3 pS at 0 mV), were activated by ≥175 nM Ca²⁺ (P _o=0.54 at 500 nM Ca²⁺) and there were two open-time components (2.1 and @70 ms) to their activity. These properties of single SK_Ca channels are similar to those of slow after-hyperpolarization channels (sAHP) previously inferred from fluctuation analysis of the sAHP current. It is concluded that the SK_Ca channel reported here may be the channel that generates the sAHP in hippocampal pyramidal neurones. Received: 9 July 1998 / Received after revision: 5 October 1998 / Accepted: 7 October 1998     

ATP suppresses activity of Ca2+ -activated K+ channels by Ca2+ chelation

Ca²⁺-activated maxi K⁺ channels were studied in inside-out patches from smooth muscle cells isolated from either porcine coronary arteries or guinea-pig urinary bladder. As described by Groschner et al. (Pflügers Arch 417:517, 1990), channel activity (NP _o) was stimulated by 3 M [Ca²⁺]_c (1 mM Ca-EGTA adjusted to a calculated pCa of 5.5) and was suppressed by the addition of 1 mM Na₂ATP. The following results suggest that suppression of NP _o by Na₂ATP is due to Ca²⁺ chelation and hence reduction of [Ca²⁺]_c and reduced Ca²⁺ activation of the channel. The effect was absent when Mg ATP was used instead of Na₂ATP. The effect was diminished by increasing the [EGTA] from 1 to 10 mM. The effect was absent when [Ca²⁺]_c was buffered with 10 mM HDTA (apparent pK _Ca 5.58) instead of EGTA (pK _Ca 6.8). A Ca²⁺-sensitive electrode system indicated that 1 mM Na₂ATP reduced [Ca²⁺]_c in 1 mM Ca-EGTA from 3 M to 1.4 M. Na₂ATP, Na₂GTP, Li₄AMP-PNP and NaADP reduced measured [Ca²⁺]_c in parallel with their suppression of NP _o. After the Na₂ATP-induced reduction of [Ca²⁺]_c was re-adjusted by adding either CaCl₂ or MgCl₂, the effect of Na₂ATP on NP _o disappeared. In vivo, intracellular [Mg²⁺] exceeds free [ATP^4–], hence ATP modulation of maxi K⁺ channels due to Ca²⁺ chelation is without biological relevance.