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     

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     

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     

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     

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 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     

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     

Effects of ruthenium red on membrane ionic currents in urinary bladder smooth muscle cells of the guinea-pig

 Three major ionic currents, Ca²⁺-dependent K⁺ current (I _K-Ca), delayed rectifier type K⁺ current (I _kd) and Ca²⁺ current (I _Ca), were activated by depolarization under whole-cell clamp in single smooth muscle cells isolated from guinea-pig urinary bladder. Externally applied ruthenium red (RuR) reduced the amplitude of I _K-Ca and I _Ca at 0 mV (IC₅₀ values were 4.2 and 5.6 μM, respectively), but did not affect I _Kd. Spontaneous transient outward currents (STOCs) and caffeine-induced outward currents (I _caf) at –30 mV were reduced by external 10 μM RuR. When 10 μM RuR was added to the pipette solution, I _K-Ca during depolarization, STOCs and I _caf significantly decreased with time. RuR did not change the unitary current amplitude of the large-conductance Ca²⁺-dependent K⁺ (BK) channels, but reduced the open probability of the channel under excised patch-clamp recording mode. RuR reduced the channel activity more effectively from the cytosolic face than from the other. This inhibition decreased when the cytosolic Ca²⁺ concentration was increased. These results indicate that RuR blocks BK and Ca²⁺ channels in urinary bladder smooth muscle cells. The decrease in I _K-Ca, STOCs and I _caf by RuR is attributable to the direct inhibition of BK channel activity, probably in addition to the inhibition of Ca²⁺ release from storage sites. The direct inhibition of BK channel activity by RuR may be related to the interaction of RuR with the Ca²⁺-binding sites of the channel protein. Received: 15 October 1997 / Received after revision and accepted: 25 November 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     

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     

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     

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     

Ruthenium red reduces the Ca2+ sensitivity of Ca2+ uptake into cardiac sarcoplasmic reticulum

 Ruthenium red inhibits mitochondrial Ca²⁺ uptake and is widely used as an inhibitor of ryanodine-sensitive Ca²⁺ channels that function to release Ca²⁺ from the sarcoplasmic reticulum (SR) of muscle cells. It also has effects on other Ca²⁺ channels and ion transporters. To study the effects of ruthenium red on Ca²⁺ transport into the SR of cardiac muscle cells, fluorescence measurements of Ca²⁺ uptake into cardiac SR vesicles were made. Ruthenium red significantly decreased the Ca²⁺ sensitivity of SR uptake in a dose-dependent manner at concentrations ranging from 5 μM to 20 μM. There were no significant effects of ruthenium red on the maximum velocity or the Hill coefficient of SR Ca²⁺ uptake. Received: 14 January 1998 / Received after revision: 12 March 1998 / Accepted: 16 March 1998     

Regulation of calcium release by calcium inside the sarcoplasmic reticulum in ventricular myocytes

 To study the effects of changes in sarcoplasmic reticulum (SR) intraluminal Ca²⁺ on the Ca²⁺ release mechanism, we correlated the activity of single cardiac ryanodine receptor (RyR) channels, monitored in planar bilayers, with the properties of spontaneous elementary Ca²⁺ release events (sparks) in intact ventricular myocytes, monitored by scanning confocal microfluorimetry. Under both normal conditions and Ca²⁺ overload, induced by elevation of extracellular [Ca²⁺], Ca²⁺ sparks represented single populations of events. During Ca²⁺ overload, the frequency of sparks increased from 0.8 to 3.1 events per second per 100 μm line scanned, and their amplitude increased from 100 nM to 400 nM. The duration of the Ca²⁺ sparks, however, was not altered. Changes in the properties of Ca²⁺ sparks were accompanied by only an ≈ 30% increase in the SR Ca²⁺ content, as determined by emptying the intracellular Ca²⁺ stores using caffeine. When single Ca²⁺ release channels were incorporated into lipid bilayers and activated by cytoplasmic Ca²⁺ (≈ 100 nM) and ATP (3 mM), elevation of Ca²⁺ on the luminal side from 20 μM to 0.2–20 mM resulted in a 1.2-fold to 7-fold increase, respectively, in open probability (P _o). This potentiation of P _o was due to an increase in mean open time and frequency of events. The relative effect of luminal Ca²⁺ was greater at low levels of cytoplasmic [Ca²⁺] than at high levels of cytoplasmic [Ca²⁺], and no effect of luminal Ca²⁺ was observed to occur in channels activated by 0.5–50 μM cytoplasmic Ca²⁺ in the absence of ATP. Our results suggest that SR Ca²⁺ release channels are modulated by SR intraluminal Ca²⁺. These alterations in properties of release channels may account for, or contribute to, the mechanism of spontaneous Ca²⁺ release in cardiac myocytes Received: 15 May 1996 / Received after revision: 5 June 1996 / Accepted: 8 July 1996     

Glibenclamide suppresses stretch-activated ANP secretion: involvements of K+ ATP channels and L-type Ca2+ channel modulation

 The mechanism by which glibenclamide regulates mechanically activated atrial natriuretic peptide (ANP) secretion was investigated using isolated perfused rat atria. A reduction in atrial pressure from an experimentally imposed distending pressure stimulated the secretion of ANP and caused concomitant translocation of extracellular fluid (ECF) into the atrial lumen. The activation of ANP secretion and ECF translocation were closely correlated with atrial volume changes and the increase in ANP secretion was a function of the ECF translocation. Glibenclamide (1, 10, 100 μM), an ATP-sensitive K⁺ (K⁺ _ATP) channel blocker, had no effect on the basal secretion of ANP, suppressed the stimulation of stretch-activated ANP secretion in a dose-dependent manner, but not the translocation of the ECF. Glipizide (100 μM) and tolbutamide (100 μM), other K⁺ _ATP channel blockers, had similar effects to those of glibenclamide. Suppression by glibenclamide (100 μM) of the stretch-induced ANP secretion was not observed in atria that had been pretreated with pinacidil (200 μM), an ATP-sensitive K⁺ channel opener: pinacidil alone had no effect on ECF translocation and ANP secretion. Furthermore, blocking Ca²⁺ influx by using the Ca²⁺ channel blocker diltiazem (10 nM), or a Ca²⁺-depleted medium prevented the suppression of stretch-induced ANP secretion by glibenclamide. In other experiments, atrial distension produced a slight membrane depolarization of cardiomyocytes; this was accentuated in the presence of glibenclamide. Furthermore, in single cardiomyocytes, glibenclamide increased the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in a dose-dependent manner. From these results, we suggest that glibenclamide suppresses atrial release of ANP by blocking K⁺ _ATP channels and increasing Ca²⁺ influx and that the K⁺ _ATP channels are associated with the regulation of the mechanically activated ANP secretion from the atria. Received: 13 May 1996 / Received after revision: 10 February 1997 / Accepted: 5 March 1997     

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     

Dual modulation of calcium channel current via recombinant α2-adrenoceptors in pheochromocytoma (PC-12) cells

 The ability of recombinant rat α_2D-and α_2B-adrenoceptors expressed in nerve-growth-factor-differentiated pheochromocytoma PC-12 cells to modulate Ca²⁺ currents, recorded by the whole-cell patch-clamp technique, has been studied. Ca²⁺ currents in different cells were either reversibly reduced or increased by dexmedetomidine, an α₂-adrenergic agonist, in a concentration-dependent manner. Pertussis toxin pretreatment reduced the number of cells that showed an inhibitory response and reduced the magnitude of inhibition. In cells expressing the α_2B-adrenoceptor, pertussis toxin increased the proportion of cells from which a stimulatory effect on Ca²⁺ currents could be recorded. The magnitude of the inhibitory responses was unaffected but the stimulatory responses were considerably reduced by the dihydropyridine Ca²⁺ channel blocker nifedipine (5 μM). All effects of dexmedetomidine were reversible upon wash-out and inhibited by the antagonist rauwolscine. The results support the idea that modulation of voltage-dependent Ca²⁺ channels in transfected PC-12 cells is mediated by activation of recombinant α_2D- and α_2B-adrenoceptors. This receptor activation predominantly causes inhibition of dihydropyridine-insensitive Ca²⁺ channels via pertussis-toxin-sensitive G proteins. Additionally receptor activation can also lead to stimulation of dihydropyridine-sensitive Ca²⁺ channels via pertussis-toxin-insensitive mechanisms. Received: 25 March 1997 / Received after revision: 27 August 1997 / Accepted: 12 September 1997     

Effect of acidosis on Ca2+-activated K+ channels in cultured porcine coronary artery smooth muscle cells

 Although acidosis induces vasodilation, the vascular responses mediated by large-conductance Ca²⁺-activated K⁺ (K_Ca) channels have not been investigated in coronary artery smooth muscle cells. We therefore investigated the response of porcine coronary arteries and smooth muscle cells to acidosis, as well as the role of K_Ca channels in the regulation of muscular tone. Acidosis (pH 7.3–6.8), produced by adding HCl to the extravascular solution, elicited concentration-dependent relaxation of precontracted, endothelium-denuded arterial rings. Glibenclamide (20 μM) significantly inhibited the vasodilatory response to acidosis (pH 7.3-6.8). Charybdotoxin (100 nM) was effective only at pH 6.9–6.8. When we exposed porcine coronary artery smooth muscle cells to a low-pH solution, K_Ca channel activity in cell-attached patches increased. However, pretreatment of these cells with 10 or 30 μM O, O′-bis(2-aminophenyl)ethyleneglycol-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl)ester (BAPTA-AM), a Ca²⁺ chelator for which the cell membrane is permeable, abolished the H⁺-mediated activation of K_Ca channels in cell-attached patches. Under these circumstances H⁺ actually inhibited K_Ca channel activity. When inside-out patches were exposed to a [Ca²⁺] of 10^–6 M [adjusted with ethyleneglycolbis(β-aminoethylester)-N,N,N′,N′-tetraacetic acid (EGTA) at pH 7.3], K_Ca channels were activated by H⁺ concentration dependently. However, when these patches were exposed to a [Ca²⁺] of 10^–6 M adjusted with BAPTA at pH 7.3, H⁺ inhibited K_Ca channel activity. Extracellular acidosis had no significant direct effect on K_Ca channels, suggesting that extracellular H⁺ exerts its effects after transport into the cell, and that K_Ca channels are regulated by intracellular H⁺ and by cytosolic free Ca²⁺ modulated by acute acidosis. These results indicate that the modulation of K_Ca channel kinetics by acidosis plays an important role in the determination of membrane potential and, hence, coronary arterial tone. Received: 20 January 1998 / Received after revision: 9 April 1998 / Accepted: 22 April 1998     

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