Effects of membrane depolarization and changes in extracellular [K<Superscript>+</Superscript>] on the Ca<Superscript>2+</Superscript> transients of fast skeletal muscle fibers. Implications for muscle fatigue

Repetitive activation of skeletal muscle fibers leads to a reduced transmembrane K⁺ gradient. The resulting membrane depolarization has been proposed to play a major role in the onset of muscle fatigue. Nevertheless, raising the extracellular K⁺ ( \textK_\texto ⁺ {\text{K}}_{\text{o}}^{ + } ) concentration ( [ \textK ⁺ ]_\texto [ {\text{K}}^{ + } ]_{\text{o}} ) to 10 mM potentiates twitch force of rested amphibian and mammalian fibers. We used a double Vaseline gap method to simultaneously record action potentials (AP) and Ca²⁺ transients from rested frog fibers activated by single and tetanic stimulation (10 pulses, 100 Hz) at various [ \textK ⁺ ]_\texto [ {\text{K}}^{ + } ]_{\text{o}} and membrane potentials. Depolarization resulting from current injection or raised [ \textK ⁺ ]_\texto [ {\text{K}}^{ + } ]_{\text{o}} produced an increase in the resting [Ca²⁺]. Ca²⁺ transients elicited by single stimulation were potentiated by depolarization from −80 to −60 mV but markedly depressed by further depolarization. Potentiation was inversely correlated with a reduction in the amplitude, overshoot and duration of APs. Similar effects were found for the Ca²⁺ transients elicited by the first pulse of 100 Hz trains. Depression or block of Ca²⁺ transient in response to the 2nd to 10th pulses of 100 Hz trains was observed at smaller depolarizations as compared to that seen when using single stimulation. Changes in Ca²⁺ transients along the trains were associated with impaired or abortive APs. Raising [ \textK ⁺ ]_\texto [ {\text{K}}^{ + } ]_{\text{o}} to 10 mM potentiated Ca²⁺ transients elicited by single and tetanic stimulation, while raising [ \textK ⁺ ]_\texto [ {\text{K}}^{ + } ]_{\text{o}} to 15 mM markedly depressed both responses. The effects of 10 mM \textK_\texto ⁺ {\text{K}}_{\text{o}}^{ + } on Ca²⁺ transients, but not those of 15 mM \textK_\texto ⁺ {\text{K}}_{\text{o}}^{ + } , could be fully reversed by hyperpolarization. The results suggests that the force potentiating effects of 10 mM \textK_\texto ⁺ {\text{K}}_{\text{o}}^{ + } might be mediated by depolarization dependent changes in resting [Ca²⁺] and Ca²⁺ release, and that additional mechanisms might be involved in the effects of 15 mM \textK_\texto ⁺ {\text{K}}_{\text{o}}^{ + } on force generation.     

Persistent sodium current and Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchange contributes to the augmentation of the reverse Na<Superscript>+</Superscript>/Ca<Superscript>2+</Superscript> exchange during hypoxia or acute ischemia in ventricular myocytes

The increases in persistent sodium currents (I _Na.P) and Na⁺/H⁺ exchange (NHE) causes intracellular Ca²⁺ overload. The objective of this study was to determine the contribution of I _Na.P and NHE on the hypoxia- or acute ischemia-induced increase in the reverse Na⁺/Ca²⁺ exchange current (HIR- or AIR-I _NCX). I _Na.P and I _NCX in rabbit ventricular myocytes were recorded during hypoxia or acute ischemia, combination of acidosis (pH values were 6.0 intracellularly and 6.8 extracellularly) and hypoxia, using whole-cell patch-clamp techniques. The results indicate that (1) under hypoxic condition, the augmentation of both HIR-I _NCX and I _Na.P was inhibited by TTX (2 to 8 μM) in a concentration-dependent manner. The inhibitions of I _Na,P and HIR-I _NCX reached maximum in the presence of either 4 μM TTX or 10 μM KR-32568 (a NHE inhibitor), respectively. The maximal inhibitions of HIR-I _NCX by 4 μM TTX and 10 μM KR-32568 were 72.54% and 16.89%, respectively. (2) Administration of 2 μM TTX and 10 μM KR-32568 in either order in the same cells decreased HIR-I _NCX by 64.83% and 16.94%, respectively. (3) I _Na.P and the reverse I _NCX were augmented during acute ischemia. TTX (4 μM) and KR-32568 (10 μM) reduced AIR-I _NCX by 73.39% and 24.13%, respectively. (4) Under normoxic condition, veratridine (20 μM) significantly increased I _Na.P and the reverse I _NCX, which was reversed by 4 μM TTX. In conclusion, during hypoxia or acute ischemia, both increased I _Na.P and NHE contribute to the HIR- or AIR-I _NCX with the former playing a major role comparing with the latter.     

The GPR55 agonist lysophosphatidylinositol directly activates intermediate-conductance Ca<Superscript>2+</Superscript>-activated K<Superscript>+</Superscript> channels

Lysophosphatidylinositol (LPI) was recently shown to act both as an extracellular mediator binding to G protein-coupled receptor 55 (GPR55) and as an intracellular messenger directly affecting a number of ion channels including large-conductance Ca²⁺ and voltage-gated potassium (BK_Ca) channels. Here, we explored the effect of LPI on intermediate-conductance Ca²⁺-activated K⁺ (IK_Ca) channels using excised inside-out patches from endothelial cells. The functional expression of IK_Ca was confirmed by the charybdotoxin- and TRAM-34-sensitive hyperpolarization to histamine and ATP. Moreover, the presence of single IK_Ca channels with a slope conductance of 39 pS in symmetric K⁺ gradient was directly confirmed in inside-out patches. When cytosolically applied in the range of concentrations of 0.3–10 μM, which are well below the herein determined critical micelle concentration of approximately 30 μM, LPI potentiated the IK_Ca single-channel activity in a concentration-dependent manner, while single-channel current amplitude was not affected. In the whole-cell configuration, LPI in the pipette was found to facilitate membrane hyperpolarization in response to low (0.5 μM) histamine concentrations in a TRAM-34-sensitive manner. These results demonstrate a so far not-described receptor-independent effect of LPI on the IK_Ca single-channel activity of endothelial cells, thus, highlighting LPI as a potent intracellular messenger capable of modulating electrical responses in the vasculature.     

Ca<Superscript>2+</Superscript> regulation of endocochlear potential in marginal cells

We examined the effect of the cytosolic Ca²⁺ concentration ([Ca²⁺]_c) in marginal cells on the asphyxia- or furosemide-induced decrease in the endocochlear potential (EP) by perfusing the endolymph with or without a Ca²⁺ chelator or inhibitors of Ca²⁺-permeable channels or Ca²⁺-pump during transient asphyxia or intravenous administration of furosemide. We obtained the following results. (1) Endolymphatic administration of SKF96365 (an inhibitor of TRPC and L-type Ca²⁺ channels) or EGTA-acetoxymethyl ester (EGTA-AM) significantly inhibited both the transient asphyxia-induced decrease in EP (TAID) and the furosemide-induced decrease in EP (FUID). (2) Endolymphatic perfusion with nifedipine significantly inhibited the TAID but not the FUID. (3) The recovery from the FUID was significantly suppressed by perfusing the endolymph with EGTA-AM, nifedipine, or SKF96365. (4) Endolymphatic administration of thapsigargin inhibited both the FUID and TAID. (5) The recovery rate from the FUID was much slower than that from the TAID, indicating that furosemide may inhibit the Ca²⁺-pump. (6) A strong reaction in immunohistochemical staining for TRPC channels was observed in the luminal and basolateral membranes of marginal cells. (7) A positive staining reaction for the γ subunit of epithelial Na⁺ channels was observed in the luminal and basolateral membranes of marginal cells. (8) Positive EP was diminished toward 0 mV by the endolymphatic perfusion with 10 μM amiloride or 10 μM phenamil. Taken together, these findings suggest that [Ca²⁺]_c regulated by endoplasmic Ca²⁺-pump and Ca²⁺-permeable channels in marginal cells may regulate the positive EP, which is partly produced by the diffusion potential of Na⁺ across the basolateral membrane in marginal cells.     

A reappraisal of the Ca<Superscript>2+</Superscript> dependence of fast inactivation of Ca<Superscript>2+</Superscript> release in frog skeletal muscle

Two drugs, 2-APB and SKF-96365, commonly used to block Store Operated Ca²⁺ Entry (SOCE) were found to have inhibitory effects at different levels of the Excitation Contraction Coupling (ECC) process in frog skeletal muscle fibers. Treatment with either drug suppressed Ca²⁺ release from the Sarcoplasmic Reticulum, but this effect was not due to inhibition of SOCE as it occurred in Ca²⁺-free conditions. 2-APB applied extracellularly at 100 μM, the usual concentration to suppress SOCE, reversibly reduced the charge movement elicited by pulses in the range between −45 and −35 mV from 7.99 ± 0.73 nC/μF (N = 17) before drug application to 6.27 ± 0.68 nC/μF in the presence of 2-APB. This effect was mostly on the delayed Q_γ component. In fibers treated with the SERCA ATPase inhibitor CPA the Q_γ component disappeared, under this condition the application of 2-APB did not suppress the remaining charge movement. Thus the effect of 2-APB on charge movement currents seemed to be secondary to the suppression of Ca²⁺ release, likely occurring directly on the release channels. No significant suppression of ECC was observed for concentration below 20 μM. 2-APB also inhibited the L-type Ca²⁺ current (20 ± 4%, N = 8). On the other hand SKF-96365 had a direct effect on the voltage sensor promoting its voltage dependent inactivation. Applied at 20 μM, a typical concentration used for inhibiting SOCE, to fibers held at −80 mV inhibited the charge moved in response to pulses ranging −45 to −30 mV from 7.95 ± 2.59 nC/μF to 3.48 ± 0.9 nC/μF (N = 12). A parallel reduction of Ca²⁺ release was observed. Wash out was drastically increased by hyperpolarization of the holding potential to −100 mV. SKF-96365 also inhibited the L-type Ca²⁺ current (41 ± 8%, N = 4) and increased its rate of inactivation.     

cGMP reduces the sarcoplasmic reticulum Ca<Superscript>2+</Superscript> loading in airway smooth muscle cells: a putative mechanism in the regulation of Ca<Superscript>2+</Superscript> by cGMP

Ca²⁺ and cGMP have opposite roles in many physiological processes likely due to a complex negative feedback regulation between them. Examples of opposite functions induced by Ca²⁺ and cGMP are smooth muscle contraction and relaxation, respectively. A main Ca²⁺ storage involved in contraction is sarcoplasmic reticulum (SR); nevertheless, the role of cGMP in the regulation of SR-Ca²⁺ has not been completely understood. To evaluate this role, intracellular Ca²⁺ concentration ([Ca²⁺]i) was determinated by a ratiometric method in isolated myocytes from bovine trachea incubated with Fura-2/AM. The release of Ca²⁺ from SR induced by caffeine was transient, whereas caffeine withdrawal was followed by a [Ca²⁺]i undershoot. Caffeine-induced Ca²⁺ transient peak and [Ca²⁺]i undershoot after caffeine were reproducible in the same cell. Dibutyryl cGMP (db-cGMP) blocked the [Ca²⁺]i undershoot and reduced the subsequent caffeine peak (SR-Ca²⁺ loading). Both, the opening of SR channels with ryanodine (10 μM) and the blockade of SR-Ca²⁺ ATPase with cyclopiazonic acid inhibited the [Ca²⁺]i undershoot as well as the SR-Ca²⁺ loading. The addition of db-cGMP to ryanodine (10 μM) incubated cells partially restored the SR-Ca²⁺ loading. Cyclic GMP enhanced [Ca²⁺]i undershoot induced by the blockade of ryanodine channels with 50 μM ryanodine. In conclusion, the reduction of SR-Ca²⁺ content in airway smooth muscle induced by cGMP can be explained by the combination of SR-Ca²⁺ loading and the simultaneous release of SR-Ca²⁺. The reduction of SR-Ca²⁺ content induced by cGMP might be a putative mechanism limiting releasable Ca²⁺ in response to a particular stimulus.     

Late sodium current contributes to diastolic cell Ca<Superscript>2+</Superscript> accumulation in chronic heart failure

We elucidate the role of late Na⁺ current (I_NaL) for diastolic intracellular Ca²⁺ (DCa) accumulation in chronic heart failure (HF). HF was induced in 19 dogs by multiple coronary artery microembolizations; 6 normal dogs served as control. Ca²⁺ transients were recorded in field-paced (0.25 or 1.5 Hz) fluo-4-loaded ventricular myocytes (VM). I_NaL and action potentials were recorded by patch-clamp. Failing VM, but not normal VM, exhibited (1) prolonged action potentials and Ca²⁺ transients at 0.25 Hz, (2) substantial DCa accumulation at 1.5 Hz, and (3) spontaneous Ca²⁺ releases, which occurred after 1.5 Hz stimulation trains in ~31% cases. Selective I_NaL blocker ranolazine (10 μM) or the prototypical Na⁺ channel blocker tetrodotoxin (2 μM) reversibly improved function of failing VM. The DCa accumulation and the beneficial effect of I_NaL blockade were reproduced in silico using an excitation-contraction coupling model. We conclude that I_NaL contributes to diastolic Ca²⁺ accumulation and spontaneous Ca²⁺ release in HF.     

Voltage-gated Ca<Superscript>2+</Superscript> channel mRNAs and T-type Ca<Superscript>2+</Superscript> currents in rat gonadotropin-releasing hormone neurons

Gonadotropin-releasing hormone (GnRH) neurons play a pivotal role in the neuroendocrine regulation of reproduction. We have previously reported that rat GnRH neurons exhibit voltage-gated Ca²⁺ currents. In this study, oligo-cell RT-PCR was carried out to identify subtypes of the α₁ subunit of voltage-gated Ca²⁺ channels in adult rat GnRH neurons. GnRH neurons expressed mRNAs for all five types of voltage-gated Ca²⁺ channels. For T-type Ca²⁺ channels, α_1H was dominantly expressed in GnRH neurons. Electrophysiological analysis in acute slice preparations revealed that GnRH neurons from adult rats exhibited T-type Ca²⁺ currents with fast inactivation kinetics (~20 ms at −30 mV) and a time constant of recovery from inactivation of ~0.6 s. These results indicate that rat GnRH neurons express subtypes of the α₁ subunit for all five types of voltage-gated Ca²⁺ channel, and that α_1H was the dominant subtype in T-type Ca²⁺ channels.     

KB-R7943 inhibits Na<Superscript>+</Superscript>-dependent Mg<Superscript>2+</Superscript> efflux in rat ventricular myocytes

Na⁺-dependent Mg²⁺ efflux activity was studied with the fluorescent Mg²⁺ indicator furaptra in the presence of various potential antagonists known to inhibit other transporters and channels. Among the compounds tested, KB-R7943, an inhibitor of Na⁺/Ca²⁺ exchange, most potently inhibited the Na⁺/Mg²⁺ exchange with half inhibitory concentrations (IC₅₀) of 21 μM (25°C) and 16 μM (35°C). These IC₅₀ values were a factor of three to four lower than those of imipramine, a widely used inhibitor of Na⁺/Mg²⁺ exchange. Apart from the inhibitory effect on Na⁺/Mg²⁺ exchange, relatively high concentrations of KB-R7943 (100 μM at 25°C and ≥20 μM at 35°C), in combination with prolonged UV-illumination, caused cell shortening, probably because of the phototoxicity of the compound and the formation of rigor crossbridges. We conclude that KB-R7943 may be a useful tool to study cellular Mg²⁺ homeostasis if care is taken to minimize its phototoxicity.     

Residual sarcoplasmic reticulum Ca<Superscript>2+</Superscript> concentration after Ca<Superscript>2+</Superscript> release in skeletal myofibers from young adult and old mice

Contrasting information suggests either almost complete depletion of sarcoplasmic reticulum (SR) Ca²⁺ or significant residual Ca²⁺ concentration after prolonged depolarization of the skeletal muscle fiber. The primary obstacle to resolving this controversy is the lack of genetically encoded Ca²⁺ indicators targeted to the SR that exhibit low-Ca²⁺ affinity, a fast biosensor: Ca²⁺ off-rate reaction, and can be expressed in myofibers from adult and older adult mammalian species. This work used the recently designed low-affinity Ca²⁺ sensor (Kd = 1.66 mM in the myofiber) CatchER (calcium sensor for detecting high concentrations in the ER) targeted to the SR, to investigate whether prolonged skeletal muscle fiber depolarization significantly alters residual SR Ca²⁺ with aging. We found CatchER a proper tool to investigate SR Ca²⁺ depletion in young adult and older adult mice, consistently tracking SR luminal Ca²⁺ release in response to brief and repetitive stimulation. We evoked SR Ca²⁺ release in whole-cell voltage-clamped flexor digitorum brevis muscle fibers from young and old FVB mice and tested the maximal SR Ca²⁺ release by directly activating the ryanodine receptor (RyR1) with 4-chloro-m-cresol in the same myofibers. Here, we report for the first time that the Ca²⁺ remaining in the SR after prolonged depolarization (2 s) in myofibers from aging (~220 μM) was larger than young (~132 μM) mice. These experiments indicate that SR Ca²⁺ is far from fully depleted under physiological conditions throughout life, and support the concept of excitation–contraction uncoupling in functional senescent myofibers.     

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     

Altered intracellular Ca<Superscript>2+</Superscript> regulation in chronic rat heart failure

Altered intracellular Ca²⁺ handling by the sarcoplasmic reticulum (SR) plays a crucial role in the pathogenesis of heart failure (HF). Despite extensive effort, the underlying causes of abnormal SR Ca²⁺ handling in HF have not been clarified. To determine whether the diastolic SR Ca²⁺ leak along with reduced Ca²⁺ reuptake is required for decreased contractility, we investigated the cytosolic Ca²⁺ transients and SR Ca²⁺ content and assessed the expression of ryanodine receptor (RyR2), FK506 binding protein (FKBP12.6), SR-Ca²⁺ ATPase (SERCA2a), and L-type Ca²⁺ channel (LTCC) using an SD-rat model of chronic HF. We found that the cytosolic Ca²⁺ transients were markedly reduced in amplitude in HF myocytes (ΔF/F ₀ = 12.3 ± 0.8) compared with control myocytes (ΔF/F ₀ = 17.7 ± 1.2, P < 0.01), changes paralleled by a significant reduction in the SR Ca²⁺ content (HF: ΔF/F ₀ = 12.4 ± 1.1, control: ΔF/F ₀ = 32.4 ± 1.9, P < 0.01). Moreover, we demonstrated that the expression of FKBP12.6 associated with RyR2, SERCA2a, and LTCC was significantly reduced in rat HF. These results provide evidence for phosphorylation-induced detachment of FKBP12.6 from RyRs and down-regulation of SERCA2a and LTCC in HF. We conclude that diastolic SR Ca²⁺ leak (due to dissociation of FKBP12.6 from RyR2) along with reduced SR Ca²⁺ uptake (due to down-regulation of SERCA2a) and defective E-C coupling (due to down-regulation of LTCC) could contribute to HF.     

HCO<Subscript>3</Subscript><Superscript>−</Superscript>-dependent transient acidification induced by ionomycin in rat submandibular acinar cells

Ionomycin (IM, 5 μM), which exchanges 1 Ca²⁺ for 1 H⁺, changed intracellular pH (pH_i) with Ca²⁺ entry into rat submandibular acinar cells. IM-induced changes in pH_i consisted of two components: the first is an HCO₃ ⁻-dependent transient pH_i decrease, and the second is an HCO₃ ⁻-independent gradual pH_i increase. IM (1 μM), which activates store-operated Ca²⁺ channels, induced an HCO₃ ⁻-dependent and transient pH_i decrease without any HCO₃ ⁻-independent pH_i increase. Thus, a gradual pH_i increase was induced by the Ca²⁺/H⁺ exchange. The HCO₃ ⁻-dependent and transient pH_i decrease induced by IM was abolished by acetazolamide, but not by methyl isobutyl amiloride (MIA) or diisothiocyanatostilbene disulfonate (DIDS), suggesting that the Na⁺/H⁺ exchange, the Cl⁻/HCO₃ ⁻ exchange, or the Na⁺-HCO₃ ⁻ cotransport induces no transient pH_i decrease. Thapsigargin induced no transient pH_i decrease. Thus, IM, not Ca²⁺ entry, reduced pH_i transiently. IM reacts with Ca²⁺ to produce H⁺ in the presence of \textCO ₂ /\textHCO ₃ ^- :  [ \textH - \textIM ] ^- + \text Ca ²⁺  + \textCO ₂ \rightleftarrows [ \textH-\textCa - \textIM ] ⁺ ·\textHCO ₃ ^- + \textH ⁺ {\text{CO}}_{ 2} /{\text{HCO}}_{ 3}{^{ - }} : \, \left[ {{\text{H}} - {\text{IM}}} \right]^{ - } + {\text{ Ca}}^{ 2+ } \,+ {\text{CO}}_{ 2} \rightleftarrows \left[ {{\text{H}}-{\text{Ca}} - {\text{IM}}} \right]^{ + } \cdot {\text{HCO}}_{ 3}{^{ - } }+ {\text{H}}^{ + } . In this reaction, a monoprotonated IM reacts with Ca²⁺ and CO₂ to produce an electroneutral IM complex and H⁺, and then H⁺ is removed from the cells via CO₂ production. Thus, IM transiently decreased pH_i. In conclusion, in rat submandibular acinar cells IM (5 μM) transiently reduces pH_i because of its chemical characteristics, with HCO₃ ⁻ dependence, and increases pH_i by exchanging Ca²⁺ for H⁺, which is independent of HCO₃ ⁻.     

Ca2+-transients induced by K+ channel openers in isolated coronary capillaries

 To study the role of endothelial ATP-sensitive K⁺ channels in the regulation of vascular tone we examined the intracellular calcium concentration ([Ca²⁺]_i) in coronary capillaries consisting only of endothelial cells. Coronary capillary fragments were isolated enzymatically from the guinea-pig heart and [Ca²⁺]_i was determined by microfluorometry of fura-2 loaded cells. Low concentrations of the K⁺ channel opener diazoxide, which caused pronounced glibenclamide-sensitive hyperpolarization in capillaries, induced a rapid, transient rise in [Ca²⁺]_i followed by a sustained elevation of [Ca²⁺]_i (19 of 40 experiments). [Ca²⁺]_i in the endothelial cells increased from 32 ± 7 nM at rest to 66 ± 11 nM at the peak (n = 19). One third of the [Ca²⁺]_i-transients showed irregular oscillations of [Ca²⁺]_i. No significant difference in the [Ca²⁺]_i-response induced by 100 nM or 1 μM diazoxide was found. Similar results were obtained with the K⁺ channel opener rilmakalim. Simultaneous measurements of the membrane potential and [Ca²⁺]_i with fluorometric methods indicated that the hyperpolarization but not the [Ca²⁺]_i-transient could be repeatedly induced in a single capillary by the K⁺ channel openers. Electrophysiological recordings of the membrane potential using the ”perforated patch” method (n = 4), showed that rilmakalim (1 μM) induced hyperpolarization of capillaries towards the K⁺ equilibrium potential, confirming our fluorometric measurements. In conclusion, for the first time, these data indicate that K⁺ channel openers induce [Ca²⁺]_i-transients in microvascular endothelial cells. This raises the possibility that these drugs not only act as synthetic vasoactive factors via hyperpolarizing smooth muscle cells but also via NO release of microvascular endothelial cells. Interestingly, only 100 nM diazoxide was sufficient for a maximal response, suggesting the expression of a new type of K_ATP-channel in coronary capillaries characterised by high sensitivity to diazoxide. Received: 22 August 1997 / Received after revision and accepted: 7 November 1997     

Effects of extracellular K+ and Ca2+ on membrane potential,contraction and86Rb+ efflux in guinea-pig mesotubarium

The effects of varying extracellular concentrations of K⁺ and Ca²⁺ [K⁺]_o and [Ca²⁺]_o on force development and membrane potential were investigated in the guinea-pig mesotubarium. At [K⁺]_o up to 40 mM, spontaneous action potentials were present, while higher [K⁺]_o gave sustained contractures at a stable membrane potential (−24 to −12 mV for [K⁺]_o from 60 to 120 mM). Tension decreased successively with increasing [K⁺]_o from 30 to 120 mM. The relaxing potency of the dihydropyridine Ca²⁺ antagonist, felodipine, increased as the membrane was depolarized with increasing [K⁺]_o and action potentials ceased. These results are compatible with the existence of Ca²⁺ channels showing voltage-dependent affinity with dihydrophyridines. Increasing [Ca²⁺]_o from 2.5 to 10 mM caused membrane hyperpolarization by about 11 mV and was accompanied by a lower frequency of spontaneous contractions and a longer duration of the relaxation between contractions.⁸⁶Rb⁺ efflux measurements in 60 mM K⁺ in the absence and presence of felodipine revealed a Ca²⁺-dependent component of the voltage-activated efflux. In normal solution (5.9 mM K⁺), efflux in the presence of felodipine was similar to the minimal value during normal spontaneous activity. The results indicate regulation of the permeability of K⁺ channels by the intracellular Ca²⁺ concentration ([Ca²⁺]_i) and suggest participation of such channels in the generation of the regularly occurring bursts of action potentials characteristic of spontaneous activity in the mesotubarium.     

Intracellular Na+ modulates large conductance Ca2+-activated K+ currents in human umbilical vein endothelial cells

We studied the effects of Na⁺ influx on large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels in cultured human umbilical vein endothelial cells (HUVECs) by means of patch clamp and SBFI microfluorescence measurements. In current-clamped HUVECs, extracellular Na⁺ replacement by NMDG⁺ or mannitol hyperpolarized cells. In voltage-clamped HUVECs, changing membrane potential from 0 mV to negative potentials increased intracellular Na⁺ concentration ([Na⁺]_i) and vice versa. In addition, extracellular Na⁺ depletion decreased [Na⁺]_i. In voltage-clamped cells, BK_Ca currents were markedly increased by extracellular Na⁺ depletion. In inside-out patches, increasing [Na⁺]_i from 0 to 20 or 40 mM reduced single channel conductance but not open probability (NPo) of BK_Ca channels and decreasing intracellular K⁺ concentration ([K⁺]_i) gradually from 140 to 70 mM reduced both single channel conductance and NPo. Furthermore, increasing [Na⁺]_i gradually from 0 to 70 mM, by replacing K⁺, markedly reduced single channel conductance and NPo. The Na⁺–Ca²⁺ exchange blocker Ni²⁺ or KB-R7943 decreased [Na⁺]_i and increased BK_Ca currents simultaneously, and the Na⁺ ionophore monensin completely inhibited BK_Ca currents. BK_Ca currents were significantly augmented by increasing extracellular K⁺ concentration ([K⁺]_o) from 6 to 12 mM and significantly reduced by decreasing [K⁺]_o from 12 or 6 to 0 mM or applying the Na⁺–K⁺ pump inhibitor ouabain. These results suggest that intracellular Na⁺ inhibit single channel conductance of BK_Ca channels and that intracellular K⁺ increases single channel conductance and NPo. GH Liang and MY Kim contributed equally to this publication and therefore share the first authorship.     

Trifluoperazine protects brain plasma membrane Ca<Superscript>2+</Superscript>-ATPase from oxidative damaging

In the central nervous system (CNS), a number of different pathological processes such as necrosis, Parkinson’s and Alzheimer’s diseases are related to disturbance in calcium homeostasis associated with oxidative stress. Here we compare the susceptibility of rat brain plasma membrane Ca²⁺-ATPase (PMCA) and sarco-endoplasmic reticulum Ca²⁺-ATPase (SERCA) isoforms to in vitro oxidative stress, and investigate a putative role of trifluoperazine (TFP), an antipsychotic drug that is also a powerful inhibitor of Ca²⁺-transporter proteins, in protecting these enzymes. It is shown that, in rat brain, PMCA is very sensitive to the damage induced by preincubation with Fe²⁺-ascorbate, or Fe²⁺-ascorbate plus H₂O_2, while SERCA is resistant. Inhibition of PMCA activity promoted by Fe²⁺/ascorbate medium is fully prevented by the presence of μM concentrations of either butylated hydroxytoluene (BHT) or TFP, but only partially protected, or reversed, by dithiothreitol (DTT), pointing to some protein cysteine(s) as one of the main targets for a lipid peroxidation-dependent damaging mechanism. However, when 0.5–1 mM H₂O₂ is added together with Fe²⁺/ascorbate, both BHT and TFP only partially prevent ATPase activity inhibition, and DTT does not confer any protection, suggesting two possible additional mechanisms involving both lipid peroxidation and direct damage to PMCA at amino acid residues other than cysteines. A possible use of μmolar concentrations of TFP as a direct antioxidant protector for PMCA under oxidative stress conditions is discussed.     

A role for the Ca<Superscript>2+</Superscript>-dependent tyrosine kinase Pyk2 in tonic depolarization-induced vascular smooth muscle contraction

Depolarization of the plasma membrane is a key mechanism of activation of contraction of vascular smooth muscle. This is commonly achieved in isolated, de-endothelialized vascular smooth muscle strips by increasing extracellular [K⁺] (replacing Na⁺ by K⁺) and leads to a rapid phasic contraction followed by a sustained tonic contraction. The initial phasic contractile response is due to opening of voltage-gated Ca²⁺ channels and entry of extracellular Ca²⁺, which binds to calmodulin, leading to activation of myosin light chain kinase, phosphorylation of the regulatory light chains of myosin II at Ser19 and cross-bridge cycling. The subsequent tonic contractile response involves, in addition to myosin light chain kinase activation, Ca²⁺-induced Ca²⁺ sensitization whereby Ca²⁺ entry activates the RhoA/Rho-associated kinase pathway leading to phosphorylation of MYPT1 (the myosin targeting subunit of myosin light chain phosphatase) and inhibition of the phosphatase. Investigations into the mechanism of activation of RhoA by Ca²⁺ have implicated a genistein-sensitive tyrosine kinase, and recent evidence indicates this to be the Ca²⁺-dependent tyrosine kinase, Pyk2.     

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.     

Effect of caffeine on K+ efflux in frog skeletal muscle

 The exposure of frog skeletal muscle to caffeine (3–4 mM) generates an increase of the K⁺ (⁴²K⁺) efflux rate coefficient (k _K,o) which exhibits the following characteristics. First it is promoted by the rise in cytosolic Ca²⁺ ([Ca²⁺]_i), because the effect is mimicked by ionomycin (1.25 μM), a Ca²⁺ ionophore. Second, the inhibition of caffeine-induced Ca²⁺ release from the sarcoplasmic reticulum (SR) by 40 μM tetracaine significantly reduced the increase in k _K,o (Δk _K,o). Third, charybdotoxin (23 nM), a blocker of the large-conductance Ca²⁺-dependent K⁺ channels (BK_Ca channels) reduced Δk _K,o by 22%. Fourth, apamin (10 nM), a blocker of the small-conductance Ca²⁺-dependent K⁺ channels (SK_Ca channels), did not affect Δk _K,o. Fifth, tolbutamide (800 μM), an inhibitor of K_ATP channels, reduced Δk _K,o by about 23%. Sixth, Ba²⁺, a blocker of most K⁺ channels, did not preclude the caffeine-induced Δk _K,o. Seventh, omitting Na⁺ from the external medium reduced Δk _K,o by about 40%. Eight, amiloride (5 mM) decreased Δk _K,o by 65%. It is concluded that the caffeine-induced rise of [Ca²⁺]_i increases K⁺ efflux, through the activation of: (1) two channels (BK_Ca and K_ATP) and (2) an external Na⁺-dependent amiloride-sensitive process. Received: 13 March 1998 / Received after revision: 17 June 1998 / Accepted: 14 September 1998