Functional coupling between 'R-type' Ca2+ channels and insulin secretion in the insulinoma cell line INS-1.

Among voltage-gated Ca2+ channels the non-dihydropyridine-sensitive alpha1E subunit is functionally less well characterized than the structurally related alpha1A (omega-agatoxin-IVA sensitive, P- /Q-type) and alpha1B (omega-conotoxin-GVIA sensitive, N-type) subunits. In the rat insulinoma cell line, INS-1, a tissue-specific splice variant of alpha1E (alpha1Ee) has been characterized at the mRNA and protein levels, suggesting that INS-1 cells are a suitable model for investigating the function of alpha1Ee. In alpha1E-transfected human embryonic kidney (HEK-293) cells the alpha1E-selective peptide antagonist SNX-482 (100 nM) reduces alpha1Ed- and alpha1Ee-induced Ba2+ inward currents in the absence and presence of the auxiliary subunits beta3 and alpha2delta-2 by more than 80%. The inhibition is fast and only partially reversible. No effect of SNX-482 was detected on the recombinant T-type Ca2+ channel subunits alpha1G, alpha1H, and alpha1I showing that the toxin from the venom of Hysterocrates gigas is useful as an alpha1E-selective antagonist. After blocking known components of Ca2+ channel inward current in INS-1 cells by 2 microM (+/-)-isradipine plus 0.5 microM omega-conotoxin-MVIIC, the remaining current is reduced by 100 nM SNX-482 from -12.4 +/- 1.2 pA/pF to -7.6 +/- 0.5 pA/pF (n = 9). Furthermore, in INS-1 cells, glucose- and KCl-induced insulin release are reduced by SNX-482 in a dose-dependent manner leading to the conclusion that alpha1E, in addition to L-type and non-L-type (alpha1A-mediated) Ca2+ currents, is involved in Ca2+ dependent insulin secretion of INS-1 cells.     

氨甲酰胆碱通过M2毒蕈碱受体增加豚鼠心肌细胞钠钙交换电流

本文旨在研究氨甲酰胆碱(carbachol, CCh)对豚鼠心肌的正性变力性机制。用Axon200A膜片钳放大器观察CCh 对电压钳制下的豚鼠心肌细胞L-型钙电流(ICa)和钠钙交换电流(INa/Ca)的效应。结果表明, CCh(100 μmol/L)分别使正向INa/Ca从对照组的(1.2 ± 0.1) pA/pF 增加到(2.0 ± 0.3) pA/pF,使反向 INa/Ca 从对照组的(1.3 ± 0.5) pA/pF 增加到(2.1 ± 0.8) pA/pF (P<0.01)。CCh对ICa无影响。CCh 对INa/Ca的激动作用可被阿托品和methoctramine所阻断。以上结果提示, CCh 对豚鼠心脏的正性变力作用是通过激动了钠钙交换,而且是 M2 毒蕈碱受体所介导的。     

The effect of adrenomedullin on the L-type calcium current in myocytes from septic shock rats: signaling pathway

Adrenomedullin (ADM) is upregulated in cardiac tissue under various pathophysiological conditions, particularly in septic shock. The intracellular mechanisms involved in the effect of ADM on adult rat ventricular myocytes are still to be elucidated. Ventricular myocytes were isolated from adult rats 4 h after an intraperitoneal injection of lipopolysaccharide (LPS, 10 mg/kg). Membrane potential and L-type calcium current (I(Ca,L)) were determined using whole cell patch-clamp methods. APD in LPS group was significantly shorter than control values (time to 50% repolarization: LPS, 169 +/- 2 ms; control, 257 +/- 2 ms, P < 0.05; time to 90% repolarization: LPS, 220 +/- 2 ms; control, 305 +/- 2 ms, P < 0.05). I(Ca,L) density was significantly reduced in myocytes from the LPS group (-3.2 +/- 0.8 pA/pF) compared with that of control myocytes (-6.7 +/- 0.3 pA/pF, P < 0.05). The ADM antagonist ADM-(22-52) reversed the shortened APD and abolished the reduction of I(Ca,L) in shock myocytes. In myocytes from control rats, incubating with ADM for 1 h induced a marked decrease in peak I(Ca,L) density. This effect was reversed by ADM-(22-52). The G(i) protein inhibitor, pertussis toxin (PTX), the protein kinase A (PKA) inhibitor, KT-5720, and the specific cyclooxygenase 2 (COX-2) inhibitor, nimesulide, reversed the LPS-induced reduction in peak I(Ca,L). The results suggest a COX-2-involved PKA-dependent switch from G(s) coupled to PTX-sensitive G(i) coupling by ADM in adult rat ventricular myocytes. The present study delineates the intracellular pathways involved in ADM-mediated effects on I(Ca,L) in adult rat ventricular myocytes and also suggests a role of ADM in sepsis.     

S100A1 decreases calcium spark frequency and alters their spatial characteristics in permeabilized adult ventricular cardiomyocytes

S100A1, a Ca2+-sensor protein of the EF-hand type, exerts positive inotropic effects in the heart via enhanced cardiac ryanodine receptor (RyR2) activity. Here we report that S100A1 protein (0.1microM) interacts with the RyR2 in resting permeabilized cardiomyocytes at free Ca2+-levels comparable to diastolic Ca2+-concentrations ( approximately 150nM). Alterations of RyR2 function due to S100A1 binding was assessed via analysis of Ca2+-spark characteristics. Ca2+-spark frequency, amplitude and duration were all reduced upon perfusion with 0.1microM S100A1 protein by 38%, 14% and 18%, respectively. Most likely, these effects were conveyed through the S100A1 C-terminus (S100A1-ct; amino acids 75-94) as the corresponding S100A1-ct peptide (0.1microM) inhibited S100A1 protein binding to the RyR2 and similarly attenuated frequency, amplitude and duration of Ca2+-sparks by 52%, 8% and 26%, respectively. Accordingly, the sarcoplasmic reticulum (SR) Ca2+-content was slightly increased but the stoichiometry of other accessory RyR2 modulators (sorcin/FKBP12.6) remained unaltered by S100A1. Hence, we propose S100A1 as a novel inhibitory modulator of RyR2 function at diastolic Ca2+-concentrations in rabbit ventricular cardiomyocytes.     

Reverse engineering the L-type Ca2+ channel alpha1c subunit in adult cardiac myocytes using novel adenoviral vectors

The alpha(1c) subunit of the cardiac L-type Ca(2+) channel, which contains the channel pore, voltage- and Ca(2+)-dependent gating structures, and drug binding sites, has been well studied in heterologous expression systems, but many aspects of L-type Ca(2+) channel behavior in intact cardiomyocytes remain poorly characterized. Here, we develop adenoviral constructs with E1, E3 and fiber gene deletions, to allow incorporation of full-length alpha(1c) gene cassettes into the adenovirus backbone. Wild-type (alpha(1c-wt)) and mutant (alpha(1c-D-)) Ca(2+) channel adenoviruses were constructed. The alpha(1c-D-) contained four point substitutions at amino acid residues known to be critical for dihydropyridine binding. Both alpha(1c-wt) and alpha(1c-D-) expressed robustly in A549 cells (peak L-type Ca(2+) current (I(CaL)) at 0 mV: alpha(1c-wt) -9.94+/-1.00pA/pF, n=9; alpha(1c-D-) -10.30pA/pF, n=12). I(CaL) carried by alpha(1c-D-) was markedly less sensitive to nitrendipine (IC(50) 17.1 microM) than alpha(1c-wt) (IC(50) 88 nM); a feature exploited to discriminate between engineered and native currents in transduced guinea-pig myocytes. 10 microM nitrendipine blocked only 51+/-5% (n=9) of I(CaL) in alpha(1c-D-)-expressing myocytes, in comparison to 86+/-8% (n=9) of I(CaL) in control myocytes. Moreover, in 20 microM nitrendipine, calcium transients could still be evoked in alpha(1c-D-)-transduced cells, but were largely blocked in control myocytes, indicating that the engineered channels were coupled to sarcoplasmic reticular Ca(2+) release. These alpha(1c) adenoviruses provide an unprecedented tool for structure-function studies of cardiac excitation-contraction coupling and L-type Ca(2+) channel regulation in the native myocyte background.     

S100B(betabeta) inhibits the protein kinase C-dependent phosphorylation of a peptide derived from p53 in a Ca2+-dependent manner.

S100B(betabeta) is a dimeric Ca2+-binding protein that is known to inhibit the protein kinase C (PKC)-dependent phosphorylation of several proteins. To further characterize this inhibition, we synthesized peptides based on the PKC phosphorylation domains of p53 (residues 367-388), neuromodulin (residues 37-53), and the regulatory domain of PKC (residues 19-31), and tested them as substrates for PKC. All three peptides were shown to be good substrates for the catalytic domain of PKC. As for full-length p53 (Baudier J, Delphin C, Grunwald D, Khochbin S, Lawrence JJ. 1992. Proc Natl Acad Sci USA 89:11627-11631), S100B(betabeta) binds the p53 peptide and inhibits its PKC-dependent phosphorylation (IC50 = 10 +/- 7 microM) in a Ca2+-dependent manner. Similarly, phosphorylation of the neuromodulin peptide and the PKC regulatory domain peptide were inhibited by S100B(betabeta) in the presence of Ca2+ (IC50 = 17 +/- 5 microM; IC50 = 1 +/- 0.5 microM, respectively). At a minimum, the C-terminal EF-hand Ca2+-binding domain (residues 61-72) of each S100beta subunit must be saturated to inhibit phosphorylation of the p53 peptide as determined by comparing the Ca2+ dependence of inhibition ([Ca]IC50 = 29.3 +/- 17.6 microM) to the dissociation of Ca2+ from the C-terminal EF-hand Ca2+-binding domain of S100B(betabeta).     

Deranged Kv channel regulation in fibroblasts from mice lacking the serum and glucocorticoid inducible kinase SGK1

Coexpression of the serum and glucocorticoid inducible kinase 1 (SGK1) up-regulates Kv channel activity in Xenopus oocytes and human embryonic kidney cells. To investigate the physiological impact of SGK1 dependent Kv channel regulation, we recorded whole-cell currents in lung fibroblasts from SGK1 knockout mice (sgk1-/-) and wild-type littermates (sgk1+/+). Serum-grown mouse lung fibroblasts (MLF) from both genotypes exhibited voltage-gated outwardly rectifying K(+)-currents with time-dependent activation (tau(act) approximately 3 msec), slow inactivation (tau(inact) approximately 700 msec), use-dependent inactivation, and (partial) inhibition by K(+) channel blockers TEA, 4-AP, and margatoxin. In serum grown MLF peak Kv current density at +100 mV was significantly lower in sgk1-/- (14 +/- 2 pA/pF, n = 13) than in sgk1+/+ (31 +/- 4 pA/pF, n = 16). PCR amplification of different Kv1 and Kv3 subunits from mouse fibroblasts demonstrated the expression of Kv1.1-1.7, Kv3.1, and Kv3.3 mRNA in both sgk1+/+ and sgk1-/- cells. Upon serum deprivation Kv currents almost disappeared in sgk1+/+ (4 +/- 1 pA/pF, n = 11) but not in sgk1-/- (10 +/- 1 pA/pF, n = 6) MLF. Accordingly, following serum deprivation Kv current density was significantly lower in sgk1+/+ than in sgk1-/-. Stimulation of serum-depleted cells with dexamethasone (dex) (1 microM, 1 day), IGF-1 (6.7 microM, 4-6 h) or both, significantly activated Kv currents in sgk1+/+ but not in sgk1-/- MLF. In the presence of both, dex and IGF-1, the Kv current density was significantly larger in sgk1+/+ (27 +/- 3 pA/pF, n = 12) than in sgk1-/- (13 +/- 3 pA/pF, n = 10) cells. Similar to MLF, Kv currents were significantly higher in sgk1+/+ mouse tail fibroblasts (MTF). In sgk1+/+ but not sgk1-/- MTF the Kv currents were inhibited upon serum deprivation and reincreased after stimulation of serum deprived MTF with dex (1 microM, 1 day) and afterwards with IGF-1 (6.7 microM, 4-6 h). According to Fura-2-fluorescence capacitative Ca(2+) entry was lower in sgk1-/- MTF compared to sgk1+/+ MTF. Upon serum deprivation capacitative Ca(2+) entry decreased significantly in sgk1+/+ but not in sgk1-/- MTF. Stimulation of depleted cells with dex (1 microM, 1 day) and afterwards with IGF-1 (6.7 microM, 4-6 h) reincreased capacitative Ca(2+) entry in sgk1+/+ MTF, whereas in sgk1-/- cells it remained unchanged. In conclusion, lack of SGK1 does not abrogate Kv channel activity but abolishes regulation of those channels by serum, glucocorticoids and IGF-1, an effect influencing capacitative Ca(2+) entry.     

Extracellular S100A1 protein inhibits apoptosis in ventricular cardiomyocytes via activation of the extracellular signal-regulated protein kinase 1/2 (ERK1/2)

S100A1 is a Ca2+-binding protein of the EF-hand type that belongs to the S100 protein family. It is specifically expressed in the myocardium at high levels and is considered to be an important regulator of cardiac contractility. Because the S100A1 protein is released into the extracellular space during ischemic myocardial injury, we examined the cardioprotective potential of the extracellular S100A1 protein on ventricular cardiomyocytes in vitro. In this report we show that extracellularly added S100A1 protein is endocytosed into the endosomal compartment of neonatal ventricular cardiomyocytes via a Ca2+-dependent clathrin-mediated process. S100A1 uptake protects neonatal ventricular cardiomyocytes from 2-deoxyglucose and oxidative stress-induced apoptosis in vitro. S100A1-mediated anti-apoptotic effects involve specific activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) pro-survival pathway, including activation of phospholipase C, protein kinase C, mitogen-activated protein kinase kinase 1, and ERK1/2. In contrast, neither transsarcolemmal Ca2+ influx via the L-type channel nor protein kinase A activity seems to take part in the S100A1-mediated signaling pathway. In conclusion, this study provides evidence for the S100A1 protein serving as a novel cardioprotective factor in vitro. These findings warrant speculation that injury-dependent release of the S100A1 protein from cardiomyocytes may serve as an intrinsic mechanism to promote survival of the myocardium in vivo.     

Involvement of phosphorylation of beta-subunit in cAMP-dependent activation of L-type Ca2+ channel in aortic smooth muscle-derived A7r5 cells

We investigated the effect of intracellular cAMP on the gating kinetics of L-type Ca2+ channel in an A7r5 smooth muscle-derived cell line using the whole-cell patch-clamp technique. Application of dibutyryl cyclic AMP (db-cAMP) to the cell increased the magnitude of Ca2+ currents through L-type Ca2+ channels (I(Ca)), and shifted the current-voltage relationship (I-V curve) for I(Ca) to the left. The magnitudes of maximum I(Ca) were 14.1 +/- 0.7 before and 16.0 +/- 1.1 pA/pF after application of 1 mM db-cAMP (P < 0.05). The values of the half-activation potential (V(1/2)) of I(Ca), estimated from activation curves, were -7.0 +/- 0.8 mV before and -10.8 +/- 1.0 mV after application of db-cAMP (P < 0.05). In cells pretreated with 10 microM Rp-cAMPS (a specific inhibitor of PKA), db-cAMP affected neither the I-V curve nor the activation curve for I(Ca). In cells pretreated with the antisense oligonucleotide for the beta-subunit of L-type Ca2+ channel, db-cAMP failed to enhance I(Ca) or alter the activation curve. On the other hand, in the cells pretreated with the nonsense oligonucleotide, application of db-cAMP caused an increase in magnitude of I(Ca) and shifted the activation curve to the left. Western blot analysis revealed that the pretreatment of cells with antisense oligonucleotide but nonsense oligonucleotide reduced the expression of the beta-subunit of the L-type Ca2+ channel. We conclude that the cAMP-dependent phosphorylation of the beta-subunit potentiates the voltage dependency of the activation kinetics of the L-type Ca2+ channel in A7r5 cells.     

严重烫伤延长大鼠心室肌细胞动作电位时程的机制

严重烫伤引起心肌细胞动作电位时程（action potential duration,APD）延长,通过加重烫伤心肌细胞钙紊乱和诱发室性心律失常,促进烫伤心功能障碍的发生,但APD延长的机制尚不清楚。通过制作约40％体表面积（total body surface area,TBSA）Ⅲ度烫伤大鼠模型,在伤后12h大鼠心功能明显减弱时分离其心肌细胞,采用膜片钳技术观察心肌细胞APD以及动作电位复极化相关的重要离子通道电流,包括瞬间外向钾电流（transient outward K^＋ current,Ito）,L-型钙电流（L-type Ca^2＋ current,ICa-L）和内向整流钾电流（inward rectifier K^＋ current,IK1）。结果显示,烫伤后12h单个心肌细胞APD明显延长,APD50和APD90在烫伤组分别为（46．02±3．78）ms、（123．24±12．48）ms（n=19）,明显长于对照组的（23．28±4．85）ms、（72．12±3．57）ms（n=17）（P〈0．01）。烫伤引起,Ito电流密度降低,＋60 mV下烫伤组的电流密度（20．39±1．98）pA／pF（n=25）明显低于对照组的（34．15±3．78）pA／pF（n=20,P〈0．01）;烫伤组在-120至-80mV电压刺激下所产生的IK1电流密度显著低于对照组：而两组之间ICa-L电流密度、电压依赖性的激活和失活无显著性差异。结果提示,烫伤引起心肌细胞APD延长的机制与瞬间外向钾通道和内向整流钾通道功能下调有关。     

Sex-based transmural differences in cardiac repolarization and ionic-current properties in canine left ventricles

The female sex is associated with longer electrocardiographic QT intervals and increased proarrhythmic risks of QT-prolonging drugs. This study examined the hypothesis that sex differences in repolarization may be associated with differential transmural ion-current distribution. Whole cell patch-clamp and current-clamp were used to study ionic currents and action potentials (APs) in isolated canine left ventricular cells from epicardium, midmyocardium, and endocardium. No sex differences in AP duration (APD) were found in cells from epicardium versus endocardium. In midmyocardium, APD was significantly longer in female dogs (e.g., at 1 Hz, female vs. male: 288 +/- 21 vs. 237 +/- 8 ms; P < 0.05), resulting in greater transmural APD heterogeneity in females. No sex differences in inward rectifier K+ current (I(K1)) were observed. Transient outward K+ current (I(to)) densities in epicardium and midmyocardium also showed no sex differences. In endocardium, female dogs had significantly smaller I(to) (e.g., at +30 mV, female vs. male: 2.5 +/- 0.2 vs. 3.5 +/- 0.3 pA/pF; P < 0.05). Rapid delayed-rectifier K+ current (I(Kr)) density and activation voltage-dependence showed no sex differences. Female dogs had significantly larger slow delayed-rectifier K+ current (I(Ks)) in epicardium and endocardium (e.g., at +40 mV; tail densities, female vs. male; epicardium: 1.3 +/- 0.1 vs. 0.8 +/- 0.1 pA/pF; P < 0.001; endocardium: 1.2 +/- 0.1 vs. 0.7 +/- 0.1 pA/pF; P < 0.05), but there were no sex differences in midmyocardial I(Ks). Female dogs had larger L-type Ca2+ current (I(Ca,L)) densities in all layers than male dogs (e.g., at -20 mV, female vs. male, epicardium: -4.2 +/- 0.4 vs. -3.2 +/- 0.2 pA/pF; midmyocardium: -4.5 +/- 0.5 vs. -3.3 +/- 0.3 pA/pF; endocarium: -4.5 +/- 0.4 vs. -3.2 +/- 0.3 pA/pF; P < 0.05 for each). We conclude that there are sex-based transmural differences in ionic currents that may underlie sex differences in transmural cardiac repolarization.     

Kir2.3 knock-down decreases IK1 current in neonatal rat cardiomyocytes

Inward rectifier potassium Kir2.x channels mediate cardiac inward rectifier potassium currents (I(K1)). As a subunit of Kir2.x, the physiological role of Kir2.3 in native cardiomyocytes has not been reported. This study shows that Kir2.3 knock-down remarkably down-regulates Kir2.3 expression (Kir2.3 protein was reduced to 19.91+/-3.24% on the 2nd or 3rd day) and I(K1) current densities (at -120 mV, control vs. knock-down: -5.03+/-0.24 pA/pF, n=5 vs. -1.16+/-0.19 pA/pF, n=7, P<0.001) in neonatal rat cardiomyocytes. The data suggest that Kir2.3 plays a potentially important role in I(K1) currents in neonatal rat cardiomyocytes.     

Activation of inward rectifier K+ channels by hypoxia in rabbit coronary arterial smooth muscle cells

We examined the effects of acute hypoxia on Ba2+-sensitive inward rectifier K+ (K(IR)) current in rabbit coronary arterial smooth muscle cells. The amplitudes of K(IR) current was definitely higher in the cells from small-diameter (<100 microm) coronary arterial smooth muscle cells (SCASMC, -12.8 +/- 1.3 pA/pF at -140 mV) than those in large-diameter coronary arterial smooth muscle cells (>200 microm, LCASMC, -1.5 +/- 0.1 pA pF(-1)). Western blot analysis confirmed that Kir2.1 protein was expressed in SCASMC but not LCASMC. Hypoxia activated much more KIR currents in symmetrical 140 K+. This effect was blocked by the adenylyl cyclase inhibitor SQ-22536 (10 microM) and mimicked by forskolin (10 microM) and dibutyryl-cAMP (500 microM). The production of cAMP in SCASMC increased 5.7-fold after 6 min of hypoxia. Hypoxia-induced increase in KIR currents was abolished by the PKA inhibitors, Rp-8-(4-chlorophenylthio)-cAMPs (10 microM) and KT-5720 (1 microM). The inhibition of G protein with GDPbetaS (1 mM) partially reduced (approximately 50%) the hypoxia-induced increase in KIR currents. In Langendorff-perfused rabbit hearts, hypoxia increased coronary blood flow, an effect that was inhibited by Ba2+. In summary, hypoxia augments the KIR currents in SCASMC via cAMP- and PKA-dependent signaling cascades, which might, at least partly, explain the hypoxia-induced coronary vasodilation.     

大鼠肥厚心肌细胞钙电流及 Na~ /Ca~(2 )交换电流的研究(英文)

本文观察了心肌肥厚对大鼠心肌细胞Na ／Ca2 交换电流的影响。我们采用Goldblatt两肾一夹方法诱发大鼠心肌肥厚,应用全细胞膜片钳技术记录电流。结果表明：肥厚心肌细胞的Ni2 -敏感Na ／Ca2 交换电流密度大于正常细胞。在钳制电压为＋50mV时,正常细胞的外向交换电流密度为1．53±0．31pA／pF,而肥厚细胞则为2．62±0．53pA／pF（P＜0．01）;钳制电压为-100mV时,正常细胞的内向交换电流密度为0．42±0．14pA／pF,肥厚细胞达1．12±0．33pA／pF（P＜0．001）。这些结果提示,肥厚心肌细胞的Na ／Ca2 交换电流发生了改变,其意义有待进一步探讨。     

Intracellular Ca(2+) regulates responsiveness of cardiac L-type Ca(2+) current to protein kinase A: role of calmodulin

The goal of this study was to determine whether the protein kinase A (PKA) responsiveness of the cardiac L-type Ca(2+) current (ICa) is affected during transient increases in intracellular Ca(2+) concentration. Ventricular myocytes were isolated from 3- to 4-day-old neonatal rats and cultured on aligned collagen thin gels. When measured in 1 or 2 mM Ca(2+) external solution, the aligned myocytes displayed a large ICa that was weakly regulated (20% increase) during stimulation of PKA by 2 microM forskolin. In contrast, application of forskolin caused a 100% increase in ICa when the external Ca(2+) concentration was reduced to 0.5 mM or replaced with Ba(2+). This Ca(2+)-dependent inhibition was also observed when the cells were treated with 1 microM isoproterenol, 100 microM 3-isobutyl-1-methylxanthine, or 500 microM 8-bromo-cAMP. The responsiveness of ICa to PKA was restored during intracellular dialysis with a calmodulin (CaM) inhibitory peptide but not during treatment with inhibitors of protein kinase C, Ca(2+)/CaM-dependent protein kinase, or calcineurin. Adenoviral-mediated expression of a CaM molecule with mutations in all four Ca(2+)-binding sites also increased the PKA sensitivity of ICa. Finally, adult mouse ventricular myocytes displayed a greater response to forskolin and cAMP in external Ba(2+). Thus Ca(2+) entering the myocyte through the voltage-gated Ca(2+) channel regulates the PKA responsiveness of ICa.     

Angiotensin II stimulates cardiac L-type Ca(2+) current by a Ca(2+)- and protein kinase C-dependent mechanism

Angiotensin II (ANG II) evokes positive inotropic responses in various species. However, the effects of this peptide on L-type Ca(2+) currents (I(Ca)) are still controversial. We report in this study that the effects of ANG II on I(Ca) differ depending on the mode of patch-clamp technique used, standard whole cell (WC) or perforated patch (PP). No significant effects of ANG II (0.5 microM) were observed when WC in cells dialyzed with high EGTA was used. However, when the intracellular milieu was preserved using PP, ANG II induced a significant 77 +/- 6% increase in I(Ca) (-2.2 +/- 0.3 in control and -3.9 +/- 0.6 pA/pF in ANG II, n = 8, P < 0.05). When WC was used in cells dialyzed with low Ca(2+) buffer capacity (EGTA 0.1 mM), ANG II was able to induce an increase in I(Ca) (-3.5 +/- 0.3 in control vs. -4.8 +/- 0.4 pA/pF in ANG II, n = 13, P < 0.05). This increase was prevented when the cells were also dialyzed with the protein kinase C (PKC) inhibitor chelerythrine (50 microM) or calphostin C (1 microM). The above results allow us to conclude that strong intracellular Ca(2+) buffering prevents the physiological actions of ANG II on cardiac I(Ca), which are also dependent on activation of PKC.     

Mechanisms of coronary artery depolarization by uridine triphosphate

We sought to define the basic mechanisms by which pyrimidine nucleotides constrict rat coronary resistance arteries. Uridine triphosphate (UTP) caused a dose-dependent constriction in coronary arteries stripped of endothelium. UTP also depolarized and increased cytosolic Ca2+ in coronary smooth muscle cells. Nisoldipine, an antagonist of voltage-operated Ca2+ channels, blocked the rise in cytosolic Ca2+ and reduced UTP-induced vasoconstriction by approximately 75% which suggests a prominent role for depolarization in this constrictor response. The ionic basis of UTP-induced depolarization was subsequently explored in coronary smooth muscle cells using whole-cell patch-clamp electrophysiology. In the absence of K+ and with CsCl in the pipette, UTP (40 microM) activated a sustained inwardly rectifying current (-0.66 +/- 0.10 pA/pF at -60 mV). A 100 mM reduction in bath Na+ shifted the reversal potential of this current (from -2 +/- 1 to -28 +/- 4 mV) and reduced the magnitude (from -2.26 +/- 0.61 to -0.51 +/- 0.11 pA/pF). In addition to activating a depolarizing cation current, UTP inhibited hyperpolarizing outward currents. Specifically, UTP inhibited ATP-sensitive and voltage-dependent K+ currents yet had no effect on inwardly rectifying and Ca2+-activated K+ channels. This study indicates that electromechanical coupling is integral to pyrimidine-induced constriction in coronary resistance arteries.     

Myocardial infarction in rat eliminates regional heterogeneity of AP profiles,I(to) K(+) currents,and [Ca(2+)](i) transients

Transient outward K(+) current density (I(to)) has been shown to vary between different regions of the normal myocardium and to be reduced in heart disease. In this study, we measured regional changes in action potential duration (APD), I(to), and intracellular Ca(2+) concentration ([Ca(2+)](i)) transients of ventricular myocytes derived from the right ventricular free wall (RVW) and interventricular septum (SEP) 8 wk after myocardial infarction (MI). At +40 mV, I(to) density in sham-operated hearts was significantly higher (P < 0.01) in the RVW (15.0 +/- 0.8 pA/pF, n = 47) compared with the SEP (7.0 +/- 1.1 pA/pF, n = 18). After MI, I(to) density was not reduced in SEP myocytes but was reduced (P < 0.01) in RVW myocytes (8.7 +/- 1.0 pA/pF, n = 26) to levels indistinguishable from post-MI SEP myocytes. These changes in I(to) density correlated with Kv4.2 (but not Kv4.3) protein expression. By contrast, Kv1.4 expression was significantly higher in the RVW compared with the SEP and increased significantly after MI in RVW. APD measured at 50% or 90% repolarization was prolonged, whereas peak [Ca(2+)](i) transients amplitude was higher in the SEP compared with the RVW in sham myocytes. These regional differences in APD and [Ca(2+)](i) transients were eliminated by MI. Our results demonstrate that the significant regional differences in I(to) density, APD, and [Ca(2+)](i) between RVW and SEP are linked to a variation in Kv4.2 expression, which largely disappears after MI.     

激活δ阿片受体可抑制大鼠心室肌细胞L-型钙电流和瞬时外向钾电流

本研究采用全细胞膜片钳技术观察了SNCl62(一种选择性δ阿片受体激动剂)对人鼠心室肌细胞L型钙电流(L-type Ca2 current,ICa-L)和瞬时外向钾电流(transient outward K current,Ito)的影响.结果显示,SNCl62明显抑制大鼠心室肌细胞,Ica L和Ica L,对Ica L.和k的最大抑制率分别为(46.13±4.12)%和(36.53±10.57)%.1x10-4mol/L SNCl62使,Ica L的甲均电流密度从(8.98±0.40)pA/pF下降到(4.84±0.44)pA/pF(P<0.01,n=5),Ito的平均电流密度从(18.69±2.42)pA/pF降低到(11.73±1.67)pA/pF(P<0.01,n=5).单独应用naltrindole(一种选择性δ阿片受体拮抗剂)对大鼠心室肌细胞Ica L和Ito无显著作用,但预先应用naltrindole可以消除SNCl62对Ica L和Ito的抑制作用.结果表明,通过δ阿片受体,SNCl62(1x10-6～1x10-4mol/L)浓度依赖性地抑制人鼠心室肌细胞Ica L和Ito这可能是激动δ阿片受体产生抗心律失常效应的重要机制.     

氨甲酰胆碱通过M2胆碱能受体对大鼠心肌细胞发挥正性肌力作用

为研究氨甲酰胆碱（carbachol,CCh）对大鼠心肌细胞的正性肌力作用机制,利用电压钳方法观察CCh对急性分离的单个大鼠心肌细胞L-型钙电流（足扎）和钠,钙交换电流（INa/Ca）的影响。细胞负载Fura-2／AM后,用离子成像系统测定场刺激下单个大鼠心肌细胞的钙瞬变和细胞缩短。结果表明,100ILmol／LCCh使正向INa/Ca从（1．18±0．57）pA／pF增加到（1．65±0．52）pA/pF（P〈O．01）,反向,INa/Ca从（1．11±0．49）pA/pF增加到（1．53±0．52）pA/pF（P〈O．01）,但不影响ICa,L。阿托品（非选择性M胆碱受体拮抗剂）和methoctramine（选择性M2胆碱受体拮抗剂）可阻断这种增加作用。100μmol／LCCh使钙瞬变从对照组的203．8±50．0增加到234．8±64.3,使细胞缩短从对照组的（3．00±0．67）μm增加到（3．55±1．21）μm。KB-R7943（选择性反向INa/Ca抑制剂）不影响钙瞬变和细胞缩短的基础水平,却完全阻断CCh引起的钙瞬变和细胞缩短的增加。尼卡地平（ICa,L抑制剂）抑制钙瞬变和细胞缩短。CCh在尼卡地平存在下仍可增加钙瞬变和细胞缩短值,提示其正性肌力作用是通过刺激钠,钙交换实现的。CCh不改变钙敏感性。阿托品和methoctramine阻断CCh的这种激动作用,说明CCh的正性肌力作用是通过M2受体实现的。以上结果提示,CCh对大鼠心肌细胞有正性肌力作用,这种作用是通过激动反向钠／钙交换实现,由M2受体介导。