Enhanced inhibition of L-type Ca2+ current by beta3-adrenergic stimulation in failing rat heart

beta3-adrenergic receptors (AR) have recently been identified in mammalian hearts and shown to be up-regulated in heart failure (HF). beta3-AR stimulation reduces inotropic response associated with an inhibition of L-type Ca2+ channels in normal hearts; however, the effects of beta3-AR activation on Ca2+ channel in HF remain unknown. We compared the effects of beta(3)-AR activation on L-type Ca2+ current (ICa,L) in isolated left ventricular myocytes obtained from normal and age-matched rats with isoproterenol (ISO)-induced HF (4 months after 340 mg/kg s.c. for 2 days). ICa,L was measured using whole-cell voltage clamp and perforated-patch recording techniques. In normal myocytes, superfusion of 4-[-[2-hydroxy-(3-chlorophenyl)ethylamino]propyl]phenoxyacetate (BRL-37,344; BRL), a beta3-AR agonist, caused a dose-dependent decrease in ICa,L with maximal inhibition (21%, 1.1 +/- 0.2 versus 1.4 +/- 0.1 nA) (p < 0.01) at 10(-7) M. In HF myocytes, the same concentration of BRL produced a proportionately greater inhibition (31%) in ICa,L (1.1 +/- 0.2 versus 1.6 +/- 0.2 nA) (p < 0.05). A similar inhibition of ICa,L was also observed with ISO (10(-7) M) in the presence of a beta1- and beta2-AR antagonist, nadolol (10(-5) M). Inhibition was abolished by the beta3-AR antagonist (S)-N-[4-[2-[[3-[3-(acetamidomethyl)phenoxy]-2-hydroxypropyl]amino]ethyl]phenyl]benzenesulfonamide (L-748,337; 10(-6) M), but not by nadolol. The inhibitory effect of BRL was attenuated by a nitric-oxide synthase (NOS) inhibitor, N(G)-nitro-L-arginine methyl ester (10(-4) M), and was prevented by the incubation of myocytes with pertussis toxin (PTX; 2 microg/ml, 36 degrees C, 6 h). In conclusion, beta3-AR activation inhibits L-type Ca2+ channel in both normal and HF myocytes. In HF, beta3-AR stimulation-induced inhibition of Ca2+ channel is enhanced. These effects are likely coupled with PTX-sensitive G-protein and partially mediated through a NOS-dependent pathway.     

Direct contact between sympathetic neurons and rat cardiac myocytes in vitro increases expression of functional calcium channels.

To test the hypothesis that direct contact between sympathetic neurons and myocytes regulates expression and function of cardiac Ca channels, we prepared cultures of neonatal rat ventricular myocytes with and without sympathetic ganglia. Contractile properties of myocytes were assessed by an optical-video system. Contractility-pCa curves showed a 60% greater increase in contractility for innervated myocytes compared with control cells at 6.3 mM [Ca]0 (n = 8, P less than 0.05). Cells grown in medium conditioned by growth of ganglia and myocytes were indistinguishable physiologically from control cells. [Bay K 8644]-contractility curves revealed a 60 +/- 10% enhancement of the contractility response at 10(-6) M for innervated cells compared with control cells. The increased response to Bay K 8644 was not blocked by alpha- or beta-adrenergic antagonists. Moreover, increased efficacy of Bay K 8644 was maintained for at least 24 h after denervation produced by removal of ganglia from the culture. Dihydropyridine binding sites were assessed with the L channel-specific radioligand 3[H]PN200-110. PN200-110 binding sites were increased by innervation (51 +/- 5 to 108 +/- 20 fmol/mg protein, P less than 0.01), with no change in KD. Peak current-voltage curves were determined by whole-cell voltage clamp techniques for myocytes contacted by a neuron, control myocytes, and myocytes grown in conditioned medium. Current density of L-type Ca channels was significantly higher in innervated myocytes (10.5 +/- 0.4 pA/pF, n = 5) than in control myocytes (5.9 +/- 0.3 pA/pF, n = 8, P less than 0.01) or myocytes grown in conditioned medium (6.2 +/- 0.2 pA/pF, n = 10, P less than 0.01). Thus, physical contact between a sympathetic neuron and previously uninnervated neonatal rat ventricular myocytes increases expression of functional L-type calcium channels as judged by contractile responses to Ca0 and Bay K 8644, as well as by electrophysiological and radioligand binding properties.     

Effects of captopril treatment of renovascular hypertension on beta-adrenergic modulation of L-type Ca(2+) current

beta-Adrenergic stimulation of cardiac L-type Ca(2+) channels is severely impaired in hypertrophied and failing hearts of both experimental animals and humans. The aim of this study was to test the hypothesis that chronic treatment of renovascular hypertension with captopril restores normal beta-adrenergic responsiveness of L-type Ca(2+) channels in cardiac myocytes. Left ventricular hypertrophy was induced in rabbits by unilateral renal artery banding and contralateral nephrectomy. Beginning at 3 months after banding, hypertensive rabbits were treated with captopril for 3 months. The responsiveness of L-type Ca(2+) current (I(Ca,L)) to (+/-)-isoproterenol was investigated with the whole-cell patch-clamp technique. (+/-)-Isoproterenol (1 microM) induced an increase of I(Ca,L) at 0 mV of 126 +/- 20% (n = 13) in control myocytes versus 69 +/- 11% (n = 18) in hypertrophied myocytes from rabbits 3 months after banding. The half-maximal activation concentration of (+/-)-isoproterenol was similar between control and hypertrophied myocytes. Forskolin (10 microM) induced a similar percentage of increase of I(Ca,L) in control and hypertrophied myocytes, 109 +/- 13% (n = 12) versus 120 +/- 14% (n = 11) at 0 mV. The responsiveness of I(Ca,L) to (+/-)-isoproterenol remained depressed in untreated hypertensive rabbits. (+/-)-Isoproterenol (1 microM) increased I(Ca, L) at 0 mV by 64 +/- 8% (n = 14) in myocytes isolated from rabbits 6 months after banding versus 111 +/- 15% (n = 16) in age-matched controls. In captopril-treated rabbits, 1 microM (+/-)-isoproterenol increased I(Ca,L) by 110 +/- 11% (n = 17). We conclude that the maximal response of I(Ca,L) to (+/-)-isoproterenol was severely depressed in hypertrophied myocytes. Chronic treatment of renovascular hypertension with captopril can restore normal responsiveness of I(Ca,L) to (+/-)-isoproterenol in cardiac myocytes.     

Effects of FK506 on [Ca2+]i differ in mouse and rabbit ventricular myocytes

FK506 binding proteins (FKBPs 12 and 12.6) interact with ryanodine receptor (RyR) and modulate its functions. FK506 binds to and reverses effects of FKBP on RyR, thus increasing RyR sensitivity to Ca2+, decreasing RyR cooperativity, and increasing RyR open probability. FK506 would thus be expected to have an effect on excitation-contraction coupling, but which of these FK506 effects predominates and how the [Ca2+]i transient would be altered are difficult to predict. FK506 has been reported to increase the [Ca2+]i transient in rat myocytes, but effects in other species have not been described. We compared the effects of FK506 on [Ca2+]i transients, L-type Ca2+ channel and Na/Ca exchange currents, membrane potential, and sarcoplasmic reticulum (SR) Ca2+ content in adult mouse and rabbit ventricular myocytes (VM). FK506 (10 microM) increased the [Ca2+]i transient in mouse VM (656 +/- 116 to 945 +/- 144 nM, p < 0.001) but decreased the amplitude of [Ca2+]i transients in rabbit VM (627 +/- 61 to 401 +/- 37 nM, p < 0.001). Similar effects were observed with rapamycin. The effects of FK506 and rapamycin on [Ca2+]i transients in VM of both species were reversible upon washout. FK506 did not alter SR Ca2+ content in mouse VM (0.79 +/- 0.1 versus 0.78 +/- 0.1 pC/pF) but reduced the SR Ca2+ content in rabbit VM (0.43 +/- 0.05 versus 0.30 +/- 0.04 pC/pF, P < 0.05) [pC = the integral (pA. s) of the caffeine-induced inward I(Na/Ca) normalized by cell capacitance (pF)]. FK506 had no effects on membrane potential, I(Ca,L) and outward I(Na/Ca) in either mouse or rabbit VM. These results indicate that alteration of the functions of RyR by FK506-mediated dissociation of FKBP from RyR has different species-dependent effects on SR Ca2+ load and thus [Ca2+]i transients. This difference may result from the fact that [Na+]i is low in rabbit myocytes, allowing extrusion by Na+/Ca2+ exchange of Ca2+ released by FK506-induced dissociation of FKBP12.6 from SR RyR.     

Carbachol inhibits the L-type Ca2+ current augmented by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid in guinea pig ventricular myocytes: calcium-sensitivity hypothesis for muscarinic inhibition.

The L-type Ca2+ current [I(Ca(L))] increases with time after patch rupture in guinea pig ventricular myocytes dialyzed with pipette solutions containing > or =20 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid ([BAPTA]pip). I(Ca(L)) progressively increases because BAPTA chelates subsarcolemmal Ca2+ to disinhibit cardiac adenylyl cyclase (AC) activity. We studied inhibition by carbachol (CCh) of I(Ca(L)) (22-24 degrees C). At 40 mM [BAPTA]pip, 100 microM CCh reversibly suppressed I(Ca(L)) maximally by 42%; half-maximal inhibition (20%) required 1 microM. Atropine antagonized the CCh effect on BAPTA-stimulated I(Ca(L),) as did dialysis with 50 microM guanosine-5'-O-(3-thio)triphosphate. At 20, 30, and 40 mM [BAPTA]pip, I(Ca(L)) increased by 6.7 +/- 1.8, 10.1 +/- 1.4, and 11.3 +/- 1.2 pA/pF, respectively. Inhibition by 100 microM CCh averaged -1.8 +/- 0.6, -2.3 +/- 0.4, and -4.1 +/- 0.4 pA/pF at 20, 30, and 40 mM [BAPTA](pip), respectively. Dialysis of the AC inhibitor 2'-dAMP (100 microM) suppressed I(Ca(L)) run up in 40 mM BAPTA and its inhibition by CCh. Replacing 1.8 mM external Ca2+ with Ba2+, which lacks high-affinity regulatory sites on AC, suppressed CCh-induced inhibition. Neither I(Ca(L)) run up nor its inhibition by CCh occurred when 40 mM EGTA, a slower chelator, replaced BAPTA. Our results support the AC disinhibition hypothesis for BAPTA. We propose that CCh inhibits I(Ca(L)) in BAPTA by increasing either AC sensitivity to inhibition by ambient Ca2+ or the activity of the inhibitory guanine nucleotide binding protein.     

beta3-adrenergic receptor activation increases human atrial tissue contractility and stimulates the L-type Ca2+ current

beta3-adrenergic receptor (beta3-AR) activation produces a negative inotropic effect in human ventricles. Here we explored the role of beta3-AR in the human atrium. Unexpectedly, beta3-AR activation increased human atrial tissue contractility and stimulated the L-type Ca2+ channel current (I Ca,L) in isolated human atrial myocytes (HAMs). Right atrial tissue specimens were obtained from 57 patients undergoing heart surgery for congenital defects, coronary artery diseases, valve replacement, or heart transplantation. The I(Ca,L) and isometric contraction were recorded using a whole-cell patch-clamp technique and a mechanoelectrical force transducer. Two selective beta3-AR agonists, SR58611 and BRL37344, and a beta3-AR partial agonist, CGP12177, stimulated I(Ca,L) in HAMs with nanomolar potency and a 60%-90% efficacy compared with isoprenaline. The beta3-AR agonists also increased contractility but with a much lower efficacy (approximately 10%) than isoprenaline. The beta3-AR antagonist L-748,337, beta1-/beta2-AR antagonist nadolol, and beta1-/beta2-/beta3-AR antagonist bupranolol were used to confirm the involvement of beta3-ARs (and not beta1-/beta2-ARs) in these effects. The beta3-AR effects involved the cAMP/PKA pathway, since the PKA inhibitor H89 blocked I(Ca,L) stimulation and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) strongly increased the positive inotropic effect. Therefore, unlike in ventricular tissue, beta3-ARs are positively coupled to L-type Ca2+ channels and contractility in human atrial tissues through a cAMP-dependent pathway.     

The amiodarone derivative KB130015 [2-methyl-3-(3,5-diiodo-4-carboxymethoxybenzyl)benzofuran] induces an Na+-dependent increase of [Ca2+] in ventricular myocytes

KB130015 [KB; 2-methyl-3-(3,5-diiodo-4-carboxymethoxybenzyl)benzofuran] is a novel amiodarone derivative designed to retain the antiarrhythmic effects without the side effects. Unlike amiodarone, KB slows Na(+) current inactivation and could, via an increase in [Na(+)](i), potentially lead to Ca(2+) overload. Therefore, we studied the effects of KB on Na(+) and Ca(2+) handling in single pig ventricular myocytes using the whole-cell ruptured patch-clamp technique and K(5)fluo-3 as [Ca(2+)](i) indicator. KB at 10 microM did not prolong action potential duration but slightly increased the early plateau; spontaneous afterdepolarizations were not observed. The amplitude of the [Ca(2+)](i) transient was larger (434.9 +/- 37.2 versus 326.8 +/- 39.8 nM at baseline, n = 13, P < 0.05), and the time to peak [Ca(2+)](i) was prolonged. During voltage-clamp pulses, [Ca(2+)](i) transient peak was also larger (578.1 +/- 98.9 versus 346.4 +/- 52.6 nM at baseline, P < 0.05). Although L-type Ca(2+) current was reduced (by 21.9% at +10 mV, n = 9, P < 0.05), sarcoplasmic reticulum Ca(2+) content was significantly enhanced with KB. Forward Na(+)/Ca(2+) exchange was significantly decreased after KB application, but reverse mode of the Na(+)/Ca(2+) exchanger was significantly larger, suggesting an increase in [Na(+)](i) with KB. This was confirmed by a 2-fold increase of the [Na(+)]-dependent current generated by the Na/K-ATPase (from 0.17 +/- 0.02 to 0.38 +/- 0.06 pA/pF, P < 0.05). In conclusion, as predicted from the slowing of I(Na) inactivation, KB130015 leads to an increase in [Na(+)](i) and consequently in cellular Ca(2+) load. This effect is partially offset by a decrease in I(CaL) resulting in a mild inotropic effect without the signs of Ca(2+) overload and related arrhythmias usually associated with Na(+) channel openers.     

Electrophysiological effects of prucalopride, a novel enterokinetic agent, on isolated atrial myocytes from patients treated with beta-adrenoceptor antagonists

Prucalopride is a selective 5-hydroxytryptamine type 4 (5-HT4) receptor agonist developed for the treatment of gastrointestinal disorders. The endogenous agonist 5-HT acting via 5-HT4 receptors increases the L-type Ca2+ current (I(CaL)) with potentially proarrhythmic consequences (Pau et al., 2003). The aims of this study were to investigate the effects of prucalopride on I(CaL), action potentials, refractory period, and arrhythmic activity in human atrial myocytes, and to compare these with the effects of 5-HT, using the whole-cell perforated patch-clamp technique. Prucalopride (10(-9) to 10(-4) M) produced a concentration-dependent increase in I(CaL) amplitude, with a maximum response at 10 microM, from -5.3 +/- 0.6 to -10.9 +/- 1.5 pA/pF (p < 0.05; n = 22 cells, 10 patients), without affecting its voltage-dependence. Subsequent application of 10 microM 5-HT further increased I(CaL) to -17.7 +/- 2.8 pA/pF (p < 0.05; n = 16 cells, 9 patients). The increase in I(CaL) by prucalopride, 98 +/- 15%, was significantly smaller than that by 5-HT, 233 +/- 26% (p < 0.05). Prucalopride (10 microM) significantly increased the action potential duration at 50% repolarization (APD50) from 12 +/- 2 to 17 +/- 3 ms (p < 0.05; n = 22 cells, 9 patients). Following washout of prucalopride, 5-HT (10 microM) increased APD50, to a greater extent, from 14 +/- 3 to 32 +/- 7 ms (p < 0.05; n = 11 cells; 8 patients). The APD75, APD90, and effective refractory period were unaffected by prucalopride or 5-HT. Furthermore, 5-HT induced abnormal depolarizations in 27% of the cells studied, whereas prucalopride induced none (p < 0.05). In conclusion, in human atrial cells, prucalopride, at concentrations markedly above those used therapeutically, acted as partial agonist on I(CaL) and APD50, with no effect on late repolarization or refractory period, and was devoid of arrhythmic activity.     

Angiotensin II Receptor Regulates Ionic Currents in Guinea Pig Ventricular Myocytes

BACKGROUND: Previous studies have shown that angiotensin II (Ang II) receptors are preset in a wide variety of target tissues and that Ang II regulates the target tissue functions through Ang II receptors. However, the action of Ang II receptors on transsarcolemmal currents in ventricular myocytes has not been elucidated. METHODS AND RESULTS: We performed whole-cell voltage clamp and patch clamp experiments to determine the effects of Ang II-receptor agonists and antagonists on ionic currents in single isolated guinea pig ventricular myocytes. We found that extracellular perfusion of Ang II (30 nM) increased the L-type Ca(2+) current from 581 +/- 27 to 837 +/- 42 pA (n = 5, P <.01). Ang II also prolonged the Ca(2+) current activation and inactivation time constants. These were reversible by losartan (100 nM), a type 1 Ang II receptor (AT(1)) blockade. On the other hand, perfusion of 30 nM Ang II decreased K(+) current (I(K)) from 1543 +/- 28 to 1194 +/- 50 pA (n = 5, P <.05) and K(+) tail current (I(K-tail)) from 275 +/- 24 to 206 +/- 29 pA (n = 5, P <.05). These effects were also abolished by perfusion of losartan. However, perfusion of Ang II resulted in an increase of inward rectified K(+) current (I(K1)) in whole-cell recordings. Single channel recordings showed that the increase in I(K1) was attributed to a burst opening current with a larger unit of amplitude. These effects were reversed by saralasin but not losartan, indicating possible type 2 Ang II receptor (AT(2)) involvement. CONCLUSIONS: Our results provide evidence that Ang II receptors regulate the transsarcolemmal currents in single guinea pig ventricular myocytes. Therefore, Ang II regulation of ionic currents is mediated through the different subtypes of Ang II receptors.     

Diminished inotropic response to amrinone in ventricular myocytes from myopathic hamsters is linked to depression of high-gain Ca2+-induced Ca2+ release

This study investigates whether amrinone (100-1000 microM), a phosphodiesterase-III inhibitor, can alleviate depression of contractions in ventricular myocytes from prefailure cardiomyopathic (CM) hamsters (80-100 days). Cell shortening and ion currents were measured in voltage-clamped cells at 37 degrees C. Normal myocytes exhibited low-gain Ca(2+)-induced Ca(2+) release (CICR) initiated by test steps from -40 mV and high-gain CICR initiated from more negative potentials. In normal myocytes, amrinone selectively increased contractions initiated by high-gain CICR (fractional shortening increased from 3.6 +/- 0.5% to 5.3 +/- 0.6%, 300 microM amrinone) but had no effect on low-gain CICR. Amrinone decreased L-type Ca(2+) current (I(Ca-L); -5.5 +/- 0.8 to -3.7 +/- 0.5 picoAmp/picoFarad, 300 microM amrinone). In contrast, in CM myocytes, high-gain CICR was virtually absent, and amrinone had no inotropic effect. Amrinone inhibited I(Ca-L) less in CM than normal myocytes. Sarcoplasmic reticulum (SR) Ca(2+) stores, assessed by caffeine, were significantly increased by amrinone in normal but not CM myocytes. Thus, the positive inotropic effect of amrinone in normal hamster myocytes was mediated by selective enhancement of high-gain CICR. This effect was not mediated by stimulation of I(Ca-L) because I(Ca-L) is inhibited by this drug in hamster. High-gain CICR, which is depressed in CM myocytes, cannot be restored by amrinone. However, minimal stimulation of adenylyl cyclase with forskolin restored the positive inotropic effect of amrinone in CM cells. This positive inotropic effect of amrinone may reflect increased SR Ca(2+) stores because increased stores accompanied the positive inotropic effect in normal myocytes but were absent in CM myocytes.     

β3受体激动剂对心力衰竭大鼠β3-肾上腺素能受体基因表达及其对细胞凋亡的影响

目的　研究β3受体激动剂 (BRL 37344 )对异丙肾上腺素诱导的心力衰竭 (心衰 )大鼠β3肾上腺素能受体 (β3AR)基因表达水平的影响 ,探讨β3AR在心衰中的作用。方法　将 Wistar大鼠随机分为 : 组(正常对照组 10只 )、 组 (正常用药组 10只 )、 组 (心衰组 30只 )和 组 (心衰用药组 35只 )。 组和 组尾静脉注射 BRL 37344为 0 .4 nm ol· kg- 1 · min- 1 ,每周 2次 ,连续 6周 ; 组和 组尾静脉注射等量生理盐水。 6周后检查以下指标 :血流动力学变化 ;免疫组织化学和 Western Blot法测定 β3AR蛋白表达 ;逆转录聚合酶链反应 (RT PCR)方法测定 β3AR m RNA表达 ;原位缺口末端标记法 (TUNEL)检测心肌细胞凋亡。结果　1与 组比较 , 组、 组、 组左室收缩末压 (PES)、左室压力最大上升速率 (dp/ dtm ax)和左室压力最大下降速率 (dp/ dtmin)逐渐减小 ,左室舒张末压 (PED)和左室等容舒张时间常数 (Tc)逐渐增加。 组与 组比较仅 PED出现统计学差异 (P<0 .0 5 ) ,其余各组间两两比较均有差异 (P<0 .0 1或 P<0 .0 5 ) ;2β3AR蛋白及其 m RNA表达 、 组较 、 组明显增高 , 、 组间比较无显著性差异 ; 组较 组升高更明显。 3 、 组较 、 组心肌细胞凋亡数明显增加 (P均 <0 .0 1) , 、 组间比较无显著性差异     

兔心肌梗死后2个月肥大心室肌细胞的电生理研究

背景心肌梗死后电重构与梗死后时间和区域相关.目的研究陈旧性心肌梗死非梗死区肥大心室肌细胞离子通道电流的变化,探讨心肌梗死后肥大心肌发生心律失常的可能的离子机制.设计随机对照实验研究.地点、材料和干预所有实验过程在石家庄市白求恩国际和平医院心内科中心实验室完成.新西兰纯种大耳白兔20只,按随机抽签法分为两组心肌梗死动物模型组和正常对照组.采用结扎兔冠状动脉左前降支的方法建立急性心肌梗死动物模型,应用膜片钳全细胞记录方法.主要观察指标梗死后2个月心外膜远离梗死区组与梗死区组及正常对照组心室肌细胞L-钙通道电流(L-calcium current,ICa-L)、瞬间外向钾电流(transient outward current,Ito)的变化.结果①远离梗死区组细胞电容[(155.7±5.8)pF,n=41]明显大于对照组[(120.3±6.2)pF,n=35]和梗死区组[(130.4±7.8)pF,n=38](t=2.642,2.613,P均＜0.01).②ICa-L远离梗死区组ICa-L电流峰值(0 mV)为[(826.12±121.31)pA,n=21],较对照组[(670.21±183.32)pA,n=10]和梗死区组[(629.43±172.12)pA,n=11]明显增大(t=2.451,2.732,P均＜0.05).但远离梗死区组电流密度峰值为(5.32±0.78)pA/pF,较对照组[(5.58±1.53)pA/pF]略下降,与梗死区组[(4.84±1.48)pA/pF]和对照组比较无显著性意义(P均＞0.05).③Ito远离梗死区组[(13.21±4.13)pA/pF,n=23]和梗死区组[(10.61±4.12)pA/pF,n=18]的Ito电流密度(+60mV时)均明显低于对照组[(17.39±5.24)pA/pF,n=16](t=3.591,2.725,P均＜0.01).但远离梗死区组明显高于梗死区组(t=2.429,P＜0.05).结论心肌梗死后2个月,远离梗死区心室肌细胞电容明显增大,反映心肌细胞发生代偿性肥大.远离梗死区心室肌细胞ICa-L幅值升高,但Ito电流密度明显下降,ICa-L电流密度轻度下降,可导致动作电位平台期延长,复极异常,异常自律性的增加,并且心室不同部位存在电生理异质性,可能是导致陈旧性心肌梗死出现室性心律失常的离子基础.     

Functional adenylyl cyclase inhibition in murine cardiomyocytes by 2'(3')-O-(N-methylanthraniloyl)-guanosine 5'-[gamma-thio]triphosphate

Beta1-adrenergic receptor activation stimulates cardiac L-type Ca2+ channels via adenylyl cyclases (ACs), with AC5 and AC6 being the most important cardiac isoforms. Recently, we have identified 2'(3')-O-(N-methylanthraniloyl)-guanosine 5'-[gamma-thio-]triphosphate (MANT-GTPgammaS) as a potent competitive AC inhibitor. Intriguingly, MANT-GTPgammaS inhibits AC5 and -6 more potently than other cyclases. These data prompted us to study the effects of MANT-GTPgammaS on L-type Ca2+ currents (ICa,L) in ventricular myocytes of wild-type (WT) and AC5-deficient (AC5-/-) mice by whole-cell recordings. In wild-type myocytes, MANT-GTPgammaS attenuated ICa,L stimulation following isoproterenol application in a concentration-dependent manner (control, +77+/-13%; 100 nM MANT-GTPgammaS, +43+/-6%; 1 microM MANT-GTPgammaS, +21+/-9%; p<0.05). The leftward shift of current-voltage curves was abolished by 1 microM but not by 100 nM MANT-GTPgammaS. In myocytes from AC5-/- mice, the residual stimulation of ICa,L was not further attenuated by the nucleotide, indicating AC5 to be the major AC isoform mediating acute beta-adrenergic stimulation in WT mice. Interestingly, basal ICa,L was lowered by 1 microM but not by 100 nM MANT-GTPgammaS. The decrease was less pronounced in myocytes from AC5-/- mice compared with wild types (-23+/-1 versus -40+/-7%), indicating basal ICa,L to be partly driven by AC5. Collectively, we found a concentration-dependent inhibition of ICa,L by MANT-GTPgammaS, both under basal conditions and following beta-adrenergic stimulation. Comparison of data from wild-type and AC5-deficient mice indicates that AC5 plays a major role in ICa,L activation and that MANT-GTPgammaS predominantly acts via AC5 inhibition.     

Mesenteric lymph from rats with thermal injury prolongs the action potential and increases Ca2+ transient in rat ventricular myocytes

Although gut-derived mesenteric lymph from animals with thermal injury appears to lead to myocardial contractile dysfunction, the cellular mechanisms remain unclear. We examined the direct effects of intestinal lymph on excitation-contraction coupling in rat ventricular myocytes. Lymph from rats receiving burn injury (burn lymph), but not from sham-burned rats, rapidly enhanced myocyte contraction and the amplitude of Ca2+ transient; the average percentage of shortening was increased from 5.5 +/- 0.3% to 10.5 +/- 0.9%. 90% and the Ca2+ transients increased by 80% +/- 20%. Burn lymph had no effect on the amplitude of L-type Ca2+ current (ICa) or the inward rectifier K+ current, but the transient outward K+ currents (Ito) were reduced significantly by burn lymph. Inhibition of Ito was not altered by an alpha1-adrenergic receptor (AR) antagonist, prazosin, indicating that the block was not mediated via alpha1-AR signaling pathway. Action potential (AP) duration, measured at 50% and 90% repolarization, was prolonged by burn lymph. Stimulation of myocytes with AP voltage-clamp waveforms derived from prolonged AP induced by burn lymph revealed a 1.7-fold increase in Ca2+ influx via ICa compared with the Ca2+ influx induced by control AP. Blocking of Ito by 4-aminopyridine prolonged AP duration and increased Ca2+ transients, mimicking the effects of burn lymph. Burn lymph did not affect Na+/Ca2+ exchange currents or caffeine-induced SR Ca2+ release. Thus, acute exposure of normal cardiac myocytes to burn lymph increases Ca2+ transients by a prolongation of AP as a result of a reduction of Ito with no intrinsic change in ICa or exchanger. The electrophysiological changes are similar to those that occur during compensated cardiac hypertrophy, suggesting a common mechanistic link between burn lymph- and hypertrophy-induced cardiac dysfunction.     

花生四烯酸对L-型钙通道的作用及其抗心律失常机制

目的 研究花生四烯酸(arachidonic acid,AA)对家兔单个心室肌细胞L-广型钙通道的作用及其抗心律失常作用的机制.方法 采用酶解法分离得到家兔单个心室肌细胞,全细胞膜片钳技术记录单个心室肌细胞L-型钙电流(L-type calcium current,Ica-L),用累积给药的方法在灌流液中加入不同浓度的AA,观察给药前后L-型钙电流的变化,统计学方法采用单因素方差分析.结果 不同浓度的从均能明显抑制心室肌细胞,Ica-L.3 μmol/L,μmol/L,20,μmol/L的AA使Ica-L峰电流密度从(10.79±0.93)pA/pF分别减少剑(8.99 ±0.43)pA/pF、(7.60 ±0.35)pA/pF和(5.60±0.30)pA/pF(n=7,P<0.05),经冲洗后Ica-L可部分恢复,并且AA可使Ica-L的I-V关系曲线上移,其形状和峰值电压保持不变;20 μmol/L的AA使Ica-L失活曲线左移,失活后恢复时间明显延长,但对激活曲线无明显影响.结论 花生四烯酸可通过加快L-型钙通道失活,延长其失活后的恢复过程而减少细胞外钙离子的内流,延长有效不应期,从而发挥抗心律失常作用.     

Comparison of L-type calcium channel blockade by nifedipine and/or cadmium in guinea pig ventricular myocytes

We tested the assumption that nifedipine blocks L-type calcium current [I(Ca(L))] at +10 mV and unmasks Na(+)/Ca(2+) exchange-triggered contractions in guinea pig isolated ventricular myocytes. Voltage-clamp pulses elicited I(Ca(L)) at +10 mV and evoked contractions in myocytes superfused with Tyrode's solution (35 degrees C). Nifedipine blocked I(Ca(L)) with an IC(50) of 0.3 microM; this decreased to 50 nM at a holding potential of -40 mV, indicating preferential block of inactivated L-type Ca(2+) channels. Use-independent block of I(Ca(L)) increased with concentration (10-100 microM) and application time when nifedipine was rapidly applied (t(1/2) = approximately 0.2 s) during rest intervals (5-30 s). The fraction of use-dependent block of I(Ca(L)) diminished with increasing drug concentration. Nifedipine also accelerated I(Ca(L)) inactivation on the first test pulse. The combination of 30 microM nifedipine/30 microM Cd(2+) (Nif 30/Cd 30) was as effective as 100 microM nifedipine to suppress I(Ca(L)) on the first test pulse at +10 mV. The incidence of complete block of contractions, as for complete block of I(Ca(L)), increased as a function of nifedipine concentration and application time. Neither nifedipine nor Nif 30/Cd 30 affected Na(+)/Ca(2+) exchange current at +10 to +100 mV. Contractions at +100 mV, although as large as those at +10 mV, were delayed in onset and resistant to nifedipine or Nif 30/Cd 30. We conclude that nifedipine-sensitive I(Ca(L)) triggers contractions at +10 mV, whereas nifedipine-resistant Na(+)/Ca(2+) exchange current initiates those at +100 mV.     

Modulation of sarcoplasmic reticulum function by PST2744 [istaroxime; (E,Z)-3-((2-aminoethoxy)imino) androstane-6,17-dione hydrochloride)] in a pressure-overload heart failure model

PST2744 [Istaroxime; (E,Z)-3-((2-aminoethoxy)imino) androstane-6,17-dione hydrochloride)] is a novel inotropic agent that enhances sarco(endo)plasmic reticulum Ca(2+) ATPase (SERCA) 2 activity. We investigated the istaroxime effect on Ca(2+) handling abnormalities in myocardial hypertrophy/failure (HF). Guinea pig myocytes were studied 12 weeks after aortic banding (AoB) and compared with those of sham-operated animals (sham). The gain of calcium-induced Ca(2+) release (CICR), sarcoplasmic reticulum (SR) Ca(2+) content, Na(+)/Ca(2+) exchanger (NCX) function, and the rate of SR reloading after caffeine-induced depletion (SR Ca(2+) uptake, measured during NCX blockade) were evaluated by measurement of cytosolic Ca(2+) and membrane currents. HF characterization: AoB caused hypertrophy and failure in 100 and 25% of animals, respectively. Although CICR gain during constant pacing was preserved, SR Ca(2+) content and SR Ca(2+) uptake were strongly depressed. Resting Ca(2+) and the slope of the Na(+)/Ca(2+) exchanger current (I(NCX))/Ca(2+) relationship were unchanged by AoB. Istaroxime effects: CICR gain, SR Ca(2+) content, and SR Ca(2+) uptake rate were increased by istaroxime in sham myocytes and, to a significantly larger extent, in AoB myocytes; this led to almost complete recovery of SR Ca(2+) uptake in AoB myocytes. Istaroxime increased resting Ca(2+) and the slope of the I(NCX)/Ca(2+) relationship similarly in sham and AoB myocytes. Istaroxime failed to increase SERCA activity in skeletal muscle microsomes devoid of phospholamban. Thus, clear-cut abnormalities in Ca(2+) handling occurred in this model of hypertrophy, with mild decompensation. Istaroxime enhanced SR function more in HF myocytes than in normal ones; almost complete drug-induced recovery suggests a purely functional nature of SR dysfunction in this HF model.     

Mechanism of myricetin stimulation of vascular L-type Ca2+ current

An in-depth analysis of the mechanism of the L-type Ca(2+) current [I(Ca(L))] stimulation induced by myricetin was performed in rat tail artery myocytes using the whole-cell patch-clamp method. Myricetin increased I(Ca(L)) in a frequency-, concentration-, and voltage-dependent manner. At holding potentials (V(h)) of -50 and -90 mV, the pEC(50) values were 4.9 +/- 0.1 and 4.2 +/- 0.1, respectively; the latter corresponded to the drug-apparent dissociation constant for resting channels, K(R), of 67.6 microM. Myricetin shifted the maximum of the current-voltage relationship by 10 mV in the hyperpolarizing direction but did not modify the threshold for I(Ca(L)) or the T-type Ca(2+) current. The Ca(2+) channel blockers nifedipine, verapamil, and diltiazem antagonized I(Ca(L)) in the presence of myricetin. Myricetin increased the time to peak of I(Ca(L)) in a voltage- and concentration-dependent manner. Washout reverted myricetin effect on both current kinetics and amplitude at V(h) of -90 mV while reverting only current kinetics at V(h) of -50 mV. At the latter V(h), myricetin shifted the voltage dependence of inactivation and activation curves to more negative potentials by 6.4 and 13.0 mV, respectively, in the mid-potential of the curves. At V(h) of -90 mV, myricetin shifted, in a concentration-dependent manner, the voltage dependence of the inactivation curve to more negative potentials with an apparent dissociation constant for inactivated channels (K(I)) of 13.8 muM. Myricetin induced a frequency- and V(h)-dependent block of I(Ca(L)). In conclusion, myricetin behaves as an L-type Ca(2+) channel agonist that stabilizes the channel in its inactivated state.     

Fatty acid block of the transient outward current in adult human atrium

Fatty acids represent an essential source of fuel for the heart and play an important role in the mechanical, electrical, and synthetic activities of cardiac cells. Under pathological conditions, such as ischemia followed by reperfusion, the myocardium is exposed to very high levels of fatty acids, in particular the monounsaturated fatty acid, oleic acid. Elevated plasma fatty acids have been linked to an increased risk for cardiac arrhythmias. In other species, fatty acids have been shown to modulate several cardiac ion channels, most notably potassium channels. Virtually nothing is known about the actions of oleic acid on potassium channels in human heart. We therefore characterized the effects of oleic acid on the transient outward current, sustained current, and inwardly rectifying current, some of the major potassium channels present in human atrium, using the whole-cell patch clamp method. Exposure of cells to oleic acid (5 microM) reduced the transient outward potassium current to 3.7 +/- 0.8 pA/pF (n = 4) compared with 7.0 +/- 0.7 pA/pF (n = 4) (P <. 05) for cells not exposed. In contrast, oleic acid had little effect on either the sustained current (4.3 +/- 0.3 pA/pF, n = 4 for oleic acid versus 4.8 +/- 0.5, n = 5 for control) present after the decay of the transient outward current or on the amplitude of IK1 measured at -100 mV (1.4 +/- 0.4 pA/pF, n = 4 for oleic acid versus 1.3 +/- 0. 4 pA/pF, n = 6 for control). In addition, oleic acid significantly slowed the rate of recovery of the transient outward current, which is predicted to result in a use-dependent reduction in current amplitude in the beating heart. These results suggest a possible contributing role for oleic acid block of the transient outward current in the pathological consequences of myocardial ischemia.     

Painful neuropathy decreases membrane calcium current in mammalian primary afferent neurons

Hyperexcitability of the primary afferent neuron leads to neuropathic pain following injury to peripheral axons. Changes in calcium channel function of sensory neurons following injury have not been directly examined at the channel level, even though calcium is a primary second messenger-regulating neuronal function. We compared calcium currents (I(Ca)) in 101 acutely isolated dorsal root ganglion neurons from 31 rats with neuropathic pain following chronic constriction injury (CCI) of the sciatic nerve, to cells from 25 rats with normal sensory function following sham surgery. Cells projecting to the sciatic nerve were identified with a fluorescent label applied at the CCI site. Membrane function was determined using patch-clamp techniques in current clamp mode, and in voltage-clamp mode using solutions and conditions designed to isolate I(Ca). Somata of peripheral sensory neurons from hyperalgesic rats demonstrated decreased I(Ca). Peak calcium channel current density was diminished by injury from 3.06+/-0.30 pS/pF to 2. 22+/-0.26 pS/pF in medium neurons, and from 3.93+/-0.38 pS/pF to 2. 99+/-0.40 pS/pF in large neurons. Under these voltage and pharmacologic conditions, medium-sized neuropathic cells lacked obvious T-type calcium currents which were present in 25% of medium-sized cells from control animals. Altered Ca(2+) signalling in injured sensory neurons may contribute to hyperexcitability leading to neuropathic pain.