Enhanced cardiac L-type calcium current response to beta2-adrenergic stimulation in heart failure.

The beta2-adrenergic receptor (beta2-AR)-mediated increase in cardiac L-type Ca2+ current (I(Ca,L)) has been documented in normal subjects. However, the role and mechanism of beta2-AR activation on I(Ca,L) in heart failure (HF) are unclear. Accordingly, we compared the effect of zinterol (ZIN), a highly selective beta2-AR agonist, on I(Ca,L) in isolated left ventricular cardiomyocytes obtained from normal control and age-matched rats with HF induced by left coronary artery ligation (4 months). I(Ca,L) was measured by using the whole-cell voltage-clamp technique. In normal myocytes, superfusion of ZIN (10(-5) M) caused a 21% increase in I(Ca,L) (9.21 +/- 0.24 versus 7.59 +/- 0.20 pA/pF) (p < 0.05). In HF myocytes, the same concentration of ZIN produced a significantly greater increase (30%) in I(Ca,L) (6.20 +/- 0.24 versus 4.75 +/- 0.17 pA/pF) (p < 0.01). This ZIN-induced increase in I(Ca,L) was further augmented in both normal and HF myocytes (normal: 59 versus 21%; HF: 71 versus 30%) after the incubation of myocytes with pertussis toxin (PTX, 2 microg/ml, 36 degrees C, 6 h). These effects were not modified by the incubation of myocytes with CGP-20712A (3 x 10(-7) M), a beta1-AR antagonist, but were abolished by pretreatment of myocytes with ICI-118551 (10(-7) M), a beta2-AR antagonist. In addition, all of the effects induced by ZIN were completely prevented in the presence of an inhibitory cAMP analog, Rp-cAMPS (100 microM, in the patch-pipette solution). In conclusion, beta2-AR activation stimulates L-type Ca2+ channels and increases I(Ca,L) in both normal and HF myocytes. In HF, beta2-AR activation-induced augmentation of I(Ca,L) was increased. These effects are likely to be mediated through a cAMP-dependent mechanism and coupled with both stimulatory G protein and PTX-sensitive G 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.     

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.     

Inhibition of L-type Ca2+ channel current in Xenopus oocytes by amiodarone.

BACKGROUND: Although amiodarone has been referred to as a class III antiarrhythmic agent, it also possesses electrophysiologic characteristics of the three other classes (classes I and IV and minor class II effects). Previous studies have demonstrated that amiodarone inhibits Ca2+ channel current in intact cardiac myocytes. However, it is not clear whether this response reflects a pure class IV effect (direct Ca2+ channel inhibition) or a class II effect (beta-adrenergic receptor blockade) of amiodarone. METHODS: In the current study, the effects of amiodarone on Ca2+ current were studied in the absence of sympathetic regulation using a Xenopus oocyte expression system. The L-type Ca2+ channel alpha1C subunit was coexpressed with the alpha2delta and beta2a subunits in enzymatically digested Xenopus oocytes. Ca2+ currents were recorded using the cut-open oocyte preparation. RESULTS: We found that perfusion of 10 microM isoproterenol produced no significant change in peak Ca2+ current (from 223+/-33 to 210+/-29 nA, mean+/-SEM, n=5, P=not significant), indicating the absence of a functional stimulatory sympathetic signal pathway in these oocytes. After 10 minutes of exposure to 10 microM amiodarone, Ca2+ current amplitude was significantly decreased from 174+/-33 to 100+/-26 nA (n=8, P<0.01; control group: 220+/-33 to 212+/-29 nA, n=5, P=not significant). These effects were similar to those of 10 microM nifedipine (201+/-48 to 108+/-48 nA, n=6, P<0.05), a typical Ca2+ channel blocker. On the other hand, neither amiodarone nor nifedipine significantly altered the Ca2+ current activation or inactivation kinetics. CONCLUSIONS: These results demonstrate that amiodarone inhibits Ca2+ current in the absence of a functional intrinsic beta-adrenergic stimulatory system and, therefore, represents a true class IV effect.     

cGMP-stimulated cyclic nucleotide phosphodiesterase regulates the basal calcium current in human atrial myocytes.

EHNA (Erythro-9-[2-hydroxy-3-nonyl]adenine) is a wellknown inhibitor of adenosine deaminase. Recently, EHNA was shown to block the activity of purified soluble cGMPstimulated phosphodiesterase (PDE2) from frog, human, and porcine heart with an apparent Ki value of approximately 1 microM and with negligible effects on Ca2+/calmodulin PDE (PDE1), cGMP-inhibited PDE (PDE3), and low Km cAMP-specific PDE (PDE4) (Méry, P.F., C. Pavoine, F. Pecker, and R. Fischmeister. 1995. Mol. Pharmacol. 48:121-130; Podzuweit, T., P. Nennstiel, and A. Muller. 1995. Cell. Signalling. 7:733- 738). To investigate the role of PDE2 in the regulation of cardiac L-type Ca2+ current (ICa), we have examined the effect of EHNA on ICa in freshly isolated human atrial myocytes. Extracellular application of 0.1-10 microM EHNA induced an increase in the amplitude of basal ICa ( approximately 80% at 1 microM) without modification of the current-voltage or inactivation curves. The maximal stimulatory effect of EHNA on ICa was comparable in amplitude with the maximal effect of isoprenaline (1 microM), and the two effects were not additive. The effect of EHNA was not a result of adenosine deaminase inhibition, since 2'-deoxycoformycin (1-30 microM), another adenosine deaminase inhibitor with no effect on PDE2, or adenosine (1-10 microM) did not increase ICa. In the absence of intracellular GTP, the substrate of guanylyl cyclase, EHNA did not increase ICa. However, under similar conditions, intracellular perfusion with 0.5 microM cGMP produced an 80% increase in ICa. As opposed to human cardiomyocytes, EHNA (1-10 microM) did not modify ICa in isolated rat ventricular and atrial myocytes. We conclude that basal ICa is controlled by PDE2 activity in human atrial myocytes. Both PDE2 and PDE3 may contribute to keep the cyclic nucleotides concentrations at minimum in the absence of adenylyl and/or guanylyl cyclase stimulation.     

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.     

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.     

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.     

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     

Selectivity and potency of agonists for the three subtypes of cloned human beta-adrenoceptors expressed in Chinese hamster ovary cells

The selectivities, potencies and efficacies of beta3-adrenoceptor (beta3-AR) agonists on human three beta-AR subtypes expressed in Chinese hamster ovary (CHO) cells were investigated using radioligand binding assay and cyclic AMP (cAMP) accumulation assay. The three beta-AR subtypes showed the nature of G protein-coupled receptors with the constitutive activity. BRL37344, CL-316,243 and a newly synthesized beta3-AR agonist N-5984, 6-[2-(R)-[[2-(R)-(3-chlorophenyl)-2-hydroxyethyl]amino]propyl]-2,3-dihydro-1,4-benzodioxine-2-(R)-carboxylic acid, were compared for the potency and selectivity for the beta3-AR. In the radioligand binding assay, the affinity of N-5984 for beta3-ARs was 14, 70 and 220 times more potent than those of BRL37344, isoproterenol and CL-316,243, respectively. N-5984 had higher selectivity than BRL37344 for human beta3-ARs compared with either for beta1-ARs or beta2-ARs. N-5984 showed higher potency and intrinsic activity of cAMP production than BRL37344 in CHO cells expressing the beta3-ARs. CL-316,243 had almost no activity of cAMP production in CHO cells expressing any subtype of beta-ARs. These results indicate that N-5984 is the most potent and selective agonist for human beta3-ARs than any other agonists tested.     

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.     

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.     

Verapamil-induced blockade of voltage-activated K+ current in small-cell lung cancer cells.

Verapamil, Ca++ channel antagonist, has proven clinically useful in the reversal of multiple drug resistance, which is a major detriment to chemotherapy. Recently, verapamil alone has been shown to diminish proliferation in a variety of neoplastic cell lines. Using the patch-clamp technique, the action of verapamil on voltage-gated K+ channels in two cell lines of human small-cell carcinoma of the lung, NCI-H146 and NCI-H82, was investigated. With inward Na+ current suppressed, virtually all control cells exhibited a slowly inactivating outward current that was insensitive to alterations in the external Ca++ concentration. Externally applied verapamil enhanced the rate and extent of outward K+ current (IK) inactivation. Verapamil at a concentration of 20 microM diminished peak IK, evoked by a test pulse to +60 mV from a holding potential of -80 mV, from 1.38 +/- 0.11 nA (mean +/- S.E.M., n = 29 cells) to 0.56 +/- 0.13 nA (n = 11) and caused IK to decay to less than 20% of the peak current within 60 msec. After blocking IK and Na+ current, Ca++ current (ICa) was measured in the presence of 10 mM Ca++. The addition of 100 microM verapamil to the external bath resulted in a 53% reduction of H146 ICa. Peak ICa fell from 81 +/- 9 pA (n = 22) to 38 +/- 8 pA (n = 12). Examination of the whole-cell K+ current on single cells before and immediately after the addition of 100 microM verapamil clearly revealed that the drug had no effect on the initial activation phase of IK, suggesting that K+ channels first open before interacting with the drug.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of dichlorobenzamil on calcium currents in clonal GH3 pituitary cells

Membrane currents were recorded from voltage-clamped clonal (GH3) pituitary cells, using the whole-cell patch clamp method. Under conditions in which currents through Na+ and K+ channels were abolished, two distinct Ca++ channel currents were identified. Dichlorobenzamil (DCB), an amiloride analog and potent inhibitor of Na-Ca exchange, inhibited both the T-type Ca++ current (ICa,t) and slowly-inactivating L-type current carried by either Ca++ (ICa,s) or Ba++ (IBa,s). The blockade was dose-dependent (1-25 microM) and ICa,t was more sensitive to inhibition by DCB than the L-type channel currents. Although the inhibition of ICa,t was not accompanied by changes in the time course of inactivation of the T-type channels, the blockade of L-type Ca++ channel currents was time-dependent, increasing throughout the depolarizing test pulse (300 msec). Repetitive stimulation at 1.0 Hz or the application of depolarizing prepulses augmented the blockade of ICa,s and IBa,s by DCB. It is proposed that the blockade is modulated by the functional state of the L-type channel, being enhanced by the channel opening. Currents conveyed by Na+ ions through both types of Ca++ channels in GH3 cells equilibrated with Ca++-free salines were inhibited by DCB concentrations (1-5 microM) comparable to those required for blocking the currents conveyed by Ca++ or Ba++ ions.     

Activation of β3-adrenergic receptor inhibits ventricular arrhythmia in heart failure through calcium handling

Ventricular arrhythmia in chronic heart failure (CHF) is considered to be associated with stimulation of β-adrenergic receptors (β-ARs). Three classes of β-ARs have been identified; importantly, distinct from β1 and β2 subtypes, β3-AR could inhibit arrhythmia. Intracellular Ca2+ is considered as a predominant effecter of arrhythmia during heart failure. However, the exact role of β3-AR in arrhythmia and Ca2+ regulation in CHF is not clear yet. Therefore, we studied the effect of BRL37344, a specific β3-AR activator, on CHF-related ventricular arrhythmia and cellular Ca2+ transport. Rabbits with CHF induced by combined aortic insufficiency and aortic constriction were treated with BRL37344 in the presence or absence of β1-AR and β2-AR stimulation. We then evaluated the current produced by sodium calcium exchanger (INCX), an electrical marker of abnormal Ca2+ removal through ion transporter protein sodium calcium exchanger (NCX), Ca2+ transient, a sign of Ca2+ entering the cell, concentration of Ca2+ in sarcoplasmic reticulum (SR) (SR Ca2+ load) and its abnormal release (SR Ca2+ leak). After treatment with BRL37344, the incidence of ventricular arrhythmias induced by infusion of a β1-AR or β2-AR activator decreased significantly. Similarly, β3-AR stimulation remarkably inhibited increase of INCX, Ca2+ transient, SR Ca2+ load and leak induced by activation of β1-AR or β2-AR. SR59230A, a specific β3-AR blocker, abolished the inhibitory effects of BRL37344. These results suggest that β3-AR activation could inhibit ventricular arrhythmia through regulating intracellular Ca2+. Thus, β3-AR is a feasible therapeutic target that holds promise in the treatment of ventricular arrhythmias in CHF.     

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.     

Age-associated changes in beta-adrenergic modulation on rat cardiac excitation-contraction coupling.

Previous studies have demonstrated that the ability of beta-adrenergic receptor (beta AR) stimulation to increase cardiac contractility declines with aging. In the present study, the control mechanisms of excitation-contraction (EC) coupling, including calcium current (ICa), cytosolic Ca2+ (Cai2+) transient and contraction in response to beta AR stimulation were investigated in ventricular myocytes isolated from rat hearts of a broad age range (2, 6-8, and 24 mo). While the baseline contractile performance and the Cai2+ transient did not differ markedly among cells from hearts of all age groups, the responses of the Cai2+ transient and contraction to beta-adrenergic stimulation by norepinephrine (NE) diminished with aging: the threshold concentration and the ED50 increased in rank order with aging; the maximum responses of contraction and Cai2+ transient decreased with aging. Furthermore, the efficacy of beta AR stimulation to increase ICa was significantly reduced with aging, and the diminished responses of the contraction and Cai2+ transient amplitudes to NE were proportional to the reductions in the ICa response. These findings suggest that the observed age-associated reduction in beta AR modulation of the cardiac contraction is, in part at least, due to a deficit in modulation of Cai2+, particularly the activity of L-type calcium channels.     

Nitric oxide regulates the calcium current in isolated human atrial myocytes.

Cardiac Ca2+ current (ICa) was shown to be regulated by cGMP in a number of different species. Recently, we found that the NO-donor SIN-1 (3-morpholino-sydnonimine) exerts a dual regulation of ICa in frog ventricular myocytes via an accumulation of cGMP. To examine whether NO also regulates Ca2+ channels in human heart, we investigated the effects of SIN-1 on ICa in isolated human atrial myocytes. An extracellular application of SIN-1 produced a profound stimulatory effect on basal ICa at concentrations > 1 pM. Indeed, 10 pM SIN-1 induced a approximately 35% increase in ICa. The stimulatory effect of SIN-1 was maximal at 1 nM (approximately 2-fold increase in ICa) and was comparable with the effect of a saturating concentration (1 microM) of isoprenaline, a beta-adrenergic agonist. Increasing the concentration of SIN-1 to 1-100 microM reduced the stimulatory effect in two thirds of the cells. The stimulatory effect of SIN-1 was not mimicked by SIN-1C, the cleavage product of SIN-1 produced after liberation of NO. This suggests that NO mediates the effects of SIN-1 on ICa. Because, in frog heart, the stimulatory effect of SIN-1 on ICa was found to be due to cGMP-induced inhibition of cGMP-inhibited phosphodiesterase (cGI-PDE), we compared the effects of SIN-1 and milrinone, a cGI-PDE selective inhibitor, on ICa in human. Milrinone (10 microM) induced a strong stimulation of ICa (approximately 150%), demonstrating that cGI-PDE controls the amplitude of basal ICa in this tissue. In the presence of milrinone, SIN-1 (0.1-1 nM) had no stimulatory effect on ICa, suggesting that the effects of SIN-1 and MIL were not additive. We conclude that NO may stimulate ICa in human atrial myocytes via inhibition of the cGI-PDE.     

Effects of N(G)-monomethyl-L-arginine on Ca(2+) current and nitric-oxide synthase in rat ventricular myocytes

The effects of N(G)-monomethyl-L-arginine (L-NMMA), a nitric-oxide synthase (NOS) inhibitor, on the L-type Ca(2+) current (ICa) and NO effects on NOS were determined in rat ventricular myocytes. L-NMMA (10 and 100 microM) had no significant effect on basal ICa, but in a cAMP-stimulated condition due to forskolin (1 microM) or milrinone (10 microM), a cGMP-inhibited cAMP-phosphodiesterase (PDE), L-NMMA (10 and 100 microM) concentration dependently augmented ICa. The enhancing effects of L-NMMA (10 and 100 microM) on ICa were not seen in the presence of either a nonselective inhibitor of PDE, 3-isobutyl-1-methylxanthine (20 microM), resulting in a stimulated ICa condition or a cGMP-dependent protein kinase activator, 8-bromo-cGMP (200 microM). 8-Bromo-cGMP (200 microM) inhibited 100 microM L-NMMA-induced ICa increase in the simultaneous application of forskolin (1 microM). Acetylcholine (ACh; 1 and 3 microM) inhibited 1 microM forskolin-stimulated ICa in a concentration-dependent manner, but this inhibitory action of ACh was significantly attenuated by the additional application of L-NMMA (100 microM). In the continuing presence of both L-NMMA (100 microM) and forskolin (1 microM), ACh (6 microM) had no inhibitory effect on ICa. In another series of experiments with isolated ventricular myocytes, we obtained both the positive staining of NADPH-diaphorase activity and the expression of the endothelial isoform of NOS. These data suggest that the effect of L-NMMA on ICa in a cAMP-stimulated condition with or without cholinergic inhibition is due to inhibition (acute effects) of a cGMP-stimulated cAMP-PDE via inhibition of the endothelial isoform of NOS.