Is there an A-type K+ current in guinea pig ventricular myocytes?

A unique transient outward K(+) current (I(to)) has been described to result from the removal of extracellular Ca(2+) from ventricular myocytes of the guinea pig (15). This study addressed the question of whether this current represented K(+)-selective I(to) or the efflux of K(+) via L-type Ca(2+) channels. This outward current was inhibited by Cd(2+), Ni(2+), Co(2+), and La(3+) as well as by nifedipine. All of these compounds were equally effective inhibitors of the L-type Ca(2+) current. The current was not inhibited by 4-aminopyridine. Apparent inhibition of the outward current by extracellular Ca(2+) was shown to result from the displacement of the reversal potential of cation flux through L-type Ca(2+) channels. The current was found not to be K(+) selective but also permeant to Cs(+). The voltage dependence of inactivation of the outward current was identical to that of the L-type Ca(2+) current. It is concluded that extracellular Ca(2+) does not mask an A-type K(+) current in guinea pig ventricular myocytes.     

Sarcoplasmic reticulum calcium defect in Ras-induced hypertrophic cardiomyopathy heart

The small G protein Ras-mediated signaling pathway has been implicated in the development of hypertrophy and diastolic dysfunction in the heart. Earlier cellular studies have suggested that the Ras pathway is responsible for reduced L-type calcium channel current and sarcoplasmic reticulum (SR) calcium uptake associated with sarcomere disorganization in neonatal cardiomyocytes. In the present study, we investigated the in vivo effects of Ras activation on cellular calcium handling and sarcomere organization in adult ventricular myocytes using a newly established transgenic mouse model with targeted expression of the H-Ras-v12 mutant. The transgenic hearts expressing activated Ras developed significant hypertrophy and postnatal lethal heart failure. In adult ventricular myocytes isolated from the transgenic hearts, the calcium transient was significantly depressed but membrane L-type calcium current was unchanged compared with control littermates. The expressions of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)2a and phospholamban (PLB) were significantly reduced at mRNA levels. The amount of SERCA2a protein was also modestly reduced. However, the expression of PLB protein and gross sarcomere organization remained unchanged in the hypertrophic Ras hearts, whereas Ser(16) phosphorylation of PLB was dramatically inhibited in the Ras transgenic hearts compared with controls. Hypophosphorylation of PLB was also associated with a significant induction of protein phosphatase 1 expression. Therefore, our results from this in vivo model system suggest that Ras-induced contractile defects do not involve decreased L-type calcium channel activities or disruption of sarcomere structure. Rather, suppressed SR calcium uptake due to reduced SERCA2a expression and hypophosphorylation of PLB due to changes in protein phosphatase expression may play important roles in the diastolic dysfunction of Ras-mediated hypertrophic cardiomyopathy.     

哌替啶对心室肌收缩的抑制作用及其机制

为明确哌替啶对心脏收缩的直接效应 ,并探讨其相关机制。采用Langendorff灌流心脏模型 ,观察了哌替啶对大鼠心室收缩功能的影响 ,并用荧光测钙技术和膜片钳技术探讨了哌替啶作用的钙离子机制。结果显示 ,哌替啶剂量依赖性地降低离体灌流心脏的LVDP×HR、 +dP/dt和 -dP/dt,而升高LVEDP。在酶解分离的心室肌细胞上 ,哌替啶剂量依赖性地降低细胞收缩时的钙瞬变幅度 ,并升高舒张末期的钙水平。哌替啶不影响高浓度咖啡因诱导的内钙释放。哌替啶使L 型钙电流强度降低到给药前的 67 4± 10 1% ,而不改变钙通道的激活和失活电位。哌替啶减弱钙电流的作用并不能被阿片受体阻断剂纳洛酮所阻断。以上结果表明 ,哌替啶能通过非阿片受体介导的途径阻断细胞外钙离子的内流 ,对心室收缩产生直接的抑制作用     

SR Ca2+ refilling upon depletion and SR Ca2+ uptake rates during development in rabbit ventricular myocytes

While it has been reported that a sparse sarcoplasmic reticulum (SR) and a low SR Ca(2+) pump density exist at birth, we and others have recently shown that significant amounts of Ca(2+) are stored in the neonatal rabbit heart SR. Here we try to determine developmental changes in SR Ca(2+) loading mechanisms and Ca(2+) pump efficacy in rabbit ventricular myocytes. SR Ca(2+) loading (load(SR)) and k(0.5) (Ca(2+) concentration at half-maximal SR Ca(2+) uptake) were higher and lower, respectively, in younger age groups. Inhibition of the L-type calcium current (I(Ca)) with 15 microM nifedipine dramatically reduced load(SR) in older but not in younger age groups. In contrast, subsequent inhibition of the Na(+)/Ca(2+) exchanger (NCX) with 10 microM KB-R7943 strongly reduced load(SR) in the younger but not the older age groups. Accordingly, the time integral of the inward NCX current (tail I(NCX)) elicited on repolarization was highly sensitive to nifedipine in the older groups and sensitive to KB-R7943 in the younger groups. Interestingly, slow SR loading took place in the presence of both nifedipine and KB-R7943 in all age groups, although it was less prominent in the older groups. We conclude that the SR loading capacity at the earliest postnatal stages is at least as large as that of adult myocytes. However, reverse-mode NCX plays a prominent role in SR Ca(2+) loading at early postnatal stages while I(Ca) is the main source of SR Ca(2+) loading at late postnatal and adult stages.     

Caveolin-3 regulates protein kinase A modulation of the Ca(V)3.2 (alpha1H) T-type Ca2+ channels

Voltage-gated T-type Ca(2+) channel Ca(v)3.2 (α(1H)) subunit, responsible for T-type Ca(2+) current, is expressed in different tissues and participates in Ca(2+) entry, hormonal secretion, pacemaker activity, and arrhythmia. The precise subcellular localization and regulation of Ca(v)3.2 channels in native cells is unknown. Caveolae containing scaffolding protein caveolin-3 (Cav-3) localize many ion channels, signaling proteins and provide temporal and spatial regulation of intracellular Ca(2+) in different cells. We examined the localization and regulation of the Ca(v)3.2 channels in cardiomyocytes. Immunogold labeling and electron microscopy analysis demonstrated co-localization of the Ca(v)3.2 channel and Cav-3 relative to caveolae in ventricular myocytes. Co-immunoprecipitation from neonatal ventricular myocytes or transiently transfected HEK293 cells demonstrated that Ca(v)3.1 and Ca(v)3.2 channels co-immunoprecipitate with Cav-3. GST pulldown analysis confirmed that the N terminus region of Cav-3 closely interacts with Ca(v)3.2 channels. Whole cell patch clamp analysis demonstrated that co-expression of Cav-3 significantly decreased the peak Ca(v)3.2 current density in HEK293 cells, whereas co-expression of Cav-3 did not alter peak Ca(v)3.1 current density. In neonatal mouse ventricular myocytes, overexpression of Cav-3 inhibited the peak T-type calcium current (I(Ca,T)) and adenovirus (AdCa(v)3.2)-mediated increase in peak Ca(v)3.2 current, but did not affect the L-type current. The protein kinase A-dependent stimulation of I(Ca,T) by 8-Br-cAMP (membrane permeable cAMP analog) was abolished by siRNA directed against Cav-3. Our findings on functional modulation of the Ca(v)3.2 channels by Cav-3 is important for understanding the compartmentalized regulation of Ca(2+) signaling during normal and pathological processes.     

Nonneurogenic hypoxia sensitivity in rat adrenal slices.

A change in the intracellular Ca(2+) ([Ca(2+)](i)) level induced by hypoxia was detected in rat adrenal slices by use of fura-2/AM. After hypoxic stress, an increase in [Ca(2+)](i) was observed only in the adrenal medulla. This increase was inhibited by nifedipine, but not modified by the cholinergic receptor blockers. The hypoxia-induced increase in [Ca(2+)](i) was observed in all postnatal developmental stages to a similar extent, whereas the nicotine and high K(+) sensitivities increased along with postnatal development. A 10 nM ryanodine enhanced the hypoxia-induced [Ca(2+)](i) increase in adult but not in neonatal rat slices. These results suggest the existence of an oxygen-sensing mechanism in adult rat adrenals even after sympathetic innervation. Hypoxic responses seemed to be similar both in neonate and in adult rat adrenals and were triggered by the influx of Ca(2+) via L-type voltage-sensitive Ca(2+) channels. However, the sustained [Ca(2+)](i) increase caused by hypoxia might depend on postnatal development and be triggered by Ca(2+)-induced Ca(2+) release (CICR).     

Abolition of reperfusion-induced arrhythmias in hearts from thiamine-deficient rats

Extensive work has been done regarding the impact of thiamine deprivation on the nervous system. In cardiac tissue, chronic thiamine deficiency is described to cause changes in the myocardium that can be associated with arrhythmias. However, compared with the brain, very little is known about the effects of thiamine deficiency on the heart. Thus this study was undertaken to explore whether thiamine deprivation has a role in cardiac arrhythmogenesis. We examined hearts isolated from thiamine-deprived and control rats. We measured heart rate, diastolic and systolic tension, and contraction and relaxation rates. Whole cell voltage clamp was performed in rat isolated cardiac myocytes to measure L-type Ca(2+) current. In addition, we investigated the global intracellular calcium transients by using confocal microscopy in the line-scan mode. The hearts from thiamine-deficient rats did not degenerate into ventricular fibrillation during 30 min of reperfusion after 15 min of coronary occlusion. The antiarrhythmogenic effects were characterized by the arrhythmia severity index. Our results suggest that hearts from thiamine-deficient rats did not experience irreversible arrhythmias. There was no change in L-type Ca(2+) current density. Inactivation kinetics of this current in Ca(2+)-buffered cells was retarded in thiamine-deficient cardiac myocytes. The global Ca(2+) release was significantly reduced in thiamine-deficient cardiac myocytes. The amplitude of caffeine-releasable Ca(2+) was lower in thiamine-deficient myocytes. In summary, we have found that thiamine deprivation attenuates the incidence and severity of postischemic arrhythmias, possibly through a mechanism involving a decrease in global Ca(2+) release.     

B-type natriuretic peptide (BNP) attenuates the L-type calcium current and regulates ventricular myocyte function

A fundamental question in physiology is how hormones regulate the functioning of a cell or organ. It was therefore the aim of this study to investigate the effect(s) of BNP-32 on calcium handling by ventricular myocytes obtained from the rat left ventricle. We specifically tested the hypothesis that BNP-32 decreased the L-type calcium current (I(Ca,L)). Perforated patch clamp technique was used to record I(Ca,L) and action potential (AP) in voltage and current clamp mode, respectively. Myocyte shortening was measured using a photodiode array edge-detection system and intracellular calcium transients were measured by fluorescence photometry. Western blotting was used to determine the relative change in the expression of proteins. At the concentrations tested, BNP-32 significantly decreased cell shortening in a dose-dependent manner; increased the phase II slope of the AP by 53.0%; increased the APD(50) by 16.9%; reduced the I(Ca,L) amplitude with a 22.9% decrease in the peak amplitude and reduced Ca(2+)-dependent inactivation; increased the V(1/2) activation of the L-type calcium channel by 51.1% and decreased V(1/2) inactivation by 31.8%; and, intracellular calcium transient amplitude was significantly decreased by 32.0%, whereas the time to peak amplitude and T(1/2) were both significantly increased by 38.7% and 89.4% respectively. Sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a) protein expression was reduced by BNP-32. These data suggest that BNP-32 regulates ventricular myocyte function by attenuating I(Ca,L), altering the AP and reducing SERCA2a activity and/or expression. This study suggests a novel constitutive mechanism for the autocrine action of BNP on the L-type calcium channel in ventricular myocytes.     

Calcium-induced calcium release in smooth muscle: loose coupling between the action potential and calcium release

Calcium-induced calcium release (CICR) has been observed in cardiac myocytes as elementary calcium release events (calcium sparks) associated with the opening of L-type Ca(2+) channels. In heart cells, a tight coupling between the gating of single L-type Ca(2+) channels and ryanodine receptors (RYRs) underlies calcium release. Here we demonstrate that L-type Ca(2+) channels activate RYRs to produce CICR in smooth muscle cells in the form of Ca(2+) sparks and propagated Ca(2+) waves. However, unlike CICR in cardiac muscle, RYR channel opening is not tightly linked to the gating of L-type Ca(2+) channels. L-type Ca(2+) channels can open without triggering Ca(2+) sparks and triggered Ca(2+) sparks are often observed after channel closure. CICR is a function of the net flux of Ca(2+) ions into the cytosol, rather than the single channel amplitude of L-type Ca(2+) channels. Moreover, unlike CICR in striated muscle, calcium release is completely eliminated by cytosolic calcium buffering. Thus, L-type Ca(2+) channels are loosely coupled to RYR through an increase in global [Ca(2+)] due to an increase in the effective distance between L-type Ca(2+) channels and RYR, resulting in an uncoupling of the obligate relationship that exists in striated muscle between the action potential and calcium release.     

Expression of Ca(2+) channel subunits during cardiac ontogeny in mice and rats: identification of fetal alpha(1C) and beta subunit isoforms

Functional cardiac L-type calcium channels are composed of the pore-forming alpha(1C) subunit and the regulatory beta(2) and alpha(2)/delta subunits. To investigate possible developmental changes in calcium channel composition, we examined the temporal expression pattern of alpha(1C) and beta(2) subunits during cardiac ontogeny in mice and rats, using sequence-specific antibodies. Fetal and neonatal hearts showed two size forms of alpha(1C) with 250 and 220 kDa. Quantitative immunoblotting revealed that the rat cardiac 250-kDa alpha(1C) subunit increased about 10-fold from fetal days 12-20 and declined during postnatal maturation, while the 220-kDa alpha(1C) decreased to undetectable levels. The expression profile of the 85-kDa beta(2) subunit was completely different: beta(2) was not detected at fetal day 12, rose in the neonatal stage, and persisted during maturation. Additional beta(2)-stained bands of 100 and 90 kDa were detected in fetal and newborn hearts, suggesting the transient expression of beta(2) subunit variants. Furthermore, two fetal proteins with beta(4) immunoreactivity were identified in rat hearts that declined during prenatal development. In the fetal rat heart, beta(4) gene expression was confirmed by RT-PCR. Cardiac and brain beta(4) mRNA shared the 3 prime region, predicting identical primary sequences between amino acid residues 62-519, diverging however, at the 5 prime portion. The data indicate differential developmental changes in the expression of Ca(2+) channel subunits and suggest a role of fetal alpha(1C) and beta isoforms in the assembly of Ca(2+) channels in immature cardiomyocytes.     

Anion channels influence ECC by modulating L-type Ca(2+) channel in ventricular myocytes.

Anion channels are extensively expressed in the heart, but their roles in cardiac excitation-contraction coupling (ECC) are poorly understood. We, therefore, investigated the effects of anion channels on cardiac ventricular ECC. Edge detection, fura 2 fluorescence measurements, and whole cell patch-clamp techniques were used to measure cell shortening, the intracellular Ca(2+) transient, and the L-type Ca(2+) current (I(Ca,L)) in single rat ventricular myocytes. The anion channel blockers 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and niflumic acid reversibly inhibited the Ca(2+) transients and cell shortening in a dose-dependent manner. Comparable results were observed when the majority of the extracellular Cl(-) was replaced with the relatively impermeant anions glutamate (Glt(-)) and aspartate (Asp(-)). NPPB and niflumic acid or the Cl(-) substitutes did not affect the resting intracellular Ca(2+) concentration but significantly inhibited I(Ca,L). In contrast, replacement of extracellular Cl(-) with the permeant anions NO, SCN(-), and Br(-) supported the ECC and I(Ca,L), which were still sensitive to blockade by NPPB. Exposure of cardiac ventricular myocytes to a hypotonic bath solution enhanced the amplitude of cell shortening and supported I(Ca,L), whereas hypertonic stress depressed the contraction and I(Ca,L). Moreover, cardiac contraction was completely abolished by NPPB (50 microM) under hypotonic conditions. It is concluded that a swelling-activated anion channel may be involved in the regulation of cardiac ECC through modulating L-type Ca(2+) channel activity.     

DMA增加正常大鼠心肌细胞钙瞬变和收缩

实验观察了钠氢交换或钠钙交换抑制剂 5 (N ,N 二甲基 )氨氯吡咪 (DMA)对正常和心肌肥厚大鼠分离心室肌细胞钙瞬变和细胞收缩的影响。通过负载荧光染料Fura 2 /Am ,应用离子影像分析系统 (IonImagingSystem)同步测定离体大鼠心肌细胞钙瞬变和细胞长度。结果表明 :DMA 10 μmol/L分别使钙瞬变和细胞缩短从对照组的 2 0 9.6 0± 5 4.96和 3.0 7± 0 .97μm增加到 2 38.5 0± 80 .41和 4.0 7± 1.0 2 μm (P <0 .0 5 ,n =7)。应用特异性反向钠钙交换阻断剂KB R7943可完全阻断DMA的激动作用。DMA还可使尼卡地平抑制L 型钙通道后的钙瞬变和细胞收缩增加。在肥厚心肌细胞 ,DMA表现出相同的药理作用 ,但对钙瞬变和细胞缩短的刺激作用更强。结果表明 :DMA可通过反向钠钙交换途径增加正常和肥厚大鼠心肌细胞钙瞬变和细胞收缩 ,且对肥厚心肌细胞的影响比对正常心肌细胞大。     

Developmental changes of intracellular Ca2+ transients in beating rat hearts

Postnatal maturation of the rat heart is characterized by major changes in the mechanism of excitation-contraction (E-C) coupling. In the neonate, the t tubules and sarcoplasmic reticulum (SR) are not fully developed yet. Consequently, Ca(2+)-induced Ca(2+) release (CICR) does not play a central role in E-C coupling. In the neonate, most of the Ca(2+) that triggers contraction comes through the sarcolemma. In this work, we defined the contribution of the sarcolemmal Ca(2+) entry and the Ca(2+) released from the SR to the Ca(2+) transient during the first 3 wk of postnatal development. To this end, intracellular Ca(2+) transients were measured in whole hearts from neonate rats by using the pulsed local field fluorescence technique. To estimate the contribution of each Ca(2+) flux to the global intracellular Ca(2+) transient, different pharmacological agents were used. Ryanodine was applied to evaluate ryanodine receptor-mediated Ca(2+) release from the SR, nifedipine for dihydropyridine-sensitive L-type Ca(2+) current, Ni(2+) for the current resulting from the reverse-mode Na(+)/Ca(2+) exchange, and mibefradil for the T-type Ca(2+) current. Our results showed that the relative contribution of each Ca(2+) flux changes considerably during the first 3 wk of postnatal development. Early after birth (1-5 days), the sarcolemmal Ca(2+) flux predominates, whereas at 3 wk of age, CICR from the SR is the most important. This transition may reflect the progressive development of the t tube-SR units characteristic of mature myocytes. We have hence directly defined in the whole beating heart the developmental changes of E-C coupling previously evaluated in single (acutely isolated or cultured) cells and multicellular preparations.     

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.     

A mechanism distinct from the L-type Ca current or Na-Ca exchange contributes to Ca entry in rat ventricular myocytes

The aim of this paper was to characterize the pathways that allow Ca(2+) ions to enter the cell at rest. Under control conditions depolarization produced an increase of intracellular Ca concentration ([Ca(2+)](i)) that increased with depolarization up to about 0 mV and then declined. During prolonged depolarization the increase of [Ca(2+)](i) decayed. This increase of [Ca(2+)](i) was inhibited by nifedipine and the calculated rate of entry of Ca increased on depolarization and then declined with a similar time course to the inactivation of the L-type Ca current. We conclude that this component of change of [Ca(2+)](i) is due to the L-type Ca current. If intracellular Na was elevated then only part of the change of [Ca(2+)](i) was inhibited by nifedipine. The nifedipine-insensitive component increased monotonically with depolarization and showed no relaxation on prolonged depolarization. This component appears to result from Na-Ca exchange (NCX). When the L-type current and NCX were both inhibited (nifedipine and Na-free solution) then depolarization decreased and hyperpolarization increased [Ca(2+)](i). These changes of [Ca(2+)](i) were unaffected by modifiers of B-type Ca channels such as chlorpromazine and AlF(3) but were abolished by gadolinium ions. We conclude that, in addition to L-type Ca channels and NCX, there is another pathway for entry of Ca(2+) into the ventricular myocyte but this is distinct from the previously reported B-type channel.     

Postnatal development and activation of L-type Ca2+ currents in locus ceruleus neurons: implications for a role for Ca2+ in central chemosensitivity

Little is known about the role of Ca(2+) in central chemosensitive signaling. We use electrophysiology to examine the chemosensitive responses of tetrodotoxin (TTX)-insensitive oscillations and spikes in neurons of the locus ceruleus (LC), a chemosensitive region involved in respiratory control. We show that both TTX-insensitive spikes and oscillations in LC neurons are sensitive to L-type Ca(2+) channel inhibition and are activated by increased CO(2)/H(+). Spikes appear to arise from L-type Ca(2+) channels on the soma whereas oscillations arise from L-type Ca(2+) channels that are distal to the soma. In HEPES-buffered solution (nominal absence of CO(2)/HCO(3)(-)), acidification does not activate either oscillations or spikes. When CO(2) is increased while extracellular pH is held constant by elevated HCO(3)(-), both oscillation and spike frequency increase. Furthermore, plots of both oscillation and spike frequency vs. intracellular [HCO(3)(-)]show a strong linear correlation. Increased frequency of TTX-insensitive spikes is associated with increases in intracellular Ca(2+) concentrations. Finally, both the appearance and frequency of TTX-insensitive spikes and oscillations increase over postnatal ages day 3-16. Our data suggest that 1) L-type Ca(2+) currents in LC neurons arise from channel populations that reside in different regions of the neuron, 2) these L-type Ca(2+) currents undergo significant postnatal development, and 3) the activity of these L-type Ca(2+) currents is activated by increased CO(2) through a HCO(3)(-)-dependent mechanism. Thus the activity of L-type Ca(2+) channels is likely to play a role in the chemosensitive response of LC neurons and may underlie significant changes in LC neuron chemosensitivity during neonatal development.     

beta2-Adrenergic receptor agonists stimulate L-type calcium current independent of PKA in newborn rabbit ventricular myocytes

Selective stimulation of beta(2)-adrenergic receptors (ARs) in newborn rabbit ventricular myocardium invokes a positive inotropic effect that is lost during postnatal maturation. The underlying mechanisms for this age-related stimulatory response remain unresolved. We examined the effects of beta(2)-AR stimulation on L-type Ca(2+) current (I(Ca,L)) during postnatal development. I(Ca,L) was measured (37 degrees C; either Ca(2+) or Ba(2+) as the charge carrier) using the whole-cell patch-clamp technique in newborn (1 to 5 days old) and adult rabbit ventricular myocytes. Ca(2+) transients were measured concomitantly by dialyzing the cell with indo-1. Activation of beta(2)-ARs (with either 100 nM zinterol or 1 microM isoproterenol in the presence of the beta(1)-AR antagonist, CGP20712A) stimulated I(Ca,L) twofold in newborns but not in adults. The beta(2)-AR-mediated increase in Ca(2+) transient amplitude in newborns was due exclusively to the augmentation of I(Ca,L). Zinterol increased the rate of inactivation of I(Ca,L) and increased the Ca(2+) flux integral. The beta(2)-AR inverse agonist, ICI-118551 (500 nM), but not the beta(1)-AR antagonist, CGP20712A (500 nM), blocked the response to zinterol. Unexpectedly, the PKA blockers, H-89 (10 microM), PKI 6-22 amide (10 microM), and Rp-cAMP (100 microM), all failed to prevent the response to zinterol but completely blocked responses to selective beta(1)-AR stimulation of I(Ca,L) in newborns. Our results demonstrate that in addition to the conventional beta(1)-AR/cAMP/PKA pathway, newborn rabbit myocardium exhibits a novel beta(2)-AR-mediated, PKA-insensitive pathway that stimulates I(Ca,L). This striking developmental difference plays a major role in the age-related differences in inotropic responses to beta(2)-AR agonists.