Reduced calcium current density in single myocytes isolated from hypertrophied failing guinea pig hearts

The present study explored the possibility that an alteration in the transmembrane calcium current (ICa), through its ability to modulate Ca2+ release from the sarcoplasmic reticulum, could contribute to the depressed peak [Ca2+]i we previously observed in hypertrophied failing myocardium. Whole-cell patch clamp was used to measure ICa in single guinea pig ventricular myocytes isolated from hearts of normal guinea pigs and from guinea pig hearts in which hypertrophy and failure were induced by gradually developing left ventricular pressure overload subsequent to ascending aortic banding of young animals. Membrane capacitance (Cm) was significantly greater. and ICa, normalized for Cm, was significantly lower in myocytes from hypertrophied failing hearts. Myocytes from hypertrophied failing hearts did not differ significantly from normal myocytes in terms of the voltage-dependence of the activation variable (d) of ICa (except at -30 mV), the time course of removal of inactivation of ICa, and the time constant of decay of ICa. Measurement of the voltage dependence of the inactivation variable (f) of ICa showed that significantly more steady-state inactivation was present at 0, -10, and -20 mV in myocytes from hypertrophied failing hearts. Multiple regression analysis of all data indicated that ICa density decreased with increasing myocyte membrane area (as reflected by Cm) irrespective of any specific effects of hypertrophy and heart failure. We conclude that ICa, normalized for Cm, is significantly reduced in myocytes isolated from hypertrophied failing hearts, probably by a process associated with increased cell size, per se.     

Inhibition of myocardial L- and T-type Ca2+ currents by efonidipine: possible mechanism for its chronotropic effect

Effects of efonidipine, a dihydropyridine phosphonate Ca2+ channel antagonist, on the guinea-pig heart were compared with those of nifedipine. In the sino-atrial node, 1 microM efonidipine produced increase in cycle length accompanied by prolongation of the phase 4 depolarization which was not prominent with 0.1 microM nifedipine. In ventricular myocytes, both efonidipine and nifedipine produced inhibition of the L-type Ca2+ current, nifedipine being tenfold more potent than efonidipine. Efonidipine also inhibited the T-type Ca2+ current at higher concentrations but nifedipine did not. Both Ca2+ channel antagonists had no or only a weak effect on K+ currents. In addition, 40 microM Ni2+, which selectively inhibited the T-type Ca2+ current, had no effect on myocardial Ca2+ transients and contractile force. In conclusion, efonidipine was shown to have inhibitory effects on both L- and T-type Ca2+ currents, which may contribute to its high negative chronotropic potency.     

Regional differences in action potential characteristics and membrane currents of guinea-pig left ventricular myocytes

Regional differences in action potential characteristics and membrane currents were investigated in subendocardial, midmyocardial and subepicardial myocytes isolated from the left ventricular free wall of guinea-pig hearts. Action potential duration (APD) was dependent on the region of origin of the myocytes (P < 0.01, ANOVA). Mean action potential duration at 90 % repolarization (APD90) was 237 +/- 8 ms in subendocardial (n = 30 myocytes), 251 +/- 7 ms in midmyocardial (n = 30) and 204 +/- 7 ms in subepicardial myocytes (n = 36). L-type calcium current (ICa) density and background potassium current (IK1) density were similar in the three regions studied. Delayed rectifier current (IK) was measured as deactivating tail current, elicited on repolarization back to -45 mV after 2 s step depolarizations to test potentials ranging from -10 to +80 mV. Mean IK density (after a step to +80 mV) was larger in subepicardial myocytes (1.59 +/- 0.16 pA pF-1, n = 16) than in either subendocardial (1.16 +/- 0.12 pA pF-1, n = 17) or midmyocardial (1. 13 +/- 0.11 pA pF-1, n = 21) myocytes (P < 0.05, ANOVA). The La3+-insensitive current (IKs) elicited on repolarization back to -45 mV after a 250 ms step depolarization to +60 mV was similar in the three regions studied. The La3+-sensitive tail current, (IKr) was greater in subepicardial (0.50 +/- 0.04 pA pF-1, n = 11) than in subendocardial (0.25 +/- 0.05 pA pF-1, n = 9) or in midmyocardial myocytes (0.38 +/- 0.05 pA pF-1, n = 11, P < 0.05, ANOVA). The contribution of a Na+ background current to regional differences in APD was assessed by application of 0.1 microM tetrodotoxin (TTX). TTX-induced shortening of APD90 was greater in subendocardial myocytes (35.7 +/- 7.1 %, n = 11) than in midmyocardial (15.7 +/- 3. 8 %, n = 10) and subepicardial (20.2 +/- 4.3 %, n = 11) myocytes (P < 0.05, ANOVA). Regional differences in action potential characteristics between subendocardial, midmyocardial, and subepicardial myocytes isolated from guinea-pig left ventricle are attributable, at least in part, to differences in IK and Na+-dependent currents.     

Cellular basis of contractile derangements of hypertrophied feline ventricular myocytes

The objective of this study was to further explore the cellular basis of the reduced rate and magnitude of contraction of feline left ventricular myocytes with severe hypertrophy induced by slow progressive pressure overload. A 3.0 mm internal diameter band was placed around the ascending aorta of 12 young (8-10 weeks old) cats, and sham operations were performed in 13 others. This caused no major pressure overload initially, but 15 weeks later there was a significant pressure gradient across the band (56+/-14 mmHg) and the heart weight to body weight ratio had increased from 4.2-7 gm/kg. Contraction rates and magnitudes of myocytes isolated from the hearts with hypertrophy (LVH) were significantly slower and smaller, respectively, than those from control (C) animals. Indo-1 fluorescence transients in LVH myocytes were significantly smaller in magnitude and longer in duration than in C, suggesting that contractile defects result from Ca2+ derangements. Elevation of bath Ca2+ increased the peak Indo-1 fluorescence and the rate and magnitude of contraction in all myocytes. At the bath Ca2+ which had maximal inotropic effects there were no differences in the peak Indo-1 fluorescence in LVH and C myocytes, but contraction magnitude remained significantly smaller in LVH. This suggests that there are Ca2+-independent contractile derangements in LVH. In support of this hypothesis, the relationship between contraction magnitude and the peak Indo-1 fluorescence (index of myofibrillar Ca2+ sensitivity) was significantly shifted in LVH myocytes, suggesting that myofibrillar Ca2+ sensitivity was reduced. There was also a significant shift of the terminal portions of hysteresis loops of cell length v indo-1 fluorescence ratio, providing additional support for this idea. Experiments with isoproterenol suggest that it can reduce myofibrillar Ca2+ sensitivity in C, but not LVH myocytes. The idea that increased internal resistance to shortening (internal load) is responsible for the contractile defects of LVH myocytes was examined by defining the relationship between the rate of relengthening and the magnitude of shortening. There was no significant difference in this relation between C and LVH myocytes. In addition, colchicine (which depolymerizes microtubular tubulin) had no significant effect on contraction magnitude in either C or LVH myocytes. These results suggest that the contractile properties of feline LVH myocytes result from changes in cellular Ca2+ regulation and myofibrillar Ca2+ sensitivity, but not from changes in the internal loading.     

Selectivity of felodipine for depolarized ventricular myocytes: a study at the single-cell level

We studied the effects of felodipine (a second-generation dihydropyridine Ca2+ channel blocker) on excitation-contraction coupling (E-C coupling) in single isolated guinea-pig ventricular myocytes, using the whole-cell perforated patch-clamp technique or the Ca indicator, indo-1. Felodipine inhibited both L-type Ca2+ channel currents (ICa) and cell contractions in a concentration-dependent manner (10 pM to 100 nM) when we used a holding potential of -80 mV or -40 mV. The potency of felodipine was sharply dependent on a holding potential. Namely, use of a more depolarized holding potential markedly increased the potency of felodipine for inhibition of ICa and cell contraction. Next we current-clamped cells and obtained the resting membrane potential of -82 +/- 8 mV. When cells were current-injected at 0.1 Hz, exposure to 10 nM felodipine slightly but significantly diminished the amplitude of cell contractions (7.2 +/- 1.6 to 6.7 +/- 1.7 microns, P < 0.05) within 10 min. When cells were field stimulated, exposure of cells to 10 nM felodipine also slightly diminished the amplitude of cell shortening (5.1 +/- 2.0 to 4.6 +/- 1.9 microns, P < 0.05) and [Ca2+]i transients. We observed clear voltage-dependent blockade of E-C coupling by felodipine in ventricular myocytes. Thus, therapeutic concentrations (1-10 nM) of felodipine could inhibit E-C coupling in depolarized ventricular myocytes, which might simulate an ischemic or failing heart.     

Ca2+ currents in compensated hypertrophy and heart failure

Transmembrane voltage-gated Ca2+ channels play a central role in the development and control of heart contractility which is modulated by the concentration of free cytosolic calcium ions (Ca2+). Ca2+ channels are closed at the normal membrane resting potential of cardiac cells. During the fast upstroke of the action potential (AP), they are gated into an open state by membrane depolarisation and thereby transduce the electrical signal into a chemical signal. In addition to its contribution to the AP plateau, Ca2+ influx through L-type Ca2+ channels induces a release of Ca2+ ions from the sarcoplasmic reticulum (SR) which initiates contraction. Because of their central role in excitation-contraction (E-C) coupling, L-type Ca2+ channels are a key target to regulate inotropy [1]. The role of T-type Ca2+ channels is more obscure. In addition to a putative part in the rhythmic activity of the heart, they may be implicated at early stages of development and during pathology of contractile tissues [2]. Despite therapeutic advances improving exercise tolerance and survival, congestive heart failure (HF) remains a major problem in cardiovascular medicine. It is a highly lethal disease; half of the mortality being related to ventricular failure whereas sudden death of the other patients is unexpected [3]. Although HF has diverse aetiologies, common abnormalities include hypertrophy, contractile dysfunction and alteration of electrophysiological properties contributing to low cardiac output and sudden death. A significant prolongation of the AP duration with delayed repolarisation has been observed both during compensated hypertrophy (CH) and in end-stage HF caused by dilated cardiomyopathy (Fig. 1A) [4-8]. This lengthening can result from either an increase in inward currents or a decrease in outward currents or both. A reduction of K+ currents has been demonstrated [6,9]. Prolonged Na+/Ca2+ exchange current may also be involved [9]. In contrast, there is a large variability in the results concerning Ca2+ currents (ICa). The purpose of this paper is to review results obtained in various animal models of CH and HF with special emphasis on recent studies in human cells. We focus on: (i) the pathophysiological role of T-type Ca2+ channels, present in some animal models of hypertrophy; (ii) the density and properties of L-type Ca2+ channels and alteration of major physiological regulations of these channels by heart rate and beta-adrenergic receptor stimulation; and (iii) recent advances in the molecular biology of the L-type Ca2+ channel and future directions.     

L- and T-type calcium currents differ in finch and rat ventricular cardiomyocytes

We compared L-type Ca current (ICaL) and T-type Ca current (ICaT) in finch and rat myocytes, using whole-cell patch clamp techniques. Cell capacitance averaged 50 +/- 4 pF in finch (n = 25) v 145 +/- 8 pF in rat (n = 38) cells, P < 0.001. In cells bathed in 1 mM Cao at 22 degrees C, peak ICaL amplitude, during a voltage clamp step (10 mM EGTA in pipette) from -45 mV to -5 mV, averaged 10.5 +/- 0.3 pA/pF in finch v 6.9 +/- 0.6 pA/pF, P < 0.001 in rat cells. ICaL inactivation kinetics were faster in finch (4.6 +/- 0.3 ms) than in rat (13.4 +/- 1.3 ms) cells. P < 0.001. ICaT was not detectable in rat cells (2 mM bathing [Ca]); but in finch cells, a large ICaT which averaged 6.8 +/- 1.4 pA/pF was activated at -30 mV and was relatively insensitive to nitrendipine (0.1 microM). The distinctive features of ICaL and ICaT in finch cells may have a role in the ability of the finch to achieve a very rapid heart rate. They may also facilitate excitation-Ca2+ release coupling in finch ventricular cells which are devoid of T tubules and have relatively few junctions between the sarcolemma and the sarcoplasmic reticulum.     

Expression of calcium channels in adult cardiac myocytes is regulated by calcium

In addition to playing a significant role in cardiac excitation-contraction coupling, intracellular Ca2+ ([Ca2+]i) can regulate gene expression. While the mechanisms regulating expression of Ca2+ channels are not entirely defined, some evidence exists for Ca2+-dependent regulation. Using an adult ventricular myocyte culture system, we determined the effects of Ca2+ on: (1) abundance of mRNA for L-type Ca2+ channel alpha1 subunit (DHP receptor); (2) amount of DHP receptors; and (3) whole-cell Ca2+ current (ICa). Rat ventricular myocytes were cultured for 1-3 days in serum-free medium containing either normal (1.8 mM) or high (4.8 mM) Ca2+. Exposing myocytes to high Ca2+ rapidly elevated [Ca2+]i as determined by fura-2. Northern blot analysis revealed that culturing cells in high Ca2+ produced 1.5-fold increase in mRNA levels for the DHP receptor. The abundance of DHP receptors, determined by ligand binding, was two-fold greater in myocytes after 3 days in high Ca2+. Moreover, peak ICa was larger in myocytes cultured for 3 days in high Ca2+ (-17.8+/-1.5 pA/pF, n=26) than in control cells (-11.0+/-1.0 pA/pF, n=23). Voltage-dependent activation and inactivation, rates of current decay, as well as percent increases in ICa elicited by Bay K8644 were similar in all groups. Therefore, larger ICa is likely to represent a greater number of functional channels with unchanged kinetics. Our data support the conclusion that transient changes in [Ca2+]i can modulate DHP receptor mRNA and protein abundance, producing a corresponding change in functional Ca2+ channels in adult ventricular myocytes.     

Effects of the nitric oxide donor sodium nitroprusside on intracellular pH and contraction in hypertrophied myocytes

BACKGROUND: We compared the effects of the nitric oxide donor sodium nitroprusside (SNP) on intracellular pH (pHi), intracellular calcium concentration ([Ca2+]i) transients, and cell contraction in hypertrophied adult ventricular myocytes from aortic-banded rats and age-matched controls. METHODS AND RESULTS: pHi was measured in individual myocytes with SNARF-1, and [Ca2+]i transients were measured with indo 1 simultaneously with cell motion. Experiments were performed at 37 degrees C in myocytes paced at 0.5 Hz in HEPES-buffered solution (extracellular pH = 7.40). At baseline, calibrated pHi, diastolic and systolic [Ca2+]i values, and the amplitude of cell contraction were similar in hypertrophied and control myocytes. Exposure of the control myocytes to 10(-6) mol/L SNP caused a decrease in the amplitude of cell contraction (72 +/- 7% of baseline, P < .05) that was associated with a decrease in pHi (-0.10 +/- 0.03 U, P < .05) with no change in peak systolic [Ca2+]i. In contrast, in the hypertrophied myocytes exposure to SNP did not decrease the amplitude of cell contraction or cause intracellular acidification (-0.01 +/- 0.01 U, NS). The cGMP analogue 8-bromo-cGMP depressed cell shortening and pHi in the control myocytes but failed to modify cell contraction or pHi in the hypertrophied cells. To examine the effects of SNP on Na(+)-H+ exchange during recovery from intracellular acidosis, cells were exposed to a pulse and washout of NH4Cl. SNP significantly depressed the rate of recovery from intracellular acidosis in the control cells compared with the rate in hypertrophied cells. CONCLUSIONS: SNP and 8-bromo-cGMP cause a negative inotropic effect and depress the rate of recovery from intracellular acidification that is mediated by Na(+)-H+ exchange in normal adult rat myocytes. In contrast, SNP and 8-bromo-cGMP do not modify cell contraction or pHi in hypertrophied myocytes.     

Formation of spin trap adducts during the decomposition of peroxynitrite

To investigate Ca2+ handling in compensated hypertrophied cardiomyocytes, we measured Ca2+ transients and contraction of hypertrophied rat left ventricular myocytes induced by aortic constriction (AC). The fluorescence ratio (I405/I480) after indo-1/AM loading and circumferential length were simultaneously measured in isolated myocytes. The amplitude of Ca2+ transients (Ca-Amp) was higher in rats with AC than in sham-operated rats (Sham) (0.25 +/- 0.08 vs 0.17 +/- 0.05). There was a positive correlation between Ca-Amp and fractional shortening (FS) in both AC and Sham rats, whereas the ratio of FS/Ca-Amp was smaller in AC rats. These observations suggest that compensated hypertrophied cardiomyocytes exhibit an adaptive increase in Ca-Amp, associated with reduced myofilament responsiveness to an increase in Ca2+. Isoproterenol and forskolin increased Ca-Amp and FS, and decreased time to 50% decline of Ca2+ transients. Although myocytes from AC rats exhibited reduced responsiveness to isoproterenol, responses to forskolin did not differ between the 2 groups. The reduced beta-adrenergic response in Ca2+ handling was probably due to altered beta-adrenoceptor numbers, G-protein function and/or their coupling process.     

Potassium currents in freshly dissociated uterine myocytes from nonpregnant and late-pregnant rats

In freshly dissociated uterine myocytes, the outward current is carried by K+ through channels highly selective for K+. Typically, nonpregnant myocytes have rather noisy K+ currents; half of them also have a fast-inactivating transient outward current (ITO). In contrast, the current records are not noisy in late pregnant myocytes, and ITO densities are low. The whole-cell IK of nonpregnant myocytes respond strongly to changes in [Ca2+]o or changes in [Ca2+]i caused by photolysis of caged Ca2+ compounds, nitr 5 or DM-nitrophene, but that of late-pregnant myocytes respond weakly or not at all. The Ca2+ insensitivity of the latter is present before any exposure to dissociating enzymes. By holding at -80, -40, or 0 mV and digital subtractions, the whole-cell IK of each type of myocyte can be separated into one noninactivating and two inactivating components with half-inactivation at approximately -61 and -22 mV. The noninactivating components, which consist mainly of iberiotoxin-susceptible large-conductance Ca2+-activated K+ currents, are half-activated at 39 mV in nonpregnant myocytes, but at 63 mV in late-pregnant myocytes. In detached membrane patches from the latter, identified 139 pS, Ca2+-sensitive K+ channels also have a half-open probability at 68 mV, and are less sensitive to Ca2+ than similar channels in taenia coli myocytes. Ca2+-activated K+ currents, susceptible to tetraethylammonium, charybdotoxin, and iberiotoxin contribute 30-35% of the total IK in nonpregnant myocytes, but <20% in late-pregnant myocytes. Dendrotoxin-susceptible, small-conductance delayed rectifier currents are not seen in nonpregnant myocytes, but contribute approximately 20% of total IK in late-pregnant myocytes. Thus, in late-pregnancy, myometrial excitability is increased by changes in K+ currents that include a suppression of the ITO, a redistribution of IK expression from large-conductance Ca2+-activated channels to smaller-conductance delayed rectifier channels, a lowered Ca2+ sensitivity, and a positive shift of the activation of some large-conductance Ca2+-activated channels.     

Regulation of mouse skeletal muscle L-type Ca2+ channel by activation of the insulin-like growth factor-1 receptor

We investigated the modulation of the skeletal muscle L-type Ca2+ channel/dihydropyridine receptor in response to insulin-like growth factor-1 receptor (IGF-1R) activation in single extensor digitorum longus muscle fibers from adult C57BL/6 mice. The L-type Ca2+ channel activity in its dual role as a voltage sensor and a selective Ca2+-conducting pore was recorded in voltage-clamp conditions. Peak Ca2+ current amplitude consistently increased after exposure to 20 ng/ml IGF-1 (EC50 = 5.6 +/- 1.8 nM). Peak IGF-1 effect on current amplitude at -20 mV was 210 +/- 18% of the control. Ca2+ current potentiation resulted from a shift in 13 mV of the Ca2+ current-voltage relationship toward more negative potentials. The IGF-1-induced facilitation of the Ca2+ current was not associated with an effect on charge movement amplitude and/or voltage distribution. These phenomena suggest that the L-type Ca2+ channel structures involved in voltage sensing are not involved in the response to the growth factor. The modulatory effect of IGF-1 on L-type Ca2+ channel was blocked by tyrosine kinase and PKC inhibitors, but not by a cAMP-dependent protein kinase inhibitor. IGF-1-dependent phosphorylation of the L-type Ca2+ channel alpha1 subunit was demonstrated by incorporation of [gamma-32P]ATP to monolayers of adult fast-twitch skeletal muscles. IGF-1 induced phosphorylation of a protein at the 165 kDa band, corresponding to the L-type Ca2+ channel alpha1 subunit. These results show that the activation of the IGF-1R facilitates skeletal muscle L-type Ca2+ channel activity via a PKC-dependent phosphorylation mechanism.     

Termination of Ca2+ release by a local inactivation of ryanodine receptors in cardiac myocytes

In heart, a robust regulatory mechanism is required to counteract the regenerative Ca2+-induced Ca2+ release from the sarcoplasmic reticulum. Several mechanisms, including inactivation, adaptation, and stochastic closing of ryanodine receptors (RyRs) have been proposed, but no conclusive evidence has yet been provided. We probed the termination process of Ca2+ release by using a technique of imaging local Ca2+ release, or "Ca2+ spikes", at subcellular sites; and we tracked the kinetics of Ca2+ release triggered by L-type Ca2+ channels. At 0 mV, Ca2+ release occurred and terminated within 40 ms after the onset of clamp pulses (0 mV). Increasing the open-duration and promoting the reopenings of Ca2+ channels with the Ca2+ channel agonist, FPL64176, did not prolong or trigger secondary Ca2+ spikes, even though two-thirds of the sarcoplasmic reticulum Ca2+ remained available for release. Latency of Ca2+ spikes coincided with the first openings but not with the reopenings of L-type Ca2+ channels. After an initial maximal release, even a multi-fold increase in unitary Ca2+ current induced by a hyperpolarization to -120 mV failed to trigger additional release, indicating absolute refractoriness of RyRs. When the release was submaximal (e.g., at +30 mV), tail currents did activate additional Ca2+ spikes; confocal images revealed that they originated from RyRs unfired during depolarization. These results indicate that Ca2+ release is terminated primarily by a highly localized, use-dependent inactivation of RyRs but not by the stochastic closing or adaptation of RyRs in intact ventricular myocytes.     

The possible function of stone ramparts at the nest entrance of the blackstart

The origin of Ibetanull, the Ca2+ current of myotubes from mice lacking the skeletal dihydropyridine receptor (DHPR) beta1a subunit, was investigated. The density of Ibetanull was similar to that of Idys, the Ca2+ current of myotubes from dysgenic mice lacking the skeletal DHPR alpha1S subunit (-0.6 +/- 0.1 and -0.7 +/- 0.1 pA/pF, respectively). However, Ibetanull activated at significantly more positive potentials. The midpoints of the GCa-V curves were 16.3 +/- 1.1 mV and 11.7 +/- 1.0 mV for Ibetanull and Idys, respectively. Ibetanull activated significantly more slowly than Idys. At +30 mV, the activation time constant for Ibetanull was 26 +/- 3 ms, and that for Idys was 7 +/- 1 ms. The unitary current of normal L-type and beta1-null Ca2+ channels estimated from the mean variance relationship at +20 mV in 10 mM external Ca2+ was 22 +/- 4 fA and 43 +/- 7 fA, respectively. Both values were significantly smaller than the single-channel current estimated for dysgenic Ca2+ channels, which was 84 +/- 9 fA under the same conditions. Ibetanull and Idys have different gating and permeation characteristics, suggesting that the bulk of the DHPR alpha1 subunits underlying these currents are different. Ibetanull is suggested to originate primarily from Ca2+ channels with a DHPR alpha1S subunit. Dysgenic Ca2+ channels may be a minor component of this current. The expression of DHPR alpha1S in beta1-null myotubes and its absence in dysgenic myotubes was confirmed by immunofluorescence labeling of cells.     

Calcium content of the sarcoplasmic reticulum in isolated ventricular myocytes from patients with terminal heart failure

Systolic [Ca2+]i-transients have been shown to be depressed in isolated ventricular myocytes from patients with terminal heart failure compared to controls. Experiments were performed in human ventricular cells to investigate whether this reduced systolic [Ca2+]i-transient may be due to a decreased Ca(2+)-content of the sarcoplasmic reticulum (SR). Single myocytes were isolated from left ventricular myocardium of patients with terminal heart failure undergoing cardiac transplantation. These results were compared to those obtained from cells of healthy donor hearts that were not suitable for transplantation for technical reasons. [Ca2+]i-transients were recorded from isolated cells under voltage clamp perfused internally with the Ca(2+)-indicator fura-2. The Ca(2+)-content of the SR was estimated by rapid extracellular application of caffeine (10 mM) to open the Ca(2+)-release channel of the SR and comparison of the caffeine-induced [Ca2+]i-transients in cells from patients with heart failure and from controls without heart failure. Upon steady-state depolarizations to +10 mV (maximum of the Ca(2+)-current), [Ca2+]i-transients in cells from patients with heart failure were significantly smaller than in myocytes from undiseased hearts (333 +/- 26 v 596 +/- 80 nM, P < 0.05). Application of caffeine caused a [Ca2+]i-transient that was always larger than during depolarization. Caffeine-induced [Ca2+]i-transients were significantly smaller in cells from diseased hearts compared with controls (970 +/- 129 v 2586 +/- 288 nM, P < 0.01). A positive correlation was found between left ventricular ejection fraction and caffeine-induced [Ca2+]i-transients in these cells. It is concluded, that depressed [Ca2+]i-transients in myocytes from patients with heart failure may be caused by a decreased Ca(2+)-content of the SR possibly due to an altered Ca(2+)-ATPase activity in these hearts. It is not necessary to postulate an additional defect of the Ca(2+)-release function of the SR to account for the alterations of intracellular (Ca2+]i-handling.     

Expression and activity of 3beta-hydroxysteroid dehydrogenase/delta5-delta4-isomerase in the rat Purkinje neuron during neonatal life

Diadenosine tetraphosphate (AP4A) is an endogenous compound and exerts diverse physiological effects in animal systems. However, the effects of AP4A on inotropy in ventricular cardiac preparations have not yet been studied. The effects of AP4A on force of contraction (FOC) were studied in isolated electrically driven guinea pig and human cardiac preparations. Furthermore, the effects of AP4A on L-type calcium current and [Ca]i were studied in isolated guinea pig ventricular myocytes. In guinea pig left atria, AP4A (0.1-100 microM) reduced FOC maximally by 36.5 +/- 4.3%. In guinea pig papillary muscles, AP4A (100 microM) alone was ineffective, but reduced isoproterenol-stimulated FOC maximally by 29.3 +/- 3.4%. The negative inotropic effects of AP4A in atria and papillary muscles were abolished by the A1-adenosine receptor antagonist 1, 3-dipropyl-cyclopentylxanthine. In guinea pig ventricular myocytes, AP4A (100 microM) attenuated isoproterenol-stimulated L-type calcium current and [Ca]i. In human atrial and ventricular preparations, AP4A (100 microM) alone increased FOC to 158.3 +/- 12.4% and 167.5 +/- 25.1%, respectively. These positive inotropic effects were abolished by the P2-purinoceptor antagonist suramin. On the other hand, AP4A (100 microM) reduced FOC by 27.2 +/- 7.4% in isoproterenol-stimulated human ventricular trabeculae. The latter effect was abolished by 1,3-dipropyl-cyclopentylxanthine. In summary, after beta adrenergic stimulation AP4A exerts negative inotropic effects in animal and human ventricular preparations via stimulation of A1-adenosine receptors. In contrast, AP4A alone can exert positive inotropic effects via P2-purinoceptors in human ventricular myocardium. Thus, P2-purinoceptor stimulation might be a new positive inotropic principle in the human myocardium.     

Contrasting biophysical and pharmacological properties of T-type and R-type calcium channels

In contrast to other kinds of voltage-gated Ca2+ channels, the underlying molecular basis of T-type and R-type channels is not well-understood. To facilitate comparisons with cloned Ca2+ channel subunits, we have carried out a systematic analysis of the properties of T-type currents in undifferentiated NG108-15 cells and R-type currents in cerebellar granule neurons. Marked differences were found in their biophysical and pharmacological features under identical recording conditions. T-type channels became activated at potentials approximately 25 mV more negative than R-type channels; however, T-type channels required potentials approximately 15 mV less negative than R-type channels to be available. Accordingly, T-type channels display a much larger overlap between the curves describing inactivation and activation, making them more suitable for generating sustained Ca2+ entry in support of secretion or pacemaker activity. In contrast, R-type channels are not equipped to provide a steady current, but are very capable of supplying transient surges of Ca2+ influx. In response to a series of increasingly strong depolarizations T-type and R-type Ca2+ channels gave rise to very different kinetic patterns. T-type current records crossed each other in a characteristic pattern not found for R-type currents. These biophysical distinctions were independent of absolute membrane potential and were, therefore, complementary to the conventional categorization of T- and R-type Ca2+ channels as low- and high-voltage activated. R-type channels deactivated approximately eight-fold more quickly than T-type channels, with clear consequences for the generation of divalent cation influx during simulated action potentials. Pharmacological comparisons revealed additional contrasts. R-type current was responsive to block by omega-Aga IIIA but not nimodipine, while the opposite was true for T-type current. Both channel types were potently inhibited by the non-dihydropyridine compound mibefradil. In all respects examined, R-type currents were similar to currents derived from expression of the alpha1E subunit whereas T-type currents were not.     

Developmental features of human striatal tissue transplanted in a rat model of Huntington''s disease

The actions of the novel calcium (Ca2+) channel antagonist mibefradil (Ro 40-5967), a selective T-type channel blocker in myocardium, were investigated in embryonic rat spinal motoneurones maintained in culture. Whole-cell currents were recorded with the patch-clamp technique. Motoneurones displayed transient, low-voltage-activated (LVA) and, more sustained, high-voltage-activated (HVA) Ca2+ currents. The LVA currents were small and preferentially blocked by amiloride and low doses of nickel. Most of the HVA Ca2+ current flowed through N-type Ca2+ channels, while L-, and P/Q-type channels represented a smaller fraction. Mibefradil caused a rapid and reversible dose-dependent block of inward Ca2+ channel currents. Inhibition was nearly complete at 10 microM, suggesting mibefradil blockade of all subclasses of Ca2+ channels. The IC50 was approximately 1.4 microM on currents measured at 0 mV, from a holding potential of -90 mV. Inhibition of LVA Ca2+ current occurred over the same contraction range. Slow tail currents induced by the dihydropyridine agonist Bay K 8644 were also blocked by mibefradil, although with a slightly lower potency (IC50 = 3.4 microM). These broad inhibitory effects of mibefradil on Ca2+ influx were also supported by the strong inhibition of depolarization-induced intracellular calcium transients, measured from Indo-1 loaded motoneurones imaged with confocal microscopy. We conclude that mibefradil has potent blocking effects on Ca2+ channels in mammalian motoneurones. We hypothesize that therapeutic and pharmacological effects of mibefradil may involve actions on Ca2+ channels other than type T.     

Characterization of the maize prolamin box-binding factor-1 (PBF-1) and its role in the developmental regulation of the zein multigene family

Voltage-dependent Ca2+ channels (VCCs) represent one of the main routes of Ca2+ entry into neuronal cells. Changes in intracellular Ca2+ dynamics and homeostasis can cause long-lasting cellular changes via activation of different Ca2+ dependent signalling pathways. We have investigated the properties of VCCs in human hippocampal dentate granule cells (DGCs) using the whole-cell configuration of the patch-clamp method. Classical high-threshold Ca2+ currents were composed mainly of omega-CgTx-sensitive N-type and nifedipine-sensitive L-type currents that were present in similar proportions. In addition, a Ca2+ current component that was sensitive to low concentrations of Ni2+, but not to nifedipine or omega-conotoxin GVIA (omega-CgTx GVIA) was present. This latter component showed a half-maximal inactivation at more hyperpolarized potentials than high-threshold currents and a more rapid time-dependent inactivation. This current was termed T-type Ca2+ current. Current components with similar pharmacological and kinetic characteristics could be elicited in acutely isolated control rat DGCs. The current density of high threshold and T-type Ca2+ components was significantly larger in human DGCs and in the kainate model compared to DGCs isolated from adult control rats. These differences in current density were not accompanied by parallel differences in the voltage-dependence of VCCs. Taken together, these data suggest that an up-regulation of Ca2+ current density may occur in hippocampal epileptogenesis without consistent changes in Ca2+ current properties.     

Mibefradil, A T-type calcium channel antagonist in Y1 cells

The effect of mibefradil, a new nondihydropyridine Ca2+ channel antagonist, was investigated on Y1 cells which exhibited T-and L-type Ca2+ currents. In the great majority of these cells, mibefradil rapidly and selectively blocked T-type Ca2+ current in a dose-dependent manner with a half maximum action at 10-7 M. Furthermore, the specific L-type Ca2+ channel inhibitor, nifedipine, blocked the Ca2+ inward current remaining after the action of mibefradil. Mibefradil does not modify the voltage-dependent characteristics of the current/voltage relationship. However, mibefradil is more effective at depolarized membrane potential.