Effect of metabolic inhibition on couplon behavior in rabbit ventricular myocytes

We investigated the effect of combined inhibition of oxidative and glycolytic metabolism on L-type Ca²⁺ channels (LCCs) and Ca²⁺ spikes in isolated patch-clamped rabbit ventricular myocytes. Metabolic inhibition (MI) reduced LCC open probability, increased null probability, increased first latency, and decreased open time but left conductance unchanged. These results explain the reduction in macroscopic Ca²⁺ current observed during MI. MI also produced a gradual reduction in action potential duration at 90% repolarization (APD₉₀), a clear decline in spike probability, and an increase in spike latency and variance. These effects are consistent with the changes we observed in LCC activity. MI had no effect on the amplitude or time to peak of Ca²⁺ spikes until APD₉₀ reached 10% of control, suggesting preserved sarcoplasmic reticulum Ca²⁺ stores and ryanodine receptor (RyR) conductance in those couplons that remained functioning. Ca²⁺ spikes disappeared completely when APD₉₀ reached <2% of control, although in two cells, spikes were reactivated in a highly synchronized fashion by very short action potentials. This reactivation is probably due to the increased driving force for Ca²⁺ entry through a reduced number of LCCs that remain open during early repolarization. The enlarged single channel flux produced by rapid repolarization is apparently sufficient to trigger RyRs whose Ca²⁺ sensitivity is likely reduced by MI. We suggest that loss of coupling fidelity during MI is explained by loss of LCC activity (possibly mediated by Ca²⁺-calmodulin kinase II activity). In addition, the results are consistent with loss of RyR activity, which can be mitigated under conditions likely to enlarge the trigger.     

Endothelin contraction in pig coronary: Receptor types and Ca2+-mobilization

Endothelin is one of the most potent vasoconstrictors known. It plays an important role in the regulation of vascular tone and in the development of many cardiovascular diseases. This study focuses on the receptor types and the Ca²⁺ mobilization responsible for endothelin-1 (ET-1) contraction in de-endothelialized pig coronary artery rings. ET-1 contracted the artery rings with an EC₅₀ = 6.5 ± 1 nM and a maximum contraction which was 98.6 ± 9% of the contraction produced by 60 mM KCl. BQ123 (5 µM), an ET_A antagonist, reversed 78 ± 3% of the ET-1 contraction (50 nM). IRL1620, a selective ET_B agonist, produced 23 ± 3% of the total ET-1 contraction with an EC₅₀ = 12.7 ± 2 nM. More than 85% of the contraction due to 100 nM IRL 1620 was inhibited by 200 nMBQ788, an ET_B antagonist. Therefore, approximately 80% of the ET-1 contraction in this artery occurred via ET_A receptors, and the other 20% was mediated by ET_B receptors. To assess the Ca²⁺ pools utilized during the ET-1 response, ET-1 contraction was also examined in medium containing an L-type Ca²⁺ channel blocker nitrendipine, and in Ca²⁺ free medium containing 0.2 mM EGTA. In Ca²⁺ containing medium the contraction elicited by ET-1 was 98.6 ± 9% of the KCl contraction, however, in the presence 10 µM nitrendipine the ET-1 induced contraction was 54 ± 7% of the KCl contraction, and in Ca²⁺-free medium it was 13 ± 2%. Similarly, the IRL 1620 contractions in Ca²⁺ containing medium, in the presence of nitrendipine and in Ca²⁺-free medium were 22.4 ± 3%, 12 ± 3% and 11 ± 2% of the KCl response respectively. Thus, both ET_A and ET_B contractions utilize extracellular Ca²⁺ pools via L-type Ca²⁺ channels and other undefined route(s), as well as intracellular Ca²⁺ pools. In the pig coronary artery smooth muscle, ET-1 contractions occur predominantly via ET_A receptors, with ET_B receptors using similar Ca²⁺ mobilization pathways, but the ET_B receptors appear to use the intracellular Ca²⁺ stores to a greater extent.     

Blockade of insulin sensitive steady-state R-type Ca2+ channel by PN 200-110 in heart and vascular smooth muscle

The effect of high K^– concentration, insulin and the L-type Ca^2– channel blocker PN 200-110 on cytosolic intracellular free calcium ([Ca²⁺]_i) was studied in single ventricular myocytes of 10-day-old embryonic chick heart, 20-week-old human fetus and rabbit aorta (VSM) single cells using the Ca²⁺-sensitive fluorescent dye, Fura-2 microfluorometry and digital imaging technique. Depolarization of the cell membrane of both heart and VSM cells with continuous superfusion of 30 mM [K⁺]_o induced a rapid transient increase of [Ca²⁺]_i that was followed by a sustained component. The early transient increase of [Ca²⁺]_i by high [⁺]_o was blocked by the L-type calcium channel antagonist nifedipine. However, the sustained component was found to be insensitive to this drug. PN 200-110 another L-type Ca²⁺ blocker was found to decrease both the early transient and the sustained increase of [Ca²⁺]_i induced by depolarization of the cell membrane with high [K⁺]_o. Insulin at a concentration of 40 to 80 U/ml only produced a sustained increase of [Ca²⁺]_i that was blocked by PN 200-110 or by lowering the extracellular Ca²⁺ concentration with EGTA. The sustained increase of [Ca²⁺], induced by high [K⁺]_o or insulin was insensitive to metabolic inhibitors such as KCN and ouabain as well to the fast Na⁺ channel blocker, tetrodotoxin and to the increase of intracellular concentrations of cyclic nucleotides. Using the patch clamp technique, insulin did not affect the L-type Ca²⁺ current and the delayed outward K⁺ current. These results suggest that the early increase of (Ca²⁺]_i during depolarization of the cell membrane of heart and VSM cells with high [K⁺]_o is due to the opening and decay of an L-type Ca ²⁺ channel. However, the sustained increase of [Ca²⁺]_i during a sustained depolarization is due to the activation of a resting (R) Ca ²⁺ channel that is insensitive to lowering [ATP]_i and sensitive to insulin.     

Physiologic gating properties of unitary cardiac L-type Ca channels

The contraction of adult mammalian ventricular cardiomyocytes is triggered by the influx of Ca²⁺ ions through sarcolemmal L-type Ca²⁺ channels (LCCs). However, the gating properties of unitary LCCs under physiologic conditions have remained elusive. Towards this end, we investigated the voltage-dependence of the gating kinetics of unitary LCCs, with a physiologic concentration of Ca²⁺ ions permeating the channel. Unitary LCC currents were recorded with 2 mM external Ca²⁺ ions (in the absence of LCC agonists), using cell-attached patches on K-depolarized adult rat ventricular myocytes. The voltage-dependence of the peak probability of channel opening (Po vs. Vm) displayed a maximum value of 0.3, a midpoint of −12 mV, and a slope factor of 8.5. The maximum value for Po of the unitary LCC was significantly higher than previously assumed, under physiologic conditions. We also found that the mean open dwell time of the unitary LCC increased twofold with depolarization, ranging from 0.53 ± 0.02 ms at −30 mV to 1.08 ± 0.03 ms at 0 mV. The increase in mean LCC open time with depolarization counterbalanced the decrease in the single LCC current amplitude; the latter due to the decrease in driving force for Ca²⁺ ion entry. Thus, the average amount of Ca²⁺ ions entering through an individual LCC opening (∼300-400 ions) remained relatively constant over this range of potentials. These novel results establish the voltage-dependence of unitary LCC gating kinetics using a physiologic Ca²⁺ ion concentration. Moreover, they provide insight into local Ca²⁺-induced Ca²⁺ release and a more accurate basis for mathematical modeling of excitation-contraction coupling in cardiac myocytes.     

Cardiac action potential duration and calcium regulation in males and females

Adult women have longer QT intervals compared with men of a similar age, indicating differences in the speed of repolarisation of the ventricles. We investigate the influences of gender on ventricular electrophysiology and intracellular Ca²⁺ regulation of the guinea pig heart. Comparing sexually mature animals, females exhibited a significantly longer APD. Peak L-type Ca²⁺ current (I_CaL) was larger in females and when this current was inhibited with nifedipine the gender differences in APD were removed. APD differences also disappeared when the SR was depleted of Ca²⁺. Inactivation of I_CaL during a clamp step is faster in females but slower during an action potential and SR Ca²⁺ content is larger. We suggest that gender differences in APD result from variation in the kinetics of I_CaL stemming from alterations to Ca²⁺ release.     

External bioenergy-induced increases in intracellular free calcium concentrations are mediated by Na+/Ca2+ exchanger and L-type calcium channel

External bioenergy (EBE, energy emitted from a human body) has been shown to increase intracellular calcium concentration ([Ca²⁺]_i, an important factor in signal transduction) and regulate the cellular response to heat stress in cultured human lymphoid Jurkat T cells. In this study, we wanted to elucidate the underlying mechanisms. A bioenergy specialist emitted bioenergy sequentially toward tubes of cultured Jurkat T cells for one 15-minute period in buffers containing different ion compositions or different concentrations of inhibitors. [Ca²⁺]_i was measured spectrofluorometrically using the fluorescent probe fura-2. The resting [Ca²⁺]_i in Jurkat T cells was 70 ± 3 nM (n = 130) in the normal buffer. Removal of external calcium decreased the resting [Ca²⁺]_i to 52 ± 2 nM (n = 23), indicating that [Ca²⁺] entry from the external source is important for maintaining the basal level of [Ca²⁺]_i. Treatment of Jurkat T cells with EBE for 15 min increased [Ca²⁺]_i by 30 ± 5% (P 0.05, Student t-test). The distance between the bioenergy specialist and Jurkat T cells and repetitive treatments of EBE did not attenuate [Ca²⁺]_i responsiveness to EBE. Removal of external Ca²⁺ or Na⁺, but not Mg²⁺, inhibited the EBE-induced increase in [Ca²⁺]_i. Dichlorobenzamil, an inhibitor of Na⁺/Ca²⁺ exchangers, also inhibited the EBE-induced increase in [Ca²⁺]_i in a concentration-dependent manner with an IC50 of 0.11 ± 0.02 nM. When external [K⁺] was increased from 4.5 mM to 25 mM, EBE decreased [Ca²⁺]_i. The EBE-induced increase was also blocked by verapamil, an L-type voltage-gated Ca²⁺ channel blocker. These results suggest that the EBE-induced [Ca²⁺]_i increase may serve as an objective means for assessing and validating bioenergy effects and those specialists claiming bioenergy capability. The increase in [Ca²⁺]_i is mediated by activation of Na⁺/Ca²⁺ exchangers and opening of L-type voltage-gated Ca²⁺ channels. (Mol Cell Biochem 271: 51–59, 2005)     

Neuroprotective effects of ginseng saponins against L-type Ca2+ channel-mediated cell death in rat cortical neurons

In the present study, we have examined any possible involvement of L-type Ca²⁺ channels in ginseng-mediated neuroprotective actions. Exposure to a 50 mM KCl (high-K) produced neuronal cell death, which was blocked by a selective L-type Ca²⁺ channel blocker in cultured cortical neurons. When cultured cells were co-treated with ginseng total saponin (GTS) and high-K, GTS reduced high-K-induced neuronal death. Using Ca²⁺ imaging techniques, we found that GTS inhibited high-K-mediated acute and long-term [Ca²⁺]_i changes. These GTS-mediated [Ca²⁺]_i changes were diminished by nifedipine. Furthermore, GTS-mediated effects were also diminished by a saturating concentration of Bay K (10 μM). After confirming the protective effect of GTS using a TUNEL assay, we found that ginsenosides Rf and Rg₃ are active components in ginseng-mediated neuroprotection. These results suggest that inhibition of L-type Ca²⁺ channels by ginseng could be one of the mechanisms for ginseng-mediated neuroprotection in cultured rat cortical neurons.     

Intracellular Ca2+ Inhibits Smooth Muscle L-Type Ca2+ Channels by Activation of Protein Phosphatase Type 2B and by Direct Interaction with the Channel

Modulation of L-type Ca²⁺ channels by tonic elevation of cytoplasmic Ca²⁺ was investigated in intact cells and inside-out patches from human umbilical vein smooth muscle. Ba²⁺ was used as charge carrier, and run down of Ca²⁺ channel activity in inside-out patches was prevented with calpastatin plus ATP. Increasing cytoplasmic Ca²⁺ in intact cells by elevation of extracellular Ca²⁺ in the presence of the ionophore A23187 inhibited the activity of L-type Ca²⁺ channels in cell-attached patches. Measurement of the actual level of intracellular free Ca²⁺ with fura-2 revealed a 50% inhibitory concentration (IC₅₀) of 260 nM and a Hill coefficient close to 4 for Ca²⁺- dependent inhibition. Ca²⁺-induced inhibition of Ca²⁺ channel activity in intact cells was due to a reduction of channel open probability and availability. Ca²⁺-induced inhibition was not affected by the protein kinase inhibitor H-7 (10 μM) or the cytoskeleton disruptive agent cytochalasin B (20 μM), but prevented by cyclosporin A (1 μg/ ml), an inhibitor of protein phosphatase 2B (calcineurin). Elevation of Ca²⁺ at the cytoplasmic side of inside-out patches inhibited Ca²⁺ channels with an IC₅₀ of 2 μM and a Hill coefficient close to unity. Direct Ca²⁺-dependent inhibition in cell-free patches was due to a reduction of open probability, whereas availability was barely affected. Application of purified protein phosphatase 2B (12 U/ml) to the cytoplasmic side of inside-out patches at a free Ca²⁺ concentration of 1 μM inhibited Ca²⁺ channel open probability and availability. Elevation of cytoplasmic Ca²⁺ in the presence of PP2B, suppressed channel activity in inside-out patches with an IC₅₀ of ∼380 nM and a Hill coefficient of ∼3; i.e., characteristics reminiscent of the Ca²⁺ sensitivity of Ca²⁺ channels in intact cells. Our results suggest that L-type Ca²⁺ channels of smooth muscle are controlled by two Ca²⁺-dependent negative feedback mechanisms. These mechanisms are based on (a) a protein phosphatase 2B-mediated dephosphorylation process, and (b) the interaction of intracellular Ca²⁺ with a single membrane-associated site that may reside on the channel protein itself.     

Response of the electrochromic dye,merocyanine 540, to membrane potential in rat liver mitochondria

Summary We have previously shown that pertussis toxin (PTX) stimulates delayed-onset, [Ca^2–] _a -dependent catecholamine (CA) release from bovine chromaffin cells. We now show that this effect of PTX is inhibited in part (50%) by dihydropyridine Ca^2–-channel antagonists niludipine and nifedipine, and is potentiated by the dihydropyridine Ca²⁺-channel agonist Bay K-8644. We and others have shown that pretreatment of chromaffin cells with PTX results in enhanced catecholamine secretion in response to high [K^–] _a , nicotine and muscarine, and here we extend these observations by showing that toxin pretreatment also enhances the secretory response to [Ba²⁺] _a . All these data are consistent with the concept that PTX may act on Ca^2– channels. To examine the possibility of a direct action of the toxin on the voltage-gated L-type Ca²⁺ channel known to be present in these cells, we studied the effects of the toxin on whole cell Ca²⁺ currents. We found and report here that spontaneous electrical activity was considerably increased in PTX-treated cells. Our measurements of whole cell inward Ca²⁺ currents indicate that the underlying mechanism is a marked shift of the activation curve of the L-type Ca²⁺ current along the voltage axis towards more negative potentials. While treatment of the cells with PTX had no effect on L-type Ca²⁺-channel conductance (6 nS/cell at 2.6mm [Ca²⁺] _a ). PTX evoked the activation of a new class of Ca²⁺-selective channels (5 pS in 25mm [Ca²⁺]_pipet), which are rather insensitive to membrane potential. We have termed theseG-type calcium channels. These data suggest that treatment with PTX not only increases the probability of L-type Ca²⁺-channel activation at more negative potentials, but also increases the probability of opening of an entirely new, voltage-independent, Ca²⁺ channel. These actions of PTX should promote Ca²⁺ entry and might explain the stimulation by the toxin of CA secretion from medullary chromaffin cells in culture.     

Increases of T-type Ca2+ current in heart cells of the cardiomyopathic hamster

In the present study, the whole-cell voltage clamp technique was used in order to record the T- and L-type Ca²⁺ currents in single heart cells of newborn and young normal and hereditary cardiomyopathic hamsters. Our results showed that the I/V relationship curve as well as the kinetics of the L-type Ca²⁺ currents (I_Ca(L)) in both normal and cardiomyopathic heart cells were the same. However, the proportion of myocytes from normal heart hamster that showed L-type I_Ca was less than that of heart cells from cardiomyopathic hamster. The I/V relationship curve of the T-type I_Ca (I_Ca(T)) was the same in myocytes of both normal and cardiomyopathic hamsters. The main differences between I_Ca(T) of cardiomyopathic and normal hamster are a larger window current and the proportion of ventricular myocytes that showed this type of current in cardiomyopathic hamster. The high density of I_Ca(T) as well as the large window current and proportion of myocytes showing I_Ca(T) may explain in part Ca²⁺ overload observed in cardiomyopathic heart cells of the hamster.     

Cardiodepressive effect elicited by the essential oil of Alpinia speciosa is related to L-type Ca current blockade

This study was undertaken to elucidate the effect of the essential oil from Alpinia speciosa (EOAs) on cardiac contractility and the underlying mechanisms. The essential oil was obtained from Alpinia speciosa leaves and flowers and the oil was analyzed by GC-MS method. Chemical analysis revealed the presence of at least 18 components. Terpinen-4-ol and 1,8-cineole corresponded to 38% and 18% of the crude oil, respectively. The experiments were conducted on spontaneously-beating right atria and on electrically stimulated left atria isolated from adult rats. The effect of EOAs on the isometric contractions and cardiac frequency in vitro was examined. EOAs decreased rat left atrial force of contraction with an EC₅₀ of 292.2 ± 75.7 μg/ml. Nifedipine, a well known L-type Ca²⁺ blocker, inhibited in a concentration-dependent manner left atrial force of contraction with an EC₅₀ of 12.1 ± 3.5 μg/ml. Sinus rhythm was diminished by EOAs with an EC₅₀ of 595.4 ± 56.2 μg/ml. Whole-cell L-type Ca²⁺ currents were recorded by using the patch-clamp technique. EOAs at 25 μg/ml decreased I_Ca,L by 32.6 ± 9.2% and at 250 μg/ml it decreased by 89.3 ± 7.4%. Thus, inhibition of L-type Ca²⁺ channels is involved in the cardiodepressive effect elicited by the essential oil of Alpinia speciosa in rat heart.     

Alteration of channel characteristics by exchange of pore-forming regions between two structurally related Ca2+ Channels

Several types of structurally homologous high voltage-gated Ca²⁺ channels (L-, P-and N-type) have been identified via biochemical, pharmacological and electrophysiological techniques. Among these channels, the cardiac L-type and the brain BI-2 Ca²⁺ channel display significantly different biophysical properties. The BI-2 channel exhibits more rapid voltage-dependent current activation and inactivation and smaller single-channel conductance compared to the L-type Ca²⁺ channel. To examine the molecular basis for the functional differences between the two structurally related Ca²⁺ channels, we measured macroscopic and single-channel currents from oocytes injected with wild-type and various chimeric channel ₁ subunit cRNAs. The results show that a chimeric channel in which the segment between S5-SS2 in repeat IV of the cardiac L-type Ca²⁺ channel, was replaced by the corresponding region of the BI-2 channel, exhibited macroscopic current activation and inactivation time-courses and single-channel conductance, characteristic of the BI-2 Ca²⁺ channel. The voltage-dependence of steady-state inactivation was not affected by the replacement. Chimeras, in which the SS2-S6 segment in repeat III or IV of the cardiac channel was replaced by the corresponding BI-2 sequence, exhibited altered macroscopic current kinetics without changes in single-channel conductance. These results suggest that part of the S5-SS2 segment plays a critical role in determining voltage-dependent current activation and inactivation and single-channel conductance and that the SS2-S6 segment may control voltage-dependent kinetics of the Ca²⁺ channel.     

Effects of sustained low-flow ischemia and reperfusion on Ca2+ transients and contractility in perfused rat hearts

We investigated changes in cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) and in left ventricular contractility during sustained ischemia and reperfusion in isolated beating rat hearts. Hearts from male Sprague-Dawley rats were perfused retrogradely and were loaded with 4 M fura-2. Low-flow global ischemia was induced by reducing perfusion flow to 10% and by electric pacing. The hearts were exposed to ischemia for 10 min or 30 min and then reperfused. [Ca²⁺]_i was measured by monitoring the ratio of 500 nm fluorescence excited at 340 and 380 nm while simultaneously measuring left ventricular pressure (LVP). To determine diastolic [Ca²⁺]_i, background autofluorescence was subtracted. LVP rapidly decreased from 82.3 ± 8.2 to 17.1 ± 2.9 mmHg , whereas the amplitude of the Ca²⁺ transient did not change significantly during the first 1 min of ischemia. After 10 min of ischemia, the amplitude decreased to 60.8 ± 10.6% (p < 0.05) and diastolic [Ca²⁺]_i increased by 26.3 ± 2.9% (p < 0.001) compared with the pre-ischemic value (n = 8). When the hearts were reperfused after 10 min of ischemia, the amplitude of the Ca²⁺ transient and LVP recovered to 79.0 ± 7.2% and 73.2 ± 7.5 mmHg, respectively. Whereas diastolic [Ca²⁺]_i decreased to the pre-ischemic value. In the hearts exposed to 30 min of ischemia (n = 10), diastolic [Ca²⁺]_i increased even further by 32.7 ± 5.3% at the end of ischemia and continued increasing during the 10 min of reperfusion by 42.6 ± 15.6%. Six of 10 hearts developed ventricular fibrillation (VF) and intracellular Ca²⁺ overload after reperfusion. Recovery of LVP after reperfusion was significantly smaller in the hearts exposed to 30 min of ischemia than in the hearts exposed to 10 min of ischemia (58.9 ± 11.7 vs. 97.2 ± 3.0% of pre-ischemic value, p < 0.05). Diastolic [Ca²⁺]_i also increased under hypoxic conditions (N₂ bubbling) in this model. These results suggest that increases in diastolic [Ca²⁺]_i might play an important role in myocardial contractile dysfunction and viability in ischemia-reperfusion injury.     

Contribution of spontaneous L-type Ca2+ channel activation to the genesis of Ca2+ sparks in resting cardiac myocytes

Ca²⁺ sparks are the elementary events of intracellular Ca²⁺ release from the sarcoplasmic reticulum in cardiac myocytes. In order to investigate whether spontaneous L-type Ca²⁺ channel activation contributes to the genesis of spontaneous Ca²⁺ sparks, we used confocal laser scanning microscopy and fluo-4 to visualize local Ca²⁺ sparks in intact rat ventricular myocytes. In the presence of 0.2 mmol/L CdCI₂ which inhibits spontaneous L-type Ca²⁺ channel activation, the rate of occurrence of spontaneous Ca²⁺ sparks was halved from 4.20 to 2.04 events/(100 μm · s), with temporal and spatial properties of individual Ca²⁺ sparks unchanged. Analysis of the Cd²⁺-sensitive spark production revealed an open probability of ~10 ^-5 for L-type channels at the rest membrane potentials (-80 mV). Thus, infrequent and stochastic openings of sarcolemmal L-type Ca²⁺ channels in resting heart cells contribute significantly to the production of spontaneous Ca²⁺ sparks.     

Mechanism of activation of K++ channels by minoxidil-sulfate in Madin-Darby canine kidney cells

Summary We studied the mechanism of K⁺⁺ channel activation by minoxidil-sulfate (MxSO₄) in fused Madin-Darby canine kidney (MDCK) cells. Patch-clamp techniques were used to assess single channel activity, and fluorescent dye techniques to monitor cell calcium. A Ca⁺²⁺-dependent inward-rectifying K⁺⁺ channel with slope conductances of 53±3 (negative potential range) and 20±3 pS (positive potential range) was identified. Channel activity is minimal in cell-attached patches. MxSO₄ initiated both transient channel activation and an increase of intracellular Ca⁺²⁺ (from 94.2±9.1 to 475±12.6 nmol/liter). The observation that K⁺⁺ channel activity of excised inside-out patches was detected only at Ca⁺²⁺ concentrations in excess of 10 mol/liter suggests the involvement of additional mechanisms during channel activation by MxSO₄.Transient K⁺⁺ channel activity was also induced in cell-attached patches by 10 mol/liter of the protein kinase C activator 1-oleoyl-2-acetyl-glycerol (OAG). OAG (10 mol/liter in the presence of 1.6 mmol/liter ATP) increased the Ca⁺² sensitivity of the K⁺ channel in inside-out patches significantly by lowering the K _mfor Ca⁺² from 100 mol/liter to 100 nmol/liter. The channel activation by OAG was reversed by the protein kinase inhibitor H8. Staurosporine, a PKC inhibitor, blocked the effect of MxSO₄ on K⁺ channel activation. We conclude that MxSO₄-induced K⁺ channel activity is mediated by the synergistic effects of an increase in intracellular Ca⁺² and a PKC-mediated enhancement of the K⁺ channel's sensitivity to Ca⁺².A. Schwab was recipient of a Feodor-Lynen-Fellowship from the Alexander von Humboldt-Stiftung. This work was supported by NIH grant DK 17433. The authors thank Nikon Instruments Partners in Research Program for their support and generous use of equipment during the course of this study. Minoxidil-sulfate was kindly provided by Upjohn, Kalamazoo, MI.     

Ultraviolet Photoalteration of Late Na+ Current in Guinea-pig Ventricular Myocytes

UV irradiation has multiple effects on mammalian cells, including modification of ion channel function. The present study was undertaken to investigate the response of membrane currents in guinea-pig ventricular myocytes to the type A (355, 380 nm) irradiation commonly used in Ca²⁺ imaging studies. Myocytes configured for whole-cell voltage clamp were generally held at −80 mV, dialyzed with K⁺-, Na⁺-free pipette solution, and bathed with K⁺-free Tyrode’s solution at 22°C. During experiments that lasted for ≈ 35 min, UVA irradiation caused a progressive increase in slowly-inactivating inward current elicited by 200-ms depolarizations from −80 to −40 mV, but had little effect on background current or on L-type Ca²⁺ current. Trials with depolarized holding potential, Ca²⁺ channel blockers, and tetrodotoxin (TTX) established that the current induced by irradiation was late (slowly-inactivating) Na⁺ current (I_Na). The amplitude of the late inward current sensitive to 100 μM TTX was increased by 3.5-fold after 20–30 min of irradiation. UVA modulation of late I_Na may (i) interfere with imaging studies, and (ii) provide a paradigm for investigation of intracellular factors likely to influence slow inactivation of cardiac I_Na.     

Dietary and physiological studies to investigate the relationship between calcium and magnesium signalling in the mammalian myocardium

This study employs both dietary and physiological studies to investigate the relationship between calcium (Ca²⁺) and magnesium (Mg²⁺) signalling in the mammalian myocardium. Rats maintained on a low Mg²⁺ diet (LMD; 39 mg Kg^-1 Mg²⁺ in food) consumed less food and grew more slowly than control rats fed on a control Mg²⁺ diet (CMD; 500 mg Kg^-1 Mg²⁺ in food). The Mg²⁺ contents of the heart and plasma were 85 ± 3% and 34 ± 6.5%, respectively relative to the control group. In contrast, Ca²⁺ contents in the heart and plasma were 177 ± 5% and 95 ± 3%. The levels of potassium (K⁺) was raised in the plasma (129 ± 16%) and slightly decreased in the heart (88 ± 6%) compared to CMD. Similarly, sodium (Na⁺) contents were slightly higher in the heart and lowered in the plasma of low Mg²⁺ diet rats compared to control Mg²⁺ diet rat. Perfusion of the isolated Langendorff's rat heart with a physiological salt solution containing low concentrations (0-0.6 mM) of extracellular magnesium [Mg²⁺]₀ resulted in a small transient increase in the amplitude of contraction compared to control [Mg²⁺]₀ (1.2 mM). In contrast, elevated [Mg²⁺]₀ (2-7.2 mM) caused a marked and progressive decrease in contractile force compared to control. In isolated ventricular myocytes the L-type Ca²⁺ current (I_Ca,L was significantly (p < 0.001) attenuated in cells dialysed with 7.1 mM Mg²⁺ compared to cells dialysed with 2.9 µM Mg²⁺. The results indicate that hypomagnesemia is associated with decrease levels of Mg²⁺ and elevated levels of Ca²⁺ in the heart and moreover, internal Mg²⁺ is able to modulate the Ca²⁺ current through the L-type Ca²⁺ channel which in turn may be involved with the regulation of contractile force in the heart.     

Non-Markovian Gating of Ca2+-Activated K+ Channels in Cultured Kidney Cells Vero. Rescaled Range Analysis

Using the patch-voltage clamp technique and the rescaled range method, activity of single large conductance Ca²⁺-activated K⁺ channels (K_Ca channels) was studied. For the sequences of alternating open and shut time intervals, the dependence R/S vs. N in the double logarithmic coordinates presented a curve with two slopes, H₁ =0.60 ± 0.04, and H₂ = 0.88 ± 0.21, where H₁ and H₂ characterized the Hurst exponents for shot and long time ranges, respectively. Similar results were obtained for reduced data sets consisting of only open or only shut intervals. Randomization of the experimental data resulted in a single slope, H, of 0.52 ± 0.02. Simulations were performed with eight-state Markovian model without memory. The calculated Hurst exponent presented in average 0.54 ± 0.02. The results suggest that the activity of single Ca²⁺-activated K⁺ channel exhibits two regimes, with slight positive correlation at short time ranges (H₁ =0.6), and strong positive correlation at long time ranges (H₂ = 0.88); therefore the channel gating as a whole is not a steady-state Markovian process.     

Endothelin-1 induces intracellular [Ca2+] increase via Ca2+ influx through the L-type Ca2+ channel, Ca2+-induced Ca2+ release and a pathway involving ETA receptors, PKC, PKA and AT1 receptors in cardiomyocytes

Using fura-2-acetoxymethyl ester (AM) fluorescence imaging and patch clamp techniques, we found that endothelin-1 (ET-1) significantly elevated the intracellular calcium level ([Ca²⁺]_i) in a dose-dependent manner and activated the L-type Ca²⁺ channel in cardiomyocytes isolated from rats. The effect of ET-1 on [Ca²⁺]_i elevation was abolished in the presence of the ET_A receptor blocker BQ123, but was not affected by the ET_B receptor blocker BQ788. ET-1-induced an increase in [Ca²⁺]_i, which was inhibited 46.7% by pretreatment with a high concentration of ryanodine (10 μmol/L), a blocker of the ryanodine receptor. The ET-1-induced [Ca²⁺]_i increase was also inhibited by the inhibitors of protein kinase A (PKA), protein kinase C (PKC) and angiotensin type 1 receptor (AT1 receptor). We found that ET-1 induced an enhancement of the amplitude of the whole cell L-type Ca²⁺ channel current and an increase of open-state probability (NPo) of an L-type single Ca²⁺ channel. BQ123 completely blocked the ET-1-induced increase in calcium channel open-state probability. In this study we demonstrated that ET-1 regulates calcium overload through a series of mechanisms that include L-type Ca²⁺ channel activation and Ca²⁺-induced Ca²⁺ release (CICR). ETA receptors, PKC, PKA and AT1 receptors may also contribute to this pathway. Supported by the National Natural Science Foundation of China (Grant No. 200830870910).     

Muscarinic activation causes biphasic inotropic response and decreases cellular Na+ activity in canine cardiac purkinje fibers

In this study, the effects of carbachol (CCh) on twitch tension, intracellular Na⁺ activity (a _Na ⁱ ), and action potential were simultaneously measured in canine cardiac Purkinje fibers in order to examine the regulation of inotropy through muscarinic receptors and its relation to a _Na ⁱ . In fibers driven at 1 Hz, CCh (10 µM) initially and transiently decreased and then increased the twitch tension by 36±8%. The action potential showed a significant elevation of the plateau and a significant shortening of the duration at 90% repolarization (APD₉₀), from 403±7 to 389±7 ms. The a _Na ⁱ decreased from 7.4±0.4 to 6.7±0.3 mM (n=23, p<0.05). Atropine (1 µM) decreased the twitch tension by 21±6% (n=7, p<0.05) without significant effects on the action potential and a _Na ⁱ , and inhibited the effects of CCh. Cs⁺ (20 mM) increased the plateau height and APD₉₀, enhanced the twitch tension by 66±24%, but decreased a _Na ⁱ from 7.3±0.3 to 6.3±0.4 mM (n=6, p<0.05). In the presence of 20 mM Cs⁺, some fibers generated slow responses. The addition of 10 µM CCh further increased the twitch tension and APD₉₀, and decreased a _Na ⁱ from 6.3±0.4 to 5.3±0.3 mM. Ouabain (0.3 µM) increased the twitch tension and a _Na ⁱ , and inhibited the CCh-induced decrease of a _Na ⁱ . In the presence of ouabain, 20 mM Cs⁺ depolarized the fiber and generated slow responses with a decreased a _Na ⁱ . The addition of 10 µM CCh enhanced the slow action potential, and increased a _Na ⁱ although there was a transient decrease during early exposure. These results suggest that activation of muscarinic receptors in canine Purkinje fibers results in an enhancement of the Na⁺-K⁺ pump activity and a biphasic inotropic response, probably via different receptor subtypes. The inhibitory effect, most likely through M₂ receptors, is associated with the activation of K⁺ channels. The stimulatory effect, on the other hand, is probably due to the action on the M₁ receptors, resulting in increases in Ca²⁺ currents.