Role of PKC in the effects of α1-adrenergic stimulation on Ca2+ transients, contraction and Ca2+ current in guinea-pig ventricular myocytes

 The effects of α₁-adrenoceptor stimulation on intracellular Ca²⁺ transients, contractility and L-type Ca²⁺ current (I _Ca,L) were studied in single cells isolated from ventricles of guinea-pig hearts. The aim of our study was to elucidate the mechanisms of the positive inotropic effect of α₁-adrenergic stimulation by focussing on the role of protein kinase C (PKC). Phenylephrine, an α₁-adrenergic agonist, at concentrations of 50–100 μM elicited a biphasic inotropic response: a transient negative inotropic response (22.9±6.0% of control) followed by a sustained positive inotropic response (61.0±8.4%, mean±SE, n=12). The Ca²⁺ transient decreased by 10.2±3.9% during the negative inotropic phase, while it increased by 67.7±10% (n=12) during the positive inotropic phase. These effects were inhibited by prazosin (1 μM), a α₁-adrenergic antagonist. Phenylephrine increased the I _Ca,L by 60.8±21% (n=5) during the positive inotropic phase. To determine whether activation of PKC is responsible for the increases in Ca²⁺ transients, contractile amplitude and I _Ca,L during α₁-adrenoceptor stimulation, we tested the effects of 4β-phorbol 12-myristate 13-acetate (PMA), a PKC activator, and of bisindolylmaleimide I (GF109203X) and staurosporine, both of which are PKC inhibitors. PMA mimicked phenylephrine’s effects on Ca²⁺ transients, contractile amplitude and I _Ca,L. PMA (100 nM) increased the Ca²⁺ transient, contractile amplitude and I _Ca,L by 131±17%, 137±25% (n=8), and 81.1±26% (n=5), respectively. Prior exposure to GF109203X (1 μM) or staurosporine (10 nM) prevented the phenylephrine-induced increases in Ca²⁺ transients, contractile amplitude and I _Ca,L. Our study suggests that during α₁-adrenoceptor stimulation increase in I _Ca,L via PKC causes an increase in Ca²⁺ transients and thereby in the contractile force of the ventricular myocytes. Received: 16 July 1998 / Received after revision and accepted: 20 October 1998     

Decrease in sodium–calcium exchange and calcium currents in diabetic rat ventricular myocytes

This study was designed in order to gain insight into possible changes in the inward sodium–calcium exchange current (I_Na–Ca) and the L -type calcium current (I_Ca), in ventricular myocytes isolated from streptozotocin-induced diabetic rats. Recordings were made using the nystatin-perforated patch technique which minimizes interference with the normal intracellular Ca²⁺ buffering mechanisms. The averaged I_Na–Ca current density elicited by Ca²⁺ current was smaller in diabetic than in normal myocytes at all potentials tested. I_Na–Ca activated by rapid application of caffeine was significantly reduced and the decay phase was prolonged. The density of I_Ca was also significantly reduced by diabetes in the range of test potentials between –10 and +50 mV. In addition, the fast time constant of I_Ca inactivation, which represents mainly the sarcoplasmic reticulum (SR) Ca²⁺ release-induced inactivation, was significantly higher in diabetic than in normal myocytes. The decrease in I_Ca, which is the main source of trigger Ca²⁺ for SR Ca²⁺ release, may explain the significantly lowered peak systolic [Ca²⁺]_i previously shown in diabetic myocytes. As activation of I_Ca is essential for subsequent stimulation of I_Na–Ca, reduced I_Ca may contribute to decreasing activation of the Na⁺–Ca²⁺ exchanger.     

Activation of Ca2+-sensitive Cl– current by reverse mode Na+/Ca2+ exchange in rabbit ventricular myocytes

 We investigated how Ca²⁺-sensitive transient outward current, I _to(Ca), is activated in rabbit ventricular myocytes in the presence of intracellular Na⁺ (Na⁺ _i) using the whole-cell patch-clamp technique at 36°C. In cells dialysed with Na⁺-free solutions,the application of nicardipine (5 μM) to block L-type Ca²⁺ current (I _Ca) completely inhibited I _to(Ca). In cells dialysed with a [Na⁺]_i≥5 mM, however, I _to(Ca) could be observed after blockade of I _Ca, indicating the activity of an I _Ca-independent component. The amplitude of I _Ca-independent I _to(Ca) increased with voltage in a [Na⁺]_i-dependent manner. The block of Ca²⁺ release from the sarcoplasmic reticulum by caffeine, ryanodine or thapsigargin blocked I _Ca-independent I _to(Ca). In Ca²⁺-free bath solution I _to(Ca) was completely abolished. The application of 2 mM Ni²⁺ or the newly synthesized compound KBR7943, a selective blocker of the reverse mode of Na⁺/Ca²⁺ exchange, or perfusion with pipette solution containing XIP (10 μM), a selective blocker of the exchanger, blocked I _Ca-independent I _to(Ca). From these results we conclude that, in the presence of Na⁺ _i, I _to(Ca) can be activated via Ca²⁺-induced Ca²⁺ release triggered by Na⁺/Ca²⁺ exchange operating in the reverse mode after blockade of I _Ca. Received: 20 January 1998 / Received after revision: 6 July 1998 / Accepted: 25 July 1998     

Ca2+ entry through cardiac L-type Ca2+ channels modulates β-adrenergic stimulation in mouse ventricular myocytes

 β-adrenergic receptor (β-AR) stimulation increases cardiac L-type Ca²⁺ channel (CaCh) currents via cAMP-dependent phosphorylation. We report here that the affinity and maximum response of CaCh to isoproterenol (Iso), in mouse ventricular myocytes were significantly higher when Ba²⁺ was used as the charge carrier (I _Ba) instead of Ca²⁺ (I _Ca). The EC₅₀ and maximum increase of peak currents were 43.7 ± 7.9 nM and 1.8 ± 0.1-fold for I _Ca and 23.3 ± 4.7 nM and 2.4 ± 0.1-fold for I _Ba. When cells were dialyzed with the faster Ca²⁺ chelator, BAPTA, both sensitivity and maximum response of I _Ca to Iso were significantly augmented compared to cells with EGTA (EC₅₀ of 23.1 ± 5.2 nM and maximal increase of 2.2 ± 0.1-fold). Response of I _Ca to forskolin was also significantly increased when cells were dialyzed with BAPTA or when currents were measured in Ba²⁺. In contrast, depletion of the sarcoplasmic reticulum (SR) Ca²⁺ stores by ryanodine did not alter sensitivity of I _Ca to Iso or forskolin. These results suggest that the Ca²⁺ entering through CaCh regulates cAMP-dependent phosphorylation, and such negative feedback may play a significant role in cellular Ca²⁺ homeostasis and contraction in cardiac cells during β-AR stimulation. Received: 10 December 1997 / Received after revision: 19 January 1998 / Accepted: 21 January 1998     

[Ca2+]i elevation evoked by Ca2+ readdition to the medium after agonist-induced Ca2+ release can involve both IP 3-, and ryanodine-sensitive Ca2+ release

 Sustained Ca²⁺ elevation (”Ca²⁺ response”), caused by subsequent readdition of Ca²⁺ to the medium after application of adenosine 5’-triphosphate (ATP, 15 μM) in a Ca²⁺-free medium, was studied using single bovine aortic endothelial (BAE) cells. In cells in which the resting intracellular Ca²⁺ concentration ([Ca²⁺]_i) was between about 50 and 110 nM, a massive Ca²⁺ response occurred and consisted of phasic and sustained components, whereas cells with a resting [Ca²⁺]_i of over 110 nM displayed small plateau-like Ca²⁺ responses. An increase of internal store depletion resulted in loss of the phasic component. When the store was partly depleted, the dependence of the Ca²⁺ response amplitude on resting [Ca²⁺]_i was biphasic over the range of 50 to 110 nM. The greatest degree of store depletion was associated with small monophasic Ca²⁺ responses, the amplitudes of which were almost constant and in the same range as resting [Ca²⁺]_i. Ni²⁺, known to partly block Ca²⁺ entry, caused no change in the half-decay time of [Ca²⁺]_i down to the level of the sustained phase [57 ± 4 s in control and 54 ± 3 s (n = 13) in the presence of 10 mM Ni²⁺] when added at the peak of the phasic component of the Ca²⁺ response. However, it lowered the sustained phase of the Ca²⁺ response by 42%. When applied at the start of the readdition of Ca²⁺, Ni²⁺ blocked the phasic component of the Ca²⁺ response, there being a threefold decrease in the initial rate of [Ca²⁺]_i rise. In cells with a resting [Ca²⁺]_i of 75–80 nM, pre-treatment with ryanodine (10 μM) did not affect the peak amplitude of the Ca²⁺ response, but it did increase the level of the sustained component. In some cells, ryanodine caused an oscillatory Ca²⁺ response. In conclusion, partial depletion of the inositol 1,4,5-trisphosphate-(IP ₃-) sensitive store by a submaximal concentration of agonist (in Ca²⁺-free medium) was followed, on readdition of Ca²⁺, by Ca²⁺ entry, which appeared to trigger IP ₃-sensitive Ca²⁺ release (IICR) which, in turn, initiated Ca²⁺-sensitive Ca²⁺ release (CICR), thus resulting in a massive elevation of [Ca²⁺]_i. Received: 3 July 1996 / Received after revision and accepted: 9 September 1996     

Accentuated antagonism by angiotensin II on guinea-pig cardiac L-type Ca-currents enhanced by β-adrenergic stimulation

 To examine mechanism(s) underlying the accentuated antagonism by angiotensin II (A-II) on twitch tension, we recorded L-type Ca²⁺ currents (I _Ca,L) using conventional patch-clamp techniques in single, guinea-pig, ventricular myocytes. I _Ca,L was recorded by a step-pulse protocol after eliminating K⁺ conductances (internal Cs⁺ plus tetraethylammonium chloride and K⁺-free extracellular solution). A-II (100 nM) did not affect basal I _Ca,L, but inhibited I _Ca,L that had been enhanced (approximately 200% of control) by (ISO, isoproterenol 100 nM). The inhibitory action of A-II was concentration dependent (concentration eliciting 50% inhibition 88±9 pM, n=41) and the ISO-enhanced component of I _Ca,L was completely blocked by A-II at concentrations above 10 nM. CV-11974 (500 nM), an A-II type-1 receptor (AT₁) antagonist, prevented the inhibitory action of A-II. Pre-incubation with pertussis toxin (PTX) abolished the inhibitory effect of A-II. A-II also inhibited the I _Ca,L enhanced by histamine (500 nM) and forskolin (1 μM), but failed to affect I _Ca,L enhanced by intracellular cyclic adenosine monophosphate (1 mM). The inhibitory action of A-II may therefore involve AT₁ receptors/PTX-sensitive, guanine nucleotide-binding (G) proteins (Gi)/adenylate cyclase and partially explains the A-II-dependent accentuated antagonism of inotropy.     

Voltage dependence of the Fura-2 transient in rabbit left atrial myocytes at 37°C

 We used the whole-cell patch-clamp technique and monitoring of Fura-2 fluorescence to investigate the voltage dependence of the L-type Ca current (I _Ca,L) and intracellular Ca (Ca_i) transient in rabbit atrial myocytes at 37°C. Imaging the atrial cell membrane with Di-4-ANNEPS showed (in contrast to ventricular cells) that atrial cells had very few transverse tubules. We measured I _Ca,L using a Cs-based internal dialysis solution to eliminate interfering K currents. The voltage dependence of peak I _Ca,L amplitude was bell-shaped: I _Ca,L was maximal at +10 mV and declined at more negative and positive potentials. For measuring the Fura-2 (Ca_i) transient, we used a K-based internal dialysis solution to preserve normal excitation–contraction coupling. Ryanodine (20 μM) plus thapsigargin (2 μM) (blockers of the sarcoplasmic reticulum, SR) abolished the phasic component of the Fura-2 transient (n = 5), demonstrating that the phasic Fura-2 transient provided an index of the magnitude of SR release. The Fura-2 transient also showed bell-shaped voltage dependence, but this was different from that for I _Ca,L. The Fura-2 transient peaked at +30 mV and partially declined at more positive potentials; but at potentials where inward I _Ca,L was small (if not absent), the phasic Fura-2 transient still attained a significant amplitude. We used a rapid application of nifedipine (32 μM), and of nifedipine plus 5 mM Ni, to assess the ability of I _Ca,L and reverse-mode Na-Ca exchange to trigger SR Ca release. With test pulses to +10 mV and +60 mV, a rapid switch to nifedipine (which blocked I _Ca,L) produced no significant reduction in phasic Fura-2 transient amplitude. This suggests that in the absence of I _Ca,L, another mechanism was able to trigger SR release. With pulses to +10 and +60 mV, a single beat switch to nifedipine plus 5 mM Ni almost completely abolished the phasic transient. Since 5 mM Ni inhibits Na-Ca exchange, this suggests that, in the absence of I _Ca,L, trigger Ca entry via reverse Na-Ca exchange was able to activate SR Ca release in atrial cells at 37°C. The mechanisms underlying the Fura-2 transient in atrial cells, and differences with pre-existing data from rabbit ventricular cells, are discussed. Received: 24 September 1996 / Received after revision and accepted: 19 December 1996     

Effects of ruthenium red on membrane ionic currents in urinary bladder smooth muscle cells of the guinea-pig

 Three major ionic currents, Ca²⁺-dependent K⁺ current (I _K-Ca), delayed rectifier type K⁺ current (I _kd) and Ca²⁺ current (I _Ca), were activated by depolarization under whole-cell clamp in single smooth muscle cells isolated from guinea-pig urinary bladder. Externally applied ruthenium red (RuR) reduced the amplitude of I _K-Ca and I _Ca at 0 mV (IC₅₀ values were 4.2 and 5.6 μM, respectively), but did not affect I _Kd. Spontaneous transient outward currents (STOCs) and caffeine-induced outward currents (I _caf) at –30 mV were reduced by external 10 μM RuR. When 10 μM RuR was added to the pipette solution, I _K-Ca during depolarization, STOCs and I _caf significantly decreased with time. RuR did not change the unitary current amplitude of the large-conductance Ca²⁺-dependent K⁺ (BK) channels, but reduced the open probability of the channel under excised patch-clamp recording mode. RuR reduced the channel activity more effectively from the cytosolic face than from the other. This inhibition decreased when the cytosolic Ca²⁺ concentration was increased. These results indicate that RuR blocks BK and Ca²⁺ channels in urinary bladder smooth muscle cells. The decrease in I _K-Ca, STOCs and I _caf by RuR is attributable to the direct inhibition of BK channel activity, probably in addition to the inhibition of Ca²⁺ release from storage sites. The direct inhibition of BK channel activity by RuR may be related to the interaction of RuR with the Ca²⁺-binding sites of the channel protein. Received: 15 October 1997 / Received after revision and accepted: 25 November 1997     

A calcium-dependent chloride current in insulin-secreting βTC-3 cells

 Ca²⁺-dependent conductances have been hypothesized to play a role in the bursting pattern of electrical activity of insulin-secreting β cells in response to high plasma glucose. A Maxi K⁺ channel has received the most attention, while a low-conductance Ca²⁺-activated K⁺ current has also been identified. We used an increasingly popular β cell model system, the βTC-3 cell line, and the perforated-patch technique to describe the properties of a novel Ca²⁺-dependent Cl^–current [I _Cl(Ca)] in insulin-secreting pancreatic β cells. The reported I_Cl(Ca) could be activated under physiological Ca²⁺ concentrations and is the first of its kind to be described in pancreatic insulin-secreting cells. We found that long depolarizing steps above –20 mV elicited an outward current which showed slow inward relaxation upon repolarization to negative membrane potentials. Both the outward currents and the inward tails showed dependence on Ca²⁺ influx: their current/voltage (I/V) relations followed that of the ”L-like” Ca²⁺ current (I _Ca) present in these cells; they were blocked completely by the removal of external Ca²⁺ or application of Cd²⁺ at concentrations sufficient for complete block of I _Ca; and their magnitude increased with the depolarizing step duration. Moreover, the inward tail decayed monoexponentially with a time constant which at voltages negative to activation of I _Ca showed a weak linear voltage dependence, while at voltages positive to activation of I _Ca it followed the voltage dependence of I _Ca. This Ca²⁺-dependent current reversed at –21.5 mV and when the external Cl^–concentration was reduced from 159 mM to 62 mM the reversal potential shifted by ≈+20 mV as predicted by the Nernst relation for a Cl^–-selective current. Cl^–channel blockers such as DIDS (100 μM) and niflumic acid (100 μM) blocked this current. We concluded that this current was a Ca²⁺-dependent Cl^–current [I _Cl(Ca)]. From substitution of the external Cl^–with various monovalent anions and from the reversal potentials we obtained the following permeability sequence for I _Cl(Ca): I ^–>NO₃ ^–>Br^–>Cl^–>Acetate^–. Received: 10 October 1996 / Received after revision and accepted: 19 December 1996     

Estimation of myofibrillar responsiveness to Ca2+ in isolated rat ventricular myocytes

 To estimate myofibrillar responsiveness to Ca²⁺, we used the relation between cell length and intracellular [Ca²⁺] ([Ca²⁺]_i) during tetanic contractions of isolated ventricular myocytes. Enzymatically isolated rat ventricular myocytes were loaded with fura-2 AM (4 μM for 10 min) and excited alternately at 340 nm and 380 nm. The ratio (R) of fura-2 fluorescence at these wavelengths [F(340)/F(380), an index of [Ca²⁺]_i] and cell length (L) were measured simultaneously. Following treatment with thapsigargin (0.2 μM), myocytes were stimulated at 10 Hz for 10 s to produce a tetanic contraction every min and an instantaneous plot of R vs L (R-L trajectory) was constructed. The R-L trajectory followed the same path during cell shortening and re-lengthening, suggesting that dynamic equilibrium between R and L was achieved during tetanus. Increasing the extracellular [Ca²⁺] from 1 to 8 mM extended the R-L trajectory without a substantial shift of the relation. The Ca²⁺-sensitizing thiadiazinone derivative, EMD57033 (1 μM), shifted the R-L trajectory to the left (sensitization of the myofibrils to Ca²⁺), whereas the non-selective phosphodiesterase inhibitor, 3-isobutyl-1-methylxantine (200 μM), shifted the R-L trajectory to the right (desensitization of the myofibrils to Ca²⁺), in agreement with previous results obtained using skinned preparations. We conclude that the R-L trajectory is useful for estimating the myofibrillar responsiveness to Ca²⁺ in isolated myocytes and may be beneficial for the evaluation of inotropic agents. Received: 13 March 1998 / Received after revision: 4 May 1998 / Accepted: 2 June 1998     

Regulation of calcium release by calcium inside the sarcoplasmic reticulum in ventricular myocytes

 To study the effects of changes in sarcoplasmic reticulum (SR) intraluminal Ca²⁺ on the Ca²⁺ release mechanism, we correlated the activity of single cardiac ryanodine receptor (RyR) channels, monitored in planar bilayers, with the properties of spontaneous elementary Ca²⁺ release events (sparks) in intact ventricular myocytes, monitored by scanning confocal microfluorimetry. Under both normal conditions and Ca²⁺ overload, induced by elevation of extracellular [Ca²⁺], Ca²⁺ sparks represented single populations of events. During Ca²⁺ overload, the frequency of sparks increased from 0.8 to 3.1 events per second per 100 μm line scanned, and their amplitude increased from 100 nM to 400 nM. The duration of the Ca²⁺ sparks, however, was not altered. Changes in the properties of Ca²⁺ sparks were accompanied by only an ≈ 30% increase in the SR Ca²⁺ content, as determined by emptying the intracellular Ca²⁺ stores using caffeine. When single Ca²⁺ release channels were incorporated into lipid bilayers and activated by cytoplasmic Ca²⁺ (≈ 100 nM) and ATP (3 mM), elevation of Ca²⁺ on the luminal side from 20 μM to 0.2–20 mM resulted in a 1.2-fold to 7-fold increase, respectively, in open probability (P _o). This potentiation of P _o was due to an increase in mean open time and frequency of events. The relative effect of luminal Ca²⁺ was greater at low levels of cytoplasmic [Ca²⁺] than at high levels of cytoplasmic [Ca²⁺], and no effect of luminal Ca²⁺ was observed to occur in channels activated by 0.5–50 μM cytoplasmic Ca²⁺ in the absence of ATP. Our results suggest that SR Ca²⁺ release channels are modulated by SR intraluminal Ca²⁺. These alterations in properties of release channels may account for, or contribute to, the mechanism of spontaneous Ca²⁺ release in cardiac myocytes Received: 15 May 1996 / Received after revision: 5 June 1996 / Accepted: 8 July 1996     

Regulation of L- and N-types of Ca2+ channels by intracellular ATP4– in frog dorsal root ganglion neurons

The roles of free Mg²⁺ ions, ATP^4– ions and Mg-ATP complexes in the regulation of N- and L-types of Ca²⁺ channels were studied in frog dorsal root ganglion (DRG) neurons using the whole-cell patch-clamp technique. Because Mg²⁺ ions interact with ATP^4– ions to form Mg-ATP complexes, addition of one species can influence the concentrations of the other two. In this study their concentrations were carefully controlled by varying the concentrations of two constituents at a time while keeping the third constant. The effects of each of the three species on barium currents through L-type (I _BaL) and N-type (I _BaN) Ca²⁺ channels were plotted against its concentrations. The dose-response curves for ATP^4– show that I _BaL and I _BaN proportionally increased with ATP^4– concentrations up to 1 mM at three different Mg²⁺ concentrations. At a fixed concentration of ATP^4–, I _BaL and I _BaN remained unchanged even when pMg changed from 3 to 5. Dose-response curves for I _BaL and I _BaN plotted against Mg-ATP concentration did not show a consistent pattern. H-7 and Mg²⁺ ions did not exert any blocking effect on the activity of either Ca²⁺ channel type, and neither dibutyryl-cAMP nor NKH-477 had any stimulating effect, suggesting that phosphorylation is not likely to be involved in ATP-induced potentiation. From these observations, it is concluded that L-type and N-type Ca²⁺ channels in frog DRG neurons are regulated by ATP^4– ions alone, and that the neuronal Ca²⁺ channels are regulated by mechanisms that are different from those regulating the cardiac Ca²⁺ channels. Received: 30 October 1998 / Received after revision: 19 February 1999 / Accepted: 8 March 1999     

Modulation of Ca2+ channels, charge movement and Ca2+ transients by heparin in frog skeletal muscle fibres

Summary This study is an investigation into the modulatory effects of heparin, a component of the extracellular matrix that binds to dihydropyridine receptors, on contraction and Ca²⁺ channels in frog skeletal muscle. Using tension and Ca²⁺ signal measurements in single intact skeletal muscle cells we have found that heparin (100–200 g ml^-1) substantially potentiates twitch and tetanic tension (55% and 28%, respectively). In contrast, heparin reduces the amplitude of K⁺ contractures. Heparin most likely potentiates twitch tension by prolonging action potentials. The ionic basis of this effect was investigated in voltage-clamp experiments. Membrane currents were monitored in voltage-clamped segments of single fibres using the triple Vaseline gap technique. We found that heparin partially blocks delayed rectifier potassium channels. The depressive effects of heparin on K⁺ contractures prompted us to investigate the effects of heparin on charge movement and Ca²⁺ currents (I _Ca) under voltage-clamp. Charge movement was measured using a subtraction procedure that employed a -20 mV control pulse from a holding potential of 100 mV. Heparin depresses the total charge by 25%. We propose that the reduction in the amplitude of potassium contractures is related to a partial blockade of charge movement. Extracellular heparin shifts the I _Ca-V relation toward more negative voltages and delays the deactivation of tail currents. Double pulse experiments revealed that conditioning depolarizations speed the activation of I _Ca during test depolarizations. Heparin does not affect this process. The primary action of heparin is to accelerate the activation of I _Ca during pulses not preceded by conditioning depolarizations. Overall, the kinetic effects of heparin on I _Ca would increase the Ca²⁺ influx associated with action potentials. However, mechanical and optical experiments performed in Ca²⁺-free solutions and in the presence of Ca²⁺ channel blockers revealed that twitch and tetanic potentiation occur even in the absence of Ca²⁺-influx.     

The contribution of the sarcoplasmic reticulum Ca2+-transport ATPase to caffeine-induced Ca2+ transients of murine skinned skeletal muscle fibres

The present study was carried out to investigate the contribution of the Ca²⁺-transport ATPase of the sarcoplasmic reticulum (SR) to caffeine-induced Ca²⁺ release in skinned skeletal muscle fibres. Chemically skinned fibres of balb-C-mouse EDL (extensor digitorum longus) were exposed for 1 min to a free Ca²⁺ concentration of 0.36 μM to load the SR with Ca²⁺. Release of Ca²⁺ from the SR was induced by 30 mM caffeine and recorded as an isometric force transient. For every preparation a pCa/force relationship was constructed, where pCa = −log₁₀ [Ca²⁺]. In a new experimental approach, we used the pCa/force relationship to transform each force transient directly into a Ca²⁺ transient. The calculated Ca²⁺ transients were fitted by a double exponential function: Y ₀ + A ₁⋅exp (−t/t ₁) + A ₂⋅exp(t/t ₂), with A ₁ < 0 < A ₂, t ₁ < t ₂ and Y ₀, A ₁, A ₂ in micromolar. Ca²⁺ transients in the presence of the SR Ca²⁺-ATPase inhibitor cyclopiazonic acid (CPA) were compared to those obtained in the absence of the drug. We found that inhibition of the SR Ca²⁺-ATPase during caffeine-induced Ca²⁺ release causes an increase in the peak Ca²⁺ concentration in comparison to the control transients. Increasing CPA concentrations prolonged the time-to-peak in a dose-dependent manner, following a Hill curve with a half-maximal value of 6.5 ± 3 μM CPA and a Hill slope of 1.1 ± 0.2, saturating at 100 μM. The effects of CPA could be simulated by an extended three-compartment model representing the SR, the myofilament space and the external bathing solution. In terms of this model, the SR Ca²⁺-ATPase influences the Ca²⁺ gradient across the SR membrane in particular during the early stages of the Ca²⁺ transient, whereas the subsequent relaxation is governed by diffusional loss of Ca²⁺ into the bathing solution. Received: 2 February 1996/Accepted: 1 April 1996     

Effect of endurance training and acute exercise on sarcoplasmic reticulum function in rat fast- and slow-twitch skeletal muscles

Following 10 weeks of endurance training and in age-matched sedentary rats, sarcoplasmic reticulum (SR) Ca²⁺-uptake, Ca²⁺-release, and Ca²⁺-stimulated adenosinetriphosphatase (ATPase) activity were examined in homogenates of the plantaris and soleus muscles from rats subjected to moderate-intensity treadmill running to exhaustion. In order to examine the effects of acute exercise and/or training on SR Ca²⁺-handling capacity, comparisons between exhausted and non-exercised rats and between trained and untrained rats were performed. Our data confirm that Ca²⁺-sequestration by the SR from fast-twitch muscles is depressed after training. Immediately after exhaustive running, decreases in SR function occurred in both muscles, but were more pronounced in the soleus. In the plantaris, reductions in SR Ca²⁺-uptake rate and Ca²⁺-ATPase activity were observed in untrained rats only, while in the soleus they were adversely affected irrespective of training status. Although the average run time to exhaustion varied markedly between untrained and trained animals (untrained: 253.0 min; trained: 559.4 min), no differences existed with regard to the magnitude of decreases in SR function in the soleus after exercise. The mean rate of decline in SR Ca²⁺-handling capacity during acute exercise, as estimated from the run time and the extent of the decline, was more than twofold higher in untrained than in trained soleus. From the present study, it is unclear whether there exists a causal relationship between muscular fatigue and SR function because the run time to exhaustion was not significantly correlated with any of parameters indicative of SR Ca²⁺-handling capacity, but suggested that endurance training may be capable of delaying a progression of the deterioration in SR function that occurs during exercise. Electronic Publication     

Role of Ca2+‐activated Cl− current during proarrhythmic early afterdepolarizations in sheep and human ventricular myocytes

Objectives: The proarrhythmic early afterdepolarizations (EADs) during phase‐2 of the cardiac action potential (phase‐2 EADs) are associated with secondary Ca²⁺‐release of the sarcoplasmic reticulum. This makes it probable that the Ca²⁺‐activated Cl^? current [I_Cl(Ca)] is present during phase‐2 EADs. Activation of I_Cl(Ca) during phase‐2 of the action potential will result in an outwardly directed, repolarizing current and may thus be expected to prevent excessive depolarization of phase‐2 EADs. The present study was designed to test this hypothesis. Methods and Results: The contribution of I_Cl(Ca) during phase‐2 EADs was studied in enzymatically isolated sheep and human ventricular myocytes using the patch‐clamp methodology. EADs were induced by a combination of a low stimulus frequency (0.5 Hz) and exposure to 1 μm noradrenaline. In sheep myocytes, the I_Cl(Ca) blocker 4,4′‐diisothiocyanostilbene‐2,2′‐disulfonic acid (DIDS, 0.5 mm ) abolished phase‐1 repolarization of the action potential in all myocytes tested. This indicates that I_Cl(Ca) is present in all sheep myocytes. However, DIDS had no effect on phase‐2 EAD characteristics. In human myocytes, DIDS neither affected phase‐1 repolarization nor phase‐2 EAD characteristics. Conclusion: In sheep ventricular myocytes, but not in human ventricular myocytes, I_Cl(Ca) contributes to phase‐1 repolarization of the action potential. In both sheep and human myocytes, I_Cl(Ca) plays a limited role during phase‐2 EADs.     

Kinetic components of the gating currents of human cardiac L-type Ca2+ channels

 It has been reported previously that the β subunit increases both the ionic current and the gating charge movement of the human cardiac L-type Ca²⁺ channel α₁ subunit, and that steady-state measurements reveal the presence of two distinct components of the charge movement [Josephson IR, Varadi G (1996) Biophys J 70:1285–1293]. The present work identifies and characterizes the kinetic properties of the components of the human cardiac L-type Ca channel gating currents (I _g), and determines the relationship of these components to the activation of the Ca channel ionic current (I _Ca). Cloned human cardiac L-type α₁+α₂+β₃ subunits were transiently expressed in HEK293 cells and calcium channel gating currents were recorded following the addition of 5 mM Co²⁺. The steady-state charge integrals of the gating currents (Q _ON-V _m) were fit by a sum of two Boltzmann components: Q _ON1, which ranged over more negative potentials, and Q _ON2, which ranged over more positive potentials. The kinetic components of the ON and OFF gating currents were identified using bi-exponential curve fitting. Reconstruction of the two kinetic components of charge (Q _ONfast and Q _ONslow) yielded distributions that were similar in their voltage dependence and relative proportion to those measured directly by steady-state integration of Q _ON1 and Q _ON2. Changes in the initial conditions were found to affect Q _ON1 and Q _ON2 differently. The time constants of the ON gating current decays were similar to those of the activation of I _Ca. The results suggest that: (1) the activation of the human cardiac L-type Ca channel involves the movements of at least two, functionally distinct gating structures; (2) a fast charge movement (≈1/4 of the total charge; Q _ON1 or Q _ONfast) precedes a slower charge movement (≈3/4 of the total charge; Q _ON2 or Q _ONslow); and (3) channel opening is associated with the conformational change(s) producing Q _ONslow. Received: 7 June 1996 / Received after revision: 24 September 1996 / Accepted: 1 October 1996     

The cation selectivity of the sarcoball Ca2+ channel in frog muscle fibres

 We have measured single-channel currents from sarcoplasmic reticulum (SR) blebs (sarcoballs) of frog skeletal muscle fibres using conventional patch-clamp electrodes with excised patches. With both the pipette and bath solutions containing 50 mM Ca(gluconate)₂ the slope conductance of the single channels was 39.2 pS for the most commonly seen state, with a reversal potential of –0.4 mV. The cation selectivity of this channel was investigated by replacing the bathing solution with either gluconate or HEPES salts of selected cations. The Goldman permeability ratios, calculated from the reversal potentials, were found to be P(Ca²⁺)/P(K⁺)=2.4, P(Ca²⁺)/ P(Na⁺)=2.7, P(Ca²⁺)/P(Tris⁺)=3.1, P(Ca²⁺)/P(Mg²⁺)=1.0 and P(Ca²⁺)/P(Ba²⁺)=1.1. Each value for the monovalent ions was found to be less than the corresponding value reported for the SR ryanodine receptor channel from skeletal and cardiac muscle. Single-channel activity could be recorded when the preparation was bathed in symmetrical 50 mM Mg(gluconate)₂ solutions, and these channels had a similar conductance and open probability to that measured when the preparation was bathed in symmetrical Ca(gluconate)₂ solution. The channel activity in symmetrical 50 mM Ca(gluconate)₂ solution was insensitive to bath-applied caffeine (5 mM) and ryanodine (10 μM). The results are in agreement with the conclusion that the sarcoball Ca²⁺ channel is not the ryanodine receptor release channel, but possibly a form of the SR Ca²⁺-ATPase which is uncoupled from the catalytic events of the pump and acts as a passive ion channel. Received: 13 February 1998 / Received after revision: 6 April 1998 / Accepted: 7 April 1998     

Mechanisms underlying changes of tetanic [Ca2+]i and force in skeletal muscle

Force development in skeletal muscle is driven by an increase in myoplasmic free [Ca²⁺] ([Ca²⁺]_i) due to Ca²⁺ release from the sarcoplasmic reticulum (SR). The magnitude of [Ca²⁺]_i elevation during stimulation depends on: (a) the rate of Ca²⁺ release from the SR, (b) the rate of Ca²⁺ uptake by the SR, and (c) the myoplasmic Ca²⁺ buffering. We have used fluorescent Ca²⁺ indicators to measure [Ca²⁺]_i in intact, single fibres from mouse and Xenopus muscles under conditions where one or more of the above factors are changed. The following interventions resulted in increased tetanic [Ca²⁺]_i: β-adrenergic stimulation, which potentiates the SR Ca²⁺ release; application of 2,5-di(tert-butyl)-1,4-benzohydroquinone, which inhibits SR Ca²⁺ pumps; application of caffeine, which facilitates SR Ca²⁺ release and inhibits SR Ca²⁺ uptake; early fatigue, where the rate of SR Ca²⁺ uptake is reduced; acidosis, which reduces both the myoplasmic Ca²⁺ buffering and the rate of SR Ca²⁺ uptake. Reduced tetanic [Ca²⁺]_i was observed in late fatigue, due to reduced SR Ca²⁺ release, and in alkalosis, due to increased myoplasmic Ca²⁺ buffering. Force is monotonically related to [Ca²⁺]_i, but depends also on the myofibrillar Ca²⁺ sensitivity and the maximum force cross-bridges can produce. This is clearly illustrated by changes of intracellular pH where, despite a lower tetanic [Ca²⁺]_i, tetanic force is higher in alkalosis than acidosis due to increases of myofibrillar Ca²⁺ sensitivity and maximum cross-bridge force.     

The Fura-2 transient can show two types of voltage dependence at 36°C in ventricular myocytes isolated from the rat heart

We used the whole-cell patch-clamp method to investigate the voltage dependence of the L-type Ca current (I _Ca,L) and intracellular Ca (Ca_i) transient in ventricular myocytes isolated from the rat heart. Intracellular Ca was monitored using Fura-2 and the experiments were carried out at 36° C. We measured I _Ca,L by using a caesium-based internal dialysis solution to eliminate interfering K currents. The voltage dependence of peak I _Ca,L amplitude was bell-shaped: I _Ca,L was maximal at +10 mV and declined at more positive potentials. When I _Ca,L was integrated over the first 25 ms to estimate the magnitude of Ca entry, this had a very similar voltage dependence to peak I _Ca,L. In all cells, phasic Fura-2 transients were abolished by 5 μM ryanodine (a blocker of the sarcoplasmic reticulum, SR) showing that the Fura-2 transient provided an index of the magnitude of SR Ca release. For experiments measuring the Ca_i transient, we used a K-based internal dialysis solution to preserve normal excitation-contraction coupling. In 30–40% of cells, we found that the Fura-2 transient had a bell-shaped voltage dependence. This suggests that, in these cells, the primary trigger mechanism for Ca-induced Ca-release might have been Ca entry via I _Ca,L. In the remaining 60–70% of cells, the voltage dependence of the Fura-2 transient was not bell-shaped. The Fura-2 transient reached a maximum with a pulse to +10 mV, and the amplitude of the transient did not decline significantly at more positive potentials to this. In cells with a non-bell-shaped voltage dependence of the Fura-2 transient, pulses to potentials as far positive as +140 mV elicited phasic Fura-2 transients. Since this potential exceeded the Nernst potential for Ca, it was unlikely there was any tigger Ca entry via I _Ca,L at this potential. This would suggest that, in these cells, another trigger for SR Ca release (in addition to I _Ca,L) might be present. We conclude that rat ventricular myocytes, produced using a standard isolation technique and under standard recording conditions, can show either a bell-shaped or a sigmoidal voltage dependence of the Fura-2 transient. Received: 13 October 1995/Received after revision and accepted: 10 January 1996