Non-steady-state calcium handling in failing hearts from the spontaneously hypertensive rat

It is generally agreed that changes in Ca²⁺ cycling are often associated with heart failure, yet the impact of these changes on a beat-to-beat basis remains unclear. Measurements of isometric force and [Ca²⁺]_i were made at 37°C in left ventricular trabeculae from failing spontaneously hypertensive rat (SHR) hearts, and their normotensive Wistar–Kyoto (WKY) controls. At 1 Hz, peak stress was reduced in SHR (14.5?±?2.4 mN mm^?2 versus 22.5?±?6.7 mN mm^?2 for WKY), although the Ca²⁺ transients were bigger (peak [Ca²⁺]_i 0.60?±?0.08 μM versus 0.38?±?0.03 μM for WKY) with a slower decay of fluorescence (time constant 0.105?±?0.005 s versus 0.093?±?0.002 s for WKY). To probe dynamic Ca²⁺ cycling, two experimental protocols were used to potentiate force: (1) an interval of 30 s rest, and (2) a 30-s train of paired-pulses, and the recirculation fraction (RF) calculated for recovery to steady-state. No difference was found between rat strains for RF calculated from either peak force or Ca²⁺, although the RF was dependent on potentiation protocol. Since SR uptake is slower in SHR, the lack of change in RF must be due to a parallel decrease in trans-sarcolemmal Ca²⁺ extrusion. This view was supported by a slower decay of caffeine-induced Ca²⁺ transients in SHR trabeculae. Confocal analysis of LV free wall showed t-tubules were distorted in SHR myocytes, with reduced intensity of NCX and SERCA2a labelling in comparison to WKY.     

Effects of thapsigargin and cyclopiazonic acid on sarcoplasmic reticulum Ca2+ uptake,spontaneous force oscillations and myofilament Ca2+ sensitivity in skinned rat ventricular trabeculae

Thapsigargin (TG) and cyclopiazonic acid (CPA) have been reported to be potent inhibitors of the sarcoplasmic reticulum (SR) Ca²⁺ uptake in isolated SR vesicles and cells. We have examined the effect of TG and CPA on (1) the Ca²⁺ uptake by the SR in saponin-skinned rat ventricular trabeculae, using the amplitude of the caffeine-induced contraction to estimate the Ca²⁺ content loaded into the SR, (2) the spontaneous Ca²⁺ oscillations at pCa 6.6 using force oscillation as the indicator, and (3) the myofilament Ca²⁺ sensitivity in Triton X-100-treated preparations. Inhibition of Ca²⁺ loading by TG and CPA increased with time of exposure to the inhibitor over 18–24 min. TG and CPA produced half inhibition of Ca²⁺ loading at 34.9 and 35.7 μM respectively, when 18–24 min were allowed for diffusion. The spontaneous force oscillations were more sensitive to the inhibitors: 10 μM TG and 30 μM CPA both abolished the oscillations in this time. The myofilament Ca²⁺ sensitivity was not affected by 10 and 300 μM TG or CPA. The results show that the concentrations of TG and CPA necessary to inhibit the SR Ca²⁺ uptake of skinned ventricular trabeculae are much higher than the reported values for single intact myocytes. One reason for this may be slow diffusion of the inhibitors into the multicellular trabecula preparation. Received: 28 July 1995/Received after revision: 11 December 1995/Accepted: 18 December 1995     

The sarcolemmal mechanisms involved in the control of diastolic intracellular calcium in isolated rat cardiac trabeculae

 We performed experiments using the calcium indicator Indo-1 to determine the relative roles of the sarcolemmal mechanisms involved in the regulation of diastolic intracellular calcium concentration ([Ca²⁺]_i) in trabeculae from the rat heart. Ryanodine was used to eliminate sarcoplasmic reticulum (SR) function. In the functional absence of the SR, 76.8 ± 3.9% of the calcium was extruded by the Na-Ca exchange carrier in the [Ca²⁺]_i range of diastolic concentration ± 200–400 nM. This was assessed by measuring the recovery of [Ca²⁺]_i from small perturbations in the presence and absence of extracellular sodium. The steady-state relationship between [Ca²⁺]_o and [Ca²⁺]_i was linear over the range of 1–40 mM, a 20-fold increase of [Ca²⁺]_o produced a 1.97-fold ± 0.13-fold increase in [Ca²⁺]_i (n = 5). In the absence of extracellular sodium raising [Ca²⁺]_o had a variable effect. In some preparations there was little change of [Ca²⁺]_i while in others the response was almost as large as in control conditions. We conclude that the Na-Ca exchanger contributes ≈ 77% of sarcolemmal calcium extrusion following small perturbations in [Ca²⁺]_i and that this fraction does not diminish as the [Ca²⁺]_i declines. In addition we have shown a sodium-independent entry of calcium into quiescent cardiac muscle under resting conditions. Received: 16 May 1996 / Received after revision and accepted: 28 June 1996     

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     

Measurement of SR Ca2+ content in the presence of caffeine in permeabilised rat cardiac trabeculae

 This study was designed to measure the Ca²⁺ content of rat cardiac sarcoplasmic reticulum (SR) after equilibration with normal diastolic levels of Ca²⁺ (100 nM), in the absence and presence of caffeine. Measurements of [Ca²⁺] based on Fura-2 fluorescence were made from a limited bath volume (230 nl) containing individual saponin-permeabilised rat cardiac trabeculae. Injection of caffeine (5–40 mM) into this volume caused an initial release of Ca²⁺ from the SR, but within 30 s the SR was able to re-accumulate a significant proportion of the Ca²⁺. Ca²⁺ re-accumulation into the SR could be prevented by removal of ATP to inhibit the SR Ca²⁺ pump. Incubation of the preparation in an ATP-containing solution containing caffeine (5–40 mM) and 100 nM Ca²⁺ indicated that the SR’s ability to retain Ca²⁺ depends inversely on the dose of caffeine. The relative Ca²⁺ content of the SR after preincubation with caffeine was 86.7±3.5% at a caffeine concentration of 5 mM, 62.5±5.1% at 10 mM caffeine, 37.8±8.1% at 20 mM caffeine and 7.1±1.9% at 40 mM caffeine. Measurement of the SR Ca²⁺ release in the presence of different BAPTA concentrations was used to calculate (1) the Ca²⁺-binding capacity of the preparation (equivalent to 245±10 μM BAPTA) and (2) the Ca²⁺ content of the SR accessed by caffeine after equilibration with 100 nM Ca²⁺ (186±11 μmol/l cell volume or 5.6 mmol/l SR volume). Received: 9 June 1998 / Received after revision: 29 July 1998 / Accepted: 31 July 1998     

Mechanisms of reduced mitochondrial Ca2+ accumulation in failing hamster heart

Mitochondrial Ca²⁺ plays important roles in the regulation of energy metabolism and cellular Ca²⁺ homeostasis. In this study, we characterized mitochondrial Ca²⁺ accumulation in Syrian hamster hearts with hereditary cardiomyopathy (strain BIO 14.6). Exposure of isolated mitochondria from 70 nM to 30 μM Ca²⁺ ([Ca²⁺]_o) caused a concentration-dependent increase in intramitochondrial Ca²⁺ concentrations ([Ca²⁺]_m). The [Ca²⁺]_m was significantly lower in cardiomyopathic (CMP) hamsters than in healthy hamsters when [Ca²⁺]_o was higher than 1 μM and a decrease of about 52% was detected at [Ca²⁺]_o of 30 μM (916 ± 67 nM vs 1,932 ± 132 nM in control). A possible mechanism responsible for the decreased mitochondrial Ca²⁺ uptake in CMP hamsters is the depolarization of mitochondrial membrane potential (Δψ _m). Using a tetraphenylphosphonium (TPP⁺) electrode, the measured Δψ _m in failing heart mitochondria was −136 ± 1.5 mV compared with −159 ± 1.3 mV in controls. Analyses of mitochondrial respiratory chain demonstrated a significant impairment of complex I and complex IV activities in failing heart mitochondria. In summary, a less negative Δψ _m resulting from defects in the respiratory chain may lead to attenuated mitochondrial Ca²⁺ accumulation, which in turn may contribute to the depressed energy production and myocardial contractility in this model of heart failure. In addition to other known impairments of ion transport in sarcoplasmic reticulum and plasma membrane, results from this paper on mitochondrial dysfunctions expand our understanding of the molecular mechanisms leading to heart failure.     

Protective effect of oxymatrine on chronic rat heart failure

Oxymatrine is one of the alkaloids extracted from the Chinese herb Sophora japonica (Sophora flavescens Ait.) with anti-inflammatory, immune reaction inhibiting, antiviral, and hepatocyte and antihepatic fibrosis protective activities. However, the effect of oxymatrine on heart failure is not yet known. In this study, the effect of oxymatrine on heart failure was investigated using a Sprague-Dawley rat model of chronic heart failure. Morphological findings showed that in the group treated with 50 and 100 mg/kg of oxymatrine; intermyofibrillar lysis disappeared, myofilaments were orderly, closely and evenly arranged; and mitochondria contained tightly packed cristae compared with the heart failure group. We investigated the cytosolic Ca²⁺ transients and sarcoplasmic reticulum (SR) Ca²⁺ content, and assessed the expression of ryanodine receptor (RyR2), SR-Ca²⁺ ATPase (SERCA2a), and L-type Ca²⁺ channel (dihydropyridine receptor, DHPR). We found that the cytosolic Ca²⁺ transients were markedly increased in amplitude in the medium- (ΔF/F ₀ = 26.22 ± 2.01) and high-dose groups (ΔF/F ₀ = 29.49 ± 1.17) compared to the heart failure group (ΔF/F ₀ = 12.12 ± 1.35, P < 0.01), with changes paralleled by a significant increase in the SR Ca²⁺ content (medium-dose group: ΔF/F ₀ = 32.20 ± 1.67, high-dose group: ΔF/F ₀ = 32.57 ± 1.29, HF: ΔF/F ₀ = 17.26 ± 1.05, P < 0.01). Moreover, we demonstrated that the expression of SERCA2a and cardiac DHPR was significantly increased in the medium- and high-dose group compared with the heart failure rats. These findings suggest that oxymatrine could improve heart failure by improving the cardiac function and that this amelioration is associated with upregulation of SERCA2a and DHPR.     

Altered intracellular Ca<Superscript>2+</Superscript> regulation in chronic rat heart failure

Altered intracellular Ca²⁺ handling by the sarcoplasmic reticulum (SR) plays a crucial role in the pathogenesis of heart failure (HF). Despite extensive effort, the underlying causes of abnormal SR Ca²⁺ handling in HF have not been clarified. To determine whether the diastolic SR Ca²⁺ leak along with reduced Ca²⁺ reuptake is required for decreased contractility, we investigated the cytosolic Ca²⁺ transients and SR Ca²⁺ content and assessed the expression of ryanodine receptor (RyR2), FK506 binding protein (FKBP12.6), SR-Ca²⁺ ATPase (SERCA2a), and L-type Ca²⁺ channel (LTCC) using an SD-rat model of chronic HF. We found that the cytosolic Ca²⁺ transients were markedly reduced in amplitude in HF myocytes (ΔF/F ₀ = 12.3 ± 0.8) compared with control myocytes (ΔF/F ₀ = 17.7 ± 1.2, P < 0.01), changes paralleled by a significant reduction in the SR Ca²⁺ content (HF: ΔF/F ₀ = 12.4 ± 1.1, control: ΔF/F ₀ = 32.4 ± 1.9, P < 0.01). Moreover, we demonstrated that the expression of FKBP12.6 associated with RyR2, SERCA2a, and LTCC was significantly reduced in rat HF. These results provide evidence for phosphorylation-induced detachment of FKBP12.6 from RyRs and down-regulation of SERCA2a and LTCC in HF. We conclude that diastolic SR Ca²⁺ leak (due to dissociation of FKBP12.6 from RyR2) along with reduced SR Ca²⁺ uptake (due to down-regulation of SERCA2a) and defective E-C coupling (due to down-regulation of LTCC) could contribute to HF.     

Ca2+ uptake by cardiac sarcoplasmic reticulum ATPase in situ strongly depends on bound creatine kinase

The role of creatine kinase (CK) bound to sarcoplasmic reticulum (SR), in the energy supply of SR ATPase in situ, was studied in saponin-permeabilised rat ventricular fibres by loading SR at pCa 6.5 for different times and under different energy supply conditions. Release of Ca²⁺ was induced by 5 mM caffeine and the peak of relative tension (T/T _max) and the area under isometric tension curves, S _T, were measured. Taking advantage of close localisation of myofibrils and SR, free [Ca²⁺] in the fibres during the release was estimated using steady state [Ca²⁺]/tension relationship. Peak [Ca²⁺] and integral of free Ca²⁺ transients (S[Ca²⁺]_f) were then calculated. At all times, loading with 0.25 mM adenosine diphosphate, Mg²⁺ salt (MgADP) and 12 mM phosphocreatine (PCr) [when adenosine triphosphate (ATP) was generated via bound CK] was as efficient as loading with both 3.16 mM MgATP and 12 mM PCr (control conditions). However, when loading was supported by MgATP alone (3.16 mM), T/T _max was only 40% and S[Ca²⁺]_f 31% of control (P < 0.001). Under these conditions, addition of a soluble ATP-regenerating system (pyruvate kinase and phosphoenolpyruvate), did not increase loading substantially. Both S _T and S[Ca²⁺]_f were more sensitive to the loading conditions than T/T _max and peak [Ca²⁺]. The data suggest that Ca²⁺ uptake by the SR in situ depends on local ATP/ADP ratio which is effectively controlled by bound CK. Received: 23 January 1996/Received after revision: 19 April 1996/Accepted: 3 May 1996     

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     

Effect of caffeine on K+ efflux in frog skeletal muscle

 The exposure of frog skeletal muscle to caffeine (3–4 mM) generates an increase of the K⁺ (⁴²K⁺) efflux rate coefficient (k _K,o) which exhibits the following characteristics. First it is promoted by the rise in cytosolic Ca²⁺ ([Ca²⁺]_i), because the effect is mimicked by ionomycin (1.25 μM), a Ca²⁺ ionophore. Second, the inhibition of caffeine-induced Ca²⁺ release from the sarcoplasmic reticulum (SR) by 40 μM tetracaine significantly reduced the increase in k _K,o (Δk _K,o). Third, charybdotoxin (23 nM), a blocker of the large-conductance Ca²⁺-dependent K⁺ channels (BK_Ca channels) reduced Δk _K,o by 22%. Fourth, apamin (10 nM), a blocker of the small-conductance Ca²⁺-dependent K⁺ channels (SK_Ca channels), did not affect Δk _K,o. Fifth, tolbutamide (800 μM), an inhibitor of K_ATP channels, reduced Δk _K,o by about 23%. Sixth, Ba²⁺, a blocker of most K⁺ channels, did not preclude the caffeine-induced Δk _K,o. Seventh, omitting Na⁺ from the external medium reduced Δk _K,o by about 40%. Eight, amiloride (5 mM) decreased Δk _K,o by 65%. It is concluded that the caffeine-induced rise of [Ca²⁺]_i increases K⁺ efflux, through the activation of: (1) two channels (BK_Ca and K_ATP) and (2) an external Na⁺-dependent amiloride-sensitive process. Received: 13 March 1998 / Received after revision: 17 June 1998 / Accepted: 14 September 1998     

Ca2+ store determines gating of store operated calcium entry in mammalian skeletal muscle

This work describes the gating of the store operated calcium entry (SOCE) in adult mammalian skeletal muscle. Flexor digitorum brevis fibers (FDB) were isolated from adult mice and exposed to conditions to deplete the sarcoplasmic reticulum (SR). A transient SR depletion caused either by repetitive depolarizations, chlorocresol (CMC) or, cyclopiazonic acid (CPA) induced a bell shaped calcium entry that raised the [Ca²⁺]_i to a maximum of 27.09 ± 4.35 nM from the resting value. The activation time to reach 10–90% of the maximum amplitude was 112 ± 10 s (n = 22). On the other hand, any mechanism that caused a permanent SR depletion (like thapsigargin, continuous CPA, or continuous CMC) triggered a calcium entry pathway that lasted 325 ± 23 s and raised the [Ca²⁺]_i to 129.50 ± 13.05 nM from the resting level (n = 28). Then, a prolonged depletion triggered an increase in [Ca²⁺]_i to higher values and for a longer time than when the SR is transiently depleted (p < 0.001). Our results, in skeletal muscle, showed that calcium store depletion was the signal for SOCE activation and how the SR got depleted was not relevant. Also, we found that SOCE deactivation was not caused by [Ca²⁺]_i but by the SR content. Our results suggest that the SR calcium content plays an important role in SOCE gating in mammalian skeletal muscle and a calcium sensor is located inside the SR.     

Effects of cyclopiazonic acid on Ca2+ regulation by the sarcoplasmic reticulum in saponin-permeabilized skeletal muscle fibres

 The effects of the sarcoplasmic reticulum (SR) Ca²⁺ pump inhibitor cyclopiazonic acid (CPA) were studied in saponin-permeabilized frog skeletal muscle fibres. Release of Ca²⁺ from the SR was triggered by brief (2 s) applications of 40 mM caffeine at 2-min intervals. Changes in [Ca²⁺] within the fibre were monitored continuously using Fura-2 fluorescence. At a bathing [Ca²⁺] of 100 nM, introduction of 20 μM CPA induced a slow release of Ca²⁺ from the SR. The following one to two caffeine-induced Ca²⁺ transients were markedly increased in amplitude and duration. Thereafter, the caffeine-induced Ca²⁺ transients decreased progressively and were barely detectable 6–7 min after introduction of CPA. However, increasing the bathing [Ca²⁺] or increasing the Ca²⁺ loading period resulted in a partial recovery of the caffeine-induced Ca²⁺ transients, suggesting that pump inhibition is incomplete, even in the presence of 100 μM CPA. The slow Ca²⁺ efflux induced by CPA was insensitive to ryanodine, but absent following abolition of SR Ca²⁺ pump activity by ATP withdrawal. These results suggest that the caffeine-induced Ca²⁺ transient reflects a balance between efflux via the SR Ca²⁺ channel and reuptake by the Ca pump. Ca²⁺ release upon addition of CPA may result from inhibition of SR Ca²⁺ uptake, which reveals a tonic Ca²⁺ efflux that is independent of the Ca²⁺ release channels. Received: 26 November 1997 / Received after revision: 12 January 1998 / Accepted: 13 January 1998     

Involvement of the IP3 cascade in the damage to guinea-pig ventricular myocytes induced by cytotoxic T lymphocytes

 We have shown previously that the interaction between cytotoxic T lymphocytes (CTL) and ventricular myocytes, an in vitro model for heart transplant rejection, results in electrophysiological and morphological alterations indicative of overload of the intracellular [Ca²⁺] ([Ca²⁺]_i). Since these deleterious effects cannot be accounted for by increased L-type Ca²⁺ current (I _Ca,L), we hypothesize that [Ca²⁺]_i overload due to Ca²⁺ release from intracellular stores, e.g. sarcoplasmic reticulum (SR), is initiated by CTL-induced activation of the inositol trisphosphate (IP₃) cascade. Patch-clamp and fura-2-fluorescence techniques were utilized to record transmembrane potentials and [Ca²⁺]_i from ventricular myocytes bound to peritoneal exudate CTL (PEL). In ventricular myocyte-PEL conjugates (after 60 min), resting potential was reduced (compared with the nonconjugated state) from –80.9 ± 0.7 to –59.9 ± 2.5 mV, action potential amplitude from 139.5 ± 1.4 to 80.6 ± 1.7 mV and action potential duration to 50% repolarization (APD₅₀) from 797 ± 97 to 52 ± 12 ms. The ratio of fluorescence at 340 and 380 nm (R _340/380) increased from a control value (in nonconjugated myocytes) of 0.71 ± 0.02 to 2.07 ± 0.03, 30 min after conjugate formation, and exceeded 4.0 at 60 min, before myocyte destruction. Heparin (50 μg/ml), an antagonist of IP₃-induced Ca²⁺ release from SR channels, or U-73122 (2 μM), a phospholipase C (PLC) inhibitor (drugs were included in the pipette solution), prevented PEL-induced morphological and electrophysiological alterations. Accordingly, heparin attenuated the PEL-induced increase in [Ca²⁺]_i; after 60 min of PEL-myocyte interaction, R _340/380 was 1.15 ± 0.09 (compared with approximately 4.0 in the absence of heparin). The results indicate that CTL-mediated damage to ventricular myocytes is, at least partially, mediated by PLC activation and IP₃-induced Ca²⁺ release from intracellular stores. Pharmacological targeting of IP₃ in heart transplant rejection is thus suggested. Received: 3 July 1996 / Received after revision: 21 October 1996 / Accepted: 3 December 1996     

Presence of two Ca2+ influx components in internal Ca2+-pool-depleted rat parotid acinar cells

The molecular mechanism(s) involved in mediating Ca²⁺ entry into rat parotid acinar and other non-excitable cells is not known. In this study we have examined the kinetics of Ca²⁺ entry in fura-2-loaded parotid acinar cells, which were treated with thapsigargin to deplete internal Ca²⁺ pools (Ca²⁺-pool-depleted cells). The rate of Ca²⁺ entry was determined by measuring the initial increase in free cytosolic [Ca²⁺] ([Ca²⁺]_i) in Ca²⁺-pool-depleted, and control (untreated), cells upon addition of various [Ca²⁺] to the medium. In untreated cells, a low-affinity component was detected with K _Ca = 3.4 ± 0.7 mM (where K _Ca denotes affinity for Ca²⁺) and V _max = 9.8 ± 0.4 nM [Ca²⁺]_i/s. In thapsigargin-treated cells, two Ca²⁺ influx components were detected with K _Ca values of 152 ±  79 μM (V _max = 5.1 ± 1.9 nM [Ca²⁺]_i/s) and 2.4 ±  0.9 mM (V _max = 37.6 ± 13.6 nM [Ca²⁺]_i/s), respectively. We have also examined the effect of Ca²⁺ and depolarization on these two putative Ca²⁺ influx components. When cells were treated with thapsigargin in a Ca²⁺-free medium, Ca²⁺ influx was higher than into cells treated in a Ca²⁺-containing medium and, while there was a 46% increase in the V _max of the low-affinity component (no change in K _Ca), the high-affinity component was not clearly detected. In depolarized Ca²⁺-pool-depleted cells (with 50 mM KCl in the medium) the high-affinity component was considerably decreased while there was an apparent increase in the K _Ca of the low-affinity component, without any change in the V _max. These results demonstrate that Ca²⁺ influx into parotid acinar cells (1) is increased (four- to five-fold) upon internal Ca²⁺ pool depletion, and (2) is mediated via at least two components, with low and high affinities for Ca²⁺. Received: 30 October 1995/Received after revisionand accepted: 13 December 1995     

Exercise-induced decreases in sarcoplasmic reticulum Ca2+–ATPase activity attenuated by high-resistance training

Muscle biopsies were performed on the vastus lateralis muscle prior to and during a high-resistance training (HRT) programme in order to examine the effects of hypertrophy on sarcoplasmic reticulum Ca²⁺ ATPase activity at rest and during exercise. In six male untrained volunteers (peak aerobic power, V˙O ₂ peak = 3.39 ± 0.13 L min^?1, mean ± SE), the resting Ca²⁺ ATPase activity (μmol min^?1 g wet wt^?1) at 0 (4.89 ± 0.20), 4 (5.62 ± 0.56), 7 (5.15 ± 0.41) and 12 (4.82 ± 0.11) weeks was unchanged by HRT. During cycle ergometer exercise, prior to training, Ca²⁺-ATPase was reduced (P < 0.05) by 14% during the initial 30 min at 58% VO ₂ peak and (P < 0.05) a further 19% during 30 min at 72% VO ₂ peak. Following 7 and 12 weeks of training, the decreases in SR Ca²⁺-ATPase were less pronounced (P < 0.05). These results indicate that muscle hypertrophy, although incapable of altering Ca²⁺-ATPase pump activity at rest, can attenuate the decrease observed in exercise by mechanism(s) as yet unknown.     

Caffeine-induced depolarization in amphibian skeletal muscle fibres: role of Na+/Ca2+ exchange and K+ release

Caffeine (4 mM ) produces a depolarization of about 10 mV in frog muscle fibres (Leptodactylus ocellatus). The aim of this work was to study the mechanisms of this effect. An approximately threefold rise in membrane resistance [Cl⁻-free (SO₄^2–) medium] substantially increased, and both Na⁺-free medium and Ni²⁺ (5 mM ) reduced, the caffeine-induced depolarization. In voltage-clamped (–60 mV) short fibres from lumbricalis muscle of the toad (Buffo arenarum), caffeine generated an inward current of 4.13 ± 0.48 μA cm^–2. This caffeine-induced current was reduced by 60% in Na⁺-free medium, 44% in the presence of 5 mM amiloride and 48% by 5 mM Ni²⁺, suggesting that the activation of the Na⁺–Ca²⁺ exchanger in its forward mode may play a role in the observed electrical effects of the drug. Caffeine also produced a marked release of K⁺. Net K⁺ efflux increased from 3.5 ± 0.2 (control) to 22.1 ± 2.3 pmol s^–1 cm^–2 (caffeine). It is shown that in the presence of the drug, [K⁺] in the lumen of the T tubules may well increase to levels which could produce, in part, both the observed depolarization and the caffeine-induced current under voltage clamp conditions. The caffeine-induced K⁺ efflux was not reduced by 5 mM Ni²⁺. At a holding potential of 30 mV the caffeine-induced current was reversed (outward) and roughly halved by 5 mM Ni²⁺. The Ni²⁺-sensitive fraction of the caffeine-induced current, assumed to represent the Na⁺–Ca²⁺ exchanger current, had an estimated reversal potential close to 12 mV ([Na⁺]_o=115 mM ; [Ca²⁺]_o=1 mM ). In conclusion, the depolarizing effect of caffeine described here would be produced by two mechanisms: (a) an inward current generated by the activation of the Na⁺–Ca²⁺ exchanger in its forward mode, and (b) the rise of the external [K⁺] in restricted spaces like the T tubules.     

Modulation of Ca2+ release through ryanodine receptors in vascular smooth muscle by protein kinase Cα

The role of protein kinase C (PKC) in Ca²⁺ release through ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (SMCs) is not well understood. Caffeine was used to activate RyRs and the intracellular Ca²⁺ concentration ([Ca²⁺]_i) was measured in both freshly isolated and cultured mouse aortic SMCs (ASMCs). Pre-activation of PKC with 1,2-dioctanoyl-sn-glycerol (DOG) prevented caffeine-induced [Ca²⁺]_i transients. Application of the PKC inhibitor calphostin C caused [Ca²⁺]_i transients which were not blocked by nifedipine or by removing extracellular Ca²⁺ but were abolished after inhibition of the SR Ca²⁺–ATPase with thapsigargin or after inhibition of RyRs with ryanodine. In addition, chelerythrine and GF109203X also elevated resting [Ca²⁺]_i but no further [Ca²⁺]_i increase was seen with subsequent application of caffeine. Selective inhibition of PKCα with safingol blocked caffeine-induced [Ca²⁺]_i transients, but the PKCε inhibitory peptide V1-2 did not. In cells expressing a EGFP-tagged PKCα, caffeine-induced [Ca²⁺]_i transients were associated with a rapid focal translocation near the cell periphery, while application of ionomycin and DOG caused translocation to the plasma membrane. Western blot showed that caffeine increased the relative amount of PKCα in the particulate fraction in a time-dependent manner. Co-immunoprecipitation of RyRs and PKCα indicated that they interact. In conclusion, our studies suggest that PKC activation can inhibit the gating activity of RyRs in the SR of ASMCs, and this regulation is most likely mediated by the Ca²⁺-dependent PKCα isoform.     

Effects of thapsigargin and cyclopiazonic acid on the sarcoplasmic reticulum Ca2+ pump of skinned fibres from frog skeletal muscle

Thapsigargin has been reported to inhibit ATP-dependent Ca²⁺ uptake by isolated sarcoplasmic reticulum (SR) vesicles of vertebrate skeletal muscle fibres at nanomolar concentrations. There have been no reports confirming this effect in skinned muscle fibre preparations. We have examined the ability of thapsigargin to inhibit the uptake of Ca²⁺ by the SR in mechanically skinned fibres of frog iliofibularis muscles, using the size of the caffeine-induced contracture to assess the Ca²⁺ content of the SR. The SR was first depleted of Ca²⁺ and then reloaded for 1 min at pCa 6.2 in the presence and absence of thapsigargin. When 5 min were allowed for diffusion, a thapsigargin concentration of at least 131 M was required to inhibit Ca²⁺ loading by 50%. In contrast, another SR Ca²⁺ uptake inhibitor, cyclopiazonic acid, was more effective, producing 50% inhibition at 7.0 M and total inhibition at 50 M. When cyclopiazonic acid (100 M) was applied after, rather than during, Ca²⁺ loading, the caffeine-induced contracture was not changed. Thapsigargin (300 M), on the other hand, caused some reduction in the peak amplitude of the caffeine-induced contracture when applied after Ca²⁺ loading. The poor effectiveness of thapsigargin in the skinned fibres, compared with in SR vesicles, is attributed to its slow diffusion into the skinned fibres, perhaps as a result of binding to myofibrillar components.     

A study of the mechanisms of excitation–contraction coupling in frog skeletal muscle based on measurements of [Ca<Superscript>2+</Superscript>] transients inside the sarcoplasmic reticulum

[Ca²⁺] transients inside the sarcoplasmic reticulum (SR) were recorded in frog skeletal muscle twitch fibers under voltage clamp using the low affinity indicator Mag Fluo 4 (loaded in its AM form) with the purpose of studying the effect on Ca²⁺ release of extrinsic Ca²⁺ buffers (i.e. BAPTA) added at high concentration to the myoplasm. When the extrinsic Ca²⁺ buffer is added to the myoplasm, part of the released Ca²⁺ binds to it, reducing the Ca²⁺ signal reported by a myoplasmic indicator. This, in turn, hinders the quantification of the amount of Ca²⁺ released. Monitoring release by measuring [Ca²⁺] inside the SR avoids this problem. The application of extrinsic buffers at high concentration reduced the resting [Ca²⁺] in the SR ([Ca²⁺]_SR) continuously from a starting value close to 400 μM reaching the range of 100 μM in about half an hour. The effect of reducing resting [Ca²⁺]_SR on the Ca²⁺ permeability of the SR activated by voltage clamp depolarization to 0 mV was studied in cells where the myoplasmic [Ca²⁺] ([Ca²⁺]_myo) transients were simultaneously recorded with Rhod2. The Ca²⁺ release flux was calculated from [Ca²⁺]_myo and divided by [Ca²⁺]_SR to obtain the permeability. Peak permeability was significantly reduced, from 0.026?±?0.005 ms^?1 at resting [Ca²⁺]_SR?=?372?±?5 μM to 0.021?±?0.004 ms^?1 at resting [Ca²⁺]_SR?=?120?±?16 μM (n?=?4, p?=?0.03). The time averaged permeability was not significantly changed (0.009?±?0.003 and 0.010?±?0.003 ms^?1, at the higher and lower [Ca²⁺]_SR respectively). Once the cells were equilibrated with the high buffer intracellular solution, the change in [Ca²⁺]_SR (Δ[Ca²⁺]_SR) in response to voltage clamp depolarization (0 mV, 200 ms) in 20 mM BAPTA was significantly lower (Δ[Ca²⁺]_SR?=?30.2?±?3.5 μM from resting [Ca²⁺]_SR?=?88.8?±?13.6 μM, n?=?5) than in 40 mM EGTA (Δ[Ca²⁺]_SR?=?72.2?±?10.4 μM from resting [Ca²⁺]_SR?=?98.2?±?15.6 μM, n?=?4) suggesting that a Ca²⁺ activated component of release was suppressed by BAPTA.