Maximal voluntary contraction force,SR function and glycogen resynthesis during the first 72 h after a high-level competitive soccer game

The aim of this study was to examine maximal voluntary knee-extensor contraction force (MVC force), sarcoplasmic reticulum (SR) function and muscle glycogen levels in the days after a high-level soccer game when players ingested an optimised diet. Seven high-level male soccer players had a vastus lateralis muscle biopsy and a blood sample collected in a control situation and at 0, 24, 48 and 72 h after a competitive soccer game. MVC force, SR function, muscle glycogen, muscle soreness and plasma myoglobin were measured. MVC force sustained over 1 s was 11 and 10% lower (P < 0.05) after 0 and 24 h, respectively, compared with control. The rate of SR Ca²⁺ uptake at 800 nM [Ca²⁺]_free was lower (P < 0.05) after 0 h (2.5 μmol Ca²⁺ g prot⁻¹ min⁻¹) than for all other time points (24 h: 5.1 μmol Ca²⁺ g prot⁻¹ min⁻¹). However, SR Ca²⁺ release rate was not affected. Plasma myoglobin was sixfold higher (P < 0.05) immediately after the game, but normalised 24 h after the game. Quadriceps muscle soreness (0–10 VAS-scale) was higher (P < 0.05) after 0 h (3.6), 24 h (1.8), 48 h (1.1) and 72 h (1.4) compared with control (0.1). Muscle glycogen was 57 and 27% lower (P < 0.001) 0 and 24 h after the game compared with control (193 and 328 vs. 449 mmol kg d w⁻¹). In conclusion, maximal voluntary contraction force and SR Ca²⁺ uptake were impaired and muscle soreness was elevated after a high-level soccer game, with faster recovery of SR function in comparison with MVC force, soreness and muscle glycogen.     

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     

Effects of cytosolic Ca2+ on the Ca2+ content of the sarcoplasmic reticulum in saponin-permeabilized rat ventricular trabeculae

 Rat ventricular trabeculae were mounted for isometric tension recording, and then permeabilized with saponin. The Ca²⁺ concentration ([Ca²⁺]) within the permeabilized preparation (cytosolic [Ca²⁺]) was monitored continuously using Indo-1 and the integrals of Ca²⁺ transients resulting from brief caffeine application used as an index of the sarcoplasmic reticulum (SR) Ca²⁺ content. The relationship between SR Ca²⁺ content and cytosolic [Ca²⁺] was studied within the reported physiological range (i.e. 50–250 nmol · l^–1 Ca²⁺). Increasing cytosolic [Ca²⁺] from 50 nmol · l^–1 to 250 nmol · l^–1 increased the steady-state SR Ca²⁺ content about threefold. However, increasing [Ca²⁺] above 250 nmol · l^–1 typically resulted in spontaneous SR Ca²⁺ release, with no further increase in SR Ca²⁺ content. The SR Ca²⁺ content increased only slowly when cytosolic [Ca²⁺] was increased; it was unchanged 20 s after a rapid increase in cytosolic [Ca²⁺], but increased progressively to a new steady-state level during the following 1–2 min. In a parallel series of experiments using intact papillary muscles, increasing extracellular [Ca²⁺] (from 0.5 to 5 mmol · l^–1) significantly increased twitch tension within 20 s of the solution change. These results support previous suggestions that the SR Ca²⁺ content may increase when diastolic cytosolic [Ca²⁺] rises during inotropic interventions such as increased stimulus rate or extracellular [Ca²⁺]. However, the rate at which SR Ca²⁺ responds to changes in cytoplasmic [Ca²⁺] within the diastolic range does not appear rapid enough to explain the early potentiation of twitch tension in intact preparations after an increase in extracellular [Ca²⁺]. Received: 26 August 1997 / Accepted: 28 October 1997     

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     

Cross-bridge movement in rat slow skeletal muscle as a function of calcium concentration

A single fibre bundle from rat soleus muscle was chemically skinned with saponin and the transfer of myosin heads from the thick filaments to the thin filaments at a sarcomere length of 2.4 μm was measured as a function of Ca²⁺ concentration using an x-ray diffraction method at 4–7 °C. In the relaxed state, the 1,0 spacing was 42.08 nm. The spacing showed no significant decrease when the Ca²⁺ concentration was below the threshold (−log₁₀ [Ca²⁺] or pCa 5.8). No significant transfer of the myosin heads occurred when the Ca²⁺concentration was below the threshold (pCa 5.8). When the muscle was maximally activated at pCa 4.4, the spacing decreased to 40.35 nm. During the maximum isometric contraction at pCa 4.4, 54.9 ± 6.5% (±SE of the mean) of the myosin heads were transferred to the thin filaments. The transfer of the myosin heads was approximately proportional to relative tension. These results suggest that myosin heads of both fast-twitch and slow-twitch skeletal muscles transferred on the common movement as a function of Ca²⁺ concentration. Received: 1 December 1995/Received after revision and accepted: 20 May 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     

Sodium-dependent adenosine transport is diminished in brush border membrane vesicles from hypothyroid rat kidney

 Studies of the uptake of [3H]adenosine ([³H]ADO) were performed using brush border membrane vesicles (BBMV) from normal (N) and hypothyroid (Tx) rat kidneys, to test if decreased Na⁺ reabsorption in hypothyroidism might be associated with abnormalities in ADO transport. [³H]ADO uptake (1–10 μmol) for both conditions was measured in the presence of Na⁺ (10–150 mmol/l); the effects of dipyridamole (10 μmol/l) and 1,3-dipropyl-8-(2-amino-4-chlorophenyl)xanthine (PACPX, 10 μmol/l) were also studied. Na⁺-stimulated ADO uptake was decreased in Tx BBMV. Michaelis–Menten constants showed a decreased ADO carrier affinity (K _m 2.46 ± 0.14 in N, vs K _m 4.46 ± 0.88 μmol/l in Tx, P<0.05), with no change in the number of carriers (V _max 295 ± 25 in N, vs 229.2 ± 56 pmol·min^–1·mg protein in Tx). Na⁺ affinity remained unchanged (K _Na+ 11.5 ± 3.5 in N, vs K _Na+ 12.72 ± 0.7 mmol/l in Tx). Inhibition of Na⁺-dependent ADO transport was 50% in N as opposed to 58% in Tx with dipyridamole, and 72% in N versus 47% in Tx with PACPX. These results suggest that decreased Na⁺-dependent ADO cotransport contributes to the diminished tubular reabsorption that occurs in hypothyroidism. Received: 17 June 1996 / Received after revision: 9 September 1996 / Accepted: 16 September 1996     

Residual sarcoplasmic reticulum Ca<Superscript>2+</Superscript> concentration after Ca<Superscript>2+</Superscript> release in skeletal myofibers from young adult and old mice

Contrasting information suggests either almost complete depletion of sarcoplasmic reticulum (SR) Ca²⁺ or significant residual Ca²⁺ concentration after prolonged depolarization of the skeletal muscle fiber. The primary obstacle to resolving this controversy is the lack of genetically encoded Ca²⁺ indicators targeted to the SR that exhibit low-Ca²⁺ affinity, a fast biosensor: Ca²⁺ off-rate reaction, and can be expressed in myofibers from adult and older adult mammalian species. This work used the recently designed low-affinity Ca²⁺ sensor (Kd = 1.66 mM in the myofiber) CatchER (calcium sensor for detecting high concentrations in the ER) targeted to the SR, to investigate whether prolonged skeletal muscle fiber depolarization significantly alters residual SR Ca²⁺ with aging. We found CatchER a proper tool to investigate SR Ca²⁺ depletion in young adult and older adult mice, consistently tracking SR luminal Ca²⁺ release in response to brief and repetitive stimulation. We evoked SR Ca²⁺ release in whole-cell voltage-clamped flexor digitorum brevis muscle fibers from young and old FVB mice and tested the maximal SR Ca²⁺ release by directly activating the ryanodine receptor (RyR1) with 4-chloro-m-cresol in the same myofibers. Here, we report for the first time that the Ca²⁺ remaining in the SR after prolonged depolarization (2 s) in myofibers from aging (~220 μM) was larger than young (~132 μM) mice. These experiments indicate that SR Ca²⁺ is far from fully depleted under physiological conditions throughout life, and support the concept of excitation–contraction uncoupling in functional senescent myofibers.     

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     

Ruthenium red reduces the Ca2+ sensitivity of Ca2+ uptake into cardiac sarcoplasmic reticulum

 Ruthenium red inhibits mitochondrial Ca²⁺ uptake and is widely used as an inhibitor of ryanodine-sensitive Ca²⁺ channels that function to release Ca²⁺ from the sarcoplasmic reticulum (SR) of muscle cells. It also has effects on other Ca²⁺ channels and ion transporters. To study the effects of ruthenium red on Ca²⁺ transport into the SR of cardiac muscle cells, fluorescence measurements of Ca²⁺ uptake into cardiac SR vesicles were made. Ruthenium red significantly decreased the Ca²⁺ sensitivity of SR uptake in a dose-dependent manner at concentrations ranging from 5 μM to 20 μM. There were no significant effects of ruthenium red on the maximum velocity or the Hill coefficient of SR Ca²⁺ uptake. Received: 14 January 1998 / Received after revision: 12 March 1998 / Accepted: 16 March 1998     

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.     

Telokin mediates Ca<Superscript>2+</Superscript>-desensitization through activation of myosin phosphatase in phasic and tonic smooth muscle

Telokin, a 17 kDa smooth muscle specific protein, consists of the C-terminal domain of MLCK, is phosphorylated by PKA and PKG at Ser13 in vivo (Wu et al. (1998) J Biol Chem 273: 11362–11369; Walker et al. (2001) J. Biol Chem 276: 24519–24524) and is proposed to induce Ca²⁺-desensitization through activation of myosin phosphatase (Wu et al. (1998) J. Biol Chem 273: 11362–11369). Telokin is reported to be highly expressed in phasic with only trace amounts in tonic smooth muscle. In α-toxin permeabilized femoral artery, 5 μM 8-Br-cGMP induced a two-fold increase in telokin phosphorylation and a maximal 30% relaxation of Ca²⁺-activated force compared to a 90% relaxation in phasic ileum muscle consistent with the relative amounts of telokin expressed in ileum, 27 ± 4.6 μM SEM compared to 6 ± 1.7 μM SEM, in femoral artery. Recombinant Wt telokin and the phospho-telokin mutant, S13D relaxed telokin-depleted femoral artery, by 38 ± 8% SEM and 60 ± 20% SEM, respectively. 8-Br-cGMP increased the rate and decreased the amplitude of force development initiated by photolysis of caged ATP in α-toxin permeabilized ileum and femoral artery smooth muscle, consistent with a cGMP-induced increase in phosphatase activity. Similarly, in telokin depleted ileum, recombinant S13D mutant telokin significantly increased the rate (0.08 ± 0.01 s⁻¹ vs. 014 ± 0.02 s⁻¹) and decreased force amplitude. In conclusion, our data support a role for telokin in cyclic nucleotide-induced relaxation of not only phasic, but also tonic smooth muscle and that this relaxation is mediated by activation of myosin phosphatase activity leading to a decrease in myosin light chain phosphorylation.     

Activation of non-selective cation conductance by carbachol in freshly isolated bovine ciliary muscle cells

 In smooth muscle cells freshly isolated from the bovine ciliary body, effects of carbachol (CCh) on the membrane potential and current were examined by the whole-cell clamp method. The resting membrane potential of the muscle cells used was –60 ± 1 mV (n=111). Extracellular application of CCh (2 μM) depolarized the cells to –15 ± 5 mV (n=50) with an apparent increase in membrane conductance. Under voltage-clamp conditions, CCh (2 μM) evoked an inward current which exhibited inward-going rectification and reversed the polarity at about 0 mV. Removal of Na⁺ from the external solution caused a reduction of the amplitude of the current and a shift of the reversal potential to the negative direction. CCh was able to elicit an inward current even under a condition where Ca²⁺ was the only cation producing an inwardly directed electrochemical gradient. The current was not affected by verapamil or by tetrodotoxin. The CCh-induced current was inhibited by antimuscarinic agents with the affinity sequence: atropine ≈4–DAMP >> pirenzepine > AF-DX116, indicating that the response is mediated by a muscarinic cholinoceptor that belongs to the M₃-subtype. Unlike the non-selective cation channel current in intestinal smooth muscles, which is activated by elevation of the intracellular Ca²⁺ concentration ([Ca²⁺]_i), the current of the ciliary muscle was inactivated when the [Ca²⁺]_i was increased. The conductance, which admits Ca²⁺, may serve as a pathway for Ca²⁺ entry required for contraction. Received: 2 December 1996 / Received after revision: 7 January 1997 / Accepted: 8 January 1997     

Characterization of specific GTP binding sites in C2C12 mouse skeletal muscle cells

Receptor sites, specific for guanosine 5′-triphosphate (GTP) were characterised in myoblasts and myotubes of C2C12 mouse skeletal muscle cells, using binding experiments and measurements of intracellular Ca²⁺ concentration ([Ca²⁺]_i). We identified two GTP binding sites in myoblasts membranes: a high affinity site (K _d = 15.4 ± 4.6 μM; B _max = 1.7 ± 0.5 nmol mg⁻¹ protein); and a low affinity site (K _d = 170 ± 94.5 μM; B _max = 14.2 ± 3.9 nmol mg⁻¹ protein). In myotube membranes only a low affinity binding site for GTP (K _d = 169 ± 39 μM; B _max = 12.3 ± 1.4 nmol mg⁻¹ protein) was detected. In myoblasts GTP binding was not displaced by ATP or UTP, even at high concentrations (up to of 1 mM), but it was affected by treatments with suramin or Reactive Blue 2 (RB2), the non-selective purine receptor antagonists. In contrast, in myotubes GTP binding was partially displaced by high concentrations of ATP, but treatments with the non-selective purine receptor antagonists, suramin or RB2, and with UTP had no effect on GTP binding. The addition of GTP to myoblasts, and to myotubes, resulted in elevations of [Ca²⁺]_i. The patterns of Ca²⁺ response however, were different in the two cell phenotypes. In myoblasts the addition of GTP induced two types of Ca²⁺ responses: (1) a fast increase in [Ca²⁺]_i, followed by a sustained [Ca²⁺]_i elevation, and (2) a slow raising and steady prolonged increase in [Ca²⁺]_i. In myotubes, however only fast Ca²⁺ responses were observed following the addition of 500 μM GTP. In the myoblasts and myotubes GTP-stimulated [Ca²⁺]_i increases were abolished by treatments with suramin or RB2 at concentrations which had no effect on the ATP-induced Ca²⁺ responses. We conclude, that C2C12 cells express two distinct binding sites for GTP before differentiation, but only one after, the low affinity binding site. These results suggest a possible role of the high affinity GTP binding site in early stage of development of skeletal muscle. This revised version was published online in July 2006 with corrections to the Cover Date.     

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     

Sarcoplasmic reticulum Ca2+ loading in rabbits 8 and 15 weeks after coronary artery ligation

 Calcium uptake by cardiac sarcoplasmic reticulum (SR) is reported to be reduced in heart failure in the human and in a number of animal models. However, the majority of studies have examined end-stage heart failure in the human and few animal studies have taken account of the duration and severity of left ventricular dysfunction. In this study we have compared SR Ca²⁺ loading in a haemodynamically assessed, coronary artery ligation model of heart failure at 8 and 15 weeks after ligation. Trabeculae were isolated from the right ventricle and mounted for isometric tension measurement. They were treated with saponin to permeabilize the sarcolemma but retain SR function and bathed in a mock intracellular solution including adenosine triphosphate (ATP) and buffered Ca²⁺. Caffeine was used to release Ca²⁺ from the SR. The amplitude of the caffeine-induced contracture was used as a quantitative gauge of the Ca²⁺ content of the SR. Eight weeks after ligation, trabeculae demonstrated enhanced SR Ca²⁺ uptake as manifest by larger caffeine-induced contractures (e.g. 200 nM [Ca²⁺], 120 s loading – 38.2±9.2 versus 67.3±10.1% of maximum Ca²⁺-activated force, F _{Ca, max}, P=0.03). At 15 weeks, trabeculae from ligated hearts were not significantly different from controls with SR Ca²⁺ loading returning to control levels (e.g. 200 nM [Ca²⁺], 120 s loading – 47.3±9.6 versus 30.2±12.8% F _{Ca, max}, P=0.12). These data suggest that SR Ca²⁺ loading may increase in the early stages of heart failure and fall back to normal with an increasing duration of left ventricular dysfunction. Increased incidence of spontaneous Ca²⁺ release observed from the SR at 8 weeks and not at 15 weeks may represent an arrhythmogenic mechanism specific to the early phase of heart failure. Received: 21 January 1998 / Received after revision and accepted: 3 April 1998     

Replacement of troponin-I in slow-twitch skeletal muscle alters the effects of the calcium sensitizer EMD 53998

 We extracted troponin-I (TnI) from skinned rat and rabbit soleus muscle fibres using a modification of the method described by Strauss et al. (FEBS Lett 310:229–234, 1992) for replacement of TnI in cardiac preparations. Incubation of soleus muscle fibres with 10 mmol/l vanadate virtually completely abolished the Ca²⁺dependence of force. Immunoblot analysis revealed that more than 80% of TnI had been extracted from the preparations. The Ca²⁺dependence of force was restored by incubation with a complex of cardiac TnI (cTnI) and troponin-C (cTnC). We examined the effects of the Ca²⁺-sensitizing compound EMD 53998 on isometric tension in native porcine cardiac and rabbit soleus skinned fibres as well as soleus in which the endogenous slow skeletal TnI (ssTnI) had been replaced by cTnI (soleus–cTnI). It was found that 10 μmol/l EMD 53998 in native soleus increased maximum Ca²⁺-activated force to 120±1.4% of control. In soleus–cTnI fibres, maximum force was increased to only 105±0.9%, which was similar to the effect observed in cardiac muscle (108±0.6%). In cardiac muscle, 10 μmol/l EMD 53998 induced a leftward shift of the pCa-tension relation by 0.65 log units. In native soleus, ΔpCa was only 0.40. Again, the effect of EMD 53998 on soleus–cTnI (ΔpCa=0.56) more closely resembled the response found in cardiac muscle than that observed in native soleus muscle. The apparent TnI-isoform dependence of the effects elicited by EMD 53998 suggests that its actions are modulated by the regulatory proteins of the thin filament. Received: 15 December 1997 / Accepted: 16 March 1998     

Effects of intracellular calcium on GABAA receptors in mouse cortical neurons

 Using the patch-clamp technique, we studied the effect of intracellular Ca²⁺ on Cl^– current gated by type A γ-aminobutyric acid receptors (GABA_A) in mouse cortical neurons. When the rapid Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid (BAPTA) was in the pipette solution, the GABA-activated Cl^– current amplitude decreased over time to 49 ± 7% of control. In contrast, equimolar replacement of BAPTA with ethylenebis(oxonitrilo)tetraacetate (EGTA) caused a 60 ± 10% increase in GABA current. An increased intracellular Ca²⁺ concentration caused a transient augmentation of the GABA current. This effect of Ca²⁺ was concentration dependent (10 nM to 34 μM). Ca²⁺ increased the amplitude of the current by enhancing the maximal response to GABA rather than by changing the affinity of the receptor to GABA (EC₅₀ = 5 ± 0.4 μM vs. 7 ± 0.3 μM). Both calmodulin (CaM) and a CaM kinase II inhibitor (200 μM) blocked the potentiating effect of Ca²⁺ suggesting that it was mediated by activation of CaM kinase II. We found that regulation of GABA_A receptors by intracellular Ca²⁺ in cortical neurons has important physiological implications since the potentiating effect of increasing the intracellular Ca²⁺ on responses to GABA was mimicked by activating excitatory receptors with 100 μM N-methyl-D-aspartate (NMDA). These findings suggest that modulation of GABA_A receptor activity by glutamate may be brought about via changes in intracellular Ca²⁺. Received: 20 May 1997 / Received after revision: 12 August 1997 / Accepted: 1 September 1997     

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     

Factors affecting SOCE activation in mammalian skeletal muscle fibers

Enzymatically dissociated mouse FDB muscle fibers, loaded with Fura-2 AM, were used to study the effect of mitochondrial uncoupling on the capacitative Ca²⁺ entry, SOCE. Sarcoplasmic reticulum (SR) Ca²⁺ stores were depleted by repetitive exposures to high K⁺ or 4-chloro-m-Cresol (4-CmC) in the absence of extracellular Ca²⁺. SR Ca²⁺ store replenishment was substantially reduced using 5 μM cyclopiazonic acid (CPA). Readmission of external Ca²⁺ (5 mM) increased basal [Ca²⁺]_i under two modalities. In mode 1 [Ca²⁺]_i initially increased at a rate of 0.8 ± 0.1 nM/s and later at a rate of 12.3 ± 2.6 nM/s, reaching a final value of 477.8 ± 36.8 nM in 215.7 ± 25.9 s. In mode 2, [Ca²⁺]_i increased at a rate of 0.8 ± 0.1 nM/s to a value of 204.9 ± 20.6 nM in 185.4 ± 21.1 s. FCCP, 2 μM, reduced this Ca²⁺ entry. In nine FCCP-poisoned fibers, the initial rate of Ca²⁺ increase was 0.34 ± 0.1 nM/s (mean ± SEM), reaching a plateau of 149.2 ± 14.1 nM in 217 ± 19 s. The results may likely be explained by the hypothesis that SOCE is inhibited by mitochondrial uncouplers, pointing to a possible mitochondrial role in its activation. Using time-scan confocal microscopy and the dyes CaOr-5N AM or Rhod-2 AM to label mitochondrial Ca²⁺, we show that during depletion [Ca²⁺]_mito initially increases and later diminishes. Finally, we show that the increase in basal [Ca²⁺]_i, associated with SOCE activation, diminishes upon external Na⁺ withdrawal. Na⁺ entry through the SOCE pathway and activation of the reversal of Na⁺/Ca²⁺ exchanger could explain this SOCE modulation by Na⁺.