Adrenoceptor-mediated regulation of myofibrillar Ca2+ sensitivity through the GTP-binding protein-related mechanisms: tension recording in β-escin-skinned single rat cardiac cells with preserved receptor functions

 To investigate the mechanisms of receptor-mediated regulation of heart muscle contraction, we developed a tension-recording system using β-escin-skinned single cardiac cells of rats and studied the effects of agonists on myofibrillar Ca²⁺ sensitivity and Ca²⁺ release from the sarcoplasmic reticulum (SR). In pCa/tension relations, 1 μM isoproterenol plus 100 μM guanosine 5′-triphosphate (GTP) decreased the myofibrillar Ca²⁺ sensitivity (pCa₅₀, the [Ca²⁺] required for half-maximal tension, as an indicator of the sensitivity; from 6.07 to 5.92); this effect was blocked by 1 μM metoprolol or 1 mM guanosine 5′-O-(2-thiodiphosphate) (GDPβS). Phenylephrine (10 μM) plus 100 μM GTP increased the Ca²⁺ sensitivity (pCa₅₀; from 6.12 to 6.28), and this effect was blocked by 1 μM phentolamine or 1 mM GDPβS. After Ca²⁺ loading into the SR, 10 μM phenylephrine plus 100 μM GTP in a low-ethylene- glycol-bis(β-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA, 0.1 mM) relaxing solution induced oscillatory contractions that were attenuated by either 1 μM phentolamine or pre-treatment with 10 μM inositol 1,4,5-trisphosphate. Our results demonstrate that β₁-adrenergic stimulation decreases myofibrillar Ca²⁺ sensitivity and that α₁-adrenergic stimulation both increases the Ca²⁺ sensitivity and activates Ca²⁺ release from the agonist-sensitive SR through GTP-binding protein-related mechanisms. Received: 17 August 1998 / Received after revision: 23 October 1998 / Accepted: 15 December 1998     

Effects of halothane on functionally skinned rabbit soleus muscle fibers: A correlation between tension transient and45Ca release

The mechanism(s) involved in the halothane-induced increase in skeletal muscle contraction was studied using functionally skinned soleus muscle fibers from rabbits: For the tension study, single functionally skinned fibers were individually mounted on two pairs of forceps, with one end attached to a photodiode tension transducer. Ca²⁺-activated tension development of the contractile proteins, and Ca²⁺ uptake and release from the sarcoplasmic reticulum (SR) using caffeine-induced tension transients were studied. To measure the amount of calcium, skinned fibers at 0.1 g/ml were used and 0.075 Ci⁴⁵Ca/ml was spiked in the solution 3 (pCa 6.5 and 1 mM [EGTA]) which promoted rapid loading of Ca²⁺. Halothane (1–3%) did not change the [Ca²⁺]-tension relationship; 2 and 3% halothane reduced the maximum Ca²⁺-activated tension by 6–7%. Halothane (1–3%) added to the solution 3, reduced⁴⁵Ca uptake by 3, 22 and 23%; however, the subsequent caffeine-induced tension transient and⁴⁵Ca release were increased by 10–40%. During the release phase only halothane increased both caffeine-induced tension transient and⁴⁵Ca release by 20–60%. The effects of halothane on the tension transient and on the⁴⁵Ca release were comparable. There was no dose-response relationship to the effects of halothane on the above parameters. It is concluded that halothane affects the SR by increasing its membrane permeability to Ca²⁺, resulting in an increase in myoplasmic [Ca²⁺] and thus in the twitch tension in skeletal muscle.     

Effects of magnesium on contractile activation of skinned cardiac cells.

1. In the presence of a slight buffering of the free [Ca2+] with 0.050 mM total EGTA cyclic contractions were induced by a Ca2+-triggered release of Ca2+ on skinned (sarcolemma-free) segments of single cardiac cells from rat ventricle. The threshold of the free [Ca2+] trigger was elevated when the free [Mg2+] was increased. 2. At a suprathreshold free [Ca2+] increasing the free [Mg2+] resulted in a decrease in frequency and in an increase in amplitude of the phasic contractions. Addition of caffeine at a specified interval after a cyclic contraction produced a larger contraction when free [Mg2+] was higher. It was concluded that an increase of free [Mg2+] increased the capacity and the rate of binding for Ca2+ by the sarcoplasmic reticulum (SR). 3. Small skinned fibres of skeletal muscle which were perfused with 10 mM caffeine yielded results similar to those obtained in skinned cardiac cells. It was concluded that the mechanism of action of free Mg2+ was similar in both preparations, but that the SR of skeletal muscle had a higher capacity and rate of binding for Ca2+ than the cardiac SR. 4. With a strong buffering of the free [Ca2+] with 4-0 mM total EGTA, a smaller tonic tension was developed for a given pCa in the presence of a higher free [Mg2+]. This result was nearly identical in skinned cells from cardiac and skeletal muscle tissue. 5. A decrease of the [MgATP2-] produced a tension in the skinned cardiac cells that were perfused in Ca2+ free media. The maximum tension was observed for [MgATP2-] 10(-5-50)M as in skinned fibres of skeletal muscle. A further decrease of [MgATP2-] resulted in a decrease of tension.     

A comparison of the abilities of CO2/HCO 3 − , protonophores and changes in solution pH to release Ca2+ from the SR of barnacle myofibrillar bundles

Increases in solution pH from 6.5 to 7.0 to 7.5 at 0.1 M free Ca²⁺ concentration had no effect on the isometric tension of barnacle myofibrillar bundles in relaxing solutions containing 0.1–0.16 mM BAPTA. Decreases in pH in the same range were also without effect. Under the same conditions CO₂-induced Ca²⁺ release from the SR could be readily obtained by replacing the Cl^–-containing relaxing solution with one containing HCO ₃ ^– and 100% CO₂ at the same pH. At a higher free Ca²⁺ of 2.5 M, there was a contraction on increasing the pH of the Cl^–-containing solution from 7.0 to 7.5. This reponse could be abolished by 1 mM procaine suggesting that it was due to Ca²⁺ release from the SR. The protonophores monensin, gramicidin, CCCP and FCCP at concentrations of 10–100 M had no effect on resting tension at either free Ca²⁺ concentration and did not inhibit the response to 100% CO₂. It is concluded that dissipation of a possible pH gradient across the SR membrane by protonophores does not release Ca²⁺ from the SR of barnacle muscle. Since both CO₂ (by possibly lowering SR pH) and an increase in solution pH can release Ca²⁺ at 2.5 M free Ca²⁺, the existence of a Ca²⁺ release channel which is opened by a change in the trans-SR pH gradient cannot be discounted. In a separate series of experiments, the CO₂-releasable Ca²⁺ store was first depleted by exposure of bundles to 100% CO₂ in the presence of 1 mM BAPTA for 10 min and then partially reloaded by 15 s exposure to a free Ca²⁺ of 0.6 M buffered by 2 mM EGTA (total) in the Cl^– relaxing solution. The same level of reloading was obtained when the loading solution contained HCO ₃ ^–/100% CO₂; this result tends to discount inhibition of the Ca²⁺ uptake pump as a possible mechanism for CO₂-induced Ca²⁺ release. Loading at 2.6 M free Ca²⁺ for 1 min resulted in almost complete recovery of the CO₂ response to its value before depletion of the store.     

Spontaneous tension oscillation in skinned bovine cardiac muscle

 Skinned fibres from bovine ventricles exhibited spontaneous tension oscillations when MgADP and inorganic phosphate (Pi) were added to the solution bathing fibres in the relaxed state (ADP-SPOC). A similar type of oscillation was observed at intermediate concentrations of free Ca²⁺ in the absence of MgADP and Pi (Ca-SPOC). To investigate the correlation between ADP-SPOC and Ca-SPOC, we constructed two-dimensional state diagrams of cardiac muscle using different concentrations of Pi (0–20 mM) and free Ca²⁺ [pCa=around 5 (+Ca²⁺), pCa=5.15–6.9 and +EGTA (–Ca²⁺)], with varying concentrations of MgADP (0–10 mM), with 2 mM MgATP and 2 mM free Mg²⁺ maintaining ionic strength at 0.15±0.01 M, pH 7.0, 25 °C. The three-dimensional (pCa-Pi-MgADP) state diagram thus obtained was divided into three regions, i.e. the contraction region in which tension oscillation was undetectable, the spontaneous tension oscillation (SPOC) region and the relaxation region. We found that the regions of ADP-SPOC and Ca-SPOC were continuously connected by a single oscillation region sandwiched between the contraction and relaxation regions. The state diagram, which encompasses physiological conditions, shows that the probability of SPOC is higher in cardiac muscle than in skeletal muscle. From these results, we suggest that, despite distinct ionic conditions, the molecular state of cross-bridges during SPOC is common to both ADP-SPOC and Ca-SPOC. Received 19 February 1996 / Received after revision: 16 July 1996 / Accepted: 14 August 1996     

Effects of halothane on Ca2+-activated tension development in mechanically disrupted rabbit myocardial fibers

The effect of halothane on maximal and submaximal Ca²⁺-activated tension in mechanically disrupted right ventricular papillary muscle from rabbits was studied. Steady-state isometric tension generation was measured in the muscle bundle. The relaxing solution contained (in mM) [mg²⁺]=1, [K⁺]=70, [MgATP^¨-]=2, [creatine phosphate^¨-]=15, [EGTA total]=7 and imidazole proprionate. The contracting solution contained in addition Ca²⁺ in various concentrations. In all solutions ionic strength was maintained at 0.15 and pH at 7.00±0.02 at 20°C. Each fiber bundle was immersed in control solutions equilibrated with 100% N₂ and test solutions equilibrated with various concentrations of halothane-N₂ mixture. Increasing doses of halothane (1–4%) significantly shifted the relationship between Ca²⁺ and tension towards higher [Ca²⁺] and depressed the maximum Ca²⁺-activated tension. The maximum tension generated atpCa=3.8 was depressed 5% per 1% increase in halothane concentration. The percentage of maximum tension at submaximum Ca²⁺ concentrations (pCa=5.6–5.0) was not significantly decreased until halothane concentration was greater than 2%. It is concluded that halothane slightly but significantly depressed the interactions of contractile proteins and to a lesser degree Ca²⁺-activation of the regulatory proteins. The halothane-induced depression was completely reversible.     

Calcium-induced inactivation of calcium release from the sarcoplasmic reticulum of skeletal muscle

The ability of myofilament space Ca²⁺ to modulate Ca²⁺ release from the sarcoplasmic reticulum (SR) of skeletal muscle was investigated. Single fibers of the frog Rana pipiens belindieri were manually skinned (sarcolemma removed). Following a standard load and pre-incubation in varying myoplasmic Ca²⁺ concentrations, SR Ca²⁺ release was initiated by caffeine. Ca²⁺ release rates were calculated from the changes in absorbance of a Ca²⁺ sensitive dye, antipyrylazo III. An apparent dissociation constant (K _d) for dye-Ca²⁺ binding of 8000 M² was determined by comparing the buffering action of the dye with that of ethylenebis(oxonitrilo)tetraacetate (EGTA) using the contractile proteins of the skinned fiber as a measure of free Ca²⁺. This value for K _d was used in the calculation of Ca²⁺ release rates. As the myoplasmic space Ca²⁺ was increased from pCa 7.4, Ca²⁺ release rates declined sharply such that at pCa 6.9 the calculated release rate was 72±3% (mean ± SEM) of control (pCa 8.4). Further increases in myoplasmic Ca²⁺ from pCa 6.9 to pCa 6.1 did not result in a further decline in release rate. The effect of a decreased driving force on Ca²⁺ ions was investigated to determine whether it could account for the change in release rates observed. At pCa 6.9, where the greatest degree of inactivation occurred, the measured effects of a change in driving force could account for at most 40% of the observed inactivation. Varying concentrations of Ba²⁺ and Sr²⁺ in the myofilament space had no inactivating effect on the SR Ca²⁺ release rates. The ability of myofilament Ca²⁺ to inhibit SR Ca²⁺ release at concentrations normally encountered during muscle activation suggests a role for released Ca²⁺ as a modulator of the SR Ca²⁺ channel.     

Influence of caffeine,Ca2+, and Mg2+ on ryanodine depression of the tension transient in skinned myocardial fibers of the rabbit

Ryanodine, a blocker for Ca²⁺-release channels of the sarcoplasmic reticulum (SR Ca²⁺-release channels), induces depression of myocardial contraction in isolated intact muscle, which is consistent with depression of the caffeine-induced tension transient in skinned muscle fibers. In isolated SR, ryanodine binds to a specific receptor with high affinity, and this binding is enhanced by caffeine and increasing Ca²⁺ and decreased by increasing Mg²⁺. The aim of this study was to test the hypothesis that depression of myocardial contraction is mediated by changes in ryanodine-receptor binding properties. Accordingly, factors (caffeine, Ca²⁺, and Mg²⁺) affecting ryanodine-receptor binding properties in the isolated SR membrane were studied in skinned myocardial fibers from adult rabbits. The depression of the caffeine-induced tension transient by ryanodine (ryanodine depression) influenced by these three factors was measured. In a dose-dependent manner, increasing caffeine or Ca²⁺ concentrations enhanced the ryanodine depression. The concentrations for 50% ryanodine depression (IC₅₀) approximated 7mM for caffeine, and pCa 5.25 for Ca²⁺. When 1 M ryanodine and 25 mM caffeine were combined, ryanodine depression was independent of Ca²⁺ at low Ca²⁺ concentrations (20%–30% at pCa>8 and 7.5) and was a direct function of Ca²⁺ at higher concentrations (pCa 7.5–6.0 with IC₅₀ approx. pCa 6.75). In contrast, increasing Mg²⁺ reduced the ryanodine depression with IC₅₀ approximately equal to pMg 3.3. In conclusion, the caffeineor Ca²⁺-enhanced, and Mg²⁺-reduced ryanodine depression observed in this study is consistent with known ryanodinereceptor binding properties.     

Effects of Ca antagonist on the contractile force in glycerinated dog heart muscles

The effect of Ca antagonist on the contractile apparatus was investigated in glycerinated cardiac muscle preparations obtained from canine hearts. Each muscle preparation had three consecutive isometric contractions. The 1st and 3rd contractions were produced with a control contraction solution, and compared with the 2nd contraction which was induced with a contraction solution containing verapamil. The results showed that maximal developed tension (Po) was enhanced significantly by 1.02×10^–2 mM of verapamil, and the augmentation of contractility was dependent on the concentrations of verapamil. Thus, not only Po, but also dT/dt increased tremendously at 1.02 mM of verapamil. Such contractile potentiation by verapamil was also ascertained by another Ca antagonist, Diltiazem hydrochloride. The developed tension was maximum at pCa 4.0, and no developed tension was found at pCa 8.0. The relationship between pCa and tension with verapamil shifted to the left from that without verapamil, showing higher sensitivity to Ca²⁺. From these results, it was strongly indicated that Ca antagonist is a potentiating agent of the contractile force.     

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     

Effects of ryanodine on acetylcholine-induced Ca2+ mobilization in single smooth muscle cells of the porcine coronary artery

To study the essential features of acetylcholine (ACh)-and caffeine-sensitive cellular Ca²⁺ storage sites in single vascular smooth muscle cells of the porcine coronary artery, the effects of ryanodine on both ACh- and caffeine-induced Ca²⁺ mobilization were investigated by measuring intracellular Ca²⁺ concentration ([Ca²⁺]_i) using Fura 2 in Ca²⁺-containing or Ca²⁺-free solution. The resting [Ca²⁺]_i of the cells was 122 nM in normal physiological solution and no spontaneous activity was observed. In a solution containing 2.6 mM Ca²⁺, 10 M ACh or 128 mM K⁺ produced a phasic, followed by a tonic, increase in [Ca²⁺]_i but 20 mM caffeine produced only a phasic increase. In Ca²⁺-free solution containing 0.5 mM ethylenebis(oxonitrilo)tetraacetate (EGTA), the resting [Ca²⁺]_i rapidly decreased to 102 nM within 5 min, and 10 M ACh or 20 mM caffeine (but not 128 mM K⁺) transiently increased [Ca²⁺]_i. Ryanodine (50 M) greatly inhibited the phasic increase in [Ca²⁺]_i induced by 10 M ACh or 5 mM caffeine and increased the time to peak and to the half decay after the peak in the presence or absence of extracellular Ca²⁺. By contrast, ryanodine (50 M) enhanced the tonic increase in [Ca²⁺]_i induced by 128 mM K⁺ and also by 10 M ACh in Ca²⁺-containing solution. In Ca²⁺-free solution containing 0.5 mM EGTA, ACh (10 M) failed to increase [Ca²⁺]_i following application of 20 mM caffeine. The level of [Ca²⁺]_i induced by 20 mM caffeine was greatly reduced, but not abolished, following application of 10 M ACh in Ca²⁺-free solution. These results suggest that both ACh and caffeine release Ca²⁺ from the ryanodine-sensitive sarcoplasmic reticulum (SR) in smooth muscle cells of the porcine coronary artery. The finding that ryanodine significantly increased the resting [Ca²⁺]_i and inhibited the rate of decline of [Ca²⁺]_i following wasthout of high K⁺ or ACh in Ca²⁺-containing solution suggests that SR may negatively regulate the resting [Ca²⁺]_i in smooth muscle cells of the porcine coronary artery.     

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     

Stretch-induced increase in the Ca2+ sensitivity of myofibrillar ATPase activity in skinned fibres from pig ventricles

The increase in force development in the heart with increase in end-diastolic pressure (Frank-Starling mechanism) has been ascribed to an increase in contractile responsiveness of the myofibrils to calcium. We now show that this calcium sensitization is also associated with an increase in calcium responsiveness of the myofibrillar ATPase. Thus, at submaximal Ca activation (pCa 6.0), the ATPase activity of skinned fibres from pig right ventricles is increased from 57.9±4.4% to 70.6±4.4% of the maximal Ca²⁺ activation of ATPase by stretching (by 15%l _o). At maximal Ca²⁺ activation, ATPase was barely altered by stretching. The relationship between ATPase activity of skinned trabecula of pig right ventricle and ATPase-Ca²⁺ concentrations is shifted (by 0.1 pCa unit) to higher pCa values after a stretch-induced increase of the sarcomere length from 2.1 μm to 2.4 μm. The relationship between force and pCa was affected in a similar way by extension. This increased calcium sensitivity is, however, not associated with an alteration in the relationship between ATPase activity and force development (tension cost). In accordance with Brenner's hypothesis, we propose therefore that stretch activation of ATPase is associated with an increase in the apparent rate constant of crossbridge attachment rather than with a decrease in the apparent rate constant of crossbridge detachment.     

Effects of ryanodine on skinned myocardial fibers of the rabbit

Skinned fiber bundles from papillary muscle of rabbits were used to study the effects of ryanodine (1) on direct Ca²⁺ activation of the contractile proteins, and (2) on direct Ca²⁺ uptake and release from the sarcoplasmic reticulum (SR). Caffeine (25 mM) was used to release Ca²⁺ from the SR and to generate a tension transient. Each tension transient occurred after sequential immersion of the fiber bundles into five solutions: loading (uptake phase, [U]) and releasing (release phase, [R]). The height of free Ca²⁺-activated tension development of the contractile proteins, and the area of the tension transient generated by caffeine were assessed. (1) The direct free Ca²⁺-activated tension development of the contractile proteins was not significantly affected by ryanodine up to 0.1 mM, either at the submaximal or maximal free Ca²⁺ concentrations. (2) Ryanodine (1 nM–1 M), in U, R, or in U and R, did not significantly change the immediate caffeine-induced tension transients. In the same preparation after ryanodien treatments, the second control caffeine-induced tension transients (C₂, no ryanodine) were decreased in a dose-dependent manner (IC₅₀=50, 10 nM, 10 nM for R, U, and U and R, respectively). The depression caused by ryanodine on the SR was activity-dependent and not readily reversible. Total calcium content in the SR of C₂ was not significantly changed by small quantities of ryanodine (<0.1 M) and was decreased with greater amounts of ryanodine (0.1 M). Thus, at low concentrations of ryanodine, the negative inotropic action is due to decrease Ca²⁺ release from the SR, at high concentration of ryanodine, it is due to decrease in calcium accumulation in the SR.     

Effects of caffeine on Ca-activated force production in skinned cardiac and skeletal muscle fibres of the rat

Skinned fibres prepared by mechanical and/or chemical means from cardiac and skeletal muscles of the rat were activated in solution strongly buffered for Ca²⁺ (with 50 mM EGTA) in the absence or presence of caffeine 5–40 mM. In all preparations caffeine was found to reversibly shift the relation between steady-state force and free [Ca²⁺] toward lower free [Ca²⁺] in a dose dependent manner. This increase in apparent Ca²⁺ sensitivity was not antagonized by procaine and was the same, within each muscle type, irrespective of the manner in which the skinned fibre was prepared, and consequently the degree to which it retained cellular membranes. The effect was more pronounced in cardiac and slow-twitch than in fast-twitch, myofibrillar preparations. At equivalent concentrations theophylline mimicked this effect of caffeine in all preparations, however, addition of exogenous cyclic AMP neither mimicked nor modified, in any way, the effect. Maximum Ca²⁺-activated force production was not affected by caffeine below 20 mM but was depressed by concentrations of 20 mM and above. The increase in apparent Ca²⁺ sensitivity produced by caffeine can not be the result of a mobilization of some cellular store of Ca²⁺ but must arise from a direct effect of caffeine on the myofilaments which leads to a change in the apparent affinity constant of the force controlling sites for Ca²⁺.     

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     

Free calcium in sheep cardiac tissue and frog skeletal muscle measured with Ca2+-selective microelectrodes

Microelectrodes filled with neutral carrier selective to Ca²⁺ were used to measure the free intracellular Ca²⁺ concentration ([Ca²⁺]_i) in sheep cardiac tissue and frog skeletal muscle. Calibration of the electrodes was performed in the presence of a solution resembling the cationic composition of the cytoplasm. [Ca²⁺]_i at rest in normal physiological saline (20–22° C) was 240 nM in Purkinje fibres, 270 nM in ventricular muscle, and 52 nM in skeletal muscle. In Purkinje fibres, elevation of [Ca²⁺]_o from 1.8 mM to 5.4 mM produced a 1.7-fold increase in [Ca²⁺]_i. Elevation of [Ca²⁺]_o from 1.8 mM to 18 mM induced a 2.6-fold increase in [Ca²⁺]_i. Exposure to Na⁺-free solution (Li⁺-substituted) gave rise to elevation of [Ca²⁺]_i by factors of 5.8 and 14 in ventricular muscle and Purkinje fibres, respectively. These latter changes in [Ca²⁺]_i were associated with the development of contractures which reached 34% and 172% of the corresponding twitch tension.     

Effects of tetracaine and procaine on skinned muscle fibres depend on free calcium

Summary The local anaesthetics, tetracaine and procaine have previously been found to block, induce or potentiate Ca²⁺ release from the sarcoplasmic reticulum (SR) of skeletal muscle depending on the preparation, experimental conditions and design. We now show that low concentrations of tetracaine and procaine block SR Ca²⁺ release whereas high concentrations induce release from the SR of amphibian and mammalian skinned fibres. Both actions depend on pCa, such that a shift in pCa can alter their effect from blocking to releasing Ca²⁺. In skinned fibres with Ca²⁺-loaded SR, tetracaine (1mm) produced a tonic contraction with a time to half-peak of 15–20 s and a magnitude reaching 80% of maximum force. Ca²⁺ release by tetracaine or procaine occured at pCa 6.5 and was not blocked by Ruthenium Red (RR) (25 m). This action of tetracaine was attributed to SR Ca²⁺ release rather than to a displacement of bound Ca²⁺ because fibres lacking a functional SR due to pre-treatment with quercetin (100 m), A 23187 (100 g ml^–1) or Triton X-100 (1%) did not contract after additions of tetracaine. Lower concentrations of tetracaine (0.5mm) and procaine (10mm) blocked contractions due to caffeine (at pCa 6.73), sulphydryl oxidizing agents, or Ca²⁺-induced Ca²⁺ release (CICR). The inhibition of CICR as a function of pCa was difficult to measure quantitatively since lowering pCa to elicit CICR twitches was sufficient to initiate tetracaine-induced tonic contractions.Experiments with isolated SR vesicles showed that 1mm tetracaine inhibited CICR, over a wide range of pCa but 3–5mm tetracaine induced rapid Ca²⁺ release. The opposite effects of tetracaine and procaine depend mostly on their concentration in SR vesicles and/or pCa in skinned fibres. Blockade of release seems to occur via the CICR pathway, and induction of release through an increase in SR membrane permeability.Abbreviations SR sarcoplasmic reticulum - HEPES N-2-hydroxy-ethylpiperazine-N¹-2-ethanesulphonic acid - EGTA ethylene glycol bis (-aminoethyl ether)-N,N,N¹,-N¹-tetraacetic acid - CICR Ca²⁺-induced Ca²⁺ release - MOPS morpholinopropane sulphonic acid - RR Ruthenium Red     

Effects of sodium on the calcium paradox in rat hearts

Reduction of the Na concentration in the Ca-free perfusion solution reduces the amount of myoglobin released by the cells when Ca is readmitted if sucrose is used to replace NaCl under mild hypothermia. When salts like cholinechloride or LiCl are used instead of sucrose, no protection is seen at any temperature. The temperature threshold above which myoglobin loss sharply increases is lowered by prolonged Ca depletion or by the addition of EGTA to the Ca-free solution. Protection by sucrose does not occur in the presence of EGTA. An increase of cell Na induced by strophanthidin during the Ca depletion phase has no effect on myoglobin release. The exponential decline in twitch tension in the early phase of Ca deprivation has the same half-live (T _1/2) for Ca-free solutions containing 145 mM Na or 35 mM Na (110 mM Li or choline), but itsT _1/2 is prolonged if sucrose is used to replace NaCl. When 5 mM EGTA is added to the Ca-free solutions, theT _1/2 is shortened and is not changed by the replacement of NaCl with sucrose. The rate of washout of Ca within the first 20 s of Ca depletion has a similar time course in a normal Na or in a Li and low Na solution. In a sucrose and low Na solution the rate of the Ca efflux is reduced. The addition of EGTA increases this rate and abolishes the slowing effect of a sucrose and low Na solution. Therefore myoglobin release during the Ca paradox does not depend on the Na gradient across the sarcolemma. Na⁺, like other cations, probably enhances the displacement of Ca²⁺ from critical binding sites during Ca-free perfusion, which predisposes the cells to the paradox.     

Activation by intracellular calcium of a potassium channel in cardiac sarcoplasmic reticulum

The effects of low (pCa 7.5 to 3) concentrations of intracellular calcium ion on a single potassium channel in the sarcoplasmic reticulum of canine heart ventricular muscle were investigated using a planar lipid bilayer technique. The low concentrations were obtained by mixing EGTA and calcium chloride. By varying the pCa of the cytoplasmic face between 3 to 7.5, two novel effects were observed. First, an increase in the intracellular Ca²⁺ concentration produced an increase in the unit current amplitude of open states; the voltage-current relationship was ohmic at these concentrations. Second, an increase in the Ca²⁺ concentration increased the open probability. Both these effects of Ca²⁺ were dose-dependent, and were consistently observed in all channels tested. Thus, the SR potassium channel observed appears to belong to the class of Ca²⁺ -activated potassium channels.