2,3‐Butanedione monoxime increases speed of relaxation in single muscle fibres of frog

The effects of 2,3‐butanedione monoxime (BDM) on intracellular Ca²⁺ transient and cross‐bridge function were studied in frog single fibres from the anterior tibialis muscle of Rana temporaria (sarcomere length, 2.2 μm; temperature, 2–4 °C). The fluorescent dye fluo‐3 was used to monitor the intracellular free calcium concentration ([Ca²⁺]_i) during isometric contractions. BDM (1–5 mM ) reduced the amplitude of the Ca²⁺ transient during twitches, but this effect was too small to explain the marked inhibition of BDM on twitch force. [Ca²⁺]_i reached at the end of 1‐s tetanic stimulation was not significantly affected by BDM (1.0 and 1.8 mM ) while the maximum tetanic tension was substantially reduced. The rate of relaxation during isometric tetanus was increased by BDM whereas the rate of decay of the Ca²⁺ transient was reduced in the presence of BDM. The results strongly suggest that BDM, under the experimental conditions used, mainly affects the contractile machinery resulting in altered performance of the cross‐bridges. These effects of BDM were evaluated in terms of the cross‐bridge model of 17 which was fitted to the experimental force–velocity data in the presence and absence of BDM.     

Effects of 2,3-butanedione monoxime on smooth-muscle contraction of guinea-pig portal vein

Effects of 2,3-butanedione-2-monoxime (BDM) on the contraction of intact and skinned smooth muscles from guinea-pig portal vein were examined. In intact preparations loaded with fura-2, 5–10 mM BDM markedly suppressed Ca²⁺ transients and force developments induced by 154 mM potassium and by phenylephrine (0.1 mM). On the other hand, in Ca²⁺-free depolarizing solution, BDM did not suppress phenylephrine (0.1 mM)-induced Ca²⁺ transient and force development. In skinned preparations obtained with Staphylococcus aureus -toxin treatment, BDM did not markedly affect active force development. The above results indicate that BDM suppresses contraction of the portal vein mainly by the inhibition of voltage-dependent cytosolic Ca²⁺ transients. An additional result suggests that BDM suppresses the force-enhancing effect of ₁-adrenergic agents on the contractile elements.     

Effects of 2,3-butanedione monoxime on activation of contraction and crossbridge kinetics in intact and chemically skinned smooth muscle fibres from guinea pig taenia coli

Summary The effects of 2,3-butanedione monoxime (BDM) were studied in smooth muscle fibres from guinea pig taenia coli. In intact muscle, active force during contractions induced by high-K⁺ was inhibited by about 10% in 1 mM BDM and by approximately 70% in 10 mM BDM. Intracellular [Ca²⁺] during contraction, measured with the fura-2 technique, was reduced in the presence of BDM. The reduction in force and [Ca²⁺] in the presence of 1 and 10 mM BDM could be reproduced by reduction in extracellular Ca²⁺, suggesting that BDM influences the Ca²⁺ entry or release. In skinned muscle preparations, BDM decreased the Ca²⁺ sensitivity of active force. This change could be explained by a decreased level of myosin light chain phosphorylation. In fibres maximally activated by thiophosphorylation, the effect of BDM on force occurred at higher concentrations; 10 mM gave no reduction of force and 60 mM 15% reduction. The maximal shortening velocity (V _max) and force were unaffected by 30 mM BDM in thiophosphorylated muscle and decreased almost in parallel in Ca²⁺-activated contractions. The present results suggest that BDM inhibits myosin light chain phosphorylation, directly decreases force generation at the crossbridge level and inhibits the Ca²⁺ translocation in smooth muscle. The effect on force in skinned fibres is observed at higher BDM concentrations than those reported to be required for inhibition of force in striated muscle. The inhibition of force in intact smooth muscle could be explained by an influence on Ca²⁺ translocation.     

Dissociation of force decline from calcium decline by preload in isolated rabbit myocardium

It is well known that the rate of intracellular calcium ([Ca²⁺]_i) decline is an important factor governing relaxation in unloaded myocardium. However, it remains unclear to what extent, under near physiological conditions, the intracellular calcium transient amplitude and kinetics contribute to the length-dependent increase in force and increase in duration of relaxation. We hypothesize that myofilament properties rather than calcium transient decline primarily determines the duration of relaxation in adult mammalian myocardium. To test this hypothesis, we simultaneously measured force of contraction and calibrated [Ca²⁺]_i transients in isolated, thin rabbit trabeculae at various lengths at 37°C. Time from peak tension to 50% relaxation (RT_50(tension)) increases significantly with length (from 49.8 ± 3.4 to 83.8 ± 7.4 ms at an [Ca²⁺]_o of 2.5 mM), whereas time from peak calcium to 50% decline (RT_50(calcium)) was not prolonged (from 124.8 ± 5.3 to 107.7 ± 11.4 ms at an [Ca²⁺]_o of 2.5 mM). Analysis of variance revealed that RT_50(tension) is significantly correlated with length (P < 0.0001). At optimal length, varying the extracellular calcium concentration increased both developed force and calcium transient amplitude, but RT_50(tension) remained unchanged (P = 0.90), whereas intracellular calcium decline actually accelerated (P < 0.05). Thus, an increase in muscle length will result in an increase in both force and duration of relaxation, whereas the latter is not primarily governed by the rate of [Ca²⁺]_i decline.     

Free cytosolic calcium during spontaneous contractions in smooth muscle of the guinea-pig mesotubarium

The free intracellular calcium ion concentration ([Ca²⁺]_i) was measured simultaneously with isometric force in strips of guinea-pig mesotubarium using the Fura-2 technique. During the relaxed period (5–15 min) between spontaneous contractions [Ca²⁺]_i continues to decrease after full mechanical relaxation to reach a minimal level of 86±8 nM (n=9) just before the start of the next contraction. During the spontaneous contractions (5–15 min) [Ca²⁺]_i reached a maximum of 211±19 nM and then oscillated between 155±16 nM and 194±9 nM. Increased extracellular Ca²⁺ concentration to 10 mM from the standard concentration of 1.5 mM caused a decreased frequency of spontaneous contractions and an increase in [Ca²⁺]_i both in the relaxed and contracted states. In 10 mM extracellular Ca²⁺, addition of AlF₄ ^–, as 1 mM NaF + 10 M AlCl₃, caused a sustained increase in [Ca²⁺]_i and maintained force. Addition of verapamil (10 M) in this situation decreased [Ca²⁺]_i to the resting level. The results suggest that the cyclic appearance of trains of action potentials is related to variation in [Ca²⁺]_i, possibly via inactivation of Ca²⁺-dependent K⁺ channels.     

Contractile properties of rabbit psoas muscle fibres inhibited by beryllium fluoride

The structure of truncated, recombinant Dictyostelium myosin motor domain complexed with Mg · ADP and slowly dissociating analogues of Pi has previously been characterized as two main states (S1-MgADP plus BeF vs. AlF ₂ ^– or Vi). The BeF bound state is thought to mimic the weak actin-binding M · ATP complex, while the states with AlF ₂ ^– or Vi bound mimic the M · ADP · Pi state. While the effects of AlF ₂ ^– and Vi on fibre mechanics have been previously described (Chase etal., 1994, 1993), the effects of BeF have not been characterized in detail. At pCa 4.5 (12°C), we measured (i) steady-state isometric tension, (ii) stiffness (K_S; 1 kHz sinusoids), and (iii)unloaded shortening velocity (V_u; slack test) in single skinned muscle fibres from rabbit psoas. Results were compared when tension was inhibited with either BeF or 2,3-butanedione-monoxime (BDM) or modulated by altering myoplasmic [Ca²⁺]. With 3 mM total fluoride, 1 mM BeF inhibited both tension and K_S by 50% (compared to 7–10 mM BDM and 50–100 M AlF ₂ ^– ). Increasing [BeF] to 10 mM further reduced tension to 15% P₀, but had little further effect on K_S; with BDM and altered [Ca²⁺], K_S scaled more proportionately with tension. Inhibition of tension and K_S by BeF was more rapidly reversible, compared with slow recovery from tension inhibition with AlF ₂ ^– or Vi. V_u exhibited a complex dependence on [BeF], being relatively unaffected by concentrations 1 mM, and becoming inhibited steeply for [BeF] above this level. With BDM, V_u co-varied more directly with force. Our results suggest that BeF may induce a different cross-bridge state in fibres than do AlF ₂ ^– or Vi, but all three analogues of Pi form complexes that mimic crossbridge states that follow ATP hydrolysis.     

Potentiation of sarcoplasmic reticulum Ca2+ release by 2,3-butanedione monoxime in crustacean muscle

The effect of the chemical phosphatase 2,3-butanedione monoxime (BDM) on various aspects of excitation/contraction coupling in crustacean muscle was investigated. Despite having a depressant effect on vertebrate skeletal and cardiac muscle, BDM was a potentiator of contraction in crustacean muscle. At concentrations of 1–3 mM BDM caused an increase of potassium contractures in bundles of fibers isolated from crayfish muscle. At higher concentrations BDM caused oscillatory contractions by itself. In single voltage-clamped cut muscle fibers loaded with rhod-2, BDM (0.5–2 mM) potentiated the magnitude and duration of intracellular Ca²⁺ transients elicited by depolarization. At the same time BDM did not affect the rate of Ca²⁺ removal from the myoplasm under conditions where Ca²⁺ release was blocked by tetracaine. Nor did BDM increase Ca²⁺ entry; in fact it caused a decrease in the amplitude of the inward Ca²⁺ current (I _Ca). In microsomes isolated from lobster muscle, BDM also potentiated Ca²⁺ release induced by caffeine and at higher concentrations (above 3 mM) induced release by itself. At the same time it had little effect on Ca²⁺ uptake. These results indicate that BDM potentiates Ca²⁺ release in crustacean muscle possibly by dephosphorylation of the Ca²⁺-release channel.     

2,3-Butanedione monoxime (BDM) decreases sarcoplasmic reticulum Ca content by stimulating Ca release in isolated rat ventricular myocytes

 The effects of 2,3-butanedione monoxime (BDM) were examined using rat ventricular myocytes loaded with Indo-1 to measure the intracellular Ca concentration ([Ca²⁺]_i). BDM (10 mM) produced a transient increase of the systolic Ca transient with no steady-state effect on its magnitude. This transient increase was more marked when BDM was applied after having decreased the external Ca concentration from 1 to 0.1 mM. There was a transient increase of resting [Ca²⁺]_i in both quiescent and electrically stimulated cells. Prior application of BDM decreased the rise of [Ca²⁺]_i produced by caffeine. In voltage-clamped cells the rise of [Ca²⁺]_i produced by BDM was accompanied by a transient inward current attributed to the electrogenic Na-Ca exchange. The amount of Ca lost from the cell upon application of 10 mM BDM could be estimated either from the integral of the BDM-evoked current or from the reduction of the integral of a caffeine-evoked current and corresponded to about 50% of the sarcoplasmic reticulum (s.r.) Ca content. The decrease of s.r. Ca content and the transient potentiation of the systolic Ca transient suggest that BDM acts by stimulating Ca-induced Ca release. These effects must be allowed for when using BDM. Received: 27 March 1998 / Received after revision: 12 May 1998 / Accepted: 13 May 1998     

Effects of β-adrenoceptor stimulation on intracellular Ca transients and tension in rat ventricular muscle

Effects of -adrenoceptor stimulation on intracellular Ca²⁺ transients and tension were explored in rat ventricular muscles injected with aequorin. Adrenaline (0.05–5.0 M) and isoproterenol (0.05–1.0 M) increased the peak of twitch tension and accelerated relaxation. The former effect depended on Ca²⁺ concentration in Tyrode's solution ([Ca²⁺]_o) and the stage of the experiment. Low concentrations of these drugs added to normal Tyrode's solution containing 2 mM [Ca²⁺]_o did not potentiate twitch tension in the early stage of the experiments. These drugs increased the peak of the aequorin light signal and slightly accelerated the falling phase of the light especially the tail. Effects of dibutyryl-cyclic AMP (DB-cAMP) (0.1–5.0 mM) and 3-isobutyl-1-methylxanthine (IBMX) (0.01–0.5 mM) were qualitatively similar to those of adrenaline and isoproterenol.Isoproterenol applied at the peak of Na-deficient contracture decreased tension without significantly changing the light signal; similar results were obtained in the presence of ryanodine (1 M).The results were interpreted as follows: The increase of intracellular cAMP induced by -adrenoceptor stimulation facilitated Ca²⁺ uptake by sarcoplasmic reticulum (SR) and decreased Ca²⁺ sensitivity of contractile elements. Faster relaxation induced by cAMP was considered to be due to the decrease of Ca²⁺ sensitivity of contractile elements and faster Ca²⁺ uptake by SR. The slightly faster falling phase of light transient might be due to the faster Ca²⁺ uptake by SR, which predominates over the slower fall of [Ca²⁺]_i induced by the decreased Ca²⁺ sensitivity of the contractile element.     

Contractile activation and the effects of 2,3-butanedione monoxime (BDM) in skinned cardiac preparations from normal and dystrophic mice (129/ReJ)

(1) Small cardiac myofibrillar preparations were obtained from the right ventricle of normal (129/ReJ) and dystrophic (129/ReJ dy/dy) mice and were chemically skinned in a relaxing solution by exposure to Triton X-100 (3% v/v). (2) The isometric force produced in these skinned cardiac preparations at different sarcomere lengths was measured in solutions of different [Ca²⁺] and ionic strength. The effect of the negative inotropic drug 2,3-butanedione monoxime (BDM), which is known to act at the myofibrillar level was also investigated. (3) The murine cardiac preparation from normal animals was found to develop 50% maximal force at a pCa (=–log₁₀[Ca²⁺]) of 5.59±0.08 and 5.94±0.03 (mean ±SD) under physiological (ionic equivalents concentration, I=154 mM; pH 7.10; [Mg²⁺] 1 mM) and low ionic strength (I=94 mM; pH 7.10; [Mg²⁺] 1 mM) conditions respectively. The isometric force curves were significantly shallower at low ionic strength (Hill coefficient, 1.8±0.1) than at physiological ionic strength (Hill coefficient, 2.6±0.3) and the sarcomere length effect on the force-pCa relation was markedly reduced at lower ionic strength. (4) Increasing BDM concentrations in solutions up to 100 mM reduced the maximum Ca²⁺-activated force of cardiac preparations from normal mice to less than 6% of the control values in a dose dependent fashion. BDM also rendered the cardiac preparations less sensitive to Ca²⁺ by a factor of up to 1.5 in a process which showed saturation at BDM concentrations higher than 15 mM. (5) Cardiac preparations from dystrophic animals compared with those from normal mice were significantly more sensitive to Ca²⁺ under physiological conditions, were more sensitive to the action of BDM at concentrations higher than 15 mM, changed sensitivity to Ca²⁺ less following a change in sarcomere length and in general were less affected by a decrease in ionic concentration. (6) The results indicate that dystrophy in mice affects the characteristics of both the contractile and regulatory systems of cardiac muscle and that BDM directly affects the Ca²⁺-activated contractile response possibly by binding to saturable sites on the myofilaments.     

The effects of the myosin-II inhibitor N-benzyl-p-toluene sulphonamide on fatigue in mouse single intact toe muscle fibres

Aim: This study determined whether fatigue in skeletal muscle is primarily due to the repeated elevations of myoplasmic free calcium concentration ([Ca²⁺]_i) or to metabolite accumulation. Methods: We examined the effects of N‐benzyl‐p‐toluene sulphonamide (BTS) which is a potent and specific inhibitor of fast muscle myosin‐II on the development of fatigue in mouse flexor digitorum brevis (FDB) muscle fibres. Single intact FDB fibres were micro‐injected with indo‐1 to monitor changes in [Ca²⁺]_i and stimulated repeatedly for a maximum of 150 tetani or until force declined to 40%. Results: BTS markedly reduced tetanic force but had no effect on the tetanic [Ca²⁺]_i transients. When fatigue was induced in the presence of BTS, the reduction in [Ca²⁺]_i and force transients occurred much more slowly than in the absence of BTS. The extent of force depression was similar after induction of fatigue in fibres exposed to Tyrode only or to BTS and force recovered to the same extent. Conclusion: The results suggest that the decrease in tetanic [Ca²⁺]_i and force caused during fatigue are due mainly to accumulated metabolic changes.     

Intracellular free calcium concentration / force relationship in rabbit inferior vena cava activated by norepinephrine and high K+

The changes in isometric force and the underlying fluctuations in intracellular free calcium concentration ([Ca²⁺]_i) were monitored simultaneously in thin sheets of rabbit inferior vena cava loaded with the fluorescent Ca²⁺ indicator fura-2. In resting tissues bathed in physiological saline solution, the estimated [Ca²⁺]_i was approximately 105 nM. The -adrenergic agonist norepinephrine (10 M) caused an initial rise in [Ca²⁺]_i to 264 nM during force development, which dropped to 216 nM during force maintenance. The maintained norepinephrine-induced increase in force and [Ca²⁺]_i was reversed in Ca²⁺-free (2 mM EGTA) solution. Membrane depolarization by high K⁺ (80 mM) significantly increased [Ca²⁺]_i to 234 nM. Compared to norepinephrine, high K⁺ caused about the same steady-state increase in [Ca²⁺]_i, but a smaller increase in force. [Ca²⁺]_i/force curves were constructed at different concentrations of extracellular Ca²⁺, with either norepinephrine or high K⁺ as a stimulant. The curve generated with norepinephrine was located to the left of that generated with high K⁺.     

Paradoxical decrease in cytosolic calcium with increasing depolarization by potassium in guinea-pig mesotubarium smooth muscle

The free intracellular Ca²⁺ concentration ([Ca²⁺]_i) was measured simultaneously with isometric force in strips of guinea-pig mesotubarium using the Fura-2 technique. [Ca²⁺]_i and force were maximal at a relatively low (30 mM) concentration of extracellular K⁺ ([K⁺]_o), and declined at 90 and 140 mM K⁺. Plateau values of both [Ca²⁺]_i and force were higher in the presence of 5 · 10^–6 M ryanodine, indicating that the sarcoplasmic reticulum (SR) contributes to the decline with depolarization. Force and [Ca²⁺]_i at 90 mM K⁺ were both lower then the high-K⁺ solution was applied after a period in 30 mM K⁺ than after a period in normal solution (5.9 mM K⁺), consistent with inactivation of Ca²⁺ channels during prolonged depolarization. Addition of carbachol to the depolarized muscle caused a maintained increase in force without maintained increase in [Ca²⁺]_i. We conclude that the decrease in force at increased [K⁺]_o (the calcium-potassium paradox) is due to a membrane-potential-mediated decrease in [Ca²⁺]_i and, to a lesser extent, to desensitization of the contractile-regulatory apparatus to Ca²⁺.     

The effects of 2,3-butanedione monoxime (BDM) on the force-velocity relation in single muscle fibres of the frog

The effects of 2,3-butanedione monoxime (BDM) on the forcevelocity relaion were studied in single fibres from the anterior tibialis muscle of Rana temporaria (2.2 μm sarcomere length, temperature 1.9-2.4 d?C). BDM (1.0 and 1.8 mM) suppressed the maximum tetanic force (P_o) and the maximum speed of shortening (V_max), and increased the main curvature of the force-velocity relation. The biphasic shape of the forcevelocity curve was maintained well in the presence of BDM, but the interrelation between the two portions of the forcevelocity relation was significantly changed. Caffeine (0.5 mM) added in the presence of BDM increased the initial rate of rise of force during twitch and tetanus, increased the twitch amplitude, but did not affect the maximum tetanic force. The latter finding suggests that the contractile system was fully activated during tetanus in the presence of BDM. The results support the view that BDM affects the cross-bridge function by exerting a direct action upon the contractile apparatus. The decrease in tetanic force and the change of the force-velocity relation induced by BDM may be interpreted to show that a larger fraction of the attached cross-bridges is in a state of low force production under the influence of BDM. This view is further supported by the observation that the instantaneous stiffness of the muscle fibre is reduced proportionally less by BDM than the tetanic force.     

Agonist-specific myosin phosphorylation and intracellular calcium during isometric contractions of arterial smooth muscle

The relationship between phosphorylation of the 20-kDa myosin light chain, intracellular calcium levels ([Ca²⁺]_i), and isometric force was studied during prolonged activation of arterial smooth muscle. Aequorin, preloaded into ferret aortic strips, was used as a [Ca²⁺]_i indicator. Two dimensional polyacrylamide gel electrophoresis was used to determine the phosphorylation levels of the 20-kDa myosin light chain (LC20). During the 30-min depolarization of arterial smooth muscle by K⁺ (21 mM), both LC20 phosphorylation and [Ca²⁺]_i increased significantly at all time points examined as did the steady state stress. A transient rise in LC20 phosphorylation and [Ca²⁺]_i occurred within 30 s, followed by suprabasal levels through the 10-min period during a sustained alpha₁-mediated activation by 10^–5 M phenylephrine whereas a higher force was developed at a shorter time compared to K⁺. An active phorbol ester 12-deoxyphorbol 13-isobutyrate 20-acetate (DPBA, 10^–6 M) induced a slow contraction of similar magnitude to that induced by K⁺ without significantly changing either [Ca²⁺]_i or LC20 phosphorylation over a 90-min period. These results demonstrate that the amount of LC20 phosphorylation correlates with the [Ca²⁺]_i in all three types of activation. The initial levels of [Ca²⁺]_i and LC20 phosphorylation correlate with the onset of force development but not the magnitude of steady state stress, suggesting a role for [Ca²⁺]_i and LC20 phosphorylation in regulating the cross bridge cycling rate during tension development. The lack of a detectable increase in [Ca²⁺]_i and LC20 phosphorylation during DPBA activation suggests that sites other than LC20, phosphorylated by protein kinase C, may be involved in regulating smooth muscle contraction.     

The effect of ionic strength on the kinetics of rigor development in skinned fast-twitch skeletal muscle fibres

 Recent atomic 3-D reconstructions of the acto-myosin interface suggest that electrostatic interactions are important in the initial phase of cross-bridge formation. Earlier biochemical studies had also given strong evidence for the ionic strength dependence of this step in the cross-bridge cycle. We have probed these interactions by altering the ionic strength (Γ/2) of the medium mainly with K⁺, imidazole⁺ and EGTA^2– to vary charge shielding. We examined the effect of ionic strength on the kinetics of rigor development at low Ca²⁺ (experimental temperature 18–22°C) in chemically skinned single fast-twitch fibres of mouse extensor digitorum longus (EDL) muscle. On average the delay before rigor onset was 10 times longer, the maximum rate of rigor tension development was 10 times slower, the steady-state rigor tension was 3 times lower and the in-phase stiffness was 2 times lower at high (230 mM) compared to low (60 mM) ionic strength. These results were modelled by calculating ATP depletion in the fibre due to diffusional loss of ATP and acto-myosin Mg.ATPase activity. The difference in delay before rigor onset at low and high ionic strength could be explained in our model by assuming a 15 times higher Mg.ATPase activity and a threefold increase in K _m in relaxing conditions at low ionic strength. Activation by Ca²⁺ induced at different time points before and during onset of rigor confirmed the calculated time course of ATP depletion. We have also investigated ionic strength effects on rigor development with the activated troponin/tropomyosin complex. ATP withdrawl at maximum activation by Ca²⁺ induced force transients which led into a ”high rigor” state. The peak forces of these force transients were very similar at low and high ionic strength. The subsequent decrease in tension was only 10% slower and steady-state ”high rigor” tension was reduced by only 27% at high compared to low ionic strength. Addition of 10 mM phosphate to lower cross-bridge attachment strongly suppressed the transient increases in force at high ionic strength and reduced the steady-state rigor tension by 17%. A qualitatively similar but smaller effect of phosphate was observed at low ionic strength where steady-state rigor force was reduced by 10%. The data presented in this study show a very strong effect of ionic strength on rigor development in relaxed fibres whereas the ionic strength dependence of rigor development after thin filament activation was much less. The data confirm the importance of electrostatic interactions in cross-bridge attachment and cross-bridge-attachment-induced activation of thin filaments. Received: 3 September 1997 / Received after revision and accepted: 12 December 1997     

The role of [Ca2+]i, membrane potential and pHi in the relaxation of rat mesenteric arteries to hyperosmolar acetate

 In vitro both acetate and hyperosmolarity cause vasodilation, which could be physiologically important during food ingestion and during peritoneal dialysis. The purpose of this study was to investigate the role of the intracellular calcium concentration ([Ca²⁺]_i, measured with fura-2), membrane potential (measured with glass microelectrodes) and intracellular pH [pH_i, measured with bis-carboxyethylcarboxyfluorescein (BCECF)] in the vasodilation. Hyperosmolar sodium acetate (30 mM) concentration dependently relaxed noradrenaline-precontracted arteries. This response was associated with hyperpolarization and a fall in [Ca²⁺]_i. In arteries precontracted with 50 mM K⁺ the relaxation was associated with a decrease of [Ca²⁺]_i but no change in membrane potential. Isoosmolar sodium acetate neither relaxed or affect [Ca²⁺]_i of K⁺-precontracted arteries, but induced a small relaxation with no reduction in [Ca²⁺]_i in noradrenaline-precontracted arteries. Hyperosmolar acetate caused a transient reduction of pH_i that was unrelated to relaxation. It is concluded that the mechanisms responsible for the relaxation to hyperosmolar acetate involve a decrease of [Ca²⁺]_i, which is only partly explained by hyperpolarization and probably a decrease in the sensitivity of the contractile proteins to [Ca²⁺]_i. pH_i seems not to play a role in these effects. Received: 14 January 1998 / Received after revision: 22 April 1998 / Accepted: 11 May 1998     

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.     

The influence of ionic strength upon relaxation from rigor induced by flash photolysis of caged-ATP in skinned murine skeletal muscle fibres

The influence of ionic strength upon relaxation kinetics from rigor in skinned murine extensor digitorum longus (EDL) skeletal muscle fibres was examined using photolysis of caged-ATP at low Ca²⁺. The ionic strength was adjusted with either KMeSO₃ or ethylene glycol bis-(-aminoethyl ether)N,N,N,N-tetraacetic acid, dipotassium salt (K₂EGTA) in the range of /2=65–215 mM, or I.E. 49–194 mM, where I.E. denotes ionic equivalent. Following rigor development at a/2 of 165–215 mM (I.E. 144–194 mM), the liberation of approximately 0.5 mM ATP resulted in an initial 6-to 10-ms detachment phase with a decline in force of approximately 10–20% followed by a 10-to 30-ms reattachment with up to a 60% increase compared to the corresponding rigor level and a final detachment leading to complete relaxation. Interestingly, when similar ATP concentrations were liberated at lower ionic strengths between a /2 of 65 mM and 110 mM (I.E. 60–100 mM), the initial detachment phase was shortened and force decreased by only approximately 5–10%, while the following reattachment phase was lengthened and led to an increased steady-state force of approximately 20–80% without final relaxation. ATP-induced detachment and subsequent reattachment were mainly determined by the currently present ionic strength and were relatively independent of the preceding rigor state which had been developed at higher or lower ionic strengths. The effects of phosphate and apyrase on the force transient suggest that reattachment of ADP- binding crossbridges may contribute to the increase in tension at high and even more at low ionic strengths. The study shows that the kinetics of initial fast relaxation and subsequent redevelopment of force following flash photolysis of similar ATP concentrations are markedly modified by the ionic strength in the narrow range of between 65 mM and 215 mM.     

The effects of 2,3-butanedione monoxime on initial heat,tension, and aequorin light output of ferret papillary muscles

At low concentrations (up to 5 mM) the compound 2,3-butanedione monoxime (BDM) was found to reduce twitch tension and initial heat production in isolated papillary muscles without significantly affecting the size of the intracellular Ca transient measured with aequorin luminescence. Higher concentrations of BDM caused further inhibition of twitch tension and heat production with a fall in the size of the Ca⁺ transient. The size of the aequorin transient was 50% of the control value at 15 mM BDM while twitch tension was negligible. These results suggest that BDM selectively inhibits Ca²⁺ activated force in cardiac muscle at low concentrations with additional effects on intracellular calcium at concentrations above 5 mM.