Measurements of the perivascular PO2 in the vicinity of the pial vessels of the cat

The effects of halothane on caffeine-induced tension transients in functionally skinned myocardial fibers were investigated. Fiber bundles from mechanically disrupted rabbit right ventricular papillary muscles were mounted on a tension transducer. The fiber preparation was loaded with Ca²⁺; Ca²⁺ was then released by the use of caffeine (25 mM); and the area of the resulting tension transient was measured. Each preparation was sequentially transferred from control to test to control solution. The control solutions were equilibrated with 100% N₂, and the test solutions with a mixture of N₂ and various halothane concentrations. The preparation was exposed to halothane during the Ca²⁺ uptake or the release phase only, or during both Ca²⁺ uptake and release phases. The areas of the test tension transients were compared with those of the two control tension transients. It was found that halothane depressed the caffeine-induced tension transient either during the uptake phase or the combined-uptake-and-release phase but not during the release phase. The halothane-induced depression was dose-dependent, reversible, and comparable to the depression observed in intact isolated papillary muscles. We conclude that halothane could induce myocardial depression by inhibiting Ca²⁺ uptake by the sarcoplasmic reticulum.Supported by Research Grants HL 20754 and AM 17081 from the National Institutes of Health and a Research Starter Grant from the Pharmaceutical Manufactures Association Foundation. Halothane was supplied by Ayerst Laboratories, Inc.     

Mechanism of adenosine 3′,5′-monophosphate (cAMP)-induced increase in Ca2+ uptake by the sarcoplasmic reticulum in functionally skinned myocardial fibers

The increased rate of Ca²⁺ uptake and ATPase activity in isolated cardiac sarcoplasmic reticulum (SR) by adenosine 3,5-monophosphate (cAMP) has been shown to be activated by a cAMP-dependent protein kinase (cAMP kinase). Functionally skinned myocardial fiber preparations were used to study the mechanisms of cAMP action on the SR at the same time that tension was monitored. cAMP effects were studied on Ca²⁺-activated tension of the contractile proteins, and on Ca²⁺ uptake and release from the SR using caffeine-induced tension transients. Neither cyclic AMP (0.1–5 M) nor the catalytic subunit of cAMP kinase (0.1–1 M) (PK-C) significantly changed either the maximal or the submaximal Ca²⁺-activated tension. The areas of the tension transients were unchanged when cAMP was present in the releasing solution (release phase), and were significantly increased up to a mean of about 80% when cAMP or PK-C was present in the Ca²⁺ loading solutions (uptake phase). The increased tension transient was blocked by the heat-stable inhibitor of cAMP kinase. We conclude that cAMP-induced increases in Ca²⁺ uptake by the SR could play an important role in the positive inotropic effect. cAMP kinase could thus play a crucial role in the regulation of myocardial contractility.     

Series elastic properties of skinned muscle fibres in contraction and rigor

Summary Isometric tension of skinned fibres from the frog semitendinosus muscle is sigmoidally related to Ca²⁺ concentration betweenpCa 7 and 6. Stiffness measurements showed that the Ca²⁺-activated tension may be due to recruitment of attached cross-bridges. In the absence of ATP (rigor solution) the skinned fibre develops a rigor tension which reaches about 80–110% of the maximum Ca²⁺-activated tension.However, stiffness measurements showed that in rigor many more cross-bridges are attached to actin at any one moment than in contraction. It was concluded that the force per cross-bridge is 37% smaller in rigor than in contraction.     

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.     

Skeletal muscle excitation-contraction coupling I

Porcine skeletal muscle fibers were studied to determine if the defect in malignant hyperthermia involves transverse tubule (TT) to sarcoplasmic reticulum (SR) communication. Pelled (mechanically skinned) skeletal muscle fibers from normal and malignant hyperthermia susceptible (MHS) pigs were stimulated with Cl^– to ionically depolarize transverse tubules and thereby trigger Ca²⁺ release from SR. Caffeine was used to directly stimulate the Ca²⁺-induced Ca²⁺ release mechanism (CaIR) of the sarcoplasmic reticulum. Calcium released from internal fiber stores was monitored as Ca²⁺-activated isomeric force generation in the form of tension transients. Cl^–-induced tension transients result from a primary component of Ca²⁺ release which triggers a secondary CaIR component; CaIR and caffeine contractures were eliminated by procaine. The primary component of Cl^–-induced SR Ca²⁺ release was indistinguishable for MHS and normal skeletal peeled fibers at all TT resting and Cl^– stimulation conditions. Only the magnitude of the secondary CaIR component was significantly larger in MHS fibers. The [Ca²⁺] threshold for secondary CaIR was lowered by resting TT depolarization in both normal and MHS fibers. Conditions for resting TT hyperpolarization selectively reduced the magnitude of the secondary CaIR component of MHS fibers, making them indistinguishable from normal.     

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.     

Modulation of sarcoplasmic reticulum Ca2+-release channels by caffeine,Ca2+, and Mg2+ in skinned myocardial fibers of fetal and adult rats

Ryanodine causes depression of the caffeine-induced tension transient (ryanodine depression) in skinned muscle fibers, because it blocks the sarcoplasmic reticulum (SR) Ca²⁺-release channels [Su, J. Y. (1988) Pflügers Arch 411:132–136, 371–377; (1992) Pflügers Arch 421:1–6]. This study was performed to examine the sensitivity of SR Ca²⁺-release channels to ryanodine in fetal compared to adult myocardium and to investigate the influence of Ca²⁺, caffeine, and Mg²⁺ on ryanodine depression in skinned fibers. Ryanodine (0.3 nM–1 M) caused a dose-dependent depression in skinned myocardial fibers of the rat, and the fetal fibers (IC₅₀74 nM) were 26-fold less sensitive than those of the adult (IC₅₀2.9 nM). The depression induced by 0.1 M or 1 M ryanodine was a function of [caffeine], or [Ca²⁺] (pCa<6.0), which was potentiated by caffeine, and an inverse function of [Mg²⁺]. At pCa>8.0 plus 25 mM caffeine, a 20% ryanodine depression was observed in both the fetal and adult fibers, indicating independence from Ca²⁺. Ryanodine depression in skinned fibers of the fetus was less affected than that seen in the adult by pCa_i, [caffeine]_i, or 25 mM caffeine plus pCa_i or plus pMg_i (IC₅₀pCa 4.5 versus 5.1; caffeine 12.7 mM versus 2 mM; pCa 6.7 versus 7.3; and pMg 3.9 versus 3.3 respectively). The results show that the SR Ca²⁺-release channel in both fetal and adult myocardium is modulated by Ca²⁺, caffeine, and Mg²⁺. It is concluded that less ryanodine depression seen in the skinned fibers of the fetus, indicating a relatively insensitive SR Ca²⁺-release channel, could contribute to the resistance of intact myocardium to ryanodine.     

Mechanisms of ryanodine-induced depression of caffeine-induced tension transients in skinned striated rabbit muscle fibers

Evidence suggests that ryanodien affects ligandgated calcium channels in the sarcoplasmic reticulum (SR) resulting in depressed muscle contraction. In skinned fibers from striated muscle the effects of ryanodine were examined (1) on Ca²⁺ uptake and on Ca²⁺ release to differentiate whether the effects are on the pump or channel, and (2) during the tension transient, with ryanodine exposure at various times either simultaneous with or directly after exposure to caffeine. Of total calcium content in the SR, 25 mM caffeine released>90% in papillary muscle (PM), 25% in soleus (SL), and 20% in adductor magnus (AM). Ryanodine (100 M for 1–3 s for AM and SL; 1 M for 7–10 s for PM), in the initial loading phase, did not significantly change, and in the initial release phase, markedly depressed the subsequent control caffeine-induced tension transients (C₂) in all three muscle types. The depression increased with increasing time of exposure to ryanodine (10 M) in the order of PM>AM>SL. Upon introduction of ryanodine after caffeine-induced tension transients, maximal depression was observed at half-maximum rise of the tension transient, followed by recovery of depression to completion in SL, and only partially in AM and PM at steady state of relaxation. The extent of recovery was in the order of SL>AM>PM. The data suggest that ryanodine affects Ca²⁺ releasing channel as a result of its binding to open channels.     

MgADP− increases maximum tension and Ca2+ sensitivity in skinned rabbit soleus fibers

Increasing concentrations of MgADP^– or MgCDP^– in the millimolar range cause an increase in the maximum Ca²⁺-activated tension that a skinned rabbit soleus muscle fiber can develop in the presence of 2 mM MgATP^2– or MgCTP² respectively. In contrast, the maximal Ca²⁺-activated ATPase activity of the fiber decreases in the presence of MgADP^–. As the nucleoside diphosphate (MgADP^– or MgCDP^–) concentration is increased, the Ca²⁺ concentration required for half-maximal activation of tension is reduced.MgADP^– has a similar effect on the Ca²⁺ concentration required to half-maximally activate the fiber ATPase. The effects on tension are due to magnesium nucleoside diphosphate and not some other form of nucleoside diphosphate since the effects occur at both low (pMg 4) and control (pMg 3) Mg²⁺ concentrations. Cooperativity, as judged by the Hill n value relating isometric tension and Ca²⁺, is less in the presence of 5 mM MgADP^– as compared to a control (no added MgADP^–) n value. Increasing concentrations of inorganic phosphate (Pi) in the millimolar range decrease maximum Ca²⁺-activated tension, and increase the concentration of Ca²⁺ required to half-maximally activate tension, effects opposite to those of MgADP. These data are consistent with the hypothesis that cooperative interactions between actin and myosin can affect the affinity of troponin for Ca²⁺.A preliminary report of this work was given at the Biophysical Society Meeting, February 1985, W. G. L. Kerrick, P. E. Hoar (1985) Effects of nucleotide diphosphate and inorganic phosphate on tension in skinned soleus and smooth muscle cells. Biophys J 47:296a     

Ruthenium red affects the contractile apparatus but not sarcoplasmic reticulum Ca2+ release of skinned papillary muscle

Ruthenium red has been shown to have a positive inotropic effect on isolated perfused hearts. The cellular mechanism of this action is not clear. Ruthenium red is able to block the Ca²⁺ release channel in isolated sarcoplasmic reticulum (SR) vesicle and reconstituted channel preparations. However, the effect of ruthenium red on SR Ca²⁺ release has not been studied in skinned cardiac muscle preparations. In the present study we investigated the actions of ruthenium red on both the characteristics of force generation by the contractile apparatus and Ca²⁺ release from the SR in chemically skinned rat papillary muscle. Ruthenium red (2 and 10 M) significantly increased the Ca²⁺ sensitivity of the contractile apparatus (decreasing Ca²⁺ required for the half-maximal response from 1.56±0.04 M to 1.46±0.05 M) but had no effect on the maximal Ca²⁺-activated force in triton X-100 treated fibers. This result may suggest one explanation for the positive inotropic effect of ruthenium red on the heart. On the other hand, ruthenium red had no significant effect on either caffeine-induced Ca²⁺ release or Ca²⁺-induced Ca²⁺ release from the SR in saponin-skinned muscle fibers. Lack of a blocking effect on SR Ca²⁺ release by ruthenium red in skinned fibers suggests that the SR Ca²⁺ channels in intact preparations have characteristics that are different from those of either vesicular or reconstituted channel preparations.     

The role of the adrenergic innervation to the pancreatic islets in the control of insulin release during exercise in man

Ca²⁺-activated isometric force was recorded in skinned (sarcolemma mechanically removed) segments of frog skeletal muscle fibers immersed in bathing solutions of different pH (5.0–10.5) and Ca²⁺ concentrations. Force in maximally activated fibers was near zero at pH 5.5, increased as pH increased to 7.5 remained relatively constant until pH 9.0 and then rapidly declined to zero by pH 10.5. The Ca²⁺ concentration at which 50% of maximum force was developed decreased 25-fold as pH increased from 5.5–7.5. The data also indicate that, while the fibers remain viable with acidosis, they deteriorate rapidly with alkalosis. These observations may be relevant clinically, since the parallel known effects of acidosis on cardiac contractility. The possible sites of action of H⁺ on the Ca²⁺-activated force generating mechanisms are discussed.     

Effects of ryanodine on skinned skeletal muscle fibers of the rabbit

The mechanism(s) of ryanodine-induced contracture of skeletal muscle were studied in skinned fibers from soleus (SL) and adductor magnus (AM) (slow- and fast-twitch skeletal muscles) of rabbits. Pieces of SL or AM were homogenized (sarcolemma disrupted). Single fibers were dissected from the homogenate and mounted on photodiode force transducers. At concentrations 1–50 M, ryanodine slightly but significantly increased the submaximal Ca²⁺-activated tension development of the contractile proteins in skinned fibers of AM but not of SL. Ryanodine in uptake phase or release phase increased caffeine-induced tension transients in the SR of both muscle types; however, no dose-response relation was found. Ryanodine 1 M decreased, however, the second control tension transients in a dose-dependent manner. The depression was nearly irreversible and activity-dependent. The concentrations of ryanodine that inhibited the second control tension transients by 50% were 10 M and 5 M for SL and AM, respectively, following ryanodine administration in the release phase, and 100 M and 30 M, respectively, for these preparations after the drug was present in the uptake phase. The quantity of calcium released from the SR by Triton X-100 and caffeine in the second control tension transient was unchanged by ryanodine at all concentrations tested when compared with that of the absence of ryanodine. The present findings suggest that the ability of ryanodine to increase immediate calcium release from the SR, and in AM but not SL, to increase the sensitivity of the contractile proteins to Ca²⁺ underlies the contracture caused by this agent in intact skeletal muscles. The delayed decreased Ca²⁺ efflux by caffeine, as evidenced by depression of tension transient with no change in the calcium content may be responsible for the decreased twitch tension caused by this agent.     

[Ca2+]i-dependent membrane currents in guinea-pig ventricular cells in the absence of Na/Ca exchange

Transient inward currents (I _ti) during oscillations of intracellular [Ca²⁺] ([Ca²⁺]_i) in ventricular myocytes have been ascribed to Na/Ca exchange. We have investigated whether other Ca²⁺-dependent membrane currents contribute to I _ti in single guinea-pig ventricular myocytes, by examining membrane currents during [Ca²⁺]_i oscillations and during caffeine-induced Ca²⁺ release from the sarcoplasmic reticulum in the absence of Na⁺. Membrane currents were recorded during whole-cell voltage clamp and [Ca²⁺]_i measured simultaneously with fura-2. In the absence of Na/Ca exchange, i.e., with Li⁺, Cs⁺ or N-methyl-D-glucamine (NMDG⁺) substituted for Na⁺, the cell could be loaded with Ca²⁺ by repetitive depolarizations to +10 mV, resulting in spontaneous [Ca²⁺]_i oscillations. During these oscillations, no inward currents were seen, but instead spontaneous Ca²⁺ release was accompanied by a shift of the membrane current in the outward direction at potentials between –40 mV and +60 mV. This [Ca²⁺]_i-dependent outward current shift was not abolished when NMDG⁺ was substituted for internal monovalent cations, nor was it sensitive to substitution of external Cl^–. It was however, sensitive to the blockade of I_Ca by verapamil. These results suggest that the transient outward current shift observed during spontaneous Ca²⁺ release represents [Ca²⁺]_idependent transient inhibition of I _Ca. Similarly, during the [Ca²⁺]_i transients induced by brief caffeine (10 mM) applications, we could not detect membrane currents attributable to a Ca²⁺-activated nonselective cation channel, or to a Ca²⁺-activated Cl^– channel; however, transient Ca²⁺-dependent inhibition of I _Ca was again observed. We conclude that neither the Ca²⁺-activated nonselective cation channel nor the Ca²⁺-activated Cl^– channel contribute significantly to the membrane currents during spontaneous [Ca²⁺]_i oscillations in guineapig ventricular myocytes. However, in the voltage range between –40 mV and +60 mV Ca²⁺-dependent transient inhibition of I _Ca will contribute to the oscillations of the membrane current.     

Caffeine and histamine-induced oscillations of K(Ca) current in single smooth muscle cells of rabbit cerebral artery

In the present experiment, we characterized the intracellular Ca²⁺ oscillations induced by caffeine (1 mM) or histamine (1–3 M) in voltage-clamped single smooth muscle cells of rabbit cerebral (basilar) artery. Superfusion of caffeine or histamine induced periodic oscillations of large whole-cell K⁺ current with fairly uniform amplitudes and intervals. The oscillatory K⁺ current was abolished by inclusion of ethylenebis(oxonitrilo)tetraacetate (EGTA, 5 mM) in the pipette solution. Caffeine- and histamine-induced periodic activation of the large-conductance Ca²⁺-activated K⁺ [K_(Ca)] channel was recorded in the cell-attached patch mode. These results suggest that the oscillations of K⁺ current are carried by the K_(Ca) channel and reflect the oscillations of intracellular Ca²⁺ concentration ([Ca²⁺]_i). Ryanodine (1–10 M) abolished both caffeine- and histamine-induced oscillations. Caffeine- induced oscillations were abolished by the sarcoplasmic reticulum Ca²⁺-adenosine 5-triphosphatase (Ca²⁺-ATPase) inhibitor, cyclopiazonic acid (10 M), and a high concentration of caffeine (10 mM). Inclusion of heparin (3 mg/ml) in the pipette solution blocked histamine-induced oscillations, but did not block caffeine-induced oscillations. By the removal of extracellular Ca²⁺, but not by the addition of verapamil and Cd²⁺, the caffeine-induced oscillations were abolished. Increasing Ca²⁺ influx rate increased the frequencies of caffeine-induced oscillations. Spontaneous oscillations were also observed in cells that were not superfused with agonists, and had similar characteristics to the caffeine-induced oscillations. From the above results, it is concluded, that in smooth muscle cells of the rabbit cerebral (basilar) artery, ryanodine-sensitive Ca²⁺-induced Ca²⁺ release pools play key roles in the generation of caffeine- and histamine-induced intracellular Ca²⁺ oscillations.     

Caffeine-induced calcium release from isolated sarcoplasmic reticulum of rabbit skeletal muscle

The essential conditions for the Ca²⁺ releasing action of caffeine from isolated sarcoplasmic reticulum (SR) of rabbits were evaluated by an investigation into the effects of Ca²⁺, Mg²⁺, MgATP^2–, and ATP concentration, ionic strength, and degree of loading. The heavy fraction (4,500×g) of the reticulum was used. Except for the study on degree of loading, 0.2 mg protein·ml^–1 SR was loaded actively with 0.02 mM⁴⁵CaCl₂, resulting in >90 nmol·mg protein^–1 at steady state, and then the effects of various parameters with or without (control) caffeine were tested.It was found that (1) caffeine induces a transient, dosedependent release of Ca²⁺, (2) the absolute amount of Ca²⁺ released by caffeine increases with the Ca²⁺ load of the SR, (3) increasing the ionic strength () from 0.09 to 0.3 lowers the threshold concentration of caffeine, (4) the SR is refractory to a repeated challenge by a caffeine concentration causing maximal effect, (5) caffeine-induced Ca²⁺ release increases with increasing (a) external Ca²⁺ concentrations up to 5 M total Ca²⁺ (or 3 M free Ca²⁺) and (b) free ATP concentrations up to 0.45 mM, and (6) caffeine-induced Ca²⁺ release is not affected by changes of either the Mg²⁺ or the MgATP^2– concentration.     

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 Ba2+ on the mechanical properties of glycerinated heart muscle

At a muscle length, L₀ (just taut), isometric tension and tension transients in response to rapid step stretches in length (mostly less than 1.2% of L₀ within 2 ms) were measured at constant levels of Ba²⁺ activation of varying magnitude in glycerinated cat right-ventricular papillary muscles (2–3 mm long, 130–200 m in diameter). The majority of the experiments were carried out at room temperature (26–27°C) and at a pH of 6.8 The steady isometric tension increased as Ba²⁺ was varied from slightly below pBa 6 to about pBa 4. The concentration range of Ba²⁺-activated muscle was roughly 10 times higher than that of Ca²⁺-activated muscle. Maximum Ba²⁺-activated isometric tension was 79.0±4.2% (mean±SD,n=8) of that activated with Ca²⁺. The tension transients in Ba²⁺-activated muscle were characterized by at least three distinct phases; an immediate tension increase coincident with the stretch, a rapid exponential tension decrease (time constant, ₁ = 7.3 ± 1.0 ms,n = 5) and a delayed exponential tension rise (₂ = 104 ± 5.7 ms,n=5). The profile of tension response was quite similar to that of Ca²⁺-activated muscle. These results suggest that in Ba²⁺-activated glycerinated heart muscle the cross-bridge turnover is taking place as in the Ca²⁺-activated muscle, but the number of active cross-bridges at maximally activated state is smaller than that of Ca²⁺-activated muscle.     

Bradykinin and inositol 1,4,5-trisphosphate-stimulated calcium release from intracellular stores in cultured bovine endothelial cells

The relative importance of intracellular and extracellular Ca²⁺ in the release of endothelium-derived relaxing factor (EDRF) and the mechanisms involved in the release of intracellular Ca²⁺ were investigated in cultured bovine endothelial cells. The release of EDRF by bradykinin, determined by bioassay, was dose-dependent showing an EC₅₀ of 4×10^–10 M. The bradykinin-induced EDRF release from endothelial cells was maintained in the presence of extracellular Ca²⁺. However, in the absence of external Ca²⁺, bradykinin-induced EDRF release was both attenuated and transient. In cells loaded to isotopic equilibrium with⁴⁵Ca, bradykinin increased the⁴⁵Ca efflux into both calcium-containing and calcium-free solutions, with an EC₅₀ for the increase in⁴⁵Ca efflux induced by bradykinin of 1.3×10^–9 M. The involvement of an intracellular Ca²⁺ store and the participation of a second messenger in its release were investigated in saponin-permeabilized endothelial cells. In saponin-permeabilized cells, ATP-sensitive calcium uptake was Ca²⁺,Mg²⁺-ATPase-dependent. The ATP-sensitive uptake of calcium at different free Ca²⁺ concentrations showed at least two compartments involved in the uptake of Ca²⁺. The⁴⁵Ca uptake into the compartment with the lowest affinity and highest capacity could be inhibited by sodium azide, suggesting that this uptake was into mitochondria. The majority of the⁴⁵Ca uptake into the azide-insensitive store could be released by inositol-1,4,5-trisphosphate (IP₃). The IP₃-induced release was not affected by apyrase or exogenous GTP. The EC₅₀ for the release of Ca²⁺ by IP₃ was 1.0 M and was unaffected by an inhibitor of IP₃ breakdown (2,3-diphosphoglyceric acid). The results suggest that the release of EDRF is dependent on extracellular Ca²⁺ influx and the release of intracellular Ca²⁺. The release of calcium from one of the high affinity intracellular Ca²⁺ stores is mediated by the intracellular second messenger, IP₃.     

Modulation of the ryanodine receptor sarcoplasmic reticular Ca2+ channel in skinned fibers of fast- and slow-twitch skeletal muscles from rabbits

This study was performed to compare skinned fibers from rabbit adductor magnus (AM) and soleus (SL) muscles with regard to the influence of caffeine, Ca²⁺ and Mg²⁺ on the depressive effects of ryanodine (RYA) on the caffeine-induced tension transients. Single skinned fibers were immersed in solution: to load Ca²⁺ into, and release Ca²⁺ from the SR (a load-release cycle). Three cycles were sequentially performed in each skinned fiber: (1) a control (no RYA) (2) a conditioning period in which activation was car ried out in the presence of ryanodine plus various con centrations of the modulators, i.e. caffeine, Ca²⁺ or Mg²⁺, and (3) a test (no RYA) which monitored the release activity retained after the conditioning cycle. The depressive effect of RYA was found to be a function of [ryanodine], [caffeine], or [Ca²⁺], and an inverse function of [Mg²⁺], where [caffeine] denotes concentration. The half-maximal effects of RYA in AM (5 M RYA) and SL (10 M RYA), respectively, occurred at a pCa₅₀ of 5.32 versus 5.43 without caffeine, or pCa₅₀ of 7.24 versus 6.88 and pMg₅₀ of 3.29 versus 3.61 with 25 mM caffeine, at a [caffeine] of 4.96 versus 7.29 mM, and at a [ryanodine] of 31.0 versus 101.6 M. Thus, the RYA depression in skinned muscle fibers is modulated by caffeine, Ca²⁺, and Mg²⁺ in both muscle types, and AM is at least two- to fourfold more sensitive than SL.     

Fura-2 detected myoplasmic calcium and its correlation with contracture force in skeletal muscle from normal and malignant hyperthermia susceptible pigs

Fura-2 was used to estimate myoplasmic [Ca²⁺] in intact intercostal muscle fibers from normal and malignant hyperthermia susceptible (MHS) pigs. Small muscle bundles (20–50 fibers) were loaded with the membrane-permeant form of the dye. Resting myoplasmic [Ca²⁺] were not significantly different in normal and MHS muscles. Halothane produced increases in myoplasmic Ca²⁺ with associated contractures in MHS muscles, but not in normal muscles. These halothane effects were reversible. Caffeine produced increases in myoplasmic Ca²⁺ and contractures in both MHS and normal muscles. The threshold concentrations were lower in the MHS muscles. The correlations between myoplasmic [Ca²⁺] and force in MHS and normal muscles were similar.