Effect of low-molecular nonelectrolytes on caffeine skeletal muscle contracture in the rat]

Contractures induced in rat fast (EDL) and slow (SOL) skeletal muscles by 0.03--3 mM of caffeine in conjunction with rapid cooling of muscle from 30 to 0 degrees C (rapid cooling contructures, RCC) were studied. Uprising speed and tension of RCC were dependent on caffeine concentration and cooling gradient. The minimal necessary temperature, below which contractures still developed, was +6 degrees. The initial temperature did not play any important role. Optimal conditions for RCC (when its tension reached 80--200% of twitch) were: cooling from 30 to 0 degrees, and concentrations of caffeine being 5 mM for SOL, and 6--7 mM for EDL. Disruption of T tubules caused by the removal of glycerol and urea (400--600 mM) from muscle fibers did not influence the RCC tension. During the first hour of the removal, relaxation rate of RCC was lowered. In the presence of 400 mM of urea and 600 mM of 1.3-dimethylurea (the latter did not disrupt the T-system), RCC was depressed by 90%, and the rate of tension development was greatly lowered, while twitches remained unchanged. This effects could be reversed during non-electrolyte removal. This may suggest that Ca2+ release is inhibited selectively by urea and by dimethylurea.     

Tension in Isolated Frog Muscle Fibers Induced by Hypertonic Solutions

The effect of hypertonic solutions on the tension of isolated twitch muscle fibers of the frog has been investigated. Increased tonicity up to about 1.7 times normal (1.7 T) caused a very small, graded, maintained tension increase. Above about 1.7 T a large, transient contracture response was superimposed on the small tension. The contracture response was graded with tonicity and reached a maximum at 2.5 T of 108 ± 25 mN·mm² a third of the maximum tetanic tension in isotonic solution. Contracture tension developed with a delay which decreased with increased tonicity. The contracture threshold was lower and the delay shorter in small fibers than in large. Contractures were obtained equally well in depolarized as in polarized fibers. They were completely suppressed by 0.1–0.5 mM tetracaine. The possible mechanism responsible for the tension-inducing effect of hypertonic solutions is discussed in terms of the close similarity between the properties of these contractures and those caused by caffeine, and it is suggested that the effect is due to a release of calcium from internal stores.     

Caffeine treatment inhibits drug-induced calcium release from sarcoplasmic reticulum and caffeine contracture but not tetanus in frog skeletal muscle

Effects of pretreatment with caffeine on Ca2+ release induced by caffeine, thymol, quercetin, or p-chloromercuriphenylsulfonic acid (pCMPS) from the heavy fraction of sarcoplasmic reticulum (SR) were studied and compared with those effects on caffeine contracture and tetanus tension in single fibers of frog skeletal muscle. Caffeine (1-5 mM) did induce transient Ca2+ release from SR vesicles, but subsequent further addition of caffeine (10 mM, final concentration) induced little Ca2+ release. Ca2+ release induced by thymol, quercetin, or pCMPS was also inhibited by pretreatment with caffeine. In single muscle fibers, pretreatment with caffeine (1-5 mM) partially reduced the contracture induced by 10 mM caffeine. However, tetanus tension was almost maximally induced by electrical stimulus in caffeine-treated fibers. These results indicate that SR, which becomes less sensitive to caffeine, thymol, quercetin, or pCMPS by pretreatment with caffeine, can still respond to a physiological signal transmitted from transverse tubules.     

Excitation-contraction coupling in cardiac Purkinje fibers. Effects of caffeine on the intracellular [Ca2+] transient, membrane currents, and contraction

The effects of caffeine on tension, membrane potential, membrane currents, and intracellular [Ca2+], measured as the light emitted by the Ca2+-activated photoprotein aequorin, were studied in canine cardiac Purkinje fibers. An initial, transient, positive inotropic effect of caffeine was accompanied by a transient increase in the second component of the aequorin signal (L2) but not the first (L1). In the steady state, 4 or 10 mM caffeine always decreased twitch tension and greatly reduced both L1 and L2. At a concentration of 2 mM, caffeine usually reduced but occasionally increased the steady state twitch tension. However, 2 mM caffeine always reduced both L1 and L2. Caffeine eliminated the diastolic oscillations of intracellular [Ca2+] induced by high extracellular [Ca2+]. In voltage-clamp experiments, 10 mM caffeine reduced the transient outward current and the peak tension elicited by step depolarization from a holding potential of -45 mV. In the presence of 20 mM Cs+, 10 mM caffeine reduced slow inward current. However, the time course of this reduction was far slower than that in tension and light observed in separate experiments. The simplest explanation of the results is that caffeine inhibits the sequestration of Ca2+ by the sarcoplasmic reticulum. The results also suggest that in Purkinje fibers caffeine increases the sensitivity of the myofilaments to Ca2+.     

Twitch potentiation and caffeine contractures in isolated rat soleus muscle

1. Electrically-evoked twitch and tetanic tension were measured in isolated rat soleus muscle after exposure to caffeine. 2. Between 0.01 and 2.5 mM caffeine twitch tension was potentiated, reaching a peak of 150% of Resting Tension at 0.5 mM. 3. Biphasic Tension development with relaxation was observed at 2.5 mM caffeine with maximal contractures (110% tetanic tension) occurring at 20 mM. 4. Creatine phosphate and ATP stores were maintained throughout the period of tension development and relaxation. 5. In contrast with amphibian muscle, the isolated soleus is very sensitive to low doses of caffeine and produces biphasic caffeine contractures which relax in the presence of caffeine.     

Effects of Verapamil and Gadolinium on Caffeine-Induced Contractures and Calcium Fluxes in Frog Slow Skeletal Muscle Fibers

In this work, we tested whether L-type Ca²⁺ channels are involved in the increase of caffeine-evoked tension in frog slow muscle fibers. Simultaneous net Ca²⁺ fluxes and changes in muscle tension were measured in the presence of caffeine. Isometric tension was recorded by a mechanoelectrical transducer, and net fluxes of Ca²⁺ were measured noninvasively using ion-selective vibrating microelectrodes. We show that the timing of changes in net fluxes and muscle tension coincided, suggesting interdependence of the two processes. The effects of Ca²⁺ channel blockers (verapamil and gadolinium) were explored using 6 mm caffeine; both significantly reduced the action of caffeine on tension and on calcium fluxes. Both caffeine-evoked Ca²⁺ leak and muscle tension were reduced by 75% in the presence of 100 μm GdCl₃, which also caused a 92% inhibition of net Ca²⁺ fluxes in the steady-state condition. Application of 10 μm verapamil to the bath led to 30% and 52% reductions in the Ca²⁺ leak caused by the presence of caffeine for the peak and steady-state values of net Ca²⁺ fluxes, respectively. Verapamil (10 μm) caused a 30% reduction in the maximum values of caffeine-evoked muscle tension. Gd³⁺ was a more potent inhibitor than verapamil. In conclusion, L-type Ca²⁺ channels appear to play the initial role of trigger in the rather complex mechanism of slow fiber contraction, the latter process being mediated by both positive Ca²⁺-induced Ca²⁺ release and negative (Ca²⁺ removal from cytosol) feedback loops. Lana Shabala and Xóchitl Trujillo contributed equally to this study.     

An examination of the ability of inositol 1,4,5-trisphosphate to induce calcium release and tension development in skinned skeletal muscle fibres of frog and crustacea

We have examined the ability of inositol 1,4,5-trisphosphate (InsP3) to cause contractions of mechanically skinned muscle fibres of frog and barnacle. InsP3 (10-500 microM) did not cause any tension development in 25 frog skinned fibres and 26 barnacle myofibrillar bundles, although contractions could be readily evoked by caffeine and by replacement of an impermeant anion by Cl-, treatments known to release calcium from the sarcoplasmic reticulum (SR). Four barnacle bundles did give responses to InsP3. InsP3 did not modify responses to caffeine or calcium-induced calcium release. Free Mg2+ was lowered to 40 microM and 15 mM D-2,3-diphosphoglycerate was added in order to inhibit the possible breakdown of InsP3 by inositol trisphosphatase. Neither measure revealed a response to InsP3. Arsenazo III absorbance measurements failed to detect any binding of Mg2+ (0-0.5 mM) by 0.35 mM InsP3 in our solutions. Inhibitors of SR calcium uptake (cadium, quercetin, furosemide), omission of EGTA from the solution and varying the temperature from 4 degrees to 22 degrees C also failed to reveal a response of frog skinned fibres to InsP3. The nucleotide GTP, which has been reported to enhance InsP3-induced calcium release from rat liver microsomes, had no effect at 50 microM on the response of frog fibres to InsP3. It is concluded that under conditions in which other calcium release mechanisms operate well, InsP3 is relatively ineffective at releasing calcium from the SR in amounts sufficient to induce contraction. Although we have been unable to find evidence to support the proposed role of InsP3 as an essential link in excitation-contraction coupling of skeletal muscle, we cannot entirely reject its role if essential cofactors are lost in the skinned preparations.     

Properties of sodium pumps in internally perfused barnacle muscle fibers

To study the properties of the Na extrusion mechanism, giant muscle fibers from barnacle (Balanus nubilus) were internally perfused with solutions containing tracer 22Na. In fibers perfused with solutions containing adenosine 5'-triphosphate (ATP) and 30 mM Na, the Na efflux into 10 mM K seawater was approximately 25-30 pmol/cm2.s; 70% of this efflux was blocked by 50-100 microM ouabain, and approximately 30% was blocked by removal of external K. The ouabain-sensitive and K-dependent Na effluxes were abolished by depletion of internal ATP and were sigmoid-shaped functions of the internal Na concentration ([Na]i), with half-maxima at [Na]i approximately or equal to 20 mM. These sigmoid functions fit the Hill equation with Hill coefficients of approximately 3.5. Ouabain depolarized ATP-fueled fibers by 1.5-2 mV ([Na]i greater than or equal to 30 mM) but had very little effect on the membrane potential of ATP-depleted fibers; ATP depletion itself caused a 2-2.5- mV depolarization. When fueled fibers were treated with 3,4- diaminopyridine or Ba2+ (to reduce the K conductance and increase membrane resistance), application of ouabain produced a 4-5 mV depolarization. These results indicate that an electrogenic, ATP- dependent Na-K exchange pump is functional in internally perfused fibers; the internal perfusion technique provides a convenient method for performing transport studies that require good intracellular solute control.     

Stimulation of cyclic nucleotides of calcium efflux in barnacle muscle fibers.

Internal application of 10⁻⁴, 10⁻⁵, 10⁻⁶ and 10⁻⁷M cGMP and cAMP caused an increase in ⁴⁵Ca efflux in barnacle muscle fibers. Stimulation by either nucleotide occurred in the absence of external calcium and could be prevented by external application of 10 mM procaine or by prior internal treatment of these fibers with EGTA. The results indicate that cyclic nucleotides increase calcium efflux by releasing calcium from internal stores.     

Effects of sulfhydryl inhibitors on depolarizations-contraction coupling in frog skeletal muscle fibers

We have studied the effects of the sulfhydryl reagents on contractile responses, using either electrically stimulated single muscle fibers or short muscle fibers that were voltage-clamped with a two-microelectrode voltage-clamp technique that allows the fiber tension in response to membrane depolarization to be recorded. The sulfhydryl inhibitors para- chloromercuribenzoic acid (PCMB) and parahydroximercuriphenyl sulfonic acid (PHMPS), at concentrations from 0.5 to 2 mM, cause loss of the contractile ability; however, before this effect is completed, they change the fiber contractile behavior in a complex way. After relatively short exposure to the compounds, < 20 min, before the fibers lose their contractile capacity, secondary tension responses may appear after electrically elicited twitches or tetani. After losing their ability to contract in response to electrical stimulation, the fibers maintain their capacity to develop caffeine contractures, even after prolonged periods (120 min) of exposure to PHMPS. In fibers under voltage-clamp conditions, contractility is also lost; however, before this happens, long-lasting (i.e., minutes) episodes of spontaneous contractile activity may occur with the membrane polarized at -100 mV. After more prolonged exposure (> 30 min), the responses to membrane depolarization are reduced and eventually disappear. The agent DTT at a concentration of 2 mM appears to protect the fibers from the effects of PCMB and PHMPS. Furthermore, after loss of the contractile responses by the action of PCMB or PHMPS, addition of 2 mM DTT causes recovery of tension development capacity.     

Swelling of Skinned Muscle Fibers of the Frog: Experimental Observations

Frog skeletal muscle fibers, mechanically skinned under water-saturated silicone oil, swell upon transfer to aqueous relaxing medium (60 mM KCl; 3 mM MgCl₂; 3 mM ATP; 4 mM EGTA; 20 mM Tris maleate; pH = 7.0; ionic strength 0.15 M). Their cross-sectional areas, estimated with an elliptical approximation, increase 2.32-fold (±0.54 SD). Sarcomere spacing is unaffected by this swelling. Addition of 200 mM sucrose to relaxing medium had no effect on fiber dimensions, whereas decreasing pH to 5.0 caused fibers to shrink nearly to their original (oil) size. Decreasing MgCl₂ to 0.3 mM caused fibers to swell 10%, and increasing MgCl₂ to 9 mM led to an 8% shrinkage. Increasing ionic strength to 0.29 M with KCl caused a 26% increase in cross-sectional area; decreasing ionic strength to 0.09 M had no effect. Swelling pressure was estimated with long-chain polymers, which are probably excluded from the myofilament lattice. Shrinkage in dextran T10 (number average mol wt 6,200) was transient, indicating that this polymer may penetrate into the fibers. Shrinkage in dextran T40 (number average mol wt 28,000), polyvinylpyrrolidone (PVP) K30 (number average mol wt 40,000) and dextran T70 (number average mol wt 40,300) was not transient, indicating exclusion. Maximal calcium-activated tension is decreased by 21% in PVP solutions and by 31% in dextran T40 solutions. Fibers were shrunk to their original size with 8 × 10^-2 g/cm³ PVP K30, a concentration which, from osmometric data, corresponds to an osmotic pressure (II/RT) of 10.5 mM. As discussed in the text, we consider this our best estimate of the swelling pressure. We find that increasing ionic strength to 0.39 M with KCl decreases swelling pressure slightly, whereas decreasing ionic strength to 0.09 M has no effect. We feel these data are consistent with the idea that swelling arises from the negatively charged nature of the myofilaments, from either mutual filamentary repulsion or a Donnan-osmotic mechanism.     

Effects of resting membrane potential and intactness of the T-tubules on caffeine contractures in rat skeletal muscle

We have studied the effects of changes in the resting membrane potential (Vm) and T-tubules on caffeine contracture (25 mM) elicited in rat soleus muscle in vitro at 34 degrees C. In high [K]o (30-140 mM, [K]o X [Cl]o constant) caffeine contractures were reduced by about 40-50% and had a faster time course than in normal Krebs ([K]o = 5 mM). Detubulation of the muscles by an osmotic treatment produces a reduction of about 30% in the caffeine contracture tension. Our results with high K solutions suggest a reduced sensitivity of the myofibrils to calcium released by caffeine. The effects of detubulation on caffeine contracture suggest that caffeine may have a direct effect on sarcolemma in addition to its well known action on the sarcoplasmic reticulum (SR). However, a depletion of the calcium content in the SR of depolarized muscle fibres as well as an anatomical damage produced by the osmotic treatment can not be ruled out as an explanation for the reduced caffeine contracture.     

In vitro correlation between force and energy metabolism in porcine malignant hyperthermic muscle studied by 31P NMR.

A comparative study of mechanical and energetic parameters of superfused muscle strips from normal pigs and malignant hyperthermia susceptible (MHS) pigs has been conducted. Phosphorus nuclear magnetic resonance spectroscopy at 80.9 MHz and mechanical measurements were used to assess muscle metabolic state. At rest, biceps femoris biopsies of MHS pigs displayed reduced phosphocreatine level, higher inorganic phosphate, and a more acidic internal pH. In normal stimulated fibers, caffeine infusion (8 or 16 mM) induced twitch potentiation and contracture while twitch tension was reduced and contracture more pronounced in malignant fibers. In normal and malignant fibers, calcium ionophore A23187 produced effects similar to those of caffeine, with the exception of twitch potentiation, which was not observed. With caffeine or A23187, the ATP level remained constant throughout the rest-stimulation-recovery protocol for normal and malignant fibers but phosphocreatine dropped to undetectable levels upon stimulation of malignant fibers. In both treatments some heterogeneity in the resonances of inorganic phosphate was observed in malignant fibers together with a more severe acidosis which might play a role in the impairment of the excitation-contraction process.     

Modulation of caffeine contractures in mammalian skeletal muscles by variation of extracellular potassium

Caffeine contractures were induced after K⁺ -conditioning of skeletal muscles from pigs and mice. K⁺ -conditioning is defined as the partial depolarization caused by increasing external potassium (K) with [K⁺]×[Cl^?] constant. Conditioning depolarizations that rendered muscles refractory to brief electrical stimulation still enhanced the contracture tension elicited by subsequent direct caffeine stimulation of sarcoplasmic reticulum (SR) calcium release. The effects of K⁺ -conditioning on caffeine-induced contractures of intact cell bundles reached a maximum at 15–30 mM K and then progressively declined at higher [K⁺]₀. Conditioning with 30 mM K⁺ for 5 min, which inactivates excitation-contraction (EC) coupling in response to action potentials, both increased the magnitude of caffeine contractures 2–10-fold and shifted the contracture threshold toward lower caffeine concentrations. Enhanced sensitivity to caffeine was inhibited by dantrolene (20 μM) and its watersoluble analogue azumolene (150 μM). These drugs decreased caffeine-induced contractures following depolarization with 4–15 mM K⁺ to 25–50% of control tension. The inorganic anion perchlorate (CIO), which like caffeine potentiates twitches, increased caffeine-induced contractures ～? twofold after K⁺ -conditioning (>4 mM). The results suggest that CIO and dantrolene, in addition to caffeine, also influence SR calcium release either directly or by mechanism(s) subsequent to depolarization of the sarcolemma. Moreover, since CIO is known to shift the voltage-dependence of intramembrane charge movement, CIO may exert effects on the transverse-tubule voltage sensors as well as the SR. © 1995 Wiley-Liss, Inc.     

Influence of caffeine on movement characteristics, fertilizing capacity and ability to penetrate cervical mucus of human spermatozoa

When added to frozen-thawed human semen, the 3 doses of caffeine tested (2, 5 and 10 mM) induced a significant increase in the percentage of motile spermatozoa but did not influence the quality of movement. Considerable variability was noted between samples in their responsiveness to caffeine which, at the 5 and 10 mM doses, was significantly correlated with the degree of motility lost during cryostorage. Caffeine treatment of frozen-thawed human spermatozoa also increased the number of spermatozoa penetrating cervical mucus in unit time, by increasing the frequency rather than the success of collisions between spermatozoa and the cervical mucus interface. When caffeine-stimulated spermatozoa were washed free of seminal plasma containing this compound they were no longer at an advantage with respect to their motility or fertilizing ability. When 2 mM-caffeine was added to washed suspensions of capacitated spermatozoa it failed to stimulate motility but did significantly enhance the fertilizing ability of the spermatozoa, indicating a possible clinical role for this compound in in-vitro fertilization therapy.     

Modulation of ryanodine-induced Ca2+ release in amphibian skeletal muscle

We examined effects of ryanodine on tension in intact and skinned amphibian skeletal muscle. 100 microM ryanodine (RY) alone in the frog Ringer's solution (FR) produced tension in the intact muscle reaching its peak by 1 h; 10 min treatment with RY augmented depolarization-induced tension and prevented a subsequent caffeine-induced contraction. In contrast, RY in Ca2+-free FR was unable to produce tension, after which caffeine produced irreversible tension. In skinned fibers, RY at pCa 6.5 produced tension and abolished a subsequent caffeine-induced contraction; while Ry in 2 mM EGTA did not produce tension. These data indicate that RY, in the presence of CA2+, releases CA2+ from the SR resulting in subsequent depletion of CA in the SR.     

Ca2+ dependence of stimulated 45Ca efflux in skinned muscle fibers

Stimulation of sarcoplasmic reticulum Ca release by Mg reduction of caffeine was studied in situ, to characterize further the Ca2+ dependence observed previously with stimulation by Cl ion. 45Ca efflux and isometric force were measured simultaneously at 19 degrees C in frog skeletal muscle fibers skinned by microdissection; EGTA was added to chelate myofilament space Ca either before or after the stimulus. Both Mg2+ reduction (20 or 110 microM to 4 microM) and caffeine (5 mM) induced large force responses and 45Ca release, which were inhibited by pretreatment with 5 mM EGTA. In the case of Mg reduction, residual efflux stimulation was undetectable, and 45Ca efflux in EGTA at 4 microM Mg2+ was not significantly increased. Residual caffeine stimulation at 20 microM Mg2+ was substantial and was reduced further in increased EGTA (10 mM); at 600 microM Mg2+, residual stimulation in 5 mM EGTA was undetectable. Caffeine appears to initiate a small Ca2+-insensitive efflux that produces a large Ca2+-dependent efflux. Additional experiments suggested that caffeine also inhibited influx. The results suggest that stimulated efflux is mediated mainly or entirely by a channel controlled by an intrinsic Ca2+ receptor, which responds to local [Ca2+] in or near the channel. Receptor affinity for Ca2+ probably is influenced by Mg2+, but inhibition is weak unless local [Ca2+] is very low.     

Genetic heterogeneity in patients having ataxia telangiectasia

UV-irradiated Chinese hamster cells on post-irradiation treatment with caffeine in growth medium for 24 h gave rise to biphasic UV-survival curves. At caffeine concentrations between 0.001 and 0.1 mM, control and caffeine-grown cells had similar survival curves initially from 0 to 30 J/m². At fluences greater than 30 J/m², there was effectively only little further killing of caffeine-grown cells beyond that observed at 30 J/m². At concentrations of caffeine greater than 0.5 mM, there was a gradual sensitization in the early part of the survival curve with increasing caffeine concentrations; but at fluences greater than 3 J/m², the slopes in the survival curves decreased very much.It has been proposed that the initial sensitization observed at low UV fluences is due to the suppression of post-replication repair by caffeine. After high fluences of UV exposures in these excision-deficient cells, in the presence of caffeine, the possibility of an induced ‘SOS’-like repair process has been suggested. This suggestion was supported by the observation that caffeine increased the yield of the UV-induced 8-azaguanine-resistant mutants only for the cell population exposed to UV fluences greater than 30 J/m².     

Radial spread of aequorin Ca2+ signal in single frog skeletal muscle fibers

The Ca²⁺-sensitive photoprotein aequorin was injected into single frog skeletal muscle fibers, and the intracellular aequorin light intensity during muscle activation with different maneuvers was mapped with digital imaging microscopy. During 50 Hz electrical activation (tetanus), the aequorin light intensity from different locations in the muscle fiber rose with very similar time course. Caffeine (10 mM) application, on the other hand, caused aequorin light signals to show significantly different time courses, with an earlier increase in Ca²⁺ concentration near the surface of the fiber than near the core. The non-uniform rise of intracellular Ca²⁺ concentration with caffeine treatment is consistent with the slow inward diffusion of caffeine and subsequent Ca²⁺ release from sarcoplasmic reticulum.     

The Effect of Bathing Solution Tonicity on Resting Tension in Frog Muscle Fibers

Resting tension and short-range elastic properties of isolated twitch muscle fibers of the frog have been studied while bathed by solutions of different tonicities. Resting tension in isotonic solution at 2.3-µm sarcomere spacing averaged 0.46 mN·mm^-2 and was proportional to the fiber cross-section area. Hypertonic solutions, containing 0.1–0.5 mM tetracaine to block contracture tension, caused a small sustained tension increase, which was proportional to the fiber cross-section area and which reached 0.9 mN·mm^-2 at two times normal tonicity (2T). Further increases in tonicity caused little increase in tension. Hypotonic solutions decreased tension. Thus, tension at 2.3 µm is a continuous, direct function of tonicity. The dependence of tension on tonicity lessened at greater sarcomere lengths. At 3.2 µm either a very small rise or, in some fibers, a fall in tension resulted from an increase in tonicity. Hypertonic solutions also decreased the tension of extended sarcolemma preparations. In constant-speed stretch experiments the elastic modulus, calculated from the initial part of the stretch response, rose steeply with tonicity over the whole range investigated (1–2.5T). The results show that tension and stiffness of the short-range elastic component do not increase in parallel in hypertonic solutions.