Inhibition by adriamycin of calmodulin-sensitive and phospholipid-sensitive calcium-dependent phosphorylation of endogenous proteins from heart.

Adriamycin, a lipid-interacting anti-cancer agent, was found to inhibit phospholipid-sensitive Ca2+-dependent phosphorylation of endogenous proteins from the cytosol of the guinea-pig heart. The drug, unexpectedly, also inhibited phosphorylation of separate endogenous proteins in the cardiac cytosol and membranes catalysed by the calmodulin-sensitive species of Ca2+-dependent protein kinase. In both phosphorylation systems, the inhibition by adriamycin was reversed by either phospholipid (phosphatidylserine or cardiolipin) or calmodulin respectively. Adriamycin also inhibited phosphorylation of histone (exogenous protein) catalysed by purified cardiac phospholipid-sensitive Ca2+-dependent protein kinase, but not that by cyclic AMP-dependent and cyclic GMP-dependent protein kinases. It appears that Ca2+-dependent protein phosphorylation systems, regulated either by phospholipid or calmodulin, may represent hitherto unrecognized sites of action of adriamycin. It remains to be seen whether inhibition by adriamycin of these systems is related to the severe cardiotoxicity, the major adverse effect of the drug that limits its clinical usefulness.     

Early Onset of Lipofuscin Accumulation in Dystrophin-Deficient Skeletal Muscles of DMD Patients and mdx Mice

Lipofuscin, the so-called ageing pigment, is formed by the oxidative degradation of cellular macromolecules by oxygen-derived free radicals and redox-active metal ions. Usually it accumulates in post-mitotic, long-lived cells such as neurons and cardiac muscle cells. In contrast, it is rarely seen in either normal or diseased skeletal muscle fibres. In this paper, we report that lipofuscin accumulates at an early age in both human and murine dystrophic muscles. Autofluorescent lipofuscin granules were localized, using confocal laser scanning microscopy and electron microscopy, in dystrophin-deficient skeletal muscles of X chromosome-linked young Duchenne muscular dystrophy (DMD) patients and of mdx mice at various ages after birth. Age-matched normal controls were studied similarly. Autofluorescent lipofuscin granules were observed in dystrophic biceps brachii muscles of 2-7-year-old DMD patients where degeneration and regeneration of myofibres are active, but they were rarely seen in age-matched normal controls. In normal mice, lipofuscin first appears in diaphragm muscles nearly 20 weeks after birth but in mdx muscles it occurs much earlier, 4 weeks after birth, when the primary degeneration of dystrophin-deficient myofibres is at a peak. Lipofuscin accumulation increases with age in both mdx and normal controls and is always higher in dystrophic muscles than in age-matched normal controls. At the electron microscopical level, it was confirmed that the localisation of autofluorescent granules observed by light microscopy in dystrophin-deficient skeletal muscles coincided with lipofuscin granules in myofibres and myosatellite cells, and in macrophages accumulating around myofibres and in interstitial connective tissue. Our results agree with previous biochemical and histochemical data implying increased oxidative damages in DMD and mdx muscles. They indicate that dystrophin-deficient myofibres are either more susceptible to oxidative stress, or are subjected to higher intra- or extracellular oxidative stress than normal controls, or both.     

Effect of low extracellular calcium and ryanodine on muscle contraction of the mouse during postnatal development.

We have examined the effects of low Ca2+ solutions, Co2+, and ryanodine on the isometric tension and contraction speed of isolated, developing mouse EDL muscles. Twitch responses of young muscles (7-14 days postnatal) were more sensitive to lowered [Ca2+]o than those of more fully developed muscles (22-35 days postnatal). Responses of EDL muscles from a middle-aged group (15-21 days postnatal) were intermediate between the two other groups. Overall, the time course of contraction in a single twitch was accelerated by low [Ca2+]o. Ca(2+)-free solution induced a 7.95 and 9.25 mV depolarization in young and "old" muscle fibres, respectively. The presence of cobalt ions (5 mM) in the Krebs solution had a similar effect as Ca(2+)-free Krebs in terms of reduction of the isometric twitch and tetanic tensions of EDL muscles from the various age groups. In contrast, the shortening of the contraction time seen with Ca(2+)-free solution did not take place following exposure to Co(2+)-containing solutions. Finally, young (7-14 days postnatal) muscles were less sensitive to the inhibitory action of ryanodine on the twitch compared with more fully developed muscles (22-35 days postnatal). Taken together, our results indicate that from birth to maturity, there is a gradual change in the spectrum of calcium utilization for the contractile process.     

Positive inotropic effect of ryanodine on rabbit ventricular muscle: dependence on the intracellular calcium load

Two types of electrical and mechanical responses to 1 mumol/l ryanodine, depending on the intracellular calcium load, were observed in rabbit papillary muscles. In a normal calcium solution, ryanodine induced a transient decline followed by a stable increase in the developed force (by 20 +/- 5% of the pretreatment level; n = 30) and prolonged the action potential (AP). The positive ryanodine response showed an increased time-to-peak force and was completely suppressed by 2 mumol/l nifedipine, partially blocked by 50 mumol/l tetracaine (Ca2+ release blocker), but greatly potentiated by 20 mmol/l CsCl or (-) Bay R 5414 which prolonged the AP. The prolonged time-to-peak force of the positive ryanodine response was shortened by procedures raising the content of Ca2+ in the sarcoplasmic reticulum (SR). It is suggested that the initial decline in the force amplitude results from Ca2+ leakage from the SR which is further compensated for by an elevation of both the transmembrane Ca2+ entry and intracellular Ca2+ release. In calcium overloaded myocardium, 1 mumol/l ryanodine caused irreversible contracture and dramatic AP shortening, explained by a massive Ca2+ release from the overloaded SR into the cytoplasm. It is concluded that the calcium content in the SR is the main modulator of the electrical and mechanical effects of ryanodine in ventricular myocardium.     

Desensitization and muscarinic re-sensitization of force and myosin light chain phosphorylation to cytoplasmic Ca2+ in smooth muscle

In alpha-toxin-permeabilized guinea-pig ileum smooth muscle, a step increase in Ca2+ caused a rapid rise in force and myosin light chain (LC20) phosphorylation, followed by their spontaneous decline to a low steady level even though Ca2+ remained constant. Carbachol resensitized the muscles to Ca2+, causing an increase in both the steady state force and LC20 phosphorylation at constant Ca2+. In beta-escin permeabilized preparations, calmodulin and okadaic acid converted the phasic responses to Ca2+ to more tonic ones. We conclude that Ca2(+)-sensitivity of force is modulated through changes in LC20 kinase/phosphatase activity ratio by Ca2+ itself (desensitization) and by agonists (sensitization).     

Occurrence of intramitochondrial Ca2+ granules in a hypertrophied heart exposed to adriamycin

Left ventricular hypertrophy was produced in rabbits by narrowing the abdominal aorta in the subdiaphragmatic region. Six weeks after the surgery, sham control as well as hypertrophied animals were treated with adriamycin. Myocardial cell damage resulting from a total cumulative dose of 5 mg/kg of adriamycin was seen only in hypertrophied hearts. Alterations in muscle cells of these hearts included prominent "contraction bands" and perinuclear edema. Mitochondria were characterized by swelling and accumulation of electron-opaque granules. Energy-dispersive x-ray analysis of the mitochondria revealed the presence of calcium in these granules. The study confirms that the hypertrophied heart is more vulnerable to adriamycin-induced cell damage and this may be due to an increased susceptibility of these hearts to the occurrence of Ca2+ overload in the cell.     

Ultrastructure of testicular macrophages in aging mice

Testicular macrophages of aging mice were studied by TEM. Testicular macrophages retained with Leydig cells the close morphological relationships observed in the adult young animals, but digitations were not found. Lipofuscin granules like those of the Leydig cells from aging mice were observed in the cytoplasm. These organelles were generally absent in the testicular macrophages of young adult mice. Testicular macrophages did not display phagocytosis of the lipofuscin granules. In addition, the latter were not found in the intercellular spaces. These observations indicated that lipofuscin granules were formed, at least in a great part, within testicular macrophages as a consequence of metabolic changes occurring with age. Fine lamellar organization was seen in the lipofuscin granules of both Leydig cells and testicular macrophages. Frequently, lipofuscin granules originated from secondary lysosomes containing lipidic vacuoles only. Together with accumulation of the lipofuscin granules, changes of testicular macrophage fine morphology were observed. Endoplasmic reticulum and Golgi apparatus became poorly developed, and coated vesicles were rarely found. Fewer mitochondria were encountered, but their ultrastructure was not altered. These results suggest that in testicular macrophages lipofuscin accumulation is associated with a functional involution.     

Changes with senescence in the fine structure of the granular convoluted tubule of the submandibular gland of the mouse

Cells of the granular convoluted tubules (GCTs) of the submandibular gland of senescent male mice show structural changes indicative of functional decline. In order to define the nature of these age-related changes more clearly, the fine structure of GCT cells of 12- and 28-month-old males was compared. In old mice, there was cell-to-cell variation in the extent of these changes, with some cells of senescent males appearing no different from those of young adults. In affected cells the most striking alterations were seen in secretion granules and lysosomal elements. Secretion granules varied greatly in size, with some GCT cells having only very fine apical granules. Secondary lysosomes and large lipofuscin granules were frequent in the basal cytoplasm. Very large dense bodies (3-5 micron) occurred in many cells. These possibly represent intracellular pools of released secretory materials, as they were occasionally seen in continuity with the luminal contents. Structures whose appearance was intermediate between the very large dense bodies and lipofuscin granules were common, suggesting crinophagic activity. There was an apparent decrease in numbers of polysomes and in the extent of the Golgi apparatus. These fine structural changes are consistent with impairments with advanced age in synthesis and posttranslational processing of secretory products by affected GCT cells. In addition to cell-to-cell variation in any one male, there was also interanimal variation in the degree and extent of these senescent changes.     

Effect of inotropic stimulation on mitochondrial calcium in cardiac muscle.

Ca(2+)-dependent activation of citric acid cycle enzymes has been demonstrated in isolated cardiac mitochondria. These observations led to the hypothesis that Ca2+ is the signal coupling myofibrillar energy use to mitochondrial energy production in vivo. To test this hypothesis we have measured mitochondrial Ca2+ content during increased energy demand, using electron probe microanalysis. Mitochondrial Ca2+ was measured in hamster papillary muscles rapidly frozen at the peak rate of tension rise under control conditions and after stimulation with the beta-adrenergic agonist isoproterenol (10(-6) M). A third group of muscles was frozen after incubation in low (46.5 mM) Na+ solution to Ca2+ load the cells. Pyruvate dehydrogenase activity was measured in each of the muscles. Isoproterenol caused a 39% increase in force and a 43% increase in pyruvate dehydrogenase activity but no change in mitochondrial Ca2+ (0.46 +/- 0.19 (S.E.) mmol of Ca2+/kg, dry weight) compared with control (0.54 +/- 0.12). In contrast, low Na+ increased pyruvate dehydrogenase activity by 56% and also elevated mitochondrial Ca2+ to 1.28 +/- 0.31 (p less than 0.02). These results demonstrate that mitochondrial Ca2+ is not elevated after inotropic stimulation of cardiac muscle by beta-adrenergic agonists although pyruvate dehydrogenase activity is increased. We conclude that Ca2+ uptake by mitochondria is not a requirement for activation of mitochondrial respiration after increased energy demand.     

The kinetics relating calcium and force in skeletal muscle.

The kinetics relating Ca2+ transients and muscle force were examined using data obtained with the photoprotein aequorin in skeletal muscles of the rat, barnacle, and frog. These data were fitted by various models using nonlinear methods for minimizing the least mean square errors. Models in which Ca2+ binding to troponin was rate limiting for force production did not produce good agreement with the observed data, except for a small twitch of the barnacle muscle. Models in which cross-bridge kinetics were rate limiting also did not produce good agreement with the observed data, unless the detachment rate constant was allowed to increase sharply on the falling phase of tension production. Increasing the number of cross-bridge states did not dramatically improve the agreement between predicted and observed force. We conclude that the dynamic relationship between Ca2+ transients and force production in intact muscle fibers under physiological conditions can be approximated by a model in which (a) two Ca2+ ions bind rapidly to each troponin molecule, (b) force production is limited by the rate of formation of tightly bound cross-bridges, and (c) the rate of cross-bridge detachment increases rapidly once tension begins to decline and free Ca2+ levels have fallen to low values after the last stimulus. Such a model can account not only for the pattern of force production during a twitch and tetanus, but also the complex, nonlinear pattern of summation which is observed during an unfused tetanus at intermediate rates of stimulation.     

On the nature and function of yellow aging pigment lipofuscin

A comparison of lipofuscin granules from warm-blooded animals and carotenoid-containing granules (cytosomes) from molluscoid neurons was carried out. It was confirmed that the carotenoids are a component of the lipofuscin granules. Data in literature and the experimental data obtained showed that the lipofuscin granules contained carotenoids, myoglobin and some respiratory enzymes.On the basis of identification of the properties of carotenoid-containing granules (cytosomes) of molluscoid neurones and the lipofuscin granules, it is proposed that the functions of the lipofuscin are those of forming the intracellular oxygen stock and providing the energy requirements of the cells under the conditions of low rate oxygen penetration into tissues.     

Adriamycin depresses in vivo and in vitro phosphatidylethanolamine N-methylation in rat heart sarcolemma

Adriamycin, an effective anticancer chemotherapeutic agent, causes an insidious and delayed cardiotoxicity. Different subcellular abnormalities including calcium transport changes in the sarcolemma (SL) as well as downregulation of the adrenergic system have been shown to be associated with the development of this cardiomyopathy. Since both of these activities are influenced by phospholipid methylation, effects of adriamycin on the three catalytic sites of SL phosphatidylethanolamine N-methyltransferase were examined. Rats were administered with a cumulative dose of adriamycin (15 mg/kg) over 2 weeks and examined after 3 weeks. Vehicle injected animals served as controls. Dyspnea, high mortality rate, ascites and decrease in aortic and left ventricular systolic pressure, as well as increase in left ventricular end diastolic pressure were seen in the adriamycin group. Myocardial cell damage typical of adriamycin cardiomyopathy, i.e. sarcotubular swelling, vacuolization and myofibrillar drop-out, was also apparent. Total methyl group incorporation into SL phosphatidylethanolamine using radiolabeled S-adenosyl-L-methionine as the donor was significantly depressed in the 3 week group at catalytic sites II and III. Decreased production of methylated intermediates, phosphatidyl-N-monomethylethanolamine and phosphatidyl-N,N-dimethylethanolamine as well as phosphatidylcholine (PC) was seen. Depression of phosphatidylethanolamine N-methylation was also noticed when SL, isolated from untreated hearts, was exposed in vitro to different concentrations (10, 100 and 1000 µM) of adriamycin. Inhibition of phosphatidylethanolamine N-methylation appears to be mediated by adriamycin-induced increase in the oxidative stress and may contribute in the pathogenesis of subcellular changes associated with this cardiomyopathy.     

Relationship between force and intracellular [Ca2+] in tetanized mammalian heart muscle

To determine features of the steady state [Ca2+]-tension relationship in intact heart, we measured steady force and intracellular [Ca2+] ([Ca2+]i) in tetanized ferret papillary muscles. [Ca2+]i was estimated from the luminescence emitted by muscles that had been microinjected with aequorin, a Ca2+-sensitive, bioluminescent protein. We found that by raising extracellular [Ca2+] and/or by exposing muscles to the Ca2+ channel agonist Bay K 8644, tension development could be varied from rest to an apparently saturating level, at which increases in [Ca2+]i produced no further rise in force. 95% of maximal Ca2+-activated force was reached at a [Ca2+]i of 0.85 +/- 0.06 microM (mean +/- SEM; n = 7), which suggests that the sensitivity of the myofilaments to [Ca2+]i is far greater than anticipated from studies of skinned heart preparations (or from previous studies using Ca2+-sensitive microelectrodes in intact heart). Our finding that maximal force was reached by approximately 1 microM also allowed us to calculate that the steady state [Ca2+]i-tension relationship, as it might be observed in intact muscle, should be steep (Hill coefficient of greater than 4), which is consistent with the Hill coefficient estimated from the entire [Ca2+]i-tension relationship derived from families of variably activated tetani (6.08 +/- 0.68; n = 7). Finally, with regard to whether steady state measurements can be applied directly toward understanding physiological contractions, we found that the relation between steady force and [Ca2+]i obtained during tetani was steeper than that between peak force and peak [Ca2+]i observed during physiological twitches.     

Physiology and excitation-contraction coupling in the intestinal muscle of the crayfish Procambarus clarkii

The intestinal muscles of Procambarus clarkii are striated and yet they are specialized to produce slow peristaltic waves of contraction, not unlike those seen in vertebrate visceral smooth muscle. These muscles cannot be tetanized either by repetitive stimulation or by elevated potassium saline. The excitation-contraction (E-C) coupling mechanism was explored and compared with that known in crustacean skeletal muscle. Contraction is dependent on external Ca2+ which triggers the release of intracellular calcium from the sarcoplasmic reticulum (SR) via calcium-induced calcium release (CICR). Whereas contraction force is proportional to [Ca2+]o up to that in normal saline (13.4 mM), higher levels of Ca2+ reduce force. Ryanodine, which blocks calcium release from the SR, abolishes electrically stimulated contractions and CICR. Relaxation is achieved by removal of calcium from the cytosol in at least two ways, first by the re-loading of calcium into the SR by Ca2+-ATPases and second by the movement of calcium out of the cell by extruding it across the sarcolemma via Na+/Ca2+-exchangers. It is hypothesized that the inability of this muscle to show tetanus arises from inactivation of the voltage-gated calcium channels by high calcium. This is supported by the result that caffeine application causes an increase in tonus and size of phasic contractions by circumventing the sarcolemma and dumping SR calcium stores.     

Study of the adriamycin-cardiolipin complex structure using attenuated total reflection infrared spectroscopy

Adriamycin plays a prominent role in the treatment of leukemia and solid tumors in man. The mode of interaction of adriamycin with its nuclear target, responsible for its therapeutic effect, is known [Berman, H. M., & Young, P.R. (1981) Annu. Rev. Biophys. Bioeng. 10, 87-114]. The planar anthracycline moiety of adriamycin intercalates between the base pairs whereas the sugar moiety fits into the DNA large groove. However, the cardiotoxicity of adriamycin places a limit on the total dose that may be given [Minow, R. A., Banjamin, R.S., & Gottlieb, J. A. (1975) Cancer Chemother. Rep. 6, 195-202]. Much evidence suggests that the mitochondrial membrane could be the target responsible for adriamycin cardiotoxicity. The formation of a very stable complex between adriamycin and cardiolipin, a phospholipid specific to the inner mitochondrial membrane, has been shown to inhibit several mitochondrial membrane enzymes whose activities depend on the presence of cardiolipin. Using attenuated total reflection infrared spectroscopy, we demonstrate here that, in the adriamycin-cardiolipin complex, both cardiolipin and adriamycin structures are modified as compared with the pure substances. Dichroism values indicate a slight reorientation of the cardiolipin molecule toward a normal to the plane of the bilayer whereas adriamycin, which shows no ordering in a pure phase, is highly ordered in the complex, the anthracycline moiety titled at about 40 degrees with respect to the normal to the plane of the bilayer. The partial disappearance of NH3+ characteristic bands indicates the involvement of the positively charged amino group of adriamycin in the complex formation.     

Modulation of the calcium release channel of sarcoplasmic reticulum by adriamycin and other drugs

The calcium release channel of sarcoplasmic reticulum mediates Ca2+ release which triggers muscle contraction in excitation-contraction coupling. The channels have been identified morphologically with the feet structures, which are involved in junctional association of terminal cisternae of sarcoplasmic reticulum with the transverse tubules to form the triad junction. In this study, we further characterize the action of drugs on the calcium release channel from sarcoplasmic reticulum fused into planar bilayers. Adriamycin is an effective cancer chemotherapeutic drug, which is limited by its cardiotoxicity. The drug, when added to the myoplasmic side (cis side), activates channel opening at microM concentrations in a dose dependent manner. Adriamycin together with ATP (mM) gives optimal activation, with an open probability (Po) of approximately 1.0. Ruthenium red added to the cis side, equivalent to the cytoplasmic (myoplasmic) domain, completely blocks channel opening. Qualitatively similar results are obtained with adriamycinol, the major metabolite of adriamycin. The inhibition by adriamycin is not reversed by reperfusion to wash out the drug. Silver ions are also found to activate the channel. The conductance of the channel activated by adriamycin, adriamycinol or Ag+ is approximately 100 ps, similar to that previously reported for activation of the channel with Ca2+ and ATP. Ruthenium red has previously been observed to block channel activation from the cytoplasmic side.(ABSTRACT TRUNCATED AT 250 WORDS)     

Some characteristics of Ca2+- regulated force production in EGTA- treated muscles from rat heart

McClellan and Winegrad (1980, J. Gen. Physiol., 75:283-295) have reported that in rat ventricular muscles that have reportedly been made "hyperpermeable" to small ions such as Ca2+, CaEGTA2-, and MgATP2- by a soak in EGTA, the maximum Ca2+-regulated force can be permanently increased by a short exposure to positively inotropic drugs, such as epinephrine or cAMP plus theophylline, in the presence of the detergent Triton X-100. The experiments reported here were begun as an attempt to repeat and extend this important observation. However, no evidence could be found for a potentiation of force that was not merely produced by Triton alone. In addition, the thickest muscles used (250-440 microns diameter) exhibited very low values for force per unit cross- sectional area, which suggested that either Ca2+ reached only a fraction of the myofibrils or the myofibrils were in a state of low contractility. The results of further experiments that were designed to test the permeability characteristics of these EGTA-treated muscles indicated that the movement of certain ions into these preparations was restricted, even in thin muscles (80-200 microns diameter). The rate of development of Ca2+-regulated force was slow (t1/2 approximately equal to 1-3 min), but was greatly accelerated after the muscles had been superfused with Triton X-100 (t1/2 approximately equal to 10-20 s). Removal of creatine phosphate (CP) in the presence of MgATP produced a partial rigor contracture in the EGTA-treated muscles. The results were consistent with the suggestion that the EGTA-treated muscles were permeable to some extent to Ca2+ and HCP2- ions but not to CaEGTA2- and MgATP2-. Thus, it would seem unlikely that the [Ca2+], [MgATP2-], and [Mg2+] in the immediate vicinity of the myofibrils in these preparations can be adequately controlled by the solution bathing the muscles.     

Fine structural changes in the epididymal epithelium of moles (Talpa europaea) throughout the year.

The epididymis of the European mole (Talpa europaea) was studied by light and electron microscopy. In the sexually active animal, spermatozoa mature during their passage through the epididymis and the structure of the cells lining the duct suggests a clear regional division into initial, middle and terminal segments. Numerous intra-epithelial vesicles were present in the distal part of the middle segment of sexually active moles and the lining epithelium in the terminal segment appeared to be secretory. Variation in the sensitivity of different regions of the epididymis to androgens was apparent: the principal cells of the initial segment were morphologically active only during the peak of the breeding season in spring, while the cells of the terminal segment became active earlier and remained so for longer. During sexual regression, many autophagic vacuoles were found in the principal cells, and these became transformed into lipofuscin pigment granules. Cells heavily laden with these granules appeared concurrently in the lining epithelium. It is suggested that such cells may be involved in the regression of principal cells by means of heterophagic activity. A similar situation was also observed, but to a lesser extent, at the beginning of the breeding season. Outside the breeding season, the organelles of the principal cells were poorly developed throughout the epididymis, and lipofuscin pigment granules remained in the principal and basal cells of adults. Such granules were seldom seen in immature animals.     

Characteristics of functioning of electromechanical coupling in striated muscles of higher and lower vertebrates

A comparative pharmacological analysis of relative contributions of different signal transduction pathways in the activation of contraction (excitation-contraction coupling, ECC) in intact fast striated muscles of frog and lamprey was performed. It was found that the major mechanism responsible for the ECC in muscles of both animals is Ca2+ release from the sarcoplasmic reticulum through the ryanodine-sensitive channels. However, the ECC in lamprey muscle displays some important differences in the units of electromechanical coupling, which precede the calcium release from sarcoplasmic reticulum. The maximum contraction force in frog muscle develops during caffeine-induced contracture, which indicates that all Ca2+ stored in sarcoplasmic reticulum is released through ryanodine-sensitive channels. In contrast, in lamprey muscle, the maximum force develops not in response to high caffeine concentration, but in response to repetitive electrical stimulation. Hence, in addition to stores liberated by ryanodine-sensitive channels, some other sources of calcium ions should exist, which contribute to the contraction activation. A source of this additional Ca2+ ions can be external medium, because acetylcholine contracture is abolished in a calcium-free medium. In frog muscle, the acetylcholine contracture was abolished in a Na(+)-free solution. It was concluded that in frog muscle ECC can be triggered by changes in the transmembrane potential (depolarization-induced calcium release), while in lamprey muscle the entry of calcium ions into myoplasm as the trigger in ECC (calcium-induced calcium release). The lamprey muscle was found to be more resistant to tetrodotoxin and tetracaine, which is indicative of a role in the activation of contraction of tetrodotoxin-resistant Na+ and/or Ca2+ channels. It was concluded, that ECC mechanism in striated muscles of low vertebrates is not limited by the generally accepted scheme of depolarization-induced calcium release but can include some other schemes, which require the Ca2+ influx into the cell.     

Scattered-light intensity fluctuations in diastolic rat cardiac muscle caused by spontaneous Ca++-dependent cellular mechanical oscillations

Laser light scattered by nonstimulated rat cardiac muscle bathed in physiological saline containing a [Ca++] of 0.4-2.5 mM displays scattered-light intensity fluctuations (SLIF); the frequencies of both SLIF and resting force are Ca++ dependent. Direct inspection of these muscles by phase-contrast microscopy under incoherent illumination revealed the presence of spontaneous asynchronous cellular motions that are also Ca++ dependent. The physical properties of the scattered light are compatible with the hypothesis that SLIF are due to the diastolic motion, except for the dependence on scattering angle, which may be perturbed because the muscles are optically thick. To determine whether diastolic SLIF and motion are an intrinsic property of activated myofilaments, photon-counting auto-correlation of the scattered light was performed both in rat right-ventricular papillary muscles skinned with the detergent Triton X-100 (1%) and in muscles with intact membranes under conditions that alter cellular Ca++ fluxes. In skinned muscles activated over a range of Ca++ from threshold to maximum force production, neither SLIF nor asynchronous motion was observed when Ca++ was buffered to constant values. In intact muscles the frequency of SLIF and the amplitude of diastolic motion were (a) markedly increased by substituting K+ or Li+ for Na+ in the bath; (b) not altered by verapamil (1 microM); and (c) reversibly abolished by caffeine (greater than or equal to 10 mM). These properties are exactly those of mechanical oscillations that have been observed in isolated cardiac cell fragments, which are the result Ca++ oscillations caused by Ca++ release from the sarcoplasmic reticulum (SR). We infer that mechanical oscillations caused by spontaneous Ca++-induced Ca++ release from the SR occur in intact nonstimulated cardiac muscle even in the absence of Ca++ overload and are the principle cause of SLIF, and that myoplasmic [Ca++] in "resting" muscle is not in a microscopic steady state.