Effects of excitation-contraction uncoupling by stretch and hypertonicity on metabolism and tension in single frog muscle fibers

Metabolism and tension were examined in single fibers of the semitendinosus muscle of Rana pipiens at 15 degree C after excitation- contraction uncoupling by stretch and hypertonicity. Interrupted tetanic stimulation at 20 HZ for 150 s, of control fibers in isotonic Ringer at a rest sarcomere length (SL) of 2.3 micrometers, resulted in a steadily declining tension, stimulated glycolysis, and significantly reduced fiber phosphocreatine (PCr) and ATP concentrations. Stretching resting muscle fibers to an SL of 4.7 micrometers did not alter metabolite concentrations, but glucose-6-phosphate rose and PCr fell markedly when the stretched fibers were stimulated tetanically, although tension was absent. Immersion of untetanized fibers in 2.5 X isotonic Ringer produced a transient rise in resting tension, an increase in glucose-6-phosphate, and a significant reduction in PCr. During the transient rise in resting tension, PCr consumption per unit of tension-time integral was the same as that in fibers stimulated tetanically in isotonic Ringer. Tetanization of fibers in hypertonic solution did not further alter metabolite concentrations or produce tension. The results indicate that exposure to hypertonicity induces an increase in both tension and consumption of high-energy phosphate bonds (approximately P) in resting fibers, but stretch does not. during tetanic stimulation, stretch interferes with contraction but does not prevent activation, whereas hypertonicity inhibits activation as well as contraction.     

Propagation of Action Potentials and the Structure of the Nexus in Cardiac Muscle

The hypothesis that the nexus is a specialized structure allowing current flow between cell interiors is corroborated by concomitant structural changes of the nexus and changes of electrical coupling between cells due to soaking in solutions of abnormal tonicity. Fusiform frog atrial fibers are interconnected by nexuses. The nexuses, desmosomes, and regions of myofibrillar attachment of this muscle are not associated in a manner similar to intercalated discs of guinea pig cardiac muscle. Indeed, nexuses occur wherever cell membranes are closely apposed. Action potentials of frog atrial bundles detected extracellularly across a sucrose gap change from monophasic to diphasic when the gap is shunted by a resistor. This indicates that action potentials are transmitted across the gap when sufficient excitatory current is allowed to flow across the gap. When the sucrose solution in the gap is made hypertonic, propagation past the gap is blocked and the resistance between the cells in the gap increases. Electron micrographs demonstrate that the nexuses of frog atrium and guinea pig ventricle are ruptured by hypertonic solutions.     

Two components of contraction in guinea pig papillary muscle

Biphasic contractions were produced in guinea pig papillary muscle by inducing partial depolarization in a K+ -rich solution (22 mM) containing 10(-6) M isoproterenol. However, when the same conditions were applied to frog and rat, monophasic contractions were obtained. In the case of guinea pig, an increase in the beating frequency produced an increase in amplitude of the first component and a reduction of the second, while in frog and rat, only a decrease in the amplitude of contractions was recorded. Caffeine (10(-3) M) eliminated the first component and increased the second in guinea pig, while in the case of rat and frog it decreased the amplitude of contractions. Procaine (10(-3) M) suppressed the first component and decreased the second one. The contraction in frog appears to be similar to the second component of contraction in guinea pig, while in rat, the contraction is comparable with the first component in guinea pig. It is suggested that the calcium ions which activate the two components of contraction in guinea pig under the given experimental conditions may arise from two different sources.     

Ouabain potentiation and Ca release from sarcoplasmic reticulum in cardiac and skeletal muscle cells

In this article, we describe a possible mechanism of ouabain potentiation in heart based on the following findings in cardiac and skeletal muscles of various species. (1) In heart ventricle muscles of frog and guinea pig, the ouabain potentiation is produced without an effect on Ca influx. In both frog and cat heart ventricle muscles, ouabain potentiates the rapid cooling contracture with or without caffeine in a Ca-deprived medium. It follows, therefore, that the ouabain potentiation is produced by an "intracellular" mechanism. (2) In crab single muscle fibers, contractile responses such as twitch, potassium-induced contracture, caffeine-induced contracture, and water-induced contracture are remarkably potentiated if ouabain is present within the fibers by microinjection, whereas the situation is reversed if the drug is given extracellularly. (3) The ouabain potentiated the Ca release from fragmented sarcoplasmic reticulum (FSR) isolated from cat, guinea pig, and frog heart and from skeletal muscles as a result of the procedures used, such as changing the ionic environment. (4) In frog, cat, and guinea pig heart ventricle muscles, a reduction of contractility as a result of pretreatment with urea--Ringer's was completely cancelled by ouabain almost without influencing the membrane depolarization. Based on these findings and others, the deduction was made that the positive inotropic effect of cardiac glycosides on the heart is brought about by potentiation of contraction - Ca release from the intracellular store sites, namely the sarcoplasmic reticulum.     

Adrenergic innervation of various sections of the heart in laboratory animals and man

By means of incubating slices in 2% glyoxylic acid solution, distribution of adrenergic fibers in the myocardium of various cardiac parts has been studied in the white rat, rabbit, cat, guinea pig and in the man. Both in the animals and in the man the distribution density of the adrenergic fibers of the myocardial plexuses in the auricle is higher than in the ventricle, and in the left half of the heart it is lower than in the right one. There are certain species differences in distribution of the adrenergic fibers. The density of the adrenergic fibers in the guinea pig myocardium is the highest, and in the white rat is is the lowest.     

Effects of low electrolyte media on salt loss and hemolysis of mammalian red blood cells.

Cation loss and hemolysis of various mammalian red cells suspended in isotonic non-electrolyte media were investigated. Sucrose buffered with 10 mM Tris-Hepes, pH 7.4 was used as the non-permeable non-electrolyte. Mammals from which the red cells were derived include the human, guinea pig, rat, rabbit, newborn calf, newborn piglet and pig, all of which contain K as the predominant cation species (HK type) and the dog, cat, sheep and cow, all of which possess Na as the predominant cation species (LK type). Of HK cells, a rapid efflux of K takes place from humans, rats and guinea pigs. Of LK type cells, the dog and cat exhibit an augmented membrane permeability to Na. The governing factors which influence cation permeability are the change in pH, temperature, and ionic strength. In response to increase in pH, the red cells of humans, dogs and cats become more permeable to cations, whereas the red cells of rat and rabbit are unaffected. In response to increase in temperature, HK type cells exhibit augmented K efflux, while the Na loss from the dog and cat cells manifest a well-defined maximum at near 37 degrees C. In all cases, a small substitution of sucrose by an equal number of osmoles of salts results in a dramatic decrease in cation loss. By contrast, the red cells of the rabbit, newborn calf, adult cow, newborn piglet, adult pig and sheep display no discernible increase in ion-permeability under the conditions alluded to above. In some species including the newborn calf, dog, and cat, an extensive hemolysis occurs usually within an hour in isotonic buffered sucrose solution. The osmolarity of sucrose solution affects these cells differently in that as the osmolarity increases from 200--500 mM, hemolytic rates of the calf and dog reach a saturation near 300 mM sucrose, whereas the hemolytic rate of the cat decreases progressively. Common features pertaining to this hemolysis are (1) the intracellular alkalinization process; and (2) the diminution of the cell volume which take place prior to and onset of hemolysis. SITS, a potent anion transport inhibitor, completely protects the cells from hemolysis by inhibiting chloride flux and the concomitant rise in intracellular pH.     

Caffeine- and Potassium-Induced Contractures of Frog Striated Muscle Fibers in Hypertonic Solutions

The effect of hypertonic solutions on the caffeine- and KCl-induced contractures of isolated fibers of frog skeletal muscle was tested. Hypertonic solutions, twice the normal osmotic strength, prepared by adding NaCl or sucrose, potentiate the caffeine-induced contractures. The fibers may develop tensions of 3.6 kg/cm² of fiber transverse section. The same hypertonic medium reduced the peak tension of KCl-induced contractures. Thus the hypertonic condition does not affect the contractile mechanism itself. These findings give further support to the view that the differential effect of hypertonic solution is on the excitation-contraction coupling mechanism. Extracellular calcium is not essentially required for the first few of a series of caffeine-induced contractures either in hypertonic or in isotonic solutions.     

Wavelength dependence of the optically recorded action potentials in guinea pig atrial muscles.

The optically recorded action potentials of the frog atrial muscles which lack transverse tubules showed different features from those reported by Heiny and Vergara (Heiny JA, Vergara J. Optical signals from surface and T system membranes in skeletal muscle fibers. J Gen Physiol 1982;80:203-230) in skeletal muscles (Fujishiro N, Kawata H. The wavelength dependence of optically recorded action potentials in the atrial muscles of the bullfrog (Rana catesbeiana). Comp Biochem Physiol 1996;114A:153-157). We examined whether or not the differences were consistent in other atrial muscles which lack transverse tubules with guinea pig atrial muscles. Two dyes (merocyanine rhodanine and merocyanine oxazolone) were used, and the dependence of the maximum rising phase of the optical signals on the wavelength of the incident beam was analyzed. No dependence was observed between them, and this finding was consistent with the structure of the membrane system of the guinea pig atrial muscles. The optical signals recorded at 718 nm of the incident beam from the guinea pig atrial muscles which stained with merocyanine oxazolone showed a more prominent second rising phase after the initial rapid rising phase of the optical signal than that recorded in the frog atrial muscles. This phase was not observed in the optical signals recorded at other wavelengths. The features of the optically recorded action potentials in guinea pig atrial muscles were consistent with those recorded in frog atrial muscles. Nifedipine did not affect the second rising phase.     

Penetration of horseradish peroxidase into the terminal cisternae of frog skeletal muscle fibers and blockade of caffeine contracture by Ca++ depletion

Summary Horseradish peroxidase, an extracellular marker, was given intravenously to frogs, and 40 min later the sartorius muscles were removed. The isolated muscles were exposed for an additional hour to Ringer solution containing peroxidase, then fixed with glutaraldehyde. Peroxidase activity was found in the T tubules, in some of the terminal cisternae (TC) of the SR, and occasionally in the longitudinal tubules of the SR. In transverse sections, the structures containing tracer formed a pattern of approximately parallel columns reaching to the cell surface; the statistical distribution of their spacing was nearly the same as that of the interdistances between the current-sensitive spots on the Z-line which triggered localized contraction (Huxley and Taylor, 1958). The caffeine contracture of frog sartorius muscles remained unchanged in isotonic Ringer solutions which were Ca⁺⁺-free or contained Mn⁺⁺ or La⁺⁺⁺; however, contracture was blocked by prior exposure of the muscles to the same solutions made 2 × hypertonic with sucrose (known to produce swelling of T tubules and (TC). Since Mn⁺⁺ and La⁺⁺⁺ are known to depress Ca⁺⁺ influx, these results suggest that washout of Ca⁺⁺ from the TC, and penetration of La⁺⁺⁺ or Mn⁺⁺ into it, occur more rapidly due to the swelling of T tubules and TC associated with hypertonicity. It is concluded that at least some of the terminal cisternae are open to the interstitial fluid via the T tubules. Thus, depolarization of the T tubules could readly depolarize the cisternae and lead to Ca⁺⁺ influx into the myoplasm.Supported by grants from the Public Health Service (HE-11155, HE-05815, and HE-10384) and from the American Heart Assocation. The authors are indebted to Mrs. Jan Redick for expert technical assistance.     

Penetration of horseradish peroxidase into the terminal cisternae of frog skeletal muscle fibers and blockade of caffeine contracture by Ca++ depletion

Summary Horseradish peroxidase, an extracellular marker, was given intravenously to frogs, and 40 min later the sartorius muscles were removed. The isolated muscles were exposed for an additional hour to Ringer solution containing peroxidase, then fixed with glutaraldehyde. Peroxidase activity was found in the T tubules, in some of the terminal cisternae (TC) of the SR, and occasionally in the longitudinal tubules of the SR. In transverse sections, the structures containing tracer formed a pattern of approximately parallel columns reaching to the cell surface; the statistical distribution of their spacing was nearly the same as that of the interdistances between the current-sensitive spots on the Z-line which triggered localized contraction (Huxley and Taylor, 1958). The caffeine contracture of frog sartorius muscles remained unchanged in isotonic Ringer solutions which were Ca⁺⁺-free or contained Mn⁺⁺ or La⁺⁺⁺; however, contracture was blocked by prior exposure of the muscles to the same solutions made 2 × hypertonic with sucrose (known to produce swelling of T tubules and (TC). Since Mn⁺⁺ and La⁺⁺⁺ are known to depress Ca⁺⁺ influx, these results suggest that washout of Ca⁺⁺ from the TC, and penetration of La⁺⁺⁺ or Mn⁺⁺ into it, occur more rapidly due to the swelling of T tubules and TC associated with hypertonicity. It is concluded that at least some of the terminal cisternae are open to the interstitial fluid via the T tubules. Thus, depolarization of the T tubules could readly depolarize the cisternae and lead to Ca⁺⁺ influx into the myoplasm.Supported by grants from the Public Health Service (HE-11155, HE-05815, and HE-10384) and from the American Heart Assocation. The authors are indebted to Mrs. Jan Redick for expert technical assistance.     

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.     

The origin of two components in contraction of guinea pig papillary muscle in the presence of noradrenaline.

In experiments on isolated guinea pig papillary muscles the effects of verapamil (5 mg/L) and caffeine (1 g/L) on the two-component contraction were investigated. The muscles were continuously superfused with normal Tyrode's solution containing 2 mg of noradrenaline/L at 20--22 degrees C. The first derivative of contractile response and transmembrane action potential were simultaneously recorded. Verapamil suppressed the amplitude of the second component and had no influence on the first component of contraction. Caffeine eliminated the first component and increased the second component of contraction. It was suggested that in the activation of two-component contraction calcium ions from two different pools take part: (1) those released from sarcoplasmic reticulum, and (2) those that crossed a cell membrane during the plateau of the action potential.     

Electrophysiological studies of the smooth muscle cell membrane of the rabbit common carotid artery

The electrical responses of the smooth muscle cells of the rabbit common carotid artery to extracellular stimulation were studied in isotonic and hypertonic solution (1.7 times normal tonicity) with microelectrodes. No spontaneous electrical or mechanical activity was recorded when the tissue was in either isotonic or hypertonic solution. The voltage-current relation of smooth muscle cells in the common carotid artery showed marked rectification in both isotonic and hypertonic solutions. In isotonic and hypertonic solutions mean values for membrane potentials were -44.5 and -51.5 mv, for space constants 1.13 and 1.21 mm, and for time constants 212.2 and 238.2 msec, respectively. Addition of 34.3 mM TEA to the solutions caused spontaneous action potentials in the common carotid artery. The action potentials recorded simultaneously from two microelectrodes showed good synchronization. It was concluded that there is electrical transmission between cells of this artery.     

Differential regulation of adenine nucleotide translocators by hypertonicity in the brain

To determine the gene(s) induced by hypertonicity in the brain, we performed a differential display analysis using RNA isolated from isotonic and hypertonic rat astrocytes. One cDNA rapidly up-regulated by hypertonicity was isolated, and the DNA sequence revealed that it was identical to adenine nucleotide translocator (ANT)2. ANT2 protein exchanges intramitochondrial ATP for cytoplasmic ADP. Among three ANT isoforms, only ANT2 mRNA was up-regulated markedly from 1 to 4 h after exposure to hypertonicity. Induction of the mRNA did not require de novo protein synthesis. Furthermore, ADP translocase activity in mitochondria of astrocytes was increased significantly by hypertonicity. To see the localization and regulation of ANT2 mRNA in the brain, we performed in situ hybridization of rat brain after intraperitoneal injection of a high concentration of NaCl. Although there were only weak signals in the control, intense hybridization signals were seen in hypertonic rat whole brain. Microscopic examination showed that ANT2 signals were present in the neurons, as well as glial cells. These results suggest that ANT2 may play a role in brain cells to adapt to the hypertonic environment.     

Expression of cell cycle related proteins—proliferating cell nuclear antigen (PCNA) and statin—during adaptation and de-adaptation of EUE cells to a hypertonic medium

EUE cells adapted to grow for long times in a hypertonic medium have a longer cell cycle than those growing in isotonic medium. To elucidate whether this lengthening involves specific cycle phases to differing extents, the expression of two cycle-related protein, PCNA and statin, was studied by dual parameter flow cytometry of indirect immunofluorescence protein labelling and DNA content. In isotonic medium, most cells, in all the cycle phases, were PCNA positive; in contrast, PCNA negative cells and statin positive cells were very few in number and only fell in the G0/1 range of DNA contents. In hypertonic medium, the frequency of PCNA positive cells was lower, and that of statin positive cells higher, than in isotonic medium, particularly in the G0/1 range of DNA contents: this suggests that a G0 block occurs under long-term hypertonic stress. Consistently, dual parameter flow cytometric measurement of BrdUrd immunofluorescence labelling and DNA content showed that fewer cells entered S phase in hypertonic medium and their progression through the S phase was slower; evidence was also found for the occurrence of a G2 block. These kinetics changes were fully reversible in isotonic medium, thus indicating the adaptive nature of the EUE response to hypertonicity.     

An interspecies comparison of gangliosides and neutral glycolipids in adrenal glands

Gangliosides and neutral glycolipids of adrenal glands of mouse, rat, guinea pig, rabbit, cat, pig, cow, monkey, and chicken were analyzed by thin layer chromatography (TLC). The major gangliosides common to all species had lactosylceramide in their core structure. GM3 containing N-acetylneuraminic acid (NeuAc) was the major ganglioside in rat, guinea pig, rabbit, and cat, whereas GM3 containing N-glycolylneuraminic acid (NeuGc) was the major one in mouse, cow, and monkey. GD3 was also detected in all species except mouse and GD3(NeuAc)2 was the major in pig adrenal gland. GM4(NeuAc) was detected in the adrenal glands of guinea pig and chicken but not in those of the other species. In the neutral glycolipid fractions, galactosylceramide, glucosylceramide, lactosylceramide, globotriaosylceramide and globotetraosylceramide were detected and the proportions of these glycolipids varied among the species. Guinea pig and chicken adrenal glands contained large amounts of galactosylceramide, this being consistent with the presence of GM4 in these two species. Globopentaosylceramide was detected in mouse, guinea pig, cat, and chicken by the TLC-immunostaining procedure.     

Entrance of colloidal ThO2 tracer into the T tubules and longitudinal tubules of the guinea pig heart

Summary Colloidal ThO₂ particles (diameter of 60 Å) were used as electron-opaque markers to trace the intracellular compartments continuous with the bulk interstitial fluid of guinea pig ventricular muscle. Beating and quiescent hearts in a Langendorff preparation were perfused for 30 min with oxygenated Ringer solution containing 1% ThO₂. The hearts were immediately fixed by perfusing with glutaraldehyde solution. The colloidal ThO₂ particles entered into many of the T tubules and into longitudinallyrunning tubules. No differences in distribution of ThO₂ were observed in a heart which was not exposed to ThO₂ until after it was fixed. Tracer did not penetrate into the intercalated disk clefts in the guinea pig hearts and one frog heart used for comparison. Tubular profiles filled with ThO₂ were not seen in frog heart, an observation which confirms the absence of T tubules in this amphibian. It is concluded that, in mammalian cardiac muscle, the lumens of the longitudinal tubules are continuous with the lumens of the T tubules, forming an extensively interconnected T-L tubular system. Hence, every myofibril has close access to a fluid-filled space which is continuous with the interstitial fluid and which may be of similar cationic composition; such an arrangement should facilitate excitation-contraction coupling.Supported by grants from the American Heart Association and from the Public Health Service (HE-11155, HE-05815 and HE-10384). The authors wish to acknowledge the expert technical assistance of Mrs. Jan Redick and to thank Dr. James Smith of Marquette University for the supply of Thorotrast used in these studies.     

The kinetics of cytosolic calcium in the right ventricular myocardium of guinea pigs and rats

The characteristics of tension increase and decline, as well as those of the calcium transients, have been measured in the trabeculae of the right cardiac ventricle of the guinea pig and rat in the isometric contraction mode with different preloads. Measurements were performed at different temperatures of physiological saline and the effects of inhibition of calcium removal from the cytosol mediated by Na⁺–Ca²⁺ exchange and the ATP-dependent Ca²⁺ pump of the sarcoplasmic reticulum (SERCA2a) were analyzed. Emergence of the “bump” phase (a phase of brief deceleration of the decay of the calcium transient) was observed in the guinea pig myocardium as the temperature was increased from 25 to 30°C; earlier observations of this phenomenon were reported only for rats. As the temperature was elevated to 35°C, the “bump” phase in the guinea pig myocardium transformed into a “plateau” phase of the calcium transient. The effect of temperature on the course of the decay of the calcium transient in the rat myocardium was negligible. In contrast, a gradual stretching of the right ventricular trabecula of the rat was accompanied by the emergence of the “bump” phase and a gradual increase of its parameters (amplitude, integral intensity, and duration), whereas preload did not exert a similar effect on the guinea pig myocardium. Selective inhibition of the reverse mode of Na⁺–Ca²⁺ exchange did not affect the characteristics of the decay of the calcium transient in guinea pig myocardium. Selective inhibition of SERCA2a in the guinea pig and rat myocardium had a significant modifying effect on the decay phase of the calcium transient and resulted in emergence of the “bump” phase or an increase in the intensity of this phase in the myocardium of these animal species. The characteristics of this phase can be used to quantify the length-dependent activation of myocardial contraction.