[Myoplasmic fixed charges of the barnacle muscle fiber (author's transl)]. / Charges fixés du protoplasme des fibres musculaires de balane.

The experimental model used to study diffusion and electrical conduction in the cytoplasm of large muscle fibers was adapted to evaluate the myoplasmic density of fixed charges. Membranes of myoplasm were prepared and phi X, the myoplasmic thermodynamically effective charge density, was calculated from the membrane potential (Kamo, N., Toyoshima, Y. and Kobatake, Y. (1971) Kolloid Z.u.Z. Polymère 1061--1068) when these membranes were used as the partition between two electrolyte solutions. The dilution of KCl in the external solutions reduced phi X, which increases with the reduction of the water content in the membrane of myoplasm. With a water content of 73.0 ml/100 g KCl concentration in the external medium equal to 0.15 M, phi X was evaluated to 0.058 equiv/l. The substitution of KCl by NaCl introduces a reduction in phi X of 20--50% depending on E1KCl] in the external solutions. The addition of ATP, Mg2+, and Ca2+ also causes a reduction of phi B by 30--50% according to the experimental conditions. These results indicate that the fraction of counterions dissociated from the myoplasmic macromolecules is reduced when the concentration of the counterions is diminished or when CKl is replaced by Nal. It also suggests a reduction of phi X during muscular contraction.

The myoplasmic fixed charges of the barnacle muscle fiber and the free Ca2+ concentration.

An experimental model was recently developed to measure the myoplasm density of fixed charges (Caillé J.P. (1979) Biochim. Biophys. Acta 585, 300-313). The effect of ATP, Mg2+ and Ca2+ suggests that the myoplasmic thermodynamically effective charge density (phi X) decreases during muscular contraction. In order to determine if this reduction was due to either ATP, Mg2+ or Ca2+, the phi X dependence on those substances was studied. The results indicated that the addition of MgCl2 (2 mM) reduced phi X by 10 to 30% depending on the water content of the myoplasm. A reduction of phi X was also observed with the addition of (10(-4) M) Ca2+, but the presence of ATP was necessary for this reduction to occur. Finally when EGTA was added to the external solution in order to control the level of free Ca2+, an important reduction of phi X (40%) was observed for a pCa equal to 5.7 (water content 76.1 +/- 0.3 ml/100 g). These results allowed a correlation between the reduction of the myoplasmic fixed charges and the turning on of the contractile machinery.

Laser Raman investigations of intact single muscle fibers. Protein conformations.

Raman spectra, in the frequency region of the protein vibrations, of intact single muscle fibers of the giant barnacle are presented. Strong bands at 1521 and 1156 cm-1 in the spectra are attributed to resonance-enhanced Raman bands of membrane-bound beta-carotene. Many bands of the myofibrillar proteins are also observed, and at least three spectral features confirm that these proteins adopt a predominantly alpha-helical structure: (1) the amide I band at 1648 cm-1, (2) the weak scattering in the amide III region, and (3) a strong skeletal C-C stretching band at 939 cm-1. Deuterated fibers have also been examined in order to find the exact shape of the amide III band. The presence in the fibers of paramyosin, which is only found in catch muscles, is also apparent from the spectra.

Laser Raman study of internally perfused muscle fibers. Effect of Mg2+, ATP and Ca2+.

Raman spectra of an intact muscle fiber and of internally perfused fibers in capillary tubes have been obtained. The use of internal perfusion has insured a good control of the concentration of Ca2+, Mg2+ and ATP. The comparison of the spectra obtained with the two types of fibers shows that the muscle structure is well preserved in capillary tubes. In addition, it appears that the sarcomere length has no significant effect on the Raman spectrum of muscle fibers. Our results on perfused fibers demonstrate that a fiber can be kept in the relaxed state for several hours, then displaying an intact fiber spectrum, when the concentration of ATP, Mg2+ and Ca2+ is maintained at 5, 2 and 0 mM, respectively. Therefore ATP and Mg2+ do not affect the Raman spectrum of muscle fibers. When one of these components is removed, or when Ca2+ is added, contraction occurs and causes major spectral changes. These results are interpreted as being due to strong electrostatic interactions between basic and acidic residues during contraction, and to a change of the alpha-helical content, or of the orientation, of some of the contractile proteins.

Calorimetric study of water in muscle tissue.

Differential scanning calorimetry has been used to investigate the state of water in intact single muscle fibers of the giant barnacle, Balanus nubilus. The shapes of the melting curves suggest the presence of three types of water: unfrozen (or bound), free (or bulk) and intermediate water. The amount of unfrozen water per g protein was constant within experimental error. An increase in water content changed almost exclusively the amounts of free water. The amount of intermediate water varies only slightly with the fiber water content.

Laser Raman investigation of intact single muscle fibers. On the state of water in muscle tissue.

Laser Raman spectroscopy has been used to investigate the state of water in intact single muscle fibers of the giant barnacle (Balanus nubilus). The spectra in the region of the O-H (or O-2H) stretching modes of water in unfrozen fibers show that there is no appreciable difference between the shape and relative intensity of the Raman bands due to the water molecules located inside a muscle fiber and those of the corresponding bands in the spectrum of pure water. The presence of significant amounts of "structured" intracellular water, greater than approx. 5% of the total water content, in these fibers is thus excluded. The Raman spectra of frozen fibers have also been recorded in order to evaluate the amount of intracellular water which remains unfrozen at temperatures below the normal freezing point of water. We have been able to reproduce these spectra by assuming that the spectrum of a frozen fiber is the sum of the individual spectra of water and ice. To calculate the amount of unfrozen water from these curve fittings, it was also necessary to determine the intensities of the water and ice Raman bands relative to one another. We have found the I(ice)/I(water) ratio is 1.07 +/- 0.01 for H2O and 1.05 +/- 0.03 for 2H2O With these figures, we have calculated that for a fiber with a normal water content of 80%, 20% of the water molecules remain in the supercooled state of -5 degrees C, which corresponds to 1 g of water per g of fiber dry weight. This amount of bound water was also found to be independent of the water content of the fibers.

[The fixed charge of protoplasm in barnacle muscle fiber; effect of the length of the sarcomeres]. / Charges fixes du protoplasme des fibres musculaires de balane; effet de la longueur des sarcomères.

The thermodynamically effective charge density (phi X) of myoplasm was measured on barnacle muscle fibers. A small section of a muscle cell formed a liquid junction between two electrolyte solutions and the electrical potential between the solutions permitted to evaluate phi X. If the sarcomere length was kept constant (Ls = 12.5 micron), phi X increased from 0.068 to 0.090 equiv./L when the water content was reduced from 75 to 65 g/100 g wet weight. This increment of X resulted from an increase of X when the concentration of macromolecules is raised in the sample of cytoplasm. But it also indicates that the counterions of the fixed charges concur less to the activity of the counterions when X is increased by lowering the water content. If the water content is maintained constant, phi X increased from 0.068 to 0.084 equiv./L for sarcomere lengths equal to 9.6 and 11.5 micron but phi X remained constant for Ls greater than 12 micron. Thus, when the length of the sarcomere is greater than 12 micron the contribution of the counterions of the fixed charges to the activity of the counterion is larger than for Ls = 9.6 micron.

[Efflux of 36Cl and the distribution of chlorine in the papillary muscles of rabbits and the effect of ouabain]. / Efflux du 36Cl et distribution du Cl dans le muscle papillaire du lapin, effet de l'ouabaïne.

The 36Cl efflux "in vivo" was measured in the rabbit papillary muscle to determine the Cl distribution in the muscle and to evaluate the effect of ouabain on this parameter. The results obtained for the 36Cl efflux are analyzed using either a two-compartment model or a model including diffusion in the extracellular space in series with one compartment. The Cl exchange with 36Cl, [Cl]i (intracellular Cl content which has participated in exchange, Cl in equilibrium with 36Cl) is computed from the exponential terms of the models. A time exposure of 40 and 80 min to the 36Cl-containing solution led to the same exchange Cl content: 20.5 and 23.9 mmol/kg cells. Addition of ouabain (10(-6) M) slightly increased the rate constant of the cellular compartment, but did not influence the [Cl]i. In the presence of ouabain (10(-6) M), there was a significant increase in the efflux component with a rapid rate constant. These results can be interpreted as follows: the Cl intracellular concentration is not affected by ouabain; thus, the increase in total Cl content induced in the papillary muscle by ouabain is located in a compartment having a very rapid exchange velocity with the extracellular medium.

Intracellular chloride activity in rabbit papillary muscle: effect of serum.

The intracellular chloride activity (aiCl), measured with Cl-selective microelectrodes on stimulated rabbit papillary muscles (1 Hz) incubated in serum, was 7.2 +/- 2.2 mM (48 measurements). Under the same condition, on the quiescent muscle, aiCl was 7.5 +/- 2.8 mM (45 measurements). The membrane potential of quiescent papillary muscles and diastolic potential of stimulated papillary muscles were -79.0 +/- 0.7 (50 measurements) and -83.5 +/- 0.5 mV (50 measurements), respectively. The experimental conditions were chosen to reproduce the in vivo conditions where the Cl equilibrium potential is close to the membrane potential or to the diastolic potential. After correcting for cytoplasmic interference (4 mM) on the aiCl measurements, the Cl equilibrium potential (ECl) was -84 mV. In conclusion, the Cl distribution in cardiac cells bathed in serum is passive as for in vivo cardiac cells.

Myoplasmic impedance of the barnacle muscle fiber.

Myoplasmic impedance was measured on a barnacle (Balanus nubilus) single muscle fiber that was placed in a cylindrical cavity to limit the volume and prevent the hydration of the myoplasm. At both ends of the cavity, the myoplasm was in direct contact with an electrolyte solution. When equilibrium with the external medium was reached, the myoplasmic impedance was measured at 10 degrees C with an impedance bridge at 1000 Hz. The results indicated that the myoplasmic impedance of the muscle fiber is mainly resistive. Treating the myoplasm as a suspension of small conductive particles, we deduced the specific conductivity of the contractile filaments kf and their volume fraction rho (kf = 2.78 X 10(-3) omega-1cm-1, and rho = 0.48). The experimental technique permits an estimate of the specific myoplasmic conductivity in vivo (6.27 X 10(-3) omega-1cm-1). Finally, a decrease in the pH of the external solution from 10.1 to 4.0 lowered the myoplasmic conductivity by 16%. This may be considered as indirect evidence that the conductivity of the contractile filaments is associated with the protein counter-ions, since Hinke et al. (1973. Ann. N.Y. Acad. Sci. 204, 274-296.) reported evidence that a lowering of pH decreases the number of counter-ions.

Freezing point and melting point of barnacle muscle fibers.

The freezing point and the melting point of myoplasm were measured with two experimental models. In all samples, a supercooled stage was reached by lowering the temperature of the sample to approximately - 7 degrees C, and the freezing of the sample was mechanically induced. The freezing process was associated with a phase transition in the interstices between the contractile filaments. In intact muscle fibers, the freezing point showed a structural component (0.43 degrees C), and the melting point indicated that the intracellular and the extracellular compartments are isotonic. When the sample of myoplasm, previously inserted in a cylindrical cavity was incubated in an electrolyte solution, the freezing point showed a structural component similar to that of the intact muscle fiber, but the melting point was lower than the freezing and the melting points of the embedding solution. This was interpreted as evidence that the counterions around the contractile filaments occupied a nonnegligible fraction of the intracellular compartment.

[Ouabain and intracellular potassium activity in the papillary muscle of the rabbit]. / Ouabaïne et activité intracellulaire du K dans le muscle papillaire du lapin.

Ionic activity on both sides of the cardiac cell membrane is an essential parameter for determining the mechanism responsible for the positive inotropic effect of cardiotonic glucosides. The intracellular activity of K (aiK) was therefore measured using selective microelectrodes, in the papillary muscles of a rabbit, for both a low concentration (10(-8) M) and a toxic concentration (10(-6) M) of ouabain. The ouabain at low concentration (10(-8) M) had no significant effect with respect to aiK, the values derived with and without ouabain being 85.4 mequiv./L and 84.2 mequiv./L respectively. The use of selective microelectrodes for measuring K, paired with a microelectrode filled with KC1, made it possible to study aiK in terms of time in the presence of a toxic concentration of ouabain (10(-6) M). In such a case, ouabain results in a reversible lessening of aiK.

Fixed charges of the heart muscle interstitium.

The interstitium of the heart muscle is primarily composed of ground substance. The glycoproteins and proteoglycans that formed the ground substance bore negative charges at neutral pH like the glycosaminoglycans and proteoglycans of the water-rich phase of the interstitium. Microelectrodes were used to look for the existence of an electrical potential between the interstitium of the rabbit papillary muscle and an ambient medium. Evidence is presented for the existence of such an electrical potential. When the ambient solution was a Krebs solution, this potential was evaluated at -5.7 mV. This electrical potential is dependent on the filling solution of the microelectrodes and on the ambient medium; in rabbit serum, the electrical potential diminished to -0.6 mV. Assuming that this potential is a measure of the Donnan potential, the Cl and Na activities in the interstitium were evaluated to 76 and 135 mM when the rabbit papillary muscle was superfused with a Kreb's solution.

Effects of organic solutes on the Raman spectra of barnacle muscle fibers.

The Raman spectra observed from barnacle muscle fibers are quite complex because the cytoplasm of these cells contains several proteins and solutes. An extraction procedure was used to separate organic solutes from the contractile proteins. Glycine, trimethylamine oxide, taurine, and alanine were found to contribute to the Raman spectra of barnacle muscle fibers, while spectra of lobster fibers reveal the presence of betaine in addition. We have observed that the increase in osmolarity of the intracellular fluid caused by the augmentation of the salinity of sea water (density, 1.023-1.030) in which the barnacles were kept, induces a reduction of intensity of the amide I band. To distinguish among the different parameters which are modified by the sea water salinity, observations were made on glycerinated barnacle muscle fibers. The reduction of intensity of the amide I band in the Raman spectra of glycerinated muscle fibers was also observed with the addition of taurine (0.08 M) in the external relaxing solution. Therefore, under these experimental conditions, the Raman scattering intensity in the amide I region assigned to the alpha-helix conformation (1645-1650 cm-1) is increased when the concentration of organic electrolytes is reduced. However, as no significant decrease of the scattering intensity in the 1660-1670 cm-1 region where the amide I bands of either beta-sheet or disordered conformations normally appear was observed, the increase of intensity of the amide I band centered at 1645 cm-1 is assigned to a change of orientation of alpha-helical segments of the myosin molecules. Our results suggest that organic solutes influence the position of the S-2 segments relative to the thick filaments.

Raman spectroscopy of cytoplasmic muscle fiber proteins. Orientational order.

The polarized Raman spectra of glycerinated and intact single muscle fibers of the giant barnacle were obtained. These spectra show that the conformation-sensitive amide I, amide III, and C-C stretching vibrations give Raman bands that are stronger when the electric field of both the incident and scattered radiation is parallel to the fiber axis (Izz). The detailed analysis of the amide I band by curve fitting shows that approximately 50% of the alpha-helical segments of the contractile proteins are oriented along the fiber axis, which is in good agreement with the conformation and composition of muscle fiber proteins. Difference Raman spectroscopy was also used to highlight the Raman bands attributed to the oriented segments of the alpha-helical proteins. The difference spectrum, which is very similar to the spectrum of tropomyosin, displays amide I and amide III bands at 1,645 and 1,310 cm-1, respectively, the bandwidth of the amide I line being characteristic of a highly alpha-helical biopolymer with a small dispersion of dihedral angles. A small dichroic effect was also observed for the band due to the CH2 bending mode at 1,450 cm-1 and on the 1,340 cm-1 band. In the C-C stretching mode region, two bands were detected at 902 and 938 cm-1 and are both assigned to the alpha-helical conformation.

[Fibrocystic deterioration of the popliteal artery. Apropos of 1 case]. / La dégénérescence fibro-kystique de l'artère poplitée. A propos d'un cas.

Set off by a sports movement, a subacute ischemia of the lower limb allowed the delayed discovery of a segmental stenosis of the popliteal artery in a young man. It involved a compression by fibro-cystic deterioration of the wall of the artery. Separation of the cyst from the diseased arterial wall was followed by the restoration of circulation by means of an internal saphenous vein graft.

Intracellular chloride activity in rabbit papillary muscle: effect of ouabain.

Intracellular chloride activity (aiCl) and membrane potential (Vm) were measured in rabbit papillary muscle under in vitro conditions. The cellular chloride concentration (Cli) was estimated from measurements of total water content, extracellular space, and total chloride concentration. The effects of therapeutic (10(-8) M) and toxic (10(-6) M) concentrations of ouabain on these parameters were tested. The chloride-sensitive microelectrodes were of the liquid-ion exchanger type. Selectivity for HCO-3 was taken into account in the calculation of aiCl. In 11 control experiments made with two different protocols aiCl was determined in subendocardial and in deeper cells. The mean membrane potentials were -78.7 and -78.0 mV and the mean cytoplasmic chloride activities were 17.5 and 17.7 mM, respectively. The chloride equilibrium potentials were -43.5 and -43.2 mV. These results indicated that chloride is not passively distributed in rabbit papillary muscle. Ouabain (10(-8) M) did not change Vm or aiCl. At a toxic concentration of ouabain, Vm fell to -68.0 mV in superficial cells and to -67.8 mV in deeper cells, but aiCl remained unchanged . These results suggested that under in vitro conditions intracellular chloride is distributed within more than one cellular compartment.