Lactic acid efflux from white skeletal muscle is catalyzed by the monocarboxylate transporter isoform MCT3.

The newly cloned proton-linked monocarboxylate transporter MCT3 was shown by Western blotting and immunofluorescence confocal microscopy to be expressed in all muscle fibers. In contrast, MCT1 is expressed most abundantly in oxidative fibers but is almost totally absent in fast-twitch glycolytic fibers. Thus MCT3 appears to be the major MCT isoform responsible for efflux of glycolytically derived lactic acid from white skeletal muscle. MCT3 is also expressed in several other tissues requiring rapid lactic acid efflux. The expression of both MCT3 and MCT1 was decreased by 40-60% 3 weeks after denervation of rat hind limb muscles, whereas chronic stimulation of the muscles for 7 days increased expression of MCT1 2-3-fold but had no effect on MCT3 expression. The kinetics and substrate and inhibitor specificities of monocarboxylate transport into cell lines expressing only MCT3 or MCT1 have been determined. Differences in the properties of MCT1 and MCT3 are relatively modest, suggesting that the significance of the two isoforms may be related to their regulation rather than their intrinsic properties.

Tensile strength and dilatational elasticity of giant sarcolemmal vesicles shed from rabbit muscle.

1. Mechanical properties of the surface membrane of skeletal muscle were determined on sarcolemmal vesicles (mean diameter, 71 microns) shed by rabbit psoas muscle swelling in 140 mM KC1 containing collagenase. 2. Vesicles were stressed by partial aspiration into parallel bore pipettes. The isotropic membrane tension so created caused an increase in membrane area which expresses itself in an elongation of the vesicle projection into the pipette. 3. For individual vesicles, a linear relationship between membrane tension and membrane area increase was found up to the point when the vesicle burst, i.e. sarcolemmal vesicles behaved as perfectly elastic structures. 4. The maximum tension sarcolemmal vesicles could sustain before bursting was 12.4 +/- 0.2 mN m-1 (median +/- 95% confidence interval), and the corresponding fractional increase in membrane area was 0.026 +/- 0.005 (median +/- 95% confidence interval). The elastic modulus of area expansion was 490 +/- 88 mN m-1 (mean +/- S.D.). 5. In conformity with cited comparable work on red blood cells and artificial lipid vesicles, the strength and area elasticity of the skeletal muscle membrane are considered properties of the fluid lipid matrix of the membrane and of the degree to which the bilayer is perturbed by lipid-protein interaction.

Ca2+ loading reduces the tensile strength of sarcolemmal vesicles shed from rabbit muscle.

1. Sarcolemmal vesicles shed by rabbit muscle were loaded with Ca2+ by means of A23187 or ionomycin. [Ca2+]0 was buffered between 0.8 and 20 microM. Membrane strength was measured by pipette aspiration. 2. At 20 microM Ca2+ many vesicles underwent autolysis, or were so weak that they burst instantly on aspiration. Between 10 and 2 microM Ca2+ a graded decrease in membrane strength was demonstrable. At 0.8 microM Ca2+ the mechanical properties of the sarcolemma remained unaltered. 3. Mg2+ carried by A23187 does not mimic the effect of Ca2+. The ionophore itself similarly did not cause a decrease in membrane tensile strength. 4. Pre-treatment with BAPTA-AM, so as to buffer internal Ca2+, partly protected vesicles against the decrease in membrane strength produced by Ca2+ loading. 5. Membrane strength was not restored by adding excess BAPTA to the bathing solution, so as to reverse the Ca2+ gradient. An irreversible degradation of the membrane consequent upon raised [Ca2+]1 seems indicated. 6. These findings are discussed in relation to the mechanisms which have been advanced to account for the role of elevated [Ca2+]1 in cell death. 7. An attempt to use staphylococcal alpha-toxin as an alternative means to permeabilize the sarcolemma led to the incidental finding that this pore-forming protein itself greatly weakens the membrane in doses lower than required for effective permeabilization.

The membrane hypothesis of Duchenne muscular dystrophy: quest for functional evidence.

(1) The location of dystrophin in normal muscle, its molecular structure and associations, characterize it as a component of the submembrane cytoskeleton. When dystrophin is missing the cytoskeleton will therefore be defective, and it has been supposed that this renders the muscle membrane more vulnerable to mechanical damage. With the discovery of animal strains lacking in dystrophin, this hypothesis has been put to experimental tests. Contradictory results have been obtained by workers using different exercise regimens and different indices of fibre damage. (2) Direct measurements of the tensile strength of the membrane have been made on patches of cultured myotubes or isolated muscle fibres, and on sarcolemmal vesicles by pipette aspiration. Neither method has revealed a difference in the tensile strength between normal and dystrophic membrane. The most plausible explanation is that the tensile strength of the membrane is a property more of the lipid bilayer than of the cytoskeleton. (3) In another experimental approach tensile membrane stress has been produced by exposing isolated muscle fibres and myotubes in culture to hypotonic solutions. In such experiments fibres and myotubes lacking dystrophin have been found to lyse more readily than do normal ones. This difference does not conflict with the similarity in tensile strength of normal and dystrophic fibre membranes noted above. Rather, the predisposition to osmotic lysis of dystrophic fibres and myotubes may signify a lower ratio of membrane surface to cell volume, perhaps as a result of loss of some of the spare membrane normally possessed by skeletal muscle fibres and myotubes. (4) In red blood cells the membrane cytoskeleton functions to maintain membrane deformability and stability. Deficiency in spectrin, the main cytoskeletal component, predisposes red cells to cytoskeletal rupture and membrane loss when they experience shear stress. Skeletal muscle fibres, especially long fibres contracting eccentrically, are susceptible to shear stress as a result of uneven contraction along their length. In that event, fibres lacking dystrophin may similarly shed membrane more readily.

Mechanical properties of normal and mdx mouse sarcolemma: bearing on function of dystrophin.

The tensile strength of the muscle fibre surface membrane was estimated (1) from the suction required to burst membrane patches and (2) by aspiration of sarcolemmal vesicles into micropipettes of uniform bore. Each method gave an average value close to 60 microN cm-1 for the maximum tension sustainable by normal mouse sarcolemma and only slightly lower values for sarcolemma from mdx mice which lack dystrophin. The elastic modulus of area expansion, as measurable by pipette aspiration of sarcolemmal vesicles, was found to have an average value of 3160 microN cm-1 for normal and 2770 microN cm-1 for mdx mouse sarcolemma. The tensile strength of the sarcolemma is much too small for any differences in it to be the basis for the different osmotic behaviour of normal and mdx muscle fibres reported recently (Menke & Jockusch, 1991). By analogy with the better understood origin of the osmotic fragility of different types of red blood cells, the higher osmotic fragility of mdx muscle fibres is suggested to be of morphological origin. We postulate that dystrophin functions as an element of the submembrane cytoskeleton so as to maintain the normal folding which safeguards the sarcolemma against mechanical damage.

Dystrophin is tightly associated with the sarcolemma of mammalian skeletal muscle fibers.

Sarcolemmal vesicles with right-side-out configuration were prepared from normal fresh human and rabbit skeletal muscle bundles by incubation in 140 mM KCl solution containing collagenase. The vesicles were used to examine the association of dystrophin, the protein product of the Duchenne muscular dystrophy gene, with the sarcolemma. Western blot analysis, indirect immunofluorescence, and immunoperoxidase staining using specific antibodies raised against the N-terminal and the C-terminal domains show that dystrophin remains associated with the membrane of sarcolemmal vesicles. Indirect immunofluorescence microscopy using permeabilized and unpermeabilized vesicles indicated that both the N-terminus and the C-terminus of dystrophin are localized to the cytoplasmic surface of the sarcolemma. These results suggest that dystrophin has much stronger attachment to the surface membrane than it has to the internal domain of skeletal muscle fibers. Sarcolemmal vesicles thus represent a new system for studying the function of dystrophin and the molecular basis of its association with the sarcolemma.

Sensitivity to flow of intrinsic gating in inwardly rectifying potassium channel from mammalian skeletal muscle.

1. Current through inwardly rectifying K+ channels was measured in inside-out patches from rat and human sarcolemmal vesicles and from dispersed rat flexor digitorum brevis muscle fibres. The patches were positioned so as to face the aperture of a large-diameter pipette from which solution of the same composition as the bath solution could be ejected. The solution within the patch pipette and the bath solution both contained principally 140 mM-KCl. 2. The kinetic behaviour of the inwardly rectifying channel was found to vary according to whether the patch was in static or flowing solution. At negative holding potentials, when the channel is open most of the time in static solution, flow produced a reversible and repeatable decrease in open probability. 3. In Mg2(+)-free solution the inwardly rectifying channel allows outward current to pass at positive holding potentials. This allows the kinetic behaviour of the channel in static and flowing solution to be compared over a wider voltage range. 4. In both static and flowing solution, the open probability-voltage relation is sigmoidal and can be fitted by a Boltzmann curve. As a result of flow, the maximum open probability at negative potentials is decreased and the mid-point of the relation is shifted to the right by more than 20 mV. 5. No evidence could be found for the existence of a local concentration gradient sensitive to flow. Application of suction to the patch pipette showed the inwardly rectifying channels not to be sensitive to membrane stretch.(ABSTRACT TRUNCATED AT 250 WORDS)

Single-channel activity in sarcolemmal vesicles from human and other mammalian muscles.

Segments of mammalian, including human, skeletal muscle 1-2 cm long can be induced to shed vesicles by treatment with collagenase in a high-KCl solution containing no added calcium. The vesicles are encompassed by clean sarcolemma so that the gigaseal necessary for patch-clamping is readily formed. The properties of inwardly rectifying potassium channels and of calcium-activated potassium channels in patches detached from such vesicles are shown to be consistent with expectations based on earlier studies on intact muscle fibers and with patch clamp results on the same type of channels in other tissues. A chloride channel which rectifies outwardly with a conductance ranging from 15 to 50 pS is also described. The utility of sarcolemmal vesicles for the study of ion channels in human biopsy material is discussed.

A randomized study of a semisynthetic heparin analogue and heparin in prophylaxis of deep vein thrombosis.

The prophylactic effect of a semisynthetic heparin analogue (SSHA) on deep vein thrombosis was investigated in a prospective double-blind multicentre trial. 440 major general surgical and gynaecological patients were randomized to one of three treatment groups: 50 mg SSHA, 37.5 mg SSHA and 5000 units sodium heparin subcutaneously 12-hourly. Deep venous thrombosis (DVT) was diagnosed with the fibrinogen uptake test and verified with phlebography. Bleeding complications and other side-effects were carefully monitored. There were no significant differences between the three treatment groups of patients in age, sex, type of operation or risk factors. A DVT was diagnosed in 16 patients (12 per cent) in the SSHA 50 mg group, in 21 patients (15 per cent) in the SSHA 37.5 mg group and 21 patients (14 per cent) in the heparin-treated group. No significant differences were found in the number of patients who bled unexpectedly in the postoperative period, required transfusion or developed wound haematomas. Blood loss at operation was similar in all three groups. Three pulmonary emboli were diagnosed by pulmonary scintigraphy, one in each group.

Ionic basis of the hyperpolarizing action of adenyl compounds on sinus venosus of the tortoise heart.

The ionic mechanism underlying the hyperpolarizing action of adenosine and adenine nucleotides was studied by measuring the efflux of 43K or 86Rb from sinus venosus and interauricular septum of tortoise heart. Preparations rendered quiescent by high-K (27 mM) Ringer solution were used. Adenosine and ATP increased the efflux of 43K and 86Rb from sinus venosus. The magnitude of the responses varied from preparation to preparation, but in the same tissue adenosine and ATP were of equal efficacy. When dose-response relationships could be determined, the adenyl compounds were found to be of similar potency. Km for adenosine was 6.2 X 10(-6) M, for ATP 8.3 X 10(-6) M. Regional variations in the magnitude of the responses were observed. The largest responses were obtained from the muscular strip of sinus venosus near its junction with atrium, and from the right horn of the sinus venosus. In interauricular septum the adenyl compounds caused only a slight increase in isotope efflux. Acetylcholine, by contrast, produced large increases in 86Rb efflux from all these preparations. Thus the distribution of the purinoceptors in the tortoise heart is more confined than that of the muscarinic receptors. Antagonism of the response to adenyl compounds by theophylline and 8-phenyltheophylline was studied. The apparent Ki for theophylline was 10(-5) M; that for 8-phenyltheophylline about 10(-6) M. Atropine did not inhibit the responses to the adenyl compounds. These results indicate that the changes in K permeability produced by adenosine and ATP are mediated by P1-purinoceptors. The adenosine transport inhibitors, dipyridamole and nitrobenzylthioinosine (NBMPR), had no effect on the adenyl-induced responses, indicating that adenosine uptake is of little importance in tortoise sinus venosus. The effects of phosphate-modified ATP analogues were studied. Adenylimidodiphosphate (APPNP) produced increases in 86Rb efflux similar to those found with ATP, confirming that breakdown of ATP to adenosine is not obligatory for its action at P1-purinoceptors. Alpha-beta methylene ATP (APCPP) and beta-gamma methylene ATP (APPCP) produced much smaller effects, which may be explained by their structural and chemical differences from ATP. The use of 86Rb as a tracer (Rb: K less than 0.01 in load solution) gives qualitatively similar results to those obtained when 43K is used to study the permeability increases produced by the adenyl compounds or acetylcholine. Quantitative differences in the measures obtained with the two isotopes, however, become apparent when the efflux of both is studied simultaneously.(ABSTRACT TRUNCATED AT 400 WORDS)

Potassium current noise induced by barium ions in frog skeletal muscle.

Ba2+ was used to introduce K+ current fluctuations in addition to those arising from the intrinsic 'gating' mechanism studied in the preceding paper. In the presence of 0.5 to 2.0 mM-Ba2+, Lorentzian spectra were observed at potentials between -10 and -63 mV. The amplitude and corner frequency of these Lorentzian spectra varied with voltage in the general manner to be expected from the voltage- and time-dependent blocking action of Ba2+, but the microscopically determined time constant was consistently faster than that determined from the intensification of block upon hyperpolarization. The variance of the Ba2+-induced fluctuations could be used to calculate the limiting single-channel conductance, gamma, without the need to assume a specific model for inward rectification. The value of gamma obtained for preparations in symmetrical 120 mM-K+ was about 8 pS (20-24 degrees), in good agreement with that found in the preceding paper. Some fibres underwent a spontaneous, large increase in membrane conductance. This conductance showed inward rectification in the positive quadrant. At negative potentials this high conductance was blocked by 1 mM-Ba2+ in a voltage- and time-dependent manner. The block became faster and more complete at more negative potentials. Analysis of current noise in the high conductance state in the presence of Ba2+ gave Lorentzian spectra with corner frequencies in general agreement with the time constant of macroscopic current relaxations. The unitary conductance in that state was estimated to be at least 70 pS.

Inward rectifier current noise in frog skeletal muscle.

Inwardly rectifying K+ currents were studied in cut muscle fibres from frogs using the Vaseline-gap voltage-clamp method. Both faces of the membrane were exposed to 120 mM-K+ methylsulphate solution. At small negative potentials, -10 and -21 mV, the current noise spectrum, after subtraction of a control spectrum at the zero current potential, could be fitted by a Lorentzian spectral component, usually with an additional 1/f component, where f is the frequency. At more negative potentials the 1/f component predominated. The zero frequency amplitude of the Lorentzian averaged 2.6 X 10(-24) A2 Hz-1 at -10 mV and 4.6 X 10(-24) A2 Hz-1 at -21 mV, with a mean half-power frequency, fc, of 34 Hz and 45 Hz, respectively. The time constant of the K+ current activation upon hyperpolarization agrees with that calculated from fc, and the Lorentzian disappears upon replacement of external K+ by tetraethylammonium (TEA+) or Rb+. Thus, the Lorentzian component appears to be ascribable to fluctuations originating in the inwardly rectifying mechanism. The noise spectra and macroscopic currents were interpreted by assuming that the inwardly rectifying K+ conductance is proportional to the product of two parameters: ps representing the state of the mechanism that gives rise to the observable macroscopic current relaxations and to the current fluctuations resulting in the observed Lorentzian spectra, and pf describing the instantaneous rectification of the single-channel conductance. Alternatively, pf may represent another mechanism in series with ps, but which fluctuates too rapidly to measure. Using this model the limiting single-channel conductance, gamma, was found to be approximately 9 pS. The corresponding specific density of channels is about 1 micron-2, assuming uniform distribution over all regions of the membrane. A preliminary value for gamma ( DeCoursey & Hutter , 1982) was derived without consideration of instantaneous rectification. Systematic errors in these results due to voltage decrement in the T-tubules are evaluated in an Appendix, and are found to be tolerably small in the voltage range studied.

[Missed tendon and nerve injuries in the newly traumatized hand - a prospective study]. / Ubersehene Sehnen- und Nervenverletzungen bei der frisch traumatisierten Hand - eine prospektive Studie.

In a series of 94 open hand injuries the preoperative findings are compared to those found during the operative procedure. Tendon and nerve injuries were diagnosed preoperatively only in 60% of the cases. The importance of the correct operative revision is emphasized.

A dual effect of formaldehyde on the inwardly rectifying potassium conductance in skeletal muscle.

1. The inwardly rectifying potassium conductance of the membrane of frog sartorius muscle fibres is greatly reduced by treatment of muscles for 30 min with a solution containing formaldehyde (10 mM). 2. A transient increase in the conductance of the inward rectifier is observed early during formaldehyde action. 3. Analysis of the biphasic time course of the conductance changes, as determined under controlled voltage conditions, suggests that treatment with formaldehyde alters simultaneously, but in opposite ways, two factors that determine the conductance of the inward rectifier. 4. The linear component of the current-voltage relation, which dominates the relation at strongly positive potentials, is not affected while the above changes occur. But on prolonged exposure to formaldehyde the leak conductance increases. 5. The effects of formaldehyde on the inward rectifier are reversible on prolonged superfusion with normal Ringer solution. 6. The slight inward rectification remaining after most of the extracellular K is replaced by Rb, is similarly reduced by treatment with formaldehyde. 7. The results are interpreted in terms of the chemical properties of formaldehyde and present views of the mechanisms of inward rectification.

[Dextran 40 in the prophylaxis of deep vein thrombosis in surgery (author's transl)]. / Dextran 40 zur Prphylaxe tiefer Venenthrombosen in der Chirurgie

The effectiveness of 4 X 500 ml dextran-40 infusions (Rheomacrodex) for the prevention of postoperative deep vein thrombosis was evaluated in a prospective, controlled, randomized study in urological and general surgical patients. The 125I-fibrinogen test was performed daily in all patients. 36 of 100 control patients developed deep vein thrombosis, but only 20 out of 92 (21.7%) did so in the dextran group (P less than 0.05). The number of patients showing bilateral deep vein thrombosis was significantly reduced under dextran prophylaxis (P less than 0.01). No side effects of dextran prophylaxis were seen.