Beta-agonists enhance the lactic dehydrogenase (LDH) expression in serum and ventricular myocytes of mice.

Beta-agonists have skeletal muscle specific protein anabolic effects and are also known to cause cardiac hypertrophy. Changed total LDH and its isozymic patterns are conveniently employed for the detection of different pathophysiological states of the tissues. The purpose of this study is to confirm total LDH and its isozymic expression in ventricular tissue and serum in mice following oral administration of single but higher dose of isoproterenol (Iso) and clenbuterol (Cl) (100 mg/kg body wt. and 20 mg/kg body wt., respectively), after 4, 8 and 20 hours of drug administration. Mice heart witnessed increased total LDH levels with time. Serum on the other hand showed decline in total LDH concentrations at the initial points of the drug treatment. No doubt, total LDH expression increased towards 20th h post-drug treatment but this increase is mainly due to anaerobic isozymes, i.e. LDH4 and LDH5. The findings of the present study suggest that tissue damage is definitely caused by two beta-agonists after giving single dose for shorter time span (20 hours) and the impact of the damage varies from drug to drug. Increase in total LDH in serum is not due to release from heart but from some other tissues having anaerobic metabolism.

Tissue specific and variable collagen proliferation in Swiss albino mice treated with clenbuterol.

Chronic administration of clenbuterol, a beta-adrenoceptor agonist (2 mg/kg body weight/day for 30 days) to mice resulted in an increased body mass. Measurement of dry tissue mass suggested a protein anabolic effect in the gastrocnemius and heart. Quantitative estimation of collagen content, a non-contractile element as calculated from hydroxyproline assay revealed its proliferation in the gastrocnemius, cardiac ventricle, intestine and to some extent also in the kidney. Clenbuterol did not induce collagen proliferation in non-muscle tissues such as the lungs and liver. Histopathological examination of sections from treated ventricles showed an extensive collagen infiltration in the subendocardium and at myonecrosis sites.

Early onset of the maximum protein anabolic effect induced by isoproterenol in chick skeletal and cardiac muscle.

Prolonged (120 days) oral administration of a beta adrenoceptor agonist, isoproterenol hydrochloride (dose = 1.5 mg/kg body weight) resulted in an increase in the live weight of growing chicks (Callus domesticus). Measurement of dry muscle mass and total proteins in muscle homogenates from M. pectoralis major. M. petoralis minor suggested a muscle hypertrophy largely responsible for this live weight increase. Further, an increase in organ weight and total tissue proteins supported cardiac hypertrophy in chicks as a result of isoproterenol administration. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed alterations in actin myosin profiles implying a drug induced change in phenotypic expression of myofibrillar component of both skeletal and cardiac muscle. The results suggest that prolonged treatment of chicks produced changes that were not much different from those recorded immediately within a fortnight.

Ca(2+)- and protein kinase C-dependent stimulation of mitogen-activated protein kinase in detergent-skinned vascular smooth muscle.

Protein kinase C and mitogen-activated protein (MAP) kinase are expressed in all smooth muscle cells and believed to be important in several physiologically relevant properties of this muscle. Our goal was to determine if protein kinase C and MAP kinase are activated by a simple increase in cellular Ca(2+) and to determine if protein kinase C is an upstream activator of MAP kinase. These studies were performed in the Triton X-100 detergent-skinned preparation of the swine carotid artery, which allows control of the intracellular environment without influence from membrane or receptor-mediated modulation. The p42 and p44 isoforms of MAP kinase were activated in a concentration-dependent fashion by an increase in Ca2+. This was shown by in-the-gel kinase assay and direct measurement of MAP kinase phosphotransferase activity. Protein kinase C was also activated by an increase in Ca2+, as shown by a novel assay that measures total active protein kinase C in the tissue. Inhibition of protein kinase C activity completely abolished MAP kinase activity. Additionally, inhibition of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) also abolished MAP kinase activity. Using intact swine carotid arteries, we showed p42 and p44 MAP kinase to be activated by both histamine and phorbol dibutyrate, but only the p42 isoform was calcium-sensitive. Our results suggest that a Ca(2+)-dependent isoform of protein kinase C and CaM kinase II are upstream activators of MAP kinase in the swine carotid artery.

Changes in myosin ATPase activity in skeletal muscles of rat during cold stress.

Three skeletal muscles viz., gastrocnemius, pectoralis and diaphragm from rats acclimated to a low temperature (4 +/- 1 degrees C; 16 hr daily; maximum for 8 weeks) exhibit an increased myosin ATPase activity. An analysis of native myosin from these muscles under non-dissociating conditions reveals two myosin isozymes instead of a single isozyme expressed in control muscles. Isoelectric focusing (IEF) coupled with two dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2-D SDS-PAGE) confirms an increased phosphorylation of myosin light chain 2 (MLC2) in muscles from cold acclimated rats.

Myosin isoforms in uterine smooth muscle during pregnancy in rat.

Effects of uterine stretching and physiological hypertrophy on myosin isozyme were investigated in rat during pregnancy. Both nonpregnant and pregnant rat uteri express a single myosin band on native gels. Analysis of native myosin under denaturing conditions revealed two myosin heavy chains (MHCs) with molecular mass of 204 and 200 kDa respectively. Filamin, a 240 kDa protein co-electrophoreses with myosin on native gels. No correlation is found between regulatory myosin light chain phosphorylation and pattern of myosin isozymes or the MHC. The results suggest that uterine stretching and physiological hypertrophy during pregnancy do not induce any changes in uterine myosin isozyme.

Mechanism of galanin-induced contraction of longitudinal smooth muscle of the rat jejunum.

Intact and alpha-toxin-permeabilized longitudinal smooth muscle were mounted for measurement of force and myosin light chain phosphorylation. Galanin contracted intact jejunum with a half-maximum effective concentration of 9.2 +/- 0.1 nM. Neither atropine, hexamethonium, guanethidine, nor tetrodotoxin affected the contraction. The contraction was also unaffected by depletion of intracellular Ca2+ or by addition of thapsigargin; removal of extracellular Ca2+ or addition of nifedipine abolished the contraction. Galanin increased myosin light chain phosphorylation levels concomitantly with force. During continued tissue stimulation, force fell to suprabasal values, whereas myosin light chain phosphorylation levels remained elevated. Galanin increased Ca2+ sensitivity of contraction in alpha-toxin-permeabilized tissues, and this was reversed by either guanosine 5'-O-(2-thiodiphosphate) or pertussis toxin. These results suggest that galanin-induced contraction of longitudinal jejunal smooth muscle is dependent on a pertussis toxin-sensitive G protein that is apparently not coupled to the release of intracellular Ca2+ but to the influx of extracellular Ca2+ and involves an initial myofilament Ca2+ sensitization followed by Ca2+ desensitization.

Changes in myosin heavy chain (MHC) expression during pregnancy in rat uterus.

Expression of myosin heavy chain (MHC) was investigated in rat uterus during pregnancy. Two MHC isoforms, SM MHCI (204 kDa) and SM MHCII (200 kDa), were resolved following the analysis of Guba Straub extract of non-pregnant and pregnant uterus using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Whereas SM MHCI showed an increase from 69.26 +/- 3.26% in nonpregnant uterus to 81.40 +/- 5.36% in pregnant uterus, the SM MHCII exhibited a corresponding decline from 30.73 +/- 3.29 to 18.59 +/- 5.36%. The native myosin separated under non-dissociating conditions and subsequently analyzed in denaturing SDS-PAGE also demonstrated two MHC isoforms with identical electrophoretic mobilities. A SDS-PAGE analysis of native myosin from pregnant rats extracted at room temperature and in the absence of proteolytic inhibitors revealed a characteristically increased proteolysis of MHC into two peptide products of 153 and 140 kDa during pregnancy. Such a proteolysis of MHC, but in very low proportions, was noticed in nonpregnant uterus too.

Changes in uterine myosin isozymes from goat during early pregnancy.

Changes in the expression of native myosin, myosin heavy chains (MHCs) and myosin light chains (MLCs) were investigated in goat uterus during early pregnancy. Electrophoresis of native myosin under non dissociating conditions displayed two isozymes differing in their proportions during gestation. Three MHC isoforms were obtained following sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Whereas 204 kDa (a smooth muscle MHC) and 196 kDa (a non muscle MHC) were recorded in uterus from non pregnant goat, uterus of pregnant goat displayed a third 200 kDa smooth muscle MHC. Two conspicuous proteins (154 and 140 kDa respectively) in addition to MHCs were also obtained when the myofibrillar extracts were analysed either in the presence or absence of proteolytic inhibitors. Non pregnant goat uterus showed a basal level (ca.5%) of phosphorylation of regulatory myosin light chain. Uterine myosin from pregnant goat was recorded in a completely dephosphorylated state.

Differential effects of a K+ channel agonist and Ca2+ antagonists on myosin light chain phosphorylation in relaxation of endothelin-1-contracted tracheal smooth muscle.

Smooth muscle contraction and relaxation are generally considered to be associated with phosphorylation and dephosphorylation of the 20-kDa regulatory myosin light chain (LC20). Thus, contractions of lamb tracheal smooth muscle induced by Bay K 8644 and relaxed by calcium channel blockers (verapamil, D-600 and nitrendipine) are accompanied by an increase and decrease, respectively, of LC20 phosphorylation. Similarly, endothelin-1 (ET-1) induces a sustained contraction, which is coupled with elevated LC20 phosphorylation and reversed by LC20 dephosphorylation after application of a potassium channel agonist (EMD 52692). In contrast, calcium channel blockers relax ET-1-induced contraction without any dephosphorylation of myosin light chains (MLC), suggesting that MLC phosphatase is inhibited in this case. Obviously, MLC dephosphorylation is not a prerequisite for smooth muscle relaxation. The variable relationship between MLC phosphorylation and force during relaxation suggests that there are mechanisms other than MLC phosphorylation that are important for regulation of contraction and relaxation in smooth muscle.

Myosin light chains (MLCs) heterogeneity in mammalian smooth and cardiac muscle.

A comparative study of myosin light chains (MLCs) has been made in the aorta, uterine and cardiac muscles (auricle, ventricle) of mice, pig, sheep and goat. Analysis of myosin light chains by sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE) has revealed that (a) aorta myosin from mice, goat and pig has identical myosin light chains profile but pig aorta myosin lacks LC-2f; (b) uterine smooth muscle myosin depicts absence of LC-1f in sheep. Whereas satellite bands of LC-1f and LC-2f fractions are absent in pig uterine myosin, mice shows duplets for both the myosin light chains; (c) the auricular myosin in pig and goat is identical to chicken gizzard myosin used as reference and exhibits ALC-2s and ALC-1s fractions only while sheep auricular myosin lacks in ALC-1f; (d) the mice ventricular myosin depicts two satellite MLCs associated with fast migrating VLC-1s.

Agonist and membrane depolarization induced activation of MAP kinase in the swine carotid artery.

Caldesmon phosphorylation has been proposed to be involved in regulation of smooth muscle contraction. Mitogen-activated protein (MAP) kinase has been suggested to be the caldesmon kinase; stimulation-induced MAP kinase activation in intact vascular smooth muscle, however, has not been demonstrated. We measured temporal profiles of MAP kinase activation in response to histamine stimulation and membrane depolarization in intact swine carotid artery. Phosphotyrosine levels of 42- and 44-kDa MAP kinases were elevated during contraction in response to histamine or KCl. The temporal profile of MAP kinase activation/inactivation was similar to that for contraction/relaxation of the vascular tissue in response to KCl or histamine stimulation. MAP kinase activated during contractile stimulation phosphorylates caldesmon with a specific activity significantly greater than that for myelin basic protein-(95-98). We propose that MAP kinase is activated in response to all forms of contractile stimulation. We also suggest that activated MAP kinase phosphorylates and disinhibits the effects of caldesmon on actin-myosin interactions. This disinhibition allows an inherent level of myosin ATPase activity to be expressed.

Effects of denervation and nerve crushing on chicken muscle.

When compared with the standard free muscle grafts, predenervated and nerve crushed chick gastrocnemius muscle grafts followed an identical and ordered sequence of tissue degeneration-regeneration. Atrophy of the fibres during ischaemic degeneration was accompanied by loss of weight, noncollagenous proteins and increment in lytic enzymes (acid and alkaline phosphatase), glucose 6-phosphatase, glycogen and succinate dehydrogenase. Regeneration was more rapid in standard free muscle grafts. Trophic insults to muscle via denervation and nerve crushing prior to transplantation did not influence graft renewal when compared with standard free muscle grafts though relatively increased recovery was shown by predenervated muscle grafts.

Endothelin-1 and okadaic acid induced contractions in tracheal smooth muscle of lamb.

Stimulation of lamb tracheal smooth muscle fibre strips with endothelin-1 and okadaic acid results in the development of isometric tensions which are long lasting. However, endothelin-1 is more potent constrictor than okadaic acid. An analysis of 20,000 Da regulatory myosin light chain by two-dimensional gel electrophoresis shows an identical phosphorylation pattern.

Endothelin-1 and carbachol: differences in contractile effects and myosin phosphorylation in lamb tracheal smooth muscle.

Endothelin-1 is a new potent vasoconstrictor peptide produced by the endothelial cells. The contractile effects of endothelin-1 (ET-1) were compared with those of cabachol in lamb tracheal smooth muscle. Equimolar concentrations (10(-6)M) of endothelin 1 and carbachol elicit rapidly rising isometric tension which is maintained indefinitely in a steady state when fibres are stimulated with carbachol. Fibre strips exposed to ET-1 cannot maintain peak isometric force beyond 15-20 min and instead these exhibit a decline in tension towards near relaxed state. In addition to an early transient relaxation, ET-1 stimulation results in a 20,000 Da myosin light chain phosphorylation pattern different from that of carbachol stimulation.

Reversal of endothelin-1-induced contractions by isoproterenol without myosin dephosphorylation in tracheal smooth muscle.

Effects of isoproterenol on isometric force, and 20,000 Da myosin light chain (LC20) phosphorylation were examined in smooth muscle fibre strips from lamb trachea stimulated with endothelin-1 (ET-1). ET-1 induced a rapidly rising isometric tension which was coupled with a multiple site phosphorylation of LC20. Isoproterenol addition at the time of peak isometric force resulted in a brisk relaxation of the fibre strips. Myosin light chain phosphorylation, however, remained unaffected.

Skeletal muscle metabolism in mice bearing adenocarcinoma. III. Activities of alanine and aspartate aminotransferases.

Activities of alanine and aspartate aminotransferases in six skeletal muscles demonstrate 114-223% and 86-146% increase respectively, over the normal by the 10th week after adenocarcinoma transplantation in mice. The white (anaerobic) muscles reveal greater increment in these two enzymes than the red (aerobic) ones. The significance of elevated aminotransferase activities in energy metabolism of the host muscles is discussed.

Skeletal muscle metabolism in mice bearing adenocarcinoma. I. Histochemical alterations in glycogenolytic, glycolytic, lipolytic and oxidative metabolism.

Metabolism of triceps, pectoralis (in the vicinity of tumor) and gastrocnemius (away from the tumor) muscles in Swiss albino mice bearing adenocarcinoma has been studied histochemically with regard to content of glycogen, lipids, phosphorylase, aldolase, lipase, succinate dehydrogenase and cytochrome oxidase in the constituent fibres. At 9-10 weeks after transplantation of adenocarcinoma, a negligible glycogen content and decreased phosphorylase and aldolase activities are observed in the white, intermediate and red fibre types in the three muscles. Hypertrophy of fibres and occurrence of targetoid fibres is distinct in the muscles of tumor-bearing mice. The red fibres demonstrate a general loss of lipids, lipase, succinate dehydrogenase and cytochrome oxidase whereas the hypertrophied fibres reveal intense localization of these parameters in their central zones. The results indicate that a decline in glycogenolysis, glycolysis, lipolysis and oxidative metabolism in the various fibre types may contribute to the muscle weakness and muscle wasting in the adenocarcinoma-bearing mice.

Skeletal muscle metabolism in mice bearing adenocarcinoma. II. Histochemical alterations in acid and alkaline phosphatases.

In Swiss albino mice bearing adenocarcinoma the degree of pathological damage as revealed by the activities of acid and alkaline phosphatases is greater in skeletal muscles closely proximal to the tumor site than in those away from it. Acid and alkaline phosphatases in myofibres of triceps, pectoralis and gastrocnemius muscles show a direct relationship between myofibre necrosis and activities of these phosphatases.