Pflügers Archiv

, Volume 412, Issue 3, pp 231–239 | Cite as

Influence of pH on isometric force development and relaxation in skinned vascular smooth muscle

  • Jeffrey P. Gardner
  • F. P. J. Diecke
Heart, Circulation, Respiration and Blood; Environmental and Exercise Physiologh

Abstract

The effects of pH (from pH values 6.50–7.10) on isometric tension development and relaxation were investigated in Triton X-100 “skinned” rat caudal artery. Helically cut skinned strips contracted in 21 μM Ca2+ were studied with respect to maximal isometric tension (Po) and rate of contraction (T0.5C), and following relaxation in 18 nm Ca2+, the rate of relaxation (T0.5R). Acidic pH (pH 6.50) decreased Po to 87% of isometric force obtained at pH 6.90, and increased the rate of contraction as shown by a decrease of T0.5C to 80%. In contrast, T0.5R increased 4.5-fold, indicating that with a change of only 0.40 pH units, relaxation rates were dramatically decreased. pCa-tension curves at pH values 6.50, 6.70, 6.90 and 7.10 indicated no significant shift in half maximal activation (pCa50) between pH 6.50 and 6.70, but a significant (P<0.01) shift in pCa50 between pH 6.70 ([Ca2+]=0.46 μM) and pH 7.10 ([Ca2+]=0.87 μM). Compared to contractions at pH 6.90, myosin light chain (LC20) phosphorylation at pH 6.50 was significantly greater at 30 and 60 s into contraction but not significantly different at 3–10 min. At both pH 6.50 and 6.90, dephosphorylation was rapid and substantially preceded relaxation; LC20 dephosphorylation and relaxation occurred more rapidly at pH 6.90 than at 6.50. At pH 6.50 and 6.90, relax solutions made with increased Ca2+ buffering capacity showed no effect in enhancing T0.5R, suggesting the difference between relaxation rates was not due to Ca2+ diffusion limitations from the skinned strip. We suggest pH changes can after the contractile and relaxation responses in vascular smooth muscle and these effects may be related to LC20 phosphorylation/dephosphorylation regulatory mechanisms.

Key words

Intracellular pH Vascular smooth muscle Chemically skinned muscle Isometric force 20,000 Da myosin light chain phosphorylation “Ca2+ jumps” 

Abbreviations

PIPES

piperazine-N,N′-bis-(2-ethanesulfonic acid)

EGTA

5-ethyleneglycol-bis-(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid

DTT

dithiothreitol

HDTA

1,6-diaminohexane-N,N,N′,N′-tetraacetic acid

SDS

sodium dodecyl sulfate

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Adelstein RS, Klee CB (1981) Purification and characterization of smooth muscle myosin light chain kinase. J Biol Chem 256: 7501–7509Google Scholar
  2. Aickin CC (1984) Direct measurement of intracellular pH and buffering power in smooth muscle cells of guinea-pig vas deferens. J Physiol (Lond) 349:571–585Google Scholar
  3. Bialojan C, Merkel L, Rüegg JC, Gifford D, Di Salvo J (1985) Prolonged relaxation of detergent-skinned smooth muscle involves decreased endogenous phosphatase activity. Proc Soc Exp Biol Med 178:648–652Google Scholar
  4. Blumenthal DK, Stull JT (1982) Effects of pH, ionic strength, and temperature on activation by calmodulin and catalytic activity of myosin light chain kinase. Biochemistry 21:2386–2391Google Scholar
  5. Chatterjee M, Murphy RA (1983) Calcium-dependent stress maintenance without myosin phosphorylation in skinned smooth muscle. Science 221:464–466Google Scholar
  6. Diecke FPJ, Gardner J, Hausser R (1986) Mathematical modelling of the simultaneous diffusion of EGTA, CaEGTA, and Ca2+ in a two-compartmental system representing skinned smooth muscle. Biophys J 49:73a (abstr)Google Scholar
  7. Dillon PF, Aksoy MO, Driska SP, Murphy RA (1981) Myosin phosphorylation and the cross-bridge cycle in arterial smooth muscle. Science 211:495–497Google Scholar
  8. Driska S, hartshorne DJ (1975) The contractile properties of smooth muscle. Properties and components of a Ca2+-sensitive actomyosin from chicken gizzard. Arch Biochem Biophys 167:203–212Google Scholar
  9. Driska SP, Aksoy MO, Murphy RA (1981) Myosin light chain phosphorylation associated with contraction in arterial smooth muscle. Am J Physiol 240:C222-C233Google Scholar
  10. Fisher MJ, Dillon PF (1986) Graded metabolic impairment of smooth muscle. Biophys J 49:101a (abstr)Google Scholar
  11. Gardner JP (1984) Relations among myosin light chain phosphorylation, Ca2+ and isometric force in skinned porcine carotid artery. Ph. D. thesis dissertation, University of Cincinnati University Microfilms Int., Ann Arbor, MIGoogle Scholar
  12. Gordon AR (1978) Contraction of detergent-treated smooth muscle. Proc Natl Acad Sci USA 75:3527–3530Google Scholar
  13. Hellstrand P, Vogel HJ (1985) Phosphagens and intracellular pH in intact rabbit smooth muscle studied by31P-NMR. Am J Physiol 248:C320-C329Google Scholar
  14. Herlihy JT (1980) Helically cut vascular strip preparation: geometrical considerations. Am J Physiol 238:H107-H109Google Scholar
  15. Hoar PE, Kerrick WGL, Cassidy PS (1979) Chicken gizzard: relation between calcium-activated phosphorylation and contraction. Science 204:503–506Google Scholar
  16. Hoar PE, Pato MD, Kerrick WGL (1985) Myosin light chain phosphatase: effect on the activation and relaxation of gizzard smooth muscle skinned fibers. J Biol Chem 260:8760–8764Google Scholar
  17. Hoar PE, Kenney RE, Kerrick WGL (1986) Mg2+ affects maximum tension and relaxation rates in skinned smooth muscle cells, but not the pCa-tension relationship. Biophys J 51:332a (abstr)Google Scholar
  18. Iino M (1981) Tension responses of chemically skinned fibre bundles of the guinea-pig taenia caeci under varied ionic environments. J Physiol (Lond) 320:459–467Google Scholar
  19. Kamm KE, Stull JT (1985a) The function of myosin and myosin light chain kinase phosphorylation in smooth muscle. Annu Rev Pharmacol Toxicol 25:593–620Google Scholar
  20. Kamm KE, Stull JT (1985b) Myosin phosphorylation, force and maximal shortening velocity in neurally stimulated tracheal smooth muscle. Am J Physiol 249:C238-C247Google Scholar
  21. Kato S, Ogasawara T, Osa T (1982) Calcium diffusion in uterine smooth muscle sheets. J Gen Physiol 80:257–277Google Scholar
  22. Kato S, Osa T, Ogasawara T (1984) Kinetic model for isometric contraction in smooth muscle on the basis of myosin phosphorylation hypothesis. Biophys J 46:35–44Google Scholar
  23. Keller CH, Olwin BB, La Porte DC, Storm DR (1982) Determination of the free energy coupling for binding of calcium ions and troponin I to calmodulin. Biochemistry 21:156–162Google Scholar
  24. Kulbertus H (1964) Etude des échanges d'ions H+ au cours de la contraction des parios carotidiennes soumises à différents agents vasomoteurs. Angiologica 1:275–278Google Scholar
  25. Marston SB (1982) The regulation of smooth muscle contractile proteins. Prog Biophys Mol Biol 41:1–41Google Scholar
  26. Moisescu DG (1976) Kinetics of reaction in calcium-activated skinned muscle fibres. Nature 262:610–613Google Scholar
  27. Mrwa U, Hartshorne DJ (1980) Phosphorylation of smooth muscle myosin and myosin light chains. Fed Proc 39:1564–1568Google Scholar
  28. Mrwa U, Achtig I, Rüegg JC (1974) Influences of calcium concentration and pH on the tension development and ATPase activity of the arterial actomyosin contractile system. Blood Vessels 11:277–286Google Scholar
  29. Murphy RA (1971) Arterial actomyosin: effects of pH and temperature on solubility and ATPase activity. Am J Physiol 220: 1494–1500Google Scholar
  30. Murphy RA (1980) Mechanics of vascular smooth muscle. In: Bohr DF, Somlyo AP, Sparks HV, Jr (eds) Handbook of physiology — vascular smooth muscle, vol 2. American Physiological Society, Bethesda, MD, pp 325–351Google Scholar
  31. Murphy RA, Aksoy MO, Dillon PF, Gerthoffer WT, Kamm KE (1983) The role of myosin light chain phosphorylation in regulation of the crossbridge cycle. Fed Proc 42:51–56Google Scholar
  32. Nakayama S, Tomita T, Seo Y, Watari H (1986) P-31 NMR studies in smooth muscles of guinea-pig stomach and taenia coli. Proc Int Union Physiol Sci XVI:67(abstr)Google Scholar
  33. Oakley BR, Kirsch DR, Morris NR (1980) A simple ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal Biochem 98:231–237Google Scholar
  34. Ogawa Y, Tanokura M (1984) Calcium binding to calmodulin: effects of ionic strength, Mg2+, pH and temperature. J Biochem 95:19–28Google Scholar
  35. Pato MD, Adelstein RS (1983) Characterization of a Mg2+-dependent phosphatase from turkey gizzard smooth muscle. J Biol Chem 258:7055–7058Google Scholar
  36. Perrin DD, Sayce IG (1967) Computer calculation of equilibrium concentrations in mixtures of metal ions and complexing species. Talanta 14:833–842Google Scholar
  37. Peterson JW 3d (1982) Rate-limiting steps in the tension development of freeze-glycerinated vascular smooth muscle. J Gen Physiol 79:437–452Google Scholar
  38. Schädler M (1967) Proportionale Aktivierung von ATPase-Aktivität und Kontraktions-Spannung durch Calciumionen in isolierten contractilen Strukturen verschiedener Muskelarten. Pflügers Arch 296:70–90Google Scholar
  39. Schneider M, Sparrow M, Rüegg JC (1981) Inorganic phosphate promotes relaxation of chemically skinned smooth muscle of guinea-pig Taenia coli. Experientia 37:980–982Google Scholar
  40. Siegman MJ, Butler TM, Mooers SU, Michalek A (1984) Ca2+ can affectV maxwithout changes in myosin light chain phosphorylation in smooth muscle. Pflügers Arch 401:385–390Google Scholar
  41. Sillen LG, Martell AE (1971) Stability constants of metal-ion complexes. Suppl No 1. Burlington House, London, pp 731–732Google Scholar
  42. Sobieszek A (1987) MgATPase activity of vertebrate smooth muscle actomyosin: stimulation by tropomyosin is modified by myosin phosphorylation and its conformational state. In: Siegman MJ, Somlyo AP, Stephens NL (eds) Progress in clinical and biological research: regulation and contraction of smooth muscle, vol 245. Liss, New York, pp 159–181Google Scholar
  43. Stephens NL, Kroeger EA, Low W (1977) Intracellular pH in hypoxic smooth muscle. Am J Physiol 232:E330-E335Google Scholar
  44. Stout MA, Diecke FPJ (1983)45Ca sequestration and release in chemically skinned strips of vascular smooth muscle. J Pharmacol Exp Ther 225:102–111Google Scholar
  45. Vermuë NA, Nicolay K (1983) Energetics of smooth muscle taenia caecum of guinea-pig: a31P-NMR study. FEBS Lett 156: 293–297Google Scholar

Copyright information

© Springer-Verlag 1988

Authors and Affiliations

  • Jeffrey P. Gardner
    • 1
  • F. P. J. Diecke
    • 1
  1. 1.Department of PhysiologyUMDNJ-New Jersey Medical SchoolNewarkUSA

Personalised recommendations