Differences in calmodulin and calmodulin-binding proteins in phasic and tonic smooth muscles.

To determine whether densities of calmodulin (CaM) and CaM-binding proteins are related to phasic and tonic behavior of smooth muscles, we quantified these proteins in the opossum esophageal body (EB) and lower esophageal sphincter (LES), which represent phasic and tonic smooth muscles, respectively. Gel electrophoresis, immunoprecipitation, Western blot, and hemagglutinin epitope-tagged CaM (HA-CaM) overlay assay with quantitative scanning densitometry and phosphorylation measurements were used. Total protein content in the two smooth muscles was similar (approximately 30 mg protein/g frozen tissue). Total tissue concentration of CaM was significantly (25%) higher in EB than in LES (P < 0.05). HA-CaM-binding proteins were qualitatively similar in LES and EB extracts. Myosin, myristoylated alanine-rich C kinase substrate protein, Ca(2+)/CaM kinase II, and calponin contents were also similar in the two muscles. However, content and total activity of myosin light chain kinase (MLCK) and content of caldesmon (CaD) were three- to fourfold higher in EB than in LES. Increased CaM and MLCK content may allow for a wide range of contractile force varying from complete relaxation in the basal state to a large-amplitude, high-velocity contraction in EB phasic muscle. Increased content of CaD, which provides a braking mechanism on contraction, may further contribute to the phasic contractile behavior. In contrast, low CaM, MLCK, and CaD content may be responsible for a small range of contractile force seen in tonic muscle of LES.

[1]  R. A. Murphy,et al.  Actin and tropomyosin variants in smooth muscles. Dependence on tissue type. , 1984, The Journal of biological chemistry.

[2]  J. A. Thomas,et al.  A rapid filter paper assay for UDPglucose-glycogen glucosyltransferase, including an improved biosynthesis of UDP-14C-glucose. , 1968, Analytical biochemistry.

[3]  Toshio Kitazawa,et al.  Reconstitution of protein kinase C‐induced contractile Ca2+ sensitization in Triton X‐100‐demembranated rabbit arterial smooth muscle , 1999, The Journal of physiology.

[4]  M. O'Connor,et al.  Construction of an epitope-tagged calmodulin useful for the analysis of calmodulin-binding proteins: addition of a hemagglutinin epitope does not affect calmodulin-dependent activation of smooth muscle myosin light chain kinase. , 1997, Analytical biochemistry.

[5]  S. Taniguchi,et al.  Contractile properties and proteins of smooth muscles of a calponin knockout mouse , 2000, The Journal of physiology.

[6]  R. A. Murphy,et al.  Myosin phosphorylation and contraction of feline esophageal smooth muscle. , 1985, The American journal of physiology.

[7]  R. Adelstein,et al.  An insert of seven amino acids confers functional differences between smooth muscle myosins from the intestines and vasculature. , 1993, The Journal of biological chemistry.

[8]  Y. Goldman,et al.  Kinetics of contraction initiated by flash photolysis of caged adenosine triphosphate in tonic and phasic smooth muscles , 1989, The Journal of general physiology.

[9]  P. Greengard,et al.  Stimulus-dependent myristoylation of a major substrate for protein kinase C , 1988, Nature.

[10]  K. Trybus,et al.  An insert in the motor domain determines the functional properties of expressed smooth muscle myosin isoforms , 1997, Journal of Muscle Research & Cell Motility.

[11]  C. Klee,et al.  [27] Purification of smooth muscle myosin light-chain kinase , 1982 .

[12]  D. Cafiso,et al.  Defining protein-protein interactions using site-directed spin-labeling: the binding of protein kinase C substrates to calmodulin. , 1996, Biochemistry.

[13]  J. Stull,et al.  Ca2+-dependent Phosphorylation of Myosin Light Chain Kinase Decreases the Ca2+ Sensitivity of Light Chain Phosphorylation within Smooth Muscle Cells* , 1994 .

[14]  Toshio Kitazawa,et al.  G-protein-mediated Ca2+ sensitization of smooth muscle contraction through myosin light chain phosphorylation. , 1991, The Journal of biological chemistry.

[15]  P. Huber.  Caldesmon. , 2020, The international journal of biochemistry & cell biology.

[16]  A. Means,et al.  Production and characterization of an antibody to myosin light chain kinase and intracellular localization of the enzyme , 1981, Cell.

[17]  P. Greengard,et al.  Distribution of protein I in mammalian brain as determined by a detergent-based radioimmunoassay. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[18]  J. Chalovich.  Actin mediated regulation of muscle contraction. , 1992, Pharmacology & therapeutics.

[19]  R. Goyal,et al.  Differences in contractile protein content and isoforms in phasic and tonic smooth muscles. , 1998, The American journal of physiology.

[20]  M. Bárány,et al.  Biochemistry of smooth muscle contraction , 1996 .

[21]  J. Head,et al.  CALMODULIN‐ACTIVATED PHOSPHODIESTERASE FROM VERTEBRATE SMOOTH MUSCLE * , 1980, Annals of the New York Academy of Sciences.

[22]  J. Haeberle.  Calponin decreases the rate of cross-bridge cycling and increases maximum force production by smooth muscle myosin in an in vitro motility assay. , 1994, The Journal of biological chemistry.

[23]  J. Graff,et al.  Phosphorylation-regulated calmodulin binding to a prominent cellular substrate for protein kinase C. , 1989, The Journal of biological chemistry.

[24]  A. Nairn,et al.  Tumor necrosis factor alpha modifies agonist-dependent responses in human neutrophils by inducing the synthesis and myristoylation of a specific protein kinase C substrate. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[25]  P. Cohen,et al.  Myosin light chain phosphatase activities and the effects of phosphatase inhibitors in tonic and phasic smooth muscle. , 1992, The Journal of biological chemistry.

[26]  M. Tansey,et al.  Ca 2 +-dependent Phosphorylation of Myosin Light Chain Kinase Decreases the Ca 2 + Sensitivity of Light Chain Phosphorylation within Smooth Muscle Cells , 2001 .

[27]  P. Gallagher,et al.  Molecular characterization of a mammalian smooth muscle myosin light chain kinase. , 1991, The Journal of biological chemistry.

[28]  Jackie D. Wood,et al.  Motility and circulation , 1989 .

[29]  J. Stull,et al.  Myosin light chain kinase- and PKC-dependent contraction of LES and esophageal smooth muscle. , 2001, American journal of physiology. Gastrointestinal and liver physiology.

[30]  S. Chacko,et al.  NH2-terminal-inserted myosin II heavy chain is expressed in smooth muscle of small muscular arteries. , 1997, The American journal of physiology.

[31]  G. Pfitzer,et al.  Caldesmon and a 20-kDa actin-binding fragment of caldesmon inhibit tension development in skinned gizzard muscle fiber bundles. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[32]  J. Spudich,et al.  The regulation of rabbit skeletal muscle contraction. I. Biochemical studies of the interaction of the tropomyosin-troponin complex with actin and the proteolytic fragments of myosin. , 1971, The Journal of biological chemistry.

[33]  K. Morgan,et al.  Mechanisms of smooth muscle contraction. , 1996, Physiological reviews.

[34]  R. Paul,et al.  Effects of calponin on isometric force and shortening velocity in permeabilized taenia coli smooth muscle. , 1996, The American journal of physiology.

[35]  R. A. Murphy,et al.  Chapter 26 – Regulation of Cross-bridge Cycling in Smooth Muscle , 1996 .

[36]  Andrew P. Somlyo,et al.  Signal transduction and regulation in smooth muscle , 1994, Nature.

[37]  R. Goyal,et al.  Electrical activity of the opossum lower esophageal sphincter in vivo: Its role in the basal sphincter pressure , 1978 .

[38]  U. Sohn,et al.  Signal transduction pathways in esophageal and lower esophageal sphincter circular muscle. , 1997, The American journal of medicine.

[39]  M. Walsh,et al.  Ca2+‐independent phosphorylation of myosin in rat caudal artery and chicken gizzard myofilaments , 1999, The Journal of physiology.

[40]  E. Rosenow.  Esophageal motility. , 1970, The Medical clinics of North America.

[41]  A. Means,et al.  Bacterial expression and characterization of proteins derived from the chicken calmodulin cDNA and a calmodulin processed gene. , 1985, The Journal of biological chemistry.

[42]  S. Mirzoeva,et al.  3 – Calmodulin-Regulated Protein Kinases , 1998 .

[43]  F. Plum.  Handbook of Physiology. , 1960 .

[44]  P. Wagner.  Preparation and fractionation of myosin light chains and exchange of the essential light chains. , 1982, Methods in enzymology.

[45]  U. Malmqvist,et al.  Correlation between isoform composition of the 17 kDa myosin light chain and maximal shortening velocity in smooth muscle , 1991, Pflügers Archiv.

[46]  P. Gailly,et al.  The unimportance of being (protein kinase C) epsilon1 , 1998, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[47]  K. Takahashi,et al.  Isolation and characterization of a 34,000-dalton calmodulin- and F-actin-binding protein from chicken gizzard smooth muscle. , 1986, Biochemical and biophysical research communications.