Airways obstruction is the sine qua non of asthma. Although excess mucus secretions contribute to this phenomenon, the primary cause is a decrease in airway caliber due to airway smooth muscle contraction. An increase in cellular cyclic AMP levels has been associated with the relaxation of airway smooth muscle. There are, at present, two mutually non-exclusive hypotheses for cyclic AMP-mediated relaxation. One hypothesis asserts that relaxation is mediated through the phosphorylation of myosin light chain kinase (MLCK) by cyclic AMP-dependent protein kinase; the other asserts that relaxation results from a decrease in intracellular calcium following a rise in cyclic AMP levels. We propose to use a multidisciplinary approach to determine if either, or both, of these mechanisms contribute to cyclic AMP-mediated relaxation. First, the role of cyclic AMP-mediated phosphorylation of MLCK in canine tracheal smooth muscle (canine TSM) relaxation will be studied by determining (a) the temporal relationship between MLCK phosphorylation and relaxation, (b) the sites that are phosphorylated and (c) the effect of phosphorylation on MLCK activity. Second, the interaction between MLCK phosphorylation and cytoplasmic calcium during relaxation of skinned and intact canine TSM will be studied. In these experiments, the cytoplasmic calcium concentration will be manipulated and antibodies that inhibit MLCK phosphorylation will be used to study how these processes affect each other. Finally, changes in cell calcium in response to pharmacological stimulation of intact canine TSM will be quantitated. The Ca2+ measurements will then be correlated with changes in cyclic AMP levels, MLCK phosphorylation, myosin dephosphorylation and relaxation of canine TSM. These experiments utilize physiological, pharmacological, biochemical and immunological methods. Such an approach should provide insights into the roles of two important cellular messengers (calcium and cyclic AMP) and two important enzymes (myosin and myosin light chain kinase) in regulating the contractile properties of airway smooth muscle. These studies should also indicate avenues for pharmacological manipulation of airway caliber.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL035808-01
Application #
3350140
Study Section
(SRC)
Project Start
1985-09-30
Project End
1986-09-29
Budget Start
1985-09-30
Budget End
1986-09-29
Support Year
1
Fiscal Year
1985
Total Cost
Indirect Cost
Name
University of Illinois at Chicago
Department
Type
Overall Medical
DUNS #
121911077
City
Chicago
State
IL
Country
United States
Zip Code
60612
Gandhi, S; Lorimer, D D; de Lanerolle, P (1997) Expression of a mutant myosin light chain that cannot be phosphorylated increases paracellular permeability. Am J Physiol 272:F214-21
Tripuraneni, J; Koutsouris, A; Pestic, L et al. (1997) The toxin of diarrheic shellfish poisoning, okadaic acid, increases intestinal epithelial paracellular permeability. Gastroenterology 112:100-8
Christensen, S; Verhage, H G; Nowak, G et al. (1995) Smooth muscle myosin II and alpha smooth muscle actin expression in the baboon (Papio anubis) uterus is associated with glandular secretory activity and stromal cell transformation. Biol Reprod 53:598-608
de Lanerolle, P; Gorgas, G; Li, X et al. (1993) Myosin light chain phosphorylation does not increase during yeast phagocytosis by macrophages. J Biol Chem 268:16883-6
de Lanerolle, P (1992) Airway smooth muscle and asthma. Am J Respir Cell Mol Biol 7:565-6
Wilson, A K; Pollenz, R S; Chisholm, R L et al. (1992) The role of myosin I and II in cell motility. Cancer Metastasis Rev 11:79-91
Strauss, J D; de Lanerolle, P; Paul, R J (1992) Effects of myosin kinase inhibiting peptide on contractility and LC20 phosphorylation in skinned smooth muscle. Am J Physiol 262:C1437-45
de Lanerolle, P; Paul, R J (1991) Myosin phosphorylation/dephosphorylation and regulation of airway smooth muscle contractility. Am J Physiol 261:L1-14
Wilson, A K; Horwitz, J; De Lanerolle, P (1991) Evaluation of the electroinjection method for introducing proteins into living cells. Am J Physiol 260:C355-63
De Lanerolle, P; Strauss, J D; Felsen, R et al. (1991) Effects of antibodies to myosin light chain kinase on contractility and myosin phosphorylation in chemically permeabilized smooth muscle. Circ Res 68:457-65

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