The aim of the proposed research is to define the biochemical basis of tracheal smooth muscle contraction. The prevailing model is one in which an agonist-induced increase in cytosolic free Ca2+ leads to the activation of the calmodulin-dependent enzyme myosin light chain kinase. The resulting phosphorylation of the myosin light chain (MLC) is thought to be the major event in both initiating and sustaining contraction. Recent data show that neither the increase in free Ca2+ nor in MLC phosphorylation are sustained. Based on studies of the hormonal regulation of aldosterone secretion, and preliminary studies in vascular and tracheal smooth muscle, we have developed a two phase model of the calcium messenger system function during sustained cellular response. A series of experiments are planned to test this model in tracheal smooth muscle by analyzing: 1) the effect of carbachol on the turnover of the polyphosphatidylinositols, inositol triphosphate, and diacylglycerol; 2) the time course of cellular and cytosol protein phosphorylation in response to carbacholamine; 3) the effects of the agents which specifically: a) increase intracellular free Ca2+ (A23187 or ionomycin); b) activate C-kinase (TPA, OAG and other phorbol esters); and/or c) increase plasma membrane Ca2+ (BAY K 8644) on the time course of the contractile response, and cellular protein phosphorylation; 4) the time course change in intracellular free Ca2+ in response to carbacholamine and these drugs; 5) the effect of carbacholamine and drug combinations on the intracellular distribution of C-kinase; and 6) the effects of the phosphoprotein products of the C-kinase enzyme on the contractile responses of 'chemically skinned' smooth muscle. An analysis of the mechanism by which forskolin, an activator of adenylate cyclase, causes a relaxation of agonist-and drug-induced contractions will also be carried out. As part of the planned studies, methods will be developed for the isolation of viable, agonist-responsive isolated smooth muscle cells for use in the analysis of cellular Ca2+ metabolism, and initial rates of Ca2+ influx in response to agonist and drugs. Accomplishment of these goals would provide new insights into the molecular basis of smooth muscle contraction which would be directly relevant to the clinical problems of bronchial asthma and other states of airway smooth muscle dysfunction.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
2R01HL035849-02
Application #
3350222
Study Section
(SRC)
Project Start
1985-09-30
Project End
1988-09-29
Budget Start
1986-09-30
Budget End
1987-09-29
Support Year
2
Fiscal Year
1986
Total Cost
Indirect Cost
Name
Yale University
Department
Type
Schools of Medicine
DUNS #
082359691
City
New Haven
State
CT
Country
United States
Zip Code
06520
Whitney, G; Throckmorton, D; Isales, C et al. (1995) Kinase activation and smooth muscle contraction in the presence and absence of calcium. J Vasc Surg 22:37-44
Bergh, C M; Brophy, C M; Dransfield, D T et al. (1995) Impaired cyclic nucleotide-dependent vasorelaxation in human umbilical artery smooth muscle. Am J Physiol 268:H202-12
Rasmussen, H; Isales, C M; Calle, R et al. (1995) Diacylglycerol production, Ca2+ influx, and protein kinase C activation in sustained cellular responses. Endocr Rev 16:649-81
Rasmussen, H; Kelley, G; Douglas, J S (1990) Interactions between Ca2+ and cAMP messenger system in regulation of airway smooth muscle contraction. Am J Physiol 258:L279-88
Rasmussen, H (1990) Ernst Klenk lecture, November 1989. The complexities of intracellular Ca2+ signalling. Biol Chem Hoppe Seyler 371:191-206
Rasmussen, H; Rasmussen, J E (1990) Calcium as intracellular messenger: from simplicity to complexity. Curr Top Cell Regul 31:1-109
Rasmussen, H (1989) The messenger function of Ca2+: from PTH action to smooth muscle contraction. Bone Miner 5:233-48
Paglin, S; Takuwa, Y; Kamm, K E et al. (1988) Atrial natriuretic peptide inhibits the agonist-induced increase in extent of myosin light chain phosphorylation in aortic smooth muscle. J Biol Chem 263:13117-20
Alkon, D L; Rasmussen, H (1988) A spatial-temporal model of cell activation. Science 239:998-1005
Takuwa, Y; Takuwa, N; Rasmussen, H (1988) The effects of isoproterenol on intracellular calcium concentration. J Biol Chem 263:762-8

Showing the most recent 10 out of 15 publications