Defense Date

2012

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Physiology

First Advisor

Karnam S. Murthy

Abstract

Caveolae are integral part of the smooth muscle membrane and caveolins, the defining proteins of caveolae, act as scaffolding proteins for several G protein-coupled receptor signaling molecules and regulate cellular signaling through direct and indirect interactions with signaling proteins. Caveolin-1 is the predominant isoform in the smooth muscle and drives the formation of caveolae. However, little is known about the role of caveolin-1 in the regulation of excitation-contraction and excitation-transcription coupling in gastrointestinal smooth muscle. In the present study we have characterized muscarinic m2 and m3 receptor signaling in gastric smooth muscle and tested the hypothesis that caveolin-1 positively regulates muscarinic receptor signaling and contractile protein expression in smooth muscle. The role of caveolae/caveolin-1 in the regulation of muscarinic signaling was examined using complementary approaches: a) methyl b-cyclodextrin (MbCD) to deplete cholesterol in dispersed muscle cells, b) caveolin-1 siRNA to suppress caveolin-1 expression in cultured muscle cells, and c) caveolin-1 knockout (KO) mice. RT-PCR, western blot and radioligand binding studies demonstrated the selective expression of m2 and m3 receptor in gastric smooth muscle cells. Carbachol (CCh), acting via m3 receptors caused stimulation of phosphoinositide (PI) hydrolysis, Rho kinase and ZIP kinase activity, and induced phosphorylation of MYPT1 (at Thr696) and MLC20 (at Ser19), and muscle contraction, and acting via m2 receptors caused inhibition of forskolin stimulated cAMP formation. Stimulation of PI hydrolysis, Rho kinase and ZIP kinase activities, phosphorylation of MYPT1 and MLC20 phosphorylation and muscle contraction in response to CCh was attenuated in dispersed cells treated with MbCD or in cultured cells transfected with caveolin-1 siRNA. Similar inhibition of all responses was obtained in gastric muscle cells from caveolin-1 KO mice compared to gastric muscle cells to WT mice. Although, caveolin-1 had no effect on m2 receptor signaling, agonist-induced internalization of m2, but not m3 receptors was blocked in dispersed cells treated with MbCD or in cultured cells transfected with caveolin-1 siRNA. These results suggest that caveolin-1 selectively and positively regulates Gq/13-coupled m3 receptor signaling, Gi-coupled m2 receptor internalization. The expression of contractile proteins, g-actin and caldesmone and the transcription factors SRF and myocardin that regulate the expression of contractile proteins are down regulated, whereas EGF-stimulated EGF receptor phosphorylation and ERK1/2 activity are up-regulated in cells transfected with caveolin-1 siRNA. These results suggest using pharmacological, molecular and genetic approaches provide conclusive evidence that caveolae and caveolin-1 play an important role in orchestrating G protein coupled receptor signaling to have dual pro- excitation-contraction and excitation-transcription coupling, and anti-proliferative role in gastric smooth muscle.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

November 2012

Included in

Physiology Commons

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