Date of Award

Summer 2010

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Biological Sciences

First Advisor

Thomas J. Eddinger

Second Advisor

Allison L. Abbott

Third Advisor

Robert O. Balza

Abstract

Smooth muscle contraction is a complicated process coordinated by contractile, regulatory and cytoskeletal proteins. The force generation depends on the phosphorylation of Myosin Regulatory Light Chain (MLC20). Myosin Light Chain Kinase (MLCK) and Myosin Light Chain Phosphatase (MLCP) are the two main regulators of the MLC20 phosphorylation level. MLCP is further controlled by two known pathways including the G protein coupled receptors (GPCRs)/ phospholipase C (PLC)/ diacylglycerol (DAG)/ protein kinase C (PKC)/ PKC-potentiated inhibitory protein for heterotrimeric myosin light chain phosphatase of 17 kDa (CPI-17) pathway. While messengers involved in this pathway have been proposed, studies on the details of the pathway are still controversial.

This study explored the spatial-temporal regulation and distribution of PKCα and CPI-17 in intact animal tissues. Immunohistochemical results show that the distribution of PKCα in the longitudinal and circular layers of the fundus and antrum under relaxed conditions was predominantly localized at or near the periphery of the smooth muscle cell. Stimulation of the tissues with 1μM phorbol 12,13-dibutyrate (PDBu) for 10 or 30 minutes or 1μM carbachol (CCh) for 3 minutes does not alter the distribution pattern of PKCα. Different from PKCα, CPI-17 appeared to be "uniformly" distributed throughout the smooth muscle cells under relaxed conditions. Stimulation of the tissues with 1μM PDBu or 1μM CCh for 30 minutes led to a significant distribution shift of CPI-17 from throughout the cytosol to primarily at the cell periphery. Results from double labeling of PKCα and vinculin/talin under relaxed condition or CPI-17 and vinculin/talin under stimulated condition suggested that PKCα and CPI-17 were not associated with the adherens junction. It is likely that PKCα and CPI-17 are localized at the caveolae on the plasma membrane. This study also revealed that the force generated in tonic fundus smooth muscle is much greater than that in phasic antrum tissue upon PDBu stimulation. Immunoblot analyses demonstrated that this difference was not caused by a difference in the expression of PKCα or CPI-17 between these two tissues.