RhoA Kinase and Protein Kinase C inhibition in Circular and Longitudinal Colon Muscle

Srinath Sriram
Marquette University
Mentor:  Dr. Thomas Eddinger

Smooth muscle contraction relies on a cascade of steps starting with a release of intracellular Ca2+ and influx of extracellular Ca2+.  The end result of this cascade is the interaction of two proteins, actin and myosin, binding, contracting, detaching, and rebinding, in a process known as cross-bridge cycling. However, cross-bridge cycling also requires the phosphorylation of myosin.  Regulaton of  phosphorylation  relies primarily on two proteins, myosin light chain kinase (MLCK), which phosphorylates myosin and myosin light chain phosphatase (MLCP), which dephosphorylates myosin.  The proteins Rho-kinase (ROK) and Protein Kinase C (PKC) have been reported to regulate MLCP. Therefore, the overall effect of both ROK and PKC is to elevate the amount of phosphorylated myosin in the cell, promoting cycling, and increasing force production. The pharmacological agents Y-27362 and GF-32323 are potent ROK and PKC inhibitors respectively, and their introduction to colon tissues should, theoretically, decrease force production. ROK and PKC have been observed as important modulators of contraction in other types of smooth muscle, and showing that ROK and PKC function in colon tissue as well will provide a novel model to study the function of these proteins.

Samples of colon were obtained from New Zealand white rabbits and dissected to expose the muscle sheet. Either circular or longitudinal strips were cut, incubated in physiological saline solution (PSS), and attached to an isometric force transducer. The length Lo was determined through several K+ induced membrane depolarizations. After determining Lo, The chambers were incubated with Y-27362, a ROK inhibitor, or GF-109203X, a PKC inhibitor, to see if inhibiting ROK or PKC will affect force. 

The introduction of Y-27362 to colon tissue appears to significantly decrease active force for both circular and longitudinal strips. Although the efficacy of Y-27362 is variable, the results indicate that ROK is present in both circular and longitudinal tissue.  Similarly, GF-109203X appears to decrease active force as well. This again shows that PKC, like ROK, has a significant role in force maintenance. Unlike peak force production, spontaneous contractions were not significantly affected by Y-27362 or GF-109203X. This is a plausible observation because the time of each spontaneous contraction may be too brief to allow the complex ROK and PKC pathways to have an effect. 
 

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