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UBC Theses and Dissertations

Role of cGMP-dependent protein kinase in smooth muscle relaxation Patel, Ashwinkumar Ishverbhai


Elevations in cyclic guanosine 3'-5'-monophosphate (cGMP) and activation of cGMP-dependent protein kinase (PKG) are believed to be responsible for nitrovasodilator-induced relaxation of vascular smooth muscle (VSM). In contrast to VSM, in tissues such as rat vas deferens and rat distal colon (non-VSM), cGMP elevation is not associated with relaxation. The hypotheses tested in this thesis were that (1) in VSM, cGMP elevation and PKG activation are correlated with relaxation, and (2) in non-VSM, the lack of correlation between cGMP elevation and relaxation is due to (a) lack of PKG activation and/or (b) low levels of PKG. Studies were carried out to optimize the assay conditions used to measure PKG activity. When PKG and cAMP-dependent protein kinase (PKA) were resolved by MonoQ anion exchange chromatography it was found that the assay conditions were specific for PKG since this activity was not found where Type I PKA activity was found. Furthermore immunoblotting with PKG antibodies showed that immunoreactive blots were only seen in those fractions which exhibited PKG activity. Concentration-dependent studies with sodium nitroprusside (SNP) and nitroglycerin (GTN), demonstrated a good correlation between elevation of cGMP levels, activation of PKG and relaxation in rabbit aorta. In temporal studies, PKG was activated at the earliest time point studied at which the preparations were known to be relaxed by SNP and GTN.In rat vas deferens, 0.1 mM SNP elevated cGMP levels and significantly increased PKG activity ratios but no inhibition of contraction took place. A higher concentration of SNP (5.0 mM), resulted in a more marked increase in PKG activity ratio (3-fold compared to control) but despite this increase the phenylephrineinduced contraction was not inhibited. Similarly, in the rat distal colon, atrial natriuretic factor (ANF) significantly increased cGMP levels and activated PKG but did not inhibit spontaneous contractions. Another possible reason why, despite activation of PKG, there is no inhibition in distal colon might be due to low total levels of PKG in this tissue compared to rabbit aorta. Total PKG levels in both proximal and distal colon were lower than those in the rabbit aorta and yet, when PKG was activated in these tissues by ANF, the contractions in proximal, but not in the distal colon, were inhibited. Taken together, these data suggest that the ability of some, but not all, smooth muscles to relax in response to cGMP-elevating agents cannot be explained solely on the basis of differences in total PKG activity in the muscles. It is possible that the lack of relaxation in response to PKG activation in muscles such as the rat vas deferens and distal colon may be due to (1) a lack of co-localization of PKG with a substrate, or (2) lack of critical protein substrates downstream from PKG activation.

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