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Acute and chronic effects of nitric oxide on cardiomyocyte guanylyl cyclase : implications for the modulation of the heart following environmental, physiological and clinical exposure to nitric oxide



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The nitric oxide (NO) - guanylyl cyclase (GC) - guanosine 3' ,5'-cyclic monophosphate (cGMP) signal transduction pathway has been shown to play a role in the modulation of ventricular contractility by the parasympathetic nervous system. Environmental/occupational NO exposures, the gastric production of NO from ingested nitrites, organic nitrate vasodilator therapy and sepsis are situations in which the body tissues are exposed to elevated levels of NO. This may in turn disrupt signal transduction through this pathway. NO-GC coupling in adult rat ventricular cardiomyocytes was investigated in this study following exposure of the cardiomyocytes to a "NO-rich" environment using two different experimental protocols: (i) ' in vitro' exposure of cardiomyocytes to NO-donors for short (2 hr) and long (24 hr) durations and (ii) injection of rats with bacterial endotoxin (LPS) to create a septic condition. GC activity was determined in cytosolic fractions of the cardiomyocytes and also in 'intact' cardiomyocytes following permeabilization with digitonin. Cardiomyocytes were shown to possess NO-sensitive GC which was localized in the cytosol. GC activity in the absence of added NO (basal activity) was low but was powerfully stimulated by nitroprusside (NP) and S-nitroso-d,l-acetylpenicillamine (SNAP). NO-stimulated GC was inhibited by physiologically relevant concentrations of free Ca2+. Furthermore, NO-stimulated GC in the cardiomyocytes was relatively insensitive to the NO-stimulated GC inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). In vitro exposure of cardiomyocytes to SNAP and NP was found to have dramatic effects on the properties of GC. Following treatment of the cardiomyocytes with SNAP, but not NP, basal GC activity was significantly increased. The activated state was long lived and was still present following processing of the cardiomyocytes to obtain a cytosolic fraction. The activation of GC did not result from the uptake of SNAP into the cardiomyocytes nor was it produced through cAMP or cGMP dependent mechanisms. Treatment of the cardiomyocytes with either NP and SNAP resulted in decreased NP- and SNAP-stimulated GC activity. The magnitude of the desensitization was dependent on the duration of the cardiomyocyte treatment When GC activity was determined using cytosolic fractions, treatment of the cardiomyocytes with either SNAP or NP also resulted in leftward shifts of the concentration-response curves for both the NO-donors. Investigations into the biochemical mechanisms through which the desensitization and affinity shifts occurred revealed that neither cAMP nor cGMP-dependent mechanisms were involved. Additional experiments demonstrated that superoxide anion does not play a role in the development of the observed changes to NO-stimulated GC. Basal GC activity was increased in cardiornyocytes from septic (LPS treated) rats. In contrast to the desensitization of GC produced by 'in vitro ' NO-exposure, NO-stimulated GC activity was greater in the cardiomyocytes from the LPS treated rats. These results indicate that NO-stimulated cGMP synthesis in cardiomyocytes is markedly affected by exposure to a NO-rich environment. As might be expected, NO exposure on its own resulted in down-regulation of cGMP synthesis. In the case of sepsis, despite the NO-rich environment generated by increased endogenous NO synthesis, NO-stimulated cGMP synthesis was augmented. Parasympathetic modulation of the myocardium may be compromised as a result of these alterations in NO-GC coupling. (Abstract shortened by UMI.)





Doctor of Philosophy (Ph.D.)







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