EVALUATE BLOOD FLOW LUNG, BRAIN, HEART, KIDNEY IN WILD-TYPE MICE W/ CONTRAST

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The initial goal of this project is to evaluate blood flow in lung, brain, heart, and kidney in wild-type mice using in situ contrast imaging. Responses to agonists that induce vasoconstriction and vasorelaxation may be altered in the knockout mouse. Blood pressure measurement in response to these same agonists will also be performed. Systemic adrenergic vasoconstriction is well induced by the alpha-agonist phenylephrine or the alpha1,2;beta-1 agonist norepinephrine. Dose responses to phenylephrine (0.5-8 ug/kg) and norepinephrine (100-600 ng/kg) will be performed. The maximum doses of these drugs is expected to raise mean arterial blood pressure (MAP) approximately 40mmHg above basal levels (basal MAP 90-120 mmHg), so an effect on blood flow should be apparent. Renin-angiotensin-aldosterone system dependent increases in blood pressure can be evaluated by administration of angiotensin II and vasopressin. Nitric oxide-dependent increases in blood pressure can be measured by chronic administration of L-NAME (400 ug/g IP for 7 days), a nitric oxide synthase inhibitor. For evaluation of renal blood flow, dopamine (2 ug/kg) should constrict the efferent arteriole and relax the afferent arteriole of the kidney. CHASM was identified as a very early target of the protein kinase PKG in isolated 32P labeled smooth muscle using mass spectrometry. The protein was identified by de novo sequence data as a theoretical protein in the human genome. Its function is therefore unknown. We have subsequently validated the protein with an antibody that was raised to the recombinant protein. Histological staining shows that CHASM is exclusively expressed in muscle fibers and neuro-endocrine cells innervating muscle. PKG is a major mediator of smooth muscle relaxation and deletion of the protein kinase in mouse resulted in a complete loss of all hormone mediated signaling in this tissue. As a result the animals were hypertensive and exhibited gastrointestinal disorders associated with a loss of coordinated gut motility. The molecular mechanisms by which PKG, once activated, causes smooth muscle to relax are unknown. Our hypothesis suggests that PKG mediates its muscle relaxing effects through phosphorylation of one or more specific proteins expressed in smooth muscle. Interestingly, the compliment of proteins phosphorylated by PKG varies with smooth muscle subtype. We believe this variance of expression infers specific contractile properties upon those muscles. CHASM is expressed largely in tonic smooth muscle such as femoral artery or aorta. Therefore we believe that in the CHASM null mouse there will be alterations in the normal responsiveness of vasculature in response to pharmacological agonists known to activate PKG. Responsiveness of muscles that do not normally express CHASM would be expected to be normal. We would also predict alterations in gut motility in the CHASM null mouse, since the protein is highly expressed in neuro-endocrine cells of ileum. Real time imaging studies will therefore provide a very elegant means to evaluate the physiological role of CHASM in the regulation of smooth muscle contraction by PKG. Parameters we would like to examine are agonist/antagonist induced effects on normal blood flow through out the circulation and every major organ (egg. renal and cardiovascular). We would also like to monitor the effects of agonists on gut motility. We would also like to examine the over all vascular and gut architecture to ensure that CHASM deletion does not cause any developmental abnormalities. Vasorelaxation in wild-type mice will be evaluated by administration of endothelium dependent vasorelaxants bradykinin (0.1-10 ug/kg), the nitric oxide donor, sodium nitroprusside (3 ug/kg), and acetylcholine (1 mg/kg). Determine the physiological significance of CHASM, a novel target of cyclic GMP dependant protein kinase (PKG) that is expressed in smooth muscl