This application proposes a series of studies, using cultured vascular smooth muscle and renal mesangial cells, on the ability of atriopeptins (APs) to decrease cytosolic free Ca, the mechanisms underlying this effect and its functional significance. The overall hypothesis proposed for investigation is as follows: APs decrease both resting and vasoconstrictor-elevated cytosolic free Ca by increasing cellular cyclic GMP levels. The decrease of cytosolic Ca may be the expression of decreased cellular Ca influx, increased Ca efflux, increased intracellular Ca sequestration or decreased intracellular Ca release. APs inhibit vasoconstrictor-elicited inositol trisphosphate accumulation which results in decreased vasoconstrictor-stimulated release of intracellular Ca. The decrease of cytosolic Ca is eventually expressed as inhibition of Ca-dependent processes such as cellular contraction. The following are specific questions to which answers will be elicited by the proposed experiments. Question 1: What are the effects of various APs on resting and ang II-elevated cytosolic free Ca levels in cultured vascular smooth muscle and renal mesangial cells? Question 2: Does the AP-evoked inhibition of cytosolic free Ca have a functional significance? That is, do APs inhibit the contractility of cultured vascular smooth muscle and renal mesangial cells? Question 3: Can APs modulate the increase of cytosolic free Ca elicited by norepinephrine and vasopressin in cultured vascular smooth muscle and mesangial cells? Question 4: What are the effects of APs on transcellular and intracellular Ca flux activities in cultured smooth muscle and mesangial cells? Question 5: Does cyclic GMP play a role as an intracellular mediator of APs actions on cytosolic free Ca, in cultured vascular smooth muscle and mesangial cells? Question 6: Do APs inhibit the ability of vasoconstrictor hormones to increase the accumulation of inositol 1,4,5-trisphosphate (IP3) in cultured vascular smooth muscle and mesangial cells? Cytosolic free Ca will be measured by dual-wavelength fluorescence spectroscopy, whereas Ca flux will be measured by compartmental or non- compartmental analysis of 45Ca influx or efflux. Cell contractility will be measured by semi-automatic image analysis. These experiments may provide important information on the subcellular mechanisms of action of atrial peptides in vascular and renal cells.
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