Mesangial cells (MC) are contractile cells that modulate filtration rate (GFR) by regulating glomerular capillary surface area. Like vascular smooth muscle, MC respond to volume and pressure regulatory hormones, such as angiotensin II, atrial natriuretic peptide (ANP) and nitric oxide (NO). Thus, MC have a critical physiological role in regulation of blood pressure and fluid and volume homeostasis. Our laboratory has recently discovered that large, calcium-activated K channels (BK) play a major role in both NO and ANP regulated relaxation of mesangial cells. The response of BK to vasorelaxing agents is biphasic, with an activation phase of approximately 10 to 30 seconds and an inactivation phase which can last for 60 seconds before returning to baseline levels. In the activation phase, NO and ANP, by stimulating guanylyl cyclase and generating cGMP, lead to specific cGMP-activated kinase stimulation of BK. The inactivation phase can be inhibited by okadaic acid or cantharidic acid, inhibitors of protein phosphatase 1 and 2A. The regulatory and signaling pathways controlling the BK activation/inactivation cycle will be a major focus of this proposal. The patch clamp method has revealed two populations of BK: one which responds to phosphatases and cGMP-activated protein kinase (BK1) and one that is unresponsive (BK2) to these agents. These results suggest that BK2 do not have the same phosphorylation sequence sites as BK1. It is postulated that these populations of BK have distinct messenger RNA which can be transcriptionally regulated. Preliminary electrophysiological studies show that BK2 are preferentially expressed when MC are exposed for 24 hours to DB-cGMP. Preliminary molecular studies show that there are at least two variants of message transcribed for hslo, the gene encoding human BK channels. One variant does not contain alternative exon inserts (hmc2) and another contains a 29 AA insert with two consensus PKG phosphorylation sites (hmc1). We propose to use molecular methods to establish which variant of hslo is the equivalent of BK1, the PKG phosphorylation site on BK1, and quantitate the differential expression of hmc1 and hmc2 transcripts under conditions of longterm exposure of MC to cGMP. Regulation by cGMP at the transcriptional level would provide an additional feedback mechanism to adapt to chronic exposure to nitric oxide or atrial natriuretic peptide. These results should lead to a better understanding of the regulation of mesangial K channels and GFR at the cellular and molecular levels.
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