MECHANISM OF REGULATION OF A GLOMERULAR K CHANNEL

系膜细胞(MC)是调节滤过率的收缩细胞 (GFR)通过调节肾小球毛细血管表面积。像血管一样 平滑肌、MC对容量和压力调节激素有反应， 如血管紧张素II、心钠素(ANP)和一氧化氮 (否)。因此，MC在血液调节中起着重要的生理作用。 压力、液体和体积的动态平衡。 我们的实验室最近发现，大的钙激活K 通道(BK)在NO和ANP调节的松弛中起主要作用 系膜细胞。BK对血管松弛药的反应是 两相，激活阶段约为10至30秒，以及 在返回之前可以持续60秒的停用阶段 基线水平。在活化期，通过刺激NO和ANP 鸟苷酸环化酶和cGMP的产生，导致cGMP的特异性激活 BK的激活剂。可以通过以下方法抑制失活阶段 冈田酸或斑腿草酸，蛋白磷酸酶1和 2a.调控BK的调节和信号通路 激活/停用周期将是本提案的主要重点。 膜片钳方法揭示了两种BK群体：一种是 对磷酸酶和cGMP激活的蛋白激酶(BK1)和1的反应 对这些代理没有响应(BK2)。这些结果表明， BK2不具有与BK1相同的磷酸化序列位点。它是 推测这些BK种群具有独特的信使RNA， 可以被转录调控。初步电生理学 研究表明，当MC暴露时，BK2优先表达 24小时至DB-cGMP。初步的分子研究表明， 至少有两种信息变体转录成hslo基因 对人类BK频道进行编码。一个变体不包含备选方案 一个外显子插入(Hmc2)，另一个含有一个29个氨基酸的插入，带有两个 一致的PKG磷酸化位点(Hmc1)。我们建议使用分子 方法来确定hslo的哪个变体等同于bk1， BK1上的PKG磷酸化位点，并对差异进行定量 Hmc1和hmc2转录本在长期条件下的表达 MC暴露于cGMP。CGMP在转录水平的调控 将提供额外的反馈机制以适应慢性 暴露于一氧化氮或心钠素。这些结果 应该有助于更好地理解系膜K的调节 细胞和分子水平上的通道和GFR。

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.