HYPERTENSION--CONTRIBUTION OF ARTERIAL WALL CHANGES

描述：(改编自申请)：激动剂激活 动脉平滑肌产生持续的钙内流和细胞膜 有助于维持紧张力的去极化。PI已经显示出 钙离子内流对全细胞钾电流(Ik)的调制具有特征性 提示L钙离子对电压门控性钾通道(Kv)的抑制作用 渠道(加州)流入。此外，这种机制在自发性高血压患者中似乎比 为之前的许多观察提供了解释，并且 也提供了一个研究分子机制的机会 牵涉其中。本研究项目基于以下假设：(A) 钙离子通过钙离子内流抑制Kv通道，(B)通过直接作用于 通道，提供了一种持续的膜去极化机制 激动剂激活，以及(D)哪个对自发性高血压大鼠的心肌细胞更有效 对阵WKY。以下具体目标将使用以下工具来验证这些假设 WKY大鼠肠系膜动脉肌细胞、RNA和膜的分离 和SHR：1)证明当K通道最大时，Cal内流抑制Ik 2)以证明钙依赖信号 转导机制(PKC和CaM激酶II)不参与这一过程 作用；3)测定WKY和SHR的MA中Kv亚型的表达；4)至 测定这些Kv亚型的(细胞内)钙敏感性(抑制) 在非洲爪哇卵母细胞中表达；5)测定Kv对钙离子的敏感性 以及6)确定抑制Kv的作用。 钙离子在激动剂激活的持续去极化过程中内流。IK将会是 用全细胞膜片钳方法的穿孔膜片钳方法测量。 Cd~(2+)、尼索地平或降低外源性钙离子浓度可抑制钙离子内流 Ca~(2+)，并被Bay K_(8644)和离子霉素提高。PKC和CaM激酶II将 被Calphostin C、KT5926和抑制肽抑制。KV 将通过RT-PCR和Southern和Western来确定表达 吸墨水。 钙离子对Kv在卵母细胞和天然心肌细胞中表达的影响 由全单元法和单通道法确定。私家侦探表示 钙离子内流对Kv的抑制是一种正反馈机制 在激动剂激活过程中通过膜维持紧张性收缩 去极化和持续的钙激活。这些研究将提供 首次详细研究了Kv的表达和功能性质 动脉肌细胞。此外，这些研究还将提供一种新的分子 高血压遗传连锁研究的目标和新的潜力 治疗靶点。

DESCRIPTION: (Adapted from the application): Agonist activation of arterial smooth muscle produces sustained Ca2+ influx and membrane depolarization that contribute to tonic force maintenance. The PI has shown that Ca2+ influx modulates whole cell K+ currents (Ik) with characteristics that suggest the inhibition of voltage-gated K+ channels (Kv) by L-type Ca2+ channel (CaL) influx. Also, this mechanism appears to be larger in SHR than in WKY myocytes providing an explanation for many previous observations, and also providing an opportunity to investigate the molecular mechanisms involved. This research project is based upon the hypothesis that: (a) Ca2+ influx through CaL inhibits Kv channels, (b) by a direct action on the channel, providing a mechanism for sustained membrane depolarization during agonist activation, and (d) which is more effective in myocytes from SHR versus WKY. The following specific aims will test these hypotheses using myocytes, RNA and membranes isolated from mesenteric arteries (MA) of WKY and SHR: 1) To demonstrate that CaL influx inhibits Ik when maxi K channels are blocked by iberiotoxin; 2) To demonstrate that Ca2+- dependent signal transduction mechanisms (PKC and CaM kinase II) are not involved in this effect; 3) To determine Kv isoform expression in MA from WKY and SHR; 4) To determine the (cytosolic) Ca2+ sensitivity (inhibition) of these Kv isoforms expressed in Xenopus oocytes; 5) To determine the Ca2+-sensitivity of Kv in native arterial myocytes; and 6) to determine the role of Kv inhibition by Ca2+ influx on sustained depolarization with agonist activation. Ik will be measured by perforated patch variant of the whole cell patch clamp method. Ca2+ influx will be inhibited by Cd2+, nisoldipine, or lowering external Ca2+, and increased by Bay K 8644 and ionomycin. PKC and CaM kinase II will be inhibited by calphostin C and KT5926 and inhibitory peptides. Kv expression will be determined by RT-PCR and by Southern and Western Blotting. The effects of Ca2+ on Kv expressed in oocytes and native myocytes will be determined by whole cell and single channel methods. The PI suggests that inhibition of Kv by Ca2+ influx represents a positive feedback mechanism which sustains tonic contractions during agonist activation through membrane depolarization and sustained CaL activation. These studies will provide the first detailed investigation of Kv expression and functional properties in arterial myocytes. In addition, these studies will provide a new molecular target for genetic linkage studies in hypertension as well as new potential therapeutic targets.