Published Online
on March 31, 2003

A more recent version of this article appeared on May 1, 2003

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Submitted on September 30, 2002
Revised on October 23, 2002

Mislocalization of eNOS and Upregulation of Cerebral Vascular Ca²⁺ Channel Activity in Angiotensin-Hypertension

Volodymyr Gerzanich; Svetlana Ivanova; Hui Zhou; and J. Marc Simard^*

From the Departments of Neurosurgery (V.G., S.I., H.Z., J.M.S.), Physiology (J.M.S.), and Pathology (J.M.S.), University of Maryland School of Medicine, Baltimore.

^* To whom correspondence should be addressed. E-mail: msimard{at}surgery1.umaryland.edu.

Abstract--We tested the hypothesis that endothelial dysfunction induced by angiotensin II (Ang-hypertension) would impair regulatory control of vascular smooth muscle L-type Ca²⁺ channels by endothelial nitric oxide synthase (eNOS). We studied cerebral lenticulostriate arterioles (LSAs) from control rats, from rats infused with Ang (240 µg · kg^-1 · h^-1 SQ x4 days), which were normotensive, and from Ang-hypertensive rats (AHR; 240 µg · kg^-1 · h^-1 x28 days). Patch-clamp measurements on isolated LSA smooth muscle cells (SMCs) showed a significant increase in Ca²⁺ channel availability with 4- and 28-day infusions versus controls (0.47±0.03 and 0.66±0.05 vs 0.36±0.03 pS/pF, respectively; P<0.01), with Western blots showing no change in channel protein expression, consistent with altered channel regulation. In LSAs from 28-day AHR, 4,5-diaminofluorescein diacetate imaging showed diminished NO production in response to acetylcholine stimulation in vivo, and inhibition of eNOS with N^G-nitro-L-arginine methyl ester failed to increase Ca²⁺ channel availability in isolated SMCs, indicating an abnormality with the eNOS/NO-signaling pathway regulating the channel. Immunofluorescence imaging showed that in 1 of 53, 33 of 109, and 53 of 62 LSAs from controls and from rats with 4- and 28-day infusions, respectively, eNOS was absent from its normal location at the abluminal border and was mislocalized to perinuclear Golgi. Ca²⁺ channel availability in LSA SMCs from controls and from rats with 4- and 28-day infusions was proportional to the fraction of LSAs showing eNOS mislocalization, but not blood pressure. These data provide the first evidence linking Ang-induced eNOS mislocalization, eNOS dysfunction, and Ca²⁺ channel upregulation, and they provide novel mechanistic insights into pathological changes in LSAs associated with stroke.

Key words: calcium channels • angiotensin II • nitric oxide synthase • hypertension, experimental • muscle, vascular, smooth • endothelium

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