Mislocalization of eNOS and upregulation of cerebral vascular Ca2+ channel activity in angiotensin-hypertension.

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 microg x kg(-1) x h(-1) SQ x4 days), which were normotensive, and from Ang-hypertensive rats (AHR; 240 microg x kg(-1) x 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 NG-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.