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.
Gerzanich
Volodymyr
V
Department of Neurosurgery, University of Maryland School of Medicine, 22 S Greene St, Baltimore, MD 21201-1595, USA.
Ivanova
Svetlana
S
Zhou
Hui
H
Simard
J Marc
JM
D000804:Angiotensin II / Q000494:pharmacology*; D000818:Animals; D015153:Blotting, Western; D015220:Calcium Channels / Q000378:metabolism*; D020746:Calcium Channels, L-Type / Q000378:metabolism; D002536:Cerebral Arteries / Q000187:drug effects / Q000503:physiopathology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D019793:Fluorescein / Q000378:metabolism; D006973:Hypertension / Q000139:chemically induced / Q000378:metabolism / Q000503:physiopathology*; D008564:Membrane Potentials / Q000187:drug effects; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D051381:Rats; D011921:Rats, Inbred WKY; D015854:Up-Regulation
C414689:4,5-diaminofluorescein diacetate; D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D004791:Enzyme Inhibitors; D000804:Angiotensin II; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; C496316:Nos3 protein, rat; D019793:Fluorescein; D019331:NG-Nitroarginine Methyl Ester