Published online before print September 25, 2003, doi: 10.1161/01.RES.0000097872.69043.A0
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Submitted on May 23, 2003
Revised on September 2, 2003
Accepted on September 17, 2003
Alternative Splicing of cGMP-Dependent Protein Kinase I in Angiotensin-Hypertension. Novel Mechanism for Nitrate Tolerance in Vascular Smooth Muscle
Volodymyr Gerzanich ;
From the Departments of Neurosurgery (V.G., A.I., S.I., J.B.Y., H.Z., Y.D., J.M.S.), Pathology (J.M.S.), and Physiology (J.M.S.), University of Maryland at Baltimore, Baltimore, Md.
* To whom correspondence should be addressed. E-mail: msimard{at}surgery1.umaryland.edu.
Nitrate tolerance (NT) in hypertension is attributed to reduced activity of soluble guanylyl cyclase (sGC). We examined NT in basilar artery vascular smooth muscle cells (VSMCs) from control rats, rats infused with angiotensin II (Ang; 240 µg/kg per hour for 4 days), which were normotensive, and Ang-hypertensive rats (AHR; 240 µg/kg per hour for 28 days). Ca2+-activated K+ (Maxi-K) channels in VSMCs from AHR showed reduced activation by NO donor, consistent with NT. The concentration-response relationship for 8-Br-cGMP was shifted 2.5-fold to the right, indicating that abnormal sGC alone could not account for NT. Inside-out patches from AHR showed normal activation with exogenous cGMP-dependent protein kinase I (cGKI), suggesting no abnormality downstream of cGKI. We hypothesized that the reduction in apparent affinity of 8-Br-cGMP for cGKI in AHR might be due to a change in relative amounts of cGKI
versus cGKI
, since cGKI is less sensitive to cGMP activators than cGKI. This was substantiated by showing the following in AHR: (1) reduced effect of the cGKI-selective activator 8-APT-cGMP; (2) reduced total cGKI protein (both isoforms), but an increase in cGKI protein in quantitative immunofluorescence and Western blots; (3) similar changes in cGKI isoforms immunoisolated with Maxi-K channels; and (4) a large increase in cGKI mRNA and a decrease in cGKI mRNA in real-time PCR and Northern blots. Upregulation of cytosolic cGKI was evident 4 days after Ang infusion, before development of hypertension. Our data identify a functional role for cGKI in VSMCs previously ascribed exclusively to cGKI. Ang-induced alternative splicing of cGKI represents a novel mechanism for reducing sensitivity to NO/cGMP.
Key words: nitrate tolerance • cGMP-dependent protein kinase I • alternative splicing • angiotensin II • vascular smooth muscle
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