Abstract
Vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) exhibit decreased levels of nitric oxide (NO) that may be responsible for the overexpression of Giα proteins that has been shown as a contributing factor for the pathogenesis of hypertension in SHR. The present study was undertaken to investigate if increasing the intracellular levels of NO by NO donor S-Nitroso-N-acetyl-DL-penicillamine (SNAP) could attenuate the enhanced expression of Giα proteins in VSMC from SHR and explore the underlying mechanisms responsible for this response. The expression of Giα proteins and phosphorylation of ERK1/2, growth factor receptors and c-Src was determined by Western blotting using specific antibodies. Treatment of VSMC from SHR with SNAP for 24 hrs decreased the enhanced expression of Giα-2 and Giα-3 proteins and hyperproliferation that was not reversed by 1H (1, 2, 4) oxadiazole (4, 3-a) quinoxalin-1-one (ODQ), an inhibitor of soluble guanylyl cyclase, however, PD98059, a MEK inhibitor restored the SNAP-induced decreased expression of Giα proteins towards control levels. In addition, the increased production of superoxide anion, NAD(P)H oxidase activity, overexpression of AT1 receptor, Nox4, p22phox and p47phox proteins, enhanced levels of TBARS and protein carbonyl, increased phosphorylation of PDGF-R, EGF-R, c-Src and ERK1/2 in VSMC from SHR were all decreased to control levels by SNAP treatment. These results suggest that NO decreased the enhanced expression of Giα-2/3 proteins and hyperproliferation of VSMC from SHR by cGMP-independent mechanism and involves ROS and ROS-mediated transactivation of EGF-R/PDGF-R and MAP kinase signaling pathways.
Highlights
Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) have been shown to activate various signal transduction systems, including the adenylyl cyclase/cAMP system that regulate of a variety of physiological functions including blood pressure [1]
As reported earlier (13), the expression of Giα-2 protein (A) and Giα-3 (B) was increased by ~80% and 100% respectively in vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) as compared to Wistar Kyoto rats (WKY) rats and the treatment of cells with SNAP for 24 h that increases the levels of nitric oxide (NO) by about 630% and 450% respectively (C) in VSMC from SHR and WKY rats completely attenuated the enhanced expression of Giα proteins to WKY levels
Since NO activates the soluble guanylyl cyclase activity and augments the levels of intracellular Cyclic GMP (cGMP) which has been shown to inhibit the expression of Giα proteins in aortic and A10 VSMC [45, 46], it was desirable to examine if cGMP could attenuate the enhanced expression of Giα proteins in VSMC from SHR
Summary
Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) have been shown to activate various signal transduction systems, including the adenylyl cyclase/cAMP system that regulate of a variety of physiological functions including blood pressure [1]. Several cellular functions including vascular tone, cell proliferation etc, that are implicated in the regulation of blood pressure are mediated through the activation of Giα proteins and associated adenylyl cyclase signaling [9,10,11,12]. The increased expression of Giα-2 and Giα-3 proteins and resultant decreased levels of cAMP were shown to contribute to the pathogenesis of hypertension in spontaneously hypertensive rats (SHR) and DOCA-salt hypertensive rats [19, 20]. This was further supported by the studies showing that the inactivation of Giα proteins in prehypertensive rats (2 weeks old SHR) by single injection of pertussis toxin (PT) prevented the development of high blood pressure that was associated with PTinduced decreased levels of Giα proteins [21]. A role of enhanced expression of Giα-2 and Giα-3 proteins has been shown in hyperproliferation of vascular smooth muscle cells (VSMC) [11, 12, 26] that contributes to vascular remodeling associated with hypertension [27]
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