Nitric oxide attenuates overexpression of Giα proteins in vascular smooth muscle cells from SHR: Role of ROS and ROS-mediated signaling

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.

Natriuretic peptide receptor‐C‐mediated attenuation of vascular smooth muscle cell hypertrophy involves Gqα/PLCβ1 proteins and ROS‐associated signaling

“…Confluent cells from SHR and WKY rats after starving for 24 hours in DMEM without FBS at 37°C were further incubated for 24 hours in the absence or presence of 0.1 μmol·L −1 C‐ANP 4‐23 . After incubation, the cells were washed twice with ice‐cold phosphate‐buffered saline (PBS) and lysed in a 200 μL buffer containing 25 mmol·L −1 Tris‐HCL (pH 7.5), 25 mmol·L −1 NaCl, 1 mmol·L −1 sodium orthovanadate, 10 mmol·L −1 sodium fluoride, 10 mmol·L −1 sodium pyrophosphate, 2 mmol·L −1 EDTA, 1 mmol·L −1 phenylmethylsulfonyl fluoride, 10 μg/mL aprotinin, 1% Triton X‐100, 0.1% sodium dodecyl sulfate, and 0.5 μg/mL leupeptin on ice as described earlier . The cell lysates were centrifuged at 12,000 g for 15 minutes at 4°C, and the supernatants were used for Western blot analysis.…”

“…The levels of protein expression and phosphorylation were determined by Western blotting as described previously . After SDS‐PAGE, the proteins were transferred to a nitrocellulose membranes and the blots were washed with PBS containing 0.1% Tween‐20 (PBST) and then incubated with respective primary antibodies against AT1(sc‐1173), Gqα(sc‐393), PLCβ1 (sc‐205), Nox4 (sc‐21860), p47phox (sc‐17845), p‐c‐Src (sc‐16846), c‐Src (s‐18), PDGFR (sc‐432), EGFR (sc‐03), p‐IGFR(sc‐101704), IGFR(sc‐713), p‐PDGFR (Y849), p‐EGFR (Y1006) using different dilutions ranging from 1:500 to 1:2000 at 4°C for overnight.…”

Inhibition of overexpression of Giα proteins and nitroxidative stress contribute to sodium nitroprusside-induced attenuation of high blood pressure in SHR

“…; Sarkar et al. ) that contributes to the enhanced expression of Giα proteins in SHR (Lappas et al. ).…”

Sodium nitroprusside is effective in preventing and/or reversing the development of schizophrenia‐related behaviors in an animal model: The SHR strain

“…The protective mechanisms associated with the long‐term beneficial effect of SNP are currently under investigation in our group. In this respect, alterations in the nitrergic system, increased brain oxidative stress, and neuroinflammation have been described in the SHR strain and might underlie the beneficial effect of SNP presented here.…”

“…Some functional and neurochemical alterations seen in the SHR strain underlie schizophrenia physiopathology and may lead to neurovascular changes and, putatively, be related to the behavioral abnormalities presented by this strain: an increase in brain and vascular oxidative stress, an increase in neuroinflammation, and a hyperpermeability of the blood‐brain and blood‐cerebrospinal fluid barrier . Considering specifically the nitrergic system, a decrease in brain and endothelial levels of NO and/or NOS activity have been reported in the SHR strain . In addition, we have reported in the SHR strain a decrease in the gene expression of glutamate AMPA‐R ( Gria1 ) and NMDA‐R ( Grin1 ) in the nucleus accumbens as well as in Gad2 (glutamate decarboxylase 2), Chrnb4 (cholinergic receptor, nicotinic, beta 4), Slc5a7 (choline transporter), and Qrfpr (pyroglutamylated RFamide peptide receptor) in the prefrontal cortex.…”