Oxidative stress impairs cGMP-dependent protein kinase activation and vasodilator-stimulated phosphoprotein serine-phosphorylation.

Reactive oxygen species induce vascular dysfunction and hypertension by directly interacting with nitric oxide (NO) which leads to NO inactivation. In addition to a decrease in NO bioavailability, there is evidence that oxidative stress can also modulate NO signaling during hypertension. Here, we investigated the effect of oxidative stress on NO signaling molecules cGMP-dependent protein kinase (PKG) and vasodilator-stimulated phosphoprotein (VASP) which are known to mediate vasodilatory actions of NO. Male Sprague Dawley (SD) rats were provided with tap water (control), 30 mM L-buthionine sulfoximine (BSO, a pro-oxidant), 1 mM tempol (T, an antioxidant) and BSO + T for 3 wks. BSO-treated rats exhibited high blood pressure and oxidative stress. Incubation of mesenteric arterial rings with NO donors caused concentration-dependent relaxation in control rats. However, the response to NO donors was significantly lower in BSO-treated rats with a marked decrease in pD2. In control rats, NO donors activated mesenteric PKG, increased VASP phosphorylation and its interaction with transient receptor potential channels 4 (TRPC4) and inhibited store-operated Ca2+ influx. NO failed to activate these signaling molecules in mesenteric arteries from BSO-treated rats. Supplementation of BSO-treated rats with tempol reduced oxidative stress and blood pressure and normalized the NO signaling. These data suggest that oxidative stress can reduce NO-mediated PKG activation and VASP-TRPC4 interaction which leads to failure of NO to reduce Ca2+ influx in smooth muscle cells. The increase in intracellular Ca2+ contributes to sustained vasoconstriction and subsequent hypertension. Antioxidant supplementation decreases oxidative stress, normalizes NO signaling and reduces blood pressure.

Effect of ischemia reperfusion on sodium-dependent phosphate transport in renal brush border membranes.

The effect of ischemia induced acute renal failure (ARF) on the transport of phosphate (Pi) after early (15-30 min) and prolonged (60 min) ischemia in the brush border membrane vesicles (BBMV) from rat renal cortex was studied. Sodium-dependent transport of Pi declined significantly and progressively due to ischemia. Western blot analysis of BBM from ischemic rats showed decreased expression of NaPi-2. A compensatory increase was observed in Pi uptake in BBMV from contralateral kidneys. There was no significant difference in NaPi-2 expression between BBMV from sham and contralateral kidneys. Early blood reperfusion for 15 min after 30 min ischemia caused further decline in Pi uptake. Prolonged reperfusion for 120 min caused partial reversal of transport activities in 30-min ischemic rats. However, no improvement in the transport of Pi was observed in 60-min ischemic rats after 120 min of blood reperfusion. Kinetic studies showed that the effect of ischemia and blood reperfusion was dependent on the Vmax of the Na-Pi transporter. Western blot analysis showed increased expression of NaPi-2 in the BBMs from ischemia-reperfusion animals. Further, a shift in the association of Na ions to transport one molecule of Pi was observed under different extracellular Na concentrations [Na]o. Feeding rats with low Pi diet and/or treatment with thyroid hormone (T3) prior to ischemia resulted in increased basal Pi transport. Ischemia caused similar decline in Pi transport in BBM from LPD and/or T3 animals. However, recovery in these animals was faster than the normal Pi diet fed (NPD) animals. The study suggests a change in the intrinsic properties of the Na-Pi transporter in rat kidneys due to ischemia. The study also indicates that treatment with T3 and feeding LPD prior to ischemia caused faster recovery of phosphate uptake due to ischemia-reperfusion injury.