Sex differences in renal inner medullary nitric oxide synthase regulation and nitric oxide synthase contribution to blood pressure control in hypertensio

Abstract

There are sex differences in the development of hypertension with young males developing a more severe pathology faster than age-matched females; however, with advancing age this "protection" in females is lost. The mechanisms responsible for the sex difference in hypertension are unclear but the vasodilator nitric oxide (NO)/NO synthase (NOS) pathway which 1s important 1n blood pressure (BP) regulation has been implicated. Systemic inhibition of NOS using L-NAME (2, 5, and 7 mg/kg/day at 4 days per dose in drinking water) in male and female spontaneously hypertensive rats (SHR) resulted in dose-dependent increases in BP measured via telemetry; however, females exhibited greater increases in BP than males. Treatment of male and female SHR chronically with L-NAME at a dose of 7 mg/kg/day for 2 weeks significantly increased BP in both sexes, however, a previous exposure to L-NAME increased BP sensitivity to chronic NOS inhibition in females exclusively; this confirmed our hypothesis that female SHR are more dependent on NOS for BP control compared to male. Important for BP control, the renal 1nner medulla (IM) is the only region of the kidney to exhibit sex differences in NOS enzymatic activity. Female SHR have greater total NOS activity than males and we observed that it is not due to differences in phosphorylation or protein expression. Therefore, we exam1ned potential molecular mechanisms to explain the sexual dimorphism in renal IM NOS activity. The endogenous NOS inhibitor asymmetric dimethylarginine (AOMA) has been indicated in hypertension. However, HPLC analysis of ADMA and the essential NOS substrate L-arginine were equal between the sexes in plasma and renal IM of SHR and thus do not contribute to the sex differences in renal 1M NOS activity. Tetrahydrobiopterin (BH4) is an essential NOS cofactor and decreased BH4 availability has been indicated to be elevated in pat1ents and animal models with essential hypertension. BH4 levels can be decreased via oxidation and male SHR have h1gher levels of oxidative stress compared to females HPLC analysis of biopterin levels 1n control and tempol (antioxidant) treated SHR showed that female SHR have greater total biopterin, BH4 and BH2 levels than males in the renal IM and that these sex differences were dependent on the presence of oxidative stress. Studies next examined if greater biopterin levels in females translated into greater NOS activity in females. In vitro analysis of NOS enzymatic activity confirmed that greater oxidative stress and deficiency of BH4 of male SHR in the renal IM resulted 1n lower levels of NOS activity relative to female SHR. In addition, in vitro analysis of rena liM NOS activity revealed that 1) female SHR exhibit a sex hormone-dependent 1ncrease 1n renal IM NOS activity from sexually immature, pre-hypertensive age to sexually mature, hypertensive age that is not evident in male SHR and 2) that the ability of female sex hormones to stimulate NOS activity is time-dependent. In conclusion, the combination of BH4 deficiency in males caused by elevated oxidative stress and the ability of female sex hormones to stimulate NOS activity in female SHR and not ADMA or L-arginine. contribute to the sexual dimorphism in renal IM NOS activity. In addition, differences in sensitivity to NOS levels in SHR aid in creating sex differences in BP control.