A wealth of studies support the key role of high
A wealth of studies support the key role of high NO in promoting higher blood flow and thus greater O2 delivery to working tissues in healthy Tibetans and thus partly compensate hypoxia induced low arterial O2 content [7,9,27]. We observed high eNOS mRNA, protein and enzyme activity in Ladakhi women as compared to lowland women. Availability of the substate l-arginine is one of the critical factors for eNOS activity and the arginase enzyme competes with eNOS for the same substrate [2,28]. We observed significant higher l-citrulline levels for Ladakhi women suggesting efficient utilization of the substrate by eNOS during hypoxia. Concomitantly, we observed lower arginase mRNA and protein levels suggesting lower activity of arginase . The plasma NOx (nitrite and nitrate) levels were also higher in Ladakhi women. As mentioned earlier, most plasma nitrite and nitrate is derived from eNOS and the observed high nitrite and nitrate concentrations provides indirect support for high endothelial NO formation in Ladakhi women [7,27,29]. Endothelial NO synthesis is regulated by wide variety of humoral (eg. bradykinin, insulin, adiponectin, estrogen), mechanical (eg. shear stress) and pharmacological stimuli (eg. statins). Bradykinin is an important determinant of vascular function and regulates eNOS activity and expression during hypoxia [8,30]. We have previously reported that bradykinin-mediated NO production facilitates high altitude acclimatization and GNE-317 . In the present study, we have observed significant higher levels of bradykinin in Ladakhi women. Estrogen is an important vasoprotective hormone and causes rapid dilation of blood vessels by activating eNOS through several mechanisms [11,31]. Vascular estrogen binds to nuclear estrogen receptors ERα and ERβ as well as the novel G protein-coupled estrogen receptor (GPER), also known as GPR30 and mediates non-genomic estrogen signaling . Estrogen also indirectly acts as a vasodilator by blocking the activity of vasoconstrictors, such as platelet-derived serotonin . Studying effect of chronic hypoxia on steroid hormone levels, Charles et al. have reported higher estrogen levels for multigenerational Andeans as compared to short-term European women . In the present study, we have also observed significant higher levels of estrogen in Ladakhi women. Concomitantly, we have observed significant lower levels of vasoconstrictors Angiotensin I and Angiotensin II in Ladakhi women. Chronic hypoxia exposure results in elevation of blood pressure through renin-angiotensin system (RAS). Vascular renin converts angiotensinogen to Ang I which is then converted to its active form Ang II by angiotensin converting enzyme (ACE). Ang II, the primary effector of RAS system constricts vascular smooth muscle cells, enhances myocardial contractility, and plays a primary role in the pathogenesis of hypertension through AT1 receptor . The RAS and eNOS-cGMP systems mutually regulate each other by feedback mechanisms . Using a plasma proteomics approach, we have previously reported lower levels of Ang II and consequent higher NO availability for Ladakhi men similar to the present observations . Our present observations suggests that multiple humoral factors regulate endothelial NO synthesis in Ladakhi women. It is noteworthy that ageing is an independent factor for endothelial dysfunction and reduced NO availability even in the absence of other cardiovascular risk factors. The impairment of endothelial function is a progressive phenomenon starting in the middle age and increases the risk of cardiovascular disorders (CVD) in humans [37,38]. Hence, ageing should limit NO availability and signaling in older women at high altitude. In contrast, we observed significant higher eNOS activity and protein in middle-aged Ladakhi women as compared to their lowland counterparts. We also observed significant higher levels of cGMP, NOx in middle-aged Ladakhi women further corroborating higher functionality of eNOS-NO-cGMP pathway though the levels were lower than young Ladakhi women. Interestingly, middle-aged Ladakhi women possessed higher NOS activity than young sea level women. Phosphorylation of eNOS at Ser1177 and Ser615 activates the enzyme while Thr495 inhibits the enzyme by interfering with the binding of cofactor calmodulin to eNOS [11,39]. Advancing age impairs eNOS activity and NO bioavailability via phosphorylation on Thr495 and dephosphorylation on Ser1177 [40,41]. In contrast, we observed higher levels of eNOS, eNOSSer1177, eNOSSer615 and significant lower eNOSThr495 in Ladakhi women irrespective of age as compared to lowland women. Taking into consideration that estrogen augments NO availability in women by post-translational modification of eNOS [37,42] and Ladakhi women exhibit higher estrogen levels, we further evaluated the levels of estradiol, the most abundant and potent estrogen in humans. Both young and postmenopausal Ladakhi women possessed significant higher levels of estradiol as compared to their age matched lowland women. It is noteworthy that middle aged Ladakhi women possessed similar estradiol levels as compared to young lowland women irrespective of their menopause status. These cumulative results suggest phosphorylation of eNOS by estrogen augments NO availability in Ladakhi women providing them functional advantage to survive chronic hypoxia irrespective of age. The present study is limited by not reporting the menopause phase of young women at both the altitudes. In addition, mRNA was isolated from whole blood and contribution of blood cell types to the reported expression levels was not determined though it is expected that leukocytes majorly contribute to blood mRNA levels. Similarly, we have not studied eNOS activity and phosphorylation status of older (>60 years) Ladakhi women, albeit such studies will provide additional information for age associated NO availability for high altitude women.