Sex differences in endothelial function in the porcine coronary artery

The prevalence of cardiovascular disease is lower in premenopausal women compared to age-matched males and postmenopausal females. Differences in risk may be due to sex differences in endothelial function. Therefore, this thesis examined the effects of gender on endothelium-dependent vasorelaxation in porcine isolated coronary arteries (PCAs). Distal PCAs were studied under myographic conditions and pre-contracted with U46619. Concentration-response curves to bradykinin, an endothelium-dependent vasorelaxant, were constructed in the presence of various inhibitors. Inhibition of NO and prostanoid synthesis (EDH-type response) produced greater inhibition in males compared to females. Eliminating H2O2 using PEG-catalase significantly reduced the bradykinin-induced vasorelaxation in the absence, but not in the presence of L-NAME and indomethacin in females, and had no effect in males. Inhibition of gap junctions with carbenoxolone and 18α-GA inhibited the EDH-type response in females but not in males. Inhibition of SKCa channels reduced the EDH-type response in PCAs from both sexes but inhibition of IKCa had an effect only in females but not males. Western blot did not detect any differences in the expression of Cx40, 43 or IKCa between sexes. H2O2 caused concentration-dependent vasorelaxations which were significantly inhibited by PEG-catalase, TEA, 60 mM K+ and 500 nM ouabain. Inhibition of NOS, cyclo-oxygenase, gap junctions, SKCa, IKCa, BKCa, Kir, KV, KATP, cGMP, Na+-Ca2+ exchanger or removal of endothelium had no effect on the H2O2-induced vasorelaxation. 1 mM H2O2 inhibited both KCl-induced vasorelaxation and rubidium-uptake consistent with inhibition of the Na+/K+-pump activity. The effects of the antioxidant Tiron® under different gassing conditions (95% O2/5% CO2 or 95% air/5% CO2) were investigated. The bradykinin-induced vasorelaxations in PCAs were unaffected by different levels of oxygenation. Tiron® increased the potency of bradykinin only when gassed with 95% O2/5% CO2 and the enhancement in vasorelaxation was prevented by catalase. Similarly, Tiron® enhanced the EDH-type response when gassed with 95% O2/5% CO2 in PCAs from both sexes. Biochemical analysis using Amplex Red demonstrated that H2O2 was generated in Krebs’-Henseleit solution when gassed with 95% O2/5% CO2, but not with 95% air/5% CO2. Inhibition of Nox had no effect in PCAs from females but DPI, a non-selective Nox inhibitor reduced the potency of the bradykinin-induced vasorelaxation in males. In the EDH-type responses, inhibition of Nox had no effect in females, but in males, ML-171 (a selective Nox inhibitor) and DPI enhances while VAS2870 (a selective Nox inhibitor) reduces the bradykinin-induced vasorelaxation. ML-171 had no effect on the forskolin-induced vasorelaxation but decreased the potency of U46619-induced tone in both sexes in the absence or presence of endothelium. Nox activity was reduced by DPI and ML-171, but not VAS2870 in PCAs from both sexes. Sex differences in the functional study of Nox could be attributed to the differential expression of Nox proteins where expression of Nox1 and Nox2 were greater in males but Nox4 was greater in females. This may underlie the greater oxidative stress observed in males. Bradykinin-induced EDH-type responses in PCAs from both sexes were essentially abolished by 2-APB (TRPC&TRPM antagonist). SKF96365 (TRPC antagonist) inhibited the bradykinin-induced vasorelaxation in males, and EDH-type response in both sexes. Pyr3 (TRPC3 antagonist) inhibited both the NO and EDH components of the bradykinin-induced vasorelaxation in males, but not females. RN1734 (TRPV4 antagonist) reduced the potency of the NO component of the bradykinin-induced vasorelaxation in females only, but inhibited the EDH-type response in both sexes. 2-APB, SKF96365 and RN1734 all reduced the H2O2-induced vasorelaxation, whereas Pyr3 had no effect. No differences in expression level of TRPC3 and TRPV4 between sexes were detected using Western blot. In conclusion, present study demonstrated clear sex differences in endothelial function in PCAs where H2O2, MEGJs, IKCa and TRPV4 channels play a role in the bradykinin-induced vasorelaxation only in female pigs while Nox-generated reactive oxygen species and TRPC3 channels play a role in the bradykinin-induced vasorelaxation only in male pigs. Therefore, gender-specific drug treatment for cardiovascular disease may be a novel therapeutic strategy.