Sex differences in the relative contributions of nitric oxide and EDHF to agonist-stimulated endothelium-dependent relaxations in the rat isolated mesenteric arterial bed

1 We have used the isolated, buffer-perfused, superior mesenteric arterial bed of male and female rats to assess the relative contributions of nitric oxide (NO) and the endothelium-derived hyperpolarizing factor (EDHF) to endothelium-dependent relaxations to carbachol. 2 Carbachol caused dose-related relaxations of methoxamine-induced tone in mesenteric vascular beds from male rats described by an ED50(M) of 0.43+/-0.15 nmol and a maximum relaxation (R-max(M)) of 89.6+/-1.2% (n=28) which were not significantly different from those observed in mesenteries from female rats (ED50(F)=0.72+/-0.19 nmol and R-max(F)=90.7+/-0.9%; n=22). 3 In the males, the addition of 100 mu M N-G-nitro-L-arginine methyl ester (L-NAME) caused the dose-response curve to carbachol to be significantly (P<0.001) shifted to the right 15 fold (ED50(M)=6.45+/-3.53 nmol) and significantly (P<0.01) reduced R-max(M) (79.7+/-2.8%, n=13). By contrast, L-NAME had no effect on vasorelaxation to carbachol in mesenteries from female rats (ED50(F)=0.89+/-0.19 nmol, R-max(F)=86.9+/-2.3%, n=9). 4 Raising tone with 60 mM KCl significantly reduced the maximum relaxation to carbachol in mesenteries from male rats 2 fold (R-max(M)=40.3+/-9.2%, n=4; P<0.001) and female rats by 1.5 fold (R-max(F)=55.3+/-3.3%, n=6; P<0.001), compared with methoxamine-induced tone. The potency of carbachol was also significantly reduced 1.2 fold in preparations from males (ED50(M)=0.87+/-0.26 nmol; P<0.01) but not the females (ED50(F)=4.04+/-1.46 nmol). In the presence of both 60 mM KCl and L-NAME, the vasorelaxation to carbachol was completely abolished in mesenteries from both groups. 5 The cannabinoid receptor antagonist SR141716A (1 mu M), which is also a putative EDHF antagonist, had no significant effect on the responses to carbachol in mesenteries from males or females (ED50(M)=1.41+/-0.74 nmol, R-max(M)=89.4+/-2.5%, n=7; ED50(F)=2.17+/-0.95 nmol, R-max(F)=89.9+/-1.8%, n=9). In mesenteries from male rats, in the presence of 100 mu M L-NAME, SR141716A significantly (P<0.05) shifted the dose-response curve to carbachol 8 fold further to the right than that seen in the presence of L-NAME alone (ED50(M)=53.8+/-36.5 nmol) without affecting R-max(M) (72.4+/-4.8%, n=10). In mesenteries from female rats, the combined presence of L-NAME and SR 141716A, significantly (P<0.01) shifted the dose-response curve to carbachol 7.5 fold, (ED50(F)=6.66+/-2.46 nmol), as compared to L-NAME alone and significantly (P<0.001) decreased R-max(F) (70.1+/-5.5%, n=8). 6 Vasorelaxations to the nitric oxide donor sodium nitroprusside (SNP), to the endogenous cannabinoid, anandamide (a putative EDHF) and to the ATP-sensitive potassium channel activator, levcromakalim, did not differ significantly between male and female mesenteric vascular beds. 7 The continuous presence of sodium nitroprusside (SNP; 20-60 nM) had no effect on vasorelaxation to carbachol in mesenteries from either males or females. In the presence of L-NAME, SNP significantly (P<0.05) reduced the potency of carbachol 6 fold, without affecting the maximal relaxation in mesenteries from male rats (ED50(M)=40.9+/-19.6 nmol, R-max(M)=79.4+/-2.5%, n=11). Similarly in mesenteries from female rats, the ED50(F) was also significantly (P<0.01) increased 7 fold (6.24+/-3.02 nmol), while the R-max(F) was unaffected (81.9+/-11.0%; n=4). 8 The results of the present investigation demonstrate that the relative contributions of agonist-stimulated NO and EDHF to endothelium-dependent relaxations in the rat isolated mesenteric arterial bed, differ between males and females. Specifically, although both NO and EDHF appear to contribute towards endothelium-dependent relaxations in males and females, blockade of NO synthesis alone has no effect in the female. This suggests that EDHF is functionally more important in females; one possible explanation for this is that in the absence of NO, the recently identified ability of EDHF to compensate for the loss of NO, is functionally more important in females than males.