Endothelium-dependent dilatation is essentially mediated by three principal vasodilators: nitric oxide (NO), prostacyclin (PGI₂), and endothelium-derived hyperpolarizing factor (EDHF). Since the identity of EDHF remains elusive (although several candidates have been proposed), selective antagonists of EDHF are lacking and its (patho)physiological role(s) therefore remains equivocal. Consequently, EDHF biological activity is defined predominantly as the response that persists in the presence of combined inhibition of NO and PGI₂ synthesis (i.e. by blocking NOS and COX, respectively). This definition, however, has proved problematic since NOS and COX inhibitors are often not isoform-selective, do not result in complete inhibition, and have inherent haemodynamic effects in vivo. In order to overcome this problem we have generated mice doubly-deficient (eNOS^-/-/COX-1^-/- or dKO) in the enzymes required for endothelial NO and PGI₂ production, eNOS and COX-1, respectively. Such animals have permitted in vivo investigation of EDHF function.

Male and female dKO mice (20-25g) were anaesthetized with isofluorane (2%). The left common carotid artery was cannulated for measurement of mean arterial blood pressure (MABP) and the right femoral artery for drug administration. The endothelium-dependent vasodilator bradykinin (BK; 0.1-10 µg/kg) and the endothelium-independent vasodilator sodium nitroprusside (SNP; 3µg/kg) were given as single bolus doses of 50µl. Systolic blood pressure (SBP) was also measured by tail-cuff plethysmography in conscious animals in a heated room (26-27 ^oC).

BK elicited dose-dependent hypotension in female dKO animals (ΔMABP of -4.8±1.41, -15.7±3.68 and -22.7±1.71 mmHg for 0.1, 1.0 and 10μg/kg BK respectively; n=4), but had no effect on MABP in male mice (ΔMABP of 0±0, 0±0 and -3.7±3.67 mmHg for 0.1, 1.0 and 10μg/kg BK respectively; n=3). In female dKO mice, the absence of eNOS and/or COX-1 had no effect on SBP, whilst SBP was significantly elevated in dKO males compared to wild-type (Table 1).

Table 1. Systolic blood pressure (mmHg) in mice of differing genotype as measured by tail cuff plethysmography. *P<0.05, significantly different from wild-type; n4.

These studies indicate that EDHF is the predominant endothelium-derived relaxing factor in female mice, whilst NO is the predominant mediator in male mice. This gender-specific prevalence of EDHF may underlie the protection of female eNOS^-/- /COX-1^-/- mice against hypertension and related cardiovascular disorders.