An endothelium-derived hyperpolarizing factor distinct from NO and prostacyclin is a major endothelium-dependent vasodilator in resistance vessels of wild-type and endothelial NO synthase knockout mice

Abstract

In addition to nitric oxide (NO) and prostacyclin (PGI(2)), the endothelium generates the endothelium-derived hyperpolarizing factor (EDHF). We set out to determine whether an EDHF-like response can be detected in wild-type (WT) and endothelial NO synthase knockout mice (eNOS -/-) mice. Vasodilator responses to endothelium-dependent agonists were determined in vivo and in vitro. In vivo, bradykinin induced a pronounced, dose-dependent decrease in mean arterial pressure (MAP) which did not differ between WT and eNOS -/- mice and was unaffected by treatment with N(omega)-nitro-l-arginine methyl ester and diclofenac. In the saline-perfused hindlimb of WT and eNOS -/- mice, marked N(omega)-nitro-l-arginine (l-NA, 300 micromol/liter)- and diclofenac-insensitive vasodilations in response to both bradykinin and acetylcholine (ACh) were observed, which were more pronounced than the agonist-induced vasodilation in the hindlimb of WT in the absence of l-NA. This endothelium-dependent, NO/PGI(2)-independent vasodilatation was sensitive to KCl (40 mM) and to the combination of apamin and charybdotoxin. Gap junction inhibitors (18alpha-glycyrrhetinic acid, octanol, heptanol) and CB-1 cannabinoid-receptor agonists (Delta(9)-tetrahydrocannabinol, HU210) impaired EDHF-mediated vasodilation, whereas inhibition of cytochrome P450 enzymes, soluble guanylyl cyclase, or adenosine receptors had no effect on EDHF-mediated responses. These results demonstrate that in murine resistance vessels the predominant agonist-induced endothelium-dependent vasodilation in vivo and in vitro is not mediated by NO, PGI(2), or a cytochrome P450 metabolite, but by an EDHF-like principle that requires functional gap junctions.

Figure 1

Effect of bradykinin on arterial blood pressure in anesthetized mice. (A) Original tracing showing the effect of a bolus application of bradykinin (Bk; 10 μg/kg, i.v.) on blood pressure (BP) in a diclofenac- (10 mg/kg, i.p.) and l-NAME- (30 mg/kg, i.p.) treated mouse. (B and C) Effect of bolus application of saline or bradykinin (doses indicated above the bars) on MAP in the absence (B) or in the presence (C) of diclofenac and l-NAME; n ≥ 5 each group; ∗, P < 0.05.

Figure 2

Dilator responses to endothelium-dependent and -independent agonists in the isolated perfused hindlimb of WT mice. (A) Original tracing showing the effect of bolus applications (100 μl) of ACh or SNP on perfusion pressure in the absence or presence of l-NA (300 μmol/liter). Numbers above the arrows indicate the dose of the agonist applied with the bolus (log mol); ∗ indicates a reduction in flow rate to maintain the perfusion pressure at a constant level. (B) Parameters of the perfused hindlimb in WT mice under basal conditions (shaded columns), during application of l-NA (filled columns), and after reduction of flow rate to adjust perfusion pressure during l-NA application (open columns). (C) Vasodilator responses to bolus applications (100 μl) of ACh and SNP in WT mice under control conditions (□), during the infusion of l-NA (■), and during infusion of l-NA and flow rate adjustment (●); n = 8 each group; ∗, P < 0.05. All experiments were performed in the presence of diclofenac (10 μmol/liter).

Figure 3

Effects of ACh, SNP, and l-NA on vasodilator responses of eNOS −/− mice. (A) Expression of eNOS in mouse aortic rings from WT and eNOS −/− mice as detected by Western blot analysis. (B) Original tracings showing relaxations of isolated aortic rings from WT and eNOS −/− mice. After constriction with phenylephrine (PE), cumulative dose–response curves to ACh were obtained. Numbers above the arrows indicate the final concentration of the agonist (log mol/liter). Tracings are representative of data obtained in six additional experiments. (C and D) Original tracings showing changes in hindlimb perfusion pressure in eNOS −/− mice upon bolus application of ACh or SNP (C) or bradykinin (Bk) (D) in the presence of diclofenac and presence or absence of l-NA. Numbers above the arrows indicate the dose of the agonist applied as a bolus (log mol). (E) Peripheral resistance of the perfused hindlimb of WT and eNOS −/− mice in the absence (shaded columns) and presence (filled columns) of l-NA. ns, Not significant. (F) Vasodilator responses to bolus applications (100 μl) of ACh in the perfused hindlimb of WT (□,■) and eNOS −/− mice (○,●) in the absence (open symbols) and presence (filled symbols) of l-NA (n ≥ 8).

Figure 4

Effects of K⁺ channel blockade and cytochrome P450 (CYP) inhibition on ACh-induced vasodilator responses in the hindlimb of WT mice. Experiments were performed in presence of diclofenac (10 μmol/liter) and l-NA (300 μmol/liter). (A) CTL = control, potassium channel blockade with charybdotoxin (CTX, 100 nmol/liter), apamin (APA, 100 nmol/liter), 4-aminopyridine, (4AP, 5 mmol/liter), glibenclamide, (Glib, 1 μmol/liter), and iberiotoxin (IbTX, 100 nmol/liter). (B) Inhibition of CYP enzymes with 17-octadecynoic acid (ODYA, 10 μmol/liter), miconazole (Mico, 10 μmol/liter), and sulfaphenazole (Sulfa, 10 μmol/liter) or of arachidonic acid release (S Dep, substrate depletion) by using the combination of the phospholipase A₂ inhibitors arachidonyl trifluoromethyl ketone (AACOCF₃, 3 μmol/liter) and ONO-RS-082 (10 μmol/liter) with the diacylglycerol-lipase inhibitor THC-80267 (10 μmol/liter). ∗, P < 0.05; n ≥ 4.

Figure 5

Effect of gap junction uncouplers and CB1 cannabinoid receptor agonists on ACh-induced vasodilator responses in the hindlimb of wild-type mice. Effects of the gap junction uncoupler 18α-glycyrrhetinic acid (aGA; 30 μmol/liter) and the CB1-receptor agonists Δ⁹-tetrahydrocannabinol (THC; 3 μmol/liter) and HU210 (10 μmol/liter) in the presence (+) or absence (−) of l-NA (300 μmol/liter). Diclofenac (10 μmol/liter) was continuously present during the experiment. CTL, control. n ≥ 5.