S-nitrosothiols dilate the mesenteric artery more potently than the femoral artery by a cGMP and L-type calcium channel-dependent mechanism

Abstract

S-nitrosothiols (SNOs) are metabolites of NO with potent vasodilatory activity. Our previous studies in sheep indicated that intra-arterially infused SNOs dilate the mesenteric vasculature more than the femoral vasculature. We hypothesized that the mesenteric artery is more responsive to SNO-mediated vasodilation, and investigated various steps along the NO/cGMP pathway to determine the mechanism for this difference. In anesthetized adult sheep, we monitored the conductance of mesenteric and femoral arteries during infusion of S-nitroso-l-cysteine (L-cysNO), and found mesenteric vascular conductance increased (137 ± 3%) significantly more than femoral conductance (26 ± 25%). Similar results were found in wire myography studies of isolated sheep mesenteric and femoral arteries. Vasodilation by SNOs was attenuated in both vessel types by the presence of ODQ (sGC inhibitor), and both YC-1 (sGC agonist) and 8-Br-cGMP (cGMP analog) mediated more potent relaxation in mesenteric arteries than femoral arteries. The vasodilatory difference between mesenteric and femoral arteries was eliminated by antagonists of either protein kinase G or L-type Ca(2+) channels. Western immunoblots showed a larger L-type Ca(2+)/sGC abundance ratio in mesenteric arteries than in femoral arteries. Fetal sheep mesenteric arteries were more responsive to SNOs than adult mesenteric arteries, and had a greater L-Ca(2+)/sGC ratio (p = 0.047 and r = -0.906 for correlation between Emax and L-Ca(2+)/sGC). These results suggest that mesenteric arteries, especially those in fetus, are more responsive to SNO-mediated vasodilation than femoral arteries due to a greater role of the L-type calcium channel in the NO/cGMP pathway.

Keywords: Fetus; L-type calcium channel; NO/cGMP pathway; S-nitrosothiol; Sheep.

Fig. 1.

Hemodynamic effects of L-cysNO infused in five adult sheep. L-cysNO (5 mmol L⁻¹) was continuously infused into one femoral artery, with rates increasing at 3-min intervals. Mean arterial blood pressure (MAP) falls about 50% while heart rate (HR) is unchanged. Conductance (flow/pressure) increases markedly in the mesenteric artery (Mes) with only a minimal increase in the femoral artery (Fem) contralateral to the SNO infusion (the conductance data from three of the five animals in the figure were reported previously [24]). (n = 5; *p < 0.05 mesenteric vs. contralateral femoral; #p < 0.05 vs. baseline.)

Fig. 2.

Relaxation responses of isolated adult sheep mesenteric and femoral arterial rings to S-nitrosothiols. Vessels were preconstricted with KCl (A, B, and C) or 5-HT (D, E, and F), and cumulatively exposed to L-cysNO (A, D), D-cysNO (B, E), or GSNO (C, F). The relaxation to each of the three S-nitrosothiols is more prominent in mesenteric than femoral vessels. (n ≥ 5; *p < 0.05 mesenteric vs. femoral for each S-nitrosothiol tested.)

Fig. 3.

Role of the NO/cGMP pathway in the differential vasodilatory response of the mesenteric and femoral arteries to L-cysNO. Rings were preconstricted with KCl and an average of five or more vessels is shown. A: Femoral (Fem) responses are compared to B: Mesenteric (Mes) responses to L-cysNO, with and without 10 μmol L⁻¹ ODQ; the right-shift is significant in both instances, suggesting an important role for the NO/cGMP pathway; C: Relaxation mediated by 100 μmol・L⁻¹ YC-1, an sGC agonist and D: Relaxation mediated by 500 μmol L⁻¹ 8-Br-cGMP, a cGMP analog, in mesenteric and femoral arteries. Both YC-1 and 8-Br-cGMP bring about more potent relaxation in mesenteric arteries than in femoral. E: EC₅₀ of the relaxation curves with and without 30 μmol L⁻¹ PKI, a PKG inhibitor; F: E_max of the curves with and without 30 μmol L⁻¹ PKI. Note that PKI does not change the EC₅₀ but eliminates the difference in E_max, indicating that the differential vasodilation between mesenteric and femoral arteries is PKG-dependent. (n ≥ 5; *p < 0.05, **p < 0.01 mesenteric vs. femoral.)

Fig. 4.

Role of the L-Ca²⁺ channel in the differential vasodilatory response of adult sheep mesenteric and femoral arteries to L-cysNO. A: Representative tracings from one animal comparing KCl-mediated contraction in mesenteric (Mes) and femoral (Fem) arteries in the presence of 10 μmol L⁻¹ nifedipine; B: Comparison of tensions in mesenteric and femoral arteries in the presence of 10 μmol L⁻¹ nifedipine at 15 min after contraction by KCl, expressed in relation to maximum contraction from an initial stimulation of KCl, showing that the L-Ca²⁺ channel blocker significantly decreases KCl-mediated contraction in mesenteric arteries but not femoral. C: Response curves to L-cysNO in mesenteric and femoral arteries, preconstricted with 5-HT, in the presence of 10 μmol L⁻¹ nifedipine or D: 10 μmol L⁻¹ diltiazem. Both nifedipine and diltiazem eliminate the differential vasodilation between mesenteric and femoral arteries principally by attenuating the dilation of the mesenteric arteries. (n ≥ 5; ***p < 0.001 mesenteric vs. femoral).

Fig. 5.

Role of actin polymerization and the BK_Ca channel in the differential vasodilatory response of adult sheep mesenteric and femoral arteries to L-cysNO. A: L-cysNO response curves in mesenteric (Mes) and femoral (Fem) arteries, preconstricted with KCl, in the presence of 1 μmol L⁻¹ cyto-D, an actin polymerization blocker; B: L-cysNO response curves in mesenteric and femoral arteries, preconstricted with 5-HT, in the presence of 1 mmol L⁻¹ TEA, a BK_Ca channel inhibitor. Note that neither cyto-D nor TEA eliminates the differential vasodilation between the two vessel types. (n ≥ 5; *p < 0.05 mesenteric vs. femoral.)

Fig. 6.

Expression of L-Ca²⁺ channel and sGC in adult sheep mesenteric and femoral arteries. A: The representative stains for the L-Ca²⁺ channel, sGC, and total protein in mesenteric and femoral arteries. Abundance of the L-Ca²⁺ channel, measured via Western blot, is higher in the mesenteric (Mes) than in the femoral (Fem) artery when normalized to either total protein (B) or sGC (D) (n = 4; *p < 0.05). Abundance of sGC (C) does not differ between mesenteric and femoral arteries.