Activation of eNOS by D-pinitol Induces an Endothelium-Dependent Vasodilatation in Mouse Mesenteric Artery

Abstract

D-pinitol is a cyclitol present in several edible plant species and extensively investigated for the treatment of metabolic diseases in humans, as food supplement, and demonstrated protective effects in the cardiovascular system. For these reasons, the present work aimed at investigating the mechanisms involved in the vascular effects of D-pinitol in mouse mesenteric artery. Mesenteric arteries from male C57BL/6 mice were mounted in a wire myograph. Nitrite was measured by the 2,3-diaminonaphthalene (DAN) method. Protein expression and phosphorylation were measured by Western blot. The systolic blood pressure (SBP) was measured by tail-cuff plethysmography. D-pinitol induced a concentration-dependent vasodilatation in endothelium-intact, but not in endothelium-denuded arteries. Nω-Nitro-L-arginine methyl ester (300 μM) abolished the effect of D-pinitol, while 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 μM) shifted the concentration-response curve to the right. KN-93 (1 μM) blunted the vasodilator effect of D-pinitol, but H-89 (0.1 μM) did not change it. 1-[2-(Trifluoromethyl) phenyl]imidazole (300 μM), indomethacin (10 μM), celecoxib (5 μM), wortmannin (1 μM), ruthenium red (10 μM), tiron (10 μM), MnTMPyP (30 μM), MPP (0.1 μM), PHTPP (0.1 μM), and atropine (1 μM) did not change the effect of D-pinitol. D-pinitol increased the concentration of nitrite, which was inhibited by L-NAME and calmidazolium (10 μM). D-pinitol increased the phosphorylation level of eNOS activation site at Ser¹¹⁷⁷ and reduced the phosphorylation level of its inactivation site at Thr⁴⁹⁵. In normotensive mice, the intraperitoneal administration of D-pinitol (10 mg/kg) induced a significant reduction of the SBP after 30 min. The present results led us to conclude that D-pinitol has an endothelium- and NO-dependent vasodilator effect in mouse mesenteric artery through a mechanism dependent on the activation of eNOS by the calcium-calmodulin complex, which can explain its hypotensive effect in mice.

Keywords: D-pinitol; calcium-calmodulin complex; endothelium; mesenteric artery; nitric oxide synthase.

FIGURE 1

Endothelium- and nitric oxide-dependent vasodilator effect of D-pinitol in mice mesenteric artery. The concentration-dependent vasodilator effect of D-pinitol was investigated in the presence (E+) or absence (E-) of a functional endothelium (A), in the presence of L-NAME (B), TRIM (C), and ODQ (D). The inset demonstrates a representative trace of the concentration-dependent vasodilator effect of D-pinitol, where each mark represent a concentration. All results are expressed as mean ± SEM of five experiments. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001 versus E+.

FIGURE 2

Cyclooxygenase 1 and 2, phosphatidylinositol 3-kinase, and transient receptor potential channels are not involved in the vasodilator effect of D-pinitol in mice mesenteric arteries. The concentration-dependent vasodilator effect of D-pinitol was investigated in mesenteric arteries with a functional endothelium in the absence (Control) and presence of indomethacin (A), celecoxib (B), wortmannin (C), and ruthenium red (D). All results are expressed as mean ± SEM of five experiments.

FIGURE 3

Ca²⁺/calmodulin-dependent kinase II, but not protein kinase A, is involved in the vasodilator effect of D-pinitol in mice mesenteric arteries. The concentration-dependent vasodilator effect of D-pinitol was investigated in mesenteric arteries with a functional endothelium in the absence (control) and presence of KN-93 (A) and H-89 (B). All results are expressed as mean ± SEM of five experiments. ^∗∗∗P < 0.001 versus respective control.

FIGURE 4

The vasodilator effect of D-pinitol in mice mesenteric arteries is not associated with the formation of reactive oxygen species. The concentration-dependent vasodilator effect of D-pinitol was investigated in mesenteric arteries with a functional endothelium in the absence (control) and presence of tiron (A) or MnTMPyP (B). All results are expressed as mean ± SEM of five experiments.

FIGURE 5

D-pinitol increases the formation of nitrite through a mechanism dependent on the activation of endothelial nitric oxide synthase (eNOS) by the calcium-calmodulin complex. The formation of nitrite was measured in endothelium-intact mesenteric arteries in the absence (basal) or presence of D-pinitol (control/ A) or acetylcholine (ACh; control; B). The involvement of eNOS and calmodulin was evaluated in mesenteric arteries stimulated with D-pinitol or ACh in the presence of L-NAME and calmidazolium, respectively. All results are expressed as mean ± SEM of five experiments. ^∗∗∗P < 0.001 versus basal. ^†P < 0.05, and ^†††P < 0.001 versus control.

FIGURE 6

D-pinitol activates the endothelial nitric oxide synthase (eNOS) phosphorylating and dephosphorylating its respective activation and inactivation sites. Western-blots for time-course of the phosphorylation level of Ser1177 (A), the activation site of eNOS, and the dephosphorylation level of Thr495 (B), the inactivation site of eNOS, were performed in endothelium-intact mesenteric arteries, in the absence (basal) and presence of D-pinitol (20 μM). All results are expressed as mean ± SEM of five experiments. ^∗P < 0.05 and ^∗∗P < 0.01 versus basal.

FIGURE 7

D-pinitol (10 mg/kg; i.p.) reduces the systolic blood pressure (SBP) in normotensive mice. All results are expressed as mean ± SEM of six experiments. ^∗∗P < 0.01 versus saline at 30 min. ^††P < 0.01 versus the SBP measured before the administration of D-pinitol.