1 The effect of depleting intracellular Ca 2 þ stores on flow-induced vascular dilatation and the mechanism responsible for the vasodilatation were examined in rat isolated small mesenteric arteries. 2 The arteries were pressurized to 50 mmHg and preconstricted with phenylephrine. Intraluminal flow reversed the effect of phenylephrine, resulting in vasodilatation. Flow dilatation consisted of an initial transient peak followed by a sustained plateau phase. The magnitude of dilatation was markedly reduced by removing Ca 2 þ from the intraluminal flow medium. 3 Depletion of intracellular Ca 2 þ stores with either cyclopiazonic acid (CPA, 2 mM) or 1,4-dihydroxy-2,5-di-tert-butylbenzene (BHQ, 10 mM) significantly augmented the magnitude of flow dilatation. Flow-induced endothelial cell Ca 2 þ influx was also markedly enhanced in arteries pretreated with CPA or BHQ. 4 Flow-induced dilatation was insensitive to N w -nitro-L-arginine methyl ester (100 mM) plus indomethacin (3 mM) or to oxyhemoglobin (3 mM), but was markedly reduced by 30 mM extracellular K þ or 2 mM tetrabutylammonium (TBA), suggesting an involvement of EDHF. 5 Catalase at 1200 U ml À1 abolished the flow-induced dilatation, while the application of exogenous H 2 O 2 (90-220 mM) induced relaxation in phenylephrine-preconstricted arteries. Relaxation to exogenous H 2 O 2 was blocked in the presence of 30 mM extracellular K þ , and H 2 O 2 (90 mM) hyperpolarized the smooth muscle cells, indicating that H 2 O 2 can act as an EDHF. 6 In conclusion, flow-induced dilatation in rat mesenteric arteries can be markedly enhanced by prior depletion of intracellular Ca 2 þ stores. Furthermore, these data are consistent with a role for H 2 O 2 as the vasodilator involved.

Shear stress, the frictional drag on the endothelium from blood flow, is a major determinant of vascular physiology and pathology. Due to the pulsatile nature of blood flow, blood vessels are subjected to significant variations in mechanical forces. Endothelial cells situated at the interface between blood and the vessel wall play a crucial role in detecting and responding to the mechanical forces generated by shear stress. Multiple sensing mechanisms are used by endothelial cells to detect changes in mechanical forces, leading to the activation of signaling networks. This review attempts to bring together recent findings on the mechanosensors present in the endothelial cells, and the regulation of endothelium-derived vasoactive autacoid production by shear stress.

Aim: To test the possible involvement of TRPC3 in agonist-induced relaxation and flow-induced vasodilation in rat small mesenteric arteries. Methods: Male Sprague-Dawley rats were used in the present study. After 72 h-treatment of antisense oligo via tail vein injection, isometric tension and isobaric diameter measurement were carried out with isolated mesenteric artery segments by using either a Pressure Myograph or a Multi Myograph system. Endothelial [Ca 2+ ] i changes were measured with a MetaFluor imaging system in response to flow or to 30 nmol/L bradykinin. Results: Immunohistochemical study showed that the 72 h-treatment of antisense oligo via tail vein injection markedly decreased the TRPC3 expression in mesenteric arteries, indicating the effectiveness of the antisense oligo. Isometric tension and isobaric diameter measurement showed that, although the antisense oligo treatment did not affect histamine-, ATP-, and CPA-induced relaxation, it did reduce the magnitude of flow-induced vasodilation by approximately 13% and decreased bradykinin-induced vascular relaxation with its EC 50 value raised by nearly 3-fold. Endothelial [Ca 2+ ] i measurement revealed that treatment of the arteries with antisense oligos significantly attenuated the magnitude of endothelial [Ca 2+ ] i rise in response to flow and to 30 nmol/L bradykinin. Conclusion:The results suggest that TRPC3 is involved in flow-and bradykinininduced vasodilation in rat small mesenteric arteries probably by mediating the Ca 2+ influx into endothelial cells.

Abstract:We have previously demonstrated that hydrogen peroxide (H 2 O 2 ) mediates flow-induced vascular dilation in rat small mesenteric arteries. In the present study, the source of this flow-induced H 2 O 2 was explored. The arteries were pressurized to 50 mm Hg and preconstricted with phenylephrine. Intraluminal flow reversed the effect of phenylephrine, resulting in vascular dilation. Cytochrome P450 (CYP) inhibitors N-methylsulfonyl-6-(2-proparglyoxyphenyl) hexanoic acid (MS-PPOH, 10 M) and miconazole (30 M) reduced the magnitude of peak flow dilation by ~20%-30%, and reduced the duration of dilatory response by ~70-80%. Nevertheless, sulphaphenazole (10 M), a selective inhibitor of CYP 2C9, had no effect neither on the peak flow dilation nor the duration of dilatory response. Oxypurinol (100 M), an inhibitor of xanthine oxidase, attenuated the duration of dilatory response by ~60% but exerted no effect on the magnitude of peak flow dilation. Cyclosporin A (2 M), an inhibitor for mitochondrial permeability transition pore, MitoQ (300 nM), a mitochondria-targeted antioxidant, and apocynin (1 mM), a NADPH oxidase inhibitor, had no effect neither on the magnitude of peak flow dilation nor the duration of dilatory response. To further confirm the role of CYP in flow-induced H 2 O 2 production, a fluorescent probe CM-H 2 DCFDA was used to monitor the production of H 2 O 2 in the primary endothelial cells isolated from rat small mesenteric arteries. The results showed that flow-induced H 2 O 2 production was markedly reduced in MS-PPOH and miconazole pretreated endothelial cells. Taken together, our results suggest that, during flow dilation, H 2 O 2 is generated from CYP epoxygenases and xanthine oxidase.