Shear stress-induced ATP-mediated endothelial constitutive nitric oxide synthase expression in human lymphatic endothelial cells

To clarify the roles of lymphatic endothelial cells (LEC) in the regulation of endothelial constitutive nitric oxide synthase (ecNOS) expression, we examined the effects of shear stress on ecNOS immunohistochemical staining and mRNA and protein expression in human LEC as well as on ATP release from these cells. Shear stress at 0.5 or 1.0 dyn/[cm.sup.2] increased ecNOS immunohistochemical staining and ecNOS mRNA and protein expression in cultured LEC. The same strength of shear stress produced a significant release of ATP from the LEC. Exogenous ATP ranging in concentration from [10.sup.-9] to [10.sup.-6] M produced a significant increase in ecNOS immunohistochemical expression in a dose-dependent manner. The increase in ecNOS expression mediated by [10.sup.-6]M ATP was significantly reduced by [10.sup.-5] M suramin. Suramin ([10.sup.-5] M) caused a significant reduction in the shear stress-mediated increases in ecNOS immunohistochemical staining and mRNA expression. The shear stress-mediated increases in ecNOS expression were significantly reduced by 3 mM tetraethylammonium, [10.sup.-4] M apamin, [10.sup.-9] M iberiotoxin, [10.sup.-5] M 2-aminoethoxydephenyl borate, or [10.sup.-5]M xestospongin C, but not [10.sup.-5] M glybenclamide or [10.sup.-5] M nifedipine. The shear stress-mediated increases in ecNOS expression were significantly potentiated by pinacidil or NS1619 in a dose-dependent manner. The immunohistochemical expression of small--([SK.sub.Ca]) and big-conductance ([BK.sub.Ca]) [Ca.sup.2+]-activated [K.sup.+] channels was confirmed on the surfaces of human LEC. These findings suggest that shear stress produces a significant release of ATP from LEC, which activates the purinergic P2X/2Y receptor, thereby facilitating ecNOS mRNA and protein expression through inositol 1,4,5-trisphosphatemediated release of intracellular [Ca.sup.2+] ions and the activation of [Ca.sup.2+]-activated [K.sup.+] channels in LEC. lymph flow rate; [Ca.sup.2+]-activated [K.sup.+] channel; mRNA; 2-aminoethoxydephenyl borate; xestospongin C; apamin; iberiotoxin; pinacidil doi:10.1152/ajpcell.00249.2009