16K-Prolactin inhibits activation of endothelial nitric oxide synthase, intracellular calcium mobilization, and endothelium-dependent vasorelaxation

Activation of endothelial nitric oxide synthase ( eNOS) and subsequent nitric oxide production ( NO) are events that mediate the effect of important angiogenic, vasopermeability, and vasorelaxation factors, including vascular endothelial growth factor (VEGF), bradykinin (BK), and acetylcholine (ACh). The N-terminal 16-kDa fragment of prolactin (16K-PRL) acts on endothelial cells to inhibit angiogenesis both in vivo and in vitro. Here, we show that 16K-PRL inhibits VEGF-induced eNOS activation in endothelial cells. Inhibition of eNOS activation may mediate the antiangiogenic properties of 16K-PRL, because the NO donor (Z)-1-[2-(2-aminoethyl)N-( 2-ammonio-ethyl) amino] diazen-1-ium-1,2- diolate ( DETANONOate) prevented 16K-PRL from blocking the VEGF-induced proliferation of endothelial cells. In addition, 16K-PRL inhibited eNOS activation by BK and blocked the BK-evoked transient increase in intracellular Ca2+ in endothelial cells. This finding suggests that 16K-PRL interferes with the mobilization of intracellular Ca2+, thereby inhibiting the Ca2+-dependent activation of eNOS. Blockage of eNOS activation can lead to inhibition of vasodilation. Consistently, 16K-PRL inhibited BK-induced relaxation of coronary vessels in isolated perfused guinea pig hearts. Moreover, 16K-PRL inhibited eNOS activation induced by ACh, and this action resulted in the inhibition of both ACh-evoked relaxation of coronary vessels in isolated perfused rat hearts and ACh-induced, endothelium-dependent relaxation of rat aortic segments. In conclusion, 16K-PRL can block the Ca2+-mediated activation of eNOS by three different vasoactive substances, and this action results in the inhibition of both angiogenesis and vasorelaxation.