The peptide hormone relaxin is a potent vasodilator with therapeutic potential in diseases complicated by vasoconstriction, including heart failure. However, the molecular mediators and magnitude of vasodilation may vary according to duration of exposure and artery type. The objective of these studies was to determine mechanisms of rapid (within minutes) relaxin-induced vasodilation and to examine whether relaxin dilates arteries from different animal species and vascular beds. Rat and mouse small renal, rat mesenteric, and human sc arteries were isolated, mounted in a pressure arteriograph, and treated with recombinant human relaxin (rhRLX; 1-100 ng/ml) after preconstriction with phenylephrine. Rat and mouse small renal as well as human sc arteries dilated in response to rhRLX, whereas rat mesenteric arteries did not. Endothelial removal or pretreatment with l-N(G)-monomethyl arginine (L-NMMA) abolished rapid relaxin-induced vasodilation; phosphatidylinositol-3-kinase (PI3K) inhibitors also prevented it. In cultured human endothelial cells, rhRLX stimulated nitric oxide (assessed using 4-amino-5-methylamino-2'7'-difluorofluorescein) as well as Akt and endothelial NO synthase (eNOS) phosphorylation by Western blotting but not increases in intracellular calcium (evaluated by fura-2). NO production was attenuated by inhibition of Gα(i/o) and Akt (using pertussis toxin and the allosteric inhibitor MK-2206, respectively), PI3K, and NOS. Finally, the dilatory effect of rhRLX in rat small renal arteries was unexpectedly potentiated, rather than inhibited, by pretreatment with the vascular endothelial growth factor receptor inhibitor SU5416. We conclude that relaxin rapidly dilates select arteries across a range of species. The mechanism appears to involve endothelial Gα(i/o) protein coupling to PI3K, Akt, and eNOS but not vascular endothelial growth factor receptor transactivation or increased calcium.

Relaxin is emerging as an important vasodilator of pregnancy, and is being tested for afterload reduction in acute heart failure. However, the mechanisms underlying relaxin-induced vasodilation are incompletely understood. The aims of this study were to establish a new in vitro model for relaxin-induced vasodilation, and to use this approach as well as chronically instrumented, conscious rats to investigate the role of angiogenic growth factors in the relaxin vasodilatory pathway. Incubation of rat and mouse small renal arteries with recombinant human H2 relaxin for 3hr in vitro attenuated myogenic constriction, which was blocked by inhibitors of gelatinases, the endothelin B receptor and nitric oxide synthase. These findings corroborate ex vivo observations in arteries isolated from relaxin-infused nonpregnant and midterm pregnant rats, thereby validating the new experimental approach and enabling study of human arteries. Incubation of small human subcutaneous arteries with relaxin for 3hr in vitro also attenuated myogenic constriction through the same molecular intermediates. Vascular endothelial growth factor receptor inhibitor SU5416, three different vascular endothelial growth factor and two different placental growth factor neutralizing antibodies prevented relaxin from attenuating myogenic constriction in rat and mouse small renal, and human subcutaneous arteries. SU5416 administration also prevented relaxin-induced renal vasodilation and hyperfiltration in chronically instrumented, conscious rats. Small renal arteries isolated from these rats demonstrated increased MMP-2 activity in the relaxin-infused group, which was not prevented by SU5416. We conclude that there is concordance of relaxin vasodilatory mechanisms in rats, mice and humans, and angiogenic growth factors are novel and essential intermediates.