Mechanism of 5‐Hydroxytryptamine Receptor 4 Mediated Vasorelaxation in the Isolated Bovine Lateral Saphenous Vein

ABSTRACT To better understand the mechanisms of 5‐hydroxytryptamine receptor 4 (HTR4)‐mediated vasorelaxation, lateral saphenous veins from cattle ( n = 4 to 7) were collected and assessed for vasoactivity in response to increasing concentrations of a selective HTR4 agonist (BIMU 8) in the absence and presence of inhibitors selective for downstream proteins involved with cyclic nucleotide‐mediated signaling. Vessels were pre‐contracted with 1 × 10 −4 M phenylephrine and exposed to increasing HTR4 agonist concentrations. Vasoactive response data were normalized as a percentage of the maximum contractile response induced by the phenylephrine pre‐contraction. Antagonism of HTR4 attenuated the vasorelaxation induced by BIMU 8. Removal of the endothelium did not influence HTR4 mRNA expression, HTR4 protein expression, or HTR4‐mediated vasorelaxation. In the presence of inhibitors selective for adenylate cyclase or guanylate cyclase, 66% and 87% of the maximal HTR4‐mediated vasorelaxation response was attenuated. In the presence of inhibitors selective for protein kinase A or protein kinase G, 66% and 98% of the maximal HTR4‐mediated vasorelaxation response was attenuated. In the presence of non‐selective blockers of Ca 2+ and K + channels, 65% and 64% of the maximal HTR4‐mediated vasorelaxation response was attenuated. In the presence of inhibitors selective for myosin light chain phosphatase and small heat shock protein phosphorylation, 72% and 57% of the maximal HTR4‐mediated vasorelaxation response was attenuated. HTR4‐mediated vasorelaxation occurs through an endothelium‐independent, cyclic nucleotide‐dependent mechanism. Downstream proteins involved in the signaling cascade leading to vasorelaxation include protein kinase A and G, Ca 2+ channels, K + channels, myosin light chain phosphatase, and small heat shock proteins.