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

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 Ca²⁺ 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, Ca²⁺ channels, K⁺ channels, myosin light chain phosphatase, and small heat shock proteins.