Diphenhydramine inhibits acetylcholine-induced, nitric oxide-mediated vasodilation in isolated rat aortas

Nitric oxide (NO), stimulated by the muscarinic receptor agonist acetylcholine (ACh) released from the endothelium via mitochondrial acetyl-CoA production, appears to contribute to physiological flow-mediated vasodilation. Diphenhydramine (DPH), a muscarinic receptor antagonist, may interfere with ACh-induced NO-mediated vasodilation. However, whether DPH affects ACh-induced, NO-mediated vasodilation remains unknown. This study aimed to examine the effect of DPH on ACh-induced, NO-mediated vasodilation and its underlying mechanisms, including muscarinic receptor subtypes. The effects of diphenhydramine, M₃ muscarinic receptor antagonist 4-diphenylacetoxyl-N-methylpiperidine methiodide (4-DAMP), and nitric oxide synthase (NOS) inhibitor N^w-nitro-l-arginine methyl ester (l-NAME) on ACh-induced vasodilation in endothelium-intact aortas were examined. Furthermore, the study evaluated the effects of DPH on vasodilation induced by the non-receptor-mediated endothelium-dependent vasodilator calcium ionophore A23187 and sodium nitroprusside in endothelium-intact aortas. The influence of DPH on ACh-induced cyclic guanosine monophosphate (cGMP) formation, endothelial nitric oxide synthase (eNOS) phosphorylation, calcium elevation, and nitrite (NO metabolite) production was further assessed in endothelium-intact aortas and human umbilical vein endothelial cells. DPH markedly inhibited ACh-induced vasodilation, whereas l-NAME almost abolished it. Pretreatment with 4-DAMP nearly eliminated the DPH-mediated inhibition of ACh-induced vasodilation. DPH slightly inhibited vasodilation induced by calcium ionophore A23187 and sodium nitroprusside. The DPH-induced inhibition of ACh-induced vasodilation was greater than the inhibition observed with calcium ionophore A23187 or sodium nitroprusside. DPH inhibited ACh-induced cGMP formation, eNOS phosphorylation, endothelial calcium elevation, and nitrite production. Overall, these results suggest that supratherapeutic DPH concentrations inhibit ACh-induced, NO-mediated vasodilation, likely primarily through inhibition of endothelial M₃ muscarinic receptors.