Calcitonin gene-related peptide activates different signaling pathways in mesenteric lymphatics of guinea pigs

Abstract

The effects of calcitonin gene-related peptide (CGRP) on constriction frequency, smooth muscle membrane potential ( Vm), and endothelial Vm of guinea pig mesenteric lymphatics were examined in vitro. CGRP (1–100 nM) caused an endothelium-dependent decrease in the constriction frequency of perfused lymphatic vessels. The endothelium-dependent CGRP response was abolished by the CGRP-1 receptor antagonist CGRP-(8–37) (1 μM) and pertussis toxin (100 ng/ml). This action of CGRP was also blocked by the nitric oxide (NO) synthase inhibitor NG-nitro-l-arginine (l-NNA; 10 μM), an action that was reversed by the addition of l-arginine (100 μM). cGMP, adenylate cyclase, cAMP-dependent protein kinase (PKA), and ATP-sensitive K+ (KATP+) channels were all implicated in the endothelium-dependent CGRP response because it was abolished by methylene blue (20 μM), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (10 μM), dideoxyadenosine (10 μM), N-[2-( p-bromociannamylamino)-ethyl]-5-isoquinolinesulfonamide-dichloride (H89; 1 μM) and glibenclamide (10 μM). CGRP (100 nM), unlike acetylcholine, did not alter endothelial intracellular Ca2+ concentration or Vm. CGRP (100 nM) hyperpolarized the smooth muscle Vm, an effect inhibited by l-NNA, H89, or glibenclamide. CGRP (500 nM) also caused a decrease in constriction frequency. However, this was no longer blocked by CGRP-(8–37). CGRP (500 nM) also caused smooth muscle hyperpolarization, an action that was now not blocked by l-NNA (100 μM). It was most likely mediated by the activation of the cAMP/PKA pathway and the opening of KATP+ channels because it was abolished by H89 or glibenclamide. We conclude that CGRP, at low to moderate concentrations (i.e., 1–100 nM), decreases lymphatic constriction frequency primarily by the stimulation of CGRP-1 receptors coupled to pertussis toxin-sensitive G proteins and the release of NO from the endothelium or enhancement of the actions of endogenous NO. At high concentrations (i.e., 500 nM), CGRP also directly activates the smooth muscle independent of NO. Both mechanisms of activation ultimately cause the PKA-mediated opening of KATP+ channels and resultant hyperpolarization.