Mechanisms Associated to Nitroxyl (HNO)-Induced Relaxation in the Intestinal Smooth Muscle

The pharmacological properties of nitroxyl (HNO) donors in the gastrointestinal tract are unknown. We investigated the properties of this molecule in the regulation of gastrointestinal contractility focusing on its possible interaction with other gaseous signaling molecules such as NO and H₂S. Organ bath, Ca²⁺ imaging, and microelectrode recordings were performed on rat intestinal samples, using Angeli's salt as HNO donor. Angeli's salt caused a concentration-dependent relaxation of longitudinal or circular muscle strips of the ileum and the proximal colon. This relaxation was strongly inhibited by the Rho-kinase inhibitor Y-27632 (10 μM), by the reducing agent DTT or by the inhibitor of soluble guanylate cyclase (sGC) ODQ (10 μM) alone or in combination with the inhibitors of the endogenous synthesis of H₂S β-cyano-L-alanine (5 mM) and amino-oxyacetate (5 mM). Preventing endogenous synthesis of NO by the NO synthase inhibitor L-NAME (200 μM) did not affect the relaxation induced by HNO. HNO induced an increase in cytosolic Ca²⁺ concentration in colonic myocytes. It also elicited myocyte membrane hyperpolarization that amounted to -10.6 ± 1.1 mV. ODQ (10 μM) and Apamin (1 μM), a selective inhibitor of small conductance Ca²⁺-activated K⁺ channels (SKca), strongly antagonized this effect. We conclude that HNO relaxes the gastrointestinal tract musculature by hyperpolarizing myocytes via activation of the sGC/cGMP pathway similarly to NO, not only inhibiting the RhoK and activating MLCP as do both NO and H₂S but also increasing cytosolic Ca²⁺ for activation of SK contributing to hyperpolarization.