Carbon monoxide activates human intestinal smooth muscle L-type Ca²⁺ channels through a nitric oxide-dependent mechanism

Abstract

Carbon monoxide (CO) is increasingly recognized as a physiological messenger. CO is produced in the gastrointestinal tract with diverse functions, including regulation of gastrointestinal motility, interacting with nitric oxide (NO) to mediate neurotransmission. The aim of this study was to determine the effect of CO on the human intestinal L-type Ca 2ϩ channel expressed in HEK cells and in native cells using the patch-clamp technique. Extracellular solution contained 10 mM Ba 2ϩ as the charge carrier. Maximal peak Ba 2ϩ current (IBa) was significantly increased by bath application of 0.2% CO to transfected HEK cells (18 Ϯ 3%). The NO donor S-nitroso-N-acetylpenicillamine also increased IBa, and CO (0.2%) increased NO production in transfected HEK cells. The CO-induced increase in IBa was blocked when cells were pretreated with 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one (10 M) or inhibitors of NO synthase (NOS). The PKA inhibitor KT-5720 (0.5 M) and milrinone (3 M), a phosphodiesterase (PDE) III inhibitor, blocked the effect of CO on IBa. Similar effects were seen in freshly dissociated human intestinal smooth muscle cells. The data suggest that exogenous CO can activate native and heterologously expressed intestinal L-type Ca 2ϩ channels through a pathway that involves activation of NOS, increased NO, and cGMP levels, but not PKG. Rather, the pathway appears to involve PKA, partly by reducing cAMP breakdown through inhibition of PDE III. CO-induced NO production may explain the apparent discrepancy between the low affinity of guanylyl cyclase for CO and the robust cGMP production evoked by CO. ion channels; gases; human studies; patch clamp L-TYPE CA 2ϩ CHANNELS PLAY a central role in gastrointestinal smooth muscle contractile activity (18). Block of L-type Ca 2ϩ channels reduces or abolishes intestinal contractile activity. It is, therefore, not surprising that L-type Ca 2ϩ channel activity is tightly regulated in intestinal smooth muscle. Regulatory mechanisms involve Ca 2ϩ , pH, cyclic nucleotides, G proteins and a variety of extracellular ligands (5).Carbon monoxide (CO) is a low molecular weight gas that shares similar properties with another low molecular weight gas, nitric oxide (NO). CO, like NO, is generated under physiological conditions (26). The synthetic enzymes that produce CO, heme oxygenase 1 (HO1) and heme oxygenase 2 (HO2), are widely expressed in the gastrointestinal tract; thus CO is endogenously produced in the gastrointestinal tract (15,27,28). In the gastrointestinal tract, CO mediates nonadrenergic noncholinergic neurotransmission (43), sets the smooth muscle membrane potential gradient (43), and appears to protect against the development of postoperative ileus (29).Both CO and NO activate guanylyl cyclase resulting in the generation of cGMP (34). However, the affinity of CO to guanylyl cyclase is severalfold lower than for NO, suggesting that the effects of CO on guanylyl cylase may require a sensitizing molecule (37) or that other pathways may be involved. Both CO and NO can directly...

Carbon Monoxide Induces Cardiac Arrhythmia via Induction of the Late Na+ Current

“…In light of these studies, we propose that CO exposure likely stimulates nNOS in cardiac myocytes, causing a localized rise of NO, leading to nitrosylation, augmentation of the late Na 1 current, and consequent arrhythmias. This interpretation is consistent with the fact that the only known biologic targets of CO are hemecontaining proteins (37), such as NOS isoforms, and that CO has been proposed as an activator of NO production (38,39).…”

Carbon Monoxide: Endogenous Production, Physiological Functions, and Pharmacological Applications

“…It was thus concluded that the relaxant effect of CO on this tissue was not due to inhibition of L-type Ca 2ϩ channels. One recent study, on the other hand, demonstrated that CO activated L-type Ca 2ϩ channels in an NO-dependent fashion (Lim et al, 2005). Bath application of 0.2% CO or NO donor increased the maximal peak current (I Ba ) in transfected HEK cells.…”

Nitric oxide increases carbon monoxide production by piglet cerebral microvessels

“…In intestinal smooth muscle, CO increased NO that activated L-type Ca 2ϩ channels (19). Conversely, CO dose dependently inhibited NO synthesis by rat renal arteries, although low concentrations of CO actually increased NO by causing release from a preformed pool (38).…”