Localization and activity of haem oxygenase and functional effects of carbon monoxide in the feline lower oesophageal sphincter.

1. In the feline lower oesophageal sphincter (LOS), the distribution of the carbon monoxide (CO) producing enzymes haem oxygenase (HO)-1 and -2 was studied by immunohistochemistry and confocal microscopy, the HO activity was measured and the possible role for CO as a mediator of relaxation was investigated. 2. HO-2 immunoreactivity was abundant in nerve cell bodies of the submucosal and myenteric plexus. Approximately 50% of the HO-2-containing myenteric cell bodies were also nitric oxide synthase- and vasoactive intestinal peptide (VIP)-immunoreactive. In addition, HO-2 immunoreactivity was seen in nerve fibres, in non-neuronal cells dispersed in the smooth muscle and in arterial endothelium. HO-1 immunoreactivity was confined to non-neuronal cells in the smooth muscle, similar to those positive for HO-2. 3. Activity of HO, measured as CO production, was observed in LOS homogenates at a rate of 1.00 +/- 0.05 nmol mg-1 protein h-1. This production was inhibited by the HO inhibitor, zinc protoporphyrin-IX (ZnPP). 4. In isolated circular smooth muscle strips of LOS, developing spontaneous tone, exogenously administered CO evoked a concentration-dependent relaxation reaching a maximum of 93 +/- 3%. This relaxation was accompanied by an increase in cyclic GMP, but not cyclic AMP levels. The relaxant response was attenuated by methylene blue, but unaffected by tetrodotoxin. Repeated exposure to CO resulted in a progressive reduction of the relaxant response. 5. ZnPP caused a rightward-shift of the concentration-response curves for the relaxant responses to VIP, peptide histidine isoleucine, and pituitary adenylate cyclase activating peptide 27. 6. ZnPP and tin protoporphyrin-IX (another inhibitor of HO) did not affect nonadrenergic, noncholinergic relaxations induced by electrical field stimulation. Nor did ZnPP affect relaxations induced by 3-morpholino-sydnonimine or forskolin. 7. The present findings, showing localization of HO immunoreactivity to both neuronal and nonneuronal cells of the feline LOS, ability of LOS to produce CO and a relaxant effect of CO in circular LOS muscle, suggest a role for CO as a peripheral messenger.