Role of Nitric Oxide in Anorectal Function of Normal and Neuronal Nitric Oxide Synthase Knockout Mice

Abstract

PURPOSE: In vitro data suggest that nitric oxide is an important inhibitory neurotransmitter in the internal anal sphincter, and morphologic evidence implies that it mediates the rectoanal inhibitory reflex. This study examined the anatomy, physiology, and pharmacology of the internal sphincter in control and neuronal nitric oxide synthase knockout mice. METHODS: Neuronal nitric oxide synthase, nicotinamide adenosine triphosphate dinucleotide phosphate diaphorase histochemistry, and PGP 9.5 immunohistochemistry were compared between knockout and sibling control mice. Anorectal manometry was performed with a balloon-tipped water-perfused catheter. In vitro studies were performed on both whole internal anal sphincter rings and strips. RESULTS: Staining of the myenteric plexus and nerves traversing the internal anal sphincter in sibling control mice demonstrated the presence of neuronal nitric oxide synthase and nicotinamide adenine dinucleotide phosphate diaphorase at these sites. These markers were absent in knockout mice. Maximum anal resting pressure was similar in control and knockout mice (15.6 ± 2.6 cm H2O (n = 4) vs. 14.0 ± 2.3 cm H2O (n = 7)). The rectoanal inhibitory reflex was present in all control mice (n = 4) but in only four of seven knockout mice. Field stimulation with parameters designed to activate inhibitory nerves produced relaxation of internal sphincter tissue from both control and knockout mice, which was partially attenuated in control mice only by pretreatment with the nitric oxide synthase inhibitor Nω-nitro-L-arginine. Further inhibition of nerve-induced relaxation in control mice was achieved with antagonists of vasoactive intestinal peptide, adenosine triphosphate, and heme oxygenase. CONCLUSIONS: Although in the normal mouse, nitric oxide is an inhibitory neurotransmitter in the internal sphincter, other transmitters may play a role in the rectoanal inhibitory reflex. These other inhibitory neurotransmitters can apparently compensate for the absence of nitric oxide synthase in knockout mice to maintain approximately normal function.