Cell Tissue Res (1994) 275"513-527 Cell &Tissue Research 9 Springer-Verlag 1994 Localization of nitric oxide synthase in canine ileocolonic and pyloric sphincters Sean M. Ward, Chun Xue, Kenton M. Sanders Department of Physiology, University of Nevada School of Medicine, Reno, NV 89557~0046, USA Received: 4 January 1993 / Accepted: 5 July 1993 Abstract. The distribution of neurons containing NAD- PH-diaphorase (NADPH-d) activity and nitric oxide synthase-like immunoreactivity (NOS-LI) in the canine pyloric and iteocolonic sphincters was studied. Cells within the myenteric and submucosal ganglia were posi- tive for NADPH-d. These cells generally had the mor- phology of Dogiel type-I enteric neurons, however, there was some diversity in the morphology of NADPH-d-pos- itive neurons in the myenteric plexus of the pylorus. In- tramuscular ganglia were observed in both sphincters, and NADPH-d was found in a sub-population of neu- rons within these ganglia. Dual staining with an anti- serum raised against nitric oxide synthase (NOS) demon- strated that almost all cells with NOS-LI were also NADPH-d positive. Varicose fibers within ganglia and within the circular and longitudinal muscle layers also possed NOS-LI and NADPH-d activity. Dual staining with anti-VIP antibodies showed that some of the NAD- PH-d-positive cells in the myenteric and submucosal ganglia also contained VIP-LI, but all VIP-LI-positive cells did not express NADPH-d activity. These data are consistent with recent physiological studies suggesting that nitric oxide serves as an inhibitory neurotransmitter in the pyloric and ileocolonic sphincters. The data also suggest that VIP is expressed in a sub-population of NADPH-d-positive neurons and may therefore act as a co-transmitter in enteric inhibitory neurotransmission to these specialized muscular regions. Key words: Nitric oxide synthase - NADPH-diaphorase Gastroduodenal sphincter -Ileocolonic sphincter - En- teric nervous system - Gastrointestinal tract - Dog Introduction The canine pyloric and ileocolonic (ICS) sphincters are regions of the gastrointestinal tract in which the circular muscle layer thickens, providing distinct anatomical sep- Correspondence to: K. M. Sanders arations between the terminal antrum and proximal duo- denum, and between the terminal ileum and proximal colon, respectively. The pylorus and ICS regulate gastric and ileal emptying and inhibit reflux of luminal contents from the duodenum and colon by the maintenance of tone in the circular muscle layer that creates a basal in- traluminal pressure (Behar et al. 1979; Hinder and San- Garde 1983; Ehrlein 1988; Phillips et. al. 1988). This im- pedes flow through these sphincters (Hertz 1913; Hinder and San-Garde 1983; Quigley et al. 1983, 1984, 1985; Schulze-Delrieu and Brown 1985; Kruis et al. 1987; Ehrlein 1988). A specialized nerve supply and various humoral factors affect the tone of the muscle and regulate flow (Allescher et al. 1988; Papasova 1989; Vogalis and Sanders 1990). Local neural reflexes in response to a vari- ety of stimuli regulate the contractile state of these sphincters. Among the major classes of intrinsic nerves supplying the canine ICS and pyloric sphincteric regions are: cholinergic excitatory nerves (Allescher et al. 1988; Vogalis and Sanders 1990; Ward et al. 1992 a); non- adrenergic, non-cholinergic (NANC) inhibitory nerves (Boeckxstaens et al. 1990, 1991; Bult et al. 1990; Allescher et al. 1992; Bayguinov and Sanders 1993) and non-cholinergic excitatory nerves (Ludtke et al. 1963; Edin et al. 1980). Recent work has suggested that nitric oxide (NO) is likely to mediate enteric inhibitory (NANC) neurotrans- mission in the pyloric (Bayguinov and Sanders 1993) and ileocolonic (Bult et al. 1990; Boeckxstaens et al. 1990, 1991; Ward et al. 1992 a) sphincters of the dog. Numer- ous biochemical and pharmacological studies have sup- ported a role for NO as a transmitter, but it is also impor- tant to establish that enteric nerves supplying these sphincters express the enzymatic apparatus necessary to synthesize NO. Nerves containing nitric oxide synthase (NOS) have been found elsewhere in the gastrointestinal tract (Bredt et al. 1990; Belai et al. 1992; Furness et al. 1992; Ward et al. 1992 b; Young et al. 1992), but similar reports describing NOS-containing neurons in the py- loric and ileocolonic sphincters are not available. Expres- sion of NOS has mainly been demonstrated by immuno- histochemical studies, but it was recently shown that neu-