Heme oxygenase immunoreactive neurons in the rat intestine and their relationship to nitrergic neurons

Abstract

Background: Carbon monoxide (CO), like nitric oxide (NO), is a putative gaseous neurotransmitter. CO is produced by the enzyme heme oxygenase (HO) acting on a family of heme-containing compounds. Two isomers of HO have been characterized (HO-1, HO-2). In the CNS and in peripheral ganglia HO-2 occurs in a majority of neurons. NO and CO function as transmitters of enteric neurons but the relative distribution of enteric neurons utilizing these gaseous transmitters is unknown in rodent. We have studied the distribution of HO-2 immunoreactivity and NO synthase (NOS) activity within the rat ileum. Methods: Tissue sections and primary neuronal cell cultures were incubated with a HO-2 specific antibody, and then assessed or reprocessed for NOS activity using NADPH-dependent diaphorase staining. Results: HO-2 immunoreactivity was expressed in subpopulations of myenteric and submucosal neurons. Approximately 45% of the ganglion cells in tissue section were HO-2 positive. This was similar in proportion to those found to stain for NOS activity, and 10% of HO-2 positive neurons also contained NOS. HO-2 immunoreactivity was also found in epithelial cells within the villi, and in interstitial cells around the myenteric plexus and within the smooth muscle. In culture, the distribution and colocalisation of HO-2 and NOS positive neurons was similar to that in tissue sections. We identified labelled neurons as either Dogiel Type I or II; only Type II cells colocalized NOS and HO-2. Conclusion: Neurons, endocrine-like cells and interstitial cells with the capacity for CO production are distributed throughout the ileum and some neurons have the capacity to synthesize both NO and CO as gaseous messengers.

Introduction

Nitric oxide (NO) has become recognized as a gaseous neurotransmitter in the central and peripheral nervous system (Knowles et al., 1989; Bredt et al., 1990; Snyder et al., 1998), and growing evidence suggests that carbon monoxide (CO) acts as an analogous gaseous transmitter (Maines, 1988, Maines, 1993; Verma et al., 1993; Dawson and Snyder, 1994; Snyder et al., 1998). Both NO and CO exert their effect by activating soluble guanyl cyclase in the target cell, leading to an increase in intracellular cGMP (Brune and Ullrich, 1987; Maines et al., 1993). There is a large body of literature regarding the role of NO in neural function in the central, peripheral and enteric nervous system (Boeckxstaens et al., 1991; Moncada et al., 1991; Sneddon and Graham, 1992; Rodeberg et al., 1995). However, the functional significance of CO as a possible signal molecule is less well understood. CO is proposed to mediate neural control of opossum anal sphincter (Rattan and Chadker, 1993), relaxation of human jejunum (Farrugia et al., 1993) and mouse ileum (Zachary et al., 1997). In addition, CO may be mediator of inhibitory non-adrenergic non-cholinergic (nanc) neurons controlling feline lower esophageal sphincter (Ny et al., 1995).

CO is produced in mammalian tissue via the action of heme oxygenase (HO), which is the enzyme enabling the rate limiting step in the degradation of a family of heme-containing compounds (Maines, 1993). HO catalyses the breakdown of heme to CO, biliverdin and iron (Schacter, 1988; Maines, 1992, Maines, 1993) Heme has been shown to be present in most mammalian tissues and intracellular heme plays a number of important roles in cellular regulation such as regulation of gene expression, gas transport, and as the prosthetic group in many enzymes, including the cytochrome P-450 system (Padmanaban et al., 1989).

Two isomers of HO, HO-1 and HO-2 appear to be the main enzymes for CO production. They are encoded by different genes and differ extensively in their molecular and biological properties (Cruse and Maines, 1988). HO-1 is identified as a heat shock protein (expressed in response to cellular stress), (Ewing and Maines, 1991) whereas HO-2 is constitutive (Ewing and Maines, 1992). In the CNS, HO-2 is widely distributed and in peripheral ganglia HO-2 is present in most neurons while the CNS distribution of HO-1 is more limited (Vincent et al., 1994; Vollerthun et al., 1996).

Studies in the CNS have also shown that only a small population of HO-2 positive neurons also express NO synthase (Vincent et al., 1994). Throughout the canine and feline gastrointestinal tracts, HO-2 immunoreactivity has been reported to be abundant in enteric nerve cell bodies and in non-neuronal cells of the smooth muscle layer (Ny et al., 1997; Zachary et al., 1997). In the mouse small intestine, Miller et al. (1993)report HO-2 labelling of non-neuronal cells which they characterized to be the Interstitial Cells of Cajal (ICC's). Immunohistochemical analysis of mouse intestine also demonstrated colocalization of nNOS and HO-2 in myenteric ganglia (Zachary et al., 1997). Preliminary data obtained in the human stomach and jejunum (Reed et al., 1996), suggests that less than 50% of myenteric neurons immunopostive for HO-2, colocalize NO synthase. We have now undertaken a study of the localisation of HO-2 in the enteric nervous system of the rat small intestine, as well as characterisation of these neurons colocalizing NOS.