Norepinephrine, EPI and DA activate their cognate G protein-coupled receptors expressed on neurons, epithelial cells and other types of target cells which influence overall mucosal function and alter intestinal susceptibility to infection. Receptors for NE and DA have been defined over several decades through the use of highly selective receptor agonists and antagonists in functional pharmacological investigations and more recently, through molecular cloning and structure-function studies in isolated cells and transgenic animals.

The receptor concept was developed in the early twentieth century on the translational approach of defining binding interactions of endogenous substances or their synthetic homologs with specific sites (“receptive substances”) expressed by target cells that are linked to a biological response (Rang 2006). Biochemical analyses of selective binding site interactions with catecholamine-like drugs and other ligands that were developed many years later have provided crucial information on the affinities (K_d, dissociation constant) and competitive interactions of catecholamines and their synthetic derivatives at specific binding sites. However, these biochemical studies do not measure the biological activity of the ligands examined, and therefore they do not truly define a “receptor”, which is an entity functionally coupled to intracellular signal transduction pathways that mediates a biological function. In other words, biochemical analyses of binding sites cannot distinguish between agonist and antagonist ligands. Binding studies, if carefully executed, can provide information on a specific, high-affinity binding site for an endogenous or synthetic ligand that can serve as supporting evidence for the presence of a true receptor in a biological system. Through GTPγ³⁵S binding assays (Harrison and Traynor 2003), it is possible to assess the effectiveness of ligands to activate G proteins coupled to a particular receptor and this approach affords a better approximation of ligand activity at the cellular level (e.g. it is possible to distinguish an agonist from an antagonist).

Functional criteria for a receptor-mediated effect include (1) selective antagonism, (2) a rank order of ligand potency (usually based on the 50 % effective concentration, EC₅₀ for agonists or a pK_B value for antagonists, which is determined through rightward shifts in an agonist concentration-effect relationship after pretreatment with a competitive antagonist) that is consistent with the pharmacological signature of a particular receptor type; and (3) stereospecificity (e.g. the levorotatory isomer of NE is much more potent than its dextrorotatory counterpart at ARs). The affinities and competitive interactions among ligands for ARs and dopamine receptors (DARs) measured by functional drug interactions are generally well correlated with the affinities and interactions of the same ligands determined biochemically at specific binding sites.

Presently, ARs are classified into alpha- and beta-AR types; this includes two subtypes of alpha-ARs (alpha ₁ and alpha ₂) having three isoforms each, and three subtypes of beta-ARs. An EPI-preferring beta ₂-AR was the first drug receptor to be cloned and characterized in the modern era of molecular pharmacology (Dixon et al. 1986). Compared to EPI, NE has relatively higher binding affinity for alpha-ARs and beta ₁– and beta ₃-ARs, but a lower affinity for beta ₂-ARs. Two main DAR types exist through gene duplication events in the vertebrate lineage (DA₁R and DA₂R); from these, five receptor subtypes have been defined. Our understanding of the nature of these receptors and their relationships to G proteins and downstream intracellular signaling cascades continues to grow. For example, there is accumulating evidence that ARs may form dimeric complexes on cell membranes that possess a pharmacological profile different from that of the monomeric receptor (Verburg-van Kemenade et al. 2013). Moreover, beta-ARs can signal differentially through intracellular G proteins and beta-arrestin, and thereby produce different ligand-dependent biological effects (Bock et al. 2014). This should be kept in mind when designing experiments involving the prolonged contact of catecholamine agonists with their receptors, as long-term stimulation of the receptor can often result in its phosphorylation, which in turn can increase interactions of the receptor with arrestin or other intracellular proteins associated with receptor down-regulation. The investigator is then left with the question of whether the biological effects examined were the result of receptor activation or the loss of receptors from cell membranes.

As indicated above, pharmacologically-defined catecholamine receptors influencing epithelial function may be expressed on epithelial cells or on nerves innervating these cells. Specific, high-affinity binding sites for catecholamines have been detected on mucosal epithelial cells in some species, including the guinea pig and rat. These include alpha ₁-, alpha ₂– or beta-adrenergic binding sites as well as DA and non-adrenergic imidazoline binding sites (Chang et al. 1983; Nakaki et al. 1983; Cotterell et al. 1984; Paris et al. 1990; Senard et al. 1990; Valet et al. 1993; Baglole et al. 2005). Food deprivation appears to increase alpha ₂-AR binding sites on rat jejunal epithelial cells (Lucas-Teixeira et al. 2000) and pro-inflammatory cytokines and short-chain fatty acids generated by colonic bacteria may decrease the rate of alpha ₂-AR gene transcription and hence, the number of binding sites in human HT-29 colonic epithelial cells (Devedjian et al. 1996; Cayla et al. 2008).

In other species, alpha-ARs modulating epithelial functions appear to be expressed solely on nerve membranes, at least in some regions of the intestinal tract. Synaptosomal preparations enriched in ³H-saxitoxin binding sites were obtained from the submucous neural plexuses of the canine and porcine small intestine. In these neural preparations, saturation analyses indicated that highly-selective alpha2-AR antagonists bound specifically to these enteric neural membranes with a dissociation constant (K_D; a measure of ligand affinity) of 3.5 nM [³H-rauwolscine, dog; Ahmad et al. 1989] and 0.4 nM, respectively [³H-yohimbine, pig; Hildebrand et al. 1993]. In a study with porcine small intestine, specific binding of ³H-yohimbine to intestinal epithelial cells was minimal (Hildebrand et al. 1993). On the other hand, in the porcine colonic mucosa, the functional effects of catecholamines on electrolyte transport are mediated by alpha ₁-ARs and are resistant to the neural conduction blocker tetrodotoxin, indicating that these substances may act on epithelial cell receptors in this gut segment (Traynor et al. 1991). Whether or not specific alpha1-AR binding sites exist on colonic epithelial cell membranes in pigs has not been determined.

Both NE and DA have been shown to alter the mucosal attachment or invasiveness of bacterial pathogens such as enterohemorrhagic Escherichia coli (EHEC) or serovars of Salmonella enterica by acting on the intestinal mucosa. The actions of these catecholamines on bacteria-mucosa interactions have been examined in mucosal explants from murine or porcine intestine mounted in Ussing chambers (Brown and O’Grady 2008). This apparatus has been used for decades in studies of transepithelial ion transport and more recently, in investigations of bacteria on intestinal transport (Lomasney and Hyland 2013). The Ussing chamber system extends the viability of mucosal explants under quasi-physiological conditions, allows for tangential flow of bacteria across a fixed mucosal surface area, and permits the selective contact of drugs and bacteria with the luminal or contraluminal surfaces of intestinal tissues. Epithelial cells in this system retain viability and active transport properties for periods >3 h (Söderholm et al. 1998). Alternative systems for measuring bacterial interactions with mucosal tissue, such as in vitro organ culture techniques (Hicks et al. 1996), rely on static interactions between bacteria and cells, and have often neglected to confirm the viability of mucosal epithelial cells after several hours or days in culture.