Enteric Nerves Catecholamines and IECBacteria Interactions

Both NE and DA have been shown to alter the mucosal attachment or invasiveness of bacterial pathogens such as EHEC or serovars of Salmonella enterica not always through direct contact with these bacteria, but rather by acting on cells of the intestinal mucosa (Table 5.1). The actions of these catecholamines on bacteria-mucosa interactions have been examined in mucosal explants 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-host interactions (Ding et al. 2001; Crane et al. 2006). This system extends the viability of mucosal explants under quasi-physiological conditions, allows for continuous, 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 (Fig. 5.2).

Table 5.1 Functional evidence for mucosal alpha-adrenergic receptors influencing EHEC adherence to explants of porcine cecal and colonic mucosae Pharmacological characteristic

Supporting evidence


Selective agonism

Selective antagonism

Laterality of drug action

At equimolar concentrations, UK14,304 (alpha2-adrenoceptor agonist), but neither phenylephrine (alpha1) nor isoproterenol (beta) increase EHEC adherence to cecal mucosa At equimolar concentrations in both cecum or colon and phentolamine (alpha-adrenoceptor antagonist), but not propranolol (beta), inhibits NE action. Furthermore, yohimbine (alpha2), but not prazosin (alpha1), inhibits NE action NE at low (pM) concentrations effective only when added to the contraluminal, but not luminal bathing medium, consistent with submucosal localization of adrenergic receptors

Fig. 5.2 Photograph of an Ussing chamber containing a porcine intestinal mucosa explant. Arrows indicate the locations of bacterial inoculations and drug additions as performed in many experiments such as those summarized in Table 5.1 Role of the ENS and Catecholamines in Bacterial Internalization into the Mucosa of the Small Intestine

The attachment and invasion of enteropathogenic bacteria to the intestinal mucosa appears to be modulated by the ENS. Inhibition of neural conduction by the sero-sal side addition of the neuronal sodium channel blocker saxitoxin increases inter-nalization of luminally inoculated Salmonella enterica serovar Choleraesuis and EHEC by >6-fold in Peyer's patch explants from the porcine jejunum. Internalization of a rodent commensal E. coli strain is unaffected by the toxin (Green et al. 2003) and that of S. enterica serovar Typhimurium is decreased by threefold (Brown and Price 2008). Serosal application of the neurotoxin or the local anesthetic lidocaine decreased S. Typhimurium internalization by three- to fourfold in explants of non-Peyer's patch absorptive mucosa from porcine jejunum. In contrast, electrical stimulation of enteric nerves in this preparation increased S. Typhimurium inter-nalization by 2.5-fold and this effect was inhibited by saxitoxin or lidocaine (Schreiber et al. 2007). Although these neurally mediated effects on Salmonella internalization may appear to be small, it should be emphasized that they were measured over a surface area of 2 cm2 in isolated tissues over a relatively short (90 min) time period. If extrapolated to the large surface area encompassed by the small intestine or even a segment thereof, these changes in Salmonella uptake are likely to be biomedically significant.

The cellular mechanisms underlying these neurally mediated effects on Salmonella internalization in the porcine small intestinal mucosa are undefined at present.

There is evidence that they differ for different serovars Choleraesuis and Typhimurium of S. enterica as saxitoxin increases internalization of the former and decreases that of the latter. Moreover, Salmonella internalization is inhibited by the actin polymerization inhibitor cytochalasin D in the nonfollicular absorptive mucosa and monolayers of the porcine enterocyte cell line, IPEC J2, a result that is in agreement with other studies of actin-dependent Salmonella invasion in epithelial preparations (Schreiber et al. 2007; Brown and Price 2007). In contrast, cytochalasin D has no effect on the uptake of S. enterica serovars Cholerasuis and Typhimurium into jejunal Peyer's patch mucosa explants (Green and Brown 2006; Brown and Price 2008).

Norepinephrine and DA have also been implicated in Salmonella internalization, especially in porcine jejunal Peyer's patches, where there is strong immunohis-tochemical evidence for catecholaminergic innervation (Kulkarni-Narla et al. 1999) At these inductive sites for mucosal immunity, nerve fibers immunoreactive for the catecholamine synthetic enzymes tyrosine hydroxylase and dopamine beta-hydroxylase can be seen terminating beneath epithelial cells. Enteric nerves near Peyer's patch follicles express immunoreactivities for the type 2 vesicular monoamine transporter, which transports catecholamines into synaptic vesicles, and the norepi-nephrine transporter NET, a target of cocaine action (Kulkarni-Narla et al. 1999; Green et al. 2003).

The serosal application of NE at a bath concentration of 10 |M produced a six- to ninefold increase in luminal S. enterica serovar Choleraesuis and EHEC internalization in porcine Peyer's patch explants. This effect was not mimicked by luminally-applied NE, but was inhibited in tissues pretreated with the alphaAR antagonist phentolamine. These results indicate that this NE action is mediated by alpha-ARs which are likely localized to the basolateral aspect of Peyer's patch epithelial cells (Green et al. 2003). In a study of S. enterica serovar Typhimurium internalization, the serosal administration of DA or the sympath-omimetic drugs cocaine and methamphetamine decreased Salmonella recovery from Peyer's patch explants (Brown and Price 2008). It is not known if these effects of NE and DA extend to species other than swine. Although the underlying cellular mechanisms for them must be investigated further, it is tempting to hypothesize that catecholamines may regulate the sampling function of Peyer's patches to control the entry or immune processing of pathogenic microbes at these intestinal sites. Catecholamines and EHEC Adherence to the Mucosa of the Large Intestine

When added to the medium bathing the contraluminal surface of cecal explants from mice, NE and DA increase the number of EHEC adhering to the mucosal surface. They do so at 50% effective concentrations (EC50) of 3.8 and 4.2 |M, respectively. The concentrations of NE applied to the basolateral aspect of the intestinal epithelium that are sufficient to promote EHEC adherence are somewhat lower than those necessary to promote epithelial EHEC adherence when incubated directly with the bacterium (Vlisidou et al. 2004; Bansal et al. 2007). The adherence-enhancing actions of NE and DA on the epithelium are inhibited respectively by AR and DR receptor antagonists, a result indicating that they are mediated by specific catecholamine receptors (Chen et al. 2003). This appears to differ from the mechanism by which these catecholamines produce their direct effects on bacterial function (Freestone et al. 2007b).

This phenomenon extends to species other than the mouse. Indeed, in mucosal explants of porcine cecum and colon, NE increases mucosal adherence of EHEC through interactions with alpha2-ARs that were characterized by conventional receptor criteria (Table 5.1). Increases in active anion secretion across the porcine colonic mucosa are in comparison mediated by alphat-ARs (Brown and O'Grady 1997). Therefore, it appears that the actions of NE on ion transport and EHEC adherence are not linked through a common cellular mechanism. Alpha2-ARs are negatively coupled to cyclic AMP production and a concomitant decrease in intra-cellular protein kinase A activity. In support of this receptor-effector association, the adherence-promoting action of NE in the porcine colonic mucosa is inhibited by the protein kinase A activator Sp-cAMPS and mimicked by the protein kinase A inhibitor Rp-cAMPs (Green et al. 2004). The effects of NE in the mouse and pig cecal mucosae are relatively rapid (£90 min), and experiments with EHEC eae and EspA deletion mutants strongly suggest that NE and other sympathomimetic drugs enhance early, nonintimate bacterial adherence (Chen et al. 2003, 2006). As with their effects on Salmonella internalization, although the effects of NE and other sympathomimetic drugs on EHEC adherence may appear small (<1.0 log unit increase in the number of adherent bacteria in mucosal explants with an exposed surface area of 1 or 2 cm2), they may assume considerable medical importance when extrapolated over the extensive surface area of the cecal or colonic mucosa (Snipes 1997).

The mechanisms underlying this unique catecholamine action remain to be further defined through the identification of epithelial surface factors that mediate bacterial adherence and the receptor-effector pathways that are linked to their rapid expression. Beta1-integrins are IEC surface receptors implicated in aspects of EHEC adherence (Sinclair et al. 2006). By blocking epithelial beta1-integrins, heparin has been shown to inhibit EHEC adherence to human colonic epithelial cells (Gu et al. 2008). Norepinephrine is known to enhance interactions between blood cells and the vascular endothelium by stimulating the rapid expression of beta1-integrins (Levite et al. 2001; Butta et al. 2004; Delahunty et al. 2006), and it is tempting to speculate that it may similarly do so in promoting IEC interactions with luminal bacteria. In addition to dissecting the cellular and molecular mechanisms underlying this phenomenon, studies of catecholamine action on bacterial adherence in vitro should be extended to investigations of the role of endogenous and exogenous DA and NE in isolated intestinal loops and intact animal models which encompass larger surface areas and have greater translational relevance.

Norepinephrine may play a physiological role in promoting bacterial colonization of the large intestine, perhaps as an element in host defense. This hypothesis is based in part on a finding that NE increases cecal adherence of a non-O157 strain of E. coli, which was isolated from the porcine colonic mucosa (Chen et al. 2006). One interpretation of this result is that the action of NE is not limited to a particular bacterial strain or species. Presumptive NE nerve fibers immunoreactive for dop-amine beta-hydroxylase are present throughout the submucosa and appear to terminate near the basal membranes of crypt and surface epithelial cells of the porcine distal colon and cecum (Green et al. 2004; Chen et al. 2006). Drugs capable of inhibiting the degradation (such as the monoamine oxidase inhibitor, pargyline) and neural reuptake (desipramine, cocaine) of NE at neuroepithelial junctions mimic the EHEC adherence-promoting action of NE, and their effects are inhibited by phentolamine (Green et al. 2004; Chen et al. 2006). In the porcine colonic mucosa, dopamine beta-hydroxylase-immunoreactive nerves terminate near IgA-positive B lymphocytes and neighboring IECs immunoreactive for the polymeric immuno-globulin receptor (Schmidt et al. 2007). Norepinephrine stimulates the vectorial secretion of secretory IgA in porcine colonic mucosa explants. This effect has been attributed to an alpha-AR-mediated increase in the luminally directed transport of secretory factor, a component of the polymeric Ig receptor (Schmidt et al. 2007). As noted above, constitutively produced secretory IgA is hypothesized to modulate colonization of the intestinal mucosa by commensal bacteria (Suzuki et al. 2007; Macpherson and Slack 2007).

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