Adenosine is thought to be a major effector in immunological stimulation of Cl- secretion in intestinal epithelia. Previous studies indicate that both apical and basolateral domains of intestinal epithelial cells possess functionally defined adenosine receptors. However, it is unclear whether the same receptor subclass is expressed, what the receptor subclass(es) is, or how the receptors signal the Cl- secretory response. We now characterize the intestinal epithelial adenosine receptor subtype using the model epithelium, T84. Both apical and basolateral adenosine receptor agonist response profiles revealed a hierarchy (ED50) of 5'-(N-ethylcarboxamido)adenosine > adenosine > CGS-21680. Similarly inhibition studies revealed identical ID50 hierarchies for apical and basolateral antagonism by xanthine amine congener > 1,3-diethyl-8-phenylxanthine > aminophylline. Analyses of both agonist and antagonist pharmacological hierarchies in Chinese hamster ovary cells stably expressing the A2b receptor revealed these same hierarchies. Northern blots performed on RNA extracted from polarized T84 monolayers demonstrated no detectable message for A1 or A2a adenosine receptor, but strong hybridization was detected for the A2b adenosine receptor. Subsequent Northern blots of RNA prepared from human alimentary tract revealed that A2b adenosine receptor message was heavily expressed throughout the colon, in the appendix, and more modestly expressed in the small intestine (ileum). Analyses of cAMP generation in T84 cells in response to adenosine indicated that the basolateral A2b receptor elicits Cl- secretion through this signaling pathway. Stimulation of Cl- secretion through the apical A2b receptor exhibited relatively small but significant increases in cAMP compared with basolateral stimulation. The protein kinase A inhibitor H-89, used at concentrations that did not affect short circuit current responses to the Ca(2+)-mediated agonist carbachol, effectively inhibited short circuit current elicited by either apical or basolateral adenosine. These data suggest that the major intestinal epithelial adenosine receptor is the A2b subclass, which is positively coupled to adenylate cyclase. Such observations have potentially important implications for the treatment of diarrheal diseases.
Salt and water secretion from intestinal epithelia requires enhancement of anion permeability across the apical membrane of Cl ؊ secreting cells lining the crypt, the secretory gland of the intestine. Paneth cells located at the base of the small intestinal crypt release enteric defensins (cryptdins) apically into the lumen. Because cryptdins are homologs of molecules known to form anion conductive pores in phospholipid bilayers, we tested whether these endogenous antimicrobial peptides could act as soluble inducers of channel-like activity when applied to apical membranes of intestinal Cl ؊ secreting epithelial cells in culture. Of the six peptides tested, cryptdins 2 and 3 stimulated Cl ؊ secretion from polarized monolayers of human intestinal T84 cells. The response was reversible and dose dependent. In contrast, cryptdins 1, 4, 5, and 6 lacked this activity, demonstrating that Paneth cell defensins with very similar primary structures may exhibit a high degree of specificity in their capacity to elicit Cl ؊ secretion. The secretory response was not inhibited by pretreatment with 8-phenyltheophyline (1 M), or dependent on a concomitant rise in intracellular cAMP or cGMP, indicating that the apically located adenosine and guanylin receptors were not involved. On the other hand, cryptdin 3 elicited a secretory response that correlated with the establishment of an apically located anion conductive channel permeable to carboxyf luorescein. Thus cryptdins 2 and 3 can selectively permeabilize the apical cell membrane of epithelial cells in culture to elicit a physiologic Cl ؊ secretory response. These data define the capability of cryptdins 2 and 3 to function as novel intestinal secretagogues, and suggest a previously undescribed mechanism of paracrine signaling that in vivo may involve the reversible formation of ion conductive channels by peptides released into the crypt microenvironment.
Monolayers of the human colonic epithelial cell line T84 exhibit electrogenic Cl− secretion in response to the Ca2+ agonist thapsigargin and to the cAMP agonist forskolin. To evaluate directly the regulation of apical Cl−conductance by these two agonists, we have utilized amphotericin B to permeabilize selectively the basolateral membranes of T84 cell monolayers. We find that apical anion conductance is stimulated by both forskolin and thapsigargin but that these conductances are differentially sensitive to the anion channel blocker DIDS. DIDS inhibits thapsigargin-stimulated responses completely but forskolin responses only partially. Furthermore, the apical membrane anion conductances elicited by these two agonists differ in anion selectivity (for thapsigargin, I− > Cl−; for forskolin, Cl− > I−). However, the DIDS-sensitive component of the forskolin-induced conductance response exhibits anion selectivity similar to that induced by thapsigargin (I− > Cl−). Thus forskolin-induced apical anion conductance comprises at least two components, one of which has features in common with that elicited by thapsigargin.
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