Jeremy A. Smith

Ammonia inhibits cAMP-regulated intestinal Cl- transport. Asymmetric effects of apical and basolateral exposure and implications for epithelial barrier function.

The colon, unlike most organs, is normally exposed to high concentrations of ammonia, a weak base which exerts profound and diverse biological effects on mammalian cells. The impact of ammonia on intestinal cell function is largely unknown despite its concentration of 4-70 mM in the colonic lumen. The human intestinal epithelial cell line T84 was used to model electrogenic Cl- secretion, the transport event which hydrates mucosal surfaces and accounts for secretory diarrhea. Transepithelial transport and isotopic flux analysis indicated that physiologically-relevant concentrations of ammonia (as NH4Cl) markedly inhibit cyclic nucleotide-regulated Cl- secretion but not the response to the Ca2+ agonist carbachol. Inhibition by ammonia was 25-fold more potent with basolateral compared to apical exposure. Ion substitution indicated that the effect of NH4Cl was not due to altered cation composition or membrane potential. The site of action of ammonia is distal to cAMP generation and is not due simply to cytoplasmic alkalization. The results support a novel role for ammonia as an inhibitory modulator of intestinal epithelial Cl- secretion. Secretory responsiveness may be dampened in pathological conditions associated with increased mucosal permeability due to enhanced access of lumenal ammonia to the basolateral epithelial compartment.

Modulation of intestinal chloride secretion at basolateral transport sites: opposing effects of cyclic adenosine monophosphate and phorbol ester.

BACKGROUND Although intestinal Cl secretion is largely regulated by apical Cl channels, we have shown that net secretory capacity can be controlled at a basolateral site, the Na-K-2Cl cotransporter (NKCC). Phorbol myristate acetate (PMA) was found to inhibit both cyclic adenosine monophosphate (cAMP)-regulated Cl secretion and basolateral NKCC function in parallel but not apical Cl channels. Because inhibition of NKCC function could occur by reducing the number of membrane NKCC units, we examined the effect of PMA on cAMP-regulated NKCC function and number. METHODS NKCC function and number were assessed in the human intestinal line HT29cl.19A by bumetanide-sensitive uptake of rubidium 86 and by specific binding of 3H-bumetanide. RESULTS The cAMP agonist forskolin enhanced bumetanide-sensitive 86Rb uptake and doubled the number of NKCCs. PMA decreased both basal and cAMP-stimulated uptake in time- and dose-dependent fashion. In addition, PMA down-regulated basal NKCC number and abolished cAMP-induced NKCC recruitment. CONCLUSIONS PMA opposes the action of cAMP on NKCC function by reducing both basal numbers of NKCCs and cAMP-induced recruitment of NKCCs and not by reducing ion translocation per NKCC. These data further emphasize the potential for modulation of intestinal Cl secretion at basolateral sites.

Adenosine scavenging: a novel mechanism of chloride secretory control in intestinal epithelial cells.

BACKGROUND Adenosine released by cells during ischemia typically serves as a feedback inhibitor of further organ work. However, in ischemic intestine, adenosine appears to act via stimulatory A2b receptors to increase work in the form of chloride ion (Cl-) secretion. This unusual response may contribute to luminal fluid sequestration in intestinal ischemia. In nonischemic cells feed-forward activation of Cl- secretion does not occur despite the fact that adenosine may be continuously generated during normal cell metabolism. Thus we postulated that intestinal epithelia normally control the disposition of adenosine to prevent inappropriate activation of secretion. METHODS Model T84 intestinal epithelia were studied by means of electrophysiologic and isotopic techniques. RESULTS Dipyridamole and nitrobenzylthioinosine (inhibitors of nucleoside transport) and iodotubercidin (an inhibitor of adenosine kinase) caused adenosine to accumulate extracellularly and induced a Cl- secretory response that was prevented by adenosine receptor blockade. Uptake of exogenous adenosine was restricted to the basolateral compartment and was blocked by nucleoside transport inhibitors. CONCLUSIONS Adenosine released from nonischemic intestinal epithelial cells is scavenged by a basolaterally restricted adenosine transporter. This system maintains extracellular adenosine levels below the prosecretory threshold and thus limits adenosine-elicited activation of Cl- secretion (and hence diarrhea) under normal conditions).

Dynamic role of microfilaments in intestinal chloride secretion

The importance of microfilaments in the regulation of chloride (Cl-) secretion by the human intestinal cell line T84 was investigated using the cytoskeletal probe phalloidin to bind and stabilize F-actin. Phalloidin was found to inhibit secretion mediated by cyclic adenosine monophosphate (cAMP) and the sustained secretory response to the calcium (Ca+2) ionophore ionomycin but not to affect the transient Ca+2-mediated response to carbachol and histamine. Fluorescent microscopic examination of F-actin revealed regionally restricted microfilament remodeling in cAMP- and ionomycin-treated cells. Normal regulation of apical Cl- and basolateral potassium (K+) channel functions was evident in phalloidin-loaded cells. It is concluded that prevention of cytoskeletal remodeling by actin stabilization inhibits the generation of a sustained Cl- secretory response by a mechanism that does not involve Cl- or K+ channels. Depolymerization of F-actin plays an integral role in the regulation of intestinal Cl- secretion.