NKCC1: tales from the dark side of the crypt.

Intestinal electrolyte secretion consist of several fundamental components: a basolateral uptake pathway, identified as the Na+–K+–Cl− cotransporter (NKCC1) (Payne et al. 1995) and an apical secretory pathway. NKCCs are highly conserved across organ and species, serving essential ion secretory and absorptive functions. Distinct regulatory mechanisms are present for apical, and for basolateral (‘dark side’) transporters. For example, in the colonic crypt-derived cell line HT29, Slotki et al. (1993) found that prolonged incubation with the cAMP activator forskolin increased K+ uptake attributable to cotransporter activity independently of new protein synthesis, leading them to speculate that NKCC is regulated by membrane insertion, in contrast to the new channel synthesis observed for CFTR. In HT29 cells in response to phorbol esters, another group demonstrated a rapid increase followed by a slow decline in cotransporter-associated K+ uptake, mirrored by a decline in [3H]bumetanide binding as a surrogate for plasma membrane NKCC expression. The change in Bmax with unchanged affinity suggested that regulation was via cotransporter internalization (Franklin et al. 1989). Indeed, control of insertion of the related renal NKCC paralogue NKCC2 is currently accepted as the primary means of regulating cotransporter function (Mount, 2006). Although intestinal Cl− secretory function is traditionally ascribed to the crypts, cotransporter function, measured by cell volume measurements in response to shrinkage or secretagogues in intact crypts and in vesicles prepared from the basolateral membrane of surface and crypt cells, was not only intact in the upper villous and surface, but appeared to be of increased activity (Diener, 1994; Wiener & van Os, 1989). In contrast, Flemmer et al. (2002), using an antibody recognizing phosphorylated NKCC, noted NKCC activation by adrenergic agonists only in the colonic crypt. In this issue of The Journal of Physiology, Reynolds et al. (2007) have used crypts prepared from human rectal biopsies to study regulation of NKCC1 function. Using a combination of fluorescently labelled NKCC1, Na+,K+-ATPase, and acetylcholine receptor antibodies and dynamic digital microimaging, the authors monitored NKCC1 localization, and measured cellular Ca2+ with fura-2 and pHi in response to NH4+ with BCECF. In a series of visually stunning images, the authors documented patterns of NKCC1 distribution in human rectal biopsies, demonstrating the propagation of a Ca2+ wave in response to acetylcholine. Secretion was also inferred from measurements of crypt width and the migration of calcein-labelled cells. In response to acetylcholine, crypt widening and acceleration of the plateau-phase pHi decrease indicated NKCC1 activation accompanied by fluid secretion. NKCC1 activity decreased after 30 min of ACh exposure, accompanied by a striking redistribution of NKCC1 immunofluorescence to the apical pole. With 4 h of ACh stimulation, NKCC1 re-inserted into the basolateral membrane. In contrast, prolonged forskolin exposure gradually increased NKCC1 activity while maintaining its presence in the basolateral membrane. This paper is remarkable in several respects: it reports an adaptation of the isolated colonic crypt preparation, first described 20 years ago for fluorescence measurements (Kaunitz, 1988), as a useful means of studying clinical tissue. Rather than use surrogate systems, the investigators have shown that intact organs obtained clinically can yield remarkable results. Furthermore, although the authors did not devise any single technique, the combination of measurements of crypt width, pHi in response to NH4+, cellular Ca2+, immunolocalization, and measurements of calcein-labelled cell movement represents how relatively straightforward methods, when applied judiciously, carefully and thoughtfully, can amplify the overall power of their observations. The authors indeed confirmed that as in the nephron, colonic NKCC1 activity in response to Ca2+ signalling is regulated via plasma membrane insertion and re-expression, and also provided a direct and plausible mechanism for the secretory synergy observed with combined cAMP and Ca2+ signals, and also the inhibitory effect of epidermal growth factor receptor/MAP kinase transactivation by elevation of cellular Ca2+. The results build on the observations summarized above, yielding fresh insights into intestinal NKCC1 regulation. I hope that other investigators follow the lead of this group in the study of intestinal transport regulation.