Fluid absorption in isolated perfused colonic crypts.

Abstract

A spatial segregation of ion transport processes between crypt and surface epithelial cells is well-accepted and integrated into physiological and pathophysiological paradigms of small and large intestinal function: Absorptive processes are believed to be located in surface (and villous) cells, whereas secretory processes are believed to be present in crypt cells. Validation of this model requires direct determination of fluid movement in intestinal crypts. This study describes the adaptation of techniques from renal tubule microperfusion to hand-dissect and perfuse single, isolated crypts from rat distal colon to measure directly fluid movement. Morphologic analyses of the isolated crypt preparation revealed no extraepithelial cellular elements derived from the lamina propria, including myofibroblasts. In the basal state, crypts exhibited net fluid absorption (mean net fluid movement = 0.34+0.01 nl mm-' -min-'), which was Na+ and partially HCO-dependent. Addition of 1 mM dibutyryl-cyclic AMP, 60 nM vasoactive intestinal peptide, or 0.1 mM acetylcholine to the bath (serosal) solution reversibly induced net fluid secretion (net fluid movement --0.35+0.01 nl mm-. min-'). These observations permit speculation that absorption is a constitutive transport function in crypt cells and that secretion by crypt cells is regulated by one or more neurohumoral agonists that are released in situ from lamina propria cells. The functional, intact polarized crypt described here that both absorbs and secretes will permit future studies that dissect the mechanisms that govern fluid and electrolyte movement in the colonic crypt. (J. Clin. Invest. 1995. 96:2373-2379

Calcium-sensing receptor abrogates secretagogue- induced increases in intestinal net fluid secretion by enhancing cyclic nucleotide destruction

“…Cyclic nucleotidedependent f luid loss in secretory diarrheas occurs through reductions in absorptive, and increases in secretory, processes (18,19). Colonic epithelial crypts provide a model for understanding intestinal f luid transport as they simultaneously absorb and secrete f luid with the direction of net f luid movement ( net J V ) depending on the relative magnitudes of these two processes (9,20). To study the effects of CaSR activation on responses of the intestine to bacterial secretagogues, we used isolated colonic crypts in which changes in f luid transport can be temporally monitored while providing access to both luminal and basolateral receptors.…”

“…Single, hand-dissected crypts were placed in a temperature-controlled chamber on the stage of an inverted microscope and perfused in vitro as described (20). Briefly, crypts were perfused (4-10 nl͞min) with a solution containing exhaustively dialyzed methoxy-[ 3 H]inulin (10 Ci͞ ml; PerkinElmer; 1 Ci ϭ 37 GBq).…”

Cloning and Expression of a Chloride-dependent Na+-H+ Exchanger

“…Preparation of Distal Crypt Cells-Crypt cells of the rat distal colon were prepared, as described previously (11,12). Briefly, colonic crypts were isolated by a modified rapid calcium chelation method (12).…”

“…The long-standing model of fluid and electrolyte movement in the large and small intestine is that fluid absorptive processes are present in surface/villous cells, whereas secretory ones are localized to crypt cells (10). To study crypt cell function directly in the rat distal colon, we recently established methods to perform microperfusion of colonic crypts adapting methods used previously with renal tubules and to prepare apical membrane vesicles (AMV) from isolated crypt cells (11,12). In microperfusion studies of isolated rat colonic crypts, we demonstrated sodium-dependent fluid absorption (11) and therefore sought the identity of this unexpected phenomenon.…”

“…To study crypt cell function directly in the rat distal colon, we recently established methods to perform microperfusion of colonic crypts adapting methods used previously with renal tubules and to prepare apical membrane vesicles (AMV) from isolated crypt cells (11,12). In microperfusion studies of isolated rat colonic crypts, we demonstrated sodium-dependent fluid absorption (11) and therefore sought the identity of this unexpected phenomenon. By using both crypt AMV and microperfusion, we demonstrated that both [H ϩ ] gradient-driven 22 Na ϩ uptake and sodium-dependent recovery of pH i from an acid load, respectively, had an absolute requirement for chloride (12)(13)(14).…”