The traditional paradigm of colonic fluid and electrolyte transport includes a spatial separation of absorptive and secretory processes to surface and crypt cells, respectively. Recent studies of isolated microperfused colonic crypts revealed constitutive Na-dependent fluid absorption while secretion is regulated by one or more neurohumoral agonists. One obvious reason for the difference found in microdissected crypts is their separation from the lamina propria milieu. While it has been shown that isolated crypts are devoid of obvious lamina propria elements, including pericryptal fibroblasts, detailed morphologic information of the content of isolated crypts has been lacking. To characterize the morphology of the isolated crypt, we performed transmission electron microscopy (TEM) and immunofluorescence on microdissected and Ca²⁺ chelated crypts. Crypt cell type analysis was carried out separately on intact rat colon using light microscopy. TEM revealed a complete lack of either lamina propria cells or extracellular material in crypts isolated by either technique. TEM also revealed a subtle difference between the two isolation methods, with intact basal membranes in microdissected crypts but focal disruption of basal membranes in Ca²⁺- chelated crypts. Immunofluorescent stains for two basement membrane components (laminin and collagen type IV) revealed the presence of adherent basement membrane only on microdissected crypts; evidence that the plane of separation differs in these two preparations. Crypt cell type analysis on intact rat colon revealed an equal proportion of goblet cells in the right and left colon (approximately 50%) when measuring the middle 70% of the crypts – the area studied during crypt microperfusion. This morphologic analysis will increase our understanding of the observed physiology of isolated colonic crypts.

An apical membrane ouabain-sensitive H-K exchange and a barium-sensitive basolateral membrane potassium channel are present in colonic crypt cells and may play a role in both K absorption and intracellular pH (pHi) regulation. To examine the possible interrelationship between apical membrane H-K exchange and basolateral membrane K movement in rat distal colon in the regulation of pHi, experiments were designed to assess whether changes in extracellular potassium can alter pHi. pHi in isolated rat crypts was determined using microspectrofluorimetric measurements of the pH-sensitive dye BCECF-AM (2', 7'-bis(carboxyethyl-5(6)-carboxy-fluorescein acetoxy methylester). After loading with the dye, crypts were superfused with a Na-free solution which resulted in a rapid and reversible fall in pHi (7.36 +/- 0.02 to 6.98 +/- 0.03). Following an increase in extracellular [K] to 20 mm, in the continued absence of Na, there was a further decrease in pHi (0.20 +/- 0.02, P < 0.01). K-induced acidification was blocked both by 2 mm bath barium, a K channel blocker, and by 0. 5 mm lumen ouabain. K-induced acidification was also observed when intracellular acidification was induced by a NH4Cl prepulse. These observations suggest that increased basolateral K movement increases intracellular [K] resulting in a decrease in pHi that is mediated by a ouabain-sensitive apical membrane H,K-ATPase. Our results demonstrate an interrelationship between basolateral K movement and apical H-K exchange in the regulation of pHi and apical K entry in rat distal colon.

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