NO EVIDENCE FOR CELL-TO-CELL COUPLING IN RAT COLONIC CRYPTS - STUDIESWITH LUCIFER-YELLOW AND WITH PHOTOBLEACHING

Epithelial cells of exocrine glands (pancreas, lacrimal glands, saliva ry glands, sweat glands and gastric glands) are intimately linked toge ther by gap junctions. Due to this close junctional coupling exocrine secretion occurs as the well concerted effort of a cell population. Co lonic crypts have, on the one hand, anatomical and functional properti es resembling those of exocrine glands (mostly crypt base cells) and, on the other hand, properties of absorbing cells (mostly surface cells ). In the mid-crypt, depending on the functional status, absorption an d secretion can occur. The present study was aimed at examining whethe r rat distal colonic crypt cells co-ordinate their functional status b y cell-to-cell coupling. Two types of measurements were performed: as an independent assessment of cell viability the membrane voltage (V-m) was measured with the fast whole-cell patch-clamp technique; to inves tigate cellular coupling simultaneously Lucifer Yellow (LY) (mol. wt. 443) distribution was visualized using digital video imaging. LY (500 mu mol/l) was included into the patch pipette filling solution. The re corded V-m was -73.4+/-2.3 mV in crypt base cells (n=15), -63.7+/-2.1 mV in mid-crypt cells (n=17) and -52.3+/-2.9 mV in crypt surface cells . All cells tested reversibly responded to carbachol (100 mu mol/l) wi th a persistent hyperpolarization, as previously shown. Activation of Cl- secretion by elevation of the cAMP concentration with forskolin (5 mu mol/l) led to a reversible depolarization. Throughout the duration of each individual experiment [mean experimental time in basal cells: 18.3+/-2.5 min (n=15), in mid-crypt cells 19.6+/-3.4 min (n=17) and i n crypt surface cells: 11.7+/-3.4 min (n=13)] LY dye distribution was solely confined to the patched cell. In addition bleaching of calcein fluorescence in laser scan microscopy was not followed by dye back dif fusion, whereas this was clearly the case in pancreatic acini (n=5). T hese data indicate that colonic crypt cells are not coupled by gap jun ctions under resting conditions or in the presence of secretagogues.