kallidin and piretanide

1. Colonic epithelial cells, derived from a human adenocarcinoma (HCA-7), were examined by the patch clamp technique. 2. Outwardly rectifying anion (Cl-) channels were identified in the apical membrane. The conductance was g(in) approximately 26 pS, g(out) approximately 40 pS. The open state probability of the channels increased with depolarization and the selectivity for Cl- over K+ (PCl/PK) was approximately 7.5. 3. The channels were sensitive to intracellular adenosine 3':5'-cyclic monophosphate (cyclic AMP, 0.1 mM), but not to Ca2+ (at concentrations up to 1 mM). At depolarized potentials the channels were blocked by pirentanide (1-5 microM) applied intracellularly. 4. HCA-7 monolayers loaded with 125I- (as a marker for Cl-) were used to measure I- efflux and converted to instantaneous rate constants. 5. The rate constant for I- efflux was increased by forskolin and lysylbradykinin (LBK). The effects of forskolin were not effected by BAPTA (an intracellular calcium chelator). The effects of LBK were inhibited by BAPTA and by Ba2+, indicating that LBK raised intracellular Ca2+ (Cai) which activates Ca(2+)-sensitive K-channels, the latter being blocked by Ba2+. 6. Although it cannot be conclusively proved that the outwardly rectifying chloride channels described here are solely or partially responsible for the increased anion efflux or transepithelial chloride secretion, the channels are likely to be more relevant for cyclic AMP-requiring rather than Ca(2+)-requiring secretagogues.

Topics: Adenocarcinoma; Barium; Calcium; Chloride Channels; Chlorides; Colforsin; Colon; Cyclic AMP; Egtazic Acid; Electric Conductivity; Epithelium; Humans; Ion Channels; Kallidin; Membrane Proteins; Potassium Channels; Sulfonamides; Tumor Cells, Cultured

A cultured human epithelial cell line, Colony 29, has been used to investigate the relation between anion secretion and intracellular Ca2+ concentration (Cai) in response to the secretagogues, lysylbradykinin (LBk) and histamine. Anion secretion was measured as short-circuit current (SCC) responses in epithelia cultured on previous supports. Cai was measured both in cell suspensions and epithelial monolayers using Fura-2 fluorescence. While it is concluded that raised Cai is responsible for anion secretion the relationship is complex. For both secretagogues there is a receptor reserve, that is the maximal Cai increase is greater than that required to cause a maximal secretory response. By examining the interactions between maximally effective concentrations of LBk and histamine it was shown that neither the SCC nor Cai responses behaved additively. From observations in the absence of external Ca2+ it was concluded that both secretagogues cause Ca2+ release from the same intracellular source, but that in normal conditions Ca2+ derived from intracellular and extracellular sources is responsible for the full effect.

Topics: Acetazolamide; Calcimycin; Calcium; Carbachol; Cell Line; Cells, Cultured; Colforsin; Colon; Digitonin; Drug Interactions; Epithelial Cells; Epithelium; Fura-2; Histamine; Humans; Intestinal Mucosa; Kallidin; Kinetics; Spectrometry, Fluorescence; Sulfonamides