kallidin and Colonic-Neoplasms

1. The patch-clamp technique, both cell attached and inside-out patches, was used to examine the effects of lysylbradykinin (LBK) and A23187 on ion channels in cultured Colony 29 epithelial cells derived from a human adenocarcinoma. 2. LBK and A23187 applied directly to the intact cell stimulated the opening of a number of types of ion channel including Ca(2+)-activated K+ channels. 3. By use of inside-out patches, anion channels could be stimulated to open by application of protein kinase A and ATP to the cytosolic surface. Ca(2+)-activated K+ channels were also identified in isolated membrane patches. 4. The results suggest that the anion secretion which is stimulated by LBK is a complex event, involving the activation of a number of different types of ion channel, and that part of the response is the result of hyperpolarization of the cell by activation of Ca(2+)-activated K+ channels. From the data presented in this and the accompanying papers it appears that the Ca(2+)-sensitive K+ channels would be equally effective in either the apical or basolateral membranes.

Topics: Adenocarcinoma; Adenylyl Cyclases; Calcimycin; Colforsin; Colonic Neoplasms; Electric Stimulation; Enzyme Activation; Humans; Ion Channels; Kallidin; Tumor Cells, Cultured

1. Chloride secretion in three types of cultured epithelial monolayers derived from a single human colonic adenocarcinoma was measured in terms of short circuit current. The three cell types were designated HCA-7, Colony 3 and Colony 29. 2. Responses of HCA-7 monolayers to basolaterally applied lysylbradykinin (LBK) (10-1000 nM) or carbachol (1-100 microM) were potentiated by pre-exposure to forskolin (10 microM) for 5 min. Forskolin itself increased short circuit current (SCC), so that the total response to forskolin and LBK or carbachol were non-additive. 3. Colony 3 cells did not respond to LBK on either face but did to carbachol on the basolateral side, while Colony 29 epithelia responded to LBK on both sides and to carbachol and histamine basolaterally. Unlike HCA-7 epithelia, responses in Colony 3 and Colony 29 epithelia were not potentiated by forskolin, but were attenuated by piroxicam. 4. In the presence of piroxicam, both forskolin and prostaglandin E2 were able to potentiate the action of both LBK and carbachol in Colony 29 epithelia. 5. LBK receptor activation in Colony 29 epithelia is transduced into an increase in intracellular Ca2+ and cyclic AMP, while in HCA-7 epithelia there is only an increase in intracellular Ca2+ (Cai). These conclusions are considered to apply to both apical and basolateral kinin receptors. 6. It is shown that forskolin has no effect on the elevation of Ca2+ by LBK in either HCA-7 or Colony 29 cells. 7. It is concluded that potentiation of agonist responses occurs when cyclic AMP is raised at the time that intracellular Ca2+ increases. No potentiation of LBK or carbachol by forskolin occurs in Colony 29 monolayers as these agonists increase cyclic AMP via eicosanoid production.

Topics: Adenocarcinoma; Calcium; Chlorides; Colforsin; Colonic Neoplasms; Cyclic AMP; Epithelium; Humans; Kallidin; Mutation; Phenotype; Tumor Cells, Cultured

1. Hoe-140, a potent kinin receptor antagonist, was investigated for its ability to inhibit the effects of lysylbradykinin (kallidin) on a cultured colonic epithelium, HCA-7 Colony 29, derived from a human adenocarcinoma. 2. Measurements of electrogenic chloride secretion (as short circuit current), and of intracellular Ca2+ (from Fura-2 fluorescence) were used to assess the action of lysylbradykinin in the absence and presence of Hoe 140. 3. From short circuit current data, Hoe 140 appeared to be a competitive antagonist with a Ki value of 5 nM. However, with measurements of intracellular Ca2+ Hoe 140 was apparently a non-competitive antagonist with a Ki of between 4-6 nM. 4. Because of the unexpected finding of non-competitive antagonism, measurements were made with a second antagonist pair, histamine and mepyramine. Mepyramine behaved as a competitive antagonist against responses to histamine with a Ki value of approximately 5 nM when short circuit current measurements were evaluated. However, when intracellular Ca2+ concentration was used as a measure mepyramine, 30 nM, produced a near parallel shift in the response curve, but at 100 nM the maximal response was depressed. 5. The reasons why the apparent type of antagonism depends upon the method of measurement is discussed, bearing in mind that the increase in intracellular Ca2+ is a signal which precedes the increase in short circuit current.

Topics: Adenocarcinoma; Binding, Competitive; Bradykinin; Calcium; Chlorides; Colonic Neoplasms; Epithelium; Fura-2; Histamine; Humans; Ion Channels; Kallidin; Kinins; Pyrilamine; Tumor Cells, Cultured