Fig. 8. Inositol-1,4,5-trisphosphate (InsP₃) formation in and Ca²⁺-responsiveness of IMCD cells. (A) IMCD cells were incubated with myo-[2-³H]inositol for 20 hours at 37°C in culture medium without Bt₂cAMP. Thereafter, cells were incubated with AVP (100 nM), sulprostone (1 µM) or carbachol (100 µM; 60 minutes for each agonist). Subsequently, radioactive InsP₃ was isolated as described in Materials and Methods. One out of three independent experiments yielding similar results is shown. InsP₃ is expressed as a percentage of total [2-³H]inositol incorporated by IMCD cells. *, statistically different from untreated control cells (P<0.05). (B) Cytosolic Ca²⁺ was determined in IMCD cells (for details see Materials and Methods) and the percentage of cells responding to stimulation with AVP (100 nM), sulprostone (1 µM) or PGE₂ (1 µM) by elevation of cytosolic Ca²⁺ was determined. Control cells were left untreated. If indicated, the EP₁ receptor antagonist SC19220 (10 µM) was added 10 minutes prior to the experiment. The cytosolic Ca²⁺ concentration in untreated cells was about 50 nM; stimulation by AVP, sulprostone or PGE₂ induced an elevation to 200 nM in the responding cells.