Figure 2.

Membrane-permeant Ins(1,4,5,6)P₄ reverses EGF- and PtdInsP₃-mediated inhibition of CaMCS. T₈₄ monolayers were incubated for 30 min with the indicated membrane-permeant esters (200 μM) or vehicle (dimethyl sulfoxide/5% Pluronic) in Ringer’s solution before mounting in Ussing chambers. Based on our previous findings that 1–2% of the membrane-permeant inositol polyphosphate esters enter T₈₄ cells (6), we estimate that the intracellular concentration of Ins(1,4,5,6)P₄ was ≈2–4 μM after 30 min of incubation with its membrane-permeant ester. (a) Time course of ΔI_sc. EGF (16.3 nM) was added basolaterally, followed 15 min later by carbachol (100 μM) to acutely elevate [Ca²⁺]_i levels. Controls also were stimulated with carbachol but not pretreated with EGF. Data are means of duplicate measurements from one representative of three experiments. (b-e) Peak ΔI_sc after carbachol addition. Data are means ± SEM of 4–10 experiments. Statistical significance was determined by Student’s t test (N.S., not significant). (d) DiC₁₆-BtPtdInsP₃/AM was used at 100 μM. Similar results were obtained with DiC₈-PtdInsP₃/AM (ΔI_sc in μA/cm²; controls 14.0 ± 1.9; 200 μM DiC₈-PtdInsP₃/AM 7.3 ± 1.7; DiC₈-PtdInsP₃ plus Bt₂Ins(1,4,5,6)P₄/AM 9.9 ± 1.8; n = 7–8). Not shown, treatment of T₈₄ cells with Bt₂Ins(1,4,5,6)P₄/AM, BtIns(1,3,4,5,6)P₅/AM, or up to 10 mM dipalmitoylglycerol (which was a contaminant in the DiC₁₆-BtPtdInsP₃/AM preparations) did not alter peak ΔI_sc after carbachol addition compared with control cells.