PHOSPHATIDYLINOSITOL 3-KINASE AND cAMP INVOLVEMENT IN THE GROWTH HORMONE REGULATION OF ION TRANSPORT IN CACO-2 CELLS

Abstract

9 In the rat intestine growth hormone (GH) increases basal ion transport and reduces Cholera toxin (CT)-induced ion secretion (1). In Caco-2 cells GH-induced ion absorption is dependent on tyrosine kinase(2). In T84 cells, tyrosine phosphorylation activates the enzyme phosphatidylinositol 3-kinase (PI 3-K) (3).Aims. To determine whether PI 3-K is involved in the GH effect on basal ion transport and/or on inhibition of ion secretion. Methods. Caco-2 cells, grown on permeable supports, were mounted in Ussing chambers 15 days after confluence. Cells were exposed to GH (4×10-9 M) in the absence or in the presence of CT (5 μg/ml). Cl- transepithelial transport was assessed by monitoring the modifications in short circuit current (Isc) expressed as μA/cm2/h. The same experiments were performed in cells treated with the specific inhibitor of PI 3-K, wortmannin(W) (50 nM). Similar experiments were repeated using cells grown on plates to measure intracellular cAMP concentration by a specific RIA. Results.Ussing chambers experiments: 1) CT induced an increase in Isc(5.8±.08 mA/cm2h, p<.01), 2) GH induced a decrease in basal Isc (-4.9±.05 mA/cm2/h, p<.01), 3) GH reduced the CT effect on Isc by 75% (p<.01). In the presence of W, basal Isc was slightly and transiently increased and: 1) the effect of CT was preserved, 2) the effect of GH on basal Isc was abolished, 3) the GH reduction of CT effect was preserved. These results indicated that the GH effects on basal transport, but not those on CT-induced secretion, were PI 3-K-dependent and raised the hypothesis that GH efficacy in reducing CT-induced secretion could be due to an effect on cAMP, the main effector of CT effects. To test this, cAMP intracellular concentrations were measured in the presence of GH and/or CT. cAMP experiments: 1) CT increased the intracellular cAMP concentration from 2.0±03 to 15.1±1 pmol/mg protein (p<0.1), 2) GH reduced the basal intracellular cAMP concentration from 2.0±.03 to 0.8±.01 pmol/mg protein (p<.01), 3) GH reduced the CT-induced cAMP increase by 70%(p<.01). Finally, the addition of W had no effect in any of these conditions. Conclusions. These data indicate that GH possesses at least two distinct mechanisms for its effects on basal ion transport and on CT-induced secretion. The first is PI 3-K-dependent and also involves the cAMP. The latter is indipendent on PI 3-K, but involves cAMP intracellular concentration.