addrenergic Receptors and theN a’/H’ Exchanger in the Intestinal Epithelial Cell Line, HT-29

HORACIO F. CANTIELLO AND STEPHEN M. LANIER

JOURNAL OF BIOLOGICAL CHEMISTRY

Año: 1989 p. 16000 - 16007

0021-9258

az-Adrenergic receptors (az-AR)a re negatively coupled to adenylyl cyclase via the GTP-binding protein Gi. However, inhibition of adenylylcyclase does not account for many effector cell responses to az-AR agonists, suggestingt hat the receptor caconu ple to other signal transduction pathways. One potential pathway may be the stimulationo f Na+/H+ exchange elicitebdy az-AR activation in renal proximal tubuclee lls, platelets, and the NG-108c1e5ll line. To determine whether the various receptor-effector couplinmge chanisms operate in a tissue-specific manner, we studiethd e effect of a,-AR activation on basal and stimulated Na+/H+ exchange in epithelial cells isolated from humacno lon (HT-29 adenocarcinoma cells). Na+/H+ exchange was measured by quantitation of intracellular hydrogenio n concentration (acetoxymethyl ester 2,7-biscarboxyethyl-5(6)carboxyfluorescein) and z2Na+ uptake. HT- 29 cells expressed an amiloride-sensitive Na+/H+ exchanger that was activatebdy reduction of intracellular pH (pHi) to 6.0 but was quiescent at a physiological pHi. The rapid alkalinization observeadf ter acid loading (0.57 f 0.0pH units/min/104 cells) was dependent on external sodium and was blocked by amiloride (ICi - 2.1 PM). Although epinephrine and the selective az- AR agonists clonidinea nd UK- 14304 inhibited forskolin- activated adenylylcyclase, thesec ompounds did not alter basal Na+/H+ exchange. Stimulated Na+/H+ exchange was similarly unaffected by epinephrine. In contrast, stimulated Na+/H+ exchanger activity was completely inhibited by the selective az-agonists clonidine, UK-14304, andg uanabenz. This inhibitory effect was not blocked by the az-AR antagonist rauwolscine, and it is likely due to a direct interaction with the exchanger molecule itself. Structure/activity studies indicated that the compounds inhibiting exchanger activity possess either an imidazoline or guanidinium moiety. Although these molecules bear structural similarity to amiloride, they didn ot inhibit the amiloridesensitive epithelial sodium channel in toad urinary bladder, suggesting that these compounds may be useful as “amiloride-like” ligands selective fotrh e Na+/H+ exchanger. These data indicate that in the HT-29 intestinal cell line, in contrast to observations in other tissues, az-adrenergic receptors are not coupled to the Na+/H+ exchanger, suggesting that the cell-signaling mechanisms utilized by the az-AR are tissue specific. az-Adrenergic receptors (az-AR)a re negatively coupled to adenylyl cyclase via the GTP-binding protein Gi. However, inhibition of adenylylcyclase does not account for many effector cell responses to az-AR agonists, suggestingt hat the receptor caconu ple to other signal transduction pathways. One potential pathway may be the stimulationo f Na+/H+ exchange elicitebdy az-AR activation in renal proximal tubuclee lls, platelets, and the NG-108c1e5ll line. To determine whether the various receptor-effector couplinmge chanisms operate in a tissue-specific manner, we studiethd e effect of a,-AR activation on basal and stimulated Na+/H+ exchange in epithelial cells isolated from humacno lon (HT-29 adenocarcinoma cells). Na+/H+ exchange was measured by quantitation of intracellular hydrogenio n concentration (acetoxymethyl ester 2,7-biscarboxyethyl-5(6)carboxyfluorescein) and z2Na+ uptake. HT- 29 cells expressed an amiloride-sensitive Na+/H+ exchanger that was activatebdy reduction of intracellular pH (pHi) to 6.0 but was quiescent at a physiological pHi. The rapid alkalinization observeadf ter acid loading (0.57 f 0.0pH units/min/104 cells) was dependent on external sodium and was blocked by amiloride (ICi - 2.1 PM). Although epinephrine and the selective az- AR agonists clonidinea nd UK- 14304 inhibited forskolin- activated adenylylcyclase, thesec ompounds did not alter basal Na+/H+ exchange. Stimulated Na+/H+ exchange was similarly unaffected by epinephrine. In contrast, stimulated Na+/H+ exchanger activity was completely inhibited by the selective az-agonists clonidine, UK-14304, andg uanabenz. This inhibitory effect was not blocked by the az-AR antagonist rauwolscine, and it is likely due to a direct interaction with the exchanger molecule itself. Structure/activity studies indicated that the compounds inhibiting exchanger activity possess either an imidazoline or guanidinium moiety. Although these molecules bear structural similarity to amiloride, they didn ot inhibit the amiloridesensitive epithelial sodium channel in toad urinary bladder, suggesting that these compounds may be useful as “amiloride-like” ligands selective fotrh e Na+/H+ exchanger. These data indicate that in the HT-29 intestinal cell line, in contrast to observations in other tissues, az-adrenergic receptors are not coupled to the Na+/H+ exchanger, suggesting that the cell-signaling mechanisms utilized by the az-AR are tissue specific.