ALPHA-2-ADRENERGIC RECEPTORS AND THE NA+/H+ EXCHANGER IN THE INTESTINAL EPITHELIAL-CELL LINE, HT-29

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  • 25 September 1989
    • journal article
    • research article
    • Vol. 264  (27) , 16000-16007
Abstract
.alpha.-Adrenergic receptors (.alpha.2-AR) are negatively coupled to adenylyl cyclase via the GTP-binding protein Gi. However, inhibition of adenylylcyclase does not account for many effector cell responses to .alpha.2-AR agonists, suggesting that the receptor can couple to other signal transduction pathways. One potential pathway may be the stimulation of Na+/H+ exchange elicited by .alpha.2-AR activation in renal proximal tubule cells, platelets, and the NG-10815 cell line. To determine whether the various receptor-effector coupling mechanisms operate in a tissue-specific manner, we studied the effect of .alpha.2-AR activation on basal and stimulated Na+/H+ exchange in epithelial cells isolated from human colon (HT-29 adenocarcinoma cells). Na+/H+ exchange was measured by quantitation of intracellular hydrogen ion concentration (acetoxymethyl ester 2,7-biscarboxyethyl-5(6)carboxyfluorescein) and 22Na+ uptake. HT-29 cells expressed an amiloride-sensitive Na+/H+ exchanger that was activated by reduction of intracellular pH (pHi) to 6.0 but was quiescent at a physiological pHi. The rapid alkalinization observed after acid loading (0.57 .+-. 0.07 pH units/min/104 cells) was dependent on external sodium and was blocked by amiloride (Ki .apprx. 2.1 .mu.M). Although epinephrine and the selective .alpha.2-AR agonists clonidine and UK-14304 inhibited forskolin-activated adenylylcyclase, these compounds 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 .alpha.2-agonists clonidine, UK-14304, and guanabenz. This inhibitory effect was not blocked by the .alpha.2-AR antagonist rauwolscine, and it is likely due to a direct interaction with the exchange 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 did not inhibit the amiloride-sensitive epithelial sodium channel in toad urinary bladder, suggesting that these compounds may be useful as "amiloride-like" ligands selective for the Na+/H+ exchanger. These data indicate that in the HT-29 intestinal cell line, in contrast to observations in other tissues, .alpha.2-adrenergic receptors are not coupled to the Na+/H+ exchanger, suggesting that the cell-signaling mechanisms utilized by the .alpha.2-AR are tissue specific.