Functional localization of adenosine receptor-mediated pathways in the LLC-PK1 renal cell line

Abstract

The functional localization of three adenosine receptor-mediated signal transduction pathways in the LLC-PK1 renal cell line was investigated. LLC-PK1 cells were grown on Millicell-CM filter inserts, which allow for the independent exposure of the apical or basolateral side of a confluent cell monolayer to hormones. Adenosine stimulated inositol phosphate turnover, inhibition of adenosine 3',5'-cyclic monophosphate (cAMP) accumulation (A1 receptor), and stimulation of cAMP accumulation (A2 receptor). Adenosine (10 microM) selectively applied to the basolateral side induced a significant (P < 0.05) increase in inositol phosphates, whereas apical exposure did not. The adenosine receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (1 microM), blocked the stimulation of inositol phosphate production in LLC-PK1 cells, provided support for an adenosine receptor-mediated event. When adenosine (30 nM) was selectively applied to the apical side, forskolin-stimulated cAMP levels were not significantly decreased (approximately 8%, P > 0.05). However, adenosine (30 nM) presented to the basolateral side produced a significant decrease (approximately 23%, P < 0.05) in forskolin-stimulated cAMP levels. A high dose (100 microM) of adenosine elicited a significant increase (P < 0.05) in cAMP levels when presented to either the apical or the basolateral cell surface. Adenosine (100 microM) applied to the apical side elicited significantly higher cAMP levels (P < 0.05) than the same dose applied basolaterally. LLC-PK1 cells grown on permeable supports exhibit a polarity of functional responses following activation by adenosine. These data support a topographic separation of the multiple adenosine signaling systems in a renal epithelial cell line.