Regulation of adenylyl cyclase in polarized renal epithelial cells by G protein-coupled receptors

We employed two guanine nucleotide binding protein (G protein)-coupled receptors known to be targeted to opposite domains in renal epithelial cells to test the hypothesis that the polarized receptor expression of receptors regulates the activity of the receptor's effector molecule, adenylyl cyclase. We used LLC-PK(1) cells stably transfected with cDNA encoding the alpha(2B)-adrenergic receptor (alpha(2B)-AR) or A(1)-adenosine receptor (A(1)-AdR). Immunohistochemistry and Western blot analysis confirmed the basolateral and apical expression of alpha(2B)-ARs and A(1)-AdRs, respectively. Adenylyl cyclase activity was assessed by measuring cAMP accumulation following the addition of forskolin (10 mu M) in the presence of 3-isobutyl-1-methylxanthine to apical or basolateral chambers of confluent monolayers. A five- to sixfold increase in cAMP accumulation occurred following apical (or basolateral) stimulation of LLC-PK(1) cells expressing apical (or basolateral) receptors in comparison to forskolin stimulation of corresponding domains of untransfected cells. We conclude 1) adenylyl cyclase activity is present at or near the apical and basolateral domains of LLC-PK(1) cells, and 2) factors that regulate the polarized expression of inhibitory G protein-coupled receptors may also regulate local adenylyl cyclase activity.