Alpha 2B-adrenergic receptors: immunolocalization and regulation by potassium depletion in rat kidney.

Regulation of transport processes in renal epithelial cells in response to alpha 2-adrenergic receptor (alpha 2-AR) stimulation is likely due to specific effects of the A-, B-, or C-subtype alpha 2-AR. The purpose of the present study was 1) to examine the precise localization, using subtype-specific antibodies, the B-subtype alpha 2-AR protein, the most abundant alpha 2-AR in rat kidney; and 2) to determine the effect of dietary potassium restriction, a factor known to increase alpha 2B-ARs, on the distribution of receptors. We employed a method to generate a specific antibody to the third intracellular loop of the alpha 2B-AR and demonstrated the specificity of this antibody. Our results demonstrated, for the first time, evidence for the alpha 2B-receptor protein in the basolateral membrane of proximal convoluted and straight tubules. No specific immunoreactivity was detected in other nephron segments. Finally, the increase in alpha 2B-AR expression observed previously was due to the recruitment of alpha 2B-ARs to the basolateral membrane of proximal tubules.

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-PK1 cells stably transfected with cDNA encoding the alpha 2B-adrenergic receptor (alpha 2B-AR) or A1-adenosine receptor (A1-AdR). Immunohistochemistry and Western blot analysis confirmed the basolateral and apical expression of alpha 2B-ARs and A1-AdRs, respectively. Adenylyl cyclase activity was assessed by measuring cAMP accumulation following the addition of forskolin (10 microM) 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-PK1 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-PK1 cells, and 2) factors that regulate the polarized expression of inhibitory G protein-coupled receptors may also regulate local adenylyl cyclase activity.