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.
Okusa
M D
MD
Department of Medicine, University of Virginia Health Sciences Center, Charlottesville 22908, USA.
Huang
L
L
Momose-Hotokezaka
A
A
Huynh
L P
LP
Mangrum
A J
AJ
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000262:Adenylyl Cyclases / Q000378:metabolism*; D000316:Adrenergic alpha-Agonists / Q000494:pharmacology; D000818:Animals; D000068438:Brimonidine Tartrate; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D016764:Cell Polarity; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D004847:Epithelial Cells / Q000502:physiology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D007668:Kidney; D011810:Quinoxalines / Q000494:pharmacology; D018341:Receptors, Adrenergic, alpha-2 / Q000096:biosynthesis / Q000502:physiology*; D018047:Receptors, Purinergic P1 / Q000096:biosynthesis / Q000502:physiology*; D011994:Recombinant Proteins / Q000096:biosynthesis; D014162:Transfection
D000316:Adrenergic alpha-Agonists; D011810:Quinoxalines; D018341:Receptors, Adrenergic, alpha-2; D018047:Receptors, Purinergic P1; D011994:Recombinant Proteins; D005576:Colforsin; D000068438:Brimonidine Tartrate; D000242:Cyclic AMP; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D015056:1-Methyl-3-isobutylxanthine