Proline transport in MDCK cells expressing a mutant regulatory subunit of cAMP-dependent protein kinase.
cAMP-dependent protein kinase (cAK) regulates the activity of several membrane-bound ion channels and carriers. The role of cAK in regulating the transport of osmoprotective amino acids in the distal tubule is unknown. We examined the regulation of Na(+)- and Cl(-)-dependent proline transport in MDCK cells expressing a mutant murine regulatory subunit (RIalpha(AB)) of cAK. For this purpose, MDCK cells were transfected with an expression vector encoding RIalpha(AB) driven by the metallothionein 1 promoter together with neomycin-resistance (NEO) gene. Stable G418-resistant colonies were isolated that expressed RIalpha(AB) as demonstrated by Northern hybridization analysis using a cDNA probe for RIalpha and cAK assay that showed decreased enzyme activity. A clone constitutively expressing high levels of RIalpha(AB) (M(AB)) in a Zn-independent manner and a control clone transfected with the NEO gene alone (M(neo)) were selected for transport studies. We examined the effect of the cAMP-stimulating agents forskolin (F) and IBMX on NaCl-dependent uptake of [(3)H]proline by confluent monolayers of transfected MDCK cells. While F/IBMX-induced mean inhibition of proline transport in M(neo) cells was 48 and 45% at 5 and 15 min, respectively, inhibition of proline uptake in M(AB) cells was 9% (5 min) and 0% (15 min). These data demonstrate that the inhibition of NaCl-linked proline transport in response to elevated cAMP is reversed in MDCK clones that express mutant cAK and provide evidence that cAK mediates the modulatory action of cAMP on proline transport. cAK may play an important role in controlling transport of proline and other osmoprotective amino acids in the renal tubule.
Zelikovic
I
I
Department of Pediatrics, Division of Nephrology, University of Washington School of Medicine, 4800 Sand Point Way NW, Seattle, Washington 98105, USA. i_zelikovic@rambam.health.gov.il
Wager-Miller
J
J
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport; D015152:Blotting, Northern; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000096:biosynthesis*; D018076:DNA, Complementary / Q000378:metabolism; D004285:Dogs; D004351:Drug Resistance / Q000235:genetics; D005822:Genetic Vectors; D007684:Kidney Tubules / Q000378:metabolism; D008668:Metallothionein / Q000235:genetics; D051379:Mice; D009154:Mutation; D009355:Neomycin / Q000494:pharmacology; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D011392:Proline / Q000378:metabolism* / Q000493:pharmacokinetics; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012965:Sodium Chloride / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection
D018076:DNA, Complementary; D010726:Phosphodiesterase Inhibitors; D011500:Protein Synthesis Inhibitors; D005576:Colforsin; D012965:Sodium Chloride; D008668:Metallothionein; D011392:Proline; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D009355:Neomycin; D015056:1-Methyl-3-isobutylxanthine