A novel LLC-PK1 renal epithelial cell mutant impaired in in vivo down-regulation of cAMP-mediated hormonal response.
A novel "cAMP-resistant" variant of LLC-PK1 renal epithelial cells which is impaired in in vivo down-regulation of response following hormonal stimulation of adenylate cyclase (AC) is described. Compared to parental cells, the BIB27 mutant exhibited markedly higher in vivo activation of cAMP-dependent protein kinase (cAMP-PK) in response to the hormones salmon calcitonin (SCT) or [Arg8]-vasopressin (AVP) or the AC activator forskolin. The activation of cAMP-PK subsequent to agonist stimulation also persisted much longer in the mutant than in LLC-PK1 cells, although the cAMP-PK of BIB27 cells was normal in terms of both absolute levels and regulation by cAMP in vitro. Intracellular cAMP accumulation was also much higher in BIB27 than in LLC-PK1 cells following agonist stimulation. Production of cAMP could be detected in BIB27 cells even 12 h after treatment with AVP or SCT, whereas cAMP production in LLC-PK1 had returned to basal within 1 and 8 h, respectively. High levels of free cAMP-PK catalytic (C) subunit in BIB27 persisted even 12 h after hormone addition, meaning that the higher cAMP production in BIB27 did not result in the normal down-regulation of cAMP-PK C subunit levels. In vitro AC activity in BIB27 cell homogenates could be stimulated by hormones or receptor-independent agonists, but to a lesser extent than in LLC-PK1 cell homogenates. The SCT and AVP concentrations promoting half-maximal AC activation in BIB27 cells were about 10- and 3-fold higher than parental, respectively. BIB27 accordingly appeared to possess a mutation in AC responsible for the impairment of both in vitro response to agonists and the normal in vivo down-regulation processes following hormonal stimulation.
Jans
D A
DA
Friedrich Miescher-Institut, Basel, Switzerland.
Resink
T J
TJ
Hemmings
B A
BA
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D001127:Arginine Vasopressin / Q000378:metabolism / Q000494:pharmacology*; D002116:Calcitonin / Q000378:metabolism / Q000494:pharmacology*; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism / Q000494:pharmacology*; D004351:Drug Resistance; D004789:Enzyme Activation / Q000187:drug effects; D004848:Epithelium / Q000378:metabolism; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D007668:Kidney / Q000378:metabolism*; D009154:Mutation; D011494:Protein Kinases / Q000378:metabolism*; D013552:Swine
D001127:Arginine Vasopressin; D005576:Colforsin; D006165:Guanylyl Imidodiphosphate; C028815:salmon calcitonin; D002116:Calcitonin; D000242:Cyclic AMP; D011494:Protein Kinases; D000262:Adenylyl Cyclases; D015056:1-Methyl-3-isobutylxanthine