A system to select for mutant LLC-PK1 cells affected in cAMP mediated hormonal response using a photoactivatable analogue of vasopressin.
The photoreactive analogue of vasopressin, [1-(3-mercapto)propionic acid, 8-(N6-4-azidophenyl-amidino)lysine] vasopressin (apa-LVP) could be used to elicit stimulation of cAMP production in LLC-PK renal epithelial cells, detectable up to 24 h after photoactivation by flash photolysis. This is in contrast to cells treated with vasopressin, or apa-LVP without photoactivation, where cAMP synthesis is down regulated within 4 h. The prolonged stimulation of cAMP production induced by photoactivation of apa-LVP was demonstrated to be cytotoxic to LLC-PK1 cells, whereas the vasopressin receptor negative LLC-PK1 mutant M18 was resistant to the cytotoxic effect. A selection strategy was developed for mutants resistant to this long-term stimulation of cAMP production, whereby multiple cycles of treatment with apa-LVP and photoactivation were used. Mutants so selected were then characterized using a novel screening system for detection of the production of urokinase-type plasminogen activator in response to cAMP agonists. One mutant was examined and found to be impaired in hormonal responsiveness, whereby hormone and forskolin stimulated cAMP-mediated responses were markedly reduced. It exhibited resistance to the long-term stimulation of cAMP production elicited by apa-LVP and photoactivation. This implies that apa-LVP can be used to select for novel mutants specifically impaired in cAMP metabolism and in particular down-regulation of cAMP response.
Luzius
H
H
Max-Planck-Institut für Biophysik, Frankfurt, FRG.
Jans
D A
DA
Fahrenholz
F
F
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured / Q000201:enzymology / Q000378:metabolism / Q000502:physiology; D000242:Cyclic AMP / Q000096:biosynthesis / Q000378:metabolism / Q000494:pharmacology*; D004351:Drug Resistance; D004789:Enzyme Activation; D004847:Epithelial Cells; D005343:Fibrinolytic Agents / Q000378:metabolism; D007668:Kidney / Q000166:cytology; D008236:Lypressin / Q000031:analogs & derivatives*; D009154:Mutation; D010641:Phenotype; D010777:Photochemistry; D010960:Plasminogen Activators / Q000096:biosynthesis; D013268:Stimulation, Chemical; D013552:Swine; D014568:Urokinase-Type Plasminogen Activator / Q000096:biosynthesis; D014667:Vasopressins / Q000494:pharmacology*
D005343:Fibrinolytic Agents; C053818:vasopressin, 1-(3-mercapto)propionic acid-8-(N(6)-4-azidophenylamidino)lysine-; D014667:Vasopressins; D008236:Lypressin; D000242:Cyclic AMP; D010960:Plasminogen Activators; D014568:Urokinase-Type Plasminogen Activator; D000262:Adenylyl Cyclases