A System to Select for Mutant LLC-PK₁Cells Affected in camp Mediated Hormonal Response Using a Photoactivatable Analogue of Vasopressin

Abstract

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.

Oxytocin induced cAMP‐dependent protein kinase activation and urokinase‐type plasminogen activator production in LLC‐PK1 renal epithelial cells is mediated by the vasopressin V2‐receptor

“…The V,-specific agonists dVSAVP and SAVP showed similar &s (Table IIA, The above results were elaborated by testing the various analogues for the stimulation of uPA-production in LLC-PK, cells. uPA activity was detected by spotting 8h conditioned media onto a casein plasminogen agar, and incubating at 37°C [30,31], activity being indicated by zones of plasminogen-dependent proteolytic clearing in the agar (Fig. 1).…”