ADH RESISTANCE OF LLC-PK1 CELLS CAUSED BY OVEREXPRESSION OF CAMP-PHOSPHODIESTERASE TYPE-IV

The studies of animal models of nephrogenic diabetes insipidus (NDI) s uggest that abnormally high activity of cAMP phosphodiesterase (cAMP-P DE) may cause unresponsiveness to the diuretic effect of AVP. We explo red whether overexpression of one of the cAMP-PDE type isozymes, PDE-I V, in [8-Arg]-vasopressin (AVP) sensitive renal epithelial LLC-PK1 cel ls can prevent the hormone-elicited cAMP increase. LLC-PK1 cells were stably transfected with ratPDE3.1 cDNA (which encodes for rolipram-sen sitive PDE-IV), inserted in plasmid pCMV5 and then were compared with sham-transfected LLC-PK1 cells and wild LLC-PK1 cells. In the stably t ransfected clone (LLC-PK1-S#16), the rolipram-sensitive PDE-IV activit y was about five times higher than in controls, whereas activities of other types of PDEs were not different. The presence of cognate mRNA f or PDE-IV was confirmed by Northern blot. Whereas in the control cells (wild LLC-PK, cells and sham-transfected LLC-PK1 cells), the incubati on with 10(-7) m AVP increased cAMP more than tenfold, the LLC-PK1-S#1 6 cells with overexpressed cAMP-PDE were resistant to cAMP-increasing effects of AVP and forskolin. However, in the same LLC-PK1-S#16 cells the cGMP increases in response to nitroprusside were not diminished. T he AVP-dependent cAMP accumulation in LLC-PK1-S#16 cells with overexpr essed PDE-IV was restored by addition of roliprams which decreased cAM P-PDE activity to the levels similar to those in wild LLC-PK1 cells an d sham-transfected LLC-PK1-#A1 cells. In contrast, inhibitors of other PDE isozymes (PDE-I or PDE-III) had little or no effect. Our findings show that excessive activity of cAMP-PDE, in this case of isozyme PDE -IV, can cause resistance to AVP which is analogous to that observed i n collecting ducts of mice with hereditary nephrogenic diabetes insipi dus.