ADH resistance of LLC-pk1 cells caused by overexpression of cAMP-phosphodiesterase type-IV.
The studies of animal models of nephrogenic diabetes insipidus (NDI) suggest that abnormally high activity of cAMP phosphodiesterase (cAMP-PDE), may cause unresponsiveness to the diuretic effect of AVP. We explored whether overexpression of one of the cAMP-PDE type isozymes, PDE-IV, in [8-Arg]-vasopressin (AVP) sensitive renal epithelial LLC-PK1 cells can prevent the hormone-elicited cAMP increase. LLC-PK1 cells were stably transfected with ratPDE3.1 cDNA (which encodes for rolipram-sensitive PDE-IV), inserted in plasmid pCMV5 and then were compared with sham-transfected LLC-PK1 cells and wild LLC-PK1 cells. In the stably transfected clone (LLC-PK1-S #16), the rolipram-sensitive PDE-IV activity was about five times higher than in controls, whereas activities of other types of PDEs were not different. The presence of cognate mRNA for PDE-IV was confirmed by Northern blot. Whereas in the control cells (wild LLC-PK1 cells and sham-transfected LLC-PK1 cells), the incubation with 10(-7) M AVP increased cAMP more than tenfold, the LLC-PK1-S#16 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. The AVP-dependent cAMP accumulation in LLC-PK1-S#16 cells with overexpressed PDE-IV was restored by addition of roliprams which decreased cAMP-PDE activity to the levels similar to those in wild LLC-PK1 cells and 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 in collecting ducts of mice with hereditary nephrogenic diabetes insipidus.
Nephrology Research Unit, Mayo Clinic and Foundation, Rochester, Minnesota.
M
M
Yamaki
S
S
McIntyre
J M
JM
Murphy
J V
JV
Swinnen
M
M
Conti
T P
TP
Dousa
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000096:biosynthesis* / Q000235:genetics; D000818:Animals; D001127:Arginine Vasopressin / Q000494:pharmacology*; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism*; D006152:Cyclic GMP / Q000378:metabolism*; D007527:Isoenzymes / Q000096:biosynthesis* / Q000235:genetics; D007668:Kidney / Q000187:drug effects* / Q000201:enzymology; D013552:Swine; D014162:Transfection
D007527:Isoenzymes; D001127:Arginine Vasopressin; D000242:Cyclic AMP; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D006152:Cyclic GMP