ADH Resistance of LLC-pk1 Cells Caused by Overexpression of cAMP-Phosphodiesterase Type-IV
| Title: | ADH Resistance of LLC-pk1 Cells Caused by Overexpression of cAMP-Phosphodiesterase Type-IV |
|---|---|
| Authors: | Yamaki, Mario; McIntyre, Steven; Murphy, Josie M.; Swinnen, Johannes V.; Conti, Marco; Dousa, Thomas P. |
| Publisher: | Kidney International |
| Date Published: | June 01, 1993 |
| Reference Number: | 132 |
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.
This translation by the NDI Foundation is to assist the lay reader. To provide a clear, accessible interpretation of the original article, we eliminated or simplified some technical detail and complicated scientific language. We concentrated our translation on those aspects of the article dealing directly with NDI. The NDI Foundation thanks the researchers for their work toward understanding and more effectively treating this disorder.
© Copyright NDI Foundation 2007 (JC)
Normally, AVP binds with its receptor, the vasopressin-2 receptor (V2R), in the base and sides of the principal cells of the kidney collecting duct. When AVP binds with V2R, it sets in motion a molecular sequence which results in the kidney being able to reabsorb the water flowing through the kidney collecting ducts. The liquid that is not reabsorbed becomes the concentrated urine that is later voided. In this way the kidney is able to balance body water.
This is how the molecular sequence goes: AVP bound to V2R couples with a stimulatory G protein; this raises the adenylyl cyclase level; this increases the activity of the metabolic regulator, cAMP. cAMP, through a process that is as yet not entirely clear, stimulates the water transporting protein called aquaporin-2 (AQP2) to travel to the apex of the collecting duct cell membrane and insert itself within it. The AQP2s make the cell membrane much more water permeable than normal, which is how the kidney is able to reabsorb the water flowing through its collecting ducts. A malfunction of any one of the steps in this sequence can result in NDI and its attendant symptoms: chronic, excessive thirst (polydipsia) and the chronic passage of large volumes of urine (polyuria).
Yamaki, et al., devised an experiment to verify whether cAMP-PDE caused the kidney to be unresponsive to AVP. They did not microdisect mice kidney as other studies have done. Instead they transfected cell cultures with cDNA or genomic DNA coding for PDE-IV in order to increase the activity of PDE-IV in those cell cultures. They developed kidney cell cultures and injected one group with rat PDE 3.1 (a type of PDE-IV derived from rats). This resulted in the cells in the culture having a cAMP-PDE-IV activity level about five times higher than the control culture. The authors then incubated the PDE-IV rich culture and the control cultures with AVP and measured the increase in cAMP levels.
Whereas the control cultures showed a more than 10-fold increase in cAMP levels, the culture with the overabundance of PDE-IV showed no increase. However, when the authors added rolipram to this PDE rich culture, its ability to respond to AVP, as measured by increases in cAMP activity, was restored. PDE-IV is sensitive to rolipram. The addition of it to the PDE-IV rich cell culture decreased the PDE-IV activity to a level similar to the control cultures. This finding helped confirm that abnormally high levels of PDE-IV can indeed cause resistance to AVP. The authors suggest that since PDE-IV participates in breaking down cAMP, perhaps when the PDE-IV is overabundant cAMP gets broken down too rapidly to adequately participate in the molecular sequence that leads to urine concentration.