High Activity of Low-Michaelis-Menten Constant 3′,5′-Cyclic Adenosine Monophosphate-Phosphodiesterase Isozymes in Renal Inner Medulla of Mice With Hereditary Nephrogenic Diabetes Insipidus*

Abstract

Our previous studies on microdissected kidney tubule segments indicate that the failure of vasopressin (VP) to increase cAMP content in collecting ducts of mice with hereditary nephrogenic diabetes insipidus (NDI mice) is due to abnormally rapid cAMP catabolism via cyclic-3',5'-nucleotide phosphodiesterases (PDE). Furthermore, the VP-stimulated cAMP accumulation can be restored by addition of PDE isozyme-specific inhibitors. To elucidate the biochemical basis of the NDI syndrome, we analyzed PDE activities in extracts from inner medullary tissues of NDI mice and from control mice separated with the use of ionex fast protein liquid chromatography on a Mono-Q column. In extracts of inner medullary tissues from either control or NDI mice, the low Michaelis-Menten constant (Km) cAMP-PDE activity specific for cAMP as a substrate (cAMP-PDE) was eluted from a Mono-Q column with linear sodium acetate gradient as peak 3 at Na-acetate concentration (0.75-0.93 M) and was well separated from fractions containing the Ca(2+)-calmodulin sensitive PDE. The cAMP-PDE activity in peak 3 was significantly higher in NDI mice (greater than delta + 100%) than in controls. The sensitivity to effect of cAMP-PDE isozyme-specific inhibitors, rolipram and cilostamide, indicates that peak 3 consists predominantly (approximately 75%) of the rolipram-sensitive PDE-IV isozyme and a minor portion (approximately 25%) of cilostamide-sensitive PDE-III isozyme in both control and NDI mice. Higher activity of PDE-IV in NDI mice was due to 2.4 times higher apparent maximum velocity compared to controls, whereas the apparent Km for cAMP was not different. Our results show that low Km cAMP-PDE activities, predominantly PDE-IV, are higher in inner medulla of NDI mice. We suggest that the higher activity of PDE-IV, and to a lesser degree perhaps also PDE-III, accounts for rapid cAMP hydrolysis, which prevents the increase of cAMP generated in the response to VP in collecting ducts of NDI mice.

A comprehensive analysis of gene expression profiles in distal parts of the mouse renal tubule

“…A significant expression of 3-isobutyl-1-methylxanthine (IBMX)-insensitive Pde8a and Pde8b provides a molecular basis for the IBMXinsensitive fraction of phosphodiesterase activity. The rolipram-sensitive cAMP-specific Pde4b and Pde4a have been proposed as important regulators of Avpr2-induced signaling cascade [23]. However, in the DCT/CNT and the CCD transcriptomes presented here, they are expressed only at moderate levels.…”

Generation and phenotypic characterization of Pde1a mutant mice

“…Contrary to this expectation, we found that at 12 months of age these mice had a mild urine concentration defect after 24 hrs dehydration (3375±312 and 3394±273 compared to 4057±255 mOsm/L in wild-type, P<0.01, Fig 3D ) and were able to excrete a water load faster (54±29 and 77±35 compared to 37±25% of 2 ml ip over 6 hrs in wild-type mice, P<0.01, Fig 3D ). Since PDE4D controls a cAMP pool that regulates the expression, phosphorylation and translocation of AQP2 to the apical membrane of the collecting duct principal cells [ 18 ] and constitutive activation of PDE4 was found to be responsible for a mouse model of nephrogenic diabetes insipidus [ 19 ], we wondered whether the increased activity of PDE4 in the kidneys of the Pde1a mutants could be responsible for the mild concentration defect observed in these mice. Consistent with this, the expression of pSer269-AQP2 was decreased in the kidneys of Pde1a Del15 and Pde1a InsA homozygous mice compared to those of wild-type controls ( Fig 3D ).…”

Physiology and pathophysiology of the vasopressin-regulated renal water reabsorption

“…After its cAMP-induced activation and subsequent phosphorylation of PKA target proteins like AQP2, PKA signaling is abrogated by PDE-mediated cAMP degradation. The activity of PDE can have profound physiological effects because increased PDE activity was found to cause hereditary nephrogenic diabetes insipidus (NDI) in mice [ 88 , 95 , 209 ]. Activated PKA directly phosphorylates and activates the PDE4D3 isoform, thereby confining its own activity [ 28 , 201 ].…”