Identification and characterization of isoenzymes of cyclic nucleotide phosphodiesterase in human kidney and heart, and the effects of new cardiotonic agents on these isoenzymes

Abstract

The present study was done to identify and characterize the isoenzymes of cyclic nucleotide phosphodiesterase (PDE) and to determine their intracellular distribution in human kidney and heart. The in vitro effects of new cardiotonic agents, namely, NSP-805 (4,5-dihydro-5-methyl-6-[4-[(2-methyl-3-oxo-1-cyclopentenyl)amino] phenyl]-3(2H)-pyridazinone), TZC-5665 (6-[4-[2-[3-(5-chloro-2-cyanophenoxy)-2-hydroxypropylamino]-2-methylpropylamino]phenyl]-5-methyl-4,5-dihydro-3(2 H)-pyridazinone) and its metabolites, OPC-18790 ((±)-6-[3-(3,4-dimethoxybenzylamino)-2-hydroxypropoxy]-2-(1H)-quinolinone), MS-857 (4-acetyl-l-methyl-7-(4-pyridyl)-5,6,7,8-tetrahydro-3(2H)-isoquinolinone) and E-1020 (1,2-dihydro-6-methyl-2-oxo-5-(imidazo[1,2-a]pyridin-6yl)-3-pyridine carbonitrile hydrochloride monohydrate), on these human PDE isoenzymes were also investigated.

PDE isoenzymes were separated from cytosolic and particulate fractions of homogenates of human kidney and heart by DEAE-Sepharose chromatography. PDE isoenzymes were identified by their elution characteristics, substrate specificities, sensitivities to regulation by effectors and by the use of isoenzyme-specific inhibitors.

In a cytosolic fraction from kidney, Ca²⁺/calmodulin-dependent PDE (CaM-PDE), cyclic GMP-stimulated PDE (cGS-PDE), cyclic GMP-inhibited PDE (cGI-PDE) and two forms of cyclic AMP-specific PDE (cAMP-PDE) were resolved. One form of cAMP-PDE (cAMP-PDEα), which was eluted at a lower ionic strength than cGI-PDE during DEAE-Sepharose chromatography, was newly recognized in human tissues, though the other form (cAMP-PDEβ), which eluted later than cGI-PDE, had been previously isolated. Both of these cAMP-specific PDEs had similar properties in that they predominantly hydrolyzed cAMP with similar K _m values for cAMP and were inhibited to almost equal extents by 3-isobutyl-l-methylxanthine (IBMX) but were hardly inhibited by cGMP. However, cAMP-PDEα was inhibited about 10 times more weakly (on the basis of IC₅₀ values) by rolipram (4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone) and Ro 20-1724 (4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone than was cAMP-PDEβ. In a cytosolic fraction from heart ventricle, four distinct PDE isoenzymes, CaM-PDE, cGS-PDE, cGI-PDE and cAMP-PDEβ, were recognized. cAMP-PDEβ was the major component of the cAMP-hydrolyzing activity in the cytosolic fraction from human kidney, while CaM-PDE and cGI-PDE represented more than 90% of the total cAMP phosphodiesterase activity in the cytosolic fraction from human heart. In the particulate fractions from human kidney and heart, three activities, those of cGI-PDE and of two forms of cAMP-PDE, were identified. In kidney, cAMP/PDEα was the main cAMP-hydrolyzing PDE, while in heart cGI-PDE accounted for most of the activity. Furthermore, we evaluated the inhibitory effects on these human PDE isoenzymes of newly synthesized compounds with inotropic effects, namely, NSP-805, metabolites of TZC-5665 referred to as M-1 (6-(4-aminophenyl)-5-methyl-4,5-dihydro-3(2H)-pyridazinone) and M-2 (6-(4-acetyl-aminophenyl)-5-methyl-4,5-dihydro-3(2H)-pyridazinone), OPC-18790, MS-857 and E-1020. These drugs potently inhibited the activity of cGI-PDE and very weakly inhibited the activities of CaM-PDE and cGS-PDE. With respect to inhibitory effects on cardiac cAMP-PDEβ, there were some differences between the pyridazinone derivatives, for example NSP-805 and M-2, and the nonpyridazinone derivatives OPC-18790, MS-857 and E-1020. From the IC₅₀ values, it was clear that the pyridazinone derivatives inhibited the activity of cGI-PDE at concentrations that were two to four orders of magnitude lower than that required for the inhibition of the activity of cAMP-PDEβ, while for the nonpyridazinone derivatives the difference was about one order of magnitude. The inhibition of the activity of human cardiac cGI-PDE by NSP-805, M2, OPC-18790, MS-857 and E-1020 was competitive with respect to cAMP with K _i values of 0.012, 0.32, 0.42, 1.3 and 0.15 μmol/l, respectively.

These results suggest that there may be two isoforms of cAMP-PDE, which exist not only in the particulate fraction but also in the cytosolic fraction of human tissues, and that PDE inhibitors, which exert their cardiotonic effects by inhibiting cGI-PDE, have different selectivities with respect to the inhibition of the other human PDE isoenzymes.