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The biochemical and pharmacological characteristics in human proinflammatory cells of BRL 50481 [3-(N,N-dimethylsulfonamido)-4-methyl-nitrobenzene], a novel and selective inhibitor of phosphodiesterase (PDE) 7, are described. BRL 50481 inhibited the activity of hrPDE7A1 expressed in baculovirus-infected Spodoptera frugiperda 9 cells in a competitive manner (Ki value of 180 nM) and was 416 and 1884 times less potent against PDE3 and 38 and 238 times less potent against PDE4 at a substrate concentration of 1 microM and 50 nM cAMP, respectively. Western blotting identified HSPDE7A1 but not HSPDE7A2 in three human cell types that are implicated in the pathogenesis of chronic obstructive lung disease, namely, CD8+ T-lymphocytes, monocytes, and lung macrophages. BRL 50481 had no effect on the proliferation of CD8+ T-lymphocytes and only marginally (approximately 2-11%) reduced the generation of tumor necrosis factor (TNF)alpha from blood monocytes and lung macrophages. However, in the presence of BRL 50481 the inhibitory effect of rolipram was enhanced on all three cell types. The expression of HSPDE7A1 was increased in a time-dependent manner in monocytes that were "aged" in culture medium. Under this condition, BRL 50481 now inhibited TNFalpha generation in a concentration-dependent manner. In aged monocytes, rolipram, Org 9935 (a PDE3 inhibitor), and prostaglandin E2 inhibited TNFalpha generation in a concentration-dependent manner and interacted additively with BRL 50481. BRL 50481 is the first fully documented PDE7 inhibitor that has acceptable selectivity for in vitro studies. Furthermore, although BRL 50481 had only a modest inhibitory effect per se on the proinflammatory cells studied, it acted at least additively with other cAMP-elevating drugs, especially when HSPDE7A1 was up-regulated.

1. Of the four major phosphodiesterase 4 (PDE4) subtypes, PDE4A, PDE4B and PDE4D are widely expressed in human inflammatory cells, including monocytes and T lymphocytes. We explored the functional role of these subtypes using ten subtype-selective PDE4 inhibitors, each belonging to one of two classes: (i) dual PDE4A/PDE4B inhibitors or (ii) PDE4D inhibitors. 2. These compounds were evaluated for their ability to inhibit antigen-stimulated T-cell proliferation and bacterial lipopolysaccharide (LPS)-stimulated tumour necrosis factor alpha (TNFalpha) release from peripheral blood monocytes. 3. All compounds inhibited T-cell proliferation in a concentration-dependent manner; with IC50 values distributed over an approximately 50 fold range. These compounds also inhibited TNFalpha release concentration-dependently, with a wider ( approximately 1000 fold) range of IC50 values. 4. In both sets of experiments, mean IC50 values were significantly correlated with compound potency against the catalytic activity of recombinant human PDE4A or PDE4B when analysed by either linear regression of log IC50 values or by Spearman's rank-order correlation. The correlation between inhibition of inflammatory cell function and inhibition of recombinant PDE4D catalytic activity was not significant in either analysis. 5. These results suggest that PDE4A and/or PDE4B may play the major role in regulating these two inflammatory cell functions but do not rule out PDE4D as an important mediator of other activities in mononuclear leukocytes and other immune and inflammatory cells. Much more work is needed to establish the functional roles of the PDE4 subtypes across a broader range of cellular functions and cell types.

Phosphodiesterase 4 (PDE4) inhibitors are novel anti-inflammatory compounds. Unfortunately, the archetypal PDE4 inhibitor rolipram produces central nervous system and gastrointestinal side-effects. To exploit these agents, we need to identify PDE4 inhibitors that retain the anti-inflammatory activity with a reduced potential to elicit unwanted side-effects. PDE4 possesses both cyclic AMP catalytic activity that is inhibitable by rolipram and a high affinity binding site for rolipram. The function of this high affinity rolipram binding site is unclear; however, certain pharmacological effects of PDE4 inhibitors are associated with competition for this site. Since PDE4 inhibitors suppress both monocyte and neutrophil activation, the present experiments were carried out to establish a correlation between suppression of monocyte activation [tumor necrosis factor alpha (TNF alpha) formation] or suppression of neutrophil activation (degranulation) with inhibition of either PDE4 catalytic activity or [3H] rolipram binding. Suppression of TNF alpha formation demonstrated a strong correlation with inhibition of PDE4 catalytic activity (r=0.87; P0.01; Spearman's Rho = 0.79, P0.05), whereas there was no correlation with inhibition of [3H]rolipram binding(r=0.21, P0.5; Spearman's Rho=0.16, P0.5). Suppression of neutrophil degranulation was not associated with inhibition of PDE4 catalytic activity (r=0.25, P0.4; Spearman's Rho=0.33, P0.2), but was associated with inhibition of [3H]rolipram binding (r=0.68, P0.05; Spearman's Rho=0.6, P=0.06). These results indicate that anti-inflammatory effects of PDE4 inhibitors can be associated with either inhibition of PDE4 catalytic activity or high affinity rolipram binding.

Recombinant baculoviruses were constructed to express cDNAs encoding two distinct subtypes of human cAMP-specific phosphodiesterase (hPDE4A and hPDE4B). Infection of Spodoptera frugiperda insect cells with the appropriate recombinant baculoviruses resulted in high level production of biologically-active protein as measured by enzymatic activity and immunoblotting using subtype-specific anti-hPDE4 antisera. Both recombinant proteins showed catalytic activity with a low Km (approximately 3 microM) for cAMP (with no cGMP hydrolyzing activity) and were inhibited by R-rolipram with apparent Kis of 0.38 and 0.25 microM, respectively. The recombinant enzymes also contained saturable, stereoselective and high-affinity rolipram-binding sites (Kd approximately 2 nM). Thus, insect cell-derived hPDE4s possess kinetic properties analogous to native enzymes as well as to recombinant enzymes produced in yeast.

We have cloned a Candida albicans gene, which encodes a cyclic nucleotide phosphodiesterase (PDEase), by complementation in a Saccharomyces cerevisiae PDEase-deficient mutant. The deduced amino acid sequence is similar to that of the low-affinity PDEase of S. cerevisiae (PDE1) and the cyclic nucleotide PDEase (PD) of Dictyostelium discoideum. Biochemical analysis of recombinant protein produced in S. cerevisiae indicated that the enzyme behaves as a PDE1 homologue: it hydrolyses both cAMP (Km = 0.49 mM) and cGMP (Km = 0.25 mM), does not require divalent cations for maximal activity and is only moderately inhibited by millimolar concentrations of standard PDEase inhibitors. Based on these data, we designate the C. albicans we have cloned, PDE1. Low-stringency genomic Southern blots showed cross-hybridization between C. albicans PDE1 and DNA from Candida stellatoidea, but not with DNA from S. cerevisiae or several closely related Candida species.

We have isolated cDNA clones from human frontal cortex cDNA libraries that encode a unique subtype of the low-Km, cAMP-specific phosphodiesterases (PDEs IV). The 564-amino acid sequence of the protein (human brain PDE IV (hPDE IVB)) shows significant homology to a PDE IV subtype expressed in human monocytes (hPDE IVA), particularly within the approximately 300-amino acid PDE IV catalytic domain. The degree of protein sequence identity is much greater between hPDE IVB and a homolog derived from rat brain (92% over 562 amino acids) than between hPDE IVB and hPDE IVA (76% over 538 amino acids), suggesting a greater subtype-specific versus species-specific conservation of protein sequence. Analysis of the distribution of hPDE IVB mRNA expression revealed a restricted pattern, with an approximately 4-kilobase mRNA detected in brain, heart, lung, and skeletal muscle and not in placenta, liver, kidney, or pancreas. An additional approximately 5-kilobase hPDE IVB-related mRNA species was detected in brain tissue. Recombinant hPDE IVB displayed all of the expected kinetic characteristics for a PDE IV, including sensitivity to the isozyme-selective inhibitor rolipram (Ki = 0.085 microM). Scatchard analysis of (R)-[3H]rolipram binding data suggested the presence of two noninteracting high affinity rolipram-binding sites (Kd = 0.4 and 6 nM) or a negatively cooperative interaction among multiple binding sites.

The role of the phosphodiesterase type IV isozyme (PDE IV) in the regulation of cerebrovascular tone was investigated in the canine basilar artery in vitro and in vivo. The PDE isozymes extracted from the canine basilar artery were isolated by diethylaminoethanol (DEAE)-Sepharose affinity chromatography and identified based on sensitivity to tsozyme-selective PDE inhibitors. [3H]cAMP hydrolysis was observed in one major and one minor peak of activity. The predominant peak was inhibited by the addition of cGMP (25%), siguazodan (26%), rolipram (39%), and the combination of siguazodan and rolipram (95%). Selective PDE IV inhibitors BRL 61063, rolipram, and denbufylline were equieffective inhibitors of [3H]-ccAMP hydrolysis mediated by PDE IV isolated from the canine basilar artery [concentrations producing 50% inhibition (IC50s) = 0.21 ± 0.05 μM, 0.67 ± 0.23 μM, and 0.73 ±0.16 μM, respectively]. In precontracted isolated ring segments of the canine basilar artery, selective PDE IV inhibitors produced potent and complete relaxation (IC50s 50s = 4.5 μM and >10 μM, respectively). Vasorelaxation produced by PDE IV inhibitors was not altered by removing the endothelium, l-NAME, or adenosine receptor antagonism. In a canine model of acute cerebral vasospasm, all three selective PDE IV inhibitors reversed basilar artery spasm produced by autologous blood without altering mean arterial blood pressure. In contrast, prolonged treatment with BRL 61063 failed to alter the development of basilar spasm in the two hemorrhage canine models of chronic cerebral vasospasm. Denbufylline-induced relaxation in vitro was also significantly impaired in basilar arteries obtained from the model of chronic vasospasm. In conclusion, PDE IV appears to be the predominant isozyme regulating vascular tone mediated by cAMP hydrolysis in cerebral vessels. In addition, vasorelaxation modulated by PDE IV is compromised in chronic cerebral vasospasm associated with subarachnoid hemorrhage.

We have isolated cDNA clones representing cyclic AMP (cAMP)-specific phosphodiesterases (PDEases) from a human monocyte cDNA library. One cDNA clone (hPDE-1) defines a large open reading frame of ca. 2.1 kilobases, predicting a 686-amino-acid, ca. 77-kilodalton protein which contains significant homology to both rat brain and Drosophila cAMP PDEases, especially within an internal conserved domain of ca. 270 residues. Amino acid sequence divergence exists at the NH2 terminus and also within a 40- to 100-residue domain near the COOH-terminal end. hPDE-1 hybridizes to a major 4.8-kilobase mRNA transcript from both human monocytes and placenta. The coding region of hPDE-1 was engineered for expression in COS-1 cells, resulting in the overproduction of cAMP PDEase activity. The hPDE-1 recombinant gene product was identified as a low-Km cAMP phosphodiesterase on the basis of several biochemical properties including selective inhibition by the antidepressant drug rolipram. Known inhibitors of other PDEases (cGMP-specific PDEase, cGMP-inhibited PDEase) had little or no effect on the hPDE-1 recombinant gene product. Human genomic Southern blot analysis suggests that this enzyme is likely to be encoded by a single gene. The presence of the enzyme in monocytes may be important for cell function in inflammation. Rolipram sensitivity, coupled with homology to the Drosophila cAMP PDEase, which is required for learning and memory in flies, suggests an additional function for this enzyme in neurobiochemistry.

Studies by various investigators have demonstrated that the low Km, cAMP-specific phosphodiesterase (PDE IV) is selectively inhibited by a group of compounds typified by rolipram and Ro 20-1724. In addition to inhibiting the catalytic activity of PDE IV, rolipram binds to a high affinity binding site present in brain homogenates. Although it has been assumed that the high affinity rolipram-binding site is PDE IV, no direct evidence has been produced to support this assumption. The present studies were undertaken to determine whether the rolipram-binding site is coexpressed with PDE IV catalytic activity in Saccharomyces cerevisiae genetically engineered to express human recombinant monocytic PDE IV (hPDE IV). Expressing hPDE IV cDNA in yeast resulted in a 20-fold increase in PDE activity that was evident within 1 h of induction and reached a maximum by 3-6 h. The recombinant protein represented hPDE IV as judged by its immunoreactivity, molecular mass (approximately 88 kDa), kinetic characteristics (cAMP Km = 3.1 microM; cGMP Km greater than 100 microM), sensitivity to rolipram (Ki = 0.06 microM), and insensitivity to siguazodan (PDE III inhibitor) and zaprinast (PDE V inhibitor). Saturable, high affinity [3H] (R)-rolipram-binding sites (Kd = 1.0 nM) were coexpressed with PDE activity, indicating that both binding activity and catalytic activity are properties of the same protein. A limited number of compounds were tested for their ability to inhibit hPDE IV catalytic activity and compete for [3H](R)-rolipram binding. Analysis of the data revealed little correlation (r2 = 0.35) in the structure-activity relationships for hPDE IV inhibition versus competition for [3H] (R)-rolipram binding. In fact, certain compounds (e.g. (R)-rolipram Ro 20-1724) possessed a 10-100-fold selectivity for inhibition of [3H] (R)-rolipram binding over hPDE IV inhibition, whereas others (e.g. dipyridamole, trequinsin) possessed a 10-fold selectivity for PDE inhibition. Thus, although the results of these studies demonstrate that hPDE IV activity and high affinity [3H](R)-rolipram binding are properties of the same protein, they do not provide clear cut evidence linking the binding site with the PDE inhibitory activity of rolipram and related compounds.