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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.

Chiral HPLC resolution of the phosphodiesterase IV (PDE IV) inhibitor rolipram (1) provided (-)-1, and this enantiomer was converted into its 1-(4-bromobenzyl) derivative, (+)-2. X-ray structural analysis of (+)-2 established the absolute configuration as R, which provides the first direct evidence for a previously assumed assignment of configuration. The crystal structure of (+)-2 and the PDE inhibitory activity of both enantiomers of 2 are discussed in the context of a previously proposed topological model.

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.

We attempted to identify and establish the role of cyclic nucleotide phosphodiesterase (PDE) isozymes in human basophils by using standard biochemical techniques as well as describing the effects of isozyme-selective and nonselective inhibitors of PDE. The nonselective PDE inhibitors, theophylline and 3-isobutyl-1-methylxanthine, inhibited anti-IgE-induced release of histamine and leukotriene C4 (LTC4) from basophils. This inhibition was accompanied by elevations in cAMP levels. Rolipram, an inhibitor of the low Km cAMP-specific PDE (PDE IV), inhibited the release of both histamine and LTC4 from activated basophils and increased cAMP levels in these cells. In contrast, mediator release from basophils was not inhibited by either siguazodan or SK&F 95654, inhibitors of the cGMP-inhibited PDE (PDE III) or zaprinast, an inhibitor of the cGMP-specific PDE (PDE V). SK&F 95654 failed to elevate basophil cAMP in these experiments whereas zaprinast induced significant increases in cAMP content. The inhibitory effect of rolipram on mediator release was potentiated by siguazodan or SK&F 95654, but not by zaprinast. SK&F 95654 also enhanced the ability of rolipram to increase cAMP content. Forskolin, a direct activator of adenylate cyclase, inhibited IgE-dependent release of mediators from basophils and increased cAMP levels in these cells. These effects were enhanced by rolipram, but not by SK&F 95654 or zaprinast. The cell permeant analog of cAMP, dibutyryl cAMP, inhibited mediator release from these cells, a property not shared by either dibutyryl-cGMP or sodium nitroprusside, an activator of soluble guanylate cyclase. The presence of both PDE III and PDE IV was confirmed by partially purifying and characterizing PDE activity in broken cell preparations. Overall, these data lend support to the hypothesis that cAMP inhibits mediator release from basophils and suggest that the major PDE isozyme responsible for regulating cyclic AMP content in these cells is PDE IV, with a minor contribution from PDE III. However, the finding that zaprinast caused increases in cAMP without inhibiting mediator release indicates that cAMP accumulation is not invariably linked to an inhibition of basophil activation.

Fibroblasts, as a major source of extracellular interstitial connective tissue matrix, play an important role in wound healing and the development of fibrosis. The phosphodiesterase (PDE) 4 inhibitor cilomilast inhibits fibroblast chemotaxis and fibroblast-mediated gel contraction. Using the Boyden blindwell chamber chemotaxis assay and the type I collagen gel contraction model, this study investigated whether specific cytokines modulate cilomilast's inhibitory effect through regulation of endogenous PGE(2) production. Human recombinant IL-1beta stimulated PGE(2) production and shifted the cilomilast concentration-dependence curve to the left in both assay systems, indicating increased sensitivity to cilomilast. In contrast, human recombinant IL-4 inhibited PGE(2) production and shifted the cilomilast concentration-dependence curve to the right in both systems. In summary, the inhibitory effect of cilomilast on fibroblast migration and collagen gel contraction is modulated by IL-1beta and IL-4 through regulation of PGE(2) production.

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.

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.

Human peripheral blood monocytes were treated for 4 h with a combination of the beta-agonist salbutamol (3 microM) and the low-Km cAMP-specific phosphodiesterase (PDE4) inhibitor rolipram (30 microM) to produce a prolonged elevation of cAMP and consequent increase in PDE activity. After this treatment, isozyme-selective PDE inhibitors were used to characterize the cAMP PDE profiles of high-speed supernatants before and after DEAE-Sepharose column chromatography. These experiments, in which total soluble PDE activity was increased by 58%, showed that the increased PDE activity is due to up-regulation of PDE4 and that at least two of the four subtypes are up-regulated. Experiments in whole cells demonstrated that this relatively modest increase in PDE4 activity has significant functional consequences, reducing cAMP accumulation in response to both PGE2 and lower, though not maximal, concentrations of rolipram. Further characterization of PDE4 subtype expression in control and treated monocytes, using polymerase chain reaction and Western blotting with subtype-specific peptide antibodies, showed that resting monocytes express both mRNA and protein for PDE4A, PDE4B and PDE4D. The amount of message for PDE4A and PDE4B appeared to increase upon up-regulation, whereas mRNA for PDE4D was not detected in treated cells. Western blots showed increases in the amount of protein for both PDE4A and PDE4B after treatment. We conclude that the PDE4 subtypes are differentially regulated upon prolonged exposure to elevated cAMP, with the consequence that the PDE4 profiles of control and treated cells differ not only in total activity but also in the relative proportions of the subtypes represented.

Elevation of cyclic AMP (cAMP) content inhibits eosinophil function. Because phosphodiesterase IV (PDE IV) appears to be the major PDE isozyme present in eosinophils, inhibitors of this isozyme should suppress eosinophil activation. Previous studies on PDE IV have revealed that this enzyme possesses both cAMP catalytic activity that is inhibitable by rolipram, a prototypical PDE IV inhibitor, and a high-affinity binding site for rolipram. The function of this high-affinity rolipram binding site relative to the inhibitory action of compounds is not clear because the rank order potency of PDE IV inhibitors for competing with [3H]-rolipram binding is distinct from that for inhibiting cAMP hydrolysis. Consequently, the present experiments were carried out to fulfill the following objectives: 1) to determine whether PDE IV inhibitors suppress eosinophil function and, if so, 2) to establish a correlation between this functional activity and inhibition of PDE IV catalytic activity or interaction with the high-affinity rolipram binding site. Various PDE inhibitors produced approximately 60% maximal inhibition of formylmethionine-leucine-phenylalanine-induced superoxide anion production, so that IC30 concentrations were used as a basis to compare the potency of various PDE inhibitors. Selective PDE IV inhibitors were the most potent compounds tested. PDE inhibitors selective for other isozymes were devoid of activity or considerably less potent.(ABSTRACT TRUNCATED AT 250 WORDS)

In the present study phosphodiesterase (PDE) isozymes in human airway smooth muscle were isolated, identified and characterized, and the functional roles of these isozymes in intact bronchi were evaluated by using isozyme-selective PDE inhibitors. PDE isozymes in human trachealis were isolated by using a combination of DEAE-Sepharose and calmodulin-Sepharose affinity chromatography, and were identified based upon their kinetic characteristics as well as their sensitivity to allosteric modulators and isozyme-selective PDE inhibitors. By using this approach, six distinct isozymes were identified: two calmodulin-stimulated PDEs (PDE I alpha and PDE I beta), cyclic GMP (cGMP)-stimulated PDE (PDE II), cGMP-inhibited PDE (PDE III), cyclic AMP (cAMP)-specific PDE (PDE IV) and cGMP-specific PDE (PDE V). PDEs III and IV were the major cAMP-hydrolyzing enzymes present, whereas PDEs I alpha, I beta and V accounted for most of the cGMP-hydrolytic activity. In carbachol-precontracted small (0.5-2 mm diameter) or large (4-15 mm diameter) human bronchus, zaprinast (10 nM-30 microM), the selective PDE V inhibitor, was without marked relaxant activity (13%), whereas rolipram (30 microM), the selective PDE IV inhibitor, produced approximately 25% relaxation in both preparations. Siguazodan was a significantly more effective relaxant than zaprinast or rolipram in large bronchus, producing a maximum relaxation of 77 +/- 15% at a concentration of 30 microM, whereas in small bronchus 30 microM siguazodan elicited 20 +/- 6% relaxation. Similar results were obtained in large bronchi contracted with leukotriene (LT) D4 (0.1 microM). The ability of isozyme-selective PDE inhibitors to potentiate agonist-induced relaxation was studied in LTD4-contracted large bronchi. Siguazodan (10 microM), but not rolipram (10 microM) or zaprinast (10 microM), potentiated the relaxant response in LTD4-contracted large bronchus to isoproterenol, a beta adrenoceptor agonist thought to induce relaxation via a cAMP-mediated mechanism. In contrast, zaprinast (10 microM), but not siguazodan (10 microM), potentiated relaxation induced by sodium nitroprusside, a nitrovasodilator that relaxes airway smooth muscle via a cGMP-mediated mechanism. The most striking observation from functional studies was that the combination of rolipram and siguazodan produced a much greater relaxation of small or large human bronchi than either agent alone, indicating an interaction between PDE III and PDE IV inhibitors that was at least additive and, in some cases, synergistic.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Experiments were conducted using undifferentiated U937 cells, a human monocytic cell line, to establish an in vitro model to examine the hormonal regulation of the cyclic AMP (cAMP)-specific phosphodiesterase (PDE IV). Standard chromatographic techniques, coupled with the use of inhibitors and activators that are selective for various phosphodiesterase (PDE) isozymes, were used to establish the PDE isozyme profile in supernatant fractions of U937 cells. When PDE activity was assessed using 1 microM [3H]cAMP as a substrate, 70 to 90% of the total U937 cell supernatant activity in the major peak eluting from anion-exchange columns was inhibited by 30 microM rolipram, a selective inhibitor of PDE IV. The remaining activity was nearly abolished by 10 microM siguazodan or 10 microM cyclic GMP (cGMP,) selective inhibitors of the cGMP-inhibited PDE. Kinetic analyses of the enzyme activity contained within this major peak of PDE activity revealed a cAMP Km = 3 microM and a rolipram Ki = 0.5 microM, values characteristic of PDE IV. Additional studies revealed the presence of a small amount of Ca++/calmodulin-stimulated PDE, but no cGMP-stimulated PDE or cGMP-specific PDE activity. In an effort to induce PDE activity in intact U937 cells by producing a sustained increase in cAMP content, cells were treated for 4 hr with salbutamol (1 microM), rolipram (30 microM) or a combination of both agents. The combination of salbutamol and rolipram produced a 2- to 3-fold increase in PDE activity in U937 cells; when used alone, rolipram was without effect whereas salbutamol induced an increase that was approximately one-half of that observed with the combination. Isozyme isolation and characterization revealed that the overall elevation of cellular PDE activity could be accounted for by a 2- to 3-fold increase in the Vmax of PDE IV with no change in its Km. The induction of PDE IV by salbutamol was: 1) concentration- and time-dependent; 2) detectable only after prolonged (2-4 hr) agonist exposure; 3) preceded by an increase in cAMP content and an activation of cAMP-dependent protein kinase; 4) mimicked by 8-bromo-cAMP and prostaglandin E2; 5) reversible within 3 hr of salbutamol removal; and 6) abolished by cycloheximide or actinomycin D. Collectively, these results indicate that the major PDE isozyme in the soluble fraction of U937 cells is PDE IV and that the activity of this enzyme is increased markedly in cells after prolonged exposure to agents that increase cAMP content.

We attempted to identify and establish the role of cyclic nucleotide phosphodiesterase (PDE) isozymes in human basophils by using standard biochemical techniques as well as describing the effects of isozyme-selective and nonselective inhibitors of PDE. The nonselective PDE inhibitors, theophylline and 3-isobutyl-1-methylxanthine, inhibited anti-IgE-induced release of histamine and leukotriene C4 (LTC4) from basophils. This inhibition was accompanied by elevations in cAMP levels. Rolipram, an inhibitor of the low Km cAMP-specific PDE (PDE IV), inhibited the release of both histamine and LTC4 from activated basophils and increased cAMP levels in these cells. In contrast, mediator release from basophils was not inhibited by either siguazodan or SKF 95654, inhibitors of the cGMP-inhibited PDE (PDE III) or zaprinast, an inhibitor of the cGMP-specific PDE (PDE V). SKF 95654 failed to elevate basophil cAMP in these experiments whereas zaprinast induced significant increases in cAMP content. The inhibitory effect of rolipram on mediator release was potentiated by siguazodan or SKF 95654, but not by zaprinast. SKF 95654 also enhanced the ability of rolipram to increase cAMP content. Forskolin, a direct activator of adenylate cyclase, inhibited IgE-dependent release of mediators from basophils and increased cAMP levels in these cells. These effects were enhanced by rolipram, but not by SKF 95654 or zaprinast. The cell permeant analog of cAMP, dibutyryl cAMP, inhibited mediator release from these cells, a property not shared by either dibutyryl-cGMP or sodium nitroprusside, an activator of soluble guanylate cyclase. The presence of both PDE III and PDE IV was confirmed by partially purifying and characterizing PDE activity in broken cell preparations. Overall, these data lend support to the hypothesis that cAMP inhibits mediator release from basophils and suggest that the major PDE isozyme responsible for regulating cyclic AMP content in these cells is PDE IV, with a minor contribution from PDE III. However, the finding that zaprinast caused increases in cAMP without inhibiting mediator release indicates that cAMP accumulation is not invariably linked to an inhibition of basophil activation.

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.

The low-Km cAMP-specific phosphodiesterases (PDEases) are of great pharmacological significance because of their involvement in regulating cAMP concentrations, which, in turn, are responsible for mediating the cellular response to extracellular signals such as hormones and neurotransmitters. We recently reported the isolation of a cDNA clone that encodes a human monocyte low-Km, rolipram-sensitive, cAMP PDEase (isozyme IV). We have engineered the inducible expression of this human PDEase in yeast. Cells of Saccharomyces cerevisiae contain two genes that encode cAMP PDEases. PDEase-deficient mutants are viable but exhibit specific growth arrest phenotypes associated with elevated intracellular cAMP content; these phenotypes include heat shock sensitivity and the inability to grow on acetate as a carbon source. We show that functional expression of our human cAMP PDEase in a genetically engineered PDEase-deficient strain of S. cerevisiae reverses these aberrant phenotypes. Furthermore, under conditions for growth arrest, rolipram is cytotoxic to PDEase-deficient mutants expressing the human cAMP PDEase, indicating that it is capable of inhibiting the human recombinant enzyme in vivo. This system can be used in the development of a yeast cell-based assay for isozyme-selective inhibitors of the human recombinant cAMP PDEase.

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.

Cyclic nucleotide phosphodiesterases (PDEs) from canine trachealis were characterized with respect to their kinetic properties, sensitivity to selective inhibitors, and subcellular distribution. Extracts from whole tissue homogenates were applied to DEAE-Sepharose anion exchange columns and eluted with a linear sodium acetate gradient. Three major peaks of PDE activity were resolved. The first (PDE I), which eluted at 0.2 M sodium acetate, was applied to a calmodulin (CaM)-Sepharose affinity column and resolved into CaM-insensitive and CaM-sensitive PDEs. The CaM-insensitive isozyme (PDE Ia) had apparent Km values of 135 microM (cAMP) and 4 microM (cGMP) and was potently inhibited by zaprinast (Ki = 0.1 microM). The CaM-sensitive isozyme (PDE Ic) had apparent Km values of 1 microM (cAMP) and 2 microM (cGMP) and was inhibited by zaprinast with an apparent Ki of 35 microM. The second peak of activity (PDE II) from the anion exchange column eluted at 0.3 M sodium acetate and had apparent Km values of 93 microM (cAMP) and 60 microM (cGMP). The enzyme displayed positive cooperativity with respect to the hydrolysis of cAMP (nH = 1.7). Low concentrations of cGMP (0.1-1 microM) reduced cooperativity (nH = 1.1) and increased the hydrolysis of 1 microM cAMP. The third peak of activity from the anion exchange column eluted at 0.6 M sodium acetate and displayed anomalous kinetics that suggested the presence of two isozymes. This was supported by the observation that enzyme activity was only partially inhibited by SKF 94120 or Ro 20-1724 but was abolished by the combination of the two PDE inhibitors. Subsequent studies confirmed the existence of two isozymes. The first, PDE III, had apparent Km values of 0.3 microM (cAMP) and 8 microM (cGMP) and was inhibited by cGMP (IC50 = 0.1 microM), SKF 94120 (Ki = 7.8 microM), and SKF 94836 (Ki = 0.4 microM). The second, PDE IV, had apparent Km values of 4 microM (cAMP) and 40 microM (cGMP) and was inhibited by Ro 20-1724 (Ki = 5.2 microM) and rolipram (Ki = 0.5 microM) but not by cGMP. Assessment of the 100,000 x g soluble and particulate PDE activity revealed that all five isozymes were present in the soluble fraction, but only four isozymes (PDEs Ia, Ic, III, and IV) were present in the particulate fraction. These results indicate that five distinct PDE isozyme exist in canine trachealis and that these isozymes differ in their kinetic characteristics, sensitivity to activators and inhibitors, and subcellular distribution.