Cilostazol (pletal): a dual inhibitor of cyclic nucleotide phosphodiesterase type 3 and adenosine uptake.

Cilostazol (Pletal), a quinolinone derivative, has been approved in the U.S. for the treatment of symptoms of intermittent claudication (IC) since 1999 and for related indications since 1988 in Japan and other Asian countries. The vasodilatory and antiplatelet actions of cilostazol are due mainly to the inhibition of phosphodiesterase 3 (PDE3) and subsequent elevation of intracellular cAMP levels. Recent preclinical studies have demonstrated that cilostazol also possesses the ability to inhibit adenosine uptake, a property that may distinguish it from other PDE3 inhibitors, such as milrinone. Elevation of interstitial and circulating adenosine levels by cilostazol has been found to potentiate the cAMP-elevating effect of PDE3 inhibition in platelets and smooth muscle, thereby augmenting antiplatelet and vasodilatory effects of the drug. In contrast, elevation of interstitial adenosine by cilostazol in the heart has been shown to reduce increases in cAMP caused by the PDE3-inhibitory action of cilostazol, thus attenuating the cardiotonic effects. Cilostazol has also been reported to inhibit smooth muscle cell proliferation in vitro and has been demonstrated in a clinical study to favorably alter plasma lipids: to decrease triglyceride and to increase HDL-cholesterol levels. One, or a combination of several of these effects may contribute to the clinical benefits and safety of this drug in IC and other disease conditions secondary to atherosclerosis. In eight double-blind randomized placebo-controlled trials, cilostazol significantly increased maximal walking distance, or absolute claudication distance on a treadmill. In addition, cilostazol improved quality of life indices as assessed by patient questionnaire. One large randomized, double-blinded, placebo-controlled, multicenter competitor trial demonstrated the superiority of cilostazol over pentoxifylline, the only other drug approved for IC. Cilostazol has been generally well-tolerated, with the most common adverse events being headache, diarrhea, abnormal stools and dizziness. Studies involving off-label use of cilostazol for prevention of coronary thrombosis/restenosis and stroke recurrence have also recently been reported.

Membrane localization of cyclic nucleotide phosphodiesterase 3 (PDE3). Two N-terminal domains are required for the efficient targeting to, and association of, PDE3 with endoplasmic reticulum.

Subcellular localization of cyclic nucleotide phosphodiesterases (PDEs) may be important in compartmentalization of cAMP/cGMP signaling responses. In 3T3-L1 adipocytes, mouse (M) PDE3B was associated with the endoplasmic reticulum (ER) as indicated by its immunofluorescent colocalization with the ER protein BiP and subcellular fractionation studies. In transfected NIH 3006 or COS-7 cells, recombinant wild-type PDE3A and PDE3B isoforms were both found almost exclusively in the ER. The N-terminal portion of PDE3 can be arbitrarily divided into region 1 (aa 1-300), which contains a large hydrophobic domain with six predicted transmembrane helices, followed by region 2 (aa 301-500) containing a smaller hydrophobic domain (of approximately 50 aa). To investigate the role of regions 1 and 2 in membrane association, we examined the subcellular localization of a series of catalytically active, Flag-tagged N-terminal-truncated human (H) PDE3A and MPDE3B recombinants, as well as a series of fragments from regions 1 and 2 of MPDE3B synthesized as enhanced green fluorescent (EGFP) fusion proteins in COS-7 cells. In COS-7 cells, the localization of a mutant HPDE3A, lacking the first 189 amino acids (aa) and therefore four of the six predicted transmembrane helices (H3A-Delta189), was virtually identical to that of the wild type. M3B-Delta302 (lacking region 1) and H3A-Delta397 (lacking region 1 as well as part of region 2) retained, to different degrees, the ability to associate with membranes, albeit less efficiently than H3A-Delta189. Proteins that lacked both regions 1 and 2, H3A-Delta510 and M3B-Delta604, did not associate with membranes. Consistent with these findings, region 1 EGFP-MPDE3B fusion proteins colocalized with the ER, whereas region 2 EGFP fusion proteins were diffusely distributed. Thus, some portion of the N-terminal hydrophobic domain in region 1 plus a second domain in region 2 are important for efficient membrane association/targeting of PDE3.

Functions of the N-terminal region of cyclic nucleotide phosphodiesterase 3 (PDE 3) isoforms.

The N-terminal portion of phosphodiesterase (PDE) 3 was arbitrarily divided into region 1 (amino acids 1-300), which contains a large hydrophobic domain with six predicted transmembrane helices, and region 2 (amino acids 301-500), with a smaller hydrophobic domain ( approximately 50 residues). To analyze these regions, full-length human (H)PDE3A and mouse (M)PDE3B and a series of N-terminal truncated mutants were synthesized in Sf9 cells. Activities of HPDE3A, H3A-Delta189, MPDE3B, and M3B-Delta196, which retained all or part of the hydrophobic domain in region 1, were recovered almost entirely in particulate fractions. H3A-Delta321 and M3B-Delta302, containing region 2, were recovered essentially equally in particulate and cytosolic fractions. H3A-Delta397 and H3A-Delta457, lacking both hydrophobic domains, were predominantly cytosolic. H3A-Delta510 and M3B-Delta604, lacking both regions 1 and 2, were virtually completely cytosolic. M3B-Delta196 eluted as a large aggregated complex during gel filtration. With removal of greater amounts of N-terminal sequence, aggregation of PDE3 decreased, and H3A-Delta607, H3A-Delta721, and M3B-Delta604 eluted as dimers. Truncated HPDE3A proteins were more sensitive than full-length HPDE3A to inhibition by lixazinone. These results suggest that the hydrophobic domains in regions 1 and 2 contain structural determinants important for association of PDE3 with intracellular membranes, as well for self-association or aggregation during gel filtration and sensitivity to a specific inhibitor.

Intracellular localization of the PDE4A cAMP-specific phosphodiesterase splice variant RD1 (RNPDE4A1A) in stably transfected human thyroid carcinoma FTC cell lines.

Cells of two human follicular thyroid carcinoma cell lines (FTC133, FTC236) were stably transfected with a cDNA encoding the PDE4A cAMP-specific phosphodiesterase (PDE) splice variant RD1 (RNPDE4A1A) so as to generate the cloned cell lines, FTC133A and FTC236A. This allowed the expression of a novel rolipram-inhibited cAMP-specific PDE activity in these cells. Unlike the parent cell lines in which Ca2+/calmodulin caused a profound activation (approx. 3-4-fold) of homogenate PDE activity, no such stimulation was evident in the RD1-expressing cell lines, indicating loss of PDE1 activity. Reverse transcriptase-PCR analysis indicated that this was due to the down-regulation of the PDE1C isoform. The novel PDE4 activity in transfected cells was located exclusively in the membrane fraction, as was immunoreactive RD1. Low concentrations of the detergent Triton X-100, but not high NaCl concentrations, allowed RD1 to be solubilized. Laser scanning confocal immunofluorescence analyses identified RD1 immunoreactivity in a discrete perinuclear region of these RD1-expressing transfected cell lines. A similar pattern of labelling was observed using the antiserum Tex1, which specifically identified the Golgi apparatus. Treatment of FTC133A cells with the Golgi-perturbing agents monensin and brefeldin A led to a similar redistribution of immunoreactive species detected using both the Tex1 and anti-RD1 antisera. It is suggested that the PDE4A splice variant RD1 contains a membrane-association signal which allows the targeted expression of RD1 within the Golgi complex of these human follicular thyroid carcinoma cell lines.

Identification and characterization of the type-IVA cyclic AMP-specific phosphodiesterase RD1 as a membrane-bound protein expressed in cerebellum.

An antiserum was generated against a dodecapeptide whose sequence is found at the C-terminus of a cyclic AMP (cAMP)-specific, type-IVA phosphodiesterase encoded by the rat 'dunc-like' cyclic AMP phosphodiesterase (RD1) cDNA. This antiserum identified a single approximately 73 kDa protein species upon immunoblotting of cerebellum homogenates. This species co-migrated upon SDS/PAGE with a single immunoreactive species observed in COS cells transfected with the cDNA for RD1. Native RD1 in cerebellum was found to be predominantly (approximately 93%) membrane-associated and could be found in isolated synaptosome populations, in particular those enriched in post-synaptic densities. Fractionation of lysed synaptosomes on sucrose density gradients identified RD1 as co-migrating with the plasma membrane marker 5'-nucleotidase. Laser scanning confocal and digital deconvolution immunofluorescence studies done on intact COS cells transfected with RD1 cDNA showed RD1 to be predominantly localized to plasma membranes but also associated with the Golgi apparatus and intracellular vesicles. RD1-specific antisera immunoprecipitated phosphodiesterase activity from solubilized cerebellum membranes. This activity had the characteristics expected of the type-IV cAMP phosphodiesterase RD1 in that it was cAMP specific, exhibited a low Km cAMP of 2.3 microM, high sensitivity to inhibition by 4-[3-(cyclopentoxyl)-4-methoxyphenyl]-2-pyrrolidone (rolipram) (Ki approximately 0.7 microM) and was unaffected by Ca2+/calmodulin and low concentrations of cyclic GMP. The phosphodiesterase activities of RD1 solubilized from both cerebellum and transfected COS cell membranes showed identical first-order thermal denaturation kinetics at 50 degrees C. Native RD1 from cerebellum was shown to be an integral protein in that it was solubilized using the non-ionic detergent Triton X-100 but not by either re-homogenization or high NaCl concentrations. The observation that hydroxylamine was unable to cause the release of RD1 from either cerebellum or COS membranes and that [3H]palmitate was not incorporated into the RD1 protein immunoprecipitated from COS cells transfected with RD1 cDNA, indicated that RD1 was not anchored by N-terminal acylation. The engineered deletion of the 25 residues forming the unique N-terminal domain of RD1 caused both a profound increase in its activity (approximately 2-fold increase in Vmax) and a profound change in intracellular distribution. Thus, immunofluorescence studies identified the N-terminal truncated species as occurring exclusively ion the cytosol of transfected COS cells. The cDNA for RD1 thus appears to encode a native full-length type-IVA phosphodiesterase that is expressed in cerebellum.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of two splice variant forms of type-IVB cyclic AMP phosphodiesterase, DPD (rPDE-IVB1) and PDE-4 (rPDE-IVB2) in brain: selective localization in membrane and cytosolic compartments and differential expression in various brain regions.

In order to detect the two splice variant forms of type-IVB cyclic AMP phosphodiesterase (PDE) activity, DPD (type-IVB1) and PDE-4 (type-IVB2), anti-peptide antisera were generated. One set ('DPD/PDE-4-common'), generated against a peptide sequence found at the common C-terminus of these two PDEs, detected both PDEs. A second set was PDE-4 specific, being directed against a peptide sequence found within the unique N-terminal region of PDE-4. In brain, DPD was found exclusively in the cytosol and PDE-4 exclusively associated with membranes. Both brain DPD and PDE-4 activities, isolated by immunoprecipitation, were cyclic AMP-specific (KmcyclicAMP: approximately 5 microM for DPD; approximately 4 microM for PDE-4) and were inhibited by low rolipram concentrations (K1rolipram approximately 1 microM for both). Transient expression of DPD in COS-1 cells allowed identification of an approx. 64 kDa species which co-migrated on SDS/PAGE with the immunoreactive species identified in both brain cytosol and membrane fractions using the DPD/PDE-4-common antisera. The subunit size observed for PDE-4 (approx. 64 kDa) in brain membranes was similar to that predicted from the cDNA sequence, but that observed for DPD was approx. 4 kDa greater. Type-IV, rolipram-inhibited PDE activity was found in all brain regions except the pituitary, where it formed between 30 and 70% of the PDE activity in membrane and cytosolic fractions when assayed with 1 microM cyclic AMP, PDE-4 formed 40-50% of the membrane type-IV activity in all brain regions save the midbrain (approx. 20%). DPD distribution was highly restricted to certain regions, providing approx. 35% of the type-IV cytosolic activity in hippocampus and 13-21% in cortex, hypothalamus and striatum with no presence in brain stem, cerebellum, midbrain and pituitary. The combined type-IVB PDE activities of DPD and PDE-4 contributed approx. 10% of the total PDE activity in most brain regions except for the pituitary (zero) and the mid-brain (approx. 3%. The isolated cDNAs for DPD and PDE-4 appear to reflect transcription products which are expressed in vivo in brain. The unique N-terminal domain of PDE-4 is suggested to target this PDE to membranes in brain. Type-IVB PDEs are differentially expressed in various brain regions, indicating that there are tissue-specific controls on both the expression of the gene and the splicing of its products.

Molecular cloning and expression, in both COS-1 cells and S. cerevisiae, of a human cytosolic type-IVA, cyclic AMP specific phosphodiesterase (hPDE-IVA-h6.1).

Screening a human T lymphocyte cDNA library with a phosphodiesterase (PDE) specific probe resulted in the isolation of two overlapping cDNA clones, h2.2 and h6.1, that encode a type IV, rolipram inhibited cAMP-specific PDE. Clones h2.2 and h6.1 were 1015 bp and 2288 bp in length, respectively, and overlapped for 984 bp with only one nucleotide difference. The h6.1 cDNA was extended at the 5'-end by 1304 bp, with respect to h2.2, and encoded an incomplete ORF (lacking an initiation codon) of 668 amino acids. The merged nucleotide sequence of h6.1/h2.2 exhibited 99.5% homology in the ORF (ten nucleotide changes resulting in six amino acid changes), and 95% homology in the 3'-untranslated region, with the previously reported human PDE-IVA cDNA [Livi G. P., Kmetz P., Mchale M. M., Cieslinski L. B., Sathe G. M., Taylor D. P., Davis R. L., Torphy T. J. and Balcarek J. M. (1990) Mol. Cell Biol. 10, 2678-2686]. The sequence reported for h6.1/h2.2 matched that found for IVA clones isolated from three other human cDNA libraries, a human genomic cosmid clone and pcr amplified products of the exon covering these differences in two individuals. The h6.1 cDNA was engineered to generate a complete ORF by building in the 56 bp, including the initiation codon, present in hPDE-IVA-Livi and missing from the 5'-end of h6.1, producing a cognate ORF encoding a protein of 687 amino acids but differing in five amino acids which lay in or adjacent to the putative catalytic domain. The complete h6.1 ORF was engineered for expression in both Saccharomyces cerevisiae and in COS-1 cells. Integration of a single copy of the engineered ORF of h6.1, under the transcriptional control of a constitutive yeast promoter, at the pep4 locus of a S. cerevisiae strain lacking both yeast PDE genes resulted in functional complementation of the yeast pde-phenotype. Yeast strains with functional PDE were a light creamy white colour, while strains devoid of PDE activity were a dull brown colour. Expression of h6.1 in COS-1 cells led to the production of a typical type IV PDE activity in that cAMP, but not cGMP, served as substrate and its activity was insensitive to either Ca2+/CaM or cGMP but was inhibited by low concentrations of rolipram.(ABSTRACT TRUNCATED AT 400 WORDS)

Engineered deletion of the unique N-terminal domain of the cyclic AMP-specific phosphodiesterase RD1 prevents plasma membrane association and the attainment of enhanced thermostability without altering its sensitivity to inhibition by rolipram.

Full-length cDNA for the rat brain rolipram-sensitive cyclic AMP phosphodiesterase (PDE), RD1 was introduced into the expression vector pSVL. COS cells transfected with the recombinant vector pSVL-RD1 exhibited a 30-55% increase in homogenate PDE activity, which was abolished by rolipram (10 microM). Removal of the first 67 nucleotides of the RD1 cDNA yielded a truncated enzyme called Met26-RD1 which lacked the N-terminal first 25 amino acids. Whereas approx. 75% of RD1 activity was membrane-associated, Met26-RD1 activity was found exclusively in the cytosol fraction. Expression of RD1 nearly doubled membrane-associated PDE activity, while expression of Met26-RD1 increased cytosolic activity by approx. 30%. Membrane RD1 activity was found to be primarily associated with the plasma membrane, was not released by either high concentrations of NaCl or by a 'hypotonic shock' treatment, but was solubilized with low concentrations of Triton X-100. Phase separation of membrane components with Triton X-114 showed partition of RD1 into both the aqueous and detergent-rich phases, whereas Met26-RD1 partitioned exclusively into the aqueous phase. Both RD1 and Met26-RD1 specifically hydrolysed cyclic AMP; were unaffected by either Ca2+/calmodulin or by low cyclic GMP concentrations; exhibited linear Lineweaver-Burke plots with similar Km values for cyclic AMP (4 microM); both were potently and similarly inhibited by rolipram (Ki approx. 0.5 microM) and were similarly inhibited by cilostamide and 3-isobutyl-1-methylxanthine. Thermal inactivation, at 50 degrees C, showed that while the cytosolic-located fraction of RD1 (t0.5 approx. 3 min) and Met26-RD1 (t0.5 approx 3 min) were similarly thermolabile, membrane-bound RD1 was considerably more thermostable (t0.5 approx. 11 min). Treatment of both cytosolic RD1 and Met26-RD1 with Triton X-100 did not affect their thermostability, but solubilization of membrane RD1 activity with Triton X-100 markedly decreased its thermostability (t0.5 approx. 5 min). The N-terminal domain of RD1 appears not to influence either the substrate specificity or inhibitor sensitivity of this enzyme, but it does contain information which can allow RD1 to become plasma membrane-associated and thereby adopt a conformation which has enhanced thermostability.

A major C-peptide deletion prevents secretion of a mutant human proinsulin from transfected monkey kidney cells.

The biosynthesis and secretion of human proinsulin and a mutant human proinsulin with a major deletion in the C-peptide, (des 38-62)proinsulin, was studied in monkey kidney cells (Cos-7) transfected with cDNAs encoding the respective normal or mutant human preproinsulins. Transfected cells were labelled with [3H]leucine, and insulin-like material was immunoprecipitated and analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. It was found that the prepeptide was removed from both the normal and mutant preproinsulins, and that there was no further processing to insulin. The normal proinsulin was rapidly released from the transfected cells, with little intracellular accumulation, while the mutant proinsulin was retained within the cell, with only small quantities of radio-labelled material in the medium. The intracellular mutant proinsulin was membrane bound and located predominantly within a microsomal fraction. These results suggest that C-peptide plays an important role in the efficient transfer of proinsulin through the early stages of the secretory pathway.