3T3 Mouse Fibroblast Cells Research Articles

Improving cell affinity of ­poly(D,L‐lactide) film modified by anhydrous ammonia plasma treatment

AbstractAnhydrous ammonia gaseous plasma treatment was used for improving cell affinity of the poly(D,L‐lactide) film surface. The contact angle was measured and the surface energy was calculated through harmonic mean equations. The surface chemical composition of the sample was identified by X‐ray photoelectron spectroscopy (XPS) analysis. It was demonstrated that the O‐containing and N‐containing groups such as hydroxyl and amine were incorporated onto the surface of films. The effect of preserving methods for maintaining the hydrophilicity was also investigated. The result indicated that preserving at low temperature (0–4 °C) was sufficient to maintain the improvements. It could resolve the problem of hydrophilicity being partially lost with preserving time after plasma treatment. Mouse 3T3 fibroblast cells were used as model cells to compare the cell affinity of the modified films and control. The ammonia plasma treatment could promote the cells attachment and cells growth. After 4 days culture, the plasma treated films yielded almost two magnitude cells compared with the control. Copyright © 2002 John Wiley & Sons, Ltd.

PDGF-regulated rab4-dependent recycling of αvβ3 integrin from early endosomes is necessary for cell adhesion and spreading

Background: It has been postulated that the regulation of integrin vesicular traffic facilitates cell migration by internalizing integrins at the rear of the cell and transporting them forward within vesicles for exocytosis at the leading edge to form new contacts with the extracellular matrix. The rab family of GTPases control key targeting events in the endo/exocytic pathway; therefore, these GTPases may be involved in the regulation of cell-matrix contact assembly.Results: The endo/exocytic cycle of αvβ3 and α5β1 integrins was studied using mouse 3T3 fibroblast cell lines. In serum-starved cells, internalized integrins were transported through rab4-positive, early endosomes and arrived at the rab11-positive, perinuclear recycling compartment approximately 30 min after endocytosis. From the recycling compartment, integrins were recycled to the plasma membrane in a rab11-dependent fashion. Following treatment with PDGF, αvβ3 integrin, but not α5β1, was rapidly recycled directly back to the plasma membrane from the early endosomes via a rab4-dependent mechanism without the involvement of rab11. This rapid recycling pathway directed αvβ3 to numerous small puncta distributed evenly across the dorsal surface of the cell, and the integrin only became localized into focal complexes at later times following PDGF addition. Interestingly, inhibition of PDGF-stimulated αvβ3 recycling using dominant-negative rab4 mutants compromised cell adhesion and spreading on vitronectin (a ligand for αvβ3), but adhesion to fibronectin (a ligand for α5β1 and αvβ3) was unchanged.Conclusions: We propose that growth factor-regulated, rab4-dependent recycling of αvβ3 integrin from early endosomes to the plasma membrane is a critical upstream event in the assembly of cell-matrix contacts.

Intracellular Measurement of Prostaglandin E2: Effect of Anti-inflammatory Drugs on Cyclooxygenase Activity and Prostanoid Expression

Cyclooxygenase (COX) converts arachidonic acid to prostaglandin (PG) H2, which is further metabolized to various prostaglandins, prostacyclin and thromboxane A2. COX exists in at least two different isoforms. COX-1 is constitutively expressed, whereas COX-2 is induced by proinflammatory stimuli. Prostaglandin E2 is a major metabolite of COX activation. In order to compare the activity of target ligands and COX inhibitors on PGE2 synthesis and release, the responsiveness of several cell lines to the calcium ionophore A23187, bacterial lipopolysaccharide (LPS), nonsteroidal anti-inflammatory drugs (NSAIDs), and the glucocorticoid, dexamethasone, were investigated. For intracellular measurements, the culture supernatant was aspirated, and the cells were thoroughly washed and lysed with dodecyltrimethylammonium bromide. Intracellular and secreted PGE2 were measured with an enzyme immunoassay. A23187 and LPS increased intracellular PGE2 in a dose-dependent manner. Kinetic experiments with A23187-stimulated mouse 3T3 fibroblast cells revealed a distinct biphasic response in COX activity. In the presence of NSAIDs or dexamethasone, there was a dose-dependent inhibition in intracellular PGE2 with A23187-stimulated 3T3 cells. Inhibitory studies demonstrated an apparent increased sensitivity of COX activity to the action of inhibitors when measuring intracellular PGE2 compared with using cell culture supernatants. Indeed, intracellular PGE2 levels were comprehensively reduced in the presence of low concentrations of inhibitor. The utilization of cell culture lysates and, in particular, measurement of intracellular PGE2 should prove useful for identifying new COX inhibitors.

The effect of Insulin like growth factor-I (IGF-I) and IGF binding protein-3 (IGFBP-3) on cellular proliferation in mouse 3T3 fibroblast cells

Background: Cell growth is a balance between cell proliferation and cell death. Insulin-like growth factor-I(IGF-I), which binds IGF-I receptor(IGF-IR), mediates cellular proliferation as a potent mitogen. IGF binding protein-3(IGFBP-3) as a circulating major IGFBP can inhibit or enhance the effects of IGF-I on cellular growth by binding IGFs. Methods: We investigated the expressions of mRNA of IGF-I and IGF-IR by northern blot and phosphorylation of IGF-IR with the treatment of IGF-I by western blot in 3T3 fibroblast cells. The cellular proliferations of 3T3 cells with the treatments of IGF-I were evaluated using -thymidine incorporation and MTT assay. Also to observe the effect of IGFBP-3 on cellular proliferation, 3T3 cells were treated with anti-IGFBP-3 and (monoclonal antibody to IGF-IR) alone or in combination. Results: Our results demonstrated that 3T3 cells showed mRNA expressions of IGF-I and IGF-IR and the IGF-I increased phosphorylation of IGF-IR. The treatments of 3T3 cells with IGF-I increased cellular proliferation in 5 % and 1 % seruma-containing media, not in serum-free media. The addition of anti-IGFBP-3 to neutralize IGFBP-3 showed 2-fold increase of cellular proliferation, and also co-incubation of anti-IGFBP-3 and together showed similar increase of cellular proliferation in 3T3 cells. Interestingly, when the cells were pretreated with for 4 hr, prior to the simultaneous addition of and anti-IGFBP-3, anti-IGFBP-3-mediated cellular proliferation was decreased to control level. All of these results suggest that free IGF-I released from IGF-I/IGFBP-3 complex would be involved in the cellular proliferation. Conclusion: IGF-I is a mitogen through the activation of IGF-IR in 3T3 cells, and IGFBP-3 could be a potent inhibitor for IGF-I action by binding IGF-I.

High-level expression of human interleukin-17 in the yeast Pichia pastoris.

Human interleukin-17 (hIL-17) gene without the signal sequence was isolated from activated peripheral blood lymphocytes by RT-PCR, then highly expressed in the yeast Pichia pastoris in the form of the glycosylated monomer. The monomer of rhIL-17 stimulated mouse fibroblast 3T3 cells to secrete IL-6 and was specifically bound to its receptors on 3T3 cells.

A human B cell line AF10 expressing HIL-17.

AF10, a human B cell line, is a mycoplasma-free variant cloned from the human IgE myeloma cell line U266. Total RNA isolated from the AF10 cells was used as template for RT-PCR, and a specific product of about 411bp corresponding to the coding region of hIL-17 lack of a leading sequence obtained. The sequence of the RT-PCR product is the same to that reported in the literature. The expressed rhIL-17 in E. coli can induce 10- to 15-fold increase of IL-6 secretion by mouse fibroblast 3T3 cells. It is for the first time until now that hIL-17 messager has been detected in B cell. There may exist some potential relationship between hIL-17 and myeloma cells.

Antitumor activity of (10 E, 12 Z)-9-hydroxy-10,12-octadecadienoic acid from rice bran

Two types of hydroxy acids, (10 E, 12 Z)-9-hydroxy-10,12-octadecadienoic acid [1] and (9 Z, 11 E)-13-hydroxy-9,11-octadecadienoic acid [2], were obtained as cytotoxic compounds from a water extract of rice bran. The cytotoxicity of acid 1 was stronger than that of acid 2 in vitro, against P388 mouse leukemia cells. Acid 1 slightly inhibited the growth of a Balb c mouse 3T3 fibroblast cells ( BALB c 3T3), but strongly inhibited that of simian virus 40-transformed 3T3 cells (SV-T2); 1 was therefore, found to have cytotoxicity against cancer cells. On the other hand, 2 inhibited neither normals cells nor transformed cells. Intraperitoneal injection of acid 1 effectively prolonged the survival time of C3H HeJ mice after intraperitoneal transplantation of MM46 mammary tumor cells. The difference in antitumor activity between 1 and 2 was also observed in vivo.

Persistent Echovirus Infection of Mouse Cells Expressing the Viral Receptor VLA-2

Mouse cells are not susceptible to infection with echovirus 1 (EV-1) because they lack the viral receptor, human VLA-2. Two mouse fibroblast cell lines, L cells and 3T3 cells, were made susceptible to EV-1 infection after transformation with cDNAs of human VLA-2. After EV-1 infection, L cell transformants of human VLA-2 (α2β1 L cells) develop cytopathic effect (CPE) as expected, while 3T3 cell transformants of human VLA-2 (α2β1 3T3 cells) or the α2 subunit of human VLA-2 (α2 3T3 cells) become persistently infected. The distinct outcome is not a result of differential virus growth on these transformants because one-step growth curve analysis reveals little difference in EV-1 replication in both cell lines. In addition, 3T3 cell transformants expressing the poliovirus receptor (Pvr 3T3 cells) are lysed during poliovirus infection, suggesting that 3T3 cells are not intrinsically resistant to CPE caused by enterovirus infection. The results of limit dilution assays indicate that all EV-1-infected α2 3T3 cells produce infectious virus. All EV-1-infected α2 3T3 cells remain viable after EV-1 infection, and the kinetics of cell growth were not altered. FACS analysis reveals that receptor down-regulation is not involved in the establishment of persistent infection. Furthermore, inhibition of host protein synthesis was not observed in EV-1-infected α2 3T3 or α2β1 L cells. Since α2β1 L cells are lysed by EV-1 infection, these findings suggest that virus-induced translation inhibition is not a determinant of cell killing.

Induction of the Mouse Ribonucleotide Reductase R1 and R2 Genes in Response to DNA Damage by UV Light

Ribonucleotide reductase is responsible for the production of deoxyribonucleotides required for DNA synthesis and consists of two nonidentical subunits, proteins R1 and R2. Here we show that the R1 promoter can be induced up to 3-fold, and the R2 promoter is induced up to 10-fold by UV light in a dose-dependent manner. This was demonstrated using serum-starved, synchronized G0/G1 mouse fibroblast 3T3 cells stably transformed with different R1 and R2 promoter-luciferase reporter gene constructs. R2 promoter activation requires a minimal promoter, containing a TTTAAA element plus the transcription start, and either three upstream DNA-protein binding regions or one proximal, NF-Y binding region. This is different from proliferation-specific activation of the R2 promoter. Using Northern blotting we show a preferential accumulation of the minor, 1. 6-kilobase R2 transcript in irradiated cells, whereas the levels of the major 2.1-kilobase transcript are unchanged. No R2 promoter activation was observed after treatment of mouse cells with agents reported to induce the ribonucleotide reductase genes in Saccharomyces cerevisiae such as hydroxyurea or methylmethane sulfonate. This indicates that activation of ribonucleotide reductase gene expression is specific for nucleotide excision repair in mammalian cells and not part of a general response to DNA damage.

IN VIVO AND IN VITRO ANALYSIS OF THE HUMAN LONG CHAIN ACYL COA DEHYDROGENASE(LCAD) GENE PROMOTER REGION. † 889

LCAD is a mitochondrial flavoenzyme encoded on nuclear genome which catalizes the first reaction in fatty acid beta-oxidation. We characterized 2 kb of upstream 5′-flanking region of the human LCAD gene by sequence analysis. This promoter region lacks TATA or CAAT boxes, is extremely GC-rich, contains SP1 binding sites, and has several nuclear response element (NRRE) consensus hexamers. Chimeric plasmids containing various 5′-flanking genomic fragments fused upstream of the chloramphenicol acetyl transferase(CAT) reporter gene were transfected into human hepatoma cells, neonatal rat cardiomyocytes, and mouse 3T3 fibroblast cells. Minimal promoter is -200 to +1 upstream of the Cap site. Two suppressor sequences, at -288 to -259, and -1048 to -1010 respectively, both contain two NRRE consensus binding sites, and interact with COUP-TF on gel shift analysis. To further define the promoter region of the human LCAD gene by transgenic mouse analysis, constructs containing the LCAD 5′-flanking regions, -1,800/+1 and -200/+1, linked with luciferase reporter gene excised from pT3/T7-LUC luciferase expression vector were micro-injected into mouse oocytes by standard techniques. Founder animals incorporating the LCAD-luciferase chimeric gene identified by PCR and tail DNA blot experiments were bred with normal C57BL/6J mice. F1 mice positive for the incorporated LCAD-luciferase fusion gene have been tested for expression by luciferase assay from various tissue extracts, and immunohistochemistry. Distribution of luciferase activity in various tissues of progeny of the human LCAD -200 or -1800/ luciferase indicated LCAD -200/+1 region contains an active promoter with some tissue specification; -1800/+1 region contains suppressor element. Thus, both in vitro andin vivo studies showed that LCAD NRRE-281 and LCAD NRRE-1032 contain suppressor activity, and LCAD -200/+1 contains promoter function with some tissue specificity. Funded by NIH grant #PO1-DK33487.

In vitro and in vivo gene transfer to pulmonary cells mediated by cationic liposomes

Cationic liposomes have been proposed as alternative to adenovirus in the treatment of cystic fibrosis lung desease. Therefore, we have investigated the efficiency of two lipid mixtures in mediating gene transfer in in vitro and in vivo models. The cationic lipid DOTMA (N-(1-(2,3(dioleyloxy)propyl)-n,n,n-trimethylammoniumchloride) and DOGS (dioctadecylamidoglycylspermine) were used in combination with the neutral lipid DOPE (dioleoylphosphatidylethanolamine). The relative transfection efficiencies of the two cationic liposomes were tested using the bacterial β-galactosidase (lacZ) and the firefly luciferase genes. Gene expression was detected in both cell lines and primary culture of rhesus monkey airway epithelium after transfection with plasmid DNA complexed with DOGS/DOPE or DOTMA/DOPE. Transfection efficiency of both types of lipids was higher in the mouse fibroblast 3T3 cell line as compared to human carcinoma A549 cells and primary epithelial cultures. Administration of DNA-liposome complexes via intratracheal instillation resulted in expression of the lacZ and luciferase marker gene in the mouse airways. In vivo transfection mediated by both types of liposomes were proven to be far less efficient than adenovirus treatment.

Angiostatic role of astrocytes: suppression of vascular endothelial cell growth by TGF-beta and other inhibitory factor(s).

Our previous in vivo analyses have suggested that astrocytes play a key role in retinal vascularization by inducing endothelial cell differentiation. Here we demonstrate that medium conditioned by cultured rat brain astrocytes (ACM) contains factors, including transforming growth factor-beta (TGF-beta), that inhibit endothelial cell growth. Serum-free medium conditioned for 1-3 days was tested on exponentially growing bovine retinal microvascular endothelial, aortic endothelial, mink lung epithelial CCL-64, and Swiss mouse 3T3 fibroblast cells. The growth of all four cell types was inhibited in a dose- and time-dependent manner. CCL cells, which are used as a model for assaying TGF-beta activity, were more sensitive than the endothelial cells, suggesting that ACM contains TGF-beta. Moreover, acid treatment significantly increased the inhibitory activity of ACM, indicating that TGF-beta in ACM is predominantly in the latent form. Mouse fibroblasts, which are not affected by TGF-beta treatment under the same conditions, were also inhibited by ACM. This suggests that other inhibitory factors in addition to TGF-beta may be involved. Adsorption by an anti-TGF-beta polyclonal antibody column substantially reduced but did not eliminate the inhibitory activity of ACM for CCL and endothelial cells. Western blot analysis of ACM and proteins eluted from the affinity column revealed a 25 kDa band that co-migrates with TGF-beta. Comparative densitometry of the 25 kDa bands on Western blot indicated that the amount of TGF-beta in ACM is not sufficient to account for the total growth-inhibitory activity. These experiments demonstrate directly that rat brain astrocytes express TGF-beta. They also indicate that astrocytes may produce other growth-inhibitory factor(s) yet to be identified.

On the photodynamic therapy action spectrum of zinc phthalocyanine tetrasulphonic acid in vivo

The photodynamic therapy (PDT) activity of zinc phthalocyanine tetrasulphonic acid in a rodent tumour model was shown to be critically dependent on the wavelength of the excitation laser light over a relatively small wavelength range. Thus the sensitizer showed a doubling of the PDT activity with fibrosarcoma LSBD 1 in BDIX rats when the wavelength of the illuminant was displaced from 680 to 692 nm. Under these conditions, the sensitizer is approximately three times more effective than polyhaematoporphyrin, whereas previously it has been considered to be of low PDT activity. This wavelength effect is attributed to a red shift of the absorption spectrum of the sensitizer in cells compared with that in solution. Fluorescence excitation studies with sensitizer absorbed in mouse 3T3 fibroblast cells are consistent with such a red shift.

Different cells and cell lines produce factors that modulate Sertoli cell function

Peritubular myoid cells derived from immature rat testes produce factors that modulate Sertoli cell function (P-Mod-S). The secretion of these factors is controlled in part by androgens. Cultured prostatic stromal cells strongly resemble peritubular myoid cells and produce mediators with similar activity. Here we investigated whether myoid cell lines can be used as a source of P-Mod-S-like factors. Rat kidney fibroblast (NRK) and mouse fibroblast (3T3) cell lines were used as non-myoid controls. Surprisingly, serum-free media conditioned by all cell lines studied modulated Sertoli cell function in a similar fashion as media conditioned by peritubular cells (PTCM) or stromal cells (STCM). Using Sertoli cell transferrin secretion as an endpoint for P-Mod-S-like activity, the nature of the active principles involved was further explored. The observed activity could not be explained by residual contamination with fetal calf serum. Moreover, the effects of the conditioned media could not be mimicked by classical growth factors (IGF-I, bFGF, EGF, TGF-β, NGF, PDGF-BB) added singly or in combination with submaximally effective concentrations of PTCM. Finally, the possibility that conditioned media might indirectly enhance Sertoli cell function by promoting the production or deposition of extracellular matrix elements was made unlikely by the demonstration that the observed effects were not mimicked by Matrigel and were unaffected when Sertoli cells were seeded on Matrigel. Superdex 75 chromatography after analytical reversed-phase chromatography indicates that the factors from different origin have a similar size (45–50 kDa). It is concluded that mediators with P-Mod-S-like activity are produced by various cells and cell lines both with and without smooth muscle cell characteristics. Whether the active principles involved are really identical requires further investigation.

Characterization of Antinuclear Autoantibodies Present in the Serum from Nonobese Diabetic (NOD) Mice

The nonobese diabetic (NOD) mouse develops insulin-dependent type 1 diabetes in response to autoantibodies and T-cell attack directed against pancreatic islet cell antigens. Sera obtained from nondiabetic and diabetic female mice demonstrated a 1.4-fold increase in IgG levels when compared to BALB/c control animals. Nondiabetic and diabetic male mice had a 2.1- and 3-fold increase, respectively, in serum IgG levels over that of control mice. Seven of 11 nondiabetic and 7/10 diabetic sera from female NOD mice contained antibody to cytoplasmic or nuclear components of HEp-2 cells. Cytoplasmic staining revealed reaction against cytoskeletal and midbody structures. Punctate nuclear staining patterns with HEp-2 cells showed antibody reaction to the centriole, mitotic chromosomes, and nuclear rim. On the other hand, sera from BALB/c mice were negative for antibody staining of HEp-2 cells. Confirmation of the autoantibody nature of the NOD sera was obtained by antibody staining of nuclear structures from the mouse 3T3 fibroblast cell line, and by staining of salivary gland tissue sections. The nuclear and cytoplasmic staining patterns of diabetic NOD sera were reminiscent of the autoantibody staining patterns observed in Sjögren's syndrome and other inflammatory autoimmune connective tissue diseases.

Phosphorylation and redistribution of the phosphatidylinositol-transfer protein in phorbol 12-myristate 13-acetate- and bombesin-stimulated Swiss mouse 3T3 fibroblasts.

By immunofluorescence microscopy it was shown that the phosphatidylinositol-transfer protein (PI-TP) becomes associated with the Golgi membranes when confluent (quiescent) Swiss mouse 3T3 fibroblast cells are stimulated with phorbol 12-myristate 13-acetate (PMA) and bombesin. Dibutyryl cyclic AMP or dexamethasone had no effect on the intracellular redistribution of PI-TP. In exponentially growing cells and in serum-starved (semi-quiescent) cells, PI-TP is already associated with Golgi structures. Stimulation of semi-quiescent cells by PMA resulted in a rapid redistribution of PI-TP. A similar yet slower response was observed after stimulation with bombesin. Stimulation of semi-quiescent 3T3 cells by PMA significantly increased the phosphorylation of PI-TP, as shown by immunoprecipitation of PI-TP from pre-labelled cells. No significant increase in phosphorylation of PI-TP was observed after stimulation of these cells by bombesin. Purified PI-TP was shown to be a substrate for protein kinase C in vitro. The possibility that the phosphorylation of PI-TP after activation of protein kinase C is involved in the observed redistribution of PI-TP is discussed.

Transforming growth factor(TGF)‐β1 induces leukemic cell‐growth‐promoting activity in fibroblast cells.

The production of the leukemic cell-growth-promoting factor (LGF) in TGF-beta 1-treated fibroblast cells was studied. BALB/c3T3 mouse fibroblast (3T3) cells cultured in Eagle's medium containing a low concentration of TGF-beta 1 (0.04-1 ng/ml) secreted 3-5 times more LGF than the cells cultured in the absence of TGF-beta 1. The amount of LGF secretion was dose-dependent on the concentration of post-cultured medium and time-dependent after the addition of TGF-beta 1. Similar findings were obtained in human diploid fibroblasts, WI-38 cells. LGF is a 18KD glycoprotein that is acid-stable but heat-unstable.

Local opening of the DNA double helix in eukaryotic cells detected by osmium probe and adduct-specific immunofluorescence.

The structure of DNA in mouse fibroblast 3T3 cells has been investigated with the single-strand-selective probe, the complex of osmium tetroxide and 2,2'-bipyridine (Os,bipy). DNA-Os,bipy adducts in the cells were detected by immunofluorescence using a highly specific, affinity-purified polyclonal antibody. Treatment of living cells with the chemical probe led to a distinct but nonuniform nuclear staining. We attribute the positive nuclear staining to the existence of single-stranded and distorted DNA regions in the living cell. Confocal laser scanning microscopy revealed dark areas corresponding to nucleoli and regions of condensed chromatin. These conclusions were supported by the results of experiments in which the chemical probe was applied to fixed cells treated with 45% acetic acid or with acidic buffers (pH 1.8-2.5). An inverse staining pattern was obtained, characterized by intense immunofluorescence of the condensed chromatin regions. Thus, the structural transitions and/or chemical alterations (e.g. depurination) induced by acid treatment increase the accessibility of normally unreactive DNA bases. We conclude that open DNA structures recognized by the chemical probe in the cells prior to their fixation are contained mainly in decondensed and transcriptionally active chromatin, but are virtually absent from nucleoli and condensed chromatin.

Possible direct role of reactive oxygens in the cause of cutaneous phototoxicity induced by five quinolones in mice.

The mechanisms of phototoxicity induced in mice by five quinolone antibacterial agents were investigated using mouse 3T3 fibroblast cells and Balb/c mice. In the in vitro study, the cultured cells were exposed to ultraviolet-A (UVA) in the presence of the five quinolones lomefloxacin, enoxacin, ciprofloxacin, ofloxacin and DR-3355 (the s-isomer of ofloxacin). Cytotoxicity after irradiation was assayed by the neutral red and MTT assay methods, both of which revealed dose-dependent phototoxicity for all five quinolones. Phototoxicity was inhibited by the addition of catalase, and was augmented by the addition of superoxide dismutase. Dimethylthiourea (a hydroxyl radical scavenger) protected against phototoxicity induced by four quinolones, but not against that by enoxacin. These results indicated that superoxide anions, hydrogen peroxide and hydroxyl radicals were generated in solutions of these quinolones under UVA irradiation. In the in vivo study, mice were injected in the auricle with hydrogen peroxide. Ear swelling reactions appeared dose dependently. When irradiated, these reactions were significantly augmented. These data suggested that cutaneous phototoxicity in Balb/c mice is initiated by the generation of reactive oxygens in the target tissue, especially of hydroxyl radicals.

Properties of base-substituted and carboxyl-esterified analogues of griseolic acid, a potent cAMP phosphodiesterase inhibitor

Griseolic acid (GA) is a potent cyclic AMP (cAMP) phosphodiesterase (PDE) inhibitor that has an adenine base and two carboxyl groups in its molecule (Nakagawa F, Okazaki T, Naito A, Iijima Y and Yamazaki M, J Antibiot 38: 823–829, 1985). GA analogues were synthesized in which the adenine group was substituted with guanine (6-deamino-2-amino-6-hydroxygriseolic acid, G-GA) or hypoxanthine (6-deamino-6-hydroxygriseolic acid, H-GA). Their inhibitory activities to cyclic GMP (cGMP) PDE and cAMP PDE were compared with GA. For cGMP PDE from rod outer segments of bovine retina, the IC 50 values of GA, G-GA and H-GA were 18, 0.040 and 0.12 μM, respectively, with 0.25 μM cGMP as substrate. For type IV PDE isozyme from mouse 3T3 fibroblast cells, the IC 50 values of GA, G-GA and H-GA were 0.021, 15 and 11 μM, respectively, with 0.25 μM cAMP as substrate. Thus, GA and G-GA were found to be base-selective inhibitors of type IV PDE of 3T3 cells and type V PDE of bovine retinas, respectively. Esters of carboxylic acids of GA were synthesized in order to increase permeability into cells, and their efficacy was tested by measuring the accumulation of cAMP in 3T3 cells. The dipivaloyloxymethyl ester of GA was found to increase cAMP levels at 0.1 μM, while GA and 3-isobutyl-1-methylxanthine were active only above 100 μM, and the dimethyl ester of GA was inactive. The dipivaloyloxymethyl ester of GA seems to exert its activity after conversion to GA in the cell, since the pivaloyloxymethyl ester was easily hydrolysed by the enzyme action and the dipivaloyloxymethyl ester of GA itself was much less potent an inhibitor of PDE. The dipivaloyloxymethyl ester of GA inhibited thrombin-induced aggregation of platelets and stimulated lipolysis of adipocytes at low concentrations.