Adenylyl cyclase P-site ligands accelerate differentiation in Ob1771 preadipocytes

Azeddine Ibrahimi, Nada Abumrad, Hengameh Maghareie, Michael Golia, Ilana Shoshani, Laurent Désaubry, and Roger A. Johnson

Department of Physiology and Biophysics, State University of New York, Stony Brook, New York 11794-8661


    ABSTRACT
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Abstract
Introduction
Experimental procedures
Results
Discussion
References

Differentiation of Ob1771 preadipocytes to adipocytes was characterized by morphological changes and elevated expression of the specific marker enzyme, glycerol-3-phosphate dehydrogenase. A differentiation response substantially more complete and rapid than that obtained with insulin and 3,5,3'-triiodothyronine was observed with established inhibitors of adenylyl cyclases: 2',5'-dideoxyadenosine (2',5'-dd-Ado), 9-(cyclopentyl)adenine (9-CP-Ade), and 9-(arabinofuranosyl)adenine (9-Ara-Ade), coincident with decreased cellular cAMP levels. These ligands inhibit adenylyl cyclases noncompetitively, via a domain referred to as the P-site because of its requirement for an intact purine moiety. Differentiation was not induced by inosine, a nucleoside known not to act at the P-site, or by N6-(2-phenylisopropyl)adenosine or 1,3-diethyl-8-phenylxanthine, agonist and antagonist, respectively, for adenosine A1 receptors. Also ineffective were IBMX or forskolin, agents that can raise intracellular cAMP levels. Potency of the differentiation response followed the order 2',5'-dd-Ado (1-20 µM) > 9-CP-Ade (10-100 µM) = 9-Ara-Ade (10-100 µM) >> inosine, consistent with their potencies to inhibit adenylyl cyclases. The data suggest that inhibition of adenylyl cyclase via the P-site and the consequent reduction in cell cAMP levels facilitate the induction of differentiation in Ob1771 cells. The findings raise the question whether the known endogenous P-site ligands participate in the differentiation response induced by hormones.

adipocytes; P-site inhibition; 2',5'-dideoxyadenosine; 9-(cyclopentyl)adenine; signal transduction; adenosine 3',5'-cyclic monophosphate


    INTRODUCTION
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Abstract
Introduction
Experimental procedures
Results
Discussion
References

DIFFERENTIATION OF THE Ob1771 clonal cell line is a controlled multistep process characterized by emergence of early, late, and very late markers (2). Adipose precursor Ob1771 cells, defined as preadipocytes, divide until confluence and then become committed to differentiate by acquiring early markers but do not yet accumulate triacylglycerol. These early markers include lipoprotein lipase and the alpha 2-chain collagen VI (3). Further differentiation leads to immature adipose cells (~6 days after confluence). These cells acquire the protein machinery for triacylglycerol synthesis and hydrolysis (~20 days after confluence) and begin accumulation of triacylglycerol droplets. Terminal differentiation leads to the formation of triacylglycerol-filled, mature adipose cells with the emergence of specific genes, one of which is for glycerol-3-phosphate dehydrogenase (2, 3).

Transition to terminal differentiation is dependent on the sensitivity of the preadipocyte to mitogenic and adipogenic stimuli (1). These include growth hormone, glucocorticoid, prostacyclin, or 3,5,3'-triiodothyronine (T3) and long-chain fatty acids (1-3). Ob1771 cells undergo spontaneous differentiation to adipocytes when cultured in medium supplemented with 8% FCS. However, the pattern of spontaneous differentiation seen with FCS is usually incomplete and clustered. This process can be promoted by insulin and T3 (12, 24). One of the main adipogenic factors in FCS is thought to be arachidonic acid, which increases cellular cAMP levels and induces polyphosphoinositide breakdown (21). It has been suggested that the cAMP and IGF-I pathways are essential to terminal differentiation, whereas diacylglycerol and insulin pathways play a modulating role (3). Insulin is well known to lower cell cAMP levels (7) by activation of type III phosphodiesterases (4, 9).

cAMP concentration can be regulated through changes in its formation, catalyzed by adenylyl cyclases, or changes in its degradation, catalyzed by cyclic nucleotide phosphodiesterases. Numerous drugs have been developed that act on cyclic nucleotide phosphodiesterases (4, 9), whereas those that act directly on adenylyl cyclases have been less well explored (36); the main class of such pharmacological agents comprises forskolin and its analogs (15). Adenylyl cyclase is potently and directly inhibited by analogs of adenosine via a configuration distinct from that of the catalytic active site (27, 29, 37, 41). It is referred to as the P-site from pharmacological characterizations of inhibition that demonstrate a requirement for a purine moiety (26, 28, 30, 40). The most potent cell-permeable P-site ligands include 2',5'-dideoxyadenosine (2',5'-dd-Ado; IC50 ~3 µM) (30), 9-(cyclopentyl)adenine (9-CP-Ade; IC50 ~20 µM) (40), and 9-(arabinofuranosyl)adenine (9-Ara-Ade; IC50 ~100 µM) (28, 39).

We utilized direct inhibitors of adenylyl cyclases to determine whether and to what extent such agents may influence the differentiation process in Ob1771 cells. The cell-permeable P-site ligands used in this study included 2',5'-dd-Ado, 9-CP-Ade, and 9-Ara-Ade. This report compares differentiation responses in Ob1771 preadipocytes typically induced by insulin and T3 with those observed in the presence of these nucleosides.


    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Experimental procedures
Results
Discussion
References

Materials. 9-CP-Ade was synthesized as described by Montgomery and Temple (31), and 2',5'-dd-Ado was synthesized by the procedure developed by Désaubry et al. (14). The bicinchoninic acid (BCA) protein assay reagent kit was from Pierce; culture cell media were from GIBCO; FCS was from Atlanta Biologicals; protein kinase A was isolated from bovine muscle and enriched by ammonium sulfate precipitation and chromatography on DEAE-Sephadex by established procedures (34); kemptide was from Sigma Chemical (K1127); the RIA kit for cAMP was from New England Nuclear; and [gamma -32P]ATP was from ICN. All the other chemicals were from Sigma.

Cell culture and treatment. Ob1771 preadipocyte cells were originally obtained after the subcloning of Ob17 cells established from the periepididymal adipose tissue of genetically obese B57BL6J mice (32). Cells were plated to a density of ~2,000/cm2 and grown in DMEM supplemented with 8% fetal bovine serum, 200 units penicillin/ml, 50 µg streptomycin/ml, 33 µM biotin, and 17 nM pantothenate. Confluence was reached within 5 days, after which differentiation was promoted by the addition of the indicated concentrations of nucleoside or of 17 nM insulin and 2 nM T3 to the medium. Nucleosides were added to prewarmed medium from concentrated stocks in ethanol. Media were changed every other day, and terminal differentiation of Ob1771 was followed for 2-4 wk, typically being complete in 3-4 wk.

Stability of 9-CP-Ade and 2',5'-dd-Ado. Cells were exposed to either 30 µM 2',5'-dd-Ado or 30 µM 9-CP-Ade. At various times, a 200-µl portion of the medium above the cells was removed and frozen. The collected samples were thawed and passed through 0.22-µm syringe filters and then were subjected to HPLC by reverse-phase chromatography (Beckman Ultrasphere 5 mm C-18; 4.6 × 250 mm) developed with a linear gradient from water to 100% methanol. Baseline separations for both nucleosides were obtained. Nucleosides were quantified as areas under peaks determined with a Waters 996 photo-diode array detector and the accompanying Millennium software (version 2.10).

Glycerol-3-phosphate dehydrogenase. Glycerol-3-phosphate dehydrogenase (GPDH) was assayed spectrophotometrically from cell homogenates (12, 21) obtained at the indicated day after confluence. Interassay variability as well as variability among mean values from separate culture dishes maintained under identical culture conditions never exceeded 7% within the same series of cells. GPDH specific activity was expressed in milliunits per milligram (nmol · min-1 · mg protein-1). Protein content was determined according to Smith et al. (35) with the BCA protein assay.

Cell cAMP. Levels of cAMP were determined on extracts of cells exposed to control medium or to medium supplemented with adenine nucleosides. Samples were handled in either of two ways. One involved extraction and a two-step chromatographic purification before assay, and the other involved simply extraction and dilution. For samples to be purified, medium was decanted from culture dishes and cells were lysed by the successive addition and removal of two 0.6-ml portions of 0.3 M HClO4, containing ~25,000 counts/min (cpm) of [3H]cAMP, included to allow the recovery of sample cAMP to be calculated. These extracts were pooled and neutralized with K2CO3. The resulting precipitate of protein and KClO4 was removed by centrifugation. The supernatant fractions were then chromatographically purified by sequential chromatography on Al2O3 and Dowex-50 (H+ form). Samples, eluted from a Dowex-50 column with water, were collected, lyophilized, and then reconstituted with buffer. A portion of this solution was used to determine sample recovery, which was typically >60%. For the simpler extraction, medium was decanted from culture dishes and cells were lysed by the addition of a solution containing 10 mM potassium phosphate, pH 6.8, 10 mM EDTA, and 0.1 mM IBMX. cAMP was determined by either of two techniques: by protein kinase A activation (10) or by RIA. Protein kinase A was as per Corbin et al. (10) in a buffer containing 20 mM magnesium acetate, 0.1 mM IBMX, 1 mg BSA/ml, 0.2 mM ATP, ~200,000 cpm [gamma -32P]ATP, 10 mM potassium phosphate buffer, pH 6.8, and 130 µM kemptide as phosphate acceptor. For assays utilizing the RIA kit from New England Nuclear, the procedure followed that described by the manufacturer. For both assays, samples were assayed in triplicate, with at least two dilutions of sample, and also with added internal standard (usually 100 fmol) with which to assess and adjust for any assay interference that may have occurred.

RNA preparation. RNA was prepared with the RNA STAT-60 kit. RNA was electrophoresed on denaturing agarose gel, transferred to Hybond-N+ membranes, and hybridized at 42°C with randomly primed 32P-labeled DNA probes. After washing, the membranes were exposed to Kodak Hyperfilm at -75°C. The mRNA for GPDH was used as an internal standard.


    RESULTS
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Abstract
Introduction
Experimental procedures
Results
Discussion
References

Effects of various P-site inhibitors on cell differentiation. Several adenine nucleosides and adenine 3'-nucleotides known to inhibit adenylyl cyclases directly in vitro and in intact cells (Fig. 1) were tested for their effects on differentiation of Ob1771 cells. Both 2',5'-dd-Ado (20 µM) and 9-Ara-Ade (30 µM) increased the number and the density of clusters of differentiated cells well above levels seen in control cells and in cells treated with insulin (17 nM) plus T3 (2 nM; Fig. 2). By comparison, inosine, a nucleoside structurally close to the class of inhibitory compounds designated as P-site ligands but known not to affect adenylyl cyclase activity (28, 30, 40), was without effect, even at the relatively high concentration of 100 µM (Fig. 2). This lack of response was consistent with its lack of effect on the cyclase.


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Fig. 1.   Structures of several inhibitors of adenylyl cyclases. A: 2',5'-dideoxyadenosine (2',5'-dd-Ado). B: 9-(cyclopentyl)adenine (9-CP-Ade). C: 9-(arabinofuranosyl)adenine (9-Ara-Ade).


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Fig. 2.   Comparison of differentiation responses in Ob1771 cells elicited by 2',5'-dd-Ado, 9-Ara-Ade, inosine, and insulin/triiodothyronine (T3). At confluence, Ob1771 cells were exposed to medium + insulin/T3 or medium + nucleoside, as indicated, with retreatment every other day. Concentrations were 30 µM 9-Ara-Ade; 20 µM 2',5'-dd-Ado; 100 µM inosine; and 17 nM insulin + 2 nM T3. Photographs were taken after 22 days of treatment and are representative of 3 experiments.

The acceleration of differentiation was concentration dependent, as evident in Fig. 3 for 2',5'-dd-Ado, with effects being noted with concentrations as low as 1 µM. Although 2',5'-dd-Ado was more potent than 9-Ara-Ade, the extent of the differentiation responses seen with these nucleosides at their respectively optimal concentrations appeared to be comparable. The effects of both nucleosides occurred at concentrations within their ranges for inhibition of adenylyl cyclases. This effect on differentiation was not related to the Ob1771 clone, since similar effects were obtained in 3T3-F442A preadipocyte cells after treatment with 2',5'-dd-Ado (not shown).


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Fig. 3.   Concentration dependence for induction of differentiation in Ob1771 cells by 2',5'-dd-Ado. Ob1771 cells were exposed to the indicated concentrations of 2',5'-dd-Ado at confluence and were maintained by retreatment every other day. Photographs were taken after 16 days of treatment and are representative of 3 experiments.

cAMP levels. Noteworthy was the consistent observation that basal cAMP levels were also reduced under conditions eliciting accelerated differentiation. This occurred with 2',5'-dd-Ado in separate experiments (Table 1), assayed by different techniques, and with 9-CP-Ade in a concentration-dependent manner (Fig. 4). For comparison, the effect of 30 µM 2',5'-dd-Ado in this experiment is also shown. In these experiments cells were fixed after 3 wk of treatment with these nucleosides and indicate that chronic treatment can reduce already low cellular levels of cAMP in these unstimulated cells.

                              
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Table 1.   Effects of 2',5'-dideoxyadenosine on cAMP levels in Ob1771 cells



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Fig. 4.   Changes in cAMP levels in Ob1771 cells after chronic exposure to 9-CP-Ade. Ob1771 cells were exposed to the indicated concentrations of 9-CP-Ade or 2',5'-dd-Ado for 22 days. Cellular cAMP levels were then determined on purified samples by the protein kinase activation method described in EXPERIMENTAL PROCEDURES. Individual values obtained with 9-CP-Ade (bullet , down-triangle) or 2',5'-dd-Ado () are shown; line shows averages of values for 9-CP-Ade.

Ligand stability. Because both 9-Ara-Ade and 2',5'-dd-Ado are ribose derivatives containing the furanosyl oxygen, they are susceptible to enzymatic and nonenzymatic depurination, which could affect the efficacy of these compounds in experiments involving chronic treatments. Depurination does not occur with 9-CP-Ade, which contains the chemically and enzymatically much more stable adenine-cyclopentyl bond (Fig. 1). 9-CP-Ade is also a P-site ligand and exhibits IC50 values for inhibition of types I and VI adenylyl cyclases of ~20 to 100 µM, respectively (25). 9-CP-Ade induced a more dramatic differentiation in Ob1771 cells than that noted with either insulin plus T3, 9-Ara-Ade, or 2',5'-dd-Ado (Fig. 5). In this experiment, cells were exposed to either 10 or 100 µM 9-CP-Ade, and the response was concentration dependent. Note also the very dense concentration of lipid droplets in areas surrounding the several dark spots. At higher magnification, the dark spots were actually floating upwellings of cells that could be seen to be full of lipid droplets (not shown). Although the concentrations of 9-CP-Ade necessary to elicit this response were greater than those used to elicit responses with 2',5'-dd-Ado, the extent of differentiation seen with 9-CP-Ade was greater than that seen with 2',5'-dd-Ado or 9-Ara-Ade.


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Fig. 5.   Accelerated differentiation in Ob1771 cells induced by 9-CP-Ade. Ob1771 cells were exposed at confluence to 9-CP-Ade at 10 or 100 µM, or vehicle (control) for 16 days. Photographs represent 1 of 2 experiments.

The response of these cells to 9-CP-Ade, when compared with the response to 2',5'-dd-Ado, was consistent with both the differences in potency of these adenine derivatives to inhibit adenylyl cyclases and with their expected differences in chemical and metabolic stability. 9-CP-Ade is the less potent but chemically more stable ligand. To establish whether the stability of 9-CP-Ade or 2',5'-dd-Ado differed in this cell culture system as expected, cells were exposed to either compound and samples were taken over 2 days and analyzed by HPLC. The concentration of 9-CP-Ade was not altered over several days, whereas 2',5'-dd-Ado exhibited a half-life of ~24 h (Fig. 6). In these experiments, no distinction was made whether disappearance of 2',5'-dd-Ado was due to medium, to cells, or both. Thus 9-CP-Ade at any given concentration would be effectively maintained longer than would 2',5'-dd-Ado.


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Fig. 6.   Fate of 2',5'-dd-Ado and 9-CP-Ade on exposure to Ob1771 cells and medium. To cells plated in 10-ml standard medium on 100-mm culture plates was added either 30 µM 2',5'-dd-Ado or 30 µM 9-CP-Ade. Thereafter, 200-µl portions were taken every hour for the 1st day and then at additional times for 2 days. Nucleoside was purified and quantified as described in EXPERIMENTAL PROCEDURES. Values are relative concentrations of nucleoside at sampling time (t) to concentrations 1 min after addition of nucleoside (t = 0).

Effects of a single exposure to nucleoside. To determine whether a single exposure of Ob1771 cells to nucleoside was sufficient to promote differentiation, cells were exposed for 48 h to 9-CP-Ade and 2',5'-dd-Ado. The cells were then washed with fresh, nucleoside-free medium and were maintained on the normal 2-day refeeding schedule but without additional exposure to either compound. With either compound the differentiation response of Ob1771 cells occurred at a rate substantially faster than that which would have occurred had cells been unexposed or exposed only to vehicle (not shown). This suggests that a single, albeit 2-day, exposure to these P-site ligands was sufficient to initiate the chain of events leading to the adipogenic response in Ob1771 cells.

Induction of GPDH. Terminal differentiation implies the capacity to accumulate triacylglycerol and the enzymes necessary for this, one of which is GPDH. The activity of GPDH correlates very well with the proportion of differentiated, triacylglycerol-containing cells (21). To substantiate that the effects of the nucleosides on cell morphology noted above were in fact indicative of terminal differentiation to adipocytes, the activity of GPDH was determined. The activity of expressed GPDH increased with 2',5'-dd-Ado and 9-CP-Ade at concentrations that elicited the accelerated differentiation response, but not by inosine, even at concentration up to 1 mM (Fig. 7). Thus the morphological changes induced by these adenine derivatives were also corroborated by elevated activity of GPDH.


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Fig. 7.   Increased expression of glycerol-3-phosphate dehydrogenase (GPDH) by 2',5'-dd-Ado or 9-CP-Ade. At confluence, Ob1771 cells were exposed to 2',5'-dd-Ado, 9-CP-Ade, or the mixture of insulin/T3, as indicated. For both A and B, nucleoside concentrations were as indicated, and 0 (solid bar) indicates addition of the mixture of 17 nM insulin + 2 nM T3. A (Expt. 1): concentrations were millimolar and cells were harvested after 22 days of treatment for determination of GPDH activities. B (Expt. 2): concentrations were millimolar and cells were harvested after 16 days of treatment for determining GPDH activities.

Effect of ligands for adenosine receptors. Given that the adenine derivatives eliciting the accelerated differentiation response above also share structural characteristics with ligands known to act on cell-surface receptors for adenosine, such ligands were also tested for their effects on differentiation. Neither N6-(2-phenylisopropyl)adenosine, an adenosine receptor agonist, nor 1,3-diethyl-8-phenylxanthine (DPX), an adenosine receptor antagonist, induced changes in the differentiation response of the Ob1771 cells (Fig. 8). This lack of response is similar to that reported by Borglum et al. (5) with 5'-(N-ethylcarboxamido)adenosine, an adenosine receptor agonist. These adenosine receptor ligands altered neither the rate of spontaneous differentiation nor that induced by 2',5'-dd-Ado, suggesting no role of adenosine receptors in the accelerated differentiation noted with the adenine derivatives we have tested.


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Fig. 8.   Comparison of differentiation responses in Ob1771 cells elicited by 2',5'-dd-Ado, 1,3-diethyl-8-phenylxanthine (DPX), or (-)N6-(2-phenylisopropyl)adenosine (PIA). At confluence, Ob1771 cells were exposed to medium + agents, as indicated, with retreatment every other day. Concentrations were: 10 µM 2',5'-dd-Ado, 5 µM DPX, or 1 µM PIA. Photographs were taken after 22 days of treatment and are representative of two experiments.

Effect of cAMP-elevating agents. The acccelerated differentiation noted with the adenine derivatives above was consistent with their effects on adenylyl cyclases via the inhibitory P-site domain. These effects would suggest that elevations in cellular cAMP levels by other agents would interfere with the differentiation response to P-site ligands. Ob1771 cells were exposed chronically to forskolin, an established activator of adenylyl cyclases in vitro and in vivo, or IBMX, an established inhibitor of cyclic nucleotide phosphodiesterases (Fig. 9). In this experiment, as with those with the adenine derivatives, exposure was followed for several weeks, with refeeding every other day, and morphological changes were monitored. Neither forskolin nor IBMX promoted differentiation (Fig. 9), without or with 2',5'-dd-Ado (not shown).


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Fig. 9.   Comparison of differentiation responses in Ob1771 cells elicited by 2',5'-dd-Ado, forskolin, or IBMX. At confluence, Ob1771 cells were exposed to medium + agents, as indicated, with retreatment every other day. Concentrations were 20 µM 2',5'-dd-Ado, 100 µM IBMX, and 50 µM forskolin. Photographs were taken after 22 days of treatment and are representative of 3 experiments.

Changes in cell morphology were substantiated by analysis of the expression of adipocyte markers under these conditions (Fig. 10). RNA analysis by Northern blotting showed a high expression of genes for GPDH and fatty-acyl-CoA synthase in cells treated with 2',5'-dd-Ado or insulin plus T3. 2',5'-dd-Ado appeared to upregulate the expression of these genes, in correlation with the morphological changes of the cells. In contrast, the expression of these genes was unaffected by treatment with either forskolin or IBMX. These data are in line with the previously described argument that increasing cAMP concentration by these two agents did not affect adipocyte differentiation (38). Moreover, the data are fully consistent with both biochemical and morphological changes induced by the adenine derivatives reported here.


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Fig. 10.   Expression of adipocyte marker genes for GPDH and acyl-CoA synthase (ACS). At confluence, Ob1771 cells were exposed to medium + agents, as indicated, with retreatment every other day for 14 days. Concentrations were: 10 µM 2',5'-dd-Ado, 100 µM IBMX, 50 µM forskolin, and 17 nM insulin + 2 nM T3. Similar data were observed in 3 experiments. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.


    DISCUSSION
Top
Abstract
Introduction
Experimental procedures
Results
Discussion
References

The present studies demonstrate a concentration-dependent acceleration in terminal differentiation of Ob1771 cells by several adenine derivatives belonging to a class of noncompetitive inhibitors of adenylyl cyclases. The differentiation response induced by 2',5'-dd-Ado was significantly more rapid and more complete than that induced by insulin and T3, agents typically used to promote differentiation of preadipocytes. This was noted by changes in cell morphology, increased GPDH activity, and induced expression of marker genes. In general, the efficacy and rank order of potency of the adenine derivatives for inducing the differentiation response followed that of P-site-mediated inhibition of adenylyl cyclases: 2',5'-dd-Ado > 9-CP-Ade > 9-Ara-Ade >> inosine. Inosine does not inhibit adenylyl cyclases or induce adipocyte differentiation. Consistent with its greater chemical and metabolic stability, 9-CP-Ade caused a differentiation response that was more dramatic than that seen with 2',5'-dd-Ado.

In 3T3-L1 cells, a fetal mouse fibroblast cell line, a dissociation between cellular cAMP levels and the differentiation response was noted (38); impaired expression or function of Gs was associated with enhanced differentiation, while elevations in cell cAMP levels did not have the opposite effect. In line with that, we also found that neither forskolin nor IBMX influenced adipocyte differentiation. The data we present, that P-site ligands accelerate differentiation, would be consistent with reduced cellular cAMP levels promoting the process. This was supported by the observations with 2',5'-dd-Ado and 9-CP-Ade noted in several experiments (Table 1 and Fig. 4). These reductions in basal cAMP levels occurred following chronic exposure to these nucleosides. These data are presented on the basis of cAMP levels per dish and may represent an underestimation of actual cellular changes, since both cell number and protein levels approximately double when confluent cultures undergo differentiation (11, 19, 21). It is thus probable that the effects of this class of compounds are mediated through reduction in cAMP and the cAMP-protein kinase A signaling pathway subsequent to adenylyl cyclase inhibition. We and others have used P-site ligands with a variety of mammalian tissues and isolated cells (8, 16, 18, 20, 22, 23, 33) and have demonstrated effects on different aspects of cell function. Rat epididymal fat cells (18), isolated hepatocytes (8), primary cultures of thyroid follicles (22), dorsal root ganglion neurons (23), bone organ cultures (33), and cortical collecting tubules (16) have been used, to name but a few. End points included changes in water conductance (16), action potential afterhyperpolarization (23), parathyroid hormone-stimulated bone resorption (33), glycerol production (18), altered enzyme activities (8), and DNA synthesis and cell growth (22). In this last example, 2',5'-dd-Ado suppressed thyroid-stimulating hormone-stimulated cAMP levels while enhancing the stimulation of cell growth and causing a two- to sevenfold increase in the [3H]thymidine incorporation into DNA caused by insulin, epidermal growth factor, and FCS (22). In each of these instances the effects of 2',5'-dd-Ado on cell function and on cellular cAMP levels were uniformly consistent with P-site inhibition of adenylyl cyclase.

It is possible that effects of P-site ligands on differentiation and cell cAMP levels reflect cell-specific action(s) of the nucleoside and/or the particular adenylyl cyclase isozyme(s) expressed. Sensitivity of adenylyl cyclases to P-site-mediated inhibition is isozyme dependent (25), and the tissue-dependent level of expression of different forms of adenylyl cyclase may influence the regulatory importance of P-site ligands, whether intracellular or extracellular in origin. Although the simplest interpretation of the data is that nucleoside-induced differentiation was due to an inhibition of adenylyl cyclase, P-site ligands may act selectively on other proteins. It has been known for some time that some of these compounds can bind to DNA polymerase (17) but are without effect on other enzymes (13). Potent P-site ligands occur naturally within cells, e.g., 3'-AMP and 2'-d-3'AMP, and we have shown that levels of these 3'-nucleotides change in a chronic fashion (6). Therefore, while 2',5'-dd-Ado and 9-CP-Ade may affect cell differentiation by inhibiting adenylyl cyclase and lowering cAMP, it is not only their effects per se that are important but also the putative natural intracellular regulatory processes and ligands that they may mimic.

The ligands used in these studies represent a class of compounds proving to be very useful as pharmacological tools for studies of cell physiology and pathophysiology. The most effective compounds for work with intact cells have been, in rank order of potency as inhibitors of adenylyl cyclases, 2',5'-dd-Ado, 9-CP-Ade, 9-Ara-Ade, and 9-(2-tetrahydrofuryl)adenine (9-THF-Ade; SQ-22536). These compounds are fairly potent (IC50 approx  3-100 µM), soluble in water (e.g., to 10 mM), stable at neutral to alkaline pH, and are readily taken up by cells. Because of the cyclic oxygen of the ribose ring, 2',5'-dd-Ado, 9-Ara-Ade, and 9-THF-Ade are subject to depurination at acidic pH, whereas 9-CP-Ade is stable. Subsequent metabolism by cells is also less likely with 9-CP-Ade (cf. Fig. 6), due to the lack of the ribosyl structure and its hydroxyl groups. Thus, although 9-CP-Ade is less potent than 2',5'-dd-Ado, for example, its chemical and pharmacological properties may make it the more useful compound for many applications. It is likely, though, that if inhibition of adenylyl cyclase is involved in the differentiation response we note here, previous work indicates that 3-phosphorylated P-site ligands will prove to be much more potent pharmacophores (6, 13, 14, 25, 27, 28). Once some of these ligands become available with protected phosphate groups for use as prodrugs, effects at substantially lower concentrations can be expected and these will become the agents of choice.

We believe that the differentiation response noted here, being a derivative of the complex mechanisms regulating cell growth, division, and development, will likely include effects of these ligands on proteins other than adenylyl cyclase. Moreover, cells may produce endogenous ligands to interact with adenylyl cyclase and specific other proteins and thereby bring about changes in cell growth/differentiation in response to stimuli or as part of a developmental event. The identification of the naturally occurring ligands, the proteins with which they interact, and the circumstances under which one or both change, will allow an understanding of the mechanisms regulating cell development to be delineated and then utilized to modify pathophysiological conditions.


    ACKNOWLEDGEMENTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-38828 to R. A. Johnson and by a grant from the Taher Fund to N. Abumrad.


    FOOTNOTES

Address for reprint requests: R. A. Johnson, Dept. of Physiology and Biophysics, State Univ. of New York, Stony Brook, NY 11794-8661.

Received 30 September 1997; accepted in final form 14 October 1998.


    REFERENCES
Top
Abstract
Introduction
Experimental procedures
Results
Discussion
References

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Am J Physiol Cell Physiol 276(2):C487-C496
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society




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