Introduction

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Cyclic nucleotide phosphodiesterases (PDEs) hydrolyze the second messengers cyclic AMP and cyclic GMP. The mammalian PDEs have been classified into 11 families (Manganiello et al., 1995; Conti and Jin, 1999; Soderling and Beavo, 2000; Francis et al., 2001). The PDE4 family, encoded by four genes (PDE4A-D), is characterized by its low K_m value for cAMP and its sensitivity to inhibition by rolipram (Beavo et al., 1994, 1995; Bolger, 1994). PDE4 inhibitors show promising pharmacological effects in a variety of disease models, particularly asthma and depression (Torphy and Undem, 1991; O'Donnell, 1993; Giembycz, 1996; O'Donnell and Frith, 1999). However, central nervous system and gastrointestinal side effects remain a problem, limiting clinical development. It has been speculated that some of the side effects may be mediated through inhibitor interaction with the high-affinity rolipram binding site (HARBS) on PDE4 (Barnette et al., 1995; Duplantier et al., 1996); rolipram and a number of other PDE4 inhibitors have been shown to bind with low nanomolar affinity to this site (Schneider et al., 1986; Torphy et al., 1992; Jacobitz et al., 1996).

In addition to binding to the HARBS, PDE4 inhibitors also bind to a low-affinity state, termed the low-affinity rolipram binding site (LARBS). It should be noted that the terminology of HARBS and LARBS refers specifically to rolipram binding. Some inhibitors bind with high affinity to both the HARBS and the LARBS (e.g., piclamilast). A study using truncated PDE4A mutants indicated that inhibitor binding to both the HARBS and the LARBS is to the catalytic site (Jacobitz et al., 1996). Binding to the HARBS, but not the LARBS, depends on the presence of the N-terminal region. Although their exact natures are still unclear, it appears the HARBS and the LARBS are more accurately described as two distinct binding affinity states, rather than separate sites (Schneider et al., 1986; Torphy et al., 1992; Souness and Scott, 1993; Jacobitz et al., 1996).

Interestingly, the rank-order potency of a variety of compounds for inhibiting PDE4 catalytic activity differs from their rank-order potency for competing with [³H]rolipram binding to the HARBS (Torphy et al., 1992; Baures et al., 1993). Although the function of the HARBS is unknown, the potency of PDE4 inhibitors to produce certain effects, such as emesis and increased gastric acid secretion, correlates with their ability to displace [³H]rolipram from this site. Other effects are more related to their ability to inhibit PDE4 catalytic activity in a cell-free system, which provides an index of inhibitor interaction with the LARBS (Harris et al., 1989; Barnette et al., 1995, 1996; Duplantier et al., 1996; Souness et al., 1996). These findings suggest that the pharmacological effects that result from the interaction of inhibitors with the HARBS and the LARBS are distinct. This is particularly evident when one compares the effects of piclamilast (RP 73,401) and rolipram. Piclamilast is about 1000-fold more potent than rolipram for inhibiting guinea pig smooth muscle PDE4, but only about 4-fold more potent for inhibiting methacholine-induced contraction of guinea pig trachealis muscle or enhancing isoproterenol-stimulated cyclic AMP formation in guinea pig eosinophils (Ashton et al., 1994; Souness et al., 1995).

Piclamilast is a selective and potent PDE4 inhibitor (Karlsson et al., 1993; Ashton et al., 1994). PDE4 isolated from various cell types is inhibited by piclamilast with a K_i value of about 1 nM (Ashton et al., 1994). Piclamilast displays similar potency for inhibition of PDE4 regardless of the source and the procedure used to prepare the enzyme; rolipram potency, by contrast, is dependent on such factors (Schwabe et al., 1976; Fredholm et al., 1979; Ruckstuhl and Landry, 1981; Lugnier et al., 1983; Davis, 1984; Schneider et al., 1986). Piclamilast does not exhibit differential affinity for the HARBS and the LARBS; it binds with low nanomolar affinity to both states of PDE4 (Souness et al., 1995). Rolipram, by contrast, exhibits approximately 500-fold greater affinity for the HARBS relative to the LARBS (Schneider et al., 1986; Torphy et al., 1992; Jacobitz et al., 1996). Thus, [³H]piclamilast appears to be a useful tool to directly study inhibitor interaction with the high- and low-affinity binding sites on PDE4. The present study was conducted to characterize the binding of [³H]piclamilast in rat brain, to compare it with [³H]rolipram binding, and to determine the affinity of PDE4 inhibitors for the HARBS and the LARBS using these two radioligands.

	Materials and Methods

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Animals. Male Sprague-Dawley rats (Harlan, Indianapolis, IN) were housed in a temperature- (22-24°C) and light- (on 6:00 AM-6:00 PM) controlled room and were allowed free access to food pellets and water. Their use in studies reported in this article have been carried out in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health and have been approved by the Animal Care and Use Committee of the University of Tennessee Health Science Center.

Radioligand Binding Assays. Rats were killed by decapitation. Brain regions (cerebral cortex, hippocampus, amygdala, hypothalamus, neostriatum, cerebellum, and brain stem) and peripheral tissues (heart, liver, and skeletal muscle) were dissected on ice and homogenized in binding buffer (50 mM Tris-HCl, 5 mM MgCl₂, pH 7.5) using a Polytron homogenizer (Brinkman Instruments, Westbury, NY). Separation of supernatant and particulate fractions was achieved by centrifugation at 15,000g for 15 min; the pellet then was resuspended in binding buffer.

Statistical Analysis. Data were analyzed by nonlinear regression to determine whether inhibitor binding to PDE4 was better described by an interaction with one or two binding sites (Draper and Smith, 1966; O'Donnell et al., 1984). Binding to a single site was assumed unless the data were better described by a two-site binding equation. This was indicated when the residual sum of square was reduced significantly (F ratio, p < 0.01).

Materials. [³H]Rolipram was purchased from American Radiolabeled Chemicals Inc. (St. Louis, MO). [³H]Piclamilast was a gift from GlaxoSmithKline (Uxbridge, Middlesex, UK). Piclamilast was provided by Aventis (Strasbourg, France). Desmethylpiclamilast, cilomilast, and roflumilast were provided by Memory Pharmaceuticals (Montvale, NJ). Rolipram was provided by Schering AG (Berlin, Germany). Milrinone and zaprinast were purchased from Calbiochem (San Diego, CA). Other drugs and chemicals were obtained from Fisher Scientific (Pittsburgh, PA) or Sigma-Aldrich (St. Louis, MO).

	Results

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Association and Dissociation. Specific binding of [³H]piclamilast at 30°C increased rapidly to reach maximal binding in about 2 min, with a half-time of association of 24 s, after which binding remained stable up to 2 h (Fig. 1). Dissociation of [³H]piclamilast, determined following the addition of 1 mM rolipram, was equally rapid, with a half-time of 11 s (Fig. 1). The amount of radioactivity bound reached the level of nonspecific binding within 3 min of the addition of rolipram. By contrast, the association of [³H]rolipram was better described by a two-phase model, with half-times of association of 24 s and 40 min. The dissociation of [³H]rolipram was relatively slow, with a half-time of 6 min (Fig. 1).

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Time course of association (A, C) and dissociation (B, D) of [3H]piclamilast and [3H]rolipram binding. Rat cerebral cortical membranes were incubated at 30°C with either 2 nM [3H]piclamilast or [3H]rolipram. Association was started by the addition of membranes. Dissociation was started by the addition of 1 mM rolipram after 1 h preincubation with either 2 nM [3H]piclamilast or [3H]rolipram. Binding was terminated by rapid filtration at different times. Results are from at least three independent experiments (mean ± S.E.M.).

Mg²⁺ Dependence. Binding of [³H]piclamilast (2 nM) and [³H]rolipram (2 nM) was determined at three concentrations of Mg²⁺ (Fig. 2). Mg²⁺ increased the specific binding of both radioligands in a concentration-dependent manner. In the presence of 5 mM Mg²⁺, the specific binding of [³H]piclamilast was about 4-fold higher than the specific binding in the absence of Mg²⁺; for rolipram, the Mg²⁺-dependent increase was about 3-fold.

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Mg2+-dependent binding of [3H]piclamilast and [3H]rolipram. Rat cerebral cortical membranes were incubated with either 2 nM [3H]piclamilast (A) or 2 nM [3H]rolipram (B) in the presence of different concentrations of Mg2+ at 30°C for 1 h. Binding was terminated by rapid filtration. Results are from at least three independent experiments (mean ± S.E.M.). *, significantly different from value in the absence of Mg2+, p < 0.05; **, p < 0.01.

Saturation Binding. Saturation binding of [³H]piclamilast and [³H]rolipram was carried out using whole homogenates of rat cerebral cortex and crude membrane and soluble (i.e., cytosolic) fractions (Fig. 3; Table 1). Specific binding of both [³H]piclamilast and [³H]rolipram to homogenate, membrane, and cytosolic preparations of rat cerebral cortex was saturable and of high affinity. Using nonlinear regression analysis, the binding of each radioligand was found to be adequately described by a one-site model. Adjusted for protein content, the B_max values for [³H]piclamilast binding to membranes and whole homogenate were similar; the B_max value for binding to the cytosolic fraction was slightly less, but the difference was not significant.

View larger version (10K): [in this window] [in a new window]   Fig. 3.   Saturation analysis of [3H]piclamilast (A) and [3H]rolipram (B) binding to membrane preparations of rat cerebral cortex. For [3H]piclamilast binding, concentrations used were 0.01 to 20 nM. For [3H]rolipram, concentrations used were 0.5 to 50 nM. Binding was carried out at 30°C for 1 h. Results are from three independent experiments (mean ± S.E.M.). Bmax and Kd values, determined by nonlinear regression analysis, are shown in Table 1.

Distribution of High- and Low-Affinity Sites. [³H]Piclamilast binding to preparations of various regions of brain and peripheral tissues was determined in the presence of different concentrations of rolipram. Data were analyzed by nonlinear regression to assess potential one- or two-site binding. Rolipram inhibition using preparations of the brain regions was better described by a two-site model, while the inhibition using the peripheral tissue preparations was adequately described by a one-site model (Fig. 4; Table 2). In all the brain regions examined, approximately 60% of the total specific binding was to the high-affinity binding site; this was quite consistent across the different regions as well as for homogenate, membrane, and cytosolic preparations. High-affinity binding was not detected in peripheral tissues (Fig. 4; Table 2). The affinity values for the binding of rolipram to the low-affinity binding site were consistent for all the brain regions and the peripheral tissues, except for liver, for which an almost 100-fold lower affinity was observed.

View larger version (11K): [in this window] [in a new window]   Fig. 4.   Rolipram inhibition of [3H]piclamilast binding to membranes prepared from rat cerebral cortex, skeletal muscle, liver, and heart. Membranes were incubated with 2 nM [3H]piclamilast and various concentrations of rolipram at 30°C for 1 h. Data shown are from one of three experiments. The competition curve obtained using cerebral cortical membranes was better described by a two-site model, while the competition curves using the membranes prepared from peripheral tissues were adequately described by a one-site model. Ki values are shown in Table 2.

Inhibition of [³H]Piclamilast and [³H]Rolipram Binding. A variety of specific PDE4 inhibitors and inhibitors of other PDE families were tested for their potency to inhibit [³H]piclamilast and [³H]rolipram binding to rat cerebral cortical membranes; the K_i values are shown in Table 3. For [³H]piclamilast binding (Fig. 5), the competition curves for rolipram, desmethylpiclamilast, ICI 63,197, Ro 20-1724, and cilomilast were better described by a two-site model; the competition curves for piclamilast, roflumilast, and CDP 840 were adequately described by a one-site model. For those inhibitors that bound to two sites, the K_i values for the high-affinity interaction were all in the nanomolar range, while the K_i values for low-affinity interaction were all in the low micromolar range.

View this table: [in this window] [in a new window]   TABLE 3 Ki values for PDE4 inhibitors and non-PDE4 inhibitors determined from competition assays using [3H]piclamilast and [3H]rolipram binding to rat cerebral cortical membranes Rat cerebral cortical membranes were incubated with 2 nM [3H]piclamilast or 2 nM [3H]rolipram in the presence of different concentrations of PDE4 inhibitors and non-PDE4 inhibitors. Ki values were calculated from IC50 values (Cheng and Prusoff, 1973), which were determined by nonlinear regression (Draper and Smith, 1966; O'Donnell et al., 1984). Results are from at least three separate experiments (mean ± S.E.M.).

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Inhibition of [3H]-piclamilast binding by PDE4 inhibitors. Rat cerebral cortical membranes were incubated with 2 nM [3H]-piclamilast and various concentrations of inhibitors at 30°C for 1 h. Data shown are from one of three experiments. The competition curves for rolipram, desmethylpiclamilast (DMP), ICI 63,197, and Ro 20-1724 were better described by a two-site model, while the competition curves for piclamilast, cilomilast, roflumilast, and CDP 840 were adequately described by a one-site model. Ki values are shown in Table 3.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Inhibition of [3H]rolipram binding by PDE4 inhibitors. Rat cerebral cortical membranes were incubated with 2 nM [3H]rolipram and various concentrations of inhibitors at 30°C for 1 h. Data shown are from one of three experiments. The competition curves for the PDE4 inhibitors were adequately described by a one-site model. Ki values are shown in Table 3.

View larger version (17K): [in this window] [in a new window]   Fig. 7.   Inhibition of [3H]piclamilast binding in the presence of 1 µM rolipram (to isolate the LARBS). Rat cerebral cortical membranes were incubated with 2 nM [3H]piclamilast and 1 µM rolipram, together with various concentrations of different inhibitors at 30°C for 1 h. Data shown are from one of three experiments; 100% binding was defined as that in the presence of 2 nM [3H]piclamilast and 1 µM rolipram. The competition curves for the PDE4 inhibitors were adequately described by a one-site model. Ki values are shown in Table 3.

	Discussion

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In the present study, the binding of [³H]piclamilast to preparations of rat brain was compared with that of [³H]rolipram. The results indicated that [³H]piclamilast labels both the LARBS and the HARBS with high affinity. [³H]rolipram, by contrast, at the concentration range examined, bound only to the HARBS.

Studies have investigated the relationship between the biological effects of PDE4 inhibitors and their interaction with the HARBS and the LARBS (Souness et al., 1995, 1996; Barnette et al., 1996; Duplantier et al., 1996; Kelly et al., 1996; Souness et al., 1999). In most of these studies, inhibition of [³H]rolipram binding is used to evaluate affinity for the HARBS, while inhibition of PDE4 hydrolytic activity in a cell-free system is used to evaluate affinity for the LARBS. The LARBS is difficult to study using [³H]rolipram binding.

Jacobitz and colleagues (1996) used an equilibrium filtration technique to detect both low- and high-affinity rolipram binding; by using this method, the filter washing protocol is eliminated. Because the ligand/receptor complex is not diluted during the separation procedure, the equilibrium condition is maintained and the ligand bound to the low-affinity site does not dissociate. However, as a consequence, nonspecific binding in this assay is highly variable. Equilibrium dialysis is another method used to study the LARBS (Rocque et al., 1997). A true equilibrium binding constant can be measured by using this method. However, a high protein concentration is required to obtain an adequate signal-to-noise ratio.

Recombinant systems that express either high- or low-affinity conformations of PDE4 have been developed to evaluate inhibitors (Bardelle et al., 1999; Allen et al., 1999). PDE4A330, a PDE4A truncate, expressed in Chinese hamster ovary cells exhibits only the low-affinity conformation, while PDE4A4, 4B2, 4C2, and 4D3 all adopt a high-affinity binding conformation for rolipram. These systems can be used to screen and optimize inhibitors against the low-affinity conformation of PDE4.

Piclamilast is a selective and very potent PDE4 inhibitor (Karlsson et al., 1993; Ashton et al., 1994). In contrast to rolipram, piclamilast binds with high affinity to both the HARBS and the LARBS (Souness et al., 1995). Thus, [³H]piclamilast is a potential tool to study the LARBS and the HARBS. The association and dissociation of [³H]piclamilast were both fast and exhibited first-order kinetics, which is consistent with the saturation binding data. By contrast, the association of [³H]rolipram binding was better described by a two-component model; its dissociation, however, was adequately described by a one-site model. Previously, Schneider and coworkers (1986) reported that both the association and dissociation of rolipram binding are biphasic.

At the active site of PDE4 there are two divalent metal ions in a binuclear motif that are involved in both cAMP binding and catalysis (Laliberte et al., 2000; Xu et al., 2000). [³H]rolipram binding is Mg²⁺-dependent (Schneider et al., 1986). It has been suggested that the HARBS and the LARBS are the consequence of PDE4 binding to its metal cofactor, such as Mg²⁺ (Laliberte et al., 2000). Mg²⁺, Mn²⁺, and Co²⁺ all stabilize high-affinity rolipram binding to the PDE4 holoenzyme. In the absence of the divalent cations, only low-affinity rolipram binding to the apoenzyme is detected (Liu et al., 2001). Furthermore, in vitro, protein kinase A phosphorylation of PDE4A4, which shifts the potencies of (R)/(S)-rolipram toward their holoenzyme binding affinities, activates the enzyme by increasing its sensitivity to the Mg²⁺ cofactor. Piclamilast exhibits equal affinity for the HARBS and the LARBS. However, in the present study, piclamilast binding to PDE4 was increased in the presence of Mg²⁺. Huang and coworkers (2000) also found that piclamilast binds preferentially to the holoenzyme. This suggests that Mg²⁺ dependence of inhibitor binding does not fully predict the nature of inhibitor interaction with the HARBS and the LARBS.

[³H]Piclamilast binding was saturable and reversible. The B_max value for [³H]piclamilast was about 1.5-fold greater than that of [³H]rolipram. Jacobitz and coworkers (1997) reported that the B_max value for [³H]piclamilast binding to human recombinant PDE4 was 2- to 3-fold greater than that for [³H]rolipram binding. These results likely reflect the fact that [³H]piclamilast binds to both the HARBS and the LARBS at nanomolar concentrations, while [³H]rolipram only binds to HARBS at these concentrations. The saturation curve for [³H]piclamilast binding, however, was consistent with a one-site model, suggesting that piclamilast binds to the two sites with similar affinity.

Schneider and the coworkers (1986) reported that the maximal [³H]rolipram binding to membranes was higher than that to the cytosolic preparations. In the present study, the B_max value for both [³H]piclamilast and [³H]rolipram binding to membranes tended to be higher than to the cytosolic fractions; however, these difference were not significant. The K_d values for [³H]piclamilast binding to whole homogenate, membrane, and cytosolic preparations were similar, while [³H]rolipram exhibited a lower K_d value for binding to the membrane fraction than to the cytosolic fraction or whole homogenate. Souness and coworkers (1992) reported that the binding of rolipram, but not piclamilast, to eosinophils is altered by solubilization or treatment with vanadate/glutathione. These results suggest that the affinity of rolipram binding, but not that of piclamilast, is sensitive to the conformational state of PDE4. The difference in K_d values for [³H]rolipram binding to membrane and cytosolic fractions may be due in part to the differential expression pattern of PDE4 variants. For example, PDE4A1 is expressed solely in membrane fractions, while PDE4A5 is present in both membrane and cytosolic fractions (McPhee et al., 1995; Huston et al., 1996; Pooley et al., 1997). Slight differences in affinity for the differentially distributed variants could translate into a difference in K_d values. In addition, association of PDE4 with membrane-associated proteins such as receptor for activated c-kinase (RACK) might result in a different rolipram affinity for membrane and cytosolic preparations containing PDE4 (Yarwood et al., 1999).

All the brain regions tested exhibited the HARBS, with the percentage varying from 47 to 66%. By contrast, the HARBS was not detected in peripheral tissues. The HARBS in rat brain was first demonstrated by Schneider and coworkers (1986). They reported that the peripheral organs tested showed either no detectable [³H]rolipram binding or a very low specific binding capacity. Although the nature of the HARBS is not yet clear, it has been suggested to be one of the two distinct conformations of PDE4 (Torphy et al., 1992; Souness and Scott, 1993). Consistent with this interpretation, the present results showed that the rolipram competition curve for [³H]piclamilast is biphasic, indicating [³H]piclamilast labels two sites on PDE4 that exhibit different affinities for rolipram. [³H]piclamilast showed the highest total binding in hippocampus. The binding also was high in hypothalamus, neostriatum, cerebellum, and amygdala. An autoradiographic study using [³H]rolipram binding revealed high binding densities for the HARBS in the cerebellum, olfactory bulb, frontal cortex, subiculum, and the CA1 region of the hippocampus (Kaulen et al., 1989).

For [³H]piclamilast binding, the competition curves for rolipram, desmethylpiclamilast, ICI 63,197, and Ro 20-1724 were better described by a two-site model, while the competition curves for piclamilast, cilomilast, roflumilast, and CDP 840 were adequately described by a one-site model. For those inhibitors that bound differentially to two states, there was approximately 100-fold greater affinity for the HARBS compared with the LARBS. K_i1 values, which represent binding to the HARBS, exhibited more variability compared with K_i2 values, which represent binding to the LARBS. The competition curve for piclamilast was adequately described by a one-site model. This is consistent with the finding from saturation analyses that piclamilast binds to both sites with equal affinity. The potency order of PDE4 inhibitors tested in this study was in agreement with the potency orders of these inhibitors reported previously for inhibition of enzyme activity (Schneider et al., 1986; Souness et al., 1997, 1999; Saldou et al., 1998). The competition curves for [³H]rolipram binding all were adequately described by a one-site model, with a potency order that was the same as that for inhibition of [³H]piclamilast binding. The inhibition of [³H]piclamilast and [³H]rolipram binding by non-PDE4 inhibitors was less potent and showed no significant differences between the two radioligands. Consistent with their low binding affinities, these drugs also have been shown to be very poor inhibitors of PDE4 enzymatic activity (Schneider et al., 1986; Souness and Scott, 1993; Makhay et al., 2001).

The LARBS can be studied directly using [³H]piclamilast binding in the presence of 1 µM rolipram, which blocked >90% of the ligand binding to the HARBS. The remaining [³H]piclamilast binding was further inhibited by PDE4 inhibitors with K_i values in the micromolar range and with competition curves consistent with a one-site model. These K_i values were close to the K_i2 values from the competition study carried out in the absence of 1 µM rolipram, which represent the binding affinity for the LARBS. These results indicate that while [³H]piclamilast binds to both the LARBS and the HARBS, inhibitor affinity for the LARBS can be evaluated using [³H]piclamilast in the presence of a proper concentration of rolipram.

In summary, it has been shown that [³H]piclamilast labels both the LARBS and the HARBS of PDE4 in rat brain with similar affinity. [³H]piclamilast binds only to the LARBS, when the HARBS is blocked with a low concentration of rolipram. All these properties should make [³H]piclamilast a useful tool to study the interaction of PDE4 inhibitors with the LARBS to assess the relationship between inhibitor interactions with this site and their pharmacological effects.