DOI 10.1007/s00702-001-0684-1 J Neural Transm (2002) 109: 1139–1149

Effects of rolipram on in vivo dopamine receptor binding R. Hosoi1, M. Ishikawa1, K. Kobayashi1, A. Gee2, M. Yamaguchi3, and O. Inoue1 Department of Medical Physics, School of Allied Health Sciences, Faculty of Medicine, Osaka University, Osaka, Japan 2 GlaxoSmithkline, Clinical Research Unit, ACCI, Addenbrookes Hospital, Cambridge, United Kingdom 3 Faculty of Pharmaceutical Sciences, Fukuoka University, Fukuoka, Japan 1

Received August 16, 2001; accepted November 13, 2001 Published online June 20, 2002; © Springer-Verlag 2002

Summary. In order to clarify whether changes in brain concentrations of the second messenger cyclic AMP (cAMP) affect in vivo receptor binding in the brain, the effects of rolipram, a selective inhibitor of phosphodiesterase type 4 (PDE4), on dopamine receptor binding in the mouse brain were studied. Rolipram significantly decreased in vivo 3H-SCH 23390 (dopamine D1 selective radioligand) binding in the mouse striatum in a dose-dependent manner. In vivo saturation experiments together with the kinetic analysis of 3H-SCH 23390 binding revealed that the apparent association rate constant (kon) for 3 H-SCH 23390 binding rather than the maximum number of binding sites available (Bmax) was decreased by rolipram. 3H-N-methylspiperone (NMSP, dopamine D2 selective radioligand) binding in the mouse striatum was also decreased by rolipram whereas no significant changes in 3H-raclopride (dopamine D2 selective radioligand) binding were observed. As 3H-raclopride binding has been reported to be much more sensitive than 3H-NMSP binding to competition by endogenous dopamine, the decreases in 3H-SCH 23390 and 3 H-NMSP binding cannot be attributed to competitive inhibition by endogenous dopamine. These results indicate that changes in second messenger cAMP concentrations may affect the apparent bimolecular association rate constant (kon) of dopamine receptor binding in intact brain. This may be mediated by changes in the receptor micro-environment and altered actual free ligand concentration surrounding the receptors. Keywords: Rolipram, cyclic AMP, dopamine receptor, SCH 23390, Nmethylspiperone, raclopride. Abbreviations cAMP cyclic AMP, PDE4 phosphodiesterase type 4, NMSP N-methylspiperone, PKA protein kinase A

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Introduction

There have been several reports that have indicated an important role for cyclic AMP (cAMP) on receptor binding and receptor function. For example, agonist mediated down regulation of dopamine D1 receptors has been well documented (Black et al., 1994) and exposure to dopamine agonists causes time-dependent phosphorylation of dopamine D1 receptors. The number of binding sites of D1 receptors has been shown to be decreased by exposure to dopamine agonists due to phosphorylation of D1 receptors. This down regulation of dopamine D1 receptors induced by cAMP mediated protein kinase A (PKA) might have a physiological role as a positive feed back system in regulation of signal transmission in the brain. With regard to dopamine D2 receptors, the effect of cAMP is controversial. Ivins (1991) reported agonist induced up-regulation of D2 receptors in vitro. However, in contrast downregulation of D2 receptors exposured to agonists in vitro has also reported (Barton and Sibley, 1990). It is of interest whether changes in cAMP content in the living brain also affect in vivo dopamine receptor binding. As previously reported, acute treatment with desipramine and other types of antidepressants significantly decreased in vivo both 3H-SCH 23390 and 3H-N-methylspiperone (NMSP) binding in mouse striatum (Suhara et al., 1990). As most of antidepressants interact with serotonergic and noradrenergic systems in the brain, in vivo levels of cAMP, the major second messenger for these receptor systems, might be altered by acute and/or chronic treatment with antidepressants. In this study, rolipram, a selective inhibitor of phosphodiesterase type 4 (PDE4) (Reeves et al., 1987), was used. Rolipram inhibits cAMP metabolism (Watchel, 1982) and increases basal intracellular cAMP levels (Stone and John, 1990; DeLapp and Eckels, 1992) in the brain. 3 H-SCH 23390, 3H-NMSP and 3H-raclopride were chosen as selective radioligands for probing the response of the rolipram-induced cAMP elevation at dopamine D1 or D2 receptors, respectively. Material and methods Animals Male ddY mice (8 weeks old) were obtained from SLC (Hamamatsu, Japan) and housed at 23°C on a 12 hr light-dark cycle. All mice were given free access to food and water. The studies were performed under the permission of the Institutional Animal Care and Use Committee, School of Allied Health Sciences, Osaka University.

Chemicals 3 H-SCH 23390, specific radioactivity 2.6 TBq/mmol, 3H-N-methylspiperone (NMSP), specific radioactivity 3.1 TBq/mmol and 3H-raclopride, specific radioactivity 2.9 TBq/ mmol were obtained from New England Nuclear (Boston, MA, USA). Rolipram (Tocris Cookson Ltd., Bristol, UK) was suspended in saline containing 5% plant oil (HCO-60, Nikkol). SCH 23390 hydrochloride was purchased from Research Biological Institute (Natick, MA, U.S.A.).

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Methods Time courses of radioactivity concentration in the brain following injection of radiotracers Mice were intravenously injected with 74 kBq of either 3H-SCH 23390 (dopamine D1 selective radioligand), 3H-NMSP (dopamine D2 selective radioligand) or 3H-raclopride (dopamine D2 selective radioligand), lightly anesthetized with ether and decapitated at various time intervals after the tracer injection. Their brains were quickly removed and dissected into cerebral cortex, striatum and cerebellum. The samples were weighed and dissolved with 1 ml of tissue solubilizer (Soluene-350, Packard) and incubated for 24 hrs. After adding 5 ml scintillation fluid (Hionic-Fluor, Packard), the radioactivity of each sample was measured with a liquid scintillation counter. The values were expressed as percent injected dose per gram of wet tissue (%dose/g). The time courses of radioactivity concentration in the brain of mice pretreated with rolipram (3 mg/kg, i.p. injection 15 min prior to the tracer injection) were determined by the method described above.

Kinetic analysis For all radioligands the cerebellum was used as a reference region for estimation of the free ligand concentration and the non-specific binding in the brain. Specific binding in the cerebral cortex and striatum was calculated by subtracting the radioactivity concentration in the cerebellum from the total radioactivity concentration in the region of interest. A simplified two-compartment model as previously reported (Kobayashi and Inoue, 1993) was employed for the determination of k3 and k4 (the input and output rate constants of specific binding component) of 3H-SCH 23390. A graphical method (Patlak plot, Patlak and Blasberg, 1985) was applied for the quantification of 3H-NMSP binding. The input rate constant (k3) is parallel with products of two components, the maximum number of binding sites available (Bmax) and the bimolecular association rate constant (kon). The output rate constant (k4) is euqal to the dissociation rate constant (koff).

Saturation experiments of 3H-SCH 23390 binding in vivo Saline or rolipram pretreated (3 mg/kg i.p. injection 15 min prior to the tracer injection) mice were intravenously injected with 3H-SCH 23390 (less than 0.3 µg/kg) together with increasing amounts of non-labeled SCH 23390 (from 3 to 3000 µg/kg). Thirty min after the tracer injection, mice were decapitated and radioactivity concentrations in the striatum and the cerebellum were determined by the method described above. The cerebellum was used as a reference region for free and non-specifically bound ligand. Specific binding in the striatum was determined as described above.

Statistical analysis The differences between control and rolipram treated groups were examined by twofactor factorial ANOVA. Group differences after significant ANOVAs were measured by Student’s t-test.

Results Effect of rolipram on 3H-SCH 23390 binding in vivo

The time courses of total radioactivity concentration in the cerebellum and the specific binding in the striatum and cerebral cortex following 3H-SCH 23390 injection in control or rolipram pretreated mice are shown in Fig. 1. The specific binding in the cerebral cortex (Fig. 1B) and the striatum (Fig. 1A)

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Fig. 1. The time courses of radioactivity concentration (specific binding) in the striatum (A) and cerebral cortex (B) and total radioactivity concentration in the cerebellum following i.v. injection of 3H-SCH 23390. The open and closed symbols indicate control and rolipram-pretreated mice, respectively. Results are mean ⫾ S.D. of 3 animals. Rolipram caused significant differences for the striatum and cerebral cortex [F(1, 24) ⫽ 34.02 and 29.68, respectively, p ⬍ 0.01] but not for the cerebellum [F(1, 24) ⫽ 2.60, p ⬎ 0.05]. *P ⬍ 0.05 as compared to control mice at the same time determined by Student’s t-test

reached a plateau at 10 and 20 min post injection of the tracer, respectively. The specific binding in mice pretreated with rolipram was significantly decreased in the striatum and cerebral cortex. One mg/kg of rolipram also decreased in 3H-SCH 23390 binding 30 min after tracer injection in the striatum and cerebral cortex, but not significant (data not shown). The results of the kinetic analysis of 3H-SCH 23390 binding are summarized in Table 1. A decrease in the input rate constant (k3) in the striatum was produced by rolipram. Effect of rolipram on 3H-NMSP binding in vivo

The time courses of total radioactivity concentration in the cerebellum and the specific binding in the striatum and cerebral cortex following injection of 3H-NMSP in control or rolipram pretreated mice are shown in Fig. 2. A significant decrease in 3H-NMSP binding in the striatum was observed in rolipram pretreated mice. On the other hand, a decrease in the total radioactivity concentration in the cerebellum was observed at 1 min after the tracer injection. The input rate constant (k3) in the striatum estimated by the Patlak plot was decreased 11% by rolipram (Table 1). In the cerebral cortex, a slight decrease in the specific binding in the early phase post tracer injection was observed.

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Fig. 2. The time courses of radioactivity concentration (specific binding) in the striatum (A) and cerebral cortex (B) and total radioactivity concentration in the cerebellum following i.v. injection of 3H-NMSP. The open and closed symbols indicate the control and rolipram-pretreated mice, respectively. Results are mean ⫾ S.D. of 3 to 4 animals. Rolipram caused significant differences for the striatum, cerebral cortex and cerebellum [F(1, 35) ⫽ 42.06, 27.19 and 9.19, respectively, p ⬍ 0.01] *P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍ 0.001 as compared to control mice at the same time determined by Student’s t-test

The effect of rolipram on 3H-raclopride binding in vivo

The time courses of total radioactivity concentration in the cerebellum and the specific binding in the striatum and cerebral cortex of 3H–raclopride in control or rolipram pretreated mice are shown in Fig. 3. In all regions were examined, there are no significant differences between control and rolipram pretreated mice. Saturation of 3H-SCH 23390 binding in vivo

The results of the saturation study on in vivo 3H-SCH 23390 binding in control and rolipram pretreated mice are shown in Fig. 4. The specific binding of 3HSCH 23390 in the striatum was significantly decreased by pretreatment with

Table 1. Results of kinetics analysis for striatal binding in control and rolipram-pretreated mice H-SCH 23390

3

k3 (min⫺1)

k4 (min⫺1)

H-NMSP k3 (min⫺1)

0.258 0.209

0.0230 0.0238

0.117 0.104

3

Control Rolipram

Calculated from date of Fig. 1 and Fig. 2

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Fig. 3. The time courses of radioactivity concentration (specific binding) in the striatum (A) and cerebral cortex (B) and total radioactivity concentration in the cerebellum following i.v. injection of 3H-raclopride. The open and closed symbols indicate the control and rolipram-pretreated mice, respectively. Results are mean ⫾ S.D. of 3 animals. There are no significant differences between control and rolipram-pretreated mice for the striatum, cerebral cortex and cerebellum [F(1, 24) ⫽ 0.19, 1.45 and 2.91, respectively, p ⬎ 0.1]

Fig. 4. In vivo saturation of 3H-SCH 23390 binding in the striatum. Mice were injected with 3H-SCH 23390 together with various doses of non-labeled SCH 23390 and decapitated 30 min after the injection. The radioactivity concentration (specific binding) in the striatum is shown. The open and closed circles indicate the control and roliprampretreated mice, respectively. Results are mean ⫾ S.D. of 3 to 6 animals. Rolipram caused significant differences for the striatum when carrier SCH 23390 concentration were below 30 µg/kg [F(1, 30) ⫽ 54.69, p ⬍ 0.01]. *P ⬍ 0.05, **P ⬍ 0.01 as compared to control mice determined by Student’s t-test

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rolipram for various injected doses of SCH 23390 (from 0.3 µg/kg to 30 µg/kg). And apparent positive cooperativity of binding was observed, particularly in rolipram pretreated mice. In other words, the effect of rolipram was more pronounced when carrier SCH 23390 concentrations were very low (below 30 µg/kg). At an injected dose of 100 µg/kg SCH 23390, no significant differences in specific binding were observed in the striatum. The estimated maximum number of binding sites available (Bmax) for 3H-SCH 23390 was not significantly altered by rolipram (Control; 308.8 pmol/g tissue, Rolipram 300.4 pmol/g tissue). Discussion

Rolipram is a potent and selective PDE4 inhibitor (Schneider et al., 1986; Kaulen et al., 1989; McLaughlin et al., 1993). Regional distribution studies of 3H-rolipram binding sites in rat brain have shown a widespread distribution of PDE4 throughout the brain (Perez et al., 2000). Acute treatment with rolipram has been reported to significantly increase the cAMP content in the brain. For example, Schneider (1984) reported that 3 mg/kg of rolipram elevated the cAMP levels by 190% and 230% in the rat frontal cortex and striatum, respectively. In the present study, rolipram significantly decreased both 3H-SCH 23390 and 3H-NMSP binding in the mouse striatum. There are several factors, which may explain these apparent changes in in vivo receptor binding, such as changes in regional blood flow, levels of endogenous dopamine, alterations in the maximum number of binding sites available (Bmax) and changes in the bimolecular association (kon) and dissociation rate constants (koff) as well as the affinity constant (Kd). Changes in the delivery process, including blood flow, of radioligands into the brain is a possible mechanism to explain the reduction in apparent 3HSCH 23390 and 3H-NMSP binding induced by rolipram. However, no significant alteration in the time course of total radioactivity concentration in the cerebellum was observed following injection of either 3H-SCH 23390 or 3Hraclopride, indicating that blood flow was not significantly changed at least in the cerebellum. Further detailed studies on the effects of blood flow on in vivo receptor binding are necessary. 11 C-raclopride and 11C-NMSP are the most commonly used radioligands for dopamine D2 receptor mapping. The former has been reported to be much more sensitive against competitive inhibition both in vitro and in vivo (Seeman et al., 1989; Rose and Jackson, 1989; Hall et al., 1990; Young et al., 1991). The present results indicate that factors other than competitive inhibition by endogenous dopamine might play an important role on changes in receptor binding in vivo. Rolipram has been reported to enhance dopamine release in brain slices (Schoffelmeer et al., 1985) or to induce significant increases of basal dopamine levels by local infusion in the rat striatum (West et al., 1996). As rolipram had a significant effect on SCH 23390 and NMSP binding but not raclopride binding, it is difficult to rationalize the results on the basis of competitive inhibition by endogenous dopamine.

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In this study, rolipram decreased 3H-SCH 23390 binding in mouse striatum in a dose-dependent manner (data not shown) without any changes in the maximum number of binding sites available (Bmax) values measured by in vivo saturation experiments. The results of the kinetic analysis indicate that the rolipram-induced changes in radioligand binding are due to the decreases of the apparent bimolecular association rate constants (kon) of both 3H-SCH 23390 and 3H-NMSP probably due to increase in cAMP caused by rolipram. Dopamine D1 receptors are coupled to Gs proteins and dopamine D2 receptors to Gi proteins. These results indicate that linkage of Gs or Gi proteins to receptors does not influence second messenger-induced changes in receptor binding in intact brain. In our previous report on the effects of antidepressants on dopamine D1 and D2 receptor binding in intact brain, significant reduction in the binding of both radioligands was observed in vivo (Suhara et al., 1990). The decline of dopamine receptor binding induced by desipramine was mainly due to the decrease of the bimolecular association rate constants (kon) of both radioligands. Desipramine is a selective noradrenaline reuptake inhibitor, and increases endogenous noradrenaline in synaptic cleft, resulting in stimulation of beta receptors and increased second messenger cAMP content. Therefore, a mechanism similar to that observed in this study might have caused a decrease in the bimolecular association rate constants (kon) of 3H-SCH 23390 and 3H-NMSP binding in desipramine treated animals. It is also of interest that desipramine and other kinds of antidepressants significantly decrease the in vivo binding of 3H-QNB and 3H-NMPB in the mouse cerebral cortex and striatum. These results together with our finding that rolipram also significantly decreases the in vivo binding of 3H-NMPB in mouse cerebral cortex and striatum (manuscript in preparation) suggest that global changes in the micro-environmental architecture surrounding receptors throughout the brain might be induced by increases in cAMP. Phosphorylation of proteins induced by cAMP-PKA systems seems to be a possible mechanism for changes in micro-environmental architecture including membrane fluidity. Another interesting finding is that an apparent positive cooperativity of 3 H-SCH 23390 binding was observed in mice treated with rolipram as shown in Fig. 4. Apparent positive cooperativity of binding has been observed for muscarinic acethylcholine receptors (Repke and Maderspach, 1982; Inoue et al., 1998), beta-noradrenergic receptors (Maderspach and Fajszi, 1982, 1983) as well as mu-opioid receptors (Maderspach and Solomonia, 1988) but only in the intact system, and not in vitro system. Apparent positive cooperativity is abolished by tissue homogenization or other post mortem assays. Cooperativity might have an important physiological role in synaptic signal transmission. The role of cAMP on the apparent cooperativity is unclear, but might be related to amplification of signal transmission in the synapse. In this experiment, the radioligands used were antagonists both for dopamine D1 or D2 receptors. As yet it is not known whether the binding of D1 or D2 agonists, or dopamine itself is also affected by rolipram. As the intact brain is an extremely complex system to study, the use of living brain slices

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and positron emitting radioligands (Matsumura et al., 1995) may be of use in further revealing the role of cAMP on ligand-receptor interactions. Several reports indicate that rolipram affects the dopaminergic system. For example, the mesolimbic autoreceptors have been reported to regulate by inhibition of the presynaptic adenylate cyclase system (Onali and Olianas, 1989). Rolipram suppresses oro-facial movements (Sasaki et al., 1995a,b), and has been proposed to have a therapeutic effect on tardive dyskinesia. Treatment with rolipram also suppresses the supersensitivity caused by repeated treatment with amphetamine (Iyo et al., 1995, 1996). On the other hand, biochemical studies in vivo show that rolipram increases dopamine synthesis but decreases dopamine utilization in the brain (Kehr et al., 1985). The relationship between pharmacological effects and kinetic changes in receptor binding induced by rolipram is a future subject to be analyzed. In conclusion, rolipram significantly decreased in vivo binding of both dopamine D1 and D2 receptor radioligands. The results indicate that second messenger cAMP plays an important role in the regulation of ligand-receptor binding in intact brain.

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Wachtel H (1982) Characteristic behavioural alterations in rats induced by rolipram and other selective adenosine cyclic 3⬘, 5⬘-monophosphate phosphodiesterase inhibitors. Psychopharmacology Berl 77: 309–316 West AR, Galloway MP (1996) Regulation of serotonin-facilitated dopamine release in vivo: the role of protein kinase A activating transduction mechanisms. Synapse 23: 20–27 Young LT, Wong DF, Goldman S, Minkin E, Chen C, Matsumura K, Scheffel U, Wagner HN Jr (1991) Effects of endogenous dopamine on kinetics of [3H]N-methylspiperone and [3H]raclopride binding in the rat brain. Synapse 9: 188–194 Authors’ address: O. Inoue, Ph. D., Department of Medical Physics, School of Allied Health Sciences, Faculty of Medicine, Osaka University, 1-7 Yamada-oka, Suita, Osaka 565-0871, Japan, e-mail: [email protected]