Phosphodiesterase type 4 isozymes expression in human brain examined by in situ hybridization histochemistry and [3H]rolipram binding autoradiography: Comparison with monkey and rat brain

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Abstract

We have examined the distribution of four different cyclic AMP-specific phosphodiesterase isozyme (PDE4A, PDE4B, PDE4C and PDE4D) mRNAs in the brain of different species by in situ hybridization histochemistry and by autoradiography with [3H]rolipram. We have compared the localization of each isozyme in human brain with that in rat and monkey brain. We have found that the four PDE4 isoforms display a differential expression pattern at both regional and cellular level in the three species. PDE4A, PDE4B and PDE4D are widely distributed in human brain, with the two latter appearing more abundant. In contrast, PDE4C in human brain, presents a more restricted distribution, limited to cortex, some thalamic nuclei and cerebellum. This is at variance with the distribution of PDE4C in rat brain, where it is found exclusively in olfactory bulb. In monkey brain, the highest expression for this isoform is found in the claustrum, and at lower levels in cortical areas and cerebellum. PDE4B presented a broad distribution, being expressed in both neuronal and non neuronal cell populations. In general, the distribution of binding sites visualized with [3H]rolipram correlated well with the expression of each PDE4 isozyme.

Introduction

Intracellular cyclic adenosine 3′,5′-monophosphate (cAMP) plays an important role as a second messenger molecule controlling multiple cellular processes. It serves to transduce the action of a wide variety of neurotransmitters, hormones and can modulate signal transduction processes regulated by a range of growth factors, cytokines, and other agents. Some of the known functions of cAMP are gene transcription (Lalli and Sassone-Corsi, 1994), neurotransmitter biosynthesis (Kaufman, 1995) and release (Kandel and Schwartz, 1982), as a response regulator (Morimoto and Koshland, 1991), survival of dopaminergic neurons (Yamashita et al., 1997), synaptic facilitation and potentiation (Zhong and Wu, 1991) among others.

In order to understand the basic mechanisms underlying these functions, it is necessary to define how levels of cAMP are regulated. Its synthesis is controlled in neurons, by a variety of membrane neurotransmitter receptors, including subtypes of the dopamine, serotonin and noradrenergic receptors. These act by means of coupling of GTP-binding (G) proteins to adenylyl cyclase either to stimulate or inhibit formation of cAMP (Houslay et al., 1998). The hydrolysis of cAMP is catalyzed by a family of enzymes called phosphodiesterases (PDE). Eleven members of this family have been described until present, and classified on the basis of substrate specificity, kinetic properties, sensitivity to specific inhibitors, tissue distribution and sequence derived information (Fawcett et al., 2000, Soderling and Beavo, 2000).

In this work, we have focused on the cAMP-specific phosphodiesterase 4 family (PDE4). Four kinds of PDE4 isozymes (PDE4A, PDE4B, PDE4C and PDE4D) are encoded by different gene loci, and each of them has been shown to produce several mRNAs by alternative splicing (Bolger et al., 1994). This group of isozymes is widely expressed in many tissues. These include the brain, where PDE4 is likely to be involved in processes such as control of mood, emesis and olfactory sensory transduction (Beavo, 1995). They are characterized by a low Km, Ca2+-insensitivity, specificity for cAMP as a substrate and sensitivity to the specific inhibitor rolipram.

The widespread distribution and highly tissue-specific patterns of expression of PDE4 isozymes in human tissues has opened the opportunity for the development of PDE4 inhibitors that would be useful in the treatment of human disease. Over the past decade, PDE4 inhibitor research has focused on two broad therapeutic areas, notably the CNS, with emphasis on depression, and in disorders of the immune and inflammatory systems.

Rolipram is one of the most potent and widely studied compound in the group of neuroactive cAMP-PDE inhibitors. Rolipram is a selective inhibitor of PDE4 that preferentially inhibits cAMP in brain, thus enhancing the intracellular availability of cerebral cAMP in the absence of direct stimulation of neurotransmitter receptors (Schneider, 1984, Schneider et al., 1986). Rolipram was described as a novel pharmacological approach for the treatment of endogenous depression (Watchel, 1983). Rolipram exhibits anti-depressant effects in models predictive of anti-depressant activity (Watchel and Schneider, 1986) and has been shown to be clinically effective (Zeller et al., 1984). However, its side effects (specially gastrointestinal actions, like nausea, pyrosis and emesis) due to the low degree of selectivity for the different PDE4 isoforms (Horowski and Sastre-y-Hernandez, 1985), have been the drawback for clinical application.

The aim of the present study was to map in detail the regional and cellular distribution of the four transcripts of PDE4 in human brain, in comparison with experimental animal (rodent and non-human primate). We have also studied how their distribution correlate with that of [3H]rolipram binding sites (a measurement of protein expression). From the comparison between the distribution of mRNA and corresponding binding sites, inferences could be drawn on the localization of these cAMP PDEs in many brain regions.

Recent studies present evidence on the fact that specific structural differences among various members of the PDE4 family account for markedly different regulatory properties (Torphy and Page, 2000). The exquisite degree of tissue selectivity presented by PDE4 isozymes can help to obtain new classes of inhibitors by targeting to a single PDE4 isozyme.

Section snippets

Specimens

Adult male Wistar rats (n=4) (200–300 g) were purchased from Iffa Credo (Lyon, France). The animals were killed by decapitation, brains were removed quickly, frozen on dry ice and kept at −20°C. Two male and one female monkey brains (Macaca fascicularis, age between 3 and 7 years old, weight range 2.4–2.7 kg, mean postmortem delay 1 h) were used. The animals were sacrificed by administration of an overdose of sodium pentobarbital. All the procedures conformed to the European Communities Council

Results

The specificity of labeling of [3H]rolipram binding sites has been earlier documented for rat brain (Kaulen et al., 1989). [3H]rolipram binding to all mammalian brain regions examined (see an example in Fig. 1A1 and C1) was completely blocked by rolipram (Fig. 1A2 and C2), in good agreement with the earlier report. Human and monkey brain sections were hybridized with the same specific oligonucleotide probes complementary to each of the PDE4 human isozyme mRNAs. Rat brain coronal sections were

Discussion

By using oligonucleotide probes that selectively recognize the transcripts of the four isozyme forms of the PDE4 family (PDE4A, PDE4B, PDE4C and PDE4D), we have examined their regional distribution and compared them in the brain of human, monkey and rat. Our results show that in the brain of these three species, PDE4A, PDE4B and PDE4D mRNA expression is abundant and widely distributed, not only in neuronal cells but also in white matter cells, especially PDE4B and PDE4D. An important finding is

Acknowledgements

This work was supported, by grants from Fundació La Marató de TV3 (#1017/97), CICYT (SAF1999-0123) and CICYT (#2FD97-0395). S.P.-T. is a recipient of a fellowship from CIRIT (Generalitat de Catalunya) and X.M. from CIRIT (Centre de Referència de la Generalitat de Catalunya).

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