Research article
In vivo selective binding of (R)-[11C]rolipram to phosphodiesterase-4 provides the basis for studying intracellular cAMP signaling in the myocardium and other peripheral tissues

https://doi.org/10.1016/j.nucmedbio.2006.10.002Get rights and content

Abstract

Introduction

Phosphodiesterase-4 (PDE4) enzymes specifically break down the second messenger cAMP, thereby terminating the intracellular signaling cascade that plays an essential role in neurohormonal modulation of many physiological systems. PDE4 activity and expression are regulated by cAMP levels, suggesting that measurement of PDE4 provides an index of intracellular cAMP signaling.

Methods

Male Sprague–Dawley rats were administered (R)- or the less active enantiomer (S)-[11C]rolipram and sacrificed 30 min later with tracer retention measured in various tissues. Co-injections with saturating doses of unlabeled (R)-rolipram, (S)-rolipram and Ro 20-1724, as well as subtype-selective PDE inhibitors vinpocetine, Bay 60-7550, cilostazol and zaprinast were used to establish binding selectivity for PDE4 over PDE1, PDE2, PDE3 and PDE5 subtypes, respectively. Autoradiography was performed to substantiate results of biodistribution studies in the myocardium.

Results

In vivo (R)-[11C]rolipram retention was dose-dependently reduced by co-injections of (R)-rolipram and (S)-rolipram (ED50 values of 0.03 mg/kg and 0.2 mg/kg, respectively). Vinpocetine, Bay 60-7550, cilostazol and zaprinast had no effect on (R)-[11C]rolipram binding, while (R)-rolipram and Ro 20-1724 reduced the tracer uptake to nonspecific levels in PDE4-rich tissues.

Conclusions

In addition to the brain, (R)-[11C]rolipram binds selectively to PDE4 across all cardiac regions, skeletal muscle, lungs and pancreas, but not in the adipose tissues. In vivo findings were confirmed by in vitro autoradiography studies, suggesting that (R)-[11C]rolipram can be applied to evaluate alterations in central and peripheral PDE4 levels and cAMP-mediated signaling.

Introduction

Cyclic 3′,5′-adenosine monophosphate (cAMP) second messenger is an important component of signaling cascades initiated by a number of neurohumoral pathways modulating cardiac [1] and metabolic functions [2]. Alterations in cAMP-mediated signaling have been reported in various disorders affecting a number of physiological systems, including asthma [3], heart failure [4], dementia and depression [5].

Phosphodiesterase-4 (PDE4) is one of the main enzymes that specifically hydrolyse cAMP (Vmax=0.3–11 μmol/(min mg), Km=2–8 μM) [6], thereby terminating the intracellular signaling following stimulation of the G subunit of various G protein-coupled receptors including β-adrenergic (β-AR), adenosine A2 and histamine H2 receptors [1], [3], [7]. PDE4 enzymes are distributed throughout the body, being present in all major organs [6], [8], [9], with the greatest concentrations expressed in the brain [9], [10]. High concentrations of PDE4 are also contained in the lungs, heart [4], [7], [8] and skeletal muscle [11]. The pancreas is reported to express PDE4 enzymes [6], [12], as do types of cells in the immune system [3]; however, platelets and red blood cells are devoid of PDE4 activity [13]. PDE4 has been shown to be expressed in white and brown adipose tissues (WAT and BAT), but to a much lesser extent than PDE3 [14], [15]. The four distinct PDE4 genes, PDE4A, B, C and D, are widely expressed in the human brain and skeletal muscle, whereas PDE4C is absent in the remainder of the above-mentioned tissues [6], [16]. PDE4 activity and density are regulated acutely by intracellular cAMP levels via phosphorylation by protein kinase A (PKA) and extracellular signal-related kinase (short-term) [17], as well as by de novo protein synthesis following prolonged elevation in cAMP levels (long-term) [3], [8], [16], [18].

The more active enantiomer of the widely used selective PDE4 inhibitor rolipram ((R)-4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidinone) binds reversibly and stereospecifically with high affinity (Kd=1–5 nM) to all subtypes of the Mg2+-induced PDE4 conformation, which is the active holoenzyme complex [16], [17], [19]. The less active enantiomer (S)-rolipram binds to PDE4 with 10- to 30-fold lower affinity [6], [10], [17], [19]. From in vitro studies, binding of rolipram to PDE4 has been reported to occur at two sites, called the high- and low-affinity rolipram binding site (HARBS and LARBS, respectively) [20], [21].

Both enantiomers of rolipram have been radiolabeled in our laboratory with C-11 and tested for potential use in positron emission tomography (PET) imaging of brain disorders [9], [22], [23], [24]. The most active stereoisomer (R)-[11C]rolipram has already found wide application in imaging brain cAMP signaling in rats [7], [25], monkeys [26], [27], [28], pigs [29] and humans [30]. Due to their function in the regulation of cardiac contraction, lipolysis and thermogenesis (conversion of food-derived energy into heat) [2], [14], [15], [31], the cAMP-specific PDE4 enzymes also present great interest for PET imaging in the periphery. (R)-[11C]Rolipram and PET imaging would allow serial assessment of PDE4 and yield insight into the mechanisms underlying disorders such as heart failure, obesity and diabetes. This paper presents studies exploring in vivo binding selectivity of (R)-[11C]rolipram in peripheral organs, with the goal of establishing its potential for use in evaluation of PDE4 regulation in myocardial and metabolic disorders.

Section snippets

Materials

(R)- and (S)-[11C]Rolipram were synthesized as previously described [22], [23] with high radiochemical purity (>95%) and specific activity (10.7–58.5 GBq/μmol; 290–1580 mCi/μmol) at the time of first injection. All drugs were purchased from Sigma-Aldrich (Canada), except for Bay 60-7550, which was purchased from Axxora (San Diego, CA), and (R)-rolipram from Tocris (Ellisville, MI). Unlabeled (R)- and (S)-rolipram, as well as Bay 60-7550, Ro 20-1724 and vinpocetine, were dissolved in

(R)-[11C]Rolipram in vivo binding selectivity studies

Co-injections of (R)-rolipram dose-dependently blocked (R)-[11C]rolipram retention in the brain, skeletal muscle, lungs (Fig. 1A) and all cardiac regions (Fig. 1B). Since cardiac uptake was uniform across all cardiac compartments (Fig. 1B), for clarity and simplicity of expressing these data, the left ventricle is presented in the following figures as the representative heart region. At a saturating dose of unlabeled (R)-rolipram (1 mg/kg iv), radiotracer retention was blocked by 81% in the

Discussion

Previous studies have shown the potential for using (R)-[11C]rolipram as a radiotracer in the evaluation of the cAMP-mediated signaling in the brain [7], [9], [24], [25], [26], [27], [28], [29]. Work performed to date has established binding selectivity of (R)-[11C]rolipram for PDE4 over PDE1 in the brain and established the presence of specific binding in rat brain, lungs and the whole heart [9], with retention levels responding to treatments altering cAMP signaling [7]. Imaging of

Acknowledgments

The authors acknowledge the contributions of the University of Ottawa Heart Institute Cardiac PET Centre radiochemistry staff (Samantha Mason, Paul Colletta, Anthony DiNardo and Jeff Collins) in the preparation of radiotracers and would like to thank Elissa Strome and Doris Doudet (University of British Columbia/TRIUMF PET Group) for help with the autoradiography methods, as well as the University of Ottawa Heart Institute Animal Care and Veterinary Staff (Rick Seymour and Dan de Vette) for

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