Quantitative comparison of phosphodiesterase mRNA distribution in human brain and peripheral tissues

doi:10.1016/j.neuropharm.2010.05.004

Neuropharmacology

Volume 59, Issue 6, November 2010, Pages 367-374

https://doi.org/10.1016/j.neuropharm.2010.05.004 Get rights and content

Abstract

Cyclic nucleotide-specific phosphodiesterases (PDEs) play a critical role in signal transduction by regulating the level of adenosine 3′,5′-cyclic monophosphate (cAMP) and guanosine 3′,5′-cyclic monophosphate (cGMP) in cells. The gene expression pattern of a PDE provides important information regarding its role in physiological and pathological processes. In this study, we have established the mRNA expression profile all PDE isoenzymes (PDE1A/B/C, 2A, 3A/B, 4A/B/C/D, 5A, 6A/B/C, 7A/B, 8A/B, 9A, 10A, 11A) in a human cDNA collection consisting of 10 brain regions (parietal, frontal, temporal cortex, hippocampus, striatum, thalamus, hypothalamus, substantia nigra, nucleus accumbens, cerebellum), spinal cord, dorsal root ganglia and 12 peripheral tissues (skeletal muscle, heart, thyroid, adrenal gland, pancreas, bladder, kidney, liver, lung, small intestine, spleen, and stomach). Using quantitative real-time polymerase chain reaction and parallel analysis of a carefully selected group of reference genes, we have determined the relative expression of each PDE isoenzyme across the 24 selected tissues, and also compared the expression of selected PDEs to each other within a given tissue type. Several PDEs show strikingly selective expression (e.g. PDE10A and PDE1B mRNA levels in the caudate nucleus are 20-fold higher than in most other tissues; PDE1C and PDE3A are highly expressed in the heart and PDE8B is expressed very strongly in the thyroid gland). This comprehensive approach provides a coherent and quantitative view of the mRNA expression of the PDE gene family and enables an integration of data obtained with other non-quantitative methods.

Introduction

The cyclic nucleotides cAMP and cGMP act as intracellular second messengers for many signal transduction pathways. Synthesis of cAMP by membrane bound adenylyl cyclases (AC) is stimulated or inhibited by various G-protein coupled receptors (Hanoune and Defer, 2001). In addition, some AC isoforms are sensitive to the intracellular Ca²⁺ concentration. The generation of cGMP by soluble guanylyl cyclase is stimulated by nitric oxide (NO), which in turn is synthesized by the Ca²⁺/calmodulin sensitive enzyme nitric oxide synthase (Mullershausen et al., 2005). Several particulate guanylyl cyclase isoforms with an extracellular ligand binding domain and an intracellular catalytic domain are activated by atrionatriuretic peptide (ANP) and related hormones (Garbers et al., 2006).

Both cAMP and cGMP can open cyclic nucleotide gated ion channels and stimulate cAMP and cGMP-activated protein kinases (PKA and PKG) (Francis and Corbin, 1999, Kaupp and Seifert, 2002). In addition, cAMP binds to EPAC (exchange protein activated by cAMP) (Bos, 2006). The downstream targets of PKA and PKG include receptors, ion channels, cytoskeletal proteins and transcription factors resulting in the modulation of neuronal excitability, metabolism, cytoskeleton and gene expression.

The intracellular concentration of cyclic nucleotides is determined by the activity of phosphodiesterases (PDEs), a class of enzymes encoded by 21 genes that are grouped in 11 families according to their structural similarity (Bender and Beavo, 2006). Phosphodiesterases can either hydrolyse both cyclic nucleotides (PDE1A,B,C, PDE2A, PDE3A,B, PDE10A, PDE11A) or are specific for cAMP (PDE4A,B,C,D, PDE7A,B, PDE8A,B) or cGMP (PDE5A, PDE6A,B,C, PDE9). Their activity is regulated by phosphorylation, changes in gene expression, Ca²⁺/calmodulin (PDE1A-C) and by the levels of cGMP that stimulate PDE2 and inhibit PDE3. PDE expression is tissue- and cell-specific. Different isoenzymes and splicing isoforms are targeted to specific cell compartments and protein complexes allowing spatiotemporal integration of multiple hormone and neurotransmitter signals.

Absolute quantification of individual PDE activities in different tissues and cell types is hampered by the lack of selective inhibitors for many isoenzymes and the challenge of determining enzyme activity at a cellular level. The protein expression levels of a number of PDEs have been studied using immunohistochemistry but comparisons between different isoenzymes are difficult. Several studies reported conflicting results depending on the quality of the available antibodies (see Bender and Beavo, 2006). The mRNA distribution of some PDE isoenzymes has been examined using in situ hybridization. This technique provides valuable information on the relative expression of one mRNA type in different regions with excellent spatial resolution, but is not suitable for a direct quantitative comparison between different PDE isoenzymes. To allow quantitative comparison of the expression levels of all PDEs in different human tissues, we examined mRNA levels by quantitative real-time PCR. We used a set of three reference genes to minimize the variation across tissues and to allow direct comparison between different tissues and different genes. This study provides a quantitative and coherent view of the mRNA distribution of the PDE gene family, in different brain regions and other human tissues.

Section snippets

RNA from CNS and other human tissues

Total RNA samples for 24 human tissues (pooled from 4 to 64 individuals) were obtained from Clontech Europe (Saint-Germain-en-Laye, France, Premium Total RNA) and Ambion Europe (Huntingdon, UK, FirstChoice Human Total RNA). To ensure that the samples represent normal human tissues, they were carefully selected to avoid donors with a cause of death that could affect the quality of the given tissue (i.e. avoiding Alzheimer’s disease for brain samples) (Table 1.) The RNA integrity of the samples

Results

PDE1A mRNA is present in all brain regions examined, but at lower levels than PDE1B and PDE1C with the exception of cerebellum, where the expression of all three PDE1 isoenzymes is equally low (Fig. 2A). Particularly striking is the very strong expression of PDE1B in the caudate nucleus (with expression levels 10 or 100 fold higher than PDE1C or PDE1A, respectively) and nucleus accumbens (Fig. 2A). In the nucleus accumbens, PDE1B is the most prevalent of all PDE mRNA species, while PDE1B and

Discussion

Our results quantify and extend Northern blot studies of PDE1A-C mRNA distribution (Loughney et al., 1996, Fidock et al., 2002). The high levels of PDE1B mRNA in caudate and nucleus accumbens and of PDE1C in the substantia nigra point to an important role in signal transduction in these brain regions. PDE1B knockout mice show changes in locomotor function, confirming a functional role of the high PDE1B levels in the caudate (Reed et al., 2002). PDE1A was the most prevalent PDE isoenzyme in

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Quantitative comparison of phosphodiesterase mRNA distribution in human brain and peripheral tissues

Abstract

Introduction

Section snippets

RNA from CNS and other human tissues

Results

Discussion

Phosphodiesterase 8B gene variants are associated with serum TSH levels and thyroid function

Am. J. Hum. Genet.

Cyclic nucleotide phosphodiesterases: molecular regulation to clinical use

Pharmacol. Rev.

Differentiation of human monocytes in vitro with granulocyte-macrophage colony-stimulating factor and macrophage colony-stimulating factor produces distinct changes in cGMP phosphodiesterase expression

Cell Signal

Inhibition of phosphodiesterase 2 increases neuronal cGMP, synaptic plasticity and memory performance

Neuropharmacology

Epac proteins: multi-purpose cAMP targets

Trends Biochem. Sci.

Milrinone, a selective phosphodiesterase 3 inhibitor, stimulates lipolysis, endogenous glucose production, and insulin secretion

Metabolism

Alterations in regulation of energy homeostasis in cyclic nucleotide phosphodiesterase 3B-null mice

J. Clin. Invest.

Isolation and differential tissue distribution of two human cDNAs encoding PDE1 splice variants

Cell Signal

Isolation and characterization of PDE9A, a novel human cGMP-specific phosphodiesterase

J. Biol. Chem.

Cyclic nucleotide-dependent protein kinases: intracellular receptors for cAMP and cGMP action

Crit. Rev. Clin. Lab. Sci.

Cloning and characterization of a novel human phosphodiesterase that hydrolyzes both cAMP and cGMP (PDE10A)

J. Biol. Chem.

Membrane guanylyl cyclase receptors: an update

Trends Endocrinol. Metab.

Identification and characterization of a novel cyclic nucleotide phosphodiesterase gene (PDE9A) that maps to 21q22.3: alternative splicing of mRNA transcripts, genomic structure and sequence

Hum. Genet.

Regulation and role of adenylyl cyclase isoforms

Annu. Rev. Pharmacol. Toxicol.

HugeIndex: a database with visualization tools for high-density oligonucleotide array data from normal human tissues

Nucleic Acids Res.

Molecular cloning and characterization of human PDE8B, a novel thyroid-specific isozyme of 3′,5′-cyclic nucleotide phosphodiesterase

Biochem. Biophys. Res. Commun.

Cyclic nucleotide-gated ion channels

Physiol. Rev.

Characterization and phosphorylation of PDE10A2, a novel alternative splice variant of human phosphodiesterase that hydrolyzes cAMP and cGMP

Biochem. Biophys. Res. Commun.

CD3- and CD28-dependent induction of PDE7 required for T cell activation

Science

Isolation and characterization of cDNAs corresponding to two human calcium, calmodulin-regulated, 3′,5′-cyclic nucleotide phosphodiesterases

J. Biol. Chem.

Isolation and characterization of PDE10A, a novel human 3′, 5′-cyclic nucleotide phosphodiesterase

Gene

High concentrations of a cGMP-stimulated phosphodiesterase mediate ANP-induced decreases in cAMP and steroidogenesis in adrenal glomerulosa cells

J. Biol. Chem.