SNP genotyping in the beta(2)-adrenergic receptor by electronic microchip assay, DHPLC, and direct sequencing.

The beta(2)-adrenergic receptor (beta2AR) is the key target for the beta(2)-agonist drugs used for bronchodilation in asthma and chronic obstructive pulmonary disease. To detect four SNPs with amino acid variations at positions -47T/C (CysBUP19Arg), 46A/G (Gly16Arg), 79C/G (Gln27Glu), and 491C/T (Thr164Ile) in the beta 2AR gene, we used the electronic microchip assay, denaturing high-performance liquid chromatography (DHPLC), and direct sequencing. Genomic DNA samples were obtained from the blood of 84 Japanese healthy volunteers.

Global analysis of differentially expressed genes during progression of calcium oxalate nephrolithiasis.

The process of nephrolithiasis development is poorly understood at the molecular level. Here, we constructed a cDNA microarray from a rat kidney normalized cDNA library, and investigated the pattern of gene expression in rat kidneys from a calcium oxalate (CaOx) nephrolithiasis model. One hundred and seventy-three genes were found to be at least 2-fold regulated at one or more time points during progression of nephrolithiasis.

Role of Dbl's big sister in the anti-mitogenic pathway from alpha1B-adrenergic receptor to c-Jun N-terminal kinase.

We previously reported that the alpha1B-adrenergic receptor leads to activation of Rho family small GTPases, and in turn, c-Jun N-terminal kinase (JNK), which results in the inhibition of cell proliferation. Here, we show the involvement of the Rho family guanine nucleotide exchange factor (GEF) Dbl's Big Sister (Dbs) in the signaling pathway. Transfection of a Dbl-homology (DH) and pleckstrin-homology (PH) domain-deficient form of Dbs into cells blocked the alpha1B-adrenergic receptor-induced activation of JNK.

Role of the alpha1D-adrenergic receptor in the development of salt-induced hypertension.

In an attempt to elucidate whether there is a specific alpha1-adrenergic receptor (alpha1-AR) subtype involved in the genesis or maintenance of hypertension, the alpha1D-AR subtype was evaluated in a model of salt-induced hypertension. The alpha1D-AR-deficient (alpha1D-/-) and control (alpha1D+/+) mice (n=8 to 14 in each group) were submitted to subtotal nephrectomy and given 1% saline as drinking water for 35 days. Blood pressure (BP) was monitored by tail-cuff readings and confirmed at the end point by direct intraarterial BP recording.

Two novel transcripts for human endothelin B receptor produced by RNA editing/alternative splicing from a single gene.

Hirschsprung disease is a heterogeneous genetic disorder, causative genes of which include the endothelin B receptor (ETB). To investigate the mutations of ETB in Hirschsprung disease, expression of the ETB gene in lymphoblastoid cells from patients and normal healthy adults was examined, and novel mutant transcripts were found. The mutant ETB gene transcripts lacked a 134-bp nucleotide sequence corresponding to exon 5, and some also contained a substitution from A to G at position 950 in exon 4, resulting in an amino acid substitution from glutamine (Q) to arginine (R).

Differences in the subcellular localization of alpha1-adrenoceptor subtypes can affect the subtype selectivity of drugs in a study with the fluorescent ligand BODIPY FL-prazosin.

G protein-coupled receptor (GPCR) subtypes are differentially distributed in the cell; however, it remains unclear how this affects the subtype selectivity of particular drugs. In the present study, we used flow cytometry analysis with the fluorescent ligand, BODIPY FL-prazosin, to study the relationship between the subcellular distribution of subtype receptors and the subtype-selective character of ligands using alpha1a and alpha1b-adrenoceptors (ARs). Alpha1a-ARs predominantly localize inside the cell, while alpha1b-ARs on the cell surface.

Recent progress in alpha 1-adrenoceptor pharmacology.

The adrenoceptors (ARs) play a key role in the modulation of sympathetic nervous system activity and are a site of action for many clinically important therapeutic agents. The alpha1-adrenoceptor subtypes (alpha1A-, alpha1B-, and alpha1D-AR) play a prominent role in regulating vascular tone and hypertrophic growth of smooth muscle and cardiac cells. Their functional characteristics with respect to ligand binding and second messenger utilization have been well described.

Genomic analysis of a mouse model of immunoglobulin A nephropathy reveals an enhanced PDGF-EDG5 cascade.

The molecular mechanism of immunoglobulin A nephropathy (IgAN), the most common primary renal glomerular disease worldwide, is unknown. HIGA (high serum IgA) mouse is a valid model of IgAN showing almost all of the pathological features, including mesangial cell proliferation. Here we elucidate a pattern of gene expression associated with IgAN by analyzing the diseased kidneys on cDNA microarrays.

Beta(1)-selective agonist (-)-1-(3,4-dimethoxyphenetylamino)-3-(3,4-dihydroxy)-2-propanol [(-)-RO363] differentially interacts with key amino acids responsible for beta(1)-selective binding in resting and active states.

(-)-1-(3,4-Dimethoxyphenetylamino)-3-(3,4-dihydroxy)-2-propanol [(-)-RO363] is a highly selective beta(1)-adrenergic receptor (beta(1)AR) agonist. To study the binding site of beta(1)-selective agonist, chimeric beta(1)/beta(2)ARs and Ala-substituted beta(1)ARs were constructed. Several key residues of beta(1)AR [Leu(110) and Thr(117) in transmembrane domain (TMD) 2], and Phe(359) in TMD 7] were found to be responsible for beta(1)-selective binding of (-)-RO363, as determined by competitive binding.

Genome medicine promised by microarray technology.

The human genome project has now been completed, which markedly changes the way to analyze gene functions. Recently developed DNA microarray technologies enable us to explore genome-wide gene expression in the diseased tissues. In this review, we introduce the principles and applications of microarray technologies (such as DNA, tissue and cell microarrays) to molecular diagnostics, drug target discovery and validation of drug effects.

The alpha(1D)-adrenergic receptor directly regulates arterial blood pressure via vasoconstriction.

To investigate the physiological role of the alpha(1D)-adrenergic receptor (alpha(1D)-AR) subtype, we created mice lacking the alpha(1D)-AR (alpha(1D)(-/-)) by gene targeting and characterized their cardiovascular function. In alpha(1D)-/- mice, the RT-PCR did not detect any transcript of the alpha(1D)-AR in any tissue examined, and there was no apparent upregulation of other alpha(1)-AR subtypes. Radioligand binding studies showed that alpha(1)-AR binding capacity in the aorta was lost, while that in the heart was unaltered in alpha(1D)-/- mice.

Functional genomic research of alpha 1-adrenoceptors.

The Human Genome Project is now almost completed, and we are about to move into the post-genome sequence era of functional genomics. The advent of genome science has markedly changed the way life science research including pharmacological study is conducted; thus, systematic and integrated 'genome-wide' survey is feasible. The stream of 'Genome-->Transcriptome--> Proteomics' is logical and, in each aspect, approaches for functional genomics are now pursued at a high pace.

Novel green fluorescent protein-based ratiometric indicators for monitoring pH in defined intracellular microdomains.

To measure pH in defined intracellular microdomains of living cells, we developed ratiometric indicators based on fusing in tandem two green fluorescent protein (GFP) variants having different pH sensitivities. The indicators function in a single-excitation/dual-emission mode involving fluorescence resonance energy transfer, as well as in a dual-excitation/single-emission mode. The fluorescence ratio from GFpH and YFpH showed pH dependency and pK(a) values were 6.

Galphaq-dependent activation of mitogen-activated protein kinase kinase 4/c-Jun N-terminal kinase cascade.

G-protein-coupled receptors (GPCRs) typically activate c-Jun N-terminal kinase (JNK) through the G protein betagamma subunit (Gbetagamma), in a manner dependent on Rho family small GTPases, in mammalian cells. Here we show that JNK activation by the prototypic Gq-coupled alpha1B-adrenergic receptor is mediated by the alpha subunit of Gq (Galphaq), not by Gbetagamma, using a transient transfection system in human embryonic kidney cells. JNK activation by the alpha1B-adrenergic receptor/Galphaq was selectively mediated by mitogen-activated protein kinase kinase 4 (MKK4), but not MKK7.

Molecular monitoring of bleomycin-induced pulmonary fibrosis by cDNA microarray-based gene expression profiling.

Pulmonary fibrosis is a progressive disorder whose molecular pathology is poorly understood. Here we developed an in-house cDNA microarray ("lung chip") originating from a lung-normalized cDNA library. By using this lung chip, we analyzed global gene expression in a murine model of bleomycin-induced fibrosis and selected 82 genes that differed by more than twofold intensity in at least one pairwise comparison with controls.

['Millennium Project' of MHLW].

Human Genome Project (information and technology) provides insights so profound that it has the ability to change the way we understand, predict, prevent, diagnose, and treat disease. Because SNPs, single nucleotide polymorphisms, are the most common type of polymorphism, they can have significant effects on both susceptibility to disease as well as drug response. In Japan, 'Millennium Project' based on SNPs has started in 2000, and National Research Centers of MHLW are performing genome-wide association study on five common diseases (dementia, cancer, diabetes mellitus, hypertension, and asthma/allergy).

[New strategy on medical research after completion of genome sequencing].

Real advances in biotechnology made it possible to complete human whole genome sequencing within a short duration. Although the genome includes a huge amount of information about biological functions and the interest is now directed to the study using genomic information, the genomic strategy is not clearly understood. The following 4 studies were therefore presented and discussed about the strategy after the completion of the genomic sequence in the 74th Annual Meeting of Japanese Pharmacological Society: 1) Asthma and atopic dermatitis: models for genetic and genomic investigations of complex genetic diseases, by W.

Electrophysiologic effects of an antiarrhythmic agent, bidisomide, on sodium current in isolated rat ventricular myocytes: comparison with mexiletine and disopyramide.

The effects of bidisomide, an antiarrhythmic agent, on sodium current (I(Na)) in isolated rat ventricular myocytes were investigated using a whole cell voltage clamp method. Bidisomide blocked I(Na) with a Ki of 214 microM at a holding potential of -140 mV. The blockade of I(Na) was enhanced at a less negative holding potential of -100 mV with a Ki of 21 microM.

Beta2-adrenergic receptor/cyclic adenosine monophosphate (cAMP) leads to JNK activation through Rho family small GTPases.

Gi- and Gq-coupled G protein-coupled receptors (GPCRs) have been shown to activate c-Jun N-terminal kinase (JNK), a subfamily of mitogen-activated protein kinases (MAPKs), through Rho family small GTPases in mammalian cells. We investigated the signaling pathway linking the Gs-coupled beta2-adrenergic receptor with JNK, using smooth muscle DDT1 MF-2 cells, which natively express the beta2-adrenergic receptor. Stimulation of the beta2-adrenergic receptor activated JNK in a time-dependent manner, and a cell-permeable cyclic adenosine monophosphate analogue (8-Br-cAMP) activated JNK.

Regulation of subcellular localization of alpha1-adrenoceptor subtypes.

Alpha1-adrenergic receptors (AR) are members of the superfamily of G protein-coupled receptors (GPCRs) which mediate the effects of the sympathetic nervous system. Alpha1-AR comprise a heterogeneous family of three distinct isoforms of alpha1A, alpha1B and alpha1D; however, very little is known about their difference in physiological role or regulation. We have recently observed a subtype-specific differences in subcellular localization of alpha1-ARs; thus, alpha1A-AR predominantly localize intracellularly, while alpha1B-AR on the cell surface.

Parallel regulation of mitogen-activated protein kinase kinase 3 (MKK3) and MKK6 in Gq-signaling cascade.

Heterotrimeric G protein G(q) stimulates the activity of p38 mitogen-activated protein kinase (MAPK) in mammalian cells. To investigate the signaling mechanism whereby alpha and betagamma subunits of G(q) activate p38 MAPK, we introduced kinase-deficient mutants of mitogen-activated protein kinase kinase 3 (MKK3), MKK4, and MKK6 into human embryonal kidney 293 cells. The activation of p38 MAPK by Galpha(q) and Gbetagamma was blocked by kinase-deficient MKK3 and MKK6 but not by kinase-deficient MKK4.

MCM3AP, a novel acetyltransferase that acetylates replication protein MCM3.

The MCM proteins are essential for the initiation of DNA replication. We have isolated an MCM3-associated protein (MCM3AP) in a two-hybrid screen using MCM3. Here we demonstrate that MCM3AP is an acetyltransferase which acetylates MCM3 and that chromatin-bound MCM3 is acetylated in vivo.

Involvement of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase in alpha1B-adrenergic receptor/Galphaq-induced inhibition of cell proliferation.

Certain G protein-coupled receptors (GPCRs) stimulate the activities of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), members of the MAPK family. We investigated the role of JNK and p38 MAPK activation induced by the alpha1B-adrenergic receptor in the proliferation of human embryonic kidney 293T cells. Activation of the alpha1B-adrenergic receptor resulted in inhibition of cell proliferation.