Masaaki Tamura

Brain Actin-associated Protein Phosphatase 1 Holoenzymes Containing Spinophilin, Neurabin, and Selected Catalytic Subunit Isoforms

We previously characterized PP1bp134 and PP1bp175, two neuronal proteins that bind the protein phosphatase 1 catalytic subunit (PP1). Here we purify from rat brain actin-cytoskeletal extracts PP1A holoenzymes selectively enriched in PP1γ1 over PP1β isoforms and also containing PP1bp134 and PP1bp175. PP1bp134 and PP1bp175 were identified as the synapse-localized F-actin-binding proteins spinophilin (Allen, P. B., Ouimet, C. C., and Greengard, P. (1997) Proc. Natl. Acad. Sci. U.u2009S.u2009A. 94, 9956–9561; Satoh, A., Nakanishi, H., Obaishi, H., Wada, M., Takahashi, K., Satoh, K., Hirao, K., Nishioka, H., Hata, Y., Mizoguchi, A., and Takai, Y. (1998) J. Biol. Chem. 273, 3470–3475) and neurabin (Nakanishi, H., Obaishi, H., Satoh, A., Wada, M., Mandai, K., Satoh, K., Nishioka, H., Matsuura, Y., Mizoguchi, A., and Takai, Y. (1997)J. Cell Biol. 139, 951–961), respectively. Recombinant spinophilin and neurabin interacted with endogenous PP1 and also with each other when co-expressed in HEK293 cells. Spinophilin residues 427–470, or homologous neurabin residues 436–479, were sufficient to bind PP1 in gel overlay assays, and selectively bound PP1γ1 from a mixture of brain protein phosphatase catalytic subunits; additional N- and C-terminal sequences were required for potent inhibition of PP1. Immunoprecipitation of spinophilin or neurabin from crude brain extracts selectively coprecipitated PP1γ1 over PP1β. Moreover, immunoprecipitation of PP1γ1 from brain extracts efficiently coprecipitated spinophilin and neurabin, whereas PP1β immunoprecipitation did not. Thus, PP1A holoenzymes containing spinophilin and/or neurabin target specific neuronal PP1 isoforms, facilitating efficient regulation of synaptic phosphoproteins.

Purification and characterization of two types of atrial natriuretic factor receptors from bovine adrenal cortex: Guanylate cyclase-linked and cyclase-free receptors

Atrial natriuretic factor (ANF) receptors with and without guanylate cyclase activity were simultaneously purified to apparent homogeneity from bovine adrenal zona glomerulosa cell membrane fractions. The particulate guanylate cyclase which co-purified with the ANF receptor showed one of the highest specific activity reported. The receptors with or without the guanylate cyclase activity showed high affinities to ANF (99-126). The receptor without the cyclase showed a high affinity to truncated ANF analogs, ANF (103-123) and ANF (105-121), whereas the cyclase-linked receptor had a much lower affinity to these analogs. Both of the receptors migrated as a single band with a molecular weight of 135,000 daltons on SDS-gel electrophoresis under non-reducing conditions. The 135,000 daltons band of the receptor without the cyclase was shifted to a 62,000 daltons band under reducing conditions, but the band for the cyclase-linked receptor was not shifted. These results demonstrated the presence of two subtypes of ANF receptor in bovine adrenal cortex and indicate two different modes of intracellular action of ANF.

Specific endogenous Na, K-ATPase inhibitor purified from bovine adrenal.

The endogenous inhibitor of Na, K-ATPase has been implicated in the pathogenesis of salt-induced hypertension. In spite of an intensive search the inhibitor has long remained elusive. We have been able to purify such an inhibitor from methanol extract of bovine adrenal glands by multiple steps of high-performance liquid chromatography (HPLC). This compound with a molecular mass of 336 showed striking similarity to the cardiac glycoside ouabain in its dose dependency in the inhibition of Na, K-ATPase and Na-pump activity, competitive binding to the ouabain-binding site, and dependence of these effects on K+ concentration. These inhibitory activities were potentiated with reduced K+ concentration in parallel with ouabain. These results indicates that vertebrate animals possess a regulator of Na, K-ATPase and suggests the possibility that it may be the mediator of salt-induced high blood pressure.

Angiotensin II AT2 receptor localization in cardiovascular tissues by its antibody developed in AT2 gene-deleted mice

In contrast to well-established physiological roles of the angiotensin II type 1 receptor (AT1), the significance of the type 2 receptor (AT2) remains largely unclear. AT2-knockout (AT2KO) mice have a phenotype associated with mild hypertension. This implies that AT2 has a role for the regulation of blood pressure. To gain insight into the mechanism by which AT2 regulates systemic blood pressure, we have investigated the expression of the AT2 receptor protein in adult rat cardiovascular tissues, using a newly developed polyclonal anti-AT2 antiserum that was successfully obtained in the AT2KO mice by immunizing with a peptide fragment of the receptor protein. In blood vessels, a stronger immunoreactivity was observed in endothelial cells than in the muscular media of resistant arteries. In the thoracic aorta, AT2 was observed only in muscular media. Abundant AT2 immunoreactivity was detected in perivascular nerve fibers. In the heart, positive immunostaining for AT2 was restricted to the coronary blood vessels. These data suggest that AT2 expressed in the vascular endothelial cells and muscular media in resistant arteries may play a pivotal role in systemic blood pressure regulation. AT2 was observed for the first time in the perivascular nerve fibers and may also play a role in neuronal blood pressure regulation.

Angiotensin II bi-directionally regulates cyclooxygenase-2 expression in intestinal epithelial cells

We previously demonstrated that angiotensin II (Ang II) receptor signaling is involved in azoxymethane-induced mouse colon tumorigenesis. In order to clarify the role of Ang II in COX-2 expression in the intestinal epithelium, the receptor subtype-specific effect on COX-2 expression in a rat intestinal epithelial cell line (RIE-1) has been investigated. Ang II dose- and time-dependently increased the expression of COX-2, but not COX-1 mRNA and protein. This stimulation was completely blocked by the AT1 receptor antagonist but not the AT2 receptor antagonist. Ang II and lipopolysaccharide (LPS) additively induced COX-2 protein in RIE-1 cells, whereas the LPS-induced COX-2 expression was significantly attenuated by low concentrations of Ang II or the AT2 agonistic peptide CGP-42112A only in AT2 over-expressed cells. These data indicate that Ang II bi-directionally regulates COX-2 expression via both AT1 and AT2 receptors. Control of COX-2 expression through Ang II signaling may have significance in cytokine-induced COX-2 induction and colon tumorigenesis.

Intracellular Sodium Modulates the Expression of Angiotensin II Subtype 2 Receptor in PC12W Cells

Although the angiotensin II subtype 2 receptor (AT2-R) is expressed abundantly in the adrenal medulla, its physiological significance has not yet been determined. To obtain fundamental knowledge of the regulation of AT2-R expression in the adrenal medulla, we investigated the effects of modulating several ion channels on AT2-R expression in PC12W cells. Experiments were performed after 24 hours of serum depletion under subconfluent conditions. After 48 hours of treatment with various agonists or antagonists, the receptor density and mRNA level of AT2-Rs were quantified by 125I-[Sar1, Ile8]angiotensin II binding and Northern blot analysis. Ouabain (10 to 100 nmol/L) and insulin (10 to 100 nmol/L) dose-dependently increased receptor density and mRNA level. Analysis of the binding characteristics revealed that the ouabain-dependent increase in AT2-R levels was due to an increase in binding capacity without a change in the Kd value. These increases were blocked by lowering the Na+ concentration in the medium. A low concentration of the sodium ionophore monensin (10 nmol/L), the K+-channel blocker quinidine (10 micromol/L), and the ATP-sensitive K+-channel blockers tolbutamide (100 micromol/L) and glybenclamide (10 micromol/L) also significantly increased receptor density, but the ATP-sensitive K+-channel agonist cromakalim (100 micromol/L) decreased receptor density significantly (P<0.01). Nifedipine (10 micromol/L) decreased basal receptor density and completely blocked the increase in receptor density caused by these agents. The increase in receptor density caused by an increase in intracellular Na+ was accompanied by an increase in mRNA level, whereas the ATP-sensitive K+-channel blockers did not change mRNA level. Nifedipine slightly decreased mRNA level. These results suggest that AT2-R expression is sensitively regulated by intracellular cation levels. The change in intracellular Na+ level transcriptionally regulates AT2-R expression, whereas the K+-channel blocker-dependent upregulation appears to be at least in part posttranslational.

Lipopolysaccharides and cytokines downregulate the angiotensin II type 2 receptor in rat cardiac fibroblasts.

The present study examines the effect of lipopolysaccharides and proinflammatory cytokines on the expression of the second isoform of the angiotensin II receptor (AT(2)), which may have a role in lowering collagen deposition in cardiac tissue. Cardiac fibroblasts express high levels of both angiotensin II type 1 (AT(1)) and type 2 receptors. Incubation with lipopolysaccharides for 24 h dose- and time-dependently decreased angiotensin II AT(2) receptor expression with no apparent difference in the affinity. Actinomycin D, cycloheximide, N(omega)-nitro-L-arginine methyl ester and the protein tyrosine kinase inhibitor herbimycin A, but not the protein kinase C inhibitors bisindolylmaleimide and calphostin C, abolished the inhibitory action of lipopolysaccharides. The cytokines interleukin-1beta and tissue necrosis factor-alpha mimicked the effect of lipopolysaccharides. All three compounds induced inducible nitric oxide synthase (iNOS). The nitric oxide donor sodium nitroprusside and the cGMP analog 8-bromoguanosine cyclic monophosphate downregulated angiotensin II AT (2) receptor expression. The findings are consistent with the pathway in which lipopolysaccharides or cytokines induce iNOS. The data suggest that lipopolysaccharide- or cytokine-dependent induction of iNOS and resultant production of nitric oxide leads to the production of cGMP, which in turn downregulates expression of the angiotensin II AT (2) receptor in cardiac fibroblasts.

Purification and Characterization of Specific Endogenous Ouabainlike Substance from Bovine Adrenal

Endogenous inhibitors of Na, K-ATPase have been implicated in the pathogenesis of salt-induced hypertension. Despite an intensive search, the inhibitor(s) have long remained elusive. We have been able to purify such an inhibitor from methanol extracts of bovine adrenal glands by multiple steps of high-performance liquid chromatography (HPLC). This compound showed striking similarity to the cardiac glycoside ouabain in its dose dependency in the inhibition of Na, K-ATPase and Na-pump activity, competitive binding to the ouabain-binding site, and dependence of these effects on K+ concentration. These results indicate that vertebrate animals contain a regulator of Na, K-ATPase.

Specific single chain variable fragment (ScFv) antibodies to angiotensin II AT(2) receptor: evaluation of the angiotensin II receptor expression in normal and tumor-bearing mouse lung.

To gain insight into the mechanism by which angiotensin II type 2 receptor (AT2) regulates carcinogen-induced lung tumorigenesis, we have newly developed anti-AT2 single chain variable fragment (ScFv) antibodies using a rodent phage-displayed recombinant antibody library with various peptide fragments of the receptor protein, and investigated the expression of the AT2 receptor protein. The specificity of the antibodies was verified using AT2 over-expressing COS-7 cells and AT2 naturally expressing PC12W cells. In control wild type mouse lung, a stronger immunoreactivity was observed in bronchial epithelial cells. A moderate immunoreactivity was detected in pulmonary vascular walls and vascular endothelial cells. In the lungs possessing tobacco-specific nitrosamine (NNK)-induced tumors, significantly increased AT2 and AT1 immunostaining was observed in adenomatous lesions. These data suggest that the increase in both receptors’ expression in the alveolar epithelial cells may be accompanied with the onset of NNK-induced tumorigenesis and hence play important roles in lung tumorigenesis.

Isolation of an endogenous Na-pump specific inhibitor from normal pig urine: characterization and comparison with the inhibitor purified from bovine adrenal glands

An endogenous Na-pump specific inhibitor has been purified to homogeneity from normal pig urine using Amberlite XAD-2 adsorption chromatography followed by five steps of reverse phase HPLC. Although most of the dose response curves for this purified Na-pump inhibitor, designated uroxin, in the various assay systems paralleled those of authentic ouabain and the specific Na-pump inhibitor previously purified from bovine adrenal glands (designated adrexin C), the cross-reactivity curve with anti-ouabain antibodies did not. The retention times of uroxin on various types of reverse phase HPLC columns were also different from those of plant-derived cardiotonic steroids and adrexin C. The cross-reaction curve of adrexin C was superimposable with that of ouabain, and adrexin C coeluted with ouabain from all of the HPLC columns tested. The results from physical and chemical characterization of both purified inhibitors suggest that uroxin is a novel Na-pump inhibitor which is structurally different from any of the known cardiotonic steroids or other substances previously reported to exhibit Na-pump inhibitory activity. The results also indicate that adrexin C is indistinguishable from ouabain. These results suggest that there are at least two different types of endogenous Na+,K(+)-ATPase inhibitors in the mammalian body.