Takehiro Tsukada

Leiomodin-2 is an antagonist of tropomodulin-1 at the pointed end of the thin filaments in cardiac muscle

Regulation of actin filament assembly is essential for efficient contractile activity in striated muscle. Leiomodin is an actin-binding protein and homolog of the pointed-end capping protein, tropomodulin. These proteins are structurally similar, sharing a common domain organization that includes two actin-binding sites. Leiomodin also contains a unique C-terminal extension that has a third actin-binding WH2 domain. Recently, the striated-muscle-specific isoform of leiomodin (Lmod2) was reported to be an actin nucleator in cardiomyocytes. Here, we have identified a function of Lmod2 in the regulation of thin filament lengths. We show that Lmod2 localizes to the pointed ends of thin filaments, where it competes for binding with tropomodulin-1 (Tmod1). Overexpression of Lmod2 results in loss of Tmod1 assembly and elongation of the thin filaments from their pointed ends. The Lmod2 WH2 domain is required for lengthening because its removal results in a molecule that caps the pointed ends similarly to Tmod1. Furthermore, Lmod2 transcripts are first detected in the heart after it has begun to beat, suggesting that the primary function of Lmod2 is to maintain thin filament lengths in the mature heart. Thus, Lmod2 antagonizes the function of Tmod1, and together, these molecules might fine-tune thin filament lengths.

Involvement of Drinking and Intestinal Sodium Absorption in Hyponatremic Effect of Atrial Natriuretic Peptide in Seawater Eels

Abstract Atrial natriuretic peptide (ANP) decreases plasma Na+ concentration and promtes seawater (SW) adaptation in eels. The hyponatremia may most probably be caused by increased branchial extrusion of Na+, but the mechanism has not been determined yet. The present study examined initially the effects of ANP on branchial Na+ efflux in vivo using isotopic 22Na. However, the efflux rate was not altered by infusion of a hyponatremic dose of ANP (5 pmol·kg−1·min−1). Therefore, we sought to examine whether the ANP-mediated hyponatremia is caused by a decrease in the uptake of Na+ from the environment. Since a decrease in drinking was highly correlated with a degree of hyponatremia, conscious SW eels were infused with dilute SW into the stomach at a normal drinking rate to offset the antidipsogenic effect of ANP. Under this regimen, the hyponatremic effect of ANP was abolished. Then, we examined the site of Na+ absorption in the alimentary tract by measuring the changes in ion composition of intraluminal fluid along the tract. Since Na+ was absorbed at the esophagus and anterior/middle intestine, a sac was prepared at each site and the effects of ANP were examined in situ in conscious SW eels. ANP infusion did not alter Na+ absorption at the esophagus, but it profoundly reduced the absorption at the intestine. Together with our previous finding that ANP does not alter renal Na+ excretion, we propose that ANP reduces plasma Na+ concentration in SW eels by inhibiting drinking and subsequent absorption of Na+ by the intestine.

Identification of residues within tropomodulin-1 responsible for its localization at the pointed ends of the actin filaments in cardiac myocytes.

Tropomodulin is a tropomyosin-dependent actin filament capping protein involved in the structural formation of thin filaments and in the regulation of their lengths through its localization at the pointed ends of actin filaments. The disordered N-terminal domain of tropomodulin contains three functional sites: two tropomyosin-binding and one tropomyosin-dependent actin-capping sites. The C-terminal half of tropomodulin consists of one compact domain containing a tropomyosin-independent actin-capping site. Here we determined the structural properties of tropomodulin-1 that affect its roles in cardiomyocytes. To explore the significance of individual tropomyosin-binding sites, GFP-tropomodulin-1 with single mutations that destroy each tropomyosin-binding site was expressed in cardiomyocytes. We demonstrated that both sites are necessary for the optimal localization of tropomodulin-1 at thin filament pointed ends, with site 2 acting as the major determinant. To investigate the functional properties of the tropomodulin C-terminal domain, truncated versions of GFP-tropomodulin-1 were expressed in cardiomyocytes. We discovered that the leucine-rich repeat (LRR) fold and the C-terminal helix are required for its proper targeting to the pointed ends. To investigate the structural significance of the LRR fold, we generated three mutations within the C-terminal domain (V232D, F263D, and L313D). Our results show that these mutations affect both tropomyosin-independent actin-capping activity and pointed end localization, most likely by changing local conformations of either loops or side chains of the surfaces involved in the interactions of the LRR domain. Studying the influence of these mutations individually, we concluded that, in addition to the tropomyosin-independent actin-capping site, there appears to be another regulatory site within the tropomodulin C-terminal domain.

Expression of Chemokine CXCL12 and Its Receptor CXCR4 in Folliculostellate (FS) Cells of the Rat Anterior Pituitary Gland: The CXCL12/CXCR4 Axis Induces Interconnection of FS Cells

The anterior pituitary gland is composed of five types of hormone-producing cells plus folliculostellate (FS) cells, which do not produce classical anterior pituitary hormones. FS cells are interconnected by cytoplasmic processes and encircle hormone-producing cells or aggregate homophilically. Using living-cell imaging of primary culture, we recently reported that some FS cells precisely extend their cytoplasmic processes toward other FS cells and form interconnections with them. These phenomena suggest the presence of a chemoattractant factor that facilitates the interconnection. In this study, we attempted to discover the factor that induces interconnection of FS cells and succeeded in identifying chemokine (CXC)-L12 and its receptor CXCR4 as potential candidate molecules. CXCL12 is a chemokine of the CXC subfamily. It exerts its effects via CXCR4, a G protein-coupled receptor. The CXCL12/CXCR4 axis is a potent chemoattractant for many types of neural cells. First, we revealed that CXCL12 and CXCR4 are expressed by FS cells in rat anterior pituitary gland. Next, to clarify the function of the CXCL12/CXCR4 axis in FS cells, we observed living anterior pituitary cells in primary culture with specific CXCL12 inhibitor or CXCR4 antagonist and noted that extension of cytoplasmic processes and interconnection of FS cells were inhibited. Finally, we examined FS cell migration and invasion by using Matrigel matrix assays. CXCL12 treatment resulted in markedly increased FS cell migration and invasion. These data suggest that FS cells express chemokine CXCL12 and its receptor CXCR4 and that the CXCL12/CXCR4 axis evokes interconnection of FS cells.

Area postrema, a brain circumventricular organ, is the site of antidipsogenic action of circulating atrial natriuretic peptide in eels

SUMMARY Accumulating evidence indicates that circulating atrial natriuretic peptide (ANP) potently reduces excess drinking to ameliorate hypernatremia in seawater (SW) eels. However, the cerebral mechanism underlying the antidipsogenic effect is largely unknown. To localize the ANP target site in the brain, we examined the distribution of ANP receptors (NPR-A) in eel brain immunohistochemically using an antiserum specific for eel NPR-A. The immunoreactive NPR-A was localized in the capillaries of various brain regions. In addition, immunoreactive neurons were observed mostly in the medulla oblongata, including the reticular formation, glossopharyngeal-vagal motor complex, commissural nucleus of Cajal, and area postrema (AP). Trypan Blue, which binds serum albumin and does not cross the blood–brain barrier, was injected peripherally and stained the neurons in the AP but not other NPR-A immunopositive neurons. These histological data indicate that circulating ANP acts on the AP, which was further confirmed by physiological experiments. To this end, the AP in SW eels was topically destroyed by electric cauterization or were by chemical lesion of its neurons by kainic acid, and ANP (100 pmol kg–1) was then injected into the circulation. Both heat-coagulative and chemical lesions to the AP greatly reduced an antidipsogenic effect of ANP, but the ANP effect was retained in sham-operated eels and in those with lesions outside the AP. These results strongly suggest that the AP, a circumventricular organ without a blood–brain barrier, serves as a functional window of access for the circulating ANP to inhibit drinking in eels.

Caveolin 3-mediated integrin β1 signaling is required for the proliferation of folliculostellate cells in rat anterior pituitary gland under the influence of extracellular matrix

Folliculostellate (FS) cells in the anterior pituitary gland are believed to have multifunctional properties. Using transgenic rats that express green fluorescent protein (GFP) specifically in FS cells in the anterior pituitary gland (S100b-GFP rats), we recently revealed that FS cells in primary culture exhibited marked proliferation in the presence of laminin, an extracellular matrix (ECM) component of the basement membrane. In a process referred to as matricrine action, FS cells receive ECM as a signal through their receptors, which results in morphological and functional changes. In this study, we investigated matricrine signaling in FS cells and observed that the proliferation of FS cells is mediated by integrin β1, which is involved in various signaling pathways for cell migration and proliferation in response to ECM. Then, we analyzed downstream events of the integrin β1 signaling pathway in the proliferation of FS cells and identified caveolin 3 as a potential candidate molecule. Caveolin 3 is a membrane protein that binds cholesterol and a number of signaling molecules that interact with integrin β1. Using specific small interfering RNA of caveolin 3, the proliferation of FS cells was inhibited. Furthermore, caveolin 3 drove activation of the mitogen-activated protein kinase (MAPK) signaling cascades, which resulted in upregulation of cyclin D1 in FS cells. These findings suggest that matricrine signaling in the proliferation of FS cells was transduced by a caveolin 3-mediated integrin β1 signaling pathway and subsequent activation of the MAPK pathway.

Relative Potency of Three Homologous Natriuretic Peptides (ANP, CNP and VNP) in Eel Osmoregulation

Abstract Evidence has accumulated that atrial natriuretic peptide (ANP) plays important roles in sea-water adaptation in eels. However, the roles of the other two natriuretic peptides (CNP and VNP) in osmo-regulation have not been examined yet. In the present study, the effects of homologous ANP, CNP and VNP were compared on plasma Na+ concentration (an indicator of plasma osmolality), hematocrit (an approximate indicator of blood volume) and drinking rate in freshwater- and seawater-adapted eels. In seawater eels, ANP and VNP, but not CNP, infused at 5 pmol/kg/min decreased plasma Na+ concentration and drinking rate and increased hematocrit. In freshwater eels, ANP and VNP failed to decrease plasma Na+ concentration but increased hematocrit to the same extent as in seawater eels. Inhibition of drinking was not detectable in freshwater eels because of little drinking before NP infusions. These results show that the effects of NPs on plasma Na+ concentration, drinking rate and hematocrit are mediated by NPR-A, since only ANP and VNP that bind with higher affinity to NPR-A are effective in seawater eels. The mechanisms of regulation of plasma Na+ concentration and hematocrit are unknown, but NPR-A is present in the responsible tissues for regulation of hematocrit in both freshwater and seawater eels. However, NPR-A may be absent in the tissues of freshwater eels that are responsible for regulation of plasma Na+ concentration.

Ambient Temperature Regulates Drinking and Arterial Pressure in Eels

Abstract Ambient temperature exerts strong influences on physiological processes in ectothermic animals. Thus, we have examined the effects of changes in water temperature on drinking and arterial blood pressure in seawater-adapted eels, Anguilla japonica. Temperature dependence was also examined with respect to the effects of atrial natriuretic peptide (ANP) on drinking and arterial pressure. When water temperature was altered abruptly from 18°C to 11°C or to 25°C, drinking rate consistently decreased or increased, respectively, in all fish examined. The temperature-response relationship was highly linear (p<0.001) between 11°C and 25°C with a Q10 value of 2.45. Arterial pressure was significantly lower at 11°C compared with the level at 18°C, but no difference was observed between 18°C and 25°C. The change in hematocrit did not parallel that of temperature. Plasma Na+ concentration did not change at different temperatures. Bolus injections of eel ANP at 10, 100, and 1000 pmol/kg decreased drinking rate and arterial pressure dose-dependently. The effects of ANP did not differ in terms of potency and efficacy at different temperatures except that the effects continued longer at lower temperatures. These results show that the ambient temperature causes profound effects on drinking rate in seawater eels even though Na+ balance and blood volume were well maintained. The sensitivity to ANP, a physiological regulator of drinking in eels, was not altered by the temperature, suggesting an involvement of other factors in the thermogenic drinking that is demonstrated in this study.

Expression of small leucine-rich proteoglycans in rat anterior pituitary gland.

Proteoglycans are components of the extracellular matrix and comprise a specific core protein substituted with covalently linked glycosaminoglycan chains. Small leucine-rich proteoglycans (SLRPs) are a major family of proteoglycans and have key roles as potent effectors in cellular signaling pathways. Research during the last two decades has shown that SLRPs regulate biological functions in many tissues such as skin, tendon, kidney, liver, and heart. However, little is known of the expression of SLRPs, or the characteristics of the cells that produce them, in the anterior pituitary gland. Therefore, we have determined whether SLRPs are present in rat anterior pituitary gland. We have used real-time reverse transcription with the polymerase chain reaction to analyze the expression of SLRP genes and have identified the cells that produce SLRPs by using in situ hybridization with a digoxigenin-labeled cRNA probe. We have clearly detected the mRNA expression of SLRP genes, and cells expressing decorin, biglycan, fibromodulin, lumican, proline/arginine-rich end leucine-rich repeat protein (PRELP), and osteoglycin are located in the anterior pituitary gland. We have also investigated the possible double-staining of SLRP mRNA and pituitary hormones, S100 protein (a marker of folliculostellate cells), desmin (a marker of capillary pericytes), and isolectin B4 (a marker of endothelial cells). Decorin, biglycan, fibromodulin, lumican, PRELP, and osteoglycin mRNA have been identified in S100-protein-positive and desmin-positive cells. Thus, we conclude that folliculostellate cells and pericytes produce SLRPs in rat anterior pituitary gland.

Isolation of dendritic-cell-like S100β-positive cells in rat anterior pituitary gland

S100β-protein-positive cells in the anterior pituitary gland appear to possess multifunctional properties. Because of their pleiotropic features, S100β-positive cells are assumed to be of a heterogeneous or even a non-pituitary origin. The observation of various markers has allowed these cells to be classified into populations such as stem/progenitor cells, epithelial cells, astrocytes and dendritic cells. The isolation and characterization of each heterogeneous population is a prerequisite for clarifying the functional character and origin of the cells. We attempt to isolate two of the subpopulations of S100β-positive cells from the anterior lobe. First, from transgenic rats that express green fluorescent protein (GFP) driven by the S100β protein promoter, we fractionate GFP-positive cells with a cell sorter and culture them so that they can interact with laminin, a component of the extracellular matrix. We observe that one morphological type of GFP-positive cells possesses extended cytoplasmic processes and shows high adhesiveness to laminin (process type), whereas the other is round in shape and exhibits low adherence to laminin (round type). We successfully isolate cells of the round type from the cultured GFP-positive cells by taking advantage of their low affinity to laminin and then measure mRNA levels of the two cell types by real-time polymerase chain reaction. The resultant data show that the process type expresses vimentin (mesenchymal cell marker) and glial fibrillary acidic protein (astrocyte marker). The round type expresses dendritic cell markers, CD11b and interleukin-6. Thus, we found a method for isolating dendritic-cell-like S100β-positive cells by means of their property of adhering to laminin.