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Biomedical subjects

A Ichikawa

Publications and source records attributed to A Ichikawa.

At least 127 records · Page 7Linked to original sources
PubMed1996

Prostaglandin E receptor EP3gamma isoform, with mostly full constitutive Gi activity and agonist-dependent Gs activity.

We recently demonstrated that two exclusively Gi-coupled isoforms of the mouse EP3 receptor, EP3alpha and beta, with different carboxyl-terminal tails, differed in agonist-independent constitutive Gi activity, and the carboxyl-terminal tail-truncated receptor showed full constitutive activity (Hasegawa, H., Negishi, M., and Ichikawa, A. (1996) J. Biol. Chem. 271, 1857-1860). Here we further examined Gi and Gs activities of the third isoform, EP3gamma, coupled to both Gi and Gs. The My receptor showed mostly full constitutive Gi activity and agonist-dependent Gs activity. The truncated receptor also showed agonist-dependent Gs activity, but the level was lower than that of the EP3gamma receptor. Thus, the carboxyl-terminal tail would differentially regulate Gi and Gs activities of the EP3 receptor.

Adenylyl Cyclases↗
PubMed1996

Induction of protein disulfide isomerase mRNA by delta 12-prostaglandin J2.

Protein disulfide isomerase (PDI) catalyzes the correct protein disulfide formation and is a key regulator for protein folding. We examined the effect of delta 12-prostaglandin (PG) J2, a thiol-reactive stress PG, on the expression of the PDI gene. Delta 12-PGJ2 transiently induced the PDI mRNA in HeLa cells. Other stress inducers, tunicamycin, A23187, and heat shock, which caused an accumulation of unfolded proteins, also induced the PDI mRNA, indicating that the unfolded protein accumulation response induces PDI gene expression. Cycloheximide by itself strongly induced the PDI mRNA, but did not inhibit the effect of delta 12-PGJ2, suggesting that the pathway of delta 12-PGJ2-induced PDI gene expression does not involve unfolded protein accumulation. On the other hand, dithiothreitol, a reducing agent, induced the PDI mRNA, and delta 12-PGJ2 did not exert additive induction. Thus, delta 12-PGJ2 induces PDI gene expression through a pathway independent of de novo protein synthesis.

Calcimycin↗
PubMed1996

Mapping of the genes encoding mouse prostaglandin D, E, and F and prostacyclin receptors.

Prostaglandins and prostacyclin are metabolites of arachidonic acid and exert a variety of actions to maintain local homeostasis in the body. Their actions are mediated by cell surface receptors specific to the respective ligands. Using a panel of interspecific back-cross mice, we have mapped the prostaglandin D receptor gene (Ptgdr), prostaglandin E receptor subtype EP(1) gene (Ptgerepl), prostaglandin F receptor gene (Ptgfr), and prostacyclin receptor gene (Ptgir). Ptgdr mapped to proximal Chr 14, Ptgfr mapped to distal Chr 3, Ptgerepl mapped to middle Chr 8, and Ptgir mapped to proximal Chr 7.

Animals↗
PubMed1996

Two isoforms of the prostaglandin E receptor EP3 subtype different in agonist-independent constitutive activity.

We previously identified two isoforms of the mouse prostaglandin E receptor EP3 subtype, EP3 alpha and EP3 beta, with different carboxyl-terminal tails, produced through alternative splicing and showing different efficiency in inhibition of adenylate cyclase (Sugimoto, Y., Negishi, M., Hayashi, Y., Namba, T., Honda, A., Watabe, A., Hirata, M., Narumiya, S., and Ichikawa, A. (1993) J. Biol. Chem. 268, 2712-2718). To assess the role of the carboxyl-terminal tails in the G protein coupling properties of the EP3 receptor, we examined the Gi activities of EP3 alpha, EP3 beta, and the mutant receptor, in which the carboxyl-terminal tail was truncated at the splicing site. The EP3 alpha receptor showed marked agonist-independent constitutive inhibition of adenylate cyclase, while EP3 beta receptor had no agonist-independent inhibition. On the other hand, the truncated receptor showed only agonist-independent constitutive inhibition. The constitutive activity of these receptors on the stimulation of GTPase activity of Gi was also observed. Thus, alternative splicing produced two isoforms with different carboxyl-terminal tails and with different constitutive activity, and the truncation of the carboxyl-terminal tail caused full constitutive activity.

Adenylate Cyclase Toxin↗
PubMed1996

Gene expression of the human prostaglandin E receptor EP4 subtype: differential regulation in monocytoid and lymphoid lineage cells by phorbol ester.

We isolated a cDNA clone encoding the human prostaglandin (PG) E receptor EP4 subtype and examined the gene expression in human blood cells. Northern blot analysis revealed that the EP4 gene is expressed at a high level in peripheral blood mononuclear cells, and at lower levels in cultured human blood cell lines, THP-1 and U937 (monocytoid cell lines), MOLT-4 and Jurkat (T-cell lines), and Raji (B-cell line). To examine regulation of the EP4 gene expression in the immune system, we studied the effects of phorbol 12-myristate 13-acetate (PMA) on these cell lines. Gene expression was upregulated in THP-1, U937, and Raji cells by PMA, and was downregulated in MOLT-4 and Jurkat cells. In THP-1 cells the effects of PMA were further analyzed, and the upregulation of the EP4 gene was shown to be followed by an increase in PGE2 binding sites and in PGE2-induced cAMP accumulation. In the striking contrast, other PGE receptor subtypes (EP1, EP2 and EP3) and other prostanoid receptors (IP and DP) were shown not to be upregulated by PMA. Therefore, this is the first demonstration of a highly specific upregulation of the EP4 subtype in THP-1 cells treated with PMA, suggesting the importance of the EP4 subtype in the immune system. In the present study we also clarified that EP4 gene expression is regulated differently among human monocytoid and lymphoid lineage cells, thus leading to the better understanding of the regulatory mechanisms for the human EP4 gene expression in the immune system.

Burkitt Lymphoma↗
PubMed1996

Membrane depolarization by activation of prostaglandin E receptor EP3 subtype of putative serotonergic neurons in the dorsal raphe nucleus of the rat.

A whole-cell current-clamp study using a thin slice preparation of the rat brain was carried out to elucidate the function of prostaglandin E (PGE) receptor EP3 subtype in the dorsal raphe nucleus (DR), where mRNA of this subtype is highly expressed. Bath application of PGE2 or M&B 28767, an EP3 agonist, depolarized the membrane of the large DR neurons in a concentration-dependent manner between 10(-9) and 10(-6) M. These neurons showed hyperpolarization of membrane potential to 10 or 50 microM serotonin. Neither an EP2 receptor agonist, butaprost, an EP2/EP4 receptor agonist, 11-deoxy-PGE1, nor an EP1 receptor agonist, 17-phenyl-PGE2, had any effect on large DR neurons between 10(-9) and 10(-6) M. The M&B 28767-induced depolarization was observed in a Ca(2+)-free, high Mg2+ (5 mM) solution containing 0.3 microM tetrodotoxin, and occurred equally well when intracellular Cl- was replaced by gluconate. These results suggest that direct agonist-activation of EP3 receptor depolarizes the membrane by a cationic conductance, leading to excitation of DR neurons, and indicate a physiological implication that EP3 receptor may modulate the serotonergic inhibition of neuronal activities.

Alprostadil↗
PubMed1996

In vitro characterization of radioiodinated (-)-m-iodovesamicol in rat cerebral membranes.

We investigated the binding characteristics of 125I-(-)-m-iodovesamicol (125I-(-)-mIV), radioiodinated at the meta position of the 4-phenylpiperidine moiety, in cerebral membranes of the rat brain. The receptor binding affinity of (-)-mIV (Ki = 37 nM) was comparable to that of (-)-vesamicol (Ki = 30 nM). The stereoselectivity of (-)-mIV and (-)-vesamicol for the vesamicol receptor was very high [both (-)-mIV and (-)-vesamicol binding more than 20-fold more active than their (+)isomers], and 125I-(-)-mIV had low affinity for the sigma, dopamine, serotonin, adrenaline and acetylcholine receptors. Furthermore, in a saturation binding study using cerebral membrane preparations, (-)-mIV exhibited a Kd of 18.2 nM with maximum number of binding site Bmax of 660 fmol/mg of protein. These results showed that the characteristics of binding between (-)-mIV and (-)-vesamicol to cholinergic binding sites in the rat brain were similar.

Animals↗
PubMed1996

Molecular aspects of the structures and functions of the prostaglandin E receptors.

Prostaglandin (PG) E2 exerts a variety of biological activities for the maintenance of local homeostasis in the body. The effects of PGE2 are exerted by a variety of PGE receptors which are different in their signal transduction properties and are classified into four subtypes, EP1, EP2, EP3 and EP4. We have isolated the mouse cDNAs for these PGE receptors and characterized the cloned receptors. EP1, EP2, EP3 and EP4 receptors consist of 405, 362, 365 and 513 amino acid residues with a putative seven hydrophobic domains, respectively. When expressed in mammalian cells, EP1 showed elevation of intracellular [Ca2+], EP2 and EP4 stimulated adenylate cyclase and EP3 inhibited the enzyme. Northern blot and in situ hybridization analyses have shown that these subtypes are differently localized to specific tissues and cells. We have identified multiple isoforms of the EP3 receptor (EP3 alpha, EP3 beta, and EP3 gamma) which differ in their carboxy-terminal domains. These isoforms displayed identical agonist binding properties, but were functionally different in the efficiency of G protein activation, the specificity of G protein coupling, and sensitivity to agonist-induced desensitization. The diverse physiological actions of PGE2 are elicited by the molecular diversity of the receptor subtypes and isoforms distributed differently in the body.

Amino Acid Sequence↗
PubMed1996

Reg gene expression is increased in rat gastric enterochromaffin-like cells following water immersion stress.

BACKGROUND & AIMS: Reg gene has been isolated from regenerating rat pancreatic islets, and subsequent studies have shown a trophic effect of Reg protein on islet cells. However, little is known about the role of Reg protein in the stomach. The aim of this study was to clarify the localization of Reg messenger RNA (mRNA) and its product in the stomach and to examine changes in the level of their expression during regeneration of gastric mucosal cells. METHODS: Gastric lesions were experimentally induced in Sprague-Dawley rats by water immersion stress. Northern blot analysis and in situ hybridization studies were performed to examine changes in mRNA levels. Immunohistochemical studies were performed to identify the cellular localization and to investigate the change in Reg protein level. RESULTS: Reg mRNA and its product were distributed in the basal part of the oxyntic mucosa and were expressed mainly in enterochromaffin-like cells. Levels of both Reg mRNA and its product were markedly increased in the water immersion-induced gastric lesions. CONCLUSIONS: Reg mRNA and its product are expressed in gastric enterochromaffin-like cells, and their levels are increased during the healing process of water immersion-induced gastric lesions.

Animals↗
PubMed1996

Prostaglandin E receptor subtypes in mouse osteoblastic cell line.

PGE2 is one of the key molecules in the osteoblast. It is the major prostanoid in the bone, and its production is under the control of both systemic and local factors. PGE2 has been reported to have multiple actions in the osteoblast, such as growth promotion and cell differentiation. To better understand the action of PGE2 in the osteoblast, we determined the PGE receptor subtypes in MC3T3-E1, an osteoblastic cell line derived from the normal mouse calvaria. Northern blot analysis revealed that EP1 and EP4 subtypes are expressed in MC3T3-E1. In contrast, EP3 subtype was not detected by either Northern blot analysis or RT-PCR. The contribution of each subtype was evaluated by studying the effects of subtype-specific analogs on osteoblastic function at confluency and 5 days after confluency. An EP1 agonist, 17-phenyl-omega-trinor PGE2, increased DNA synthesis and decreased alkaline phosphatase activity. 11-Deoxy-PGE1, and EP2 and EP4 agonist, decreased DNA synthesis and increased alkaline phosphatase activity at both stages. Butaprost, an EP2-selective agonist, showed effects similar to those of 11-deoxy-PGE1 only at confluency. Another and more differentiated osteoblastic marker, osteocalcin production, was detectable and was stimulated by 11-deoxy-PGE1 only 5 days after confluency. The exposure of these cells to EP1 agonist changed the cell shape to a more fibroblastic appearance. These results indicate that EP1, EP4, and probably EP2 are present in MC3T3-E1 cells; EP1 promotes cell growth, and EP2 and EP4 mediate differentiation of the osteoblast. Furthermore, the decreased response to EP2-specific agonist 5 days after confluency suggests that the expression of PGE receptor subtype is dependent on the stage of osteoblastic differentiation. This is the first report to determine PGE receptor subtypes in the bone.

Alprostadil↗
PubMed1996

Restoration of endothelial cell function by chronic cicletanine treatment in Dahl salt-sensitive rats with salt-induced hypertension.

The effects of chronic cicletanine (CICL) treatment on endothelial cell function were investigated in Dahl salt-sensitive (Dahl S) rats. Forty-four six-week-old Dahl S rats were divided into four groups: i) 10 Dahl S rats fed a low-salt (0.3% NaCl) diet and given vehicle, ii) 12 Dahl S rats fed a high-salt (4% NaCl) diet and given vehicle, iii) 11 low-dose (10 mg/kg body weight/d) CICL-treated Dahl S rats fed a high-salt diet, and iv) 11 high-dose (30 mg/kg body weight/d) CICL-treated Dahl S rats fed a high-salt diet. The rats were maintained on the respective salt regimen for 12 wk and treated with cicletanine for the last 6 wk, after which various parameters of endothelial cell function were determined. Systolic blood pressure, measured by the tail-cuff method, was reduced significantly by high-dose cicletanine (223 vs. 195 mmHg, p < 0.01). Scanning electron microscopy revealed that high-dose CICL attenuated endothelial injury in the aorta of Dahl S rats. Arterial lesions in the heart and glomerulosclerosis in the kidney were significantly reduced by treatment with high-dose CICL. Moreover, prostacyclin (PGI2) and prostaglandin E2 (PGE2) generation in the aortic wall was significantly increased by 28% (p < 0.005) and by 149% (p < 0.001), respectively, by high-dose CICL. Nitric oxide (NO) generation in the aortic walls was significantly increased by high-dose CICL (0.38 vs. 15.4 pmol/cm2/30 min, p < 0.001). This effect was accompanied by a 47% increase in cGMP synthesis in the vascular walls. In contrast, the synthesis of PGI2, PGE2, and NO in the kidney slices did not differ significantly among the four experimental groups. In addition, the generation of vasodilatory substances inversely correlated with the score of vascular lesions in the heart and kidney. The results suggested that the blood pressure reduction by chronic cicletanine treatment in Dahl S rats is associated with an improvement in endothelial cell function. The increased release of vasodilatory substances from endothelial cells may contribute to the blood pressure reduction and attenuation of vascular injury observed with cicletanine treatment.

Animals↗
PubMed1996

Overexpression of the MDM2 oncogene in leukemia and lymphoma.

A cellular phosphoprotein that binds to and inactivates p53 has recently been identified as a product of the oncogene MDM2. Amplification of the MDM2 gene was found in more than a third of sarcomas and in a subset of malignant gliomas. Despite the absence of amplification, the MDM2 gene was overexpressed in some types of leukemias and lymphomas. Overexpression was significantly more frequent in the low-grade type of B-cell non-Hodgkin's lymphoma (B-NHL) than in the intermediate/high grade types of lymphoma and the overexpression was also significantly more frequent in the advanced rather than the earlier stages of B-cell chronic lymphocytic leukemia (B-CLL) and B-NHL. This suggests that MDM2 could play a role, via the p53 pathway, in tumorigenicity and/or in disease progression in some hematological malignancies. However, in the light of our findings that, in a few cases, both the overexpression of MDM2 and mutant-type p53 was seen, it is possible that MDM2 overexpression may also promote neoplastic growth by mechanisms other than inactivation of the p53 protein.

Animals↗
PubMed1996

Radioiodinated (-)-2-[4-(3-iodophenyl) piperidino] cyclohexanol: a potential radioligand for mapping presynaptic cholinergic neurons.

We synthesized the (-)-enantiomer of radioiodinated 2-[4-(3-iodophenyl) piperidino] cyclohexanol ((-)-[125I]-m-iodovesamicol [(-)-[125I]mIV]) and evaluated its in vivo binding specificity and affinity for the vesamicol receptor in the rat brain. Significant amounts (approximately 3% of the injected dose) of (-)-mIV accumulated and retention was prolonged. The accumulation of (-)-[125I]mIV in the brain was significantly reduced by the pre- and post-administration of unlabelled vesamicol (0.5 mumol kg -1). Blocking and displacement studies in vivo showed that the binding affinity of (-)-[125I]mIV for the vesamicol receptor was very high, but that for other receptors was very low. Autoradiographic studies showed that the regional distribution of radioactivity, as revealed by coronal sections of the rat brain, was similar for both (-)-[125I]mIV and (-)-[3H]vesamicol. The results demonstrated that the characteristics of (-)-[125I]mIV for binding sites in rat brain were similar in vivo and in vitro. Radioiodinated (-)-mIV may potentially be useful for studying presynaptic cholinergic neurons in the brain.

Animals↗
PubMed1996

[Molecular structures of eicosanoid receptors].

Prostanoids exert versatile actions in diverse tissues and cells through specific cell surface receptors. Molecular biological studies have revealed the primary structure of eight types and subtypes of prostanoid receptors from various species. They are coupled to different signal transduction systems and show different tissue distribution. In addition, multiple isoforms of prostaglandin E receptor EP3 subtype have been identified in various species. They are produced through alternative RNA splicing from a single gene and differ only in their carboxyl-terminal tails. These isoforms differ in the efficiency of G protein activation, in the specificity of coupling to G proteins or in sensitivity to desensitization. This molecular characterization is useful for understanding the diverse physiological role of prostanoids.

Amino Acid Sequence↗
PubMed1996

Two Gs-coupled prostaglandin E receptor subtypes, EP2 and EP4, differ in desensitization and sensitivity to the metabolic inactivation of the agonist.

There are at least four subtypes of prostaglandin E (PGE) receptors. The EP1 and EP3 receptors are coupled to Ca2+ mobilization and the inhibition of adenylate cyclase, respectively, and the EP2 and EP4 receptors are coupled to the same signal transduction pathway, stimulation of adenylate cyclase. To identify the functional differences between EP2 and EP4 receptors, we examined agonist-induced desensitization of these two receptors using Chinese hamster ovary cells, which stably express these receptors. The EP4 receptor underwent short term agonist-induced desensitization, but no such desensitization was observed for the EP2 receptor. In contrast, the EP2 and EP4 receptors displayed similar patterns of down-regulation in response to prolonged exposure to PGE2. On the other hand, PGE2 is rapidly metabolized to 15-keto-PGE2 and, subsequently, to 13,14-dihydro-15-keto-PGE2. Thus, we compared the sensitivities of the two receptors to these two metabolites. The EP4 receptor markedly lost the response at the first metabolism, whereas the EP2 receptor gradually lost the response according to the degree of metabolism, having higher sensitivity to the first metabolite, 15-keto-PGE2, than the EP4 receptor. Therefore, the physiological significance of EP2 and EP4 may lie in their different sensitivities to agonist-induced short term desensitization and their differential susceptibilities to the metabolic inactivation of the agonist.

Animals↗
PubMed1995

Processing and activation of recombinant mouse mastocytoma histidine decarboxylase in the particulate fraction of Sf9 cells by porcine pancreatic elastase.

Mature 53 kDa histidine decarboxylase (HDC) peptide is produced from a precursor 74 kDa peptide. The mechanism of specific cleavage by processing enzyme is unknown. Using the recombinant mouse 74 kDa HDC, we found that porcine pancreatic elastase specifically converted the inactive 74 kDa HDC to its active form of 53 kDa HDC.

Animals↗
PubMed1995

Molecular mechanisms of diverse actions of prostanoid receptors.

This review summarizes recent advances in the molecular characterization of prostanoid receptors. Prostanoids exert versatile actions in diverse tissues and cells through specific cell surface receptors. Molecular biological studies revealed the primary structure of eight types and subtypes of prostanoid receptor from various species. These include the thromboxane A2 receptor, prostacyclin receptor, prostaglandin (PG) F receptor, PGD receptor and four subtypes of PGE receptors. They are coupled to different signal transduction systems. In addition, multiple isoforms of PGE receptor EP3 subtype have been identified in various species. They are produced through alternative RNA splicing from a single gene and differ only in their carboxy-terminal tails. These isoforms differ in the efficiency of G protein activation, in the specificity of coupling to G proteins or in sensitivity to desensitization. This molecular characterization is useful for understanding the diverse physiological roles of prostanoids.

Amino Acid Sequence↗