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Biomedical subjects

G Tsujimoto

Publications and source records attributed to G Tsujimoto.

At least 73 records · Page 4Linked to original sources
PubMed1998

Differential mechanism for the cell surface sorting and agonist-promoted internalization of the alpha1B-adrenoceptor.

1. Alpha1B-adrenoceptors are localized at a steady state in the plasma membrane in untreated cells, and internalize to intracellular vesicles when exposed to agonist. Flow cytometry analysis with an anti-N-terminus-antibody (1B-N1-C, (Hirasawa et al., 1996)) facilitated the quantification of cell surface alpha1B-adrenoceptor. Also, the cellular distribution of alpha1B-adrenoceptors was visually monitored by immunocytochemical confocal microscopy. 2. Utilizing this combined approach, we have examined the molecular mechanism for cellular trafficking of alpha1B-adrenoceptors, including the process of sorting of the synthesized receptor protein to the cell surface, and the agonist-induced internalization. The two processes were separately examined by using alpha1B-adrenoceptor inducible DDT1MF-2 cells for the sorting process and CHO cells stably expressing alpha1B-adrenoceptors for the agonist-promoted internalization. 3. We examined the effects of cytochalasin D and mycalolide B (actin depolymerization agents), demecolcine (a microtubule disrupting agent), brefeldin A (an inhibitor of vesicular transport and Golgi function), bafilomycin A1 (a specific inhibitor of the vacuolar proton pump) or hyperosmotic sucrose treatment (that may inhibit clathrin-mediated endocytosis) on these processes. 4. We found that the agonist-promoted internalization was blocked by cytochalasin D and mycalolide B, while the cell surface sorting process was specifically blocked by brefeldin A, indicating that the two processes involve different components of the cellular endocytic machinery. 5. The experimental approach as exemplified in this study would provide a valuable system to study further the molecular mechanism(s) of cellular trafficking of G protein-coupled receptors.

Adrenergic alpha-1 Receptor Agonists↗
PubMed1998

Real-time optical monitoring of ligand-mediated internalization of alpha1b-adrenoceptor with green fluorescent protein.

The study of G protein-coupled receptor signal transduction and behavior in living cells is technically difficult because of a lack of useful biological reagents. We show here that a fully functional alphalb-adrenoceptor tagged with the green fluorescent protein (alphalbAR/GFP) can be used to determine the molecular mechanism of intemalization of alphalbAR/ GFP in living cells. In mouse alphaT3 cells, alpha1bAR/GFP demonstrates strong, diffuse fluorescence along the plasma membrane when observed by confocal laser scanning microscope. The fluorescent receptor binds agonist and antagonist and stimulates phosphatidylinositol/Ca2+ signaling in a similar fashion to the wild receptor. In addition, alpha1bAR/ GFP can be internalized within minutes when exposed to agonist, and the subcellular redistribution of this receptor can be determined by measurement of endogenous fluorescence. The phospholipase C inhibitor U73,122, the protein kinase C activator PMA, and inhibitor staurosporine, and the Ca2+-ATPase inhibitor thapsigargin were used to examine the mechanism of agonist-promoted alphalbAR/GFP redistribution. Agonist-promoted internalization of alphalbAR/GFP was closely linked to phospholipase C activation and was dependent on protein kinase C activation, but was independent of the increase in intracellular free Ca2+ concentration. This study demonstrated that real-time optical monitoring of the subcellular localization of alphalbAR (as well as other G protein-coupled receptors) in living cells is feasible, and that this may provide a valuable system for further study of the biochemical mechanism(s) of agonist-induced receptor endocytosis.

Adrenergic alpha-Agonists↗
PubMed1998

[Application of genetically engineered animals in pharmacology: the use of green fluorescent protein for selective production of transgenic animals].

Transgenic animals are a very important tool not only for basic science but also for the pharmaceutical industry. The use of genetically engineered farm animals are suitable as a disease model. Production of a therapeutic protein such as human clotting factors by transgenic animals will reduce the risk of infection with human immunodeficiency virus and hepatitis virus. The efficiency of transgenic animal production, however, has been low and thus limited its application. We improved the efficiency using a green fluorescent protein (GFP) as a marker of gene integration. Using this method, we obtained 12 fetuses, and Southern blot analysis showed eight of them were transgenic, indicating transgenic embryos were successfully selected at the preimplantation stage. We also observed similar GFP expression in rat and bovine blastocysts. Application of this GFP selection method should improve the efficiency of transgenic livestock production.

Animals↗
PubMed1998

Vascular alpha1-adrenoceptor subtype selectivity and alpha1-blocker-induced orthostatic hypotension.

Newly developed alpha1-adrenoceptor antagonists including naftopidil are free from the "prazosin-like" side effect of orthostatic hypotension and associated symptoms. We investigated the mechanism for the differential effects of naftopidil and prazosin on the development of postural hypotension, with special attention on their selectivity for the alpha1-adrenoceptor subtype. We observed that head-up tilt caused a similar extent of drop in mean arterial pressure in control, naftopidil (1 mg/kg)- or prazosin (10 microg/kg)-treated rats; however, the tilt-induced postural hypotension was recovered within 2 min in the naftopidil-treated group, but not in the prazosin-treated group. Comparing an inhibitory effect on noradrenaline-induced contraction in the rat aorta and portal vein, we found that naftopidil was sixfold less potent in the portal vein, while prazosin showed similar potency in both tissues. Reverse transcription-polymerase chain reaction analysis showed that the expression of alpha1d-adrenoceptor mRNA predominated in the aorta, while that of alpha1b-adrenoceptor mRNA predominated in the portal vein. Using cloned rat alpha1-adrenoceptor subtypes, we found that naftopidil was selective for the alpha1d-subtype with approximately ninefold higher affinity than at the other subtypes. These results show that the pharmacological character of naftopidil, combined with the differential expression of the alpha1-adrenoceptor subtype in the artery and the vein, may partly explain the differential effect of naftopidil and prazosin on head-up tilt-induced hemodynamic responses.

Adrenergic alpha-Antagonists↗
PubMed1998

OPC-41061, a highly potent human vasopressin V2-receptor antagonist: pharmacological profile and aquaretic effect by single and multiple oral dosing in rats.

The pharmacological profile and the acute and chronic aquaretic effects of OPC-41061, a novel nonpeptide human arginine vasopressin (AVP) V2-receptor antagonist, were respectively characterized in HeLa cells expressing cloned human AVP receptors and in conscious male rats. OPC-41061 antagonized [3H]-AVP binding to human V2-receptors (Ki = 0.43 +/- 0.06 nM) more potently than AVP (Ki = 0. 78 +/- 0.08 nM) or OPC-31260 (Ki = 9.42 +/- 0.90 nM). OPC-41061 also inhibited [3H]-AVP binding to human V1a-receptors (Ki = 12.3 +/- 0.8 nM) but not to human V1b-receptors, indicating that OPC-41061 was 29 times more selective for V2-receptors than for V1a-receptors. OPC-41061 inhibited cAMP production induced by AVP with no intrinsic agonist activity. In rats, OPC-41061 inhibited [3H]-AVP binding to V1a-receptors (Ki = 325 +/- 41 nM) and V2-receptors (Ki = 1.33 +/- 0. 30 nM), showing higher receptor selectivity (V1a/V2 = 244) than with human receptors. A single oral administration of OPC-41061 in rats clearly produced dose-dependent aquaresis. In treatment by multiple OPC-41061 dosing for 28 days at 1 and 10 mg/kg p.o. in rats, significant aquaretic effects were seen throughout the study period. As the result of aquaresis, hemoconcentration was seen at 4 hr postdosing although, no differences were seen in serum osmolality, sodium, creatinine and urea nitrogen concentrations at 24 hr postdosing. Furthermore, there was no difference in serum AVP concentration, pituitary AVP content or the number and affinity of AVP receptors in the kidney and liver at trough throughout the study period. These results demonstrate that OPC-41061 is a highly potent human AVP V2-receptor antagonist and produces clear aquaresis after single and multiple dosing, suggesting the usefulness in the treatment of various water retaining states.

Animals↗
PubMed1997

Day-night variation of pituitary adenylate cyclase-activating polypeptide (PACAP) level in the rat suprachiasmatic nucleus.

Adenylate cyclase-activating polypeptide (PACAP) is synthesized in the retinal ganglion cells which terminate on vasoactive intestinal polypeptide neurons in the suprachiasmatic nucleus (SCN), the location of circadian clock. To examine whether PACAP exhibits daily variations in the rat SCN, we measured endogenous PACAP contents throughout the day under 12:12 h light-dark or constant dark conditions. PACAP level was low during the light periods, high during the dark periods, and was stable under constant dark conditions. In the periventricular nucleus of the hypothalamus and cerebral cortex, PACAP content did not show any significant variation throughout the day. Our findings suggest that PACAP content in the SCN may be changed by lighting conditions. Thus, PACAP-containing neurons may play certain roles in the entrainment of circadian rhythms.

Animals↗
PubMed1997

Modulation of the arterial baroreceptor reflex by the vasopressin receptor in the area postrema of the hypertensive rats.

The role of arginine8-vasopressin (AVP) in regulation of the baroreceptor reflex in the area postrema was examined in anesthetized hypertensive rats. The sensitivity of the baroreceptor reflex in a one-kidney one clip (1K1C) hypertensive rats was increased in only the initial stage (2 weeks), in association with increase in blood pressure, and then returned to the normal level. This increase in the sensitivity of the baroreceptor reflex in the initial stage was reversed by microinjection of a V1 or V2 antagonist (1 microg) into the area postrema. AVP V2 receptor mRNA was expressed temporarily in the area postrema in this period. These results suggest that vasopressin receptors in the area postrema is important in regulating the sensitivity of the baroreceptor reflex.

Animals↗
PubMed1997

Selective production of transgenic mice using green fluorescent protein as a marker.

The low efficiency of transgenic animal production by microinjection has been a serious problem especially for the production of transgenic livestock. We developed a method to selectively produce transgenic mice using green fluorescent protein (GFP) as a marker. Using this method, we obtained eight fetuses and four live-born mice derived from 55 GFP-positive blastocysts. PCR analysis showed 11 out of 12 mice (fetuses and newborn mice) were transgenic. Southern blot analysis showed that 8 out of 12 were transgenic. GFP expression was also observed in bovine blastocysts, suggesting that this method should contribute to the efficient production of transgenic livestock.

Animals↗
PubMed1997

Quantification and distribution of alpha 1-adrenoceptor subtype mRNAs in human vas deferens: comparison with those of epididymal and pelvic portions.

1. This study was intended to quantify the amounts of the alpha 1-adrenoceptor subtype mRNAs in human vas deferens, and demonstrate the receptor subtype responsible for the vas contraction. 2. The RNase protection assay showed that the mean total amount of alpha 1a mRNA was 7.4 +/- 2.2 pg/5 micrograms of poly (A)+ RNA (97.0% of the total alpha 1 mRNA) in the epididymal portion (E-vas) and 4.9 +/- 0.8 pg/5 micrograms of poly (A)+ RNA (96.3% of the total) in the pelvic portion (P-vas). The E-vas showed a tendency to have a greater alpha 1a mRNA abundance than the P-vas (P = 0.11). The alpha 1b and alpha 1d mRNAs were absent or of extremely low abundance. 3. By an in situ hybridization, the alpha 1a and alpha 1d mRNAs were recognized in the smooth muscle cells of the E-vas and the P-vas, and the distribution pattern the same in both tissue. The alpha 1b mRNA positive site was scarcely detectable in both vas portions. 4. In a functional study, l-phenylephrine produced concentration-dependent contraction in the E-vas (Emax = 2.24 +/- 0.70 g; pD2 = 5.32 +/- 0.09) and the P-vas (Emax = 2.46 +/- 0.46 g; pD2 = 5.07 +/- 0.12). KMD-3213, a novel alpha 1A-adrenoceptor-selective antagonist, caused parallel rightward shifts of the concentration-response curves for l-phenylephrine. Apparent pKB values were 9.90 +/- 0.16 for the E-vas and 9.71 +/- 0.17 for the P-vas. There was no significant difference in Emax, pD2 or pKB estimates between the two portions. 5. We have found that alpha 1a mRNA is predominant in the human vas deferens, and confirmed that contraction of this organ is mediated by the alpha 1A-adrenoceptor.

Aged↗
PubMed1997

Subtype-specific differences in subcellular localization of alpha1-adrenoceptors: chlorethylclonidine preferentially alkylates the accessible cell surface alpha1-adrenoceptors irrespective of the subtype.

Selective inactivation of alpha1B-adrenoceptor (AR) by the site-directed alkylating agent chlorethylclonidine (CEC) has been used as one of major pharmacological criteria to subclassify alpha1-AR; however, the mechanism for the differential CEC sensitivity of the two subtypes is uncertain, and the extent of CEC inactivation varies depending on the treatment employed. In this study, we examined the correlation between the subcellular localization of alpha1-AR subtypes (alpha1A and alpha1B) and CEC sensitivity. Constructing alpha1-AR tagged with the FLAG epitope at the amino terminus and/or green fluorescent protein (GFP) at the carboxyl terminus, we examined the subcellular distribution of alpha1-ARs expressed in COS-7 cells. Flow cytometry analysis showed that most populations of GFP-expressing alpha1B-AR cells, but very few GFP-expressing alpha1A-AR cells, were detected by the anti-amino terminus antibodies. The immunocytochemical and GFP-fluorescence confocal micrographs showed that alpha1A-ARs predominantly localize intracellularly, whereas alpha1B-ARs localize on the cell surface. Furthermore, CEC (10 microM) treatment of intact cells resulted in an inactivation of approximately 42% of alpha1A-ARs and 93% of alpha1B-ARs, whereas treatment of the membrane preparations resulted in an inactivation of approximately 83% of alpha1A-ARs and 88% of alpha1B-ARs, respectively. Together, the results showed that a hydrophilic alkylating agent CEC preferentially inactivates alpha1-AR on the cell surface irrespective of its subtype, and that the subtype-specific subcellular localization rather than the receptor structure is a major determinant for CEC inactivation of alpha1-AR. Subtype-specific subcellular localization suggests an additional class of functional properties that provide new insight into drug action.

Adrenergic alpha-Antagonists↗
PubMed1996

Alpha 1b-adrenoceptor-mediated calcium oscillation is specific for the S phase in cell cycle and dependent on the extracellular calcium.

Using Chinese hamster ovary cells stably expressing alpha 1b-adrenoceptor (alpha 1bAR) as a model, we examined the effect of the cell cycle on the agonist-promoted intracellular [Ca2+]i oscillation. In cells synchronized into either the G1 state or the M phase, no oscillatory behavior was observed under any extracellular Ca2+ concentrations ([Ca2+]o < or = 3 mM), whereas in cells synchronized into the S phase, norepinephrine caused [Ca2+]i oscillation in a [Ca2+]o-dependent manner, indicating that alpha 1AR-mediated [Ca2+]i oscillation is specific for the S phase in cell cycle and dependent on [Ca2+]o. The S phase-specific occurrence of alpha 1AR-mediated [Ca2+]i oscillation is not associated with changes in alpha 1AR density. As the cells consistently developed [Ca2+]i oscillation in the S phase, the cells would provide a valuable system to study further the biochemical mechanism for agonist-induced [Ca2+]i oscillation phenomenon.

Animals↗
PubMed1996

Selective growth of human mast cells induced by Steel factor, IL-6, and prostaglandin E2 from cord blood mononuclear cells.

To establish the method for generating a large number of mature human mast cells, we cultured cord blood mononuclear cells (CBMC) in several conditions in the presence of Steel factor (SF). Among several cytokines tested, IL-6 enhanced SF-dependent mast cell growth from purified CD34+ cells for more than 8 wk in culture. When CBMC were cultured instead of CD34+ cells, IL-6 enhanced the mast cell development in the presence but not in the absence of PGE2. PGE2 enhanced the SF- and IL-6-dependent development of mast cells from CBMC probably by blocking granulocyte-macrophage CSF (GM-CSF) secretion from accessory cells, because 1) PGE2, or anti-GM-CSF enhanced the mast cell development induced by SF and IL-6 from CBMC, but not from CD34+ cells; 2) GM-CSF inhibited the enhancing effect of IL-6 on the mast cell development from CD34+ cells; and 3) PGE2 inhibited GM-CSF secretion from CBMC. The mast cells cultured in the presence of SF, IL-6, and PGE2 for >10 wk were 99% pure, and seemed to be functionally mature, because 1) they contained 5.62 micrograms of histamine and 3.46 micrograms of tryptase per 10(6) cells; and 2) when sensitized with human IgE and then challenged with anti-human IgE, the cells released a variety of mediators such as histamine, and an increase in intracellular Ca2+ was found in advance of the activation of membrane movement by using a confocal laser-scanning microscope. Electron-microscopic analysis revealed that some of the cultured mast cells are morphologically mature since they filled with scroll granules and contained crystal granules.

Base Sequence↗
PubMed1996

Flow cytometry analysis of alpha1-adrenoceptor subtypes.

To characterize the alpha1-adrenoceptor subtypes, we developed a flow cytometry method using the fluorescent ligand BODIPY-FL prazosin and the anti-peptide antibody against the alpha1b-adrenoceptor amino terminus (designated 1B-N1-C) as probes. Three alpha1-adrenoceptors (alpha1a, alpha1b and alpha1d) expressed in CHO cells were detected by BODIPY-FL prazosin; however, only alpha1b-adrenoceptor subtype was detected by the anti-peptide antibody 1B-N1-C. Furthermore, the flow cytometry analysis with 1B-N1-C specifically identified alpha1b-adrenoceptor in native cells of hamster DDT1-MF2 cells, rat hepatocytes and cardiomyocytes.

Amino Acid Sequence↗
PubMed1996

Detection of alpha 1-adrenoceptor subtypes in human hypertrophied prostate by in situ hybridization.

Adrenergic stimulation induces contraction of hypertrophied prostatic tissue via the alpha 1 adrenoceptor, and the results of pharmacological studies suggested the existence of adrenoceptor subtypes. Recently three subtypes (alpha 1a, alpha 1b, and alpha 1d) were cloned. Using probes for these subtypes, we demonstrated their expression in the tissues of ten cases of benign prostatic hypertrophy, using in situ hybridization. To determine the ratio between these subtypes, an RNase protection assay was also performed in three cases. Expression of the alpha 1a and alpha 1d adrenoceptors was diffuse in the smooth muscles of the interstitium, but was absent in glandular epithelial cells. On the contrary, the alpha 1b adrenoceptor was hardly detectable. The RNase protection assay confirmed the absence of the alpha 1b adrenoceptor, the ratio of alpha 1a and alpha 1d being 4:1. These results supported the idea that the differences in prostatic contractile response to several adrenergic drugs are based on the affinities of these drugs for the different subtypes.

Aged↗
PubMed1996

Oxytocin receptors expressed and coupled to Ca2+ signalling in a human vascular smooth muscle cell line.

1. In a human vascular smooth muscle cell line (HVSMC), binding experiments with [3H]-arginine8-vasopressin (AVP) have shown the existence of a homogeneous population of binding sites with affinity (Kd value) of 0.65 nM and a maximum number of binding sites (Bmax) of 122 fmol mg-1 protein. 2. Nonlabelled compounds compete for [3H]-AVP binding in the HVSMC membrane with an order of potency of oxytocin > lyspressin > or = AVP > Thr4, Gly7-oxytocin > (beta-mercapto-beta-beta-cyclopentamethylenepropionyl-O-Me Tyr2, Arg8) vasopressin > desmopressin > OPC21268 > OPC31260. This order was markedly different from that observed in rat vascular smooth muscle cells (A10), a well-established V1A receptor system. 3. In HVSMC both oxytocin and AVP increased inositol 1,4,5-trisphosphate (IP3) production and [Ca2+]i response, but the efficacy of the responses was greater for oxytocin than AVP. 4. Reverse transcription-polymerase chain reaction (RT-PCR) assay detected only oxytocin receptor but not V1A or V2 receptors in HVSMC, whereas only V1A receptors were found in A10 cells. 5. In conclusion, in HVSMC only oxytocin receptors are expressed among the vasopressin receptor family, and they coupled to phosphatidyl inositol (PI) turnover/Ca2+ signalling. This unexpected observation should provide new insight into the functional role of the oxytocin receptor in a human vascular smooth muscle cell line.

Animals↗
PubMed1996

Alpha 1a-adrenoceptor polymorphism: pharmacological characterization and association with benign prostatic hypertrophy.

1. Two restriction fragment length polymorphisms of the human alpha 1a-adrenoceptor gene digested with PstI restriction enzyme exist; the nucleotide change causes the substitution of C residue for T at nucleotide 1441, thereby Arg492 to Cys492 transition, which might confer an additional putative palmitoylation site in the carboxy-terminal segment of the alpha 1a-adrenoceptor. In the present study, we compared their pharmacological properties and examined whether this alpha 1a-adrenoceptor polymorphism is associated with benign prostatic hypertrophy (BPH). 2. The frequency of alpha 1a-adrenoceptor polymorphism was not differently distributed between patients with benign prostatic hypertrophy (BPH) and normal subjects in Japan; thus, the relative frequencies of the C and T alleles were 0.90 : 0.10 in normal male subjects (n = 45) and 0.87 : 0.13 in BPH patients (n = 222), respectively. However, the frequency distribution of this polymorphism was significantly different between the Japanese and U.S. populations; thus, C and T alleles were 0.34 and 0.66 in U.S. populations. 3. Utilizing Chinese hamster ovary (CHO) cells stably expressing the two polymorphic alpha 1a-adrenoceptors (Arg492 and Cys492), we compared their binding affinity and signal transduction. Radioligand binding studies with 2-[beta-(4-hydroxy-3[125I]-iodophenyl) ethylamino-methyl]tetralone ([125I]-HEAT) showed no marked difference in the antagonist or agonist binding affinities between the two receptors. Also, both receptors were found to be coupled to the calcium signaling, and the concentration-cytosolic Ca2+ concentrations ([Ca2+]i) response relationships for noradrenaline were similar for the two polymorphic receptors. Furthermore, the receptor-mediated [Ca2+]i response was markedly desensitized after a 2 h exposure of phenylephrine (10 microM), and the extent of the desensitization was not significantly different between the two receptors. 4. In summary, the results showed that the two alpha 1a-adrenoceptors generated by genetic polymorphism have similar pharmacological characteristics, and the receptor-mediated [Ca2+]i response can be desensitized in a similar manner. The study did not provide any evidence to support the hypothesis that alpha 1a-adrenoceptor gene polymorphism is associated with BPH.

Aged↗
PubMed1996

Quantification and distribution of alpha 1-adrenoceptor subtype mRNAs in human prostate: comparison of benign hypertrophied tissue and non-hypertrophied tissue.

1. There are at least three alpha 1-adrenoceptor subtypes, alpha 1a, alpha 1b and alpha 1d, in human tissues. Using an RNase protection assay, we have now determined the amount of each subtype mRNA in human prostatic tissue, for both benign prostatic hypertrophy (BPH) and non-BPH. In all tissue samples examined, the predominant subtype mRNA was alpha 1a. The total abundance of alpha 1-adrenoceptor mRNA in BPH samples was over six times that in non-BPH samples. This increase was mostly accounted for by alpha 1a, which was almost nine times as abundant in BPH samples as in non-BPH samples. The abundance of alpha 1b was almost the same between BPH and non-BPH samples, and the abundance of alpha 1d in BPH samples was about three times that in non-BPH samples. The ratio of the numbers of the subtype mRNAs, alpha 1a: alpha 1b: alpha 1d, was 85:1:14 in BPH samples and 63:6:31 in non-BPH samples. 2. In situ hybridization studies showed no significant differences in the tissue localization of alpha 1-adrenoceptor subtype mRNAs between BPH and non-BPH samples. alpha 1a and alpha 1d were clearly detected in the interstitium of the prostate, where alpha 1a was stained more intensely than alpha 1d, and the positive sites were primarily smooth muscle cells. In contrast, alpha 1b staining was very faint. 3. This increase in mRNA abundance may be directly related to the contraction of prostatic tissue that leads to obstruction of the urinary tract in BPH patients. Specifically, our data suggest that increased expression of the alpha 1a subtype may be primarily responsible for the contraction of the prostate.

Aged↗