Prostaglandin F2α FP receptor inhibitor reduces demyelination and motor dysfunction in a cuprizone-induced multiple sclerosis mouse model.

Previously, we have demonstrated that prostamide/PGF synthase, which catalyzes the reduction of prostaglandin (PG) H2 to PGF2α, is constitutively expressed in myelin sheaths and cultured oligodendrocytes, suggesting that PGF2α has functional significance in myelin-forming oligodendrocytes. To investigate the effects of PGF2α/FP receptor signaling on demyelination, we administrated FP receptor agonist and antagonist to cuprizone-exposed mice, a model of multiple sclerosis. Mice were fed a diet containing 0.2% cuprizone for 5 weeks, which induces severe demyelination, glial activation, proinflammatory cytokine expression, and motor dysfunction. Administration of the FP receptor antagonist AL-8810 attenuated cuprizone-induced demyelination, glial activation, and TNFα expression in the corpus callosum, and also improved the motor function. These data suggest that during cuprizone-induced demyelination, PGF2α/FP receptor signaling contributes to glial activation, neuroinflammation, and demyelination, resulting in motor dysfunction. Thus, FP receptor inhibition may be a useful symptomatic treatment in multiple sclerosis.

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 6.1 and 6.8, respectively. Using these indicators expressed in cultured cells, we measured and visualized pH changes in the cytosol and nucleus. Furthermore, by tethering the indicator to a membrane protein (the alpha(1B) adrenergic receptor), we visualized the pH in the vicinity of the protein during internalization caused by endocytosis after agonist stimulation. These novel probes will serve as a useful tool for monitoring pH in the defined organelle and in the microenvironment of a target protein, to analyze cellular function.

Expression of a green fluorescent protein variant in mouse oocytes by injection of RNA with an added long poly(A) tail.

In oocytes, cytoplasmic 3' polyadenylation regulates translational activation of dormant mRNA during meiotic maturation. Thus exogenous proteins are hardly expressed after injection of conventional RNA. To circumvent this, we synthesized a long polyadenylated (approximately 250 A) tail to encode RNA with an enhanced yellow fluorescent protein targeted to mitochondria (EYFP-mito), and injected it into mouse oocytes at the germinal vesicle (GV) stage. From this transcript, EYFP-mito was clearly expressed in approximately 80% of oocytes, while scarce expression from a transcript with only 30 A was observed. In strongly expressing oocytes, fluorescence was detected within 1-3 h after RNA injection, increased linearly up to 12 h, and reached a maximum at 12-15 h. The distribution of EYFP-mito matched the staining of mitochondria in these oocytes. About 80% of these oocytes underwent GV breakdown and 60% matured in vitro, comparable to non-expressing or non-RNA-injected oocytes. Some of the oocytes which strongly expressed EYFP-mito remained at the GV stage. Thus, the expression was not always accompanied by meiotic maturation, nor did it suppress the maturation process. Mature oocytes expressing EYFP-mito possessed normal fertilizability associated with intracellular Ca(2+) oscillations, and developed into 2-cell embryos. Thus, polyadenylated RNA is a useful tool applicable to the expression of EYFP-fused functional proteins or of indicator protein probes for studies of mammalian fertilization.

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. To examine the molecular mechanism for the subtype-specific differences in subcellular localization, we conducted a search for novel proteins that interact with the alpha1B-AR, specifically focusing on the carboxyl-terminal cytoplasmic domain. Using interaction cloning and biochemical techniques, we demonstrate that gC1q-R interacts with alpha1B-AR in vitro and in vivo through the specific site, and that in cells which co-express alpha1B-AR and gC1q-R, the subcellular localization of alpha1B-AR is markedly altered and its expression is down-regulated. These results suggest that gC1q-R plays a role in the regulation of the subcellular localization as well as the function of alpha1B-ARs.

Differential subcellular localization of DNA-dependent protein kinase components Ku and DNA-PKcs during mitosis.

The Ku protein is a complex of two subunits, Ku70 and Ku80. Ku plays an important role in DNA-PKcs-dependent double-strand break repair and V(D)J recombination, and in growth regulation, which is DNA-PKcs-independent. We studied the expression and the subcellular localization of Ku and DNA-PKcs throughout the cell cycle in several established human cell lines. Using immunofluorescence analysis and confocal laser scanning microscopy, we detected Ku70 and Ku80 in the nuclei in interphase cells. In mitotic cells (1) most of Ku protein was found diffused in the cytoplasm, (2) a fraction was detected at the periphery of condensed chromosomes, (3) no Ku protein was present in the chromosome interior. Association of Ku with isolated chromosomes was also observed. On the other hand, DNA-PKcs was detected in the nucleus in interphase cells and not at the periphery of condensed chromosomes during mitosis. Using indirect immunoprecipitation, we found that throughout the cell cycle, Ku70 and Ku80 were present as heterodimers, some in complex with DNA-PKcs. Our findings suggest that the localization of Ku at the periphery of metaphase chromosomes might be imperative for a novel function of Ku in the G(2)/M phase, which does not require DNA-PKcs.

Regulatory roles of complexins in neurotransmitter release from mature presynaptic nerve terminals.

Complexins are presynaptic proteins whose functional roles in synaptic transmission are still unclear. In cultured rat hippocampal neurons, complexins are distributed throughout the cell bodies, dendrites and axons, whereas synaptotagmin I and synaptobrevin/VAMP-2, essential proteins for neurotransmitter release, accumulated in the synaptic-releasing sites as early as 1 week in culture. With a maturation of synapses in vitro, complexins also accumulated in the synaptic release sites and co-localized with synaptotagmin I and synaptobrevin/VAMP-2 after 3-4 weeks in culture. Complexins I and II were expressed in more than 90 and 70% of the cultured neurons, respectively; however, they were largely distributed in different populations of synaptic terminals. In the developing rat brain, complexins were distributed in neuronal cell bodies in the early stage of postnatal development, but gradually accumulated in the synapse-enriched regions with development. In mature presynaptic neurons of Aplysia buccal ganglia, injection of anticomplexin II antibody caused a stimulation of neurotransmitter release. Injection of recombinant complexin II and alphaSNAP caused depression and facilitation of neurotransmitter release from nerve terminals, respectively. The effect of complexin was reversed by a subsequent injection of recombinant alphaSNAP, and vice versa. These results suggest that complexins are not essential but have some regulatory roles in neurotransmitter release from presynaptic terminals of mature neurons.

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.

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.

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.

Subtype-specific differences in subcellular localization and chlorethylclonidine inactivation of alpha1-adrenoceptors.

Chlorethylclonidine (CEC) inactivation has been used as one criterion to subclassify the alpha1-adrenoceptors (AR); however, the extent of CEC inactivation can vary depending on the CEC treatment. By constructing the FLAG-tagged (N-terminus) and green fluorescent protein (GFP)-fused (C-terminus) alpha1-ARs, we have determined the relationship between CEC sensitivity and the cellular localization of alpha1-AR subtypes using COS-7 cells. In GFP-expressing cells, flow cytometry analysis with anti-FLAG N-terminus antibody detected strong fluorescent signals in most of alpha1B-AR-expressing cells, but low signals in alpha1A-AR-expressing cells. Further examination with confocal microscopy showed that fluorescent signals densely localized intra-cellularly in alpha1A-AR-expressing cells, while most of alpha1B-AR localized on the cell surface. Furthermore, radioligand binding studies with [125I]HEAT showed that CEC (10 microM) treatment of intact cells inactivated approximately 30-40% of alpha1A-AR and >90% of alpha1B-AR, while the CEC treatment of membrane preparations resulted in >80% decrease in the alpha1A-AR density and >90% of alpha1B-AR density, respectively. The results showed that the hydrophilic alkylating agent CEC inactivated only alpha1-AR on the cell surface irrespective of its subtype, and that the subtype-specific sorting is a major determinant for CEC inactivation of alpha1-AR. Subtype-specific cellular localization suggests a new class of functional properties that may explain the signal and functional diversity of homologous alpha1-AR (as well as other G protein-coupled receptors) subtypes.

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.

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.

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.

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.

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.

Acute antiarrhythmic effects of intravenously administered amiodarone on canine ventricular arrhythmia.

We investigated antiarrhythmic effects of intravenously (i.v.) administered amiodarone using four canine ventricular arrhythmia models. Bolus injections of amiodarone 3 mg/kg suppressed epinephrine (EPI)-induced arrhythmia and 5-mg/kg bolus injections of amiodarone suppressed digitalis- and two-stage coronary ligation-induced arrhythmia models, but the antiarrhythmic effects did not correlate with the amiodarone plasma concentrations. The infusion of amiodarone 6.67 mg/kg/h did not prolong the QTc interval or produce antiarrhythmic effects in coronary ligation and reperfusion experiments. Amiodarone significantly decreased the mean blood pressure (MAP), and this effect lasted throughout the observation period. The results indicate that the antiarrhythmic effects of intravenously administered amiodarone may not be due to its class III action, but to other actions, such as class I, II and IV actions.

[Flow cytometry and laser scanning confocal microscopy analysis of the receptor distribution].

To study the physiological regulation of the receptor protein, a fluorescent probe and detection system for alpha 1B-adrenergic receptors have been developed. By using the anti-peptide antibody developed against the alpha 1B-adrenergic receptor NH2-terminus, we have examined the agonist-regulated alpha 1B-adrenergic receptor redistribution in desensitized cells. Flow cytometry analysis showed that anti-peptide antibody against alpha 1B-adrenergic receptor specifically identifies the receptor in CHO cells, COS-7 cells that were transfected with alpha 1B-adrenergic receptor cDNA and rat hepatocytes. Using a fluoro-labeled receptor ligand, BODIPY FL-prazosin, as a probe, cell surface alpha 1-adrenergic receptor subtypes can be detected by flow cytometry. Laser scanning confocal microscopy visualized the agonist-regulated redistribution process of alpha 1B-adrenergic receptor in living cells; thus, following phenylephrine (10(-6) M) stimulation, receptor antigen at the cell surface rapidly internalized and clustered together in a cell within 30 min. The results showed that the antibody and fluoro-labeled ligand are valuable tools for studying the localization and functional role of the alpha 1-adrenergic receptor subtype.

Plasma methylguanidine and creatinine concentrations in cats with experimentally induced acute renal failure.

Plasma methylguanidine (MG) and creatinine (CRN) concentrations were measured in 11 cats with experimentally induced acute renal failure by a two-stage surgical procedure. According to the progression of renal failure, both plasma MG and CRN levels increased. A significant positive correlation (y = 0.187X - 0.379, lambda = 0.9176, P < 0.001) was found between plasma MG and CRN levels. These results suggested that the increase in plasma MG level was an available indicator for uremic status in cats.

Effects of the new class III antiarrhythmic drug MS-551 and d-sotalol on canine coronary ligation-reperfusion ventricular arrhythmias.

The antiarrhythmic effects of a new class III antiarrhythmic agent, MS-551 [1,3-dimethyl-6-(2-[N-(2-hydroxyethyl)-3-(4-nitrophenyl) propylamino]ethylamino)-2,4(1H,3H)-pyrimidinedione hydrochloride], were investigated using canine coronary ligation-reperfusion arrhythmia models under slow and fast heart rate conditions and compared with those of d-sotalol. Slow and fast heart rate conditions were produced by using different anesthetics; i.e., halothane anesthesia for the slow heart rate condition and pentobarbital Na anesthesia for the fast heart rate condition. MS-551 prolonged QTc and suppressed the occurrence of fatal ventricular fibrillation (VF) on coronary reperfusion under either halothane or pentobarbital anesthesia. However, it also showed proarrhythmic effects, i.e., induction of torsades de pointes-like arrhythmia in 1 of 6 halothane anesthetized dogs before coronary ligation. d-Sotalol did not suppress the reperfusion VF in halothane anesthetized animals, nor did it show proarrhythmic effects. However, in the pentobarbital anesthetized animals, d-sotalol suppressed reperfusion VF accompanied by proarrhythmic effects in 1 of 7 dogs. d-Sotalol did not show reverse rate dependent QT prolongation. These results indicate that although both these class III drugs have similar electrophysiological properties, such as QTc prolongation, they have different antiarrhythmic effects. Also, antifibrillatory effects of class III drugs on coronary reperfusion apparently can not be explained solely by their QT prolonging effects.

Effects of a new forskolin derivative, NKH477, on canine ventricular arrhythmia models.

Using two-stage coronary ligation-, digitalis- and epinephrine-induced canine ventricular arrhythmia models, we examined whether a new positive inotropic agent, NKH477, 6-(3-dimethylaminopropionyl)forskolin hydrochloride, a water-soluble derivative of forskolin, had deleterious effects on arrhythmias. NKH477 increased heart rate (HR) and decreased blood pressure (BP) in dogs with all the arrhythmia models. Unexpectedly, NKH477 suppressed digitalis- and epinephrine-induced arrhythmias, but did not suppress two-stage coronary ligation arrhythmia or aggravate it. These results indicate that NKH477, unlike other new positive inotropic agents such as amrinone, milrinone, sulmazole and vesnarinone, did not worsen these arrhythmias; thus, NKH477 may be a useful positive inotropic agent with little arrhythmogenic effect.