Akira Karasawa

Modulation of erythropoietin production by selective adenosine agonists and antagonists in normal and anemic rats.

Hypoxia or anemia is the fundamental stimulus for erythropoietin (EPO) production. Recent in vitro studies suggest that EPO secretion in response to hypoxia is regulated by adenosine in the kidney. In order to examine the in vivo effect of adenosine on EPO production, we determined the effects of adenosine receptor agonists and antagonists on serum EPO concentration in normal and anemic rats. In normal rats, intravenous injection of adenosine agonists (NECA, CHA and CGS-21680) dose-dependently stimulated EPO production. Pretreatment with KW-3902, an adenosine A1 antagonist with modest A2b antagonistic action, or KF17837, an adenosine A2a antagonist, inhibited the NECA (0.1 mg/kg, i.v.)-stimulated EPO production. Anemic hypoxia, induced by 2% (v/w body weight) blood withdrawal, increased serum EPO concentration from 38 +/- 2 to 352 +/- 76 mU/ml, with the increased serum adenosine concentration in the renal vein. KF17837 (0.1 mg/kg, i.v.), but not KW-3902 (0.1 mg/kg, i.v.), inhibited the anemic hypoxia-induced increase in EPO production. The present findings support the notion that adenosine mediates the EPO production in response to hypoxia in the kidney.

KF24345, an adenosine uptake inhibitor, ameliorates the severity and mortality of lethal acute pancreatitis via endogenous adenosine in mice.

Adenosine protects against cellular damage and dysfunction under several adverse conditions including inflammation and ischemia. In this study, we examined the effects of 3-[1-(6,7-diethoxy-2-morpholinoquinazolin-4-yl)piperidin-4-yl]-1,6-dimethyl-2,4(1H,3H)-quinazolinedione hydrochloride (KF24345), an adenosine uptake inhibitor, on experimental acute pancreatitis induced by choline-deficient and ethionine-supplemented diet in mice. KF24345, administered with the diet onset and every 24 h thereafter, prevented hyperamylasemia, acinar cell injury and serum tumor necrosis factor-alpha elevation and ultimately decreased mortality. Therapeutic treatment with KF24345, which started 32 h after the diet onset, also decreased mortality. The beneficial effect of KF24345 on mortality was abolished by the pretreatment with 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385), a selective adenosine A(2A) receptor antagonist. An intravenous injection of KF24345 at 48 h after the diet onset increased plasma adenosine concentrations in mice with acute pancreatitis. These results suggest that KF24345 shows anti-pancreatitis effects via endogenous adenosine and adenosine A(2A) receptors. The adenosine uptake inhibition could be a new therapeutic approach for acute pancreatitis.

Roles of mast cells and sensory nerves in cutaneous vascular hyperpermeability and scratching behavior induced by poly-L-arginine in rats.

We investigated whether the polycation poly-L-arginine elicited cutaneous vascular hyperpermeability and scratching behavior and, if so, whether these responses involved mast cells and sensory nerves in rats. Intradermal injections of poly-L-arginine induced vascular hyperpermeability and scratching behavior. Combined treatment with chlorpheniramine and methysergide almost completely suppressed the poly-L-arginine (50 microg/site)-induced plasma leakage. Capsaicin desensitization and the tachykinin NK(1) receptor antagonist LY303870, (R)-1-[N-(2-methoxybenzyl)acetylamino]-3-(1H-indol-3-yl)-2-[N-(2-(4-(piperidin-1-yl)piperidin-1-yl)acetyl)amino]propane, partially inhibited the leakage. In mast cell-deficient rats, poly-L-arginine only minimally induced plasma leakage. On the other hand, capsaicin desensitization and LY303870, but not chlorpheniramine or methysergide, suppressed the poly-L-arginine (200 microg/site)-induced scratching. Moreover, poly-L-arginine elicited the scratching even in mast cell-deficient rats. These results suggest that substance P is at least partly involved in both the cutaneous plasma leakage and the scratching behavior induced by poly-L-arginine. Moreover, mast cell-derived amines are suggested to be involved in the plasma extravasation but scarcely, if any, in the scratching behavior.

Effect of Olopatadine and Other Histamine H1 Receptor Antagonists on the Skin Inflammation Induced by Repeated Topical Application of Oxazolone in Mice

Histamine H1 receptor antagonists have long been prescribed for atopic dermatitis as an adjuvant therapy with topical therapy by local applied steroids. Olopatadine is one of the second-generation histamine H1 receptor antagonists that are treated for allergic disorders. We investigated that the effect of olopatadine on oxazolone-induced chronic contact hypersensitivity response in BALB/c mice compared with other histamine H1 receptor antagonists loratadine, cetirizine and fexofenadine. The chronic contact hypersensitivity induced by repeated application of oxazolone was treated with olopatadine and other histamine H1 receptor antagonists at the effective doses on histamine-induced paw edema in mice. The effects of these drugs in the oxazolone-induced model were quantified by measurements of ear swelling, and levels of cytokines in the lesioned ear. Olopatadine significantly inhibited the ear swelling and the increased production of IL-4, IL-1β, IL-6, GM-CSF and NGF in the lesioned ear. On the other hand, the other histamine H1 receptor antagonists did not significantly suppress the increase in ear thickness. Moreover, they did not affect the production of cytokines in the lesioned ear. These results indicate that olopatadine appears to exert additional biological effects besides its blockade of the histamine H1 receptor.

Non-prostanoid thromboxane A2 receptor antagonists with a dibenzoxepin ring system. 1

A series of 11-[[2-[(arylsulfonyl)amino]ethyl]thio]-6,11- dihydrodibenz[b,e]oxepin-2-carboxylic acids and related derivatives were synthesized. The compounds were tested for their antagonizing effects on guinea pig platelet TXA2/PGH2 receptors. Structure-activity relationships are discussed. (+/-)-11-[[2-[(Styrylsulfonyl)amino]ethyl]-thio]-6,11- dihydrodibenz[b,e]oxepin-2-carboxylic acid (41) and (+/-)-11-[[2-[(phenylsulfonyl)amino]ethyl]thio]-6,11- dihydrodibenz[b,e]thiepin-2-carboxylic acid (4af) were the most promising compounds with K(i) values of 6.5 +/- 0.29 and 3.7 +/- 0.31 nM, respectively, for the TXA2/PGH2 receptor. These compounds also significantly inhibited U-46619-induced guinea pig platelet aggregation ex vivo (10 mg/kg po). Compound 41 was resolved into its optically active form. The (-)-isomer was 60-fold more potent than the (+)-isomer in the TXA2/PGH2 receptor binding assay. Some compounds tested in this study showed both TXA2/PGH2 receptor antagonizing and TXA2 synthase inhibitory effects.

Butanol extracts from myelin fragments. VI. Lack of specificity of 5-hydroxytryptamine binding to recombinate fraction with sulphatides and phosphatidylinositol

Abstract To clarify the participation of sulphatides and PI for the binding of 5-HT to myelin butanol extracts, binding experiments of 14C·5-HT to the recombinate fraction of these two acidic lipids were performed. The recombinate fraction was incubated with 5 × 10−7 M of 14C·5-HT, and elution profile of 14C·5-HT, sulphatides and PI was examined by Sephadex LH20 column chromatography. Three components were eluted with CM 4:1 and elution areas of those relatively corresponded to each other. On the other hand, when 3 fold excess volumes of solvents were used 14C·5-HT was also eluted with CM 4:1 but the clear difference was observed in the elution pattern of sulphatides and PI. In the myelin butanol extracts, we have reported that the 5-HT binding macromolecules present in the myelin extracts would be composed of saturable and non-saturable components, and ACh, DA and tryptamine specifically inhibited the saturable 5-HT binding. Therefore, on the recombinate system of two acidic lipids the specificity of 14C·5-HT binding was investigated by displacement experiments. The results indicated that only ACh slightly inhibited the 14C·5-HT binding but 5-HT, DA and tryptamine had no effect. All these observations suggest that other component(s) besides sulphatides and PI may be implicated in the binding of 5-HT to myelin butanol extracts.

Treatment with an adenosine uptake inhibitor attenuates glomerulonephritis in mice

This study evaluated the effects of KF24345 (3-[1-(6,7-diethoxy-2-morpholinoquinazolin-4-yl)piperidin-4-yl]-1,6-dimethyl-2,4(1H,3H)-quinazolinedione hydrochloride), a novel adenosine uptake inhibitor, on experimental glomerulonephritis induced in mice by two intravenous injections of rabbit anti-mouse glomerular basement membrane antiserum. Mice with glomerulonephritis showed continuous proteinuria and the histological evaluation revealed glomerular and tubular damage at 7 weeks after the first antiserum injection. KF24345 as well as prednisolone and cyclophosphamide significantly inhibited proteinuria and glomerular damage when it was orally administered once a day from 2 to 7 weeks. Prednisolone elevated plasma bilirubin and glutamic-pyruvic transaminase levels, and cyclophosphamide decreased erythrocytes. Moreover, both prednisolone and cyclophosphamide decreased spleen and thymus weights. KF24345 did not show this kind of side effects. These results demonstrate that KF24345 ameliorates glomerulonephritis with minimal side effects in mice, suggesting that the adenosine uptake inhibitor may be useful for the treatment of glomerulonephritis.

Modulation of oligosaccharide structure of a pro-urokinase derivative (pro-UKΔGS1) by changing culture conditions of a lymphoblastoid cell line Namalwa KJM-1 adapted to serum-free medium

Pro-UKΔGS1 was designed as a long-life and thrombin-resistant derivative of pro-urokinase (pro-UK) by deleting the growth factor domain of pro-UK and introducing a glycosylation site near the thrombin cleaving site for thrombin-resistance using site-directed mutagenesis. An expression plasmid for pro-UKDGS1, pIH1UKΔGS1SEd1–5 was constructed and introduced into Namalwa KJM-1, a lymphoblastoid cell line adapted to serum-free medium, and cells resistant to G418 and Methotrexate (MTX) were obtained. Amongst them, the highest pro-UKΔGS1 producer (resistant to 200 nM of MTX), clone 2–9, was selected and used for further studies.Under the conventional conditions, i.e. at 37°C in serum-free ITPSGF medium (based on RPMI-1640 medium), the oligosaccharide structure of pro-UKΔGS1 produced by clone 2–9 mainly consisted of fucose (Fuc)-containing biantennary complex-type oligosaccharide. Addition of dexamethasone (Dex), changed the carbohydrate contents in the media, and a shift down of incubation temperature caused a change in oligosaccharide structure of pro-UKΔGS1 from mainly Fuc-containing biantennary to mainly Fuc-containing tri-and tetraantennary complex-type oligosaccharide. The modulated pro-UKΔGS1 showed superiorin vivo activity for a canine femoral thrombosis formed by inserting a copper-coil.

Adenosine uptake inhibition ameliorates cerulein-induced acute pancreatitis in mice

Introduction and Aims Adenosine shows protective effects against cellular damage and dysfunction under several adverse conditions such as inflammation and ischemia. In the current study, we examined the effects of 3-[1-(6,7-diethoxy-2-morpholinoquinazolin-4-yl)piperidin-4-yl]-1,6-dimethyl-2,4(1 H,3 H)-quinazolinedione hydrochloride (KF24345), an adenosine uptake inhibitor, on cerulein-induced acute pancreatitis in mice to investigate whether inhibition of adenosine uptake could ameliorate the severity of acute pancreatitis. Methodology Acute pancreatitis was induced in mice with six intraperitoneal injections of cerulein (50 &mgr;g/kg each) at hourly intervals. Results The cerulein injection increased activities of serum amylase and lipase and caused pathologic changes such as interstitial edema, polymorphonuclear cell infiltration, and acinar cell necrosis in the pancreas. KF24345 (10 mg/kg p.o.) ameliorated all these changes observed in mice with acute pancreatitis, and the suppressing effect of KF24345 on the elevation in serum amylase activity was abolished by the treatment with 8-(p-sulfophenyl)theophylline, an adenosine receptor antagonist. In addition, 2-(aminocarbonyl)-N-(4-amino-2,6-dichlorophenyl)-4-[5,5-bis-(4-fluorophenyl)pentyl]-1-piperazineacetamide (R75231) and dipyridamole, other adenosine uptake inhibitors, also decreased the elevated serum amylase activity. Conclusions These are the first demonstrations that the adenosine uptake inhibitors ameliorate cerulein-induced acute pancreatitis in mice, and these data suggest that adenosine uptake inhibition could ameliorate the severity of acute pancreatitis in vivo.

Temperature-sensitive high affinity [3H]tryptamine binding sites in rat brain

The influence of prior incubation on [3H]tryptamine binding was investigated in rat brain synaptic plasma membranes. A 55 min preincubation of the membranes at 37 degrees C induced an approx. 2.4-fold increase in the specific binding of [3H]ligand to the subsequently washed preparations and this phenomenon was quite temperature-dependent. On the other hand, the proportion of nonspecific binding sites was significantly decreased by 70% of the original sites within 20 min of the start of preincubation. Pargyline, ascorbic acid, EGTA, metal ions (Ca2+, Mg2+, Na+) and guanine nucleotides, included in the preincubation buffer, were all inactive on the stimulation of [3H]tryptamine binding, while the pretreatment of membranes with glutaraldehyde antagonized the augmentation of this binding. Furthermore, it was revealed that the Scatchard plot of the [3H]tryptamine binding preincubated at 0 degree C conformed to a straight line (KD = 33.1 nM, Bmax = 543 fmoles/mg protein), whereas a curvilinear Scatchard plot was obtained at 37 degrees C preincubation. Nonlinear regression analysis of the latter resulted in apparent KD (nM) & Bmax (fmoles/mg protein) values of 0.45 & 102.7 and 33.7 & 603.4 for the high and low affinity sites, respectively. All these observations lead to the inference that the preincubation-induced increase in [3H]tryptamine binding (i.e., nearly high affinity proportion of sites) may occur as a result of temperature-sensitive interconvertible conformational changes.