Role of daunorubicin in the induction therapy for adult acute myeloid leukemia.

PURPOSE: To evaluate the relationship of total-dose of daunorubicin (DNR) to the induction therapy and treatment outcome, we have administered individualized doses of DNR during induction treatment to patients with acute myelogenous leukemia (AML). PATIENTS AND METHODS: Ninety-two previously untreated adult patients with AML who entered our hospital were analyzed for the dose of DNR required to achieve complete remission (CR), the CR rate, disease-free survival (DFS), and overall survival (OS). Induction therapy consisted of DNR 40 mg/m2 daily intravenously from day 1 until the marrow was hypoplastic, cytarabine (Ara-C), prednisolone (PRD), and/or 6-thioguanine (6-TG). RESULTS: Eighty-three of 92 patients with adult AML were assessable for this study. Sixty-three (76%) patients achieved CR. Fifty-two of 63 CR patients achieved the CR in the first course of induction therapy, and 11 patients required the second course of induction therapy. The 5-year and 10-year DFS rates were 31.2% and 5-year and 10-year OS rates were 45.1% and 42.3%, respectively. The median total dose of DNR in the induction therapy was 280 mg/m2 (120 to 480 mg/m2). DNR dose did not influence the response to therapy and was not influenced by the initial WBC count or French-American-British (FAB) system classification. CONCLUSION: These results indicated that when the dose was linked to observed tumor response, the optimal dose of DNR in the induction therapy was approximately 280 mg/m2 (40 mg/m2 for 7 days), which is greater than the conventional dose of 40 to 60 mg/m2 for 3 days.

Approaches to the development of novel antihypertensive drugs: crucial role of the renal kallikrein-kinin system.

The renal kallikrein-kinin system has been shown to be important in the development of hypertension, but its precise role remains to be determined. The recent use of mutant rats that are deficient in plasma kininogens and hence incapable of generating kinin in the renal tubules has facilitated elucidation of the involvement of kinin in hypertension. In this article, Masataka Majima and Makoto Katori provide evidence that the renal kallikrein-kinin system plays a crucial role in the development of hypertension and discuss a novel rationale for developing effective new antihypertensive drugs.

Heavy chain ferritin acts as an antiapoptotic gene that protects livers from ischemia reperfusion injury.

Heme oxygenase-1 (HO-1) is induced under a variety of pro-oxidant conditions such as those associated with ischemia-reperfusion injury (IRI) of transplanted organs. HO-1 cleaves the heme porphyrin ring releasing Fe2+, which induces the expression of the Fe2+ sequestering protein ferritin. By limiting the ability of Fe2+ to participate in the generation of free radicals through the Fenton reaction, ferritin acts as an anti-oxidant. We have previously shown that HO-1 protects transplanted organs from IRI. We have linked this protective effect with the anti-apoptotic action of HO-1. Whether the iron-binding properties of ferritin contributed to the protective effect of HO-1 was not clear. We now report that recombinant adenovirus mediated overexpression of the ferritin heavy chain (H-ferritin) gene protects rat livers from IRI and prevents hepatocellular damage upon transplantation into syngeneic recipients. The protective effect of H-ferritin is associated with the inhibition of endothelial cell and hepatocyte apoptosis in vivo. H-ferritin protects cultured endothelial cells from apoptosis induced by a variety of stimuli. These findings unveil the anti-apoptotic function of H-ferritin and suggest that H-ferritin can be used in a therapeutic manner to prevent liver IRI and thus maximize the organ donor pool used for transplantation.

Inhibition site of dexamethasone on extravasation of polymorphonuclear leukocytes in the hamster cheek pouch microcirculation.

A video system was used to investigate the inhibitory effect(s) of the glucocorticoid dexamethasone (DEX) on polymorphonuclear leukocyte (PMN) extravasation in the microcirculation of hamster cheek pouch. DEX was given intraperitoneally at 0.1 or 0.3 mg/kg body weight 2 h before induction of extravasation by topical application of leukotriene B4 (LTB4) or formyl-methionyl-leucyl-phenylalanine (fMLP) on the microvasculature. The number, time course, and behavior of PMNs were examined in the following five steps of extravasation: (1) rolling on the venular endothelium, (2) adhesion on the endothelium, (3) passage between the endothelial cells, (4) staying in the venular wall, and (5) migration from the venular wall into the interstitial space. In either the presence or absence of DEX, topical application of LTB4 (15 pmol/50 microliters) or fMLP (10 nmol/50 microliters) caused an increase in the number of PMNs that adhered to the venules. Thus, DEX did not inhibit the adhesion of PMNs on the endothelial cells. The adhered PMNs induced by these chemoattractants became gradually smaller, finally disappearing in the vascular lumen, as observed on the monitor screen. The whole process took about 10 min. This passage of PMNs between the endothelial cells was also not inhibited by DEX, as over 90% of the adhered PMNs passed through the endothelial cells and advanced to the next step of extravasation in the presence or absence of DEX. When these chemoattractants were applied to DEX-untreated animals, the PMNs that passed through the endothelial cells stayed for about 30 min in the venular wall. Thereafter, PMNs migrated into the interstitial space. The numbers of PMNs in the interstitial space were counted at 30, 60, and 90 min after the application of chemoattractants. In the DEX-untreated animals, PMNs in the interstitial space started to appear by 30 min, and thereafter the number further increased. However, in the DEX-treated animals, the number of PMNs at 60 and 90 min was significantly suppressed. Since DEX may inhibit the synthesis and/or release of a proteinase(s) in the PMN granules, which would normally degrade the basement membrane, this inhibition may be due to the inability of PMNs to penetrate the basement membrane.

Detection of leukotriene B4 in cardiac tissue and its role in infarct extension through leucocyte migration.

The main left coronary artery of rats was ligated near its origin under light ether anaesthesia and the infarction observed for 48 h. The ischaemic area was determined after an intravenous injection of pontamine sky blue dye 1 h before induction of cardiac arrest with potassium chloride. The unstained area (true ischaemic area) decreased with time to 27.1% of the left ventricle at 48 h, whereas the intensely stained area between the normal and the true ischaemic areas increased with time, suggesting that blood was flowing to the border from the normal surrounding tissue. The infarcted area, identified by its lack of triphenyltetrazolium chloride staining, became evident after 3 h and stabilised at 12 h (42% of left ventricle). The polymorphonuclear leucocyte counts in the hearts, differentiated by staining of their chloroacetate esterase, increased gradually up to 5500 cells per section at 24 h. The leukotriene B4 concentration, determined by radioimmunoassay after freezing of the beating heart in liquid nitrogen, increased to eight times that of the sham operated hearts and peaked at 8 h (9.4(0.6) ng per heart, mean(SEM) n = 5) before the leucocyte counts reached their maximum. A single oral dose of a selective 5-lipoxygenase inhibitor (AA-861, 80 mg.kg-1, 1 h before ligation) lowered the leukotriene B4 concentration to that of the sham operated hearts and decreased the leucocyte count by 49.4% and 41.2% at 12 and 24 h respectively. The inhibitor also reduced the infarct size at 48 h by 34.4%. It was concluded that leukotriene B4, generated in ischaemic cardiac tissue, may increase infarct size through migration of polymorphonuclear leucocytes.

Hypertension induced by a nonpressor dose of angiotensin II in kininogen-deficient rats.

Brown Norway Katholiek rats with very low levels of plasma kininogens excreted a much smaller amount of kinin in the urine than normal rats of the same strain. The systolic blood pressure of 7-week-old kininogen-deficient rats (132 +/- 2 mmHg, n = 7) was not different from that of normal rats. Angiotensin II (Ang II) (20 micrograms/d SC) from 7 weeks of age for 2 weeks with a micro-osmotic pump caused significant increases in blood pressure (181 +/- 5 mm Hg, n = 7, 9 weeks old) in the deficient rats, although the same treatment induced no blood pressure increase in the normal rats. Also during this period, the deficient rats had significantly higher heart rates, tended to excrete less urinary sodium, and showed significantly higher sodium levels in serum, erythrocytes, and cerebrospinal fluid compared with the normal rats. Ang II increased urinary excretion of aldosterone in both deficient and normal rats (P < .05). Spironolactone treatment (50 mg/kg per day) for 7 days in deficient rats restored blood pressure and heart rate to normal levels and significantly reduced sodium levels in erythrocytes and cerebrospinal fluid. Subcutaneous infusion of bovine low-molecular-weight kininogen with an osmotic pump in Ang II-treated deficient rats induced significant reductions in blood pressure, heart rate, and erythrocyte sodium levels. By contrast, subcutaneous infusion of the bradykinin antagonist Hoe 140 in Ang II-treated normal rats induced a hypertensive response in parallel with significant increases in heart rate and erythrocyte sodium level. These results suggest that the lack of kinin generation observed in the kininogen-deficient rats may cause the hypertensive response during the administration of a nonpressor dose of Ang II mainly through sodium retention probably caused by aldosterone release.

High sensitivity to salt in kininogen-deficient brown Norway Katholiek rats.

Brown Norway Katholiek rats, which have very low levels of plasma kininogens, excreted a much smaller amount of kinin in the urine than normal rats of the same strain. The systolic blood pressure of 7-week-old kininogen-deficient rats fed low (0.3%) NaCl diets (131 +/- 4 mm Hg, n = 12) was not different from that in normal rats. Two percent NaCl diets given from 7 weeks of age for 4 weeks caused rapid increases in blood pressure (167 +/- 4 mm Hg, n = 12, 9 weeks old) in deficient rats, although the same diets induced no blood pressure increase in normal rats. Urinary excretion of active kallikrein and prokallikrein remained constant in both rat groups throughout NaCl loading. During this period, the deficient rats secreted less urine (9 weeks old, P < .05) and less urinary sodium (11 weeks old, P < .05). Serum levels of sodium in deficient rats were higher (P < .05) than in normal rats at 9 weeks of age. Intracellular concentrations of sodium in the erythrocytes of deficient rats were higher (P < .05) than in normal rats throughout NaCl loading. Subcutaneous infusion of bovine low molecular weight kininogen with an osmotic pump in NaCl-loaded deficient rats induced a reduction (P < .01) in blood pressure and increases (P < .05) in urine volume and urinary sodium and kinin levels. By contrast, subcutaneous infusion of the bradykinin antagonist Hoe 140 or of aprotinin in NaCl-loaded normal rats induced a hypertensive response. This antagonist treatment reduced urine volume and urinary sodium. These results indicate that the lack of kinin generation observed in the kininogen-deficient rats was related through sodium retention to the hypertensive response to NaCl loading.

Suppression of rat deoxycorticosterone-salt hypertension by kallikrein-kinin system.

Brown Norway kininogen-deficient rats had very low levels of plasma kininogens and lower levels of plasma prekallikrein, compared with those of normal rats of the same strain. Systolic blood pressure, determined by the tail-cuff method, of 5-week-old kininogen-deficient rats (106 +/- 0.4 mm Hg, n = 7) and the rate of systolic blood pressure increase with age were not different from those in normal rats. Weekly injections of deoxycorticosterone acetate (5 mg/kg s.c.) with 1% sodium chloride solution in drinking water after uninephrectomy at 7 weeks of age caused a gradual increase in the blood pressure of normal rats, reaching a plateau at 18 weeks of age, whereas that of deficient rats rose rapidly to 158 +/- 6 mm Hg 2 weeks after the start of treatment and continued to increase slightly, becoming significantly higher than normal rats at 8, 9, 10, 11, and 12 weeks of age (p less than 0.05 or 0.01). The levels of urinary prokallikrein and active kallikrein were slightly higher in deficient rats before deoxycorticosterone acetate-salt treatment but were not significantly increased after this treatment, whereas these levels in normal rats were increased 3.6- and 4.7-fold by this treatment. Urinary free kinin, collected from the ureter in untreated deficient rats, was below the detection limit. The plasma level of low molecular weight kininogen, the substrate of glandular kallikrein, was decreased in normal rats during the treatment. Continuous subcutaneous injection of aprotinin by an osmotic pump to normal rats induced significant increase in blood pressure. These results indicate that glandular kallikrein may play a suppressive role in deoxycorticosterone acetate-salt hypertension.

Prior induction of heat shock proteins by a nitric oxide donor attenuates cardiac ischemia/reperfusion injury in the rat.

BACKGROUND: Recent studies have demonstrated that nitric oxide (NO) releasers considerably increase heat shock proteins (HSPs) in the in vitro cell system, providing resistance to oxidant damage. This study was designed to examine the cellular responses of HSPs induced by prior administration of an NO releaser, FK409 (FK), in an in vivo transplantation model. METHODS: Lewis rats received either saline or FK solution intravenously administered at different time points before graft harvesting (10 micromol/kg) or for 15 min during reperfusion (0.66 micromol/kg/min). Tissue specimens were taken to determine HSP70 and heme oxygenase-1/HSP32 (HO-1) expression, and glutathione content. After 24-hr preservation with University of Wisconsin solution, heterotopic cardiac transplantations were performed, and graft survival was determined at 14 days. Tissue samples for end labeling of nuclear DNA fragments (TdT-mediated d-uridine triphosphate biotin nick end labeling; TUNEL) and propidium iodide staining were taken 15 min after reperfusion. RESULTS: The gene and protein expression of HSP70 after FK administration peaked at 12 min and 60-90 min, whereas those of HO-1 peaked at 6 min and 90 min, respectively. Then, representative cardiac grafts taken 60 min after FK treatment were examined for further assay. Localization of induced HSP70 and HO-1 molecules were observed in the myocardium and vascular endothelium, respectively. Prior treatment of FK was effective in preventing the reduction of tissue glutathione contents compared with control (P<0.05). Fewer TUNEL and propidium iodide-positive cells were also observed in the FK group (P<0.0005, vs. control). The graft survival rate was higher in the FK group (9/10 vs. 1/10 of control; P<0.001), whereas the groups either harvested 10 min after FK pretreatment or continuously infused for 15 min during reperfusion were inferior, similar to that of control. CONCLUSION: Prior induction of HSP70 and HO-1 with a relatively low dose of FK administration attenuates ischemia and reperfusion injury, which was due to antioxidant and antiapoptotic activities augmented by such stress proteins. Thus, NO releasers as a pharmacological maneuver may provide an innovative approach for the prevention of ischemia and reperfusion injury.

Immediate inhibitory effect of methylprednisolone suleptanate (U-67590A) on antigen-induced cutaneous and airway anaphylactic responses in guinea-pigs.

1. Inhibitory effects of water-soluble glucocorticoids administered intravenously were examined on skin and airway reactions caused by antigen challenge or chemical mediators in guinea-pigs. 2. Methylprednisolone suleptanate (U-67590A) which is an analogue of methylprednisolone, produced immediate inhibition of 3-h and 7-day homologous passive cutaneous anaphylaxis (PCA) reactions, but not of histamine- or bradykinin-induced cutaneous vascular permeability, when administered 10 min before antigen challenge. In contrast, methylprednisolone succinate (MP) or dexamethasone (DXM) administered 10 min before antigen challenge failed to show an immediate inhibitory effect on the PCA or mediator-induced reactions. When administered 1 to 5 h before antigen challenge, all the steroids used in this study reduced both PCA and mediator-induced reactions. 3. Pretreatment with cycloheximide almost completely abolished the late inhibition of 3-h PCA and histamine reactions produced by U-67590A or MP, but it did not affect the immediate inhibition of 3-h PCA produced by U-67590A. 4. U-67590A also demonstrated immediate inhibitory effects on antigen-induced bronchoconstriction in guinea-pigs actively sensitized with ovalbumin even when administered 10 min before antigen challenge, whereas MP and DXM failed to show the immediate inhibitory effect. When administered 3 h before antigen challenge, all the steroids used in this study reduced the response to antigen. 5. The late inhibitory effect of U-67590A administered 3 h before antigen challenge was almost completely abolished by treatment with cycloheximide or 17 alpha-methyltestosterone, whereas the immediate inhibition produced by U-67590A administered 10 min before challenge was not affected by this treatment. 6. U-67590A administered 10 min or 3 h before challenge did not affect the bronchoconstriction induced by histamine or leukotriene D4.7. Release of histamine from lung fragments of sensitized guinea-pigs in vitro was inhibited by U-67590A.8. The present experiments indicate that U-67590A demonstrated dual, immediate and late, inhibitory effects. The former are independent of protein synthesis and may be associated with non-genomic direct action on the mediator-releasing process without affecting mediator-induced reactions. The latter share common inhibitory actions with other glucocorticoids which are dependent on protein synthesis through gene expression.

Effects of an orally active non-peptide bradykinin B2 receptor antagonist, FR173657, on plasma exudation in rat carrageenin-induced pleurisy.

1. Effects of an orally active non-peptide (BK) B2 receptor antagonist, FR173657 ((E)-3-(6-acetamido-3-pyridyl)-N-[N-[2,4-dichloro-3-[(2-methyl-8-quinoli nyl) oxymethyl]phenyl]-N-methylaminocarbonylmethyl] acrylamide) on the plasma exudation in rat carrageenin-induced pleurisy were investigated. 2. Plasma exudation induced by intrapleural injection of bradykinin (BK, 3 nmol per rat) into male SD strain rats (SPF, 8 weeks old) were significantly inhibited by oral administration of novel B2 receptor antagonist FR173657 (3-30 mg kg-1, 1 h before BK injection) in a dose-dependent manner, whereas that induced by histamine was not. 3. The inhibitory effect of 30 mg kg-1 FR173657 persisted for more than 4 h. 4. Intrapleural injection of lambda-carrageenin (2% (w/v), 0.1 ml per rat) caused marked plasma exudation and accumulation of exudates from 1 h after carrageenin injection. The maximum plasma exudation response was observed 5 h after carrageenin. The oral administration of FR173657 to rats (30 mg kg-1, 1 h before carrageenin) significantly (by 50-77%) blunted the plasma exudation 1, 3, 5, and 7 h after carrageenin, causing a significant parallel reduction (by 42-57%) in the volume of exudates. 5. The anti-inflammatory effect of FR173657 on rat carrageenin-induced pleurisy was almost equipotent with that of the peptide B2 antagonist Hoe140 (1 mg kg-1, i.v.), a plasma kallikrein inhibitor, soy bean trypsin inhibitor (0.3 mg per rat, intrapleural injection) and bromelain (10 mg kg-1, i.v.). 6. In pleurisy induced by intrapleural injection of a histamine releaser, compound 48/80, the plasma exudation was observed only within 20 min after the injection. This plasma exudation was not affected by FR173657, although it was completely inhibited by a mixture of pyrilamine (5 mg kg-1, i.v.) and methysergide (3 mg kg-1, i.v.). 7. These results indicate that FR173657 is an orally active, promising anti-inflammatory agent for kinin-dependent inflammation.

Increased secretion of glandular-kallikrein in the bronchial washings induced by intravenous injection of leukotriene C4 in guinea-pigs.

1. Intravenous administration of leukotriene C4 (LTC4) and LTD4 (1-10 nmol kg-1) caused a dose-dependent increase in secretion of glandular-kallikrein in the bronchial washings of guinea-pigs, as measured by cleavage of a synthetic substrate and the formation of kinin. LTC4 was more potent than LTD4 and pilocarpine was much less potent than peptide leukotrienes on a molecular basis. 2. The increases in levels of glandular-kallikrein in the bronchial washings that were induced by LTC4 (3 nmol kg-1, i.v.) were almost completely inhibited by pretreatment with an antagonist of leukotrienes (ONO-1078), with an antagonist of thromboxane (S-1452), with an inhibitor of thromboxane synthetase (OKY-046), with indomethacin, with atropine or with scopolamine. These results indicate that the LTC4-induced increase in levels of glandular-kallikrein may have been mediated by the formation of thromboxane and the release of acetylcholine. 3. The increases in levels of glandular-kallikrein in the bronchial washings induced by STA2 (20 pmol kg-1, i.v.), a stable analogue of thromboxane A2, were completely blocked by pretreatment with atropine, whereas increases induced by pilocarpine (41 mumol kg-1, i.v.) were not blocked by pretreatment with indomethacin, although such increases were inhibited by atropine. This result indicates that secretion of kallikrein stimulated by LTC4 may have been mediated by the successive formation of thromboxane A2 and release of acetylcholine. 4. Intravenous administration of bradykinin (3-30 nmol kg-1) caused a dose-dependent increase in levels of glandular-kallikrein in the bronchial washings. This increase was completely inhibited by pretreatment with atropine, with indomethacin or with an antagonist of thromboxane.5. The increases in levels of glandular-kallikrein in the bronchial washings induced by LTC4 (3 nmol kg'- , i.v.) and pilocarpine (41 flmol kg- 1, i.v.) were significantly inhibited by pretreatment with an antagonist of bradykinin. These results suggest that intravenous LTC4 may increase secretion of glandular-kallikrein via formation of thromboxane A2 and release of acetylcholine in that order, and kinin released by kallikrein may enhance the rate of secretion of glandular-kallikrein.

Evidence for the involvement of a plasma kallikrein-kinin system in the immediate hypotension produced by endotoxin in anaesthetized rats.

1. In vitro incubation of normal rat plasma with endotoxin from E. coli (3-10 mg ml-1) in the incubation mixture) caused a dose-dependent increase in levels of free kinin and plasma kallikrein in the presence of o-phenanthroline, together with a mirror-image, dose-dependent decrease in the residual levels of the precursors, plasma prekallikrein and high-molecular-weight kininogen. Low-molecular-weight kininogen levels were not modified. 2. Intravenous injection of endotoxin (3-30 mg kg-1) into the femoral vein of anaesthetized rats resulted in dose-dependent hypotension. In blood collected up to 15 min after injection, the levels of prekallikrein and high-molecular-weight kininogen in plasma were decreased while levels of the active forms, plasma kallikrein and free kinin, showed a transient increase in the blood 1 min after administration of endotoxin. 3. A degradation product of bradykinin, des-Phe8-Arg9-bradykinin, as measured by a newly developed enzyme immunoassay, was detectable up to 5 min after administration of endotoxin. 4. Intravenous infusion of soybean trypsin inhibitor inhibited both the formation of bradykinin and des-Phe8-Arg9-bradykinin and the initial hypotension. 5. It can be concluded from our results that plasma prekallikrein is activated in the blood immediately after administration of endotoxin to rats and that bradykinin is a major cause of the immediate hypotension.

Effect of prolonged administration of a urinary kinase inhibitor, ebelactone B on the development of deoxycorticosterone acetate-salt hypertension in rats.

The effect of prolonged administration of a carboxypeptidase Y-like kininase inhibitor, ebelactone B (EB) (2-ethyl-3, 11-dihydroxy-4, 6, 8, 10, 12-pentamethyl-9-oxo-6-tetradecenoic 1, 3-lactone), on the development of deoxycorticosterone acetate (DOCA)-salt hypertension was tested. The systolic blood pressure (SBP) of non-treated 6-week-old Sprague-Dawley strain rats was gradually increased by DOCA-salt treatment from 137+/-2 mmHg (n=11) to 195+/-7 mmHg at 10 weeks of age. With daily oral administration of lisinopril (5 mg kg(-1), twice a day), which is an inhibitor of angiotensin converting enzyme, a major kininase in plasma, the development of hypertension was not suppressed. By contrast, administration of EB (5 mg kg(-1), twice a day), completely inhibited the development of hypertension (SBP: 146+/-1 mmHg, n=5, 10 weeks old). The reduced SBP at 10 weeks of age was equal to the SBP before any treatment (142+/-1 mmHg, n=5). Direct determination of mean blood pressure (MBP) in conscious, unrestrained rats confirmed that MBP elevation was completely inhibited by EB. Continuous subcutaneous infusion (5 mg kg(-1) day(-1)) of HOE140, a bradykinin B2 receptor antagonist, restored the elevation of SBP, which was suppressed by EB. The weights of left ventricle of DOCA-salt treated rats 10-weeks-old (0.36+/-0.02 g 100 g body weight(-1), n=11) was significantly reduced by EB (0.27+/-0.01, n=5), as were the sodium levels in serum, cerebrospinal fluid and erythrocyte. These findings suggested that EB is effective in preventing salt-related hypertension presumably by eliminating sodium retention.

Cyclo-oxygenase-2 enhances basic fibroblast growth factor-induced angiogenesis through induction of vascular endothelial growth factor in rat sponge implants.

Angiogenesis is reportedly enhanced by prostaglandins (PGs). In the present study, we investigated whether or not cyclo-oxygenase (COX)-2 mediated angiogenesis in chronic and proliferate granuloma. In rat sponge implants, angiogenesis was gradually developed over a 14-day experimental period as granuloma formed. This angiogenesis was enhanced by topical injections of human recombinant basic fibroblast growth factor (bFGF). In sponge granuloma, mRNA of COX-1 was constitutively expressed, whereas that of COX-2 was increased with neovascularization in parallel with that of vascular endothelial growth factor (VEGF). Topical injections of bFGF increased the expression of COX-2 mRNA. bFGF-stimulated angiogenesis was inhibited by indomethacin or selective COX-2 inhibitors, NS-398, nimesulide, and JTE-522. The levels of PGE(2) and 6-keto-PGF(1alpha) in the sponge granuloma were increased with bFGF 13 fold and 9 fold, respectively, and these levels were markedly reduced by NS-398. The expression of VEGF mRNA in the granuloma was also enhanced by bFGF, and was reduced by NS-398. Topical injections of PGE(2) and beraprost sodium, a PGI(2) analogue, increased the expression of VEGF mRNA, with angiogenesis enhancement. The enhanced angiogenesis by bFGF was significantly inhibited by topical injections of VEGF anti-sense oligonucleotide. These results suggested that COX-2 may enhance bFGF-induced neovascularization in sponge granuloma by PG-mediated expression of VEGF, and that a COX-2 inhibitor would facilitate the management of conditions involving angiogenesis.

Detection of the degradation products of bradykinin by enzyme immunoassays as markers for the release of kinin in vivo.

We developed an enzyme immunoassay (EIA) specific for Arg1-Pro2-Pro3-Gly4-Phe5 ([1-5]-BK) for determination of the levels of this peptide in biological fluids. Previously developed EIAs for bradykinin (BK) and for des-Phe8-Arg9-BK ([1-7]BK) were also used. Incubation of rat plasma with glass powder resulted in the transient appearance of BK. A degradation product, [1-7]BK, could be detected in the incubation mixture for a longer period of time. When compared with BK and [1-7]BK, a larger amount of [1-5]BK was detectable even longer. In carrageenan-induced pleurisy in rats, which was associated with a peak rate of plasma exudation 5 hr after administration of carrageenan, BK was undetectable (< 160 pg/rat) in the pleural exudates. By contrast, [1-7]BK was detectable over the entire course of the inflammatory response. A larger amount of [1-5]BK was detectable. The peak level of [1-5]BK was 6050 +/- 1050 pg/rat, 5 hr after administration of carrageenan. Inhibition of the generation of BK by intrapleural administration of soy bean trypsin inhibitor (0.3 mg/rat) 30 min before collection of pleural fluid resulted in significant reductions in the levels of both [1-7]BK (by 51-65%) and [1-5]BK (by 63-79%) in the exudates 3, 7 and 19 hr after administration of carrageenan. Intraperitoneal administration of captopril (10 mg/kg) caused a marked reduction (by 98%) in levels of [1-5]BK in exudates 3 hr after administration of carrageenan. The reduction was accompanied by an increase in the level of BK up to 1250% of that in untreated rats. These results indicate that the newly developed EIA for [1-5]BK might be a useful tool for verifying the release of kinin in vivo.

Mechanism of prevention by capsaicin of ethanol-induced gastric mucosal injury--a study in the rat using intravital microscopy.

BACKGROUND: Capsaicin acts specifically on primary afferent neurones to release neuropeptides, including calcitonin gene-related peptide (CGRP), and prevents ethanol-induced mucosal injury. AIM: To investigate the microvascular changes in the gastric mucosa in response to ethanol using intravital microscopy to elucidate the mechanism of capsaicin-induced gastroprotection. METHODS: The posterior gastric wall in the rat was secured in an observation chamber and perfused with Tyrode's solution. The microcirculation was observed through a window made by removing a limited area of smooth muscle. RESULTS: Ethanol (50%) applied to the mucosa constricted the collecting venules and venules but dilated arterioles. The constriction of the collecting venules resulted in mucosal congestion, which caused mucosal injury. Application of capsaicin to the mucosa dilated the arterioles but not the collecting venules or venules. Arteriolar dilation was inhibited by a CGRP antagonist, CGRP-(8-37). Prior application of capsaicin prevented ethanol-induced constriction of the collecting venules, and the action of capsaicin was inhibited by prior application of CGRP-(8-37). CONCLUSIONS: The results suggest that the inhibition of ethanol-induced gastric injury by capsaicin is attributable to the suppression of collecting venule constriction, via CGRP release.

Dilatation and constriction of rat gastric mucosal microvessels through prostaglandin EP2 and EP3 receptors.

BACKGROUND: Prostaglandin (PG)E2 has both a vasodilating action and a protective function in the gastric mucosa. There are four subtypes of PGE2-sensitive, or EP, receptors. AIM: To identify the subtype of EP receptors in the microvessels of the rat gastric mucosa using EP2 and EP3 receptor agonists. METHODS: The posterior wall of the anaesthetized rat stomach was secured in a chamber and superfused with Tyrode's solution, and the gastric microcirculation of the mucosal base was observed through a window with transillumination. PGE2 and its derivatives (20 microL) were applied topically in the window. RESULTS: PGE2 (0.001-10 micromol/L), misoprostol (EP2/EP3 receptor agonist; 0.01-100 micromol/L) and butaprost (EP2 receptor agonist; 1-1000 micromol/L) dilated the arterioles dose-dependently, but M&B 28 767 (EP3 receptor agonist; 0.001-10 micromol/L) did not alter their diameters. M&B 28 767 constricted the venules and collecting venules dose-dependently whereas butaprost dilated them. PGE2 and misoprostol had bell-shaped dose-response curves: constriction by low doses of PGE2 and misoprostol (0.001-0.1 micromol/L and 0.01-1 micromol/L) and dilation by high doses of PGE2 and misoprostol (0.1-100 micromol/L and 1-100 micromol/L). CONCLUSIONS: These results suggest that PGE2 dilated both arterioles and venules in the rat gastric mucosa through the EP2 receptors and constricted the venules through the EP3 receptors.

Release site of TNF alpha after intravenous and intraperitoneal injection of LPS from Escherichia coli in rats.

Lipopolysaccharide (LPS) concentrations in the portal vein after intraperitoneal (i.p.) injection were slightly higher than those in the arteries. The tumor necrosis factor (TNF alpha) levels in arterial serum were higher after i.p. injection than after i.v. injection and rose to a peak at 90 min after some delay. Infusion of LPS into the portal vein increased the TNF alpha levels in the arterial serum. Pretreatment with indomethacin further increased the arterial levels of TNF alpha after portal infusion, but did not after them after i.p. injection, because of the reduction by indomethacin of LPS absorption after i.p. Injection of LPS. TNF alpha was also generated in the peritoneal cavity after i.p. injection of LPS. The TNF alpha concentrations in the arterial serum and in the peritoneal cavity were accelerated by mast cell degradation. In conclusion, TNF alpha was generated mainly in the liver, but also in the peritoneal cavity, after i.p. injection of LPS, and was negatively regulated by prostaglandins.

Preferential consumption of coagulation factors I, V, and VIII in rat endotoxemia.

To ascertain the time course of prolonged coagulation time and the coagulation factors that were consumed preferentially after injection of Escherichia coli endotoxin (ETX, 3 mg/kg, intravenously) in rats, the activated partial thromboplastin time (aPTT) and prothrombin time (PT) were measured. Using aPTT and PT, the residual levels of the major coagulation factors were quantified by partial replacement of ETX-injected rat plasma with individual factor-deficient human plasma. The residual levels of prekallikrein and high molecular weight (HMW) kininogen were also measured. After ETX injection, aPTT and PT showed gradual increasing prolongation, which was marked at 3-5 h after the injection. The residual level of fibrinogen was markedly reduced between 1 and 3 h after ETX injection and dropped to the determination limit 7 h after the injection. Ratios of the consumed coagulation factors, prekallikrein, and HMW kininogen in rat plasma collected 7 h after intravenous injection of ETX were obtained as follows: prekallikrein (18.0 +/- 4.8%), HMW kininogen (36.2 +/- 1.9 %), factor XII (54.0 +/- 0.7%), factor VIII (86.1 +/- 1.8%), factor VII (35.6 +/- 7.7%), factor V (90.6 +/- 0.8%), and factor I (fibrinogen) (>89.6 +/- 0.0%). Thus, coagulation factor I (fibrinogen) and factors V and VIII (cofactors) were consumed preferentially. The extrinsic coagulation pathway was dominantly activated, whereas the intrinsic coagulation pathway, including plasma kallikrein-kinin system, played less important role in the ETX-induced consumption coagulopathy in rat.