Dihydropyrimidine dehydrogenase activity in 150 healthy Japanese volunteers and identification of novel mutations.

PURPOSE: Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme catalyzing the metabolic degradation of the anticancer drug 5-fluorouracil (5-FU). Population studies of DPD activity in peripheral blood mononuclear cells (PBMC) were reported in healthy volunteers and cancer patients. Although these studies were done in mainly Caucasian and African American populations, only a little information is available for a Japanese population. EXPERIMENTAL DESIGN: One hundred fifty healthy Japanese volunteers were screened for a population distribution of PBMC-DPD activity. Genetic analysis of a volunteer with very low DPD activity was carried out by reverse transcriptase-PCR and genomic sequencing. Bacterially expressed recombinant mutant DPD proteins were purified and characterized. RESULTS: Mean and median values of PBMC-DPD activity for 5-FU reduction in the study population were 0.173 and 0.166 nmol/min/mg protein, respectively. A 57-year-old female volunteer (proband in this study) had very low DPD activity (0.014 nmol/min/mg protein) with a very low level of expression of DPD protein. Two novel nucleotide substitutions, at nucleotide positions 1097 (1097G > C) and 2303 (2303C > A), resulting in amino acid substitutions at positions 366 (G366A) and 768 (T768K), respectively, were identified. The G366A mutation caused not only a marked decrease in the affinity of the enzyme to cofactor NADPH but also reduced Vmax for 5-FU-reducing activity to approximately 0.5. T768K mutant lost its activity much faster than did wild DPD. CONCLUSIONS: We found one healthy volunteer (0.7% of the population) with very low PBMC-DPD activity due to heterozygosity for a mutant allele of the DPYD gene in a population of 150 Japanese.

Upregulation by retinoic acid of transforming growth factor-beta-stimulated heat shock protein 27 induction in osteoblasts: involvement of mitogen-activated protein kinases.

We investigated whether transforming growth factor-beta (TGF-beta) stimulates the induction of heat shock protein (HSP) 27 and HSP70 in osteoblast-like MC3T3-E1 cells and the mechanism underlying the induction. TGF-beta increased the level of HSP27 but had no effect on the HSP70 level. TGF-beta stimulated the accumulation of HSP27 dose-dependently, and induced an increase in the level of mRNA for HSP27. TGF-beta induced the phosphorylation of p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase. The HSP27 accumulation induced by TGF-beta was significantly suppressed by PD98059, an inhibitor of the upstream kinase of p44/p42 MAP kinase, or SB203580, an inhibitor of p38 MAP kinase. PD98059 and SB203580 suppressed the TGF-beta-stimulated increase in the level of mRNA for HSP27. Retinoic acid, a vitamin A (retinol) metabolite, which alone had little effect on the HSP27 level, markedly enhanced the HSP27 accumulation stimulated by TGF-beta. Retinoic acid enhanced the TGF-beta-induced increase of mRNA for HSP27. The amplification of TGF-beta-stimulated HSP27 accumulation by retinoic acid was reduced by PD98059 or SB203580. Retinoic acid failed to affect the TGF-beta-induced phosphorylation of p44/p42 MAP kinase or p38 MAP kinase. These results strongly suggest that p44/p42 MAP kinase and p38 MAP kinase take part in the pathways of the TGF-beta-stimulated HSP27 induction in osteoblasts, and that retinoic acid upregulates the TGF-beta-stimulated HSP27 induction at a point downstream from p44/p42 MAP kinase and p38 MAP kinase.

Leukemia inhibitory factor enhances bFGF-induced IL-6 synthesis in osteoblasts: involvement of JAK2/STAT3.

We previously showed that basic fibroblast growth factor (bFGF) stimulates release of vascular endothelial growth factor (VEGF) and synthesis of interleukin-6 (IL-6) in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effects of leukemia inhibitory factor (LIF) on the release of VEGF and IL-6 in these cells. LIF did not affect the bFGF-stimulated VEGF release. On the contrary, LIF, which alone had little effect on IL-6 release, significantly enhanced the bFGF-stimulated IL-6 release. The amplifying effect of LIF on the IL-6 release was dose dependent in the range between 0.01 and 10 ng/ml. AG490, an inhibitor of JAK2, suppressed the amplifying effect of LIF. LIF induced the phosphorylation of STAT3. AG490 inhibited the LIF-induced STAT3 phosphorylation. Taken together, our results strongly suggest that LIF enhances bFGF-stimulated IL-6 synthesis via JAK2/STAT3 pathway in osteoblasts.

Sphingomyelinase amplifies BMP-4-induced osteocalcin synthesis in osteoblasts: role of ceramide.

We previously reported that extracellular sphingomyelinase induces sphingomyelin hydrolysis in osteoblast-like MC3T3-E1 cells and that mitogen-activated protein (MAP) kinases are involved in bone morphogenetic protein (BMP)-4-stimulated osteocalcin synthesis in these cells. In the present study, we investigated whether sphingomyelinase affects BMP-4-stimulated synthesis of osteocalcin in osteoblast-like MC3T3-E1 cells. Sphingomyelinase significantly enhanced the BMP-4-stimulated osteocalcin synthesis. Among sphingomyelin metabolites, C(2)-ceramide enhanced the BMP-4-stimulated osteocalcin synthesis while sphingosine and sphingosine 1-phosphate had little effect on the synthesis. D-erythro-MAPP, an inhibitor of ceramidase, amplified the sphingomyelinase-effect on the osteocalcin synthesis. C(2)-ceramide suppressed the BMP-4-induced phosphorylation of p44/p42 MAP kinase, while having little effect on the phosphorylation of Smad1 and p38 MAP kinase. Taken together, our results strongly suggest that extracellular sphingomyelinase enhances the BMP-stimulated osteocalcin synthesis via ceramide in osteoblasts and that the effect of ceramide is exerted at a point upstream from p44/p42 MAP kinase.

Function of tissue-type plasminogen activator releaser on vascular endothelial cells and thrombolysis in vivo.

The effect of monosodium[2-(6-hydroxynaphthalen-2-yl)-6-methylpyrimidin-4-yloxy]acetate dihydrate (JTV-926) on fibrinolysis was investigated in vitro and in vivo. JTV-926 released tissue-type plasminogen activator (t-PA) from human vascular endothelial cells in a dose-dependent manner. The thrombolytic effect of JTV-926 was studied using three animal thrombosis models; a photo-irradiation-induced mouse carotid artery thrombosis model, a photo-irradiation-induced rat femoral artery thrombosis model and a thrombin-induced rat venous thrombosis model. In the mouse thrombosis model, t-PA deficient mice (t-PA-/- mice) and their wild-type (t-PA+/+) were used. JTV-926 was injected as a bolus 30 min after the interruption of blood flow by an occlusion thrombi. Blood flow was continuously monitored for 180 min after intravenous administration of JTV-926 (1 mg/kg). Although the recanalization rate of the occluded artery was 37.5% in t-PA +/+ mice with the vehicle control, it increased to 75% in t-PA+/+ mice after JTV-926 administration. However, when JTV-926 was administrated in t-PA-/- mice, vascular recanalization was not observed in any arteries. In the photo-irradiation-induced rat femoral artery thrombosis model, intra-duodenal administration of JTV-926 induced thrombolysis. Moreover, in the thrombin-induced rat venous thrombosis model, the dose-dependent thrombolysis was also observed by oral administration of JTV-926. It was suggested that JTV-926 revealed a sufficient thrombolytic effect through the absorption from the intestine. Thus, a newly synthesized compound, JTV-926 induced t-PA release from vascular endothelial cells and effective thrombolysis in vivo.

Plasmin generation plays different roles in the formation and removal of arterial and venous thrombus in mice.

The role of plasminogen (Plg) and alpha2-antiplasmin (alpha2-AP) in vascular thrombolysis in vivo was investigated in mice deficient in plasminogen (Plg-/-) or a2-AP (alpha2-AP-/-) or their wild type (PAI-1+/+, alpha2-AP+/+). A thrombus was induced in the murine carotid artery or the internal jugular vein by endothelial injury. Blood flow was continuously monitored for 90 min and for 6 h 30 min after the initiation of endothelial injury. The times to occlusion by the developing thrombus in the carotid artery and the jugular vein of wild type mice were 12+/-1.8 and 7.2+/-1.9 min, respectively. The arterial thrombus formation in alpha2-AP-/- mice was indistinguishable from the one in wild type mice, whereas the time to occlusion in Plg-/- was significantly shortened to 5.9+/-1.7 min. Vascular patency after spontaneous reperfusion was markedly improved in alpha2-AP-/- mice. On the contrary, arteriarpatency in Plg-/- mice was aggravated. In venous thrombus formation, the time to occlusion in alpha2-AP-/- mice was significantly prolonged (27.1+/-5.2 min), whereas in Plg-/- it was slightly shortened to 6.5+/-2.5 min. Vascular patency after spontaneous reperfusion was also improved in alpha2-AP-/- mice, but not in Plg-/- mice. Histological observations using SEM indicated that fibrin nets were firmly fixed on the injured area in Plg-/- mice, but not in alpha2-AP-/- mice. The tail bleeding time was not different in any type of mice. However, re-bleeding time using a template bleeding device was significantly prolonged in alpha2-AP-/- as compared with that of wild type mice. In conclusion, lack of plasminogen markedly reduces the antithrombotic activities in vivo, whereas alpha2-AP plays a more important role in the formation and removal of venous thrombus in mice. Consequently, the inhibition of alpha2-AP could be a useful tool for the therapy of venous thrombosis and the prevention of re-thrombus formation.

Pharmacokinetic and pharmacodynamic properties of a new thromboxane receptor antagonist (Z-335) after single and multiple oral administrations to healthy volunteers.

The pharmacokinetics and pharmacodynamics of a new oral thromboxane (TX) A2 receptor antagonist, Z-335, were studied in healthy male volunteers following single doses (0.5-40 mg, PO) in a dose-escalating manner and multiple doses (40 mg, PO, once daily for 7 consecutive days) with a single-blind, placebo-controlled design. Serial blood and urine samples were analyzed for Z-335 and its metabolites to obtain key pharmacokinetic parameters. In the single-dose (10, 20, and 40 mg) study, the maximum plasma concentration (Cmax) and area under the plasma concentration versus time curve (AUC) increased in proportion to the dose when administered afterfasting, while the mean elimination half-life (t1/2beta) was essentially unchanged (7.79-7.93 h). Recovery of the unchanged and taurine-conjugated drugs in the urine within 24 hours was 6.5% to 8.4% and 11.9% to 14.2%, respectively. These parameters essentially remained unchanged when the effect of meal intake was evaluated at the dose of 20 mg with a crossover design. Ex vivo platelet aggregation in the plasma by a TXA2 analogue, U46619, was completely inhibited within 2 hours after all doses, and complete inhibition was maintained for 12 to 14 hours, depending on the dose. The aggregation induced by collagen was also inhibited to a lesser extent, whereas that by adenosine diphosphate was hardly influenced. In the multiple-dose study, Cmax and AUC0-24 were increased by 34% after the last dose compared with the first dose. Z-335 afforded extensive inhibition of platelet aggregation by U46619 throughout the administration period, which returned, however, almost to the control level 48 hours after the last dose. The agent was well tolerated without any abnormalities in subjective and objective symptoms, blood biochemistry, hematology, and urinalysis definitely attributable to the agent, except for the changes expected from its TXA2 receptor-antagonizing actions. Z-335 was concluded to be safe and to provide long-lasting blockade of TXA2 receptors on the basis of a once-daily regimen, promoting further clinical evaluation.

Involvement of p38 mitogen-activated protein kinase in heat shock protein 27 induction in human neutrophils.

We investigated whether tumor necrosis factor-alpha (TNF-alpha) stimulates the induction of heat shock protein 27 (HSP27) in human neutrophils and the mechanism underlying this induction. In intact neutrophils, almost no HSP27 was detected. Stimulation of neutrophils by TNF-alpha increased the levels of HSP27 in the presence, but not in the absence, of cycloheximide. Reverse transcription-polymerase chain reaction (RT-PCR) experiments showed that TNF-alpha also induced HSP27 mRNA in the presence of cycloheximide. TNF-alpha induced the phosphorylation of p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase. The HSP27 accumulation induced by TNF-alpha was significantly suppressed by 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole (SB203580) or 4-(4-fluorophenyl)-2-(4-nitrophenyl)-5-(4-pyridyl)-1H-imidazole (PD169316); both are specific inhibitors of p38 MAP kinase, but not by 2'-amino-3'-methoxyflavone (PD098059, a specific inhibitor of the upstream kinase that activates p44/p42 MAP kinase). The accumulation of HSP27 induced by TNF-alpha plus cycloheximide was also suppressed by pretreatment with a specific protein kinase C (PKC) inhibitor. Furthermore, phorbol myristate acetate (PMA), a PKC stimulant, but not dibutyryl cyclic AMP, a protein kinase A stimulant, stimulated the accumulation of HSP27. Interestingly, SB203580 did not inhibit PMA-stimulated HSP27 induction. These results strongly suggest that TNF-alpha may act as the regulator of HSP27 induction in neutrophils. p38 MAP kinase (but not p44/p42 MAP kinase) and PKC take part in TNF-alpha-stimulated HSP27 induction in human neutrophils.

Correlation of angiographic circulation time and cerebrovascular reserve by acetazolamide-challenged single photon emission CT.

BACKGROUND AND PURPOSE: Although cerebral circulation time (CCT) is one of the main parameters in cerebral blood flow measurements, its clinical significance is controversial. To assess the importance of CCT by using a nondiffusible indicator, we studied the relationship between angiographic CCT and cerebrovascular reserve. METHODS: Twenty-eight patients, each with a unilateral occlusive lesion in the internal carotid artery or middle cerebral artery, were examined. To assess the CCT, the regional arteriocapillary circulation time (rACCT) was measured by angiography and the ratio of the value on the occlusive side to the value on the contralateral side was calculated as the rACCT ratio. To estimate the cerebrovascular reserve, acetazolamide-challenged single photon emission CT was used. Patients with a decreased cerebrovascular reserve were defined as the "poor reserve" group, and those without a decrease were defined as the "normal reserve" group. The ratio of the radioactivity count on the occlusive side to the count on the contralateral side was calculated as the asymmetry index, and the proportion of the acetazolamide-challenged asymmetry index to the baseline asymmetry index was defined as the regional reactivity index. RESULTS: The rACCT ratio in the poor reserve group (n = 19) was significantly (P <.001) larger than that in the normal reserve group (n = 9), and a significant correlation (r = -0.83, P <.01) was found between the rACCT ratio and the regional reactivity index. CONCLUSION: The angiographic CCT and the cerebral vasoreactivity to acetazolamide on single photon emission CT were well correlated, suggesting that measurement of the CCT by using a nondiffusible indicator could be used as an index of cerebrovascular reserve.

HSP20, low-molecular-weight heat shock-related protein, acts extracellularly as a regulator of platelet functions: a novel defense mechanism.

We previously showed that a dissociated form of a low-molecular-weight heat shock-related protein 20 (HSP20) but not an aggregated form of HSP20 suppresses platelet aggregation. In the present study, we investigated the behavior of HSP20 in response to endothelial injury and the possible mechanism of HSP20 in platelet functions. The levels of HSP20 in vessel wall after endothelial injury were markedly reduced. This observation was supported by the results of Western blotting analysis and immunohistochemical analysis. Additionally, the plasma levels of HSP20 in cardiomyopathic hamsters were markedly elevated. Centrifugation on sucrose density gradients allowed detection mainly of the dissociated form of plasma HSP20 in these hamsters. Human platelets showed specific binding sites for HSP20. Moreover, HSP20 markedly reduced thrombin-induced phosphoinositide hydrolysis by phospholipase C in human platelets. Taken together, our results strongly suggest that HSP20, which immediately responds to pathological events, acts extracellularly as a regulator of platelet functions.

Neuronal degradation in mouse retina after a transient ischemia and protective effect of hypothermia.

Temporal profile of neuronal deaths in the mouse retina evoked by a transient retinal ischemia and the protective effect of hypothermia on such deaths were evaluated. A transient ischemic insult was induced in the mouse retina by elevating the intra-ocular pressure. The retina tissue responses after reperfusion were histopathologically detected by monitoring the retinal cell death in the ganglion cell layer and inner nuclear layer, using a sequential TUNEL-staining technique, and by measuring the inner retinal thickness. Elevation of intra-ocular pressure induced a time-related appearance of TUNEL-positive cells in the mouse inner retinas. Peak TUNEL staining occurred 12 h after reperfusion. Lowering mice body temperature to 35 degrees C, 33 degrees C and 29 degrees C during the ischemia period significantly inhibited DNA fragmentation of retinal neurons in a lowering temperature dependent manner. In this experiment, the inner retinal thickness was preserved in 29 degrees C group compared with that in 37 degrees C group. From these results, the 45-min transient ischemia and histopathological examination 12 h later provided a reproducible number of retinal neuronal deaths. Furthermore, hypothermic intervention showed a protective effect to salvage retinal neuronal cells from a transient ischemic insult.

Prediction of pharmacokinetics of antibiotics in patients with end-stage renal disease.

A novel and convenient method to predict the pharmacokinetics of several kinds of antibiotic agents in patients with end-stage renal disease (ESRD) was examined based on the in vitro extraction ratios and pharmacokinetic parameters in healthy volunteers. The dializability of 17 antibiotic agents in 4% human serum albumin solution were determined using a high-performance hemodialytic membrane for clinical use. We assumed that the off-hemodialysis clearance approximated the non-renal clearance, while the on-hemodialysis clearance was considered to be sum of the off-hemodialysis clearance and the hemodialytic clearance. The estimated on- and off-hemodialysis clearances were compared with the ones observed in ESRD patients. In order to confirm the method prospectively, an in vivo pharmacokinetic study was performed in dogs with mercury chloride-induced experimental renal failure. The in vitro extraction ratios of 9 beta-lactams were broadly ranged from 10.9 to 75.6% depending on their physicochemical properties. In contrast, those of the other antibiotics were consistent with their chemical classes: 60.5-63.2% for fluoroquinolone, 48.8-51.1% for aminoglycoside and 18.7-25.6% for glycopeptide. Both the estimated on- and off-hemodialysis clearances of the 17 antibiotics coincided well with the observed values in the literature, regardless of their physicochemical and pharmacokinetic properties. The validity and applicability of this method to three cefems, cefmetazole, cefotaxime and cefoperazone, was prospectively confirmed in the animal study. In conclusion, this new method enables the prediction of the on- and off-hemodialysis clearances of several kinds of antibiotics in ESRD patients from minimal information of their pharmacokinetics in healthy subjects and their in vitro dializability.

Pharmacokinetics of panipenem/betamipron in patients with end-stage renal disease.

Panipenem/betamipron (Carbenin), a parenteral carbapenem antibiotic, is used for the treatment of severe and intractable bacterial infections caused by gram-positive and gram-negative bacteria. Because 30% of panipenem and most of the betamipron are excreted in the urine in an unchanged form, renal function is the important determinant of the dosage regimen of panipenem/betamipron. In this study, the pharmacokinetics of panipenem/betamipron were investigated in patients with end-stage renal disease (ESRD) undergoing hemodialysis treatment to establish an appropriate dose regimen. We further attempted to predict the in vivo clearance in patients undergoing hemodialysis based on the in vitro dializability. The pharmacokinetics of panipenem/betamipron were investigated in eight patients after a 1-h intravenous infusion of panipenem/betamipron (500 mg/500 mg). The in vitro extraction ratios of panipenem/betamipron through a high-flux dialyzer were obtained, and compared with those obtained in vivo. The clearances of panipenem in patients were 9.53 +/- 1.26 l/h with hemodialysis, and 2.92 +/- 0.238 l/h without hemodialysis. In contrast, those of betamipron were 4.18 +/- 0.643 l/h and 0.615 +/- 0.511 l/h, respectively. The clearance of panipenem with hemodialysis were predicted well from in vitro extraction ratios, while that of betamipron was overestimated about 1.4-fold, probably due to high plasma protein binding and the binding difference between patients and healthy subjects. After comparing the pharmacokinetic behavior of panipenem in patients with ESRD and that of a surrogate marker of efficacy, we recommend that these patients be treated with 500 mg/500 mg of panipenem/betamipron once daily, which gives a similar clinical result in a patient with normal renal function.

Divergent regulation by p44/p42 MAP kinase and p38 MAP kinase of bone morphogenetic protein-4-stimulated osteocalcin synthesis in osteoblasts.

In the present study, we investigated whether the mitogen-activated protein (MAP) kinase superfamily is involved in the bone morphogenetic protein (BMP)-4-stimulated synthesis of osteocalcin in osteoblast-like MC3T3-E1 cells. BMP-4 dose-dependently stimulated osteocalcin synthesis. BMP-4 markedly induced the phosphorylation of p44/p42 MAP kinase and p38 MAP kinase, while having little effect on SAPK (stress-activated protein kinase)/JNK (c-Jun N terminal kinase) phosphorylation. SB203580 and PD169316, specific inhibitors of p38 MAP kinase, significantly reduced the osteocalcin synthesis stimulated by BMP-4. In contrast, PD98059 and U0126, inhibitors of upstream kinase of p44/p42 MAP kinase, markedly enhanced the BMP-4-stimulated osteocalcin synthesis. The BMP-4-induced phosphorylation of p44/p42 MAP kinase was suppressed by PD98059, which did not, however, affect the BMP-4-induced phosphorylation of p38 MAP kinase. Taken together, our results strongly suggest that p38 MAP kinase takes part in BMP-4-stimulated osteocalcin synthesis as a positive regulator in osteoblasts, whereas p44/p42 MAP kinase acts as a negative regulator in the synthesis.

Effects of SCH27899 (Ziracin), an oligosaccharide everninomicin antibiotic, on urate kinetics in humans.

OBJECTIVE: Intravenous administration of an everninomicin antibiotic, SCH27899 (Ziracin), in healthy subjects caused a marked decrease in serum urate by increasing its urinary excretion, as well as an increase in serum bilirubin in a dose-dependent manner. To clarify the underlying mechanism, a crossover study and an in vitro study were conducted. METHODS: Crossover study was performed in nine healthy male volunteers over three periods by administering SCH27899 (1-h i.v. infusion of 3 mg/kg) alone, probenecid (2000 mg, p.o.) alone and their combination. Also, an in vitro experiment was conducted using rat brush-border membrane vesicles to elucidate the effect of SCH27899 on urate transport across renal tubular epithelium. RESULTS: SCH27899 alone and probenecid alone showed a uricosuric, serum urate-lowering effect, and, when given in combination, the effects on serum urate appeared to be additive, as indicated in the earlier phase, prior to the peaks of respective drug effects. Serum and urinary concentrations of SCH27899 were not influenced by the co-administration of probenecid. Serum bilirubin was also significantly increased by both SCH27899 alone and in combination with probenecid. The SCH27899-probenecid combination additive effect on serum bilirubin did not reach significance. SCH27899, probenecid and losartan, an angiotensin-II-receptor antagonist possessing a uricosuric effect, significantly inhibited (14)C-urate uptake into the vesicles, which was dependent on the pH gradient across the membrane; whereas, vancomycin did not. CONCLUSION: It is concluded that SCH27899 itself contributes, at least in part, to a uricosuric effect following i.v. infusion. However, some metabolite(s) may also contribute to this, since the degree of urate-uptake inhibition by SCH27899 was less than probenecid and losartan, and the serum urate-lowering effect was delayed and prolonged compared with the time profile of serum concentration.

Hair analysis of flecainide for assessing the individual drug-taking behavior.

OBJECTIVE: The concentration of flecainide in hair was measured to determine its value as an index of individual drug-taking history. METHODS: Hair samples obtained from 15 patients treated with flecainide for more than 1 month were cut into 1-cm-long portions successively from its scalp end. The concentration of flecainide in each hair portion was measured using high-performance liquid chromatography. RESULTS: Flecainide was detected in the 1-cm-long hair portion at the scalp end in the concentration range of 38.0-411.9 ng x mg(-1), which significantly correlated with the area under the plasma flecainide concentration versus time curve. The axial centimeter-by-centimeter distribution of flecainide along the hair shaft well reflected the individual history of drug use. CONCLUSION: The present study suggests the usefulness of determining flecainide in hair to provide retrospective information on the individual drug-taking behavior qualitatively.

Inhibitors of fibrinolytic components play different roles in the formation and removal of arterial thrombus in mice.

Plasminogen activator inhibitor-1 (PAI-1) and alpha2-anti-plasmin (alpha2-AP) may contribute to arterial thrombolysis resistance. The role of these components on thrombus evolution in vivo was investigated in mice deficient for PAI-1 (PAI-1(-/-)) or alpha2-AP (alpha2-AP(-/-)) or their wild-type counterparts (PAI-1(+/+), alpha2-AP(+/+)). Moreover, the influence of either PAI-1 or alpha2-AP deficiency on the results of pharmacologic inhibition of glycoprotein IIb/IIIa of platelets or thrombin was also investigated. A thrombus was induced in the murine carotid artery by endothelial injury. The alpha2-AP(-/-) mice were indistinguishable from wild-type, whereas the time to occlusion in PAI-1(-/-) was significantly prolonged to 24.9 +/- 3.7 min. Vascular patency was markedly increased in both PAI-1- and alpha2-AP-deficient mice. In separate animals, either a glycoprotein IIb/IIIa antagonist or a thrombin inhibitor was applied. The time required to occlusion was prolonged in a dose-dependent manner in all types of mice. When each compound was administered to PAI-1(-/-) mice, significant changes were observed. In conclusion, lack of PAI-1 prolongs the time to occlusion and accelerates clot lysis, whereas alpha2-AP only has an effect on spontaneous reperfusion. Consequently, the inhibition of PAI-1, but not of alpha2-AP, could enhance the effects of anti-thrombotic therapy.

Zinc suppresses IL-6 synthesis by prostaglandin F2alpha in osteoblasts: inhibition of phospholipase C and phospholipase D.

We previously reported that prostaglandin F2alpha (PGF2alpha) induces phosphoinositide hydrolysis by phospholipase C and phosphatidylcholine hydrolysis by phospholipase D through heterotrimeric GTP-binding protein, resulting in the activation of protein kinase C (PKC) in osteoblast-like MC3T3-E1 cells and that PGF2alpha stimulates the synthesis of interleukin-6 (IL-6) via PKC-dependent p44/p42 mitogen-activated protein (MAP) kinase activation. In the present study, we investigated whether zinc affects the PGF2alpha-induced IL-6 synthesis in these cells. Zinc complex of l-carnosine (l-CAZ) dose-dependently suppressed the PGF2alpha-stimulated IL-6 synthesis. In addition, zinc alone reduced the IL-6 synthesis. L-CAZ suppressed the PGF2alpha-induced p44/p42 MAP kinase phosphorylation. However, the p44/p42 MAP kinase phosphorylation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), a direct activator of PKC, or NaF, a direct activator of GTP-binding protein, was not affected by l-CAZ. l-CAZ reduced the PGF2alpha-stimulated formation of inositol phosphates and choline. However, l-CAZ did not affect the formation of inositol phosphates or choline induced by NaF. These results strongly suggest that zinc reduces PGF2alpha-induced IL-6 synthesis via suppression of phosphoinositide-hydrolyzing phospholipase C and phosphatidylcholine-hydrolyzing phospholipase D in osteoblasts.

Priming by grepafloxacin on respiratory burst of human neutrophils: its possible mechanism.

Grepafloxacin is a broad-spectrum fluoroquinolone derivative that has good tissue penetration. We demonstrated that grepafloxacin showed a priming effect on neutrophil respiratory burst, triggered by either a chemotactic factor N-formyl-methionyl-leucyl-phenylalanine (fMLP) or leukotriene B4 (LTB4), but not by the phorbol ester phorbol 12-myristate 13-acetate (PMA). The priming effect of grepafloxacin on fMLP-stimulated superoxide generation by human neutrophils correlated with the penetration of grepafloxacin into cells. Removal of extracellular grepafloxacin did not inhibit the priming effect on fMLP-stimulated superoxide generation. Furthermore, grepafloxacin induced the translocation of p47-phox and p67-phox to the membrane fraction of neutrophils, whereas tyrosine phosphorylation was hardly observed in neutrophils exposed to grepafloxacin. The priming effect of grepafloxacin on superoxide generation from neutrophils was not inhibited by treatment with pertussis toxin, a protein-tyrosine kinase inhibitor (ST-638) or a protein kinase C inhibitor (calphostin C), or chelation of extracellular calcium. Grepafloxacin did not change the fMLP receptor-binding properties. Taken together, these findings suggest that grepafloxacin evokes a priming effect on neutrophil superoxide generation intracellularly through the translocation of p47-phox and even p67-phox protein to the membrane fractions. GTP binding protein, protein-tyrosine phosphorylation and protein kinase C activation are not involved in the priming effect.

The calcimimetic agent KRN 1493 lowers plasma parathyroid hormone and ionized calcium concentrations in patients with chronic renal failure on haemodialysis both on the day of haemodialysis and on the day without haemodialysis.

AIMS: Treatment with vitamin D sterols can lower plasma parathyroid hormone (PTH) in patients with secondary hyperparathyroidism; however, hypercalcaemia, hyperphosphataemia, or both, often develop. Calcimimetic agents, employed in alternative therapeutic approaches, directly inhibit PTH secretion by activating the calcium-sensing receptor in the parathyroid glands. METHODS: In this study, patients were given orally 25, 50, and 100 mg doses of the calcimimetic agent KRN 1493 each on two occasions, on the day of haemodialysis and on the day without haemodialysis. RESULTS: In the pharmacokinetic results, because the clearance of KRN 1493 by haemodialysis was much smaller than the systemic clearance, the influence of haemodialysis was not remarkable. In the pharmacodynamic study, on both the days with or without haemodialysis, plasma PTH concentrations decreased in a dose-dependent manner. Serum calcium concentrations decreased in association with the decrease in plasma PTH concentrations. Mild dose-dependent adverse effects (mainly nausea) were seen after the administration of KRN 1493 on both the day of haemodialysis and the day without haemodialysis. CONCLUSIONS: We conclude that the pharmacokinetics of KRN 1493 after a single administration were similar on the day of haemodialysis and the day without haemodialysis. KRN 1493 is safe and effective in suppressing PTH secretion and serum calcium concentrations on the day of haemodialysis and on the day without haemodialysis in patients with secondary hyperparathyroidism.