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Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta superfamily, which regulate the differentiation of osteoprogenitor cells. Here we show that among members of the BMP family, BMP-4 and growth/differentiation factor 5 (GDF-5) induce osteoblast differentiation through the activation of three receptor-regulated Smads (i.e. Smad1, Smad5 and Smad8). By contrast, BMP-6 and BMP-7 induce alkaline phosphatase activity through Smad1 and Smad5, but not through Smad8. Consistent with these findings, BMP-4 induced phosphorylation and nuclear translocation of Smad1, Smad5 and Smad8, but BMP-6 activated only Smad1 and Smad5. BMP-4 and GDF-5 are known to bind to activin receptor-like kinase 3 (ALK-3) and/or ALK-6 (also termed BMP type IA and type IB receptors, respectively), whereas BMP-6 and BMP-7 preferentially bind to ALK-2. Compared with the effects induced by only one of the type I receptors, the combination of constitutively active forms of ALK-2 and ALK-3 (or ALK-6) more strongly induced alkaline phosphatase activity in C2C12 cells. Moreover, addition of BMP-4 and BMP-6 to C2C12 cells resulted in higher alkaline phosphatase activity than that of only one of these BMPs. The combination of ALK-2 and ALK-3 also induced higher transcriptional activity than either receptor alone. Thus, ALK-2 and ALK-3 (or ALK-6) might synergistically induce osteoblast differentiation of C2C12 cells, possibly through efficient activation of downstream signaling pathways.

4-Hydroxynonenal (HNE), a diffusible product of lipid peroxidation, has been suggested to be a key mediator of oxidative stress-induced cell death. In this study, we partially characterized the mechanism of HNE-mediated cytotoxicity. Incubation of human T lymphoma Jurkat cells with 20-50 microM HNE led to cell death accompanied by DNA fragmentation. Western blot analysis showed that HNE-treatment induced time- and dose-dependent activation of caspase-8, caspase-9 and caspase-3. HNE-induced caspase-3 processing was confirmed by a flow cytometric demonstration of increased catalytic activity on the substrate peptide. HNE treatment also led to remarkable cleavage of poly(ADP-ribose) polymerase (PARP), which was prevented by pretreatment of cells with DEVD-FMK as a caspase-3 inhibitor. The HNE-mediated activation of caspases, cleavage of PARP and DNA fragmentation were blocked by antioxidants cysteine, N-acety-L-cysteine and dithiothreitol, but not by two other HNE-reactive amino acids lysine and histidine, or by cystine, the oxidized form of cysteine. HNE rapidly decreased levels of intracellular reduced glutathione (GSH) and its oxidized form GSSG, and these were also attenuated by the reductants. Coincubation of Jurkat cells with a blocking anti-Fas antibody prevented Fas-induced but not HNE-induced activation of caspase-3. HNE also activated caspase-3 in K562 cells that do not express functional Fas. Our results thereby demonstrate that HNE triggers oxidative stress-linked apoptotic cell death through activation of the caspase cascade. The results also suggest a possible mechanism involving a direct scavenge of intracellular GSH by HNE.

The c-RET proto-oncogene encodes a receptor-type tyrosine kinase, and its mutations in the germ line are responsible for the inheritance of multiple endocrine neoplasia type 2A (MEN2A) and 2B (MEN2B). Ret kinases are constitutively activated as a result of MEN2A mutations (Ret-MEN2A) or MEN2B mutations (Ret-MEN2B). Here we demonstrate that UV light (UV) irradiation induces superactivation of the constitutively activated Ret-MEN2A and Ret-MEN2B as well as activation of c-Ret. Before UV irradiation, small percentages of c-Ret (3-4%) and Ret-MEN2B (1-2%) and large percentages of Ret-MEN2A (30-40%) were dimerized through disulfide bonds. These dimerized Ret proteins were preferentially autophosphorylated, suggesting a close relation between up-regulated kinase activity and disulfide bond-mediated dimerization of Ret proteins. We found that UV irradiation promotes the disulfide bond-mediated dimerization of the Ret proteins, in close association with activation and superactivation of Ret kinases. UV irradiation also induced dimerization and activation of the extracellular domain-deleted mutant Ret (Ret-PTC-1). Interestingly, the levels of basic kinase activity and dimerization of Ret-PTC-1-C376A, in which cysteine 376 in the tyrosine kinase domain of Ret-PTC-1 was replaced by alanine, were low and were not increased by UV irradiation. These results suggest that Ret-PTC-1 cysteine 376 is one of possibly multiple critical target amino acids of UV for Ret kinase activation. Overexpression of Cu/Zn superoxide dismutase in cells as a result of gene transfection prevented both the UV-mediated promotion of dimerization and the superactivation of Ret-MEN2A kinase. These results suggest that the UV-induced free radicals in cells attack intracellular domains of Ret to dimerize the kinase proteins for superactivation.

A steady-state pharmacokinetic analysis was performed to investigate the overall elimination and extraction of hepatocyte growth factor (HGF) by its target organs, including liver, kidney, and lung, during its constant i.v. infusion in rats. The plasma clearance of HGF became saturated as the steady-state plasma concentration (Cpss) increased, but complete saturation was not achieved, even when the Cpss ( approximately 1000 pM) was much higher than the dissociation constant for the HGF receptor (20-40 pM), which has been identified as one of the major clearance sites for HGF. This result suggests that there is a low-affinity and high-capacity clearance mechanism, other than receptor-mediated endocytosis, involved in its elimination from the body. The hepatic extraction ratio of HGF, assessed by determining the HGF concentration in both the circulating blood and hepatic vein, was 40 to 60%, whereas the HGF extraction both in kidney and lung was always less than 10%. Hepatic clearance accounted for approximately 70% of the plasma clearance at any Cpss. Thus, the present study shows that HGF in circulating plasma is efficiently extracted by the liver compared with other HGF target organs, the liver being involved in 70% of the overall elimination both under linear and nonlinear conditions. Biliary excretion of HGF was observed, but this accounted for only 0.1 to 0. 2% of the infusion rate, indicating that the nonlysosomal pathway of HGF, which avoids the lysosomal enzymes and transcytoses HGF directly into the bile, is very minor indeed.

The present study examined the role of the rostral ventrolateral medulla (RVLM) in the modulation of acetylcholine (ACh) release by morphine. We examined the effect of morphine on the release of ACh in the RVLM of freely moving rats using the in vivo microdialysis method. The basal level of ACh was 303.0 +/- 28.2 fmol/20 microliter/15 min in the presence of neostigmine (10 microM). Morphine at a low dose of 5 mg/kg (i.p.) increased ACh release by the RVLM by 42.4%. A higher morphine dose (10 mg/kg i.p.) significantly increased the release of ACh by 75.4%, with a maximal effect (86.4%) at 75 min. This enhancement following i.p. administration of morphine was reversed by naloxone (1 mg/kg i.p.). Addition of morphine (10(-4) M) to the perfusion medium increased the ACh release by 85.8% of the predrug values. The increased ACh release induced by local application of morphine was reversed by pretreatment with naloxone (1 mg/kg i.p.). The antinociceptive effect of locally applied morphine into the RVLM was assessed using the hot-plate test and tail immersion test in unanesthetized rats. Local application of morphine (10(-4) M) via a microdialysis probe induced an increase in both tail withdrawal and hot-plate response. These findings suggest that morphine seems to exert a direct stimulatory effect on ACh release by the RVLM and that morphine-induced nociception is, in part, activated by the release of ACh in freely moving rats.

The biological effects of type I serine/threonine kinase receptors and Smad proteins were examined using an adenovirus-based vector system. Constitutively active forms of bone morphogenetic protein (BMP) type I receptors (BMPR-IA and BMPR-IB; BMPR-I group) and those of activin receptor-like kinase (ALK)-1 and ALK-2 (ALK-1 group) induced alkaline phosphatase activity in C2C12 cells. Receptor-regulated Smads (R-Smads) that act in the BMP pathways, such as Smad1 and Smad5, also induced the alkaline phosphatase activity in C2C12 cells. BMP-6 dramatically enhanced alkaline phosphatase activity induced by Smad1 or Smad5, probably because of the nuclear translocation of R-Smads triggered by the ligand. Inhibitory Smads, i.e., Smad6 and Smad7, repressed the alkaline phosphatase activity induced by BMP-6 or the type I receptors. Chondrogenic differentiation of ATDC5 cells was induced by the receptors of the BMPR-I group but not by those of the ALK-1 group. However, kinase-inactive forms of the receptors of the ALK-1 and BMPR-I groups blocked chondrogenic differentiation. Although R-Smads failed to induce cartilage nodule formation, inhibitory Smads blocked it. Osteoblast differentiation induced by BMPs is thus mediated mainly via the Smad-signaling pathway, whereas chondrogenic differentiation may be transmitted by Smad-dependent and independent pathways.

T-794 is a new reversible inhibitor of MAO type A. In order to predict its clinical utility as an antidepressant, we examined its pharmacological profile (i.e., MAO inhibitory activity, antidepressant-like activity and safety) in vivo in rodents. The p.o. administration of T-794 potentiated L-5-hydroxytryptophan-induced symptoms with ED50 = 1.01 mg/kg (mice) or 1.15 mg/kg (rats), and L-dopa-induced behavior with ED50 = 5.90 mg/kg (mice), whereas it did not alter the effect of beta-phenylethylamine even at 100 mg/kg (mice). In the L-5-hydroxytryptophan test in rats, the activity of T-794 (at twice the dose of ED50) disappeared by 8 h; the duration of action was similar to that of moclobemide. These results confirm the previous biochemical results that MAO-A inhibition by T-794 is highly selective and of short duration. T-794 was effective in three animal models of depression: reserpine reversal (mice, rats), behavioral despair test (mice) and learned helplessness (rats). In these tests, it had potency similar to or greater than moclobemide, tranylcypromine or imipramine. The p.o. administration of T-794 (30 mg/kg) did not affect the pressor effect of tyramine in anesthetized rats, whereas moclobemide (30 mg/kg) and tranylcypromine (6 mg/kg) potentiated the effect. Acute toxicity of T-794 proved to be very low (maximal tolerated dose > 2 g/kg p.o.) in contrast to brofaromine (maximal tolerated dose = 150 mg/kg p.o.). Unlike tricyclic antidepressants, T-794 did not prevent the oxotremorine-induced tremor even at 100 mg/kg p.o.; in this it demonstrated a lack of the anticholinergic activity. These results suggest that T-794 is an effective and particularly safe antidepressant and that it may make an important contribution in the treatment of depressive disorders.

The contribution of erythropoietin- (EPO) receptors in target tissues, such as bone marrow and spleen, to the nonlinear pharmacokinetics of recombinant human EPO (rh-EPO) was evaluated in rats. The total body clearance after i.v. administration of rh-EPO (0.2-5 microg/kg) decreased as the dose of rh-EPO increased, approaching a plateau at high doses. The uptake clearance of 125I-rh-EPO by the target tissues, bone marrow and spleen, exhibited clear saturation. The Km values ranged from 240 to 450 pM, which are comparable with the reported value for the dissociation constant of EPO binding to EPO-receptors (180 pM) in rat bone marrow cells. A single s.c. administration of a large dose of rh-EPO (1 microg/kg) caused a reduction in tissue uptake clearance of 125I-rh-EPO by bone marrow and spleen (down-regulation). Furthermore, repeated intravenous injection of rh-EPO caused up-regulation of the tissue uptake clearance of 125I-rh-EPO, especially by the spleen, in a dose-dependent manner. Hematopoietic parameters such as hematocrit and hemoglobin concentration were also increased by repeated rh-EPO treatment and significantly correlated with the sum of the tissue uptake clearances in bone marrow and spleen. These findings suggest that the pharmacological receptor could be an important factor in defining the nonlinear pharmacokinetics of rh-EPO.

The NADP+-dependent dehydrogenase activity of a predominant isoenzyme of human liver 3alpha-hydroxysteroid dehydrogenase was activated by antihyperlipidemic drugs, such as bezafibrate and clinofibrate, and by clofibric acid and fenofibric acid (active metabolites of clofibrate and fenofibrate, respectively). The optimal pH of the activation by the drugs was about 7.5, and the concentrations giving maximum stimulation (1.8- to 2.4-fold) were 100, 50, 400 and 50 microM for bezafibrate, clinofibrate, clofibric acid and fenofibric acid, respectively. Clofibrate and fenofibrate acted as weak inhibitors, and the clofibric acid derivatives that lack the chloro group, methyl group on the alpha-carbon or carboxyl group greatly decreased the stimulatory effects. The activation by the drugs increased both Km and kcat (turnover number) values for the coenzyme and substrates. Kinetic analysis with respect to NADP+ showed that bezafibrate, clinofibrate, clofibric acid and fenofibric acid were nonessential activators showing dissociation constants of 32, 6, 103 and 11 microM, respectively. The combined activators experiments with one of the above drugs and sulfobromophthalein, a known activator specific for this enzyme, and comparison of their effects on the activities of mutant enzymes (with Met replacing Lys-270 or Arg-276) indicated that sulfobromophthalein and the drugs bind to an identical site on the enzyme. These results suggest that the long-term therapy with the antihyperlipidemic drugs influences the metabolism of steroid hormones, bile acids and several ketone-containing drugs mediated by the enzyme.

(+/-)-(E)-4-Ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (FK409) shows both potent in vitro vasorelaxant and antiplatelet activities via nitric oxide (NO) generated spontaneously from the compound. In this study, we measured spontaneous NO-releasing rates of a series of FK409 derivatives, of which chain lengths or substituents were systematically modified, in sodium-phosphate buffer solution at pH 7.4. Furthermore, we studied their in vitro antiplatelet and vasorelaxant effects to evaluate relationships between spontaneous NO-releasing activities of FK409 analogs and their biological activities. FK409 derivatives were found to possess different spontaneous NO-releasing rates and biological activities according to their structural modification. In addition, these studies revealed a close correlation between NO-releasing rates of FK409 derivatives and their in vitro antiplatelet activities in human platelet-rich plasma, whereas the in vitro vasorelaxant activities of these compounds in isolated rat aorta did not correlate with the rates of NO liberation. The vasorelaxant effects were supposed to be affected by the structural properties of FK409 derivatives as well as their NO-releasing abilities.

We examined the pharmacokinetics of recombinant human erythropoietin (rh-EPO) in genetically anemic mice (W/Wv genotype) to clarify its disposition mechanism in hematopoietic injury such as occurs in aplastic anemia. After rh-EPO was administered to W/Wv and control (+/+ genotype) mice once a day for 1 week at different doses, both the hematocrit (Hct) and tissue uptake clearance (CLup) of 125I-rh-EPO by spleen and bone marrow in the femur were estimated on the eighth day. The hematocrit increased on eighth day, depending on the dose administered. Compared with +/+ mice 10 times more rh-EPO was needed in W/Wv mice to produce an almost equivalent pharmacological effect. In +/+ mice, the CLup of 125I-rh-EPO by spleen increased to 4-fold that of controls after treatment with rh-EPO, 4.8 microg/kg, whereas that by bone marrow remained unchanged, irrespective of the dose administered. On the other hand, the increase in both the Hct and CLup in spleen was minimal in W/Wv mice. The CLup by bone marrow and spleen in both types of mice showed saturation with similar Km values (389-619 pM), comparable with the dissociation constant of the EPO receptor. In addition, the Hct correlated with the sum of the CLup by bone marrow and spleen in both types of mice, and the correlation lines were superimposable. These results suggest that the pharmacological receptors govern the saturable tissue uptake not only in normal mice but also in those that are anemic.

We studied changes in the pharmacokinetics of 125I-recombinant human erythropoietin (125I-rh-EPO) after repeated subcutaneous administration once a week for 4 weeks. The plasma level of trichloroacetic acid-precipitable radioactivity after the fourth administration of 125I-rh-EPO was minimal in 8 of 10 rats, whereas in the other two rats, the plasma level was almost the same or somewhat higher than that in control rats that had received the vehicle solution 3 times instead of the first three sequential administrations. Antibody against rh-EPO in serum was detected in all 10 rats receiving multiple administrations of 125I-rh-EPO. However, the binding capacity for 125I-rh-EPO in the latter two rats, assessed by an in vitro serum binding study, was lower than for the other eight rats, suggesting that the antibody level in these two was lower. The effect of intravenous preinjection of various volumes of anti-rh-EPO antiserum on the pharmacokinetics of 125I-rh-EPO was examined. The half-life in the beta-phase was prolonged at lower doses of antiserum. When the pretreatment dose of antiserum was further increased, the half-life in the beta-phase rather shortened and the total body clearance (CL(total) increased. These results suggest that repeated administration of rh-EPO induces the production of antibody against rh-EPO that affects the pharmacokinetics of rh-EPO in a biphasic manner; CL(total) was reduced when a small amount of antibody was produced, and CL(total) was increased when a large amount of antibody was produced.