Biomedical subjects

M Nakashima

Publications and source records attributed to M Nakashima.

At least 271 records · Page 15Linked to original sources

PubMed1995

Vessel wall injury and arterial thrombosis induced by a photochemical reaction.

Arterial thrombosis may be initiated in an experimental animal by a photochemical reaction between transmural green light and i.v. administered Rose Bengal, a photosensitizer dye. In this study, scanning electron microscopy has been used to reveal the nature of vessel injury and the cellular composition of the photochemically induced thrombus. A 5 mm segment of the guinea pig femoral artery was occluded by a thrombus about 10 min after irradiation with green light in the presence of systemically administered Rose Bengal. Electron microscopy revealed that following photochemical reaction, endothelial cells first contract and, with further irradiation, become detached from the vessel wall, with their cell membrane being destroyed at the irradiated site where an occlusive platelet-rich thrombus was formed. Endothelial cell injury and vessel occlusion could be completely inhibited by the aminothiol, DL-cysteine administered i.v. 1 min after Rose Bengal. The mechanism of endothelial injury in this model appears to be by singlet molecular oxygen, 1O2 formed by energy transfer from the photo-excited dye to O2.

Animals↗

PubMed1995

Comparison of antithrombotic effects of GPIIb-IIIa receptor antagonist and TXA2 receptor antagonist in the guinea-pig thrombosis model: possible role of TXA2 in reocclusion after thrombolysis.

The effects of Ro 44-9883, a new specific antagonist of platelet glycoprotein IIb-IIIa receptor, on thrombus formation and reocclusion after thrombolysis induced by tissue-type plasminogen activator (t-PA) were compared with those of vapiprost, a thromboxane (TX) A2 receptor antagonist, using a photochemically-induced thrombosis model in the guinea-pig femoral artery. Pretreatment with Ro 44-9883 (5, 10 and 20 micrograms/kg/min, i.v.) prolonged the time required to occlude the artery in a dose-dependent manner. Ro 44-9883 at 10 and 20 micrograms/kg/min significantly inhibited ex vivo platelet aggregation in whole blood induced by collagen, ADP or U46619. Vapiprost 0.3 mg/kg inhibited thrombus formation and platelet aggregation induced by collagen or U46619, to the same extent as Ro 44-9883 at the higher doses. In the thrombolysis study, Ro 44-9883 at the higher doses given as comedication with t-PA reduced the time to achieve reperfusion and increased the vascular patency after successful reperfusion. Vapiprost also significantly reduced the time to reperfusion and prevented reocclusion. However, the vascular patency after thrombolysis by t-PA with vapiprost was significantly increased compared with Ro 44-9883. Ro 44-9883 inhibited platelet aggregation, but did not prevent TXA2 formation in platelets. Thus, vascular contraction mediated by platelet-derived TXA2 may be responsible for lower efficacy of Ro 44-9883 against reocclusion compared with vapiprost. These results indicate that not only platelet aggregation but also vasoconstriction may contribute to reocclusion after t-PA-induced thrombolysis in the guinea-pig.

Acetates↗

PubMed1995

Isoproterenol causes hyperpolarization through opening of ATP-sensitive potassium channels in vascular smooth muscle of the canine saphenous vein.

Experiments were designed to determine how isoproterenol affects the cell membrane potential of smooth muscle cells of the canine saphenous vein. Measurements of membrane potential were performed using glass microelectrodes. Isoproterenol (10(-9) to 10(-6) M) caused sustained, concentration-dependent membrane hyperpolarizations in tissues with and without endothelium. ICI 118,551 (a selective beta 2-adrenoceptor antagonist), but not atenolol (a selective beta 1-adrenoceptor antagonist), abolished the hyperpolarization to isoproterenol. Forskolin, an activator of adenylate cyclase, produced sustained hyperpolarizations, which were not mimicked by 1,9-dideoxyforskolin. Incubation with either ouabain or extracellular K(+)-free solution did not inhibit the electrical response to isoproterenol. Glibenclamide (a selective ATP-sensitive K+ channel antagonist) attenuated the hyperpolarizations induced by either isoproterenol or forskolin, whereas charybdotoxin (an inhibitor of large conductance Ca(++)-activated K+ channels) did not. These findings suggest that isoproterenol opens ATP-sensitive K+ channels indirectly through activation of adenylate cyclase in the smooth muscle of the canine saphenous vein. The adrenergic receptor involved belongs to the beta 2-adrenoceptor subtype.

Animals↗

PubMed1995

Previous exposure to bradykinin unmasks an endothelium-dependent relaxation to the converting enzyme inhibitor trandolaprilat in isolated canine coronary arteries.

Experiments were designed to investigate the mechanism underlying the endothelium-dependent relaxation to trandolaprilat, a converting enzyme inhibitor, in canine coronary arteries previously exposed to bradykinin. Rings suspended in organ chambers were exposed to bradykinin for 3 min and washed repeatedly for 150 min. Trandolaprilat caused relaxations [IC50(-log M)8.59] in rings with endothelium previously exposed to bradykinin. This response was observed already at concentrations where trandolaprilat did not augment relaxations to bradykinin. When the rings were exposed to acetylcholine or to Des-Arg9-bradykinin (B1 agonist) trandolaprilat caused only a minimal response. Carboxypeptidase B but not aprotinin impaired the relaxation to trandolaprilat, suggesting a contribution of bradykinin. After exposure to [3H]-bradykinin, no detectable amounts of the peptide were released by trandolaprilat or found in the preparations. The relaxation to trandolaprilat was not affected by the B1 antagonist Leu8-des-Arg9-bradykinin. HOE-140, a B2 antagonist impaired the maximal response to trandolaprilat, while exhibiting competitive antagonism against bradykinin (pA2 9.00). The maximal relaxation to trandolaprilat was impaired in the presence of nitro-L-arginine and methylene blue. The potency, but not the maximal effect of bradykinin was reduced by these inhibitors. Exogenous bradykinin, but not trandolaprilat, caused an endothelium-dependent hyperpolarization. At equipotent submaximal concentrations, bradykinin released both nitric oxide and endothelium-derived hyperpolarizing factor although trandolaprilat stimulated the production of nitric oxide only. These findings suggest that acute endothelium-dependent relaxations to trandolaprilat in preparations previously exposed to bradykinin are mediated by nitric oxide and may be due partially to protection of bound bradykinin but also to some other unresolved mechanism(s) as well.

Animals↗

PubMed1995

Parathyroid hormone-related peptide expression in endocrine tumors.

Parathyroid hormone-related peptide (PTHrP) expression is associated with the histological type in pituitary tumor, but not in thyroid carcinoma. However invasive and metastatic tumors showed intense PTHrP staining in both pituitary and thyroid tumors. Analysis of the established metastatic rat pituitary tumor cell line showed that PTHrP plays a crucial role in in vivo cell proliferation closely related to worsening of malignant transformation and neovascularization in a paracrine fashion.

Animals↗

PubMed1995

Establishment of a melanoma cell line with metastatic characteristics.

We report here the establishment and characterization of a human melanoma cell line derived from the ascitic fluid of a patient with metastatic malignant melanoma with peritonitis carcinomatosa. This cell line was easily transplanted into nude mice. Flow cytometry and immunocytochemical analyses demonstrated that the cells expressed melanotransferrin and S-100 protein. Expression of cell adhesion and HLA molecules on the cells was also investigated. The results indicate that the cell line possesses some metastatic characteristics.

Aged↗

PubMed1995

The nitrate ester ITF 296 relaxes isolated canine arteries and veins.

Experiments were designed to compare the relaxant activity of the new nitrate ester ITF 296 in isolated arteries and veins of the dog and to determine the extent of modulation by the endothelium of the response to the compound. Rings (with and without endothelium) of coronary, basilar, mesenteric, and femoral arteries and of mesenteric, femoral, and saphenous veins were suspended in organ chambers for measurement of changes in isometric tension. In all blood vessels without endothelium, ITF 296 caused concentration-dependent relaxation. The order of potency (from measurements of ED50) was: basilar artery > or = coronary artery > femoral artery > mesenteric vein > femoral vein > saphenous vein > mesenteric artery. The maximal relaxation to ITF 296 was greater in the arteries (with the exception of the mesenteric) than in veins. In all blood vessels except for the basilar artery, nitroglycerin caused larger relaxations than ITF 296: The ED50 for nitroglycerin was comparable in all blood vessels studied except for the mesenteric artery, where it was less. ITF 296 did not cause hyperpolarization of vascular smooth muscle in coronary arteries either with or without endothelium. The presence of endothelial cells blunted the relaxation to ITF 296 in basilar and coronary arteries, but not in the other blood vessels studied. These results demonstrate that ITF 296 has a direct inhibitory effect on vascular smooth muscle, which is most pronounced in coronary and cerebral arteries. These findings are in line with the vascular selectivity profile reported for the compound in vivo.

Animals↗

PubMed1994

Enhancement of thrombolytic efficacy of tissue-type plasminogen activator by adjuvants in the guinea pig thrombosis model.

Reocclusion following thrombolysis is a major limitation of thrombolytic therapy with recombinant tissue-type plasminogen activator (rt-PA). We investigated the effects of vapiprost ((1R-(1 alpha(Z),2 beta,3 beta,5 alpha))-7-(5-((1,1'-biphenyl)-4-yl-methoxy)- 3-hydroxy-2-(1-piperidinyl)cyclopentyl)-4-heptenoic acid, a thromboxane A2 receptor antagonist); argatroban ((2R,4R)-4-methyl-1-[N2-(3-methyl-1,2,3,4-tetrahydro-8-quinolinyl)sulfon yl] - L-arginyl)]-2-piperidine-carboxylic acid, a specific thrombin inhibitor) and MK-886 (3-[1-(4-chlorobenzyl)-3-t-butyl-thio-5-isopropylindol-2-yl]-2,2- dimethylpropanoic acid, a specific leukotriene biosynthesis inhibitor) on the thrombolytic efficacy of rt-PA. The guinea pig femoral artery was thrombotically occluded by photochemical reaction between rose bengal and green light. Thirty min after the occlusion, rt-PA was administered and the time (T1) for reopening of the vessel and the frequency of reocclusion (Fro) 24 h after thrombolysis were monitored. With rt-PA alone, T1 was 28 +/- 7 min (n = 10) and Fro was 70%. T1 was reduced to 9 and 20 min by a combination of rt-PA with vapiprost and argatroban respectively. Fro was reduced by all three adjuvants. Histological observations revealed extensive adherence of polymorphonuclear leucocytes to the damaged endothelium at the site of thrombolysis. It is concluded that thromboxane A2, thrombin and leucocytes are involved in reocclusion after thrombolysis.

Adjuvants, Pharmaceutic↗

PubMed1994

Thrombolytic efficacy of a modified tissue-type plasminogen activator, SUN9216, in the rat middle cerebral artery thrombosis model.

We have developed a model whereby the middle cerebral artery in an experimental animal can be occluded by a photochemical reaction between rose bengal and green light. This causes endothelial injury followed by platelet adhesion, aggregation and formation of a platelet-rich thrombus at the site of the photochemical reaction. SUN9216, a modified tissue-type plasminogen activator, is a new thrombolytic agent which consists of the fibrin kringle 1 domain of plasminogen and the two kringles, the serine protease domains of the native tissue-type plasminogen activator. The mannose glycosylation site on the kringle 1 of tissue-type plasminogen activator is modified to yield a compound with a longer half-life in the blood than native tissue-type plasminogen activator. We evaluated the thrombolytic effects of recombinant tissue-type plasminogen activator and SUN9216 in the thrombotically occluded rat middle cerebral artery. SUN9216 was administered by continuous infusion or as a single bolus injection 30 min after the middle cerebral artery had been occluded by a thrombus. Both SUN9216 and recombinant tissue-type plasminogen activator caused reopening of the middle cerebral artery by thrombolysis. The efficacy of SUN9216 was higher than that of recombinant tissue-type plasminogen activator. Further, the area of ischaemic cerebral damage caused by the middle cerebral artery occlusion was significantly (P < 0.05) reduced by SUN9216, but in this respect, recombinant tissue-type plasminogen activator was ineffective.

Analysis of Variance↗

PubMed1994

In vitro expansion of tumor-specific, HLA-restricted human CD8+ cytolytic T lymphocytes.

The effect of cytokines with human recombinant interleukin-2 (rIL-2) on cytolytic T cell (CTL) generation was studied. Lymphocytes were isolated from involved lymph nodes of melanoma patients and expanded in medium containing rIL-2 alone or in combination with other human cytokines (rIL-1, rIL-4, rIL-6, and recombinant human tumor necrosis factor-alpha (rTNF alpha)). Lymphocytes incubated with rIL-2 alone did not grow, whereas addition of the other cytokines augmented IL-2-mediated lymphocyte proliferation. In all cultures, the majority of expanded lymphocytes were CD3+, CD56- T cells. Lymphocytes cultured with rIL-1, rIL-2, rIL-4, and rIL-6 exhibited cytolytic activity specific for autologous melanoma, which increased during the culture period (24.08 and 58.18% at 16 and 30 days in culture, respectively) without detectable changes in cell surface phenotype and remained high even after 100 days in culture. Moreover, the cytolytic activity was inhibited by monoclonal antibodies (mAbs) against HLA-class I, CD3, and CD8 molecules but not by mAbs against HLA-class II or CD4 molecules. Lymphokine-activated killer (LAK) activity was detected in lymphocytes cultured with rIL-1, rIL-2, and rIL-6 in the presence or absence of rTNF alpha. These data indicate that lymphocytes derived from melanoma-invaded lymph nodes and cultured in the presence of rIL-1, rIL-2, rIL-4, and rIL-6 offers an efficient system to expand CD8+ CTLs with HLA-restricted cytolytic specificity against autologous tumor cells.

Antigens, CD↗

PubMed1994

Simultaneous determination of ofloxacin, norfloxacin and ciprofloxacin in human hair by high-performance liquid chromatography and fluorescence detection.

A high-performance liquid chromatographic method for the simultaneous determination of ofloxacin, norfloxacin and ciprofloxacin in human hair is described. A reversed-phase C18 column and a fluorescence detector with switching fluorescence wavelengths were used together with solid-phase extraction of the drugs from hair dissolved in 1 M sodium hydroxide. Reproducibility and linearity studies yielded coefficients of variation of 0.2-2.2, 1.4-3.1 and 1.5-3.4%, and correlation coefficients of 1.000, 0.999 and 0.999 within the concentration range 0.3-100 ng/ml for ofloxacin, norfloxacin and ciprofloxacin, respectively. For validation, hair samples were obtained from six subjects who had been taking one or two of the three fluoroquinolones. Assuming a hair growth-rate of 1 cm per month fluoroquinolones could be detected in the hair section(s) that had grown approximately between the dates of drug administration and hair sampling.

Adult↗

PubMed1994

Effect of 21-aminosteroid lipid peroxidation inhibitor, U74006F, in the rat middle cerebral artery occlusion model.

The aim of this study was to evaluate the effect of 21-aminosteroid lipid peroxidation inhibitor, U74006F, on ischaemic brain tissue damage using the rat middle cerebral artery occlusion model. Under anaesthesia, the left middle cerebral artery was exposed without cutting the dura mater via a subtemporal craniotomy, under an operating microscope. Photo-illumination (wave length; 540 nm) was applied to the middle cerebral artery and then rose bengal (20 mg/kg) was administered intravenously. The middle cerebral artery was completely occluded by thrombus about 6 min after the administration of rose bengal. U74006F (1.0 mg/kg) was then injected intravenously just after the cessation of illumination. Twenty four hours after the operation, the extent of ischaemic damage was measured by magnetic resonance imaging technique. After measuring the extent of ischaemic damage, the brain was immediately removed from animals treated with or without U74006F for determination of lipid peroxidation, and the generation of free arachidonic acid in the brain. U74006F significantly (P < 0.01) reduced the size of ischaemic damage. Twenty-four hours after the operation, lipid peroxidation and the concentration of free arachidonic acid in the left hemisphere (infarction side) were significantly (P < 0.05) higher than in the right hemisphere. U74006F significantly (P < 0.05) decreased the content of lipid peroxidation products and free arachidonic acid. There was a significant (P < 0.05) correlation between the extent of ischaemic damage and the concentration of lipid peroxidation products in the left hemisphere 24 h after the operation. In conclusion, U74006F might reduce the extent of ischaemic damage by inhibiting lipid peroxidation in the brain, thus minimizing oxidative damage to neural tissues.

Animals↗

PubMed1994

Phase I study of E1077, a novel parenteral cephem antibiotic.

The safety and pharmacokinetics of E1077, a new injectable cephem antibiotic, were evaluated in healthy male adult volunteers. In the single-dose studies, 100, 250, 500, 1,000, and 2,000 mg of E1077 were administered by intravenous infusion at a constant rate for 60 minutes, then 1,000 mg of the drug by intravenous infusion at a constant rate for 5 minutes. The Cmax were 6.4, 15.7 +/- 12.0, 34.7 +/- 4.6, 63.2 +/- 4.6, 142.7 +/- 5.6, and 131.6 +/- 36.0 (means +/- SD) micrograms/mL, respectively, and the Cmax and AUC increased linearly with the dose. Plasma concentration-time curves were well described by a two-compartment open model. The plasma elimination half life of the drug was 1.88 +/- 0.15 hours. The mean urinary recovery within the first 24 hours was 94.1 +/- 5.1% of the dose. In the multiple-dose study, 2,000 mg of E1077 was intravenously administered at a constant rate over 60 minutes every 12 hours for 4.5 days (a total of nine times). The Cmax after the first and ninth doses were 134.0 +/- 17.4 and 135.5 +/- 15.5 micrograms/mL, respectively, and trough levels in day 1 and day 5 (at 12 hours after the first and ninth administration, respectively) were 2.2 +/- 0.8 and 1.9 +/- 0.4 micrograms/mL, respectively. No accumulation of the drug in plasma was observed. There were no significant differences in plasma levels or in the urinary recoveries between the single- and multiple-dose regimens.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1994

Pharmacokinetic and pharmacodynamic profiles of CS-518, a selective, long-lasting thromboxane synthase inhibitor, after single and multiple oral administration to healthy volunteers.

A selective thromboxane (TX) synthase inhibitor, CS-518, was orally administered to healthy male Japanese volunteers and the pharmacokinetic and pharmacodynamic properties were investigated. The time profile of drug concentrations in plasma was determined, and the effects of the drug on platelet aggregation in plasma induced by arachidonic acid (AA) and adenosine diphosphate (ADP) ex vivo were examined. The production of TXB2 and 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) in serum during whole blood coagulation ex vivo also were examined. In the single-dose study (50, 100, and 200 mg), plasma concentrations of the drug were well fitted to a one-compartment open model with first-order absorption. The area under plasma concentration curve (AUC) and maximum plasma concentration (Cmax) showed dose-related increases, whereas the mean elimination half-lives remained rather constant (.68-.92 hour). The drug was recovered in urine by 32 to 37% and 62 to 65% as unchanged and conjugated forms (acylglucuronide), respectively, showing almost complete absorption of CS-518. The effect of food intake on the pharmacokinetics of CS-518 was determined at the dose of 100 mg. The time to reach Cmax was prolonged from .42 to 2.08 hours and the Cmax was decreased by about 66%, whereas the AUC and urinary recovery showed no significant changes. The platelet aggregation in plasma induced by AA was markedly inhibited, whereas the secondary aggregation induced by ADP was inhibited to a much less degree. Platelet aggregation by AA was almost completely inhibited 2 hours after administration of any dose and the duration for maintaining the significant inhibition tended to depend on the dose ranging from 48 to 72 hours after administration, which was much longer than expected from the plasma concentration of drug.(ABSTRACT TRUNCATED AT 250 WORDS)

6-Ketoprostaglandin F1 alpha↗

PubMed1994

Pharmacokinetics and safety of NM441, a new quinolone, in healthy male volunteers.

The safety and pharmacokinetics of NM441, a prodrug of a new thiazeto-quinoline carboxylic acid derivative, NM394, were evaluated in healthy male volunteers given the drug orally in single doses of 20, 50, 100, 200, and 400 mg, and multiple doses of 300 mg twice daily for 6.5 days. No remarkable abnormalities were observed in symptoms, physical tests, laboratory tests, electrocardiogram (ECG), electroencephalogram (EEG), or equilibrium test. The mean plasma concentrations of active metabolite NM394 peaked between 0.5 and 1.0 hours, and the maximum concentrations were 0.68, 1.09, and 1.88 micrograms/mL at doses of 100, 200, and 400 mg, respectively. The mean half-lives were 7.7 to 8.9 hours and were not affected by dose. The mean urinary excretion rates of NM394 were 46.0, 38.3, and 30.6% of the doses within 48 hours, respectively, and other metabolites were excreted in urine by 7% of the doses. The mean salivary concentrations of NM394 were approximately 20% of the plasma concentrations. The mean fecal excretion rates of NM394 and NM441 were 52.9 and 4.2%, respectively within 72 hours after dosing of 400 mg. The Cmax, AUC, and urinary excretion rates were not altered by food intake, whereas the Tmax was prolonged slightly. In the multiple-dose study, the steady state of plasma concentration of NM394 was achieved on day 3 or 4, and further accumulation did not occur thereafter. The mean urinary excretion rate of NM394 was 49.0% during and 48 hours after the multiple administration. The acceptable safety and tolerance and defined pharmacokinetic characteristics of NM441 support further testing.

Administration, Oral↗

PubMed1994

Measurement of temafloxacin in human scalp hair as an index of drug exposure.

Scalp hair samples were obtained at 1-month intervals up to 3 months from healthy male volunteers participating in a phase I study of a new antimicrobial quinolone, temafloxacin. Hair was sectioned into 1-cm lengths from the scalp end. After corresponding portions from 10 pieces of hair were dissolved in 1 N NaOH, temafloxacin was extracted by chloroform and measured by HPLC equipped with fluorescence detection (excitation and detection wavelengths, 280 and 460 nm, respectively). In all subjects taking a single oral dose (600 mg; n = 6) or repeated oral doses (900 mg/day, b.i.d., for 6.5 days; total, 5850 mg; n = 6), the drug was detected in hair. The 1-cm length of hair, in which temafloxacin was peaked, was shown to move at the rate of 1 to 1.3 cm/month when the hair of growing stage was analyzed. The largest sum of the concentrations of temafloxacin found in any set of measurements in hair in each subject was 31.7 +/- 15.0 (mean +/- SD, n = 6) and 226.3 +/- 99.4 ng/mg hair (n = 6) for the single and repeated doses, respectively. The drug concentration in hair increased by a factor of 7.1, whereas the ratios between the single and repeated doses were 9.8, 1.3, and 10.7 for the total given dose, observed maximal plasma concentration (Cmax), and area under the plasma concentration curve, respectively. It was obvious that the Cmax was the least likely factor determining temafloxacin concentration in hair.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1994

Pharmacokinetic properties of a novel gastric proton pump inhibitor, (+/-)-2-[(4-methoxy-6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9- yl)sulfinyl]-1H-benzimidazole sodium salt, in healthy subjects.

The pharmacokinetics and safety of TY-11345 [(+/-)-2-[(4-methoxy-6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-9- yl)sulfinyl]-1H-benzimidazole sodium salt], a novel gastric proton pump inhibitor, were studied in healthy male volunteers after single (20, 40, and 80 mg) and repeated oral doses (60 mg, once daily for 7 days) as enteric-coated tablet. At the single doses of 20 and 40 mg, intragastric pH was continuously monitored in each of two subjects. No abnormal findings definitely attributable to the test drug were observed throughout the study. In the single-dose study, the maximum plasma concentration (Cmax) and area under the plasma concentration-time curve of TY-11345 increased in a dose-proportional manner. The time to reach Cmax was about 3 h after dosing and plasma concentrations declined thereafter with a half-life of about 1 h irrespective of dose. The effect of food intake on the pharmacokinetic parameters of TY-11345, which was evaluated at the dose of 40 mg in a cross-over design, was not significant. TY-11345 was not detected in urine unchanged, while a main metabolite and its conjugate were identified in urine as 32-38% of the dose. An intragastric pH value over 4 was obtained about 3 h after the administration of 40 mg and maintained for more than 5 h, despite the fall of plasma concentration. This effect was less obvious at a dose of 20 mg. In the multiple-dose study, the pharmacokinetics exhibited no substantial difference between the first and last doses.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Oral↗

PubMed1994

Regulatory role of transforming growth factor-beta, bone morphogenetic protein-2, and protein-4 on gene expression of extracellular matrix proteins and differentiation of dental pulp cells.

The expression of developmental stage-specific genes during pulp cell differentiation into preodontoblasts was examined in bovine adult pulp cell culture. When proliferation was down-regulated after 14 days of primary culture, expression of fibronectin and type I and type III collagen mRNAs was increased. Expression of alkaline phosphatase was gradually increased, and mRNA for osteocalcin, a marker of preodontoblast, appeared just before the onset of mineralization. Contrarily, in expanded culture, the expression of mRNA for the extracellular matrix proteins was gradually increased from the beginning of culture up to Day 28. Similarly, mRNA levels of alkaline phosphatase and osteocalcin were also increased gradually. Expression of TGF-beta 1 mRNA disappeared on Day 21 in the primary culture when expression of alkaline phosphatase mRNA was increased. BMP-4 mRNA was expressed on Day 14 when the expression of the extracellular matrix proteins was increased. BMP-2 mRNA was expressed on Day 28 when osteocalcin appeared. Recombinant TGF-beta 1 inhibited alkaline phosphatase activity, while BMP-2 and BMP-4 stimulated it. BMP-4 increased expression of alpha 1(I) collagen mRNA, and BMP-2 increased osteocalcin synthesis. These results demonstrate the regulatory role of these TGF-beta superfamily members on the gene expression of extracellular matrix proteins and the differentiation of pulp cells into preodontoblasts.

Alkaline Phosphatase↗