Biomedical subjects

T Kumada

Publications and source records attributed to T Kumada.

At least 55 records · Page 3Linked to original sources

PubMed1999

[Clinical effectiveness of degradable starch microspheres (DSM) in patients with liver cancer].

Recently, degradable starch microspheres (DSM) have become available for use in patients with liver cancer in Japan. When DSM combined with a cytotoxic drug are infused through the hepatic artery, the steep drug concentration gradient to the tumor tissue results in a higher tissue drug concentration, which may elicit an increased antitumor response by blocking regional blood flow. Furthermore, the reduced systemic exposure of a coinjected drug can be translated into an increased regional extraction ratio due to blood flow reduction. DSM is infused via a catheter connecting to a subcutaneously implanted reservoir in outpatients. Pain is experienced by all patients. Other frequently observed adverse reactions are nausea and vomiting. However, these symptoms improve within a few hours. These observations indicate that intra-arterial chemotherapy combined with DSM may provide a more potent anticancer effect than a cytotoxic drug alone.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed1998

High-dose (9 MU) long-term (60 weeks) alfa-interferon therapy for chronic hepatitis patients infected with HCV genotype 1b.

Efficacy of standard regimens (e.g., 3-6 MU for 24 weeks) of alfa-IFN therapy for chronic hepatitis C has been limited, particularly in patients with HCV/1b. To see if higher-dose longer term treatment is more effective, we tried a 9 MU 60-week regimen. HCV/1b-infected chronic hepatitis patients received 9 MU IFN alpha 2a everyday but Sunday for 2 weeks and thrice a week for next 10 weeks, and 76 patients became HCV RNA-negative while 81 remained positive. The RNA-negative patients were then randomized to receive 3 MU (group I, n = 37) or 9 MU (group II, n = 39) for 48 weeks. Of the RNA-positive patients, only those with normal ALT received another 9 MU 48-week treatment (group III, n = 45). Sustained responders (SR) were defined as those with negative RNA and normal ALT 6 months after the therapy. SR rates based on intent-to-treat principle did not differ significantly between groups I and II (30% vs 41%), but those based on the protocol-compatible cases showed a significantly lower than those in group II. Adverse effects of IFN, developed more frequently in groups II and III than in group I, were mostly reversible. In conclusion, our results encourage 9 MU 60-week IFN alpha treatment in HCV/1b-infected patients with careful attention to adverse effects, and suggest that the treatment should be discontinued if HCV RNA does not disappear within 12 weeks.

Genotype↗

PubMed1998

Characteristics of patients with chronic infection due to hepatitis C virus of mixed subtype: prevalence, viral RNA concentrations, and response to interferon therapy.

We studied 349 patients with chronic infection due to hepatitis C virus (HCV), including 83 with coagulopathy, 122 with community-acquired infection, 20 with chronic posttransfusion hepatitis C, and 124 for whom the transmission mode was unknown. The prevalence of mixed-subtype HCV infection was investigated in each group of patients. The serum HCV RNA concentration and the response to interferon (IFN) therapy were evaluated. HCV infection with mixed subtypes was more frequent in patients who had been at high risk for exposure to HCV, such as those with coagulopathy or community-acquired infection. HCV RNA concentrations were lower in patients infected with mixed subtypes, except for those with subtypes 1a and 1b. Ten of 11 patients with mixed subtypes (not including those with 1a and 1b) achieved complete clearance of HCV RNA with IFN therapy.

Adolescent↗

PubMed1998

Quasispecies nature of hepatitis C virus (HCV) in patients with chronic hepatitis C with mixed HCV subtypes.

The quasispecies nature of hepatitis C virus (HCV) in patients with mixed HCV subtype infection was compared with that in patients with single HCV subtype infection. The number of HCV quasispecies was compared between 35 patients with mixed HCV subtype infection and 83 patients with single subtype infection. Subtype was determined by primers deduced from the core region and by line probe assay respectively. The number of quasispecies was evaluated by polymerase chain reaction amplification of hypervariable region 1 and by fluorescence single-strand conformation polymorphism analysis. There was no difference in clinical background between patients with mixed subtype infection and patients with single subtype infection. The number of quasispecies in patients with multiple subtype HCV infection was larger than in patients with single subtype HCV infection. The immunologic environments which allow the coexistence of more HCV quasispecies in patients with multiple HCV subtype infection differs from that in patients with single HCV subtype infection.

Adult↗

PubMed1998

[The usefulness of percutaneous ethanol injection therapy under guidance with carbon dioxide contrast enhanced ultrasound sonography].

We evaluated the usefulness of percutaneous ethanol injection therapy (PEIT) under Carbon dioxide (CO2) contrast enhanced ultrasound sonography (CEUS) guidance during digital subtraction angiography (DSA) in 21 cases of hepatocellular carcinoma (HCC) with 28 nodules that could not be detected by plain (non-contrast enhanced) US (PUS). In all cases of HCC that could not be visualized by PUS, PEIT could be performed successfully under CEUS guidance. Tumor size was below 10 mm in most cases, in 2 cases tumor size was more than 20 mm. Tumor location was roughly divided into 5 areas: just below the diaphragm and it's vicinity, liver surface, edge of the liver, around the portal and hepatic vein, and around the inferior vena cave. The detection rate of the nodules that could not be visualized with PUS was 35.7% for CT and 32.1% for DSA. PEIT was performed 1-9 times for each lesion, 3.32 times on an average. The effectiveness of PEIT was judged by CT. All cases were confirmed as LDA. We concluded that the range of indication of PEIT can be expanded by this method.

Carbon Dioxide↗

PubMed1997

Quasispecies nature of hepatitis C virus and response to alpha interferon: significance as a predictor of direct response to interferon.

BACKGROUND/AIMS: We evaluated the significance of the quasispecies nature of HCV as a predictor of the response to alpha interferon therapy in patients with chronic hepatitis C. METHODS: Natural alpha interferon was administered in 62 patients for 24 weeks (daily for 2 weeks, then three times weekly for 22 weeks) and factors were analyzed that could affect the response. HCV subtype, HCV RNA concentrations and the number of HCV quasispecies were evaluated before treatment. HCV RNA concentrations were measured by branched DNA probe assay. The number of HCV quasispecies was measured by fluorescence single-strand conformation polymorphism analysis. RESULTS: The HCV RNA concentration (p < 0.0001), HCV subtype (p = 0.0076), and the number of HCV quasispecies (p = 0.0024) were significantly associated with a complete response. Multivariate analyses showed that the number of HCV quasispecies was an independent predictor of the disappearance of HCV RNA during the administration of alpha interferon, but did not predict a relapse after its completion. Pretreatment concentration of HCV RNA was the only factor that was related to a long-term disappearance of HCV RNA. CONCLUSIONS: The number of HCV quasispecies was significantly related to the response to alpha interferon early in its administration. The pretreatment concentration of HCV RNA was mainly related to a relapse following completion of treatment.

Adult↗

PubMed1997

Interferon-alpha therapy in patients dually infected with hepatitis C virus and GB virus C/hepatitis G virus--virological response of HGV and pretreatment HGV viremia level.

BACKGROUND/AIMS: The response to interferon-alpha (IFN) therapy of recently isolated GB virus C and hepatitis G virus (HGV) is still unclear. To investigate the biochemical and virological response to IFN therapy in patients with chronic hepatitis C virus (HCV) infection concomitantly infected with HGV, 196 patients with HCV who had received IFN therapy were retrospectively studied. METHODS: HGV and HCV RNA were detected by reverse transcription nested polymerase chain reaction (RT-PCR). Serum HGV RNA levels were quantified by competitive RT-PCR. The HGV genotype was detected by restriction fragment length polymorphism analysis using the PCR products. RESULTS: Of 196 patients, 16 (8.2%) were positive for both HCV and HGV RNA before IFN therapy. There were no significant clinical and virological differences between the patients with dual infection and those with only HCV infection. During the therapy, a decrease or loss of serum HGV RNA level was observed in these patients. Six months after cessation of the therapy, five of 16 patients became negative for HGV RNA by RT-PCR. The pretreatment HGV RNA level of the patients who lost HGV RNA after cessation of IFN was low (median=10(3) copies/ml), compared to the level (median=10(7) copies/ml, p<0.01) in the patients with positive HGV RNA after the therapy. The HGV genotype of these 16 patients was the same type. CONCLUSIONS: These data suggest that: 1) there is no significant difference in response to IFN therapy between patients with dual and single infection; 2) HGV shows sensitivity to IFN therapy; and 3) in the patients who show a low pretreatment HGV RNA level, serum HGV RNA becomes undetectable by RT-PCR after cessation of IFN therapy.

Adult↗

PubMed1997

Changes in blood supply in small hepatocellular carcinoma: correlation of angiographic images and immunohistochemical findings.

BACKGROUND/AIMS: To assess the changes occurring in blood flow with growth in small hepatocellular carcinomas, we analyzed the angiographic features and immunohistochemical findings in 35 hepatocellular carcinomas less than 2 cm in diameter. METHODS: Hepatocellular carcinomas were evaluated by digital subtraction angiography (DSA), ultrasound angiography with intraarterial CO2 microbubbles (USAG), and computed tomography during arterial portography (CTAP). Immunohistochemically, hepatocellular carcinomas were evaluated using QB-end/10 (QB) monoclonal antibody. RESULTS: All 18 moderately-differentiated hepatocellular carcinomas stained positively with QB antibody. No hepatocellular carcinomas without attenuation on CTAP were positive by immunohistochemistry, and two hepatocellular carcinomas with attenuation on CTAP also lacked staining. We observed four hepatocellular carcinomas without hypervascularity on DSA or USAG, which stained positively with QB antibody; these hepatocellular carcinomas had fatty metamorphosis. CONCLUSIONS: 1. Immunohistochemical findings are closely associated with angiographic findings regarding changes in blood supply. 2. All moderately-differentiated hepatocellular carcinomas have characteristics of hypervascularity, both by angiographic images and by immunohistochemistry. 3. The increase in arterial blood supply occurs later than the decrease in portal perfusion, which may indicate that the decrease in portal perfusion may not be the direct result of replacement by angiogenesis. 4. Some hepatocellular carcinomas with fatty metamorphosis, which are often hypovascular by angiographic evaluation, have hypervascular immunohistochemical characteristics.

Aged↗

PubMed1997

Patterns of recurrence after initial treatment in patients with small hepatocellular carcinoma.

To assess intrahepatic metastasis (IM) and multicentric occurrence (MO) after initial treatment of small hepatocellular carcinomas (HCC) < or = 2 cm in diameter, we performed clinical and pathological studies in 112 patients who underwent percutaneous ethanol injection therapy (PEIT) or hepatic resection for HCC from January 1985 to December 1994. Patients with intrahepatic recurrences were classified into two groups based on the type of recurrence: the IM group (n = 29, 50.9%) and the MO group (n = 28, 49.1%). Overall recurrence rates after initial treatment were 23.7% at 1 year, 64.5% at 3 years, and 76.1% at 5 years. In patients with IM, the majority of intrahepatic recurrences were observed within 3 years of initial treatment and the primary HCC lesions were closely related to the degree of tumor cell differentiation. Alternatively, intrahepatic recurrences occurred throughout the follow-up period in patients with MO, and the evidence of underlying liver disease (anti-HCV [antibody to hepatitis C virus] positive) and elevated serum alfa-fetoprotein (AFP) concentrations were closely associated with intrahepatic recurrence. Prognoses following additional treatment in MO group patients were superior to those in IM group patients. These results suggest that differentiation between IM and MO in patients with HCC is important for understanding the development and biological behavior of the tumor. That is, the early detection of intrahepatic recurrence and the institution of appropriate additional therapy (PEIT or hepatic resection) may prolong survival in patients with MO.

Adult↗

PubMed1997

Significance of early measurement of serum hepatitis C virus RNA in predicting response to interferon therapy in patients with chronic hepatitis C.

BACKGROUND: Interferon can induce complete clearance of hepatitis C virus (HCV) in some patients with chronic hepatitis C. However, various side effects often require cessation of administration during the treatment period. Early prediction of response to interferon would be helpful. We evaluated measurement of serum HCV RNA 2 weeks after starting interferon as a predictor of response. METHODS: The presence of HCV RNA was measured in serum 2 weeks after starting therapy in 85 patients receiving natural interferon a (total 480 MU). RESULTS: Twenty-seven of 38 patients (71.1%) in whom serum HCV RNA had disappeared at 2 weeks achieved a sustained response. Only two out of 47 patients (4.3%) in whom serum HCV RNA had not disappeared at 2 weeks achieved a sustained response. Of 42 patients with pre-treatment HCV RNA concentrations less than 1 x 10(6) eq/ml, 26 of 30 patients (86.7%) whose HCV RNA had disappeared at 2 weeks achieved a sustained response, while only one of 12 patients (8.3%) whose HCV RNA was still detectable at 2 weeks had a sustained response. CONCLUSION: Clearance of serum HCV RNA after 2 weeks of treatment with interferon was more likely in patients with lower pre-treatment HCV RNA concentrations and they had a high likelihood of achieving a sustained response. In patients in whom serum HCV RNA was still detectable after 2 weeks of interferon therapy, a sustained response was most unlikely.

Adult↗

PubMed1997

Astrocytic lineage analysis by detection of GFAP promoter activity in vitro.

To survey the emergence and onset of differentiation of the astrocytic lineage in the developing mouse cerebral wall, the promoter activity of a 2.5 kb 5'-flanking region of glial fibrillary acidic protein (GFAP) was measured in individual developing brain cells using a retrovirus-mediated gene transfer system. We identified precursors for astrocytes in primary culture of embryonic mouse cerebral wall cells by detection of GFAP promoter activity, which was detected approximately 3 days prior to the appearance of GFAP immunoreactivity. Since retroviruses only integrate into the chromosomes of actively proliferating cells, cells detected by this method should have been mitotically active at the time of retroviral infection on day 15 postfertilization (E15). Furthermore, we observed that cells activating GFAP promoter were located near the ventricular surface of cultured cerebral wall slices as a cluster of spherical cells. These results demonstrate that precursor cells for astrocytes exist within the germinative zone of developing cerebral wall, and that these cells are mitotically active on day E15, which is a late stage of neuronal production period in the mouse cerebral wall. The morphology, location and mitotic activity of these cells suggest that they are unlikely to be cells that have been transformed from radial glial cells.

Animals↗

PubMed1997

Determining the optimum dose for the intravenous administration of nicardipine in the treatment of acute heart failure--a multicenter study. The Nicardipine Heart Failure Study Group.

Nicardipine is a potent arteriolar vasodilator with a negligible negative inotropic effect. Although intravenous administration of this drug has been reported to be effective in the treatment of heart failure, the optimal dose by this route is not clear. This study was designed to determine the optimum dose for the intravenous infusion of nicardipine in the treatment of heart failure. In Trial 1, nicardipine was administered intravenously at a dose of 0.5 microgram/kg per min to 14 patients with acute heart failure. The dose was increased to 1.0 microgram/kg per min in 13 cases with marked improvement at 2 h. In Trial 2, nicardipine was administered in a double-blind manner to 53 patients at 3 different rates of infusion for 2 h: 1.0 (Group 1, n = 19), 2.0 (Group 2, n = 15), and 3.0 (Group 3, n = 19) micrograms/kg per min. Neither heart rate nor mean right atrial pressure changed in any of the 3 groups. Favorable hemodynamic effects were evident in all groups beginning 30 min after the start of infusion, with an increase in cardiac index (control vs 2 h after infusion, L/min per m2) (Group 1: 2.2 +/- 0.4 vs 3.1 +/- 0.8, Group 2: 2.2 +/- 0.4 vs 2.9 +/- 0.5, Group 3: 2.3 +/- 0.3 vs 3.1 +/- 0.7, all p < 0.01 compared to the control) and a decrease in diastolic pulmonary artery pressure (Group 1: 26 +/- 10 vs 19 +/- 7, Group 2: 27 +/- 10 vs 20 +/- 8, Group 3: 26 +/- 7 vs 18 +/- 5 mmHg, all p < 0.01). The decrease in systolic pressure was greatest in Group 3 (Group 1: 141 +/- 31 vs 119 +/- 18, Group 2: 149 +/- 25 vs 118 +/- 17, Group 3; 147 +/- 27 vs 107 +/- 14 mmHg, all p < 0.01 compared to control, and p < 0.05 between Groups 1 and 3). The intravenous drip infusion of nicardipine is effective in the treatment of heart failure by inducing an increase in cardiac output and a decrease in pulmonary artery wedge pressure. The optimal dose in this study was 1.0 microgram/kg per min.

Acute Disease↗

PubMed1996

Change in aortic end-systolic pressure by alterations in loading sequence and its relation to left ventricular isovolumic relaxation.

BACKGROUND: A brief, sustained constriction of the descending and the ascending aortas produces systolic loads at different times during ejection, and descending intervention prolongs left ventricular (LV) relaxation more than ascending intervention. Although alterations in the sequence of loading the ventricle have been suggested as a cause of such load-induced relaxation abnormalities, the relation of the loading system to relaxation has been unclear. METHODS AND RESULTS: LV peak systolic pressure was elevated by approximately 40 mm Hg by constricting the descending and ascending aortas in seven anesthetized dogs. The descending intervention increased aortic end-systolic pressure (AoESP, 110.4 +/- 9.3 to 150.8 +/- 11.5 mm Hg; P < .05), reduced aortic flow (P < .05), and prolonged LV relaxation (time constant [T], 31.9 +/- 4.4 to 69.8 +/- 12.8 ms; P < .05). LV ejection time was reduced, but the systolic time interval was unchanged. In contrast, ascending intervention decreased AoESP (111.9 +/- 11.4 to 101.5 +/- 10.3 mm Hg; P < .05), reduced aortic flow (P < .05), and prolonged T (31.2 +/- 5.4 to 42.2 +/- 8.3 ms; P < .05), whereas ejection time and systolic time interval increased (both P < .01). Prolongation of T was significantly greater during descending intervention (P < .05) and was associated with an increase in AoESP during descending intervention but a decrease in AoESP during ascending intervention. CONCLUSIONS: Descending intervention induced greater prolongation of T than ascending intervention. Prolongation of T was closely related to an increase in AoESP in the descending intervention but a decrease in AoESP in the ascending intervention. These data suggest that not only the loading sequence but also the pressure level at the onset of isovolumic relaxation determines LV relaxation.

Animals↗

PubMed1996

Allelic-expression imbalance of the insulin-like growth factor 2 gene in hepatocellular carcinoma and underlying disease.

It has been well documented that the liver is an exceptional organ in which the monoallelic expression of insulin-like growth factor 2 (IGF2) due to genomic imprinting is relaxed during the postnatal period, resulting in biallelic expression thereafter. In the present study, changes in the status of genomic imprinting were examined in 15 hepatocellular carcinomas (HCCs) as well as in 29 liver biopsies of chronic hepatitis or liver cirrhosis without clinical evidence of HCC, following screening for heterozygotes with an ApaI polymorphism in IGF2 in 34 HCCs and 80 such non-HCC cases. Extreme allelic-expression imbalance, leading to restoration of monoallelic IGF2 expression, was observed in 15 (100%) of 15 informative HCCs for the polymorphism with this monoallelic IGF2 expression appearing to be non-random from the paternal allele. Interestingly, the same allelic-expression imbalance was also present in a significant fraction of noncancerous liver specimens of patients with underlying disease known to be associated with HCC development. In contrast, the status of genomic imprinting of H19, another gene closely mapped at 11p15 under opposite imprinting, was strictly maintained in seven (100%) of seven cases informative for an RsaI polymorphism of H19. Together with the previous reports on altered genomic imprinting of IGF2 and H19 in embryonal lesions such as Wilms tumors as well as in lung cancers, the results suggest that perturbations of imprinting status occur as locus and tumor-type specific events in the development of human cancers.

Alleles↗

PubMed1996

Comparison of serum hepatitis C virus RNA concentration by branched DNA probe assay with competitive reverse transcription polymerase chain reaction as a predictor of response to interferon-alpha therapy in chronic hepatitis C patients.

A study was carried out to assess the correlation between the serum concentration of hepatitis C virus RNA (HCV-RNA) in patients with chronic hepatitis, as measured by competitive reverse transcription polymerase chain reaction (cRT-PCR) and branched DNA probe assay (bDNA), and response to interferon-alpha (IFN alpha) therapy. The serum HCV-RNA concentration was evaluated by both cRT-PCR and bDNA in 54 patients who had received a total dose of 480 MU of IFN alpha. HCV subtypes were also identified in all patients. The measurement of serum HCV-RNA concentration by bDNA correlated significantly with that of cRT-PCR. The concentration of HCV-RNA in subtype 1 patients was significantly higher than that in subtype 2 patients when measured by bDNA, but not when measured by cRT-PCR. The correlation of HCV-RNA concentration between bDNA and cRT-PCR was associated with both subtypes 1 and 2. The difference in serum HCV-RNA concentration between complete and incomplete responders was more significant when measured by bDNA probe assay than by cRT-PCR. Moreover, only 1 of 26 patients with a HCV-RNA concentration of more than 1 x 10(6) eq/ml as measured by bDNA probe assay attained a complete response, while 19 of 28 patients with that of less than 1 x 10(6) eq/ml achieved it. Measurement of serum HCV-RNA concentration by bDNA probe assay was a better predictor of clinical response of IFN alpha therapy than measurement by cRT-PCR.

Adult↗

PubMed1996

Early diastolic regional function of the hypertrophied left ventricle.

We analyzed cardiac catheterization data from 7 patients with aortic stenosis and 10 patients with nonobstructive hypertrophic cardiomyopathy to compare left ventricular regional diastolic function. Left ventriculogram in the right anterior oblique projection was analyzed by the area method, and regional wall stress and regional area were computed for 4 regions in the mid-portion of the left ventricle. For each region, we assessed the percent area changes (normalized by end-diastolic regional area) and time constant for regional wall stress decrease during the isovolumic relaxation period. Regional non-uniformity during the isovolumic relaxation period was then evaluated by standard deviations for the percent area changes and for regional time constants of the 4 ventricular regions. In patients with hypertrophic cardiomyopathy, both the standard deviations for the percent area changes and the regional time constants were greater (P < 0.05) than those in patients with aortic stenosis, suggesting the presence of pronounced non-uniformity of regional relaxation in hypertrophic cardiomyopathy. The time constant of left ventricular pressure decrease during early relaxation phase was significantly greater (P < 0.01), and the early diastolic peak filling rate of the global left ventricle was significantly smaller (P < 0.05) in patients with hypertrophic cardiomyopathy. Thus, early diastolic left ventricular regional non-uniformity was more pronounced in hypertrophic cardiomyopathy than in aortic stenosis, which was associated with the impairment of relaxation and early filling of the global left ventricle. These findings suggest that different mechanisms are responsible for diastolic dysfunction in primary versus secondary myocardial hypertrophy.

Adult↗

PubMed1996

Brefeldin A inhibits antigen- or calcium ionophore-mediated but not PMA-induced phospholipase D activation in rat basophilic leukemia (RBL-2H3) cells.

Recent reports have indicated that ADP-ribosylation factor (ARF) plays a role in the regulation of phospholipase D (PLD) activity in the in vitro assay system. Since a fungal metabolite brefeldin A (BFA) is known to interfere with ARF function, the effect of BFA on antigen-induced PLD activation was examined in rat basophilic leukemia (RBL-2H3) cells. BFA inhibited the antigen-induced formation of phosphatidylbutanol (PBut), a specific and stable metabolite produced by PLD activity in a concentration-dependent manner. The maximal inhibition obtained at 10 micrograms/ml of the drug was nearly 70% and further inhibition was not observed at higher concentrations. Ca(2+)-ionophore A23187-mediated PLD activation was also prevented by BFA. In contrast, BFA failed to inhibit PLD activation in response to 4 beta-phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC). This indicates that there are BFA-sensitive and BFA-insensitive pathways leading to PLD activation in RBL-2H3 cells and also that the PKC-mediated pathway may be insensitive to BFA treatment, suggesting the existence of PLD isozymes. BFA inhibited Ag-induced serotonin release at a concentration 20-fold lower than that needed for the inhibition of PLD. Moreover, PMA caused a marked production of PBut, but it failed to elicit secretory response. This implies that PLD may be not a crucial element for secretory responses.

Animals↗

PubMed1996

Phenylarsine oxide (PAO)-mediated activation of phospholipase D in rat basophilic leukemia (RBL-2H3) cells: possible involvement of calcium and protein kinase C.

Addition of phenylarsine oxide (PAO) to [3H]oleic acid-labeled rat basophilic leukemia (RBL-2H3) cells gave rise to the remarkable formation of [3H]phosphatidylbutanol (PBut), a specific product of phospholipase D (PLD) activation. Preincubation of cells with 2,3-dimercaptopropanol (DMP) or dithiothreitol (DTT), compounds containing sulfhydryls, prevented PAO-stimulated [3H]PBut formation, indicating that PAO-stimulated PLD through interacting with vicinal thiol groups. Treatment of cells with PAO resulted in increase in intracellular Ca2+ concentration without significant production of inositol phosphates. Removal of extracellular free Ca2+ by chelating with EGTA was found to inhibit [3H]PBut formation by PAO. Incubation of cells with 20 nM phorbol 12-myristate 13-acetate (PMA) for 6 h caused down-regulation of protein kinase C (PKC) alpha and beta isozymes, whereas it had no effect on PKC delta, epsilon and zeta isozymes. Under this condition, decrease in PAO-stimulated [3H]PBut formation was observed to occur with a concomitant decrease in the level of PKC alpha and beta isozymes. These results suggest that a covalent bridge between vicinal thiol groups of cell surface proteins induced by PAO potentiates PLD activation and that PAO-induced PLD activation is regulated by Ca2+ and PKC alpha and/or beta isozymes.

Animals↗