Lack of ethnic differences of moxifloxacin and metabolite pharmacokinetics in East Asian men.

This study was designed to investigate ethnic differences in the pharmacokinetics (PKs) of moxifloxacin and its metabolites, M1 (sulfo conjugate) and M2 (acyl-glucuronate), among Japanese, Chinese, and Korean populations, following oral administration. We used a population PK modeling approach using data from a clinical study involving 79 healthy male volunteers. A comprehensive population PK model considering the PK mechanism of moxifloxacin and its metabolites was newly built. The structures of the final model were two-compartment for moxifloxacin and one-compartment for M1 and M2, with first-order absorption with lag time for all three compounds. The formation of M1 and M2 from moxifloxacin via a first-pass effect and subsequent metabolic clearance in the system were also modeled. Lean body mass on the central volume of distribution (V c ) and estimated glomerular filtration rate on renal clearance (CL r ) were identified as covariates of PKs of moxifloxacin. Additionally, bioavailability was slightly higher in Koreans, whereas CL r , non-renal clearance (CL nr ), and V c were slightly lower. Regarding M1 and M2, body surface area on CL r of M2 and UGT1A1*6 on F of M2 were modeled. Korean ethnicity was observed to influence CL nr of M2, F of M2, and the metabolic clearance of moxifloxacin to M2. However, the exposure levels of moxifloxacin, M1, and M2 in Koreans were comparable to those in Japanese and Chinese because the effects of Korean ethnicity on some PK parameters were counterbalanced. These results suggest that PKs for moxifloxacin and its metabolites among East Asian populations are essentially similar.

Ethnic Difference in the Pharmacodynamics-efficacy Relationship of Dipeptidyl Peptidase-4 Inhibitors Between Japanese and non-Japanese Patients: A Systematic Review.

A systematic review of the differences in the efficacy of dipeptidyl peptidase-4 (DPP-4) inhibitors between Japanese and non-Japanese subjects was conducted. We searched for randomized controlled trials in patients with type 2 diabetes mellitus (T2DM) that studied the intervention of a DPP-4 inhibitor once-daily vs. placebo, as monotherapy or as add-on therapy. Data regarding placebo-corrected HbA1c reduction and trough DPP-4 inhibition rate after ≥12 weeks' treatment were extracted. In the 12 eligible studies, linear regression analysis revealed that the hemoglobin A1c (HbA1c) reduction at each DPP-4 inhibition level was larger in studies involving Japanese patients than in studies involving non-Japanese patients, with statistical significance between the two groups (P < 0.0001). Sensitivity analysis excluding studies of add-on therapies supported the robustness of the result. Our study indicated that DPP-4 inhibitors show greater efficacy in Japanese patients than in non-Japanese patients, which may be an important consideration in the global development strategy of new diabetic medications.

Evaluation of two Japanese regulatory actions using medical information databases: a 'Dear Doctor' letter to restrict oseltamivir use in teenagers, and label change caution against co-administration of omeprazole with clopidogrel.

WHAT IS KNOWN AND OBJECTIVE: The implementation of appropriate epidemiological methodology using medical information databases (MIDs) to evaluate the effects of regulatory actions has been highly anticipated. To assess scientific methods for active pharmacovigilance using MIDs, we conducted a quantitative assessment of the impact of two regulatory actions by the Japanese government: (i) restriction of use of oseltamivir in teenagers in March 2007 and (ii) caution against the co-administration of omeprazole (OPZ) with clopidogrel (CPG) in April 2010. METHODS: Data were obtained from four hub hospitals in Japan. We measured the seasonal proportion of patients prescribed oseltamivir to those prescribed neuraminidase inhibitors for the 2002/2003 to 2010/2011 seasons. The monthly proportion of patients co-administered OPZ and CPG (OPZ+CPG) to those prescribed CPG was measured from May 2009 to April 2011. We evaluated the changes observed with implementation of the regulatory actions. To estimate the impact of the actions, we conducted segmented regression analysis using interrupted time series data. The impact was assessed by two parameter estimates of the regression model: the change in level for short-term effects and change in trend for long-term effects. RESULTS AND DISCUSSION: The use of oseltamivir in the target 10-19 years age group showed a significant and large decline (63·16%) immediately after the intervention (P = 0·0008). No change was observed in OPZ+CPG, although there was a relative inhibitory trend for OPZ+CPG compared with co-administration of lansoprazole or rabeprazole with CPG as the control group. When restricted to new users of CPG, the stratified results were consistent with the overall results. WHAT IS NEW AND CONCLUSION: The current analysis demonstrates the effectiveness of two regulatory actions. The results of the current study indicate that MID research can contribute to assessing and improving pharmacovigilance activities.

An algorithm for the identification of heparin-induced thrombocytopenia using a medical information database.

WHAT IS KNOWN AND OBJECTIVE: Using effective algorithms for extracting relevant drug and patient information from electronic medical information data systems is likely to form an increasingly important aspect of pharmacovigilance. To this end, we aimed to develop and validate a novel algorithm for detecting heparin-induced thrombocytopenia (HIT) using a medical information database (MID) and for identifying possible risk factors for HIT. METHODS: We developed a new algorithm for detecting HIT based on platelet count at distinct time-points and diagnostic information from patients treated with unfractionated heparin (UFH) from April 2008 through March 2012 at Hospital of Hamamatsu University School of Medicine, Japan. Definitive diagnoses of HIT were made through reviews of the medical records by a skilled haematologist. The performance of the algorithm was assessed using the positive predictive value (PPV). Multivariate logistic regression analysis was used to identify possible risk factors for HIT. RESULTS AND DISCUSSION: This algorithm detected 47 patients with suspected HIT in the source population (n = 2875). Of these, 41 were identified as patients with definitive HIT after review of the medical records. The PPV for the algorithm was 87·2%, and the frequency of definitive HIT was 1·4%. Longer-term treatment (≥4 days) with UFH was identified as a risk factor for HIT, with an odds ratio of 5·38 (95% CI: 2·35-12·32) for definitive HIT. WHAT IS NEW AND CONCLUSION: We developed a novel, high-PPV detection algorithm for HIT and identified longer-term treatment with UFH as a risk factor for HIT. Our results support the utility of MIDs for improving pharmacovigilance.

Development of a detection algorithm for statin-induced myopathy using electronic medical records.

WHAT IS KNOWN AND OBJECTIVE: Demonstration of the utility of electronic medical records (EMRs) for pharmacovigilance (PV) has been highly anticipated. Analysis using appropriately selected EMRs should enable accurate estimation of adverse drug event (ADE) frequencies and thus promote appropriate regulatory actions. Statin-induced myopathy (SIM) is a clinically important ADE, but pharmacoepidemiological methodology for detecting this ADE with high predictability has not yet been established. This study aimed to develop a detection algorithm, highly selective for SIM using EMRs. METHODS: We collected EMRs on prescriptions, laboratory tests, diagnoses and medical practices from the hospital information system of Kobe University Hospital, Japan, for a total of 5109 patients who received a statin prescription from April 2006 to March 2009. The current algorithm for extracting SIM-suspected patients consisted of three steps: (i) event detection: increase in creatine kinase (CK) and subsequent statin discontinuation, (ii) filtration by exclusion factors (disease diagnosis/medical practices) and (iii) refinement by the time course of CK values (baseline, event and recovery). A causal relationship between the event and statin prescription (probable/possible/unlikely) was judged by review of patient medical charts by experienced pharmacists. The utility of the current algorithm was assessed by calculating the positive predictive value (PPV). In a comparative analysis, subjects screened in step 1 were extracted by the diagnostic term/code for 'myopathy/rhabdomyolysis', and the PPV of this diagnostic data approach was also estimated. RESULTS AND DISCUSSION: Five subjects with suspected SIM were identified using our proposed algorithm, giving a frequency of 0·1% for the adverse event. Review of the medical charts revealed that the causal association of SIM with statin use was judged as 'Likely (probable/possible)' for all five suspected patients; thus, the PPV was estimated as 100% (95% confidence interval: 56·6-100%). The higher utility of the current algorithm compared with the diagnostic data approach was also shown by assessing the PPV (100 vs. 33·3%). WHAT IS NEW AND CONCLUSION: We report on a detection algorithm with high predictability for SIM using EMRs. Combined use of exclusion criteria for disease, medical practice data and time course of CK values contributes to better prediction of SIM. The utility of the proposed algorithm should be further confirmed in a larger study.

A whole-genome association study of major determinants for allopurinol-related Stevens-Johnson syndrome and toxic epidermal necrolysis in Japanese patients.

Stevens-Johnson syndrome and toxic epidermal necrolysis (SJS/TEN) are severe, cutaneous adverse drug reactions that are rare but life threatening. Genetic biomarkers for allopurinol-related SJS/TEN in Japanese were examined in a genome-wide association study in which Japanese patients (n=14) were compared with ethnically matched healthy controls (n=991). Associations between 890 321 single nucleotide polymorphisms and allopurinol-related SJS/TEN were analyzed by the Fisher's exact test (dominant genotype mode). A total of 21 polymorphisms on chromosome 6 were significantly associated with allopurinol-related SJS/TEN. The strongest association was found at rs2734583 in BAT1, rs3094011 in HCP5 and GA005234 in MICC (P=2.44 × 10(-8); odds ratio=66.8; 95% confidence interval, 19.8-225.0). rs9263726 in PSORS1C1, also significantly associated with allopurinol-related SJS/TEN, is in absolute linkage disequilibrium with human leukocyte antigen-B*5801, which is in strong association with allopurinol-induced SJS/TEN. The ease of typing rs9263726 makes it a useful biomarker for allopurinol-related SJS/TEN in Japanese.

Targeted disruption of the nuclear receptor FXR/BAR impairs bile acid and lipid homeostasis.

Mice lacking the nuclear bile acid receptor FXR/BAR developed normally and were outwardly identical to wild-type littermates. FXR/BAR null mice were distinguished from wild-type mice by elevated serum bile acid, cholesterol, and triglycerides, increased hepatic cholesterol and triglycerides, and a proatherogenic serum lipoprotein profile. FXR/BAR null mice also had reduced bile acid pools and reduced fecal bile acid excretion due to decreased expression of the major hepatic canalicular bile acid transport protein. Bile acid repression and induction of cholesterol 7alpha-hydroxylase and the ileal bile acid binding protein, respectively, did not occur in FXR/BAR null mice, establishing the regulatory role of FXR/BAR for the expression of these genes in vivo. These data demonstrate that FXR/BAR is critical for bile acid and lipid homeostasis by virtue of its role as an intracellular bile acid sensor.

Targeted disruption of soluble epoxide hydrolase reveals a role in blood pressure regulation.

Renal microsomal cytochrome P-450 monooxygenase-dependent metabolism of arachidonic acid generates a series of regioisomeric epoxyeicosatrienoic acids that can be further metabolized by soluble epoxide hydrolase to the corresponding dihydroxyeicosatrienoic acids. Evidence exists that these metabolites affect renal function and, in particular, blood pressure regulation. To examine this possibility, blood pressure and renal arachidonic acid metabolism were examined in mice with a targeted disruption of the soluble epoxide hydrolase gene. Systolic blood pressure of male soluble epoxide hydrolase-null mice was lower compared with wild-type mice in both the absence and presence of dietary salt loading. Both female soluble epoxide hydrolase-null and wild-type female mice also had significantly lower systolic blood pressure than male wild-type mice. Renal formation of epoxyeicosatrienoic and dihydroxyeicosatrienoic acids was markedly lower for soluble epoxide hydrolase-null versus wild-type mice of both sexes. Although disruption of soluble epoxide hydrolase in female mice had minimal effects on blood pressure, deletion of this gene feminized male mice by lowering systolic blood pressure and altering arachidonic acid metabolism. These data provide the first direct evidence for a role for soluble epoxide hydrolase in blood pressure regulation and identify this enzyme as a novel and attractive target for therapeutic intervention in hypertension.

Aryl hydrocarbon receptor is required for p300-mediated induction of DNA synthesis by adenovirus E1A.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates the biological responses to environmental contaminants such as 2,3,7, 8-tetrachlorodibenzo-p-dioxin. Embryonic fibroblast (EF) isolated from AHR-null mice exhibited slow cell growth compared with wild-type EF. Reintroduction of AHR into AHR-null EF increased cell growth, suggesting that AHR is involved in cell cycle control. The role of the AHR in cell cycle control was examined using the adenovirus oncoprotein E1A. EF, derived from wild-type and AHR-null mice, were transfected with two mutant E1A expression plasmids that inactivate either p300/CBP or retinoblastoma protein (pRb). Although DNA synthesis of wild-type EF was induced by both E1A mutants, DNA synthesis in the AHR-null EF was induced only by the mutant that binds pRb, not by the mutant to p300/CBP. These data show that both pRb and p300/CBP were the target of E1A-induced DNA synthesis in wild-type EF. In AHR-null mice, however, only pRb was the target of E1A-induced DNA synthesis and p300/CBP cannot be inactivated by E1A in the absence of AHR. Immunoprecipitation revealed that AHR directly bound to p300, thus suggesting the intriguing possibility that AHR is involved in control of the cell cycle via interaction with p300.

Cloning and expression analysis of a new member of the cytochrome P450, CYP2A15 from the Chinese hamster, encoding testosterone 7alpha-hydroxylase.

We cloned a new cytochrome P450 cDNA encoding testosterone 7alpha-hydroxylase in the Chinese hamster, designated CYP2A15 which shares significant amino acid sequence homology with members of the CYP2A subfamily. The CYP2A15 cDNA was isolated by screening a liver cDNA library and the sequence contains an open reading frame of 1482 nucleotides encoding a polypeptide of 493 amino acids with a calculated molecular mass of 56,295 Da. This is flanked by a 5'-untranslated region of 2 bp and a 3' untranslated region of 191 bp including the poly(A) tail. We determined the catalytic activity of CYP2A15 using microsomes obtained by transient expression of its cDNA in transfected COS-7 cells. The heterologously expressed CYP2A15 was found to hydroxylate testosterone at position 7alpha in a reconstituted system. RT-PCR experiments revealed that the mRNA of CYP2A15 was expressed in liver, but not detected in kidney, lung, or small intestine. The expression of CYP2A15 mRNA was slightly induced by treatment with either rifampicin or 3-methylcholanthrene.

Molecular cloning, heterologous expression, and characterization of a novel member of CYP2A in the Syrian hamster.

The cDNA clone coding for a novel cytochrome P-450 2A subfamily member (CYP2A16) was isolated from a Syrian hamster liver cDNA library. The deduced amino acid sequence of CYP2A16 showed more than 90% identity with those of rat CYP2A3 and mouse CYP2A4/5. The catalytic activity of CYP2A16 was determined by transient expression of its cDNA in transfected COS7 cells and CYP2A16 was found to have the testosterone 2 beta-, 15 alpha-, and 15 beta-hydroxylases, coumarin 7-hydroxylase, and ethoxycoumarin O-deethylase activities. These enzymatic characteristics of CYP2A16 are different from those of other Syrian hamster CYP2A subfamily members, CYP2A8 and CYP2A9. Northern blot analysis showed that CYP2A16 was expressed in kidney and lung while most of the other CYP2A subfamily members have been reported to be expressed in liver and olfactory. These observations indicated that the Syrian hamster CYP2A16 had unique properties compared with those of other CYP2A subfamily members.

A novel positive regulatory element that enhances hamster CYP2A8 gene expression mediated by xenobiotic responsive element.

CYP2A8 is a major form of cytochrome P-450 inducible by 3-methylcholanthrene in Syrian hamster liver. To identify DNA elements necessary for the transcriptional activation of the CYP2A8 gene, we analyzed the regulatory region of the CYP2A8 gene and conducted transient transfection experiments of CYP2A8-luciferase fusion plasmids in primary cultures of hamster hepatocytes. We analyzed up to -5 kb of the 5'-flanking region and found the region sufficient for the 3-methylcholanthrene-inducible gene expression. This region contained a consensus sequence for xenobiotic responsive element (XRE) between -2366 and -2349, which was shown to be essential for induction of the gene expression. Furthermore, we found a novel positive regulatory element for XRE-mediated gene expression (PREX) located upstream of the XRE. This element is not identified in any genes inducible by 3-methylcholanthrene so far reported. Without PREX, the XRE-mediated promoter activity was enhanced nearly 10-fold, whereas with PREX, the activity was enhanced 20-fold over the basal level. Gel mobility shift assays revealed specific binding of nuclear proteins to PREX. Mutations and deletions of PREX caused a loss of the binding and promoter-enhancing activities, respectively. Moreover, transient expression experiments showed that the enhancing activity of PREX was not observed in Drosophila Schneider's line 2 cells, which were shown to lack the PREX binding proteins.

Evidence for involvement of cAMP-dependent pathway in the phenobarbital-induced expression of a novel hamster cytochrome P450, CYP3A31.

Recently, we isolated a novel Syrian hamster cDNA clone that encodes a protein which has been named CYP3A31. In primary hepatocyte cultures, CYP3A31 is dramatically induced by phenobarbital. To elucidate the mechanism of this induction, we first studied the effects of cAMP on phenobarbital-induced CYP3A31 expression using forskolin and N6,O2'-dibutyryl cAMP in hepatocyte cultures. At 100 microM, forskolin significantly inhibited both the phenobarbital-induced CYP3A31 mRNAs expression and the testosterone 6beta-hydroxylation activity related to the CYP3A subfamily in rats, whereas 0.1 microM forskolin potentiated the phenobarbital induction of CYP3A31 mRNA and the testosterone 6beta-hydroxylation activity. Treatment with N6,O2'-dibutyryl cAMP resulted in an inhibition of phenobarbital-induced CYP3A31 gene expression and testosterone 6beta-hydroxylation activity. Increasing amounts of transfected cAMP-response element binding proteins (CREB) or CREB-binding proteins in hamster hepatocytes reduced the phenobarbital-induction of CYP3A31 mRNAs expression. These results suggest that in vitro induction of CYP3A31 by phenobarbital in Syrian hamster hepatocytes is regulated by a cAMP-dependent pathway.

cDNA cloning and expression of a novel CYP3A from the Syrian hamster, CYP3A31.

A clone, encoding a cytochrome P450 protein consisting of 501 amino acids, was isolated from a cDNA library constructed from mRNA of Syrian hamster liver. The deduced amino acid sequence of this clone showed a high homology (65 to 81%) with other mammalian CYP3As and hence, this novel isozyme was named CYP3A31. By Northern blotting, using an oligonucleotide specific to CYP3A31, the mRNA for this isozyme was shown to be expressed constitutively in liver and induced by treatment with phenobarbital but repressed by 3-methylcholanthrene or dexamethasone treatments. The increase in mRNA expression by phenobarbital and decrease by dexamethasone corresponded to changes in CYP3A protein as analysed by Western blotting. These indicate that CYP3A31 might constitute one of the major CYP3A isozymes in the hamster.

Cloning and characterization of Syrian hamster testosterone 7 alpha-hydroxylase, CYP2A9.

We report here the cloning of a cDNA encoding testosterone 7 alpha-hydroxylase in the Syrian hamster, designated CYP2A9. Overlapping clones encoding Syrian hamster CYP2A9 were isolated by screening a liver cDNA library and by performing rapid amplification of cDNA ends polymerase chain reaction on the cDNA library. The sequence of the CYP2A9 cDNA contains an open reading frame of 1482 nucleotides encoding a polypeptide of 493 amino acids with a calculated molecular mass of 56,295 Da. The sequence is flanked by a 5'-untranslated region of 10 bp and a 3'-untranslated region of 178 bp including the poly(A) tail. The deduced amino acid sequence shares significant homology with members of CYP2A subfamily, notably with CYP2A1 and CYP2A12 which have testosterone 7 alpha-hydroxylase activity. We characterized the catalytic activity of CYP2A9 using microsomes obtained by transient expression of its cDNA in transfected COS-7 cells. CYP2A9 was found to hydroxylate testosterone at position 7 alpha. In Syrian hamster livers, a higher level of testosterone 7 alpha-hydroxylase activity as well as the mRNA of CYP2A9 in male than in female was obtained. The testosterone 7 alpha-hydroxylase activity and the mRNA level in liver were both decreased moderately by administration of 3-methylcholanthrene and slightly by administration of phenobarbital. In contrast, in kidney, both 3-methylcholanthrene and phenobarbital significantly decreased the mRNA level. These facts indicate that the regulation of the hamster testosterone 7 alpha-hydroxylase (CYP2A9) expression is different from that of the rat (CYP2A1) and hamster reported previously by other workers.

Okadaic acid potentiates 3-methylcholanthrene-induced CYP2A8 gene expression in primary cultures of Syrian hamster hepatocytes: possible involvement of activator protein-1.

In Syrian hamster liver, treatment with 3-methylcholanthrene (3-MC) markedly induces an isozyme of cytochrome P450 (CYP), CYP2A8. To elucidate the mechanism of this induction, we studied the effect of okadaic acid (OA), an inhibitor of serine threonine protein phosphatases 1 and 2A, on 3-MC-induced CYP2A8 expression in primary cultures of Syrian hamster hepatocytes. The addition of OA to the cultured hepatocytes at a concentration of 1 nM potentiated 3-MC- (0.1 and 1 microM) induced expression of mRNA and protein of CYP2A8 and its associated coumarin 7-hydroxylase activity. In addition, OA not only induced c-fos and jun-D mRNA, components of transcription factor activator protein-1 (AP-1), with an increase in AP-1 binding activity in the nucleus, but also activated AP-1-dependent gene transcription in the hepatocytes. The dose-dependent effect of OA on 3-MC-induced CYP2A8 expression corresponded to that of OA on c-fos and jun-D mRNA induction and on the activation of AP-1-dependent gene transcription. The expression of c-fos and jun-D mRNA induced by OA preceded the expression of CYP2A8 mRNA potentiated by co-treatment with 3-MC and OA. Treatment with anisomycin and cycloheximide also potentiated 0.1 microM 3-MC-induced coumarin 7-hydroxylase activity, induced c-fos and jun-D mRNA expression, and activated AP-1-dependent gene transcription in the hepatocytes. Furthermore, 3-MC-induced CYP2A8 expression was potentiated in the hepatocytes transfected with c-Jun expression plasmid. These results suggest that AP-1, inducible by serine threonine protein kinase, may be one of the components of the signal transduction system from 3-MC to CYP2A8 gene expression.

Androgens directly stimulate mineralization and increase androgen receptors in human osteoblast-like osteosarcoma cells.

5 alpha-Dihydrotestosterone (DHT), an active form of testosterone, was shown to stimulate the mineralization of osteoblasts in a time- and dose-dependent manner in vitro in the presence of inorganic phosphate, as well as transforming growth factor beta and 1 alpha, 25 dihydroxyvitamin D3. However, DHT, stimulated the mineralization by a different mechanism from that of transforming growth factor beta and 1 alpha, 25 dihydroxyvitamin D3. We found that DHT increased the number of androgen receptors. These findings suggest that androgens directly stimulate mineralization in osteoblasts associated with an increase in the number of their receptors.

Comparative study of inhibitory effects by murine interferon gamma and a new bisphosphonate (alendronate) in hypercalcemic, nude mice bearing human tumor (LJC-1-JCK).

The inhibitory effect of murine interferon gamma (muIFN gamma) on humoral hypercalcemia in nude mice bearing lower-jaw cancer (LJC-1-JCK), in which parathyroid-hormone(PTH)-related protein is responsible for causing humoral hypercalcemia by activating bone resorption, was examined in comparison with that of a new bisphosphonate, 4-amino-1-hydroxybutylidene-1,1-bisphosphonate (alendronate). muIFN gamma was injected into tumor-bearing nude mice for 5 days before the establishment of hypercalcemia. The increase of plasma calcium concentration was delayed and this effect continued for more than 6 days even after the injection was stopped. Alendronate markedly suppressed hypercalcemia in tumor-bearing nude mice but this inhibitory effect continued for less than 6 days. Neither muIFN gamma nor alendronate affected the tumor volume or serum PTH-related protein concentration. Injection of muIFN gamma into mice for 3 days almost completely abolished the formation of multinucleated osteoclast-like cells from bone marrow cells in vitro, whereas injection of alendronate into mice had no effect. These findings suggested that muIFN gamma suppressed the formation of osteoclasts, resulting in the prolonged decrease of plasma calcium concentration in hypercalcemic tumor-bearing nude mice, whereas alendronate is cytotoxic to functionally mature osteoclasts and inhibited osteoclastic bone resorption, resulting in a marked decrease in the plasma calcium concentration in tumor-bearing hypercalcemic nude mice.

The involvement of the stimulatory G protein in sexual dimorphism of beta-adrenergic receptor-mediated functions in rat liver.

In rat hepatocytes, beta-adrenergic receptor (beta-AR)-mediated cAMP generation was found to be higher in the female than in the male. As compared to the male, the number of beta-AR, detected by [125I]iodocyanopindolol, was elevated in the female. In agonist competition experiments, the proportion of beta-AR in the high-affinity state was promoted in the female than in the male. The alpha subunit of the stimulatory G protein (Gs alpha) was quantified using ADP-ribosylation catalyzed by cholera toxin. The amount of Gs alpha, both small, 42 kDa (Gs alpha S), and large, 47 kDa (Gs alpha L), forms increased in parallel with enhancement of catecholamine-sensitive adenylate cyclase activity in the female. The female showed a disproportionate increase in Gs alpha L, which is preferentially coupled to beta-AR, compared with Gs alpha S. In addition, 17 beta-estradiol facilitated isoproterenol-induced cAMP generation in both male and female rats, whereas castration or testosterone had no effect on this response. It is proposed that the cellular sites for sexual dimorphism in hepatic beta-adrenergic functions are the coupling state of beta-AR to Gs and the amount of Gs alpha as well as the level of beta-AR.

Pancreatic-type phospholipase A2 stimulates prostaglandin synthesis in mouse osteoblastic cells (MC3T3-E1) via a specific binding site.

Previously, we have reported a novel proliferative action of pancreatic group I phospholipase A2 (PLA2-I) via a specific binding site in Swiss 3T3 fibroblasts, vascular smooth muscle cells, and chondrocytes. In this study, we characterized the PLA2-I specific binding site in osteoblastic cell line (MC3T3-E1 cells) with an equilibrium binding constant (Kd) value of 1.13 nM and maximum binding capacity of 40.1 fmol/10(6) cells. PLA2-I stimulated prostaglandin E2 (PGE2) production in a concentration-dependent manner in MC3T3-E1 cells, and its EC50 value was similar to the Kd value for PLA2-I binding. This effect of PLA2-I was type-specific and did not depend on its hydrolytic activity. PLA2-I increased the activity of prostaglandin endoperoxide synthase (PES), and PLA2-I-stimulated PGE2 synthesis was inhibited by cycloheximide. Northern blot analysis showed the increase in both type-1 and type-2 PES mRNAs. These findings indicated that PLA2-I stimulated PGE2 synthesis by induction of PES via a specific binding site in osteoblastic cells.