Presence Of Isobutylmethylxanthine Research Articles

Polyphenol-Rich Leaf of Annona squamosa Stimulates Insulin Release from BRIN-BD11 Cells and Isolated Mouse Islets, Reduces (CH2O)n Digestion and Absorption, and Improves Glucose Tolerance and GLP-1 (7-36) Levels in High-Fat-Fed Rats.

Annona squamosa, commonly known as custard apple, is traditionally used for the treatment of various diseases including diabetes, cardiovascular disease (CVD), and gastritis. This study was undertaken to investigate the effects of an ethanolic (80% v/v) extract of A. squamosa (EEAS) leaves in vitro on insulin secretion from clonal pancreatic BRIN BD11 β-cells and mouse islets, including mechanistic studies on the effect of EEAS on membrane potential and intracellular calcium ion concentration. Additional in vitro glucose-lowering actions were assessed. For in vivo studies, high-fat-fed (HFF) obese/normal rats were selected. EEAS increased insulin secretion in vitro in a dose-dependent manner. This effect was linked to β-cell membrane depolarisation and cytoplasmic Ca2+ influx. In the presence of isobutyl methylxanthine (IBMX), tolbutamide, or KCl, the insulin-releasing effect of EEAS was increased, suggesting its effect was also mediated via a KATP-independent pathways. EEAS inhibited insulin glycation, glucose absorption, and DPP-IV enzyme activity in vitro and enhanced glucose uptake and insulin action in 3T3L1 cells. In vivo, gut motility, food intake, glucose tolerance, plasma insulin, and active GLP-1 (7-36) levels were improved, whereas plasma DPP-IV levels were reduced in HFF rats. EEAS attenuated the absorption of sucrose and glucose as well as decreased serum glucose levels after sucrose loading and in situ intestinal perfusion in non-diabetic rats. Rutin, proanthocyanidin, and squafosacin G were putatively identified as the anti-hyperglycaemic phytomolecules in EEAS using HPLC followed by LC-MS analysis. This study illustrates the potential of A. squamosa and its phytoconstituents as a source of potential antidiabetic agents.

Expression of developmental genes in brown fat cells grown in vitro is linked with lipid accumulation.

En1, Nr2f1, Gpc4, Sfrp2, Shox2, Tbx15 and Thbd are among the genes involved in development process of an organism in a number of tissues, in particular adipose tissue. Considering the involvement of isobutyl-methyl-xanthine (IBMX), indomethacin, dexamethasone (DEX), triiodothyronine (T3), and insulin in adipocyte differentiation, we propose that these differentiation-inducing agents may regulate differentiation in brown adipose tissue through a developmental process. Stromavascular cells isolated from interscapular brown fat of mice were cultured in DMEM-LG medium. Proliferating brown preadipocytes were differentiated in the presence of IBMX, indomethacin, DEX, T3 and insulin. Pref1 (marker of proliferation stage) and uncoupling protein 1 (UCP1, marker for differentiation stage) were upregulated during proliferation and differentiation, respectively. Relative expression of Pref1, UCP1 and developmental genes was determined in different stages of adipogenesis. En1, Gpc4, Nr2f1, Sfrp2 and Shox2 were upregulated during differentiation. Differentiation of preadipocytes in the absence of IBMX, indomethacin, and DEX resulted in drastic reduction in fat accumulation in differentiated adipocytes with simultaneous decrease in En1, Gpc4, Nr2f1, Sfrp2, Shox2 and Tbx15 gene expression. T3 upregulated the expression of En1, Gpc4, Sfrp2 and Tbx15 genes during differentiation and downregulated Shox2 expression as compared to proliferated state. Insulin upregulated the expression of Shox2.

Activation of Protein Kinase A and Exchange Protein Directly Activated by cAMP Promotes Adipocyte Differentiation of Human Mesenchymal Stem Cells

Human mesenchymal stem cells are primary multipotent cells capable of differentiating into several cell types including adipocytes when cultured under defined in vitro conditions. In the present study we investigated the role of cAMP signaling and its downstream effectors, protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac) in adipocyte conversion of human mesenchymal stem cells derived from adipose tissue (hMADS). We show that cAMP signaling involving the simultaneous activation of both PKA- and Epac-dependent signaling is critical for this process even in the presence of the strong adipogenic inducers insulin, dexamethasone, and rosiglitazone, thereby clearly distinguishing the hMADS cells from murine preadipocytes cell lines, where rosiglitazone together with dexamethasone and insulin strongly promotes adipocyte differentiation. We further show that prostaglandin I2 (PGI2) may fully substitute for the cAMP-elevating agent isobutylmethylxanthine (IBMX). Moreover, selective activation of Epac-dependent signaling promoted adipocyte differentiation when the Rho-associated kinase (ROCK) was inhibited. Unlike the case for murine preadipocytes cell lines, long-chain fatty acids, like arachidonic acid, did not promote adipocyte differentiation of hMADS cells in the absence of a PPARγ agonist. However, prolonged treatment with the synthetic PPARδ agonist L165041 promoted adipocyte differentiation of hMADS cells in the presence of IBMX. Taken together our results emphasize the need for cAMP signaling in concert with treatment with a PPARγ or PPARδ agonist to secure efficient adipocyte differentiation of human hMADS mesenchymal stem cells.

Adenylyl cyclase/cAMP-PKA-mediated phosphorylation of basal L-type Ca 2+ channels in mouse embryonic ventricular myocytes

In fetal mammalian heart, constitutive adenylyl cyclase/cyclic AMP-dependent protein kinase A (cAMP-PKA)-mediated phosphorylation, independent of β-adrenergic receptor stimulation, could under such circumstances play an important role in sustaining the L-type calcium channel current ( I Ca,L) and regulating other PKA dependent phosphorylation targets. In this study, we investigated the regulation of L-type Ca 2+ channel (LTCC) in murine embryonic ventricles. The data indicated a higher phosphorylation state of LTCC at early developmental stage (EDS, E9.5–E11.5) than late developmental stage (LDS, E16.5–E18.5). An intrinsic adenylyl cyclase (AC) activity, PKA activity and basal cAMP concentration were obviously higher at EDS than LDS. The cAMP increase in the presence of isobutylmethylxanthine (IBMX, nonselective phosphodiesterase inhibitor) was further augmented at LDS but not at EDS by chelation of intracellular Ca 2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA)-acetoxymethyl ester (BAPTA-AM). Furthermore, I Ca,L increased with time after patch rupture in LDS cardiomyocytes dialyzed with pipette solution containing BAPTA whereas not at EDS. Thus we conclude that the high basal level of LTCC phosphorylation is due to the high intrinsic PKA activity and the high intrinsic AC activity at EDS. The latter is possibly owing to the little or no effect of Ca 2+ influx via LTCCs on AC activity, leading to the inability to inhibit AC.

Immunohistochemical assessment of cyclic guanosine monophosphate (cGMP) and soluble guanylate cyclase (sGC) within the rostral ventrolateral medulla

Functional evidence suggests that nitric oxide (NO) signalling in the rostral ventrolateral medulla (RVLM) is cGMP-dependent and that this pathway is impaired in hypertension. We examined cGMP expression as a marker of active NO signalling in the C1 region of the RVLM, comparing adult (>18 weeks) Wistar-Kyoto (WKY, n = 4) and spontaneously hypertensive rats (SHR, n = 4). Double label immunohistochemistry for cGMP-immunoreactivity (IR) and C1 neurons [as identified by phenylethanolamine N-methyltransferase (PNMT-IR) or tyrosine hydroxylase TH-IR)], or neuronal NO synthase (nNOS) neurones, failed to reveal cGMP-IR neurons in the RVLM of either strain, despite consistent detection of cGMP-IR in the nucleus ambiguus (NA). This was unchanged in the presence of isobutylmethylxanthine (IBMX; 0.5 mM, WKY, n = 4, SHR n = 2) and in young animals (WKY, 10-weeks, n = 3). Incubation of RVLM-slices (WKY, 10-weeks, n = 9) in DETA-NO (100 mum; 10 min) or NMDA (10 muM; 2 min) did not uncover cGMP-IR. In all studies, cGMP was prominent within the vasculature. Soluble guanylate cyclase (sGC)-IR was found throughout neurones of the RVLM, but did not co-localise with PNMT, TH or nNOS-IR neurons (WKY, 10-weeks, n = 6). Results indicate that within the RVLM, cGMP is not detectable using immunohistochemistry in the basal state and cannot be elicited by phosphodiesterase inhibition, NMDA receptor stimulation or NO donor application.

Different roles of α 2-adrenoceptor subtypes in non-pregnant and late-pregnant uterine contractility in vitro in the rat

The roles of the α 2-adrenoceptor (α 2-AR) subtypes (α 2A-, α 2B- and α 2C-AR) in uterine contractility have not been investigated. The aims of this study were to identify these receptors in the non-pregnant and the late-pregnant rat myometrium and to determine their roles in contractions. We found that the myometrial α 2-AR subtypes are involved differently in the control of late-pregnant contractions, while they have no influence on the contractions of the non-pregnant myometrium. The myometrial expressions of the α 2-AR subtypes were determined by RT-PCR and Western blotting techniques. In vitro contractions were stimulated with noradrenaline, and its effect was modified with the selective antagonists BRL 44408 (α 2A), ARC 239 (α 2B/C) and spiroxatrine (α 2C). cAMP production was followed by noradrenaline stimulation in the presence of isobutylmethylxanthine and forskolin, and alterations induced in it by the antagonists were determined with an Enzyme Immunoassay Kit. The most effective antagonist was tested on labour-induced uteri in vitro. All the α 2-AR subtypes were identified in both non-pregnant and pregnant uteri. Noradrenaline was not able to contract the non-pregnant tissue in the presence of propranolol and doxazosin, while its contracting effect in the pregnant uteri was enhanced by BRL 44408, spiroxatrine and the combination BRL 44408 + spiroxatrine. ARC 239 exerted a strong inhibitory effect on noradrenaline-stimulated contractions. The increasing and the decreasing effects of the compounds were confirmed by the changes in the intracellular cAMP levels. The effect of ARC 239 on the labour-induced myometrium was similar to that on the 22-day-pregnant myometrium. The stimulation of α 2-ARs does not evoke contractions in the non-pregnant uterus. The α 2A- and α 2C-ARs mediate decreases, while the α 2B-AR mediates an increase in the contractions in the 22-day-pregnant myometrium. These differences may offer new targets for drugs against premature contractions in pregnancy.

Role of versican and hyaluronan in the differentiation of 3T3-L1 cells into preadipocytes and mature adipocytes

We have previously shown that during the adipose conversion of these cells the culture medium changed its viscoelastic properties due to the presence of hyaluronan and a chondroitin sulfate proteoglycan [Calvo, J.C., Rodbard, D., Katki, A., Chernick, S., and Yanagishita, M., 1991. Differentiation of 3T3-L1 preadipocytes with 3-isobutyl-1-methylxanthine and dexamethasone stimulates cell-associated and soluble chondroitin 4-sulfate proteoglycans. J. Biol. Chem. 266, 11237–11244., Calvo, J.C., Gandjbakhche, A.H., Nossal, R., Hascall, V.C., and Yanagishita, M., 1993. Rheological effects of the presence of hyaluronic acid in the extracellular media of differentiated 3T3-L1 preadipocyte cultures. Arch. Biochem. Biophys. 302, 468–475]. Here, we analyze the time course for the appearance of these molecules during drug-induced cell differentiation. The synthesis of both hyaluronan and the proteoglycan, was maximal at 48 h in the presence of isobutylmethylxanthine and dexamethasone, but while hyaluronan remained high after changing the culture medium, the proteoglycan dropped to almost basal levels after a few days. Northern analysis revealed the presence of message for a “versican-like” molecule as well as the possibility of alternative splicing. Three major bands of 9.39, 8.48, and 7.69 kb appeared in the analysis. These bands showed a dramatic increase in intensity when RNA from non-differentiated cells was compared to differentiating 3T3-L1 cells. In addition, when the time course of appearance for this message was analyzed, it perfectly correlated the metabolic labeling of the glycosaminoglycans during cell culture. The nucleotide sequencing of two exons revealed between a 100–94% homology with proteoglycan PG-M from murine endothelial cells. At least 13% of the proteoglycan was able to bind hyaluronan. Disruption of the synthesis of the proteoglycan molecule by exogenous addition of xyloside, did not prevent triglyceride accumulation but was inhibitory to preconfluent 3T3-L1 cell proliferation. Coating of plastic culture dishes with conditioned medium from differentiating 3T3-L1 cells, resulted in decreased cell adhesion. Cell adhesion was partially recovered after degradation of hyaluronan and chondroitin sulfate by enzymatic treatment. All these results indicate a possible role of these molecules in the observed changes in the viscoelastic properties of the culture medium, as well as open the field for a more thorough study of their role in 3T3-L1 cell proliferation and/or differentiation.

Potentiation of cyclic AMP-mediated proopiomelanocortin gene promoter activity by calcium channel blockers in a pituitary cell line

Although it is known that the expression of proopiomelanocortin, a precursor protein of adrenocorticotropic hormone, can be affected by a variety of drugs, the effects of calcium channel blockers have not been studied. This study examined the effect of calcium channel blockers on proopiomelanocortin gene expression. Mouse pituitary tumor cells stably transfected with approximately 0.7 kb of the rat proopiomelanocortin 5′ promoter–luciferase fusion gene were stimulated by potassium chloride, corticotropin-releasing hormone (CRH) or forskolin, in the presence or absence of calcium channel blockers (nifedipine, verapamil and diltiazem). Assessments were made of proopiomelanocortin gene promoter activity and cyclic adenosine 3′,5′-monophosphate (cyclic AMP) efflux. A dose-dependent enhancement of CRH- or forskolin-stimulated proopiomelanocortin promoter activity was observed with nifedipine and verapamil, but not diltiazem. Cyclic AMP efflux induced by CRH or forskolin was also enhanced by nifedipine and verapamil. In the presence of isobutylmethylxanthine, a phosphodiesterase inhibitor, enhancement of proopiomelanocortin promoter activity and cyclic AMP efflux by nifedipine and verapamil was not observed. It was concluded that the inhibition of phosphodiesterase is a probable mechanism for the effect of nifedipine and verapamil on CRH or forskolin induction of proopiomelanocortin gene expression.

Effects of (R)‐ and (S)‐isomers of β‐adrenergic agonists on eosinophil response to interleukin‐5

Racemic beta2-adrenergic receptor agonists (beta2-agonists) are used frequently to treat patients with asthma. Potential differences in the biological activities and clinical efficacies among racemic beta2-agonists and their isomers are controversial, and research into these possible differences is limited. We hypothesized that the (S)- and the (R)-isomers of beta2-agonists have opposing effects on the activation of inflammatory cells. Isolated human eosinophils were pretreated with 1:1 racemic (R,S)-, (R)- or (S)-albuterol, isobutyl methylxanthine (IBMX), and stimulated with IL-5. The kinetics of superoxide production were examined by reduction of cytochrome c, and the effects of pharmacological agents on superoxide production were monitored for 180 min. (R,S)-albuterol inhibited IL-5-induced superoxide production. This inhibition was enhanced by a cyclic adenosine monophosphate (cAMP) phosphodiesterase inhibitor, IBMX, and was reversed by the selective beta2-adrenergic receptor antagonist, ICI 118, 551, verifying the involvement of both cAMP and the beta2-adrenergic receptor. In addition, (R)-albuterol alone, similarly to (R,S)-albuterol, significantly inhibited IL-5-induced superoxide production up to 60 min (P<0.05, n=4), but the inhibition was lost with longer incubation. In contrast, (S)-albuterol with IBMX did not inhibit IL-5-induced superoxide production before 60 min, but it significantly enhanced IL-5-mediated superoxide production after 60 min (P<0.05, n=4). When both were present as racemic (R,S)-albuterol, the inhibitory effect of (R)-albuterol was not affected by (S)-albuterol. When incubated with IL-5-activated eosinophils, (R)-albuterol shows anti-inflammatory effects and (S)-albuterol shows pro-inflammatory effects in the presence of IBMX. The kinetics of these effects are different, and when used simultaneously, (R)-albuterol predominates. When marked usage of the (S)-isomer is anticipated, racemic (R,S)-albuterol should be used clinically with caution.

Changes in expression of cGMP selective phosphordiesterses 2,5 and 9 in the rat brain during aging

Nitric oxide (NO)-stimulated cGMP synthesis is present in the adult rat brain in close proximity to the NO-synthase containing structures [1]. Intracellular cGMP concentration is under very precise control and depends on parameters of its synthesis by guanylyl cyclase and degradation by phosphodiesterases (PDE). It is known that in the senescent brain the concentration of cGMP decreased [2]. When brain slices were incubated in the presence of isobutylmethylxanthine (IBMX), a nonselective phosphodiesterase inhibitor, sildenafil as a selective PDE5 inhibitor and BAY 60-7550 as a selective PDE2 inhibitor and DEANONOate as an NO donor, we have previously found that old brains are unresponsive to sildenafil treatment. Therefore in this study we wanted to investigate how the expression of cGMP selective phosphodiesterases changes in the brain during aging by using mRNA in situ hybridization.

Hyperinsulinaemia triggered by dietary conjugated linoleic acid is associated with a decrease in leptin and adiponectin plasma levels and pancreatic beta cell hyperplasia in the mouse

Dietary supplementation with conjugated linoleic acids (CLA) has a fat-reducing effect in various species, but induces severe hyperinsulinaemia and hepatic steatosis in the mouse. This study aimed to determine the causes of the deleterious effects of CLA on insulin homeostasis. The chronology of adipose and liver weight, hepatic triglyceride accumulation and selected blood parameters, including lipids, insulin, leptin and adiponectin, was determined in C57BL/6J female mice fed a 1% isomeric mixture of CLA for various periods of time ranging from 2 to 28 days. Insulin secretion was measured in 1-h static incubations of pancreatic islets, and pancreas morphometric parameters were determined in mice fed CLA for 28 days. Plasma levels of leptin and adiponectin sharply decreased after 2 days of CLA feeding, although adipose tissue mass only decreased after day 6. Hyperinsulinaemia developed at day 6 and consistently worsened up to day 28, in parallel with increases in hepatic lipid content. Islets from CLA-fed mice displayed three- to four-fold increased rates of glucose-stimulated insulin secretion, both in the absence and presence of isobutyl methylxanthine or carbachol. The increased insulin-releasing capacity of islets from CLA-fed mice was explained by an increase in beta cell mass and number. The data suggest that CLA supplementation induces a profound reduction of leptinaemia and adiponectinaemia, followed by hyperinsulinaemia due to the increased secretory capacity of pancreatic islets, leading, in turn, to liver steatosis. These observations cast doubt on the safety of dietary supplements containing CLA.

The effect of a nitric oxide donor on the synthesis of cGMP in Hymenolepis diminuta: a radiometric study

The formation of cGMP in homogenates of the adult rat-tapeworm Hymenolepis diminuta was followed with a radiometric assay during 3 h after stimulation with the nitric oxide donor sodium nitroprusside (SNP) and in the presence of isobutylmethylxanthine (IBMX). The level of cGMP was stable in worms incubated with IBMX during the first hour. After 3 h of incubation, the level of cGMP had declined by 27%. Addition of SNP stimulated the formation of cGMP during the first hour of incubation. After 3 h of incubation, a two-fold decline in cGMP formation was observed. The rate of nitric oxide (NO) release by the worm was determined by a spectrophotometric assay for the accumulation of nitrites and nitrates, the stable degradation products of NO, using the Griess reaction. The results are discussed from the perspective of the current concept on the role of the nitrergic mechanisms in the flatworm nervous system.

Spontaneous differentiation of mesenchymal stem cells obtained from fetal rat circulation

Mesenchymal stem cells (MSCs) are thought to be multipotential, capable of differentiating into multiple lineages. We attempted to characterize rat cells derived from fetal circulating blood (FCBCs) that displayed a fibroblastic morphology and differentiated into osteoblastic and chondrocytic lineages. Notably, they differentiated into a chondrocyte-specific phenotype on plastic culture dishes in medium supplemented only with 10% fetal bovine serum (FBS) without the use of a three-dimensional culture substrate. Bone marrow-derived cells did not convey such phenotypic expression under the same conditions. The characteristic features of these cells were analyzed by reverse transcription polymerase chain reaction, immunohistological and von Kossa staining, and by immuno-dot blotting. In one population, expression of collagen types II and X was detected in differentiated cells at the same levels as observed in chondrocytes derived from rat rib cartilage. In another population, parathyroid hormone receptor, alkaline phosphatase, and osteocalcin were also expressed at levels almost equal to those observed in long bone-derived osteoblasts. After 3 weeks in culture, extensively condensed cell masses, stained with anti-type II collagen antibody, could be distinguished histologically from small, multilayered, von Kossa-positive nodules, which stained with anti-osteocalcin, but not with anti-type II collagen antibody. In addition, the FCBCs differentiated into adipogenic cells in the presence of methyl-isobutyl xanthine, dexamethasone, insulin, and indomethacin. These cells expressed PPARγ2 mRNA and accumulated lipid vesicles detectable by Oil red-O staining. Our findings suggest that FCBCs have the potential to readily differentiate into multiple lineages and that they are distinct from mesenchymal stem cells derived from bone marrow or circulating blood from more mature and adults in their spontaneous differentiation in the absence of specific factors such as transforming growth factor-β (TGF-β) or dexamethasone, or a three-dimensional culture environment.

Potentiation of proopiomelanocortin gene expression in cultured pituitary cells by benzodiazepines.

Benzodiazepines are frequently used not only as a part of general anesthesia but also for the purpose of sedation during regional anesthesia. Effects of these drugs on the hypothalamic-pituitary-adrenal axis activity have been studied, but are still controversial. It is not known whether benzodiazepines affect expression of proopiomelanocortin, precursor protein of adrenocorticotropic hormone and related peptides. AtT20PL cell line, a clone of AtT20/D16v mouse corticotroph tumor cells stably transfected with approximately 0.7 kilobases (kb) of the rat proopiomelanocortin 5' promoter-luciferase fusion gene, was used. In the presence or absence of diazepam or midazolam, cells were stimulated by corticotropin-releasing hormone (CRH) or forskolin. Proopiomelanocortin gene expression was estimated by measurement of luciferase activity. Furthermore, to study the mechanism of benzodiazepine effects, cyclic adenosine 3',5'-monophosphate (cyclic AMP) efflux was measured by enzyme immunoassay. Diazepam and midazolam dose-dependently increased the proopiomelanocortin gene expression induced by CRH or forskolin. The potentiating effect was not affected by benzodiazepine receptor antagonists flumazenil and PK11195, but was abolished by a cyclic AMP-dependent protein kinase inhibitor H89. Cyclic AMP efflux induced by CRH or forskolin was also enhanced by diazepam and midazolam. In the presence of isobutylmethylxanthine, a nonspecific phosphodiesterase inhibitor, potentiation of proopiomelanocortin gene expression and enhancement of cyclic AMP efflux by benzodiazepines were not observed. Benzodiazepines potentiate the effect of CRH or forskolin on proopiomelanocortin gene expression. The potentiating effect is not mediated by the benzodiazepine receptors, but its mechanism probably involves inhibition of phosphodiesterase.

Troponin I phosphorylation enhances crossbridge kinetics during beta-adrenergic stimulation in rat cardiac tissue.

Inotropic agents that increase the intracellular levels of cAMP have been shown to increase crossbridge turnover kinetics in intact rat ventricular muscle, as measured by the parameter f(min) (the frequency at which dynamic stiffness is minimum). These agents are also known to increase the level of phosphorylation of two candidate myofibrillar proteins: myosin binding protein C (MyBPC) and Troponin I (TnI), but have no effect on myosin light chain 2 phosphorylation (MyLC2). The aim of this study was to investigate whether the phosphorylation of TnI and/or MyBPC was responsible for the increase in crossbridge cycling kinetics (as captured by f(min)) seen with the elevation of cAMP within cardiac tissue. Using barium-activated intact rat papillary muscle, we investigated the actions of isobutylmethylxanthine (IBMX), an inhibitor of cAMP-dependent phosphatase, which simulates the action of beta-adrenergic agents, and the chemical phosphatase 2,3-butanedione monoxime (BDM), which has been shown to dephosphorylate a number of contractile proteins. The presence of 0.6 mM IBMX approximately doubled the f(min) value of intact rat papillary muscle. This action was unaffected by the addition of BDM. In the presence of IBMX and BDM, the level of phosphorylation of MyBPC was unchanged, that of MyLC2 was reduced to 60 % of control, yet that of TnI was markedly increased (to 30 % above control levels). We conclude that TnI phosphorylation, mediated by cAMP-dependent protein kinase A, is the molecular basis for the enhanced crossbridge cycling seen during beta-adrenergic stimulation of the heart.

Inhibiting Phosphodiesterase Activity to Maintain Germinal Vesicle Stage Arrest Inhibits DNA Strand Breaks in Germinal Vesicle Stage Mouse Oocytes

Objective: Oocyte maturation in vitro offers many advantages to women undergoing ART, but the success rate remains low. One possibility is premature entry into meiosis in culture. Inhibitors of phosphodiesterase block meiosis in mouse oocytes, presumably by elevating cAMP and activating protein kinase A. In contrast, inhibitors of DNA topoisomerase II (TOPO II), required for the DNA strand breaks and repair necessary for chromosome condensation and separation, lead to germinal vesicle (GV) breakdown and extrusion of the entire chromosomal complement (“chemical enucleation”) of mouse oocytes. These opposing effects imply that maintaining the GV may stabilize TOPO II activity. We have tested this possibility. Design: Mouse oocytes were cultured without and with the phosphodiesterase inhibitor, isobutylmethylxanthine (IBMX) to maintain GV arrest and examined for DNA strand breaks as evidence of TOPO II activity. Some experiments included the antibiotic, distamycin, which stimulates TOPO II activity by exposing AT-rich regions of DNA. Oocytes from postpubertal females either not stimulated or stimulated with gonadotropin were examined. Materials and Methods: Oocytes were fixed and subjected to a sensitive terminal-uridine-nucletide-end-labeling (TUNEL) assay to detect DNA strand breaks. Two assay conditions were employed, without and with exposure to 2N HCL to remove chromatin histones. The incorporation of biotinylated uridine was detected by streptavidin-bound horseradish peroxidase reacting with the azo-dye, AEC. The oocytes were mounted under coverslips and counterstained with Hoechst to visualize DNA. Results: Forty two percent of GV eggs not pre-treated with HCl from both classes of postpubertal females were TUNEL assay positive at the time of collection from ovarian follicles. The frequency increased to 90% if the oocytes were pre-treated with HCl, implying histone masking of some of the DNA strand breaks. The TUNEL assay staining corresponded to Hoechst staining patterns, implying involvement of all regions of the chromatin. In contrast, following 24 hours of culture in IBMX, the frequency of TUNEL assay positive GV stage eggs decreased to 15 to 17% without or with pre-treatment with HCl. Co-culture with distamycin resulted in a return to 88% TUNEL-positive germinal vesicles. Overnight culture without IBMX and/or following transfer to IBMX-free conditions resulted in the resumption of meiosis and TUNEL-positive metaphase chromosomes. Conclusions: DNA strand breaks are common in the chromatin of mouse oocytes. IBMX exposure eliminates the strand breaks, apparently by inhibiting the cleavage, but not the repair activity of TOPO II. The inhibition is reversible. This novel finding supports the efficacy of extending oocyte culture in the presence of IBMX. Supported by the Assisted Reproduction Foundation and the Urologic Research Fund.

Inhibition of Osf2/Cbfa1 expression and terminal osteoblast differentiation by PPARγ2

Cells of the bone marrow stroma can reversibly convert among different phenotypes. Based on this and on evidence for a reciprocal relationship between osteoblastogenesis and adipogenesis, we have isolated several murine bone marrow-derived clonal cell lines with phenotypic characteristics of osteoblasts or adipocytes, or both. Consistent with a state of plasticity, cell lines with a mixed phenotype synthesized osteoblast markers like type I collagen, alkaline phosphatase, osteocalcin, as well as the adipocyte marker lipoprotein lipase, under basal conditions. In the presence of ascorbic acid and β-glycerophosphate—agents that promote osteoblast differentiation—they formed a mineralized matrix. In the presence of isobutylmethylxanthine, hydrocortisone, and indomethacin—agents that promote adipocyte differentiation—they accumulated fat droplets, but failed to express adipsin and aP2, markers of terminally differentiated adipocytes. Furthermore, they were converted back to matrix mineralizing cells when the adipogenic stimuli were replaced with the osteoblastogenic ones. A prototypic cell line with mixed phenotype (UAMS-33) expressed Osf2/Cbfa1—a transcription factor required for osteoblast differentiation, but not PPARγ2—a transcription factor required for terminal adipocyte differentiation. Stable transfection with a PPARγ2 expression construct and activation with the thiazolidinedione BRL49653 stimulated aP2 and adipsin synthesis and fat accumulation, and simultaneously suppressed Osf2/Cbfa1, α1(I) procollagen, and osteocalcin synthesis. Moreover, it rendered the cells incapable of forming a mineralized matrix. These results strongly suggest that PPARγ2 negatively regulates stromal cell plasticity by suppressing Osf2/Cbfa1 and osteoblast-like biosynthetic activity, while promoting terminal differentiation to adipocytes. J. Cell. Biochem. 74:357–371, 1999. © 1999 Wiley-Liss, Inc.

Inhibition of Osf2/Cbfa1 expression and terminal osteoblast differentiation by PPAR?2

Cells of the bone marrow stroma can reversibly convert among different phenotypes. Based on this and on evidence for a reciprocal relationship between osteoblastogenesis and adipogenesis, we have isolated several murine bone marrow-derived clonal cell lines with phenotypic characteristics of osteoblasts or adipocytes, or both. Consistent with a state of plasticity, cell lines with a mixed phenotype synthesized osteoblast markers like type I collagen, alkaline phosphatase, osteocalcin, as well as the adipocyte marker lipoprotein lipase, under basal conditions. In the presence of ascorbic acid and beta-glycerophosphate-agents that promote osteoblast differentiation-they formed a mineralized matrix. In the presence of isobutylmethylxanthine, hydrocortisone, and indomethacin-agents that promote adipocyte differentiation-they accumulated fat droplets, but failed to express adipsin and aP2, markers of terminally differentiated adipocytes. Furthermore, they were converted back to matrix mineralizing cells when the adipogenic stimuli were replaced with the osteoblastogenic ones. A prototypic cell line with mixed phenotype (UAMS-33) expressed Osf2/Cbfa1-a transcription factor required for osteoblast differentiation, but not PPARgamma2-a transcription factor required for terminal adipocyte differentiation. Stable transfection with a PPARgamma2 expression construct and activation with the thiazolidinedione BRL49653 stimulated aP2 and adipsin synthesis and fat accumulation, and simultaneously suppressed Osf2/Cbfa1, alpha1(I) procollagen, and osteocalcin synthesis. Moreover, it rendered the cells incapable of forming a mineralized matrix. These results strongly suggest that PPARgamma2 negatively regulates stromal cell plasticity by suppressing Osf2/Cbfa1 and osteoblast-like biosynthetic activity, while promoting terminal differentiation to adipocytes.

Rat angiotensin-converting enzyme promoter regulation by beta-adrenergics and cAMP in endothelium.

To shed light on mechanisms of angiotensin-converting enzyme (ACE) upregulation, we used a rabbit endothelial cell model to characterize intracellular pathways of beta-adrenergic stimulation. In these cells, ACE activity is increased by isoproterenol (ISO). The stably transfected 1273-bp ACE promoter is stimulated by ISO in the presence of isobutyl methylxanthine. This effect is abolished by propranolol. Promoter stimulation is mimicked by cholera toxin, forskolin, and 8BrcAMP, but not by 8BrcGMP. Promoter stimulation by ISO and isobutyl methylxanthine is blocked by protein kinase A inhibitors, indicating that beta-adrenergic stimulation of the ACE gene depends on phosphorylation of protein kinase A targets. Activation by cAMP, resistance to phorbol ester, and lack of synergism between cAMP and phorbol ester suggest that promoter regulation is due to cAMP responsive element rather than to activating protein-2 sequences. Okadaic acid potentiation of 8BrcAMP induction indicated that promoter activation by cAMP is regulated by phosphatases controlling activation of typical cAMP responsive element regulated genes. In summary, beta-adrenergic activation of rat ACE promoter is specific; uses G(s) proteins, adenylyl cyclase, protein kinase A; and probably includes cAMP responsive element-like sequences.

Regulation of adenylyl cyclase activity in human peripheral blood mononuclear cells: effects of protein kinase inhibitors and of a calcium ionophore

In a previous paper we presented evidence for a negative regulation of adenylyl cyclase activity by tyrosine protein kinase(s) in the human leukemic T cell line Jurkat. In order to examine this point in non malignant cells, we conducted the present study in human peripheral blood mononuclear cells (PBMC). In these cells, staurosporine, a broad spectrum protein kinase inhibitor, enhanced not only the receptor-mediated, induced by prostaglandin E2 (PGE2), but also the direct (forskolin-induced) stimulation of adenylyl cyclase activity. Herbimycin A, a specific protein tyrosine kinase inhibitor, reproduced only in part the effect of staurosporine, whereas bisindolylmaleimide, the most specific protein kinase C (PKC) inhibitor known at present time, was ineffective. All these observations were made both in the absence and presence of isobutylmethylxanthine, a phosphodiesterase inhibitor, indicating that the effects of staurosporine and herbimycin A on cAMP accumulation were not due to phosphodiesterase inhibition. The calcium ionophore A 23187 also enhanced the PGE2-induced cAMP accumulation, and this effect was not additive to that of staurosporine, but additive to that of herbimycin A. These results confirm and extend those obtained in Jurkat cells. Taken together, they indicate that in human PBMC the adenylyl cyclase activity is negatively regulated by tyrosine kinase(s) and not by PKC, and positively regulated by Ca 2. They also suggest that the major enhancement by staurosporine of the PGE2-induced cAMP accumulation, although chiefly mediated by protein tyrosine kinase inhibition, also depends on another, presently undetermined, effect of the drug simulating that of Ca 2.