Comparative Analysis of Dibutyric cAMP and Butyric Acid on the Differentiation of Human Eosinophilic Leukemia EoL-1 Cells

Purification of enough numbers of circulating eosinophils is difficult because eosinophils account for less than 5% peripheral blood leukocytes. Human eosinophilic leukemia EoL-1 cells have been considered an in vitro source of eosinophils as they can differentiate into mature eosinophil-like cells when incubated with dibutyryl cAMP (dbcAMP) or butyric acid. In this study, the viability and phenotypic maturation of EoL-1 cells stimulated by either dbcAMP or butyric acid were comparatively analyzed. After treatment with 100 microM dbcAMP or 0.5 microM butyric acid, EoL-1 cells showed morphological signs of differentiation, although the number of nonviable EoL-1 cells was significantly increased following butyric acid treatment. Stimulation of EoL-1 cells with 0.5 microM butyric acid more effectively induced the expression of mature eosinophil markers than stimulation with dbcAMP. These results suggest that treatment of EoL-1 cells with 0.5 microM butyric acid for limited duration could be an effective strategy for inducing their differentiation. Considering that expression of CCR3 was not sufficient in EoL-1 cells stimulated with 0.5 microM butyric acid, treatment of the chemically stimulated EoL-1 cells with cytokines, which primarily support eosinophil maturation, would help to obtain differentiated EoL-1 cells with greater functional maturity.

Human SNF2L Gene Is Regulated Constitutively and Inducibly in Neural Cells via a cAMP-Response Element

PURPOSE: SNF2L belongs to Imitation Switch family and plays an essential role in neural tissues and gonads. In our previous studies, we have demonstrated that the basal transcription of human SNF2L gene is regulated by two cis-elements, cAMP response element (CRE)- and Sp1-binding sites. Recent studies suggested that cyclic adenosine monophosphate (cAMP) stimulation significantly up-regulated SNF2L expression in ovarian granulose cells. These data suggested that protein kinase-mediated signal pathways might also regulate SNF2L expression in neural cells. We therefore investigated the effects of agents that activate protein kinases A on SNF2L gene expression in neural cells. MATERIALS AND METHODS: To increase intracellular cAMP levels, all neural cells were treated with forskolin and dbcAMP, two cAMP response activators. We exmined the effects of cAMP on the promoter activity of human SNF2L gene by luciferase reporter gene assays, and further examined the effects of cAMP on endogenous SNF2L mRNA levels by qPCR. RESULTS: Transient expression of a luciferase fusion gene under the control of the SNF2L promoter was significantly increased by treatment of rat primary neurons with forskolin or dbcAMP, but not PC12, C6 and SH-SY5Y cells. Consistently, treatment with forskolin or dbcAMP could enhance endogenous SNF2L mRNA levels also only in rat primary neurons. CONCLUSION: These results suggest that the CRE consensus sequence in the SNF2L proximal promoter most likely confers constitutive activation and regulation by cAMP in neural cells.

Role of cGMP and cAMP in the hemodynamic response to intrathecal sildenafil administration

INTRODUCTION: Results from our laboratory have demonstrated that intracerebroventricular administration of sildenafil to conscious rats promoted a noticeable increase in both lumbar sympathetic activity and heart rate, with no change in the mean arterial pressure. The intracerebroventricular administration of sildenafil may have produced the hemodynamic effects by activating sympathetic preganglionic neurons in the supraspinal regions and spinal cord. It is well documented that sildenafil increases intracellular cGMP levels by inhibiting phosphodiesterase type 5 and increases cAMP levels by inhibiting other phosphodiesterases. OBJECTIVE: To examine and compare, in conscious rats, the hemodynamic response following the intrathecal administration of sildenafil, 8-bromo-cGMP (an analog of cGMP), forskolin (an activator of adenylate cyclase), or dibutyryl-cAMP (an analog of cAMP) in order to elucidate the possible role of the sympathetic preganglionic neurons in the observed hemodynamic response. RESULTS: The hemodynamic responses observed following intrathecal administration of the studied drugs demonstrated the following: 1) sildenafil increased the mean arterial pressure and heart rate in a dose-dependent manner, 2) increasing doses of 8-bromo-cGMP did not alter the mean arterial pressure and heart rate, 3) forskolin did not affect the mean arterial pressure but did increase the heart rate and 4) dibutyryl-cAMP increased the mean arterial pressure and heart rate, similar to the effect observed following the intrathecal injection of the highest dose of sildenafil. CONCLUSION: Overall, the findings of the current study suggest that the cardiovascular response following the intrathecal administration of sildenafil to conscious rats involves the inhibition of phosphodiesterases other than phosphodiesterase type 5 that increase the cAMP level and the activation of sympathetic preganglionic neurons.

Efecto del extracto acuoso de hojas de Bauhinia megalandra sobre la glucogenólisis hepática en ratas / Effect of aqueous extract of leaves of Bauhinia megalandra glucogenólisis on the liver in rats

El extracto acuoso de las hojas de Bauhinia megalandra ha sido muy empleado en Venezuela en el tratamiento empírico de la diabetes mellitus. En el presente trabajo se estudió el efecto del extracto acuoso de B. megalandra sobre la glucogenolísis hepática estimulada por adrenalina o dibutiril AMPc. La administración oral del extracto de la planta, a ratas alimentadas, disminuyó de una manera estadísticamente significativa el incremento de la glicemia promovido por la adrenalina. De igual manera, rebanadas de hígado de ratas alimentadas incubadas en presencia del extracto de B. megalandra produjeron menos glucosa en respuesta a la adrenalina o al dibutiril AMPc que los controles. Estos resultados indican una disminución de la glucogenolísis hepática por efecto del extracto acuoso de hojas de B. megalandra, probablemente por inhibición de la enzima glucosa-6-fosfatasa.

Slow Ca2+ channels neither contribute to upstroke of action potential nor to pacemaker potential in spontaneously active freshly isolated three day embryonic chick ventricle.

Contribution of slow Ca2+ channels to the upstroke of action potential (AP) and pacemaker potential was studied by observing the effects of Ca2+ channel activators- high [Ca2+]0, Bay-K-8644, isoproterenol, forskolin and dibutyryl-cAMP on spontaneous AP of freshly isolated 3 day embryonic chick ventricle (3 day ECV). The spontaneous APs showed maximal upstroke velocity (+Vmax), maximum diastolic potential (MDP), overshoot (Eov) and AP duration at -20 mv (APD20) of 42.60 +/- 2.40 V/sec, -59.05 +/- 0.95 my, 16.30 +/- 0.53 mv and 70.32 +/- 4.60 msec, respectively (an average value of 35 preparations). Bay-K-8644 (0.1-0.8 microM), isoproterenol (5-10 pM) and forskolin (0.1-2.0 microM) induced a concentration-dependent increase in APD20 and Eov without affecting +Vmax. Dibutyryl-cAMP (1 microM) also enhanced the APD20 and Eov and had no effect on +Vmax. Elevation of [Ca2+]0 from 0.6 mM to 9.6 mM caused a concentration-dependent increase in APD20 and Eov leaving +Vmax unaltered. Elevated [Ca2+] and the other Ca2+ channel activators had no significant effect on MDP in above concentration range. Increase in APD20 and Eov could be explained at least by activation of slow Ca2+ channels but the lack of any change in +Vmax clearly suggests that the slow Ca2+ channels do not contribute to the upstroke of AP. All these interventions reduced the rate of spontaneous firing without any noticeable effect on MDP. This finding shows that the slow Ca2+ channels also do not contribute directly to the generation of pacemaker potential in spontaneously active freshly isolated 3 day ECV.

Study of effect and mechanism of c-myb on the fertilization in mouse / 中国应用生理学杂志

<p><b>AIM</b>To investigate the distribution of c-myb, an oncoprotein, in mouse oocytes-cumulus cell complex and sperm immunohistochemically.</p><p><b>METHODS</b>To study the effect of c-myb on mouse fertilization in vitro, various concentration of c-myb antisense-oligodeoxynucleotides (c-myb ASODNs) were incubated with sperms and oocytes during fertilization. To explore the possible mechanism involved in fertilization, the relationship between c-myb ASODNs and GABA or dbcAMP or Verapamil or Progesterone in fertilization was also observed by immunohistochemical methods.</p><p><b>RESULTS</b>c-myb oncoprotein was observed on the nucleus of cumulus cell and head of sperm. c-myb ASODNs inhibited the rate of fertilization in vitro in a dose-dependent way. The fertilization rates of the control group, low (5 micromol/L), medium (10 micromol/L), high (20 micromol/L) concentration c-myb ASODNs groups and nonsense tat oligodeoxynucleotides (20 micromol/L) group were 34.97%, 30.89%, 20.14%, 16.68%, 34.47%, respectively. All of GABA, Progesterone and dbcAMP inversed the c-myb ASODNs inhibition effects on fertilization rate, but neither of them showed significant effect on the percentages of immunohistochemical stain of Myb on sperm and cumulus cells. By contrast, Verapamil inhibited the fertilization rate. Co-treated with c-myb ASODNs, Verapamil showed synergic inhibiting effects on the fertilization with c-myb ASODNs. Verapamil also inhibited the expression of Myb on head of sperm. The fertilization rates of the control group, medium (10 micromol/L) concentration c-myb ASODNs group, GABA group, P4 group, Verapamil group, dbcAMP group were 34.81%, 22.96%, 40.83%, 39.12%, 7.46%, 40.61%, respectively.</p><p><b>CONCLUSION</b>c-myb ASODNs is closely correlated with fertilization. Verapamil can inhibit fertilization in vitro through regulating Myb expression of sperm, while GABA, dbcAMP and Verapamil may affect the process of fertilization through the way other than Myb expression.</p>

Phosphorylation of phospholipase D1 and the modulation of its interaction with RhoA by cAMP-dependent protein kinase

Agents that elevate cellular cAMP are known to inhibit the activation of phospholipase D (PLD). We investigated whether PLD can be phosphorylated by cAMP-dependent protein kinase (PKA) and PKA-mediated phosphorylation affects the interaction between PLD and RhoA, a membrane regulator of PLD. PLD1, but not PLD2 was found to be phosphorylated in vivo by the treatment of dibutyryl cAMP (dbcAMP) and in vitro by PKA. PKA inhibitor (KT5720) abolished the dbcAMP-induced phosphorylation of PLD1, but dibutyryl cGMP (dbcGMP) failed to phosphorylate PLD1. The association between PLD1 and Val14RhoA in an immunoprecipitation assay was abolished by both dbcAMP and dbcGMP. Moreover, RhoA but not PLD1 was dissociated from the membrane to the cytosolic fraction in dbcAMP-treated cells. These results suggest that both PLD1 and RhoA are phosphorylated by PKA and the interaction between PLD1 and RhoA is inhibited by the phosphorylation of RhoA rather than by the phosphorylation of PLD1.

Effects of Melatonin on the Meiotic Maturation of Mouse Oocytes in vitro / 대한불임학회지

OBJECTIVE: Melatonin, which is secreted by pineal gland play an important role in the regulation of ovarian function via seasonal rhythm and sleep in most mammals. It also has a role in the protection of cells by removing toxic oxygen free radicals brought about by metabolism. In the present study, effects of melatonin on the mouse oocyte maturation were examined using two different culture conditions provided with 5% or 21% oxygen concentration. MATERIAL AND METHOD: Immature mouse oocytes were obtained from the ovarian follicles of 3~4 weeks old ICR strain mice intraperitoneally injected with 5 I.U. PMSG 44 hour before. Under stereomicroscope, morphologically healthy oocytes with distinct germinal vesicle (GV) were liberated from the graafian follicles and collected using mouth-controlled micropipette. They were then cultured for 17 hour at 37degrees C, 5% CO2 and 21% O2 (95% air) or 5% CO2, 5% O2 and 90% N2. New modified Hank's balanced salt solution (New MHBS) was used as a culture medium throughout the experiments. Effects of melatonin were examined at a concentration of 0.0001 micrometer, 0.01 micrometer or 1.0 micrometer. For the prevention of spontaneous maturation of immature oocytes during culture, dibutyryl cyclic AMP (dbcAMP) and/or hypoxanthine were included in the medium. RESULTS: Under 21% oxygen condition, oocytes cultured in the presence of 0.01 micrometer melatonin showed a significantly higher maturation rates, in terms of germinal vesicle breakdown (95.0% vs 89.0%) and polar body formation (88.1% vs 75.4%), compared to those cultured with 0.0001 micrometer or 1.0 micrometer melatonin. However, no difference was observed in oocytes cultured under 5% oxygen whether they were treated with melatonin or not. In the presence of 0.01 micrometer melatonin, oocytes either cultured under 21% or 5% oxygen exhibited no difference in the polar body formation (85.6% vs 86.7%). However, in the absence of melatonin, oocytes cultured under 21% oxygen exhibited lower polar body formation (74.7%). When oocytes were cultured in the presence of dbcAMP alone or with varying concentrations of melatonin, those treated with both compounds always showed better maturation, i.e., germinal vesicle breakdown and polar body formation, compared to those cultured with dbcAMP alone. At the same concentration of melatonin, however, oocytes exposed to 21% oxygen showed poor maturation than those to 5% oxygen. Similar results were obtained from the experiments using hypoxanthine instead of dbcAMP. CONCLUSION: Based upon these results, it is suggested that melatonin could enhance the meiotic maturation of mouse oocytes under 21% oxygen concentration, and release oocytes from the meiotic arrest by dbcAMP or hypoxanthine regardless of the concentration of oxygen, probably via the removal of oxygen free radicals.

CD99 type II is a determining factor for the differentiation of primitive neuroectodermal cells

CD99 is a 32-kDa cell surface molecule present on thymocytes, peripheral T cells, many other hematopoietic stem cells and somatic cells were implicated in cell-cell adhesion and cell-activation phenomena. Two major subtypes have been identified so far, designated CD99 type I and type II. We have investigated the correlation between the degree of neural differentiation and the expression of CD99 subtypes in three differentially differentiated cell lines such as CADO-ES1, RD-ES, and SH-N-SY5Y, in order of differentiation. In addition, we induced differentiation of the RD-ES cell line by N(6),2'-dibutyryl-cAMP (db-cAMP). Six days after treatment with db-cAMP, RD-ES cell line has changed its morphology from uniform round cells to cells with neurites, and initially CD99 type II-overexpressed RD-ES cells showed significant down-regulation of CD99 type II, whereas CD99 type I expression remained constant. When RD- ES cells were transfected with the cDNA encoding for CD99 type I-green fluorescence protein (GFP) and type II-GFP, CD99 type II transfected RD-ES cell line remained unchanged with morphology of undifferentiated form. Our data suggest that CD99 type II acts as a negative regulator in the neural differentiation of precursor cells that might occur during nerve system development.

Experimental study of c-erbB2 on the fertilization in mouse / 中国应用生理学杂志

<p><b>AIM AND METHODS</b>The distribution of ErbB2 in mouse testis, epididymidis, ovaries, oocyte-cumulus cells-complexes in oviducts and sperms was investigated immunohistochemically. To study the effect of c-erbB2 on mouse fertilization in vitro, various concentrations of c-erbB2 antisense oligonucleotides (c-erbB2 ASODNs) were incubated with sperms and oocyte-cumulus cells-complexes during fertilization in vitro. To explore possible mechanisms involved in fertilization, the relationship between c-erbB2 ASODNs and GABA, or dbcAMP, or verapamil during fertilization in vitro was also observed.</p><p><b>RESULTS</b>ErbB2 oncoprotein was observed in epithelial cells in epididymis, sperms and cumulus cells. C-erbB2 ASODNs inhibited the rate of fertilization in vitro in a dose-dependent way. The fertilization rate of the control group, low (5 micromol/L), medium (10 micromol/L), high (20 micromol/L) concentration c-erbB2 ASODNs group, and nonsense at oligonucleotides group (20 micromol/L) was 38.3%, 19.6%, 10.7%, 5.0%, and 33.8% respectively. Integral optical density immunostaining of ErbB2 in sperms was notably reduced. Medium and high concentration of c-erbB2 ASODNs notably inhibited cumulus cells adhering to inner wall of Petri dish. Treated alone with GABA or dbcAMP, the rate of fertilization was increased. Both GABA and dbcAMP partially inversed the ASODNs inhibition effect on fertilization rate, but neither of them showed significant effect on sperm integral optical density of ErbB2 immunostaining. In contrast, verapamil inhibited fertilization rate. Co-treated with c-erbB2 ASODN, verapamil showed synergic inhibiting effect on fertilization with c-erbB2 ASODN. Verapamil also inhibited the expression of c-erbB2 in sperms.</p><p><b>CONCLUSION</b>It is suggested that c-erbB2 is closely correlated with fertilization. Ca2+ may inhibit fertilization in vitro through regulation the expression of c-erbB2 gene in sperm cells, while both of GABA and dbcAMP may affect the process of fertilization through the way other than c-erbB2 expression in sperm cells.</p>

Influences of dibutyryl cyclic adenosine monophosphate and forskolin on human sperm motility in vitro / 亚洲男科学杂志(英文版)

<p><b>AIM</b>To study the influences of dibutyryl cyclic adenosine monophosphate (dbcAMP) and forskolin on human sperm motility in vitro.</p><p><b>METHODS</b>Semen samples, aseptically obtained by masturbation and prepared by swim-up technique from 20 fertile men, were incubated with different concentrations of dbcAMP and forskolin at 37 deg. Measurements were carried out after 10 min, 20 min, 30 min and 60 min incubation. Motility parameters were estimated by using an automatic analyzing system.</p><p><b>RESULTS</b>Treatment with dbcAMP or forskolin resulted in a significant increase in sperm motility and progressive motility. The larger the concentrations of dbcAMP or forskolin, the greater the effect appeared. The straight linear velocity and curvilinear velocity were not affected by both agents.</p><p><b>CONCLUSION</b>dbcAMP and forskolin increase the motility and progressive motility of human sperm in vitro.</p>

Influence of dibutyryl cyclic adenosine monophosphate on motility of sperm / 中华男科学杂志

<p><b>OBJECTIVES</b>To investigate the influence of cAMP/PKA signal transduction on human sperm motility, and to study the effect of dibutyryl cyclic adenosine monophosphate dibutyryl cyclic adenosine monophosphate (dbcAMP) on human sperm motility in vitro.</p><p><b>METHODS</b>Sperm aseptically obtained by masturbation and prepared by swim-up technique from 10 healthy fertile men were incubated with different concentrations of dbcAMP. Measurement of mobility were carried out at 20, 30, and 60 min in all specimens.</p><p><b>RESULTS</b>The sperm treated with dbcAMP showed a significant increase in sperm progressive motility and the percentage of motile cells. The effect seemed enhanced with the increasing of dbcAMP concentration. VSL and VCL were not affected by dbcAMP.</p><p><b>CONCLUSIONS</b>dbcAMP can activate the mobility of human sperm in vitro.</p>

Relationship between bicarbonate and cyclic nucleotide in the promoting effects on head-to-head agglutination in boar spermatozoa / 亚洲男科学杂志(英文版)

<p><b>AIM</b>To clarify the relationship between bicarbonate and cAMP in the promoting effects on the sperm agglutination.</p><p><b>METHODS</b>Spermatozoa were collected from mature boars, washed and resuspended in a modified Krebs-Ringer HEPES lacking calcium chloride (mKRH). The sperm suspensions were incubated in a water bath (38.5 degrees C) for 60 min and then the percentage of head-to-head agglutinated spermatozoa was determined.</p><p><b>RESULTS</b>Supplementation of the mKRH with sodium bicarbonate (5-10 mM) significantly raised the percentage of head-to-head agglutinated spermatozoa in the samples. The addition of selective inhibitors for calcium/calmodulin-dependent phosphodiesterases (type 1: 8-methoxymethyl-IBMX and vinpocetine, 25-50 micro M) or for cAMP-specific phosphodiesterases (type 4: Ro20-1724 and rolipram, 25-50 microM) enhanced the effect of bicarbonate on sperm agglutination as highly as did the addition of non-selective inhibitors for phosphodiesterases (IBMX and papaverine, 25-50 microM). A calmodulin antagonist (W-7, 2 microM), that potentially blocks the stimulator of the calcium/calmodulin-dependent phosphodiesterases, significantly enhanced the effect of bicarbonate on sperm agglutination. Moreover, a phosphodiesterase-resistant cAMP analogue (cBiMPS, 0.1 mM) markedly induced agglutination in more spermatozoa (76%) after the incubation without bicarbonate and phosphodiesterase inhibitors than did a less potent cAMP analogue (dibutyryl cAMP, 1 mM) (21%), while three kinds of cGMP analogues (0.1-1 mM) had no effect on sperm agglutination. In addition, a cAMP antagonist (Rp-cAMPS, 1 mM) significantly reduced the sperm agglutination resulting from the actions of bicarbonate and IBMX. On the other hand, the effect of bicarbonate was abolished by a change of incubation temperature from 38.5 degrees C to 25 degrees C.</p><p><b>CONCLUSION</b>These findings demonstrate that the bicarbonate-induced agglutination of boar spermatozoa is controlled via the cAMP-mediated, temperature-dependent signaling cascade. This cascade is suppressed by the action of the phosphodiesterase (at least types 1 and 4).</p>

Theiler virus-GDVII strain (TMEV-GDVII) infection of cultured astrocytes. An image analysis of its effects on cell activation

Our original aim was to determine whether dBcAMP-induced activation of cultured astrocytes affected the course of subsequent viral infection. After 2 h exposure of 2-day-old first subculture of mouse astrocytes to dBcAMP 1 mM, cell monolayers grown in glass coverslips of Leighton tubes were inoculated with 10(3) PFU of Theiler virus-GDVII strain (TMEV-GDVII). At 9 days post-infection (pi), viral infectivity persisted in supernatants from dBcAMP-treated cultures, but was no longer detectable in non-stimulated controls. The relatively spared astroglial monolayer at day 1 pi, hardly affected by progressive viral cytolytic effect, was chosen for immunolabeled cell count, whether by viral antigen or GFAP. To this end, 20 fields for each coverslip were digitalized at 250x final magnification. In dBcAMP treated cultures, viral antigen(+) cells were fewer and lower in percentage versus infected cultures lacking stimulation. As regards GFAP staining, stimulation or infection per se induced a greater number and percentage of labeled astrocytes. According to morphometric characterization, such increase was due to a greater number of process-bearing astrocytes. It may be concluded that, regardless of previous dBcAMP treatment, early TMEV-GDVII infection enhanced immunocytochemical and morphological differentiation in cultured astrocytes.

Time sequence of changes in the responsiveness of glycogen breakdown to adrenergic agonists in perfused liver of rats with insulin-induced hypoglycemia

The time-course changes of the responsiveness of glycogen breakdown to a- and Beta-adrenergic agonists during insulin-induced hypoglycemia (IIH) were investigated. Blood glucose levels were decreased prior to the alteration in the hepatic responsiveness to adrenergic agonists. The activation of hepatic glucose production and glycogenolysis by phenylephrine (2 µM) and isoproterenol (20 µM) was decreased in IIH. The changes in the responsiveness of glycogen catabolism were first observed for isoproterenol and later for phenylephrine. Hepatic ß-adrenergic receptors showed a higher degree of adrenergic desensitization than did a-receptors. Liver glycogen synthase activity, glycogen content and the catabolic effect of dibutyryl cyclic AMP (the Beta-receptor second messenger) were not affected by IIH.

Immunocytochemical and morphological effects of short-term stimulation of cultured astrocytes

Both image analysis at light microscopy level and ultrastructural characterization by transmission electron microscopy were employed to evaluate the differentiation stage in young cultured mouse astrocytes after 1-day exposure to dBcAMP, a chemical compound known to induce cell activation. The aim was to validate an experimental model of stimuled astrocytes preserving the properties of recently seeded cells, thus avoiding the overlapping effects of in vitro aging. Differentiated astrocytes, as evidenced by GFAP labeling by streptavidin-perioxidase, doubled their number in treated cultures (45 per cent) versus controls (23 per cent). In addition, a significant increase in processing-bearing astrocytes (elongated forms) to the detriment of immature polygonal astrocytes, was recorded. No noticeable changes were found in cell perimeter, but cell area displayed a significant reduction in labeled surface of astrocytes undergoing morphological differentiation. Concomitantly, electron microscopy showed that radially organized bundles of numerous intermediate filaments compatible with GFAP replaced the few scattered structures observed in control cultures. However methodological caution is advisable as regards the relevance of the in vitro counterpart in situ reactive astrocytes, since cell plasicity is recognized to depend on culture conditions. At any rate, present quantitative results demonstrate that GFAP-positive cell percentage and cell area measurement are adequate parameters of early immunocytochemical and morphological differentiation, respectively, and thus contribute to a better histometric characterization of an easily available substrate to discriminate the wide variety of factors involved in CNS response to injury.

Physiological Role of PGE2 and DBcAMP in Bone Cell Metabolism / 대한면역학회지

One of the primary functions for which bones have evolved is to act as a structural support. To achieve this, bones remodel throughout life so that their structure remains optimal for the prevailing mechanical environment. Bone remodeling consists of an initial phase of osteoclastic bone resorption followed by a bone formation period. Prostaglandins are potent regulators of bone formation and bone resorption that can have both stimulatory and inhibitory effects. Elevation of intracellular cAMP is an important intracellular signaling mechanism involved in the regulation of the expression of many proteins. In this study we examine whether PGE or DBcAMP affects osteoblastic activation or osteoclastic differentiation in mouse bone marrow cells and osteosarcoma ROS 17/2.8 cells. The effect of PGE and DBcAMP on the cell proliferation was measured by the incorporation of [3H]- thymidine into DNA. As a result, PGE2 (0.5-1 ug/ml) and DBcAMP (0.1-0.5 mM) inhibited the [3H]-thymidine incorporation into DNA in a dose dependent manner. The effect of PGE2 and DBcAMP on the induction of alkaline phosphatase (ALP) was investigated in ROS 17/2.8 cells cultured in medium containing 0.4% fetal bovine serum. PGE and DBcAMP stimulated ALP activity in the cells in a dose- dependent manner. PGE2 also increased the intracellular cAMP content in a dose- dependent fashion with a maximal effect at 0.5 ug/ml. ROS 17/2.8 cells release nitric oxide upon stimulation of PGE2 or DBcAMP with interferon-r. PGE2 and DBcAMP increase the phosphorylation level of CREB (cAMP response element binding protein) without any change on the amount of CREB protein. Also, PGE (10-6 M) and DBcAMP (10-4 M) significantly increase the generation of osteoclasts in mouse bone marrow cell culture system. In conclusion, the results of this study suggested that cAMP appears to be an important regulatory molecule in the processes of bone formation and resorption.

Cholera toxin mediated regulation of the expression of Gq alpha and G11 alpha GTP binding proteins

Previously it has been shown that persistent activation of the stimulatory adenylyl cyclase pathway with cholera toxin (CT) downregulates the Gs alpha polypeptide (80%) in a cAMP-independent manner in C6 glioma cells (Shah, 1997). This study was conducted to examine the short and long term effects of CT on the regulation of pertussis toxin-sensitive and -insensitive G proteins and their transcripts in C6 glioma cells. Treatment of C6 cells with CT (100 ng/ml) up to 16 h had no effect on either Gi or Gq/11 alpha proteins. However, prolonged exposure (24-48 h) caused increased expression of Gi (20-30%) and Gq/11 alpha proteins (40%). Urea gradient gels, which can separate Gq alpha and G11 alpha proteins, revealed that prolonged CT treatment increased the expression of both of these G proteins. The CT-mediated enhanced expression of Gq alpha and G11 alpha proteins was accompanied by increased mRNA levels of these proteins as determined by RT/PCR. Cyclic-AMP elevating agents like forskolin (10 microM) and db-cAMP (1 mM) mimicked the effect of CT on Gi but not Gq/11 alpha proteins. These studies show long term cAMP-dependent regulation of Gi and cAMP-independent expression of Gq/11 alpha proteins in C6 glioma cells.

Effects of Nitric Oxide on the Maturation of Mouse Oocyte in vitro / 대한산부인과학회잡지

OBJECTIVE: Nitric oxide (NO) produced in ovary may contribute to follicle maturation, ovulation, oocyte maturation and luteinization. In this study, the effect of nitric oxide on the spontaneous maturation of mouse oocyte was observed. Method: The index of oocyte maturation was checked by the germinal vesicle breakdown (GVBD) and appearance of polar body (PB) under microscope in the denuded oocytes and oocyte-cumulus complexes (OCCs) from mouse ovarian follicles after 24 hours pregnant-mare serum gonadotropin treatment. RESULTS: The GVBD appeared 50 %, 1 hour and 80 %, 2 hrs after changes of oocytes from dibutyryl cAMP (dbcAMP, 0.5 mM) contained media into dbcAMP-free media. dbcAMP (0.5 mM) completely blocked the GVBD until 24 hrs but dbcGMP (5 mM) delayed the GVBD by 1 hr. Sodium nitroprusside, the NO generator, inhibited the GVBD dose-dependently at 2 hr incubation in denuded and OCCs. The appearance of GVBD was not different between control and dbcGMP or SNP in denuded oocytes and OCCs at 24 hrs incubation. The guanylate cyclase activity in denuded oocyte cytosol was not detected whereas the guanylate cyclase activity in OCCs cytosol was 1.3 nmole/min/mg protein which was increased about 3 times by SNP (100 micrometer). CONCLUSION: These results suggest that the NO in ovary may delay the spontaneous oocyte maturation in early stage by acting on the maturation signaling protein as well as guanylate cyclase.