Presence Of dbcAMP Research Articles

The Modulation of Functional Status of Bovine Spermatozoa by Progesterone.

Simple SummaryProgesterone is an endogenous steroid hormone, which can induce capacitation and/or acrosome reactions in semen of certain mammalian species. Our study aimed to investigate the effect of progesterone on the functional status of fresh bovine spermatozoa using a chlortetracycline fluorescent probe. Results showed that heparin induced capacitation in spermatozoa incubated with or without progesterone. The destruction of microfilaments by an inhibitor of cytochalasin D blocked the stimulating effect of heparin. Steroid hormone in mixture with prolactin stimulated the acrosome reaction in spermatozoa, which was blocked by an inhibitor of microtubule polymerization (nocodazole). At the acrosome stage, prolactin provided the undergoing of acrosome reaction in male gametes. This effect was noted both in the presence and absence of progesterone and inhibited by nocodazole. The supplementation of dibutyryl cyclic adenosine monophosphate during the acrosome reaction to progesterone-untreated spermatozoa did not cause changes in proportion of acrosome-reacted cells. However, when progesterone was added during capacitation, a significant increase in the proportion of capacitated cells was noted, which was inhibited by nocodazole. Thus, progesterone under the action of prolactin and dibutyryl cyclic adenosine monophosphate determines the functional status of fresh spermatozoa, which indicates progesterone-modulating effect on the indicators of post-ejaculatory maturation of male gametes.The aim of this study is to identify the effects of progesterone (PRG) on the capacitation and the acrosome reaction in bovine spermatozoa. The fresh sperm samples were incubated with and without capacitation inductors (heparin, dibutyryl cyclic adenosine monophosphate (dbcAMP)), hormones (prolactin (PRL), PRG), inhibitors of microfilaments (cytochalasin D) and microtubules (nocodazole) during capacitation and acrosome reactions. The functional status of spermatozoa was examined using the chlortetracycline assay. Supplementation of heparin stimulated capacitation in the presence and absence of PRG. Cytochalasin D blocked the stimulating effect of heparin on capacitation. The addition of PRL during capacitation (without PRG) did not affect the functional status of spermatozoa, while in PRG-treated cells PRL stimulated the acrosome reaction. PRL (with and without PRG) increased the acrosome reaction in capacitated cells. These PRL-dependent effects were inhibited by nocodazole. During the acrosome reaction, in presence of dbcAMP, PRG decreased the proportion of acrosome-reacted cells compared to PRG-untreated cells. This effect in PRG-treated cells was canceled in the presence of nocodazole. In conclusion, PRG under the action of PRL and dbcAMP determines the changes in the functional status of native sperm cells, which indicates PRG modulating effect on the indicators of post-ejaculatory maturation of spermatozoa.

Effects of meiotic inhibitors and gonadotrophins on porcine oocytes in vitro maturation, fertilization and development.

This study evaluated the effect of three reversible meiotic inhibitors (MINs) and their interaction with gonadotrophins (Gns) on the meiotic maturation and developmental competence of porcine oocytes. In experiment 1, the oocytes were matured for 22hr in the presence or absence of dbcAMP (1mM), cycloheximide (7μM) or cilostamide (20μM) with or without Gns, and for an additional 22hr in the absence of MINs and Gns. At 22hr of maturation, regardless of the presence of Gns, a higher proportion (p<.001) of oocytes cultured in the presence of MINs were effectively arrested at the germinal vesicle stage compared with the oocytes cultured without MINs. At 44hr of maturation, the proportion of oocytes that reached MII was higher (p<.05) in groups with Gns compared with groups without Gns. In experiment 2, oocytes that were matured as in experiment 1 were inseminated and cultured for 7days to evaluate fertilization parameters and blastocyst formation. Only oocytes from the dbcAMP+Gns group had higher (p<.05) efficiency of fertilization compared with the other treatment groups. The presence of dbcAMP during maturation also increased (p<.05) blastocyst formation and efficiency of blastocyst formation in both the presence and absence of Gns. These results indicate that the interaction of Gns with the tested MINs improved meiotic progression. In addition, regardless of supplementation with Gns, the presence of dbcAMP during the first maturation period increased and even doubled the capacity of oocytes to develop to the blastocyst stage.

204 INADEQUACY OF VASCULAR ENDOTHELIAL GROWTH FACTOR IN CULTURE MEDIUM REDUCES THE VIABILITY OF CUMULUS CELLS AND PREVENTS IN VITRO MATURATION OF PORCINE OOCYTES

Recently, vascular endothelial growth factor (VEGF) has been regarded as an important factor associated with not only follicle development but also meiotic competence of oocytes. However, the mechanism of how VEGF works is poorly understood. In this study, we investigated in vitro maturation (IVM) of oocytes from different sizes in the absence or presence of a VEGF receptor inhibitor, Axitinib. Cumulus-oocyte complexes (COC) were obtained from small follicles (SF; l &lt; 3 mm in diameter) and medium follicles (MF; 3–6 mm in diameter). Each group of 30–40 COC with at least 3 layers of clear and compact cumulus cells (CC) was cultured in 500 μL of modified porcine oocyte medium (POM-β-mercaptoethanol) supplemented with 10 IU mL–1 eCG, 10 IU mL–1 hCG and 1mM dibutyryl-cyclic-adenosine monophosphate (dbc-AMP) for the first 20 h and then without those supplements for another 24 h at 39°C, 5% CO2 in air. During the first 20 h of IVM, culture medium was also supplemented with or without 1.25 nM Axitinib. At 20 h and 44 h after the start of IVM, the oocytes were denuded and stained with 4′6-diamidino-2-phenylindole (DAPI) to observe the nuclear stages. At 20 h after the start of IVM, some COC were also stained with PI and SYBR Green I to evaluate the ratio of live/dead cumulus cells. Statistical analyses of data from 5 replications were analysed by ANOVA and Tukey’s multiple comparison test. As compared with controls at 20 h after the start of IVM, the number of dead cumulus cells increased significantly in the groups treated with Axitinib, regardless of COC derived from MF and SF (16.8 v. 43.1% in MF and 25.3 v. 57.7% in SF, P &lt; 0.01, respectively). At that time, a majority of oocytes from MF and SF remained at the germinal vesicle (GV) stage in controls (89.8 and 84.6%, respectively), but the percentage significantly reduced in the presence of Axitinib (57.9 and 48.9% of oocytes from MF and SF, respectively) and proceeded around the metaphase-I stage (37.5 and 44.8% of oocytes from MF and SF, respectively). At 44 h after the start of IVM, lower maturation rates were observed in oocytes treated with Axitinib than controls (35.0 v. 81.2% in MF; 20.1 v. 49.0% in SF; P &lt; 0.01). In conclusions, VEGF plays an important role in maitaining the viability of cumulus cells. The presence of VEGFR inhibitor caused the oocytes to develop uncontrollably, even in the presence of dbc-AMP. Moreover, the deficiency of VEGF prevented oocytes fully competent to resume meiosis and arrest to metaphase II.

CAMP-dependent proteolysis of GATA-6 is linked to JNK-signaling pathway

A JNK inhibitor SP600125 inhibited cAMP-dependent proteolysis of GATA-6 by proteasomes around its IC50. We further examined the effects of SP600125 on the degradation of GATA-6 in detail, since an activator of JNK (anisomycin) is available. Interestingly, anisomycin immediately stimulated the export of nuclear GATA-6 into the cytoplasm, and then the cytoplasmic content of GATA-6 decreased slowly through degradation by proteasomes. Such an effect of anisomycin was inhibited by SP600125, indicating that the observed phenomenon might be linked to the JNK signaling pathway. The inhibitory effect of SP600125 could not be ascribed to the inhibition of PKA, since phosphorylation of CREB occurred in the presence of dbcAMP and SP600125. The nuclear export of GATA-6 was inhibited by leptomycin B, suggesting that CRM1-mediated export could be activated by anisomycin. Furthermore, it seems likely that the JNK activated by anisomycin may stimulate not only the nuclear export of GATA-6 through CRM1 but also the degradation of GATA-6 by cytoplasmic proteasomes. In contrast, A-kinase might activate only the latter process through JNK.

Enhancement of carbachol-induced amylase secretion in parotid glands from rats with experimental periodontitis

ObjectiveIn a previous study we observed that parotid glands from rats with experimental periodontitis showed an increase in basal amylase release as a result of an increase in cAMP accumulation induced by PGE2 production. The aim of this work was to study whether this change in amylase release influences the secretory effect of carbachol. DesignExperimental periodontitis was induced through placing a black thread around the cervix of the two lower first molars. Experiments were done 22 days after ligature induced periodontitis. Amylase release was evaluated in vitro and determined using a colorimetric method which uses starch as substrate. ResultsThe effect of carbachol was increased in parotid glands from periodontitis rats. The effect of 10−6M carbachol was inhibited by 4-DAMP (10−6M), U-73122 (5×10−6M) and trifluoperazine (5×10−6M) in both groups. No changes were observed in the binding sites and affinity in parotid membranes from rats with experimental periodontitis. The inhibition of the adenylyl cyclase and the cyclooxygenase induced a right shift of the carbachol concentration–response curve in periodontitis group whilst the opposite effect was observed in control group in the presence of db-cAMP and PGE2. ConclusionsParotid glands from rats with experimental periodontitis release more amylase in response to carbachol suggesting an interaction between Ca2+ and cAMP in the fusion/exocytosis step of secretory vesicles.

Neural Stem/Progenitor Cells Differentiate In Vitro to Neurons by the Combined Action of Dibutyryl cAMP and Interferon-γ

Transplantation of neural stem/progenitor cells (NSPCs) is a promising strategy for repair of the diseased/injured central nervous system (CNS); however, controlling their differentiation remains a significant hurdle. This study is aimed at controlling differentiation and specifically at screening exogenous factors to direct NSPC differentiation into neurons in vitro. In this study, adult rat SVZ-derived NSPCs were treated with several factors and screened individually and in combination for changes in cellular morphology, neuronal marker expression, quantitative real-time qRT-PCR, and electrophysiological properties. These in vitro screens showed that of all the different treatments, dibutyryl cyclic AMP (dbcAMP) and interferon-gamma (IFN-gamma) enhanced neuronal differentiation most significantly compared to the 1% fetal bovine serum (FBS) controls. Importantly, the combined treatment of NSPCs with dbcAMP and IFN-gamma promoted greater neuronal differentiation as reflected by an increase in beta-III tubulin expression and morphological differentiation. Interestingly, the neurons that were generated from the NSPCs in vitro in the presence of dbcAMP and IFN-gamma, alone or in combination, responded to exogenous glutamate (Glu), but not gamma-aminobutyric acid (GABA), indicating that these neurons express glutamate receptors. These NSPC-derived neurons may be promising for neural regenerative strategies in the CNS.

Effect of follicle size and dibutyryl cAMP on the cAMP content and gap junctional communication of porcine prepubertal cumulus - oocyte complexes during IVM

The lower ability of oocytes from prepubertal pigs to yield viable embryos than those from adult pigs appears, in part, a consequence of their reduced ability to accumulate cAMP during IVM. The present study examined the cAMP content of oocytes and cumulus-oocyte complexes (COCs), cumulus expansion and gap junctional communication (GJC) in COCs from 3- and 5-8-mm follicles during IVM. The effect of 1 mm dibutyryl cAMP (db-cAMP) treatment for the first 22 h of IVM was also examined for both follicle size classes. The cAMP concentration of oocytes from 5-8-mm follicles was threefold greater than that in oocytes from 3-mm follicles following 11 h of IVM (11.9 +/- 5.9 v. 3.6 +/- 1.8 fmol, respectively; P < 0.05). In the presence of db-cAMP, the cAMP content of oocytes from 3- and 5-8-mm follicles was no longer significantly different at 11 h IVM. The cAMP concentration of intact COCs from 5-8-mm follicles was significantly higher than that in COCs from 3-mm follicles at 11 h (1110.6 +/- 318.0 v. 116.9 +/- 55.7 fmol, respectively; P < 0.05). Despite maturation with db-cAMP, the cAMP content in COCs from 3- and 5-8-mm follicles at 11 h of IVM remained significantly different (15.1 +/- 4.9 v. 196.2 +/- 33.3 fmol, respectively; P < 0.05). The COCs from 3-mm follicles displayed lower cumulus expansion than did COCs from 5-8-mm follicles at both 11 h (cumulus expansion index (CEI) 1.0 +/- 0.1 v. 1.8 +/- 0.1, respectively; P < 0.01) and 22 h (CEI 1.9 +/- 0.3 v. 2.9 +/- 0.2, respectively; P < 0.05) of IVM. The level of cumulus cell-oocyte GJC decreased during IVM, with the number of GJC significantly greater in COCs from 3-mm compared with 5-8-mm follicles at both 6 h (613 +/- 55 v. 304 +/- 44 fluorescence intensity (FI), respectively; P < 0.05) and 11 h (644 +/- 99 v. 337 +/- 38 FI, respectively; P < 0.05) of IVM. By 22 h of IVM, the GJC of COCs from 3-mm follicles had decreased (227 +/- 18 FI) and was no longer significantly different to that of COCs from 5-8-mm follicles (139 +/- 15 FI; P > 0.05). Dibutyryl cAMP had no effect on the cAMP content, cumulus expansion or GJC of the whole COC. In conclusion, the results of the present study demonstrate that COCs from 3-mm follicles accumulate less intraoocyte and inter-COC cAMP, display lower cumulus expansion and maintain their cumulus cell-oocyte GJC for longer during IVM than do COCs from 5-8-mm follicles.

Cyclic AMP‐dependent proteolysis of GATA‐6 expressed on the intracellular membrane

Cyclic AMP-dependent proteolysis of GATA-6 was characterized by fusing GATA-6 with the carboxyl-terminal membrane domain of SREBP-2. When the fusion protein was stably expressed in CHO-K1 cells, it was recovered in the ER membrane. This protein was processed in a similar manner to SREBP-2 upon cholesterol starvation, and the GATA-6 moiety moved into the nucleus. The GATA-6 moiety on the membrane became undetectable in the presence of dbcAMP or cholera toxin. However, H-89, K-252a, MG115 and lactacystin inhibited this decrease, suggesting that the cytoplasmic GATA-6 moiety of the fusion protein was degraded by proteasomes though A-kinase upon elevation of the cellular cAMP concentration.

The involvement of epoxygenase metabolites of arachidonic acid in cAMP-stimulated steroidogenesis and steroidogenic acute regulatory protein gene expression

The essential role of arachidonic acid (AA) in steroidogenesis has been previously demonstrated. The present study continues the investigation into how AA regulates steroidogenesis by examining the effects of epoxygenase-derived AA metabolites on cAMP-stimulated steroidogenic acute regulatory (StAR) gene expression and steroid hormone production in MA-10 mouse Leydig cells. The HPLC analysis of cell extracts from MA-10 cells treated with the cAMP analog dibutyryl cAMP (dbcAMP) demonstrated an increase in three epoxygenase-generated AA metabolites: 5,6-epoxyeicosatrienoic acid (EET), 8,9-EET, and 11,12-EET. Incubating MA-10 cells with each of the EETs induced a dose-dependent increase in StAR protein expression and steroid hormone production in the presence of dbcAMP. These metabolites also significantly enhanced StAR gene transcription as determined by luciferase assays of StAR promoter activity and reverse transcriptase-PCR analysis of StAR mRNA levels. While the EETs enhanced steroidogenesis, inhibiting the activity of protein kinase A (PKA) abolished the stimulatory effects of these AA metabolites on StAR expression and steroid hormone production. This study suggests that cAMP stimulation of MA-10 cells increases epoxygenase-generated AA metabolites and the co-action of these metabolites with PKA significantly increases StAR gene expression and steroid hormone production.

Characterization of cAMP-dependent proteolysis of GATA-6

Cyclic AMP-dependent proteolysis of GATA-6(Δ50) was characterized using inhibitors for intracellular signaling pathways. Among these kinase inhibitors, only H-89 and K252a inhibited the proteolysis induced by dbcAMP, a membrane permeable cAMP analogue, others such as PD98059, SB203580, calphostine C, PP1, and KN-93 did not do so. These results suggest that A-kinase, but not C-kinase, MEK, P38 MAP-kinases or Src kinase, could participate in the observed phenomenon. We further demonstrated that an inhibitor for ubiquitin isopeptidase (Δ12-PGJ2) inhibited the degradation of GATA-6(Δ50) in the presence of dbcAMP, suggesting that the cAMP-dependent proteolysis could be mediated through the ubiquitin–proteasome pathway, although proteasome activity did not change significantly during dbcAMP treatment. The full-length GATA-6 was also responsive to the induced degradation. Furthermore, mutation of a potential phosphorylation site (Ser-290 → Ala) for A- and C-kinases, and deletion of the PEST sequence of GATA-6 did not abolish the degradation. All these results suggest that cellular factor(s) may play a crucial role in mediating the activation of the cAMP-dependent process.

Isolation of CHO-K1 clones defective in cAMP-dependent proteolysis, as determined by the stability of exogenously expressed GATA-6

Degradation of the GATA-6(Δ50) protein expressed in a CHO-K1 clone (tc1-17a) is stimulated in the presence of dbcAMP through proteasome without new protein synthesis [FEBS Lett. 408 (1997) 301], whereas the intrinsic GC-box-binding protein was stable. To examine the cellular mechanism responsible for this specific degradation of GATA-6(Δ50), we initially introduced the blasticidin-S deaminase gene carrying a promoter with GATA motifs that are recognized by GATA-6. The resulting cell line (tc2G2) grew in the presence of blasticidin S. However, the presence of both blasticidin S and dbcAMP was lethal due to degradation of GATA-6. Cells resistant to such lethality were isolated by chemical mutagenesis. The GATA-6(Δ50) in these resistant cells was stable in the presence of dbcAMP in contrast to that in the parent tc2G2 cells, as determined by gel-mobility shift analysis and Western blotting. These clones could be beneficial for identification and characterization of the components participating in the signaling pathway for both protein degradation and cAMP-dependent biological processes.

Protective effects of Δ 9-tetrahydrocannabinol against N-methyl- d-aspartate-induced AF5 cell death

The neuroprotective effects of Δ 9-tetrahydrocannabinol (THC) were examined using an in vitro model in which the AF5 CNS cell line was exposed to toxic levels of N-methyl- d-aspartate (NMDA), an agonist of the NMDA glutamate receptor. NMDA toxicity was reduced by THC, but not by the more specific cannabinoid receptor agonist, WIN55,212-2. Addition of dibutyryl cAMP (dbcAMP) to the culture medium did not alter the neuroprotective effect of THC and did not unmask a neuroprotective effect of WIN55,212-2. The cannabinoid antagonist SR141716A did not inhibit the neuroprotection induced by THC or alter the response to WIN55,212-2, even in the presence of dbcAMP, indicating that the neuroprotective effect of THC was cannabinoid receptor-independent. On the other hand, both THC and WIN55,212-2 produced cellular toxicology at higher dosages, an effect which was blocked in part by SR141716A. Capsaicin, an antioxidant and vanilloid receptor agonist, also produced a protective effect against NMDA toxicology. The protective effect of capsaicin was blocked by co-application of ruthenium red, but was not blocked by the specific vanilloid receptor antagonist capsazepine, and the transient receptor potential vanilloid type 1 (TRPV1) and ANKTM1 transcripts were not detected in AF5 cells. Thus, the neuroprotective effects of THC and capsaicin did not appear to be mediated by TRP ion channel family receptors. The antioxidant α-tocopherol prevented neurotoxicity in a dose-dependent manner. Therefore, THC may function as an antioxidant to increase cell survival in NMDA-induced neurotoxicity in the AF5 cell model, while higher dosages produce toxicity mediated by CB1 receptor stimulation.

283 EFFECTS OF CUMULUS CELL REMOVAL ON IN VITRO OOCYTE MATURATION, FERTILIZATION, AND EMBRYONIC DEVELOPMENT IN PIGS

It is generally accepted that cumulus cells (CCs) support the nuclear maturation of immature oocytes in mammals. However, the precise mechanism of interaction between cumulus cells and oocytes has not been clarified. Furthermore, the role of cumulus cells in embryonic development has not been reported. In the present study, the effect of denuding cumulus cells from porcine oocytes on oocyte maturation, ertilization, and their subsequent development to the blastocyst stage was examined in vitro. In vitro maturation, fertilization, and culture were carried out as previously reported (Kikuchi et al. 2002 Biol. Reprod. 66, 1033-1041). Porcine cumulus-oocyte complexes (COCs) were collected; some of them were completely denuded of cumulus cells immediately after the collection (DO-0 group). The remaining intact COCs and the DO-0 oocytes were cultured for 24 h in the presence of dbcAMP and hormones. After the initial culture, some of the intact COCs were denuded either completely (DO-24 group) or partially (H-DO-24 group). Additionally, some of DO-24 oocytes were co-cultured with the cumulus cells removed at 0 h and pre-cultured for 24 h (DO-24 + CCs group). The denuded oocytes in each experimental group and intact COCs (control) were further cultured for total 46 h. The remaining oocytes with a first polar body were either examined for the levels of intracellular glutathione (GSH) or fertilized in vitro with frozen-thawed boar spermatozoa. The inseminated oocytes were cultured and examined for their fertilization status after 10 h and for their developmental competence after 6 days. Data were analyzed by ANOVA, followed by the Duncan's multiple range tests. The maturation rates of all denuded groups were significantly lower (P &lt; 0.05; 34.3 to 45.0%) than that of the control group (64.5%). Intracellular GSH concentrations of all denuded groups were also significantly lower (P &lt; 0.05; 4.03 to 7.00 pmol/oocyte) than that of the control group (9.60 pmol/oocyte); however, the GSH level of H-DO-24 oocytes was significantly higher (P &lt; 0.05) than the GSH levels in the other denuded groups. Male pronuclear formation rates of completely denuded oocytes (DO-0, DO-24, and DO-24 + CCs groups) were significantly lower (P &lt; 0.05; 41.4 to 59.3%) than those of the control (89.4%) and the H-DO-24 (80.0%) groups. The blastocyst rate of the control group was significantly higher (P &lt; 0.05; 19.9%) than that of H-DO-24 group (11.6%), and these rates were significantly higher (P &lt; 0.05) than those of the completely denuded groups (3.0 to 4.5%). The results suggest that the presence of cumulus cells during maturation culture improves nuclear maturation of oocytes and plays an important role in embryonic development to the blastocyst stage in vitro.

Mouse oocyte meiotic resumption and polar body extrusion in vitro are differentially influenced by FSH, epidermal growth factor and meiosis-activating sterol.

In this study, we compared the relative ability of FSH (100 mIU/ml), epidermal growth factor (EGF) (10 ng/ml), and follicular-fluid meiosis-activating sterol (FF-MAS, 10 micromol/l) to induce meiotic resumption and polar body I (PBI) extrusion in mouse oocytes. Cumulus-enclosed oocytes (CEO) were co-incubated with meiosis-arresting agents, including 4 mmol/l hypoxanthine (Hx), 0.3 mmol/l dibutyryl cAMP (dbcAMP), and 8.5 micromol/l cilostamide, a selective inhibitor of the oocyte-specific phosphodiesterase 3 (PDE 3). In Hx-treated oocytes, FSH, EGF and FF-MAS induced meiosis resumption at very high rates, but only FSH and EGF also promoted PBI extrusion with high frequency. In experiments conducted in the presence of dbcAMP, FF-MAS was unable to promote an increase in germinal vesicle breakdown (GVBD) rate, whereas FSH and EGF generated a response similar to the Hx groups. Neither FSH, EGF nor FF-MAS caused any change in the meiotic status of CEO when meiotic arrest at the germinal vesicle (GV) stage was maintained by cilostamide. In the presence of Hx, naked oocytes (NkO) co-cultured with their cumulus cells were able to respond to the GVBD-inducing effect of FSH and EGF by resuming meiosis at high rate. Collectively, these results indicate that: (i) a signal triggered in cumulus cells by either FSH or EGF, but not necessarily coincident with FF-MAS, may contribute to meiotic maturation, supporting GVBD and extrusion of PBI; (ii) the transmission of this signal can occur in a paracrine fashion, at least with reference to the breakdown of the GV. It also appears that concomitant regulation of intra-oocyte cAMP degradation is a prerequisite for meiosis resumption.

Dibutyryl c-AMP as an inducer of sporidia formation: biochemical and antigenic changes during morphological differentiation of Karnal bunt (Tilletia indica) pathogen in axenic culture.

Effect of dibutyryl adenosine 3',5'-cyclic monophosphate (dbc-AMP), an analogue of c-AMP, was investigated on growth and morphological differentiation of Tilletia indica. Exponential growth was observed up to 21 days in both presence and absence of dbc-AMP; however, increasing concentration of dbc-AMP was deleterious to mycelial growth in liquid culture. A slow increase of mycelial biomass up to 21 days and decline at 30 days in the presence of 2.5 mM dbc-AMP was observed, therefore, this concentration was chosen in subsequent investigations. The inhibitory influence of dbc-AMP was further substantiated by decrease in soluble protein. The fungus on exposure to dbc-AMP experienced morphological differentiation from vegetative mycelial phase to sporogenous mycelial phase, and was induced to produce filiform sporidia. Use of quantitative ELISA further suggested that sporidia formation took more than 21 days in the presence of dbc-AMP. Variations of proteins during different stages of T. indica grown in the presence and absence of dbc-AMP suggested the expression of stage-specific proteins or differential expression of proteins induced by dbc-AMP. The changes in expression of cell surface antigens as evidenced from decrease and increase binding of anti-mycelial and anti-sporidial antibodies in dbc-AMP treated culture by ELISA was further interpreted on the basis of morphological differentiation from mycelial to sporidial phase

Signal transduction in the ductuli efferentes testis of the rat: inhibition of fluid reabsorption by cyclic adenosine 3', 5'-monophosphate.

It is important to identify the signal transduction pathway involved in the regulation of fluid reabsorption by the ductuli efferentes of the testis because they reabsorb most of the fluid leaving the testis and are essential for male fertility. Microperfusion studies of the ducts in vivo showed that 0.1 or 1.0 mM dibutyryl (db)-cGMP in the perfusate had no effect on fluid reabsorption, but 0.1 mM db-cAMP significantly reduced fluid reabsorption, 0.25 mM abolished reabsorption, and 0.5-1.0 mM caused secretion. The inhibitory effect of db-cAMP was reversible. Although the presence of db-cAMP in the perfusate did not affect the concentration of Na+ in the collectate, the concentrations of K(+) and Cl(-) increased, indicating that their transport is at least partly regulated by cAMP. Including the phosphodiesterase inhibitor pentoxifylline in the perfusate decreased fluid reabsorption by the ducts in a dose-dependent manner, and it also increased the concentration of cAMP (5.5-fold) in collectate. Pentoxifylline also increased the production of cAMP (4-fold) by ducts incubated in vitro. It is concluded that cAMP, but probably not cGMP, is an intracellular messenger regulating fluid reabsorption in the efferent ducts.

Ca2+]i oscillations induced by high [K+]o in acetylcholine-stimulated rat submandibular acinar cells: regulation by depolarization, cAMP and pertussis toxin.

Maintaining the extracellular K(+) concentration ([K(+)](o)) between 15 and 60 mM induced oscillations in the intracellular Ca(2+) concentration ([Ca(2+)](i)) in rat submandibular acinar cells during stimulation with acetylcholine (ACh, 1 micro M). These [Ca(2+)](i) oscillations were also induced by 1 micro M thapsigargin and were inhibited by 50 micro M La(3+), 1 micro M Gd(3+), or the removal of extracellular Ca(2+), indicating that the [Ca(2+)](i) oscillations were generated by store-operated Ca(2+) entry (SOC). The frequency of the ACh-evoked [Ca(2+)](i) oscillations increased from 0.8 to 2.3 mHz as [K(+)](o) was increased from 15 to 50 mM. TEA (an inhibitor of K(+) channels) also induced [Ca(2+)](i) oscillations at [K(+)](o) of 4.5 or 7.5 mM in ACh-stimulated cells. These data suggest that depolarization causes [Ca(2+)](i) to oscillate in ACh-stimulated submandibular acinar cells. Pertussis toxin (PTX, an inhibitor of G proteins) caused [Ca(2+)](i) to be sustained at a high level in ACh-stimulated cells at 25 mM or 60 mM [K(+)](o). This suggests that the [Ca(2+)](i) oscillations are generated by a periodic inactivation of the SOC channels via PTX-sensitive G proteins, which are stimulated by depolarization. Moreover, in the presence of DBcAMP or forskolin which accumulated cAMP the frequency of the [Ca(2+)](i) oscillations remained constant (approximately 1.2 mHz) when [K(+)](o) was maintained in the range 25-60 mM. Based on these observations in ACh-stimulated submandibular acinar cells, we conclude that depolarization stimulates the PTX-sensitive G proteins, which inactivate the SOC channels periodically ([Ca(2+)](i) oscillation), while hyperpolarization or PTX inhibits the G proteins, maintaining the activation of the SOC channels. Accumulation of cAMP is likely to modulate the PTX-sensitive G proteins.

Different mechanisms for the induction of copper-zinc superoxide dismutase and manganese superoxide dismutase by progesterone in human endometrial stromal cells.

The present study was undertaken to investigate the cAMP-dependent regulation of copper-zinc superoxide dismutase (Cu,Zn-SOD) and manganese SOD (Mn-SOD) by ovarian steroids in human endometrial stromal cells (ESC). To examine the effect of cAMP on SOD expression, ESC were incubated with dibutyryl-cAMP (db-cAMP, 0.5 mmol/l), forskolin (25 micromol/l), or estradiol (E(2), 10(-8) mol/l) + medroxyprogesterone acetate (MPA, 10(-6) mol/l), for 18 days. E(2) + MPA significantly increased Cu,Zn-SOD activity and mRNA concentrations, whereas db-cAMP and forskolin had no effect. On the other hand, Mn-SOD activity and mRNA concentration were significantly increased by all of these treatments. Insulin-like growth factor-binding protein-1, a marker of decidualization, was clearly induced by db-cAMP, forskolin or E(2) + MPA, accompanied by morphological changes characteristic of decidualization. To study whether the increase in Mn-SOD by db-cAMP or E(2) + MPA was mediated by cAMP-dependent protein kinase A (PKA), ESC were incubated with protein kinase inhibitor (PKI) (10 microg/ml), an inhibitor of PKA, in the presence of db-cAMP or E(2) + MPA. The increase in Mn-SOD activity following db-cAMP or E(2) + MPA was completely inhibited by PKI. In the process of decidualization, E(2) + MPA increases Mn-SOD expression via a cAMP-dependent pathway. Cu,Zn-SOD is also up-regulated by E(2) + MPA, but via a different pathway from that involving cAMP.

Multipathways for transdifferentiation of human prostate cancer cells into neuroendocrine-like phenotype

The neuroendocrine (NE) cell is a minor cell population in normal human prostate glands. The number of NE cells is increased in advanced hormone-refractory prostate carcinomas (PCA). The mechanism of increased NE cell population in these advanced tumors is poorly understood. We examined molecular mechanisms which may be involved in the regulation of the transdifferentiation process of human PCA cells leading to a NE phenotype. We compared PCA cell lines LNCaP and PC-3 in the following medium conditions: steroid-reduced (SR), interleukin-6 (IL-6)-supplemented, or dibutyrate cAMP (db-cAMP)-supplemented. We found that androgen-responsive C-33 LNCaP cells responded to all treatments, having a neuronal-like morphology. In contrast, C-81 LNCaP cells, having a decreased androgen responsiveness, had a less pronounced effect although followed a similar trend. Androgen-unresponsive PC-3 cells showed little change in their morphology. Grown in the SR condition, the level of neuron-specific enolase (NSE), a marker of neuronal cells, was upregulated in C-33 LNCaP cells, while to a lesser degree in the presence of IL-6. In the presence of db-cAMP, the NSE level in C-33 cells was decreased, lower than that in control cells. An opposite effect was observed for C-81 LNCaP cells. Nevertheless, the NSE level was only elevated in db-cAMP-treated PC-3 cells, but no change was found in PC-3 cells grown in the SR- or IL-6-supplemented medium. Thus, a similar gross phenotypic change may correlate with differential molecular expressions. We also analyzed the expression of protein tyrosine phosphatase α (RPTPα) since it plays a critical role in normal neuronal differentiation and signaling. Our results showed that the expression of RPTPα correlates with the NE phenotypic change of LNCaP cells in the SR condition. In summary, our data clearly show that the molecular process by which cultured human prostate cancer cells undergo a transdifferentiation process to a NE cell-like phenotype is accompanied by differential expressions of different markers, and a gross NE cell-like phenotype can occur by exposing PCA cells to different pharmacological agents.

The regulation of leptin gene expression in the C6 glioblastoma cell line

The hormone leptin is implicated in the regulation of appetite and body weight in rodents, primates and humans. We reported that the leptin gene ( ob) is expressed in the brain, but the factors which control ob expression in the central nervous system are not known. We previously showed that brain-derived rat C6 glioblastoma cells express ob mRNA and protein. In the present study we examined the regulation of ob expression in C6 cells. Leptin and leptin receptor immunoreactivity was detected in C6 cells, suggesting a possible autocrine role for leptin. The identity of the leptin immunoreactivity (OB-ir) in C6 cells was confirmed by immunoprecipitation and Western blotting using two leptin specific polyclonal antibodies. Using RT-PCR analysis a product of the expected size for the short, but not the long, leptin receptor isoform was detected in C6 cells. Cells were maintained in serum-free (SF) media for 0–24 h in the presence of various regulators of leptin expression. Leptin mRNA levels were significantly higher in cells treated with dbcAMP (1 mM), IGF 1 (100 ng/ml) or insulin (5 μg/ml) compared to SF controls. In contrast, corticosterone (10 −7M) reduced leptin mRNA. In the presence of dbcAMP, C6 cells undergo a dramatic alteration in morphology which is coincident with an apparent increase in the number of leptin-ir nuclei and an increase in leptin immunoreactivity. In contrast to C6 cells, glucocorticoids are reported to increase leptin levels in adipocytes/adipose tissue, while increases in intracellular cAMP levels are reported to reduce leptin levels. Overall, our in vitro data suggest that the regulation of leptin gene expression in C6 glioblastoma cells is different from that in adipocytes.