Messenger Analogs Research Articles

Abstract P146: Dopaminergic Stimulation Increases Gastrin Secretion via a PKA-PPAR-Alpha Pathway

Dietary sodium stimulates renal excretion of sodium long before the sodium is enterally absorbed. Moreover, intravenously administered sodium does not stimulate as large an excretory response as sodium taken by mouth. We previously showed that sodium stimulates an increase in gastrin mRNA and protein in stomach G-cells via PPAR-α and dopamine D1 receptor (D 1 R). Studies have shown that PKA pathway is important in the regulation of PPAR-α activity, and PKA is also involved in the D 1 R pathway. Thus, we hypothesized that D 1 R may regulate PPAR-α through PKA in human G-cells in response to sodium. Human G-cells were treated with 8-Br-cAMP, a cell-permeable second messenger analog, and Rp-cAMP (RPCA), a PKA inhibitor. Image analysis showed there was more PPAR-α in the nucleus with 8-Br-cAMP treatment, and less PPAR-α in the nucleus with RPCA treatment. Therefore, PKA is involved in regulating PPAR-α (VEH, 1194±74; 8-Br-cAMP, 1490±44; RPCA, 740±75; 8-Br-cAMP+ RPCA, 1037±54; VEH vs 8-Br-cAMP, P<0.01; 8-Br-cAMP vs 8-Br-cAMP+ RPCA, P<0.001; one-way ANOVA, n=13). Moreover, gastrin mRNA was increased by 8-Br-cAMP stimulation while the increase was blocked by RPCA (VEH, 1.01±0; 8-Br-cAMP, 1.32±0.07; RPCA, 0.82±0.08; 8-Br-cAMP+ RPCA, 0.9±0.05; VEH vs 8-Br-cAMP, P<0.05; 8-Br-cAMP vs 8-Br-cAMP+ RPCA, p<0.01; one-way ANOVA, n=3). Gastrin protein level was also increased by 8-Br-cAMP treatment with image analysis (VEH, 1.14±0.01; 8-Br-cAMP, 1.28±0.01; RPCA, 1.17±0.01; 8-Br-cAMP+ RPCA, 1.38±0.03; VEH vs 8-Br-cAMP, P<0.01; one-way ANOVA, n=3). These data indicate that PKA is involved in the D 1 R and PPAR-α pathways to regulate G-cell gastrin expression stimulated by sodium.

The Dopamine D1 Receptor and Angiotensin II Type-2 Receptor are Required for Inhibition of Sodium Transport Through a Protein Phosphatase 2A Pathway.

Activation of the renal D1R (dopamine D1-like receptor) or AT2R (angiotensin II type-2 receptor), individually or both, simultaneously, is necessary in the normal regulation of renal sodium (Na+) transport and blood pressure. However, little is known regarding the precise mechanism of this interaction. Pharmacological stimulation, membrane biotinylation, and cell surface immunofluorescence were used to study the effect of the D1R/AT2R interaction in human renal proximal tubule cells. D1R activation of GαS stimulates AC (adenylyl cyclase) and induces apical plasma membrane recruitment of AT2Rs. We now show for the first time the reciprocal reaction, AT2R stimulation with Ang III (angiotensin III) leads to the apical plasma membrane recruitment of the D1R. The cell-permeable second messenger analogs of cAMP (8-Br-cAMP) or cGMP (8-Br-cGMP) induce translocation of both D1R and AT2R to the plasma membrane. Inhibition of PKA (protein kinase A) with Rp-cAMPS and PKG (protein kinase G) with Rp-8-CPT-cGMPS blocks D1R and AT2R recruitment, respectively, indicating that both PKA and PKG are necessary for D1R and AT2R trafficking. Both 8-Br-cAMP and 8-Br-cGMP activate PP2A (protein phosphatase 2A), which is necessary for both plasma membrane recruitment of D1R and AT2R and the inhibition of sodium hydrogen exchanger 3-dependent Na+ transport. These studies provide insights into the D1R/AT2R transregulation mechanisms that play a crucial role in maintaining Na+ and ultimately blood pressure homeostasis.

Structural and functional characteristics of cGMP-dependent methionine oxidation in Arabidopsis thaliana proteins

BackgroundIncreasing structural and biochemical evidence suggests that post-translational methionine oxidation of proteins is not just a result of cellular damage but may provide the cell with information on the cellular oxidative status. In addition, oxidation of methionine residues in key regulatory proteins, such as calmodulin, does influence cellular homeostasis. Previous findings also indicate that oxidation of methionine residues in signaling molecules may have a role in stress responses since these specific structural modifications can in turn change biological activities of proteins.FindingsHere we use tandem mass spectrometry-based proteomics to show that treatment of Arabidopsis thaliana cells with a non-oxidative signaling molecule, the cell-permeant second messenger analogue, 8-bromo-3,5-cyclic guanosine monophosphate (8-Br-cGMP), results in a time-dependent increase in the content of oxidised methionine residues. Interestingly, the group of proteins affected by cGMP-dependent methionine oxidation is functionally enriched for stress response proteins. Furthermore, we also noted distinct signatures in the frequency of amino acids flanking oxidised and un-oxidised methionine residues on both the C- and N-terminus.ConclusionsGiven both a structural and functional bias in methionine oxidation events in response to a signaling molecule, we propose that these are indicative of a specific role of such post-translational modifications in the direct or indirect regulation of cellular responses. The mechanisms that determine the specificity of the modifications remain to be elucidated.

Signal transduction mechanism for potentiation by α1- and β2-adrenoceptor agonists of l-ascorbic acid-induced DNA synthesis and proliferation in primary cultures of adult rat hepatocytes

We investigated the effects of α- and β-adrenoceptor agonists on l-ascorbic acid-induced hepatocyte DNA synthesis and proliferation in primary cultures of adult rat hepatocytes. The results showed that phenylephrine (10−6M) and metaproterenol (10−6M) alone did not induce hepatocyte DNA synthesis and proliferation. However, when combined with l-ascorbic acid (10−6M), these adrenoceptor agonists potentiated the hepatocyte DNA synthesis and proliferation induced by l-ascorbic acid. Then intracellular signal transduction mechanisms for the effects of phenylephrine and metaproterenol on l-ascorbic acid-induced hepatocyte mitogenesis were examined. Western blot analysis showed that phenylephrine and metaproterenol did not potentiate l-ascorbic acid-induced insulin-like growth factor I receptor tyrosine kinase phosphorylation. In contrast, they both significantly potentiated l-ascorbic acid-induced extracellular-signal regulated kinase-2 (ERK2) phosphorylation within 5min. Moreover, cell-permeable second messenger analogs phorbol ester (10−7M) and 8-bromo cAMP (10−7M) mimicked the effects of phenylephrine and metaproterenol on l-ascorbic acid-induced ERK2 phosphorylation. The effects of these adrenoceptor agents were specifically antagonized by GF109203X and H-89, respectively. These results indicate that activation of ERK2 via protein kinas C and protein kinase A represents a mechanism for potentiation of l-ascorbic acid-induced hepatocyte DNA synthesis and proliferation in primary cultures of adult rat hepatocytes.

Ablation of the Sam68 gene impairs female fertility and gonadotropin-dependent follicle development

Sam68 is a multifunctional RNA-binding protein highly expressed in the gonads, whose ablation causes male infertility. Herein, we have investigated Sam68 expression in the adult ovary and its function in female fertility. Immunohistochemistry showed that Sam68 was localized in the nucleus of oocytes and follicular cells at all stages of folliculogenesis. Sam68(-/-) females were severely subfertile, and they showed a delay in the age of first pregnancy, increased breeding time for successful pregnancy and yielded smaller litters. Morphological analyses indicated a significant reduction in the number of secondary and pre-antral follicles in the ovary. These defects were associated with alteration of oestrous cycles and a reduced number of ovulated oocytes, which were only partially restored by the administration of exogenous gonadotropins. Crosslinking/immunoprecipitation experiments showed that Sam68 directly binds the mRNAs for the follicle-stimulating hormone (FSH) and the luteinizing hormone receptors (Fshr and Lhcgr), which were downregulated in ovaries of adult knockout females. Stimulation of immature females with FSH-like pregnant mare serum gonadotropin (PMSG), or of follicular cells with the FSH second messenger analogue 8Br-cAMP, caused the upregulation of Sam68. The increase in Sam68 levels paralleled that of the Fshr and Lhcgr mRNAs in the pre-ovulatory follicle and was required to allow accumulation of these transcripts in follicular cells. These studies identify a new crucial function for Sam68 in the regulation of female fertility and indicate that this protein is required to insure proper expression of the gonadotropin receptor transcripts in pre-ovulatory follicles in adult ovary.

PTTH-stimulated ERK phosphorylation in prothoracic glands of the silkworm, Bombyx mori: Role of Ca2+/calmodulin and receptor tyrosine kinase

Our previous studies showed that the prothoracicotropic hormone (PTTH) stimulated extracellular signal-regulated kinase (ERK) phosphorylation in prothoracic glands of Bombyx mori both in vitro and in vivo. In the present study, the signaling pathway by which PTTH activates ERK phosphorylation was further investigated using PTTH, second messenger analogs, and various inhibitors. ERK phosphorylation induced by PTTH was partially reduced in Ca2+-free medium. The calmodulin antagonist, calmidazolium, partially inhibited both PTTH-stimulated ERK phosphorylation and ecdysteroidogenesis, indicating the involvement of calmodulin. When the prothoracic glands were treated with agents that directly elevate the intracellular Ca2+ concentration [either A23187, thapsigargin, or the protein kinase C (PKC) activator, phorbol 12-myristate acetate (PMA)], a great increase in ERK phosphorylation was observed. In addition, it was found that PTTH-stimulated ecdysteroidogenesis was greatly attenuated by treatment with PKC inhibitors (either calphostin C or chelerythrine C). However, PTTH-stimulated ERK phosphorylation was not attenuated by the above PKC inhibitors, indicating that PKC is not involved in PTTH-stimulated ERK phosphorylation. A potent and specific inhibitor of insulin receptor tyrosine kinase, HNMPA-(AM)3, greatly inhibited the ability of PTTH to activate ERK phosphorylation and stimulate ecdysteroidogenesis. However, genistein, another tyrosine kinase inhibitor, did not inhibit PTTH-stimulated ERK phosphorylation, although it did markedly attenuate the ability of A23187 to activate ERK phosphorylation. From these results, it is suggested that PTTH-stimulated ERK phosphorylation is only partially Ca2+- and calmodulin-dependent and that HNMPA-(AM)3-sensitive receptor tyrosine kinase is involved in activation of ERK phosphorylation by PTTH.

Syndecan-1 Expression in Epithelial Cells Is Induced by Transforming Growth Factor β through a PKA-dependent Pathway

Syndecans comprise a major family of cell surface heparan sulfate proteoglycans (HSPGs). Syndecans bind and modulate a wide variety of biological molecules through their heparan sulfate (HS) moiety. Although all syndecans contain the ligand binding HS chains, they likely perform specific functions in vivo because their temporal and spatial expression patterns are different. However, how syndecan expression is regulated has yet to be clearly defined. In this study, we examined how syndecan-1 expression is regulated in epithelial cells. Our results showed that among several bioactive agents tested, only forskolin and three isoforms of TGFbeta (TGFbeta1-TGFbeta3) significantly induced syndecan-1, but not syndecan-4, expression on various epithelial cells. Steady-state syndecan-1 mRNA was not increased by TGFbeta treatment and cycloheximide did not inhibit syndecan-1 induction by TGFbeta, indicating that TGFbeta induces syndecan-1 in a post-translational manner. However, TGFbeta induction of syndecan-1 was inhibited by transient expression of a dominant-negative construct of protein kinase A (PKA) and by specific inhibitors of PKA. Further (i) syndecan-1 cytoplasmic domains were Ser-phosphorylated when cells were treated with TGFbeta and this was inhibited by a PKA inhibitor, (ii) PKA was co-immunoprecipitated from cell lysates by anti-syndecan-1 antibodies, (iii) PKA phosphorylated recombinant syndecan-1 cytoplasmic domains in vitro, and (iv) expression of a syndecan-1 construct with its invariant Ser(286) replaced with a Gly was not induced by TGFbeta. Together, these findings define a regulatory mechanism where TGFbeta signals through PKA to phosphorylate the syndecan-1 cytoplasmic domain and increases syndecan-1 expression on epithelial cells.

Activation of the Lutropin/Choriogonadotropin Receptor in MA-10 Cells Leads to the Tyrosine Phosphorylation of the Focal Adhesion Kinase by a Pathway that Involves Src Family Kinases

We show that activation of the endogenous or recombinant lutropin/choriogonadotropin receptor (LHR) in mouse Leydig tumor cells (MA-10 cells) leads to the tyrosine phosphorylation of the focal adhesion kinase (FAK) and one of its substrates (paxillin). Using specific antibodies to the five tyrosine residues of FAK that become phosphorylated, we show that activation of the LHR increases the phosphorylation of Tyr576 and Tyr577, but it does not affect the phosphorylation of Tyr397, Tyr861, or Tyr925. Because FAK is a prominent substrate for the Src family of tyrosine kinases (SFKs) we tested for their involvement in the LHR-mediated phosphorylation of FAK-Tyr576. Src is not detectable in MA-10 cells, but two other prominent members of this family (Fyn and Yes) are present. The LHR-mediated phosphorylation of FAK-Tyr576 is readily inhibited by PP2 (a pharmacological inhibitor of SFKs) and by dominant-negative mutants of SKFs. Moreover, activation of the LHR in MA-10 cells results in the stimulation of the activity of Fyn and Yes, and overexpression of either of these two tyrosine kinases enhances the LHR-mediated phosphorylation of FAK-Tyr576. Studies involving activation of other G protein-coupled receptors, overexpression of the different Galpha-subunits, and the use of second messenger analogs suggest that the LHR-induced phosphorylation of FAK-Tyr576 in MA-10 cells is mediated by SFKs, and that this family of kinases is, in turn, independently or cooperatively activated by the LHR-induced stimulation of Gs and Gq/11-mediated pathways.

PGE 2 induces the gene expression of bone matrix metalloproteinase-1 in mouse osteoblasts by cAMP-PKA signaling pathway

Prostaglandin E 2 (PGE 2), an abundant eicosanoid in bone, has been implicated in a number of pathological states associated with bone loss, and is also known to stimulate matrix metalloproteinase (MMP)-1 synthesis and secretion in rat and human osteoblast cells, although the nature of the intracellular reaction remains unclear. Although MMP-1 plays a critical role in bone-remodeling, it would be of interest to examine whether PGE 2 regulates MMP-1 expression by mouse osteoblasts or not. Here we demonstrate that PGE 2 is a potent inducer of MMP-1 production in fetal osteoblasts and show that PGE 2 stimulates the activity of the MMP-1 promoter in osteoblasts, suggesting that PGE 2 controls MMP-1 gene expression at least at the transcriptional level. PGE 2 induced MMP-1 messenger RNA (mRNA) expression in the cells within 4 h, and this expression was maintained for 36 h. The increase in MMP-1 production with 0.1–2.0 μM PGE 2 was dose-dependent. We also found that PGE2 (1.5 μM) up-regulated MMP-1 protein levels in cultured mouse osteoblasts, as evidenced by ELISA. To examine whether PGE 2-mediated response and signal pathway are involved in the intracellular action, the PGE 2-mediated expression of the MMP-1 gene was investigated in mouse osteoblast cells. A Northern blot analysis showed that PGE 2 and PGE 1 were potent stimulators of MMP-1 transcription, and the presence of thromboxane B 2 had no effect. The increase in MMP-1 transcript after PGE 2 treatment was observed at 4 h, reaching a maximum at 6 h, and persisted for 24 h. This response was dose-dependent. Cycloheximide, an inhibitor of protein synthesis, completely blocked this effect by PGE 2, indicating that the expression of other genes is also required. The second messenger analog, 8-bromo-cAMP, mimicked the effects of PGE 2 by stimulating a dose-dependent increase in MMP-1 mRNA levels, with a maximal effect that was quantitatively similar to that observed with PGE 2. Thus, the present results strongly suggest that the PGE 2 stimulation of MMP-1 synthesis is due to the activation of MMP-1 gene transcription and a subsequent marked increase in MMP-1 transcription. This effect is dependent on de novo protein synthesis and is mimicked by protein kinase A activation. The findings suggest that PGE 2 is involved in the cAMP-PKA signaling pathway in regulating MMP-1 gene expression in osteoblasts.

ATRIAL NATRIURETIC PEPTIDE REDUCES PRESERVATION AND REPERFUSION INJURY IN RAT LIVER: ROLE OF CGMP AND HEME OXYGENASE-1

O255 Aims: Preconditioning with atrial natriuretic peptide (ANP) markedly reduces hepatic ischemia and reperfusion (I/R) injury via the inhibition of proinflammatory response and the induction of cytoprotective molecules. However, molecular mechanisms are as yet widely unknown. Heme oxygenase (HO)-1 is well recognized as an antioxidant and cytoprotective gene. The aim of this study is to investigate the involvement of HO-1 induced by ANP in rat liver I/R model. Methods: Lewis male rats were intravenously pretreated with saline (as control), ANP (4 mg/kg) or 8-Bromo cyclic guanosine monophosphate (8-Br-cGMP; 10 mg/kg) 6 hrs before liver harvesting. HO-1 mRNA expression, protein synthesis and enzyme activity were determined by reverse transcription polymerase chain reaction, Western immunoblotting, bilirubin formation, and immunohistochemical analyzes. In a different group, liver grafts were stored with chilled University of Wisconsin solution for 30 hrs and transplanted orthotopically. Immediately after revascularization a potent inhibitor of HO enzyme activity, zinc protoporphyrin IX (ZnPP; 1.5 mg/kg) was intravenously administered in animals which had received ANP or 8-Br-cGMP pretreated livers. Following reperfusion serum level of tumor necrosis factor (TNF)-αlpha and immunohistochemical expression of vascular cell adhesion molecule (VCAM)-1 were assessed. Propium iodide (PI)- and in situ nick-end labeling (TUNEL)-positive cells were determined by immunohistochemical methods. Animal survival was also monitored. Results: At 24 hrs after treatment, ANP or 8-Br-cGMP induced a significant increase in hepatic HO-1 gene and protein expression with high enzyme activity. HO-1 was dominantly expressed in Kupffer-like dendritic cells. Following reperfusion, the expression of TNF-αlpha mRNA and VCAM-1 protein as well as the numbers of necrotic and apoptotic cells were markedly reduced in ANP or 8-Br-cGMP pretreated livers. Higher animal survival rates were observed in both experimental groups (p<0.05, ANP; 7/10, 8-Br-cGMP; 6/10 vs. Saline; 0/10). These cytoprotective effects were abrogated in the presence of HO enzyme inhibitor, ZnPP, introduced immediately after reperfusion. Conclusions: These results suggest that prior induction of HO-1 by ANP and its second messenger analogue 8-Br-cGMP may lead to anti-inflammatory and anti-apoptotic effects on hepatic I/R injury depending on its enzyme activity. Thus, clinically used and less-toxic ANP would be potentially applicable to donor preconditioning or pharmacologic treatment for pathologic conditions that lead to oxidative stress including I/R injury.

Atrial natriuretic peptide preconditioning protects against hepatic preservation injury by attenuating necrotic and apoptotic cell death

Background/Aims: Preconditioning of livers with the atrial natriuretic peptide (ANP) markedly reduces hepatic ischemia-reperfusion injury. Aim of this study was to characterize the influence of ANP preconditioning on necrotic and apoptotic cell death and on proliferation. Methods: Rat livers were perfused with Krebs-Henseleit buffer with or without ANP or its second messenger analogue 8-Bromo cyclic guanosine monophosphate (8-Br cGMP) for 20 min, stored in cold University of Wisconsin solution (24 h), and reperfused for up to 120 min. Apoptosis and necrosis were determined using biochemical and morphological criteria, proliferation was assessed by Ki67 histochemistry. Results: Apoptosis peaked after 24 h of cold ischemia. Preconditioning with both ANP and 8-Br-cGMP significantly reduced caspase-3-like activity and the number of triphosphate nick-end labelling-positive cells. Reduction of apoptosis was significant for hepatocytes, but not for endothelial cells. After ischemia, degenerative cell changes were clearly reduced in ANP pretreated livers. After reperfusion, ANP preconditioning led to a significant reduction of necrotic hepatocytes and endothelial cells in periportal zones. Cell proliferation was not affected by preconditioning. Conclusions: ANP reduces necrotic and apoptotic cell death without affecting the proliferation status. The protection takes place mainly in the periportal area and seems to be most prominent against necrosis of hepatocytes and endothelial cells during reperfusion.

Prothoracicotropic hormone stimulated extracellular signal-regulated kinase (ERK) activity: the changing roles of Ca 2+- and cAMP-dependent mechanisms in the insect prothoracic glands during metamorphosis

The synthesis of ecdysteroids by the lepidopteran prothoracic gland is regulated by a brain neuropeptide hormone, prothoracicotropic hormone (PTTH). In Manduca sexta glands, PTTH stimulates several events including Ca 2+ influx, Ca 2+-dependent cAMP generation and the activation of several protein kinases. In the present study, the path by which PTTH stimulates extracellular signal-activated regulated kinase (ERK) phosphorylation was investigated using PTTH and second messenger analogs. The results indicate that Ca 2+-dependent processes, other than cAMP generation, play the major role in PTTH stimulation of ERK phosphorylation in larval prothoracic glands, that cAMP-dependent events increase in importance during later development and that PTTH-stimulated ERK phosphorylation is highest in larval glands. The decline in PTTH-stimulated ERK phosphorylation associated with metamorphosis results from decreased ERK levels and an increased basal rate of ERK phosphorylation. The data suggest that the role or importance of components of the PTTH signal transduction cascade are not fixed and can change during development.

Ultrastructural changes in the Malpighian tubules of the house cricket, Acheta domesticus, at the onset of diuresis: A time study

The Malpighian tubules (Mt) of insects are responsible for maintaining osmotic homeostasis and eliminating waste from the hemolymph. When stimulated by diuretic factors the tubule cells are able to transport extraordinary volumes of fluid over short periods of time. We have been studying the changes that occur within the cells that accompany and facilitate this phenomenon. We present the ultrastructural changes that occur in the mid-tubule of the house cricket, Acheta domesticus, following exposure to the second messenger analog, dibutyryl cAMP, over the period from 15–420 sec. Vacuolation of the cytoplasm begins as early as 30 sec poststimulation with a significant increase in vacuolation occurring after 120 sec. As expected, there is an increase in the surface area of the basolateral membrane to facilitate the rapid movement of fluid into the cells. Other ultrastructural changes noted to accompany the onset of diuresis include the movement of mitochondria into areas adjacent to transport membranes, the vesiculation of Golgi, mobilization of CaPO4 spherites, and a direct interaction of these spherites with active mitochondria. We discuss several possible roles for these changes in terms of rapid fluid transport. J. Morphol. 247:80–92, 2001. © 2001 Wiley-Liss, Inc.

Cell biology of human salivary secretion.

We treated surgical specimens of human parotid and submandibular glands in vitro to manipulate the receptor-signaling cascade pharmacologically and analyzed cellular responses by light microscopy on epoxy embedded sections. Treatment of specimens with the b-agonist, isoproterenol, and with the second messenger analog, dibutyryl cyclic AMP, stimulated serous acinar cells to engage in exocytosis and degranulation. The muscarinic agonist, carbachol, and the calcium ionophore, A23187, on the other hand, elicited formation of "vacuoles" in the cytoplasm of serous acinar cells. Taking previous in vivo human and animal studies into account, these changes are suggested as the morphological expression of enzyme release and fluid secretion, respectively. Specimens obtained from patients over 70 years old exhibited poor response even though their morphological appearance remained intact. Aged salivary glands are thus suggested to experience a decline in their secretory activity at the cellular level, probably by impairment of the signaling processes downstream to the receptor activation and second messenger production.

Involvement of osteoprotegerin/osteoclastogenesis inhibitory factor in the stimulation of osteoclast formation by parathyroid hormone in mouse bone cells.

Recently, osteoprotegerin (OPG)/osteoclastogenesis inhibitory factor (OCIF) has been shown to inhibit osteoclast differentiation. On the other hand, we have reported that parathyroid hormone (PTH) stimulated osteoclast formation, presumably through a PTH-responsive cAMP-dependent protein kinase (PKA) pathway, in mouse bone cells. The present study was performed to examine how OPG/OCIF expression is regulated by PTH and to further investigate the possible involvement of OPG/OCIF in the stimulation of osteoclast formation by PTH in mouse bone cells. OPG/OCIF mRNA expression was analyzed by Northern hybridization after 24h treatments of mouse whole bone cells and mouse stromal cell line, ST2 cells with PTH or various second messenger analogs. Human (h) PTH(1-34) (10(-10) and 10(-8)mol/l) but not 10(-8)mol/l hPTH(3-34) down-regulated OPG/OCIF mRNA expression in mouse bone cells. Dibutyryl cAMP, but not phorbol ester, an activator of protein kinase C, or A23187, a calcium ionophore, down-regulated it. The same was also observed in ST2 cells, suggesting that stromal cells are responsible for the inhibitory effect of PTH and cAMP analogs on OPG/OCIF mRNA expression in mouse bone cells. The present study indicates that PTH down-regulates OPG/OCIF mRNA expression through the PKA pathway in stromal cells, which would result in the stimulation of osteoclast formation.

Ultrastructural changes in the Malpighian tubules of the house cricket, Acheta domesticus, at the onset of diuresis: A time study.

The Malpighian tubules (Mt) of insects are responsible for maintaining osmotic homeostasis and eliminating waste from the hemolymph. When stimulated by diuretic factors the tubule cells are able to transport extraordinary volumes of fluid over short periods of time. We have been studying the changes that occur within the cells that accompany and facilitate this phenomenon. We present the ultrastructural changes that occur in the mid-tubule of the house cricket, Acheta domesticus, following exposure to the second messenger analog, dibutyryl cAMP, over the period from 15-420 sec. Vacuolation of the cytoplasm begins as early as 30 sec poststimulation with a significant increase in vacuolation occurring after 120 sec. As expected, there is an increase in the surface area of the basolateral membrane to facilitate the rapid movement of fluid into the cells. Other ultrastructural changes noted to accompany the onset of diuresis include the movement of mitochondria into areas adjacent to transport membranes, the vesiculation of Golgi, mobilization of CaPO(4) spherites, and a direct interaction of these spherites with active mitochondria. We discuss several possible roles for these changes in terms of rapid fluid transport.

Nitric Oxide–Mediated Effects on Myometrial Contractility at Term During Prelabor and Labor

In Brief Objective To assess the existence of a nitric oxide (NO) system in the human myometrium and the effects of mediators of this system on contractile activity in vitro. Methods Myometrial tissue was obtained before the onset of labor and during labor at term. Production of NO was assessed by the use of nicotinamide dinucleotide phosphate diaphorase staining and by immunoblots for NO. Effects of NO were examined by adding L-arginine (the substrate for NO synthesis); NG-nitro-L-arginine methyl ester (an inhibitor of NO synthase); two NO donors, sodium nitroprusside and spermine NONOate; as well as 8-bromo cyclic guanosine monophosphate (8-bromo cGMP) (a second messenger analogue) to organ baths. Results Myometrial NO production was indicated by positive nicotinamide adenine dinucleotide phosphate diaphorase staining. Immunoblots detected endothelial NO synthase, whereas only a weak signal for inducible NO synthase was seen. The addition of L-arginine (10−4–10−3 mol/L) did not result in any change of contractility. NG-nitro-L-arginine methyl ester (10−3 mol/L) caused a minor increase of contractility in half of the specimens. Sodium nitroprusside, spermine NONOate, and 8-bromo cGMP resulted in a concentration-dependent inhibition of contractility (10−7–10−6 mol/L for sodium nitroprusside, 10−6–10−5 mol/L for spermine NONOate, and 10−5–10−3 mol/L for 8-bromo cGMP). However, at 10−5–10−4 mol/L, sodium nitroprusside exhibited a dose-dependent increase in the frequency of contractions. Women in prelabor did not differ from those in active labor. Conclusion The myometrium produces NO at term. Nitric oxide inhibits myometrial contractile activity. The responsiveness to NO is similar in nonlaboring and laboring women. Nitric oxide synthase is present in human myometrial tissue, and nitric oxide–donating substances inhibit spontaneous contractility of myometrial strips.

The p38 mitogen-activated protein kinase inhibitor SB203580 antagonizes the inhibitory effects of interleukin-1β on long-term potentiation in the rat dentate gyrus in vitro

Levels of the pro-inflammatory cytokine interleukin-1β are known to be elevated in patients with chronic disorders such as Alzheimer's disease. We have investigated the effects of interleukin-1β on long-term potentiation and N-methyl- d-aspartate receptor-mediated field potentials in the rat dentate gyrus in vitro utilizing field extracellular recordings obtained from the middle third of the molecular layer of the dentate gyrus. Presynaptic stimulation was applied to the commissural/association pathway at a frequency of 0.05 Hz and at a distance of 50 μm from the granule cell body layer. As previously reported, interleukin-1β (1 ng/ml) caused an inhibition of long-term potentiation (108±2% of baseline 1 h following application of tetanic stimulation compared with 145±5% in vehicle control slices). This action of interleukin-1β on long-term potentiation, as well as an inhibition of N-methyl- d-aspartate receptor-mediated field potentials, was attenuated by pre-treatment of slices with the p38 mitogen-associated protein kinase inhibitor SB203580 (1 μM). SB203580 alone had no significant affect on long-term potentiation, but did cause an increase in baseline synaptic transmission [107±2% of baseline, 1 h after SB203580 (1 μM) treatment]. The p42/44 mitogen-activated protein kinase cascade inhibitor PD98059 (50 μM) did not inhibit the interleukin-1β-induced inhibition of N-methyl- d-aspartate receptor-mediated field potentials. The cyclooxygenase inhibitor indomethacin (50 μM) was found to attenuate the interleukin-1β-induced effects on both long-term potentiation and N-methyl- d-aspartate receptor-mediated field potentials. The lipid second messenger analogue C2 ceramide (20 μM) was found to attenuate the expression of long-term potentiation (108±3% of baseline 1 h following tetanic stimulation), and this effect was not blocked by pre-treatment with SB203580. To investigate a possible role for interleukin-1β in the normal expression of long-term potentiation, the interleukin-1 receptor antagonist (25 ng/ml) was applied during the maintenance phase of long-term potentiation. This was found to depress the sustained expression of long-term potentiation (116±6% of baseline 1 h following tetanic stimulation). Our results indicate possible signalling mechanisms by which interleukin-1β at pathophysiological concentrations may serve to inhibit long-term potentiation, and also suggests a role for IL-1β in the physiological expression of synaptic plasticity in the rat dentate gyrus in vitro.

Nitric oxide–mediated effects on myometrial contractility at term during prelabor and labor

Objective: To assess the existence of a nitric oxide (NO) system in the human myometrium and the effects of mediators of this system on contractile activity in vitro. Methods: Myometrial tissue was obtained before the onset of labor and during labor at term. Production of NO was assessed by the use of nicotinamide dinucleotide phosphate diaphorase staining and by immunoblots for NO. Effects of NO were examined by adding l-arginine (the substrate for NO synthesis); N G-nitro- l-arginine methyl ester (an inhibitor of NO synthase); two NO donors, sodium nitroprusside and spermine NONOate; as well as 8-bromo cyclic guanosine monophosphate (8-bromo cGMP) (a second messenger analogue) to organ baths. Results: Myometrial NO production was indicated by positive nicotinamide adenine dinucleotide phosphate diaphorase staining. Immunoblots detected endothelial NO synthase, whereas only a weak signal for inducible NO synthase was seen. The addition of l-arginine (10 −4–10 −3 mol/L) did not result in any change of contractility. N G-nitro- l-arginine methyl ester (10 −3 mol/L) caused a minor increase of contractility in half of the specimens. Sodium nitroprusside, spermine NONOate, and 8-bromo cGMP resulted in a concentration-dependent inhibition of contractility (10 −7–10 −6 mol/L for sodium nitroprusside, 10 −6–10 −5 mol/L for spermine NONOate, and 10 −5–10 −3 mol/L for 8-bromo cGMP). However, at 10 −5–10 −4 mol/L, sodium nitroprusside exhibited a dose-dependent increase in the frequency of contractions. Women in prelabor did not differ from those in active labor. Conclusion: The myometrium produces NO at term. Nitric oxide inhibits myometrial contractile activity. The responsiveness to NO is similar in nonlaboring and laboring women.

Invasion of cytotrophoblastic JEG-3 cells is stimulated by hCG in vitro

Trophoblast invasion into the uterine wall is controlled by many factors. Previously, a human chorionic gonadotropin (hCG) receptor has been found to be expressed on invasive trophoblast as well as on choriocarcinoma cells implying a possible role for the hormone in trophoblast invasion. Therefore, this study examined the role of hCG in the invasion of trophoblastic (JEG-3) cells. Increasing hCG concentrations were applied in a trophoblast invasion model, JEG-3, through matrigel-coated filters. The proliferation was quantified by WST-1 cleavage assay. Cell migration was studied by examining the number of cells that had passed the uncoated porous (8-μn pore size) filters. After staining, filters were examined microscopically for cells on the underside of the membrane. A quantitative protease assay was also performed. Flow cytometric analysis of a5 and a6 integrin subunits, which are essential for interactions between cells and extracellular matrix, was performed. hCG increased significantly ( P<0.01) the in vitro invasion of trophoblastic JEG-3 cells in a dose-dependent manner. Migration was also increased by hCG ( P<0.01). However, cell proliferation remained unchanged. The second messenger analogue dibutyryl CAMP (db cAMP) and the CAMP elevating factor (forskolin) mimicked the effects of hCG by stimulating a dose-dependent increase of trophoblastic cell (JEG-3) invasion. The collagenolytic activity of trophoblastic cells (IEG-3) was increased by hCG stimulation. No changes were shown in the expression of α5 and α6 integrin subunits on JEG-3 cells. In vitro hCG is a regulatory factor of invasion and migration in trophoblastic JEG-3 cells, whereas proliferation is not influenced. The endogenous production of hCG by the trophoblast in vivo implies an autocrine control of invasion processes by hCG.