Expression of the cholinergic gene locus in the rat placenta

High amounts of acetylcholine (ACh) and its synthesising enzyme choline acetyltransferase (ChAT) have been detected in the placenta. Since the placenta is not innervated by extrinsic or intrinsic cholinergic neurons, placental ACh and ChAT originate from non-neuronal sources. In neurons, cytoplasmic ACh is imported into synaptic vesicles by the vesicular acetylcholine transporter (VAChT), and released through vesicular exocytosis. In view of the coordinate expression of VAChT and ChAT from the cholinergic gene locus in neurons, we asked whether VAChT is coexpressed with ChAT in rat placenta, and investigated this issue by means of RT-PCR, in situ hybridisation, western blot and immunohistochemistry. Messenger RNA and protein of the common type of ChAT (cChAT), its splice variant peripheral ChAT (pChAT), and VAChT were detected in rat placenta with RT-PCR and western blot. ChAT in situ hybridisation signal and immunoreactivity for cChAT and pChAT were observed in nearly all placental cell types, while VAChT mRNA and immunolabelling were detected in the trophoblast, mesenchymal cells and the visceral yolk sac epithelial cells. While ChAT is nearly ubiquitously expressed in rat placenta, VAChT immunoreactivity is localised cell type specifically, implying that both vesicular and non-vesicular ACh release machineries prevail in placental cell types.     

Regulation of the two forms of glycogen phosphorylase by cAMP and its analogs in Dictyostelium discoideum

Summary We have recently reported the existence of two forms of glycogen phosphorylase (1,4--D-glucan: orthophosphate--glucosyltransferase; EC 2.4.1.1) in Dictyostelium discoideum. During development the activity of the glycogen phosphorylase b form decreased as the activity of the a form increased. The total phosphorylase activity remained constant. The physical and kinetic properties of the Dictyostelium enzyme were similar to those of the mammalian enzyme. In mammals, cAMP regulates the conversion of the two forms by a cAMP dependent protein kinase (cAMPdPK). We report here that if cAMP is added to a single cell suspension, the Dictyostelium phosphorylase activity becomes independent of 5AMP and a 104kd peptide appears. We also show the effect of several cAMP analogs on the phosphorylase activity in these single-cell suspensions. The cAMP analogs were selected on the basis of their affinities for the membrane-bound cAMP receptor or the cytoplasmic cAMPdPK. We found that relatively low levels, 100 M, of cAMP or 2'd-cAMP added to aggregation-competent cells in shaking culture caused a loss of phosphorylase b activity and the appearance of phosphorylase a activity. The analog, 2'd-cAMP, has a high affinity for the cAMP receptor but a low affinity for the cAMPdPK. Two other analogs, Bt₂-cAMP and 8-Br-cAMP, which have low affinities for the cAMP receptor but high affinities for the cAMPdPK, required high levels (500 M) for b to a conversion. cDNAs to three cAMP-regulated genes-PL3, Dll, and D3-were used as controls in the above experiments. In order to determine if intracellular levels of cAMP were involved in the regulation of phosphorylase activity, both the phosphorylase and the PL3, D11 and D3 mRNA levels were examined in cells suspended in a glucose/albumin mixture - a medium in which adenylate cyclase is inhibited. Under these conditions, neither gene regulation nor a change in the phosphorylase b to a activity occurred in response to added extra cellular cAMP. The results suggest that an intracellular increase in cAMP is involved in the regulation of the two forms of glycogen phosphorylase in Dictyostelium.Abbreviations EGTA Ethyleneglycol-bis-(-aminoethyl ether) - N,N,N N-tetra acetic acid - SDS Sodium Dodecyl Sulfate - PAGE Polyacrylamide Gel Electrophoresis     

Adenosine 3′, 5′- Cyclic Monophosphate Increases Processing of Amyloid Precursor Protein (APP) to β-Amyloid in Neuroblastoma Cells Without Changing APP Levels or Expression of APP mRNA

-Amyloid (A), a 39–43 residue peptide generated by splicing of the amyloid precursor protein (APP), is one of the major components of senile plaques which are the hallmark of Alzheimer's disease (AD); and therefore, a role of A in neuronal degeneration has been proposed. The factors which regulate the levels of A have not been fully identified. Since an elevation of the intracellular levels of adenosine, 3, 5-cyclic monophosphate (cAMP) in neuroblastoma cells (NB) induces terminal differentiation, and since these differentiated NB cells undergo spontaneous degeneration, the role of cAMP in the regulation of A levels in these cells have been investigated. In order to determine the specificity of the effect of cAMP on nerve cells, rat glioma cells (C-6) were investigated in a similar manner. Results showed that an elevation of the levels of cAMP in NB cells enhances the intensity of A immunostaining without changing the levels of APP or APP mRNA. This suggests that the rate of processing of APP to A increases following an elevation of cAMP level in NB cells. Data also revealed that an elevation of cAMP level in glioma cells did not alter the intensity of staining with APP or A.     

δ and κ Opiate receptors in primary astroglial cultures from rat cerebral cortex

The effects of , , and receptor-agonists on forskolin stimulated cyclic adenosine-3, 5-monophosphate (cAMP) formation were examined in astroglial enriched primary cultures from the cerebral cortex of newborn rats. Intracellular cAMP accumulation was quantified by radioimmunoassay. Morphine was used as a -receptor agonist, D-Ala-D-Leu-Enkephalin (DADLE) as a -receptor agonist and dynorphine 1–13 (Dyn) as a -receptor agonist. Basal cAMP levels were unaffected by either the opiate agonists or the antagonists used. In the presence of the cAMP stimulator forskolin, morphine had no significant effect on the cytoplasmic cAMP levels. DADLE caused a dose related inhibition of the forskolin stimulated cAMP accumulation. The effects of this receptor stimulation was blocked with the selective antagonist ICI 174.864. In the presence of Dyn, the forskolin stimulated cAMP accumulation was inhibited in a dose related manner. This receptor stimulation was blocked with the selective antagonist MR 2266. Co-administration of DADLE and Dyn resulted in a non additive inhibition of the forskolin stimulated accumulation of cAMP. These findings indicate that astroglial enriched cultures from the cerebral cortex of rats express and -receptors co-localized ont he same population of cells, and that these receptors are inhibitory coupled to adenylate cyclase.     

Role of histamine and cyclic nucleotides in implantation in the rabbit

Summary The effect of histamine on cAMP and cGMP levels in day 6 (144 h post coitum) rabbit blastocysts was determined. Histamine at 200 M and 1000 M concentrations stimulated the increased formation of cAMP in vitro, whereas stimulation of cGMP occurred only in the presence of 1000 M histamine. Furthermore, intrauterine injection of RMI-12330A (50 g or 500 g/uterine horn), an inhibitor of adenylyl cyclase, on day 5 of pregnancy interrupted embryro development and implantation of the embryo. The drug was also effective in reducing the cAMP level in the endometrial cells in vitro. A relationship between histamine and cyclic nucleotide changes in embryo development and implantation is suggested.     

Effects of Sex Hormones,Forskolin, and Nicotine on Choline Acetyltransferase Activity in Human Isolated Placenta

The activity of choline acetyltransferase (ChAT) was investigated in the human placenta before and after long-term incubation (24 h) to test the effects of sex hormones, nicotine and forskolin. ChAT activity differed considerably between the amnion (0.03 mol/mg protein/h) and the villus (0.56). After long-term incubation, ChAT activity persisted in the latter but declined in the amnion. Neither sex hormones (-estradiol, testosterone, progesterone; 10 or 100 nM each) nor follicle stimulating hormone and luteinizing hormone (FSH/LH; 8.4 U/ml each) modified ChAT activity. Also nicotine (1 nM–100 M) did not affect ChAT activity. Forskolin, an activitor of adenylyl cyclase, reduced ChAT activity in the villus but not in amnion. The present model offers the possibility to investigate ChAT regulation in intact tissue under long-term incubation. The risks of maternal smoking during pregnancy cannot be attributed to an effect of nicotine on placental ChAT activity. Differences in the regulation of ChAT appear to exist between neuronal and nonneuronal cells.     

In vivo effects of insulin and bis(maltolato)oxovanadium (IV) on PKB activity in the skeletal muscle and liver of diabetic rats

In this study, the in vivo effects of insulin and chronic treatment with bis(maltolato)oxovanadium (IV) (BMOV) on protein kinase B (PKB) activity were examined in the liver and skeletal muscle from two animal models of diabetes, the STZdiabetic Wistar rat and the fatty Zucker rat. Animals were treated with BMOV in the drinking water (0.75–1 mg/ml) for 3 (or 8) weeks and sacrificed with or without insulin injection. Insulin (5 U/kg, i.v.) increased PKB activity more than 10fold and PKB activity more than 3fold in both animal models. Despite the development of insulin resistance, insulininduced activation of PKB was not impaired in the STZdiabetic rats up to 9 weeks of diabetes, excluding a role for PKB in the development of insulin resistance in type 1 diabetes. Insulin-induced PKB activity was markedly reduced in the skeletal muscle of fatty Zucker rats as compared to lean littermates (fatty: 7fold vs. lean: 14fold). In contrast, a significant increase in insulinstimulated PKBa activity was observed in the liver of fatty Zucker rats (fatty: 15.7fold vs. lean: 7.6fold). Chronic treatment with BMOV normalized plasma glucose levels in STZdiabetic rats and decreased plasma insulin levels in fatty Zucker rats but did not have any effect on basal or insulininduced PKB and PKB activities. In conclusion (i) in STZdiabetic rats PKB activity was normal up to 9 weeks of diabetes; (ii) in fatty Zucker rats insulininduced activation of PKB (but not PKB) was markedly altered in both tissues; (iii) changes in PKB activity were tissue specific; (iv) the glucoregulatory effects of BMOV were independent of PKB activity.     

Effects of losartan on the collagen degradative enzymes in hypertrophic and congestive types of cardiomyopathic hamsters

The present study was undertaken to determine the effects of AT1 receptor blockade which occurred in response to losartan, on the extracellular matrix (ECM) degradation process in the Bio 14.6 (n = 12) and Bio 53.58 (n = 12) strains which are referred as models of hypertrophic and dilated cardiomyopathy, respectively. The administration of losartan (30 mg/kg/day) in hamsters from 10–20 weeks of age reduced the accumulation of the left ventricular collagen matrix in both of the Bio 14.6 and the Bio 53.58 strains. According to the RTPCR, the levels of mRNA for matrix metalloproteinase (MMP) and the tissue inhibitor of MMP (TIMP) were examined. MMP1, 2, 3, and 9 were more enhanced in both myopathic strains than in the control F1 strains. With losartan, the levels of MMP1, 2, 9, TIMP1 and 2 decreased in the both strains but those for MMP3 did not in Bio 14.6 strains. TIMP3 and 4 mRNA levels did not change in any of the experimental hamsters, whether treated or untreated with losartan. The Western blots also showed similar observations in the both strains as seen in mRNA expressions although MMP2 in the Bio 53.58 strains did not differ between treated and untreated with losartan. Although losartan has an inhibitory effect on collagen accumulation in the development of cardiomyopathy, MMPs (1, 2, 9) and TIMPs (1, 2) seem to be susceptible to responding to losartan in Bio cardiomyopathic hamsters.     

Adrenergic and muscarinic receptor regulation and therapeutic implications in heart failure

In end-stage heart failure the expression of different myocardial regulatory proteins involved in the -adrenergic cAMP signalling pathway is altered. The downregulation of -adrenoceptors and their uncoupling from the effector as well as an increased expression of the inhibitory GTP-binding protein seem to be the most important alterations. Since catecholamine levels are elevated in these patients and since some alterations can be restored after treatment with -adrenoceptor antagonists it was hypothesized that excessive -adrenergic stimulation could be involved in these alterations.In this article the changes of -adrenergic receptors, GTP-binding proteins, sarcoplasmic reticulum Ca²⁺-ATPase and of phospholamban found in heart failure are addressed with its possible therapeutic implications.     

Ethanol-induced changes in properties of rat brain ribosomes

Altered in vivo and in vitro brain protein metabolism have been demonstrated in rodents following long-term ethanol ingestion. In the present study, ethanol effects were examined on properties of brain ribosomes of male Sprague-Dawley rats ingesting a specially formulated Lieber-DeCarli liquid diet. The development of physical dependence was demonstrated by the presence of withdrawal reactions within 24 hr of ethanol abstinence. Data showed significant inhibition of in vitro protein synthesis by ribosomes from the ethanol and 1-day-withdrawn groups. Partial reversal of inhibition occurred by using a control brain pH 5 enzymes source instead of the matched source. The observed [¹⁴C]leucine-incorporating activity was temperature dependent, with the optimum temperature being 37°C. The determination of the state of ribosomal aggregation showed an increased monosomes-disomes ratio in the ethanol group. The ratio was even more increased in the 1-day-withdrawn group. Data suggest that reduced ribosomal binding to stable mRNA may be a contributing factor in the ethanol-induced effects on protein synthesis.     

Inhibitory effect of menaquinone-7 (vitamin K2) on osteoclast-like cell formation and osteoclastic bone resorption in rat bone tissues in vitro

Heme oxygenase1, the major inducible isoform of heme oxygenase (HO), can be induced by heme and numerous other physical and chemical factors, many of which cause cellular stress. This has led to the realization that HO1 is a major highly conserved stress or heat shock protein. Recent work has implicated activation of mitogenactivated protein kinases and other kinases in the mechanism of induction of HO1, and suggested that signal transduction pathways through tyrosine kinases are involved in induction of HO1 gene expression by stress inducers. We hypothesized that phenylarsine oxide (PAO), an inhibitor of protein tyrosine phosphatases (PTPs), might up-regulate the HO1 gene. Here, we show that a remarkably brief (1–15 min) exposure of normal hepatocytes to low concentrations (0.5–3 M) of PAO produces a marked increase in mRNA and protein of HO1. This increase is comparable to the level obtained by addition of heme (20 M), and occurs without producing changes in cellular glutathione levels or stabilization of HO1 message. Preincubation of cells with inhibitors of protein synthesis decreased the ability of PAO to increase levels of HO1 mRNA, suggesting that the inductive effect requires de novo protein synthesis. Addition of thiol donors abrogated the PAOmediated induction of HO1 in a dose dependent fashion. Addition of genistein, a tyrosine kinase inhibitor, blunted the induction produced by both PAO and heme. After brief incubations with PAO or heme, cell extracts showed comparable increases in levels of protein tyrosine phosphorylation in general, and specifically in ZAP70 kinase. Our results are consistent with the proposition that induction of HO1 by PAO involves inhibition of specific PTP(s), and that the mechanisms of induction of HO1 by PAO and by heme may share some common pathways.     

Phytohormone-regulated β-amylase gene expression in rice

The expression of -amylase genes in rice (Oryza sativa) and its regulation by phytohormones gibberellic acid (GA) and abscisic acid (ABA) were examined. Upon germination -amylase is synthesizedde novo in aleurone cells and (GA) is not required. Exogenous addition of GA does not enhance the -amylase activity, while ABA inhibits the -amylase activity, mRNA accumulation, and the germination of rice seeds. GA can reverse ABA inhibition of -amylase expression, but not ABA inhibition of seed germination. Such regulation represents a new interaction of ABA and GA.     

Binding of the galactose-specificPseudomonas aeruginosa lectin,PA-I,to glycosphingolipids and other glycoconjugates

The carbohydrate-binding specificity ofPseudomonas aeruginosa lectin I (PA-I) in iodinated or biotinylated form was studied. A large number of glycosphingolipids, as well as some glycoproteins and neoglycoproteins were used as ligands. Also, inhibition by free saccharides of PA-I binding to glycosphingolipids was tested. It was found that the lectin binds most strongly to terminal and nonsubstituted Gal3Gal- or Gal4Gal-structures.Abbreviations PA-I Pseudomonas aeruginosa lectin I - Cer ceramide - lactosylceramide Gal4GlcCer - iso globotriaosylcerami Gal3Gal4GlcCer - globotriaosylceramide Gal4Gal4GlcCer - globoside or globotetraosylceramide GalNAc3Gal4Gal4GlcCer - Forssman glycolipid GalNAc3GalNAc3Gal4Gal4GlcCer - P1 glycolipid Gal4Gal4GlcNAc3Gal4GlcCer - lactoneotetraosylceramide Gal4GlcNAc3Gal4GlcCer - B5 glycolipid Gal3Gal4GlcNAc3Gal4GlcCer - gangliotetraosylceramide Gal3GalNAc4Gal4GlcCer - GM1 Gal3GalNAc4(NeuAc3)Gal4GlcCer - RBC red blood cells - BSA bovine serum albumin - PBS phosphate-buffered saline - SDS sodium dodecyl sulfate - TLC thin-layer chromatography - HPLC high pressure liquid chromatography - MS mass spectrometry - FAB fast-atom bombardment - EI electron impact     

Hormonal effects on the regulation of hepatic heme biosynthesis

Summary Drug-induced porphyrin accumulation occurs in chick embryo liver cells maintained in serum-free Waymouth MD 705/1 medium. Addition of insulin and thyroxine to the medium results in a marked enhancement of porphyrin accumulation. The addition of hydrocortisone results in a further enhancement of porphyrin accumulation.Several agents which are reported to increase intracellular adensosine 3:5-monophosphate (cAMP) levels, viz. glucagon, sodium fluoride, cAMP or its dibutyryl derivative, 3-isobutyl-1-methylxanthine and papaverine enhanced drug-induced porphyrin biosynthesis. On the other hand, agents which are reported to decrease intra-cellular cAMP levels, viz. alloxan and imidazole, diminished drug-induced porphyrin accumulation. cAMP appears to enhance, but not to function as a second messenger in drug-induced porphyrin biosynthesis.Drug-induced porphyrin accumulation in chick embryo liver cells depend upon the insulin to glucagon ratio. A low level of porphyrin accumulation occurs at insulin to glucagon ratios similar to those found following glucose administration in vivo, suggesting a possible explanation for the therapeutic effect of glucose in hepatic porphyria.The 5H(A:B trans) and 5H(A:B cis) steroids are equipotent in inducing -aminolevulinic acid synthetase and porphyrin accumulation in chick embryo liver cells maintained in serum-free culture medium. Thus, there is no specific steric requirement for porphyrin-inducing activity in steroids.This work was supported by the Medical Research Council of Canada.     

Task Relevance Enhances Early Transient and Late Slow-Wave Activity of Distributed Cortical Sources

The primary purpose of these studies was to link together concepts related to attention/working memory and feedforward/feedback activity using MEG response profiles obtained in humans. Similar to recent studies of attention in monkeys, we show early spike-like activity (<200 ms poststimulus), most likely reflecting an early transient excitatory response mixed with feedback influences, followed by slow-wave activity (>200 ms poststimulus) in MEG cortical response profiles evoked by a visual working memory task. We experimentally dissociated the early transient activity from the later sustained activity (predominately feedback) by conducting an auditory size classification task. Words, representing common objects, evoked activity in occipital cortex (presumably due to imagery) even though visual stimuli were not present in this task. The initial spike was absent from the response profile obtained from occipital cortex, leaving only slow-wave activity, thereby allowing us to characterize or profile feedback activity in this situation. Attention or task relevance enhanced the initial spike and slow-wave activity in visually responsive areas. Prefrontal activity, along the superior frontal sulcus, evoked by the working memory task, was active later in time than initial activity in visual cortex and later than the earliest effect of attention modulation in visual cortex.     

Ozone-induced changes of mRNA levels of β-1,3-glucanase,chitinase and ‘pathogenesis-related’ protein 1b in tobacco plants

Treatment of the ozone-sensitive tobacco cultivar Bel W3 with an ozone pulse (0.15 l/l, 5 h) markedly increased the mRNA level of basic -1,3-glucanase and to a lower degree that of basic chitinase. The increase of -1,3-glucanase mRNA level occurred within 1 h and showed a transient maximum. Seventeen hours after ozone treatment, the -1,3-glucanase mRNA level decreased to lower values. The increase of basic chitinase mRNA level was delayed and was less pronounced than that of -1,3-glucanase mRNA. Cultivar Bel B showed only a small increase of -1,3-glucanase mRNA level after the same ozone treatment, whereas its basic chitinase mRNA was more strongly induced. Prolonged ozone treatment for 2 days of tobacco Bel W3 led to a persistent level of -1,3-glucanase and basic chitinase mRNAs, as well as to an increase of acidic chitinase and pathogenesis-related (PR) 1b mRNA levels. The results indicate that genes so far considered to code for PR proteins may also be involved in the plant response to oxidative stress.     

Regulation of low density lipoprotein receptor mRNA levels by estradiol 17β and chorionic gonadotropin in human placenta

Inhibition of synthesis of estradiol 17 by the addition of inhibitors of aromatase, a key enzyme in the biosynthesis of estradiol 17, or addition of tamoxifen - an estrogen receptor antagonist, to human placental minces resulted in an increase in the level of LDL-receptor mRNA. This increase could be blocked by the simultaneous addition of estradiol 17. A concentration dependent effect of estradiol 17 on the level of LDL-receptor mRNA was seen both in first trimester, and term placenta. Addition of human chorionic gonadotropin (hCG) to term placental minces also increased the LDL-receptor mRNA levels. When hCG and cycloheximide were added together, an additive effect was observed. The results obtained in this study suggest that the LDL-receptor mRNA levels in the human placenta are regulated by estradiol 17 and hCG.     

Short- and long-term effects of ACTH on the adrenal zona glomerulosa of the rat

Summary Short-term ACTH treatment provoked a decrease in volume of the lipid-droplet compartment in rat zona glomerulosa cells, and a rise in plasma and intracellular concentrations of corticosterone and aldosterone. It enhanced activities of 3-hydroxysteroid dehydrogenase (3HSD), 11-hydroxylase (11OH) and 18-hydroxylase (18OH). Long-term ACTH administration produced a hypertrophy of the zona glomerulosa and its parenchymal cells, a result of the increase in volume of the smooth endoplasmic reticulum and the mitochondrial compartment. The surface area per cell of mitochondrial inner membranes increased; the tubular cristae were transformed into a homogeneous population of vesicles. The plasma and intracellular concentrations of corticosterone further increased, whereas those of aldosterone fell below basal levels (the aldosterone-escape phenomenon). The activities of 3HSD and 11OH were enhanced, that of 180H decreased. Therefore, ACTH stimulates zona glomerulosa growth and transforms parenchymal elements into zona fasciculata celltypes. Cyanoketone nullified acute ACTH effects on plasma and intracellular concentrations of corticosterone and aldosterone, but did not affect the activities of 11OH and 18OH. Chronic ACTH treatment produced similar results, although 18OH activity was not suppressed. The mechanism underlying the aldosterone-escape phenomenon may thus involve a rise in the intracellular concentration of corticosterone, caused by the enhanced synthesis and activation of 3HSD and 11OH.     

Modulation of inner mitochondrial membrane channel activity

Three classes of inner mitochondrial membrane (IMM) channel activities have been defined by direct measurement of conductance levels in membranes with patch clamp techniques in 150 mM K Cl. The 107 pS activity is slightly anion selective and voltage dependent (open with matrix positive potentials). Multiple conductance channel (MCC) activity includes several levels from about 40 to over 1000 pS and can be activated by voltage or Ca²⁺. MCC may be responsible for the Ca²⁺-induced permeability transition observed with mitochondrial suspensions. A low conductance channel (LCC) is activated by alkaline pH and inhibited by Mg²⁺. LCC has a unit conductance of about 15 pS and may correspond to the inner membrane anion channel, IMAC, which was proposed from results obtained from suspension studies. All of the IMM channels defined thus far appear to be highly regulated and have a low open probability under physiological conditions. A summary of what is known about IMM channel regulation and pharmacology is presented and possible physiological roles of these channels are discussed.     

Role of mitochondria in ethanol tolerance of Saccharomyces cerevisiae

The presence of active mitochondria and oxidative metabolism is shown to be essential to maintain low inhibition levels by ethanol of the growth rate (), fermentation rate (v) or respiration rate () of Saccharomyces cerevisiae wild type strain S288C. Cells which have respiratory metabolism show K _i (ethanol inhibition constant) values for , v and , higher (K _i>1 M) than those of petite mutants or grande strains grown in anaerobiosis (K _i=0.7 M). In addition, the relationship between or v and ethanol concentration is linear in cells with respiratory metabolism and exponential in cells lacking respiration. When functional mitochondria are transferred to petite mutants, the resulting strain shows K _i values similar to those of the grande strain and the inhibition of and v by increasing ethanol concentrations becomes linear.