Modulation of biliary lipid secretion by forskolin and cyclic AMP analogues.
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ABSTRACT:
SUBMITTER: Hamlin S
PROVIDER: S-EPMC1133713 | biostudies-other | 1990 Feb
REPOSITORIES: biostudies-other
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Project description:Forskolin is a diterpene that activates adenylate cyclase in a variety of mammalian cells. In addition of forskolin to blowfly salivary glands increased cyclic AMP accumulation and salivary secretion. There was a small increase in transepithelial movement of labelled Ca2+. Forskolin did not induce breakdown of labelled phosphatidylinositol or inhibit the stimulation of phosphatidylinositol breakdown caused by 5-hydroxytryptamine. These data indicate that forskolin can mimic all the effects of 5-hydroxytryptamine on salivary-gland secretion that have been attributed to cyclic AMP.
| S-EPMC1158326 | biostudies-other
Project description:Forskolin is a unique structurally complex labdane-type diterpenoid used in the treatment of glaucoma and heart failure based on its activity as a cyclic AMP booster. Commercial production of forskolin relies exclusively on extraction from its only known natural source, the plant Coleus forskohlii, in which forskolin accumulates in the root cork. Here, we report the discovery of five cytochrome P450s and two acetyltransferases which catalyze a cascade of reactions converting the forskolin precursor 13R-manoyl oxide into forskolin and a diverse array of additional labdane-type diterpenoids. A minimal set of three P450s in combination with a single acetyl transferase was identified that catalyzes the conversion of 13R-manoyl oxide into forskolin as demonstrated by transient expression in Nicotiana benthamiana. The entire pathway for forskolin production from glucose encompassing expression of nine genes was stably integrated into Saccharomyces cerevisiae and afforded forskolin titers of 40 mg/L.
| S-EPMC5388535 | biostudies-literature
Project description:Cholangiocarcinoma is a lethal disease with scarce response to current systemic therapy. The rare occurrence and large heterogeneity of this cancer, together with poor knowledge of its molecular mechanisms, are elements contributing to the difficulties in finding an appropriate cure. Cholangiocytes (and their cellular precursors) are considered the liver component giving rise to cholangiocarcinoma. These cells respond to several hormones, neuropeptides and molecular stimuli employing the cAMP/PKA system for the translation of messages in the intracellular space. For instance, in physiological conditions, stimulation of the secretin receptor determines an increase of intracellular levels of cAMP, thus activating a series of molecular events, finally determining in bicarbonate-enriched choleresis. However, activation of the same receptor during cholangiocytes' injury promotes cellular growth again, using cAMP as the second messenger. Since several scientific pieces of evidence link cAMP signaling system to cholangiocytes' proliferation, the possible changes of this pathway during cancer growth also seem relevant. In this review, we summarize the current findings regarding the cAMP pathway and its role in biliary normal and neoplastic cell proliferation. Perspectives for targeting the cAMP machinery in cholangiocarcinoma therapy are also discussed.
| S-EPMC8303798 | biostudies-literature
Project description:This study was performed to determine the effects of various organic anions on biliary lipid secretion in rats. We infused bile-salt-pool-depleted rats with sodium taurocholate at a constant rate, with or without various organic anions: Indocyanine Green (ICG), bromosulphophthalein (BSP), BSP-glutathione and Phenol Red (PR). BSP decreased biliary secretion of cholesterol and phospholipids in a dose-dependent manner without affecting bile salt secretion (uncoupling), and this change was fully reversible. In contrast, ICG, BSP-glutathione and PR did not cause such an uncoupling of biliary lipids. In addition, the distribution pattern of each organic anion to various lipid particles was determined by gel-permeation chromatography. BSP was predominantly associated with bile salt micelles, whereas vesicular association was dominant for ICG, and both BSP-glutathione and PR formed only self-aggregations. From these data, we concluded that the uncoupling of biliary lipids from bile salt secretion by BSP resulted from the interaction between BSP and bile salt micelles in the bile canaliculus, and that this interaction inhibited the capacity of bile salts to induce the secretion of phospholipids and cholesterol.
| S-EPMC1133038 | biostudies-other
Project description:Multiple short pulses of taurocholate (TC) brought about, in isolated perfused rat livers, the secretion of phospholipid and cholesterol into bile; the lipids showed an appreciable lag period behind the bile-salt secretion, and there was considerable variability in response, both between low and high dose pulses of TC and, at the higher dose, even between individual livers. When a background continuous infusion of taurodehydrocholate (a hydrophilic non-micelle-forming bile-salt analogue) was superimposed upon the short TC pulses, the lipid secretion showed much better control, and the lipid peaks were of more uniform size, following more closely, or more coincident with, the bile-salt output peaks. Taurodehydrocholate may provide a signal for the control of the supply and delivery of lipid vesicles to the bile-canalicular membrane, from where the lipid vesicles are then removed by the action of the pulses of TC.
| S-EPMC1148154 | biostudies-other
Project description:Biochemical and immunochemical studies were undertaken to quantify the effects of cyclic AMP on cyclic AMP-dependent protein kinase subunit levels in nuclei of H4IIE hepatoma cells. Dibutyryl cyclic AMP (10 microM) caused a significant biphasic (10 and 120 min after stimulation) increase in total nuclear protein kinase activity. The increase observed 10 min after dibutyryl cyclic AMP stimulation was primarily due to an approx. 3-fold increase of catalytic (C) subunit activity, whereas the change observed 120 min after stimulation consisted of an increase in both C subunit and cyclic AMP-independent protein kinase activities. Analysis of nuclear protein extracts by photoaffinity labelling with 8-azido cyclic [32P]AMP identified only the type II regulatory subunit (RII), but not the type I regulatory subunit (RI). Analysis of nuclear RII variants by two-dimensional gel electrophoresis demonstrated that dibutyryl cyclic AMP caused the appearance of two RII variant forms which were not present in the nuclei of unstimulated cells. Using affinity-purified polyclonal antibodies and immunoblotting procedures, we identified an approx. 2-fold increase in the RII and C subunits in nuclear extracts of dibutyryl cyclic AMP-treated hepatoma cells. Finally, the RI, RII and C subunits were quantified by an e.l.i.s.a. which indicated that dibutyryl cyclic AMP increased nuclear RII and C subunits levels biphasically, reaching peak values 10 and 120 min after the initial stimulation. Nuclear RI subunit levels were not affected. These results provide qualitative as well as quantitative evidence for a modulation by cyclic AMP of the nuclear RII and C subunit levels in rat H4IIE hepatoma cells, and indicate a relatively rapid but temporarily limited dibutyryl cyclic AMP-induced translocation of the RII and C subunits to nuclear sites.
| S-EPMC1138730 | biostudies-other
Project description:A follicular spike in cyclic AMP (cAMP) and its subsequent degradation to AMP promotes oocyte maturation and ovulation. In vitro matured (IVM) oocytes do not receive the cAMP increase that occurs in vivo, and artificial elevation of cAMP in IVM cumulus-oocyte complexes improves oocyte developmental potential. This study examined whether mouse oocytes can use the cAMP degradation product AMP to generate ATP via the adenosine salvage pathway, and examined whether pharmacological elevation of cAMP in IVM cumulus-oocyte complexes alters ATP levels. Oocytes cultured with isotopic 13C5-AMP dose-dependently produced 13C5-ATP, however total cellular ATP remained constant. Pharmacological elevation of cAMP using forskolin and IBMX prior to IVM decreased oocyte ATP and ATP:ADP ratio, and promoted activity of the energy regulator AMPK. Conversely, cumulus cells exhibited higher ATP and no change in AMPK. Culture of oocytes without their cumulus cells or inhibition of their gap-junctional communication yielded lower oocyte 13C5-ATP, indicating that cumulus cells facilitate ATP production via the adenosine salvage pathway. In conclusion, this study demonstrates that mouse oocytes can generate ATP from AMP via the adenosine salvage pathway, and cAMP elevation alters adenine nucleotide metabolism and may provide AMP for energy production via the adenosine salvage pathway during the energetically demanding process of meiotic maturation.
| S-EPMC6895058 | biostudies-literature
Project description:Autocrine/paracrine factors generated in response to 17β-estradiol (E2) within the fallopian tube (FT) facilitate fertilization and early embryo development for implantation. Since cyclic AMP (cAMP) plays a key role in reproduction, regulation of its synthesis by E2 may be of biological/pathophysiological relevance. Herein, we investigated whether cAMP production in FT cells (FTCs) is regulated by E2 and environmental estrogens (EE's; xenoestrogens and phytoestrogens). Under basal conditions, low levels of extracellular cAMP were detectable in bovine FTCs (epithelial cells and fibroblasts; 1:1 ratio). Treatment of FTCs with forskolin (AC; adenylyl cyclase activator), isoproterenol (β-adrenoceptor agonist) and IBMX (phosphodiesterase (PDE) inhibitor) dramatically (>10 fold) increased cAMP; whereas LRE1 (sAC; soluble AC inhibitor) and 2',5'-dideoxyadenosine (DDA; transmembrane AC (tmAC)) inhibitor decreased cAMP. Comparable changes in basal and stimulated intracellular cAMP were also observed. Ro-20-1724 (PDE-IV inhibitor), but not milrinone (PDE-III inhibitor) nor mmIBMX (PDE-I inhibitor), augmented forskolin-stimulated cAMP levels, suggesting that PDE-IV dominates in FTCs. E2 increased cAMP levels and CREB phosphorylation in FTCs, and these effects were mimicked by EE's (genistein, 4-hydroxy-2',4',6'-trichlorobiphenyl, 4-hydroxy-2',4',6'-dichlorobiphenyl). Moreover, the effects of E2 and EE were blocked by the tmAC inhibitor DDA, but not by the ERα/β antagonist ICI182780. Moreover, BAPTA-AM (intracellular-Ca2+ chelator) abrogated the effects of E2, but not genistein, on cAMP suggesting differential involvement of Ca2+. Treatment with non-permeable E2-BSA induced cAMP levels and CREB-phosphorylation; moreover, the stimulatory effects of E2 and EEs on cAMP were blocked by G15, a G protein-coupled estrogen receptor (GPER) antagonist. E2 and IBMX induced cAMP formation was inhibited by LRE1 and DDA suggesting involvement of both tmAC and sAC. Our results provide the first evidence that in FTCs, E2 and EE's stimulate cAMP synthesis via GPER. Exposure of the FT to EE's and PDE inhibitors may result in abnormal non-cyclic induction of cAMP levels which may induce deleterious effects on reproduction.
| S-EPMC8158772 | biostudies-literature
Project description:The effect of the adenylate cyclase activator forskolin on bone resorption and cyclic AMP accumulation was studied in an organ-culture system by using calvarial bones from 6-7-day-old mice. Forskolin caused a rapid and fully reversible increase of cyclic AMP, which was maximal after 20-30 min. The phosphodiesterase inhibitor rolipram (30 mumol/l), enhanced the cyclic AMP response to forskolin (50 mumol/l) from a net cyclic AMP response of 1234 +/- 154 pmol/bone to 2854 +/- 193 pmol/bone (mean +/- S.E.M., n = 4). The cyclic AMP level in bones treated with forskolin (30 mumol/l) was significantly increased after 24 h of culture. Forskolin, at and above 0.3 mumol/l, in the absence and the presence of rolipram (30 mumol/l), caused a dose-dependent cyclic AMP accumulation with an calculated EC50 (concentration producing half-maximal stimulation) value at 8.3 mumol/l. In 24 h cultures forskolin inhibited spontaneous and PTH (parathyroid hormone)-stimulated 45Ca release with calculated IC50 (concentration producing half-maximal inhibition) values at 1.6 and 0.6 mumol/l respectively. Forskolin significantly inhibited the release of 3H from [3H]proline-labelled bones stimulated by PTH (10 nmol/l). The inhibitory effect by forskolin on PTH-stimulated 45Ca release was significant already after 3 h of culture. In 24 h cultures forskolin (3 mumol/l) significantly inhibited 45Ca release also from bones stimulated by prostaglandin E2 (1 mumol/l) and 1 alpha-hydroxycholecalciferol (0.1 mumol/l). The inhibitory effect of forskolin on spontaneous and PTH-stimulated 45Ca release was transient. A dose-dependent stimulation of basal 45Ca release was seen in 120 h cultures, at and above 3 nmol of forskolin/l, with a calculated EC50 value at 16 nmol/l. The stimulatory effect of forskolin (1 mumol/l) could be inhibited by calcitonin (0.1 unit/ml), but was insensitive to indomethacin (1 mumol/l). Forskolin increased the release of 3H from [3H]proline-labelled bones cultured for 120 h and decreased the amount of hydroxyproline in bones after culture. Forskolin inhibited PTH-stimulated release of Ca2+, Pi, beta-glucuronidase and beta-N-acetylglucosaminidase in 24 h cultures. In 120 h cultures forskolin stimulated the basal release of minerals and lysosomal enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)
| S-EPMC1147447 | biostudies-other
Project description:Forskolin, a naturally occurring activator of adenylate cyclase, inhibits total and high-affinity cyclic AMP phosphodiesterase activity in soluble and particulate fractions of cultured LLC-PK1 renal epithelial cells. The naturally occurring forskolin analogue 1,9-dideoxyforskolin, which does not stimulate adenylate cyclase activity, is a more potent inhibitor of cyclic AMP phosphodiesterase activity than forskolin. To clarify the structural feature of the forskolin molecule responsible for inhibition of cyclic AMP phosphodiesterase activity, the effects of two agents which share structural identity with portions of the forskolin ring were tested. The steroid 5-pregnenolone, but not the hexose alpha-D-galactose, inhibited cyclic AMP phosphodiesterase activity in LLC-PK1 cells. Forskolin and 1,9-dideoxyforskolin both stimulate protein kinase C activity in LLC-PK1 cells. The effect of 1,9-dideoxyforskolin in stimulating LLC-PK1 protein kinase C activity can be attenuated by staurosporine. Both 5-pregnenolone and alpha-D-galactose also stimulate protein kinase C activity in LLC-PK1 cells. 5-Pregnenolone and the phorbol ester phorbol 12-myristate 13-acetate cause translocation of protein kinase C from a soluble to a particulate fraction, while both 1,9-dideoxyforskolin and alpha-D-galactose increase protein kinase C activity in both soluble and particulate fractions. Our results demonstrate that forskolin exerts diverse enzymic effects in cultured LLC-PK1 cells.
| S-EPMC1151541 | biostudies-other