Forskolin as an activator of cyclic AMP accumulation and secretion in blowfly salivary glands.
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ABSTRACT:
SUBMITTER: Litosch I
PROVIDER: S-EPMC1158326 | biostudies-other | 1982 Apr
REPOSITORIES: biostudies-other
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Project description:The salivary glands of adult blowflies (Calliphora erythrocephala) contain enzymes that hydrolyse phosphatidylinositol, predominantly by a Ca2+-independent deacylation, though a Ca2+-dependent phosphodiesterase (phospholipase C) activity could be detected. The deacylating enzymes could also hydrolyse phosphatidylcholine and phosphatidylethanolamine, and were secreted in the saliva. Homogenization of salivary glands prelabelled with [3H]inositol resulted in a rapid deacylation of the endogenous 3H-labelled phosphatidylinositol; this hydrolysis was unaffected by addition of 5-hydroxytryptamine to the homogenate.
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Project description:Exposure of isolated perfused rat livers to either 100 microM-forskolin, a potent activator of adenylate cyclase, or to 0.5 mM-concentrations of the cAMP analogues chlorophenylthio cAMP (CPTcAMP), dibutyryl cAMP (dbcAMP) and 8-bromo cAMP (8BrcAMP), to provoke increases in intracellular concentrations of cAMP, resulted in marked changes in bile volume and composition. Bile flow reached a peak after 10 min, before declining towards control levels, and an increase in several secretory parameters was also observed at this time. At 20 min, a substantial decrease in the output of both phospholipid and cholesterol was evident, and this suppression of secretion was maintained throughout the remainder of the experiment. The order of effectiveness of the cAMP-elevating agents at decreasing biliary lipid output was CPTcAMP greater than forskolin greater than dbcAMP greater than 8BrcAMP. Biliary output of bile acids was essentially unaltered compared with controls; similarly, no decrease in the secretion of protein and triacylglycerols into the perfusion medium was observed. This suggests that the elevation of intracellular levels of cAMP may cause a selective inhibition of biliary lipid output rather than a more general inhibition of hepatic secretion.
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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.
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Project description:Drug-induced taste disturbance is a common adverse drug reaction often triggered by drug secretion into saliva. Very little is known regarding the molecular mechanisms underlying salivary gland transport of xenobiotics, and most drugs are assumed to enter saliva by passive diffusion. In this study, we demonstrate that salivary glands selectively and highly express OCT3 (organic cation transporter-3), a polyspecific drug transporter in the solute carrier 22 family. OCT3 protein is localized at both basolateral (blood-facing) and apical (saliva-facing) membranes of salivary gland acinar cells, suggesting a dual role of this transporter in mediating both epithelial uptake and efflux of organic cations in the secretory cells of salivary glands. Metformin, a widely used anti-diabetic drug known to induce taste disturbance, is transported by OCT3/Oct3 in vitro. In vivo, metformin was actively transported with a high level of accumulation in the salivary glands of wild-type mice. In contrast, active uptake and accumulation of metformin in salivary glands were abolished in Oct3(-/-) mice. Oct3(-/-) mice also showed altered metformin pharmacokinetics and reduced drug exposure in the heart. These results demonstrate that OCT3 is responsible for metformin accumulation and secretion in salivary glands. Our study uncovered a novel carrier-mediated pathway for drug entry into saliva and sheds new light on the molecular mechanisms underlying drug-induced taste disorders.
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Project description:Salivary glands (SG) arise from ectodermal tissue between 6 and 12th weeks of intrauterine life through finely regulated epithelial-mesenchymal interactions. For this reason, different types of structural congenital anomalies, ranging from asymptomatic anatomical variants to alterations associated with syndromic conditions, have been described. Notable glandular parenchyma anomalies are the SG aplasia and the ectopic SG tissue. Major SG aplasia is a developmental anomaly, leading to variable degrees of xerostomia, and oral dryness. Ectopic SG tissue can occur as accessory gland tissue, salivary tissue associated with branchial cleft anomalies, or true heterotopic SG tissue. Among salivary ducts anomalies, congenital atresia is a rare developmental anomaly due to duct canalization failure in oral cavity, lead to salivary retention posterior to the imperforate orifice. Accessory ducts originate from the invagination of the developing duct in two places or from the premature ventral branching of the main duct. Heterotopic ducts may arise from glandular bud positioned in an anomalous site lateral to the stomodeum or from the failure of the intraoral groove development, hindering their proximal canalization. These anomalies require multidisciplinary approach to diagnosis and treatment. While ectopic or accessory SG tissue/ducts often do not require any treatment, patients with SG aplasia could benefit from strategies for restoring SG function. This article attempts to review the literature on SG parenchyma and ducts anomalies in head and neck region providing clinicians with a comprehensive range of clinical phenotypes and possible future applications of bioengineered therapies for next-generation of regenerative medicine.
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Project description:Salivary-gland homogenates contain 5-hydroxytryptamine-stimulated adenylate cyclase. Half-maximal stimulation was obtained with 0.1 microM-5-hydroxytryptamine in the presence of added guanine nucleotides. Gramine antagonized the stimulation of cyclase caused by 5-hydroxytryptamine. In the presence of hormone, guanosine 5'-[gamma-thio]triphosphate produced a marked activation of adenylate cyclase activity. Stimulation of adenylate cyclase by forskolin or fluoride did not require the addition of guanine nucleotides or hormone. In the presence of EGTA, Ca2+ produced a biphasic activation of cyclase activity. Ca2+ at 1-100 microM increased activity, whereas 2000 microM-Ca2+ inhibited cyclase activity. The neuroleptic drugs trifluoperazine and chlorpromazine non-specifically inhibited adenylate cyclase activity even in the absence of Ca2+. The cyclic AMP phosphodiesterase activity in homogenates was not affected by Ca2+ or exogenous calmodulin. This enzyme was also inhibited by trifluoperazine in the absence of Ca2+. These results indicate that Ca2+ elevates adenylate cyclase activity, but had no effect on cyclic AMP phosphodiesterase of salivary-gland homogenates.
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Project description:Blowfly salivary glands, previously exposed to 10 microM-5-hydroxytryptamine for 30 min, demonstrated a rapid compensatory resynthesis of [3H]inositol-labelled phosphatidylinositol 4,5-bisphosphate when allowed to recover in medium containing 3-5 microM-inositol. Phosphatidylinositol 4,5-bisphosphate comprised 70% of the total [3H]-phosphoinositide, and there was a corresponding decrease in the formation of [3H]-phosphatidylinositol. Subsequent addition of 5-hydroxytryptamine produced an equivalent breakdown of the newly synthesized phosphoinositides but little 45Ca2+ gating. Increasing the inositol concentration in the medium to 300 microM produced a 14-fold stimulation of phosphatidylinositol synthesis but only a 5-fold increase in phosphatidylinositol 4,5-bisphosphate synthesis. Increasing the inositol concentration in the medium from 3 microM to 300 microM resulted in a progressively greater recovery of the 45Ca2+-gating response. At 300 microM-inositol there was an 85% recovery of 45Ca2+-gating response. These results indicate that conversion of phosphatidylinositol into phosphatidylinositol 4,5-bisphosphate occurs in blowfly salivary glands and is secondary to an initial breakdown of the phosphoinositides. Recovery of Ca2+ gating is dependent on the restoration of both phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate to appropriate concentrations.
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Project description:Summary Salivary gland cells, which secrete water in response to neuronal stimulation, are closely connected to other neurons. Transcriptomic studies show that salivary glands also express some proteins responsible for neuronal function. However, the physiological functions of these common neuro-exocrine factors in salivary glands are largely unknown. Here, we studied the function of Neuronal growth regulator 1 (NEGR1) in the salivary gland cells. NEGR1 was also expressed in mouse and human salivary glands. The structure of salivary glands of Negr1 knockout (KO) mice was normal. Negr1 KO mice showed tempered carbachol- or thapsigargin-induced intracellular Ca2+ increases and store-operated Ca2+ entry. Of interest, the activity of the large-conductance Ca2+-activated K+ channel (BK channel) was increased, whereas Ca2+-activated Cl− channel ANO1 channel activity was not altered in Negr1 KO mice. Pilocarpine- and carbachol-induced salivation was decreased in Negr1 KO mice. These results suggest that NEGR1 influence salivary secretion though the muscarinic Ca2+ signaling. Graphical abstract Highlights • Negr1 knockout mice show reduced salivation• NEGR1 is essential for muscarinic Ca2+ signaling in the submandibular gland• The study provides an explanation for dry mouth among depression patients Biological sciences; Molecular biology; Molecular neuroscience; Neuroscience
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Project description:Secretion in blowfly (Calliphora vicina) salivary glands is stimulated by the biogenic amine serotonin (5-hydroxytryptamine, 5-HT), which activates both inositol 1,4,5-trisphosphate (InsP(3))/Ca(2+) and cyclic adenosine 3',5'-monophosphate (cAMP) signalling pathways in the secretory cells. In order to characterize the signal-inducing 5-HT receptors, we cloned two cDNAs (Cv5-ht2?, Cv5-ht7) that share high similarity with mammalian 5-HT(2) and 5-HT(7) receptor genes, respectively. RT-PCR demonstrated that both receptors are expressed in the salivary glands and brain. Stimulation of Cv5-ht2?-transfected mammalian cells with 5-HT elevates cytosolic [Ca(2+)] in a dose-dependent manner (EC(50) = 24 nM). In Cv5-ht7-transfected cells, 5-HT produces a dose-dependent increase in [cAMP](i) (EC(50) = 4 nM). We studied the pharmacological profile for both receptors. Substances that appear to act as specific ligands of either Cv5-HT(2?) or Cv5-HT(7) in the heterologous expression system were also tested in intact blowfly salivary gland preparations. We observed that 5-methoxytryptamine (100 nM) activates only the Cv5-HT(2?) receptor, 5-carboxamidotryptamine (300 nM) activates only the Cv5-HT(7) receptor, and clozapine (1 µM) antagonizes the effects of 5-HT via Cv5-HT(7) in blowfly salivary glands, providing means for the selective activation of each of the two 5-HT receptor subtypes. This study represents the first comprehensive molecular and pharmacological characterization of two 5-HT receptors in the blowfly and permits the analysis of the physiological role of these receptors, even when co-expressed in cells, and of the modes of interaction between the Ca(2+)- and cAMP-signalling cascades.
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Project description:Cyclic di-AMP is an essential second messenger in many Gram-positive bacteria, including the model organism Bacillus subtilis. Here, we analyzed the transcriptome of a strain accumulating c-di-AMP in vivo. Our results demonstrate that accumulation of c-di-AMP affects the expression of several hundred genes, among them many mother-cell specific sporulation genes. Additionally, the two major biofilm operons, epsA-O and tapA-sipW-tasA, are affected. High levels of c-di-AMP abolish transcription of genes responsible for biofilm formation which in turn leads to a defect in complex colony formation in B. subtilis.
2016-05-25 | GSE79716 | GEO