Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:4.6.1.1 (adenylate cyclase)
19,190 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To detect heterologous expression of receptors coupled via G proteins to the stimulation of adenylate cyclase in Xenopus laevis oocytes, the receptor of interest is coexpressed with the cystic fibrosis transmembrane conductance regulator (CFTR)--a cAMP-dependent Cl- channel. The binding of an agonist to the expressed receptor stimulates adenylate cyclase resulting in intracellular cAMP elevation, which in turn activates the CFTR. The CFTR-mediated Cl- current response is then measured using the standard two-electrode voltage-clamp technique. This method has allowed us to detect functional expression in oocytes of the human EP2 and IP prostanoid receptors. This method should prove valuable for expression and identification of putative G protein-coupled receptors signaling through stimulation of adenylate cyclase, for structure/function studies, and for analysis of receptor antagonists and agonists.
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PMID:Detection of adenylate cyclase-coupled receptors in Xenopus oocytes by coexpression with cystic fibrosis transmembrane conductance regulator. 754 56

Chloride channel activity of cystic fibrosis transmembrane conductance regulator (CFTR) requires activation of protein kinase A (PKA) by 3'-5'-cyclic adenosine monophosphate (cAMP). The level of cAMP is controlled by the balance between cAMP synthesis and hydrolysis by adenylate cyclase and phosphodiesterases (PDEs), respectively. CFTR channel activity appears to be most sensitive to the activity of type III cyclic nucleotide PDEs in Calu-3 and 16HBE cells, both derived from airway epithelium and expressing wild-type CFTR. Type III PDEs can be identified by their sensitivity to specific inhibitors such as milrinone and amrinone. In Calu-3 cells, specific inhibition of type III PDEs increased chloride efflux up to 13.7-fold, whereas neither rolipram nor Ro20-1724 (type IV PDE inhibitors) nor 3-isobutyl-1-methylxanthine (IBMX, a nonspecific PDE inhibitor) elicited significant increases. None of these compounds had an appreciable effect on total cellular cAMP levels, yet the effects of milrinone and amrinone on chloride efflux were blocked by treatment of cells with Rp-cAMPS, a cAMP analog that inhibits PKA at the site of cAMP binding. Similarly, H-8, an inhibitor of PKA, reduced milrinone-stimulated chloride efflux, indicating that efflux is mediated through the cAMP/PKA pathway. Whole-cell patch clamp analysis revealed that milrinone generated chloride conductances with properties consistent with those of CFTR. Milrinone elicited chloride currents in a dose-dependent manner and induced CFTR activity in the absence of adenylate cyclase agonists. These data suggest that type III PDEs are specifically involved in CFTR activation in airway epithelial cells and that PDE regulation of CFTR may involve subcellular compartments of cAMP.
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PMID:CFTR-mediated chloride permeability is regulated by type III phosphodiesterases in airway epithelial cells. 757 3

We have sought to determine the mechanisms driving fluid secretion by the cystic epithelium in autosomal dominant polycystic kidney disease (ADPKD). We have performed in vitro experiments on intact cysts dissected from discarded ADPKD kidneys, on monolayers of cells cultured from the cystic epithelium and on microcysts clonally derived from single cultured cells. These preparations absorb fluid in the control state but secrete fluid in response to native cyst fluid, to adenylate cyclase agonists and to permeant analogues of cAMP. Measurements of short-circuit current and transepithelial voltage in the monolayers indicate that anion secretion must drive the fluid secretion. Fluid secretion by the intact cysts was inhibited by basolateral application of ouabain but not by apical application. The effect of ouabain on fluid secretion and short-circuit current in the monolayers followed the same pattern. Thus the functional Na,K-ATPase enzyme complex is located only in the basolateral membrane of the cystic cells and serves to maintain the transmembrane chemical and electrical gradients that drive anion secretion by other transport mechanisms. Fluid secretion and short-circuit current in the cultured monolayers was inhibited by the basolateral application of the Na-K-2Cl cotransporter inhibitors, bumetanide and furosemide, and by apical application of the chloride channel blocker, diphenylamine-2-carboxylate (DPC). These data suggest that chloride is the anion that is actively secreted. Preliminary experiments utilizing the monolayers and the microcysts and measuring cell chloride concentration and chloride efflux across the apical membrane support this conclusion. Other preliminary data indicate that the cystic fibrosis transmembrane conductance regulator is present in the apical membrane. Thus active chloride transport generates fluid secretion by the cystic epithelium.
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PMID:Mechanisms of fluid secretion by polycystic epithelia. 874 60

Many heterologously expressed mutants of the cystic fibrosis transmembrane conductance regulator (CFTR) exhibit residual chloride channel activity that can be stimulated by agonists of the adenylate cyclase/protein kinase A pathway. Because of clinical implications for cystic fibrosis of activating mutants in vivo, we are investigating whether deltaF508, the most common disease-associated CFTR mutation, can be activated in airway epithelial cells. We have found that, 36Cl- efflux can be stimulated 19-61% above baseline by beta-adrenoreceptor agonists and cGI-phosphodiesterase inhibitors in transformed nasal polyp (CF-T43) cells homozygous for the deltaF508 mutation. The increase in 36Cl- permeability is diminished by protein kinase A inhibitors and is not mediated by an increase in intracellular calcium concentrations. Preincubation of CF-T43 cells with CFTR anti-sense oligonucleotides prevented an increase in 36Cl- efflux in response to beta-agonist and phosphodiesterase inhibitor. Primary cells isolated from CF nasal polyps gave similar results. These data indicate that endogenous levels of deltaF508 protein can be stimulated to increase 36Cl- permeability in airway epithelial cells.
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PMID:Activation of endogenous deltaF508 cystic fibrosis transmembrane conductance regulator by phosphodiesterase inhibition. 875 64

Transepithelial fluid secretion promotes the progressive enlargement of cysts in autosomal dominant polycystic kidney disease (ADPKD). Recent indirect evidence indicated that active chloride transport may drive net fluid secretion in cultures of epithelia derived from ADPKD cysts. We now report that forskolin, which stimulates adenylate cyclase, increased the efflux rate constant for 36Cl in monolayers of ADPKD cells in vitro from 0.23 +/- 0.02 min-1 to 0.44 +/- 0.05 min-1 (N = 4) and that diphenylamine 2-carboxylate (DPC), which blocks chloride channels, eliminated the forskolin-stimulated chloride efflux from these cells. To establish whether the cAMP-regulated chloride transporter, cystic fibrosis transmembrane conductance regulator (CFTR), may potentially be involved in the chloride transport and fluid secretion of ADPKD epithelia, we examined CFTR mRNA and protein in these cultures. Northern blot hybridization using a human (h) CFTR cDNA probe demonstrated the presence of an approximately 6.5 kb transcript in total RNA from polarized cultures of ADPKD, normal human kidney cortex (HKC), and T84 cells. Utilizing several antibodies to hCFTR, immunocytochemistry and confocal fluorescence microscopy localized an immunoreactive protein primarily in the apical region of forskolin-stimulated ADPKD cells grown on permeable supports. This immunoreactivity could be eliminated by preincubation of antibody with immunizing peptide. To determine the effect of CFTR abundance on the magnitude of net fluid secretion, polarized ADPKD cultures were treated with deoxyoligonucleotides that were either complementary (antisense), homologous (sense), or partially complementary (misantisense) to a sequence near the translation initiation site in hCFTR mRNA. Treatment with 5.0 microM antisense oligonucleotide resulted in a 73% reduction in forskolin-stimulated fluid secretion and a comparable reduction in the abundance of CFTR as detected by immunocytochemistry. By contrast, treatment with 5.0 microM sense oligonucleotide reduced fluid secretion by only 34% and had less of an effect on CFTR abundance, while the effects of 5.0 microM misantisense oligonucleotide on both fluid secretion and CFTR abundance were insignificant. On the basis of these results we suggest that CFTR is a major mediator of forskolin-stimulated chloride and fluid secretion by epithelial cells of human polycystic kidneys in vitro.
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PMID:The cystic fibrosis transmembrane conductance regulator mediates transepithelial fluid secretion by human autosomal dominant polycystic kidney disease epithelium in vitro. 880 90

Pancreatic duct cell lines have been isolated from a number of animal and human tumors, but none appear to express ion transport properties expected for differentiated pancreatic duct epithelial cells. We sought to generate an immortalized ductal cell line from well-differentiated primary cultures of bovine pancreatic duct epithelium. Epithelial cells from the main duct of the bovine pancreas were isolated and immortalized by transfection with a DNA construct encoding simian virus 40 large T antigen. A single clone (BPD1) survived negative selection and was maintained in culture for > 100 passages over 2 yr. The cells grow readily in culture as monolayers and express several properties characteristic of differentiated pancreatic ductal epithelium. The cells do not appear to form a functional tight junction complex, since the transepithelial resistance of the monolayer cultures grown on a permeable support is < 10 omega.cm2. Northern blot analysis revealed that the cells continue to express simian virus 40 large T antigen and contain significant levels of mRNA for proteins thought to be important in transepithelial bicarbonate secretion [carbonic anhydrase II, Cl-/HCO3- exchanger, Na+/H+ exchanger, and cystic fibrosis transmembrane conductance regulator (CFTR)]. In vivo pancreatic ductal secretion is stimulated by the peptide hormone secretin. The secretin receptor is expressed and functionally coupled to adenylate cyclase in the immortalized cells, since secretin caused a dose-dependent accumulation of adenosine 3'5'-cyclic monophosphate (cAMP; approximately 20-fold increase over basal levels) with a mean effective concentration of 15 nM. Elevation of intracellular cAMP by exposure of the cells to forskolin (10 microM) or secretin (0.1 microM) increase plasma membrane Cl- permeability, most likely mediated by activation of CFTR. The results of these studies demonstrate that the pancreatic duct cell line (BPD1) retains several properties exhibited by the secretory epithelial cells that line the pancreatic ductal tree. This cell line should prove useful for studies of expression, function, and regulation of pancreatic duct cell proteins.
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PMID:Immortalization of bovine pancreatic duct epithelial cells. 892 98

We have described a preparation of Necturus maculosus gallbladder (NGB) epithelium yielding isolated cells that retain structural and functional polarity ("figure-eight" cells). These cells have a normal membrane voltage and remain polarized for several hours after isolation. Apical and basolateral membrane domains are differentially labeled with hydrophobic fluorescent dyes; freeze-fracture electron microscopy reveals two distinct membrane domains separated by tight junctions; ZO-1, Na+/H+ exchanger (NHE3), and Na(+)-K(+)-ATPase are present in the junctional, apical, and basolateral region, respectively; and cell-attached patch-clamp experiments reveal different K+ currents in the two membrane domains [R. J. Torres, G. A. Altenberg, J. A. Copello, G. Zampighi, and L. Reuss, Am. J. Physiol. 270 (Cell Physiol. 39): C1864-C1874, 1996]. Here, we show that NGB epithelial cells express a protein cross-reactive with an antibody against human cystic fibrosis transmembrane conductance regulator (CFTR). In figure-eight cells, immunoreactivity was restricted to the apical membrane domain. Using intracellular microelectrodes and a novel method of regional superfusion, we found that control cells have high K+ conductances in both membranes and a small basolateral Cl- conductance, similar to findings in the epithelium. Activation of adenylate cyclase with forskolin elicited a large apical membrane Cl- conductance and membrane depolarization. Whole cell patch-clamp studies yielded a forskolin-activated linear Cl- current, with high Cl-/aspartate selectivity. In conclusion, 1) figure-eight cells maintain the conductive membrane properties present in the epithelium, including polarized expression of adenosine 3',5'-cyclic monophosphate (cAMP)-activated Cl- channels, and 2) the cAMP-activated Cl- conductance is underlied by a CFTR homologue.
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PMID:Polarized expression of cAMP-activated chloride channels in isolated epithelial cells. 894 41

C-type natriuretic peptide (CNP), a hormone which stimulates particulate guanylate cyclase activity, was studied for its ability to stimulate chloride permeability through the cystic fibrosis transmembrane conductance regulator (CFTR) in airway epithelial cells. Two cell lines, Calu-3 and CF-T43, were used as models of normal and cystic fibrosis (CF) airway epithelial cells, respectively. Calu-3 cells, derived from a lung carcinoma, express relatively high levels of wild-type CFTR. CF-T43 is a transformed line derived from a nasal polyp and expresses the mutant CFTR, deltaF508. Calu-3 cells exposed to the nucleotide guanosine-3',5'-monophosphate (cGMP) analogue 8-Br-cGMP exhibit increased 36Cl- efflux, demonstrating that cGMP can mediate changes in chloride permeability. CNP induces a bumetanide-sensitive short circuit current across Calu-3 monolayers. Whole-cell currents stimulated by CNP display linear current-voltage relationships and have inhibitor pharmacology and ion selectivity consistent with CFTR channel activity. Sodium nitroprusside (SNP), an activator of soluble guanylate cyclase, and CNP both increase cGMP levels and short circuit current in Calu-3 cells. In contrast, exposure of CF-T43 cells to CNP resulted in an increased 36Cl- efflux rate only when combined with the adenylate cyclase agonist isoproterenol and the response was sensitive to kinase inhibitors. CF-T43 cells exposed to isoproterenol and SNP showed no increase in chloride efflux. Together, these data indicate that CNP can activate wild-type and mutant CFTR through a cAMP-dependent protein kinase pathway and that the sensitivity of Calu-3 cells for this stimulation is greater than that of the CF-T43 cells.
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PMID:C-type natriuretic peptide increases chloride permeability in normal and cystic fibrosis airway cells. 911 58

Inhibitors of guanosine 3',5'-cyclic monophosphate (cGMP)-inhibited phosphodiesterases stimulate Cl- transport across the nasal epithelia of cystic fibrosis mice carrying the delta F508 mutation [cystic fibrosis transmembrane conductance regulator (CFTR) (delta F/delta F)], suggesting a role for cGMP in regulation of epithelial ion transport. Here we show that activation of membrane-bound guanylate cyclases by C-type natriuretic peptide (CNP) stimulates hyperpolarization of nasal epithelium in both wild-type and delta F508 CFTR mice in vivo but not in nasal epithelium of mice lacking CFTR [CFTR(-/-)]. With the use of a nasal transepithelial potential difference (TEPD) assay, CNP was found to hyperpolarize lumen negative TEPD by 6.1 +/- 0.6 mV in mice carrying wild-type CFTR. This value is consistent with that obtained with 8-bromoguanosine 3',5'-cyclic monophosphate (6.2 +/- 0.9 mV). A combination of the adenylate cyclase agonist forskolin and CNP demonstrated a synergistic ability to induce Cl- secretion across the nasal epithelium of CFTR(delta F/delta F) mice. No effect on TEPD was seen with this combination when used on CFTR(-/-) mice, implying that the CNP-induced change in TEPD in CFTR(delta F/delta F) mice is CFTR-dependent.
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PMID:In vivo activation of CFTR-dependent chloride transport in murine airway epithelium by CNP. 937 36

The effects of genistein, a protein tyrosine kinase inhibitor, on the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel were studied in guinea pig ventricular myocytes and in NIH3T3 mouse fibroblasts stably transfected with CFTR cDNA by the whole-cell patch-clamp technique. Genistein did not activate whole-cell Cl- currents when applied to the intracellular (pipette) solution. In contrast, when applied to the extracellular solution, genistein alone promptly activated the Cl- current in a fully reversible manner. Also, extracellular genistein reversibly potentiated the forskolin-activated Cl- current. However, both basal and forskolin-activated Cl- currents were not affected by other protein tyrosine kinase inhibitors, including herbimycin A, lavendustin A, tyrphostin 21, tyrphostin 47, and tyrphostin 51. A nonspecific inhibitor of protein phosphatases, orthovanadate, had no effect on the genistein-induced activation of CFTR. Pretreatment with a protein kinase inhibitor, either H-89 or H-7, or with an adenylate cyclase inhibitor, SQ 22536, also had no effect on the genistein-induced response. Thus, it is concluded that genistein alone activates CFTR by a protein tyrosine kinase-independent and protein phosphatase-independent mechanism from the extracellular side, but not from the intracellular side.
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PMID:Tyrosine kinase-independent extracellular action of genistein on the CFTR Cl- channel in guinea pig ventricular myocytes and CFTR-transfected mouse fibroblasts. 985 48


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