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:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In amphibians and mammals, luminal H+ secretion by the stomach requires Cl-. It is widely accepted that a basolateral Cl-/HCO3- exchanger in the acid-secreting oxyntic cell restores the Cl- deficit resulting from apical HCl secretion. In this study, we used reverse transcriptase-polymerase chain reaction (RT-PCR) to generate a 1,200-bp fragment specific to a basolateral isoform of the Na(+)-K(+)-Cl- cotransporter in the gastric fundus of Necturus maculosus. By Northern analysis, we observed that gastric mucosa expresses greater than fivefold higher levels of mRNA encoding this cotransporter than any other tissue in the gastrointestinal tract. Feeding results in > 100% increases in mRNA levels in acid-secreting fundic mucosa but does not alter mRNA levels in the neighboring and non-acid-secreting antral mucosa or duodenum. Flux measurements using in vitro fundic mucosae indicate that acid secretion requires Na+ in the nutrient (i.e., serosal side) perfusate, is modulated by changes in nutrient K+ levels, and is inhibited by nutrient solutions containing 50 microM bumetanide, a recognized blocker of Na(+)-K(+)-Cl- cotransport. These findings suggest that this basolateral transporter plays a dominant and previously unsuspected role in secretion of HCl across the apical membrane.
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PMID:Role of basolateral Na(+)-K(+)-Cl- cotransport in HCl secretion by amphibian gastric mucosa. 763 51

The cortical collecting duct of the kidney contains two types of intercalated cells that transport HCO3 in opposite directions. HCO3 reabsorption takes place in the alpha-type intercalated cells, which express a Cl/HCO3 exchanger on the basolateral membrane. This exchanger is the product of the anion exchanger 1 (AE1) or band 3 gene. HCO3 secretion occurs in the beta-intercalated cells, which have a Cl/HCO3 exchanger on the apical membrane. Based on studies in an immortalized cell line, recently it was proposed that the apical anion exchanger of beta-intercalated cells is also AE1 (van Adelsberg, J. S., Edwards, J. C., and Al-Awqati, Q. (1993) J. Biol. Chem. 268, 11283-11289). In the present study we reinvestigated this issue by determining the distribution of AE1 mRNA and protein in the two intercalated cell types using cells freshly isolated from the native epithelium. Using quantitative reverse transcriptase polymerase chain reaction, we found that alpha-intercalated cells, isolated from rabbit kidney by fluorescence-activated cell sorting, have high levels of AE1 mRNA, whereas beta-intercalated cells express very low levels. The ratio of AE1 mRNA levels in alpha- versus beta-intercalated cells averaged 10.1 +/- 2.6. In addition, metabolic acidosis increased the levels of AE1 mRNA by 3-5-fold in cortical collecting duct cells. This difference was confirmed by Northern blotting. Western blotting using an antibody against rabbit AE1 revealed a major immunoreactive product with a molecular weight of approximately 110 kDa in cortical collecting duct cells. Deglycosylation reduced the size of the immunoreactive product to approximately 90 kDa, which is compatible with the presence of a truncated form of AE1. Metabolic acidosis increased the intensity of the AE1 immunoreactive band. The level of AE1-immunoreactive protein was significantly higher in alpha-intercalated cells than in beta-intercalated cells. In aggregate, these data provide evidence for the differential expression of AE1 in HCO3-reabsorbing versus HCO3-secreting renal intercalated cells both at the mRNA and at the protein level. These results give no support to the concept that AE1 functions both as a basolateral and an apical anion exchanger in cortical collecting duct cells.
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PMID:Differential expression of AE1 in renal HCO3-secreting and -reabsorbing intercalated cells. 792 5

We have characterized the Na+/H+ exchanger (NHE) isoforms expressed in rat renal cortical tubule fragments. Amiloride sensitivity of the Na(+)-dependent intracellular pH (pHi) recovery in suspended tubules that had been acid loaded by an NH4+ prepulse was determined in nominally CO2/HCO3(-)-free solution, using the fluorescent pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. In the presence of 140 mM extracellular Na+, 800 microM amiloride inhibited the rate of Na(+)-dependent pHi recovery by only 65%, demonstrating the presence of a Na(+)-dependent amiloride-insensitive H+ extrusion system. This system was not affected by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid but was activated by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Lowering extracellular Na+ concentration permitted 300 microM amiloride to completely inhibit Na(+)-dependent pHi recovery. These results can be explained by the expression of a Na+/H+ exchange with the pharmacological properties of NHE4. Using reverse transcriptase-polymerase chain reaction, we found specific mRNA for NHE1, NHE2, NHE3, and NHE4 isoforms in the renal cortex. Immunohistochemical studies using polyclonal antibodies against rat NHE4 peptide demonstrated that NHE4 is heterogeneously expressed on basolateral membrane domains of cortical tubules. These results strongly suggest that amiloride-insensitive Na+/H+ exchange expressed in renal cortical tubule suspensions is mediated by NHE4.
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PMID:Evidence for an amiloride-insensitive Na+/H+ exchanger in rat renal cortical tubules. 931 28

The balance between the concentrations of free ionized Ca2+ and bicarbonate in pancreatic juice is of critical importance in preventing the formation of calcium carbonate stones. How the pancreas regulates the ionic composition and the level of Ca2+ saturation in an alkaline environment such as the pancreatic juice is not known. Because of the tight cause-effect relationship between Ca2+ concentration and lithogenicity, and because hypercalcemia is proposed as an etiologic factor for several pancreatic diseases, we have investigated whether pancreatic tissues express a Ca2+-sensing receptor (CaR) similar to that recently identified in parathyroid tissue. Using reverse transcriptase-polymerase chain reaction and immunofluorescence microscopy, we demonstrate the presence of a CaR-like molecule in rat pancreatic acinar cells, pancreatic ducts, and islets of Langerhans. Functional studies, in which intracellular free Ca2+ concentration was measured in isolated acinar cells and interlobular ducts, show that both cell types are responsive to the CaR agonist gadolinium (Gd3+) and to changes in extracellular Ca2+ concentration. We also assessed the effects of CaR stimulation on physiological HCO3- secretion from ducts by making measurements of intracellular pH. Luminal Gd3+ is a potent stimulus for HCO3- secretion, being equally as effective as raising intracellular cAMP with forskolin. These results suggest that the CaR in the exocrine pancreas monitors the Ca2+ concentration in the pancreatic juice, and might therefore be involved in regulating the level of Ca2+ in the lumen, both under basal conditions and during hormonal stimulation. The failure of this mechanism might lead to pancreatic stone formation and even to pancreatitis.
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PMID:Molecular and functional identification of a Ca2+ (polyvalent cation)-sensing receptor in rat pancreas. 1040 Jun 86

Although bicarbonate transport in corneal endothelium has been suggested to be coupled to Na+, the underlying molecular mechanism has not been clarified. In the present study we investigated whether a recently cloned Na(+)-HCO3- cotransporter (NBC-1) is responsible for this process, and, if so, whether the endothelium expresses a separate isoform or one of the other two isoforms that have recently been identified (kNBC-1 from kidney and pNBC-1 from pancreas). Using primers designed for specific and common regions we demonstrated by reverse transcriptase polymerase chain reaction (RT-PCR) that both kNBC-1 and pNBC-1 are expressed in cultured human corneal endothelial cells. In addition functional studies with a pH-sensitive fluorescence probe were performed. In the presence of HCO3-/CO2 a pH regulatory process was demonstrated which depends on the presence of Na+ and membrane potential, but is independent of Cl- and is inhibited by the disulfonic stilbene DIDS. These results support the presence of NBC-1 as the major bicarbonate transport system in corneal endothelium.
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PMID:Functional and molecular evidence for Na(+)-HCO3- cotransporter in human corneal endothelial cells. 1051 38

In mammalian germ cells, cAMP signaling is dependent on two forms of adenylyl cyclase, the conventional membrane-bound ACIII and a soluble form of adenylyl cyclase (sAC). Recent elucidation of the sAC sequence indicates that this enzyme is phylogenetically distinct from the membrane-bound AC, does not interact with G proteins, and its activity is regulated by bicarbonate ions. Here we have investigated the properties and regulation of this enzyme during spermatogenesis. Two different transcripts encoding a full-length and truncated sAC were identified by reverse transcriptase-polymerase chain reaction and RNase protection analysis. The truncated sAC transcript lacks exon 11 with a premature termination of the open reading frame after the catalytic domain. Reverse transcriptase-polymerase chain reaction with testis RNA from adult mouse and rat of different ages, as well as RNase protection, showed that both transcripts are absent at 11 days of age, appear between 20 and 30 days of age, and are retained in the adult testis. The presence of corresponding proteins in testis, germ cells, and spermatozoa was demonstrated by fast protein liquid chromatography and differential immunoprecipitation with full-length sAC-specific antibodies. Bicarbonate ions activated both sAC forms and increased cAMP levels in germ cells isolated from 25- and 50-day-old rats and adult rats in a concentration-dependent manner. These findings provide evidence that full-length and truncated sAC are generated by alternate splicing. Both forms are active in spermatids, and the bicarbonate present in the seminiferous tubule may be a signal that regulates cAMP levels in these cells.
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PMID:Identification and functional analysis of splice variants of the germ cell soluble adenylyl cyclase. 1142 34

We investigated the transport systems that can sustain Na+ and Cl- movements across bovine gall bladder epithelium, focusing on the Na+-H+ exchanger (NHE) family and chloride conductive pathways. Experiments conducted using the fluorescent probe acridine orange (AO) with brush-border membrane vesicles (BBMV) or vesicles obtained from the total epithelium (EMV) demonstrated the presence of a Na+-H+ exchange in both preparations. The use of specific inhibitors indicated the presence of an apical NHE3 exchanger and a NHE1 isoform which should reside in the basolateral membrane. Using reverse transcriptase (RT) PCR, we identified cDNA fragments corresponding to the NHE1, NHE3, Cl--HCO3- (AE2a) transporters and to the CFTR channel. Using the patch-clamp technique, we investigated Cl- conductances on cultured epithelial cells. We found a 5 pS Cl- channel with a voltage-independent open probability, insensitive to stilbenes (SITS), Zn2+ and cAMP. The results suggest that absorption and secretion coexist in calf gall bladder epithelium. A Na+-H+-Cl--HCO3- double exchange may, at least partially, sustain the absorptive function, and a Cl- apical conductive pathway may be involved in secretion. The conductance we observed does not seem to be cAMP-regulated, unlike other mammalian gall bladders.
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PMID:The presence of NHE1 and NHE3 Na+-H+ exchangers and an apical cAMP-independent Cl- channel indicate that both absorptive and secretory functions are present in calf gall bladder epithelium. 1157 84

Syntheses of [carbonyl-11C]2-(2-benzoylphenoxy)-N-phenylacetamide, a radiolabeled inhibitor of human immunodeficiency virus type 1 (HIV 1) reverse transcriptase, were achieved by applying palladium-mediated cross-coupling reactions with insertion of [11C]carbon monoxide. Our interest was focused for the present on a comparison of the Stille and Suzuki methods, using trimethylphenylstannane or phenylboronic acid as alternative coupling reagents, respectively. The Suzuki variant gave a much higher amount of [11C]CO radioactivity trapped in the reaction mixture, but a significant loss of product occurred due to adsorption phenomena on the potassium carbonate present in the heterogeneous reaction mixture. The labeled product was isolated in only 20% yield (based on trapped [11C]CO, not corrected for decay). According to Stille, the reaction provided a product that could be isolated more easily but it did not increase the final yield of the target compound due to a low trapping efficiency for [11C]CO. Both methods were performed in an overall synthesis time of 30min, starting from [11C]CO2, and gave a product with a specific radioactivity of at least 30GBq/micromol. The Stille method as well as the Suzuki reaction allowed the synthesis of a radiochemically pure product in aqueous acetonitrile.
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PMID:Syntheses of [carbonyl-11C]2-(2-benzoylphenoxy)-N-phenylacetamide from [11C]carbon monoxide by the Suzuki and the Stille reactions. 1243 42

Electrolyte transport by nasal epithelia has been suggested to be important for controlling the quantity and composition of the nasal fluid and may play an important role in the development of nasal polyps. One of a number of mechanisms involving translocation of Na+ and Cl- across cell membranes includes electroneutral processes, such as Na+/H+ exchange (NHE) and Cl-/HCO3- exchange (AE). The present study evaluated the presence of mRNAs for various members of the human NHE and AE gene families in human inferior turbinate mucosa and nasal polyp using reverse transcriptase polymerase chain reaction and in situ hybridization. The mRNA for NHE1 was detected in human turbinate mucosa and nasal polyp while the mRNAs for NHE2 and NHE3 could not be detected in any of the samples examined. Of the AE isoforms, AE2 mRNA was expressed in inferior turbinate mucosa but not in nasal polyp. In situ hybridization revealed that NHE1 mRNA in the turbinate mucosa and nasal polyp was localized in the epithelial layer and submucosal glands. AE2 mRNA was also expressed in the epithelial layer and submucosal glands of inferior turbinate mucosa. Taken together, these results indicate that the expression of AE2 mRNA is altered in nasal polyp compared with inferior turbinate mucosa, suggesting that the altered expression of these genes in nasal polyp may cause impaired electrolyte and water transport across the epithelial cells.
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PMID:Expression of mRNA transcripts of the Na+/H+ and Cl-/HCO3- exchanger isoforms in human nasal mucosa. 1254 7

Lung cells import iron across the plasma membrane as ferrous (Fe2+) ion by incompletely understood mechanisms. We tested the hypothesis that human bronchial epithelial (HBE) cells import non-transferrin-bound iron (NTBI) using superoxide-dependent ferri-reductase activity involving anion exchange protein 2 (AE2) and extracellular bicarbonate (HCO3-). HBE cells that constitutively express AE2 mRNA by reverse transcriptase-polymerase chain reaction and AE2 protein by Western analysis avidly transported NTBI after exposure to either Fe2+ or Fe3+, but reduction of Fe3+ to Fe2+ was first required. The ability of HBE cells to reduce Fe3+ and transport Fe2+ was inhibited by active extracellular superoxide dismutase (SOD). Similarly, HBE cells that overexpress Cu,Zn SOD after adenoviral infection with AdSOD1 showed diminished iron uptake. The role of AE2 in iron uptake was indicated by three lines of evidence: (i) lack of both iron reduction and iron transport in bicarbonate-free buffer at controlled pH, (ii) failure of HBE cells treated with stilbene AE inhibitors to reduce Fe3+ or transport iron, and (iii) inhibition of iron uptake in HBE cells by inhibition of AE2 protein expression with antisense oligonucleotides. We thus disclose a novel ferri-reductase mechanism of NTBI uptake by human lung cells that employs superoxide exchange for HCO3- by AE2 protein in the plasma membrane.
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PMID:Superoxide-dependent iron uptake: a new role for anion exchange protein 2. 1279 78


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