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:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Neutral endopeptidase (NEP; enkephalinase, EC 3.4.24.11) is a cell membrane associated zinc metalloprotease, which cleaves peptides like atrial natriuretic peptide (ANP) on the amino-side of hydrophobic amino acids. Although NEP is mainly located in reabsorptive epithelia (kidney proximal tubule), it is also present in non-epithelial cells like neuronal cells. As the renal NEP cannot account for the entire ANP metabolism, other locations were postulated. The present experiments show its expression in endothelial cells (EC) from arterial (bovine pulmonary, porcine and human aorta) and venous (human umbilical, rabbit ear marginal) origins. Three different methods were used to demonstrate the presence of the protein and its mRNA: 1) NEP enzymatic activity was estimated using both a synthetic ([D-Ala2, Leu5] enkephalin) and a natural substrate (bradykinin). Using the synthetic substrate, the enzymatic activity in EC was completely blocked by thiorphan, a specific NEP inhibitor with an IC50 value in the nM range. In contrast, captopril, bestatin, GEMSA, inhibitors of angiotensin-converting enzyme, aminopeptidases and carboxypeptidases, respectively, were 10,000 times less active, revealing an inhibition profile similar to that of the purified enzyme. Bradykinin, a natural substrate of NEP, was in part metabolized by NEP, in presence of captopril, since 50% of the formation of the major metabolite bradykinin 1-7 was inhibited by thiorphan. 2) Immunoreactive NEP was detected on the plasma membrane of rabbit EC using a monoclonal antibody directed against the homologous renal enzyme. 3) NEP mRNA was detected by Northern blot analysis on rabbit EC as a major transcript of 3.9 kb. Reverse transcriptase PCR amplification showed the presence of a specific transcript in all EC tested. Therefore, endothelial NEP could play an important role in the inactivation of ANP, bradykinin and endothelins by its localization facing the circulating vasoactive peptides.
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PMID:[Identification and characterization of neutral endopeptidase in endothelial cells of arterial or venous origin]. 133 90

Neutral endopeptidase (NEP; enkephalinase, EC 3.4.24.11) is a cell membrane-associated zinc metalloprotease, which cleaves peptides like atrial natriuretic peptide (ANP) on the amino side of hydrophobic amino acids. Although NEP is mainly located in reabsorptive epithelia (kidney proximal tubule), it is also present in non-epithelial cells such as neuronal cells. As the renal NEP cannot account for the entire ANP metabolism, other locations were postulated. The present experiments show its expression in endothelial cells (EC) from arterial (bovine pulmonary, porcine, and human aorta) and venous (human umbilical, rabbit ear marginal) origins. Three different methods were used to demonstrate the presence of the protein and its mRNA. 1) NEP enzymatic activity was estimated using both a synthetic ([D-Ala2,Leu5]enkephalin) and a natural substrate (bradykinin). Using the synthetic substrate, the enzymatic activity in EC was completely blocked by thiorphan, a specific NEP inhibitor with an IC50 value in the nanomolar range. In contrast, captopril, bestatin, [2-guanidinoethylmercapto]succinic acid, inhibitors of angiotensin-converting enzyme, aminopeptidases, and carboxypeptidases, respectively, were 10,000 times less active, revealing an inhibition profile similar to that of the purified enzyme. Bradykinin, a natural substrate of NEP, was in part metabolized by NEP, in the presence of captopril, since 50% of the formation of the major metabolite bradykinin 1-7 was inhibited by thiorphan. 2) Immunoreactive NEP was detected on the plasma membrane of rabbit EC using a monoclonal antibody directed against the homologous renal enzyme. 3) NEP mRNA was detected by Northern blot analysis of rabbit EC as a major transcript of 3.9 kilobases. Reverse transcriptase polymerase chain reaction amplification showed the presence of a specific transcript in all EC tested. Therefore, endothelial NEP may play an important role in the inactivation of ANP, bradykinin, and endothelins by its localization facing the circulating vasoactive peptides.
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PMID:Identification and characterization of neutral endopeptidase in endothelial cells from venous or arterial origins. 162 99

Rat kidney proximal tubule epithelial cells (RPTE) in primary culture express acidic fibroblast growth factor 1 (FGF-1). Transformation of RPTE by SV40 (SV-RPTE) suppressed FGF-1 expression but activated secretion of FGF-like factor(s). SV-RPTE conditioned medium contained growth-promoting activity for SV-RPTE and human umbilical vein endothelial cells, indicating that both autocrine and angiogenic factors were secreted. Reverse transcriptase-polymerase chain reaction and Northern analysis for various FGFs showed that only FGF-3, also known as int-2, mRNA was expressed in SV-RPTE. In addition, expression of mRNA for the heparin-binding angiogenic factor vascular endothelial growth factor (VEGF) increased dramatically in SV-RPTE. Physical characterization of the activity in the SV-RPTE conditioned medium suggested that FGF-3 and VEGF contributed the autocrine and angiogenic activities, respectively. We also investigated FGF-3 and VEGF secretion in temperature-sensitive (ts) SV40-transformed RPTE. tsSV-RPTE had transformed properties resembling those of SV-RPTE only at the permissive temperature (33 degrees C), e.g., increased growth potential and anchorage-independent growth. FGF-1 was expressed only at the nonpermissive temperature. VEGF mRNA levels and secretion of the human umbilical vein endothelial cell growth-promoting activity were reduced by switching tsSV-RPTE cells from 33 degrees to 39 degrees C. However, FGF-3 mRNA levels were not affected significantly by the temperature switch suggesting that activation of VEGF and FGF-3 occurs through different mechanisms. These results indicate that FGF-1 expression in RPTE is suppressed by SV40 transformation.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Stable and temperature-sensitive transformation of rat kidney epithelial cells suppresses expression of acidic fibroblast growth factor 1 but activates secretion of fibroblast growth factor 3 (int-2) and vascular endothelial growth factor. 751 42

Renal secretion of organic cations involves at least two distinct transporters, located in the basolateral and apical membranes of proximal tubule cells. Whereas the basolateral transporter has recently been cloned, sequence information about the apical type was not yet available. An organic cation transporter, OCT2p, was cloned from LLC-PK1 cells, a porcine cell line with properties of proximal tubular epithelial cells. OCT2p was heterologously expressed and characterized in human embryonic kidney 293 cells. OCT2p-mediated uptake of the prototypical organic cation [14C]tetraethylammonium ([14C]TEA) into 293 cells was saturable. There was a highly significant correlation between the Ki values for the inhibition of apical [14C]TEA uptake into LLC-PK1 cells and 293 cells transfected with OCT2p (r = 0.995; p < 0.001; n = 6). Although OCT2p is structurally related to OCT1r, the basolateral organic cation transporter from rat kidney, the transporters could be clearly discriminated pharmacologically with corticosterone, decynium22, and O-methylisoprenaline. The findings at hand suggest that OCT2 corresponds to the apical type of organic cation transporter. Reverse transcriptase-polymerase chain reaction indicates that mRNA of OCT1r is limited to non-neuronal tissue, whereas OCT2r, the OCT2p homologue from rat, was found in both the kidney and central nervous regions known to be rich in the monoamine transmitter dopamine.
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PMID:Primary structure and functional expression of the apical organic cation transporter from kidney epithelial LLC-PK1 cells. 909 81

While there is considerable evidence for phosphate (Pi) reabsorption in the distal tubule, Pi transport and its regulation have not been well characterized in this segment of the nephron. In the present study, we examined Na+-dependent Pi transport in immortalized mouse distal convoluted tubule (MDCT) cells. Pi uptake by MDCT cells is Na+-dependent and, under initial rate conditions, is inhibited by phosphonoformic acid (41 +/- 3% of control), a competitive inhibitor of Na+-Pi cotransport. The transport system has a high affinity for Pi (Km = 0.46 mM) and is stimulated by lowering the extracellular pH from 7.4 to 6.4 and inhibited by raising the pH from 7.4 to 8.4. Exposure to Pi-free medium for 21 h increased Na+-Pi cotransport from 2.1 to 5.5 nmol/mg of protein/5 minutes (p < 0.05) while parathyroid hormone, forskolin, and phorbol 12-myristate 13-acetate failed to alter Pi uptake in MDCT cells. Reverse transcriptase polymerase chain reaction of MDCT cell RNA provided evidence for the expression of the Npt1 but not the Npt2 Na+-Pi cotransporter gene. However, preincubation of MDCT cells with Npt1 antisense oligonucleotide led to only 20% inhibition of Na+-Pi cotransport, suggesting that other Na+-Pi cotransporters are operative in MDCT cells. Indeed, we showed, by ribonuclease protection assay, that MDCT cells express the ubiquitous cell surface receptors for gibbon ape leukemia virus (Glvr-1) and amphoteric murine retrovirus (Ram-1) that also function as Na+-Pi cotransporters. In summary, we demonstrate that the pH dependence and regulation of Na+-Pi cotransport in MDCT cells is distinct from that in the proximal tubule and suggest that different gene products mediate Na+-Pi cotransport in the proximal and distal segments of the nephron.
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PMID:Na+ -phosphate cotransport in mouse distal convoluted tubule cells: evidence for Glvr-1 and Ram-1 gene expression. 955 59

Delta-aminolevulinic acid (ALA) is the precursor of porphyrin synthesis and has been recently used in vitro and in clinical studies as an endogenous photosensitizer for photodynamic therapy in the treatment of various tumors. For this purpose, ALA is given topically, systemically, or orally. When administered by the oral route, it shows excellent intestinal absorption. ALA is also efficiently reabsorbed in the renal proximal tubule after glomerular filtration. However, the pathways and mechanisms for its transmembrane transport into epithelial cells of intestine and kidney are unknown. Here we demonstrate that ALA uses the intestinal and renal apical peptide transporters for entering into epithelial cells. Kinetics and characteristics of ALA transport were determined in Xenopus laevis ooyctes and Pichia pastoris yeast cells expressing either the cloned intestinal peptide transporter PEPT1 or the renal form PEPT2. By using radiolabeled ALA and electrophysiological techniques in these heterologous expression systems, we established that: (a) PEPT1 and PEPT2 translocate 3H-ALA by saturable and pH-dependent transport mechanisms, (b) that ALA and di-/tripeptides, but not GABA or related amino acids, compete at the same substrate-binding site of the carriers, and (c) that ALA transport is electrogenic in nature as a consequence of H+/ALA cotransport. Reverse transcriptase-PCR analysis performed with specific primers for PEPT1 and PEPT2 in rabbit tissues demonstrates that, in particular, the PEPT2 mRNA is expressed in a variety of other tissues including lung, brain, and mammary gland, which have been shown to accumulate ALA. This suggests that these tissues could take up the porphyrin precusor via expressed peptide transporters, providing the endogenous photosensitizers for efficient photodynamic therapy.
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PMID:Delta-aminolevulinic acid transport by intestinal and renal peptide transporters and its physiological and clinical implications. 963 10

The subcellular distribution of doxorubicin was evaluated in living non-fixed LLC-PK1 cells, which maintain the structural and functional characteristics of the kidney proximal tubule epithelium and also express P-glycoprotein. After 10 min incubation, doxorubicin fluorescence was detectable in the nucleus. The intensity of nuclear fluorescence progressively increased, reaching the maximum at the end of the first hour. Then, the nuclear signal started to decrease and, at 2 h, doxorubicin fluorescence disappeared almost completely from the cell nucleus. Cytoplasmic fluorescent vesicles first appeared in the perinuclear region after 10 min doxorubicin exposure and increased in number and size over a period of 2 h. From 2 to 5 h, fluorescent vesicles moved unidirectionally to the cell periphery. Disappearance of doxorubicin punctate fluorescence in LLC-PK1 cells treated with methylamine or monensin demonstrated that drug accumulation occurred inside acidic compartments. In addition, the cytoplasmic pattern of doxorubicin fluorescence was very similar to that observed upon exposure to the acidotropic tracer LysoSensor Blue. Involvement of P-glycoprotein in doxorubicin handling by LLC-PK1 cells was suggested by modified intracellular doxorubicin distribution after cell incubation with verapamil and vinblastine. Moreover, the fluorescent P-glycoprotein substrate Bodipy FL Verapamil was shown to accumulate in LLC-PK1 cells in a manner that is quite similar to that observed for doxorubicin. P-glycoprotein expression was evaluated by immunoblot using the JSB-1 and C219 monoclonal antibodies. Immunofluorescence analysis was performed using the JSB-1 monoclonal antibody. P-glycoprotein immuno-reactivity was found both on the plasma membrane and intracytoplasmically in a perinuclear position. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed that MDR1 gene was expressed. This study indicates that a rapid intracellular redistribution accompanies the process of doxorubicin uptake by LLC-PK1 cells. Although these cells are non-tumour cells derived from the normal epithelium of the proximal renal tubule, they display a model of doxorubicin redistribution which is characteristic of doxorubicin-resistant tumour cells.
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PMID:Kinetics of doxorubicin handling in the LLC-PK1 kidney epithelial cell line is mediated by both vesicle formation and P-glycoprotein drug transport. 1057 12

The properties and functions of gamma-aminobutyric acid (GABA(A)) receptors in the mammalian central nervous system are well studied. However, the presence and significance of GABA(A) receptors in nonneural tissue is less clear. The goal of this study was to examine the expression and localization of the GABA(A) receptor beta(2) and beta(3) subunits in the kidney. Reverse transcriptase products from RNA isolated from rat and rabbit kidney cortex and cerebellum and rabbit S(2) segments were amplified by use of PCR and GABA(A) beta(2) and beta(3) subunit-specific primers. Sequencing of the kidney PCR products revealed that the rat kidney cortex and rat neuronal GABA(A) receptor beta(2) subunit were identical in nucleotide composition. The rabbit kidney and rabbit neuronal GABA(A) receptor beta(2) subunit were 99% identical in nucleotide composition. Sequencing of the kidney PCR products revealed that the rat kidney cortex and rat neuronal GABA(A) receptor beta(3) subunits were 93% and 95% identical in nucleotide and amino acid composition, and rabbit kidney cortex and rabbit neuronal GABA(A) receptor beta(3) subunits were 95% and 98% identical in nucleotide and amino acid composition, respectively. PCR screening of a human kidney cDNA library and sequencing revealed that the human kidney cortex and neuronal beta(3) subunits were identical in nucleotide composition. Immunoblot analysis of rat kidney cortex and brain identified immunoreactive proteins in the 55 to 57 kD region, corresponding to the GABA(A) receptor beta(2) and beta(3) subunits. Immunohistochemistry revealed cytosolic and basolateral staining of the proximal convoluted and straight tubule. These results provide compelling evidence for the expression of the GABA(A) receptor beta(2) and beta(3) subunits in the kidney of multiple species and the localization of the beta(2)/beta(3) subunits to the renal proximal tubule.
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PMID:Identification of the gamma-aminobutyric acid receptor beta(2) and beta(3) subunits in rat, rabbit, and human kidneys. 1137 33

Dysregulated renal water handling is a cardinal feature of nephrotic syndrome that has been shown in animal models of experimental nephrosis to mediate renal aquaporin (AQP) expression. However, data on the effect of proteinuria on the proximal tubule, which is heavily vested with AQP1 and therefore may participate in water homeostasis, are limited. To investigate this, we exposed primary human proximal tubular epithelial cells (PTECs) to two key proteinuric components shown to perturb tubule function: human serum albumin and transferrin. Using reverse-transcriptase polymerase chain reaction and immunocytochemical techniques, PTECs in the quiescent state were found to express AQP3 in addition to AQP1 gene and protein, which was also validated in a human proximal tubule cell line, HK-2. Immunohistochemical staining localized AQP1 synthesis to the apical and basolateral membranes and AQP3 synthesis to the basolateral membrane of proximal tubule epithelium. Transferrin in doses reaching nephrotic range upregulated PTEC transcription and translation of both AQP1 and AQP3 in a time- and dose-dependent manner. After 24 hours of stimulation, transferrin led to a 2.4- and 2.2-fold increase in AQP1 and APQ3 messenger RNA expression, whereas protein synthesis surged by 40.7% +/- 2.48% and 24.2% +/- 0.9% compared with control, respectively. These effects were not observed with albumin challenge and were not caused by osmolality fluctuation with transferrin treatment. In summary, our novel finding of AQP3 in PTECs indicates a role for AQP3 in proximal tubule water reabsorption. The pathophysiological significance of heightened AQP1 and AQP3 expression in PTECs on protein challenge as occurs in the nephrotic state requires further investigation.
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PMID:In vitro studies of aquaporins 1 and 3 expression in cultured human proximal tubular cells: upregulation by transferrin but not albumin. 1147 58

Progression of renal disease is closely correlated to the degree of renal interstitial fibrosis, and evidence is increasing that epithelial cells of the renal proximal tubule (PTCs) may contribute to its pathogenesis. Such cytokines as basic fibroblast growth factor (FGF-2) have been implicated in progressive renal injury, and we previously showed that PTCs are a source of this cytokine. FGF-2 is characterized by its high affinity for heparin, and numerous studies have suggested that heparin may modify the progression of renal disease. The current study examined how heparin influenced FGF-2 generation and bioactivity in the human renal epithelial PTC line, HK-2. Incubation of HK-2 cells with heparin led to a dose- and time-dependent increase in FGF-2 concentration in the culture supernatant that was not accompanied by alterations in FGF-2 messenger RNA expression, assessed by reverse-transcriptase polymerase chain reaction and Northern analysis. The heparin-induced increase in FGF-2 concentration was accompanied by a decrease in the amount of FGF-2 bound to the extracellular matrix, although this accounted for only a small proportion of the total FGF-2 generated. Induction of FGF-2 by 2-O-desulfated heparin, together with a reduction in total cell-associated FGF-2 and anti-FGF-2 antibody binding to fixed permeabilized cells after the addition of heparin, suggested that the FGF-2 released was mainly derived from a preformed intracellular source. That FGF-2 was predominantly derived from an intracellular pool was also confirmed by pulse chase experiments. The addition of heparin resulted in the generation of bioinactive FGF-2, judged by in vitro fibroblast proliferation. Conversely, heparitinase treatment of supernatant samples from heparin-treated cells and the addition of 2-O-desulfated heparin resulted in the generation of active FGF-2, suggesting that the generation of bioinactive FGF-2 was related to binding of FGF-2 by extracellular heparin after its release from cells. These data show that heparin depletes both the cell and surrounding matrix of FGF-2 and suggest that FGF-2 released from cells was mainly derived from a preformed intracellular source. Furthermore, FGF-2 released from epithelial PTCs after the application of heparin was bioinactive. This likely resulted from released FGF-2 binding to an excess of extracellular heparin. Results presented here therefore suggest a mechanism by which heparin, through its effect on depletion of matrix and cells of FGF-2 and its generation in an inactive form, may influence progressive renal interstitial fibrosis.
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PMID:Regulation of renal proximal tubular epithelial cell fibroblast growth factor-2 generation by heparin. 1153 94


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