Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:4.6.1.1 (adenylate cyclase)
 19,190 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Primary cultures of renal rabbit proximal tubule cells were initiated from a pure suspension of proximal tubule fragments. Proximal tubule cells were grown in a hormone-supplemented, serum-free medium containing low concentrations of antibiotics. Confluent monolayers exhibited multicellular dome formation, indicating the presence of transepithelial solute and water transport. Ultrastructural examination revealed a monolayer of polarized epithelial cells with tight junctions and sparse membraneous microvilli facing the culture medium. Time course biochemical characterization was performed using a palette of 12 enzymes, representative of important metabolic functions or pathways. Brush-border-associated enzymes (gamma-glutamyl transpeptidase and alanine aminopeptidase) were moderately reduced throughout the culture whereas alkaline phosphatase was markedly decreased at confluency. Mitochondrial and lysosomal marker enzymes were well preserved over the culture period. Glutathione-S-transferase activity remained stable during the 16-day culture period investigated. Glycolysis enzyme activities (lactate dehydrogenase and hexokinase) were enhanced, as a function of culture age. Na(+)-K(+)-ATPase activity rise was concomitant with the increase of glycolysis marker enzymes. In contrast, the gluconeogenesis marker enzyme, glucose-6-phosphatase, fell dramatically to reach a low level equivalent to 4% of the activity measured in isolated proximal tubules. Primary cultures exhibited several differentiated functions of the proximal tubule cell: (a) PTH alone was able to induce a significant stimulation of adenylate cyclase activity, unlike isoproterenol, thyrocalcitonin, and arginine vasopressin, and (b) sodium-dependent alpha-methylglucoside (AMG) transport was detected. This AMG uptake was selectively inhibited by phlorizin (5 X 10(-3) M), which is a competitive inhibitor of glucose uptake at the apical membrane. Complete characterization made it possible to investigate hitherto unexplored aspects of in vitro cultured proximal tubule cells. This primary culture model could provide a useful and reliable tool to investigate in vitro renal proximal tubule function, under normal conditions or after a drug-induced toxicity.
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PMID:Biochemical, functional, and morphological characterization of a primary culture of rabbit proximal tubule cells. 167

The effect of forskolin on the biosynthesis and intracellular transport of pig intestinal aminopeptidase N (EC 3.4.11.2) was studied in organ cultured mucosal explants. The drug which activates adenylate cyclase and hence the cAMP-dependent glycogenolytic pathway did not affect the explant content nor microvillar enrichment of the enzyme. Forskolin, however, caused a decrease in the microvillar expression of aminopeptidase N which developed in a time-dependent manner from about 40% by 80 min to 80% by 4 h of labeling. The intracellular pool size of the transient, high mannose glycosylated form of aminopeptidase N was unaffected by forskolin, indicating a normal synthesis in the rough endoplasmic reticulum. The decrease in surface expression is therefore caused by an induced posttranslational degradation of the enzyme, most likely taking place in the Golgi complex. The degradatory effect on newly synthesized aminopeptidase N was not accompanied by any morphological alterations of the enterocyte; in particular, the microvillar membrane appeared entirely unaffected by forskolin. The results obtained provide evidence for the existence of a posttranslational mechanism, whereby a polarized cell is capable of regulating its expression of apical proteins.
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PMID:Biosynthesis of intestinal microvillar proteins. Forskolin reduces surface expression of aminopeptidase N. 289 8

The human colon cancer line Caco-2 exhibits after confluency a concomitant increase of glycogen accumulation and an enterocytic differentiation. The purpose of this work was to investigate whether forskolin (FK), an activator of adenylate cyclase, would induce a permanent glycogenolysis and, if so, whether it would result in modifications of the differentiation pattern of the cells. FK activates adenylate cyclase in Caco-2 cells with an ED50 of 7 X 10(-6)M. Three different treatment protocols with FK (10(-5)M) were applied: 1) the cells were treated during all the time in culture (20 days); 2) the treatment was started after confluency; 3) the treatment was interrupted after confluency. The presence of FK results in a permanent stimulation of cAMP accumulation (10 to 20 fold the basal values) and in a permanently reduced glycogen content (30 or 50% of the control values). The rates of glucose consumption are increased three and five fold in protocols 1 and 3 respectively. These metabolic changes are associated with morphological changes (tightening of the intercellular spaces and shortening of the brush border microvilli) and with a dual inhibition of the activities of brush border hydrolases: a) an inhibition of the post-confluent increase of activity of sucrase, aminopeptidase N and alkaline phosphatase in the brush border enriched fraction; b) an inhibition of the post-confluent increase of activity of sucrase in the cell homogenate. A comparison of the results obtained in each protocol shows that the morphological modifications and the decrease of the enzyme activities in the brush border fraction are regularly associated with an increased cAMP accumulation, whereas the inhibition of the differentiation of sucrase is a direct consequence of the increase in glucose consumption and decrease in glycogen stores.
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PMID:Enterocytic differentiation and glucose utilization in the human colon tumor cell line Caco-2: modulation by forskolin. 298 31

This study concerns whether the pancreatic beta cell expresses cell-surface ectopeptidases that are capable of proteolysis of peptide hormones and neuropeptides that modify glucose-dependent insulin release. These biochemical investigations of the RINm5F cell line found that these cells express ectopeptidases. We have characterized the limited endoproteolysis of GLP-1 (7-36) amide that occurs in the presence of RINm5F plasma membranes. The products and the sensitivity to specific peptidase inhibitors of the proteolysis is characteristic of neutral endopeptidase (NEP) 24.11. Vasoactive intestinal polypeptide (VIP), pituitary adenylate cyclase-activating peptide (PACAP), amylin, glucagon, glucose-dependent insulinotropic polypeptide (GIP), and exendin-4 also undergo proteolysis in the presence of RIN cell membranes. NEP 24.11-activity in RIN cell membranes was confirmed using a specific fluorogenic assay, by histochemistry, and by comparison with the recombinant enzyme with respect to the kinetics of proteolysis of GLP-1 (7-36) amide and of a fluorogenic substrate. Specific fluorogenic assays revealed the presence of aminopeptidase N and the absence of aminopeptidase A and of dipeptidylpeptidase IV.
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PMID:Endoproteolysis of glucagon-like peptide (GLP)-1 (7-36) amide by ectopeptidases in RINm5F cells. 921 54

After collagenase digestion and Percoll density gradient centrifugation of human renal tissue, tubular epithelial cells of the proximal and the distal segments were isolated with an immunomagnetic method using MACS microbeads. To enrich proximal tubular (PT) cells we used a monoclonal antibody (mAb) against aminopeptidase M (APM, CD 13), specific of the proximal tubule. Distal tubular (DT) cells were isolated through a mAb recognizing Tamm-Horsfall glycoprotein (THG), a specific antigen for the thick ascending limb and the early distal convoluted tubule. Cells of the proximal primary isolate were histochemically strongly positive for aminopeptidase M (98.6%), however, cells of the distal portion were negative (98.7%). Ultrastructural analysis of PTC primary isolates revealed highly preserved brush border microvilli, well-developed endocytosis apparati and numerous mitochondria, whereas DTC primary isolates showed smaller cells with basolateral invaginations and less apical microvilli. Characterization by immunofluorescence indicated the coexpression of cytokeratin and vimentin, whereas staining for desmin, smooth muscle actin, a fibroblast-specific marker and von Willebrand factor was negative. Cultured PT and DT cells displayed different adenylate cyclase responsiveness to hormonal stimulation. PTH (10(-6) M) increased cAMP production in distal cells up to 32.8-fold of the basal level and in proximal only up to 3.5-fold (10(-8) M, DT 14.4x and PT 2.25x). Calcitonin stimulated adenylate cyclase in DT in a dose dependent fashion (10(-6) M, 4.3x; 10(-8) M, 2.25x), whereas only a low calcitonin response was found in PT cells (10(-6) M, 1.6x; 10(-8) M, 1.4x). AVP (10(-6) M) activated the distal cAMP-production only up to 1.9x of the basal level, but the proximal cAMP-production was negligible (only 1.3x the basal level). The data of this study indicate the proximal and distal tubule origin of the cultured cells that were isolated according to their segment-specific antigens.
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PMID:Isolation of proximal and distal tubule cells from human kidney by immunomagnetic separation. Technical note. 935 Jun 55

The effects of prostaglandin E2, forskolin, and phorbol 12-myristate 13-acetate on cell proliferation, cell surface antigen expression, vitamin D-24-hydroxylase activity and vitamin D receptor (VDR) expression have been studied in an adherent variant (Ad-HL60) of the human HL60 promyelomonocytic leukemia cell line. Ad-HL60 cells have a more differentiated phenotype than the nonadherent HL60 cells from which they were derived and, unlike the parent cell line, constitutively express vitamin D-24-hydroxylase activity. Treatment of Ad-HL60 cells with 1 microM PGE2 resulted in a decrease in the rate of cell proliferation (cell numbers were approximately 23% of control values after 72 h treatment), a change in expression of leukocyte surface antigens (decreased CD13 and CD14, increased CD11b and CD49d expression), an increase in the synthesis of 24,25-dihydroxyvitamin D3 from substrate 25-hydroxyvitamin D3 (control 5.76 +/- 0.17, 72 h PGE2-treated cells 12.10 +/- 1.90 pmol/h/10(6) cells), and an increase in receptors for the active metabolite of vitamin D, 1 alpha,25-dihydroxyvitamin D3, from 3910 to 11285 receptors per cell in control and 7-day treated cells, respectively. Prostaglandin E2 may be acting via a mechanism involving cyclic AMP in these cells, as we have also demonstrated that 10 microM forskolin, an adenylate cyclase activator, has similar effects. Phorbol 12-myristate 13-acetate had little effect on any of the parameters measured in this cell line.
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PMID:Prostaglandin E2 regulates vitamin D receptor expression, vitamin D-24-hydroxylase activity and cell proliferation in an adherent human myeloid leukemia cell line (Ad-HL60). 1041 Mar 79

Human renal proximal and distal (thick ascending limb and early distal convoluted tubule) epithelial cells have been isolated according to their specific antigen expression. The cells were well characterized by flow cytometry, enzyme cytochemistry and electron microscopy and cultured for up to 3 months. Cultured tubular cells coexpressed cytokeratin and vimentin as intermediate filament proteins. While primary isolated cells, proximal as well as distal, revealed the phenotypic characteristics of their nephron origin, cultured distal cells showed the tendency to dedifferentiate/transdifferentiate. Distal cells lost their characteristic expression of Tamm-Horsfall glycoprotein and started de novo expression of the proximal marker proteins aminopeptidase M, gamma-glutamyl transferase and dipeptidyl peptidase IV. The expression of these antigens by distal cells could be shown by flow-cytometric analysis and fluorescence microscopy. Enzyme activity assays revealed the activity of aminopeptidase M, gamma-glutamyl transferase and dipeptidyl peptidase IV, but not of the proximal marker enzyme alkaline phosphatase. This antigenic shift could not be prevented in different culture media, and the original phenotype could not be restored. Cultured cells displayed characteristic hormonal stimulation patterns indicative of their proximal and distal origins, as shown by activation of adenylate cyclase by different peptide hormones. These results indicate that distal tubular cells possibly transdifferentiate to a more proximal phenotype in view of loss of the distal marker enzyme Tamm-Horsfall protein and de novo expression of proximal marker enzymes like dipeptidyl peptidase IV and aminopeptidase M.
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PMID:Transdifferentiation of distal but not proximal tubular epithelial cells from human kidney in culture. 1045 18

Changes in human endometrium are essential to allow the establishment of pregnancy. These changes are induced in vivo by progesterone, and include appearance within the tissue of a specific uterine natural killer cell, characterized by an abundant expression of CD56. Changes also occur in the stromal cells, which undergo a characteristic decidualization reaction. Decidualized stromal cells are derived from the fibroblast-like cells within the endometrium, which maintain their progesterone receptors in the presence of progesterone. Prolonged exposure to progesterone induces a rounded cell characterized by release of prolactin and insulin-like growth factor binding protein-1 (IGFBP-1), and expression of tissue factor. Additional changes include the secretion of interleukin (IL)-15, vascular endothelial growth factor, and surface expression of zinc dependent metalloproteinases such as CD10 and CD13. In vitro, elevated intracellular cAMP as well as progesterone is necessary for decidualization. In vivo, these conditions may be provided by progesterone from the corpus luteum, by prostaglandin E, a stimulator of adenyl cyclase, and relaxin, which has recently been shown to be a phosphodiesterase inhibitor. Given the co-distribution of uterine natural killer cells and decidualized stromal cells, a mutual interaction might provide the correct regulatory environment for successful implantation, and penetration of the maternal blood vessels by trophoblastic cells.
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PMID:Decidualization of the human endometrial stromal cell: an enigmatic transformation. 1456 82

Bacillus thuringiensis (Bt) bacteria produce Cry toxins that are able to kill insect pests. Different models explaining the mode of action of these toxins have been proposed. The pore formation model proposes that the toxin creates pores in the membrane of the larval midgut cells after interaction with different receptors such as cadherin, aminopeptidase N and alkaline phosphatase and that this pore formation activity is responsible for the toxicity of these proteins. The alternative model proposes that interaction with cadherin receptor triggers an intracellular cascade response involving protein G, adenylate cyclase (AC) and protein kinase A (PKA). In addition, it was shown that Cry toxins induce a defense response in the larvae involving the activation of mitogen-activated kinases such as MAPK p38 in different insect orders. Here we analyzed the mechanism of action of Cry1Ab and Cry1Ac toxins and a collection of mutants from these toxins in the insect cell line CF1 from Choristoneura fumiferana, that is naturally sensitive to these toxins. Our results show that both toxins induced permeability of K+ ions into the cells. The initial response after intoxication with Cry1Ab and Cry1Ac toxins involves the activation of a defense response that involves the phosphorylation of MAPK p38. Analysis of activation of PKA and AC activities indicated that the signal transduction involving PKA, AC and cAMP was not activated during Cry1Ab or Cry1Ac intoxication. In contrast we show that Cry1Ab and Cry1Ac activate apoptosis. These data indicate that Cry toxins can induce an apoptotic death response not related with AC/PKA activation. Since Cry1Ab and Cry1Ac toxins affected K+ ion permeability into the cells, and that mutant toxins affected in pore formation are not toxic to CF1, we propose that pore formation activity of the toxins is responsible of triggering cell death response in CF1cells.
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PMID:Toxicity of Cry1A toxins from Bacillus thuringiensis to CF1 cells does not involve activation of adenylate cyclase/PKA signaling pathway. 2786 74