scholarly journals Induction of tyrosine aminotransferase in H-35 hepatoma cells by cAMP captured in phospholipid vesicles.

1981 ◽  
Vol 88 (1) ◽  
pp. 89-95 ◽  
Author(s):  
J A Culpepper ◽  
A Y Liu
Keyword(s):  
Cyclic Nucleotide ◽  
Lipid Vesicles ◽  
Tyrosine Aminotransferase ◽  
Hepatoma Cells ◽  
Receptor Protein ◽  
Regulatory Subunit ◽  
Phospholipid Vesicles ◽  
Intracellular Camp ◽  
Intact Cells ◽  
Relationship Of

The uptake, metabolism, and action of cAMP, captured within phospholipid vesicles, in H-35 hepatoma cells were studied. Sonication of lipids in buffer containing cAMP resulted in the formation of 300-A unilamellar lipid vesicles, capturing cAMP in the internal aqueous cavity. Incubation of H-35 hepatoma cells with vesicles containing cAMP (vesicle-cAMP) resulted in rapid incorporation of the vesicle content; apparent saturation of uptake was reached after approximately 30 min of incubation at 37 degrees C. Uptake of vesicle-cAMP was linear over a 10-fold vesicle concentration range. Pretreatment of cells with combined inhibitors of glycolysis and respiration inhibited vesicle uptake by 27%, suggesting vesicle fusion with the cell membrane as a predominant pathway of vesicle uptake. Studies on the metabolism of incorporated cAMP indicated that greater than 50% of the cell-associated radioactivity, derived from vesicle-[3H]cAMP, was preserved as cAMP at the end of a 20-min incubation at 37 degrees C. The incorporation of vesicle-cAMP by H-35 hepatoma cells resulted in increased tyrosine aminotransferase (TAT) activity. The concentration of vesicle-cAMP needed to produce a half-maximal increase in TAT activity was 10 microM, approximately two orders of magnitude lower than that of exogenously added dbcAMP. cAMP was ineffective when added extracellularly. The kinetic relationship of the cAMP-induced increase in TAT activity and the binding of cAMP to its receptor protein, in intact H-35 cells, was examined using vesicle-trapped 8-N3-cAMP, a photoaffinity labeling analogue of cAMP. Incubation of H-35 hepatoma cells with vesicle-8-N3-cAMP resulted in increased TAT activity, preceded by the binding of 8-N3-cAMP to the regulatory subunit of type II cAMP-dependent protein kinase. The use of lipid vesicles provides a means of modulating intracellular cAMP concentration without adding cyclic nucleotide in the millimolar concentration range to the extracellular medium. The increased efficiency of intracellular delivery of cyclic nucleotide with retention of biological activity, provides a useful technique in examining the relationship of occupancy of specific cAMP-receptor protein(s) and the occurrence of a cAMP-mediated biological response in intact cells.

scholarly journals Cyclic 3',5'-AMP relay in Dictyostelium discoideum III. The relationship of cAMP synthesis and secretion during the cAMP signaling response.

1980 ◽  
Vol 86 (2) ◽  
pp. 537-544 ◽  
Author(s):  
M C Dinauer ◽  
S A MacKay ◽  
P N Devreotes
Keyword(s):  
Adenylate Cyclase ◽  
Time Course ◽  
Camp Signaling ◽  
Intracellular Camp ◽  
Intact Cells ◽  
Camp Synthesis ◽  
Transient Activation ◽  
Direct Measurements ◽  
Precipitous Decline ◽  
Relationship Of

Refinement of a perfusion technique permitted the simultaneous measurement of cAMP-elicited [3H]cAMP secretion and intracellular [3H]cAMP levels in sensitive D. discoideum amoebae. These data were compared with measurements of the rate of [32P]cAMP synthesis by extracts of amoebae sonicated at different times during the cAMP signaling response. cAMP stimulation of intact cells led to a transient activation of adenylate cyclase, which was blocked if 10(-4) M NaN3 was added with the stimulus. During responses elicited by 10(-6) M cAMP, 10(-8) M cAMP, and an increment in cAMP from 10(-8) M to 10(-7) M, the rate of cAMP secretion was proportional to the intracellular cAMP concentration. Removal of a 10(-6) M cAMP stimulus 2 min after the initiation of the response led to a precipitous decline in intracellular cAMP. This decline was more rapid than could be accounted for by secretion alone, suggesting intracellular phosphodiesterase destruction of newly synthesized cAMP. Employing these data and a simple rate equation, estimates of the time-course of the transient activation of adenylate cyclase and the rate constants for cAMP secretion and intracellular phosphodiesterase activity were obtained. The calculated rate of cAMP synthesis rose for approximately 1 to 2 min, peaked, and declined to approach prestimulus levels after 3 to 4 min. This time-course agreed qualitatively with direct measurements of the time-course of activation, indicating that the activation of adenylate cyclase is a major in determining the time-course of the cAMP secretion response.

The distal enhancer implicated in the developmental regulation of the tyrosine aminotransferase gene is bound by liver-specific and ubiquitous factors

1993 ◽  
Vol 13 (8) ◽  
pp. 4494-4504
Author(s):  
D Nitsch ◽  
G Schütz
Keyword(s):  
Thymidine Kinase ◽  
Specific Binding ◽  
Regulatory Element ◽  
Hepatoma Cell ◽  
Tyrosine Aminotransferase ◽  
Hepatoma Cells ◽  
Hepatoma Cell Line ◽  
Distal Enhancer ◽  
Binding Studies ◽  
Enhancer Sequences

Tyrosine aminotransferase gene expression is confined to parenchymal cells of the liver, is inducible by glucocorticoids and glucagon, and is repressed by insulin. Three enhancers control this tissue-specific and hormone-dependent activity, one of which, located at -11 kb, is implicated in establishing an active expression domain. We have studied in detail this important regulatory element and have identified a 221-bp fragment containing critical enhancer sequences which stimulated the heterologous thymidine kinase promoter more than 100-fold in hepatoma cells. Within this region, we have characterized two essential liver-specific enhancer domains, one of which was bound by proteins of the hepatocyte nuclear factor 3 (HNF3) family. Analyses with the dedifferentiated hepatoma cell line HTC suggested that HNF3 alpha and/or -gamma, but not HNF3 beta, are involved in activating the tyrosine aminotransferase gene via the -11-kb enhancer. Genomic footprinting and in vitro protein-DNA binding studies documented cell-type-specific binding of ubiquitous factors to the second essential enhancer domain, which by itself stimulated the thymidine kinase promoter preferentially in hepatoma cells. These results will allow further characterization of the role of these enhancer sequences in developmental activation of the tyrosine aminotransferase gene.

scholarly journals The Lantibiotic Nisin Induces Transmembrane Movement of a Fluorescent Phospholipid

1998 ◽  
Vol 180 (24) ◽  
pp. 6565-6570 ◽  
Author(s):  
Gert N. Moll ◽  
Wil N. Konings ◽  
Arnold J. M. Driessen
Keyword(s):  
Antimicrobial Peptide ◽  
Rate Constant ◽  
Lipid Vesicles ◽  
Phospholipid Vesicles ◽  
Nisin Concentration ◽  
Outer Leaflet ◽  
Target Membrane

ABSTRACT Nisin is a pore-forming antimicrobial peptide. The capacity of nisin to induce transmembrane movement of a fluorescent phospholipid in lipid vesicles was investigated. Unilamellar phospholipid vesicles that contained a fluorescent phospholipid (1-acyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]caproyl}-sn-glycero-3-phosphocholine) in the inner leaflet of the bilayer were used. Nisin-induced movement of the fluorescent phospholipid from the inner leaflet to the outer leaflet of the membrane reached stable levels, which were dependent on the concentration of nisin added. The rate constant k of this nisin-induced transmembrane movement increased with the nisin concentration but was not dependent on temperature within the range of 5 to 30°C. In contrast, the rate constant of movement of fluorescent phospholipid from vesicle to vesicle strongly depended on temperature. The data indicate that nisin transiently disturbs the phospholipid organization of the target membrane.

scholarly journals Thermodynamics of Cyclic Nucleotide Binding to the cAMP Receptor Protein and Its T127L Mutant

1995 ◽  
Vol 270 (37) ◽  
pp. 21679-21683 ◽  
Author(s):  
Inna Gorshkova ◽  
Julie L. Moore ◽  
Keith H. McKenney ◽  
Frederick P. Schwarz
Keyword(s):  
Cyclic Nucleotide ◽  
Nucleotide Binding ◽  
Receptor Protein ◽  
Camp Receptor Protein ◽  
Cyclic Nucleotide Binding ◽  
Camp Receptor

Differential activity of a tissue-specific extinguisher locus in hepatic and nonhepatic cells

1989 ◽  
Vol 9 (5) ◽  
pp. 1813-1822
Author(s):  
H Gourdeau ◽  
T C Peterson ◽  
R E Fournier
Keyword(s):  
Mouse Chromosome ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Genetic Locus ◽  
Cell Types ◽  
Tyrosine Aminotransferase ◽  
Hepatoma Cells ◽  
Chromosome 11 ◽  
Tissue Specific ◽  
Primary Mouse ◽  
Mouse Chromosome 11

Tissue-specific extinguisher 1 (Tse-1) is a genetic locus on mouse chromosome 11 that can repress expression of several liver genes in trans. This locus is clearly active in fibroblasts, as hepatoma cells retaining fibroblast chromosome 11 are extinguished for both tyrosine aminotransferase and phosphoenolpyruvate carboxykinase gene expression. To assess the activity of Tse-1 in other tissues, we transferred mouse chromosome 11 from several different cell types into rat hepatoma recipients. Tse-1 was active in nonhepatic cell lines derived from each primary germ layer, but Tse-1 activity was not apparent in hybrids between hepatoma cells and primary mouse hepatocytes. These differences in the genetic activity of murine Tse-1 were apparently heritable in cis.

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