TY - JOUR
T1 - Induction of tyrosine aminotransferase in H-35 hepatoma cells by cAMP captured in phospholipid vesicles
AU - Culpepper, J. A.
AU - Liu, Alice
N1 - Copyright:
Copyright 2018 Elsevier B.V., All rights reserved.
PY - 1981
Y1 - 1981
N2 - 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-Å unilamellar lipid vesicles, capturing cAMP in the internal aqueous cavity. Incubation of H-35 hepatoma cells with vesicles containing cAMP (versicle-cAMP) resulted in rapid incorporation of the vesicle content; apparent saturation of uptake was reached after ~30 min of incubation at 37°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 >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°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 μM, 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-N30cAMP, 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.
AB - 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-Å unilamellar lipid vesicles, capturing cAMP in the internal aqueous cavity. Incubation of H-35 hepatoma cells with vesicles containing cAMP (versicle-cAMP) resulted in rapid incorporation of the vesicle content; apparent saturation of uptake was reached after ~30 min of incubation at 37°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 >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°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 μM, 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-N30cAMP, 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.
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U2 - 10.1083/jcb.88.1.89
DO - 10.1083/jcb.88.1.89
M3 - Article
C2 - 6110670
AN - SCOPUS:0019366110
VL - 88
SP - 89
EP - 95
JO - Journal of Cell Biology
JF - Journal of Cell Biology
SN - 0021-9525
IS - 1
ER -