Regulation of hamster alpha 1B-adrenoceptors expressed in Chinese hamster ovary cells.
Chinese hamster ovary (CHO) cells were stably transfected to express the hamster alpha 1B-adrenoeceptor, and the function and agonist-induced regulation of the binding properties of these receptors were characterized. The cells expressed approximately 230,000 receptors per cell, with a KD for [3H]prazosin of 140 pM. In assays of competition by epinephrine for [3H]prazosin binding to receptors on intact cells, 88% of the receptors were in a low affinity form. The protein kinase C activator phorbol 12-myristate, 13-acetate (PMA) did not further increase the fraction in the low affinity form, but the protein kinase C inhibitor staurosporine reduced the low affinity fraction to 51%. In sucrose density gradient centrifugation assays of receptor internalization, the percentage of receptors in the light vesicle fraction was 25% for control cells, 53% for epinephrine-pretreated cells, 44% for PMA-pretreated cells, and 53% for cells pretreated with epinephrine plus PMA. Staurosporine completely blocked PMA-induced internalization, but only partially inhibited epinephrine-induced internalization. These results suggest a relationship between low affinity binding and internalization for alpha 1B-adrenoceptors and the involvement of protein kinase C in both processes. Longer-term (24 h) exposure of cells to epinephrine induced an unexpected up-regulation of receptor density of approximately 2-fold that was accompanied by an increase in maximal agonist-stimulated phosphoinositide turnover. These studies document several regulatory differences between alpha 1B-adrenoceptors expressed in transfected CHO cells and those natively expressed in DDT1 MF-2 hamster smooth muscle cells, and they provide additional information on the molecular mechanisms involved in agonist-induced regulation of alpha 1B-adrenoceptors.
Zhu
S J
SJ
Department of Pharmacology, University of Nebraska Medical Center, Omaha 68198-6260, USA.
Cerutis
D R
DR
Anderson
J L
JL
Toews
M L
ML
D000316:Adrenergic alpha-Agonists / Q000378:metabolism / Q000494:pharmacology*; D000818:Animals; D016466:CHO Cells / Q000187:drug effects / Q000378:metabolism; D006224:Cricetinae; D004837:Epinephrine / Q000494:pharmacology*; D010716:Phosphatidylinositols / Q000096:biosynthesis*; D011224:Prazosin / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D018340:Receptors, Adrenergic, alpha-1 / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D019311:Staurosporine / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection
D000316:Adrenergic alpha-Agonists; D010716:Phosphatidylinositols; D018340:Receptors, Adrenergic, alpha-1; D011994:Recombinant Proteins; D011493:Protein Kinase C; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate; D011224:Prazosin; D004837:Epinephrine