Alpha 1 adrenergic receptor mediated polyphosphoinositide breakdown in DDT1-MF2 cells. Lack of evidence of desensitization after prolonged exposure to epinephrine.
The DDT1-MF2 cell line is a transformed smooth muscle cell line which is known to possess both alpha 1 and beta 2 adrenergic receptors. We have utilized these cells to compare the effects of epinephrine pretreatment on the functional capabilities of these two different adrenergic receptors. Pretreatment of the cells grown in suspension with 10(-7) M epinephrine for 6 hr resulted in desensitization of beta receptor stimulated cyclic AMP accumulation. The maximal response to isoproterenol was decreased to 46 +/- 6% of the value in controls (P less than 0.05); there was also a decrease in the sensitivity of the cells to isoproterenol (log EC50 = -6.65 +/- 0.22 vs -7.26 +/- 0.11 in controls, P less than 0.05). Also, there was a decrease in the number of beta receptors from 257 +/- 29 to 163 +/- 22 fmol/mg protein. In contrast, pretreatment with 10(-6) M epinephrine for 6 hr failed to induce a loss of sensitivity in the ability of the alpha 1 receptor agonist phenylephrine to stimulate inositol triphosphate accumulation (log EC50 = -5.59 +/- 0.18 vs -5.42 +/- 0.44 in control cells). A 2-fold increase in basal inositol monophosphate accumulation was observed after epinephrine pretreatment (P less than 0.05); however, there was no change in maximal phenylephrine-stimulated inositol monophosphate accumulation in these cells. There was a small decrease in the alpha 1 receptor number after epinephrine pretreatment (Bmax = 457 +/- 89 fmol/mg protein vs 540 +/- 94 in control cells, P less than 0.05). In contrast to epinephrine pretreatment, pretreatment of cells in suspension with 10(-7) M 12-O-tetradecanoylphorbol-13-acetate (TPA) for 15 min resulted in a nearly complete blunting in the ability of both norepinephrine and phenylephrine to stimulate inositol phosphate accumulation: after norepinephrine stimulation, 774 +/- 34 dpm in TPA-pretreated cells vs 2590 +/- 10 in control cells; inositol monophosphate accumulation after phenylephrine stimulation 576 +/- 25 dpm in TPA-pretreated cells vs 1660 +/- 27 in control cells. Basal levels of inositol monophosphate remained unchanged at 544 +/- 28 dpm vs 505 +/- 31 in TPA-pretreated cells compared to control cells. These data indicate that protein kinase C may exert a negative feedback control on the alpha 1 receptor in these cells and that direct activation of protein kinase C by phorbol esters may have a different effect on the alpha 1 adrenergic receptor system in DDT1-MF2 cells than does prolonged exposure to epinephrine.
Rosenbaum
J S
JS
Department of Medicine, Stanford University School of Medicine, CA.
Azhar
S
S
Hoffman
B B
BB
D002460:Cell Line; D000242:Cyclic AMP / Q000096:biosynthesis; D004305:Dose-Response Relationship, Drug; D004837:Epinephrine / Q000494:pharmacology*; D007295:Inositol Phosphates / Q000378:metabolism*; D007545:Isoproterenol / Q000494:pharmacology; D009130:Muscle, Smooth; D010656:Phenylephrine / Q000494:pharmacology; D010716:Phosphatidylinositols / Q000378:metabolism*; D011942:Receptors, Adrenergic, alpha / Q000187:drug effects / Q000502:physiology*; D011943:Receptors, Adrenergic, beta / Q000187:drug effects / Q000378:metabolism; D013403:Sugar Phosphates / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D007295:Inositol Phosphates; D010716:Phosphatidylinositols; D011942:Receptors, Adrenergic, alpha; D011943:Receptors, Adrenergic, beta; D013403:Sugar Phosphates; D010656:Phenylephrine; D000242:Cyclic AMP; D007545:Isoproterenol; D013755:Tetradecanoylphorbol Acetate; D004837:Epinephrine