Abstract
The effect of lysophosphatidic acid on the phosphorylation and function of α1b-adrenoceptors transfected into rat-1 fibroblasts was studied. This phospholipid mitogen increased in a concentration-dependent fashion (EC50 ∼50 nM) the phosphorylation of these adrenoceptors. Lysophosphatidic acid-induced α1b-adrenoceptor phosphorylation was relatively rapid (t1/2 ∼1 min), intense (2.5-fold), and sustained for at least 60 min. The effect of lysophosphatidic acid was blocked by pretreatment with pertussis toxin. The α1b-adrenoceptor phosphorylation induced by lysophosphatidic acid was not blocked by genistein, a tyrosine kinase inhibitor, but it was inhibited by inhibitors of protein kinase C (bisindolylmaleimide I, staurosporine, and Ro 31-8220) and phosphoinositide 3-kinase (wortmannin and LY 294002). The ability of norepinephrine to increase cytosol calcium concentration was markedly decreased in cells previously challenged with lysophosphatidic acid. Norepinephrine-induced [35S]GTPγS binding in membrane preparations was used as an index of the functional coupling of the α1b-adrenoceptors and G proteins. Norepinephrine-stimulated [35S]GTPγS binding was markedly decreased in membranes from cells pretreated with lysophosphatidic acid. This effect of lysophosphatidic acid was blocked by pretreatment with wortmannin or staurosporine. Our data indicate that: 1) activation of lysophosphatidic acid receptors induce phosphorylation of α1b-adrenoceptors; 2) this effect is mediated through pertussis toxin-sensitive G proteins, phosphatidylinositol 3-kinase, and protein kinase C; and 3) the phosphorylation of α1b-adrenoceptors induced by the lipid mitogen is associated to adrenoceptor desensitization.
Footnotes
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Send reprint requests to: J. Adolfo Garcı́a-Sáinz, M.D., Ph.D., Inst. Fisiologı́a Celular, UNAM, Ap. Postal 70–248, México, D. F. 04510. E-mail: agarcia{at}ifisiol.unam.mx
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This research was partially supported by grants from Dirección General de Asuntos del Personal Académico (IN 200596 and IN 205199) and Consejo Nacional de Ciencia y Tecnologı́a (27569N).
- Abbreviations:
- PKC
- protein kinase C
- AR
- adrenoceptor
- LPA
- lysophosphatidic acid
- DMEM
- Dulbecco's modified Eagle's medium
- GRK
- G protein-coupled receptor kinase
- PI3K
- phosphoinositide 3-kinase
- [Ca2+]i
- cytosol calcium concentration
- NE
- norepinephrine
- TPA
- tetradecanoyl phorbol acetate
- PDK1
- phosphoinositide-dependent protein kinase-1
- Received August 30, 1999.
- Accepted January 20, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
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