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Beta-adrenergic receptor desensitization of wild-type but not cyc lymphoma cells unmasked by submillimolar Mg2+
R B Clark1, J Friedman, J A Johnson
1Graduate School of Biomedical Sciences, University of Texas Health Science Center at Houston 77225.
Abstract:
Treatment with low physiological concentrations of epinephrine (5-50 nM) rapidly desensitizes beta-adrenergic stimulation of cAMP formation in S49 wild-type (WT) lymphoma cells. Previous attempts to detect this early phase of desensitization in cell-free assays of adenylate cyclase (EC 4.6.1.1) after intact cell treatment were unsuccessful. We have now found that reducing the Mg2+ concentrations in the adenylate cyclase assays to less than 1.0 mM unmasked this rapid phase of desensitization of the WT cells, and that high Mg2+ concentrations (5-10 mM) largely obscured the desensitization. Submillimolar Mg2+ conditions also revealed a two- to threefold decrease in the affinity of epinephrine binding to the beta-adrenergic receptor after desensitization with 20 nM epinephrine. Detection of 4 beta-phorbol 12-myristate 13-acetate (PMA) desensitization of the WT beta-adrenergic receptor was also dependent on low Mg2+ as measured either by the decrease in epinephrine stimulation of adenylate cyclase or by the reduction in the affinity of epinephrine binding. Unexpectedly, when cyc- cells were pretreated with 50 nM epinephrine, the beta-adrenergic stimulation of reconstituted adenylate cyclase was not desensitized. The characteristics of the Mg2+ effect on epinephrine- and PMA-induced desensitizations suggest a similar mechanism of action with the most likely events being phosphorylations of the beta-adrenergic receptors. Our data indicate that cAMP-dependent protein kinase (EC 2.7.1.37) may play a role in the desensitization caused by low epinephrine concentrations inasmuch as this phase of desensitization did not occur in the cyc-. For the PMA-induced desensitization, the phosphorylation may be mediated by protein kinase C (EC 2.7.1.37).
Insights
Low epinephrine concentrations rapidly desensitize beta-adrenergic receptors in lymphoma cells. This desensitization, previously undetectable in cell-free assays, is now observable under low magnesium conditions, suggesting phosphorylation as a key mechanism.
Area of Science:
- Cellular signaling
- Adrenergic receptor pharmacology
- Enzyme kinetics
Background:
- Beta-adrenergic receptor desensitization is a critical cellular response to prolonged agonist exposure.
- Previous cell-free assays failed to detect rapid desensitization of adenylate cyclase by low epinephrine concentrations in S49 WT lymphoma cells.
- Understanding the early phase of receptor desensitization is crucial for elucidating cellular regulatory mechanisms.
Purpose of the Study:
- To identify conditions that allow detection of rapid beta-adrenergic receptor desensitization in cell-free assays.
- To investigate the role of magnesium concentration in the desensitization process.
- To explore the potential mechanisms underlying epinephrine- and PMA-induced receptor desensitization.
Main Methods:
- Adenylate cyclase activity assays were performed on S49 WT lymphoma cells treated with epinephrine under varying magnesium (Mg2+) concentrations.
- Epinephrine binding affinity to beta-adrenergic receptors was measured after cell treatment.
- Desensitization experiments were conducted using both epinephrine and phorbol ester (PMA) in wild-type and cyc- S49 cells.
Main Results:
- Reducing Mg2+ concentration to <1.0 mM in cell-free assays unmasked rapid beta-adrenergic receptor desensitization by low epinephrine (5-50 nM).
- Low Mg2+ conditions also revealed decreased epinephrine binding affinity and enabled detection of PMA-induced desensitization.
- Desensitization by low epinephrine concentrations did not occur in cyc- cells, suggesting a role for cAMP-dependent protein kinase.
Conclusions:
- Magnesium concentration critically influences the detectability of beta-adrenergic receptor desensitization in cell-free systems.
- The findings suggest that phosphorylation of beta-adrenergic receptors is a key event in both epinephrine- and PMA-induced desensitization.
- Low epinephrine-induced desensitization likely involves cAMP-dependent protein kinase, while PMA-induced desensitization may involve protein kinase C.