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Desensitization of LLC-PK1 cells by vasopressin results in receptor down-regulation
Abstract:
The molecular mechanism of desensitization of antidiuretic hormone receptors is not well understood. Preincubation of LLC-PK1 cells with lysine vasopressin (LVP) (10(-6) M, 5 h) decreased subsequent LVP-stimulated cAMP accumulation in cells by 83% and reduced the Vmax of LVP-stimulated adenylate cyclase by 81%. Such preincubation also reduced by 90% the binding of [3H]LVP to both intact cells and isolated plasma membranes, suggesting a loss of vasopressin receptors. Both the reduction in cAMP response and the apparent loss of receptors showed similar dose and time dependence. Monensin (33 microM) did not alter [3H]LVP binding or stimulation of cAMP by LVP, nor did it prevent desensitization. However, membranes prepared from cells preincubated with LVP in the presence of monensin did not show a decrease in [3H]LVP binding. Forskolin preincubation, at 0.1, 1, 10 and 100 microM, did not alter [3H]LVP binding or accumulation of cellular cAMP by LVP, nor did it induce desensitization to LVP. Cells desensitized with varying LVP concentrations in the presence of 10 microM forskolin displayed the same loss of [3H]LVP binding and LVP responsiveness as observed in the absence of forskolin. LVP-desensitized cells, upon removal from LVP-containing medium, recovered cAMP responsiveness to LVP and specific binding of [3H]LVP at the same rate, achieving control levels after 50 h. Recovery was prevented by cycloheximide (25 micrograms/ml). These findings are consistent with a desensitization process involving LVP-mediated receptor internalization, and a recovery process requiring protein synthesis.
Insights
Lysine vasopressin (LVP) causes desensitization of antidiuretic hormone receptors by promoting receptor internalization. Recovery of receptor function requires protein synthesis.
Area of Science:
- Endocrinology
- Molecular Pharmacology
- Cell Biology
Background:
- The molecular mechanisms underlying the desensitization of antidiuretic hormone receptors remain incompletely understood.
- Antidiuretic hormone receptors play a crucial role in regulating water balance and blood pressure.
Purpose of the Study:
- To elucidate the molecular mechanism of antidiuretic hormone receptor desensitization.
- To investigate the role of receptor internalization and protein synthesis in receptor recovery.
Main Methods:
- LLC-PK1 cells were preincubated with varying concentrations of lysine vasopressin (LVP) and forskolin.
- Radioligand binding assays using [3H]LVP were performed on intact cells and isolated plasma membranes.
- Cyclic adenosine monophosphate (cAMP) accumulation was measured following LVP stimulation.
- The effect of monensin and cycloheximide on LVP receptor binding and cAMP accumulation was assessed.
Main Results:
- Preincubation with LVP significantly reduced LVP-stimulated cAMP accumulation and [3H]LVP binding, indicating receptor desensitization and apparent receptor loss.
- Monensin did not prevent desensitization but prevented the loss of [3H]LVP binding in membranes from LVP-preincubated cells.
- Forskolin did not affect LVP receptor binding or desensitization.
- Recovery of LVP responsiveness and [3H]LVP binding was observed upon removal of LVP and was inhibited by cycloheximide, suggesting a requirement for protein synthesis.
Conclusions:
- These findings support a model where LVP-induced receptor desensitization involves receptor internalization.
- The recovery of receptor function necessitates new protein synthesis, highlighting the dynamic nature of receptor regulation.