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Agents that decrease gonadotropin-releasing hormone (GnRH) receptor internalization do not inhibit GnRH-mediated
Abstract:
Exposure of pituitary cell cultures to GnRH causes gonadotropin release, receptor capping, internalization, and loss as well as altered responsiveness of the target cell. In the present study, the relationship between loss of gonadotrope secretory responsiveness to GnRH (desensitization) and internalization of the GnRH-receptor complex was examined. Pituitary cell cultures were pretreated (30 min) with vinblastine (100 microM, a concentration that prevents measurable receptor internalization) or with medium containing carrier only, incubated with 10(-7) M GnRH (a desensitizing concentration) with or without vinblastine or with medium alone for 60 min, and finally washed and rechallenged for 3 h with increasing concentrations of GnRH to assess the degree of desensitization as determined by LH release. Results indicate that vinblastine had no measurable effect on the ability of GnRH to stimulate LH release or desensitize the cells. In a second series of studies, a GnRH analog (D-Lys6-GnRH) was immobilized to a cross-linked agarose matrix. The covalent link was shown to be stable by biological, immunological, and physical criteria. This product bound to the GnRH receptor and provoked LH release, but was not internalized, as determined by GnRH receptor binding assays. Cultured cells were treated with either 10(-9) M free analog or an equivalent concentration of coupled analog (as measured by LH release) for 3 h. Cells were washed, then rechallenged with GnRH to assess desensitization. Both the free and coupled analogs provoked an equivalent degree of desensitization. While a significant degree of desensitization also occurred in the presence of 3 mM EGTA (conditions that totally inhibited GnRH-stimulated LH release), the loss of responsiveness was not as great as in the absence of EGTA, indicating that partial depletion of available LH may play a role in GnRH-stimulated gonadotrope desensitization. The present findings suggest that GnRH receptor internalization and LH release can be uncoupled and that loss of the GnRH receptor by internalization is not a sufficient explanation for GnRH-mediated desensitization of the gonadotrope.
Insights
Gonadotrope desensitization to GnRH is not solely explained by receptor internalization. Immobilized GnRH analogs that are not internalized still cause desensitization, uncoupling receptor internalization from LH release.
Area of Science:
- Endocrinology
- Molecular Cell Biology
- Reproductive Biology
Background:
- Exposure to Gonadotropin-Releasing Hormone (GnRH) in pituitary cell cultures triggers gonadotropin release, receptor capping, internalization, and loss.
- This exposure also leads to altered responsiveness of the target cell, a phenomenon known as desensitization.
Purpose of the Study:
- To investigate the relationship between the loss of gonadotrope secretory responsiveness to GnRH (desensitization) and the internalization of the GnRH-receptor complex.
- To determine if GnRH receptor internalization is a necessary mechanism for GnRH-mediated desensitization.
Main Methods:
- Pituitary cell cultures were pretreated with vinblastine (to prevent receptor internalization) or a control medium.
- Cells were then incubated with GnRH or a GnRH analog (free or immobilized on agarose) to induce desensitization.
- Receptor binding assays and Luteinizing Hormone (LH) release measurements were used to assess desensitization and receptor internalization.
Main Results:
- Vinblastine did not affect GnRH-stimulated LH release or desensitization, indicating internalization is not required for initial response or desensitization.
- An immobilized GnRH analog, which bound to the receptor but was not internalized, caused desensitization equivalent to the free analog.
- Partial depletion of LH stores contributed to desensitization, particularly under conditions inhibiting GnRH-stimulated LH release (EGTA).
Conclusions:
- GnRH receptor internalization and LH release can occur independently, suggesting these processes can be uncoupled.
- The loss of GnRH receptor by internalization is not sufficient to explain GnRH-mediated desensitization of gonadotropes.
- Desensitization involves mechanisms beyond receptor internalization, potentially including intracellular signaling pathways and LH store depletion.