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Phorbol ester causes desensitization of gonadotropin-responsive adenylate cyclase in a murine Leydig tumor cell line
Abstract:
The murine Leydig tumor cell line, MLTC-1, contains gonadotropin receptors and a gonadotropin-responsive adenylate cyclase system that became refractory (desensitized) when exposed to human chorionic gonadotropin (hCG). MLTC-1 cells also contain phorbol ester receptors with a Kd of 53 nM for [3H]phorbol dibutyrate. Exposing cells to 12-O-tetradecanoyl phorbol 13-acetate (TPA) also causes desensitization of the hCG response. TPA-induced desensitization was similar to hCG-induced desensitization by every criteria tested. Both TPA- and hCG-induced desensitization caused approximately 50% loss of the hormone response within 30 min. Neither TPA or hCG altered receptor affinity for hCG. The dose response of adenylate cyclase to hCG or GTP in isolated membranes was not affected by either hCG- or TPA-induced desensitization. Similarly the dose response to hCG of cAMP accumulation in intact cells was not altered by desensitization with hCG or TPA. It was determined that MLTC-1 cells have Ca2+/phospholipid-dependent protein kinase activity that displayed a dose-dependent response to TPA. The concentration of TPA required to activate the protein kinase was similar to that required for desensitization. Phorbol esters that were unable to activate protein kinase C were also unable to desensitize MLTC-1 cells. The protein kinase from MLTC-1 cells was also activated by diacylglycerol. In addition, diacylglycerols caused desensitization of the hCG response. TPA- and diacylglycerol-induced desensitization is probably mediated by protein kinase C, and the similarities between hCG- and TPA-induced refractoriness suggests a convergence of mechanisms at some point of MLTC-1 cell desensitization.
Insights
Human chorionic gonadotropin (hCG) and phorbol ester (TPA) desensitize Leydig cells similarly. This suggests a shared pathway, likely involving protein kinase C, in regulating cellular response to these stimuli.
Area of Science:
- Endocrinology
- Cell Biology
- Molecular Pharmacology
Background:
- Murine Leydig tumor cells (MLTC-1) possess gonadotropin receptors and a responsive adenylate cyclase system.
- Exposure to human chorionic gonadotropin (hCG) leads to desensitization of this system.
- MLTC-1 cells also exhibit phorbol ester receptors, suggesting potential interactions with signaling pathways.
Purpose of the Study:
- To investigate the mechanism of desensitization in MLTC-1 cells induced by hCG and phorbol esters.
- To compare the effects of hCG and 12-O-tetradecanoyl phorbol 13-acetate (TPA) on cellular responsiveness.
- To elucidate the role of protein kinase C in mediating TPA-induced desensitization.
Main Methods:
- Desensitization experiments using MLTC-1 cells exposed to hCG and TPA.
- Assessment of receptor affinity and adenylate cyclase activity in isolated membranes.
- Measurement of cAMP accumulation in intact cells.
- Characterization of Ca2+/phospholipid-dependent protein kinase activity and its response to TPA and diacylglycerols.
Main Results:
- Both hCG and TPA induced rapid desensitization (approx. 50% loss of response in 30 min) without altering receptor affinity.
- Neither hCG nor TPA affected the dose-response of adenylate cyclase to hCG or GTP in membranes.
- TPA activated a Ca2+/phospholipid-dependent protein kinase in MLTC-1 cells, with activation concentrations similar to those causing desensitization.
- Phorbol esters unable to activate protein kinase C did not cause desensitization.
- Diacylglycerols also activated the protein kinase and induced desensitization.
Conclusions:
- TPA-induced desensitization is likely mediated by protein kinase C activation.
- The similar desensitization profiles of hCG and TPA suggest a convergence of signaling pathways.
- These findings indicate a shared mechanism in the refractoriness of MLTC-1 cells to hormonal and phorbol ester stimulation.