| Secretion forms the basic for signal transmission between the neurons and hormone release from endocrine cells, which are important in remaining normal physiological function of organism. Abnormity in secretion will result in the appearance of desease. The anterior pituitary gonadotropes secret pituitary gonadotropins and serve as a control center for integration of hormonal signals in the reproductive axis, deficiency in hormone secretion from gonadotrops will impact development and mature of sex. The study on the mechanism of secretion and its regulation is important to further understand the essence of secretion and to treat secretion-relevant deseases. We have proved that PKC activation could sensitize the Ca2+-dependent secretion in primary gonadotrops, but the regulation mechanism remain unclear. In the present study, we applied advanced biophysical techniques, such as microfluoremetric technique, membrane capacitance measurement, UV flash technique, modeling and statistic to systemic investigate on the the heterogeneity of the Ca2+-sensitivity of secretion in single gonadotrope cell line, LβT2, and the mechanism of protein kinase regulated exocytosis in primary pituitary gonadotropes.Main result of the study are as follows:(1) By employing high time resolution measurement of membrane capacitance (Cm) stimulated with step-like or ramp [Ca2+]i elevation, we have identified the co-existence of both high and low Ca2+-sensitive exocytosis in an immortal pituitary gonadotrope cell line, LβT2. Ramp [Ca2+]i elevation generated by slow uncaging of nitrophenyl-EGTA elicited a biphasic Cm response. The first phase of response, which may represent a highly calcium sensitive pool (HCSP) of vesicles, began to secrete at low [Ca2+]i concentration (<1μM) with low Ca2+ cooperativity. Whereas the second phase, which may represent a low Ca2+-sensitive pool (LCSP) of vesicles, only exocytozed at higher [Ca2+]i (> 5μM ) and displayed a steep [Ca2+]i dependence. The co-existence of vesicle populations with different Ca2+ dependence was further confirmed by flash photolysis stimuli. The size of the HCSP was ~30 fF under resting conditions, but was dramatically increased (~ 3 fold) by application of PMA. Forskolin, however, exerted no significant effect on the size of both HCSP and LCSP. GnRH (gonadotropin releasing hormone), a physiological stimulus of gonadotrope, augmented the size of both pools to a larger extend (5 and 1.7 fold increase for HCSP and LCSP, respectively). The shifting of more vesicles to a highly Ca2+-sensitive status by GnRH signaling and PKC would dramatically increase the release in response to spatially averaged [Ca2+]i. This mechanism may thus play an important role in the stimulus-secretion coupling of gonadotropin secretion. The heterogeneity of Ca2+-sensitivity from different pools of vesicles and its differential modulation by intracellular signals manifest LβT2 cells as an ideal model to further unravel the mechanism underlying the modulation of Ca2+-sensing machineries for exocytosis. (2) PKC increase the apparent Ca2+-affinity for exocytosis by augmenting the size of the HCSP, but it is not known how the Ca2+-sensor be modulated by protein kinase. We have previously reported that protein kinase C (PKC) activation sensitizes the Ca2+-sensor for exocytosis in pituitary gonadotropes. To further unravel the underlying mechanism of how the Ca2+-sensor is modulated by protein phosphorylation, we have performed kinetic modeling of the exocytotic burst and investigated how the kinetic parameters of Ca2+-triggered fusion are affected by PKC activation. We propose that PKC sensitizes exocytosis by reducing the number of calcium binding sites on the Ca2+ sensor (from 3 to 2) without significantly altering the Ca2+-binding kinetics. The reduction in the number of Ca2+-binding steps lowers the threshold for release and up-regulates release of fusion-competent vesicles distant from Ca2+ channels.
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