Magnocellular neurosecretory cells (MNCs) are neuroendocrine cells with somata located in the hypothalamus and nerve terminals in the posterior pituitary. They receive neuronal inputs from the brain and release vasopressin and oxytocin into the blood to regulate many important functions such as water balance, lactation, and parturition. The process of hormone release depends on Ca2+ influx mediated by voltage-gated Ca2+ channels (VGCCs) on the plasma membranes of neuroendocrine cells. To better understand the cellular and molecular components that are involved in regulating secretory vesicle exocytosis, this thesis work was conducted to investigate the expression and function of different subtypes of VGCCs in MNCs and pituicytes (the glial cells surrounding MNC nerve terminals).<p> Molecular biology, immunohistochemistry and cellular biology were used to detect expression and alternative splicing of different VGCC subtypes in MNCs, neurons, and pituicytes. First, the presence of CaV2.2 and CaV2.3 channels were detected on the pituicytes in situ. When the pituicytes were isolated and cultured for 14 days, more VGCC subtypes were expressed including CaV1.2 channels. Regulation of VGCC expression was measured in normal and dehydrated rats, and CaV1.2 channels were found to be selectively up-regulated in pituicytes after 24 hours of dehydration.<p> Second, two splice variants of CaV2.1 channels (CaV2.1Ä1 and Ä2) that lack a large portion of the synprint (synaptic protein interaction) site were detected in the rat brain. To determine whether the splice variants were expressed in MNCs, we did immunocytochemistry using two antibodies (the selective and the inclusive antibody) that recognized the carboxyl-terminus of channels and the synprint site, respectively, in different cell types. We found that vasopressin MNCs, but not the oxytocin MNCs, and one type of endocrine cell (the melanotropes of the pituitary gland) expressed the synprint site deleted variants, whereas the hippocampal neurons mainly expressed the full-length isoform. The splice variants were properly distributed on the plasma membrane of the somata and nerve terminals of the MNCs, suggesting the synprint site is not essential for CaV2.1 channel targeting into the nerve terminals of neuroendocrine cells.<p> Third, expression and distribution of CaV2.2 channels were studied in the MNCs. All CaV2.2 isoforms we detected contained the full-length synprint site. To test the importance of the CASK/Mint1 binding site for CaV2.2 channel targeting, we over-expressed a peptide that inhibits the interaction between CaV2.2 channels and CASK/Mint1 in differentiated PC12 cells (a neuroendocrine cell line). We found that the distribution of CaV2.2 channels in the growth cones of PC12 cells were significantly decreased, suggesting that the CASK/Mint1 interaction is important for CaV2.2 channel targeting into the neuroendocrine terminals.<p> In conclusion, these results provide insights of VGCC expression in neuroendocrine cells, and also give rise to a better understanding of the molecular components that are involved in forming the exocytotic machinery in these cells.

Magnocellular neurosecretory cells (MNCs) are neuroendocrine cells with somata located in the hypothalamus and nerve terminals in the posterior pituitary. They receive neuronal inputs from the brain and release vasopressin and oxytocin into the blood to regulate many important functions such as water balance, lactation, and parturition. The process of hormone release depends on Ca2+ influx mediated by voltage-gated Ca2+ channels (VGCCs) on the plasma membranes of neuroendocrine cells. To better understand the cellular and molecular components that are involved in regulating secretory vesicle exocytosis, this thesis work was conducted to investigate the expression and function of different subtypes of VGCCs in MNCs and pituicytes (the glial cells surrounding MNC nerve terminals).<p> Molecular biology, immunohistochemistry and cellular biology were used to detect expression and alternative splicing of different VGCC subtypes in MNCs, neurons, and pituicytes. First, the presence of CaV2.2 and CaV2.3 channels were detected on the pituicytes in situ. When the pituicytes were isolated and cultured for 14 days, more VGCC subtypes were expressed including CaV1.2 channels. Regulation of VGCC expression was measured in normal and dehydrated rats, and CaV1.2 channels were found to be selectively up-regulated in pituicytes after 24 hours of dehydration.<p> Second, two splice variants of CaV2.1 channels (CaV2.1Ä1 and Ä2) that lack a large portion of the synprint (synaptic protein interaction) site were detected in the rat brain. To determine whether the splice variants were expressed in MNCs, we did immunocytochemistry using two antibodies (the selective and the inclusive antibody) that recognized the carboxyl-terminus of channels and the synprint site, respectively, in different cell types. We found that vasopressin MNCs, but not the oxytocin MNCs, and one type of endocrine cell (the melanotropes of the pituitary gland) expressed the synprint site deleted variants, whereas the hippocampal neurons mainly expressed the full-length isoform. The splice variants were properly distributed on the plasma membrane of the somata and nerve terminals of the MNCs, suggesting the synprint site is not essential for CaV2.1 channel targeting into the nerve terminals of neuroendocrine cells.<p> Third, expression and distribution of CaV2.2 channels were studied in the MNCs. All CaV2.2 isoforms we detected contained the full-length synprint site. To test the importance of the CASK/Mint1 binding site for CaV2.2 channel targeting, we over-expressed a peptide that inhibits the interaction between CaV2.2 channels and CASK/Mint1 in differentiated PC12 cells (a neuroendocrine cell line). We found that the distribution of CaV2.2 channels in the growth cones of PC12 cells were significantly decreased, suggesting that the CASK/Mint1 interaction is important for CaV2.2 channel targeting into the neuroendocrine terminals.<p> In conclusion, these results provide insights of VGCC expression in neuroendocrine cells, and also give rise to a better understanding of the molecular components that are involved in forming the exocytotic machinery in these cells.