| dc.description.abstract | The regulation of exocytosis and endocytosis in the insulin secreting pancreatic b-cell was studied by microfluorimetry and digital imaging, whole-cell and single-channel recordings of voltage-gated Ca2+-channels, measurements of changes in cell capacitance and flash release of caged compounds. It is well established that insulin secretion is dependent on an increase in [Ca2+]i. Previous work has suggested that exocytosis in the b-cell is principally determined by [Ca2+]i in the vicinity of the Ca2+-channels where the concentrations are likely to be higher than indicated by microfluorimetry. This possibility was explored by combining digital imaging of granule distribution and depolarization-evoked changes of [Ca2+]i with whole-cell/single-channel recordings of Ca2+-channel activity. It could be demonstrated that L-type Ca2+-channels co-localize with the secretory granules and that the [Ca2+]i-transient during the time required to elicit exocytosis is confined to this region of the cell; measured [Ca2+]i being 5-10-fold higher in this part of the cell than in the opposite pole of the cell. An increase in [Ca2+]i is certainly required to initiate exocytosis in the b-cell. However, from this study it has emerged that the b-cell has evolved extensive systems for the modulation of its exocytosis. These systems operate via changes in the extent of phosphorylation of, as yet, unidentified exocytosis-regulating proteins. In general, conditions promoting protein phosphorylation, such as activation of protein kinases or inhibition of protein phosphatases, result in stimulation of exocytosis. Measurements of cell capacitance also enabled us to demonstrate that the hypoglycaemic sulphonylureas, in addition to closing ATP-sensitive K+-channels in the plasma membrane of the b-cell, also are insulinotropic by a more direct interaction with the exocytotic machinery. In this context it is also of interest that up to 90% of the sulphonylurea binding in the b-cell is localized to the secretory granules. The cellular/molecular mechanisms mediating the effect remain elusive but some evidence implicates protein kinase C in the process. The b-cell contains more than 10000 secretory granules but, as in other neuroendocrine cells, only a fraction of these are immediately available for Ca2+- or GTP-dependent release (the readily releasable pool). Recent evidence suggests that the secretory granule needs to be chemically modified prior to release: a process referred to as priming and supposed to involves hydrolysis of ATP by the protein NSF. We demonstrate that manoeuvres which prevent ATP-hydrolysis suppress a late (>200 ms) component of exocytosis whereas the initial phase (<200 ms) is almost unaffected. The amplitude of the capacitance increase observed after replacement of the stable analogue AMP-PCP for ATP or after inhibition of NSF by NEM suggests that the number of primed granules is ª50 in the pancreatic b-cell; much lower than corresponding values in other neuroendocrine cells. The latter value compares favourably with that which can be calculated to be released during first phase glucose-induced insulin secretion in vivo. After exocytosis, the secreted membranes are recaptured by endocytosis. Capacitance measurements offer the unique possibility to monitor endocytosis. Endocytosis follows on exocytosis after a variable delay and is initiated by an increase in [Ca2+]i, but does not require a sustained elevation to continue. In most cases, the amount of endocytosis matches the preceding exocytosis suggesting that the cell has means of recognizing the granular membrane. Contrary to exocytosis, endocytosis is independent of access to metabolic energy and is unaffected by activation of protein kinases A and C. These feature lead us to conclude that exo- and endocytosis, although being interdependent, are regulated by different cellular mechanisms. | en |
