Mechanisms Underlying a Unique Form of Neuroendocrine Adaptation in Osmosensitive Supraoptic Neurons
The neurohormonal mechanisms underlying the regulation of extracellular osmolality are of critical physiological importance. These mechanisms act to maintain the osmolality of human plasma close to a “set-point” of about 290 milliosmoles per litre. The magnocellular neurosecretory cells (MNCs) of the supraoptic nucleus (SON), synthesize and secrete the neurohypophysial hormones vasopressin (VP) and oxytocin (OT). The primary hormonal regulator of osmolality is VP, which is released by the MNCs as a function of plasma osmolality and acts by controlling water reabsorption at the kidneys. MNCs decrease their volume and thus plasma membrane tension in response to acute increases in external osmolality and lack the compensatory mechanisms that limit volume changes in most cell types. This enables them to transduce changes in osmolality into changes in excitability via a mechanosensitive cation channel. It has been shown in vivo that sustained increases in plasma osmolality, however, cause marked hypertrophy of the MNCs that is part of a structural and functional adaptation that is thought to enable the MNCs to secrete large quantities of VP for prolonged periods. The mechanism of this important structural and functional adaptation of MNCs is difficult to address in an in vivo preparation and so an in vitro model of acutely isolated MNCs was used to pharmacologically assess the hypertrophy. It was observed that MNCs exposed to sustained hypertonic solutions, underwent an immediate shrinkage followed by a hypertrophy over 90 minutes and quickly recovered when reintroduced to isotonic conditions. This effect was found to depend on the size of the increase in osmolality, as smaller increases in osmolality resulted in smaller shrinkage and hypertrophy of the MNCs. Hypertrophy was shown to be independent of cell volume regulatory processes as inhibitors of the Na+-K+-Cl- cotransporter did not affect hypertrophy. Hypertrophy was also shown to be dependent on activation of phospholipase C (PLC) and protein kinase C (PKC), as adding inhibitors of these enzymes to the hypertonic solution prevented hypertrophy. Hypertrophy could occur in isotonic conditions by inducing cell depolarization, increasing intracellular calcium ([Ca2+]i) and by activating PKC, thus showing each of these processes are involved in hypertrophy. Recovery from hypertrophy depends upon dynamin-mediated endocytosis as blocking dynamin function prevented recovery. In addition, exposing the MNCs to hypotonic solution resulted in an immediate enlargement followed by a sustained decrease in cell size. Finally, exposing acutely isolated MNCs to hypertonic solution for two hours resulted in a 37% increase in the immunolabeling of the L-type Ca2+ channel CaV1.2 subunit. This increase in CaV1.2 immunolabeling does not depend on action potential firing as adding tetrodotoxin (TTX) to the hypertonic solution failed to prevent the increase. This project will help to elucidate the mechanisms underlying this interesting example of neuroendocrine adaptation and will help us to understand the regulation of body fluid balance during chronic challenges as seen in the elderly and chronically ill.
osmosensitivity, neuroendocrine adaptation, dehydration
Master of Science (M.Sc.)