Stimulus-Synthesis/Secretion Coupling: The Tilapia Prolactin Cell as a Model Osmoreceptor

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Prolactin (PRL) is a highly versatile hormone of the pituitary gland of all vertebrates. It has essential actions in the regulation of salt and water balance (osmoregulation) in fish, including the tilapia, Oreochromis mossambicus, a species which can move freely between fresh water and seawater (it is euryhaline). In the diluting freshwater environment, blood ion levels tend to fall and PRL acts to counter this by increasing blood osmolality and reducing the water and ion permeability of skin and other osmoregulatory surfaces. Consistent with these actions, PRL cells show greater activity in tilapia held in fresh water than in those held in seawater. Blood PRL also rises and remains elevated as blood osmolality declines when tilapia move from seawater to freshwater. The tilapia PRL cell appears to be directly responsive to changes in extracellular osmolality inasmuch as PRL release increases as medium osmolality is reduced when tilapia pituitary glands are incubated in vitro. The term "osmoreceptor" refers to cells that respond to osmotic signals with an appropriate physiological response that is evoked through mechanisms called signal transduction pathways. Calcium (Ca+2) is widely used in these pathways as a second messenger that mediates the induction of cellular responses to specific external signals. The osmoreceptive properties of the tilapia PRL cells offer an excellent and possibly unique model system for investigating the pathways through which an osmotic signal is transduced into an osmoregulatory response (i.e., PRL secretion). In teleosts, unlike in other vertebrates, PRL cells are segregated into the anterior portion of the pituitary as a nearly homogeneous mass, which is easily separated for organ and dispersed-cell culture. This anatomical arrangement greatly facilitates the characterization of secretory mechanisms, and provides a highly useful model that can augment our general understanding of the regulation of other less-accessible osmosensitive (and osmoregulatory) endocrine and neuroendocrine systems (e.g. the hypothalamo-neurohypophysial magnocellular systems involved in vasopressin and oxytocin release in mammals).The long-term objective of this research is to identify the mechanisms involved in the regulation of the production and release of PRL by changes in the osmotic environment of the tilapia PRL cell. The present studies build on previous work in this laboratory, and are designed to construct a more detailed picture of signal transduction in osmoreceptive cells. The specific aim is to determine whether the rise in PRL release in response to reduced extracellular osmolality is triggered by an increase in cell size that leads to an increase in intracellular free Ca+2 ([Ca2+]i). The hypothesis is that a fall in extracellular osmolality leads to the passive influx of water into the PRL cell that increases cell volume; this initiates an influx of extracellular Ca2+ and a rise in [Ca2+]i, which triggers the increase in PRL release. In order to address this question, experiments will be conducted to determine: 1) whether an increase in cell size leads to an increase in [Ca2+]i; 2) whether an increase in cell size initiates a rise in [Ca2+]i and/or an increase in PRL release; and 3) whether an increase in cell size and PRL release following exposure to hyposmotic medium is dependent on the entry of extracellular Ca2+.To achieve these aims, in vitro perifusion-incubation techniques will be used which allow measurements of individual cell size, PRL release, and intracellular calcium concentration ([Ca2+]i) through video-captured images, homologous radioimmunoassays, and microspectrofluorometry, respectively. The general approach for testing the proposed hypotheses consists of utilizing endogenous regulators of PRL release and pharmacological agents to manipulate the various steps putatively involved in the transduction of the osmotic signal. The proposed research will provide a better understanding of the signal transduction processes involved in osmoreception, a sensory modality whose full understanding has been hampered by the lack of a suitable model system. The use of the tilapia PRL cell as a model offers distinct investigative advantages unavailable to researchers addressing similar questions in other vertebrate osmoregulatory systems.