EFFECT OF WATER DEPRIVATION ON KCC2 EXPRESSION IN HYPOTHALAMIC VASOPRESSIN NEURONS IN RAT

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2013-04-12

Authors

Knapp, Blayne

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Abstract

Purpose: Argininge Vasopressin (AVP) is a neurohypophyseal hormone that contributes to body fluid homeostasis by regulating plasma fluid and electrolyte composition. It is released from the posterior pituitary (PP) by magnocellular neurosecretory cells (MNCs) located within the supraoptic (SON) and paraventricular (PVN) nuclei of the hypothalamus. The regulation of AVP release is critical for maintaining blood volume and blood pressure, and although the molecular mechanisms of AVP regulation are not fully understood, we do know that it is partially due to the vital balance of synaptic excitatory and inhibitory inputs that determine the relationship between plasma osmolality and AVP release. Dysregulation of this system and the resulting disturbances of water and electrolyte balance can lead to increased morbidity associated with disease states such as heart and liver failure. This is why it is critical for us to elucidate the molecular mechanisms involved with its physiological control and its inappropriate release in disease states. Models of water deprivation represent a physiological challenge that requires sustained release of AVP from the PP into circulation as a means of maintaining body fluid homeostasis. We attempt to address the gaps that remain controversial or unexplained in our current understanding of AVP regulation by measuring changes in the expression of extruder KCC2 in water deprived and euhydrated adult male rats and measuring its effects on inhibitory GABAergic neurotransmission in AVP MNCs. Methods: Using laser capture microdissection (LCM), AVP MNCs were harvested from the SON and PVN of euhydrated and 48 hour water deprived adult male rats and reverse transcriptase polymerase chain reaction (RT-PCR) studies were used to test for changes in KCC2 message. Western blot protocols were used to measure changes in protein expression. Results: We observed a significant elevation in KCC2 mRNA expression in AVP cells of the SON (WD 2.5 ± 0.52; Control 1.0 ± 0.06, p<0.05) but not in the PVN (WD 3.5 ± 1.7; Control 1.0 ± 0.1). Immunofluorescence demonstrated the colocalization of KCC2 and AVP in the SON. Conclusions: Increased expression of KCC2 could be associated with decreased intracellular Cl- in AVP neurons in the SON, thereby serving to maintain or enhance the inhibitory tone of AVP neurons in the SON but not the PVN during WD.

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