Abstract
Although the steps involved in biosynthesis and secretion of the neuropeptide vasopressin (AVP) have been extensively studied, the factors which regulate AVP gene expression remain unknown. Therefore, we sought to determine the dynamics of AVP mRNA accumulation in response to a strong stimulus for AVP release, i.e. during salt imbibition and the ensuing period of rehydration. AVP mRNA levels were determined in terms of absolute amounts by a novel quantitative densitometric hybridization assay, using in vitro synthesized sense-strand RNA as a quantitative standard and complementary anti-sense RNA as a specific probe. The template used for RNA transcription consisted of a 196-base pair genomic DNA fragment corresponding to exon C of the rat AVP gene. Determination of basal hypothalamic AVP mRNA levels yielded 12.5 +/- 2.7 fmol/hypothalamus. Salt imbibition, which induced a 6% rise in blood osmolality and an 82% loss of pituitary AVP, resulted in a 3-fold increase of AVP mRNA to 35 +/- 5 fmol/hypothalamus. Following rehydration, plasma osmolality returned to control levels by day 2, pituitary AVP by day 6, and hypothalamic AVP by day 14. By contrast, AVP mRNA levels remained significantly elevated throughout the 30-day rehydration period. Furthermore, pituitary AVP reached a level of 177% of control by day 14 of rehydration. These data show that osmotic stimulation results in a long-lasting elevation of hypothalamic AVP mRNA; during rehydration, these elevated mRNA levels direct AVP biosynthesis at a rate which surpasses secretory demands; AVP mRNA accumulation does not appear to be directly regulated by either pituitary or hypothalamic AVP. Therefore, either an unusually long half-life of greater than or equal to 7 days must be assumed for AVP mRNA or, alternatively, a continued stimulation of AVP gene transcription must occur, even in the absence of a secretory stimulus and following complete repletion of cellular AVP stores.
Highlights
The steps involved in biosynthesis and secretion of the neuropeptide vasopressin (AVP) have been extensively studied, the factors which regulate AVP gene expression remain unknown
As we show in the presentstudy, rat preprovasopressin neurophysin I1 mRNA (mRNA) levelsremain high and rose significantly above control to reach, by day 14, a during the period of rehydration and result in a continued level whichcorresponded to 177%of control (Fig. 2), indicat- biosynthetic activity which surpasses largely the secretory ing that thepituitary AVP stores hadbeen amply replenished demands
Quuntitation of A VP mRNA-The techniques currently used for the measurement of tissue levels of specific mRNA species suffer from the disadvantage that they are nosttrictly quantitative
Summary
The steps involved in biosynthesis and secretion of the neuropeptide vasopressin (AVP) have been extensively studied, the factors which regulate AVP gene expression remain unknown. In order to investigate the regulation of AVP gene expression in response to a secretory stimulus, we determined, in the present study, the levels of rat hypothalamic AVP mRNA before, during, and following osmotic stimulation, using aquantitativeand specific AVP mRNA assay technique. In order to define the observed regulatory response of AVP gene expression within the physiological context of the entire homeostatic system, we have measured the following system parameters concomitantly: blood osmolality and Na+ concentration (stimulatory input to thesystem), aswell as AVP tissue stores in the hypothalamus (site of biosynthesis) and in the neural lobe of the pituitary (storage site).
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