Regulation of vasopressin gene expression in rat hypothalamic ...

THEJOURNALOF BIOLOGICAL CHEMl8TRY 0 1986 by The American Societyof Biological Chemists, Inc.

Vol. 261, No. 28, Issue of October 5,pp. 12956-12959.1986 Printed in U.S.A.

Regulation of Vasopressin Gene Expressionin Rat Hypothalamic Neurons RESPONSETO

OSMOTIC STIMULATION* (Received for publication, May 23,1986)

Hans H. Zinggl, DianaLefebvre, and Guillermina Almazang From the Laboratoly of Moleculnr Endocrinolopy, Royal - Victoria Hospital, McGiEl University, Montreal, Quebec, C a d a H3A 1A1 ”

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 hybridizationassay, using in vitro synthesized sense-strand RNA as a quantitative standard andcomplementary anti-sense RNA as a specific probe. The template used for RNA transcription consisted of a 196-base pair genomic DNA fragment corresponding toexon C of the ratAVP gene. Determination of basal hypothalamic AVP mRNA levels yielded 12.5 F 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, plasmaosmolality 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-dayrehydration period. Furthermore,pituitary AVP reached a level of 177% of control by day 1 4 of rehydration. These data show that 1) osmotic stimulation results in a long-lasting elevation of hypothalamic AVP mRNA; 2) during rehydration, these elevated mRNA levels direct AVP biosynthesis at a rate which surpasses secretory demands; 3) AVP mRNA accumulation does not appear to be directly regulated by either pituitary or hypothalamic AVP. Therefore, either an unusually long half-life of 27 days must be assumed for AVP mRNA or, alternatively, a continued stimulation of AVP gene transcription must occur, even in following comthe absence of a secretory stimulus and plete repletionof cellular AVP stores.

The mechanisms involved in the regulation of peptide biosynthesis in neurons are still poorly understood. In partkular, it is unclear to what extent electrophysiological activity, secretory activity, or changes in peptide stores may play a role in the regulation of the biosynthetic processes. Since the discovery of the neurosecretory cell by Scharrer (I), the magnocellular hypothalamoneurohypophyseal system has served as animportant model for the study of peptide biosynthesis, processing, and release. Over the last several decades, this system has been extensively studied using electrophysiological, anatomical, and biochemical approaches (2), culminating in the recent elucidation of the vasopressin and oxytocin gene structure in man, rat, and cow (3-5). Therefore, the hypothalamic magnocellular neurons should provide an ideal model system for studies designed to deepen our understanding of the mechanisms controlling gene expression in neurons. As first described by Verney (6), a rise in plasma osmolality represents a powerful stimulus for vasopressin (AVP’) secretion by hypothalamic neurons. In addition, prolonged osmotic stimulation by either water deprivation or salt imbibition leads to a marked depletion of the AVP stores inthe posterior pituitary (7). 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), as well as AVP tissue stores in the hypothalamus (site of biosynthesis) and in the neural lobe of the pituitary (storage site). Our results indicate that, during dehydration, AVP mRNA levels are directly correlated with the rising levels of plasma osmolality and inversely correlated with the decreasing AVP levels in pituitary and hypothalamus. By contrast, our study further indicates that neither of these correlations is maintained duringthe ensuing period of rehydration. EXPERIMENTAL PROCEDURES~

*This work was supported by the Medical Research Council of Canada and by the Fonds de laRecherche en Santi. du Qubbec. The work was carried out in accordance to theGuidelines for the Handling of Recombinant DNA Molecules and Animal Viruses and Cells published by the Medical Research Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18U.S.C. Section 1734 solelyto indicate this fact. $ Supported by a Scholarship from the Fonds de la Recherche en Santb du Qubbec. 8 Supported by a Fellowship from the Medical Research Council of Canada.

Abbreviations used AVP, [Ar$]vasopressin; AVP mRNA, rat preprovasopressin neurophysin I1 mRNA; kb, kilobase; bp, base pair; DTT, dithiothreitol; U, unit; RIA, radioimmunoassay; SDS, sodium dodecyl sulfate. Portions of this paper (including “Experimental Procedures” and Fig. 4) are presented in miniprint at the endof this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 86M-1723, cite the authors, and include a check or money order for $2.00 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

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Regulation of Vasopressin Gene Expression 1

A

2

3

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* L

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hybridized with labeled AVP anti-sense RNA were scanned densitometrically, and the absorbance of the hybrid image was recorded as described earlier (9). Quantitative interpretation of the densitometric measurements was rendered possible by the inclusion of a set of serially diluted sense-RNA standards in each blot (Fig. 1B). The linear range of the standard curve extended from 63 pg to 1 ng (Fig. IC). This range was therefore used for the conversion of the densitometric values of autoradiographic signals into absolute values (nanograms or femtomoles). Based on the assay shown inFig. 1 plus three additional independent assays, the amount of AVP mRNA per hypothalamus was estimated at 12.5 2.7 fmol. The lowest dose that gave an autoradiographic signal corresponded to %2 of one hypothalamic equivalent or 0.39 fmol of AVP mRNA. Regulation of A V P mRNA Levels Following Osmotic Stimulation-Replacement of drinking water by 2% NaCl over a 6-day period resulted in a 19% lossof body weight and a 6% increase in both plasma osmolality and serum [Na+] (Fig. 2). Concomitantly, the AVP stores in the pituitary decreased to 18%of the control values, whereas the levels of AVP in the hypothalamus, the siteof AVPbiosynthesis,decreased to 61% of control levels. Measurements of hypothalamicAVP mRNA, on the other hand, revealed a steady increase in levelsof gene transcripts from day 1 of dehydration onwards. Over the entire 6-day periodof dehydration, the mRNA levels increased 3-foldto reach a maximum valueof 35f5 fmol/hypothalamus (Figs. 2 and 3). Following rehydration, body weight, serum osmolality, and [Na’] returned to control within 2 days. The levels of AVP in the pituitary returned to normal by day 6 of rehydration

*

C

sense-strand RNA standard (pg)

FIG. 1. Quantitative AVP mRNA assay. A, autoradiograph of serially diluted rat hypothalamic AVP mRNA. Lanes 1-4 represent %,%,%, and %6 of a normal rat hypothalamus, respectively. Arrowheads indicate the position of RNA size markers. From top to bottom: 28 S RNA, 18 S RNA, and tRNA. B, serially diluted sense-strand RNA standards. Lanes 1-4 contain 1,0.5,0.25, and 0.125 ng of sensestrand RNA, respectively. The RNA was electrophoresed, transferred to a membrane, and hybridized with labeled AVP anti-sense RNA as described under“Experimental Procedures.” The membrane was exposed for 36 h to Kodax XAR-1 film using an intensifying screen. C, plot of sense-strand RNA standard ( x axis) against the area under the curve of densitometric scans of the hybridization signal (y axis). The autoradiogram shown in B as well as three additional autoradiograms were used. Each point, therefore, represents the mean & S.E. of four independent determinations.

A

DAYS OF DEWYORATION

RESULTS

Quantitative Measurement of HypothalamicAVP mRNATissue extracts from adult rat hypothalami contained a single species ofAVP transcripts which hybridized specifically to the anti-sense RNA probe employed (Fig. 1A). Using 28 S RNA, 18 S RNA, and tRNA as size markers, the size of the transcript was estimated at 0.7 kilobase, in accordance to data reported by others (4, 8). In order to study the regulation of AVP gene expression, a quantitative densitometric hybridization assay was developed. Autoradiographs ofRNA blots

DAYS O f REHYDRATION

FIG.2. Effects of salt imbibition and rehydration on hypothalamic AVP mRNA and hypothalamic as well as pituitary AVP content (upperpunel)and on plasma osmolality, plasma [Na+], and body weight (lower panel). The time points representedare (from left to right): control, 1, 2, and 6 days of salt imbibition and 2, 6, 14, 22, and 30 days of rehydration. Each data point represents the mean f S.E. of three to six independent determinations ( n = 6 for control, 6 days salt imbibition and 14 days rehydration; n = 3 for all other time points). Standard errors have been partially omitted for clarity in the curves depicting plasma osmolality and plasma [Na’]. C.L., control level. Asterisks denote values which differ significantly from control a t a level of p < 0.05.

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Regulation of Vasopressin Gene Expression

moles rather than in nanograms, the actuallength of the RNA species measured is irrelevant for the calculations. AVP Transcription in Response to Osmotic StimuhtionSeveral indirect approaches have earlier been used for the estimation of net hypothalamic AVP synthesis under control conditions and under osmotic stress. Valtin and co-workers (11)based their calculations on data pertainingto changes in pituitary AVP stores and the rate of AVP secretion during dehydration (11).They estimated a &fold increase in AVP synthesis following prolonged water deprivation. Additional findings which attested to an increased biosynthetic activity of magnocellular vasopressinergic neurons following dehydration include ultrastructural changes at the level of the endoplasmatic reticulum (12), augmented cell and nucleolar volume (13), and increased [35S]cysteineincorporation into AVP FIG. 3. Effect of salt imbibition on hypothalamic AVP precursor molecules (14). The finding of an increase in hymRNA. RNA extracted from individual rat hypothalami or sense- pothalamic AVP mRNA levels followingonset of dehydration strand RNA standards were electrophoresed and hybridized to labeled is, therefore, consistent with these earlier observations and anti-sense VP-C RNA as described under “ExperimentalProcedures.” demonstrates that the regulation of AVP synthesis involves In A, each lane contains the equivalent of one-half rat hypothalamus. control mechanisms operating at apretranslational level, Lane 1, control; lanes 2-4, 1, 2, and 6 days of salt imbibition; lanes 5 most likely at thelevel of AVP gene transcription. These data and 6 , 6 and 14 days of rehydration following a 6-day salt imbibition period. B, serial dilution of sense-strand RNA standards, run on the are also consistent with our recent findings using in situ same gel as A. Lanes 1-6 contain 2, 1, 0.5, 0.25, 0.125, and 0.063 ng hybridization techniques (15) as well as with reports using a of sense-strand RNA. Exposure time, 36 h. liquid hybridization assay (16) or blot hybridization (17, 18). As we show in the presentstudy, 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. This is evidenced by a rise of AVP stores in the by this point in time. Replenishment of hypothalamic AVP pituitary which exceeds the control level by 77% byday 14 of followed a somewhat slower time course, but was complete by rehydration. This continued biosynthesis occurs after that the day 14 of rehydration. By contrast, thelevels of AVP mRNA original stimulus for secretion has been shut off for a t least 7 did not show any significant change following the onset of days. Since AVP mRNA levels remain significantly elevated rehydration and remained significantly elevated (250-280% following complete repletion of tissue stores, it seems unlikely that either the rise in blood osmolality or the depletion of of control) over the entire30-day rehydration period. cellular AVP stores has a direct action on the regulation of AVP genetranscription. Alternatively, an unusually long halfDISCUSSION life, well in excess of 7 days, would have to be assumed for Quuntitation of A VP mRNA-The techniques currently AVP mRNA. Although there is currently no information on used for the measurement of tissue levels of specific mRNA the half-life of any mammalian neuropeptide mRNA species, species suffer from the disadvantage that they are notstrictly data are available on the stability on some abundant mRNA quantitative. Single stranded sense-strand DNA fragments species, including globin, ovalbumin, casein, and vitellogenin cannot provide accurate standards, since the melting temperature of DNA.DNA hybrids differs from that of DNA- RNA (19-22). None of these mRNA species seems to possess a halflife in excess of 2 days. Therefore, it appears reasonable to hybrids (10). Thus, only pure sense-strand RNA can serve as assume that AVP gene transcription remains activated bean appropriate standard. However, it is generally impossible to isolate the mRNA species to be measured in pure form and yond the period of acute cellular stimulation andcellular AVP in sufficient quantities from a given tissue. By contrast, it is depletion. A definite answer, however, must await the availfeasible to generate considerable amounts of pure RNA of ability of data on AVP mRNA stability. defined sequence and length by in vitro transcription of a REFERENCES cloned DNA template using specific viral RNA polymerases. 1. 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