Detection of vasopressin mRNA in the neurointermediate lobe of the rat pituitary

Molecular Brain Research, 8 (1990) 325-329

325

Elsevier BRESM 70240

Detection of vasopressin mRNA in the neurointermediate lobe of the rat pituitary Joseph T. McCabe 1, Elke Lehmann 2, Nicole Chastrette 1, Jorg H/inze 2, Rudolf E. Lang 2, Detlev Ganten 2 and Donald W. Pfaff 1 1Laboratory of Neurobiology and Behavior, The Rockefeller University, New York, NY 10021-6399 (U.S.A.) and 2Department of Pharmacology and Institute for High Blood Pressure Research, University of Heidelberg, Heidelberg (F.R. G.) (Accepted 15 May 1990)

Key words: Vasopressin; Antidiuretic hormone; mRNA; Gene expression; Posterior pituitary; Neurointermediate lobe; Hypothalamus; Nucleic acid hybridization

A messenger ribonucleic acid (mRNA) homologous to the transcript that encodes vasopressin (VP) was detected in the neurointermediate lobe (NIL) of the rat pituitary. The abundance of this transcript is approximately 1/100th the amount detected in the hypothalamus. In rats drinking 2% NaCl-water for 0, 2, 4, or 10 days, or for 10 days and then tap water for 14 days, the levels of VP mRNA in the NIL were altered in a fashion that paralleled changes in the hypothalamus.

INTRODUCTION

MATERIALS AND METHODS

T h e h y p o t h a l a m o - n e u r o h y p o p h y s i a l system is comp o s e d of m a g n o c e l l u l a r secretory neurons that p r o j e c t to the p o s t e r i o r pituitary lobe (neurohypophysis). The n e u r o h y p o p h y s i a l h o r m o n e s , vasopressin (VP) and oxytocin ( O T ) , as well as o t h e r n e u r o p e p t i d e s , are synthesized in the p e r i k a r y a of these neurons, and are delivered via axonal t r a n s p o r t to the p o s t e r i o r pituitary for secretion into the general circulation. Peripherally secreted VP and O T then subserve m a n y h o m e o s t a t i c functions including w a t e r balance, stress, lactation, and parturition 1A1. T h e sequence of events that leads to the p r o d u c t i o n of VP and O T has been an excellent m o d e l system of n e u r o s e c r e t o r y protein synthesis 6,1°, and the cloning and sequencing of the genes encoding VP and O T clarified their p r i m a r y structure 25. Recently, V P o r in s o m e cases its messenger R N A , has b e e n d e t e c t e d in p e r i p h e r a l organs, including thymus, testes, corpus luteum, anterior pituitary, dorsal r o o t ganglion, and the a d r e n a l gland 7'16'31. D a t a p r e s e n t e d initially by L e h m a n n 14 indicates some vasopressin m R N A is also localized in p o s t e r i o r pituitary tissue. The p r e s e n t r e p o r t is a confirmation and extension of these initial findings and was briefly r e p o r t e d in abstract form 20.

Animal treatments Male, Long-Evans strain rats (Charles River) were randomly assigned to one of five treatment conditions: maintenance on tap water (0 Days condition), or maintenance on 2% sodium chloride water as drinking solution for 2, 4, 10 days, or for 10 days followed by 14 days on tap water. Food was provided ad libitum. At the end of the treatment condition, rats were sacrificed with an overdose of sodium pentobarbital (60 mg/kg), were decapitated, and the brain and pituitary were removed and placed upon a chilled glass plate. The anterior pituitary lobe was separated from the neurointermediate lobe with the aid of two fine-needle forceps, and the separated tissues were rapidly frozen by placement on separate pieces of aluminum foil. Before freezing the hypothalamus, it was blocked from the rest of the brain (with the aid of a clean razor blade)at the following extents: rostrally, at the level of the anterior commissure; caudally, at the level of the ventromedial nucleus, and dorsally, at the mid-dorsoventral extent of the thalamus. The hypothalamic blocks weighed between 150 and 250 mg. After blocking, the following tissue samples were frozen in separate pieces of aluminum foil: the hypothalamic blocks, a sample of the cortex which overlies the diencephalon (100-200 rag), and the cerebellum (200-280 rag). All samples were placed in 50-ml plastic tubes for storage at -70 °C.

RNA preparation Tissue RNA from the pituitary, hypothalamus, cortex, and cerebellum was extracted with LiC1/urea according to the procedure of Auffray and Rougeon 2. Briefly, the frozen tissue was placed in the bottom of SorvaU tubes (DuPont, Wilmington, DE) and 0.01 ml/mg tissue (v/w) of cold homogenization buffer was added to the tube. The homogenization buffer contained 3 M LiC1, 6 M urea, 10

Correspondence: J.T. McCabe, Department of Anatomy, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814-4799, U.S.A. 0169-328X/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

326 mM sodium acetate (pH 5.0), 200 ktg/ml heparin, and 0.1% SDS. Brain tissue was dispersed with a tissue homogenizer (Brinkmann Instruments, Westbury, NY) for 90 s. For pituitary samples, the tissue was placed in 250 /~1 of the homogenization buffer and homogenized for 30 s. After storage overnight at 4 °C, the samples were centrifuged for 1-3 h (4-8 °C, 25,000 rpm). The supernatant was then discarded and the pellets were vortexed with 1 ml (or 300 /~1 for pituitary samples) of 4 M LiCI and 8 M urea, and centrifuged for approximately 15 min (4-8 °C, 25,000 rpm). The supernatant was then discarded, and tubes were inverted to remove excess supernatant. For brain tissue samples, 300 /~1 SP/SDS (50 mM sodium acetate, pH 5.0/1% SDS) and 300/d phenol were sequentially added (200/A solutions for pituitary samples), to transfer the pellets by pipette to 1.5 ml sterile microfuge tubes. The capped tubes were then placed on a shaker for 30 min, allowed to then settle for 15 min, spun (10,000 rpm, Brinkmann microfuge, 25 °C), and the aqueous phase was carefully pipetted to a second set of sterile microfuge tubes. Cold ethanol (2.5x vol.) and 0.3 M sodium acetate (pH 5.5) was added to the tube before overnight storage (-20 °C). RNA was then precipitated by centrifugation in a microfuge (30 min, 14,000 rpm, 4 °C), the supernatant discarded, the pellet rinsed with 80% ethanol, re-centrifuged (15 min, 4 °C), and dried under vacuum. Brain RNA samples were re-dissolved in 150/~1 diethylpyrocarbonate-treated, autoclaved water (pituitary samples in 35/~1) and frozen (-20 °C). Optical density measures determined total RNA content and the relative yield of each sample.

Solution hybridization The solution hybridization procedure was conducted, with modifications outlined below, as previously described 14'17A9. In brief, two aliquots of each sample were diluted in separate tubes to a final concentration of 40% formamide, 0.4 M NaCI, 1 mM EDTA, 40 mM PIPES (pH 6.7). Two microgram RNA samples from hypothalamus were always used, while samples of cerebellum and cortex were 2-100 ktg. Pituitary samples were - 5 ktg RNA/tube. In order to make the amount of total RNA equivalent in each tube, yeast tRNA (Sigma Chemical Co., Type-X: -<43/~g/tube) was added to each sample 13. Approximately 30,000 cpm of 32p-labeled cRNA probe 2~ complementary to exon-C of the rat vasopressin gene (SstI-DraI fragment, 242 base pairs (bp) in length) was added per tube (specific activity 3-9 x l0 s cpm//~g), and hybridizations were for approximately 44 h at 73 °C. For the particular probe used in these investigations, the time, temperature, and formamide con-

centration of the hybridization buffer were all parameters which previous work had empirically demonstrated would produce stringent and complete hybridization 17. After hybridization, singlestranded RNA was digested (1 h at 30 °C) by the addition of buffer containing 40 pg/ml RNAse-A (Boehringer-Mannheim) and 2/~g/ml RNAse-T1, 0.3 M NaCI, 5 mM EDTA, 10 mM Tris-HCl pH 7.5. The remaining hybrids were precipitated with 10% trichloroacetic acid-l.5% sodium pyrophosphate, filtered over nitrocellulose (Grade BA85: Schleicher and Schuell, Keene, NH), dried (1 h, 70 °C), and the radioactivity was counted in liquid scintillation fluid. The assay was calibrated by executing parallel hybridizations with a known standard amount of hypothalamic RNA, and results were compared by utilizing the linear region of the standard curve to determine the amount of cpms/hybridization (see below).

Controls and analysis of solution hybridization results As noted earlier, samples from the same stock of hypothalamic RNA were incorporated into each experiment. This permitted comparisons of results (expressed as cpms/tube) across hybridization trials since the precise amount of probe, probe specific activity, and hybridization conditions could vary from one experiment to another. Known standard amounts of hypothalamic RNA were hybridized at increasingly greater concentrations to establish the relationship between input RNA and amount of detected signal. The linear portion of the curve (see Results below) was then used to calculate the amount of VP mRNA per sample from measured cpms, as well as to measure background (the y-intercept) due to the formation of hybrids resistant to RNAse treatment. For pituitary samples, background was calculated with respect to counts obtained from 45 ktg samples of tRNA. No more than 2 pg of hypothalamic RNA was used in a single hybridization tube to ensure the measured cpms was within the linear range (see below) of measurement under the present experimental conditions. The results reported below are from a total of 6 hybridization runs, but never more than 60% of the data for any tissue or treatment group were from a single hybridization experiment. In order to determine the validity of the hybridization procedure, 2.5 /~g of hypothalamic RNA was hybridized in the presence of increasingly larger amounts of probe. Utilizing the procedure described by Bishop 3, one can then estimate the amount of VP mRNA in hypothalamic tissue. The results obtained from the different durations of salt-loading were compared using a single-factor analysis of variance design 32

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Fig. 1. Left: hybridization of the VP-specific probe as a function of input RNA. Under the same hybridization conditions, hybridization of increasingly greater amounts of hypothalamic total RNA (abscissa) with a fixed amount of 32p-labeled antisense probe results in a linear relationship for 1-8/~g of input RNA. Right: hybridization of varying amounts of labeled VP antisense probe with a fixed amount (2.5 #g) of hypothalamic RNA. A plot of increasingly greater amounts of antisense probe (denoted as R on the abscissa) and the ratio of input antisense probe (R) to amount of probe that hybridizes (RH) generates a linear function. (For convenience in plotting, both of these variables were plotted as R/IO00 and RH/IO00.) The reciprocal of the slope determines the 'saturation value' (see Bishop 3, equation 13, Fig. 2) as -7.1 fM per hypothalamus. Linear equation: y" = 0.15x + 2.97, regression coefficient r = 0.99.

327 and post hoc comparisons of the treatment conditions utilized the Newman-Keuls test (P < 0.05).

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Analysis of solution hybridization results Fig. 1 (left) demonstrates the results of titration of a constant amount of probe with different amounts of hypothalamic RNA. As expected, the first component of the curve (1-8 /tg input RNA) is essentially a linear function of input RNA. Amounts of hypothalamic RNA greater than - 8 / ~ g results in the apparent elimination of all unhybridized VP probe from the solution 13. We have determined the amount of VP mRNA in the hypothalamus by a saturation kinetic experiment 3. Utilizing a constant amount of input tissue, Fig. 1 (right) plots the relationship between increasing amounts of the labeled VP probe and the ratio of the amount of input VP probe to the amount of probe that hybridizes to the tissue RNA. The reciprocal of the slope gives one an estimate of the 'saturation value', and in conjunction with estimates of probe specific activity, to then determine the concentration of VP mRNA in hypothalamic tissue blocks as - 7 . 1 fM. This estimate is in line with our previous determination 19 (Long-Evans rats: - 6 . 7 fM), as well as estimates by Ludwig TM (Wistar rats: - 5 . 9 fM) and Zingg 33 (Sprague-Dawley rats: -12.5 fM). As demonstrated in many previous investigations, the maintenance of separate groups of rats on 2% salt water

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as drinking solution for increasingly longer durations results in a graded increase in VP mRNA content in the hypothalamus (Fig. 2). By Day 10 of salt loading, rats

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Fig. 3. Left: solution hybridization of samples of neurointermediate lobe (NIL, black bars) and anterior lobe (AL, striped bars) to the VP antisense probe where samples were obtained from groups of rats that received tap water to drink (denoted 0 Days), or salt water to drink for 2, 4, or 10 days, or 10 days followed by 14 days of water drinking. The number of rats per group were 8, 6, 8, 9, and 7, respectively. The histograms denote the group means and bars indicate the standard error of the mean. For NIL samples, the Newman-Keuls test indicated the 10 Day condition was significantly different from the 0 Day and 10 Day + 14 Day conditions (P < 0.05). The analysis of variance for the anterior lobe data (AL) was not statistically significant (P > 0.10). The higher means observed for both the 2 Day and 10 Day + 14 Day groups are, in each case, the result of a single AL sample that exhibited a spuriously high count (1042 and 2526 cpm, respectively). The means (+ S.E.M.) for these groups without these data are 108.0 _+ 108 and 67.3 + 67 cpm, respectively. Right: a scatterplot of the relationship between the group mean percent change in VP mRNA content in the hypothalamus and in the neurointermediate lobe for the five treatment conditions. The point at x and y equals 100% and refers to the tap water drinking (control) group. The calculated Pearson correlation coefficient was 0.93 (P < 0.01).

328 exhibited a signifiant 2.4-fold increase in hypothalamic content of VP m R N A compared to animals maintained on tap water. In rats that were first maintained on salt water for 10 days, but then received tap water for 14 days (10 Days + 14 Days Water), levels of VP m R N A in the hypothalamus exhibited a dramatic diminution compared with the 10 day salt-treated animals (P < 0.05) and were equivalent to levels seen in the control rats (see also ref. 5). Akin to what was observed in the samples of hypothalamic RNA, increasingly longer durations of salt water drinking lead to higher amounts of VP m R N A in samples of the neurointermediate lobe (Fig. 3, left). After 10 days of salt water drinking, rats exhibited an approximately 6.3-fold increase in VP m R N A content in the neurointermediate lobe. Returning salt-loaded rats to tap water drinking resulted in a precipitous fall in VP mRNA content to levels not significantly different from animals that were never maintained on salt water. We have estimated the amount of VP m R N A in the neurointermediate lobe to be approximately 1/100th the amount of VP m R N A in the hypothalamus. This determination is based upon the results of the magnitude of the signal detected in hypothalamus and pituitary samples and the total RNA mass of these tissues. We have found no evidence for comparable or even lower levels of VP m R N A in the anterior lobe samples, since the levels estimated from the solution hybridization results are the same magnitude seen in samples of cortex, cerebellum, and tRNA samples. No reliable alteration in this background level of hybridization signal was seen in anterior pituitary lobe samples as a result of the salt loading treatment (analysis of variance: P > 0.10). The alteration in VP m R N A in the hypothalamus and neurointermediate lobe follow the same general response pattern: namely, an increase in VP m R N A content with salt loading and a similar decrease in m R N A content following a return to water drinking. Fig. 3 (right illustration) depicts the relationship between the percent change in VP m R N A content relative to control rats and there would appear to be a parallel change at both sites as a function of the osmotic stimulation condition. DISCUSSION The solution hybridization method was employed to detect VP m R N A in the neurointermediate lobe of the pituitary. This work confirms the initial observations of Lehmann TM, and is consonant with the independent reports of others (refs. 15, 22, 31 and D. Richter, personal communication). We estimate VP m R N A levels in the posterior lobe to be far less than levels in the hypothalamus, where they may be less than 1/100th the

amounts seen in the latter tissue. Using Northern blotting, we have also observed (data not shown) this message is shorter in nucleotide length compared to what exists in the hypothalamus. Utilizing RNAse-H digests, Murphy and coworkers 22 have shown this is due to the apparently shorter poly-A tail of the pituitary message. The present investigation examined how VP m R N A content in the hypothalamus and the pituitary changed as a function of osmotic state. Solution hybridization results (summarized in Fig. 3, right illustration) show a strikingly similar change in message levels in both tissues. It would seem the pituitary lobe vasopressin m R N A could be derived from at least 3 sources. One possibility is that there are solitary vasopressinergic neurons in the posterior pituitary. To our knowledge no papers have reported such an observation. A second possibility is that the m R N A is transported by vasopressinergic axons to the terminal sites in the neural lobe. This does not concur with the prevailing view that axons do not contain or transport RNA s'12, but at least two recent papers were published that add some plausibility to this idea. Rapallino 24 and Capano 4, and their coworkers, have reported the squid giant axon contains R N A that could represent mRNA. A portion of their R N A samples were of the size (nucleotide length) that is consistent with mRNA, and they found part of their samples from the squid axon are also polyadenylated. In addition, Dirks and colleagues 9 have reported the presence of m R N A encoding the neuropeptide, egg-laying hormone, in axons of the pond snail (Lymnaea stagnalis). The cellular content of vasopressin m R N A is similarly high in terms of copies/cell, and it is also localized in morphologically similar - - that is, large, metabolically active - - neurosecretory cells. A third, and more likely, possibility is that the vasopressin message is derived from pituicytes or resident endothelial cells. As observed in peripheral organs, m R N A levels are low, and the message is shorter 14'22, due to an apparent difference in poly-A tail length 22. This may reflect the fact that the processes which control transcription and polyadenylation do not arise from a hypothalamic neurosecretory cell. This possibility is also in line with the recent report of Schafer and colleagues 27 who have detected proenkephalin m R N A in rat pituicytes by in situ hybridization. Finally, neuropeptide gene expression in cultured astrocytes was recently described by Schwartz, Shinoda, and their coworkers 2s' 29 This is relevant to the present observations since pituicytes are a type of astrocyte 26'3°, and it may be VP m R N A is expressed during the proliferative stage of pituicyte or endothelial cell mitosis 23. Further study is warranted to determine the precise source of this pituitary VP m R N A , as well as its functional significance.

329

Acknowledgements. The authors thank Drs. David Murphy, David Carter, and Dietmar Richter for discussion of their results. Supported by NIH Grants NS25913 to J.T.M. and HD05751 to D.W.P. The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or

reflecting the views of the Department of Defense of the USUHS. The research reported herein was conducted according to the principles set forth in the Guide for Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, National Research Council, HHS Pub. No. (NIH) 85-23, revised 1985.

REFERENCES

18 Ludwig, G., H/inze, J., Lehmann, E., Lang, R.E. and Ganten, D., Measurement of mRNA for arginine vasopressin (AVP) in spontaneously hypertensive rats (SHRSP) by a newly developed liquid hybridization assay, Abstract, llth Sci. Meeting Int. Soc. Hypertens., Heidelberg, p. 214. 19 McCabe, J.T., Almasan, K., Lehmann, E., Hanze, J., Lang, R.E., Pfaff, D.W. and Ganten, D., In situ hybridization demonstrates vasopressin gene transcription in hypothalamic neurons of crossbred hypertensive × diabetes insipidus rats, Neuroscience, 27 (1988) 159-167. 20 McCabe, J.T., Lehmann, E., H/inze, J., Lang, R., Ganten, D. and Pfaff, D.W., Detection of vasopressin mRNA in the posterior pituitary by solution hybridization and Northern blotting, Soc. Neurosci. Abstr., 15 (1989) 344. 21 Melton, D.A., Krieg, P.A., Rebagliati, M.R., Maniatis, T., Zinn, K. and Green, M.R., Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage Sp6-promoter, Nucl. Acids Res., 12 (1984) 7035-7056. 22 Murphy, D., Levy, A., Lightman, S. and Carter, D., Vasopressin RNA in the neural lobe of the pituitary: dramatic accumulation in response to salt loading, Proc. Natl. Acad. Sci. U.S.A., 86 (1989) 9002-9005. 23 Patterson, J.A. and Leblond, C.P., Increased proliferation of neuroglia and endothelial cells in the supraoptic nucleus and hypophysial neural lobe of young rats drinking hypertonic sodium chloride solution, J. Comp. Neurol., 175 (1977) 373-390. 24 Rapillino, M.V., Cupelio, A. and Giuditta, A., Axoplasmic RNA species synthesized in the isolated giant axon, Neurochem. Res., 13 (1988) 625-631. 25 Richter, D., Molecular events in expression of vasopressin and oxytocin and their cognate receptors, Am. J. Physiol., 255 (1988) F207-F219. 26 Salm, A.K., Hatton, G.I. and Nilaver, G., Immunoreactive glial fibrillary acidic protein in pituicytes of the rat neurohypophysis, Brain Res., 236 (1982) 471-476. 27 Sch/ifer, M.K.-H., Day, R., Ortega, M.R., Akil, H. and Watson, S.J., Proenkephalin messenger RNA is expressed both in the rat anterior and posterior pituitary, Neuroendocrinology, 51 (1990) 444-448. 28 Schwartz, J.P., Shinoda, H., Marini, A.M. and Cosi, C., Neuropeptide gene expression in cultured astrocytes: brain region and gene specificity, Soc. Neurosci. Abstr., 15 (1989) 690. 29 Shinoda, H., Marini, A.M., Cosi, C. and Schwartz, J.P., Brain region and gene specificity in cultured astrocytes, Science, 245 (1989) 415-417. 30 Suess, U. and Pli~ka, V., Identification of pituicytes as astroglial cells by indirect immunofluorescence staining for glial fibrillary acidic protein, Brain Res., 221 (1981) 27-33. 31 Tracer, H.L. and Loh, Y.P., Vasopressin (AVP) gene expression in the rat pituitary: localization and preliminary characterization of the transcripts, Endocr. Soc. Abstr., (1989) Abstract #55. 32 Winer, B.J., Statistical Principles of Experimental Design, 2nd edn., McGraw-Hill, New York, 1971, 907 pp. 33 Zingg, H.H., Lefebvre, D. and Almazan, G., Regulation of vasopressin gene expression in rat hypothalamic neurons: response to osmotic stimulation, J. Biol. Chem., 261 (1986) 12956-12959.

1 Amico, J.A. and Robinson, A.G., Oxycotin, Excerpta Medica, Amsterdam, 1985, 444 pp. 2 Auffrey, Ch. and Rougeon, E, Purification of mouse immunoglobulin heavy-chain messenger RNAs from total myeloma tumor RNA, Eur. J. Biochem., 107 (1980) 303-314. 3 Bishop, J.O., DNA-RNA hybridization, Acta Endocrinol., Suppl. 168 (1972) 247-276. 4 Capano, C.P., Giuditta, A., Castigli, E. and Kaplan, B.B., Occurrence and sequence complexity of polyadenylated RNA in squid axoplasm, J. Neurochem., 49 (1987) 698-704. 5 Carrazana, E.J., Pasieka, K.B. and Majzoub, J.A., The vasopressin mRNA poly(A) tract is unusually long and increases during stimulation of vasopressin gene expression in vivo, Mol. Cell. Biol., 8 (1988) 2267-2274. 6 Castel, M., Gainer, H. and Dellman, H.-D., Neuronal secretory systems, Int. Rev. Cytol., 88 (1984) 304-359. 7 Clements, J.A. and Funder, J.W., Arginine vasopressin (AVP) and AVP-like immunoreactivity in peripheral tissue, Endocr. Rev., 7 (1986) 449-479. 8 Davis, L., Banker, G.A. and Steward, O., Selective dendritic transport of RNA by hippocampal neurons in culture, Nature, 330 (1987) 477-479. 9 Dirks, R.W., Raap, A.K., Van Minnen, J., Vreugdenhil, E., Smit, A.B. and van der Ploog, M., Detection of mRNA molecules coding for neuropeptide hormones of the pond snail Lymnaea stagnalis by radioactive and non-radioactive in situ hybridization, J. Histochem. Cytochem., 37 (1989) 7-14. 10 Gainer, H., Altstein, M., Whitnall, M.H. and Wray, S., The biosynthesis and secretion of oxytocin and vasopressin. In E. Knobil and J. Neill (Eds.), The Physiology of Reproduction, Raven, New York, 1988, pp. 2265-2282. 11 Gash, D.M. and Boer, G.J., Vasopressin: Principles and Properties, Plenum, New York, 1988, 635 pp. 12 Lasek, R.J. and Brady, S.T., The axon: a prototype for studying expressional cytoplasm, Cold Spring Harbor Syrup. Quant. Biol., 46 (1981) 113-124. 13 Lee, J.L. and Costlow, N.A., A molecular titration assay to measure transcript prevalence levels, Methods Enzymol., 152 (1987) 633-648. 14 Lehmann, E., Untersuchungen zur Expression des Vasopressingens in der Ratte, Doctoral Dissertation, University of Heidelberg, 1988. 15 Lehmann, E., Hanze, J., Pauschinger, M., Ganten, D. and Lang, R.E., Vasopressin mRNA in the neuroiobe of the rat pituitary, Neurosci. Lett., 111 (1990) 170-175. 16 Loh, Y.P., Castro, M.G., Zeng, E-J. and Patel-Vaidya, U., Presence of pro-vasopressin mRNA, neurophysin and arginine vasopressin in mouse anterior pituitary cells and AtT-20 corticotrophic tumour cell line, J. Mol. Endocrinol., 1 (1988) 39-48. 17 Ludwig, G., H/inze, J., Lehmann, E., Lang, R.E., Burbach, J.H.P. and Ganten, D., Measurement of mRNA by solution hybridization with 32p-labelled single stranded cRNA probe ('SP6 test'). Comparison with a 32p-labelled single stranded cDNA as hybridization probe ('S~ test') for measurement of AVP mRNA, Clin. Exp. Hypertens. Theor. Pract., A10 (1988) 467-483.

Note added in proof The cited work of Richter et al. has now been published: Mohr, E., Zhou, A., Thorn, N.A. and Richter, D., Rats with physically disconnected hypothalamo-pituitary tracts no longer contain vasopressin-oxytocin gene transcripts in the posterior pituitary lobe, FEBS Lett., 263 (1990) 332-336.