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Endocrinology Vol. 148, No. 7 3041-3052
Copyright © 2007 by The Endocrine Society

Dehydration-Induced Proteome Changes in the Rat Hypothalamo-Neurohypophyseal System

S. S. Gouraud, K. Heesom, S. T. Yao, J. Qiu, J. F. R. Paton and D. Murphy

Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology (S.S.G., S.T.Y., J.Q., D.M.) and Department of Biochemistry Proteomics Facility (K.H.) and Department of Physiology, Bristol Heart Institute (J.F.R.P.), University of Bristol, Bristol, United Kingdom

Address all correspondence and requests for reprints to: David Murphy, Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology, Dorothy Hodgkin Building, Whitson Street, Bristol BS1 3NY, United Kingdom. E-mail: d.murphy{at}bristol.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The hypothalamo-neurohypophyseal system (HNS) mediates neuroendocrine responses to dehydration through the action of the antidiuretic hormone vasopressin (VP). VP is synthesized as part of a prepropeptide in magnocellular neurons of the hypothalamic supraoptic nucleus (SON) and paraventricular nucleus. This precursor is processed during transport to axon terminals in the posterior pituitary gland, in which biologically active VP is stored until mobilized for secretion by electrical activity evoked by osmotic cues. During release, VP travels through the blood stream to specific receptor targets located in the kidney in which it increases the permeability of the collecting ducts to water, reducing the renal excretion of water, thus promoting water conservation. The HNS undergoes a dramatic function-related plasticity during dehydration. We hypothesize that alterations in steady-state protein levels might be partially responsible for this remodeling. We investigated dehydration-induced changes in the SON and pituitary neurointermediate lobe (NIL) proteomes using two-dimensional fluorescence difference gel electrophoresis. Seventy proteins were altered by dehydration, including 45 in the NIL and 25 in the SON. Using matrix-assisted laser desorption/ionization mass spectrometry, we identified six proteins in the NIL (four down, two up) and nine proteins in the SON (four up, five down) that are regulated as a consequence of chronic dehydration. Results for five of these proteins, namely Hsp1{alpha} (heat shock protein 1{alpha}), NAP22 (neuronal axonal membrane protein 22), GRP58 (58 kDa glucose regulated protein), calretinin, and ProSAAS (proprotein convertase subtilisin/kexin type 1 inhibitor), have been confirmed using independent methods such as semiquantitative Western blotting, two-dimensional Western blotting, enzyme-linked immunoassay, and immunohistochemistry. These proteins may have roles in regulating and effecting HNS remodeling.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE HYPOTHALAMO-neurohypophyseal system (HNS) is a central integrative structure that regulates co-coordinated responses to perturbations in water balance and osmotic stability (1). The HNS consists of the large peptidergic magnocellular neurons (MCNs) (20–40 µm cell body diameter) of the supraoptic (SON) and paraventricular (PVN) hypothalamic nuclei, the axons of which course though the internal zone of the median eminence and terminate on blood capillaries of the posterior lobe of the pituitary gland (PP) (2).

The antidiuretic hormone vasopressin (VP) is synthesized as part of a prepropeptide precursor in the cell bodies of SON and PVN MCNs (3). This precursor is processed during anterograde axonal transportation to terminals in the PP in which biologically active VP is stored until mobilized for secretion into the circulation by MCN electrical activities evoked by hyperosmolality (4, 5). A rise in plasma osmolality is detected by intrinsic MCN osmoreceptor mechanisms (6, 7) and by specialized osmoreceptive neurons in the circumventricular organs that project to, and regulate, SON MCNs (4, 5, 8, 9). During release, VP travels through the blood stream to specific receptor targets located in the kidney in which it increases the permeability of the collecting ducts to water, reducing the renal excretion of water, thus promoting water conservation.

The HNS also produces other neuropeptides in addition to VP, for example, the closely related hormone oxytocin (OT), well known for its roles in parturition and lactation. Single-cell RT-PCR enables VP and OT transcripts to be detected in the same MCN (10), but the expression levels of each neuropeptide RNA differ by orders of magnitude. Only a few percent of MCNs express high, equivalent levels of both peptides (11), although the proportion increases after dehydration (12).

Dehydration evokes a dramatic functional remodeling of the SON, a process known as function-related plasticity (13, 14). A plethora of activity-dependent changes in the morphology, electrical properties, and biosynthetic and secretory activity of the HNS have all been described (15), which may contribute to the facilitation of hormone production and delivery and, hence, the survival of the organism. For example, alterations in the relationship between MCNs and glia, the extent of terminal contact with the basal lamina in the neurohypophysis, the type and weight of synaptic inputs, and the extent of electrotonic coupling between MCNs have all been documented (13, 14, 15, 16, 17). This plasticity appears to be governed by a complex and dynamic interplay between the intrinsic properties of the MCN, interactions between MCNs, interactions with glia, and the influences of extrinsic synaptic inputs. However, the molecular mechanics of these processes are not well understood.

We have begun to investigate the osmotic plasticity of the HNS using high throughput techniques. These have initially focused on the transcriptome, using techniques such as microarray global gene expression profiling to address the question of how many genes are used by the HNS (particularly the SON) and how the overall pattern of gene expression is altered by osmotic cues (18, 19, 20, 21, 22). It is thus apparent that changes in the abundance of many hundreds of mRNA species accompanies physiological stimulation of the HNS, and it has been suggested that such differentially expressed genes are candidate regulators and effectors of HNS activity and remodeling (15, 19, 23).

The relative ease of transcriptome analysis belies a number of problems with the extrapolation of the data to an understanding of biological systems as complex as the HNS. First, the steady-state levels of a transcript and the protein that it encodes are not necessarily directly related (24). Second, a single gene can, through alternative transcriptional initiation points, alternative splicing, and alternative termination, give rise to several transcripts that encode different proteins (25). Third, a protein can be subject to many different types of posttranslational modification that can have profound effects on its activity. Finally, it is the proteins, rather than the mRNAs, that are at the "business end" of the gene expression pathway, being the principal biological effector molecules of a cell. Thus, we extended our global analysis of the dehydration-induced changes in the HNS to encompass the proteome using two-dimensional (2D) fluorescence difference gel electrophoresis (DIGE) combined with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) (26, 27, 28) to identify proteins that change in abundance in the SON and the neurointermediate lobe (NIL) of the pituitary (which encompasses the PP and the intermediate lobe) after 3 d of dehydration in the rat.

Here, we report the results of our interrogation of the HNS proteome. In contrast to the dramatic and numerous changes seen in our microarray analyses of the SON and NIL transcriptomes from euhydrated and dehydrated rats (21, 22), we did not find many proteome changes, and those that we did find were rather small. Seventy proteins were found being regulated in dehydrated rats, including 45 in the NIL and 25 in the SON. Using MS, we identified six proteins in the NIL (four down, two up) and nine proteins in the SON (four up, five down) that are regulated as a consequence of chronic dehydration. Results for five of these proteins, namely heat shock protein 1{alpha} (Hsp1{alpha}), neuronal axonal membrane protein 22 (NAP22), 58 kDa glucose regulated protein (GRP58), calretinin, and proprotein convertase subtilisin/kexin type 1 inhibitor (ProSAAS), have been confirmed using independent methods such as semiquantitative Western blotting, 2D Western blotting, enzyme-linked immunoassay (EIA), and immunohistochemistry.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult male Sprague Dawley rats (10- to 12-wk old; Harlan, Bicester, UK) were maintained at standardized temperature (22 ± 1 C), humidity (50 ± 5%), and diurnal conditions (10-h light, 14-h dark cycle; lights on at 0700 h). Dehydration involved complete fluid deprivation for 3 d (D), whereas control animals (C) had free access to drinking water (tap). Both groups had access to food (standard laboratory rat chow) ad libitum. Three days before tissue extraction, water bottles were removed from the dehydration group at 1100 h. Dehydration for 3 d results in an approximately 12% loss in weight (before, 389.5 ± 1.3 g; after, 339.5 ± 1.3 g; n = 4; P = 3.6 x 10–6). After 72 h of total water deprivation, the rats were killed, and the tissue was extracted and processed as described below. Control animals were also killed at the same time of day. All experimental procedures were approved by the University of Bristol Ethical Review Committee and were performed under government license in accord with the Animals (Scientific Procedures) Act, 1986.

Tissue collection
Rats were stunned and decapitated with a small animal guillotine (Harvard Apparatus, Edenbridge, UK). The brain was rapidly removed from the cranium and placed in an ice-cold brain matrix (ASI Instruments, Warren, MI). Two or three sections of approximately 1-mm thickness were taken, and the SON was carefully dissected on a Petri dish placed above a bed of ice. For the PP, we removed the NIL with a pair of fine forceps from the anterior pituitary. This includes the intermediate lobe as well as the PP. After isolation, the samples were stored for no longer than 1 month at –80 C before additional processing. A single operative performed all dissections.

Sample preparation for 2D-DIGE
Each sample was prepared by pooling the SON or NIL from 10 C or 10 D rats. The tissues were solubilized in 200-µl 2D sample buffer [7 M urea, 2 M thiourea, 30 mM TrisHCl, and 4% (wt/vol) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) (pH 8.5)], a buffer compatible with the 2D-DIGE technique that allows the extraction of the proteins from the whole cell (excepting some plasma membrane proteins). Each sample, 50 µg, was then labeled for DIGE analysis using fluorescent cyanine (Cy) dyes according to the guidelines of the manufacturer (GE Healthcare, Little Chalfont, UK). Samples were labeled using Cy5 (C) or Cy3 (D) N-hydroxysuccinamide (NHS) ester DIGE dyes freshly dissolved in anhydrous dimethylformamide by mixing 50 µg protein with 1 µl CyDye (400 pmol/µl, 5 nmol). In each case, the labeling reaction was allowed to proceed on ice in the dark for 30 min. The reaction was terminated by the addition of 10 nmol lysine and subsequent incubation on ice in the dark for an additional 10 min.

2D gel electrophoresis
For the first dimension, Cy3- and Cy5-labeled samples were combined with 25 µg of each unlabeled sample and rehydration buffer [7 M urea, 2 M thiourea, 4% (wt/vol) CHAPS, 40 mM dithiothreitol (DTT), 0.5% (vol/vol) immobilized pH-gradient (IPG) buffer (pH 3–11 nonlinear), and 0.002% (wt/vol) bromophenol blue] was added to give a total volume of 450 µl. This was loaded onto a 24-cm Immobiline DryStrip gel (pH 3–11 nonlinear) by passive rehydration for a minimum of 12 h. After rehydration, the DryStrip gel was transferred to an Ettan IPGPhor system (GE Healthcare), and isoelectric focusing was performed by applying 500 V for 1 h, 1000 V for 1 h, and 8000 V for 10.5 h until a total of 64,000 Vh had been achieved. After isoelectric focusing, strips were equilibrated in SDS equilibration buffer [50 mM Tris-HCl (pH 8.8), 6 M urea, 30% (vol/vol) glycerol, 2% (wt/vol) SDS, and 0.002% (wt/vol) bromophenol blue] containing 1% (wt/vol) DTT for 15 min at room temperature, followed by an incubation in SDS equilibration buffer containing 2.5% (wt/vol) iodoacetemide for 15 min at room temperature. After equilibration, strips were applied to a 12.5% (wt/vol) SDS-PAGE gel and run at 20 W/gel on an Ettan DALT-12 separation unit (GE Healthcare) until the blue dye front reached the bottom of the gel. The inverted labeling of the samples was run in parallel and confirmed the similar efficiency of labeling for both Cy3 and Cy5 (data not shown).

Image acquisition and analysis
After protein separation, the gel was scanned at two different wavelengths using a Typhoon 9400 variable mode imager (GE Healthcare) to obtain images of the Cy3- and Cy5-labeled proteins. The laser power was chosen so that no saturated signal was obtained. Images were then analyzed using DeCyder Differential In Gel Analysis version 4.0 software (GE Healthcare) to identify spot fluorescence intensities that were increased or decreased after dehydration. Spots to be analyzed were picked from a separate preparative gel on which 150 µg each of unlabeled C and D proteins had been run and stained for total protein using SYPRO ruby protein gel stain, according to the instructions of the manufacturer (Invitrogen, Paisley, UK).

Spot picking, protein processing, and MS
Selected protein spots were cut from the gel, using the Investigator ProPic Automated 2D spot picker (PerkinElmer Life Sciences, Beaconsfield, UK) and digested with trypsin using the ProGest automated digestion unit (both from PerkinElmer Life Sciences). The resulting peptides were analyzed by MS using a Voyager DE-STR mass spectrometer (Applied Biosystems, Foster City, CA) to give a peptide mass fingerprint, which was searched against various databases using the Mascot search program (www.matrixscience.com) to identify the protein present in the gel spot.

Antibodies
Primary antibodies used were as follows: polyclonal goat anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnologies, Santa Cruz, CA), monoclonal mouse anti-NAP22 (kindly provided by Prof. S. Maekawa, Kobe University, Kobe, Japan), polyclonal rabbit anti-HSP90{alpha} (Stressgen Bioreagents, Assay Design, Ann Arbor, MI), polyclonal rabbit anti-GRP58/Erp57 (Stressgen Bioreagents, Assay Design), polyclonal rabbit anti-calretinin (Santa Cruz Biotechnologies), polyclonal rabbit anti-Big-LEN (ProSAAS 245–260) (rabbit number 2766) and polyclonal rabbit anti-Little-SAAS (ProSAAS 42–59) (rabbit number 85b) (kindly provided by Prof L. Fricker, Albert Einstein College of Medicine, New York, NY) (29), monoclonal mouse anti-neurophysin II (VP-derived; PS41) and monoclonal mouse anti-neurophysin I (OT-derived; PS38) (both kindly provided by Prof. H. Gainer, National Institutes of Neurological Diseases and Stroke, National Institutes of Health, Bethesda, MD) (30, 31), polyclonal rabbit anti-neurophysin II (Sigma, St. Louis, MO), and polyclonal goat anti-glial fibrillary acidic protein (GFAP) (Santa Cruz Biotechnologies). Secondary antibodies used were as follows: antimouse IgG horseradish peroxidase-linked secondary antiserum raised in sheep (GE Healthcare), antirabbit IgG horseradish peroxidase-linked secondary antiserum raised in sheep (GE Healthcare), peroxidase-labeled horse antigoat IgG second antibody (Vector Laboratories, Burlingame, CA), biotinylated goat antirabbit or horse antigoat IgG (Vector Laboratories), streptavidin-conjugated Alexa Fluor 594 (Invitrogen), antirabbit Alexa Fluor 594, and antimouse Alexa Fluor 488 (Invitrogen).

Western blotting
Frozen SON and NIL tissue samples from C and D animals were incubated (one animal per sample) in homogenizing buffer [PBS, 1.5% (vol/vol) Nonidet P-40, 0.5% (wt/vol) sodium deoxycholate, 0.1% (wt/vol) SDS, 100 mM sodium orthovanadate, and protease inhibitor cocktail (Sigma)], on ice for 30 min and homogenized by intermittent sonication (two times for 10 s). The homogenates were centrifuged at 8100 rpm in a minicentrifuge (Biofuge Fresco; Heraeus Instruments, Hanau, Germany) for 10 min. The supernatant representing the total cell lysate was collected and stored at –20 C until used for immunoblotting studies. Homogenates were solubilized in sample buffer [17.5% (vol/vol) glycerol, 8.7% (vol/vol) mercaptoethanol, 5% (wt/vol) SDS, 217 mM Tris-HCl, and blue bromophenol] at 100 C for 10 min and subjected to 12.5% (wt/vol) SDS-PAGE. The gel was transferred to Immobilon 45 µm (GE Healthcare) using a mini transblot electrophoretic transfer cell (Bio-Rad, Hercules, CA), and the membrane was blocked in blotting buffer [150 mM NaCl, 20 mM Tris-HCl (pH 7.4), and 0.1% (vol/vol) Tween 20] containing 5% (vol/vol) BSA for 1 h before incubation with the different primary antisera (monoclonal anti-NAP22, diluted 1:1000), polyclonal rabbit anti-HSP90{alpha} (diluted 1:1000), polyclonal rabbit anti-GRP58/Erp57 (diluted 1:1000), and polyclonal rabbit anti-calretinin (diluted 1:500) for 2 h at room temperature or overnight at 4 C in blotting buffer containing 5% (wt/vol) BSA. The membrane was washed for a total 30 min in three changes of the same blotting buffer before incubation in the appropriate horseradish peroxidase-linked secondary antiserum (antimouse IgG horseradish peroxidase-linked secondary antiserum raised in sheep, 1:8000 dilution, or antirabbit IgG horseradish peroxidase-linked secondary antiserum raised in sheep, 1:8000 dilution) for 1 h at room temperature. Membranes were then rinsed several times in blotting buffer (three times for 10 min), and immunoreactive proteins were revealed with ECL plus chemiluminescence reaction (GE Healthcare). Comparing GAPDH band intensity with Coomassie blue staining of C and D total proteins from SON and NIL, we found that the amount of GAPDH is not altered by dehydration in the SON and NIL (data not shown), thus allowing us to use GAPDH as an internal control. The signals were thus normalized with the signal of the GAPDH protein in the same samples. The blot was stripped in stripping buffer [62.5 mM Tris-HCl (pH 6.7), 2% SDS (vol/vol), and 0.7% mercaptoethanol (vol/vol)] at 50 C for 30 min and washed stringently in blotting buffer. The membrane was then processed in exactly the same manner as that described above for visualization of GAPDH protein using a goat anti-GAPDH primary antibody at a dilution of 1:200 and a peroxidase-labeled horse antigoat IgG second antibody at a dilution of 1:100,000. Relative quantitation of the band densities from immunoblots films was performed by using NIH Image J software (version 10.2). We normalized the densitometry values of the tested proteins signal to GAPDH signal, and we defined the mean for the control group as the value 1. P < 0.05 was considered statistically significant (unpaired Student’s t test). We note that quantification by enhanced chemiluminescence is difficult because of the narrow linear range of the signal produced. However, we ensured that assessments were performed using exposures within these limits.

2D Western blotting
As for 2D-DIGE, tissue from the SON (two rats per sample) and NIL (one rat per sample) were solubilized in 200 µl 2D sample buffer [7 M urea, 2 M thiourea, 30 mM TrisHCl, and 4% (wt/vol) CHAPS (pH 8.5)]. Samples were cleaned using the 2D Sample Clean-up kit (GE Healthcare) according the guidelines of the manufacturer. Pellets were resuspended in 100 µl of 7 M urea, 2 M thiourea, and 4% (wt/vol) CHAPS. For the first dimension, 50 or 25 µg of each sample were made up to 125 µl with rehydration buffer [7 M urea, 2 M thiourea, and 4% (wt/vol) CHAPS, 1.5 µl deStreak reagent, 0.5% (vol/vol) IPG buffer pH 4–7 nonlinear, and 0.002% (wt/vol) bromophenol blue]. After 3 h incubation at room temperature, this was loaded onto a 7-cm Immobiline DryStrip gel (pH 4–7 nonlinear) by passive rehydration for a minimum of 12 h. After rehydration, the DryStrip gel was transferred to an Ettan IPGPhor system (GE Healthcare), and isoelectric focusing was performed by applying 500 V for 1 h, 1000 V for 1 h, and 8000 V for 10.5 h until a total of 64,000 Vh had been achieved. After isoelectric focusing, strips were equilibrated in SDS equilibration buffer [50 mM Tris-HCl (pH 8.8), 6 M urea, 30% (vol/vol) glycerol, 2% (wt/vol) SDS, and 0.002% (wt/vol) bromophenol blue] containing 1% (wt/vol) DTT for 15 min at room temperature, followed by an incubation in SDS equilibration buffer containing 2.5% (wt/vol) iodoacetemide for 15 min at room temperature. After equilibration, strips were applied to a 12.5% (wt/vol) SDS-PAGE gel and run at 20 W/gel on a Mini-PROTEAN 3 electrophoresis cell (Bio-Rad) until the blue front reached the bottom of the gel. The gel was transferred to Immobilon 45 µm (GE Healthcare) using a mini semidry transblot electrophoretic transfer cell (Bio-Rad). For antigen detection, the membranes were treated exactly as described above for Western blotting.

Competitive EIA
Homogenization of frozen NILs was performed in 10 vol of boiling water followed by incubation at 100 C for 10 min. The homogenates were subjected to centrifugation (13,000 x g for 30 min). The supernatants were dried in a SpeedVac Plus concentrator (Savant SC11OA; Thermo Fisher Scientific, Waltham, MA) and stored at –20 C. Before EIA, samples were resuspended in StartingBlock T20 (PBS) blocking buffer (pH 7.5) (Pierce, Rockford, IL). The quantification of peptides by competitive EIA was performed using antisera generated against Big-LEN (ProSAAS 245–260) (rabbit number 2766) and Little-SAAS (ProSAAS 42–59) (rabbit number 85b). Synthetic standard peptides and competitor biotinylated peptides, respectively, ProSAAS(245–260)/Big-PEN (rat) (catalog no. 004-56), Pro-SAAS (42–59)/Little SAAS (rat) (catalog no. 044-54), and Biotinyl-Pro-SAAS (245–260)/Big-PEN (rat) (catalog no. B-004-56), and Biotinyl-Pro-SAAS (42–59)/Little SAAS (rat) (catalog no. B-044-54) were produced by Phoenix Pharmaceuticals (Karlsruhe, Germany). The 96-well microplates used to run the EIA were Pre Blocked Reacti-Bind Goat Anti-Rabbit IgG-Coated plates (Pierce via Perbio Science, Cramlington, UK) and StartingBlock T20 Blocking Buffer (Pierce via Perbio Science) was used to dilute all of the components. Plates were used according to the instructions of the manufacturer. Wells were washed using PBS wash buffer containing 0.05% (vol/vol) Tween 20. The capture antibody concentrations used were 1:3000 for anti-Little-SAAS and 1:1500 for anti-Big-LEN. The concentration of competitor biotinylated peptides that was found to prevent 50% of peptide binding was 125 ng/ml for Big-PEN and 25 ng/ml for Little-SAAS. Protein, 160 µg/well (obtained by pooling two NILs), was used to detect Little-SAAS, whereas 600 µg/well (obtained by pooling eight NILs) was used to detect Big-LEN. The enzyme-labeled detection antibody streptavidin-conjugated horseradish peroxidase (Vector Laboratories) was used at a dilution of 1:500. The detection of signal was obtained using Immunopure TMB Substrate kit (Pierce via Perbio Science) following the instructions of the manufacturer, and the absorbances were read at 450 nm in a microplate reader (Jencons-PLS, East Grinstead, UK). Data were analyzed using the Prism software (GraphPad, San Diego, CA) and unpaired Student’s t test. P < 0.05 was considered as statistically significant.

Double-fluorescence immunohistochemistry
Rats were anesthetized with sodium pentobarbitone (100 mg/kg, ip) and transcardially perfused with 100 ml of 0.1 M PBS (pH 7.4) at room temperature, followed by 300 ml of 4% (wt/vol) paraformaldehyde in 0.1 M PBS. Brains were removed, stored, and cryoprotected in fixative containing 20% (wt/vol) sucrose overnight at 4 C. The following morning, brains were rapidly frozen in liquid nitrogen, and four sets of coronal sections (40 µm) of the entire rostrocaudal axis of the forebrain were cut on a cryostat (Cryocut CM3050; Leica Microsystems, Milton Keynes, UK). The free-floating sections were collected in 24-well tissue culture plates containing PBS before being processed for immunohistochemical detection. To determine whether validated proteins and VP-containing neurons have an overlapping or different topography, we used double-labeling fluorescence immunohistochemistry. Free-floating rat hypothalamic sections were incubated for 15 min in a preblocking solution comprising 10% (vol/vol) normal goat or horse serum (Sigma) and 0.3% (vol/vol) Triton X-100 (Sigma) in 0.1 M PBS, followed by rinses in PBS (3–10 min). Sections were then incubated in a monoclonal mouse anti-neurophysin II primary antiserum (1:100 dilution) and polyclonal rabbit anti-HSP90{alpha} (1:500 dilution) or polyclonal rabbit anti-GRP58/Erp57 (1:500 dilution) or polyclonal rabbit anti-Big-LEN (1:500 dilution) in PBS containing 1% (vol/vol) normal goat or horse serum and 0.3% (vol/vol) Triton X-100 for 48 h at 4 C. For double-labeling fluorescence immunohistochemistry of NAP22 and VP, we used the polyclonal rabbit anti-neurophysin II antibody (diluted 1:1000; Sigma) or the polyclonal goat anti-GFAP (diluted 1:100) and the monoclonal anti-NAP22 (diluted 1:1000) in PBS containing 1% (vol/vol) normal horse serum and 0.3% (vol/vol) Triton X-100 for 48 h at 4 C. Sections were rinsed in PBS (three times for 10 min) before a 1 h incubation in PBS containing biotinylated goat antirabbit or horse antigoat IgG (1:500 dilution), 10% (vol/vol) normal goat or horse serum, and 0.3% (vol/vol) Triton X-100 at room temperature. Sections were rinsed in PBS (three times for 10 min), incubated for 1 h in streptavidin-conjugated Alexa Fluor 594 (1:500 dilution) or antirabbit Alexa Fluor 594 and antimouse Alexa Fluor 488 (1:500 dilution) in PBS containing 1% (vol/vol) normal goat or horse serum and 0.3% (vol/vol) Triton X-100. Subsequent to additional washes (three times for 10 min), sections were then mounted onto glass microscope slides with 0.5% (vol/vol) gelatin and allowed to air dry for 10–15 min before being coverslipped using an antifade fluorescent mountant (VectorShield; Vector Laboratories). Confocal images of the sections were acquired using a Leica TCS-NT scanning laser confocal microscope housing a Leica DM IRBE inverted epifluorescence with a two-line krypton/argon laser.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Dehydration-induced differentially expressed proteins in the SON and the NIL
We used 2D-DIGE combined with MALDI-TOF MS to identify proteins that change in abundance in the rat SON and the NIL after 3 d of dehydration. Figures 1Go (NIL) and 2Go (SON) show the superimposition of the Cy5 (C) and the Cy3 (D) labeling. Down-regulated spots appear as red, up-regulated proteins appear as green, whereas unchanged proteins appear as yellow. Using DeCyder Differential In Gel Analysis version 4 software, a total of 25 spots were found decreased or increased by a fold change >1.5 in SON sample and 45 spots for NIL samples. The 1.5-fold cutoff is arbitrary, but it is probably true to say that anything less than this would be questionable. The corresponding spots were identified in the SYPRO stained gel, excised, and digested by trypsin, and the digested peptides were subjected to MALDI-TOF MS. The amino acid sequences for the peptides were identified using Mascot computer searches. Proteins identification was significant for six spots for NIL samples (Fig. 1Go and Table 1AGo) and nine spots (Fig. 2Go and Table 1BGo) for SON samples (Table 1Go); the three-dimensional (3D) pixel density plots of some of them are shown in the respective figures. The theoretical molecular weight, theoretical isoelectric point (pI) values, the locus, and the score of the search (all significant with P < 0.05) are also indicated. Note that we only listed those proteins whose identification was classed as significant. Other spots were identified with a lower degree of confidence (probably reflecting the presence or multiple proteins in a "single" spot, which will reduce the confidence of the Mascot protein identification).


Figure 1
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  		FIG. 1. 2D-DIGE analysis of protein changes in the NIL after 3 d of dehydration. Superimposed images from Cy3- and Cy5-labeled samples run in a 12-cm pH 3–11 IPG strip are shown. Red and green spots represent proteins that are down- and up-regulated after dehydration, respectively, whereas yellow spots represent proteins that are equally abundant in both samples in NIL. The spots shown have a volume change of more than 1.5-fold. 3D representations of some of the spots are shown.

 

Figure 2
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  		FIG. 2. 2D-DIGE analysis of protein changes in the SON after 3 d of dehydration. 2D gels were run from samples separated in a 12-cm pH 3–11 IPG strip (A) and in a 12-cm pH 4–5 IPG strip (B). Images from Cy3- and Cy5-labeled samples were superimposed. Red and green spots represent proteins that are down- and up-regulated after dehydration, respectively, whereas yellow spots represent proteins that are equally abundant in both samples in SON. The spots shown have a volume change of more than 1.5-fold. 3D representations of some of the spots are shown.

 

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  		TABLE 1. Characterization of NIL (top) and SON (bottom) proteins fractionated by 2D-DIGE and identified by MALDI-TOF MS

 
The first step of identification confirmation was the matching of the pI and the molecular weight of the gel spot with that of the protein identified by MS (Table 1Go). These matched except for ProSAAS, ATP6AP2 protein, ubiquitous mitochondrial creatine kinase 1, and NAP22 [also known as Basp1 (brain abundant membrane attached signal protein 1)]. We identified ProSAAS at a molecular mass of 18–21 kDa rather than 30 kDa, perhaps suggesting that we had identified a cleavage product of the native full-length protein. Additionally, we identified a spot corresponding to ATP6AP2 protein at a molecular mass lower (31 kDa) than the expected size at 39 kDa, again suggesting protein cleavage. NAP22 was found at a molecular mass much higher (43 kDa) than predicted (22 kDa). This discrepancy has been reported previously, with the migration of this protein varying with the acrylamide composition of the gel (32).

Although 2D-DIGE combined with MS can provide us with large lists of candidate proteins, these data need to be confirmed using independent methods with independent protein samples. We thus performed semiquantitative Western blotting or EIA experiments using protein samples from SON or NIL of C and D rats on selected proteins for which we could obtain working antibodies. We also analyzed the anatomy of the expression of each protein in the HNS and compared it with VP expression using double-fluorescence immunohistochemistry.

Hsp1{alpha}
2D-DIGE revealed that Hsp1{alpha} (also known as HSP90, spot 449) was decreased by about 1.67-fold change in the SON after dehydration (Fig. 2Go and Table 1BGo). Semiquantitative Western blotting (Fig. 3AGo) also revealed a significant decrease in protein expression in D SON samples (C, 1 ± 0.078; D, 0.735 ± 0.078; n = 9; P = 0.028). Using immunohistochemistry, Hsp1{alpha} were found enriched in the SON and expressed in the cytosol of VP (Fig. 3BGo) and OT (Fig. 3CGo) MCN cell bodies.


Figure 3
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  		FIG. 3. Hsp1{alpha} expression in the SON. A, Semiquantitative immunoblots of Hsp1{alpha}. Western blots were performed using 30 µg total cell protein extract in each lane (n = 9). The signal of the bands intensity was normalized relative to GAPDH and is presented graphically (arbitrary units; *, P < 0.05). B and C, Hsp1{alpha} immunostaining in the C SON. Hsp1{alpha} immunostaining was compared with VP neurophysin II (B) and OT neurophysin I (C) immunostaining. Alexa Fluor 488 and Alexa Fluor 594 fluorescent secondary antibodies were used to reveal VP (PS41, green) or OT (PS38, green) and Hsp1{alpha} (red) immunoreactivities. Yellow cells are both VP/OT and Hsp1{alpha} immunoreactive. Scale bars: left column, 80 µm; right column, 10 µm.

 
NAP22
The 2D-DIGE experiment results showed a dehydration-induced decrease of 1.84-fold (Fig. 2Go and Table 1BGo) for NAP22 (also known as the Basp1, spot 1351) in the SON. The monoclonal antibody against NAP22 detected a band at about 43 kDa rather than 22 kDa, as expected in a 12.5% (wt/vol) acrylamide gel (Fig. 4AGo) (33). Consistent with the 2D-DIGE results, the analysis of band densitometry showed a significant decrease in the NAP22 signal in D samples compared with the SON from C samples (C, 1 ± 0.111; D, 0.6055 ± 0.107; n = 8; P = 0.0294) (Fig 4AGo). Immunohistochemical examination revealed that there was no overlap between the NAP22 and VP staining. Rather, NAP22-like material seemed to surround the intracellular VP inclusions (Fig. 4Go, B and C). Double staining with GFAP, a glial cell marker, suggested that the GFAP fibrillary structure was distinct from the NAP22 staining (Fig. 4DGo). It is thus possible that NAP22 is localized to synapses.


Figure 4
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  		FIG. 4. NAP22 expression in the SON. A, Semiquantitative immunoblots of NAP22. Western blots were performed using 15 µg total cell protein extract in each lane (n = 8). The signal of the bands intensity was normalized relative to GAPDH and is presented graphically (arbitrary units; *, P < 0.05). B, NAP22 immunostaining in the C SON. NAP22 immunostaining was compared with VP neurophysin II immunostaining (B and C) and to GFAP immunostaining (D). Alexa Fluor 488 and Alexa Fluor 594 fluorescent secondary antibodies were used to reveal, respectively, VP or GFAP (green) and NAP22 (red) immunoreactivities. Scale bars: B, 80 µm; C and D, 20 µm.

 
GRP58
The 2D-DIGE experiments revealed that the GRP58 (also known as protein disulfide isomerase associated 3, ER-60, and Erp57, spot 1001) is up-regulated by 1.7-fold change in the SON after dehydration (Fig. 2Go and Table 1BGo). Using semiquantitative Western blotting, the 58-kDa band for GRP58 was also found to be significantly up-regulated by dehydration in the SON (C, 1 ± 0.132; D, 1.593 ± 0.187; n = 4; P = 0.041) (Fig. 5AGo). Using immunohistochemistry, GRP58 was found to be enriched in the SON and expressed in VP (Fig. 5BGo) and OT (Fig. 5CGo) neurons.


Figure 5
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  		FIG. 5. GRP58 expression in the SON. A, Semiquantitative immunoblots of GRP58. Western blots were performed using 15 µg total cell protein extract in each lane (n = 4). The signal of the bands intensity was normalized relative to GAPDH and is presented graphically (arbitrary units; *, P < 0.05). B and C, GRP58 immunostaining in the C SON. GRP58 immunostaining was compared with VP neurophysin II (B) and OT neurophysin I (C) immunostaining. Alexa Fluor 488 and Alexa Fluor 594 fluorescent secondary antibodies were used to reveal VP (PS41, green) or OT (PS38, green) and GRP58 (red) immunoreactivities. Yellow cells are both VP/OT and GRP58 immunoreactive. Scale bars: left column, 80 µm; right column, 10 µm.

 
Calretinin
The 2D-DIGE experiments showed that calretinin (spot 840) is up-regulated by about 1.5-fold change in the NIL of D rats (Fig. 1Go and Table 1AGo). Semiquantitative Western blotting also showed that the protein is significantly up-regulated in D NIL compared with C NIL (C, 1 ± 0.117; D, 1.548 ± 0.127; n = 12; P = 0.004) (Fig. 6AGo), consistent with the 2D-DIGE data. Calretinin has been demonstrated to migrate at a pI of 5.3 in a 2D gel, which corresponds to the most abundant spot we detected in 2D Western blotting (Fig. 6BGo). However, calretinin is also present in another form with a more basic pI (34, 35). Experiments on recombinant calretinin have shown that the pI of the protein is calcium dependent. In the presence of calcium, the protein migrates with a pI of 6.4, whereas in the presence of EGTA, a calcium chelator, the protein has a pI of 5.0 (35). Interestingly, analysis of calretinin by 2D Western blotting of NIL samples from C and D rats using the same antibody revealed two spots at a molecular mass of 31 kDa (Fig. 6BGo). The major spot found at the expected pI of 5 may represent the calcium-depleted form of calretinin, and a less abundant spot detected at a pI of around 6 might represent a calcium-bound form of calretinin. The ratio of the signal for the pI of 6 spot against the pI of 5 spot was found to be significantly increased in the D NIL sample compared with C NIL sample (C, 1 ± 0.21; D, 2.8 ± 0.56; n = 9; P = 0.0087) (Fig. 6BGo).


Figure 6
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  		FIG. 6. Expression of calretinin in the NIL. A, Semiquantitative immunoblots of calretinin in the NIL. Western blots were performed using 15 µg total cell protein extract in each lane (n = 12). The signal of the bands intensity was normalized relative to GAPDH and is presented in the graphic (arbitrary units; *, P < 0.001). B, 2D immunoblot of calretinin in the NIL from C and D rats. The 2D electrophoresis was performed using 50 µg total cell protein extract in each condition (n = 9). The graph represents the ratio in arbitrary units between the intensity of the boxed spot (high pI) and the intensity of the main spot (pI of 4.94) in C and D samples (*, P < 0.05).

 
ProSAAS
Using 2D-DIGE coupled with MS, we found that spots at 18–20 kDa corresponding to a fragment of ProSAAS are down-regulated in the NIL (spots 1140 and 1142, –1.99- and –2.91-fold change, respectively) (Fig. 1Go and Table 1AGo) and up-regulated in the SON (spot 594, +1.5-fold change) (Fig. 2Go and Table 1BGo). Note that spots 1140 and 1142 found in the NIL have similar molecular weights but slightly different pI values, the basis of which is not known.

The ProSAAS precursor protein is known to be processed into a number of small peptides: 34–40 KEP, 42–59 Little-SAAS, 34–59 Big-SAAS, 221–242 PEN, 245–260 Big-LEN, 245–254 Little-LEN, 221–260 Big-PEN-LEN, and 221–254 Little-PEN-LEN (29). The identified ProSAAS protein sequence aligned to amino acids 66–77, 78–87, 91–104, 93–104, 104–111, 118–138, and 202–216, corresponding to the central sequence of the ProSAAS protein between the Little-SAAS and the Big-LEN peptides (data not shown).

To quantify ProSAAS peptide abundance in the NIL from C and D rats, we performed competitive EIA using two specific antibodies against two different amino acid sequences within ProSAAS: Big-LEN (ProSAAS 245–260) and Little-SAAS peptides (ProSAAS 42–59) and the respective synthetic biotinylated peptides as competitors. We detected a significant decrease in immunoreactivity for the Big-LEN peptide (C, 1 ± 0.22; D, 0.23 ± 0.04; n = 4; P = 0.014) (Fig. 7AGo) and Little-SAAS (C, 1 ± 0.12; D, 0.37 ± 0.04; n = 6; P < 0.001) in the NIL from D animals compared with C animals (Fig. 7BGo).


Figure 7
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  		FIG. 7. Expression of ProSAAS peptides in the HNS. Quantitative EIA for Big-LEN (A) and Little-SAAS (B) peptides in the NIL from C and D rats. The EIA was performed using 600 µg (A; n = 4) and 160 µg (B; n = 6) total cell protein extract in each well. The intensity of the signal for each immunoreactive peptide is presented graphically (arbitrary units; *, P < 0.05; ***, P < 0.001). B and C, Big-LEN (LEN) immunostaining in the C SON. Big-LEN immunostaining was compared with VP neurophysin II and OT neurophysin I (C) immunostaining. Alexa Fluor 488 and Alexa Fluor 594 fluorescent secondary antibodies were used to reveal VP (PS41, green) or OT (PS38, green) and Big-LEN (red) immunoreactivities. Yellow cells are both VP/OT and Big-LEN immunoreactive. Scale bars: left column, 80 µm; right column, 10 µm.

 
To localize ProSAAS peptides in the HNS, we used the antibody against the C-terminal portion of ProSAAS (ProSAAS 245–260, Big-LEN) for which efficacy in immunohistochemistry has been validated (36). We show that Big-LEN peptide, an intermediary peptide between the full-length ProSAAS and a small peptide from the C-terminal sequence of ProSAAS, is enriched in the SON and is expressed in the cell bodies of VP and OT (Fig. 7CGo) MCNs.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
To better understand the remodeling that occurs in the HNS as a consequence of osmotic stress, we identified proteins that are up- or down-regulated in the SON and NIL from C and D rats using 2D-DIGE and MALDI-TOF MS. Forty-five spots were found to be changed more than 1.5-fold in the NIL, whereas 25 spots were altered in the SON (Figs. 1Go and 2Go, respectively). Protein identification was significant for six NIL spots (Table 1Go) and nine SON spots (Table 1Go).

Two of the identified proteins (Table 1Go), GFAP and calmodulin, have already been reported as being regulated in the HNS after an osmotic disturbance. We found that the GFAP was increased in the NIL from D rats (Fig. 1Go, spot 2, and Table 1Go), consistent with previous studies reporting an increase in PP immunoreactivity after osmotic stimulation (37). Furthermore, Affymetrix (Santa Clara, CA) microarray analysis (21) has shown that GFAP mRNA is up-regulated by 5.678-fold after 3 d of dehydration in the NIL. Calmodulin, involved in calcium-mediated signal transduction, is increased in the SON after dehydration (Fig. 2Go, spot 631, and Table 1Go). This result is consistent with previous findings showing expression of calmodulin in the hypothalamus (38) and, comparing hyperosmolar and hypoosmolar rats, more calmodulin mRNA in the SON of the former (39). Array analysis has also suggested that 3 d of dehydration increases the abundance of calmodulin transcripts (21, 22). It has been suggested that calmodulin agent may be involved in the regulation of both osmotic- and angiotensin II-induced VP release (40).

The remaining newly identified proteins were validated using independent methodologies and are discussed below in the context of their possible roles in HNS physiology.

Hsp1{alpha}
Hsp1{alpha} colocalizes with VP and OT in SON MCNs and is significantly decreased in abundance after dehydration. Hsp1{alpha} is a 90 kDa protein belonging to the family of the Hsp molecular chaperones, a group of proteins that act principally in the prevention of target protein aggregation and the promotion of their correct folding and assembly (41). Hsp1{alpha} interacts mainly with proteins involved in transcription regulation and signal transduction pathways (42). Its role in the HNS remains to be determined.

NAP22
We found a significant decrease in the abundance of NAP22 protein in the SON of D rats compared with C rats. We note that Affymetrix microarray analysis has shown that the NAP22 mRNA (Basp1) increases by a small but significant degree (1.137 ± 0.043-fold) after 3 d of dehydration, indicating that there is no correlation between the mRNA and the protein steady-state level after dehydration in the SON (Table 1Go), (21, 22). NAP22 has been reported to be expressed throughout the brain, with localization to the synaptic terminals, dendritic spines, and thin nerves fibers associated with synaptic vesicles, presynaptic and postsynaptic membranes, and microtubules (43). NAP22 shows a consistent localization in the SON. NAP22 has a consensus sequence of myristoylation on its N-terminal region, and, once posttranslationally covalently modified, it can bind to the membrane despite a very hydrophilic amino acid sequence (32, 44), explaining its location in the membrane fraction of the brain and on the synaptic vesicles (45). NAP22 appears to be important for neuronal sprouting and plasticity (46), and it has calmodulin-binding activities that can be inhibited by phosphorylation with protein kinase C (32, 47). Whether NAP22 is involved in osmotically induced synaptic remodeling in the SON (15) remains to be determined.

GRP58
2D-DIGE and semiquantitative Western blotting have both shown that the GRP58 is up-regulated in the SON of dehydrated rats. There is clear colocalization of GRP58 with VP and OT in SON MCNs. GRP58 is a member of the protein disulfide isomerase family and is also a glucose-regulated protein induced by a variety of cellular stress conditions. GRP58 is mainly located in the endoplasmic reticulum but has also been found in the cytoplasm and in the nucleus, in which it can bind DNA and may be involved in gene regulation (48). Whether GRP58 is involved in mediated signals from the endoplasmic reticulum to the nucleus in osmotically stressed VP and OT neurons remains to be determined.

Calretinin
Using 2D-DIGE and semiquantitative Western blotting, we found that calretinin is up-regulated in the NIL after dehydration. Although calretinin had been found previously to be expressed in OT SON MCNs (49) and is up-regulated in the SON after salt loading (50), these are the first data on its expression in the NIL. Calretinin is a 28 kDa protein that is widely used as a neuronal marker (51). The protein is characterized by the presence of an evolutionary well-conserved helix-loop-helix motive, which binds Ca2+ ions with high affinity, thus suggesting that calretinin is a regulator of calcium pools critical for synaptic activity (52). Our study using 2D Western blotting of the posttranslational modification of calretinin revealed a significant up-regulation of its basic, possibly calcium-associated form in the NIL of dehydrated rats. Because calcium is an important modulator of VP release HNS (53), calretinin expression level might be modified to modulate the changing intracellular calcium.

ProSAAS
We have shown that fragments of ProSAAS are down-regulated in the NIL and up-regulated in the SON after dehydration. In the latter, ProSAAS peptides colocalize with both VP and OT. ProSAAS mRNA has widespread pattern of expression in the rat brain (medial hypothalamus, arcuate nucleus, SON, and hippocampus), and the protein is expressed abundantly in neuroendocrine tissues, such as the pituitary gland and hypothalamus (36). Within the cell, ProSAAS has been shown to coexist with proprotein convertase 1 (PC1), and PC1 mRNA is known to be expressed in both VP and OT MCNs of the PVN and SON (54). Pro-VP is processed by PC1 (55), and ProSAAS is known to inhibit the activity of PC1 (56, 57, 58). Thus, ProSAAS is potentially an indirect actor in VP and OT processing. PC1 expression is up-regulated in the HNS after dehydration, perhaps to cope with increased VP and OT biosynthesis, and a reduction in ProSAAS levels in the NIL would be consistent with this. Furthermore, ProSAAS-derived peptides have also been shown to be secreted and have endocrine activities related to feeding and obesity (29, 59, 60). An up-regulation of ProSAAS in the SON, along with a decrease in the NIL, might suggest transport of peptides from the SON to the NIL after dehydration, followed by subsequent secretion from PP nerve terminals. Its function in the HNS remains to be determined.

In contrast to the dramatic and numerous changes seen in our microarray analyses of the SON and NIL transcriptomes from C and D rats (21, 22), we did not find many proteome changes, and those that we did find were rather small. It is pertinent at this point to consider the limitations of the technology used. 2D-DIGE is only able to resolve the most abundant 2000 or so proteins in the proteome. The dynamic range of the technique is limited, and most proteins are undetectable or hidden by the noise of the abundant minority. Thus, it is unlikely that the interesting proteins, known to be modulated by dehydration, such as transcription factors (19) or signaling molecules such as IL-6 (18), would be detected by this method. We used whole-cell extracts for our studies; previous cell fractionation might reveal such proteins. The technique further selects for the most soluble proteins and excludes proteins with extreme pIs or very low or very high molecular weights; thus, low-molecular-weight neuropeptides, such as VP and OT, would be excluded. Furthermore, the technique presents a snapshot of the steady-state levels of the detected proteins. An increase in protein activity, and protein turnover, might necessitate an increase in protein synthesis and hence mRNA biogenesis, but there may not be a change in the steady-state level of that protein.

To summarize, 2D-DIGE experiments on SON/NIL from C and D rats combined with MS showed that dehydration affects protein levels. Semiquantitative Western blotting and EIA confirmed that at least five proteins are changed in abundance/posttranslational modification in SON/NIL after chronic dehydration, ProSAAS peptides, NAP22, GRP58, calretinin, and Hsp1{alpha}. Future studies will use gene transfer into whole organisms to elucidate the functions of these proteins in HNS physiology.


   Footnotes
 
This work was supported by Biotechnology and Biological Sciences Research Council Grant 7/S18346.

Disclosure Statement: The authors have nothing to disclose.

First Published Online April 5, 2007

Abbreviations: Basp1, Brain abundant membrane attached signal protein 1; C, control animals; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; Cy, cyanine; D, animals deprived of fluids for 3 d; 2D, two-dimensional; 3D, three-dimensional; DIGE, fluorescence difference gel electrophoresis; DTT, dithiothreitol; EIA, enzyme-linked immunoassay; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFAP, glial fibrillary acidic protein; GRP58, 58 kDa glucose regulated protein; HNS, hypothalamo-neurohypophyseal system; Hsp1{alpha}, heat shock protein 1{alpha}; IPG, immobilized pH-gradient; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; MCN, magnocellular neuron; MS, mass spectrometry; NAP22, neuronal axonal membrane protein 22; NIL, neurointermediate lobe; OT, oxytocin; PC1, proprotein convertase 1; pI, isoelectric point; PP, posterior lobe of the pituitary gland; ProSAAS, proprotein convertase subtilisin/kexin type 1 inhibitor; PVN, paraventricular nucleus; SON, supraoptic nucleus; VP, vasopressin.

Received February 7, 2007.

Accepted for publication March 26, 2007.


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 Introduction
 Materials and Methods
 Results
 Discussion
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