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. 2010 Sep 22;151(11):5403–5414. doi: 10.1210/en.2010-0345

Hypothalamic Paraventricular Nucleus Gαq Subunit Protein Pathways Mediate Vasopressin Dysregulation and Fluid Retention in Salt-Sensitive Rats

Richard D Wainford 1, Daniel R Kapusta 1
PMCID: PMC2954710  PMID: 20861238

Abstract

Central Gαz and Gαq protein-gated pathways play a pivotal role in modulating (inhibiting vs. stimulating, respectively) vasopressin release and urine output; these studies examined the role of brain Gαz/Gαq proteins in the regulation of vasopressin secretion during high-salt challenge. We examined the effects of 21-d normal or high salt intake on plasma vasopressin levels, daily sodium and water balance, and brain Gαz and Gαq protein levels in male Sprague–Dawley (SD), Dahl salt-resistant (DSR), and Dahl salt-sensitive (DSS) rats. Additionally, the effect of central Gαq protein down-regulation on these parameters and the diuretic response evoked by pharmacological [nociceptin/orphanin FQ; 5.5 nmol intracerebroventricularly (icv)] and physiological stimuli (isotonic-saline volume expansion, 5% bodyweight, iv) was examined. After 21 d of high salt intake, DSS, but not SD or DSR rats, exhibited vasopressin dysregulation, as evidenced by elevated plasma vasopressin levels (P < 0.05), marked positive water (and sodium) balance (P < 0.05), and an impaired diuretic response to pharmacological and physiological stimuli (P < 0.05). Chronic high salt intake (21 d) evoked down-regulation of Gαq (P < 0.05), but not Gαz, proteins in the hypothalamic paraventricular nucleus of SD and DSR, but not DSS rats. In salt-challenged (21 d) DSS rats, acute oligodeoxynucleotide-mediated down-regulation of central Gαq proteins returned plasma vasopressin to control levels (P < 0.05), decreased salt-induced water retention (P < 0.05), and restored the profound diuretic responses to pharmacological and physiological stimuli (P < 0.05). Therefore, the down-regulation of PVN Gαq proteins plays a critical counter-regulatory role in preventing vasopressin hypersecretion in salt-resistant phenotypes and may represent a new therapeutic target in pathophysiological states featuring vasopressin dysregulation.

Central Gαz and Gαq proteins represent a novel molecular mechanism that regulates the appropriate secretion of vasopressin in response to increased dietary salt intake.

The nonapeptide, vasopressin (AVP), plays an essential role in regulating plasma osmolality and fluid and electrolyte homeostasis by enhancing water reabsorption in the collecting ducts of the kidneys (1). Within the central nervous system (CNS), the endocrine hormone AVP is synthesized in the magnocellular neurons of the paired paraventricular (PVN) and supraoptic (SON) nuclei of the hypothalamus (1,2). Vasopressin is then secreted into the systemic circulation from the posterior pituitary in response to increased plasma osmolality/sodium concentration (predominant) or decreased blood volume/pressure (3). Changes in body fluid osmolality/sodium concentration are sensed within the CNS by osmo-sodium receptors located in the lamina terminalis, which has direct neural projections to the PVN and SON (4). These integrated neural pathways regulate sympathetic outflow via the hypothalamic parvocellular neurons and appropriate AVP secretion via the PVN magnocellular neurons to maintain osmoregulatory homeostasis.

Several disease states are associated with elevated plasma AVP levels and marked water retention, including hepatic cirrhosis (5), congestive heart failure (6), and certain models of hypertension (including salt-sensitive hypertension) (7,8). However, little is known about the central molecular mechanisms that mediate AVP dysregulation. We have demonstrated that brain GTP-binding protein coupled receptor (GPCR) Gαz and Gαq subunit protein signaling pathways have a novel physiological gating role in controlling AVP secretion (inhibitory vs. stimulatory, respectively) (9). Whether alterations in brain Gαz and/or Gαq subunit protein pathways participate in manifesting AVP dysregulation in different disease states remains to be investigated.

In addition to the development of hypertension, Dahl salt-sensitive (DSS) rats manifest marked elevations in plasma AVP levels upon consumption of a chronic high NaCl diet (8,10,11). In contrast, in salt-resistant rats [e.g. Sprague–Dawley (SD) and Dahl salt-resistant (DSR) rats] increased dietary salt intake fails to alter circulating AVP levels, total body water balance, or systemic arterial blood pressure (7,12). Based on our recent findings (9), it is possible that high NaCl intake differentially affects central Gαz and/or Gαq subunit protein pathways and thus, AVP secretion, in SD and DSR rats vs. DSS rats. We hypothesize that in animals which remain normotensive when faced with an elevated dietary salt intake (SD and DSR rats), chronic high NaCl intake triggers a change in brain Gαz and/or Gαq subunit protein levels, which favors an inhibitory influence on AVP secretion and thereby prevents water retention. Alternatively, in salt-sensitive animals (DSS rats), we predict that this central molecular mechanism is not functional and leads to AVP hypersecretion.

Materials and Methods

Animals

Male Sprague–Dawley (SD), male SR/JrHsd Dahl salt-resistant (DSR), and male SS/JrHsd Dahl salt-sensitive (DSS) rats (Harlan Laboratories Inc., Indianapolis, IN), age matched at 7–8 weeks at time of purchase, were housed individually under a 12-h light, 12-h dark cycle and randomly assigned to experimental treatment groups. Rats were allowed tap water ad libitum and were fed either standard control rodent diet which contained a total NaCl content of 0.4% (174 meq Na+/kg), or a modified high-salt rodent diet with a total NaCl content of 8% (1,378 meq Na+/kg); both diets were purchased from Test Diet (Richmond, IN). All animals were maintained on either a control or high-salt diet for a 21-d period, and all experimental protocols were performed on d 21 of the dietary salt intake period. All procedures were conducted in accordance with National Institutes of Health and the Louisiana State University Health Sciences Center Institutional Animal Care and Use Committee guidelines for the Care and Use of Animals.

Surgical procedures

Sprague–Dawley, DSR, and DSS rats were maintained on either a control diet (0.4% NaCl) or a high-salt (8% NaCl) diet for 21 d, the latter of which induces salt-sensitive hypertension in DSS rats (8,13). In certain experimental groups a stainless steel cannula was stereotaxically implanted into the right lateral cerebral ventricle of anesthetized rats (30 mg/kg im ketamine in combination with 3 mg/kg im xylazine) 5–7 d before experimentation (i.e. 5–7 d before d 21 of dietary salt intake) as previously described (9,16,17). In experiments in which the measurement of systemic arterial blood pressure, heart rate, and/or renal function was required on the day of study rats were anesthetized with sodium methohexital (20 mg/kg ip, supplemented with 10 mg/kg iv as required) and instrumented with catheters in the left femoral artery, left femoral vein, and bladder, as described previously (9,16,17). After surgical preparation rats were placed in a rat holder and an iv infusion of isotonic saline was initiated and continued for the duration of the experiment. The experimental protocol commenced after the animal regained full consciousness, and cardiovascular and renal excretory functions stabilized. Mean arterial pressure (MAP) and heart rate (HR) were continuously recorded via the surgically implanted femoral artery cannula using computer-driven BIOPAC data acquisition software (MP100 and AcqKnowledge 3.8.2) connected to an external pressure transducer (P23XL; Viggo Spectramed Inc., CA).

Metabolic balance procedures

All rats were housed in individual metabolic cages (model 18cv, Fenco, Cataumet, MA) with external food containers and water bottles. Metabolic cages were equipped with a double-fine mesh screen that allowed separation of food and feces contamination from urine that was collected in vials which contained a layer of mineral oil to prevent urine evaporation. Rats were fed either a control diet (0.4% NaCl) or a high-salt diet (8% NaCl) and allowed tap water ad libitum via external trays and bottles, respectively. All animals were allowed to adapt to the metabolic cages for 3 d before data collection commenced, which occurred on d 21 of the diet regimens. Measurements were made for body weight, food and water intake, and urine output during a 24-h period enabling calculation of daily sodium and water balance. After completion of the metabolic balance study the same animals were then either decapitated for measurement of plasma AVP, or surgically instrumented (see Surgical Procedures above) for measurement of systemic arterial blood pressure. After the completion of all experimental protocols, brains were collected and frozen for measurement of Gα-subunit proteins.

Oligodeoxynucleotide administration procedures

As outlined in specific protocols, rats previously instrumented with an icv cannula were randomly assigned to receive an acute single icv injection (delivered over a 60-second period) of a scrambled (SCR) (25 μg/5 μl, 5′-GGGCGAAGTAGGTCTTGG-3′) or a Gαq (25 μg/5 μl, 5′-GCTTGAGCTCCCGGCGGGCG-3′) phosphodiesterase oligodeoxynucleotide (ODN) probe dissolved in isotonic saline (9,14,15). All ODN sequences were purchased from the Midland Certified Reagent Company Inc. (Midland, TX). After an NCBI Basic Local Alignment Search Tool (blast) search of the Rattus norvegicus RefSeq protein database, it was confirmed that the Gαq ODN is specific for Gαq and will not interact with any other rat protein gene sequence and that the SCR ODN sequence does not match any rat protein sequence.

CNS tissue collection

Animals which underwent metabolic balance studies were killed by decapitation; whole brains were removed and frozen at −80 C. Frontal brain cortex (BC), PVN, and ventrolateral medulla (VLM) samples were extracted from frozen brains cut on a cryostat using a brain punch tool (Stoelting, Kiel, WI). Brain cortex and PVN samples were taken using a punch diameter of 1.00 mm, VLM samples were taken using a punch diameter of 0.76 mm and stored at −80 C. The location of the PVN and VLM was determined using visual landmarks (12,18) and by identification of neuron populations in sections examined under a light microscope.

Gα-protein immunoblotting

Tissue lysates were prepared from brain punch samples, and protein levels were quantified using the BCA assay as per manufacturers’ instruction (Thermo Scientific, Waltham, MA). Lysates were resolved on 10% SDS-PAGE gels and transferred to nitrocellulose membrane (GE Healthcare, Piscataway, NJ). Gαz, Gαq and GAPDH protein levels were determined using anti-Gαz antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA) (1:3000) (9), anti-Gαq antibody (Santa Cruz Biotechnology Inc.) (1:2000) (9), and an anti-GAPDH antibody (Abcam Inc., Cambridge, MA) (1:1000) (9,12) and Gαz and Gαq protein levels were normalized to GAPDH. Chemiluminescent immunoreactive bands were detected by a horseradish peroxidase-conjugated secondary antibody; data were imaged and semi-quantified using Bio-Rad Quantity One image analysis software. Probing with each antibody was performed sequentially following stripping of the membrane with a commercially available stripping reagent as per manufacturers’ instruction (Bio-Rad Laboratories, Hercules, CA).

Experimental protocols

24-hr metabolic studies

Groups of SD, DSR, and DSS rats (n = 6 per group) maintained for 21 d on a normal (0.4%) or high (8%) NaCl diet underwent an acute 24-hr metabolic study before either plasma AVP measurement or surgical instrumentation for blood pressure measurement before collection of brain tissue (see Metabolic balance procedures above).

Groups of DSS rats maintained for 21 d on a high (8%) NaCl diet received a SCR or Gαq ODN pretreatment via icv injection (25 μg/5 μl each). Immediately after icv ODN administration, a 24-h metabolic data study was performed (n = 6 per ODN group) before either plasma AVP or surgical instrumentation for blood pressure measurement before collection of brain tissue (see Metabolic balance procedures above).

AVP studies

Groups of SD, DSR, and DSS rats were fed a control diet (0.4% NaCl) or a high-salt (8% NaCl) diet for 21 d. Animals were then killed by decapitation, and trunk blood was collected for determination of plasma AVP using an AVP enzyme-linked immunosorbent assay kit as per manufacturers’ instruction (Assay Designs Inc, MI) (n = 6 per group).

Groups of DSS rats were maintained on a high-salt (8% NaCl) diet for 21 d. On the morning of d 21 rats randomly received an acute icv injection of a SCR or Gαq ODN (25 μg/5 μl each). Twenty-four hours after ODN administration animals were decapitated and plasma AVP levels were determined (n = 6 per group).

Intracerebroventricular nociceptin/orphanin FQ (N/OFQ) studies

DSR and DSS rats previously implanted with an icv cannula and maintained for 21 d on a normal (0.4% NaCl) or a high-salt (8% NaCl) diet were acutely instrumented with arterial, venous, and bladder catheters (see Surgical procedures) (n = 6 per group per dietary intake per rat strain). After surgical preparation and initiation of an iv isotonic saline infusion (55 μl/min) and the stabilization of cardiovascular and renal excretory function (approximately 6 h), systemic cardiovascular parameters were measured and urine was collected during a 20-min control period. After this, N/OFQ (5.5 nmol; a peptide inhibitor of AVP) (19) or isotonic saline vehicle was injected icv (n = 6 per group). Cardiovascular function was then measured and urine samples collected during consecutive 10-min experimental periods for 90 min. Urine volume was determined gravimetrically. Urine sodium concentration was measured by flame photometry (model 943; Instrumentation Laboratories, Lexington, MA) and expressed as urinary sodium excretion (UNaV).

Groups of DSS rats, previously implanted with an icv cannula, were maintained on a high-salt (8% NaCl) diet for 21 d. On the morning of d 21 rats received a SCR or Gαq ODN pretreatment via icv injection (25 μg/5 μl each). Twenty-four hours after ODN administration animals underwent an acute icv N/OFQ injection study as described above (n = 6 per group per ODN pretreatment).

Volume expansion studies

Groups of DSS rats maintained for 21 d on a normal (0.4% NaCl) or a high-salt (8% NaCl) diet were acutely instrumented with arterial, venous, and bladder catheters (see Surgical procedures) (n = 6 per group). After surgical preparation an iv isotonic saline infusion (20 μl/min) was started. After a 2-hr recovery period, urine was collected and cardiovascular parameters were monitored during two consecutive 10-min control periods. Rats then received an acute iv isotonic saline load, equivalent to 5% of the rats body weight over 30 min. Continuous 10-min urine samples were collected during the volume expansion period. The volume expansion was then stopped, and the rate of isotonic saline infusion returned to 20 μl/min. Consecutive 10-min urine samples were then taken during a 90-min recovery period.

Groups of DSS rats (n = 6 per group), previously implanted with an icv cannula, were maintained for 21 d on a high (8%)-NaCl diet and received a SCR or Gαq ODN pretreatment via icv injection (25 μg/5 μl each). Twenty-four hours after ODN administration, an iv isotonic saline volume expansion study was performed as described above.

Additional isotonic saline volume expansion experiments were performed in which DSS rats maintained for 21 d on high salt intake were administered a continuous iv infusion of the AVP V2-receptor antagonist, OPC-31260, during the experimental protocol (32 nmol/kg/min) (20).

Analytical techniques

Total 24-h sodium intake was calculated from weight of food consumed over 24 h. Dietary sodium intake (meq/24 h) = dietary sodium content (meq/g) × daily food intake (g/24 h). Urinary sodium concentration was measured by flame photometry (model 943, Instrumentation Laboratories). Urinary sodium excretion (meq/24 h) = 24-h urine output (ml) × urinary sodium concentration (meq/liter). Sodium balance (meq/24 h) = dietary sodium intake (meq/24 h) − urinary sodium excretion (meq/24 h). Water balance (ml/24 h) = water intake (ml/24 h) − urine output (ml/24 h). Urinary osmolality was measured by vapor pressure osmometer (model 5500; WESCOR, Logan, UT). Free water clearance (CH20) was calculated as a difference between the rate of urine volume (ml) per 24 h and the osmolar clearance.

Statistical analysis

All data are expressed as mean ± sem. The magnitude of the changes in cardiovascular and renal excretory parameters at different time points after icv injection of N/OFQ or acute iv volume expansion were compared with respective group control values by a one-way repeated-measures ANOVA with subsequent Dunnett’s test. Differences occurring between treatment groups (e.g. 0.4% NaCl diet and 8% NaCl diet) were assessed by a two-way repeated measure ANOVA with treatment group being one fixed effect and time the other, with the interaction included. The time (min) was then the repeated factor. Post hoc analysis was performed using Bonferroni’s test. Where appropriate, a Student’s t test was also used to compare means between two groups. In each case, statistical significance was defined as probability (P) < 0.05.

Results

High salt intake elevates plasma AVP and total body water balance in DSS rats

DSS, but not SD or DSR, rats developed hypertension when subjected to a high-salt challenge [DSS MAP (mm Hg); 0.4% NaCl, 130 ± 1.8 vs. 8% NaCl, 163 ± 2.8, P < 0.05, Fig. 1A]. When fed a high-NaCl diet, DSS rats had a 2.6-fold increase in basal plasma AVP levels [DSS plasma AVP (pg/ml); 0.4% NaCl, 2.14 ± 0.3 vs. 8% NaCl, 5.63 ± 0.2, P < 0.05, Fig. 1B]. In contrast, a high-NaCl diet did not alter plasma AVP levels in SD or DSR rats (Fig. 1B). Concurrent with increased circulating AVP, DSS rats on a high-NaCl diet had significantly increased 24-h water balance [DSS H2O balance (ml/24-h); 0.4% NaCl, 13.8 ± 1.5 vs. 8% NaCl, 37 ± 1.9, P < 0.05] (Fig. 1C). When treated with a high-NaCl diet, DSS rats also had a 24-fold increase in 24-hr sodium balance, which increased from 0.34 ± 0.1 meq (0.4% NaCl) to 8.16 ± 0.6 meq (8% NaCl) (P < 0.05) (Fig. 1D). While high NaCl intake also elevated daily total sodium, but not water, balance in DSR rats, this represented a 2.5-fold increase. In DSS rats on high NaCl, free-water clearance was increased compared with DSR rats (P < 0.05; Fig. 1E). The maintenance of SD, DSR, or DSS rats on a high-salt diet led to increased daily water intake (of comparable magnitude) across all three rat strains and no change in daily food intake (please see Supplemental Table 1 published on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org).

Figure 1.

Figure 1

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Effect of elevated dietary salt consumption on resting MAP (mm Hg; A), circulating plasma AVP levels, expressed as pg/ml (B), 24-h total body water balance, expressed as ml (C), 24-h total body sodium balance, expressed as meq (D), and 24-h free water clearance (CH20), expressed as ml/min (E), in conscious male Sprague–Dawley (SD), Dahl salt-resistant (DSR), and Dahl salt-sensitive (DSS) rats maintained for 21 d on a control (0.4% NaCl) or high-salt (8% NaCl) diet (n = 6 group). The values are the means ± se of the mean. *, P < 0.05 compared with respective rat strain group value maintained on a 0.4% NaCl diet. τ, P < 0.05 compared with DSR group maintained on an 8% NaCl diet. N.D., not determined.

High salt intake blunts the diuretic response to a peptide inhibitor of AVP secretion in DSS rats

In DSR rats, icv injection of the neuropeptide N/OFQ, which has a central action to inhibit AVP secretion (9,21), produced a profound diuretic response [from a stable baseline pretreatment urine output (μl/min); 0.4% NaCl, 52.4 ± 3 vs. 8% NaCl, 53.1 ± 4], with the magnitude of the cumulative urine output not altered by a high salt intake [DSR 90-min cumulative urine output (μl); 0.4% NaCl, 5,238 ± 412 vs. 8% NaCl, 4,910 ± 256, Fig. 2A]. In DSS rats fed a normal (0.4%) NaCl diet, central N/OFQ produced a robust diuresis [from a stable baseline pretreatment urine output (μl/min); 0.4% NaCl, 51.5 ± 4 vs. 8% NaCl, 55.6 ± 5] that was not significantly different from that observed in DSR rats on a normal or high NaCl diet. However, when DSS rats were maintained on a high-salt diet, the magnitude of the cumulative diuretic response to icv N/OFQ was significantly blunted [DSS 90-min cumulative urine output (μl); 0.4% NaCl, 4,600 ± 398 vs. 8% NaCl, 1,670 ± 108, P < 0.05, Fig. 2A]. In both DSR and DSS rats, N/OFQ produced a profound drop in heart rate, the magnitude of which was unaltered by high salt intake in either strain. However, the bradycardic response to icv N/OFQ was blunted in DSS rats compared with DSR rats irrespective of the dietary salt content (Fig. 2B). The icv injection of N/OFQ also produced a transient (approximately 40-min), but significant, decrease in MAP of comparable magnitude in both DSR and DSS rat fed a normal NaCl diet. Moreover, the hypotensive response to icv N/OFQ was not affected in DSR rats by pretreatment of rats with a high salt intake; in contrast there was a small but significant increase in the hypotensive response to icv N/OFQ in DSS rats on a high-salt diet (Fig. 2C). In DSS and DSR rats on either normal or high NaCl diet, icv administration of isotonic saline vehicle did not alter HR, MAP, or urine output (data not shown).

Figure 2.

Figure 2

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Cumulative urine output (μl) over 90-min (A), peak change in HR (BPM; B), and peak change in MAP (mm Hg; C) produced by icv injection of the neuropeptide N/OFQ (5.5 nmol/5 μl) in conscious male DSR and DSS rats maintained for 21 d on a control (0.4% NaCl) or high-salt (8% NaCl) diet (n = 6 per group). The values are the mean ± se of the mean. *, P < 0.05 compared with response observed in respective rat strain group maintained on a 0.4% NaCl diet. τ, P < 0.05 compared with DSR group maintained on an 8% NaCl diet. ≠, P < 0.05 compared with DSR group maintained on a 0.4% NaCl diet.

Influence of salt intake on brain Gαq and Gαz subunit protein expression

In both SD and DSR rats, Gαq and Gαz subunit proteins were expressed at comparable levels in BC and the VLM, with increased expression of Gαq in the PVN (Fig. 3, A and B). Twenty-one-day high salt intake produced a selective sixfold down-regulation of Gαq proteins within the PVN in SD rats [SD Gαq protein (ODU/mm2 normalized to GAPDH); 0.4% NaCl diet, 0.95 ± 0.07 vs. 8% NaCl diet, 0.15 ± 0.05, P < 0.05] (Fig. 3, A and C), and a threefold down-regulation in the PVN of DSR rats [DSR Gαq protein (ODU/mm2 normalized to GAPDH); 0.4% NaCl diet, 1.42 ± 0.08 vs. 8% NaCl diet, 0.43 ± 0.06, P < 0.05] (Fig. 3, A and C). In SD and DSR rats, there was no change in Gαz protein levels in any brain site examined after high salt intake (Fig. 3, B and C). In DSS rats on a normal NaCl intake, Gαq and Gαz proteins were expressed at comparable basal levels throughout the brain (Fig. 3, A and B). In contrast to SD and DSR rats, DSS rats did not down-regulate Gαq protein levels following consumption of a high salt diet (Fig. 3, A and C). High NaCl treatment did not alter Gαz protein levels in any brain region in DSS rats (Fig. 3, B and C).

Figure 3.

Figure 3

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Effect of dietary salt intake on Gαq protein levels normalized to GAPDH and expressed as optical density units/mm2 (A), Gαz protein levels normalized to GAPDH and expressed as optical density units/mm2 (B) in the BC, hypothalamic PVN and VLM in male Sprague–Dawley, Dahl salt-resistant, and Dahl salt-sensitive rats maintained for 21 d on a control (0.4% NaCl) or high-salt (8% NaCl) diet (n = 6 per group), and representative immunoblots illustrating GAPDH, Gαq, and Gαz protein levels in BC, PVN, and VLM tissues (C) from male Sprague–Dawley, Dahl salt-resistant, and Dahl salt-sensitive rats maintained for 21 d on a control (0.4% NaCl; denoted NS) or a high-salt (8% NaCl; denoted HS) diet. The values are the mean ± se of the mean. *, P < 0.05 compared with respective rat strain group value observed in animals maintained on a 0.4% NaCl diet. τ, P < 0.05 compared with comparative rat strain Gαq protein level observed in brain cortex and VLM tissue on a 0.4% NaCl diet.

ODN-mediated down-regulation of brain Gαq proteins in hypertensive DSS rats

Scrambled ODN icv pretreatment did not alter Gαz or Gαq protein expression in any brain region examined in DSS rats fed a high NaCl diet (Fig. 4, A and C). Administration of a Gαq targeted ODN (icv) to DSS rats on a high-NaCl diet significantly decreased Gαq protein expression in all brain sites examined 24-h post ODN administration (Fig. 4, B and C). In all brain sites Gαq protein was decreased at least by 85%, indicating ODN efficacy and dispersal throughout the brain 24-h after a single icv ODN pretreatment. Intracerebroventricular Gαq ODN pretreatment did not alter brain Gαz protein expression demonstrating Gαq ODN specificity and selectivity (Fig. 4, B and C).

Figure 4.

Figure 4

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Effect of (A) icv SCR ODN pretreatment (24 h; 25 μg/5 μl), or (B) icv Gαq ODN pretreatment (24 h; 25 μg/5 μl) on Gαq and Gαz protein levels normalized to GAPDH expressed as optical density units/mm2 in the BC, hypothalamic PVN, and VLM in male DSS rats maintained for 21 d on a high-salt (8% NaCl) diet (n = 6 per group) and, (C) representative immunoblots illustrating GAPDH, Gαq, and Gαz subunit protein levels in BC, PVN, and VLM tissues from male DSS rats maintained for 21 d on a high-salt (8% NaCl; denoted HS) diet or maintained on a high-salt (8% NaCl) diet then pretreated (24 h) with a SCR ODN (25 μg; denoted HS + SCR ODN) or a Gαq ODN (25 μg; denoted HS + Gαq ODN). The values are the mean ± se of the mean. *, P < 0.05 compared with 8% NaCl intake group value.

Central Gαq subunit protein pathways regulate AVP secretion and influence water balance in hypertensive DSS rats

In DSS rats maintained on a high-salt diet, 24-hr icv pretreatment with a SCR ODN did not alter any of the physiological parameters under investigation (Fig. 5, A–E). In DSS rats maintained on a high-salt diet, 24-h icv Gαq ODN pretreatment did not alter basal MAP (Fig. 5A). However, plasma AVP levels, when measured 24 h post-Gαq ODN administration, were significantly reduced [DSS plasma AVP (pg/ml); 8% NaCl + SCR ODN, 5.18 ± 0.3 vs. 8% NaCl + Gαq ODN, 2.7 ± 0.15, P < 0.05; Fig. 5B]. In parallel with reduced plasma AVP levels, 24-hr icv Gαq ODN pretreatment decreased the amount of water retained by high salt–treated DSS rats. This was indicated by a decrease in the magnitude of 24-hr positive water balance without altering fluid intake [DSS H2O balance (ml/24 h); 8% NaCl + SCR ODN, 35 ± 2.1 vs. 8% NaCl + Gαq ODN, 24 ± 2.4, P < 0.05; Fig. 5C]. In contrast to the effects on water excretion, central Gαq ODN treatment had no effect on 24-h total body sodium balance (Fig. 5D). In DSS rats on a high-NaCl diet, ODN-mediated down-regulation of Gαq proteins produced a significant increase in 24-h free water clearance [DSS CH2O (ml/min); 8% NaCl + SCR ODN, −131 ± 8 vs. 8% NaCl + Gαq ODN, −98 ± 5.2, P < 0.05; Fig. 5E]. It should be noted that icv administration of a SCR or Gαq ODN did not alter 24-h food or water intake compared with DSS rats maintained on a high-salt diet (Supplemental Table 1).

Figure 5.

Figure 5

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Effect of icv SCR or Gαq ODN pre-treatment (24 h; 25 μg/5 μl) on resting MAP (mm Hg; A), circulating plasma AVP levels, expressed as pg/ml (B), 24-h total body water balance, expressed as ml (C), 24-h total body sodium balance, expressed as meq (D), and 24-h free water clearance (CH20), expressed as ml/min (E), in Dahl salt-sensitive rats maintained for 21 d on a high-salt (8% NaCl) diet (n = 6 per group). The values are the means ± se of the mean. Data for 0.4% and 8% NaCl diet alone are reproduced from Fig. 1 for clarity. *, P < 0.05 compared with respective group on a 0.4% NaCl diet. τ, P < 0.05 compared with DSS group maintained on an 8% NaCl diet.

Central Gαq protein pathways contribute to the impaired diuretic response to pharmacological and physiological stimuli in hypertensive DSS rats

Intracerebroventricular N/OFQ administration resulted in a transient decrease in HR of similar magnitude and duration that persisted for approximately 50 min in DSS rats on either normal or high salt that was not altered by a SCR or Gαq ODN pretreatment (Fig. 6A). Further, the time course and magnitude of the hypotensive response to central N/OFQ was not affected by a SCR or Gαq ODN pre-treatment [peak Δ MAP (mm Hg); DSS 8% NaCl + SCR ODN, −29 ± 2.1 vs. 8% NaCl + Gαq ODN, −28 ± 1.8; Fig. 6B]. In contrast N/OFQ administration produced a blunted diuretic response in DSS rats maintained on a high salt intake in terms of both peak and cumulative urine output [peak urine flow rate (μl/min); 0.4% NaCl, 161 ± 20 50-min post-injection vs. 8% NaCl, 103 ± 12 30-min post-injection; P < 0.05; Fig. 6C]. Gαq ODN pretreatment restored the maximal diuretic response and increased the duration of diuresis to icv N/OFQ (168 ± 18 μl/min 50 min postinjection), while increasing cumulative urine output (5,056 ± 201 μl/90 min) (Fig. 6, C and D).

Figure 6.

Figure 6

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Effect of icv SCR or Gαq ODN pretreatment (24 h; 25 μg/5 μl) in conscious male Dahl salt-sensitive rats maintained for 21 d on a high-salt (8% NaCl) diet on HR (bpm; A), MAP (mm Hg; B), the diuretic response (urine flow rate expressed as μl/min) produced by icv N/OFQ (5.5 nmol/5 μl; C), and cumulative urine output over 90-min (μl) produced by icv N/OFQ (5.5 nmol/5 μl; D) (n = 6 per group). The values are the mean ± se of the mean. V, urine flow rate. *, P < 0.05 compared with baseline pretreatment group value (designated C). τ, P < 0.05 compared with respective group value in Dahl salt-sensitive rats maintained on a 0.4% NaCl diet. ≠, P < 0.05 compared with respective group value in Dahl salt-sensitive rats maintained on an 8% NaCl diet.

In separate studies, the physiological stimulus of an acute isotonic saline volume expansion, which does not alter HR or MAP in conscious rats (Fig. 7, A and B), produced a profound diuretic response in DSS rats on a normal salt diet (Fig. 7C). However, when fed a high-salt diet DSS rats had an impaired ability to excrete the volume load [peak diuresis (μl/min); 0.4% NaCl, 426 ± 33 vs. 8% NaCl, 136 ± 14, P < 0.05; Fig. 7C]. Central Gαq ODN (but not SCR ODN) pretreatment partially restored the ability of high salt–treated DSS rats to excrete urine during an acute isotonic saline volume load [peak diuresis (μl/min); 8% NaCl + SCR ODN, 136 ± 14 vs. 8% NaCl + Gαq ODN, 260 ± 17, P < 0.05; Fig. 7C]. In a separate group of high salt–maintained DSS rats, concurrent iv infusion of the AVP V2-receptor antagonist OPC-31260 did not further increase the peak diuresis to acute iv isotonic saline volume expansion in DSS rats on a high-salt diet [peak diuresis (μl/min); 8% NaCl + OPC-31260, 273 ± 23 vs. 8% NaCl + Gαq ODN, 260 ± 17; Fig. 7C].

Figure 7.

Figure 7

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Effect of icv SCR or Gαq ODN pretreatment in conscious male Dahl salt-sensitive rats maintained for 21 d on a high (8% NaCl) diet on HR (bpm; A), MAP (mm Hg; B), and the diuretic response (urine flow rate expressed as μl/min; C) to an acute (5% body weight) isotonic saline volume expansion compared with effects of an iv infusion of the V2-receptor antagonist OPC-31260 (32 nmol/kg/min). The values are the mean ± se of the mean of six conscious rats per group. V, urine flow rate. *, P < 0.05 compared with baseline pretreatment group value (designated C). τ, P < 0.05 compared with respective group value in Dahl salt-sensitive rats maintained on a 0.4% NaCl diet. ≠, P < 0.05 compared with respective group value in Dahl salt-sensitive rats maintained on an 8% NaCl diet.

Discussion

We report that the chronic consumption of a high-salt diet down-regulates Gαq protein levels selectively in the hypothalamic PVN of SD and DSR rats. This site-specific down-regulation of PVN Gαq proteins was correlated with the ability of SD and DSR rats to maintain unchanged plasma AVP levels and fluid/electrolyte balance in the face of a high salt challenge. In contrast, after high salt treatment of DSS rats, Gαq protein levels in the PVN remained unchanged. The inability of PVN Gαq protein levels to down-regulate in response to a high salt intake is of high physiological significance because in these hypertensive DSS rats, plasma AVP levels and water balance were markedly elevated by high salt intake. Together, these findings provide evidence that in salt-resistant animals the down-regulation of PVN Gαq protein levels acts as a site-specific counter-regulatory mechanism to prevent salt-induced increases in AVP secretion and water retention. Furthermore, the lack of suppression of PVN Gαq protein pathways, as which occurs in DSS rats on a high-salt diet, leads to sustained elevation in plasma AVP levels (i.e. AVP dysregulation), thereby contributing to the water retention observed in these salt-sensitive animals (7,8,22). On one hand, these data suggest that the PVN Gαq counter-regulatory response is intrinsically abnormal in DSS rats and therefore fails to prevent high salt-induced excess AVP secretion. Alternatively, we propose that the lack of endogenous down-regulation of PVN Gαq proteins in DSS rats, which have an inherited functional derangement to excrete sodium at the level of the kidneys, occurs as an adaptive compensatory response to facilitate AVP secretion and thereby enhance water retention as a means to prevent hypernatremia.

The proposed stimulatory role of Gαq protein pathways in the brain, particularly in the PVN, on AVP release and subsequent water retention in DSS rats consuming a high-sodium diet is supported by several lines of evidence. As demonstrated in these and other studies (7,8,22), DSS rats maintained on a high-salt diet have elevated basal plasma AVP levels. Intracerebroventricular Gαq ODN administration resulted in widespread selective down-regulation of Gαq proteins in multiple brain sites within 24 h without altering daily food or water intake as previously reported in SD rats (9,14,15). In agreement with our previous findings in SD rats (9), we demonstrate that icv scrambled ODN pretreatment does not alter any physiological parameter under investigation or brain Gαz/Gαq protein levels in conscious DSS rats maintained on a high-salt diet. Twenty-four hours after selective down-regulation of brain Gαq proteins (via central Gαq ODN administration), plasma AVP levels in DSS rats on a high salt intake were decreased to a level similar to those detected in DSS rats on a normal salt intake. In addition to the reduction in plasma AVP levels, the magnitude of positive water balance in high salt–treated DSS rats was significantly reduced and free water clearance significantly increased. These central Gαq ODN-mediated effects on plasma AVP and the renal handling of water occurred independent of changes in 24-hr sodium balance, which remained elevated despite central Gαq ODN treatment. Of physiological merit, central ODN-mediated down-regulation of Gαq proteins also completely/partially restored the ability of high salt–treated DSS rats to evoke a marked diuretic response to pharmacological (icv N/OFQ administration) and physiological (iv acute isotonic saline volume expansion) stimuli, respectively. This is important because these stimuli are known to increase urine output exclusively (N/OFQ) (9,21,23), or in part (volume expansion) (24,25), through suppression of AVP secretion. Together, these findings provide evidence that sustained central (presumably PVN) Gαq protein levels, and thus downstream signaling pathways, play a critical role in mediating excessive AVP secretion, impaired diuretic capacity, and the marked water retention which occurs during consumption of a high-salt diet in DSS rats and potentially other models of salt-sensitivity. This premise is further supported by a reported link between reduced regulator of G protein signaling 2 (RGS2) function and the development of hypertension in animal models (26) and human subjects (27). The development of hypertension in these models is suggested to be a result of an impaired ability of RGS2 to inhibit Gαq signaling (28).

In the current study, DSS rats on a high salt intake remained hypertensive 24 hrs after central Gαq ODN administration. However ODN-mediated down-regulation of central Gαq proteins significantly decreased basal plasma AVP levels and the salt-induced water retention which occurred in these DSS rats. These findings are of interest because fluid retention is hypothesized to be a critical step in the development of salt-sensitive hypertension (29,30,31). To explain this discrepancy, it is possible that the observed decrease in plasma AVP levels produced by Gαq ODN pretreatment (24-hrs) caused an increase in urine output (and decrease in positive water balance) that only transiently lowered arterial blood pressure in hypertensive DSS. This is suggested because in telemetered DSS hypertensive rats, oral administration of RWJ-676070, a dual vasopressin V1A/V2 receptor antagonist, produced a significant but only transient decrease in arterial pressure, which began within the first 30 min of dosing but only lasted for approximately 3 h (11). Another important consideration is that the kidneys of DSS rats have an inherited functional derangement that causes avid NaCl retention which leads to hypertension (13,32). Thus, despite even a complete correction of a deranged central AVP secretory system, it is likely that DSS rats fed a high salt intake will remain hypertensive. Further, it is likely that in response to increased dietary salt intake hypothalamic PVN parvocellular neurons contribute to sympathetic hyperactivity (32,33) and subsequent sodium retention and hypertension as has been documented in multiple models of salt-sensitive hypertension (34,35,36). This is suggested because avid renal sodium retention, which we show is central Gαq ODN-insensitive, is likely to impact diverse cardiovascular regulatory pathways that lead to hypertension and allow pressure natriuresis in an attempt to restore body sodium balance (33,37). In this regard, a decrease in the circulating levels (icv Gαq ODN pretreatment) or action (iv OPC-31260 treatment) of AVP improved, but did not fully restore, the ability of DSS rats to excrete an acute water load. Together, these findings demonstrate that there are other non-AVP dependent pathways (i.e. impaired ability of the kidneys to excrete sodium) that contribute to the retention of water in DSS rats on high salt intake.

In conclusion, when faced with a high-salt diet, DSS, but not SD or DSR rats, exhibit AVP dysregulation (i.e. increased plasma AVP levels), marked positive water (and sodium) balance, and an impaired diuretic response to pharmacological and physiological stimuli. In contrast to SD and DSR rats, a key finding of these investigations is that in response to a high salt intake, DSS rats did not down-regulate Gαq protein levels within the hypothalamic PVN, this being a major brain site involved in AVP synthesis and release. Future studies are required to establish whether the observed changes in Gαq protein expression in response to high salt intake are limited to the PVN of salt-resistant phenotypes or are also occurring within the hypothalamic SON, this being another key brain site involved in AVP secretion (1,2). It is likely that in SD and DSR rats subjected to a high-salt diet, down-regulation of PVN Gαq protein signaling pathways reduces the ability of native central GPCR ligands, including catecholamines (8,38,39,40) and angiotensin II (41,42), to stimulate AVP release. We speculate that in salt-resistant rats, the down-regulation of central Gαq (AVP-stimulatory) protein pathways, presumably within the PVN, acts as a critical counter-regulatory system to augment the inhibitory influence of central (PVN) Gαz protein pathways on AVP secretion (9). In this manner, the effects of high salt on AVP secretion, water retention, and potentially the development of high blood pressure are attenuated/prevented. Together, these findings support a novel role for PVN Gαq subunit protein-gated pathways in the integrated endocrine regulation of AVP secretion and highlight the importance of this pathway in central AVP dysregulation in salt-sensitive subjects. Further investigations in this area may provide a molecular/cellular target in which new therapies can be directed to prevent AVP dysregulation in other pathological states in which excess AVP secretion and water overload occur (e.g. hepatic cirrhosis with ascites, congestive heart failure, syndrome of inappropriate AVP secretion).

Footnotes

This work was supported by American Heart Association Grant 09BGIA2250585 (to R.D.W.), and National Institutes of Health Grants NIDDK DK43337, NHLBI HL071212, and NCRR COBRE P20 RR018766 and American Heart Association Grant 0855293E (to D.R.K.).

Disclosure Summary: The authors have nothing to declare.

First Published Online September 22, 2010

Abbreviations: AVP, Vasopressin; BC, brain cortex; CNS, central nervous system; DSR, Dahl salt-resistant; DSS, Dahl salt-sensitive; GPCR, GTP-binding protein coupled receptor; HR, heart rate; icv, intracerebroventricular; MAP, mean arterial pressure; N/OFQ, nociceptin/orphanin FQ; ODN, oligodeoxynucleotide; PVN, paraventricular nucleus; SCR, scrambled; SD, Sprague–Dawley; SON, supraoptic; VLM, ventrolateral medulla.

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