Deletion of proton-sensing receptor GPR4 associates with lower blood pressure and lower binding of angiotensin II receptor in SFO

Abstract

Diets rich in grains and meat and low in fruits and vegetables (acid-producing diets) associate with incident hypertension, whereas vegetarian diets associate with lower blood pressure (BP). However, the pathways that sense and mediate the effects of acid-producing diets on BP are unknown. Here, we examined the impact of the deletion of an acid sensor GPR4 on BP. GPR4 is a proton-sensing G protein-coupled receptor and an acid sensor in brain, kidney, and blood vessels. We found that GPR4 mRNA was higher in subfornical organ (SFO) than other brain regions. GPR4 protein was abundant in SFO and present in capillaries throughout the brain. Since SFO partakes in BP regulation through the renin-angiotensin system (RAS), we measured BP in GPR4−/− and GPR4+/+ mice and found that GPR4 deletion associated with lower systolic BP: 87 ± 1 mmHg in GPR4−/− (n = 35) vs. 99 ± 2 mmHg (n = 29) in GPR4+/+; P < 0.0001, irrespective of age and sex. Angiotensin II receptors detected by ¹²⁵I-Sarthran binding were lower in GPR4−/− than GPR4+/+ mice in SFO and in paraventricular nucleus of hypothalamus. Circulating angiotensin peptides were comparable in GPR4−/− and GPR4+/+ mice, as were water intake and excretion, serum and urine osmolality, and fractional excretion of sodium, potassium, or chloride. A mild metabolic acidosis present in GPR4−/− mice did not associate with elevated BP, implying that deficiency of GPR4 may preclude the effect of chronic acidosis on BP. Collectively, these results posit the acid sensor GPR4 as a novel component of central BP control through interactions with the RAS.

diet-dependent, subtle fluctuations of acid-base balance may influence blood pressure (BP) over a prolonged period of time (12, 30). Our Western diet is referred to as an “acid-producing diet” because it elicits net endogenous, metabolic acid production (5): Western diet is rich in acid-producing proteins from grains and meat and low in base-producing organic K⁺-salts (e.g., K⁺-citrate) from fruits and vegetables. Acid-producing diets associate with higher BP (12, 20, 30), whereas vegetarian diets associate with lower BP (29). Although these observations have important implications for public health, pathophysiology underlying the effect of acid-producing diets on BP has not been elucidated.

To explore potential contributory mechanisms, we focused on an acid sensor, GPR4, a member of a family of G-protein-coupled receptors (GPCRs) activated by protons in the physiological pH range (8, 16). These GPCRs (OGR1, TDAG8, and GPR4) function as acid sensors in bone (11), kidney (23, 24), and brain (13, 27) as well as in several cell types, including endothelial cells (2, 28).

GPR4 is abundant in brain, lung, and kidney relative to other tissues (17). We showed that GPR4 deficiency impairs acid excretion by the kidney, eliciting a mild non-gap metabolic acidosis similar to distal renal tubular acidosis in humans (23, 24). Kumar et al. (13) demonstrated that GPR4 expression in chemosensory neurons of the mouse retrotrapezoid nucleus is required for CO₂-stimulated breathing. Yang et al. (28) reported that GPR4 functions as an acid sensor during blood vessel development, regulates outgrowth of small capillaries, and mediates an inflammatory response in endothelium exposed to acidic environments (2, 4). Together, these studies provide a physiological context for the acid-sensing function of GPR4.

Here, we report on expression of GPR4 in the brain capillaries and its potential role in BP regulation. Abundant expression of GPR4 was detected in the subfornical organ (SFO), a sensory circumventricular organ that plays a prominent role in electrolyte and fluid homeostasis, exhibits high concentration of angiotensin II (Ang II) receptors, and has a local renin-angiotensin system (RAS). Deficiency of GPR4 associated with lower systolic BP and lower Ang II receptor binding in SFO, suggesting that this acid sensor may be involved in BP regulation by affecting central RAS.

METHODS

Animals.

GPR4−/− mice (on C57BL/6 background) were a gift from Drs. Witte (University of California, Los Angeles, and Howard Hughes Medical Institute) and Yang (East Carolina University) (28). Mice ate standard laboratory diet (Prolab RMH 3000 by LabDiet; composition: http://www.labdiet.com/cs/groups/lolweb/@labdiet/documents/web_content/mdrf/mdi4/∼edisp/ducm04_028406.pdf) and drank freely. Prior to euthanasia, an arterial blood sample was measured with a blood gas analyzer (IRMA TruPoint, International Technidyne, Edison, NJ). For measurements of water intake, urine excretion, osmolarity, and fractional sodium excretion, samples were collected in metabolic cages as previously described (24). All animal procedures were approved by the Institutional Animal Care and Use Committee of Wake Forest School of Medicine and University of Cincinnati College of Medicine.

Brain RNA isolation and RT-PCR.

Specific brain regions were dissected, and RNA was extracted with RNeasy Mini Kit (Qiagen); 0.2 μg RNA was reverse-transcribed to the first-strand cDNA with oligo(dT)_12–18 primer by using SuperScript First-Strand Synthesis System (Invitrogen, Carlsbad, CA) and analyzed in triplicates on 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA). Data from each brain area represent a pool of tissue from three different animals. Primers were the following: GPR4 (GenBank:NM_175668); 5′-CTA CCT GGC TGT GGC TCA T-3′ (sense); 5′-CAA AGA CGC GGT ACA GAT TCA-3′ (antisense); β-actin (GenBank:NM_007393): 5′-TTG CTG ACA GGA TGC AGA AG-3′ (sense); and 5′-CAG TGA GGC CAG GAT GGA GC-3′ (antisense); size of PCR products was 218 and 122, respectively. The absolute mRNA per sample was calculated from a standard curve and normalized by using β-actin as gene/β-actin ratio.

Isolation of SFO RNA and AT₁ receptor RT-PCR.

After necropsy, brains were flash frozen, positioned on a cryostat at −20°C, and 500-μm coronal slices were cut from the anterior brain until the third ventricle combined with the left and right lateral ventricles. A 0.5-mm brain punch tool (Stoelting, Wood Dale, IL) was positioned over SFO superior to dorsal third ventricle at bregma −0.22 and the tissue was extracted and kept in RNAlater. RNA was processed as above and, because of the small amount of tissue, one sample from a pool of three different animals was analyzed for GPR4+/+ and one sample from a pool of three animals for GPR4−/−; samples were run in triplicate. Ang II receptor type 1 primers were based on mouse AT_1a (GenBank:NM_177322) and AT_1b (GenBank:NM_175086) sequences, including an overlapping sequence: 5′-ACA GCA TCA TCT TTG TGG TG-3′ (sense), 5′-TGG CGT AGA GGT TGA AAC TG-3′ (antisense) (RT-PCR product = 240 bp).

Immunohistochemistry.

We mapped the expression of GPR4 protein with green fluorescent protein (GFP) because the lack of reliable antibodies has so far precluded precise tissue localization of GPR4. GFP was included in a construct used to generate GPR4−/− mice (28) so that GPR4 promoter drove the expression of GFP. Therefore, GFP-labeled cells, that would normally express GPR4, were detected in GPR4−/− (n = 4). GPR4+/+, which did not express GFP, served as controls (n = 4). We amplified GFP signal with rabbit monoclonal anti-GFP antibody (1:2,000 dilution; Molecular Probes, Eugene, OR) on 30-μm brain cryosections cut from tissues fixed with periodate-lysine-2% paraformaldehyde (23). The labeling was visualized using 3,3′-diaminobenzidine or a fluorescent secondary antibody (conjugated with AlexaFluor 488 or 594). When using fluorescence, nuclei were counterstained with Hoechst 33342 at final concentration of 1 μg/ml.

To confirm expression in endothelial cells, cryosections were incubated with a standard endothelial cell marker, mouse anti-CD31 antibody (22) (BD Biosciences, San Jose, CA) in addition to anti-GFP antibody; the antibodies were diluted 1:250 and 1:1,000, respectively. Images were acquired on a Zeiss Laser Scanning Confocal Microscope 710 (Carl Zeiss Vision, San Diego, CA) as 0.5-μm “optical sections,” by using ZEN software (Zeiss).

BP measurements.

BP was measured with tail-cuff plethysmography in conscious mice by using 8-Chanel Non-Invasive BP system and v2.2.5 software from Columbus Instruments (Columbus, OH). Before measurements, mice were acclimated to restrainers for 3 days and on day 4, BP was measured for 10 min at 60-s intervals. The systolic BP was expressed as the average of all stable measurements.

Angiotensin II receptor autoradiography.

Binding of [¹²⁵I-sarcosine¹-threonine⁸]ANG II (¹²⁵I-Sarthran) in GPR4−/− vs. +/+ was measured in adjacent coronal 14-μm cryosections of mouse forebrain as reported (19). To protect the ligand during the incubation, the procedure was modified with the following additions: 10 μM lisinopril, an Ang-converting enzyme inhibitor; 200 μM phenylmethanesulfonyl-fluoride, a serine protease inhibitor; 10 μM Sch-39370, a neprilysin inhibitor; and 1 μM amastatin/bestatin, aminopeptidase inhibitors. Nonspecific binding was defined in the presence of excess (10 μM) unlabeled Sarthran. Receptor subtypes were discriminated by absence/presence of competitors: 3 μm losartan (AT₁) or 3 μm PD123,319 (AT₂) or 10 μm Sarthran to determine specific binding. A low concentration of ¹²⁵I-Sarthran (0.2 nM) was used for competition and a higher concentration near saturation (0.6 nM) for maximal binding. Images were exposed to films with standards and quantified with a densitometric system (MCID; InterFocus Imaging, Cambridge, UK) (19).

Angiotensin peptide assays.

Ang I, II, and -(1–7) were measured in GPR4−/− vs. GPR4+/+ (n = 12 each) by the Biomarker Analytical Core of Wake Forest School of Medicine using standard protocols (1, 19) Blood was collected immediately after euthanasia in prechilled sample tubes with a cocktail of angiotensin/renin peptidase inhibitors. Plasma samples were subsequently stored at −80°C until analyzed with three separate radioimmunoassays.

Statistical analysis.

Data are presented as means ± SE. Comparisons were performed by unpaired two-tailed Student's t-test; P < 0.05 was considered significant.

RESULTS

GPR4 mRNA was abundant in SFO.

The expression profile of GPR4 mRNA in brain suggested that GPR4 mRNA was higher in SFO vs. other regions examined (Fig. 1A).

Fig. 1.

Expression of GPR4 in brain. A: expression of GPR4 mRNA in each brain region was analyzed in a single sample pooled from three wild types by using real-time PCR. B: GFP-mapped expression of GPR4 was analyzed in GPR4−/− mice (n = 4), which express GFP under control of GPR4 promoter. GFP-mapped expression was compared with controls (GPR4+/+; n = 4), which do not express GFP. The cryosections were stained with 3,3′-diaminobenzidine (A–C). GFP-mapped GPR4 was detected in subfornical organ (SFO) (B) and adjacent small blood vessels (C) in GPR4−/−, but not in controls (A). D shows a representative 0.5-μm confocal “optical section” of a capillary illustrating GFP-mapped GPR4 and nuclear labeling. (GFP-mapped GPR4 fluorescence is amplified by an anti-GFP primary antibody and a fluorescent secondary and pseudostained in green. Hoechst 33342-labeled nuclei are shown in blue.) This labeling pattern is corroborated by a video of a series of optical sections spanning the entire SFO section (please see Supplemental Material, which is accessible on the journal web site). C: to confirm expression in blood vessels, double labeling, using anti-GFP antibody (with a red fluorescent secondary) and anti-CD31 antibody (with a green fluorescent secondary), was performed on cryosections from GPR4−/− and GPR4+/+mice (n = 4 of each). Images were acquired on a confocal microscope from the region adjacent to SFO. A representative 0.5-μm optical section illustrates colocalization of GFP-mapped GPR4, and CD31, an endothelial cell marker, in brain capillaries. Sections from controls (GPR4+/+) showed only CD31 labeling (data not shown). D: representative 0.5-μm optical section shows a capillary within SFO double labeled with CD31 and GFP-mapped GPR4.

GPR4 protein was expressed in brain capillaries and prominent in SFO.

GFP-mapped GPR4 (see methods) (28) was detected in GPR4−/− mice in the cells that would normally express GPR4 protein (Fig. 1B, panels B–D); GPR4+/+ mice, which lack GFP, served as controls (Fig. 1B, panel A). GFP mapping of GPR4 indicated that GPR4 labeling was particularly prominent in SFO (consistent with mRNA expression; Fig. 1B, panel B) and abundant in adjacent small blood vessels (Fig. 1B, panels C and D; and video in Supplemental Material; Supplemental Material for this article is available online at the Journal website). Expression in blood vessels was corroborated with double labeling with an endothelial cell marker anti-CD31 antibody (Fig. 1C). CD31-labeling and GFP-mapped GPR4 are also shown in a capillary within SFO (Fig. 1D).

GPR4−/− mice had lower BP than GPR4+/+.

High expression of an acid sensor GPR4 in SFO, a pivotal center integrating cardiovascular actions of RAS, prompted us to measure BP. GPR4−/− mice exhibited lower systolic BP (SBP): 87 ± 1 mmHg in GPR4−/− (n = 35) vs. 99 ± 2 mmHg (n = 29) in GPR4+/+ (P < 0.0001). In those same animals, we also ascertained that the effect of GPR4 deficiency on SBP did not depend on age or sex (Fig. 2, A and B).

Fig. 2.

Deletion of GPR4 associated with lower systolic blood pressure (SBP). SBP was measured in conscious mice with tail-cuff plethysmography. A: SBP was lower in younger (2–4 mo old) GPR4−/− (n = 14) vs. GPR4+/+ (n = 19) and in older (11–15 mo old) GPR4−/− (n = 13) vs. GPR4+/+ (n = 12) mice, regardless of sex. B: SBP was lower in male GPR4−/− (n = 20) vs. male GPR4+/+ (n = 14) and in female GPR4−/− (n = 15) vs. female GPR4+/+ (n = 5), regardless of age. **P < 0.01. ***P < 0.001.

GPR4 deficiency was associated with lower binding of angiotensin receptor in SFO and paraventricular nucleus.

High density of angiotensin receptors in SFO and its downstream projection site paraventricular nucleus (PVN) is illustrated in representative images of ¹²⁵I-Sarthran binding (Fig. 3A). Lower signal overall is also present in broader regions, such as cortex, thalamus, and hippocampus. Quantification of specific ¹²⁵I-Sarthran binding revealed substantially less signal intensity (Fig. 3A, arrows) in SFO and PVN of the GPR4−/− vs. +/+ mice (Fig. 3B, quantification summarized). AT₁ and AT₂ receptors each represented ∼50% of the specific binding in SFO or PVN based on competition with receptor subtype-selective ligands (Fig. 3C). The percentage of AT₁ or AT₂ subtype was comparable in GPR4+/+ and GPR4−/− mice. Nonspecific binding defined in the presence of excess (10 μM) unlabeled Sarthran was higher in GPR4−/− mice (48 ± 5% in SFO and 62 ± 7% in PVN in the two nuclei of GPR4−/− vs. 26 ± 7% in SFO and 38 ± 8% in PVN of GPR4+/+; P < 0.05), suggesting a possible reduction in receptor affinity rather than number. A reduction in receptor affinity was also suggested by comparable expression of AT_1(a&b) receptor mRNA in SFO in GPR4−/− vs. GPR4+/+: AT1/α-actin mRNA ratio × 10⁻³ was 1.8 in GPR4+/+ (n = 3) vs. 1.9 in GPR4−/− (n = 3).

Fig. 3.

Deletion of GPR4 is associated with lower angiotensin receptor binding in SFO and paraventricular nucleus (PVN). A: representative computerized images from autoradiograms of ¹²⁵I-Sarthran binding illustrate differences between GPR4−/− and GPR4+/+ mice in SFO and PVN (high densities shown in red and yellow and lower in green and blue). B: quantification of specific ¹²⁵I-Sarthran binding revealed lower binding density in SFO and PVN from GPR4−/− mice [n = 12 GPR4+/+ (5 female, 7 male) and n = 13 GPR−/− (4 female, 9 male); *P < 0.05]. C: AT₁ receptors defined by competition with losartan (3 μM) and AT₂ receptors defined by competition with PD123319 (3 μM). Each receptor represents ∼50% of the ¹²⁵I-Sarthran binding in the SFO and PVN of both GPR4+/+ and GPR4−/− mice.

Plasma angiotensin peptides were comparable in GPR4+/+ and GPR −/− mice.

Angiotensin peptides were measured in plasma with three separate radioimmunoassays. No difference was detected between GPR4−/− vs. GPR4+/+ mice: Ang I (in pg/ml) was 57.1 ± 11.4 in GPR4−/− vs. 54.8 ± 13.4 in GPR4+/+, P = 0.9; Ang II (in pg/ml) was 16.0 ± 4 in GPR4−/− vs. 16.0 ± 4 in GPR4+/+, P = 0.8; and Ang-(1–7) (in pg/ml) was 37.3 ± 10.1 in GPR4−/− vs. 40.9 ± 6.6 in GPR4+/+, P = 0.8.

Deletion of GPR4 did not affect baseline water and sodium balance.

To estimate water and electrolyte balance, we determined fractional excretion of sodium, potassium, chloride, water intake, urine output, and urine and serum osmolarity; all of these variables were similar in GPR4−/− vs. GPR4+/+ (Fig. 4). We also confirmed our previous finding of mild metabolic acidosis (23, 24) in GPR4−/−, which had lower blood bicarbonate (20.3 ± 0.4 mM/l, n = 15) than GPR4+/+ (22 ± 0.3 mM/l, n = 14, P < 0.01).

Fig. 4.

Deletion of GPR4 does not affect baseline water and sodium balance. Water intake and urine output were measured over 24 h. A: water intake was comparable in GPR4+/+ (n = 43, 20 male, 23 female) vs. GPR4−/− (n = 49, 24 male, 25 female). B: urine output was similar in GPR4+/+ (n = 43, 20 male, 23 female) vs. GPR4−/− (n = 49, 24 male, 25 female). C: urine osmolality of 24-h samples was comparable in GPR4+/+ (n = 28, 11 male, 17 female) vs. GPR4−/− (n = 34, 14 male, 20 female). D: serum osmolality was comparable in GPR4+/+ (n = 18, 9 male, 9 female) vs. GPR4−/− (n = 15, 8 male, 7 female). Fractional excretion of sodium, potassium, and chloride was comparable in GPR4−/− vs. GPR4+/+ mice (E, F, and G, respectively).

DISCUSSION

The main findings of this report are the following: 1) an acid sensor GPR4 is abundant in endothelial cells of brain capillaries and in highly vascularized SFO, 2) GPR4 deficiency associated with lower SBP (∼10 mmHg reduction), 3) AT₁ and AT₂ receptor binding in SFO and PVN were lower in GPR4−/− vs. GPR4+/+ mice, and 4) circulating Ang peptides and baseline water and salt reabsorption were comparable in GPR4−/− vs. GPR4+/+ mice.

Localization of GPR4 in brain capillaries is consistent with reported expression in cultured endothelial cells (4) and may be important in conditions that associate with lower pH, such as brain ischemia or inflammation. In this study, we focused on abundance of GPR4 in SFO, a highly vascularized chemosensory circumventricular organ involved in BP regulation and fluid homeostasis (9), and found that SBP was lower in GPR4−/− than in GPR4+/+ mice.

SFO and neuronal projections from SFO to PVN express Ang II receptors abundantly. Both AT₁ and AT₂ receptors are implicated in physiological responses to Ang II at these sites (3, 6, 10, 15, 19). In the present study, Ang II receptors, detected with ¹²⁵I-Sarthran binding, were lower in both SFO and PVN of GPR4−/− vs. GPR4+/+ mice. Based on competition with selective ligands, 50% of Ang II receptors detected were AT₁ and 50% AT₂ subtype_, consistent with physiological actions of the peptide involving both subtypes. The subtype profile was not different between GPR4−/− and GPR4 +/+ mice, implying fewer AT₁ and AT₂ receptors within SFO and PVN of GPR4−/− mice. The nonspecific binding, however, was higher in GPR4−/− than in GPR4+/+ mice. The higher nonspecific binding, coupled with no difference in AT₁ mRNA in GPR4−/− vs. GPR4+/+ mice (assessed with a nonselective primer to recognize both AT_1a and AT_1b), may indicate a desensitized state of Ang II receptors and potentially explain the reduced binding of AT₁ and AT₂ receptors that we observed (18). AT₁ receptors primarily increase sympathetic nervous system outflow to elevate BP, whereas AT₂ receptors have minimal role in BP regulation. Therefore, the lower AT₁ receptor density may contribute to the lower SBP in GPR4−/− mice.

SFO and PVN contribute to the regulation of thirst and water intake (3, 9, 10, 15, 19), so lower extracellular volume could account for lower SBP in GPR4−/− mice. However, plasma vasopressin and aldosterone are not different in GPR4−/− vs. GPR4+/+ mice (24). Water intake, urine output, serum and urine osmolality and fractional excretion of sodium, potassium, and chloride were also comparable between GPR4−/− and GPR4+/+ mice. It is, therefore, unlikely that lower SBP in GPR4−/− resulted from salt wasting and volume contraction.

Decreased peripheral resistance, if present, may also lower SBP in GPR4−/− mice. Our preliminary ex vivo experiments did not show increased vasodilatation in mesenteric arteries isolated from GPR4−/− mice (21). However, the preliminary nature of these findings and potential differences in the ex vivo and in vivo conditions preclude definitive conclusions about the state of peripheral resistance in GPR4−/− and indicate the need to conduct these ex vivo and in vivo studies in parallel.

Severe metabolic acidosis, as in life-threatening conditions such as diabetic ketoacidosis or hypoxic lactic acidosis, elicits vasodilation by affecting vascular smooth muscle directly (14, 26). In contrast, mild subclinical acidosis associates with higher BP and incident hypertension over time (12, 30). Mild acidosis in GPR4−/− mice (23, 24), however, was not associated with elevated BP. Moreover, SBP was lower in younger GPR4−/− and remained lower even at a relatively advanced age (15 mo), implying that the GPR4 receptor deficiency may preclude the effect of chronic acidosis on SBP. Whether metabolic acidosis affects the affinity of angiotensin receptors for their ligand(s) is currently unknown. Based on our present experiments, we cannot determine whether the affinity of the angiotensin receptors was influenced by systemic pH directly or, alternatively, by a physical interaction of the two receptors (AT1 and GPR4), interaction of their respective signaling pathways, and/or other adaptive changes during development. Tissue-specific and inducible mouse models or localized perturbations of GPR4 expression with siRNA or oligonucleotides are needed to resolve these possibilities.

The significance of our report lies in its translational potential and implications that may clarify the effects of acid-producing diets and subclinical changes in acid-base balance on BP. Acid-producing diets were linked to incident hypertension in adult women followed for 15 yr (30) by the Nurses′ Health Study II and to higher BP in young boys followed for 7 yr by the DONALD Study (12). Moreover, supplementation with base KHCO₃ lowered BP in spontaneously hypertensive stroke-prone rats, as it did in hypertensive humans in subsequent small clinical trials (7, 25). We propose that the acid sensor GPR4 and its effects on Ang II receptors and BP may be part of a mechanism translating subclinical fluctuations in acid-base status into cardiovascular outcomes.

GRANTS

Support for this study was provided by Scientist Development Grant 0835313N, American Heart Association (to S. Petrovic); Research and Career Development Scholar Funds (to S. Petrovic) from Claude D Pepper Older Americans Independence Center of WFSM, National Institute on Aging Grant P30 AG-21332 (S. Kritchevsky, PI); Cross-Campus Collaborative Award and Center for Molecular and Cell Signaling, WFU (to S. Petrovic and Glen Marrs); Leon Goldberg, RJ Reynolds Postdoctoral Fellowship (to D. Molina); and Hypertension and Vascular Research Center and Farley Hudson Foundation of Jacksonville (to D. Diz).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

X.S., R.S., D.I.D., and S.P. conceived and designed research; X.S., E.T., D.M., R.S., and S.P. performed experiments; X.S., E.T., R.S., K.B.B., D.I.D., and S.P. analyzed data; X.S., R.S., K.B.B., D.I.D., and S.P. interpreted results of experiments; X.S., D.I.D., and S.P. prepared figures; X.S. and S.P. drafted manuscript; X.S., D.M., R.S., K.B.B., D.I.D., and S.P. edited and revised manuscript; X.S., E.T., D.M., R.S., K.B.B., D.I.D., and S.P. approved final version of manuscript.