Bicarbonate signalling via G protein-coupled receptor regulates ischaemia-reperfusion injury

Homoeostatic regulation of the acid–base balance is essential for cellular functional integrity. However, little is known about the molecular mechanism through which the acid–base balance regulates cellular responses. Here, we report that bicarbonate ions activate a G protein-coupled receptor (GPCR), i.e., GPR30, which leads to Gq-coupled calcium responses. Gpr30-Venus knock-in mice reveal predominant expression of GPR30 in brain mural cells. Primary culture and fresh isolation of brain mural cells demonstrate bicarbonate-induced, GPR30-dependent calcium responses. GPR30-deficient male mice are protected against ischemia-reperfusion injury by a rapid blood flow recovery. Collectively, we identify a bicarbonate-sensing GPCR in brain mural cells that regulates blood flow and ischemia–reperfusion injury. Our results provide a perspective on the modulation of GPR30 signalling in the development of innovative therapies for ischaemic stroke. Moreover, our findings provide perspectives on acid/base sensing GPCRs, concomitantly modulating cellular responses depending on fluctuating ion concentrations under the acid–base homoeostasis.


Cckar
Cholecystokinin receptor type A cholecystokinin Mediates pancreatic growth and enzyme secretion, smooth muscle contraction of the gall bladder and stomach. x

Agtr1b
Type-1B angiotensin II receptor angiotensin II Mediates smooth muscle contraction of vasculature, cell growth, secretion of catecholamines and aldosterone.
x x
Promotes formation of synapses in neurons. x
x x

Retinoic acid-induced protein 3 orphan
Interacts with frizzled GPCRs and activates the Wnt signaling pathway.A negative modulator of EGFR signaling. x
Stimulates neuronal migration and axon outgrowth.
x x
x Step 1: Of the 353 murine GPCRs, 50 and 33 were highly expressed in stomach and pancreas, respectively.From these, 10 GPCRs that were commonly highly expressed in both the tissues were selected as potential candidates.Among the 10 candidate GPCRs, GPR30 was selected for the neurovascular unit (NVU) -specific expression in the brain.e) Expression of each indicated candidate GPCR in single-cell components of the NVU.PC, pericytes; SMC, smooth muscle cells; MG, microglia; FB, vascular fibroblast-like cells; OL, oligodendrocytes; EC, endothelial cells; AC, astrocytes.
Data were obtained from the single-cell RNA-sequencing database of mouse brain vascular and perivascular cells (http://betsholtzlab.org/VascularSingleCells/database.html).Supplementary Fig. 6 CAG promoter Neo GCaMP6        (The Jackson Laboratory) male male male male male male male male male male male male male male 5 7 7 8

Mock
TGFα shedding assay using mock-and human GPR30 (hGPR30)-transfected HEK293 cells treated with oestradiol (E2).PMA (100 nM) was used as the positive control.b) E2-dependent accumulation of oestrogen receptor α (ERα).COS-7 cells expressing ERα fused with EGFP (ERα-EGFP) or mock-EGFP were treated with vehicle or E2 (1 µM) and examined using confocal microscopy.Mock-EGFP served as the negative control.Scale bar, 20 µm.c, d) The calcium mobilization assay (c) and the TGFα shedding assay (d) using hGPR30-transfected HEK293 cells treated with a putative synthetic agonist, G-1.ATP (25 µM) and PMA (100 nM) were used as positive controls.e-h) Inorganic components of DMEM activate hGPR30.Amino acids and glucose (e), vitamins, and trace elements (f) in DMEM, and their combination (g) did not induce calcium mobilization in MCF-GPR30.A mixed solution of the inorganic components of Dulbecco's modified Eagle's medium (DMEM) elevated intracellular calcium (h).i) hGPR30-transfected HEK293 cells were treated with 5.5 mM sodium bicarbonate and the indicated concentrations of E2 in the TGFα shedding assay.j-l) TGFα shedding assay using HEK293 cells transfected with rat (j), mouse (k), and zebrafish (l) GPR30, and treated with physiological concentrations of bicarbonate ions.Statistical analysis: two-tailed unpaired t-test with Bonferroni's correction after two-way ANOVA (a, c, d, i-l).In i, # indicates no significant difference compared with the hGPR30 column treated with the vehicle.Two-tailed unpaired t-test with Bonferroni's correction after one-way ANOVA (e).Two-tailed unpaired t-test with Holm-Šídák's correction (f-h).Data are presented as mean values ± SEM.P values are shown if significant.ns indicates no significant difference.Source data are provided as a Source Data file.
Whole cell expression of HA-tagged mutants analysed using western blotting.b) Cell surface expression of HA-tagged mutants, using cell surface biotinylation and avidin immunoprecipitation, analysed using western blotting.c) Expression of HAtagged GPR30 in membrane fractions analysed using western blotting.Org, organellar membrane fraction including endoplasmic reticulum membrane; PM, plasma membrane fraction; MVB, multivesicular body fraction.d) Scintillation proximity assay (SPA) using the plasma membrane fraction incubated with sodium bicarbonate-[ 14 C] and SPA beads.The plasma membrane fractions of HEK293 cells transiently expressing mock, hGPR30-HA, E115A-HA, Q138A-HA, or H307A-HA were analysed.Statistical analysis: two-tailed unpaired t-test with Tukey's correction after two-way ANOVA (d).P values are shown if significant.Source data are provided as a Source Data file.Intracellular calcium imaging using Fura-2-loaded mouse myoblast C2C12 cells stably expressing control shRNA (a) or Gpr30-shRNA (b), sequentially treated with indicated concentrations of sodium bicarbonate and ATP.Frozen sections of the brain (a), pituitary (b), heart (c), kidney (d), and muscle (e) obtained from heterozygous Gpr30-Venus-KI mice were analysed using confocal microscopy.In (b), A, I, and P indicate the anterior, intermediate, and posterior lobes of the pituitary, respectively.The scale bar on the right panel of (a), 5 µm.The other scale bars, 50 µm.
Design of the Gpr30-codon-improved Cre recombinase (iCre) knock-in (Gpr30-iCre-KI) construct.The coding sequence of Gpr30 was replaced in frame with that of iCre.The conditional GCaMP knock-in mice were generated by intercrossing Gpr30-iCre-KI mice and B6-Gt(Rosa)26Sor<tm1(CAG-GCaMP6,-mCherry)Shi>.b-d) Visualisation of Gpr30 (b, d) and iCre (c, d) using multiple in situ hybridisation analyses of the Gpr30-iCre-KI brain cortex.Scale bars, 50 µm.Western blot analyses of neuronal (neuron-specific bIII tubulin, GAD-67), synaptic (synaptophysin and PSD-95), and astrocytic (GFAP) markers in the brain cortex (a) and hippocampus (b).For each target, all the samples were analysed on the same blot and normalized by reprobing the same membrane with anti-b-actin antibody.c) Quantification of pericyte coverage.Left, immunohistochemistry of CD13 (pericytes) and Col-IV (basement membrane) followed by 3D reconstructions of confocal image z-stacks of Gpr30 +/Venus and Gpr30 −/Venus brain slices.The lower images are the magnification of the white dashed square in the upper image.Scale bars, 100 µm.Right, pericyte coverage quantified as CD13 + Col-IV + volume/Col-IV + volume.d) Quantification of endothelial cell coverage.Left, immunohistochemistry of CD31 (endothelial cell) and Col-IV (basement membrane) followed by 3D reconstructions of confocal image z-stacks of Gpr30 +/Venus and Gpr30 −/Venus brain slices.The lower images are the magnification of the white dashed square in the upper image.Scale bars, 100 µm.Right, endothelial coverage quantified as CD31 + Col-IV + volume/Col-IV + volume.The endothelial coverage of Gpr30 +/Venus and Gpr30 −/Venus mice were significantly different.The difference was no longer significant when analysed in the septal area and cortex subgroups.e) Electron microscopy analysis of brain cortices of Gpr30 +/+ , Gpr30 −/− , Gpr30 +/Venus , and Gpr30 Venus/Venus mice.Scale bars, 2 µm.f-h) Mice were intravenously injected with 3 (f), 10 (g), or 70 kDa (h) dextran-Tetramethylrhodamine (TMR) and perfused with PBS after 3 h (f), 1 h (g), or 16 h (h).Vascular permeability was evaluated using the relative fluorescence units (RFU) of the leaked TMR per mg of wet tissue weight.NT indicates mice that were not injected with dextran-TMR.Statistical analysis: two-tailed unpaired t-test with Bonferroni's correction after two-way ANOVA (a-d).Two-tailed unpaired t-test with Bonferroni's correction after one-way ANOVA (f-h).Data are presented as mean values ± SEM.P values are shown if significant.ns indicates no significant difference.Source data are provided as a Source Data file.Modified Neurological Severity Score (mNSS) of Gpr30 +/+ and Gpr30 −/− mice 1 to 7 days after reperfusion.b, c) Evaluation of the blood-brain barrier (BBB) impairment.Representative images of IgG immunostaining of Gpr30 +/+ and Gpr30 −/− mouse brains at 3 and 7 days after MCAO (b) and quantification of IgG staining of whole brain sections 7 days after MCAO (c).Scale bars, 1 mm.d, e) Evaluation of infarct volume.Representative images of cresyl violet staining of Gpr30 +/+ and Gpr30 −/− mouse brains 3 and 7 days after MCAO (d) and quantification of cresyl violet staining of whole brain sections 7 days after MCAO (e).Scale bars, 1 mm.f, g) Evaluation of apoptosis using TUNEL staining of whole brain sections 3 days after MCAO.Representative images of TUNEL staining (f) show that TUNEL-positive apoptotic cells were hardly detectable in Gpr30 −/− mice.The right images are the magnification of the white dashed square in the left images.Scale bars, 1 mm (the left) and 330 µm (the right).Statistical analysis: two-tailed mixed-effects analysis with Bonferroni's correction (a).Two-tailed unpaired t-test (c, e).Two tailed Mann-Whitney test (g).Data are presented as dot plots with medians.All p values are shown.Source data are provided as a Source Data file.

Expression profiles of various GPCRs and GPR30 in mouse tissues.
x a) Public data from the PDSP database (https://pdsp.unc.edu/databases/ShaunCell/heatmaps.php) that covers the expression of 353 murine GPCRs in various tissues.The red line represents GPR30.b) Candidate GPCRs that are specifically expressed in the stomach and pancreas.c) Receptor names, ligands, and characteristics of the 10 candidate GPCRs.'High expression in the brain' indicates that the brain expresses a particular GPCR over the 10th highest level among other tissues.d) Two-step selection of candidate GPCRs.