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Activation of 2-oxoglutarate receptor 1 (OXGR1) by α-ketoglutarate (αKG) does not detectably stimulate Pendrin-mediated anion exchange in Xenopus oocytes.
Heneghan JF
,
Majmundar AJ
,
Rivera A
,
Wohlgemuth JG
,
Dlott JS
,
Snyder LM
,
Hildebrandt F
,
Alper SL
.
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SLC26A4/Pendrin is the major electroneutral Cl- /HCO3- exchanger of the apical membrane of the Type B intercalated cell (IC) of the connecting segment (CNT) and cortical collecting duct (CCD). Pendrin mediates both base secretion in response to systemic base load and Cl- reabsorption in response to systemic volume depletion, manifested as decreased nephron salt and water delivery to the distal nephron. Pendrin-mediated Cl- /HCO3- exchange in the apical membrane is upregulated through stimulation of the β-IC apical membrane G protein-coupled receptor, 2-oxoglutarate receptor 1 (OXGR1/GPR99), by its ligand α-ketoglutarate (αKG). αKG is both filtered by the glomerulus and lumenally secreted by proximal tubule apical membrane organic anion transporters (OATs). OXGR1-mediated regulation of Pendrin by αKG has been documented in transgenic mice and in isolated perfused CCD. However, aspects of the OXGR1 signaling pathway have remained little investigated since its original discovery in lymphocytes. Moreover, no ex vivo cellular system has been reported in which to study the OXGR1 signaling pathway of Type B-IC, a cell type refractory to survival in culture in its differentiated state. As Xenopus oocytes express robust heterologous Pendrin activity, we investigated OXGR1 regulation of Pendrin in oocytes. Despite functional expression of OXGR1 in oocytes, co-expression of Pendrin and OXGR1 failed to exhibit αKG-sensitive stimulation of Pendrin-mediated Cl- /anion exchange under a wide range of conditions. We conclude that Xenopus oocytes lack one or more essential molecular components or physical conditions required for OXGR1 to regulate Pendrin activity.
FIGURE 1. Functional expression of OXGR1 in xenopus oocytes. (a). Confocal immunolocalization of MYC‐tagged OXGR1 in a representative oocyte previously injected with 50 ng OXGR1 cRNA (left panel) and a representative uninjected xenopus oocyte (right panel). Median intensity images are shown (b). MYC immunostaining intensity at oocyte periphery in OXGR1 cRNA‐injected oocytes and in uninjected oocytes. Values are means ± s.e.m. for n = 11 oocytes. One of two experiments with similar results. (c). Anti‐MYC immunoblot documenting MYC‐tagged OXGR1 polypeptide expression in Xenopus oocytes (lysate from 10 oocytes). (d). Unidirectional 45Ca2+ influx into uninjected oocytes and oocytes previously injected with 10 ng MYC‐OXGR1 cRNA, in the absence (light gray bars) and presence of 1 mM αKG (dark gray bars). Values are means ± s.e.m. for n = 10 oocytes; *p = 0.01 by two‐way t‐test. (e). Representative immunoblot depicting 1 mM αKG stimulation of ERK phosphorylation in uninjected oocytes or in oocytes expressing OXGR1. One of three similar experiments.
FIGURE 2. αKG (1 mM) does not stimulate Pendrin‐mediated Cl− uptake into Xenopus oocytes. Unidirectional 36Cl− influx at pH 7.4 into uninjected oocytes or oocytes previously injected with cRNA encoding Pendrin, with or without OXGR1 cRNA at the indicated ratios. Values are means ± s.e.m. for n = 10 oocytes in each condition. *p < 0.02; **p < 0.001 versus 1 ng PDS without OXGR1, both by ANOVA %
p < 0.001 versus 0.3 ng PDS without OXGR1, by two‐tailed unpaired t‐test. One of two experiments with similar results.
FIGURE 3. αKG (3 mM) does not stimulate Pendrin‐mediated Cl‐ efflux from Xenopus oocytes co‐injected with a range of ratios of co‐injected cRNAs encoding Pendrin and OXGR1. (a). 36Cl− efflux traces from individual oocytes previously uninjected or coinjected with pendrin cRNA (1 ng) without or with co‐injected OXGR1 cRNA (10 ng). Oocytes were subjected sequentially to baths containing NaCl (ND96), Na cyclamate +3 mM αKG (Cl−‐free αKG), ND96 + 3 mM αKG, and terminating in Na cyclamate (Cl−‐free) as indicated. (b). 36Cl− efflux rate constants during each efflux time period (see key, corresponding to periods of panel A) for each experimental group of oocytes as indicated for (n) oocytes. *p < 0.05 versus ND96, two‐tailed paired t‐test. (c). 36Cl− efflux traces from individual oocytes previously uninjected or coinjected with Pendrin cRNA (0.3 ng) without or with co‐injected OXGR1 cRNA (10 ng). Oocytes were subjected sequentially to baths containing ND96, Na cyclamate +3 mM αKG, ND96 + 3 mM αKG and terminating with Na cyclamate as indicated. (d). 36Cl− efflux rate constants during each efflux time period (see key, corresponding to periods of panel C) for each experimental group of oocytes as indicated for (n) oocytes. *p < 0.03 versus same condition without OXGR1; two‐tailed unpaired t‐test. (e). 36Cl− efflux traces from individual oocytes previously uninjected or coinjected with Pendrin cRNA (0.3 ng) without or with co‐injected OXGR1 cRNA (20 ng or 40 ng as indicated). Oocytes were subjected sequentially to baths containing ND96, Na cyclamate +3 mM αKG, ND96 + 3 mM αKG and terminating with Na cyclamate as indicated. (f). 36Cl− efflux rate constants during each efflux time period (see key, corresponding to periods of panel E) for each indicated experimental group of (n) oocytes. *p = 0.055; **p < 0.03; each versus Pds without OXGR1 in ND96, each by ANOVA.
FIGURE 4. Protein kinase C activator phorbol 12‐myristate 13‐acetate (PMA) does not stimulate Pendrin‐mediated 36Cl− efflux from Xenopus oocytes. (a). Representative 36Cl− efflux traces from individual oocytes previously uninjected (upper trace with “x” time points) or injected with Pendrin cRNA (2 ng, three lower traces with black square time points), exposed sequentially to baths containing ND96, Na cyclamate containing PMA (100 nM), NaCl in the continued presence of PMA, and a terminal bath of Na cyclamate. (b). 36Cl− efflux rate constants during each efflux time period (see key, corresponding to periods of panel A) for each indicated experimental group of (n) oocytes.
FIGURE 5. αKG does not stimulate Pendrin‐mediated 36Cl− efflux from Xenopus oocytes co‐expressing Gαq, Ga11, or Gα15. (a). Representative 36Cl− efflux traces from individual uninjected oocytes or oocytes previously injected with pendrin cRNA (1 ng) alone or with co‐injected OXGR1 cRNA (10 ng) without or with one of the coinjected cRNAs encoding Gαq, Gα11, or Gα15 (20 ng). Oocytes were subjected to sequential baths containing ND96, Na cyclamate + αKG (3 mM), ND96 + αKG (3 mM) and a terminal bath of Na cyclamate. (b). 36Cl− efflux rate constants during each efflux time period (see key corresponding to periods of panel A) for each indicated experimental group of oocytes (n = 3). (*), p = 0.055 versus PDS/OXGR1 in ND96, by ANOVA across all ND96 samples.
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