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ABSTRACT: The reported sequences of the human and mouse Na+-driven Cl-/HCO3(-) exchangers (NDCBEs) differ greatly in their extreme cytosolic COOH termini (Ct). In human NDCBE (NDCBE-B), a 17-amino acid (aa) sequence replaces 66 aa at the equivalent position in mouse NDCBE (NDCBE-A). We performed 5'- and 3'-rapid amplification of cDNA ends (RACE) on human brain cDNA, followed by PCR of full-length cDNAs to determine whether the human SLC4A8 gene was capable of producing the mouselike Ct sequence. Our study confirmed the presence in human cDNA of mouse NDCBE-like transcripts (human NDCBE-A) and also disclosed the existence of three further novel NDCBE transcripts that we have called NDCBE-C, NDCBE-D, and NDCBE-D'. The novel NDCBE-C/D/D' transcripts initiate at a novel "exon 0" positioned approximately 35 kb upstream of the first exon of NDCBE-A/B. NDCBE-C/D/D' protein products are predicted to be truncated by 54 aa in the cytosolic NH(2) terminus (Nt) compared with NDCBE-A/B. Our data, combined with a new in silico analysis of partial transcripts reported by others in the region of the human SLC4A8 gene, increase the known extent of the SLC4A8 gene by 49 kb, to 124 kb. A functional comparison of NDCBE-A/B/C/D expressed in Xenopus oocytes demonstrates that the Nt variation does not affect the basal functional expression of NDCBE, but those with the shorter Ct have a 25-50% reduced functional expression compared with those with the longer Ct. By comparison with an artificially truncated NDCBE that contains neither 17-aa nor 66-aa Ct cassette, we determined that the functional difference is unrelated to the 66-aa cassette of NDCBE-A/C, but is instead due to an inhibitory effect of the 17-aa cassette of NDCBE-B/D.
Physiological Genomics 07/2008; 34(3):265-76. · 2.73 Impact Factor
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ABSTRACT: The proximal tubule (PT) is major site for the reabsorption of filtered HCO(3)(-). Previous work on the rabbit PT showed that 1) increases in basolateral (BL) CO(2) concentration ([CO(2)](BL)) raise the HCO(3)(-) reabsorption rate (J(HCO(3))), and 2) the increase that luminal angiotensin II (ANG II) produces in J(HCO(3)) is greatest at 0% [CO(2)](BL) and falls to nearly zero at 20%. Here, we investigate the role of angiotensin receptors in the [CO(2)](BL) dependence of J(HCO(3)) in isolated perfused PTs. We found that, in rabbit S2 PT segments, luminal 10(-8) M saralasin (peptide antagonist of ANG II receptors), lowers baseline J(HCO(3)) (5% CO(2)) to the value normally seen at 0% in the absence of inhibitors and eliminates the J(HCO(3)) response to changes in [CO(2)](BL). However, basolateral 10(-8) M saralasin has no effect. As with saralasin, luminal 10(-8) M candesartan (AT(1) antagonist) reduces baseline J(HCO(3)) and eliminates the [CO(2)](BL) dependence of J(HCO(3)). Luminal 10(-7) M PD 123319 (AT(2) antagonist) has no effect. Finally, we compared PTs from wild-type and AT(1A)-null mice of the same genetic background. Knocking out AT(1A) modestly lowers baseline J(HCO(3)) and, like luminal saralasin or candesartan in rabbits, eliminates the J(HCO(3)) response to changes in [CO(2)](BL). Our accumulated evidence suggests that ANG II endogenous to the PT binds to the apical AT(1A) receptor and that this interaction is critical for both baseline J(HCO(3)) and its response to changes in [CO(2)](BL). Neither apical AT(2) receptors nor basolateral ANG II receptors are involved in these processes.
American journal of physiology. Renal physiology 08/2007; 293(1):F110-20. · 3.68 Impact Factor
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ABSTRACT: Colony-stimulating factor-1 (CSF-1) promotes the survival of osteoclasts, short-lived cells that resorb bone. Although a rise in intracellular pH (pH(i)) has been linked to inhibition of apoptosis, the effect of CSF-1 on pH(i) in osteoclasts has not been reported. The present study shows that, in the absence of CO(2)/HCO(3)(-), CSF-1 causes little change in osteoclast pH(i). In contrast, exposing these cells to CSF-1 in the presence of CO(2)/HCO(3)(-) causes a rapid and sustained cellular alkalinization. The CSF-1-induced rise in pH(i) is not blocked by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, an inhibitor of HCO(3)(-) transporters but is abolished by removing extracellular sodium. This inhibition profile is similar to that of the electroneutral Na/HCO(3) cotransporter NBCn1. By RT-PCR, NBCn1 transcripts are present in both osteoclasts and osteoclast-like cells (OCLs), and by immunoblotting, the protein is present in OCLs. Moreover, CSF-1 promotes osteoclast survival in the presence of CO(2)/HCO(3)(-) buffer but not in its absence. Preventing the activation of NBCn1 markedly attenuates the ability of CSF-1 to 1) block activation of caspase-8 and 2) prolong osteoclast survival. Inhibiting caspase-3 or caspase-8 in OCLs prolongs osteoclast survival to the same extent as does CSF-1. This study provides the first evidence that osteoclasts express a CSF-1-regulated Na/HCO(3) cotransporter, which may play a role in cell survival.
Endocrinology 03/2007; 148(2):831-40. · 4.46 Impact Factor
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ABSTRACT: A previous study demonstrated that proximal tubule cells regulate HCO(3)(-) reabsorption by sensing acute changes in basolateral CO(2) concentration, suggesting that there is some sort of CO(2) sensor at or near the basolateral membrane (Zhou Y, Zhao J, Bouyer P, and Boron WF Proc Natl Acad Sci USA 102: 3875-3880, 2005). Here, we hypothesized that an early element in the CO(2) signal-transduction cascade might be either a receptor tyrosine kinase (RTK) or a receptor-associated (or soluble) tyrosine kinase (sTK). In our experiments, we found, first, that basolateral 17.5 microM genistein, a broad-spectrum tyrosine kinase inhibitor, virtually eliminates the CO(2) sensitivity of HCO(3)(-) absorption rate (J(HCO(3))). Second, we found that neither basolateral 250 nM nor basolateral 2 microM PP2, a high-affinity inhibitor for the Src family that also inhibits the Bcr-Abl sTK as well as the Kit RTK, reduces the CO(2)-stimulated increase in J(HCO(3)). Third, we found that either basolateral 35 nM PD168393, a high-affinity inhibitor of RTKs in the erbB (i.e., EGF receptor) family, or basolateral 10 nM BPIQ-I, which blocks erbB RTKs by competing with ATP, eliminates the CO(2) sensitivity. In conclusion, the transduction of the CO(2) signal requires activation of a tyrosine kinase, perhaps an erbB. The possibilities include the following: 1) a TK is simply permissive for the effect of CO(2) on J(HCO(3)); 2) a CO(2) receptor activates an sTK, which would then raise J(HCO(3)); 3) a CO(2) receptor transactivates an RTK; and 4) the CO(2) receptor could itself be an RTK.
American journal of physiology. Renal physiology 09/2006; 291(2):F358-67. · 3.68 Impact Factor
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ABSTRACT: Previous authors showed that, at low doses, both basolateral and luminal ANG II increase the proximal tubule's HCO(3)(-) reabsorption rate (J(HCO(3))). Using out-of-equilibrium CO(2)/HCO(3)(-) solutions, we demonstrated that basolateral CO(2) increases J(HCO(3)). Here, we examine interactions between ANG II and CO(2) in isolated, perfused rabbit S2 segments. We first used equilibrated 5% CO(2)/22 mM HCO(3)(-)/pH 7.40 in bath and lumen. At 10(-11) M, basolateral (BL) ANG II increased J(HCO(3)) by 41%, and luminal ANG II increased J(HCO(3)) by 35%. At 10(-9) M, basolateral ANG II decreased J(HCO(3)) by 43%, whereas luminal ANG II was without effect. Second, we varied [CO(2)](BL) from 0 to 20% at fixed [HCO(3)(-)](BL) and pH(BL). Fractional stimulation produced by BL 10(-11) M ANG II falls when [CO(2)](BL) exceeds 5%. Fractional inhibition produced by BL 10(-9) M ANG II tends to rise when [CO(2)](BL) exceeds 5%. Regarding luminal ANG II, fractional stimulation produced by 10(-11) M ANG II fell monotonically as [CO(2)](BL) rose from 0 to 20%. Fractional inhibition produced by 10(-9) M ANG II rose monotonically with increasing [CO(2)](BL). Viewed differently, ANG II at 10(-11) M tended to reduce stimulation by CO(2), and at 10(-9) M, produced an even greater reduction. In conclusion, the mutual effects of 1) ANG II on the J(HCO(3)) response to basolateral CO(2) and 2) basolateral CO(2) on the J(HCO(3)) responses to ANG II suggest that the signal-transduction pathways for ANG II and basolateral CO(2) intersect or merge.
American journal of physiology. Renal physiology 04/2006; 290(3):F666-73. · 3.68 Impact Factor
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ABSTRACT: Respiratory acidosis, a decrease in blood pH caused by a rise in [CO(2)], rapidly triggers a compensatory response in which the kidney markedly increases its secretion of H(+) from blood to urine. However, in this and other acid-base disturbances, the equilibrium CO(2) + H(2)O HCO(3)(-) + H(+) makes it impossible to determine whether the critical parameter is [CO(2)], [HCO(3)(-)], and/or pH. Here, we used out-of-equilibrium CO(2)/HCO(3)(-) solutions to alter basolateral (BL) [HCO(3)(-)], [CO(2)], or pH, systematically and one at a time, on isolated perfused S2 rabbit proximal tubules. We found that increasing [HCO(3)(-)](BL) from 0 to 44 mM, at a fixed [CO(2)](BL) of 5% and a fixed pH(BL) of 7.40, caused HCO(3)(-) reabsorption (J(HCO(3))) to fall by half but did not significantly affect volume reabsorption (J(V)). Increasing [CO(2)](BL) from 0% to 20%, at a fixed [HCO(3)(-)](BL) of 22 mM and pH(BL) of 7.40, caused J(HCO(3)) to rise 2.5-fold but did not significantly affect J(V). Finally, increasing pH(BL) from 6.80 to 8.00, at a fixed [HCO(3)(-)](BL) of 22 mM and [CO(2)](BL) of 5%, did not affect either J(HCO(3)) or J(V). Analysis of the J(HCO(3)) and J(V) data implies that, as the tubule alters J(HCO(3)), it compensates the reabsorption of other solutes to keep J(V) approximately constant. Because the cells cannot respond acutely to pH changes, we propose that the responses of J(HCO(3)) and the reabsorption of other solutes to changes in [HCO(3)(-)](BL) or [CO(2)](BL) involve sensors for basolateral HCO(3)(-) and CO(2).
Proceedings of the National Academy of Sciences 04/2005; 102(10):3875-80. · 9.68 Impact Factor
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ABSTRACT: Previous reports suggest that an important characteristic of chemosensitive neurones is an unusually large change of steady-state intracellular pH in response to a change in extracellular pH (DeltapH(i)/DeltapH(o)). To determine whether such a correlation exists between neurones from the medullary raphe (a chemosensitive brain region) and hippocampus (a non-chemosensitive region), we used BCECF to monitor pH(i) in cultured neurones subjected to extracellular acid-base disturbances. In medullary raphe neurones, respiratory acidosis (5%--> 9% CO(2)) caused a rapid fall in pH(i) (DeltapH(i) approximately 0.2) with no recovery and a large DeltapH(i)/DeltapH(o) of 0.71. Hippocampal neurones had a similar response, but with a slightly lower DeltapH(i)/DeltapH(o) (0.59). We further investigated a possible link between pH(i) regulation and chemosensitivity by following the pH(i) measurements on medullary raphe neurones with an immunocytochemistry for tryptophan hydroxylase (a marker of serotonergic neurones). We found that the DeltapH(i)/DeltapH(o) of 0.69 for serotonergic neurones (which are stimulated by acidosis) was not different from either the DeltapH(i)/DeltapH(o) of 0.75 for non-serotonergic neurones (most of which are not chemosensitive), or from the DeltapH(i)/DeltapH(o) of hippocampal neurones. For both respiratory alkalosis (5%--> 3% CO(2)) and metabolic alkalosis (22 mm--> 35 mm HCO(3)(-)), DeltapH(i)/DeltapH(o) was 0.42-0.53 for all groups of neurones studied. The only notable difference between medullary raphe and hippocampal neurones was in response to metabolic acidosis (22 mm--> 14 mm HCO(3)(-)), which caused a large pH(i) decrease in approximately 80% of medullary raphe neurones (DeltapH(i)/DeltapH(o)= 0.71), but relatively little pH(i) decrease in 70% of the hippocampal neurones (DeltapH(i)/DeltapH(o)= 0.09). Our comparison of medullary raphe and hippocampal neurones indicates that, except in response to metabolic acidosis, the neurones from the chemosensitive region do not have a uniquely high DeltapH(i)/DeltapH(o). Moreover, regardless of whether neurones were cultured from the chemosensitive or the non-chemosensitive region, pH(i) did not recover during any of the acid-base stresses.
The Journal of Physiology 08/2004; 559(Pt 1):85-101. · 4.72 Impact Factor
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ABSTRACT: Working with isolated perfused S2 proximal tubules, we asked whether the basolateral CO2 sensor acts, in part, by raising intracellular Ca2+ concentration ([Ca2+]i), monitored with the dye fura 2 (or fura-PE3). In paired experiments, adding 5% CO2/22 mM HCO3- (constant pH 7.40) to the bath (basolateral) solution caused [Ca2+]i to increase from 57 +/- 3 to 97 +/- 9 nM(n = 8, P < 0.002), whereas the same maneuver in the lumen had no effect. Intracellular pH (pHi), measured with the dye BCECF, fell by 0.54 +/- 0.08 (n = 14) when we added CO2/HCO3- to the lumen. In 14 tubules in which we added CO2/HCO3- to the bath, pHi fell by 0.55 +/- 0.11 in 9 with a high initial pHi, but rose by 0.28 +/- 0.07 in the other 5 with a low initial pHi. Thus it cannot be a pHi change that triggers the [Ca2+]i increase. Introducing to the bath an out-of-equilibrium (OOE) solution containing 20% CO2/no HCO3-/pH 7.40 caused [Ca2+]i to rise by 62 +/- 17 nM (n = 10), whereas an OOE solution containing 0% CO2/22 mM HCO3-/pH 7.40 caused only a trivial increase. Removing Ca2+ from the lumen and bath, or adding 10 microM nifedipine (L- and T-type Ca2+-channel blocker) or 2 microM thapsigargin [sarco-(endo) plasmic reticulum Ca2+-ATPase inhibitor] or 4 microM rotenone (mitochondrial inhibitor) to the lumen and bath, failed to reduce the CO2-induced increase in [Ca2+]i. Adding 10 mM caffeine (ryanodine-receptor agonist) had no effect on [Ca2+]i. Thus basolateral CO2, presumably via a basolateral sensor, triggers the release of Ca2+ from a nonconventional intracellular pool.
American journal of physiology. Renal physiology 10/2003; 285(4):F674-87. · 3.68 Impact Factor
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ABSTRACT: For almost a century it was generally assumed that the lipid phases of all biological membranes are freely permeable to gases. However, recent observations challenge this dogma. The apical membranes of epithelial cells exposed to hostile environments, such as gastric glands, have no demonstrable permeability to the gases CO2 and NH3. Additionally, the water channel protein aquaporin 1 (AQP1), expressed at high levels in erythrocytes, can increase membrane CO2 permeability when expressed in Xenopus oocytes. Similarly, nodulin-26, which is closely related to AQP1, can act as a conduit for NH3. A key question is whether aquaporins, which are abundant in virtually every tissue that transports O2 and CO2 at high levels, ever play a physiologically significant role in the transport of small volatile molecules. Preliminary data are consistent with the hypothesis that AQP1 enhances the reabsorption of HCO3- by the renal proximal tubule by increasing the CO2 permeability of the apical membrane. Other preliminary data on Xenopus oocytes heterologously expressing the electrogenic Na+-HCO3- cotransporter (NBC), AQP1 and carbonic anhydrases are consistent with the hypothesis that the macroscopic cotransport of Na+ plus two HCO3- occurs as NBC transports Na+ plus CO3(2-) and AQP1 transports CO2 and H2O. Although data - obtained on AQP1 reconstituted into liposomes or on materials from AQP1 knockout mice - appear inconsistent with the model that AQP1 mediates substantial CO2 transport in certain preparations, the existence of unstirred layers or perfusion-limited conditions may have masked the contribution of AQP1 to CO2 permeability.
The Journal of Physiology 08/2002; 542(Pt 1):17-29. · 4.72 Impact Factor
