17 research outputs found
Identification of Functionally Distinct Na-HCO3 Co-Transporters in Colon
Na-HCO3 cotransport (NBC) regulates intracellular pH (pHi) and HCO3 secretion in rat colon. NBC has been characterized as a 5,5′-diisothiocyanato-2-2′-stilbene (DIDS)-sensitive transporter in several tissues, while the colonic NBC is sensitive to both amiloride and DIDS. In addition, the colonic NBC has been identified as critical for pHi regulation as it is activated by intravesicular acid pH. Molecular studies have identified several characteristically distinct NBC isoforms [i.e. electrogenic (NBCe) and electroneutral (NBCn)] that exhibit tissue specific expression. This study was initiated to establish the molecular identity and specific function of NBC isoforms in rat colon. Northern blot and reverse transcriptase PCR (RT-PCR) analyses revealed that electrogenic NBCe1B or NBCe1C (NBCe1B/C) isoform is predominantly expressed in proximal colon, while electroneutral NBCn1C or NBCn1D (NBCn1C/D) is expressed in both proximal and distal colon. Functional analyses revealed that amiloride-insensitive, electrogenic, pH gradient-dependent NBC activity is present only in basolateral membranes of proximal colon. In contrast, amiloride-sensitive, electroneutral, [H+]-dependent NBC activity is present in both proximal and distal colon. Both electrogenic and electroneutral NBC activities are saturable processes with an apparent Km for Na of 7.3 and 4.3 mM, respectively; and are DIDS-sensitive with apparent Ki of 8.9 and 263.8 µM, respectively. In addition to Na-H exchanger isoform-1 (NHE1), pHi acidification is regulated by a HCO3-dependent mechanism that is HOE694-insensitive in colonic crypt glands. We conclude from these data that electroneutral, amiloride-sensitive NBC is encoded by NBCn1C/D and is present in both proximal and distal colon, while NBCe1B/C encodes electrogenic, amiloride-insensitive Na-HCO3 cotransport in proximal colon. We also conclude that NBCn1C/D regulates HCO3-dependent HOE694-insensitive Na-HCO3 cotransport and plays a critical role in pHi regulation in colonic epithelial cells
Identification of functionally distinct Na-HCO3 co-transporters in colon.
Na-HCO3 cotransport (NBC) regulates intracellular pH (pHi) and HCO3 secretion in rat colon. NBC has been characterized as a 5,5'-diisothiocyanato-2-2'-stilbene (DIDS)-sensitive transporter in several tissues, while the colonic NBC is sensitive to both amiloride and DIDS. In addition, the colonic NBC has been identified as critical for pHi regulation as it is activated by intravesicular acid pH. Molecular studies have identified several characteristically distinct NBC isoforms [i.e. electrogenic (NBCe) and electroneutral (NBCn)] that exhibit tissue specific expression. This study was initiated to establish the molecular identity and specific function of NBC isoforms in rat colon. Northern blot and reverse transcriptase PCR (RT-PCR) analyses revealed that electrogenic NBCe1B or NBCe1C (NBCe1B/C) isoform is predominantly expressed in proximal colon, while electroneutral NBCn1C or NBCn1D (NBCn1C/D) is expressed in both proximal and distal colon. Functional analyses revealed that amiloride-insensitive, electrogenic, pH gradient-dependent NBC activity is present only in basolateral membranes of proximal colon. In contrast, amiloride-sensitive, electroneutral, [H(+)]-dependent NBC activity is present in both proximal and distal colon. Both electrogenic and electroneutral NBC activities are saturable processes with an apparent Km for Na of 7.3 and 4.3 mM, respectively; and are DIDS-sensitive with apparent Ki of 8.9 and 263.8 µM, respectively. In addition to Na-H exchanger isoform-1 (NHE1), pHi acidification is regulated by a HCO3-dependent mechanism that is HOE694-insensitive in colonic crypt glands. We conclude from these data that electroneutral, amiloride-sensitive NBC is encoded by NBCn1C/D and is present in both proximal and distal colon, while NBCe1B/C encodes electrogenic, amiloride-insensitive Na-HCO3 cotransport in proximal colon. We also conclude that NBCn1C/D regulates HCO3-dependent HOE694-insensitive Na-HCO3 cotransport and plays a critical role in pHi regulation in colonic epithelial cells
Immunocytochemical localization of NBCn1-like proteins in rat proximal and distal colon.
<p>[<b>A, a’, a”</b>] Rat proximal colon demonstrates weak epithelial staining for NBCn1 along the crypt to surface axis. The connective tissue underlying the epithelial cells is strongly fluorescent. Higher magnification of the surface cells [<b>a’</b>] shows a rather punctuate apical signal [*]. High magnification of epithelial cells in the crypt region (a”) demonstrates a weak diffuse localization. [<b>B, b’, b”</b>] Rat distal colon shows stronger staining for NBCn1. High magnifications of surface cells [<b>b’</b>] and crypt cells [<b>b”</b>] clearly demonstrate basolateral membrane localization [<b>arrows</b>]. Smooth muscle cells were also strongly stained (data not shown).</p
Effect of extracellular HCO<sub>3</sub> in the presence and absence of HOE694 on intracellular pH change in crypt glands from proximal colon.
<p>Images of a crypt gland before [<b>A</b>] and after BCECF loading [<b>B</b>]. The BCECF loaded crypts were excited at 490 nm and 440 nm and the emission signal was monitored at 535 nm to derive intracellular pH (pHi). [<b>C</b>] Under basal condition, bath HCO<sub>3</sub> increased pHi, while the increased pHi was completely reversed by HCO<sub>3</sub> removal. [<b>D</b>] In the presence of amiloride, bath HCO<sub>3</sub> reduced the pHi, while the reduced pHi was partially reversed by HCO<sub>3</sub> removal. [<b>E</b>] In the presence of HOE694, bath HCO<sub>3</sub> increased pHi, while the increased pHi was completely reversed by HCO<sub>3</sub> removal. Typical tracing presented is representative of observations in 4 different crypts from 5 different rats.</p
DIDS-inhibition kinetics of Na-HCO<sub>3</sub> cotransport activities in basolateral membrane vesicles from proximal colon.
<p>Increasing DIDS concentrations inhibit both amiloride-insensitive and amiloride-sensitive proton/HCO<sub>3</sub> gradient-driven <sup>22</sup>Na uptake with half maximal inhibitory concentration (Ki) of approximately 8.9±1.2 and 263.8±26.4 µM, respectively. Proton/HCO<sub>3</sub> gradient-driven <sup>22</sup>Na uptake (0.1 mM) was measured as described in the Method section with increasing extravesicular DIDS concentrations both in presence and absence of inhibitors (either 100 µM EIPA or 1 µM HOE694). Uptake obtained in presence of amiloride represented amiloride-insensitive proton/HCO<sub>3</sub> gradient-driven uptake. Amiloride-sensitive uptake (which also includes HOE694 sensitive Na-H exchange) was calculated by subtracting uptake obtained in presence of EIPA from that of uptake in the absence of inhibitors. HOE694-sensitive uptake was calculated by subtracting uptake obtained in presence of HOE694 from that of uptake in the absence of inhibitors. Amiloride-sensitive proton/HCO<sub>3</sub> gradient-driven uptake was calculated by subtracting HOE694-sensitive uptake from that of EIPA-sensitive uptake. The inhibitory constants were calculated using a nonlinear regression with the EnzFitter software (Biosoft, Cambridge, UK).</p
Effect of varying outward acid pH-gradients on Na-HCO<sub>3</sub> cotransport activities in basolateral membrane vesicles from proximal colon.
<p>Proton/HCO<sub>3</sub> gradient-driven <sup>22</sup>Na uptake (0.1 mM) was measured as described in the Method section with different extravesicular pH in presence and absence of inhibitors (1 mM amiloride or 1 µM HOE694). Both one-unit (pHo/pHi: 7.5/6.5) and two-unit (pHo/pHi: 7.5/5.5) outward acid pH-gradients stimulated the amiloride-insensitive Na-HCO<sub>3</sub> cotransport. A two-unit, but not a one-unit outward acid pH gradient stimulated amiloride-sensitive Na-HCO<sub>3</sub> cotransport. Uptake obtained in presence of amiloride represented amiloride-insensitive proton/HCO<sub>3</sub> gradient-driven uptake. Amiloride-sensitive uptake (which also includes HOE694 sensitive Na-H exchange) was calculated by subtracting uptake obtained in presence of amiloride from that of uptake in the absence of inhibitors. HOE694-sensitive uptake was calculated by subtracting uptake obtained in presence of HOE694 from that of uptake in the absence of inhibitors. Amiloride-sensitive proton/HCO<sub>3</sub> gradient-driven uptake was calculated by subtracting HOE694-sensitive uptake from that of amiloride-sensitive uptake. *<i>p</i><0.02 compared to 7.5/7.5 (pHo/pHi) in amiloride-insensitive; <i><sup>$</sup>p</i><0.002 compared to 7.5/7.5 (pHo/pHi) in amiloride-insensitive; **<i>p</i><0.004 compared to 7.5/7.5 (pHo/pHi) in amiloride-sensitive; *<i>p</i><0.001 compared to 7.5/7.5 (pHo/pHi) in amiloride-sensitive; ns: not significant compared to 7.5/6.5 (pHo/pHi) in amiloride-insensitive.</p
Northern blot and RT-PCR analyses of mRNA from rat colon.
<p>[<b>A</b>] Northern blot analyses indicate that NBCe1A (7.5 kb)-like mRNA is predominantly expressed in proximal colon, while NBCn1B (7.9 kb)-like mRNA is expressed in both proximal and distal colon. Full length NBCe1A and NBCn1A cDNAs were used as probe. The blots were also probed with a GAPDH cDNA fragment. Results presented are from an individual blot that was stripped and probed sequentially with each probe (NBCe1A, NBCn1A and GAPDH). [<b>B</b>] RT-PCR analyses indicate that NBCe1B/C and NBCn1C/D specific transcripts are predominantly expressed in epithelial cells from proximal and distal colon. NBC isoform specific fragments were amplified by RT-PCR using isoform specific primers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062864#pone-0062864-t002" target="_blank">Table 2</a>) and mRNA from proximal colon, distal colon, brain, heart and kidney. RT-PCR products were separated on 1% agarose gels and stained with ethidium bromide. cDNA size markers are indicated on the right side.</p
Kinetics of Na-HCO<sub>3</sub> cotransport activities in basolateral membrane vesicles from proximal colon.
<p>Increasing extravesicular Na concentrations (1–50 mM) saturate both amiloride-insensitive and amiloride-sensitive proton/HCO<sub>3</sub> gradient-driven <sup>22</sup>Na uptake with apparent Km for Na of 7.3±0.6 and 4.3±0.3 mM, respectively. Proton/HCO<sub>3</sub> gradient-driven <sup>22</sup>Na uptake was measured as described in the Method section with increasing extravesicular Na concentrations both in presence and absence of inhibitors (either 1 mM amiloride or 1 µM HOE694). Uptake obtained in presence of amiloride represented amiloride-insensitive proton/HCO<sub>3</sub> gradient-driven uptake. Amiloride-sensitive uptake (which also includes HOE694 sensitive Na-H exchange) was calculated by subtracting uptake obtained in presence of amiloride from that of uptake in the absence of inhibitors. HOE694-sensitive uptake was calculated by subtracting uptake obtained in presence of HOE694 from that of uptake in the absence of inhibitors. Amiloride-sensitive proton/HCO<sub>3</sub> gradient-driven uptake was calculated by subtracting HOE694-sensitive uptake from that of amiloride-sensitive uptake. Best fit curves were drawn using Michaelis-Menten equation. The kinetic constants were calculated using a nonlinear regression with the EnzFitter software (Biosoft, Cambridge, UK).</p
Effect of one-unit pH-gradient with varying proton concentrations on Na-HCO<sub>3</sub> cotransport activities in basolateral membrane vesicles from proximal colon.
<p>Proton/HCO<sub>3</sub> gradient-driven <sup>22</sup>Na uptake (0.1 mM) was measured as described in the Method section with one unit acid outward pH gradient at different extravesicular and intravesicular pH in presence and absence of inhibitors (1 mM amiloride or 1 µM HOE694). One-unit outward acid pH-gradients with both low (pHo/pHi: 8.0/7.0) and high (pHo/pHi: 7.0/6.0) proton concentrations stimulated amiloride-insensitive Na-HCO<sub>3</sub> cotransport, while amiloride-sensitive Na-HCO<sub>3</sub> cotransport was substantially stimulated only at high proton concentrations. Uptake obtained in presence of amiloride represented amiloride-insensitive proton/HCO<sub>3</sub> gradient-driven uptake. Amiloride-sensitive uptake (which also includes HOE694 sensitive Na-H exchange) was calculated by subtracting uptake obtained in presence of amiloride from that of uptake in the absence of inhibitors. HOE694-sensitive uptake was calculated by subtracting uptake obtained in presence of HOE694 from that of uptake in the absence of inhibitors. Amiloride-sensitive proton/HCO<sub>3</sub> gradient-driven uptake was calculated by subtracting HOE694-sensitive uptake from that of amiloride-sensitive uptake. *<i>p</i><0.001 - compared to 8.0/7.0 (pHo/pHi) in amiloride-sensitive; ns: not significant - compared to 8.0/7.0 (pHo/pHi) in amiloride-insensitive.</p
Effect of HOE694 on intracellular pH (pHi; proton extrusion) in crypt gland from distal colon.
<p>Rate of change in pHi in presence of extracellular HCO<sub>3</sub> was examined in the presence (<b>HOE694</b>) and absence (<b>Control</b>) of 1 µM HOE694. Rate of pHi in the presence of HOE694 was also measured in additional presence of 0.1 mM amiloride (<b>HOE694/Amil</b>). *<i>p</i><0.0003 - compared to control; <sup>$</sup><i>p</i><0.0001– compared to control; <sup>†</sup><i>p</i><0.0001– compared to HOE694.</p