Acid-base disturbances have serious clinical consequences and are particularly critical in patients whose cardiopulmonary function is compromised. Cellular transport of NH3 and NH4+ has important physiological significance in the regulation of acid-base balance. In the kidney, production and excretion of NH3/NH4+ is critical for net acid excretion. Recently, two non-erythroid glycoproteins (Rhbg and Rhcg) belonging to the Rh family were suggested to be involved in NH3/NH4+ transport. Thus far, the functional properties of these membrane proteins as transport mechanisms are not resolved. In this study, we expressed Rh proteins in Xenopus oocytes and demonstrated that they transport both NH4+ and NH3. As such, the Rh transporters are unique in being able to transport both the ionic and the neutral gaseous components of ammonia. Previous studies have shown that DIDS, a stilbene derivative known to inhibit anion exchangers, was shown to inhibit CO2 transport by AQP1. This led us to hypothesize that DIDS might also inhibit transport of other gases such as NH3 by Rh proteins. We therefore conducted the present study to test the effects of DIDS on NH4+ and NH3 transport by Rh glycoproteins. To do so we used ion-selective microelectrodes and two-electrode voltage clamp to measure changes in surface pH (pHs) and whole cell currents (I) induced by NH3/NH4+ and methyl ammonium (MA/MA+) with or without DIDS. All experiments were conducted in Xenopus oocytes expressing Rhbg. Rhbg was expressed by injecting the oocytes with cRNA of the cloned genes. Control oocytes were injected with H2O. Our results indicate that in oocytes expressing Rhbg, exposure to 5mM NH4Cl (NH3/NH4+) caused a decrease in surface pH (pHs) and an inward current. The decrease in pHs is caused by NH3 influx whereas the inward current is due to electrogenic NH4+ influx. In the presence of DIDS, exposure to 5mM NH4Cl caused a significantly smaller decrease in pHs and current. The %inhibition of pHs and ΔI were 33% and 49%, respectively (P<0.05). Similarly, exposing oocytes expressing Rhbg to 5mM MA/MA+ (a substitute to NH3/NH4+) caused a decrease in pHs and an inward current. In the presence of DIDS, the MA/MA+ induced changes in pHs and current were also inhibited (37% and 63%, respectively; P<0.05). DIDS had no effect on NH3/NH4+ transport in H2O-injected oocytes (not expressing Rhbg). In summary, our data support the following conclusions: 1) RhAG and Rhbg transport both the ionic NH4+ and the neutral NH3 species. 2) Transport of NH4+ is electrogenic. 3) RhAG and Rhbg expression both enhance MA transport, an electroneutral component. 4) Like Rhbg, RhAG also transports MA+, an electrogenic component. The charged MA+ seems to be a direct substrate for RhAG whose transport likely resembles that of NH4 +. 5) Rhcg is likely to be a predominantly NH3 transporter. 6) RhAG and Rhbg are unlikely to be NH4 +/H+ exchangers. Regarding the effect of DIDS, our data also indicate that 1) DIDS partially inhibits the transport of NH3 and MA by Rhbg without affecting endogenous NH3 and MA transport. 2) DIDS also inhibits the electrogenic transport of NH4 + and MA+ by Rhbg. 3) DIDS is the only inhibitor shown to block both gas (NH3) and ionic (NH4 +) transport by Rhbg. / acase@tulane.edu
| Identifer | oai:union.ndltd.org:TULANE/oai:http://digitallibrary.tulane.edu/:tulane_27866 |
| Date | January 2014 |
| Contributors | Caner, Tolga (Author), Nakhoul, Nazih (Thesis advisor) |
| Publisher | Tulane University |
| Source Sets | Tulane University |
| Language | English |
| Detected Language | English |
| Format | 111 |
| Rights | Copyright is in accordance with U.S. Copyright law |
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