ABSTRACT
We have examined the effects of oxidizing and reducing agents on a number of subtypes of N-methyl-D-aspartate (NMDA) receptors expressed in Xenopus oocytes. Oocytes were injected with cRNA for the zeta 1 subunit from mouse to express homomeric receptors or with zeta 1 in combination with either epsilon 1, epsilon 2, epsilon 3 or epsilon 4 subunits to express heteromeric receptors. All heteromeric combinations resulted in receptors that were affected by the redox reagents, dithiothreitol (DTT) and 5-5-dithio-bis-2-nitrobenzoic acid (DTNB). However, the effects on the small currents from homomeric receptors were quite variable. The zeta 1/epsilon 3 combination showed a greater enhancement by DTT than any of the other combinations. All four receptors expressed showed both a component of persistent potentiation and a slowly reversible component. The reversible component was largest for zeta 1/epsilon 3. Additional experiments were done with S-nitrosocysteine (SNOC), a nitric oxide donor that may affect NMDA receptors by oxidation. SNOC had transient effects on the four heteromeric subunit combinations. The different sensitivities of particular subunit combinations may have pharmacological and clinical significance.
Subject(s)
Receptors, N-Methyl-D-Aspartate/metabolism , Animals , Dithionitrobenzoic Acid/pharmacology , Dithiothreitol/pharmacology , Electrochemistry , Female , In Vitro Techniques , Mice , Oocytes/metabolism , Oxidation-Reduction , Protein Conformation , RNA, Complementary/genetics , Receptors, N-Methyl-D-Aspartate/drug effects , Receptors, N-Methyl-D-Aspartate/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sulfhydryl Reagents/pharmacology , Xenopus laevisABSTRACT
We have examined the effects of oxidizing and reducing agents and sodium nitroprusside (SNP) on currents evoked by NMDA (N-methyl-D-aspartate) using the Xenopus oocyte expression system. Oocytes were injected with RNA prepared from either whole rat brain or from the NMDAR1 clone recently isolated from rat brain. Bath application of 1-1000 microM SNP, which releases nitric oxide and ferrocyanide, caused a rapid inhibition of NMDA-evoked current in both preparations. The inhibitory effect reversed spontaneously within 15 min. Kainate responses were not affected by SNP. Exposure to the reducing agent, dithiothreitol (DTT), enhanced NMDA currents; the oxidant, 5,5-dithio-bis-2-nitrobenzoic acid (DTNB), inhibited NMDA responses, as has been observed in other preparations. The site of action of SNP appeared to be different than the DTT/DTNB redox site for several reasons: SNP and DTNB inhibitions were additive at high doses, DTT did not rapidly reverse SNP effects, and SNP and DTT treatments did not show the same susceptibility to block by the NMDA antagonist, aminophosphonovaleric acid (APV). The results demonstrate that modulation of NMDA receptors by SNP is a property of homomeric channels and is retained when the NMDAR1 subunit is expressed in oocytes.
Subject(s)
Nitroprusside/pharmacology , Oocytes/drug effects , Receptors, N-Methyl-D-Aspartate/drug effects , Animals , DNA, Complementary , Female , Ferrocyanides/pharmacology , Indicators and Reagents , Ligands , Oocytes/metabolism , Oxidation-Reduction , Xenopus laevisABSTRACT
This study examines the cellular mechanisms of acid secretion by the in vitro perfused late distal tubule of Amphiuma kidney. Acidification of tubule fluid occurred against an electrochemical gradient of 16 mV; thus H+ secretion was active. Amiloride (1 mM) or a reduction of sodium in the perfusion fluid (from 83.7 to 7.7 mM) partially reduced acidification. Amiloride, in the presence of low sodium, completely inhibited acidification. Furthermore, acetazolamide and ouabain in the bath solution (0.1 mM) also inhibited acidification. Conductive properties of the epithelium and of individual cell membranes were determined by means of cable analysis of the tubule and intracellular voltage recordings. The transepithelial voltage and resistance averaged -0.4 +/- 0.4 mV, lumen negative, and 7,147 +/- 845 omega X cm, respectively. Two functionally different cell types were identified by intracellular microelectrodes. Type I cells had a basolateral membrane voltage (Vbl) of -67.7 mV. As determined by ion substitution experiments, the basolateral membrane was conductive to K+ and Cl-. This cell also had a 4-acetamido-4'-isothiocyanostilbene-2-2'-disulfonic acid (SITS)-sensitive Na+-dependent HCO3- exit pathway in the basolateral membrane. Type II cells had a Vbl of -76.1 mV (P less than 0.05 vs. type I) and the basolateral membrane was conductive to K+ and Cl- but not to HCO3-. HCO3- movement across the basolateral membrane in this cell may occur by electroneutral Cl- -HCO3- exchange. The apical cell membrane of both cell types did not contain measurable ionic conductances, as evidenced by a high value of apical membrane fractional resistance (0.98 +/- 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)
