Vijay Lyall

A modified technique for the measurement of sulfhydryl groups oxidized by reactive oxygen intermediates.

This paper suggests a simple modification of the Ellman procedure when used to measure accurate changes in sulfhydryl (-SH) content induced by reactive oxygen intermediates (ROI). This modification became necessary when we found that the standard technique did not produce time invariant results in the presence of ROI-generating systems. Cysteine (cys; 20-100 microM) in 20 mM imidazole buffer (pH 7.0) containing 1.0 mM EDTA was reacted with excess (0.2 mM) 5,5-dithiobis(2-nitrobenzoic acid), DTNB. The absorbance of the product (p-nitrothiophenol anion) was recorded at 412 nm (A412). This A412 was stable for 60 min and gave a linear relationship with cys concentrations used. ROI were generated either by 0.01 U xanthine oxidase (XO) + 0.01-1.0 mM hypoxanthine (HX), 0.01-1.0 mM H2O2, or H2O2 + 100 microM FeSO4. In the presence of ROI, A412 decreased with time and its rate of decrease was dependent upon the concentration of components of the ROI-generating system. This time-dependent decrease in A412 was prevented completely by the addition of 100 U of catalase (CAT). Therefore, we modified the DTNB method as follows: -SH groups were reacted with ROI for 30 min; this was followed by the addition of 100 U of CAT to scavenge the excess unreacted ROI before the addition of DTNB to generate the product. Using this modification the ROI-induced decrease in A412 was stable with time and was linearly related to the cys concentration. We further tested the modified procedure using metallothionein (MT) as a substrate for the ROI-induced changes in -SH content. MT, at concentrations of 2.5, 5.0, and 7.5 microM, was treated with XO + 100 microM HX.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of apical Na+ conductive transport in epithelia by pH

Alterations in extracellular (pHo) and/or intracellular pH (pHi) have significant effects on the apical Na+ conductive transport in tight epithelia. They influence apical membrane Na+ conductance via a direct effect on amiloride-sensitive apical Na+ channel activity and indirectly through effects on the basolateral Na+/K(+)-ATPase. Changes in pH also modulate the hormonal regulation of apical Na+ conductive transport. The pH sensitive steps in hormone action include: (i) hormone-receptor binding, (ii) increase in intracellular cyclic 3,5-adenosine monophosphate (cAMP), (iii) mobilization of intracellular free Ca2+ ([Ca2+]i), and (iv) incorporation of new channels into the apical membrane or recruitment of existing channels. Alternately, changes in pH induce secondary effects via alterations in [Ca2+]i. A reciprocal relationship between pHi and [Ca2+]i has been demonstrated in renal epithelial cells. Natriferic hormones induce a significant increase in pHi. There is a strong temporal relation between hormone-induced increase in pHi and overall increase in transepithelial Na+ transport. This suggests that changes in pHi act as an intermediate in the second messenger cascade initiated by the hormones. Several natriferic hormones activate Na(+)-H+ exchanger, H(+)-ATPase, H+/K(+)-ATPase, H+ conductive pathways in cell membranes or potential-induced changes in pHi. However, changes in pHi do not seem to be essential for the hormone effect on Na+ conductive transport. It is suggested that the role of pHi changes during hormone action is permissive rather than strictly obligatory.

pH modulates cAMP-induced increase in Na+ transport across frog skin epithelium

Apical membrane potential (Va), fractional apical membrane resistance (FRa), and/or intracellular pH (pHi) were measured in principal cells of isolated frog (Rana pipiens) skin with microelectrodes under short-circuit conditions. Apical exposure to 0.33 mM 8-(4-chlorophenylthio)adenosine 3,5-cyclic monophosphate (cAMP) depolarized Va, decreased FRa and increased short-circuit current (Isc). cAMP-induced 50% larger effects on Va and Isc at external pH (pHo) of 8.0 than at pHo 6.4. Increasing pHo from 6.4 to 8.0 in presence of cAMP further depolarized Va and increased Isc. cAMP-induced effects on Va and Isc were observed in the absence of Cl- and HCO3- and in the presence of 1 mM 4,4-diisothiocyanostilbene-2,2-disulfonic acid (DIDS) or 10 microM 5-(N-ethyl-N-isopropyl)amiloride (EIPA) or 1 microM 5-(N-methyl-N-isobutyl)amiloride (MIA). These data indicate that Na(+)-H+ exchange, Cl(-)-HCO3- exchange, and electrogenic Na(+)-(HCO3-)n cotransport are not involved in cAMP-induced increase in Isc. Apical exposure to 2 mM Cd2+ or Zn2+ depolarized Va, decreased FRa, increased Isc and increased pHi. In HCO(3-)-free solutions containing DIDS, unilateral replacement of apical Cl- by NO3- induced a fast transient depolarization of Va and an increase in Isc. These data suggest that potential-dependent changes in pHi are involved in increases in Isc. However, when changes in Va were minimized by pretreating the basolateral membrane with 25 or 75 mM K+, the cAMP-induced increase in Isc was not blocked. These data indicate that changes in pHi do not play a strict regulatory role but are only permissive in cAMP-induced effects on Isc.