ISSN:
1432-1424
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
,
Chemistry and Pharmacology
Notes:
Summary Previously we have shown that the inhibition of active transport by amiloride is noncompetitive with sodium inRana catesbeiana skin, suggesting that amiloride acts at a site separate from the sodium entry site (Benos, D.J., Mandel, L.J., Balaban, R.S. 1979,J. Gen Physiol. 73: 307). In the present study, the effects of a number of sulfhydryl, amino, and carboxyl group selective reagents were studied on short-circuit current (I sc) as well as the efficacy of amiloride in bullfrog skin, to determine those functional ligands which may be involved with either of these processes. Addition of the sulfhydryl reagent PCMBS (1mm) to the outside bathing medium produced biphasic effects, initially reversibly increasingI sc by an average 56% followed by a slower, irreversible decay to levels below baseline. In contrast, the addition of 0.1mm PCMB always resulted in a rapid, irreversible decrease inI sc. When a 40,000 mol wt dextran molecule was attached to PCMB, a stable, reversible increase inI sc was observed. These observations suggest that at least two populations of-SH groups are involved in Na translocation through the entry step. Amiloride was equally effective in inhibitingI sc before and after treatment with PCMBS both during the stimulatory as well as the inhibitory phase. The sulfhydryl reducing agent DTT, and oxidizing agent DTNB had only minor influence onI sc and did not alter the effectiveness of amiloride. Similarly, the amino reagents, SITS and TNBS did not affectI sc. However, TNBS decreased the ability of amiloride to inhibit Na entry. These results suggest that an amino group may be involved in the interaction of amiloride and its site, without affecting Na entry. The carboxyl reagents EEDQ, TMO, and three separate carbodiimides were without effect onI sc or amiloride inhibition. Methylene blue (MB), a molecule that interacts with both carboxyl and hydroxylspecific groups, inhibitedI sc by 20% and decreased amiloride's ability to inhibitI sc. These effects, however, are likely to occur from the cytoplasmic side as MB appears to enter into the cells. These results support the notion that amiloride and Na interact with the entry protein at different regions on the membrane.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/BF01875966
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