Amiloride-sensitive Channel Activity Research Articles

Volume measurements and fluorescent staining indicate an increase in permeability for organic cation transporter substrates during apoptosis

Extensive membrane blebbing is one of the earliest observable changes in HeLa cells stimulated with apoptosis inducers. Blebbing caused by actinomycin D or camptothecin, but not by anti-Fas antibody, is accompanied by an almost 10% volume increase as measured by transmission-through-dye microscopy. When the experiment is carried out in DMEM medium, the swelling appears to result from activation of amiloride-sensitive channels. Low-sodium choline-, but not N-methyl−D-glucamine-based, medium, also supports swelling during the blebbing phase of apoptosis; this indicates that the membrane becomes permeable to choline as well. Because choline can enter the cells through organic cation transporters (OCT), we tested three fluorescent dyes (2-[4-(dimethylamino)styryl]-1-methylpyridinium iodide, rhodamine 123 and ethidium bromide) that have been reported to utilize OCT for cell entry. Intact HeLa cells are poorly permeable for these fluorophores, and initially they accumulate on the plasma membranes. Blebbing results in an enhanced penetration of these dyes into the cell interior, as was demonstrated both by direct observation and by FRET. The increased membrane permeability is specific for OCT substrates; the other tested cationic dyes apparently cross the membrane by other routes and exhibit a markedly different behavior. Our results reveal a previously unknown feature of apoptosis and the utility of cationic dyes for studying membrane transport.

Epithelial Na channels and short-term renal response to salt deprivation.

To test the role of epithelial Na channels in the day-to-day regulation of renal Na excretion, rats were infused via osmotic minipumps with the Na channel blocker amiloride at rates that achieved drug concentrations of 2-5 microM in the lumen of the distal nephron. Daily Na excretion rates were unchanged, although amiloride-treated animals tended to excrete more Na in the afternoon and less in the late evening than controls. When the rats were given a low-Na diet, Na excretion rates were elevated in the amiloride-treated group within 4 h and remained higher than controls for at least 48 h. Adrenalectomized animals responded similarly to the low-Na diet. In contrast, rats infused with polythiazide at rates designed to inhibit NaCl transport in the distal tubule were able to conserve Na as well as did the controls. Injection of aldosterone (2 microg/100 g body wt) decreased Na excretion in control animals after a 1-h delay. This effect was largely abolished in amiloride-treated rats. On the basis of quantitative analysis of the results, we conclude that activation of amiloride-sensitive channels by mineralocorticoids accounts for 50-80% of the immediate natriuretic response of the kidney to a reduction in Na intake. Furthermore, the channels are necessary to achieve minimal rates of Na excretion during more chronic Na deprivation.

Transport Characteristics of the Colonic Epithelium of the Japanese Quail ( Coturnix coturnix)

The colon of the domestic fowl sustains a reabsorptive Na + current on both high- and low-sodium diets. However, there is a marked shift in the apical transport step under these two extreme conditions, from amino acid/hexose cotransport on high-salt diets to amiloride-sensitive Na + channels on low-salt diets. The present experiments were performed to study colonic Na + transport in another galliform species, the Japanese quail ( Coturnix coturnix). Birds were maintained on a commercial game feed containing 0.18% Na + (78 μmoles/g), an intermediate level of salt intake. Experiments were performed on unstripped colons in standard Ussing chambers with bicarbonate/CO 2 buffer solution on both sides. Baseline values ( n = 11) for PD (3.13 ± 0.68 mV) and short circuit current (SCC, 30.87 ± 7.79 μA/cm 2) were lower than those reported for chickens on a similar diet, whereas tissue resistance (76.06 ± 4.19 Ω·cm 2) was similar. Addition of amino acids (4 mM leucine + lysine) increased SCC by 10.85 ± 1.97 μA/cm 2. Both phloridzin (1 mM) and amiloride (10 −5M) decreased SCC, by 7.05 ± 1.26 and 9.64 ± 2.68 μA/cm 2, respectively. Thus, on this diet the quail colonic epithelium maintains both amino acid/hexose cotransporter activity and amiloride sensitive channel activity. Arginine vasotocin (10 −6 M) caused a small, but consistent decrease in SCC, while acetazolamide increased SCC. Aldosterone (128 μg/kg), given 4 hr prior to the experiment ( n = 4) significantly reduced the amino acid stimulated SCC. These results confirm, for the Japanese quail, the presence of multiple apical Na + entry mechanisms in colonic epithelium. Amino acid cotransporter activity, in particular, appears to be highly sensitive to aldosterone suppression.

The amiloride-sensitive sodium channel

Net Na+ movement across the apical membrane of high-electrical resistance epithelia is driven by the electrochemical potential energy gradient. This entry pathway is rate limiting for transepithelial transport, occurs via a channel-type mechanism, and is specifically inhibited by the diuretic drug amiloride. This channel is selective for Na+, Li+, and H+, saturates with increasing extracellular Na+ concentration, and is not affected, at least in frog skin epithelium, by changes in apical membrane surface potential. There also appears to be multiple inhibitory regions associated with each Na+ channel. We discuss the possible implications of a voltage-dependent block by amiloride in terms of macroscopic inhibitory phenomena. We describe the use of cultured epithelial systems, in particular, the toad kidney-derived A6 cell line, and the preparation of apical plasma membrane vesicles to study the Na+ entry process. We discuss experiments in which single, amiloride-sensitive channel activity has been detected and summarize current experimental approaches directed at the biochemical identification of this ubiquitous Na+ transport system.