Dale J. Benos

Activation of Amiloride‐Sensitive Sodium Transport in C6 Glioma Cells

Abstract: We have characterized, in C6 cells, an amiloride‐sensitive Na+ entry pathway that can exchange for H+. In this report we demonstrate that this cation‐exchange system can be induced within 24–36 h by either serum removal or by dibutyryl cyclic AMP; however, these modes of induction are not additive and are manifest only after activation by serum. In these glioma cells we found that activation by serum can be mimicked in part by specific serum factors, i.e., epidermal growth factor and bradykinin. We attempted to characterize this activation process further using several cell biologic probes. We had previously shown that that activation process involves a calcium‐dependent step with full activation obtained in the presence of the calcium iono‐phore A23187. The activation by serum was inhibited by preincubation with colchicine but not with dihydrocyto‐chalasin B, suggesting a cytoskeletal involvement in the activation process. Activation by epidermal growth factor and bradykinin was found to be unaffected by colchicine, suggesting that other factors must be present in serum that confer sensitivity to colchicine. Incubation of the cells with phorbol myristoyl acetate results in the activation of amiloride‐sensitive transport, suggesting that stimulation of protein kinase C may be integral to the activation process. Unlike the effects of serum, activation by phorbol myristoyl acetate is not inhibited by colchicine, indicating that this drug works in a way that bypasses the cytoskeletal‐dependent step. Since diacyl‐glycerol is the presumed endogenous activator of protein kinase C., we studied the effects of dioleylglycerol. This intermediate of phosphoUpid turnover was found to increase specifically the amiloride‐sensitive sodium pathway. We feel therefore that Na+‐H+ exchange in glial cells as well as in other cells may be tightly linked to phospholipid turnover and to calcium flux and that these two processes are integral to glial cell acidification and ion homeostasis.

Ouabain binding to preimplantation rabbit blastocysts

Fluid accumulation which accompanies blastocyst development is an important prerequisite for implantation. Little is known concerning the underlying epithelial transport processes responsible for this swelling, although net Na transport mediated by ouabain-sensitive, Na−K pumps has been implicated. The total number of ouabain binding sites as well as the ouabain-sensitive component of Na influx have been determined for different-aged, preimplantation rabbit blastocysts. In addition, the appearance of an amiloride-sensitive Na transport system between Day 6 and Day 7 post coitus (p.c.) was observed. The results of this work demonstrated that (1) the total number of ouabain binding sites per trophoblast cell increased between 4 and 5 days p.c. (1.2 to 9×105 sites/cell), but thereafter remained relatively constant; (2) the ouabain-inhibited Na influx increased from 0.20 μmole·cm−2·hr−1 on Day 5 p.c. to 0.33 μmole·cm−2·hr−1 on Day 6 p.c.; and (3) both 22Na influx as well as the rate of ouabain binding were inhibited by the diuretic drug amiloride in 7-day p.c. blastocysts but not in 6-day p.c. embryos.

Amiloride fluxes across erythrocyte membranes

Amiloride is known to inhibit both the influx of Na+ and the activation of mitogenesis in many cultured cell lines. This paper describes experiments in which the permeability coefficient of amiloride was determined from measurements of tracer fluxes across human erythrocytes and resealed ghosts. From an analysis of these fluxes, a permeability coefficient of 10(-7) cm/s for the uncharged form of amiloride was deduced. Based upon this measured permeability value, we present calculations of intracellular accumulation times of amiloride in cells of differing surface-to-volume ratio.

Effects of chemical group specific reagents on sodium entry and the amiloride binding site in frog skin evidence for separate sites

SummaryPreviously 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 (Isc) 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 increasingIsc 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 inIsc. When a 40,000 mol wt dextran molecule was attached to PCMB, a stable, reversible increase inIsc 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 inhibitingIsc 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 onIsc and did not alter the effectiveness of amiloride.Similarly, the amino reagents, SITS and TNBS did not affectIsc. 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 onIsc or amiloride inhibition. Methylene blue (MB), a molecule that interacts with both carboxyl and hydroxylspecific groups, inhibitedIsc by 20% and decreased amilorides ability to inhibitIsc. 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.

Changes in Interfacial Potentials Induced by Carbonylcyanide Phenylhydrazone Uncouplers: Possible Role in Inhibition of Mitochondrial Oxygen Consumption and Other Transport Processes

The charged and uncharged forms of carbonylcyanide phenylhydrazone uncouplers bind to phosphatidylcholine monolayers in a dose-dependent fashion, inducing changes in the interfacial potential of these model membranes. The interfacial potential change produced by the charged uncoupler is composed of a double-layer potential and an internal electrostatic potential (boundary and/or dipole). Changes in double-layer potential induced by the uncouplers in mitochondrial membranes can explain both the inhibition of oxygen consumption (QO2) caused by the uncouplers and the competition shown by succinate when mitochondria are respiring in the presence of rotenone. From these results and from dose-response curves of QO2 versus uncoupler concentrations, we conclude that 1 microM is an upper limit for free uncoupler concentration in the medium to avoid unwanted side effects during cell physiology studies that require total mitochondrial uncoupling.

BLASTOCYST FLUID FORMATION

All mammalian blastocysts undergo some expansion prior to implantation, but the amount varies between species. Blastocysts of the human, mouse, and rat display minimal volume changes, while those of the rabbit and pig undergo enormous increases. Figure 1 presents growth curves for preimplantation rabbit blastocysts. The blastocoelic cavity increases from about 70 nl on Day 4 postcoitus (p.c.) to almost 80 μl on Day 7 p.c., the day of implantation. This increase in size stems largely from fluid accumulation, although cell multiplication also occurs (Daniel, 1964). Because the blastocyst consists almost entirely of a single-cell-thick epithelium, the trophectoderm. it is ideally suited for the study of developmental aspects of epithelial solute and water movements.

Synthesis and characterization of methylbromoamiloride, a potential biochemical probe of epithelial Na+ channels

SummaryWe report the synthesis of a radioactive, methylated analog of bromoamiloride which inhibits the amiloride-sensitive, epithelial Na+ channel reversibly and with high affinity. This synthesis was achieved by methylation of a nitrogen in the acylguanidinium moiety with tritiated methyliodide of high specific activity. This methylated bromoamiloride molecule (CH3BrA) was purified by both thin layer and high performance liquid chromatography. Proton nuclear magnetic resonance and mass spectroscopy techniques were used to determine the structure of this analog. This compound inhibited both short-circuit current ofin vitro frog skin and22Na+ influx into apical plasma membrane vesicles made from cultured toad kidney cells (line A6) with the same or lower apparent inhibitory dissociation constant as bromoamiloride. Irradiation with ultraviolet light rendered this inhibition irreversible in both A6 vesicles and frog skin. Preparation of radioactive CH3BrA yielded specific activities in excess of 1 Ci/mmol. We suggest that this compound will be useful in the isolation and purification of this ubiquitous Na+ channel.

Cation movements across mouse red blood cells

This paper describes some features of the Na and K transport systems in red cells obtained from B10.A mice. When mouse erythrocytes were incubated in a plasma-like control medium, the scillaren-sensitive Na efflux was 3.6 +/- 0.4 mmol/l red blood cells per h while the scillaren-sensitive K influx was 3.1 +/- 0.3, values not significantly different from ech other. Scillaren had no significant effect on either Na influx or K efflux. There was a large (approx. 3 mmol/l red blood cells per h) scillaren-sensitive, Na-Na exchange diffusion component present under K-free conditions. When K was present in the incubation medium, this exchange system was suppressed.

Acidification and sodium entry in frog skin epithelium

Acidification of the external medium by isolated frog skin epithelium (Rana catesbeiana, Rana temporaria, and Caudiververa caudiververa) and its relationship to Na+ uptake was studied. Acidification was measured by the pH-stat technique under short-circuit or open-circuit conditions. The results of this study demonstrate that (a) acidification by these species of in vitro frog skins is not directly coupled to Na+ or anion transport; (b) acidification can be inhibited by the diuretic drug amiloride, but only at high external Na+ concentrations; (c) acidification rate in these species of frog skin is controlled in part by the metabolic production of CO2; and (d) the positive correlation between net Na+ absorption and net acidification observed in whole animal studies could not be replicated in the in vitro skin preparation, even when the frogs were first chronically stressed by salt depletion, a physiological state comparable to that used in the in vivo experiments.

Amiloride-Sensitive Epithelial Sodium Channels

Electrically high-resistance epithelia actively transport sodium from the luminal side to the blood (Macknight et al., 1980). The first step in this transepithelial movement of Na+ is the facilitated diffusion of this ion across the luminal or apical membrane down its electrochemical potential energy gradient. This particular transport pathway is rate limiting for the overall transport, is regulated hormonally, and is inhibited by the diuretic drug amiloride. Single-site turnover numbers deduced from current-noise experiments (106 ionsJsec) are consistent with a channel or pore-type mechanism (Lindemann and Van Driessche, 1977). Fuchs et al. (1977) and Van Driessche and Lindemann (1979) found that under their experimental conditions, Na+ permeation through these channels could be adequately described by an electrodiffusion model in which the passive movement of Na+ obeys the independence principle.