Autoregulation of apical membrane Na+ permeability of tight epithelia. Noise analysis with amiloride and CGS 4270

Noise analysis of the Na+ channels of the apical membranes of frog skin bathed symmetrically in a Cl-HCO3 Ringer solution was done with amiloride and CGS 4270. Tissues were studied in their control states and after inhibition of transepithelial Na+ transport (Isc) by addition of quinine or quinidine to the apical solution. A critical examination of the amiloride-induced noise indicated that the single channel Na+ currents (iNa) were decreased by quinine and quinidine, probably because of depolarization of apical membrane voltage. Despite considerable statistical uncertainty in the methods of estimation of the Na+ channel density with amiloride-induced noise (NA, see text), the striking observation was a large increase of NA with amiloride inhibition of the rate of Na+ entry into the cells. NA was increased to 406% of control, whereas Isc was inhibited to 8.6% of control by 6 microM amiloride. Studies were done also with the Na+ channel blocker CGS 4270. Noise analysis with this compound was advantageous, permitting iCGSNa and NCGS to be measured in individual tissues with a relatively small inhibition of Isc. As with amiloride, inhibition of Isc with CGS 4270 caused large increases of the Na+ channel density (approximately 200% at approximately 35% inhibition of the Isc). Quinine and quinidine caused an approximately 50% increase of Na+ channel density while inhibiting iNa by approximately 60-70%. As inhibition of Na+ entry leads to an increase of Na+ channel density, a mechanism of autoregulation appears to be a major factor in adjusting the apical membrane Na+ permeability of the cells.     

The mechanism of action of Cu2+ on the frog skin.

The mechanism of action of Cu2+ when applied to the external side of the frog skin preparation was investigated. Cu2+ acts most probably on the external barrier of this preparation, since it increases the transport pool of Na+ proportionally to the increase in the short circuit current (Isc). Cu2+ does not open new routes for the Na+ entry since the stimulated Isc is still completely abolished by amiloride. The Isc dependence of Na+ concentration in the external medium is modified by copper, since the Km value increases in addition to changes in V. It is suggested that copper acts at the external barrier Na channels in a way similar to that proposed by Zeiske and Lindemann ((1974) Biochim. Biophys. Acta 352, 323--326) for benzoylimidazole-2 guanidine and benzoylthiazole-2 guanidine and by Dick and Lindemann ((1975) Pflügers Arch. ges. Physiol. 355, R72) for para-chloromercuribenzenosulfonate and para-chloromercuribenzoate.     

Possible role of Ca2+-binding sites in the regulation of Na+ transport in toad urinary bladder

La3+ was used to assess the role of membrane-bound Ca2+ in the regulation of basal and antidiuretic hormone (ADH)-induced Na+ transport by the isolated toad urinary bladder. Na+ transport was monitored by means of a short-circuit current (Isc) device. Mucosal La3+ (0.5-5 mM) increased Isc, while serosal La3+ (5 mM) produced a biphasic response (stimulation followed by inhibition). The stimulatory effects of La3+ were additive when present on both sides and were suppressed by mucosal amiloride or serosal ouabain. The action of mucosal La+ was reversible but the inhibition produced by serosal La3+ was not. In the presence of serosal La3+ the natriferic effect of ADH was abolished, but Theophylline, dibutyryl-cAMP, Amphotericin B, mucosal La3+, mucosal low pH, and phospho(enol) pyruvate, were able to increase Isc. These results suggest that Ca2+ binding sites in apical and basolateral membranes may play a key role in the modulation of both basal and ADH-induced Na+ transport. Serosal La3+ apparently inactivates the hormone-receptor interaction and/or the link between the ADH-receptor complex and the activation of adenylate cyclase, but does not interfere with the operation of the Na+ "pump", the basal activity of adenylate cyclase or any of the intracellular events that mediate the effect of ADH on Na+ transport.     

Interaction of heptanol and pressure on sodium and chloride transport by toad skin

We examined the interaction of heptanol and hydrostatic pressure on Na+ and Cl- transport in isolated toad skin. In the presence of Cl-, heptanol decreased short-circuit current (Isc) and total transepithelial resistance (Rt). However, in the absence of Cl- in the mucosal bath, heptanol increased Rt, although it retained the same inhibitory effect on Isc. When transepithelial active Na+ transport was blocked by amiloride, heptanol had no effect on Isc whether or not Cl- was present, whereas it decreased the shunt resistance (Rs) only in the presence of Cl- in the mucosal bath. Moreover, this effect of heptanol on Rs was significantly smaller in the presence of diphenylamine-2-carboxylate (DPC), a known Cl- channel blocker. Pressure also decreased Isc through inhibition of active Na+ transport, but it increased Rs. When heptanol and pressure were applied together, their inhibitory effects on Isc were additive, but their effects on Rs were antagonistic. Furthermore, when a transepithelial Cl- current was produced by reducing the Cl- concentration of the serosal bath, heptanol stimulated this current, which was reversibly inhibited by pressure or DPC addition to the mucosal bath. When the heptanol-stimulated Cl- current was first inhibited by pressure, subsequent DPC addition had less or no effect. These results suggest that one site of an antagonistic interaction of heptanol and pressure in toad skin is an apical membrane Cl- conductance.     

Effect of amiloride and some of its analogues of cation transport in isolated frog skin and thin lipid membranes

The inhibition of short-circuit current (Isc) in isolated frog skin and the induction of surface potentials in lipid bilayer membranes produced by the diuretic drug amiloride and a number of its chemical analogues was studied. The major conclusions of our study are: (a) The charged form of amiloride is the biologically active species. (b) Both the magnitude of Isc and the amiloride inhibitory effect are sensitive to the ionic milieu bathing the isolated skin, and these two features are modulated at separate and distinct regions on the transport site. (c) Amiloride is very specific in its inhibitory interaction with the Na+ transport site since slight structural modifications can result in significant changes in drug effectiveness. We found that substitutions at pyrazine ring position 5 greatly diminish drug activity, while changes at position 6 are less drastic. Alterations in the guanidinium moiety only diminish activity if the result is a change in the spatial orientation of the amino group carrying the positive charge. (d) Amiloride can bind to and alter the charge on membrane surfaces, but this action cannot explain its highly specific effects in biological systems.     

Invertebrate epithelial Na+ channels: amiloride-induced current-noise in crab gill.

Epithelial sheets (including cuticle) from posterior gills of the freshwater-adapted euryhaline crab Eriocheir sinensis were obtained according to the method of Schwarz and Graszynski ((1989) Comp. Biochem. Physiol. 92A, 601-604; (1989) Verh. Dtsch. Zool. Ges. 82, 211 and (1989) Arch. Int. Physiol. Biochim. 97, C45). With external NaCl-saline, the outward-directed short-circuit current (Isc) could hardly be influenced by external amiloride up to 100 mumol/l but was, on the contrary, strictly dependent on apical Cl- (Onken, Graszynski and Zeiske (1991) J. Comp. Physiol. B 161, 293-301). In absence of external chloride an inward-directed, amiloride-inhibitable Isc was observed which depended on external Na+ (thus, Isc approximately INa) in a two-step, saturating mode. The Isc-block by amiloride obeyed saturation kinetics (half-maximal at less than or equal to 1 mumol/l, suggesting apical Na(+)-channels). Only for Na+ concentrations below 100 mmol/l we found an indication for a competitive interaction between Na+ and amiloride at the channel. Current fluctuation analysis revealed the presence of an amiloride-induced relaxation (Lorentzian) component in the Isc-noise (so-called 'blocker-noise'). The Lorentzian parameter-shifts with increasing amiloride concentration indicate first-order kinetics of the blocker with its apical receptor. Using a 'two-state' blocking model we calculated, for amiloride concentrations between 2 and 5 mumol/l, a mean single-channel current of 0.46 pA and a mean channel density of 250.10(6) cm-2.     

Cation selectivity of the apical membrane of the turtle colon: sodium entry in the presence of lithium

Exposure of the apical surface of the isolated turtle colon to Li produced a marked transient in short-circuit current (ISC) and total tissue conductance (GT) which was abolished by amiloride but was unaffected by ouabain or by removing Na or Cl from the mucosal bathing solution. Despite marked changes in Isc, Na uptake across the apical membrane was a linear function of time during exposure to Li-containing solutions, and except at very high Li concentrations, the initial rate of Na uptake, JiNa, was identical to its pre-Li value. In the presence of Li, however, JiNa was significantly less than the total Isc. The apparent "transference number" for Na in the apical membranes was a function of the Li:Na concentration ratio in the mucosal bathing solution. These results suggest that Li can carry substantial amounts of current through amiloride-sensitive channels in the apical membrane of the colon without having any effect on the rate coefficient for Na entry. This behavior is not consistent with "competition" of Na and Li for a membrane "carrier" but rather suggests that the Na entry mechanism may be a population of pores or channels through which Na and Li may pass with negligible interaction.     

Binding of [3H]ouabain to split frog skin: the role of the Na,K-ATPase in the generation of short circuit current

The binding of [3H]ouabain to the serosal side was studied in a chambered preparation of frog skin, free of connective tissue, while the short circuit (Isc) was concurrently monitored. Both ouabain binding and Isc inhibition proceeded as hyperbolic functions of time. A plot of the number of ouabain molecules bound vs. the corresponding values of Isc inhibition (percent) yielded a straight line, yet showed that one-third of the binding occurred before any inhibition of Isc. Upon separation of the skins into two groups based upon initial Isc(Isci)--high, greater than 20 microamperemeter/cm2 and low, less than 10 microamperemeter/cm2, we observed two distinct populations. The high Isci skins bound very little ouabain before inhibition of Isc whereas low Isci skins bound one-half of the total number of sites before exhibiting any inhibition of Isc. These observations strongly suggest that (a) the Na,K-ATPase is directly involved in the generation of Isc, and (b) at low Isc, inhibition of some pumps by ouabain causes a "recruitment" of other pumps to increase their turnover rate and maintain Isc relatively unaffected. In addition, the binding of ouabain also displayed various characteristics that were consistent with known properties of the Na,K-ATPase: (a) increased intracellular K/Na concentrations, whether achieved through the addition of amiloride or removal of Na from the outside medium, led to a significant decrease in ouabain binding rate relative to paired controls; and (b) ouabain binding, either with normal or decreased intracellular Na, was significantly reduced in the presence of elevated K in the serosal bathing medium. Finally, the number of ouabain molecules bound to the frog skins was not correlated with their initial Isc values, indicating that the spontaneous skin-to-skin variation in Isc was not related to the number of functional pump sites but, rather, to their turnover rate.     

Bioelectric properties and ion flow across excised human bronchi

Bioelectric properties and ion transport of excised human segmental/subsegmental bronchi were measured in specimens from 40 patients. Transepithelial electric potential difference (PD), short-circuit current (Isc), and conductance (G), averaged 5.8 mV (lumen negative), 51 microA X cm-2, and 9 mS X cm-2, respectively. Na+ was absorbed from lumen to interstitium under open- and short-circuit conditions. Cl- flows were symmetrical under short-circuit conditions. Isc was abolished by 10(-4) M ouabain. Amiloride inhibited Isc (the concentration necessary to achieve 50% of the maximal effect = 7 X 10(-7) M) and abolished net Na+ transport. PD and Isc were not reduced to zero by amiloride because a net Cl- secretion was induced that reflected a reduction in Cl- flow in the absorptive direction (Jm----sCl-). Acetylcholine (10(-4) M) induced an electrically silent, matched flow of Na+ (1.7 mueq X cm-1 X h-1) and Cl- (1.9 mueq X cm-12 X h-1) toward the lumen. This response was blocked by atropine. Phenylephrine (10(-5) M) did not affect bioelectric properties or unidirectional ion flows, whereas isoproterenol (10(-5) M) induced a small increase in Isc (10%) without changing net ion flows significantly. We conclude that 1) Na+ absorption is the major active ion transport across excised human bronchi, 2) Na+ absorption is both amiloride and ouabain sensitive, 3) Cl- secretion can be induced by inhibition of the entry of luminal Na+ into the epithelia, and 4) cholinergic more than adrenergic agents modulate basal ion flow, probably by affecting gland output.     

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

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.     

Electrogenic ion transport in the intestine of the Australian common brushtail possum, <Emphasis Type="Italic">Trichosurus vulpecula</Emphasis>: indications of novel transport patterns in a marsupial

In this study, electrogenic ion transport in the intestine of the Australian common brushtail possum, Trichosurus vulpecula was investigated. In the ileum, a Na(+)-dependent, phloridzin- and amiloride-insensitive short-circuit current ( Isc) was present. Mucosal glucose stimulated a further phloridzin-sensitive, dose-dependent increase in Isc. A Na(+)-dependent, ouabain-sensitive Isc was also present in the caecum and colon. In the proximal and distal colon, amiloride (100 micro mol l(-1), mucosal) inhibited this Isc by 81+/-4% and 65+/-3%, respectively and the Ki for amiloride (approximately 1 micro mol l(-1)) was consistent with the inhibition of a classical epithelial Na(+) channel. In the caecum, 50% of the Isc was inhibited by amiloride (100 micro mol l(-1), mucosal). The amiloride-insensitive Isc in the colon was not due to electrogenic Cl(-) secretion, as serosal bumetanide (100 micro mol l(-1)) had no effect on the Isc. Furthermore, the secretagogues forskolin (10 micro mol l(-1)), carbachol (100 micro mol l(-1)) and dibutyryl-cAMP or dibutyryl-cGMP (100 micro mol l(-1)) did not stimulate electrogenic Cl(-) secretion by the colon. These results indicate that the transport properties of the hindgut of the possum differ significantly from those of eutherian mammals and may be associated with different functions of the hindgut of possums when compared to eutherian mammals.     

Cationic selectivity and competition at the sodium entry site in frog skin

The cation selectivity of the Na entry mechanism located in the outer membrane of the bullfrog (Rana catesbeiana) skin epithelium was studied. This selectivity was determined by measuring the short-circuit current when all of the external sodium was replaced by another cation and, also, by noting the relative degree of inhibition that the alkali metal cations produced on Na influx. The ability of the Group Ia cations to permeate the apical membrane was determined from the tracer uptake experiments. The results demonstrate that (a) only Li and Na are actively transported through the epithelium; (b) the alkali cations K, Rb, and Cs do not enter the epithelium through the apical border and, therefore, Na and Li are the only alkali cations translocated through this membrane; (c) these impermeable cations are competitive inhibitors of Na entry; (d) the cations NH4 and Tl exhibit more complex behavior but, under well-defined conditions, also inhibit Na entry; and (e) the selectivity of the cation binding site is in the sequence Li congruent to Na > Tl > NH4 congruent to K > Rb > Cs, which corresponds to a high field strength site with tetrahedral symmetry.     

Changes in cell membrane fluidity affect the sodium transport across frog skin and its sensitivity to amiloride

1. 1-5 mM n-hexanol added to the outer (mucosal) medium of isolated skin of the frog Rana temporaria increases the short circuit current (Isc) across it. 2. This effect shows a saturable dependency on the outer sodium concentration, also when NaCl is replaced by Na2SO4. 3. n-Hexanol at a concentration of 1 mM, and cold acclimation of the frogs, which increases the fluidity of epidermal cell membranes, do not affect the sensitivity of Isc to the inhibiting effect of amiloride. 4. n-Hexanol at a concentration (5 mM) which causes a fluidization of cell membrane preparations from isolated frog epidermis also increases the sensitivity of Isc to amiloride. 5. The effects of low concentrations of n-hexanol and of cold acclimation probably depend on an increase of the permeability of apical membranes of epidermal cells to sodium caused by membrane fluidization. At higher concentrations of n-hexanol, a further disordering of the membrane structure occurs with a better access of amiloride to its action sites.     

The stimulation of Na+ uptake in frog skin by uranyl ions.

1. The Na+ uptake in the isolated from skin of Rana esculenta was measured by the short-circuit current (Isc). Uranyl ions increase at pH 5.5 the Isc up to 200% at concentrations of 10 mM. The half-maximal value for this effect is at about 1 mM uranyl salt. 2. The effect is (a) specific for the Na+-selective membrane, (b) fully reversible. No stimulation can be seen in presence of 1 mM H+ or 0.1 mM amiloride. 3. The decrease of the sodium permeability of the apical membrane (PNa), normally induced by increasing concentrations of Na+ in the mucosal solution, %Na]o, is partially prevented by uranyl ions. The apparent Michaelis constant of the saturable Na+ uptake is shifted to much higher values. 4. A comparison between the uranyl effect and similar effects of the other drugs leads to the conclusion that uranyl ions might act in a polar hydrophobic environment, possibly by combining with phosphate groups (of phospholipids), and, thus, enhancing Na+ permeability by changes in tertiary structure near each Na channel. The interaction of mucosal Na+ with their receptor, normally triggering the [Na]o-dependent decrease of PNa, is thought to be diminished by uranyl association in a neighbouring region, causing a noncompetitive stimulation of the Na+ translocation though the apical frog skin membrane.     

Ion transport in an immortalized rat submandibular cell line SMG-C6

The immortalized rat submandibular epithelial cell line, SMG-C6, cultured on porous tissue culture supports, forms polarized, tight-junction epithelia facilitating bioelectric characterization in Ussing chambers. The SMG-C6 epithelia generated transepithelial resistances of 956+/-84Omega.cm2 and potential differences (PD) of -16.9 +/- 1.5mV (apical surface negative) with a basal short-circuit current (Isc) of 23.9 +/- 1.7 microA/cm2 (n = 69). P2 nucleotide receptor agonists, ATP or UTP, applied apically or basolaterally induced a transient increase in Isc, followed by a sustained decreased below baseline value. The peak DeltaIsc increase was partly sensitive to Cl- and K+ channel inhibitors, DPC, glibenclamide, and tetraethylammonium (TEA) and was completely abolished following Ca2+ chelation with BAPTA or bilateral substitution of gluconate for Cl-. The major component of basal Isc was sensitive to apical Na+ replacement or amiloride (half-maximal inhibitory concentration 392 nM). Following pretreatment with amiloride, ATP induced a significantly greater Isc; however, the poststimulatory decline was abolished, suggesting an ATP-induced inhibition of amiloride-sensitive Na+ transport. Consistent with the ion transport properties found in Ussing chambers, SMG-C6 cells express the rat epithelial Na+ channel alpha-subunit (alpha-rENaC). Thus, cultured SMG-C6 cells produce tight polarized epithelia on permeable support with stimulated Cl- secretory conductance and an inward Isc accounted for by amiloride-sensitive Na+ absorption.     

Sodium arsenite affects Na+ transport in the isolated skin of the toad Pleurodema thaul

Arsenic, applied as sodium arsenite (As(III)) to either inner or outer surfaces of the isolated toad skin, dose-dependently decreased the short-circuit current (Isc), potential difference (PD) and sodium conductance (G(Na)) in the concentration range 1-1000 microM, with effects often lasting over 3 h. Maximal inhibitory effect was over 90% with an IC(50) of about 34 microM. Applied during amiloride block, As(III) did not change this effect. However, an increase in electric parameters was noted during the initial 30 min in 22 experiments, indicating a possible translocation of cytosolic protein kinase C (PKC) to the membrane within 15 min, thus stimulating sodium transport; this is followed by a progressive inhibition of kinase activity. Comparative effects of amiloride (8 microM), As(III) (100 microM, outer surface) and noradrenaline (NA, 10 microM, inner surface) showed a significant increase in the stimulatory effect of NA on the electric parameters, which could be the result of arsenite clustering of cell surface receptors and activation of ensuing cellular signal transduction pathways. Ouabain 5 microM, followed by As(III) 100 microM, also stimulated the skin response to NA (10 microM), although the duration of the two phases of the response was markedly shortened. The exact mechanism is still in doubt: however, As(III) increases cerebral metabolites of NA and ouabain can increase NA efflux from tissue slices. The amiloride test, performed with As(III) in the outer surface, confirmed significant decrease in all the parameters: the driving force (E(Na)), sodium conductance (G(Na)), and importantly, shunt conductance (G(sh)), due to the known fact that arsenic inhibits gap junctional intercellular communication.     

Effect of diuretics on intestinal transport of electrolytes, glucose, and amino acid.

The jejunal mucosal membrane of albino mice was used to study the electrical properties and ion transport. The membrane was bathed in Krebs-Ringer solution with or without glucose.When ethacrynic acid (EA), furosemide, or amiloride was added to the bathing fluid of both sides, a transient increase followed by a decrease of both potential difference (PD) and short circuit current (Isc) were observed. In glucose-containing bathing medium, EA inhibited both net Na and Cl flux and residual flux; however, EA had little effect on both Na and Cl flux in glucose-free bathing medium. Studies using everted intestinal sac technique showed that EA inhibited both glucose and L-tyrosine across the mucosal membrane against concentration gradients. Furosemide and amiloride were less potent than EA in inhibiting the Na and Cl flux when the bathing solution contained glucose. But these two compounds had no effect on glucose and L-tyrosine transport across the intestinal mucosa. Furthermore, they did inhibit Cl flux even in the condition of glucose-free bathing medium. It is postulated that all three diuretics act on the brush-border membrane of the intestine. EA probably inhibits the Na-glucose cotransporting system; furosemide and amiloride inhibit the simple diffusion process of Na entry of Cl exit by decreasing the conductance of the membrane.     

Amiloride analogs inhibit L-type calcium channels and display calcium entry blocker activity

Three structural classes of commonly used amiloride analogs, molecules derivatized at the terminal guanidino-nitrogen, the five-position pyrazinoyl-nitrogen, or di-substituted at both of these positions, inhibit binding of the L-type Ca2+ channel modulators diltiazem, gallopamil, and nitrendipine to porcine cardiac sarcolemmal membrane vesicles. The rank order of inhibitory potencies among the various derivatives tested is well defined with amiloride being the least potent. Saturation binding studies indicate that inhibition of ligand binding results primarily from effects on Kd. Ligand dissociation measurements suggest that amiloride derivatives do not associate directly at any of the known sites in the Ca2+ entry blocker receptor complex. In addition, these compounds do not compete at the "Ca2+ coordination site" within the channel. However, studies with inorganic and substituted diphenylbutylpiperidine Ca2+ entry blockers reveal that amiloride analogs interact at a site on the channel where metal ions bind and occlude the pore. Photolysis experiments performed with amiloride photoaffinity reagents confirm that a specific interaction occurs between such probes and the channel protein. Upon photolysis, these agents produce concentration- and time-dependent irreversible inactivation of Ca2+ entry blocker binding activities, which can be protected against by either verapamil or diltiazem. 45Ca2+ flux and voltage-clamp experiments performed with GH3 anterior pituitary cells demonstrate that amiloride-like compounds inhibit L-type Ca2+ channels directly. Moreover, these compounds block contraction of isolated vascular tissue in pharmacological assays. Electrophysiological experiments indicate that they also inhibit T-type Ca2+ channels in GH3 cells. Taken together, these results demonstrate unequivocally that amiloride analogs display significant Ca2+ entry blocker activity in both ligand binding and functional assays. This property, therefore, can seriously complicate the interpretation of many in vitro and in vivo studies where amiloride analogs are used to elicit inhibition of other transport systems (e.g. Na-Ca and Na-H exchange).     

Aldosterone upregulates purinergic responses in larval amphibian skin epithelium

Bullfrog tadpoles respond to apical application of 100 microM amiloride, acetylcholine (ACh) or ATP with a sharp transient inward (apical to basolateral) cation current. In adult skin, amiloride blockable transepithelial Na+ transport is upregulated by the hormone aldosterone. Tadpoles were treated in vivo with aldosterone and changes in short circuit current (Isc) in response to apical application of ATP were determined. Bullfrog tadpoles were exposed to aldosterone (10(-6) M) for periods ranging from 3 h to 60 h. Skins from 60-h aldosterone-treated animals showed a two- to three-fold increase in apical ATP-activated short circuit current when compared to animals treated with vehicle alone. Sodium replacement with a large, nonpermeable cation resulted in no measurable increase in Isc after exposure to ligand, consistent with ATP activation of an inward cation current and not chloride efflux. Activation/desensitization time courses and treatment with blockers revealed no measurable differences between aldosterone-treated and non-treated skins. Activation by amiloride and ACh gave essentially identical results. Studies with RT/PCR showed significant increases over controls of levels of mRNA associated with P2X channels. Given these data, our working hypothesis is that all three ligands activate the same process that exhibits both purinergic and cholinergic characteristics. These data are consistent with aldosterone upregulation of ATP gated channels expressed in the apical membrane of larval frog skin.     

The role of the Na+/H+ exchange system in cardiac cells in relation to the control of the internal Na+ concentration. A molecular basis for the antagonistic effect of ouabain and amiloride on the heart

Cardiac cells in culture (from rat and chick heart) have a membrane Na+/H+ exchange system that is inhibited by amiloride (K0.5 = 5 microM) and by its more potent N-5-disubstituted derivatives dimethylamiloride (K0.5 = 300 nM) and ethylisopropylamiloride (K0.5 = 30 nM). The properties of the cardiac Na+/H+ exchange system are similar to those found for the Na+/H+ exchanger in other cellular types. The Na+/H+ exchange system is a major pathway for Na+ uptake by cardiac cells. Ouabain which inhibits the (Na+,K+)-ATPase, a major pathway for Na+ efflux, is known to provoke Na+ accumulation and to stimulate 45Ca2+ entry via the Na+/Ca2+ exchange mechanism, thereby producing an inotropic effect. N-5-Disubstituted amiloride derivatives, by blocking Na+ entry into cardiac cells, antagonize both ouabain-induced intracellular Na+ accumulation and the ouabain-induced acceleration of 45Ca2+ uptake.