Biomedical subjects

E Cragoe

Publications and source records attributed to E Cragoe.

At least 19 recordsLinked to original sources

PubMed1999

[Dependence of myocardial damage on the anion composition and osmotic pressure in the extracellular fluid during "calcium paradox" in rats].

The data obtained reveal that elevation of extracellular osmolarity with sucrose during reintroduction of Ca-containing medium after 10 minutes of Ca2+ removal prevents loss of haemoglobin in a concentration-dependent mode. Reducing the extracellular osmolarity of the reperfusion medium by means of decreasing the concentration of sodium chloride and calcium chloride exacerbates the loss of haemoglobin from the cardiomyocytes. There is a close correlation between the water contents in tissues and the loss of haemoglobin during the "calcium paradox". The findings suggest dependence of the heart damage during the "calcium paradox" on anionic composition of extracellular space and activity of anionic transporters.

Animals↗

PubMed1997

An aldosterone regulated chicken intestine protein with high affinity to amiloride.

The pattern of chicken intestine amiloride-binding proteins was determined using the photoreactive amiloride analogue 2'-methoxy-5'-nitrobenzamil (NMBA) and a polyclonal anti-amiloride antibody. At 10(-7)M, NMBA inhibits approximately 62% of the Na+ channel activity. At this concentration the amiloride analogue labels a number of membrane proteins, and in particular a 40-45 kDa polypeptide denoted ABP40. Incorporation of NMBA into ABP40 could be prevented by a 100-fold excess of benzamil, but not by a 1000-fold excess of 5-(N-ethyl-N-isopropyl)-amiloride. Labeling of ABP40 was intense in membranes derived from salt-deprived chickens and approximately 5-fold weaker in membranes from salt-repleted animals. Because of its small size, ABP40 is not likely to be an avian Na+ channel subunit, yet this amiloride-binding protein could be involved in the response to aldosterone.

Aldosterone↗

PubMed1997

IGF-I regulation of Na(+)-K(+)-ATPase in rat arterial smooth muscle.

Insulin-like growth factor I (IGF-I) is vasodilatory and mitogenic for vascular smooth muscle cells (VSMC). Alteration in VSMC Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase) activity is hypothesized to underlie abnormal vascular tone and growth in hypertension and diabetes. Therefore, we investigated effects of IGF-I on Na(+)-K(+)-ATPase activity in rat aortic VSMC. IGF-I increases pump activity in a dose- and time-dependent manner: the minimal dose required was 10(-10) M, and the minimal time required was 20 min (at 10(-8) M) to increase activity. Similar effects persisted through 12 h. In Na(+)-loaded cells, IGF-I does not further stimulate activity. Blockade of Na+/H+ exchange attenuates IGF-I-induced increases in activity after 30 min but has no effect after 12 h. Northern blot analyses reveal that expression of the alpha 1- and the alpha 2-subunits of the pump were unaffected by IGF-I. Plasma membrane alpha 1- and alpha 2-protein were also unaffected, suggesting translocation of preformed pools was not responsible for the increases. Inhibitors revealed that neither tyrosine kinase activity, RNA transcription, protein synthesis, nitric oxide synthase activity, or protein kinase C activity mediated this IGF-I effect. Therefore, IGF-I regulates Na pump activity in the short term by an Na+/H+ exchange-dependent but transcription/translocation-independent mechanism. These data suggest that IGF-I, known to be produced by VSMC, may regulate tone and growth responses abnormal in disease states such as hypertension and diabetes.

Animals↗

PubMed1995

The osmolality-sensitive taurine channel in flounder erythrocytes is strongly stimulated by noradrenaline under hypo-osmotic conditions

Stimulation of flounder erythrocytes by noradrenaline under isosmotic conditions (330 mosmol kg-1) and physiological Na+ concentration (113 mmol l-1) caused swelling of the cells. The EC50 of this cell swelling was 0.65 µmol l-1 noradrenaline. The effect of the noradrenaline-induced cell swelling on the taurine channel under isosmotic conditions was negligible. However, when the cells were stimulated by noradrenaline (1.0 µmol l-1) before, simultaneously with or after reduction of osmolality (255 mosmol kg-1), the volume regulatory efflux of taurine mediated by the taurine channel was transiently accelerated. The rate coefficient for taurine efflux was more than four times higher than in osmolality-stimulated cells not exposed to noradrenaline. The present paper deals with the accelerating effect of noradrenaline on the taurine channel under hypo-osmotic conditions and the lack of effect of noradrenaline-induced cell swelling on the channel under iso-osmotic conditions. Noradrenaline initiated the cell swelling by interacting with ß-receptors which appeared to be more related to the mammalian ß1-receptors than to the ß2-receptors. The receptor interaction activated the adenylate cyclase system and, in the presence of 1.0 µmol l-1 noradrenaline, the cellular cyclic AMP concentration increased about 23 times. Noradrenaline also stimulated the Na+/H+ and Cl-/HCO3- antiporters and this affected the extracellular pH as well as the cell volume. Depending on the extracellular Na+ concentration, the incubation medium was acidified (113 mmol l-1 Na+) or alkalized (2.7 mmol l-1 Na+). Under these two conditions, the accelerating effects of noradrenaline on the taurine efflux were of similar magnitude. Similar effects on the cell volume, the extracellular pH and the volume regulatory taurine efflux were obtained in the presence of the cyclic AMP analogue 8-bromo-cyclic AMP. Under hypo-osmotic conditions in the absence of noradrenaline, the cellular level of cyclic AMP was not elevated. There was no significant positive correlation between the water content of the cells (cell volume) under different conditions in the presence or absence of noradrenaline and the state of activation of the osmolality-sensitive taurine channel. We conclude that the mechanism(s) which activate(s) the osmolality-sensitive taurine channel in flounder erythrocytes is transiently and strongly accelerated by noradrenaline, but not triggered by the noradrenaline-induced events. The acceleration does not appear to be due to increased activity of the antiporters, but to increased cellular levels of cyclic AMP.

Journal Article↗

PubMed1993

An epithelial high-affinity amiloride-binding site, different from the Na+ channel.

Specific binding of the radioactive amiloride analogues [3H]phenamil and [3H]benzamil was studied in plasma membrane from chicken lower intestine. A single population of sites whose affinities and specificities towards pyrazinecarboxamides roughly resemble those of the epithelial Na+ channel, was identified. However, a matched comparison of pyrazinecarboxamide binding and Na+ transport inhibition revealed substantial differences between the high-affinity [3H]phenamil-binding site detected, and the site whose occupancy by phenamil blocks Na+ transport. First, 5-(N-ethyl-N-isopropyl)-amiloride was found to displace bound [3H]phenamil at concentrations that are at least 10-fold lower than those needed to block the channel. Second, the rates at which [3H]phenamil associates and dissociates from this site are lower than the rates at which Na+ channels are inhibited and reactivated, under similar conditions. A site with high affinity to both amiloride and 5-(N-ethyl-N-isopropyl)-amiloride was detected also in membranes from other epithelia. We conclude that tight epithelia contain a major high-affinity amiloride receptor other than the Na(+)-conducting channel, the Na+/H+ antiport or the Na+/Ca2+ exchanger. This site could be associated with a pool of nonconducting channels, another (but structurally related) channel, or a totally unrelated protein.

Aldosterone↗

PubMed1992

Hormonal and pharmacologic regulation of sodium absorption in rabbit cecum in vitro.

The rabbit cecum is a moderately tight epithelium with amiloride-resistant but phenamil-sensitive electrogenic Na absorption. We performed flux and electrical studies under short-circuit conditions in vitro to further characterize the mechanisms of ion transport in cecum in normal and animals pretreated with methylprednisolone (MP) and deoxycorticosterone acetate (DOCA). MP treatment increased Na absorption and decreased tissue conductance. In contrast, DOCA increased Isc but did not significantly alter Na or Cl fluxes. Amiloride analogs with primary specificity for Na channel and Na/H exchanger both inhibited Isc and Na absorption. Ethacrynic acid, but not bumetanide, inhibited Isc. Nystatin and amphotericin B increased Isc. We conclude that: (1) Steroids have a differential effect on cecal ion transport; methylprednisolone increases Na absorption, but DOCA does not. (2) The response to amiloride analogs is different from other electrogenic transport systems, suggesting a distinct mechanism of Na transport in cecum. (3) The effect of ethacrynic acid was unexpected, suggesting an inhibitory response on an alternate transport system. (4) The effects of polyene antibiotics are similar to those found in other tight epithelia. Electrogenic Na absorption in rabbit cecum represents a distinct transport system, significantly different from Na absorptive mechanisms in other segments of the gut.

Amiloride↗

PubMed1992

5-(N-ethyl-N-isopropyl)amiloride and mild acidosis protect cultured cerebellar granule cells against glutamate-induced delayed neuronal death.

In the experiments on the primary cerebellar granule cell cultures, delayed neuronal death was induced by 15 min treatment of the cells with 50 microM glutamate. 5-(N-ethyl-N-isopropyl)amiloride (10 microM) known as a potent inhibitor of the Na+/H+ exchanger, when added to the glutamate-containing Mg(2+)-free solution caused a considerable (approximately by 40%) decrease in the number of dead cells counted 4 h after the termination of glutamate treatment. Patch-clamp experiments with freshly isolated rat hippocampal neurons have shown that the neuroprotective effect of 5-(N-ethyl-N-isopropyl)amiloride can be explained by its ability to block N-methyl-D-aspartate channels (receptors) at micromolar concentrations. A similar mechanism apparently underlies neuroprotective effect of external acidosis (reduction of pH from 7.6-7.8 to 6.7-6.8) during glutamate application. 5-(N-ethyl-N-isopropyl)amiloride (10 microM) and low pH (6.7) also proved capable of exhibiting neuroprotective effects upon application during the post-glutamate period. In this instance, however, the number of dead cells was decreased by no more than 20%. This neuroprotective effect of 5-(N-ethyl-N-isopropyl)amiloride and low pH is interpreted as resulting from inhibition of Na+/H+ exchange, since a direct blockade of N-methyl-D-aspartate receptors by 1 mM DL-2-amino-5-phosphonovalerate after termination of glutamate treatment did not attenuate the delayed neuronal death. Finally, we have established that the addition of 10 microM 5-(N-ethyl-N-isopropyl)amiloride to the cultures both during glutamate treatment and after its termination results in a complete protection of cultured cerebellar granule cells.

Amiloride↗

PubMed1991

Inhibition of Na+/Ca2+ exchange enhances delayed neuronal death elicited by glutamate in cerebellar granule cell cultures.

Experiments have been carried out on the primary cerebellar granule cell cultures from 7- to 8-day-old Wistar rats. To study a possible contribution of Na+/Ca2+ exchange to the toxic effect of glutamate, two amiloride derivatives, 3',4'-dichlorobenzamil (DCB) and 5-(N-4-chlorobenzyl)-2',4'-dimethylbenzamil (CBDMB), known to be the potent inhibitors of this exchange system, were used. Addition of DCB or CBDMB (at 30 and 10 microM, respectively) to a 25 microM glutamate solution dramatically enhanced the delayed neuronal death observed during the 4 h after termination of glutamate treatment. Similar but insignificantly smaller effects were obtained when these agents were added to the cultures in the post-glutamate period. Removal of Na+ (by substituting for choline chloride) from the external Mg2+-free solution in the post-glutamate period also enhanced a delayed neuronal damage. The data obtained suggest that Na+/Ca2+ exchanger does not constitute the route for Ca2+ entry during the post-glutamate period but, on the contrary, attenuates glutamate neurotoxicity providing Ca2+ extrusion from the cells under the conditions of a sustained Ca2+ influx.

Amiloride↗

PubMed1990

Evidence for a DIOA-sensitive [K+,Cl-]-cotransport system in cultured vascular smooth muscle cells.

The existence of a [K+,Cl-]-cotransport system in vascular smooth muscle cells was investigated in the A10 cell line by studying the effect of DIOA (dihydroindenyl-oxy-alkanoic acid, a potent inhibitor) on K+, Rb+ and Cl- fluxes. Hypotonic medium (150 mOsm) increased initial rates of ouabain and bumetanide-resistant (OBR) Rb+ uptake by 100%, bumetanide and DIDS-resistant Cl- uptake by 200%, and OBR net K+ efflux by 130%. DIOA inhibited 40 to 100% of the Rb+ influx and net K+ efflux stimulation with an IC50 of 4 X 10(-5) mol/L. DIOA-sensitive Rb+ influx was a sigmoidal function of the decrease in osmolarity, with a threshold at about 230 mOsm. Our results suggest that vascular smooth muscle cells have a DIOA-sensitive [K+,Cl-]-cotransport system. Dissipation of the outwardly directed Cl- gradient with an apparent [Cl- to K+] stoichiometry much higher than one may provide the energy to ensure net KCl (and osmotic water) extrusion and cell volume regulation in these cells.

Animals↗

PubMed1990

Refined estimation of kinetic parameters of the Na+/H+ antiport in human fibroblasts and platelets.

A technique is presented to estimate the initial rates of Na(+)-dependent alkalinization of acidified human fibroblasts and platelets and assess the kinetics of the Na+/H+ antiport in these cells. Cytosolic pH (pHi) exhibits an exponential recovery following cellular acidification. Thus, the length of the time interval selected to monitor changes in pHi (delta pHi) is critical to estimating the kinetics of the Na+/H+ antiport. We compared kinetic parameters of the Na+/H+ antiport, using computed and observed changes in delta pHi, for arbitrarily selected time intervals following Na(+)-dependent activation. In both cells, significant increases in both the [Na+] for half-maximal activation (K0.5) and maximal velocities (Vmax) were observed as delta pHi was decreased. We conclude that kinetic parameters derived from initial rate determinations enable a more accurate characterization of the Na+/H+ antiport.

Biological Transport, Active↗

PubMed1988

Intracellular pH and cell adhesion to solid substrate.

It was shown that activation of the Na+/H+ antiporter resulting in an increase of intracellular pH (pHi) by 0.2-0.3 is a necessary stage of cell stimulation by soluble growth factors. Solid substrate can also be formally regarded as a growth factor since adhesion stimulates proliferation of various cell types. In the present study we have found that the attachment of mouse embryo fibroblasts to solid substrate is followed by an increase of pHi by approx. 0.3 units. pH shift occurs after the cell attaches to the substrate and is obligatory for cell spreading. The evidence for Na+/H+ antiporter involvement in the increase of pHi in substrate-attached cells is presented. It is suggested that signals for cell proliferation by chemical (soluble ligands) and physical (solid substrate) growth factors are transmitted similarly.

Animals↗

PubMed1988

A study of the interaction between the Na+, K+ pump and Na+:Ca2+ exchange in macrophages and vascular smooth muscle cells.

Blaustein (Am J Physiol 1977;232:C165-C173) postulated that Na+:Ca2+ exchange in vascular smooth muscle plays a key role in the link between sodium and hypertension. Investigation of this hypothesis was facilitated by the use of: a) Sr2+, a slowly transported Ca2+ analogue, and b) new quasispecific inhibitors of Na+:Ca2+ exchange such as 2',4'-dimethylbenzamil. Preliminary experiments in mouse macrophages showed that the initial rate of Sr2+ uptake lasted for at least 15 minutes and was therefore easier to measure than the initial rate of unidirectional isotopic Ca2+ influx (which lasted less than 30 seconds). In cells with normal Na+ content, basal Sr2+ influx (432 +/- 77 mumol [L cells X h]-1; mean +/- SEM of seven experiments) exhibited properties compatible with a ground membrane leak for divalent cations (quasilinear dependence on the external Sr2+ concentration, partial or full resistance to external Ca2+, Ba2+, verapamil, and 2',4'-dimethylbenzamil). Membrane depolarization by external K+ was unable to modify basal Sr2+ uptake. Conversely, a 100% increase in cell Na+ content by preincubation with ouabain increased the rate of Sr2+ uptake by 233 +/- 48 mumol (L cells X h)-1 (mean +/- SEM of seven experiments). 2',4'-Dimethylbenzamil, but not the Ca2+ antagonists diltiazem or methoxyverapamil, inhibited ouabain-stimulated Sr2+ influx (IC50 of about 3 X 10(-5) M). 2',4'-Dimethylbenzamil was also able to inhibit Na+ efflux (by 3.05 +/- 0.98 mmol (L cells X h)-1; mean +/- SEM of three experiments) suggesting the existence of Na+:Sr2+ exchange.(ABSTRACT TRUNCATED AT 250 WORDS)

Amiloride↗

PubMed1988

Angiotensin II effect on 22Na+ transport in vascular smooth muscle cells.

It is well established that angiotensin II (AII) rapidly increases free cytosolic Ca2+ in vascular smooth muscle cells (VSMCs). Several studies have indicated that the hormone also plays a role in Na+-K+ regulation of these cells. In this study, we explored the mechanism of AII effect on 22Na+ transport in cultured rat VSMCs. The 22Na+ washout from these cells was described by three exponents with exponential factors k1 greater than k2 greater than k3. In 1.8 mM Ca2+ medium, AII (10(-9)-10(-6) M) increased (in a dose response manner) the k1 value, and consequently the initial washout rate constant (kei) for the isotope. AII had no effect on kei in Ca2+-deficient medium or in the presence of ouabain. Amiloride (10(-3) M) and verapamil (10(-5) M) abolished the AII induced increase in kei. These findings are consistent with angiotensin II stimulation of an amiloride-sensitive Na+ transport, which is likely to represent the Na+/H+ antiport. In cultured VSMCs, the sustained stimulation by AII of this transport system requires the presence of extracellular Ca2+ and its influx into these cells.

Amiloride↗

PubMed1987

Amiloride derivatives that block Na+/Ca2+ exchange inhibit spontaneous inward currents in Na+-loaded cardiac myocytes.

Spontaneous electrical and mechanical activity was observed when single myocytes from guinea-pig hearts were loaded with sodium by direct intracellular application. Transient membrane depolarizations were found to be due to spontaneous inward currents (Isp). Both Isp and spontaneous contractions were abolished by 2',3'-benzobenzamil or 3',4'-dichlorobenzamil, two compounds that were previously reported to inhibit Na+/Ca2+ exchange. These findings suggest that the spontaneous membrane currents in Na+-loaded myocytes could be generated by the Na+/Ca2+ exchange mechanism.

Amiloride↗

PubMed1987

Angiotensin II-stimulated Na+/H+ exchange in cultured vascular smooth muscle cells. Evidence for protein kinase C-dependent and -independent pathways.

Angiotensin II, a potent vasoconstrictor, is known to stimulate Ca2+ mobilization and Na+ influx in vascular smooth muscle cells (VSMC). The fact that the Na+/H+ exchange inhibitor, amiloride, blocks angiotensin II-stimulated Na+ influx and is itself a vasodilator suggests that Na+/H+ exchange may play a role in the angiotensin II-mediated effects on VSMC. We have used a pH-sensitive fluorescent dye to study Na+/H+ exchange in cultured rat aortic VSMC. Basal intracellular pH was 7.08 in physiological saline buffer. Angiotensin II stimulation caused an initial transient acidification, followed by a Na+-dependent alkalinization. Angiotensin II increased the rate of alkalinization with apparent threshold, half-maximal, and maximal effect of 0.01, 3, and 100 nM, respectively. Angiotensin II stimulation appeared to be mediated by a shift in the Km of the Na+/H+ exchanger for extracellular Na+. Since angiotensin II activates phospholipase C in VSMC, we tested the possibility that angiotensin II increased Na+/H+ exchange by activation of protein kinase C via stimulation of diacylglycerol formation. The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), stimulated Na+/H+ exchange in VSMC cultured for 24 h in serum-free medium, and the subsequent angiotensin II response was inhibited. However, VSMC grown in serum and treated for 24 h with TPA to decrease protein kinase C activity showed no inhibition of angiotensin II-stimulated Na+/H+ exchange. TPA caused no intracellular alkalinization of VSMC grown in serum, while the angiotensin II response was actually enhanced compared to VSMC deprived of serum for 24 h. We conclude that angiotensin II stimulates an amiloride-sensitive Na+/H+ exchange system in cultured VSMC which is mediated by protein kinase C-dependent and -independent mechanisms. Angiotensin II-mediated Na+ influx and intracellular alkalinization may play a role in excitation-response coupling in vascular smooth muscle.

Angiotensin II↗

PubMed1987

Angiotensin II effect on cytosolic pH in cultured rat vascular smooth muscle cells.

This study investigated fluctuations of cytosolic pH (pHi) of cultured rat vascular smooth muscle cells (VSMCs) in reaction to metabolic alterations induced by angiotensin II (AII). Serially passed VSMCs from Wistar rat aortae were grown on coverslips and loaded with the pH-sensitive fluorescent indicator 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein. A biphasic reaction was seen after exposure of these cells to AII (1 nM to 1 microM); an initial and relatively brief phase of acidification was followed by sustained alkalinization. The rate of acidification and magnitude of alkalinization were dose-dependent. This biphasic effect of AII was also demonstrated in Ca2+-free medium and was mimicked by subjecting VSMCs to the calcium ionophore A23187 (5 microM) in Ca2+-containing medium but not in Ca2+-free medium. Verapamil (10 microM) almost entirely eliminated the AII-induced acidification, whereas amiloride analogues 5-(N-methyl-N-isobutyl)amiloride and 5-(N-ethyl-N-isopropyl)amiloride (100 microM) as well as Na+-deficient medium abolished the subsequent (alkalinization) phase produced by the hormone. Activation of the Na+/H+ antiport by subjecting VSMCs to phorbol 12-myristate 13-acetate (100 nM) prevented a subsequent effect of AII on the pHi profile. This resistance to a further action of the hormone was not mediated via cytoplasmic alkalinization. AII produced a dramatic redistribution in the cellular compartments of 45Ca2+ associated with accelerated 45Ca2+ washout. These findings suggest that the AII-induced acidification phase may relate to activation of the Ca2+ pump (Ca2+/H+ exchange) and that this process can take place in the presence and absence of extracellular Ca2+. The alkalinization phase is the consequence of stimulation of the Na+/H+ antiport, which in cultured VSMCs can be activated by a rise in cytosolic free Ca2+ as well as other mechanisms.

Amiloride↗

PubMed1986

Isolation and properties of fibroblast mutants overexpressing an altered Na+/H+ antiporter.

A new method based on the toxicity of low intracellular pH (pHi) was developed to isolate fibroblast variants overexpressing Na+/H+ antiport activity. Chinese hamster lung fibroblasts (CCL39) were incubated for 60 min in medium containing 50 mM NH4Cl. Removal of external NH+4 induced a rapid and lethal intracellular acidification when the Na+/H+ antiporter was inhibited during the 60 min of the pHi recovery phase. The inhibition was provoked either by adding 5-(N-methyl,N-propyl)amiloride (MPA, LD50 = 0.3 microM) or by reducing external [Na+] (LD50 = 25 mM). Progressively increasing the MPA concentration during the acid-load selection led to the isolation of two stable variants: AR40 and AR300, resistant, respectively, to 40 and 300 microM MPA. In response to an acid-load, these variants display a much higher rate of pHi recovery due to an overexpression of Na+/H+ antiport activity. In addition, AR40 and AR300 have an altered Na+/H+ antiporter: in AR300 cells K0.5 of MPA for inhibiting Na+/H+ exchange is shifted from 5 X 10(-8) to 1.5 X 10(-6) M, Km (Na+) is decreased 2-fold, and Vmax is increased 4.5-fold. Alternatively reducing Na+ concentration of the pHi recovery saline medium in a stepwise manner led to the selection of another class of variants (DD8 and DD12) also characterized by an altered Na+/H+ antiporter and an increased expression level. The 10-fold increased rate of amiloride-sensitive Na+ influx of DD12 is accounted for by a 4-fold increase in Vmax and a 2.5-fold increase in affinity for Na+ or Li+ at the external site. Interestingly, the affinity for the amiloride analog MPA and for external H+ is unchanged in DD12. In conclusion, the genetic approach presented here: provides a general and specific method for selecting variants of the Na+/H+ antiporter with increased expression levels and/or with structural alterations and demonstrates that the external Na+- and amiloride-binding sites are not identical, since they can be genetically altered independently of each other.

Amiloride↗

PubMed1986

Photoaffinity labeling of the epithelial sodium channel.

Sodium enters tight epithelia across the apical plasma membrane through a sodium channel, a process inhibited by submicromolar concentrations of amiloride and benzamil. Using membrane vesicles from bovine kidney cortex, we found that sodium transport through the sodium channel was inhibited by benzamil with an IC50 of 4 nM. Amiloride (IC50 = 400 nM) was a weaker inhibitor of sodium transport. [3H]Benzamil bound to the vesicles at a single class of high affinity binding sites with a Kd of 5 nM, the similarity of which to the IC50 suggests that these binding sites are associated with the sodium channel. Amiloride displaced bound [3H]benzamil with a Ki of 2,500 nM. Bromobenzamil is a photoactive amiloride analog with potency similar to benzamil in inhibiting sodium transport (IC50 = 5 nM) and binding to the sodium channel (Kd = 6 nM). [3H]Bromobenzamil was specifically photoincorporated into three molecular weight classes of polypeptides with apparent Mr values of 176,000, 77,000, and 47,000. The photoincorporation of [3H]bromobenzamil into these three classes of polypeptides was blocked by addition of excess benzamil and by amiloride in a dose-dependent manner. These data suggest that these polypeptides are components of the epithelial sodium channel.

Affinity Labels↗