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E Cragoe

Publications and source records attributed to E Cragoe.

18 recordsLinked to original sources
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↗
PubMed1984

Blockade of the Na+/H+ antiport abolishes growth factor-induced DNA synthesis in fibroblasts. Structure-activity relationships in the amiloride series.

We have previously characterized in Chinese hamster lung fibroblasts a growth factor activatable and amiloride-sensitive Na+/H+ antiport (Pouysségur, J., Chambard, J. C., Franchi, A., Paris, S., and Van Obberghen-Schilling, E. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 3935-3939). In this report, we compared the affinity of 28 analogs of amiloride for inhibition of the Na+/H+ antiport and inhibition of growth factor-induced DNA synthesis. We showed that the guanidino moiety of amiloride must be protonated to elicit inhibition of the Na+/H+ exchange. Substitutions within this moiety by methyl, phenyl, or benzyl groups reduced the activity 20- to 1000-fold. On the contrary, substitution of the proton(s) of the 5-amino group of amiloride with alkyl or alkenyl groups increases potency up to 100-fold (5-N,N-diethylamiloride has a KI of 4 X 10(-8) M). In HCO-3-free medium and at lower [Na+]0 (25 or 50 mM) to reduce competition with amiloride, we found that growth factor-stimulated DNA synthesis of G0-arrested cells is inhibited by amiloride and its analogs with the same rank order as that for Na+/H+ antiporter inhibition. Over a range of 3 logs of concentration, a tight correlation was established between IC50 for the blockade of both processes, Na+/H+ exchange and percentage of cells entering the S phase upon growth factor action. These findings indicate that, in HCO-3-free medium, the functioning of the Na+/H+ exchange system is required for growth factor-induced DNA synthesis.

Amiloride↗
PubMed1984

Amiloride potentiation of differentiation of human promyelocytic cell line HL-60.

Cells of the human acute promyelocytic cell line HL-60 undergo differentiation when exposed to dimethyl sulfoxide (DMSO); in this report, amiloride, an inhibitor of passive intracellular Na+ flux, potentiated the DMSO-induced differentiation of HL-60 cells. This effect was seen at several concentrations of DMSO. Amiloride alone did not affect HL-60 differentiation. Various analogues of amiloride were tested for their ability to potentiate differentiation of HL-60 cells. The synergistic induction of myeloid differentiation by a membrane solvent (DMSO) and an Na+ transport inhibitor (amiloride) suggested membrane cation flux as being important in initiating differentiation.

Amiloride↗
PubMed1983

Inhibition of Na+ influx and DNA synthesis in human fibroblasts and neuroblastoma-glioma hybrid cells by amiloride analogs.

Identification of a Na+ influx inhibitor that is significantly more potent than amiloride and devoid of the nonspecific effects of amiloride would be of great value in determining the validity of the hypothesis that mitogen-stimulated Na+ influx acts as a signal for induction of cell proliferation. In this study, we evaluated a number of amiloride analogs for potency of Na+ influx inhibition in human fibroblasts (HSWP). One analog, benzamil, was found to exhibit a 60-fold enhanced potency relative to amiloride. We also assessed the relative efficacies with which amiloride and benzamil inhibit Na+ influx and DNA synthesis in HSWP cells and neuroblastoma-glioma hybrid cells (NG108-15). Concentrations of benzamil required for 50% inhibition (ID50) of Na+ influx and DNA synthesis of HSWP cells are in excellent agreement (15 and 18 microM, respectively), an observation which, on the surface, is supportive of the hypothesis in question. Benzamil also inhibits Na+ influx of NG108-15 cells with an ID50 comparable to that for HSWP cells (18 microM) and suppresses DNA synthesis with a slightly higher ID50 (38 microM). Although the benzamil concentrations needed to inhibit cell growth and Na+ influx are in reasonable agreement, caution should be exercised in interpreting the effects of benzamil on cell growth with respect to the role of Na+ influx as we also observed that an analog of benzamil with a reduced ability to inhibit Na+ influx gave inhibition of DNA synthesis at concentrations which do not inhibit Na+ influx.

Amiloride↗