Amiloride Derivatives Research Articles

Amiloride derivatives are potent blockers of KATP channels.

In cardiomyocytes sarcolemmal KATP channels open massively when the cytosolic [ATP] drops into the range of tens of micromolar, as during acute ischemia. The diuretic drug amiloride and related derivatives are well established as drugs blocking the Na+/H+- and the Na+/Ca2+-exchange, protecting the ischemic heart. Herein, the blocking action of amiloride and its derivatives 2',4'-dichlorobenzamil (DCB) and 5-(N-ethyl-N-isopropyl)amiloride (EIPA) on KATP channels was tested. In inside-out patches of mouse cardiac myocytes, amiloride, DCB, and EIPA reversibly blocked the KATP channels with the IC50 values 102, 1.80, and 2.14 micromol/l (-80 mV), respectively. Similar IC50 values were obtained in recombinant channels when coexpressing the KIR6.2 subunit with one of the sulfonylurea receptors SUR1 and SUR2A. All three drugs also blocked currents generated by the C-terminus deletion mutant KIR6.2delta26 in the absence of SUR. Amiloride blocked outward currents more effectively than inward currents whereas the block by DCB and EIPA was voltage independent. In cardiomyocytes, also whole-cell IKATP was blocked by the three drugs. In conclusion, amiloride, EIPA, and DCB block the pore-forming KIR6.2 subunit of cardiac KATP channels with higher potency than the Na+/H+- and the Na+/Ca2+-exchange, precluding a specific block of the exchanges under ischemic conditions.

Inhibition of Na +/H + exchange by SM-20220 attenuates free fatty acid efflux in rat cerebral cortex during ischemia–reperfusion injury

The Na +/H + exchanger (NHE) is activated during ischemia–reperfusion in an effort to restore intracellular pH to normal levels. Inhibition of NHE with non-selective amiloride derivatives has been shown to be neuroprotective and to attenuate free fatty acid efflux during ischemia–reperfusion. We evaluated the effects of SM-20220 (20 μM), a highly selective and specific NHE inhibitor, applied topically onto rat cerebral cortex prior to and during a 20-min period of ischemia. SM-20220 application significantly reduced the ischemia-evoked efflux of myristic, palmitic, and arachidonic acids during both ischemia and reperfusion with significant decreases in linoleic and docosahexaenoic levels during reperfusion. This study confirms the importance of NHEs in eliciting free fatty acid efflux, inhibition of which may be an essential component of the neuroprotective benefits of NHE inhibitors in ischemia–reperfusion injury.

Novel inhibitors of the sodium–calcium exchanger: benzene ring analogues of N-guanidino substituted amiloride derivatives

A series of N-guanidino substituted 2,4-diamino-5-carbonylguanidine molecules related to amiloride were synthesised and evaluated for their ability to inhibit the sodium–calcium exchanger in rat insulinoma cells (RINm5F) and human platelets. Specific chemical pathways were used to prepare the benzene derivatives designed as bioisosteric analogues of the pyrazine derivatives of amiloride. Several so-called ‘simplified analogues’, where some substituents of amiloride were omitted or replaced, were also prepared and included in the biological evaluation. The inhibitory potency of the sodium–calcium exchanger was screened on both cell types by measuring their effect on 45Ca 2+ uptake. Among the most active compounds, N-(2-amino-5-chloro-4-nitrobenzoyl)- N′-(1-naphtylmethyl)guanidine (IC 50=3.4 μM) was found more active than amiloride (IC 50=690 μM) and 3,4-dichlorobenzamil (IC 50=15.2 μM), the reference inhibitor.

The myocardial Na+/H+ exchanger: a potential therapeutic target for the prevention of myocardial ischaemic and reperfusion injury and attenuation of postinfarction heart failure.

The myocardial Na+/H+ exchange (NHE) represents a major mechanism for pH regulation during normal physiological processes but especially during ischaemia and early reperfusion. However, there is now very compelling evidence that its activation contributes to paradoxical induction of cell injury. The mechanism for this most probably reflects the fact that activation of the exchanger is closely coupled to Na+ influx and therefore to elevation in intracellular Ca2+ concentrations through the Na+/Ca2+ exchange. The NHE is exquisitely sensitive to intracellular acidosis; however, other factors can also exhibit stimulatory effects via phosphorylation-dependent processes. These generally represent various autocrine and paracrine as well as hormonal factors such as endothelin-1, angiotensin II and alpha1-adrenoceptor agonists, which probably act through receptor-signal transduction processes. Thus far, 6 NHE isoforms have been identified and designated as NHE1 through NHE6. All except NHE6, which is located intracellularly, are restricted to the sarcolemmal membrane. In the mammalian myocardium the NHE1 subtype is the predominant isoform, although NHE6 has also been identified in the heart. The predominance of NHE1 in the myocardium is of some importance since, as discussed in this review, pharmacological development of NHE inhibitors for cardiac therapeutics has concentrated specifically on those agents which are selective for NHE1. These agents, as well as the earlier nonspecific amiloride derivatives have now been extensively demonstrated to possess excellent cardioprotective properties, which appear to be superior to other strategies, including the extensively studied phenomenon of ischaemic preconditioning. Moreover, the salutary effects of NHE inhibitors have been demonstrated using a variety of experimental models as well as animal species suggesting that the role of the NHE in mediating injury is not species specific. The success of NHE inhibitors in experimental studies has led to clinical trials for the evaluation of these agents in high risk patients with coronary artery disease as well as in patients with acute myocardial infarction (MI). Recent evidence also suggests that NHE inhibition may be conducive to attenuating the remodelling process after MI, independently of infarct size reduction, and attenuation of subsequent postinfarction heart failure. As such, inhibitors of NHE offer substantial promise for clinical development for attenuation of both acute responses to myocardial as well as chronic postinfarction responses resulting in the evolution to heart failure.

Inhibition of Synaptosomal Uptake of 3H-L-glutamate and 3H-GABA by Hyperforin, a Major Constituent of St. John's Wort The Role of Amiloride Sensitive Sodium Conductive Pathways

Extracts of St. John's Wort are widely used for the treatment of depressive disorders. The active principles have not yet been finally elucidated. We have recently shown that hyperforin, a major active constituent of St. John's Wort, not only inhibits the neuronal uptake of serotonin, norepinephrine and dopamine, but also that of L-glutamate and GABA. No other antidepressant compound exhibits a similar broad uptake inhibiting profile. To investigate this unique kind of property, kinetic analyses were performed regarding the uptake of 3H-L-glutamate and 3H-GABA into synaptosomal preparations of mouse brain. Michaelis-Menten kinetics revealed a reduction of Vmax (8.27 to 1.80 pmol/mg/min for 3H-L-glutamate, 2.76 to 0.77 pmol/mg/min for 3H-GABA) while Km was nearly unchanged in both cases, suggesting non-competitive inhibition. The unselective uptake inhibition by hyperforin could be mimicked by the Na+-ionophore monensin and by the Na+-K+-ATPase inhibitor ouabain. However, both mechanisms can be discarded for hyperforin. Several amiloride derivatives known to affect sodium conductance significantly enhance 3H-GABA and 3H-L-glutamate uptake and inhibit the uptake inhibition by hyperforin, while monensin or ouabain inhibition were not influenced. Selective concentrations of benzamil for amiloride sensitive Na+-channels and selective concentrations of 5'-ethylisopropylamiloride (EIPA) for the Na+-H+-exchangers both had an attenuating effect on the hyperforin inhibition of L-glutamate uptake, suggesting a possible role of amiloride sensitive Na+-channels and Na+-H+-exchangers in the mechanism of action of hyperforin.

A Novel Topology Model of the Human Na+/H+ Exchanger Isoform 1

The membrane topology of the human Na(+)/H(+) exchanger isoform 1 (NHE1) was assessed by substituted cysteine accessibility analysis. Eighty-three cysteine residues were individually introduced into a functional cysteineless NHE1, and these mutants were expressed in the exchanger-deficient PS120 cells. The topological disposition of introduced cysteines was determined by labeling with a biotinylated maleimide in the presence or absence of preincubation with the membrane-impermeable sulfhydryl reagent, 2-trimethylammoniumethyl-methanethiosulfonate in streptolysin O-permeabilized or nonpermeabilized cells. We proposed a new model for the topology of NHE1 that is significantly different from the model derived from hydropathy analysis. In this model, NHE1 is composed of 12 transmembrane segments (TMs) with the N and C termini located in the cytosol. The large, last extracellular loop in the membrane domain of the original model was suggested to comprise an intracellular loop, a new transmembrane segment (TM11), and an extracellular loop in the new model. Interestingly, cysteines at 183 and 184 and at 324 and 325 mapped to intracellular loops connecting TMs 4 and 5 (IL2) and TMs 8 and 9 (IL4), respectively, were accessible to sulfhydryl reagents from the outside. Furthermore, exchange activities of two mutants, R180C and Q181C, within IL2 were markedly inhibited by external MTSET. These data suggest that part of IL2 or IL4 may be located in a pore-lining region that is accessible from either side of the membrane and involved in ion transport.

Essential Requirement of Cytosolic Phospholipase A2for Activation of the H+ Channel in Phagocyte-like Cells

The NADPH oxidase-producing superoxide is the major mechanism by which phagocytes kill invading pathogens. We previously established a model of cytosolic phospholipase A(2) (cPLA(2))-deficient differentiated PLB-985 cells (PLB-D cells) and demonstrated that cPLA(2)-generated arachidonic acid (AA) is essential for NADPH oxidase activation (Dana, R., Leto, T., Malech, H., and Levy, R. (1998) J. Biol. Chem. 273, 441-445). In the present study, we used this model to determine the physiological role of cPLA(2) in the regulation of both the H(+) channel and the Na(+)/H(+) antiporter and to study whether NADPH oxidase activation is regulated by either of these transporters. PLB-D cells and two controls: parent PLB-985 cells and PLB-985 cells transfected with the vector only (PLB cells) were differentiated using 1.25% Me(2)SO or 5 x 10(-8) M 1, 25-dihydroxyvitamin D(3). Activation of differentiated PLB cells resulted in a Zn(2+)-sensitive alkalization, indicating H(+) channel activity. In contrast, differentiated PLB-D cells failed to activate the H(+) channel, but the addition of exogenous AA fully restored this activity, indicating the role of cPLA(2) in H(+) channel activation. The presence of the H(+) channel inhibitor Zn(2+) caused significant inhibition of NADPH oxidase activity, suggesting a role of the H(+) channel in regulating oxidase activity. Na(+)/H(+) antiporter activity was stimulated in differentiated PLB-D cells, indicating that cPLA(2) does not participate in the regulation of this antiporter. These results establish an essential and specific physiological requirement of cPLA(2)-generated AA for activation of the H(+) channel and suggest the participation of this channel in the regulation of NADPH oxidase activity.

Characterization of rabbit kidney and brain pancreatic polypeptide-binding neuropeptide Y receptors: differences with Y 1 and Y 2 sites in sensitivity to amiloride derivatives affecting sodium transport

Sites sensitive to human and rat pancreatic polypeptides (hPP and rPP) accounted for more than 30% of the specific binding of [ 125I](Leu 31,Pro 34) human peptide YY (LP-PYY) in particulates from rabbit kidney cortex, and about 10% of the specific binding in membranes from rabbit hypothalamus. The binding of [ 125I]hPP or [ 125I]rPP showed a high-affinity displacement with either hPP, rPP, LP-PYY, neuropeptide Y or peptide YY (K i below 50 pM for all), while being quite insensitive to Y 2-selective ligands. The PP binding had a high sensitivity to alkali cations and inhibitors of phospholipase C, very similar to that of LP-PYY binding ‘masked’ by excess cold hPP. However, as different from the Y 1-like LP-PYY binding, but similar to the binding of the Y 2-selective ligand [ 125I] human peptide YY(3–36) (hPYY(3–36)), the PP binding showed a low sensitivity to guanosine polyphosphates. The PP binding was much more sensitive to N5-substituted amiloride inhibitors of Na + transport than the binding of LP-PYY, or that of hPYY(3–36). The inhibition of PP binding by N5-substituted amilorides was not enhanced by guanine nucleotides or by phospholipase C blockers. However, pairing of N5-substituted amilorides disproportionately increased the inhibition of hPP binding. Thus, in rabbit kidney or hypothalamus, the high-affinity PP-responding sites share some of the basic properties of the Y 1 and Y 2 sites, but are distinguished from both by a high sensitivity to compounds affecting sodium transport. These PP/NPY receptors could associate with membrane structures involved in the control of ion balance and osmotic responses.

Cellular uptake of chloroquine is dependent on binding to ferriprotoporphyrin IX and is independent of NHE activity in Plasmodium falciparum.

Here we provide definitive evidence that chloroquine (CQ) uptake in Plasmodium falciparum is determined by binding to ferriprotoporphyrin IX (FPIX). Specific proteinase inhibitors that block the degradation of hemoglobin and stop the generation of FPIX also inhibit CQ uptake. Food vacuole enzymes can generate cell-free binding, using human hemoglobin as a substrate. This binding accounts for CQ uptake into intact cells and is subject to identical inhibitor specificity. Inhibition of CQ uptake by amiloride derivatives occurs because of inhibition of CQ-FPIX binding rather than inhibition of the Na+/H+ exchanger (NHE). Inhibition of parasite NHE using a sodium-free medium does not inhibit CQ uptake nor does it alter the ability of amilorides to inhibit uptake. CQ resistance is characterized by a reduced affinity of CQ-FPIX binding that is reversible by verapamil. Diverse compounds that are known to disrupt lysosomal pH can mimic the verapamil effect. These effects are seen in sodium-free medium and are not due to stimulation of the NHE. We propose that these compounds increase CQ accumulation and overcome CQ resistance by increasing the pH of lysosomes and endosomes, thereby causing an increased affinity of binding of CQ to FPIX.

Synthesis of Macromolecular Conjugates of a Urokinase Inhibitor: Amiloride

Amiloride is a potent inhibitor of a urokinase type plasminogen activator which is involved in the invasive process of cancer cells leading to the initiation of metastasis. Synthesis, characterization and in vitro tests of four macromolecular conjugates of Amiloride are presented. One of them is a degradable derivative, HPMA-Gly-D,l-Phe-Leu-Gly-amiloride. In this case the in vitro release of Amiloride was monitored. Other conjugates are stable containing a new amiloride derivative, 6-aminohexyl amiloride [AHA], coupled to different polymeric carriers: a branched polypeptide, poly-[Lys(AcGlu1.0-D,l-Ala4.5)] [AcEAK], poly-[N-(2-hydroxy propyl) metacrylamide] [HPMA] and poly-[1-vinyl-2-pyrrolidone- co-maleic acid] [NVP MA]. Inhibition of uPA, plasminogen activation and proteinases secreted by cancer cells was measured as well as basement membrane degradation in vitro. Each amiloride AHA and the corresponding conjugates retained their activity in these experiments.

Regulation of intracellular pH in hamster preimplantation embryos by the sodium hydrogen (Na+/H+) antiporter.

This study was an investigation of the mechanisms for the regulation of intracellular pH (pHi) by hamster preimplantation embryos. The resting pH values of hamster embryos were similar at the 1-cell (7. 19 +/- 0.34), 2-cell (7.21 +/- 0.21), and 8-cell (7.22 +/- 0.41) stages. Cleavage-stage hamster embryos alleviated intracellular acidosis by activity of the Na+/H+ antiporter. The rate of recovery from acidosis was similar for embryos at 1-cell, 2-cell, and 8-cell stages. When Na+/H+ antiporter activity was inhibited by either incubation in Na+-free medium or the presence of an inhibitor, pHi was unable to recover to initial levels. Instead, pHi remained acidic. The Na+/H+ antiporter was also found to contribute to baseline pH regulation, as incubation in Na+-free medium resulted in an immediate intracellular acidification. The set point for Na+/H+ antiporter was pH 7.14. There was no evidence at any developmental stage for activity of either Na+-dependent HCO3-/Cl- exchanger or H+-ATPase in the regulation of pHi. Inhibition of the Na+/H+ antiporter by an amiloride derivative significantly reduced the ability of 2-cell embryos to develop in culture when challenged with acidosis, indicating that the Na+/H+ antiporter is an essential regulator of pHi.

Interactions between Benzylamiloride and Fura-2: Studiesin Vitroand in Cardiac Myocytes

Amiloride derivatives are commonly used inhibitors of Na+/H+- and Na+/Ca2+-exchange. Because they are fluorescent molecules the use of benzylamiloride (BZA), an inhibitor of Na+/Ca2+exchange, in conjunction with Fura-2, a commonly used fluorescent Ca2+indicator, might complicate interpretation of fluorescence data obtained.In vitrodata show that BZA decreases the Fura-2 fluorescence at all useful wavelengths in a concentration-dependent manner. The Fura-2 ratio 340/380 (used to estimate intracellular Ca2+([Ca2+]in)) also decreased with increasing BZA concentrations. The Stern–Volmer relation suggests that this phenomenon is due to either static or dynamic quenching. Varying temperatures from 4 to 37°C did not alter Stern–Volmer constants, consistent instead with fluorescence resonance energy transfer (FRET). Thein siturelevance of these interactions was evaluated in adult rat cardiac myocytes which exhibit Na+/Ca2+exchange reflected by rapid [Ca2+]inincrease following Na+removal. Pretreatment with BZA ≥ 25 μM decreased the magnitude of Fura-2 changes induced by Na+removal. Analysis of the individual Fura-2 useful wavelengths indicated that ≥ 25 μM BZA altered the Fura-2 signal in a manner consistent with the quenching effects notedin vitro.Together, these data show that BZA interacts with Fura-2in vitroandin situand suggest caution when interpreting Fura-2 fluorescence data derived in conjunction with BZA.

Amiloride Analogs Inhibit Chronic Hypoxic Pulmonary Hypertension

Na+/H+ exchange regulation of intracellular pH may play a permissive role in pulmonary artery smooth muscle cell (PASM) proliferation. Our laboratory has demonstrated that dimethyl amiloride (DMA), an amiloride derivative with enhanced selectivity as an inhibitor of the Na+/H+ antiporter, can inhibit bovine PASM proliferation in vitro. We hypothesized that DMA would inhibit development of hypoxic pulmonary hypertension by interfering with PASM growth in vivo. Sprague-Dawley rats were exposed to 10% O2 for 14 d without (n 9) or with (n = 7) DMA continuous infusion 3 mg/ kg/d. The animals treated with DMA had significant reductions in pulmonary artery pressure and total pulmonary vascular resistance index (TPVRI) when compared with hypoxic control rats (p < 0.05). Pulmonary vascular remodeling was significantly reduced in animals treated with DMA as measured by percent wall thickness and percentage of thick-walled intra-acinous vessels (p < 0.05). We used a second Na+/H+ exchange inhibitor, ethylisopropyl amiloride (EIPA, 3 mg/kg/d, n = 9), and found similar reductions in pulmonary artery pressure, TPVRI, and pulmonary vascular remodeling. Polycythemia during hypoxia was unchanged by treatment with DMA or EIPA. In conclusion, despite the hypertensive effects of polycythemia, DMA and EIPA can significantly reduce pulmonary vascular remodeling induced by chronic hypoxia.

Inhibition of Bicarbonate Transport Protects Embryonic Heart Against Reoxygenation-induced Dysfunction

It has not been well established whether the mechanisms participating in pH regulation in the anoxic–reoxygenated developing myocardium resemble those operating in the adult. We have specially examined the importance of Na+/H+exchange (NHE) and HCO3-dependent transports in cardiac activity after changes in extracellular pH (pHo). Spontaneously contracting hearts isolated from 4-day-old chick embryos were submitted to single or repeated anoxia (1 min) followed by reoxygenation (10 min). The chronotropic, dromotropic and inotropic responses of the hearts were determined in standard HCO3−buffer at pHo7.4 and at pHo6.5 (hypercapnic acidosis). In distinct experiments, acidotic anoxia preceded reoxygenation at pHo7.4. NHE was blocked with amiloride derivative HMA (1μmol/l) and HCO3-dependent transports were inactivated by replacement of HCO3or blockade with stilbene derivative DIDS (100μmol/l). Anoxia caused transient tachycardia, depressed mechanical function and induced contracture. Reoxygenation temporarily provoked cardiac arrest, atrio-ventricular (AV) block, arrhythmias and depression of contractility. Addition of DIDS or substitution of HCO3at pHo7.4 had the same effects as acidosisper se, i.e. shortened contractile activity and increased incidence of arrhythmias during anoxia, prolonged cardioplegia and provoked arrhythmias at reoxygenation. Under anoxia at pHo6.5/reoxygenation at pHo7.4, cardioplegia, AV block and arrhythmias were all markedly prolonged. Interestingly, in the latter protocol, DIDS suppressed AV block and arrhythmias during reoxygenation, whereas HMA had no effect. Thus, intracellular pH regulation in the anoxic–reoxygenated embryonic heart appears to depend predominantly on HCO3availability and transport. Furthermore, pharmacological inhibition of anion transport can protect against reoxygenation-induced dysfunction.

Sodium-Calcium Exchanger in Cultured Human Retinal Pigment Epithelium

Regulation of intracellular free Ca2+concentration ([Ca2+]i) by an Na+/Ca2+exchanger was studied in cultures of human retinal pigment epithelial cells using Ca2+-indicator dyes (fura-2 and fluo-3) and digital fluorescence imaging. Mean resting [Ca2+]iof cultured RPE in a control Ringer solution was 189±16 nm. Replacing extracellular Na+withN-methyl-d-glucamine elicited a two-fold rise in [Ca2+]i; the magnitude of the [Na+]o-free-induced rise in [Ca2+]ivaried as a function of extracellular [Ca2+]. The [Na+]o-free response was not significantly affected by the Ca2+channel blocker nifedipine, or by pretreatment with thapsigargin which depletes intracellular Ca2+stores. By contrast, the [Na+]o-free-induced rise in [Ca2+]iwas significantly reduced by CBDMB, an amiloride derivative that is highly selective for Na+/Ca2+exchange inhibition. These findings indicate that removal of extracellular Na+promotes net [Ca2+]igain via Na+/Ca2+exchange. Western and Northern blot analyses, respectively, confirmed the presence of Na+/Ca2+exchanger protein and mRNA in cultures of human RPE. Specifically, Western blot analysis of whole cell lysates of cultured RPE using a polyclonal antibody made against the canine cardiac exchanger identified a major band at ∼126 kD. Northern blot analysis of total human RPE RNA using a restriction fragment cRNA probe coding for the canine cardiac Na+/Ca2+exchanger showed that the major exchanger-related transcript was ∼6.8 kb. In sum, our findings demonstrate the presence of a cardiac-type Na+/Ca2+exchanger in cultures of human RPE.

Intracellular pH Increase Driven by an Na+/H+Exchanger upon Activation of Surf Clam Oocytes

Intracellular pH (pHi) measurements were performed in surf clam (Spisula solidissima) oocytes before and after artificial activation or fertilization [evidenced by germinal vesicle breakdown (GVBD)] by the dimethyloxazolidinedione (DMO) and 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) methods. Results using both methods showed increases of pHiof 0.3 pH unit after activation by excess K+. Using BCECF, we found an increase of similar magnitude after fertilization or after the addition of serotonin. By contrast, GVBD did not occur when the pHiwas increased to similar or even higher levels by exposing the oocytes to ammonia. In sodium-free seawater, excess K+induced GVBD but the pHiof K+-activated oocytes decreased significantly below the resting level of unactivated oocytes. The pHiincreases in K+-activated oocytes were otherwise proportional to the external Na+concentration. The amiloride derivatives dimethylamiloride and hexamethylene amiloride (at 10–50 μM) efficiently inhibited the K+-induced increase of pHibut did not block GVBD. These two derivatives were able, however, to retard K+-induced GVBD, hexamethylene amiloride being the more efficient. This retardation of K+-induced GVBD could be abolished by the simultaneous addition of ammonia. Taken altogether, these results show that a pHiincrease, driven by a typical Na+/H+exchanger, follows activation of surf clam oocytes but that this pHiincrease is neither sufficient nor required for GVBD, though it does allow its progression at an optimal rate.

Na+/H+ exchange and osmotic shrinkage in isolated trout hepatocytes

The ability of rainbow trout liver cells to regulate their intracellular pH (pHi) was studied using two methods on hepatocytes isolated by collagenase digestion: (i) by monitoring pHi with the fluorescent dye BCECF-AM, and (ii) by measuring the amiloride-sensitive uptake of 22Na, which represents Na+/H+ exchange. In low-Na+ medium (&frac34;16mmoll-1), Na+ uptake was reduced by approximately 70% in the presence of amiloride derivatives (DMA or MPA, 10(-4)moll-1). Changing separately either the extracellular pH (pHe) or the intracellular pH (pHi, clamped by treating the cells with nigericin in the presence of 140mmoll-1 K+) between 6 and 8 induced an increase in the rate of Na+ uptake when pHe was raised or when pHi was reduced. When transferred to hypertonic medium, hepatocytes shrank to nearly 72% of their initial volume, and thereafter a slow and partial regulatory volume increase phase was observed, with an increase in the amiloride-sensitive rate of Na+ uptake and an increase in intracellular pH. As DIDS-sensitive Cl- uptake was concomitantly enhanced, it is suggested that hypertonic stress activates Na+/H+ and Cl-/HCO3- exchange.

Sodium/calcium exchanger in rat olfactory neurons

The chemo-electrical transduction process in olfactory neurons is accompanied by a rapid and transient increase in intracellular calcium concentrations. The notion that Na +/Ca 2+ exchanger activities may play a major role in extruding calcium ions out of the cell and maintaining Ca 2+ homeostasis in olfactory receptor cells was assessed by means of laser scanning confocal microscopy in combination with the fluorescent indicators Fluo-3 and Fura-Red. The data indicate that high exchanger acitivity, which was inhibited by amiloride derivatives, is located in the dendritic knob and probably in the olfactory cilia. This result was supported by experiments using specific antiserum raised against retinal Na +/Ca 2+ exchanger protein which labelled an immunoreactive protein of 230 kDa in Western blots from olfactory tissue and strongly stained the ciliary layer of the olfactory epithelium.

Pharmacological evidence that the activation of the Na+‐Ca2+ exchanger protects C6 glioma cells during chemical hypoxia

1. In C6 glioma cells exposed to chemical hypoxia a massive release of lactate dehydrogenase (LDH) occurred at 3 and 6 h, coupled with an increased number of propidium-iodide positive dead cells. 2. Extracellular Na+ removal, which activates the Na(+)-Ca2+ exchanger as a Na+ efflux pathway and prevents Na+ entrance, significantly reduced LDH release and the number of propidium iodide positive C6 cells. 3. During chemical hypoxia, in the presence of extracellular Na+ ions, a progressive increase of [Ca2+]i occurred; in the absence of extracellular Na+ ions [Ca2+]i was enhanced to a greater extent. 4. The blockade of the Na(+)-Ca2+ exchanger by the amiloride derivative 5-(N-4-chlorobenzyl)-2',4'-dimethylbenzamil (CB-DMB), lanthanum (La3+) and the Ca2+ chelator EGTA, completely reverted the protective effect exerted by the removal of Na+ ions on C6 glioma cells exposed to chemical hypoxia. 5. The inhibition of the Na(+)-Ca2+ antiporter enhanced chemical hypoxia-induced LDH release when C6 glioma cells were incubated in the presence of physiological concentrations of extracellular Na+ ions (145 mM), suggesting that the blockade of the Na(+)-Ca2+ antiporter during chemical hypoxia can lead to increased cell damage. 6. Collectively, these results suggest that activation of the Na(+)-Ca2+ exchanger protects C6 glioma cells exposed to chemical hypoxia, whereas its pharmacological blockade can exacerbate cellular injury.

Antagonists of the cGMP-gated conductance of vertebrate rods block the photocurrent in scallop ciliary photoreceptors.

1. Hyperpolarizing scallop photoreceptors, like vertebrate rods, use cGMP as an internal messenger and their light-sensing structure is also of ciliary origin. To ascertain possible functional similarities between the light-sensitive conductances in the two classes of visual cells, we examined in scallop photoreceptors the effects of several antagonists of the photocurrent of rods. 2. Extracellular application of L-cis-diltiazem rapidly and reversibly suppressed the photocurrent. The effect was stereospecific and dose dependent, with a K1/2 of approximately 400 microM. Intracellular dialysis at lower doses (100-200 microM) also induced a substantial inhibition. 3. L-cis-Diltiazem reduced the light-activated conductance without shifting the intensity-response curve. Furthermore, the drug also blocked the current directly evoked by application of cGMP. These observations indicate that the inhibitory effects result from blockage of the conductance, rather than from impairment of the activating cascade. 4. The fractional blockage increased e-fold per approximately 55 mV depolarization, regardless of the side of drug application, as if the charged form of L-cis-diltiazem can only access the blocking site from the intracellular compartment. 5. The amiloride derivative 3',4'-dichlorobenzamil potently suppressed the photocurrent (K1/2 approximately 5 microM), without affecting its kinetics or operating range. Amiloride itself was also effective at higher concentrations. 6. The pharmacological resemblance of these light-dependent channels to those of rods and cones indicates that significant aspects of the transduction cascade are conserved across disparate sensory cells of ciliary origin.