Inhibitory Effect Of Amiloride Research Articles

Contribution of the Amiloride-Sensitive Component and the Na+/H+ Exchanger to Renal Responsiveness to Vasoconstrictors

Aims: This study analyzed the role of the amiloride-sensitive component and the participation of the Na⁺/H⁺ exchanger in renal responsiveness to vasoconstrictors in the isolated perfused rat kidney. Methods: The renal responses to vasoconstrictors (angiotensin II, phenylephrine, vasopressin and KCl) were studied under baseline conditions and after the administration of amiloride (10 and 100 µmol/l) or the specific Na⁺/H⁺ exchange inhibitor ethylisopropylamiloride (EIPA, 10 µmol/l). The effects of amiloride and EIPA on renal responsiveness to vasoconstrictors were also analyzed in endothelium-denuded preparations. Results: Amiloride reduced renal responsiveness to all vasoconstrictors in a dose-related manner, whereas EIPA did not affect the renal pressor response to KCl. The inhibitory effects of amiloride and EIPA on renal responsiveness to vasoconstrictors persisted after endothelium removal. Conclusion: These results indicate that the amiloride-sensitive component and the Na⁺/H⁺ exchanger play an important role in responsiveness to the main endogenous vasoconstrictors in the renal vasculature. These results also suggest that amiloride might be useful as an inhibitor of renal vasoconstriction, even in diseases with endothelial dysfunction.

Inhibitory effects of amiloride on the current mediated by native GABAA receptors in cultured neurons of rat inferior colliculus

1. The diuretic amiloride is known to modulate the activity of several types of ion channels and membrane receptors in addition to its inhibitory effects on many ion transport systems. However, the effects of amiloride on some important ion channels and receptors, such as GABA(A) receptors, in the central nervous system have not been characterized. 2. In the present study, we investigated the functional action of amiloride on native GABA(A) receptors in cultured neurons of rat inferior colliculus using whole-cell patch-clamp recordings. 3. Amiloride reversibly inhibited the amplitude of the GABA-induced current (I(GABA)) in a concentration-dependent manner (IC(50) 454 +/- 24 micromol/L) under conditions of voltage-clamp with a holding potential at -60 mV. The inhibition depended on drug application mode and was independent of membrane potential. Amiloride did not change the reversal potential of I(GABA). Moreover, amiloride induced a parallel right-ward shift in the concentration-response curve for I(GABA) without altering the maximal value and Hill coefficient. 4. The present study shows that amiloride competitively inhibits the current mediated by native GABA(A) receptors in the brain region, probably via a direct action on GABA-binding sites on the receptor. The findings suggest that the functional actions of amiloride on GABA(A) receptors may result in possible side-effects on the central nervous system in the case of direct application of this drug into the cerebrospinal fluid for treatment of diseases such as brain tumours.

Role of Na(+)-glucose cotransport in fluid absorption across alveolar epithelium in isolated rat lungs

To evaluate the role of Na(+)-glucose cotransport in fluid absorption across alveolar epithelial walls in isolated rat lungs, we measured the inhibitory effects of amiloride (a Na+ channel blocker) and phlorizin (a Na(+)-glucose cotrasport blocker) on the fluid absorption rate in fluid-filled lungs. Amiloride (10(-5)-10(-4) M) reduced alveolar fluid absorption by 30%. This value was similar to that obtained in the presence of 10(-3) M phlorizin. The coefficient of Na(+)-glucose cotransport was estimated to be 2.5. The strong correlation between Na+ escape and fluid absorption (r = 0.907) was not affected by phlorizin. These findings suggest that the impact of Na(+)-glucose cotransport was similar to that of Na+ transport alone, and that glucose molecules transported by Na(+)-glucose cotransport do not play an important role in alveolar fluid absorption across rat alveolar epithelium.

Inhibitory effect of amiloride and gadolinium on fine afferent nerves in the rat knee: evidence of mechanogated ion channels in joints

Synovial joints are complex sensory organs which provide continuous feedback regarding position sense and degree of limb movement. The transduction mechanisms which convert mechanical forces acting on the joint into an electrochemical signal which can then be transmitted to the central nervous system are not well understood. The present investigation examined the effect of the mechanogated ion channel blockers amiloride and gadolinium on knee joint mechanosensitivity. In deeply anaesthetised rats (sodium thiopental: 100-120 mg/kg, i.p.), single unit extracellular recordings were made from knee joint group III (Adelta) and group IV (C) primary afferents in response to mechanical rotation of the joint. Afferent firing rate was measured before and after topical application of either amiloride (0.1 mM, 1 mM) or gadolinium (250 microM) onto the receptive field of the sensory unit and recording was continued every 10 min up to a total of 50 min. With normal rotation of the knee, joint mechanosensitivity was significantly reduced by both amiloride (P<0.0001; n=10-21) and gadolinium (P=0.001; n=12) and this effect was sustained throughout the recording period. This investigation provides the first in vivo electrophysiological evidence that joint mechanotransduction involves the activation of amiloride and gadolinium-sensitive mechanogated ion channels. Future studies to determine the mechanogated ion channel subtypes present in joints and the modulation of their gating properties during inflammation may yield novel approaches for the control of arthritis pain.

Amiloride and KB-R7943 in Outward Na+/Ca2+ Exchange Current in Guinea Pig Ventricular Myocytes

The reverse mode of Na+/Ca2+ exchange represents an important pathway in inducing Ca2+ overload during ischemia and reperfusion. The inhibitory effects of amiloride and KB-R7943 on Na+/Ca2+ exchange current (INa/Ca) were investigated in guinea pig ventricular myocytes. Whole-cell patch clamp techniques were used under bidirectional ionic conditions and 25 mM of Na+ in pipette solution. At +50 mV, amiloride 10, 30, and 100 microM inhibited the outward INa/Ca by 15, 23, and 41%, respectively; at -80 mV, it inhibited inward INa/Ca by 6, 15, and 23%, respectively. Its inhibitory effect on outward INa/Ca was greater than that on inward INa/Ca. At +50 mV, KB-R7943 1 and 10 microM inhibited the outward INa/Ca by 29 and 61%, respectively; at -80 mV, it inhibited inward INa/Ca by 22 and 57%, respectively. KB-R7943 inhibited both directions of the exchange current with an equal potency. The data suggest that KB-R7943 is not a selective inhibitor on reverse mode of Na+/Ca2+ exchange.

The effects of amiloride on the labellar taste receptor cells of the fleshfly Boettcherisca peregrina

Amiloride is known to inhibit the taste response of vertebrates to salt by blocking the amiloride-sensitive sodium channel. In this study, we investigated electrophysiologically the effect of amiloride on the taste response of the fleshfly Boettcherisca peregrina. When 0.5 mM amiloride was included in taste solutions, the response of the salt receptor cell (salt response) to sodium chloride (NaCl) was not depressed but those of the sugar receptor cell (sugar responses) to sucrose, glucose, fructose, l-valine ( l-Val) and l-phenylalanine ( l-Phe) were strongly depressed. An inhibitory effect of amiloride on the concentration-response relationship for both sucrose and l-Phe was clearly revealed, but not at high concentrations of sucrose. After pretreatment of a chemosensory seta with 0.15 mM amiloride for 10 min, the salt response to NaCl was not affected. On the other hand, the sugar responses to sucrose, fructose, l-Val and l-Phe were depressed just after amiloride pretreatment. The sugar response to adenosine 5’-diphosphate (ADP) mixed with 0.5 mM amiloride was not depressed, but the response to ADP alone was depressed after amiloride pretreatment. It was therefore observed that amiloride depressed the responses to all stimulants that react with each of the receptor sites of the sugar receptor cell.

Effects of amiloride on gustatory neural responses to salts in the frog.

In frogs, the glossopharyngeal nerve (GL) innervates taste receptors on almost the entire tongue. The mandibular branch (MBF) and palatine branch (PN) of the facial nerve innervate taste receptors on a very small area at the base of the tongue and on the palate, respectively. In the present study, effects of amiloride, an epithelial sodium channel blocker, on the tonic responses of the GL, MBF and PN in frogs to NaCl, LiCl, KCl and CaCl(2) were investigated. In three nerves, amiloride at 0.5 mM, a relatively high concentration, did not affect the responses to 0.15 (concentration just above threshold)-0.5 M NaCl, 0.5 M LiCl and 0.3 M KCl, whereas it almost completely inhibited the response to 1.0 mM CaCl(2). Amiloride may exert an inhibitory action on the response to CaCl(2) by a competitive antagonism between Ca(2+) and a monovalent cation of amiloride, because the response to Ca(2+) is competitively inhibited by other cations such as Na(+) and Mg(2+). The lack of inhibitory effect of amiloride on the responses in the GL, MBF and PN to NaCl suggests that amiloride-sensitive sodium channels in the apical membrane of taste receptor cells are not involved in sodium taste transduction in frogs.

Inhibition of the Na +/H + antiporter suppresses IL-12 p40 production by mouse macrophages

The amiloride-inhibitable Na +/H + antiporter plays an important role in macrophage activation. The intracellular pathways leading to interleukin (IL)-12 p40 production by activated macrophages are incompletely understood. In the present study, we examined the contribution of the Na +/H + antiporter to the production of IL-12 p40. Amiloride or its analogs decreased the production of IL-12 p40 in macrophages stimulated with bacterial lipopolysaccharide and interferon-γ. The order of potency of amiloride analogs was consistent with the proposition that the effect of amiloride is mediated by the inhibition of the Na +/H + antiporter. The effect of amiloride was post-transcriptional, as IL-12 p40 mRNA levels induced by lipopolysaccharide and interferon-γ were not affected by this inhibitor. Furthermore, the inhibitory effect of amiloride on IL-12 p40 production was not a result of interference with the activation of the p38 and p42/44 mitogen-activated protein kinases or c-Jun kinase. In summary, the production of IL-12 p40 requires a functional Na +/H + antiporter.

The inhibitory effect of amiloride on the growth of human stomach cancer cells in vitro

The inhibitory effect of amiloride on the growth of human stomach cancer cells in vitro

Inhibitory Effect of Amiloride on the Urokinase Plasminogen Activators in Prostatic Cancer

The diuretic drug amiloride (AMLD), which competitively inhibits the catalytic activity of urokinase plasminogen activators (UPA), was used to study its effects on the proteolytic enzymes implicated in the invasiveness and metastases in a prostatic tumor model carrying two different sublines of adenocarcinoma of the prostate. Our data showed that UPA activity was significantly higher, both in the cytosol and pellet of R3327-AT3, a fast-growing highly metastatic and androgen-insensitive tumor, as compared to the G3327-G subline, a slow-growing nonmetastatic tumor of the prostate. The UPA activity in AT3 tumor dropped when the rats were treated with AMLD for 3 weeks. The UPA activity in the sera and tumor effusions from rats with AT3 tumor was significantly higher as compared to those with G subline tumor. The number of pulmonary metastatic foci was the same in untreated rats as compared to those treated with AMLD. The lymph node inspection after 3 weeks revealed no secondary tumor in the AMLD-treated group. The role of UPA in the metastases of prostate cancer is discussed.

Intracellular Mg 2+ is regulated by [formula omitted] antiport in neurones of an invertebrate central nervous system

Triple-barrelled, ion-sensitive microelectrodes were used to investigate the regulation of intracellular Mg 2+ in Retzius neurones of the medicinal leech ( Hirudo medicinalis). There is strong evidence that Mg 2+ extrusion from Mg 2+-loaded Retzius neurones is mediated by an amiloride-sensitive Na + Mg 2+ - antiport , possibly working at a stoichiometry of 1: 1. In contrast, Mg 2+ influx pathways are more complex. Mg 2+ influx could be partially inhibited by the Ca 2+ channel blockers Ni 2+, Co 2+, La 3+, and Gd 2+. However, inhibition was especially prominent under conditions where the voltage-sensitive Ca 2+ channels of Retzius neurones are closed, so that these results indicate the existence of specific Mg 2+ influx pathways. For thermodynamic reasons a 1 Na + 1 Mg 2+ antiport should reverse its mode of action in solutions with high Mg 2+ content and contribute to Mg 2+ influx, while a 2 or 3 Na + 1 Mg 2+ antiport would still be able to extrude Mg 2+ from the cell. As Mg 2+ influx into Retzius neurones was Na +-independent and hardly affected by amiloride, a contribution of Na + Mg 2+ - antiport , if present at all, is very small. However, an inhibitory effect of amiloride was detectable when, in addition, Mg 2+ influx was reduced by the application of Ni 2+ or Co 2+. This reduction of Mg 2+ influx by amiloride indicates a 1: 1 stoichiometry of the Na + Mg 2+ antiport.

Salt taste responses of mouse chorda tympani neurons: evidence for existence of two different amiloride-sensitive receptor components for NaCl with different temperature dependencies.

1. Inhibitory effects of amiloride on salt responses of single fibers of the chorda tympani nerve of the C57BL/6 strain of mice were examined at two different temperatures (approximately 12 and 24 degrees C). 2. Of 36 single fibers that responded to NaCl, 20 fibers showed strong suppression of responses to NaCl actuated by lingual treatment with amiloride (amiloride-sensitive fibers), whereas the remaining 16 fibers showed no such amiloride inhibition (amiloride-insensitive fibers). 3. Twenty amiloride-sensitive fibers were further classified into two subgroups according to the temperature dependency of their NaCl responses. In 15 of 20 fibers, amiloride-inhibitable NaCl responses were larger at 24 degrees C than at 12 degrees C, whereas the reverse was true for the remaining 5 fibers. All amiloride-insensitive fibers showed smaller responses to NaCl at 12 degrees C. 4. These results suggest that there exist two different amiloride-sensitive receptor components for NaCl with different temperature dependencies in mouse taste cells: one is more sensitive to NaCl at the higher temperature, and the other is more sensitive at the lower temperature.

Role of Na(+)-H(+)-antiport in restitution of isolated guinea pig gastric epithelium after superficial injury.

In addition to its pHi regulatory function Na(+)-H(+)-antiport is also involved in volume regulation of epithelial cells, particularly in neutral conditions. It is also known that the antiport is activated after ligand binding following growth factor receptor activation. The aim of the present study was to evaluate the role of the antiport in restitution of gastric mucosa and whether its activity is dependent on the type of superficial injury. Therefore the fundic epithelium of guinea pig stomach was perfused in an Ussing chamber in neutral conditions. Na(+)-H(+)- and Cl(-)-HCO3(-)-antiports were inhibited with 1.0 mM amiloride, 1.0 mM SITS, or with HCO3- removal and Na(+)-K(+)-2Cl(2-)-cotransporter with 0.3 M furosemide during 4 hr of restitution after superficial injury induced either by 1.25 M NaCl or by 1.0% Triton. Luminal exposure of the epithelium to amiloride had no effect on restitution but serosal application abolished the process completely. The inhibitory effect of amiloride was similar after both NaCl and Triton injury. The inhibition of Cl(-)-HCO3(-)-antiport with SITS interfered with the process as well, while HCO3- removal had no significant inhibitory effect, nor did the inhibition of Na(+)-K(+)-2Cl(-)-cotransporter. The morphologic findings were in accordance with the electrophysiologic measurements in each pair of tissues. It is concluded that the Na(+)-H(+)-antiport is essential for the epithelial cells during restitution even in neutral conditions, but a functional Cl(-)-HCO3(-)-antiport is also required. The activity of Na(+)-H(+)-antiport is sensitive to basolateral amiloride and is necessary regardless of the type of chemical injury.

Amiloride inhibition of chorda tympani responses to NaCI and its temperature dependency in mice

Inhibitory effects of amiloride on salt responses of the chorda tympani nerve and its temperature dependency were compared among three inbred strains of mice (C57BL, BALB and 129). In C57BL mice, lingual treatment with amiloride significantly suppressed responses to 0.1–1.0 M NaCI at two different temperatures, 24 ± 2°C and 12 ± 2°C. The magnitude of the amiloride-inhibited component of NaCl response was slightly larger at the higher temperature. In contrast, in BALB mice, amiloride suppression of NaCl responses was observed only at the lower temperature. No such suppression was exhibited by 129 mice at either temperature levels. These results suggest that there exist at least two different amiloride-sensitive receptor components for NaCl in mice: one is more sensitive to NaCl at the higher temperature, and the other is more sensitive at the lower temperature. It is hypothesized, C57BL mice possess the former (or both) component(s), whereas BALB mice have the latter one. The 129 strain may be lacking both components.

Developmental changes in Ca(2+)-uptake, Na+,Ca(2+)-exchange and Ca(2+)-ATPase in freshly isolated embryonic, newborn and adult chicken heart.

Developmental changes in cellular Ca(2+)-transport mechanisms were studied in chick heart by determining cellular Ca(2+)-uptake and Na+,Ca(2+)-exchange activity in freshly isolated ventricular tissues of embryonic (5-18 days old), newborn (1-2 days old) and young adult (90-100 days old) heart by monitoring 45Ca influx. Ca(2+)-ATPase activity was determined in microsomal fractions at different stages of development. The Ca(2+)-uptake (per g wet tissue weight) increased with the development of embryonic as well as post-hatch chick heart, reaching a maximum in the young adult chicken. The overall increase in Ca(2+)-uptake, from embryonic day 5 to young-adult stage, was more than 3 fold. The Na+,Ca(2+)-exchange activity, determined as Na(+)-gradient-induced Ca(2+)-uptake in presence of either ouabain or zero [Na+]0, showed a 6-fold increase during development of heart from the embryonic day 5 to the young adult stage. Amiloride, an inhibitor of Na+,Ca(2+)-exchange, caused a dose-dependent reduction in a ouabain-induced rise in 45Ca influx at different stages of development. The inhibitory effect of amiloride was, however, greater during later stages of development. A progressive increase in Ca(2+)-ATPase activity was also seen during development. Ca(2+)-ATPase exhibited about a 4-fold increase in activity from embryonic day 7 to the young adult. The concomitant increase in Ca(2+)-uptake, Na+,Ca(2+)-exchange and Ca(2+)-ATPase activities suggests age-dependent changes in Ca(2+)-transport and storage systems of developing heart during embryogenesis and post-embryonic life. During embryogenesis the developmental increase in Na+,Ca(2+)-exchange activity was greater than that during post-hatch development of heart. However, the increase in Ca(2+)-ATPase activity was greater during post-hatch development than during embryogenesis. It is suggested that Na+,Ca(2+)-exchange and Ca(2+)-ATPase play a prominent role in maintaining cellular Ca2+ homeostasis during embryogenesis and after hatching.

Amiloride modulates urokinase gene expression at both transcription and post-transcription levels in human colon cancer cells

Activity of receptor-bound urokinase plasminogen activator (uPA) on the surface of colon cancer cells appears to be a function of the number of uPA receptors. The regulation of uPA therefore may determine the invasive phenotype. The effects of amiloride on the modulation of uPA mRNA and protein induced by phorbol ester (PMA) and cycloheximide (CHX) were studied in four colon cancer cell lines, HCT116, KM12SM, LIM1215 and LS123. Northern blot analyses showed that PMA induced uPA mRNA that peaked at 2-48 h in HCT116 cells. In all colon cancer cell lines tested, the expression of uPA mRNA by PMA was super-induced after the addition of the protein synthesis inhibitor CHX, suggesting that stimulation of uPA gene expression does not require de novo protein synthesis. uPA mRNA was also induced by CHX alone, indicating that there may be a labile protein which inhibits uPA mRNA processing. Amiloride profoundly inhibited uPA mRNA production at concentrations between 0.1-1 mM in the presence or absence of PMA or CHX. uPA protein levels on the colon cancer cell surface reflected PMA induction and amiloride inhibition of uPA mRNA levels. Transcriptional elongation experiments using isolated nuclei indicated that while the induction effects of PMA or CHX on uPA gene expression were mediated at the post-transcriptional level, amiloride acted at both transcription and post-transcription levels. The inhibitory effects of amiloride on uPA gene expression reported in this paper may offer the prospect of developing new therapeutic approaches to the prevention of invasion and metastasis by adenocarcinomas.

Inhibitory effect of amiloride on glucose transport in isolated rat adipocytes

The effect of amiloride on 3- O-methylglucose (3-O-MG) uptake was studied in isolated rat adipocytes to define to what extent amiloride inhibited the process of insulin action or glucose transport. Amiloride (1 mM), which did not change the intracellular water space of adipocytes, inhibited by 43.3% the insulin-stimulated uptake of 3-O-MG, while it did not appear to inhibit the basal uptake. To distinguish the inhibitory effect on glucose transport activity from that on the process of insulin action, the effect of amiloride was evaluated in the transport system using adipocytes deprived of ATP, in which glucose transporters were considered immobile. Amiloride (1 mM) inhibited this transport by 32.8% in an insulin-stimulated state, which was obtained using adipocytes that had been treated with 20 nM insulin and exposted to 2 mM KCN, whereas it did not inhibit the transport system at the basal state. In the inhibitory effect, 76% was thus attributable to the inhibition of glucose transport activity recruited by insulin and 24% to the inhibition of the action of 20 nM insulin itself. These results indicate that amiloride can not be used as a specific inhibitor of the insulin action itself.

Amiloride blockage of Na+ channels in amphibian epithelia does not require external Ca2+.

Noise analysis was used to study the influence of external Ca2+ on the blockage of Na+ transport by amiloride. Experiments were done using frog skin (Rana temporaria and Rana catesbeiana), toad urinary bladder (Bufo marinus) and epithelia of A6 cells. In nondepolarized skins and bladders, removal of Ca2+ from the mucosal bath diminished markedly the inhibitory effect of amiloride. Ca2+ depletion also gave rise to the appearance of an additional noise component related to cation movement through the poorly selective cation channel in the apical membrane [Aelvoet I, Erlij D, Van Driessche W (1988) J Physiol (Lond) 398:555-574; Van Driessche W, Desmedt L, Simaels J (1991) Pflügers Arch 418:193-203]. The amplitude of this Ca(2+)-blockable noise component was elevated by amiloride and markedly exceeded the amiloride-induced Lorentzian noise levels as recorded in the presence of Ca2+. On the other hand, in K(+)-depolarized skins and bladders as well as in nondepolarized epithelial of A6 cells, the Ca(2+)-blockable noise was absent or of much smaller amplitude. Depolarization of frog skin and toad urinary bladder apparently inactivated the poorly selective channels, whereas in A6 cells they were not observed. Under these conditions the typical amiloride-induced blocker noise could also be analysed in the absence of Ca2+ and demonstrated that the on and off rates for amiloride binding were not significantly altered by external Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Blockage of Na+ currents through poorly selective cation channels in the apical membrane of frog skin and toad urinary bladder.

The blockage of Na+ movements through the poorly selective cation channels in the apical membrane of frog skin (Rana temporaria) and toad urinary bladder (Bufo marinus) was investigated with noise, impedance analysis and microelectrode techniques. Na+ currents through this pathway were studied with NaCl Ringer solutions on both sides. After removal of Ca2+ and other divalent cations from the mucosal compartment, a considerable part of Isc became insensitive to amiloride. In frog skin, the inhibitory effect of amiloride in mucosal Ca(2+)-free solutions was highly variable. In some experiments a complete lack of inhibition was observed. Similarly, in the absence of amiloride, the inhibitory effect of mucosal Ca2+ varied strongly among frogs. In the absence of mucosal Ca2+, analysis of the fluctuation in Isc revealed a Lorentzian component in the power density spectrum. The corner frequency (fc) of this spontaneous Lorentzian was 12.3 Hz in frog skin and 347 Hz in the toad urinary bladder. In frog skin, nanomolar concentrations of mucosal Ca2+ induced an additional Lorentzian noise component. Its corner frequency shifted upwards with increasing mucosal Ca2+ concentration ([Ca2+]m). The relation between 2 pi fc and [Ca2+]m was linear at small [Ca2+]m whereas a parabolic increase of fc was observed at the highest [Ca2+]m. In the bladder, nanomolar concentrations of mucosal Ca2+ did not induce an additional noise component but modified the spontaneous Lorentzian noise by increasing fc proportionally with [Ca2+]m. Microelectrode recordings demonstrated that at least part of the Ca(2+)-blockable current passes through the granulosum cells and confirmed the apical localization of the poorly selective cation channel. The lack of the inhibitory effect of amiloride in Ca(2+)-free solutions seems to originate from the parallel arrangement of the amiloride- and Ca(2+)-blockable pathways and from influences of the blockage of apical channels on the basolateral membrane conductances. The latter cross-talk seems to find its origin in the voltage dependence of the basolateral membrane conductance [Garty H (1984) J Membr Biol 77:213-222; Nagel W (1985) Pflügers Arch 405 [Suppl 1]:S39-S43].

Amiloride blocks glutamate-operated cationic channels and protects neurons in culture from glutamate-induced death

The diuretic amiloride has been suggested as a specific inhibitor of T-type neuronal Ca 2+ channels. The effects of amiloride on glutamate receptor-gated cationic channels and glutamate-induced, Ca 2+-dependent neuronal death were investigated in primary neuronal cultures from neonatal rats. In primary cultures of cerebellar granule neurons of the rat, receiving 50 μM glutamate for 15 min, at 22°C, in the absence of Mg 2+, about 80% of neurons were killed in about 24 hr. Exposure of neurons to such a pulse of glutamate, in the presence of various concentrations of amiloride, resulted in a dose-dependent protection from neurotoxicity (EC 50 300 μM, complete protection 1 mM). In voltage-clamped cortical and cerebellar neurons of neonatal rats in primary culture, 100 μM amiloride diminished (by about 25%) glutamate- and/or NMDA-evoked cationic currents, recorded in the whole-cell mode. About 80% of the NMDA- (20 μM) stimulated current was inhibited by 700 μM amiloride. The inhibitory effect of amiloride was not voltage-dependent. In outside-out membrane patches, excised from granule cells and held at −50 mV, 100 μM amiloride changed the NMDA-elicited single channel activity into a fast flickering between the open and closed states. The noise analysis of the data revealed that, although resembling the Mg 2+-induced flickering, the amiloride-induced channel block was more similar to the effects described for the action of local anaesthetics on the nicotinic cholinergic channel. The pharmacological relevance of this action of amiloride requires further characterization; the data point out the necessity of a cautious use of amiloride in studying neuronal function.