Diuretic Amiloride Research Articles

Block of Human CaV3 Channels by the Diuretic Amiloride

Previous studies in native T-type currents have suggested the existence of distinct isoforms with dissimilar pharmacology. Amiloride was the first organic blocker to selectively block the native T-type calcium channel, but the potency and mechanism of block of this drug on the three recombinant T-type calcium channels (Ca(V)3.1, Ca(V)3.2, and Ca(V)3.3) have not been systematically determined. The aim of the present study was to investigate whether there is differential block of Ca(V)3 channels by amiloride, to establish the mechanism of block, and to obtain insights into the amiloride putative binding sites in Ca(V)3 channels. By performing whole-cell patch-clamp recordings of human embryonic kidney 293 cells stably expressing human Ca(V)3 channels, we found that amiloride blocked the human Ca(V)3 channels in a concentration-response manner; the IC₅₀ for Ca(V)3.2 channels (62 μM) was 13-fold lower than that for Ca(V)3.1 and Ca(V)3.3. Block is voltage-independent (except for Ca(V)3.3 channels) and targets mainly closed-state channels, although a small use-dependent component was observed in Ca(V)3.1 channels. In addition, amiloride block of Ca(V)3.2 channels is mainly due to an extracellular effect, whereas in Ca(V)3.1 and Ca(V)3.3 channels, the amiloride inhibition is equally effective from both sides of the membrane. The results demonstrate that amiloride blocks human Ca(V)3 channels differentially through a mechanism involving mainly the closed state of the channel and suggest a negative allosteric interaction with at least two putative binding sites with different affinities. The preferential block of Ca(V)3.2 channels labels amiloride as the only organic blocker to be selective for any T-type channel.

2-Amidino analogs of glycine–amiloride conjugates: Inhibitors of urokinase-type plasminogen activator

The relative non-toxicity of the diuretic amiloride, coupled with its selective inhibition of the protease urokinase plasminogen activator (uPA), makes this compound class attractive for structure–activity studies. Herein we substituted the C(2)-acylguanidine of C(5)-glycyl-amiloride with amidine and amidoxime groups. The data show the importance of maintaining C(5)-hydrophobicity. The C(5)-benzylglycine analogs containing either C(2)-acylguanidine or amidine inhibited uPA with an IC50 ranging from 3 to 7μM and were cytotoxic to human U87 malignant glioma cells.

Anti‐tumour/metastasis effects of the potassium‐sparing diuretic amiloride: An orally active anti‐cancer drug waiting for its call‐of‐duty?

Amiloride.HCl is clinically used as an oral potassium-sparing diuretic, but multiple studies in biochemical, cellular and animal models have shown that the drug also possesses anti-tumour and anti-metastasis activities. The additional effects appear to arise through inhibition of two discrete targets: (i) the sodium-hydrogen exchanger 1 (NHE1), a membrane protein responsible for the characteristically low extracellular pH of tumours and (ii) the urokinase-type plasminogen activator (uPA), a serine protease mediator of cell migration, invasion and metastasis and well-known marker of poor prognosis in cancer. This mini-review summarises for the first time the reported anti-tumour/metastasis effects of amiloride in experimental models, discusses the putative molecular mechanisms responsible for these effects and concludes by commenting on the pros and cons of trialling amiloride or one of its structural analogues as potential new anti-tumour/metastasis drugs.

Alteration in Mg2+ content of red blood cells from patients with diuretic therapy

Hypomagnesemia can cause ventricular tachycardia, cardiac necrosis, or torsades de pointes. Diuretic treatment can result in Mg2+ depletion, as many diuretics inhibit the reabsorption of filtered Mg2+ in the proximal or distal tubule. As plasma Mg2+ constitutes only about 3% of total body Mg2+ stores, intracellular Mg2+ determinations may be more useful for assessing total body Mg2+ content. Therefore we studied the effect of a thiazide diuretic (trichlormethiazide 4 mg/day), a combination of a thiazide and a potassium-sparing diuretic (trichlormethiazide and amiloride 2 mg/day each), and the loop diuretic piretanide (6 mg/day) on intracellular Mg2+ in patients with mild essential hypertension before and after 6 and 10 weeks of therapy. Mg2+ measurements were performed in blood plasma and in red blood cells by atomic absorption spectroscopy, using a Video 12 apparatus. There was a significant decrease in intracellular Mg2+ content under trichlormethiazide therapy (p<0.05). Furthermore, our results show that diuretic treatment with a combination of a thiazide and a potassium-sparing diuretic or with the loop diuretic piretanide may have the advantage of avoiding intracellular Mg2+ depletion.

Effect on ex vivo platelet aggregation and in vivo cyclic flow with Na+/H+ exchange inhibition

BIIB 513 and EMD 85131 are selective inhibitors of the Na+/H+ exchanger-1 (NHE-1) that are benzoylguanidine derivatives of the clinically employed diuretic amiloride. Prior studies have suggested a role for NHE-1 activity in platelet activation and aggregation using amiloride or its non- benzoylguanidines derivatives. However, the concentrations employed in these prior studies were at levels known to exert effects on other ion transport systems besides the NHE-1. Therefore, the purpose of this study was to examine the effects of more selective NHE-1 inhibitors, BIIB 513 and EMD 85131, on platelet aggregation and in vivo cyclic flow following arterial injury. BIIB 513 and EMD 85131 effects on ex vivo canine and human platelet aggregation in response to various agents was monitored via platelet aggregation. For analysis of in vivo thrombus formation, a femoral artery crush injury model was employed and a flow meter was used to monitor the effect of BIIB 513 on cyclic blood flow. Treatment of either canine or human platelets with up to 1 mM of BIIB 513 had no effect on aggregation induced by platelet activating factor (PAF), thrombin receptor activator peptide (TRAP), or adenosine diphosphate (ADP). Additionally, the structurally related compound EMD 85131 at up to 1 mM failed to inhibit TRAP induced platelet aggregation. In vivo administration of up to 9 mg/kg of BIIB 513 intravenously failed to affect cyclic flow in a canine model of femoral artery injury. These data demonstrate that the specific and selective NHE-1 inhibitors BIIB 513 or EMD 85131 have no effect on ex vivo platelet aggregation or in vivo cyclic flow following arterial injury.

A Single Amino Acid Tunes Ca2+ Inhibition of Brain Liver Intestine Na+ Channel (BLINaC)

Ion channels of the degenerin/epithelial Na(+) channel gene family are Na(+) channels that are blocked by the diuretic amiloride and are implicated in several human diseases. The brain liver intestine Na(+) channel (BLINaC) is an ion channel of the degenerin/epithelial Na(+) channel gene family with unknown function. In rodents, it is expressed mainly in brain, liver, and intestine, and to a lesser extent in kidney and lung. Expression of rat BLINaC (rBLINaC) in Xenopus oocytes leads to small unselective currents that are only weakly sensitive to amiloride. Here, we show that rBLINaC is inhibited by micromolar concentrations of extracellular Ca(2+). Removal of Ca(2+) leads to robust currents and increases Na(+) selectivity of the ion pore. Strikingly, the species ortholog from mouse (mBLINaC) has an almost 250-fold lower Ca(2+) affinity than rBLINaC, rendering mBLINaC constitutively active at physiological concentrations of extracellular Ca(2+). In addition, mBLINaC is more selective for Na(+) and has a 700-fold higher amiloride affinity than rBLINaC. We show that a single amino acid in the extracellular domain determines these profound species differences. Collectively, our results suggest that rBLINaC is opened by an unknown ligand whereas mBLINaC is a constitutively open epithelial Na(+) channel.

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.

An acid‐sensing ion channel from shark (Squalus acanthias) mediates transient and sustained responses to protons

Acid-sensing ion channels (ASICs) are proton-gated Na(+) channels. They are implicated in synaptic transmission, detection of painful acidosis, and possibly sour taste. The typical ASIC current is a transient, completely desensitizing current that can be blocked by the diuretic amiloride. ASICs are present in chordates but are absent in other animals. They have been cloned from urochordates, jawless vertebrates, cartilaginous shark and bony fish, from chicken and different mammals. Strikingly, all ASICs that have so far been characterized from urochordates, jawless vertebrates and shark are not gated by protons, suggesting that proton gating evolved relatively late in bony fish and that primitive ASICs had a different and unknown gating mechanism. Recently, amino acids that are crucial for the proton gating of rat ASIC1a have been identified. These residues are completely conserved in shark ASIC1b (sASIC1b), prompting us to re-evaluate the proton sensitivity of sASIC1b. Here we show that, contrary to previous findings, sASIC1b is indeed gated by protons with half-maximal activation at pH 6.0. sASIC1b desensitizes quickly but incompletely, efficiently encoding transient as well as sustained proton signals. Our results show that the conservation of the amino acids crucial for proton gating can predict proton sensitivity of an ASIC and increase our understanding of the evolution of ASICs.

Effect of canrenone and amiloride on the prooxidative effect induced by aldosterone in human mononuclear leukocytes in vitro

Clinical studies have demonstrated that aldosterone receptor antagonists do improve the survival of patients with chronic heart diseases and in vitro studies have shown that canrenone blocks the proinflammatory effect of aldosterone in mononucler leukocytes (MNL). The aim of the study was to compare, in the model of human MNL, the effect of potassium-sparing diuretics amiloride and canrenone, on the protein expression of p22phox, a NADPH-oxidase system subunit, that is a principal marker of production of superoxide anions. MNL were isolated from 10 informed healthy volunteers (5 males and 5 females, age range 24-36 yr) and the proteins extracted. p22phox protein expression was evaluated by Western blot and quantified using a densitometric semiquantitative analysis. The experiments showed that aldosterone (10(-8) M) enhances the protein expression of p22phox and that its effect is reversed by co-incubation with canrenone (10(-6) M), while incubation with amiloride (10(-6) M) reduced the prooxidative effect of aldosterone at a significantly lower extent than canrenone. Co-incubation with canrenone, amiloride, and aldosterone together produced the same effect as aldosterone plus canrenone. Incubation with cortisol (40(-8) M) was not effective. These data confirm the prooxidative effect of aldosterone in MNL. The addition of aldosterone-receptor antagonist canrenone produced a higher inhibition than sodium channel blocker amiloride on the effect of aldosterone on p22phox protein expression.

K+ secretion in the rat kidney: Na+ channel-dependent and -independent mechanisms

Renal Na(+) and K(+) excretion was measured in rats with varying dietary K(+) intake. The requirement for channel-mediated distal nephron Na(+) reabsorption was assessed by infusing the animals with the K(+)-sparing diuretic amiloride via osmotic minipumps. At infusion rates of 2 nmol/min, the concentration of amiloride in the urine was 38 microM, corresponding to concentrations of 9-23 microM in the distal tubular fluid, sufficient to block >98% of Na(+) transport through apical Na(+) channels (ENaC). With a control K(+) intake (0.6% KCl), amiloride reduced K(+) excretion rates (U(K)V) from 0.85 +/- 0.15 to 0.05 +/- 0.01 micromol/min during the first 2 h of infusion, suggesting that distal nephron K(+) secretion was completely dependent on the activity of Na(+) channels. When K(+) intake was increased by feeding overnight with a diet containing 10% KCl, amiloride reduced U(K)V from 7.5 +/- 0.7 to 1.3 +/- 0.1 micromol/min despite an increased plasma K(+) of 9 mM, again suggesting a major but not exclusive role for the Na(+) channel-dependent pathway of K(+) secretion. The maximal measured rates of amiloride-sensitive K(+) excretion correspond well with estimates based on apical K(+) channel activity in distal nephron segments. However, when the animals were adapted to the high-K(+) diet for 7-9 days, the diuretic decreased U(K)V less, from 6.1 +/- 0.6 to 3.0 +/- 0.8 micromol/min, indicating an increasing fraction of K(+) excretion that was independent of Na(+) channels. This indicates the upregulation of a Na(+) channel-independent mechanism for secreting K(+).

A synaptic DEG/ENaC ion channel mediates learning in C. elegans by facilitating dopamine signalling

An important component of learned behaviour is the ability to forecast positive or negative outcomes based on specific sensory cues. Predictive capacity is typically manifested by appropriate behavioural patterning. However, the molecular mechanisms underlying behavioural plasticity are poorly understood. Caenorhabditis elegans displays experience-dependent behavioural responses by associating distinct environmental signals. We find that ASIC-1, a member of the degenerin/epithelial sodium channel family, which localizes at presynaptic terminals of dopaminergic neurons, is required for associative learning in C. elegans. ASIC-1 functions in these neurons to amplify normal dopaminergic signalling, necessary for associative learning. Our results reveal a novel role of DEG/ENaC ion channels in neuronal communication by enhancing the activity of dopaminergic synapses. Similar mechanisms may facilitate synaptic plasticity in vertebrates.

Amiloride is neuroprotective in an MPTP model of Parkinson's disease

The diuretic amiloride has recently proven neuroprotective in models of cerebral ischemia, a property attributable to the drug's inhibition of central acid-sensing ion channels (ASICs). Given that Parkinson's disease (PD), like ischemia, is associated with cerebral lactic acidosis, we tested amiloride in the MPTP-treated mouse, a model of PD also manifesting lactic acidosis. Amiloride was found to protect substantia nigra (SNc) neurons from MPTP-induced degeneration, as determined by attenuated reductions in striatal tyrosine hydroxylase (TH) and dopamine transporter (DAT) immunohistochemistry, as well as smaller declines in striatal DAT radioligand binding and dopamine levels. More significantly, amiloride also preserved dopaminergic cell bodies in the SNc. Administration of psalmotoxin venom (PcTX), an ASIC1a blocker, resulted in a much more modest effect, attenuating only the deficits in striatal DAT binding and dopamine. These findings represent the first experimental evidence of a potential role for ASICs in the pathogenesis of Parkinson's disease.

Small Molecule Activator of the Human Epithelial Sodium Channel

The epithelial sodium channel (ENaC), a heterotrimeric complex composed of alpha, beta, and gamma subunits, belongs to the ENaC/degenerin family of ion channels and forms the principal route for apical Na(+) entry in many reabsorbing epithelia. Although high affinity ENaC blockers, including amiloride and derivatives, have been described, potent and specific small molecule ENaC activators have not been reported. Here we describe compound S3969 that fully and reversibly activates human ENaC (hENaC) in an amiloride-sensitive and dose-dependent manner in heterologous cells. Mechanistically, S3969 increases hENaC open probability through interactions requiring the extracellular domain of the beta subunit. hENaC activation by S3969 did not require cleavage by the furin protease, indicating that nonproteolyzed channels can be opened. Function of alphabetaG37Sgamma hENaC, a channel defective in gating that leads to the salt-wasting disease pseudohypoaldosteronism type I, was rescued by S3969. Small molecule activation of hENaC may find application in alleviating human disease, including pseudohypoaldosteronism type I, hypotension, and neonatal respiratory distress syndrome, when improved Na(+) flux across epithelial membranes is clinically desirable.

Molecular Proximity of Cystic Fibrosis Transmembrane Conductance Regulator and Epithelial Sodium Channel Assessed by Fluorescence Resonance Energy Transfer

We present the evidence for a direct physical association of cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial sodium channel (ENaC), two major ion channels implicated in the pathophysiology of cystic fibrosis, a devastating inherited disease. We employed fluorescence resonance energy transfer, a distance-dependent imaging technique with capability to detect molecular complexes with near angstrom resolution, to estimate the proximity of CFTR and ENaC, an essential variable for possible physical interaction to occur. Fluorescence resonance energy transfer studies were complemented with a classic biochemical approach: coimmunoprecipitation. Our results place CFTR and ENaC within reach of each other, suggestive of a direct interaction between these two proteins.

Vitamin B1 (thiamine) uptake by human retinal pigment epithelial (ARPE-19) cells: mechanism and regulation.

Retinal abnormality and visual disturbances occur in thiamine-responsive megaloblastic anaemia (TRMA), an autosomal recessive disorder caused by mutations in the human thiamine transporter-1 (hTHTR-1). Human retinal pigment epithelial cells play a pivotal role in supplying thiamine to the highly metabolically active retina but nothing is known about the mechanism, regulation or biological processes involved in thiamine transport in these cells. To address these issues, we used human-derived retinal pigment epithelial ARPE-19 cells to characterize the thiamine uptake process. Thiamine uptake is energy- and temperature-dependent, pH-sensitive, Na+-independent, saturable at both the nanomolar (apparent Km, 30 +/- 5 nM) and the micromolar (apparent Km, 1.72 +/- 0.3 microM) concentration ranges, specific for thiamine and sensitive to sulfhydryl group inhibition. The diuretic amiloride caused a concentration-dependent inhibition in thiamine uptake, whereas the anti-trypanosomal drug, melarsoprol, failed to affect the uptake process. Both hTHTR-1 and hTHTR-2 are expressed in ARPE-19 cells as well as in native human retinal tissue with expression of the former being significantly higher than that of the latter. Uptake of thiamine was adaptively regulated by extracellular substrate level via transcriptionally mediated mechanisms that involve both hTHTR-1 and hTHTR-2; it was also regulated by an intracellular Ca2+-calmodulin-mediated pathway. Confocal imaging of living ARPE-19 cells expressing TRMA-associated hTHTR-1 mutants (D93H, S143F and G172D) showed various expression phenotypes. These results demonstrate for the first time the existence of a specialized and regulated uptake process for thiamine in a cellular model of human retinal pigment epithelia that involves hTHTR-1 and hTHTR-2. Further, clinically relevant mutations in hTHTR-1 lead to impaired cell surface expression or function of the transporter in retinal epithelial ARPE-19 cells.

Aldosterone antagonists: Silver bullet or just sodium excretion and potassium retention?

ACE inhibitors or AT(1) antagonists are powerful therapeutic strategies to slow the progression of renal disease. However, they provide only imperfect protection since they cannot always prevent end-stage renal failure. Innovative approaches are needed to keep patients with chronic kidney disease off dialysis. Blockade of the aldosterone pathway may prove to be such a beneficial therapeutic concept.

Gain-of-Function Mutations in the MEC-4 DEG/ENaC Sensory Mechanotransduction Channel Alter Gating and Drug Blockade

MEC-4 and MEC-10 are the pore-forming subunits of the sensory mechanotransduction complex that mediates touch sensation in Caenorhabditis elegans (O'Hagan, R., M. Chalfie, and M.B. Goodman. 2005. Nat. Neurosci. 8:43–50). They are members of a large family of ion channel proteins, collectively termed DEG/ENaCs, which are expressed in epithelial cells and neurons. In Xenopus oocytes, MEC-4 can assemble into homomeric channels and coassemble with MEC-10 into heteromeric channels (Goodman, M.B., G.G. Ernstrom, D.S. Chelur, R. O'Hagan, C.A. Yao, and M. Chalfie. 2002. Nature. 415:1039–1042). To gain insight into the structure–function principles that govern gating and drug block, we analyzed the effect of gain-of-function mutations using a combination of two-electrode voltage clamp, single-channel recording, and outside-out macropatches. We found that mutation of A713, the d or degeneration position, to residues larger than cysteine increased macroscopic current, open probability, and open times in homomeric channels, suggesting that bulky residues at this position stabilize open states. Wild-type MEC-10 partially suppressed the effect of such mutations on macroscopic current, suggesting that subunit–subunit interactions regulate open probability. Additional support for this idea is derived from an analysis of macroscopic currents carried by single-mutant and double-mutant heteromeric channels. We also examined blockade by the diuretic amiloride and two related compounds. We found that mutation of A713 to threonine, glycine, or aspartate decreased the affinity of homomeric channels for amiloride. Unlike the increase in open probability, this effect was not related to size of the amino acid side chain, indicating that mutation at this site alters antagonist binding by an independent mechanism. Finally, we present evidence that amiloride block is diffusion limited in DEG/ENaC channels, suggesting that variations in amiloride affinity result from variations in binding energy as opposed to accessibility. We conclude that the d position is part of a key region in the channel functionally and structurally, possibly representing the beginning of a pore-forming domain.

Chapter 13 - Insights into the Pore of the Hair Cell Transducer Channel from Experiments with Permeant Blockers

This chapter presents recent experiments designed to infer the properties of the ion-conducting pore of the mechanoelectrical transducer channel of sensory hair cells using permeant blockers. By combining results from experiments with three classes of large cationic permeant blockers, the fluorescent dye FM1-43, the aminoglycoside antibiotics, and the potassium-sparing diuretic amiloride, information has been obtained on the free energy profile along the transducer channel's pore as sensed by these blocker molecules. These energy profiles provide information about the position of the negatively charged binding site for the blockers as well as about positively charged barriers near the extracellular and intracellular faces of the channel that impede the blockers' permeation. The extracellular barrier is relatively modest and allows almost diffusion-limited entry of blockers from the extracellular side. A larger intracellular energy barrier effectively prevents exit of the blocking molecules from the intracellular side, trapping the blockers inside the hair cells. A putative geometrical model of the transducer channel pore is presented that draws on results from all the three classes of permeant blockers. The pore contains a large vestibule that is easily accessible from the extracellular side.

Regulation of ENaC by intracellular chloride in mammalian taste receptor cells

The transduction of sodium salts (e.g. “salty taste”) has been shown to involve permeation of sodium ions through epithelial sodium channels (ENaC) in the gustatory system. Though numerous studies have demonstrated complex regulation of ENaC in other sodium transporting epithelia, there have been few studies that have focused on the regulation of ENaC in the taste system. To understand the regulatory capacity of the gustatory system we have begun to investigate the ability of ENaC to be regulated by intracellular Cl ions (Schrieber et al. J. Memb. Biol. 199:85, 2004). To identify expression of various transport proteins for Cl in the gustatory system we have used RT-PCR and immunocytochemistry. Taste cells express a variety of Cl transport proteins including several members of the ClC family of Cl channels, the cystic fibrosis transmembrane conductance regulator (CFTR) and the sodium-potassium-chloride cotransporter, NKCC. Patch clamp recording on mouse taste cells revealed that both the magnitude of ENaC currents and their sensitivity to the diuretic amiloride is inversely correlated with intracellular Cl concentration. In solutions high in extracellular Cl, inhibition of Cl transport through NKCC with bumetanide leads to increases in ENaC-mediated sodium influx in taste cells. We are currently investigating the role of other Cl transport proteins in ENaC regulation in taste cells. Supported by NIH DC02507 (TAG).

Thiazolidinediones expand body fluid volume through PPARγ stimulation of ENaC-mediated renal salt absorption

Thiazolidinediones (TZDs) are widely used to treat type 2 diabetes mellitus; however, their use is complicated by systemic fluid retention. Along the nephron, the pharmacological target of TZDs, peroxisome proliferator-activated receptor-gamma (PPARgamma, encoded by Pparg), is most abundant in the collecting duct. Here we show that mice treated with TZDs experience early weight gain from increased total body water. Weight gain was blocked by the collecting duct-specific diuretic amiloride and was also prevented by deletion of Pparg from the collecting duct, using Pparg (flox/flox) mice. Deletion of collecting duct Pparg decreased renal Na(+) avidity and increased plasma aldosterone. Treating cultured collecting ducts with TZDs increased amiloride-sensitive Na(+) absorption and Scnn1g mRNA (encoding the epithelial Na(+) channel ENaCgamma) expression through a PPARgamma-dependent pathway. These studies identify Scnn1g as a PPARgamma target gene in the collecting duct. Activation of this pathway mediates fluid retention associated with TZDs, and suggests amiloride might provide a specific therapy.