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Respiratory complex I has an L-shaped structure formed by the hydrophilic arm responsible for electron transfer and the membrane arm that contains protons pumping machinery. Here, to gain mechanistic insights into the role of subunit NuoL, we investigated the effects of Mg 2+ , Zn 2+ and the Na + /H + antiporter inhibitor 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) on proton pumping activities of various isolated NuoL mutant complex I after reconstitution into Escherichia coli double knockout (DKO) membrane vesicles lacking complex I and the NADH dehydrogenase type 2. We found that Mg 2+ was critical for proton pumping activity of complex I. At 2 µM Zn 2+ , proton pumping of the wild-type was selectively inhibited without affecting electron transfer; no inhibition in proton pumping of D178N and D400A was observed, suggesting the involvement of these residues in Zn 2+ binding. Fifteen micromolar of EIPA caused up to ∼40% decrease in the proton pumping activity of the wild-type, D303A and D400A/E, whereas no significant change was detected in D178N, indicating its possible involvement in the EIPA binding. Furthermore, when menaquinone-rich DKO membranes were used, the proton pumping efficiency in the wild-type was decreased significantly (∼50%) compared with NuoL mutants strongly suggesting that NuoL is involved in the high efficiency pumping mechanism in complex I., (© The Authors 2016. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Background: Diabetic nephropathy (DN) is associated with hypertension, expanded extracellular volume and impaired renal Na(+) excretion. It was hypothesized that aberrant glomerular filtration of serine proteases in DN causes proteolytic activation of the epithelial sodium channel (ENaC) in the kidney by excision of an inhibitory peptide tract from the γ subunit., Methods: In a cross-sectional design, urine, plasma and clinical data were collected from type 1 diabetic patients with DN (n = 19) and matched normoalbuminuric type 1 diabetics (controls, n = 20). Urine was examined for proteases by western immunoblotting, patch clamp and ELISA. Urine exosomes were isolated to elucidate potential cleavage of γENaC by a monoclonal antibody directed against the 'inhibitory' peptide tract., Results: Compared with control, DN patients displayed significantly higher blood pressure and urinary excretion of plasmin(ogen), prostasin and urokinase that correlated directly with urine albumin. Western blotting confirmed plasmin, prostasin and urokinase in urine from the DN group predominantly. Urine from DN evoked a significantly larger amiloride-sensitive inward current in single collecting duct cells compared with controls. Immunoblotting of urine exosomes showed aquaporin 2 in all patient samples. Exosomes displayed a virtual absence of intact γENaC while moieties compatible with cleavage by furin only, were shown in both groups. Proteolytic cleavage by the extracellular serine proteases plasmin or prostasin was observed in DN samples predominantly., Conclusion: DN is associated with increased urinary excretion of plasmin, prostasin and urokinase and proteolytic activation of ENaC that might contribute to impaired renal Na(+) excretion and hypertension., (© The Author 2015. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.)

Amilorides, well-known inhibitors of Na(+)/H(+) antiporters, have also shown to inhibit bacterial and mitochondrial NADH-quinone oxidoreductase (complex I). Since the membrane subunits ND2, ND4, and ND5 of bovine mitochondrial complex I are homologous to Na(+)/H(+) antiporters, amilorides have been thought to bind to any or all of the antiporter-like subunits; however, there is no direct experimental evidence in support of this notion. Photoaffinity labeling is a powerful technique to identify the binding site of amilorides in bovine complex I. Commercially available amilorides such as 5-(N-ethyl-N-isopropyl)amiloride are not suitable as design templates to synthesize photoreactive amilorides because of their low binding affinities to bovine complex I. Thereby, we attempted to modify the structures of commercially available amilorides in order to obtain more potent derivatives. We successfully produced two photoreactive amilorides (PRA1 and PRA2) with a photolabile azido group at opposite ends of the molecule.

Somatic mutations of the potassium channel KCNJ5 are found in 40% of aldosterone producing adenomas (APAs). APA-related mutations of KCNJ5 lead to a pathological Na(+) permeability and a rise in cytosolic Ca(2+), the latter presumably by depolarizing the membrane and activating voltage-gated Ca(2+) channels. The aim of this study was to further investigate the effects of mutated KCNJ5 channels on intracellular Na(+) and Ca(2+) homeostasis in human adrenocortical NCI-H295R cells. Expression of mutant KCNJ5 led to a 2-fold increase in intracellular Na(+) and, in parallel, to a substantial rise in intracellular Ca(2+). The increase in Ca(2+) appeared to be caused by activation of voltage-gated Ca(2+) channels and by an impairment of Ca(2+) extrusion by Na(+)/Ca(2+) exchangers. The mutated KCNJ5 exhibited a pharmacological profile that differed from the one of wild-type channels. Mutated KCNJ5 was less Ba(2+) and tertiapin-Q sensitive but was inhibited by blockers of Na(+) and Ca(2+)-transporting proteins, such as verapamil and amiloride. The clinical use of these drugs might influence aldosterone levels in APA patients with KCNJ5 mutations. This might implicate diagnostic testing of APAs and could offer new therapeutic strategies.

Two stability-indicating chromatographic methods are described for simultaneous determination of amiloride hydrochloride (AMI), atenolol (ATE), and chlorthalidone (CHL) in combined dosage forms. The first method was based on HPTLC separation of the three drugs followed by densitometric measurements of their bands at 274 nm. The separation was carried out on Merck HPTLC silica gel 60F254 aluminum sheets using chloroform-methanol-ammonia 27%, w/w (9 + 2 + 0.3, v/v/v) mobile phase. Analysis data was used for the linear regression graph in the range of 0.1-0.5, 0.8-5.0, and 0.3-1.5 microg/band for AMI, ATE, and CHL, respectively. The second method was based on an RP-HPLC separation of the cited drugs performed on an RP stainless steel C18 analytical column (250 x 4.6 mm id) with a gradient elution system of methanol and 0.05 M aqueous phosphate buffer adjusted to pH 4 as the mobile phase, at the flow rate of 1.0 mL/min. Quantitation was achieved with photodiode array detection at 275 nm for AMI and 225 nm for ATE and CHL. The calibration graphs for each drug were rectilinear in the range of 2-50, 25-150, and 2-100 microg/mL for AMI, ATE, and CHL, respectively. The proposed chromatographic methods were successfully applied for determination of the investigated drugs in pharmaceutical preparations. Both methods were validated in compliance with International Conference on Harmonization guidelines in terms of linearity, accuracy, precision, robustness, LOD, and LOQ.

Maillard reacted peptides (MRPs) were synthesized by conjugating a peptide fraction (1000-5000 Da) purified from soy protein hydrolyzate with galacturonic acid, glucosamine, xylose, fructose, or glucose. The effect of MRPs was investigated on human salt taste and on the chorda tympani (CT) taste nerve responses to NaCl in Sprague-Dawley rats, wild-type, and transient receptor potential vanilloid 1 (TRPV1) knockout mice. MRPs produced a biphasic effect on human salt taste perception and on the CT responses in rats and wild-type mice in the presence of NaCl + benzamil (Bz, a blocker of epithelial Na+ channels), enhancing the NaCl response at low concentrations and suppressing it at high concentrations. The effectiveness of MRPs as salt taste enhancers varied with the conjugated sugar moiety: galacturonic acid = glucosamine > xylose > fructose > glucose. The concentrations at which MRPs enhanced human salt taste were significantly lower than the concentrations of MRPs that produced increase in the NaCl CT response. Elevated temperature, resiniferatoxin, capsaicin, and ethanol produced additive effects on the NaCl CT responses in the presence of MRPs. Elevated temperature and ethanol also enhanced human salt taste perception. N-(3-methoxyphenyl)-4-chlorocinnamid (a blocker of TRPV1t) inhibited the Bz-insensitive NaCl CT responses in the absence and presence of MRPs. TRPV1 knockout mice demonstrated no Bz-insensitive NaCl CT response in the absence or presence of MRPs. The results suggest that MRPs modulate human salt taste and the NaCl + Bz CT responses by interacting with TRPV1t.

The integrated responses to gustatory stimuli applied to the soft palate were recorded from the greater superficial petrosal nerve (GSP) and were compared with those from the chorda tympani nerve (CT) innervating the anterior part of the tongue in the rat. Stimuli included various concentrations of NaCl, sucrose, HCl and quinine hydrochloride, and 0.5 M of six sugars. The inhibitory effects of amiloride on the responses to sodium salts, including various concentration of NaCl, 0.1 M sodium acetate and 0.01 M sodium saccharin, were also tested. Both the phasic and tonic responses to sugars in the GSP were significantly larger than those in the CT, whereas both responses to NaCl in the GSP were significantly smaller than those in the CT. Although amiloride at 50 microM significantly depressed the phasic and tonic responses to NaCl with a wide range of concentration in the CT, little inhibitory effect was observed in the GSP. The tonic response to sodium acetate, when dissolved in amiloride solution, was depressed to 15% of the control in the CT, and slightly but significantly depressed to 70% in the GSP. These response characteristics of the GSP may play important roles in the processing of gustatory information.