Effect of Maillard reacted peptides on human salt taste and the amiloride-insensitive salt taste receptor (TRPV1t).

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.

Hormonal regulation of the epithelial Na+ channel: from amphibians to mammals.

High-resistance epithelia derived from amphibian sources such as frog skin, toad urinary bladder, and the A6 Xenopus laevis kidney cell line have been widely used to elucidate the underlying mechanisms involved in the regulation of vectorial ion transport. More recently, the isolation of high-resistance mammalian cell lines has provided model systems in which to study differences and similarities between the regulation of ion transporter function in amphibian and mammalian renal epithelia. In the present study, we have compared the natriferic (Na+ retaining) responses to aldosterone, insulin, and vasotocin/vasopressin in the A6 and mpkCCDcl4 (mouse principal cells of the kidney cortical collecting duct) cell lines. The functional responses of the epithelial Na+ channel (ENaC) to hormonal stimulation were remarkably similar in both the amphibian and mammalian lines. In addition, insulin- and aldosterone-stimulated, reabsorptive Na+ transport in both cell lines requires the presence of functional PI3-kinase.

Dependence of Plasmodium falciparum in vitro growth on the cation permeability of the human host erythrocyte.

Intraerythrocyte growth of the malaria parasite Plasmodium falciparum induces a Ca2+-permeable unselective cation conductance in the host cell membrane which is inhibited by ethylisopropylamiloride (EIPA) and is paralleled by an exchange of K+ by Na+ in the host cytosol. The present study has been performed to elucidate the functional significance of the electrolyte exchange. Whole-cell patch-clamp experiments confirmed the Ca2+ permeability and EIPA sensitivity of the Plasmodium falciparum induced cation channel. In further experiments, ring stage-synchronized parasites were grown in vitro for 48 h in different test media. Percentage of Plasmodium-infected and phosphatidylserine-exposing erythrocytes was measured with FACS analysis by staining with the DNA-dye Syto16 and annexin V, respectively. The increase of infected cells was not significantly affected by an 8 h replacement of NaCl in the culture medium with Na-gluconate but was significantly blunted by replacement of NaCl with KCl, NMDG-Cl or raffinose. Half maximal growth was observed at about 25 mM Na+. The increase of infected cells was further inhibited by EIPA (IC50< 10 microM) and at low extracellular free Ca2+. Infected cells displayed significantly stronger annexin binding, an effect mimicked by exposure of noninfected erythrocytes to oxidative stress (1 mM T-butylhydroperoxide for 15 min) or to Ca2+ ionophore ionomycin (1 microM for 60 min). The observations indicate that parasite growth requires the entry of both, Na+ and Ca2+ cations into the host erythrocyte probably through the EIPA sensitive cation channel. Ca2+ entry further induces break-down of the phospholipid asymmetry in the host membrane.

Inhibition by xipamide of amiloride-induced acidification in cultured rat cardiocytes.

The diuretic drug xipamide improves myocardial relaxation in hypertensive patients with left ventricular hypertrophy, but its mechanism of action is unknown. Here, xipamide was tested in cultured rat heart myogenic H9c2 cells and newborn cardiomyocytes for its effects on cell acidification (and Ca2+ mobilization). In H9c2 cells, blocking Na+/H+ exchange with amiloride (2 mM) provoked cell acidification with rate = 0.82 +/- 0.17 pH units/h (n = 6). Xipamide 1 microM maximally inhibited 50 +/- 7% (n = 9) of cell acidification. The action of xipamide required the presence of HCO3- and was antagonized by the HCO3(-)-transport blocker DIDS (4,4'-diisothiocyanostilbene-2.2'-disulfonic acid). Conversely, the carbonic anhydrase (EC 4.2.1.1) inhibitor acetazolamide failed to prevent xipamide action. Finally, xipamide was without significant effect on the Ca2+ signals induced by endothelin-1, vasopressin or the Ca2+ ionophore ionomycin. In newborn rat cardiomyocytes, xipamide reduced amiloride-induced cell acidification at similar concentrations as in H9c2 cardiocytes, but with a slightly higher extent of maximal inhibition (70-80%). In conclusion, xipamide reduced amiloride-dependent cell acidification in the rat heart myogenic H9c2 cell line and in newborn rat cultured cardiomyocytes. This action of xipamide seems to be related to a complex interaction with DIDS-sensitive HCO3- movements. Prevention of cell acidification by xipamide could be involved in the beneficial effects of this compound in myocardial relaxation and left ventricle filling in hypertensive patients with left ventricular hypertrophy.

Regulation of cell pH by K+/H+ antiport in renal epithelial cells.

Acid-loaded opossum kidney (OK) cells secrete H+ by Na+/H+ exchange and by a Na(+)- and HCO3(-)-independent pathway that has not been fully characterized. We studied the Na(+)-independent component by measuring H+ flux using the pH-sensitive trapped indicator 2',7'-bis(2-carboxyethyl)-5(6)- carboxyfluorescein. Two Na(+)-independent H(+)-transport systems were identified in acid-loaded cells perfused with HCO3(-)-free buffers. The minor component appears to be a conductive pathway for H+, over 90% inhibitable by 5 mM barium. The major component is stimulated by extracellular K+ and was fully active in the presence of barium, amiloride, ouabain, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, and bumetanide and in the absence of Cl-. Ammonium inhibited the H+ flux by 72% at 50 mM, and the H+ flux could be accelerated two- to threefold by limited proteolysis of intact cells using kallikrein or papain. In cells pretreated with barium, the K(+)-induced H+ flux caused no change of bis-oxonol fluorescence, suggesting an electroneutral pathway. The H+ flux was a saturable function of extracellular K+ (Michaelis constant 55 mM), and flux reversed when the K+ gradient was reversed. Similarly, the H+ flux was a linear function of the H+ gradient and reversed when the H+ gradient reversed. Evidence for ongoing K(+)-induced H+ flux was also found in nonacidified cells. First, changing perfusate K+ from 5 to 50 mM alkalinized baseline cell pH, an effect not reproduced by barium despite an equivalent depolarizing effect. Second, increasing perfusate K+ from 5 to 50 mM completely eliminated the acidification produced by 1 mM amiloride. We conclude that the OK cell expresses two Na(+)-independent acid-base transport systems. One is a barium-sensitive electrogenic H+ conductance and the other functions as an electroneutral K+/H+ antiporter. The antiporter is capable of H+ extrusion from acid-loaded cells but in normal cells functions in the reverse direction, as an H+ loader. The K+/H+ antiporter appears to be one of the major systems regulation cell pH in these cells, balancing the H+ efflux mediated by Na+/H+ exchange.

Characterization and subtype identification of the Na(+)-H+ exchanger in bovine corneal epithelium.

Amiloride analogues with N5-alkyl substitutions are specific high-affinity ligands for the Na(+)-H+ exchanger in various tissues. As a means to characterize the Na(+)-H+ exchanger in the bovine corneal epithelium, we determined the binding properties of [3H] methylisobutylamiloride (MIA) to a fraction enriched in plasma membrane from this tissue. [3H]MIA bound to these membranes in a time, -a temperature-, and -a pH-dependent manner. The binding was optimal at 4 degrees C and at pH 8.5 and it reached equilibrium at 60 min. Under these conditions, specific binding, which was inhibitable by excess unlabeled MIA, was about 85%. Scatchard analysis of this specific binding revealed a single saturable binding component with a Kd of 61 nM and a Bmax of 271 pmoles/mg protein. Inhibition of [3H]MIA specific binding by amiloride analogues showed the following order of potency: MIA > dimethylamiloride (DMA) > benzamil > amiloride. Na+ did not compete with MIA for binding. The effectiveness of clonidine, an alpha 2 agonist, and cimetidine, an H2 receptor antagonist, as inhibitors of Na(+)-H+ exchange activity was also determined because these compounds are used to distinguish between the exchanger subtypes. At concentrations higher than those needed for receptor interaction, clonidine was more effective than cimetidine in decreasing MIA binding. The activity of Na(+)-H+ exchanger, which was measured as the uptake of 22Na+ in the presence of an outwardly directly H+ gradient, was also inhibited by DMA, benzamil and amiloride with the same order of potency as obtained in the binding studies.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of amiloride-sensitive Na+ channels of hen lower intestine in Xenopus oocytes: electrophysiological studies on the dependence of varying NaCl intake.

Epithelial Na+ channels were incorporated into the plasma membrane of Xenopus laevis oocytes after micro-injection of RNA from hen lower intestinal epithelium (colon and coprodeum). The animals were fed either a normal poultry food which contained NaCl (HS), or a similar food devoid of NaCl (LS). Oocytes were monitored for the expression of amiloride-sensitive sodium channels by measuring membrane potentials and currents. Oocytes injected with poly(A)+RNA prepared from HS animals or non-injected control oocytes showed no detectable sodium currents, whereas oocytes injected with LS-poly(A)+RNA had large amiloride-blockable sodium currents. These currents were almost completely saturated by sodium concentrations of 20 mM with a Km of about 2.6 mM sodium. Amiloride (10 microM) inhibits the expressed sodium channels entirely and examination of dose response relationships yielded a half-maximal inhibition concentration (Ki) of 120 nM amiloride. I-V difference curves in the presence or absence of sodium or amiloride (10 microM) indicate a potential dependence of the sodium transport which can be described by the Goldman equation. When Na+ is replaced by K+, no amiloride response was detected indicating a high selectivity for Na+ over K+. These results provide strong evidence that intestinal Na+ channels are regulated by dietary salt intake on the RNA level.

Calcium channel blockers inhibit amiloride-stimulated short-circuit current in frog tadpole skin.

The larval frog skin has a very high electrical resistance and a corresponding low rate of transepithelial ion transport. Amiloride, a blocker of sodium transport in adult skin, transiently stimulates rather than inhibits short-circuit current (Isc) across larval skin. The time course and concentration response to amiloride and the effects of calcium channel blockers on Isc were studied with larval frog skin mounted in modified Ussing chambers. The amiloride (1 mM) transient was markedly blunted if the skin was previously exposed to low amiloride (0.01-0.1 mM) concentrations. The calcium channel blockers verapamil, nitrendipine, diltiazem, W-7, and lanthanum all blocked the amiloride transient. Diltiazem itself caused a rapid transient in Isc, indicating that it may be a partial agonist. These data suggest that the amiloride-stimulated cation channels rapidly desensitize in a manner similar to the acetylcholine receptor. The decline in Isc after amiloride stimulation could be caused by amiloride block of the open channel. Blockade of amiloride stimulation by well-known calcium channel blockers suggests that these larval cation channels may have some characteristics in common with calcium channels.

Atrial natriuretic peptide-induced inhibition of amiloride-sensitive 22Na+ uptake into cerebral capillaries of spontaneously hypertensive rats.

22Na+ uptake into capillaries isolated from the cerebral cortex of adult (20- to 26-week-old) sustained-hypertensive spontaneously hypertensive rats (SHR) and stroke-prone SHR (SHRSP) was compared with findings in age-matched Wistar Kyoto rats (WKY). In the presence of 1.0 mM ouabain, 1.0 mM furosemide and 2.0 mM LiCl, 22Na+ uptake into the isolated cerebral capillaries of WKY and SHR was significantly reduced to 38% and 65% of the control values, respectively, when 0.1 microM alpha-rat atrial natriuretic peptide (rANP) was added to the uptake buffer. The rANP-induced inhibition observed in SHR was significantly less, as compared with that in the WKY. Noteworthy was the observation that the Na+ uptake into the cerebral capillaries of SHRSP was not inhibited by rANP. As this peptide is thought to regulate amiloride-sensitive Na+ transport from the blood to brain by interacting with specific receptors, the present finding may relate to the etiology of dysfunction of the blood-brain barrier, in the presence of hypertension.

Characterization of Na+-H+ antiport in type II alveolar epithelial cells.

The presence of a Na+-H+ exchange pathway in the plasma membrane of type II alveolar epithelial cells was explored using the pH-sensitive fluorescent probe 2,7-biscarboxyethyl-5,6-carboxyfluorescein (BCECF) to monitor changes in cytosolic pH. Freshly prepared pneumocytes suspended in medium at pH 7.4 had an intracellular pH of 7.07 +/- 0.07. Acid-loaded cells equilibrated in sodium-free buffer showed rapid cytoplasmic alkalinization when exposed to sodium. This response to sodium was inhibited greater than 90% by 10(-4) M amiloride. The presence of the K+ ionophore, valinomycin, had no effect on the rate of Na+-dependent alkalinization, indicating the electroneutrality of the system. Li+ partially supported the alkalinization process, but other monovalent cations, notably K+, Rb+, and Cs+, were without effect. Kinetic analysis for Na+ at the external binding site yielded KNat (dissociation constant) = 62 +/- 3 mM. Hill equation analysis of the data derived a Hill coefficient (n) = 1.2 +/- 0.1 for Na+, consistent with a 1:1 stoichiometry for Na+ and H+ for the transporter. The Ki for amiloride inhibition of proton efflux at the external locus was 0.45 microM. These findings define the transport pathway as Na+-H+ antiport, with kinetic parameters somewhat similar to those described for other cell types. Antiport activity was detected at intracellular pH (pHi) values of 6.8 or below, with no activity observed at pHi 7.0-7.2. It is suggested that Na+-H+ exchange is a major mechanism whereby pneumocytes recover from an acid load and that this transport pathway may play an important role in vectorial reabsorption of Na+ from the alveolar air spaces.

Effects of detergents on sodium transport in toad urinary bladder.

The effects of ionic, nonionic and zwitterionic detergents (zwittergents) on apical sodium permeability of toad urinary bladder were investigated. Applied to the mucosal side at concentrations less than 1/100 of their respective critical micellar concentration (CMC), sodium lauryl sulfate, Triton X-100 and a series of sulfobetaine zwittergents reversibly increased amiloride-sensitive sodium current. These detergents decreased the current at concentrations higher than 1/100 of the CMC and caused large, irreversible increases of tissue electrical conductance at a concentration close to the CMC. Nonionic detergent Tween 80, however, stimulated the current at concentrations from 1/100 to 10,000 of its CMC. The maximum stimulation of current by each zwittergent occurred at 1/100 of its CMC, and the larger the CMC, the greater the stimulation attainable. Analysis of the dependence of current on mucosal sodium concentration in the presence of stimulating doses of a zwittergent and Tween 80 showed that the increase in current was not the result of increased apical maximum sodium permeability, but the consequence of removal of sodium self-inhibition. The amiloride dose-response curve in the presence of stimulating dose of zwittergent was shown to shift to the left and yield a smaller apparent inhibition constant as predicted on the basis of such a removal and an unaltered intrinsic amiloride blocking kinetic.

Transport in cultured epithelia.

Recent progress in studying epithelia in culture indicates that techniques already available and applied to naturally occurring epithelia can be applied effectively to cultured epithelia. Studies using this approach are summarized as well as new information the studies have yielded regarding the mechanism of action of aldosterone and the regulation of expression of the sodium-coupled hexose transporter. In addition, special features of cultured epithelia should lead to new approaches to understanding epithelial biology as well as epithelial transport.

Modification of cation permeability of rabbit descending colon by sulphydryl reagents.

1. Addition of the organic mercurials mersalyl, p-chloromercuribenzoate, and p-chloromercuribenzene sulphonate to the Ringer solution (140 mM-Na) bathing the luminal side of isolated epithelia of rabbit descending colon increases short-circuit current (Isc) and tissue conductance (Gt) when the spontaneous Isc is below 2-3 muequiv/cm2 hr. 2. The stimulation of Isc by mersalyl is due to an increase in Na absorption, simultaneously K secretion is induced, whereas Cl absorption is not affected. 3. Mersalyl inhibits Isc at Na concentrations below 50 mM. The Na concentration at which Isc is half-maximal (KNa) is shifted by mersalyl from 25 to 133 mM. The overshoot in Isc to a peak volume of 5 muequiv/cm2 hr observed when Na-depleted tissues are suddenly exposed to Na is markedly depressed by mersalyl. 4. Mersalyl inhibits non-competitively the blocking effect of amiloride on Isc. Both the stimulation of Isc and the inhibition of the amiloride effect by mersalyl have the same time course (half-time of the effects 30-40 min) and similar concentration-response curve (half-maximal effects with 2.0-2.6 x 10(-4) M), indicating a common mechanism. 5. The mersalyl effects on Isc and on the amiloride action are only partially reversed by dimercaptopropanol. p-Chloromercuribenzoate conjugated with dextran (mol. wt. 10,000) elicited the same effects as mersalyl. 6. The stoichiometry of the mersalyl-amiloride interaction, estimated by use of the Hill plot, is 1:1; a Hill coefficient of 1 was also obtained for the stimulating effect of mersalyl on Isc. 7. It is concluded that one sulphydryl group per luminal Na entry site controls both its Na conductance and cation selectivity. Titration of these sulphydryl groups by organic mercurials appear to fix the conductance of the luminal Na entry mechanism in a submaximal position and prevent its modulation by amiloride or variations in intra- and/or extracellular Na concentrations.

P-chloromercuribenzene sulfonate blocks and reverses the effect of amiloride on sodium transport across rabbit colon in vitro.

The addition of 10(-3) M p-chloromercuribenzene sulfonate (PCMBS) to the solution bathing the mucosal surface of rabbit colon has no effect on the rate of active Na transport but blocks or reverses the inhibitory action of amiloride. The tissue must be exposed to PCMBS for 20-30 min for a complete blocking effect, and removal of PCMBS from the mucosal solution after this period of exposure does not restore the sensitivity of the tissue to amiloride. The slow time-courses of the blocking and reversal effects suggest that PCMBS does not irreversibly interact with groups directly involved in the binding of amiloride.