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PubMed2002

The effect of amiloride on operantly conditioned performance in an NaCl taste detection task and NaCl preference in C57BL/6J mice.

A 2-response operant taste discrimination procedure, modified to assess taste sensitivity in water-restricted C57BL/6J mice, revealed a detection threshold of 0.065 M sodium chloride. Amiloride increased the threshold by approximately 1 log10 unit. These results are the first to demonstrate the necessity of the amiloride-sensitive taste transduction pathway in the normal detection of low concentrations of sodium chloride in mice and provide a functional context in which to evaluate electrophysiological findings. Two-bottle preference tests performed with these mice and additional naive mice revealed only marginal, if any, effects of amiloride on salt intake behavior, highlighting the importance of considering the relative attributes and limitations of different behavioral assays of taste function.

Amiloride↗
PubMed1996

Responses of gustatory cells in the nucleus of the solitary tract of the hamster after NaCl or amiloride adaptation.

1. The responses of single nucleus of the solitary tract (NST) neurons in the hamster were recorded to an array of Na+ and non-Na+ stimuli under each of three adaptation conditions: distilled H2O, 0.032 M NaCl, and 10 microM amiloride. Each adapting solution flowed for 60 s before delivery of one of seven test stimuli: 0.032 M NaCl, NaNO3, and Na-gluconate, 0.1 M KCl and sucrose, 1 mM HCl, and 3 mM quinine hydrochloride (QHCl). Stimuli were dissolved in distilled H2O (H2O and NaCl adaptation conditions) or 10 microM amiloride (amiloride adaptation condition). 2. Both amiloride treatment and NaCl adaptation reduced responses to the Na+ stimuli. The effects of NaCl adaptation were generally greater than those of amiloride, and the responses to the Na+ salts were reduced by NaCl adaptation in every cell that responded to NaCl, regardless of its best-stimulus classification. Amiloride treatment suppressed the responses to Na+ salts with larger anions (NaNO3 and Na-gluconate) more than the response to NaCl. 3. Unlike amiloride treatment, NaCl adaptation also reduced responses to several non-Na+ stimuli (KCl, HCl, and QHCl). This effect occurred primarily in the NaCl-best neurons that were most highly responsive to NaCl and that showed a postexcitatory suppression after NaCl. This suppression has been observed in recordings from the chorda tympani nerve in both rats and hamsters and in taste receptor cell responses recorded in situ in the rat. If it is a receptor phenomenon, these data would imply that some NaCl-sensitive receptor cells are also responsive to these non-Na+ electrolytes. 4. The effects of amiloride on the responses to Na+ stimuli were not limited to NaCl-best neurons, but occurred in sucrose-best cells as well. These results suggest that the sucrose-best cells in the NST receive converging input from sucrose- and NaCl-best chorda tympani fibers, because there is little Na+ sensitivity in the peripheral sucrose-best fibers and the amiloride sensitivity is restricted to NaCl-best chorda tympani fibers. The responses to NaCl in the few HCl- and QHCl-best NST neurons were not affected by amiloride. 5. Rinsing the tongue with amiloride for 60 s resulted in a reduction in the baseline response rate of NST cells. This effect occurred primarily in NaCl- and sucrose-best NST neurons and implies that much of the spontaneous activity in these brain stem cells arises from amiloride-sensitive channel activity in the peripheral receptor cells. 6. The results of human psychophysical studies show very different effects of NaCl adaptation and amiloride treatment. Adaptation to NaCl produces a robust and specific reduction in the saltiness of all salts. The present results show that NaCl adaptation reduces the responses of all cells sensitive to NaCl. Treatment of the human tongue with amiloride produces a proportionately smaller reduction in the response to NaCl than it does in rodents, and it appears to have no effect on saltiness. Rather, amiloride has been shown to specifically reduce the sour side taste of NaCl, Nagluconate, and LiCl. Therefore conclusions about the effects of amiloride on taste quality based on rodent electrophysiology are questionable.

Adaptation, Physiological↗
PubMed2001

Tolerance to NaCl induces changes in plasma membrane lipid composition, fluidity and H+-ATPase activity of tomato calli.

Two tomato (Lycopersicon esculentum Mill. cv. Pera) callus lines tolerant to NaCl were obtained by successive subcultures of NaCl-sensitive calli in 50 and 100 mM NaCl-supplemented medium. Growth and ion content, as well as plasma membrane lipid composition, fluidity and H+-ATPase (EC 3.6.1.35) activity, were studied in both NaCl-sensitive and NaCl-tolerant calli. Although calli tolerant to 100 mM NaCl exhibited a reduced growth relative to calli sensitive to NaCl or tolerant to 50 mM NaCl, growth of calli tolerant to 100 mM NaCl was higher than that of NaCl-sensitive calli grown for one subculture in 100 mM NaCl. Growth in the presence of 100 mM NaCl provoked an increase of Na+ and Cl- content, but no significant changes in K+ and Ca2+. As compared with NaCl-sensitive and 50 mM NaCl-tolerant calli, plasma membrane vesicles isolated from calli tolerant to 100 mM NaCl exhibited a higher phospholipid and sterol content as well as a lower phospholipid/free sterol ratio and a lower double bond index (DBI) of phospholipid fatty acids. The changes in plasma membrane lipid composition were correlated with a decrease of plasma membrane fluidity in calli tolerant to 100 mM NaCl, as indicated by fluorimetric studies using diphenylhexatriene (DPH) as probe. Plasma membrane-enriched vesicles isolated from calli tolerant to 100 mM NaCl showed lower ATP hydrolysis and ATP-dependent H+-pumping activities, as well as a lower passive permeability to H+ than plasma membrane from NaCl-sensitive and 50 mM NaCl-tolerant calli. The involvement of the changes in plasma membrane lipid content and composition, fluidity and H+-ATPase activity in salt tolerance of tomato calli is discussed.

Journal Article↗
PubMed2001

Plasma membrane H-ATPase activity is involved in adaptation of tomato calli to NaCl.

A tomato (Lycopersicon esculentum Mill. cv. Pera) callus culture tolerant to NaCl was obtained by successive subcultures of NaCl-sensitive calli in medium supplemented with 50 mM NaCl. NaCl-tolerant calli grew better than NaCl-sensitive calli in media supplemented with 50 and 100 mM NaCl. Analysis of callus ion content showed a strong increase in Na+ and Cl- both in NaCl-tolerant and -sensitive calli grown in media containing NaCl for one subculture. Cells from NaCl-tolerant calli showed a higher H+ extrusion activity than those from NaCl-sensitive calli grown for one subculture in the presence of NaCl. The inhibition of H+ extrusion by NaCl-sensitive cells was correlated with an inhibition of microsomal vanadate-sensitive H+-ATPase (EC 3.6.1.35) and ATP-dependent H+ transport, while the stimulation of H+ extrusion by cells tolerant to 50 mM NaCl was correlated with an increase in plasma membrane ATP-dependent H+ transport. The increase of ATP-dependent H+ extrusion in plasma membranes isolated from 50 mM NaCl-tolerant calli was not a result of stimulation of a vanadate-sensitive ATP hydrolytic activity or an increase in passive permeability to H+. Relative to NaCl-sensitive calli, plasma membrane H+-ATPase from calli tolerant to 50 mM NaCl showed a lower Km for Mg2+-ATP. Our results indicate that tolerance of tomato calli to 50 mM NaCl increases the affinity of plasma membrane H+-ATPase for the substrate ATP and stimulates the H+-pumping activity of this enzyme without modifying its phosphohydrolytic activity.

Journal Article↗
PubMed1990

Lesions of the anterior hypothalamic area increase arterial pressure in NaCl-sensitive spontaneously hypertensive rats.

In NaCl-sensitive spontaneously hypertensive rats, diets high in NaCl increase arterial pressure and peripheral sympathetic nervous system activity and decrease the sympatho-inhibition mediated by the anterior hypothalamic area. To test the importance of the defect in anterior hypothalamic area-mediated sympatho-inhibition in the pathogenesis of NaCl-sensitive hypertension, bilateral ibotenic acid lesions of the anterior hypothalamic area were made in NaCl-sensitive spontaneous hypertensive rats, in NaCl-resistant spontaneously hypertensive rats and in normotensive, NaCl-resistant Wistar Kyoto rats. In NaCl-sensitive spontaneous hypertensive rats on a basal NaCl diet, the anterior hypothalamic area lesions caused a rapid rise in arterial pressure within the first week after surgery; by 21 days after surgery, mean systolic arterial pressure of the lesion group was 24 mmHg higher than that of the sham-operated group. In a second experiment, NaCl-sensitive spontaneous hypertensive rats were placed on an 8% NaCl diet 1 day after the lesion of the anterior hypothalamic area. 5 days after the operation, the lesion group of NaCl-sensitive spontaneous hypertensive rats on the 8% NaCl diet had a significantly higher arterial pressure than the sham-operated group, but by 1 week after the lesion, arterial pressures were not significantly different between the lesion and sham-operated NaCl-sensitive spontaneous hypertensive rats on the high NaCl diet. In Wistar Kyoto rats on a basal NaCl diet, lesions of the anterior hypothalamic area resulted in a small, transient elevation of arterial pressure, but no sustained effect. In NaCl-resistant spontaneous hypertensive rats, the anterior hypothalamic area lesions did not affect arterial pressure.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

The perception of saltiness is eliminated by NaCl adaptation: implications for gustatory transduction and coding.

The tastes of salts to humans are complex. NaCl is the most purely salty of all salts, but even this stimulus tastes sweet at low concentrations and somewhat sour at mid-range intensities. Other salts taste significantly sour or bitter in addition to salty. Previous studies have shown that the saltiness of simple halide salts is reduced by adaptation to NaCl, suggesting that a single mechanism might be responsible for the salty taste of these stimuli. In electrophysiological studies in rodents, the response to NaCl is reduced by application to the tongue of the Na(+)-channel blocker amiloride. Organic Na+ salts are more heavily dependent on this amiloride-sensitive transduction component than NaCl, and are generally less salty and more sour. In order to investigate the relationship between NaCl saltiness and that evoked by other salts, we adapted the tongue to distilled H2O and to 0.1 M NaCl and obtained direct magnitude estimates of the taste intensity of 15 organic and inorganic Na+, Li+, K+ and Ca2+ salts, matched for total intensity. Subjects divided these magnitude estimates among the component taste qualities. Adaptation to NaCl abolished the taste of NaCl and LiCl, and eliminated the saltiness of all other salts. The magnitude estimates of the bitterness and sourness of many salts increased after NaCl adaptation. Since recent biophysical data suggest that adaptation in taste receptors may involve whole-cell mechanisms, we propose that saltiness is reduced by NaCl adaptation because it originates in the subset of taste receptors responsive to NaCl. This implies that saltiness is coded within the CNS in cells whose receptive fields include the NaCl-sensitive receptor cells and that the degree to which any salt tastes salty is determined by its ability to drive these receptors. This model proposes, for example, that KCl has a salty component because it stimulates some of the same receptor cells as NaCl, even though the transduction mechanisms for KCl are different than those engaged by NaCl. Adaptation to NaCl blocks the saltiness of KCl and other salts because they stimulate NaCl-sensitive receptor cells.

Adaptation, Psychological↗
PubMed1993

Excitatory sympathetic reflex in NaCl-sensitive spontaneously hypertensive rats.

We have previously demonstrated blunted reflex responses of lumbar sympathetic nerve activity during volume expansion in NaCl-sensitive spontaneously hypertensive rats maintained on basal (1% NaCl) diets compared with NaCl-resistant spontaneously hypertensive rats, Wistar-Kyoto rats, and Sprague-Dawley rats. The current study tested the hypothesis that chronic ingestion of a high (8%) NaCl diet further blunts cardiopulmonary reflex function in the NaCl-sensitive spontaneously hypertensive rat. After 3 weeks of a 1% or 8% NaCl diet, male rats of all four strains were instrumented with femoral arterial and venous cannulas and lumbar nerve recording electrodes at 10 weeks of age. Two days later, conscious rats were infused with whole blood to expand blood volume. NaCl-sensitive spontaneously hypertensive rats maintained on a 1% NaCl diet had blunted responses of nerve activity to acute volume expansion compared with control strains. NaCl-sensitive spontaneously hypertensive rats maintained on an 8% NaCl diet had increases in nerve activity responses to volume expansion. In a second experiment, the volume expansion protocol was repeated in anesthetized NaCl-sensitive spontaneously hypertensive rats that had been subjected to sinoaortic denervation after 3 weeks of a 1% or 8% NaCl diet. After sinoaortic denervation, an increase in nerve activity was again observed during volume expansion in animals fed the 8% NaCl diet. In animals fed the 1% NaCl diet, changes in nerve activity were variable. The excitatory response was significantly reduced after bilateral vagotomy. These studies suggest that blood pressure regulation in NaCl-sensitive spontaneously hypertensive rats is a complex interaction of excitatory and inhibitory sympathetic reflex systems that is altered by high dietary NaCl exposure.

Animals↗
PubMed1986

Functional and morphological alterations in the rat stomach following exposure to hypertonic NaCl solution.

We examined the effects of 1 M NaCl as a mild irritant on gastric potential difference (PD), acid secretion, mucosal blood flow (MBF), and DNA synthetic activity in anesthetized rat stomachs and compared these effects with those of 4 M NaCl as a strong irritant. Both 1 M and 4 M NaCl produced a PD reduction (mucosal injury), but the reduced PD recovered faster in the mucosa exposed to 1 M NaCl as compared to 4 M NaCl. Acid secretion ceased after exposure to these hypertonic NaCl solutions, but histamine infusion (8 mg/kg/hr) stimulated acid secretion only in the mucosa exposed to 1 M NaCl. The MBF was significantly increased in response to 1 M NaCl, while exposure to 4 M NaCl had no effect on the MBF. These changes in acid secretion and MBF induced by 1 M NaCl were significantly antagonized by pretreatment with indomethacin (5 mg/kg, s.c.). The levels of PGE2 and 6-keto PGF1 alpha in the corpus mucosa were significantly increased in the stomach exposed to both 1 M and 4 M NaCl, and these increases disappeared in the presence of indomethacin. The rate of [3H]-thymidine incorporation was significantly reduced in the mucosa after exposure to 4 M NaCl, but remained unaltered in the stomach exposed to 1 M NaCl. These results suggest that although both 1 M and 4 M NaCl produced mucosal injury (PD reduction) and enhanced PGs formation, a variety of functional alterations mediated by PGs occurred in response to injury in the stomach exposed to 1 M NaCl. The presence or absence of these functional responses may be associated with the biphasic actions on the gastric mucosa of these hypertonic NaCl solutions as mild and strong irritants.

Adaptation, Physiological↗
PubMed2002

Rearing on basal or high dietary NaCl modifies chorda tympani nerve responses in rats.

Two experiments were conducted to determine the influence of dietary NaCl level on the integrated responses of the chorda tympani (CT) nerve to salt stimulation alone and mixed with the sodium-channel blocker, amiloride hydrochloride. Five groups of adult male rats were reared on regular chow containing either basal 0.1%, intermediate 1.0%, or high 3.0% NaCl from conception to postnatal day (PD) 30 or from conception to adulthood. Adult rats reared from conception to adulthood on basal dietary NaCl demonstrated a reduction in the CT nerve response to NaCl due to a decrease in the amiloride sensitive transduction mechanism. However, the CT nerve responses of adult rats reared on basal dietary NaCl to PD30 and then switched to intermediate dietary NaCl were similar to those of rats reared for a lifetime on intermediate dietary NaCl. Similarly, the CT nerve responses to NaCl in animals reared on high dietary NaCl from conception to PD30 and then switched to an intermediate NaCl diet were comparable to animals reared on intermediate and basal dietary NaCl. However, we found that exposure to high dietary NaCl led to a greater amiloride inhibition of NaCl responses. Thus, there is critical association between dietary NaCl level over two different exposure periods and CT nerve responsiveness to NaCl specifically regarding the degree of amiloride inhibition.

Amiloride↗
PubMed1983

Comparison between a Stable NaCl-Selected Nicotiana Cell Line and the Wild Type : K, Na, and Proline Pools as a Function of Salinity.

An NaCl-resistant line has been developed from suspension-cultured tobacco cells (Nicotiana tabacum/gossii) by stepwise increases in the NaCl concentration in the medium. Resistance showed stability through at least 24 generations in the absence of added NaCl.Above an external NaCl concentration of 35 millimolar, proline concentration in the selected cells rose steeply with external NaCl, particularly so above 100 millimolar NaCl. Proline accumulation in the wild type was far slighter. Selected cells which had been grown for 24 generations in the absence of added NaCl accumulated proline strongly on re-exposure to NaCl medium, indicating stability of this character. Proline accumulation was fully reversible with a half-time of about 6 hours. When selected cells were transferred sequentially to lower and lower NaCl concentrations, their proline content fell to the level corresponding to the new NaCl concentration. The NaCl-selected cells responded to water stress (i.e. added mannitol) by accumulating markedly more proline than did the wild type.The addition of Ca(2+) to the growing and rinsing media minimized Na(+) and K(+) binding in the Donnan free space of cell walls and thus allowed assessment of intracellular Na(+) and K(+). In both cell types, internal Na(+) content rose steadily as a function of external NaCl concentration. In the course of 7 days in NaCl media, the wild type cells lost a considerable part of their K(+) content, the extent of the loss increasing with rise in external NaCl concentration. The selected cells, by contrast, lost no K(+) at external NaCl concentrations below 50 millimolar external NaCl, and at higher concentrations lost less than the wild type.

Journal Article↗
PubMed1991

Mechanism of proximal NaCl reabsorption in the proximal tubule of the mammalian kidney.

In the mammalian proximal tubule NaCl reabsorption occurs by both passive and active transport processes. Passive NaCl reabsorption occurs in the presence of a high luminal chloride and a low luminal bicarbonate concentration. These anion gradients provide the driving forces for diffusive Na and Cl movement. Na is driven by the lumen positive PD effected by the greater permeability of the tubular wall to Cl than to HCO3. Cl is driven by its high tubular concentration. Passive NaCl reabsorption accounts for only about 10% to 15% of total proximal NaCl transport. The remaining proximal NaCl is reabsorbed by active transport processes and occurs both in the presence or absence of anion gradients reabsorption. Two mechanisms of active NaCl reabsorption participate in active NaCl reabsorption along the proximal tubule. Firstly, active NaCl reabsorption is electrogenic. In the early proximal tubule Na enters to cell coupled to organic solute transport. This Na reabsorption generates a lumen negative PD and effects "coupled" electrogenic NaCl reabsorption. This mechanism is limited by the supply of organic solutes and is blunted by the greater Na than Cl permeability in the proximal tubule; it probably can account for no more than 10% of proximal NaCl reabsorption. In the terminal proximal tubule, the proximal straight tubule, the apical membrane appears to possess a channel for Na entry. This Na reabsorption also generates a lumen negative PD and effects "simple" electrogenic NaCl reabsorption. This mechanism is limited by the low transport capacity of this segment and probably accounts for no more than 5% to 10% of total proximal NaCl reabsorption. The great bulk of proximal NaCl reabsorption occurs along the entire proximal tubule by active, transcellular electroneutral NaCl reabsorption. The precise cellular transport mechanisms responsible for this process are only recently being defined. At the apical membrane parallel ion exchangers are responsible for NaCl entry into the cell. Na enters via the apical membrane Na-H antiporter. Cl most likely crosses the apical membrane by some combination of Cl-OH and Cl-HCO2 exchangers but not via a Cl-HCO3 exchanger. The relative contributions of Cl-OH and Cl-HCO2 exchange have not been defined. There are two important considerations in this question. First is the availbility of OH versus HCO2. Although there is an infinite supply of OH and a small equilibrium supply of HCO2, it is possible that the luminal concentration of HCO2 could be increased by an USL that raises the concentration of HCO2 to a degree sufficient to supply H2CO2 recycling for physiological transcellular Cl transport rates.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

The importance of NaCl concentration in a chemically defined medium for the development of bovine oocytes matured and fertilized in vitro.

Bovine oocytes matured and fertilized in vitro were cultured in a chemically defined medium (modified Tyrode's solution) without glucose. When different concentrations of NaCl were added to the medium, the proportions of embryos developed to the >or=8-cell, morula and blastocyst stages 96, 144 and 192 h post insemination, respectively, were significantly higher at 89 to 114 mM than 64 to 76 and 126 to 139 mM NaCl. A high proportion (28%) of blastocyst-stage embryos 192 h post insemination was obtained at 89 mM NaCl. When calculated osmolarity in the medium with 64 mM NaCl was varied by adding D-sorbitol, significantly higher proportions of morula-stage embryos were obtained at 265 to 315 mOsm (27 to 38%) than 215 (9%) and 365 (2%) mOsm, but the development to the blastocyst stage was difficult at any osmolarities (215 to 365 mOsm) tested. In the medium with a fixed osmolarity (315 mOsm) but with different concentrations (64 to 114 mM) of NaCl, there were no differences in the proportions (29 to 33%) of morula-stage embryos among different NaCl concentrations. However, significantly higher proportions of embryos developed to the blastocyst stage at 89 to 101 mM (22 to 23%) than 64 to 76 (0 to 9%) and 114 (11%) mM NaCl. When Cl- concentration in the medium with 64 mM NaCl was adjusted by adding choline chloride, significantly higher proportions of embryos developed to the morula stage at 97 to 122 mM (32 to 40%) than 72 (6%) and 147 (2%) mM Cl-, but few embryos developed to the blastocyst stage at any Cl- concentrations (72 to 147 mM) tested. In the medium with 64 or 114 mM NaCl and each with 2 different Na+/K+ ratios, there were no differences in the proportions of morula- and blastocyst-stage embryos between different Na+/K+ ratios (31 and 39 at 64 mM NaCl, and 39 and 47 at 114 mM NaCl) at each NaCl concentration. When glucose was added to the medium with 89 mM NaCl 120 h post insemination, there were no significant differences in the proportions (40 to 48%) of morula-stage embryos 144 h post insemination among different concentrations (0 to 6.95 mM) of glucose. The proportion (33%) of blastocysts 192 h post insemination at 2.78 mM glucose was significantly higher than the values at 0 (22%), 5.56 (19%) and 6.95 (15%) mM but not different compared with the values at 1.39 (23%) and 4.17 (28%) mM. In conclusion, NaCl concentration in a defined medium is one of the most important factors for the development of bovine embryo to the blastocyst stage, but the development of embryos up to the morula stage is also regulated by osmolarity and/or Cl-concentration.

Journal Article↗