Ethanol Modulates the VR-1 Variant Amiloride-insensitive Salt Taste Receptor. I. Effect on TRC Volume and Na+ Flux

The effect of ethanol on the amiloride- and benzamil (Bz)-insensitive salt taste receptor was investigated by the measurement of intracellular Na+ activity ([Na+]i) in polarized rat fungiform taste receptor cells (TRCs) using fluorescence imaging and by chorda tympani (CT) taste nerve recordings. CT responses were monitored during lingual stimulation with ethanol solutions containing NaCl or KCl. CT responses were recorded in the presence of Bz (a specific blocker of the epithelial Na+ channel [ENaC]) or the vanilloid receptor-1 (VR-1) antagonists capsazepine or SB-366791, which also block the Bz-insensitive salt taste receptor, a VR-1 variant. CT responses were recorded at 23°C or 42°C (a temperature at which the VR-1 variant salt taste receptor activity is maximally enhanced). In the absence of permeable cations, ethanol induced a transient decrease in TRC volume, and stimulating the tongue with ethanol solutions without added salt elicited only transient phasic CT responses that were insensitive to elevated temperature or SB-366791. Preshrinking TRCs in vivo with hypertonic mannitol (0.5 M) attenuated the magnitude of the phasic CT response, indicating that in the absence of mineral salts, transient phasic CT responses are related to the ethanol-induced osmotic shrinkage of TRCs. In the presence of mineral salts, ethanol increased the Bz-insensitive apical cation flux in TRCs without a change in cell volume, increased transepithelial electrical resistance across the tongue, and elicited CT responses that were similar to salt responses, consisting of both a transient phasic component and a sustained tonic component. Ethanol increased the Bz-insensitive NaCl CT response. This effect was further enhanced by elevating the temperature from 23°C to 42°C, and was blocked by SB-366791. We conclude that in the presence of mineral salts, ethanol modulates the Bz-insensitive VR-1 variant salt taste receptor.

Modulation of Rat Chorda Tympani NaCl Responses and Intracellular Na+ Activity in Polarized Taste Receptor Cells by pH

Mixture interactions between sour and salt taste modalities were investigated in rats by direct measurement of intracellular pH (pHi) and Na+ activity ([Na+]i) in polarized fungiform taste receptor cells (TRCs) and by chorda tympani (CT) nerve recordings. Stimulating the lingual surface with NaCl solutions adjusted to pHs ranging between 2.0 and 10.3 increased the magnitude of NaCl CT responses linearly with increasing external pH (pHo). At pH 7.0, the epithelial sodium channel (ENaC) blocker, benzamil, decreased NaCl CT responses and inhibited further changes in CT responses induced by varying pHo to 2.0 or 10.3. At constant pHo, buffering NaCl solutions with potassium acetate/acetic acid (KA/AA) or HCO3−/CO2 inhibited NaCl CT responses relative to CT responses obtained with NaCl solutions buffered with HEPES. The carbonic anhydrase blockers, MK-507 and MK-417, attenuated the inhibition of NaCl CT responses in HCO3−/CO2 buffer, suggesting a regulatory role for pHi. In polarized TRCs step changes in apical pHo from 10.3 to 2.0 induced a linear decrease in pHi that remained within the physiological range (slope = 0.035; r2 = 0.98). At constant pHo, perfusing the apical membrane with Ringer's solutions buffered with KA/AA or HCO3−/CO2 decreased resting TRC pHi, and MK-507 or MK-417 attenuated the decrease in pHi in TRCs perfused with HCO3−/CO2 buffer. In parallel experiments, TRC [Na+]i decreased with (a) a decrease in apical pH, (b) exposing the apical membrane to amiloride or benzamil, (c) removal of apical Na+, and (d) acid loading the cells with NH4Cl or sodium acetate at constant pHo. Diethylpyrocarbonate and Zn2+, modification reagents for histidine residues in proteins, attenuated the CO2-induced inhibition of NaCl CT responses and the pHi-induced inhibition of apical Na+ influx in TRCs. We conclude that TRC pHi regulates Na+-influx through amiloride-sensitive apical ENaCs and hence modulates NaCl CT responses in acid/salt mixtures.

Lack of Inhibition by Inhalational Anesthetics of Myocardial Contraction Dependent on Intracellular Sodium Activity

Background When cardiac muscle is stimulated at a high frequency after rest, the peak force increases rapidly at first and then slowly. The slow phase reflects Ca2+ accumulation dependent on an increase in intracellular Na+ activity. The positive inotropic effect of high concentrations of cardiac glycosides also involves Ca2+ accumulation dependent on an increase in intracellular Na+ activity. The effect of isoflurane on the component of myocardial contraction dependent on an increase in intracellular Na+ activity is not well understood. Methods The isometric force of rabbit papillary muscle contractions was measured. The authors studied the effects of isoflurane on the force increase that occurs when the muscles are stimulated after a rest and on the force increase induced by ouabain. Results Isoflurane (1.5%, 2.4%) had no statistically significant effect on the magnitude of the slow phase of force increase when the muscles were stimulated at 2 Hz after a rest. Isoflurane (1.5%) did not decrease the magnitude of force increase induced by ouabain (1 microM). Conclusions Isoflurane has little effect on myocardial contractions that depend on Ca2+ accumulation after an increase in intracellular Na+ activity. This may partly account for the smaller cardiac depressant effect of isoflurane observed at high frequencies rather than at low frequencies. The results of the present study and an earlier study with halothane suggest that the lack of inhibition of contractions dependent on Ca2+ accumulation after an increase in intracellular Na+ activity may be a common property of inhalational anesthetics.

Distribution and functional properties of glutamate receptors in the leech central nervous system

1. The effect of kainate and other glutamatergic agonists on the membrane potential (Em), the intracellular Na+ activity (aNai), and the intracellular free Ca2+ concentration ([Ca2+]i) of identified leech neurons and neuropile glial cells was measured with conventional and ion-sensitive microelectrodes, as well as with the use of the iontophoretically injected fluorescent indicators sodium-binding benzofuran isophthalate and Fura-2. 2. In Retzius neurons, AE, L, 8, and 101 motoneurons, and in the unclassified 50 neurons (Leydig cells) and AP neurons, as well as in neuropile glial cells, bath application of 100 microM kainate evoked a marked membrane depolarization and an increase in aNai and [Ca2+]i. The kainate-induced aNai increase persisted in solutions with high Mg2+ concentration in which synaptic transmission is blocked. 3. A membrane depolarization as well as an increase in aNai and [Ca2+]i was also evoked by L-glutamate, quisqualate, and L-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA). The agonist-induced [Ca2+]i increase was inhibited by 6,7-dinitroquinoxaline-2,3-dione (DNQX). 4. In Ca(2+)-free solution, the kainate-induced [Ca2+]i increase was abolished in the neurons and in neuropile glial cells, whereas membrane depolarization and aNai increase were unchanged. In Na(+)-free solution, kainate had no effect on Em, aNai, or [Ca2+]i in the neurons. 5. In the mechanosensory T, P, and N neurons, kainate induced considerably smaller membrane depolarizations than in the other neurons or in neuropile glial cells, and it had no significant effect on aNai or [Ca2+]i. 6. It is concluded that in leech segmental ganglia the majority of the neurons and the neuropile glial cells, but probably not the mechanosensory neurons, possess glutamate receptors of the AMPA-kainate type. In the neurons, the [Ca2+]i increase caused by glutamatergic agonists is due to Ca2+ influx through voltage-dependent Ca2+ channels that are activated by the agonist-induced membrane depolarization.

Thyroid status and Na(+)-K+ pump current, intracellular sodium, and action potential duration in rabbit heart

Thyroid status influences the abundance of Na(+)-K+ pumps in the heart. To evaluate whether this phenomenon may contribute to a dependence of the action potential duration (APD) on thyroid status, we induced hypothyroidism in a group of New Zealand White rabbits. Another group was treated similarly but also received triiodothyronine (T3). Right ventricular myocytes were isolated and voltage clamped at -40 mV. We identified Na(+)-K+ pump current (Ipump) as a ouabain-induced shift in holding current. Mean Ipump, measured using patch pipettes containing 10 mM Na+, was 0.24 +/- 0.02 pA/pF in 9 cells from 4 hypothyroid rabbits and 0.48 +/- 0.05 in 10 cells from 4 rabbits treated with T3 (P < 0.001). Because thyroid status influences Na+ influx, we measured intracellular Na+ activity (aiNa) in right ventricular papillary muscles. We found that aiNa was 5.20 +/- 0.42 mM in nine papillary muscles from seven hypothyroid rabbits and 7.62 +/- 0.69 mM in nine papillary muscles from six rabbits treated with T3 (P < 0.01). The effect of thyroid-induced changes in Ipump and aiNa on APD was stimulated with a computer model. The simulations predicted that thyroid-induced changes in Ipump can influence APD. The predicted changes were similar to changes in APD measured in isolated papillary muscles.

Angiotensin-converting enzyme inhibition, intracellular Na+, and Na(+)-K+ pumping in cardiac myocytes

Angiotensin-converting enzyme (ACE) inhibitors can reduce cardiac mass in both clinical and experimental cardiac hypertrophy. Because cytoplasmic Na+ and pH have been implicated as regulators of cell growth, we examined the effect of treatment with an ACE inhibitor on intracellular Na+ activity (alpha iNa) and pH (pHi) in the heart. After treatment of rabbits with captopril for 8 days alpha iNa was reduced relative to controls (3.6 +/- 0.4, n = 8, vs. 8.2 +/- 0.4 mM, n = 9, P < 0.001), whereas pHi was unchanged. To account for the difference in alpha iNa we measured electrogenic Na(+)-K+ pump activity in single isolated myocytes. Treatment with captopril increased pump currents at near-physiological levels of intracellular Na+ but had no effect at near-saturating levels of Na+. A similar increase in Na(+)-K+ pump activity occurred in rabbits treated with another ACE inhibitor, enalapril, but not with the vasodilator, hydralazine. We speculate that a decrease in alpha iNa after treatment with captopril may contribute to the well-documented ability of ACE inhibitors to reduce cardiac mass.

Effect of norepinephrine on cellular sodium transport in Ambystoma kidney proximal tubule

The effect of norepinephrine (NE) on mechanisms of cellular Na+ transport in the isolated, perfused proximal tubule of Ambystoma tigrinum was examined. Single-barreled voltage and ion-selective microelectrodes were used to determine basolateral (V1), luminal (V2), and transepithelial (V3) membrane potentials and intracellular Na+ activity (alpha Nai). In CO2/HCO3- control solution, addition of NE (10(-6) M) to the bath caused depolarizations of V1, V2, and V3 are decreased alpha Nai. These effects were mimicked by isoproterenol and inhibited by propranolol. Addition of NE in the absence of luminal Na+ and substrates did not cause any changes in V1, V2, V3, or alpha Nai. NE did not affect the changes in membrane potential difference (PD) or alpha Nai caused by removal and readdition of luminal substrates and/or Na+. To study the effect of NE on Na-K-adenosinetriphosphatase (Na-K-ATPase), the pump was inhibited by external K+ removal and then reactivated by readdition of 12 mM K+ to the bath in the presence and absence of NE. Reactivation of the pump caused hyperpolarization of membrane PDs, and alpha Nai recovered monotonically in 3-5 min. The peak hyperpolarizations of V1 and V2 (approximately 1 min) were significantly larger in the presence of NE. During the first 3 min, and also at the same alpha Nai, the rate of decrease of alpha Nai was significantly faster in the presence of NE. In conclusion, these results show a direct effect of NE on cell membrane PDs and alpha Nai in the kidney proximal tubule. Most likely, beta-receptors are involved in mediating the action of NE. Neither Na/H exchange nor Na-substrate cotransport at the luminal membrane are affected by NE. On the other hand, NE activates Na-K-ATPase.

Changes in potassium contractures due to simulated weightlessness in rat soleus muscle

Some contractile properties of soleus muscle (SOL) fibers isolated from tail-suspended (21 days) rats were compared with those determined in the slow-twitch SOL and the fast-twitch extensor digitorum longus muscle (EDL) of control rats. In SOL from suspended rats, the resting membrane potential and the intracellular Na+ activity were typical of fast-twitch muscles. The relationship between the amplitude of K+ contractures and the membrane potential was steeper for control SOL than for control EDL and suspended SOL. The inactivation curve was also shifted to more positive potentials after suspension. In the presence of perchlorate anions, the tension activation curves of control EDL and suspended SOL were similarly shifted to more negative potentials. Thus, in the present study, modifications induced by suspension in SOL, mainly related to changes in the voltage-sensing process involved in the excitation-contraction coupling mechanism, were that the SOL assumed some of the characteristics of fast-twitch muscles.