Acid-induced responses in hamster chorda tympani and intracellular pH tracking by taste receptor cells

HCl- and NaCl-induced hamster chorda tympani nerve responseswere recorded during voltage clamp of the lingual receptive field. Voltage perturbations did not influence responses to HCl. In contrast, responses to NaCl were decreased by submucosal-positive and increased by submucosal-negative voltage clamp. Responses to HCl were insensitive to the Na⁺ channel blockers,amiloride and benzamil, and to methylisobutylamiloride (MIA), anNa⁺/H⁺exchange blocker. Responses to NaCl were unaffected by MIA but weresuppressed by benzamil. Microfluorometric and imaging techniques wereused to monitor the relationship between external pH(pH_o) and the intracellular pH(pH_i) of fungiform papilla tastereceptor cells (TRCs) following2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein loading.TRC pH_i responded rapidly andmonotonically to changes in pH_o.This response was unaffected byNa⁺ removal or the presence ofamiloride, benzamil, or MIA. The neural records and the data fromisolated TRCs suggest that the principal transduction pathway for acidtaste in hamster is similar to that in rat. This may involve themonitoring of changes in TRC pH_i mediated through amiloride-insensitiveH⁺ transport across TRC membranes.This is an example of cell monitoring of environmental pH through pHtracking, i.e., a linear change inpH_i in response to a change inpH_o, as has been proposed for carotid bodies. In taste, the H⁺transport sites may be concentrated on the basolateral membranes ofTRCs and, therefore, are responsive to an attenuatedH⁺ concentration from diffusion ofacids across the tight junctions.

     

Expression of amiloride-sensitive epithelial sodium channels in mouse taste cells after chorda tympani nerve crush

Our previous electrophysiological study demonstrated that amiloride-sensitive (AS) and -insensitive (AI) components of NaCl responses recovered differentially after the mouse chorda tympani (CT) was crushed. AI responses reappeared earlier (at 3 weeks after the nerve crush) than did AS ones (at 4 weeks). This and other results suggested that two salt-responsive systems were differentially and independently reformed after nerve crush. To investigate the molecular mechanisms of formation of the salt responsive systems, we examined expression patterns of three subunits (alpha, beta and gamma) of the amiloride-sensitive epithelial Na(+) channel (ENaC) in mouse taste cells after CT nerve crush by using in situ hybridization (ISH) analysis. The results showed that all three ENaC subunits, as well as alpha-gustducin, a marker of differentiated taste cells, were expressed in a subset of taste bud cells from an early stage (1-2 weeks) after nerve crush, although these taste buds were smaller and fewer in number than for control mice. At 3 weeks, the mean number of each ENaC subunit and alpha-gustducin mRNA-positive cells per taste bud reached the control level. Also, the size of taste buds became similar to those of the control mice at this time. Our previous electrophysiological study demonstrated that at 2 weeks no significant response of the nerve to chemical stimuli was observed. Thus ENaC subunits appear to be expressed prior to the reappearance of AI and AS neural responses after CT nerve crush. These results support the view that differentiation of taste cells into AS or AI cells is initiated prior to synapse formation.     

Regulation of intracellular pH in LLC-PK1 cells by Na+/H+ exchange

Summary Suspensions of LLC-PK₁ cells (a continuous epitheliod cell line with renal characteristics) are examined for mechanisms of intracellular pH regulation using the fluorescent probe BCECF. Initial experiments determine suitable calibration procedures for use of the BCECF fluorescent signal. They also determine that the cell suspension contains cells which (after 4 hr in suspension) have Na⁺ and K⁺ gradients comparable to those of cells in monolayer culture. The steady-state intracellular pH (7.05±0.01,n=5) of cells which have recovered in (pH 7.4) Na⁺-containing medium is not affected over several minutes by addition of 100 M amiloride or removal of extracellular Na⁺ (Na _o ⁺ /H _i ⁺ and Na _i ⁺ /H _o ⁺ exchange reactions are functionally inactive (compared to cellular buffering capacity). In contrast, Na _o ⁺ /H _i ⁺ exchange is activated by an increased cellular acid load. This activation may be observed directly either as a stimulation of net H⁺ efflux or net Na⁺ influx with decreasing intracellular pH. The extrapolation of this latter data suggests a set point of Na⁺/H⁺ exchange of approximately pH 7.0, consistent with the observed resting intracellular pH of approximately 7.05.     

Modulation of gating of a metabolically regulated,ATP-dependent K+ channel by intracellular pH in B cells of the pancreatic islet

Summary Membrane-permeant weak acids and bases, when applied to the bath, modulate the resting membrane potential and the glucose-induced electrical activity of pancreatic B cells, as well as their insulin secretion. These substances alter the activity of a metabolite-regulated. ATP-sensitive K⁺ channel which underlies the B-cell resting potential. We now present several lines of evidence indicating that the channel may be directly gated by pH _i . (1) The time course of K⁺(ATP) channel activity during exposure to and washout of NH₄Cl under a variety of experimental conditions, including alteration of the electrochemical gradient for NH₄Cl entry and inhibition of the Na _o ⁺ H _i ⁺ exchanger, resembles the time course of pH _i measured in other cell types that have been similarly treated. (2) Increasing pH _o over the range 6.25–7.9 increases K⁺(ATP) channel activity in cell-attached patches where the cell surface exposed to the bath has been permeabilized to H⁺ by the application of the K⁺/H⁺ exchanger nigericin. (3) Increasing pH _i over a similar range produces similar effects on K⁺(ATP) channels in inside-out excised patches exposed to small concentrations of ATP _i . The physiological role of pH _i in the metabolic gating of this channel remains to be explored.     

The candidate sour taste receptor, PKD2L1, is expressed by type III taste cells in the mouse

The transient receptor potential channel, PKD2L1, is reported to be a candidate receptor for sour taste based on molecular biological and functional studies. Here, we investigated the expression pattern of PKD2L1-immunoreactivity (IR) in taste buds of the mouse. PKD2L1-IR is present in a few elongate cells in each taste bud as reported previously. The PKD2L1-expressing cells are different from those expressing PLCbeta2, a marker of Type II cells. Likewise PKD2L1-immunoreactive taste cells do not express ecto-ATPase which marks Type I cells. The PKD2L1-positive cells are immunoreactive for neural cell adhesion molecule, serotonin, PGP-9.5 (ubiquitin carboxy-terminal transferase), and chromogranin A, all of which are present in Type III taste cells. At the ultrastructural level, PKD2L1-immunoreactive cells form synapses onto afferent nerve fibers, another feature of Type III taste cells. These results are consistent with the idea that different taste cells in each taste bud perform distinct functions. We suggest that Type III cells are necessary for transduction and/or transmission of information about "sour", but have little or no role in transmission of taste information of other taste qualities.     

Amiloride inhibition on NaCl responses of the chorda tympani nerve in two 129 substrains of mice, 129P3/J and 129X1/SvJ

Amiloride, a sodium channel blocker, is known to suppress NaCl responses of the chorda tympani (CT) nerve in various mammalian species. In mice, the NaCl suppressing effect of amiloride is reported to differ among strains. In C57BL mice, amiloride inhibits NaCl responses to about 50% of control, whereas no such clear suppression was evident in prior studies with 129 mice. However, evidence from behavioral studies is not entirely consistent with this. Recently, it has been found that genetic backgrounds of 129 mice differ within substrains. 129X1/SvJ (formerly 129/SvJ) mice differ from the 129P3/J (formerly 129/J) strain by 25% of sequence length polymorphisms. Therefore, we examined possible substrain difference between 129P3/J and 129X1/SvJ mice in the amiloride sensitivity of electrophysiologically recorded NaCl responses. Amiloride significantly suppressed CT responses to NaCl without affecting responses to KCl both in 129P3/J and 129X1/SvJ mice. However, the magnitude of the amiloride inhibition was significantly larger (approximately 50% of control in response to 0.01-1.0 M NaCl by 100 microM amiloride) in 129X1/SvJ than in 129P3/J mice (approximately 20% of control in response to 0.03-0.3 M NaCl by 100 microM amiloride). Threshold amiloride concentration for suppression of responses to 0.3 M NaCl was 30 microM in 129P3/J mice, which was higher than that in 129X1/SvJ mice (10 microM). In 129X1/SvJ mice, the threshold amiloride concentration eliciting inhibition of NaCl responses and the magnitude of the inhibition were comparable with those in C57BL/6 mice. These results suggest that amiloride sensitivity of NaCl responses differs even among the 129 substrains, 129P3/J and 129 X1/SvJ, and the substrain difference of 129 mice in amiloride sensitivity is as large as that between two inbred strains (129P3/J and C57BL/6).     

Modulation of apical Na permeability of the toad urinary bladder by intracellular Na,Ca, and H

Summary The Na conductance of the apical membrane of the toad urinary bladder was measured at different concentrations of Na both in the external medium and in the cell. Bladders were bathed in high K-sucrose medium to reduce basal-lateral resistance and voltage, and the transepithelial currents measured under voltage-clamp conditions. Amiloride was used as a specific blocker of the apical Na channel. At constant external Na, the internal Na concentration was increased by blocking the basallateral Na pump with ouabain. With high Na activity in the mucosal medium (86mm), increases in intracellular Na activity from 10 to over 40mm increased the amiloride-sensitive slope conductance at zero voltage while apical Na permeability, estimated from current-voltage plots using the constant field equation, decreased by less than 20%. Lowering the serosal Ca concentration from 1 to 0.1mm had no effect on the change inP _Na with increasing Na_c, but increasing serosal Ca to 5mm enhanced the reduction inP _Na with increasing Na _c , presumably by increasing Ca influx into the cell.P _Na was also reduced by serosal vanadate (0.5mm), a putative blocker of ATP-dependent Ca extrusion from the cell, and by acute exposure to CO₂, which presumably acidifies the cytoplasm. Current-voltage relationships of the amiloridesensitive transport pathway were also measured in the absence of a Na gradient across the apical membrane. These plots show that outward current passes through the channels somewhat less easily than does inward current. The shape of theI-V relationships was not significantly altered by changes in cellular Na, Ca or H, indicating that the effects of these ions onP _Na are voltage independent.     

H+ transport and the regulation of intracellular pH in ehrlich ascites tumor cells

Summary The intracellular pH (pH _i ) of Ehrlich ascites tumor cells, both in the steady state and under conditions of acid loading or recovery from acid loading, was investigated by measuring the transmembrane flux of H⁺ equivalents and correlating this with changes in the distribution ratio of dimethyloxazolidine-2,4-dione (DMO). The pH _i of cells placed in an acidic medium (pH _o below 7.15) decreases and reaches a steady-state value that is more alkaline than the outside. For example when pH _o is acutely reduced to 5.5, pH _i falls exponentially from 7.20 ± 0.06 to 6.29 ± 0.04 with a halftime of 5.92 ± 1.37 min, suggesting a rapid influx of H⁺. The unidirectional influx of H⁺ exhibits saturation kinetics with respect to extracellular [H⁺]; the maximal flux is 15.8 ± 0.05 mmol/(kg dry wt · min) andK _m is 0.74 ± 0.09 × 10^–6 m.Steady-state cells with pH _i above 6.8 continuously extrude H⁺ by a process that is not dependent on ATP but is inhibited by anaerobiosis. Acid-loaded cells (pH _i 6.3) when returned to pH _o 7.3 medium respond by transporting H⁺, resulting in a rapid rise in pH _i . The halftime for this process is 1.09 ± 0.22 min. The H⁺ efflux measured under similar conditions increases as the intracellular acid load increases. An ATP-independent as well as an ATP-dependent efflux contributes to the restoration of pH _i to its steady-state value.     

Differences in nucleotide effects on intracellular pH, Na+/H+ antiport activity, and ATP-binding proteins in endothelial cells

Summary Bovine (BPAEC) and human (HPAEC) pulmonary artery endothelial cell monolayers were incubated with either ATP, ATP analogues, or UTP, followed by measurement of intracellular pH (pHi) and the rate of recovery from acidosis. ATP increased baseline pHi and the rate of acid recovery in BPAEC. This response was inhibited by the amiloride analogue, methyisobutylamiloride, demonstrating that activation of the Na⁺/H⁺ antiport was responsible for the increase in baseline pHi and the recovery from acidosis. This response had the features of both a P_2Y and P_2U purinergic receptor, based on the responses to a series of ATP analogues and UTP. In contrast, none of the nucleotides had any significant effect on pHi and Na⁺/H⁺ antiport activity in HPAEC. This difference in the response to extracellular nucleotides was not due to a difference in ATP metabolism between cell types, since the ectonucleotidase-resistant analogue, ATPγS, also had no effect on HPAEC. Analogues of cAMP had no effect on pHi or acid recovery in either cell type. Incubation of BPAEC and HPAEC with the photoaffinity ligand [³²P] 8-AzATP indicated that both BPAEC and HPAEC possess an ATP-binding protein of 48 kDa. However, BPAEC exhibited an additional binding protein of 87 kDa. Thus, the contrasting response to extracellular ATP between bovine and human pulmonary artery endothelial cells may be related to differences in the signal transduction pathway leading to antiport activation, including different ATP-binding sites on the cell membrane.     

Flow cytometric analysis of intracellular pH in cultured opossum kidney (OK) cells

Summary Suspensions of OK cells (a continuous renal epithelial cell line originating from the opossum kidney) were examined by flow cytometry. Three parameters were evaluated simultaneously; cell integrity as assayed by propidium iodide fluorescence, cell size as measured by time-of-flight, and intracellular pH as measured by fluorescence of 2,7-bis-(2-carboxyethyl)-5,6 carboxyfluorescein (BCECF). The suspension was shown to be composed of both intact singlets and doublets of cells, and no difference was noted in the behavior of these two populations with respect to the resting intracellular pH, or of the response of intracellular BCECF to changes in pH. Evidence suggests that using NH₄ prepulses to create an acid load broadens the intracellular pH distribution. The population of OK cells demonstrates a recovery from this acid load which is very homogeneous with respect to its sensitivity to Na⁺ removal or EIPA (ethylisopropyl-amiloride), suggesting that virtually all cells utilize Na⁺/H⁺ exchange for this recovery. The data also suggest heterogeneity in the cellular pH recovery from an acid load with respect to the observed rates of Na⁺/H⁺ exchange. Despite this heterogeneity, the Na⁺/H⁺ exchanger is observed to focus the resting intracellular pH of the population to approximately pH 7.4–7.5. The response of the population to PTH suggests that the majority of cells respond to the hormone, and that the total Na⁺/H⁺ exchange in individual cells is only partially inhibited even in the presence of saturating PTH concentrations.     

Two members of the TRPP family of ion channels, Pkd1l3 and Pkd2l1, are co-expressed in a subset of taste receptor cells

Taste receptors cells are responsible for detecting a wide variety of chemical stimuli. Several molecules including both G protein coupled receptors and ion channels have been shown to be involved in the detection and transduction of tastants. We report on the expression of two members of the transient receptor potential (TRP) family of ion channels, PKD1L3 and PKD2L1, in taste receptor cells. Both of these channels belong to the larger polycystic kidney disease (PKD or TRPP) subfamily of TRP channels, members of which have been demonstrated to be non-selective cation channels and permeable to both Na(+) and Ca(2+). Pkd1l3 and Pkd2l1 are co-expressed in a select subset of taste receptor cells and therefore may, like other PKD channels, function as a heteromer. We found the taste receptor cells expressing Pkd1l3 and Pkd2l1 to be distinct from those that express components of sweet, bitter and umami signal transduction pathways. These results provide the first evidence for a role of TRPP channels in taste receptor cell function.     

Na+/K+-ATPase inhibition during cardiac myocyte swelling: Involvement of intracellular pH and Ca2+

Previous studies in chick embryo cardiac myocytes have shown that the inhibition of Na⁺/K⁺-ATPase with ouabain induces cell shrinkage in an isosmotic environment (290 mOsm). The same inhibition produces an enhanced RVD (regulatory volume decrease) in hyposmotic conditions (100 mOsm). It is also known that submitting chick embryo cardiomyocytes to a hyperosmotic solution induces shrinkage and a concurrent intracellular alkalization. The objective of this study was to evaluate the involvement of intracellular pH (pH_i), intracellular Ca²⁺ ([Ca²⁺]_i) and Na⁺/K⁺-ATPase inhibition during hyposmotic swelling. Changes in intracellular pH and Ca²⁺ were monitored using BCECF and fura-2, respectively. The addition of ouabain (100 M) under both isosmotic and hyposmotic stimuli resulted in a large increase in [Ca²⁺]_i (200%). A decrease in pH_i (from 7.3 ± 0.09 to 6.4 ± 0.08, n = 6; p < 0.05) was only observed when ouabain was applied during hyposmotic swelling. This acidification was prevented by the removal of extracellular Ca²⁺. Inhibition of Na⁺/H²⁺ exchange with amiloride (1 mM) had no effect on the ouabain-induced acidification. Preventing the mitochondrial accumulation of Ca²⁺ using CCCP (10 M) resulted in a blockade of the progressive acidification normally induced by ouabain. The inhibition of mitochondrial membrane K⁺/H⁺ exchange with DCCD (1 mM) also completely prevented the acidification. Our results suggest that intracellular acidification upon cell swelling is mediated by an initial Ca²⁺ influx via Na⁺/Ca²⁺ exchange, which under hyposmotic conditions activates the K⁺ and Ca²⁺ mitochondrial exchange systems (K⁺/H⁺ and Ca²⁺/H⁺).Deceased     

Growth responses and ion accumulation in the halophytic legume Prosopis strombulifera are determined by Na2SO4 and NaCl

Halophytes are potential gene sources for genetic manipulation of economically important crop species. This study addresses the physiological responses of a widespread halophyte, Prosopis strombulifera (Lam.) Benth to salinity. We hypothesised that increasing concentrations of the two major salts present in soils of central Argentina (Na₂SO₄, NaCl, or their iso‐osmotic mixture) would produce distinct physiological responses. We used hydroponically grown P. strombulifera to test this hypothesis, analysing growth parameters, water relations, photosynthetic pigments, cations and anions. These plants showed a halophytic response to NaCl, but strong general inhibition of growth in response to iso‐osmotic solutions containing Na₂SO₄. The explanation for the adaptive success of P. strombulifera in high NaCl conditions seems to be related to a delicate balance between Na ⁺ accumulation (and its use for osmotic adjustment) and efficient compartmentalisation in vacuoles, the ability of the whole plant to ensure sufficient K⁺ supply by maintaining high K ⁺ /Na ⁺ discrimination, and maintenance of normal Ca^{2 +} levels in leaves. The three salt treatments had different effects on the accumulation of ions. Findings in bi‐saline‐treated plants were of particular interest, where most of the physiological parameters studied showed partial alleviation of SO₄^2?‐induced toxicity by Cl^?. Thus, discussions on physiological responses to salinity could be further expanded in a way that more closely mimics natural salt environments.     

Functional Roles of Clusters of Hydrophobic and Polar Residues in the Epithelial Na+ Channel Knuckle Domain

The extracellular regions of epithelial Na⁺ channel subunits are highly ordered structures composed of domains formed by α helices and β strands. Deletion of the peripheral knuckle domain of the α subunit in the αβγ trimer results in channel activation, reflecting an increase in channel open probability due to a loss of the inhibitory effect of external Na⁺ (Na⁺ self-inhibition). In contrast, deletion of either the β or γ subunit knuckle domain within the αβγ trimer dramatically reduces epithelial Na⁺ channel function and surface expression, and impairs subunit maturation. We systematically mutated individual α subunit knuckle domain residues and assessed functional properties of these mutants. Cysteine substitutions at 14 of 28 residues significantly suppressed Na⁺ self-inhibition. The side chains of a cluster of these residues are non-polar and are predicted to be directed toward the palm domain, whereas a group of polar residues are predicted to orient their side chains toward the space between the knuckle and finger domains. Among the mutants causing the greatest suppression of Na⁺ self-inhibition were αP521C, αI529C, and αS534C. The introduction of Cys residues at homologous sites within either the β or γ subunit knuckle domain resulted in little or no change in Na⁺ self-inhibition. Our results suggest that multiple residues in the α subunit knuckle domain contribute to the mechanism of Na⁺ self-inhibition by interacting with palm and finger domain residues via two separate and chemically distinct motifs.     

Interactions of intracellular pH and intracellular calcium in primary cultures of rabbit corneal epithelial cells

Summary Homeostasis of intracellular calcium ([Ca⁺⁺]_i) and pH (pH_i) is important in the cell's ability to respond to growth factors, to initiate differentiation and proliferation, and to maintain normal metabolic pathways. Because of the importance of these ions to cellular functions, we investigated the effects of changes of [Ca⁺⁺]_i and pH_i on each other in primary cultures of rabbit corneal epithelial cells. Digitized fluorescence imaging was used to measure [Ca⁺⁺]_i with fura-2 and pH_i with 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Resting pH_i in these cells was 7.37±0.05 (n=20 cells) and resting [Ca⁺⁺]_i was 129±10 nM (n=35 cells) using a nominally bicarbonate-free Krebs Ringer HEPES buffer (KRHB), pH 7.4. On exposure to 20 mM NH₄Cl, which rapidly alkalinized cells by 0.45 pH units, an increase in [Ca⁺⁺]_i to 215±14 nM occurred. Pretreatment of the cells with 100 μM verapamil or exposure to 1 mM ethylene bis-(oxyethylenenitrilo)-tetraacetic acid (EGTA) without extracellular calcium before addition of 20 mM NH₄Cl did not abolish the calcium increase, suggesting that the source of the calcium transient was from intracellular calcium stores. On removal of NH₄Cl or addition of 20 mM sodium lactate, there were minimal changes in calcium even though pH_i decreased. Treatment of CE cells with the calcium ionophores, ionomycin and 4-bromo A23187, increased [Ca⁺⁺]_i, but produced a biphasic change in pH_i. Initially, there was an acidification of the cytosol, and then an alkalinization of 0.10 to 0.11 pH units above initial values. When [Ca⁺⁺]_i was decreased by treating the cells with 5 mM EGTA and 20 μM ionomycin, pH_i decreased by 0.35±0.02 units. We conclude that an increase in pH_i leads to an increase in [Ca⁺⁺]_i in rabbit corneal epithelial cells; however, a decrease in pH_i leads to minor changes in [Ca⁺⁺]_i. The ability of CE cells to maintain proper calcium homeostasis when pH_i is decreased may represent an adaptive mechanism to maintain physiological calcium levels during periods of acidification, which occur during prolonged eye closure.     

Acetylcholine-Induced Na+ influx in the mouse lacrimal gland acinar cells: Demonstration of multiple Na+ transport mechanisms by intracellular Na+ activity measurements

Summary In the isolated, superfused mouse lacrimal gland, intracellular Na⁺ activities (aNa _i ) of the acinar cells were directly measured with double-barreled Na⁺-selective microelectrodes. In the nonstimulated conditionaNa _i was 6.5±0.5 mM and membrane potential (V _m ) was –38.9±0.4 mV. Addition of 1 mM ouabain or superfusion with a K⁺-free solution slightly depolarized the membrane and caused a gradual increase inaNa _i . Stimulation with acetylcholine (ACh, 1 M) caused a membrane hyperpolarization by about 20 mV and an increase inaNa _i by about 9 mM in 5 min. The presence of amiloride (0.1 mM) reduced the ACh-induced increase inaNa _i by approximately 50%, without affectingV _m and input resistance in both nonstimulated and ACh-stimulated conditions. Acid loading the acinar cells by an addition/withdrawal of 20 mM NH₄Cl or by replacement of Tris⁺-buffer saline solution with HCO ₃ ^– /CO₂-buffered solution increasedaNa _i by a few mM. Superfusion with a Cl^–-free NO ₃ ^– solution or 1 mM furosemide or 0.5 mM bumetanide-containing solution had little effect on the restingaNa _i levels, however, it reduced the ACh-induced increase inaNa _i by about 30%. Elimination of metabolite anions (glutamate, fumarate and pyruvate) from the superfusate reduced both the restingaNa _i and the ACh-induced increase inaNa _i .The present results suggest the presence of multiple Na⁺ entry mechanisms activated by ACh, namely, Na⁺/H⁺ exchange, Na-K-Cl cotransport and organic substrate-coupled Na⁺ transport mechanisms.     

Na+/H+ exchanger NHE1 and NHE2 have opposite effects on migration velocity in rat gastric surface cells

Following superficial injury, neighbouring gastric epithelial cells close the wound by rapid cell migration, a process called epithelial restitution. Na⁺/H⁺ exchange (NHE) inhibitors interfere with restitution, but the role of the different NHE isoforms expressed in gastric pit cells has remained elusive. The role of the basolaterally expressed NHE1 (Slc9a1) and the presumably apically expressed NHE2 (Slc9a2) in epithelial restitution was investigated in the nontransformed rat gastric surface cell line RGM1. Migration velocity was assessed by loading the cells with the fluorescent dye DiR and following closure of an experimental wound over time. Since RGM1 cells expressed very low NHE2 mRNA and have low transport activity, NHE2 was introduced by lentiviral gene transfer. In medium with pH 7.4, RGM1 cells displayed slow wound healing even in the absence of growth factors and independently of NHE activity. Growth factors accelerated wound healing in a partly NHE1‐dependent fashion. Preincubation with acidic pH 7.1 stimulated restitution in a NHE1‐dependent fashion. When pH 7.1 was maintained during the restitution period, migratory speed was reduced to ～10% of the speed at pH 7,4, and the residual restitution was further inhibited by NHE1 inhibition. Lentiviral NHE2 expression increased the steady‐state pH_i and reduced the restitution velocity after low pH preincubation, which was reversible by pharmacological NHE2 inhibition. The results demonstrate that in RGM1 cells, migratory velocity is increased by NHE1 activation, while NHE2 activity inhibit this process. A differential activation of NHE1 and NHE2 may therefore, play a role in the initiation and completion of the epithelial restitution process.     

Effects of voltage perturbation of the lingual receptive field on chorda tympani responses to Na+ and K+ salts in the rat: implications for gustatory transduction

Taste sensory responses from the chorda tympani nerve of the rat were recorded with the lingual receptive field under current or voltage clamp. Consistent with previous results (Ye, Q., G. L. Heck, and J. A. DeSimone. 1993. Journal of Neurophysiology. 70:167-178), responses to NaCl were highly sensitive to lingual voltage clamp condition. This can be attributed to changes in the electrochemical driving force for Na+ ions through apical membrane transducer channels in taste cells. In contrast, responses to KCl over the concentration range 50-500 mM were insensitive to the voltage clamp condition of the receptive field. These results indicate the absence of K+ conductances comparable to those for Na+ in the apical membranes of taste cells. This was supported by the strong anion dependence of K salt responses. At zero current clamp, the potassium gluconate (KGlu) threshold was > 250 mM, and onset kinetics were slow (12 s to reach half-maximal response). Faster onset kinetics and larger responses to KGlu occurred at negative voltage clamp (-50 mV). This indicates that when K+ ion is transported as a current, and thereby uncoupled from gluconate mobility, its rate of delivery to the K+ taste transducer increases. Analysis of conductances shows that the paracellular pathway in the lingual epithelium is 28 times more permeable to KCl than to KGlu. Responses to KGlu under negative voltage clamp were not affected by agents that are K+ channel blockers in other systems. The results indicate that K salt taste transduction is under paracellular diffusion control, which limits chemoreception efficiency. We conclude that rat K salt taste occurs by means of a subtight junctional transducer for K+ ions with access limited by anion mobility. The data suggest that this transducer is not cation selective which also accounts for the voltage and amiloride insensitive part of the response to NaCl.     

The fusicoccin-stimulated phosphorylation of a 33 KDa polypeptide in cells of Acer pseudoplatanus as influenced by extracellular and intracellular pH

Abstract In a previous study, it was shown that the fungal toxin fusicoccin (FC) is able to stimulate the in vivo phosphorylation of a 33 KDalton polypeptide (33 KP) independently of protein synthesis. Here we show that the stimulation by FC of both proton efflux and 33 KP phosphorylation are strongly enhanced when the external medium contains K⁺ or Na⁺, suggesting that the two phenomena are related. The stimulatory effect of FC is higher in unbuffered than in buffered media; moreover, in the absence of FC, 33 KP is more phosphorylated at an acidic than at a basic pH of the medium, suggesting that the effect of FC may depend, to a certain extent, on the acidification of the free space caused by FC-promoted proton efflux. Treatments reported to alter the intracellular pH influence 33 KP phosphorylation even more strongly than the external pH does. The acidifying agents isobutyric acid and trimethylacetic acid decrease 33 KP phosphorylation, while the alkalinizing agents, ammonia and procaine, increase it. FC partially counteracts the inhibition by the weak acids, whereas the stimulatory effect of FC is not additive with that of the weak bases. The results indicate that 33 KP phosphorylation senses both the external and internal pH. The stimulatory effect of cytoplasm-alkalinizing treatments, which mimics that of FC, agrees with the reported capacity of FC to cause cytoplasmic alkalinization, following activation of the plasmalemma proton pump.     

Modulation of cytosolic and nuclear Ca2+ and Na+ transport by taurine in heart cells

Brain membranes contain tubulin that can be isolated as a hydrophobic compound by partitioning into Triton X-114. We have previously postulated: (a) that this kind of tubulin is a peripheral membrane protein that arises from microtubules that in vivo interact with membranes and (b) that the hydrophobic behaviour is due to the interaction of tubulin with a membrane component. Here we report the in vitro conversion of hydrophilic into hydrophobic tubulin by incubating microtubule associated proteins (MAPs) free taxol-stabilized microtubules with Triton X-100 solubilized membranes. After incubation, the microtubules were sedimented, depolymerized and subjected to partition into Triton X-114. Part of the tubulin was isolated in the detergent phase and contained, as observed in native membranes, a high proportion of the acetylated isotype. Because of the high proportion of acetylated tubulin the in vitro conversion resembles the in vivo interaction. Electrophoretic analysis of the detergent phase shows, besides tubulin, two major protein bands of 29 and 100 kDa molecular mass. The ability of the solubilized membranes to convert hydrophilic into hydrophobic tubulin is greatly diminished if the solubilized membrane preparation is preincubated in the presence of trypsin or heated at 90°C for 5 min, indicating that the membrane component that confers the hydrophobic behaviour to tubulin is of proteinaceous nature.