Akinori Nishiyama

Na(+)-Ca2+ exchange in the isolated cochlear outer hair cells of the guinea-pig studied by fluorescence image microscopy.

The outer hair cell isolated from the guinea-pig was superfused in vitro and the cytosolic calcium concentration ([Ca2+]i) and sodium concentration ([Na+]i) were measured using fluorescence indicators. Under the resting condition, [Ca2+]i and [Na+]i were 91±9 nM (n = 51) and 110±5 mM (n = 12), respectively. Removal of external Na+ by replacing with N-methyl-D-glucamine (NMDG+) increased [Ca2+]i by 270±79% (n = 27) and decreased [Na+]i by 23±4 mM (n = 6). Both changes in [Ca2+]i and [Na+]i were totally reversible on returning external Na+ to the initial value and were inhibited by addition of 0.1 mM La3+ or 100 μM amiloride 5-(N,N-dimethyl) hydrochloride. Elevation of external Ca2+ ions to 20 mM reversibly decreased [Na+]i by 8±6 mM (n = 5). Moreover, the chelation of the intracellular Ca2+ with 1,2-bis (2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA) exerted an inhibitory action on the NMDG+-induced reduction in [Na+]i. Exposure to 5 mM NaCN for 2 min significantly and reversibly increased [Ca2+]i by 290±37% (n = 5), but did not affect the [Ca2+]i elevation induced by the NMDG+ solution. The rise in [Ca2+]i induced by the NMDG+ solution was not enhanced by ouabain pretreatment. Addition of ouabain did not alter the [Na+]i. The present results are best explained by the presence of an Na+-Ca2+ exchanger in cell membrane and indicate that the activity of Na+/K+ pump is poor in outer hair cells.

Characterization of ryanodine-sensitive Ca2+ release from microsomal vesicles of rat parotid acinar cells: regulation by cyclic ADP-ribose.

Abstract. We have measured ryanodine (caffeine)-sensitive 45Ca2+ release from isolated microsomal vesicles of endoplasmic reticulum prepared from rat parotid acinar cells. After a steady state of ATP-dependent 45Ca2+ uptake, the addition of caffeine (40 mm), ryanodine (10∼500 μm) or an NAD+ metabolite, cyclic ADP-ribose (cADPR, 4 μm) released about 10% of the 45Ca2+ that had been taken up. The 45Ca2+ release was not inhibited by heparin, an antagonist of IP3 receptor. The effects of caffeine, ryanodine and cADPR on 45Ca2+ release were also tested in the presence of thapsigargin (TG), an inhibitor of microsomal Ca2+-ATPase. When caffeine (10∼40 mm), ryanodine (10 μm) or cADPR (1∼10 μm) was added in the medium with 100 nm TG, a significant 45Ca2+ release was seen, while higher concentrations of ryanodine (>100 μm) did not cause any 45Ca2+ release in the presence of TG. The initial rate of caffeine (40 mm)-induced 45Ca2+ release was increased by a pretreatment with 10 μm ryanodine, whereas the caffeine-induced 45Ca2+ release was strongly inhibited by the presence of a higher concentration (500 μm) of ryanodine. cADPR-induced 45Ca2+ release was also inhibited by 500 μm ryanodine. Caffeine (40 mm)- or cADPR (4 μm)-induced 45Ca2+ release was abolished by a presence of ruthenium red (50∼100 μm). The presence of a low concentration (0.5 μm) of cADPR shifted the dose-response curve of caffeine-induced 45Ca2+ release to the left. These results indicate the presence of a ryanodine sensitive Ca2+ release mechanism in the endoplasmic reticulum of rat parotid acinar cells that is distinct from the IP3-sensitive Ca2+ channel and is activated by caffeine, cADPR and a low concentration (10 μm) of ryanodine, but is inhibited by higher concentrations (>100 μm) of ryanodine and ruthenium red. The properties of the ryanodine-sensitive mechanism are similar to that of the ryanodine receptor as described in muscle cells.

Acetylcholine-induced change in intracellular Cl− activity of the mouse lacrimal acinar cells

Using double-barreled Cl−-sensitive microelectrodes, intracellular Cl− activity (ACli) in the mouse lacrimal acinar cells in vitro was determined in both resting and secretory phases. In the resting stateACli was 31 mmol/l which was 1.4 times higher than that predicted for the passive distribution according to the membrane potential (Vm) of −41 mV. Addition of acetylcholine (ACh, 1μM) hyperpolarizedVm to −63 mV and decreasedACli to 20 mmol/l which was still twice the equilibrium activity. A-23178 produced similar changes inVm andACli to those induced by ACh. It was concluded that Cl− was actively accumulated in the acinar cells and, in the secretory phase, Cl− efflux was enhanced by the increased driving force and Ca2+-mediated increase in the Cl− permeability across the cell membrane.

Effect of Neuroregulators on the Intracellular Calcium Level in the Outer Hair Cell Isolated from the Guinea Pig

The cytosolic calcium concentration [( Ca2+]i) of the isolated outer hair cell of the guinea pig was measured using fluorescence imaging microscopy and the effects of efferent neuroregulators such as acetylcholine, ATP, GABA, substance P, enkephalin, calcitonin gene-related peptide, serotonin, dopamine, norepinephrine, and glutamate were investigated. Among the drugs tested only ATP induced an elevation of the [Ca2+]i of the outer hair cell. In the resting condition, [Ca2+]i averaged 104.5 +/- 31.1 nM (n = 27), while 100 microM ATP significantly increased [Ca2+]i to 146.3 +/- 43.5 nM (n = 19). Superfusion with Ca2(+)-free solution (pCa = 7.5) abolished the increase in [Ca2+]i induced by ATP, suggesting that ATP causes an entry of external Ca2+. The relevance of [Ca2+]i to the inhibitory actions of efferent neuroregulators is discussed.

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

SummaryIn the isolated, superfused mouse lacrimal gland, intracellular Na+ activities (aNai) of the acinar cells were directly measured with double-barreled Na+-selective microelectrodes. In the nonstimulated conditionaNai was 6.5±0.5 mM and membrane potential (Vm) 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 inaNai. Stimulation with acetylcholine (ACh, 1 μM) caused a membrane hyperpolarization by about 20 mV and an increase inaNai by about 9 mM in 5 min. The presence of amiloride (0.1 mM) reduced the ACh-induced increase inaNai by approximately 50%, without affectingVm 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 HCO3−/CO2-buffered solution increasedaNai by a few mM. Superfusion with a Cl−-free NO3− solution or 1 mM furosemide or 0.5 mM bumetanide-containing solution had little effect on the restingaNai levels, however, it reduced the ACh-induced increase inaNai by about 30%. Elimination of metabolite anions (glutamate, fumarate and pyruvate) from the superfusate reduced both the restingaNai and the ACh-induced increase inaNai.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.

Intracellular pH regulation in the mouse lacrimal gland acinar cells

SummaryIntracellular pH (pHi) of the acinar cells of the isolated, superfused mouse lacrimal gland has been measured using pH-sensitive microelectrodes. Under nonstimulated condition pHi was 7.25, which was about 0.5 unit higher than the equilibrium pH. Alterations of the external pH by ±0.4 unit shifted pHi only by ±0.08 unit. The intracellular buffering value determined by applications of 25mm NH4+ and bicarbonate buffer solution gassed with 5% CO2/95% O2 was 26 and 46mm/pH, respectively Stimulation with 1 μm acetylcholine (ACh) caused a transient, small decrease and then a sustained increase in pHi. In the presence of amiloride (0.1mm) or the absence of Na+, application of ACh caused a significant decrease in pHi and removal of amiloride or replacement with Na+-containing saline, respectively, rapidly increased the pHi. Pretreatment with DIDS (0.2mm) did not change the pHi of the nonstimulated conditions; however, it significantly enhanced the increase in pHi induced by ACh. The present results showed that (i) there is an active acid extrusion mechanism that is stimulated by ACh; (ii) stimulation with ACh enhances the rate of acid production in the acinar cells; and (iii) the acid extrusion mechanism is inhibited by amiloride addition to and Na+ removal from the bath solution. We suggest that both Na+/H+ and HCO3−/Cl− exchange transport mechanisms are taking roles in the intracellular pH regulation in the lacrimal gland acinar cells.

The effect of acetylcholine on chloride transport across the mouse lacrimal gland acinar cell membranes

The mechanisms of Cl− transport and the effects of acetylcholine (ACh) and electrochemical Cl− potential changes across the basolateral plasma membrane on intracellular Cl− activity in the acinar cells of isolated mouse lacrimal glands were studied using double-barreled Cl−-selective microelectrodes. In the resting state, the basolateral membrane potential (Vm) was about −40 mV and intracellular Cl− activity was about 35 mmol/l. Addition of ACh (10−9∼10−6 mol/l) hyperpolarizedVm and decreased the Cl− activity in a dose-dependent manner. ACh (10−6 mol/l) hyperpolarizedVm by 20 mV and decreased the cytosolic Cl− activity with an initial rate of 16.0 mmol/l · min. Reduction of the perfusate Cl− concentration to 1/9 control depolarizedVm and decreased cytosolic Cl− activity at a rate of 1.9 mmol/l · min. AVm hyperpolarization of 20 mV produced by DC injection to the adjacent cell decreased Cl− activity at a rate of 4.6 mmol/l · min. DIDS (1 mmol/l) hyperpolarizedVm by 8 mV with little change in Cl− activity and increased the input resistance of the cells by 25%. DIDS decreased the rate of change in Cl− activity induced by low-Cl− Ringer to 35% of control, but had no effect on the ACh-evoked decrease in the Cl− activity. Furosemide (1 mmol/l) slightly hyperpolarizedVm and decreased Cl− activity at a slow rate but affected Cl− movements induced by ACh or low-Cl− Ringer only slightly. Cl− uptake into the cells was inhibited partially by furosemide. The present results showed that ACh induces an increase in the Cl− permeability across the luminal plasma membrane and that the basolateral membrane possesses a DIDS-sensitive Cl− conductance pathway and a furosemide-sensitive Cl− uptake mechanism.

Ensemble noise and current relaxation analysis of K+ current in single isolated salivary acinar cells from rat.

The K+ channel in rat parotid gland acinar cells were investigated by ensemble current noise analysis in single isolated cells employing the giga-seal whole cell current recording mode. Sets of 20–40 identical de- and hyperpolarization voltage steps were applied and the resultant current records were processed by computer to obtain the mean and the variance of the current. The time-course of the mean current could be fitted by the sum of two exponentials, suggesting a 3-state model. The simplest plausible hypothesis is a model with one open and two closed states. Assuming this model, the relationship between the variance (σ2) and the mean current (I) could be fitted by the function σ2/I=i−I/N. The estimated single channeli/V-relations were similar to those taken from single channel current recordings, and the size of the population of channels per cell (N) was 76±26 (n=12). The validity of the model was tested by a successful simulation of the time-course of the variance.

Membrane potential measurement in isolated outer hair cells of the guinea pig cochlea using conventional microelectrodes

Membrane potential of the isolated outer hair cells (OHCs) from the guinea pig cochlea was measured using conventional microelectrodes filled with 200 mM KCl. The resting membrane potential during superfusion with the standard physiological saline solution containing 3.5 mM K+ was -47.3 +/- 1.4 mV (N = 72), which was higher than those previously reported for isolated OHCs studied by using microelectrodes. Addition of ouabain (10(-5)-10(-3) M), the specific Na+, K+ ATPase inhibitor, depolarized the cell slowly and progressively, indicating the presence of low but definite Na+, K+ ATPase activity in the plasma membrane of OHCs. The magnitude of membrane potential was mainly dependent on the extracellular K+ concentration ([K+]O). A ten-fold increase of [K+]O depolarized the membrane potential by 49.6 +/- 1.0 mV (N = 58). A decrease of [Na+]O to one tenth of the control hyperpolarized the membrane potential by about 2 mV. Decreasing extracellular Cl- from 131.3 mM to 27.5 mM did not cause a significant change in the membrane potential. Using the Goldman-Hodgkin-Katz equation, assuming a negligible contribution of Cl- to the membrane potential and total monovalent cat ion concentration of the cytosol similar to the extracellular fluid, we calculated the permeability ratio of K+ versus Na+ to 131 +/- 19 and intracellular K+ concentration to 33.3 +/- 1.9 mM.

Microfluorimetric imaging study of the mechanism of activation of the Na+/H+ antiport by muscarinic agonist in rat mandibular acinar cells

The mechanism of regulation of intracellular pH (pHi) in dispersed acini from the rat mandibular salivary gland has been studied with a microfluorimetric imaging method and the pH probe 2′,7′-bis(2-carboxyethyl)-5(and −6)-carboxyfluorescein. The pHi in the TRIS/HEPES-buffered standard solution was 7.29±0.01. Addition of 1 μmol/l acetylcholine (ACh) or ionomycin caused a sustained increase in the pHi. These agents decreased pHi in the absence of external Na+ or in the presence of amiloride. The rate of pHi recovery from an acid load after NH4+prepulse was a linear function of pHi and increased as pHi became more acidic. Addition of ACh shifted the relationship towards a more alkaline pHi range. The increase in pHi induced by ACh or ionomycin was not inhibited by the protein kinase C inhibitors staurosporine (10 nM) and 1-(5-isoquinolinesulfonyl)-1-methylpiperazine (50 μmol/l). Addition of 0.1–1 μmol/l phorbol 12-myristate 13-acetate (TPA) had little effect on pHi within 10 min; however, exposure to TPA for 120 min resulted in a significant rise in pHi. In Ca2+-free solution with 50 μmol/l 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate, the ACh-induced rise in both pHi and cytosolic Ca2+ concentration was suppressed. ACh and ionomycin caused an increment of amiloride-sensitive acid output into the extracellular fluid, while 20 μmol/l 1-oleoyl-2-acetylglycerol had little effect on it. It was concluded that (a) stimulation with ACh activated the Na+/H+ antiport in the plasma membrane, (b) ACh also stimulated the intracellular acid production but acid extrusion by the Na+/H+ antiport prevented the cell from intracellular acidification, and (c) the major route of signal transduction for the ACh-induced activation of the Na+/H+ antiport was independent of protein kinase C but was dependent on the rise in cytosolic Ca2+ concentration. The implication of the cytosolic acidification and cell volume change in pHi regulation is discussed.