Terutaka Ozawa

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.

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.

Modulation of ryanodine receptor Ca2+ channels (Review).

Ryanodine-sensitive Ca2+ release channels (ryanodine receptors, RyRs) play a crucial role in the mobilization of Ca2+ from the sarcoplasmic reticulum (SR) during the excitation-contraction coupling of muscle cells. In skeletal muscle, depolarization of transverse tubules activates the RyR, whereas in cardiac muscle, a Ca2+ influx through an L-type Ca2+ channel activates the RyR. The RyR is also activated by caffeine, a low concentration (<10 µM) of ryanodine or cyclic ADP-ribose. RyR activity is inhibited by Mg2+, ruthenium red, or higher concentrations (≥100 µM) of ryanodine. The activity of RyR channels is modulated by phosphorylation and by associated proteins, including calmodulin (CaM), calsequestrin (CSQ) and FK506-binding proteins (FKBPs). In muscle cells, apoCaM (Ca2+-free CaM) activates the RyR channel, and Ca2+ CaM (Ca2+-bound CaM) inhibits the channel. CSQ can bind approximately 40 moles of Ca2+/mole of CSQ in the SR lumen of muscle cells, and interacts functionally with RyR protein. When the RyR is stimulated, Ca2+ released from the lumen is dissociated from the CSQ- Ca2+ complex. A 12-kDa or 12.6-kDa FK506-binding protein (FKBP12 or FKBP12.6, respectively) is associated with RyR protein. When FKBP12 or FKBP12.6 is dissociated from the FKBP-RyR complex, the RyR is modulated (activated). Phosphorylation of the RyR by cAMP-dependent protein kinase (PKA) and Ca2+/calmodulin-dependent protein kinase II modulates the channel. PKA phosphorylation of the RyR on the skeletal and cardiac muscle SR dissociates FKBP12 or FKBP12.6 from the RyR complex. This review deals with the modulation mechanisms of RyR proteins by associated proteins and phosphorylation.

Mechanism of uphill chloride transport of the mouse lacrimal acinar cells: studies with Cl−-sensitive microelectrode

The mechanism of uphill Cl− accumulation by mouse lacrimal acinar cells was studied using double-barrelled Cl−-selective microelectrodes. When measured in standard tris-buffered saline solution, the membrane potential (Vm) was −39.2±0.4 mV and intracellular Cl− activity (ACli) was 34.6±0.7 mmol/l which was 1.4 times higher than the equilibrium level. In Na+-free solution,ACli decreased from 34 mmol/l to 19 mmol/l in 100 min, a level that was close to the equilibrium activity. Return to the standard solution restored the normal level ofACli in 5 min. In the presence of furosemide (1 mmol/l), Cl− uptake induced by Na+-readmission was inhibited by 44%. Superfusion with a K+-free solution gradually decreasedACli until it was close to the equilibrium level after 75 min; superfusion with a high-K+ (29.5 mmol/l) solution increasedACli significantly. In the presence of ouabain (1 mmol/l), switching the superfusing solutions from K+-free to high-K+ and from high-K+ to K+-free at timed intervals of 15 min caused, respectively, an increase (+9 mmol/l) and a decrease (−7 mmol/l) inACli. These changes inACli were inhibited by furosemide respectively by 61% and 24%. In the presence of furosemide, DIDS (1 mmol/l) or furosemide plus DIDS, the initial rate of Cl− uptake after cessation of acetylcholine (ACh 1 μmol/l) stimulation was inhibited by 47%, 37% or 74%, respectively. Present results show that the characteristics of the uphill chloride uptake by the mouse lacrimal acinar cells are consistent with those of Na+−K+−Cl− cotransport. The additional inhibitory effect of DIDS to furosemide inhibition suggests an involvement of anion exchange transport, in parallel with the cotransport, in uphill Cl− uptake into the cells.

Ryanodine-sensitive Ca2+ release mechanism of rat pancreatic acinar cells is modulated by calmodulin

The effects of calmodulin (CaM) and CaM antagonists on microsomal Ca(2+) release through a ryanodine-sensitive mechanism were investigated in rat pancreatic acinar cells. When caffeine (10 mM) was added after a steady state of ATP-dependent (45)Ca(2+) uptake into the microsomal vesicles, the caffeine-induced (45)Ca(2+) release was significantly increased by pretreatment with ryanodine (10 microM). The presence of W-7 (60 microM), a potent inhibitor of CaM, strongly inhibited the release, while W-5 (60 microM), an inactive CaM antagonist, showed no inhibition. Inhibition of the release by W-7 was observed at all caffeine concentrations (5-30 mM) tested. The presence of exogenously added CaM (10 microg/ml) markedly increased the caffeine (5-10 mM)-induced (45)Ca(2+) release and shifted the dose-response curve of caffeine-induced (45)Ca(2+) release to the left. Cyclic ADP-ribose (cADPR, 2 microM)-induced (45)Ca(2+) release was enhanced by the presence of ryanodine (10 microM). cADPR (2 microM)- or ryanodine (500 microM)-induced (45)Ca(2+) release was also inhibited by W-7 (60 microM), but not by W-5 (60 microM), and was stimulated by CaM (10 microg/ml). These results suggest that the ryanodine-sensitive Ca(2+) release mechanism of rat pancreatic acinar cells is modulated by CaM.

Solvent dependence of gel swelling in the system of merckogel OR-2000 and various organic solvents

Abstract The amount of internal solvent in Merckogel OR-2000 has been determined for ten different solvents using polystyrene (mol. wt., 10,000) as the excluded solute. In this method, dry gel is equilibrated with an organic solution containing a known concentration of a solute which is excluded by the gel. The internal solvent volume of the swollen gel is calculated from the concentration change due to mixing. There are distinct relations between the amount of internal solvent, a solubility parameter and the molar volume of the solvent.

Effects of intra- and extracellular H+ and Na+ concentrations on Na+-H+ antiport activity in the lacrimal gland acinar cells

Kinetic properties of the Na+-H+ antiport in the acinar cells of the isolated, superfused mouse lacrimal gland were studied by measuring intracellular pH (pHi) and Na+ activity (aNai) with the aid of double-barreled H+- and Na+-selective microelectrodes, respectively. Bicarbonate-free solutions were used throughout. Under untreated control conditions, pHi was 7.12±0.01 and aNai was 6.7±0.6 mmol/l. The cells were acid-loaded by exposure to an NH4+solution followed by an Na+-free N-methyl-d-glucamine (NMDG+) solution. Intracellular Na+ and H+ concentrations were manipulated by changing the duration of exposure to the above solutions. Subsequent addition of the standard Na+ solution rapidly increased pHi. This Na+-induced increase in pHi was almost completely inhibited by 0.5 mmol/l amiloride and was associated with a rapid, amiloride-sensitive increase in aNai. The rate of pHi recovery induced by the standard Na+ solution increased in a saturable manner as pHi decreased, and was negligible at pHi 7.2–7.3, indicating an inactivation of the Na+-H+ antiport. The apparent Km for intracellular H+ concentration was 105 nmol/l (pH 6.98). The rate of acid extrusion from the acid-loaded cells increased proportionally to the increase in extracellular pH. Depletion of aNai to less than 1 mmol/l by prolonged exposure to NMDG+ solution significantly increased the rate of Na+-dependent acid extrusion. The rate of acid extrusion increased as the extracellular Na+ concentration increased following Michaelis-Menten kinetics (Vmax was 0.55 pH/min and the apparent Km was 75 mmol/l at pHi 6.88). The results clearly showed that the Na+-H+ antiport activity is dependent on the chemical potential gradient of both Na+ and H+ ions across the basolateral membrane, and that the antiporter is asymmetric with respect to the substrate affinity of the transport site. The data agree with the current model of activation and inactivation of the antiporter by an intracellular site through changes in the intracellular Na+ and H+ concentrations.