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ABSTRACT: Ionomycin (IM) at 5μM mediates the Ca2+/H+ exchange, while IM at 1μM activates the store-operated Ca2+ entry channels (SOCs). In this study, the effects of depolarization on both pathways were examined in rat submandibular acinar
cells by increasing extracellular K+ concentration ([K+]o). IM (5μM, the Ca2+/H+ exchange) increased the intracellular Ca2+ concentration ([Ca2+]i) to an extremely high value at 151mM [K+]o. However, with increasing [K+]o, the rates of Ca2+ entry decreased in a linear relationship. The reversal potential (E
rev) for the Ca2+/H+ exchange was +93mV, suggesting that IM (5μM) exchanges 1 Ca2+ for 1 H+. Thus, depolarization decreases the Ca2+ influx via the Ca2+/H+ exchange because of its electrogenicity (1 Ca2+ for 1 H+). On the other hand, IM (1μM, the SOCs) abolished an increase in [Ca2+]i at 151mM [K+]o. With increasing [K+]o, the rate of Ca2+ entry immediately decreased linearly. The E
rev for the SOC was +3.7mV, suggesting that the SOCs are nonselective cation channels and less selective for Ca2+ over Na+ (P
Ca/P
Na=8.2). Moreover, an increase in extracellular Ca2+ concentration (20mM) enhanced the Ca2+ entry via the SOCs at 151mM [K+]o, suggesting depolarization does not inhibit the SOCs and decreases the driving force for the Ca2+ entry. This suggests that membrane potential changes induced by a secretory stimulation finely regulate the [Ca2+]i via the SOCs in rat submandibular acinar cells. In conclusion, IM increases [Ca2+]i via two pathways depending on its concentration, the exchange of 1 Ca2+ for 1 H+ at 5μM and the SOCs at 1μM.
KeywordsIonomycin-Thapsigargin-Gd3+
-Intracellular Ca2+ concentration-Store-operated Ca2+ entry
The Journal of Physiological Sciences 04/2012; 60(5):363-371. · 1.61 Impact Factor
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ABSTRACT: Ionomycin (IM) at 5 microM mediates the Ca(2+)/H(+) exchange, while IM at 1 microM activates the store-operated Ca(2+) entry channels (SOCs). In this study, the effects of depolarization on both pathways were examined in rat submandibular acinar cells by increasing extracellular K(+) concentration ([K(+)](o)). IM (5 microM, the Ca(2+)/H(+) exchange) increased the intracellular Ca(2+) concentration ([Ca(2+)](i)) to an extremely high value at 151 mM [K(+)](o). However, with increasing [K(+)](o), the rates of Ca(2+) entry decreased in a linear relationship. The reversal potential (E (rev)) for the Ca(2+)/H(+) exchange was +93 mV, suggesting that IM (5 microM) exchanges 1 Ca(2+) for 1 H(+). Thus, depolarization decreases the Ca(2+) influx via the Ca(2+)/H(+) exchange because of its electrogenicity (1 Ca(2+) for 1 H(+)). On the other hand, IM (1 microM, the SOCs) abolished an increase in [Ca(2+)](i) at 151 mM [K(+)](o). With increasing [K(+)](o), the rate of Ca(2+) entry immediately decreased linearly. The E (rev) for the SOC was +3.7 mV, suggesting that the SOCs are nonselective cation channels and less selective for Ca(2+) over Na(+) (P (Ca)/P (Na) = 8.2). Moreover, an increase in extracellular Ca(2+) concentration (20 mM) enhanced the Ca(2+) entry via the SOCs at 151 mM [K(+)](o), suggesting depolarization does not inhibit the SOCs and decreases the driving force for the Ca(2+) entry. This suggests that membrane potential changes induced by a secretory stimulation finely regulate the [Ca(2+)](i) via the SOCs in rat submandibular acinar cells. In conclusion, IM increases [Ca(2+)](i) via two pathways depending on its concentration, the exchange of 1 Ca(2+) for 1 H(+) at 5 muM and the SOCs at 1 microM.
The Journal of Physiological Sciences 09/2010; 60(5):363-71. · 1.61 Impact Factor
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ABSTRACT: Ionomycin (IM, 5 microM), which exchanges 1 Ca2+ for 1 H+, changed intracellular pH (pHi) with Ca2+ entry into rat submandibular acinar cells. IM-induced changes in pHi consisted of two components: the first is an HCO3--dependent transient pHi decrease, and the second is an HCO3--independent gradual pHi increase. IM (1 microM), which activates store-operated Ca2+ channels, induced an HCO3--dependent and transient pHi decrease without any HCO3--independent pHi increase. Thus, a gradual pHi increase was induced by the Ca2+/H+ exchange. The HCO3--dependent and transient pHi decrease induced by IM was abolished by acetazolamide, but not by methyl isobutyl amiloride (MIA) or diisothiocyanatostilbene disulfonate (DIDS), suggesting that the Na+/H+ exchange, the Cl-/HCO3- exchange, or the Na+-HCO3- cotransport induces no transient pHi decrease. Thapsigargin induced no transient pHi decrease. Thus, IM, not Ca2+ entry, reduced pHi transiently. IM reacts with Ca2+ to produce H+ in the presence of CO2/HCO3-: [H-IM]-+Ca2++CO2<-->{H-Ca-IM]+.HCO3-+H+. In this reaction, a monoprotonated IM reacts with Ca2+ and CO2 to produce an electroneutral IM complex and H+, and then H+ is removed from the cells via CO2 production. Thus, IM transiently decreased pHi. In conclusion, in rat submandibular acinar cells IM (5 microM) transiently reduces pHi because of its chemical characteristics, with HCO3- dependence, and increases pHi by exchanging Ca2+ for H+, which is independent of HCO3-.
The Journal of Physiological Sciences 07/2010; 60(4):273-82. · 1.61 Impact Factor
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ABSTRACT: Using patch-clamp technique, we studied the role of the Ca2+/calmodulin kinase II (CaMK II)-mediated phosphorylation process on the K+ channel with an inward conductance of 90 pS in opossum kidney proximal tubule cells (OKPCs). The intracellular Ca2+ concentration ([Ca]i) was measured by use of the fluorescent dye fura 2. The following results were obtained: (i) In cell-attached patches, the channel activity was inhibited by a decrease in [Ca]i induced by perfusion with low Ca2+ (10(-8) M), La3+ (100 microM), or EGTA/AM (100 microM) contained in the bath solution. The application of KN-62 (10 microM) or KN-93 (5 microM), inhibitors of CaMK II, also inhibited the channel activity. (ii) The membrane potential measured with nystatin-perforated patches was significantly decreased by the fall in [Ca]i induced by the perfusion with EGTA- or La(3+)-containing solution. Also, the application of KN-62 (10 microM) or KN-93 (5 microM) to the bath significantly decreased the membrane potential. (iii) In inside-out patches, the channel activity was significantly stimulated by the application of CaMK II (300 pM) at 10(-7) M Ca2+ in the bath. Furthermore, the application of KN-62 (10 microM) to the bath significantly decreased the channel activity. Our findings show that the constitutive activity of inwardly rectifying K+ channel at physiological [Ca]i is mediated by the Ca2+/CaMK II pathway in OKPCs.
The Journal of Physiological Sciences 07/2008; 58(3):199-207. · 1.61 Impact Factor
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Sachiko Tokuda,
Chikao Shimamoto, Hideyo Yoshida,
Hitoshi Murao,
Gen-ichi Kishima,
Shigenori Ito,
Takahiro Kubota,
Toshiaki Hanafusa,
Tohru Sugimoto,
Naomi Niisato,
Yoshinori Marunaka,
Takashi Nakahari
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ABSTRACT: Intracellular pH (pHi) after the NH+4 pulse addition and its removal were measured in isolated alveolar type II cells (ATII cells) using BCECF fluorescence. In the absence of HCO(-3), the NH+4 pulse addition increased pHi (alkali jump) and its removal decreased pH(i) (acid jump) to the control level (no overacidification). This pHi change was induced by reaction 1 (NH3 + H+ <--> NH+4). However, in the presence of HCO(-3), the NH+4 pulse removal decreased pHi (acid jump) with overacidification. The extent of overacidification was decreased by acetazolamide (a carbonic anhydrase inhibitor), bumetanide (an inhibitor of Na+/K+/2Cl(-) cotransporter [NKCC]), and NPPB (an inhibitor of Cl(-) channel). The NH+4 pulse addition led to the accumulation of NH+4 in ATII cells via reaction 1 and NKCC, and the NH+4 pulse removal induced reaction 2 (NH+4 + HCO(-3) --> NH3 + H+ HCO(-3)) in addition to the reversal of reaction 1. Thus, NH+4 that entered via NKCC reacts with HCO(-3) (reaction 2) to produce H+, which induces overacidification in the acid jump. After the overacidification, the pH(i) recovery consisted of a rapid recovery (first phase) followed by a slow recovery (second phase). The first phase was inhibited by NPPB, glybenclamide, amiloride, and an Na+-free solution, and the second phase was inhibited by DIDS, MIA, and an Na+-free solution. Both phases were accelerated by a high extracellular HCO(-3) concentration. These observations indicate that the first phase was induced by HCO(-3) entry via Cl(-) channels coupled with Na+ channels activities, and that the second phase was induced by H+ extrusion via Na+/H+ exchanger and by HCO(-3) entry via HCO(-3) cotransporter. Thus, in ATII cells, HCO(-3) entry via Cl(-) channels is essential for recovering pHi after overacidification during the acid jump and for removing NH+4 that entered via NKCC from ATII cells, suggesting HCO(-3)-dependent NH3 excretion from lungs.
Pflügers Archiv - European Journal of Physiology 11/2007; 455(2):223-39. · 4.46 Impact Factor
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ABSTRACT: Perfluorooctane sulfonate (PFOS) is one of the emerging persistent organic pollutants, ubiquitously found in the global environment, even in human serum. PFOS has been reported to perturb Ca(2+) homeostasis in Paramecium, cardiomyocytes and neurons. Since ciliary beat frequency (CBF) in the trachea is known to be increased by cytoplasmic Ca(2+) elevation, the effects of PFOS on CBF were evaluated in a slice preparation using video-enhanced contrast microscopy. PFOS increased CBF by 11% (P<0.05) at 100 microM, while it did not do so at 30 microM. At 100 microM, it increased intracellular Ca(2+) concentration ([Ca(2+)](i)) in mouse tracheal ciliary cells. In Ca(2+)-free solution, PFOS at 100 microM failed to increase CBF (0.96-fold of vehicle control). The addition of Gd(3+) (1 microM), a store-operated Ca(2+) channel blocker, did not prevent the increase in CBF (1.09-fold (P<0.01) of vehicle control). High extracellular K(+) concentration (50 mM), which causes depolarization of the plasma membrane potential and a transient increase in [Ca(2+)](i), increased CBF by 20% (P<0.05). This observation indicates involvement of voltage-dependent Ca(2+) channels (VDCCs) in stimulation of CBF. Nifedipine (30 microM), a selective VDCC blocker, antagonized the effects of high K(+) (0.92-fold of high K(+) solution) and PFOS (0.96-fold of vehicle control) on CBF. In cells from peroxisome proliferator-activated receptor alpha (PPARalpha)-null mice, PFOS still increased CBF (1.12-fold (P<0.05) of vehicle control), indicating that the actions of PFOS are not mediated via PPARalpha. These findings collectively suggest that PFOS stimulates CBF by increasing cytoplasmic Ca(2+) through VDCC.
Toxicology 08/2007; 236(3):190-8. · 3.68 Impact Factor
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ABSTRACT: Ciliary beat frequency (CBF) was measured by video-optical microscopy in rat tracheal and distal airway ciliary cells using a slice preparation. In tracheal ciliary cells (tracheal slice), ATP or 2-methylthio ATP (MeSATP) increased CBF, which was inhibited by suramin (100 microm, an inhibitor of purinergic receptor). Ionomycin (5 microm) or thapsigargin (2 microm) increased CBF similarly. Ca2+-free solution or addition of Ni2+ (1 mm) decreased CBF gradually by approximately 25% and subsequent stimulation with ATP (10 microm) increased CBF transiently. The purinergic agonist experiments demonstrated that ATP increases CBF in tracheal ciliary cells via both P2X and P2Y receptors. ATP increased the intracellular calcium concentration ([Ca2+]i) in tracheal ciliary cells. However, in distal airway ciliary cells (lung slice), ATP did not increase CBF and [Ca2+]i, although a Ca2+-free solution decreased CBF, and ionomycin (5 microm) or thapsigargin (2 microm) increased it. Moreover, acetylcholine (100 microm) did not increase CBF in distal airway ciliary cells, although it increased CBF in tracheal ciliary cells. Terbutaline (10 microm), a selective beta2-adrenergic agonist, increased CBF in both tracheal and distal airway ciliary cells. These observations suggest that the Ca2+-mobilization mechanisms via purinergic or muscarinic receptors of the distal airway ciliary cell may be different from those of the tracheal ciliary cell. In conclusion, the CBF increase is differently regulated in the tracheal and distal airway epithelia of the rat.
Experimental Physiology 08/2005; 90(4):535-44. · 3.21 Impact Factor
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Hitoshi Murao,
Akira Shimizu,
Keita Hosoi,
Akitaka Iwagaki,
Kyong-Yob Min,
Gen-ichi Kishima,
Toshiaki Hanafusa,
Takahiro Kubota,
Masumi Kato, Hideyo Yoshida,
Takashi Nakahari
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ABSTRACT: The effects of intracellular Ca2+ concentration, [Ca2+]i, on the volume of rat alveolar type II cells (AT-II cells) were examined. Perfusion with a Ca2+-free solution induced shrinkage of the AT-II cell volume in the absence or presence of amiloride (1 microm, an inhibitor of Na+ channels); however, it did not in the presence of 5-(N-methyl-N-isobutyl)-amiloride (MIA, an inhibitor of Na+-H+ exchange). MIA decreased the volume of AT-II cells. Inhibitors of Cl(-)-HCO3- exchange, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) also decreased the volume of AT-II cells. This indicates that the cell shrinkage induced by a Ca2+-free solution is caused by a decrease in NaCl influx via Na+-H+ exchange and Cl(-)-HCO3- exchange. Addition of ionomycin (1 microm), in contrast, induced cell swelling when AT-II cells were pretreated with quinine and amiloride. This swelling of the AT-II cells is not detected in the presence of MIA. Intracellular pH (pHi) measurements demonstrated that the Ca2+-free solution or MIA decreases pHi, and that ionomycin increases it. Ionomycin stimulated the pHi recovery after an acid loading (NH4+ pulse method), which was not noted in MIA-treated AT-II cells. Ionomycin increased [Ca2+]i in fura-2-loaded AT-II cells. In conclusion, the Na+-H+ exchange activities of AT-II cells, which maintain the volume and pHi, are regulated by [Ca2+]i.
Experimental Physiology 04/2005; 90(2):203-13. · 3.21 Impact Factor
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ABSTRACT: The ciliary beat frequency (CBF) of rat tracheal ciliary cells in a slice preparation was measured using video-enhanced contrast (VEC) microscopy. Acetylcholine (ACh) increased CBF mediated via intracellular Ca2+ concentration ([Ca2+]i) in a dose-dependent manner. An adequate hypo-osmotic stress (-40 mosM) potentiated ACh-stimulated CBF increase in tracheal ciliary cells and shifted the ACh dose-response curve to the left (lower concentration side). This potentiation was independent of hypo-osmotic stresses applied ranging from -20 mosM to -90 mosM. A hypo-osmotic stress induces ATP release in many cell types. The present study demonstrated that suramin (an inhibitor of purinergic receptors) and apyrase (an ATPase/ADPase) eliminate the hypo-osmotic potentiation of ACh-stimulated CBF increase and that ATP increased [Ca2+]i and CBF, as well as potentiating ACh-stimulated rises in [Ca2+]i and CBF increase. Moreover, the apical surface of tracheal ciliary cells were stained immunopositive for the P2X4 purinergic receptor. A hypo-osmotic stress (-40 mosM) transiently increased [Ca2+]i and potentiated the ACh-stimulated [Ca2+]i increase. The hypo-osmotic potentiation of ACh-stimulated CBF increase was not detected under Ca2+-free conditions. These observations suggest that a hypo-osmotic stress stimulates ATP release from the trachea. The released ATP may induce further increases in [Ca2+]i and CBF in ACh-stimulated tracheal ciliary cells, which may be mediated by purinergic receptors, such as P2X4.
Experimental Physiology 12/2004; 89(6):739-51. · 3.21 Impact Factor
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ABSTRACT: Maintaining the extracellular K(+) concentration ([K(+)](o)) between 15 and 60 mM induced oscillations in the intracellular Ca(2+) concentration ([Ca(2+)](i)) in rat submandibular acinar cells during stimulation with acetylcholine (ACh, 1 micro M). These [Ca(2+)](i) oscillations were also induced by 1 micro M thapsigargin and were inhibited by 50 micro M La(3+), 1 micro M Gd(3+), or the removal of extracellular Ca(2+), indicating that the [Ca(2+)](i) oscillations were generated by store-operated Ca(2+) entry (SOC). The frequency of the ACh-evoked [Ca(2+)](i) oscillations increased from 0.8 to 2.3 mHz as [K(+)](o) was increased from 15 to 50 mM. TEA (an inhibitor of K(+) channels) also induced [Ca(2+)](i) oscillations at [K(+)](o) of 4.5 or 7.5 mM in ACh-stimulated cells. These data suggest that depolarization causes [Ca(2+)](i) to oscillate in ACh-stimulated submandibular acinar cells. Pertussis toxin (PTX, an inhibitor of G proteins) caused [Ca(2+)](i) to be sustained at a high level in ACh-stimulated cells at 25 mM or 60 mM [K(+)](o). This suggests that the [Ca(2+)](i) oscillations are generated by a periodic inactivation of the SOC channels via PTX-sensitive G proteins, which are stimulated by depolarization. Moreover, in the presence of DBcAMP or forskolin which accumulated cAMP the frequency of the [Ca(2+)](i) oscillations remained constant (approximately 1.2 mHz) when [K(+)](o) was maintained in the range 25-60 mM. Based on these observations in ACh-stimulated submandibular acinar cells, we conclude that depolarization stimulates the PTX-sensitive G proteins, which inactivate the SOC channels periodically ([Ca(2+)](i) oscillation), while hyperpolarization or PTX inhibits the G proteins, maintaining the activation of the SOC channels. Accumulation of cAMP is likely to modulate the PTX-sensitive G proteins.
Experimental Physiology 06/2003; 88(3):369-79. · 3.21 Impact Factor
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ABSTRACT: The effects of cAMP accumulation evoked by acetylcholine (ACh) stimulation were studied in rat submandibular acinar cells by observing the exocytotic events, swelling of intercellular canaliculi (IC) and intracellular Ca2+ concentration ([Ca2+]i), which were monitored using an optical microscope. ACh stimulation evoked transient increases followed by sustained increases in the frequency of exocytotic events and IC swelling, while isoproterenol (isoprenaline; IPR) stimulation evoked sustained increases in these parameters. BAPTA treatment reduced the frequency of exocytotic events evoked by 5 μM ACh in the absence of extracellular Ca2+, and further addition of Rp-cAMPS or H-89 (protein kinase A (PKA) inhibitors) eliminated the remaining ACh-evoked responses (50%). Addition of PKA inhibitors in the presence of extracellular Ca2+ reduced the frequency of exocytotic events evoked by 500 μM ACh in non-BAPTA-loaded cells. However, IC swelling evoked by 5 μM ACh was not affected by addition of PKA inhibitors, and was eliminated in BAPTA-loaded cells perfused with Ca2+-free solution. These results indicate that the IC swelling is regulated by [Ca2+]i and the frequency of exocytotic events is regulated by both [Ca2+]i and [cAMP]i during ACh stimulation. Addition of H-89 inhibited the capacitative Ca2+ entry into ACh-stimulated acinar cells. Biochemical analysis revealed that ACh stimulation increased the cAMP content in perfused submandibular glands. These results indicate that ACh stimulates the accumulation of cAMP in submandibular acinar cells and that this accumulation of cAMP modulates Ca2+-regulated exocytosis.
Experimental physiology 02/2000; 85(2):159 - 169. · 3.17 Impact Factor
