HCO(3) (-)-dependent transient acidification induced by ionomycin in rat submandibular acinar cells

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-.

Fig. 1

Intracellular pH (pH_i) changes induced by ionomycin (IM) in the presence of HCO₃ ⁻. a 5 μM IM in HCO₃ ⁻-containing solution. IM (5 μM) induced no pH_i decrease in Ca²⁺-free solution. The re-introduction of Ca²⁺ induced a large transient pH_i decrease followed by a rapid pH_i increase. The plateau pH_i was slightly higher than that before re-introduction of Ca²⁺. b τ Plot of a. The pH_is for 4 min after re-introduction of Ca²⁺ were used for the τ plot. The τ plot clearly shows that the pH_i increase following the rapid pH_i decrease consisted of two phases: the first phase followed by the second phase. c 1 μM IM in HCO₃ ⁻-containing solution. The re-introduction of Ca²⁺ induced a large transient pH_i decrease followed by a rapid pH_i increase. The plateau pH_i was slightly lower than that before the re-introduction of Ca²⁺. d τ plot of c. The pH_is for 4 min after re-introduction of Ca²⁺ were used. The τ plot revealed that the pH_i increase following the rapid pH_i decrease consisted of the single component during 1 μM IM stimulation

Fig. 2

Intracellular pH (pH_i) changes induced by ionomycin (IM) in the absence of HCO₃ ⁻. a 5 μM IM in HCO₃ ⁻-free solution. IM (5 μM) induced no change in pH_i in Ca²⁺-free solution. The re-introduction of Ca²⁺ induced a rapid pH_i decrease followed by a gradual pH_i increase. b 1 μM IM in HCO₃ ⁻-free solution. When control solution was switched to HCO₃ ⁻-free solution, pH_i transiently increased from 7.4 to 7.7 and then plateaued (pH_i 7.5). The switch to Ca²⁺-free solution and addition of IM (1 μM) did not change pH_i. The re-introduction of Ca²⁺ induced a rapid pH_i decrease without any pH_i increase

Fig. 3

Intracellular pH (pH_i) changes induced by thapsigargin (TG). a HCO₃ ⁻ containing solution. TG (4 μM) induced no pH_i decrease in Ca²⁺-free solution. The re-introduction of Ca²⁺ induced a gradual pH_i decrease without any rapid pH_i increase. b HCO₃ ⁻-free solution. The switch to HCO₃ ⁻-free solution, increased pH_i transiently, and it then plateaued (pH_i 7.5). The switch to Ca²⁺-free solution and addition of IM (1 μM) did not change pH_i. The re-introduction of Ca²⁺ induced a gradual pH_i decrease without any pH_i increase

Fig. 4

Effects of 100 μM acetazolamide (ACZ, an inhibitor of carbonic anhydrase) on pH_i changes induced by IM. a 5 μM IM. The addition of ACZ and the further addition of 5 μM IM have no effect on pH_i in Ca²⁺-free solution. The re-introduction of Ca²⁺ induced a small pH_i decrease followed by a gradual pH_i increase. b τ plot of a. The pH_is for 4 min after the re-introduction of Ca²⁺ were used. The τ plot revealed that the pH_i increase after re-introduction of Ca²⁺ consisted of the single component with ACZ. c 1 μM IM. ACZ abolished the pHi changes induced by 1 μM IM

Fig. 5

Effects of MIA and DIDS on pHi changes induced by 5 μM IM. a MIA (10 μM, an inhibitor of Na⁺/H⁺ exchange) has no effect on pH_i. Re-introduction of Ca²⁺ induced a rapid pH_i decrease followed by a rapid pH_i increase. The pH_i decrease stimulated by 5 μM IM was enhanced by MIA in submandibular acinar cells. b DIDS (200 μM, an inhibitor of Cl⁻/HCO₃ ⁻ exchange and Na⁺-HCO₃ ⁻ cotransport). DIDS did not inhibit the rapid transient pHi decrease. The pH_i increase after the pH_i decrease seems to be faster with DIDS than that without DIDS

Fig. 6

IM-induced [Ca²⁺]_i increases after re-introduction of Ca²⁺. a IM (5 μM). Experiments were carried out using HCO₃ ⁻-containing solution. Addition of 5 μM IM induced a large transient increase in fura 2 fluorescence ratio (F ₃₄₀/F ₃₈₀) in Ca²⁺-free solution. Re-introduction of Ca²⁺ increased F ₃₄₀/F ₃₈₀, which plateaued within 3 min. The plateau level was extremely high. This increase was not inhibited by 1 μM Gd³⁺. b and c IM (1 μM, b) and TG (4 μM, c). Addition of 1 μM IM or 4 μM TG induced a small transient increase in F ₃₄₀/F ₃₈₀ in Ca²⁺-free solution. Re-introduction of Ca²⁺ induced a biphasic increase in F ₃₄₀/F ₃₈₀, which was inhibited by 1 μM Gd³⁺

Fig. 7

Effects of extracellular pH (pH_o) on the rate of F ₃₄₀/F ₃₈₀ increase (Ca²⁺ influx) stimulated by IM. Experiments were carried out using a HCO₃ ⁻-containing solution. a 5 μM IM: increases in F ₃₄₀/F ₃₈₀ after re-introduction of Ca²⁺ at pH_o of 8.0 and 6.8. Increases in F ₃₄₀/F ₃₈₀ were enhanced at a pH_o of 8.0. b 1 μM IM. Increases in F ₃₄₀/F ₃₈₀ after re-introduction of Ca²⁺ at pH_o of 8.0 and 6.8. Increases in F ₃₄₀/F ₃₈₀ were not affected by pH_o. c Rates of the F ₃₄₀/F ₃₈₀ increase were plotted against pH_o. Upon stimulation with 5 μM IM, the rates increased with increasing pH_o. However, upon stimulation with 1 μM IM, the rates remained almost constant with increasing pH_o. The rates of F ₃₄₀/F ₃₈₀ increase obtained in HCO₃ ⁻-free solution (data calculated from Fig. 8) are also plotted in this figure

Fig. 8

IM-induced [Ca²⁺]_i increase after re-introduction of Ca²⁺ in HCO₃ ⁻-free solution. The re-introduction of Ca²⁺ increases [Ca²⁺]_i in rat submandibular acinar cells stimulated with 5 μM or 1 μM IM. Increases in [Ca²⁺]_i in the absence of HCO₃ ⁻ were similar to those in the presence of HCO₃ ⁻. a 5 μM IM, b 1 μM IM