Membrane potential modulation of ionomycin-stimulated Ca²⁺ entry via Ca²⁺/H⁺ exchange and SOC in rat submandibular acinar cells

Abstract

Ionomycin (IM) at 5 μM mediates the Ca²⁺/H⁺ exchange, while IM at 1 μM 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 (5 μM, the Ca²⁺/H⁺ exchange) increased the intracellular Ca²⁺ concentration ([Ca²⁺]_i) to an extremely high value at 151 mM [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 +93 mV, suggesting that IM (5 μM) 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 (1 μM, the SOCs) abolished an increase in [Ca²⁺]_i at 151 mM [K⁺]_o. With increasing [K⁺]_o, the rate of Ca²⁺ 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 Ca²⁺ over Na⁺ (P _Ca/P _Na = 8.2). Moreover, an increase in extracellular Ca²⁺ concentration (20 mM) enhanced the Ca²⁺ entry via the SOCs at 151 mM [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 5 μM and the SOCs at 1 μM.

Introduction

Ionomycin (IM) is known to increase intracellular Ca²⁺ concentration ([Ca²⁺]_i) in many cell types, including salivary acinar cells, and is used in a variety of experiments. The [Ca²⁺]_i increase stimulated by IM was reported to be due to the exchange of 1 Ca²⁺ for 1 H⁺, which was concluded to be electrogenic [1–5].

On the other hand, IM is also known to activate store-operated Ca²⁺ channels (SOCs), that is, IM depletes Ca²⁺ from intracellular stores [4, 6, 7], which triggers Ca²⁺ entry via SOCs [4, 6–10]. The electrophysiological characteristics of the SOC in salivary cells have been reported to be different from those of the typical calcium release-activated Ca²⁺ channels (CRAC) in leukemia cells [8–14]. The transient receptor potential canonical 1 (TRPC1) of ion channel proteins is also reported to be an essential component of the SOC in salivary acinar cells [11–13]. Patch clamp studies demonstrated that the SOCs are nonselective cation channels in salivary gland cells. In human submandibular gland (HSG) cells, the SOC current (I _SOC) was relatively inwardly rectifying and relatively selective for Ca²⁺ over Na⁺ [reversal potential (E _rev) = +25 mV] [13, 14]. In human parotid gland (HSY) cells and mouse submandibular acinar cells, the I _SOC is almost linear and less selective for Ca²⁺ over Na⁺ (E _rev = +3 mV) [13, 14]. Thus, two types of SOCs were reported in salivary acinar cells.

The SOCs in salivary cells are modulated by many cellular events stimulated by [Ca²⁺]_i increases, including membrane potential. In salivary acinar cells, depolarization was reported to decrease store-operated Ca²⁺ entry (SOCE) [8, 15] and to induce [Ca²⁺]_i oscillations (at 20–30 mM [K⁺]_o) [16, 17].

To examine the effects of depolarization on SOCE, we measured the [Ca²⁺]_i in IM-stimulated rat submandibular acinar cells with increasing extracellular K⁺ concentration ([K⁺]_o). In the course of experiments, a high [K⁺]_o, for example 150 mM, did not suppress increases in [Ca²⁺]_i stimulated by 5 μM IM, whereas it abolished those stimulated by 1 μM IM. Moreover, Gd³⁺ (1 μM) inhibited an increase in [Ca²⁺]_i stimulated by 1 μM, but not by 5 μM [5]. Thus, IM has two actions depending on its concentration, suggesting that 5 μM IM mediates the Ca²⁺/H⁺ exchange and 1 μM IM activates the SOCs [5].

In this study, we examined the effects of depolarization on the IM-stimulated Ca²⁺ entry pathways (the Ca²⁺/H⁺ exchange and the SOC) by measuring the rate of [Ca²⁺]_i increase (Ca²⁺ influx) with increasing [K⁺]_o. The goal of this study is to confirm IM increases [Ca²⁺]_i via two pathways depending on its concentration, the electrogenic Ca²⁺/H⁺ exchange at 5 μM and the SOCs at 1 μM.

Materials and methods

Solutions and chemicals

The control solution contained (in mM): NaCl 121, KCl 4.5, MgCl₂ 1, CaCl₂ 1.5, NaHCO₃ 25, NaHEPES 5, HHEPES 5, and glucose 5; the KCl solution contained (in mM): KCl 125.5, MgCl₂ 1, CaCl₂ 1.5, KHCO₃ 25, KHEPES 5, HHEPES 5, and glucose 5. The pH of the solutions was adjusted to 7.4 by adding 1 M HCl. To increase the K⁺ concentration of test solutions, an appropriate amount of KCl solution was added to the control solution. The solutions were aerated with a gas mixture of 95% O₂ and 5% CO₂. To prepare a Ca²⁺-free solution, CaCl₂ was excluded from the solutions (nominal Ca²⁺-free). We did not use EGTA to chelate Ca²⁺, since EGTA also chelates Gd³⁺. There was no difference between [Ca²⁺]_i increases following the reintroduction of Ca²⁺, when we used a Ca²⁺-free solution with or without EGTA [5]. A high Ca²⁺ solution (20 mM Ca²⁺, HCO₃ ⁻-free) contained (in mM): NaCl 146, KCl 4.5, MgCl₂ 1, CaCl₂ 20, NaHEPES 5, HHEPES 5, and glucose 5; the KCl solution containing high Ca²⁺ contained (in mM): KCl 151.5, MgCl₂ 1, CaCl₂ 20, KHEPES 5, HHEPES 5, and glucose 5. The pH of the solutions was adjusted to 7.4 by adding 1 M HCl. To prepare a Ca²⁺ free solution for high Ca²⁺ experiments, 20 mM CaCl₂ was excluded from the high Ca²⁺ solutions, and 30 mM NaCl or 30 mM KCl was added to adjust the osmolarity. The high Ca²⁺ solutions (HCO₃ ⁻-free) were aerated with 100% O₂. IM, thapsigargin (TG), bovine serum albumin (BSA), and collagenase were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). The reagents were diluted to their final concentrations just before the experiments. All the experiments were performed at 37°C.

Cell preparation and fluorescence measurements

Male rats (Slc: Wistar/ST, Japan SLC Inc., Hamamatsu, Japan) weighing 150–200 g were purchased and fed a standard pellet diet and water. The rats were anesthetized by intraperitoneal injection of pentobarbital sodium (Nembutal 60–70 mg/kg). The submandibular glands were removed from the animals for cell isolation, and then the animals were killed by an additional injection of pentobarbital sodium (Nembutal 100 mg/kg). Experiments were approved by the Animal Research Committee of Osaka Medical College, and the animals were cared for according to the guidelines of this committee. The procedures for the cell preparations have already been described in detail [5, 16]. The submandibular glands were washed with the control solution to remove blood. Collagenase (0.1%) was dissolved in the control solution containing 2% BSA. The glands were minced in a collagenase solution with fine forceps and then incubated (37°C) for 15 min. The digested tissue was filtered through a nylon mesh with a pore size of 150-μm squares to remove undigested tissue. The filtrate was centrifuged at 300 rpm (10g) for 2 min, washed three times with centrifugation between each wash, and suspended in the control solution containing 2% BSA.

The isolated cells were loaded with fura 2-AM (2.5 μM, Dojindo, Kumamoto, Japan) or BCECF-AM (2.5 μM, Dojindo, Kumamoto, Japan) for 25 min at room temperature (23°C) and washed with the control solution. They were then mounted on a coverslip precoated with neutralized Cell-Tak (Becton-Dickinson Labware, Bedford, MA) to allow the cells to firmly adhere to the coverslip. The coverslip with cells was set in a perfusion chamber, which was mounted on the stage of an inverted microscope (TE2000, Nikon, Tokyo, Japan) connected to an image analysis system (Aqua Cosmos, Hamamatsu Photonics, Hamamatsu, Japan). The capacity of the chamber was approximately 100 μl, and the perfusion rate was 500 μl/min. The fura 2 fluorescence ratios (340:380 nm) were calculated and stored in the image analysis system. One experiment was performed using 6–12 coverslips from 2 to 4 animals. A typical response obtained from five to seven cells on a coverslip is shown in the figures (mean ± SEM). To compare the values among experiments, averaged values calculated from five to six coverslips obtained from two to three animals were used.

Activation of Ca²⁺ entry

To observe Ca²⁺ entry, Ca²⁺ was reintroduced (reintroduction of Ca²⁺) [5, 6]. The procedure for “the reintroduction of Ca²⁺” was as follows. Before the start of experiments, the cells were perfused with control solution for 5 min. Following the control perfusion, the cells were perfused with a Ca²⁺-free solution for 5 min and stimulated with IM for a further 10 min. Then, the Ca²⁺-free solution was suddenly switched to a Ca²⁺-containing solution, keeping the IM concentration (the reintroduction of Ca²⁺). The reintroduction of Ca²⁺ is a method for stimulating a rapid Ca²⁺ entry without Ca²⁺ release from stores.

Membrane potential measurements

Membrane potential was measured by a gramicidin-perforated whole cell patch method. The details have already been described in a previous report [18]. Briefly, gramicidin D (Sigma) was dissolved in methanol (10 mg/ml) and diluted 100 times with a standard KCl-rich pipette solution. The KCl-rich pipette solution contained (in mM): KCl 150 and HEPES 10. The pH of the solution was adjusted to 7.4 using KOH. After making the perforated patch configuration, the membrane potential was measured using a patch clamp amplifier (model CEZ-2400, Nihon Kohden, Tokyo, Japan). The membrane potentials of the cells were measured under an unstimulated condition and 5 μM IM- or 4 μM TG-stimulated condition with 4.5 mM [K⁺]_o and 151 mM [K⁺]_o.

The statistical significance of the difference between the mean values was assessed using Student’s t test. Differences were considered significant at p < 0.05.

Results

Effects of high [K⁺]_o on IM-stimulated [Ca²⁺]_i

During the control perfusion, the F340/F380 of the submandibular acinar cells were 1.2–1.3. The switch to a Ca²⁺-free solution slightly decreased and plateaued [Ca²⁺]_i within 2 min in rat submandibular acinar cells (data not shown). The addition of 1 μM IM induced a small transient increase in [Ca²⁺]_i. The reintroduction of Ca²⁺ induced a rapid transient increase followed by a sustained increase in [Ca²⁺]_i (Fig. 1a). Experiments were carried out using a KCl solution ([K⁺]_o = 151 mM). The KCl solution abolished the [Ca²⁺]_i increase following the reintroduction of Ca²⁺ in cells stimulated with 1 μM IM (Fig. 1a). Gd³⁺ (1 μM) similarly inhibited the [Ca²⁺]_i increase following the reintroduction of Ca²⁺ in cells stimulated with 1 μM IM (data not shown), as previously reported [5].

Fig. 1

[Ca²⁺]_i increases following the reintroduction of Ca²⁺ during IM stimulation. a IM (1 μM). The addition of 1 μM IM in the Ca²⁺-free solution increased [Ca²⁺]_i slightly and transiently. The reintroduction of Ca²⁺ induced a transient increase followed by a sustained increase in [Ca²⁺]_i. An increase of [K⁺]_o (151 mM) abolished the [Ca²⁺]_i increase following the reintroduction of Ca²⁺. b IM (5 μM). The addition of 5 μM IM in the Ca²⁺-free solution increased [Ca²⁺]_i transiently. The reintroduction of Ca²⁺ induced a sustained increase in [Ca²⁺]_i. The final [Ca²⁺]_is were much higher than that of 1 μM IM. An increase of [K⁺]_o (151 mM) still increased [Ca²⁺]_i following the reintroduction of Ca²⁺, although the final [Ca²⁺]_i decreased by 10%. c, d Effects of 1 or 5 μM IM on internal Ca²⁺ stores. The addition of 1 or 5 μM IM increased [Ca²⁺]_i transiently, and then the further addition of 10 μM ACh did not induce any [Ca²⁺]_i increase. The reintroduction of Ca²⁺ increased [Ca²⁺]_i

The experiments were carried out using 5 μM IM. The switch to Ca²⁺-free solution decreased [Ca²⁺]_i slightly, and then, the addition of 5 μM IM induced a large transient [Ca²⁺]_i increase. The reintroduction of Ca²⁺ immediately increased [Ca²⁺]_i to an extremely high value (F340/F380 = ~5) (Fig. 1b). Experiments were carried out using the KCl solution ([K⁺]_o = 151 mM). The KCl solution did not inhibit the [Ca²⁺]_i increase following the reintroduction Ca²⁺ in cells stimulated by 5 μM IM, although the plateaus [Ca²⁺]_is were decreased by 10–15% (F340/F380 = ~4.4) (Fig. 1b). However, the rate of [Ca²⁺]_i increase was decreased by ~50%, compared with those of the control experiments. Gd³⁺ (1 μM) did not inhibit the [Ca²⁺]_i increase following the reintroduction of Ca²⁺ in cells stimulated by 5 μM IM (data not shown), as previously reported [5].

The effects of 1 and 5 μM IM on the intracellular Ca²⁺ stores were examined (Fig. 1c, d). The addition of 1 or 5 μM IM induced a transient [Ca²⁺]_i increase in a Ca²⁺ free solution, and then, the further addition of 10 μM ACh induced no [Ca²⁺]_i increase. Thus, IM at both concentrations completely depletes Ca²⁺ from internal stores, indicating that IM (1 and 5 μM) activates the SOCs. Then, the reintroduction of Ca²⁺ increased [Ca²⁺]_i in both cells stimulated with 1 and 5 μM IM (Fig. 1c, d). Increases in [Ca²⁺]_i shown in panels c and d were similar to those shown in panels a and b.

Effects of high [K⁺]_o on TG-stimulated [Ca²⁺]_i

Experiments were also carried out using 4 μM TG (Fig. 2). The addition of TG (4 μM) in a Ca²⁺-free solution induced a slight and transient [Ca²⁺]_i increase. The reintroduction of Ca²⁺ induced a biphasic increase in [Ca²⁺]_i in cells stimulated with 4 μM TG, similarly to those stimulated with 1 μM IM. Experiments were carried out using the KCl solution. The KCl solution abolished the [Ca²⁺]_i increase following the reintroduction of Ca²⁺ in cells stimulated with 4 μM TG (Fig. 2). The [Ca²⁺]_i increase stimulated with 4 μM TG was inhibited by 1 μM Gd³⁺ (data not shown), as previously reported [5]. Thus, [Ca²⁺]_i increases stimulated by 4 μM TG are similar to those stimulated by 1 μM IM, indicating that 1 μM IM activates SOCs.

Fig. 2

[Ca²⁺]_i increases following the reintroduction of Ca²⁺ during 4 μM TG stimulation. The addition of 4 μM TG in the Ca²⁺-free solution increased [Ca²⁺]_i slightly and transiently. The reintroduction of Ca²⁺ induced a transient increase followed by a sustained increase in [Ca²⁺]_i. An increase of [K⁺]_o (151 mM) abolished the [Ca²⁺]_i increase following the reintroduction of Ca²⁺

Effects of [K⁺]_o on [Ca²⁺]_i increase

The rates of [Ca²⁺]_i increase following the reintroduction of Ca²⁺ were measured at various [K⁺]_os in cells stimulated with 1 μM IM. The [K⁺]_os used ranged from 4.5 to 151 mM. The experimental protocol used is shown in Fig. 1. Cells were treated with 1 μM IM for 10 min in a Ca²⁺-free solution, and then the Ca²⁺ was reintroduced. Figure 3 shows increases in [Ca²⁺]_i for 1.5 min following the reintroduction of Ca²⁺ at three [K⁺]_os (4.5, 25 and 151 mM). In cells stimulated by 1 μM IM, the rate of Ca²⁺ entry and the peak [Ca²⁺]_i decreased to ~30 and ~50% at 25 mM [K⁺]_o, respectively. No [Ca²⁺]_i increase was detected at 151 mM [K⁺]_o. Similar results were obtained in cells stimulated by 4 μM TG instead of 1 μM IM (data not shown).

Fig. 3

Effects of depolarization on [Ca²⁺]_i increase following the reintroduction of Ca²⁺ during IM stimulation. The experimental protocol is shown in Figs. 1 and 2. Cells were perfused with a Ca²⁺-free solution for 5 min prior to the IM addition. Cells were treated with IM for 10 min, and then Ca²⁺ (1.5 mM) was reintroduced. In these figures, changes in the [Ca²⁺]_i increase for 2 min were shown (9.5–11.5 min from the start of IM stimulation). The figure clearly shows decreases in the rate of [Ca²⁺]_i increase following the reintroduction of Ca²⁺ with increasing [K⁺]_o. a IM (1 μM). With increasing [K⁺]_o, the rate of [Ca²⁺]_i increase decreased, and the final [Ca²⁺]_i also decreased. b IM (5 μM). With increasing [K⁺]_o, the rate of [Ca²⁺]_i increase decreased. The final [Ca²⁺]_is decreased by 10% at 151 mM [K⁺]_o

Experiments were also carried out using 5 μM IM (Fig. 3b). Cells were treated with 5 μM IM for 10 min in a Ca²⁺-free solution, and then the Ca²⁺ was reintroduced. The reintroduction of Ca²⁺ increased [Ca²⁺]_i to a high value (F340/F380 = 4.5–5). However, the rates of Ca²⁺ entry stimulated by 5 μM IM decreased with increasing [K⁺]_os from 4.5 to 151 mM (Fig. 3b). The peak [Ca²⁺]_i (5 min after the reintroduction of Ca²⁺) was decreased by ~10% at 151 mM [K⁺]_o (Figs. 1b, 3b).

The results were summarized in Fig. 4. In panel a, the rates of Ca²⁺ entry following the reintroduction of Ca²⁺ were plotted against [K⁺]_os. In cells stimulated with 5 μM IM, the rates of Ca²⁺ entry decreased in a linear relationship with increasing [K⁺]_o. The intercept of the x axis ([K⁺]_o) is 4.5 M. [K⁺]_i is 125 mM, which was calculated from the membrane potential at 151 mM [K⁺]_o (+5 mV) as shown in Figs. 6 and 7. The E _rev was calculated to be +93 mV [=−60 × log(0.125/4.5)]. In cells stimulated with 1 μM IM, the rates of Ca²⁺ entry also linearly decreased with increasing [K⁺]_o, and the intercept of the x axis ([K⁺]_o) was 144 mM (Fig. 4a). The E _rev was calculated to be +3.7 mV. Experiments were carried out using 4 μM TG. In cells stimulated with 4 μM TG, the rates of Ca²⁺ entry also linearly decreased, and the intercept of the x axis ([K⁺]_o) was 132 mM. The E _rev was calculated to be +1.4 mV. These results suggest that the SOCs of rat submandibular acinar cells are nonselective cation channels and less selective for Ca²⁺ over Na⁺ [12–14]. Depolarization markedly decreased the driving force for Ca²⁺ entry via SOCs in submandibular acinar cells.

Fig. 4

Effects of [K⁺]_o on [Ca²⁺]_i increase stimulated by 1 and 5 μM IM. a Effects of [K⁺]_o on the rate of F340/F380 increase (Ca²⁺ influx). During 5 μM IM stimulation, the rate of [Ca²⁺]_i increase linearly decreased. The intercept of the x axis was 4.5 M. During 1 μM IM and 4 μM TG stimulation, the rates of [Ca²⁺]_i increase linearly decreased. The intercepts of the x axis were 144 mM during IM stimulation and 131 mM during TG stimulation. b Effects of [K⁺]_o on the sustained F340/F380. With increasing [K⁺]_o, the final [Ca²⁺]_is are almost constant during 5 μM IM stimulation. However, with increasing [K⁺]_o, the final [Ca²⁺]_i linearly decreased

The peak [Ca²⁺]_is within 5 min following the reintroduction of Ca²⁺ were plotted against [K⁺]_os in Fig. 4b. In cells stimulated with 5 μM IM, the peak [Ca²⁺]_is were decreased slightly with increasing [K⁺]_o. In cells stimulated with 1 μM IM or 4 μM TG, the peak [Ca²⁺]_is decreased in a linear relationship with increasing [K⁺]_o. The regression line of 1 μM IM shifted upward compared with that of 4 μM TG, although the x intercepts of both lines were not significantly different (p > 0.05) (Fig. 4b). IM (1 μM) may mediate the Ca²⁺/H⁺ exchange, although the [Ca²⁺]_i increases mediated by the Ca²⁺/H⁺ exchange were negligibly small in cells stimulated by 1 μM IM.

Effects of high [Ca²⁺]_o on Ca²⁺ entry

To examine the effects of an increase in the driving force for Ca²⁺ entry via SOCs during depolarization, a high Ca²⁺ solution ([Ca²⁺]_o = 20 mM) was used. In cells stimulated with 4 μM TG, a high [Ca²⁺]_o (20 mM) enhanced the [Ca²⁺]_i increase following the reintroduction of Ca²⁺ at 151 mM [K⁺]_o, suggesting that depolarization does not inhibit SOCs (Fig. 5a). The rate of Ca²⁺ entry was plotted against [K⁺]_o in Fig. 5b. With increasing [K⁺]_o, the rates of Ca²⁺ entry linearly decreased (Fig. 5b). The intercept of the x axis ([K⁺]_o) was 188 mM. Assuming the intracellular osmolarity was the same as the extracellular osmolarity, [K⁺]_i was estimated to be 150 mM (=125 × 180/151). The E _rev calculated was +5.9 mV.

Fig. 5

Effects of a high [Ca²⁺]_o (20 mM). Experiments were carried out using 4 μM TG in a HCO₃ ⁻-free solution. In these experiments, the [Ca²⁺]_o used was 20 mM to increase the driving force for Ca²⁺ entry. a An increase in [Ca²⁺]_o (20 mM) enhanced the [Ca²⁺]_i increase following the reintroduction of Ca²⁺ at 151 mM [K⁺]_o. b Effects of [K⁺]_o on the rate of F340/F380 increase (Ca²⁺ influx). With increasing [K⁺]_o, the rates of [Ca²⁺]_i increase linearly decreased, and the intercept of the x axis was 188 mM

Membrane potential measurement

The membrane potential of submandibular acinar cells was measured using a whole cell patch-clamp technique [18]. The resting membrane potential was approximately −40 to −50 mV. The addition of 5 μM IM induced depolarization following an immediate hyperpolarization (−80 mV), and the final membrane potential was −10 to −20 mV (Fig. 6a). However, the addition of 4 μM TG induced a gradual depolarization following an immediate hyperpolarization (−80 mV), and the final membrane potential was similar to that before the addition of 4 μM TG (−40 mV) (Fig. 6c). The switch to a high-K⁺ solution (151 mM) induced an immediate depolarization (+5 mV), and the further addition of 4 μM TG or 5 μM IM had no effect on the membrane potential. On returning to the control solution, the membrane potential shifted to −10 mV during stimulation with 5 μM IM (Fig. 6b) and to −60 mV during stimulation with 4 μM TG (Fig. 6d). The final membrane potentials (12 min after the addition of IM or TG) are summarized in Fig. 7 (mean ± SEM, 5 cells obtained from 2 to 3 animals). IM (5 μM) and TG (4 μM) depolarized the membrane potentials to −18 and −37 mV, respectively. The depolarization induced by 5 μM IM was significantly larger than that induced by TG (p < 0.05), suggesting that the 5 μM IM mediated an electrogenic Ca²⁺/H⁺ exchange. With increasing [K⁺]_o from 4.5 to 151 mM, the membrane potentials depolarized immediately to +5 mV, and the further addition of IM or TG had no effect on the membrane potentials (Fig. 7). From the membrane potential at 151 mM [K⁺]_o, we evaluated the [K⁺]_i to be 125 mM.

Fig. 6

Membrane potential changes induced by 4 μM TG and 5 μM IM. Membrane potentials were measured using a gramicidin-perforated whole cell patch clamp technique. a Stimulation with 5 μM IM at 4.5 mM [K⁺]_o. b Stimulation with 5 μM IM at 151 mM [K⁺]_o. c Stimulation with 4 μM TG at 4.5 mM [K⁺]_o. d Stimulation with 4 μM TG at 151 mM [K⁺]_o

Fig. 7

Membrane potential changes induced by 4 μM TG and 5 μM IM. Membrane potentials before stimulation (a), 12 min after the addition of TG or IM at 4.5 mM [K⁺]_o (b) and at 151 mM [K⁺]_o (c) were shown in this figure. IM (5 μM) significantly depolarized the membrane potential. Asterisks indicate significantly different (p < 0.05)

Discussion

This study demonstrated that IM increases [Ca²⁺]_i via two pathways in rat submandibular acinar cells depending on its concentration. One is the Ca²⁺/H⁺ exchange, which is mediated by a high concentration of IM, such as 5 μM. The Ca²⁺ entry via the Ca²⁺/H⁺ exchange is insensitive to 1 μM Gd³⁺ and depolarization, and accompanied by membrane depolarization [3–5, 19]. A previous report demonstrated that an increase in extracellular pH enhances the rate of Ca²⁺ entry stimulated by 5 μM IM [5]. The Ca²⁺/H⁺ exchange increased [Ca²⁺]_is to an extremely high value. The other is the SOCs, which are activated by a low concentration of IM, such as 1 μM. The Ca²⁺ entry via SOCs is sensitive to 1 μM Gd³⁺ [5] and decreased by depolarization. Changes in extracellular pH have been shown to have no effect on the rate of Ca²⁺ entry [5]. [Ca²⁺]_i increases stimulated by 1 μM IM were similar to those stimulated by TG, which activated SOCs by depletion of Ca²⁺ from stores [6, 15].

In the Ca²⁺/H⁺ exchange (5 μM IM), Ca²⁺ influx linearly decreased with developing depolarization. The reversal potential (E _rev) was estimated from the rates of Ca²⁺ entry with increasing [K⁺]_o. The [K⁺]_o, which abolished the driving force for Ca²⁺ entry via the Ca²⁺/H⁺ exchange, is estimated to be 4.5 M from the observation shown in Fig. 4a (the intercept of x axis), and [K⁺]_i is evaluated to be 125 mM from the membrane potential (+5 mV) at 151 mM [K⁺]_o (Fig. 7). The E _rev for the Ca²⁺/H⁺ exchange calculated was +93 mV [=−60 × log(0.125/4.5)].

In the simplest model for the IM-mediated Ca²⁺/H⁺ exchange, the E _rev was expressed using conductances of Ca²⁺ (g_Ca) and H⁺ (g_H) as Eq. 1.

$$ {\text{g}}_{\text{Ca}} \left( {E_{\text{rev}} -E_{\text{Ca}} } \right) = {\text{g}}_{\text{H}} \left( {E_{\text{H}} -E_{\text{rev}} } \right). $$

(1)

The equilibrium potential for Ca²⁺ (E_Ca) is calculated to be +140 mV, when [Ca²⁺]_i is 100 nM and [Ca²⁺]_o is 1.5 mM. The equilibrium potential for H⁺ (E _H) is 0 mV, because intracellular pH (pH_i) is similar to extracellular pH (pH_o).

This leads to Eq. 2, because E _H is 0 mV.

$$ E_{\text{rev}} = \left[ {{\text{g}}_{\text{Ca}} /\left( {{\text{g}}_{\text{Ca}} + {\text{g}}_{\text{H}} } \right)} \right] \times E_{\text{Ca}} $$

(2)

E _rev for Ca²⁺/H⁺ exchange mediated by 5 μM IM is +91 mV, which was 65% of E _Ca. This indicates that the g_Ca/g_H is approximately 2/1. Since Ca²⁺ has two positive charges and H⁺ has one positive charge, Ca²⁺ and H⁺ appear to be equally contributed to the E _rev, suggesting that IM exchanges 1 Ca²⁺ for 1 H⁺.

On the other hand, in the SOC stimulated by 1 μM IM or 4 μM TG, the Ca²⁺ influx decreased linearly with increasing [K⁺]_o from 4.5 to 151 mM. The E _revs are +3.7 and +1.4 mV in 1 μM IM- and 4 μM TG-stimulated cells, respectively. Moreover, in TG-stimulated cells, an increase in [Ca²⁺]_o (20 mM) enhanced Ca²⁺ influx significantly at 151 mM [K⁺]_o, indicating that depolarization does not inhibit SOCs, and the E _rev was +5.9 mV. Thus, an increase in [Ca²⁺]_o did not shift the E _rev significantly (p > 0.05). These E _revs suggest that the SOCs are permeable to monovalent cations, such as Na⁺ and K⁺. The previous patch clamp studies demonstrated that the SOCs are permeable to not only Ca²⁺, but also Na⁺ and Cs⁺ in salivary cells (P _Cs/P _Na = 1 in HSG cells and 1.3 in HSY cells) [11, 14].

In this study, we calculated P _Ca/P _Na [20], assuming that the SOCs are permeable to K⁺ similarly to Cs⁺ and the P _Na/P _K is 1.

$$ \begin{aligned} P_{\text{Ca}} /P_{\text{Na}} & = \left( {1 + \exp (E_{\text{rev}} F/RT)} \right) \\ & \quad \times \left( {([{\text{Na}}^{ + } ]_{i} + \alpha [{\text{K}}_{ + } ]_{\text{i}} ) \times \exp (E_{\text{rev}} \times F/RT) - ([{\text{Na}}^{ + } ]_{\text{o}} + \alpha [{\text{K}}^{ + } ]_{\text{o}} )} \right)/4/[{\text{Ca}}^{2 + } ]_{\text{o}} \\ \end{aligned} $$

(3)

In Eq. 3, α is P _K/P _Na (=1). We also assumed that the intracellular osmolarity is equal to the extracellular osmolarity. The calculated ratio (P _Ca/P _Na) was 8.2 at 1.5 mM [Ca²⁺]_o in rat submandibular acinar cells stimulated with 1 μM IM. In cells stimulated with 4 μM TG, the P _Ca/P _Nas were 2.6 at 1.5 mM [Ca²⁺]_o and 2.3 at 20 mM [Ca²⁺]_o. Previous studies showed that the E _revs have been reported to be +25 mV in HSG cells, +5 mV in HSY cells, and +3 mV in mouse submandibular acinar cells [13, 14], and that the permeability ratio of Ca²⁺ over Na⁺ (P _Ca/P _Na) is 40 in HSG cells and 4.6 in HSY cells [13, 14]. The characteristics of the SOC in rat submandibular acinar cells are similar to those in HSY cells and mouse submandibular acinar cells, that is, the SOCs are less selective for Ca²⁺ over Na⁺ [13, 14].

The SOCs of rat submandibular acinar cells have a linear current-voltage (I–V) relationship. Moreover, they are less selective for Ca²⁺ over Na⁺ (P _Ca/P _Na = 2–8) compared with the typical CRAC of rat basophilic leukemia cells [8, 9, 14]. I _CRAC showed strong inward rectification and high selectivity for Ca²⁺ over Na⁺ $(P_{{{\text{Ca}}^{2 + } }} /P_{{{\text{Na}}^{ + } }} > 500)$. Thus, the SOCs of rat submandibular acinar cells are distinct from those of CRAC channels of T lymphocytes and other hematopoietic cells. The SOCs of rat submandibular acinar cells are likely to be TRPCs type, as shown in HSY cells and mouse submandibular acinar cells [11–14].

A dependence of IM-induced Ca²⁺ entry on the magnitude of depolarization has been studied mainly in artificial liposomes [3, 19], since artificial liposomes have simple properties unlike cells containing complicated biological properties. The extents of Ca²⁺ transport mediated via IM (Ca²⁺/H⁺ exchange) are reported to be a function of IM concentration [19]. The ratio of the Ca²⁺/H⁺ exchange in IM was varied according to the experimental conditions, such as the IM concentration and the external pH, especially in living cells [1–4, 19]. The experimental conditions making IM exchange 1 Ca²⁺ for 1 H⁺ in living cells are still unknown. The present study demonstrated that a high concentration of IM (5 μM) exchanges 1 Ca²⁺ for 1 H⁺ in rat submandibular acinar cells and that the stoichiometry is consistent with that obtained from studies of artificial liposomes or vesicles [1–3, 19].

The present study also showed that the peak values stimulated by 1 μM IM are slightly higher than those stimulated by 4 μM TG (Fig. 4b), and the P _Ca/P _Na in 1 μM IM stimulated cells (=8.2) was higher than those in TG stimulated cells (2.2–2.6). This may suggest that 1 μM IM also mediates the Ca²⁺/H⁺ exchange, although the Ca²⁺/H⁺ exchange contributed little to the increase [Ca²⁺]_i in cells stimulated with 1 μM IM. At present, the reason why IM (1 μM) does not exchange Ca²⁺ for H⁺ via the plasma membrane is unknown. IM (1 μM) transports Ca²⁺ via the ER membrane, since it completely depletes intracellular stores. IM may accumulate intracellular vesicles, not cytoplasma in cells stimulated by 1 μM IM.

In rat submandibular acinar cells, ACh stimulation induces a hyperpolarization followed by a depolarization [21, 22], as shown in the whole cell patch clamp recording of this study. These changes in membrane potential affect [Ca²⁺]_i by altering the driving force for Ca²⁺ entry via SOCs. This indicates that changes in membrane potential induced by ACh stimulation finely control [Ca²⁺]_i via SOCs, which regulates fluid secretion in the rat submandibular gland.

On the other hand, previous reports [16, 17] demonstrated that an increase in [K⁺]_o, such as 25–30 mM, stimulates [Ca²⁺]_i oscillations in rat submandibular acinar cells with a long interval (~10 min). These oscillations were induced by changes in Ca²⁺ entry via the SOCs [16]. However, an increase in [K⁺]_o (30 mM) induced no oscillation in the membrane potential (data not shown). Depolarization affects many cellular events, such as ion fluxes and cell volume. These cellular events may modulate SOCs, which may cause [Ca²⁺]_i oscillations. Further studies will be needed to answer this.