Membrane potential modulation of ionomycin-stimulated Ca(2+) entry via Ca (2+)/H (+) exchange and SOC in rat submandibular acinar cells

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.

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

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⁺

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

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

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

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)