The properties of ryanodine-sensitive Ca(2+) release in mouse gastric smooth muscle cells

Abstract

1. Under voltage-clamped conditions, gastric smooth muscle cells of BALB/c mice developed spontaneous (STOCs) and caffeine- (I(CAF)) and carbachol-induced (I(CCh)) transient outward currents. 2. In fura-2 microscopic measurements of intracellular Ca(2+) concentration ([Ca(2+)](i)), caffeine and carbachol (CCh) provoked similar transient [Ca(2+)](i) elevations. 3. Both I(CCh) and CCh-induced [Ca(2+)](i) elevation of single smooth muscle cells occurred in an 'all-or-nothing' fashion in contrast to the reproducible caffeine responses. 4. On the basis of the suppression of STOCs and I(CAF) by nicardipine, tetraethylammonium and iberiotoxin, but not by charybdotoxin nor apamin, it was suggested that both currents were generated by large conductance type Ca(2+)-activated K(+) channels. 5. In measurements of isometric tension, caffeine produced relaxation of gastric smooth muscle strips in a concentration-dependent manner (0.1 -- 3 mM). The concentration-dependent relaxation with caffeine was mimicked by dibutyryl cyclic AMP which produced potentiation of contraction triggered by 50 mM KCL. 6. At caffeine concentrations >3 mM, a transient contraction followed by relaxation was provoked as the quasi maximal response to caffeine. In the quasi maximal response, caffeine acted as a potent relaxant in smooth muscle strips precontracted with 50 mM KCl or 3 microM CCh. 7. The relaxation with caffeine was significantly accelerated in those strips precontracted with KCl or CCh. All these results suggest that ryanodine-sensitive Ca(2+) release, which is triggered by caffeine, is an important modifier of Ca(2+) homeostasis in the cytoplasm and the contractility of gastric smooth muscle cells of mice.

Figure 1

I_CAF and STOCs of mouse gastric smooth muscle cells. (a) I_CAF and current responses to a voltage-ramp stimulation to +40 mV and −70 mV from a V_H of −20 mV in the absence (open triangle) and in the presence (closed triangle) of caffeine. The same current recording is shown at different time scales. (b) STOCs and I_CAF at different V_H. Horizontal closed bars indicate application of caffeine. Dotted lines indicate the zero current level. The zero current level was obtained by application of 10 mM TEA. When the frequency of STOCs was comparatively low, the baseline current was easily determined without application of TEA and was not affected by TEA. (c) Instantaneous I-V relationships of STOCs and I_CAF. Abscissa: V_H, ordinate: amplitude of STOCs and I_CAF. Symbols and error bars indicate the mean±s.e.mean (n=11).

Figure 2

Pharmacological properties of I_CAF and STOCs. (a) Effects of TEA and IbTx on STOCs and I_CAF at a V_H of −20 mV. (b) I_CAF evoked at various concentrations of caffeine at −20 mV. Horizontal closed bars indicate application of caffeine, while open and striped bars indicate application of the K⁺ channel blockers, TEA and IbTx. Dotted lines indicate the zero current level. (c) Caffeine concentration-I_CAF relationship. Abscissa: concentrations of caffeine, ordinate: relative I_CAF which was normalized to that evoked at 1 mM caffeine. Symbols and error bars indicate the mean±s.e.mean (n=11).

Figure 3

Effects of Ca²⁺-free media on STOCs and caffeine-induced responses. (a) Effects of nominally Ca²⁺-free external solutions on STOCs and I_CAF at a V_H of −20 mV. (b) Effects of nominally Ca²⁺-free external solutions on fura-2 microscopic [Ca²⁺]_i signals in the response to caffeine. All traces in (b) are the records from separate gastric smooth muscle cells tested simultaneously. Horizontal closed bars indicate application of caffeine, while open bars indicate application of the Ca²⁺-free external solution. Dotted lines indicate the zero current level.

Figure 4

Effects of nicardipine-containing media on STOCs and caffeine-induced responses. (a) Effects of nicardipine-containing external solutions on STOCs and I_CAF at a V_H of −20 mV. (b) Effects of nicardipine-containing external solutions on fura-2 microscopic [Ca²⁺]_i signals in the response to caffeine. All traces in (b) are the records from separate gastric smooth muscle cells tested simultaneously. Horizontal closed bars indicate application of caffeine, while striped bars indicate application of nicardipine. Dotted lines indicate the zero current level.

Figure 5

Effects of ryanodine on STOCs and caffeine-induced responses. (a) Effects of ryanodine on STOCs and I_CAF at a V_H of −20 mV. Dotted lines indicate the zero current level. (b) Effects of ryanodine on fura-2 microscopic [Ca²⁺]_i signals in the response to caffeine. All traces in (b) are the records from separate gastric smooth muscle cells tested simultaneously. Horizontal closed and open bars indicate application of caffeine and ryanodine.

Figure 6

Responsiveness to caffeine of gastric smooth muscle strips under (a) control conditions and pretreatment conditions with a (b) 50 mM KCl-containing solution, and (c) dB-cAMP-containing solution. (d) High K⁺ contraction before (left panel) and after (right panel) treatment with dB-cAMP. Inset in (a) enlarges 1 and 3 mM caffeine-induced effects. Arrows indicate cumulative application of caffeine and dB-cAMP. Fifty mM KCl was applied at indicated period.

Figure 7

Difference between the effects of caffeine and CCh. (a) Caffeine- and CCh-induced outward currents at a V_H of −20 mV in gastric smooth muscle cells. (b) Caffeine- and CCh-induced [Ca²⁺]_i elevations measured with fura-2. All traces in (b) are the records from separate gastric smooth muscle cells tested simultaneously. Horizontal closed and open bars indicate application of caffeine and CCh. (c) Effects of caffeine on the contracted gastric smooth muscle with CCh. Arrows indicate cumulative applications of CCh and caffeine and removal of the drugs.

Figure 8

(a) Contraction and relaxation kinetics of high concentration (3 mM) caffeine-induced effects on the tonus under control (upper left panel), TEA (upper right panel)-, 50 mM KCl (lower left panel)- and CCh (lower right panel)-treated conditions. Horizontal closed and open bars indicate application of caffeine and other conditioning media. Dotted lines indicate the baseline. In each panel except for CCh, the same recording is presented at different time scales and both traces are superimposed. (b) τ_C and τ_R under preconditioning with TEA, IbTx, 50 mM KCl and CCh. Numerals in brackets indicate the number of strips tested. Each column represents mean±s.e.mean. ^* Denotes a statistically significant decrease in the time constant (Student's unpaired t-test, P<0.05); ^**denotes a statistically significant decrease in the time constant (Student's unpaired t-test, P<0.01).