Carbachol triggers RyR-dependent Ca(2+) release via activation of IP(3) receptors in isolated rat gastric myocytes

Abstract

Possible interactions between different intracellular Ca(2+) release channels were studied in isolated rat gastric myocytes using agonist-evoked Ca(2+) signals. Spontaneous, local Ca(2+) transients were observed in fluo-4-loaded cells with linescan confocal imaging. These were blocked by ryanodine (100 microM) but not by the inositol 1,4,5-trisphosphate receptor (IP(3)R) blocker, 2-aminoethoxydiphenyl borate (100 microM), identifying them as Ca(2+) sparks. Caffeine (10 mM) and carbachol (10 microM) initiated Ca(2+) release at sites which co-localized with each other and with any Ca(2+) spark sites. In fura-2-loaded cells extracellular 2-aminoethoxydiphenyl borate and intracellular heparin (5 mg ml(-1)) both inhibited the global cytoplasmic [Ca(2+)] transient evoked by carbachol, confirming that it was IP(3)R-dependent. 2-Aminoethoxydiphenyl borate and heparin also increased the response to caffeine. This probably reflected an increased Ca(2+) store content since 2-aminoethoxydiphenyl borate more than doubled the amplitude of transients evoked by ionomycin. Ryanodine completely abolished carbachol and caffeine responses but only reduced ionomycin transients by 30 %, suggesting that blockade of carbachol transients by ryanodine was not simply due to store depletion. Double labelling of IP(3)Rs and RyRs demonstrated extensive overlap in their distribution. These results suggest that carbachol stimulates Ca(2+) release through co-operation between IP(3)Rs and RyRs, and implicate IP(3)Rs in the regulation of Ca(2+) store content.

Figure 1. Spontaneous Ca²⁺ release events in a fluo-4-loaded myocyte

Fluo-4 fluorescence was recorded from the scanline indicated on the cell outline to the right of the top panel and normalized to initial, control fluorescence levels (F/F₀, see colour code). Distance is plotted vertically and time horizontally. A, images from a cell showing spontaneous, localized Ca²⁺ transients were seen in 3 mm external [Ca²⁺] (upper panel). These were completely blocked following 2 min exposure to ryanodine (100 μm, lower panel). B, images from a different cell showing spontaneous Ca²⁺ transient activity under control conditions (3 mm external Ca²⁺, upper panel) and in the presence of 2-aminoethoxydiphenyl borate (100 μm for 2 min, lower panel).

Figure 5. Effects of ryanodine on store release by carbachol and caffeine

Evoked [Ca²⁺]_c responses in a fura-2-loaded myocyte before and during application of ryanodine (100 μm).

Figure 2. Comparison of Ca²⁺ spark sites with carbachol and caffeine-sensitive release sites in a single myocyte

A, normalized fluo-4 fluorescence (F/F₀, see colour code) was recorded from the scanline indicated on the cell outline. Spontaneous Ca²⁺ sparks were seen (top panel). Applications of carbachol (10 μm in zero Ca²⁺, middle panel) and caffeine (10 mm in zero Ca²⁺, bottom panel) were separated by a wash period (100 s) to allow store refilling. B, F/F₀ plotted against distance along the scanline at four time points (i-iv in A). Early responses to carbachol (middle graph) and caffeine (bottom graph) correspond closely in position. Peripheral peaks also correspond to the Ca²⁺ spark sites (top graph).

Figure 3. Effects of 2-aminoethoxydiphenyl borate on store release by carbachol and caffeine

2-Aminoethoxydiphenyl borate (2APB, 100 μm) inhibited the global [Ca²⁺]_c response to carbachol (cch, 10 μm in zero Ca²⁺) but enhanced the caffeine (caff, 10 mm in zero Ca²⁺) response in a myocyte loaded with fura-2.

Figure 4. Effects of heparin on store release by carbachol and caffeine

Heparin was dialysed into fura-2-loaded myocytes from a patch electrode (5 mg ml⁻¹ in pipette solution). Cells were exposed to either carbachol (cch, 10 μm, record A) or caffeine (caff, 10 mm, record B) immediately after rupture of the membrane subjacent to the patch pipette and at approximately 100 s intervals thereafter. C, summary of the mean amplitude (± s.e.m.) of carbachol- (○) and caffeine (•)-dependent transients, normalized to the initial evoked transient in each case. The [Ca²⁺]_c response evoked by carbachol was inhibited within 5 min while the response to caffeine increased in amplitude with time after membrane rupture.

Figure 6. Effects of 2-aminoethoxydiphenyl borate and ryanodine on store content as assessed by ionomycin-evoked release

A, the [Ca²⁺]_c transient evoked from a fura-2-loaded myocyte by ionomycin (25 μm in zero Ca²⁺) was increased in the presence of 2-aminoethoxydiphenyl borate (2ABP, 100 μm). B, bar chart summarizing the mean amplitude (± s.e.m.) of the ionomycin-dependent transient in six cells under control conditions and in the presence of 2-aminoethoxydiphenyl borate (* P < 0.005). C, ionomycin-evoked [Ca²⁺] transients in a myocyte before (control) and during superfusion with ryanodine (100 μm). D, summary data from nine cells exposed to the protocol illustrated in C (* P < 0.02).

Figure 7. Distribution of Ca²⁺ release channels

Distribution of IP₃Rs and RyRs in a single x,y plane from a gastric myocyte double labelled with polyclonal anti-IP₃R antibody (green) and fluorescently labelled ryanodine (orange).