Control of Ca²⁺ wave propagation in mouse pancreatic acinar cells

Abstract

We have investigated control mechanisms involved in the propagation of agonist-induced Ca2+ waves in isolated mouse pancreatic acinar cells. Using a confocal laser-scanning microscope, we were able to show that maximal stimulation of cells with acetylcholine (ACh, 500 nM) or bombesin (1 nM) caused an initial Ca2+ release of comparable amounts with both agonists at the luminal cell pole. Subsequent Ca2+ spreading to the basolateral membrane was faster with ACh (17.3 ± 5.4 μm/s) than with bombesin (8.0 ± 2.2 μm/s). The speed of bombesin-induced Ca2+ waves could be increased up to the speed of ACh-induced Ca2+waves by inhibition of protein kinase C (PKC). Activation of PKC significantly decreased the speed of ACh-induced Ca2+ waves but had only little effect on bombesin-evoked Ca2+waves. Within 3 s after stimulation, production of inositol 1,4,5-trisphosphate [Ins(1,4,5) P 3] was higher in the presence of ACh compared with bombesin, whereas bombesin induced higher levels of diacylglycerol (DAG) than ACh. These data suggest that the slower propagation speed of bombesin-induced Ca2+ waves is due to higher activation of PKC in the presence of bombesin compared with ACh. The higher increase in bombesin- compared with ACh-induced DAG production is probably due to activation of phospholipase D (PLD). Inhibition of the PLD-dependent DAG production by preincubation with 0.3% butanol led to an acceleration of the bombesin-induced Ca2+ wave. In further experiments, we could show that ruthenium red (100 μM), an inhibitor of Ca2+-induced Ca2+ release in skeletal muscle, also decreased the speed of ACh-induced Ca2+ waves. The effect of ruthenium red was not additive to the effect of PKC activation. From the data, we conclude that, following Ins(1,4,5) P 3-induced Ca2+ release in the luminal cell pole, secondary Ca2+ release from stores, which are located in series between the luminal and the basal plasma membrane, modifies Ca2+spreading toward the basolateral cell side by Ca2+-induced Ca2+ release. Activation of PKC leads to a reduction in Ca2+release from these stores and therefore could explain the slower propagation of Ca2+ waves in the presence of bombesin compared with ACh.

Agonist-evoked Mitochondrial Ca2+ Signals in Mouse Pancreatic Acinar Cells

“…From the distance between the areas and the delay in the fluorescence increase, we determined the speed of Ca 2ϩ waves. In one set of experiments (n ϭ 13; 38 cells) the mean propagation rate of Ca 2ϩ signals in response to 500 nM ACh was 16.5 Ϯ 0.9 m/s, which is close to the values found in previous studies (8,29). When cells from the same preparation were preincubated for 5 min with 100 M Ruthenium Red, which is an inhibitor of the mitochondrial Ca 2ϩ uniporter (12,24), not a faster but a slower spreading of Ca 2ϩ waves (12.7 Ϯ 0.6 m/s, n ϭ 12; 28 cells) could be observed.…”

“…Cytosolic Ca 2ϩ signals were monitored using the Ca 2ϩ -sensitive dye fluo-3. Cells were loaded with fluo-3/AM at a concentration of 4 M for 30 min at room temperature (8). For simultaneous registration of cytosolic and mitochondrial Ca 2ϩ signals, cells were double-loaded with rhod-2 and fluo-3.…”

“…The same protocol was used for cells double-loaded with rhod-2 and MitoTracker TM Green FM. The propagation rate of ACh-induced Ca 2ϩ waves was determined as described previously (8,29). Cells loaded solely with fluo-3 were excited at 488 nm, and fluorescence images (emission Ͼ 515 nm) of 128 ϫ 128 pixels were taken every 0.25 s. In the luminal and basal cell pole, small rectangular areas were selected, and the speed of ACh-induced Ca 2ϩ waves was calculated from the distance between these areas and the time lag between the increase in [Ca 2ϩ ] cyt at the luminal and basal cell region.…”

Mitochondrial Reactive Oxygen Species Regulate Spatial Profile of Proinflammatory Responses in Lung Venular Capillaries

“…Two agents that inhibit Ca 2ϩ release from ER, namely XeC that inhibits InsP3 receptors (13) and t-BHQ that depletes the stores by blocking ER Ca 2ϩ -ATPase (12,14), inhibited the TNF-␣-induced Ca 2ϩ…”

Induction by bombesin of peritoneal metastasis of gastric cancers induced by N -methyl- N ?-nitro- N -nitrosoguanidine in Wistar rats

“…One possibility is the effect of bombesin on intracellular calcium. Bombesin induced a transient rise in cytoplasmic calcium levels in various normal and tumor cells [14][15][16][17][18][19][20]. Elevated intracellular calcium is thought to be closely related to tumor cell invasion.…”