Src Dependent Pancreatic Acinar Injury Can Be Initiated Independent of an Increase in Cytosolic Calcium

Abstract

Several deleterious intra-acinar phenomena are simultaneously triggered on initiating acute pancreatitis. These culminate in acinar injury or inflammatory mediator generation in vitro and parenchymal damage in vivo. Supraphysiologic caerulein is one such initiator which simultaneously activates numerous signaling pathways including non-receptor tyrosine kinases such as of the Src family. It also causes a sustained increase in cytosolic calcium- a player thought to be crucial in regulating deleterious phenomena. We have shown Src to be involved in caerulein induced actin remodeling, and caerulein induced changes in the Golgi and post-Golgi trafficking to be involved in trypsinogen activation, which initiates acinar cell injury. However, it remains unclear whether an increase in cytosolic calcium is necessary to initiate acinar injury or if injury can be initiated at basal cytosolic calcium levels by an alternate pathway. To study the interplay between tyrosine kinase signaling and calcium, we treated mouse pancreatic acinar cells with the tyrosine phosphatase inhibitor pervanadate. We studied the effect of the clinically used Src inhibitor Dasatinib (BMS-354825) on pervanadate or caerulein induced changes in Src activation, trypsinogen activation, cell injury, upstream cytosolic calcium, actin and Golgi morphology. Pervanadate, like supraphysiologic caerulein, induced Src activation, redistribution of the F-actin from its normal location in the sub-apical area to the basolateral areas, and caused antegrade fragmentation of the Golgi. These changes, like those induced by supraphysiologic caerulein, were associated with trypsinogen activation and acinar injury, all of which were prevented by Dasatinib. Interestingly, however, pervanadate did not cause an increase in cytosolic calcium, and the caerulein induced increase in cytosolic calcium was not affected by Dasatinib. These findings suggest that intra-acinar deleterious phenomena may be initiated independent of an increase in cytosolic calcium. Other players resulting in acinar injury along with the Src family of tyrosine kinases remain to be explored.

Figure 2. Pervanadate induced F-actin reorganization and antegrade Golgi fragmentation is inhibited by Dasatinib.

Fluorescence imaging of acini under basal conditions (Basal, A, E), after stimulation with 100 µM pervanadate (PV) for 10 minutes (B,F) or pervanadate treated acini in the presence of Dasatinib (PV+DAS, C,G) showing F-actin (Red), the Golgi marker GM130 (green) and nuclei (Blue). Under basal conditions F-actin (A) is enriched in the sub-apical area, and the stacks of Golgi (E) in the supranuclear area occupying <25% of the apical-basal axis length (H). 100 µM pervanadate within 10 minutes reorganizes the F-actin to the basal surface (B) and induces antegrade fragmentation of the Golgi (F). The pervanadate induced changes in F-actin (C) and Golgi (G) are prevented in acini pre-incubated with 10 µM Dasatinib. Quantification of integrated density of apical to basal ratios of F-actin staining (D) and ratio of Golgi length in the apical-basal axis to that of the acinar cell (H) from 3 different experiments are shown. The measurement bar (left lower corner) depicts 10 µm.

Figure 1. Src is activated by pervanadate and supramaximal Caerulein.

Western-blot of immunoprecipitated Src after treatment of acini with 100 µM pervanadate (PV) for various times (A), 2 minutes of 100 µM pervanadate with or without pre-incubation with 10 µM Dasatinib (DAS) (B) or 100 nM caerulein (CER) with or without pre-incubation with 10 µM Dasatinib (Das) (C). These were blotted for antibodies to Src PY416 (i.e. active Src, upper panel), and then stripped and blotted for Total Src (SC-18, Lower panel). Corresponding graphs shown on the right show active Src levels (PY-416) as a ratio to total Src (SC-18) depicted as fold change over basal (BAS). Each data point was calculated from 3 or more experiments. For figure 1A, the graph depicts fold increase over BAS at the time of adding the stimulus and the asterisks in the graph depict a p value of ≤0.02. p values for the graphs corresponding to figure 1B, C are mentioned above these.

Figure 3. Dasatinib reduces pervanadate and caerulein induced trypsinogen activation and acinar cell injury.

Trypsin activity is increased in cell homogenates from acini treated with 100 µM pervanadate (PV) (A), 100 nM caerulein (CER) (B) for 30 minutes. Lactate dehydrogenase (LDH) leakage is increased from acini treated with 100 µM pervanadate (PV) (C), 100 nM caerulein (D) for 4 hours. Preincubation with 10 µM Dasatinib (DAS) prevents these phenomena in response to both pervanadate and caerulein (A, B, C, D). BAS; Basal conditions. p-values mentioned in the figure were calculated using the Student’s t-test. Each bar representing mean ± SEM, was calculated from at least 3 different experiments.

Figure 4. Pervanadate and Dasatinib do not affect resting or caerulein induced changes in cytosolic calcium.

Cytosolic calcium levels in Fura-2AM loaded acini measured over 10 minutes. Arrow indicates time of addition of the stimulus. 100 µM pervanadate (PV, blue diamonds) does not result in an increase in resting cytosolic calcium levels, which remain unchanged in its presence, similar to basal acini (BASAL, black triangles). 100 nM Caerulein (100 nM CER, red squares), causes a prompt increase in cytosolic calcium levels, which is not reduced in the presence of 10 µM Dasatinib (100 nM CER+10 µM DAS, pink squares). Each data point represents mean value calculated over multiple (n≥3) experiments, in each of which 7–25 acini per field were analyzed. Standard error of mean is depicted as a bar. Asterisks correspond to time points at which calcium levels were significantly different (p<0.05) from basal levels compared to either conditions with 100 nM Caerulein alone, or to the same in the presence of 10 µM Dasatinib. Calcium levels in conditions with 100 µM Pervanadate were not significantly different from basal.