Rho and Rac promote acinar morphological changes, actin reorganization, and amylase secretion

Yan Bi, Sophie Le Page, John A. Williams

Abstract

Supramaximal stimulation of isolated pancreatic acini with specific agonists such as CCK induces the formation of large basolateral blebs, redistributes filamentous actin, and inhibits secretion. Rho family small G proteins are well documented for their function in actin reorganization that determines cell shape and have been suggested to play a role in secretion. Here, we determined whether Rho and Rac are involved in the morphological changes, actin redistribution, and inhibition of amylase secretion induced by high concentrations of CCK. Introduction of constitutively active RhoV14 and RacV12 but not Cdc42V12 in mouse pancreatic acini by adenoviral vectors stimulated acinar morphological changes including basolateral protrusions, increased the total amount of F-actin, and reorganized the actin cytoskeleton. Dominant-negative RhoN19, Clostridium botulinum C3 exotoxin, which inhibits Rho, and dominant-negative RacN17 all partially blocked CCK-induced acinar morphological changes and actin redistribution. To study the correlation between actin polymerization and acinar shape changes, two marine toxins were employed. Jasplakinolide, a reagent that facilitates actin polymerization and stabilizes F-actin, stimulated acinar basolateral protrusions, whereas latrunculin, which sequesters actin monomers, blocked CCK-induced acinar blebbing. Unexpectedly, RhoV14, RacV12, and jasplakinolide all increased amylase secretion by CCK from 30 pM to 10 nM. The data suggest that Rho and Rac are involved in CCK-evoked changes in acinar morphology, actin redistribution, and secretion and that inhibition of secretion by high concentrations of CCK is not directly coupled to the changes in acinar morphology.

  • pancreas
  • latrunculin
  • jasplakinolide

pancreatic acinar cells synthesize and release digestive enzymes and have been used for decades as a model to study secretion. Freshly isolated resting acinar cells are pyramidal shaped with a well-defined apical region filled with zymogen granules (ZGs) that contain digestive enzymes. Filamentous actin (F-actin) is mainly located in the subapical region surrounding the lumen where it forms the terminal web with a small amount under the basolateral membrane (36). Cholecystokinin (CCK) acts on acinar cells as a secretagogue through receptors coupled to Gq to increase intracellular calcium and elicit amylase secretion. Secretion follows a bell-shaped dose-response curve, as increasing the concentration of secretagogues stimulates amylase release until the maximal release of amylase is obtained; higher concentrations of secretagogues cause a progressive decrease in amylase release (51). In addition, supramaximal concentrations of CCK induce massive morphological changes in isolated acini with one of the earliest being prominent basolateral blebs, balloon-like basolateral protrusions of the cytoplasm containing rough endoplasmic reticulum, free ribosomes, and mitochondria (1, 8, 37). Superphysiological stimulation also elicits obvious F-actin reorganization, including dissociation of actin filaments in the terminal web and in microvilli, redistribution of F-actin to basal lateral membrane to form basolateral F-actin rings (8, 36, 37, 41), and, ultimately, loss of cell polarity. This basolateral bleb formation and actin reorganization have been proposed to have pathophysiological significance, because high doses of CCK or the CCK analog caerulein, which induce acute pancreatitis in rodents (26), also lead to actin cytoskeleton reorganization and loss of cell polarity (21).

Several lines of evidence suggest that actin cytoskeletal reorganization is involved in the process of bleb formation induced by high concentrations of CCK. Cytochalasin B and cytochalasin D, agents that cap the barbed end of actin filaments thereby inhibiting association of subunits and disrupting microfilaments, are able to block bleb formation and retraction (8, 46). Bleb formation is also inhibited by the myosin ATPase inhibitor butanedione monoxime and the myosin light chain kinase inhibitor ML-9, suggesting that myosin II is also required (46). However, the precise molecular mechanisms responsible for these agonist-induced morphological changes remain undefined.

Rho family small G proteins, which can be activated by a variety of growth factors and hormones including CCK, act as molecular switches to relay extracellular stimuli in the cell to a large number of downstream effectors. Among them, RhoA, Rac1, and Cdc42 are best characterized for their ability to reorganize the actin cytoskeleton. In fibroblasts, Rho regulates the formation of actin stress fibers and focal adhesion, Rac is involved in membrane ruffling and lamellipodia, and Cdc42 participates in receptor-induced filopodia (35, 39, 40). We have demonstrated that Rho and Rac are activated by CCK in both NIH3T3 cells stably transfected with the CCK-A receptor (28) and in pancreatic acinar cells (4). However, their functions in regulating acinar morphology still remain largely unknown, although one report in permeabilized acinar cells implicated Rho involvement in CCK-induced acinar morphological changes based on the blockage with C3 toxin (24).

Actin has been proposed to play both positive and negative roles in secretion. Rho, Rac, and Cdc42 have been implicated in the regulation of exocytosis in a variety of cells, including pancreatic β-cells, mast cells, parietal cells, PC-12 cells, chromaffin cells, and neurons (6, 12, 15, 25, 45). In pancreatic acini, introduction of low concentrations of the actin monomer-binding protein β-thymosin in permeablilized acini elicited rapid and robust apical exocytosis independent of Ca2+ mobilization, whereas introduction of the F-actin-stabilizing agent phalloidin into streptolycin O (SLO)-permeabilized acinar cells or treatment of intact cells with jasplakinolide abolished regulated secretion (32). These data suggest that actin depolymerization is sufficient to induce secretion and that actin plays a negative role in secretion. On the other hand, cytochalasin B causes disappearance of the apical “terminal web” and reduces stimulated amylase release, suggesting that actin is necessary for secretion and may play an active role in secretion (50). Dominant-negative RhoN19, dominant-negative RacN17, and C3 exotoxin all significantly inhibited physiological amylase secretion induced by CCK and carbachol (4), suggesting that Rho and Rac are both involved in CCK-induced amylase release, possibly through an effect on the actin cytoskeleton. Whether Rho family G proteins play a role in the inhibition of amylase secretion in response to supramaximal CCK is unclear. Therefore, the aim of this study was to elucidate the role of Rho family G proteins in high-dose CCK-induced acinar morphological changes, including actin reorganization, blebbing, and the accompanying inhibition of amylase secretion.

EXPERIMENTAL PROCEDURES

Materials.

Collagenase was purchased from Worthington (Lakewood, NJ) and Crescent Chemical (Islandia, NY). Rhodamine phalloidin, jasplakinolide, and latrunculin were from Molecular Probes (Eugene, OR), and sulfated CCK octapeptide was from Research Plus (Bayonne, NJ). Triple hemaglutinin-tagged RhoAN19, RhoAV14, Rac1N17, Rac1V12, and Cdc42V12 in the PKH3 plasmid (30) were obtained from Ian Macara (University of Virginia, Charlottesville, VA) and C3 plasmid from Silvio Gutkind (NIH, Bethesda, MD). All other chemical reagents were obtained from Sigma.

Construction of recombinant adenoviruses.

The recombinant adenoviruses encoding RhoN19, RhoV14, RacN17, RacV12, Cdc42V12, and C3 exotoxin were constructed according to the method of He et al. (18), as described previously (9). The titers of the viral stocks were estimated by counting enhanced green fluorescent protein (EGFP)-expressing cells as pAdTrack-CMV encodes EGFP driven by a separate cytomegalovirus (CMV) promoter. An adenovirus expressing β-galactosidase (β-Gal) and EGFP was used as a control.

Preparation, short-term culture, and viral infection of pancreatic acini.

Mouse pancreatic acini from ICR mice were prepared by enzymatic digestion with collagenase followed by mechanical shearing, as previously described (52, 53). Acini were cultured in suspension without shaking at low density in 10-cm petri dishes in DMEM enriched with 0.5% BSA, 0.01% soybean trypsin inhibitor, and antibiotics and incubated at 37°C with 5% CO2 for up to 16 h. For the viral infection experiments, 106 plaque-forming units (PFU)/ml constitutively active or 107 PFU/ml dominant-negative mutants and C3 exotoxin were added to the culture medium at the beginning of the incubation unless otherwise described.

Quantitation of blebs.

Freshly isolated acini or overnight adenovirus-infected acini were stimulated with or without CCK for 30 min. Digital acinar images were obtained in a plastic dish by means of a Nikon inverted microscope with a ×20 objective and a Spot camera (Diagnostic Instruments, Sterling Heights, MI). Blebs were counted by hand, and the perimeter of acini was measured using ImageJ software (Wayne Rasband, NIH). The density of blebbing was expressed as the number of blebs per millimeter perimeter of acini. Results were presented as means ± SE from at least three independent experiments in each of which at least 30 acini were analyzed.

F-actin staining.

Isolated mouse pancreatic acini were fixed with 4% paraformaldehyde in PBS, pelleted, and frozen. Cryostat sections were then rehydrated on slides and permeabilized in 0.1% Triton X-100, and F-actin was stained with rhodamine-phalloidin diluted 1:400 (41). Images were taken with an Olympus Fluoview 500 confocal laser scanning microscope utilizing an argon laser and a ×63 objective to produce an approximate optical section thickness of 0.1 μm. Laser energy and parameters of intensity detection were kept constant for all slides within an experiment, and images were processed under similar conditions using Photoshop 6.0 (Adobe Systems, Mountain View, CA). The images in this study represent typical acini from at least three cell preparations.

F-actin quantitation.

Changes in F-actin content were measured as described previously (41). Briefly, acini were fixed for 15 min with 3.7% formaldehyde. Acini were then pelleted, and resuspended in 0.1% saponin, and incubated with 0.7 μM rhodamine-conjugated phalloidin for 60 min in darkness on a rotator. Stained pellets were washed three times with buffer containing saponin after which the rhodamine-conjugated phalloidin was extracted with methanol. Protein was measured with the Bio-Rad protein assay. The fluorescence of extracts was measured using excitation at 541 nm and emission at 565 nm with a Perkin-Elmer LS 55 luminescence spectrometer (Perkin-Elmer, Wellesley, MA). The relative F-actin content was calculated as the ratio of the fluorescence emission per microgram of protein divided by the fluorescence emission per microgram of protein of the control sample.

Analysis of agonist-stimulated amylase secretion.

After incubation for 16 h with adenovirus or preincubation for 30 min for fresh acini, acini were allowed to settle by gravity, resuspended in HEPES-Ringer buffer, and then incubated with various concentrations of CCK or carbachol at 37°C in 1-ml aliquots in plastic vials that were shaken at 50 cycles/min. After 30 min, the acinar suspension was centrifuged for 30 s in a microcentrifuge, and supernatant was assayed for amylase activity using Infinity Amylase reagent (Thermo Electron, Woburn, MA) in a μQuant plate reader (Bio-tek instruments, Winooski, VT). Amylase release was expressed as a percentage of total acinar amylase content.

Statistical analysis.

Results are expressed as means ± SE. Multiple comparisons in the dose-response curves were performed using an ANOVA with the Dunnett's test for comparison with control using Graphpad Prism software. P < 0.05 represents significance.

RESULTS

High concentrations of CCK induce acinar morphological changes in fresh and overnight-cultured pancreatic acini.

In the resting state, freshly isolated mouse pancreatic acinar cells are highly polarized, with a smooth and well-defined basolateral membrane (Fig. 1A). Supramaximal concentrations of CCK (1 and 10 nM) induced dramatic morphological changes by forming numerous basolateral protrusions often referred to as blebs (Fig. 1, B and C), as have been reported previously (1, 8, 37). Of note, 10 nM CCK also stimulated pinching off of blebs. As an effort to determine the underlying molecular mechanism of this phenomenon, we used adenovirus to express dominant-active and -negative mutant Rho family proteins in overnight-cultured pancreatic acini. First, we demonstrated that a high concentration of CCK stimulated acinar shape changes in overnight-cultured cells. Isolated pancreatic acini were cultured with or without adenovirus expressing β-Gal for 16 h and then stimulated with 1 or 10 nM CCK for 30 min at 37°C before morphological changes were visualized using an inverted microscope. In overnight-cultured acini infected with adenovirus expressing β-Gal (Fig. 1D) or left uninfected (data not shown), the basolateral membrane remained smooth and well defined, although occasional blebs were observed. After CCK stimulation (30 min), both infected (Fig. 1, E and F) and uninfected acini (data not shown) developed massive basolateral protrusions. Again, some floating blebs were observed when cells were treated with 10 nM CCK. These results suggested that adenovirus itself had minimal effects on acinar morphology and the response to CCK. Because 10 nM CCK had a damaging effect on acinar cells and 1 nM induced a similar response, we chose the latter concentration for further study.

Fig. 1.

Suprastimulation by CCK induces acinar morphological changes in fresh and overnight-cultured pancreatic acini. Freshly isolated mouse pancreatic acini were stimulated without CCK (A) or with 1 nM (B) or 10 nM (C) CCK for 30 min. Isolated acini were also cultured with adenovirus expressing β-galactosidase (β-Gal) for 16 h (D) before stimulation with 1 nM CCK (E) or 10 nM CCK (D) for 30 min. Representative light microscope images are shown. Arrows denote the basolateral protrusions. Bar = 100 μm.

Active Rho and Rac both induce acinar morphological changes.

Rho and Rac have been reported to promote reorganization of the actin cytoskeleton and to regulate cell morphology in a variety of cells. Therefore, we hypothesized that Rho and Rac were involved in CCK-mediated acinar morphological changes in pancreatic acini. Isolated acini were cultured with adenovirus-expressing β-Gal, active Rho V14, or active Rac V12 for 16 h, and cell images were obtained. To infect ∼50% of the cells, we reduced the amount of adenovirus to 0.5 × 106 PFU/ml compared with 106 PFU/ml used for other experiments where homogeneous infection was required. The efficiency of viral infection and protein expression were monitored by EGFP-expressing cells, because each adenovirus possesses a second CMV promoter to drive EGFP expression. There was no difference between EGFP-expressing cells and non-EGFP-expressing cells when they were infected with adenovirus expressing β-Gal; all acini showed normal morphology with well-defined basolateral membrane (Fig. 2). However, cells strongly expressing EGFP and therefore active RhoV14 or Rac V12 showed obvious basolateral protrusions compared with uninfected cells (Fig. 2). By contrast, cells expressing active Cdc42V12 did not show obvious acinar morphological changes (data not shown).

Fig. 2.

RhoV14 and RacV12 both induce acinar basolateral protrusions. Isolated acini were cultured with 0.5 × 106 plaque-forming units (PFU)/ml adenovirus expressing β-Gal, RhoV14, or RacV12 for 16 h. Top: representative microscopic images. Bottom: respective fluorescence images. Enhanced green fluorescent protein (EGFP) expression indicates adenoviral infection of cells. White arrows indicate acini that strongly express EGFP and therefore the indicated protein β-Gal, RhoV14, or RacV12. Bar = 100 μm.

With the use of 106 PFU/ml to infect essentially 100% of cells, in three experiments, RhoV14 induced a bleb density of 11.4 ± 2.1 blebs/mm and RacV12 13.4 ± 3.0 blebs/mm. When RhoV14 and RacV12 were added simultaneously, the bleb density was increased to 16.1 ± 4.4 blebs/mm, although no significant difference was detected between either RhoV14 or RacV12 alone and the combination of RhoV14 and RacV12.

C3 greatly blocks active Rho- but only modestly blocks CCK-induced acinar morphological changes.

To further determine if Rho is involved in acinar blebbing, we employed C3 toxin, which specifically ADP ribosylates and inactivates Rho (43). C3 transferase alone had minimal effects on acinar morphology and bleb density in control acini, but almost totally blocked the effect of RhoV14 to induce morphological changes (Fig. 3A). By contrast, C3 had no effect on acini expressing RacV12 (data not shown). Quantitation revealed that C3 decreased RhoV14-induced bleb density by 85% from 10.4 ± 2.0 to 1.5 ± 0.2 blebs/mm. However, C3 only decreased the number of blebs induced by CCK by 61% from 24.1 ± 3.6 to 9.4 ± 2.2 blebs/mm (Fig. 3B). These data suggest that both Rho and Rac may be involved in the CCK effect but may not fully explain the effect of CCK. Higher amounts of both adenovirus and CCK increased the number of blebs and caused their detachment, making quantitation difficult.

Fig. 3.

C3 greatly blocks active Rho but only partially blocks CCK-induced acinar morphological changes. Isolated acini were cultured with adenovirus expressing β-Gal, C3, RhoV14, or the combination of RhoV14 and C3 for 16 h before some of them were stimulated with 1 nM CCK for 30 min. Top: representative microscopic images. Bottom: quantitation of the number of blebs/mm acinar perimeter from 3 independent experiments. In this and the following experiments, isolated pancreatic acini were cultured with 106 PFU/ml adenovirus, and all acini strongly express EGFP. *P <0.05 compared with RhoV14; †P < 0.05 compared with β-Gal + CCK. Bar = 100 μm.

Dominant-negative Rho and Rac partially block CCK-induced acinar morphological changes.

To further test the role of Rho and explore the role of Rac in acinar shape changes induced by high concentrations of CCK, we examined the effects of dominant-negative RhoN19 and dominant-negative RacN17. Acini cultured with adenovirus expressing RhoN19 or RacN17 did not significantly increase the bleb density compared with acini cultured with adenovirus expressing β-Gal. However, dominant-negative RhoN19 inhibited CCK-induced acini blebbing and decreased the density of blebbing by 59%, similar to that of C3 exotoxin (Fig. 4). RacN17 also reduced the density of blebbing stimulated by 1 nM CCK by 43.3% compared with acini expressing β-Gal, suggesting that Rac also plays a role in CCK-induced morphological changes. Inhibition of Rho and Rac together by using the combination of C3 and RacN17 or RhoN19 and RacN17, however, showed no further decrease in blebbing density (Fig. 4).

Fig. 4.

RhoN19 and RacN17 partially block acinar morphological changes induced by high concentrations of CCK. A: isolated mouse pancreatic acini were cultured with adenovirus expressing the indicated proteins for 16 h (top) before some of them were stimulated with 1 nM CCK (bottom) for 30 min. Representative microscopic images are shown. B: quantitation of the number of blebs/mm perimeter of acini from 3 independent experiments. *P < 0.05 compared with acini infected with β-Gal and stimulated with CCK. Bar = 100 μm.

Jasplakinolide alone induces acinar basolateral protrusions whereas latrunculin blocks high-dose CCK-stimulated blebs.

Because the major function of Rho and Rac is to stimulate actin polymerization, we hypothesized that Rho and Rac may induce acinar blebbing by facilitating actin polymerization. To manipulate actin dynamics without activating other signal transduction pathways, two toxins were employed. Jasplakinolide is a membrane-permeable toxin from the red sea sponge that stimulates actin polymerization and stabilizes F-actin (7). Latrunculin, another marine toxin, specifically binds to G-actin near the nucleotide-binding site, thereby preventing G-actin polymerization and ultimately leading to actin depolymerization (10, 44). Acini showed prominent basolateral protrusions after 30 min of incubation with 1 μM jasplakinolide (Fig. 5). Latrunculin (3 μM) treatment alone altered the conformation of the basolateral membrane but did not stimulate bleb formation. However, latrunculin prevented the basolateral protrusions induced by 30 min of stimulation with 1 nM CCK (Fig. 5). Prevention of blebs by latrunculin was also observed in confocal images from cryostat sections stained with rhodamine phalloidin (data not shown).

Fig. 5.

Jasplakinolide (Jas) alone induces blebs but latrunculin (Lat) blocks blebs stimulated by high concentrations of CCK. Freshly isolated acini were left as control or pretreated with latrunculin (3 μM) or jasplakinolide (1 μM) for 30 min (top) before being stimulated with 1 nM CCK for 30 min (bottom). Representative microscopic images are shown. White arrows indicate basolateral blebs. Bar = 100 μm.

High-dose CCK, RhoV14, and RacV12 all induce actin rearrangement.

The actin cytoskeleton is one of the major determinants of cell morphology. In pancreatic acini, F-actin redistribution has been suggested to correlate with cell shape changes (46). To study the effect of high concentrations of CCK or active Rho and active Rac on actin cytoskeleton reorganization, F-actin was stained with rhodamine phalloidin and evaluated using fluorescence confocal microscopy. Isolated acini were cultured with virus expressing either β-Gal, RhoV14, or RacV12 for 16 h before the acini cultured with β-Gal were stimulated with CCK (1 nM) for 30 min. Control overnight-cultured acini expressing β-Gal showed normal distribution of F-actin, which is mainly located to the apical region of the acini, with the basolateral region having much less F-actin (Fig. 6). Both RhoV14 and RacV12 significantly changed the actin cytoskeleton by enhancing the basolateral F-actin staining, altering cell polarity and increasing the total F-actin of the acini (Fig. 6). By contrast, no significant actin reorganization was observed in acini infected with adenovirus expressing Cdc42V12 (data not shown). Because of the difference in sizes among acini, it was difficult to quantitate the amount of total F-actin using the confocal images. We therefore extracted rhodamine phalloidin from acini as described in experiment procedures to evaluate the total amount of F-actin after different treatments. CCK (1 nM), RhoV14, and RacV12 but not Cdc42V12 increased the total amount of F-actin in acini compared with control β-Gal (Fig. 7). However, none of the constitutively active G proteins affected the total actin determined by Western blotting (data not shown) after overnight culture, suggesting that Rho and Rac alter actin polymerization dynamics without changing the total actin content.

Fig. 6.

RhoV14 and RacV12 induce acinar actin reorganization, and C3 blocks RhoV14 but not RacV12 effects on actin rearrangement. Isolated mouse pancreatic acini were cultured with adenovirus expressing β-Gal, C3 exotoxin, RhoV14 or RacV12, and RhoV14 or RacV12 with C3 for 16 h. Acini were collected, and frozen secretion were cut and stained with rhodamine phalloidin. Representative confocal microscope images are shown. C3 itself had minimal effects on actin redistribution, whereas RhoV14 and RacV12 induced basolateral protrusions (denoted by arrows) and enhanced basolateral actin staining (denoted by arrowheads). C3 blocked bleb formation by RhoV14 but not RacV12; however, C3 failed to inhibit actin enhancement at the basolateral membrane. Bar = 100 μm.

Fig. 7.

C3, RhoV14, and RacV12 but not Cdc42V12 increase the total amount of F-actin of acini. Isolated mouse pancreatic acini were cultured with adenovirus expressing the indicated proteins for 16 h before some infected with β-Gal control virus were stimulated with 1 nM CCK for 30 min. Total F-actin content was quantitated based on the binding of rhodamine phalloidin, as described in experimental procedures. Results are expressed as means ± SE from 3 independent experiments. **P < 0.01 and ***P < 0.001 compared with β-Gal.

C3 blocks RhoV14 but not RacV12 effects on actin reorganization.

To determine whether Rac activates Rho to alter the actin cytoskeleton, C3 was used to specifically inhibit Rho but not Rac. C3 alone had minimal effects on the actin cytoskeleton compared with control acini; however, it greatly blocked the alteration in cell structure and loss of subapical actin induced by RhoV14, although not preventing the increase in basolateral actin staining as shown in Fig. 6. By contrast, C3 had minimal effects on RacV12-induced acinar reorganization and blebbing as indicated in Fig. 6. Thus the effects of Rho and Rac are most likely independent, and Rac does not act downstream of Rho. We also determined the effects of C3 on total F-actin induced by RhoV14 and RacV12, and C3 failed to significantly reduce the increase in F-actin induced by either RhoV14 or RacV12. However, C3 totally blocked the effects of RhoV14 on serum response element activity measured by a luciferase reporter (Bi and Williams, unpublished data).

C3 and dominant-negative Rho and Rac partially block CCK-induced acinar actin reorganization.

To determine whether Rho or Rac is involved in high-dose CCK-induced acinar actin reorganization, we infected acini with either C3 or dominant-negative Rho or Rac for 16 h followed by CCK stimulation. C3, RhoN19, RacN17, and the combination of C3 or RhoN19 with RacN17 all partially prevented high-dose CCK-induced actin reorganization (Fig. 8) by preserving cell polarity and inhibiting basolateral protrusions.

Fig. 8.

RhoN19 and RacN17 partially block high-dose CCK-induced acinar actin reorganization. Isolated mouse pancreatic acini were cultured with adenovirus expressing the indicated proteins for 16 h before being stimulated with 1 nM CCK for 30 min. F-actin was stained with rhodamine phalloidin. Representative confocal microscope images are shown. Bar = 100 μm.

Overexpression of RhoV14 and RacV12 enhances high-dose CCK-stimulated amylase release.

We next studied the effects of active Rho and Rac on CCK-induced amylase release. RhoV14 and RacV12 did not affect basal release but enhanced amylase release by concentrations of CCK from 30 pM to 10 nM. At a CCK concentration of 1 nM, RhoV14 and RacV12 increased stimulated amylase secretion by 113 and 104%, respectively (Fig. 9). By comparison, Cdc42V12 slightly inhibited amylase secretion throughout the entire CCK concentration-response curve (data not shown). These data were in agreement with earlier data showing that dominant-negative Rho and Rac decreased acinar secretory response to CCK (4) and further suggested that Rho and Rac play a positive role in regulated secretion in acini.

Fig. 9.

RhoV14 and RacV12 increase amylase release induced by CCK. Isolated mouse pancreatic acini were cultured with adenovirus expressing the indicated proteins for 16 h before stimulation with CCK at the indicated concentration. Amylase release as a percentage of total content is shown as mean ± SE of 3 or 4 independent experiments in each of which amylase release was measured in duplicate at each concentration. *P < 0.05 and **P < 0.01 compared with β-Gal.

Jasplakinolide mimics the effect of RhoV14 and RacV12 on secretagogue-induced amylase secretion.

We have shown that both RhoV14 and RacV12 stimulated the actin polymerization and increased the total F-actin content. Therefore, Rho and Rac may affect amylase secretion by increasing actin polymerization or stabilizing F-actin. We then evaluated the effect of jasplakinolide (1 μM) on CCK-induced amylase release and found that jasplakinolide had a similar effect on amylase secretion to RhoV14 or Rac V12 in that it had no effect on basal release, minimally affected low-dose CCK-induced amylase secretion, but significantly enhanced supramaximal CCK-induced amylase release from 300 pM to 10 nM CCK (Fig. 10). However, increasing the concentration of jasplakinolide to 3 μM inhibited amylase secretion across the dose curve (data not shown), as reported previously (49). Thus the effects of jasplakinolide are very concentration dependent. We also studied the action of latrunculin, an actin monomer sequester, on amylase secretion and found that latrunculin decreased amylase release stimulated by CCK throughout the dose-response curve, as reported previously (4, 47).

Fig. 10.

Jasplakinolide can mimic the effects of RhoV14 and RacV12 on amylase release. Freshly isolated acini were preincubated with jasplakinolide (1 μM) for 30 min before stimulation with the indicated concentrations of CCK for amylase assay. Results are expressed as means ± SE from 4 independent experiments. *P < 0.05 and **P < 0.01 compared with control.

DISCUSSION

High concentrations of CCK induce significant actin cytoskeletal reorganization, alter cell morphology, and inhibit secretion in pancreatic acini (8, 36, 37, 41). However, the underlying molecular basis remains poorly understood. Rho and Rac are well characterized for their role in rearranging the actin cytoskeleton, which is closely related with cell shape, and have been suggested to be involved in secretion. To determine whether Rho and Rac play a role in the effects induced by high concentrations of CCK, we expressed dominant-active and -negative mutant Rho and Rac proteins in pancreatic acini by means of adenoviral vectors. Constitutively active Rho and Rac each stimulated acinar morphological changes, including blebbing and actin redistribution, and increased the total amount of F-actin in acini, whereas dominant-negative Rho, Rac, and C3 exotoxin all partially blocked acinar shape changes and actin rearrangement induced by high concentrations of CCK. Similar acinar shape changes were induced by jasplakinolide, a reagent that polymerizes and stabilizes F-actin, whereas CCK-induced changes were blocked by latrunculin, a reagent that prevents actin polymerization. These data suggest that actin polymerization is critical for acinar morphological changes and that Rho and Rac may cause acinar shape changes by inducing actin polymerization. We also studied the effects of active Rho and Rac on amylase secretion induced by CCK. RhoV14, RacV12, and jasplakinolide all enhanced secretion induced by concentrations of CCK from 30 pM to 10 nM. We concluded that Rho and Rac are involved in CCK-evoked acinar morphological changes, actin reorganization, and amylase secretion and that inhibition of secretion by high concentrations of CCK is not directly coupled to the changes in acinar morphology.

Supramaximal stimulation of pancreatic acinar cells with specific agonists such as CCK induces the formation of large basolateral blebs (1, 8, 37). The actin cytoskeleton is one of the major determinants of cell morphology and has been suggested to be critical for membrane blebbing, since cytochalasin B and D reduced F-actin and decreased bleb formation and retraction (8, 17, 46). CCK (1 nM) induced a rapid decrease in total F-actin content that was maximal by 1 min followed by a significant increase in total F-actin content (41). In the present study, we used two marine toxins that manipulate actin dynamics to further study the role of actin in acinar morphological changes. Jasplakinolide, a reagent that stimulates actin polymerization (7), induced massive basolateral protrusions. By contrast, the blebs evoked by high concentrations of CCK were blocked by latrunculin, which sequesters actin monomers and thereby blocks polymerization. Our data confirmed and extended previous observations and suggested that actin polymerization is sufficient and necessary for bleb formation induced by high concentrations of CCK.

Rho and Rac have been linked to membrane blebbing in other cells. Constitutively active RhoA stimulated blebbing in lens cells (20). C3 exotoxin was shown to inhibit blebbing induced by ATP in macrophages (38) and nucleotide receptor-transfected 293 cells (31). Expression of an active Rac mutant Q61L/F37A in NIH3T3 cells also induced extensive blebbing (42). In pancreatic acinar cells, Rho and Rac appear to be involved in high-dose CCK-induced acinar morphological changes and actin cytoskeleton reorganization, because dominant-active RhoV14 or RacV12 stimulated acinar basolateral protrusions and enhanced actin redistribution, whereas C3 exotoxin, dominant-negative RhoN19, or dominant-negative RacN17 all partially decreased the blebbing density and actin rearrangement evoked by CCK. In addition, RhoV14 and RacV12 both increased the total amount of F-actin in pancreatic acini to an even greater extent than CCK, which had a similar effect to that previously reported in fresh acini (41). Thus RhoV14 and RacV12 may promote basolateral protrusions through facilitating actin polymerization and redistribution. In our experiments, C3 exotoxin did not totally block CCK-induced acinar blebbing, which differs from a previous study where C3 protein was reported to completely inhibit CCK effects in permeabilized acini (23). The different experimental systems used may explain the discrepancy between results: we used intact overnight-cultured pancreatic acini infected with adenovirus expressing C3 exotoxin, and the previous study used digitonin-permeabilized acini (23) in which key regulatory proteins that are normally tethered at intracellular locations may leak out, as has been reported for SLO-permeabilized cells (6). The failure of C3 exotoxin to totally block CCK effects on acinar morphology also indicates that other regulators, including Rac, may also play a role, which is supported by our data. However, other mediators besides Rho and Rac may also be involved in bleb formation, because the effect of CCK is greater than active Rho or Rac and the combination of dominant negatives do not fully inhibit. Hydrostatic pressure has been reported to be responsible for bleb formation and motility in Walker carcinoma cells (14). In addition, the weak connection between cytoskeleton and membrane is also suggested to play a role in bleb formation, because mutant cells lacking actin-binding protein (an actin cross-linking protein) showed prolonged blebbing (11). In isolated pancreatic acini, the degradation of plectin, a member of the plakin family of cytoskeletal proteins that act as molecular bridges within the cytoskeleton and between the cytoskeleton and membrane adhesion complexes, has been reported to precede the F-actin breakdown (2).

Blebbing has also been widely observed in both physiological and pathological situations, such as during mitosis (5, 27, 33), cell spreading (3, 11, 13, 19) and migration (22, 48), apoptosis, and necrosis (27). However, it is still under debate whether the blebbing induced by high concentrations of CCK is apoptotic or not. Apoptosis has been reported in both human pancreatitis and rodent pancreatitis models induced by administration of high-dose CCK or its analog cerulean. In vitro studies showed that high concentrations of CCK may induce apoptosis by activating caspase-3, -8, and -9, releasing cytochrome c from the mitochondria and inducing DNA fragmentation (2, 16). These data suggested that the blebbing induced by high concentrations of CCK is a pathophysiological phenomenon. On the contrary, a study by Torgenson and McNiven (46) showed that the blebs induced by high concentrations of CCK are reversible, dynamic, and require ATP for bleb formation and suggests that the acinar blebs are not apoptotic blebs but a response to physiological stimulation. More experiments are needed to determine the property of the blebs after supramaximal CCK stimulation.

In addition to modifying the actin cytoskeleton, Rho and Rac have been shown to be involved in regulated secretion in a variety of cells, including pancreatic β-cells (25), mast cells (6), parietal cells (45), and PC-12 cells and neurons (12). In pancreatic acini, the observations that dominant-negative RhoN19, RacN17, and C3 exotoxin all significantly inhibited physiological amylase secretion induced by CCK and carbachol (4), whereas constitutively active RhoV14 and RacV14 increased amylase secretion induced by both physiological and superphysiological concentrations of CCK indicate that Rho and Rac are playing a positive role in amylase secretion. This function may be because of their effects on actin cytoskeletal rearrangement, because the effects of active mutants on secretion can be mimicked by jasplakinolide and the effects of dominant-negative mutants on amylase secretion can be mimicked by latrunculin (4). However, it remains to be elucidated why high concentrations of CCK progressively inhibit secretion in acinar cells. Because high concentrations of secretagogue disrupt the apical F-actin network and Rho and Rac promote actin polymerization, it is possible that the inhibition of secretion induced by high concentrations of CCK is the result of overdisruption or depolymerization of actin cytoskeleton. The data that jasplakinolide, which polymerizes actin, also partially reduced the inhibition of secretion induced by high concentrations of CCK support this hypothesis. It is also supported by other studies showing that blockage of actin redistribution induced by high concentrations of CCK by use of a caspase inhibitor also partially blocked the inhibition of secretion by CCK (2, 16). Inhibition of Src family kinases also was reported to partially reverse the actin breakdown and partially restore amylase secretion in the face of supramaximal CCK stimulation (29). In addition, latrunculin treatment induced great actin depolymerization, whereas apical actin is more resistant and inhibited amylase release. This suggests that it is the local actin rearrangement, especially at the apical region, and not the amount of total F-actin that determines or affects the process of secretion. These observations in total suggest that actin structure is important for amylase secretion, and actin polymerization may play a positive role in secretion. Therefore, the decrease in secretion by high concentrations of CCK may be due in part to disassembly or prevention of actin polymerization.

The exact molecular mechanism by which Rho and Rac regulate the actin cytoskeleton to affect secretion is unclear. Several recently studies showed that filamentous actin associates with ZGs in the course of exocytosis (34, 49). Moreover, one study showed that latrunculin or C3 exotoxin was able to decrease F-actin at the apical region and prevented ZG coating upon secretagogue stimulation (34), suggesting that Rho is involved in the apical actin polymerization and that Rho-dependent actin polymerization occurs at the apical membrane at a slow rate.

The present study was designed to evaluate the roles of small G proteins Rho and Rac during the response to high concentrations of CCK. We found that both Rho and Rac are involved in acinar morphological changes, including blebbing, probably through facilitating actin polymerization. Both can also play a role by increasing amylase secretion by concentrations of CCK from 30 pM to 10 nM, probably through polymerizing or stabilizing F-actin. These results along with our earlier study showing a requirement for Rho and Rac in physiological secretion (4) support the active involvement of Rho family G proteins in acinar cell function. Further studies are necessary to determine both how these Rho family G proteins are regulated and their downstream mediators.

GRANTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-41122 and DK-52860 to J. A. Williams, P30 DK-34933 to the Michigan Gastrointestinal Peptide Center, and P60 DK-20572 to the Michigan Diabetes Research and Training Center.

Acknowledgments

We thank Stephen Lentz for help with confocal microscopy and Baoan Ji for helpful discussion during the course of this study and the preparation of this manuscript. Special thanks go to Edward Stuenkel and Craig D. Logsdon for critically reading the manuscript.

Footnotes

  • The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

REFERENCES

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