Endoplasmic reticulum Ca2+-ATPase inhibitors stimulate membrane guanylate cyclase in pancreatic acinar cells

Anna S. Gukovskaya, Sofiya Gukovsky, and Stephen J. Pandol

Departments of Medicine, Veterans Affairs Greater Los Angeles Healthcare System and University of California, Los Angeles, California 90073


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we show that particulate guanylate cyclase (GC) is present in rat pancreatic acinar cells and is located both on plasma membrane and membranes of endoplasmic reticulum (ER). Western blot analysis indicates that the enzyme isoform GC-A is present in the acinar cell membranes. The specific inhibitors of ER Ca2+-ATPase thapsigargin, 2,5-di-(t-butyl)-1,4-hydroquinone (BHQ), and cyclopiazonic acid all activated particulate GC in pancreatic acini, both in membrane fractions and intact cells. These inhibitors also induced dephosphorylation of GC. Dose dependencies of Ca2+-ATPase inhibition and GC activation by BHQ are very similar, and those for thapsigargin partially overlap. ER Ca2+-ATPase and GC are coimmunoprecipitated both by antisera against membrane GC and by antisera against ER Ca2+-ATPase, suggesting a physical association between the two enzymes. The results suggest that thapsigargin and the other inhibitors act through ER Ca2+-ATPase to activate membrane GC in pancreatic acinar cells, although their direct effect on GC cannot be excluded.

calcium transport; cyclopiazonic acid; 2,5-di-(t-butyl)-1,4-hydroquinone; guanosine 3',5'-cyclic monophosphate


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

BOTH SOLUBLE AND PARTICULAR guanylate cyclases (GC) mediate cGMP production in cells (7, 8, 17, 39). Three isoforms of mammalian membrane GC (GC-A, GC-B, and GC-C) have already been cloned (3, 5, 31, 32, 39), and they serve as receptors for natriuretic peptides, heat-stable enterotoxin, and guanylin (7). Membrane GC consists of a one-polypeptide chain (7, 8). Isoforms A, B, and C have high percentages of homology in cytoplasmic domains but differ in extracellular, ligand-binding domains (7, 8, 17, 39). The family of membrane GC receptors also includes sea urchin GC, which is activated by egg peptide (9), and retina GC, which is involved in the phototransduction process (35). Little is known about intrinsic mechanisms of the regulation of particulate GC. Dephosphorylation (24, 25) and oligomerization (20, 39) of GC receptors, as well as association of GC with a regulatory phosphatase (4), were suggested to regulate GC activity. Retina GC is negatively regulated by Ca2+ through a Ca2+-binding protein (11, 16). Regulation by Ca2+ of other isoforms of particulate GC has not been reported.

To our knowledge, membrane GC in the pancreas has not yet been characterized. Some observations suggest, however, that it can play a role in the physiology of exocrine pancreas, in particular, in regulating acinar cell growth (28).

In this study, we show that GC-A is present and functional in pancreatic acinar cells. We found that specific inhibitors of endoplasmic reticulum (ER) Ca2+-ATPase, namely thapsigargin, 2,5-di-(t-butyl)-1,4-hydroquinone (BHQ), and cyclopiazonic acid (14, 15, 29, 33), all activated GC in crude membrane preparations and in ER fractions of rat pancreatic acinar cells. These inhibitors also induced dephosphorylation of GC. GC and ER Ca2+-ATPase were coimmunoprecipitated both by antisera against membrane GC and by antisera against ER Ca2+-ATPase, suggesting that there is a physical association between these two enzymes. The results suggest that thapsigargin and the other inhibitors act through ER Ca2+-ATPase to activate membrane GC in pancreatic acinar cells, although their direct effect on GC cannot be excluded.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. A-23187 was from Calbiochem (La Jolla, CA), thapsigargin was from Molecular Probes (Eugene, OR), and BHQ was from Aldrich (Milwaukee, WI). Protein A-Sepharose was from Pierce (Rockford, IL), precast Tris-glycine gels were from Novex (San Diego, CA), and [32P]orthophosphate was from DuPont NEN (Boston, MA). Cyclopiazonic acid, aprotinin, antipain, chymostatin, leupeptin, pepstatin, phenylmethylsulfonyl fluoride (PMSF), and all other chemicals were from Sigma Chemical (St. Louis, MO).

Preparation of pancreatic acinar cell membranes. Dispersed pancreatic acini were isolated from rats, using the standard collagenase method (12, 13). To prepare a crude membrane fraction, the dispersed acini were homogenized with 20 strokes in a Dounce homogenizer in an ice-cold homogenization buffer containing 25 mM Tris (pH 7.2), 0.32 M sucrose, 1 mM EDTA, 1 mM dithiothreitol, and 0.1 mM PMSF, as well as 5 µg/ml each of the protease inhibitors pepstatin, leupeptin, chymostatin, antipain, and aprotinin. The homogenate was centrifuged at 150,000 g for 40 min at 4°C. The pellet was collected, washed five times with the homogenization buffer, suspended in the same buffer, aliquoted, and kept frozen at -80°C.

The solubilized protein was obtained from crude membrane preparations by incubating them for 20 min at 4°C in lysis buffer containing 0.15 M NaCl, 50 mM Tris (pH 7.2), 1% deoxycholic acid (wt/vol), 1% Triton X-100 (wt/vol), 0.1% SDS (wt/vol), and 1 mM PMSF, as well as the protease inhibitor cocktail specified above. The lysates were centrifuged for 20 min at 15,000 g at 4°C, and the supernatants were aliquoted and stored at -80°C.

Preparation of ER fractions. Fractions of ER were purified as we described previously (23). Briefly, the pancreatic acinar homogenate was centrifuged at 2,000 g for 5 min at 4°C to pellet cell debris and nuclei. Then sucrose gradient density centrifugation was performed at 100,000 g for 2 h. The heavy sucrose gradient consisted of 1.5 ml of an 80% (wt/vol) sucrose cushion and an 11.0-ml linear sucrose gradient of 50-25% (wt/vol) sucrose. The light sucrose gradient consisted of 1.5 ml of a 70% (wt/vol) sucrose cushion and an 11.0-ml linear sucrose gradient of 25-4% sucrose. Three discrete bands, designated H-1, H-2, and H-3, were observed for the heavy sucrose gradient, and two bands, designated L-1 and L-2, were observed for the light sucrose gradient. The following were the sucrose concentrations for each band: H-1 and H-2 at 37% sucrose, H-3 at 25% sucrose, L-1 at 12.5% sucrose, and L-2 at 10% sucrose. Electron microscopic observations demonstrated that H-1, H-2, and H-3 fractions consisted of rough membrane vesicles, L-1 contained a mixture of smooth and rough ER, and L-2 consisted of entirely smooth vesicles (23). All five subcellular fractions demonstrated ATP-dependent 45Ca2+ uptake and contained ER Ca2+-ATPase (23).

Immunoprecipitation. To extract proteins, freshly prepared pancreatic acini or cell crude membrane preparations were lysed by incubation for 20 min at 4°C in lysis buffer containing 0.15 M NaCl, 50 mM Tris (pH 7.2), 1% deoxycholic acid (wt/vol), 1% Triton X-100 (wt/vol), 0.1% SDS (wt/vol), and 1 mM PMSF, as well as 5 µg/ml each of the protease inhibitors pepstatin, leupeptin, chymostatin, antipain and aprotinin. Then the lysates were centrifuged for 20 min at 15,000 g at 4°C, and the supernatants were used for immunoprecipitation.

For immunoprecipitation, the supernatants from cell membrane lysates were incubated at 4°C overnight with primary antibodies and then for 1 h with protein A-Sepharose. The protein A-Sepharose/antigen precipitates were separated by centrifugation, washed three times with the lysis buffer, and resuspended in a sample buffer containing 10% glycerol (vol/vol), 2% SDS (wt/vol), and 0.0025% (wt/vol) bromphenol blue in 63 mM Tris (pH 6.8). The antigen was eluted from protein A-Sepharose by heating for 5 min at 100°C. Samples were centrifuged, and supernatants containing the antigen were collected. GC was immunoprecipitated with a 1:200 dilution of rabbit antiserum A034 against rat GC-A, kindly provided by the laboratory of Dr. D. Garbers (Dallas, TX). The specific peptide against which the antibody was raised was also obtained from the Garbers laboratory. ER Ca2+-ATPase was immunoprecipitated with a 1:1,000 dilution of rabbit antiserum R-2 against sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2b (SERCA2b), kindly provided by Dr. F. Wuytack (Guthuisberg, Belgium).

Western blot analysis. Immunoprecipitated proteins were separated by SDS-PAGE at 110 V, using precast Tris-glycine gels and a Mini-Cell apparatus (Novex). Separated proteins were electrophoretically transferred to a nitrocellulose membrane for 2 h at 30 V using a Novex blot module. Nonspecific binding was blocked by a 1-h incubation of the membrane in 5% (wt/vol) nonfat dry milk in Tris-buffered saline (TBS; pH 7.5). Blots were then incubated for 2 h with primary antibodies in an antibody buffer containing 1% (wt/vol) nonfat dry milk in TTBS (0.05% vol/vol Tween-20 in TBS), washed with TTBS, and finally incubated for 1 h with a peroxidase-labeled secondary antibody in the antibody buffer. Blots were developed for visualization using an enhanced chemiluminescence detection kit (ECL, Amersham).

Measurement of GC phosphorylation state. Pancreatic acini were isolated from three rats and suspended in 10 ml of buffer Q containing (in mM) 20 HEPES (pH 7.4), 120 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 glucose, 10 sodium pyruvate, and 10 ascorbic acid, as well as 0.1% (wt/vol) BSA, 0.01% (wt/vol) soybean trypsin inhibitor, and 5 mCi of [32P]orthophosphate. Acini were incubated for 90 min in a tissue culture incubator (37°C, 5% CO2 humidified atmosphere). Then the cells were equally divided into three flasks and incubated for 30 min in the tissue culture incubator with 2.5 µM thapsigargin or 10 µM BHQ or without inhibitors. Cells were then washed twice with PBS and lysed by incubation for 20 min in an ice-cold modified "phosphatase inhibitor" lysis buffer (described in Ref. 25) that contained 50 mM Tris · HCl (pH 7.2), 20% (vol/vol) glycerol, 50 mM NaCl, 100 mM NaF, 80 µM beta -glycerophosphate, 10 mM NaPO4, 1% (wt/vol) deoxycholic acid, 1% (wt/vol) Triton X-100, 0.1% (wt/vol) SDS, 1 mM PMSF, and pepstatin, leupeptin, chymostatin, antipain, and aprotinin, each at 5 µg/ml. Then the cell lysates were centrifuged at 150,000 g for 40 min at 4°C. Supernatants were adjusted to equal protein concentrations, and GC-A was immunoprecipitated as described above. Immunoprecipitated proteins were fractionated by SDS-PAGE and visualized by autoradiography. The 32P content was quantified using PhosphorImager (Molecular Dynamics, Sunnyvale, CA).

Measurement of GC activities. GC assays were conducted as previously described (12, 13). cGMP production was measured in crude membrane preparations, in ER fractions, or using immunoprecipitated proteins.

Unless indicated otherwise, GC assays were performed at 37°C for 1-10 min in a buffer containing 25 mM Tris (pH 7.1), 5 mM MgCl2, 1 mM GTP, 1 mM 3-isobutyl-1-methylxanthine (IBMX), 100 µM NG-monomethyl-L-arginine (L-NMMA), and other components as indicated. Fifty microliters of membrane protein or immunoprecipitated protein were added in a total volume of 0.2 ml to a solution of the above reagents kept on ice. Samples were transferred to a 37°C water bath (time 0), and cGMP levels in the samples were measured at 1, 3, 5, and 10 min. The reaction was stopped by adding 50 µl of 30% TCA and cooling on ice. After centrifugation at 4°C (12,000 g) for 15 min, supernatants were collected and washed four times with 5 vol of diethyl ether. cGMP was determined in aliquots of the supernatants using the Amersham RIA kit. cGMP accumulation increased linearly with an increase in protein amount in the assay sample from 10 to 70 µg (r2 = 0.988). Also, cGMP production increased linearly between 1 and 10 min (r2 = 0.985).

Measurement of cellular cGMP. After isolation, dispersed acini from one rat were suspended in 10 ml of buffer Q. Before experiments, cells were transferred in buffer Q supplemented with 1 mM L-NMMA and incubated for 10 min at room temperature. Then cells were transferred to 37°C and incubated with indicated agents or vehicle only (control). At the indicated time, 0.5-ml aliquots were removed from both control and treated cell suspensions and centrifuged at 13,000 g for 20 s. The supernatant was aspirated, and 0.5 ml of iced ethanol was added to the pellet. After a 40-min extraction with ethanol, samples were centrifuged (13,000 g, 20 min), and cGMP was determined in aliquots of the supernatant using the Amersham RIA kit.

Measurement of (Ca2+-Mg2+)-ATPase activity. (Ca2+-Mg2+)-ATPase activity was measured as described in Ref. 6 as the difference in ATPase activity in the presence and in the absence of 0.7 µM free Ca2+ maintained with Ca2+-EGTA buffer (1 mM EGTA and 0.5 mM CaCl2, pH 6.8). Measurements of ATPase activity were performed for 10 min at 37°C in a buffer containing 25 mM Tris (pH 6.8), 5 mM MgCl2, 5 mM ATP, 1 mM IBMX, 100 µM L-NMMA, and 1 mM EGTA in the presence or absence of 0.5 mM CaCl2 and indicated amount of thapsigargin or BHQ. The reaction was started by addition of 50 µl of crude membrane protein (~1 mg/ml) into the specified buffer in a total volume of 0.2 ml. The reaction was stopped by addition of 50 µl 30% TCA. After 10 min on ice, the precipitate was centrifuged, and the supernatant was assayed for Pi by the method of LeBel et al. (19).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Membrane GC is present in the pancreatic acinar cell. We found GC activity in crude membrane preparations and ER fractions from rat pancreatic acinar cells (Fig. 1A). To determine whether particulate GC is present in membranes of ER, crude membrane preparations were fractionated using gradient centrifugation, and five fractions were separated as described in Preparation of ER fractions. We previously showed (23) that those fractions contained intracellular agonist-sensitive store of Ca2+. Furthermore, there was minimal contamination of these ER subfractions with plasma membrane except for L-1 (23). The results (Fig. 1A) indicate that GC activity was present in both rough (H-1, H-2, H-3) and smooth ER (L-1, L-2) and that it was particularly enriched in the L-1 fraction.


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Fig. 1.   Membrane guanylate cyclase (GC)-A is present in rat pancreatic acinar cells. A: GC activity was measured in crude membrane preparations and in subfractions of endoplasmic reticulum (ER) designated H-1, H-2, H-3, L-1, and L-2 (see MATERIALS AND METHODS). ER fractions H-1, H-2, and H-3 contain rough ER, L-1 contains smooth and rough ER, and L-2 contains mainly smooth ER. Values are means ± SE of 3 determinations. B: characterization of pancreatic GC-A by immunoprecipitation and Western blot analysis. Proteins were extracted from isolated pancreatic acinar cells as described in MATERIALS AND METHODS. Extracts were adjusted to equal protein concentration, and GC-A was immunoprecipitated with either anti-GC-A antiserum (lanes a and c) or with anti-GC-A antiserum preabsorbed with a GC-specific peptide (lane b). Precipitates were solubilized, fractionated on 4-20% (lanes a and b) or 8% (lane c) SDS-polyacrylamide gels, and immunoblotted with anti-GC-A antiserum. Immunoreactive bands (arrows) were visualized by enhanced chemiluminescence (ECL). Representative of 3 experiments that gave similar results. Protein standard masses at left are in kDa. C: activation of membrane GC under action of 1 µM atrial natriuretic peptide (ANP), 1 µM ANP and 1 mM adenosine 5'-O-(3-thiotriphosphate) (ATPgamma S), or 1 µM natriuretic peptide C (CNP) and 1 mM ATPgamma S. Values are means ± SE of 5 determinations. * P < 0.05 compared with basal activity.

The NO-releasing compound nitroprusside (0.1 mM) did not induce any increase in GC activity in our crude membrane preparations (data not shown). Nitroprusside (0.1 mM) was shown to stimulate the activity of soluble GC in cytosolic fractions of pancreatic acinar cells (12, 13), but it does not affect the membrane GC (7, 17). These data indicate that our membrane preparations were not contaminated with soluble GC.

To test whether the GC-A isoform is present in acinar cell membranes, we applied Western blot analysis using an antibody that specifically recognizes GC-A but not the other isoforms of particulate GC (24, 25). The concentration of the protein was first enriched by immunoprecipitation with this antibody, and then immunoprecipitated proteins were separated using a 4-20% gradient gel and transferred to a nitrocellulose membrane. A prominent broad band in the region from 140 to 180 kDa was specifically recognized by the antiserum against particulate GC-A; it was blocked by inclusion of the blocking peptide (Fig. 1B, lanes a and b). Another specific band at 60 kDa, which was abolished by inclusion of the blocking peptide, may represent a product of GC proteolysis. The band at 50 kDa is probably nonspecific, since it was retained in the presence of the peptide (Fig. 1B, lanes a and b). The broad 140- to 180-kDa band was further resolved on an 8% gel into three separate bands seen on the immunoblot as ~140-, 160-, and 180-kDa proteins (Fig. 1B, lane c). These three bands may represent forms of GC-A with different glycosylation levels (18, 37). The immunoprecipitate retained GC activity (see below).

GC activity in crude membrane preparations from acinar cells was stimulated by atrial natriuretic peptide (ANP; Fig. 1C), confirming that the pancreatic acinar cell contains isoform A of particulate GC (7, 39). Furthermore, GC stimulation by ANP increased in the presence of ATP or nonhydrolyzable adenosine 5'-O-(3-thiotriphosphate) (ATPgamma S), which is characteristic of particulate GC (2).

Although Western blot analysis revealed the presence of GC-A in all ER subfractions from the acinar cell membrane preparations, we could not detect GC responses to ANP in the ER fractions (data not shown). Particulate GC is known to easily undergo desensitization to the action of ANP. Such desensitization was reported previously for both sea urchin and mammalian GC-A (7, 24, 25). Although the mechanism of this phenomenon is unclear, it is thought to be related to dephosphorylation of GC (24-26).

Natriuretic peptide C (CNP) did not produce any significant activation of GC in our crude membrane preparations, either in the presence (Fig. 1C) or in the absence (not shown) of ATPgamma S. This argues against a functional role of GC-B, which is a receptor for CNP (7), in the pancreatic acinar cell. No activation of GC by CNP was detected in the ER subfractions (data not shown).

The results presented in Fig. 1 demonstrate the presence of functional GC-A in membranes of the pancreatic acinar cell.

Ca2+-ATPase inhibitors potentiate GC activity in membranes of the pancreatic acinar cell. GC activity in membrane preparations of pancreatic acinar cells was potentiated by specific inhibitors of ER Ca2+-ATPase: thapsigargin, BHQ, and cyclopiazonic acid (Fig. 2, A-D). This potentiation was observed during the first minutes after addition of thapsigargin (Fig. 2A) or the other inhibitors of ER Ca2+-ATPase. The effect was present in both crude membrane preparations and solubilized protein (Fig. 2B) and also in the GC-A immunoprecipitate (Fig. 2C). Thapsigargin- and BHQ-induced GC responses were also observed in all ER fractions (Fig. 2D and data not shown). We showed previously (23) that Ca2+-ATPase is present in all the subfractions of ER. Although the basal GC activity was higher in the L-1 subfraction (Figs. 1A and 2D), the extent of GC potentiation by the Ca2+-ATPase inhibitors was similar in all ER fractions (Fig. 2D).


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Fig. 2.   Inhibitors of ER Ca2+-ATPase [thapsigargin (THP), 2,5-di-(t-butyl)-1,4-hydroquinone (BHQ), and cyclopiazonic acid (CA)], but not Ca2+-mobilizing agents [inositol 1,4,5-trisphosphate (IP3) and A-23187], potentiate activity of particulate GC in membrane preparations from pancreatic acinar cells. A: GC activity was measured in crude membrane preparations in presence (shaded bars) or absence (open bars) of thapsigargin 1, 3, and 10 min after addition of thapsigargin (120 nmol/mg protein) or vehicle. Values are means ± SE of 3 determinations. * P < 0.05 compared with GC activity in absence of thapsigargin. B: GC activity was measured 3 min after addition to samples of cyclopiazonic acid (1 µmol/mg protein), BHQ (400 nmol/mg protein), A-23187 (2 µM), or IP3 (3 µM). Samples contained crude membrane preparations or solubilized protein. Values are means ± SE of 3 determinations. * P < 0.05 compared with control. C: GC activity was measured in GC-A immunoprecipitate 3 min after addition to samples of BHQ (400 nmol/mg protein) or vehicle. cGMP accumulation was expressed as a ratio to basal GC activity, which in GC-A immunoprecipitate was 75 ± 11 pmol · mg protein-1 · min-1. Values are means ± SE of 3 determinations. * P < 0.05 compared with control. D: GC activity was measured in ER subfractions 3 min after addition of thapsigargin (120 nmol/mg protein) or BHQ (400 nmol/mg protein). Values are means ± SE of 3 determinations. * P < 0.05 compared with control.

The effects of Ca2+-ATPase inhibitors on GC could be mediated by changes in the internal Ca2+ content of the ER. However, neither the Ca2+ ionophore A-23187 nor exogenous inositol 1,4,5-trisphosphate had any effect on GC activity (Fig. 2B). We did not find any significant difference between the basal GC activities at external Ca2+ concentrations of ~10 µM (regular medium) and ~20 nM (medium supplemented with 2 mM EGTA). Neither did EGTA affect GC responses to thapsigargin or BHQ. These data indicate that changes in Ca2+ do not mediate GC stimulation by the Ca2+-ATPase inhibitors.

Effects of Ca2+-ATPase inhibitors on the activity and phosphorylation state of particulate GC in whole pancreatic acinar cells. To study the effects of Ca2+-ATPase inhibitors on particulate GC in whole cells, we used two approaches. First, we tested whether the inhibitors could stimulate cGMP production in conditions that eliminate contribution of soluble GC. Earlier, we and others (10, 12, 40, 41) showed that increasing cytosolic Ca2+ in pancreatic acinar cells stimulated NO synthase, which led to activation of soluble GC. In particular, such activation was observed with thapsigargin. To eliminate the contribution of soluble GC, we preincubated pancreatic acinar cells for 10 min with 1 mM L-NMMA, an inhibitor of NO synthase. As we showed previously (12), this procedure completely prevented activation of soluble GC. However, under these conditions, both thapsigargin and BHQ increased cGMP level in acinar cells (Fig. 3A).


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Fig. 3.   Inhibitors of Ca2+-ATPase stimulate activity and induce dephosphorylation of membrane GC in whole acinar cells. A: effects of thapsigargin and BHQ on cellular cGMP level. After isolation, pancreatic acinar cells were suspended in a medium containing 1 mM NG-monomethyl-L-arginine and 1 mM IBMX (buffer Q; see MATERIALS AND METHODS), incubated for 10 min at room temperature, transferred to 37°C, and incubated for 20 min without (control) and with 2.5 µM thapsigargin or 10 µM BHQ. Reaction was stopped by addition of ice-cold ethanol, and cellular cGMP levels were measured using Amersham RIA kit. Values are means ± SE of 3 determinations. * P < 0.05 compared with control. B-D: decrease in 32P content of GC under action of thapsigargin and BHQ. Pancreatic acinar cells were metabolically labeled with 32PO4 and then incubated without inhibitor (lane a), with 2.5 µM thapsigargin (lane b), or with 10 µM BHQ (lane c) for 30 min. Cells were then lysed, extracts were adjusted to equal protein concentrations, and GC-A was immunoprecipitated, fractionated by SDS-PAGE, and visualized by autoradiography. To show that thapsigargin- or BHQ-induced decreases in GC-A 32P content were not due to losses in protein, same samples were blotted on nitrocellulose filters after being fractionated by SDS-PAGE, and amount of GC-A protein was then detected by immunoblot analysis and visualized by ECL (immunoblot). Numbers shown to right of autoradiograph and immunoblot are protein standard masses in kDa. Amount of 32P associated with GC bands of 180, 160, and 140 kDa (bands 1-3 on immunoblot) was quantified using PhosphorImager (Molecular Dynamics). Background correction was done by subtracting nonspecific counts obtained from equal size regions of gel above and below GC-A bands. Experiment was repeated twice with same results. Quantitation of 32P content of GC-A bands represents means ± SE of 2 determinations.

Membrane GC and, in particular, GC-A exist as phosphoproteins and undergo dephosphorylation under the actions of various stimuli (24, 25). Therefore, we asked whether thapsigargin and BHQ affect the phosphorylation state of particulate GC in pancreatic acinar cells (Fig. 3B-D). On the 32P autoradiograph of GC-A immunoprecipitate from untreated cells, there was one broad band of 140-180 kDa (Fig. 3B, lane a). The amount of 32P in this broad band drastically decreased in cells treated with thapsigargin or BHQ (Fig. 3B, C lanes b and c).

The arrows on the autoradiograph show the positions of separate 140-, 160-, and 180-kDa bands resolved on the immunoblot of the same samples (Fig. 3D). Densitometric quantitation of these three bands (Fig. 3C) confirmed a drastic decrease of their 32P content in both thapsigargin- and BHQ-treated cells. The immunoblot verified that the decrease in 32P content was not due to a loss in the amount of immunoprecipitated GC-A protein in the samples. Interestingly, the 160- and 180-kDa protein bands contained more 32P than did the 140-kDa band (Fig. 3B lane a). This observation correlates with the previous findings that more glycosylated forms of particulate GC with a higher molecular mass are more heavily phosphorylated (18, 27).

The results in Fig. 3 show that thapsigargin and BHQ activate GC-A and decrease its phosphorylation level in pancreatic acinar cells.

Coimmunoprecipitation of membrane GC and SERCA2b Ca2+-ATPase. To test a possible physical association between GC and Ca2+-ATPase, we measured whether these two proteins coimmunoprecipitate. ER Ca2+-ATPase was immunoprecipitated from acinar cell lysate or crude membrane preparation with an antiserum against the SERCA2b Ca2+-ATPase, and the immunoprecipitate was analyzed via Western blotting with the same antibody, which recognized a major band of 100-110 kDa (Fig. 4A, lane a). This band was blocked when immunoprecipitation was performed with the SERCA2b antibody preabsorbed with a specific peptide (Fig. 4A, lane b). These data are in agreement with Ref. 6, which showed that SERCA2b is present in pancreatic acinar cells. The same immunoprecipitates (obtained with SERCA2b antibody or with SERCA2b antibody preabsorbed with the specific peptide) were subjected to Western blot analysis with an antiserum against GC-A. The GC-A antibody recognized three specific GC-A bands of about 140, 160, and 180 kDa in the SERCA2b immunoprecipitate (Fig. 4A, lane c), which were greatly inhibited in the immunoprecipitate obtained in the presence of the peptide (Fig. 4A, lane d). Thus the immunoprecipitate of SERCA2b contained membrane GC as well as the ER Ca2+-ATPase.


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Fig. 4.   Coimmunoprecipitation of ER Ca2+-ATPase and membrane GC. A: ER Ca2+-ATPase was immunoprecipitated (IP) with either sarco(endo)plasmic reticulum Ca2+-ATPase 2b (SERCA2b) antiserum (lanes a and c) or with SERCA2b antiserum preabsorbed with specific peptide against which this antibody was raised (lanes b and d). Immunoprecipitates were immunoblotted (IB) with antibody against SERCA2b (lanes a and b) or against GC-A (lanes c and d). B: GC was immunoprecipitated with either GC-A antiserum (lanes a and c) or with GC-A antiserum preabsorbed with specific peptide against which GC-A antibody was raised (lanes b and d). Immunoprecipitates were immunoblotted with antibody against GC-A (lanes a and b) or SERCA2b (lanes c and d). Representative of 3 independent experiments that gave similar results.

Furthermore, the SERCA2b immunoprecipitate displayed both basal and thapsigargin- or BHQ-induced GC activity. The basal GC activity in the SERCA2b immunoprecipitate was 2.05 ± 0.1 times higher than in crude membrane preparations (n = 3, P < 0.05). The extent of GC stimulation by thapsigargin or BHQ in the SERCA2b immunoprecipitate was similar to that observed in crude membrane preparations (Fig. 2, A and B) or GC-A immunoprecipitate (Fig. 2C).

In the reverse experiments, we immunoprecipitated GC-A from acinar cell lysate with a specific antibody against GC-A and probed this immunoprecipitate with the antibody against SERCA2b (Fig. 4B). In agreement with the results shown in Fig. 4A, the immunoprecipitate of GC-A displayed an immunoreactive SERCA2b band (Fig. 4B, lane c) as well as GC-A (Fig.4B, lane a). Both SERCA2b and GC-A bands were inhibited when the GC-A antiserum was preabsorbed with a specific peptide (Fig. 4B, lanes b and d).

Dose dependencies of the effects of thapsigargin and BHQ on the activities of ER Ca2+-ATPase and GC. Figure 5 shows the dose dependencies of the effects of thapsigargin and BHQ on the activities of ER Ca2+-ATPase and particulate GC in crude membrane preparations from pancreatic acinar cells. The EC50 value for Ca2+-ATPase inhibition by thapsigargin at a constant free Ca2+ concentration of 0.7 µM was ~0.3 nmol/mg protein (or 60 nM for the protein concentration used in the experiments). This is close to the EC50 value reported for thapsigargin inhibition of ER Ca2+-ATPase in these cells (6). The EC50 for thapsigargin stimulation of membrane GC was ~80 times higher (Fig. 5A). However, the dose dependencies of thapsigargin effects on the activities of ER Ca2+-ATPase and membrane GC partially overlap (Fig. 5A).


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Fig. 5.   Dose dependence curves for effects of thapsigargin (A) and BHQ (B) on activities of particulate GC and ER Ca2+-ATPase in pancreatic acinar cells. Measurements of GC and ATPase activities were performed as described in MATERIALS AND METHODS on aliquots of same samples of crude membranes in absence and presence of indicated amounts of thapsigargin or BHQ. Reaction was stopped 10 min after application of thapsigargin or BHQ. Supernatants were collected and assayed for cGMP and for Pi. Maximum activation of GC and maximum inhibition of Ca2+-ATPase, compared with control values, were set to 100%. Values are means ± SE of 5 determinations.

With BHQ, the EC50 values for Ca2+-ATPase inhibition and GC activation were very close: 30 and 73 nmol/mg protein, respectively. Thus BHQ activates GC and inhibits Ca2+-ATPase in the same concentration range, whereas higher concentrations of thapsigargin are required for GC stimulation than for Ca2+-ATPase inhibition. For cyclopiazonic acid, the dose dependencies for Ca2+-ATPase inhibition and GC activation also overlap significantly, based on our measurements and literature data. The EC50 for cyclopiazonic acid-induced GC activation in acinar cells was ~150 nmol/mg protein (data not shown). The EC50 for cyclopiazonic acid-induced Ca2+-ATPase inhibition in acinar cells is close to that observed for BHQ (21).

Thus, for all three inhibitors of Ca2+-ATPase, the dose dependencies for Ca2+-ATPase inhibition and GC activation overlap partially or significantly. The order of potency for these inhibitors is the same for activation of GC as for inhibition of Ca2+-ATPase: cyclopiazonic acid < BHQ < thapsigargin.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The results demonstrate that particulate GC is present in rat pancreatic acinar cells both on the plasma membrane and on the intracellular membranes, particularly on ER membranes. Western blot analysis indicates that the enzyme isoform GC-A is present in the acinar cell membranes. In agreement with this, the enzyme is activated by ANP.

A surprising finding in the present study is that the specific inhibitors of ER Ca2+-ATPase (thapsigargin, BHQ, and cyclopiazonic acid) all activate particulate GC. We also showed that thapsigargin and BHQ induced a decrease in the 32P content of GC-A by stimulating dephosphorylation or by inhibiting phosphorylation of the enzyme. Thus the activation of particulate GC could be associated with its dephosphorylation, as was demonstrated on transfected 3T3 cells (25).

We found that GC-A and Ca2+-ATPase from pancreatic acinar cells coimmunoprecipitate, suggesting a physical association between these two enzymes. Hence, the Ca2+-ATPase inhibitors could, in principle, stimulate membrane GC by changing the conformation of ER Ca2+-ATPase (29, 30). Ca2+-ATPase inhibition by thapsigargin is very rapid (29, 30), so it precedes activation of GC. Another possibility is that the Ca2+-ATPase inhibitors directly interact with membrane GC. However, the following data weigh against this possibility. The three inhibitors have different structures and interact with different sites on Ca2+-ATPase (14, 15, 29, 33), but they all cause a similar degree of GC stimulation: 2.5- to 4-fold. Analysis that we performed showed no homology between primary structures of GC and Ca2+-ATPase. The spatial structures of GC and Ca2+-ATPase are also very different (7, 8, 14, 29).

The order of potency to activate GC is the same as that to inhibit Ca2+-ATPase: cyclopiazonic acid < BHQ < thapsigargin. Dose dependencies of the effects of BHQ or cyclopiazonic acid on Ca2+-ATPase and GC significantly overlap. For thapsigargin, these dose dependencies overlap only partially, and thapsigargin inhibited Ca2+-ATPase at lower concentrations than it stimulated GC in acinar cell membranes. A difference in effective concentrations, however, does not necessarily mean that thapsigargin effects on Ca2+-ATPase and on GC are independent. GC-Ca2+-ATPase protein-protein interaction may be less sensitive to the action of thapsigargin than Ca2+-ATPase enzymatic activity, and hence higher concentrations of thapsigargin may be required to stimulate GC activity.

Our data show that membrane GC is present and activated by the Ca2+-ATPase inhibitors in all the ER subfractions that we previously characterized in the pancreatic acinar cell (23). We did not find a correlation between the total amount of Ca2+-ATPase in ER subfractions (as determined by Western blot; Ref. 23) and GC activity. However, such correlation should not be expected, since ER Ca2+-ATPase is much more abundant than GC and therefore only a small portion of ER Ca2+-ATPase is likely to be involved in interaction with GC.

In summary, the results suggest that the inhibitors act through ER Ca2+-ATPase to activate membrane GC in pancreatic acinar cells, although their direct effect on GC cannot be excluded.

The fact that specific inhibitors of ER Ca2+-ATPase at micromolar concentrations activate particulate GC in acinar cells should be taken into account when interpreting their effects. In more general terms, coupling between GC and Ca2+-ATPase activities may provide one more mechanism coordinating Ca2+ and cGMP responses. Several such mechanisms have been reported. Elevation of cytosolic Ca2+ activates NO synthase, which results in stimulation of soluble GC and cGMP rise (22, 34). We and others (12, 13, 40, 41) previously demonstrated this pathway in pancreatic acinar cells. On the other hand, cGMP stimulates Ca2+ influx in acinar cells, (1, 10, 12, 40, 41). cGMP is also known to stimulate plasma membrane Ca2+-ATPase (36, 38, 42). Our data suggest that a decrease in ER Ca2+-ATPase activity results in activation of particulate GC and an increase in cGMP, which in turn may regulate Ca2+ transport through the mechanisms specified above.


    ACKNOWLEDGEMENTS

We thank Dr. Timothy Fitzsimmons for preparation of the ER fractions and Margaret Chu for preparation of the manuscript.


    FOOTNOTES

This work was supported by the Department of Veterans Affairs and National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-33010.

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: A. S. Gukovskaya, West Los Angeles VA Medical Center, Bldg. 258, Rm. 340, 11301 Wilshire Blvd., Los Angeles, CA 90073 (E-mail: agukovsk{at}ucla.edu).

Received 25 February 1999; accepted in final form 21 September 1999.


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