Calcium signaling in rat pancreatic acinar cells: a role for Galpha q, Galpha 11, and Galpha 14

David I. Yule, Christopher W. Baker, and John A. Williams

Department of Physiology, University of Michigan Medical School, Ann Arbor, Michigan 48105


    ABSTRACT
Top
Abstract
Introduction
Methods and materials
Results
Discussion
References

Stimulus-secretion coupling in the pancreatic acinar cell is initiated by the secretagogues CCK and ACh and results in the secretion by exocytosis of the contents of zymogen granules. A key event in this pathway is the G protein-activated production of second messengers and the subsequent elevation of cytosolic-free Ca2+. The aim of this study was therefore to define the heterotrimeric G protein alpha -subunits present and participating in this pathway in rat pancreatic acinar cells. RT-PCR products were amplified from pancreatic acinar cell mRNA with primers specific for Galpha q, Galpha 11, and Galpha 14 but were not amplified with primers specific for Galpha 15. The sequences of these PCR products confirmed them to be portions of the rat homologues of Galpha q, Galpha 11, and Galpha 14. The pancreatic-derived cell line AR42J similarly expressed Galpha q, Galpha 11, and Galpha 14; however, the Chinese hamster ovary (CHO) cell line only expressed Galpha 11 and Galpha q. These data indicate that caution should be exercised when comparing signal transduction pathways between different cell types. The expression of these proteins in acinar cells was confirmed by immunoblotting samples of acinar membrane protein using specific antisera to the individual G protein alpha -subunits. The role of these proteins in Ca2+ signaling events was investigated by microinjecting a neutralizing antibody directed against a homologous sequence in Galpha q, Galpha 11, and Galpha 14 into acinar cells and CHO cells. Ca2+ signaling was inhibited in acinar cells and receptor-bearing CHO cells in response to both physiological and supermaximal concentrations of agonists. The inhibition was >75% in both cell types. These data indicate a role for Galpha q and/or Galpha 11 in intracellular Ca2+ concentration signaling in CHO cells, and in addition to Galpha q and Galpha 11, Galpha 14 may also fulfill this role in rat pancreatic acinar cells.

Gq proteins; microinjection


    INTRODUCTION
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Abstract
Introduction
Methods and materials
Results
Discussion
References

THE MAJOR PHYSIOLOGICAL role of the pancreatic acinar cells is to synthesize, store in zymogen granules, and secrete by regulated exocytosis digestive enzymes (26). Secretion is stimulated physiologically, primarily by the hormone CCK and the neurotransmitter ACh. A key event in acinar stimulus-secretion coupling is the elevation of intracellular Ca2+ as a consequence of the action of the second-messenger inositol 1,4,5-trisphosphate (IP3). IP3 is formed as a result of the heterotrimeric G protein-activated hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C (PLC). Heterotrimeric G proteins are composed of three subunits (alpha , beta , gamma ) and are usually defined on the basis of their alpha -subunit. In pancreatic acinar cells, the heterotrimeric G protein alpha -subunits believed responsible for stimulation of PLC activity are members of the Gq family. This contention is based on the observations (13, 24) that the production of IP3 and Ca2+ signaling events are not sensitive to pertussis or cholera toxin, which modify the effects of many other alpha -subunits. In addition, Galpha q-directed antisera, when introduced through a patch pipette, inhibits the agonist stimulation of a Ca2+-activated current in acinar cells (31).

Genes for four members of the Gq family have been cloned and have been named Galpha 11, Galpha q, Galpha 14, and Galpha 15. Although the four proteins appear to have widespread distribution in neonatal tissue, the expression of Galpha 14 and Galpha 15 is restricted in adult tissue (1, 23). Both proteins have been shown to be abundant in hematopoietic tissue, and, in addition, Galpha 14 is highly expressed in kidney and testis. In contrast, Galpha q and Galpha 11 are thought to be expressed almost ubiquitously, although the level of expression can vary widely (14). An exception to this rule is that Galpha q but not Galpha 11 is expressed in platelets (14). Presumably, as a result of the high sequence identity of Galpha q and Galpha 11 (>85% amino acid identity), it appears, at least in overexpression systems, that receptors coupled to this pathway cannot distinguish between these proteins and the extent of activation of PLC-beta is not markedly different (17, 18). In contrast to Galpha q and Galpha 11, evidence (2, 9) suggests that differences exist in the ability of agonist receptors to couple to Galpha 14 and Galpha 15 and the efficacy with which these G proteins activate PLC-beta differs from Galpha 11 and Galpha q. While it is known that individual members of a G protein family can have a restricted distribution, it is not clear whether in an individual cell type multiple forms of a particular protein are expressed. In this study, the Gq family alpha -subunit complement of acinar cells and the pancreatic-derived cell line AR42J is defined and compared with the Chinese hamster ovary (CHO) cell line, which is often used for the study of signal transduction pathways (5, 22, 30). Using immunoneutralization, we demonstrate that expression of G protein heterotrimers containing these alpha -subunits can account for Ca2+ signaling events in pancreatic acinar cells.


    METHODS AND MATERIALS
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Abstract
Introduction
Methods and materials
Results
Discussion
References

Materials. Fura 2-AM and sulforhodamine B (Lissamine rhodamine B 200) were purchased from Molecular Probes (Eugene, OR), collagenase (CLSPA grade) from Worthington Biochemicals (Freehold, NJ), BSA (fraction V) from ICN Immunobiologicals (Lisle, IL), and minimal essential amino acid supplement from GIBCO (Grand Island, NY). All other materials were obtained from Sigma Chemical (St. Louis, MO). Galpha q-COOH terminus (Galpha q-CT) and internal sequence Galpha 11-specific antisera were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The Gq-specific antiserum was obtained from Calbiochem. Molecular biology reagents are from sources indicated.

Preparation of pancreatic acini. Acini were prepared by methods previously described (12, 25, 30). In brief, pancreata were excised from freely fed adult male Sprague-Dawley rats (200-250 g). Acini were prepared by enzymatic digestion with purified collagenase, followed by mechanical shearing. Acini were then filtered through 150-µm Nitex mesh, purified by sedimentation through 4% BSA in HEPES-Ringer, and then suspended in a physiological saline solution (PSS) containing 10 mg/ml BSA, 0.1 mg/ml soybean trypsin inhibitor, and (in mM) 137 NaCl, 4.7 KCl, 0.56 MgCl2, 1.28 CaCl2, 1.0 Na2HPO4, 10 HEPES, 2 L-glutamine, and 5.5 D-glucose and essential amino acids. The pH was adjusted to 7.4 and equilibrated with 100% O2. Enriched plasma membrane fractions were prepared as previously described (3).

Western blotting. Membrane samples (8-50 µg) were subjected to electrophoresis on 12% SDS-PAGE gels. The resolved proteins were transferred to nitrocellulose (Schleicher & Schuell, Keene, NH). Immunoreactivity was visualized using peroxidase-conjugated secondary antibodies followed by detection using the enhanced chemiluminescence (ECL) detection system exposed on ECL Hyperfilm (Amersham Life Science, Arlington Heights, IL), as previously described (22). Immunoprecipitations were performed as follows: enriched membranes were sonicated in 0.5 ml of ice-cold lysis buffer (100 mM NaF, 50 mm Tris, 150 mM NaCl, 10 mM EDTA, 1 mM benzamidine, 1% Triton X-100, 1% 2-mercaptoethanol, 10 µg/ml leupeptin, and 10 µg/ml pepstatin, at pH 7.4) and then sonicated. After 30 min on ice, the samples were centrifuged for 30 min at 100,000 g in a Beckman bench top ultracentrifuge. The supernatant was assayed for protein concentration. Samples of equal protein concentration were then incubated with antiserum overnight at 4°C, after which immobilized protein A beads (Pierce) were added to each sample. As a control, a sample was processed with no cellular lysate, which would eventually be used to ascertain specific bands on the immunoblot. After the samples were rotated for 2 h, they were then microcentrifuged and the supernatant was discarded. The beads were washed four times (50 mM Tris, 150 mM NaCl, 1 mM EDTA, and 1% Triton X-100), suspended in SDS-PAGE sample buffer, and boiled for 3 min. The proteins were then separated by SDS-PAGE and transferred to nitrocellulose before immunoblotting.

RT-PCR analysis of Gq family alpha -subunits. Total RNA was isolated after disruption of acini, cultured cells, or whole pancreas with guanidinium thiocyanate and centrifugation through a CsCl gradient, according to the method developed by Chomczynski and Sacchi (4), using a total RNA isolation kit (Invitrogen, San Diego, CA). We reverse transcribed ~1 µg of RNA at 42°C for 50 min with 200 units of Superscript II RNase H- RT and 50 ng random hexamers (Superscript preamplification system, GIBCO). PCR reactions were performed in 100-µl vol containing 10 mM Tris · HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 200 µM of each dNTP, 0.2 µM of each primer, and 2.5 units of AmpliTaq DNA polymerase (GeneAmp PCR core reagents, Perkin-Elmer, Foster City, CA). The temperature parameters for 40 cycles were 2 min at 94°C and 2 min at the annealing temperature followed by another 3 min at 72°C. The cDNA product was used as a template in PCR with primers specific for the alpha -subunits (Galpha q, Galpha 11, Galpha 14, Galpha 15), using mouse pancreas cDNA as a positive control and including a negative control with no template. The RT-PCR also included a negative control with no RT to rule out amplification of DNA from contaminating sources. The amplification primers were designed against the known mouse mRNA sequences with appropriate restriction sites engineered onto the 5' ends. Primers for Galpha q were designed to amplify a 997-bp region from bases 68 to 1065 of the mouse sequence (GQ: 5'-CTGAGCGAGGAGGCCAAGGAAG-3' and 5'-TTGTAGGCGAAGCAGAAACGTC-3'; annealing temperature, 55°C). Primers for Galpha 11 were designed to amplify a 346-bp region from bases 339 to 685 of the mouse sequence (G11: 5'-AGACGCTCAAGATCCTCTACAAGT-3' and 5'-CACCTACACCCGCCCGTCTC-3'; annealing temperature, 57°C). A primer pair was also designed to amplify bases 216-791, which is a region common to mouse Galpha q, Galpha 11, and Galpha 14 (GQF: 5'-GATCATCCACGGGTCGGGCTACT-3' and 5'-TCTTGGCGTACCTCCTCCTCTCGTT-3'; annealing temperature, 55°C). Primers for Galpha 14 were designed to amplify a 424-bp insert spanning bases 641-1065 of the mouse sequence (G14: 5'-GTATCGCCATGCCCTCTTTCGTG-3' and 5'-TTCTACAGTTTCGCCGGTCCCT-3'; annealing temperature, 63°C). Primers for Galpha 15 were designed to amplify bases 188-898 of mouse Galpha 15 (G15: 5'-GGCCTGGTGAGAGCGGGAAGAGTA-3' and 5'-TAAGTGTGGAGGGTGGACCGGTGT-3'; annealing temperature, 67°C) and against a 253-bp region of the rat spleen PCR product (rG15: 5'-CGCCGAGGACGACTACATC-3' and 5'-GTCCTCTTGGCATACTTCCTCTCA-3'; annealing temperature, 67°C). PCR products were analyzed by electrophoresis in 1% agarose gels containing ethidium bromide. The PCR products were purified (Geneclean II kit, BIO 101, Vista, CA) in preparation for cloning and sequencing. The purified double-stranded fragments were restriction digested and ligated into pBluescript II (KS-) (Stratagene, La Jolla, CA) that had been likewise cut. The plasmids containing inserts were sequenced using Sequenase quick-denature plasmid sequencing kit (Amersham). Both strands of the double-stranded templates were sequenced. The rat sequences were identified as homologues of mouse Gq family alpha -subunit mRNA sequences using the BLAST service of the National Center for Biotechnology Information (NCBI) database. These sequences were then translated, aligned, and compared with available mouse, human, and bovine counterparts (retrieved from the NCBI database) using DNA-STAR LaserGene programs (Madison, WI).

Measurement of intracellular Ca2+ concentration. Isolated acini were incubated with 2.5 µM fura 2-AM at ambient temperature for 30 min and then washed and resuspended in fresh PSS without BSA. For measurement of intracellular Ca2+ concentration ([Ca2+]i), fura 2-loaded acini were transferred to a chamber, mounted on the stage of a Zeiss Axiovert 35 microscope, and continuously superfused at 1 ml/min with PSS without BSA. CHO cells were grown on glass coverslips, loaded for 45 min with 4 µM fura 2-AM, and similarly superfused. Solution changes were rapidly accomplished by means of a valve attached to an eight-chambered superfusion reservoir, which was maintained at 37°C. Determination of [Ca2+]i was performed using digital imaging microscopy with an Attofluor (Rockville, MD) ratiovision system, as previously described (7, 29). Briefly, the 340 nm-to-380 nm excitation ratio was alternately achieved by a computer-controlled filter and shutter system, and the resultant emission at 505 nm was recorded at the rates indicated in the figures, by an intensified charge-coupled device (CCD) camera, and subsequently digitized. Mean gray values obtained by excitation at 340 and 380 nm in user-defined areas of interest were used to compute 340 nm-to-380 nm ratios. Calibration of fluorescence ratio signals was accomplished as previously described according to the equation of Grynkiewicz et al. (7) by comparing the fluorescence of known standard Ca2+ buffers containing fura 2.

Microinjection of antisera. Microinjection of antibodies was performed using a Burleigh Piezoelectric cell penetrator system (Fishers, NY) mounted on the stage of an Axiovert 35 microscope/Attofluor imaging system, which was itself positioned on a vibration isolation platform. Briefly, pipettes of tip diameter of <0.1 µm were pulled on a micropipette forge (Sutter Instruments, Novato, CA) from 1.0-mm glass tubing containing a filament (World Precision Instruments, Sarasota, FL). Pipettes were filled with test agents together with 0.25 µM sulforhodamine B as an indicator of the efficacy of injection in a buffer solution containing 120 mM potassium glutamate and 20 mM HEPES adjusted to pH 7.2. The pipette solution was filtered through a 0.22-µm filter. The pipettes were then mounted in a Perspex holder connected to a pressure injector (Medical Systems, Greenvale, NY). Aliquots of acinar cells were seeded onto coverslips that had been coated with Cell-Tak (Collaborative Research, Bedford, MA) to aid firm adherence of the cells. The final approach and penetration of a single acinar cell was accomplished by making multiple 3-µm jumps toward the cell using the piezoelectric actuator. After penetration, the pipette was rapidly withdrawn by making a single reverse jump of 15 µm. During this procedure and 10 min postinjection, the cells were maintained in PSS containing no added Ca2+. After this 10-min period, the cells were incubated for a further 5 min in a similar solution but containing 10% FCS before they were returned to the normal PSS containing Ca2+ before the monitoring of [Ca2+]i. The success of the injection was assessed by monitoring the morphology of the cell pre- and postinjection, together with the capacity of the cell to retain the injected dye or previously loaded indicator and the ability to maintain an appropriate basal [Ca2+]i. When dye loss from the injected cell was rapid or the cell suffered obvious insult from the injection, the experiment was discarded. The excitation and emission characteristics of sulforhodamine B (excitation, 560 nm; emission, 630 nm) and fura 2 (excitation, 340 nm-to-380 nm ratio; emission, 505 nm) allowed simultaneous monitoring of injection efficacy and [Ca2+]i measurement by virtue of a four-position filter and shutter system. Images at the respective wavelengths were captured by intensified CCD camera and subsequently digitized for analysis.


    RESULTS
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Abstract
Introduction
Methods and materials
Results
Discussion
References

Characterization of Gq family alpha -subunits by RT-PCR in pancreatic acini The expression of alpha -subunits of the Gq family in rat pancreatic acini was investigated by PCR analysis of reverse-transcribed RNA prepared from the rat exocrine pancreas. As the exact nucleotide sequence of each of the Gq family alpha -subunits is known for murine tissue, oligonucleotide primers were designed against conserved regions in these genes to use RT-PCR to amplify from reverse-transcribed rat acinar RNA the cDNA for the Gq homologues that are expressed in rat pancreatic acinar cells. RNA prepared from rat spleen together with that from the cell line AR42J, which is derived from pancreatic tissue, were included for comparison. Initially, primers were designed to recognize a sequence that is unique to Galpha 11. RT-PCR resulted in products of the expected size amplified from RNA derived from rat pancreatic acinar cells, whole pancreas, and AR42J cells (Fig. 1A). As controls, PCR reactions were performed routinely in which no RT was included to exclude the possibility that products had been amplified from contaminating DNA. In addition, reactions were performed in which no template was added. In both of these cases, no product was ever amplified. The products amplified from rat pancreatic acinar cells were purified, ligated into pBluescript, cloned, and sequenced. The products from multiple clones were completely sequenced in both directions, and, compared with sequences in GenBank, the sequence amplified most closely resembled mouse Galpha 11. In all, 530 bases were sequenced from clones amplified utilizing the G11 and GQF primer pairs. The translated region of the mouse gene consists of 1364 bases. At the nucleotide level, the portion of the rat Galpha 11 homologue was 95% identical to that of the mouse and the predicted amino acid identity between mouse and rat protein was >99%. In a similar fashion, a primer pair was designed to target a 997-bp region of Galpha q. As shown in Fig. 1B, products of appropriate size were amplified from rat pancreatic acini, whole rat pancreas, and AR42J cells. The product amplified from acini was cloned and then sequenced as described. The nucleotide sequence was most highly homologous to the GenBank sequence corresponding to mouse Galpha q and thus in all probability represents the rat Galpha q homologue, with the portion of the rat sequence being 95% identical at the nucleotide and 98% identical at the amino acid level (953 bases were sequenced, from a predicted total of 1460, based on the mouse translated region).


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Fig. 1.   PCR analysis of Galpha q family subunits in pancreatic tissue. Specific oligonucleotide primers were designed to recognize regions of Galpha 11, Galpha q, Galpha 14, and Galpha 15. RT-PCR reactions were performed, and products electrophoresed on 1% agarose gels in a buffer containing ethidium bromide. Representative experiments are shown using primers specific for Galpha 11 (A), Galpha q (B), Galpha 14 (C), and Galpha 15 (D). Products of predicted size were amplified from pancreatic-derived reverse-transcribed RNA (pancreas, acinar cells, and AR42J) that represented portions of Galpha 11, Galpha q, and Galpha 14 (A-C, respectively). A product could not be amplified from pancreatic samples using oligonucleotides designed to amplify a portion of Galpha 15, although products were readily amplified from reverse-transcribed RNA prepared from rat spleen (D).

In addition, a primer pair designed to a conserved sequence in Galpha 11, Galpha q, and Galpha 14 was utilized. This primer set was designed to amplify a 576-bp product covering bases 216-791 of the mouse cDNAs. Utilizing this primer set, we again isolated individual clones that were highly identical to mouse Galpha 11 or Galpha q, confirming the expression of these Galpha q family members in rat acini. No clones corresponded to the mouse Galpha 14 sequence using these primers, and therefore specific primers were designed to recognize a 424-bp region of the mouse Galpha 14 gene. With the use of this primer pair, PCR resulted in a single product of predicted size from acinar tissue, whole pancreas, and the AR42J cell (Fig. 1C). When sequenced, the products most closely resembled mouse Galpha 14, being 95% identical at the nucleotide level and 97% identical in terms of the predicted amino acid sequence. In all, 578 of the 1503 bases of the predicted translated region of the gene were sequenced. These products were derived from inserts amplified with G14 primers and a primer pair designed to amplify a slightly larger but overlapping region of the Galpha 14 gene. These data indicate the expression of Galpha 14 in rat acinar tissue. In a similar fashion, primers were designed to amplify a 734-bp region of Galpha 15; however, with these primers no product was amplified from RNA of pancreatic origin. A product of predicted size was amplified from rat spleen (Fig. 1D). These data are consistent with the expression of Galpha 15 being largely confined to hematopoietic tissue. The rat Galpha 15 RT-PCR product was sequenced and proved to be 91% identical to mouse Galpha 15 (520 bases were sequenced from a total of 1353 bases of the predicted mouse translated region). As further evidence for the lack of expression of Galpha 15 in rat pancreatic acini, primers were designed based on the exact sequence of the rat spleen Galpha 15 PCR product. PCR using these primers again only yielded products in rat spleen and a mouse B cell line (data not shown). These data are consistent with the expression at the mRNA level of the three members of the Galpha q family, Galpha 11, Galpha q, and Galpha 14, in rat acini and AR42J cells.

RT-PCR analysis of Gq family alpha -subunits expressed in CHO cells. Because CHO cells are often used for expression studies defining signaling pathways activated by G protein-coupled receptors, including studies of pancreatic secretagogues (5, 22, 30), the Galpha q family complement of this cell type was investigated. Using the primer pairs designed to amplify alpha -subunits from pancreatic tissue, we performed similar PCR reactions utilizing reverse-transcribed RNA prepared from CHO cells, transfected with the CCK-A receptor (CHO-CCK-A). PCR products of appropriate size were amplified using primers designed against both Galpha q and Galpha 11 but not amplified using primers against Galpha 14 and Galpha 15 (Fig. 2). These data would imply that the CHO cell expresses Galpha q and Galpha 11 but not Galpha 14 and Galpha 15 and thus expresses a more restricted complement of Galpha q family alpha -subunits compared with the pancreas.


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Fig. 2.   PCR analysis of Galpha q family subunits in Chinese hamster ovary (CHO) cells. RT-PCR reactions were performed utilizing primers designed to specifically recognize individual Galpha q family proteins with RNA prepared from CHO cells. Products of appropriate size, which represented hamster homologues of mouse Galpha q and Galpha 11, were amplified. No products were amplified using primers designed to recognize Galpha 14 and Galpha 15.

Western analysis of Galpha q family proteins. Although preparation of pancreatic acinar cells by these methods is known to de-enrich nonacinar cells significantly (25), PCR has the capability of amplifying message from a very small number of copies of a particular mRNA. It is therefore possible that the Galpha q family fragments amplified by PCR could have been derived from a contaminating cell type, other than the predominating acinar cell. To confirm that Galpha 11, Galpha q, and Galpha 14 proteins are expressed in acinar cells, we performed immunoblots of acinar membrane proteins, utilizing an antiserum directed against individual Galpha q family members. Initially, acinar proteins separated by electrophoresis and transferred to nitrocellulose were probed with an antiserum (Galpha q-CT) directed against a 19-amino-acid region that is identical in the COOH terminus of Galpha 11 and Galpha q and highly conserved in Galpha 14 (2 amino acid substitutions). As shown in Fig. 3, a single strong band of ~42 kDa was resolved in acinar cells, indicating the presence of Galpha q family proteins. Similar procedures using membranes prepared from CHO cells and AR42J cells also resolved a band of 42 kDa, which in all probability represents Galpha q family proteins (data not shown). Similar samples were probed with antisera specific to Galpha 11 and Galpha q. Because these antisera failed to unequivocally resolve Galpha 11 and Galpha q by conventional immunoblots, experiments were performed to enrich these proteins by first immunoprecipitating Galpha q family proteins using the Galpha q-CT antiserum before electrophoresis and transfer to nitrocellulose and subsequent probing with specific antisera. In this case, bands of appropriate molecular mass were resolved, confirming the presence of Galpha 11 and Galpha q (Fig. 3). Immunoblots utilizing an antiserum specific for Galpha 14 (23) (kindly provided by Dr. Danquing Wu, University of Rochester) also resolved a protein of ~42 kDa, indicating the expression of Galpha 14 in acinar cells. The specificity of these antisera was confirmed, as they did not cross-react inappropriately with recombinant Galpha 11 and Galpha q standards. These data confirm the expression of Galpha 11, Galpha q, and Galpha 14 in pancreatic acinar cells.


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Fig. 3.   Immunoblot analysis of Galpha q subunits in pancreatic acinar membranes. Pancreatic membrane proteins were separated by electrophoresis on 12% SDS-PAGE gels. Resolved proteins were subsequently transferred to nitrocellulose and probed with antisera designed to recognize Galpha q family proteins. Left lane, proteins were probed with an antibody that recognizes a conserved region in Galpha 11, Galpha q, and Galpha 14 (Galpha q-COOH terminus, Galpha q-CT). A single band of ~42 kDa was resolved. Center lanes, proteins were first immunoprecipitated as described in MATERIALS AND METHODS using the Galpha q-CT antiserum (indicated by IP). This maneuver was performed to enrich Galpha q family proteins before separation by SDS-PAGE. After transfer of immunoprecipitated proteins, the membrane was probed with antisera specific for Galpha 11 or Galpha q. A band representing Galpha 11 or Galpha q of molecular mass of ~42 kDa was resolved. Right lane, an antiserum specific for Galpha 14 reveals a band of ~42 kDa representing Galpha 14 protein in pancreatic acinar membranes. * Antibody chains.

Functional characterization of Galpha q subunits. To demonstrate a role for Galpha q subunits in stimulating PLC activity and the resulting signaling cascade in acinar cells, we performed experiments to monitor the effect of functionally ablating Galpha q proteins, while monitoring intracellular Ca2+ signaling events. Antiserum Galpha q-CT is directed against a region of the Galpha q subunit that is thought to be involved in interaction between the alpha -subunit and intracellular domains of cell surface receptors. Moreover this region of the alpha -subunit is thought to be exposed in the intact heterotrimeric complex (21). For these reasons, it is a good candidate for a neutralizing antibody, and indeed antibodies directed against this general region of the alpha -subunit have proved to be effective in reducing PLC-mediated responses in other cell types (8, 22, 27, 31).

Initially, experiments were performed by injecting Galpha q-CT antiserum into CHO cells stably transfected with CCK-A receptors (30), which express only Galpha q and Galpha 11. Cells were injected with Galpha q-CT antiserum from a micropipette containing affinity-purified antibody at a concentration of 0.5 mg/ml. Through estimating the concurrent dilution of dye fluorescence from injection pipette into the cell, we found the approximate dilution of antibody to be between 1:100 and 1:500, giving a cellular antibody concentration of ~1-5 µg/ml. In 22 cells in nine experiments, the response of antibody-injected cells was reduced by 78 ± 12% (Fig. 4A). In six cells injected with injection buffer alone the agonist-induced elevation in [Ca2+]i was not significantly inhibited (456 ± 80 vs. 520 ± 40 nM; means ± SD, unpaired t-test, P > 0.1), indicating that the trauma of injection per se did not account for the reduction in response to agonist. As a further control, CHO cells were injected with an antiserum at a similar concentration (0.2 mg/ml) directed against an unrelated heterotrimeric G protein, Galpha i-3, and the response to agonist stimulation was monitored. As shown in Fig. 4B, injection of antiserum against this alpha -subunit did not markedly perturb Ca2+ signaling events, implying that inhibition seen on injection of Galpha q-CT antibody was specific to this antiserum. Responses to bombesin in CHO cells transfected with neuromedin C receptor and to carbachol (CCh) in cells transfected with M3 muscarinic receptors were similarly affected (data not shown). Individual cells were also injected with antisera directed specifically against Galpha q or Galpha 11 (4 and 6 cells, respectively). In both cases, no marked inhibition of the response was seen. Furthermore, in three additional cells a combination of Galpha q and Galpha 11 antisera also proved not to attenuate the agonist-induced signal. These data indicate that the specific Galpha q and Galpha 11 antisera are ineffective in immunoneutralizing the function of these proteins. This is not surprising as these antibodies are directed against internal sequences of the G protein that are not thought to be exposed in the native, nondenatured protein. In addition, these regions are not thought to be important for receptor or effector-alpha -subunit interactions.


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Fig. 4.   Injection of Galpha q-CT antiserum inhibits Ca2+ signaling events in CHO cells. Single fura 2-loaded CHO cells that stably express CCK-A receptor were injected with antiserum directed against Galpha q family proteins. Cells were subsequently stimulated by superfusion with 0.5 nM CCK, and elicited Ca2+ response in injected cells was compared with noninjected neighbors. A: typical experiments in which 3 cells (in red) were injected. Response in injected cells is markedly inhibited. B: a typical experiment in which cells were injected with an antiserum directed against Galpha i-3. Injection of this antiserum did not markedly alter Ca2+ signaling events in CHO cells.

Further experiments were performed to determine if Galpha q-CT antiserum was capable of inhibiting Ca2+ signaling in acinar cells that express Galpha 14 in addition to Galpha 11 and Galpha q. Preliminary experiments (29) microinjecting the antiserum into single cells in situ within the acinus proved ineffective, presumably since acinar cells are coupled to their neighbors by gap junctions through which Ca2+ signaling molecules can pass, effectively "rescuing" the injected cell. Experiments were therefore performed in single isolated acinar cells that maintained distinct polarity. The response in the injected cell to a maximal concentration of agonist (0.1 mM CCh or 1 nM CCK), known to induce a "peak and plateau" type of Ca2+ profile was compared with noninjected cells in the same field. In 12 experiments in which 14 cells were injected with Galpha q-CT antibody, the response of the injected cell to 0.1 mM CCh was reduced from a peak of 460 ± 40 nM above basal compared with 97 ± 35 nM above basal for noninjected cells; an inhibition of ~80% percent. A representative experiment is shown in Fig. 5A. Experiments were also performed to investigate if responses to physiological concentrations of agonists are mediated as a result of activation of the heterotrimeric G protein complex. At these low concentrations of secretagogues (10-50 pM CCK), it is well established that stimulation of the activity of PLC cannot be measured (12, 19). In eight cells injected with Galpha q-CT antibody, the response to concentrations of agonists that induce the oscillatory physiological profile of Ca2+ signal was markedly inhibited to close to basal levels, as shown in Fig. 5B. These data indicate that, in addition to superphysiological doses of agonist that result in a peak and plateau type of response, oscillatory changes in Ca2+ stimulated by physiological concentrations of agonist are the result of coupling involving Galpha q family proteins. Injection of an antiserum directed against Galpha i-3 had no marked effect on Ca2+ signaling events in single acinar cells (Fig. 5C).


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Fig. 5.   Injection of Galpha q-CT antiserum inhibits Ca2+ signaling events in single pancreatic acinar cells. Single fura 2-loaded pancreatic acinar cells were injected with Galpha q-CT antiserum, and Ca2+ signals in injected cells (in red) were compared with noninjected cells in same field. A: response of a single acinar cell to a superphysiological concentration of carbachol (CCh) is markedly inhibited in Galpha q-CT-injected cells. B: response to a physiological concentration of CCK that induces repetitive Ca2+ oscillations is severely attenuated. In contrast, when single cells are injected with a similar concentration of an antiserum directed against Galpha i-3, the response to a physiological (oscillatory) concentration of CCK is not altered (C).


    DISCUSSION
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Abstract
Introduction
Methods and materials
Results
Discussion
References

Activation of intracellular Ca2+ signaling events in nonexcitable cells, such as pancreatic acinar cells, is believed to be predominately mediated through a cascade that involves the Galpha q family of heterotrimeric GTP binding proteins. Because this family consists of four related proteins, the aim of this study was to define which members of the Galpha q family are expressed in pancreatic acinar cells and to implicate expression of these individual proteins in mediating Ca2+ signaling events on stimulation by secretagogues. As a secondary goal, the complement of Galpha q proteins expressed in acinar cells was compared with that expressed in two model cell lines, the pancreas-derived AR42J cell and the CHO cell transfected with pancreatic secretagogue receptors.

In pancreatic acinar cells, RT-PCR and immunoblotting techniques demonstrated that Galpha 11, Galpha q, and Galpha 14 are expressed, both at the level of mRNA and protein. The expression of both Galpha 11 and Galpha q is consistent with the almost ubiquitous expression of these proteins, while the expression of Galpha 14 is somewhat more surprising based on its limited reported distribution. Galpha 15 does not appear to be expressed in pancreatic tissue, and its expression appears limited to blood cells or tissue involved in the genesis of, or rich in, blood cells (1, 23). The expression of Galpha q family proteins was also defined in AR42J cells and CHO cells. In a manner similar to that of the native pancreatic acinar cells, AR42J cells expressed Galpha q, Galpha 11, and Galpha 14 but not Galpha 15. In contrast, by PCR techniques, CHO-CCK-A cells expressed Galpha q and Galpha 11 but not Galpha 14 or Galpha 15. These data have relevance to the use of these cell lines for study of acinar signal transduction. Although it appears that use of the AR42J cell line for such studies would seem appropriate based on the expression of Galpha q family members, studies utilizing CHO cells should be treated with some caution since the cell line does not express the full complement of acinar Galpha q family members and thus the potential for altered downstream signaling events is a possibility.

The physiological impetus underlying expression of multiple members of this G protein family in a particular cell type is unclear. It has been postulated that molecular diversity expressed in a particular cell type either may be related to achieving signal specificity or alternatively may simply be related to redundancy. Because Galpha 11 and Galpha q do not differ markedly in their distribution, recognition by receptors, or activation of PLC-beta , it would appear that their coexpression in the majority of cells may represent evolutionary redundancy. Levels of Galpha q and Galpha 11 can, however, vary widely in particular cell types; for example, in the cerebellum, levels of Galpha q are up to five times higher than Galpha 11. In this particular case, cerebellar function is disrupted in transgenic knockouts of Galpha q but not of Galpha 11, indicating that the level of a particular protein can be critical for coupling effectively (15). Because the present study did not address the relative abundance of Galpha q vs. Galpha 11 in the acinar cell, further study will be necessary to determine if these proteins serve an identical functional role in this cell type.

Galpha 14 and Galpha 15, in contrast, appear to couple effectively to only a subset of cell-surface receptors and differ in their efficacy for activating PLC (2, 9, 18). For example, in RBL-1 cells M1 receptors fail to mount a Ca2+ response in cells injected with antisense oligonucleotides directed against Galpha q, but the Ca2+ response is unaffected in cells injected with antisense RNA directed against Galpha 14 (9). These data can be interpreted as indicating that coupling in cells expressing Galpha 11/Galpha q and Galpha 14 cannot be regarded as promiscuous and that expression of Galpha 14 allows a cell type expressing numerous PLC-coupled receptors to gain specificity. This is indeed an attractive possibility in the pancreatic acinar cell, since this cell type expresses multiple PLC-coupled receptors. In addition, these receptors appear not to be coupled identically, since activation of individual receptors results in markedly different patterns of Ca2+ signaling events (16, 28). The coupling of individual receptors to specific members of the Galpha q family when multiple proteins are present in a particular cell type has not been reported. Precedent exists, however, for specificity of interaction between members of a Galpha family and an effector. For example, the coupling of receptors to Ca2+ channels by individual Goalpha family members, presumably to achieve signal specificity, is well documented in GH3 pituitary cells. In this case, inhibition of Ca2+ channels through muscarinic receptor stimulation is mediated through a heterotrimer that includes a Goalpha 1 subunit, while somatostatin-induced inhibition is mediated through a Goalpha 2 subunit-containing complex (10).

Ablation of protein function by immunoneutralization by specific antisera is a useful technique for probing the function of a particular protein. Using this technique, it has been demonstrated that Ca2+ signaling stimulated by agonists, such as thyrotropin-releasing hormone in GH3 cells, thromboxane A2 in platelets, and angiotensin in rat liver, is a result of receptors for these agonists interacting with Galpha q family proteins (8, 20, 27). In a similar fashion, this study has demonstrated that both CCh and CCK-induced Ca2+ signaling in single pancreatic acinar cells is markedly inhibited by injection of antiserum directed against Galpha q family members. This inhibition is evident even at physiological concentrations of agonist where it is impossible to detect changes in IP3 concentration. It has been presumed that signaling at physiological concentrations of agonists is the result of the activity of Galpha q proteins since Ca2+ signaling events can be inhibited by high concentrations of heparin, an antagonist of the IP3 receptor (6, 29). This study thus provides direct evidence that physiological concentrations of agonist result in the activation of Galpha q family proteins.

As a consequence of the epitope that the antiserum is directed against, the mechanism of immunoneutralization of this particular antiserum-protein interaction is likely to be a result of the antiserum "masking" the region of the protein that is essential for liganded-receptor recognition (21). Because the extreme COOH terminus of Galpha q proteins is predicted to be exposed in the intact (nonactivated) heterotrimer, the most plausible mechanism for inhibition of Ca2+ signaling would be that binding of the antiserum to the intact heterotrimer inhibits the dissociation of heterotrimer, thus preventing activation of the effector. Alternatively, it is possible that binding of antiserum to the activated alpha -subunit sterically hinders its interaction with PLC-beta . The lack of inhibition of responses in cells injected with Galpha q and Galpha 11 (or combination)-specific antisera is likely due to the antiserum either not recognizing the protein in its native form or binding to a region not important in its action.

In this study, it was also demonstrated that the Galpha q-CT antiserum would inhibit CCK-induced signaling in CHO cells to a similar marked extent (>75%) compared with acini. This is of interest since this study has demonstrated that acini but not CHO cells express Galpha 14 in addition to Galpha q/Galpha 11. The similarity in the degrees of inhibition argues that the antiserum also recognizes and neutralizes Galpha 14 in acini, despite the two amino acid substitutions in this region between Galpha q/Galpha 11 and Galpha 14. In support of the contention that the Galpha q-CT antiserum recognizes Galpha 14, Milligan and colleagues (14) in a study designed to separate Galpha q/Galpha 11 on urea gradient gels showed that a similar antiserum would reveal three bands in some cells, which the authors speculated probably represented Galpha q, Galpha 11, and Galpha 14.

In conclusion, this study has demonstrated the expression of individual members of the Galpha q heterotrimeric G protein family and shown that activation of these proteins is a key step in the generation of a Ca2+ signal in pancreatic acinar cells, stimulated by both physiological and pathophysiological concentrations of agonists. Further studies are needed to determine if Galpha q, Galpha 11, and Galpha 14 contribute equally and interchangeably to the response to each pancreatic secretagogue or whether specificity exists in signaling through a particular secretagogue receptor.


    ACKNOWLEDGEMENTS

We thank Nellie Park for technical support during the project. We also thank Dr. Craig Logsdon and Dr. Linda Samuelson for helpful discussion throughout the project.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-41122 (J. A. Williams) and DK-54568 (D. I. Yule), Michigan Diabetes Research and Training Center Grant P60-DK-20572, and Michigan Gastrointestinal Peptide Center Grant P30-DK-34933.

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: D. I. Yule, Dept. of Pharmacology and Physiology, School of Medicine and Dentistry, Univ. of Rochester, Box 711, Rochester, NY 14642-8711.

Received 10 August 1998; accepted in final form 26 September 1998.


    REFERENCES
Top
Abstract
Introduction
Methods and materials
Results
Discussion
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