ARTICLE |
Correspondence to: Lisa M. Matovcik, Surgical Service (112), VA CT Healthcare System, 950 Campbell Ave. West Haven, CT 06516.
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Summary |
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Ca2+/calmodulin-dependent protein kinases I and II, initially identified in brain on the basis of their ability to phosphorylate synapsin I, have been implicated in the regulation of Ca2+-dependent synaptic neurosecretion. Specific recombinant and synthetic peptide antibodies were used to examine the distribution of CaM kinases I and II in the rat pancreas and other tissues. The CaM kinase I antibodies detected a doublet of cytosolic proteins of ~38 and ~42 kD by immunoblot. CaM kinase I was observed in glucagon-containing A-cells at the periphery of the islet of Langerhans but had little or no overlap with pancreatic polypeptide or somatostatin cells. In contrast, CaM kinase II was localized to somatostatin-containing D-cells. CaM kinase I co-localized with glucagon secretory granules. CaM kinase II was not associated with the somatostatin granule but rather was enriched in areas of the cells that contained relatively little somatostatin. Because glucagon secretion is Ca2+-dependent, it is attractive to speculate that CaM kinase I may play a regulatory role in glucagon secretion. Glucagon and somatostatin cells both utilize intracellular Ca2+ for signaling. Therefore, specific CaM kinases may act as effectors of Ca2+ in these different cell types. (J Histochem Cytochem 46:519526, 1998)
Key Words: Ca2+/calmodulin-dependent, protein kinase I, Ca2+/calmodulin-dependent, protein kinase II, cell signaling, calcium, exocytosis, glucagon, somatostatin, pancreas, parathyroid, neuroendocrine cells
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Introduction |
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CALCIUM IS A MEDIATOR of intracellular signals in all eucaryotic cells, regulating such diverse functions as proliferation, gene expression, degradation, and exocytosis. The complex spatial and temporal changes in intracellular calcium concentration in response to external events are sensed by proteins that act as effectors of the calcium signals. Some effectors are specific, leading to one defined cellular response, and some are more general, influencing many related functions.
Most of the intracellular effects of calcium are mediated by binding to calmodulin. In turn, the calcium/calmodulin complex interacts with many classes of effector molecules, including the calcium/calmodulin-dependent protein kinases (CaM kinases), resulting in their activation (
Like CaM kinase II, CaM kinase I catalytic activity and mRNA have been found in all tissues examined; the highest levels of the enzyme are present in brain, adrenals, liver and lung ( and ß, have recently been isolated from an embryonic rat brain cDNA library (
The substrate recognition motif for CaM kinase I, Hyd- Xaa-Arg-Xaa-Xaa-(Ser/Thr)-Xaa-Xaa-Xaa-Hyd (
In this study we compared the distributions of two effectors of calcium signaling, CaM kinases I and II, in gastrointestinal tissues using an immunocytochemical approach. The results indicate that the two enzymes have distinct cellular distributions with little overlap. The greatest differences are observed in the pancreatic islet, in which CaM kinase I immunoreactivity is concentrated in glucagon-containing cells and CaM kinase II immunoreactivity is found exclusively in somatostatin-containing cells. We also compared the localization pattern of CaM kinase I to previously described localization patterns of CFTR (
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Materials and Methods |
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Antibodies
CC77 is an affinity-purified rabbit antibody to CaM kinase I and is expressed in E. coli as a glutathione S-transferase fusion protein (-subunit of rat brain CaM kinase II (
Immunoblot Analysis
Tissues were cut into 5-10 mm cubes in ice-cold minimal Eagle's Medium (Gibco; Grand Island, NY) and were homogenized with a mechanical homogenizer in the presence of protease inhibitors (
Immunofluorescence Localization
Rats were perfused with DMEM, followed by 2% formaldehyde (made from paraformaldehyde) in 0.075 M NaPO4, pH 7.4. Rats were treated in accordance with local institutional guidelines. Tissues were removed, fixation was continued for a total of 1 hr, and the tissues were cryopreserved in 15% sucrose in PBS overnight at 4C, then cryosectioned to approximately 3 µm in thickness. After incubation in 3% goat serum in PBS for 1 hr, then for 2 hr with primary antibody, FITC-goat anti-rabbit immunoglobulin F(ab')2 (1:200) (Biosource; Camarillo, CA) or TRITC-goat-anti-mouse F(ab')2 (1:300) (Biosource) was used to detect the antigen.
Slides were photographed on a Zeiss Axiophot microscope (Thornwood, NY) or a Zeiss Axiovert 10 microscope equipped with a Bio-Rad (Richmond, CA) MRC-600 confocal attachment.
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Results |
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Immunoblot Survey of CaM Kinase I in Non-neuronal Tissues
Rat tissues were homogenized and postnuclear supernatants were subjected to immunoblot analysis using antibodies CC77 and CC102 (Figure 1). In the pancreas, CC77, raised against a fusion protein encoding the entire 42-kD -isoform, (
-isoform, preferentially detected the larger band in all tissues examined. In addition, an ~38-kD band was also observed in the pancreas, stomach, adrenal and submandibular glands. Interestingly, in the parathyroid gland, this antibody recognized an ~42-kD band even though CC77 did not. In some of the tissues examined the antibodies recognized other unidentified nonspecific bands. CC77 reacted with minor higher molecular weight bands in stomach and parathyroid. Antibody CC102 detected prominent high molecular weight bands in the stomach and minor bands in the adrenal gland. These observations are consistent with the presence of at least two isoforms of CaM kinase I in various ratios in all of the non-neuronal tissues surveyed.
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Immunocytochemical Localization of CaM Kinase I in Non-neuronal Tissues
Immunofluorescence microscopy was used to examine the distribution of CaM kinase I in tissues outside the central nervous system. The choroid plexus has previously been shown to be immunoreactive with CC77 and to have high levels of CaM kinase I (
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In the duodenum, the most prominent staining by antibodies CC77 (Figure 2D) and CC102 (Figure 2E and Figure 2F) was in mononuclear cells of the lamina propria. There was also prominent staining of either capillary endothelium or lacteals at the core of the villus. A distinct population of mucosal cells were labeled by antibodies CC77 (data not shown) and CC102 (Figure 2F). CaM kinase I immunoreactivity was restricted to the basal region of these cells; phase microscopy demonstrated that they were mucous-secreting goblet cells. Only goblet cells in the mid-villous region, not those at the tip, were immunoreactive. Notably, CaM kinase I immunoreactivity was detected neither in the apical membrane region of intestinal mucosal cells where CFTR is found (
CaM Kinase I and II Are Soluble Proteins in Pancreas
Rat pancreas postnuclear supernatant was centrifuged at 100,000 x g to obtain a soluble and a particulate pellet fraction. Each of these was examined for immunoreactivity with CaM kinase I (CC77 and CC102) and CaM kinase II (G301) by immunoblotting (Figure 3). CaM kinase I was recovered in the soluble fraction but not in the pellet, and therefore is a soluble cytosolic protein in the pancreas, consistent with its predominantly cytosolic localization in the cortex of the brain (-subunit of CaM kinase II.
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Immunocytochemical Localization of CaM Kinases I and II in Pancreas
The localization of CaM kinase I and CaM kinase II in the pancreas was determined by immunofluorescence microscopy (Figure 4). Both CaM kinases were predominantly expressed in the islet of Langerhans. Antibodies to CaM kinase I (CC77, Figure 4A, and CC102, Figure 4B) detected a signal at the periphery of the islet, with the majority of cells at the mantle of the islet being immunoreactive. A cytoplasmic pattern of labeling was observed; labeling was excluded from the nucleus. Little or no CaM kinase I staining was seen in acinar cells or in the pancreatic duct system. Immunoabsorption with the fusion protein antigen completely blocked CC77-generated immunofluorescence in the pancreatic islet and greatly diminished CC102 staining (data not shown). G301, the antibody specific for CaM kinase II, labeled a subset of cells in the mantle of the islet, but these cells were less numerous than the CaM kinase I-positive cells (Figure 4C). With a slightly higher antibody concentration and longer photographic exposure times, CaM kinase II was also observed to be prominent in the apical region of pancreatic ducts (Figure 4D).
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CaM Kinase I Co-localizes with Glucagon in the Islet of Langerhans
Double label studies using islet cell markers were performed to identify the cells immunoreactive for CaM kinase I. Glucagon immunoreactivity was observed in the majority of the cells at the periphery of the islet, and CaM kinase I was co-localized to this cell population (Figure 5A). At higher magnification it was apparent that CaM kinase I had a granular cytoplasmic distribution and co-localized with glucagon granules in the A-cells (Figure 5B). Simultaneous labeling for pancreatic polypeptide and CaM kinase I revealed that these two antigens primarily reside in different cell types (Figure 5C and Figure 5D) but rarely do they co-localize to the same cell (Figure 5C). Double labeling for somatostatin and CaM kinase I revealed no overlap between the two antigens (Figure 5E and Figure 5F).
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CaM Kinase II Co-localizes with Somatostatin in the Islet of Langerhans
Double label studies with rabbit antibody 301 and mouse monoclonal anti-somatostatin demonstrated that CaM kinase II in the islet is restricted to D-cells (Figure 6A and Figure 6B). Although CaM kinase II was localized to virtually all cells with somatostatin immunoreactivity, the intensity of CaM kinase labeling was variable. Somatostatin was prominent at the cell periphery and in the processes that extend from these neuroendocrine cells; CaM kinase II was concentrated at the center of the cell. There was no co-localization of CaM kinase II with pancreatic polypeptide (Figure 6C) or with glucagon (Figure 6D).
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Discussion |
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The cellular and subcellular distributions of CaM kinase I were examined in rat gastrointestinal tissues. Unexpectedly, the most prominent immunoreactivity was observed in the pancreatic islets of Langerhans. Double label immunolocalization demonstrated that CaM kinase I was localized to the glucagon-containing A-cells and occasional pancreatic polypeptide cells, and was not observed in insulin or somatostatin cells. Within A-cells, CaM kinase I was specifically localized to the glucagon-containing secretory granule. Glucagon secretagogues increase intracellular Ca2+, which leads to vesicle release. Because glucagon secretion is Ca2+-dependent it is attractive to speculate that CaM kinase I may play a regulatory role in glucagon secretion. It may integrate input from spatial and temporal changes in intracellular Ca2+ concentration, such as the Ca2+ oscillations that have been observed to occur spontaneously in a clonal glucagon-secreting cell line derived from hamster endocrine pancreas (
The distributions of CaM kinases I and II were compared in the rat pancreas and were found to be distinct. CaM kinase II was detected at high levels in the pancreatic islet but in a different cell population. It was specifically found in somatostatin-secreting D-cells, but there was no evidence for co-localization of somatostatin and CaM kinase II to the same intracellular compartment within the D-cell.
A regulatory role for CaM kinase II in insulin secretion has been observed, and recently the 2 isoform has been identified on the insulin secretory granule of B-cells (
2-isoform is identical to the 301 antigen with the exception of one conservative amino acid substitution (
291K/
2292R) (
CaM kinase I is apparently encoded for by only one gene in both rat (, ß, and
, that show considerable homology throughout their catalytic and regulatory regions (
and ß differ at their C-terminus (the C-terminus of
has not yet been defined). The predicted molecular weights of
and ß are 41,643 and 38,463 daltons, respectively. Therefore, it is likely that the protein bands that we are detecting represent different isoforms of CaM kinase I. Although both antibodies labeled bands of approximately 42 and 38 kD, they demonstrated tissue specificity, as illustrated in the pancreas and the parathyroid.
CaM kinase I has been found to efficiently phosphorylate CFTR, a chloride ion channel, although the physiological relevance of this is unknown (
Another in vitro substrate for CaM kinase I is the cAMP response element binding protein (CREB), a nuclear phosphoprotein that regulates transcription (
In summary, CaM kinases I and II are found in distinct cell types in the endocrine pancreas that are specialized for the secretion of glucagon and somatostatin, respectively. CaM kinase I is associated with the glucagon secretory granule. On occasion it was observed in pancreatic polypeptide cells but not in acinar or duct cells. In contrast, CaM kinase II was observed predominantly in D-cells of the islet and in the pancreatic duct system. Because CaM kinases I and II are found in different islet cells and have different intracellular localizations, they are likely to have different substrates and different effector mechanisms.
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Acknowledgments |
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Supported by Merit Review Awards from the Veterans Administration to FSG and LMM.
We thank Lillemor Wallmark for performing the immunofluorescence experiments, Michael Lipcan and Steven Rhee for the immunoblots, and Gloria Bertuzzi for technical assistance.
Received for publication June 5, 1997; accepted November 4, 1997.
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