Individual Subtypes of Enteroendocrine Cells in the Mouse Small Intestine Exhibit Unique Patterns of Inositol 1,4,5-trisphosphate Receptor Expression
Department of Internal Medicine, Division of Endocrinology, Metabolism, and Lipid Research, Washington University School of Medicine, Saint Louis, Missouri
Correspondence to: Burton M. Wice, PhD, Dept. of Internal Medicine, Division of Endocrinology, Metabolism and Lipid Research, Washington U. School of Medicine, Campus Box 8127, 660 South Euclid Avenue, St Louis, MO 63110. E-mail: bwice{at}im.wustl.edu
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Summary |
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Key Words: enteroendocrine cells K-cells GIP hormone secretion inositol 1,4,5-trisphosphate receptor IP3R
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Introduction |
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GIP is produced almost exclusively by a specific subpopulation of EE cells in the proximal small intestine, called intestinal K-cells. This hormone is released in response to nutrients present in the lumen of the gut but not to those circulating in the blood (Fehmann et al. 1995; Schmier 1995
; Drucker 1998
). Surprisingly, hormone release from GIP-producing cell lines is independent of ATP-regulated potassium channels (Ramshur et al. 2002
). Consistent with this result, IHC studies revealed that GIP-producing EE cells in the mouse small intestine do not express detectable levels of inward rectifying potassium channel 6.1 (Kir 6.1) or inward rectifying potassium channel 6.2 (Kir 6.2) (Wang et al. 2003
). Therefore, ATP-regulated potassium channels are not major regulators of cell depolarization and subsequent calcium mobilization in GIP-producing EE cells in vivo. A detailed survey of the mouse small intestinal epithelium revealed that EE cells that produce GLP-1, CCK, or SST also do not express detectable levels of Kir 6.1 or Kir 6.2 (Wang et al. 2003
). Conversely, most of the EE cells that produce chromogranin A (CGA), serotonin, and/or substance P (sub P) also express Kir 6.2, suggesting that these particular subpopulations of EE cells utilize ATP-regulated potassium channels to control hormone release. About 40% of EE cells that produced secretin also expressed Kir 6.2. Therefore, different subtypes of EE cells exhibit remarkable heterogeneity with respect to expression of subunits required to generate functional ATP-regulated potassium channels.
Endocrine cells can also release hormones independent of ATP-regulated potassium channels by mobilizing calcium from intracellular stores (Rutter 2001). Release of ER-derived calcium stores can be regulated by ryanodine receptors (RyR) (Islam 2002
) and/or inositol 1,4,5-trisphosphate receptors (IP3Rs) (Blondel et al. 1995
; Taylor et al. 1999
; Hagar and Ehrlich 2000
; Thrower et al. 2001
). IP3Rs are expressed in secretory granules of neuroendocrine cells and are believed to facilitate secretion by controlling calcium release from the granules (Blondel et al. 1995
). There are at least three different IP3Rs. Type 1, 2, and 3 receptors (IP3R-1, IP3R-2, and IP3R-3, respectively) are encoded by different genes and exhibit a high degree of overall sequence homology. IP3Rs assemble into homo- or heterotetrameric complexes (Nucifora et al. 1996
). Because each isoform exhibits unique regulatory properties, complexes of different IP3Rs display different calcium-gating properties. IP3R-1 and -3 are expressed in isolated intestinal epithelial cells (Matovcik et al. 1996
; Nakanishi et al. 1996
), suggesting that they are present in EE cells. However, the specific subtypes of EE cells that express IP3Rs have not been identified. Therefore, double-label immunofluorescence studies were performed to determine the pattern of IP3R expression in EE cells in the mouse intestinal epithelium. This analysis revealed unexpected levels of complexity and heterogeneity in IP3R expression by different subtypes of EE cells.
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Materials and Methods |
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Antibodies
Information concerning the primary and secondary antibodies is presented in Table 1. To generate our own antibodies against GIP, human GIP was purchased from California Peptide Research (Napa, CA), conjugated to KLH using EDAC, and then used to immunize guinea pigs. The specificity of the antisera was confirmed by double-label immunofluorescence. Briefly, a single paraffin-embedded section of mouse small intestine was labeled with commercial rabbit anti-GIP plus the guinea pig anti-GIP antiserum (Table 1). Bound primary antibodies were detected using FITC-conjugated donkey anti-rabbit IgG plus Cy3-conjugated donkey anti-guinea pig IgG secondary antibodies. Three hundred labeled cells were counted under the fluorescent microscope and all positive cells were labeled with both antibodies.
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When both primary antibodies were raised in the same species, a two-step procedure was conducted for double-label studies (Shindler and Roth 1996). First, tyramide signal amplification (TSA) was performed using the TSA-Plus Fluorescein System (PerkinElmer Life Sciences; Boston, MA) to detect the EE cell hormone. Briefly, tissues sections were re-hydrated, treated with hydrogen peroxide, subjected to antigen retrieval required for subsequent detection of the appropriate IP3R, and then incubated only with the rabbit antibody against the indicated EE cell hormone at a dilution greatly increased over the optimal dilution for the standard method (Table 1). After buffer washes, the tissue section was incubated with HRP-conjugated donkey anti-rabbit IgG antibodies (30 min at RT), washed with buffer, and then incubated with FITC-conjugated tyramide (10 min at RT). This protocol labeled the EE cell hormone with FITC (green fluorescence). Next, the tissue section was incubated with the second primary antibody, which was then detected with Cy3-conjugated donkey anti-rabbit IgG secondary antibodies (red fluorescence). Because the TSA amplifies the signal from the highly diluted first primary antibody to such a great extent, the first primary antibody is not detected by the Cy3-conjugated donkey anti-rabbit IgG antibodies used to detect the second primary antibody. This was confirmed for each primary antibody by omitting the second primary antibody. For example, the rabbit anti-GIP antibody, when used at a 1:300,000 dilution, was detected with FITC-labeled tyramide but not with Cy3-conjugated donkey anti-rabbit IgG antibodies. Because of variability in the degree of antigen retrieval and tyramide amplification on each occasion, several concentrations of the first primary antibody were used each time labeling was performed.
Quantification of Double-labeled EE Cells
EE cells expressing a particular hormone were identified with fluorescent microscope. The filters were then switched to determine whether the same EE cells also expressed the second fluorescently labeled antigen. At least 100 EE cells were identified per section and the numbers of those cells that also expressed the indicated IP3R were determined. Sections prepared from at least five different mice were counted to determine the number of GIP-producing vs CGA-producing EE cells that expressed each IP3R isoform. For statistical analyses, data for each tissue section were treated as a single sample (i.e., n=5 for each pair of antigens). For additional double-label studies, a single section was quantified. However, qualitatively similar results were noted on at least two additional occasions.
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Results |
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Discussion |
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The unique pattern of IP3R expression in different subtypes of EE cells in the mouse small intestine has important biological implications. First, IP3R-1 and IP3R-3 appear to localize to distinct subcellular compartments (basal vs supranuclear, respectively), suggesting that these two different IP3Rs may regulate calcium flux from distinct intracellular pools. Second, the open probabilities for each of the IP3Rs vary as a function of intracellular IP3 and calcium concentrations (Thrower et al. 2001). Therefore, differential expression of these channels would allow different subtypes of EE cells to secrete hormones in response to different concentrations of intracellular signaling molecules. Remarkably, individual cells that produce sub P or secretin express different combinations of Kir 6.2, IP3-1, and IP3R-3. Because these observations were noted in single tissue sections prepared from fed animals, this heterogeneity can not be explained simply by differences in the nutritional state of different mice. Therefore, a single ligand could elicit multiple secretory responses for sub P and secretin.
GIP-producing EE cells reside predominantly in the proximal small intestine and are located in the crypts as well as on the villi. Intestinal L-cells, which produce GLP-1 plus additional hormones, are located principally in the distal intestine. Neither subtype of EE cells expresses detectable levels of Kir 6.2, IP3R-1, or IP3R-3. These results indicate that the patterns of expression of these regulatory molecules are not simply due to the geographic location of different subpopulations of EE cells. Rather, expression of Kir 6.2, IP3R-1, and IP3R-3 is differentially regulated along with different gut hormones.
Our results suggest that EE cells that produce GIP, GLP-1, CCK, or SST do not express CGA. It has been previously reported by many groups that SST-expressing EE cells produce little if any CGA (Varndell et al. 1985; Buffa et al. 1989
; Cetin and Grube 1991
; PortelaGomes et al. 1997
). However, there are conflicting reports in the literature concerning CGA expression in other subtypes of EE cells (Varndell et al. 1985
; Buffa et al. 1989
; Cetin et al. 1989
; Cetin and Grube 1991
; PortelaGomes et al. 1997
). It has been reported that CGA is not expressed in GIP-producing EE cells (Buffa et al. 1989
) or is expressed in only a subset of these cells (Varndell et al. 1985
; Cetin and Grube 1991
; PortelaGomes et al. 1997
). Fixation artifacts could account for some of the conflicting results in the literature. However, Cetin and co-workers (1989)
surveyed expression of CGA plus different EE cell hormones in the gastrointestinal tract of human, pig, cat, guinea pig, and cattle. They observed that CGA was produced in specific subtypes of EE cells in one species but not in others (e.g., CCK). Furthermore, individual subtypes of EE cells in a single species could express either none or very high levels of CGA. Because all of the tissues used for their study were similarly prepared and analyzed using the same set of antibodies, it appears unlikely that tissue fixation could account for the conflicting results. ProCGA can be processed to at least 10 different bioactive peptides that (a) inhibit hormone release from many cell types, including islet ß-cells, parathyroid chief cells, and parietal cells, (b) regulate intermediary metabolism in peripheral tissues, (c) inhibit vasoconstriction, (d) trigger apoptosis, and (e) exhibit antimicrobial activity (Taupenot et al. 2003
). Therefore, an alternative explanation for the conflicting results is that CGA is processed to different peptides by various EE cells and that individual antibodies to CGA can recognize the intact CGA molecule plus only specific subsets of CGA-derived peptides. As shown in Figure 5, we did not detect CGA and GIP in any of the same EE cells. This result was obtained using two different antibodies to GIP and two different antibodies to CGA. One of the antibodies to CGA was raised against the intact protein, whereas the other was generated against a synthetic peptide (see Table 1). When tissue sections were labeled with the sheep anti-CGA plus the rabbit anti-CGA antibodies, EE cells were labeled either with both antibodies or with neither antibody. Therefore, we feel that we are detecting intact CGA in EE cells that express detectable levels of this molecule. Thus, our results could be interpreted to suggest that EE cells that produce GIP, GLP-1, CCK, or SST do not produce detectable levels of intact CGA but may contain CGA-derived peptides.
Intact chromogranins are low-affinity, high-capacity calcium-binding proteins that aggregate at acidic pH. This aggregation facilitates the sequestration of hormones and other proteins that enter the regulated secretory pathway (Taupenot et al. 2003). It has been proposed that CGA is an "on/off" switch that controls the biogenesis of dense-core granules in endocrine and neuroendocrine cells (Kim et al. 2001
). Because certain subtypes of EE cells do not express detectable levels of intact CGA, other chromogranins or chromogranin-like molecules must play a role in secretory granule formation in these cells. Chromogranin B was not detected in GIP-producing EE cells in humans (PortelaGomes et al. 1997
) or guinea pigs (Cetin and Grube 1991
), whereas chromogranin C was detected in GIP-producing EE cells in guinea pigs (Cetin and Grube 1991
), but not in humans (PortelaGomes et al. 1997
). However, it is important to keep in mind that, as has been observed with CGA, studies using antibodies to different regions of chromogranin B have yielded conflicting results (Norlen et al. 2001
; PortelaGomes and Stridsberg 2002a
,b
). Therefore, we can not exclude the possibility that GIP-producing EE cells express chromogranin B.
Our results raise an important question: what molecules control cell depolarization and calcium mobilization from EE cells that do not express detectable levels of Kir 6.2 or IP3Rs? RyRs, as well as IP3Rs, can regulate release of ER-derived calcium stores (Islam 2002). Nicotinic acid adenine dinucleotide phosphate is a newly identified regulator of calcium mobilization from lysosomal stores (Lee and Aarhus 1995
; Lee 2003
; Patel et al. 2001
). This calcium store has been shown to be important for calcium mobilization by islet ß-cells (Johnson and Misler 2002
; Masgrau et al. 2003
; Mitchell et al. 2003
), exocrine pancreas (Cancela et al. 1999
; Burdakov and Galione 2000
), T-lymphocytes (Berg et al. 2000
), and sea urchin eggs (Churchill et al. 2002
). Studies are under way to determine whether RyRs or lysosomal calcium stores are involved in calcium mobilization from intracellular stores in GIP-producing EE cells.
EE cells that produce GIP, GLP-1, CCK, or SST do not express detectable levels of IP3Rs, Kir 6.2, or Kir 6.1. Therefore, do these subtypes of EE cells express the same repertoire of regulatory proteins or will a detailed molecular analysis reveal additional heterogeneity and complexity in their secretory machineries? Recent observations have demonstrated that GIP plays a critical role in promoting high fat diet-induced obesity and insulin resistance (Miyawaki et al. 2002). PYY, produced by GLP-1-producing L-cells, has been shown to inhibit food intake (Batterham et al. 2002
). Therefore, compounds that inhibit GIP secretion and/or promote PYY release represent potential therapeutic drugs that could reduce obesity and insulin resistance. Because hormone release from different subtypes of EE cells appears to be regulated by distinct machineries, an understanding of the unique nature of each type of EE cell should provide important information to lay the groundwork for the development of novel drugs to treat human diseases by targeting the secretory machineries of specific subtypes of EE cells.
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Acknowledgments |
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We also wish to thank Dr David Kipnis for helpful discussions.
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Footnotes |
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