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Cholecystokinin Cells Purified by Fluorescence-Activated Cell Sorting Respond to Monitor Peptide with an Increase in Intracellular Calcium RODGER A

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Cholecystokinin Cells Purified by Fluorescence-Activated Cell Sorting Respond to Monitor Peptide with an Increase in Intracellular Calcium RODGER A Proc. Nati. Acad. Sci. USA Vol. 89, pp. 5147-5151, June 1992 Medical Sciences Cholecystokinin cells purified by fluorescence-activated cell sorting respond to monitor peptide with an increase in intracellular calcium RODGER A. LIDDLE*tt, MARY ANN MISUKONIS*t, LYNNE PACY§, AND ANDREW E. BALBER§¶ Departments of *Medicine and TMicrobiology and Immunology, and §Flow Cytometry Facility, Comprehensive Cancer Center, Duke University Medical Center, Durham, NC 27710; and tDurham Veterans Administration Medical Center, Durham, NC 27705 Communicated by Viktor Mutt, February 21, 1992 ABSTRACT Cholecystokinin (CCK) is secreted from spe- upper small intestine and appears to be responsible for the cific enteroendocrine cells of the upper small intestine upon pronounced increase in CCK secretion that occurs with ingestion of a meal. In addition to nutrients, endogenously diversion of bile and pancreaticjuice (13, 14). Therefore, it is produced factors appear to act within the gut lumen to stim- likely that this intestinal CCK releasing factor is physiolog- ulate CCK release. One such factor is a trypsin-sensitive ically important in the regulation of CCK secretion. How- CCK-releasing peptide found in pancreatic juice, known as ever, this factor has not yet been isolated and its chemical monitor peptide. This peptide is active within the intestinal structure is not known. A second releasing factor named lumen and is hypothesized to stimulate CCK secretion by monitor peptide has been identified in high amounts in interacting directly with the CCK cell. We have found that pancreatic juice (15-17). Monitor peptide is a 61-amino acid monitor peptide releases CCK from isolated rat intinal peptide that is structurally related to a 56-amino acid protein mucosal cells and that this effect is dependent upon extracel- that possesses trypsin inhibitor-like activity-hence, the lular calcium. In the present study, we used monitor peptide as name pancreatic secretory trypsin inhibitor (PSTI). How- a tool for isolating CCK cells from a population of small ever, the ability of monitor peptide to stimulate CCK release intestinal mucosal cells. Dispersed rat intestinal mucosal cells is independent of its trypsin inhibitor activity and the 56- were loaded with the calcium-sensitive fluorochrome Indo-1, amino acid PSTI itself does not stimulate CCK secretion. and CCK secretory cells were identified spectrofluorometri- Therefore, monitor peptide appears to directly stimulate the cally by their change in fluorescence when stimulated with CCK cell by interacting with the apical surface possibly monitor peptide. Cells demonstrating a change in their emis- through a membrane receptor. sion fluorescence ratio were sorted using a fluorescence- Most studies of CCK secretion have been conducted in activated cell sorter. More than 90% of the sorted cells stained whole animals, intestinal segments, or only partially enriched positively for CCK with immunohistochemical staining. Fur- preparations of CCK cells (2, 18-20). Accordingly, it has not thermore, sorted cells secreted CCK when stimulated with been possible to study intracellular mechanisms regulating membrane-depolarizing concentrations of potassium chloride, CCK release without possible interference from other hor- dibutyryl cAMP, calcium ionophore, and monitor peptide. mones, neurotransmitters, or paracrine agents. We have re- These findings indicate that functional intestinal CCK cells can cently developed a method for studying hormone secretion be highly enriched using fluorescence-activated cell sorting. from rat intestinal mucosal cells in a perifusion apparatus. In Furthermore, monitor peptide appears to interact directly with this preparation, CCK cells represent <1% ofthe mucosal cell CCK cells to signal CCK release through an increase in population. Nevertheless, cells release CCK in response to intracellular calcium. calcium ionophores and monitor peptide. Moreover, the abil- ity of monitor peptide to stimulate CCK secretion was depen- Cholecystokinin (CCK) is an established gastrointestinal dent upon extracellular calcium. Therefore, we postulated that hormone that is localized in discrete mucosal endocrine cells monitor peptide is a specific secretagogue for the CCK cell and of the upper small intestine (1). CCK is secreted from the that one mechanism by which monitor peptide affects the CCK intestine in response to eating and has effects on many cell is by increasing intracellular calcium ([Ca2+]i). digestive processes including gallbladder contraction, pan- Calcium-sensitive fluorochromes that accumulate intra- creatic secretion, bowel motility, and perhaps satiety (2). cellularly are sensitive to changes in [Ca2+]1 (21, 22). We have Although much is known about the actions of CCK, many utilized these properties to identify CCK cells among a aspects regarding the cellular mechanisms regulating its mixture of rat intestinal mucosal cells loaded with a calcium- secretion have not been characterized. CCK release can be sensitive dye, the acetoxymethyl ester of Indo-1 (Indo-1 regulated in several different ways. It is hypothesized that AM), by measuring the change in calcium fluorescence of foods in the intestinal lumen that come in contact with the CCK cells when stimulated with monitor peptide. By using apical surface of the CCK cell may affect CCK release fluorescence-activated cell sorting, it was then possible to directly. In addition, it is likely that neural and hormonal isolate a highly enriched population of biologically respon- factors also modulate CCK secretion (3-9). CCK secretion is sive CCK cells that could be used for studying CCK secretion also under negative feedback regulation by pancreatic secre- in vitro. tion whereby active proteases in the intestinal lumen inhibit MATERIALS AND METHODS CCK secretion, and inactivation of intestinal proteases or Materials. The following materials were purchased. Hepes, binding of proteases by substrates such as protein or trypsin EDTA, EGTA, and goat serum were from Sigma; minimal inhibitor stimulates CCK secretion (10-12). Eagle's medium amino acid supplement and Dulbecco's Recently, it has been proposed (13-16) that CCK secretion bo- is regulated by two types oftrypsin-sensitive "CCK releasing modified Eagle's medium (DMEM) were from GIBCO; factors." One type of releasing factor is produced in the Abbreviations: CCK, cholecystokinin; [Ca2+]j, intracellular calcium ion concentration; Indo-1 AM, the acetoxymethyl ester of Indo-1. The publication costs of this article were defrayed in part by page charge *To whom reprint requests should be addressed at: Department of payment. This article must therefore be hereby marked "advertisement" Medicine, P.O. Box 3083, Duke University Medical Center, in accordance with 18 U.S.C. §1734 solely to indicate this fact. Durham, NC 27710. 5147 Downloaded by guest on September 29, 2021 5148 Medical Sciences: Liddle et al. Proc. Natl. Acad Sci. USA 89 (1992) vine serum albumin, fraction V, was from Miles; octadecyl- For perifusion of cells after cell sorting, =105 cells were silylsilica (C18 Sep-Pak) cartridges were from Waters; Seph- incubated in DMEM supplemented with 10%6 (wt/vol) bovine adex G-50 medium resin was from Pharmacia; collagenase serum albumin and layered over 1 ml ofSephadex G-50 resin. was from Worthington; Indo-1 AM and Pluronic F-127 were Cells were incubated at 370C in 95% 02/5% CO2 for 18 h. The from Molecular Probes; CCK antibody AB 1972 was from cells and Sephadex mixture were then pipetted into a 3-ml Chemicon; goat anti-rabbit antibody fluorescein-conjugate column and perifused as described above. was from Tago; A23187 and ionomycin were from Calbio- Flow Cytometric Analysis of Intracellular Ca2+ and Cell chem; and rats were from Charles River Breeding Labora- Sorting. Indo-1 AM was used to measure [Ca2+]i in intestinal tories. The following materials were gifts. Monitor peptide mucosal cells before and after exposure to monitor peptide. was from Gary Green (University of Texas, San Antonio); The AM group of Indo-1 AM allows this compound to enter MK-329 was from Merck Sharp & Dohme; and CCK-8 was cells (21, 22). Intracellular esterases then cleave the AM from Squibb. group, rendering the dye impermeable and, thus, trapped Cell Preparation. Male Sprague-Dawley rats, weighing within cells. Binding ofCa2+ to Indo-1 alters its fluorescence 250-300 g, were sacrificed, and the proximal 20 cm of small spectrum. [Ca2+], can be measured by comparing the ratio of intestine beginning 3 cm distal to the pylorus was removed. Indo-1 emission at 405 nm and 485 nm when excited at 350 The intestine was everted and washed for 1 min with saline nm. This ratio increases as Ca2+ enters cells and complexes at room temperature to remove mucus. The tissue was then with Indo-1 (26, 27). incubated for 5 min at room temperature under agitation in 15 From 1 to 5 x 107 intestinal mucosal cells were loaded with 5 ,uM Indo-1 AM for 45 min at 240C in 1 ILM Pluronic F-127 ml of calcium-free modified Krebs-Henseleit bicarbonate in Hepes buffer as recommended by the manufacturer and buffer (KHB) containing 2.5 mM EDTA (23). This buffer was washed twice. Loading of these cells with Indo-1 AM was enriched with mixed amino acids and was equilibrated to pH measured by the dual emission method using an EPICS 753 7.4 in an atmosphere of 95% 02/5% Co2. Tissue was then flow cytometer equipped with an argon ion laser operating in placed in 10 ml of KHB with 1.2 mM CaCl2 containing 1 mg the ultraviolet at 40 mW. Forward and orthogonal light of collagenase and agitated by gentle inversion for 10 min. scattering were used to gate analysis to signals from intact After this incubation, the suspension was centrifuged for 3 cells. Fluorescence emission ratios for intracellular Indo-1 min at 100 x g. The supernate was discarded and the pellet were calculated in real time for each cell (21, 22). (containing cells) was resuspended in 2 ml of Hepes buffer Once baseline fluorescence ratios were established, cells containing 10 mM Hepes, 1.2 mM CaCl2, 103 mM NaCl, 1 were stimulated with monitor peptide (10 nM).
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