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 ability 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). Cells that mM NaH2PO4, 4.7 mM KCl, 0.56 mM MgCl2, 11.1 mM exhibited an increased fluorescence ratio, reflecting an in- glucose, 0.1% bovine serum albumin, and mixed amino acids creased [Ca2+]i, were sorted into tissue culture medium at (adjusted to pH 7.4). At this point, single cells were identified rates of 500-1000 cells per sec. microscopically. For perifusion studies, these cells were Immunohistochemical Stining. Cells were prepared in sus- added to a vial containing 2.5 ml of Sephadex G-50 medium pension for immunohistochemical labeling by using previ- resin that had been preswollen in saline overnight at 4°C. The ously described methods (28). From 1 to 50 x 105 cells were cells and resin were mixed for 1 min. fixed using 2% (wt/vol) paraformaldehyde. Prior to staining, Perifusion of Intestinal Mucosal Cells. A perifusion appa- cells were incubated with 10% (vol/vol) goat serum for 1 h ratus was modified as described (24, 25). Cells were loaded and then washed three times with phosphate-buffered saline into a perifusion chamber consisting of a 3-ml disposable (PBS). Cells were stained for CCK content using antibody plastic syringe whose plunger was perforated with a 19-gauge AB 1972 (which is directed against the midportion of CCK- needle connected to polyethylene tubing. Two to 2.5 ml of 33) at 1:100 dilution (28). Goat anti-rabbit antiserum was used Sephadex resin/cell mixture was layered over a double layer as secondary antibody at a 1:40 dilution in PBS. To confirm of 5-,um nylon filters. Perifusion solutions were made of the specificity ofantibody staining for CCK-containing cells, Hepes buffer (pH 7.4) supplemented as described above and AB 1972 was preabsorbed with 10 nmol ofCCK-33 (Peninsula bubbled with 100% 02. Solutions were maintained in a 37°C Laboratories). This preabsorption eliminated specific cell water bath and pumped through silicone-coated polyethylene staining. In addition, staining with antibodies OAL-656 tubing (0.02 inch, inner diameter; 0.037 inch, outer diameter; (which is specific for the sulfated region ofCCK) (29) and AB 1 inch = 2.54 cm) by a peristaltic pump through the perifusion 1972 yielded similar results. column at a rate of 1 ml/min. The tip ofthe syringe was fitted CCK Assay. CCK values were measured by bioassay as with a 19-gauge needle, which, in turn, was attached to described (11, 30). In brief, Sep-Pak cartridges (containing polyethylene tubing that emptied into a fraction collector. CCK from the column fractions) were eluted with 1 ml of Once loaded into the column, cells were equilibrated for 1 ethanol/1% trifluoroacetic acid, 4:1 (vol/vol), into incuba- h with Hepes buffer to obtain a steady baseline level of CCK tion vials and dried under nitrogen. Extracts were then release. The perifusion medium could be changed instantly incubated with isolated rat pancreatic acinifor 30 min at 37C, by means of switching flasks. In this way, perifusion medium and the amylase released into the incubation medium was containing different reagents were sequentially perifused assayed using procion yellow-coupled starch as substrate. through the chamber containing the mucosal cells. Column Amylase release expressed as percent of total amylase con- fractions were passed through octadecylsilylsilica (C18 Sep- tent was compared with a dose-response curve for CCK-8. Pak) cartridges prewashed with 5 ml of methanol and 20 ml With this method, CCK concentrations in the original column ofdeionized water, to concentrate CCK (11). Cartridges were effluent as low as 1 fmol per fraction could be detected. then washed with 20 ml of deionized water and stored at The use of animals for these studies was approved by the -20°C or used immediately for assay. Duke University Institutional Animal Care and Use Com- All periods of stimulation with secretagogues were 15 min mittee. long followed by a period of at least 20 min during which the column was perifused with Hepes buffer. Fractions for assay RESULTS were collected at 5-min intervals. The dead space of the Effect of Monitor Peptide on CCK Relae. Dispersed system was 4.6 ml; therefore, the effect of a test substance intestinal mucosal cells were mixed with Sephadex G-50 lagged approximately one fraction behind its initiation. All resin, introduced into the perifusion column, and perifused solutions to be perifused were maintained at 370C in a water with Hepes buffer at 1 ml/min. Fractions were collected at bath and were oxygenated with 10O% 02. 5-min intervals and measured for CCK activity. Monitor Downloaded by guest on September 29, 2021 Medical Sciences: Liddle et al. Proc. Natl. Acad. Sci. USA 89 (1992) 5149

peptide produced a prompt increase in CCK release that Monitor persisted for the duration of exposure (Fig. 1). The ability of peptide monitor peptide to stimulate CCK release was dependent upon extracellular calcium ion concentration since monitor 1 peptide had no effect on CCK release in calcium-free buffer. Several controls were used to confirm that the CCK activity measured by bioassay was an accurate estimate of 0 the amount of CCK bioactivity present in column fractions. (i) Perifusion buffer either with or without EGTA, after passage through Sep-Pak cartridges (identical to the manner a) 0.5 in which column fractions were processed), did not modify U basal or CCK-stimulated amylase release. (ii) Monitor pep- (11 tide in concentrations of up to 1 AM, when incubated with 0 pancreatic acini, had no effect on amylase release. (iii) The CCK receptor antagonist MK-329 (1 AM), when added to column fractions immediately before bioassay, completely 0 blocked the bioactivity of column fractions. 0 5 10 Cell Sorting with Monitor Peptide. Dispersed mucosal cells were loaded with Indo-1 AM, washed, and resuspended in Time (minutes) Hepes buffer containing 1.2 mM CaC12. Basal fluorescence ratios ranged between 0 and 0.4 (n = 10) and remained stable FIG. 2. Flow cytometric analysis of [Ca2+]i. Cells were loaded for up to 1 h. A change in fluorescence ratio was observed with Indo-1 AM and analyzed. The fluorescence ratio is shown on the with addition of monitor peptide. A concentration of 10 nM y axis in relation to time on the x axis. Each point shows the ratio for was sufficient to detect a in a single cell. Density of shading is proportional to cell number. Basal change fluorescence ratio in 15 fluorescence is shown at the far left and preceded the administration of 17 cell preparations. The fluorescence signal increased of monitor peptide, which was added to the incubation buffer at the within 30 sec ofadding monitor peptide and was sustained for indicated time. Cells with a fluorescence ratio (405 nm/485 nm) at least 30 min during which time cells were separated by >0.33 were sorted. One percent of cells in the experiment shown selecting a gating ratio over that ofbasal (Fig. 2). The number demonstrated an increase in their fluorescence ratio. Similar results of dispersed intestinal mucosal cells that responded to mon- were obtained in 15 other experiments. itor peptide by demonstrating a change in fluorescence ratio (thus reflecting an increase in [Ca2+]j) averaged 0.86 ± 0.16% cells) stained positively for CCK (Fig. 3 A and B). However, (mean ± SEM; n = 15) of the total number of mucosal cells after sorting 92.9% of sorted cells (n = 225 cells) stained loaded with Indo-1. This compared with >95% of cells positively for CCK (Fig. 3C). responding with a change in fluorescence ratio when stimu- CCK Secretion from Purified CCK Cells. The ability of lated with 1 ,uM ionomycin in the presence of 1.2 mM CaCl2. enriched CCK cells to secrete hormone in response to Immunohistochemical Staining of Sorted Cells. As a test of secretagogue stimulation was tested by placing cells into cell viability after sorting, >90% of cells excluded trypan short-term overnight culture with Sephadex G-50 beads. The blue. To determine the purity of CCK cells in this prepara- cell and resin mixture was then introduced into a perifusion tion, cells were analyzed by immunohistochemistry using a column for measuring dynamic hormone secretion. Enriched CCK-specific antibody to stain CCK-containing cells. In CCK cells were stimulated with four types of potential preparations of cells prior to sorting, 0.66% of cells (n = 455 secretagogues (Fig. 4). Cells were continuously perifused with Hepes buffer and then stimulated with 30 nM monitor M MP3 nM) MP3nM ) MP 3n) peptide. After an intervening period with perifusion of buffer 10, alone, cells were exposed to buffer to which 5 ,uM dibutyryl cAMP was added. This was in which Calcium-free 1.2 mM followed by Hepes 52 Calcium. Calcium: mM KCl was substituted for NaCl. Finally, the effects of the 8. calcium ionophore A23187 (1 .M) on CCK release was ..T.I...... tested. All four agents produced significant increases in CCK c

._ release. In between stimulations, CCK levels returned to u 6 basal levels, indicating that hormone release was not due to toxic effects on the CCK cells. Effects similar to those of A23187 were also achieved using 1 LM ionomycin. Control 4- 4- substances such as buffer alone, saline, and 1 ,uM bovine cd serum albumin had no effect on CCK release. 2. DISCUSSION In the present study we used a specific CCK secretagogue to ...... :...... identify and purify a population of CCK cells from intestinal o- mucosa. These experiments tested the hypothesis that mon- 0 20 40 60 80 100 120 itor peptide selectively stimulated CCK cells and, in so doing, increased [Ca2+]J. This hypothesis suggests that, if a mixed Time (minutes) population ofintestinal mucosal cells was loaded with the dye Indo-1, then stimulation with monitor peptide would increase FIG. 1. Effect of monitor peptide and extracellular calcium on [Ca2+]J, only in CCK cells. The increase in [Ca2+], would then CCK release from dispersed intestinal mucosal cells. Dispersed change the emission fluorescence within cells responding to intestinal mucosal cells were perifused for 1 h with Hepes buffer containing 1.2 mM CaC12 before beginning experiments at time 0. monitor peptide. These changes could then be detected by a Monitor peptide (MP, 3 nM) was perifused for 15-min periods as fluorescence-activated cell sorter and CCK cells could be designated by arrows. From 25 to 70 min, perifusion buffer was separated from other cells in the mucosal preparation. Im- calcium-free Hepes containing 1 mM EGTA. The data shown are munofluorescent antibody studies indicated that >90%o of representative of three experiments. sorted cells stained positively for CCK. Therefore, it appears Downloaded by guest on September 29, 2021 5150 Medical Sciences: Liddle et al. Proc. Natl. Acad. Sci. USA 89 (1992)

Mcoi:10' Di!D:Iutyry peptide cAMP 3C) n M; 5 FiM

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4 -

3 -

In 2 4 80 ' 1'

Time (minutes)

FIG. 4. Perifusion of enriched CCK cells after cell sorting. Cells sorted by monitor peptide stimulation were layered over Sephadex G-50 resin overnight and then transferred into the perifusion column. Cells were then perifused continuously with Hepes buffer to which 30 nM monitorpeptide, 5 AM dibutyryl cAMP, 52 mM KCI, and 1 A.M A23187 were tested in sequential order. CCK released into the column buffer is expressed as fmol per fraction. These results are representative of three experiments. is the first luminal factor selective for stimulating CCK release to be purified and sequenced, to our knowledge, other CCK releasing factors appear to exist (13, 14, 31). Further- more, the relative physiologic importance of each type of releasing factor has yet to be determined. Characterization of specific releasing factors could be facilitated by the use of a pure population of responsive CCK cells. Other approaches to studying CCK secretion in vitro have included enriching CCK cells by counterflow elutriation (20) and perfusion of isolated intestinal segments (18, 19). Barber et al. (20) performed the first studies on isolated canine CCK cells, which were enriched =20-fold. Elevated extracellular potassium, dibutyryl cAMP, forskolin, and tryptophan were all shown to stimulate CCK release. The present data using a preparation of CCK cells enriched >100-fold support the findings that membrane depolarization with high concentrations of KCl stimulate CCK release. Furthermore, cAMP also appears to function as a second messenger pathway mediating CCK secretion since dibutyryl cAMP stimulated secretion from highly enriched CCK cells. Our studies with calcium ionophore have extended earlier observations by invoking intracellular calcium as an important intracellular regulator of CCK secretion. These findings raise the possi- FIG. 3. Intestinal mucosal cells before and after cell sorting. bility that an increase in [Ca2+]i occurs by activation of the Isolated intestinal mucosal cells were stained with AB 1972. Light inositol phospholipid cascade through a membrane-receptor- photomicrograph of cells prior to sorting is shown in A. The initiated event (32, 33). Additional studies on purified CCK corresponding immunofluorescence photomicrograph of the same cells should clarify the precise steps involved in CCK release. field is shown in B. Immunofluorescence photomicrograph of cells Cuber et al. (18) have recently utilized an isolated vascu- sorted by monitor peptide stimulation is shown in C. (x280.) larly perfused duodenojejunum from rat to study CCK secretion. Intraluminal administration of monitor peptide was that monitor peptide signals an increase in [Ca2+]i only in found to stimulate CCK release (19). A theoretical advantage CCK cells. This selectivity of monitor peptide for CCK cells of the intestinal segment model is that cells maintain their suggests that the major physiologic action of monitor peptide orientation to the luminal surface and potential secretagogues in the intestinal lumen is to stimulate CCK secretion. can be administered either intravascularly or on the luminal The ability to highly enrich CCK cells from other cells of surface. These studies indicated that monitor peptide was the enteric mucosa was dependent upon identifying a secre- acting on the luminal surface of the CCK cell to stimulate tagogue specific for the CCK cell. Although monitor peptide release. Downloaded by guest on September 29, 2021 Medical Sciences: Liddle et al. Proc. Natl. Acad. Sci. USA 89 (1992) 5151

There are several advantages to studying a highly enriched 9. Funakoshi, A., Nakano, I. & Miyazaki, K. (1991) Tohoku J. population ofhormone-secreting cells instead of a mixture of Exp. Med. 157, 55-59. types. The major advantage is that direct effects 10. Green, G. M. & Lyman, R. L. (1972) Proc. Soc. Exp. Biol. different cell Med. 140, 6-12. of secretagogues can be determined in a homogeneous pop- 11. Liddle, R. A., Goldfine, I. D. & Williams, J. A. (1984) Gas- ulation of CCK cells. In a mixed population of intestinal troenterology 87, 542-549. mucosal cells, it is extremely difficult to evaluate whether 12. Owyang, C., Louie, D. S. & Tatum, D. (1986) J. Clin. Invest. agents are acting directly on the CCK cell or indirectly on 77, 2042-2047. another cell type that in turn affects CCK secretion. In 13. Miyasaka, K., Guan, D., Liddle, R. A. & Green, G. M. (1989) addition, an enriched population of CCK cells also provides Am. J. Physiol. 257, G175-G181. 14. Lu, L., Louie, D. & Owyang, C. (1989) Am. J. Physiol. 256, a means for studying intracellular second messenger systems. G430-G435. By being able to measure enzyme and protein products that 15. Iwai, K., Fukuoka, S. I., Fushiki, T., Tsujikawa, M., Hirose, are activated or generated during secretagogue stimulation, M., Tsunasawa, S. & Sakiyama, F. (1987) J. Biol. Chem. 262, this preparation of cells should be useful for studying stim- 8956-8959. ulus-secretion coupling. 16. Fukuoka, S. I., Fushiki, T., Tsujikawa, M. & Iwai, K. (1987) The ability of monitor peptide to stimulate CCK release Agric. Biol. Chem. 51, 1091-1097. from isolated CCK cells indicates that it is acting directly on 17. Iwai, K., Fukuoka, S.-I., Fushiki, T., Kodaira, T. & Ikei, N. the CCK cell. Because monitor peptide increases it (1986) Biochem. Biophys. Res. Commun. 136, 701-706. [Ca2+]i, 18. Cuber, J. C., Vilas, F., Charles, N., Bernard, C. & Chayvialle, is likely that monitor peptide binds to membrane receptors. J. A. (1989) Am. J. Physiol. 256, G989-G996. In animals, monitor peptide is found in pancreatic juice and 19. Cuber, J. C., Bernard, G., Fushiki, T., Bernard, C., Yaman- is secreted into the lumen of the small intestine. Therefore, ishi, R., Sugimoto, E. & Chayvialle, J. A. (1990) Am. J. for monitor peptide to regulate CCK release in vivo, it is likely Physiol. 259, G191-G197. to interact with specific receptors on the luminal surface of 20. Barber, D. L., Walsh, J. H. & Soll, A. H. (1986) Gastroenter- the CCK cell. Further studies using autoradiographic tech- ology 91, 627-636. niques will be required to definitively address this issue. 21. Tsien, R. Y. (1989) Annu. Rev. Neurosci. 12, 227-253. 22. Tsien, R. Y. (1988) Trends Neurosci. 11, 419-424. 23. Williams, J. A., Korc, M. & Dormer, R. L. (1978) Am. J. This work was supported by National Institutes of Health Grant Physiol. 235, E517-E524. DK 38626, the Department of Veterans Affairs, and the Sarah W. 24. Evans, W. S., Cronin, M. J. & Thorner, M. J. (1983) Methods Stedman Center for Nutritional Studies. Enzymol. 103, 294-305. 25. Richelsen, B., Rehfeld, J. F. & Larsson, L.-I. (1983) Am. J. 1. Buchan, A., Polak, J., Solcia, E., Capella, C., Hudson, D. & Physiol. 245, G463-G469. Pearse, A. (1978) Gut 19, 403-407. 26. Tsien, R. Y. & Poenie, M. (1986) Trends Biochem. Sci. 11, 2. Mutt, V. (1988) in Gastrointestinal Hormones, ed. Mutt, V. 450-455. (Academic, San Diego), pp. 251-320. 27. Parks, D. R. & Herzenberg, L. A. (1984) Methods Enzymol. 3. Hopman, W. P. M., Jansen, J. B. M. J. & Lamers, 108, 197-241. C. B. H. W. (1984) Digestion 29, 19-25. 28. Watkins, S. (1987) in Current Protocols in Molecular Biology, 4. Levan, V. H. & Green, G. M. (1986) Am. J. Physiol. 251, eds. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, G64-G69. D. D., Seidman, J. G., Smith, J. A. & Struhl, K. (Greene and 5. De Jong, A. J. L., Klamer, M., Jansen, J. B. M. J. & Lamers, Wiley, New York), pp. 14.0.1-14.6.13. C. B. H. W. (1987) Regul. Pept. 17, 285-293. 29. Tateishi, K., Hamaoka, T., Sugiura, N., Yanaihara, C. & 6. Nakano, I., Tawil, T., Spannagel, A. W., Liddle, R. A. & Yanaihara, N. (1981) J. Immunol. Methods 47, 249-258. Green, G. M. (1990) Pancreas 5, 621-625. 30. Liddle, R. A., Goldfine, I. D., Rosen, M. S., Taplitz, R. A. & 7. Nakano, I., Miyazaki, K., Funakoshi, A., Tateishi, K., Ha- Williams, J. A. (1985) J. Clin. Invest. 75, 1144-1152. maoka, T. & Yajima, H. (1988) Regul. Pept. 23, 153-159. 31. Fushiki, T. & Iwai, K. (1989) FASEB J. 3, 121-126. 8. Kanayama, S. & Liddle, R. A. (1990) Am. J. Physiol. 258, 32. Hokin, L. E. (1985) Annu. Rev. Biochem. 54, 205-235. G358-G364. 33. Rasmussen, H. & Barrett, P. Q. (1984) Physiol. Rev. 64, 938. Downloaded by guest on September 29, 2021