Identification of nonsulfated cholecystokinin-58 in canine intestinal extracts and its biological properties
Joseph R. Reeve, Jr.,1
Rodger A. Liddle,2
Douglas C. McVey,2
Steven R. Vigna,2
Travis E. Solomon,1
David A. Keire,1
Grace Rosenquist,3
John E. Shively,4
Terry D. Lee,4
Peter Chew,1
Gary M. Green,5 and
Tamer Coskun1
1CURE, Digestive Diseases Research Center, Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles 90073; and Digestive Diseases Division, University of California Los Angeles School of Medicine, Los Angeles 90024; 3Section of Neurobiology, Physiology and Behavior, University of California, Davis 95616; 4Division of Immunology, Beckman Research Institute of the City of Hope, Duarte, California 91010; 2Division of Gastroenterology, Duke University Medical Center and Durham Veterans Affairs Medical Centers, Durham, North Carolina 22210; and 5University of Texas Health Science Center at San Antonio, Department of Physiology, San Antonio, Texas 78229
Submitted 15 December 2003
; accepted in final form 8 February 2004
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ABSTRACT
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Nonsulfated CCK58 [CCK58(ns)] has not been considered to be of biological importance because CCK58(ns) binds poorly to the CCKA receptor and has only been identified once in intestinal extracts. In this work, a radioimmunoassay specific for the COOH-terminal region of gastrin and CCK (antibody 5135) was used to monitor the purification of CCK molecular forms from canine intestinal extracts. A minor immunoreactive peak was associated with a major absorbance peak during an ion-exchange, HPLC step. Characterization of this minor immunoreactive peak demonstrated that it was CCK58(ns). CCK58(ns) is 14% as immunoreactive as sulfated CCK8 [CCK8(s)]. Amino acid analysis demonstrated that CCK58(ns) was present at 50% the amount of CCK58(s). In addition, we found that CCK58(ns) does not potently displace an 125I-labeled CCK10 analog from the CCKA receptor in mouse pancreatic membranes and does not stimulate amylase release from isolated pancreatic acini, or stimulate pancreatic secretion in an anesthetized rat model. By contrast, CCK58(ns) does bind to CCKB receptors and stimulates gastric acid secretion via this receptor. The presence of CCK58(ns) and its ability to selectively stimulate the CCKB receptor without stimulation of the CCKA receptor suggest that CCK58(ns) may have unique physiological properties, especially tissues where the nonsulfated peptide can act as a paracrine or neurocrine agent.
gallbladder; pancreas; gastric acid; receptor binding; CCKA; CCKB
NO BIOLOGICAL RELEVANCE HAS been attributed to nonsulfated molecular forms of CCK because 1) nonsulfated forms have not been routinely detected in brain or intestinal extracts and 2) nonsulfated CCK forms cannot bind and activate the CCKA receptor. However, if a nonsulfated molecular form of CCK is present in tissue, this form could act at CCKB receptors through endocrine, neurocrine, or paracrine pathways.
Recently, a report (2), contrary to the conventional idea that all molecular forms of CCK are fully sulfated (22), described the purification and characterization of nonsulfated CCK58 [CCK58(ns)] from porcine intestinal extracts. Sulfated CCK58 [CCK58(s)] was 35 times more potent than CCK58(ns) for contraction of the guinea-pig gallbladder, whereas sulfated CCK8 [CCK8(s)] was 150 times more potent than nonsulfated CCK8 [CCK8(ns)] for contraction of gallbladder (28). This led the authors to conclude that "the NH2-terminal end of CCK58(ns) partially compensates for the decrease in activity arising from the lack of sulphated tyrosine" (2).
CCK binds to two receptors. The CCKA (CCK-1) receptor regulates pancreatic secretion (44), gallbladder contraction (8), gastric motility (25), and is one component of satiety (19). The CCKB (CCK2 or gastrin/CCKB) receptor regulates gastric acid secretion (42). We have studied the binding of CCK58(s) to these receptors (34), but we are unaware of any other binding studies with CCK58(ns).
Sulfation of prohormones (e.g., pro-CCK) could influence the expression of their physiological activities at several levels. The sulfate moiety could influence processing of the prohormone as shown for progastrin, where the extent of proteolytic processing has been correlated with sulfation (3, 35, 41). In addition, sulfation could also alter the bioavailability of a hormone by changing its circulating half-life (29) or the susceptibility of the hormone to degradation after leaving the circulation (5, 6). Furthermore, sulfation has been shown to greatly increase the potency of CCK8 for binding and activation of the CCKA receptor (21).
If the amino terminus of CCK58(ns) is able to partially compensate for the lack of the tyrosine sulfate on its COOH terminus [as suggested by its actions on the gallbladder (2)], then binding and activation of the rat pancreatic CCKA receptor may be influenced by the NH2 terminus of CCK58(ns). We have previously shown that the NH2 terminus of CCK58(s) decreases the ability of this molecular form to stimulate release of amylase compared with CCK8(s) in a purified acinar cell preparation (32). However, CCK8(s) and CCK58(s) were equipotent for stimulation of amylase release in an in vivo anesthetized rat model (30), suggesting that the greater bioavailability (20) of CCK58(s) offsets its lower intrinsic potency to release amylase from purified acinar cells.
In this report, we demonstrated that CCK58(ns) is present in the intestine of dog and that CCK58(ns) binds and activates the CCKA receptor on rat pancreas in vivo or in vitro only at high doses. Therefore, if CCK58(ns) is of physiological relevance at CCKA receptors, it must act in a neurocrine or paracrine manner to achieve the concentrations necessary for activation of the CCKA receptor. However, we also demonstrate that CCK58(ns) potently binds to mouse brain CCKB receptors. Furthermore, CCK58(ns) stimulates gastric acid in an anesthetized rat model, demonstrating its ability to stimulate CCKB receptor in vivo. The presence of CCK58(ns) in intestinal extracts and its ability to bind and activate CCKB receptors suggest that CCK58(ns) may be of physiological relevance.
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METHODS
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Radioimmunoassay.
A radioimmunoassay based on antibody 5135 was performed as previously described (36). Tracer was radiolabeled gastrin 17 (I) purified by reverse-phase HPLC. This antiserum reacts equally with CCK8 and CCK33 (13), but about 50 and 20% with canine CCK58 (31) and rat CCK58 (33), respectively.
Purification of nonsulfated canine [CCK58(ns)].
Canine intestines from dogs used for other purposes were kindly provided by Dr. A. Soll. Briefly, canine intestinal mucosa was obtained after boiling unopened, freshly dissected intestine in water for 510 min. The cooled intestine was cut open, and the mucosa was scraped away from the muscle. The mucosa was then stored at 70°C until extraction. The frozen mucosa (140 g) was blended with 1.4 liters of 2% trifluoroacetate in a Waring blender and then stirred for 30 min. The extract was centrifuged at 3,000 g for 30 min and filtered through an Amicon HL10,000 filter (10,000 MW cut off). The filtrate was loaded directly onto a Rainin C18 preparative HPLC column (42 mm x 25 cm). After rinsing with 1 liter of 0.1% trifluoroacetate CCK, immunoreactivity was eluted with increasing concentrations of acetonitrile (see Table 1 for gradients used in all HPLC purification steps). All purification eluates were monitored by absorbance at 220 and 280 nm and CCK radioimmunoassay. Fractions containing CCK immunoreactivity were pooled and diluted threefold with 0.1% trifluoroacetate (TFA) and loaded onto a Rainin C8 preparative HPLC column (21.4 mm x 25 cm). The pooled immunoreactive fractions were diluted threefold with 0.1% TFA and loaded onto a Rainin C4 preparative HPLC column (21.4 mm x 25 cm). The immunoreactive fractions were loaded directly onto an Aspartamide anion-exchange HPLC column (Nest Group) equilibrated in potassium phosphate (0.01 M, pH 3, containing 25% acetonitrile). The column was eluted with increasing concentrations of KCl. The pooled CCK immunoreactivity from this column was diluted threefold with 0.1% TFA and loaded onto an analytical C4 reverse-phase HPLC column (Vydac) equilibrated in aqueous 0.1% TFA. The column was eluted with increasing concentrations of acetonitrile. Finally, the peptide was stored at 4°C for 10 wk before dilution and repurification on the same C4 column with a slightly different gradient 1 wk before commencing the bioassays.
Amino acid analysis of sulfated and canine CCK58(ns).
A small aliquot of the purified peptides corresponding to
5 nmol was placed into a pyrolozed hydrolysis tube. The solvent was removed under vacuum, 6 N HCl was added to the container holding the tubes, the container was sealed under a vacuum, and the mixture was hydrolyzed for 20 h at 110°C. The resulting amino acids were dried, dissolved, and analyzed on a Beckman 126 amino acid analyzer by manufacturer recommended procedures.
Mass spectral analysis.
Mass spectrometric analyses were performed at the Beckman Research Institute of the City of Hope (Duarte, CA) on a TSQ-700 triple-sector quadrapole mass spectrometer (Finnigan-MAT, San Jose, CA) equipped with a standard electrospray ion source. Online separations were performed using a City of Hope-built capillary LC system described previously (10). Mass assignments were made using the Finnigan MAT BIOMASS data-reduction software.
Tryptic mapping of canine sulfated and CCK58(ns).
Approximately 200 pmol of the peptides were vacuum centrifuged until the volume was <50% of the original sample volume to remove acetonitrile. The volume was adjusted to 50 µl with 1 M ammonium bicarbonate. Trypsin (5 pmol in 50 µl of 1 M ammonium bicarbonate) was added, and the mixture was incubated at 37°C for 18 h. A portion (5 µl) of the digestion mixture was loaded onto the liquid chromatography-mass spectrometer.
Membrane preparation for radioreceptor binding assays.
Mouse brain membranes were used as a source of CCKB receptors and mouse pancreatic membranes as a source of CCKA receptors as previously described using the CCK10 analog I125-labeled dTyr-Gly-Asp-Tyr(SO3H)-Nle-Gly-Trp-Nle-Asp-Phe-amide (Research Plus, Bayonne, NJ) as a tracer. Freshly prepared membranes (75 µg protein) were incubated in plastic tubes in 1 ml of incubation buffer with 100 pM 125I-labeled CCK10 analog for 1 h at 22°C. Nonsaturable binding was determined in the presence of 1 µM CCK8. After incubation, triplicate 300-µl aliquots were removed from each tube and centrifuged at 10,000 g in a microcentrifuge for 30 s. The supernatants were aspirated to waste, and the pellets were washed with 300 µl of ice-cold incubation buffer. After centrifugation and aspiration of the supernate, the radioactivity in the tips was determined in a gamma counter. Saturation binding curves were analyzed at equilibrium to determine concentrations of CCK peptides that inhibited 50% of saturable 125I-labeled CCK10 analog binding (IC50). Saturation binding values were analyzed by the program Equilibrium Binding Data Analysis (26) to obtain estimates of IC50, and the differences among IC50 for the CCK peptides were tested for significance by paired t-test.
In vitro stimulation of amylase from pancreatic acini.
The stimulation of amylase from isolated pancreatic acini by CCK58(ns) was compared with CCK8(s) and -(ns) as previously described (32). Briefly, amylase release from isolated rat pancreatic acini was assessed in response to natural canine CCK58(ns), synthetic CCK8(ns), and synthetic CCK8(s). Pancreatic acini were prepared as previously described (23, 44) from Sprague-Dawley rats weighing 250280 g, according to the method of Williams et al. (44). Krebs-Henseleit bicarbonate buffer containing 0.1 mg/ml soybean trypsin inhibitor and 0.1 mg/ml purified collagenase was injected into the pancreatic parenchyma, and the tissue was incubated at 37°C for 50 min. The acini were dissociated by passage through pipettes with restrictive orifices and purified by centrifugation through step gradients of buffer containing 4% BSA. Acini were then incubated for 30 min at 37°C in 40 mM Tris-Ringer buffer with (in mM) 103 NaCl, 1 NaH2PO4, 4.7 KCl, 1.28 CaCl2, 0.56 MgCl2, and 11.1 glucose with 0.1 mg/ml soybean trypsin inhibitor enriched with Eagle's medium amino acid supplement, and 5 mg/ml BSA with various concentrations of the CCK peptides.
In vivo studies: animals.
Male Sprague-Dawley albino rats weighing 280350 g (Harlan Laboratory, San Diego, CA) were housed in group cages under conditions of controlled temperature (2224°C) and illumination (12:12-h light-dark cycle starting at 06:00 AM) for at least 7 days before experiments. Animals are maintained on Purina Laboratory Chow (Ralson Purina, St. Louis, MO) ad libitum and tapwater. Experiments were performed in urethane-anesthetized rats deprived of food for 18 h but given free access to water up to the beginning of the study. Experimental protocols were approved by the Animal Research Committee of the Veteran Affairs Greater Los Angeles Healthcare System.
Surgery and comparison of in vivo stimulation of pancreatic amylase secretion.
Exocrine pancreatic secretory responses to canine CCK58(ns) and CCK8(s) and CCK8(ns) were determined by previously described methods (37). Briefly, the right jugular vein of anesthetized rats (280400 g body wt, fasted for 2024 h) was catheterized; intravenous fluids were delivered at an infusion rate of 1.08 ml/h. The trachea was intubated to facilitate breathing and to prevent aspiration. Through a midline celiotomy, the common bile-pancreatic duct was catheterized in two different locations: 1) at the level of sphincter of Oddi (close to duodenum) and 2) distal to the pancreas (close to the liver) to collect bile and pancreatic juice separately. Collections were begun after a 2 h postsurgical period of stabilization. Collections were made at 30-min intervals. Previously collected bile-pancreatic juice diluted 1:1 with saline was infused into the duodenum at 1.08 ml/h to prevent the release of endogenous CCK.
After unstimulated pancreatic secretion was collected for 1 h, either CCK8(s) or CCK8(ns) or CCK58(ns) was administered intravenously for 2 h at a dose of 1 nmol·kg1·h1. Only one dose was given to each rat. The volume of collected pancreatic juice was measured, and amylase concentration in each sample was determined.
Surgery and gastric acid secretion measurement.
Rats were anesthetized with urethane (1.25 g/kg ip). The trachea was cannulated (PE-240, IntraMedic) to facilitate the breathing, and the cervical esophagus was ligated. For CCK8(ns) or CCK58(ns) or gastrin-17(ns) infusion, an intravenous line (PE-50, IntraMedic) was placed into the right jugular vein. After the completion of the neck surgery, the surgical wounds were sutured. The abdomen was opened through a ventral median celiotomy. The pylorus was ligated, and a double-lumen cannula was inserted through a small incision into the nonglandular forestomach. The plastic gastric cannula was secured in the stomach and exited the abdomen through the midline incision. The surgical wound was covered by cotton soaked in saline. The body temperature of the animal was kept at 37°C by a heating pad. The experiments were started 23 h after completion of the surgery. During this stabilization period, the stomach lumen was flushed with pH 7.0 saline at room temperature.
Gastric effluent was collected every 10 min by flushing the gastric lumen through the gastric cannula twice with 3-ml boluses of saline under gravity drainage and followed once with 3-ml boluses of air at 10-min intervals. Gastric samples were back-titrated to pH 7.0 with 0.01 N NaOH using an automatic titrator (Radiometer, Copenhagen, Denmark). After a 30-min basal period, CCK8(ns) or CCK58(ns) at 0.1, 0.3 1, 3, 10, or 30 nmol·kg1·h1, or gastrin-17 at 10 nmol·kg1·h1 was infused for 2 h. Intravenous infusions were performed in a volume of 1 ml/h.
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RESULTS
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Purification of canine CCK58(s) and -(ns).
Three peaks of immunoreactivity (a total of 33 nmol) eluted during the first reverse-phase HPLC step (results not shown). The immunoreactivity in the first peak tube eluted in a similar position to sulfated CCK8 during analytical reverse-phase chromatography (results not shown). No nonsulfated CCK8 was observed in this series of experiments or in previous evaluations of CCK forms from canine intestinal extracts (15, 31). The immunoreactivity in the second peak was not characterized, because it did not bind to the ion-exchange column used in a subsequent step of purification. The elution position of this CCK immunoreactivity during reverse-phase chromatography suggested that it may be CCK22. The third peak contained 11 nmol of CCK immunoreactivity and was purified as shown in Table 1. There was no clear resolution of two CCK immunoreactive peaks until the ion-exchange step (Fig. 1). The ion-exchange step contained two immunoreactive CCK peaks (7.1 and 1.4 nmol of CCK-like immunoreactivity) that were associated with absorbance peaks of similar heights. Further purification of the two peaks resulted in the purification of CCK58(s) (results not shown) and a new CCK molecular form. The final step (Table 1) in the purification of the new molecular form of CCK is shown in Fig. 2.

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Fig. 1. Ion-exchange separation of canine sulfated [CCK58(s)] and nonsulfated CCK58 [CCK58(ns)]. The CCK immunoreactivity from the C4 reverse-phase chromatography (step 4, Table 1) was loaded and eluted as described in the text. The absorbance at 220 nm and CCK immunoreactivity are plotted against fraction number.
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Fig. 2. Final step in purification of CCK58(ns). Ion-exchange purified CCK from the minor immunoreactive peak (Fig. 1) was loaded onto a reverse-phase C-18 column and eluted with increasing concentrations of acetonitrile.
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Chemical characterization of the new molecular form of CCK.
The amino acid composition of the new molecular form of CCK was the same as that determined for CCK58(s) (Table 2). The new molecular form was cleaved by trypsin, dried, and characterized by liquid chromatography coupled to a mass spectrometer. Mass spectral analysis of the tryptic fragments of the two purified peptides showed that the earliest eluting, major peak from the ion-exchange column was CCK58(s) (Fig. 1), whereas that of the latter eluting peak was CCK58(ns) (Fig. 3, Table 3).

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Fig. 3. Structural analysis of CCK58(ns). The new molecular form of CCK (Figs. 1 and 2) was digested by trypsin, dried, and chromatographed by HPLC. The effluent from the HPLC was introduced into a mass spectrometer by electrospray ionization. All anticipated tryptic peptides of CCK58(s) and CCK58(ns) were observed except for T4, which was not detected from either peptide. T7 is the COOH-terminal dodecapeptide of CCK58. The masses of this peptide from CCK58(s) and CCK58(ns) are shown in Table 3. These masses identified the intact peptides as CCK58(s) and CCK58(ns)
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Table 3. Mass spectral analysis of the COOH-terminal peptide of canine CCK58(s) and -(ns) after tryptic digestion
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Immunoreactivity of CCK58(s) and -(ns).
Amino acid analysis and radioimmunoassay of the purified peaks demonstrated that the sulfated and nonsulfated peptides were 59 and 14% as immunoreactive as CCK8(s), respectively, showing that CCK58(s) is four times more cross-reactive than CCK58(ns) (Table 4). Because the immunoreactivity of antisera 5135 is based on a shared homology between CCK and gastrin, factors other than primary sequence must account for the decreased immunoreactivity of the CCK58(s) and -(ns) molecular forms. All data presented here did not adjust for the lower immunoreactivity of canine CCK58(ns) or -(s) compared with CCK8. Figure 1 shows that the absorbance at 280 nm for CCK58(ns) was about one-half the height of CCK58(s), but the peak for immunoreactivity of the nonsulfated peptide was only one-eighth that of the sulfated peptide. This cross-reactivity agrees well with the cross-reactivity calculated for CCK58(s) and -(ns) when their amounts were determined by amino acid analysis (Table 4).
Binding to CCKA and CCKB receptors.
Both sulfated and nonsulfated peptides were potent at CCKB receptors (Fig. 4A). CCK58(s) was about fivefold more potent than CCK8(ns) and
20-fold more potent than CCK58(ns) at mouse brain CCKB receptors (Table 5). Only sulfated CCK58 exhibited high affinity binding at CCKA receptors in mouse pancreatic membranes, but some displacement was observed at the highest concentrations of CCK8(ns) (Fig. 4B). CCK58(s) was
300-fold more potent than CCK8(ns) for displacement of the label at CCKA receptor. We anticipate that if higher concentrations of CCK58(ns) had been available for testing, then some label would have been displaced as observed for CCK8(ns).

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Fig. 4. Relative affinities of CCK58(s), CCK8(ns), and CCK58(ns) at mouse CCKA and CCKB receptors. A: competitive inhibition of saturable CCK label binding to mouse brain membranes (CCKB receptors). B: competitive inhibition of saturable CCK label binding to mouse pancreas membranes (CCKA receptors).
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Table 5. Relative affinities expressed as Ki (nM) of CCK8(s), CCK8(ns), CCK58(s), and CCK58(ns) at CCKA and CCKB receptors in mouse pancreas and brain membranes
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In vitro stimulation of pancreatic secretion.
CCK58(ns) was a poor stimulant of amylase release from isolated pancreatic acini. The potency of CCK58(ns) was <1% of CCK8(s) and
1% that of CCK58(s) (Fig. 5). There was not enough of the natural peptide for evaluation of amylase secretion at higher doses.

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Fig. 5. Stimulation of amylase release from isolated rat pancreatic acini by CCK8(ns), CCK8(s), CCK58(s), and CCK58(ns).
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In vivo stimulation of pancreatic secretion.
The basal in vivo amylase output was 399.7 ± 64.6 U/30 min. CCK8(s) was a potent stimulant of amylase (
increase = 6,559.7 ± 680.0 U/30 min; P < 0.05 vs. basal) at 1 nmol·kg1·h1 iv. However, the same intravenous dose of nonsulfated CCK8 did not cause any stimulation in amylase output (32.2 ± 114.1 U/30 min). CCK58(ns) (1 nmol·kg1·h1 iv) caused a slight but significant increase in amylase output (
increase = 480.6 ± 148.4 U/30 min; P < 0.05 vs. basal; Fig. 6). The ratio of activity between canine CCK58(ns) and -(s) (0.02) for pancreatic output was similar to the ratio of activity between porcine CCK58(ns) and -(s) (0.03) for gallbladder contraction (2).
In vivo stimulation of gastric acid secretion.
Basal gastric acid output was 1.88 ± 0.16 µmol/10 min in urethane-anesthetized rats. Both CCK8(ns) and CCK58(ns) were able to stimulate gastric acid (Fig. 7) starting from a dose of 3 nmol·kg1·h1 iv. Gastric acid stimulation with either CCK8(ns) or CCK58(ns) at a dose of 10 nmol·kg1·h1 iv reached plateau level in the first hour (13.0 ± 1.5 µmol/10 min or 16.6 ± 2.0 µmol/10 min; P < 0.05 vs. basal), and the level of stimulation remained the same for both peptides as in the first hour. CCK8(ns) and CCK58(ns) were equipotent (change from the basal values 17.6 ± 5.6 µmol/10 min and 14.0 ± 1.6 µmol/10 min, respectively) but not as potent as gastrin (change from the basal values 39.7 ± 4.0 µmol/10 min). The level of gastric acid stimulation by CCK8(ns) or CCK58(ns) at 10 nmol·kg1·h1 iv was 47.8 ± 15.1 or 38.1 ± 4.4%, respectively, of the gastric acid output caused by gastrin-17(ns) stimulation.
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DISCUSSION
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This work reports the presence of major amounts of CCK58(ns) in canine small intestinal extracts, that CCK58(ns) weakly displaced CCK label from mouse pancreatic CCKA receptors, and that CCK58(ns) is a very weak stimulant of amylase from isolated pancreatic acini or from the in vivo pancreas. In contrast, CCK58(ns) is active at CCKB receptors as shown by mouse brain CCKB receptor binding and its stimulation of gastric acid secretion in vivo. The presence of CCK58(ns) in canine intestinal extracts and its ability to stimulate gastric acid suggests that it may be of physiological relevance, especially for neurocrine and paracrine actions mediated by CCKB receptors.
Importantly, CCK58(ns) has now been characterized from two species using two different methods of extraction and purification. In pig, the CCK58(s) to CCK58(ns) ratio is 1:1 (2), and for dogs, this ratio is
2:1. The absence of detectable CCK58(ns) in other studies may be due to either the poor sensitivity of the assay used to detect CCK forms or the difficulty of separating CCK58(s) and -(ns). The presence of CCK58(ns) in pig and dog suggests that this peptide may be characterized from other species if appropriate methods are used for its detection, which include chromatographic separation from sulfated CCK58 and appropriate measurement with a CCK58(ns) standard. CCK58(s) has been detected in extracts of dog (17), pig (39), and cow (12) brain, but the ratio of CCK58(s) to -(ns) in brain is not known. Likewise, CCK58(s) is a major endocrine form of CCK in the blood of human (11), dog (14), and rat (33), but it is not known whether CCK58(ns) is present in the blood of any of these species.
The extent of gastrin sulfation in mammals varies from 2480% (1). For example, in dogs and pigs, the percentage of gastrin that is sulfated is 24 and 57%, respectively (1). Of note, this work shows that 67% of canine CCK is sulfated. Sulfation of propeptides occurs in the trans-golgi network (40), and the consensus sequence for sulfation is an acidic or neutral group one residue before or a basic group two residues after the tyrosine that is sulfated (4). The mechanism that reproducibly results in different extents of gastrin and CCK sulfation in mammals (1) is not known. It is unlikely that the presence of both sulfated and nonsulfated forms is an artifact of sample extraction and purification, because synthetic CCK58(s) can be cleaved from its synthesis resin under much harsher acidic conditions (91% trifluoroacetate for 2 h) than extraction conditions (1% trifluoroacetate for 2 h), with only partial desulfation of the tyrosine. In addition, to our knowledge, no studies have shown enzymatic desulfation of CCK. In fact, one study (38) has shown that mammalian aryl sulfatases cannot desulfate sulfated tyrosyl residues. Further evidence for presence of the nonsulfated peptide is observed for porcine CCK58(ns), which was purified using very different extraction and purification techniques (2).
Natural canine CCK58(ns) does not potently displace CCK label from mouse CCKA receptors at the concentrations available (Fig. 4). Similarly, CCK58(ns) does cause stimulation of amylase secretion (CCKA receptor mediated) in vitro or in vivo, although at greatly diminished potency (Figs. 5 and 6). Similarly, other studies (2) have shown that porcine CCK58(ns) contracted guinea pig gallbladder (CCKA receptor mediated). The concentrations of CCK58(ns) required for minimal actions at the CCKA receptor suggest that the peptide does not express its endocrine activity through the CCKA receptor. We cannot evaluate whether CCK58(ns) acts in an endocrine fashion to stimulate the CCKB receptor because the circulating levels of the peptide are not known. However, the presence of CCKB receptors in vagal afferent neurons suggests that CCK58(ns) may act in a paracrine or neurocrine manner, in which much higher concentrations of the peptide are possible (27).
CCK58(ns) and CCK8(ns) have similar potencies for binding to CCKB receptors and stimulation of gastric acid secretion. In this work CCK8(ns) and CCK58(ns) were about one-tenth as potent as gastrin for stimulation of gastric acid. The greater potency of gastrin-17(ns) is most likely due to its higher potency at the CCKB (21) receptor relative to the nonsulfated CCK peptides and the longer circulating half-life of gastrin (16). The similarity of CCK58(ns) and CCK8(ns) for in vivo gastric acid stimulation is unexpected because this action is an integration of several components including circulating half-life, resistance to degradation after leaving the circulation, receptor binding, and receptor activation. Therefore, CCK58(ns) could be a valuable reagent for the evaluation of how each of these components contributes to the integrated response for stimulation of CCKB receptors.
The presence of CCK58(ns) solves an apparent inconsistency between processing of progastrin and pro-CCK. The processing of progastrin results in both sulfated and nonsulfated peptides (1). Processing of pro-CCK was formerly thought to result in only sulfated peptides (22). This work and the results from Bonetto et al. (2) demonstrate that CCK, similar to gastrin, exists as sulfated as well as nonsulfated peptides in pigs and dogs (present work). The degree of sulfation of CCK in other tissues, such as brain and blood, in other species and in varied physiological states will aid our understanding of the physiological relevance of sulfation for pro-CCK.
The existence of CCK58(ns) in tissue and its presumed ability to reach neurocrine, paracrine, and endocrine targets suggest that regulation of processing can cause pro-CCK gene products to act exclusively at CCKB receptors (nonsulfated peptide) or at both CCKA and CCKB receptors (sulfated peptide). Maeda et al. (24) demonstrated that CCK8(ns) is a potent stimulant of gastric acid, whereas CCK8(s) did not stimulate gastric acid. The difference between CCK8(s) and -(ns) effects is that the nonsulfated peptide can only act at CCKB receptors on gastric enterchromaffin-like and parietal cells to stimulate gastric acid, whereas the CCK8(s) reacts at these receptors and also at the CCKA receptor on gastric D cells to release somatostatin. Somatostatin inhibits the release of acid through actions on the parietal cell (18). Thus, for CCK8(ns), the stimulatory effects at CCKB receptors and inhibitory actions at CCKA receptors result in no net stimulation of gastric acid (24). Of note, a similar pattern of concurrent stimulation and inhibition of gastric acid secretion probably explains the fact that CCK-33(s) stimulates much less gastric acid than gastrin (9).
In this work, 10 nmol·kg1·h1 gastrin stimulated about threefold more gastric acid secretion than CCK58(ns) infused at the same rate (Fig. 7B). This increased response could result from gastrin's higher affinity binding at the CCKB receptor and/or a lower metabolic clearance rate. In this work, studies of the pharmokinetics of CCK58(ns) were not performed because of the limited amounts of natural peptide available. However, previously reported data for sulfated and nonsulfated gastrins show conflicting conclusions for the effect of sulfation on peptide half-lives. Cantor et al. (7) showed that there is little difference in the rates of metabolism for sulfated and nonsulfated forms of gastrin-17. By contrast, Pauwels et al. (29) have reported a two- to fivefold longer half-life for sulfated gastrin-17 compared with the nonsulfated form. Therefore, because of the limited amounts of natural peptide available and the difficulty of performing half-life studies in the rat (due to limited blood volumes), we have not evaluated whether the decreased potency of CCK58(ns) resulted from lower affinity binding or lower plasma concentrations.
Tertiary structural differences between CCK58(s) and CCK58(ns) may influence how they expresses their biological activity by altering their metabolism in the circulation and interstitial fluid. We have previously demonstrated (34) that CCK58(s) is much more stable than CCK8(s) to digestion by neural endopeptidase. Similar differences in stability may exist for CCK58(ns) and CCK8(ns). This could account for the equal potency of the nonsulfated peptides for gastric acid secretion, even though CCK8(ns) binds more potently to the CCKB receptor than CCK58(ns) (Table 5).
Regulation of sulfation alters the pattern of physiological activity of CCK by changing an exclusive CCKB agonist into an agonist for both CCKA and CCKB receptors. Therefore, the physiological relevance of CCK58(ns) can be evaluated by determining 1) its existence in blood, brain, and peripheral nerves, 2) whether there are physiological states that alter the ratio of sulfated and nonsulfated forms of CCK in these tissues, and 3) whether the concentration of CCK58(ns) is sufficient to activate CCKB receptors in an endocrine, paracrine, or neurokine manner.
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GRANTS
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This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants RO1-DK-33580 (to J. R. Reeve, Jr.), RO1-DK-38626 (to R. A. Liddle), and RO1-DK-37482 (to G. M. Green), City of Hope National Institutes of Health (NIH) Cancer Center Core Grant CA-3572 (to J. E. Shively), NIH Grant RO1-RR-06217 (to T. D. Lee), CURE Digestive Diseases Research Center Grant DK-41301, and by the Department of Veterans Affairs Research Service.
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ACKNOWLEDGMENTS
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Support from the Peptide Biochemistry and Molecular Probes Core and the Animal Core of the CURE Center Grant is gratefully acknowledged.
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FOOTNOTES
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Address for reprint requests and other correspondence: J. R. Reeve Jr., CURE: Digestive Diseases Research Center, Bldg. 115, Rm. 115, VA Greater Los Angeles Health Care, Los Angeles, CA 90073 (E-mail: jreeve{at}ucla.edu).
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. Section 1734 solely to indicate this fact.
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