Cholecystokinin-mediated ileal electrolyte transport in the guinea pig

GASTROENTEROLOGY

BRIEF

1991;101:1428-1431

REPORTS

Cholecystokinin-Mediated Ileal Electrolyte Transport in the Guinea Pig Characterization

of Receptor Subtype

JAMES F. KACHUR, SHU-XIAN and TIMOTHY

S. GAGINELLA

Gastrointestinal Diseases Research, National Institutes of Pharmaceutical China

Searle Research and Development, Skokie, Illinois; and Research and Development, Beijing, People’s Republic

Cholecystokinin (CCK) receptors are currently divided into at least two subtypes: a CCK-A subtype, responsive to the sulfated form of cholecystokinin octapeptide (CCK-8) and selectively antagonized by L-384,718, and a CCK-B subtype, which shares equal affinities for gastrin and CCK-8. In the present study the receptor subtype that mediates guinea pig ileal secretion by evaluating the potencies of CCK- and gastrin-related peptides to evoke increases in transmucosal short-circuit current was characterized. The antagonist potencies of L-365,260(CCK-B selective) and L-364,718(CCK-A selective) against CCK-8 were also determined. Both CCK-8 and cerulein, when added to the serosal side of the tissue, evoked increases in the short-circuit current, having EC,, values of 0.8 and 0.2 nmol/L, respectively. Desulfated (SO,) CCK-8, CCK-4, gastrin,,-I, pentagastrin, gastrin,,-11, and gastrin,,-I were relatively weak agonists (EC,, > 1000 nmol/L. Cholecystokinin octapeptide-induced short-current responses were competitively antagonized by L-364,718 (PA,, 10.3) and L-365,260 (PA,, 7.4). The high selectivity of the tissue for sulfated CCK-8 suggests that the secretory effect of CCK-8 on guinea pig ileal electrolyte transport is mediated by a CCK-A receptor. The potent effect of L-364,718 against CCK-8 is also consistent with an action at the A-subtype receptor.

erecently

found that segments of ileal mucosa mounted in Ussing chambers respond to cholecystokinin octapeptide (CCK-8), when studied under short-circuited conditions (1). Adding CCK-8 to the serosal side of the tissue leads to a transient stimulation of electrogenic anion secretion. These changes in electrolyte transport seem to be mediated, in part, by the effect of CCK-8 at neuronal cholecystokinin (CCK)

W

WANG, GARY W. GULLIKSON,

of

receptors linked to the release of acetylcholine. Because cholecystokinin-like immunoreactivity has been shown for enteric nerves that innervate the mucosa of the guinea pig (2), CCK-related peptides may have an important function in the control of mucosal electrolyte transport. More than two subtypes of CCK receptors are postulated to exist (3). In the gut CCK receptors are classified as CCK-A (alimentary) and in the brain as CCK-B (4). Pancreatic acinar cells and cerebral cortex are known to express the CCK-A and CCK-B subtypes, respectively. Cholecystokinin A receptors show considerable selectivity for sulfated rather than desulfated CCK-8 and bind L-364,718 with nanomolor affinity. Cholecystokinin B receptors bind L-364,718 less readily and show little selectivity between sulfated and nonsulfated CCK-8 (3,5). The CCK-B receptor also shares strong similarities with gastrin receptors (6). In the present study, we have defined the principal subtype of CCK receptor that mediates guinea pig ileal electrolyte secretion by evaluating the agonist potencies of CCK- (sulfated and nonsulfated) and gastrinrelated peptides to evoke increases in transmucosal short-circuit current (1s~); we have also defined the antagonist potencies of L-364,718 (CCK-A selective) and L-365,260 (CCK-B selective) against CCK-8 (7,8) in this system.

Abbreviations used in this paper: CCK-4, cholecystokinin tetrapeptide; CCK-8, cholecystokinin octapeptide; DMSO, dimethy1 sulfoxide; G,, tissue conductance; Isc, short-circuit current. o 1991 by the American Gastroentemlogical Association 0016~5085/91/$3.00

November 1991

CCK-A RECEPTORS

Methods Ion Transport Studies Intestinal tissues were obtained from male Hartley (CRL:COBS) guinea pigs (350-400 g; Charles River Laboratories, Wilmington, MA) maintained on a standard laboratory diet with free access to water before they were killed by CO, asphyxiation. A midline laparotomy was performed, and a -20-cm segment of ileum was removed. Four adjacent segments were then mounted muscularis intact, as a flat sheet between two Ussing-type half chambers, with an exposed area of 0.64 cm*. Tissues were bathed on both sides by a bicarbonate-buffered saline solution circulated by gas lift and maintained at 37°C by water jacketed reservoirs. The ionic composition of the Ringer’s solution (in mmol/L) was: NaCl. 120.2; KCl, 5.9; CaCl,, 2.5; MgCl,, 1.2; NaH,PO,, 1.2; and NaHCO,, 25. Electrical measurements were monitored with an automatic voltage clamp (TRlOO-F; JWT engineering, Overland Park, KS). Direct connecting voltage and current-passing electrodes (World Precision Instruments Inc., New Haven, CT) were used to measure transepithelial potential difference (PD) and Isc. Transepithelial PD was periodically measured, and tissue conductance (G,) was calculated from Ohm’s law. Short-circuit current was continuously recorded on a Gould model 2800s recorder (Gould Inc., Cleveland, OH). Tissues were equilibrated under shortcircuit conditions until Isc had stabilized (usually 30-45 minutes). Basal conductance values averaged 22-26 mS/ cm’ 30 minutes after the tissues were mounted. Concentration-response curves to agonists (alone or in presence of antagonist] were constructed by their cumulative addition to the serosal side of the tissue. Antagonists (unless specified otherwise) were added to the serosal side of the tissue 5 minutes before CCK-8. The maximal response was defined as the last incremental concentration of CCK-8 that produced no further increase in Isc.

Materials Cholecystokinin octapeptide (sulfated), gastrin,,-I (human), gastrin,,-II (sulfated), gastrin,,-I (mini gastrin-I, human), and cerulein (sulfated) were purchased from Sigma Chemical Company (St. Louis, MO). Cholecystokinin tetrapeptide (CCK-41, CCK-8 (desulfated), and pentagastrin were purchased from Peninsula Laboratories Inc. (Belmont, CA). L-364,718 and L-365,260 were gifts from Merck Sharp & Dohme Laboratories (West Point, NY). Agents not soluble in distilled water or saline were dissolved in dimethyl sulfoxide (DMSO). Equivalent amounts of DMSO (without drug) added to the serosal side of the tissue had no effect on Isc.

Results Agonist Studies fourfold

The amphibian-derived more potent than

peptide cerulein was CCK-8 at evoking an in-

MEDIATE ILEAL ELECTROLYTE

1429

TRANSPORT

crease in Isc. Removal of the CCK-8 sulfate moiety [e.g., desulfated (SO,) CCK-81 reduced potency by at least lOOO-fold (Table 1). All other peptides had weak agonist effects (EC,, > 1000 nmol/L). Cholecystokinin tetrapeptide, desulfated (SO,) CCK-8, pentagastrin, gastrin,,-I, and gastrin,,-II were not active at concentrations < 1.0 p,mol/L, but all produced a response at 1.0 p,mol/L (Table 1 and Figure 1). Their weak effects and prohibitive cost precluded testing at higher concentrations to obtain full concentrationresponse curves. Antagonist Studies After the addition of L-364,718 and L-365,260, the concentration-response curves to CCK-8 were shifted to the right in a parallel manner, without depression of the maxima, suggesting competitive antagonism. Neither L-364,718 nor L-365,260 affected basal Isc or G, at the concentrations tested. Calculated pA, values (Figure 2) showed that the CCK-Aselective anatagonist, L-364,718, was 820-fold more potent against CCK-8 than the CCK-B selective antagonist, L-365,260; pA, values were 10.3 and 7.4, respectively. Desulfated SO, CCK-8, at concentrations that showed no agonist activity, was also tested for antagonist activity against CCK-8. We found that a z-minute pretreatment with either 10 or 100 nmol/L des (SO,) CCK-8 did not shift the concentration-response curve to CCK-8 (data not shown). Discussion The major finding of the present study was that CCK-like peptides alter guinea pig ileal electrolyte transport in a manner consistent with the involvement of a CCK-A subtype receptor. This conclusion is supported by agonist studies showing that mucosal CCK receptors are preferentially sensitive to sulfated

Table 1. Agonist Potencies of Cholecystokinin Octapeptide and Related Peptides on Guinea Pig Ileal Electrolyte Transport No. of

EC,,,

Agonist

animals

aSEM (nmol/L)

CCK-8 Cerulein Desulfated (SO,) CCK-8 Gastrin,,-I Gastrin,,-II CCK-4 Pentagastrin Gastrin,,-I

11 6 6 3 4 4 3 3

0.8 t 0.2 0.2 + 0.02 > 1000 6300 2 200 >lOOO > 1000 > 1000 > 1000

“Submaximal

response at 1.0 KmoI/L.

E rndk

(&cm’1 62 64 14 41 12 13 19 10

2 8 + 10 2 5” + 11 + 9" f 8" It 13" rt 5"

GASTROENTEROLOGY

1430 KACHUR ET AL.

A

2min

II

II ,o-lo

,o-9

&

10-6

10“

[CCK-81,

M

6

CCK-8, I o-6

‘wym2

2min

I 10-19

I

I

I

10-g 10-8

[des(S03)

I

10-7 10-6

CCK-81, M

Figure 1. Representative effect of increasing concentrations of CCK-8 (A] and des (SO,) CCK-8 (B) on Isc in guinea pig ileal mucosa.

CCK-8 and by antagonist studies showing the high affinity of these receptors for the CCK-A selective antagonist L-364,718. Cholecystokinin analogs, which possess a sulfate moiety at the seventh position from the terminal COOH group, interact with high affinity with pancreatic CCK-A receptors (5). The amphibian decapeptide cerulein (which like CCK-8 is sulfated at the seventh position) shares the biological properties of CCK-8. Of the analogs tested (Table l), only these two compounds showed potent activity; cerulein was fourfold more potent. Cerulein was previously reported to be twofold more potent than CCK-8 in its ability to displace Y-labled CCK from pancreatic acinar cells (9). Desulfated CCK-8 and gastrin,,-I, which share a common COOH terminal with CCK-8 but exist in the unsulfated form, interact with CCK-A receptors with low affinity (5). Gastrin,,-II, which is sulfated at position six from the COOH terminus, also interacts with CCK-A receptors with low affinity. Cholecystokinin tetrapeptide, the minimal fragment needed for

Vol. 101, No. 5

biological activity (lo), is reported to be inactive at the A site (3). Pentagastrin behaves much like CCK-4 and des (SO,) CCK-8 in other assays (11). Thus, CCKrelated peptides, which are known to be selective for gastrin or CCK-B receptors and show little preference for CCK-A receptors, were relatively weak in our system. In addition, des (SO,) CCK was inactive as an antagonist of sulfated CCK-8. A series of benzodiazepine analogs with CCK antagonist activity have been described (12,13). One (L365,260) shows 100-300-fold selectivity for gastrin and CCK-B receptors, and the other (L-364,718) is reported to have 120-X300-fold selectivity for CCK-A receptors (5). L-365,260 shows a high affinity (PA,, 8.2-9.0) for CCK-B or gastrin receptors and low affinity (PA,, 6.6) for CCK-A receptors. In contrast, L-364,718 shows a high affinity (PA,, 9.0-10.0) for CCK-A receptors and a low affinity (PA,, 6.6) for CCK-B or gastrin receptors (5). Based on our calculated pA, values of 10.3 for L-364,718 and 7.4 for L-365,260 and comparisons with their effects in other assays, the receptor that mediates intestinal electrolyte transport in the guinea pig more closely fits the CCK-A subtype. Characterizing the CCK-A subtype by comparing the different antagonist potencies of L364,718 and L365,260 against CCK-8 assumes that the receptors in the guinea pig ileal mucosa are the same as those previously defined in the brain, stomach, and pancreas. The amino acid derivative lorglumide (CR 1409) is also a potent antagonist at CCK-A receptors (13). We

Log

(Antagonist),

M

Figure 2. Schild plots (17) for L-384,718 and L-365,260 against CCK-8. L-364,718 and L-365,260 showed slopes of 0.98 and 1.07, respectively. Neither of the slopes were significantly different (P > 0.05, general linear test) from unity. Slopes of unity are usually indicative of competitive antagonism. Respective pA, values and (95% confidence limits) of 10.3 (11.7,9.7) and 7.4 (7.7, 7.2) were obtained for L-364,716 and L-365,260. The PA, is taken as the intercept on the abscissa and refers to the -log (Kb). The Kb [dissociation constant) is equal to the concentration of antagonist required to shift the CCK-8 concentration-response curve twofold.

November

1991

CCK-A RECEPTORS

previously found that lorglumide was relatively potent (PA,, 7.7) against CCK-8 in this tissue, again supporting a role for the A subtype (1). Cholecystokinin octapeptide may act as an excitatory neurotransmitter in the myenteric plexus. Studies by Nemeth et al. (14) showed that CCK-8 and cerulein (but not pentagastrin) enhanced the excitability of guinea pig myenteric neurons. Similarly, pentagastrin was not active in our study. Because we used segments of ileum with the enteric nerves intact in our study, we cannot discount the possibility that mucosal secretion is influenced by the action of CCK-8 at receptors situated within the myenteric plexus, receptors which also mediate contraction of longitudinal muscle. Contraction of the guinea pig longitudinal muscle is thought to be mediated by both CCK-A and CCK-B receptors (15). The presence of CCK-B receptors was suggested by the study of Lucaites et al. (15) partly as a result of their observation that CCK-4, desulfated (SO.,) CCK-8 and gastrin were relatively potent agonists, with EC,,, < 50 nmol/L. However, this does not seem to be the case in our mucosal preparation because the EC,, for CCK-4, gastrin, and desulfated (SO:,) CCK-8 were far less potent (estimated to be > 1000 nmol/L). A dissociation constant of 0.06 nmol/L was recently determined for L-364,718 antagonism of the acetylcholine-releasing effect of CCK in guinea pig myenteric neurons (16).Moreover, a pA, value for L-364,718 as an antagonist of CCK-8-induced contractions of the guinea pig longitudinal muscle was reported to be 9.9 (7). This value is in good agreement with the value of 10.3 we obtained for L-364,718 as an antagonist of CCK-8 on electrolye transport. The CCK receptor that mediates mucosal ion secretion closely resembles the CCK-A subtype-associated ileal longitudinal muscle contraction.

References Kachur JF, Phillips GS, Gaginella TS. Neuromodulation of guinea pig intestinal electrolyte transport by cholecystokinin octapeptide. Gastroenterology 1991;100:344-349. Furness JB, Costa M. Keast JR. Choline-acetyltransferase and peptide immunoreactivity of submucous neurons in the small intestine of the guinea pig. Cell Tissue Res 1984;237:329-336. Moran TH. Robinson PH. Goldrich MS, McHugh PR. Two brain

MEDIATE

cholecystokinin 4. 5.

6. 7.

8.

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11.

12.

ILEAL ELECTROLYTE

receptors:

implications

TKANSPORT

for behavioral

l-131

actions.

Brain Res 1986;362:175-179. Yu D-H, Huang SC, Wank SA. Mantey S. Gardner JD, Jensen RT. Am J Physiol 1990;258:G86-G95. Jensen RT. Wank SA, Rowley WH. Soto S, Gardner JD. Interaction of CCK with pancreatic acinar ceils. Trends Pharmaco1 Sci 1989;10:418-423. Beinfeld MC. Cholecystokinin in the central nervous system: a minireview. Neuropeptides 1983:3:411-427. Chang RSL. Lotti VJ. Biochemical and pharmacological characterization of an extremely potent and selective nonpeptide cholecystokinin antagonist. Proc Nat1 Acad Sci 1986:83:49234926. Lotti VJ. Chang RSL. A new potent and selective non-peptide gastrin antagonist and brain cholecystokinin receptor (CCK-Bl l&and: L-365,260. Eur J Pharmacol 1989;162:273-280. Jensen RT, Lemp GF, Gardner JD. Interaction of cholecystokinin with specific membrane receptors on pancreatic acinar cells. Proc Nat1 Acad Sciences IJSA 1980:77:2079-2083. Jensen RT, Lemp CF. Gardner JD. Interactions of COOHterminal fragments of cholecystokinin with receptors on dispersed acini from guinea pig pancreas. J Biol Chem 1982:257: 5554-5559. Yu D-H, Noguchi M. Zhou Z-C, \‘illanueva ML, Gardner JII. Jensen RT. Characterization of gastrin receptors on guinea pig pancreatic acini. Am J Physiol 1987;253:G793-G801. Bock MG, DiGardo RM, Evans BE, Rittle KE. Whitter WE, Veber DF. Anderson PS, Freidinger RM. Benzodiazepine gastrin and brain cholecystokinin ligands: L-365.260. J Med Chem 1989:32: 13.-16.

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RM. Cholecystokinin and gastrin antagonists. Med Res Rev 1989;9:271-290. Nemeth PR, Zafirov DH. Wood JD. Effects of cholecystokinin. caerulein and pentagastrin on electrical behavior of myenteric neurons. Eur J Pharmacol 1985;116:263-269. Lucaites VL, Mendelsohn LG, Mason NR, (,ohen ML. CCK-8, CCK-4 and gastrin-induced contractions of guinea pig ileum: evidence for differential release of acetylcholine and substance P by CCK-A and CCK-B receptors. J Pharmacol Exp Ther 1991:256:695-703. Zelles T. Harsing LG. Vizi ES. Characterization of neuronal cholecystokinin receptor by L364-718 in Auerbachs plexus. Eur 1Pharmacol 1990;178:101-104. Arunlakshana 0, Schild HO. Some quantitative uses of drug antagonists. Br J Pharmacol 1959:14:48-58.

Received September 18, 1990. Accepted March 27, 1991. Address requests for reprints to: James F. Kachur. Ph.D., Gastrointestinal Diseases Research, Searle Research and Development, 4901 Searle Parkway, Skokie. Illinois 60077. The expert secretarial assistance of Jeanette Fraser and Mary Lasquety is greatly appreciated. We are also indebted to Mike Carniello for his help with the statistical analysis. We also thank Xie Yue for her expert technical assistance. Dr. Shu-Xian Wang was a visiting scientist from the Institute of Pharmacology, Beijing, People’s Republic of China,