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Ceruletide, ceruletide analogues and cholecystokinin octapeptide (CCK-8): Effects on isolated intestinal preparations and gallbladders of guinea pigs and mice
Peptides. Vol. 5, pp. 729--736, 1984. e Ankho International Inc. Printed in the U.S.A.
0196-9781/84$3.00 ÷ .00
Ceruletide, Ceruletide Analogues and Cholecystokinin Octapeptide (CCK-8): Effects on Isolated Intestinal Preparations and Gallbladders of Guinea Pigs and Mice G. Z E T L E R Institut fiir Pharmakologie, Medizinische Hochschule Liibeck, D-2400 Liibeck Federal Republic o f Germany R e c e i v e d 19 J a n u a r y 1984 ZETLER, G. Ceruletide. ceruletide analogues and cholecystokinin octapeptide (CCK-8): Effects on isolated intestinal preparations and gallbladders of guinea pigs and mice. PEPTIDES 5(4) 729--736, 1964.raThe smooth muscle stimulatory effects of cholecystokinin octapeptide (CCK-8), ceruletide (CER), ten analogues of CER, and carbaehoi were studied in isolated organs of the guinea pig and the mouse (stomach," ileum, duodenum, colon and gallbladder). On a molar basis, CCK-8 and CER had in all organs except stomach greater potency (lower ECso) than carbachol. The effectiveness (Emax)of CCK-8 and CER was in the gut less than that of carhaehoi, in the guinea pig gallbladder equal with and in the mouse gallbladder superior to that of carbachol. The alteration of peptide structure was virtually without influence on effectiveness: however, it greatly modified the potency and the organ selectivity of the effect. There was no clear-cut correlation between the potency to stimulate smooth muscle and to alter the behavior of the mouse. Cholecystokinin octapeptide Ceruletide Ceruletide analogues Colon Smooth muscle stimulation Structure-activity relation
Carbachol
Gallbladder
Ileum
ioral effects was studied almost exclusively in mice for which presently only peptide effects on the isolated urinary bladder are known [12].
CHOLECYSTOKININ-like peptides such as CCK-8, ceruletide (CER) and some of its analogues exert in mice and rats many behavioral effects that were supposed to be of central origin [13, 16, 17, 19, 20, 21, 22, 30, 37, 38, 39, 40]. These peptides are also powerful stimulants for the smooth muscle of the gastrointestinal tract (except for the sphincter of Oddi) and the gallbladder [ l, 2, 7, 8, 9]. Conceivably then, at behaviorally active systemic doses, CER strongly influenced gastrointestinal propulsion in mice [26,27]. The possibility emerged that some neuropharmacological effects seen after peripheral administration of these peptides may be consequences of abdominal signals running via vagal afferent nerves to the brain. This question has been studied for the inhibitory effects of CCK-8 on rat and mouse exploratory behaviors [3, 4, 5, 6] and for the depression by CER of the crossed extensor reflex of the rat [24]. The present approach to this problem evaluates the structure-activity relationships that should run in parallel for smooth-muscle stimulatory and behavioral effects, provided that abdominal events were the primary cause for changes in behavior. This study concerns the actions of the peptides on isolated intestinal preparations and gallbladders of both guinea pigs and mice. It was important to include the latter species, since the relationship between structure and behav-
METHOD
Isolated Organs Segments (2-3 cm in length) of ileum and descending colon were taken from male guinea pigs (250-350 g) and male NMRI-mice (25-30 g). Strips of the same length were prepared from the fundal part of the mouse and guinea pig stomach, as described for the rat stomach [29]. The whole mouse gallbladder was used, but a strip was made from the guinea pig gallbladder [41]. These isolated preparations were suspended in 5 ml of a solution containing (mM) NaCI 137, KCI 2.68, CaCi2 1.8 (0.9 for guinea pig ileum and colon), MgSO~ 0.49, NaH~PO4 0.42, NaHCO2 11.9, glucose 5.5. This fluid was maintained at 32°C and gassed with 95% 02 and 5% CO2. Contractile responses were recorded by means of an isotonic transducer, the resting load being 0.75 and 1.0 g for the guinea pig ileum and colon, respectively; 0.3 g for the mouse intestines; 0.4 and 0.8 g for the fundus strips of mouse and guinea pig, respectively: 0.2 and 0.25 g for the gallbladder preparations of mouse and guinea pig, respectively. The
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FIG. 1. Concentration-response curves to CCK-8 (CCK), ceruletide {CER), carbachol (CAR) and some ceruletide analogues (No. 1, 2, 4, 8) in the isolated colon of the mouse. Ordinate, E,,,~ relative to the maximal effect of carbachol (= 100). Abscissa, log riM. Shown are the plots of the Hill equation (see the Method section) and for each curve the ECho from Table 2.
FIG. 2. Effects ofcarbachoi and some peptides in the isolated mouse gallbladder. For details see legend to Fig. 1. Shown are mean concentration-response curves from those experiments in which the sensitivity for carbachol was high enough to compare the maximal response to a peptide with that to carbachol (= 100) in the same preparation (see text). The curves refer to carbachol (CAR. n=8), merged results obtained with CCK-8 and CER (n--6, see text), analogue No. 9 (n=5) and analogue No. 10 (n=10). Note that abscissa and ordinate are not the same as in Fig. 1.
experiment began aider an equilibration period (1 hr) during which several doses o f carbachol were appfied. To allow for the differences in time needed by the effect to develop, the contact time was with ileum, duodenum and colon 1 rain for carbachol and 3 win for each concentration of a given peptide, before the bath fluid was changed by overflow (washing); in the other preparations the contact time was 5 rain throughout. The time between washing and the next dose was with ileum and colon 2 rain for carbachoi (with duodenum, 5 rain) and 10 rain for peptides (to prevent tachyphylaxis), 5 min throughout with the stomach strip preparations; with the isolated mouse gallbladder, this time was 20 rain for both carbachol and the peptides.
power to excite the smooth muscles under study. A period of 30--40 rain was allowed for recovery between two concentration-response curves, however, with the mouse gallbladder only one curve was made per preparation. The EC~ was calculated only if the coefficient of correlation, r, of the log concentration---Iogit regression was greater than 0.95 (only very few experiments did not meet this condition). The parameters Emax, E C ~ and s permitted to calculate and plot the sigmoid log concentration-response curves according to the Hill equation y = Ema~ • conc~/(ECso s +concS); examples are shown in Figs. 1 and 2.
Concentration-Response Curves The doses of a given agonist were not standardized but rather adapted to the sensitivity o f the individual preparation by first determining the minimal effective concentration and then increasing it further by a constant factor, mostly of 2 (this procedure does not allow to calculate means of actual responses to the drug concentrations applied). The relationship between concentration and effect was determined by dosing each agonist in the non-cumulative way until the maximal response was reached. Each submaximal effect was expressed as percentage (y) of the corresponding maximal response and transformed into l o o t , i.e., log [y/(100-y)]. The resulting log concentration-logit relationships were linear and permitted the estimation (by means of the least-squares method) o f the regression line, its slope (s), and the EC.~,, i.e., the concentration that elicits half the maximal response at Iogit=0.000. Each determination of a concentrationresponse curve was followed by increasing doses o f carbachol (up to 0. I-8/~g/mi, depending on the type of tissue and its sensitivity) in order to appraise the effectiveness Em~x 1321, i.e., the maximal effect of a given peptide (top of its concentration-response curve) relative to that of carbachol. This cholinergic drug was used as a reference agent characterized by non-peptidic nature, specific receptors, and high
Statistics The results are expressed as arithmetic means, however, the ECs.-values are geometric means as recommended by others [18]. p < 0 . 0 5 was the threshold for the verdict of statistical significance.
Drugs All drugs were dissolved in physiological NaCI solution. Ceruletide (caerulein diethylammonium hydrate) and its analogues were produced by Farmitalia Carlo Erba (Milan/Italy) and kindly given by Professor R. De Castiglione. CCK-8 was from Bachem (CH-4416 Bubendoff/Switzerland) and carbachol (Doryl) from Hoffmann-LaRoche (commercial ampoules). RESULTS
Ileum and Cohm Like carbachol, all peptides except Phe(OHP°-CER stimulated the longitudinal muscle of the intestines of both the guinea pig and the mouse in a concentration-dependent manner, which resulted in sigmoid concentration-response curves and permitted the calculation of parameters presented in the Tables 1-4. Examples of the concentrationresponse curves are shown only for the isolated colon of the
CCK-LIKE PEPTIDES
731
TABLE 1 STIMULATORY POTENCY OF PEPTIDES AND CARBACHOL (EC.~, AND 95%-CONFIDENCE RANGE) IN ISOLATED PREPARATIONS OF ILEUM, COLON AND GALLBLADDEROF GUINEA PIG Ileum Analogue N r.
Compound Ceruletide (CER)
EC.~,, nM
Colon Relative potency*
2
100
2
( 1. I-2.7)
I
Desulphated CER
2
NIeK-CER
3
VaP,NIe~-CER
4
Met(OP-CER
5
(/3-AspP-CER
6 7
Phe(OHP°-CER Boc-LeuS-CER-(4-10)
8
Boc-Nles-CER-(4-10)
9
NIe~-CER-(4-10)
10 lI
Tyr(SOyH)e-CER-(6-10) CCK-8 Carbachol
460 (255-824) 6 (3.7-10) 7 (5.6-9.7) 180 (84--382) 96 (75-122)
inactive'~ 12 (7.%20) 4 ( I. %8.7) 14 (8.8-21) 7420 (3980-13820) 4 (2.3---6.4) 120
(97-151 )
EC:,, nM
Gallbladder Relative potency* 100
( 1.5-4.1 )
0.4 33 29 I.1 2
17 [50] 14 0.03 [50] 1.7
406 (290-569) 9 (4.5-17) 14 (8.4-22) 140 (66-296) 57 (40-81)
inactivet 10 (4.%20) 6 (2.2-14) 27 (18-41) 6510 (4345-9740) 6 (3.0-12) 167
(108--259)
EC:~, nM 1.5
Relative potency* 100
( 1.09-1.97)
0.5 22 14 1.4 4
20 [33] 7 0.03 [33] 1.2
960 (459-2009) 1.7 (I.14--2.49) 1.4 (0.80-2.31 ) 95 (48.8-197) 28 (18.9-41.9)
inactive~ 3 ( 1.78-5.39) 3.2 (2.25-4.65) 19 (10.8-34.6) 3570 (2262-5628) 1.6 (1.05-2.34) 570 (479-685)
0.16 [88] [I07] 1.6 5.4
48 47 8 0.04 [94] 0.26
Values of EC:,, are geometric means and their 95%-confidence limits in= 10 per compound and preparation). Relative potency: (EC.~ of CER/EC.~, of compound) × 100. *Parentheses indicate that the difference from CER is not statistically significant. ~Effect of 30--60 ~zM was less than that of 20--40 nM carbaehol. CEffect of 30 ~.M was less than that of 270 nM carbachol.
mouse (Fig. 1), because (a) this is a very unusual smoothmuscle preparation and (b) the mouse is the main object of this study. It is obvious that potency (EC.~) and effectiveness (Emax) must be considered separately. In both ileum and colon of guinea pig and mouse, the peptidic effects had less effectiveness than those of carbachol, and there were no important differences between peptides in this respect. Therefore, it may suffice to present values of Ema~ only for three selected peptides (Table 3). Emax mostly was in the colon somewhat greater than in the ileum, however, this difference was large only with CER in the mouse because of a very low Em~ in the ileum. Differences in Emax between guinea pig and mouse occurred only in the ileum with lower values for the mouse, most clearly with C E R (Table 3) and analogues No. 2 and 9 (not shown). Regarding potency (EC.~,), however, many peptides were superior to carbachol and a strong influence of chemical structure was evident. Features common to the results from ileum and colon of both species are potency highest with CER and lowest with analogue No. 10, followed by that of No. 1: furthermore, statistical significance was missing for the difference from CER in the cases o f CCK-8 and analogue
No. 8. However, species differences occurred: in the colon of the guinea pig, analogues No. 2, 3, 7 and 9 were clearly less potent than CER, whereas this was not so in the colon of the mouse. The slopes of the concentration-response curves showed no statistically significant differences between either stimulants or preparations (Table 4). This applies to both mouse and guinea pig. Very similar slopes were found with the analogues of CER. Gallbladder
In both species, the gallbladder was less sensitive to carbachol than the intestinal preparations (Tables 1, 2 and 3; Fig. 2). Conversely, the potency o f the peptides was higher in the gallbladder than in the gut, and this was for the mouse more obvious than for the guinea pig (Table 5). Differences in potency between species occurred also with carbachol and with the CER analogues No. 1, 9 and 10 which were, in contrast to carbachol, more potent in the mouse than in the guinea pig. This is the first time that the mouse gallbladder was studied. This organ had a very low responsiveness to car-
732
ZETLER
TABLE
2
S T I M U L A T O R Y P O T E N C Y OF PEPTIDES A N D C A R B A C H O L (ECsu A N D 95%-CONFIDENCE R A N G E ; FOR R E L A T I V E P O T E N C I E S SEE T A B L E 6) IN I S O L A T E D P R E P A R A T I O N S O F ILEUM. C O L O N A N D GALLBLADDER OF T H E M O U S E Analogue Nr.
Ileum nM
Colon
4 ( 1.6-11.5) 672 (446-1014) 32 (17-63) 20 (5.7-68) 55 (25-122) 175 (73-418) inactive*
7 (2.7-18) 543
Compound Ceruletide (CER)
1
Desulphated CER
2
Nles-CER
3
ValS,Nles-CER
4
Met(OP-CER
5
(/3-Asp)~-CER
6
Phe(OH)'°-CER
7
Boc-Leu~-CER-(4--10)
nM
(395-747) 12 (5.7-27) 12 (4.7-33) 184 129-263) 98 (48--201 ) reactive*
Gallbladder nM 1.0 (0.44-2.O8) 193 (118--315) 2.7 ( I.1 I-6.33) 0.7 (0.39-1.33) I00 (56.5-176) 14 (7.1-28.9) 3430
(2283--5156)
8
29 (15-57) 6 (2.8--I1.3) 20
Boc-Nles-CER-(4-10)
9
Nlen-CER-(4--10)
10
Tyr(SO~H)e-CER-(6-- I0)
11
CCK-8 Carbachol
24
(11-50) 3
(1.3-6.3) 21
(9.5-44)
(10-43)
3799 (1954-.7385) 6 (2.9-12) 590 (456--763)
6750 (4301-10592) 3 ( 1.4--7.4) 244 (155-384)
1.7 (0.97-3.08) 1.2 (0.51-2.68) 0.7 (0.35-I .36) 1385 807-2377) 0.7 10.41-I. 14) 1480 (875-2495 )
Values of EC.~, are geometric means and their 9f:'~-confidence limits, n= 10 per compound and preparation: exception: n=8 for carbachol on gallbladder (see text). *Effect of 15-30 ~M was less than that of 40--50 nM carbachoi.
TABLE
3
E F F E C T I V E N E S S O F T H R E E PEPrIDES R E L A T I V E TO T H A T O F C A R B A C H O L (= 100) IN S O M E I S O L A T E D O R G A N S O F T H E M O U S E A N D T H E G U I N E A PIG Animal
Organ
Mouse
Duodenum Ileum Colon Gallbladder
Guinea pig
Ileum Colon Gallbladder
CER
CCK-8
36 (22--49)* 33 (27-38) 49 (38-59) 64 (54--74) 68 (51-84) 220 ( 122-320)~; 66 (56-75) 78 (55-101) I00 (83-I 19)
63 (56-70) 77 (66--89) 87 (79-96)
NIe"-CER-(4-10 41 (37-45)t 43 (33--52) 63 (51-76) 380 (254-506)§ 69 (60-78) 69 (62-76) 92 (80-104)
Figures are arithmetic means and their 95%-confidence ranges (n= I0). *From combined results of the experiments with both peptides (see text). ¢n=3. ~From combined results obtained with CER and CCK-8 (n=3 each, see text). §n=5 out of I0 (see text).
C C K - L I K E PEPTIDES
733 TABLE 4
SLOPES OF THE REGRESSION LINES FOR LOG1TS VERSUS LOG CONCENTRATION OF CER. CCK-8 AND CARBACHOL, AS DETERMINED IN SOME ISOLATED ORGANS OF THE MOUSE AND THE GUINEA PIG
Animal
Organ
CER
CCK-8
Carbachol
Mouse
Duodenum Ileum Colon Gallbladder
1.47 ( 1.11-1.83)* 1.28 (1.02-1.55) 1.36 (1.09-1.63) 1.45 ( I. 15-1.75) 1.52 ( 1.26-- 1.79) 1.80 (!.39-2.20)'t 1.96 (1.48-2.44)
1.50 1.49 1.57 2.54
Guinea pig
Ileum Colon Gallbladder
1.27 (1.03--1.52) 1.26 ( 1.05-1.48) 1.64 (1.36--1.92)
1.42 (1.20-1.64) 1.58 (1.15-2.02) 2.15 (1.89-2.41)
1.36 (0.94-1.77) 1.55 ( 1.34-1.76) 1.74 (I.51-1.97)
(I.21-1.80) (1.23--I.74) (1.26--1.89) (1.99-3.09)
Figures are arithmetic means and their 95%-confidence ranges (n = 10; mouse gallbladder, n--8). *From combined results of the experiments with both peptides (see text). tDifference from carbachol is statistically significant (p<0.05); statistical significance exists also for the difference between CER plus CCK-8 (combined) and carbachol (p<0.01, ANOVA).
bachoi, which permitted in only 8 out o f 21 preparations the determination of a dose-response curve (Fig. 2). Another consequence was that in only 3 out of 10 experiments each with CER and CCK-8 a dose-response curve for carbachol could be established in addition to that for a peptide. Only in these cases could the effectiveness (Enaax) of the peptides be determined, and the resulting six values were combined (see Table 3). In the guinea pig gallbladder all peptides had virtually the same effectiveness as carbachol, which is shown for three examples in Table 3. In the mouse preparation, the effectiveness of the peptides was by far larger than that of carbachol. This is also demonstrated by Fig. 2 which also shows that analogue No. 10, it is true, had nearly the same low potency as carbachol but was clearly more effective (note that No. 10 was active in 10/10 preparations, but carbachol only in 8/21). The slopes (for examples see Table 4) showed no statistically significant differences between peptides. The only important difference was observed in the mouse gallbladder between CER + CCK-8 and carbachol (compare with Fig. 2).
Stomach Strip This preparation reacted to carbachol in a concentration-dependent manner with an EC.~o (nM) o f 270 (95%-confidence range, 184--398) in the guinea pig and of 295 (203--427) in the mouse. The potency o f the peptides, however, was very low and permitted only the determination of minimal effective concentrations (compared with carbachoi by "'bracketing"). In the mouse fundus strip, the effect of 150-190 nM of the peptides (CER, CCK-8, NIeS-CER and Boc-Nie"-CER-(4--10); other peptides not tested) was less than that o f 50-100 nM of carbachol. In the guinea-pig preparation, these concentrations of peptides and carbachoi were equiactive.
Duodemmt This organ of the mouse was only incompletely studied in order to get just an impression of whether it differs in sensitivity from the other intestinal preparations. Some EC.~,, values (nM) were: carbachol, 566 (350-884, n= 10); CER, 4 (2.9--5.9. n=8): CCK-8. 3 (1.8--4.7, n=2); (/~-Asp)9-CER, 87
TABLE 5 INDEX OF SELECTIVITY OF THE GALLBLADDER-STIMULATING EFFECT Analogue Nr.
1 2 3 4 5 7 8 9 10 11
Compound
Ceruletide (CER) Desuiphated CER Nies-CER ValS,NIes-CER Met(O)8-CER (/3-Asp)'-CER Boc-Leu~-CER-(4-- 10) Boc-Nies-CER-(4-- 10) Nles-CER-(4-- 10) Tyr(SO.~H)s-CER46-10) CCK-8 Carbachol
Guinea pig
Mouse
1.3 0.5 4.4 7 1.7 2.6 3.7 1 1 2 3 0.25
5.3 3.1 7.3 22 i 9.4 15.5 3.5 29 3.7 6 0.26
To calculate the index of selectivity, the geometric mean of the EC.~,, for ileum and colon was divided by the EC.~ for gallbladder. The difference between both columns (carbachol neglected) is statistically significant (p<0.01, Wiicoxon-Mann-Whitney rank sum t~st). The ranking orders of both columns are different (Spearman's r=0.502, p >0.05).
(39--195, n=5); NIeS-CER-(4--10), 5 (3.4--8.2, n=3). Additional data are presented in Tables 3 and 4. These results together suggest that the duodenum has virtually the same sensitivity for CCK-like peptides as ileum and colon. DISCUSSION
The present results obtained with CER and CCK-8 on the guinea-pig ileum are nearly identical with those observed on the isolated longitudinal muscle (separated from the circular muscle) o f the same organ [33]. The results on five analogues o f CER (Nos. 1, 4, 7, 8, 11) can be compared with those of other authors on the guinea-pig gallbladder and dog jejunum, both in situ [1, 2, 7, 8]. There is agreement that structures No. 1, 4 and 10 are very inactive, whereas analogues No. 7
734
ZETLER TABLE 6
RELATIVE POTENCIES OF CERULETIDE (CER) ANALOGUES, REGARDING BEHAVIOURAL AND PERIPHERAL EFFECTS IN THE MOUSE
Behavioural Effects
Effects on Isolated Organs
No.
Compound
REA
PTO
CAT
HEX
ANA
M1G
Gallbladder
Ileum
Colon
1 2 3 4 5 "6 7 8 9 10 11
Desulfated Nies-CER ValS,Nles-CER Met(O)8-CER (~-AspP-CER Pbe(OH)'O-CER Boc-LeuB-CER-(4-10) Boc-Nles-CER-(4--10) Nles-CER-(4--10) TyP(SO.~H)~-CER-(6--10) CCK-8
0 [78] 29 7 7 0 14 33 5 0 6
0 [200] [801 4 6 0 14 27 11 0 [561
0.3 [157] [921 15 5 0.6 29 26 13 nt 8
0 39 [64] 4 3 0 9 13 4 0 12
<3 36 [641 8 4 0 28 23 26 0 [30]
0 409 [94] [94] [681 0 [551 298 249 0 30
0.5 [37] [1431 1 7 0.03 [591 [831 11431 0.07 11431
0.6 13 [201 7 2 0 14 167] 20 0. I 1671
1.3 [581 [581 4 7 0 [291 12331 [331 0. I 12331
Figures are relative potencies (ceruletide= 100) as calculated from values of ED~,. or EC.~,, respectively. The data on behavioural effects are from previous studies [37,38], those on effects on isolated organs are from Table 2 of the present study. Figures in parentheses mean that the difference between the absolute value and the corresponding value for CER was not statistically significant (nt, not tested). REA, inhibition of exploratory rearing activity; PTO, production of ptosis; CAT, production catalepsy (akinesia on a vertical rod); HEX. prolongation of sleep induced by hexobarbital (50 mg/kg IV); ANA, antinociception on the hot plate; MIG. antistereotypic effect against gnawing induced by methylphenidate (50 mg/kg SC).
and 8 have retained much CER-like activity. The present findings in isolated guinea-pig gallbladder and ileum confirm the previous observation of only small differences in potency between CER and CCK-8 [33]. The results obtained with ileum and colon did not reveal dramatic differences between guinea pig and mouse, especially as far as compounds with low potency are concerned. However, for the problems to be discussed below, it is important that in the mouse colon six out of ten active C E R analogues had relative potencies which were not clearly different from that of CER, whereas in the guinea-pig colon this applied only to two peptides. The gallbladder of the mouse had a greater sensitivity to CCK-like peptides than the gallbladder o f the guinea pig. This was more pronounced with some analogues than with either CER or CCK-8: desulfated CER, factor 5; analogue No. 9, factor 27; No. 10, factor 2.6. This species difference is perhaps specific for some peptides, because it is inverted with carbachoi which was 2.5 times less potent in the mouse gallbladder (if potent at all, see Results). CCK-iike peptides stimulate many types of smooth muscle. Therefore, the question of whether the effect on the gallbladder is specific deserves attention. According to Table 5, all peptides had greater selectivity than carbachol. Furthermore, all peptides, except No. 4, had greater selectivity in the mouse than in guinea pig preparation. Changes in chemical structure modified the selectivity, however, with results differing with species (ranking orders of indices of selectivity are not correlated; see Table 5). For example, with analogue No. 3 selectivity was increased 3.4 times in the guinea pig but 1.4 times in the mouse; or, with No. 8 and 9 it was unchanged in the guinea pig but in the mouse decreased 1.5 times and increased 5.5 times, respectively. Hence, the isolated gallbladder of the mouse is useful for bioassays on CCK-like peptides, especially since in this preparation, in contrast to the guinea-pig gallbladder, the
peptides were superior to carbachol not only in potency but also in effectiveness and dependability (Table 3). The question of whether there exists a correlation between peripheral (i.e., smooth muscle stimulatory) and central (i.e., behavioral) effects can be discussed with the aid of Table 6 which contains only results obtained in NMRI-mice. Those o f the known behavioral effects were chosen which either are " s e d a t i v e " or were also observed after either intracerebroventricular or intracerebral administration to rats. The latter were sedation [20, 22.23], antinociception [22] and antagonism o f apomorphine-induced effects [I 1,30]. However. the intracerebroventricular injection may not warrant an exclusively central origin of a behavioral effect, because CCK-8 has been found (in the rabbit) to diffuse rapidly into the blood when administered into the lateral ventricle [25]. It is obvious that a very unfavorable modification of chemical structure abolishes both peripheral and central activities (compounds No. 1.6 and 10). However, peptides that do not differ in potency from CER on gallbladder, ileum and colon (Nos. 3, 8 and 11) often have reduced central potencies: this very clearly applies to Nos. 8 and 11 with the notable exception of an increased antistereotypic (MIG) potency of No. 8. On the other hand, a large decrease in peripheral potency (Nos. 4 and 5) is not matched by similar losses of oil central effects. Therefore. it is important to note that with analogue No. 5 not only the antistereotypic but also the tremorolytic potency (against the harmine-induced tremor, not shown in Table 6) was virtually the same as that of CER [391. The increase in antistereotypic potency of Nos. 2, 8 and 9 was not parailelled by a comparable change in peripheral potency. On the other hand. CCK-8 had nearly the same peripheral potency as No. 8. but was less antistereotypic. Likewise, Nos. 2, 7 and 9 are similar as regards peripheral effects (low ileal potency) but very dissimilar concerning central effects. It is important that CCK-8, which has the same peripheral potency as CER, had
C C K - L I K E PEPTIDES
735
less central potency than CER not only after systemic administration (Table 6) but also after injection either into the ventricular system [21 ] and the spinal subarachnoid space [22] or even into circumscript parts of the rat brain [22,30]. Finally, CER was more potent than CCK-8 in facilitating the K +evoked GABA release from tissue slices of rat parietal cortex [27], which may be relevant to the similar difference in anticonvulsive potency against picrotoxin, barman and thiosemicarbazide [35,36]. The relations between chemical structure and pharmacological effect of CCK-like peptides are obviously different for the behavioral effects and the smooth muscle stimulator3' actions studied. Hence, the present study does not support the notion that CCK-like peptides owe their behavioral effects to their powerful peripheral actions (see Introduction). This agrees with the similar conclusion drawn from the finding that nonsulfated CCK-8, in spite of its very
weak peripheral potency, produced the same effect as CCK-8 on active and passive avoidance behaviour in rats 114, 15, 30]. The peptide effects on the stomach fundus preparations were too weak as to hint at the stomach as an important source of peptide-induced abdominal signals to the brain. Other organs besides those studied here can be considered as possible peripheral sites of CCK-peptidic effects that may modify the behavior: the pancreas and the vagus nerve which both have receptors for CCK-like peptides !10,341.
ACKNOWLEDGEMENTS
The author would like to thank Professor Roberto De Castiglionc (Farmitalia Carlo Erba, Milan) for the generous gift of the peptides; Ms. Gisela Schmidt for skilled technical assistance, and Ms. Gerda Mundhenke for typing the manuscript.
REFERENCES I. Anastasi, A., L. Bernardi, G. Bertaccini, G. Bosisio, R. De Castigiione, V. Erspamer, O. Goffredo and M. Impicciatore. Synthetic peptides related to caerulein. Experientia 24: 771-773, 1968. 2. Bertaccini, G. Peptides: Gastrointestinal hormones, in: Mediators and Drugs in Gastrointestinal Motility H, Handbook Exp. Pharmacol. vol 59/II, edited by G. Bertaccini. Berlin: Springer-Verlag, 1982, pp. 11-83. 3. Crawley, J. N.. S. E. Hays, T. L. O'Donohue, S. M. Paul and F. K. Goodwin. Neuropeptide modulation of social and exploratory behaviors in laboratory rodents. Peptides 2: Suppl. !, 123129, 1981. 4. Crawley. J. N.. S. E. Hays and S. M. Paul. Vagotomy abolishes the inhibitory effects of cholecystokinin on rat exploratory behaviors. Ear J Pharmacol 73: 379--380, 1981. 5. Crawley, J. N., S. E. Hays. S. M. Paul and F. K. Goodwin. Cholecystokinin reduces exploratory behavior in mice. Physiol Behar 27:407--411, 1981. 6. Crawley. J. N.. J. A. Rojas-Ramirez and W. B. Mendelson. The role of central and peripheral cbolecystokinin in mediating appetitive behaviors. Peptides 3: 535-538, 1982. 7. De Castiglione. R. Structure-activity relationships in ceruletide-like peptides. In: First International Symposium on Hormonal Receptor.s in Digestive Tract Physiology. INSERM Symposium. No. 3. edited by S. Bonfils, P. Fromageot and G. Rosselin. Amsterdam: Elsevier/North-Holland Biomedical Press. 1977, pp. 33--42. 8. De Castigiione. R. Structural requirements of ceruletide-like peptides for activity on gut and brain. Peptides 2: Suppl. 2, 61-63, 1981. 9. De Castiglione, R. and E. Di Salle. Is commercial "'cerulein'" always true cerulein? Gastroenterology 78: i ! 13-1 i 14, 1980. 10. Deschodt-Lanckman. M., P. Robberecht. J. Camus and J. Christophe. The interaction of caerulein with the rat pancreas. 1. Specific binding of [3Hlcaerulein on plasma membranes and evidence for negative cooperativity. Ear J Biochem 91: 21-29, 1978. 1I. Ellinwood. E. H.. Jr.. K. Rockwell and N. Wagoner. Apomorphine behavioral effect is facilitated by dibutyryl/cAMP and inhibited by caerulein. Psychopharmacol Bull 19: 351-354, 1983. 12. Falconieri Erspamer. G., L. Negri and D. Piccinelli. The use of preparations of urinary bladder smooth muscle for bioassay of and discrimination between polypeptides. Naunyn Schmiedebergs Arch Pharmacol 279: 61-74. 1973. 13. Faris. P. L.. B. R. Komisaruk. L. R. Watkins and D. J. Mayer. Evidence for the neuropeptide cholecystokinin as an antagonist of opiate analgesia. Science 219:310--312, 1983.
14. Fekete, M., M. Bokor, B. Penke and G. Telegdy. Effects of cholecystokinin octapeptide sulphate ester and unsulphated cholecystokinin octapeptide on active avoidance behaviour in rats. Acta Physiol Acad Sci Hang 60: 57--64, 1983. 15. Fekete, M., T. Kfid~r, B. Penke and G. Tolegdy. Modulation of passive avoidance behaviour by cholecystokinin octapeptide in rats. Nearopeptides 1: 301-307, 1981. 16. Fekete, M., A. l.,engyel, B. HegedOs, B. Penke, M. Za~ndy, G. K. T6th and G. Telegdy. Further analysis of the effects of cholecystokinin octapeptides on avoidance behaviour in rats. Fur J Pharraacol 98: 79--91, 1984. 17. Fekete, M., A. Rentzsch, H. Schwarzberg and G. Telegdy. Effect of cholecystokinin on self-stimulation behavior in rats. Fur J Pharmacol 91: 77-82, 1983. 18. Fleming, W. W., D. P. Wesffali, 1. S. De La Lande and L.B. Jellett. Log-normal distribution of equieffective doses of norepinephrine and acetylcholiue in several tissues. J Pharmacol Exp Ther 181: 339--345, 1972. 19. ltoh, S. and G. Katsuura. Suppressive effect of cholecystokinin and its related peptides on B-endorphin-induced catalepsy in rats. Fur J Pharraacol 74: 381-384, 1981. 20. Itoh, S. and G. Katsuura. Suppressive action ofcholecystokinin octapeptide on the behavioral effects of L-dopa in the rat. Fur J Pharrnacol 75: 313-316, 1981. 21. ltoh, S., G. Katsuura and Y. Maeda. Caerulein and cholecystokinin suppress ~-¢ndorphin-induced analgesia in the rat. Eur J Pharrnacol 80: 421--425, 1982. 22. Jurna, I. and G. Zetler. Antinociceptive effect of centrally administered caerulein and cholecystokinin octapeptide (CCK-8). Eur J Pharraacol 73: 323-331, 1981. 23. Katsuura, G. and S. Itoh. Sedative action of cholecystokinin octapept/de on behavioral excitation by thyrotropin releasing hormone and methamphetamine in the rat. Jpn J Physiol 32: 83-91, 1982. 24. Kawasaki, K., M. Kodama and A. Matsushita. Caerulein, a cholecystokinin-related peptide, depresses somatic function via the vagal afferent system. Life Sci 33: 1045-1050, 1983. 25. Passaro, E., Jr., H. Debas, W. Oldendorf and T. Yamada. Rapid appearance of intraventricularly administered neuropeptides in the peripheral circulation. Brain Res 241: 335-340, 1982. 26. Piceinelli, D., F. Ricciotti, A. Catalani and P. Sale. The action of caerulein on gastro-intestinal propulsion in mice. Naunyn Schmiedebergs Arch Pharmacol 279: 75-82, 1973. 27. Scheufler, E. and G. Zetler. Interactions of caerulein and morphine on gastrointestinal propulsion in the mouse. Naunyn Schmiedebergs Arch Pharmacol 318: 66-69, 1981. 28. Sheehan. M. J. and J. De Belleroche. Facilitation of gaba release by cholecystokinin and caerulein in rat cerebral cortex. Neuropeptides 3: 429-434, 1983.
736 29. Vane, J. R. A sensitive method for the assay of 5-hydroxytryptamine. Br J Pharmacol 12: 344-349, 1957. 30. Van Ree, J. M., O. Gaffori and D. De Wied. In rats, the behavioral profile of CCK-8 related peptides resembles that of antipsychotic agents. Eur J Pharmacol 93: 63-78, 1983. 31. Vizi, E. S., G. Bertaccini, M. Impicciatore, P. Mantovani, J. Zs~li and J. Knoll. Structure-activity relationship of some analogues of gastrin and cholecystokinin on intestinal smooth muscle of the guinea pig. Naunyn Schmiedebergs Arch Pharmacol 21M:233-243, 1974. 32. Waud, D. R. Pharmacological receptors. Pharmacol Rev 20: 49-88, 1968. 33. Yau, W. M., G. M. Makhlouf, L. E. Edwards and J. T. Farrar. The action of cholecystokininand related peptides on guinea pig small intestine. Can J Physiol Pharmacol 52: 298--303, 1974. 34. Zarbin, M. A., L K. Wamsley, R. B. Innis and M. J. Kuhar. Cholecystokinin receptors: Presence and axonal flow in the rat vagus nerve. Life Sci 29: 697-705, 1981. 35. Zetler, G. Central depressant effects of caerulein and cholecystokinin octapeptide (CCK-8) differ from those of diazepam and haloperidol. Neuropharmacology 20: 277-283, 1981.
ZETLER 36. Zetler, G. Anticonvulsant effects of caerulein, cholecystokinin octapeptide (CCK-8) and diazepam against seizures produced in mice by harman, thiosemicarbazide and isoniazid. Neurosci Lett 24: 175--180, 1981. 37. Zetler, G.. Central effects of ceruletide analogues. Peptide.s 2: Suppl 2, 65-69, 1981. 38. Zetler, G. Ceruletide, ceruletide analogues and cholecystokinin octapeptide (CCK-8): Effects on motor behaviour. hexobarbital-induced sleep and harman-induced convulsions. Peptides 3: 701-704, 1982. 39. Zetler, G. Cholecystokinin octapeptide (CCK-SL ceruletide and analogues of ceruletide: effects on tremor induced by oxotremorine, harmine and ibogaine. A comparison with prolylleucylglycine amide (MIF), anti-parkinsonian drugs and clonazepam. Neuropharmacology 22: 757-766, 1983. 40. Zetler, G. Behavioral pharmacology of CCK and analogs. Ps.vchopharmacol Bull 19: 347-351, 1983. 41. Zetler, G., D. Cannon, D. Powell, P. Skrabanek and J. J. Vanderhaeghen. A cholecystokinin-like peptide in crude substance P from human and bovine brain. Arch lnt Pharmacodyn 238: 128-141, 1979.
0196-9781/84$3.00 ÷ .00
Ceruletide, Ceruletide Analogues and Cholecystokinin Octapeptide (CCK-8): Effects on Isolated Intestinal Preparations and Gallbladders of Guinea Pigs and Mice G. Z E T L E R Institut fiir Pharmakologie, Medizinische Hochschule Liibeck, D-2400 Liibeck Federal Republic o f Germany R e c e i v e d 19 J a n u a r y 1984 ZETLER, G. Ceruletide. ceruletide analogues and cholecystokinin octapeptide (CCK-8): Effects on isolated intestinal preparations and gallbladders of guinea pigs and mice. PEPTIDES 5(4) 729--736, 1964.raThe smooth muscle stimulatory effects of cholecystokinin octapeptide (CCK-8), ceruletide (CER), ten analogues of CER, and carbaehoi were studied in isolated organs of the guinea pig and the mouse (stomach," ileum, duodenum, colon and gallbladder). On a molar basis, CCK-8 and CER had in all organs except stomach greater potency (lower ECso) than carbachol. The effectiveness (Emax)of CCK-8 and CER was in the gut less than that of carhaehoi, in the guinea pig gallbladder equal with and in the mouse gallbladder superior to that of carbachol. The alteration of peptide structure was virtually without influence on effectiveness: however, it greatly modified the potency and the organ selectivity of the effect. There was no clear-cut correlation between the potency to stimulate smooth muscle and to alter the behavior of the mouse. Cholecystokinin octapeptide Ceruletide Ceruletide analogues Colon Smooth muscle stimulation Structure-activity relation
Carbachol
Gallbladder
Ileum
ioral effects was studied almost exclusively in mice for which presently only peptide effects on the isolated urinary bladder are known [12].
CHOLECYSTOKININ-like peptides such as CCK-8, ceruletide (CER) and some of its analogues exert in mice and rats many behavioral effects that were supposed to be of central origin [13, 16, 17, 19, 20, 21, 22, 30, 37, 38, 39, 40]. These peptides are also powerful stimulants for the smooth muscle of the gastrointestinal tract (except for the sphincter of Oddi) and the gallbladder [ l, 2, 7, 8, 9]. Conceivably then, at behaviorally active systemic doses, CER strongly influenced gastrointestinal propulsion in mice [26,27]. The possibility emerged that some neuropharmacological effects seen after peripheral administration of these peptides may be consequences of abdominal signals running via vagal afferent nerves to the brain. This question has been studied for the inhibitory effects of CCK-8 on rat and mouse exploratory behaviors [3, 4, 5, 6] and for the depression by CER of the crossed extensor reflex of the rat [24]. The present approach to this problem evaluates the structure-activity relationships that should run in parallel for smooth-muscle stimulatory and behavioral effects, provided that abdominal events were the primary cause for changes in behavior. This study concerns the actions of the peptides on isolated intestinal preparations and gallbladders of both guinea pigs and mice. It was important to include the latter species, since the relationship between structure and behav-
METHOD
Isolated Organs Segments (2-3 cm in length) of ileum and descending colon were taken from male guinea pigs (250-350 g) and male NMRI-mice (25-30 g). Strips of the same length were prepared from the fundal part of the mouse and guinea pig stomach, as described for the rat stomach [29]. The whole mouse gallbladder was used, but a strip was made from the guinea pig gallbladder [41]. These isolated preparations were suspended in 5 ml of a solution containing (mM) NaCI 137, KCI 2.68, CaCi2 1.8 (0.9 for guinea pig ileum and colon), MgSO~ 0.49, NaH~PO4 0.42, NaHCO2 11.9, glucose 5.5. This fluid was maintained at 32°C and gassed with 95% 02 and 5% CO2. Contractile responses were recorded by means of an isotonic transducer, the resting load being 0.75 and 1.0 g for the guinea pig ileum and colon, respectively; 0.3 g for the mouse intestines; 0.4 and 0.8 g for the fundus strips of mouse and guinea pig, respectively: 0.2 and 0.25 g for the gallbladder preparations of mouse and guinea pig, respectively. The
729
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ZETLER , mmr
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8
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FIG. 1. Concentration-response curves to CCK-8 (CCK), ceruletide {CER), carbachol (CAR) and some ceruletide analogues (No. 1, 2, 4, 8) in the isolated colon of the mouse. Ordinate, E,,,~ relative to the maximal effect of carbachol (= 100). Abscissa, log riM. Shown are the plots of the Hill equation (see the Method section) and for each curve the ECho from Table 2.
FIG. 2. Effects ofcarbachoi and some peptides in the isolated mouse gallbladder. For details see legend to Fig. 1. Shown are mean concentration-response curves from those experiments in which the sensitivity for carbachol was high enough to compare the maximal response to a peptide with that to carbachol (= 100) in the same preparation (see text). The curves refer to carbachol (CAR. n=8), merged results obtained with CCK-8 and CER (n--6, see text), analogue No. 9 (n=5) and analogue No. 10 (n=10). Note that abscissa and ordinate are not the same as in Fig. 1.
experiment began aider an equilibration period (1 hr) during which several doses o f carbachol were appfied. To allow for the differences in time needed by the effect to develop, the contact time was with ileum, duodenum and colon 1 rain for carbachol and 3 win for each concentration of a given peptide, before the bath fluid was changed by overflow (washing); in the other preparations the contact time was 5 rain throughout. The time between washing and the next dose was with ileum and colon 2 rain for carbachoi (with duodenum, 5 rain) and 10 rain for peptides (to prevent tachyphylaxis), 5 min throughout with the stomach strip preparations; with the isolated mouse gallbladder, this time was 20 rain for both carbachol and the peptides.
power to excite the smooth muscles under study. A period of 30--40 rain was allowed for recovery between two concentration-response curves, however, with the mouse gallbladder only one curve was made per preparation. The EC~ was calculated only if the coefficient of correlation, r, of the log concentration---Iogit regression was greater than 0.95 (only very few experiments did not meet this condition). The parameters Emax, E C ~ and s permitted to calculate and plot the sigmoid log concentration-response curves according to the Hill equation y = Ema~ • conc~/(ECso s +concS); examples are shown in Figs. 1 and 2.
Concentration-Response Curves The doses of a given agonist were not standardized but rather adapted to the sensitivity o f the individual preparation by first determining the minimal effective concentration and then increasing it further by a constant factor, mostly of 2 (this procedure does not allow to calculate means of actual responses to the drug concentrations applied). The relationship between concentration and effect was determined by dosing each agonist in the non-cumulative way until the maximal response was reached. Each submaximal effect was expressed as percentage (y) of the corresponding maximal response and transformed into l o o t , i.e., log [y/(100-y)]. The resulting log concentration-logit relationships were linear and permitted the estimation (by means of the least-squares method) o f the regression line, its slope (s), and the EC.~,, i.e., the concentration that elicits half the maximal response at Iogit=0.000. Each determination of a concentrationresponse curve was followed by increasing doses o f carbachol (up to 0. I-8/~g/mi, depending on the type of tissue and its sensitivity) in order to appraise the effectiveness Em~x 1321, i.e., the maximal effect of a given peptide (top of its concentration-response curve) relative to that of carbachol. This cholinergic drug was used as a reference agent characterized by non-peptidic nature, specific receptors, and high
Statistics The results are expressed as arithmetic means, however, the ECs.-values are geometric means as recommended by others [18]. p < 0 . 0 5 was the threshold for the verdict of statistical significance.
Drugs All drugs were dissolved in physiological NaCI solution. Ceruletide (caerulein diethylammonium hydrate) and its analogues were produced by Farmitalia Carlo Erba (Milan/Italy) and kindly given by Professor R. De Castiglione. CCK-8 was from Bachem (CH-4416 Bubendoff/Switzerland) and carbachol (Doryl) from Hoffmann-LaRoche (commercial ampoules). RESULTS
Ileum and Cohm Like carbachol, all peptides except Phe(OHP°-CER stimulated the longitudinal muscle of the intestines of both the guinea pig and the mouse in a concentration-dependent manner, which resulted in sigmoid concentration-response curves and permitted the calculation of parameters presented in the Tables 1-4. Examples of the concentrationresponse curves are shown only for the isolated colon of the
CCK-LIKE PEPTIDES
731
TABLE 1 STIMULATORY POTENCY OF PEPTIDES AND CARBACHOL (EC.~, AND 95%-CONFIDENCE RANGE) IN ISOLATED PREPARATIONS OF ILEUM, COLON AND GALLBLADDEROF GUINEA PIG Ileum Analogue N r.
Compound Ceruletide (CER)
EC.~,, nM
Colon Relative potency*
2
100
2
( 1. I-2.7)
I
Desulphated CER
2
NIeK-CER
3
VaP,NIe~-CER
4
Met(OP-CER
5
(/3-AspP-CER
6 7
Phe(OHP°-CER Boc-LeuS-CER-(4-10)
8
Boc-Nles-CER-(4-10)
9
NIe~-CER-(4-10)
10 lI
Tyr(SOyH)e-CER-(6-10) CCK-8 Carbachol
460 (255-824) 6 (3.7-10) 7 (5.6-9.7) 180 (84--382) 96 (75-122)
inactive'~ 12 (7.%20) 4 ( I. %8.7) 14 (8.8-21) 7420 (3980-13820) 4 (2.3---6.4) 120
(97-151 )
EC:,, nM
Gallbladder Relative potency* 100
( 1.5-4.1 )
0.4 33 29 I.1 2
17 [50] 14 0.03 [50] 1.7
406 (290-569) 9 (4.5-17) 14 (8.4-22) 140 (66-296) 57 (40-81)
inactivet 10 (4.%20) 6 (2.2-14) 27 (18-41) 6510 (4345-9740) 6 (3.0-12) 167
(108--259)
EC:~, nM 1.5
Relative potency* 100
( 1.09-1.97)
0.5 22 14 1.4 4
20 [33] 7 0.03 [33] 1.2
960 (459-2009) 1.7 (I.14--2.49) 1.4 (0.80-2.31 ) 95 (48.8-197) 28 (18.9-41.9)
inactive~ 3 ( 1.78-5.39) 3.2 (2.25-4.65) 19 (10.8-34.6) 3570 (2262-5628) 1.6 (1.05-2.34) 570 (479-685)
0.16 [88] [I07] 1.6 5.4
48 47 8 0.04 [94] 0.26
Values of EC:,, are geometric means and their 95%-confidence limits in= 10 per compound and preparation). Relative potency: (EC.~ of CER/EC.~, of compound) × 100. *Parentheses indicate that the difference from CER is not statistically significant. ~Effect of 30--60 ~zM was less than that of 20--40 nM carbaehol. CEffect of 30 ~.M was less than that of 270 nM carbachol.
mouse (Fig. 1), because (a) this is a very unusual smoothmuscle preparation and (b) the mouse is the main object of this study. It is obvious that potency (EC.~) and effectiveness (Emax) must be considered separately. In both ileum and colon of guinea pig and mouse, the peptidic effects had less effectiveness than those of carbachol, and there were no important differences between peptides in this respect. Therefore, it may suffice to present values of Ema~ only for three selected peptides (Table 3). Emax mostly was in the colon somewhat greater than in the ileum, however, this difference was large only with CER in the mouse because of a very low Em~ in the ileum. Differences in Emax between guinea pig and mouse occurred only in the ileum with lower values for the mouse, most clearly with C E R (Table 3) and analogues No. 2 and 9 (not shown). Regarding potency (EC.~,), however, many peptides were superior to carbachol and a strong influence of chemical structure was evident. Features common to the results from ileum and colon of both species are potency highest with CER and lowest with analogue No. 10, followed by that of No. 1: furthermore, statistical significance was missing for the difference from CER in the cases o f CCK-8 and analogue
No. 8. However, species differences occurred: in the colon of the guinea pig, analogues No. 2, 3, 7 and 9 were clearly less potent than CER, whereas this was not so in the colon of the mouse. The slopes of the concentration-response curves showed no statistically significant differences between either stimulants or preparations (Table 4). This applies to both mouse and guinea pig. Very similar slopes were found with the analogues of CER. Gallbladder
In both species, the gallbladder was less sensitive to carbachol than the intestinal preparations (Tables 1, 2 and 3; Fig. 2). Conversely, the potency o f the peptides was higher in the gallbladder than in the gut, and this was for the mouse more obvious than for the guinea pig (Table 5). Differences in potency between species occurred also with carbachol and with the CER analogues No. 1, 9 and 10 which were, in contrast to carbachol, more potent in the mouse than in the guinea pig. This is the first time that the mouse gallbladder was studied. This organ had a very low responsiveness to car-
732
ZETLER
TABLE
2
S T I M U L A T O R Y P O T E N C Y OF PEPTIDES A N D C A R B A C H O L (ECsu A N D 95%-CONFIDENCE R A N G E ; FOR R E L A T I V E P O T E N C I E S SEE T A B L E 6) IN I S O L A T E D P R E P A R A T I O N S O F ILEUM. C O L O N A N D GALLBLADDER OF T H E M O U S E Analogue Nr.
Ileum nM
Colon
4 ( 1.6-11.5) 672 (446-1014) 32 (17-63) 20 (5.7-68) 55 (25-122) 175 (73-418) inactive*
7 (2.7-18) 543
Compound Ceruletide (CER)
1
Desulphated CER
2
Nles-CER
3
ValS,Nles-CER
4
Met(OP-CER
5
(/3-Asp)~-CER
6
Phe(OH)'°-CER
7
Boc-Leu~-CER-(4--10)
nM
(395-747) 12 (5.7-27) 12 (4.7-33) 184 129-263) 98 (48--201 ) reactive*
Gallbladder nM 1.0 (0.44-2.O8) 193 (118--315) 2.7 ( I.1 I-6.33) 0.7 (0.39-1.33) I00 (56.5-176) 14 (7.1-28.9) 3430
(2283--5156)
8
29 (15-57) 6 (2.8--I1.3) 20
Boc-Nles-CER-(4-10)
9
Nlen-CER-(4--10)
10
Tyr(SO~H)e-CER-(6-- I0)
11
CCK-8 Carbachol
24
(11-50) 3
(1.3-6.3) 21
(9.5-44)
(10-43)
3799 (1954-.7385) 6 (2.9-12) 590 (456--763)
6750 (4301-10592) 3 ( 1.4--7.4) 244 (155-384)
1.7 (0.97-3.08) 1.2 (0.51-2.68) 0.7 (0.35-I .36) 1385 807-2377) 0.7 10.41-I. 14) 1480 (875-2495 )
Values of EC.~, are geometric means and their 9f:'~-confidence limits, n= 10 per compound and preparation: exception: n=8 for carbachol on gallbladder (see text). *Effect of 15-30 ~M was less than that of 40--50 nM carbachoi.
TABLE
3
E F F E C T I V E N E S S O F T H R E E PEPrIDES R E L A T I V E TO T H A T O F C A R B A C H O L (= 100) IN S O M E I S O L A T E D O R G A N S O F T H E M O U S E A N D T H E G U I N E A PIG Animal
Organ
Mouse
Duodenum Ileum Colon Gallbladder
Guinea pig
Ileum Colon Gallbladder
CER
CCK-8
36 (22--49)* 33 (27-38) 49 (38-59) 64 (54--74) 68 (51-84) 220 ( 122-320)~; 66 (56-75) 78 (55-101) I00 (83-I 19)
63 (56-70) 77 (66--89) 87 (79-96)
NIe"-CER-(4-10 41 (37-45)t 43 (33--52) 63 (51-76) 380 (254-506)§ 69 (60-78) 69 (62-76) 92 (80-104)
Figures are arithmetic means and their 95%-confidence ranges (n= I0). *From combined results of the experiments with both peptides (see text). ¢n=3. ~From combined results obtained with CER and CCK-8 (n=3 each, see text). §n=5 out of I0 (see text).
C C K - L I K E PEPTIDES
733 TABLE 4
SLOPES OF THE REGRESSION LINES FOR LOG1TS VERSUS LOG CONCENTRATION OF CER. CCK-8 AND CARBACHOL, AS DETERMINED IN SOME ISOLATED ORGANS OF THE MOUSE AND THE GUINEA PIG
Animal
Organ
CER
CCK-8
Carbachol
Mouse
Duodenum Ileum Colon Gallbladder
1.47 ( 1.11-1.83)* 1.28 (1.02-1.55) 1.36 (1.09-1.63) 1.45 ( I. 15-1.75) 1.52 ( 1.26-- 1.79) 1.80 (!.39-2.20)'t 1.96 (1.48-2.44)
1.50 1.49 1.57 2.54
Guinea pig
Ileum Colon Gallbladder
1.27 (1.03--1.52) 1.26 ( 1.05-1.48) 1.64 (1.36--1.92)
1.42 (1.20-1.64) 1.58 (1.15-2.02) 2.15 (1.89-2.41)
1.36 (0.94-1.77) 1.55 ( 1.34-1.76) 1.74 (I.51-1.97)
(I.21-1.80) (1.23--I.74) (1.26--1.89) (1.99-3.09)
Figures are arithmetic means and their 95%-confidence ranges (n = 10; mouse gallbladder, n--8). *From combined results of the experiments with both peptides (see text). tDifference from carbachol is statistically significant (p<0.05); statistical significance exists also for the difference between CER plus CCK-8 (combined) and carbachol (p<0.01, ANOVA).
bachoi, which permitted in only 8 out o f 21 preparations the determination of a dose-response curve (Fig. 2). Another consequence was that in only 3 out of 10 experiments each with CER and CCK-8 a dose-response curve for carbachol could be established in addition to that for a peptide. Only in these cases could the effectiveness (Enaax) of the peptides be determined, and the resulting six values were combined (see Table 3). In the guinea pig gallbladder all peptides had virtually the same effectiveness as carbachol, which is shown for three examples in Table 3. In the mouse preparation, the effectiveness of the peptides was by far larger than that of carbachol. This is also demonstrated by Fig. 2 which also shows that analogue No. 10, it is true, had nearly the same low potency as carbachol but was clearly more effective (note that No. 10 was active in 10/10 preparations, but carbachol only in 8/21). The slopes (for examples see Table 4) showed no statistically significant differences between peptides. The only important difference was observed in the mouse gallbladder between CER + CCK-8 and carbachol (compare with Fig. 2).
Stomach Strip This preparation reacted to carbachol in a concentration-dependent manner with an EC.~o (nM) o f 270 (95%-confidence range, 184--398) in the guinea pig and of 295 (203--427) in the mouse. The potency o f the peptides, however, was very low and permitted only the determination of minimal effective concentrations (compared with carbachoi by "'bracketing"). In the mouse fundus strip, the effect of 150-190 nM of the peptides (CER, CCK-8, NIeS-CER and Boc-Nie"-CER-(4--10); other peptides not tested) was less than that o f 50-100 nM of carbachol. In the guinea-pig preparation, these concentrations of peptides and carbachoi were equiactive.
Duodemmt This organ of the mouse was only incompletely studied in order to get just an impression of whether it differs in sensitivity from the other intestinal preparations. Some EC.~,, values (nM) were: carbachol, 566 (350-884, n= 10); CER, 4 (2.9--5.9. n=8): CCK-8. 3 (1.8--4.7, n=2); (/~-Asp)9-CER, 87
TABLE 5 INDEX OF SELECTIVITY OF THE GALLBLADDER-STIMULATING EFFECT Analogue Nr.
1 2 3 4 5 7 8 9 10 11
Compound
Ceruletide (CER) Desuiphated CER Nies-CER ValS,NIes-CER Met(O)8-CER (/3-Asp)'-CER Boc-Leu~-CER-(4-- 10) Boc-Nies-CER-(4-- 10) Nles-CER-(4-- 10) Tyr(SO.~H)s-CER46-10) CCK-8 Carbachol
Guinea pig
Mouse
1.3 0.5 4.4 7 1.7 2.6 3.7 1 1 2 3 0.25
5.3 3.1 7.3 22 i 9.4 15.5 3.5 29 3.7 6 0.26
To calculate the index of selectivity, the geometric mean of the EC.~,, for ileum and colon was divided by the EC.~ for gallbladder. The difference between both columns (carbachol neglected) is statistically significant (p<0.01, Wiicoxon-Mann-Whitney rank sum t~st). The ranking orders of both columns are different (Spearman's r=0.502, p >0.05).
(39--195, n=5); NIeS-CER-(4--10), 5 (3.4--8.2, n=3). Additional data are presented in Tables 3 and 4. These results together suggest that the duodenum has virtually the same sensitivity for CCK-like peptides as ileum and colon. DISCUSSION
The present results obtained with CER and CCK-8 on the guinea-pig ileum are nearly identical with those observed on the isolated longitudinal muscle (separated from the circular muscle) o f the same organ [33]. The results on five analogues o f CER (Nos. 1, 4, 7, 8, 11) can be compared with those of other authors on the guinea-pig gallbladder and dog jejunum, both in situ [1, 2, 7, 8]. There is agreement that structures No. 1, 4 and 10 are very inactive, whereas analogues No. 7
734
ZETLER TABLE 6
RELATIVE POTENCIES OF CERULETIDE (CER) ANALOGUES, REGARDING BEHAVIOURAL AND PERIPHERAL EFFECTS IN THE MOUSE
Behavioural Effects
Effects on Isolated Organs
No.
Compound
REA
PTO
CAT
HEX
ANA
M1G
Gallbladder
Ileum
Colon
1 2 3 4 5 "6 7 8 9 10 11
Desulfated Nies-CER ValS,Nles-CER Met(O)8-CER (~-AspP-CER Pbe(OH)'O-CER Boc-LeuB-CER-(4-10) Boc-Nles-CER-(4--10) Nles-CER-(4--10) TyP(SO.~H)~-CER-(6--10) CCK-8
0 [78] 29 7 7 0 14 33 5 0 6
0 [200] [801 4 6 0 14 27 11 0 [561
0.3 [157] [921 15 5 0.6 29 26 13 nt 8
0 39 [64] 4 3 0 9 13 4 0 12
<3 36 [641 8 4 0 28 23 26 0 [30]
0 409 [94] [94] [681 0 [551 298 249 0 30
0.5 [37] [1431 1 7 0.03 [591 [831 11431 0.07 11431
0.6 13 [201 7 2 0 14 167] 20 0. I 1671
1.3 [581 [581 4 7 0 [291 12331 [331 0. I 12331
Figures are relative potencies (ceruletide= 100) as calculated from values of ED~,. or EC.~,, respectively. The data on behavioural effects are from previous studies [37,38], those on effects on isolated organs are from Table 2 of the present study. Figures in parentheses mean that the difference between the absolute value and the corresponding value for CER was not statistically significant (nt, not tested). REA, inhibition of exploratory rearing activity; PTO, production of ptosis; CAT, production catalepsy (akinesia on a vertical rod); HEX. prolongation of sleep induced by hexobarbital (50 mg/kg IV); ANA, antinociception on the hot plate; MIG. antistereotypic effect against gnawing induced by methylphenidate (50 mg/kg SC).
and 8 have retained much CER-like activity. The present findings in isolated guinea-pig gallbladder and ileum confirm the previous observation of only small differences in potency between CER and CCK-8 [33]. The results obtained with ileum and colon did not reveal dramatic differences between guinea pig and mouse, especially as far as compounds with low potency are concerned. However, for the problems to be discussed below, it is important that in the mouse colon six out of ten active C E R analogues had relative potencies which were not clearly different from that of CER, whereas in the guinea-pig colon this applied only to two peptides. The gallbladder of the mouse had a greater sensitivity to CCK-like peptides than the gallbladder o f the guinea pig. This was more pronounced with some analogues than with either CER or CCK-8: desulfated CER, factor 5; analogue No. 9, factor 27; No. 10, factor 2.6. This species difference is perhaps specific for some peptides, because it is inverted with carbachoi which was 2.5 times less potent in the mouse gallbladder (if potent at all, see Results). CCK-iike peptides stimulate many types of smooth muscle. Therefore, the question of whether the effect on the gallbladder is specific deserves attention. According to Table 5, all peptides had greater selectivity than carbachol. Furthermore, all peptides, except No. 4, had greater selectivity in the mouse than in guinea pig preparation. Changes in chemical structure modified the selectivity, however, with results differing with species (ranking orders of indices of selectivity are not correlated; see Table 5). For example, with analogue No. 3 selectivity was increased 3.4 times in the guinea pig but 1.4 times in the mouse; or, with No. 8 and 9 it was unchanged in the guinea pig but in the mouse decreased 1.5 times and increased 5.5 times, respectively. Hence, the isolated gallbladder of the mouse is useful for bioassays on CCK-like peptides, especially since in this preparation, in contrast to the guinea-pig gallbladder, the
peptides were superior to carbachol not only in potency but also in effectiveness and dependability (Table 3). The question of whether there exists a correlation between peripheral (i.e., smooth muscle stimulatory) and central (i.e., behavioral) effects can be discussed with the aid of Table 6 which contains only results obtained in NMRI-mice. Those o f the known behavioral effects were chosen which either are " s e d a t i v e " or were also observed after either intracerebroventricular or intracerebral administration to rats. The latter were sedation [20, 22.23], antinociception [22] and antagonism o f apomorphine-induced effects [I 1,30]. However. the intracerebroventricular injection may not warrant an exclusively central origin of a behavioral effect, because CCK-8 has been found (in the rabbit) to diffuse rapidly into the blood when administered into the lateral ventricle [25]. It is obvious that a very unfavorable modification of chemical structure abolishes both peripheral and central activities (compounds No. 1.6 and 10). However, peptides that do not differ in potency from CER on gallbladder, ileum and colon (Nos. 3, 8 and 11) often have reduced central potencies: this very clearly applies to Nos. 8 and 11 with the notable exception of an increased antistereotypic (MIG) potency of No. 8. On the other hand, a large decrease in peripheral potency (Nos. 4 and 5) is not matched by similar losses of oil central effects. Therefore. it is important to note that with analogue No. 5 not only the antistereotypic but also the tremorolytic potency (against the harmine-induced tremor, not shown in Table 6) was virtually the same as that of CER [391. The increase in antistereotypic potency of Nos. 2, 8 and 9 was not parailelled by a comparable change in peripheral potency. On the other hand. CCK-8 had nearly the same peripheral potency as No. 8. but was less antistereotypic. Likewise, Nos. 2, 7 and 9 are similar as regards peripheral effects (low ileal potency) but very dissimilar concerning central effects. It is important that CCK-8, which has the same peripheral potency as CER, had
C C K - L I K E PEPTIDES
735
less central potency than CER not only after systemic administration (Table 6) but also after injection either into the ventricular system [21 ] and the spinal subarachnoid space [22] or even into circumscript parts of the rat brain [22,30]. Finally, CER was more potent than CCK-8 in facilitating the K +evoked GABA release from tissue slices of rat parietal cortex [27], which may be relevant to the similar difference in anticonvulsive potency against picrotoxin, barman and thiosemicarbazide [35,36]. The relations between chemical structure and pharmacological effect of CCK-like peptides are obviously different for the behavioral effects and the smooth muscle stimulator3' actions studied. Hence, the present study does not support the notion that CCK-like peptides owe their behavioral effects to their powerful peripheral actions (see Introduction). This agrees with the similar conclusion drawn from the finding that nonsulfated CCK-8, in spite of its very
weak peripheral potency, produced the same effect as CCK-8 on active and passive avoidance behaviour in rats 114, 15, 30]. The peptide effects on the stomach fundus preparations were too weak as to hint at the stomach as an important source of peptide-induced abdominal signals to the brain. Other organs besides those studied here can be considered as possible peripheral sites of CCK-peptidic effects that may modify the behavior: the pancreas and the vagus nerve which both have receptors for CCK-like peptides !10,341.
ACKNOWLEDGEMENTS
The author would like to thank Professor Roberto De Castiglionc (Farmitalia Carlo Erba, Milan) for the generous gift of the peptides; Ms. Gisela Schmidt for skilled technical assistance, and Ms. Gerda Mundhenke for typing the manuscript.
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