The sites of action of cholecystokinin in decreasing meal size in pigs

Physiology & Behavior, Vol. 31, pp. 6930698. Pergamon Press Ltd., 1983. Printed in the U.S.A.

The Sites of Action of Cholecystokinin in Decreasing Meal Size in Pigs T. R I C H A R D H O U P T Departtnent o f Physioh)gy, N e w York State College o f Veterinary Medicine Cornell Unit,ersity, lthaca, N Y 14853 R e c e i v e d 31 M a r c h 1983 HOUVF, T, R. The sites of action of cholecystokinin in decreasing meal size ~f pigs. PHYSIOL BEHAV 31(5) 693--698, 1983.--The synthetic octapeptide of cholecystokinin (CCK-8) was infused at the rate of 67 ng/kg-minute into the jugular vein, carotid artery, portal vein, gastric branch of the splenic artery, and aortic artery both craniad and caudad to the origins of the cranial mesenterie and coeliac arteries of 39 young female pigs. Jugular. carotid and cranial arotic infusions resulted in significant reduction of meal size respectively to 65, 67 and 71% of control meal size (p<0.05). Gastric artery infusion resulted in a significant increase of meal size to 122"%of control meal size (p<0.05). Portal vein and caudal aortic artery infusions had no significant effects on meal size. The results were interpreted as indicating a major site of action of CCK in reducing meal size in the bed of the cranial mesenteric or coeliac arteries, but excluding the stomach and liver. Cholecystokinin

Meal size

Satiety

Food intake

THE presence o f c h y m e in the small intestine has long been believed to play a role in determining meal size, initiating inhibitory signals that act ultimately on the brain integrating systems that directly control feeding behavior. Shortly after purified cholecystokinin (CCK) became available, the inhibitory effect of exogenous CCK on meal size was demonstrated in rats [6], and the hypothesis was formulated that CCK released from duodenal mucosa during a meal acts as one of the negative feedback signals to the CNS that bring a meal to an end. Until recent years it was generally assumed (although often not articulated) that the CCK released from intestinal mucosa entered the mesenteric blood stream, passed via the portal system, liver, and hepatic vein to reach the general arterial circulation, and thence to the brain where it was presumed to act on the feeding control systems in the hypothalamus. The finding that appreciable amounts of CCK are synthesized in various regions of the brain [17] suggests a role for CCK as a CNS neurotransmitter largely, but not necessarily, independent of its role in the control of food intake. Failure o f systemic CCK to pass the blood brain barrier [15] indicates a separation of brain and gut CCK activities. Further, when part o f the nerve supply to (and from) the stomach and small intestine is sectioned, the meal depressing action of CCK is greatly attenuated [10, 12, 18, 19]. This has led to the hypothesis that CCK exerts its " s a t i e t y " action peripherally, probably in the upper gastrointestinal tract [5]. Studies on the inhibitory effect of CCK on gastric emptying have led to the companion hypothesis that by delaying gastric emptying and thus prolonging the stretch due to the presence of food, the afferent neural influences originating in stretch receptors cause the inhibition of food intake [13]. Among the possible sites o f this CCK action are the brain, liver, stomach and small intestine. In the experiments

Pigs reported here exogenous CCK was infused continuously during meals into the specific blood supply to these tissues, as well as to the serosal surface of the duodenum and ileum. The results point to the small intestine as a major site of CCK satiety action. METHOD Thirty-nine young female Yorkshire pigs initially weighing from 8 to 15 kg were the subjects of the experiments. They were fed a pelleted, nutritionally complete commercial feed (Squealer, Agway, Syracuse, NY) ad l i b e x c e p t for 3 or 4 hours (1100 to 1400 or 1500 hr) before the experiment and for an hour afterwards. This premeal fast period approximates the normal intermeal interval for free feeding pigs of this age and ensured that a meal would be consumed when feed was presented. The pigs were housed individually in large stainless steel cages in an air conditioned room ( 2 3+- l ~ C) with a 12 hr on-off light cycle (on at 0700 hr). A dim red light was on during the dark phase. Each pig was allowed out o f its cage daily for a 10 to 15 minute exercise period while the cage was being cleaned. The test meals, during which infusions were made, were taken in a specially constructed wooden cabinet that facilitated manipulation of the catheters and weighing o f the feed. Pigs were conditioned to the p r o c e d u r e - - t h e fast period, the infusion through implanted catheter, and the test m e a l A b y being subjected to the routine (with isotonic saline as the infusion) for several days before beginning the CCK infusions. Silastic catheters were implanted at the sites of infusion under halothane anesthesia using sterile surgical techniques. Catheter size was either 1.0 mm i.d.-2.2 mm o.d. (jugular, aortic, portal, ileal, duodenal) or 0.8 mm i . d . - l . 7 mm o.d. (gastric, carotid). Cuffs (l cm) of larger silastic tubing were cemented 6 to 12 cm from the tip and used to anchor the

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FIG. I. Effects on meal size ofcholecystokinin octapeptide infused continuously during a 10 minute meal at the rate of 67 ng/kg-minute Meal size is expressed as a percentage of mean meal size on adjacent control days when saline was infused. Asterisks indicate that meal size was significantly different from paired control means: ***p<0.001; **p<0.01; *p<0.05. Route of infusion is indicated beneath each column, as well as the number of pigs used and total number of individual measurements (in parentheses).

catheter in place. The jugular catheter was placed with the tip lying in the anterior vena cava. The carotid catheter was placed with the tip directed toward the brain, almost to the division of the common carotid artery into internal and external carotid arteries. The portal vein was approached by feeding the catheter into the splenic vein until the tip entered the portal vein. The gastric catheter was inserted directed toward the stomach into the gastric branch of the splenic artery which in the pig is a substantial artery running in the gastrosplenic ligament. This artery supplies much of the greater curvature of the stomach and anastomoses with the other arteries supplying the stomach. The aortic catheters were inserted into the right carotid artery and fed in until the tip was located in the desired position. The " s h o r t " aortic catheters were placed with the tip a few centimeters craniad to the origins of the coeliac and cranial mesenteric arteries. The tip of the "long" aortic catheter was caudad to the origins of those arteries. Position of the catheter tip was estimated during implantation by body measurements and was confirmed by radiographic examination while radio-opaque solution was being injected and by postmortum dissection. These intravascular catheters were kept filled with heparinized saline (1000 units/ml). The supraileal and supraduodenal catheters were prepared with an 8 to 10 c m end section perforated at 1 cm intervals from the tip to the cuffs. This end portion of the catheter was tacked to the serosal surface of the ileum with interrupted nylon sutures at about 1 centimeter intervals. This positioned the perforated section over most of the length of the ileum from the beginning of the ileocecal fold to the ileocecal junction. Similarly the supraduodenal catheter was tacked to the serosai surface of the initial 10 cm of the duodenum, beginning 2 or 3 centimeters beyond the pylorus. The intraduodenal catheter was inserted through a small in-

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FIG. 2. Effect on meal size of infusion of cholecystokinin octapeptide onto the serosal surface of the duodenum and ileum and into the lumen of the duodenum at the rate of 67 ng/kg-minute. Meal size for the 10 minute test period is expressed as a percentage of mean meal size on adjacent control days when only saline was infused. Number of pigs used at each site is indicated beneath each column with total number of measurements given in parentheses.

cision in the duodenum just beyond the pylorus, the ,-a.theter tip then lay in the lumen 4-5 cm aborally. These catheters were kept filled with isotonic saline. All catheters were brought to exit from the dorsum of the neck and the external length of silastie tubing and plug were attached with a collar of adhesive tape. The daily infusion procedure was usually begun a day or two after surgery with infusions of isotonic saline being given during the short test meals. When the size of the daily test meals had stabilized, the CCK experiments were begun. The typical daily procedure was as follows. Feed, but not water, was removed at 1100 hr; at 1500 hr the pig was allowed to walk from its cage into the holding box for the test meal and infusion. The only time water was not available was during the test meal. The silastic catheter was attached to a syringe pump, and the infusion begun at a rate of 6 ml/10 minutes. At the time when the infusion solution was calculated to have reached the implanted tip of the catheter, and therefore the infusion into the body actually began, a weighed container of the pelleted feed was presented. After 10 minutes, the feed container was reweighed to determine intake and returned to the pig. The infusion was continued, but changed to isotonic saline, during the second 10 minutes. At the end of the second I0 minutes, the infusion was discontinued and the feed was reweighed. The pig then returned to its cage. Feed in the cage was returned an hour later. During the test meals the pigs were under observation and their behavior was noted for signs of discomfort. During 128 test meals eating behavior was monitored either by recording

C H O L E C Y S T O K I N I N AND MEAL SIZE

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FIG. 3. Meal patterns during cholecystokinin octapeptide infusion at 67 ng/kg-min into the jugular vein. Upper record: Each horizontal line shows the time during which the pig was eating for 67 individual control meals (left) and 61 individual CCK infusion meals (right). The brief time break between the first and second I0 minute periods of the meals represents the fraction of a minute during which feed was being weighed and the infusion apparatus adjusted. Lower graph: Mean meal size (ordinate). duration (abscissa), and position in time for the two 10 minute feeding test periods for control meals (left) and CCK infusion meals (right). Mean values derived from the mean values for each pig (n= 17 pigs).

the interruptions of a light beam whenever the pig put its snout into the feed container or by direct observation of times of initiation and termination of bouts of eating. The rate of intrajugular chotecystokinin infusion used (67 ng/kg-min=27 pmoles/kg-min) was one we had found previously to reduce reliably meal size to 60-70% of control meal size [1]. The effects of CCK-8 infusion at 33 and 134 ng/kgminute were also measured in 7 of these pigs. The synthetic octapeptide of choleystokinin (CCK-8, Squibb) was used. The CCK-8 was supplied dry in vials that also contained a small amount of NaCI. During all infusions the volume was kept constant at 6 ml/10 minutes, consequently as the CCK-8 dose varied with body weight, the amount of NaCI and hence osmolality also varied. Control saline infiJsions were, therefore, prepared so as to be isosmotic with the CCK-8 infusion solutions. Saline and CCK-8 infusions were given on alternate days. The effects of CCK-8 infusion on test meal size was calculated as a percentage of the average meal size of the two adjacent control test meals when saline was infused, The CCK infusion experiments at each site were repeated on each pig for l to 7 times with a mean of 3.4 times. The results on each pig were averaged to give a best value for the CCK effect on meal size at that infusion site. These mean values from individual pigs were then used to calculate the overall mean effect of CCK on meal size when infused at that site ( n = n u m b e r of pigs). For statistical analysis the mean intake in grams during CCK-8 infusion test meals and during the saline control meals were calculated lbr each pig. The signif-

icance of any differences from control level was then assessed by the paired t test (two-tailed) using n as the number ofp~s. RESULTS The effects of CCK-8 infusion during meals are summarized for the intravascular infusions in Fig. I. Jugular infusion of 67 ng/kg-min CCK-8 significantly reduced meal size to 65% of control. Intracarotid arterial infusion had a similar effect, significantly reducing meal size to 67% of control. CCK-8 infusion into the portal vein surprisingly had no significant effect on meal size, Even more unexpected, the infusion into the gastric branch of the splenic artery resulted in a consistent and significant increase in meal size to 122% of control intake. Infusion of CCK-8 into the aorta caudad to the origins of the coeliac and cranial mesenteric arteries (long catheter) had only a slight and non-significantdepressing effect on intake, but a similar infusion cr;:~iad to the origin of those arteries (short catheter) significantly reduced intake to 71% of control. The effects on intake of CCK-8 infused during meals onto the serosal surfaces of the duodenum and ileum were inconsistent. In 2 pigs supraduodenal infusion resulted in a slight depression of intake, in 2 others an apparent stimulation of intake, and in a fifth, no effect: giving a mean intake not significantly different from control (Fig. 2). In 4 pigs in l I experiments suprialeal infusion always caused a considerable depression of intake, but in three experiments on a fifth

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pig all resulted in an increase in meal size. Overall mean intake was greatly depressed [Fig. 2), but statistically the difference was not quite significant at the 0.05 level, t(4)=2.70, p<0.06. Infusion of CCK into the lumen had no significant effect. In these experiments the pigs generally ate meals of about 10 minutes duration, and if the meal extended beyond the first 10 minute period, it simply continued into the second l0 minute period. In those experiments where CCK-8 infusion reduced meal size, meal duration was also reduced, because rate of eating tended to be constant. However, a few minutes after the CCK-8 infusion was discontinued, the pig often resumed eating during the second I0 minute period. This pattern of response to CCK-8 infusion at 67 ng/kg-min is shown in Fig. 3 for the 128 meals in which eating time was recorded during intrajugular infusions. Shown are both the actual timing of individual meals (Fig. 3, top) and mean meal size and duration (bottom) during the first and second 10 minute measurement periods. The mean interval between the end of the CCK-8 infusion and resumption of eating during the 38 meals in which feeding did resume (top, right, Fig. 3) was 5.8~0.4 minutes (• The pigs exhibited a typical postprandial pattern o f behavior, rubbing the snout against and licking or chewing the wooden floor or sides of the cabinet, with occasional soft grunting. This calm exploratory activity might continue to the end o f the test meal or the pig might sit down. lay down and even doze off. The younger pigs tended to be more restless for the first few days. There were no apparent differences between the postprandial behavior in pigs receiving saline infusions and those receiving CCK-8 during meals except that the latter often resumed eating before the end of the second 10 minute period. There were no signs of discomfort. When the rate o f CCK-8 infusion into the jugular vein or carotid artery was halved or doubled (from 67 to 33 or 134 ng/kg-min), meal size tended to increase o r decrease respectively, as did meal duration (Fig. 4). Meal duration here

consistently effective in reducing meal size (Figs. 1,3 and 4). Presumably thorough mixing occurred during the passages of blood through the heart and lungs, and arterial blood of uniform CCK concentration was then conducted to all other tissues, including the specific site or sites of C C K ' s satiety action. The infusion o f CCK was stopped at the end of the first 10 minute test period, and feeding often resumed late in the second 10 minute period (Fig. 3). The half-life of CCK is 2 to 3 minutes in the systenlic circulation [16], and the average latency to resume eating of 5.8 minutes during the second 10 minute period was ample to allow catabolism of most o f the infused CCK. Apparently this inhibition of eating is related directly to blood CCK level and ceases as soon as that lever falls below a threshold value. Baldwin t,t al. [2] have found a similar duration o f inhibition of food intake following bolus injections of CCK-8. Infusion of CCK at the same dose rate into one carotid artery towards the brain reduced meal size to the same extent as did jugular infusion (Figs. I and 4), Infusion by this route should have produced a higher concentration of CCK in cerebral blood flow than when infused into the jugular vein, and if the primary site of CCK action were in the circulatory bed o f the carotid artery, a greater depression of meal size would have been expected. Notice that the dose rate of CCK deli'~ery (67 ng/kg-min) was not maximal for reduction of meal size (Fig. 4), i.e., a high dose rate as was present in the carotid artery would have been capable of inducing a greater meal reduction if the site of action was there. If the site of action were elsewhere, a decreased effectiveness might have been expected. The characteristics of cerebral circulation may explain why carotid infusion resulted in a reduction of meal size apparently identical to that during jugular infusion. First, because CCK does not cross the blood-brain barrier, extraction of CCK from carotid blood is nil and all CCK infused into the carotid artery should appear in the jugular blood [ 15]. Second, cerebral blood flow is relatively high and the vascular pathway short, circulation time from carotid to jugular must be brief---of the order of a few seconds. Thus, ff all CCK infused into the carotid artery quickly appears in jugular blood, the effectiveness o f either route in reducing meal size should be nearly equal. That CCK infused into the carotid artery did not have a greater effect than when infused into the jugualr vein suggests that there was no additional effect on the brain directly. However, the variability of the results could obscure a minor effect of CCK, perhaps at a site where the blood-brain barrier is weak. Such a higher permeability has been suggested in structures near the hypothalamic areas [15]. An action of CCK within the liver would be appropraite because during digestion endogenous CCK released from intestinal mucosal ceils enters the portal vein bed where the highest concentrations should be found. Failure of CCK-8 infusions into the portal vein to influence meal size was surprising, but definite, based on 36 experiments in 9 pigs. This

C H Q L E C Y S T O K I N I N AND M E A L SIZE result suggests that CCK does not act upon a receptor site within the liver. Why CCK, however, had no effect at all upon food intake is puzzling. One might expect that after passage ~.~ough ttne *,~er irffused CCK would join the vena t a r a , via the hepatic vein, and then have an effect similar to jugular infusion. One possible explanation is that CCK is inactivated during its passage through the liver. However, porcine CCK-33 is not so inactivated [3,161 and the kidneys are probably the major site of inactivation or removal [TL Another exp}anation is suggested by the anatomical fact that a large p o ~ of b l ~ 6 is eonVained within the liver, about 10 to 15% of total blood volume [41, and this may result in a long time delay in the passage of CCK through the liver. This interpretation was supported by a happenstance. Occasionally there was reason to doubt whether an intravascular catheter was still in position within the vessel (as for example, ifblood could not be withdrawn). One method of testing a catheter utus t~ ir~ject rapir~y a dose of sodium pentobarbital that would cause the pig to collapse within I0 to 20 seconds if it were intravascular. However, when injected into the portal vein, "there was always a long pause before the typical cerebral effects of this anesthetic appeared--50 to 60 seconds before any sign of cerebral depression and then a slow collapse 3t) to 90 seconds later. The most likely interpretation (s th~,t the 9e~xtoba~bitat enters the pool of blood within the liver, mixes there, and then gradually leaves at a slow rate into the hepatic vein. Sodium pentobarbital is not rapidly rne~.ab~tized in the liver and so its full anesthetic effects eventually appear. If portally infused CCK also trickles out of the hepatic pool of blood into the hepatic vein flow, the rise in systemic CCK concentration would be slow, perhaps to,a sto,,u ;.v,ttxe far of its rapid catabolism to allow a rise to effective levels during the 10 minute test meal. There has been a preliminary report of investigations similar to these, but on sheep. Orovum [8] infused porcine CCK-33 at a similar dose rate (approximately equivalent to 80 ng CCK-8/kg-min) via implanted catheters into the jugular vein, carotid artery, portal vein, and aortic arch of fasted sheep begi~uxirtg ~O mir~utes before mad then during a meal. There was a significant depression o f meal size during jugular, carotid and portal infusions o f CCK, but not during infusions in~.o 'the aortic arch. The reduction of meal size during jugular, carotid and portal infusions were not different from one another. The absence of an enhanced effect via the carotid artery is in good agreement with the pig results. The effectiveness of portal infusion in the sheep may have been due to the long premeal infusion of CCK that may have compensated for the long delay of CCK passage through the liver seen in pigs. Ther~ is no obvious explanation for failure o f CCK infusions into the aorta of sheep--equivalent to the "short" aortic infusions in pigs--to reduce meal size significantly. The great differences in gastrointestinal function between the herbivorous sheep and omivorpus pig can be invoked as a general explanation. For the moment, however, as Grovum points out, his results suggest. *,b,e. I,,a~gs as the site ~f Q C K ' s action in reducing meal size. Failure of CCK-8 intraportal infusions in these experiments to cause any reduction of meal size is not in agreement with our earlier work in pigs in ",vhich bolus ir~jections of both porcine CCK-33 and CCK-8 redoced meal size signiticantly [1]. Those bo]u~ injections mast have momentarily cau,~ed a very high c~n~ent~'ation irL the portal blood that probably overcame the time delay encountered by the slow, low dose infusions.

697 The slight, but consistent and significant, stimulation of food intake when CCK was infused into the arterial SUl~ply of the stomach was unexpected. There have been reports that CCK in vivo causes a relaxation of gastric musculature [20]. /t is conceivable that such inhibition of gastric tension in these experiments also depressed excitation of gastric stretch receptors and hence decreased afferent flow to the CNS. Such stretch stimuli have been suggested as the basis of inhibition of food intake [13]. At any rate these results do not support the hypothesis that the stomach is the site of CCK aetiota in reducing meal size. It seems possible that the block of CCK's effect on food intake when vagal branches were sectioned in rats [19] was due to interruption of innervation to the small intestine rather than of that to the stomach. That at least part of the satiety action of CCK is on the small intestine was also suggested by the results of the aortic catheter infusions (Fig. I L When CCK was infused at a point in the aorta craniad to the origins of the coeliac and cranial mesenteric arteries, reduction of meal size was about the same as when CCK was infused into the jugular vein. However. infusion with the tip of the catheter caudad to the origin of the cranial mesenteric artery caused only a small, nonsignificant depression of meal size. The coeliac and cranial mesenterie arteries supply the stomach and small intestine. as well as the spleen, pancreas and gall bladder, and all of these tissues, save the stomach, are possible sites for CCK satiety action. The more caudad infusion ~ould sx~pply the kidneys, large intestine, urogenital organs and the musculoskeletal structures of the hind part of the animal. Presumably, the small variable satiety action of CCK infused by this caudal route was due to residual CCK which survived passage through those organs. During these experiments meal duration was the latency of the CCK infusion to end the meal, and it was thought that comparison of these latencies might reveal a more rapid onset of action when the site of infusion was closest to the site of action. The data are summarized in Fig. 4 for jugular, carotid and aortic (short) infusions at 67 ng/kg-mit~ute and also for 33 and 134 ng/kg-minute for jugular and carotid infusions. Although there is a dose-related effect on latency to end the meal, there was no appreciable difference between routes of infusion. However, the variability of these latency measurements make it impossible to discriminate differences less than 1 minute, and differences of circulation time would be of that magnitude. The correspondence of meal size and meal duration in Fig. 4 indicates that the effect of CCK infusion is predominantly on duration of meal rather than on rate of eating during the meal. This response of growing pigs to intestinal stimuli that reduce meal size has been noted also when the stimulus was a hypertonic solution injected into the duodenum during meals [9]. The results point to a group of tissues supplied by the coeliac and cranial mesenteric arteries (excepting the stomach and liver) as being sites for this CCK action. Two sections of the intestine are limited in size and surgically accessible: the duodenum and ileum. Simply dropping CCK solution onto the serosal surface of these parts of the intestine can cause increases in motility (personal observation; and also see [14~), and this suggests that suprafusion of CCK might also elicit satiety effects. The results were variable, but duodenal suprafusion resulted in a slight, but nonsignificant, stimulation of intake, while ileal supraftision resulted in a depression of meal size (Fig. 2). The depression of intake was not quite significant (p<0.0(i), bat in 11 expert-

698

H O U Irr

m e n t s on 4 pigs all ileal s u p r a f u s i o n s r e s u l t e d in c o n s i d e r a b l e d e p r e s s i o n o f meal size. while in 3 e x p e r i m e n t s on a fifth pig n o n e c a u s e d a d e p r e s s i o n o f intake. A significant role o f the ileum in inhibition o f i n t a k e has r e c e n t l y b e e n r e p o r t e d [111. T h e failure o f C C K d e l i v e r e d to the l u m e n o f the d u o d e n u m to influence meal size (Fig. 2) was e x p e c t e d b e c a u s e C C K is p r e s u m a b l y d i g e s t e d , and the m a t t e r was not p u r s u e d further. In s u m m a r y , the results of the e x o g e n o u s C C K infusion e x p e r i m e n t s indicate: (I) that the s t o m a c h a n d liver are unlikely to be sites o f C C K satiety a c t i o n ; (2) that the brain is p r o b a b l y not a m a j o r site for this C C K a c t i o n , a l t h o u g h a m i n o r role c a n n o t be e x c l u d e d ; a n d ~3) t h a t the p r i m a r y sites o f a c t i o n lie in t h e v a s c u l a r b e d o f t h e cranial m e s e n t e r i c a n d / o r coeliac a r t e r i e s , excluding the s t o m a c h and liver.

S u p r a f u s i o n s of C C K o n t o the serosal surface o f the d u o d e n u m and ileum were i n c o n c l u s i v e , but l h e results hint at a role o f the ileum in intestinally m e d i a t e d c o n t r o l o f meal size and call for f u r t h e r s t u d y o f this p o r t i o n o f the small intestine. Finally, it s e e m s that C C K m a y h a v e m o r e than one site o f action Ibr i n f l u e n c i n g meal size.

ACKNOWI,EI)GF.MENTS The technic,'d assistance of Arlene Sw;m and Judith A. Bigelow in currying out these experiments is gratefully acknowledged as is the advice of K. A. Houpt with the manuscript. We thank l)r. M. A. Ondetti lbr arranging for the supply of CCK-8 from Squibb Institute t~f Medical Research, F'rinceton. NJ. This research was supported in part by the U. S. D. A. Formula Funds Program.

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