Actions of centrally administered neuropeptides on rat intestinal transport: enhancement of ileal absorption by angiotensin II

European Journal of Pharmacology, 148 (1988) 411-418 Elsevier

411

EJP 50220

Actions of centrally administered neuropeptides on rat intestinal transport: enhancement of ileal absorption by angiotensin II D a v i d R. B r o w n * a n d M i c h a e l A . G i l l e s p i e University of Minnesota, College of Veterinary Medicine, Department of Veterinary Biology and Neuroscience Graduate Program, 1988 Fitch Avenue, St. Paul, MN 55108, U.S.A.

Received 21 September 1987, revised MS received 14 January 1988, accepted 26 January 1988

Recent evidence suggests that opioids and other peptides may act within the CNS to modulate intestinal fluid and ion transport. In this study, the brain peptides bombesin and angiotensin II were examined for their ability to alter water flux across the small intestine after their intracerebroventricular (i.c.v.) administration to rats. In addition, changes in mean arterial pressure and respiratory frequency were determined after peptide treatment to assess the physiological specificity of their CNS actions. Bombesin, administered by i.c.v, bolus injections (10-1000 ng/rat) or continuous infusion (100 ng/min), rapidly elevated blood pressure and respiration, but had no significant effect on water transport in proximal jejunum or distal ileum in situ (as measured by single-pass perfusion with [14C]polyethylene glycol as non-absorbed water marker). Angiotensin II rapidly increased blood pressure and enhanced ileal absorption 30 rain after its i.c.v, bolus injection at 0.1-1/zg, but had no effect on jejunal transport or respiration. These effects were inhibited in rats pretreated with either the angiotensin antagonist [Sarl,ValS,AlaS]angiotensin II (5 /~g i.c.v.) or the a-adrenoceptor antagonist, phentolamine (1 m g / k g i.v.). In contrast, atropine methylnitrate (0.1 m g / k g i.v.) pretreatment inhibited the proabsorptive, but not the pressor effects of angiotensin. These results indicate for the first time that angiotensin II promotes fluid absorption in the rat intestine by an action within the CNS. The mechanisms underlying this novel action of angiotensin appear to differ from those responsible for its hypertensive action. Bombesin; Angiotensin II; Arterial pressure (mean); CNS; Ileum: Water absorption; Respiration

1. Introduction

A c c u m u l a t i n g evidence indicates that s o m e classes of n e u r o p e p t i d e s m a y act directly at b r a i n sites to alter ion a n d water a b s o r p t i o n in the m a m m a l i a n small intestine. In the first studies of this p h e n o m e n o n , m e t a b o l i c a l l y stable e n k e p h a l i n derivatives, a d m i n i s t e r e d b y the i n t r a c e r e b r o ventricular (i.c.v.) route, were f o u n d to a t t e n u a t e

* To whom all correspondence should be addressed: Department of Veterinary Biology, University of Minnesota, College of Veterinary Medicine, 1988 Fitch Avenue, St. Paul, MN 55108, U.S.A.

or reverse fluid a c c u m u l a t i o n in the p r o x i m a l j e j u n u m elicited b y c h o l e r a toxin or p r o s t a g l a n d i n E 1 in a n e s t h e t i z e d rats (Brown a n d Miller, 1983; 1984; Q u i t o a n d Brown, 1987). These initial findings have b e e n r e p l i c a t e d in conscious dogs (Primi et al., 1986). T h e a n t i s e c r e t o r y a c t i o n of centrally a d m i n i s t e r e d o p i o i d s m a y be involved in the wellk n o w n a n t i d i a r r h e a l or c o n s t i p a t i n g activity of this d r u g class. I n c o n t r a s t to the opioids, s a l m o n c a l c i t o n i n a n d s o m a t o s t a t i n - 1 4 r e p o r t e d l y decrease fluid a n d electrolyte a b s o r p t i o n in the dog j e j u n u m d u r i n g their i.c.v, infusion, a l t h o u g h their p h y s i o l o g i c a l roles in r e g u l a t i n g intestinal transp o r t have not yet b e e n c h a r a c t e r i z e d (Primi a n d Bueno, 1986; 1987).

0014-2999/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

412

It is of considerable interest to identify and characterize the mechanisms of action of other classes of brain-gut peptides that may affect intestinal fluid and ion transport after their CNS administration. In this study, we examined the CNS-mediated actions of two structurally unrelated peptides, angiotensin II (A-II) and bombesin, on water absorption from the small intestine of urethane-anesthetized rats. The octapeptide A-II acts in the periphery and at central nervous system sites to maintain the volume and composition of the extracellular fluid (Phillips, 1987) and its systemic administration is associated with increases in intestinal salt and water absorption (Levens, 1985). Bombesin, a tetradecapeptide, alters gastrointestinal function through CNS sites of action. For example, the i.c.v, administration of bombesin decreases gastric acid secretion, gastric emptying and intestinal transit in rats (Tache et al., 1982; Porreca and Burks, 1983). Local application of bombesin stimulates active anion secretion across the rodent intestinal mucosa in vitro (Kachur et al., 1982) and i.v. infusions reduce fluid and electrolyte absorption in the canine jejunum in situ (Barbezat and Reasbeck, 1983). We monitored simultaneously the effects of each peptide on intestinal water flux, blood pressure and respiration in order to assess the physiological specificity of its actions. Our results suggest that i.c.v. A-II selectively increases water absorption in the distal small intestine of the rat.

2. Materials and methods

2.1. Animals and surgical procedures Male Sprague-Dawley rats (Biolab, Inc., White Bear Township, MN), weighing between 275-400 g, were deprived of food 18-24 h prior to anesthesia and surgery. They were anesthetized with urethane (3 g / k g s.c.) and midline laparotomy was performed to expose the small intestine. A 15-20 cm segment of the proximal jejunum (beginning 3 cm from the ligament of Trietz) or distal ileum (ending 2 cm from ileocecal junction) was isolated, rinsed free of luminal debris, ligated at its extreme ends and cannulated for intraluminal perfusion.

Care was taken to insure neural and vascular continuity in each segment. Cannulated segments were replaced into the abdominal cavity and the laparotomy incision was closed. Rats rested on heating pads in order to maintain their core temperature at 3 7 ° C throughout each experiment. Core temperature was measured via a rectal thermistor probe connected to a electronic telethermometer (Yellow Springs Instruments Model 42). The carotid artery was cannulated to permit continuous measurement of arterial blood pressure using a Statham pressure transducer connected to a Gilson Duograph chart recorder. Respiratory frequency was measured by impedance pneumography. In some experiments, a jugular vein was isolated and cannulated to permit the i.v. drug administration. Prior to i.c.v, injections, animals were positioned stereotaxically and a small hole was drilled through the skull. I.c.v. injections of peptides were made using the following coordinates relative to bregma: AP = 0.8 mm, L = 1.8 m m and V = 4.0 mm; upper incisor bar set 5 m m above the interaural line. At the end of each experiment, a 4 /~1 bolus of Evans Blue dye was administered at the injection site to verify needle placement in the ventricles.

2.2. Gut perfusion Gut segments were perfused with one of two buffered solutions which were maintained at 37 ° C and p H 7.4. In our initial experiments using bombesin, a modified oxygenated Ringer-HCO 3 solution of composition (in mmol/1): Na ÷ 142.6, K + 5.0, C1- 121.2, Mg 2+ 1.1, Ca 2+ 1.3, H C O 3 25.0, HPO42- 1.7, H 2 P O 4- 0.3 and glucose 5.0 was employed. In subsequent studies with A-II, a bicarbonate-buffered saline solution of simpler composition (in mmol/1), N a + 143, K + 5.0, C1- 123, H C O ~ 25.0 and D-glucose 5.0 was used. Both perfusate solutions also contained 5 /xCi/1 of [14C]polyethylene glycol (MW 4000) as a non-absorbed dilution marker of water absorption and 5 g/1 of unlabelled polyethylene glycol 4000 as a carrier. Each segment was perfused by single-pass at a constant rate of 22.6 ml h -1 and perfusate exiting the segment was collected over 15 min time intervals. Peptides (i.c.v. route) were administered

413

after a 60 min equilibration period and a 30 rain baseline period in which all physiological parameters stabilized. Drugs were administered i.v. 60 min before peptide treatment, although in experiments of bombesin action on blood pressure and respiration which followed those of water transport, drugs were administered 15 rain prior to i.c.v, peptide injections. 14C activity in intestinal perfusate collections was determined by liquid scintillometry and net water flux occurring over each 15 min time interval was calculated by the following formula:

(1 - DDpM tp P M x ] ' L = net fluid flux ( # l / c m × h ) where DPM S and DPM x =14C activity in standard and perfusate sample, respectively. P = perfusion rate (22 600 ttl/h), L = length of intestinal segment in cm. Water flux occurring over two successive 15 min intervals was averaged to obtained an average absorption rate over 30 min periods. Positive values for fluid flux denote increases in net intestinal absorption; negative values indicate decreases in absorption. The total 14C recovery in intestinal perfusates averaged 97 +_ 2% across all experiments, with the residual amount of 14C activity recoverable from the luminal surface of the intestine.

2.3. Drugs and peptides Bombesin (mol. wt. 1620), [Ile 5] A-II (mol. wt. 1046) and [Sarl,ValS,AlaS]A-II (mol. wt. 912; all purchased from Peninsula Laboratories, Belmont, CA) were stored as frozen aliquots in an aqueous solution containing 10 mM acetate and 0.1% bovine serum albumin (pH 7.35) and diluted to final concentrations in 0.9% saline titrated to pH 7.4 prior to injection. Control animals received the peptide vehicle diluted in pH-adjusted saline. Peptides or saline were administered i.c.v, in a total volume of 4 /~1 as a bolus or by continuous infusion (0.167 / d / m i n ) over 30 rain. Atropine methylnitrate (Sigma Chemical Co., St. Louis, MO) and phentolamine hydrochloride (Ciba-Geigy, Summit, N J) were dissolved in saline and administered at a doses of 0.1 and 1.0 m g / k g respectively.

2.4. Data analysis Net water fluxes in 15 min intervals before and after peptide administration were measured; in some cases, the results of two 15 min flux periods preceding and succeeding peptide administration were averaged to reflect water fluxes in 30 min intervals flanking peptide treatments. Peak changes in mean arterial pressure (MAP) and respiratory frequency occurring in successive 15 min intervals after peptide administration were calculated relative to preinjection equilibrated values in each rat. Data representing differences in physiological parameters occurring before and after peptide treatment within individual animals were analyzed by a paired t-test and differences between control and peptide-treated groups were analyzed by an unpaired t-test for one treatment mean and Duncan's multiple range test or Dunnett's test for multiple treatment means. The lower limit of statistical significance was set at P < 0.05.

3. Results

3.1. Effects of i. c.v. bombesin The i.c.v, administration of saline by either bolus injection or continuous infusion had negligible effects on basal water flux in either the proximal jejunum or distal ileum and produced only small and non-significant changes in mean arterial pressure (MAP) or respiration rate (RR; table 1). Bombesin given by i.c.v, bolus injections produced marked dose-related increases in both MAP and RR. In concentrations as low as 10 ng/rat, the peptide produced significant elevations in MAP relative to those observed in rats treated with vehicle (AMAP after vehicle and 10 ng bombesin, respectively was 5 + 2 and 32 + 9 mm Hg, P < 0.01, unpaired t-test). At higher concentrations, bombesin significantly increased respiration. The peptide, administered i.c.v, at 300 ng/rat, increased R R by 40 + 8 inspirations/min. In contrast, injection of vehicle increased RR by 3 + 3 inspirations/min (P < 0.01, vehicle vs. 300 ng bombesin condition, unpaired t-test). The cardiopulmonary actions of bombesin occurred within

414 TABLE 1

TABLE 3

Actions of bombesin on intestinal fluid transport after i.c.v. bolus injection. Differences in water fluxes before and after i.c.v, injections were not significant under all conditions (P > 0.05, unpaired t-test).

Changes in mean arterial pressure, respiration rate, and jejunal fluid transport during continuous i.c.v, infusion of bombesin %

Proximal jejunum

Saline Bombesin a

Pre-inj ection baseline ~

Distal ileum

Before a

After b

n ~ Before

After

n

77±13 75±18

81±10 74-+15

7 7

66-+ 8 60_+15

6 5

53-+10 45±15

a Average net water flux determined in one 15 min time interval before i.c.v, injections, b Average net water flux determined in one 15 min time interval after i.c.v, injection, c n represents number of animals tested, a Injected i.c.v, in bolus dose of 1 ~g/rat.

Saline (n=5) d Bombesin (n=5)

83+_11 78+ 4

w a s i n h i b i t e d b y t h e p r i o r i.v. a d m i n i s t r a t i o n o f the peripherally selective muscarinic acetylcholine receptor mg/kg;

antagonist

atropine

methylnitrate

(0.1

t a b l e 2). I.c.v. i n f u s i o n s o f b o m b e s i n a t

TABLE 2 Effects of atropine methylnitrate on bombesin-induced changes in mean arterial pressure and respiratory rate. n rats

Pre-injection baseline

A after drug/ peptide administration a

30

45min

4+2 25+8 g

1_+ 4 23+ 9 g

-5+

3

16_+ 7 g

Respiratory rate (breaths/min) Saline (n=4-5) Bombesin (n=5)

101_+10 99_+13

1_+3

3_+ 6

16+ 4

15_+3 g

36-+ 9 g

51±15

Jejunal water flux ( ~ l / c m per h)

T h e e f f e c t o f i.c.v, b o l u s i n j e c t i o n s o f b o m b e s i n or R R , b u t n o t M A P

15

Mean arterial pressure (mm Hg)

15 m i n o f its a d m i n i s t r a t i o n a n d r a p i d l y s u b s i d e d . (300 n g o r 0.19 n m o l / r a t )

Change after start of infusion b

Saline (n=6) Bombesin (n-6)

Average flux e

Change in flux f

63+ 6

8_+10

64_+ 8

4+12

Infused at rate of 100 ng/rat per min i.c.v, for 30 min. b Means +_S.E. of changes in average MAP and RR at each time point relative to pre-injection baseline value. ~Average values determined within 30 min before start of infusion, a n indicates number of rats tested in each condition, e Means ± S.E. of transport rate averaged over two 15 min periods prior to infusion, f Means +_S.E. of changes from averaged 30 rain transport rates before and during i.c.v, infusion, g P < 0.05 between means of saline- vs. bombesin-treated animals (unpaired t-test). a

Mean arterial pressure (mm Hg) Vehicle Bombesin b Atropine c Atropine plus bombesin d

11 7 6

88_+ 5 77 ± 5 82 _+ 6

5_+1 26_+ 6 e -- 6 ± 3

4

90 -+ 8

22 ± 2 ¢

Respiratory rate (breaths/min) Saline Bombesin b Atropine c Atropine plus bombesin a

11 5 6

110 ± 8 84 ± 4 103± 7

3± 3 40 ± 7 ¢ --2_+4

100 n g / m i n produced MAP

and

p e r r a t o v e r a 30 m i n p e r i o d a l s o

w i t h i n 15 m i n s i g n i f i c a n t i n c r e a s e s in RR

in c o m p a r i s o n

to saline-infused

c o n t r o l s ( t a b l e 3). I n c o n t r a s t , n e i t h e r b o l u s i n j e c t i o n s o v e r a w i d e d o s e r a n g e (3-3000 n g / r a t ; d a t a n o t s h o w n ) n o r t h e c o n t i n u o u s i.c.v, i n f u s i o n o f bombesin

significantly

altered

intestinal

fluid

t r a n s p o r t ( t a b l e s 1 a n d 3). 4

88 ± 12

5± 5

a Difference in values obtained in each rat measured for 15 min before and after drug or peptide administration. b Bombesin administered at 300 ng/rat in an i.c.v, bolus. CAtropine methylnitrate administered at 0.1 mg/kg, d Atropine methylnitrate administered at 0.1 mg/kg i.v. 15 rain prior to i.c.v, bombesin injection. ¢ P < 0.01 relative to change in saline-treated animals after drug or peptide administration; Dunnett's test.

3.2. Effects o f i.c.v. A - H T h e i n j e c t i o n o f A - I I (1 ~ g o r 0.96 n m o l / r a t ) produced a rapid, statistically significant elevation i n M A P w i t h 15 m i n a f t e r its i n j e c t i o n , a n e f f e c t w h i c h quickly s u b s i d e d in s u b s e q u e n t time interv a l s ( a v e r a g e M A P b e f o r e a n d in f i r s t 15 m i n a f t e r

415

[]

100

E

Jejunum Jm

80 co

60 O o~ ..Q

,'3-

40 20

~0

m

0 -30-0 0-30 Time after i.cv. A-II injection (min)

Fig. 1. Action of angiotensin II (1 f i g / r a t i.c.v.) on fluid absorption from the proximal jejunum and distal ileum perfused in situ in urethane-anesthetized rats. Bars represent the means -+ S.E. of the rate of net water absorption in each intestinal segment 30 min before (depicted as - 3 0 - 0 ) and 30 rain after (depicted as 0-30) peptide administration in 7 (ileum) and 8 (jejunum) rats. * P < 0.05 vs. pre-injection ( - 30-0 min) value; paired t-test.

A-II injection: 84.8 +_ 5.2 and 99.4 _+ 4.8 mm Hg, P < 0.01, paired t-test). The peptide at this dose had no significant effects on respiration at any time point after its injection. A-II increased the basal net absorption of water from the lumen of the distal ileum, but not the proximal jejunum in the first 30 min after its administration (fig. 1). This ileal proabsorptive action of the peptide was dose-dependent over a 10-fold dose range (fig. 2). The specific A-II antagonist analog [Sarl,Val 5, Ala s] A-II did not significantly affect either blood pressure or ileal absorption when administered

TABLE 4 Effects of receptor blockers on ileal proabsorptive and pressor actions of i.c.v, angiotensin II (1 #g). A after administration a

nb

13+ 4

16

16-+ 5 5 -+ 10

4 6

Ileal net water flux (btl/cm per h) Saline, i.c.v. + [Sarl,ValS,AlaS]angiotensin If, 5/~g, i.c.v. + phentolamine, 1 m g / k g , i.v. + atropine methylnitrate, 0.1 m g / k g i.v. Angiotensin II +[Sarl ValS,Ala8I_A_II d + phentolamine +atropine

50 el

E 40 .o O

" 20 ,'7" m

3 9e 7 7 4

5 9 5 6 10

Zl after administration e

n

0+ 1 - 3 _+ 4 - 2 _+ 2 4+ 2 15 5 : 4 e 2+ 2 3+ 1 12+ 3 e

14 4 6 7 10 5 8 12

Mean arterial pressure (rnm Hg)

*'1 10

0

4_+ 44 -+ 13_+ 6 -+ 9-+

I

Saline

0.1

0.5 A-II,

ug

1.0 i.cv.

Fig. 2. Dose-effect relationship for angiotensin II (A-II) in elevating net fluid absorption from the rat ileum perfused in situ. Points represent the means + S.E. of differences in ileal fluid absorption averaged over 30 rain before and after the i.c.v, injection of saline or A-II in 8-12 animals. Significant differences between pre- and post-injection transport values in each rat under each experimental condition are indicated as * P < 0.05 and * * P < 0.01 (paired t-test).

Saline, i.c.v. + [Sarl,ValS,AlaS]_A_ii a + phentolamine +atropine methylnitrate Angiotensin II +[Sarl,ValS,AlaS]_A_ii d + phentolamine +atropine

Represents m e a n s + S.E. of differences in the average rate in ileal transport during 30 min intervals before and after saline, peptide or drug injection, b N u m b e r of animals tested, c Represents means_+ S.E. of differences in average blood pressure determined 15 min before and after saline, peptide or drug administration, d Drug and peptide doses are the same as above. Significant differences between saline control mean (i.e. saline i.c.v.) and all other treatment means are indicated as e p < 0.01 as determined by Duncan's multiple range test. a

416 alone, but inhibited both actions of A-II when administered i.c.v, immediately prior to A-II injection (table 4). The ileal proabsorptive action of A-II was also inhibited in rats pretreated i.v. with either atropine methylnitrate (0.1 m g / k g ) or the a-adrenoceptor blocker phentolamine (1.0 mg/kg; table 4). The centrally mediated pressor effects of the peptide were also inhibited by phentolamine pretreatment, but were not significantly altered by atropine methylnitrate administration (table 4).

4. Discussion

The present experiments were designed to test the hypothesis that bombesin and A-II act at brain sites to alter water flux in the rat intestinal tract. Our results indicate for the first time that A-II appears to promote fluid absorption in the lower small intestine upon i.c.v, administration in pmol concentrations. This phenomenon is consistent with other CNS actions of the peptide which are directed towards the maintenance of extracellular fluid volume (Phillips, 1987). Moreover, A-II joins a growing list of neuropeptides which seem to alter intestinal transport processes through interactions at sites within both the gut and brain. Among its many CNS-mediated actions, the brain-gut peptide bombesin produces rapid, but long-lasting increases in respiration in rats and cats (Holtman et al., 1983; Hedner et al., 1985) and elevations in mean arterial pressure in pithed and freely moving, conscious rats after its i.c.v. administration in nmol concentrations (Fisher and Brown, 1984; Bayorh and Feuerstein, 1985). The respiratory stimulation produced by the peptide consists mainly of an increase in tidal volume and is abolished by cervical vagotomy (Hedner et al., 1985). As impedance pneumography was employed in the present investigation, bombesin-induced changes in tidal volume could not be determined specifically. Nevertheless, bombesin appeared to rapidly increase the rate of respiration at i.c.v, doses as low as 30 ng (19 pmol). This effect was completely blocked by prior administration of the peripherally acting acetylcholine receptor antagonist atropine methylnitrate, sug-

gesting that peripheral cholinergic neurons, possibly in the vagus, contribute to the respiratory stimulant effects of the peptide. In contrast, the pressor action of i.c.v, bombesin was not affected by atropine methylnitrate, as result in agreement with that of a previous study utilizing conscious rats (Fisher and Brown, 1984). Thus, the respiratory stimulant and pressor activities of bombesin appear to be mediated through different physiological mechanisms. Bombesin has generally been found to increase blood pressure in conscious animals after i.c.v, injection, although at least one research group has failed to observe this action (Fisher and Brown, 1984; Bayorh and Feuerstein, 1985; Hedner et al., 1985). It has been reported that i.c.v, bombesin infusion has no effect on MAP in urethane-anesthetized rats, a finding at variance with the present results (Brown and Tache, 1981). This discrepancy may possibly stem from differences in the level of urethane anesthesia employed in the two studies; indeed, the dose of urethane and its route of administration can affect resting blood pressure and pressor responses to drugs (Maggi and Meli, 1986). Despite its prominent pressor and respiratory stimulant activities, neither i.c.v, bolus injections nor continuous infusion of bombesin appeared to affect basal water flux in the small intestine. In pilot studies, the peptide also did not alter luminal fluid accumulation produced by the i.v. infusion of prostaglandin El, suggesting that it does not possess intestinal antisecretory activity which might have been obscured under basal conditions of high fluid absorption. From these results, we tentatively conclude that bombesin does not act at periventricular brain sites to alter small intestinal fluid transport. There is convincing evidence that A-f1 subserves a neurotransmitter role in the CNS (Phillips, 1987). The administration of A-II via CNS routes has been associated with increases in water and salt consumption (Fitzsimons, 1979), hypertension (Bickerton and Buckley, 1961) and hypophyseal vasopressin release (Keil et al., 1975). In this investigation, A-II rapidly increased fluid absorption from the rat ileum after its i.c.v, injection. This response was inhibited by prior i.c.v. administration of the selective angiotensin

417

antagonist, [Sarl,ValS,AlaS]A-II indicating that it is mediated by CNS A-II receptors. This proabsorptive action of A-II differs from that previously reported to be produced by i.c.v, enkephalins with respect to the intestinal region affected. Opioids enhance basal fluid absorption from isolated loops of the rat jejunum and medial ileum, but have no effect on transport in the distal ileum (Brown and Miller, 1983). Moreover, enkephalin analogs such as [D-Ala2,MetS]enkephalinamide, given by the i.c.v, route, reduce the anti-absorptive or prosecretory actions of cholera toxin or prostaglandin E 1 in the upper small intestine (Brown and Miller, 1984; Quito and Brown, 1987). The intestinal transport effects of A-II and enkephalins are attenuated significantly in rats pretreated with phentolamine, indicating an involvement of peripheral catecholamines in their activity. Norepinephrine is known to enhance the absorption of fluid and ions from the small intestine (Tapper, 1983) and the CNS antisecretory actions of enkephalin analogs appear to be mediated through the release of norepinephrine from sympathetic nerves projecting to the jejunum (Brown and Miller, 1984). It is, however, unclear in the present study whether A-II either activates the gut sympathetic innervation or evokes the release of catecholamines from the adrenal medulla to produce its intestinal action. In the latter case, increases in circulating levels of catecholamines might be expected to enhance absorption in both the jejunum and ileum. As A-II appears to have a rather specific action in the distal small intestine, this possibility is an unlikely one. Moreover, the finding that i.c.v. A-II increases absorption in an intestinal region different from that affected by opioids suggests that the former peptide does not produce gut effects secondary to the release of brain enkephalins. We have not, in the present study, attempted to ascertain whether A-II administered into the peripheral circulation could act at brain sites to enhance absorption, although experiments are in progress to address this possibility. Nevertheless, it is tempting to speculate that the opioids and A-II present within the CNS act at discrete brain sites having different neural projections to the intestinal tract. In addition to its proabsorptive effects, A-II

was found to produce a small increase in MAP after its i.c.v, injections. Hypertensive responses to i.c.v. A-II injections are well documented and have been attributed to interactions of the peptide with A-II receptors in the circumventricular organs which modulate sympathetic nervous activity and vasopressin release (Phillips, 1987). Both the intestinal and pressor actions of A-II were inhibited after blockade of a-adrenoceptors by phentolamine. It might be inferred from this result and the similar times of onset for the two activities that A-II-elicited increases in absorption might be directly linked to MAP elevations. Two lines of evidence do not support this conclusion. First, our results indicate that bombesin is even more effective than A-II in increasing blood pressure, yet is ineffective in altering intestinal transport. This finding suggests that MAP elevations alone cannot account for changes in intestinal water absorption. Second, atropine methylnitrate inhibits A-II-induced absorption, but does not affect the increase in MAP produced by the peptide. Thus, it appears that blockade of peripheral muscarinic acetylcholine receptors by atropine dissociates these two A-II-mediated actions. Although we have not examined the possible involvement of vasopressin in A-II actions on intestinal absorption and blood pressure, the complete inhibition of ileal proabsorptive responses to A-II observed in rats pretreated with either phentolamine or atropine suggests that this action is not hormonally mediated. Nevertheless, we cannot exclude an A-II-evoked vasopressinergic component in the gut responses to i.c.v. A-II at present. We can, however, hypothesize at this early stage of investigation that the CNS actions of A-II on blood pressure and ileal absorption are produced through different mechanisms. Psychological factors, such as emotional stress or depression, have been implicated in the pathogenesis of several functional disorders of the intestinal tract, including irritable bowel syndrome, certain forms of chronic diarrhea, and idiopathic constipation (Clouse and Alpers, 1986). Although it is clear that disturbances in intestinal motor function underlie many of these disorders, alterations in intestinal fluid and electrolyte transport may also play a role in their expression.

418

Moreover, exposure of human subjects to auditory stressors has been reported to decrease jejunal salt and water absorption (Woodmansey et al., 1983; Barclay and Turnberg, 1987). These findings, in concert with the present results, offer a suggestion that sites within the CNS may participate in the extrinsic neural control of fluid transport in discrete regions of the intestinal tract. The precise brain areas involved and the interactions of A-II and other CNS peptides with them remain to be elucidated.

Acknowledgement This work was funded by U.S.P.H.S. Grant DK-35260.

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