Proc. Natl. Acad. Sci. USA Vol. 77, No. 5, pp. 2753-2756, May 1980 Cell Biology

Control of guinea pig intestinal electrolyte secretion by a 5- receptor (/short circuit current/intestinal mucosa/chloride transport) JAMES F. KACHUR*, RICHARD J. MILLERt, AND MICHAEL FIELD* Departments of *Pharmacological and Physiological Sciences and Medicine, University of Chicago, Chicago, Illinois 60637 Communicated by Josef Fried, February 8, 1980

ABSTRACT The effects of on transepithelial po- are known to exist. For , four receptor types have been tential difference and short-circuit current across guinea pig suggested. These have been designated K, 6, and a. Of these ileum stripped of one muscle layer were measured in vitro in A,. Ussing chambers. peptides such as [DA6a2, DLeu5Jen- four, the properties of u and 3 receptors are best defined. kephalin and [DAla2, DMet5Jenkephalin, which are primarily Typical for the ju receptor include ; for the agonists at -opiate receptors, were able to reduce transepi- a receptor, agonists include [Leu5]enkephalin and some of its thelial potential difference and short-circuit current at con- stable analogues. Some agents (e.g., f3-endorphin and etorphine) centrations as low as 1 nM. The drug etorphine was also appear to have appreciable effects on both types of receptor. very potent in reducing short-circuit current, but and In the present series of studies we have found that only opiates morphine, which are primarily agonists at -opiate receptors, were almost complete y ineffective. Ketocyclazocine was rel- and opioid peptides that have effects on the 3-opiate receptor atively ineffective, and P-endorphin had intermediate potency. are able to produce antisecretory effects in guinea pigs. All opioid effects could be reversed by the opiate antagonist . Somatostatin also reduced short-circuit current, but METHODS AND MATERIALS its effect was not reduced by naloxone. Chloride flux measure- ments indicated that the effect of etorphine on short-Circuit Materials. Synthetic somatostatin was a gift of J. E. Shields current is associated with an enhancement of active Cl- ab- (Lilly). f3-Endorphin was obtained from Peninsula Labs (San sorption. The relative effects of opioids in this system suggest Carlos, CA). Naloxone, morphine, etorphine, fentanyl, and that their actions are being mediated by a specific 5-opiate re- ketocyclazocine were given by Bruce Wainer (Department of ceptor. In contrast, opioid effects on guinea pig intestinal smooth muscle seem to be primarily mediated by a i-opiate Pediatrics, University of Chicago). Atropine was obtained from receptor. Sigma; [Met5]enkephalin, [Leu5]enkephalin, and enkephalin analogues were gifts from Sam Wilkinson (Wellcome, Beck- Opiates and opiate-like drugs such as and lop- enham, Kent, England). 36CI (6 mCi/g) was obtained as NaCl eramide are widely used in the treatment of diarrhea (1-4). from New England Nuclear. Opiates are generally believed to produce their antidiarrheal Methods. Female Hartley guinea pigs weighing between 500 action by stimulating intestinal circular muscle, thereby im- and 600 g were killed by cervical dislocation and the terminal peding the progress of material through the gut (5-7). In ad- 8 cm of ileum was quickly cut open along the mesenteric bor- dition, in the guinea pig myenteric plexus, opioids also decrease der, rinsed clean of intestinal contents, and placed in oxygen- the stimulated release of acetylcholine from cholinergic neu- ated ice-cold Ringer's solution. After the serosa and the rons. This action is the basis for a well established pharmaco- underlying longitudinal muscle layer were stripped off, tissues logical assay for opiates (8). The recent discovery of the en- were mounted between two half chambers, the exposed area dogenous opioid peptides, the , has provided a being 0.64 cm2. Mucosal and serosal surfaces were bathed in neuroanatomical basis for much of the classical pharmacology a HCO3-/Ringer's solution, maintained at 370C, and gassed of the opiates (9). In particular, both [Met5]enkephalin and with 5% CO2 in 02 (pH 7.4). The ionic composition of the [Leu5]enkephalin have been found in intestinal enteric ganglia Ringer's solution in mmol/liter was: Na+, 141; K+, 5; Ca 1.25; and in endocrine cells of the gut mucosa, suggesting a physio- Mg2+, 1.1; Cl-, 125; HCO3-, 25; H2PO4-, 0.3; and HP042-, logical role for the enkephalins in the gastrointestinal tract 1.65. The apparatus for measuring transmural electrical po- (10-12). One possibility is that they act as neurotransmitters and tential and short-circuit current (Isc) was similar to that previ- influence intestinal motility. Another possibility is that the ously described (13, 14). Unidirectional and net Cl- fluxes were enkephalins may have "paracrine" effects on the intestinal measured under short-circuit conditions as described (14). mucosa. Enkephalin released locally from endocrine cells or Fluxes were calculated from least-squares regression lines for neuronal elements might act upon the intestinal mucosa to total accumulated radioactivity against time. Four 10-min regulate the release of other bioactive substances or, more di- samples were taken beginning 10 min after addition of 36C1. rectly, the transport of ions across the mucosa. We have recently demonstrated that somatostatin may modulate electrolyte RESULTS transport across rabbit ileal mucosa (13). The present paper demonstrates that opioids have an antisecretory effect on guinea The transepithelial potential difference (PD) reached a plateau pig mucosa. At the present time there is considerable discussion of about 6 mV (serosa positive) after about 30 min in vitro. In as to the possible existence of multiple opiate receptors. This some tissues the PD then declined slowly over the next hour. would be analogous to the situation with acetylcholine, for ex- The effects of various opioids and other substances were assayed ample, where both nicotinic and muscarinic types of receptors as soon as the plateau was reached. As illustrated in Fig. 1, the PD rapidly decreased when a low concentration of the en- The publication costs of this article were defrayed in part by page kephalin analogue [DA6a2, DLeu5]enkephalin (6-receptor charge payment. This article must therefore be hereby marked "ad- vertisement" in accordance with 18 U. S. C. §1734 solely to indicate Abbreviations: PD, potential difference; I,,, short-circuit current. this fact. t To whom reprint requests should be addressed. 2753 Downloaded by guest on October 5, 2021 2754 Cell Biology: Kachur et al. Proc. Natl. Acad. Sci. USA 77 (1980)

[DAla2, DLeU5]_ [DAIa2, DLeu5]- enkephalin Somatostatin enkephalin 5- 4. E I 8- 4- X 0 3 .c E 7. 2 3- a- 6- :._ 1I 2- 5- 1- Atropine 0 coC 4- Naloxone c] -1* Naloxone 0- 0 0 0 0 5 10 0 5 10 0 10 Time, min FIG. 1. Effects of a stable enkephalin analogue, [DAla2, DLeu5]- enkephalin, and somatostatin on PD across guinea pig ileal mucosa. -9 -8 -7 -6 -5 (Left) Effect of [DAla2, DLeu5]enkephalin (1 ,qM). (Center) Inhibition log [drug] of the effect of [DAla2, DLeu5jenkephalin (10 MM) by naloxone (50 ,uM). (Right) Inhibition of transepithelial PD by somatostatin (0.5 FIG. 3. Effect of increasing concentrations of different opioid peptides on ISC in guinea pig ileum. Point and brackets are means and ,uM); note lack of effect of atropine (1 ,uM) or naloxone (1 ,M). SEM of 3-10 experiments. 0, [DAla2, DMet5]enkephalin; 0, f3-en-

dorphin; A, [Leulenkephalin. specificity) was added to the serosal bathing medium. Although PD changes are shown in Fig. 1 rather than ISC, the latter is by naloxone. With respect to narcotic drugs, however, morphine equal to the PD divided by the tissue resistance. Because the (,-receptor specificity) proved relatively ineffective, greater agents tested caused a less than 10% change in tissue resistance, than 10 ,uM being required to produce any significant change the shapes of dose-response relationships are essentially the in the PD across tissues that showed large responses to the same whether PD or IC is plotted. The effect of the stable en- subsequent addition of [DAla2, DLeu5]enkephalin (Figs. 2 and kephalin analogue (15) was rapidly reversed by addition of the 4). The potent opiate , fentanyl (,-receptor specificity), opiate antagonist naloxone (Fig. 1). Naloxone produced no ef- was also relatively ineffective (Fig. 4). Ketocyclazocine (K- fects when added by itself. Somatostatin also caused a rapid receptor specificity) was more effective than fentanyl or decrease in Is, (AIsc = 35 IA/cm2 at 0.5 ,uM somatostatin), as morphine (Fig. 4), but only at concentrations several orders of previously noted in rabbits (13). However, the effect of 0.5 ,uM magnitude higher than those required for its effect in the guinea somatostatin was not reversed by 1 ,uM naloxone. Atropine (1 pig ileum/myenteric plexus/smooth muscle preparation (Table ,uM) did not alter the responses of the tissue to either [DAla2, 1). Opiate-mediated effects in this latter preparation are mainly DLeu5]enkephalin or somatostatin. [DAla2, DMet5]Enkephalin mediated through ,u- and K-opiate receptors. In contrast to also produced effects similar in magnitude and potency to those morphine, etorphine (,t and specificity) was even more potent produced by [DAla2, DLeu5]enkephalin (Fig. 2). (3-Endorphin in decreasing PD and ISC than were the stable enkephalin ana- (,u and 6 specificity) decreased transepithelial PD but was less logues (Fig. 4). The action of etorphine and also of the other less potent than either of the stable enkephalin analogues (Fig. 3). effective could be reversed by naloxone. Unlike the effects of [DAla2, DMet5]enkephalin, the effects of In order to examine the ionic basis for the observed decrease [Leu5]enkephalin and [Met5]enkephalin were frequently in IC, we measured unidirectional Cl- fluxes across the short- transient. Rapid metabolism of the latter compounds may limit circuited intestine in the presence and absence of 1 ,uM etor- their effects. This has been observed in many other cases in phine (Table 2). In control tissues there was a net Cl- secretion which the effects of the enkephalins have been examined (9). of -1.2 ,ueq-cm-2*hr-'; etorphine reversed this secretion to a We were unable to enhance the effect of [Met5]enkeph~in or mean net absorption of +0.6 geq-cm-2-hr-1. The effect of [Leu5]enkephalin with bacitracin (100 ,ug/ml), which protects etorphine on ISC appears to be somewhat less in Table 2 than that enkephalins from degradation in binding assays in vitro (15). shown in Fig. 4 because of the tendency for the control tissue All effects produced by the opioid peptides could be reversed

40 cY NE 0E 50- I 30 Z3. <. 40 < c 30 20 0 co' 20- 0) M10

0 10- a 0

I

-10 -9 -8 -7 -6 -5 log [drug] 0 -10 -9 -8 -7 -6 -5 log [drug] FIG. 2. Effect of increasing concentrations of two stable en- kephalin analogues on IS, in guinea pig ileum. The effect of morphine FIG. 4. Effect of increasing concentrations of different narcotics is shown in comparison. Points and brackets are means and SEM of on ISC in guinea pig ileum. Points and brackets are means and SEM 3-10 experiments. 0, [DAla2, DLeu5]enkephalin; 0, [DAla2, DMet5]- of 3-10 experiments. 0, Etorphine; O, ketocyclazocine; A, fentanyl;

enkephalin; A, morphine. A, morphine. Downloaded by guest on October 5, 2021 Cell Biology: Kachur et al. Proc. Natl. Acad. Sci. USA 77 (1980) 2755 Table 1. Comparison of mean inhibitory concentrations of three of active solute absorption. The effects of a agonists, somatos- opioids in inhibiting the contractions of electrically stimulated tatin, and the opioids are essentially the same, suggesting a guinea pig ileum and in suppressing guinea pig ileal ISC common mode of action. The molecular basis for these ion Mean inhibitory transport changes is presently unknown. A decrease in cytosol concentration, M Ca2+ may be responsible; Ca2+ is a secretory stimulus in the Ileum Ileum ileum (18) and, in the case of somatostatin at least, changes in Receptor smooth transepi- concentrations of cyclic AMP and cyclic GMP have not been Opioid specificity muscle* thelial Ict observed (13). In a preliminary report, has been shown mucosa. Ketocyclazocine K 1.5 X 10-9 4 X 10-6 to enhance Na+ and Cl- absorption by rabbit ileum [DAla2, DLeu5]Enkephalin a 5.5 X 10-8 8 X 10-9 These effects were reversible by naloxone (19). Morphine As 1.5 X 10-7 >10-4 Currently, there is considerable debate over the possibility that multiple types of opiate receptors may exist (20,21). Four * Data courtesy of A. Killian (Department of Pediatrics, University have been these K, (, and of Chicago) (n = 3-5). receptor subtypes suggested, being ,, t This 6. In the guinea pig ileum myenteric plexus/smooth muscle study. preparation, the modulation of acetylcholine release by opioids appears to be mediated mainly by ,u receptors (and possibly K to decline spontaneously during the 40 min period of flux receptors) (22, 23). Thus, morphine (it specificity) and keto- measurement. (K specificity) are relatively potent in this tissue, whereas [DAla2, DLeu5]enkephalin (a-receptor specificity) is DISCUSSION relatively weak. In the mouse vas deferens, however, the op- Two main points of interest emerge from the present series of ioid-induced modulation of noradrenaline release appears to experiments. The first is that opioids may alter ion transport in be mediated mainly by 6 receptors (20, 22, 23). Thus, [DAla2, the small intestine and the second is the pharmacological DLeu5]enkephalin (3-receptor specificity) is very effective and specificity of this effect. In general, the constipating effect of morphine (,a-receptor specificity) relatively weak. From the opiates has been attributed to their stimulating effects on in- use of these tissues and also receptor-binding assays in brain and testinal circular muscle (5-7). Such actions lead to increased clonal cell lines in culture, the relative potency of opioids should segmentation of the gut which disrupts the overall propulsive be [DAla2, DLeu5]enkephalin > f3-endorphin > morphine for movements required for the intestinal transit of material. This a a receptor (22, 24, 25). Thus, in the mouse vas deferens, which action may be mediated by a serotoninergic mechanism (7). In is thought to be relatively rich in 3 receptors, the relative guinea pigs an additional finding is that opiates can reduce the potencies of these agents are 29.0/0.32/0.03 (22). Etorphine, field-stimulated release of acetylcholine from myenteric neu- an narcotic, appears to have high affinity for both 3 rons (8). The present results suggest, however, that in some cases and ,t receptors (25). In addition, the related antagonist, di- the antidiarrheal effects of opiates may be attributable, at least prenorphine, appears more effective than naloxone as an an- in part, to inhibition of active secretion. This may not be the case tagonist in the mouse vas deferens (26). Morphine and fentanyl for opiates such as morphine, which have very little effect in may be predicted to have low affinity for the 6 receptor (24). the present system. However, there is some evidence that cer- It is therefore remarkable that, of all the narcotics tested, only tain antidiarrheal agents of the opiate type may produce anti- etorphine was effective in the present study. The almost com- secretory effects. This is true for , for example, plete ineffectiveness of morphine and fentanyl suggests that which is effective in reducing the secretory effects of prosta- they have a low affinity for the receptor-mediating opioid ef- glandins in some circumstances (16). It is not known whether fects in the present studies. The true potencies of [Leu5]en- or not opioids can produce effects on ileal electrolyte transport kephalin and [Met5]enkephalin themselves are impossible to in humans as reported here in guinea pigs. However, if this does judge because their effects are generally transient, suggesting prove to be the case, then this suggests a novel pharmacological rapid metabolism by the tissue. This observation is in keeping strategy for producing antidiarrheal agents-i.e., opioids with with previous observations in other tissues (15). The relative a 3-opiate action (see below) that do not easily enter the potency series found in the present study-i.e., [DAla2, brain. DLeu5]enkephalin, [DAla2, DMet5]enkephalin, and etorphine Based on the results in Table 2, the effects of opioids on ISC > f3-endorphin >> morphine and fentanyl-seems to fit in with can be attributed to suppression of the net Cl- secretion. These predicted potencies for the 6 receptor and contrasts to that in effects are similar to those previously demonstrated in rabbit the guinea pig ileal myenteric plexus/smooth muscle prepa- ileum with a-adrenergic agonists (17) and somatostatin (13). ration. It therefore appears that the opiate receptor mediating In rabbit ileum, enhancement of active Na+ absorption and changes in ion fluxes across the guinea pig ileal mucosa is quite inhibition of HCO3 secretion were also observed. In view of different from that mediating the opioid-induced modulation the similarities between rabbits and guinea pigs in the measured of acetylcholine release in the myenteric plexus. changes in Cl- fluxes and IS, it is very likely that these other Enkephalin-containing endocrine cells have been observed changes also occur in guinea pig ileum. The overall impact of in the intestinal mucosa of many species, including humans (10). these several ion transport changes is a marked enhancement It is interesting to speculate that modulation of intestinal transport may represent a paracrine action of enkephalin re- Table 2. Effect of 1 IAM etorphine on chloride fluxes leased from such cells. It therefore seems as though two types of opiate receptor may exist in the guinea pig ileum. The first JS-m jcm-s JnCt Isc type appears to modulate the release of acetylcholine from Control 8.6 + 0.60 7.4 + 0.48 -1.2 + 0.25 3.3 + 0.30 neurons and has u-receptor specificity. The second type, pos- Etorphine 7.8 + 0.60 8.4 i 0.81 +0.6 I 0.44* 2.6 + 0.29* sessing 6-receptor specificity, may exist on mucosal cells and Values are Aeq./hr-l.cm-2 i SEM for eight paired experiments. modulate intestinal electrolyte transport. Fluxes were measured over a 40-min period beginning 10 min after addition of etorphine. We thank Mr. Tony Killian (Department of Pediatrics, University * P < 0.05 compared to control. of Chicago) for experiments on the guinea pig myenteric plexus/ Downloaded by guest on October 5, 2021 2756 Cell Biology: Kachur et al. Proc. Natl. Acad. Sci. USA 77 (1980)

smooth muscle preparation. This work was supported by National 13. Guandalini, S., Kachur, J. F., Miller, R. J., Smith, P. & Field, M. Institutes of Health Grants DA-02121 and AM-21345. J.F.K. was (1980) Am. J. Physiol. 238,67-74. supported by Mental Health Training Grant 1-T32MH 1442704. 14. Field, M., Fromme, D. & McColl, I. (1971) Am. J. Physiol. 220, 1388-1396. 1. Burks, T. F., Castro, G. A. & Weisbrodt, N. W. (1976) in Opiates 15. Miller, R. J., Chang, K.-J., Cuatrecasas, P. & Wilkinson, S. (1977) and Endogenous Opioid Peptides, ed. Kosterlitz, H. (Elsevier/ Biochem. Biophys. Res. Commun. 74,1311-1318. North Holland, Amsterdam), pp. 369-376. 16. Karim, S. M. & Aduikan, P. G. (1977) Prostaglandins 13, 2. Reynolds, A. K. & Randall, L. 0. (1957) Morphine and Allied 321-326. Drugs (Univ. of Toronto Press, Toronto). 17. Field, M. & McColl, I. (1973) Am. J. Physiol. 225,852-857. 3. Bass, P., Kennedy, J. A., Wiley, J. N., Villereal, J. & Butler, D. E. 18. Bolton, J. E. & Field, M. (1977) J. Membr. Biol. 35, 159-173. (1973) J. Pharmacol. Exp. Ther. 186, 183-198. 19. Racusen, L. C., H. J. & Dobbins, J. W. (1978) Castroen- 4. Stahl, K. D., Van Bever, W., Janssen, P. & Simon, E. J. (1977) Eur. Binder, J. Pharmacol. 46, 199-205. terology 74, 1081A (abstr.). 5. Burks, T. F. & Long, J. P. (1967) J. Pharmacol. Exp. Ther. 158, 20. Lord, J. A. H., Waterfield, A., Hughes, J. & Kosterlitz, H. W. 264-274. (1977) Nature (London) 267,495-499. 6. Burks, T. F. & Grubb, M. N. (1974) J. Pharmacol. Exp. Ther. 191, 21. Martin, W. R., Eades, L. G., Thompson, J. A., Huppler, R. E. & 518-526. Gilbert, P. E. (1976) J. Pharmacol. Exp. Ther. 197,517-532. 7. Burks, T. F. & Long, J. P. (1967) J. Pharmacol. Exp. Ther. 156, 22. Kosterlitz, H. W. (1979) in Mechanisms of Pain and 267-274. Compounds, eds. Beers, R. F. & Bassett, E. G. (Raven, New 8. Kosterlitz, H. W. & Robinson, J. A. (1957) J. Physiol: 136, York), pp. 707-716. 249-262. 23. Hutchinson, M., Kosterlitz, H. W., Leslie, F. M., Waterfield, A. 9. Miller, R. J. & Cuatrecasas, P. (1979) in Vitamins and Hormones, A. & Terenius, L. (1975) Br. J. Pharmacol. 55,541-546. ed. Munson, P. (Academic, New York), Vol. 36, pp. 297-382. 24. Chang, K.-J., Miller, R. J. & Cuatrecasas, P. (1979) Mol. Phar- 10. Alumets, J., Hakanson, R., Sundler, F. & Chang K.-J. (1978) macol. 14, 961-972. Histochemistry 56,187-196. 25. Chang, K.-J. & Cuatrecasas, P. (1979) J. Biol. Chem. 254, 11. Furness, J. B. & Costa, M. (1980) Neuroscience 5, 1-21. 2610-2620. 12. Linnoilla, I., DiAugustine, R., Miller, R. J., Chang, K.-J. & Cua- 26. Waterfield, A. A., Smokcum, R. W. J., Hughes, J., Kosterlitz, H. trecasas, P. (1978) Neuroscience 3, 1187-1196. W. & Henderson, G. (1977) Eur. J. Pharmacol. 43, 107-116. Downloaded by guest on October 5, 2021