Peptidergic (Enkephalin) innervation of the mammalian esophagus

GASTROENTEROLOGY

76732-737, 1966

Peptidergic (Enkephalin) Innervation of the Mammalian Esophagus R. UDDMAN, B. WALLES

J. ALUMETS,

R. HAKANSON,

F. SUNDLER, and

Department of Otolaryngology, University Hospital, Malmo, and Departments of Histology and Pharmacology, University of Lund, Lund, Sweden

Enkephalin-like immunoreactivity was demonstrated in nerves in the esophagus of guinea-pig, opossum, cat, pig, monkey, and humans. Immunoreactive nerves were found in the external muscle layer and in the muscularis mucosae. In the myenteric plexus immunoreactive nerve fibers were numerous; immunoreactive nerve cell bodies were observed only occassionally. Enkephalin nerves appeared early (the 7th wk of gestation) in porcine fetuses; at this stage the nerves were confined to the myenteric plexus. Motor effects of enkephalin were studied on segments from circular smooth muscle of cat esophagus. Enkephalin inhibited electrically induced contractions in a dose-dependent manner. This effect was blocked by naloxone. The results suggest that enkephalin nerves may be involved in the regulation of esophageal motility. Like enkephaJin, bretylium (a blocker of adrenergic transmission) and phentolamine (an a-adrenergic blocker) prevented the contractile response to electrical stimulation. With smooth muscle strips from reserpinized cats the response to electrical field stimulation was abolished, but not the response to norepinephrine. Taken together, the results suggest that neuronal enkephalin in the esophagus functions as a modulator of adrenergic transmission. The esophagus smooth muscle is richly supplied with adrenergic, choline@, and peptidergic (VIPcontaining) nerves.lm4 In 1975, Hughes” isolated from brain tissue an opiate receptor agonist, identified as a pentapeptide and given the name enkephalin. In the same report enkephalin-like material was demonstrated in extracts from the gut. Enkephalin in the Received December 1, 1976. Accepted November 13, 1979. Address requests for reprints to: Rolf Uddman, M.D., University of Lund, ENT-Department, Malmb General Hospital, S-214 01 Malmo, Sweden. This study was supported by a grant from the Swedish Medical Research Council (No. 64X 4499) and Pahlsson’s Foundation. 0 1966 by the American Gastroenterological Association 6616-5065/66/046732-66562.25

brain was shown to occur in two varieties, one having leucine, the other methionine in the COOH-terminal position. Immunohistochemical studies have revealed a prominent network of enkephalin immunoreactive nerves in the intestinal wall.“” The present study deals with the occurrence and distribution of enkephalin immunoreactive nerve fibers in the esophagus of various mammals and with the in vitro effects of enkephalin on cat esophagus motor activity.

Materials and Methods Jmmunohistochemistry Tissue specimens were collected from the upper, middle, and lower parts of the esophagus from adult guinea pigs, opossums, cats, pigs, and monkeys, at least five of each species. Two cats were given reserpine (Serpasil, CIBA-Geigy AG, Basle, Switzerland), 5 mg/kg intraperitoneally. They were killed 24 hr later. The animals were killed by exsanguination under light diethyl ether (guinea pigs) or sodium pentobarbitone (cats, opossums, and monkeys) anesthesia. Specimens were also collected from 21 fetal (4-26 cm crown-rump length) and 5 newborn pigs. The gestational age of the fetal pigs was from 6 to 16 wk (parturition occurs at about 17 wk after conception).’ Material from adult pigs was obtained from a local abattoir. Specimens of smooth muscle from lower esophagus were obtained from 4 patients undergoing surgery due to carcinoma of the stomach. The specimens were frozen to the temperature of liquid nitrogen in a propane-propylene mixture and freeze-dried. They were then fixed by exposure to diethyl-pyrocarbonate vapor (if not otherwise stated) at 55“C for 3 hr” and embedded in paraffin in vacua. Sections, 5 pm in thickness, were deparaffinized, hydrated, and subjected to the indirect immunofluorescence method” or to the peroxidase-antiperoxidase (PAP) procedure12 for the demonstration of enkephalin. The enkephalin antiserum, kindly provided by Dr. K.-J. Chang (Wellcome Research Labs., Research Triangle Park, N.C.), was raised in rabbits against synthetic leu-enkepha-

April 1999

ENKEPHALIN

NERVES IN ESOPHAGUS

733

Figure 1. Enkephalin immunoperoxidase (PAP) staining of lower esophagus of a. and b. cat c. guinea-pig, and d. fetal pig (crown-rump length 14 cm). a. Immunoreactive nerve terminals in the muscularis mucosae (MM). Epithelium (EP) (X ZOO).b. Numerous coarse nerves in external smooth muscle (X ZOO).c. Nerve terminals in the myenteric plexus (arrows) (X 250). d. Intensely immunoreactive nerve cell bodies (arrows) and nerve terminals in myenteric plexus. Scattered nerve terminals also in longitudinal smooth muscle (LM) (X 250).

lin.13 In immunochemical as well as in immunohistochemical test systems the antiserum has been found to crossreact to some degree with met-enkephalin but not at all with the endorphins (a-, /3-, and y-endorphin) or /?-lipointestinal tropin.‘.13 Other peptides such as vasoactive polypeptide, substance P, neurotensin, CCK-8, and somatostatin, did not react with the antiserum. For brevity enkephalin immunoreactivity is referred to as enkephalin, although it cannot be excluded that the antiserum demonstrates also hitherto unidentified enkephaiin-like peptides. The antiserum was used in dilution 1:80 (immunofluorescence) or 1:480 (PAP procedure). In the immunofluorescence technique the site of antigen-antibody reaction was revealed by fluorescein isothiocyanate-labeled sheep antirabbit IgG (SBL, Stockholm, Sweden) diluted 120. In the PAP procedure, PAP complex (Cappel Labs., Downington, Pa.) was used in dilution 1:320. For controls in the immunohistochemical procedures we used sections incubated with the enkephalin antiserum inactivated by the addition of antigen in excess (10 pg of leu-

enkephalin/ml

diluted antiserum).

Formaldehyde-Induced

Fluorescence

Freeze-dried specimens (see above) from normal and reserpinized cats were treated with formaldehyde vapor at 80°C for 1 hr and embedded in paraffin.14 Sections were deparaffinized and mounted in Entellan. The formaldehyde-induced fluorescence of adrenergic nerves were examined in a fluorescence microscope.‘*

In Vitro Studies Segments of the esophagus from 8 normal cats and 2 reserpinized cats were dissected out 1 cm above the esophagogastric junction. After removing adherent nonmuscle tissue, strips of circular muscle (10 X 4 mm) were immersed in a modified Krebs-Ringer solution with the following composition (mM Gncentration): NaCl, 118; KCl, 4.5: CaCl, x ZH,O, 2.5; MgSO, x 7H,O, 1.0; NaHCO,, 2.5; KH,PO,, 1.0; and glucose, 6.0. The solution was continuously aerated by a mixture of 95% 0, and 5% CO, at a flow rate giving a pH of 7.4. The temperature was maintained at 37 f 0.2”C.

734

GASTROENTEROLOGY

UDDMAN ET AL.

The muscle strips were mounted between two metal holders in an organ bath. At least two strips were studied simultaneously. The strips were given an initial tension of 400 dyn and allowed to accommodate for 1.5 hr before

of all the species studied. They were not found in the striated muscle. Generally, immunoreactiye nerves were found in the muscularis mucosae (Figure la) and in the external muscle coat (Figure lb). In the latter location enkephalin nerves occurred only in the distal part of the esophagus where smooth muscle predominated; there was no conspicuous accumulation of nerves in the region of the sphincter. In the submucosa, single immunoreactive nerves occurred close to small blood vessels. The nerves were frequent in the myenteric plexus and seen to surround nonimmunoreactive cell bodies (Figure lc). Occasionally, single or clustered immunoreactive nerve cell bodies were detected in this location (Figure Id). In the fetal pig enkephalin-immunoreactive nerves were first observed at a gestational age of 7 wk; at this stage the nerves were restricted to the myenteric plexus. In later stages (13-16 wk gestational age) enkephalin nerves were found among bundles of smooth muscle outside the plexus. Immunoreactive cell bodies were very scarce in the myenteric plexus at the earliest stages studied. They became more frequent in later stages (15 wk gestational age). Reserpine was found to deplete the adrenergic nerve terminals of their norepinephrine content without visibly affecting the enkephalin nerve sup-

starting the experiments. The mechanical activity was measured by force-displacement transducers and amplified and recorded on a Grass model 7B polygraph. The strips were stimulated electrically by biphasic pulses generated from a Grass S44 stimulator via two platinum electrodes, one on each side of the preparation. The distance between the electrodes was 6 mm. The stimulation was given at supramaximal voltage (4.0-5.0 V over the preparation) and the pulse was kept short (0.6 msec). The pulse frequency was 2-4 pulses per second. Each stimulation period lasted for 5 set and was followed by a 2-min pause which included rinsing. After the experiments the muscle strips were fixed in Bouin solution, dehydrated, embedded in paraffin, sectioned and stained with hematoxylin and eosin for examination of the presence of striated muscle. The following drugs were used: acetylcholine (Sigma Chemical Co., St. Louis, MO.), L-arterenol HCl (Sigma), atropine sulphate (Vitrum AB, Sweden), bretylium tosylate (Burroughs Wellcome, England), carbamoylcholine chloride (EGAChemie AG, West Germany), hexamethonium bromide (May and Baker Ltd., England), DL-isoproterenol hydrochloride (Sigma), naloxone chloride (Winthrop Labs, USA), phentolamine methansulfon (Ciba-Geigy AG, Switzerland), tetrodotoxin (Astra Pharmaceut. Prod. Inc., Worcester, Mass.), leu-enkephalin and met-enkephalin (Beckman, Switzerland). The substances were dissolved in 0.9% saline.

PlY. In Vitro Studies The muscle strips, suspended in an organ bath, did not exhibit any spontaneous motor activity. Upon electrical field stimulation, lasting for 5 set, the strips usually responded with a biphasic contraction. The first contraction, which was inconsistent and variable, occurred l-2 set after the beginning of the stimulation period. Following this ini-

Results Distribution Nerves activity

of Enkephalin

Immunoreactive

Nerves displaying enkephalin immunoreoccurred in the esophageal smooth muscle

Figure 2.

CCh

TTX

d

1 I

I

I

I

Vol. 78, No. 4

I

I

I

The biphasic response to electrical field stimulation of a strip of smooth muscle from cat esophagus taken 1 cm above the esophagogastric juncare tion. 0. Both contractions blocked by tetrodotoxin (TTX; 3 x lo-' M). b). Carbamoylcholine (CCh; lo-’ M). b. Carbamoylcholine (CCh; the presence of tetrodotoxin. Figure 2c and d illustrate the contractile responses of muscle strips from c. a control cat and from d. a reserpinized cat recorded simultaneously. Pulse duration: 1 msec; length of stimulation periods: 5 set (indicated by rectangles); frequency: 4 pulses per second; supramaximal voltage. Vertical bar: 1000 dyn, horizontal bar: 1 min.

ENKEPHALIN

April 1960

-

I

NERVES IN ESOPHAGUS

735

60-

Phentolamine

b

2

0

I

I

I

I

I

I

0-l lo-‘0

10-g

t Figure 5.

Atropine

1.11 i

I

I

1

i

I

I

I

I

l

I

t

Hexamethonlum

Figure 3.

Contractile response to electrical field stimulation in the presence of various drugs. Each tracing is a representative example of 4 experiments. a. phentolamine (lo-’ M), b. atropine (10e5 M), and c. hexamethonium (3 x 10m5M). Vertical bar: 1000 dynes, horizontal bar: 1 min.

tial contraction the muscular tone returned to the base level. Immediately after cessation of the stimulation, a second contraction of short duration was noted. The addition of tetrodotoxin (3 x 10d7 M) abolished the electrically induced biphasic response, whereas it was still possible to induce contraction with carbamoylcholine (10e5 M) (Figure 2a and b). Muscle strips from reserpinized cats did not contract

Effects of stepwise increasing doses of met-enkephalin on the electrically induced contractions expressed as percent of maximal response. Pulse duration: 0.6 msec; length of stimulation periods: 5 set; frequency: 4 pulses per second; supramaximal voltage. Each value is the mean of 3 experiments. Vertical bars give SEM.

upon electrical field stimulation (Figure 212 and d). With muscle strips from normal cats the contractile responses to electrical field stimulation were inhibited by phentolamine (lo-’ M) and bretylium (2.4 x 10e5 M), while atropine (low5 M) and hexamethonium (3 x 10e5 M) had little or no effect (Figure 3). Both leu- and met-enkephalin (4 X lo-” - 4 x lo-’ M) caused a dose-dependent inhibition of the electrically induced contractions (Figures 4 and 5). The two phases of contraction were influenced to about the same extent. The contraction induced by acetylcholine (lo-’ M) (which persisted in specimens from reserpinized cats) was not counteracted by enkephalin (met-enkephalin), while the beta-adrenergic agonist isoproterenol (lo-’ M) induced a marked relaxation (Figure 6). With muscle strips from both

I3 4.10-7 -

I

I

I

I

,

I

I

I

I

t

Leu-enkephalin

Figure 4.

Effect of leu-enkephalin (3.5 x lOA M) on the contractile response to electrical field stimulation of esophagus smooth muscle. Pulse duration: 0.6 msec; length of stimulation periods: 5 set; frequency: 4 pulses per second; supramaximal voltage. Vertical bar: 1000 dyn; horizontal bar: 1 min.

2.10-6

Met-enkephalin

I

Figure 6.

1.10-6

1.10-6

(MI

lsoproterenol

Effect of met-enkephalin and isoproterenol on acetylcholine-induced contraction of esophagus smooth muscle. Acetylcholine (lo-’ M) was given 15 min before met-enkephalin was first added to the bath. The sustained acetylcholine contraction (9000 dyn) was not affected by met-enkephalin, while isoproterenol effectively reduced the tension. Vertical bar: 5000 dyn; horizontal bar: I min.

736

UDDMAN ET AL.

GASTROENTEROLOGY

normal and reserpinized cats norepinephrine induced a marked dose-dependent contraction, which was not prevented by pretreatment with tetrodotoxin nor inhibited by enkephalin (Figure 7). The enkephalin-mediated inhibition of the response to electrical field stimulation was totally blocked by naloxone (7.6 x lo+'M) (Figure 8). If anything, naloxone per se slightly enhanced the contraction that followed upon cessation of electrical field stimulation.

Controct,on

Vol. 78. No. 4

I%1

loo-

80-

:

a

Discussion Previous studies have shown that the esophagus smooth muscle is richly supplied with autonomic nerves.‘-4 Stimulation of cholinoceptors and a-adrenoceptors causes contraction,“*” whereas stimulation of P-adrenoceptors and VIP receptors seems to cause relaxation of the lower esophageal sphincter.‘5.‘7 In the present study of a number of mammalian species, enkephalin immunoreactive nerve fibers were regularly observed in the myenteric plexus; nerve cell bodies occurred only occasionally. Immunoreactive nerve fibers occurred also in the smooth muscle. Nerve fibers displaying enkephalin immunoreactivity have previously been observed in the gut wall”’ and recent immunohistochemical evidence indicate that immunoreactive enkephalin in the gut is indistinguishable from brain enkephalin.‘” The enkephalin immunoreactive nerves in the esophagus seem to have roughly the same topographic distribution as those in the gut in that they predominate in the smooth muscle and in the myenteric plexus. In contrast to VIP nerves3 there was no conspicuous accumulation of enkephalin nerves in the region of the sphincter. As studied in the pig, enkephalin nerves appeared early in fetal development, i.e., at about the same time as the VIP containing nerve.9 but much earlier than those storing substance P (Uddman et al., to be published). Electrical field stimulation resulted in a biphasic contractile response in the esophageal circular muscle strips from normal cats.1g’20No response could be elicited with strips from reserpinized cats, in which animals the adrenergic nerves had been depleted of their norepinephrine content (see reference 21). A short pulse duration was used in order to avoid direct stimulation of the smooth muscle.” In a previous report it was argued that only the second contractile response was neurogenic since the initial response was not abolished by tetrodotoxin.m In our studies, both contractions were invariably abolished by tetrodotoxin suggesting a neural mediation of both responses.23 The ability of esophagus smooth muscle to contract upon stimulation with norepinephrine and choline esters was not affected by tet-

0 10

B

x

8

o

10-7 lo” 10’5 Norep,nephrme concentrator” IMI

lo-’

Figure 7. Contractile effect of stepwise increasing doses of norepinephrine alone (0) or in the presence of 10” M met-enkephalin (0) using muscle strips from 2 control cats, and of norepinephrine alone on muscle strips from 2 reserpinized cats (x). Each point is’the mean of 3 separate experiments.

rodotoxin or by reserpinization. The contrations induced by electrical field stimulation seemed to depend primarily on adrenergic nerves and a-receptors since reserpinization prevented them and since bretylium, which is known to block adrenergic transmission, and phentolamine, an a-adrenergic blocker, markedly inhibited the contractions. These observations contrast with previous findings that the contractile responses to electrical field stimulation were unaffected by a-blockers.” However, we and others agree that blockade of muscarinic receptors or of ganglionic transmission had little or no effect on the contractile responses to field stimulation.” Both leu- and met-enkephalin were found to inhibit

I

l~lllllll t Met-enkephalin

Figure 8.

I

I

I

I

t NalOXOfle

Naloxone (7.6 x 10-s M) antagonized the effect of metenkephalin (1.7 X lo-’ M) on the contractile response of esophagus smooth muscle to’electrical field stimulation. This tracing is representative’of a series of 3 experiments. Pulse duration: 0.6 msec; length of stimulation periods: 5 set; frequency: 2 pulses per second; supramaximal voltage. Vertical bar: 5ooO dyn; horizontal bar: 1 min.

April 1980

the contractions induced by electrical field stimulation. The enkephalins had no effect on the contraction induced by choline esters or norepinephrine indicating the absence of a direct relaxing effect on esophagus smooth muscle. Together these findings suggest that the inhibitory action of enkephalin is exerted via presynaptic blockade of adrenergic transmission. Naloxone, which effectively and selectively inhibits the effects of morphine and other opiate receptor agonists,“.” blocked the inhibitory action of enkephalin. Studies performed on the guinea pig ileum have shown enkephalin to inhibit neurally mediated motor activity’~” as well as the firing rate of single myenteric neurons.” In other smooth muscle preparations the enkephalins have been claimed to lower the release rate of transmitters such as norepinephrine and acetylcholine.“~‘” In conclusion, the characteristic distribution of enkephalin immunoreactive nerves in the esophagus suggests that they participate in regulating esophagus motility. Since our results indicate that enkephalin is a potent presynaptic blocker of adrenergic nerve-mediated contraction of esophagus smooth muscle it is conceivable that neuronal enkephalin exerts its activity by modulating the release of the adrenergic transmitter.

References 1. Baumgarten

2.

3. 4.

5.

6.

7.

8.

HG, Lange W: Adrenergic innervation of the oesophagus in the cat (Felis domestica) and rhesus monkey (Macacus rhesus). Z Zellforsch Mikrosk Anat 95529-545, 1969 Gonella J, Niel JP, Roman C: Vagal control of lower oesophageal sphincter motility in the cat. J Physiol273:647-664, 1977 Uddman R, Alumets J, Edvinsson L, et al: Peptidergic (VIP) innervation of the esophagus. Gastroenterology 755-8, 1978 Gonella J, Niel JP, Roman C: Sympathetic control of lower oesophageal sphincter motility in the cat. J Physiol 287:177-190, 1979 Hughes J: Isolation of an endogenous compound from the brain with pharmacological properties similar to morphine. Brain Res 88:295-308, 1975 Elde R, Hakfelt T, Johansson 0, et al: lmmunohistochemical studies using antibodies to leucine-enkephalin: initial observations on the nervous system of the rat. Neuroscience 1:349351, 1976 Polak JM, Sullivan SN, Bloom SR, et al: Enkephalin-like immunoreactivity in the human gastrointestinal tract. Lancet 1:972-974, 1977 Alumets J, Hakanson R, Sundler F, et al: Leu-enkephalin-like material in nerves and enterochromaffin cells in the gut: an immunohistochemical study. Histochemistry 56:187-196,1978

ENKEPHALIN

NERVES IN ESOPHAGUS

737

Book SA, Bustad LK: The fetal and neonatal pig in biomedical research. J Animal Sci 38997-1002, 1974 10.Pearse AGE, Polak JM, Adams C, et al: Diethylpyrocarbonate, a vapour-phase fixative for immunofluorescence studies on polypeptide hormones. Histochem J 6347-352, 1974 11. Coons AH, Leduc EH, Conolly JM, et al: Studies on antibody production. A method for the histochemical demonstration of specific antibody and its application to a study of the hyperimmune rabbit. J Exp Med 102:49-60,1955 12. Sternberger LA: lmmunocytochemistry. Englewood Cliffs, NJ, Prentice-Hall Inc., 1974 13. Miller RJ, Chang K-J, Cooper B, et al: Radioimmunoassay and characterization of enkephalins in rat tissue. J Biol Chem 253:531-538, 1978 14. Bjiirklund A, Falck B, Owman Ch: Fluorescence microscopic and microspectrofluorometric techniques for the cellular localization and characterization of biogenic monoamines. In: Berson SA. (Ed). Methods of Investigative and Diagnostic Endocrinology, Vol. 1. Edited by JE Rall, IJ Kopin. The Thyroid and Biogenic Amines. Amsterdam, North-Holland Publishing Company, 1972, p 318-368 15. Christensen J, Daniel EE: Effects of some autonomic drugs on circular esophageal smooth muscle. J Pharmacol Exp Ther 159243-249, 1968 16. Christensen J, Dons RF: Regional variations in response of cat esophageal muscle to stimulation with drugs. J Pharmacol Exp Ther 161:55-58, 1968 17. Rattan S, Said Sl, Goyal RK: Effect of vasoactive intestinal polypeptide (VIP) on the lower esophageal sphincter pressure (LESP). Proc Sot Exp Biol Med 155:40-43, 1977 18. Linnola RI, DiAugustine RP, Miller RJ, et al: An immunohistochemical and radioimmunological study of the distribution of (Met)- and (Leu)-enkephalin in the gastrointestinal tract. Neuroscience 3:1187-1196, 1978 19. Christensen J, Lund G: Esophageal responses to distension and electrical stimulation. J Clin Invest 48:408-418, 1969 20. Lund G, Christensen J: Electrical stimulation of esophageal smooth muscle and effects of antagonists. Am J Physiol 217:1369-1374. 1969 21. Carlsson A, Rosengren E. Bertler A, et al: Effect of reserpine on the metabolism of catechol amines. In: Psychotropic Drugs. Edited by S Garattini, V Ghetti. Amsterdam, Elsevier, 1957, p 363-372 22. Swedin G: Studies on neurotransmission mechanisms in the rat and guinea-pig vas deferens. Acta Physiol Stand (Suppl 369):1-34, 1971 23. Ganong WF: Review of Medical Physiology. Menlo Park, Calif., Lange Medical Publications, 1977 24. Kosterlitz HW, Watt AJ: Kinetic parameters of narcotic agonists and antagonists, with particular reference to N-allylnoroxymorphone (Naloxone). Br J Pharmac Chemother 33:266276, 1968 25 North RA, Williams JT: Extracellular recording from the guinea-pig myenteric plexus and the action of morphine. Eur J Pharmacol45:23-33, 1977 26. Day AR, Luj&n M, Dewey WL, et al: Structure-activity relationships of enkephalin in the stimulated guinea pig ileum. Res Commun Chem Path01 Pharmacol 14:597-603, 1976 27. Enero MA: Properties of the peripheral opiate receptors in the cat nicitating membrane. Eur J Pharmacol45:349-356, 1977 28. Knoll J. Ill& P, Medzihradszky K, et al: The action of enkephalins and enkephalin analogues on neurotransmission in the isolated nicitating membrane of the cat. J Pharm Pharmaco1 30:394-395, 1978 9.