Download Location of enkephalinase and functional effects of

Clinical Science (1992) 82, 169-173 (Printed in Great Britain)
I69
location of enkephalinase and functional effects of
[leu5]enkephalin and inhibition of enkephalinase in the
feline main pancreatic and bile duct sphincters
A. THUNE, L. JIVEGARD, H. POLLARD, J. MOREAU, J.C. SCHWARTZ and J. SVANVIK
Department of Surgery, University of Goteborg, Sahlgrenska Hospital, Goteborg, Sweden,
and Unit of Neurobiology, Centre Paul Broca de I’INSERM, Paris, France
(Received I5 Aprill9 August I99 I; accepted 27 August I99 I )
1. Morphological studies have demonstrated enkephalinergic nerve fibres in proximity to the sphincter of
Oddi, and opiates are known to contract this sphincter. In
this study, the flow resistances in the common bile duct
and main pancreatic duct sphincters were studied simultaneously in anaesthetized cats using a perfusion technique.
2. Naloxone did not affect the activity of these sphincters
under basal conditions, indicating that there is no basal
enkephalinergic tone.
3. The response to [Leu’jenkephalin (0.015-15 pg/kg),
morphine (1 mg/kg) and ketamine (10 mg/kg) was a
naloxone-sensitive increased activity in the sphincters
with a raised frequency of phasic contractions. The
threshold dose for an effect of [Leu’lenkephalin on the
sphincter of Oddi was 0.015 pg/kg and a maximal
response was observed at 0.75 pg/kg. There were no differences in the response of the main pancreatic duct
sphincter and the bile duct sphincter to the different
drugs.
4. Immunoautoradiographic studies demonstrated enkephalinase in the spincter of Oddi.
5. Acetorphan (3 mg/kg intravenously), which inhibits
endogenous enkephalinase both in the peripheral and the
central nervous system when administered parenterally,
caused a naloxone-sensitive contraction, whereas
thiorphan (3-20 mg/kg), an enkephalinase inhibitor that
does not easily penetrate the blood-brain barrier, had no
effect on the sphincter of Oddi.
6. These results show that endogenous and exogenous
opiates influence the function of the feline sphincter of
Oddi and that enkephalins may be involved in the physiological control of this sphincter, although not under basal
conditions.
INTRODUCTION
Previous research has suggested that the endogenous
opiate [Leu’lenkephalin has both excitatory and inhibitory effects on the feline sphincter of Oddi (SO)[l], and
morphological studies have demonstrated enkephalinergic nerve fibres in proximity to the smooth muscle
fibres in this sphincter [2]. Endogenous enkephalins are
inactivated partly by enkephalinase, a membrane metalloendopeptidase (EC 3.4.24.1 l ) , and this enzyme has been
located in several peripheral organs, including those of
the gatrointestinal tract [3]. Enkephalinase activity may be
blocked with the drugs acetorphan and thiorphan [4]. The
effects induced by these drugs on the gastrointestinal tract
are blocked by naloxone [5, 61. Naloxone is an opiatereceptor antagonist with no agonistic effect at low concentrations [7]. Ketamine, a commonly used anaesthetic
agent, has central and peripheral opiate agonist actions
and anticholinergic effects [S, 91 besides its anaesthetic
actions.
The aim of this study was to study the role and
mechanism of action of endogenous and intravenously
administered [Leu’]enkephalin on the feline SO. Since
ketamine has been used to induce or maintain anaesthesia
in several previous studies on the SO, the effect on the
feline SO of this drug was also tested.
METHODS
Experimental procedures
Experiments were performed in cats anaesthetized
with chloralose (60 mg/kg body weight intraperitoneally)
and maintained with 10 mg/kg intravenously as needed.
The effects of opiates were studied in both sphincteric
segments of the SO by simultaneous perfusion of the main
pancreatic duct sphincter (PDS) and the bile duct
sphincter (BDS). The flow resistance (FR) exerted by
these sphincteric segments was calculated from the
pressure gradient between the distal common bile duct
and the duodenum, the pancreatic duct and the duodenum, respectively, and the flow rate. The mean FR for
each segment was estimated with the aid of integrators.
The technical details of this method and the validity of the
measurements are described in detail in previous reports
[lo, 111.
Key words: enkephalin, enkephalinase, ketamine, opiate, sphincter of Oddi.
Abbreviations: BDS, bile duct sphincter; FC, frequency of contractions; FR, flow resistance; mAb, monoclonal antibody; PBS, phosphate-bufferedsaline; PDS, pancreatic
duct sphincter; SO, sphincter of Oddi; TTX, tetrodotoxin.
Correspondence: D r ]oar Svanvik, Department of Surgery I, Sahlgrenska Hospital, S-413 45 Goteborg, Sweden.
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A. Thune et al.
Mean FR values were calculated during 5 rnin periods
before and after administration of the drugs. When
responses after blocking with naloxone were tested, the
investigated drug was injected 5 rnin after naloxone. The
left femoral artery was cannulated and was connected to a
pressure transducer, and the left femoral vein was used
for intravenous injections of all drugs except for the
nerve-blocking agent tetrodotoxin (TTX), which was
given as a bolus dose into the celiac artery via the splenic
artery. Physiological saline (150 mmol/l NaCI), given by
the same intra-arterial route, was used as a control.
Sweden; [Leus]enkephalin was supplied by Fluka AG,
Buchs, Switzerland; TTX was obtained from Sigma
Chemicals, St Louis, MO, U.S.A.
Statistical methods
Statistical analyses were performed by using the paired
or unpaired t-test and linear regression analysis. A P
value of less than 0.05 was considered significant. All
results are given as means t- SEM.
RESULTS
Immuno-autoradiographic studies
Specimens, including the common bile duct and the
duodenum, were taken from three cats and were immediately frozen in liquid nitrogen. The region of the SO was
cut (20 p m thick) on a cryostat (Ames) at - 18"C, thawmounted on gelatin-coated glass slides and stored at
-20°C until used. The sections were warmed to room
temperature, dried for 30 rnin and washed twice for 30
min in 0.1 mol/l phosphate-buffered saline (PBS),pH 7.4,
immediately before incubation for 3 h at 20°C with 0.2 ml
of an 12sI-labelledmonoclonal antibody (mAb) known to
recognize cat enkephalinase (mAb 135, 2.5 x 106 c.p.m./
ml) [GI dissolved in PBS containing 0.2'10 (w/v) gelatin and
0.1"/0 (v/v) Tween 20. The slides were rinsed twice for
3 rnin in the same supplemented buffer, followed by two
3 rnin rinses in pure PBS. Finally, they were dipped into
distilled water, dried and apposed to H3-sensitive hyperfilm (Amersham) in X-ray cassettes and the films were
developed 2-3 days later.
Non-specific labelling was assessed by using a control,
similarly "'I-iodinated, mAb (mAb 85 A2)known for its
inability to recognize cat enkephalinase.
Function of the SO during basal conditions and in
response to TTX and naloxone
During basal conditions, FR was 4.7 +- 0.7 cmH,O hml-l in the BDS and 5.2k0.7 cmH,O h-' ml-I in the
PDS in 18 cats. Spontaneous synchronous contractions
were registered in both the PDS and the BDS and no
difference in the frequency of contractions (FC) was
observed. FC was 4.9 k 0.6 min- under basal conditions.
Administration of TTX (9 pg/kg body weight) in six
animals significantly increased FR (change in FR: BDS,
6.19 k 1.27 cmH,O h- I ml- I, P < 0.01; PDS, 3.89 k 0.68
cmH,O h-I ml-l, P<O.01) and (change in FC: 4.2k0.9
min-I, P < 0.01). Injection of 2 ml of physiological saline
into the celiac artery neither affected FC nor FR.
Naloxone (1 mg/kg intravenously) given under basal
conditions did not affect FR in either of the sphincters
(change in F R BDS, 0.2OkO.18 cmH,O h - ' ml-'; PDS,
0.1 1 k0.12 cmH,O h - l ml-I). Neither did this drug cause
any change in FC (change in FC: 0.0 k 0.3 min- I, 17 = 7).
Effects of exogenous opiate agonists
Drugs
Acetorphan and thiorphan were provided by Laboratoire Bioproject, Paris, France; Nalonee (naloxone hydrochloride) was purchased from Du Pont, Wilmington, DE,
U.S.A.; Ketalar (ketamine hydrochloride) was obtained
from Parke Davis, Morris Plains, NJ, U.S.A.; Morfin
(morphine chloride) was purchased from ACO, Solna,
Intravenous injection of morphine (1 mg/kg) significantly increased FR in both sphincters (change in FR:
BDS, 3.17k1.09 cmH,O h - ' m1-I; PDS, 2.35k0.58
cmH,O h-I ml-I). A similar response was noted in
response to ketamine (10 mg/kg) and [Leu5]enkephalin
(15 pg/kg) (Table 1).The response to the different drugs
were of comparable magnitude. The increment in FR
Table I. Change in FR calculated from the change in mean pressure during a 5 min period before and after an
intravenous injection of [Leu']enkephalin, acetorphan or ketamine. The increase in FR was significant (P <0.05. paired
r-test. n = 5 for each drug) for all three drugs. After pretreatment with naloxone the responses were all significantly reduced
(P<O.O5. unpaired t-test, n = 5 for each drug). Values are meanstsm.
Change in FR (cmH,O h-l m1-I)
Acetorphan (3 mglkg)
(15 yglkg)
[Le~~lenkephalin
Ketamine (10 mglkg)
Alone
After pretreatment
with naloxone
Alone
After pretreatment
with naloxone
Alone
After pretreatment
with naloxone
BDS
3.51k0.81
1.481l.lI
2.7610.76
0.39f0.43
2.22k0.70
0.42t0.19
PDS
4.37 k0.75
I .39 f I .O
2.77 f0.93
0.3410.35
1.29k0.29
0.51 t O . 1 I
[Leus]enkephalin and t h e sphincter of O d d i
induced by [Leus]enkephalin or ketamine was of short
duration (10-20 min).
After pretreatment with naloxone, the response to
[Leu5]enkephalin and ketamine were significantly reduced
(Table 1). Morphine (change in FC: 3.5f1.0 min-I,
PcO.05, n = 5 ) and ketamine (change in FC: 2.4k0.6
min-I, P<O.O5, n = 5 ) significantly increased FC in the
SO. F R correlated significantly ( P < 0.001) with the
increase in FC (Fig. 1).
171
doses of [Leu'lenkephalin above 0.75 ,ug/kg, a tonic
contraction lasting several minutes was recorded and FC
could not be determined.
Pretreatment with TTX (9 pg/kg) significantly reduced
the increment in FR caused by an injection of
[Leus]enkephalin (15 pg/kg) (change in F R PDS,
0.88 k0.75 cmH,O h-' ml-I, P<O.O5; BDS, 0.26f0.75
cmH,O h-' ml-', P < 0.05) in five experiments.
Localization of enkephalinase in the feline SO
Dose-response analysis and mode of action of
[Leus]enkephalin
[Leu5]enkephalin was administered in doses ranging
from 1.5 X
to 15 pg/kg. FR increased progressively
from 15 x
,ug/kg, and a maximal response was
observed at a dose of 0.75 pug/kg (Fig. 2).
[Leus]enkephalin (0.15 pglkg) significantly increased FC
in the SO (change in FC: 2.41.0.5 min-', P<0.05). At
Autoradiographs generated with '251-labelledmAb 135
demonstrated a distribution of autoradiographic grains
located in the smooth muscle surrounding the distal
common bile duct and the distal main pancreatic duct
(Fig. 3). On the same transverse section, labelling of the
epithelium of the small intestinal mucosa is also observed.
Non-specific labelling obtained using '251-labelled mAb
85 A2 under the same conditions was negligible (Fig. 3).
Effects of inhibition of enkephalinase by acetorphan and
thiorphan
Injection of acetorphan ( 3 mg/kg) caused a naloxonesensitive, significant increase in FR in the SO (Table 1)as
well as a significant increase in the FC (change in F C
3.3 f 0.9 min- I , P < 0.05, n = 5).
The effects of thiorphan were analysed at doses from 3
to 20 mg/kg in seven animals. No change in FR ( r< 0.01,
linear regression dose-response analysis) or in FC
( r < 0.01, linear regression dose-response analysis) was
observed.
/
0
0
0%
0
,
I
-2
.
I
0
0.
0
.
I
.
2
I
-
4
I
-
I
8
6
Change in FC (min-')
Fig. I. Correlation between the change in FC and the change in
FR obtained by use of linear regression analysis. n=43, r=0.70,
P<O.OOI.
1 . 5 ~ 1 0 - ~ 1 . 5 ~ 110.-5~~ 1 0 -0.15
~
0.75
1.5
15
[Leu'lenkephalin (pglkg)
Fig. 2. Dose-response analysis for [Leu'lenkephalin in the PDS
(a) and the BDS (0).The change in FR was calculated from the
change in mean pressure during a 5 min period before and after an intravenous injection. The difference between the response t o doses of 0.75
pglkg and higher was significant (P <0.05) compared with the response
to the lowest dose (1.5 X
pglkg). Values are means with bars
indicating SEN.
DISCUSSION
The results of this study confirm previous findings of
opiates having excitatory properties on the feline SO [ 1,
121. The opiate agonists morphine, [Leus]enkephalin and
ketamine thus induced a contraction of the PDS and the
BDS. The response to [Leus]enkephalin was dosedependent and was significantly inhibited by pretreatment
with TTX as well as by pretreatment with naloxone.
There were no significant differences in the responses of
the main PDS and BDS to the opiate agonists, indicating a
common sphincteric complex with the same actions on
FR and FC in both systems. These results concord with
those obtained in a recent study [lo].
TTX blocks nerve excitability by blocking sodium
channels [13], and the administered dose is known to
block the effects of electrical stimulation of the splanchnic
nerves in the feline biliary tract [14]. The increase in SO
activity produced by administration of TTX confirms
previous results [15] and may be explained by increased
spontaneous activity in the SO when denervated.
Naloxone, which blocks the opiate receptors, by itself
neither affected the basal F R nor FC in the sphincters,
suggesting that there is no basal enkephalinergic tone
under the conditions studied. This disagrees with earlier
findings by Behar & Biancani [l]. The discrepancy may
A. Thune e t al.
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2mm
SM
.2mm
O.bmm
H
..
Fig. 3. Autoradiographic localization of enkephalinase immunoreactivity i n t h e cat SO. ( a ) Haemalinierythrosinstained section from which the autoradiogram ( b ) was generated. ( b ) Labelling obtained with 'lsl-labelled mAb 135, which
recognizes cat enkephalinase. The immunoreactivity is found in the epithelium of the small intestinal mucosa (SIM) and also in
the smooth muscle (SM) surrounding the distal common bile duct (CBD) and main pancreatic duct (PD). (c) Non-specific
labelling determined on an adjacent section incubated with 'lsl-labelled mAb 85A2, which does not recognize cat enkephalinase.
be explained by the repeated doses of ketamine used for
anaesthesia in the quoted study.
Ketamine was shown to exhibit opiate-like properties
in this study with naloxone-sensitive increases in FR and
FC in the SO of the same magnitude as those produced by
morphine. These effects exclude ketamine as an appropriate anaesthetic agent when studying opiates in vivo.
Dose-response analysis for [Leu'lenkephalin demonstrated that the feline SO is highly sensitive to this drug
and that the effect is mediated by opiate receptors as the
response to a maximal dose was significantly reduced by
naloxone. Pretreatment with TTX also significantly
decreased the change in FR in the SO by a maximal dose
of [Leu']enkephalin, indicating that the response of the
SO to [Leus]enkephalin was influenced by nervous
activity.
An increase in FC in the SO was observed after injection of [Le~~lenkaphalin,
ketamine and morphine in this
study. These results agree with those of Helm et al. [lG],
who found an increased FC in the human SO after injec-
tion of morphine. In the present study, FC was also
increased in response to TTX. This may be explained by
blocking of activity in inhibitory nerves to the SO. Further,
the increase in FC in the SO was accompanied by an
increase in FR. It thus seems that, in the cat, an increased
FC is associated with reduced flow through the sphincter
rather than an increased flow due to propulsion of bile
and pancreatic juice, as has been postulated for other
species. The present findings correspond to those
obtained earlier in the guinea pig [ 171and in the cat [ 181.
This study demonstrates the presence of enkephalinase
in the smooth muscle of the SO. This finding suggests that
enkephalinergic neurons may be active in the regulation
of the smooth muscle activity of the SO. To further
investigate the activity of endogenous enkephalins the
effect on the SO by enkephalinase inhibitors were studied.
These drugs reduce the inactivation of endogenous
enkephalins. In previous studies the prodrug acetorphan
was shown to have actions on the central nervous system
as well as on the peripheral tissues [4].Thiorphan, on the
[Leus]enkephalin and t h e sphincter
other hand, is only effective in peripheral tissues owing to
poor passage across the blood-brain barrier [4]. The
contraction observed in response to acetorphan demonstrates that endogenous enkephalins have a stimulating
effect on the SO. This effect is probably of central
nervous system origin, as no response by the SO was seen
with the administration of thiorphan under basal conditions. However, these results do not exclude the possibility that endogenous enkephalins in peripheral tissue
may be active under conditions other than basal.
This study also shows that administration of the opiate
agonist [Leu5]enkephalin contracts the feline SO. As
enkephalins d o not pass the blood-brain barrier [ 191, the
site of action of [Leus]enkephalin is suggested to be in the
intrinsic neural network of the SO. This assumption is
supported by the observation of enkephalinase activity in
the SO. The results suggest that both central and peripheral enkephalinergic mechanisms may be implicated in
a possible excitatory regulation of the activity of the feline
SO. The lack of effect of naloxone per se indicates that
there is no basal tone in the enkephalinergic system under
basal conditions.
ACKNOWLEDGMENTS
This study was supported by grants from the Goteborg
Medical Society, the Swedish Medical Research Council
(17X-04984)and the University of Goteborg.
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