JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2004, 55, 1, 279288 www.jpp.krakow.pl
G.B. STEFANO , W. ZHU , P. CADET , T.V. BILFINGER , K. MANTIONE 1
1
1
2
1
MORPHINE ENHANCES NITRIC OXIDE RELEASE IN THE MAMMALIAN GASTROINTESTINAL TRACT VIA THE µ3 OPIATE RECEPTOR SUBTYPE: A HORMONAL ROLE FOR ENDOGENOUS MORPHINE
1
Neuroscience Research Institute, State University of New York / College at Old Westbury,
2
Cardiothoracic Division, Department of Surgery, Health Sciences Center, State University
Old Westbury, New York, 11568
of New York at Stony Brook, Stony Brook, New York, 11794. U.S.A.
Studies from our laboratory have revealed a novel µ opiate receptor, µ3, which is
expressed in both human vascular tissues and leukocytes. The µ3 receptor is selective for opiate alkaloids, insensitive to opioid peptides and is coupled to constitutive nitric
oxide
(cNO)
release.
We
now
identify
the
µ3
receptor
characteristics
in
mammalian gut tissues. It appears that the various regions of the mouse gut release low levels of NO (0.02 to 4.6 nM ) in a pulsatile manner. We demonstrate that
morphine stimulates cNO release (peak level 17 nM) in the mouse stomach, small intestine and large intestine in a naloxone and L-NAME antagonizable manner.
Opioid peptides do not exhibit cNO-stimulating capabilities in these tissues. Taken together, we surmise morphine acts as a hormone to limit gut activity via µ3 coupled
to NO release since µ opiate receptors are found in the gut and endogenous morphine is not but is found in blood.
Key
w o r d s : morphine, stomach, small intestine, large intestine, nitric oxide, mu3 opiate receptor
Abbreviations: SNAP, arginine
S-nitroso-N-acetyl-DL-penicillamine; methyl
enkephalin.
ester;
DAMGO,
L-NAME,
(Tyr-D-Ala , 2
N
omega-nitro-L-
Gly-N-Me-Phe , 4
Gly(ol) )5
280
INTRODUCTION
The demonstration of the multiplicity of opiate receptor types has led to the understanding that, depending on their site of action, opioid peptides, as well as opiate alkaloids, may bind to more than one opiate receptor subtype (1- 4). Different degrees of selectivity have been recognized for the various ligands. For example, Pasternak and Snyder (5) reported both high and low affinity binding sites for [ H]-dihydromorphine ( DHM) and [ H]-naloxone in the rat brain. The 3
3
3
higher affinity type was designated µ1 and the lower affinity morphine-selective
type was designated µ2 (6, 7). In
addition
to
the
two
main
µ
opiate
receptor
subtypes,
µ1
and
µ2,
our
laboratory has demonstrated a third µ opiate receptor (µ3) that is selective for
opiate alkaloids, but insensitive to opioid peptides (8, 9). Functional studies have shown that µ3 is expressed on immunocytes and neural tissues of the invertebrate
Mytilus edulis as well as on human monocytes, granulocytes, vascular endothelial cells and other cells including those of the nervous system (8, 10, 11). Recently, using RT-PCR, we have amplified transcript fragments with 100% homology to µ1 that are constitutively expressed (12, 13). We have now identified the human
µ3 opiate receptor subtype at the molecular level as a truncated µ splice variant,
which exhibits the expected functionality: nitric oxide (NO) release in an opiate alkaloid-selective
and
opioid
peptide-insensitive
manner,
as
well
as
the
previously described binding and physiological characteristics of the µ3 receptor (9, 14, 15).
In the current report we extend these µ3 observations into the mammalian gut,
which is known to contain both NO signaling as well as mu opiate receptors (16 - 21). We propose the hypothesis, given the current results, that morphine, an endogenous signaling molecule is involved with controlling gut motility via NO release.
Furthermore,
we
surmise
this
morphinergic
signaling
process
is
protective in nature.
MATERIALS AND METHODS
Gut tissue Mouse ( ICRHA mouse, Taconic, Germantown, NY) gut (small intestine, large intestine, and stomach) was immediately separated, washed and stored in an electrolyte solution at 4°C (500 cc plasmalyte with 5000 U heparin and 60 mg papaverine), and immediately transported on ice to the laboratory for processing as described in detail elsewhere (22). Study protocol was approved by Ethical Committee of the State University New York, New York, USA.
Nitric oxide determination For NO determination, approximately 300 mg (wet weight) of stomach, small or large intestine fragments from freshly sacrificed mice were placed in 1 ml PBS. NO release from the respective
281
gastrointestinal tissues was directly measured using an NO-specific amperometric probe (200 µm flex, World Precision Instruments, Sarasota, FL). A micromanipulator (World Precision Instruments, Sarasota, Fl), which was attached to the stage of an inverted microscope (Nikon Diaphot, Melville, NY), was used to position the amperometric probe 15 µm above the tissues. The system was calibrated
daily
by
nitrosothiol
donor
S-nitroso-N-acetyl-DL-penicillamine
(SNAP),
(World
Precision Instruments) resulting in the liberation of a known quantity of NO. The amperometric probe was allowed to equilibrate for at least 10 min prior to being transferred to the tube containing the respective tissues. The detection limit of this system is 500 pM. Morphine-stimulated NO release was evaluated at final concentrations of 10
-6
to 10
-10
M. The receptor antagonist, naloxone (10
-6
M),
was added 30 min prior to morphine addition. N omega-nitro-L-arginine methyl ester (L-NAME), a potent nitric oxide synthase inhibitor, was administered at 10 M and 5 min prior to monitoring basal -4
NO levels. The addition of the opioid peptide Met-enkephalin was also evaluated at 10
M. Each
-7
experiment was repeated four times along with a control that was just exposed to vehicle.
RESULTS
We demonstrate that the gastrointestinal tissues examined appear to release low levels of NO (1-4 nM) in a pulsatile manner without exogenous stimulation (Fig. 1). This pulsatile release of NO is inhibited by L-NAME (10
-4
M) exposure,
substantiating the nature of the material detected (Fig. 1). To demonstrate the expression of a functional µ3 receptor-like mechanism in
the gut tissues, we measured NO release in response to the addition of morphine (10
M) (Figs 2, 3). Following exposure to 10
and
large
-6
-6
originates
intestine from
fragments
constitutive
released NO
an
synthase
M morphine, the stomach, small
average
of
15-18
stimulation,
nM
given
NO,
its
which
immediate
Fig. 1.
Fig.
Representative
1.
illustration pulsatile
of
nitric
from
small
(A.)
and
spontaneous, oxide
intestine its
release
fragment
inhibition
by
exposure to L-NAME (10
M;
-4
B.). Y axis = nM NO
282
Fig. 2.
Fig. 2. Representative real time amperometric
analysis
of
NO
release in the mouse gut and its stimulation
morphine.
via
Bar
scale on the left of each reading varies
so
activity Basal
that
can
NO
mouse
any
be
underlying
observed.
release
small
Morphine (10
A.
from
the
intestine.
B.
M) stimulation
-6
(added at X) of cNOS derived NO
release
intestine Exposure NAME
from
tissue of
(10
-4
a
small
fragment.
the
tissue
M)
for
C.
to
10
L-
min
antagonizes morphine (10
-6
M)
NO release (morphine added at time
0
following
exposure
to
a
10
min
L-NAME).
Small
intestine
exposed
to
D.
fragments
naloxone
(10
-6
M)
for 10 min and then exposed to morphine (at X).
increase over basal levels and its decrease within minutes of morphine exposure (Fig. 2; Table 1). The morphine stimulated NO release in these tissues occurs in a concentration dependent manner (Fig. 3). Furthermore, naloxone (10 M), the -6
opiate receptor antagonist, blocks morphine-stimulated NO release from these tissues, demonstrating a classic receptor mediated process (Fig. 2; Table 1). The addition of Met-enkephalin does not stimulate NO release in any of the tissues, demonstrating the µ3 nature of the process (Table 1). L-NAME, a cNOS inhibitor,
pretreatment of the various gastrointestinal tissues also antagonized morphine stimulated NO production, further demonstrating the validity of the observations (Fig. 2; Table 1). These results demonstrate that gut tissues exhibit the expected opiate alkaloid selective and opioid peptide insensitive characteristics of the µ3
opiate receptor, as well as its coupling to constitutive NO release as demonstrated in other tissues, including human (12 - 15).
DISCUSSION
Fig. 3.
The opiate alkaloid selectivity of the G protein coupled µ3 opiate receptor
subtype and its opioid peptide insensitivity, including to endomorphin-1, -2, correlates
with
the
coupling
of
µ3
to
cNOS-derived
NO
release
(14).
This
2
283
Table 1. Morphine stimulated cNOS derived NO in the mouse gut. Basal NO release is observed in all segments of the mouse gut. L-NAME (10
-4
M) significantly reduces basal NO release, further
demonstrating that we are measuring NO release. Naloxone (10 in all segments. Morphine (10
-6
-6
M) does not alter basal NO release
M) significantly enhances cNOS derived NO release in all segments
in a naloxone antagonizable manner. Furthermore, L-NAME antagonizes morphine's ability to release NO, demonstrating it is working via NO release. Peak nM NO ± SEM Treatment
Stomach
Small Intestine
Large Intestine
Untreated (basal)
1.2 ± 0.5
4.1 ± 1.0
2.0 ± 0.2
Morphine
13.0 ± 2.4
17.8 ± 2.5
14.3 ± 2.8
L-NAME (basal)
1.0 ± 0.5
2.1 ± 0.8
2.0 ± 1.1
Naloxone +Morphine
3.0 ± 0.5
3.5 ± 0.9
2.8 ± 0.6
L-NAME + Morphine
4.2 ± 1.7
3.0 ± 1.5
3.3 ± 0.5
Naloxone
0.8 ± 0.2
2.0 ± 0.4
1.9 ± 0.5
None detected
1.4 ± 0.7
0.7 ± 0.2
Met-enkephalin
morphine-stimulated NO release is naloxone-sensitive, antagonized by the NOS inhibitors N-nitro-L-arginine and L-NAME, and occurs in human immunocytes and endothelial cells and as in the vasculature of the median eminence (23, 24). In the present report, we now extend these observations to the mammalian gut. The selectivity of the µ3 opiate receptor subtype, therefore, provides further evidence for the status of morphine as an endogenous signaling molecule (15).
In examining the pharmacology of the gut, we find reports demonstrating that morphine inhibits the release of acetylcholine in the mesenteric plexus, increasing muscle tone and reducing peristalses, which in a postoperative setting prolongs
22
Nitric Oxide nM + SEM
20 18 16
Fig.
14
3.
Representative
data
demonstrating
the
concentration
12
dependent
nature of morphine stimulation
10
of
8
cNOS-derived
small
NO
in
intestine.
the
Each
experiment was repeated three
6
times
4 6
8
Concentration (10 -N M)
10
and
the
SEM
graphed.
Each point represents the mean of
3
readings
stomach,
small
obtained intestine
large intestine combined.
from and
284
ileus (25, 26). The substrate on which these actions depend appears to be the presence of the mu and kappa opioid receptor subtypes in the gut (16 - 18). These, and other investigations, conclude with the fact that opiate and opioid compounds are strongly involved with the ileus phenomenon. The involvement of NO as a gut signaling molecule has also been extensively studied. Several authors have concluded that inducible NO has a role in surgically induced
ileus
colleagues
(19
(20)
-
21).
Using
demonstrated
inducible
that
the
NO
NOS
knockout
activity
may
mice,
come,
in
Kalff part,
and from
circulating leukocytes, expanding the sphere of influence of the gut assault to immune components. Others demonstrate that a NOS inhibitor will potentiate carbachol-stimulated rat ileum activity, demonstrating NO involvement in down regulation (19). Additionally, iNOS involvement in an ileus only takes place after injury to the tissue creating a proinflammatory event (27). Inhibition of iNOS activity, in this regard, was found to improve gastric emptying and transit (28). Taken
together,
we
now
surmise
the
following:
1)
Morphine,
and
its
precursors, cannot be detected at the sensitivity of the assay employed in the mouse gut, whereas they are present in the central nervous system, adrenal gland and plasma (29); 2) The µ3 opiate receptor subtype appears to be found in gut
tissues, where it is coupled to constitutive NO release. Thus, the potential for endogenous morphine to represent a major pathway for ileus generation via NO becomes significant. Additionally, in this regard, morphine may exert this action acting as a hormone that emerges, i.e., increases its plasma concentration, after trauma, including surgical (8, 15, 23, 30-32). Thus, the trauma of manipulation may
trigger
an
ileus,
explaining
why
NOS
inhibitors
and
naloxone
may
alleviate/shorten the condition (19, 27, 28). When considering this hypothesis further, one must ask why these processes are present in the first place? The answer lies in the phenomenon of trauma and/or the "fight or flight" response of the autonomic nervous system, as well as the fact that morphine is endogenous (15, 23, 31). Indeed, under certain circumstances, perception/belief phenomenon.
In
may these
also
generate
reports,
we
an
have
input
into
surmised
this
that
stress-associated
morphine
generally
emerges to dampen, or diminish, the level of activation caused by trauma, helping to restore homeostasis (Fig. 4). Thus, we surmise morphine's normal role in the gut is to decrease activity, i.e., propulsion, potentially to divert energy/attention to where it is needed most. Thus, by limiting this vegetative function for a moment, the "fight" component of the sympathetic response becomes stronger. Morphine exerts this action via cNOS-derived NO release. How, then, does an ileus emerge? Taking the above into account, we surmise it emerges in a counter-intuitive manner, through the inhibition of cNOS. In past investigations, we have demonstrated that cNOS-derived NO, via autoregulation, can limit its own efficacy (33, 34). In this instance, too much signal molecule stimulation, i.e., morphine, causes cNOS-derived NO to become uncoupled in its regulation of iNOS activity, freeing iNOS-derived NO from this inhibition (22,
285
Fig. 4
Brain + Adrenal Gland Plasma Morphine (increases with trauma/stress) A. NO Normal Activity
Gut Decrease excitability, i.e., iNOS inhibition, Maintain Rhythmic Movement
cNOS-derived NO
B. DisturbanceMorphine Enhanced NO
Greater decrease in excitability, i.e., iNOS inhibition via cNOS-derived NO, and slow down gut motility, i.e., flight or fight response.
C. Extreme Trauma (proinflammatory) Morphine desensitization (tolerance)
cNOS-derived NO mediated processes become desensitized, i.e., iNOS up regulation, Allowing for iNOS-derived NO to induce an ileus.
Fig. 4. Speculative illustration of how gut activity may be controlled via nitric oxide and morphine. A. As demonstrated in Fig. 1 elements of the mammalian gut release NO at low levels and in an apparent rhythmic patter, i.e., in a pulsatile manner. We surmise this pulsatile nature is similar to that found in blood vessels see (36, 37). Thus the low and high components of the pulse have regulatory functions, allowing for excitability or diminishing it, respectively (37). In part this control may actually stem from the ability of NO to degranulate actin, controlling the very ability of the muscles in the gut wall to contract (36, 37). B. Under this scenario cNOS gets stimulated via morphine whose plasma levels becomes elevated due to some type of stimulation. This morphine originates from either the brain and/or the adrenal gland, which contains this material. Now, the enhanced morphine stimulates cNOS derived NO via the mu receptors found in the gastrointestinal tract to a higher level of production and subsequent release, providing for a greater period of down regulation, leading to a much lower level of gut motility. This scenario may work as a result of sympathetic stimulation, decreasing general vegetative functions, i..e., gut motility. C. In cases of extreme trauma, i.e., sepsis, the sustained and enhanced morphine levels exert no activity due to tolerance. This can also be observed in the lack of inhibition of iNOS expression following exposure to cNOS derived NO (22, 34, 35, 39, 40). Thus, counter intuitively, iNOS expression predominates allowing for an ileus to develop.
34, 35). Under these circumstances any trauma would activate iNOS, including endogenous fauna of the gut or various proinflammatory cytokines, giving rise to an ileus. In essence, the gut becomes free of the disinhibition action of cNOSderived NO (36) (Fig 4). This can easily be understood given the basal level of
4
NO that is released from gut tissues. Here, basal NO limits the degree of tissues
286
excitation on a second by second basis, allowing for the pulsatile regulatory effect of NO (36, 37). All in all, the role of endogenous morphine in gut regulation appears to complement
the
brain-gut
axis
via
local
modulation
of
NO
generation.
Supporting this communication is the presence of endogenous morphine in the brain
and
receptors,
adrenal but
morphine
not
tissues,
and
morphine,
functions
as
a
blood, in
the
gut
as
well
gut.
as
This
hormone.
the
presence
alone
In
of
strongly
part,
also
mu
opiate
suggests
supporting
that this
communication, is the presence of intestinal parasites, i.e., Ascaris suum, that produce
and
liberate
morphine
in
the
gut
(38).
Here,
the
worm
enters
this
communication to, again, slow down gut movements, preserving its environment. Acknowledgements: This work was supported in part by NIDA grant number DA09010. µ3 Opiate Receptor, GenBank Accession # AY195733.
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R e c e i v e d : December 15, 2003 A c c e p t e d : January 30, 2004
Authors address: George B. Stefano, Ph.D., Neuroscience Research Institute, State University of New York, College at Old Westbury, Old Westbury NY 11568, USA, Phone: 516-876-2732; Fax: 516-876-2727 E-mail:
[email protected]
