(Received for publication, October 12, 1995)
From the
Initial confinement of opiate receptors to the nervous system
has recently been broadened to several other cell types. Based on the
well established hypotensive effect of morphine, we hypothesized that
endothelial cells may represent a target for this opiate substance.
Endothelial cells (human arterial and rat microvascular) contain a high
affinity, saturable opiate binding site presumed to mediate the
morphine effects that is stereoselectively and characteristically
antagonized by naloxone. This opiate alkaloid-specific binding site is
insensitive to opioid peptides. It is, therefore, considered to be the
same subtype of opiate receptor (designated µ) used in
the mediation of morphine in other cell types exhibiting the same
binding profile. Experiments with endothelial cultures and the aortic
ring of rats cultured in vitro demonstrate that morphine
exerts direct modulatory control over the activities of endothelial
cells, which leads to vasodilation. It induces the production of nitric
oxide, a process that is sensitive to naloxone antagonism and nitric
oxide synthase inhibition. In contrast with that of opiates, the
administration of opioid peptides does not induce nitric oxide
production by endothelial cells. In conclusion, the data presented
above reveal a novel site of morphine action, endothelial cells, where
a µ
receptor is coupled to nitric oxide release and
vasodilation.
The vast majority of pharmacological studies of the properties
of opiate substances have long been almost exclusively concerned with
their effects on analgesic and antinociceptive phenomena. More
recently, a number of experiments have demonstrated that morphine
modulates the activity of varieties of cell types, among them the
immunocytes of several mammalian and invertebrate
species(1, 2) . In addition, this largely
down-regulating effect was found to be mediated by a highly specific,
opiate alkaloid-sensitive receptor used selectively by
opiates(1, 2) . In the present case, the receptor
(µ) accomplishes this by counteracting the cellular
responsiveness to a number of immunoexcitatory molecules, e.g. lipopolysaccharides and some cytokines (see (3) ).
In this context, morphine was found to be quite potent in lowering or terminating the activation of human granulocytes and monocytes exposed to the stimulatory activity of plasma obtained from cardiopulmonary bypass patients(4, 5, 6, 7) . From these observations, we surmised that a proportion of these cells may have been derived from intravascular immune cells whose adhesiveness to the vascular lining may have been altered by the presence of morphine in this tissue.
The present study was aimed at the exploration of
the possibility that endothelial cells may be under the direct control
of the opiates. It provided evidence for the specific binding of
morphine to endothelial cells, resulting in stimulation of nitric oxide
(NO) ()production in a naloxone-reversible manner and
relaxation of blood vessels. It also demonstrated that these activities
are mediated by the special opiate receptor µ
present
in the endothelial cells.
Immediately
prior to the binding experiment, the S1` supernatant was centrifuged at
30,000 g for 15 min, and the resulting pellet (P2) was
washed once by centrifugation in 50 volumes of the sucrose/Tris-HCl.
The P2 pellet was then resuspended with a Dounce hand-held homogenizer
(10 strokes) in 100 volumes of buffer. Binding analysis was then
performed on the cell membrane suspensions.
Aliquots of membrane
suspensions from these cells were incubated with nonradioactive
compounds at six concentrations for 10 min at 22 °C and then with
[H]dihydromorphine (3DHM) for 60 min at 4 °C.
One hundred percent binding is defined as bound 3DHM in the presence of
10 µM dextrorphan minus bound 3DHM in the presence of 10
µM levorphanol. K
is defined as the
concentration of drug that elicits half-maximal inhibition of specific
binding. The mean ± S.E. for three experiments is given. The
displacement analysis data indicate the potency of various opioid
(Met-enkephalin and D-Ala
-Met
-enkephalin, Sigma) and
opiate substances in displacing 3DHM (58 Ci/mM, DuPont NEN) and may
give specific information on different receptor populations. The
incubation medium for Met-enkephalin contained phosphoramidon (100
µM) and bestatin (100 µM) to inhibit enzyme
degradation.
NO release was monitored with an NO-selective microprobe manufactured by Inter Medical Co. (Nagoya, Japan). The working electrode made of platinum/iridium alloy was coated with a film containing KCl, NO-selective nitrocellulose resin (pyroxyline lacquer), and a gas-permeable silicon membrane(10) . A counter electrode was made of carbon fiber. The redox current was detected by a current-voltage converter circuit and continuously recorded. Tip diameter of the probe (25 µm) permitted the use of a micromanipulator (Zeiss-Eppendorff) attached to the stage of an inverted microscope (Nikon Diaphot) to position the sensor, which was enclosed in a Faraday's chamber, 3-5 µm above the cell surface. Calibration of the electrochemical sensor was performed by use of different concentrations of a nitrosothiol donor S-nitroso-N-acetyl-DL-penicillamine, as previously detailed(10) .
Rings of thoracic aorta (3 mm) were placed in PSS, and NO release in response to 1 µM morphine was evaluated by use of a computer-interfaced NO-specific amperometric probe as described above.
Membrane homogenates of human arterial and rat microvascular
endothelial cells revealed opiate binding sites and their ligand
specification ( Fig. 1and Table 1). Saturation and
Scatchard analysis showed a single, relatively high affinity binding
site with K values of 38 and 19 nM, with B
values of 1,167 and 1,098 fmol/mg membrane
protein for human and rat-derived endothelial cells, respectively ( Fig. 1and Table 1). Nonspecific binding increased linearly
with respect to the concentration of the binding ligand (Fig. 1, inset). Furthermore, a variety of opioid peptides was found to
be ineffective in displacing specifically bound 3DHM (Table 1).
By contrast, the opiate alkaloid ligands were the most potent, and
ligands dynorphin 1-17 and ethylketocyclazocine were weak.
Interestingly, fentanyl was quite poor in this regard. Naloxone was
found to be less potent than naltrexone in counteracting 3DHM binding.
Of interest was the finding that the µ opioid peptide DAGO was
ineffective in displacing 3DHM (Table 1). Of equal interest was
that the displacement profile for all ligands in both types of
endothelial cells was identical. This profile demonstrating opiate
alkaloid sensitivity and opioid peptide insensitivity is characteristic
of the presence of the µ
opiate receptor (1) .
Figure 1: Scatchard analysis of 3DHM binding to human arterial endothelial cells. Inset, circle represents saturation of specific sites and box represents nonspecific binding, which continues in a linear fashion.
Experiments with endothelial cells and aortic rings examined in
vitro have revealed a direct modulating influence of morphine on
the activities of these cells. Morphine resulted in a dose-dependent
release of NO from endothelial cells ( Fig. 2and Fig. 3).
The effect was blocked by naloxone. By contrast, opioid peptides or the
µ opioid receptive DAGO (10 µM) did not effect any
changes in NO release (data not shown). For comparison, the effect of a
well established stimulator of NO release, bradykinin, is presented.
The effect of morphine is qualitatively and quantitatively similar to
that of bradykinin, with no delay in NO release (Fig. 2).
Furthermore, pretreatment of the human endothelial cells with L-nitroarginine methyl ester (10M for 5 min before morphine exposure), a nitric oxide synthase
inhibitor, also abolished morphine-induced (10
M) NO release (data not shown).
Figure 2:
In vitro stimulation of NO
production by morphine. Representative recordings of NO in the
incubation medium prior to and following opiate and opioid exposure are
shown (arrow, M, morphine). a, 50 nM morphine; b, 100 nM morphine; c, 200
nM morphine; d, 200 nM naloxone + 200
nM morphine. ME, 100 nM Met-enkephalin; DAME, 100 nM
[D-Ala,Met
]enkephalin.
Bradykinin (BK, 10
M) was added
for comparison.
Figure 3: Morphine-induced relaxation of rat aorta. Morphine (1 µM) was added to aortic rings in the absence (boldface line) or presence (narrow line) of naloxone (5 µM). The data shown are representative of n = 4 experiments, which did not vary. Inset, stimulation of aortic rings with morphine (1 µM) resulted in a marked increase in NO release (boldface line) that was abrogated by pretreatment with naloxone (5 µM, narrow line). The data shown are representative of n = 4 experiments, which did not vary.
In intact aortic rings, morphine (1 µM) induced relaxation, a phenomenon that was attributable to NO and that was naloxone-reversible (Fig. 3). DAGO (10 µM) did not cause relaxation of the vascular rings (data not shown). In aortic rings denuded of the endothelial layer all opiate actions were lost.
In view of the
distinct vasorelaxing action of morphine (12) and the presence
of the µ opiate receptors on endothelial cells
expressed with comparative density at the level of conduit and
resistance arteries, the effects of morphine on vascular endothelium
can be considered to be direct and not via interaction with the neural
or neuroendocrine systems.
The present report demonstrates the following. 1) Endothelial
cells (human arterial and rat microvascular) contain a high affinity
saturable opiate binding site that is stereoselectively and
characteristically antagonized by naloxone. 2) This binding site is
opiate alkaloid-specific and opioid peptide-insensitive. 3) The binding
to this site as well as to other cell types exhibiting this novel
binding profile (Table 1) is designated to be of the µ opiate receptor subtype. 4) Morphine can induce the production of
NO from MVE cells and rat aortic ring endothelial cells in
vitro, a phenomenon that is sensitive to naloxone antagonism. 5)
Aortic rings respond to morphine by relaxation which was endothelial
dependent. 6) Opioid peptides do not induce in vitro NO
production or relaxation of the aortic ring. 6) Endothelial NO
production, therefore, appears to be mediated by the µ
opiate alkaloid-specific receptor.
With regard to the
interaction of NO and opiate substances mentioned earlier, recent
studies suggest a definite link. Nitric oxide has been associated with
antinociception (13, 14, 15) as well as the
states of tolerance and dependence(16) . Peripheral morphine
analgesia involves NO-stimulating cGMP(17) . Morphine-depressed
concanavalin A stimulated macrophage NO production(18) .
Morphine and NO have been linked in gastrointestinal regulation (19) and in food intake(20, 21) . Thus, the
present report further documents this interaction, for the first time,
in endothelial cells mediated by way of the µ receptor.
The specificity of the opiate receptor subtype (µ)
in mediating endothelial NO production substantiates the strict
involvement of opiate alkaloids or similar substances in this process
and simultaneously excludes the involvement of opioid peptides. The
µ
opiate-selective receptor has now been found on human
monocytes and granulocytes(1, 2) , invertebrate and
vertebrate microglia(22, 23) , neuronal cell lines (24) , and, in a preliminary study, on specific invertebrate
neurons (25) (see Table 1). Clearly, the presence of
such a highly selective opiate receptor strongly suggests that
endogenous substances exist that make use of this receptor. In this
regard, naturally occurring morphine appears to be a strong
candidate(3) .
The present study demonstrates that opiate
alkaloids have the potential to mediate vasodilation by way of
regulation of NO production. Many reports suggest that opiates, in this
regard, mediate their effects by way of the central nervous system.
Effects of intravenously injected morphine in the rat include depressor
response and bradycardia(12) . These investigators have
demonstrated that the specific µ opioid receptor agonist, DAGO,
reproduced hemodynamic effects of morphine. Effects of either agonist
were attenuated by the µ-specific antagonist
-funalfrexamine, whereas naloxonazine, the
µ
-specific antagonist, inhibited the effects of DAGO
but not those induced by morphine(12) . In conscious
chronically instrumented pigs, morphine at a high dose (1 mg/kg bolus
intravenously) induced tachycardia, elevation in mean systemic and
pulmonary arterial pressure, but did not change stroke volume or
peripheral vascular resistance(26) . These data indicate that
in pigs, the species that shows an excitatory response to morphine,
hemodynamic changes were largely induced by tachycardia. Apart from its
well established hemodynamic effects, morphine has been shown to
attenuate vasopressor responses to angiotensin II or substance
P(27) . Furthermore, the pressor effect of social deprivation
during 1-15 days of isolation in rats was inhibited by
administration of morphine; however, 7 days after morphine withdrawal
elevation of blood pressure occurred in these rats(28) . In a
study on morphine-induced hypotension, Calignano et al.(29) suggested that it was mediated by adenosine. Based on
the results of the present study, endothelial cells are capable of
mediating a direct action of morphine or morphine-like molecules. Thus,
future studies must be concerned with this phenomenon and its
involvement in the regulation of vasomotor responsiveness.