Morphine
Down Regulates Human Vascular Tissue Estrogen Receptor Expression
Determined by Real-Time RT-PCR
Abstract
| Introduction | Materials
and Methods:
Material; Treatment; Isolation of Total RNA; Reverse Transcription;
Taq-Man Probe Real-Time Reverse Transcription-Polymerase Chain
Reaction (RT-PCR ); Sequencing of the cloned PCR Products
| Results | Discussion
| Acknowledgements
Patrick
Cadet (1),
Kirk Mantione (1),
Thomas V. Bilfinger (1,2)
George
B. Stefano
(1,2)
1. Neuroscience Research Institute, State University of New
York at Old Westbury, NY 11568, USA.
2. Division of Cardiothoracic Surgery, Department of Surgery,
State University of New York at Stony Brook, Stony Brook,
NY
Corresponding
author: George B. Stefano, Neuroscience Research Institute,
State University of New York Old Westbury, P.O. Box 210 Old
Westbury, NY 11568
Key words:
morphine, human vascular endothelial cells, estrogen, estrogen
receptors, nitric oxide
Abstract
Human vascular endothelial cells express the estrogen receptor-b
(ER-b), which can be modulated
by the opiate alkaloid morphine.
OBJECTIVES: To determine if morphine is capable of down regulating
the ER-b receptor in a similar
fashion as the m opiate receptor since they are both coupled
to constitutive nitric oxide synthase derived nitric oxide
release.
METHODS AND RESULTS: Endothelial cells obtain from human vascular
tissues (saphenous vein, atria and primary saphenous vein
cells) were treated with 1 mM morphine plus or minus the m
opiate receptor antagonist naloxone or CTOP (10 µM) for 24
h at 37 °C. Total RNA was isolated from treated and untreated
primary endothelial cells, and specific primers and a probe
were used to determine the ER-b
gene expression by real-time RT-PCR. Cells treated with morphine
exhibited a down-regulation of ER-b,
whereas naloxone and CTOP were able to partially block the
morphine effect. In addition, the 266 bp fragment generated
by RT-PCR using the same primers as in the real-time PCR was
sequenced and revealed a 100% sequence identity as the authentic
ER-b gene sequence.
CONCLUSIONS: These results indicate that ER-b
is expressed in human vascular endothelial cells, and morphine
appears to regulate this receptor in a similar fashion as
the m opiate receptor.
Introduction
Recently, we have demonstrated the expression of the estrogen
receptor (ER) subtype b on human endothelial and that it is
coupled to constitutive endothelial nitric oxide synthase
(cNOS) derived nitric oxide (NO) release[1]. This finding
also has physiological significance due to the fact that in
rats it can selectively affect median eminence neurosecretory
processes [2].
Furthermore,
over the last six years we have also demonstrated that human
endothelial cells express on a pharmacological, biochemical
and molecular level the opiate selective and opioid peptide
insensitive opiate alkaloid receptor subtype designated m3
see [3-7]. Additionally, we have demonstrated that morphine
and the NO donor S-nitroso-N-acetyl-penicillamine (SNAP) causes
a down regulation of the mu receptor gene expression in human
vascular endothelium and mononuclear cells [5,8]. These reports
also documented that morphine stimulates ceNOS derived NO
release, which may modulate median eminence neurosecretion
[9], demonstrating a common process, i.e., endothelial NO
release, mediated by estrogen and morphine.
Given
this NO convergence by both signal molecules we sought to
determine if there is a relationship between them. In this
regard, we demonstrate that morphine can down regulate the
expression of the ER-b receptors
found on human endothelial cells.
Materials
and Methods
Material
Saphenous vein (SV) segments were obtained from patients undergoing
elective coronary artery bypass grafting (CABG) for atherosclerotic
coronary artery disease. Saphenous vein segments were obtained
from patients (four post-menopausal women, mean age 74.5 ±
10.3 years and eight men mean age 57.6 ± 13.3 years).
This material is regarded as waste and the institutional review
board approved the project. Patients with chronic illnesses,
such as diabetes or cancer as well as acute processes (e.g.,
known infections, trauma), were excluded. In all patients
undergoing CABG, the analgesic management included induction
with fentanyl (fentanyl citrate adjusted for pH) or sufentanil
(N-[-4(methoxymethyl0-1-[2-(2 thienyl) ethyl]-4-piperidinyl]-N-phenylpropanamide
2 hydroxy-1,2,3 propanetricaboxylate) up to 15 ug/kg. Maintenance
was achieved with the same agents. Parts of SV that were not
used in the CABG procedure were stored in an electrolyte solution
at 4 °C (500 cc plasmalyte with 5000 U heparin), and these
specimens were immediately transported on ice to the laboratory
for processing. Immediately upon arrival, each blood vessel
was washed in phosphate buffered saline (PBS). SV specimens
were prepared as previously described [10].
Primary human saphenous vein endothelial cells were obtained
from Clonetics (Walkersville, MD). The cells were seeded in
a 75 cm2 flask at 1 x 107 cells in EGM2 media (Clonetics)
as recommended and incubated at 37 °C with 5% CO2.
Treatment
Primary human SV endothelial cells were stimulated with 1
uM of morphine. When specific receptor antagonists naloxone
(10 uM) or CTOP (10 uM) were employed they were administered
10 min prior to the agonist. Tissues were exposed to the drugs
for a total of 24 h. In the case of pure agonist exposure,
10 min prior to its administration PBS was added in the same
volume. Controls consisted of tissues not exposed to drugs.
All drugs were purchased from Sigma Chemical CO. (St. Louis,
MO).
Isolation
of Total RNA
... ...
Isolation
of Total RNA
... ...
Reverse
Transcription
... ...
Taq-Man Probe Real-Time Reverse Transcription-Polymerase Chain
Reaction (RT-PCR )
... ...
Sequencing
of the cloned PCR Products
... ...
Results
Discussion
Acknowledgements
This work was supported by the following grants: NIDA 09010,
NIMH 47392 and the Research Foundation and Central Administration
of the State University of New York and NIH Fogarty INT 00045.
