1 Divisions of Gastroenterology and Hepatology, Department of Internal Medicine, and 2 Department of Pharmacology, Virginia Commonwealth University- Medical College of Virginia, Richmond, Virginia 23298-0711
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ABSTRACT |
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The
endogenous cannabinoid anandamide causes hypotension and mesenteric
arteriolar dilation. A detailed analysis of its effects on systemic and
portal venous hemodynamics had not yet been performed. We assessed the
effects of anandamide (0.4-10 mg/kg) on systemic and portal
hemodynamics with and without prior treatment with various antagonists.
The specific antagonists used included SR-141716A, N-nitro-L-arginine methyl ester,
indomethacin, and nordihydroguaiaretic acid. Anandamide produced a
dose-dependent decrease in mean arterial pressure due to a drop in
systemic vascular resistance (SVR) that was accompanied by a
compensatory rise in cardiac output. Anandamide also elicited an
increase in both portal venous flow and pressure, along with a decline
in mesenteric vascular resistance (MVR). Pretreatment with 3 mg/kg
SR-141716A, a CB1 antagonist, prevented the decline of SVR
and MVR from the lower dose of anandamide. Antagonism of nitric oxide
synthetase, cyclooxygenase, or 5-lipoxygenase did not prevent the
systemic nor the portal hemodynamic effects of anandamide. Furthermore,
the use of R-methanandamide, a stable analog of anandamide, produced
similar hemodynamic effects on the mesenteric vasculature, thereby
implying that the effects of anandamide are not related to its
breakdown products. Anandamide produced profound, dose-dependent
alterations in both the systemic and portal circulations that could be
at least partially blocked by pretreatment with SR-141716A.
portal vein flow; portal vein pressure; cannabinoids; SR-141716A; portal hypertension; blood pressure; splanchnic blood flow; cirrhosis
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INTRODUCTION |
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THE REGULATION OF BLOOD FLOW through various circulatory beds is complex and incompletely understood. It is, however, appreciated that the blood flow and pressure in any given vascular bed depend on a balance between vasodilatory and vasoconstrictive factors. Over the last 10 years, nitric oxide (NO) has gained predominance as the major vasodilatory factor that controls vascular tone (19). However, it is possible as well as probable that other vasodilatory pathways also exist that may affect both regional and systemic blood flow.
In 1992, the first endogenous cannabinoid was isolated and identified as arachidonyl ethanolamide (anandamide) (6). Recent studies (23, 24), have shown that anandamide produces hypotension and bradycardia in anesthetized rats. It has also been shown to cause mesenteric arterial dilation in isolated mesenteric arterial preparations (28). Theoretically, increased mesenteric arterial dilation could lead to increased portal venous flow (PVF) and portal venous pressure (PVP) (10). However, the effects of anandamide on portal hemodynamics have not been previously defined. Also, other than the effects of anandamide on heart rate and blood pressure, a detailed analysis of the systemic hemodynamic effects of anandamide have not been performed.
The objective of the present study was to perform a detailed characterization of the effects of anandamide on the portal and systemic circulation in normal, anesthetized rats. This was done by simultaneous measurement of heart rate, blood pressure, cardiac output (CO), systemic venous pressure, mesenteric arterial flow, PVF, and PVP.
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MATERIALS AND METHODS |
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Animals. Male Sprague-Dawley rats (Harlan, Indianapolis, IN) weighing 250-350 g were used for all experiments. The animals were maintained on a 12:12-h light/dark cycle and allowed rat chow and water ad libitum in the animal care facility at the Virginia Commonwealth University-Medical College of Virginia. All animals were allowed to acclimate for several days before surgery. All studies were approved and performed in compliance with the guidelines of the institutional animal use and care committee.
Experimental technique.
Anesthesia was induced with the intraperitoneal administration of
pentobarbital sodium at a dose of 50 mg/kg. The animal was then placed
on a homeotherm blanket (Harvard Apparatus, South Natick, MA) to
maintain body temperature between 37.0 and 38.0°C. Once anesthesia
was induced, the right femoral vein was cannulated with PE-50 tubing
(Becton Dickinson, Franklin Lakes, NJ), and 0.9% NaCl was infused
intravenously at a rate of 0.02 cm3 · g1 · h
1
by a syringe pump (Razel Scientific Instruments, Stamford, CT) to
compensate for evaporative losses due to the surgical preparation. A
fluid-filled PE-50 tube was used to cannulate the right internal jugular vein and served as the route of drug administration as well as
the route for administration of saline for transpulmonary thermodilution measurements of CO. Supplemental pentobarbital sodium
(10 mg/kg) was given intravenously as indicated on the basis of
spontaneous muscle activity or lessening of the anesthetic plane. A
tracheostomy was performed using PE-190 tubing to maintain airway patency.
Drugs.
Anandamide was obtained from Deva Biotech (Hatboro, PA). SR-141716A
(CB1 receptor antagonist) was obtained from the National Institute of Drug Abuse.
N-nitro-L-arginine methyl ester
(L-NAME, an inhibitor of NO synthetase), indomethacin, and
nordihydroguaiaretic acid (NDGA; 0.1 mg/kg; a 5-lipooxygenase
inhibitor) were all obtained from Sigma (St. Louis, MO). The vehicle
for all drugs except L-NAME consisted of
emulphor:ethanol:saline 1:1:8. Emulphor is a polyoxyphenylated vegetable oil. L-NAME was prepared in 0.9% normal saline
before each experiment.
Study design and experimental protocols. In the first set of experiments, at the end of the stabilization period, the animals received anandamide at varying doses (0.4, 4, and 10 mg/kg). Mean arterial pressure (MAP), central venous pressure (CVP), PVP, and PVF were measured continuously for 10 min following each dose. By using the transpulmonary thermodilution technique (3), CO was obtained at 1, 2, 3, 5, and 10 min after drug administration by injecting 150 µl of saline at room temperature into the right internal jugular vein. In selected animals, the superior mesenteric arterial flow was measured as well.
Following completion of the first set of studies, a second set of experiments was performed to determine the interactions between anandamide and other known regulators of vascular tone and blood flow. Following anesthesia and surgical preparation, animals received pretreatment with one of four drugs administered 10 min before administration of anandamide (4 or 10 mg/kg): SR-141716A (3 mg/kg), L-NAME (6.25 mg/kg), indomethacin (5 mg/kg), or NDGA (0.1 mg/kg).Data acquisition and storage. All pressure transducers were connected to a analog-digital converter (Transonic Systems) that had up to 16 independent ports. The zero point for all pressure transducers was set to the level of the right heart. CO was measured using well-established thermodilution methods (3) by using a Cardiotherm 400-R instrument (Columbus Instruments, Columbus, OH) connected to the thermodilution catheter in the carotid arch. PVF and mesenteric arterial flow were directly measured using the T206 small animal flow meter. All data were recorded simultaneously in real time on a Pentium II personal computer using Windaq data acquisition software (Dataq Instruments, Akron, OH). Data were stored in electronic files using the Windaq software.
Calculations and data analysis.
MAP was calculated using the Windaq software by obtaining the average
of the arterial waveform over a 3-s period. Systemic vascular
resistance (SVR) was calculated from: [(MAP CVP)/CO] · 80 (15). Mesenteric vascular
resistance (MVR) was similarly calculated as [(MAP
PVP)/PVF] · 80. Data from Windaq software were extracted onto
a Microsoft Excel 7.0 spreadsheet for individual experiments, and
summary data from multiple experiments were analyzed on a separate
spreadsheet. Statistical analyses were performed using both Microsoft
Excel 7.0 and SPSS software (SPSS, Chicago, IL). A P value
<0.05 was considered significant.
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RESULTS |
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Detailed analyses of systemic and splanchnic hemodynamics were
performed in an initial set of five rats. Before each experiment, all
pressure transducers were calibrated against a sphygmomanometer, and
the flowmeter was calibrated via an internal calibration system per the
manufacturer's instructions. Before initiation of each experiment,
stable normal baselines were documented in each case for at least 20 min. Raw data measurements were made simultaneously, as shown in Fig.
1.
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Effects of anandamide on systemic hemodynamics.
Anandamide produced a profound, dose-dependent drop in arterial
pressure within 2 min of administration. The effects of anandamide on
systemic hemodynamics are shown in Fig.
2. Although 0.4 mg/kg of anandamide had
relatively minor effects on the MAP (Fig. 2A), both 4 and 10 mg/kg produced a marked, significant decrease in MAP
[P = 0.008 for 4 mg/kg and P = 0.01 for 10 mg/kg by paired t-test (baseline vs. maximal
decrease)]. The effects of anandamide were most pronounced within the
first 1-3 min following intravenous administration, and the MAP
returned to baseline values within 10-15 min. A transient pressor
response, as noted previously (23), lasting <1 min after
administration, was seen particularly with the higher dose of
anandamide (10 mg/kg).
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The effects of anandamide on the portal venous circulation.
At baseline, PVP varied from 6.29 to 8.9 mmHg, whereas PVF varied from
5.31 to 9.01 ml/min, which are well within published values (8,
20, 21). Anandamide produced a dose-dependent increase in PVF
and PVP (Fig. 3, A and
B). In contrast to its systemic effects (Fig. 2), an
increase in PVF was seen at even the lowest dose of anandamide (0.4 mg/kg). This was even more pronounced at higher doses of anandamide.
These effects were short-lived, and PVF returned to baseline within 5 min. The increase in PVF was accompanied by an increase in PVP.
However, at all doses, the percent increment in PVF was almost twice
that seen for PVP.
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Effects of pretreatment with specific antagonists.
To test the possibility that the anandamide effects were mediated, at
least partially, by either NO or prostaglandins, the effects of
anandamide were restudied in another set of animals (n = 9) following pretreatment with specific blocking antagonists (Fig.
4). In each given animal, pretreatment
with only a single antagonist was done. The hypotensive effects of
anandamide were not significantly blocked by L-NAME,
indomethacin, or NDGA. In animals receiving L-NAME, there
was an increase in resting MAP after L-NAME administration.
However, on subsequent administration of anandamide, a dose-dependent
hypotensive effect was obtained.
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DISCUSSION |
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The hypotensive effects of tetrahydrocannabinol (THC), the major psychoactive component of marijuana, were established almost 30 years ago (7). More recently, these properties were also described for anandamide (24), an endogenous ligand for cannabinoid receptors (6). The present study corroborated previous findings (23, 24) that anandamide produced a profound systemic hypotension and further demonstrated that the hypotensive effects of anandamide were due to a decrease in SVR.
The effects of anandamide on SVR most likely represented a composite of its effects on various regional circulatory beds. For example, THC (2, 18) has been shown to increase cerebral circulation, and anandamide has been found to increase renal blood flow by producing afferent arteriolar dilation (5). It is also recognized that mesenteric arterioles are dilated by anandamide (22, 28). However, the effects of anandamide-induced mesenteric arteriolar dilation on portal venous hemodynamics had not been previously characterized.
An important observation in the present study was that anandamide increased both PVF and PVP. It is interesting to note that the degree in increment in PVF did not translate into a similar degree of rise in PVP. A potential explanation for this observation could be that anandamide decreases resistance to flow through the liver at the level of the hepatic sinusoids. Such an effect would dampen the rise in PVP due to an increase in PVF. Indeed, we have recently demonstrated the presence of CB1 receptor mRNA (16) on both normal and cirrhotic hepatic sinusoidal endothelium, suggesting that potential targets for endogenous cannabinoids exist in the hepatic microcirculation. However, this hypothesis remains to be experimentally verified.
The nearly complete blockade of low dose (4 mg/kg) anandamide-induced decreases in SVR, MVR, and MAP as well as the increase in PVF and PVP by SR-141716A indicated that these effects were mediated via CB1 receptors. These data are in agreement with the loss of anandamide-induced hypotension in CB1 receptor knockout mice (14). Also, the preservation of the hypotensive effects with R-methanandamide further demonstrated that the observed effects were due to anandamide itself rather than its breakdown products.
In contrast, the effects of high-dose anandamide were more intriguing. Pretreatment with SR-141716A had virtually no effect on the decrease in SVR or MVR produced by subsequent administration of anandamide (10 mg/kg). A potential explanation could be that this was due to a high dose of the agonist (anandamide) in the presence of a relatively low dose of the competitive antagonist (SR-141716A). If so, one would expect similar hemodynamic changes in pretreated vs. non-pretreated animals. The near-identical drop in SVR in pretreated animals compared with non-pretreated animals was, however, associated with only a limited drop in MAP after anandamide administration. This was mainly due to a greater increase in CO than that noted in non-pretreated animals, which tended to keep the SVR down and MAP up. The basis for these observations remains to be experimentally defined.
It has been suggested (23) that anandamide acts at a
presynaptic location to decrease norepinephrine release by sympathetic neurons, thereby decreasing sympathetic vasoconstrictive tone and heart
rate. Indeed, CB1 receptors have also now been identified on sympathetic nerve fibers innervating resistance vessels
(17). The hypotensive effects of the synthetic
cannabinoids WIN-55212-2 and HU-210 are, however, retained in
sympathetomized rats receiving norepinephrine infusions
(26). Also, the maximal hypotension with HU-210 exceeds
that seen after -adrenergic blockade (13), indicating
that additional peripheral vasodilatory effects are also present. The
documentation of CB1 receptors, anandamide, and the
amidases that break down anandamide in endothelial cells and renal
tissue (5) supports such a concept.
Recent studies have focused on the role of both direct cannabinoid receptor-mediated effects on vascular smooth muscle cells (12) and indirect endothelium-dependent effects (5, 28). Binding of anandamide to CB1 receptors has been shown to increase NO in renal vasculature (5). Although the present studies could not demonstrate blockade of anandamide-induced hypotension by L-NAME, this does not completely exclude the possibility that endothelial CB1 receptors mediate localized NO-dependent vasodilation in some regional vascular beds.
A key question relates to the potential role of anandamide and other endogenous cannabinoids under normal and disease states. The failure to demonstrate major hemodynamic effects following administration of SR-141716A may not necessarily be construed to indicate a lack of substantive effects in normal rats. For example, it has been proposed that, following a meal, a rise in interstitial Ca2+ concentration causes release of anandamide from perivascular nerve terminals (4, 11), which can then produce vasodilation by both endothelium-dependent, SR-141716A-sensitive and endothelium-independent, SR-141716A-insensitive pathways. A potential mechanism for the latter pathway may involve interaction with capsaicin-sensitive vanilloid receptors on sensory nerve terminals, which cause release of calcitonin gene-related peptide (CGRP) and cause CGRP-mediated vasodilation (29). The current study was not designed to examine such effects, and these now require further research.
It has also been shown that exposure to endotoxin (Escherichia coli lipopolysaccharide) stimulates release of both anandamide and 2-arachidonoyl glycerol from platelets and macrophages (25). Under conditions in which platelets and/or macrophages are activated, it is possible that the release of anandamide may cause vasodilation and contribute to hypotension. Indeed, anandamide has been shown to contribute to severe irreversible hemorrhagic shock (27). Theoretically, the release of anandamide may also contribute to the local vasodilation seen in areas of inflammation as well as the mesenteric and systemic arterial vasodilation present in cirrhosis (9). Although much additional work is necessary, the current studies provide an important basic step in the right direction and should provide the groundwork for future studies to define the role of endogenous cannabinoids under normal and pathological circumstances.
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ACKNOWLEDGEMENTS |
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This work was supported in part by grants from the National Institute of Diabetes and Digestive and Kidney Diseases (DK-02755 and DK-07150-23).
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FOOTNOTES |
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Address for reprint requests and other correspondence: A. J. Sanyal, MCV Box 980711, Medical College of Virginia, Richmond, VA 23298-0711 (E-mail:ajsanyal{at}hsc.vcu.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 3 March 2000; accepted in final form 26 July 2000.
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