Involvement of central β 2 -adrenergic, NMDA and thromboxane A 2 receptors in the pressor effect of anandamide in rats - PDF

Naunyn-Schmied Arch Pharmacol (2010) 381: DOI /s ORIGINAL ARTICLE Involvement of central β 2 -adrenergic, NMDA and thromboxane A 2 receptors in the pressor effect of anandamide

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Naunyn-Schmied Arch Pharmacol (2010) 381: DOI /s ORIGINAL ARTICLE Involvement of central β 2 -adrenergic, NMDA and thromboxane A 2 receptors in the pressor effect of anandamide in rats B. Malinowska & A. Zakrzeska & C. M. Kurz & M. Göthert & G. Kwolek & P. Wielgat & J. J. Braszko & E. Schlicker Received: 9 January 2010 / Accepted: 6 February 2010 / Published online: 3 March 2010 # Springer-Verlag 2010 Abstract Intravenous (i.v.) injection of the endocannabinoid anandamide induces triphasic cardiovascular responses, including a pressor effect mediated via unknown central and peripheral mechanism(s). The aim of the present study was to determine the central mechanism(s) responsible for the pressor response to anandamide. For this purpose, the influence of antagonists at thromboxane A 2 TP (sulotroban, daltroban, SQ 29548), NMDA (MK-801) and β 2 -adrenergic receptors (ICI ) on the pressor effect induced by i.v. and intracerebroventricularly (i.c.v.) administered anandamide was examined in urethane-anaesthetized rats. Anandamide (1.5 3 µmol/kg, i.v.) or its stable analogue methanandamide (0.75 µmol/kg, i.v.) increased blood pressure by 25%. Anandamide (0.03 μmol per animal i.c.v.) caused a pure pressor effect (by 20%) but only in the presence of antagonists of CB 1 and TRPV1 receptors. The effects of cannabinoids (i.v. or i.c.v.) were diminished by i.v. daltroban, sulotroban (10 μmol/kg each), and/or SQ (1 μmol/kg). The effect of anandamide i.v. was reduced by SQ (0.02 μmol per animal i.c.v.) and by the thromboxane A 2 synthesis inhibitor furegrelate i.c.v. (1.8 µmol per animal). ICI , MK-801 (1 µmol/kg i.v. each), and bilateral adrenalectomy diminished the effect of anandamide i.c.v. Sulotroban (i.v.) failed to affect the response to anandamide (i.v.) in pithed rats, and anandamide and methanandamide did not bind to TP receptors in rat platelets. The present study suggests that central β 2 -adrenergic, NMDA and thromboxane A 2 receptors are involved in the anandamide-induced adrenal secretion of catecholamines and their pressor effect in urethane-anaesthetized rats. Keywords Anandamide. β 2 -adrenoceptors. Cannabinoid receptors. NMDA receptors. Thromboxane A 2 receptor. TRPV1 receptor B. Malinowska (*) : A. Zakrzeska : M. Göthert : G. Kwolek Zakład Fizjologii Doświadczalnej, Uniwersytet Medyczny w Białymstoku, ul. Mickiewicza 2A, Białystok, Poland C. M. Kurz : M. Göthert : E. Schlicker Institut für Pharmakologie und Toxikologie, Biomedizinisches Zentrum, Universität Bonn, Sigmund-Freud-Strasse 25, Bonn, Germany P. Wielgat : J. J. Braszko Zakład Farmakologii Klinicznej, Uniwersytet Medyczny w Białymstoku, ul. Waszyngtona 15A, Białystok, Poland Introduction Anandamide is one of the endogenous ligands of the endocannabinoid system and plays an important role under various physiological and pathophysiological conditions (for review, see Di Marzo 2009). In the cardiovascular system, anandamide elicits both hypo- and hypertensive responses and acts both via cannabinoid receptor-dependent and receptor-independent sites (for review, see Pacher et al. 2008). In order to disclose its numerous facets of cardiovascular effects, anaesthetized rodents are used frequently. Intravenous (i.v.) injection of anandamide to anaesthetized normotensive rats (Varga et al. 1995; 1996; Lake et al. 1997; Malinowska et al. 2001a; Kwolek et al. 2005) and mice (Pacher et al. 2004) causes a triphasic 350 Naunyn-Schmied Arch Pharmacol (2010) 381: cardiovascular response, namely an initial rapid and shortlasting bradycardia and hypotension, known as the Bezold- Jarisch reflex (phase I), a subsequent increase in blood pressure (phase II), and a delayed and prolonged hypotension (phase III). The mechanisms underlying phases I and III have been clarified. Thus, phase I involves the activation of vanilloid TRPV1 receptors located on sensory vagal nerves in the heart (Malinowska et al. 2001a). Phase III involves several mechanisms including the activation of (1) presynaptic cannabinoid CB 1 receptors innervating the sympathetic neurones supplying blood vessels and heart (Malinowska et al. 1997, 2001b; Niederhoffer et al. 2003), (2) of CB 1 receptors causing a decrease in cardiac contractility (Bátkai et al. 2004), (3) of TRPV1 receptors in the spinal cord (del Carmen Garcia et al. 2003), and (4) of non- CB 1 vascular receptors sensitive to O-1918 (Zakrzeska et al. 2010). Unlike in the anaesthetized rat, anandamide induces only the initial phase I and the subsequent pressor response in conscious rats (Lake et al. 1997; Gardiner et al. 2009). The mechanisms underlying the hypertensive effect, i.e. phase II, of the triphasic response to anandamide in the anaesthetized rat, have not been fully disclosed. In experiments in urethane-anaesthetized rats, we found (Kwolek et al. 2005) that reflex- and CB 1 receptor-independent peripheral and central components are involved in the induction of this phase. One component is located most probably in blood vessels (Kwolek et al. 2005) and was not further considered in the present paper. A second component, which involves the central nervous system, was inhibited by the non-selective β-adrenoceptor antagonist propranolol, the β 2 -adrenoceptor antagonist ICI , and the non-selective NMDA receptor antagonist MK-801. In continuation of our previous work (Kwolek et al. 2005), this central mechanism of action was studied in more detail in the present work. In particular, a prostanoid system capable of increasing blood pressure but not yet investigated in the context of the anandamide-induced pressor effect is the thromboxane A 2 (TXA 2 )-TP receptor system. TXA 2 is one of the most potent peripheral vasoconstrictors and aggregators of thrombocytes (e.g. Sellers and Stallone 2008) and increasing evidence suggests that TXA 2 acts also as central neuromodulator of cardiovascular function (Gao et al. 1997; Murakami et al. 2002; Okada et al. 2000, 2008). Therefore, the first step of the present study was to incorporate experiments with TP receptor antagonists into the same scheme as applied with the drugs studied previously (Kwolek et al. 2005). Accordingly, we decided to examine in intact and pithed rats whether TP receptor antagonists inhibit the anandamide-induced pressor response. In fact, this response was diminished by TP receptor antagonists in intact, but not in pithed rats, suggesting a central site of action. Therefore, in a second step, we studied the underlying central mechanism in more detail by intracerebroventricularly injecting anandamide and appropriate pharmacological tools such as relevant receptor antagonists. In addition, radioligand binding experiments were carried out in order to study whether anandamide directly interacts with the TP receptor. Methods Experiments on whole animals Male Wistar normotensive rats weighing g were used in the present experiments. The animals were maintained at a 12/12 h light dark cycle and housed in a special room at constant temperature (22±2 C) and humidity (50%) and had free access to water and standard rat chow. All surgical procedures and experimental protocols were approved by the local Animal Ethics Committee in Białystok (Poland). They have been carried out in accordance with the Guide for the Care and Use of Laboratory Animals. Surgical procedure preparing for intracerebroventricular drug administration Animals were anaesthetized intraperitoneally (i.p.) with pentobarbitone sodium (300 μmol/kg). They were prepared for intracerebroventricular injections exactly as described earlier (Braszko et al. 1991). Briefly, a circular piece of skin, 7 mm in diameter, was cut off the scalp, and the underlying skull surface was cleaned from soft tissue. A burr hole, 0.5 mm in diameter, was drilled in the skull 2.5 mm laterally and 1 mm caudally from the bregma on the right side of the head. The operation took about 2 min. Animals were housed in separate cages until the wound was completely dry, and the animal behaved normally. Intracerebroventricular injections were made freehand into the right cerebral ventricle with a 10-μl Hamilton syringe, using a KF 730 needle cut 4.5 mm from its base. This procedure allowed the tip of the needle to be lowered about 0.5 mm below the ceiling of the lateral cerebral ventricle. The injection volume was 2 μl per animal (or 5 µl per animal in the case of furegrelate), administered over 3 s. Upon completion of each experiment, rats were sacrificed and the sites of injections were verified microscopically after brain sectioning. Anaesthetized rats At least 72 h later, rats were anaesthetized i.p. with urethane (14 mmol/kg). The trachea was cannulated. Mean, systolic and diastolic blood pressure (MBP, SBP and DBP, respectively) were measured from the right carotid artery via a transducer (ISOTEC; Hugo Sachs Elektronik, March Hugstetten, Naunyn-Schmied Arch Pharmacol (2010) 381: Germany). We have mainly concentrated on the examination of DBP since this parameter reflects changes in vascular resistance. Moreover, i.v. and i.c.v. injection of anandamide and methanandamide induces more marked increases in DBP when compared with MBP and SBP (see Table 1 in Zakrzeska et al and Fig. 1 in the present paper, respectively). Heart rate (HR) was measured by a rate meter triggered from the pressure record. The left femoral vein was cannulated for i.v. injection of drugs administered in a volume of 0.5 ml/kg. Since the extent of vasopressor/ vasodepressor effects is dependent on the basal level of DBP (Malinowska and Schlicker 1993), vasopressin ( IU kg/min) was infused into the right femoral vein in some animals to have a DBP of mmhg in each animal (vasopressin administration was necessary in all pithed (see below) and bilaterally adrenalectomized rats and in some intact anaesthetized animals). Body temperature was kept constant at about C using a heating pad (Bio-Sys- Tech, Białystok, Poland) and monitored by a rectal probe (Physitemp BAT10, Clifton, NJ, USA). After surgical procedures, animals were gently placed on their abdomen. Fifteen to 30 min later, during which the cardiovascular parameters were allowed to stabilise, experiments were performed. Pithed rats Rats were anaesthetized i.p. with urethane (14 mmol/kg) and then injected i.p. with atropine (2 μmol/kg). After cannulation of the trachea, the animals were pithed by inserting a stainless-steel rod (1.5 mm diameter and 190 mm length) through the right orbit and the foramen magnum and down to the vertebral canal. Artificial respiration (1 ml/100 g, 60 strokes/min) with room air was immediately started using a respirator (7025 Rodent respirator, Hugo Sachs Elektronik, March Hugstetten, Germany). Both vagal nerves were cut. Blood pressure, heart rate and body temperature were measured as described above. After 30 min of equilibration, during which the cardiovascular parameters were allowed to stabilise, experiments were performed. Experimental protocol Anandamide or methanandamide was injected twice i.v. (S 1 and S 2, 15 min apart) or only once i.c.v. (S 1 ) (anandamide only). Since individual differences in responses to anandamide were noticed, we applied i.v. anandamide at doses of µmol/kg. We have chosen a dose of anandamide that increased DBP during phase II by about 20 30% of the basal value. Methanandamide was administered i.v. at 0.75 µmol/kg and anandamide i.c.v. at 0.03 µmol per animal. The TP receptor antagonists sulotroban (10 µmol/kg, i.v., Stegmeier et al. 1984), daltroban (10 µmol/kg, i.v., Bertolino et al. 1997) and SQ (1 µmol/kg, i.v., Bertolino et al or 0.02 µmol per animal i.c.v., Yalcin et al. 2005), the NMDA receptor antagonist MK-801 (1 µmol/kg, i.v., Kwolek et al. 2005) and the β 2 -adrenoceptor antagonist ICI (1 µmol/kg, i.v., Kwolek et al. 2005) or their solvents were administered i.v. or i.c.v. 5 min before the second (S 2 ) or the only (S 1 ) dose of anandamide (i.v. or i.c.v.) or methanandamide (i.v.), Fig. 1 Typical traces showing the changes in systolic (SBP), mean (MBP), diastolic (DBP) blood pressure and heart rate (HR) induced by i.c.v. injection of anandamide (AEA) in a urethane-anaesthetized rat a, b without and c, d after intravenous (i.v.) injection of AM 251 plus ruthenium red (AM 251+R. Red; 3 µmol/kg each, given 5 min before AEA). Note that the short-lived decrease in HR shown in panel b and d also occurred when the solvent for anandamide was used. Arrows indicate drug application 352 Naunyn-Schmied Arch Pharmacol (2010) 381: respectively. All experiments with i.c.v. administration of anandamide were performed in the presence of AM 251 (3 µmol/kg, Baranowska et al. 2008) and ruthenium red (3 µmol/kg, Malinowska et al. 2001a), given i.v. 5 min before anandamide i.c.v. AM 251 is a selective CB 1 receptor antagonist; ruthenium red is a non-selective TRPV1 receptor antagonist but was preferred over the selective TRPV1 receptor antagonist capsazepine due to its more marked and much longer antagonistic effect in the anaesthetized rat (Malinowska et al. 2001a). There are two exceptions from the above protocol. The inhibitor of thromboxane A 2 synthase, furegrelate (1.8 µmol per animal, Okada et al. 2008), was given i.c.v. 10 min after S 1 and 30 min before S 2. In some experiments, bilateral acute adrenalectomy or a sham operation was performed at the end of other surgical preparations described in the part Anaesthetized rats. These rats received intramuscular (i.m.) injections of cortisol 3 μmol/kg or its solvent (250 µl saline per animal) together with anaesthesia (according to Okada et al. 2008). Binding studies Binding studies were carried out according to the method described by Hedberg et al. (1988) (modified). Wistar rats (Charles River, Sulzfeld, Germany) were killed by an overdose of 120 mg/kg pentobarbitone i.p. Blood was withdrawn from the vena cava by venipuncture and collected in a tube containing K-EDTA (1.2 2 mg EDTA/ml blood; Sarstedt, Nümbrecht, Germany). Needles (Sarstedt) were heparinised (heparin 25,000 IE/ml) prior to use to prevent immediate clotting. The tube was centrifuged immediately at 200 g for 20 min. The pellet was discarded, the supernatant recentrifuged at 1,000 g for 15 min, and the platelets (pellet) were resuspended gently in platelet buffer (concentration in millimolars: NaCl 145, HEPES 10, Na 2 HPO 4 500, KCl 10, MgCl 2 4, glucose 10, bovine serum albumin 45 μm). After centrifugation at 1,000 g for 15 min, the supernatant was discarded and the washing procedure was repeated in Tris-saline (50 mm Tris, 154 mm NaCl, ph 7.4). The resulting pellet was resuspended in Tris-saline to a final concentration of µg/100 µl protein ( platelets/100 µl). Fresh cell suspension was used for receptor binding assays. Protein content was determined using the method of Bradford; cell count was determined in a Neubauer chamber. The binding assay was performed in Tris-saline buffer in a final volume of 0.5 ml containing µg protein. 3 H-SQ 29,548 was used at eight concentrations ranging from 0.15 to 30 nm for saturation experiments and at a concentration of 3 nm for displacement experiments. The incubation was terminated after 30 min by filtration through polyethyleneimine (0.3%)-pretreated Whatman GF/C filters (Whatman, Maidstone, UK). All steps were carried out at room temperature. Unspecific binding was determined in the presence of 50 μm unlabeled SQ 29,548. Calculations and statistics Results are given as mean±standard error of the mean (SEM); n refers to the number of rats (whole animal experiments) and to the number of separate experiments in triplicate (binding studies). In order to quantify the effects of antagonists on the anandamide- and methanandamideinduced changes in cardiovascular parameters, S 1 and S 2 values were calculated as percent of the basal diastolic blood pressure immediately before injection of that particular agonist dose. In the case of two administrations of agonists (S 1 and S 2 ), the final results are S 2 expressed as a percentage of S 1. For comparison of the mean values, the t test for paired and unpaired data was used, as appropriate. When two or more groups were compared with the same control, the one-way analysis of variance (ANOVA) followed by the Dunnett test was used. Differences were considered as significant when P 0.05. Radioligand binding curves were analysed by nonlinear curve fitting using the GraphPad Prism 5.0 software (GraphPad, San Diego, CA, USA). Drugs used AM 251 [N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide]; anandamide; (R)-(+)-methanandamide; ICI (erythro-(±)-1- (7-methylindan-4-yloxy)-3-isopropylaminobutan-2-ol; Tocris Cookson, Bristol, UK); atropine sulphate; ruthenium red; urethane; [Arg 8 ]-vasopressin; daltroban; cortisol; MK-801 ((5R,10 S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo(a,d) cyclohepten-5,10-imine hydrogen maleate; Sigma, München, Germany); furegrelate (sodium salt; Cayman Chemical, Ann Arbor, MI, USA); U 46,619 (9,11-dideoxy-9α,11α-methanoepoxy prostaglandin F 2α ; Biomol, Hamburg, Germany); SQ 29,548 ([1 S-[1α, 2α(Z), 3α, 4α]]-7-[3-[[2-[(phenyl- amino)carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1]hept- 2-yl]-5-heptenoic acid; Cayman or Biomol); pentobarbitone sodium (Biowet, Puławy, Poland or Abbott, Ludwigshafen, Germany); sulotroban (Boehringer Mannheim, Mannheim, Germany); heparin sodium (ratiopharm, Ulm, Germany); 3 H-SQ 29,548 (specific activity 43.6 Ci/mmol; PerkinElmer, Boston, MA, USA). Drugs used for the whole animal experiments were dissolved in saline with the following exceptions: AM 251 in a mixture of ethanol, Cremophor El, DMSO and saline (1:1:1:9.5); daltroban in a mixture of 2 mm Na 2 CO 3 and 2 mm NaOH (50:1); sulotroban in a mixture of saline (0.9% NaCl) and 2 M NaOH (50:1); SQ in a mixture of saline and DMSO (20:1); cortisol in a mixture of CHCl 3 Naunyn-Schmied Arch Pharmacol (2010) 381: and ethanol (1:1). Anandamide and methanandamide were purchased from Tocris Cookson as 10 mg/ml emulsion in soya water (1:4). Vasopressin was provided by the manufacturer as an aqueous stock solution (100 IU/ml), which subsequently was diluted (1:74) in isotonic saline before the experiment. Intravenous injection of saline or the solvents for sulotroban, daltroban and SQ first decreased and then increased DBP by about 10 30% each; the alterations were short-lived (maximally by about 30 s). Basal HR was not affected. Intracerebroventricular administration of solvents did not change basal cardiovascular parameters, with the exception of a slight and short-lived decrease in HR. The drugs used for the binding studies were dissolved in ethanol (SQ 29548, U-46,619) or in ethanol plus bovine serum albumin 0.5% (anandamide, methanandamide). The solvents did not affect binding by themselves. Results Experiments on whole animals In urethane-anaesthetized rats, the basal diastolic blood pressure measured immediately before the administration of the first (or only one) dose of agonist was in the range of mmhg in most anaesthetized rats or was brought to this level by vasopressin in all pithed and adrenalectomized rats and in some intact anaesthetized animals (for the exact values, see later Figs. 3, 4, 5, 6 and 7; bottom of the columns). Basal HR was 369±6 (n=123), 357±9 (n=9), 349±30 (n=5) and 341±9 (n=6) beats/min in intact, pithed, bilaterally adrenalectomized and sham-operated rats, respectively. Antagonists (given i.v. or i.c.v.) did not affect basal DBP or HR. Influence of intravenous and intracerebroventricular administration of anandamide and/or methanandamide on blood pressure Intravenous injection of anandamide (1.5 3 µmol/kg) induced typical triphasic changes in cardiovascular parameters in urethane-anaesthetized rats as described by us in detail earlier (for typical traces see Fig. 1 in Kwolek et al. 2005; Malinowska et al. 2001a; Zakrzeska et al. 2010). Thus, the
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