Barbara Malinowska

Cannabinoid CB1 receptor-mediated inhibition of the neurogenic vasopressor response in the pithed rat

Abstract The effects of two cannabinoid receptor agonists, R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]-pyrrolo[1, 2, 3-de]-1,4-benzoxazin-yl]-(1-naphthalenyl)methanone (WIN 55,212-2) and (-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol (CP-55,940), were studied on (i) the vasopressor response elicited in pithed rats by electrical stimulation of the sympathetic outflow and (ii) the release of 3H-noradrenaline and the vasoconstriction elicited in isolated rat tail arteries by transmural electrical stimulation. In pithed rats, the electrical (1Hz for 10s) stimulation of the preganglionic sympathetic nerve fibres increased diastolic blood pressure by about 30mmHg. This neurogenic vasopressor response (which under the conditions of our study was almost exclusively due to the release of catecholamines) was decreased by WIN 55-212,2 and CP-55,940 in a dose-dependent manner (inhibition by WIN 55,212-2 and CP-55,940, 0.1μmol/kg each, about 25–30%). The inhibition was identical in adrenalectomized rats and in animals with intact adrenals. The inhibitory action of WIN 55,212-2 and CP-55,940 was abolished by a dose of 0.03μmol/kg of the CB1 receptor antagonist N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlo- rophenyl)-4-methyl-3-pyrazole-carboxamide (SR 141716), which, by itself, had no effect. WIN 55,212-2, CP-55,940 and SR 141716 failed to affect the vasopressor response to exogenous noradrenaline (1nmol/kg), which also increased diastolic blood pressure by about 30mmHg. In isolated rat tail arteries, the electrically (0.4Hz) evoked tritium overflow and vasoconstriction were not modified by WIN 55,212-2 and CP-55,940 (1μmol/l each). In conclusion, the neurogenic vasopressor response in the pithed rat can be modulated via cannabinoid CB1 receptors probably located presynaptically on the postganglionic sympathetic nerve fibres innervating resistance vessels.

Mediation of the positive chronotropic effect of CGP 12177 and cyanopindolol in the pithed rat by atypical β‐adrenoceptors, different from β3‐adrenoceptors

1 The influence of β1, β2, and β3‐adrenoceptor agonists and of CGP 12177 and cyanopindolol on heart rate and diastolic blood pressure was studied in the pithed rat. 2 The β1‐adrenoceptor agonist, prenalterol, increased heart rate and the β2‐adrenoceptor agonist, fenoterol, caused a fall in blood pressure. The effect of prenalterol was antagonized by the β1‐adrenoceptor antagonist, CGP 20712 0.1 μmol kg−1 and the action of fenoterol was attenuated by the β2‐adrenoceptor antagonist, ICI 118551 0.1 μmol kg−1. Both effects were markedly diminished by the non‐selective β‐adrenoceptor antagonist, bupranolol 0.1 μmol kg−1. 3 The non‐selective β‐adrenoceptor agonist, isoprenaline, three β3‐agonists as well as CGP 12177 and cyanopindolol elicited a positive chronotropic effect, exhibiting the following pEDA60 values (negative log values of the doses increasing heart rate by 60 beats min−1): isoprenaline 10.4, CGP 12177 8.3, cyanopindolol 7.2, BRL 37344 6.9, ZD 2079 5.2 and CL 316243 <5. 4 CGP 20712 0.1 μmol kg−1, given together with ICI 118551 0.1 μmol kg−1, markedly attenuated the positive chronotropic effect of isoprenaline, BRL 37344, ZD 2079 and CL 316243 without affecting the increase in heart rate produced by CGP 12177 and cyanopindolol. 5 The positive chronotropic effect of CGP 12177 and cyanopindolol was attenuated by CGP 20712, 1 and 10 μmol kg−1 and bupranolol, 10 μmol kg−1 but was not affected by ICI 118551, 10 μmol kg−1. The effect of CGP 12177 was also not changed by BRL 37344 1 μmol kg−1, ZD 2079 10 μmol kg−1, CL 316243 10 μmol kg−1, the α1‐adrenoceptor antagonist, prazosin 1 μmol kg−1 and the 5‐hydroxytryptamine 5‐HT2A receptor antagonist, ketanserin 3 μmol kg−1. 6 CGP 12177 0.002 μmol kg−1 and cyanopindolol 0.003 μmol kg−1 shifted to the right the dose‐response curve of prenalterol for its positive chronotropic effect. The ‐log values of the doses causing a twofold shift to the right were 9.6 and 9.5, respectively. 7 Isoprenaline 0.00001‐0.001 μmol kg−1, BRL 37344 0.01‐1 μmol kg−1 and CGP 12177 0.1 μmol kg−1 caused a fall in diastolic blood pressure which was markedly attenuated by combined administration of CGP 20712 and ICI 118551, 0.1 μmol kg−1 each. 8 CGP 12177 0.01 and 0.1 μmol kg−1 and cyanopindolol 1 μmol kg−1 elicited an increase in diastolic blood pressure. CGP 20712, ICI 118551, bupranolol and, in the case of CGP 12177, also BRL 37344, ZD 2079, CL 316243, prazosin and ketanserin did not influence this effect. 9 In conclusion, the positive chronotropic effect of CGP 12177 and cyanopindolol is not mediated via β1‐, β2‐, β3‐, α1‐adrenoceptors or 5‐HT2A receptors. This effect may involve atypical β‐adrenoceptors, similar or identical to those described by Kaumann (1989) in isolated heart preparations.

H3 receptor-mediated inhibition of the neurogenic vasopressor response in pithed rats

In pithed rats, the H3 agonist R-(-)-alpha-methylhistamine (R alpha MeHA) inhibited the electrically induced increase in blood pressure without affecting the vasopressor response to exogenous noradrenaline. The effect of R alpha MeHA was not affected by the H1 and H2 antagonists dimetindene and ranitidine, but attenuated by the H3 antagonist thioperamide. At higher doses, R alpha MeHA itself increased basal blood pressure; this effect was not affected by the H1, H2 and H3 antagonists. In conclusion, the neurogenic vasopressor response can be modulated via H3 receptors, probably located presynaptically on postganglionic sympathetic nerve fibres.

Ifenprodil influences changes in mouse behaviour related to acute and chronic ethanol administration

The aim of the present study was to examine the influence of ifenprodil (a non-competitive NMDA receptor antagonist which also blocks 5-HT3 receptors and alpha1-adrenoceptors) on the effects of ethanol in the mouse in vivo and to elucidate the role of various receptors in these actions. The ethanol (4 g/kg i.p.)-induced sleeping time was shortened by ifenprodil 1 mg/kg but was not affected by ifenprodil 0.3 mg/kg, the 5-HT3 receptor antagonist ondansetron 0.03 mg/kg and the non-competitive NMDA receptor antagonist MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo(a,d)cycloheptan-5,10-imine maleate) 0.01 mg/kg. Ifenprodil 10 mg/kg mimicked the alpha1-adrenoceptor antagonist prazosin 1 mg/kg in that it prolonged the hypnotic response to ethanol (no additive effect when both drugs were given in combination); this is compatible with an involvement of alpha1-adrenoceptors in this effect of ifenprodil. Chronic exposure to ethanol (7%) induced physical dependence. The severity of ethanol withdrawal was suppressed by ifenprodil 1 and 10 mg/kg. In conclusion, ifenprodil influences ethanol-related changes in mouse behaviour and may prove to be useful in the treatment of alcoholism.

Triphasic blood pressure responses to cannabinoids: do we understand the mechanism?

The cannabinoids comprise three major classes of substances, including compounds derived from the cannabis plant (e.g. Δ9‐tetrahydrocannabinol and the chemically related substances CP55940 and HU210), endogenously formed (e.g. anandamide) and synthetic compounds (e.g. WIN55212‐2). Beyond their psychotropic effects, cannabinoids have complex effects on blood pressure, including biphasic changes of Δ9‐tetrahydrocannabinol and WIN55212‐2 and an even triphasic effect of anandamide. The differing pattern of blood pressure changes displayed by the three types of compounds is not really surprising since, although they share an agonistic effect at cannabinoid CB1 and CB2 receptors, some compounds have additional effects. In particular, anandamide is known for its pleiotropic effects, and there is overwhelming evidence that anandamide influences blood pressure via (i) CB1 receptors, (ii) TRPV1 receptors, (iii) endothelial cannabinoid receptors and (iv) degradation products. This review is dedicated to the description of the effects of externally added cannabinoids on cardiovascular parameters in vivo. First, the cardiovascular effects of cannabinoids in anaesthetized animals will be highlighted since most data have been generated in experiments of that type. The text will follow the three phases of anandamide on blood pressure, and we will check to which extent cardiovascular changes elicited by other cannabinoids show overlap with those effects or differ. The second part will be dedicated to the cardiovascular effects of the cannabinoids in conscious animals. In the third part, cardiovascular effects in humans will be discussed, and similarities and differences with respect to the data from animals will be examined.

Identification of the vasodilatory endothelial cannabinoid receptor in the human pulmonary artery.

Background The endocannabinoid anandamide is implicated in the pathogenesis of hypotension in haemorrhagic, endotoxic, and cardiogenic shock. It has been demonstrated in animal, but not in human, vessels that the vasodilatory effects of anandamide and abnormal cannabidiol are partially mediated by an as yet unidentified endothelial cannabinoid receptor. Our study was performed to examine the influence of abnormal cannabidiol on the human pulmonary artery. Methods Isolated human pulmonary arteries were obtained from patients without clinical evidence of pulmonary hypertension during resection of lung carcinoma. Vasodilatory effects of abnormal cannabidiol were examined on endothelium-intact vessels preconstricted with serotonin or potassium chloride. Results Anandamide and abnormal cannabidiol relaxed serotonin-preconstricted vessels concentration-dependently. The effect of abnormal cannabidiol was reduced by endothelium denudation, pertussis toxin and two antagonists of the novel endothelial receptor, cannabidiol and O-1918, but not by the nitric oxide synthase inhibitor L-NAME given together with the cyclooxygenase inhibitor indomethacin. It was also diminished by blockade of calcium-activated potassium channels by the nonselective blocker tetraethylammonium or by combination of selective blockers of small (apamin) and intermediate and large (charybdotoxin) conductance calcium-activated potassium channels. The potency of abnormal cannabidiol to relax vessels was lower in potassium chloride than in serotonin-preconstriced preparations. Conclusions Abnormal cannabidiol relaxes human pulmonary arteries in an endothelium-independent and endothelium-dependent manner. The latter component is probably mediated via the putative endothelial cannabinoid receptor, activation of which may release endothelium-derived hyperpolarizing factor, which in turn acts via calcium-activated potassium channels. Abnormal cannabidiol is behaviourally inactive; it may have a therapeutic implication in vascular diseases, especially in the treatment of pulmonary hypertension.

Cannabinoid receptor-independent inhibition by cannabinoid agonists of the peripheral 5-HT3 receptor-mediated von Bezold–Jarisch reflex

On the basis of previous findings that cannabinoids inhibit the function of human and rat 5‐HT3 receptors in vitro, we investigated whether cannabinoid receptor agonists also modulate the activity of the rat peripheral 5‐HT3 receptors on the terminals of cardiopulmonary afferent C‐fibres in vivo. In urethane‐anaesthetized rats, pre‐treated intravenously (i.v.) with the CB1 receptor antagonist SR 141716A (3 μmol kg−1) and with the β1/β2 adrenoceptor antagonist propranolol (0.3–0.4 μmol kg−1), bolus injection of the serotonin 5‐HT3 receptor agonist phenylbiguanide (3–10 μg kg−1, i.v.) or the vanilloid VR1 receptor agonist capsaicin (3–10 μg kg−1, i.v.) caused an immediate decrease in heart rate and mean arterial blood pressure (the von Bezold–Jarisch reflex). The phenylbiguanide‐induced bradycardia was dose‐dependently attenuated by the cannabinoid receptor agonists CP 55,940 (0.1–1 μmol kg−1, i.v.) and WIN 55,212‐2 (0.1–3 μmol kg−1, i.v.) 20 min after injection, but not by the inactive S‐(−)enantiomer of the latter, WIN 55,212‐3 (1 μmol kg−1, i.v.). The inhibition was reversible within 30 min. The extent of inhibition by the highest doses of cannabinoid receptor agonists amounted to about 50%. Both cannabinoid receptor agonists failed to affect the capsaicin‐evoked bradycardia. In conclusion, our results demonstrate that cannabinoid receptor agonists modulate the von Bezold–Jarisch reflex by inhibiting peripheral serotonin 5‐HT3 receptors in rats in vivo. An analogous mechanism of cannabinoid receptor agonists may be assumed to be involved in other serotonin 5‐HT3 receptor‐mediated responses.

Further evidence for differences between cardiac atypical β‐adrenoceptors and brown adipose tissue β3‐adrenoceptors in the pithed rat

1 We have previously shown ( Malinowska & Schlicker, 1996 ) that the atypical β‐adrenoceptor involved in the positive chronotropic effect of the so‐called non‐conventional partial β‐adrenoceptor agonists CGP 12177 and cyanopindolol in the pithed rat possesses properties markedly different from those observed for β3‐adrenoceptors in the literature. In the present study, we have directly compared the pharmacological properties of the atypical cardiostimulant β‐adrenoceptor and of the β3‐adrenoceptor mediating the thermogenic response in the brown adipose tissue in pithed and vagotomized rats. 2 Heart rate was dose‐dependently increased by CGP 12177 and cyanopindolol by maximally 150 and 100 beats min−1, yielding pED50 values of 8.0 and 7.3, respectively (pED50, −log10 of the dose in mol kg−1 body weight i.v. causing the half‐maximum effect), but not affected by the selective β3‐adrenoceptor agonist CL 316243 (pED50>6.0). 3 CGP 12177, cyanopindolol and CL 316243 increased temperature in the brown adipose tissue by maximally 1°C (pED50 values 7.4, 6.3 and 8.6, respectively). 4 The β1‐adrenoceptor antagonist CGP 20712 10 μmol kg−1, attenuated the cardiostimulatory effect of CGP 12177 and, at a still higher dose (30 μmol kg−1), also antagonized its thermogenic effect. The −log10 values of the doses causing a two fold shift of the dose‐response curves (DRCs) of CGP 12177 to the right were 6.1 and 5.2, respectively, and were much lower than the corresponding value for the antagonism of CGP 20712 against the β1‐adrenoceptor‐mediated positive chronotropic effect which was 8.6. 5 The cardiostimulant and the thermogenic effect of CGP 12177 were not affected by the β2‐adrenoceptor antagonist ICI 118551 10 μmol kg−1. 6 The β3‐adrenoceptor antagonist SR 59230A (which, by itself, caused a β1‐adrenoceptor‐mediated increase in heart rate and, for this reason, was studied after administration of a low dose of CGP 20712) attenuated the cardiostimulant and the thermogenic effect of CGP 12177 to a similar extent. The −log10 values of the doses causing two fold rightward shifts of the DRCs of CGP 12177 were 5.9 and 5.7, respectively. 7 The non‐selective β‐adrenoceptor antagonist bupranolol diminished the cardiostimulant and thermogenic response to a very similar extent. The −log10 values causing two fold rightward shifts of the DRCs of CGP 12177 were 5.6 and 5.7, respectively, and were much lower than the corresponding values for the antagonism of bupranolol against the β1‐adrenoceptor‐mediated positive chronotropic effect and the β2‐adrenoceptor‐mediated decrease in diastolic blood pressure which were 7.6 and 8.3, respectively. 8 The rank order of agonistic potencies for the cardiostimulant effect (CGP 12177>cyanopindolol > CL 316243) differs from that for the thermogenic response in the brown adipose tissue (CL 316243>CGP 12177>cyanopindolol); furthermore, there is a difference with respect to the rank orders of antagonistic potencies for cardiostimulation (CGP 20712SR 59230Abupranolol>ICI 118551) and thermogenesis (SR 59230A=bupranolol>CGP20712>ICI 118551). 9 In conclusion, our study provides further evidence that the atypical cardiostimulant β‐adrenoceptors (causing an increase in heart rate) and β3‐adrenoceptors are pharmacologically different.

Presynaptic cannabinoid CB1 receptors are involved in the inhibition of the neurogenic vasopressor response during septic shock in pithed rats

Our study was undertaken to investigate whether bacterial endotoxin/lipopolysaccharide (LPS) affects the neurogenic vasopressor response in rats in vivo by presynaptic mechanisms and, if so, to characterize the type of presynaptic receptor(s) operating in the initial phase of septic shock. In pithed and vagotomized rats treated with pancuronium, electrical stimulation (ES) (1 Hz, 1 ms, 50 V for 10 s) of the preganglionic sympathetic nerve fibers or intravenous bolus injection of noradrenaline (NA) (1–3 nmol kg−1) increased the diastolic blood pressure (DBP) by about 30 mmHg. Administration of LPS (0.4 and 4 mg kg−1) under continuous infusion of vasopressin inhibited the neurogenic vasopressor response by 25 and 50%, respectively. LPS did not affect the increase in DBP induced by exogenous NA. The LPS‐induced inhibition of the neurogenic vasopressor response was counteracted by the cannabinoid CB1 receptor antagonist SR 141716A (0.1 μmol kg−1), but not by the CB2 receptor antagonist SR 144528 (3 μmol kg−1), the vanilloid VR1 receptor antagonist capsazepine (1 μmol kg−1) or the histamine H3 receptor antagonist clobenpropit (0.1 μmol kg−1). The four antagonists by themselves did not affect the increase in DBP induced by ES or by injection of NA in rats not exposed to LPS. We conclude that in the initial phase of septic shock, the activation of presynaptic CB1 receptors by endogenously formed cannabinoids contributes to the inhibition of the neurogenic vasopressor response.

Atypical β‐adrenoceptors, different from β3‐adrenoceptors and probably from the low‐affinity state of β1‐adrenoceptors, relax the rat isolated mesenteric artery

We examined whether β3‐ and/or atypical β‐adrenoceptors relax the rat isolated mesenteric artery. Mesenteric arteries precontracted with phenylephrine were relaxed by β‐agonists with the following potencies (pD2): nonselective agonist isoprenaline (6.00)>nonconventional partial agonist cyanopindolol (5.45)>β2‐agonist fenoterol (4.98)>nonconventional partial agonist CGP 12177 (4.19)>β3‐agonist ZD 2079 (3.72). The β3‐agonist CL 316243 1 mM relaxed the vessel only marginally. The concentration–response curves (CRCs) for cyanopindolol, CGP 12177 and ZD 2079 were not affected by the nonselective β‐antagonist propranolol 0.3 μM, the β2‐antagonist ICI 118551 1 μM and by CL 316243 60 μM, but shifted to the right by bupranolol (pA2 5.3–5.7), CGP 20712 (5.4) and SR 59230A (6.5–6.7) (the latter three drugs block atypical and/or β3‐adrenoceptors at high concentrations). The CRC for isoprenaline was shifted to the right by propranolol (pA2 7.0) but, in the presence of propranolol 0.3 μM, not affected by SR 59230A 1 μM. The CRC for fenoterol was shifted to the right by propranolol (pA2 6.9) and ICI 118551 (6.8). Removal of endothelium diminished the vasorelaxant effects of cyanopindolol, CGP 12177 and ZD 2079. Fenoterol and cyanopindolol also relaxed (endothelium‐intact) mesenteric arteries precontracted with serotonin. The relaxant effect of cyanopindolol was antagonized by bupranolol to about the same degree as in phenylephrine‐contracted vessels. In conclusion, β2‐ and atypical β‐adrenoceptors (but not β3‐adrenoceptors) relax the rat mesenteric artery. The atypical β‐adrenoceptor, which is partially located endothelially, may differ from the low‐affinity state of the β1‐adrenoceptor.