Grzegorz Godlewski

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.

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.

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.

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.

Central and peripheral components of the pressor effect of anandamide in urethane-anaesthetized rats

1 We wanted to search for the mechanism(s) responsible for the brief pressor response induced by anandamide in urethane‐anaesthetized rats. 2 The anandamide‐induced pressor effect was not modified by the antagonists of cannabinoid CB1 and vanilloid TRPV1 receptors, SR 141716A (3 μmol kg−1) and capsazepine (1 μmol kg−1), respectively, by bilateral vagotomy and by pithing. Replacement of urethane by pentobarbitone virtually abolished the pressor effect of anandamide, both in pithed and vagotomized and in ‘intact’ rats (i.e. not treated in this manner). 3 The pressor effect of anandamide was reduced by the nonselective TRPV family inhibitor ruthenium red (3 μmol kg−1) and by the blocker of L‐type calcium channels nifedipine (1 μmol kg−1), both in pithed urethane‐anaesthetized rats and in ‘intact’ urethane‐anaesthetized rats. The nonselective β‐adrenoceptor antagonist propranolol (0.1 or 0.3 μmol kg−1) and the nonselective NMDA receptor antagonist MK‐801 (1 μmol kg−1) diminished the anandamide‐induced vasopressor response in ‘intact’ but not in pithed rats. The inhibitory effect of propranolol in ‘intact’ rats was mimicked by the β2‐adrenoceptor antagonist ICI 118551 (1 μmol kg−1), but not by the β1‐adrenoceptor antagonist CGP 20712 (1 μmol kg−1). 4 The present study revealed that two mechanisms may be responsible for the anandamide‐induced pressor response in urethane‐anaesthetized rats. The first involves the central nervous system (probably the medulla oblongata) and is sensitive to propranolol and MK‐801. The second, which is located peripherally (most probably in blood vessels), is sensitive to nifedipine, ruthenium red and pentobarbitone and, hence, probably represents a Ca2+‐dependent mode of action.

INHIBITORY H3 RECEPTORS ON SYMPATHETIC NERVES OF THE PITHED RAT : ACTIVATION BY ENDOGENOUS HISTAMINE AND OPERATION IN SPONTANEOUSLY HYPERTENSIVE RATS

Abstract Our previous results demonstrate the occurrence of presynaptic inhibitory histamine H3 receptors on sympathetic neurons innervating resistance vessels of the pithed rat. The present study, in which new H3 receptor ligands with increased potency and selectivity (imetit, clobenpropit) were used, was designed to further explore the role of H3 receptors in the regulation of the rat cardiovascular system. In particular we were interested whether these receptors may be activated by endogenous histamine and whether they are detectable in an experimental model of hypertension.All experiments were performed on pithed and vagotomized rats treated with rauwolscine 1 μmol/kg. In normotensive Wistar rats the electrical (1 Hz, 1 ms, 50 V for 20 s) stimulation of the preganglionic sympathetic nerve fibres increased diastolic blood pressure by about 35 mmHg. Two H3 receptor agonists, R-(–)-α-methylhistamine and imetit, inhibited the electrically induced increase in diastolic blood pressure in a dose-dependent manner. The maximal effect (about 25%) was obtained for R-(–)-α-methylhistamine at about 10 μmol/kg and for imetit at about 1 μmol/kg. Two H3 receptor antagonists, thioperamide 1 μmol/kg and clobenpropit 0.1 μmol/kg, attenuated the inhibitory effect of imetit. The neurogenic vasopressor response was increased by about 15% by thioperamide 1 μmol/kg and clobenpropit 0.1 μmol/kg and decreased by 25% by the histamine methyltransferase inhibitor metoprine 37 μmol/kg. R-(-)-α-Methylhistamine, imetit, thioperamide, clobenpropit and metoprine did not affect the vasopressor response to exogenously added noradrenaline 0.01 μmol/kg (which increased diastolic blood pressure by about 40 mmHg). Metoprine had only a very low affinity for H3 binding sites (labelled by 3H-Nα-methylhistamine; pKi 4.46). In pithed Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats, electrical (1 Hz, 1 ms, 50 V for 10 s) stimulation increased diastolic blood pressure by 28 and 37 mmHg, respectively. Imetit inhibited the neurogenic vasopressor response to about the same extent in WKY and SHR rats (maximal effect of about 30%). The inhibitory influence of imetit was diminished by thioperamide 1 μmol/kg to about the same degree in rats of either strain.The present study confirms the occurrence of presynaptic H3 receptors on sympathetic nerve fibres involved in the inhibition of the neurogenic vasopressor response. Moreover, it demonstrates that these H3 receptors are probably activated by endogenous histamine and appear to be operative in hypertension.

Atypical cardiostimulant β-adrenoceptor in the rat heart: stereoselective antagonism by bupranolol but lack of effect by some bupranolol analogues

Atypical β‐adrenoceptors resistant to propranolol, but blocked by bupranolol, increase contractile force and/or frequency of the heart in humans and rats. We compared the potencies of the enantiomers of bupranolol and examined the possible effects of seven bupranolol analogues including bevantolol (BEV) at this receptor in pithed and vagotomized rats. CGP 12177, an agonist of the atypical β‐adrenoceptor, increased heart rate dose‐dependently. Its dose–response curve was shifted to the right by S‐(−)‐bupranolol 10 μmol kg−1 by a factor of 8.4, but not affected by the same dose of R‐(+)‐bupranolol. Desmethylbupranolol and compounds BK‐21, BK‐22, BK‐23 and BK‐25 also increased heart rate dose‐dependently. The β1‐adrenoceptor antagonist CGP 20712 given in combination with the β2‐adrenoceptor antagonist ICI 118,551 (0.1 μmol kg−1 each) reduced the positive chronotropic action of the five bupranolol analogues without affecting that of CGP 12177. The potencies of the bupranolol analogues to increase heart rate were correlated (r=0.91, P<0.05) with their affinities for β1‐adrenoceptor binding sites in rat brain cortex membranes labelled with [3H]CGP 12177 (in the presence of ICI 118,551). BK‐26 and BEV, 10 μmol kg−1 each, had only minor effects on heart rate by themselves and did not antagonize the effect of CGP 12177. However, at 1 μmol kg−1, they antagonized the increase in heart rate elicited by the β1‐adrenoceptor agonist prenalterol. In conclusion, bupranolol is a stereoselective antagonist at the atypical cardiostimulant β‐adrenoceptor. The effects of the bupranolol analogues are related to the activation or blockade of β1‐adrenoceptors, but not of atypical β‐adrenoceptors.

Identification of histamine H3 receptors in the tail artery from normotensive and spontaneously hypertensive rats.

We examined the possible existence of prejunctional histamine H3 receptors on sympathetic nerve fibers innervating rat tail artery. The stimulation-evoked tritium outflow from isolated vessels preincubated with [3H]-noradrenaline and perfused/superfused in the presence of the alpha2-adrenoceptor antagonist rauwolscine, 3 microM, was inhibited by histamine 10 microM (by 8%) and the H3 agonists R-(-)-alpha-methylhistamine, 10 microM (by 18%), and imetit, 0.1-10 microM (by < or =20%). The inhibitory effect of imetit, which did not occur in the absence of rauwolscine, was counteracted by thioperamide, 1 microM. In the presence of rauwolscine, 3 microM, the inhibitory effect of imetit also occurred when the current strength or the Ca2+ concentration in the medium was reduced to compensate for the increase in tritium overflow elicited by rauwolscine, indicating that the inhibitory action of imetit is not associated with the increase in noradrenaline release produced by rauwolscine. In spontaneously hypertensive rats (SHRs), imetit also inhibited the overflow of tritium. This inhibitory effect was comparable to that observed in Wistar-Kyoto (WKY) rats and indicates that the sympathetic nerves of the rat tail artery in SHRs, like those in normotensive rats, are endowed with prejunctional histamine H3 receptors.

Facilitation by substance P and inhibition by (+)-tubocurarine of the 5-HT3 receptor-mediated Bezold-Jarisch reflex in rats

The influence of substance P 3 (microgram/kg) and (+)-tubocurarine (850 micrograms/kg) on the Bezold-Jarisch reflex in urethane-anaesthetized rats was studied. The Bezold-Jarisch reflex was induced by the 5-HT3 receptor agonist phenylbiguanide (0.3, 1, 3 and 10 micrograms/kg i.v.) and by capsaicin (10 micrograms/kg i.v.). The 5-HT3 receptor antagonist ondansetron (10 micrograms/kg) abolished the phenylbiguanide- but not the capsaicin-stimulated bradycardia, indicating that phenylbiguanide and capsaicin act via different trigger mechanisms (5-HT3 receptor-dependent and -independent, respectively). Substance P significantly potentiated the phenylbiguanide- but not the capsaicin-induced decrease in heart rate. Also, when the phenylbiguanide-induced response was amplified by substance P, it was abolished by ondansetron. (+)-Tubocurarine inhibited the phenylbiguanide-induced bradycardia, but did not affect the capsaicin-stimulated decrease in heart rate. Our results demonstrate that substance P potentiates but (+)-tubocurarine inhibits the 5-HT3 receptor-mediated Bezold-Jarisch reflex. Both effects are probably due to direct influences of the drugs on the 5-HT3 receptors on sensory vagal nerves in the heart.

Inhibitory effect of ethanol on the 5-hydroxytryptamine-induced Bezold-Jarisch reflex — involvement of peripheral 5-HT3 receptors

The influence of ethanol (0.5, 1.0 and 2.0 g/kg i.p.) on the Bezold-Jarisch reflex in urethane-anaesthetized rats was studied. 5-Hydroxytryptamine (serotonin; 5-HT; 1, 3, 10 and 30 micrograms/kg i.v.) and capsaicin (1, 3 and 10 micrograms/kg i.v.) reflexly decreased heart rate in a dose-dependent manner. The 5-HT3 receptor antagonist ondansetron 10 micrograms/kg i.v. abolished the 5-HT- but not the capsaicin-stimulated bradycardia, indicating that 5-HT and capsaicin acted via different trigger mechanisms (5-HT3 receptor-dependent and -independent, respectively). Ethanol at 1.0 and 2.0 g/kg i.p. inhibited in a dose-dependent manner (by 20-45%) the 5-HT- but not the capsaicin-stimulated decrease in heart rate. Our results demonstrate that the inhibitory effect of ethanol on the 5-HT3 receptor-mediated Bezold-Jarisch reflex may be related to the direct effect of ethanol on 5-HT3 receptors on sensory vagal nerves in the heart.