Alessandro Bartolini

Local anaesthetic activity of β-caryophyllene ☆

Abstract In this work we studied the local anaesthetic activity of β-caryophyllene, one of the main components of clove oil obtained from the dried flower-buds of Syzygium aromaticum (Myrtaceae family). We compared its activity to a chemically related compound, caryophyllene oxide. Anaesthetic activity was evaluated in vivo in the rabbit conjunctival reflex test and in vitro in a rat phrenic nerve-hemidiaphragm preparation. β-Caryophyllene (10 −4 –1 μg/ml), but not caryophyllene oxide, was able to reduce drastically, in a dose-dependent manner, the electrically evoked contractions of the rat phrenic hemidiaphragm. In the rabbit, conjunctival reflex test treatment with a solution of β-caryophyllene (10–1000 μg/ml) allowed a dose-dependent increase in the number of stimuli necessary to provoke the reflex. As in the in vitro results, caryophyllene oxide was ineffective also in the in vivo test. In conclusion, these data evidence the local anaesthetic activity of β-caryophyllene, which appears to be strictly dependent on its chemical structure.

Role of histamine in rodent antinociception

1 Effects of substances which are able to alter brain histamine levels on the nociceptive threshold were investigated in mice and rats by means of tests inducing three different kinds of noxious stimuli: mechanical (paw pressure), chemical (abdominal constriction) and thermal (hot plate). 2 A wide range of i.c.v. doses of histamine 2HCl was studied. Relatively high doses were dose‐dependently antinociceptive in all three tests: 5–100 μg per rat in the paw pressure test, 5–50 μg per mouse in the abdominal constriction test and 50–100 μg per mouse in the hot plate test. Conversely, very low doses were hyperalgesic: 0.5 μg per rat in the paw pressure test and 0.1–1 μg per mouse in the hot plate test. In the abdominal constriction test no hyperalgesic effect was observed. 3 The histamine H3 antagonist, thioperamide maleate, elicited a weak but statistically significant dose‐dependent antinociceptive effect by both parenteral (10–40 mg kg−1) and i.c.v. (1.1–10 μg per rat and 3.4–10 μg per mouse) routes. 4 The histamine H3 agonist, (R)‐α‐methylhistamine dihydrogenomaleate was hyperalgesic, with a rapid effect (15 min after treatment) following i.c.v. administration of 1 μg per rat and 3 μg per mouse, or i.p. administration of 100 mg kg−1in mice. In rats 20 mg kg−1, i.p., elicited hyperalgesia only 4 h after treatment. 5 Thioperamide‐induced antinociception was completely prevented by pretreatment with a non‐hyperalgesic i.p. dose of (R)‐α‐methylhistamine in the mouse hot plate and abdominal constriction tests. Antagonism was also observed when both substances were administered i.c.v. in rats. 6 l‐Histidine HCl dose‐dependently induced a slowly occurring antinociception in all three tests. The doses of 250 and 500 mg kg−1, i.p. were effective in the rat paw pressure test, and those of 500 and 1500 mg kg−1, i.p. in the mouse hot plate test. In the mouse abdominal constriction test 500 and 1000 mg kg−1, i.p. showed their maximum effect 2 h after treatment. 7 The histamine N‐methyltransferase inhibitor, metoprine, elicited a long‐lasting, dose‐dependent antinociception in all three tests by both i.p. (10–30 mg kg−1) and i.c.v. (50–100 μg per rat) routes. 8 To ascertain the mechanism of action of the antinociceptive effect of l‐histidine and metoprine, the two substances were also studied in combination with the histamine synthesis inhibitor (S)‐α‐fluoro‐methylhistidine and with (R)‐α‐methylhistamine, respectively. l‐Histidine antinociception was completely antagonized in all three tests by pretreatment with (S)‐α‐fluoromethylhistidine HCl (50 mg kg−1, i.p.) administered 2 h before l‐histidine treatment. Similarly, metoprine antinociception was prevented by (R)‐α‐methylhistamine dihydrogenomaleate 20 mg kg−1, i.p. administered 15 min before metoprine. Both (S)‐α‐fluoromethylhistidine and (R)‐α‐methylhistamine were used at doses which did not modify the nociceptive threshold when given alone. 9 The catabolism product, 1‐methylhistamine, administered i.c.v. had no effect in either rat paw pressure or mouse abdominal constriction tests. 10 These results indicate that the antinociceptive action of histamine may take place on the postsynaptic site, and that its hyperalgesic effect occurs with low doses acting on the presynaptic receptor. This hypothesis is supported by the fact that the H3 antagonist, thioperamide is antinociceptive and the H3 agonist, (R)‐α‐methylhistamine is hyperalgesic, probably modulating endogenous histamine release. l‐Histidine and metoprine, which are both able to increase brain histamine levels, are also able to induce antinociception in mice and rats. Involvement of the histaminergic system in the modulation of nociceptive stimuli is thus proposed.

Role of muscarinic receptor subtypes in central antinociception

1 The ability to modify the pain threshold by the two M1‐muscarinic agonists: McN‐A‐343 and AF‐102B and by the specific M2‐agonist arecaidine was examined in mice and rats by using three different noxious stimuli: chemical (writhing test), thermic (hot‐plate test) and mechanical (paw pressure test). 2 In the mouse hot‐plate test McN‐A‐343 (20–50 μg per mouse i.c.v.) and AF‐102B (1–10 mg kg−1 i.p.) produced significant antinociception which was prevented by atropine (1 μg per mouse i.c.v.) and by the two selective M1 antagonists: pirenzepine (0.01 μg per mouse i.c.v.) and dicyclomine (0.08 μg per mouse i.c.v. or 10 mg kg−1 i.p.) but not by the specific M2‐antagonist AFDX‐116 (0.1 μg per mouse i.c.v.), naloxone (1 mg kg−1 i.p.) or by the acetylcholine (ACh) depletor hemicholinium‐3 (HC‐3) (1 μg per mouse i.c.v.). McN‐A‐343 and AF‐102B were able to increase the pain threshold also in the mouse acetic acid writhing test and in rat paw pressure test. These antinociceptive effects were completely prevented by dicyclomine (0.08 μg per mouse i.c.v. or 10 mg kg−1 i.p.) but not by AFDX‐116 (0.1 μg per mouse or rat i.c.v.). 3 In contrast with the M1‐agonists, the M2‐agonist arecaidine (0.1–2 μg per mouse or rat i.c.v.) did not induce antinociception in all three analgesic tests. However, arecaidine, at the same i.c.v. doses, was able to reduce the pain threshold in the hot‐plate and paw pressure tests. 4 The site of muscarinic control of the pain threshold is localized in the CNS since drugs which do not cross the blood‐brain barrier such as McN‐A‐343, pirenzepine and arecaidine exerted their effects only if injected i.c.v. 5 On the basis of the above findings and existing literature we suggest that the postsynaptic muscarinic receptors involved in antinociception belong to the M1 subtype. Nevertheless, presynaptic autoreceptors (M2 subtype) may play a role in pain regulation since they are involved in modulation of endogenous ACh release.

Effect of psychoactive drugs on the output of acetylcholine from the cerebral cortex of the cat

Abstract The effect of chlorpromazine (CPZ), pentobarbital and imipramine on the output of acetylcholine (ACh) from the cerebral cortex was investigated in cats transected at midbrain level. The spontaneous output and the increase of the output caused by stimulation of the reticular formation and by the administration of amphetamine were determined. The effect of the stimulation of the reticular formation on ACh output was prevented by pretreatment with pentobarbital, it was reduced by CPZ but it was not modified by imipramine. On the other hand the increase of ACh output following amphetamine was prevented by CPZ, it was not modified by pentobarbital and it was enhanced by imipramine. The experiments support the hypothesis that amphetamine stimulates cortical ACh output by acting on catecholamine nerve terminals which make synaptic connection with cholinergic neurons in the rostral part of the brain.

CGP 35348, a new GABAB antagonist, prevents antinociception and muscle-relaxant effect induced by baclofen.

1 CGP 35348, a new GABAB antagonist, was examined on antinociception induced by (±)‐baclofen by use of the hot plate and writhing tests in mice and the paw pressure test in rats. CGP 35348 was also studied in mice on (±)‐baclofen‐induced impairment of rota‐rod performance. 2 CGP 35348, injected either i.p. (60–100 mg kg−1 in mouse) or intracerebroventricularly (i.c.v.) (0.5–2.5 μg per mouse; 25 μg per rat) prevented (±)‐baclofen‐induced antinociception. 3 CGP 35348 did not modify oxotremorine‐ and morphine‐induced antinociception in mice and rats. 4 CGP 35348 (2.5 μg i.c.v. per mouse) also prevented (±)‐baclofen‐induced impairment of the rota‐rod test. 5 Two other GABAB antagonists, phaclofen (50 μg i.c.v. per mouse) and 2‐OH‐saclofen (2.5 μg‐10 μg i.c.v. per mouse) did not modify (±)‐baclofen‐induced antinociception. 7 These results suggest that, at present, CGP 35348 is the only compound able to antagonize (±)‐baclofen‐induced antinociception.

Antinociception induced by systemic administration of local anaesthetics depends on a central cholinergic mechanism.

1 The antinociceptive effects of systemically‐administered procaine, lignocaine and bupivacaine were examined in mice and rats by using the hot‐plate, writhing and tail flick tests. 2 In both species all three local anaesthetics produced significant antinociception which was prevented by atropine (5 mg kg−1, i.p.) and by hemicholinium‐3 (1 μg per mouse, i.c.v.), but not by naloxone (3 mg kg−1, i.p.), α‐methyl‐p‐tyrosine (100 mg kg−1, s.c.), reserpine (2 mg kg−1, i.p.) or atropine methylbromide (5.5 mg kg−1, i.p.). 3 Atropine (5 mg kg−1, i.p.) which totally antagonized oxotremorine (40 μg kg−1, s.c.) antinociception did not modify morphine (5 mg kg−1, s.c.) or baclofen (4 mg kg−1, s.c.) antinociception. On the other hand, hemicholinium, which antagonized local anaesthetic antinociception, did not prevent oxotremorine, morphine or baclofen antinociception. 4 Intracerebroventricular injection in mice of procaine (200 μg), lignocaine (150 μg) and bupivacaine (25 μg), doses which were largely ineffective by parenteral routes, induced an antinociception whose intensity equalled that obtainable subcutaneously. Moreover, the i.c.v. injection of antinociceptive doses did not impair performance on the rota‐rod test. 5 Concentrations below 10−10 m of procaine, lignocaine and bupivacaine did not evoke any response on the isolated longitudinal muscle strip of guinea‐pig ileum, or modify acetylcholine (ACh)‐induced contractions. On the other hand, they always increased electrically‐evoked twitches. 6 The same concentrations of local anaesthetics which induced antinociception did not inhibit acetylcholinesterase (AChE) in vitro. 7 On the basis of the above findings and the existing literature, a facilitation of cholinergic transmission by the local anaesthetics is postulated; this could be due to blockade of presynaptic muscarinic receptors.

Tiagabine antinociception in rodents depends on GABAB receptor activation : parallel antinociception testing and medial thalamus GABA microdialysis

The effects of a new antiepileptic drug, tiagabine, (R)-N-[4,4-di-(3-methylthien-2-yl)but-3-enyl] nipecotic acid hydrochloride, were studied in mice and rats in antinociceptive tests, using three kinds of noxious stimuli: mechanical (paw pressure), chemical (abdominal constriction) and thermal (hot plate). In vivo microdialysis was performed in parallel in awake, freely moving rats in order to evaluate possible alterations in extracellular gamma-aminobutyric acid (GABA) levels in a pain-modulating region, the medial thalamus. Systemic administration of tiagabine, 30 mg kg(-1) i.p., increased nearly twofold the extracellular GABA levels in rats and increased significantly the rat paw pressure nociceptive threshold in a time-correlated manner. Dose-related significant tiagabine-induced antinociception was also observed at the doses of 1 and 3 mg kg(-1) i.p. in the mouse hot plate and abdominal constriction tests. The tiagabine antinociception was completely antagonised by pretreatment with the selective GABA(B) receptor antagonist, CGP 35348, (3-aminopropyl-diethoxy-methyl-phosphinic acid) (2.5 microg/mouse or 25 microg/rat i.c.v.), but not by naloxone (1 mg kg(-1) s.c.), both administered 15 min before tiagabine. Thus, it is suggested that tiagabine causes antinociception due to raised endogenous GABA levels which in turn activate GABA(B) receptors.

Antidepressant-like effects of endogenous histamine and of two histamine H1 receptor agonists in the mouse forced swim test

Effects of substances which are able to alter brain histamine levels and two histamine H1 receptor agonists were investigated in mice by means of an animal model of depression, the forced swim test. Imipramine (10 and 30 mg kg−1, i.p.) and amitriptyline (5 and 15 mg kg−1, i.p.) were used as positive controls. Their effects were not affected by pretreatment with the histamine H3 receptor agonist, (R)‐α‐methylhistamine, at a dose (10 mg kg−1, i.p.) which did not modify the cumulative time of immobility. The histamine H3 receptor antagonist, thioperamide (2–20 mg kg−1, s.c.), showed an antidepressant‐like effect, with a maximum at the dose of 5 mg kg−1, which was completely prevented by (R)‐α‐methylhistamine. The histamine‐N‐methyltransferase inhibitor, metoprine (2–20 mg kg−1, s.c.), was effective with an ED50 of 4.02 (2.71–5.96) mg kg−1; its effect was prevented by (R)‐α‐methylhistamine. The histamine precursor, l‐histidine (100–1000 mg kg−1, i.p.), dose‐dependently decreased the time of immobility [ED30 587 (499–712) mg kg−1]. The effect of 500 mg kg−1 l‐histidine was completely prevented by the selective histidine decarboxylase inhibitor, (S)‐α‐fluoromethylhistidine (50 mg kg−1, i.p.), administered 15 h before. The highly selective histamine H1 receptor agonist, 2‐(3‐trifluoromethylphenyl)histamine (0.3–6.5 μg per mouse, i.c.v.), and the better known H1 agonist, 2‐thiazolylethylamine (0.1–1 μg per mouse, i.c.v.), were both dose‐dependently effective in decreasing the time of immobility [ED50 3.6 (1.53–8.48) and 1.34 (0.084–21.5) μg per mouse, respectively]. None of the substances tested affected mouse performance in the rota rod test at the doses used in the forced swim test. It was concluded that endogenous histamine reduces the time of immobility in this test, suggesting an antidepressant‐like effect, via activation of H1 receptors.

Loss of muscarinic antinociception by antisense inhibition of M1 receptors

The effect on cholinergic analgesia of inactivation of the M1 gene by an antisense oligodeoxyribonucleotide (aODN) was investigated in the mouse hot plate test. Mice received a single intracerebroventricular (i.c.v.) injection of anti‐M1 aODN (0.3, 1.0 or 2.0 nmol per injection), degenerate ODN (dODN) or vehicle on days 1, 4 and 7. A dose‐dependent inhibition of the antinociception induced by the muscarinic agonists oxotremorine (0.1 mg kg−1 s.c.) and McN‐A‐343 (30 μg per mouse i.c.v.) and the cholinesterase inhibitor physostigmine (0.2 mg kg−1 s.c.) was observed 24 h after the last i.c.v. injection of aODN. Time‐course experiments revealed that, after the end of the aODN treatment, sensitivity to analgesic drugs progressively appeared reaching the normal range at 96 h. The anti‐M1 aODN was selective against muscarinic antinociception since the enhancement of pain threshold produced by morphine and baclofen were not affected by the above‐mentioned treatment. dODN, used as control, did not affect muscarinic antinociception. Binding studies evidenced a selective reduction of M1 receptor levels in the hippocampus of aODN‐treated mice. Neither aODN, dODN nor vehicle produced any behavioural impairment of mice as revealed by the rota‐rod and Animex experiments. These results indicate that activation of M1 muscarinic receptor subtype is fundamental to induce central cholinergic analgesia in mice.

Caffeine induces central cholinergic analgesia.

Abstract The antinociceptive effect of caffeine was examined by using the hot-plate, abdominal constriction tests in mice and the tail flick and paw-pressure tests in rats. Caffeine (1–5 mg kg–1 s.c. in mice; 2.5–5 mg kg–1 i.p. in rats) produced significant antinociception in both species which was prevented by atropine (5 mg kg–1 i.p.), pirenzepine (0.1 μg per mouse i.c.v.), hemicholinium-3 (1 μg/ mouse i.c.v.) and N6-cyclopentyladenosine (5 μg/mouse i.c.v.), but not by naloxone (1 mg kg–1 i.p.), CGP 35348 (100 mg kg–1 i.p.), α-methyl p-tyrosine (100 mg kg–1 i.p.) and reserpine (2 mg kg–1 i.p.). Intracerebroventricular injection of caffeine in mice at doses (2.5–5 μg per mouse) which were largely ineffective by parenteral routes, induces an antinociception whose intensity equalled that obtainable s.c. or i.p. In the antinociceptive dose-range, caffeine did not produce any behavioural impairment as revealed by the rotarod and Irwing tests. On the basis of the above data, it can be postulated that caffeine exerts an antinociceptive effect mediated by central amplification of cholinergic transmission.