A. Giotti

GABAA and GABAB receptor‐mediated effects in guinea‐pig ileum

1 The effects of γ‐aminobutyric acid (GABA) and related substances were examined in guinea‐pig ileum longitudinal muscle. 2 GABA at doses ranging from 10−7 m to 3 m 10−6 m elicited a relaxation while at higher doses (3 × 10−6 m — 10−4 m), as previously described, it caused a contraction followed by relaxation. 3 GABA‐induced relaxation was bicuculline‐insensitive, was mimicked by (—)‐baclofen but not by homotaurine and muscimol. The effect of baclofen was stereospecific. GABA‐ and (—)‐baclofen‐induced relaxations were dose‐dependent and their ED50 values were similar. A specific cross‐desensitization occurred between GABA and (—)‐baclofen. 4 The bicuculline‐insensitive relaxation induced by GABA and (—)‐baclofen was prevented by tetrodotoxin and hyoscine but not by phentolamine plus propranolol, naloxone or theophylline. 5 In preparations in which the muscle tone was raised by histamine or prostaglandin F2α, GABA and (—)‐baclofen induced relaxation to the same extent as before increasing the tone. If the tone was raised by DMPP, a greater bicuculline‐insensitive relaxation occurred. 6 Contraction caused by GABA was bicuculline‐sensitive and was mimicked by homotaurine and muscimol. Contraction was dose‐dependent and muscimol was about three times more potent than GABA or homotaurine. A specific cross‐desensitization occurred between the contractile effects of GABA and those of homotaurine or muscimol. 7 Bicuculline competitively antagonized the contractile effects of GABA, homotaurine and muscimol and gave closely similar pA2 values. The slope of the Schild plot for the above drugs was near 1, confirming the competitive nature of the antagonism. 8 The bicuculline‐sensitive contraction induced by GABA, homotaurine and muscimol was abolished by tetrodotoxin and was non‐competitively antagonized by hyoscine, while it was unaffected by hexamethonium, mepyramine and methysergide. 9 It is concluded that two receptors mediate the GABA effects in guinea‐pig ileum: a bicuculline‐sensitive GABAA receptor, which elicits contraction through an excitatory action on cholinergic post‐ganglionic neurones; and a bicuculline‐insensitive GABAB receptor which causes relaxation through an inhibitory presynaptic action on cholinergic post‐ganglionic neurones. We confirm that GABA, homotaurine and muscimol are GABAA agonists, while GABA and (—)‐baclofen are GABAB agonists.

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 taurine on calcium kinetics of guinea-pig heart

Abstract Guinea-pig hearts, perfused with Tyrode containing 8 mM taurine, show a less marked decrease of contractile force when washed out with calcium-free Tyrode than control hearts. The effects of taurine cardiac contractility are explained by its interference with calcium kinetics. Total calcium content is increased in taurine-treated hearts, and greater amounts of calcium are washed out from calcium exchanging compartments, analyzed according to the model of Bayley et al. (1968). Most of the additional calcium retained by taurine-treated hearts is bound to some tissue structure, and can be partially mobilized when the concentration of easily exchanged calcium falls. The hypothesis is advanced that taurine increases the affinity of some cell structure for calcium.

Tachykinins activate guinea-pig alveolar macrophages: involvement of NK2 and NK1 receptors.

1 The effects of substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) were evaluated on superoxide anion () production by guinea‐pig alveolar macrophages (AM). 2 SP dose‐dependently (ED50 = 0.7 nm) evoked production from guinea‐pig AM; the N‐terminal heptapeptide, SP(1–7), was ineffective. In the presence of thiorphan (10−5 m), an enkephalinase inhibitor, the stimulating effects of SP were not significantly modified. NKA and NKB were both able to induce production from guinea‐pig AM, ED50 values being 0.1 and 1.3 nm, respectively. Therefore, the rank order of activity of natural tachykinins was NKA > SP > NKB. Tachykinin‐evoked effects were quantitatively similar to those elicited by the autacoid mediator PAF‐acether and less than those induced by the synthetic peptide N‐formylmethionyl‐leucyl‐phenylalanine (FMLP). 3 The NK2 receptor agonist [β‐Ala8]‐NKA (4–10) dose‐dependently evoked production from guinea‐pig AM; the NK1 receptor agonist [Pro9]‐SP sulphone acted only at high concentrations, while the NK3 receptor agonist [Me, Phe7]‐NKB was ineffective. 4 These findings indicate that guinea‐pig AM possess NK2 and possibly some NK1 tachykinin receptors and further suggest tachykinin involvement in lung pathophysiology.

Effects of noradrenaline and isoprenaline, in combination with α‐ and β‐receptor blocking substances, on the action potential of cardiac Purkinje fibres

1. The effects of noradrenaline and isoprenaline on the repolarization phase of the action potential have been studied in the Purkinje fibres of sheep heart, electrically driven at constant rates.

Effect of taurine on calcium levels and contractility in guinea-pig ventricular strips

Taurine increases the calcium levels in guinea-pig ventricular strips at external calcium concentrations of 0.45, 0.9 and 1.8 mM. At 2.7 mM calcium, however, a decrease is observed. Analogous changes occur in contractile force. It is also seen that the superfusion of ventricular strips with taurine-free medium produces a decrease in taurine content at the end of 120 min superfusion. Taurine levels can be restored by superfusion with 10 mM taurine; a linear relationship exists between external taurine and internal taurine levels.

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.

The influences of adrenolytic drugs and noradrenaline on the histamine release in cardiac anaphylaxis in vitro

1. The regional distribution (auricles, ventricles and septum) of histamine and its release during anaphylaxis were studied in isolated perfused guinea‐pig hearts.