Fat-storing cells as liver-specific pericytes. Spatial dynamics of agonist-stimulated intracellular calcium transients.

Liver perisinusoidal fat-storing cells (FSC) show morphological and ultrastructural characteristics similar to pericytes regulating local blood flow in other organs. In the present study we have analyzed whether FSC respond to local vasoconstrictors such as thrombin, angiotensin-II, and endothelin-1 with an increase in intracellular free calcium concentration ([Ca2+]i) coupled with effective cell contraction. All agonists tested induced a rapid and dose-dependent increase in [Ca2+]i followed by a sustained phase lasting several minutes in confluent monolayers of Fura-2-loaded human FSC. Pharmacological studies performed using different Ca2+ channel blockers indicated that, at least for thrombin and angiotensin-II, the sustained phase is due to the opening of voltage-sensitive membrane Ca2+ channels. To analyze the temporal and spatial dynamics of Ca2+ release in response to these agonists, we performed experiments on individual Fura-2-loaded human FSC using a dual wavelength, radiometric video imaging system. The rise in [Ca2+]i was exclusively localized to the cytoplasm, particularly in the branching processes. Increases in [Ca2+]i more than four-fold were associated with a simultaneous and transient reduction of cell area indicating reversible cell contraction. Our results indicate that the Ca(2+)-dependent contraction of human FSC in vitro may reflect a potential role in regulating sinusoidal blood flow in vivo.

Desensitization of GABAB receptors and antagonism by CGP 35348, prevent bicuculline- and picrotoxin-induced antinociception.

The effect of the GABAA antagonists, bicuculline and picrotoxin, in the hot plate and writhing tests in mice and the paw-pressure test in rats was assessed. Subconvulsant doses of bicuculline (1.3-4 mumol kg-1, s.c.) or picrotoxin (0.8-2.5 mumol kg-1, s.c.) induced a dose-related increase in latency of licking in the hot plate test in mice, whereas subconvulsant doses of strychnine and thiosemicarbazide (0.9 and 6 mg kg-1, s.c. respectively), did not modify the threshold to thermal stimuli in mice. The effects of bicuculline and picrotoxin were not modified by naloxone (3 mg kg-1, i.p., a dose which inhibited the antinociceptive effect of morphine) or by atropine (5 mg kg-1, i.p., a dose which prevented oxotremorine-induced antinociception) but were antagonized by the GABAB antagonist CGP 35348 (2.5 micrograms, i.c.v., a dose which prevented (+/-)baclofen-induced antinociception). Mice, rendered tolerant to baclofen-induced antinociception by twice daily injection of increasing doses of baclofen (5-18 mg kg-1, s.c.), were unresponsive to the antinociceptive effects of bicuculline and picrotoxin but still responded to morphine. Bicuculline and picrotoxin, in the same range of doses which affected the three models of antinociception used, inhibited pentobarbital-induced hypnosis. Large doses of bicuculline and picrotoxin (4 and 2.5 mumol kg-1, s.c. respectively), reduced locomotor activity and impaired rota-rod performance in mice. The changes in response to noxious stimuli, induced by bicuculline and picrotoxin, are interpreted as an antinociceptive effect. It is then suggested that this effect might depend on an indirect activation of GABAB receptors through release of GABA.(ABSTRACT TRUNCATED AT 250 WORDS)

Stereoselective increase in cholinergic transmission by R-(+)-hyoscyamine.

R-(+)-hyoscyamine, the dextro enantiomer of atropine, has been shown to amplify cholinergic transmission. R-(+)-hyoscyamine, unlike S-(-)-hyoscyamine, was able to increase acetylcholine release both in vitro and in vivo at a range of concentrations (10(-14) to 10(-12) M) and doses (5 microg/kg i.p.) which were inadequate for blocking muscarinic receptors. The increase over control values in ACh release was 15.9 +/- 2.1% in in vitro experiments performed in rat phrenic nerve-hemidiaphragm preparations (n = 6), and 63.3 + 16.3% in cortical microdialysis performed in free-moving rats (n = 5). The maximum ACh release was reached 60 min after R-(+)-hyoscyamine administration in in vivo experiments. At the same doses and concentrations, R-(+)-hyoscyamine was also able to elicit: antinociception of a cholinergic type (55.6-112.7% depending on the test used); complete prevention of scopolamine- and dicyclomine-induced amnesia; potentiation of muscular contractions electrically evoked in isolated guinea-pig ileum (16.7 +/- 3.6%) and in rat phrenic nerve-hemidiaphragm (19.9 +/- 3.2%) preparations. Antinociception was performed using the hot-plate and acetic acid abdominal constriction tests in mice, and the paw pressure test in rats, while prevention of induced amnesia was evaluated in mice using the passive-avoidance test. The respective affinities (pA2) for R-(+)- and S-(-)-hyoscyamine vs M1 (rabbit vas deferens), M2 (rat atrium) and M3 (rat ileum) receptor subtypes were as follows: 7.05 +/- 0.05/9.33 +/- 0.03 for M1; 7.25 +/- 0.04/8.95 +/- 0.01 for M2; 6.88 +/- 0.05/9.04 +/- 0.03 for M3. The respective pKi values for R-(+)- and S-(-)-hyoscyamine vs the five human muscarinic receptor subtypes expressed in Chinese hamster oocytes (CHO-K1) were as follows: 8.21 +/- 0.07/9.48 +/- 0.18 for m1; 7.89 +/- 0.06/9.45 +/- 0.31 for m2; 8.06 +/- 0.18/9.30 +/- 0.19 for m3; 8.35 +/- 0.11/9.55 +/- 0.13 for m4; 8.17 +/- 0.08/9.24 +/- 0.30 for m5.

Characteristics of L-type calcium channel blockade by lacidipine in guinea-pig ventricular myocytes.

1. The Ca(2+)-antagonistic properties of lacidipine were investigated in patch-clamp guinea-pig ventricular myocytes. 2. In basal conditions, 0.1 microM lacidipine reduced the action potential duration, associated with a decrease in the L-type calcium current (ICa,L) to 66 +/- 4% of the control value, without a change in the current-voltage relationship. Sodium current and background potassium currents were not affected. All the effects reached a steady state within 2 min. 3. The Ca(2+)-antagonistic effect of lacidipine was voltage-dependent: a marked negative shift (about 20 mV) of the steady-state inactivation curve was observed with long (10 s) conditioning prepulses, but not with short (350 ms) prepulses. 4. The onset of and recovery from the voltage-dependent effect caused by 0.1 microM lacidipine were significantly slower when compared to those of equiactive concentrations of nimodipine (0.5 microM) and nisoldipine (0.1 microM). ICa,L measured after prepulses at -40 mV lasting 500 ms or less was unchanged (95 +/- 5% of maximum current value) while it was reduced to 49 +/- 10% by nimodipine and 43 +/- 9% by nisoldipine (P < 0.05 vs lacidipine for both). 5. Similarly, the recovery from block in the presence of lacidipine was slower than with nimodipine and nisoldipine. After a prepulse of 1 s at -80 mV, ICa,L recovered up to 54 +/- 2% of the maximum current value in the presence of lacidipine, and up to 91 +/- 3% and 93 +/- 5% in the presence of nimodipine and nisoldipine, respectively (P < 0.05 vs lacidipine). 6. Blockade of ICa,L by lacidipine was use-dependent. After ten 200 ms long pulses (1 Hz) from -80 mV, ICa,L was reduced to 55 +/- 7% of the current measured at the first pulse. In the presence of nimodipine and nisoldipine, ICa,L elicited by the tenth pulse amounted to 93 +/- 3% and 80 +/- 6% of the first pulse value, respectively (P < 0.05 vs lacidipine). Lacidipine did not cause use-dependent blockade of ICa,L in cells stimulated with 10 ms long pulses. 7. These results demonstrate that lacidipine selectively inhibits ICa,L in isolated cardiomyocytes and suggest that this effect occurs mainly through binding to the inactivated Ca2+ channels.

Effect of verapamil and diltiazem on calcium-dependent electrical activity in cardiac Purkinje fibres.

The effects of verapamil and diltiazem on normal action potentials, abnormal automaticity at depolarized membrane potential and oscillatory afterpotentials were compared in sheep cardiac Purkinje fibres. Concentrations of verapamil and diltiazem exerting the same action on abnormal automaticity due to slow action potentials, caused different effects on action potential characteristics and on oscillatory afterpotentials. Diltiazem significantly shortened action potential duration whereas verapamil slightly lengthened it (NS). Diltiazem appeared to be more effective than verapamil in preventing the development of oscillatory afterpotentials induced by barium or by strophanthidin. In 50% of barium-treated preparations, verapamil caused the appearance of spontaneous activity due to enhanced normal diastolic depolarization, while diltiazem had no such effect. The observed differences were explained in terms of the different effects of the two drugs on currents other than the slow inward current, since diltiazem was more potent than verapamil in depressing Vmax.

GABAA and GABAB receptor-mediated effects in guinea-pig ileum.

1 The effects of gamma-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 x 10(-6) M elicited a relaxation while at higher doses (3 x 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 ED(50) 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 F(2alpha), 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 pA(2) 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 GABA(A) receptor, which elicits contraction through an excitatory action on cholinergic post-ganglionic neurones; and a bicuculline-insensitive GABA(B) receptor which causes relaxation through an inhibitory presynaptic action on cholinergic post-ganglionic neurones. We confirm that GABA, homotaurine and muscimol are GABA(A) agonists, while GABA and (-)-baclofen are GABA(B) agonists.

Homotaurine: a GABAB antagonist in guinea-pig ileum.

Homotaurine (3-aminopropane sulphonic acid) did not inhibit the twitch response in guinea-pig ileum longitudinal muscle whilst gamma-aminobutyric acid (GABA) and (-)-baclofen evoked dose-dependent inhibitions. The inhibitory effects of GABA and (-)-baclofen were prevented in the presence of homotaurine 2 X 10(-4) and 10(-3) M. The log dose-effect curves of GABA and (-)-baclofen were shifted in a parallel manner compatible with competitive antagonism. The pA2 of homotaurine with GABA (4.22 +/- 0.05) and (-)-baclofen (4.26 +/- 0.1) were the same. Homotaurine did not antagonize the inhibitory effects of morphine (ED50 4 X 10(-7) M), noradrenaline (ED50 10(-6) M) or ATP (ED50 1.5 X 10(-5) M). The inferior homologue of homotaurine, taurine 10(-3) M, did not modify the inhibitory effects of GABA and (-)-baclofen. Picrotoxin 5 X 10(-5) M antagonized GABAA receptor-mediated contraction but did not affect GABAB receptor-mediated inhibition. At the same concentration the drug did not influence the antagonistic action of homotaurine, thus showing no GABAA receptor-mediated interference. It may be concluded that homotaurine is a competitive antagonist of GABAB mediated effects in the guinea-pig ileum.

Investigation into atropine-induced antinociception.

1. The effect of atropine on the nociceptive system was examined in mice and rats by use of the hot-plate, writhing and tail-flick tests. 2. Atropine dose-dependently produced analgesia, no effect and hyperalgesia. Analgesia was observed in both species with doses ranging from 1 to 100 micrograms kg-1 while hyperalgesia was obtained with 5 mg kg-1. 3. Atropine antinociception was prevented by pirenzepine (0.1 microgram per mouse, i.c.v.), dicyclomine (10 mg kg-1, i.p.), atropine-methylbromide (0.5 microgram per mouse, i.c.v.) and hemicholinium-3 (1 microgram per mouse, i.c.v.). Naloxone (1 mg kg-1, i.p.), alpha-methyl-p-tyrosine (100 mg kg-1, s.c.) and reserpine (2 mg kg-1, i.p.) were ineffective. 4. The site of atropine analgesia is in the CNS since it exerts its antinociceptive effect also when injected i.c.v. (1-10 ng per mouse). Moreover drugs which do not cross the blood-brain barrier, such as hemicholinium-3, pirenzepine and atropine methylbromide, were unable to antagonize atropine analgesia if administered i.p. 5. Atropine also in vitro, showed a biphasic action on electrically-evoked guinea-pig ileum contractions. Concentrations between 10(-14) and 10(-12) M increased electrically and nicotine-evoked contractions but did not affect acetylcholine- and oxotremorine-evoked contractions. Concentrations above 10(-9) M inhibited both electrically- and drug (acetylcholine, nicotine and oxotremorine)-evoked contractions while they were ineffective on unstimulated ileum. 6. On the basis of the above findings, amplification of cholinergic transmission by very low doses of atropine is postulated, through a selective blockade of presynaptic muscarinic autoreceptors, as the likely mechanism of action. 7. Atropine antinociception, unlike oxotremorine antinociception, was obtained without any impairment of mouse rota-rod performance. 8. The antagonism by pirenzepine and dicyclomine of oxotremorine and atropine antinociception suggests that M1 muscarinic receptor subtypes are responsible for cholinergic analgesia.

Modulatory activity of GABAB receptors on cholinergic tone in guinea-pig distal colon.

The effect of gamma-aminobutyric acid (GABA) administration was studied in both in vitro and in vivo preparations of the guinea-pig distal colon. In in vitro preparations GABA (10(-7) - 10(-3) M) elicited a dose-dependent relaxation; a decrease in the spontaneous contractions was sometimes observed. The effect of GABA was mimicked by (-)-baclofen, which gave a dose-response curve overlapping that of GABA, while (+)-baclofen was about one hundred times less potent. The relaxation responses induced by the above drugs were antagonized by 5-aminovaleric acid (5 X 10(-4) M), which did not affect adenosine-induced relaxation, but they were insensitive to bicuculline (10(-5) M) and picrotoxin (10(-5) M). Moreover, they were prevented by tetrodotoxin (6 X 10(-7) M). In hyoscine (10(-7) M)-pretreated preparations, GABA still evoked a small relaxation response (approx. 10% of the maximum) that was bicuculline-sensitive. Desensitization to GABA (10(-5) M) was observed. A specific cross-desensitization occurred between GABA (10(-5) M) and (-)-baclofen (10(-5) M). In in vivo preparations, GABA (10 mumol kg-1) and (-)-baclofen (5 mumol kg-1) produced a dose-related inhibition of basal tone, while (+)-baclofen (5 mumol kg-1) had much less effect (about 25%). A decrease in the spontaneous contractions was sometimes observed. The relaxant effect of GABA and (-)-baclofen persisted in guinea-pigs pretreated (1-2 min) with picrotoxin (1.6 mumol kg-1), whereas it was significantly reduced in animals injected 1 min beforehand with 5-aminovaleric acid (0.2 mmol). The maximal relaxant effect induced by GABA and (-)-baclofen did not differ from that of atropine (0.9 mumol kg-1) and after atropine administration GABA had no further inhibitory effect. Relaxation responses induced by GABA and (-)-baclofen still occurred after blockade of nicotinic receptors by hexamethonium (0.17 mmol kg-1), which itself caused an increase in the basal tone. When the tone was increased by topical application of physostigmine (40 micrograms), GABA and (-)-baclofen induced a greater relaxation than that obtained in basal conditions. It is concluded that GABA, both in vitro and in vivo administration, inhibits cholinergic tone in guinea-pig distal colon and that this effect is mediated mainly by activation of GABAB receptors. Further experiments are required to ascertain the possible physiological role of a GABA-releasing neuronal system in the colon in vivo.

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 micrograms per mouse; 25 micrograms 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 micrograms i.c.v. per mouse) also prevented (+/-)-baclofen-induced impairment of the rota-rod test. 5. Two other GABAB antagonists, phaclofen (50 micrograms i.c.v. per mouse) and 2-OH-saclofen (2.5 micrograms-10 micrograms 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.

Enhanced responsiveness of ovalbumin-sensitized guinea-pig alveolar macrophages to tachykinins.

1. We have evaluated the ability of substance P (SP), neurokinin A (NKA) and the selective NK2 receptor agonist [beta-Ala8]-NKA(4-10) to induce superoxide anion (O2-) production and prostanoid (prostaglandin E2, thromboxane B2) release from alveolar macrophages (AMs) isolated from control or actively sensitized guinea-pigs. 2. The dose-response curves for NKA and SP were shifted to the left (three orders and one order of magnitude, respectively) in AMs isolated from sensitized animals, with no variation in maximal effects. 3. By evaluating the effects of [beta-Ala8]-NKA(4-10), we observed that not only was the concentration-response curve shifted to the left in both the functional parameters examined, but also maximal effects were significantly enhanced in AMs isolated from sensitized guinea-pigs. 4. This varied responsiveness seems to be specific for tachykinins, as it was not reproduced by another AM stimulant, the bacterial peptide N-formylmethionyl-leucyl-phenylalanine (fMLP). 5. Only small amounts of beta-glucuronidase were released following tachykinin or ovalbumin stimulation both in control and sensitized AMs. 6. These results indicate that AMs isolated from sensitized guinea-pigs show an increased responsiveness to NK2 receptor stimulation and further stress the role played by AMs in allergic lung diseases.

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 dose were dose-dependently antinociceptive in all three tests: 5-100 micrograms per rat in the paw pressure test, 5-50 micrograms per mouse in the abdominal constriction test and 50-100 micrograms per mouse in the hot plate test. Conversely, very low doses were hyperalgesic: 0.5 microgram per rat in the paw pressure test and 0.1-1 microgram 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 micrograms per rat and 3.4-10 micrograms per mouse) routes. 4. The histamine H3 agonist, (R)-alpha-methylhistamine dihydrogenomaleate was hyperalgesic, with a rapid effect (15 min after treatment) following i.c.v. administration of 1 microgram per rat and 3 microgram per mouse, or i.p. administration of 100 mg kg-1 in 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)-alpha-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 micrograms 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)-alpha-fluoromethylhistidine and with (R)-alpha-methylhistamine, respectively. L-Histidine antinociception was completely antagonized in all three tests by pretreatment with (S)-alpha-fluoromethylhistidine HCl (50 mg kg-1, i.p.)administered 2 h before L-histidine treatment. Similarly, metoprine antinociception was prevented by(R)-alpha-methylhistamine dihydrogenomaleate 20 mg kg-1, i.p. administered 15 min before metoprine. Both(S)-alpha-fluoromethylhistidine and (R)-alpha-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)-alpha-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.

Effects of L- and D-arginine and some related esters on the cytosolic mechanisms of alpha-thrombin-induced human platelet activation.

1. In Fura-2 preloaded human platelets, the increase in cytosolic calcium induced by alpha-thrombin was reduced by some L- and D-arginine ester compounds the IC50 (microM) values of which were 7.4 for TAEE, 56.9 for BAEE, 77.6 for TAME, 560 for T(d)AME, 656.3 for L-ArgOMe and 2206.7 for D-ArgOMe. alpha-tosyl-L-Arginine, L- and D-arginine were inactive. 2. The inhibitory activity of the L-arginine esters was not modified when platelets were pretreated with 100 microM N omega-monomethyl-L-arginine. 3. The L-arginine esters did not increase cyclic GMP content in platelets either in the presence or absence of indomethacin and apyrase at rest and after alpha-thrombin stimulation. 4. The kinetic parameters of platelet Na+/H+ antiporter (amiloride-inhibitable, evaluated after cytosolic nigericin-induced acidification) were modified by L- and D-arginine esters, while the native amino acids were ineffective. 5. The inhibitory effects of the L- and D-arginine esters on platelet activation appear to be mainly due to their inhibitory effect on Na+/H+ antiporter.

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 microgram per mouse, i.c.v.), but not by naloxone (3 mg kg-1, i.p.), alpha-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 micrograms 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 micrograms), lignocaine (150 microgram) and bupivacaine (25 micrograms), 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 effect of taurine on high potassium-and noradrenaline-induced contraction in rabbit ear artery.

1 Intraluminal administration of taurine (40 mM) did not affect the contractile tone of rabbit isolated ear artery 2 Taurine (10-80 mM) exerted a powerful concentration-dependent, vasodilator action in arteries contracted with high potassium medium. 3 In the same experimental conditions, the taurine analogues beta-alanine and homotaurine, had no effect. 4 Taurine (40-80 mM) did not affect in a significant manner the tonic component of the noradrenaline (5x10(-6 M)-induced contraction. 5 When noradrenaline (5x10(-6M) was followed by the administration of high potassium medium a further increase in intraluminal pressure was observed. Under these conditions taurine (40 mM) reversed specifically the component due to the high potassium medium.

Effect of some cyclooxygenase inhibitors on the increase in guanosine 3':5'-cyclic monophosphate induced by NO-donors in human whole platelets.

1. The effect of the NSAIDs indomethacin, indoprofen, diclofenac and acetylsalicylic acid on the increase in guanosine 3':5'-cyclic monophosphate (cyclic GMP) induced by nitric oxide-donor agents was tested in human whole platelets and in platelet crude homogenate. 2. In whole platelets, indomethacin reduced the increase in cyclic GMP induced by the nitric oxide-donors (NO-donors) sodium nitroprusside (NaNP) and S-nitroso-N-acetylpenicillamine (SNAP) in a dose-dependent way, its IC50 being 13.7 microM and 15.8 microM, respectively. 3. Of the other cyclooxygenase inhibitors tested, only indoprofen reduced the increase in cyclic GMP induced by both NO-donors in a dose-dependent way (IC50=32.7 microM, NaNP and 25.0 microM, SNAP), while acetylsalicylic acid (up to 1000 microM) and diclofenac (up to 100 microM) were ineffective. 4. However, in platelet crude homogenate neither indomethacin nor indoprofen reduced the cyclic GMP production. 5. Indomethacin (10 microM), indoprofen (30 microM), diclofenac (100 microM) and acetylsalicylic acid (1000 microM) showed a comparable efficacy in inhibiting platelet thromboxane B2 (TXB2) production, suggesting that the inhibitory effect of indomethacin and indoprofen on the increase in cyclic GMP induced by both NO-donors was not mediated by inhibition of cyclooxygenase. 6. In vitro, the NSAIDs analysed did not interfere with nitrite production of SNAP. 7. The unhomogeneous behaviour of NSAIDs on the increase in cyclic GMP induced by NO-donors in whole platelets may contribute to the different pharmacological and toxicological characteristics of the drugs, providing new knowledge on the effect of indomethacin and indoprofen.

Cardiac hypoxia and subsequent reoxygenation: sensitivity to L-arginine methylester.

The effect of L-arginine methylester (L-Arg-Me) was studied in the isolated heart of the guinea-pig perfused with hypoxic substrate-free medium for 30 min and subsequently reoxygenated with normal saline solution for 30 min. The administration of L-Arg-Me in basal conditions decreases dose-dependently heart rate without any changes in the myocardial structure. On the other hand, the administration of L-Arg-Me (5-10 mM) decreases ventricular arrhythmias, especially during reoxygenation; in fact ventricular fibrillation is abolished. L-Arg-Me treatment increases the recovery of normal electrical and mechanical activity at the end of reoxygenation and reduces the increase in basal tone. Treatment with 10 mM L-Arg-Me decreases lactate dehydrogenase (LDH) release in the effluent and lysosomal fragility in cardiac tissue, while it does not influence calcium gain. L-Arginine (L-Arg) does not mimic any of the effects of L-Arg-Me.

Electrophysiological mechanisms for the antiarrhythmic action of mexiletine on digitalis-, reperfusion- and reoxygenation-induced arrhythmias.

The antiarrhythmic potency of mexiletine was evaluated on three groups of guinea-pig isolated hearts. Arrhythmias were induced (a) with digitalis intoxication, (b) with hypoxia followed by reoxygenation and (c) with ischaemia followed by reperfusion. Mexiletine 10 microM was found to be very effective against all three types of arrhythmias in all three groups. The electrophysiological effects of mexiletine were then studied on sheep cardiac Purkinje fibres manifesting oscillatory afterpotentials and triggered automaticity induced by barium or strophanthidin. Mexiletine 10 microM consistently decreased the amplitude of oscillatory afterpotentials and blocked subsequent triggered activity in sheep Purkinje fibres. In contrast, mexiletine 10 microM had no significant effect on Vmax in normal, barium- and strophanthidin-treated preparations. The results are discussed in relation to the mechanisms of antiarrhythmic action of mexiletine.

Interference of WEB 2086 and BN 52021 with Paf-induced effects on guinea-pig trachea.

1. The thienotriazolodiazepine WEB 2086 and the gingkolide BN52021 have been evaluated as antagonists of Paf-acether (Paf) by studying their effects on Paf-induced relaxation and Paf-induced prostaglandin E2 (PGE2) production in histamine-contracted guinea-pig tracheal preparations. 2. Relaxation induced by Paf 4 microM in histamine-contracted guinea-pig tracheal preparations was 39.67 +/- 3.5% (n = 30). At the same concentration, Paf significantly increased PGE2 production from histamine-contracted guinea-pig tracheal preparations. 3. WEB 2086 inhibited in a dose-related manner (IC50 = 21.2 nM) the relaxant effect induced by Paf and, at 1 microM, suppressed Paf-induced release of PGE2. 4. BN 52021 100 microM inhibited to about 60% Paf-induced relaxation of histamine-contracted guinea-pig tracheal preparations, but completely abolished Paf-induced increase in PGE2. 5. Both antagonists had no effects on relaxations induced by arachidonic acid 10 microM or PGE2 0.1-1 microM in histamine-contracted guinea-pig tracheal preparations. 6. The results are consistent with the presence of specific Paf receptors in guinea-pig trachea and indicate that a relaxant prostanoid, namely PGE2, at least partially mediates Paf-induced relaxation in this experimental model.

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 micrograms per mouse i.c.v.) and AF-102B (1-10 mg kg-1 i.p.) produced significant antinociception which was prevented by atropine (1 microgram per mouse i.c.v.) and by the two selective M1 antagonists: pirenzepine (0.01 micrograms per mouse i.c.v.) and dicyclomine (0.08 micrograms per mouse i.c.v. or 10 mg kg-1 i.p.) but not by the specific M2-antagonist AFDX-116 (0.1 micrograms per mouse i.c.v.), naloxone (1 mg kg-1 i.p.) or by the acetylcholine (ACh) depletor hemicholinium-3 (HC-3) (1 micrograms 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 micrograms per mouse i.c.v. or 10 mg kg-1 i.p.) but not by AFDX-116 (0.1 microgram per mouse or rat i.c.v.). 3. In contrast with the M1-agonists, the M2-agonist arecaidine (0.1-2 micrograms 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.