Lucilla Zilletti

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.

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.

Agonists, antagonists and modulators of excitatory amino acid receptors in the guinea‐pig myenteric plexus

1 The receptors for glutamic acid (L‐Glu) present in the guinea‐pig myenteric plexus‐ileal longitudinal muscle preparation have been studied by measuring the muscle contraction induced by numerous putative endogenous agonists acting at these receptors. Furthermore, the actions of different concentrations of antagonists, glycine, Mg2+ and Ca2+ on the ileal contractions induced by l‐G1u have been evaluated. 2 The EC50 values of the most common putative endogenous agonists of these receptors were: L‐Glu 1.9 × 10−5 m; L‐aspartate 8 × 10−5 m; quinolinate 5 × 10−4M; L‐homocysteate 1.4 × 10−4M; the dipeptide aspartyl‐glutamate 8 × 10−5m, while N‐acetyl‐aspartyl‐glutamate was inactive. Among the molecules used to classify excitatory amino acid receptors, N‐methyl‐D‐aspartate (NMDA) was the most potent (EC50 5 × 10−4m). Kainic and quisqualic acids were almost completely inactive. 3 The responses to L‐Glu were competitively antagonized by 2‐amino‐5‐phosphonovaleric acid. They were, also, prevented by hyoscine (10−7m) and by tetrodotoxin (3 × 10−7 m), suggesting that the L‐Glu‐induced ileal contraction was in some way dependent upon an action on the myenteric cholinergic neurones. Kynurenic acid was a non‐competitive antagonist, γ‐D‐glutamyl‐taurine (10−4m) and aminophosphonobutyric acid (10−4m) did not modify the L‐Glu‐induced contractions. 4 Glycine (10−5 m) significantly potentiated the effects of glutamate especially when the ionic composition of the superfusion medium contained concentrations of Ca2+ in the range of 0.6–1.2 mM. Strychnine 3 × 10−5 m did not modify the actions of glycine. 5 The data presented here confirm the presence of NMDA receptors in the guinea‐pig myenteric plexus, and show that these receptors, similar to those present in primary neuronal cultures may be modulated by glycine.

Homotaurine: a GABAB antagonist in guinea‐pig ileum

1 Homotaurine (3‐aminopropane sulphonic acid) did not inhibit the twitch response in guinea‐pig ileum longitudinal muscle whilst γ‐aminobutyric acid (GABA) and (−)‐baclofen evoked dose‐dependent inhibitions. 2 The inhibitory effects of GABA and (−)‐baclofen were prevented in the presence of homotaurine 2 × 10−4 and 10−3 M. 3 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. 4 Homotaurine did not antagonize the inhibitory effects of morphine (ED50 4 × 10−7 M), noradrenaline (ED50 10−6 M) or ATP (ED50 1.5 × 10−5 M). 5 The inferior homologue of homotaurine, taurine 10−3 M, did not modify the inhibitory effects of GABA and (−)‐baclofen. 6 Picrotoxin 5 × 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. 7 It may be concluded that homotaurine is a competitive antagonist of GABAB mediated effects in the guinea‐pig ileum.

Effect of N-acetyl-l-cysteine on peroxynitrite and superoxide anion production of lung alveolar macrophages in systemic sclerosis

Lung macrophages may play a relevant role in oxidative processes producing both superoxide anion (O(2)(-)) and NO. In this view, an antioxidant therapy can be useful in the treatment of systemic sclerosis (SSc) patients. N-Acetylcysteine (NAC) is able to expand natural antioxidant defenses by increasing intracellular gluthatione concentration and it has been proposed as an antioxidant therapy in respiratory distress syndromes. The aim of our study was to determine whether lung macrophages obtained from SSc patient bronchoalveolar lavage (BAL) express the inducible form of nitric oxide synthase (iNOS) and whether NAC can reduce the peroxynitrite (ONOO(-)) and O(2)(-) production of these cells. Alveolar macrophages were isolated from BAL of 32 patients and used for the immunocytochemical determination of iNOS, and the production of ONOO(-) and O(2)(-) was measured by fluorimetric or spectrophotometric methods, respectively. Lung macrophages obtained from SSc patients expressed a higher level of iNOS compared to healthy subject cells. NAC preincubation (5 x 10(-5)M, 24h) significantly reduced (-21%) the ONOO(-) production in formyl Met-Leu-Phe (fMLP)-activated cells and slightly reduced it under resting conditions, whereas NAC preincubation was unable to modify the release of O(2)(-) both in basal condition and in fMLP-stimulated cells. We conclude that since SSc lung macrophages express high levels of iNOS and produce a significant quantity of ONOO(-), NAC administration reduces ONOO(-) production and can be an useful treatment to alleviate SSc symptoms.

Rhein: an anthraquinone that modulates superoxide anion production from human neutrophils.

Rhein (4,5‐dihydroxyanthraquinone‐2‐carboxylic acid), the active metabolite of diacetylrhein, which has been reported as an effective antirheumatic drug in man, inhibited superoxide anion production from human neutrophils challenged with N‐formylmethionyl‐leucyl‐phenylalanine (FMLP: IC50, 2 times 10−5 M) and A23186 (IC50, 10−5M), but not with phorbol myristate acetate. In the same concentration range (10−6‐10−3M), the drug did not affect oxy‐radical production by a cell‐free hypoxanthine‐xanthine oxidase system and exerted weak inhibitory effects on FMLP‐evoked lysosomal enzyme release. Rhein inhibitory effects on neutrophil functioning may contribute to the overall therapeutic activity of the parent drug, diacetylrhein.

The influence of rhein on the biosynthesis of prostaglandin-like substances in-vitro

It has recently been demonstrated that diacetylrhein (DAR; 1,8-diacetoxy-9,10-dioxo-dihydroanthracene-3carboxylic acid) attenuates the symptoms of osteoarthritis in man (Kay et a1 1980; Neuman 1980) and of various types of inflammatory arthritis (Neuman 1980). Diacetylrhein is well absorbed from the gastrointestinal tract and is deacetylated to rhein, which is the principal urinary metabolite in animals and in man (Friedmann, personal communication). In addition to being an antipyretic and analgesic, DAR blocks the inflammation induced by carrageenan, beer yeast, Freunds adjuvant and cotton pellet granuloma. It also protects rabbit ear and knee cartilage from retinoic acid-induced degradation. It is suggested that protection from degradation of proteoglycans occurs as a result of inhibition of the liberation and/or of the activity of lysosomal enzymes. This view has also been suggested by the capacity of the molecule and of its deacetylated metabolite to inhibit certain proteinase enzymes in-vitro (trypsin, pepsin, carboxypeptidase A, elastase) (Raimondi et al 1982). Other findings have indicated that DAR does not inhibit the synthesis of prostaglandins in-vivo. In fact it only possesses minimal effects in blocking the diarrhoea caused by castor oil using the methodology of Awouters et a1 (1978). In these conditions the anti-inflammatory drugs, which are active in inhibiting prostaglandin formation, also cause a delay in the production of diarrhoea. On the contrary, when administered to rats, DAR and its metabolite, increase the prostaglandin content in the carrageenan-induced inflammatory exudate (Panarelli et a1 1980). Bearing in mind that diacetylrhein is metabolized to rhein, which is a diphenolketone able to chelate divalent metallic ions and which possesses the biological property of specifically inhibiting NADdependent dehydrogenase (Kean 1968,1970), we examined the possibility that rhein might also interfere with prostaglandin synthesis in-vitro. In this way we also hoped to avoid variations in the concentration of the deacetylated metabolite, which might have occurred when diacetylrhein itself was used.

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 β‐alanine and homotaurine, had no effect. 4 Taurine (40–80 mm) did not affect in a significant manner the tonic component of the noradrenaline (5 × 10−6 m)‐induced contraction. 5 When noradrenaline (5 × 10−6 m) 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.

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

1 The effect of γ‐aminobutyric acid (GABA) administration was studied in both in vitro and in vivo preparations of the guinea‐pig distal colon. 2 In in vitro preparations GABA (10−7 − 10−3m) elicited a dose‐dependent relaxation; a decrease in the spontaneous contractions was sometimes observed. 3 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. 4 The relaxation responses induced by the above drugs were antagonized by 5‐aminovaleric acid (5 × 10−4m), which did not affect adenosine‐induced relaxation, but they were insensitive to bicuculline (10−5m) and picrotoxin (10−5m). Moreover, they were prevented by tetrodotoxin (6 × 10−7m). In hyoscine (10−7m)‐pretreated preparations, GABA still evoked a small relaxation response (approx. 10% of the maximum) that was bicuculline‐sensitive. 5 Desensitization to GABA (10−5m) was observed. A specific cross‐desensitization occurred between GABA (10−5m) and (—)‐baclofen (10−5m). 6 In in vivo preparations, GABA (10 μmol kg−1) and (—)‐baclofen (5 μmol kg−1) produced a dose‐related inhibition of basal tone, while (+)‐baclofen (5 μmol kg−1) had much less effect (about 25%). A decrease in the spontaneous contractions was sometimes observed. 7 The relaxant effect of GABA and (—)‐baclofen persisted in guinea‐pigs pretreated (1–2 min) with picrotoxin (1.6 μmol kg−1), whereas it was significantly reduced in animals injected 1 min beforehand with 5‐aminovaleric acid (0.2 mmol). 8 The maximal relaxant effect induced by GABA and (—)‐baclofen did not differ from that of atropine (0.9 μmol kg−1) and after atropine administration GABA had no further inhibitory effect. 9 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. 10 When the tone was increased by topical application of physostigmine (40 μg), GABA and (—)‐baclofen induced a greater relaxation than that obtained in basal conditions. 11 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.

The ACh‐induced contraction in rat aortas is mediated by the Cys Lt1 receptor via intracellular calcium mobilization in smooth muscle cells

Our previously published data indicate that an endogenously produced 5‐lipoxygenase metabolite can strongly contract isolated endothelium‐preserved rat aortic strips when cyclo‐oxygenase isoenzymes are inhibited. Therefore, we decided to investigate if cysteinyl‐containing leukotrienes (Cys Lts) are involved in this endothelium‐dependent contraction. The isometric contraction of endothelium‐preserved rat aortic strips was recorded in preparations preincubated with 5 μM indomethacin and precontracted with phenylephrine, adjusting resting tension at 0.7 g. Acetylcholine (ACh) contracted control strips. Montelukast and MK‐571, selective type 1 Cys Lts receptor (Cys Lt1) antagonists and the Cys Lt1/Cys Lt2 (type 2 Cys Lts receptor) antagonist BAYu9773 dose‐dependently prevented ACh‐induced contraction, their IC50s being 2.2, 3.1 and 7.9 nM respectively. The leukotriene B4 receptor antagonist U75302 was far less potent (IC50 1.5 μM). In rat aorta smooth muscle cells (RASMs), Western blot analysis showed the presence of Cys Lt1 and Cys Lt2 receptors, the Cys Lt1 receptor being predominantly expressed. In fura‐2 loaded RASMs, LTD4 (0.01–100 nM) and LTC4 (200–800 nM) dose‐dependently increased intracellular calcium concentration ([Ca2+]i). Montelukast (1–100 nM) reduced LTD4‐induced [Ca2+]i increase, its IC50 being approximately 10 nM. BAY u9773 exhibited significantly low effectiveness. LTD4 (10 nM) induced a redistribution of smooth muscle actin fibres throughout the cytoplasm as visualized by confocal microscopy. In conclusion, Cys Lt1 activation by endogenously produced Cys Lts, can contract rat aortas, while Cys Lt2 only marginally influences aortic tone. Intracellularly, this effect is mediated by an increase in [Ca2+]i. Therefore, Cys Lts, by inducing vascular contraction, can contribute to systemic hypertension.