Bernardo Boris Vargaftig

Mianserin hydrochloride: Peripheral and central effects in relation to antagonism against 5-hydroxytryptamine and tryptamine

Abstract Mianserin hydrochloride, a new anti-5-hydroxytryptamine agent, was compared with cyproheptadine with respect to a variety of responses evoked by 5-hydroxytryptamine and other agonists. Central effects against tryptamine responses and general depressant effect on CNS were also studied. The two antagonists induced comparable inhibition against responses in which D-receptors of 5-HT were involved. Mianserin displayed α-adrenolytic activity and did not antagonize muscarinic effects of acetylcholine; the opposite was true for cyproheptadine. Mianserin was more effective than cyproheptadine in counteracting tryptamine-induced responses in rabbits and rats. Cyproheptadine produced diffuse depression of the CNS, with marked elevation of the threshold to electric stimulation of the brain stem reticular system in rabbits, persistent block of the acoustic arousal in rats and rabbits and prolongation of barbiturate sleep in rats. Mianserin however did not produce CNS depression, other than transient inhibition of the sensory activation of the EEG in rabbits. The earlier postulated structure-activity relationship between mianserin and cyproheptadine does not appear to apply to effects other than certain 5-HT responses and central tryptamine effects.

The inhibition of cyclo-oxygenase of rabbit platelets by aspirin is prevented by salicylic acid and by phenanthrolines.

Salicylic acid, 1,10- and 1,7-phenanthroline prevented inhibition by aspirin of platelet aggregation and of generation of thromboxane A2 due to arachidonic acid, to the ionophore A21387, to thrombin and to collagen. Dithiothreitol, another drug which prevents aggregation and formation of thromboxane A2, but only reversibly, failed to interfere with the inhibition by aspirin. Irreversible inhibition by indomethacin and by the substrate analogue 5,8,11,14-tetraynoic acid was also unaffected by salicylic acid or by 1,10-phenanthroline, which thus probably exert a specific interaction with the aspirin-binding site. Inactivation of platelet cyclo-oxygenase with arachidonic acid led to inhibition of the formation of thromboxane A2 and of aggregation due to arachidonic acid itself and to collagen, but barely affected aggregation by thrombin, even though generation of thromboxane A2 was blocked. Use of salicylic acid and of reversible inhibitors of cyclo-oxygenase may help to unravel the mechanism of inhibition due to other agents.

ROLE OF PLATELETS IN ASPIRIN‐SENSITIVE BRONCHOCONSTRICTION IN THE GUINEA‐PIG; INTERACTIONS WITH SALICYLIC ACID

1 The bronchoconstriction caused in the guinea‐pig by arachidonic acid (AA), bradykinin, adenosine diphosphate (ADP) and adenosine triphosphate (ATP) was correlated with effects on platelets. ATP and ADP produced a brief thrombocytopenia and AA a more prolonged one. Bradykinin had no effect on platelets. 2 Aspirin inhibited bronchoconstriction and thrombocytopenia produced by AA and part of the bronchoconstriction produced by ATP, but had no effect against ADP. Thrombocytopenia produced by ADP and ATP was not affected by aspirin or indomethacin. 3 Platelet depletion by antiserum prevented bronchoconstriction in response to ADP and to ATP, but not in response to bradykinin or to AA, showing that platelets are not involved in aspirin‐sensitive bronchoconstriction. Infusions of ADP reduced bronchoconstriction and thrombocytopenia in response to ADP itself and to ATP, but not to AA. Bronchoconstriction by ADP or ATP involves an action on platelets. Only that due to ATP is partially dependent on the activity of prostaglandin synthetase. 4 ATP induced aggregation in vitro in guinea‐pig platelet‐rich plasma (PRP). Rabbit PRP responded only when ATP was first incubated with guinea‐pig plasma. The aggregating compound formed was probably ADP, since it was destroyed by apyrase. Its formation was not inhibited by aspirin or indomethacin, indicating that aspirin inhibits ATP‐induced bronchoconstriction by a different mechanism. 5 The aggregating effect of ATP on guinea‐pig platelets was inhibited by concentrations of apyrase that block ADP‐induced aggregation, and potentiated by lower concentrations of apyrase. 6 Adenosine 5′‐tetraphosphate did not aggregate platelets in vivo or in vitro. In vitro aggregation occurred when apyrase was added, suggesting transformation into ADP. Adenosine 5′‐tetraphosphate and apyrase inhibited aggregation due to ADP, but failed to affect that due to AA. This suggests that aggregation involving products of prostaglandin synthesis does not require ADP. 7 Salicylic acid did not interfere with bronchoconstriction or aggregation due to AA, but prevented inhibition by aspirin when the weight ratio, salicylic acid:aspirin was 4:1. Salicylic acid may be useful in studies of potential inhibitors of thromboxane A2 synthesis and of thromboxane A2‐depen‐dent processes in vivo and in vitro.

Dog platelets fail to aggregate when they form aggregating substances upon stimulation with arachidonic acid

Dog platelets are refractory to aggregation by arachidonic acid (AA) but generate an unstable activity that aggregates rabbit platelets. Formation of this activity is inhibited by indomethacin, by the peroxide scavenging enzyme catalase, by two chelating agents that bind Cu+ and Cu2+ ions, by the -SH agent dithiothreitol and is stimulated by cysteine. Agitation of dog platelets is followed by spontaneous aggregation and uncovers aggregation by AA, which is blocked by indomethacin. Neither indomethacin nor apyrase prevent spontaneous aggregation, ruling out both activation of prostaglandin synthetase and leakage of ADP as possible explanations. Complexation of plasma Ca2+ by citrate as an explanation for refractoriness to AA was ruled out by replacing citrate with heparin. Dog platelets are also refractory to PGH2 formed from AA by the cyclo oxygenase component of prostaglandin synthetase. Aggregation of rabbit platelets by PGH2 is not inhibited by indomethacin, by catalase, by dithiothreitol or by metal chelating agents and is not potentiated by cysteine. This confirms that the reagents act before PGH2 is formed. Aggregating activity generated by dog platelets is probably due to an unstable lipoperoxide whose generation involves mechanisms similar to those responsible for aggregation of rabbit platelets, since similar antagonists block both processes.

Lymphatic thoracic duct ligation modulates the serum levels of IL-1beta and IL-10 after intestinal ischemia/reperfusion in rats with the involvement of tumor necrosis factor alpha and nitric oxide.

Intestinal ischemia/reperfusion (I/R) causes local and remote injuries that are multifactorial and essentially inflammatory in nature. To study the putative influences of nitric oxide (NO) and tumor necrosis factor &agr; (TNF-&agr;) on the release of interleukin (IL) 1&bgr; and IL-10 and the involvement of lymphatic system on a systemic inflammation caused by I/R, we have quantified the serum and lymph levels of IL-1&bgr; and IL-10 in rats during I/R after treatment with inhibitors of NO synthase (N&ohgr;-nitro-L-arginine methyl ester hydrochloride [L-NAME]) or TNF-&agr; (pentoxifylline [PTX]). Intestinal I/R was performed by means of a 45-min occlusion of the mesenteric artery, followed by 2-h reperfusion; groups of rats subjected to I/R had the thoracic lymph duct ligated immediately before the procedure. The I/R caused a significant increase of the serum levels of IL-1&bgr; and IL-10 in rats with intact thoracic lymph duct, whereas the thoracic duct ligation blunted the serum release of IL-1&bgr; and elevated that of IL-10. The levels of the cytokines collected in the lymph after I/R increased, and even more increase was observed in L-NAME-treated rats. L-NAME significantly increased the lymph levels of IL-1&bgr; and IL-10; in serum, however, only IL-1&bgr; increased in rats with either intact or ligated thoracic lymph duct. The treatment with PTX reduced the serum levels of IL-1&bgr; irrespective of the lymph circulation interruption but was effective to increase the IL-10 levels in intact rats during I/R. The lymphatic levels of IL-1&bgr; of rats subjected to I/R were reduced and those of IL-10 were increased after treatment with PTX. In conclusion, during I/R, the serum levels of IL-1&bgr; seem modulated by stimulant mechanisms that could be associated with TNF-&agr; and inhibited by NO and by the integrity of the thoracic lymphatic flow. On the other hand, IL-10 seems controlled by TNF-&agr;-related, largely NO-independent mechanisms. Thus, it is reasonable to suppose that an endogenous mechanism that can limit the systemic inflammatory response ensuing an I/R splanchnic trauma exists.

Inhibition by sulfhydryl agents of arachidonic acid-induced platelet aggregation and release of potential inflammatory substances

Sulfhydryl agents (mercaptoethanol, thioglycerol, dithiotreitol, and sodium diethyldithiocarbamate) prevented aggregation of rabbit platelets and the accompanying generation of pharmacologically active substances due to arachidonic acid. Inhibition was also found after in vivo administration of the antagonists. This antagonism was suppressed if the inhibitors were removed from the platelet suspension by washing procedures, whereas inhibition by indomethacin was irreversible. Amino-thiol reagents either failed to antagonize the effects of AA or potentiated them. CuCl2 increased the amounts of pharmacologically active substances generated in incubates of intact platelets with arachidonic acid, and reversed the inhibition due to thiol agents, but did not interfere with inhibition by indomethacin. Platelets suspended in Tyrode solution generated unstable pharmacologically active substances upon incubation with arachidonic acid ; stability of these substances could be maintained at 4°C. Generation of this temperature — sensitive material was inhibited by indomethacin and by thiol agents. Interference with a copper-containing component of PG synthetase or reduction of an intermediate lipoperoxide appear as two possible mechanisms of action of thiol agents.

Blockade by metal complexing agents and by catalase of the effects of arachidonic acid on platelets: Relevance to the study of anti-inflammatory mechanisms

Metal-chelating agents inhibited platelet aggregation and the accompanying generation of rabbit aorta contracting and PG-like activities, when platelets were challenged with arachidonic acid. Inhibition required the presence of the chelating agents in the medium, and was insured by reagents avid for free or protein-bound copper. Catalase also prevented aggregation and generation of pharmacologically active substances; its activity was reversed by aminothiol agents and by Cu2+ and Zn2+, shown previously to potentiate the platelet effects of arachidonic acid. Inhibition by indomethacin was not prevented by amino-thiol drugs nor by Cu2+ or Zn2+. The catalase-induced inhibition was not affected by scavenging of thiol groups; this rules out, as a mechanism of action of catalase, the increased destruction of popoperoxides by glutathione peroxidase, which requires reduced glutathione as hydrogen donor. The results are compatible with the hypothesis that the agent that mediates platelet aggregation by arachidonic acid is a popoperoxide, requiring the presence either of H2O2 or of a similarly catalase-sensitive substance to be generated.

Acute hypotension due to carrageenan, arachidonic acid and slow reacting substance C in the rabbit: Role of platelets and nature of pharmacological antagonism

Abstract Carrageenan injected i.v. to rabbits induced thrombocytopenia, hypotension and death. The latter two phenomena, but not the former, were prevented by aspirin and by indomethacin. Immune platelet depletion protected against the effects of carrageenan, but failed to interfere with hypotension by arachidonic acid (AA) and by slow reacting substance C. Inhibition by aspirin of hypotension due to AA was short lasting (

INHIBITION BY NON‐STEROID ANTI‐INFLAMMATORY AGENTS OF RABBIT AORTA CONTRACTING ACTIVITY GENERATED IN BLOOD BY SLOW REACTING SUBSTANCE C

1 A crude and a partially purified preparation of slow reacting substance C (SRS‐C) as well as arachidonic acid decreased resistance to perfusion of the dog hind paw. This effect was suppressed by treatment with non‐steroid anti‐inflammatory drugs. 2 Injections of SRS‐C or of arachidonic acid induced marked and reproducible contractions of strips of rabbit aorta and a rat stomach which were bathed in blood from an anaesthetized dog. The effect on the rabbit aorta is attributed to formation of a rabbit aorta contracting substance (RCS). The contractions were suppressed when the dog was treated with a non‐steroid anti‐inflammatory drug. 3 Incubation of blood or of platelet‐rich plasma with SRS‐C or arachidonic acid resulted in the formation of similar materials. This formation was suppressed by anti‐inflammatory drugs. 4 SRS‐C, linoleic, linolenic, and arachidonic acids are suitable substrates for soybean lipoxidase for the generation of RCS. 5 It is suggested that RCS and prostaglandin are formed within platelets, when SRS‐C or arachidonic acid are injected into animals or added in vitro. Non‐steroid anti‐inflammatory drugs suppress these effects, possibly by inhibiting prostaglandin synthetase.

Synthesis of thromboxane A2 by non-aggregating dog platelets challenged with arachidonic acid or with prostaglandin H2.

Dog platelets challenged with arachidonic acid fail to aggregate but synthesize a substance which aggregates rabbit and human platelets, this aggregation being suppressed by dibutyryl cyclic AMP. The aggregating substance contracts strips of rabbit aorta and of coeliac and mesenteric arteries, is soluble in diethyl ether, has a half-life of about 40 seconds at 37 degrees C and of 100 seconds at 22 degrees C. Its generation is blocked by various inhibitors of prostaglandin biosynthesis. The thromboxane A2 synthetase inhibitor imidazole and its analogue benzimidazolamine also suppress generation of vessel contracting activity in incubates of dog platelets and prostaglandin H2. Since dog platelets also transform prostaglandin H2 into thromboxane A2 their failure to aggregate, when stimulated by arachidonic acid or by prostaglandin H2, is not due to lack of thromboxane synthesizing ability.