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Although thrombocytopenia associated with the use of histamine H2 receptor (H2R) antagonists has been described, a drug-dependent, platelet-reactive antibody has not previously been identified in such cases. We studied serum from a patient who developed acute, severe thrombocytopenia after exposure to the H2 receptor antagonist, ranitidine, and identified an antibody that reacted with normal platelets in the presence of this drug at pharmacologic concentrations. In flow cytometric and immunoprecipitation studies, the antibody was shown to be specific for the glycoprotein Ib/IX complex (GPIb/IX). From the pattern of monoclonal antibody (MoAb) inhibition and the reactions of antibody with Chinese hamster ovary (CHO) cells transfected with GPIX and GPIbβ, we found that the patient's antibody is specific for an epitope on GPIX close to, or identical with a site recognized by the MoAb SZ1 that is a common target for antibodies induced by quinine and quinidine, drugs structurally unrelated to ranitidine. These findings provide evidence that immune thrombocytopenia can be caused by sensitivity to an H2 R antagonist and suggest that the SZ1 binding site on GPIX may be a common target for drug-induced antibodies. Further studies of the epitope for which SZ1 is specific may provide clues to the mechanism(s) by which drugs promote tight binding of antibody to a membrane glycoprotein and cause platelet destruction in patients with drug sensitivity.

1. In sensitized guinea-pigs, the effects of gamma-aminobutyric acid (GABA) and GABAmimetic drugs have been investigated on tracheal segments contracted by cumulative application of an allergen (ovoalbumin, OA) and on serosal mast cells. The same drugs have also been tested on activation of alveolar macrophages isolated from unsensitized guinea-pigs. 2. Superfusion with GABA (1-1000 microM) reduced the contraction intensity of tracheal strips. The effect of GABA (100 microM) was not affected by the carrier blockers, nipecotic acid and beta-alanine (300 microM each). It was mimicked by the GABAB agonist (-)-baclofen (100 microM) but not 3-aminopropanephosphinic acid (100 microM, 3-APA). The GABAA agonist, isoguvacine (100 microM) did not exert any effect. GABA (10 microM)-induced inhibition of tracheal contractions was reduced by the GABAB antagonist, 2-hydroxysaclofen (100 microM, 2-HS), but not by the GABAA antagonist, bicuculline (30 microM). 3. The reduction in contraction intensity induced by GABA (100 microM) was prevented by a 40 min preincubation of tracheal strips with capsaicin (10 microM), but not tetrodotoxin (TTX, 0.3 microM). The effect of GABA (1000 microM) was absent after preincubation with indomethacin (2.8 microM) but unmodified when nordihydroguaiaretic acid (NDGA, 3.3 microM) was used. Finally, removal of the epithelium prevented the GABA effect. 4. Anaphylactic histamine release from serosal mast cells isolated from sensitized animals was not affected either by GABA (10-1000 microM) or the selective receptor agonists (-)-baclofen (0.1-1000 microM) and isoguvacine (10-1000 microM). The release of platelet-activating factor (PAF) from alveolar macrophages stimulated by formyl-Met-Leu-Phe (FMLP; 1 microM) was modified neither by GABA (100 microM)nor by (-)-baclofen (100microM).5. In conclusion, these data show that GABA can inhibit allergic phenomena in the guinea-pig airways through activation of GABAB receptors. An involvement of neuropeptidergic sensory structures is suggested but a role for epithelial cells and arachidonate metabolites is not definitely proved.

Compounds derived from glucocorticoids, 21-aminosteroids, were reported to inhibit in vitro lipid peroxidation in CNS tissue. In order to evaluate the possible scavenging and/or iron chelating activities in vivo of the 21-aminosteroid U74500A (pregna-1,4,9(11)-triene-3,20-dione, 21-(4-(5,6-bisdiethylamino)-2-pyridinyl)-1-piperazinyl)-16-methyl-, HCl (16α)), the drug was administered for seven days to rats. These rats had been induced by iron-saccharate complex injection a slow process of lipid peroxidation into their right brain hemicortex. The drug was injected also to intact rats (normal rats). Seven days after the operation the extent of iron-induced lipid peroxidation in both the hemicortices and the effect of the drug, were assessed by the evaluation of lipid-soluble fluorescence and of conjugated diene formation. The assessment was performed both in vehicle (control) and in U74500A-treated rats. In the iron-injected rat groups the drug induced a significant dose-related reduction of fluorescence values. Formation of conjugated dienes showed a significant decrease when U74500A (48 mg/kg every 48 hr) was administered to cortico-cerebrally iron-injected animals. The lipid peroxidation of cortices in normal rats was evaluated as thiobarbituric acid reactant substances in both the drug-treated and the control animals. In normal rats, U74500A (48 mg/kg every 48 hr) caused a significant decrease of TEARS values, as compared to those observed in the control group. The iron content in the iron-injected hemicortices, which was evaluated by the ferrozine method, was not modified by drug treatment. U74500A appears to have in vivo antioxidant properties and not to affect the iron content in the neural tissue. An interaction of this drug with the metal, however, cannot be excluded.

In view of the emerging role of metals and particularly iron in the pathogenesis of several ischemic or degenerative CNS diseases, via a lipid peroxidative process, a model of slow iron-induced peroxidative damage in the rat brain cortex has been carried out. Iron-carbohydrate complexes were injected in the right brain cortex, and biochemical assays were performed on ipsilateral and contralateral samples two hours or seven days after injection. Iron-saccharate caused a significant increase in the ipsilateral cortex in TBARS, conjugated dienes and fluorescent substances seven days after injection, whereas no biochemical alteration was observed two hours after treatment. In order to prevent or to limit lipid peroxidation, some drugs known for chelating and/or scavenging activity were administered to iron-injected rats. DL-alpha-tocopherol, methylprednisolone, D-penicillamine significantly decreased the value of fluorescent products formed by iron-saccharate, whereas desferrioxamine was not effective.

Further evidence is reported on the influence exerted by capsaicin on the anaphylactic reaction evoked in actively sensitized guinea-pigs. In Herxheimer microshock induced by ovalbumin aerosol, pretreatment of animals with 100 micrograms/kg i.p. capsaicin prolonged the preconvulsion time when the drug was administered 3 h before antigen challenge. In contrast, the same dose of capsaicin injected 30 min before aerosol caused a shortening of latency of the respiratory symptomatology. The influence of the drug is no longer evident after 24 h. In "in vitro" experiments desensitization to capsaicin of tracheal preparations caused a reduction of histamine and SRS-A released during antigen challenge, in comparison to controls. Moreover, anaphylactic histamine release was increased in preparations perfused with 10(-8) M substance P. In conclusion, our findings confirm that neuropeptides may be involved in the pathogenesis of asthma by affecting release of mediators.

Publisher Summary When hemoglobin (Hb) diffuses into the extracellular space of living tissues, which may occur during hemolysis or when erythrocytes escape from vessels, its catalytic potential toward several endogenous or exogenous compounds can be activated. There has been much attention focused on the capacity of Hb to metabolize polyunsaturated fatty acids (PUFAs), giving rise to the formation of products that may be important in the pathogenesis of systemic (intravascular hemolysis) or local (micro- or macrohemorrhages, inflammation, hemorrhagic infarcts) conditions in which erythrocyte lysis occurs. At low concentrations, Hb catalyzes a quasi-lipoxygenase reaction on linoleic acid with remarkably high substrate specificity. The fatty acid cyclooxygenases, which may be regarded as a special type of lipoxygenase that initiate the biosynthesis of prostaglandins and thromboxanes, require heme for their activity. Hb is a good candidate for exerting cyclooxygenase activity owing to its peroxidative properties already described in the chapter. There is also some discussion about the cyclooxygenase activity of Hb, which has been made possible by the use of either physicochemical or biological analytical method.

D-Penicillamine, a trifunctional amino acid known for its ability to form metal complexes and for being a radical scavenger, has been investigated "in vitro" and "in vivo" in the rat brain cortex. At 50 microM the drug facilitated lipid hydroperoxides and TBARS formation in brain cortex homogenates, while at higher concentrations a clear inhibition of the lipid peroxidative process was observed. The activity of the D-penicillamine (25 and 50 mg/Kg i.p.) was evaluated "in vivo" after a 7-day treatment in rats in whose brain cortex a slow process of lipid peroxidation was induced by iron-saccharate injection. Lipid hydroperoxides, lipid soluble fluorescent compounds and the iron content of both iron-injected and contralateral hemicortices showed a significant decrease in comparison to rats untreated with D-penicillamine. The higher dose also induced in normal rats a significant decrease in basal TBARS and iron content of the brain cortex. In the iron-injected cortex the observed Fe2+/Fe3+ ratio was significantly different from that of normal rats. On the contrary ratios obtained form D-penicillamine treated animals were higher in comparison to both normal and iron-injected animals. These results suggest that D-penicillamine, acting as a reducing agent, inhibits the iron redox system and, as a chelating agent, can remove metal from action sites where lipid peroxidation may occur.

The spontaneous motility of longitudinal muscle of human jejunum was recorded and the effect of γ-aminobutyric acid-ergic (GABAergic) drugs was tested. GABA and (−)-baclofen (10 −6 −10 −4 M) dose dependently reduced the amplitude and frequency of the spontaneous contractions; muscimol and 3-aminopropanesulfonic acid (3 × 10 −5 M) were ineffective. The effect of 3 × 10 −5 M GABA was reduced by 3 × 10 −3 M 5-aminovaleric acid but not by 3 × 10 −5 M picrotoxin. The dose-response curve for GABA was shifted to the right by 3 × 10 −3 M 3-aminopropanesulfonic acid. Tetrodotoxin 3 × 10 −7 M prevented the GABAergic action, whereas various receptor antagonists tested did not affect it. GABAergic drugs did not influence the spontaneous motility of either circular or longitudinal muscles of human colon. It is suggested that GABA B receptor activation induces the inhibition of human jejunum longitudinal muscle motility by a neurogenic mechanism. The possible involvement of postganglionic cholinergic neurons is to be evaluated by other techniques.

It is well known than the anaphylactic reaction can be affected by various neurotransmitters. Furthermore, exocytie processes following challenge with antigen can be modulated due to the presence of different neurotransmitter receptors located on the membranes of involved sensitized cells [23, 22]. GABA, through GABAA and GABAB receptors, appears to have a peculiar role as a neuromodulator in the neuro-vegetative system, since it can inhibit or facilitate the release of classical neurotransmitters such as acetylcholine (ACh), norepinephrine (NE) or peptides (for comprehensive and recent reviews see [5, 7]). Thus, the possibility that GABA could interfere with anaphylaxis arose, and this has been the object of our study, performed in in vivo and in vitro models. We have shown that GABAergic drugs can exert a protective effect against allergic bronchospasm in actively sensitized guinea-pigs [20] and in the Schultz-Dale reaction in isolated tissues [33].

Salmeterol (1 Nm-100μm) showed an inhibitory action on anaphylactic histamine release from mast cells, isolated from pleural and peritoneal cavities of actively sensitized guinea-pigs and stimulated by incubation with allergen. The effect is concentration-dependent and is reduced by the β-adrenoceptor antagonist propranolol (1 μm). This study supports the hypothesis of an antiinflammatory property of salmeterol, which concerns cells involved in the early phases of asthma.

Summary o 1) Responsiveness of guinea-pig ileal longitudinal smooth muscie-myenteric plexus preparations to drugs activating GABA-B receptors was studied in morphine-tolerant animals. For this purpose morphine pellets (75 mg each) were implanted subcutaneously in guinea-pigs and experiments were performed three days later in electrically-stimulated ileal strips. 2) Activation of GABA-B receptors with GABA (10 −6 −10 −3 M) or (-)-baclofen (10 −6 −10 −3 M) caused a dose-related inhibition of twitch response that was about 80% lower in preparations from morphine-tolerant animals than in controls. This was found both in preparations maintained in the presence of morphine (10 −6 M) and in morphine-free Krebs. The effect was evident also in ileal preparations from morphine-tolerant animals in which a withdrawal syndrome was induced by the administration of naloxone before sacrifice. 3) The phenomenon was specific since the dose-response curve of the adenosine-inhibitory effect was comparable in preparations from tolerant animals and controls. 4) The hyporesponsiveness to GABA-B receptor activation began 12 h after pellet implantation and was maximal on the third day. 5) It is concluded that during tolerance to and withdrawal from morphine there is a hyporesponsiveness of GABA-B receptors in “in vitro” guinea-pig ileal longitudinal muscle-myenteric plexus preparations.

The influence of capsaicin on anaphylactic reactions in the guinea-pig was studied both in vivo and in vitro. In guinea-pigs actively sensitized with ovalbumin, Herxheimer microshock was elicited by antigen aerosol and the preconvulsion time recorded. The preconvulsion time was reduced by about 30% in animals pretreated with capsaicin (1 mg/kg) injected i.p. 30 min before antigen aerosol, whereas it remained unchanged when the drug was administered two days before aerosol treatment. Capsaicin shows a partial protective effect when the provocative aerosol was administered 3 h after the last of three doses of capsaicin (100 micrograms/kg, i.p.), which had been injected for three consecutive days. Ileum longitudinal muscle strips were used for in vitro anaphylaxis studies. These were isolated from guinea-pigs actively sensitized with ovalbumin and histamine release evoked by antigen was measured. Preparations perfused with capsaicin (10(-6)-10(-4) M) and desensitized to the drug, showed a lower anaphylactic release of histamine. This effect was dose-dependent, with the histamine release reduced by 35% at higher concentrations (10(-5)-10(-4) M) of capsaicin. The mechanism of the influence of capsaicin on anaphylactic reactions is discussed briefly.