The guinea-pig lung strip: a study of the mediators of the Schultz-Dale reaction

A new in vitro preparation, the isolated lung strip of the guinea-pig, is described for investigating the Schultz-Dale reaction and the effect of drugs on the smooth muscles of the lung.Histamine caused a contraction of the preparation. This was reversed to a relaxation in the presence of the H1-blocker brompheniramine. The relaxation was blocked by the H2-blocker methiamide, indicating the presence of both H1- and H2-receptors. Brompheniramine, 10^–9–10^–5 M, delayed the onset and diminished the maximum Schultz-Dale contraction, whereas methiamide, 10^–4 M, increased the antigen-induced contraction without influencing the time of onset of the contraction.Diffusate from challenged and chopped guinea-pig lung contracted the lung strip. This was completely blocked by a combination of brompheniramine, 10^–5 M, and the SRS/A blocker FPL 55712, 10 g/ml. However, FPL 55712 did not influence either the onset or the extent of the Schultz-Dale contraction of the lung strip. Indomethacin, 0.05–5 g/ml, did not influence the Schultz-Dale contraction, whereas a high dose, 50 g/ml, decreased the height of the contraction.It is concluded that the isolated lung strip of the guinea-pig is a useful in vitro model for studying the Schultz-Dale reaction. The smooth muscles contain histamine receptors of H1 and H2 type. The anaphylactic contraction, especially during the early phase, is partially mediated by histamine. The lack of evidence of SRS-A as an important mediator depends more probably on the inability of FPL 55712 to block the effect of endogenously generated SRS-A than on the lack of SRS-A receptors on the smooth muscles of the lung.Subsidiary to AB Astra, Sweden.     

Inhibitory effect of econazole on the release of thromboxanes

The effect of econazole on the release of thromboxanes was investigated. It was found that econazole inhibited concentration-dependently the aggregation of guinea pig platelets stimulated with arachidonic acid. The compound also reduced significantly the LTB₄-induced contraction of guinea pig lung parenchyma strips and the contraction of rabbit aorta to the effluent of LTD₄-stimulated guinea pig lungs, both effects mediated mostly by thromboxane generation. The concentration of TXB₂ in the effluents from LTD₄ stimulated lungs, assayed by EIA, was significantly reduced following pretreatment of the lungs with 10^–4 M and 10^–5 M of econazole, whereas the levels of PGE₂ were increased. These results demonstrate that econazole is a selective inhibitor of thromboxane synthesis.     

The effect of Ro 21-7634 on the antigen-induced production of bronchoconstrictive arachidonic acid metabolites in the guinea pig lung

Ro 21-7634 has previously been shown to inhibit histamine and SRS-A release from actively-sensitized guinea pig lung fragments upon antigen challenge. In the studies described herein, it was observed that Ro 21-7634 does not decrease SRS-A release but instead acts to inhibit the synthesis of this mediator. This was confirmed by studying SRS-A synthesisin vitro in rat peritoneal cells after challenge with ionophore A23187. In the peritoneal cell system, Ro 21-7634 exhibited an IC₅₀ of 500 M, in comparison with 5,8,11,14-eicosatetraynoic acid, phenidone and BW755C (IC₅₀'s of 2, 100, and 100 M, respectively). When studied at 10^–4 and 10^–3 M in perfused guinea pig lung, Ro 21-7634 inhibited antigen-induced thromboxane A₂ production by 68 and 96%, respectively. In this system, antigen is believed to induce thromboxane A₂ production through the release of histamine and SRS-A from lung tissue. These mediators then interact at receptor sites in the lung parenchyma to induce thromboxane A₂ synthesis. Ro 21-7634 could thus be inhibiting thromboxane A₂ production by preventing the release of histamine and synthesis of SRS-A in the perfused lung system. Such a mechanism is suggested by the fact that although Ro 21-7634 was effective in inhibiting antigen-induced thromboxane production, it was ineffective in inhibiting thromboxane A₂ production induced in the guinea pig lung system by the direct perfusion of histamine or SRS-A through the lung.     

Inhibition of antigen-induced contractions of ileum segments and lung parenchymal strips of actively sensitized guinea pigs by different anti-allergic agentsin vitro

Different types, of anti-allergic agents have been compared for their ability to inhibit the antigen-induced contraction of isolated lung parenchymal strips and ilea of guinea pigs. Extremely low concentrations of the lipoxygenase inhibitor NDGA inhibit the contraction of the lung parenchymal strips and higher concentrations block the ileal phase II-contraction (both predominantly caused by the developing SRS-A), but do not inhibit ileal phase I-contraction (caused by released histamine). The H₁-antihistamine clemastine predominantly antagonizes phase I of the ileum contraction, and the anti-allergic drug oxatomide reduces all three types of contraction. The degranulation inhibitor DSCG does not influence the contraction of the lung strip, but produces a slight inhibition of both ileal contractions. The PAF-antagonist BN 52021 influences these three contractions in the opposite way to DSCG. As these various types of anti-allergic agents influence these three contractions in a different manner, the models can be used for judging the mechanism of action of a new anti-allergic compound.     

Effects of dithiothreitol on the isolated rabbit mesenteric artery

Rabbit mesenteric artery strips exposed to 10^–3 M dithiothreitol (DTT) were contracted with a series of concentrations of histamine and 2-pyridylethylamine (PEA). DTT exposure increased the sensitivity to histamine 100-fold but increased the sensitivity to PEA only 4-fold. DTT did not reduce dimaprit-induced relaxations, but reduced histamine-induced relaxations. Following a high concentration of histamine (10^–3 M), DTT itself produced a sustained, slowly developing contraction (29±6.8% of the maximal contraction) relaxed by 7×10^–6 M mepyramine but not by 10^–6 M phentolamine. Metiamide (3×10^–3 M) potentiated DTT-induced contractions (29±6.8 before, 57±7.5% after metiamide, as a percent of maximal contraction). Changing the bathing fluid and repeating DTT exposure slowly relaxed previously contracted strips. DTT did not prevent the increase in sensitivity of relaxant histamine receptors on exposure to cold. We conclude that DTT, in addition to potentiating histamine H₁-receptor responses, releases histamine presumably from non-mast cell pools when they are loaded with a high concentration of exogenous histamine.     

Indomethacin inhibitsin vitro immunoglobulin production by human B-lymphocytes

Immunoglobulin production from human B-lymphocytes stimulatedin vitro with pokeweed nitogen (PWM) was measured after 11 days of cell culture. IgM and IgG levels in culture supernatants were quantified using ELISA techniques. Addition of the prostaglandin cyclooxygenase inhibitor indomethacin (10^–7 M) to cell cultures concurrently with PWM, inhibited both IgM and IgG synthesis by 80–90% in the four individuals studied. Reduction of immunoglobulin production was apparently concentration-related over the range studied (10^–6–10^–8 M) and was not associated with cytotoxicity. If this action of indomethacin results from inhibition of cyclooxygenase, then prostaglandins or other arachidonic acid products could be involved in the regulation of human B-lymphocyte immunoglobulin production.     

Studies on the role of arachidonic acid metabolites in airways contraction inducedin vitro by antigen and calcium ionophore A23187

The effects of inhibitors of the cyclooxygenase and lipoxygenase pathways of arachidonic acid (AA) metabolism were studied on the contractions of guinea pig tracheal spirals and lung parenchymal strips induced by antigen and calcium ionophore A23187. Inhibition of the cyclooxygenase pathway with indomethacin results in enhancement of antigen- and A23187-induced contraction of trachea which is a result of diversion of AA into the lipoxygenase pathway and inhibition of modulatory cyclooxygenase products. Indomethacin does not enhance parenchymal contractions but partly inhibits contraction induced by a strong stimulus (5.7 μM A23187 or 100 μg/ml ovalbumin). Parenchymal contraction is inhibited by agents that block the lipoxygenase pathway of AA metabolism, phenidone and nordihydroguaiaretic acid. These agents inhibit the prolonged phase of antigen-induced tracheal contraction but in some cases enhance the early phase of tracheal contraction induced by antigen or A23187. The results suggest that contraction of parenchyma induced by antigen or A23187 are the result of a bronchoconstrictor lipoxygenase product and only have a cyclooxygenase bronchoconstrictor component following a strong stimulus. In contrast, contraction of trachea also appears to be the result of a bronchoconstrictor lipoxygenase product which may not be identical to that contracting parenchyma, and is modulated by cyclooxygenase products. The results emphasize the importance of comparing small and large airways to further our understanding of asthmatic bronchoconstriction.     

Are the anti-allergic actions of theophylline due to antagonism at the adenosine receptor

Adenosine potentiated anaphylactic histamine release from isolated rat mast cells in a dose-dependent manner between 10^–8 and 10^–5 M. Adenosine was found to be present during a normal incubation of mast cells, but the concentration was low (2×10^–8 M). In rat plasma the concentration was 1.5×10^–7 M. The effect of 10^–5 M adenosine was dose-dependently inhibited by theophylline. 50% inhibition was found at 3×10^–5 M theophylline. Cyclic nucleotide phosphodiesterase inhibition required much higher concentrations (IC₅₀10^–3 M). It is suggested that some of the anti-allergic actions of theophylline (clinical concentration range: 10^–5–10^–4 M) does not involve cyclic nucleotides but may be due to inhibition of the effects of endogenous adenosine.     

Ranitidine but not famotidine releases acetylcholine from the guinea pig myenteric plexus

The effects of the histamine H2-receptor antagonists ranitidine and famotidine on acetylcholine release have been studied in the guinea pig myenteric plexus longitudinal muscle preparation incubated with [³H]-choline. Ranitidine (3×10^–5–3×10^–4 M) dose-dependently increased the resting release of acetylcholine and that evoked by electrical stimulation. The effect was present only in strips perfused with 10^–5 M physostigmine. The effect of ranitidinc was inhibited by tetrodotoxin and hexamethonium. Famotidine (10^–5–3×10^–4 M) was totally ineffective in modifying both the resting release and that evoked by field stimulation. Ranitidine did not antagonize the inhibitory effect of oxotremorine, which specifically activates negative feedback mechanisms via presynaptic muscarinic receptors.     

Release of slow-reacting substance of anaphylaxis from dispersed pig lung cells: effect of cyclo-oxygenase and lipoxygenase inhibitors

Study of the release of slow-reacting substance of anaphylaxis (SRS-A).from lung cells has been hampered by the lack of a suitable animal model. Using both immunologic and pharmacologic stimuli, we have obtained histamine and SRS-A release from dispersed pig lung cells containing 6% mast cells (with a histamine content of 1.9 pglcell). Lung cells dispersed from actively sensitized (with intratracheal Ascaris antigen) but not unsensitized pigs released both histamine (mean net release 33%) and SRS-A (mean release, 47 units/10⁷ cells) when challenged with Ascaris antigen. Greater release of histamine (mean net release 52%) and of slow-reacting substance (SRS) (mean release 701 units/10⁷ cells) was induced by challenge with the calcium ionophore A23187. The pharmacologic and physicochemical characteristics of the SRS together with its profile of enzymatic inactivation resembled those described for SRS-A released from human lung. Both antigen-induced and A23187-induced SRS(-A) release were enhanced by indomethacin, a cyclo-oxygenase inhibitor, but inhibited by both phenidone (IC₅₀ 35 μM) and eicosatetraenoic acid (IC₅₀ 15 μM), inhibitors of both cyclo-oxygenase and lipoxygenase, confirming that generation of SRS(-A) by either stimulus required an intact lipoxygenase pathway of arachidonic acid metabolism.     

A histamine-induced decrease of action potential duration in cardiac muscle mediated by H2 receptors

The effects of histamine (10^–7–10^–5 M) on the cardiac action potential have been studied in ventricular strips of guinea pig heart, electrically driven at a constant rate.A decrease of action potential was constantly observed at histamine concentrations above 10^–6 M.No significant variations of the other electrophysiological parameters were induced by the amine.The decrease in the duration of the action potential was blocked by burimamide at a concentration (10^–4 M) which induced only slight changes in action potential outline.It is suggested that the shortening of the repolarization phase induced by histamine is due to the H₂ receptor-mediated activation of the calcium inward current, which can increase the intracellular calcium concentration influencing the potassium permeability.     

Endothelium-dependent relaxing effect of histamine on the isolated guinea-pig main pulmonary artery strips

Contribution of endothelial cells (ECs) to the effects of histamine (HA) was investigated on the isolated guinea-pig main pulmonary artery (GPPA) strips precontracted with noradrenaline (NA). HA caused a dose-dependent relaxation at the concentrations ranged between 10^–8 to 10^–6 M but produced a contraction when a relatively higher concentration (10^–5 M) was used in unrubbed strips. Cimetidine partially inhibited the relaxing effect of HA without altering its constrictive action. In rubbed strips, however, HA produced a dose-dependent contraction. The constrictive effect of HA in rubbed strips enhanced after addition of cimetidine to the incubation medium. HA elicited a concentration-dependent relaxation in both unrubbed and rubbed strips in the presence of mepyramine. Impromidine produced a relaxation in the strips with and without endothelium.These data was taken as an evidence indicating that HA caused a relaxation in the isolated GPPA strips, first causing the release of endothelium derived relaxing factor (EDRF) which is triggered by H₁-receptors and secondly by the direct stimulation of H₂-receptors.     

Capsaicin and anaphylactic reactions in the guinea-pig

The influence of capsaicin on anaphylactic reactions in the guinea-pig was studied bothin vivo andin 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 g/kg, i.p.), which had been injected for three consecutive days.Ileum longitudinal muscle strips were used forin 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.     

Auranofin modulates mast cell histamine and polymorphonuclear leukocyte collagenase release

Auranofin, an orally gold preparation, effective in the treatment of rheumatoid arthritis, was found to be a potent noncytotoxic inhibitor of histamine and collagenase release from mast cells and polymorphonuclear (PMN) leukocytes respectively.Histamine release has been inhibited by auranofin in dose-dependent fashion. Auranofin at concentration of 10^–5 M inhibited 100% of the release, lower concentration 10^–6 M and 10^–7 M produced 80 and 40% decrease.The exposure of PMN-leukocytes to auranofin caused also dose-dependent inhibition of collagenase release. Auranofin at a concentration of 10^–4 M produced a marked reduction (75–100%) of enzyme release from human and rat blood PMN-leukocytes. The modest inhibition 40 and 15–20% at a concentration of 10^–5 M and 10^–6 M respectively was obtained. Auranofin more significantly suppressed collagenase release from leukocytes isolated from inflammatory exudate. Decrease of 100, 80 and 60% were observed upon addition of 10^–4 M, 10^–5 M and 10^–6 M of auranofin.These results suggest that therapeutic action of auranofin may be caused, at least in part, by the inhibition of cellular release of histamine and collagenase in the course of inflammation.     

Role of immunoglobulins G1 and G2 in anaphylactic shock in the guinea pig

Heating serum from actively sensitised guinea pigs did not remove its ability to sensitise recipient animals in vivo and parenchymal lung strips in vitro to anaphylaxis. Thermoresistant antibodies should thus account for the transferable sensitising effect, which persists for at least 9 days. IgG1 and IgG2, contained in the serum, were separated by affinity chromatography to determine the importance and the participation of these subclasses in passive anaphylactic shock. IgG1, present in smaller amounts than IgG2, was more effective in sensitising isolated lung strips. The intravenous administration of ovalbumin to guinea pigs, which had been injected with 0.8 mg/kg of IgG1 or 2 mg/kg of IgG2 9 days beforehand, induced an intense bronchoconstriction with leucopenia and moderate thrombopenia, suggesting an as yet undescribed role for IgG2 in passive tissue sensitisation. The use of mepyramine, an antagonist of the histamine H1 receptor, WEB 2086, an antagonist of platelet-activating factor, and nordihydroguaiaretic acid, a dual inhibitor of cyclooxygenase and lipooxygenase, alone or associated, demonstrated that the anaphylactic contraction of lung strips from guinea pigs sensitised by IgG1 is mediated by histamine and arachidonate derivatives, whereas that of lung strips from guinea pigs sensitised with IgG2 is mostly mediated by histamine. In addition, the association of the three potential antagonists slightly reduced the anaphylactic contraction of lung strips provided by guinea pigs sensitised by serum. Our results, using a sensitisation procedure considered until now to involve exclusively IgE antibodies, indicate that IgG1 and IgG2 are in fact the essential antibodies for passive anaphylactic shock in the guinea pig.     

Interactions of morphine with PGE1, isoproterenol,dopamine and aminophylline in rat mast cells; their effect on IgE-mediated14C-serotonin release

The formaldehyde method was used to examine the interactions of morphine with PGE₁, isoproterenol, dopamine and aminophylline in rat mast cells by their effects on IgE-mediated¹⁴C-serotonin release. PGE₁ (2×10^–8–2×10^–5 M), isoproterenol (10^–10–10^–8 M), dopamine (4×10^–8–4×10^–6 M) and aminophylline (6×10^–6–6×10^–4 M) caused dose-related inhibition of the mediator release 1 min after an antigen challenge, and propranolol (10^–7 M) blocked the inhibition by isoproterenol (10^–8 M) but not that by dopamine (4×10^–6 M), while haloperidol (4×10^–6 M) blocked that by dopamine (4×10^–6 M) but not that by isoproterenol (10^–8 M). Morphine (3×10^–7–3×10^–5 M) reversed the inhibitory effects of PGE₁ (2×10^–6 M), isoproterenol (10^–8 M) and dopamine (4×10^–6 M) dose-dependently and stereospecifically; naloxone (2×10^–4 M) antagonized these reversing actions of morphine (3×10^–5 M). Morphine (10^–6–10^–4 M) did not reverse the inhibitory action of aminophylline (6×10^–4 M). These results suggest that the inhibitory responses of mast cells to PGE₁, isoproterenol and dopamine but not to aminophylline in immunological mediator release were reversed by morphine through opioid receptors, and that the inhibition of adenylate cyclase in mast cells is one of the biochemical actions of morphine.     

Histamine release from serosal mast cells by intermediate products of arachidonic acid metabolism

In the present paper we report the results of experiments carried out to measure the release of histamine from isolated rat mast cells during the metabolic activation of arachidonic acid. Arachidonic acid (10^–8–10^–4 M) and the terminal products (10^–6 M) of the arachidonic acid pathways were devoid of any significant histamine releasing properties. A substantial amount of histamine was released from rat mast cells by low concentrations of arachidonic acid during incubation with prostanoid generating systems, such as guinea-pig lung microsomes, rat serosal macrophages and polymorphonuclear cells and prostaglandin-H-synthase from calf seminal vesicles. The release of histamine was not accompanied by a leakage of lactate dehydrogenase and was blocked byd-mannitol and by lipoxygenase and cyclo-oxygenase pathway inhibitors. The data are consistent with the hypothesis that free radical derivatives of arachidonic acid, originating from hydroperoxy fatty acids, are generated during catalysis, causing mast cell histamine release.     

Modulation of the release of histamine and arachidonic acid metabolites from human basophils and mast cells by auranofin

In these experiments the effects of pharmacological concentrations of auranofin, a new absorbable gold compound, were assessed on the release of histamine and peptide leukotriene C₄ (LTC₄) from human basophils and lung mast cells. Auranofin, at pharmacological concentrations, inhibitedin vitro histamine and LTC₄ release from human basophils induced by anti-IgE. Inhibition began at about 3×10^–7 M and was maximum at 10^–5 M. We also evaluated the effect of auranofin on the release of histamine and LTC₄ induced by anti-IgE from mast cells purified from human lung. Auranofin (3×10^–7 to 10^–5 M) dose-dependently inhibited the release of histamine and LTC₄ from human lung mast cells. Thus pharmacological concentrations of auranofin cause dose-related inhibition of histamine release andde novo synthesis of LTC₄ by human basophils and lung mast cells.Supported in part by grants from the C.N.R. (83.00430.04, 84.01756.04) and (85.00491.04) and M.P.I. (Rome, Italy).     

Prostaglandins and neurotransmission at the guinea pig and rabbit urinary bladder

High concentrations of prostaglandins (PGE₁, PGE₂, or PGE₂) (2×10^–6 M) produced a slow contraction of longitudinal strips of detrusor muscle taken from the bladders of guinea pigs and rabbits. At a lower concentration (10^–6 M) prostaglandins enhanced contractions produced by field stimulation of nerves in guinea pig but not rabbit strips. The contractions were not affected by indomethacin. Contractions of guinea pig strips in response to acetylcholine at 10^–4 M were enhanced by prostaglandins and unaffected by indomethacin. Membrane potentials of smooth muscle cells recorded with micro electrodes, were unchanged up to 10^–6 M PGE₂. Above this the cells were depolarized with an increase in frequency of spontaneous action potentials. Synchronous recording of electrical and mechanical activity with the double sucrose gap indicated a decrease in amplitude of the evoked excitatory junction potential and action potential even when the contraction was enhanced in the presence of PGE₂. Responses to repeated stimulation at 10 Hz for 1 min were progressively depressed. This trend was slightly reduced by PGE₂ but unaffected by indomethacin. It is concluded that prostaglandins are not normally released by the nerves to the urinary bladder but are able to facilitate contraction in the guinea pig. This effect is probably on the excitatory-contraction coupling, possibly by mobilizing Ca²⁺. Some modification of transmitter release by the nerves may also occur.     

The effect of substance P and related peptides on the guinea-pig lung strip

Substance P is present in sensory nerves in the lung and we report here its actions on the lung strip. Substance P was shown to produce rapid, small contractions of the lung strip at doses from 10^–9 to 10^–5 M, and there was no apparent dose-response relationship. In the presence of a mixture of three inhibitors of substance P breakdown: bacitracin, 1,4-dithio-l-threitol and ethylenediaminetetraacetic acid all at 10^–4 M, the responses to substance P were greatly increased and dose-response curves could be established. The concentrations producing half maximal effect in the presence of these inhibitors were 6×10^–6 M ± 4×10^–6 M. The inhibitors of substance P breakdown were found to have no effect on the histamine dose-response curve: any small shifts obtained were not significant.Chlorpheniramine 10^–6 M and 10^–5 M shifted the histamine dose-response curve to the right producing dose ratios of 100 and 900 respectively. At these doses chlorpheniramine had no effect on the dose-response curve to substance P. A dose of substance P not itself producing a response (5×10^–8 M) caused an increase in the size of the responses to histamine. The histamine dose-response curve was shifted to the left in the presence of substance P producing an average dose ratio of 1.4±0.2. In the presence of the peptidase inhibitors, dose-response curves have also been produced for physalaemin (10^–7–10^–5 M) and eledoisin (10^–7–10^–5 M).