Effects of C5a on guinea pig lung: histamine release and mechanism of contraction.

C5a, a product of complement system activation, causes a significant contraction of the isolated guinea pig trachea, and the antihistamine diphenhydramine does not alter the rate, amplitude, or duration of the contraction (Regal et al., 1980). The present study demonstrates that over the range of C5a concentrations investigated, the C5a-induced contractile response of the trachea maximized, whereas the contraction of lung parenchymal strips and the release of histamine from chopped lung did not. In addition, the antihistamines, diphenhydramine and pyrilamine, caused a significant delay in the onset of the C5a-induced contraction of lung parenchymal strips. When parenchymal strips from nonperfused lung were used, the maximum of the contraction was also reduced by pyrilamine. Aspirin and indomethacin effectively inhibited the C5a-induced contraction of lung parenchymal strips, and inhibition by a combination of aspirin and pyrilamine was the sum of the inhibition by aspirin and the inhibition by pyrilamine. The SRS-A antagonist, FPL 55712, caused a more rapid relaxation of the C5a-induced response over control. These studies suggest that products of the cyclo-oxygenase pathway are mediators involved in the C5a-induced contraction of lung parenchyma, with histamine and leukotrienes contributing as well.

Ultrastructural and histochemical observations of respiratory epithelium and gland.

Secretory products of epithelial cells of the human respiratory tract have been studied biochemically and by a variety of histochemical methods for differentiating and characterizing complex carbohydrates at the light and electron microscopic level. By light microscopy a majority of mucous secretory cells of the surface epithelium secret glycoprotein with terminal sialic acid, penultimate galactose residues, and variable sulfate esters. Ultrastructurally the mucous cells of the surface epithelium vary within and between regions of the respiratory tree and comprise a heterogeneous population of cells differing in the fine structure and cytochemistry of their secretory granules. Serous tubules and demilunes in glands of the lamina propria produce a secretion that contains less carbohydrate than that in the nearby mucous cells, resembles the latter in content of sulfate esters, and differs in having little or no sialic acid and no terminal or penultimate galactose. Mucous tubules produce a carbohydrate-rich secretion containing glycoconjugate with terminal sialic acids, penultimate galactose residues, and a variable degree of sulfation like the glycoconjugate of surface mucous cells. Heterogeneity of cells can be demonstrated in the serous and mucous tubules by ultrastructural, morphologic, and cytochemical methods.

C5a-induced histamine release. Species specificity.

C5a has long been known to cause histamine release from isolated perfused guinea pig lung, isolated human basophils, rat and guinea pig mast cells, and guinea pig skin. The purpose of the present study was to compare the ability of guinea pig C5a to cause histamine release from lung fragments of dog, rabbit and rat with that released from guinea pig lung fragments. In addition, C5a-induced histamine release from the skin of the guinea pig, rabbit, and rat was investigated by examining antihistamine inhibition of C5a-induced vascular permeability changes. Guinea pig C5a caused a concentration-related release of histamine from guinea pig lung fragments with only minimal amounts of histamine being released from dog, rat, and rabbit lung fragments over the same concentration range. Guinea pig C5a also caused increased vascular permeability in guinea pig skin which was markedly inhibited by H1 antagonists. In rabbit skin, guinea pig C5a increased vascular permeability in the presence of PGE2 but the vascular leakage was unaffected by antihistamines. Rat skin was virtually unresponsive to C5a with or without PGE2. This data emphasize the differences between reactivity of species to C5a and the danger of extrapolating results obtained in one animal species to models employed with another species.

Effect of extracts of Angelica polymorpha on reaginic antibody production.

An aqueous extract of Angelica polymorpha was examined for its immunoregulating properties. Its effect on the production of antibodies was tested in (C57BL/6 X DBA/2)F1 mice. When the animals were treated daily with the extract, the serum titers of reaginic antibodies normally observed after a single injection dinitrophenylovalbumin (DNP3-OA) were significantly lower, and the higher and more sustained reaginic titers induced by booster injections of DNP3-OA were also inhibited. The immunosuppressive activity was observed both by oral and intraperitoneal administration of the extract, and it was not removed by dialysis. In contrast, the serum titers of IgG were not significantly altered by the administration of the extract. The extract had little or no effect on the passive cutaneous anaphylaxis reaction or the release of histamine from sensitized rat lung fragments.

Effect of histamine agonists and antagonists on the production of murine reaginic antibodies.

At least wo histamine receptors have been pharmacologically defined. Using the appropriate agonists and antagonists, the possible involvement of these receptor types in the production of reaginic antibodies in the rodent was investigated. After injecting mice with dinitrophenyl-ovalbumin (DNP-OA), maximal serum reaginic titers occurred on day 11 as measured by heterologous passive cutaneous anaphylaxis. If the mice were dosed daily (i.p.) with the H1 agonist, 2-methylhistamine, or the H2 antagonist, metiamide, the titers of reaginic antibodies on day 11 were significantly higher than the controls. The titers were significantly lower than the controls if an H2 agonist (4-methylhistamine, dimaprit, or impromidine) or if the H1 antagonist, pyrilamine, was administered daily. None of these agents significantly affected total serum IgG titers as measured by ELISA. However, if the mice were injected with DNP-OA on day 0, then dosed daily with metiamide, pyrilamine, or 4 methylhistamine beginning on day 32, the titers of reaginic antibodies elicited by a second injection of DNP-OA given on day 36 were not significantly different from the titers of the non-drug treated mice. Thus, under these conditions, with these agents, the results suggest that histamine receptors may be involved in modulating the production of reaginic antibodies during a primary immunological response, H1 receptor agonists enhanced, while H2 receptor agonists suppressed the responses, and the reverse effect was observed with the appropriate antagonists. However, histamine receptors appear not to be measurably involved in the development of the secondary reaginic response.

Immunologic histamine release in vitro from the heart and lung of the cynomolgus monkey.

Immunologic histamine release was evoked from the sensitized fragmented cardiac and pulmonary tissue of the cynomolgus monkey by a reverse anaphylactic reaction. Ventricular and pulmonary tissue released a similar fraction (approximately 6%) of the total tissue histamine when challenged with antihuman IgE, presumably reflecting the 'active' sensitization of the monkey in vivo. Passive sensitization of these tissues in vitro resulted in significantly greater immunologic histamine release in 6 of the 14 ventricles and d a similar fraction (approximately 6%) of the total tissue histamine when challenged with antihuman IgE, presumably reflecting the 'active' sensitization of the monkey in vivo. Passive sensitization of these tissues in vitro resulted in significantly greater immunologic histamine release in 6 of the 14 ventricles and d a similar fraction (approximately 6%) of the total tissue histamine when challenged with antihuman IgE, presumably reflecting the 'active' sensitization of the monkey in vivo. Passive sensitization of these tissues in vitro resulted in significantly greater immunologic histamine release in 6 of the 14 ventricles and in the lungs. The antiallergic compounds, disodium cromoglycate and SK&F 64398, inhibited immunologic histamine release from passively sensitized monkey ventricular tissue. These results demonstrate that ventricular histamine may be immunologically released and that this release process can be pharmacologically inhibited in a manner similar to that of pulmonary tissue.

Histamine release from rat lung sensitized in vitro with mouse serum--an alternative pharmacologic method.

The ability of mouse anti-dinitropheny ovalbumin (anti-DNP-OA) serum to sensitize rat lung fragments can be demonstrated by the release of histamine from the tissue after antigenic challenge. This method can be utilized to evaluate anti-allergic agents, e.g., measuring the inhibition of histamine release by disodium cromoglycate (DSCG). In addition, the method can be utilized to evaluate antibody production, e.g., demonstrating the presence of separate antibodies against hapten and protein carrier in the sera of mice sensitized with DNP-OA. The correlation between heterologous rat passive cutaneous anaphylaxis (HPCA) and pulmonary histamine release for sera was determined by linear regression analysis. The r was greater than 0.8 with 16 observations, suggesting the utility of histamine release as a convenient alternative for pharmacologically evaluating different aspects of the immediate hypersensitivity response.

Preclinical evaluation of Antiallergic agents.

This paper examines the advantages and disadvantages of many in vivo and in vitro experimental model systems employed in the preclinical testing of antiallergic drugs.

Pyranenamines: a new series of antiallergic compounds.

Condensation of 3,5-diacylpyrantriones with various aromatic amines gave a new class of potent, orally active, antiallergic compounds, the 3-[(arylamino)ethylidene]-5-acylpyrantriones, hereafter referred to as pyranenamines, as evaluated not only in the traditional rat passive cutaneous anaphylaxis (PCA) assay but also in the in vitro fragmented rat and primate lung assay. Potencies in the PCA system, when measured intravenously, reached a maximum ID50 of 0.9 mu/kg (1000 times more potent than disodium chromoglycate) with 5-acetyl-4-hydroxy-3-[1-[(3,5-bis-glyceramoylphenyl)amino]ethylidene]-2H-pyran-2,6(3H)-dione (100), as predicted by structure-activity relationship (SAR) analysis. Potencies in the iv PCA system correlated well with potencies in the in vitro rat lung system but not with potencies in the oral PCA system or the in vitro primate lung system. Several compounds had good oral potency, and one analogue, 3-acetyl-4-hydroxy-3-[1-[3-amino-4-hydroxyphenyl)amino]ethylidene]-2H-pyran-2,6(3H)-dione hydrochloride (78), reached an oral ID50 of less than 1 mg/kg and was better than 10 times more effective than disodium chromoglycate at inhibiting the release of histamine and slow-reacting substance of anaphylaxis in the fragmented primate lung assay.

Application of quantitative structure-activity relationships in the development of the antiallergic pyranenamines.

QSAR techniques played a major role in development of the antiallergic pyranenamines (I). Graphical analysis of data resulting from an unsuccessful Topliss approach suggested that increased substituent hydrophilicity might enhance potency. The 3-NHAc-4-OH derivative which first resulted was an order of magnitude more potent than any preceding series member, and its deacylated congenar is clinical candidate SK&F 78729 (R1 = -NH2, R2 - OH, R3 = H). Further pursuit of hydrophilicity and other strategies suggested by multiple regression yielded 98 pyranenamines, the most active [R1 = R3 = NHCO(CHOH)2H, R2 = H] being 1000 times more potent than any original series member.

Pharmacologic characterization of the antiallergic activity of SK&F 78729-A.

SK&F 78729-A, one of a series of pyranenamine compounds, was studied in several laboratory models of immediate-type hypersensitivity reactions. In vivo, SK&F 78729-A, i.v./p.o., inhibited the antigen-induced rat 48-hr passive cutaneous anaphylaxis by a mechanism other than end-organ antagonism to either histamine or serotonin. In vitro, the compound produced a concentration-related inhibition of the immunologic release of histamine and slow-reacting substance of anaphylaxis from passively sensitized fragmented rhesus monkey and canine lung, and modest inhibition of histamine release from passively sensitized fragmented rhesus monkey skin. Similarly, SK&F 78729-A inhibited the antigen-induced release of histamine from passively sensitized fragmented rat lung. Under certain conditions, SK&F 78729-A inhibited antigen-induced pathophysiologic pulmonary responses in in vivo canine and rhesus monkey models of allergic asthma. The results of these studies suggest that clinically, SK&F 78729-A should exhibit a pharmacologic profile consistent with that of a prophylactic antiallergic agent.

Pharmacologic regulation of antigen-induced mediator release from canine lung.

The ascaris antigen-induced release of histamine and a slow-reacting substance of anaphylaxis (SRS-A) from passively sensitized fragmented canine lung is further characterized. Histamine and SRS-A were released within 30 sec of antigen challenge, reached a maximum at 7 and 10 min, respectively, and thereafter appeared to remain constant to 30 min. Contractions of guinea pig ileum produced by canine SRS-A were competitively antagonized by the SRS-A antagonist FPL-55712. Indomethacin and deuterium oxide enhanced antigen-induced SRS-A release from canine lung but had little effect on histamine release. The ability of several chemically novel 'antiallergic agents' to inhibit mediator release was evaluated. Inhibition of histamine release, and to a lesser extent SRS-A release, by one of these compounds was shown to vary with time and temperature. It is concluded that fragmented canine lung, while disclosing some qualitative pharmacological differences from other species, is a useful in vitro model of immediate hypersensitivity reactions.

Characterization of the adrenergic activity of carbuterol (SK&F 40383-A).

Carbuterol is a beta-adrenergic bronchodilator with selectivity for bronchial smooth muscle relative to cardiac and vascular tissues of several species including man. The present studies were undertaken to further characterize its adrenergic profile. In vitro studies demonstrated that carbuterol was a direct acting beta-adrenergic agonist, not dependent on endogenous catecholamine release, and was devoid of alpha-adrenergic agonist activity. The activity of the racemate was shown to reside primarily in the l-enantiomer. Carbuterol inhibited immunologically induced release of histamine and slow reacting substance of anaphylaxis from passively sensitized fragmented rhesus monkey lung and also inhibited passive cutaneous anaphylaxis in rats. The relatively weak stimulant activity of carbuterol on beta1 receptors mediating both rate and force of contraction was confirmed in anesthetized open-chest dogs. In the anesthetized cat, carbuterol was significantly less potent than isoproterenol in decreasing diastolic blood pressure, increasing heart rate, and decreasing the tension and degree of fusion of incomplete tetanic contraction of the soleus muscle.

The effect of metiamide in in vitro and in vivo canine models of type I hypersensitivity reactions.

The histamine H2-receptor antagonist metiamide was evaluated in in vitro and in vivo canine models of immediate-type hypersensitivity reactions. At concentrations of 2 and 4 X 10(-4) M, the antagonist significantly increased the amount of histamine present in the medium surrounding passively sensitized canine lung fragments which had been challenged with ascaris antigen. In contrast, the compound was without effect on the release of a slow reacting substance of anaphylaxis. On the basis of these observations metiamide was investigated in an in vivo canine model of allergic asthma. At doses of 10, 30 and 50 mu moles/kg, the antagonist did not enhance the ascaris antigen-induced pulmonary pathophysiology. Similarly, at 10 and 50 mu moles/kg metiamide did not alter histamine-induced increases in pulmonary resistance or decreases in dynamic lung compliance. Insofar as the canine model of allergic asthma may be predictive of the human disease, it could be anticipated that the use of histamine H2-receptor antagonists may not be deleterious to allergic asthmatics.

Studies on the mechanism of tachyphylaxis to disodium cromoglycate.

The tachyphylaxis to disodium cromoglycate's (DSCG) inhibition of antigen-induced histamine release is readily demonstrable utilizing passively sensitized rat lung fragments. This tachyphylaxis to DSCG is evident whether or not calcium is present during drug preincubation. An attempt to relate the mechanism of tachyphylaxis to the DSCG-induced release of an endogenous cellular inhibitory material was unsuccessful insofar as could be demonstrated by an effect on mediator release.

Further studies on the tachyphylaxis to DSCG. The effects of concentration and temperature.

The tachyphylaxis to DSCG's inhibition of anaphylactic histamine release has been demonstrated in passively sensitized rat lung fragments. The induction of tachyphylaxis appears to depend on the concentration of the drug and the length of pretreatment. Tachyphylaxis is relatively independent of the concentration of the second exposure to DSCG. The development of tachyphylaxis is highly temperature dependent; it can be prevented by cooling the tissues to 2-4 degrees C after a brief (30 sec) preincubation with DSCG. It is suggested that DSCG inhibits histamine release by binding to 'receptor' site(s). Once the site is occupied by DSCG, it is modified by a temperature-dependent process, thus losing the ability as a 'receptor' for inhibition of histamine release. The tachyphylaxis is the result of such a modification.

Canine airway responses to acetylcholine, prostaglandin F2alpha, histamine, and serotonin after chronic antigen exposure.

Dose-response curves were obtained for aerosols of acetylcholine (ACh), prostaglandin F2alpha (PGF2alpha), histamine (H), and 5-hydroxytryptamine (5-HT) on pulmonary resistance (Rp) and dynamic lung compliance (Cdyn) in Ascaris-hypersensitive dogs. Previously, these animals had been subjected to chronic biweekly "allergic asthmatic" episodes by aerosol administration of Ascaris antigen. When examined either one week before or after antigen provocation the airways were not hyperreactive to ACh, H, or 5-HT but did demonstrate a modest hyperreactivity to PGF2alpha. When aerosol dose-response curves were obtained for these agonists immediately following an "allergic asthmatic" episode, the airways were hyporeactive to PGF2alpha, H, and 5-HT, but not to ACh. Studies with atropine indicated that the hyporeactivity was the result of decreased airway responsiveness to both direct and indirect effect of PGF2alpha and H. It is concluded that, in dogs, chronic antigen challenge is not accompanied by a general increase in airway reactivity to pharmacologic agents.