Pulmonary responses of the guinea pig to inhaled A23187: A brief review

The pulmonary effects of inhaled A23187 are reviewed. Guinea pigs challenged with this divalent cationic ionophore rapidly develop airway obstruction, which is maintained for at least 4 h. Pulmonary inflammation and increased airway responsiveness are also observed. Pharmacologic manipulations suggest that these actions are due to the release of multiple mediators. We have found A23187 challenge to be valuable as an approach for testing potential asthma drugs.     

Calcium fluxes and contractility in isolated guinea pig atrium: effect of A23187.

The Ca2+ ionophore A23187 (10(-6) to 3 X 10(-5) M) increased the force of contraction is isolated guinea pig atria. In individual twitches, peak tension, maximum rate of tension development, time to peak tension, and total twitch duration were all increased by A23187. Tripelennamine, indomethacin, and atropine did not significantly alter the inotropic effect of A23187. Serotonin produced changes in individual twitches that differed qualitatively and quantitatively from those of A23187. Therefore, the inotropic action of A23187 is probably not mediated by release of endogenous histamine, prostaglandins, acetylcholine, or serotonin. 45Ca influx and efflux were increased by A23187. The enhanced 45Ca efflux exceeded that which would be predicted if the ionophore acted only to increase the passive Ca2+ permeability of the myocardial cell membrane. These results suggest that A23187 facilitates the entry of extracellular Ca2+ into the myocardial cell and the release of intracellular Ca2+ stores into the myoplasm. The resultant increase in intracellular Ca2+ activity could account for the positive inotropic action of A23187.     

Role of mast cells in calcium ionophore (A23187)-induced peritoneal inflammation in mice

Several lines of evidence document a critical role for mast cells in immune complex-mediated inflammatory models. However, their role in nonimmune models of acute inflammation is largely unknown. In the present investigation, the role of mast cells was examined in calcium ionophore (A23187)-induced mouse peritoneal inflammation. Intraperitoneal injection of A23187 (20)g/mouse) elicited marked and transient increases in immunoreactive levels of 6-ketoprostaglandin-F₂, leukotrienes B₄, C₄, D₄, E₄, and F₄. There were no discernible differences in levels of these mediators in male Swiss Webster mice, mast cell-deficient mice (WBB6F1-W/W), and age-matched controls (WBB6F1-+/+), suggesting a minimal role of mast cells in eicosanoid biosynthesis in this model. However W/W mice showed smaller increases in levels of myeloperoxidase, a marker for neutrophils, compared to +/+ mice. Both W/W and +/+ mice have lower constitutive levels of peritonealN-acetyl--d-glucosaminidase (NAG), a marker for mononuclear cells. Similar to the changes seen in myeloperoxidase, W/W mice exhibited a blunted NAG response compared to +/+ mice. These results suggest that mast cell products other than eicosanoids may contribute to the changes in cellular trafficking in response to intraperitoneal A23187. These results also suggest that mast cells are required for full expression of inflammatory responses.     

Leukotriene B4-induced granulocyte trafficking in guinea pig dermis

Leukotriene B₄ (LTB₄) is a proinflammatory product of arachidonic acid metabolism that has teen implicated as a mediator in a number of inflammatory diseases. When injected intradermally into the guinea pig, LTB₄ elicits a dose-dependent migration (chemotaxis) of neutrophils (PMNs) into the injection sites as assessed by the presence of a neutrophil marker enzyme myeloperoxidase. SC-41930 7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)propoxy]-3,4-dihydro-8-propyl-2H-1 -benzopyran-2-carboxylic acid, a first-generation LTB₄ receptor antagonist inhibitedthe chemotactic actions of LTB₄ when coadministered into the dermal site and when given orally with ED₅₀ values of 340 ng and 1.7 mg/kg, respectively. The secondgeneration LTB₄ receptor antagonists SC-50605 7-[3-[2(cyclopropylmethyl)-3-methoxy-4-(4-thiazolyl)phenoxy] propoxy]-3,4-dihydro-8-propyl-2H-1-benzopyran-2-carboxylic acid and SC-51146 7-[3-[2(cyclopropylmethyl)-3-methoxy-4-[(methylamino)carbonyl]phenoxy]propoxy]-3,4-dihydro-8-propyl-2H-1-benzopyran2-propanoic acid inhibited LTB₄-induced chemotaxis when coadministered with ED₅₀ values of 70 ng and 32 ng, respectively, and when given intragastrically with ED₅₀ values of 0.10 and 0.09 mg/kg, respectively. SC-41930, SC-50605, and SC-51146 had oral durations of action of 5.5, 15, and 21 h, respectively. These potent, LTB₄ receptor antagonists may well have application in the medical management of disease states such as asthma, rheumatoid arthritis, inflammatory bowel disease, contact dermatitis, and psoriasis, where LTB₄ is implicated as an inflammatory mediator.     

Phospholipase A2-induced lung edema and its mechanism in isolated perfused guinea pig lung

Lung injury induced by phospholipase A₂ (PLA₂, 0.046 IU/ml perfusate) was studied in a continuous weighing system of isolated perfused guinea pig lungs. The results revealed that lung weight increased progressively during the 30-min perfusion of PLA₂. No change of pulmonary arterial pressure was observed in the same period. Albumin permeability-surface area product, lung index, lung water content, exudate from pleura, and angiotensin-converting-enzyme activity increased significantly at the end of 30 min PLA₂ perfusion.p-Bromophenacyl bromide, a PLA₂ inhibitor, may block the above changes nearly completely. The effects of inhibitors of cyclooxygenase (indomethacin, IM), lipoxygenase (diethylcarbamaxine, DE), and platelet-activating factor (SRI 63-441) on PLA₂-induced lung injury were also studied. We found: (1) PLA₂ may induce high permeability lung edema. The role of endothelial injury in the permeability change remains to be further investigated. (2) DE ameliorated lung injury significantly within 10 min of PLA₂ treatment but showed no effect after 15 min. IM ameliorated lung injury during the whole experimental period. SRI 63-441 had no effect. It is suggested that PLA₂ may damage lung by inducing products of cyclooxy genase and lipoxygenase besides its direct effect.Project supported by Natural Science Foundation of China, NO. 3880399.     

Action of ionophore A23187 on the strength of contraction and transmembrane action potential of guinea pig papillary muscle

The calcium ionophore A23187, in a concentration of 2.5 M, caused a two- to threefold increase in the strength of the contraction of the isolated papillary muscle of the guinea pig heart, stimulated at a frequency of 0.2 Hz. The ionophore A23187 reduced the resting voltage of the preparation in the intertrial interval and reduced the total duration of contraction. The increase in the strength of contraction was not accompanied by any change in the amplitude or duration of the transmembrane action potential. In the presence of the ionophore the ascending phase of a single contraction cycle showed a discontinuity separating the development of the twitch into two phases; differentiation of the twitch gave two positive maxima. The substance D-600, which blocks the calcium current, reduced the duration of the action potential and inhibited the second phase of twitch development, but caused no change in the magnitude or rate of the first phase of contraction. It is suggested that under the influence of the ionophore the component of the twitch which is not blocked by D-600 is caused by liberation of calcium from the sarcoplasmic reticulum.Laboratory of Electrophysiology of the Heart and Laboratory of Myocardial Metabolism, All-Union Cardiologic Scientific Center, Academy of Medical Sciences of the USSR, Moscow. Department of Pharmacology, Vanderbilt University, Nashville, USA. (Presented by Academician of the Academy of Medical Sciences of the USSR V. V. Zakusov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 85, No. 6, pp. 690–693, June, 1978.     

Immunologically induced purulent anterior segment inflammation of the guinea pig eye

A single conjunctival application of ovalbumin to inbred guinea pigs (IMM/S 209) immunized with the same antigen in Freund's complete adjuvant provoked an acute purulent inflammation of the anterior segment of the eyes with a duration of up to 1 week. Intense conjunctival injection and chemosis were followed by a purulent discharge. A corneal haze was observed regularly, and a considerable proportion of the animals developed a pronounced pannus and corneal ulcers. Tear fluid cytology revealed a rapid increase in cell concentration, from the normal level (less than 10(8)/l) to greater than 10(11)/l. Seventy to 95% of the cells were polymorphonuclear leukocytes. Histological examination revealed an acute inflammatory reaction which radiated from the conjunctival fornices to the entire anterior segments of the eyes. The process was characterized by an intense oedema, vasodilation and perivascular aggregations of polymorphonuclear leukocytes, and to a lesser extent eosinophilic granulocytes which characteristically infiltrated and penetrated the epithelial layers. Neovascularization could be observed early after challenge in the stroma of all parts of the outer eye. Ulcerations of the conjunctival and corneal epithelia were observed frequently. After a number of reiterations of the antigenic challenge, a marked infiltration with lymphocytes and basophils/mast cells was observed, and significant scarring of the conjunctival mucosa developed. In several animals, a slight, but significant co-reaction of the contra-lateral, non-challenged eye was observed.     

Inhibition of antigen- and calcium ionophore A23187-induced contractions of guinea pig isolated airways with 8-(diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8)

Both antigen- and calcium ionophore A23187-induced airways contractions are dependent on increased concentrations of intracellular calcium. Challenge of isolated tracheal spirals and parenchymal strips from sensitized guinea pigs with antigen or calcium ionophore A23187 in the presence of the intracellular calcium antagonist, 8-(diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8), resulted in significantly reduced contractions. The results demonstrated that TMB-8 inhibits events that are dependent on either mobilization of intracellular calcium (i.e. antigen) or entry of calcium from extracellular sources (i.e. calcium ionophore A23187). The efficacy of TMB-8 in this model suggests that intracellular calcium antagonists may be potentially useful in the therapy of asthma.     

Effects of adenosine on guinea pig pulmonary eosinophils

Extracellular adenosine has pharmacological activity on a wide variety of cell types and may play an important role as an inflammatory modulator with both pro- and anti-inflammatory activities. These studies examine the effects of adenosine on guinea pig pulmonary eosinophils. Adenosine alone did not directly induce superoxide (O ₂ ^– ) production. Pretreatment with adenosine primed the O ₂ ^– response of guinea pig pulmonary eosinophils following the addition of 1 or 10M plateletactivating factor (PAF). Priming was seen at adenosine concentrations greater than 1 M and was maximal at 100M. At this maximal dose, adenosine priming increased the O ₂ ^– response to 1M and 10M PAF by 86% and 51%, respectively. Priming by adenosine was not seen when ionomycin or phorbol myristate acid (PMA) were used as agonists. In fura-2 loaded eosinophils, the addition of 100 M adenosine resulted in a small but significant rise in intracellular calcium of 54.4 ±9.2 nM above baseline. In contrast, similar adenosine concentrations had no effect on cytosolic calcium levels in guinea pig neutrophils. These data demonstrate a pro-inflammatory role for adenosine in elicited guinea pig pulmonary eosinophils.     

Specific plasma cell accumulation in antigen-induced chronic inflammation in the guinea pig peritoneal cavity

Specificity of plasmacellular infiltration was studied using a guinea pig peritoneal inflammation model. Acute and chronic inflammations were induced by repeated injections of either of two non-crossreacting antigens (DNP-BSA and PPD). With an enzyme-immunohistochemical sandwich procedure allowing quantitation of DNP-BSA-specific plasma cells, specificity of plasmacellular infiltration could be demonstrated. DNP-BSA-specific antibody-forming cells were found not to enter inflammatory reactions elicited by PPD. Our data support the hypothesis that virtually all plasma cells in a chronic inflammatory exudate release antibodies specific for antigens that are locally available, and that such antigens are likely to play a central role in the perpetuation of chronicity.     

Effect of BRL-35135 on LTB4-induced guinea pig eosinophil chemotaxis

The effect of an atypical -adrenoceptor agonist, BRL-35135 on leukotriene B₄-induced-guinea pig eosinophil chemotaxis was studied. BRL-35135 and SC-41930 (leukotriene B₄-antagonist) inhibited the chemotaxis in a concentration-dependent manner (IC₅₀=9.0×10^–6 and 2.6×10^–7 M, respectively). However, isoproterenol, fenoterol and another atypical -agonist, BRL-37344 had no effects. The inhibitory effect of BRL-35135 was not affected by (±)-propranolol (10^–4 M). In contrast, the nonselective -adrenoceptor antagonist, (–)-alprenolol (10^–4 M) dextrally shifted the inhibitory curve of BRL-35135. The response to BRL-35135 was antagonized in a competitive manner by (–)-alprenolol, with the slope of the Schild plot close to unity, and a pA ₂ value of 5.62. These findings suggest that guinea pig eosinophils possess an atypical receptor, which differs from either ₁, ₂ or atypical -adrenoceptor on guinea pig ileum, and through which eosinophil chemotaxis can be modulated by BRL-35135.     

Distribution and histochemical characterization of pulmonary mast cells in the rat and guinea pig

The rat and guinea pig are widely used in experimental models dealing with immuno allergic bronchoreactivity. The present study was designed to compare the distribution and heterogeneity of the mast cell population in the respiratory tract of the Sprague-Dawley rat and Hartley guinea pig. Mast cells were identified according to the sequential Alcian blue/safranin O staining method. From the trachea to the peripheral conductive airways, the density of mast cells increased in the guinea pig whereas it decreased in the rat. Although mast cells were observed in the interalveolar septa of the guinea pig, none were found in the rat. In the lung of the rat, three types of mast cells were observed: in the wall of the trachea and large bronchi, safranin-positive, mixed and Alcian-blue-positive mast cells were found, whereas only Alcian-blue-positive mast cells were observed in the small conductive airways. In contrast, only Alcian-blue-positive mast cells were observed in the lung of the guinea pig. These results show that the types and localization of the mast cell populations are distinctly different in these two species. Consequently, experimental models of bronchoreactivity in these species are testing different cellular systems.     

Photoaffinity labeling of the guinea pig pulmonary mast cell beta-adrenergic receptor

Beta-adrenergic agonists can prevent mediator release from guinea pig pulmonary mast cells. By pharmacologic characterization, this response is mediated through a beta-2 receptor. Structural characterization of this receptor on the lung mast cell, however, has been limited by methods for isolation of this pulmonary cell. In this study, the guinea pig lung mast cell was isolated to greater than 90% purity, and its beta-adrenergic receptor identified by photoaffinity labeling with [125I]iodoazidobenzylpindolol (125IABP) and separation of membrane proteins by SDS-PAGE. We found the guinea pig pulmonary mast cell beta-adrenergic receptor to electrophorese as a heterogeneous protein between 68 and 116 kD. Photoaffinity labeling with 125IABP was protectable by alprenolol and isoproterenol but not by phentolamine and norepinephrine. Using subtype-selective compounds, the pulmonary mast cell receptor was established to be of a beta-2 subtype. This is the first report of the structural identification of a lung mast cell beta-adrenergic receptor and the first report of a beta-adrenergic receptor of approximately 100 kD in mass. This mast cell receptor is considerably larger than the 65 kD beta-adrenergic receptors that have been identified in whole lung and other tissues. Data we have obtained using Northern blot analysis of mast cell RNA suggest a protein message of 45 kD for this beta-adrenergic receptor and a high degree of glycosylation most likely accounts for the large molecular size observed.     

Quantitation of bronchiolar epithelial proliferation in A23187-exposed guinea pigs

Aerosol exposure to the ionophore A23187 results in PMN accumulation, airway epithelial injury and prolonged airway constriction. Bronchiolar epithelial damage in ionophore-exposed guinea pigs was quantitated by measuring epithelial proliferation using bromodeoxyuridine (BRDU). Animals were killed at 24, 48 or 72 hours post-ionophore exposure and lungs were collected for H&E and immunostaining. Numerical scores were assigned for morphologic changes and the number of labeled cells per mm of airway was determined. Significant increases in labeled epithelial cells were evident at 48 hours. Inflammation and epithelial damage scores also were elevated. These results indicate that ionophore exposure results in pulmonaray inflammation and bronchiolar epithelial proliferation as assessed by BRDU labeling.