Effects of strain on contractile force and number of sarcomeres in series of Xenopus laevis single muscle fibres during long-term culture.

The aim of the present study is to test whether mechanical strain uniquely regulates muscle fibre atrophy/hypertrophy and adaptation of the number of sarcomeres in series within mature muscle fibres in vitro . Mature single muscle fibres from Xenopus laevis illiofibularis muscle were cultured (4-97 days) while kept at negative strain ( approximately 20% below passive slack length, 'short fibres') or at positive strain ( approximately 5% over passive slack length, 'long fibres'). Before and after culture the number of sarcomeres in series was determined using laser diffraction. During culture, twitch and tetanic force characteristics were measured every day. Survival time of long fibres was substantially less than that of short fibres. Of the long fibres 40% died or became inexcitable within 1 week, whereas this did not occur for short fibres. During culture, twitch and tetanic force of all short fibres increased substantially. Regression analysis showed that the post-culture number of sarcomeres in series was not significantly changed compared to the number before culture. It is concluded that culture at negative strain does not result in atrophy or a reduction of the number of sarcomeres in series, even after 97 days. For the long fibres we did not detect any hypertrophy as tetanic force remained stable or decreased slowly, while twitch force varied. Regression analysis of the change of the number of sarcomeres in series as a function of the culture time showed a positive slope ( P=0.054). Two out of four long fibres that were cultured for at least 2 weeks showed an increase in the number of sarcomeres of 4-5%. Compared with in vivo adaptation to mechanical stimuli this is much less than would be expected. The data suggest that strain may not be the only factor that regulates hypertrophy and the number of sarcomeres in series.

Bovine tracheal responsiveness in vitro: role of the epithelium and nitric oxide.

Airway epithelium releases inhibitory factors, such as nitric oxide (NO) and prostaglandin E2 (PGE2), which may counteract bronchoconstriction. We investigated whether epithelium-derived inhibitory substances exert a crucial influence on bovine tracheal responsiveness in vitro. Isotonic and isometric contractions in response to histamine of intact and epithelium-denuded tracheal smooth muscle strips were compared. In addition, the effects of L-arginine (L-arg), N(G)-nitro-L-arginine methyl esther (L-NAME), and N(G)-monomethyl L-arginine (L-NMMA) on histamine responsiveness were investigated. The release of NO and PGE2 from tracheal epithelium was measured. Removal of the epithelium from tracheal smooth muscle strips did not change the negative log of the concentration of histamine producing half the maximal effect (pD2) or the maximal effect (Emax). Incubation of the tissues for 25 min with L-arg or L-NAME did not influence basal tone or the contractions induced by histamine. However, incubation with L-NMMA increased the basal tone and caused a slight hyporesponsiveness to histamine. S-nitroso-N-acetyl-penicillamine (SNAP, a direct NO donor) reversed the contraction induced by histamine in a concentration-dependent manner. Stimulation of the epithelial layer by 0.1 microM histamine increased the release of NO 3-4 fold compared to basal levels; this effect was completely inhibited in the presence of L-NMMA. In addition, 1 mM histamine caused a significant increase in the release of PGE2 from the epithelial tissue. In conclusion, no functional inhibitory influence of the epithelium can be identified in bovine airways. The S-nitroso-N-acetyl-penicillamine-induced relaxation demonstrates the presence of a nitric oxide sensitive pathway in bovine airways. However, the amounts of nitric oxide and prostaglandin E2 released from bovine tracheal epithelium are probably too low to exert a significant effect on the histamine-induced contractions.

Peroxynitrite induces airway hyperresponsiveness in guinea pigs in vitro and in vivo.

Peroxynitrite (ONOO-) is a cytotoxic product of the rapid reaction between nitric oxide and superoxide that may initiate inflammation. Isolated perfused tracheas from guinea pigs were incubated from the mucosal side for 15 min with peroxynitrite (1 to 100 muM). Thereafter, concentration-response curves to histamine and methacholine were constructed on the preparations. Peroxynitrite (10 muM) caused a significant hyperresponsiveness; the maximal contractions in response to histamine and methacholine were enhanced by 30% and 40%, respectively. In the peroxynitrite-treated group, clear epithelial damage as well as eosinophil destruction were detected. Moreover, 3, 5, and 10 days after intratracheal instillation of peroxynitrite (100 nmol), a significant rise in pulmonary resistance to histamine of anesthetized animals was observed. It is suggested that the generation of peroxynitrite from nitric oxide superoxide radicals during inflammatory processes induces epithelial damage, mediator release, and hence airway hyperresponsiveness. These findings may have clinical implications, because airway inflammation, epithelial damage, and hyperresponsiveness are characteristic features in patients suffering from asthma.

Bronchoconstriction and airway hyperresponsiveness after ovalbumin inhalation in sensitized mice.

To investigate the mechanisms underlying airway hyperresponsiveness a murine model was developed with several important characteristics of human allergic asthma. Mice were intraperitoneally sensitized with ovalbumin and after 4 weeks challenge via an ovalbumin aerosol. After aerosol, lung function was evaluated with a non-invasive forced oscillation technique. The amount of mucosal exudation into the airway lumen and the presence of mast cell degranulation was determined. Tracheal responsiveness was measured at several time points after challenge. At these time points also bronchoalveolar lavage and histology were performed. Sensitization induced high antigen-specific IgE levels in serum. Inhalation of ovalbumin in sensitized mice induced an immediate but no late bronchoconstrictive response. During this immediate phase, respiratory resistance was increased (54%). Within the first hour after ovalbumin inhalation increased mucosal exudation and mast cell degranulation were observed. At 12 and 24 h after ovalbumin challenge, mice showed tracheal hyperresponsiveness (29% and 34%, respectively). However, no apparent inflammation was found in the lungs or bronchoalveolar lavage. From these results it can be concluded that hyperresponsiveness can develop via mechanisms independent of an inflammatory infiltrate. Since mast cell degranulation occurred after ovalbumin exposure, we hypothesize that mast cells are involved in the induction of airway hyperresponsiveness in this model.

Endogenous nitric oxide modulation of potassium-induced changes in guinea-pig airway tone.

1. An experimental set up is used whereby the serosal (out)side or mucosal (in)side of the guinea-pig isolated tracheal tube can be stimulated selectively with drugs and reactivity measured. 2. Potassium induces a concentration-dependent (5-70 mM) monophasic contraction of tracheal tubes when added on the outside. In contrast, on the inside, potassium induces a concentration-dependent relaxation at low concentrations (5-40 mM) which was reversed into a contraction up to approximately basal tone at higher concentrations (50-70 mM). 3. Epithelium denudation reversed the potassium-induced relaxation into a contraction. Interestingly, in the 'half' epithelium-denuded trachea the contractions were significantly (P < 0.01) reduced by 46% compared to complete epithelium-denuded tissues. 4. Incubation with the nitric oxide (NO) synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME, 120 microM) for 30 min on the inside of the tracheal tube completely prevented the relaxation. However, L-NAME did not reverse the potassium-induced relaxation into a contraction. This indicates that potassium does not penetrate through the epithelial layer. 5. It is concluded that depolarization of smooth muscle cells leads to a monophasic contraction and that depolarization of the epithelium leads to a relaxation of tracheal smooth muscle. The epithelial layer has an important barrier function and can release relaxing factors like NO.

Antibody to interleukin-5 inhibits virus-induced airway hyperresponsiveness to histamine in guinea pigs.

In humans, respiratory viral infections lead to increased airway responsiveness and exacerbations of asthma. In the present study, the role of interleukin-5 (IL-5) in virus-induced airway hyperresponsiveness and inflammation was examined in guinea pigs. In animals treated with control antibody, parainfluenza-3 virus significantly potentiated (219%) the histamine-induced increase in lung resistance compared with vehicle treatment. In addition, viral infection significantly increased (130 to 450%) the responsiveness of isolated tracheal segments to histamine in animals treated with control antibody. In guinea pigs treated with control antibody, the numbers of eosinophils (226%), neutrophils (1,380%), and monocytes (626%) in bronchoalveolar lavage fluid were significantly increased after viral infection. The level of major basic protein in bronchoalveolar lavage fluid was not altered after viral infection. In addition, electron microscopic examination of eosinophils in airway tissue and alveolar lumen did not point to increased degranulation after viral infection. In guinea pigs treated with antibody to IL-5 the virus-induced airway hyperresponsiveness to histamine both in vivo and in vitro was almost completely inhibited. In guinea pigs treated with anti-IL-5, viral infection significantly increased the numbers of eosinophils (234%), neutrophils (1,255%), and monocytes (617%) in bronchoalveolar lavage fluid. These data suggest that IL-5 plays an important role in airway hyperresponsiveness to histamine but not in the infiltration of eosinophils after respiratory viral infection.

Virus-induced changes in airway responsiveness, morphology, and histamine levels in guinea pigs.

A significant increase in airway responsiveness to histamine was observed in vitro and in vivo 4 days after intratracheal inoculation of parainfluenza Type 3 (PI-3) virus to guinea pigs. Light microscopic and ultrastructural examination of the central airways of animals inoculated with virus revealed stratification of the epithelial lining, with pronounced loss of cilia and granule-depleted goblet cells. In the peripheral airways, typical lesions of patchy alveolitis and bronchiolitis were found. The alveolar epithelium often lacked Type I alveolar cells and was lined merely by cells containing osmiophilic lamellar bodies typical of Type II alveolar cells. PI-3 virus inoculation resulted in a reduction in the number of airway mucosal mast cells, particularly in the bronchioles, and in a change of density of the granules of mast cells. Further, a significant rise (100%) in histamine concentration was observed in lung lavage fluid after virus inoculation. The prostaglandin E2 (PGE2) content in the lavage fluid was not changed. After stimulation with histamine, the tracheae of animals inoculated with control solution or PI-3 virus produced similar amounts of PGE2. These data indicate that PI-3 virus activates airway mast cells and increases the histamine content in the respiratory tract. Neither the virus-induced lung damage nor the increased levels of histamine in the airways influence the release of the epithelially derived relaxing factor PGE2. It is suggested that mast cell-derived products, in particular histamine, are involved in virus-induced airway hyperresponsiveness.

Identification of macrophages in intimal thickening of rat carotid arteries by cytochemical localization of purine nucleoside phosphorylase.

Complete desquamation of the endothelium of the rat carotid artery by balloon catheter stripping resulted within 2 weeks in the formation of a large intimal thickening. After an enzyme cytochemical technique was applied to localize cytosolic purine nucleoside phosphorylase (PNP), light microscopical evaluation indicated that this intimal thickening in normocholesterolemic rats was composed of 5.8% to 11.8% (mean 8.8%) PNP-positive cells. At the electron microscopic level, all these PNP-positive cells were identified as macrophages by the absence of a basement membrane and plasmalemmal vesicles and by the occurrence of specific intracytoplasmic granules. The nearly nonreactive intimal cells were classified as modified smooth muscle cells. Additional evidence of the macrophage nature of the PNP-stained intimal cells was obtained by differential immunogold labeling of these cells with a monoclonal antibody against rat macrophages. Moreover, in hypercholesterolemic rats, only the cells stained for PNP transformed into foam cells (between 8.5% and 11.4% of all nucleated intimal cells; mean 9.6%). This study shows that PNP cytochemistry discriminates macrophages from modified smooth muscle cells in the rat carotid intimal thickening. It further suggests that the intimal thickening in normocholesterolemic rats originates not only from migration and proliferation of smooth muscle cells, but also from a considerable number of leukocyte-derived macrophages. Whether the latter cells are actively involved in the establishment of the intimal thickening as has been suggested in dietary hypercholesterolemia, remains to be verified.