Effect of M2 and M3 muscarinic receptors on airway responsiveness to carbachol in bronchial-hypersensitive (BHS) and bronchial-hyposensitive (BHR) guinea pigs.

The expression balance of M2 and M3 muscarinic receptor subtypes on the pathogenesis of airway hyperresponsiveness was investigated by using two congenitally related strains of guinea pigs, bronchial-hypersensitive (BHS) and bronchial-hyposensitive (BHR). CCh-induced airway responses in vivo and in vitro were investigated by comparing the effects of muscarinic receptor subtype antagonists, and the relative amounts of M2 and M3 muscarinic receptor mRNA in tracheal smooth muscle and lung tissue were investigated. After treatment with muscarinic receptor subtype antagonists, the ventilatory mechanics (VT, Raw, and Cdyn) of response to CCh aerosol inhalation were measured by the bodyplethysmograph method. The effects of these antagonists on CCh-induced tracheal smooth muscle contraction were also investigated. The effects of M2 muscarinic receptor blockade were less but the effects of M3 muscarinic receptors blockade on the airway contractile responses were greater in BHS than in BHR. In M3 muscarinic receptor blockades, CCh-induced tracheal contractions in BHS were significantly greater than those in BHR. In tracheal smooth muscle from BHS, the relative amount of M2 muscarinic receptors mRNA was less but that of M3 muscarinic receptor mRNA was more than those in BHR. These results suggest that the high ACh level as a consequence of dysfunction of M2 muscarinic autoreceptors and the excessive effect of M3 muscarinic receptors on the airway smooth muscle may play an important role in the pathogenesis of airway hyperresponsiveness.

Effect of acetylcholinesterase activity on pathogenesis of airway hyperresponsiveness in guinea pigs.

To clarify the effect of acetylcholinesterase (AChE) on the pathogenesis of airway hyperresponsiveness, AChE activities in tracheal smooth muscle and lung tissue from congenitally bronchial-hypersensitive (BHS) and bronchial-hyposensitive (BHR) guinea pigs were compared. For this purpose, AChE activities were determined by measuring the rate of absorbance of tissue homogenate. Relative amounts of AChE mRNA were also evaluated by the RT-PCR method. In both tracheal smooth muscle and lung tissue from BHS, the AChE activity and the relative amount of AChE mRNA were less than those in BHR. These results suggest that the reduced AChE activity is at least a candidate for inducing airway hyperresponsiveness.

The difference in citric acid-induced cough in congenitally bronchial-hypersensitive (BHS) and bronchial-hyposensitive (BHR) guinea pigs.

Cough elicitation and major physiological factors influencing cough occurrence were investigated in congenitally bronchial-hypersensitive (BHS) and -hyposensitive (BHR) guinea pigs exposed to citric acid (0.3 M) aerosol for 10 min. The number of cough in BHS was significantly larger than in BHR, while the latency to cough in BHS was significantly shorter than in BHR. Pretreatment with atropine (0.2%), lidocaine (2%) or salbutamol (0.1%) aerosol and desensitization of C-fibers with capsaicin (100 mg/kg) decreased the cough numbers in both BHS and BHR. The salbutamol, atropine and capsaicin pretreatments prolonged the cough latency in BHS, but only salbutamol prolonged the latency in BHR. After salbutamol pretreatment all BHR guinea pigs exhibited cough, while 66.7% of BHS guinea pigs exhibited it. Vagal blocking by atropine suppressed coughing in both BHS and BHR. Only a small number (33.3%) of BHR guinea pigs and no BHR guinea pigs exhibited a cough response after capsaicin and lidocaine pretreatment whereas many BHS guinea pigs still produced cough after such pretreatment. The present study demonstrated that the cough responsiveness to citric acid aerosol was significantly higher in BHS than in BHR. It was revealed that airway smooth muscle contraction and functional and/or morphological development of airway nervous receptors, especially C-fiber endings, contributed to aggravation of coughing in BHS.

Airway responsiveness to acetylcholine in congenitally bronchial-hypersensitive (BHS) and bronchial-hyposensitive (BHR) guinea pigs in vivo and in vitro.

The characteristics of airway responsiveness to acetylcholine (ACh) in congenitally bronchial-hypersensitive (BHS) and bronchial-hyposensitive (BHR) guinea pigs were clarified in vivo and in vitro. We measured the change in ventilatory mechanics in response to ACh inhalation by means of the bodyplethysmograph and the contractile responses of isolated trachea to ACh and carbachol (CCh). Further, muscarinic receptor subtypes involved these responses were identified. The basal values for ventilatory mechanics in BHS were not significantly different from those in BHR. Respiratory resistance to ACh was progressively increased in a time- and dose-dependent manner in BHS. The contractile responses of tracheal smooth muscle to ACh in BHS were significantly greater than those in BHR, but CCh-induced responses in BHS and BHR were similar. ACh- and CCh-induced contractions were mediated via M3 receptors. These results suggested that the falling-down of BHS in response to ACh inhalation was caused by the strong constriction of the airway and the reduction in ventilation. Moreover, the airway hyperresponsiveness to ACh in BHS might be partly dependent on the change in acetylcholinesterase activity.

Histamine-induced airway contraction in congenitally bronchial-hypersensitive (BHS) and bronchial-hyposensitive (BHR) guinea pigs.

Airway hyper-responsiveness is known as an important pathogenesis of asthma. In the present study, the airway responsiveness to aerosolized and injected histamine in congenitally bronchial-hypersensitive (BHS) and bronchial-hyposensitive (BHR) guinea pigs was investigated. In addition, the role of the vagal reflex in histamine-induced airway contraction was evaluated by vagal blocking with atropine inhalation or bilateral vagotomy. A significantly higher bronchoconstrictive reaction, i.e., a decrease in tidal volume (VT) and an increase in respiratory resistance (Rrs), to histamine-inhalation was observed in BHS than in BHR. A noticeably lower reduction in VT was noted after atropine pretreatment for both BHS and BHR, whereas an increase in Rrs was inhibited only in BHS. The intravenous injection of histamine caused a noticeable bronchoconstrictive reaction in both BHS and BHR with a dose-dependent relationship, but no significant differences were observed and the bilateral vagotomy failed to induce any difference between the two animal groups. These results demonstrated that the airway responsiveness to histamine is considerably different in BHS from that in BHR, but the difference is largely dependent on the route of administration of histamine. The important role of the vagal reflex on the elicitation of airway contraction was elucidated in both animal groups, and it appeared that the BHS possessed relatively higher dependency on the vagal reflex mechanism than the BHR.

Bradykinin-induced airway contraction in two lines of guinea pigs with congenitally different airway sensitivity.

The airway responsiveness to bradykinin (0.1, 1 and 10 microg/kg, i.v.) was examined in two lines of guinea pigs, BHS (bronchial hypersensitive) and BHR (bronchial hyposensitive) lines, with different airway sensitivity to inhalation of acetylcholine (ACh)-aerosol. Normal Hartley strain guinea pigs were used as a control group. The airway contraction was measured by recording intratracheal pressure (P[IT]) and respiratory airflow (V) under the condition of artificial ventilation in anesthetized guinea pigs. The results show airway responsiveness to bradykinin in BHS guinea pigs to be significantly greater than in BHR and normal Hartley strain guinea pigs.

Characteristics of two lines of guinea pigs (BHS and BHR) differing in bronchial sensitivity to acetylcholine and histamine exposure.

In a previous study, we reported that as model animals to be used in the study of bronchial asthma in humans, two lines of guinea pigs were developed by ourselves: bronchial hypersensitive line (BHS) and bronchial hyposensitive line (BHR) as a control. Studies on biological characteristics in guinea pigs of two lines were undertaken, and the following results were obtained. 1) Airway resistance of guinea pigs of the two lines to intravenously administered acetylcholine, histamine and leukotriene D4 was found to be different between BHS and BHR. Airway resistance of BHS to the chemicals was increased compared with those of BHR. 2) The number of muscarinic acetylcholine receptors in lung membrane preparation and its affinity increased significantly in BHS compared with those of BHR. In beta-adrenergic and histamine H1 receptors, there was observed no difference between BHS and BHR. 3) No difference in IgE antibody production to ovalbumin was observed between BHS and BHR. 4) When total leukocytes and differential leukocyte count (percentage, %) in peripheral blood of BHS and BHR were examined, relative percentage of lymphocytes and eosinophils was significantly higher in BHS than in BHR, while percentage of neutrophils was significantly lower in the former than in the latter.

Selective breeding of two lines of guinea pigs differing in bronchial sensitivity to acetylcholine and histamine exposure.

We developed two lines of guinea pigs, one as model animals for bronchial asthma with bronchial hypersensitivity and the other with hyposensitivity as a control. In the last four years, the bronchial hypersensitive line (BHS) and hyposensitive line (BHR), both derived from Hartley strain guinea pigs, have been selected by using bronchial reactivity to acetylcholine and to histamine as parameters. Both lines have reached the F6 generation. The following results were obtained with the two lines: 1) Sib and cous in matings, and mating of selected consanguineous individuals were adopted in breeding BHS and BHR. The breeding started with six families, each, but in the F6 generation the number of families decreased to two in each line. 2) Appearance rates of hyper- or hyposensitivity to acetylcholine and histamine increased with successive generations in both lines, which had been completely separated by the F6 generation. 3) Coefficients of inbreeding in BHS and BHR in the F6 generation ranged from 42% to 45% in the former and 42% in the latter. 4) Heritabilities (h2) of BHS and BHR for the appearance rates of sensitivity to acetylcholine were presumed to be 0.54 in the former and 0.69 in the latter. 5) No difference in the body weight of 0, 20, and 40 day-old BHS was observed in any generation. On the other hand, the body weight of 20 and 40 day-old BHR tended to decrease with successive generations. 6) Mean litter sizes of BHS and BHR in each of the generations ranged from 2.24 to 3.47 animals in the former and from 2.63 to 3.38 animals in the latter.(ABSTRACT TRUNCATED AT 250 WORDS)

Strain differences in guinea pigs' bronchial sensitivity to acetylcholine.

The bronchial sensitivity to acetylcholine (ACh) of guinea pigs of various strains was investigated to clarify strain differences. Inbred Strain 2, Strain 13 and JY-1 and non-inbred Hartley strain (two colonies) were used in this experiment. (1) Guinea pigs were exposed to 0.08% ACh aerosol and the time needed to produce falling down (TNPFD) was determined. Mean +/- standard error of TNPFD (n = 14 per group) of animals was 182 +/- 28 sec, 148 +/- 22 sec, 210 +/- 30 sec, 342 +/- 24 sec and 406 +/- 36 sec in Strain 2, Strain 13, JY-1, Hartley (Japan SLC) and Hartley (Hitachi), respectively. There was a significant difference in TNPFD between inbred strains and non-inbred strains (P less than 0.05 or P less than 0.01), indicating that inbred strains had higher sensitivity. (2) Guinea pigs were exposed to 20-5000 micrograms/ml ACh for 2 min. The mean dose threshold as determined by transcutaneous oxygen pressure was 524 micrograms/ml, 424 micrograms/ml, 614 micrograms/ml, 1317 micrograms/ml and 1651 micrograms/ml (n = 14 per group) in Strain 2, Strain 13, JY-1, Hartley (Japan SLC) and Hartley (Hitachi), respectively. Inbred strains showed lower dose thresholds than non-inbred strains. (3) Isolated trachea-lungs of 5 guinea pigs were perfused with 10(-9)-10(-5) g/ml ACh to determine strain differences. Dose response curves of animals of inbred strains shifted to the left (lower concentrations), unlike those of non-inbred strains, suggesting that inbred strains had higher sensitivity to ACh than non-inbred strains.(ABSTRACT TRUNCATED AT 250 WORDS)

Bronchial reactivities in guinea pigs to acetylcholine or histamine exposure.

The bronchial reactivities in Hartley guinea pigs to acetylcholine (ACh) or histamine (Hist) were investigated, and the following results obtained; 1. Eight-week-old animals were exposed to ACh and Hist. A significant relationship was observed between the concentrations of the chemicals and the time needed to produce falling down (TNPFD) due to the asthmatic reaction to ACh and Hist. 2. Eight-week-old animals were exposed to 0.1% ACh and 0.05% Hist, for which the mean TNPFD +/- standard error were 377 +/- 33 sec and 122 +/- 5 sec respectivity. However, no difference in reactivity between male and female animals was noted. 3. Eight- and 9-week-old animals were exposed to 0.01% ACh and 0.025% Hist. A positive correlation was observed (r = 0.736, p less than 0.01) between the TNPFD for ACh and that for Hist. 4. Growing animals from 2 weeks to 20 weeks old were exposed to 0.08% ACh and 0.025% Hist. After inhalation of both chemicals, 6-week-old animals showed the greatest prolongation of mean TNPFD (lowest sensitivity). 5. Eight-week-old animals were exposed to 0.08% ACh and 0.025% Hist. With both of these chemicals, a positive correlation was observed between TNPFD and dose threshold (ACh r = 0.886, p less than 0.001; Hist r = 0.891, p less than 0.001).

[Reaction of various experimental animals to exposure to acetylcholine or histamine, and morphological observations of bronchial smooth muscle].

We investigated the sensitivity of the airway of various experimental animals to acetylcholine (ACh) and histamine (Hist). The experimental animals were exposed to 0.1% ACh or 0.05% Hist. Guinea pigs and rabbits exhibited an asthmatic reaction to both chemicals, but rabbits seemed to have a milder reaction than guinea pigs to both chemicals. Mice, rats and hamsters showed no reaction. We then performed a morphological study of the airways of 5 species in order to clarify the reason for their different reactivities to ACh and Hist. In the morphological study, abundant smooth muscle could be seen in the terminal bronchioles and respiratory bronchioles of guinea pigs and rabbits. In contrast, animals of other species had little smooth muscle in either site. Mice had no respiratory bronchioles. Consequently, we concluded that there is a high correlation between sensitivity to ACh and Hist and the extent of smooth muscle distribution around the airway.