Effect of sustained drug-induced vagotonia on isolated tissue sensi. tivity to carbachol and histamine in guinea-pigs

Transcription

1 Effect of sustained drug-induced vagotonia on isolated tissue sensi tivity to carbachol and histamine in guinea-pigs Sachiko TANIHATA, Akira AIZAWA, Russell CHESS-WILLIAMS* and Toshimitsu UCHIYAMA Department of Pharmacology, Toho University School of Medicine, Omori-nishi , Ota-ku, Tokyo 143, Japan *Department of Biomedical Sciences, University of Sheffield, Scheffield S10 2TN, United Kingdom Key words: carbachol, echothiophate, histamine, tissue sensitivity, vagotonia Abstract The effects of sustained vagotonia induced by 7 consecutive days of pretreatment with an anticholinesterase, echothiophate iodide (ETP, 20ug/kg/day, s.c.) on carbachol-induced responses and muscarinic receptor binding characteristics in lung, heart and ileum of guinea-pigs have been investigated. The study was aimed at elucidating the mechanisms involved in the ETP-induced airway subsensitivity to carbachol observed in our previous studies. ETP-pretreatment reduced cholinesterase activity by about % in lung, heart, ileum and serum. In the lung and heart, neither the sensitivity of isolated tissues to carbachol nor the binding of [3H] quinuclidinyl benzilate ( [3H]QNB) were influenced by ETP-pretreatment. In contrast, the sensitivity of isolated trachea and ileum to carbachol was reduced by ETP-pretreatment, and this sensitivity change of the ileum was associated with a fall in muscarinic receptor density without a change in receptor affinity, as determined in [3H]QNB binding experiments. ETP-pretreatment also reduced the sensitivity of trachea and ileum to histamine, although [3H] pyrilamine binding in the ileum was not affected. These results suggest that sustained vagotonia induced by chronic ETP-pretreatment reduces the sensitivity of isolated ileum and possibly trachea to carbachol and histamine through a reduction in muscarinic receptor density and also some alteration of receptor coupling to intracellular mechanisms. Introduction Cholinergic mechanisms are believed to play an important role in the bronchial hyperactivity observed in asthma (1), with airway sensitivity to acetylcholine being 100 times greater in asthmatic patients than in healthy volunteers (2). The cholinergic muscarinic antagonists, atropine and - ipratropium, are useful bronchodilators in the treatment of bronchial asthma (3,4) and it is now gener- Abbreviations: ETP: echothiophate iodide, QNB: quinuclidinyl benzilate ally accepted that a depression of airway muscarinic responsiveness is beneficial in the treatment of this condition. Chronic administration of organophosphorus anticholinesterases has been shown to cause a subsensitivity to carbachol in guinea-pig ileum and atrium (5), rat ileum(6,7) and mouse ileum (8). We have also previously demonstrated that the chronic administration to guinea-pigs of echothiophate iodide (ETP), an organophosphorus anticholinesterase, reduces airway sensitivity not only to carbachol but also to histamine (9). The ETP-induced subsen-

2 sitivity to carbachol and histamine is observed in both the peripheral and central airways, as reflected by changes in lung compliance and airway resistance, respectively. The ETP-induced subsensitivity in respiratory function to carbachol and histamine can also be demonstrated in guinea-pig isolated trachea (10), which reflects changes in central airway responsiveness. We have recently attempted to determine the mechanism of this airway hyposensitivity in radioligand binding studies, but could not obtain sufficient tissue to perform the experiment, even after pooling the tracheal tissue from 10 animals. The present study investigates the effects of sustained vagotonia induced by chronic ETPpretreatment on the sensitivity of guinea-pig lung parenchyma to carbachol and histamine. In addition, the effects of sustained vagotonia induced by ETP on the sensitivity of the heart and ileum to carbachol and histamine have been examined in view of existence of different receptor subtypes. Materials and Methods Animals : Male Hartley guinea-pigs (Sankyo Labo Service), weighing g, were housed in an air conditioned room (22-23 Ž, humidity 55-65%) with a 12hr ( hr) light cycle. The animals were provided with rodent laboratory chow (GC4, Oriental Yeast) and water ad libitum. ETP-pretreatment and tissue isolation: ETP was administered by subcutaneous injection, once a day for 7 days, at a dose of 20ug/kg/day in volume of 0.1ml/100g body weight (10). Animals were decapitated on the 8th day, blood was collected and trachea, heart, lung and ileum were excised. Cholinesterase activity and muscarinic receptor binding assays were determined in the same animals, while separate groups of animals were used for the histamine receptor binding assays and the pharmacodynamic examination in isolated tissues. Determination of cholinesterase activity: The cholinesterase activity in serum and tissue homogenates was determined by the method of Ellman et al.(11) using acetylthiocholine as a substrate. Approximately 100mg wet weight of tissue from lung, ventricle and ileum were homogenized in ice-cold Na/K phosphate buffer (50mM, ph 7.4). Protein content was determined by the method of Lowry et al.(12) using bovine serum albumin as a standard. Pharmacodynamic examination of isolated tissues: The trachea was cut into several ring of equal width. These were tied together to form a chain and each ring was opened by cutting the cartilage. The strip of lung parenchyma(2-3mm wide and 1.5-2cm long)was obtained from the right lower lobe. The strips of trachea and lung were mounted in an organ baths containing Tyrode solution (NaC , KCl 2.7, CaC121.8, MgCl21.1, NaH2PO40.3, NaHCO311.9, glucose 5.6mM) at 37t. The segments of ileum, approximately 2cm in length, were mounted in organ baths containing low calcium Locke-Ringer solution (NaC , KC1 5.6, CaCl21.1, MgCl22.1, NaHCO32.4, glucose 2.8mM) at 32t and the right atrium (auricle) was mounted in an organ bath containing Krebs-Henseleit solution (NaCI 118.0, KCl 4.7, CaC122.6, MgSO41.2, KH2PO41.2, NaHCO3 24.9, glucose 11.1mM) at 37t. Each preparation had a resting tension of 0.5g and the physiological salt solution was aerated with a gas mixture (95 % O2 5%CO2). The tension developed by tissues was measured with isometric force transducers and the spontaneous rate of beating of right atria was recorded with a tachograph triggered by a signal from the isometric force recording. After a 2 hr equilibration period, cumulative concentration-response curves to histamine and carbachol were obtained on each preparation. In isolated right atria, agonist potency was expressed as the EC50 value (molar concentration producing a half maximal response). The EC50 value of carbachol in right atrium was the concentration which reduced tension and rate by 50%. Agonist potency in isolated trachea, lung and ileum preparations was expressed as the pd2 value (negative logarithm of the molar EC50 value) calculated from concentration-response curves(13). Preparation of membranes for radioligand binding assays: The excised lung, ventricle and ileum

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6 sensitivity to carbachol in the guinea-pig lung. In spite of the reduced effect of carbachol on lung Discussion The present study demonstrates that in guineapig ileum, cholinesterase activity, muscarinic receptor density and isolated tissue sensitivity to carbachol are significantly reduced by chronic ETPpretreatment. The reduction in cholinesterase activity in lung and heart was similar to that observed in the ileum, but neither the muscarinic receptor density nor response to carbachol in these tissues were affected by the ETP-pretreatment. Our previous in vivo study in guinea-pigs suggested that chronic ETP-pretreatment reduced the sensitivity of both central and peripheral airways to carbachol as reflected by changes in airway resistance and lung compliance, respectively (9). We speculated that this reduction of airway responsiveness to carbachol by ETP-pretreatment might be due to a fall in muscarinic receptor density. However, we were unable to determine receptor density in membranes prepared from trachea, due to the small quantity of tissue available, even when the tissue from 10 animals was pooled. Consequently, the aim of the present study was to determine the effects of chronic ETP-pretreatment on cholinesterase activity, [3H] QNB binding and isolated tissue compliance observed in our earlier study (9), the sensitivity of isolated lung strips to carbachol was not altered by ETP-pretreatment in the present study. Lung compliance reflects the response of the peripheral airways. Lung parenchymal strips, however, contain not only the peripheral airways but also vascular smooth muscle, and carbachol has opposite effects on these two tissues: contracting respiratory smooth muscle and relaxing vascular smooth muscle. It is possible that the effects of ETP- pretreatment on the contractile response of lung strips to carbachol may be masked by simultaneous vascular relaxation. Thus, simultaneous vascular smooth muscle relaxation may explain the discrepancies between our earlier in vivo and our present in vitro results. Chronic ETP-pretreatment decreased the cholinesterase activity in guinea-pig lung and atrium by 47% and 62%, respectively, but did not affect [3H]QNB binding or the sensitivity of these tissues to carbachol. On the other hand, the pretreatment significantly reduced the sensitivity of the ileum to carbachol and reduced muscarinic receptor density in this tissue. The cholinesterase activity in all three tissues was uniformly decreased by treatment with ETP. The results are consistent with a report (17) in which the administration of an organophosphorus anticholinesterase (diisopropylfluorophosphate) significantly reduced muscarinic receptor density without affecting receptor affinity in guinea-pig ileum, but had no effect on [3H]QNB binding characteristics in lung or atrium. On the contrary, a significant decrease in muscarinic receptor density in lung and subsensitivity of guinea-pig atria to carbachol has been observed after chronic pretreatment with the organophosphates, diisopropylfluorophosphate (18) and 4-methylthiophenyl dipropylphosphate (5), respectively. Although we can not appropriately explain these contrasting results, different organophosphorus agents and different dosage and administration schedules may be responsible for the conflicting results.

7 In the present study. the sensitivity to carbachol and the density of [3H]QNB binding sites in the ileum, but not in the heart, were decreased by ETP-pretreatment. The muscarinic receptors in cardiac, respiratory and intestinal smooth muscles are heterogenous (19, 20). Muscarinic receptors have been divided into three major subtypes as M1, M2 and M3 (21). Muscarinic receptors in tracheal and ileum smooth muscle are classified as M3 receptors which are generally associated with phosphatidylinositol breakdown, whereas M2 receptors in the heart are associated with inhibitory GTPbinding protein (19, 20, 22). Consequently, the discrepancy observed between the heart and ileum following ETP-pretreatment in view of carbachol desensitization may be explained by the different M2 and M3 receptors in heart and ileum tissues. The sensitivity of the ileum to carbachol was reduced by ETP-pretreatment and this change was accompanied by a significant fall in the density of [3 H]QNB binding sites. This decrease in muscarinic receptor density in the ileum may reflect a compensatory downregulation in response to the decrease in cholinesterase activity. Chronic ETP-pretreatment resulted in a significant decrease in tracheal as well as ileum sensitivity to carbachol. This suggests that ETP-pretreatment may induce the attenuation of M3 receptor-mediated responses resulting from the reduction of receptor density in trachea. We could not accomplish [3H] QNB binding study of guinea-pig trachea due to its insufficient protein, but there remains the possibility that chronic ETPpretreatment may also reduce muscarinic receptor density in this tissue. Muscarinic receptors in smooth muscle are linked to phosphatidylinositol breakdown (22-24) and histamine contracts tracheal and ileum smooth muscles via H1 receptors, which are also linked to the phosphatidylinositol second messenger system (25-27). Chronic inhibition of cholinesterase activity induced a subsensitivity not only to carbachol but also to histamine in guinea-pig ileum and trachea. This suggests that an alteration in muscarinic receptor coupling to intracellular mechanisms may also play a role in the ETP-induced changes in tissue sensitivity, in addition to the reduction in muscarinic receptor density. In the heart, histamine-induced inotropic and chronotropic responses were not altered by chronic ETP-pretreatment. On the contrary, the pretreatment increased the sensitivity of right atrial rate responses to histamine, which acts via H2 receptors in this tissue (28). Chronic cholinesterase inhibition resulted in a desensitization of responses to both carbachol and histamine in the guinea-pig ileum, yet only [3H]QNB, but not [3H]pyrilamine binding, was reduced. These data suggest that chronic ETP-pretreatment may attenuate the physiological responses to agents which act through the phosphatidylinositol system. Consequently, a reduction in the muscarinic receptor density may be the primary mechanism for the subsensitivity observed to carbachol in the guinea-pig ileum after ETP-pretreatment, but an alteration in receptor coupling to intracellular mechanisms may also be involved. Conclusion This study suggests that sustained vagotonia induced by chronic ETP-pretreatment may reduce tissue sensitivity to both carbachol and histamine in the ileum partially through a decrease in muscarinic receptor density and also by some alteration, to be elucidated, of receptor coupling to intracellular mechanisms. Furthermore, it is suggested that the subsensitivity to carbachol and histamine in trachea may be associated with the same mechanisms demonstrated ileum. for these sensitivity changes in the Acknowledgement We would like to thank Prof. I. Takayanagi and Ass. Prof. K. Koike (Department of Pharmacology, Toho University School of Pharmacy) for their sincere advice and consultations concerning the radioligand binding studies. All correspondence should be addressed to T. Uchiyama.

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