Muscarinic receptors in human leukocytes are not coupled to phosphoinositide metabolism or adenylate cyclase

In the present study it was established, using computer-assisted fitting and simulation, that the muscarinic radioligand used ([3Hldexetimide) possesses a small selectivity for M 3 o v e r M 2 r~eptors in bovine tracheal smooth muscle membranes, and that such radioligand selectivity, if not recognized, leads to erroneous estimates of binding parameters for carclioselective muscarinic antagonists. The observed radioligand selectivity also results in an inability to detect both M 2 and M3 receptor populations using smooth muscle selective muscarinic antagonists with selectivities of up to 30-fold. To accomplish this, the smooth muscle selectivity of the displacing ligand must exceed that of the radioligand by at least a factor of about 10. Alternatively, monophasic displacement curves of muscarinic antagonists with moderate smooth muscle selectivities could be properly resolved into M 2 and M 3 receptor populations by using foreknowledge acquired with the cardioselective compounds. Using this approach, a good correlation was obtained for the first time between binding pKi-values for the minority of M 3 t y p e binding sites and functional pA2-values obtained in contraction measurements as well as in the determLnation of phosphoinositide metabolism (Roffel et al., 1990). As an important implication, the function of the majority of M 2 muscarinic receptors in smooth muscle membranes as well as the transduction mechanism involved remain to be established. Supported by a research grant from the Netherlands Asthma Foundation.

References Ladimky, H., E. Giraldo, E. Monferini, G.B. Schiavi, M.A. Vigano, L. De Conti, R. Micheletti and R. Hammer, 1988. Muscarinic receptor heterogeneity in smooth muscle: binding and functional studies with AF-DX 116, Trends Pharmacol. S~.'.9 (Sup?lement Subtypes of Muscarinic Receptors llI), 44. Roffel, A.F., C.R.S. Fblnga, ILG.M. Van Amsterdam, R.A. De Zeeuw and J. Zaagsma, 1988, Muscarinic M 2 receptors in bovine tracheal smooth muscle: discrepancies between binding and function, Eur. J. Pharmacol. 153, 73. Roffel. A.F., H. Meurs, C.R.S. Elzinga and L Zaagsma, 1990, Characterization of the muscarinic receptor subtype involved in phosphoinositide v-~abolism in bovine tracheal smooth muscle, Br. J. Pharmacol., in press.

Mmcarinic receptors in human leukocytes are not coupled to phosphoinositide metabolism or adenylate cyc|ase Meurs *'* *, H., T i m m e r m a n s * *, A., de M o n c h y * *, J.G.R., Zaagsma *, J. and K a u f f m a n * *, H . F . • Dept. of Phartr~zcologyand Therapeutics, University Centre for Pharmacy, A. Deusinglaan 2, 9713 A W Groningen and • * Dept. of Ailergology, University Hospita~ Oostersinge159, 9713 EZ Groningen, The Netherlands

Cholinergic hyperresponsiveness has been implicated in the pathogenesis of bronchial asthma, which is characterized by enhanced bronchoconstriction in response to muscarinic agonists. Muscarinic receptors in airway smooth muscle are coupled to activation of phosphoinositide (PI) hydrolysis and inhibition of adenylate cyclase (AC) as transduction mechanisms that may be involved in the pharmacomechanical coupling of contraction. Hence, a change in muscarinic receptor-induced PI metabolism or AC inhibition could play a role in the enhanced airway reactivity. Since lung tissue from asthmatic patients is difficult to obtain for in vitro investigation, we considered the possibility to use peripheral leukocytes as an easily accessible model to study possible changes in muscarinic transduction mechanisms in the.~ patients. Several investigations have indicated the presence of muscarinic binding sites in human leukocytes; however, the transduction mechanism(s) of these sites is (are) still unclear. We found that mononuclear leukocytes (MNL) from healthy donors were unable to accumulate inositol phosphates (IP, IP2, 11)3) after stimulation with methacholine (MCh) in the presence of LiCI. By contrast, the mitogen phytohaemagglutinin (PHA) caused enhanced levels of IP, IP2 and IP3 in these cells; however, with a markedly slow onset with positive responses after 2-5 min. In addition, MNL from both healthy subjects and allergic asthmatics showed no response to MCh when determined as total inositol phosphates accumulation after 30 min of incubation with agonist and LiCI, indicating that cholinergic hyperresponsiveness in asthmatics is not associated with the expression of muscarinic Pl metabolism in MNL. Moreover, normal basal and PHA-stimulated inositol phosphate

177,3 levels were found in cells of these patients. Polymorphonuclear leukocytes (PMN) from healthy donors were also unresponsive to MCh as assessed by total inositol phosphates accumulation, whereas a positive response in these cells was obtained with the chemoattractant formylmethionylleucylphenylalanine (fMLP). In both MNL and PMN, MCh had no effect on basal or isoprenaline-induced cAMP production, indicating that muscarinic receptors in these cells are not coupled to inhibition of AC either. Reevaluation of the muscarinic receptor density on MNL by radiofigand binding studies with [3HI-N-methylscopolamine surprisingly showed a very small population of high affinity binding sites (B.~,: approx. 50-100 sites/cell; Kd: approx. 50-150 pM). This is in sharp contrast to earlier reports of much larger populations of [3H]-quinuclidinyl benzilate binding sites, which, however, possessed a remarkably low affinity to the radioligand and other muscarinic antagonists. The results indicate that human leukocytes are not a suitable model to study muscarinic receptor function and dysfunction in relation to asthma. At least for MNL, this might be caused by an extremely low density of functional rw:eptors. This study was financially supported by the Netherlands Asthma Foundation.

P.th.063 1

Alterations in phosphoinositide hydrolysis associated with up-regulation of muscarinic receptor in primary cultured neurons O h k u m a , S., Ma, F.-H., Kishi, M. and Kuriyama, K.

Department of Pharmacology, Kyoto PrefecturalUniversityof Medicine, Kamikyo-Ku~ Kyoto 602, Japan Alterations in phosphoinositide (PI) hydrolysis associated with the supersensitivity of muscarinic receptor were investigated using mouse cerebral cortical neurons in primary culture possessing acetylcholine metabolizing system and muscarinic receptor (Ohkuma et al., 1987). A long-term (5 days) exposure of primary cultured neurons to atropine (10 - s M) induced a significant increase in binding of [3H]quinuclidinyl benzilate (QNB), a radiolabeled ligand specifically binding to muscarinic receptor, to prticulate fraction obtained from primary cultured neurons. This increase in [3H]QNB binding was found to be attributed to the increases in both Kd and Bmax values and was dose-dependently inhibited by atropine added into a binding assay system. In addition, atropine-induced increase in [3H]QNB binding was abolished by the addition of cycloheximide (10 -6 M) into culture medium. These results indicate that the increase in number of muscar~'_nic receptor following the exposure to atropine is due to the increased synthesis of new receptor molecules. In spite of a significant increase in number of muscarinic receptor, carbachol-stimulated PI hydrolysis was significantly decreased in atropine-treated neurons, which is in agreement with the data obtained from brain of atropine-treated rats (Goobar and Bartfai, 1988). The activities of transport of [3H]inositol, used as a pcecursor of phosphoinositides, into neurons and of incorporation of [3H]inositol into phosphoinositides were not different in both atropine-treated and non-treated neurons. The extent of GTP-induced shift to the right of carbachol competition curves for [~H]QNB binding was observed to be similar in both types of cells. Moreover, the activities of GTPase determinded in the presence and absence of carbachol significantly elevated in atropine-treated cells. Similarly, the binding of [3H]guanylylimido diphosphate (GppNHp), a radiolabeled ligand specific for GTP-binding protein (G protein), to particulate fraction obtained from atropine-treated cells also significantly increased. These results indicate that the functional interaction between muscarinic receptor and G protein, both of which showed a similar extent of increase, is not affected by the long-term exposure to atropine. On the other hand, a significant reduction in GTPrS-stimulated PI hydrolysis was observed in atropine-treated cells, although the activity of membrane-bound phospholipase C in atropine-treated cells was not signifficantly different from that in non-treated cells. These results suggest that the coupling mechanism between G protein and phospholipase C may be deteriorated in atropine-treated neurons. Conclusion: The long-term exposure of primary cultured neurons to the Low dose of atropine induces up-regulation