Subtypes of muscarinic receptors in cultured explants of the hippocampus of the rat

Neuropharmacology Vol. 26, No. 7B, pp. 1027-1029, Printed in Great Britain. All rights reserved

1987 Copyright

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0028-3908/87 $3.00 + 0.00 1987 Pergamon Journals Ltd

SUBTYPES OF MUSCARINIC RECEPTORS IN CULTURED EXPLANTS OF THE HIPPOCAMPUS OF THE RAT P. G. WASERand KARIN RIMVALL Institute of Pharmacology, Gloriastrasse 32, CH-8006 Ziirich, Switzerland Summary-Using

the tritiated, muscarinic antagonist quinuclidinyl benzilate ([3H]QNB) as a ligand, muscarinic receptors have been identified and characterized in intact, cultured explants of the hippocampus of the rat. Competition studies with scopolamine and oxotremorine indicated a certain heterogeneity in the population of muscarinic receptors, whereas atropine and pirenzepine competed with [‘H]QNB in a manner consistent with only one binding site for these substances. Thus, the observed heterogenity does not fit in with the M,/M2 receptor concept. Extended studies, with the aim of determining to what extent these putative subtypes of receptors are functional, would be of interest. Key words: muscarinic receptor subtypes, cultured explant, hippocampus, rat.

On the basis of differences in the affinities for cholinergic agonists and for the antagonist pirenzepine, it has lately become obvious that the population of muscarinic acetylcholine receptors (mAChrs) in the central nervous system is heterogeneous (e.g. Birdsall, Hulme and Stockton, 1984). With pirenzepine the MI and Mz receptors have been characterized (Hammer and Giachetti, 1982; Watson, Yamamura and Roeske, 1983). The M, receptor, with a high affinity for pirenzepine, is situated primarily in the forebrain, whereas the Mz receptor, with a low affinity for pirenzepine, is found predominantly in the heart, brain stem and in the intestinal tract (Watson et al., 1983). There is, however, also a heterogeneity in the binding of pirenzepine intrinsic to the different structures of the brain and, on the basis of binding studies with the substance AF DX-I 16 (Hammer, Giraldo, Schiavi, Monferini and Ladinsky, 1986), it has been indicated that subtypes of the Mz receptor may also exist. In the case of the hippocampus it has been suggested that the M, receptors correspond to the postsynaptic muscarinic receptors and that the Mz receptors correspond to the presynaptic muscarinic receptors which are situated on the septal cholinergic terminals and possibly involved in the release of acetylcholine (ACh) (Mash and Potter, 1986; Raiteri, Leardi and Marchi, 1984). Using the tritiated muscarinic antagonist quinuclidinyl benzilate (QNB), muscarinic receptors in cultured explants from the hippocampal formation of the rat have previously been identified and characterized (Rimvall, 1986; Rimvall, i(eller and Waser, 1986). In the present report, preliminary results, indicating the presence of subtypes of muscarinic receptors in the cultured explants of hippocampus, are presented and the use to which these kinds of cultures can be put in investigations of the functional aspects of different subtypes of muscarinic receptors are discussed.

METHODS Cultures

Explants were taken from the hippocampus of 7day old rats (Wistar) and cultured on cover slips according to the roller-tube technique as previously described (GBhwiler, 1981; Rimvall, Keller and Waser, 1985). Receptor binding

Incubation of the intact, cultured hippocampal explants with the tritiated, muscarinic, cholinergic antagonist [3H]QNB and with the other substances of interest followed at day 9 or 10 in vitro and took place directly in the roller tube. For technical reasons, the cultures had to be fixed histologically, prior to the incubations. Specific binding was determined through the subtraction of the binding determined in the presence of 10pM atropine from the total binding. After incubation and washing, the explants were scraped off the cover slips and the radioactivity was measured with liquid scintillation counting. Analysis of data

The binding constants from the direct binding data were determined with a least-squares fitting procedure as previously described (Rimvall et al., 1986). The kinetics of the interaction between [3H]QNB and the receptors was analyzed as described by Weiland and Molinoff (1981). The competition data was analyzed with the curve-fitting program LIGAND (Munson, 1983; generously supplied by Dr P. Munson, NIH: U.S.A.).

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RESULTS In direct binding studies (i.e. determination

of the

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P. G. Table

WASER and KARIN RIMVALL

I. Kinetic parameters for the specific binding of [‘HIQNB in cultured explants of hippocampus of the rat (IO DIV)

[‘HIQNB(PM)

Association constant (K,,) (M-‘min-’ x IO’)

Dissociation constant (K_ ,) (min-’ x IO-‘)

Half life

1300 520

1.40 I .43

6.44 5.19

83 96

Concentration of

specific binding of [3H]QNB, as a function of the free concentration of [3H]QNB), it was demonstrated that the specific binding of [3H]QNB to the explants of hippocampus was saturable, a dissociation constant (& equil.) in a typical series of explants amounted to 500-600pM and a B,,,,, of 65-80 fmol/explant was determined (Table 1; column 6 and 7). This direct binding data also indicated that the binding of [‘H]QNB took place to an homogeneous population of binding sites [linear Scatchard plots and Hill coefficients close to 1 (not shown)]. Kinetic analysis of the association and dissociation between [3H]QNB and the receptor, showed that the Kds determined in direct binding experiments (Kd equil.) corresponded very well with the Kds determined in kinetic experiments (Kd kin.) (Table 1). The kinetic Kds in two different series of cultures and with two different concentrations of [‘H]QNB amounted to 405 and 460pM and the Kds determined through the analysis of the data obtained from the direct binding studies with [3H]QNB in the same two series of cultures, amounted to 5 16 and 5 14 pM, respectively. Competition experiments with [3H]QNB and a number of cholinergic drugs were also performed and these experimental data were subsequently evaluated with the program LIGAND. Atropine competed with [3H]QNB in a manner consistent with one binding site for atropine, with an affinity of 19 nM (Table 2). The competition with scopolamine showed a significantly better fit with a two-site model and this was also the case with the cholinergic agonist oxotremorine. With pirenzepine, however, only one single binding site was determined, with an affinity in the micromolar range.

DISCUSSION

The affinity for [3H]QNB was smaller (Kd 50& 600pM) in the intact explant than those found in in situ in homogenates and also smaller than the value determined in homogenate preparations of the cultures (Kd = 185pM; Rimvall et al., 1986). Investigators, who have observed similar differences in the

(min)

Kd (kinetic) (K-,/K+,,

2-Site

Atropine Scopolaminef Pirenzepine Oxotremorinef

at (3) (4) (2)

I9 38 3.3 I7

f 2.9 (lO-9) i 9.8 (10-9) + 0.88 (10-6) +_ 2.8 (10-6)

du

516 514

65.1 82.4

affinity for QNB in homogenate and slice preparations (Gilbert, Hanley and Iversen, 1979; Nonaka and Moroji, 1984) have suggested that diffusion barriers and decreased accessability of the receptor in a slice may play a role. It is, however, also possible that the histological fixation of the tissue may have caused changes in affinity and, finally, it is not impossible that there are some real differences between the receptor in an intact slice and in a homogenate preparation. In this context the kinetics of the association and dissociation between the receptor and QNB was also investigated and a very good correlation between the binding parameters determined in direct binding experiments and the parameters determined in kinetic experiments was found (see Results). With competition experiments between the labeled ligand [3H]QNB and the cholinergic drugs atropine, scopolamine, pirenzepine and oxotremorine, evidence was obtained that subtypes of muscarinic receptors are present in intact explants of cultured hippocampus. There were, however, some discrepancies between these results and those of other investigators, making it difficult to fit the present data in to the concept of M, and M, receptor sites. The extremely increased goodness-of-fit (P < 0.00001) with a two-site model for scopolamine is somewhat puzzling in view of the fact that scopolamine is a so-called classical, muscarinic antagonist and as such is supposed to interact with a homogeneous population of muscarinic receptors. There have been reports, however, that methylscopolamine shows heterogeneity in its competition with [3H]QNB (Lee and El-Fakahany, 1985) and that this heterogeneity does not correlate with an interaction with the M, and Mr sites, as defined with pirenzepine (El-Fakahany, Ramkumar and Lai, 1986). It was not possible to detect a second binding site for pirenzepine in its competition with [3H]QNB. It might be that a second (lower-affinity?) site is present in the cultured explants of hippocampus in such a small amount that it cannot be detected. Autoradiographic observations by Mash and Potter (1986), indi-

I--Site model K., (M)

B (ftnol/e~~lant)

460 405

Table 2. Displacement of the specific binding of [‘H]QNB in cultured explants of the hippocampus substances Competitor

(PM)

Kd (equil.)

PM)

(M)’

5.0 f 2.1 (10-g) II If: 6.8 (10-s)

(9-IOdiv) with different cholinergic

model

% R,* Ill-conditioned 90 Ill-conditioned 19

d,

CM)

% R,

500 f 350 (10-g)

10

28 + 30 (IO@)

21

*K,,Aissociation constant for the high-affinity site: % R,-% of total amount of receptor with a high affinity for the competitor. tNumber of pooled, independent experiments (at least 25 explants per experiment). fBetter fit resulted using a 2-site model (P < 0.00001 scopolamine: P < 0.01 oxotremorine).

Subtypes of mAChrs in hippocampal cultures

cate that the M, receptors in the hippocampus are located postsynaptically, whereas the M2 sites are located on the presynaptic, septal terminals. In this context it must be taken into account that a cultured explant of hippocampus is a totally deafferented preparation and that the septal terminals are not present in explants of hippocampus, cultured alone (Rimvall ef al., 1985). Finally, the competition of oxotremorine was best fitted to a two-site model with the two binding sites having affinities around 10 and 30pM respectively. The M2 receptor supposedly corresponds to the receptors which have been found to have a high affinity for agonists (Horvath, van Rooijen, Traber and Spencer, 1986). Hence, the absence of a receptor site with a higher affinity for oxotremorine may also be explained by the absence of septal terminals in this preparation. Thus, the results indicate that subtypes of muscarinic receptors might exist in cultured explants of the hippocampus. Whether they correspond to the subtypes of muscarinic receptors described by other investigators remains to be elucidated. Also, the functionality of the receptor(s) remains to be determined. As mentioned previously, it has been suggested that the M, receptors have a postsynaptic localization and the M2 receptors a presynaptic localization. Recent studies (Keller, Rimvall and Waser, 1987) have demonstrated that cultured explants of the septum of the rat take up choline and subsequently synthesize and release acetylcholine, the latter being dependent on the presence of calcium ions. It has, furthermore, been shown that cholinergic fibers from cultured explants of septum innervate co-cultured explants of hippocampus in a specific manner (Rimvall et al., 1985) and also that the septal fibers form functioning, cholinergic synapses in the hippocampal part of these combined cultures (Gahwiler and Brown, 1985). Hence, combined cultures of the septum and hippocampus may prove to be a useful preparation with which to study the functional aspects of the putative receptor subtypes further.

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Cohen R. M., Eds), pp. 33-48. Academic Press, Orlando. Nonaka R. and Moroji T. (1984) Quantitative autoradiography of muscarinic cholinergic receptors in the rat brain. Brain Res. 296: 295-303. Raiteri M., Leardi R. and Marchi M. (1984) Heterogeneity of presynaptic muscarinic receptors regulating neurotransmitter release in the rat brain. J. Pharmac. exp. Ther. 228: 209-214.

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