Muscarinic cholinergic receptor subtypes expression by human placenta

Neuroscience Letters 221 (1997) 208–212

Muscarinic cholinergic receptor subtypes expression by human placenta Seyed Khosrow Tayebati a, Maurizio Sabbatini a, Damiano Zaccheo b, Francesco Amenta a ,* a

Sezione di Anatomia Umana, Dipartimento di Scienze Farmacologiche e Medicina Sperimentale, Universita` di Camerino, Via Scalzino 3, 62032 Camerino, Italy b Istituto di Anatomia Umana, Universita` di Genova, Via De Toni, 10, 16132 Genoa, Italy Received 2 September 1996; revised version received 6 November 1996; accepted 6 December 1996

Abstract The presence of a cholinergic system in the placenta is suggested by several data, but no information is available concerning cholinergic receptor expression by placenta. The present study was designed to investigate muscarinic cholinergic receptors in sections of human placenta using a radioligand binding techniques with [3H]N-methyl scopolamine ([3H]NMS) as a ligand. [3H]NMS was bound to sections of human placenta in a manner consistent with the labelling of muscarinic cholinergic receptors. The dissociation constant (Kd) value was 0.1 ± 0.03 nM and the maximum density of binding site (Bmax) value was 10.82 ± 0.09 fmol/mg of tissue. The binding was time-, temperature- and concentration-dependent, belonging to one class of high affinity sites. Analysis of [3H]NMS displacement curves by compounds acting on the different subtypes of muscarinic cholinergic receptor subtypes suggests that human placenta expresses the four subtypes (M1 –M4) of muscarinic cholinergic receptor assayable with radioligand binding assay techniques. The demonstration of muscarinic cholinergic recognition sites in human placenta may contribute to define the possible significance of placental cholinergic system. Moreover, human placenta can be used as an easily obtainable human source of M1 –M4 muscarinic cholinergic receptor subtypes.  1997 Elsevier Science Ireland Ltd. All rights reserved Keywords: Muscarinic cholinergic receptors; M1 receptor; M2 receptor; M3 receptor; M4 receptor; Human placenta; Radioligand binding assay

Increasing evidence indicates the presence of a cholinergic system in the placenta [22,23]. This system was characterised by the demonstration of the acetylcholine biosynthetic enzyme choline acetyltransferase [20]. Detectable amounts of acetylcholine were assayed by gas chromatography techniques [22]. The occurrence of gestational variations of acetylcholine concentrations was demonstrated as well [22]. In spite of relatively high concentrations of acetylcholine and choline acetyltransferase in placental tissue [20–23], demonstration of the acetylcholine catabolic enzyme acetylcholinesterase was controversial. Some studies denied the occurrence of acetylcholinesterase activity in the placenta [19] or were unable to clearly establish whether the enzyme activity associated with placenta is due to erythrocyte contamination or reflects the expression of an enzyme localized in * Corresponding author. Fax: +39 737 630618; e-mail: [email protected]

placental tissue [6]. Subsequent investigations, based on the use of both biochemical and enzyme histochemical techniques, have demonstrated the expression of acetylcholinesterase activity by syncytiotrophoblast, cytotrophoblast cells, endothelial cells and the tunica media of foetal blood vessels of the human placenta. These studies suggested also an increased enzyme activity in pre-eclampsia [8]. In spite of the above data on the occurrence of cholinergic system markers in the placenta, only sparse information is available concerning cholinergic receptors expressed by placental tissue. Functional studies have shown that l-nicotine increases release of acetylcholine from isolated placental villi. This phenomenon was blocked by atropine [17,21]. Radioligand binding studies on placental microvilli plasma membranes with the nonselective muscarinic cholinergic radioligand [3H]quinuclidinyl benzylate have suggested the occurrence of muscarinic cholinergic receptor binding [7] or denied it [26].

0304-3940/97/$17.00  1997 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3940 (96 )1 3311-5

S.K. Tayebati et al. / Neuroscience Letters 221 (1997) 208–212

In view of this, we decided to investigate the issue using a radioligand binding assay technique in frozen sections of human placenta. Placentas were obtained from 12 subjects (age 29 ± 4 years) after Caesarean parturition (before labour or during labour) or vaginal delivery (after spontaneous labour). Material was taken from apparently normal subjects with gestational periods ranging 35–42 weeks. Patient data relevant to this study were confidentially recorded. Placentas removed from Caesarean delivery required surgical assistance for reasons not affecting placental function such as contracted pelvis, breech presentation or previous history of Caesarean delivery. After delivery, placentas were placed in an ice-cold Krebs solution. Samples of marginal and central portions of placenta (approximate size 3 cm3) were cut and frozen quickly in isopentane cooled with liquid nitrogen. Material was then stored at −80°C until use. For radioligand binding assay, 10 mm thick sections were obtained using −25°C microtome cryostat and mounted on pre-weighed gelatine-coated microscope slides. Slides were air-dried, reweighed and processed as detailed below. The protein content of sections of human placenta was approximately 10 mg tissue protein per 10 mg dry tissue weight. Some sections were stained with toluidine blue to verify microanatomical details. Analysis of these sections revealed a normal placental morphology in all subjects examined and the absence of erythrocytes in tissues investigated (data not shown). Muscarinic cholinergic receptor assay was performed with the non-selective receptor antagonist [3H]N-methyl scopolamine ([3H]NMS) as radioligand. It was used alone or plus 1 mM atropine to define non-specific binding. Sections were incubated with [3H]NMS (0.125–2.5 nM) in a buffer composed by 1.3 M NaCl, 0.07 M Na2HPO4, 0.03 M NaH2PO4 (pH 7.4). In a series of preliminary experiments, incubation was done at different times (30, 60, 90, 120 and 150 min) and temperatures (4°C, 23°C and 37°C) to assess optimal incubation conditions. From these experiments it was found that the highest specific/non-specific binding ratio was obtained using an incubation time of 60 min and a temperature of 23°C (data not shown). At the end of incubation, sections were washed in ice-cold incubation buffer, rinsed quickly with distilled water and wiped onto glass fibre filters. Filters were placed in scintillation vials containing 5 ml of scintillation liquid (Aqualyte, J.I. Baker Italy, Milan, Italy). Vials were counted using a Beckman liquid scintillation spectrometer at an efficiency of 40%. Muscarinic cholinergic receptors were also assayed in sections of rat frontal cortex, neostriatum, heart and sub maxillary gland. These tissues were used as reference, since express muscarinic M1, M4, M2, and M3 receptor subtypes respectively [3]. The pharmacological specificity of radioligand binding to placenta or reference tissues was evaluated by incubating sections with the radioligand (see below) in the presence of increasing concentrations of

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compounds selective for the muscarinic cholinergic receptor subtypes. In these experiments, [3H]NMS was used at a concentration of 0.25 nM in human placenta, rat heart and sub maxillary gland and of 0.5 nM in rat frontal cortex and neostriatum. Dissociation constant (Kd) and maximum density of binding sites (Bmax) were calculated by linear regression analysis of Scatchard plot of saturation isotherms. The competitor dissociation constant (Ki) was determined according to the method of Cheng and Prusoff [5]. Comparative analysis of saturation and displacement curves was done with the RADLIGAND analysis of multiple data files [15]. [3H]NMS methyl chloride (specific activity, 85 Ci/ mmol) was purchased from Amersham Radiochemical Centre (Buckinghamshire, UK). Other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA) or Merck (Darmstadt, Germany). Muscarinic receptor antagonists, pirenzepine, 4-diphenylacetoxy-N-methyl piperidine methiodide (4-DAMP) and (±)-p-fluoro-hexahydro-sila-difenidol hydrochloride (p-F-HHSiD, were obtained from Research Biochemicals (Natick, MA, USA). AF-DX-116 [11-(2-[(diethyl amino)methyl]-1piperidinylacetyl)-5,11-dihydro-6h-pyrido-(2.3-b)-(1.4)benzo-diazepine-6-one)] was synthesised by the Department of Chemistry of Camerino University. Methoctramine was a gift of Professor C. Melchiorre (University of Bologna, Italy). [3H]NMS was specifically bound to sections of human placenta. The binding was time-, temperature- (data not shown) and concentration-dependent (Fig. 1A). No significant differences were found in Kd and Bmax values between marginal and central portions of placenta. The Kd value was 0.10 ± 0.03 nM in the central placenta and 0.13 ± 0.04 nM in the marginal placenta. The Bmax value was 10.82 ± 0.09 fmol/mg of tissue in the central placenta and 8.2 ± 0.16 fmol/mg of tissue in the marginal placenta. Scatchard analysis of [3H]NMS binding to human placenta revealed the labelling of a single class of high affinity sites, in the central and in the marginal placenta (Fig. 1B). Data on the pharmacological profile of [3H]NMS binding to placental muscarinic cholinergic receptors are summarized in Table 1. As shown, the non-selective muscarinic cholinergic compounds tested [atropine, (−)QNB and carbachol] displayed Ki values (Table 1) consistent with the labelling of muscarinic cholinergic receptors [3]. Analysis of Ki values obtained using compounds selective for the different subtypes of muscarinic cholinergic receptors, revealed that the most powerful displacer of [3H]NMS was the M4 receptor antagonist tropicamide (Ki = 35.4 ± 2.1 nM) followed in descending order by the M1 receptor antagonist pirenzepine (Ki = 153.2 ± 9.3 nM), by compounds active on the M3 muscarinic cholinergic receptor subtype (p-F-HHSiD and 4-DAMP-methiodide, Ki = 534.5 ± 21.2 nM and 672.3 ± 33.3 nM respectively) and on the M2 (methoctramine and AF-DX

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Fig. 1. (A) Saturation curve of [3H]NMS binding to the central part of human placenta. Sections were incubated with increasing concentrations of the radioligand alone (total binding, X) or plus 1 mM atropine to define non-specific binding (K). Specific binding (♦) was obtained by subtracting non-specific from total binding. Points are the mean ± SEM of triplicate determinations. (B) Scatchard analysis of specific [3H]NMS binding to sections of the central (B) and marginal (X) part of human placenta. The Bmax value was 10.82 ± 0.09 fmol/mg of tissue in the central part and 8.2 0 ± 0.16 fmol/mg of tissue in the marginal part of placenta. Points are the mean of triplicate determinations.

116) receptor subtypes (Table 1). Two compounds active on nicotinic cholinergic receptors (nicotine and tubocurarine) tested were ineffective as displacers of [3H]NMS binding (data not shown). [3H]NMS displacement curves by test compounds active on the four subtypes of muscarinic cholinergic receptors (pirenzepine, methoctramine, p-F-HHSiD and tropicamide) in sections of rat frontal cortex (source of M1 receptor [3]), heart (source of M2 receptor [3]), sub maxillary gland (source of M3 receptor [3]) and neostriatum (source of M4 receptor [12,13]), and in sections of the central portion of human placenta are shown in Fig. 2. The muscarinic M4 receptor antagonist tropicamide exhibited the highest competitive activity on [3H]NMS binding in sections of rat neostriatum (Ki = 14.13 ± 0.9 nM), as well as in sections of human placenta (Fig. 2D). The muscarinic M1 receptor antagonist pirenzepine was the most powerful competitor of [3H]NMS in sections of rat frontal cortex (Ki = 81.45 ± 6.8 nM), but displayed an intermediate activity, similar to that shown for neostriatum

(Ki = 179.3 ± 16.3 nM), in sections of human placenta (Fig. 2A). Analysis of [3H]NMS displacement curves by the M3 receptor antagonist p-F-HHSiD in rat sub maxillary gland (Ki = 21.5 ± 1.7 nM) (Fig. 2C) and by the M2 receptor antagonist methoctramine in rat heart (Ki = 24 ± 2.1 nM) (Fig. 2B) revealed that the compounds were the most powerful competitors of the radioligand in sub maxillary gland and heart, respectively. However, they displayed only a moderate and weak activity respectively in sections of placenta (Fig. 2B–C). In view of the close Ki values obtained with test compounds in organs source of different muscarinic cholinergic receptor subtypes and in human placenta, our data indicate that placenta expresses the M1 –M4 muscarinic cholinergic receptor subtypes in the following order of density M4 ≥ M1 . M3 . M2. Molecular biology techniques applied to muscarinic cholinergic receptor research have shown that muscarinic cholinergic receptors belong to five subtypes (m1, m2, m3, m4 and m5), cloned from pig, rat and human brains and various cell lines [2,3,9,11–13,16,18,25]. Classic pharmacological (primarily radioligand binding) techniques allow to characterise four subtypes of muscarinic cholinergic receptors (M1, M2, M3 and M4), whereas no selective compounds for the m5 receptor subtype were identified so far [3]. In view of this, studies using conventional radioligand binding techniques as the present one, should be limited to investigate the M1 –M4 receptor subtypes [25]. Placenta is a tissue devoid of innervation [23]. It could therefore represent a model for analysing the cholinergic system without the interference of neuronal uptake mechanism. From a functional point of view, placental cholinergic system is probably linked to prostaglandin generation at the parturition time. Inhibition of this system is Table 1 Pharmacological specificity of [3H]NMS binding to sections of the central part of human placenta Compound

Receptor specificity [3]

Ki (nM)

Atropine

non-selective muscarinic antagonist non-selective muscarinic antagonist non-selective muscarinic agonist m1>m2, m3, m4, m5 antagonist m2>m4>m3>m1>m5 antagonist m2>m1>m4,m5>m3 antagonist m3>m1, m4, m5>m2 antagonist m3 antagonist m4 antagonist

0.13 ± 0.01

(−)-QNB Carbachol Pirenzepine AF-DX 116 Methoctramine p-F-HHSiD 4-DAMP methiodide Tropicamide

1.95 ± 0.08 2032 ± 205 153.2 ± 9.3 793.1 ± 98.5 711 ± 79 672.3 ± 33.3 534.5 ± 21.2 35.4 ± 2.1

Values represent the competitor dissociation constant (Ki) calculated according to the method of Cheng and Prusoff [5]. Values are the mean ± SEM of data obtained from 5–8 triplicate experiments.

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Fig. 2. Influence of increasing concentrations of pirenzepine (PZP) (A), methoctramine (MET) (B), p-F-HHSiD (C) and tropicamide (TRP) (D) on specific [3H]NMS binding to sections of rat frontal cortex (source of the muscarinic M1 receptor subtype [3,12], inset A), rat heart (source of the muscarinic M2 receptor subtype [3,12], inset B), rat sub maxillary gland (source of the muscarinic M3 receptor subtype [3,12], inset C), rat neostriatum (source of the muscarinic M4 receptor subtype [3,12], inset D) and of human placenta (inset E). Binding experiments were performed as described in the text. Points are the mean ± SEM of triplicate determinations.

associated with reduced output of prostaglandins from placenta [10]. It has been also suggested that acetylcholine is involved in the regulation of placental amino-acid transport [6], whereas no conclusive evidence has been provided as whether acetylcholine has a role in the control of placental blood flow [1,8]. The large availability of placentas makes this material useful for analysing the cholinergic system directly by using human material and with minimal ethical implications. As mentioned in the introduction, although the existence of a cholinergic system in placenta has been documented long time ago [4], only a few and contradictory reports were published concerning the cholinergic receptors expressed by placental tissue [7,26]. In the present study, we have seen that human placenta expresses muscarinic cholinergic receptors. The inadequate procedures for separating radioligand bound from free and the use of membranes of placenta [7,26] instead of sections, which represent a more suitable placental preparation for radioligand binding studies [24], are the most probable reasons of inconsistency between the above studies and the present one. The density of placental muscarinic cholinergic receptors is rather low (10.82 ± 0.09 fmol/mg of tissue in the central part of placenta). However, they display a high affinity (Kd = 0.1 ± 0.03 nM in the central part of placenta), which is in the range of Kd values found in different

brain areas (0.05–0.12 nM) by several studies [25]. Moreover, human placenta apparently expresses the four muscarinic receptor subtypes assayable with conventional radioligand binding techniques. Hence, placenta can be considered as the first human tissue which can be used for characterizing subtypes of human muscarinic cholinergic receptors or compounds active on them. The placental cholinergic system probably plays a regulatory function in the transport of nutritive substances through the materno-foetal barrier [14]. The characterization of muscarinic cholinergic receptors in human placenta may contribute to evaluate the possible functional role of the muscarinic cholinergic system in placental tissue as well as in other organs not directly supplied by a cholinergic innervation. The present study was supported in part by a grant of the Italian National Research Council (C.N.R., Rome) [1] Brennecke, S.P., Chen, S., King, R.G. and Boura, A.L.A., Human placental acetylcholine content and release at parturition, Clin. Exp. Pharmacol. Physiol., 15 (1988) 715–725. [2] Buckley, N.J., Bonner, T.I. and Buckley, C.M., Antagonist binding properties of five cloned muscarinic receptors expressed in CHOK1 cells, Mol. Pharmacol., 35 (1989) 469–476. [3] Caulfield, M.P., Muscarinic receptors: characterization, coupling and function, Pharmacol. Ther., 58 (1993) 319–379. [4] Chang, H.C. and Gaddum, J.H., Cholinesterases in tissue extracts, J. Physiol. (London), 79 (1933) 255–285.

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