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Muscarinic M1 and M3 receptors are present and increase intracellular calcium in adult rat anterior pituitary gland
Brain Research Bulletin, Vol. 48, No. 4, pp. 449 – 456, 1999 Copyright © 1999 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/99/$–see front matter
PII S0361-9230(98)00169-5
Muscarinic M1 and M3 receptors are present and increase intracellular calcium in adult rat anterior pituitary gland Ildiko´ Pinte´r, Georg Moszkovszkin, Zsolt Ne´methy and Ga´bor B. Makara* Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary [Received 15 June 1998; Revised 1 December 1998; Accepted 1 December 1998] ABSTRACT: Physiological and biochemical evidence indicates the existence of functional muscarinic cholinergic receptors in the anterior pituitary. The selectivity of these receptors has been characterised by studying the binding of [3H]quinuclidinyl benzilate ([3H]QNB) and [3H]diphenyl-acetoxy-N-methyl-piperidine ([3H]4-DAMP) in membrane preparation of male rat anterior pituitary at 25°C. Competition experiments with receptor selective muscarinic antagonists were used to characterise specific selective muscarinic receptor binding. Both [3H]QNB and [3H]4DAMP bound to anterior pituitary membranes at low concentrations, binding was saturable and was potently displaced by 4-DAMP (M1, M3 subtypes selective antagonist) > atropine (general) > pirenzepine (M1). Methoctramine (M2) didn’t antagonise the [3H]QNB binding efficiently. Acetylcholine and carbachol increased the intracellular Ca21 level in 62% and 65% of cultured rat anterior pituitary cells in a dose-dependent manner, and this effect was prevented by pirenzepine. Based on these results we suggest that both M1 and M3 muscarinic receptors are present and active in the majority of cells in the rat anterior pituitary gland, but their physiological role in the adult rat remains to be examined. © 1999 Elsevier Science Inc.
potentiated the stimulatory effect of thyreotropin releasing hormone (TRH) on prolactin secretion [41]. Cholinergic agonists (carbachol and ACh) as well as the muscarinic antagonist atropine can stimulate GH secretion from the rat anterior pituitary in vitro under various conditions. These studies suggest that a complicated system involving at least 2-second messenger systems transmit the effect of muscarinic signals in the anterior pituitary gland. This suggestion is supported by functional studies [1,14,22,24,35,37, 47]. Up to now five muscarinic receptor subtypes have been identified in the rat based on their molecular structure, but only four of them have been pharmacologically characterised [11,15,20]. The discovery of subtype selective muscarinic antagonists makes it possible to characterise muscarinic receptor subtypes by pharmacological tools [27,28]. It is not known yet, which muscarinic subtypes are responsible for the effect of ACh on the regulation of pituitary hormone secretion. Based on the controversial results about the effect of ACh on hormone secretion, the existence of different receptor subtypes in the rat anterior pituitary seems likely. In this study we attempted to characterise the selectivity of muscarinic ACh receptors in rat anterior pituitary gland comparing the displacement of [3H]QNB binding by receptor selective muscarinic antagonists with the displacement of [3H]diphenyl-acetoxy-N-methyl piperidine [3H]4-DAMP) (M1, M3). We also examined the effect of ACh and cholinergic agonists and antagonists on intracellular Ca21 in an attempt to learn about the physiological role of the receptor subtypes.
KEY WORDS: Acetylcholine, Carbachol, Pirenzepine, QNB, 4-DAMP, Intracellular calcium, Fura-2, Pituitary.
INTRODUCTION Physiological and biochemical evidences indicate the existence of functional muscarinic cholinergic receptors in the anterior pituitary. Muscarinic receptors have been identified by [3H]quinuclidinyl benzilate ([3H]QNB) and [3H]N-methylscopolamine ([3H]NMS) binding on anterior pituitary membrane preparation, as well as on intact pituitary cells [1,2,6,19,29,32]. Muscarinic mechanisms seem to play a role in the control of pituitary hormone secretion, but the results are controversial [36,39]. It has been described that acetylcholine (ACh) can act directly in the pituitary as either a stimulatory or an inhibitory signal on regulation of the same hormone [growth hormone (GH) and prolactin] secretion [7,9,41,43,47]. In the rat, ACh inhibits prolactin secretion in pituitary lactotroph cells and this inhibition can be blocked by the general muscarinic antagonist atropine [4,10]. In contrast, ACh stimulates prolactin release in the bovine anterior pituitary [33]. Furthermore, ACh
MATERIALS AND METHODS Membrane Preparation Male Wistar rats weighing 250 –300 g were used in all experiments. Anterior pituitary glands were pooled and homogenised for 10 s in ice-cold 50 mM TRIS-HCl buffer (pH 7.4 at 4°C) containing 1 mM ethylenediaminetetraacetic acid (EDTA) using an UltraTurrax tissue homogenizer at maximal setting. The homogenate was centrifuged twice at 50,000 g for 15 min at 4°C. The final pellet was used immediately for ligand binding assay or rapidly frozen at 222°C and used the next day.
* Address for correspondence: G. B. Makara, Institute of Experimental Medicine, Hungarian Academy of Sciences, H-1450 Budapest, P. O. Box 67, Hungary. Fax: 1361-210-0811; E-mail: [email protected]
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450 Ligand Binding Assays Competitive displacement of [3H]4-DAMP (with nonlabeled 4-DAMP, atropine, and pirenzepine) and [3H]QNB (with nonlabeled 4-DAMP, atropine, pirenzepine, and methoctramine) have been performed according to Michel and Whiting [28], and Yamamura and Snyder [46], respectively. In all experiments the radioligands, competing drugs, and membranes were incubated in a final volume of 0.5 ml assay buffer (50 mM Tris-HCl-EDTA, pH 7.4) for 45 min at 25°C. In competition studies the final assay concentration of [3H]4-DAMP and [3H]QNB were 0.3 and 0.01 nM, respectively. For saturation studies the concentration of [3H]QNB was varied between 0.001 and 10 nM. Nonspecific binding was measured in the presence of 1 mM atropine. Protein was determined by the method of Lowry et al. [25]. In each assay 2–300 mg protein/tube was applied. Incubations were terminated by vacuum filtration through 0.1% polyethyleneimine pretreated glass fibre filters (GF/C, Whatman) using a multiunit filtration device. The filters were washed three times with 5 ml ice-cold 50 mM TRIS-HCl buffer solution and the radioactivity retained on the filters was determined by liquid scintillation spectrometry. Competition binding data were analysed using an iterative curve fitting method (Prism, Graphpad, San Diego, CA, USA). IC50 were converted to Ki using the Cheng and Prusoff approximation [13]. Monolayer Anterior Pituitary Cell Cultures Pituitaries of male Wistar rats (150 –250 g) of the CharlesRiver strain were used. Pituitaries from two rats were removed under aseptic conditions and collected in Medium 199. Neurointermediate lobe was removed under a stereomicroscope. Anterior pituitaries were washed in Medium 199 containing 10 mg/ml penicillin-streptomycin, and then in Ca21 2 Mg21-free Tyrode’s solution, and incubated for 30 min in Ca21 2 Mg21-free Hank’s solution containing 0.25% trypsin at 37°C. After the incubation, the anterior lobes were washed with Medium 199 containing 20% foetal calf serum and centrifuged with 800 g for 10 min and the sediment was resuspended in 1 ml Medium 199 containing 20% foetal calf serum and then anterior pituitaries were dispersed to individual cells by pipetting. Each 500 ml of the cell suspension (106 cell/ml) was plated on a sterile, 22 mm diameter coverslip and cultured for 2 days at 37°C in a 5% CO2 thermostat. Fluorescence Measurement of Intracellular Ca21 Concentration Cultured anterior pituitary cells on coverslips were washed three times with HEPES buffered salt solution (HBSS); composition: NaCl, 136.7 mM; KCl, 5 mM; CaCl2, 2 mM; MgCl2, 2 mM; Na2HPO4, 0.34 mM; KH2PO, 0.44 mM; NaHCO3, 4 mM; glucose, 5.55 mM; HEPES, 20 mM; pH 7.4) then loaded with 5 mM Fura-2-AM in HBSS [cell permeant acetoxymethyl ester diluted from a 1 mM stock solution in dimethyl sulfoxide (DMSO)] for 45 min at 37°C in 95% air and 5% CO2 atmosphere. At the end of the incubation, cells were washed three times in HBSS, and [Ca21]i was then measured microspectrofluorimetrically in a 37°C thermostated chamber; agonists were added usually for 10 s via a pressure ejection system, while antagonists were added directly in the medium and were also mixed with the agonist in the pipette. Microspectrofluorimetrical measurements were carried out with an inverted Nikon TDM Diaphot epifluorescence microscope equipped with a Fluor 403 objective (NA: 0.85; Nikon, Japan). Fluorescence intensity was measured at 520 nm by a photomultiplier (D104 Microscope Photometer, PTI, South Brunswick, NJ, USA) in response to excitation at 340 and 380 nm, [Ca21]i was calculated from the formula established by Grynkiewicz et al. [17]:
FIG. 1. The specific binding of [3H]QNB to rat anterior pituitary membranes. Increasing concentrations of [3H]QNB were incubated at 25°C for 60 min with membranes (0.3 mg of protein) in the presence (nonspecific binding) or absence (total binding) of 1 mM atropine. Specific binding was determined by substracting the nonspecific from the total binding. Data are expressed as the average of the triplicate determinations in a representative experiment. Replicate experiments (three times) gave similar results. (Inset) Scatchard replot of the binding data. B, femtomoles of the [3H]QNB bound/mg protein; F, free [3H]QNB (picomolar) (total added-specifically bound).
[Ca21]i 5 Kd*(R 2 Rmin)/(Rmax 2 R)*(Sf2/Sb2), where R is the ratio of F1/F2 fluorescence intensities at l1 5 340 nm and l2 5 380 nm; Rmin and Rmax represents the measured minimum and maximum ratios at zero and at saturating concentration of Ca21, respectively. The Sf2 and Sb2 are the fluorescence values for the free and bound forms of the dye. The Kd is the dissociation constant of the Ca21-Fura-2 complex. Materials [3H]4-DAMP (specific activity: 80.5 Ci/mmol) and [3H]QNB (specific activity 49 Ci/mmol) were obtained from New England Nuclear. Nonlabeled QNB, pirenzepine, tropicamide, methoctramine, and 4-DAMP were purchased from RB International (Natick, MA, USA). Atropine sulphate, Medium 199, trypsin, penicillin, and streptomycin were obtained from Sigma Chemical Company (St. Louis, MO, USA). Fura-2-AM (cell permeant acetoxymethyl ester) was purchased from Molecular Probes (Eugene, OR, USA). Foetal calf serum was obtained from Gibco (Glasgow, UK). RESULTS Saturation Studies The binding of [3H]QNB to membrane preparation of male rat anterior pituitary was saturable at 25°C. The nonspecific binding was less than 10% of the total binding. The dissociation constant (Kd) was 20.5 6 1.9 pM, Bmax was 35 6 5.4 fmol/mg protein. [3H]QNB labelled an inhomogeneous population of muscarinic receptors, as the Scatchard plot was not linear (Fig. 1). The binding of [3H]4-DAMP in membrane preparation of male rat anterior pituitary was also saturable at 25°C at quite low concentration of the radioligand (Fig. 2.). The nonspecific binding was 30%– 40% of the total binding. The Kd of this saturation plot
MUSCARINIC RECEPTORS IN THE PITUITARY
FIG. 2. The specific binding of [3H]4-DAMP to rat anterior pituitary membranes. [3H]4-DAMP was incubated at 25°C for 60 min with membranes (0.3 mg of protein) in the presence (nonspecific binding) or absence (total binding) of 1 mM atropine. Specific binding was determined by substracting the nonspecific from the total binding. Data are expressed as the average of the triplicate determinations in a representative experiment. Replicate experiments (three times) gave similar results. (Inset) Scatchard replot of the binding data. B, femtomoles of the [3H]4-DAMP bound/mg protein; F, free [3H]4-DAMP (nanomolar) (total added-specifically bound).
was higher than that of [3H]QNB (0.3 6 0.02 nM), but the Bmax (39.8 6 4.2 fmol/mg protein) was quite similar to the Bmax of the [3H]QNB saturation plot, suggesting, that [3H]4-DAMP and [3H]QNB predominantly bind to the same binding sites. Competition Studies Various muscarinic antagonists were used for displacing specific [3H]QNB binding to membrane preparation of anterior pituitary gland. 4-DAMP (Ki: 1.7 pM according to a one site model) and atropine (Ki: 7.9 pM) seemed to be the most potent inhibitors of [3H]QNB binding. Pirenzepine (Ki: 0.36 nM) was 45.5-fold less potent than atropine and 210 less potent than 4-DAMP. Methoctramine (Ki: 218 nM) was more than 600-fold less potent competitor of [3H]QNB binding than pirenzepine. The 4-DAMP displaced the [3H]QNB binding apparently labelling an inhomogeneous receptor population, with a low Hill coefficient. The two binding sites model seemed to fit the [3H]QNB displacement by DAMP ( p , 0.001). In case of methoctramine, pirenzepine, and atropine the one-site model was better than a two-site model (Fig. 3; Table 1). The results of competition studies of [3H]4-DAMP binding with muscarinic antagonists were very similar to those of QNB binding (Fig. 4). According to a one-site model the Ki of displacement of [3H]4-DAMP binding by DAMP, pirenzepine, and atropine were 0.4 pM, 4.5 nM, and 9 pM, respectively. 4-DAMP binding displayed low Hill coefficients and data were better fit to two populations of either [3H]QNB or [3H]4-DAMP binding sites. In contrast atropine, pirenzepine, and methoctramine displayed a Hill coefficient not significantly different from one (Table 1). Effect of Acetylcholine and Cholinergic Agonists on [Ca21]i The resting [Ca21]i of anterior pituitary cells was 119.8 6 14.6 nM (mean 6 SEM for 194 cells). A 10-s pulse administration of 10 mM ACh increased the [Ca21]i in 62.2% of the 127 pituitary
451
FIG. 3. Inhibition of [3H]QNB binding to rat anterior pituitary membranes by muscarinic antagonists. Membranes (0.3 mg) were incubated with 0.01 nM [3H]QNB and various concentration of antagonists in 0.5 ml Tris-HCl for 45 min at 25°C. Each point represents the mean of specific [3H]QNB binding of three independent experiments carried out in triplicate or duplicate. Inhibition curves depicted the best fit determined from nonlinear regression analyses. Values are mean 6SEM of three experiments.
cells (224.7 6 3.68% of the resting level). After administration of the drug, a rapid increase of [Ca21]i was observed and this effect proved to be dose dependent (Fig. 5). 100 nM ACh had no significant effect on [Ca21]i, 1 mM caused smaller [Ca21]i increase than 10 mM, and 100 mM ACh didn’t cause further increase. The magnitude of [Ca21]i increase or the percentage of the cells responsive to ACh was similar in the presence (228 6 13% of the resting level in 25 from 40 cells) or absence (225 6 4% in 79 from 127 cells) of 10 mM of the cholinesterase inhibitor physostigmine. Carbachol (1 mM, 10 mM) also significantly increased cytoplasmic Ca21 concentration ([Ca21]i) in 65.0% of the anterior pituitary cells. In 10% of the cells after administration of carbachol (1 mM, 10 mM) a decrease of the [Ca21]i was observed (69.4 6 2.8% of the resting level). The effect of ACh was mediated through muscarinic receptors (Fig. 6A–C; Table 2). Administration of 10 mM atropine prevented the effect of ACh (104.35 6 1.64 in 37 from 37 cells) on [Ca21]i. Administration of 10 mM muscarine or 10 mM McN-A-343 caused the same effect as 10 mM ACh (205.85 6 21.5% in 24 from 37 cells, 221.5 6 4.23% in 20 from 30 cells, respectively). Nicotine had no significant effect on [Ca21]i (114.3 6 3.3% in 25 from 40 cells). After 10 mM nicotine, a small, delayed tendency to increase in [Ca21]i appeared in 20% of the cells, but this effect didn’t exceed the standard deviation range of the baseline in any case. In order to determine the receptor subtypes responsible for this effect we tested the effect of selective muscarinic antagonists on ACh induced [Ca21]i increase (Fig. 7A–D; Table 2). In the presence of pirenzepine (M1) (1 mM or 10 mM) the stimulatory effect of the same dose of ACh was blocked (111.6 6 15.6% in 19 from 22 cells or 104.2 6 10.8% in 33 from 39 cells, respectively). In the presence of pirenzepine 15% of the cells responded to ACh (three from 22 cells or six from 39 cells, respectively, including the subpopulation unresponsive to ACh). The increase of [Ca21]i in the presence of 10 mM of pirenzepine was 152.4 6 9.9% of the resting level (six from 39 cells). In the presence of 4-DAMP (M3, M1) no [Ca21]i response to ACh was observed (111.8% 6 8.23 in
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452 TABLE 1 NONLINEAR REGRESSION ANALYSIS OF A TWO SITE MODEL INHIBITION OF [3H]QNB AND [3H]4-DAMP BINDING TO RAT ANTERIOR PITUITARY MEMBRANES Ligand
[3H]QNB [3H]QNB [3H]QNB [3H]QNB [3H]4-DAMP [3H]4-DAMP [3H]4-DAMP
atropine 4-DAMP pirenzepine methoctramine atropine 4-DAMP pirenzepine
KiH
KiL
nH
p
7.90E-12 2.370E-12 — — 9.47E-12 3.57E-13 —
— 1.96E-09 3.57E-09 2.18E-06 — 4.12E-09 4.50E-09
0.78 0.18 0.81 1.03 0.95 0.32 0.92
0.650 0.008 0.356 0.580 0.141 0.001 0.471
KiH and KiL represent the inhibition constants of each drug at high and low affinity sites, respectively. A degree of improvement of data analysis compared to the one site model was statistically evaluated by a partial F test. p means the probability of the difference between the two equation for the one and two site models.
41 from 41 cells). Methoctramine (M2) and tropicamide (M4) had no effect on ACh induced [Ca21]i increase on rat anterior pituitary cells. The ACh induced increase in [Ca21]i was similar in the presence and in the absence of the extracellular [Ca21]i, suggesting that the stimulatory effect of ACh may involve mobilisation of Ca21 from intracellular stores (Table 2). DISCUSSION The results of the present study suggest that M1 muscarinic ACh receptors are present on adult rat anterior pituitary cells and they are linked to mechanisms capable of transiently increasing cytosolic calcium levels. The presence of another muscarinic ACh receptor population (possibly M3) also seems to be likely. Muscarinic ACh receptors have been previously described on adult rat anterior pituitary homogenates [2,19,32], in pituitary tumoural cell lines [1,18] as well as on intact pituitary cells of adult rat [29], but little is known about the subtypes and/or the homogeneity of these receptors. In this study we provide the first
FIG. 4. Inhibition of [3H]4-DAMP binding to rat anterior pituitary membranes by muscarinic antagonists. Membranes (0.3 mg) were incubated with 0.01 nM [3H]4-DAMP and various concentrations of antagonists in 0.5 ml Tris-HCl for 45 min at 25°C. Each point represents the means of a specific [3H]4-DAMP binding of three independent experiments carried out in triplicate or duplicate. Inhibition curves depict the best fit determined from nonlinear regression analyses.
demonstration of the presence of M1 muscarinic receptor subtype in adult rat anterior pituitary gland. In our experiments, both [3H]QNB and [3H]4-DAMP binding to rat anterior pituitary membranes were saturable at relatively low concentration. The Bmax values from the [3H]QNB and [3H]4DAMP binding are similar, and this suggests that [3H]QNB and [3H]4-DAMP labelled the same receptor populations. The Scatchard plot of the [3H]4-DAMP binding suggested a homogenous population, but due to its lower affinity to anterior pituitary membranes and the higher nonspecific binding we could not show specific binding with acceptable error at the low concentration, which is necessary to show a probable higher affinity site. Pirenzepine can displace either [3H]QNB or [3H]4-DAMP binding with relatively high affinity that corresponds to its affinity to the M1 muscarinic receptor subtype [27], suggesting the presence of M1 muscarinic subtype in rat anterior pituitary gland. Methoctramine didn’t displace the [3H]QNB binding efficiently, therefore, it seems likely that the M2 receptor subtype is not expressed to an appreciable extent in the rat pituitary gland [28]. This finding partially conflicts with the results suggesting that carbachol inhibits cyclic adenosine monophosphate (cAMP) synthesis in cultured adult rat pituitary cells and tumoral cell lines [1,18,30,42,43,47] supposedly via M2 receptors, because we
FIG. 5. The dose related effect of 1 mM (F, n 5 62) and 10 mM ACh (E, n 5 79) on intracellular Ca21 concentration in cultured anterior pituitary cells. The dotted lines represent the duration of ACh administration.
MUSCARINIC RECEPTORS IN THE PITUITARY
453 TABLE 2 THE EFFECT OF CHOLINERGIC AGENTS ON [CA21]I ON CULTURED RAT ANTERIOR PITUITARY CELLS
Stimuli
Agonists ACh ACh 1 EDTA Carbachol Nicotine Muscarine McN-A-343 (M1) Antagonists ACh 1 atropine ACh 1 pirenzepine (M1) ACh 1 4-DAMP (M3, M1) ACh 1 methoctramine (M2) ACh 1 tropicamide (M4)
[Ca21]i after Drug Administration (% of the Resting Level)
Number of Cases
224.7 6 3.68 218.69 6 6.25 221.5 6 4.23 114.3 6 3.3 205.85 6 21.5 221.5 6 4.23
79 28 44 25 24 20
104.35 6 1.64 104.2 6 10.8 111.8 6 8.23 219.32 6 21.4 221.32 6 17.63
37 33* 41 38 42
Each drug was used in 10 mM concentration. Values are mean 6 SEM of three experiments. In the presence of pirenzepine the stimulatory effect of the same dose of ACh was blocked in 85% of the cells (in 33 from 39 cells).
FIG. 6. The effect of cholinergic agonists (10 mM) on intracellular Ca21 concentration in cultured anterior pituitary cells. Representative single cell responses. The dotted lines represent the duration of the drug administration. (A) Carbachol; (B) muscarine; (C) nicotine.
couldn’t demonstrate M2 binding sites in appreciable amount in pituitary gland. However, the number of the M2 receptors may be lower than the detection limit of our binding method. These results as well as the saturation binding studies suggest that in the adult rat pituitary most of the muscarinic binding sites are of the M1 receptor subtype. However, the M3 muscarinic subtype also seems to be present in the pituitary gland because 4-DAMP was a more
potent displacer than the selective M1 muscarinic receptor specific antagonist pirenzepine, suggesting that a high affinity population of muscarinic binding sites in male rat anterior pituitary gland may be M3 subtype. This high affinity is unusual because in most tissues 4-DAMP has the same affinity to M1 and M3 muscarinic receptors, but recent results [23,26,38,40] show the existence of a M3 muscarinic receptor subtype with high affinity to 4-DAMP. The results of Mahesh et al. [26] are very similar to our results considering either the Kd of [3H]QNB binding or the Ki of displacement of [3H]QNB by 4-DAMP. The presence of muscarinic M1 receptors is also supported by studies on intracellular calcium concentration. Looking for physiological consequences of muscarinic receptor activation we found that ACh increased the [Ca21]i in 62.2% of the pituitary cells. Receptor mediated stimuli lead to rise of [Ca21]i via two mechanisms: mobilisation of Ca21 from intracellular stores, and/or Ca21 influx induced by receptor mediated changes of Ca21 channel conductivity. M1 and M3 muscarinic receptors are known to act mainly through phosphatidyl inositol hydrolysis, but muscarinic agents also can stimulate the Ca21 influx [12,33,44]. In our experiments the ACh induced increase in [Ca21]i was similar in the presence and in the absence of the extracellular [Ca21]i, suggesting that the stimulatory effect of ACh may involve mobilisation of Ca21 from intracellular stores. Our data agree well with the findings of other investigators, who have found that cholinergic agonists can stimulate inositol trisphosphate formation in bovine and rat anterior pituitary [1,7,35,44,47]. Canonico et al. [7] found that ACh in the presence of physostigmine stimulated inositol trisphosphate formation in a concentration dependent fashion. Carbachol, but not oxotremorine produced similar effects. The cholinergic agonists induced inositol phosphates production was very rapid, and could be inhibited by atropine [7]. Shrey and Read [35] observed similar effect in rat anterior hemipituitaries. Nicotine had no significant effect on the [Ca21]i in adult rat pituitary cell culture, but muscarine caused an effect similar to that of ACh. Moreover, the general muscarinic antagonist atropine prevented the effect of ACh on [Ca21]i. All these findings suggest that ACh acts through muscarinic receptors only.
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FIG. 7. The effect of ACh (10 mM) on intracellular Ca21 concentration in the presence of cholinergic antagonists (10 mM) in cultured anterior pituitary cells. Representative single cell responses. The dotted lines represent the duration of the drug administration. (A) Pirenzepine; (B) methoctramine; (C) 4-DAMP; (D) tropicamide.
We found that the intracellular Ca21 increasing effect of ACh can be prevented by the M1 receptor selective muscarinic receptor antagonist pirenzepine. This result supports the suggestion from the receptor binding studies that the M1 receptor subtype is present in pituitary. This was also supported by the fact that an M1 muscarinic agonist McN-A-343 caused significant [Ca21]i elevation. Ten percent of the ACh sensitive cells were insensitive to pirenzepine, which suggests that another receptor population, possibly M3 also takes part in the cholinergic stimulation of intracellular Ca21. These results agree well with the receptor binding results and suggest that more than one type of ACh receptor exists in rat anterior pituitary gland and one of them is M1 receptor. We also found that carbachol decreased the [Ca21]i in 10% of
the cells in adult rat pituitary cell culture, suggesting that in a minority of cells a mechanism different from the previously mentioned may be operating. These findings agree well with the study of Schegel et al [34], who find that muscarinic receptors (presumably M2 or M4, which are coupled to cAMP) can inhibit the Ca21 influx through a cAMP dependent mechanism [45]. Whether more than one muscarinic binding site is present in the adult rat pituitary gland is debated. Schaeffer and Hsueh [32] found that a homogeneous muscarinic receptor population bound [3H]QNB with high affinity. In contrast, Avissar et al. [2] and Henish et al. [19] found an inhomogeneous receptor population with [3H]QNB equilibrium binding studies as well as with [3H]methylscopolamine and [3H]NMS. Muhkerjee et al. [29] also found two [3H]QNB sites with distinct affinity in intact rat pituitary cells. The contradiction between these results can be resolved considering that different buffers were used, and—as it is well established—muscarinic receptors show different affinity and selectivity at high and low ionic strength [5]. It is also likely that Schaeffer and Hsueh [32] could not demonstrate the second binding site because they did not use a concentration range wide enough to cover two very different Kds. Our study supports the presence of at least two distinct populations of muscarinic receptors in the anterior pituitary gland. Since our preliminary studies suggested the existence of two [3H]QNB binding sites with rather different dissociation constants (more than two order of magnitude difference between the two Kd) in rat anterior pituitary, we used a wider concentration range of the radioligand than usual in order to show the presence of the two binding sites. The present results may have physiological importance as all components of an intrinsic cholinergic system (ACh, choline acetyltransferase, acetylcholinesterase, as well as muscarinic receptors coupled to second messenger systems) are present in the pituitary of the adult rat [2,3,9,10,16,32]. In rat anterior pituitary cells ACh inhibits prolactin secretion and this inhibition can be blocked by the muscarinic antagonists atropine [4]. In contrast, ACh stimulated the prolactin release in bovine anterior pituitary [44]. Furthermore, ACh can potentiate the stimulatory effect of TRH on prolactin secretion [41]. According to Carmeliet and Denef [9,10], either muscarinic agonists (in the absence of dexamethasone) or the muscarinic antagonist atropine (in the presence of dexamethasone) may have stimulatory effect on GH secretion. In addition to this, dexamethasone pretreatment either in vivo or in vitro reverses the stimulatory effect of carbachol [8,9]. They presumed that the corticotrop cells might exert a paracrine tonic inhibitory activity on GH and prolactin release by producing ACh, which may be secreted together with adrenocorticotopin (ACTH). This endogenous ACh may act locally as a paracrine factor, and through muscarinic receptor(s) can modulate basal and stimulated GH release [9,10, 36]. It remains to be studied whether the various muscarinic receptor subtypes found in the anterior pituitary are expressed in the same cell types or the subtypes are involved in the regulation of different pituitary cell type. According to in vitro studies muscarinic agonists may have a direct hypophyseal effect on the secretion of GH, [7,9,10,21,33,42,47] prolactin, [4,10,21,30,31,33, 34,42], and ACTH [18]. Presumably, ACh through M3 receptors (which are fewer in number and have higher affinity) modulates its own release together with release of ACTH and the released ACh through M1 receptors can inhibit GH secretion. CONCLUSIONS The findings in the present study suggest that in the adult rat anterior pituitary gland at least two muscarinic receptor popula-
MUSCARINIC RECEPTORS IN THE PITUITARY tions exist and can elevate cytosolic Ca21 level. We suggest that in the adult rat anterior pituitary the main muscarinic receptor subtype may be the M1 subtype, but the presence of M3 muscarinic subtype also seems to be likely.
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22.
23.
REFERENCES 1. Akiyama, K.; Vickroy, T. W.; Watson, M.; Roeske, W. R.; Reisine, T. D.; Smith, T. L.; Yamamura, H. I. Muscarinic cholinergic ligand binding to intact mouse pituitary tumour cells (AtT-20/D16-16) Coupling with two biochemical effectors: Adenylate cyclase and phosphatidylinositol turnover. J. Pharm. Exp. Ther. 236:653– 661; 1986. 2. Avissar, S.; Egozi, Y.; Sokolovsky, M. Biochemical characterization and sexual dismorphism of muscarinic receptors in rat adenohyphophysis. Neuroendocrinology 32:303–309; 1981. 3. Barron, S. E.; Hoover, D. B. Localisation of acetylcholinesterase and cholin-acetyltransferase in the rat anterior pituitary gland. Histochem. J. 15:1087–1098; 1983. 4. Beach, J. E.; Smallridge, R. C.; Chiang, P. C.; Fein, H. G. Reversal inhibition of prolactin secretion in cultured pituitary cells by muscarinic antagonists. J. Pharm. Exp. Ther. 246:548 –552; 1989. 5. Birdsall, N. J.; Burgen, A. S. The effect of ions on the binding of agonists and antagonists to muscarinic receptors. Br. J. Pharmacol. 67:371–377; 1979. 6. Burt, D. R.; Taylor, R. L. Muscarinic receptor binding in sheep anterior pituitary. Neuroendocrinology 30:344 –349; 1980. 7. Canonico, P. L.; Jarvis, W. D.; Sortino, M. A.; Scapagnini, U.; MacLeod, R. M. Cholinergic stimulation of inositol phosphate production in cultured anterior pituitary cells. Neuroendocrinology 46:306 –311; 1987. 8. Carmeliet, P.; Baes, M.; Denef, C. Dexamethasone reverses the GH releasing property of cholinomimetic carbachol. Endocrinology 124: 2625–2633; 1989. 9. Carmeliet, P.; Denef, C. Immunocytochemical and pharmacological evidence for an intrinsic cholinomimetic system modulating prolactin and growth hormone release in rat pituitary. Endocrinology 123:1128 – 1139; 1988. 10. Carmeliet, P.; Denef, C. Synthesis and release of acetylcholine by normal and tumoral pituitary corticotrophs. Endocrinology 124:2218 – 2227; 1989. 11. Caulfield, M. P. Muscarinic receptors— characterisation, coupling and function. Pharmacol. Ther. 58:391–379; 1993. 12. Chen, C.; Vincent, J. D.; Clarke, I. J. Ion channels and the signal transduction pathways in the regulation of growth hormone secretion. Trends End. Met. 5:227–233; 1995. 13. Cheng, C.; Prusoff, W. H. Relationship between the inhibition constant and the concentration of inhibitor which causes 50 percent inhibition in enzymatic reaction. Biochem. Pharmacol. 22:3099 –3108; 1973. 14. Chou, Y. C.; Fong, J. C. Enhanced cAMP responses in GH3 pituitary cells pretreated with muscarinic receptor agonists. Cell. Signal. 6:413– 421; 1994. 15. Do¨rje, F.; Wess, J.; Lambrecht, G.; Tacke, R.; Mutschler, E.; Braun, M. R. Antagonist binding profiles of five cloned muscarinic receptors. J. Pharm. Exp. Ther. 256:727–733 1991. 16. Egozi, Y.; Kloog, Y.; Flemingerand, G. and Sokolovsky, M. Acetylcholine in the rat pituitary: A possible humoral factor. Brain Res. 475:376 –379; 1988. 17. Grynkiewicz, G.; Poenie, M.; Tsien, R. Y. A new generation of Ca21 indicators with greatly improved fluorescence properties. J. Biol. Chem. 260:3440 –3450; 1985. 18. Heisler, S.; Desjardins, D.; Ngujen, M. Muscarinic cholinerg receptors in mouse pituitary tumour cells: Prolonged agonist pretreatment decreases receptor content and increases forskolin and hormone stimulated cAMP synthesis and adrenocorticotropin secretion. J. Pharm. Exp. Ther. 232:232–239; 1985. 19. Henish, Y. J.; Galron, R.; Avissar, S.; Sokolovsky, M. Interaction between antagonist occupied binding sites in rat anterior pituitary. FEBS Lett. 140:173–178; 1982. 20. Hulme, E. C.; Birdsall, N. J. M.; Buckley, N. J. Muscarinic receptor subtypes. Ann. Rev. Pharmacol. Toxicol. 30:633– 673; 1990. 21. Ingram, C. D.; Bicknell, R. J. Synergistic interaction of GHRH and
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other hypohysiothroph factors in bovine anterior pituitary cells. J. Endocrinol. 109:67–74; 1986. Jones, P. S. V. M4 muscarinic receptor subtype activates an inwardly rectifying potassium conductance in AtT-20 cells. Neurosci. Lett. 147:125–130; 1992. Klangkalya, B.; Chan, A. Inhibition of hypothalamic and pituitary muscarinic receptor binding by progesterone. Neuroendocrinology 47: 294 –302; 1988. Kleuss, C.; Hescheler, J.; Ewel, C.; Rosenthal, W.; Schultz, Z. G.; Witting, B. Assignment of G protein subtypes to specific receptor inducing inhibition of Ca11 currents. Nature 353:43– 48; 1991. Lowry, O. H.; Rosenbrough, N. J.; Farr, A. L.; Randall, R. I. Protein measurement with the Folin reagent. J. Biol. Chem. 193:265–275; 1951. Mahesh, V. K.; Nunan, L. M.; Halonen, M.; Yamamura, H. I.; Palme, J. D.; Bloom, J. W. Muscarinic receptor binding to rat respiratory tract. Am. J. Resp. Mol. Biol. 6:279 –286; 1991. Michel, A. D.; Stefanich, E.; Whiting, R. L. Direct labelling of rat M3-muscarinic receptors by [3H]4-DAMP. Eur. J. Pharmacol. 166: 459 – 466; 1989. Michel, A. D.; Whiting, R. L. Methoctramine, a polymethylene tetraamine, differentiates three subtypes of muscarinic receptor in direct binding study. Eur. J. Pharmacol. 145:61– 66; 1988. Mukherjee, A.; Snyder, G.; McCann, S. M. Characterisation of muscarinic cholinergic receptors of intact rat anterior pituitary cells. Life Sci. 27:475– 482; 1980. Onali, P.; Eva, C.; Olianas, M. C.; Schwartz, J. P.; Costa, E. In GH3 pituitary tumour cells acetylcholine and vasoactive intestinal peptide antagonistically modulate adenylate cyclase cAMP content and prolactin secretion. Mol. Pharmacol. 24:184 –194; 1983. Rudnick, M. S.; Danner, P. S. Muscarinic receptor mediated inhibition of prolactin secretion in cultured rat pituitary cells. Biochem. Biophys. Res. Commun. 101:689 – 696; 1981. Schaeffer, J. M.; Hsueh, A. J. W. Acetylcholine receptors in the rat anterior pituitary gland. Endocrinology 106:1377–1381; 1980. Schoffield, J. G.; Khan, A. I.; Wood, A. Acetylcholine stimulated prolactin, growth hormone and Rb efflux in bovine antetrior pituitary cells. J. Endocrinol. 116:393– 401; 1988. Schlegel, W. F.; Wuarin, C.; Zbaren, G. R.; Zahn, D. B. Lowering cytosolic free Ca21 by carbachol, a muscarinic cholinergic agonist in clonal pituitary cells GH3. Endocrinology 117:976 –981; 1985. Schrey, M. P.; Read, A. M. Cholinergic stimulation of phosphoinositide hydrolysis in rat anterior pituitary. Mol. Cell. Endocrinol. 57:107– 113; 1988. Schwartz, J.; Cherny, R. Intercellular communication within the anterior pituitary influencing the secretion of hypophysial hormones. Endocrine Rev. 13:453– 475; 1992. Stojkiowitcz, S. S.; Izumi, S.; Catt, K. J. Participation of voltagesensitive calcium channels in pituitary hormone release. J. Biol. Chem. 263:13054 –13061; 1988. Swillens, S.; Waelbroeck, M.; Champeil, P. Does radiolabelled ligand bind to a homogenous receptor population of non-interacting receptor sites? Trends Pharmacol. Sci. 166:151–155; 1995. Tuomisto, J.; Mannisto, P. The neurotransmitter regulation of anterior pituitary hormones. Pharmacol. Rev. 37:249 –305; 1985. Wang, B.; Luthin, G. R.; Ruggieri, M. R. Muscarinic receptor subtypes mediating urinary bladder contractility and coupling to GTP binding proteins. J. Pharmacol. Exp. Ther. 273:959 –966; 1995. Wang, F. F.; Mo, F. E.; Yen, Y. T.; Fong, J. C. Potentiation of thyrotropin-releasing hormone-stimulated prolactin mRNA levels in GH3 cells by acetylcholine. Mol. Cell. Endocrinol. 82:117–121; 1991. Wojcikiewicz, R. J.; Dobson, P. R.; Brown, B. L. Muscarinic acetylcholine receptor activation causes inhibition of cyclic AMP accumulation, prolactin and growth hormone secretion in GH3 rat anterior pituitary tumour cells. Biochim. Biophys. Acta 805:25–29; 1984. Wojcikiewicz, R. J.; Dobson, P. R.; Irons, L.; Robinson, A., Brown, B. L. The relationship between pertussis toxin-induced ADP-ribosylation of plasma membrane protein and reversal of muscarinic inhibition of prolactin secretion in GH3 cells. Biochem. J. 224:339 –342; 1984. Wood, C. A.; Schoffield, J. G. The effect of acetylcholine on inositol
456 lipid metabolism and intracellular Ca21 concentration in bovine anterior pituitary cells. Biochim. Biophys. Acta 1013:180 –186; 1989. 45. Yagishawa, H.; Simmonds, H. S.; Hawtorne, J. N. The muscarinic receptor of rat pituitary GH3 cells is coupled with adenylate cyclase inhibition, but not with phosphoinositol turnover. Biochem. Pharmacol. 37:2675–2681; 1988.
PINTE´R ET AL. 46. Yamamura, H. I., Snyder, G. Binding of [3H]QNB in rat brain. Proc. Natl. Acad. Sci. 71:1725–1729; 1981. 47. Young, P. W.; Bicknell, R. J.; Schofield, J. G. Acetylcholine stimulates growth hormone secretion, phosphatidyl inositol labelling, Ca21 efflux, and cyclic GMP accumulation in bovine anterior pituitary glands. J. Endocrinol. 80:203–213; 1979.
PII S0361-9230(98)00169-5
Muscarinic M1 and M3 receptors are present and increase intracellular calcium in adult rat anterior pituitary gland Ildiko´ Pinte´r, Georg Moszkovszkin, Zsolt Ne´methy and Ga´bor B. Makara* Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary [Received 15 June 1998; Revised 1 December 1998; Accepted 1 December 1998] ABSTRACT: Physiological and biochemical evidence indicates the existence of functional muscarinic cholinergic receptors in the anterior pituitary. The selectivity of these receptors has been characterised by studying the binding of [3H]quinuclidinyl benzilate ([3H]QNB) and [3H]diphenyl-acetoxy-N-methyl-piperidine ([3H]4-DAMP) in membrane preparation of male rat anterior pituitary at 25°C. Competition experiments with receptor selective muscarinic antagonists were used to characterise specific selective muscarinic receptor binding. Both [3H]QNB and [3H]4DAMP bound to anterior pituitary membranes at low concentrations, binding was saturable and was potently displaced by 4-DAMP (M1, M3 subtypes selective antagonist) > atropine (general) > pirenzepine (M1). Methoctramine (M2) didn’t antagonise the [3H]QNB binding efficiently. Acetylcholine and carbachol increased the intracellular Ca21 level in 62% and 65% of cultured rat anterior pituitary cells in a dose-dependent manner, and this effect was prevented by pirenzepine. Based on these results we suggest that both M1 and M3 muscarinic receptors are present and active in the majority of cells in the rat anterior pituitary gland, but their physiological role in the adult rat remains to be examined. © 1999 Elsevier Science Inc.
potentiated the stimulatory effect of thyreotropin releasing hormone (TRH) on prolactin secretion [41]. Cholinergic agonists (carbachol and ACh) as well as the muscarinic antagonist atropine can stimulate GH secretion from the rat anterior pituitary in vitro under various conditions. These studies suggest that a complicated system involving at least 2-second messenger systems transmit the effect of muscarinic signals in the anterior pituitary gland. This suggestion is supported by functional studies [1,14,22,24,35,37, 47]. Up to now five muscarinic receptor subtypes have been identified in the rat based on their molecular structure, but only four of them have been pharmacologically characterised [11,15,20]. The discovery of subtype selective muscarinic antagonists makes it possible to characterise muscarinic receptor subtypes by pharmacological tools [27,28]. It is not known yet, which muscarinic subtypes are responsible for the effect of ACh on the regulation of pituitary hormone secretion. Based on the controversial results about the effect of ACh on hormone secretion, the existence of different receptor subtypes in the rat anterior pituitary seems likely. In this study we attempted to characterise the selectivity of muscarinic ACh receptors in rat anterior pituitary gland comparing the displacement of [3H]QNB binding by receptor selective muscarinic antagonists with the displacement of [3H]diphenyl-acetoxy-N-methyl piperidine [3H]4-DAMP) (M1, M3). We also examined the effect of ACh and cholinergic agonists and antagonists on intracellular Ca21 in an attempt to learn about the physiological role of the receptor subtypes.
KEY WORDS: Acetylcholine, Carbachol, Pirenzepine, QNB, 4-DAMP, Intracellular calcium, Fura-2, Pituitary.
INTRODUCTION Physiological and biochemical evidences indicate the existence of functional muscarinic cholinergic receptors in the anterior pituitary. Muscarinic receptors have been identified by [3H]quinuclidinyl benzilate ([3H]QNB) and [3H]N-methylscopolamine ([3H]NMS) binding on anterior pituitary membrane preparation, as well as on intact pituitary cells [1,2,6,19,29,32]. Muscarinic mechanisms seem to play a role in the control of pituitary hormone secretion, but the results are controversial [36,39]. It has been described that acetylcholine (ACh) can act directly in the pituitary as either a stimulatory or an inhibitory signal on regulation of the same hormone [growth hormone (GH) and prolactin] secretion [7,9,41,43,47]. In the rat, ACh inhibits prolactin secretion in pituitary lactotroph cells and this inhibition can be blocked by the general muscarinic antagonist atropine [4,10]. In contrast, ACh stimulates prolactin release in the bovine anterior pituitary [33]. Furthermore, ACh
MATERIALS AND METHODS Membrane Preparation Male Wistar rats weighing 250 –300 g were used in all experiments. Anterior pituitary glands were pooled and homogenised for 10 s in ice-cold 50 mM TRIS-HCl buffer (pH 7.4 at 4°C) containing 1 mM ethylenediaminetetraacetic acid (EDTA) using an UltraTurrax tissue homogenizer at maximal setting. The homogenate was centrifuged twice at 50,000 g for 15 min at 4°C. The final pellet was used immediately for ligand binding assay or rapidly frozen at 222°C and used the next day.
* Address for correspondence: G. B. Makara, Institute of Experimental Medicine, Hungarian Academy of Sciences, H-1450 Budapest, P. O. Box 67, Hungary. Fax: 1361-210-0811; E-mail: [email protected]
449
PINTE´R ET AL.
450 Ligand Binding Assays Competitive displacement of [3H]4-DAMP (with nonlabeled 4-DAMP, atropine, and pirenzepine) and [3H]QNB (with nonlabeled 4-DAMP, atropine, pirenzepine, and methoctramine) have been performed according to Michel and Whiting [28], and Yamamura and Snyder [46], respectively. In all experiments the radioligands, competing drugs, and membranes were incubated in a final volume of 0.5 ml assay buffer (50 mM Tris-HCl-EDTA, pH 7.4) for 45 min at 25°C. In competition studies the final assay concentration of [3H]4-DAMP and [3H]QNB were 0.3 and 0.01 nM, respectively. For saturation studies the concentration of [3H]QNB was varied between 0.001 and 10 nM. Nonspecific binding was measured in the presence of 1 mM atropine. Protein was determined by the method of Lowry et al. [25]. In each assay 2–300 mg protein/tube was applied. Incubations were terminated by vacuum filtration through 0.1% polyethyleneimine pretreated glass fibre filters (GF/C, Whatman) using a multiunit filtration device. The filters were washed three times with 5 ml ice-cold 50 mM TRIS-HCl buffer solution and the radioactivity retained on the filters was determined by liquid scintillation spectrometry. Competition binding data were analysed using an iterative curve fitting method (Prism, Graphpad, San Diego, CA, USA). IC50 were converted to Ki using the Cheng and Prusoff approximation [13]. Monolayer Anterior Pituitary Cell Cultures Pituitaries of male Wistar rats (150 –250 g) of the CharlesRiver strain were used. Pituitaries from two rats were removed under aseptic conditions and collected in Medium 199. Neurointermediate lobe was removed under a stereomicroscope. Anterior pituitaries were washed in Medium 199 containing 10 mg/ml penicillin-streptomycin, and then in Ca21 2 Mg21-free Tyrode’s solution, and incubated for 30 min in Ca21 2 Mg21-free Hank’s solution containing 0.25% trypsin at 37°C. After the incubation, the anterior lobes were washed with Medium 199 containing 20% foetal calf serum and centrifuged with 800 g for 10 min and the sediment was resuspended in 1 ml Medium 199 containing 20% foetal calf serum and then anterior pituitaries were dispersed to individual cells by pipetting. Each 500 ml of the cell suspension (106 cell/ml) was plated on a sterile, 22 mm diameter coverslip and cultured for 2 days at 37°C in a 5% CO2 thermostat. Fluorescence Measurement of Intracellular Ca21 Concentration Cultured anterior pituitary cells on coverslips were washed three times with HEPES buffered salt solution (HBSS); composition: NaCl, 136.7 mM; KCl, 5 mM; CaCl2, 2 mM; MgCl2, 2 mM; Na2HPO4, 0.34 mM; KH2PO, 0.44 mM; NaHCO3, 4 mM; glucose, 5.55 mM; HEPES, 20 mM; pH 7.4) then loaded with 5 mM Fura-2-AM in HBSS [cell permeant acetoxymethyl ester diluted from a 1 mM stock solution in dimethyl sulfoxide (DMSO)] for 45 min at 37°C in 95% air and 5% CO2 atmosphere. At the end of the incubation, cells were washed three times in HBSS, and [Ca21]i was then measured microspectrofluorimetrically in a 37°C thermostated chamber; agonists were added usually for 10 s via a pressure ejection system, while antagonists were added directly in the medium and were also mixed with the agonist in the pipette. Microspectrofluorimetrical measurements were carried out with an inverted Nikon TDM Diaphot epifluorescence microscope equipped with a Fluor 403 objective (NA: 0.85; Nikon, Japan). Fluorescence intensity was measured at 520 nm by a photomultiplier (D104 Microscope Photometer, PTI, South Brunswick, NJ, USA) in response to excitation at 340 and 380 nm, [Ca21]i was calculated from the formula established by Grynkiewicz et al. [17]:
FIG. 1. The specific binding of [3H]QNB to rat anterior pituitary membranes. Increasing concentrations of [3H]QNB were incubated at 25°C for 60 min with membranes (0.3 mg of protein) in the presence (nonspecific binding) or absence (total binding) of 1 mM atropine. Specific binding was determined by substracting the nonspecific from the total binding. Data are expressed as the average of the triplicate determinations in a representative experiment. Replicate experiments (three times) gave similar results. (Inset) Scatchard replot of the binding data. B, femtomoles of the [3H]QNB bound/mg protein; F, free [3H]QNB (picomolar) (total added-specifically bound).
[Ca21]i 5 Kd*(R 2 Rmin)/(Rmax 2 R)*(Sf2/Sb2), where R is the ratio of F1/F2 fluorescence intensities at l1 5 340 nm and l2 5 380 nm; Rmin and Rmax represents the measured minimum and maximum ratios at zero and at saturating concentration of Ca21, respectively. The Sf2 and Sb2 are the fluorescence values for the free and bound forms of the dye. The Kd is the dissociation constant of the Ca21-Fura-2 complex. Materials [3H]4-DAMP (specific activity: 80.5 Ci/mmol) and [3H]QNB (specific activity 49 Ci/mmol) were obtained from New England Nuclear. Nonlabeled QNB, pirenzepine, tropicamide, methoctramine, and 4-DAMP were purchased from RB International (Natick, MA, USA). Atropine sulphate, Medium 199, trypsin, penicillin, and streptomycin were obtained from Sigma Chemical Company (St. Louis, MO, USA). Fura-2-AM (cell permeant acetoxymethyl ester) was purchased from Molecular Probes (Eugene, OR, USA). Foetal calf serum was obtained from Gibco (Glasgow, UK). RESULTS Saturation Studies The binding of [3H]QNB to membrane preparation of male rat anterior pituitary was saturable at 25°C. The nonspecific binding was less than 10% of the total binding. The dissociation constant (Kd) was 20.5 6 1.9 pM, Bmax was 35 6 5.4 fmol/mg protein. [3H]QNB labelled an inhomogeneous population of muscarinic receptors, as the Scatchard plot was not linear (Fig. 1). The binding of [3H]4-DAMP in membrane preparation of male rat anterior pituitary was also saturable at 25°C at quite low concentration of the radioligand (Fig. 2.). The nonspecific binding was 30%– 40% of the total binding. The Kd of this saturation plot
MUSCARINIC RECEPTORS IN THE PITUITARY
FIG. 2. The specific binding of [3H]4-DAMP to rat anterior pituitary membranes. [3H]4-DAMP was incubated at 25°C for 60 min with membranes (0.3 mg of protein) in the presence (nonspecific binding) or absence (total binding) of 1 mM atropine. Specific binding was determined by substracting the nonspecific from the total binding. Data are expressed as the average of the triplicate determinations in a representative experiment. Replicate experiments (three times) gave similar results. (Inset) Scatchard replot of the binding data. B, femtomoles of the [3H]4-DAMP bound/mg protein; F, free [3H]4-DAMP (nanomolar) (total added-specifically bound).
was higher than that of [3H]QNB (0.3 6 0.02 nM), but the Bmax (39.8 6 4.2 fmol/mg protein) was quite similar to the Bmax of the [3H]QNB saturation plot, suggesting, that [3H]4-DAMP and [3H]QNB predominantly bind to the same binding sites. Competition Studies Various muscarinic antagonists were used for displacing specific [3H]QNB binding to membrane preparation of anterior pituitary gland. 4-DAMP (Ki: 1.7 pM according to a one site model) and atropine (Ki: 7.9 pM) seemed to be the most potent inhibitors of [3H]QNB binding. Pirenzepine (Ki: 0.36 nM) was 45.5-fold less potent than atropine and 210 less potent than 4-DAMP. Methoctramine (Ki: 218 nM) was more than 600-fold less potent competitor of [3H]QNB binding than pirenzepine. The 4-DAMP displaced the [3H]QNB binding apparently labelling an inhomogeneous receptor population, with a low Hill coefficient. The two binding sites model seemed to fit the [3H]QNB displacement by DAMP ( p , 0.001). In case of methoctramine, pirenzepine, and atropine the one-site model was better than a two-site model (Fig. 3; Table 1). The results of competition studies of [3H]4-DAMP binding with muscarinic antagonists were very similar to those of QNB binding (Fig. 4). According to a one-site model the Ki of displacement of [3H]4-DAMP binding by DAMP, pirenzepine, and atropine were 0.4 pM, 4.5 nM, and 9 pM, respectively. 4-DAMP binding displayed low Hill coefficients and data were better fit to two populations of either [3H]QNB or [3H]4-DAMP binding sites. In contrast atropine, pirenzepine, and methoctramine displayed a Hill coefficient not significantly different from one (Table 1). Effect of Acetylcholine and Cholinergic Agonists on [Ca21]i The resting [Ca21]i of anterior pituitary cells was 119.8 6 14.6 nM (mean 6 SEM for 194 cells). A 10-s pulse administration of 10 mM ACh increased the [Ca21]i in 62.2% of the 127 pituitary
451
FIG. 3. Inhibition of [3H]QNB binding to rat anterior pituitary membranes by muscarinic antagonists. Membranes (0.3 mg) were incubated with 0.01 nM [3H]QNB and various concentration of antagonists in 0.5 ml Tris-HCl for 45 min at 25°C. Each point represents the mean of specific [3H]QNB binding of three independent experiments carried out in triplicate or duplicate. Inhibition curves depicted the best fit determined from nonlinear regression analyses. Values are mean 6SEM of three experiments.
cells (224.7 6 3.68% of the resting level). After administration of the drug, a rapid increase of [Ca21]i was observed and this effect proved to be dose dependent (Fig. 5). 100 nM ACh had no significant effect on [Ca21]i, 1 mM caused smaller [Ca21]i increase than 10 mM, and 100 mM ACh didn’t cause further increase. The magnitude of [Ca21]i increase or the percentage of the cells responsive to ACh was similar in the presence (228 6 13% of the resting level in 25 from 40 cells) or absence (225 6 4% in 79 from 127 cells) of 10 mM of the cholinesterase inhibitor physostigmine. Carbachol (1 mM, 10 mM) also significantly increased cytoplasmic Ca21 concentration ([Ca21]i) in 65.0% of the anterior pituitary cells. In 10% of the cells after administration of carbachol (1 mM, 10 mM) a decrease of the [Ca21]i was observed (69.4 6 2.8% of the resting level). The effect of ACh was mediated through muscarinic receptors (Fig. 6A–C; Table 2). Administration of 10 mM atropine prevented the effect of ACh (104.35 6 1.64 in 37 from 37 cells) on [Ca21]i. Administration of 10 mM muscarine or 10 mM McN-A-343 caused the same effect as 10 mM ACh (205.85 6 21.5% in 24 from 37 cells, 221.5 6 4.23% in 20 from 30 cells, respectively). Nicotine had no significant effect on [Ca21]i (114.3 6 3.3% in 25 from 40 cells). After 10 mM nicotine, a small, delayed tendency to increase in [Ca21]i appeared in 20% of the cells, but this effect didn’t exceed the standard deviation range of the baseline in any case. In order to determine the receptor subtypes responsible for this effect we tested the effect of selective muscarinic antagonists on ACh induced [Ca21]i increase (Fig. 7A–D; Table 2). In the presence of pirenzepine (M1) (1 mM or 10 mM) the stimulatory effect of the same dose of ACh was blocked (111.6 6 15.6% in 19 from 22 cells or 104.2 6 10.8% in 33 from 39 cells, respectively). In the presence of pirenzepine 15% of the cells responded to ACh (three from 22 cells or six from 39 cells, respectively, including the subpopulation unresponsive to ACh). The increase of [Ca21]i in the presence of 10 mM of pirenzepine was 152.4 6 9.9% of the resting level (six from 39 cells). In the presence of 4-DAMP (M3, M1) no [Ca21]i response to ACh was observed (111.8% 6 8.23 in
PINTE´R ET AL.
452 TABLE 1 NONLINEAR REGRESSION ANALYSIS OF A TWO SITE MODEL INHIBITION OF [3H]QNB AND [3H]4-DAMP BINDING TO RAT ANTERIOR PITUITARY MEMBRANES Ligand
[3H]QNB [3H]QNB [3H]QNB [3H]QNB [3H]4-DAMP [3H]4-DAMP [3H]4-DAMP
atropine 4-DAMP pirenzepine methoctramine atropine 4-DAMP pirenzepine
KiH
KiL
nH
p
7.90E-12 2.370E-12 — — 9.47E-12 3.57E-13 —
— 1.96E-09 3.57E-09 2.18E-06 — 4.12E-09 4.50E-09
0.78 0.18 0.81 1.03 0.95 0.32 0.92
0.650 0.008 0.356 0.580 0.141 0.001 0.471
KiH and KiL represent the inhibition constants of each drug at high and low affinity sites, respectively. A degree of improvement of data analysis compared to the one site model was statistically evaluated by a partial F test. p means the probability of the difference between the two equation for the one and two site models.
41 from 41 cells). Methoctramine (M2) and tropicamide (M4) had no effect on ACh induced [Ca21]i increase on rat anterior pituitary cells. The ACh induced increase in [Ca21]i was similar in the presence and in the absence of the extracellular [Ca21]i, suggesting that the stimulatory effect of ACh may involve mobilisation of Ca21 from intracellular stores (Table 2). DISCUSSION The results of the present study suggest that M1 muscarinic ACh receptors are present on adult rat anterior pituitary cells and they are linked to mechanisms capable of transiently increasing cytosolic calcium levels. The presence of another muscarinic ACh receptor population (possibly M3) also seems to be likely. Muscarinic ACh receptors have been previously described on adult rat anterior pituitary homogenates [2,19,32], in pituitary tumoural cell lines [1,18] as well as on intact pituitary cells of adult rat [29], but little is known about the subtypes and/or the homogeneity of these receptors. In this study we provide the first
FIG. 4. Inhibition of [3H]4-DAMP binding to rat anterior pituitary membranes by muscarinic antagonists. Membranes (0.3 mg) were incubated with 0.01 nM [3H]4-DAMP and various concentrations of antagonists in 0.5 ml Tris-HCl for 45 min at 25°C. Each point represents the means of a specific [3H]4-DAMP binding of three independent experiments carried out in triplicate or duplicate. Inhibition curves depict the best fit determined from nonlinear regression analyses.
demonstration of the presence of M1 muscarinic receptor subtype in adult rat anterior pituitary gland. In our experiments, both [3H]QNB and [3H]4-DAMP binding to rat anterior pituitary membranes were saturable at relatively low concentration. The Bmax values from the [3H]QNB and [3H]4DAMP binding are similar, and this suggests that [3H]QNB and [3H]4-DAMP labelled the same receptor populations. The Scatchard plot of the [3H]4-DAMP binding suggested a homogenous population, but due to its lower affinity to anterior pituitary membranes and the higher nonspecific binding we could not show specific binding with acceptable error at the low concentration, which is necessary to show a probable higher affinity site. Pirenzepine can displace either [3H]QNB or [3H]4-DAMP binding with relatively high affinity that corresponds to its affinity to the M1 muscarinic receptor subtype [27], suggesting the presence of M1 muscarinic subtype in rat anterior pituitary gland. Methoctramine didn’t displace the [3H]QNB binding efficiently, therefore, it seems likely that the M2 receptor subtype is not expressed to an appreciable extent in the rat pituitary gland [28]. This finding partially conflicts with the results suggesting that carbachol inhibits cyclic adenosine monophosphate (cAMP) synthesis in cultured adult rat pituitary cells and tumoral cell lines [1,18,30,42,43,47] supposedly via M2 receptors, because we
FIG. 5. The dose related effect of 1 mM (F, n 5 62) and 10 mM ACh (E, n 5 79) on intracellular Ca21 concentration in cultured anterior pituitary cells. The dotted lines represent the duration of ACh administration.
MUSCARINIC RECEPTORS IN THE PITUITARY
453 TABLE 2 THE EFFECT OF CHOLINERGIC AGENTS ON [CA21]I ON CULTURED RAT ANTERIOR PITUITARY CELLS
Stimuli
Agonists ACh ACh 1 EDTA Carbachol Nicotine Muscarine McN-A-343 (M1) Antagonists ACh 1 atropine ACh 1 pirenzepine (M1) ACh 1 4-DAMP (M3, M1) ACh 1 methoctramine (M2) ACh 1 tropicamide (M4)
[Ca21]i after Drug Administration (% of the Resting Level)
Number of Cases
224.7 6 3.68 218.69 6 6.25 221.5 6 4.23 114.3 6 3.3 205.85 6 21.5 221.5 6 4.23
79 28 44 25 24 20
104.35 6 1.64 104.2 6 10.8 111.8 6 8.23 219.32 6 21.4 221.32 6 17.63
37 33* 41 38 42
Each drug was used in 10 mM concentration. Values are mean 6 SEM of three experiments. In the presence of pirenzepine the stimulatory effect of the same dose of ACh was blocked in 85% of the cells (in 33 from 39 cells).
FIG. 6. The effect of cholinergic agonists (10 mM) on intracellular Ca21 concentration in cultured anterior pituitary cells. Representative single cell responses. The dotted lines represent the duration of the drug administration. (A) Carbachol; (B) muscarine; (C) nicotine.
couldn’t demonstrate M2 binding sites in appreciable amount in pituitary gland. However, the number of the M2 receptors may be lower than the detection limit of our binding method. These results as well as the saturation binding studies suggest that in the adult rat pituitary most of the muscarinic binding sites are of the M1 receptor subtype. However, the M3 muscarinic subtype also seems to be present in the pituitary gland because 4-DAMP was a more
potent displacer than the selective M1 muscarinic receptor specific antagonist pirenzepine, suggesting that a high affinity population of muscarinic binding sites in male rat anterior pituitary gland may be M3 subtype. This high affinity is unusual because in most tissues 4-DAMP has the same affinity to M1 and M3 muscarinic receptors, but recent results [23,26,38,40] show the existence of a M3 muscarinic receptor subtype with high affinity to 4-DAMP. The results of Mahesh et al. [26] are very similar to our results considering either the Kd of [3H]QNB binding or the Ki of displacement of [3H]QNB by 4-DAMP. The presence of muscarinic M1 receptors is also supported by studies on intracellular calcium concentration. Looking for physiological consequences of muscarinic receptor activation we found that ACh increased the [Ca21]i in 62.2% of the pituitary cells. Receptor mediated stimuli lead to rise of [Ca21]i via two mechanisms: mobilisation of Ca21 from intracellular stores, and/or Ca21 influx induced by receptor mediated changes of Ca21 channel conductivity. M1 and M3 muscarinic receptors are known to act mainly through phosphatidyl inositol hydrolysis, but muscarinic agents also can stimulate the Ca21 influx [12,33,44]. In our experiments the ACh induced increase in [Ca21]i was similar in the presence and in the absence of the extracellular [Ca21]i, suggesting that the stimulatory effect of ACh may involve mobilisation of Ca21 from intracellular stores. Our data agree well with the findings of other investigators, who have found that cholinergic agonists can stimulate inositol trisphosphate formation in bovine and rat anterior pituitary [1,7,35,44,47]. Canonico et al. [7] found that ACh in the presence of physostigmine stimulated inositol trisphosphate formation in a concentration dependent fashion. Carbachol, but not oxotremorine produced similar effects. The cholinergic agonists induced inositol phosphates production was very rapid, and could be inhibited by atropine [7]. Shrey and Read [35] observed similar effect in rat anterior hemipituitaries. Nicotine had no significant effect on the [Ca21]i in adult rat pituitary cell culture, but muscarine caused an effect similar to that of ACh. Moreover, the general muscarinic antagonist atropine prevented the effect of ACh on [Ca21]i. All these findings suggest that ACh acts through muscarinic receptors only.
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FIG. 7. The effect of ACh (10 mM) on intracellular Ca21 concentration in the presence of cholinergic antagonists (10 mM) in cultured anterior pituitary cells. Representative single cell responses. The dotted lines represent the duration of the drug administration. (A) Pirenzepine; (B) methoctramine; (C) 4-DAMP; (D) tropicamide.
We found that the intracellular Ca21 increasing effect of ACh can be prevented by the M1 receptor selective muscarinic receptor antagonist pirenzepine. This result supports the suggestion from the receptor binding studies that the M1 receptor subtype is present in pituitary. This was also supported by the fact that an M1 muscarinic agonist McN-A-343 caused significant [Ca21]i elevation. Ten percent of the ACh sensitive cells were insensitive to pirenzepine, which suggests that another receptor population, possibly M3 also takes part in the cholinergic stimulation of intracellular Ca21. These results agree well with the receptor binding results and suggest that more than one type of ACh receptor exists in rat anterior pituitary gland and one of them is M1 receptor. We also found that carbachol decreased the [Ca21]i in 10% of
the cells in adult rat pituitary cell culture, suggesting that in a minority of cells a mechanism different from the previously mentioned may be operating. These findings agree well with the study of Schegel et al [34], who find that muscarinic receptors (presumably M2 or M4, which are coupled to cAMP) can inhibit the Ca21 influx through a cAMP dependent mechanism [45]. Whether more than one muscarinic binding site is present in the adult rat pituitary gland is debated. Schaeffer and Hsueh [32] found that a homogeneous muscarinic receptor population bound [3H]QNB with high affinity. In contrast, Avissar et al. [2] and Henish et al. [19] found an inhomogeneous receptor population with [3H]QNB equilibrium binding studies as well as with [3H]methylscopolamine and [3H]NMS. Muhkerjee et al. [29] also found two [3H]QNB sites with distinct affinity in intact rat pituitary cells. The contradiction between these results can be resolved considering that different buffers were used, and—as it is well established—muscarinic receptors show different affinity and selectivity at high and low ionic strength [5]. It is also likely that Schaeffer and Hsueh [32] could not demonstrate the second binding site because they did not use a concentration range wide enough to cover two very different Kds. Our study supports the presence of at least two distinct populations of muscarinic receptors in the anterior pituitary gland. Since our preliminary studies suggested the existence of two [3H]QNB binding sites with rather different dissociation constants (more than two order of magnitude difference between the two Kd) in rat anterior pituitary, we used a wider concentration range of the radioligand than usual in order to show the presence of the two binding sites. The present results may have physiological importance as all components of an intrinsic cholinergic system (ACh, choline acetyltransferase, acetylcholinesterase, as well as muscarinic receptors coupled to second messenger systems) are present in the pituitary of the adult rat [2,3,9,10,16,32]. In rat anterior pituitary cells ACh inhibits prolactin secretion and this inhibition can be blocked by the muscarinic antagonists atropine [4]. In contrast, ACh stimulated the prolactin release in bovine anterior pituitary [44]. Furthermore, ACh can potentiate the stimulatory effect of TRH on prolactin secretion [41]. According to Carmeliet and Denef [9,10], either muscarinic agonists (in the absence of dexamethasone) or the muscarinic antagonist atropine (in the presence of dexamethasone) may have stimulatory effect on GH secretion. In addition to this, dexamethasone pretreatment either in vivo or in vitro reverses the stimulatory effect of carbachol [8,9]. They presumed that the corticotrop cells might exert a paracrine tonic inhibitory activity on GH and prolactin release by producing ACh, which may be secreted together with adrenocorticotopin (ACTH). This endogenous ACh may act locally as a paracrine factor, and through muscarinic receptor(s) can modulate basal and stimulated GH release [9,10, 36]. It remains to be studied whether the various muscarinic receptor subtypes found in the anterior pituitary are expressed in the same cell types or the subtypes are involved in the regulation of different pituitary cell type. According to in vitro studies muscarinic agonists may have a direct hypophyseal effect on the secretion of GH, [7,9,10,21,33,42,47] prolactin, [4,10,21,30,31,33, 34,42], and ACTH [18]. Presumably, ACh through M3 receptors (which are fewer in number and have higher affinity) modulates its own release together with release of ACTH and the released ACh through M1 receptors can inhibit GH secretion. CONCLUSIONS The findings in the present study suggest that in the adult rat anterior pituitary gland at least two muscarinic receptor popula-
MUSCARINIC RECEPTORS IN THE PITUITARY tions exist and can elevate cytosolic Ca21 level. We suggest that in the adult rat anterior pituitary the main muscarinic receptor subtype may be the M1 subtype, but the presence of M3 muscarinic subtype also seems to be likely.
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23.
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