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Muscarinic receptors of the albino rabbit ciliary process
Eqp.
Eye RPS. (1989)
Muscarinic
48, 509-522
Receptors
P. MALLouoA,t Merck
Sharp
of the Albino
R. W.
BABILON,
S. BUISSON*
and Dohme Research Laboratories,
(Received13 May 1988 and accepted
Rabbit
in
AND
West Point,
revised form
Ciliary M. F.
Process SUGRUE
PA 19486,
14 September
USA
1988)
Muscarinic receptor binding sites were identified in membranes prepared from albino rabbit ciliary processes, using the muscarinic antagonist [3H]L-quinuclidinyl benzylate as the radioligand. Analysis of saturation binding experiments demonstrated that [SH]L-quinuclidinyl benzylate bound to an apparent homogeneous population of binding sites with a K, value of 64 PM and a B,,, value of 155 fmol mg-’ protein. Seventy percent (70%) of binding sites showed high affinity for pirenzepine. i.e. belonged to the M, subtype. In contrast, AF-DX 116 was unable to discriminate between subtypes of muscarinic binding sites in this tissue. Carbachol caused a dose-dependent increase in phosphatidylinositol turnover (EC,, = 154 PM) in ciliary processes. A maximum stimulation of 652% of basal activity was obtained following a 45 min incubation with 10 mM carbachol. The potency of muscarinic antagonists to block the carbachol-induced response was comparable to that found for M, receptors in other tissues. Oxotremorine and pilocarpine behaved like partial agonists in this assay. The carbachol-induced increase in phosphatidylinositol turnover was also observed in a suspension of epithelial cells from ciliary processes and it was blocked by atropine ; thus, indicating the presence of muscarinic receptors functionally coupled to phosphatidylinositol turnover in these cells. Key wol-ds : muscarinic receptors ; ciliary process ; phosphatidylinositol turnover ; pirenzepine : AF-DX 116.
1. Introduction
Functional studies, as well as radioligand binding studies using selective muscarinic antagonists like pirenzepine, have indicated the existence of subtypes of muscarinic receptors in brain and peripheral tissues (Hammer, Berrie, Birdsall, Burgen and Hulme, 1980; Hammer and Giachetti, 1982; Pagani, Schiavone, Monferine, Hammer and Giachetti, 1984; Watson, Yamamura and Roeske, 1983). Receptors with a high and a low affinity for pirenzepine were labeled M, and M,, respectively. Recently, the use of a new cardioselective muscarinic antagonist AF-DX 116 has allowed the characterization, in binding and pharmacological experiments, of subtypes of M, receptors as well as M, receptors (Hammer, Giraldo, Schiavi, Monferine and Ladinsky, 1986; Giachetti, Michelett and Montaga, 1986; Korc, Ackermann and Roeske, 1987; Bloom, Halonen, Seaver and Yamamura, 1987b). Pilocarpine and other cholinergic agonists, in addition to their established effect on aqueoushumor outflow and pupil diameter, have been shown to affect aqueoushumor secretion (Kaufman, Wiedman and Robinson, 1984; Miichi and Nagataki, 1983; Gherezghiher, March, Nordquist and Koss, 1986). Becauseof this effect, and the role of the ciliary processin the secretion of aqueoushumor, cholinergic receptors may be present in this tissue. Binding studies using [3H]quinuclidinyl benzilate as a radioligand have indicated the presence of muscarinic receptors on bovine nonpigmented ciliary epithelial ceils (Polansky, Zlock, Brasier and Bloom, 1985). In addition, carbachol is able to stimulate the labeling of phosphatidic acid and phosphatidylinositol (PI) by [““PI in the rabbit ciliary process (Akhtar and Abdel* Faculte des Sciences, t To whom all offprint 0014-4835/89/040509+14
Universiti requests $03.00/O
de Clermont-Ferrand. should be addressed.
63000
Clermont-Ferrand. 0 1989 Academic
France. Press Limited
510
P. MALLORG.4
ET
AL
Latif, 1983). However, a complete characterization of the muscarinic receptors in the ciliary process using selective antagonists has not been reported. With the aim of characterizing the muscarinic receptor population in the rabbit ciliary process, we performed binding and PI turnover studies using selective muscarinic antagonists such as pirenzepine (M,) and AF-DX 116 (Ma). The presence of muscarinic receptors in epithelial cells from this tissue was also investigated.
2. Materials
and Methods
Tritiated L-quinuclidinyl benzilate ([3H]&NB, 3040 Ci mmoll’) and myo-inositol ([3H]INS, 10-20 Ci mmoll’) were purchased from New England Nuclear Corporation (Boston, MA). Atropine, carbachol, oxotremorine sesquifumarate, pilocarpine hydrochloride, inorepinephrine bitartrate and pirenzepine were purchased from Sigma Chemical Company (St. Louis, MO). AF-DX 116 was a gift from Dr M. Wax (Universit’y of Pennsylvania School of Medicine, Philadelphia, PA). The AGl-X8 (109200 mesh) resin was in the formate form and was purchased from Bio-Rad (Richmond, CA). All other reagents were analytical grade and obtained from Sigma Chemical Company or Serva (Heidelberg, FRG). All drugs were dissolved in distilled water or ethanol at a 10 mM concentration and diluted as necessary in the appropriate buffer. Membrane preparation Membrane suspensions were prepared from frozen ciliary processes of adult New Zealand albino rabbits. The animals were sacrificed by an intravenous injection of an overdose of sodium pentobarbital and the ciliary processes dissected as previously described (Nathanson, 1980). Eyes were enucleated, separated from adhering tissue, and the optic nerve cut at its base. Each eye was opened by four incisions starting at the base of the optic nerve and ending 2-3 mm posterior to the limbus. The lens and vitreous were carefully dissected. The opened eye was inverted and placed, cornea down, on a plexiglass holder in a Petri dish filled with ice-cold phosphate-buffered saline (pH 7.4, PBS). The floating ciliary processes from four eyes were transected at their attachment to the iris and the ciliary body, collected in a polystyrene tube and centrifuged at 300 g for 10 min. The supernatant was discarded and the pellet quickly frozen in liquid nitrogen and stored at 80°C until used. Tissue was homogenized in 10 mM Na-K phosphate buffer (pH 7.4) using a Polytron homogenizer (setting 7, 30 set). The homogenate was centrifuged for 10 min at 35006g. The pellet was resuspended in buffer and recentrifuged at 35000g for 10 min. The homogenizationcentrifugation process was repeated once again and the final pellet resuspended in 15 ml of 10 mM Na-K phosphate buffer (pH 7.4) containing 5 IIIM MgCI,. The final suspension was kept on ice until used in the binding assay. Binding assay Binding was done as described previously (Gil and Wolfe, 1985) with the exception that 5 mM MgCl, was included in our assay. Briefly, membranes (109150,ug of protein) were incubated with [‘H]QNB, the drug to be tested or buffer in a final volume of 5 ml. The tubes were incubated for 120 min at 32’C. Incubations were terminated by the addition of 4 ml of cold buffer, rapid filtration under reduced pressure through Filtermats glass fiber filters (Skatron Inc., Sterling, VA) and 2 x 4 ml washes using the Skatron cell harvester (Skatron Inc.). The filters were placed in polyethylene vials containing 3 ml of PCS (Amersham Corp., Arlington Heights, IL) and counted overnight in a liquid scintillation spectrometer (Packard 2000 CA). Specific binding was defined as the difference between total binding and binding in the presence of 10 ,UM atropine. Specific binding represented 89-96 % of total [3H]QNB (50 PM) binding. The apparent dissociation constant (K,) and the maximum number of binding sites (&,) were calculated using the non-linear curve fitting program LIGAND (Munson and Rodbard, 1980). The displacement curves by agonists or antagonists were first analyzed by the non-linear curve fitting program EBDA (McPherson, Elsevier Biosoft, Cambridge, U.K.) which calculated K, values and Hill coefficients (nH). The curves were then analyzed with
OCULAR
MURCARINIC
REC’EPTORS
LIGAND according to a one-site and two-site model. Testing for difference between the models was done by comparing the residual variance of fits to the data by an F test, P < 905 was taken as indicating a significant difference. When a two-site model preferred, dissociation constants K, and K, were calculated as well as the % of high and affinity sites.
511 two and was low
Stimulation of Inositol Phosphates Formation Methodology was similar to that used for rat brain slices by Brown, Kendall and Nahorski (1984). Ciliary processes from 8 to 10 albino rabbit eyes were dissected in ice-cold PBS, as previously described (Nathanson, 1980). The processes were placed in 1 ml of Krebs bicarbonate buffer (KBB) containing 118 mrvr NaCl, 47 mM KCl, 12 mM MgSO,, 25 mM NaHCO,, 1.2 tnM KH,PO,, 1.3 tnM CaCl, and 10 mM glucose, and previously equilibrated with 95% 0,:5% CO, to a final pH of 7.4. The processes were washed twice with KBB by sedimentation and aspiration of the supernatant. After the last aspiration, 25 gl aliquots of the ciliary process suspension (20&300 yg of protein) were placed in polystyrene tubes containing 210~1 KBB. the tubes were then capped and incubated at 37’C in a gently of shaking water bath. After 40 min, [3H]INS and LiCl were added to a final concentration 93 ,UM and 10 mM respectively. After 20 min. antagonists were added and this was followed 10 min later by the addition of agonists. The vials were incubated for a further 45 min. Incubations were stopped by the addition of 694 ml of chloroform : methanol (1: 2, v : v) followed by homogenization of the suspension with a Polytron homogenizer (setting 5, 10 set). After 10 min standing, 031 ml of chloroform and @31 ml of water were sequentially added. The vials were agitated on a vortex mixer and centrifuged at 1OOOg for 5 min to separate phases. A 975 ml aliquot of the upper phase was removed and placed over an Amicon column containing 1 ml of AGi-X8 (formate form). [3H]INS was eluted with 5 ml H,O. and both glycerophosphoinositol and inositol 1: 2 cyclic phosphate eluted with 5 ml of 5 mM sodium tetraborate plus 60 mM ammonium formate. Myo-inositol-l-phosphate (IP,) was eluted with 10 ml of 92 M ammonium formate plus 91 M formic acid. Myo-inositol- 1,4diphosphate (IP,) was eluted with 10 ml of 94 M ammonium formate plus 91 M formic acid and myo-inositol-1,4,5-triphosphate (IP,) as well as myo-inositol-1,3,4-triphosphate with 6 ml of 1 M ammonium formate plus 0.1 M formic acid. No attempt was made to separate the myo-inositol-triphosphate isomers. In the majority of experiments, the inositol phosphates (IP) were not separated, and were eluted together with 5 ml of 61 M formic acid, plus 975 M ammonium formate after the elution with water and tetraborate. Aliquots of the eluates (2 ml) were added to 10 ml of Aquasol 2 (NEN) and counted for radioactivity in a liquid scintillation spectrometer (Packard 460 CD). All experiments were conducted a minimum of three times, and each experimental data point was calculated from the mean of two separate ciliary process incubations. The dose of agonist that produced half maximal stimulation (EC,,), and the dose of antagonist that produced half maximal inhibition (IC,,), were calculated using t,he non-linear regression analysis ALLFIT of the results in d.p.m. (DeLean, Munson and Rodbard, 1978). Ki values were then calculated using the following formula in whirh d is the concentration of agonist :
Ki_Ic,,, A 1+EC,, Epithdial Cell Preparation Epithelial cells from ciliary processes were isolated using a technique derived from that of Nathanson (1980). Briefly, dissected ciliary processes were incubated with agitation at 37°C for 15 min in Dulbecco’s phosphate buffered saline containing trypsin (01 %), and no calcium or magnesium (DPBS) and then allowed to stand until the ciliary processes had sedimented. The supernatant, which contained mainly red blood cells, was discarded. The pellet was resuspended in DPBS and the incubation-sedimentation process repeated twice ; the respective supernatants, which contained epithelial cells, were saved and pooled. Horse serum was added to the supernatants to a final concentration of 10% (v:v). The cell
512
P. MALLORGA
ET
AL.
suspension was filtered on a 37 p nylon mesh, to eliminate fragments of blood vessels (CocaPrados and Kondo, 1985), centrifuged at 100 g for 5 min, and the cellular pellet resuspended in KBB. A population of 7540% cells extruding trypan blue was obtained. Samples were taken for histologic study. Protein concentrations were measured by the method of Lowry, Rosebrough, Barr and Randall (1951) using bovine serum albumin as standard. 3. Results
In preliminary experiments, specific binding of [3H]QNB to membranes prepared from ciliary processeswas found to be linearly dependent on the concentration of tissue, up to @05 mg of protein per ml. Concentrations of 002-003 mg ml-’ were used in all subsequentexperiments. Equilibrium binding was achieved within 90 min (data not shown). The specific binding of [3H]QNB to a ciliary process membrane fraction was saturable and of high affinity. Scatchard analysis, as well as non-linear regression analysis of equilibrium binding data indicated that [3H]QNB (Fig. 1) bound to a single population of high affinity binding sites with a Kd of 6*4+@7 pM and a B,,, of 155IfI 2 fmol mg-l protein. The ability of several muscarinic agonists and antagonists to displace [3H]QNB from ciliary processbinding sites was tested. In addition, the effect of GTP (500 ,UM) on the displacement curves for agonists was also studied. The most potent of the three antagonists tested (Table I) was atropine with a Ki of @36 nM, followed by pirenzepine (34 nM) and AF-DX 116 (230 nM). The displacement curves for atropine and AF-DX 116 were best fitted by a one-site model. In contrast, the curve for pirenzepine, for which a Hill coefficient of @69was obtained, was best fitted by a two-site model. The inhibition constants for pirenzepine were 12 and 359 nM, and the relative number of
0.000 4 0
I
I
50
100
Bound
(fmoC
mg-’
O\ 150
proi.)
FIG. 1. Scatchard plot of [aH]&NB binding to eiliaq process membranes. [SH]QNB concentrations ranged from 2 to 1000PM. Each point is the mean of three replicates from a single experiment. The experiment wa8 repeated three times, and a Kb = 64*@7 PM and II 4.X = 155+ 2 fmol mg-’ protein were obtained.
OCULAR
MUSCARINIC
RECEPTORS
TABLE
Inhibitory
I:
potency of various antagonists for speci$c [3H]QNB ciliary process Drug
nH
4
Atropine Pirenzepine AF-DX 116
are the mean of three experimentsf % L represents the percentage of low affinity The concentration of [aH]&NS was 005 nM.
on the inhibitory
bin&q
carhachol carbachol oxotremorine oxotremorine pilocarpine pilocarpine
binding in the rabbit
%L
KL
125 1 -
359*99 --
and are expressed
S.E.M.
30+3
in no.
sites.
TABLE
Ejfect of GTP
4
0~87~004 969kO.10 1.08*@08
036&O-12 3400+900 23000 + 3900
K values
Drug
513
II
potency of various agonists for speci$c [3H]QNB in the rabbit ciliary process
GTP (500 PM)
K, bM)
nH
+ + +
540+110 2900* 1100
048 * @08 951+902
38+3 140+30
070+0.11 OS5+003
309+50 830+ 100
@79f@05 1~01+@05
Values are the mean of at least three experimentsk~.~.~. 905 nM. % L represents the percentage of low affinity sites.
4
KL
W)
NW
71+28 630f260
30000+20000 28000+9000
7f5 -
The
%L 41+9 56515
685 & 588 260,70
48k12 MO+0
330+70 650 + 50
100&O lOOjO
concentration
of [aH]QNS
was
high- and low-affinity sites in t,he ciliary processwere estimated to be 70 and 30%, respectively. Pilocarpine was the only agonist of the three tested for which the competition curve was best fitted by a one-site model (Table II). A Kd of 300 nM was obtained, which was slightly increased to 830 nM when GTP was added to the assay. The Hill coefficient of 679 became 1.01 in the presenceof GTP. Oxotremorine was the most potent of the three agonists with a K, of 38 nM. The displacement curve was best fitted by a two-site model, the inhibition constants being 7 and 685 nM, and the relative number of high- and low-affinity sites being 52 and 48%, respectively. When GTP was added to the assay, the Ki value for oxotremorine was 140nM and the displacement curve best fitted by a single site model. The Hill coefficient was increasedfrom 670 to 065 in the presenceof GTP. The inhibition curves for carbachol were best fitted by a two-site model in the absenceas well as in the presenceof GTP ; the K, value was increased, however, from 540 nM to 2900 nM in the presenceof GTP. The number of low affinity sites was concurrently increased from 41% to 56 %. It is alsoto be noted that the inhibition constant for the high affinity site for carbachol was nine times higher in the presence of GTP (630 nM vs. 71 nM). The Hill coefficient for the two inhibition curves was 648 without and 651 with GTP. A 30 min preincubation of ciliary processeswith [3H]INS and LiCl resulted in 5%
514
I’. MALLORGA
ET
.1L.
of the radioactivity being incorporated into the phospholipids. as indicated by the radioactivity recovered in the organic phase of the chloroform extraction. A preincubation time of 30 min, followed by a 45 min incubation in the presence of agonists, were the conditions chosen in this study because these conditions were found to optimize the stimulation factor by agonists. Similar conditions were used by Brown et al. (1984) for rat brain slices. Longer preincubation periods with [3H]INS (1 and 2 hr), followed by a 45 min incubation with carbachol, have resulted in values for (a) the maximum stimulation by carbachol, (b) the EC’,, for carbachol, and (c) the amount of radioactivity recovered in the IP fract,ion after the 45 min incubation with or without carbachol, comparable to those obtained with a 30 min preincubation. In preliminary experiments we found that’ the total IP labeling was increased by a factor of 1.8 after a 5 min incubation with carbachol (10 mM) (Fig. 2). A stimulation facto1 of 6-9 was obtained after a 45 min incubation (Fig. 3). The distribution of the radioactivity in IP,, IP, and IP, was 74, 23 and 3%. respectively, in the control. and 83, 15 and 2 %, respectively, in the 5 min carbachol-stimulated tissue, indicating a 2, 1.2 and 1.2 stimulation fact’or for IP,, IP, and IP,, respectively. After a 45 min incubation in the presence of carbachol, stimulation factors of 8, 1.1 and 2.0 were found for IP,, IP, and IP,, respectively (data not shown). To further characterize the carbachol response, experiments were conducted without separating IP,, IP, and IP,, which were eluted together, as described in the Materials and Methods section.
0
1
, 5 Fraction
No.
Fro. 2. Elution profile of the inositol phosphates. Ciliary processes were incubated with carbachol for 5 min and the IP extracted as described in Materials and Methods. The buffers used for the elution were : (a) 5 ml of H,O; (b) 5 ml of 5 rniu sodium tetraborate+ 60 rniv ammonium formate; (c) 10 ml of @2 M ammonium formate +@l M formic acid; (d) 10 ml of 0.4 M ammonium formate+O.l M formic acid; (e) 6 ml of 1 M ammonium formate +@l M formic acid.
OCULAR
MUSCARINIC
I 5
6
RECEPTORS
I 4
515
I 3 -Log
I 2
I
(cone)
Fm. 3. Stimulation of [3H]INS metabolism in ciliary processes by cerbachol. Ciliary processes were incubated in the presence of [sH]INS and LiCl for 30 min, and then in the presence of carbachol for 45 min. The [3H]IP were extracted and separated as described in the text. Each value is expressed as a percentage of the maximum stimulation obtained with 10 mM cerbachol. and is the mean* S.E.M. of three replicates from a single experiment. The experiment was repeated three times and a maximum stimulation of 652 + 126 % of basal. and an EC,, = 154 + 53 ,uM were obtained. A basal value of 4179k215 dpm per assay was obtained.
120 -
80 -
40 -
20 -
0 II
I
I
I
IO
9
8 -Log
7 (cone)
6
5
4
3
FIG. 4. Inhibition of carbachol stimulation of [3H]INS metabolism in ciliary processes by atropine (W-W), pirenzepine (A-A), AF-DX 116 (0-O) an d mecamylamine (0-O). Ciliary processes were incubated in the presence of the antagonists for 10 min and then in the presence of carbachol (1 m&r) for a further 45 min. Each value is the me+s.n.M. of duplicate determinations from three different experiments. Inhibition constants of 1.15f007 nM, 47 & 7 nM and 637 k 120 nM were obtained. A value of 53 149 + 7255 dpm per assay was obtained in the presence of carbachol.
516
P. MALLORGA
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AL.
Carbachol caused a dose-dependent increase in the formation of IP. A maximum stimulation of 652% of basal IP formation was obtained with 10 mM carbachol, the EC,, being 154 FM (Fig. 3). Pilocarpine and oxotremorine caused a small increase in 1P formation, reaching stimulations of 5 % and 10 % of that of carbachol, respectively, at a 1 mM concentration (data not shown). EC,, values for pilocarpine and oxotremorine were not calculated because of the small stimulation factors. The stimulation of IP formation by a submaximal concentration of carbachol (1 mM) was blocked in a dose-dependent fashion by atropine, pirenzepine and AF-DX 116, but not by the nicotinic antagonist, mecamylamine at concentrations up to 100,uM (Fig. 4). Inhibition constants of 1.15, 47 and 637 nM were obtained for atropine, pirenzepine and AF-DX 116, respectively. We have shown in a separate study (Mallorga, Buisson and Sugrue, 1908) that norepinephrine is able to increase IP formation in the ciliary process, and that a maximum stimulation (430 %) is obtained with 1 mM norepinephrine. When maximal concentrations of carbachol and norepinephrine were assayed alone or in combination (Table III), an almost perfect additivity was obtained in the effects of the two agonists on IP formation. When epithelial cells, prepared from ciliary processes were incubated with [3H]INS and carbachol (1 mM), a stimulation of 384 % in IP accumulation was obtained when compared to non-stimulated cells (Table IV). The stimulation by carbachol was blocked by 10 ,UM atropine, which was previously shown to completely block the carbachol response in the intact ciliary process (Fig. 4). III.
TABLE
Additivity
of the carbachol
and the norepinephrine stimulations in the diary process of the rabbit
Addition
(loncentration
Stimulation
(mM)
(“h of Control)
1
385 * 20
norepinephrine carbachol norepinephrine carbachoi
of [3HJZP accumulation
+
10
825k
1 10
1093+99
13
Results are the mean of three experimentsf S.E.M. Ciliary processes (300 /cg of protein) were incubated in the presence of [aH]INS and LiCl for 30 min and then in the presence of the agonists for 45 min. The [3H]IP were extracted and separated as described in the text. A basal value of 13647 + 1092 dpm per assay was obtained in the absence of agonist. TABLE
Stimulation
by carbachol
of [3HlIP
Addition carbachol carbachol+ atropine
f ormation Concentration (PM) 1000 1000 10
IV
in
epithelial
cells
of the diary
process.
Stimulation (“/ of Control) 384& 18 91&l
Results are the mean of three separate experiments? S.E.M. Cells (100 fig of protein) were incubated with [3H]INS as indicated for the ciliary process in Materials and Methods. A basal value of 12 369 dpm per assay was obtained in the absence of agonist.
OC’ULBR
MI’SC’ARINIC’
RECEPTORS
517
4. Discussion In rabbit ciliary process membranes, [3H]QNB bound with high affinity to a single population of binding sites. The K, value obtained in this study (6.4 PM) was very similar to values obtained for rabbit peripheral lung (Bloom et al., 1987a) and rat brain (Korn. Martin and Harden, 1983), but lower than the Kd values observed for the bovine non-pigmented epithelium of the ciliary process (120 PM, Polansky et al., 1985) and the rabbit iris (39 PM% Akhtar, Honkamen, Howe and Abdel-Latif, 1987). Variations in methodology, including the temperature and the receptor concentration used in the assay, may be the cause of these differences. The rabbit ciliary process was found to be richer in [3H]QNB binding sites than the rabbit aorta (Yamanaka. Hashimoto and Muramatsu, 1986) or the peripheral lung (Bloom et al., 1987a), but poorer than the rat brain (Fisher and Bartus, 1985; Luthin and Wolfe, 1984) or the rat atria (Dunlap and Brown, 1983). Of particular interest are t,he 14-17-fold great’er concentrations found in the rabbit iris sphincter (Akhtar et al.. 1987) and the primate ciliary muscle (Gabelt, Polansky and Kaufman, 1987). In the rabbit iris sphincter, the muscarinic receptors belong to the M, subtype (Akhtar et al., 1987). In contrast, the majority of the [3H]QNB binding sites of the rabbit ciliary process (70%) possess a high affinity for the M, selective antagonist pirenzepine (12 nM) and, therefore, probably belong to the M, subtype. The muscarinic M, receptors have been subdivided into two subtypes using functional (GiarhetC et al., 1986) as well as radioligand binding studies (Hammer et al., 1986). The M, cardiac receptor shows, in binding studies, a slightly lower affinity for pirenzepine (2-4-fold, Gil and Wolfe, 1985; Hammer et al., 1980) than the M, glandular subtype (rat parotid and lacrimal glands) do and, therefore, pirenzepine is not useful in distinguishing between M, receptor subtypes. AF-DX 116. a cardioselective muscarinic antagonist (Giachetti et al., 1986), has a 30-50 times greater affinity for the M, cardiac binding site than for the glandular site and an intermediate affinity for M, binding sites (Hammer et al., 1986; Bloom et al., 198713). In our study, AF-DX 116 was unable to discriminate between subtypes of muscarinic M, receptors as indicated by a Hill coefficient of 1. Thus, it is difficult,, despite the use of pirenzepine and AF-DX 116, to assign a subtype to the 30% of M, binding sites detected on ciliary process membranes. In the cerebral cortex, which is also a tissue rich in M, binding sites (65-75 %), t,he analysis of AF- DX 116 displacement curves has indicated a homogeneous population of binding sites in some studies (Hammer et al., 1986; Bloom et al., 1987b) and multiple sites in others (Korc et al., 1987; Watson, Roeske and Yamamura 1986b). It appears, therefore, t’hat in tissues rich in M, binding sit*es, AF-DX 116 is not always able to differentiate muscarinic receptor subtypes. The Ki value found for AF-DX 116 in this study (230 nM) was comparable to Ki values reported for the rat cerebral cortex (139-176 nM) when [3H]QNB was used as radioligand (Bloom et al., 1987b; Watson et al.. 1986b) as well as when [3H]Nmethylscopolamine (3H-NMS) was used (440-764 nM, Korc et al., 1987; Hammer et al., 1986). Recent studies using [“HIAF-DX 116 as radioligand have suggesbed t,he presence of M, cardiac as well as MM, glandular sites, in addition to the M, sites, in the cerebral cortex (Wang, Roeske, Gulya, Wand and Yamamura, 1987). The presence of the three binding sites in the ciliary process is also a possibility. The Hill coefficients for the inhibition of [3H]QNB binding to ciliary process membranes by muscarinic agonists were less than unity, as has been described previously in several studies using various tissues (Birdsall, Hulme and Burgen. 1980;
518
P.MALLORGAETAL.
Korn et al., 1983: Evans, Hepler, Masters, Brown and Harden, 1985; Watson, Yamamura and Roeske, 1986a). The inhibition curves were best fitted by a two-site model for carbachol and oxotremorine, but not for pilocarpine. Earlier studies have indicated that multiple muscarinic binding sites recognized by agonists (high and low) are not identical to those identified by antagonists (M, and M, subtypes) (Gillard, Waelbroeck and Christophe, 1987). M, and M, subtypes probably represent two separate proteins (Kervalage, Fraser and Venter, 1987) and the use of selective radioligands like [3H]pirenzepine and [3H]AF-DX 116 will help in their characterization. In contrast, the high and low affinity sites detected by agonists may result from the existence of different conformational states of the same receptor (Birdsall et al., 1980 ; Watson et al., 1986a) regulated by the interaction of the receptor with a GTP-binding protein. Guanine nueleotides have been shown to lower the affinity of several muscarinic agonists for [3H]QNB as well as [“H]NMS binding sites. This effect is more pronounced in M,-rich tissues, like heart and cerebellum, than in RI,-rich tissues, like the cerebral cortex or the hippocampus (Korn et al., 1983; Watson et al., 1986a). In ciliary process membranes, the observed GTP-induced shifts in affinity for agonists were slightly greater (1-3-fold) than those observed in cerebral cortex membranes, but smaller (3.lo-fold) than those observed in cardiac membranes (Korn et al., 1983; Watson et al., 1986a). This supports the hypothesis that a mixture of M, and M, binding sites are present in this tissue. In 1321Nl human astrocytoma cells and in the cerebral cortex, a GTP-binding protein is probably involved in the muscarinic receptor-mediated stimulation of a phospholipase C-induced breakdown of phosphoinositides, which results in the increased synthesis of IP (Evans et al., 1985; Gonzales and Crews, 1985). We have found in this study that carbachol causes a large increase in the formation of tritiated IP in rabbit ciliary processes prelabelled with [3H]INS. A much smaller effect was observed by Akhtar and Abdel-Latif (1983) in the rabbit ciliary process, but only one dose of agonist (50,~~) was used in their study. This carbachol-induced effect has been previously observed by many groups in several tissues (for review, see AbdelLatif, 1986) and arises from the increased hydrolysis of phosphatidylinositol-4,5bisphosphate, which generates two second messengers : IP, and 1,2-diacylglycerol. IP, is responsible for the intracellular mobilization of Ca2’ and 1,2-diacylglycerol for the stimulation of protein kinase C (Berridge, 1984). IP,, which in some cells can give rise to INS(1,3,4,5)P, and INS(1,3,4)P, (Irvine, Lechter, Heslop and Berridge, 1986), is eventually hydrolyzed to IP, which in turn is hydrolyzed to IP,. The breakdown of IP, is inhibited by lithium, and the use of this ion causes an amplification of the signal (IP accumulation) generated by the stimulation of the receptor. In our experiments, the use of a long incubation time (45 min) in the presence of LiCl may explain the large accumulation of radioactivity in the IP, peak. The EC,, value found for carbachol in this study is comparable to values obtained in Ml-containing tissues (46200 pM, Fisher and Bartus, 1985; Lazareno, Kendall and Nahorski, 1985; Gil and Wolfe, 1985) but is much greater than those found in M,containing tissues (2-13 ,UM, Akhtar et al., 1987; Woodcock, Leung and McLeod, 1987; Gil and Wolfe, 1985), as well as for a pure M, cardiac receptor expressed in hamster ovary cells (6 ,UM, Ashkenazi et al., 1987). The very small stimulation of PI turnover observed with oxotremorine in the ciliary process (10 % of carbachol maximum response) is also comparable to that reported previously for M,-rich tissues ((r22 %, Fisher and Bartus, 1985; Gil and Wolfe, 1985), in contrast to the M, cardiac tissues (27-40%, Fisher and Bartus, 1985; Woodcock et al., 1987) and the M, glandular tissues (41-lOO%, Gil and Wolfe, 1985; Jacobson, Wusterman and
OCtTLAR
MVSCARINIC
REC’EPTORS
519
Downes, 1985). Based on these results with agonists, the muscarinic receptor coupled to PI turnover in the ciliary process would appear to be of the M, subtype. This finding was confirmed using three antagonists as results from agonist studies can be affected by the presence of spare receptors. However, the somewhat lower affinity of pirenzepine for the PI turnover-coupled receptor than for M, binding sites would suggest again the presence of M, receptors in this t,issue. Although affinities of antagonists in the PI turnover assay correlated well with those found in the binding assay, discrepancies were observed with agonsits. In particular, oxotremorine and pilocarpine, which possess higher affinities than carbachol for [3H]QNB binding sites, are much less effective than carbachol in stimulating IP accumulation. In earlier studies (Fisher and Bartus, 1985; Jacobson et al., 1985), it has been shown that pilocarpine and oxotremorine, in addition to thei weak agonist activity, were able to inhibit carbachol-induced IP accumulation in rat brain cortex slices; hence, they can be considered partial agonists. We have confirmed this finding by observing inhibitions of 81+ 1% and 84+ 4 % by pilocarpine (1 mM) and oxotremorine (1 mM), respectively, of IP accumulation induced by 1 mM carbachol in the ciliary process (data not shown). Another notable difference was the lower potency of carbachol (EC,, = 154 pM) to stimulate PI turnover than to displace [3H]QNB (Ki = 3 ,UM in the presence of GTP). However, carbachol consistently showed multiple affinity states in displacing [3H]QNB from ciliary process membranes and if the low affinity state (Ki N 30 ,UM) is the one coupled to PI turnover, then only a difference of 5-fold exists between the potency of carbachol in the two assays. Differences (24 ,UM vs. 170 ,UM ; Freedman, Harley and Iversen 1988) as well as good agreement (250 ,UM vs. 345 PM: Heacock, Fisher and Agranoff. 1987) have been observed for the rat cerebral cortex. It is not clear why these anomalies exist. However, it is to be noted that binding studies were performed with membrane suspensions, whereas PI turnover studies employed slices of intact tissue. Intact tissue has to be used to study PI turnover because, in preliminary experiments, it was observed that carbachol stimulation was totally lost by homogenizing the ciliary processes (data not shown). Another possibility not tested in this study is that after a prolonged incubation with the tissue, Li+ ions may lower the potency of muscarinic agonists to stimulate PI turnover. None of these possibilities can be completely ruled out, but the good agreement observed for the muscarinic antagonists suggest that technical differences, rather than different receptors, may be the reason for these discrepancies. Muscarinic receptors of the ciliary process could be associated with epithelial cells, blood vessels or connective tissue cells. Our finding that muscarinic receptors are functionally coupled to phosphoinositide metabolism in epithelial cells suggests that these receptors may be involved in the secretion of aqueous humor. More interestingly, the presence of a majority of M, receptors in this tissue, in contrast to the presence of the M, subtype in the iris sphincter muscle, raises the possibility of a selective pharmacological action on secretion. Support to this hypothesis awaits the development of more selective M, and M, agonists and antagonists. In any event, the demonstration of muscarinic receptors on epithelial cells may help to explain why muscarinic agonists can alter aqueous humor secretion. However, they do not explain why increases as well as decreases have been observed (for review see Kaufman et al., 1984). Further studies are needed to determine which receptor subtypes are present on the epithelial cells and particularly on pigmented versus non-pigmented epithelium and whether these receptors can be associated with a physiological or pharmacological effect on aqueous humor secret,ion.
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ET AL
ACKNOWLEDGMENTS The authors wish to thank
Maryanne
Olkowski
for the preparation
of the manuscript.
REFERENCES Abdel-Latif, A.A. (1986). Calcium-mobilizing receptors, polyphosphoinositides, and the generation of second messengers. Phurmacol. Rev. 38, 227-72. Akhtar, R.A. and Abdel-Latif, A.A. (1983). Effects of acetylcholine and norepinephrine on incorporation of 32P-orthophosphate into phospholipids of rabbit iridial processes and iris smooth muscle. Ez~. Eye Res 36, 103-12, Akhtar, R.A., Honkamen, R.E., Howe, R.H. and Abdel-L&if, A.A. (1987). M, muscarinic receptor subtype is associated with inositol triphosphate accumulation, myosin light chain phosphorylation and contraction in sphincter smooth muscle of rabbit iris. J. Phurmucol. Exp. Ther. 243, 624-32. Ashkenazi, A., Winslow, J.W., Peralta, E.G., Peterson,G.L., Schimerlik, M.I., Capon, D.J. and Ramachandran, J. (1987). An M, muscarinic receptor subtype coupled to both adenyl cyclase and phosphoinositide turnover. Science 238, 672-5. Berridge, M.J. (1984). Inositol triphosphate and diacylglycerol as second messengers. B&hem. J. 220, 345-60. Birdsall, N.J.M., Hulme, E.C. and Burgen, A.S.V. (1980).The character of the muscarinic receptors in different regions of the rat brain. Proc. R. SOC. Land. B 207, l-12. Bloom, J.W., Halonen, M., Lawrence, L.J., Rould, E., Seaver, N.A. and Yamamura, H.I. (1987a). Characterization of high affinity 3H-pirenzepine and 3H-&NB binding to muscarinic cholinergic receptors in rabbit peripheral lung. J. Phxwmacol. Exp. Ther. 240, 51-8. Bloom, J.W., Halonen, M., Seaver, N.A. and Yamamura, HI. (1987b). Heterogeneity of the M, muscarinic receptor subtype between peripheral lung and cerebral cortex demonstrated by the selective antagonist AF-DX 116. Lije Sci. 41, 491-6. Brown, E., Kendall, D.A. and Nahorski, S.R. (1984). Inositol phospholipid hydrolysis in rat cerebral cortical slices: receptor characterization. J. Neurochem. 42, 1379-87. Coca-Prados, M. and Kondo, K. (1985). Separation of bovine pigmented epithelial cells by density gradient and further characterization in culture. Exp. Eye Res. 40, 731-9. De Lean, A., Munson, P.J. and Rodbard, D. (1978). Simultaneous analysis of families of sigmoidal curves : application to bioassay, radoligand assay and physiological doseresponse curves. Am. J. Physiol. 235, E97-102. Dunlap, J. and Brown, J.H. (1983). Heterogeneity of binding sites on cardiac muscarinic receptors induced by the neuromuscular blocking agents gallamine and pancuronium. Mol. Phurmacol. 24, 1522. Evans, T., Hepler, J.R., Masters, S.B., Brown, J.H. and Harden, T.K. (1985). Guanine nucleotide regulation of agonist binding to muscarinic cholinergic receptors. Biochem. J. 232, 751-7. Fisher, SK. and Bartus, R.T. (1985). Regional differences in the coupling of muscarinic receptors to inositol phospholipid hydrolysis in guinea pig brain. J. Neurochem. 45, 1085-95. Freedman, S.B., Harley, E.A. and Iversen, L.L. (1988). Biochemical measurement of muscarinic receptor efficacy and its role in receptor regulation. Trends Pharmacol Sci. (Suppl.) III, 54-60. Gabelt, A.T., Polansky, J.R. and Kaufman, P.L. (1987). Ciliary muscle muscarinic receptor content and affinity for QNB in young and old rhesus monkeys. Invest. Ophthalmol. Vie. Sci. (Suppl.) 28, 65. Gherezghiher, T., March, W.F., Nordquist, R.E. and Koss, M.C. (1986). Laser-induced glaucoma in rabbits Exp. Eye. Res. 43, 885-94. Giachetti, A., Micheletti, R. and Montaga, e5E. (1986). Cardioselective profile of AF-DX 116, a muscarinic M, receptor antagonist. Lije Sci. 38, 1663-72. Gil, D.W. and Wolfe, B.B. (1985). Pirenzepine distinguishes between muscarinic receptormediated phosphoinositide breakdown and inhibition of adenylate cyclase. J. Pharmacol. Exp. Ther. 232, 60%16.
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Gillard, M., Waelbroeck, M. and Christophe, J. (1987). Muscarinic receptor heterogeneity in rat central nervous system. II. Brain receptors labeled by SH-oxotremorine-M correspond to heterogenous M, receptors with very high affinity for agonists. Mol. Pharmucol32, 100-8. Gonzales, R.A. and Crews, F.T. (1985). Guanine nucleotides stimulate production of inositol triphosphate in rat cortical membranes. Biochem. J. 232. 79%W4. Hammer, R., Berrie, C.P., Birdsall. N.J.M., Burgen, A.S.V. and Hulme, E.C. (1980). Pirenzepine distinguishes between different classes of muscarinic receptors. Nature (London) 283, 962. Hammer, R. and Giachetti. A. (1982). Muscarinic receptor subtypes : M, and M, biochemical and functional characterization. Life Sci. 31, 2991-8. Hammer, R.? Giraldo, E., Schiavi, G.B., Monferini, E. and Ladinsky, H. (1986). Binding profile of a novel cardioselective muscarinic receptor antagonist, AF-DX 116, to membrane of peripheral tissues and brain in the rat. Li,fe Sci. 38, 1653-62. Heacock, A.M., Fisher, SK. and Agranoff, B.W. (1987). Enhanced coupling of neonatal muscarinic receptors in rat brain to phosphoinositide turnover. J. Neurochem. 48. 1904-1911. Irvine, R.F., Lechter, A.J., Heslop, J.P. and Berridge. M.J.( 1986). The inositol tris/ betrabisphosphate pathway-demonstration of INS (1,4,5)P, 3-kinase activity in animal tissues. Nature (London) 320, 6314. Jacobson. M.D.. Wusteman, M. and Downes. C.P. (1985). Muscarinic receptors and hydrolysis of inositol phospholipids in rat cerebral cortex and parotid gland. J. Seurochem.
44, 465-72.
Kaufman, P.L.. Wiedman, T. and Robinson, J.R. (1984). Cholinergics. In Pharmacology of fhe Eye (Ed. Sears, M.L.). Pp. 14991. Springer-Verlag: Berlin. Kervalage, A.R., Fraser, CM. and Venter, J.C. (1987). Muscarinic cholinergic receptor structure : molecular biological support for subtypes. Trends Pharmaeol. Sci. 8,426431. Korc. M.. Ackermann, M.S. and Roeske. W.R. (1987). A cholinergic antagonist identifies a subclass of muscarinic receptors in isolated rat pancreatic acini. J. Pharmacol. Exp. Ther. 240, 118-22. Kern. S.J., Martin, M.W. and Harden, T.K. (1983). N-ethylmaleimide-induced alteration in the interaction of agonists with muscarinir cholinergic receptors of rat brain. J. Pharmacol. Exp. Ther. 224, 118-26. Lazareno. S., Kendall, D.A. and Nahorski, S.R. (1985). Pirenzepine indicates heterogeneity of muscarinic receptors linked to cerebral inositol phospholipid metabolism. Neuropharmacology 24, 593-5. Lowry, O.H.. Rosebrough, N.J., Barr, A.L. and Randall, R.J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193. 265-75. Luthin, G.R. and Wolfe, B.B. (1984). Comparison of 3H-pirenzepine and 3H-QNB binding to muscarinic cholinergic receptors in rat brain. J. Pharmacol. Exp. Ther. 228, 648-55. Mallorga, P., Buisson, S. and Sugrue, M.F. (1988). Alpha,-adrenoceptors in the albino rabbit ciliary process. J. Ocular Pharm. 4, 203-214. Miichi. H. and Nagataki. S. (1983). Effects of pilocarpine, salbutamol and timolol on aqueous humor formation in cynomolgus monkeys. Invest. Ophthalmol. Via. Sci. 24, 126975. Munson. P.J. and Rodbard, D. (1980). Ligand: a versatile computerized approach for characterization of ligand-binding systems. Anal. Biochem. 107, 22Ck39. Nathanson, J.A. (1980). Adrenergic regulation of intraocular pressure : identification of beta,-adrenergic-stimulated adenylate cyclase in ciliary process epithelium. Proc. LIutZ. Acad. 8ci. 1T.S.A. 77, 74204. Pagani, F., Schiavone, A., Monferini, E., Hammer, R. and Giachetti, A. (1984). Distinct muscarinic receptors (M, and M,) controlling acid secretion in rodents. Trends Pharmacol. Sci. (Suppl.) 1, 66-8. Polansky. J.R.. Zlock, D.. Brasier,A. and Bloom, E. (1985). Adrenergic and cholinergic receptors in isolated non-pigmented ciliary epthelial cells. Current Eye Res. 4, 517-22. Wang, J.X.. Roeske, W.R.. Gulya, K., Wang, W. and Yamamura, H.I. (1987). ‘H-AF-DX 116 labels subsets of muscarinic cholinergic receptors in rat brain and heart. Lije Sci. 41. 1751-60. Watson, M., Roeske, W.R. and Yamamura, H.I. (1986b). 3H-Pirenzepine and 3H-QNB
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binding to rat cerebral cortical and cardiac muscarinic cholinergic sites. II. Characterization and regulation of antagonist binding to putative muscarinic subtypes. J. Phurmacol. Exp. Ther. 237, 419-27. Watson, M., Yamamura, H.I. and Roeske, W.R. (1983). A unique regulatory profile and regional distribution of 3H-pirenzepine in the rat provide evidence for distinct MM, and M, muscarinic receptor subtypes. Life Sci 32, 369-11. Watson, M., Yamamura, HI. and Roeske, W.R. (1986a). 3H-Pirenzepine and 3H-QPU’B binding to rat cerebral cortical and cardiac muscarinic cholinergic sites. 1. Characterization and regulation of agonist binding to putative muscarinic subtypes. J. Pharmacol. Exp. Ther. 237, 41 l-7. Woodcock, E.A., Leung, E. and McLeod, J.K. (1987). A comparison of muscarinic acetylcholine receptors coupled to phosphatidylinositol turnover and to adenylate cyclase in guinea-pig atria and ventricles. Eur. J. Pharmacol. 133, 283-9. Yamanaka, K., Hashimoto, S. and Muramatsu, I. (1986). Species difference in the muscarinic receptors of the aorta. J. Pharmacol. Exp. Ther. 237, 980-6.
Eye RPS. (1989)
Muscarinic
48, 509-522
Receptors
P. MALLouoA,t Merck
Sharp
of the Albino
R. W.
BABILON,
S. BUISSON*
and Dohme Research Laboratories,
(Received13 May 1988 and accepted
Rabbit
in
AND
West Point,
revised form
Ciliary M. F.
Process SUGRUE
PA 19486,
14 September
USA
1988)
Muscarinic receptor binding sites were identified in membranes prepared from albino rabbit ciliary processes, using the muscarinic antagonist [3H]L-quinuclidinyl benzylate as the radioligand. Analysis of saturation binding experiments demonstrated that [SH]L-quinuclidinyl benzylate bound to an apparent homogeneous population of binding sites with a K, value of 64 PM and a B,,, value of 155 fmol mg-’ protein. Seventy percent (70%) of binding sites showed high affinity for pirenzepine. i.e. belonged to the M, subtype. In contrast, AF-DX 116 was unable to discriminate between subtypes of muscarinic binding sites in this tissue. Carbachol caused a dose-dependent increase in phosphatidylinositol turnover (EC,, = 154 PM) in ciliary processes. A maximum stimulation of 652% of basal activity was obtained following a 45 min incubation with 10 mM carbachol. The potency of muscarinic antagonists to block the carbachol-induced response was comparable to that found for M, receptors in other tissues. Oxotremorine and pilocarpine behaved like partial agonists in this assay. The carbachol-induced increase in phosphatidylinositol turnover was also observed in a suspension of epithelial cells from ciliary processes and it was blocked by atropine ; thus, indicating the presence of muscarinic receptors functionally coupled to phosphatidylinositol turnover in these cells. Key wol-ds : muscarinic receptors ; ciliary process ; phosphatidylinositol turnover ; pirenzepine : AF-DX 116.
1. Introduction
Functional studies, as well as radioligand binding studies using selective muscarinic antagonists like pirenzepine, have indicated the existence of subtypes of muscarinic receptors in brain and peripheral tissues (Hammer, Berrie, Birdsall, Burgen and Hulme, 1980; Hammer and Giachetti, 1982; Pagani, Schiavone, Monferine, Hammer and Giachetti, 1984; Watson, Yamamura and Roeske, 1983). Receptors with a high and a low affinity for pirenzepine were labeled M, and M,, respectively. Recently, the use of a new cardioselective muscarinic antagonist AF-DX 116 has allowed the characterization, in binding and pharmacological experiments, of subtypes of M, receptors as well as M, receptors (Hammer, Giraldo, Schiavi, Monferine and Ladinsky, 1986; Giachetti, Michelett and Montaga, 1986; Korc, Ackermann and Roeske, 1987; Bloom, Halonen, Seaver and Yamamura, 1987b). Pilocarpine and other cholinergic agonists, in addition to their established effect on aqueoushumor outflow and pupil diameter, have been shown to affect aqueoushumor secretion (Kaufman, Wiedman and Robinson, 1984; Miichi and Nagataki, 1983; Gherezghiher, March, Nordquist and Koss, 1986). Becauseof this effect, and the role of the ciliary processin the secretion of aqueoushumor, cholinergic receptors may be present in this tissue. Binding studies using [3H]quinuclidinyl benzilate as a radioligand have indicated the presence of muscarinic receptors on bovine nonpigmented ciliary epithelial ceils (Polansky, Zlock, Brasier and Bloom, 1985). In addition, carbachol is able to stimulate the labeling of phosphatidic acid and phosphatidylinositol (PI) by [““PI in the rabbit ciliary process (Akhtar and Abdel* Faculte des Sciences, t To whom all offprint 0014-4835/89/040509+14
Universiti requests $03.00/O
de Clermont-Ferrand. should be addressed.
63000
Clermont-Ferrand. 0 1989 Academic
France. Press Limited
510
P. MALLORG.4
ET
AL
Latif, 1983). However, a complete characterization of the muscarinic receptors in the ciliary process using selective antagonists has not been reported. With the aim of characterizing the muscarinic receptor population in the rabbit ciliary process, we performed binding and PI turnover studies using selective muscarinic antagonists such as pirenzepine (M,) and AF-DX 116 (Ma). The presence of muscarinic receptors in epithelial cells from this tissue was also investigated.
2. Materials
and Methods
Tritiated L-quinuclidinyl benzilate ([3H]&NB, 3040 Ci mmoll’) and myo-inositol ([3H]INS, 10-20 Ci mmoll’) were purchased from New England Nuclear Corporation (Boston, MA). Atropine, carbachol, oxotremorine sesquifumarate, pilocarpine hydrochloride, inorepinephrine bitartrate and pirenzepine were purchased from Sigma Chemical Company (St. Louis, MO). AF-DX 116 was a gift from Dr M. Wax (Universit’y of Pennsylvania School of Medicine, Philadelphia, PA). The AGl-X8 (109200 mesh) resin was in the formate form and was purchased from Bio-Rad (Richmond, CA). All other reagents were analytical grade and obtained from Sigma Chemical Company or Serva (Heidelberg, FRG). All drugs were dissolved in distilled water or ethanol at a 10 mM concentration and diluted as necessary in the appropriate buffer. Membrane preparation Membrane suspensions were prepared from frozen ciliary processes of adult New Zealand albino rabbits. The animals were sacrificed by an intravenous injection of an overdose of sodium pentobarbital and the ciliary processes dissected as previously described (Nathanson, 1980). Eyes were enucleated, separated from adhering tissue, and the optic nerve cut at its base. Each eye was opened by four incisions starting at the base of the optic nerve and ending 2-3 mm posterior to the limbus. The lens and vitreous were carefully dissected. The opened eye was inverted and placed, cornea down, on a plexiglass holder in a Petri dish filled with ice-cold phosphate-buffered saline (pH 7.4, PBS). The floating ciliary processes from four eyes were transected at their attachment to the iris and the ciliary body, collected in a polystyrene tube and centrifuged at 300 g for 10 min. The supernatant was discarded and the pellet quickly frozen in liquid nitrogen and stored at 80°C until used. Tissue was homogenized in 10 mM Na-K phosphate buffer (pH 7.4) using a Polytron homogenizer (setting 7, 30 set). The homogenate was centrifuged for 10 min at 35006g. The pellet was resuspended in buffer and recentrifuged at 35000g for 10 min. The homogenizationcentrifugation process was repeated once again and the final pellet resuspended in 15 ml of 10 mM Na-K phosphate buffer (pH 7.4) containing 5 IIIM MgCI,. The final suspension was kept on ice until used in the binding assay. Binding assay Binding was done as described previously (Gil and Wolfe, 1985) with the exception that 5 mM MgCl, was included in our assay. Briefly, membranes (109150,ug of protein) were incubated with [‘H]QNB, the drug to be tested or buffer in a final volume of 5 ml. The tubes were incubated for 120 min at 32’C. Incubations were terminated by the addition of 4 ml of cold buffer, rapid filtration under reduced pressure through Filtermats glass fiber filters (Skatron Inc., Sterling, VA) and 2 x 4 ml washes using the Skatron cell harvester (Skatron Inc.). The filters were placed in polyethylene vials containing 3 ml of PCS (Amersham Corp., Arlington Heights, IL) and counted overnight in a liquid scintillation spectrometer (Packard 2000 CA). Specific binding was defined as the difference between total binding and binding in the presence of 10 ,UM atropine. Specific binding represented 89-96 % of total [3H]QNB (50 PM) binding. The apparent dissociation constant (K,) and the maximum number of binding sites (&,) were calculated using the non-linear curve fitting program LIGAND (Munson and Rodbard, 1980). The displacement curves by agonists or antagonists were first analyzed by the non-linear curve fitting program EBDA (McPherson, Elsevier Biosoft, Cambridge, U.K.) which calculated K, values and Hill coefficients (nH). The curves were then analyzed with
OCULAR
MURCARINIC
REC’EPTORS
LIGAND according to a one-site and two-site model. Testing for difference between the models was done by comparing the residual variance of fits to the data by an F test, P < 905 was taken as indicating a significant difference. When a two-site model preferred, dissociation constants K, and K, were calculated as well as the % of high and affinity sites.
511 two and was low
Stimulation of Inositol Phosphates Formation Methodology was similar to that used for rat brain slices by Brown, Kendall and Nahorski (1984). Ciliary processes from 8 to 10 albino rabbit eyes were dissected in ice-cold PBS, as previously described (Nathanson, 1980). The processes were placed in 1 ml of Krebs bicarbonate buffer (KBB) containing 118 mrvr NaCl, 47 mM KCl, 12 mM MgSO,, 25 mM NaHCO,, 1.2 tnM KH,PO,, 1.3 tnM CaCl, and 10 mM glucose, and previously equilibrated with 95% 0,:5% CO, to a final pH of 7.4. The processes were washed twice with KBB by sedimentation and aspiration of the supernatant. After the last aspiration, 25 gl aliquots of the ciliary process suspension (20&300 yg of protein) were placed in polystyrene tubes containing 210~1 KBB. the tubes were then capped and incubated at 37’C in a gently of shaking water bath. After 40 min, [3H]INS and LiCl were added to a final concentration 93 ,UM and 10 mM respectively. After 20 min. antagonists were added and this was followed 10 min later by the addition of agonists. The vials were incubated for a further 45 min. Incubations were stopped by the addition of 694 ml of chloroform : methanol (1: 2, v : v) followed by homogenization of the suspension with a Polytron homogenizer (setting 5, 10 set). After 10 min standing, 031 ml of chloroform and @31 ml of water were sequentially added. The vials were agitated on a vortex mixer and centrifuged at 1OOOg for 5 min to separate phases. A 975 ml aliquot of the upper phase was removed and placed over an Amicon column containing 1 ml of AGi-X8 (formate form). [3H]INS was eluted with 5 ml H,O. and both glycerophosphoinositol and inositol 1: 2 cyclic phosphate eluted with 5 ml of 5 mM sodium tetraborate plus 60 mM ammonium formate. Myo-inositol-l-phosphate (IP,) was eluted with 10 ml of 92 M ammonium formate plus 91 M formic acid. Myo-inositol- 1,4diphosphate (IP,) was eluted with 10 ml of 94 M ammonium formate plus 91 M formic acid and myo-inositol-1,4,5-triphosphate (IP,) as well as myo-inositol-1,3,4-triphosphate with 6 ml of 1 M ammonium formate plus 0.1 M formic acid. No attempt was made to separate the myo-inositol-triphosphate isomers. In the majority of experiments, the inositol phosphates (IP) were not separated, and were eluted together with 5 ml of 61 M formic acid, plus 975 M ammonium formate after the elution with water and tetraborate. Aliquots of the eluates (2 ml) were added to 10 ml of Aquasol 2 (NEN) and counted for radioactivity in a liquid scintillation spectrometer (Packard 460 CD). All experiments were conducted a minimum of three times, and each experimental data point was calculated from the mean of two separate ciliary process incubations. The dose of agonist that produced half maximal stimulation (EC,,), and the dose of antagonist that produced half maximal inhibition (IC,,), were calculated using t,he non-linear regression analysis ALLFIT of the results in d.p.m. (DeLean, Munson and Rodbard, 1978). Ki values were then calculated using the following formula in whirh d is the concentration of agonist :
Ki_Ic,,, A 1+EC,, Epithdial Cell Preparation Epithelial cells from ciliary processes were isolated using a technique derived from that of Nathanson (1980). Briefly, dissected ciliary processes were incubated with agitation at 37°C for 15 min in Dulbecco’s phosphate buffered saline containing trypsin (01 %), and no calcium or magnesium (DPBS) and then allowed to stand until the ciliary processes had sedimented. The supernatant, which contained mainly red blood cells, was discarded. The pellet was resuspended in DPBS and the incubation-sedimentation process repeated twice ; the respective supernatants, which contained epithelial cells, were saved and pooled. Horse serum was added to the supernatants to a final concentration of 10% (v:v). The cell
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ET
AL.
suspension was filtered on a 37 p nylon mesh, to eliminate fragments of blood vessels (CocaPrados and Kondo, 1985), centrifuged at 100 g for 5 min, and the cellular pellet resuspended in KBB. A population of 7540% cells extruding trypan blue was obtained. Samples were taken for histologic study. Protein concentrations were measured by the method of Lowry, Rosebrough, Barr and Randall (1951) using bovine serum albumin as standard. 3. Results
In preliminary experiments, specific binding of [3H]QNB to membranes prepared from ciliary processeswas found to be linearly dependent on the concentration of tissue, up to @05 mg of protein per ml. Concentrations of 002-003 mg ml-’ were used in all subsequentexperiments. Equilibrium binding was achieved within 90 min (data not shown). The specific binding of [3H]QNB to a ciliary process membrane fraction was saturable and of high affinity. Scatchard analysis, as well as non-linear regression analysis of equilibrium binding data indicated that [3H]QNB (Fig. 1) bound to a single population of high affinity binding sites with a Kd of 6*4+@7 pM and a B,,, of 155IfI 2 fmol mg-l protein. The ability of several muscarinic agonists and antagonists to displace [3H]QNB from ciliary processbinding sites was tested. In addition, the effect of GTP (500 ,UM) on the displacement curves for agonists was also studied. The most potent of the three antagonists tested (Table I) was atropine with a Ki of @36 nM, followed by pirenzepine (34 nM) and AF-DX 116 (230 nM). The displacement curves for atropine and AF-DX 116 were best fitted by a one-site model. In contrast, the curve for pirenzepine, for which a Hill coefficient of @69was obtained, was best fitted by a two-site model. The inhibition constants for pirenzepine were 12 and 359 nM, and the relative number of
0.000 4 0
I
I
50
100
Bound
(fmoC
mg-’
O\ 150
proi.)
FIG. 1. Scatchard plot of [aH]&NB binding to eiliaq process membranes. [SH]QNB concentrations ranged from 2 to 1000PM. Each point is the mean of three replicates from a single experiment. The experiment wa8 repeated three times, and a Kb = 64*@7 PM and II 4.X = 155+ 2 fmol mg-’ protein were obtained.
OCULAR
MUSCARINIC
RECEPTORS
TABLE
Inhibitory
I:
potency of various antagonists for speci$c [3H]QNB ciliary process Drug
nH
4
Atropine Pirenzepine AF-DX 116
are the mean of three experimentsf % L represents the percentage of low affinity The concentration of [aH]&NS was 005 nM.
on the inhibitory
bin&q
carhachol carbachol oxotremorine oxotremorine pilocarpine pilocarpine
binding in the rabbit
%L
KL
125 1 -
359*99 --
and are expressed
S.E.M.
30+3
in no.
sites.
TABLE
Ejfect of GTP
4
0~87~004 969kO.10 1.08*@08
036&O-12 3400+900 23000 + 3900
K values
Drug
513
II
potency of various agonists for speci$c [3H]QNB in the rabbit ciliary process
GTP (500 PM)
K, bM)
nH
+ + +
540+110 2900* 1100
048 * @08 951+902
38+3 140+30
070+0.11 OS5+003
309+50 830+ 100
@79f@05 1~01+@05
Values are the mean of at least three experimentsk~.~.~. 905 nM. % L represents the percentage of low affinity sites.
4
KL
W)
NW
71+28 630f260
30000+20000 28000+9000
7f5 -
The
%L 41+9 56515
685 & 588 260,70
48k12 MO+0
330+70 650 + 50
100&O lOOjO
concentration
of [aH]QNS
was
high- and low-affinity sites in t,he ciliary processwere estimated to be 70 and 30%, respectively. Pilocarpine was the only agonist of the three tested for which the competition curve was best fitted by a one-site model (Table II). A Kd of 300 nM was obtained, which was slightly increased to 830 nM when GTP was added to the assay. The Hill coefficient of 679 became 1.01 in the presenceof GTP. Oxotremorine was the most potent of the three agonists with a K, of 38 nM. The displacement curve was best fitted by a two-site model, the inhibition constants being 7 and 685 nM, and the relative number of high- and low-affinity sites being 52 and 48%, respectively. When GTP was added to the assay, the Ki value for oxotremorine was 140nM and the displacement curve best fitted by a single site model. The Hill coefficient was increasedfrom 670 to 065 in the presenceof GTP. The inhibition curves for carbachol were best fitted by a two-site model in the absenceas well as in the presenceof GTP ; the K, value was increased, however, from 540 nM to 2900 nM in the presenceof GTP. The number of low affinity sites was concurrently increased from 41% to 56 %. It is alsoto be noted that the inhibition constant for the high affinity site for carbachol was nine times higher in the presence of GTP (630 nM vs. 71 nM). The Hill coefficient for the two inhibition curves was 648 without and 651 with GTP. A 30 min preincubation of ciliary processeswith [3H]INS and LiCl resulted in 5%
514
I’. MALLORGA
ET
.1L.
of the radioactivity being incorporated into the phospholipids. as indicated by the radioactivity recovered in the organic phase of the chloroform extraction. A preincubation time of 30 min, followed by a 45 min incubation in the presence of agonists, were the conditions chosen in this study because these conditions were found to optimize the stimulation factor by agonists. Similar conditions were used by Brown et al. (1984) for rat brain slices. Longer preincubation periods with [3H]INS (1 and 2 hr), followed by a 45 min incubation with carbachol, have resulted in values for (a) the maximum stimulation by carbachol, (b) the EC’,, for carbachol, and (c) the amount of radioactivity recovered in the IP fract,ion after the 45 min incubation with or without carbachol, comparable to those obtained with a 30 min preincubation. In preliminary experiments we found that’ the total IP labeling was increased by a factor of 1.8 after a 5 min incubation with carbachol (10 mM) (Fig. 2). A stimulation facto1 of 6-9 was obtained after a 45 min incubation (Fig. 3). The distribution of the radioactivity in IP,, IP, and IP, was 74, 23 and 3%. respectively, in the control. and 83, 15 and 2 %, respectively, in the 5 min carbachol-stimulated tissue, indicating a 2, 1.2 and 1.2 stimulation fact’or for IP,, IP, and IP,, respectively. After a 45 min incubation in the presence of carbachol, stimulation factors of 8, 1.1 and 2.0 were found for IP,, IP, and IP,, respectively (data not shown). To further characterize the carbachol response, experiments were conducted without separating IP,, IP, and IP,, which were eluted together, as described in the Materials and Methods section.
0
1
, 5 Fraction
No.
Fro. 2. Elution profile of the inositol phosphates. Ciliary processes were incubated with carbachol for 5 min and the IP extracted as described in Materials and Methods. The buffers used for the elution were : (a) 5 ml of H,O; (b) 5 ml of 5 rniu sodium tetraborate+ 60 rniv ammonium formate; (c) 10 ml of @2 M ammonium formate +@l M formic acid; (d) 10 ml of 0.4 M ammonium formate+O.l M formic acid; (e) 6 ml of 1 M ammonium formate +@l M formic acid.
OCULAR
MUSCARINIC
I 5
6
RECEPTORS
I 4
515
I 3 -Log
I 2
I
(cone)
Fm. 3. Stimulation of [3H]INS metabolism in ciliary processes by cerbachol. Ciliary processes were incubated in the presence of [sH]INS and LiCl for 30 min, and then in the presence of carbachol for 45 min. The [3H]IP were extracted and separated as described in the text. Each value is expressed as a percentage of the maximum stimulation obtained with 10 mM cerbachol. and is the mean* S.E.M. of three replicates from a single experiment. The experiment was repeated three times and a maximum stimulation of 652 + 126 % of basal. and an EC,, = 154 + 53 ,uM were obtained. A basal value of 4179k215 dpm per assay was obtained.
120 -
80 -
40 -
20 -
0 II
I
I
I
IO
9
8 -Log
7 (cone)
6
5
4
3
FIG. 4. Inhibition of carbachol stimulation of [3H]INS metabolism in ciliary processes by atropine (W-W), pirenzepine (A-A), AF-DX 116 (0-O) an d mecamylamine (0-O). Ciliary processes were incubated in the presence of the antagonists for 10 min and then in the presence of carbachol (1 m&r) for a further 45 min. Each value is the me+s.n.M. of duplicate determinations from three different experiments. Inhibition constants of 1.15f007 nM, 47 & 7 nM and 637 k 120 nM were obtained. A value of 53 149 + 7255 dpm per assay was obtained in the presence of carbachol.
516
P. MALLORGA
ET
AL.
Carbachol caused a dose-dependent increase in the formation of IP. A maximum stimulation of 652% of basal IP formation was obtained with 10 mM carbachol, the EC,, being 154 FM (Fig. 3). Pilocarpine and oxotremorine caused a small increase in 1P formation, reaching stimulations of 5 % and 10 % of that of carbachol, respectively, at a 1 mM concentration (data not shown). EC,, values for pilocarpine and oxotremorine were not calculated because of the small stimulation factors. The stimulation of IP formation by a submaximal concentration of carbachol (1 mM) was blocked in a dose-dependent fashion by atropine, pirenzepine and AF-DX 116, but not by the nicotinic antagonist, mecamylamine at concentrations up to 100,uM (Fig. 4). Inhibition constants of 1.15, 47 and 637 nM were obtained for atropine, pirenzepine and AF-DX 116, respectively. We have shown in a separate study (Mallorga, Buisson and Sugrue, 1908) that norepinephrine is able to increase IP formation in the ciliary process, and that a maximum stimulation (430 %) is obtained with 1 mM norepinephrine. When maximal concentrations of carbachol and norepinephrine were assayed alone or in combination (Table III), an almost perfect additivity was obtained in the effects of the two agonists on IP formation. When epithelial cells, prepared from ciliary processes were incubated with [3H]INS and carbachol (1 mM), a stimulation of 384 % in IP accumulation was obtained when compared to non-stimulated cells (Table IV). The stimulation by carbachol was blocked by 10 ,UM atropine, which was previously shown to completely block the carbachol response in the intact ciliary process (Fig. 4). III.
TABLE
Additivity
of the carbachol
and the norepinephrine stimulations in the diary process of the rabbit
Addition
(loncentration
Stimulation
(mM)
(“h of Control)
1
385 * 20
norepinephrine carbachol norepinephrine carbachoi
of [3HJZP accumulation
+
10
825k
1 10
1093+99
13
Results are the mean of three experimentsf S.E.M. Ciliary processes (300 /cg of protein) were incubated in the presence of [aH]INS and LiCl for 30 min and then in the presence of the agonists for 45 min. The [3H]IP were extracted and separated as described in the text. A basal value of 13647 + 1092 dpm per assay was obtained in the absence of agonist. TABLE
Stimulation
by carbachol
of [3HlIP
Addition carbachol carbachol+ atropine
f ormation Concentration (PM) 1000 1000 10
IV
in
epithelial
cells
of the diary
process.
Stimulation (“/ of Control) 384& 18 91&l
Results are the mean of three separate experiments? S.E.M. Cells (100 fig of protein) were incubated with [3H]INS as indicated for the ciliary process in Materials and Methods. A basal value of 12 369 dpm per assay was obtained in the absence of agonist.
OC’ULBR
MI’SC’ARINIC’
RECEPTORS
517
4. Discussion In rabbit ciliary process membranes, [3H]QNB bound with high affinity to a single population of binding sites. The K, value obtained in this study (6.4 PM) was very similar to values obtained for rabbit peripheral lung (Bloom et al., 1987a) and rat brain (Korn. Martin and Harden, 1983), but lower than the Kd values observed for the bovine non-pigmented epithelium of the ciliary process (120 PM, Polansky et al., 1985) and the rabbit iris (39 PM% Akhtar, Honkamen, Howe and Abdel-Latif, 1987). Variations in methodology, including the temperature and the receptor concentration used in the assay, may be the cause of these differences. The rabbit ciliary process was found to be richer in [3H]QNB binding sites than the rabbit aorta (Yamanaka. Hashimoto and Muramatsu, 1986) or the peripheral lung (Bloom et al., 1987a), but poorer than the rat brain (Fisher and Bartus, 1985; Luthin and Wolfe, 1984) or the rat atria (Dunlap and Brown, 1983). Of particular interest are t,he 14-17-fold great’er concentrations found in the rabbit iris sphincter (Akhtar et al.. 1987) and the primate ciliary muscle (Gabelt, Polansky and Kaufman, 1987). In the rabbit iris sphincter, the muscarinic receptors belong to the M, subtype (Akhtar et al., 1987). In contrast, the majority of the [3H]QNB binding sites of the rabbit ciliary process (70%) possess a high affinity for the M, selective antagonist pirenzepine (12 nM) and, therefore, probably belong to the M, subtype. The muscarinic M, receptors have been subdivided into two subtypes using functional (GiarhetC et al., 1986) as well as radioligand binding studies (Hammer et al., 1986). The M, cardiac receptor shows, in binding studies, a slightly lower affinity for pirenzepine (2-4-fold, Gil and Wolfe, 1985; Hammer et al., 1980) than the M, glandular subtype (rat parotid and lacrimal glands) do and, therefore, pirenzepine is not useful in distinguishing between M, receptor subtypes. AF-DX 116. a cardioselective muscarinic antagonist (Giachetti et al., 1986), has a 30-50 times greater affinity for the M, cardiac binding site than for the glandular site and an intermediate affinity for M, binding sites (Hammer et al., 1986; Bloom et al., 198713). In our study, AF-DX 116 was unable to discriminate between subtypes of muscarinic M, receptors as indicated by a Hill coefficient of 1. Thus, it is difficult,, despite the use of pirenzepine and AF-DX 116, to assign a subtype to the 30% of M, binding sites detected on ciliary process membranes. In the cerebral cortex, which is also a tissue rich in M, binding sites (65-75 %), t,he analysis of AF- DX 116 displacement curves has indicated a homogeneous population of binding sites in some studies (Hammer et al., 1986; Bloom et al., 1987b) and multiple sites in others (Korc et al., 1987; Watson, Roeske and Yamamura 1986b). It appears, therefore, t’hat in tissues rich in M, binding sit*es, AF-DX 116 is not always able to differentiate muscarinic receptor subtypes. The Ki value found for AF-DX 116 in this study (230 nM) was comparable to Ki values reported for the rat cerebral cortex (139-176 nM) when [3H]QNB was used as radioligand (Bloom et al., 1987b; Watson et al.. 1986b) as well as when [3H]Nmethylscopolamine (3H-NMS) was used (440-764 nM, Korc et al., 1987; Hammer et al., 1986). Recent studies using [“HIAF-DX 116 as radioligand have suggesbed t,he presence of M, cardiac as well as MM, glandular sites, in addition to the M, sites, in the cerebral cortex (Wang, Roeske, Gulya, Wand and Yamamura, 1987). The presence of the three binding sites in the ciliary process is also a possibility. The Hill coefficients for the inhibition of [3H]QNB binding to ciliary process membranes by muscarinic agonists were less than unity, as has been described previously in several studies using various tissues (Birdsall, Hulme and Burgen. 1980;
518
P.MALLORGAETAL.
Korn et al., 1983: Evans, Hepler, Masters, Brown and Harden, 1985; Watson, Yamamura and Roeske, 1986a). The inhibition curves were best fitted by a two-site model for carbachol and oxotremorine, but not for pilocarpine. Earlier studies have indicated that multiple muscarinic binding sites recognized by agonists (high and low) are not identical to those identified by antagonists (M, and M, subtypes) (Gillard, Waelbroeck and Christophe, 1987). M, and M, subtypes probably represent two separate proteins (Kervalage, Fraser and Venter, 1987) and the use of selective radioligands like [3H]pirenzepine and [3H]AF-DX 116 will help in their characterization. In contrast, the high and low affinity sites detected by agonists may result from the existence of different conformational states of the same receptor (Birdsall et al., 1980 ; Watson et al., 1986a) regulated by the interaction of the receptor with a GTP-binding protein. Guanine nueleotides have been shown to lower the affinity of several muscarinic agonists for [3H]QNB as well as [“H]NMS binding sites. This effect is more pronounced in M,-rich tissues, like heart and cerebellum, than in RI,-rich tissues, like the cerebral cortex or the hippocampus (Korn et al., 1983; Watson et al., 1986a). In ciliary process membranes, the observed GTP-induced shifts in affinity for agonists were slightly greater (1-3-fold) than those observed in cerebral cortex membranes, but smaller (3.lo-fold) than those observed in cardiac membranes (Korn et al., 1983; Watson et al., 1986a). This supports the hypothesis that a mixture of M, and M, binding sites are present in this tissue. In 1321Nl human astrocytoma cells and in the cerebral cortex, a GTP-binding protein is probably involved in the muscarinic receptor-mediated stimulation of a phospholipase C-induced breakdown of phosphoinositides, which results in the increased synthesis of IP (Evans et al., 1985; Gonzales and Crews, 1985). We have found in this study that carbachol causes a large increase in the formation of tritiated IP in rabbit ciliary processes prelabelled with [3H]INS. A much smaller effect was observed by Akhtar and Abdel-Latif (1983) in the rabbit ciliary process, but only one dose of agonist (50,~~) was used in their study. This carbachol-induced effect has been previously observed by many groups in several tissues (for review, see AbdelLatif, 1986) and arises from the increased hydrolysis of phosphatidylinositol-4,5bisphosphate, which generates two second messengers : IP, and 1,2-diacylglycerol. IP, is responsible for the intracellular mobilization of Ca2’ and 1,2-diacylglycerol for the stimulation of protein kinase C (Berridge, 1984). IP,, which in some cells can give rise to INS(1,3,4,5)P, and INS(1,3,4)P, (Irvine, Lechter, Heslop and Berridge, 1986), is eventually hydrolyzed to IP, which in turn is hydrolyzed to IP,. The breakdown of IP, is inhibited by lithium, and the use of this ion causes an amplification of the signal (IP accumulation) generated by the stimulation of the receptor. In our experiments, the use of a long incubation time (45 min) in the presence of LiCl may explain the large accumulation of radioactivity in the IP, peak. The EC,, value found for carbachol in this study is comparable to values obtained in Ml-containing tissues (46200 pM, Fisher and Bartus, 1985; Lazareno, Kendall and Nahorski, 1985; Gil and Wolfe, 1985) but is much greater than those found in M,containing tissues (2-13 ,UM, Akhtar et al., 1987; Woodcock, Leung and McLeod, 1987; Gil and Wolfe, 1985), as well as for a pure M, cardiac receptor expressed in hamster ovary cells (6 ,UM, Ashkenazi et al., 1987). The very small stimulation of PI turnover observed with oxotremorine in the ciliary process (10 % of carbachol maximum response) is also comparable to that reported previously for M,-rich tissues ((r22 %, Fisher and Bartus, 1985; Gil and Wolfe, 1985), in contrast to the M, cardiac tissues (27-40%, Fisher and Bartus, 1985; Woodcock et al., 1987) and the M, glandular tissues (41-lOO%, Gil and Wolfe, 1985; Jacobson, Wusterman and
OCtTLAR
MVSCARINIC
REC’EPTORS
519
Downes, 1985). Based on these results with agonists, the muscarinic receptor coupled to PI turnover in the ciliary process would appear to be of the M, subtype. This finding was confirmed using three antagonists as results from agonist studies can be affected by the presence of spare receptors. However, the somewhat lower affinity of pirenzepine for the PI turnover-coupled receptor than for M, binding sites would suggest again the presence of M, receptors in this t,issue. Although affinities of antagonists in the PI turnover assay correlated well with those found in the binding assay, discrepancies were observed with agonsits. In particular, oxotremorine and pilocarpine, which possess higher affinities than carbachol for [3H]QNB binding sites, are much less effective than carbachol in stimulating IP accumulation. In earlier studies (Fisher and Bartus, 1985; Jacobson et al., 1985), it has been shown that pilocarpine and oxotremorine, in addition to thei weak agonist activity, were able to inhibit carbachol-induced IP accumulation in rat brain cortex slices; hence, they can be considered partial agonists. We have confirmed this finding by observing inhibitions of 81+ 1% and 84+ 4 % by pilocarpine (1 mM) and oxotremorine (1 mM), respectively, of IP accumulation induced by 1 mM carbachol in the ciliary process (data not shown). Another notable difference was the lower potency of carbachol (EC,, = 154 pM) to stimulate PI turnover than to displace [3H]QNB (Ki = 3 ,UM in the presence of GTP). However, carbachol consistently showed multiple affinity states in displacing [3H]QNB from ciliary process membranes and if the low affinity state (Ki N 30 ,UM) is the one coupled to PI turnover, then only a difference of 5-fold exists between the potency of carbachol in the two assays. Differences (24 ,UM vs. 170 ,UM ; Freedman, Harley and Iversen 1988) as well as good agreement (250 ,UM vs. 345 PM: Heacock, Fisher and Agranoff. 1987) have been observed for the rat cerebral cortex. It is not clear why these anomalies exist. However, it is to be noted that binding studies were performed with membrane suspensions, whereas PI turnover studies employed slices of intact tissue. Intact tissue has to be used to study PI turnover because, in preliminary experiments, it was observed that carbachol stimulation was totally lost by homogenizing the ciliary processes (data not shown). Another possibility not tested in this study is that after a prolonged incubation with the tissue, Li+ ions may lower the potency of muscarinic agonists to stimulate PI turnover. None of these possibilities can be completely ruled out, but the good agreement observed for the muscarinic antagonists suggest that technical differences, rather than different receptors, may be the reason for these discrepancies. Muscarinic receptors of the ciliary process could be associated with epithelial cells, blood vessels or connective tissue cells. Our finding that muscarinic receptors are functionally coupled to phosphoinositide metabolism in epithelial cells suggests that these receptors may be involved in the secretion of aqueous humor. More interestingly, the presence of a majority of M, receptors in this tissue, in contrast to the presence of the M, subtype in the iris sphincter muscle, raises the possibility of a selective pharmacological action on secretion. Support to this hypothesis awaits the development of more selective M, and M, agonists and antagonists. In any event, the demonstration of muscarinic receptors on epithelial cells may help to explain why muscarinic agonists can alter aqueous humor secretion. However, they do not explain why increases as well as decreases have been observed (for review see Kaufman et al., 1984). Further studies are needed to determine which receptor subtypes are present on the epithelial cells and particularly on pigmented versus non-pigmented epithelium and whether these receptors can be associated with a physiological or pharmacological effect on aqueous humor secret,ion.
520
P. MALLORGA
ET AL
ACKNOWLEDGMENTS The authors wish to thank
Maryanne
Olkowski
for the preparation
of the manuscript.
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MUSCARINIC
RECEPTORS
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