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Effects of cyproheptadine and pizotifen on central muscarinic receptors
European Journul of Phamacolog: 195 (1991) 403-405 i 1991 Elsevier Science Publishers B.V. 0014.2999/91/$03.50
403
ADONIS 0014299991003022
EIJP 20797
Short communication
and pizotifen on central Mary H. Richards Marion Merrell Dow Research Imtitutc
16 Rue d’Ankara.
67084 Strashourg
Chde.v. France
Received 29 January 1991, accepted 5 February 1991
The affinities of cyproheptadine, pizotifen and ( k)-quinuclidinyl xanthane-9-carboxylate hemioxylate (QNX) were determined at muscarinic autoreceptors and postsynaptic (IPl formation) receptors in rat hippocampal slices. The affinity values for QNX were 8.2 and 8.5 respectively. Cyproheptadine and pizotifen were less potent than QNX. Pizotifen was slightly (tfold) less active at antagonizing IPI formation than blocking the autoreceptors whereas cyproheptadine was equally active at antagonizing the two hippocampal muscarinic receptors. Muscarinic
receptors; Cyproheptadine:
Pizotifen
1. Introduction
2. Materials and methods
Recently, the anti-conflict effect of cyproheptadine, nominally a S-HTz antagonist with moderate to high affinity for muscarinic and histamine receptors (Leysen et al., 1981), was attributed to its muscarinic receptor blocking properties (Ketelaars and Bruinvels, 1989). Cyproheptadine was subsequently found to display high affinities but no selectivity among three muscarinic receptor subtypes in peripheral tissues (El&e et al., 1989). In the present communication, cyproheptadine and pizotifen. which has a binding profile similar to that of cyproheptadine (Leysen et al., 1981). are compared with a muscarinic receptor antagonist, ( + )-3.quinuclidinyl xanthane-9-carboxylate hemioxylate (QNX), for their effects on two models of central muscarinic receptors, muscarinic autoreceptors and postsynaptic receptors in rat hippocampus. Muscarinic autoreceptors were recently found to be pharmacologically similar to muscarinic M, receptors in the heart (Richards, 1990a,b). Muscarinic receptors linked to inositol monophosphate formation are located, at least in part, on postsynaptic elements in the hippocampus (Smith et al., 1989) and may include mt.dtiple subtypes (Richards, 1990a). The results obtained in these two models of central muscarinic receptors show that none of the three antagonists displayed seiectivity in these tests.
Detailed descriptions of hippocampal slice preparation and the experimental conditions for determining inositol-monophosphate (IPl) formation and muscarinic autoreceptor activation have been published (Richards. 1990a.b) and will be briefly described here.
Correspondence to: M.H. Richards. Marion Merrell Dow Research
Institute. 16 Rue d’Ankara. 67084 Strasbourg CCdex. France.
2.1. Tissue preparatiotz
Transverse prepared
hippocampal
from male
slices. 0.4 mm thick. were
Sprague-Dawley
rats (250-350
g)
and kept in a physiological buffer (see below) gassed with carbogen (5% CO,-95% 0,) to maintain the pH at 7.4. 2.1.1. A utoreceptor assqHippocampal slices from one rat were suspended in 10 ml of buffer containing 0.1 PM [‘Hlmethylcholine chloride and incubated 15 min in a shaking bath at 37°C. The slices were then rinsed and placed in 10 glass superfusion units, 2 slices per unit. After 30 min superfusion with buffer containing 10 PM hemicholinium-3. the slices were subjected to three periods of stimulation. each at 1 Hz, 120 pulses. The first stimulation period (151) was to verify that all slices were responding. Three or four concentrations of carbachol were added 14 min before S2 to the superfusing media of eight units. One concentration of antagonist was added 14 min before S3 to the carbachol-containing media and to the two units superfused with buffer. At the end of the experiment. the tritium remaining in the slices and that collected in fractions of superfusate was determined by liquid scin-
Effhtk c~~~fficie~lt~~fr~~cti~~nalrelease r:FFc’>) \tere c;~~c~iI~~Fed ~Ricbards. 1990a) and the S&‘Sl OFS-1 Sl ratios \vert determinrd. These ratios were then plotted rqainst log,,,concentration of the agonist and r’ :qparent p.4 J values were calculated (Schlicker and othert, 198 a ).
The formation of [ ‘Hlinositol monophosphate in in&vi&tol bippocampal slices. Iahelled with [ “Hlmyo-in&to1 and incubated with antagonists for 25 min before Fhe addition of carbachof. was determined by anion exchange cbr~~tl~at~~gr~ph~.The antagonist IC,,, values were determined by plotting the inhibition curves obtained wiFh increasing concentrations of an antagonist in slices exposed to 1 mM carbachol and pK, values were calculated as described by Hawcock et al. (1986).
TABLE 1 Affinity vnlurs for QNX. c~pr~~h~ptadin~ and pizotifen at muxurinic autoreceptors and postsynaptic receptors in rat hippocampus. The number of experiments (n) is indicated in parentheses. Antagonists
Affinity values Autoreceptors
IPI formation
~PP- PA 2
DK.
n,
QNX
X.lRI!zO.07(6)
8.52+0.14(4)
1.05+0.11
C‘yprohcptadinr
7.48k0.13
(4)
7.55+0.14(4)
1.05rtO.O8(4)
Pizotifen
7.5OkO.14 (4)
7.16+0.14(5)
0.94*0.12(5)
(4)
formation of 1P1 induced by 1 mM carbachol. The pKi values and Hill coefficients (n,) are listed in table 1.
4. Discussion Xfvo-[2- “H(N )]inosiFol. 15 Ci/mmol. was purchased from‘ American Radiolabeled Chemicals (U.S.A.). ]MethyI- ’ Hfchohne chloride was purchased from A~~e~harn (France). C~~rohe~tadine was a gift from Merck. Sharpe & Dohme (U.K.). Pizotifen was a gift from Sandoz Products (U.K.). ( -t )-3-Quinuclidinyl santhanc-9-carbosylate hemioxylate was from Research Biochemicafs (U.S.A.). The composition of the physioIogical buffer was fin mM): NaCI 118. KC’I 5.0, CaCl? 1.3. KH,PO, 1.0. MgSO, 1.2. NaHCOj 25. glucose 10.
3. Results Carbachol had no effect on the basal overflow of tritium but concentration dependently inhibited the electrically stimulated release of tritium from rat hippocampal slices prelabelled with [ ‘HIcholine. The maximum inhibition. at 10 PM carbachol. was 84 + 5% (n = 8). The mean pD1 value (negative log EC,,>) was 6.02 * 0.03 (n = 14). Superfusion with 100 nM cyproheptadine, 100 nM pizotifen or 5 nM QNX had no effect on either basal overflow or the stimulated release of tritium. Each of the three antagonists dextrally shifted the concentration-response curves to carbachol. The apparent pA, values are listed in table 1. Carbachol stimulated the formation of IPl in rat hippocampal slices in a concentration-dependent manner. The maximum response was obtained with 1 mM carbachol and averaged 1986 + 137% above basal formation (dpm in unstimulated slices = 654 + 115, n = 8). The mean pD, value was 4.76 + 0.10 (n = 8). The highest concentration tested of each antagonist had no effect on the basal formation of IPI. Increasing concentrations of the three antagonists inhibited the
QNX has been reported to be 16 times more active in displacing the non-selective ligand [“H]QNB from specific binding sites in ventricular muscle (dog, rat) than from rabbit striatal membranes (Gibson et al., 1983). However. Hawcock et al. (1986) found that QNX did not distinguish among muscarinic receptors in rat brain, superior cervical ganglia, ileum or atrium. In two modds of central muscarinic receptors, we found QNX to be a potent but non-seiective antagonist (table 1). Cyproheptadine and pizotifen were five times less potent than QNX at antagonizing responses due to activated muscarinic autoreceptors in rat. hippocampus (table 1). The !I-HT, antagonists were 10 to 20 times less active than QNX at in~biting the formation of IPl induced by carbachol in hippocampal slices. The data derived from the antagonism of the latter response gave rise to Hill coefficients close to unity for all three compounds. None of the antagonists displayed more than two-fold selectivity between the two hipp~amp~ receptors. Because some putatively selective muscarinic receptor antagonists can clearly distinguish between these two receptors (Richards, 1990a), it may be concluded that none of the three compounds, QNX, cyproheptadine or pizotifen, are selective in these tests. Although it has been suggested that the anti-conflict effects of cyproheptadine are due to activity at central M, receptors (Ketelaars and Bruinvels. 1989), the present results obtained with in vitro models of central muscarinic receptors appear to suggest that this may not be the case. Cyproheptadine was as effective as an antagonist at rat hippocampal muscarinic autoreceptors (putative M, receptors, Richards, 1990a,b) as at muscarinic postsynaptic receptors mediating the formation of IPl.
405
Acknowledgement The excellent technical assistance of Ms. Martine Schneider is acknowledged with pleasure.
References Eltze. M.. G. Lambrecht and E. Mutschler, 1989, Cyproheptadine displays high affinity for muscarinic receptors but does not discriminate between receptor subtypes, European J. Pharmacol. 173, 219. Gibson, R.E., W.J. Rzeszotarski, W.C. Eckelman. E.M. Jagoda, D.J. Weckstein, and R.C. Reba, 1983. Differences in affinities of muscarinic acetylcholine receptor antagonists for brain and heart receptors, B&hem. Pharmacol. 32, 1851. Hawcock, T.. S. Lazareno and F. Roberts. 1986, Assessment of the muscarinic subtype selectivity of ( f )-3-quinuclidinyl xanthene-9carboxylate hemioxalate hydrate (QNX). Br. J. Pharmacol. 88. 357P. Ketelaars. C.E.J. and J. Bruinvels, 1989, The anti-conflict effect of
cyproheptadine is not mediated by its 5-hydroxytryptamine antagonistic property, Life Sci. 44. 1743. Leysen. J.E.. F. Awouters. L. Kennis. P.M. Laduron. J. Vandenherk and P.A.J. Janssen. 1981, Receptor binding profile of R41468. a novel antagonist at 5-HT2 receptors, Life Sci. 28. 1015. Richards. M.H.. 1990a. Rat hippocampal muscarinic autoreceptors are similar to the M2 (cardiac) subtype: comparison with hippocampal Ml. atrial M2 and ileal M3 receptors. Br. J. Pharmacol. 99, 753. Richards. M.H., 1990b. Relative potencies of agonists and differential sensitivity to N-ethylmaleimide on muscarinic autoreceptors and postsynaptic receptors in rat hippocampus. J. Pharmacol. Exp. Ther. 255. 83. Schlicker, E. and M. Giithert. 1981. Antagonist properties of quipazinc at presynaptic serotonin receptors and alpha-adrenoceptors in rat brain cortex slices, Naunyn-Schmeideb. Arch. Pharmacol. 317. 204. Smith. C.J., J.A. Court. A.B. Keith and E.B. Perry, 1989. increases in muscarinic stimulated hydrolysis of inositol phospholipids in rat hippocampus following choline& deafferentation are not paralleled by alterations in choline+ receptor density. Brain Res. 415. 317.
403
ADONIS 0014299991003022
EIJP 20797
Short communication
and pizotifen on central Mary H. Richards Marion Merrell Dow Research Imtitutc
16 Rue d’Ankara.
67084 Strashourg
Chde.v. France
Received 29 January 1991, accepted 5 February 1991
The affinities of cyproheptadine, pizotifen and ( k)-quinuclidinyl xanthane-9-carboxylate hemioxylate (QNX) were determined at muscarinic autoreceptors and postsynaptic (IPl formation) receptors in rat hippocampal slices. The affinity values for QNX were 8.2 and 8.5 respectively. Cyproheptadine and pizotifen were less potent than QNX. Pizotifen was slightly (tfold) less active at antagonizing IPI formation than blocking the autoreceptors whereas cyproheptadine was equally active at antagonizing the two hippocampal muscarinic receptors. Muscarinic
receptors; Cyproheptadine:
Pizotifen
1. Introduction
2. Materials and methods
Recently, the anti-conflict effect of cyproheptadine, nominally a S-HTz antagonist with moderate to high affinity for muscarinic and histamine receptors (Leysen et al., 1981), was attributed to its muscarinic receptor blocking properties (Ketelaars and Bruinvels, 1989). Cyproheptadine was subsequently found to display high affinities but no selectivity among three muscarinic receptor subtypes in peripheral tissues (El&e et al., 1989). In the present communication, cyproheptadine and pizotifen. which has a binding profile similar to that of cyproheptadine (Leysen et al., 1981). are compared with a muscarinic receptor antagonist, ( + )-3.quinuclidinyl xanthane-9-carboxylate hemioxylate (QNX), for their effects on two models of central muscarinic receptors, muscarinic autoreceptors and postsynaptic receptors in rat hippocampus. Muscarinic autoreceptors were recently found to be pharmacologically similar to muscarinic M, receptors in the heart (Richards, 1990a,b). Muscarinic receptors linked to inositol monophosphate formation are located, at least in part, on postsynaptic elements in the hippocampus (Smith et al., 1989) and may include mt.dtiple subtypes (Richards, 1990a). The results obtained in these two models of central muscarinic receptors show that none of the three antagonists displayed seiectivity in these tests.
Detailed descriptions of hippocampal slice preparation and the experimental conditions for determining inositol-monophosphate (IPl) formation and muscarinic autoreceptor activation have been published (Richards. 1990a.b) and will be briefly described here.
Correspondence to: M.H. Richards. Marion Merrell Dow Research
Institute. 16 Rue d’Ankara. 67084 Strasbourg CCdex. France.
2.1. Tissue preparatiotz
Transverse prepared
hippocampal
from male
slices. 0.4 mm thick. were
Sprague-Dawley
rats (250-350
g)
and kept in a physiological buffer (see below) gassed with carbogen (5% CO,-95% 0,) to maintain the pH at 7.4. 2.1.1. A utoreceptor assqHippocampal slices from one rat were suspended in 10 ml of buffer containing 0.1 PM [‘Hlmethylcholine chloride and incubated 15 min in a shaking bath at 37°C. The slices were then rinsed and placed in 10 glass superfusion units, 2 slices per unit. After 30 min superfusion with buffer containing 10 PM hemicholinium-3. the slices were subjected to three periods of stimulation. each at 1 Hz, 120 pulses. The first stimulation period (151) was to verify that all slices were responding. Three or four concentrations of carbachol were added 14 min before S2 to the superfusing media of eight units. One concentration of antagonist was added 14 min before S3 to the carbachol-containing media and to the two units superfused with buffer. At the end of the experiment. the tritium remaining in the slices and that collected in fractions of superfusate was determined by liquid scin-
Effhtk c~~~fficie~lt~~fr~~cti~~nalrelease r:FFc’>) \tere c;~~c~iI~~Fed ~Ricbards. 1990a) and the S&‘Sl OFS-1 Sl ratios \vert determinrd. These ratios were then plotted rqainst log,,,concentration of the agonist and r’ :qparent p.4 J values were calculated (Schlicker and othert, 198 a ).
The formation of [ ‘Hlinositol monophosphate in in&vi&tol bippocampal slices. Iahelled with [ “Hlmyo-in&to1 and incubated with antagonists for 25 min before Fhe addition of carbachof. was determined by anion exchange cbr~~tl~at~~gr~ph~.The antagonist IC,,, values were determined by plotting the inhibition curves obtained wiFh increasing concentrations of an antagonist in slices exposed to 1 mM carbachol and pK, values were calculated as described by Hawcock et al. (1986).
TABLE 1 Affinity vnlurs for QNX. c~pr~~h~ptadin~ and pizotifen at muxurinic autoreceptors and postsynaptic receptors in rat hippocampus. The number of experiments (n) is indicated in parentheses. Antagonists
Affinity values Autoreceptors
IPI formation
~PP- PA 2
DK.
n,
QNX
X.lRI!zO.07(6)
8.52+0.14(4)
1.05+0.11
C‘yprohcptadinr
7.48k0.13
(4)
7.55+0.14(4)
1.05rtO.O8(4)
Pizotifen
7.5OkO.14 (4)
7.16+0.14(5)
0.94*0.12(5)
(4)
formation of 1P1 induced by 1 mM carbachol. The pKi values and Hill coefficients (n,) are listed in table 1.
4. Discussion Xfvo-[2- “H(N )]inosiFol. 15 Ci/mmol. was purchased from‘ American Radiolabeled Chemicals (U.S.A.). ]MethyI- ’ Hfchohne chloride was purchased from A~~e~harn (France). C~~rohe~tadine was a gift from Merck. Sharpe & Dohme (U.K.). Pizotifen was a gift from Sandoz Products (U.K.). ( -t )-3-Quinuclidinyl santhanc-9-carbosylate hemioxylate was from Research Biochemicafs (U.S.A.). The composition of the physioIogical buffer was fin mM): NaCI 118. KC’I 5.0, CaCl? 1.3. KH,PO, 1.0. MgSO, 1.2. NaHCOj 25. glucose 10.
3. Results Carbachol had no effect on the basal overflow of tritium but concentration dependently inhibited the electrically stimulated release of tritium from rat hippocampal slices prelabelled with [ ‘HIcholine. The maximum inhibition. at 10 PM carbachol. was 84 + 5% (n = 8). The mean pD1 value (negative log EC,,>) was 6.02 * 0.03 (n = 14). Superfusion with 100 nM cyproheptadine, 100 nM pizotifen or 5 nM QNX had no effect on either basal overflow or the stimulated release of tritium. Each of the three antagonists dextrally shifted the concentration-response curves to carbachol. The apparent pA, values are listed in table 1. Carbachol stimulated the formation of IPl in rat hippocampal slices in a concentration-dependent manner. The maximum response was obtained with 1 mM carbachol and averaged 1986 + 137% above basal formation (dpm in unstimulated slices = 654 + 115, n = 8). The mean pD, value was 4.76 + 0.10 (n = 8). The highest concentration tested of each antagonist had no effect on the basal formation of IPI. Increasing concentrations of the three antagonists inhibited the
QNX has been reported to be 16 times more active in displacing the non-selective ligand [“H]QNB from specific binding sites in ventricular muscle (dog, rat) than from rabbit striatal membranes (Gibson et al., 1983). However. Hawcock et al. (1986) found that QNX did not distinguish among muscarinic receptors in rat brain, superior cervical ganglia, ileum or atrium. In two modds of central muscarinic receptors, we found QNX to be a potent but non-seiective antagonist (table 1). Cyproheptadine and pizotifen were five times less potent than QNX at antagonizing responses due to activated muscarinic autoreceptors in rat. hippocampus (table 1). The !I-HT, antagonists were 10 to 20 times less active than QNX at in~biting the formation of IPl induced by carbachol in hippocampal slices. The data derived from the antagonism of the latter response gave rise to Hill coefficients close to unity for all three compounds. None of the antagonists displayed more than two-fold selectivity between the two hipp~amp~ receptors. Because some putatively selective muscarinic receptor antagonists can clearly distinguish between these two receptors (Richards, 1990a), it may be concluded that none of the three compounds, QNX, cyproheptadine or pizotifen, are selective in these tests. Although it has been suggested that the anti-conflict effects of cyproheptadine are due to activity at central M, receptors (Ketelaars and Bruinvels. 1989), the present results obtained with in vitro models of central muscarinic receptors appear to suggest that this may not be the case. Cyproheptadine was as effective as an antagonist at rat hippocampal muscarinic autoreceptors (putative M, receptors, Richards, 1990a,b) as at muscarinic postsynaptic receptors mediating the formation of IPl.
405
Acknowledgement The excellent technical assistance of Ms. Martine Schneider is acknowledged with pleasure.
References Eltze. M.. G. Lambrecht and E. Mutschler, 1989, Cyproheptadine displays high affinity for muscarinic receptors but does not discriminate between receptor subtypes, European J. Pharmacol. 173, 219. Gibson, R.E., W.J. Rzeszotarski, W.C. Eckelman. E.M. Jagoda, D.J. Weckstein, and R.C. Reba, 1983. Differences in affinities of muscarinic acetylcholine receptor antagonists for brain and heart receptors, B&hem. Pharmacol. 32, 1851. Hawcock, T.. S. Lazareno and F. Roberts. 1986, Assessment of the muscarinic subtype selectivity of ( f )-3-quinuclidinyl xanthene-9carboxylate hemioxalate hydrate (QNX). Br. J. Pharmacol. 88. 357P. Ketelaars. C.E.J. and J. Bruinvels, 1989, The anti-conflict effect of
cyproheptadine is not mediated by its 5-hydroxytryptamine antagonistic property, Life Sci. 44. 1743. Leysen. J.E.. F. Awouters. L. Kennis. P.M. Laduron. J. Vandenherk and P.A.J. Janssen. 1981, Receptor binding profile of R41468. a novel antagonist at 5-HT2 receptors, Life Sci. 28. 1015. Richards. M.H.. 1990a. Rat hippocampal muscarinic autoreceptors are similar to the M2 (cardiac) subtype: comparison with hippocampal Ml. atrial M2 and ileal M3 receptors. Br. J. Pharmacol. 99, 753. Richards. M.H., 1990b. Relative potencies of agonists and differential sensitivity to N-ethylmaleimide on muscarinic autoreceptors and postsynaptic receptors in rat hippocampus. J. Pharmacol. Exp. Ther. 255. 83. Schlicker, E. and M. Giithert. 1981. Antagonist properties of quipazinc at presynaptic serotonin receptors and alpha-adrenoceptors in rat brain cortex slices, Naunyn-Schmeideb. Arch. Pharmacol. 317. 204. Smith. C.J., J.A. Court. A.B. Keith and E.B. Perry, 1989. increases in muscarinic stimulated hydrolysis of inositol phospholipids in rat hippocampus following choline& deafferentation are not paralleled by alterations in choline+ receptor density. Brain Res. 415. 317.