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The Presence of σ Receptor Subtypes in Bovine Retinal Membranes
Exp. Eye Res. (1997) 64, 857–860
LETTER TO THE EDITORS
The Presence of σ Receptor Subtypes in Bovine Retinal Membranes
0014–4835}97}05085704 $25.00}0}ey960272
suspended in 20 volumes (W}V) of standard buffer. The suspension was recentrifuged and the pellet was resuspended in fresh standard buffer for binding assays. The protein content in the final suspension was C 0±6 mg ml−" as determined by the method of Lowry et al. (1951). Binding assays of [$H]-()-pentazocine and [$H]-1,3di-(2-tolyl)guanidine ([$H]-DTG) (in the presence of
1000
800 300 600 B/F
Specific bound (fmol mg–1 protein)
(A)
400
200 100
200
0
0
500 Bound
1000
30
10 20 [3H]-(+)-Pentazocine [nM]
1000 (B) 800 100 80
600 B/F
Specific bound (fmol mg–1 protein)
The existence of the σ receptor was first proposed by Martin et al. (1976), as a novel opioid receptor subtype responsible for the psychotomimmetic effects of benzomorphans, such as N-allylnormetazocine (SKF-10 047), pentazocine and cyclazocine. Since then, the σ receptor has been established as unique, because it was shown that the benzomorphans were not antagonized by the classic opioid receptor antagonist, naloxone, and could bind to a unique nonopioid binding site, distinct from the phencyclidine receptor (Walker et al., 1990). More recent studies suggest that the σ receptor has two distinct subtypes termed σ and σ (Quirion et al., 1992 ; McCann, " # Weissman and Su, 1994). Although the functional roles of the σ receptor subtypes have not yet been clarified, it has been suggested that the σ receptor " subtype may be involved in schizophrenia (Deutsch et al., 1988 ; Snyder and Largent, 1989 ; Walker et al., 1990) and regulate functions of the central neural system such as in the glutaminergic and cholinergic systems (De Coster et al., 1995 ; Matsuno et al., 1995). The σ receptor subtype may also be involved in motor # function (Walker et al., 1993) and K+ channels (Jeanjean et al., 1993). Intensive studies on the distribution of the σ receptor in central and peripheral tissues indicate that σ receptors exist in many organs such as the nervous system, the endocrine organs and the immune system (Walker et al., 1990 ; Su and Junien, 1994). Particularly, in the nervous system, the σ receptor was found to be distributed throughout most of the brain (Walker et al., 1990 ; Itzhak, 1994). In eye tissues, it was also reported that the σ receptor existed in the lacrimal gland (Schoenwald et al., 1993). However, it is not yet known if the σ receptor is expressed in the retina, which is rich in neurons. In the present study, we investigated the σ receptor subtypes present in bovine retinal membranes. Membranes of bovine sensory retina were prepared as described previously (Senda et al., 1995 ; 1996). Fresh bovine eyes were obtained from a local slaughterhouse (Nanko-zoki, Osaka, Japan) and transported on ice. The retina was scraped from the pigment epithelium and homogenized in eight volumes (W}V) of ice-cold 0±32 sucrose in 50 m Tris–HCl buffer (pH 7±7 at 25°C, standard buffer) using a Wheaton glass-glass homogenizer. The homogenate was centrifuged at 1000 g for 10 min at 4°C. The supernatant fraction was collected and centrifuged at 47 000 g for 20 min at 4°C. The resulting membrane pellet was
400
60 40 20
200
0
0
20
60 40 [3H]-DTG [nM]
500 Bound 80
1000
100
F. 1. Saturation curves and Scatchard plots (insert) of [$H]-()-pentazocine (A) and [$H]-DTG (B) specific binding to bovine retinal membrane. The data shown are from typical experiment representing the means of triplicate determinations. See text for details. # 1997 Academic Press Limited
858
LETTER TO THE EDITORS
T I Binding parameters of [$H]-()-pentazocine and [$H]-DTG binding to bovine retinal membrane Radiolabeled ligands
Kd (n)
Bmax (fmol mg−" protein)
[$H]-()-pentazocine [$H]-DTG
4±49³0±81 13±21³1±00
1141±73³49±90 994±45³150±76
The results are expressed as the means³... of four experiments, each conducted in triplicate. See text for details.
T II The inhibitory potencies of σ receptor ligands for [$H]-()-pentazocine or [$H]-DTG binding to bovine retinal membrane IC
&!
σ receptor ligands
[$H]-()-pentazocine
()-Pentazocine (®)-Pentazocine ()-SKF-10 047 (®)-SKF-10 047 SA4503 DTG
56±48³13±97 423±07³141±04 689±60³52±00 6374±00³1151±51 64±59³22±72 1760±33³96±74
(n) [$H]-DTG 6958±33³153±46 426±07³49±26 56800±00³7772±91 26 606±67³4502±25 337±12³60±37 18±91³6±03
The results are expressed as the means³... of three to six experiments, each conducted in triplicate. See text for details.
200 n ()-pentazocine to mask σ site) were carried " out as described previously (Matsuno et al., 1996 ; Senda et al., 1996). The reaction was initiated by the addition of 0±2 ml of the membrane preparation to a mixture containing 5 n [$H]-()-pentazocine or 5 n [$H]-DTG and unlabeled drug in a final volume of 1±0 ml. Incubations were carried out at 37°C for 150 min in the [$H]-()-pentazocine binding study and at 25°C for 90 min in the [$H]-DTG binding study. The binding reaction was stopped by a rapid vacuum filtration through Whatmann GF}B glass filters presoaked with 0±5 % polyethyleneimine. The filters were washed three times with 4 ml of ice-cold standard buffer and subjected to liquid scintillation counting. Non-specific binding was determined in the presence of 100 µ of each unlabeled ligand. Saturation experiments were conducted over a concentration range of 0±1–30 n [$H]-()-pentazocine or 0±1 to 100 n [$H]-DTG, respectively. Competition studies were carried out with 5 n [$H]-()-pentazocine or 5 n [$H]-DTG, respectively. Dissociation constants (Kd values) and maximum numbers of binding sites (Bmax values) from saturation experiments were calculated from linear-squares regression analysis using a computer program. The concentrations of test drugs causing 50 % inhibition of radioligand binding ( ) from competition experi&! ments were determined by Hill’s analysis using a computer assisted linear least-squares regression analysis. The following drugs were used : ()-SKF-10 047, (®)-SKF-10 047, DTG (Research Biochemicals
Int., Wayland, MA, U.S.A.) ; ()-pentazocine, (®)-pentazocine, 1-(3,4-dimethoxyphenethyl)-4-(3phenylpropyl)-piperazine dihydrochloride (SA4503) (synthesized in our laboratory) ; [$H]-()-pentazocine (specific activity 1169±2 GBq mmol−", New England Nuclear (NEN), Boston, MA, U.S.A.), [$H]-DTG (specific activity 1457±8 GBq mmol−", NEN). Other chemicals and reagents of analytical grade were obtained from commercial suppliers. ()-SKF-10 047 and (®)-SKF-10 047 were dissolved in distilled water. ()-Pentazocine, (®)pentazocine, DTG and SA4503 were dissolved in dimethyl sulfoxide (DMSO) containing equimolar hydrochloric acid (HCl). The final concentration of DMSO was less than 1 %. As shown in Fig. 1, the binding of both [$H]-()pentazocine and [$H]-DTG to the bovine retina was saturable. Scatchard plots from the saturation studies showed that both radiolabeled ligands bound to the bovine retina with one site (Fig. 1). The binding parameters of [$H]-()-pentazocine and [$H]-DTG indicated that both radiolabeled ligands bound with high affinity in bovine retina (Table I). In addition, the binding sites of [$H]-()-pentazocine and [$H]-DTG in bovine retina were about the same density. The ability of several σ receptor ligands to inhibit the binding of [$H]-()-pentazocine and [$H]-DTG in bovine retina is shown in Table II. The binding of both radiolabeled ligands was inhibited by all σ receptor ligands used. Particularly, σ receptor ligands, such as " ()-pentazocine and SA4503, strongly inhibited the [$H]-()-pentazocine binding. In addition, the in-
LETTER TO THE EDITORS
hibitory effects of ()-benzomorphans, such as ()pentazocine and ()-SKF-10 047, on [$H]-()pentazocine binding were about 10 times more potent than the corresponding (®)-isomers. On the other hand, the inhibitory effects of ()-benzomorphans on [$H]-DTG bindings were less than those of the corresponding (®)-isomers. The present study showed that both [$H]-()pentazocine and [$H]-DTG bound with high affinity to the bovine retinal membranes. Their binding affinities were comparable to observed in brain tissues (Cagnotto, Bastone and Mennini, 1994 ; McCann et al., 1994 ; Leitner et al., 1994). In addition, the present results showed that the binding of [$H]-()pentazocine was strongly inhibited by ()benzomorphans rather than (®)-isomers, and that of [$H]-DTG was strongly inhibited by (®)benzomorphans rather than ()-isomers. Reportedly, the classification of the σ receptor subtypes was based on the stereoselectivities of the benzomorphans. Namely, the σ receptor is characterized by a stereo" selectivity for benzomorphans ()-enantiomer, while the σ receptor is more sensitive to benzomorphans # (®)-enantiomer (Hellewell and Bowen, 1990 ; Su et al., 1991 ; Quirion et al., 1992). Furthermore, it was reported that SA4503 has a higher affinity for σ " receptor subtype than for σ receptor subtype and # showed little affinity for 36 other receptors, ion channels and second messenger systems (Matsuno et al., 1996 ; Senda et al., 1996). In agreement with these reports, the present results also showed that SA4503 strongly inhibited [$H]-()-pentazocine binding rather than [$H]-DTG binding. These findings suggested that the binding sites of [$H]-()pentazocine and [$H]-DTG correspond to the σ and σ " # receptor subtypes, respectively. Although it is clear from the present study that both σ and σ receptor subtypes exist in the bovine retina, " # the functional role of these σ receptor subtypes in retina is an unresolved question. The densities of both σ and σ receptor subtypes in bovine retina are higher " # than found in other tissues. For example, Rogers, Cecyre and Lemaire (1989) reported that the Bmax value of the binding sites of [$H]-()-SKF-10 047, a selective σ receptor ligand (Itzhak, 1994), was " 67 pmol g−" protein in the bovine adrenal medulla. In eye tissues, Schoenwald et al. (1993) reported that the Bmax value of the binding sites of [$H]-Haloperidol, a ligand for the σ and}or σ receptor subtypes " # (Hellewell and Bowen, 1990), was 588 fmol mg−" protein in the rabbit lacrimocyte. Moreover, in the nervous system, the densities of both σ and σ " # receptor subtypes in the bovine retina were more than those in the rat brain (Cagnotto et al., 1994 ; Leitner et al., 1994 ; McCann et al., 1994). These findings indicated that the retina has highest densities of σ " and σ receptor subtypes in central and peripheral # tissues. Therefore, it seems likely that σ receptor subtypes play a functional role in the retina.
859
TOSHIHIKO SENDA*, KIYOSHI MATSUNO SHIRO MITA Nara Research and Development Center, Santen Pharmaceutical Co., Ltd., 8916-16, Takayama-cho, Ikoma-shi, Nara 630-01, Japan * Address correspondence to : Toshihiko Senda, Discovery Research Division, Nara Research and Development Center, Santen Pharmaceutical Co., Ltd., 8916-16, Takayama-cho, Ikoma-shi, Nara 630-01, Japan.
References Cagnotto, A., Bastone, A. and Mennini, T. (1994). [$H]()Pentazocine binding to rat brain σ receptors. Eur. J. " Pharmacol. (Molecular Pharmacology Section) 266, 131–8. De Coster, M. A., Klette, K. L., Knight, E. S. and Tortella, F. C. (1995). σ Receptor-mediated neuroprotection against glutamate toxicity in primary rat neural cultures. Brain Res. 671, 45–53. Deutsch, S. I., Weizman, A., Goldman, M. E. and Morihisa, J. M. (1988). The sigma receptor : a novel site implicated in psychosis and antipsychotic drug efficacy. Clin. Neuropharmacol. 11, 105–19. Hellewell, S. B. and Bowen, W. D. (1990). A sigma-like binding site in rat pheochromocytoma (PC12) cells : decreased affinity for ()-benzomorphans and lower molecular weight suggest a different sigma receptor form from that of guinea pig brain. Brain Res. 527, 244–53. Itzhak, Y. (1994). Multiple sigma binding sites in the brain. In Sigma receptors (Ed. Itzhak, Y.) Pp. 113–37. Academic Press : San Diego. Jeanjean, A. P., Mestre, M., Maloteaux, J.-M. and Laduron, P. M. (1993). Is the σ receptor in rat brain related to # the K+ channel of class III antiarrhythmic drugs ? Eur. J. Pharmacol. 241, 111–16. Leitner, M. L., Hohmann, A. G., Patrick, S. L. and Walker, J. M. (1994). Regional variation in the ratio of σ to σ " # binding in rat brain. Eur. J. Pharmacol. 259, 65–9. Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–75. Martin, W. R., Eades, C. G., Thompson, J. A., Huppler, R. E. and Gilbert, P. E. (1976). The effects of morphine- and nalorphine-like drugs in the nondependent and morphine-dependent chronic spinal dogs. J. Pharmacol. Exp. Ther. 197, 517–32. Matsuno, K., Senda, T., Kobayashi, T. and Mita, S. (1995). Involvement of σ receptor in ()-N" allylnormetazocine-stimulated hippocampal cholinergic functions in rats. Brain Res. 690, 200–6. Matsuno, K., Nakazawa, M., Okamoto, K., Kawashima, Y. and Mita, S. (1996). Binding properties of SA4503, a novel and selective σ receptor agonist. Eur. J. " Pharmacol. 306, 271–9. McCann, D. J., Weissman, A. D. and Su, T.-P. (1994). Sigma-1 and sigma-2 sites in rat brain : comparison of regional, ontogenetic, and subcellular patterns. Synapse 17, 182–9. Quirion, R., Bowen, W. D., Itzhak, Y., Junien, J. L., Musacchio, J. M., Rothman, R. B., Su, T.-P., Tam, S. W. and Taylor, D. P. (1992). A proposal for the classification of sigma binding sites. Trends Pharmacol. Sci. 13, 85–6. Rogers, C., Cecyre, D. and Lemaire, S. (1989). Presence of
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LETTER TO THE EDITORS
sigma and phencyclidine (PCP)-like receptors in membrane preparations of bovine adrenal medulla. Biochem. Pharmacol. 38, 2467–72. Schoenwald, R. D., Barfknecht, C. F., Xia, E. and Newton, R. E. (1993). The presence of σ-receptors in the lacrimal gland. J. Ocular Pharmacol. 9, 125–39. Senda, T., Matsuno, K. and Mita, S. (1995). Differences in the high affinity sites of σ receptors between guinea pig and rat brain. Neurosci. Res. Commun. 17, 97–105. Senda, T., Matsuno, K., Okamoto, K., Kobayashi, T., Nakata, K. and Mita, S. (1996). Ameliorating effect of SA4503, a novel σ receptor agonist, on memory impairment " induced by cholinergic dysfunction in rats. Eur. J. Pharmacol. 315 1–10. Snyder, S. H. and Largent, B. L. (1989). Receptor mechanisms in antipsychotic drug action : focus on sigma receptors. J. Neuropsychiatry 1, 7–15. Su, T.-P., Wu, X.-Z., Spivak, C. E., London, E. D. and Bell, J.
A. (1991). Binding sites on intact NCB-20 cells suggest sigma receptor multiplicity : σ and σ . In NMDA " # Receptor related agents : biochemistry, pharmacology and behavior (Eds Kameyama, T., Nabeshima, T. and Domino, E. F.) Pp. 227–33. NPP Books : Ann Arbor. Su, T.-P. and Junien, J. L. (1994). Sigma receptor in the central nervous system and the periphery. In Sigma receptors (Ed. Itzhak, Y.) Pp. 21–44. Academic Press : San Diego. Walker, J. M., Bowen, W. D., Walker, F. O., Matsumoto, R. R., De Costa, B. and Rice, K. C. (1990). Sigma receptors : biology and function. Pharmacol. Rev. 42, 355–402. Walker, J. M., Bowen, W. D., Patrick, S. L., Williams, W. D., Mascarella, S. W., Bai, X. and Carroll, F. I. (1993). A comparison of (®)-deoxybenzomorphans devoid of opiate activity with their dexrorotatory phenolic counterparts suggests role of σ receptor in motor # function. Eur. J. Pharmacol. 231, 61–8.
(Received Seattle 10 October 1996 and accepted in revised form 9 December 1996)
LETTER TO THE EDITORS
The Presence of σ Receptor Subtypes in Bovine Retinal Membranes
0014–4835}97}05085704 $25.00}0}ey960272
suspended in 20 volumes (W}V) of standard buffer. The suspension was recentrifuged and the pellet was resuspended in fresh standard buffer for binding assays. The protein content in the final suspension was C 0±6 mg ml−" as determined by the method of Lowry et al. (1951). Binding assays of [$H]-()-pentazocine and [$H]-1,3di-(2-tolyl)guanidine ([$H]-DTG) (in the presence of
1000
800 300 600 B/F
Specific bound (fmol mg–1 protein)
(A)
400
200 100
200
0
0
500 Bound
1000
30
10 20 [3H]-(+)-Pentazocine [nM]
1000 (B) 800 100 80
600 B/F
Specific bound (fmol mg–1 protein)
The existence of the σ receptor was first proposed by Martin et al. (1976), as a novel opioid receptor subtype responsible for the psychotomimmetic effects of benzomorphans, such as N-allylnormetazocine (SKF-10 047), pentazocine and cyclazocine. Since then, the σ receptor has been established as unique, because it was shown that the benzomorphans were not antagonized by the classic opioid receptor antagonist, naloxone, and could bind to a unique nonopioid binding site, distinct from the phencyclidine receptor (Walker et al., 1990). More recent studies suggest that the σ receptor has two distinct subtypes termed σ and σ (Quirion et al., 1992 ; McCann, " # Weissman and Su, 1994). Although the functional roles of the σ receptor subtypes have not yet been clarified, it has been suggested that the σ receptor " subtype may be involved in schizophrenia (Deutsch et al., 1988 ; Snyder and Largent, 1989 ; Walker et al., 1990) and regulate functions of the central neural system such as in the glutaminergic and cholinergic systems (De Coster et al., 1995 ; Matsuno et al., 1995). The σ receptor subtype may also be involved in motor # function (Walker et al., 1993) and K+ channels (Jeanjean et al., 1993). Intensive studies on the distribution of the σ receptor in central and peripheral tissues indicate that σ receptors exist in many organs such as the nervous system, the endocrine organs and the immune system (Walker et al., 1990 ; Su and Junien, 1994). Particularly, in the nervous system, the σ receptor was found to be distributed throughout most of the brain (Walker et al., 1990 ; Itzhak, 1994). In eye tissues, it was also reported that the σ receptor existed in the lacrimal gland (Schoenwald et al., 1993). However, it is not yet known if the σ receptor is expressed in the retina, which is rich in neurons. In the present study, we investigated the σ receptor subtypes present in bovine retinal membranes. Membranes of bovine sensory retina were prepared as described previously (Senda et al., 1995 ; 1996). Fresh bovine eyes were obtained from a local slaughterhouse (Nanko-zoki, Osaka, Japan) and transported on ice. The retina was scraped from the pigment epithelium and homogenized in eight volumes (W}V) of ice-cold 0±32 sucrose in 50 m Tris–HCl buffer (pH 7±7 at 25°C, standard buffer) using a Wheaton glass-glass homogenizer. The homogenate was centrifuged at 1000 g for 10 min at 4°C. The supernatant fraction was collected and centrifuged at 47 000 g for 20 min at 4°C. The resulting membrane pellet was
400
60 40 20
200
0
0
20
60 40 [3H]-DTG [nM]
500 Bound 80
1000
100
F. 1. Saturation curves and Scatchard plots (insert) of [$H]-()-pentazocine (A) and [$H]-DTG (B) specific binding to bovine retinal membrane. The data shown are from typical experiment representing the means of triplicate determinations. See text for details. # 1997 Academic Press Limited
858
LETTER TO THE EDITORS
T I Binding parameters of [$H]-()-pentazocine and [$H]-DTG binding to bovine retinal membrane Radiolabeled ligands
Kd (n)
Bmax (fmol mg−" protein)
[$H]-()-pentazocine [$H]-DTG
4±49³0±81 13±21³1±00
1141±73³49±90 994±45³150±76
The results are expressed as the means³... of four experiments, each conducted in triplicate. See text for details.
T II The inhibitory potencies of σ receptor ligands for [$H]-()-pentazocine or [$H]-DTG binding to bovine retinal membrane IC
&!
σ receptor ligands
[$H]-()-pentazocine
()-Pentazocine (®)-Pentazocine ()-SKF-10 047 (®)-SKF-10 047 SA4503 DTG
56±48³13±97 423±07³141±04 689±60³52±00 6374±00³1151±51 64±59³22±72 1760±33³96±74
(n) [$H]-DTG 6958±33³153±46 426±07³49±26 56800±00³7772±91 26 606±67³4502±25 337±12³60±37 18±91³6±03
The results are expressed as the means³... of three to six experiments, each conducted in triplicate. See text for details.
200 n ()-pentazocine to mask σ site) were carried " out as described previously (Matsuno et al., 1996 ; Senda et al., 1996). The reaction was initiated by the addition of 0±2 ml of the membrane preparation to a mixture containing 5 n [$H]-()-pentazocine or 5 n [$H]-DTG and unlabeled drug in a final volume of 1±0 ml. Incubations were carried out at 37°C for 150 min in the [$H]-()-pentazocine binding study and at 25°C for 90 min in the [$H]-DTG binding study. The binding reaction was stopped by a rapid vacuum filtration through Whatmann GF}B glass filters presoaked with 0±5 % polyethyleneimine. The filters were washed three times with 4 ml of ice-cold standard buffer and subjected to liquid scintillation counting. Non-specific binding was determined in the presence of 100 µ of each unlabeled ligand. Saturation experiments were conducted over a concentration range of 0±1–30 n [$H]-()-pentazocine or 0±1 to 100 n [$H]-DTG, respectively. Competition studies were carried out with 5 n [$H]-()-pentazocine or 5 n [$H]-DTG, respectively. Dissociation constants (Kd values) and maximum numbers of binding sites (Bmax values) from saturation experiments were calculated from linear-squares regression analysis using a computer program. The concentrations of test drugs causing 50 % inhibition of radioligand binding ( ) from competition experi&! ments were determined by Hill’s analysis using a computer assisted linear least-squares regression analysis. The following drugs were used : ()-SKF-10 047, (®)-SKF-10 047, DTG (Research Biochemicals
Int., Wayland, MA, U.S.A.) ; ()-pentazocine, (®)-pentazocine, 1-(3,4-dimethoxyphenethyl)-4-(3phenylpropyl)-piperazine dihydrochloride (SA4503) (synthesized in our laboratory) ; [$H]-()-pentazocine (specific activity 1169±2 GBq mmol−", New England Nuclear (NEN), Boston, MA, U.S.A.), [$H]-DTG (specific activity 1457±8 GBq mmol−", NEN). Other chemicals and reagents of analytical grade were obtained from commercial suppliers. ()-SKF-10 047 and (®)-SKF-10 047 were dissolved in distilled water. ()-Pentazocine, (®)pentazocine, DTG and SA4503 were dissolved in dimethyl sulfoxide (DMSO) containing equimolar hydrochloric acid (HCl). The final concentration of DMSO was less than 1 %. As shown in Fig. 1, the binding of both [$H]-()pentazocine and [$H]-DTG to the bovine retina was saturable. Scatchard plots from the saturation studies showed that both radiolabeled ligands bound to the bovine retina with one site (Fig. 1). The binding parameters of [$H]-()-pentazocine and [$H]-DTG indicated that both radiolabeled ligands bound with high affinity in bovine retina (Table I). In addition, the binding sites of [$H]-()-pentazocine and [$H]-DTG in bovine retina were about the same density. The ability of several σ receptor ligands to inhibit the binding of [$H]-()-pentazocine and [$H]-DTG in bovine retina is shown in Table II. The binding of both radiolabeled ligands was inhibited by all σ receptor ligands used. Particularly, σ receptor ligands, such as " ()-pentazocine and SA4503, strongly inhibited the [$H]-()-pentazocine binding. In addition, the in-
LETTER TO THE EDITORS
hibitory effects of ()-benzomorphans, such as ()pentazocine and ()-SKF-10 047, on [$H]-()pentazocine binding were about 10 times more potent than the corresponding (®)-isomers. On the other hand, the inhibitory effects of ()-benzomorphans on [$H]-DTG bindings were less than those of the corresponding (®)-isomers. The present study showed that both [$H]-()pentazocine and [$H]-DTG bound with high affinity to the bovine retinal membranes. Their binding affinities were comparable to observed in brain tissues (Cagnotto, Bastone and Mennini, 1994 ; McCann et al., 1994 ; Leitner et al., 1994). In addition, the present results showed that the binding of [$H]-()pentazocine was strongly inhibited by ()benzomorphans rather than (®)-isomers, and that of [$H]-DTG was strongly inhibited by (®)benzomorphans rather than ()-isomers. Reportedly, the classification of the σ receptor subtypes was based on the stereoselectivities of the benzomorphans. Namely, the σ receptor is characterized by a stereo" selectivity for benzomorphans ()-enantiomer, while the σ receptor is more sensitive to benzomorphans # (®)-enantiomer (Hellewell and Bowen, 1990 ; Su et al., 1991 ; Quirion et al., 1992). Furthermore, it was reported that SA4503 has a higher affinity for σ " receptor subtype than for σ receptor subtype and # showed little affinity for 36 other receptors, ion channels and second messenger systems (Matsuno et al., 1996 ; Senda et al., 1996). In agreement with these reports, the present results also showed that SA4503 strongly inhibited [$H]-()-pentazocine binding rather than [$H]-DTG binding. These findings suggested that the binding sites of [$H]-()pentazocine and [$H]-DTG correspond to the σ and σ " # receptor subtypes, respectively. Although it is clear from the present study that both σ and σ receptor subtypes exist in the bovine retina, " # the functional role of these σ receptor subtypes in retina is an unresolved question. The densities of both σ and σ receptor subtypes in bovine retina are higher " # than found in other tissues. For example, Rogers, Cecyre and Lemaire (1989) reported that the Bmax value of the binding sites of [$H]-()-SKF-10 047, a selective σ receptor ligand (Itzhak, 1994), was " 67 pmol g−" protein in the bovine adrenal medulla. In eye tissues, Schoenwald et al. (1993) reported that the Bmax value of the binding sites of [$H]-Haloperidol, a ligand for the σ and}or σ receptor subtypes " # (Hellewell and Bowen, 1990), was 588 fmol mg−" protein in the rabbit lacrimocyte. Moreover, in the nervous system, the densities of both σ and σ " # receptor subtypes in the bovine retina were more than those in the rat brain (Cagnotto et al., 1994 ; Leitner et al., 1994 ; McCann et al., 1994). These findings indicated that the retina has highest densities of σ " and σ receptor subtypes in central and peripheral # tissues. Therefore, it seems likely that σ receptor subtypes play a functional role in the retina.
859
TOSHIHIKO SENDA*, KIYOSHI MATSUNO SHIRO MITA Nara Research and Development Center, Santen Pharmaceutical Co., Ltd., 8916-16, Takayama-cho, Ikoma-shi, Nara 630-01, Japan * Address correspondence to : Toshihiko Senda, Discovery Research Division, Nara Research and Development Center, Santen Pharmaceutical Co., Ltd., 8916-16, Takayama-cho, Ikoma-shi, Nara 630-01, Japan.
References Cagnotto, A., Bastone, A. and Mennini, T. (1994). [$H]()Pentazocine binding to rat brain σ receptors. Eur. J. " Pharmacol. (Molecular Pharmacology Section) 266, 131–8. De Coster, M. A., Klette, K. L., Knight, E. S. and Tortella, F. C. (1995). σ Receptor-mediated neuroprotection against glutamate toxicity in primary rat neural cultures. Brain Res. 671, 45–53. Deutsch, S. I., Weizman, A., Goldman, M. E. and Morihisa, J. M. (1988). The sigma receptor : a novel site implicated in psychosis and antipsychotic drug efficacy. Clin. Neuropharmacol. 11, 105–19. Hellewell, S. B. and Bowen, W. D. (1990). A sigma-like binding site in rat pheochromocytoma (PC12) cells : decreased affinity for ()-benzomorphans and lower molecular weight suggest a different sigma receptor form from that of guinea pig brain. Brain Res. 527, 244–53. Itzhak, Y. (1994). Multiple sigma binding sites in the brain. In Sigma receptors (Ed. Itzhak, Y.) Pp. 113–37. Academic Press : San Diego. Jeanjean, A. P., Mestre, M., Maloteaux, J.-M. and Laduron, P. M. (1993). Is the σ receptor in rat brain related to # the K+ channel of class III antiarrhythmic drugs ? Eur. J. Pharmacol. 241, 111–16. Leitner, M. L., Hohmann, A. G., Patrick, S. L. and Walker, J. M. (1994). Regional variation in the ratio of σ to σ " # binding in rat brain. Eur. J. Pharmacol. 259, 65–9. Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–75. Martin, W. R., Eades, C. G., Thompson, J. A., Huppler, R. E. and Gilbert, P. E. (1976). The effects of morphine- and nalorphine-like drugs in the nondependent and morphine-dependent chronic spinal dogs. J. Pharmacol. Exp. Ther. 197, 517–32. Matsuno, K., Senda, T., Kobayashi, T. and Mita, S. (1995). Involvement of σ receptor in ()-N" allylnormetazocine-stimulated hippocampal cholinergic functions in rats. Brain Res. 690, 200–6. Matsuno, K., Nakazawa, M., Okamoto, K., Kawashima, Y. and Mita, S. (1996). Binding properties of SA4503, a novel and selective σ receptor agonist. Eur. J. " Pharmacol. 306, 271–9. McCann, D. J., Weissman, A. D. and Su, T.-P. (1994). Sigma-1 and sigma-2 sites in rat brain : comparison of regional, ontogenetic, and subcellular patterns. Synapse 17, 182–9. Quirion, R., Bowen, W. D., Itzhak, Y., Junien, J. L., Musacchio, J. M., Rothman, R. B., Su, T.-P., Tam, S. W. and Taylor, D. P. (1992). A proposal for the classification of sigma binding sites. Trends Pharmacol. Sci. 13, 85–6. Rogers, C., Cecyre, D. and Lemaire, S. (1989). Presence of
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sigma and phencyclidine (PCP)-like receptors in membrane preparations of bovine adrenal medulla. Biochem. Pharmacol. 38, 2467–72. Schoenwald, R. D., Barfknecht, C. F., Xia, E. and Newton, R. E. (1993). The presence of σ-receptors in the lacrimal gland. J. Ocular Pharmacol. 9, 125–39. Senda, T., Matsuno, K. and Mita, S. (1995). Differences in the high affinity sites of σ receptors between guinea pig and rat brain. Neurosci. Res. Commun. 17, 97–105. Senda, T., Matsuno, K., Okamoto, K., Kobayashi, T., Nakata, K. and Mita, S. (1996). Ameliorating effect of SA4503, a novel σ receptor agonist, on memory impairment " induced by cholinergic dysfunction in rats. Eur. J. Pharmacol. 315 1–10. Snyder, S. H. and Largent, B. L. (1989). Receptor mechanisms in antipsychotic drug action : focus on sigma receptors. J. Neuropsychiatry 1, 7–15. Su, T.-P., Wu, X.-Z., Spivak, C. E., London, E. D. and Bell, J.
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(Received Seattle 10 October 1996 and accepted in revised form 9 December 1996)