Sigma-1 receptors modulate functional activity of rat splenocytes

Journal of Neuroimmunology ELSEVIER

Journal of Neuroimmunology 59 (1995) 143-154

Sigma-l receptors modulate functional activity of rat splenocytes Yuhong Liu a, Ben B. Whitlock a, Joseph A. Pultz b, Seth A. Wolfe, Jr. a,* a Department of Medical Microbiology and Immunology, The Ohio State UrGersity College of Medicine, 2078 Graxy Hall, 333 West 10th Al,enue, Columbus, OH 43210.1239, USA ’ Department of Statistics, The Ohio State lJnir,ersity, 148 Cockins Hall, 1959 West 9th Ar,enue, Columbu,

OH 43210, USA

Received 10 November 1994; revised 10 February 1995; accepted 10 February 1995

Abstract Neuroleptics, opiates, and cocaine are commonly prescribed for or abused by humans. Although primarily used for their actions at other receptors in brain, these compounds also act at sigma receptors. We have previously identified sigma-l receptors on human peripheral blood leukocytes and rat spleen, and in the present study we demonstrate a correlation between the pharmacology of these receptors and the ability of drugs to suppress concanavalin A-induced splenocyte proliferation. These results support the hypothesis that sigma-l receptors regulate functional activities of immune cells, and suggest that sigma agonists may cause changes in immune competence in vivo. Keywords:

Sigma receptor;

Neuro-immunomodulation;

Immunosuppressant;

Opiate

receptor;

Phencyclidine

receptor;

Leukocyte;

Lymphocyte

1. Introduction Sigma receptors were postulated by Martin et al. (19761, as a result of canine physiological studies with racemic mixtures of the opiate benzomorphan, N-allylnormetazocine (SKF 10,047, or NANM), and drug binding sites with the appropriate pharmacology were subsequently identified in guinea pig brain by Su (1981,1982). These receptors have since been found to be widely distributed in the central nervous system (Gundlach et al., 19861, endocrine tissues (Wolfe et al., 1989; Jansen et al., 1990; Su and Wu, 1990), gastrointestinal tract (Roman et al., 1988), liver (Samovilova et al., 1988), and immune tissues (Wolfe et al., 1988; Wolfe and De Souza, 1992). Sigma receptors bind psychotomimetic benzomorphans, guanidines, 3phenyl-piperidines, phencyclidine (PCP), cocaine and steroids (Sharkey et al., 1988; reviewed in Walker et al., 19901, yet they are distinct from opiate receptors, and from the PCP binding sites (PCP receptors) that reside in the ion channels of NMDA-type glutamate receptor complexes (Quirion et al., 1987). Little is known about the structure of sigma receptors, but sigma agonists have been associated with

* Corresponding 9805

author. Phone (614) 292 2630; Fax: (614) 292

Elsevier Science B.V. SSDI 0165-5728(95)00032-l

several physiological functions. Bowen et al. (1988) reported that sigma agonists inhibited carbachol-induced phosphoinositide turnover in rat brain membranes. From data derived in the guinea pig ileum twitch model, Campbell and colleagues inferred that sigma agonists altered cholinergic neurotransmission (Campbell et al., 1989). In addition, Paul et al. (1993) found that sigma agonists inhibited nicotine-stimulated catecholamine release from adrenal chromaffin cells. Wu et al. (1991), found that sigma agonists blocked a tonic potassium current in NCB-20 neuroblastoma cells. In the immune system, Fudenberg, Whitten and Khansari found that high concentrations of PCP (lo-’ M) could inhibit functional activities of human peripheral blood leukocytes (HPBL) in vitro, and they detected specific binding sites for [“HIPCP on HPBL (Fudenberg et al., 1984; Khansari et al., 1984). In 1987, Dornand and colleagues reported that PCP and PCP analogs suppressed mitogen-induced proliferation of murine splenocytes (Dornand et al., 1987). They found that PCP analogs caused a reduction in the resting potential of splenocytes, and reduced the ability of these cells to depolarize and elevate their intracellular calcium levels in response to stimulation with concanavalin A (ConA). However, in our previous studies, we could not detect high affinity PCP receptors (PCP,) in rat spleen or HPBL, while we did find strikingly high

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of Neuroimmunology 59 (1995) 143-154

concentrations of sigma receptors in both of these tissues (Wolfe et al., 1988; Wolfe and De Souza, 1992). Subsequently, Carr et al. (19911, reported that the sigma agonists 1,3-di(2-tolyl)guanidine (DTG), haloperidol, and ( + )-pentazocine suppressed ConA-induced proliferation of murine splenocytes. They also reported that pokeweed mitogen (PWM)-induced and bacterial lipopolysaccharide (LPSl-induced immunoglobulin production was either enhanced or suppressed, depending on the mitogen and concentration of drug (Carr et al., 1992). More recently, Garza et al. (1993), found that high concentrations (lop6 M to low5 M) of haloperidol and the novel sigma ligand (+>azidophenazocine (De Costa and Bowen, 19911 suppressed ConA-induced interferon production. However, in their hands, the sigma agonists DTG, (+ )-pentazocine, and (+ )-l-propyl-3-(3_hydroxyphenyl)piperidine (( + l-3-PPP) did not significantly affect interferon production, which suggests that this may not have been a sigma receptor-mediated effect. More recently, drug binding and modulation of several in vivo and in vitro functional assays have been reported for a novel sigma ligand, SR 31747 (Casellas et al., 1994; Paul et al., 1994). Collectively, these data suggest that sigma receptors on lymphocytes may regulate immune functions. However, the evidence for this is suggestive, rather than definitive. Sigma agonists do not act exclusively at sigma receptors, but can also be quite potent at a multitude of other sites, including ion channels, dopamine CD,, D,, D,) receptors, serotonin (5HT,,, 5-HT,) receptors, opiate (p, 6, K) receptors, phencyclidine receptors, al receptors, and uptake sites for serotonin (5HT) and dopamine (reviewed in Walker et al., 1990). In addition, it has recently been found that there are multiple forms of sigma receptors, of which nomenclature and labeling conditions have been standardized for two subtypes: (pi and u2 (Quirion et al., 19921. The subtypes of sigma receptors, which can co-exist on cells, have overlapping pharmacology, and must be distinguished from one another, and from other receptor types, by demonstrating an appropriate rank order of binding potency of a series of sigma ligands. Thus, immune modulation by putative sigma agonists might actually be the result of drug actions at a multitude of receptors on immune cells. In order to demonstrate that sigma receptors modulate a biological function, the receptors must be demonstrated, quantified, and characterized kinetically and pharmacologically in the cells and tissues of interest. Sigma agonists must modulate functional activity in those cells or tissues, and the ability of drugs to modulate function must correlate with drug binding potency at the receptor. (It is to be expected that some compounds may fall outside the correlation due to additional actions at other receptors.) This has been ac-

complished in other systems in which sigma receptors have been reported to act (Campbell et al., 1989; Matsumoto et al., 1990; Jeanjean et al., 1993; Paul et al., 19931, but these criteria have not yet been met in the immune system. In the immune system, several laboratories have demonstrated drug binding sites and/or have shown modulation of functional activities by sigma agonists (Fudenberg et al., 1984; Khansari et al., 1984; Dornand et al., 1987; Wolfe et al., 1988; Carr et al., 1991,1992; Wolfe and De Souza, 1992; Garza et al., 1993; Casellas et al., 1994; Paul et al., 19941, but a mathematical correlation between drug binding at the receptor and the pharmacology of the biological responses has not been demonstrated. In the present study, we have confirmed the presence of high numbers of ui receptors in rat spleen by labeling them with [3H]haloperidol in the presence of spiperone to block D, and 5-HT, sites. Using a panel of fourteen sigma agonists and related compounds in competition drug-binding assays, we also confirmed the pharmacology of the drug binding sites to be that of (pi receptors. These [ 3H]haloperidol-binding u1 receptors were demonstrated to be present in high concentrations on isolated splenocytes. We then tested the ability of the 15 compounds to modulate a commonly used measure of lymphocyte responsiveness: proliferation of rat splenocytes in response to the T cell mitogen, ConA. A correlation was made between drug binding potency at [“Hlhaloperidol-labeled (pi sites and the ability of the drugs to modulate splenocyte proliferation. Because these compounds act at additional, non-u, sites, many of which are known to be present on immune cells, a reference correlation was first made with the seven most sigma-selective agonists: haloperidol, haloperidol metabolite II, ( + l-pentazocine, ( + )SKF 10,047, (+I-3-PPP, DTG and (-)-butaclamol. A high correlation was obtained (Y = 0.86). Then, seven less a,-specific compounds, haloperidol metabolite I, ( - )-pentazocine, (- )-SKF 10,047, (- )-3-PPP, PCP, thienyl PCP (TCP) and (+I-butaclamol, were compared with the reference group. All but three fell within the 95% prediction interval of the reference correlation. PCP, TCP and (+ )-butaclamol fell at the limits of the prediction interval. Rimcazole, a putative sigma antagonist, was, as expected, an outlier. Significantly, no sigma agonist failed to show activity in this biological assay. We interpret these results as strong evidence that V, receptors on splenocytes are functional, and modulate proliferative responses to ConA. 2. Materials and methods 2.1. Animals

Male (150-300 g) Sprague-Dawley rats (Harlan Sprague-Dawley Inc., Indianapolis, IN) were used for

Y Liu et al. /Journal

of Neuroirnmunology 59 (1995) 143-154

all experiments. Animals were housed three to four per cage, had access to food and water ad libitum, and were treated in accordance with NIH animal care guidelines. 2.2. Con&induced

proliferation of rat splenocytes

Spleens from CO,-killed rats were removed and teased into single-cell suspensions by pressing them through a stainless steel mesh. Splenocytes were suspended in Ca*+/Mg *+-free Hank’s balanced salt solution (HBSS, GIBCO BRL Life Technologies, Inc., Gaithersburg, MD), layered over 70% Percoll (Pharmacia, LKB Biotechnology, Uppsala, Sweden), and centrifuged at 1000 X g for 15 min, at 4” C. Splenocytes were collected from the HBSS-Percoll interface, washed by centrifugation in HBSS, and suspended in RPM1 1640 medium (BioWhittaker, Inc., Walkersville, MD) containing 10% fetal bovine serum (FBS, defined grade, HyClone Laboratories, Inc., Logan, UT), 50 pg/ml gentamicin (GIBCO BRL Life Technologies, Inc.), and 5 x lop5 M 2-ME (BIO-RAD Laboratories, Richmond, CA). Splenocytes (2.5 X lo5 cells/well) were incubated in flat-bottom 96-well microtiter plates (Becton Dickinson, Lincoln Park, NJ) at 37” C for 2 h with varying concentrations of drugs, after which ConA (final concentration, 0.5 pg/ml, Sigma Chemical Co.) was added to all wells. The cells were then incubated at 37” C in 5% CO,/95% air for 48 h, and [ 3H]thymidine (0.5 pCi/well, Amersham Corp., Arlington Heights, IL) was added during the last 4-8 h of culture. Cultures were harvested on glass fiber filters, washed with distilled water, and counted in a scintillation counter to measure [ 3H]thymidine incorporation as an index of cell proliferation. 2.3. Preparation of spleen and splenocyte membranes Rats were killed with CO, and their spleens were dissected. For radioligand binding assays in which whole spleens were used, spleens were frozen rapidly on dry ice, then stored at -80” C. For assay, frozen spleens were disrupted with a Polytron tissue homogenizer (Brinkmann Instruments, Westbury, NY) in 25-50 volumes of ice-cold 50 mM Tris * HCl buffer (pH 7.7 at 4” 0. Membranes were pelleted by centrifugation at 40000 X g for 10 min at 4” C, and washed twice by resuspension in the same buffer and recentrifugation. After the second wash they were resuspended in 50 mM Tris . HCl buffer (pH 7.7 at 22” C) and kept on ice until placed in binding assays. To prepare washed splenocyte membranes for binding assays, cells were teased from freshly dissected spleens and isolated on Percoll gradients as described for proliferation assays, above. Isolated splenocytes were then frozen on dry ice and stored at -80” C. For assay, frozen cells were

145

thawed, subjected to homogenization, centrifuged, washed and resuspended in a manner identical to the procedure described for whole spleen. Protein content of the membrane suspensions was determined using a commercial protein assay kit (Sigma Chemical Co., St. Louis, MO, Cat. No. P5656). 2.4. Characterization of [3H]haloperidol sites in rat spleen

binding to a,

For saturation binding assays (Tam and Cook, 1984; Wolfe et al., 1988; Quirion et al., 1992), membrane preparations equivalent to 5.0 mg wet weight of spleen, or 2 x 10’ splenocytes/tube, were incubated at room temperature in a total volume of 2.5 ml 50 mM Tris . HCl buffer (pH 7.71, with increasing concentrations (0.2-10 nM) of [3H]haloperidol @.A., 50 Ci/mmol, New England Nuclear, Boston, MA). Spiperone (50 X the [3Hlhaloperidol concentration) was included in all incubations in order to block [3H]haloperidol binding to D, dopamine and 5-HT, receptors (Creese et al., 1979; Peroutka and Snyder, 1979). Non-specific binding was defined by the absence and presence of 10 PM DTG. After a 90-min incubation, membrane-bound [“Hlhaloperidol was separated from free radioligand in a cell harvester (Brandel Research and Development Co., Gaithersburg, MD) by rapid filtration through Whatman GF/B glass fiber filters that had been pretreated with 0.001% PEI to reduce non-specific binding. The filters were washed at room temperature for 10 s with 5 mM Tris . HCl buffer (pH 8.0). For competition binding assays, rat spleen membranes were incubated under the conditions described above with 0.7 nM [“Hlhaloperidol in the presence of 35 nM spiperone plus increasing concentrations of competing drugs. Non-specific binding again was defined by the absence and presence of 10 PM DTG. The following drugs were used in both competition binding and proliferative assays: haloperidol, haloperido1 metabolite I (4-(4-chlorophenyl)-4-hydroxypiperidine), haloperidol metabolite II ((t_ )-4-(4-chlorophenyl)-a-(4-fluorophenyl)-4-hydroxy-l-piperidinebutanol), (+I and (-)-pentazocine, (+) and (--)-butaclamol, (+> and (-l-3-PPP, (+> and (--l-SKF10,047, DTG, rimcazole, thienyl PCP (TCP), and PCP (Research Biochemicals, Inc., Natick, MA). 2.5. Data analysis Saturation binding and drug competition data were analyzed using the MacLIGAND nonlinear curve fitting program (Munson and Rodbard, 19801 in order to obtain K, (ligand concentration at which 50% of binding sites are occupied at steady state), Ki and B,,, (number of binding sites per cell or unit protein) values for drugs. A least squares fit to a logarithm-probit

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Y. Liu et al. /Journal of Neuroimmunology 59 (1995) X43-154

analysis was used to calculate EC,, values for drugs in suppression of ConA-induced proliferation assays. Analysis of the relationship between drug potencies in binding and proliferation experiments was performed using PROC REG in the SAS computer package. Because we expected relationships to be relative, rather than absolute, data were examined in a logarithmic format, comparing geometric, rather than arithmetic means. Seven reference compounds were examined with a linear regression analysis. The leverage of individual points was examined to determine their influence on the regression. Cook’s distance statistic was used as a test for outliers. The remaining drugs were then compared to the regression in order to determine whether they fell within the 95% prediction interval of the reference compounds.

3. Results 3.1. Sigma agonists modulate Cowl-induced proliferation

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Drug (M) Fig. 1. Three classical (T agonists, haloperidol, ( + I-pentazocine and DTG, suppress ConA-induced rat splenocyte proliferation in a dose-dependent manner. Conditions were as specified in Materials and methods. Data shown are from a representative experiment. Each point represents average values of three replicates, and each titration curve was repeated three times.

splenocyte

A series of 15 compounds were tested as described in Materials and methods for their ability to influence mitogen-induced proliferation in vitro. A stimulus of 0.5 pg/ml ConA was used for all experiments. This concentration was in the linear range of the ConA dose response, and induced somewhat less than 50% of the maximum proliferative response that could be obtained using higher lectin concentrations (data not shown). In this system, all 15 drugs suppressed mitogenic responses in a dose-dependent manner. Representative experiments with the prototypic sigma agonists haloperidol, (+)-pentazocine and DTG are shown in Fig. 1. All compounds were titrated in the manner illustrated in Fig. 1. As described in Materials and methods, drug potency was quantified as EC,, values, or the concentration of each drug required to produce 50% suppression of ConA-induced [ 3H]thymidine incorporation. Vehicle controls were performed for all diluents required to solubilize drugs (data not shown). The EC,, values of the drugs ranged from 2 x lo-’ M to 6 x 10e5 M. The rank order of drug potencies (from most to least potent) was: haloperidol metabolite II 2 haloperidol 2 rimcazole 2 ( + )-butaclamol 2 TCP 2 ( - )-butaclamol 2 PCP 2 ( + )-pentazocine = DTG r haloperidol metabolite I 2 (+ )-3-PPP 2 ( -)-pentazocine 2 ( + )-SKF10,047 2 (- )-SKF10,047 2 (- )-3PPP. Haloperidol, one of the most potent drugs at suppressing ConA-induced proliferation of rat splenocytes, also is a potent antipsychotic drug which acts as a D, dopamine receptor antagonist (Seeman, 1981). Since haloperidol binds to sigma and D, receptors with similar affinity, some of the immunosuppressive effects

of haloperidol might have been mediated through its actions at D, receptors. This does not appear to be the case, as (- )-sulpiride, a potent D, antagonist, had no effect on ConA-induced proliferation (data not shown). 3.2. Splenocytes have [3H]haloperidol-labelable

a, re-

ceptors

Although we previously identified and characterized g1 receptors on human PBL and in rat spleen (Wolfe et al., 1988; Wolfe and De Souza, 19921, their presence had not been demonstrated on the isolated rat splenocytes used for the present functional assays. As may be seen in Fig. 2A and B, washed membranes of splenocytes isolated on 70% Percoll gradients bound [3H]haloperidol in a manner comparable to membranes derived from whole spleen. In both tissues, binding data gave linear Rosenthal plots (insets, Fig. 2A and B), indicating the presence of a single class of sites, with K, values (mean k S.E.M) of 0.65 + 0.12 nM and 1.07 + 0.27 nM in whole spleen and splenocytes, respectively. As may be seen from the B,,, values (the number of binding sites/unit tissue) in Table 1, there was an enrichment of binding sites in splenocytes relative to whole spleen, indicating that the cells we isolated, rather than structural elements of the spleen, were a major source of g1 receptors in this tissue. 3.3. Regulation of proliferation correlates with the pharmacology of splenic a, receptors The ability of our test compounds to compete with [3H]haloperidol for binding at U, receptors was deter-

Y. Liu et al. /Journal

of Neuroimmunology 59 (1995) 143-154

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Fig. 2. Specific binding sites for [3H]haloperidol are present on membranes of rat spleen (A) and rat splenocytes (B). Isolated splenocytes had higher densities of [3H]haloperidol binding sites than the spleens from which they were obtained. As described in Materials and methods, membrane preparations were incubated with 0.078-10 nM [3H]haloperidol in the presence of 50-fold excess spiperone (3.9-500 nM) to block dopamine and serotonin receptors, and nonspecific binding was defined using 10 yM DTG. Insets are Rosenthal plots of the same data, which show straight lines, indicative of a single class of drug binding site. Data from three experiments are shown, each point being the average of three replicates within one experiment.

mined in competition binding assays, as illustrated in Fig. 3. Ki values at [3H]haloperidol-labeled U, receptors (mathematically equal to the drugs’ K, values) were determined for each compound tested in the ConA proliferative assays. In accordance with previous

Table 1 Splenic [3H]haloperidol cytes

binding

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Fig. 3. Using competition binding assays, splenic binding sites for 0.7 nM [“Hlhaloperidol (in the presence of 35 nM spiperone) were demonstrated to have the pharmacology of CT, receptors. Representative curves using the prototypic sigma ligands haloperidol, (+ )pentazocine and DTG are shown in the figure. Experiments were carried out as described in Materials and methods. Each point represents triplicate measurements, and all experiments were repeated three times.

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studies (Wolfe and De Souza, 19921, the [3H]haloperidol-labeled sites in rat spleen displayed a pattern of drug potency typical of the pharmacology of al receptors. Haloperidol was the most potent compound, while PCP and TCP were least potent. Furthermore, there was selectivity for ( + I-stereoenantiomers of pentazocine, SKF 10,047 and 3-PPP, and for (--)butaclamol. The rank order of potency in the binding assay (from most to least potent) was: haloperidol > metabolite II > ( + )-pentazocine 2 ( - >haloperidol pentazocine r ( - )-butaclamol > ( + I-3-PPP 2 DTG > (- )-3-PPP 2 ( + )-SKF 10,047 > haloperidol metabolite 10,047 2 I 2 rimcazole > (+)-butaclamol r (--)-SKF TCP > PCP. Competition binding assays were performed using membrane preparations from homogenized spleens, whereas proliferative assays utilized splenocytes teased from spleen and isolated on Percoll gradients. Since saturation binding studies demonstrated the existence of only a single class of binding sites in spleen, and this site also occurred on isolated splenocytes derived from whole spleen (Fig. 2 and Table 11, we deemed it unnecessary to repeat the pharmacological characterization of drug binding sites using isolated splenocytes as a tissue source. Of the 15 compounds tested, the seven most u,selective agonists, haloperidol, haloperidol metabolite II, DTG, ( + )-pentazocine, ( + )-SKF10,047, ( + )-3-PPP, and (-I-butaclamol, were chosen as reference compounds. Five drugs, ( - )-pentazocine, ( - )-SKF10,047, ( - )-3-PPP, ( + )-butaclamol, and haloperidol metabolite I, were chosen to serve as contrasting, less potent stereoenantiomers or metabolites of the reference

148

I’. Liu et al. /Journal of Neuroimmunology 59 (1995) 143-154

Table 2 Pharmacological specificity of [3H]haloperidol-labeled sigma receptors in rat spleen (Ki) and potency of the drugs suppressing ConAinduced proliferation of rat splenocytes (EC,,) Drug

EC,, a (/.LM)

K, a (nM)

1. Haloperidol metabolite II 2. Haloperidol 3. Rimcazole 4. (+ )-Butaclamol 5. TCP 6. (- l-Butaclamol 7. PCP 8. ( + )-Pentazocine 9. DTG 10. Haloperidol metabolite I 11. ( + )-3-PPP 12. ( - )-Pentazocine 13. f + l-SKF 10,047 14. f - l-SKF 10,047 15. ( - l-3-PPP

0.197+0.081 (41 0.260 + 0.053 (31 0.290 + 0.036 (31 4.00* 1.21 (3) 5.48+ 1.70 (4) 5.90& 1.83 (3) 9.82 +_2.44 (5) 12.82 4.8 (3) 12.9 f 2.9 (3) 20.4 + 6.4 (3) 26.5 + 17.5 (3) 30.7 + 10.5 (3) 31.2k9.9 (3) 55.6 + 36.9 (31 57.7 rf 26.7 (3)

5.67+0.16 (3) 0.654 + 0.012 b (31 448 f 117 (31 1420 + 180 (31 2530 + 280 (3) 31.0f 4.0 (3) 5980 + 1690 (3) 20.3 + 3.6 (3) 69.6 + 12.5 (3) 389 f 23 (31 68.9 f 23.8 (3) 28.5 + 3.8 (3) 233 + 20 (3) 2110+310 (3) 207 + 41 (31

a Values represent arithmetic means+S.E.M. pendent experiments). b This is a K, value.

of (number of inde-

compounds. Although it is a weak sigma agonist and is considerably more potent at modulating several ion channels in cells (Albuquerque et al., 1980; Blaustein and Ickowicz, 19831, PCP was also tested because of its historical association with sigma receptors (Quirion et al., 1987) and immune modulation (Fudenberg et al., 1984; Khansari et al., 1984; Domand et al., 1987). TCP Wignon et al., 1983) and rimcazole (Ferris et al., 1986; Kennedy et al., 1990; Matsuno et al., 1993) were selected as a PCP analog and newly described sigma antagonist, respectively. To determine whether the biological effect was due to actions at CT, receptors, drug’s EC,, values in the proliferative assay were compared on a logarithmic scale with their binding assay Ki values at ui receptors. These values are listed in Table 2. Because of the actions of sigma agonists at multiple receptors (discussed in Introduction), only the seven most ai-selective test compounds, haloperidol, haloperidol metabolite II, ( + )-pentazocine, ( + )-SKF10,047, ( + )-3-PPP, DTG and (-)-butaclamol, were used to establish this correlation, which was quite strong (adjusted r = 0.86). The slope of the correlation line (0.98) had a value approaching unity (Fig. 4). Although it is a very poor sigma agonist and has multiple (and more potent) actions at a variety of other sites, PCP was initially included in the reference group because of its historical role in the field of sigma receptors (see Introduction). By this analysis, a weak, but marginally significant regression (r = 0.63, P approx. 0.05) was obtained. However, PCP seemed to fall outside the pattern of the other compounds. The average leverage of the points was 0.25, but the leverage of PCP was 0.6, indicating that PCP exerted an unusually large influence on the position of the line. A statistical

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Ki (log Ml Fig. 4. The ability of seven reference sigma agonists, haloperidol metabolite II (11, haloperidol (2), (- l-butaclamol (61, ( + I-pentazocine (81, DTG (91, (+I-3-PPP (11) and (+I-SKF 10,047 (13), to suppress ConA-induced proliferation (EC,, values) correlated highly (r = 0.86) with their potency in binding [3H]haloperidol-labeled splenic ~+i receptors (K, values). The 95% prediction interval of the regression is indicated by the dashed lines. Numbers refer to drug’s potency rank in suppressing ConA-induced proliferation (Table 2).

test to determine whether PCP could be declared an outlier was inconclusive (P = 0.148, 1;(i,,) statistic for the size of Cook’s distance = 2.67). Because in non-immune cells PCP is known to have a variety of actions in addition to those at sigma receptors (Albuquerque et al., 1980; Blaustein and Ickowicz, 19831, we felt it should be removed from the reference group if there was any question as to its actions in this system. As discussed above, this resulted in a strong correlation (adjusted r = 0.86) between binding and function for the remaining seven compounds (Fig. 4).

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Ki (JogMl Fig. 5. Haloperidol metabolite I (101, (- )-pentazocine (121, ( - I-SKF 10,047 (14) and (-)-3-PPP (15) fell within the 95% prediction interval (dashed lines) of the regression line of the seven reference sigma agonists (circled numbers, see Fig. 4). Numbers identify drugs by their relative potencies to suppress ConA-induced splenocyte proliferation (Table 2). PCP (71, TCP (5) and (+ I-butaclamol(4), fell at the limits of the prediction interval. The putative sigma antagonist rimcazole (3) was, as expected, an outlier in this relationship.

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When the compounds not used to make the correlation were considered, four, ( - I-pentazocine, ( - ISKF10,047, ( - I-3-PPP and haloperidol metabolite I, fell within the 95% prediction interval of the seven reference standards. Three, ( + I-butaclamol, PCP and TCP, fell at the borders of the prediction interval, and rimcazole (a putative antagonist) was clearly an outlier (Fig. 5). 4. Discussion In the present study, we have three major observations: (i) ui receptors are present in rat spleen and on isolated splenocytes; (ii) a series of 15 sigma agonists and related compounds suppressed ConA-induced splenocyte proliferation in a dose-dependent manner; and (iii) drug suppression of ConA-induced proliferation correlated highly with the pharmacology of pi receptors in spleen. The regression of EC,, values in bioassay versus Ki values in binding assays had a slope approaching unity, which is suggestive of a one-for-one relationship between g1 receptor binding events and biological responses. The reliability of measurements was high for both Ki and EC,, values. On a logarithmic scale, the standard errors of K, and EC,, values were 0.07 and 0.19 orders of magnitude, whereas the measurements of K, and EC,, ranged over 4 orders and 2.5 orders of magnitude, respectively. Therefore, the correlation was not significantly biased by errors in measurement. These results strongly support the hypothesis that drugs can modulate functional activities via actions at c, receptors on immune cells. It has been reported for almost a decade that PCP and PCP analogs can depress lymphocyte proliferation and alter secretion of antibody, IL-l and IL-2 in vitro (Khansari et al., 1984; Dornand et al., 1987), and that PCP can act at sigma receptors (Quirion et al., 1981,1987). At the time of these initial observations, the presumed site of action of these drugs was referred to as the sigma/PCP receptor (Quirion et al., 1981). Subsequently, two distinct receptors were recognized that could bind PCP (Quirion et al., 1987). These are now termed sigma and PCP receptors. Although certain drugs can bind to both of these, the affinities of binding and the rank order of potency of compounds are distinctive for each (Quirion et al., 1987). With the advent of more selective labels for PCP and sigma receptors, we were able to examine the immune system and to determine that sigma receptors were present in abundance in both spleen and circulating leukocytes, while PCP receptors were absent or below the threshold of detectability in these tissues (Wolfe et al., 1988; Wolfe and De Souza, 1992). However, PCP and TCP have additional actions at a variety of other non-sigma sites, including several potassium channels (Albuquerque et al., 1980; Blaustein and Ickowicz, 1983).

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Since these drugs fell right at the confidence limit of our correlation, we can neither hypothesize, nor rule out, a contribution by other, non-g1 receptors to the inhibition of proliferation caused by PCP and TCP. Because most sigma agonists also act at other receptors, it would be unrealistic to expect a perfect correlation between modulation of cell proliferation and drug binding potency at U, receptors. Nonetheless, all but three of the 14 tested agonists clearly fell within the 95% confidence limits of the correlation, and gave a collective r value of 0.84, inclusive with the reference group (r value of the reference group alone was 0.86). Most significantly, none of the compounds tested were less potent in suppressing proliferation than was predicted by their relative binding potencies at (pi sites. We interpret this as very strong, if not definitive, evidence that u, receptors on rat splenocytes are physiologically active. In our hands, the immunosuppressive effect of sigma agonists required drug concentrations several orders of magnitude greater than their K, or Ki values in binding assays. This is common in other bioassays of sigma agonists as well (Khansari et al., 1984; Dornand et al., 1987; Carr et al., 1991; Paul et al., 1993). Five possible explanations for this are: (i) The buffer used for binding assays is different from tissue culture medium in many respects, such as pH, ionic strength, the presence of specific ions, and the presence of exogenous proteins in serum. In the tissue culture medium in which the cells encountered drugs, the binding affinities of sigma agonists may be lower than the K, and Ki values obtained under optimized binding conditions. (ii) Over the course of 2-day cultures with live cells, sigma agonists may be metabolized into inactive forms. Therefore, higher initial drug concentrations may be required in order to maintain effective drug levels for the duration of the cultures. (iii) Binding sites for sigma ligands have been reported to reside within cells, as well as on the external surfaces of the plasma membranes (McCann and Su, 1990). Proliferation assays used intact cells, while binding assays were done with disrupted cytoplasmic membranes plus internal membranes and nuclei. If the biologically active sigma receptors reside within cells, and if the cytoplasmic membrane is not completely permeable to the drugs, elevated drug concentrations in the medium may be required to reach effective levels at the site of the active receptors within cells. (iv) Since we observed an inhibition of activity, it might be a non-specific toxic, rather than a receptor-mediated effect. Sigma agonists have

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been reported to be toxic to glioma cells over periods of about 1 week in culture (Vilner and Bowen, 1993). We saw no short-term drug toxicity evidenced by Trypan blue vital dye exclusion (data not shown). However, in the absence of stimulation, lymphocytes tend to gradually die off in culture, and it is difficult to distinguish between slow toxic effects and a drug-mediated blockade of the mitogenic stimulus (ConA). Recently, Casellas et al. (19941, reported that a novel sigma ligand, SR 31747, could suppress proliferation of human lymphocytes, and argued that this suppression was not due to toxicity, because after 120 h of drug-induced suppression the cells could be restored to full mitogenic responsiveness by removal of the drug. Regardless, the correlation of biological effect with the pharmacology of (T, receptors in our hands indicates that if this is a toxic effect, it is mediated through drug actions at (+i receptors. (v) Suppression of Con-A-induced proliferation may not occur unless a very high percentage of (T, receptors are occupied. Scenario (i) seems improbable because we have performed binding studies in RPM1 1640 culture medium with the identical additives as were used for cell cultures, and we observed no significant changes in affinities of drug binding (Liu and Wolfe, unpublished). At this time, we cannot distinguish among the remaining four possibilities. c1 receptors are selective for the (+ j-stereoenantiomers of 3-PPP and the psychotomimetic benzomorphans (pentazocine and SKF 10,047), while u2 receptors display minimal, or the reverse, selectivity (Quirion et al., 1992). In the proliferation assay, ( + )-pentazocine, ( + )-SKF10,047 and ( + )-3-PPP were invariably more biologically potent than their ( - j-stereoenantiomers in all experiments, although the differences were less pronounced than expected by their relative binding affinities (Table 2 and Fig. 5). It should be emphasized that all stereoisomers of these compounds fell within the 95% prediction interval of the correlation. Therefore, no actions by these compounds at non-a, sites are predicted or supported by our data. However, it is tempting to suggest possible explanations for this reduction of stereoselectivity. One of these is that the stereoselectivity that we observed in binding assays is an artifact of the buffer conditions, and that the receptor is less stereoselective in physiological buffers. However, in our hands competition binding revealed no diminution of stereoselectivity in tissue culture medium (Liu and Wolfe, unpublished). Therefore, we consider it more probable that other receptors may have made minor contributions to our proliferative assays. Receptors of particular interest in this regard are (TV, dopamine, and opiate receptors.

There is evidence that both ui and u2 receptors are biologically active. (+i sites appear to mediate the inhibition of electrically and 5-HT-induced contractions of guinea pig ileum (Campbell et al., 19891, the modulation of muscarinic acetylcholine receptor-triggered phosphoinositide turnover in brain (Bowen et al., 1990b; Brog and Beinfeld, 1990), and nicotinic receptor function in adrenal chromaffin cells (Paul et al., 1993). On the other hand, it is most likely that the dystonia from microinjection of sigma agonists into the rat red nucleus (Matsumoto et al., 1990), and modulation of potassium channels (Quirion et al., 1992; Jeanjean et al., 1993), are mediated through a, sites. Interestingly, pi and a2 receptors can co-exist. For example, rat liver contains approximately 25% (+i sites and 75% u2 sites (Hellewell et al., 1990); rat brain also contains both sites (Bowen et al., 1992). Our preliminary experiments support the notion that a2 receptors are also present along with ui receptors in immune tissues. Using [ 3H]DTG under a, receptor-selective conditions (Quirion et al., 19921, we have labeled specific binding sites in splenic homogenates, isolated splenocytes, and T and B cell lines (Wolfe et al., unpublished). However, the data we present here show a poor correlation with the pharmacological profile of a, receptors (Vilner and Bowen, 1992) (correlation not shown). Therefore, while a minor contribution by these receptors cannot be excluded, the overall pattern that we observed cannot be accounted for on the basis of drug actions at a, sites. There is overlapping drug specificity between dopamine, 5-HT, (serotonin) receptors and sigma receptors. Haloperidol is most commonly known for its actions as a dopamine receptor antagonist (reviewed in Seeman, 19811. However, the carbonyl-reduced metabolite of haloperidol (haloperidol metabolite II) has almost the same affinity to sigma receptors as haloperidol, but an 85-fold lower affinity to dopamine receptors; the chlorophenyl-hydroxy-piperidine metabolite of haloperidol (haloperidol metabolite I) lacks affinity for dopamine receptors, but has a moderate affinity for sigma receptors (Bowen et al., 1990a). Both of these haloperidol metabolites were immunosuppressive (Table 2), and fell within our correlation (Fig. 5). Furth ermore, the D, dopamine antagonist (- )-sulpiride failed to affect proliferation (data not shown). Thus, it seems unlikely that D, receptors had a significant contribution to the immunosuppressive effect we observed. At this time, we cannot rule out a minor contribution by D, dopamine receptors (Ricci and Amenta, 19941, but it should be emphasized that our over-all correlation of suppression of proliferation with u1 receptor binding indicates that ui sites play the predominant role in the present study. We obtained highly selective labeling of ui receptors with [3H]haloperidol by blocking dopamine and 5-HT,

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(serotonin) receptors with excess spiperone in all radioligand binding assays. We demonstrated the ur pharmacology of the sites labeled in this manner (Table 2), and showed a correlation between these drug binding sites and regulation of cell proliferation. Therefore, contributions by dopamine and serotonin receptors to the present findings must be minimal. Opiate receptors are known to be present on immune cells, and to affect a variety of functional activities, but the literature in this field is inconsistent. Our data are consistent with drug actions via or receptors. However, we consider it probable that interactions of opiate compounds with (or receptors, such as was the case with (--)-pentazocine and (--)-SKF 10,047 in this study, may be responsible for some of the confusion in the opiate literature. Rimcazole is a putative sigma antagonist (Ferris et al., 1986; Kennedy et al., 1990; Matsuno et al., 1993). As such, it should have had no effect in this system unless endogenous sigma agonists were present in the culture medium, or were generated by the cells in culture. Although, as expected, rimcazole was the only clear outlier, it was not expected to affect the system in the same direction as the agonists. We must assume that this relatively uncharacterized compound has additional, non-sigma actions that have yet to be described. The mechanism by which (or receptors modulate immune function is unknown, though one might speculate an inhibitory action on T cell phosphoinositide turnover in splenocytes. Such an action would be similar to that reported for rat synaptoneurosomes (Bowen et al., 1988), rat cortical brain slices (Candura et al., 1990) and pheochromocytoma cells (Bowen et al., 1992). There have been reports of @I receptor coupling to guanine regulatory proteins (Itzhak and Khouri, 1988; Itzhak, 1989), but no evidence for this was found in other studies. Dornand et al. (19871, found that PCP prevented resting splenocytes from hyperpolarizing, and reduced subsequent mitogen-induced depolarization and calcium mobilization. However, our data indicate that PCP and TCP may have additional, non-g1 actions on splenocytes. Thus, it is not clear which receptors are responsible for the phenomena reported by Dornand and associates. It should be obvious from this discussion that a major obstacle to investigations into the physiological role of sigma receptors is the broad cross-reactivity of many or most sigma agonists and antagonists with both (or and u2 sites, as well as with other, non-sigma receptors. Much effort has recently been devoted to developing and characterizing more selective compounds (Su et al., 1991; Bonhaus et al., 1993; Bonhaus et al., 1994; and numerous articles by De Costa and colleagues, reviewed in De Costa and He, 1994). The intent of the present study was to correlate function

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with the ‘classical’ pharmacology which has been reported for sigma binding sites. Future studies are planned in the immune system, in particular examination of cell signaling and early gene activation, which will utilize several of these newly developed compounds, some of which have the added advantage of being able to better discriminate between U, and u2 binding sites. In summary, we have found that suppression of ConA-induced proliferation correlates with the pharmacology of or receptors on splenocytes. Most other physiological assays involving sigma receptors have utilized preparations containing neuronal elements, and elicited responses with electrical stimulation or by means of neurotransmitters such as norepinephrine or 5-HT (Bowen et al., 1988; Campbell et al., 1989; Massamiri and Duckles, 1990a,b,1991; Paul et al., 1993). To the best of our knowledge, the splenocyte proliferative response to the mitogen, ConA, is the first demonstration of sigma receptors modulating a function that does not involve neuronal elements or induction of responses by neurotransmitters or neuroactive drugs. This implies that sigma agonists have a direct effect on immune cells. Since we have identified sigma receptors in the nervous, endocrine and immune systems (Wolfe et al., 1988,1989; Wolfe and De Souza, 1992), it is reasonable to suggest that endogenous sigma ligands may play a role in neuroimmune regulation. The presence of endogenous substance(s) which are released by ‘physiological’ stimuli, and which can bind sigma receptors, has been elegantly demonstrated in brain by Chavkin and associates (Neumaier and Chavkin, 1989a,b; Connor and Chavkin, 1991,1992a,b). Although there are several partially characterized tissue extracts, and a few identified molecules which have been reported to bind sigma receptors (reviewed in Patterson et al., 19941, at the present time the primary endogenous sigma agonist is unknown. Endogenous substances capable of binding sigma receptors have been reported in brain (Su et al., 1986; Contreras et al., 1987; Zhang et al., 1988; Connor and Chavkin, 1992a1, liver (Nagornaia et al., 1988) and pituitary (Glamsta et al., 1991). Elemental zinc (Zn2+) (Connor and Chavkin, 1992a), h-LVV-hemoporphin-6 (Glamsta et al., 19911, neuropeptide Y (NPY) (Roman et al., 19891, and a fragment of substance P (SP) (Mousseau et al., 1992) have been reported to compete at sigma receptors, although others have reported that NPY (McCann and Su, 1991; Tam and Mitchell, 1991) and SP (Patterson et al., 1994) have no such activity. Steroids, most notably progesterone, can bind sigma receptors (Su et al., 19881, and it has been postulated by Su et al. (1988, 1989) that sigma receptors may thus play a role in the anti-inflammatory actions and psychiatric disturbances caused by steroids. Schwarz and Pohl (Schwarz, 1989; Schwarz and Pohl, 1990) have contested this notion,

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noting that progesterone concentration in CSF does not reach physiological levels for sigma receptors in the brain, and that due to the presence of binding protein, only a small portion of the total progesterone in plasma is actually free and biologically available to receptors. In rebuttal, Su et al. (1989) have noted that ‘available free’ steroid concentrations in the brain may be much higher than in CSF, and that in pregnancy, ‘available free’ progesterone in plasma and local milieus such as placenta does in fact reach high enough levels to occupy a significant fraction of sigma receptors. Thus, they argue that endogenous steroids may be physiologically significant in this regard under specific circumstances, and that some of the anti-inflammatory actions and psychiatric disturbances caused by exogenously administered steroids may be due to actions at sigma receptors in immune cells and brain, respectively. Although one can speculate from these findings, at the present time the identity, origin, and means of delivery of endogenous sigma agonists to immune cells in non-pregnant individuals is unknown. There is significant exposure of humans to compounds that can act at sigma receptors. Among these are the prescribed drugs haloperidol (haldol), chlorpromazine (thorazine), perphenizine, thioridazine, pentazocine and anti-inflammatory steroids, and the abused drugs PCP, cocaine, and possibly anabolic steroids. Except for steroids, these drugs are used primarily for their effects at other receptors in the brain. However, the widespread distribution of sigma receptors in the central nervous, endocrine and digestive systems, and our demonstration that (+i sites are present and functional in the immune system, indicates that these sigma agonists may have direct actions on multiple target organs that result in more global physiological effects. Specifically, alteration of mental, endocrine, and immune status by prescribed and abused sigma agonists may be directly relevant to human health.

Acknowledgements

The authors wish to acknowledge the gracious assistance of Dr. Dennis Pearl, of The Ohio State University Department of Statistics, for discussions and consultation regarding statistical analysis of our data. This work was supported by the following grants: Ohio State University Seed Grant 221392; Bremer Foundation Award 9107; American Cancer Society Starter Grant lRG16-30; National Institute on Drug Abuse, NIH, lR29 DA07769; and National Cancer Institute, NIH, CA-16058.

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