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Antipsychotic drugs block IP3-dependent Ca2+-release from rat brain microsomes
Antipsychotic Drugs Block IP3-Dependent Ca 2+Release From Rat Brain Microsomes Steven R. Sczekan and Felix Strumwasser
Cellular Ca2+-dysregulation has been proposed as an important mechanism in certain diseases such as bipolar affective disorder (BPAD) and malignant hyperthermia. Recently it has been found that in BPAD, the plasma membrane Ca2+-channel blockers verapamil and nimodipine are useful substitutes in Li+-treatable patients. We have investigated the effects of these drugs and the antipsychotic drugs (clozapine, fluspirilene, and haloperidol) on IP3-induced Cae+-release from Ca e+-loaded rat brain microsomes. In the presence of either the Ca2+-channel blockers or the neuroleptic drugs, Ca2+-release was blocked in a dose-dependent fashion. For the neuroleptics, the ECso ranged from 22 ~M for fluspirilene to 145 t~M for haloperidol. The ECsofor nimodipine was 160 p~M and 450 ixM for verapamil. Carbamazapine and valproic acid, anticonvulsants recently used for treating BPAD, were relatively ineffective, as were various haloperidol metabolites. The research described in this paper establishes for the first time that antipsychotic drugs, as well as certain Cae+-channel blockers, directly block the IP3-induced Ca2+-release in a rat brain microsome assay. Key Words: Ca2+-release, IP 3, antipsychotics, Ca2+-channel blockers, rat brain BIOL PSYCHIATRY
1996;40:497-502
Introduction Most antipsychotic drugs target specific membrane receptors at postsynaptic and/or presynaptic sites in the brain, while antidepressant drugs are commonly inhibitors of transmitter reuptake transporters (Kaplan and Sadock 1995; Meltzer 1987). More recently, drugs which block plasma membrane Ca2+-channels have been used in treating manic-depressive illness (Dubovsky 1993). Haloperidol and clozapine, a typical and atypical antipsychotic, respectively, used in controlling acute and chronic psyFrom the Department of Psychiatry, Uniformed Services University of the Health Sciences. Bethesda, Maryland. Address reprint requests to Felix Strumwasser, Department of Psychiatry, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814-4799. Received February 6, 1995; revised December 18, 1995.
© 1996 Society of Biological Psychiatry
chosis, are examples of drugs that mainly target different receptors that are thought to play a role in schizophrenia. Increasingly, there is evidence of some type of Ca 2+dysregulation in bipolar disorder, as judged by the fact that intracellular basal CaZ+-levels and Ca2+-responses to various ligands are significantly elevated in the platelets and lymphocytes of untreated patients (Dubovsky et al 1989, 1992; Tan et al 1990; Dubovsky 1993; Eckert et al 1994). In addition, verapamil, a CaZ+-channel blocker, has been used recently as an effective anti-manic-depressive agent (Walden et al 1992). Finally, antidepressants have been found to block spontaneous oscillations in intracellular Ca 2+ concentration in cultured rat cortical neurons (Shimizu et al 1992). These findings led us to examine the effects of both antipsychotics and Ca2+-channel blockers on Ca2---re0006-3223/96/$15.00 SSDI 0006-3223(95)00657-5
498
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S.R. Sczekan and F. Strumwasser
1996;40:497-502
lease in rat brain microsome preparations. Mammalian brain microsome preparations have been shown to take up Ca 2+ through a Ca-adenosine triphosphatase (ATPase) pump and to release Ca2+ with either inositol 1,4,5 trisphosphate (IP 3) (Stauderman et al 1988) or cyclic adenosine diphosphoribose (cADPR) (White et al 1993). In this study we concentrated on the impact of the antipsychotics and Ca2+-channel blockers on the IP 3induced release of C a 2+.
Microsomal calcium release 300
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Methods Microsomes were isolated from whole rat brains on the day of the experiment. Male Sprague-Dawley rats were decapitated, and the brain quickly removed and placed on ice. Following crude mincing, the brains were homogenized in 9 volumes of homogenization buffer, consisting of 20 mM Hepes, 100 mM KC1, 0.3 M sucrose and 1 mM MgC12, pH 7.2, containing soybean trypsin inhibitor (100 p,g/ml), leupeptin (25 p~g/ml) and aprotinin (20 Ixg/ml). The tissue was gently homogenized in a Dounce glass homogenizer, with 6 - 8 passes, until the suspension was smooth. The mixture was centrifuged at 1000 X g for 5 rain, the supernatant removed and stored on ice while the pellet was washed with another 10 mls of homogenization buffer, and recentrifuged. The supernatants were combined and centrifuged at 9000 X g for 10 min to remove nuclei and mitochondria. The pale supernatant was then spun at 100,000 X g for 40 min to pellet the microsomes. The microsomal pellet obtained was gently resuspended in 600-800 IxL of homogenization buffer containing 1 mM ATP, 10 mM phosphocreatine, and 10 U/ml creatine phosphokinase. The suspension was left on ice for 1-2 hr prior to assay. Calcium release assays were performed in 2.5 ml samples, containing 150-175 txL aliquots of the microsomal suspension (final protein concentration = 0.5-1.0 mg/ml), in Hepes/KC1/PO 4 buffer containing 2 mM NaN 3. The mix was placed in the thermostatted cuvette holder of a Photon Technology International, Inc. (South Brunswick, NJ). M-series spectrofluorimeter, and stirred at 32°C for 10-15 min prior to challenge. Calcium levels were quantitated using the fluorescent dye fura-2 at 1 IxM, in a ratiometric mode. Excitation was at 340 and 380 nm and emission was detected at 510 nM. The ratio of the 510 nm fluorescent emission from the 340/380 excitation was computed and displayed as a continuous trace by the software. Conversion to absolute calcium levels was made by transforming the data using a standard curve previously prepared with known calcium concentrations, calibrated according to Grynkiewitz et al (1985). Reagent grade drugs were purchased from Sigma (St. Louis, MO) or
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time (seconds) Figure 1. Original [Ca2+] data traces with time under two different conditions (control and 25 txM fluspirilene). The fura-2 fluorescense was obtained with alternating excitation at 340 nm and 380 nm, and monitoring emission at 510 nm. At specific time intervals, the microsomes were challenged with 100 nM IP 3 (t = 200, 500 and 800 see., control trace). The trace for the sample containing fluspirilene is offset for greater clarity. The Q control trace indicates the quenching effect of the fluspirilene on a sample containing fura-2 but no microsomes. The asterisks indicate the point of addition of fluspirilene in the sample and quench traces.
Research Biochemicals International (RBI, Natick, MA). Reduced haloperidol and haloperidol metabolites I and III were obtained from RBI. A typical experiment involved adding an aliquot of brain microsomes, followed by fura-2, to the cuvette and monitoring the baseline ratio for Ca 2÷ uptake (asymptotic decrease in 340/380 emission). When the baseline was flat, 100 nM IP 3 (without drugs) was added and the response was stored as a file on the computer for later analysis (see Fig. 1). After each IP 3 response returned to baseline, up to two more administrations of IP 3 were made during a total testing period of about 1000 sec. Such control runs were assayed at the start and end of each series in order to provide a reference standard for the calcium release. Drug testing was accomplished by administering 100 nM IP 3 in the presence of the drug to a fresh aliquot of the same rat brain microsome preparation. The percent response was calculated by taking the ratio of the peak of the IP 3 response (in the presence of drug), measured from the immediately preceding baseline, to the peak of the response (without drug). For drugs dissolved in dimethyl sulfoxide (DMSO), control runs containing only DMSO were performed. Typically, IP 3 responses were measured in the presence of seven to ten drug concentrations for each of the five effective drugs tested. Multiple challenges at the same concentration of a drug were made to ensure accuracy. The interassay variability was quite
Antipsychotics Block Ca2+-Release
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1996;40:497-502
IP3-induced calcium release
IP3 -induced calcium release 100
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log [Fluspiriline], M Figure 2. Dose-response curve for the antipsychotic drug fluspirilene on Ca2+-release from rat brain microsomes induced by IP3. The drug was added to the reaction at t = 0 sec, and the microsomes were monitored until a stable baseline of calcium concentration was achieved, at which time the I P 3 challenges were made. The measured calcium release is calculated as the percent of calcium released by the I P 3 challenge in the absence of the drug (control), and transformed as percent inhibition. The IP3 challenge used was 100 nM. At least three challenges at a given drug concentration were made to ensure accuracy. The solid line indicates the statistically fitted curve, generated as in the text.
small as can be seen from the fit of the curve to the data points in Figure 2 which presents a typical set of dose/ response points. The correlation coefficients of the dose/ response curves ranged from 0.982 to 0.996 for the five drugs tested. The data were analyzed with a four-parameter logistic equation (non-linear regression), and fitted via the Marquardt-Levenburg method, as described in Press et al (1988). An important control in these studies was to determine the degree of possible quenching of the furao2 fluorescence by the drugs themselves, i.e., in the absence of microsomes. We performed these experiments under the same buffer conditions and temperature described above (Hepes/KC1/PO4 buffer containing 2 mM NAN3), including the ATP but not the phosphocreatine and creatine phosphokinase. Ethylene glycol-bis(13-aminoethylether)N,N,N',N'-tetraacetic acid (10 p~M) was used to simulate the ambient Ca 2+ levels in the microsome assays. Two challenges of 1 ixM CaCI2 were tested in each run. The reference control was a run with no drug. The five drugs of interest were studied at their EC5o for the inhibition of IP3-induced Ca2+-release. Our results indicated that neither fluspirilene nor nimodipine quenched Ca2+-induced fura-2 fluorescence. There were small quenching effects with the other drugs: haloperidol (7%), clozapine (9%), and verapamil (14%). Some of the drugs produced a baseline decrease (clozapine > nimodipine = verapamil).
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log [drug], M Figure 3. Composite graph comparing the effects of the five antipsychotics on the IP3-induced calcium release. C, clozapine; F, fluspirilene; H, haloperidol; N, nimodipine; V, verapamil.
This artifact was not investigated but does not appear to be simply due to quenching.
Results A representative calcium release experiment is shown in Figure 1. The three larger responses represent the calcium release to successive 100 nM IP 3 pulses. This concentration of IP 3 typically released a significant amount of sequestered calcium, resulting in an overall change in the calcium concentration of 75-100 nM. At this level of IP 3 (100 nM), desensitization of the IP 3 receptor was minimized. The amount of calcium released was approximately 40% of that released by 1 ixM IP 3. In the presence of the compounds tested, e.g., fluspirilene (Fig. 1, three smaller responses), calcium release was markedly inhibited, and the inhibition was dose-dependent (Figure 2). As a rule, the amplitude of the change was most strongly affected, while the recovery time required for reuptake of the released calcium remained relatively unaffected, suggesting that the Ca2+-ATPase was not compromised. Of the five drugs we tested, clozapine, fluspirilene, haloperidol, and nimodipine were found to inhibit the IP3-dependent calcium release when present in the low micromolar range. The fifth, verapamil, also resulted in a dose-dependent inhibition of the calcium release, but the concentration required was significantly higher. Doseresponse curves of the calcium response vs drug concentration are shown in Figure 3. The response typically exhibited sigmoidal dose effects. The ECso (effective concentration for 50% inhibition) was calculated from the least squares fitted curves. Values are shown in Table 1. Correlation coefficients for the statistically fitted curves were greater than 0.98 in all cases. Fluspirilene was the most effective of the three antipsychotic drugs tested, with
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1996;40:497-502
Table 1. Concentration of Drug Required for 50% Inhibition of IP3-Induced Ca2+-Release Drug Clozapine Fluspirilene Haloperidol Nimodipine Verapamil
ECso (IxM) 140 22 145 160 450
an ECso of 22 ixM, while of the two Ca2+-channel blockers, nimodipine (ECso = 160 ~xM) was much more effective than verapamil (EC5o = 450 IxM). Neither carbamazepine, valproic acid, nor the reduced form of haloperidol (or metabolites I and III) had significant effects on IP3-induced Ca2+-release at concentrations below 1 mM.
Discussion Several lines of evidence suggest that intracellular Ca 2+ levels are perturbed in cases of bipolar disorder (Dubovsky et al 1989; Tan et al 1990; Dubovsky et al 1992, 1994; Eckert et al 1994; Kusumi et al 1994). There is a significantly increased baseline intracellular Ca 2÷ concentration in platelets and lymphocytes in patients during mania. Also, the amplitude of the responses of intracellularly-released Ca z+ in platelets stimulated with platelet-activating factor, thrombin or serotonin, as well as lymphocytes stimulated with other ligands, are significantly higher in manic and bipolar depressed patients than in controls and other groups. There has been a renewed interest recently in the use of calcium channel blockers in the treatment of bipolar disorder. In several studies (Hoschl and Kozeny 1989; Garza-Trevino et al 1992; Walden et al 1992; Dubovsky 1993), verapamil, diltiazem, and nimodipine exhibited significant antimanic properties, and verapamil was as effective as lithium in several cases. The fact that there is evidence for Ca2+-dysregulation in bipolar disorder, that Caa+-channel blockers can be successfully used to treat such patients, and that our present study finds inhibition of intracellular Ca2+-release by nimodipine and verapamil, lends support to an impairment in one or more aspects of intracellular Ca 2÷ regulation in bipolar disorder. There is evidence that bipolar affective disorder (BPAD) is genetically transmitted but with a complex mode of inheritance (Simpson et al 1992; Berrettini et al 1994). Recently, two independent studies have focused on the pericentromeric region of chromosome 18 (Berrettini et al 1994; Stine et al 1995) using linkage analysis of genetic markers by logarithm of odds score. It is too early to know how these studies will hold up and
what the susceptible genes might be, however, there is precedent for an impairment in intracellular Ca 2+ regulation in humans that is genetically-based and results in the clinical condition of malignant hyperthermia. In susceptible subjects certain volatile anaesthetics (e.g., halothane) induce the Ca2÷-release channel (ryanodine receptor) of skeletal muscle to open, inducing a sustained heat production that can prove fatal (McLennan and Phillips 1992). It is possible that the Ca2+-dysregulation associated with bipolar disorder is an example of a genetically-based alteration in some aspect of intracellular Ca 2+ regulation. Alternatively, the Ca2+-dysregulation may be a secondary consequence of another dysregulated system. Our results indicate that neither carbamazepine nor valproate, classical antiepileptics, inhibited IP3-induced Ca2+-release. These drugs have been used as adjuncts or monotherapeutics in treating BPAD with varied results (Calabrese and Woyshville 1995; Bowden and McElroy 1995). A recent meta-analysis of carbamazepine compared to lithium concludes that its prophylactic efficacy is questionable (Dardennes et al 1995). Both carbamazepine and valproate have actions facilitating GABAergic actions, although carbamazepine's actions may be indirect since it is thought to act primarily on the peripheral type of benzodiazepine receptor located in mitochondrial membranes, resulting in increased pregnenolone formation and other neuroactive steroids that then act centrally (Post 1995). Thus, these two agents may relieve manic symptoms through enhancement of GABAergic transmission, thereby countering the putative Ca2+-dysregulation. We have investigated the potential effects of various anti-psychotic agents on calcium homeostasis in rat brain microsomes by directly measuring the amount of calcium released via the IP 3 receptor in the presence of these compounds. Our results indicate that, as a general rule, the release of stored Ca 2+ from IP3-sensitive pools is inhibited by these drugs in a dose-dependent manner. Such an action may play a role in the pharmacological profiles of these drugs. The research presented here raises the possibility that inhibition of Ca 2+ release may arise as a primary or secondary effect during treatments with neuroleptic agents, however, the question of whether or not the inhibition by these drugs on the intracellular Ca 2÷ release occurs in vivo during treatment cannot currently be answered. The concentrations found which yield measurable effects, in the range of 10 ~xM-50 txM, may appear to be pharmacologically excessive when compared with therapeutic plasma levels (approximately 1 p~M for clozapine and verapamil and 0.1 ixM for haloperidol), however, since we have directly measured the microsomal IP 3 response, these data reflect the conditions found in the interior of the cell. Little is known regarding the intracel-
Antipsychotics Block Ca2+-Release
lular concentrations of these drugs. While their established actions lie on the plasma membrane, where traditionally there are either dopaminergic and other related receptors or cellular calcium channels, it is highly probable that many of these drugs are also internalized to an appreciable extent. Haloperidol, for example, is extensively metabolized by the microsomes of the liver, which is in fact the likely site for intracellular calcium storage. Hence, the microsomal microenvironment of the cell may be exposed to unusually high concentrations of the drug. Furthermore, several of these drugs are extremely hydrophobic, and, as such, can be expected to partition readily into the cellular membranes, where they may be concentrated. It is therefore plausible that the drugs could accumulate to a degree sufficient to impact on Ca 2+ release. The internal membranes of cells could be expected to act as a large sink for the more hydrophobic drugs (clozapine, fluspirilene, nimodipine), because these membranes make up 95-98% of the total membrane in mammalian cells (Alberts et al 1994). Finally, there is evidence that the antipsychotic drug chlorpromazine, a cationic amphiphilic agent, interferes with receptor recycling by trapping receptors in the endosomes (Wang et al 1993). If any of the drugs that we have tested would bind to and enrich the endosome population, the intracellular drug concentrations would be enhanced in vivo by this mechanism. While we favor the idea that hydrophobic drugs concentrate in internal membranes of the cell, further work is needed to determine the actual intracellular concentrations of the drugs used in our studies to determine clinical relevance. Previous studies have shown that various plasma membrane calcium channels are affected by a variety of
BIOLPSYCHIATRY 1996;40:497-502
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neuroleptics, including fluspirilene and haloperidol (Galizzi et al 1986; Enyeart et al 1992). Haloperidol has been found to reduce histamine-induced smooth muscle contractions dependent on intracellular Ca2+-release (Hishinuma et al 1992), and to block the twitch response in rat phrenic nerve diaphragm preparations at 1 0 - 8 0 p~M (Seth et al 1991). Hence, it seems clear that such compounds are acting potentially through a variety of targets, not merely the established dopaminergic receptors. In a direct calcium-release assay, we have found fluspirilene, haloperidol, and clozapine, which are typically thought of as dopaminergic receptor antagonists, to be either as effective or more effective at blocking the IP3-dependent calcium release than verapamil, a traditional calcium channel blocker, or nimodipine, a more recently developed calcium channel blocker (Fig. 3). The fact that plasma membrane Ca2+-channel blockers inhibited IP3-dependent cae+-release, in spite of the fact that the amino acid sequences of various plasma membrane Ca2+-channels share little homology with the IP3-dependent Ca2+-release channel, suggests that the tertiary and quaternary structures of these two rather different Ca 2+channels might share some similarities.
This research was supported by an intramural Uniformed Services University of the Health Sciences (USUHS)grant (C0-8893)to F.S. We express our appreciation to Mrs. Eleanore Gamble for all of her assistance in the laboratory. We are grateful to Dr. Martha M. Coetzee (USUHS) for very helpful discussions in the field of psychopharmacology and to Drs. B.W. Agranoff(U. of Michigan), S. K. Fisher (U. of Michigan) and J. P. Staab (USUHS) for useful comments on an earlier version of the manuscript.
References Alberts B, Bray D, Lewis J, Raft M, Roberts K, Watson JD (1994): Molecular Biology of the Cell, 3rd ed. New York: Garland Publishing, Inc. p 553. Berrettini WH, Ferraro TN, Goldin LR, Weeks DE, DeteraWadleigh S, Nurnberger Jr JI, Gershon ES (1994): Chromosome 18 DNA markers and manic-depressive illness: Evidence for a susceptibility gene. Proc Natl Acad Sci USA 91:5918-5921. Bowden C, McElroy S (1995): History of the development of valproate for treatment of bipolar disorder. J Clin Psychiatry 56 (suppl 3):3-5. Calahrese JR, Woyshville MJ (1995): A medication algorithm for treatment of bipolar rapid cycling? J Clin Psychiatry 56 (suppl 3): 11-18. Dardennes R, Even C, Bange F, Heim A (1995): Comparison of carbamazepine and lithium in the prophylaxis of bipolar disorders. Brit J of Psychiatry 166:378-381.
Dubovsky SL (1993): Calcium antagonists in manic-depressive disease. Neuropsychobiology 27:184-192. Dubovsky SL, Christiano J, Daniell LC, Franks RD, Murphy J, Adler L, Baker N, Harris RA (1989): Increased platelet intracellular calcium concentration in patients with bipolar affective disorders. Arch Gen Psychiatry 46:632-638. Dubovsky SL, Murphy J, Thomas M, Rademacher J (1992): Abnormal intracellular calcium ion concentration in platelets and lymphocytes of bipolar patients. Am J Psychiatry 149: 118-120. Dubovsky SL, Thomas M, Hijazi A, Murphy J (1994): Intracellular calcium signalling in peripheral cells of patients with bipolar affective disorder. Eur Arch Psychiatry Clin Neurosci 243:229-234. Eckert A, Gann H, Riemann D, Aldenhoff J, Mtiller WE (1994): Platelet and lymphocyte free intracellular calcium in affective disorders. Eur Arch Psychiatry Clin Neurosci 243:235-239.
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Enyeart JJ, Biagi BA, Mlinar B (1992): Preferential block of T-type calcium channels by neuroleptics in neural crestderived rat and human C cell lines. Mol Pharmacol 42:364372. Galizzi JP, Fosset M, Romey G, Laduron P, Lazdunski M (1986): Neuroleptics of the diphenylbutylpiperidine series are potent calcium channel inhibitors. Proc Natl Acad Sci 83: 7513-7517. Garza-Trevino ES, Overall JE, Hollister LE (1992): Verapamil versus lithium in acute mania. Am J Psychiatry 149:121-122. Grynkiewicz G, Poenie M, Tsien RY (1985): A new generation of Ca z+ indicators with greatly improved fluorescence properties. J Biol Chem 260:3440-3450. Hishinuma S, Hongo I, Uchida MK, Kurokawa M (1992): Haloperidol differentiates smooth muscle contractions induced by release of intracellularly stored Ca and by influx of extracellular Ca. Gen Pharmacol 23:211-215. Hoschl C, Kozeny J (1989): Verapamil in affective disorders: a controlled, double-blind study. Biol Psychiatry 25:128-140. Kaplan HI, Sadock BJ (eds) (1995): Comprehensive Textbook of Psychiatry/V1, 6th ed. Baltimore: Williams and Wilkins. Kusumi I, Koyama T, Yamashita I (1994): Serotonin-induced platelet intracellular calcium mobilization in depressed patients. Psychopharmacology 113:322-327. MacLennan DH, Phillips MS (1992): Malignant hyperthermia. Science 256:789-794. Meltzer HY (1987): Psychopharmacology: The Third Generation of Progress, New York: Raven Press. Post RM (1995): Carbamazepine. In Kaplan HI, Sadock BJ (eds), Comprehensive Textbook of Psychiatry/Vl, 6th ed. Baltimore: Williams and Wilkins, pp 1964-1972. Press WH, Flannery BP, Teukolsky SA, Vetterling WT (1988): Numerical Recipes in C, The Art of Scientific Computing, New York: Cambridge University Press.
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Seth P, Maitra KK, Ganguly DK (1991): Haloperidol on rat phrenic hemidiaphragm. Arch Int Pharmacodyn Ther 310: 87-93. Shimizu M, Nishida A, Yamawaki S (1992): Antidepressants inhibit spontaneous oscillations of intracellular Ca 2+ concentration in rat cortical cultured neurons. Neurosci Lett 146: 101-104. Simpson SG, Folstein SE, Myers DA, DePaulo JR (1992): The assessment of lineality in bipolar I linkage studies. Am J Psychiatry 149:1660-1665. Stauderman KA, Harris GD, Lovenberg W (1988): Characterization of inositol 1,4,5-trisphosphate-stimulated calcium release from rat cerebellar microsomal fractions. Comparison with [3H]inositol 1,4,5-trisphosphate binding. Biochem J 255:677-683. Stine OC, Xu J, Koskela R, McMahon FJ, Gschwend M, Friddle C, Clark CD, McInnis MG, Simpson SG, Breschel TS, Vishio E, Riskin K, Feilotter H, Chen E, Shen S, Folstein S, Meyers DA, Botstein D, Marr TG, DePaulo JR (1995): Evidence for linkage of bipolar disorder to chromosome 18 with a parentof-origin effect. Am J Hum Genet 57:1384-1394. Tan CH, Javors MA, Seleshi E, Lowrimore PA, Bowden CL (1990): Effects of lithium on platelet ionic intracellular calcium concentration in patients with bipolar (manic-depressive) disorder and healthy controls. Life Sci 46:1175-1180. Walden J, Grunze H, Olbrich H, Berger M (1992): Importance of calcium ions and calcium antagonists in affective psychoses. Fortschr Neurol Psychiatr 60:471-476. Wang L-H, Rothberg KG, Anderson RGW (1993): Mis-assembly of clathrin lattices on endosomes reveals a regulatory switch for coated pit formation. J Cell Biol 123:1107-1117. White AM, Watson SP, Galione A (1993): Cyclic ADP-riboseinduced Ca 2+ release from rat brain microsomes. FEBS Lett 318:259-263.
Cellular Ca2+-dysregulation has been proposed as an important mechanism in certain diseases such as bipolar affective disorder (BPAD) and malignant hyperthermia. Recently it has been found that in BPAD, the plasma membrane Ca2+-channel blockers verapamil and nimodipine are useful substitutes in Li+-treatable patients. We have investigated the effects of these drugs and the antipsychotic drugs (clozapine, fluspirilene, and haloperidol) on IP3-induced Cae+-release from Ca e+-loaded rat brain microsomes. In the presence of either the Ca2+-channel blockers or the neuroleptic drugs, Ca2+-release was blocked in a dose-dependent fashion. For the neuroleptics, the ECso ranged from 22 ~M for fluspirilene to 145 t~M for haloperidol. The ECsofor nimodipine was 160 p~M and 450 ixM for verapamil. Carbamazapine and valproic acid, anticonvulsants recently used for treating BPAD, were relatively ineffective, as were various haloperidol metabolites. The research described in this paper establishes for the first time that antipsychotic drugs, as well as certain Cae+-channel blockers, directly block the IP3-induced Ca2+-release in a rat brain microsome assay. Key Words: Ca2+-release, IP 3, antipsychotics, Ca2+-channel blockers, rat brain BIOL PSYCHIATRY
1996;40:497-502
Introduction Most antipsychotic drugs target specific membrane receptors at postsynaptic and/or presynaptic sites in the brain, while antidepressant drugs are commonly inhibitors of transmitter reuptake transporters (Kaplan and Sadock 1995; Meltzer 1987). More recently, drugs which block plasma membrane Ca2+-channels have been used in treating manic-depressive illness (Dubovsky 1993). Haloperidol and clozapine, a typical and atypical antipsychotic, respectively, used in controlling acute and chronic psyFrom the Department of Psychiatry, Uniformed Services University of the Health Sciences. Bethesda, Maryland. Address reprint requests to Felix Strumwasser, Department of Psychiatry, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814-4799. Received February 6, 1995; revised December 18, 1995.
© 1996 Society of Biological Psychiatry
chosis, are examples of drugs that mainly target different receptors that are thought to play a role in schizophrenia. Increasingly, there is evidence of some type of Ca 2+dysregulation in bipolar disorder, as judged by the fact that intracellular basal CaZ+-levels and Ca2+-responses to various ligands are significantly elevated in the platelets and lymphocytes of untreated patients (Dubovsky et al 1989, 1992; Tan et al 1990; Dubovsky 1993; Eckert et al 1994). In addition, verapamil, a CaZ+-channel blocker, has been used recently as an effective anti-manic-depressive agent (Walden et al 1992). Finally, antidepressants have been found to block spontaneous oscillations in intracellular Ca 2+ concentration in cultured rat cortical neurons (Shimizu et al 1992). These findings led us to examine the effects of both antipsychotics and Ca2+-channel blockers on Ca2---re0006-3223/96/$15.00 SSDI 0006-3223(95)00657-5
498
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S.R. Sczekan and F. Strumwasser
1996;40:497-502
lease in rat brain microsome preparations. Mammalian brain microsome preparations have been shown to take up Ca 2+ through a Ca-adenosine triphosphatase (ATPase) pump and to release Ca2+ with either inositol 1,4,5 trisphosphate (IP 3) (Stauderman et al 1988) or cyclic adenosine diphosphoribose (cADPR) (White et al 1993). In this study we concentrated on the impact of the antipsychotics and Ca2+-channel blockers on the IP 3induced release of C a 2+.
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Methods Microsomes were isolated from whole rat brains on the day of the experiment. Male Sprague-Dawley rats were decapitated, and the brain quickly removed and placed on ice. Following crude mincing, the brains were homogenized in 9 volumes of homogenization buffer, consisting of 20 mM Hepes, 100 mM KC1, 0.3 M sucrose and 1 mM MgC12, pH 7.2, containing soybean trypsin inhibitor (100 p,g/ml), leupeptin (25 p~g/ml) and aprotinin (20 Ixg/ml). The tissue was gently homogenized in a Dounce glass homogenizer, with 6 - 8 passes, until the suspension was smooth. The mixture was centrifuged at 1000 X g for 5 rain, the supernatant removed and stored on ice while the pellet was washed with another 10 mls of homogenization buffer, and recentrifuged. The supernatants were combined and centrifuged at 9000 X g for 10 min to remove nuclei and mitochondria. The pale supernatant was then spun at 100,000 X g for 40 min to pellet the microsomes. The microsomal pellet obtained was gently resuspended in 600-800 IxL of homogenization buffer containing 1 mM ATP, 10 mM phosphocreatine, and 10 U/ml creatine phosphokinase. The suspension was left on ice for 1-2 hr prior to assay. Calcium release assays were performed in 2.5 ml samples, containing 150-175 txL aliquots of the microsomal suspension (final protein concentration = 0.5-1.0 mg/ml), in Hepes/KC1/PO 4 buffer containing 2 mM NaN 3. The mix was placed in the thermostatted cuvette holder of a Photon Technology International, Inc. (South Brunswick, NJ). M-series spectrofluorimeter, and stirred at 32°C for 10-15 min prior to challenge. Calcium levels were quantitated using the fluorescent dye fura-2 at 1 IxM, in a ratiometric mode. Excitation was at 340 and 380 nm and emission was detected at 510 nM. The ratio of the 510 nm fluorescent emission from the 340/380 excitation was computed and displayed as a continuous trace by the software. Conversion to absolute calcium levels was made by transforming the data using a standard curve previously prepared with known calcium concentrations, calibrated according to Grynkiewitz et al (1985). Reagent grade drugs were purchased from Sigma (St. Louis, MO) or
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time (seconds) Figure 1. Original [Ca2+] data traces with time under two different conditions (control and 25 txM fluspirilene). The fura-2 fluorescense was obtained with alternating excitation at 340 nm and 380 nm, and monitoring emission at 510 nm. At specific time intervals, the microsomes were challenged with 100 nM IP 3 (t = 200, 500 and 800 see., control trace). The trace for the sample containing fluspirilene is offset for greater clarity. The Q control trace indicates the quenching effect of the fluspirilene on a sample containing fura-2 but no microsomes. The asterisks indicate the point of addition of fluspirilene in the sample and quench traces.
Research Biochemicals International (RBI, Natick, MA). Reduced haloperidol and haloperidol metabolites I and III were obtained from RBI. A typical experiment involved adding an aliquot of brain microsomes, followed by fura-2, to the cuvette and monitoring the baseline ratio for Ca 2÷ uptake (asymptotic decrease in 340/380 emission). When the baseline was flat, 100 nM IP 3 (without drugs) was added and the response was stored as a file on the computer for later analysis (see Fig. 1). After each IP 3 response returned to baseline, up to two more administrations of IP 3 were made during a total testing period of about 1000 sec. Such control runs were assayed at the start and end of each series in order to provide a reference standard for the calcium release. Drug testing was accomplished by administering 100 nM IP 3 in the presence of the drug to a fresh aliquot of the same rat brain microsome preparation. The percent response was calculated by taking the ratio of the peak of the IP 3 response (in the presence of drug), measured from the immediately preceding baseline, to the peak of the response (without drug). For drugs dissolved in dimethyl sulfoxide (DMSO), control runs containing only DMSO were performed. Typically, IP 3 responses were measured in the presence of seven to ten drug concentrations for each of the five effective drugs tested. Multiple challenges at the same concentration of a drug were made to ensure accuracy. The interassay variability was quite
Antipsychotics Block Ca2+-Release
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IP3-induced calcium release
IP3 -induced calcium release 100
,
,
100
,
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40 21
20 0 -7.0
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log [Fluspiriline], M Figure 2. Dose-response curve for the antipsychotic drug fluspirilene on Ca2+-release from rat brain microsomes induced by IP3. The drug was added to the reaction at t = 0 sec, and the microsomes were monitored until a stable baseline of calcium concentration was achieved, at which time the I P 3 challenges were made. The measured calcium release is calculated as the percent of calcium released by the I P 3 challenge in the absence of the drug (control), and transformed as percent inhibition. The IP3 challenge used was 100 nM. At least three challenges at a given drug concentration were made to ensure accuracy. The solid line indicates the statistically fitted curve, generated as in the text.
small as can be seen from the fit of the curve to the data points in Figure 2 which presents a typical set of dose/ response points. The correlation coefficients of the dose/ response curves ranged from 0.982 to 0.996 for the five drugs tested. The data were analyzed with a four-parameter logistic equation (non-linear regression), and fitted via the Marquardt-Levenburg method, as described in Press et al (1988). An important control in these studies was to determine the degree of possible quenching of the furao2 fluorescence by the drugs themselves, i.e., in the absence of microsomes. We performed these experiments under the same buffer conditions and temperature described above (Hepes/KC1/PO4 buffer containing 2 mM NAN3), including the ATP but not the phosphocreatine and creatine phosphokinase. Ethylene glycol-bis(13-aminoethylether)N,N,N',N'-tetraacetic acid (10 p~M) was used to simulate the ambient Ca 2+ levels in the microsome assays. Two challenges of 1 ixM CaCI2 were tested in each run. The reference control was a run with no drug. The five drugs of interest were studied at their EC5o for the inhibition of IP3-induced Ca2+-release. Our results indicated that neither fluspirilene nor nimodipine quenched Ca2+-induced fura-2 fluorescence. There were small quenching effects with the other drugs: haloperidol (7%), clozapine (9%), and verapamil (14%). Some of the drugs produced a baseline decrease (clozapine > nimodipine = verapamil).
I
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-4
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-3
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log [drug], M Figure 3. Composite graph comparing the effects of the five antipsychotics on the IP3-induced calcium release. C, clozapine; F, fluspirilene; H, haloperidol; N, nimodipine; V, verapamil.
This artifact was not investigated but does not appear to be simply due to quenching.
Results A representative calcium release experiment is shown in Figure 1. The three larger responses represent the calcium release to successive 100 nM IP 3 pulses. This concentration of IP 3 typically released a significant amount of sequestered calcium, resulting in an overall change in the calcium concentration of 75-100 nM. At this level of IP 3 (100 nM), desensitization of the IP 3 receptor was minimized. The amount of calcium released was approximately 40% of that released by 1 ixM IP 3. In the presence of the compounds tested, e.g., fluspirilene (Fig. 1, three smaller responses), calcium release was markedly inhibited, and the inhibition was dose-dependent (Figure 2). As a rule, the amplitude of the change was most strongly affected, while the recovery time required for reuptake of the released calcium remained relatively unaffected, suggesting that the Ca2+-ATPase was not compromised. Of the five drugs we tested, clozapine, fluspirilene, haloperidol, and nimodipine were found to inhibit the IP3-dependent calcium release when present in the low micromolar range. The fifth, verapamil, also resulted in a dose-dependent inhibition of the calcium release, but the concentration required was significantly higher. Doseresponse curves of the calcium response vs drug concentration are shown in Figure 3. The response typically exhibited sigmoidal dose effects. The ECso (effective concentration for 50% inhibition) was calculated from the least squares fitted curves. Values are shown in Table 1. Correlation coefficients for the statistically fitted curves were greater than 0.98 in all cases. Fluspirilene was the most effective of the three antipsychotic drugs tested, with
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Table 1. Concentration of Drug Required for 50% Inhibition of IP3-Induced Ca2+-Release Drug Clozapine Fluspirilene Haloperidol Nimodipine Verapamil
ECso (IxM) 140 22 145 160 450
an ECso of 22 ixM, while of the two Ca2+-channel blockers, nimodipine (ECso = 160 ~xM) was much more effective than verapamil (EC5o = 450 IxM). Neither carbamazepine, valproic acid, nor the reduced form of haloperidol (or metabolites I and III) had significant effects on IP3-induced Ca2+-release at concentrations below 1 mM.
Discussion Several lines of evidence suggest that intracellular Ca 2+ levels are perturbed in cases of bipolar disorder (Dubovsky et al 1989; Tan et al 1990; Dubovsky et al 1992, 1994; Eckert et al 1994; Kusumi et al 1994). There is a significantly increased baseline intracellular Ca 2÷ concentration in platelets and lymphocytes in patients during mania. Also, the amplitude of the responses of intracellularly-released Ca z+ in platelets stimulated with platelet-activating factor, thrombin or serotonin, as well as lymphocytes stimulated with other ligands, are significantly higher in manic and bipolar depressed patients than in controls and other groups. There has been a renewed interest recently in the use of calcium channel blockers in the treatment of bipolar disorder. In several studies (Hoschl and Kozeny 1989; Garza-Trevino et al 1992; Walden et al 1992; Dubovsky 1993), verapamil, diltiazem, and nimodipine exhibited significant antimanic properties, and verapamil was as effective as lithium in several cases. The fact that there is evidence for Ca2+-dysregulation in bipolar disorder, that Caa+-channel blockers can be successfully used to treat such patients, and that our present study finds inhibition of intracellular Ca2+-release by nimodipine and verapamil, lends support to an impairment in one or more aspects of intracellular Ca 2÷ regulation in bipolar disorder. There is evidence that bipolar affective disorder (BPAD) is genetically transmitted but with a complex mode of inheritance (Simpson et al 1992; Berrettini et al 1994). Recently, two independent studies have focused on the pericentromeric region of chromosome 18 (Berrettini et al 1994; Stine et al 1995) using linkage analysis of genetic markers by logarithm of odds score. It is too early to know how these studies will hold up and
what the susceptible genes might be, however, there is precedent for an impairment in intracellular Ca 2+ regulation in humans that is genetically-based and results in the clinical condition of malignant hyperthermia. In susceptible subjects certain volatile anaesthetics (e.g., halothane) induce the Ca2÷-release channel (ryanodine receptor) of skeletal muscle to open, inducing a sustained heat production that can prove fatal (McLennan and Phillips 1992). It is possible that the Ca2+-dysregulation associated with bipolar disorder is an example of a genetically-based alteration in some aspect of intracellular Ca 2+ regulation. Alternatively, the Ca2+-dysregulation may be a secondary consequence of another dysregulated system. Our results indicate that neither carbamazepine nor valproate, classical antiepileptics, inhibited IP3-induced Ca2+-release. These drugs have been used as adjuncts or monotherapeutics in treating BPAD with varied results (Calabrese and Woyshville 1995; Bowden and McElroy 1995). A recent meta-analysis of carbamazepine compared to lithium concludes that its prophylactic efficacy is questionable (Dardennes et al 1995). Both carbamazepine and valproate have actions facilitating GABAergic actions, although carbamazepine's actions may be indirect since it is thought to act primarily on the peripheral type of benzodiazepine receptor located in mitochondrial membranes, resulting in increased pregnenolone formation and other neuroactive steroids that then act centrally (Post 1995). Thus, these two agents may relieve manic symptoms through enhancement of GABAergic transmission, thereby countering the putative Ca2+-dysregulation. We have investigated the potential effects of various anti-psychotic agents on calcium homeostasis in rat brain microsomes by directly measuring the amount of calcium released via the IP 3 receptor in the presence of these compounds. Our results indicate that, as a general rule, the release of stored Ca 2+ from IP3-sensitive pools is inhibited by these drugs in a dose-dependent manner. Such an action may play a role in the pharmacological profiles of these drugs. The research presented here raises the possibility that inhibition of Ca 2+ release may arise as a primary or secondary effect during treatments with neuroleptic agents, however, the question of whether or not the inhibition by these drugs on the intracellular Ca 2÷ release occurs in vivo during treatment cannot currently be answered. The concentrations found which yield measurable effects, in the range of 10 ~xM-50 txM, may appear to be pharmacologically excessive when compared with therapeutic plasma levels (approximately 1 p~M for clozapine and verapamil and 0.1 ixM for haloperidol), however, since we have directly measured the microsomal IP 3 response, these data reflect the conditions found in the interior of the cell. Little is known regarding the intracel-
Antipsychotics Block Ca2+-Release
lular concentrations of these drugs. While their established actions lie on the plasma membrane, where traditionally there are either dopaminergic and other related receptors or cellular calcium channels, it is highly probable that many of these drugs are also internalized to an appreciable extent. Haloperidol, for example, is extensively metabolized by the microsomes of the liver, which is in fact the likely site for intracellular calcium storage. Hence, the microsomal microenvironment of the cell may be exposed to unusually high concentrations of the drug. Furthermore, several of these drugs are extremely hydrophobic, and, as such, can be expected to partition readily into the cellular membranes, where they may be concentrated. It is therefore plausible that the drugs could accumulate to a degree sufficient to impact on Ca 2+ release. The internal membranes of cells could be expected to act as a large sink for the more hydrophobic drugs (clozapine, fluspirilene, nimodipine), because these membranes make up 95-98% of the total membrane in mammalian cells (Alberts et al 1994). Finally, there is evidence that the antipsychotic drug chlorpromazine, a cationic amphiphilic agent, interferes with receptor recycling by trapping receptors in the endosomes (Wang et al 1993). If any of the drugs that we have tested would bind to and enrich the endosome population, the intracellular drug concentrations would be enhanced in vivo by this mechanism. While we favor the idea that hydrophobic drugs concentrate in internal membranes of the cell, further work is needed to determine the actual intracellular concentrations of the drugs used in our studies to determine clinical relevance. Previous studies have shown that various plasma membrane calcium channels are affected by a variety of
BIOLPSYCHIATRY 1996;40:497-502
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neuroleptics, including fluspirilene and haloperidol (Galizzi et al 1986; Enyeart et al 1992). Haloperidol has been found to reduce histamine-induced smooth muscle contractions dependent on intracellular Ca2+-release (Hishinuma et al 1992), and to block the twitch response in rat phrenic nerve diaphragm preparations at 1 0 - 8 0 p~M (Seth et al 1991). Hence, it seems clear that such compounds are acting potentially through a variety of targets, not merely the established dopaminergic receptors. In a direct calcium-release assay, we have found fluspirilene, haloperidol, and clozapine, which are typically thought of as dopaminergic receptor antagonists, to be either as effective or more effective at blocking the IP3-dependent calcium release than verapamil, a traditional calcium channel blocker, or nimodipine, a more recently developed calcium channel blocker (Fig. 3). The fact that plasma membrane Ca2+-channel blockers inhibited IP3-dependent cae+-release, in spite of the fact that the amino acid sequences of various plasma membrane Ca2+-channels share little homology with the IP3-dependent Ca2+-release channel, suggests that the tertiary and quaternary structures of these two rather different Ca 2+channels might share some similarities.
This research was supported by an intramural Uniformed Services University of the Health Sciences (USUHS)grant (C0-8893)to F.S. We express our appreciation to Mrs. Eleanore Gamble for all of her assistance in the laboratory. We are grateful to Dr. Martha M. Coetzee (USUHS) for very helpful discussions in the field of psychopharmacology and to Drs. B.W. Agranoff(U. of Michigan), S. K. Fisher (U. of Michigan) and J. P. Staab (USUHS) for useful comments on an earlier version of the manuscript.
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Dubovsky SL (1993): Calcium antagonists in manic-depressive disease. Neuropsychobiology 27:184-192. Dubovsky SL, Christiano J, Daniell LC, Franks RD, Murphy J, Adler L, Baker N, Harris RA (1989): Increased platelet intracellular calcium concentration in patients with bipolar affective disorders. Arch Gen Psychiatry 46:632-638. Dubovsky SL, Murphy J, Thomas M, Rademacher J (1992): Abnormal intracellular calcium ion concentration in platelets and lymphocytes of bipolar patients. Am J Psychiatry 149: 118-120. Dubovsky SL, Thomas M, Hijazi A, Murphy J (1994): Intracellular calcium signalling in peripheral cells of patients with bipolar affective disorder. Eur Arch Psychiatry Clin Neurosci 243:229-234. Eckert A, Gann H, Riemann D, Aldenhoff J, Mtiller WE (1994): Platelet and lymphocyte free intracellular calcium in affective disorders. Eur Arch Psychiatry Clin Neurosci 243:235-239.
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