Suppression of human inflammatory cell function by subtype-selective PDE4 inhibitors correlates with inhibition of PDE4A and PDE4B.

1. Of the four major phosphodiesterase 4 (PDE4) subtypes, PDE4A, PDE4B and PDE4D are widely expressed in human inflammatory cells, including monocytes and T lymphocytes. We explored the functional role of these subtypes using ten subtype-selective PDE4 inhibitors, each belonging to one of two classes: (i) dual PDE4A/PDE4B inhibitors or (ii) PDE4D inhibitors. 2. These compounds were evaluated for their ability to inhibit antigen-stimulated T-cell proliferation and bacterial lipopolysaccharide (LPS)-stimulated tumour necrosis factor alpha (TNFalpha) release from peripheral blood monocytes. 3. All compounds inhibited T-cell proliferation in a concentration-dependent manner; with IC50 values distributed over an approximately 50 fold range. These compounds also inhibited TNFalpha release concentration-dependently, with a wider ( approximately 1000 fold) range of IC50 values. 4. In both sets of experiments, mean IC50 values were significantly correlated with compound potency against the catalytic activity of recombinant human PDE4A or PDE4B when analysed by either linear regression of log IC50 values or by Spearman's rank-order correlation. The correlation between inhibition of inflammatory cell function and inhibition of recombinant PDE4D catalytic activity was not significant in either analysis. 5. These results suggest that PDE4A and/or PDE4B may play the major role in regulating these two inflammatory cell functions but do not rule out PDE4D as an important mediator of other activities in mononuclear leukocytes and other immune and inflammatory cells. Much more work is needed to establish the functional roles of the PDE4 subtypes across a broader range of cellular functions and cell types.

Expression and cellular localization of the CC chemokines PARC and ELC in human atherosclerotic plaques.

Local immune responses are thought to play an important role in the development of atherosclerosis. Histological studies have shown that human atherosclerotic lesions contain T lymphocytes throughout all stages of development, many of which are in an activated state. A number of novel CC chemokines have been described recently, which are potent chemoattractants for lymphocytes: PARC (pulmonary and activation-regulated chemokine), ELC (EBI1-ligand chemokine), LARC (liver and activation-regulated chemokine), and SLC (secondary lymphoid-tissue chemokine). Using reverse transcriptase-polymerase chain reaction and in situ hybridization, we have found gene expression for PARC and ELC but not for LARC or SLC in human atherosclerotic plaques. Immunohistochemical staining of serial plaque sections with specific cell markers revealed highly different expression patterns of PARC and ELC. PARC mRNA was restricted to CD68+ macrophages (n = 14 of 18), whereas ELC mRNA was widely expressed by macrophages and intimal smooth muscle cells (SMC) in nearly all of the lesions examined (n = 12 of 14). ELC mRNA was also found to be expressed in the medial SMC wall of highly calcified plaques (n = 4). Very low levels of ELC mRNA expression could also be detected in normal mammary arteries but no mRNA expression for PARC was detected in these vessels (n = 4). In vitro, ELC mRNA was found to be up-regulated in aortic SMC stimulated with tumor necrosis factor-a and interferon-gamma but not in SMC stimulated with serum. Both PARC and ELC mRNA were expressed by monocyte-derived macrophages but not monocytes. The expression patterns of PARC and ELC mRNA in human atherosclerotic lesions suggest a potential role for these two recently described CC chemokines in attracting T lymphocytes into atherosclerotic lesions.

1,4-Cyclohexanecarboxylates: potent and selective inhibitors of phosophodiesterase 4 for the treatment of asthma.

Evaluation of a variety of PDE4 inhibitors in a series of cellular and in vivo assays suggested a strategy to improve the therapeutic index of PDE4 inhibitors by increasing their selectivity for the ability to inhibit PDE4 catalytic activity versus the ability to compete for high affinity [3H]rolipram-binding sites in the central nervous system. Use of this strategy led ultimately to the identification of cis-4-cyano-4-[3-(cyclopentyloxy)-4-methoxyphenyl]cyclohexane-1-carboxyl ic acid (1, SB 207499, Ariflo), a potent second-generation inhibitor of PDE4 with a decreased potential for side effects versus the archetypic first generation inhibitor, (R)-rolipram.

SB 207499 (Ariflo), a potent and selective second-generation phosphodiesterase 4 inhibitor: in vitro anti-inflammatory actions.

First-generation phosphodiesterase 4 (PDE4) inhibitors, such as rolipram, inhibit the activation of immune and inflammatory cells. The clinical use of these compounds is limited by gastrointestinal side effects, such as increased acid secretion and nausea. Consequently, the challenge has been to design novel PDE4 inhibitors that maintain the anti-inflammatory actions of rolipram while achieving an improved side effect profile. Among the first of this new class of PDE4 inhibitors specifically designed to have an improved therapeutic index relative to earlier compounds is SB 207499 (Ariflo) [c-4-cyano-4-(3-cyclopentyloxy-4-methoxy-phenyl)-r-1-cyclohexanecarboxyl ic acid]. In this study, we compared the anti-inflammatory and gastric secretogogue activities of SB 207499 with those of rolipram. The cellular models used were (1) histamine release from human basophils, (2) tumor necrosis factor-alpha generation in human monocytes, (3) degranulation of human neutrophils, (4) antigen-driven proliferation and cytokine synthesis from human T cells and (5) acid secretion from isolated rabbit gastric glands. SB 207499 inhibited the activation of a variety of immune and inflammatory cells in a concentration-dependent manner: (1) histamine release in basophils [-log IC25 = 6.6 +/- 0.3 vs. 8.0 for (R)-rolipram], (2) lipopolysacchride-induced TNF-alpha formation in monocytes [-log IC50 = 7.0 +/- 0.1 vs. 7.2 +/- 0.1 for (R)-rolipram], (3) fMLP-induced degranulation in neutrophils [-log IC15 = 7.1 +/- 0.2 vs. 6.4 +/- 0.5 for (R)-rolipram], (4) house dust mite induced-proliferation of peripheral blood mononuclear cells [-log IC40 = 6.5 +/- 0.3 vs. 6.4 +/- 0.3 for (R)-rolipram] and (5) ragweed-induced production of interferon-gamma [-log IC50 = 5.4] and interleukin-5 [-log IC50 = 5.0]. Although SB 207499 inhibits the activation of a variety of immune and inflammatory cells with a potency equal to that of rolipram, it is > 100-fold less potent than the latter compound as an acid secretagogue [-log EC50 = 6.1 +/- 0.1 vs. 8.3 +/- 0.2 for (R)-rolipram]. Collectively, these data indicate that SB 207499 retains the anti-inflammatory activity of the prototypical PDE4 inhibitor rolipram but is substantially less likely to stimulate gastric acid secretion.

Characterization of functional chemokine receptors (CCR1 and CCR2) on EoL-3 cells: a model system to examine the role of chemokines in cell function.

A growing family of proteins, known as the chemokines, play an important role in the recruitment and activation of inflammatory cells. The purpose of these studies was to characterize the chemokine receptors present on human sodium butyrate differentiated EoL-3 cells (dEoL-3 cells). Using a combination of 3' rapid amplification of cDNA ends and nested polymerase chain reaction, we detected mRNA for CC chemokine receptor (CCR)1, CCR2, CCR3 and low level of CCR5. Radioligand binding studies demonstrated high-affinity saturable binding for both 125I-macrophage inflammatory protein (MIP)-1alpha and 125I-regulated upon activation normal T cell expressed and secreted (RANTES) with Kd values of 1.4 and 7 nM, respectively. Competition binding with chemokines demonstrated exactly the same rank order of potency for displacement of both ligands: MIP-1alpha approximately monocyte chemoattractant protein (MCP)-3 approximately RANTES > MIP-1beta >> MCP-1 >>> IL-8. RANTES, MCP-3 and MIP-1alpha all produced concentration-dependent transient increases in intracellular calcium concentrations in dEoL-3 cells. Desensitization studies indicated that RANTES, MIP-1alpha and MCP-3 interacted at the same receptor, which is identical in characterization to the cloned CCR1. 125I-MCP-1 also demonstrated high-affinity satuable binding to dEoL-3 cells with a Kd value of 0.4 nM. Competition studies showed that MCP-3 was slightly more potent than MCP-1 and MCP-2. MIP-1alpha, MIP-1beta and RANTES were unable to displace 125I-MCP-1. Addition of either MCP-1 or MCP-3 produced a concentration-dependent elevation of intracellular calcium with a maximun response 2-fold higher than that seen with RANTES or MIP-1alpha. Desensitization studies indicated that MCP-1 and MCP-3 function through CCR2 on these cells. Thus binding and functional studies indicate that dEoL-3 cells express functional CCR1 and CCR2 and that these cells may serve as an important system with which to study the regulation and role of these receptors.

Association of the anti-inflammatory activity of phosphodiesterase 4 (PDE4) inhibitors with either inhibition of PDE4 catalytic activity or competition for [3H]rolipram binding.

Phosphodiesterase 4 (PDE4) inhibitors are novel anti-inflammatory compounds. Unfortunately, the archetypal PDE4 inhibitor rolipram produces central nervous system and gastrointestinal side-effects. To exploit these agents, we need to identify PDE4 inhibitors that retain the anti-inflammatory activity with a reduced potential to elicit unwanted side-effects. PDE4 possesses both cyclic AMP catalytic activity that is inhibitable by rolipram and a high affinity binding site for rolipram. The function of this high affinity rolipram binding site is unclear; however, certain pharmacological effects of PDE4 inhibitors are associated with competition for this site. Since PDE4 inhibitors suppress both monocyte and neutrophil activation, the present experiments were carried out to establish a correlation between suppression of monocyte activation [tumor necrosis factor alpha (TNF alpha) formation] or suppression of neutrophil activation (degranulation) with inhibition of either PDE4 catalytic activity or [3H] rolipram binding. Suppression of TNF alpha formation demonstrated a strong correlation with inhibition of PDE4 catalytic activity (r=0.87; P<0.01; Spearman's Rho = 0.79, P<0.05), whereas there was no correlation with inhibition of [3H]rolipram binding(r=0.21, P>0.5; Spearman's Rho=0.16, P>0.5). Suppression of neutrophil degranulation was not associated with inhibition of PDE4 catalytic activity (r=0.25, P>0.4; Spearman's Rho=0.33, P>0.2), but was associated with inhibition of [3H]rolipram binding (r=0.68, P<0.05; Spearman's Rho=0.6, P=0.06). These results indicate that anti-inflammatory effects of PDE4 inhibitors can be associated with either inhibition of PDE4 catalytic activity or high affinity rolipram binding.

Prolonged beta adrenoceptor stimulation up-regulates cAMP phosphodiesterase activity in human monocytes by increasing mRNA and protein for phosphodiesterases 4A and 4B.

Human peripheral blood monocytes were treated for 4 h with a combination of the beta-agonist salbutamol (3 microM) and the low-Km cAMP-specific phosphodiesterase (PDE4) inhibitor rolipram (30 microM) to produce a prolonged elevation of cAMP and consequent increase in PDE activity. After this treatment, isozyme-selective PDE inhibitors were used to characterize the cAMP PDE profiles of high-speed supernatants before and after DEAE-Sepharose column chromatography. These experiments, in which total soluble PDE activity was increased by 58%, showed that the increased PDE activity is due to up-regulation of PDE4 and that at least two of the four subtypes are up-regulated. Experiments in whole cells demonstrated that this relatively modest increase in PDE4 activity has significant functional consequences, reducing cAMP accumulation in response to both PGE2 and lower, though not maximal, concentrations of rolipram. Further characterization of PDE4 subtype expression in control and treated monocytes, using polymerase chain reaction and Western blotting with subtype-specific peptide antibodies, showed that resting monocytes express both mRNA and protein for PDE4A, PDE4B and PDE4D. The amount of message for PDE4A and PDE4B appeared to increase upon up-regulation, whereas mRNA for PDE4D was not detected in treated cells. Western blots showed increases in the amount of protein for both PDE4A and PDE4B after treatment. We conclude that the PDE4 subtypes are differentially regulated upon prolonged exposure to elevated cAMP, with the consequence that the PDE4 profiles of control and treated cells differ not only in total activity but also in the relative proportions of the subtypes represented.

Inhibitors of phosphodiesterase IV (PDE IV) increase acid secretion in rabbit isolated gastric glands: correlation between function and interaction with a high-affinity rolipram binding site.

In this report, we describe the ability of selective inhibitors of phosphodiesterase (PDE) isozymes to increase aminopyrine accumulation in rabbit isolated gastric glands. Aminopyrine accumulation in the presence of histamine was increased by the nonselective PDE inhibitor isobutylmethylxanthine (EC50 = 4.8 microM) and by two selective PDE IV inhibitors, rolipram and Ro 20-1724 (EC50 = 0.013 and 0.07 microM, respectively) but not by selective PDE III inhibitors (siguazodan and SK&F 94120) or by a selective PDE V inhibitor (zaprinast). These results suggest that PDE IV is an important regulator of acid secretion in response to histamine. One of the more fascinating properties of PDE IV is the expression of a high-affinity binding site for [3H]-rolipram in addition to cAMP catalytic activity. Although agents that inhibit PDE IV catalytic activity also appear to bind to the high-affinity rolipram-binding site, the rank-order potencies of compounds for these two effects are poorly correlated. Also, certain pharmacological actions of PDE IV inhibitors appear to be related to an interaction with this binding site. In this study, we observed that the ability of PDE IV inhibitors to enhance acid secretion was not associated with their ability to inhibit PDE IV catalytic activity but did show a strong correlation with their ability to compete for [3H]-rolipram binding. Furthermore, we were able to detect [3H]-rolipram binding sites in gastric glands that had characteristics similar to those of the [3H]-rolipram binding sites in rat brain microsomes and human recombinant PDE IV.

Phosphodiesterase IV inhibitors as therapy for eosinophil-induced lung injury in asthma.

Asthma is a complex, multifactorial disease that is underpinned by airway inflammation. A variety of cytotoxic substances are released into the airway from infiltrating inflammatory cells, especially the eosinophil. These cytotoxic substances, including reactive oxygen metabolites, produce damage to the airway epithelium, a histologic feature of chronic asthma. Damage to the airway epithelium, in turn, is thought to be a major factor responsible for the development of airway hyperreactivity, a hallmark of asthma. One notable molecular target for novel antiasthmatic drugs is the cyclic AMP-specific phosphodiesterase (PDE) or PDE IV. This isozyme is the predominant form of cyclic nucleotide PDE activity in inflammatory cells. Thus, in view of the putative role of cyclic AMP as an inhibitory second messenger in these cells, PDE IV inhibitors have been shown to suppress inflammatory cell activity. The purpose of the present experiments was to examine the effect of the PDE IV inhibitor, R-rolipram, on three key functions of the guinea pig eosinophil: a) superoxide anion (O2-) production, b) adhesion to human umbilical vein endothelial cells (HUVECs), and c) infiltration into the airway. R-rolipram-elevated eosinophil cyclic AMP content (EC50 = 1.7 microM) and inhibited fMLP-induced O2- production in a concentration-dependent manner (IC50 = 0.3 microM). In contrast, neither siguazodan, a PDE III inhibitor, nor zaprinast, a PDE V inhibitor, had an appreciable effect. R-rolipram (30 microM) also reduced by 25 to 40% the adhesion of eosinophils to HUVECs stimulated with phorbol myristate acetate or tumor necrosis factor-alpha, particularly under conditions in which both cell types were simultaneously exposed to the PDE IV inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)

Initial biochemical and functional characterization of cyclic nucleotide phosphodiesterase isozymes in canine colonic smooth muscle.

Cyclic nucleotides mediate relaxation of gastrointestinal smooth muscle. The intracellular concentration of these second messengers is determined by a balance between their synthesis and metabolism. Because cyclic nucleotide phosphodiesterase enzymes (PDE) are the sole enzymes responsible for their degradation, it is essential to determine the role of the various PDE isozymes in regulating cyclic nucleotide content of gastrointestinal smooth muscle. To examine the role of different PDE isozymes in colonic smooth muscle motility, soluble PDE activity was measured in fractions obtained from homogenates of canine colon using DEAE sepharose chromatography. PDE activity was determined using [3H]cyclic AMP (cAMP) (1 microM) or [3H]cyclic GMP (cGMP) (1 microM) as a substrate. Results indicated that colonic smooth muscle contains at least two forms of PDE with a high affinity for cGMP. One form was stimulated by calmodulin (type I) and the other was inhibited by low concentrations of zaprinast (type V). In addition, colonic smooth muscle contains at least two isozymes that prefer cAMP as a substrate. One form was inhibited by SB 94120 and cGMP (type III) and the other by rolipram (type IV). An additional peak of PDE activity was identified. The hydrolysis of cAMP by this peak was greatly enhanced by the presence of cGMP, suggesting that this activity belonged to type II or cGMP-stimulated PDE. The functional role of these isozymes was evaluated by determining the ability of selective PDE inhibitors to antagonize a carbachol (0.3 microM)-induced contraction of isolated circular colonic muscle strips in the presence of forskolin (0.2 microM). Concentration-dependent decreases in contractile activity were observed with the following potency order: rolipram > Ro 20-1724 > isobutyl methylxanthine > SB 94120 > zaprinast. These results demonstrate that colonic smooth muscle contains several PDE isozymes and that selective inhibition of PDE isozymes can increase cyclic nucleotide content and antagonize contractile responses. Functionally, PDE IV appears to be very important in reducing contractile activity, suggesting that selective PDE IV inhibitors might be useful in the treatment of gut hypermotility disorders.

Human lower oesophageal sphincter relaxation is associated with raised cyclic nucleotide content.

Increases in cyclic adenosine monophosphate and cyclic guanosine monophosphate content accompany relaxation of isolated strips of opossum and canine lower oesophageal sphincter muscle. The aim of this investigation was to characterise these responses in isolated muscle from the human lower oesophageal sphincter. Electrical stimulation of enteric neurons produced a frequency dependent relaxation of the human lower oesophageal sphincter that was sensitive to tetrodotoxin. Furthermore, as previously shown in the opossum and canine lower oesophageal sphincter, cyclic guanosine monophosphate content was significantly raised in muscle strips frozen during maximum electrical field stimulation whereas cyclic adenosine monophosphate content was unchanged. In addition, sodium nitroprusside (EC50 = 0.1 microM) produced a concentration dependent relaxation of human lower oesophageal sphincter, significantly increased cyclic guanosine monophosphate content, but did not alter cyclic adenosine monophosphate content. Zaprinast (M&B 22948) and SK&F 94120, selective inhibitors of cyclic guanosine monophosphate and cyclic adenosine monophosphate phosphodiesterases, respectively, both relaxed human lower oesophageal sphincter with a potency similar to that seen in the dog or opossum lower oesophageal sphincter. Finally, the 8-bromo analogues of both cyclic adenosine monophosphate (EC50 = 420 microM) and cyclic guanosine monophosphate (EC50 = 100 microM) relaxed the human lower oesophageal sphincter. These studies suggest that in the human, as well as the canine and opossum lower oesophageal sphincter, increases in cyclic nucleotide content are associated with relaxation and increases in cyclic guanosine monophosphate are associated with the relaxation induced by stimulation of enteric neurons.

Lower esophageal sphincter relaxation is associated with increased cyclic nucleotide content.

Experiments were conducted to determine whether relaxation of the opossum isolated lower esophageal sphincter (LES), induced by electrical field stimulation (EFS) or various pharmacological agents, is associated with changes in cyclic nucleotide content. EFS relaxed the LES in a frequency-dependent manner with 0.7 Hz producing half-maximal relaxation. Control tissues and tissues stimulated at various frequencies were clamp-frozen and assayed for cyclic nucleotide content. EFS had no effect on adenosine 3',5'-cyclic monophosphate (cAMP) content but increased guanosine 3',5'-cyclic monophosphate (cGMP) content in a frequency-dependent manner. Tetrodotoxin eliminated both the relaxation and cGMP accumulation in response to EFS. Vasoactive intestinal polypeptide (VIP) relaxed the LES with an EC50 of 0.1 microM. In contrast to the results with EFS, VIP enhanced cAMP content but had no effect on cGMP content. Relaxation of the LES produced by sodium nitroprusside or atriopeptin II was accompanied by an increase in cGMP accumulation, whereas isoproterenol- and dopamine-induced relaxation was accompanied by an increase in cAMP content. The data indicate that, depending on the stimulus, increases in either cAMP or cGMP content can accompany LES relaxation. These results are consistent with the proposed role of cyclic nucleotides as second messengers mediating LES relaxation.

Cyclic nucleotide-dependent protein kinases in the lower esophageal sphincter.

The characteristics and regulation of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent and guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinases (PKs) in opossum, canine, and human lower esophageal sphincter (LES) were investigated. As measured by the incorporation of 32P from [gamma-32P]adenosine 5'-triphosphate (ATP) into histone, LES homogenates from all three species contained three distinct types of PK: cAMP-dependent PK, cGMP-dependent PK, and cyclic nucleotide-independent PK. In all three species, cAMP-dependent PK comprised approximately 80%, cGMP-dependent PK comprised approximately 10%, and cyclic nucleotide-independent PK comprised approximately 10% of the total PK activity in the LES. Diethylaminoethyl-sepharose column chromatography of the supernatant fraction of opossum LES homogenates revealed that of the total cAMP-dependent PK, 10% was Type I and 90% was Type II. In contrast, equal amounts of Type I and Type II cAMP-dependent PK were present in both the human and canine LES. Isoproterenol-induced relaxation of the isolated opossum LES was accompanied by an increase in cAMP content and an activation of cAMP-dependent PK. The results of this study support the proposal that cyclic nucleotides and cyclic nucleotide-dependent PKs regulate LES tone.

Inhibition of calmodulin-stimulated phosphodiesterase activity by vasoactive intestinal peptide.

The effects of certain peptides of the glucagon family on calmodulin activity were determined from their capacity to inhibit a calmodulin-dependent form of phosphodiesterase. Vasoactive intestinal peptide and secretin were potent inhibitors of calmodulin activity, having IC50 values of 0.5 microM and 2 microM, respectively. By contrast, glucagon failed to inhibit calmodulin activity even at concentrations of 100 microM. None of these compounds significantly inhibited the basal activity of phosphodiesterase at concentrations up to 100 microM. These findings support the suggestion that important structural features of peptides for anticalmodulin activity include a net positive charge and a hydrophobic surface.

Inhibition of calmodulin activity by insect venom peptides.

Several peptides found in insect venom, including melittin, apamin and mastoparan, inhibited the activity of calmodulin-stimulated phosphodiesterase at concentrations that had no appreciable effect on basal phosphodiesterase activity; the Ki value of melittin for inhibiting calmodulin activity was 30 nM. Acetylation of the peptides reduced their inhibitory effect on calmodulin, suggesting that a net positive charge was an important determinant of anti-calmodulin activity. An examination of other structural features of these peptides suggested that the most potent inhibitors of calmodulin had an alpha-helical conformation. Equilibrium dialysis experiments showed that melittin inhibited the calcium-dependent binding of 3H-chlorpromazine to calmodulin (IC50 0.9 microM); kinetic analyses of these data indicated that this inhibition was non-competitive, suggesting that melittin and chlorpromazine act at different sites on calmodulin. Since calmodulin regulates a number of processes that these peptides inhibit, our results raise the possibility that the inhibition of calmodulin activity by these insect venom peptides may explain some of their biochemical or toxicological effects.

Pharmacological regulation of calmodulin.

A number of psychotropic drugs, particularly the phenothiazines and related antipsychotic compounds, inhibit a variety of calmodulin-dependent enzymes. The mechanism by which these compounds inhibit the activity of calmodulin is through a selective calcium-dependent binding to this protein. With the notable exception of certain stereoisomers, compounds that are clinically effective antipsychotic agents showed the greatest degree of binding to calmodulin. Other classes of pharmacological agents, including aminergic agonists and antagonists, and nonspecific central nervous system depressants and stimulants, showed little or no binding to calmodulin. In fact, the specificity with which antipsychotic drugs bind to calmodulin suggests the possibility of screening for new and clinically more effective antipsychotic agents based on their selective binding to calmodulin. Certain neuropeptides that produce behavioral effects in animals also were found to inhibit the activity of calmodulin, suggesting that there may be endogenous psychotogens or antipsychotic peptides that interact with calmodulin. Although under ordinary conditions the binding of antipsychotics to calmodulin is reversible, the binding of phenothiazine antipsychotics to calmodulin can be made irreversible either photochemically by ultraviolet irradiation, or enzymatically by a hydrogen peroxide-peroxidase system. Such a labeling technique should prove to be a useful tool to study the localization and turnover of calmodulin. These results indicate that several of the diverse biochemical actions of antipsychotic agents can be explained by a common mechanism, namely, by their binding to and inhibition of calmodulin, and raise the possibility that calmodulin may serve as one of the cellular receptors for certain antipsychotic compounds. However, further studies must be completed before we can state with any degree of certainty that these in vitro biochemical findings can explain the pharmacological and clinical actions of the antipsychotics.