Regulation of signal transduction pathways by mood-stabilizing agents: implications for the delayed onset of therapeutic efficacy.

A series of investigations were performed to elucidate the mechanisms of action of lithium, valproate, and carbamazepine. We have found that lithium exerts major effects on G proteins, most likely via a posttranslational process stabilizing the inactive heterotrimeric (alpha beta gamma) form of the protein. We also find that chronic lithium and valproate exert major, very similar effects on the PKC signaling pathway, with both drugs decreasing the levels of membrane-associated PKC alpha and epsilon, and have similar effects on the DNA binding activity of the transcription factor, AP-1. By contrast, we find that carbamazepine exerts major, direct inhibitory effect at the level of adenylyl cyclases. Overall, the results suggest that signal transduction pathways are targets for the actions of mood-stabilizing agents; given their key roles in the amplification and integration of signals in the central nervous system, these findings have clear implications not only for research into the etiology/pathophysiology of manic-depressive illness, but also for the development of innovative treatment strategies.

Ethnic considerations in the pharmacotherapy of mood disorders.

In the past several years, there has been an increased appreciation for the importance of including cultural and ethnic factors in the evaluation and treatment of patients in psychiatry. Most of the focus has been on understanding the patients' psychosocial milieu and how these factors contribute to the patient's overall clinical presentation. Few studies have addressed the significance of pharmacogenetic heterogeneity among various cultural, ethnic, and racial groups. This article reviews the pharmacogenetic data and summarizes the sparse pharmacokinetic and pharmacodynamic data that pertain to the treatment of mood disorders in different ethnic and racial groups.

Regulation of corticotropin-releasing factor secretion and synthesis in the human neuroblastoma clones- BE(2)-M17 and BE(2)-C.

The BE(2)-M17 and BE(2)-C human neuroblastoma cell lines have been shown to synthesize and secrete corticotropin-releasing factor (CRF) following retinoic acid treatment. It has been demonstrated that CRF secretion and intracellular synthesis increases in response to forskolin treatment. In this report, we have further characterized these cells in response to protein kinase C activators, dexamethasone, interleukin-1 alpha, as well as various neurotransmitters and peptides. Nanomolar concentrations of the phorbol ester--phorbol 12 myristate 13--acetate (TPA), increased intracellular CRF content in both cell lines while increasing secretion only in the BE(2)-M17 cell. Nanomolar concentrations of dexamethasone were not able to alter basal levels of secretion and content in either cell type. However, in the BE(2)-M17 cell but not the BE(2)-C cell, the same concentrations of dexamethasone added to 30 microM forskolin augmented levels of CRF secretion and content. Likewise, the same augmented response in CRF secretion and content was seen only in the BE(2)-M17 cell line when nanomolar concentrations of dexamethasone were added to 20 nM TPA. Furthermore, only in the BE(2)-M17 cell line were micromolar levels of the biogenic amine serotonin able to increase levels of CRF secretion and content. No effects on CRF in both cell lines were demonstrable with picomolar levels of interleukin-1 alpha as well as micromolar levels of acetylcholine, norepinephrine, arginine-vasopressin, oxytocin, and angiotensin-II. The potential usefulness of these cells as models of central nervous system or placental CRF-containing neurons is discussed.

Clozapine-induced EEG abnormalities and clinical response to clozapine.

The authors hypothesized that patients who develop gross EEG abnormalities during clozapine treatment would have a less favorable outcome than patients who did not develop abnormal EEGs. The clinical EEGs and the Brief Psychiatric Rating Scale (BPRS) scores of 12 patients with schizophrenia and 4 patients with schizoaffective disorder were compared before and during treatment with clozapine. Eight patients developed significant EEG abnormalities on clozapine; 1 showed worsening of an abnormal pre-clozapine EEG; none of these subjects had clinical seizures. BPRS scores improved significantly in the group of patients who developed abnormal EEGs but not in the group who did not. Findings are consistent with previous reports of a high incidence of clozapine-induced EEG abnormalities and a positive association between these abnormalities and clinical improvement.

Natural killer cell activity in schizophrenia and schizoaffective disorder: a pilot study.

Natural killer cell activity was prospectively studied in 15 patients with chronic schizophrenia and in seven patients with schizoaffective disorder, depressed type. These patients were compared to individually matched normal controls. No mean differences in natural killer cell activity between the patient groups and their controls were observed.

Limbic-hypothalamic-pituitary-adrenal axis activity and ventricular-to-brain ratio studies in affective illness and schizophrenia.

Some investigators have speculated that structural brain alterations observed in some psychiatric patients might be related to increased limbic-hypothalamic-pituitary-adrenal axis (LHPA) activity. To explore this hypothesis, we prospectively studied 166 research volunteers (19 patients with research diagnostic criteria (RDC) major depression, 9 patients with RDC bipolar depression, 45 patients with RDC schizophrenia, and 94 RDC normal controls), examining the relationship between magnetic resonance image-determined ventricular-to-brain ratio (VBR) and indices of LHPA axis function (cerebrospinal fluid (CSF) corticotropin-releasing factor (CRF), CSF adrenocorticotropic hormone (ACTH), and 24-hour urinary-free cortisol secretion). We observed no significant differences in mean VBR among the three patient groups and the normal control volunteers. Of the indices of LHPA activity, only CSF CRF concentrations distinguished the four subject groups, with CSF CRF being significantly elevated in the more severely depressed major depression patients. Indices of LHPA activity were not significantly correlated with VBR in any of the three patient groups or in the normal volunteers. These preliminary results suggest that VBR is not highly associated with alterations in LHPA activity, at least as determined cross-sectionally. Further longitudinal studies with reference to diagnostic subtypes, severity, symptom profiles, and more specific neuroanatomic regions may allow the elucidation of possible relationships between LHPA pathology and structural brain alterations.

Lithium administration modulates platelet Gi in humans.

Platelet G proteins were assessed in 7 normal volunteers before and after 14 days of lithium administration at therapeutic plasma levels. Cholera and pertussis toxin catalyzed ADP-ribosylation of platelet membrane proteins were measured by SDS-PAGE. Immunoblotting with specific antibodies was used to measure platelet membrane alpha i content. There was a statistically significant 37% increase in pertussis toxin mediated ADP-ribosylation of a 40,000 Mr protein in platelet membranes after lithium administration, but cholera toxin mediated ADP-ribosylation of a 45,000 Mr protein and alpha i immunoblotting were unchanged by lithium. Increased pertussis toxin stimulated ADP-ribosylation in the absence of changes in alpha i content could be explained by a shift in platelet Gi in favor of its undissociated, inactive form. This would be consistent with increased platelet adenylyl cyclase activity found in these same subjects after lithium.

ECT treatment does not enhance neuroendocrine responses to serotonergic challenge.

We prospectively investigated the effects of a course of electroconvulsive therapy (ECT) on neuroendocrine responses to serotonergic challenge in five depressed patients. Low dose intravenous chlorimipramine (CMI) challenge produced a modest release of prolactin and significant increases in plasma adrenocorticotrophic hormone (ACTH) and cortisol. Interestingly, ECT did not alter the neuroendocrine responses to serotonergic challenge despite clinical response in four of the five patients. If anything, the modest prolactin (PRL) response to CMI, rather than being enhanced, appeared to be abolished following ECT. Using confidence intervals, we estimate that there is less than a 5% probability of a 78% increase in prolactin response to CMI after ECT. To detect this, a sample size of greater than 35 would be needed. These findings suggest that neither ECT nor the clinical response in severely depressed patients is likely to produce consistent changes in neuroendocrine response to the acute serotonergic effects of CMI infusion. The lack of effect of ECT on prolactin response to serotonergic challenge might be explained by simultaneous enhancement of both serotonergic and dopaminergic neurotransmission.

The mechanisms of action of lithium. I. Effects on serotoninergic and noradrenergic systems in normal subjects.

The effects of 2 weeks of lithium carbonate administration at therapeutic plasma levels were examined in 11 normal volunteers. Serotoninergic function before and after lithium administration was assessed using low-dose intravenous clomipramine hydrochloride challenge, while urinary and plasma metabolites of norepinephrine (NE) were used to assess noradrenergic systems. Long-term lithium administration in normal subjects did not significantly or consistently enhance serotonin-mediated neuroendocrine responses but did increase measures related to neuronal release of NE. No statistically significant effects of lithium on prolactin, corticotropin, or cortisol responses to serotoninergic challenge could be detected. The probability of a type II error was assessed, and a doubling of prolactin level was unlikely to have been missed, although more modest increases (less than 75%) could have been overlooked. After 2 weeks of lithium administration, there were significant increases in 24-hour urinary excretion of NE, normetanephrine, and fractional NE release, compatible with increased neuronal release of NE and a lithium-induced subsensitivity in alpha 2-adrenergic receptor function. These changes were not statistically significant after 1 week of administration, suggesting that increased NE release is characteristic of long- rather than short-term lithium administration. Since previous reports have demonstrated enhanced prolactin responses after short- but not long-term lithium use, the present study points to temporal specificity in lithium's effects on both serotoninergic and noradrenergic function. Lithium's effects on NE release were consistent but small (a 16% increase), while its effects on serotoninergic responses were larger (a 50% increase in prolactin responses) but quite inconsistent, suggesting that neither of these systems is the primary site of action of lithium.

The mechanisms of action of lithium. II. Effects on adenylate cyclase activity and beta-adrenergic receptor binding in normal subjects.

As part of a study of the effects of lithium carbonate on neurochemical function in man, platelet and lymphocyte adenylate cyclase activity and lymphocyte beta-adrenergic receptor binding characteristics were determined before and after 2 weeks of lithium treatment in 10 normal volunteers. Lithium had differential effects on platelet and lymphocyte adenylate cyclase activity. In platelets, basal and stimulated (guanyl imidodiphosphate [Gpp[NH]p] or cesium fluoride) adenylate cyclase activity was significantly augmented by lithium treatment. By contrast, in lymphocytes, Gpp(NH)p- and cesium fluoride-stimulated adenylate cyclase activity was unaffected, while basal activity was decreased modestly after lithium. These results are consistent with preclinical studies that suggest that lithium's effects on adenylate cyclase activity are specific with respect to tissue and brain region and that lithium may interfere with guanine nucleotide binding (G) protein function. Lithium treatment significantly increased the ratio of low- to high-affinity dissociation constants for agonist displacement of antagonist binding to lymphocyte beta-adrenergic receptors (thought to reflect coupling between the beta-adrenergic receptor and stimulatory G protein). Lithium had significant effects on measures associated with signal transduction that might be contrasted to its more subtle effects on neuronal function (norepinephrine release) and neuroendocrine systems (responses to serotoninergic challenge) in these same subjects (reported in a companion article). Lithium's primary site of action may be on signal transduction mechanisms. These effects subsequently may be manifested in changes in neurotransmitter function that may be important to lithium's mood-stabilizing actions.

Hypothalamic--pituitary--adrenal axis and monoamine transmitter activity in depression: a pilot study of central and peripheral effects of electroconvulsive therapy.

Central and peripheral measures of hypothalamic--pituitary--adrenal (HPA) axis and monoamine neurotransmitter activity were assessed in 8 depressed patients during a medication-free period and again after completion of a course of electroconvulsive therapy (ECT). Seven patients responded fully to ECT. At baseline there was corresponding activation of the HPA and noradrenergic systems, with apparent elevation in cerebrospinal fluid (CSF) concentrations of corticotropin-releasing hormone (CRH) in some patients. Neither CRH nor adrenocorticotropin (ACTH) in CSF changed significantly after ECT, with a mean 10% decline in CSF CRH. Urinary free cortisol (UFC) excretion was high both before and after treatment. Although peripheral noradrenergic hyperreactivity at baseline appeared to normalize with ECT, CSF concentrations of the principal norepinephrine metabolite, 3-methoxy-4-hydroxyphenyl-glycol (MHPG) were unaffected and remained correlated with CSF CRH. In contrast, there were increases in the CSF levels of the main metabolite of serotonin in half the patients.

Signal transduction modulation by lithium: cell culture, cerebral microdialysis and human studies.

Considerable evidence suggests that signal transduction pathways are targets of lithium (Li) action. A number of investigators have reported that Li attenuates both adenylate cyclase (AC) activity and phosphoinositide (PI) turnover in rodents and in humans, thus "dampening" these systems. We have studied selected components of these second-messenger systems in a series of clinical and preclinical investigations. To overcome confounding effects of alterations in mood state, we examined AC activity and G-protein ribosylation in peripheral blood cells from 10 healthy volunteers, prior to and following 14 days of Li administration. Basal and postreceptor [cesium fluoride (CsF) or Gpp(NH)p] stimulated AC activity were unaffected in lymphocytes. In contrast, both basal and stimulated AC activity in platelets were significantly augmented, compatible with an attenuation of Gi function. Ribosylation of platelet Gs by cholera toxin was unchanged, whereas that of Gi by pertussis toxin (PT) was increased. Given that undissociated G protein is the preferred substrate for PT, our results suggest that Li interferes with subunit dissociation and the subsequent activation of Gi. To determine if Li has similar effects on Gi in the central nervous system, we measured extracellular (EC) cyclic adenosine monophosphate (cAMP) in rat brain by in vivo microdialysis, revealing a dose-dependent increase in cAMP by norepinephrine (NE) antagonized by propranolol. Chronic (4-week) Li doubled basal EC cAMP, while decreasing the fractional response to 100 microM NE. Thus, using in vivo microdialysis, we observed the reported reduction in NE-stimulated AC activity, but only as a function of elevated basal cAMP. Increased basal AC activity has been observed following chronic Li in both humans and rat tissues but generally has not been considered relevant. The PI generating system is another proposed major target for Li that we have studied using an in vitro cell culture model of peripheral blood cells. Chronic (6-day) exposure of neutrophil-like HL60 cells to 1 mM LiCl did not affect agonist fMet-Leu-Phe (fMLP) induced PI turnover. In contrast, Li attenuated both agonist and phorbol ester stimulated Na+/H+ exchange, suggesting reduced protein kinase C (PKC) function. Western blot analysis revealed altered levels of PKC in both membrane and cytosolic fractions. The functional consequences of these complex effects on the two major signal transduction pathways and their interactions in the intact living organism remain to be elucidated.

Intravenous alprazolam challenge in normal subjects. Biochemical, cardiovascular, and behavioral effects.

Alprazolam, a novel benzodiazepine derivative is thought to be effective in the treatment of anxiety, panic, and depressive disorders. There is considerable interest in alprazolam's mechanism of action, particularly whether its profile of actions might resemble that of the alpha 2 adrenoreceptor agonist, clonidine. The present study assessed the biochemical, cardiovascular, and behavioral responses of healthy volunteers to acute intravenous infusions of alprazolam and placebo. Alprazolam reduced ACTH and cortisol while increasing growth hormone. There was a transient reduction in plasma norepinephrine and only modest effects on cardiovascular parameters. Subjects became quite sedated after intravenous alprazolam. This pharmacodynamic profile resembles that previously reported for traditional benzodiazepines, although alprazolam may be a more potent stimulator of growth hormone release. Alprazolam's effects on growth hormone resemble those of clonidine, but unlike clonidine, alprazolam has relatively little effect on plasma catecholamine and cardiovascular parameters. This suggests that alpha 2 mechanisms do not play a primary role in alprazolam's mode of action. Since alprazolam infusion affects three different measures (ACTH/cortisol, growth hormone, and plasma norepinephrine) thought to be dysregulated in depression, challenge with intravenous alprazolam may prove to be a useful "probe" in affective disorders.

Antidepressants reduce whole-body norepinephrine turnover while enhancing 6-hydroxymelatonin output.

The effects of antidepressant treatment on noradrenergic function were studied in 27 patients with a major affective disorder. Twenty-four-hour urinary excretion of 6-hydroxymelatonin and "whole-body norepinephrine (NE) turnover," ie, 24-hour urinary output of NE and its major metabolites 3-methoxy-4-hydroxyphenylglycol, vanillylmandelic acid, and normetanephrine, were measured before and after treatment with the tricyclic desipramine hydrochloride, the aminoketone bupropion hydrochloride, the nonselective monoamine oxidase (MAO) inhibitor tranylcypromine sulfate, and the specific MAO type A inhibitor clorgiline. 6-Hydroxymelatonin excretion increased following antidepressant treatment, while at the same time whole-body NE turnover was reduced. These findings support the hypothesis that antidepressant therapy increases noradrenergic "efficiency," in that functional output, as measured by 6-hydroxymelatonin, is maintained while total NE production is decreased.