Preclinical Assessment of the Abuse Potential of Purified Botanical Cannabidiol: Self-Administration, Drug Discrimination, and Physical Dependence.

Cannabidiol (CBD) is a constituent of the cannabis plant with a diverse array of pharmacological activities as well as potential therapeutic uses. An oral formulation of CBD (Epidiolex in the US; Epidyolex in Europe) is approved for treating seizures associated with rare and severe forms of epilepsy. These studies, which supported the approval of the medication, investigated abuse-related effects of CBD in rats and nonhuman primates (NHPs) using drug self-administration, drug discrimination, and physical dependence procedures and characterized its pharmacokinetics. In NHPs (n = 5) that self-administered midazolam (0.01 or 0.032 mg/kg/infusion), CBD (0.1-3.2 mg/kg/infusion) failed to maintain responding above vehicle levels. CBD maintained very modest levels of self-administration in rats (n = 7-8) that self-administered heroin (0.015 mg/kg/infusion) and did not increase drug-lever responding, up to a dose of 150 mg/kg (by mouth), in rats (n = 6) trained to discriminate 0.5 mg/kg (i.p.) midazolam. In juvenile (5-6 weeks old) and adult (10-11 weeks old) male and female rats, discontinuation of chronic treatment (twice daily for 20 days) with an oral formulation of CBD (20 or 100 mg/kg, by mouth) did not reliably produce signs of withdrawal. Pharmacokinetic studies confirmed that the dosing regimens used in these studies resulted in therapeutically relevant plasma levels. Taken together, the lack of reliable self-administration, the failure to increase drug-lever responding in rats trained to discriminate midazolam, and the absence of withdrawal signs upon discontinuation of chronic treatment indicate that CBD has very low abuse potential and is unlikely to produce physical dependence. SIGNIFICANCE STATEMENT: Legalization of cannabis across the United States and elsewhere has led to intense investigation into the safety and therapeutic potential of cannabis and its constituent materials, including cannabidiol (CBD). Results of these preclinical abuse potential studies on CBD indicate no rewarding properties, physical dependence potential, or similarity to a benzodiazepine. Together with data from in vitro pharmacology and human abuse potential studies, the abuse potential of Epidiolex in humans is likely to be negligible.

Physiological and behavioral effects of amphetamine in BACE1(-/-) mice.

ß-Site APP-cleaving Enzyme 1 (BACE1) is a protease that has been linked to schizophrenia, a severe mental illness that is potentially characterized by enhanced dopamine (DA) release in the striatum. Here, we used acute amphetamine administration to stimulate neuronal activity and investigated the neurophysiological and locomotor-activity response in BACE1-deficient (BACE1(-/-) ) mice. We measured locomotor activity at baseline and after treatment with amphetamine (3.2 and 10 mg/kg). While baseline locomotor activity did not vary between groups, BACE1(-/-) mice exhibited reduced sensitivity to the locomotor-enhancing effects of amphetamine. Using high-performance liquid chromatography (HPLC) to measure DA and DA metabolites in the striatum, we found no significant differences in BACE1(-/-) compared with wild-type mice. To determine if DA neuron excitability is altered in BACE1(-/-) mice, we performed patch-clamp electrophysiology in putative DA neurons from brain slices that contained the substantia nigra. Pacemaker firing rate was slightly increased in slices from BACE1(-/-) mice. We next measured G protein-coupled potassium currents produced by activation of D2 autoreceptors, which strongly inhibit firing of these neurons. The maximal amplitude and decay times of D2 autoreceptor currents were not altered in BACE1(-/-) mice, indicating no change in D2 autoreceptor-sensitivity and DA transporter-mediated reuptake. However, amphetamine (30 µm)-induced potassium currents produced by efflux of DA were enhanced in BACE1(-/-) mice, perhaps indicating increased vesicular DA content in the midbrain. This suggests a plausible mechanism to explain the decreased sensitivity to amphetamine-induced locomotion, and provides evidence that decreased availability of BACE1 can produce persistent adaptations in the dopaminergic system.

The cortisol awakening response predicts subclinical depressive symptomatology in Mexican American adults.

While childhood trauma appears to be a risk factor for the onset of depression and subclinical depressive symptomatology in Mexican Americans, the specific physiological mechanisms contributing to this relationship remain to be clarified. Stress-induced dysregulation of the Hypothalamic-Pituitary-Adrenal (HPA) axis is associated with depressive symptomatology in non-Hispanics. The current study assessed the extent to which the cortisol awakening response (CAR) predicts subclinical depressive symptomatology beyond the influence of childhood trauma in a sample of 55 Mexican American males and females ages 18-38 years, without a diagnosis of clinical depression. Participants were assessed for exposure to early trauma and current depressive symptomatology. Salivary cortisol samples were collected on two consecutive days at awakening, 30, 45, and 60 min thereafter, and again at 3 p.m., 6 p.m. and 9 p.m. Data were analyzed using general linear models with repeated measures at four morning time points, and again, at three afternoon and evening time points. Results indicated a significant Symptoms×Time interaction for the CAR(p<0.05). The Symptom×Time interaction was not significant for afternoon and evening cortisol concentrations. Moreover, subclinical symptomatology was associated with attenuation of the initial rise in CAR, after controlling for the total frequency of exposure to childhood traumas. Hierarchical analyses show attenuation of the initial rise in the CAR was the best predictor of greater subclinical depressive symptomatology beyond the influence of trauma, and independent of a current diagnosis of major depression in a sample of adult Mexican Americans.

Sequential combination of self-report, breath carbon monoxide, and saliva cotinine to assess smoking status.

The purpose of this analysis was to develop an algorithm for the cost effective and accurate assessment of smoking during the previous few days by combining self-report, breath carbon monoxide (BCO), and saliva cotinine (sCOT). These measurements are convenient, quantitative, and do not require invasive procedures. The data used to devise the algorithm were gathered during a treatment trial of participants seeking to stop smoking. Self-report of smoking was determined using a written questionnaire, BCO was measured with a handheld breathalyzer, and sCOT was quantified using a high sensitivity ELISA. Participants were 130 males and 97 females between the ages of 19 and 67 years who reported smoking at least 15 cigarettes a day and had a BCO level ≥ 15 ppm. Self-reports and BCO levels were collected at each of 6 visits (V0-V5) and sCOT levels were determined at V0 and V5. Based on the data collected, we recommend that the sequential determination of self-reported smoking, BCO level, and sCOT level be employed to assess smoking during the previous few days to minimize the higher cost and longer turnaround time associated with the sCOT test while maximizing accuracy.

Design of aging intervention studies: the NIA interventions testing program.

The field of biogerontology has made great strides towards understanding the biological processes underlying aging, and the time is ripe to look towards applying this knowledge to the pursuit of aging interventions. Identification of safe, inexpensive, and non-invasive interventions that slow the aging process and promote healthy aging could have a significant impact on quality of life and health care expenditures for the aged. While there is a plethora of supplements and interventions on the market that purport to slow aging, the evidence to validate such claims is generally lacking. Here we describe the development of an aging interventions testing program funded by the National Institute on Aging (NIA) to test candidate interventions in a model system. The development of this program highlights the challenges of long-term intervention studies and provides approaches to cope with the stringent requirements of a multi-site testing program.

Effect of valproic acid on serotonin-2A receptor signaling in C6 glioma cells.

Valproic acid (VPA), which has demonstrated efficacy in the treatment of bipolar disorder, has been shown to alter components of the phosphoinositide (PI) signaling cascade and to increase gene expression mediated by the transcription factor activator protein 1 (AP-1). Central serotonin-2A (5-HT2A) receptors, which have been implicated in the pathophysiology of manic-depressive illness, are coupled to PI hydrolysis. The promoter region of the 5-HT2A receptor gene contains AP-1 binding sites. We examined in C6 glioma cells the effect of VPA on 5-HT2A receptor signaling. Treatment of cells with VPA (100 microg/mL) for 20 h, but not 1.5 h, resulted in an enhancement of 5-HT2A receptor-stimulated PI hydrolysis. This effect of 20-h VPA exposure appeared not to be at the level of G protein or effector (i.e. phospholipase C: PLC) as inositol phosphate accumulation stimulated by aluminum fluoride or the PLC activator 2,4,6-trimethyl-N-(m-3-trifluromethylphenyl) benzenesulfonamide was not increased. The number of 5-HT2A receptors, as determined in saturation binding experiments using [3H]ketanserin, was increased by 20-h VPA treatment, with no change in affinity (KD). Taken together, our data suggest that the increase in 5-HT2A receptor-mediated PI hydrolysis following 20-h VPA exposure is not due to a general effect of VPA on this signaling cascade, but due to the up-regulation of 5-HT2A receptor number.

Modulatory effects of norepinephrine in the lateral bed nucleus of the stria terminalis on behavioral and neuroendocrine responses to acute stress.

The brain noradrenergic system is activated by stress, and modulates the activity of forebrain regions involved in behavioral and neuroendocrine responses to stress, such as the lateral bed nucleus of the stria terminalis (BSTL). This region of the limbic forebrain receives dense noradrenergic innervation, and has been implicated in both anxiety and regulation of the hypothalamic-pituitary-adrenal axis. We hypothesized that stress-induced release of norepinephrine in the BSTL modulates anxiety-like behavioral responses to stress and activation of the hypothalamic-pituitary-adrenal stress axis. Using microdialysis, we showed that release of norepinephrine was increased in the BSTL of male Sprague-Dawley rats during immobilization stress. In the next experiment, we then microinjected noradrenergic antagonists into the BSTL immediately prior to acute immobilization stress to examine noradrenergic modulation of behavioral stress reactivity. Either the alpha(1)-receptor antagonist benoxathian, or a cocktail of beta(1)- and beta(2)-receptor antagonists (betaxolol+ICI 118,551) blocked the anxiety-like reduction in open-arm exploration on the elevated plus-maze, but not the reduction in social behavior induced in the social interaction test. In a third experiment, benoxathian reduced plasma levels of adrenocorticotropic hormone following stress, but beta-receptor antagonists had no effect. From these results we suggest that stress-induced norepinephrine release acts on both alpha(1)- and beta-receptors in the BSTL to facilitate anxiety-like behavioral responses on the plus-maze but not the social interaction test, and modulates hypothalamic-pituitary-adrenal axis activation via alpha(1)-receptors only. Together with previous results in which adrenergic antagonists in central amygdala attenuated behavioral responses on the social interaction test but not the plus-maze, these observations suggest the two behavioral tests measure different dimensions of stress reactivity, and that norepinephrine facilitates different components of the stress response by region- and receptor-specific mechanisms.

Effect of acute administration of cocaine on pituitary gonadotrophin secretion in female rats.

The aim of this study was to characterize the acute effects of cocaine administration on pituitary gonadotrophin secretion in adult female rats. Ovariectomized, oestradiol-treated rats were infused i.v. with 0.1 ml normal saline or 2 mg cocaine hydrochloride kg(-1). Blood samples were collected immediately before cocaine infusion and at 3, 10, 30 and 60 min after cocaine infusion. Circulating LH concentrations were increased by 10 min after cocaine administration (P < 0.05 versus saline-treated controls) and decreased thereafter. Serum FSH concentrations were not significantly different from those of saline-infused controls at any time. In a second experiment, oestradiol-treated, ovariectomized rats were infused i.v. with: saline only, 2 mg cocaine hydrochloride kg(-1) in saline, 200 ng synthetic GnRH in 100 microl PBS or 200 ng synthetic GnRH plus 2 mg cocaine hydrochloride kg(-1) in PBS. Blood samples were collected immediately before drug infusions and 20 min later. Cocaine had no effect on either GnRH-stimulated LH or FSH secretion. In a third experiment, pituitary cells were obtained from oestradiol-treated, ovariectomized rats. The cell cultures were exposed to 25 ng cocaine hydrochloride ml(-1), 10(-10)-10(-7) mol GnRH (-1) with and without 25 ng cocaine hydrochloride ml(-1), or vehicle only. Medium was collected before and after exposure to GnRH to determine concentrations of secreted LH and FSH. Similar to the results of the second study, cocaine had no effect on GnRH-stimulated LH or FSH secretion from pituitary cells in vitro. On the basis of these results it is suggested that acutely administered cocaine stimulates release of hypothalamic GnRH, which, in turn, stimulates pituitary gonadotrophin secretion. Acute administration of cocaine does not appear to affect pituitary gonadotrophin secretion directly.

Combining ondansetron and naltrexone treats biological alcoholics: corroboration of self-reported drinking by serum carbohydrate deficient transferrin, a biomarker.

BACKGROUND: Recently, we showed by using self-report that combining ondansetron (4 microg/kg twice a day) and naltrexone (25 mg twice a day) was effective at reducing drinking and increasing abstinence among early-onset alcoholics (EOAs), who are characterized by a range of antisocial behaviors and high biological and familial disease predisposition. Here, we investigated whether the self-reported differences in drinking would be corroborated by measurements of serum carbohydrate-deficient transferrin (CDT) level, a sensitive, reliable, and well-validated marker of transient alcohol consumption. METHOD: An 8-week double-blind clinical trial was performed in which 20 EOAs were randomized to receive ondansetron (4 microg/kg twice a day) and naltrexone (25 mg twice a day) or placebo as an adjunct to weekly standardized cognitive behavioral therapy. Serum CDT was assessed at weeks 0 (baseline), 4, and 8. RESULTS: Log serum CDT was significantly lower in the ondansetron and naltrexone group (group mean, 1.44 +/- 0.076) compared with the placebo group (group mean, 1.82 +/- 0.113), as evidenced by a main effect of group [F(1,15) = 7.2, p = 0.017; effect size = 0.32], visit [F(1,16) = 11.2, p = 0.004; effect size = 0.41], and an interaction between group and visit [F(1,16) = 27.54, p < 0.001; effect size = 0.63]. CONCLUSIONS: The combination of ondansetron plus naltrexone was superior to placebo at reducing serum CDT. This corroborated our self-reported drinking data and demonstrated that the medication combination is an effective treatment for EOAs.

Extraction and quantification of epibatidine in plasma.

Epibatidine was extracted from human and mouse plasma into a hexane-isopropanol mixture and back-extracted into a phosphate buffer, pH 2.5, then identified by HPLC isocratically using a CN column and quantified with ultraviolet detection at a fixed wavelength of 214 nm. The percent recovery of epibatidine from spiked plasma samples was 83.6% and the percent extraction was linear between 10 and 1,000 ng/ml. Desipramine was used as the internal standard. For spiked control samples containing 50 and 750 ng/ml, between-day precisions were 20.8 and 7.2% (RSD%), respectively; accuracy was 87.0 and 99.1%, respectively. The limit of detection was 2 ng/ml. Using this method, an intraperitoneal dose of 0.1 mg/kg of epibatidine produced mean levels of 7.3 and 37.1 ng/ml in pooled male and female plasma samples from C57BL/10 J mice, respectively. This is a simple and straightforward procedure by which plasma samples may be analyzed for epibatidine.

Innovative technologies for the diagnosis of alcohol abuse and monitoring abstinence.

This article summarizes the proceedings of a workshop presented at the 2000 RSA Meeting in Denver, Colorado. The aim of this workshop was to discuss the basic methodologies, diagnostic performance, and clinical utility of three technologies: carbohydrate-deficient transferrin, the "Early Detection of Alcohol Consumption" score, and whole blood associated acetaldehyde. Each method adopts a different strategy to identify heavy alcohol consumption and offers a unique approach to determine alcohol abstinence and relapses. Appropriate application of these technologies can lead to early intervention for alcohol problems before significant tissue damage occurs. To date these methodologies have yet to be formally contrasted and compared. Sensitivity, specificity, predictive value, availability, ease of use, and interpretation of tests results are important aspects to consider when selecting the most appropriate and cost-effective system. Critical evaluation of these methodologies can enable research and clinical laboratories to choose the system that best meets their particular needs in terms of assay feasibility, budget, and goals.

Ondansetron for reduction of drinking among biologically predisposed alcoholic patients: A randomized controlled trial.

CONTEXT: Early-onset alcoholism differs from late-onset alcoholism by its association with greater serotonergic abnormality and antisocial behaviors. Thus, individuals with early-onset alcoholism may be responsive to treatment with a selective serotonergic agent. OBJECTIVE: To test the hypothesis that drinking outcomes associated with early vs late-onset alcoholism are differentially improved by the selective 5-HT(3) (serotonin) antagonist ondansetron. DESIGN: Double-blind, randomized, placebo-controlled clinical trial. SETTINGS: University of Texas Health Science Center in Houston (April 1995-June 1998) and University of Texas Health Science Center in San Antonio (July 1998-December 1999). PARTICIPANTS: A total of 321 patients with diagnosed alcoholism (mean age, 40.6 years; 70.5% male; 78.6% white) were enrolled, 271 of whom proceeded to randomization. INTERVENTIONS: After 1 lead-in week of single-blind placebo, patients were randomly assigned to receive 11 weeks of treatment with ondansetron, 1 microg/kg (n = 67), 4 microg/kg (n = 77), or 16 microg/kg (n = 71) twice per day; or identical placebo (n = 56). All patients also participated in weekly standardized group cognitive behavioral therapy. MAIN OUTCOME MEASURES: Self-reported alcohol consumption (drinks per day, drinks per drinking day, percentage of days abstinent, and total days abstinent per study week); and plasma carbohydrate deficient transferrin (CDT) level, an objective and sensitive marker of transient alcohol consumption. RESULTS: Patients with early-onset alcoholism who received ondansetron (1, 4, and 16 microg/kg twice per day) compared with those who were administered placebo, had fewer drinks per day (1.89, 1.56, and 1.87 vs 3.30; P =.03, P =.01, and P =.02, respectively) and drinks per drinking day (4.75, 4.28, and 5.18 vs 6.90; P =.03, P =.004, and P =.03, respectively). Ondansetron, 4 microg/kg twice per day, was superior to placebo in increasing percentage of days abstinent (70.10 vs 50.20; P =.02) and total days abstinent per study week (6.74 vs 5.92; P =.03). Among patients with early-onset alcoholism, there was a significant difference in the mean log CDT ratio between those who received ondansetron (1 and 4 microg/kg twice per day) compared with those who received the placebo (-0.17 and -0.19 vs 0.12; P =.03 and P =.01, respectively). CONCLUSION: Our results suggest that ondansetron (particularly the 4 microg/kg twice per day dosage) is an effective treatment for patients with early-onset alcoholism, presumably by ameliorating an underlying serotonergic abnormality. JAMA. 2000;284:963-971

Platelet serotonin uptake is higher in early-onset than in late-onset alcoholics.

The main objective of this study was to compare platelet serotonin (5-HT) uptake between early-onset alcoholics (EOA) and late-onset alcoholics (LOA). Subjects were 24 dependent male alcoholic in-patients and 21 healthy control subjects. 5-HT uptake was quantified by incubating platelets with [3H]5-HT at various concentrations (0.5 to 1000 nM). 5-HT uptake was higher in EOA, compared to both the LOA and control groups (P < 0.02) at the highest 5-HT concentration (1000 nM). No significant difference was found between LOA and controls or between EOA + LOA and controls. Previous studies have shown that 5-HT uptake was higher in platelets, lymphocytes, and brain of alcoholics vs controls, but our data suggest that higher platelet serotonin transporter function is limited to EOA, not LOA.

Effects of chronic antidepressant treatments on serotonin transporter function, density, and mRNA level.

To investigate functional changes in the brain serotonin transporter (SERT) after chronic antidepressant treatment, several techniques were used to assess SERT activity, density, or its mRNA content. Rats were treated by osmotic minipump for 21 d with the selective serotonin reuptake inhibitors (SSRIs) paroxetine or sertraline, the selective norepinephrine reuptake inhibitor desipramine (DMI), or the monoamine oxidase inhibitor phenelzine. High-speed in vivo electrochemical recordings were used to assess the ability of the SSRI fluvoxamine to modulate the clearance of locally applied serotonin in the CA3 region of hippocampus in drug- or vehicle-treated rats. Fluvoxamine decreased the clearance of serotonin in rats treated with vehicle, DMI, or phenelzine but had no effect on the clearance of serotonin in SSRI-treated rats. SERT density in the CA3 region of the hippocampus of the same rats, assessed by quantitative autoradiography with tritiated cyanoimipramine ([(3)H]CN-IMI), was decreased by 80-90% in SSRI-treated rats but not in those treated with phenelzine or DMI. The serotonin content of the hippocampus was unaffected by paroxetine or sertraline treatment, ruling out neurotoxicity as a possible explanation for the SSRI-induced decrease in SERT binding and alteration in 5-HT clearance. Levels of mRNA for the SERT in the raphe nucleus were also unaltered by chronic paroxetine treatment. Based on these results, it appears that the SERT is downregulated by chronic administration of SSRIs but not other types of antidepressants; furthermore, the downregulation is not caused by decreases in SERT gene expression.

Thrombin-stimulated increases in cytosolic Ca2+ level and gonadotropin-releasing hormone release in GT1-7 neurons.

The effects of thrombin on cytosolic calcium levels ([Ca2+]cyt), and on gonadotropin-releasing hormone (GnRH) release, were characterized in cultured GT1-7 neurons. GnRH release from GT1-7 neurons was pulsatile with an average pulse amplitude of 14.3+/-5.8 pg x min x ml(-1) and an average pulse duration of 21.3+/-4.2 min. The [Ca2+]cyt response to 0.005 to 0.2 U/ml thrombin was saturable and concentration dependent (EC50 = 0.0268 U/ml). Ethyleneglycotetraacetic acid (EGTA) chelation of extracellular Ca2+ resulted in an approximately 70% attenuation of thrombin-stimulated increase in [Ca2+]cyt. By use of a special superfusion system, a 5-min exposure to 0.1 U/ml thrombin significantly increased the amplitude (193.2+/-67.8 pg x min x ml(-1); P = 0.001) but not the duration (22.5+/-2.4 min; P = 0.8) of GnRH release. These results suggest that thrombin increases [Ca2+]cyt and GnRH release from GT1-7 neurons via specific membrane-bound receptors.

Increased norepinephrine levels during catheterization in patients with spinal cord injury.

The hypothesis for this study was that catecholamine levels increase during urinary catheterization in human patients with spinal cord injury. Catecholamine levels, blood pressure, and pulse were measured prospectively in 40 subjects at baseline and during urinary catheterization. Results showed a significant increase in norepinephrine levels from baseline 245 +/- 240 pg (standard deviation (SD)) to 314 +/- 311 pg (SD) during catheterization (P = 0.018, Wilcoxon's). Results also showed a nonsignificant increase in epinephrine levels from baseline (56 +/- 70 pg, SD) to catheterization (84 +/- 125 pg, SD; P = 0.35, Wilcoxon's). Systolic blood pressure increased from 114 to 124 mm Hg (P = 0.004, paired t test). Diastolic blood pressure increased from 75 to 78 mm Hg (P = 0.11, paired t test). There was no significant change in diastolic blood pressure or pulse (P = 0.11 and P = 0.29, respectively, paired t test). In conclusion, norepinephrine levels increased during catheterization in patients with spinal cord injury. Knowledge of catecholamine levels in this process may assist in determining both pathophysiology and potential pharmacologic treatment options in future studies.

Biphasic changes in left ventricular function during hyperdynamic endotoxemia.

Cardiac contractility was studied in a clinically relevant conscious swine model simulating human hemodynamics during endotoxemia. The slope of the end-systolic pressure-volume relationship [end-systolic elastance (EES)] was used as a load-independent contractility index. Chronic instrumentation in 10 pigs included two pairs of endocardial ultrasonic crystals for measuring internal major and minor axial dimensions of the left ventricle, a micromanometer for left ventricular pressure measurement, and a thermodilution pulmonary artery catheter. After a 10-day recovery period, control measurements of cardiac hemodynamic function were obtained. The following week, Escherichia coli endotoxin (10 micrograms . kg-1. h-1) was administered intravenously for 24 h. EES increased 1 h after endotoxin infusion and decreased beyond 7 h. The later hemodynamic changes resembled human cardiovascular performance during endotoxemia more closely than the changes during the acute phase. EES decreased in the later phase. A similar biphasic response of EES has been reported during a tumor necrosis factor-alpha (TNF) challenge. Even though plasma TNF was highest at 1 h and declined thereafter in this study, no consistent relationship between TNF and EES was identified, and TNF levels did not correlate directly with the changes in EES.

Concentration-dependent effects of muscimol to enhance pulsatile GnRH release from GT1-7 neurons in vitro.

Immortalized GT1-7 neurons were used to characterize the effect of muscimol, a GABAA receptor agonist, to enhance pulsatile gonadotropin-releasing hormone (GnRH) release. GT1-7 neurons were grown on Cytodex-3 beads and placed in special superfusion microchambers. The cells were superfused at a rate of 6.2 ml x h-1 with Media 199 (pH 7.35) using a commercially available perfusion system. After a pre-muscimol period of 120 min, the cells were exposed for 5 min to 0.35, 1, 5 or 10 microM muscimol or 5 microM muscimol+20 microM of the GABAA receptor antagonist, bicuculline. Following removal of the muscimol (and bicuculline, in the case of the latter experiment), the superfusion was continued for another 115 min. Sample fractions were collected at 5 min intervals throughout the perfusion. Basal GnRH release from the GT1-7 neurons was pulsatile with an average interpulse interval of 45.4+/-0.5 min and an average pulse amplitude of 191.5+/-22.6 pg x min x ml-1. Our results also demonstrated that the GABAA receptor agonist, muscimol, enhances pulsatile GnRH release from GT1-7 neurons in culture. The response to muscimol was saturable and concentration-dependent with an EC50 of 0.47 microM. The effects of 5 microM muscimol to increase GnRH pulsatility were blocked by co-exposure to the GABAA receptor antagonist, bicuculline. The average GnRH interpulse intervals were 41.7+/-1.8 min, 32.5+/-2.9 min, 30.6+/-0.7 min and 25.5+/-0.4 min in the period following exposure to 0.35, 1, 5 and 10 microM of muscimol, respectively (post-muscimol period). GnRH pulse amplitude (mean-area for each pulse) was increased during exposure to muscimol but not during the pre- or post-muscimol periods. The GABAA receptor antagonist, bicuculline, itself had no effect on pulsatile GnRH release. These results are consistent with previously published reports suggesting that activation of the GABAA receptor stimulates hypothalamic GnRH release in embryonic and neonatal animals.

Effects of suramin on human platelet aggregation and Ca2+ mobilization induced by thrombin and other agonists.

The purpose of this study was to investigate the effect of suramin, a polyanionic napthalene sulfonic acid, on human platelet aggregation and Ca2+ mobilization induced by various agonists. Our results show that suramin completely inhibited aggregation by thrombin, platelet activating factor (PAF), alkyllysophosphatidic acid (ALPA), or arachidonic acid in a concentration-dependent manner. The IC50 values of suramin for inhibition of aggregation by PAF, arachidonic acid, and thrombin were 76.7, 239, and 1.49 microg/ml, respectively. Ca2+ mobilization induced by thrombin was inhibited by suramin with an approximate IC50 value of 20 microg/ml. This concentration of suramin had no effect on PAF or oleic acid-induced Ca2+ mobilization. The mechanism by which suramin inhibits aggregation is not clear, but our results suggest that suramin inhibits the ligand-receptor interaction.

Incorporation of [3H]valproic acid into lipids in GT1-7 neurons.

Valproic acid (2-propylpentanoic acid, valproate, VPA), an 8-carbon, branched chain fatty acid, is effectively used in the treatment of mania and epilepsy. The biochemical mechanisms by which this drug has its therapeutic effects are not yet established. The purpose of this study was to partially characterize the incorporation of [3H]VPA into phospholipids of GT1-7 neurons, an immortalized hypothalamic cell line. GT1-7 neurons were grown to confluence in culture dishes, and then were incubated with various concentrations of [3H]VPA between 10 and 400 microg/mL for various times up to 20 hr. Total lipids were extracted and phospholipids were separated from neutral lipids using TLC. Our results indicate that [3H]VPA (10 microg/mL) was incorporated into phospholipids of GT1-7 neurons in a time-dependent and saturable manner over 300 min. Subsequent separation of the lipid fraction by TLC indicated that 44.4% of the radioactivity taken up by the cells was incorporated into phospholipids and neutral lipids. One of the phospholipids migrated with a slightly lower Rf value than authentic phosphatidylcholine. Our results show that the incorporation of VPA into phospholipids and glycerides was linear with VPA concentrations from 10 to 400 microg/mL. Finally, we synthesized 1-acyl-2-valproyl-sn-glycero-3-phosphocholine and validated its structure with nuclear magnetic resonance and electrospray mass spectrometry to verify the structure of this compound, confirming that this compound is structurally possible. We conclude that VPA is incorporated into lipids in GT1-7 neurons and discuss the possible effects of valproyl phospholipids on neuronal functional properties.