Quinazoline-based tricyclic compounds that regulate programmed cell death, induce neuronal differentiation, and are curative in animal models for excitotoxicity and hereditary brain disease.

Expanding on a quinazoline scaffold, we developed tricyclic compounds with biological activity. These compounds bind to the 18 kDa translocator protein (TSPO) and protect U118MG (glioblastoma cell line of glial origin) cells from glutamate-induced cell death. Fascinating, they can induce neuronal differentiation of PC12 cells (cell line of pheochromocytoma origin with neuronal characteristics) known to display neuronal characteristics, including outgrowth of neurites, tubulin expression, and NeuN (antigen known as 'neuronal nuclei', also known as Rbfox3) expression. As part of the neurodifferentiation process, they can amplify cell death induced by glutamate. Interestingly, the compound 2-phenylquinazolin-4-yl dimethylcarbamate (MGV-1) can induce expansive neurite sprouting on its own and also in synergy with nerve growth factor and with glutamate. Glycine is not required, indicating that N-methyl-D-aspartate receptors are not involved in this activity. These diverse effects on cells of glial origin and on cells with neuronal characteristics induced in culture by this one compound, MGV-1, as reported in this article, mimic the diverse events that take place during embryonic development of the brain (maintenance of glial integrity, differentiation of progenitor cells to mature neurons, and weeding out of non-differentiating progenitor cells). Such mechanisms are also important for protective, curative, and restorative processes that occur during and after brain injury and brain disease. Indeed, we found in a rat model of systemic kainic acid injection that MGV-1 can prevent seizures, counteract the process of ongoing brain damage, including edema, and restore behavior defects to normal patterns. Furthermore, in the R6-2 (transgenic mouse model for Huntington disease; Strain name: B6CBA-Tg(HDexon1)62Gpb/3J) transgenic mouse model for Huntington disease, derivatives of MGV-1 can increase lifespan by >20% and reduce incidence of abnormal movements. Also in vitro, these derivatives were more effective than MGV-1.

Cigarette Smoke Decreases Salivary 18 kDa Translocator Protein Binding Affinity ­ in Association with Oxidative Stress

Reactive oxygen species (ROS) generated by cigarette smoke may contribute to lung and oral cancer. The 18 kDa Translocator protein (TSPO) has been reported to be affected by ROS as well as to participate in ROS generation at mitochondrial levels, and has been implicated in pro-apoptotic and anti-carcinogenic functions. The present study reports the presence of TSPO in the cellular fraction of human saliva. In cells collected from untreated saliva, the specific TSPO ligand [(3)H]PK 11195 showed saturable binding with high affinity, with mean B(max) and K(d) values of 6,471 +/- 501 fmol/mg protein and 6.2 +/- 0.5 nM, respectively. Our study further indicates that the cellular fraction of human saliva possesses TSPO with binding characteristics similar to that of cells from other tissues of human origin. Following exposure of saliva to cigarette smoke a three-fold decrease in the affinity of salivary TSPO to its specific ligand, [(3)H]PK 11195 (p < 0.01) occurred in the cellular fraction of the saliva, in comparison to sham treated control, without significant accompanying changes in TSPO B(max), TSPO protein levels, or general protein levels. The changes in affinity of TSPO from the cellular fraction of saliva exposed to cigarette smoke were accompanied by changes in the mean levels of protein oxidation products (carbonyls) and lipid peroxides, which were three-fold higher (p < 0.01) and two-fold higher (p < 0.01), respectively, compared to those of sham treated controls. Thus, our study shows that TSPO is present in the cellular component of saliva. Interestingly, in vitro this cellular TSPO is affected by exposure of the whole saliva to cigarette smoke, in negative correlation with oxidative stress.

PK 11195 attenuates kainic acid-induced seizures and alterations in peripheral-type benzodiazepine receptor (PBR) protein components in the rat brain.

Peripheral-type benzodiazepine receptors (PBR) are located in glial cells in the brain and in peripheral tissues. Mitochondria form the primary location for PBR. Functional PBR appear to require at least three components: an isoquinoline binding protein, a voltage-dependent anion channel, and an adenine nucleotide carrier. In the present study, rats received intraperitoneal kainic acid injections, which are known to cause seizures, neurodegeneration, hyperactivity, gliosis, and a fivefold increase in PBR ligand binding density in the hippocampus. In the forebrain of control rats, hippocampal voltage-dependent anion channel and adenine nucleotide carrier abundance was relatively low, while isoquinoline binding protein abundance did not differ between hippocampus and the rest of the forebrain. One week after kainic acid injection, isoquinoline binding protein abundance was increased more than 20-fold in the hippocampal mitochondrial fraction. No significant changes were detected regarding hippocampal voltage-dependent anion channel and adenine nucleotide carrier abundance. Pre-treatment with the isoquinoline PK11195, a specific PBR ligand, attenuated the occurrence of seizures, hyperactivity, and increases in isoquinoline binding protein levels in the hippocampus, which usually follow kainic acid application. These data suggest that isoquinoline binding protein may be involved in these effects of kainic acid injections.

Acute and repeated swim stress effects on peripheral benzodiazepine receptors in the rat hippocampus, adrenal, and kidney.

Peripheral benzodiazepine receptor (PBR) density has been found to be sensitive to stress. We set out to compare the influences of acute and repeated swim stress on behavior and PBR density. Following acute and repeated swim stress, rats were tested in an elevated plus-maze and an open-field test for anxiety levels, and tissues were collected from the adrenal gland, kidney, and hippocampus for measurements of PBR density. The acute rather than the repeated stress led to robust alterations in PBR density. The largest reduction in hippocampal and adrenal gland PBR density was found one hour after acute stress. In the hippocampus, acute stress caused a biphasic change in PBR density: a robust reduction in PBR density one hour after the acute stress and a distinct elevation in PBR density at 24 hours, while 72 hours after stress the elevation in PBR density appeared to be reduced.

Hormonal regulation of peripheral benzodiazepine receptor binding properties is mediated by subunit interaction.

The peripheral benzodiazepine receptor (PBR) is composed of three subunits with molecular masses of 18, 30, and 32 kDa. Many physiological functions have been attributed to the PBR, including regulation of steroidogenesis. Furthermore, the PBR itself is under hormonal regulation. In the current study, we investigated the role of female gonadal sex hormones in the regulation of PBR expression in steroidogenic and nonsteroidogenic tissues. To accomplish this, adult female rats were pharmacologically castrated using chronic administration of the gonadotropin-releasing hormone agonist decapeptyl (triptorelin-D-Trp(6)-LHRH). Half of these rats received 17beta-estradiol as hormone replacement, while a control group received daily injections of vehicle only. We found that PBR binding capacity dropped by 40 and 48% in ovaries and adrenals, respectively, following decapeptyl administration, as opposed to no change in the kidney. This down-regulation of PBR densities was prevented by estradiol replacement. We did not find evidence for transcriptional, posttranscriptional, and translational mechanisms in this decapeptyl-induced down-regulation. In contrast, immunoprecipitation of the PBR complex, using antibodies against the 18- and 32-kDa subunits of the complex, demonstrated that there were changes in PBR subunit interactions, consistent with the down-regulation of PBR binding capacity. These findings represent a novel hormone-dependent posttranslational regulatory mechanism.

Tissue-specific regulation of the peripheral benzodiazepine receptor by antidepressants and lithium.

Peripheral benzodiazepine receptors (PBR) have been identified in peripheral organs as well as in brain glial cells. PBR differ from central benzodiazepine receptors (CBR) in their lack of coupling to the gamma-aminobutyric acid receptors and the chloride ion channels. We investigated the effect of 21 days administration, followed by 7 days withdrawal, of fluvoxamine (10 mg/kg), desipramine (10 mg/kg) and lithium carbonate (25 mg/kg) on PBR and CBR binding characteristics in male Sprague-Dawley rats. All three agents significantly increased PBR density in the testes and adrenals. All tested drugs induced a significant decrease in PBR density in the kidney and liver. After withdrawal, PBR density remained decreased in the liver in all three groups and in the kidneys of the desipramine- and lithium-treated animals. In the cerebral cortex, CBR density increased in response to all three agents, whereas PBR density decreased significantly in response to desipramine and lithium carbonate. Chronic treatment with fluvoxamine, desipramine and lithium carbonate is apparently associated with a modulation in PBR expression in the testes, adrenals, kidneys, liver and brain, and in CBR expression in brain. The relevance of these tissue-selective alterations to the antidepressive and/or anxiolytic effects of these agents, or their adverse effects, still needs to be determined.

Long-lasting effect of early handling on the peripheral benzodiazepine receptor.

The present study determined the impact of early handling (EH) in rats on behavioral response to environmental stress and on peripheral benzodiazepine receptor (PBR) binding characteristics (Bmax and Kd) in various organs. The behavioral consequences of EH in rats were expressed as increased exploratory activity in an open-field paradigm, when compared with nonhandled control rats. These findings are interpreted in terms of decreased emotionality. The biochemical consequences of EH, in both male and female rats, were expressed as the upregulation of PBR in the adrenal and kidney and the downregulation of gonadal (testis and ovary) PBR. It is possible that the long-lasting adrenal and renal changes in PBR expression in EH rats may enable better regulation of the hypothalamic-pituitary-adrenal axis, renin-angiotensin system, and autonomic nervous system responses to stress in adulthood. The significance of the EH-induced reduction in gonadal PBR for gonadal activity in adulthood is as yet unclear.

Platelet peripheral-type benzodiazepine in pregnancy and lactation.

The aim of the present study was to investigate the impact of hormonal changes during pregnancy and lactation on the expression of peripheral-type benzodiazepine receptors in platelet membranes. Platelet peripheral benzodiazepine receptor binding characteristics, Hamilton anxiety and depression rating Scores, and progesterone and prolactin (PRL) levels were evaluated during pregnancy and lactation in 17 pregnant women [first (n = 9) and third (n = 8) trimesters], 10 lactating women, and 8 nonpregnant women. A significant decrease (38-41%) in peripheral benzodiazepine receptor density was observed in women during the third trimester of pregnancy when compared to nonpregnant controls and women in their first trimester of pregnancy. The decrease is peripheral benzodiazepine receptors was parallel to the peak in progesterone and PRL secretion. The reduction in peripheral benzodiazepine receptor expression is hormone-dependent and may play a regulatory role geared to prevent pregnancy-related overactivity of the hypothalamic-pituitary-ovarian, hypothalamic-pituitary-adrenal, and hypothalamic-PRL axes.

Decreased platelet peripheral-type benzodiazepine receptors in adolescent inpatients with repeated suicide attempts.

BACKGROUND: Peripheral-type benzodiazepine receptors (PBR) are responsible for mitochondrial cholesterol uptake, the rate limiting step of steroidiogenesis. They have been shown to be increased after acute stress, and decreased during exposure to chronic stressful conditions, and in patients with generalized anxiety disorder and post-traumatic stress disorder. In view of the proven connection between adolescent suicidal behavior and stress, we hypothesized that PBR may be decreased in the suicidal adolescent population. METHODS: We measured [3H] PK 11195 binding to platelet membrane in nine adolescent (age 13-20 years) inpatients with a history of at least three suicidal attempts and ten age-matched psychiatric inpatients with no history of suicide attempts. Suicidality was assessed with the Suicide Risk Scale (SRS), and symptom severity with the Beck Depression Inventory, State-Trait Anxiety Inventory (STAI), Overt Aggression Scale (OAS), and Impulsivity Scale (IS). RESULTS: Suicide Risk Scale scores were significantly higher in the suicidal group. The suicidal group showed a significant decrease in platelet PBR density (-35%) compared to the controls (p < 0.005). CONCLUSIONS: Our results of PBR depletion in adolescent suicide are in accordance with the findings in patients with generalized anxiety disorder and posttraumatic stress disorder and lend further support to the role of PBR in human response to chronic stress in adolescent suicide.

Peripheral-type benzodiazepine receptors in the regulation of proliferation of MCF-7 human breast carcinoma cell line.

Peripheral-type benzodiazepine receptors (PBR) have been implicated in cell proliferation. The aim of the present study was to test the effect of the PBR ligands PK 11195 and Ro 5-4864 and the central-type benzodiazepine receptor ligand clonazepam on breast carcinoma cell proliferation, using [3H] thymidine incorporation. We then carried out a study to identify where the PBR-specific ligands Ro 5-4864 and PK 11195 act in the cell cycle, using flow cytometric analysis. We found PBR expression in the malignant breast cancer tumors, representing various levels of estrogen and/or progesterone receptors, as well as in the MCF-7 breast carcinoma cell line. PK 11195 and Ro 5-4864 inhibited cell proliferation at concentrations of 10(-5) to 10(-4) M, while clonazepam (the central-type benzodiazepine receptor-specific ligand) had no effect. In this same concentration range, PK 11195 and Ro 5-4864, in contrast to clonazepam, induced an accumulation of MCF-7 cells in both the G0-G1 and G2-M phases of the cell cycle. The present study demonstrates that PBR ligands play a role in regulating cell proliferation in the human breast carcinoma cell line MCF-7.

Cross-sensitivity between isoflurane and diazepam: evidence from a bidirectional tolerance study in mice.

We examined in mice the effect of chronic diazepam treatment on the sensitivity to isoflurane, and that of repeated isoflurane exposure on the sensitivity to diazepam. Mice were divided into four groups: group 1, treated with diazepam, 10 mg/kg i.p. twice daily; group 2, vehicle-treated controls; group 3, exposed to 3% isoflurane for 25 min twice daily; and group 4, untreated controls. After 14 days the effect of the treatment was assessed. Twenty-four hours after the last 10 mg/kg diazepam treatment, groups 1 and 2 received diazepam, 5 mg/kg i.p., and were subjected to the horizontal wire test (HWT). All control mice but only 10% of the diazepam-treated mice failed the HWT. Groups 1 and 2 were then exposed to increasing concentrations of isoflurane. Diazepam-treated mice (group 1) lost the HWT at 0.7+/-0.7%, compared with 0.6+/-0.1% in controls (group 2) (P<0.001); the ED50 was 0.75% vs. 0.65%. Group 1 mice lost the righting reflex at 0.94+/-0.07% isoflurane vs. 0.87+/-0.06% in group 2 (P<0.01); the ED50 was 0.93% vs. 0.82%. Recovery time was 175+/-161 s in group 1 vs. 343+/-275 s in group 2 (P<0.02). Twenty-four hours after the last of the repeated exposures to isoflurane, we examined the responses of groups 3 and 4 to increasing concentrations of isoflurane. Mice in group 3 lost the righting reflex at 1.0+/-0.06% isoflurane vs. 0.9+/-0.04% in controls (group 4) (P<0.001); the ED50 was 0.96% vs. 0.85%. Recovery time was 113+/-124 s vs. 208+/-126 s in groups 3 and 4 (P<0.09). Diazepam, 3 mg/kg i.p. administered to groups 3 and 4, caused loss of the HWT reflex in 33% of group 3 mice and in 82% of controls (group 4) (P<0.001). It appears that prolonged exposure to both diazepam and isoflurane caused reduced sensitivity to each drug separately, as well as to the other drug. This finding may strengthen the theory that inhalational anesthetics may act via the same mechanism as the benzodiazepines.

Peripheral benzodiazepine receptors in cerebral cortex, but not in internal organs, are increased following inescapable stress and subsequent avoidance/escape shuttle-box testing.

Stress-induced alterations in peripheral benzodiazepine receptor (PBR) density have been reported in humans and in rats. However, the PBR response is highly specific, and its function remains largely unexplained. The aim of the present study was to investigate the relationship between behavior in the two-way active avoidance paradigm (2WAA) and post-test PBR densities in adrenal, testis, kidney, and cerebral cortex. Adult male Wistar rats were tested in the 2WAA either in the naive state (AA) or 24 h following shock preexposure (PE), known to interfere with avoidance/escape response acquisition, and decapitated immediately after testing. Control subjects were decapitated without experimental experience. The stressful characteristic of the experiment was validated by significantly increased post-test corticosterone levels in AA and PE subjects compared with controls, with a trend towards higher corticosterone levels in PE relative to AA rats. Similarly, PE compared with AA subjects tended to show retarded acquisition of the escape/avoidance response. PBR densities in adrenal, kidney, and testis and central benzodiazepine receptors (CBR) in the cerebral cortex remained unaffected by avoidance testing. Cerebral cortex PBR density was significantly increased in PE subjects. These findings suggest that avoidance testing, although stressful to the animals, led to changes confined to cerebral cortex PBR, indicating that the hypothalamic-pituitary-adrenal (HPA) response occurs independently of the PBR response in peripheral organs, and also suggest that the opportunity for coping alters the impact of the stressor on the subject and prevents the expression of PBR response in peripheral organs.

Ovarian steroids and stress produce changes in peripheral benzodiazepine receptor density.

Although past research has described changes in the density of the peripheral benzodiazepine receptor in brain and in peripheral organs in response to stressors and steroid hormone exposure, their combined influence had yet to be determined. This study examined the effect of swim-stress as a function of ovarian hormone administration on the binding of an isoquinoline carboxamide derivative, [3H]PK 11195, in brain and peripheral tissues. In olfactory bulb and adrenal gland, stress increased peripheral benzodiazepine receptor density in ovariectomized rats with and without estradiol and progesterone replacement injection, even when compared with unstressed animals treated with hormones, where estradiol + progesterone decreased peripheral benzodiazepine receptor number in olfactory bulb, but estradiol and estradiol + progesterone increased it in adrenal gland. In frontal cortex, stress decreased peripheral benzodiazepine receptor number, an effect that was reversed by estradiol. In hippocampus estradiol decreased peripheral benzodiazepine receptor density in unstressed animals and estradiol + progesterone decreased peripheral benzodiazepine receptor number in unstressed and stressed animals. In cerebellum, stress, estradiol and estradiol + progesterone alone decreased peripheral benzodiazepine receptor density. In uterus of unstressed controls, estradiol + progesterone increased peripheral benzodiazepine receptor density, and stress produced a further increase in steroid-treated females. Stress did not affect peripheral benzodiazepine receptor density in kidney, except in animals that received estradiol + progesterone injections, where swim-stress produced a significant decrease in peripheral benzodiazepine receptor density. Thus, steroid hormones regulate peripheral benzodiazepine receptor density in endocrine organs and brain, and the hormonal state of the organism modifies the peripheral benzodiazepine receptor response to stress in a tissue- and brain region-specific manner, suggesting that the peripheral benzodiazepine receptor may play a pivotal role in an integrated response to stress.

Effects of peripheral-type benzodiazepine receptor antisense knockout on MA-10 Leydig cell proliferation and steroidogenesis.

The peripheral-type benzodiazepine receptor (PBR) is not only widely expressed throughout the body, but it is also genetically conserved from bacteria to humans. Many functions have been attributed to it, but its primary role remains a puzzle. In the current study, we stably transfected cultures of MA-10 Leydig cells with either control or 18-kDa PBR antisense knockout plasmids. The antisense knockout vector was driven by the human enkephalin promoter, which contains two cAMP response elements, such that cAMP treatment of transfected cells could superinduce 18-kDa PBR antisense RNA transcription and, hence, down-regulate endogenous 18-kDa PBR mRNA levels. Control and knockout MA-10 cell lines were then compared at the level of receptor binding, thymidine incorporation, and steroid biosynthesis. Eighteen-kilodalton PBR knockout reduced the maximal binding capacity of tritium-labeled PBR ligands, and the affinity of receptors to the ligands remained unaltered. Additionally, 24-h accumulation of progesterone was lower in the knockout cells. Exposure of the two cell types to 8-bromo-cAMP resulted in a robust increase in steroid production. However, a complex pattern of steroid accumulation was observed, in which further progestin metabolism was indicated. The later decline in accumulated progesterone as well as the synthesis of androstenedione were different in the two cell types. At the level of cell proliferation, reduction of 18-kDa PBR mRNA showed no effect. Thus, we conclude that the 18-kDa PBR may have a more important role in steroidogenesis than in proliferation in this Leydig cell line.

Peripheral-type benzodiazepine receptor ligands and serum steroid hormones.

The peripheral-type benzodiazepine receptors (PBR) are involved in various cellular functions, including steroidogenesis. The impact of these receptor ligands has been demonstrated mainly in steroidogenic cells. The aim of the present study was to assess in intact female rats the effect of chronic (21 days) administration of the PBR ligands PK 11195 (15 mg/kg) and Ro 5-4864 (5 mg/kg), the mixed ligand diazepam (5 mg/kg), and the central benzodiazepine receptor ligand clonazepam (1 mg/kg) on PBR binding characteristics in steroidogenic (ovary and adrenal) and non-steroidogenic (uterus and kidney) organs, as well as on serum hormonal steroids (estradiol, progesterone, and corticosterone). Selective and mixed PBR ligands up-regulated PBR density in the two steroidogenic organs, while Ro 5-4864 also induced elevation of the receptor density in the non-steroidogenic organs. In contrast to Ro 5-4864, PK 11195 treatment down-regulated renal PBR. Clonazepam elevated adrenal PBR. On the serum hormonal level, Ro 5-4864 suppressed estradiol secretion. The other ligands did not affect hormonal steroid levels. It appears that in female rats, at least at these doses and dosing schedules, there is no correlation between the impact of chronic in vivo exposure to these agents on PBR density and ovarian and adrenal hormone levels.