Expression of plasminogen activator inhibitors type 1 and type 3 and urokinase plasminogen activator protein and mRNA in breast cancer

BACKGROUND: The urokinase plasminogen activator (uPA) system has been involved in cancer cell invasion and in metastasis. uPA activity is controlled by its principal inhibitor, the PA inhibitor type-1 (PAI-1), but it can also be inhibited by PAI-3. Increased levels of uPA and PAI-1 are known to be associated with a poor prognosis in breast cancer. To our knowledge this is the first study of the expression and role of PAI-3 in human breast cancer tissue. MATERIALS AND METHODS: Protein and mRNA levels were evaluated for uPA, PAI-1 and PAI-3 in breast cancer tissues from 70 different patients. The localization of antigen and mRNA of these proteins was studied by immunohistochemistry and in situ hybridization, respectively. RESULTS: No significant differences were observed for PAI-3 mRNA or protein levels between the nodal status groups or the different post-surgical tumor-node-metastasis (pTNM) stages. However, uPA and PAI-1 mRNA and antigen levels significantly increased at the pTNM stage and in node-positive patients. PAI-3 antigen levels were significantly higher in early relapse-free patients, whereas PAI-1 antigen levels were significantly higher in patients who suffered a relapse. PAI-3 protein and mRNA were localized in stromal cells. PAI-1 and uPA protein were detected in cancer, endothelial and stromal cells and their mRNA mainly in stromal cells. CONCLUSIONS: Our results indicate that PAI-3 is expressed in human breast cancer tissues, and that elevated levels of PAI-3 could be a positive prognostic factor in this disease. A potential mechanism for the contribution of PAI-3 to a positive long-term outcome may involve suppression of tumor invasion through protease inhibition in stroma.     

Tamoxifen induces resistance to activated protein C

Introduction

The estrogen antagonist tamoxifen (TAM) increases the thrombotic risk similar to estrogen containing oral contraceptives (OC). In OC users this risk is attributed to alterations of hemostasis resulting in acquired resistance to activated protein C (APC). TAM-induced APC resistance has not been reported yet.

Materials and Methods

Blood samples were collected prospectively from women with breast cancer before (n = 25) and monthly after start of adjuvant TAM treatment (n = 75). APC resistance was evaluated on basis of the effect of APC on the endogenous thrombin generation potential. To detect increased in vivo APC generation APC plasma levels were measured using a highly sensitive oligonucleotide-based enzyme capture assay. Routine hemostasis parameters were measured additionally.

Results

APC sensitivity decreased by 41% (p = 0.001) compared to baseline after one month of TAM application and remained significantly decreased during the study period. Free protein S increased (p = 0.008) while other analyzed procoagulant factors, inhibitors, and activation markers of coagulation decreased or did not change significantly. In five patients the APC concentration increased to non-physiological levels but an overall significant increase of APC was not observed.

Conclusions

This is the first study showing acquired APC resistance under TAM therapy. Acquired APC resistance might explain the increased thrombotic risk during TAM treatment. Observed changes of hemostasis parameters suggest different determinants of TAM-induced APC resistance than in OC-induced APC resistance. The presence of acquired APC resistance in TAM patients warrants further evaluation if these patients may benefit from antithrombotic prophylaxis in the presence of additional thrombotic risk factors.     

Effects of TNF-alpha and curcumin on the expression of thrombomodulin and endothelial protein C receptor in human endothelial cells

The objective of this study was to elucidate the effects of tumor necrosis factor-alpha (TNF-alpha) on the expression of thrombomodulin (TM) and endothelial protein C receptor (EPCR) in human endothelial cells as well as the effect of curcumin, a spice and coloring food compound, as a potential therapeutic agent. Human umbilical vein endothelial cells (HUVECs) treated with TNF-alpha (2.0 ng/ml) showed reduced TM mRNA levels by 80%, 97%, 94%, and 97% at 3, 6, 12, and 24 h, respectively (P<0.05), by real-time PCR analysis. Dose-dependent study showed that TM mRNA levels of HUVECs were decreased by 86%, 89%, 91%, and 94% after treatment of TNF-alpha (0, 0.25, 0.5, 1, and 2 ng/ml) for 6 h, respectively (P<0.05). TM protein levels in HUVECs were significantly reduced by 69% in TNF-alpha-treated cells as compared to controls (P<0.05) by Western blot analysis. Secreted protein and activity of TM of HUVEC cultures were also significantly reduced in TNF-alpha-treated cells. In addition, EPCR mRNA levels of HUVECs were significantly reduced in TNF-alpha-treated group as compared to controls (P<0.05). Furthermore, these effects were observed in other types of endothelial cells from human coronary arteries, lung, and skin. Curcumin effectively blocked these effects of TNF-alpha on downregulation of TM and EPCR. These data demonstrate that TNF-alpha significantly decreases expression of TM and EPCR at both mRNA and protein levels in several human endothelial cells. Curcumin can effectively block TNF-alpha-induced endothelial dysfunction. This study suggests a new molecular mechanism of inflammation-induced thrombosis and a new therapeutic strategy to prevent this clinical problem.     

Differences in circadian variations of tissue factor pathway inhibitor type 1 between able-bodied and spinal cord injured

INTRODUCTION: Tissue factor pathway inhibitor type 1 (TFPI) is the physiological inhibitor of the tissue factor pathway of coagulation. TFPI is produced by endothelial cells, and most intravascular TFPI is composed of full-length TFPI associated with the endothelium. Circulating TFPI is mainly truncated and lipoprotein-associated, but a small fraction circulates in a free full-length form. Although hormonal state influences the plasma variation of TFPI between individuals, other factors like temporal variation may be important. Hence, in the current study we aimed at exploring the intra-individual variation with focus on the possible circadian variations of TFPI. MATERIALS AND METHODS: TFPI free and total antigen from 8 able-bodied and 6 tetraplegic men were measured at 12 time points during a 24 h period. RESULTS: TFPI free antigen in the able-bodied exhibited circadian variation with the highest levels (approximately 20% above mean) from 12:00 to 18:00 h and the lowest levels (approximately 15% below mean) at 09:00 and 02:00 h. In contrast, TFPI free antigen in the tetraplegic group showed no circadian variation. TFPI total antigen exhibited circadian variation in neither group, but mean TFPI total antigen was lower in the tetraplegic group compared with the able-bodied (80 versus 110 ng/mL, respectively). Notably, even if TFPI total antigen in both groups did not vary according to any specific circadian rhythm, the intra-individual variation was higher than the assay variation. CONCLUSION: TFPI free antigen exhibited circadian variations in able-bodied, but not in tetraplegic subjects and the able-bodied had higher levels of TFPI total antigen than the tetraplegic group.     

Treatment of endothelium with the chemotherapy agent vincristine affects activated protein C generation to a greater degree in newborn plasma than in adult plasma

INTRODUCTION: Activated protein C (APC) is well-established as a physiologically important anticoagulant. During development, plasma concentrations of protein C and alpha(2)macroglobulin, factors involved in APC generation, differ from adult levels. Chemotherapy drugs can perturb endothelial expression of PC-activating receptors. This study examines the effect of chemotherapy treatment of endothelium on APC generation in newborn and adult plasma. MATERIALS AND METHODS: APC generations were initiated on endothelial cells treated with vincristine or media by recalcifying defibrinated plasma with buffer containing thromboplastin. APC generation was terminated by mixing timed subsamples into FFRCMK-EDTA or heparin, followed by EDTA. APC-PCI and APC-alpha(1)AT were assayed by ELISA. APC-alpha(2)M was measured chromogenically. Since heparin converts free APC to APC-PCI, the difference between APC-PCI detected in heparin subsamples and APC-PCI detected in FFRCMK-EDTA subsamples gave the free APC. Cellular expression of EPCR and TM were measured by flow cytometry and Western blot. RESULTS: Vincristine-treated endothelium decreased free APC generation in newborn plasma to a greater degree than in adult plasma. APC-PCI levels in both adult and newborn plasma were unaffected by chemotherapy. Vincristine treatment reduced levels of APC-alpha(1) AT and APC-alpha(2) M to a greater degree in newborn plasma versus adult plasma. Expression of EPCR was reduced in cells treated with vincristine. Conversely, TM was reduced on the cell surface, but increased in whole cell lysates. CONCLUSIONS: The differential response of newborn and adult plasma PC components to chemotherapy-mediated changes in cell surface components may be a factor in the increased risk of thrombosis in children receiving chemotherapy.     

Activation of protein kinase C and inositol 1,4,5-triphosphate receptors antagonistically modulate voltage-gated sodium channels in striatal neurons

Regulation of voltage-gated sodium channels is crucial to firing patterns that constitute the output of medium spiny neurons (MSN), projecting neurons of the striatum. This modulation is thus critical for the final integration of information processed within the striatum. It has been shown that the adenylate cyclase pathway reduces sodium currents in MSN through channel phosphorylation by cAMP-dependent protein kinase. However, it is unknown whether a phospholipase C (PLC)-mediated signaling cascade could also modulate voltage-gated sodium channels within MSN. Using the whole-cell patch clamp technique, we investigated the effects of activation of two key components in PLC-mediated signaling cascades: protein kinase C (PKC) and inositol-1,4,5-triphosphate (IP(3)) receptors on voltage-dependent sodium current. Cellular dialysis with phorbol 12-myristate 13-acetate, an activator of PKC, significantly reduced peak sodium current amplitude, while adenophostin A, an activator of IP(3) receptors, significantly increased peak sodium current amplitude. This effect of adenophostin was abolished by calcium chelation or by FK506, an inhibitor of calcineurin. These results suggest an antagonistic role of PKC and IP(3) in the modulation of striatal voltage-gated sodium channels, peak current amplitude being decreased through phosphorylation by PKC and increased through dephosphorylation by calcineurin.     

Opioid mu receptor activation inhibits sodium currents in prefrontal cortical neurons via a protein kinase A- and C-dependent mechanism

Opioid transmission in the medial prefrontal cortex is involved in mood regulation and is altered by drug dependency. However, the mechanism by which ionic channels in cortical neurons are controlled by mu opioid receptors has not been elucidated. In this study, the effect of mu opioid receptor activation on voltage-dependent Na(+) currents was assessed in medial prefrontal cortical neurons. In 66 out of 98 nonpyramidal neurons, the application of 1 microM of DAMGO ([D-Ala(2), N-Me-Phe(4), Gly(5)-OL]-enkephalin), a specific mu receptor agonist, caused a decrease in the Na(+) current amplitude to approximately 79% of that observed in controls (half blocking concentration = 0.094 microM). Moreover, DAMGO decreased the maximum current activation rate, prolonged its time-dependent inactivation, and shifted the half inactivation voltage from -63.4 mV to -71.5 mV. DAMGO prolonged the time constant of recovery from inactivation from 5.4 ms to 7.4 ms. The DAMGO-evoked inhibition of Na(+) current was attenuated when GDP-beta-S (0.4 mM, Guanosine 5-[beta-thio]diphosphate trilithium salt) was included in the intracellular solution. Inhibitors of kinase A and C greatly attenuated the DAMGO-induced inhibition, while adenylyl cyclase and kinase C activators mirrored the DAMGO inhibitory effect. Na(+) currents in pyramidal neurons were insensitive to DAMGO. We conclude that the activation of mu opioid receptors inhibits the voltage-dependent Na(+) currents expressed in nonpyramidal neurons of the medial prefrontal cortex, and that kinases A and C are involved in this inhibitory pathway.     

Purification, biochemical properties and antithrombotic effect of a novel Streptomyces enzyme on carrageenan-induced mice tail thrombosis model

Introduction

The prevalence of cardiovascular diseases, one of the major causes of worldwide mortality, is being increasingly reported. Safer, more effective, and less expensive thrombolytic drugs can possibly overcome the underlying problems associate with current thrombolytic drugs.

Methods

A thrombolytic enzyme was purified and characterized from a Streptomyces strain. Carrageenan induced tail-thrombosis mice model was used to evaluate in vivo antithrombotic effect of the enzyme.

Results

First 15 N-terminal amino acids of the purified enzyme were IAGGQAIYAGGGRRS, which are significantly different from the reported fibrinolytic enzymes. The enzyme exhibited 14.3 ± 2.3-fold stronger thrombolytic activity than that of plasmin. In carrageenan induced tail-thrombosis model, the enzyme caused reduction in frequency of thrombus. Tail-thrombus of the enzyme treated group was significantly shorter than the physiological saline treated group and the thrombus decrement was correlated with the enzyme dose.

Conclusions

The enzyme purified from the Streptomyces strain can be a potential candidate for the treatment of thrombosis.     

Circulating platelet-derived microparticles and endothelium-derived microparticles may be a potential cause of microthrombosis in patients with osteonecrosis of the femoral head

Introduction

To test the hypothesis that the platelet microparticle (PMP) and endothelial microparticle (EMP) may contribute to the hypercoagulability associated with microvascular thrombosis in patients with nontraumatc osteonecrosis of the femoral head (ONFH).

Materials and methods

The study comprised 46 patients who had been diagnosed with ONFH and 20 control subjects. The plasma was ultracentrifuged, and then PMPs and EMPs were examined by the flow cytometry. The thrombotic and fibrinolytic disorders were investigated.

Results

The numbers of PMPs expressing P-selectin and CD42a and EMPs expressing E-selectin and CD31 in the ONFH patients were significantly higher than those in the controls (P < 0.001). The number of MPs was correlated with the level of the serum C-reactive protein (CRP) (r = 0.661, P < 0.001), but there was a poor correlation between the MPs counts and the risk factors for ONFH (P > 0.05). The mean levels PAI-1, F1 + 2, and TAT were higher in the patients with ONFH than in the controls (P < 0.05).

Conclusions

The elevated numbers of PMPs and EMPs may contribute to hypercoagulability in the ONFH patients. This may provide important pathophysiological insights into the hypercoagulability associated with nontraumatic ONFH and have implications for pharmacological prevention and treatment of ONFH.     

Single prolonged stress increases contextual freezing and the expression of glycine transporter 1 and vesicle-associated membrane protein 2 mRNA in the hippocampus of rats

Rats subjected to single prolonged stress (SPS) show enhanced HPA negative feedback, exaggerated acoustic startle response, and enhanced contextual freezing 7 days after SPS, and accordingly, SPS is an animal model of PTSD. To elucidate the influence of contextual fear on gene expression in the hippocampus of SPS rats, we used cDNA microarray followed by real-time quantitative PCR analyses to compare the hippocampal gene expression profiles between rats that were or were not subjected to SPS during exposure to contextual fear. In the behavioral experiments, spontaneous locomotor activity was measured 7 days after SPS. Twenty-four hours after footshock conditioning (7 days after SPS), freezing behavior was measured during re-exposure to the chamber in which footshock was delivered. Based on the behavioral analysis, rats subjected to SPS exhibited a significant enhancement of contextual freezing compared to rats not subjected to SPS, without any changes in locomotor activity. Analyses using cDNA microarray and RT-PCR showed that the hippocampal levels of glycine transporter 1 (Gly-T1) and vesicle-associated membrane protein 2 (VAMP2) mRNA in rats subjected to SPS were significantly increased relative to sham-treated rats. Administration of SPS alone did not affect the expression of these 2 genes. These findings suggest that the upregulation of Gly-T1 and VAMP2 in the hippocampus may be, at least in part, involved in the enhanced susceptibility to contextual fear in rats subjected to SPS.     

The high affinity peripheral benzodiazepine receptor ligand DAA1106 binds specifically to microglia in a rat model of traumatic brain injury: implications for PET imaging

Traumatic brain injury (TBI) is a significant cause of mortality, morbidity, and disability. Microglial activation is commonly observed in response to neuronal injury which, when prolonged, is thought to be detrimental to neuronal survival. Activated microglia can be labeled using PK11195, a ligand that binds the peripheral benzodiazepine receptor (PBR), receptors which are increased in activated microglia and sparse in the resting brain. We compared the binding properties of two PBR ligands PK11195 and DAA1106 in rats using the controlled cortical impact (CCI) model of experimental TBI. While both ligands showed relative increases with specific binding in the cortex ipsilateral to injury compared to the contralateral side, [(3)H]DAA1106 showed higher binding affinity compared with [(3)H](R)-PK11195. Combined immunohistochemistry and autoradiography in brain tissues near the injury site showed that [(3)H]DAA1106 binding co-registered with activated microglia more than astrocytes. Further, increased [(3)H]DAA1106-specific binding positively correlated with the degree of microglial activation, and to a lesser degree with reactive astrocytosis. Finally, in vivo administration of each ligand in rats with TBI showed greater retention of [(11)C]DAA1106 compared to [(11)C](R)-PK11195 at the site of the contusion as assessed by ex vivo autoradiography. These results in a rat model of TBI indicate that [(11)C]DAA1106 binds with higher affinity to microglia when compared with PK11195, suggesting that [(11)C]DAA1106 may represent a better ligand than [(11)C](R)-PK11195 for in vivo PET imaging of activated microglia in TBI.     

Clinical evaluation of a new functional test for detection of activated protein C resistance (Pefakit APC-R Factor V Leiden) at two centers in Europe and the USA

A new clotting assay, Pefakit APC-R Factor V Leiden (Pentapharm Ltd., Switzerland), for the detection of an increased resistance of coagulation factor V against degradation by activated protein C, caused mainly by the factor V Leiden mutation, was evaluated in clinical studies at two University Centers in Europe and the US. The performance was compared with the performance of the routinely used predicate device COATEST APC Resistance V (Chromogenix IL, USA). Both tests were run in parallel on a STA-R analyzer (Diagnostica Stago, France). Samples from subjects undergoing routine laboratory thrombophilia screening were examined, 187 at the Institute of Medical and Chemical Laboratory Diagnostics (IMCLD), University of Vienna, Austria, and 236 at the Duke University Medical Center (DUMC), Durham/Raleigh NC, USA. All samples were analyzed for factor V Leiden mutation and prothrombin 20210 G/A mutation using standard PCR methods. The data show that the Pefakit APC-R Factor V Leiden assay discriminates very well between healthy controls and carriers of the factor V Leiden mutation, even in patients with lupus anticoagulant or with deficiency in Protein C or Protein S. Furthermore, this new test is able to discriminate well between heterozygous and homozygous carriers of the factor V Leiden mutation. In both studies the Pefakit assay showed 100% sensitivity and 100% specificity for detection of the factor V Leiden mutation, compared to 93.1% sensitivity and 93.0% specificity for the COATEST APC Resistance V in the IMCLD study and 93.9% sensitivity and 95.6% specificity in the DUMC study. The new test has PCR-like discrimination power which will help to decrease costs by reducing the need for PCR verification of borderline cases.     

Modulation of defensive responses and anxiety-like behaviors by group I metabotropic glutamate receptors located in the dorsolateral periaqueductal gray

Glutamatergic neurotransmission in the dorsolateral periaqueductal gray (dlPAG) is related to defensive responses. However, the role of group I glutamate metabotropic receptors (mGluR) in these responses has been poorly investigated. The objective of the present study, therefore, was to test the hypothesis that interference with group I mGluR-mediated neurotransmission in dlPAG could modulate defensive responses. Male Wistar rats with cannulae aimed at the dlPAG were submitted to the following experiments: 1. intra dlPAG injections of vehicle (veh, 0.2 microL) or (RS)1-aminoindan-1,5-dicarboxylic acid (AIDA, 30-100 nmol, an mGluR1 receptor competitive antagonist) followed, 5 min later, by veh or trans-(+)-1-amino-1,3-ciclopentanedicarboxylic acid (tACPD, a group I and II mGluR agonist, 30 nmol); 2. intra-dlPAG injections of veh, AIDA (30 nmol) or 2-methyl-6-(phenylethynyl)-pyridine (MPEP, an mGluR5 receptor non-competitive antagonist, 50 nmol) followed by trans-azetidine-2,4-dicarboxylic acid (tADA, a group I mGluR agonist, 10 nmol); 3. and 4. intra-dlPAG injections of vehicle, AIDA (10-30 nmol) or MPEP (10-50 nmol) before the elevated plus maze (EPM) test; 5. intra-dlPAG injections of vehicle, AIDA (30 nmol) or MPEP (50 nmol) before the Vogel punished licking test. tACPD induced defensive responses characterized by jumps and an increased number of crossings in the observation box. These responses were attenuated by AIDA (30 nmol). tADA produced similar responses, although of lower intensity. tADA effects were prevented by AIDA and MPEP. Both drugs also produced anxiolytic-like effects in the EPM and Vogel tests when injected alone. The results suggest that group I metabotropic glutamate receptors in the dlPAG facilitate defensive responses and may also be involved in regulating anxiety-like behavior.     

Enhanced expression of the high affinity glutamate transporter GLT-1 in C6 glioma cells delays tumour progression in rat

High grade gliomas are known to release excitotoxic concentrations of glutamate, a process thought to contribute to their malignant phenotype through enhanced autocrine stimulation of their proliferation and destruction of the surrounding nervous tissue. A model of C6 glioma cells in which expression of the high affinity glutamate transporter GLT-1 can be manipulated both in vivo and in vitro was used in order to investigate the consequences of increasing glutamate clearance on tumour progression. These cells were grafted in the striatum of Wistar rats and doxycycline was administered after validation of tumour development by magnetic resonance imaging. Both GLT-1 expression examined by immunohistochemistry and glutamate transport activity measured on synaptosomes appeared robustly increased in samples from doxycycline-treated animals. Moreover, these rats showed extended survival times as compared to vehicle-treated animals, an effect that was consistent with volumetric data revealing delayed tumour growth. As constitutive deficiency in glutamate clearance at the vicinity of brain tumours is well established, these data illustrate the potential benefit that could be obtained by enhancing glutamate transport by glioma cells in order to reduce their invasive behaviour.     

Molecular basis and thrombotic manifestations of antithrombin deficiency in 15 unrelated Chinese patients

Introduction

Antithrombin (AT) deficiency is associated with an increasing risk of thrombosis.

Materials and methods

15 unrelated patients with AT deficiency defined by thrombophilic assays were recruited and detailed clinical information about patients, focusing on the personal and family history of thromboembolism (TE), were recorded. Mutation analysis was performed by direct sequencing of an AT gene (SERPINC1) in the patients and their family members.

Results

A total of 15 heterozygous causative mutations, each being identified in one family, were identified. Five mutations (33.3%) were reported here for the first time, including three null mutations (Ser36X, Lys70X and Try307X) and two missense mutations (Phe123Cys and Leu340Phe) probably impairing the structural integrity and stability of protein based on the AT structural analysis. Of the 15 patients, 33.3% (5/15) had additional risk factors and only one patient presented with additional genetic alteration causing an early onset of thrombosis. Fourteen patients (93.9%) suffered from multisite recurrent thrombotic episodes after a first episode of thrombosis. 93.3% of the patients experienced deep vein thrombosis (DVT) and 40.0% presented with mesenteric venous thrombosis (MVT). In addition, both venous and arterial thrombosis was present in two unrelated patients. 51.0% subjects with AT deficiency in the 15 unrelated pedigrees experienced TE events.

Conclusions

Prophylactic anticoagulation may be suggested in AT-deficient patients to avoid the recurrent and multisite thrombosis. The association of primary MVT and AT deficiency is highlighted.     

Spice drugs are more than harmless herbal blends: A review of the pharmacology and toxicology of synthetic cannabinoids

"K2" and "Spice" drugs (collectively hereafter referred to as Spice) represent a relatively new class of designer drugs that have recently emerged as popular alternatives to marijuana, otherwise characterized as "legal highs". These drugs are readily available on the Internet and sold in many head shops and convenience stores under the disguise of innocuous products like herbal blends, incense, or air fresheners. Although package labels indicate "not for human consumption", the number of intoxicated people presenting to emergency departments is dramatically increasing. The lack of validated and standardized human testing procedures and an endless supply of potential drugs of abuse are primary reasons why researchers find it difficult to fully characterize clinical consequences associated with Spice. While the exact chemical composition and toxicology of Spice remains to be determined, there is mounting evidence identifying several synthetic cannabinoids as causative agents responsible for psychoactive and adverse physical effects. This review provides updates of the legal status of common synthetic cannabinoids detected in Spice and analytical procedures used to test Spice products and human specimens collected under a variety of clinical circumstances. The pharmacological and toxicological consequences of synthetic cannabinoid abuse are also reviewed to provide a future perspective on potential short- and long-term implications.     

Calcium channel alpha2-delta type 1 subunit is the major binding protein for pregabalin in neocortex, hippocampus, amygdala, and spinal cord: an ex vivo autoradiographic study in alpha2-delta type 1 genetically modified mice

Pregabalin is a synthetic amino acid compound effective in clinical trials for the treatment of post-herpetic neuralgia, diabetic peripheral neuropathy, generalized anxiety disorder and adjunctive therapy for partial seizures of epilepsy. However, the mechanisms by which pregabalin exerts its therapeutic effects are not yet completely understood. In vitro studies have shown that pregabalin binds with high affinity to the alpha(2)-delta (alpha(2)-delta) subunits (Type 1 and 2) of voltage-gated calcium channels. To assess whether alpha(2)-delta Type 1 is the major central nervous system (CNS) binding protein for pregabalin in vivo, a mutant mouse with an arginine-to-alanine mutation at amino acid 217 of the alpha(2)-delta Type 1 protein (R217A mutation) was generated. Previous site-directed mutagenesis studies revealed that the R217A mutation dramatically reduces alpha(2)-delta 1 binding to pregabalin in vitro. In this autoradiographic analysis of R217A mice, we show that the mutation to alpha(2)-delta Type 1 substantially reduces specific pregabalin binding in CNS regions that are known to preferentially express the alpha(2)-delta Type 1 protein, notably the neocortex, hippocampus, basolateral amygdala and spinal cord. In mutant mice, pregabalin binding was robust throughout regions where the alpha(2)-delta Type 2 subunit mRNA is abundant, such as cerebellum. These findings, in conjunction with prior in vitro binding data, provide evidence that the alpha(2)-delta Type 1 subunit of voltage-gated calcium channels is the major binding protein for pregabalin in CNS. Moreover, the distinct localization of alpha(2)-delta Type 1 and mutation-resistant binding (assumed to be alpha(2)-delta Type 2) in brain areas subserving different functions suggests that identification of subunit-specific ligands could further enhance pharmacologic specificity.     

Association between obsessive-compulsive disorder and a variable number of tandem repeats polymorphism in intron 2 of the serotonin transporter gene

BACKGROUND: Pharmacological studies indicate a dysregulation of the serotonergic system in obsessive-compulsive disorder (OCD). A variable number tandem repeats (VNTR) polymorphism with three alleles (Stin2.9, Stin2.10, Stin2.12) has been described in intron 2 of the serotonin transporter (5-HTT) gene. This polymorphism has been associated with unipolar depression, bipolar disorder, schizophrenia, and anxiety disorders including OCD. METHODS: The association between OCD and the polymorphism is examined in 97 OCD patients, 578 psychiatric controls and 406 healthy controls, all Spanish Caucasians. RESULTS: Genotype frequencies for the polymorphism were significantly different in OCD patients, psychiatric patients and controls. There was a significant excess of 12/12 and 12/10 genotypes in OCD patients compared to psychiatric patients and controls. CONCLUSIONS: Our results indicate a possible association between the Stin2.12 allele of the VNTR polymorphism and OCD.