Activated protein C (APC) exerts endothelial protein C receptor (EPCR)-

The P2RX7 gene (coding for P2X7 purinergic receptor) has been suggested as a novel candidate gene for major depressive disorder (MDD) and bipolar disorder (BPD). The proposed risk allele (G-allele) of the rs2230912 polymorphism results in an amino acid change at the 460th position, marking this genetic variation a possibly functional one. Here we present a case-control analysis of 171 patients diagnosed with MDD or BPD and 178 controls, as well as a dimensional approach using the Hospital Anxiety and Depression Scale (HADS) for studying the Gln460Arg polymorphism of the P2RX7 gene as a genetic risk factor in depression. While case-control analysis did not show significant difference between the groups, a significant association was found between the P2RX7 polymorphism and the HADS scales in the clinical group (MANOVA P = 0.001). Both anxiety and depression scores increased as the number of G-allele increased in the genotype groups (ANOVA for HADS-anxiety: P = 0.01, HADS-depression: P < 0.001). A significant interaction of clinical status and the P2RX7 polymorphism was also found for the depression scale (MANOVA P = 0.025, subsequent ANOVA for anxiety: P = 0.252; depression: P = 0.002). Whereas patients with G-allele-present genotypes showed more elevated depression scores, level of depression in the control group was not affected by the P2RX7 genotype. In conclusion, case-control analysis did not reveal significant results, but using a symptom severity scale we could support the association between depressive disorder and the G-allele of the Gln460Arg polymorphism in the P2RX7 gene.

Summary: Both thrombin and plasmin induce contraction of brain endothelial cells, which may increase capillary perme ability thereby leading to disruption of the blood-brain barrier. Identification of thrombin receptors, as well as the influence of plasmin on their activation, in capillary endothelial cells and astrocytes are therefore essential for understanding injury related actions of thrombin in the brain. Using the reverse transcriptase-polymerase chain reaction method, the present study shows that primary cultures of rat brain capillary endo thelial (RBCE) cells and astrocytes derived from rat brain ex press two different thrombin receptors. The first is proteolyti cally activated receptor (PAR)-l, the receptor responsible for the vast majority of the thrombin's cellular activation func tions; the second is PAR-3, a receptor described to be essential for normal responsiveness to thrombin in mouse platelets. In addition to these thrombin receptors, the mRNA (messenger RNA) for PAR-2, a possible trypsin receptor, was also identi fied. Functional significance of thrombin receptors was indi cated by changes in rCa 2 +l; in response to thrombin, as mea sured by FURA-2 fluorescence in RBCE cells. Thrombin as low as 4 nmollL induced an abrupt increase in lCa 2 +1i whereas, Thrombosis is of paramount importance in the patho physiology of ischemic stroke. Thrombogenic and fibri nolytic enzymes accumulating in the occluded vascular segments may play a role in the opening of the blood brain barrier, and the subsequent exposure of cells in the brain to high amounts of thrombin and plasmin is likely to have deleterious effects. Thrombin actions are largely mediated by PAR-I, the first member of the proteolyti-

The role of Na+–Ca2+ exchange in the regulation of the cytosolic free Ca2+ concentration ([Ca2+]i) was studied in primary cultured rat brain capillary endothelial cells. [Ca2+]i was measured by digital fluorescence imaging in cells loaded with fura‐2. ATP (100 μm) applied for a short period of time (6 s) caused a rise in [Ca2+]i from 127 ± 3 (n= 290) to 797 ± 25 nm, which then declined to the resting level, with a t½time required for [Ca2+]i to decline to half of peak [Ca2+]i) of 5.4 ± 0.09 s. This effect was independent of external Ca2+ and could be abolished by previously discharging the Ca2+ pool of the endoplasmic reticulum with thapsigargin (1 μm). Application of thapsigargin (1 μm) or cyclopiazonic acid (10 μm) to inhibit the Ca2+‐ATPase of the endoplasmic reticulum 6 s prior to ATP application did not influence the peak [Ca2+]i but greatly reduced the rate of decline of [Ca2+]i, with t½ values of 15 ± 1.6 and 23 ± 3 s, respectively. In the absence of external Na+ (Na+ replaced by Li+ or N‐methylglucamine) the basal [Ca2+]i was slightly elevated (152 ± 6 nm) and the restoration of [Ca2+]i after the ATP stimulation was significantly slower (t½, 7.3 ± 0.46 s in Li+ medium, 8.12 ± 0.4 s in N‐methylglucamine medium). The external Na+‐dependent component of the [Ca2+]i sequestration was also demonstrated in cells stimulated by ATP subsequent to addition of cyclopiazonic acid; in a Na+‐free medium [Ca2+]i remained at the peak level in 88% of the cells after stimulation with ATP. Addition of monensin (10 μm) in the presence of external Na+ increased the resting [Ca2+]i to 222 ± 9 nm over ∼1 min and subsequent removal of extracellular sodium resulted in a further increase in [Ca2+]i to a peak of 328 ± 11 nm, which was entirely dependent on external Ca2+. These findings indicate that a functional Na+–Ca2+ exchanger is present at the blood‐brain barrier, which plays a significant role in shaping the stimulation‐evoked [Ca2+]i signal and is able to work in reverse mode under pharmacological conditions.

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