The expression at the protein level of plasma membrane calcium pump (PMCA) isoforms in rat brain was detected by new antibodies that distinguished the four gene products and their alternatively spliced variants. All four gene products were distributed throughout hippocampus, cortex, and cerebellum, but the alternate splices showed more distinct distribution patterns. The b splice of isoform 1 was not detectable in any of the brain regions, which makes it unlikely that this isoform performs an essential housekeeping role as is frequently proposed. The b splices of isoforms 3 and 4, although expressed in all three regions, showed evidence of proteolysis, which removed a portion of the carboxyl terminus. In contrast, isoform 2b retained its full length, indicating that PMCA2b is more resistant to proteolysis than the other b forms. Whereas substantial amounts of isoforms 1a, 2a, and 3a were expressed in all regions, 4a was found only in frontal cortex. The distinct patterns of expression of the PMCA isoforms in brain suggest that some of them play a special role in intracellular Ca regulation.The plasma membrane calcium pump is one of the mechanisms responsible for maintaining the low cytosolic calcium concentration critical to cell function. Because of its high affinity for calcium, it is believed to have a unique role in the maintenance of calcium homeostasis in eukaryotic cells. Sequencing and molecular cloning work have revealed some 20 isoforms of the pump, generated by four different genes encoding the pump and by the alternative splicing of the primary gene transcripts in two sites identified as A and C in the pump molecule. Studies on the distribution of mRNA of the pump in rat and human tissues revealed that gene products 1 and 4 are transcribed in all tissues, whereas gene products 2 and 3 were more tissue-specific and were found enriched in excitable cells. The transcription of the splicing variants was also found to be specific. In tissues where there is wide cell-type diversity, the distribution of the spliced variants has been found to be cellspecific. For example, in the pancreas, where secretion of insulin is dependent on calcium concentration, the beta cells express only isoform 4b, whereas alpha and gamma cells express both 4a and 4b. The islets do not express the a but only the b form of isoforms 1 and 2 (1). In brain, where highly regulated signal transduction events occur, the mRNAs of the spliced variants were also localized to specific cells and regions (2-4). The specific presence of the proteins of the a and b variants of the different isoforms is particularly interesting, since these variants are products of the alternate splicing at site C in the region where most of the regulatory properties of the pump are located (5, 6). In fact, Enyedi et al. (7,8) show that these proteins have different affinities for calcium and calmodulin. The protein products of the four genes encoding the pump have been identified and have confirmed the reported distribution of the mRNA (9, 10). However, informat...

During routine storage, packed red blood cells (PRBC) undergo biochemical and morphological changes including loss of red blood cell (RBC) membrane asymmetry and release of microparticles (MPs) bearing phosphatidylserine (PS), a procoagulant phospholipid. This study investigated the association between PRBC storage duration, MP profile and procoagulant activity. Leukodepleted PRBC-supernatant (PRBC-SN; n=13) was prepared at weekly intervals throughout storage. Phospholipid-dependent procoagulant activity, assessed using a factor X-activated clotting time (XACT) assay, decreased throughout storage (p<0.0001), corresponding with increased procoagulant phospholipid content. As determined by flow cytometry, total numbers of MPs and of PS-bearing MPs increased by Day 28 of storage (p<0.01 and p<0.05, respectively, versus D1), and these MPs were predominantly RBC-derived (CD235). Depletion of MPs from stored (Day 42) PRBC-SN using 0.22 μm filters reduced the number of PS-bearing MPs (p<0.01) but did not increase XACT clotting times. Furthermore, the reduction in procoagulant activity when lactadherin was used to block PS was not altered pre- or post-filtration of PRBC-SN. In conclusion, routine PRBC storage was associated with accumulation of MPs (particularly RBC-derived PS-bearing MPs) and of procoagulant phospholipids; however, depletion of PS-bearing MPs by 0.22 μm filtration did not reduce phospholipid-dependent procoagulant activity.

Background: Blood components are irradiated to inactivate lymphocytes to prevent transfusion-associated graft versus host disease. As there are little data regarding the effects of X-irradiation on red blood cell components (RBCs), the in vitro quality of stored red cells (standard, pediatric, washed, and intrauterine transfusion [IUT]) following X-or gamma-irradiation was compared. Study design and methods: RBCs were pooled, split, and processed to produce standard (<14 days and < 5 days post-collection), pediatric (<5 days post-collection), washed (<14 days post-collection), or IUT RBCs (<5 days post-collection).Standard RBCs were either X-or gamma-irradiated (n = 10 pairs). A further 10 replicates were prepared by pooling and splitting three matched RBCs (X-, gamma-, and non-irradiated). All other RBCs were either X-or gamma-irradiated (n = 20 pairs). Red cell indices, hemolysis, potassium release, metabolism, microparticles, ATP, and 2,3-DPG were measured pre-irradiation and 6 h, 1, 2, 3, 7, 10, and 14 days post-irradiation, depending on the component type. Data were analyzed using two-way repeated measures ANOVA.Results: There were no significant differences in any in vitro quality measurements, with the exception of marginally higher potassium release in washed, IUT, and RBCs <5 days old (p < .0001) following X-irradiation.Both irradiation types increased generation of microvesicles, particularly in components that were older at the time of irradiation or stored for longer post-irradiation. Conclusion: X-and gamma-irradiation have similar effects on the in vitro quality of RBCs, indicating that either technology is suitable for blood component irradiation.

Background Familial pseudohyperkalemia (FP) is a rare asymptomatic condition characterized by an increased rate of potassium leak from red blood cells (RBC) on refrigeration. Gamma irradiation compromises RBC membrane integrity and accelerates potassium leakage. Here, we compared the effect of irradiation, applied early or late in storage, on FP versus non‐FP RBC. Study Design Five FP and 10 non‐FP individuals from the National Institute for Health Research Cambridge BioResource, UK, and three FP and six non‐FP individuals identified by Australian Red Cross Lifeblood consented to the study. Blood was collected according to standard practice in each center, held overnight at 18–24°C, leucocyte‐depleted, and processed into red cell concentrates (RCC) in Saline Adenine Glucose Mannitol. On Day 1, RCC were split equally into six Red Cell Splits (RCS). Two RCS remained non‐irradiated, two were irradiated on Day 1 and two were irradiated on Day 14. RBCs were tested over cold storage for quality parameters. Results As expected, non‐irradiated FP RCS had significantly higher supernatant potassium levels than controls throughout 28 days of storage (p < .001). When irradiated early, FP RCS released potassium at similar rates to control. When irradiated late, FP RCS supernatants had higher initial post‐irradiation potassium concentration than controls but were similar to controls by the end of storage (14 days post‐irradiation). No other parameters studied showed a significant difference between FP and control. Discussion FP does not increase the rate of potassium leak from irradiated RBCs. Irradiation may cause a membrane defect similar to that in FP RBCs.