Plasma membrane Ca(2+) pumps (PMCA) that expel Ca(2+) from cells are encoded by four genes (PMCA1-4). In this study, we show that aortic endothelium and smooth muscle differ in their PMCA isoform mRNA expression: endothelium expressed predominantly PMCA1, and smooth muscle expressed PMCA4 and a lower level of PMCA1. In this study, we report a novel peptide (caloxin 1b1, obtained by screening for binding to extracellular domain 1 of PMCA4), which inhibited PMCA extracellularly, selectively, and had a higher affinity for PMCA4 than PMCA1. It inhibited the PMCA Ca(2+)-Mg(2+)-ATPase activity in leaky erythrocyte ghosts (mainly PMCA4) with a K(i) value of 46 +/- 5 microM, making it 10x more potent than the previously reported caloxin 2a1. It was isoform selective because it inhibited the PMCA1 Ca(2+)-Mg(2+)-ATPase in human embryonic kidney-293 cells with a higher K(i) value (105 +/- 11 microM) than for PMCA4. Caloxin 1b1 was selective in that it did not inhibit other ATPases. Because caloxin 1b1 had been selected to bind to an extracellular domain of PMCA, it could be added directly to cells and tissues to examine its effects on smooth muscle and endothelium. In de-endothelialized aortic rings, caloxin 1b1 (200 microM) produced a contraction. It also increased the force of contraction produced by a submaximum concentration of phenylephrine. In aortic rings with endothelium intact, precontracted with phenylephrine and relaxed partially with a submaximum concentration of carbachol, caloxin 1b1 increased the force of contraction rather than potentiating the endothelium-dependent relaxation. In cultured cells, caloxin 1b1 increased the cytosolic [Ca(2+)] more in arterial smooth muscle cells than in endothelial cells. Thus caloxin 1b1 is the first highly selective extracellular PMCA inhibitor that works better on vascular smooth muscle than on endothelium.
The addition of Ca2+ ionophore A23187 or ATP to freshly isolated or cultured pig coronary artery endothelial cells (PCEC) potentiated the release of ascorbate (Asc). Cultured PCEC were used to characterize the Ca2+‐mediated release. An increase in Ca2+‐mediated Asc release was observed from PCEC preincubated with Asc, Asc‐2‐phosphate or dehydroascorbic acid (DHAA). The effects of various ATP analogs and inhibition by suramin were consistent with the ATP‐induced release being mediated by P2Y2‐like receptors. ATP‐stimulated Asc release was Ca2+‐mediated because (a) ATP analogs that increased Asc release also elevated cytosolic [Ca2+], (b) Ca2+ ionophore A23187 and cyclopiazonic acid stimulated the Asc release, (c) removing extracellular Ca2+ and chelating intracellular Ca2+inhibited the ATP‐induced release, and (d) inositol‐selective phospholipase C inhibitor U73122 also inhibited this release. Accumulation of Asc by PCEC was examined at Asc concentrations of 10 μM (Na+‐Asc symporter not saturated) and 5 mM (Na+‐Asc symporter saturated). At 10 μM Asc, A23187 and ATP caused an inhibition of Asc accumulation but at 5 mM Asc, both the agents caused a stimulation. Substituting gluconate for chloride did not affect the basal Asc uptake but it abolished the effects of A23187. PCEC but not pig coronary artery smooth muscle cells show a Ca2+‐ mediated Asc release pathway that may be activated by agents such as ATP. British Journal of Pharmacology (2006) 147, 131–139. doi:
Allogeneic hematopoietic cell transplant (HCT) is often the only curative therapy for patients with nonmalignant diseases; however, many patients do not have an HLA-matched donor. Historically, poor survival has been seen after HLA-haploidentical HCT because of poor immune reconstitution, increased infections, graft-versus-host disease (GVHD), and graft failure. Encouraging results have been reported using a nonmyeloablative T cellÀreplete HLA-haploidentical transplant approach in patients with hematologic malignancies. Here we report the outcomes of 23 patients with various nonmalignant diseases using a similar approach. Patients received HLA-haploidentical bone marrow (n = 17) or granulocyte colony-stimulating factorÀmobilized peripheral blood stem cell (n = 6) grafts after conditioning with cyclophosphamide 50 mg/kg, fludarabine 150 mg/m 2 , and 2 or 4 Gy total body irradiation. Postgrafting immunosuppression consisted of cyclophosphamide, mycophenolate mofetil, tacrolimus, § sirolimus. Median patient age at HCT was 10.8 years. Day 100 transplant-related mortality (TRM) was 0%. Two patients died at later time points, 1 from intracranial hemorrhage/disseminated fungal infection in the setting of graft failure and 1 from infection/ GVHD. The estimated probabilities of grades II to IV and III to IV acute GVHD at day 100 and 2-year National Institutes of Health consensus chronic GVHD were 78%, 26%, and 42%, respectively. With a median follow-up of 2.5 years, the 2year overall and event-free rates of survival were 91% and 78%, respectively. These results are encouraging and demonstrate favorable disease-specific lineage engraftment with low TRM in patients with nonmalignant diseases using nonmyeloablative conditioning followed by T cellÀreplete HLA-haploidentical grafts. However, additional strategies are needed for GVHD prevention to make this a viable treatment approach for patients with nonmalignant diseases.
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