To determine the nature of intracellular Mg2+ stores and Mg2+ release mechanisms in differentiated PC12 cells, Mg2+ and Ca2+ mobilizations were measured simultaneously in living cells with KMG-104, a fluorescent Mg2+ indicator, and fura-2, respectively. Treatment with the mitochondrial uncoupler, carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), increased both the intracellular Mg2+ concentration ([Mg2+]i) and the [Ca2+]i in these cells. Possible candidates as intracellular Mg2+ stores under these conditions include intracellular divalent cation binding sites, endoplasmic reticulum (ER), Mg-ATP and mitochondria. Given that no change in [Mg2+]i was induced by caffeine application, intracellular IP3 or Ca2+ liberated by photolysis, it appears that no Mg2+ release mechanism thus exists that is mediated via the action of Ca2+ on membrane-bound receptors in the ER or via the offloading of Mg2+ from binding sites as a result of the increased [Ca2+]i. FCCP treatment for 2 min did not alter the intracellular ATP content, indicating that Mg2+ was not released from Mg-ATP, at least in the first 2 min following exposure to FCCP. FCCP-induced [Mg2+]i increase was observed at mitochondria localized area, and vice versa. These results suggest that the mitochondria serve as the intracellular Mg2+ store in PC12 cell. Simultaneous measurements of [Ca2+]i and mitochondrial membrane potential, and also of [Ca2+]i and [Mg2+]i, revealed that the initial rise in [Mg2+]i followed that of mitochondrial depolarization for several seconds. These findings show that the source of Mg2+ in the FCCP-induced [Mg2+]i increase in PC12 cells is mitochondria, and that mitochondrial depolarization triggers the Mg2+ release.
Shear stress stimulus is expected to enhance angiogenesis, the formation of microvessels. We determined the effect of shear stress stimulus on three-dimensional microvessel formation in vitro. Bovine pulmonary microvascular endothelial cells were seeded onto collagen gels with basic fibroblast growth factor to make a microvessel formation model. We observed this model in detail using phase-contrast microscopy, confocal laser scanning microscopy, and electron microscopy. The results show that cells invaded the collagen gel and reconstructed the tubular structures, containing a clearly defined lumen consisting of multiple cells. The model was placed in a parallel-plate flow chamber. A laminar shear stress of 0.3 Pa was applied to the surfaces of the cells for 48 h. Promotion of microvessel network formation was detectable after approximately 10 h in the flow chamber. After 48 h, the length of networks exposed to shear stress was 6.17 (+/-0.59) times longer than at the initial state, whereas the length of networks not exposed to shear stress was only 3.30 (+/-0.41) times longer. The number of bifurcations and endpoints increased for networks exposed to shear stress, whereas the number of bifurcations alone increased for networks not exposed to shear stress. These results demonstrate that shear stress applied to the surfaces of endothelial cells on collagen gel promotes the growth of microvessel network formation in the gel and expands the network because of repeated bifurcation and elongation.
We examined whether autotransplantation of microvessel fragments (Mvf) and/or myofibroblasts (Mf) into an in vivo skin flap model might improve the survival of the ischemic flap. If so, this could improve blood perfusion, increase blood flow, and improve the survival of the flap. A skin flap was raised on the back of each rat (n = 15 in each group). In the control group, the flap was sutured to the original bed. In the other groups (1) phosphate-buffered saline; (2) autotransplanted Mvf, Mf, or Mvf plus Mf, and (3) a homogenized mixture of Mvf plus Mf was injected into the distal part of the flap. In a further group, Mvf labeled with DiI-acetylated low-density lipoprotein were autotransplanted with Mf into the distal part of the flap, and India ink was perfused through the abdominal aorta 7 days postoperatively. The transplanted Mvf plus Mf group showed better flap survival after 7 days than the other groups (p < 0.02). Labeling with DiI-acetylated low-density lipoprotein showed that transplanted Mvf sent arborizations into the nearby tissue. India ink was found in the lumina within such arborizations. Thus, autotransplanted Mvf may improve the survival of ischemic skin flaps by promoting the early formation of patent connections between Mvf and the host’s microcirculatory system. This apparently requires the presence of Mf.
Endothelial cells (ECs) are exposed to various environmental forces, and a Ca wave is occurred in mechanical stimulated cells. Pharmacological studies reveal that the translocation of protein kinase Cα (PKCα) to the membrane is observed simultaneously with intracellular Ca wave. In this study, we investigate whether and how the kinetics of PKCα in ECs is induced in response to mechanical stress. The results show that a mechanical stimulus induced biphasic and directional PKCα translocation; PKCα initially translocated near or at the membrane and then accumulated at the stimulus point. The initial translocation occurred simultaneously with Ca increase. Initial translocation was inhibited in spite of Ca increase when the diacylglycerol (DAG) binding domain of PKCα was inhibited, suggesting that translocation requires intracellular Ca increase and DAG. On the other hand, secondary translocation was delayed, occurring after the Ca wave; however, this translocation occurred even when Ca release from the endoplasmic reticulum was inhibited, while it did not occur when the mechanosensitive (MS) channel was inhibited. These results indicated that at least Ca influx from extracellular space through MS channel is required. Our results support the implication of PKCα in the Ca signaling pathway in response to mechanical stress in ECs.
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