Presenilin-1 Established ER-Ca2+ Leak: a Follow Up on Its Importance for the Initial Insulin Secretion in Pancreatic Islets and ß-Cells upon Elevated Glucose.

BACKGROUND/AIMS: In our recent work, the importance of GSK3ß-mediated phosphorylation of presenilin-1 as crucial process to establish a Ca2+ leak in the endoplasmic reticulum and, subsequently, the pre-activation of resting mitochondrial activity in ß-cells was demonstrated. The present work is a follow-up and reveals the importance of GSK3ß-phosphorylated presenilin-1 for responsiveness of pancreatic islets and ß-cells to elevated glucose in terms of cytosolic Ca2+ spiking and insulin secretion. METHODS: Freshly isolated pancreatic islets and the two pancreatic ß-cell lines INS-1 and MIN-6 were used. Cytosolic Ca2+ was fluorometrically monitored using Fura-2/AM and cellular insulin content and secretion were measured by ELISA. RESULTS: Our data strengthened our previous findings of the existence of a presenilin-1-mediated ER-Ca2+ leak in ß-cells, since a reduction of presenilin-1 expression strongly counteracted the ER Ca2+ leak. Furthermore, our data revealed that cytosolic Ca2+ spiking upon administration of high D-glucose was delayed in onset time and strongly reduced in amplitude and frequency upon siRNA-mediated knock-down of presenilin-1 or the inhibition of GSK3ß in the pancreatic ß-cells. Moreover, glucose-triggered initial insulin secretion disappeared by depletion from presenilin-1 and inhibition of GSK3ß in the pancreatic ß-cells and isolated pancreatic islets, respectively. CONCLUSION: These data complement our previous work and demonstrate that the sensitivity of pancreatic islets and ß-cells to glucose illustrated as glucose-triggered cytosolic Ca2+ spiking and initial but not long-lasting insulin secretion crucially depends on a strong ER Ca2+ leak that is due to the phosphorylation of presenilin-1 by GSK3ß, a phenomenon that might be involved in the development of type 2 diabetes.

Calcium Signaling in ß-cell Physiology and Pathology: A Revisit.

Pancreatic beta (ß) cell dysfunction results in compromised insulin release and, thus, failed regulation of blood glucose levels. This forms the backbone of the development of diabetes mellitus (DM), a disease that affects a significant portion of the global adult population. Physiological calcium (Ca2+) signaling has been found to be vital for the proper insulin-releasing function of ß-cells. Calcium dysregulation events can have a dramatic effect on the proper functioning of the pancreatic ß-cells. The current review discusses the role of calcium signaling in health and disease in pancreatic ß-cells and provides an in-depth look into the potential role of alterations in ß-cell Ca2+ homeostasis and signaling in the development of diabetes and highlights recent work that introduced the current theories on the connection between calcium and the onset of diabetes.

Development and Application of Sub-Mitochondrial Targeted Ca2 + Biosensors.

Mitochondrial Ca2+ uptake into the mitochondrial matrix is a well-established mechanism. However, the sub-organellar Ca2+ kinetics remain elusive. In the present work we identified novel site-specific targeting sequences for the intermembrane space (IMS) and the cristae lumen (CL). We used these novel targeting peptides to develop green- and red- Ca2+ biosensors targeted to the IMS and to the CL. Based on their distinctive spectral properties, and comparable sensitivities these novel constructs were suitable to visualize Ca2+-levels in various (sub) compartments in a multi-chromatic manner. Functional studies that applied these new biosensors revealed that knockdown of MCU and EMRE yielded elevated Ca2+ levels inside the CL but not the IMS in response to IP3-generating agonists. Knockdown of VDAC1, however, strongly impeded the transfer of Ca2+ through the OMM while the cytosolic Ca2+ signal remained unchanged. The novel sub-mitochondrially targeted Ca2+ biosensors proved to be suitable for Ca2+ imaging with high spatial and temporal resolution in a multi-chromatic manner allowing simultaneous measurements. These informative biosensors will facilitate efforts to dissect the complex sub-mitochondrial Ca2+ signaling under (patho)physiological conditions.

Tracking intra- and inter-organelle signaling of mitochondria.

Mitochondria are as highly specialized organelles and masters of the cellular energy metabolism in a constant and dynamic interplay with their cellular environment, providing adenosine triphosphate, buffering Ca2+ and fundamentally contributing to various signaling pathways. Hence, such broad field of action within eukaryotic cells requires a high level of structural and functional adaptation. Therefore, mitochondria are constantly moving and undergoing fusion and fission processes, changing their shape and their interaction with other organelles. Moreover, mitochondrial activity gets fine-tuned by intra- and interorganelle H+ , K+ , Na+ , and Ca2+ signaling. In this review, we provide an up-to-date overview on mitochondrial strategies to adapt and respond to, as well as affect, their cellular environment. We also present cutting-edge technologies used to track and investigate subcellular signaling, essential to the understanding of various physiological and pathophysiological processes.

Measurement of Calcium Fluctuations Within the Sarcoplasmic Reticulum of Cultured Smooth Muscle Cells Using FRET-based Confocal Imaging.

Maintenance of steady-state calcium (Ca(2+)) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall health. A significant portion of intracellular Ca(2+) content is found within the SR stores in VSMCs. As the only intracellular organelle with a close association to the surrounding extracellular space through plasma membrane-SR junctions, the SR can be considered to constitute the first line of response to any irregularity in Ca(2+) transients, or stress experienced by the cell. Among its many functions, one of the most important is its role in the transmission of Ca(2+)-regulated signals throughout the cell to induce further cell-wide reactions downstream. The more common use of cytoplasmic Ca(2+) indicators in this regard is overall insufficient for research into the highly dynamic changes to the intraluminal SR Ca(2+) store that have yet to be fully characterized. Here, we provide a detailed protocol for the direct and clear measurement of luminal SR Ca(2+). This tool is useful for investigation into the nuanced changes in SR Ca(2+) that have significant subsequent effects on the normal function and health of the cell. Fluctuations in SR Ca(2+) content specifically can provide us with a significant amount of information pertaining to cellular responses to disease or stress conditions experienced by the cell. In this method, a modified version of a SR-targeted Ca(2+) indicator, D1SR, is used to detect Ca(2+) fluctuations in response to the introduction of agents to cultured rat aortic smooth muscle cells (SMCs). Following incubation with the D1SR indicator, confocal fluorescence microscopy and fluorescence resonance energy transfer (FRET)-based imaging are used to directly observe changes to intraluminal SR Ca(2+) levels under control conditions and with the addition of agonist agents that function to induce intracellular Ca(2+) movement.

Drop in endo/sarcoplasmic calcium precedes the unfolded protein response in Brefeldin A-treated vascular smooth muscle cells.

The present study addresses the causal relationship between induction of endo/sarcoplasmic reticulum stress and dysregulation of calcium transport, while examining whether the most widely-used experimental endo/sarcoplasmic reticulum stressors can be considered appropriate for elucidating underlying cellular mechanisms involved during the progression of the unfolded protein response in vascular smooth muscle cells. Brefeldin A is most commonly cited as inducing the stress response through an accumulation of unfolded proteins in the lumen as a result of a blockage of protein transport from the endo/sarcoplasmic reticulum to the Golgi apparatus. We investigated the effects of Brefeldin A on cellular calcium regulation during the the unfolded protein response in cultured rat vascular smooth muscle cells. Acute exposure of cells to Brefeldin A caused a small transient increase in cytoplasmic calcium, which did not cause a significant decrease in endo/sarcoplasmic reticulum calcium content. However, over the time course of 0-12 h post-treatment with Brefeldin A, we observed that the endo/sarcoplasmic reticulum of vascular smooth muscle cells exhibited an approximate fifty percent decrease in calcium concentration after the first hour of exposure, which is maintained over the next eleven hours, whereas concentrations of unfolded protein response markers only began to increase significantly around nine to twelve hours post-treatment. We have concluded that the endo/sarcoplasmic reticulum calcium drop, which up to now, has been considered as a characteristic of the late onset of cellular stress response, occurs prior to the initiation of the unfolded protein response, rather than as a result of its many corrective pathways.

Calcium regulation in aortic smooth muscle cells during the initial phase of tunicamycin-induced endo/sarcoplasmic reticulum stress.

Endo/sarcoplasmic reticulum stress and the unfolded protein response have been implicated as underlying mechanisms of cell death in many pathological conditions. We have confirmed that long-term exposure to 10µM tunicamycin induced the endo/sarcoplasmic reticulum stress in cultured vascular smooth muscle cells. Since tunicamycin is reported to induce the stress response by inhibiting protein glycosylation, we attempted to investigate a causal link between accumulation of unfolded proteins and dysregulation of cellular calcium transport. However, we found that tunicamycin caused an immediate release of calcium from the endo/sarcoplasmic reticulum, which was sensitive to thapsigargin, and an influx of calcium through the plasma membrane, resulting in a significant increase in cytoplasmic calcium and depletion of endo/sarcoplasmic reticulum calcium. Furthermore, we observed that tunicamycin also induced contraction in intact vascular smooth muscle. By applying established procedures and antagonists, we established that tunicamycin did not directly activate physiological calcium channels, such as store-operated channels, voltage gated calcium channels, ryanodine receptors or inositol trisphosphate receptors. Instead, we found that its effects on cellular calcium fluxes closely resembled those of the known calcium ionophore, ionomycin. We have concluded that tunicamycin directly permeabilizes the plasma membrane and endo/sarcoplasmic reticulum to calcium, and is, therefore, inappropriate for studying the relationship between accumulation of unfolded proteins and endo/sarcoplasmic reticulum calcium dysregulation during the endo/sarcoplasmic reticulum stress response. In contrast, we also report that two other well-known endo/sarcoplasmic reticulum stress inducers, brefeldin A and dithiothreitol, did not exhibit similar increases in calcium permeability.

Comparison of prenatal and postnatal treatments of spina bifida in Poland--a non-randomized, single-center study.

OBJECTIVE: The aim of this study was a comparison of the outcomes of intrauterine myelomeningocele (MMC) repairs (IUMR) in type II Chiari malformation (II CM) fetuses with clinical data of newborns and infants operated on postnatally. METHODS: The study group (SG) comprised 46 pregnant women whose type II CM children underwent IUMR, while 47 pregnant women whose type II CM children were operated on postnatally constituted the control group (CG). A total of 24 SG and 20 CG patients reached the endpoint of the study. RESULTS: High incidence of prelabor rupture of membranes (24 (52.2%), CI: 3.74 (1.69-8.26) (p < 0.001) was noted in the group of prenatal surgeries as compared to controls. The need for ventriculoperitoneal shunt implantation was statistically significantly lower (p < 0.008) in the group of children after IUMR as compared to controls (5 (27.8%) and 16 (80%), respectively, CI: 0.35 (0.16-0.75). None of the postnatally treated CG children can walk without adaptive equipment. In contrast, two children from the SG (2 (11.1%) CI: 1.86 (1.00-3.48) p < 0.05) are able to walk independently. CONCLUSIONS: Prenatal MMC closure significantly lowers further adverse evolution of the II CM. Further studies are needed, especially on preventive measures for preterm labor and iatrogenic preterm prelabor rupture of membranes (iPPRM) in the postoperative course of IUMR.