Ca²⁺ homeostasis in microvascular endothelial cells from an insulin dependent diabetic model: role of endosomes/Lysosomes

Cytosolic Ca²⁺ ([Ca²⁺]^cyt) regulates several functions, e.g. cell growth, contraction, secretion, etc. In many cell types, ion homeostasis appears to be coupled with glucose metabolism. In certain cell types, a strict coupling between glycolysis and the activity of Sarcoplasmic/Endoplasmic Reticulum Ca²⁺-ATPases (SERCA) has been suggested. Glucose metabolism is altered in diabetes. We hypothesize that: (a) Ca²⁺ homeostasis is altered in microvascular endothelial cells from diabetic animals due to the dysfunction of glycolysis coupling the activity of SERCA; (b) endosomal/lysosomal (E/L) compartments expressing SERCA are involved in the dysfunction associated with diabetes. Ca²⁺ ions in E/L compartments can be studied by either spectral imaging/confocal microscopy in single cells, or by fluorescence spectroscopy in cell populations. In cell populations, agonist stimulation elicited greater [Ca²⁺]^cyt increases in cells from diabetic than from normal animals. Simultaneous measurements of [Ca²⁺]^cyt and Ca²⁺ in E/L compartments ([Ca²⁺]^E/L) using fluorescence spectroscopy and spectral imaging/confocal microscopy, demonstrate that Ca²⁺ is released from E/L compartments following agonist stimulation. Immunocytochemical studies demonstrate that E/L compartments exhibit the machinery to regulate Ca²⁺: SERCA and ryanodine receptor (RyR) coupled Ca²⁺ channels. Our functional studies, using confocal/spectral imaging microscopy, indicated that E/L compartments employ SERCA and RyR coupled Ca²⁺ channels to refill and release Ca²⁺, respectively. Glucose modulates refilling of E/L compartments with Ca²⁺ via SERCA, since SERCA inhibitors suppress this refilling. The regulation of these phenomena are altered in diabetes. These data indicate that E/L compartments are important for Ca²⁺ homeostasis.