Jose D. Rojas

Impaired nitric oxide production in coronary endothelial cells of the spontaneously diabetic BB rat is due to tetrahydrobiopterin deficiency

Endothelial cells (EC) from diabetic BioBreeding (BB) rats have an impaired ability to produce NO. This deficiency is not due to a defect in the constitutive isoform of NO synthase in EC (ecNOS) or alterations in intracellular calcium, calmodulin, NADPH or arginine levels. Instead, ecNOS cannot produce sufficient NO because of a deficiency in tetrahydrobiopterin (BH(4)), a cofactor necessary for enzyme activity. EC from diabetic rats exhibited only 12% of the BH(4) levels found in EC from normal animals or diabetes-prone animals which did not develop disease. As a result, NO synthesis by EC of diabetic rats was only 18% of that for normal animals. Increasing BH(4) levels with sepiapterin increased NO production, suggesting that BH(4) deficiency is a metabolic basis for impaired endothelial NO synthesis in diabetic BB rats. This deficiency is due to decreased activity of GTP-cyclohydrolase I, the first and rate-limiting enzyme in the de novo biosynthesis of BH(4). GTP-cyclohydrolase activity was low because of a decreased expression of the protein in the diabetic cells.

Use of a new polyclonal antibody to study the distribution and glycosylation of the sodium-coupled bicarbonate transporter NCBE in rodent brain.

NCBE (SLC4A10) is a member of the SLC4 family of bicarbonate transporters, several of which play important roles in intracellular-pH regulation and transepithelial HCO(3)(-) transport. Here we characterize a new antibody that was generated in rabbit against a fusion protein consisting of maltose-binding protein and the first 135 amino acids (aa) of the N-terminus of human NCBE. Western blotting--both of purified peptides representing the initial approximately 120 aa of the transporters and of full-length transporters expressed in Xenopus oocytes--demonstrated that the antibody is specific for NCBE versus the two most closely related proteins, NDCBE (SLC4A8) and NBCn1 (SLC4A7). Western blotting of tissue in four regions of adult mouse brain indicates that NCBE is expressed most abundantly in cerebral cortex (CX), cerebellum (CB) and hippocampus (HC), and less so in subcortex (SCX). NCBE protein was present in CX, CB, and HC microdissected to avoid choroid plexus. Immunocytochemistry shows that NCBE is present at the basolateral membrane of embryonic day 18 (E18) fetal and adult choroid plexus. NCBE protein is present by Western blot and immunocytochemistry in cultured and freshly dissociated HC neurons but not astrocytes. By Western blot, nearly all NCBE in mouse and rat brain is highly N-glycosylated (approximately 150 kDa). PNGase F reduces the molecular weight (MW) of natural NCBE in mouse brain or human NCBE expressed in oocytes to approximately the predicted MW of the unglycosylated protein. In oocytes, mutating any one of the three consensus N-glycosylation sites reduces glycosylation of the other two, and the triple mutant exhibits negligible functional expression.

Plasmalemmal vacuolar H+-ATPase is decreased in microvascular endothelial cells from a diabetic model.

Angiogenesis requires invasion of extracellular matrix (ECM) proteins by endothelial cells and occurs in hypoxic and acidic environments that are not conducive for cell growth and survival. We hypothesize that angiogenic cells must exhibit a unique system to regulate their cytosolic pH in order to cope with these harsh conditions. The plasmalemmal vacuolar type H+‐ATPase (pmV‐ATPase) is used by cells exhibiting an invasive phenotype. Because angiogenesis is impaired in diabetes, we hypothesized that pmV‐ATPase is decreased in microvascular endothelial cells from diabetic rats. The in vitro angiogenesis assays demonstrated that endothelial cells were unable to form capillary‐like structures in diabetes. The proton fluxes were slower in cells from diabetic than normal model, regardless of the presence or absence of Na+ and HCO  3− and were suppressed by V‐H+‐ATPase inhibitors. Immunocytochemical data revealed that pmV‐ATPases were inconspicuous at the plasma membrane of cells from diabetic whereas in normal cells were prominent. The pmV‐ATPase activity was lower in cells from diabetic than normal models. Inhibition of V‐H+‐ATPase suppresses invasion/migration of normal cells, but have minor effects in cells from diabetic models. These novel observations suggest that the angiogenic abnormalities in diabetes involve a decrease in pmV‐ATPase in microvascular endothelial cells.

Using Simulation in Interprofessional Education

Simulation-based training (SBT) is a powerful educational tool permitting the acquisition of surgical knowledge, skills, and attitudes at both the individual- and team-based level in a safe, nonthreatening learning environment at no risk to a patient. Interprofessional education (IPE), in which participants from 2 or more health or social care professions learn interactively, can help improve patient care through the promotion of efficient coordination, dissemination of advances in care across specialties and professions, and optimization of individual- and team-based function. Nonetheless, conducting SBT IPE sessions poses several tactical and strategic challenges that must be effectively overcome to reap IPEs benefits.

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

Cytosolic Ca2+ ([Ca2+]cyt) regulates several cellular 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: (1) Ca2+ homeostasis is altered in microvascular endothelial cells from diabetic animals due to the dysfunction of glycolysis coupling the activity of SERCA; (2) endosomal/lysosomal compartments expressing SERCA are involved in the dysfunction associated with diabetes.

Proton pumps, angiogenesis, and metastatic breast cancer

We have previously shown the relationship between metastatic potential and plasmalemmal V-H+-ATPase (pmV-ATPase) expression in tumor cells. This led us to hypothesize that pmV-ATPase activity is involved in invasion. Angiogenesis involves invasion of adjacent tissues by microvascular endothelial cells, thus we hypothesized that pmV-ATPases contribute to pHin regulation and invasion in microvascular endothelial cells.

Intracellular pH (pHin) and cytosolic calcium ([Ca2+]cyt) regulation via ATPases: studies in cell populations, single cells, and subcellular compartments

Changes in pHin and (Ca2+)cyt are important in the signal transduction mechanisms leading to many physiological responses including cell growth, motility, secretion/exocytosis, etc. The concentrations of these ions are regulated via primary and secondary ion transporting mechanisms. In diabetes, specific pH and Ca2+ regulatory mechanism might be altered. To study these ions, we employ fluorescence spectroscopy, and cell imagin spectroscopy/confocal microscopy. pH and Ca2+ indicators are loaded in the cytosol with acetoxymethyl ester forms of dyes, and in endosomal/lysosomal (E/L) compartments by overnight incubation of cells with dextran- conjugated ion fluorescent probes. We focus on specific pH and Ca2+ regulatory systems: plasmalemmal vacuolar- type H+-ATPases (pm V-ATPases) and sarcoplasmic/endoplasmic reticulum Ca2+-ATPases (SERCA). As experimental models, we employ vascular smooth muscle (VSM) and microvascular endothelial cells. We have chosen these cells because they are important in blood flow regulation and in angiogenesis. These processes are altered in diabetes. In many cell types, ion transport processes are dependent on metabolism of glucose for maximal activity. Our main findings are: (a) glycolysis coupling the activity of SERCA is required for cytosolic Ca2+ homeostasis in both VSM and microvascular endothelial cells; (b) E/L compartments are important for pH and Ca2+ regulation via H+-ATPases and SERCA, respectively; and (c) pm-V- ATPases are important for pHin regulation in microvascular endothelial cells.