IU Indianapolis ScholarWorks :: Browsing by Author "Blazer-Yost, Bonnie L."

Browsing by Author "Blazer-Yost, Bonnie L."

Now showing 1 - 10 of 26

Activation of TRPV4 stimulates transepithelial ion flux in a porcine choroid plexus cell line
(American Journal of Physiology, 2018-09-01) Preston, Daniel; Simpson, Stefanie; Halm, Dan; Hochstetler, Alexandra; Schwerk, Christian; Schroten, Horst; Blazer-Yost, Bonnie L.; Biology, School of Science
The choroid plexus (CP) epithelium plays a major role in the production of cerebrospinal fluid (CSF). A polarized cell line, the porcine CP-Riems (PCP-R) line, which exhibits many of the characteristics of the native epithelium, was used to study the effect of activation of the transient receptor potential vanilloid 4 (TRPV4) cation channel found in the PCP-R cells as well as in the native epithelium. Ussing-style electrophysiological experiments showed that activation of TRPV4 with a specific agonist, GSK1016790A, resulted in an immediate increase in both transepithelial ion flux and conductance. These changes were inhibited by either of two distinct antagonists, HC067047 or RN1734. The change in conductance was reversible and did not involve disruption of epithelial junctional complexes. Activation of TRPV4 results in Ca2+ influx, therefore, we examined whether the electrophysiological changes were the result of secondary activation of Ca2+-sensitive channels. PCP-R cells contain two Ca2+-activated K+ channels, the small conductance 2 (SK2) and the intermediate conductance (IK) channels. Based on inhibitor studies, the former is not involved in the TRPV4-mediated electrophysiological changes whereas one of the three isoforms of the IK channel (KCNN4c) may play a role in the apical secretion of K+. Blocking the activity of this IK isoform with TRAM34 inhibited the TRPV4-mediated change in net transepithelial ion flux and the increased conductance. These studies implicate TRPV4 as a hub protein in the control of CSF production through stimulation by multiple effectors resulting in transepithelial ion and subsequent water movement.
Bioinformatic Analysis of Differential Protein Expression in Calu-3 Cells Exposed to Carbon Nanotubes
(MDPI, 2013-10-24) Li, Pin; Lai, Xianyin; Witzmann, Frank A.; Blazer-Yost, Bonnie L.; Biology, School of Science
Carbon nanomaterials are widely produced and used in industry, medicine and scientific research. To examine the impact of exposure to nanoparticles on human health, the human airway epithelial cell line, Calu-3, was used to evaluate changes in the cellular proteome that could account for alterations in cellular function of airway epithelia after 24 hexposure to 10 μg/mL and 100 ng/mLof two common carbon nanoparticles, single- and multi-wall carbon nanotubes (SWCNT, MWCNT). After exposure to the nanoparticles, label-free quantitative mass spectrometry (LFQMS) was used to study the differential protein expression. Ingenuity Pathway Analysis (IPA) was used to conduct a bioinformaticanalysis of proteins identified in LFQMS. Interestingly, after exposure to ahigh concentration (10 μg/mL; 0.4 μg/cm2) of MWCNT or SWCNT, only 8 and 13 proteins, respectively, exhibited changes in abundance. In contrast, the abundance of hundreds of proteins was altered in response to a low concentration (100 ng/mL; 4 ng/cm2) of either CNT. Of the 281 and 282 proteins that were significantly altered in response to MWCNT or SWCNT respectively, 231 proteins were the same. Bioinformatic analyses found that the proteins in common to both nanotubes occurred within the cellular functions of cell death and survival, cell-to-cell signaling and interaction, cellular assembly and organization, cellular growth and proliferation, infectious disease, molecular transport and protein synthesis. The majority of the protein changes represent a decrease in amount suggesting a general stress response to protect cells. The STRING database was used to analyze the various functional protein networks. Interestingly, some proteins like cadherin 1 (CDH1), signal transducer and activator of transcription 1 (STAT1), junction plakoglobin (JUP), and apoptosis-associated speck-like protein containing a CARD (PYCARD), appear in several functional categories and tend to be in the center of the networks. This central positioning suggests they may play important roles in multiple cellular functions and activities that are altered in response to carbon nanotube exposure.
Bioinformatic Analysis of Proteomic Changes That Occur in an Airway Epithelial Cell Line in Response to Exposure to Physiologically Relevant Concentrations of Carbon Nanotubes
(Office of the Vice Chancellor for Research, 2013-04-05) Li, Pin; Lewis, Shanta; Witzmann, Frank; Blazer-Yost, Bonnie L.
Carbon nanomaterials are widely produced and used in industry, medicine and scientific research. To examine the impact of acute exposure to nanoparticles on human health, the human airway epithelial cell line, Calu-3, was used to evaluate potential alterations in cellular function of airway epithelia after 24 hours exposure to different concentrations of two common carbon nanoparticles, single- and multi-wall carbon nanotubes (SWCNT, MWCNT). After exposure to the nanoparticles, label-free quantitative mass spectrometry (LFQMS) was used to study the differential protein expression in Calu-3 cells. Ingenuity Pathway Analysis (IPA) was used to conduct a bioinformatic analysis of proteins identified in LFQMS. Changes in protein abundance generated in response to 100 ng/ml exposure of both MWCNT and SWCNT suggest that cell functions of cell death and survival, cell-to-cell signaling and interaction, cellular assembly and organization, cellular growth and proliferation, infectious disease, molecular transport and protein synthesis are predicted to be effected. The majority of the protein changes represent a decrease in amount suggesting a shut down of metabolism to protect cells. The STRING database was used to analyze the protein networks in different functions. Interestingly some proteins like cadherin 1 (CDH1), signal transducer and activator of transcription 1 (STAT1), junction plakoglobin (JUP), apoptosis-associated speck-like protein containing a CARD (PYCARD), appear in several functions and tend to be in the center of the networks, which suggest they may play important roles in the cell function and activity.
Consideration of Kinase Inhibitors for the Treatment of Hydrocephalus
(MDPI, 2023-04-03) Blazer-Yost, Bonnie L.; Biology, School of Science
Hydrocephalus is a devastating condition characterized by excess cerebrospinal fluid (CSF) in the brain. Currently, the only effective treatment is surgical intervention, usually involving shunt placement, a procedure prone to malfunction, blockage, and infection that requires additional, often repetitive, surgeries. There are no long-term pharmaceutical treatments for hydrocephalus. To initiate an intelligent drug design, it is necessary to understand the biochemical changes underlying the pathology of this chronic condition. One potential commonality in the various forms of hydrocephalus is an imbalance in fluid-electrolyte homeostasis. The choroid plexus, a complex tissue found in the brain ventricles, is one of the most secretory tissues in the body, producing approximately 500 mL of CSF per day in an adult human. In this manuscript, two key transport proteins of the choroid plexus epithelial cells, transient receptor potential vanilloid 4 and sodium, potassium, 2 chloride co-transporter 1, will be considered. Both appear to play key roles in CSF production, and their inhibition or genetic manipulation has been shown to affect CSF volume. As with most transporters, these proteins are regulated by kinases. Therefore, specific kinase inhibitors are also potential targets for the development of pharmaceuticals to treat hydrocephalus.
Effect of carbon nanotubes on transepithelial resistance in barrier epithelial cells
(Office of the Vice Chancellor for Research, 2012-04-13) Lewis, Shanta; Blazer-Yost, Bonnie L.; Petrache, Horia I.; Roark, Torri
The burgeoning of the nanotechnology industry has revolutionized engineering, medicine and the fashion industry amongst many other technologies. The arrays of products that are synthesized from nanomaterials include high definition TV and computer screens, artificial organs, as well as antibacterial food containers. With these novel applications there is a paralleled concern of the negative implications of nano-material contamination in our environment and food chains. We are interested in carbon nanotubes because they are the most abundantly occurring nanoparticles that are found in the workplace. We have recently conducted studies in barrier epithelial cells to show that long single-wall and multi-wall carbon nanotubes (CNTs) caused a decrease in the transepithelial electrical resistance, a measure of the barrier function ,of renal principal cells at very low concentrations (0.4 ng/cm2- 4 g/cm2). These results suggested that nanoparticles may also cause an effect on other barrier epithelial cells such as those lining the human digestive and respiratory tracks. After 48 hours of CNT exposure, to airway and colon cell lines, Calu-3 and t84 respectively, the calculated resistances were approximately half of the control monolayers’, indicating that the barrier function of the tissue had been compromised, while the cellular monolayer remained intact. We sought to determine the mechanism of action of the nanoparticles, by investigating the interaction of CNTs with model lipid membranes using a bilayer clamp amplifier. Measurements showed that the presence of nanoparticles caused transient disruptions in lipid membranes made of phosphatidylcholine lipids. Nanotubes also caused transient interruptions in the current allowed by the ion channel reporter (gramicidin A). These results began to elucidate the mode of action of the particles and indicated that it is important to develop a complete understanding of how nanoparticles interact with cells if we are to safeguard against changes that these materials will cause in vivo.
The Effectors of the Transient Receptor Vanilloid Potential Type 4 in the Choroid Plexus
(Office of the Vice Chancellor for Research, 2016-04-08) Otun, Ayodamola; Ahmed, Shehab; Joyner, Michael; Simpson, Stephanie; Blazer-Yost, Bonnie L.
Hydrocephalus is a deadly disease that affects 1-2 births in 1000. When severe, this disease can result in irreversible brain damage. There are no drugs to treat hydrocephalus and the standard therapy is to surgically implant shunts to drain the excess cerebrospinal fluid (CSF) into other parts of the body. However this approach often results in a less than optimal outcomes. Shunt failures due to blockage, infection, and other causes are as high as 50% even in major medical centers which specialize in these procedures. Our laboratory is using a Meckel-Gruber syndrome rat model to study the development of severe hydrocephalus. Immunohistochemistry has been used to show the overexpression of Transient Receptor Vanilloid Potential Type 4, TRPV4 on the choroid plexus epithelial membrane. Because the choroid plexus is responsible for the majority of the cerebrospinal fluid that contributes to the progression of hydrocephalus, the TRPV4 calcium channel is a potential target that could contribute to the disease development. The endogenous activators of this channel in the choroid plexus are yet to be determined. Consequently, the current study is using a cultured choroid plexus cell line to identify endogenous activators of this channel. Potential activators include homovanillic acid (HVA), lysophosphatidic acid, and arachidonic acid. In addition, the introduction of novel compounds that act as sensitizers of the channel led to a set of experiments that were conducted to confirm the existence and identification of the sensitizers such as inflammatory cytokines. The effect of these compounds on the activation of the TRPV4 channel are being investigated using electrophysiological techniques in a porcine choroid plexus cell line with the characteristics of the in vivo choroid plexus. This cell line exhibits a robust increase in ion transport in response to a TRPV4 agonist. . The determination of the endogenous TRPV4 activators and sensitizers will provide important information in the development of a drug that can be used to treat hydrocephalus with minimal side effects by altering the activity of TRPV4.
Effects of Carbon Nanotubes in Barrier Epithelial Cells via Effects on Lipid Bilayers
(Office of the Vice Chancellor for Research, 2013-04-05) Lewis, Shanta; Petrache, Horia I.; Blazer-Yost, Bonnie L.; Witzmann, Frank
Carbon nanotubes (CNTs) are one of many nanoparticles (NP) which are being developed as part of the burgeoning nanotechnology. The tubes have similar physical properties to known toxic materials, such as asbestos; yet there is a lack of evidence showing that they may be hazardous to humans, specifically to our barrier epithelial cells. We measured the effects of CNTs on human airway epithelial cells (Calu-3 cell line) using electrophysiology. This is a technique which measures transepithelial electrical resistance (TEER), a measure of monolayer integrity; and short circuit current (SCC) a measure of net ion transport across the cell. Exposed cells showed significant decreases in TEER when incubated for 48 hours with physiologically relevant concentrations of 4μg/cm2 - 0.4ng/cm2 of multi-wall (MW) and 4μg/cm2 - 0.04ng/cm2 single-wall (SW) CNT. TEER is a measure of barrier function which is important in cells that maintain separate compartments in the body. The impaired barrier function, despite sustained cell viability, led us to investigate the mechanism by which the CNT were interacting with the cell when applied topically. Model lipid membranes connected to an ion channel amplifier, Planar Bilayer Workstation (BLM), were used. Membranes were formed using the neutral diphytanoylphosphatidylcholine (DPhPC) and negatively charged diphytanoyl phosphatidylserine (DPhPS) lipids. CNTs caused random, transient currents ranging from 0pA to 6479pA to traverse the membrane. In the presence of Gramicidin A, an ion channel reporter protein, the tubes induced increased gramicidin channel formation in the membrane to saturation level and then membrane lysis. This CNT- lipid interaction indicated that short MWCNTs permits unregulated ion movement across the lipid membrane. Disruption in the selective permeability of the plasmalemma may impact the tissue’s barrier function.
Effects of Lysophosphatidic Acid (LPA) and Antidiuretic Hormone (ADH) on Cl- Secretory Responses in Polycystic Kidney Disease (PKD)
(Office of the Vice Chancellor for Research, 2013-04-05) Martinez, Gabriel M.; Flaig, Stephanie; Blazer-Yost, Bonnie L.
Polycystic kidney disease (PKD) is a genetic disease that causes the formation of fluid-filled cysts in the kidney and other organs such as the liver and pancreas. Kidney function is seemingly unaltered despite substantial cyst development over the first four to six decades of life, but then the decline in renal function is precipitous often leading to complete renal failure in 5 years. Antidiuretic hormone (ADH) causes an increase in Cl- secretion into the cyst lumen, and one of the drugs in human clinical trials for treatment of PKD is an ADH receptor antagonist. The hormone works by stimulating cAMP production, which leads to the Cl- secretion. Interestingly, we have found that cyst fluid from human patients also causes a secretory Cl- flux that can lead to the growth of the remaining intact cysts. The active component of the cyst fluid is LPA, a phospholipid that acts as an extracellular signaling molecule. This secretion is important in late stage disease when large cysts are likely to leak or burst contributing to the rapid decline in renal function. Electrophysiological techniques were implemented to compare the ion fluxes stimulated by ADH and LPA. In the mpkCCDc14 (mouse principal cells of the cortical collecting duct clone 4) cell line we found that the Cl- secretory pathways stimulated by the two factors are separate and independent. Further indication of this separation is our finding that LPA stimulation does not increase cAMP levels. Therefore we have identified an additional target for potential pharmaceutical intervention in the treatment of PKD.
Epinephrine stimulation of anion secretion in the Calu-3 serous cell model
(American Physiological Society (APS), 2014-05-15) Banga, Amiraj; Flaig, Stephanie; Lewis, Shanta; Winfree, Seth; Blazer-Yost, Bonnie L.; Department of Biology, School of Science
Calu-3 is a well-differentiated human bronchial cell line with the characteristics of the serous cells of airway submucosal glands. The submucosal glands play a major role in mucociliary clearance because they secrete electrolytes that facilitate airway hydration. Given the significance of both long- and short-term β-adrenergic receptor agonists in the treatment of respiratory diseases, it is important to determine the role of these receptors and their ligands in normal physiological function. The present studies were designed to characterize the effect of epinephrine, the naturally occurring β-adrenergic receptor agonist, on electrolyte transport of the airway serous cells. Interestingly, epinephrine stimulated two anion secretory channels, the cystic fibrosis transmembrane conductance regulator and a Ca2+-activated Cl− channel, with the characteristics of transmembrane protein 16A, thereby potentially altering mucociliary clearance via multiple channels. Consistent with the dual channel activation, epinephrine treatment resulted in increases in both intracellular cAMP and Ca2+. Furthermore, the present results extend previous reports indicating that the two anion channels are functionally linked.
Excess HB-EGF, which promotes VEGF signaling, leads to hydrocephalus
(Nature Publishing Group, 2016-05-31) Shim, Joon W.; Sandlund, Johanna; Hameed, Mustafa Q.; Blazer-Yost, Bonnie L.; Zhou, Feng C.; Klagsbrun, Michael; Madsen, Joseph R.; Department of Anatomy & Cell Biology, IU School of Medicine
Heparin binding epidermal growth factor-like growth factor (HB-EGF) is an angiogenic factor mediating radial migration of the developing forebrain, while vascular endothelial growth factor (VEGF) is known to influence rostral migratory stream in rodents. Cell migratory defects have been identified in animal models of hydrocephalus; however, the relationship between HB-EGF and hydrocephalus is unclear. We show that mice overexpressing human HB-EGF with β-galactosidase reporter exhibit an elevated VEGF, localization of β-galactosidase outside the subventricular zone (SVZ), subarachnoid hemorrhage, and ventriculomegaly. In Wistar polycystic kidney rats with hydrocephalus, alteration of migratory trajectory is detected. Furthermore, VEGF infusions into the rats result in ventriculomegaly with an increase of SVZ neuroblast in rostral migratory stream, whereas VEGF ligand inhibition prevents it. Our results support the idea that excess HB-EGF leads to a significant elevation of VEGF and ventricular dilatation. These data suggest a potential pathophysiological mechanism that elevated HB-EGF can elicit VEGF induction and hydrocephalus.

Browsing by Author "Blazer-Yost, Bonnie L."

Results Per Page

Sort Options