Receptor Regulation of Volume-Sensitive Osmolyte Efflux from Neural Cells.

Cell volume regulation is a homeostatic imperative in the brain due to the restrictions of the skull. To counteract an osmolarity disturbance and restore normal cell volume, neural cells initiate volume regulatory mechanism by modifying the concentration of their intracellular inorganic and organic osmolytes. This dissertation illustrates the important ability of GPCRs to potentiate osmolyte efflux in response to hypo-osmotic stress. I discovered that the addition of sub-nanomolar concentrations of thrombin, mediated via the activation of PAR-1 receptor, can robustly enhance the release of the organic osmolyte taurine, through the volume sensitive organic osmolyte and anion channel (VSOAC) from neuro-tumor cells in response to hypo-osmotic stress. Biochemical and pharmacological studies demonstrated that both intracellular Ca2+ and PKC are required for receptor-mediated, but not basal (swelling-induced), taurine release. The results indicated that activation of PAR-1 receptors by thrombin lowers the threshold osmolarity or “set-point” for osmolyte efflux, thereby facilitating the ability of cells to respond to small reductions in osmolarity. Receptor activation (PAR-1 and mAChR) also enhanced the release of the inorganic osmolyte, Cl- (125I- used as a tracer). The kinetics and magnitude of 125I- release were greater than those of taurine. However, both osmolytes showed similar pharmacological profiles in response to VSOAC inhibitors. Although receptor stimulated-taurine efflux was inhibited by depletion of intracellular Ca2+ and/or inhibition of PKC, 125I- efflux was either unaffected or less dependent upon these two parameters. This differential regulation of the osmolyte release suggests either the presence of separate but pharmacologically similar, efflux channels or, receptor-specific activation of distinct signal transduction pathways that differentially contribute to the release of taurine and 125I-, both of which are released through a common membrane channel. I also demonstrated that cholesterol depletion with methyl-ß-cyclodextrin synergistically potentiated receptor-mediated taurine release. This stimulatory effect was not due to disruption of lipid rafts or changes in receptor signaling. Experiments with cholesterol analogs provided evidence that the potentiation by cholesterol depletion resulted from changes in the biophysical properties (fluidity) of the membrane. Together, this thesis provide new insight into mechanisms involved in the GPCR-regulation of osmolyte efflux following hypo-osmotic stress in neural cells.Ph.D.PharmacologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/57604/2/tcheema_1.pd

Cholesterol regulates volumesensitive osmolyte efflux from human SH-SY5Y neuroblastoma cells following receptor activation

ABSTRACT The ability of cholesterol to modulate receptor-mediated increases in the volume-dependent release of the organic osmolyte, taurine, has been examined. Depletion of cholesterol from SH-SY5Y neuroblastoma by preincubation of the cells with 5 mM methyl-␤-cyclodextrin (CD) for 10 min resulted in a 40 to 50% reduction in cholesterol and an enhancement of the ability of proteinase-activated receptor (PAR) 1, muscarinic cholinergic receptor (mAChR), and sphingosine 1-phosphate receptor to stimulate taurine efflux, when monitored under hypoosmotic conditions. Basal (swelling-induced) release of taurine was also enhanced by cholesterol depletion, but less markedly. Both basaland receptor-mediated increases in taurine efflux were mediated via a volume-sensitive organic osmolyte and anion channel in control and cholesterol-depleted cells. Studies with the PAR-1 and mAChR receptor subtypes indicated that the stimulatory effect of CD pretreatment could be reversed by incubation of the cells with either CD/cholesterol or CD/5-cholesten-3␣-ol donor complexes and that cholesterol depletion increased agonist efficacy, but not potency. The ability of cholesterol depletion to promote the PAR-1 receptor-mediated stimulation of osmolyte release was most pronounced under conditions of isotonicity or mild hypotonicity. In contrast to CD pretreatment, preincubation of the cells with cholesterol oxidase, a condition under which lipid microdomains are also disrupted, had no effect on either basal-or receptor-stimulated taurine efflux. Taken together, the results suggest that cholesterol regulates receptor-mediated osmolyte release via its effects on the biophysical properties of the plasma membrane, rather than its presence in lipid microdomains

Immunovirotherapy for the treatment of glioblastoma

We have recently described a new murine model of glioblastoma, generated by the implantation of syngeneic glioblastoma stem cells into immunocompetent mice, that recapitulates the salient histopathological and immunological features of the human disease. We employed this model to demonstrate the multifaceted activity of an oncolytic herpes simplex virus genetically modified to express interleukin-12, G47∆-IL12

Combination of Oncolytic Herpes Simplex Viruses Armed with Angiostatin and IL-12 Enhances Antitumor Efficacy in Human Glioblastoma Models

Oncolytic herpes simplex virus (oHSV) can potentially spread throughout the tumor, reach isolated infiltrating cells, kill them, and deliver anticancer agents. However, the host responds to oHSV by inducing intratumoral infiltration of macrophages that can engulf the virus, limiting the potential of this therapeutic strategy. Hypervascularity is a pathognomonic feature of glioblastoma (GBM) and is a promising therapeutic target. Antiangiogenic treatments have multiple benefits, including the capacity to increase oHSV efficacy by suppressing macrophage extravasation and infiltration into the tumor. Angiostatin is an antiangiogenic polypeptide, and interleukin-12 (IL-12) is an immunostimulatory cytokine with strong antiangiogenic effects. Clinical use of each has been limited by delivery issues and systemic toxicity.We tested a combination treatment strategy using oHSVs expressing angiostatin (G47Δ-mAngio) and IL-12 (G47Δ-mIL12) in two orthotopic human GBMmodels. Intratumoral injection of G47Δ-mAngio and G47Δ-mIL12 in mice bearing intracranial U87 or tumors derived from glioblastoma stem cells significantly prolonged survival compared to each armed oHSV alone. This was associated with increased antiangiogenesis and virus spread and decreased macrophages. These data support the paradigm of using oHSV expressing different antiangiogenic agents and show for the first time that oHSVs expressing angiostatin and IL-12 can improve efficacy in human GBM models