Disclosure: H.K. Ghayee: Other; Self; Royalties form the University of Texas Southwestern Medical... more Disclosure: H.K. Ghayee: Other; Self; Royalties form the University of Texas Southwestern Medical Center. N. Bala: None. Y. Xu: None. S.G. Tevosian: None. M. Kozuch: None. N.D. Denslow: None. A.A. Alli: None. Introduction: Pheochromocytoma and paraganglioma (PPGL) patients with mutations in the succinate dehydrogenase B subunit (SDHB) frequently develop metastatic lesions and have a poor prognosis. The stearoyl-CoA desaturase 1 (SCD1) protein plays an important role in lipid metabolism. SCD1 inhibitors have shown great potential as new treatments for various diseases including cancer. Hypothesis: siRNA mediated knockdown of SCD1 compared to non-targeting siRNA would result in differences in the concentrations of saturated fatty acids and monounsaturated fatty acids in SDHB KD cells compared to hPheo1 wild-type (WT) progenitor cells. Methods: Fatty acid concentrations were assessed by fatty acid methyl ester (FAME) analysis. SCD1 expression was assessed by quantitative polymerase cha...
Pheochromocytomas (PCC) and paragangliomas (PGL) are rare neuroendocrine tumors with limited cura... more Pheochromocytomas (PCC) and paragangliomas (PGL) are rare neuroendocrine tumors with limited curative treatment options outside of surgical resection. Patients with mutations in succinate dehydrogenase subunit B (SDHB) are at an increased risk of malignant and aggressive disease. As cation channels are associated with tumorigenesis, we studied the expression and activity of cation channels from the Degenerin superfamily in a progenitor cell line derived from a human PCC. hPheo1 wild-type (WT) and SDHB knockdown (KD) cells were studied to investigate whether epithelial sodium channels (ENaC) and acid-sensing ion channels (ASIC) are regulated by the activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). First, we performed targeted metabolomic studies and quantified changes in glycolysis pathway intermediates and citric acid cycle intermediates using hPheo1 WT cells and SDHB KD cells. Next, we performed protein biochemistry and electrophysiology studies to characterize the protein expression and activity, respectively, of these ion channels. Our western blot experiments show both ENaC alpha and ASIC1/2 are expressed in both hPheo1 WT and SDHB KD cells, with lower levels of a cleaved 60 kDa form of ENaC in SDHB KD cells. Single-channel patch clamp studies corroborate these results and further indicate channel activity is decreased in SDHB KD cells. Additional experiments showed a more significant decreased membrane potential in SDHB KD cells, which were sensitive to amiloride compared to WT cells. We provide evidence for the differential expression and activity of ENaC and ASIC hybrid channels in hPheo1 WT and SDHB KD cells, providing an important area of investigation in understanding SDHB-related disease.
The expression of the myristoylated alanine-rich C-kinase substrate (MARCKS) family of proteins i... more The expression of the myristoylated alanine-rich C-kinase substrate (MARCKS) family of proteins in the kidneys plays an important role in the regulation of the renal epithelial sodium channel (ENaC) and hence overall blood pressure regulation. The function of MARCKS is regulated by post-translational modifications including myristoylation, phosphorylation, and proteolysis. Proteases known to cleave both ENaC and MARCKS have been shown to contribute to the development of high blood pressure, or hypertension. Here, we investigated protein expression and proteolysis of MARCKS, protein expression of multiple protein kinase C (PKC) isoforms, and protein expression and activity of several different proteases in the kidneys of diabetic db/db mice compared to wild-type littermate mice. In addition, MARCKS protein expression was assessed in cultured mouse cortical collecting duct (mpkCCD) cells treated with normal glucose and high glucose concentrations. Western blot and densitometric analys...
Cathpesin B is a multi-functional protease that plays numerous roles in physiology and pathophysi... more Cathpesin B is a multi-functional protease that plays numerous roles in physiology and pathophysiology. We hypothesized that actin cytoskeleton proteins that are substrates of cathepsin B, various lipids, and kinases that are regulated by lipids would be down-regulated in the kidney of cathepsin B knockout mice. Here, we show by Western blot and densitometric analysis that the expression and proteolysis of the actin cytoskeleton proteins myristoylated alanine-rich C-kinase substrate (MARCKS) and spectrin are significantly reduced in kidney cortex membrane fractions of cathepsin B knockout mice compared to C57B6 wild-type control mice. Lipidomic results show that specific lipids are increased while other lipids, including lysophosphatidylcholine (LPC) species LPC (16:0), LPC (18:0), LPC (18:1), and LPC (18:2), are significantly decreased in membrane fractions of the kidney cortex from Cathepsin B null mice. Protein Kinase C (PKC) activity is significantly lower in the kidney cortex o...
Hypertension may develop before or after the onset of diabetes and it is known to increase the ri... more Hypertension may develop before or after the onset of diabetes and it is known to increase the risk of developing diabetic nephropathy. Alpha-1 antitrypsin (AAT) is a multi-functional protein with beneficial effects in various diseases but its role in reducing blood pressure in the diabetic kidney has not been thoroughly studied. Like blood pressure, epithelial sodium channels (ENaC) and its adaptor protein myristoylated alanine-rich C-kinase substrate (MARCKS) are regulated by circadian rhythms. Our hypothesis is that administration of human AAT (hAAT) reduces blood pressure in hypertensive diabetic mice by attenuating membrane expression of ENaC and its association with the actin cytoskeleton. First, we show hAAT administration results in reduced blood pressure in diabetic db/db mice compared to vehicle treatment in both the inactive and active cycles. Western blotting and immunohistochemistry analyses showed a reduction of ENaC and the actin cytoskeleton protein, MARCKS in the ki...
The C-type natriuretic peptide receptor (NPRC) is expressed in many cell types and binds all natr... more The C-type natriuretic peptide receptor (NPRC) is expressed in many cell types and binds all natriuretic peptides with high affinity. Ligand binding results in the activation or inhibition of various intracellular signaling pathways. Although NPRC ligand binding has been shown to regulate various ion channels, the regulation of endothelial sodium channel (EnNaC) activity by NPRC activation has not been studied. The objective of this study was to investigate mechanisms of EnNaC regulation associated with NPRC activation in human aortic endothelial cells (hAoEC). EnNaC protein expression and activity was attenuated after treating hAoEC with the NPRC agonist cANF compared to vehicle, as demonstrated by Western blotting and patch clamping studies, respectively. NPRC knockdown studies using siRNA’s corroborated the specificity of EnNaC regulation by NPRC activation mediated by ligand binding. The concentration of multiple diacylglycerols (DAG) and the activity of protein kinase C (PKC) w...
Extracellular vesicles (EVs) are nanosized vesicles that carry bioactive cargo from one cell to a... more Extracellular vesicles (EVs) are nanosized vesicles that carry bioactive cargo from one cell to another and play an important role in intracellular signaling, cell differentiation, and intercellular communication. We investigated and found that packaged cargo of circulating EVs from SDHB mutated pheochromocytoma/paraganglioma patients increase hypoxia-inducible factor (HIF) signaling in a cell model system compared to EVs from control subjects. Keywords: Adrenal Tumors, Endocrine Neoplasia, Neuroendocrinology, succinate dehydrogenase Presentation: Saturday, June 11, 2022 1:00 p.m. - 2:00 p.m.
Pheochromocytomas (PCC) and paragangliomas (PGL) are rare neuroendocrine tumors with limited cura... more Pheochromocytomas (PCC) and paragangliomas (PGL) are rare neuroendocrine tumors with limited curative treatment options outside of surgical resection. Patients with mutations in succinate dehydrogenase subunit B (SDHB) are at an increased risk of malignant and aggressive disease. As cation channels are associated with tumorigenesis, we studied the expression and activity of cation channels from the Degenerin superfamily in a progenitor cell line derived from a human PCC. hPheo1 wild-type (WT) and SDHB knockdown (KD) cells were studied to investigate whether epithelial sodium channels (ENaC) and acid-sensing ion channels (ASIC) are regulated by the activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). First, we performed targeted metabolomic studies and quantified changes in glycolysis pathway intermediates and citric acid cycle intermediates using hPheo1 WT cells and SDHB KD cells. Next, we performed protein biochemistry and electrophysiology studies to characterize the prot...
Disclosure: H.K. Ghayee: Other; Self; Royalties form the University of Texas Southwestern Medical... more Disclosure: H.K. Ghayee: Other; Self; Royalties form the University of Texas Southwestern Medical Center. N. Bala: None. Y. Xu: None. S.G. Tevosian: None. M. Kozuch: None. N.D. Denslow: None. A.A. Alli: None. Introduction: Pheochromocytoma and paraganglioma (PPGL) patients with mutations in the succinate dehydrogenase B subunit (SDHB) frequently develop metastatic lesions and have a poor prognosis. The stearoyl-CoA desaturase 1 (SCD1) protein plays an important role in lipid metabolism. SCD1 inhibitors have shown great potential as new treatments for various diseases including cancer. Hypothesis: siRNA mediated knockdown of SCD1 compared to non-targeting siRNA would result in differences in the concentrations of saturated fatty acids and monounsaturated fatty acids in SDHB KD cells compared to hPheo1 wild-type (WT) progenitor cells. Methods: Fatty acid concentrations were assessed by fatty acid methyl ester (FAME) analysis. SCD1 expression was assessed by quantitative polymerase cha...
Pheochromocytomas (PCC) and paragangliomas (PGL) are rare neuroendocrine tumors with limited cura... more Pheochromocytomas (PCC) and paragangliomas (PGL) are rare neuroendocrine tumors with limited curative treatment options outside of surgical resection. Patients with mutations in succinate dehydrogenase subunit B (SDHB) are at an increased risk of malignant and aggressive disease. As cation channels are associated with tumorigenesis, we studied the expression and activity of cation channels from the Degenerin superfamily in a progenitor cell line derived from a human PCC. hPheo1 wild-type (WT) and SDHB knockdown (KD) cells were studied to investigate whether epithelial sodium channels (ENaC) and acid-sensing ion channels (ASIC) are regulated by the activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). First, we performed targeted metabolomic studies and quantified changes in glycolysis pathway intermediates and citric acid cycle intermediates using hPheo1 WT cells and SDHB KD cells. Next, we performed protein biochemistry and electrophysiology studies to characterize the protein expression and activity, respectively, of these ion channels. Our western blot experiments show both ENaC alpha and ASIC1/2 are expressed in both hPheo1 WT and SDHB KD cells, with lower levels of a cleaved 60 kDa form of ENaC in SDHB KD cells. Single-channel patch clamp studies corroborate these results and further indicate channel activity is decreased in SDHB KD cells. Additional experiments showed a more significant decreased membrane potential in SDHB KD cells, which were sensitive to amiloride compared to WT cells. We provide evidence for the differential expression and activity of ENaC and ASIC hybrid channels in hPheo1 WT and SDHB KD cells, providing an important area of investigation in understanding SDHB-related disease.
The expression of the myristoylated alanine-rich C-kinase substrate (MARCKS) family of proteins i... more The expression of the myristoylated alanine-rich C-kinase substrate (MARCKS) family of proteins in the kidneys plays an important role in the regulation of the renal epithelial sodium channel (ENaC) and hence overall blood pressure regulation. The function of MARCKS is regulated by post-translational modifications including myristoylation, phosphorylation, and proteolysis. Proteases known to cleave both ENaC and MARCKS have been shown to contribute to the development of high blood pressure, or hypertension. Here, we investigated protein expression and proteolysis of MARCKS, protein expression of multiple protein kinase C (PKC) isoforms, and protein expression and activity of several different proteases in the kidneys of diabetic db/db mice compared to wild-type littermate mice. In addition, MARCKS protein expression was assessed in cultured mouse cortical collecting duct (mpkCCD) cells treated with normal glucose and high glucose concentrations. Western blot and densitometric analys...
Cathpesin B is a multi-functional protease that plays numerous roles in physiology and pathophysi... more Cathpesin B is a multi-functional protease that plays numerous roles in physiology and pathophysiology. We hypothesized that actin cytoskeleton proteins that are substrates of cathepsin B, various lipids, and kinases that are regulated by lipids would be down-regulated in the kidney of cathepsin B knockout mice. Here, we show by Western blot and densitometric analysis that the expression and proteolysis of the actin cytoskeleton proteins myristoylated alanine-rich C-kinase substrate (MARCKS) and spectrin are significantly reduced in kidney cortex membrane fractions of cathepsin B knockout mice compared to C57B6 wild-type control mice. Lipidomic results show that specific lipids are increased while other lipids, including lysophosphatidylcholine (LPC) species LPC (16:0), LPC (18:0), LPC (18:1), and LPC (18:2), are significantly decreased in membrane fractions of the kidney cortex from Cathepsin B null mice. Protein Kinase C (PKC) activity is significantly lower in the kidney cortex o...
Hypertension may develop before or after the onset of diabetes and it is known to increase the ri... more Hypertension may develop before or after the onset of diabetes and it is known to increase the risk of developing diabetic nephropathy. Alpha-1 antitrypsin (AAT) is a multi-functional protein with beneficial effects in various diseases but its role in reducing blood pressure in the diabetic kidney has not been thoroughly studied. Like blood pressure, epithelial sodium channels (ENaC) and its adaptor protein myristoylated alanine-rich C-kinase substrate (MARCKS) are regulated by circadian rhythms. Our hypothesis is that administration of human AAT (hAAT) reduces blood pressure in hypertensive diabetic mice by attenuating membrane expression of ENaC and its association with the actin cytoskeleton. First, we show hAAT administration results in reduced blood pressure in diabetic db/db mice compared to vehicle treatment in both the inactive and active cycles. Western blotting and immunohistochemistry analyses showed a reduction of ENaC and the actin cytoskeleton protein, MARCKS in the ki...
The C-type natriuretic peptide receptor (NPRC) is expressed in many cell types and binds all natr... more The C-type natriuretic peptide receptor (NPRC) is expressed in many cell types and binds all natriuretic peptides with high affinity. Ligand binding results in the activation or inhibition of various intracellular signaling pathways. Although NPRC ligand binding has been shown to regulate various ion channels, the regulation of endothelial sodium channel (EnNaC) activity by NPRC activation has not been studied. The objective of this study was to investigate mechanisms of EnNaC regulation associated with NPRC activation in human aortic endothelial cells (hAoEC). EnNaC protein expression and activity was attenuated after treating hAoEC with the NPRC agonist cANF compared to vehicle, as demonstrated by Western blotting and patch clamping studies, respectively. NPRC knockdown studies using siRNA’s corroborated the specificity of EnNaC regulation by NPRC activation mediated by ligand binding. The concentration of multiple diacylglycerols (DAG) and the activity of protein kinase C (PKC) w...
Extracellular vesicles (EVs) are nanosized vesicles that carry bioactive cargo from one cell to a... more Extracellular vesicles (EVs) are nanosized vesicles that carry bioactive cargo from one cell to another and play an important role in intracellular signaling, cell differentiation, and intercellular communication. We investigated and found that packaged cargo of circulating EVs from SDHB mutated pheochromocytoma/paraganglioma patients increase hypoxia-inducible factor (HIF) signaling in a cell model system compared to EVs from control subjects. Keywords: Adrenal Tumors, Endocrine Neoplasia, Neuroendocrinology, succinate dehydrogenase Presentation: Saturday, June 11, 2022 1:00 p.m. - 2:00 p.m.
Pheochromocytomas (PCC) and paragangliomas (PGL) are rare neuroendocrine tumors with limited cura... more Pheochromocytomas (PCC) and paragangliomas (PGL) are rare neuroendocrine tumors with limited curative treatment options outside of surgical resection. Patients with mutations in succinate dehydrogenase subunit B (SDHB) are at an increased risk of malignant and aggressive disease. As cation channels are associated with tumorigenesis, we studied the expression and activity of cation channels from the Degenerin superfamily in a progenitor cell line derived from a human PCC. hPheo1 wild-type (WT) and SDHB knockdown (KD) cells were studied to investigate whether epithelial sodium channels (ENaC) and acid-sensing ion channels (ASIC) are regulated by the activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). First, we performed targeted metabolomic studies and quantified changes in glycolysis pathway intermediates and citric acid cycle intermediates using hPheo1 WT cells and SDHB KD cells. Next, we performed protein biochemistry and electrophysiology studies to characterize the prot...
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