Papers by Geoffrey Woodard
Current topics in developmental biology, 2005
American journal of physiology. Cell physiology, 2008
Ca+ release-activated Ca2+ (CRAC) channels are activated when free Ca2+ concentration in the intr... more Ca+ release-activated Ca2+ (CRAC) channels are activated when free Ca2+ concentration in the intracellular stores is substantially reduced and mediate sustained Ca2+ entry. Recent studies have identified Orai1 as a CRAC channel subunit. Here we demonstrate that passive Ca2+ store depletion using the inhibitor of the sarcoendoplasmic reticulum Ca2+-ATPase, thapsigargin (TG), enhances the surface expression of Orai1, a process that depends on rises in cytosolic free Ca2+ concentration, as demonstrated in cells loaded with dimethyl BAPTA, an intracellular Ca2+ chelator that prevented TG-evoked cytosolic free Ca2+ concentration elevation. Similar results were observed with a low concentration of carbachol. Cleavage of the soluble N-ethylmaleimide-sensitive-factor attachment protein receptor, synaptosomal-assiciated protein-25 (SNAP-25), with botulinum neurotoxin A impaired TG-induced increase in the surface expression of Orai1. In addition, SNAP-25 cleaving by botulinum neurotoxin A reduces the maintenance but not the initial stages of store-operated Ca2+ entry. In aggregate, these findings demonstrate that store depletion enhances Orai1 plasma membrane expression in an exocytotic manner that involves SNAP-25, a process that contributes to store-dependent Ca2+ entry.
International review of cell and molecular biology, 2008
Four major natriuretic peptides have been isolated: atrial natriuretic peptide (ANP), brain natri... more Four major natriuretic peptides have been isolated: atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), and Dendroaspis-type natriuretic peptide (DNP). Natriuretic peptides play an important role in the regulation of cardiovascular homeostasis maintaining blood pressure and extracellular fluid volume. The classical endocrine effects of natriuretic peptides to modulate fluid and electrolyte balance and vascular smooth muscle tone are complemented by autocrine and paracrine actions that include regulation of coronary blood flow and, therefore, myocardial perfusion; modulation of proliferative responses during myocardial and vascular remodeling; and cytoprotective anti-ischemic effects. The actions of natriuretic peptides are mediated by the specific binding of these peptides to three cell surface receptors: type A natriuretic peptide receptor (NPR-A), type B natriuretic peptide receptor (NPR-B), and type C natriuretic peptide receptor (NPR-C). NPR-A and NPR-B are guanylyl cyclase receptors that increase intracellular cGMP concentration and activate cGMP-dependent protein kinases. NPR-C has been presented as a clearance receptor and its activation also results in inhibition of adenylyl cyclase activity. The wide range of effects of natriuretic peptides might be the base for the development of new therapeutic strategies of great benefit in patients with cardiovascular problems including coronary artery disease or heart failure. This review summarizes current literature concerning natriuretic peptides, their receptors and their effects on fluid/electrolyte balance, and vascular and cardiac physiology and pathology, including primary hypertension and myocardial infarction. In addition, we will attempt to provide an update on important issues regarding natriuretic peptides in congestive heart failure.
Molecular and cellular biology, 2010
In model organisms, resistance to inhibitors of cholinesterase 8 (Ric-8), a G protein ␣ (G␣) subu... more In model organisms, resistance to inhibitors of cholinesterase 8 (Ric-8), a G protein ␣ (G␣) subunit guanine nucleotide exchange factor (GEF), functions to orient mitotic spindles during asymmetric cell divisions; however, whether Ric-8A has any role in mammalian cell division is unknown. We show here that Ric-8A and G␣ i function to orient the metaphase mitotic spindle of mammalian adherent cells. During mitosis, Ric-8A localized at the cell cortex, spindle poles, centromeres, central spindle, and midbody. Pertussis toxin proved to be a useful tool in these studies since it blocked the binding of Ric-8A to G␣ i , thus preventing its GEF activity for G␣ i . Linking Ric-8A signaling to mammalian cell division, treatment of cells with pertussis toxin, reduction of Ric-8A expression, or decreased G␣ i expression similarly affected metaphase cells. Each treatment impaired the localization of LGN (GSPM2), NuMA (microtubule binding nuclear mitotic apparatus protein), and dynein at the metaphase cell cortex and disturbed integrin-dependent mitotic spindle orientation. Live cell imaging of HeLa cells expressing green fluorescent protein-tubulin also revealed that reduced Ric-8A expression prolonged mitosis, caused occasional mitotic arrest, and decreased mitotic spindle movements. These data indicate that Ric-8A signaling leads to assembly of a cortical signaling complex that functions to orient the mitotic spindle.
International Review of Neurobiology, 2004
This chapter outlines the chemosensory G-Protein coupled receptor signaling in the brain. The bra... more This chapter outlines the chemosensory G-Protein coupled receptor signaling in the brain. The brain is highly representative of the full spectrum of G-protein coupled-receptor regulation throughout the body. The brain contains all the master regulatory feedback and control mechanisms in the body that manage vision, taste, odor sensing, fluid balance, reproduction, emotions, memory, pain, sleep cycle, and appetite, with G-protein coupled receptors and their respective G-proteins being the true arbiters of all the effectors involved in these human functions. The chapter outlines the chemosensing G-protein coupled receptors in the brain, such as (1) odorant and pheromone smell receptors, (2) pheromones and the vomeronasal system, and (3) umami and glutamate taste receptors. In an unexpected location, the taste receptors could transduce chemosensory stimuli for modulating respiration and digestion. The taste receptors are appearing complex with patterns of gene expression among the taste receptors that suggests other potential major or minor pathways of execution. Future work is needed to correlate the physiological response results with molecularly typed single-cell studies that look at native taste receptor cells from a homologous receptor perspective.
Methods in Enzymology, 2004
Heterodimeric complexes between individual members of the R7 subfamily of regulators of G-protein... more Heterodimeric complexes between individual members of the R7 subfamily of regulators of G-protein signaling proteins and the Gβ5 isoform of the heterotrimeric G-protein β subunit family are strongly expressed in the cell nucleus in neurons and brain, as well as in the cytoplasm and plasma membrane. Native and recombinant Gβ5 and⧸or R7 expression have been studied in model systems like rat pheochromocytoma PC12 cells where their nuclear localization can be studied by fluorescence microscopy and⧸or subcellular fractionation. Nucleic acid counterstains chosen for compatibility with the fluorescent tags on secondary antibodies can facilitate the assay of R7⧸Gβ5 nuclear localization by epifluorescence or confocal laser microscopy. Subcellular fractionation allows isolation of a purified nuclear fraction that can be probed for the presence of Gβ5 and⧸or R7 subunits by immunoblots or immunoprecipitation and compared to other subcellular fractions. While the function of nuclear R7⧸Gβ5 complexes is unknown, comparison with the properties of other RGS proteins that localize to the cell nucleus may suggest modes of action. Models are offered in which the reversible post-translational modification of R7⧸Gβ5 complexes regulates their nuclear localization and signaling activity, whether the target of such signaling activity is in the nucleus, at the plasma membrane, or both.
Oncogene, 2005
Parafibromin is the 531-amino-acid protein product encoded by HRPT2, a putative tumor suppressor ... more Parafibromin is the 531-amino-acid protein product encoded by HRPT2, a putative tumor suppressor gene recently implicated in the autosomal dominant hyperparathyroidism-jaw tumor familial cancer syndrome, sporadic parathyroid cancer, and a minority of families with isolated hyperparathyroidism. Parafibromin contains no identified functional domains but bears sequence homology to Cdc73p, a budding yeast protein component of the RNA polymerase II-associated Paf1 complex. This study addressed the expression and functional properties of human parafibromin. A survey of human and mouse tissues analysed with polyclonal antibodies to parafibromin showed specific immunoreactivity in adrenal and parathyroid glands, kidney, heart, and skeletal muscle. Subcellular fractionation and laser confocal microscopy of normal human parathyroid gland demonstrated expression of parafibromin in both the cytoplasmic and nuclear compartments. Parafibromin was expressed in four parathyroid adenomas but was absent from two parathyroid carcinomas. Transient overexpression of wild-type parafibromin, but not its Leu64Pro missense mutant implicated in parathyroid cancer and familial isolated hyperparathyroidism, inhibited cell proliferation, and blocked expression of cyclin D1, a key cell cycle regulator previously implicated in parathyroid neoplasia. These results demonstrate that human parafibromin is a nucleocytoplasmic protein with functions consistent with its postulated role as a tumor suppressor protein.
Biochimica et biophysica acta, Jan 21, 2015
STIM1 is a ubiquitous Ca(2+) sensor of the intracellular, agonist-sensitive, Ca(2+) stores that c... more STIM1 is a ubiquitous Ca(2+) sensor of the intracellular, agonist-sensitive, Ca(2+) stores that communicates the filling state of the Ca(2+) compartments to plasma membrane store-operated Ca(2+) (SOC) channels. STIM1 has been presented as a point of convergence between store-operated and voltage-operated Ca(2+) influx, both inducing activation of SOC channels while suppressing Cav1.2 channels. Here we report that Homer proteins play a relevant role in the communication between STIM1 and Cav1.2 channels. HEK-293 cells transiently expressing Cav1.2 channel subunits α1, β2 and α2δ-1 exhibited a significant Ca(2+) entry upon treatment with a high concentration of KCl. In Cav1.2-expressing cells, treatment with thapsigargin (TG), to induce passive discharge of the intracellular Ca(2+) stores, resulted in Ca(2+) influx that was significantly greater than in cells not expressing Cav1.2 channels, a difference that was abolished by nifedipine and diltiazem. Treatment with TG induces co-immunoprecipitation of Homer1 with STIM1 and the Cav1.2 α1 subunit. Impairment of Homer function by introduction of the synthetic PPKKFR peptide into cells, which emulates the proline-rich sequences of the PPXXF motif, or using siRNA Homer1, reduced the association of STIM1 and the Cav1.2 α1 subunit. These findings indicate that Homer is important for the association between both proteins. Finally, treatment with siRNA Homer1 or the PPKKFR peptide enhanced the nifedipine-sensitive component of TG response in Cav1.2-expressing cells. Altogether, these findings provide evidence for a new role of Homer1 supporting the regulation of Cav1.2 channels by STIM1.
PloS one, 2014
Here we show that distinct subpopulations of cells exist within traumatic human extremity wounds,... more Here we show that distinct subpopulations of cells exist within traumatic human extremity wounds, each having the ability to differentiate into multiple cells types in vitro. A crude cell suspension derived from traumatized muscle was positively sorted for CD29, CD31, CD34, CD56 or CD91. The cell suspension was also simultaneously negatively sorted for either CD45 or CD117 to exclude hematopoietic stem cells. These subpopulations varied in terms their total numbers and their abilities to grow, migrate, differentiate and secrete cytokines. While all five subpopulations demonstrated equal abilities to undergo osteogenesis, they were distinct in their ability to undergo adipogenesis and vascular endotheliogenesis. The most abundant subpopulations were CD29+ and CD34+, which overlapped significantly. The CD29+ and CD34+ cells had the greatest proliferative and migratory capacity while the CD56+ subpopulation produced the highest amounts of TGFß1 and TGFß2. When cultured under endothelial differentiation conditions the CD29+ and CD34+ cells expressed VE-cadherin, Tie2 and CD31, all markers of endothelial cells. These data indicate that while there are multiple cell types within traumatized muscle that have osteogenic differentiation capacity and may contribute to bone formation in post-traumatic heterotopic ossification (HO), the major contributory cell types are CD29+ and CD34+, which demonstrate endothelial progenitor cell characteristics.
International Review of Cytology-a Survey of Cell Biology, 2007
The transient receptor potential (TRP) family of ion channels is composed of more than 50 functio... more The transient receptor potential (TRP) family of ion channels is composed of more than 50 functionally versatile cation‐permeant ion channels expressed in most mammalian cell types. Considerable research has been brought to bear on the members of this family, especially with regard to their possible role as store‐operated calcium channels, although studies have provided evidence that TRP channels exhibit a number of regulatory and functional aspects. Endogenous and transiently expressed TRP channels can be activated by different mechanisms grouped into four main categories: receptor‐operated activation, store depletion‐mediated activation, ligand‐induced activation, and direct activation. This article reviews the biochemical characteristics of the different members of the TRP family and summarizes their involvement in a number of physiological events ranging from sensory transduction to development, which might help in understanding the relationship between TRP channel dysfunction and the development of several diseases.
Journal of Cellular and Molecular Medicine, 2007
• Introduction• Natriuretic peptides as a ‘biomarker’ of congestive heart failure and acute renal... more • Introduction• Natriuretic peptides as a ‘biomarker’ of congestive heart failure and acute renal failure/insufficiency• Renoprotective effect of natriuretic peptides after acute renal failure• Nesiritide – current benefits/hazards of drug usage• Introduction• Natriuretic peptides as a ‘biomarker’ of congestive heart failure and acute renal failure/insufficiency• Renoprotective effect of natriuretic peptides after acute renal failure• Nesiritide – current benefits/hazards of drug usageIntroductionNatriuretic peptides as a ‘biomarker’ of congestive heart failure and acute renal failure/insufficiencyRenoprotective effect of natriuretic peptides after acute renal failureNesiritide – current benefits/hazards of drug usageAbstractThe natriuretic peptides are a family of related hormones that play a crucial role in cardiovascular and renal homeostasis. They have recently emerged as potentially important clinical biomarkers in heart failure. Natriuretic peptides, particularly brain natriuretic peptide (BNP) and the inactive N-terminal fragment of BNP, NT-proBNP, that has an even greater half-life than BNP, are elevated in heart failure and therefore considered to be excellent predictors of disease outcome. Nesiritide, a recombinant human BNP, has been shown to provide symptomatic and haemodynamic improvement in acute decompensated heart failure, although recent reports have suggested an increased short-term risk of death with nesiritide use. This review article describes: the current use of BNP and its inactive precursor NT-proBNP in diagnosis, screening, prognosis and monitoring of therapy for congestive heart failure, the renoprotective actions of natriuretic peptides after renal failure and the controversy around the therapeutic use of the recombinant human BNP nesiritide.The natriuretic peptides are a family of related hormones that play a crucial role in cardiovascular and renal homeostasis. They have recently emerged as potentially important clinical biomarkers in heart failure. Natriuretic peptides, particularly brain natriuretic peptide (BNP) and the inactive N-terminal fragment of BNP, NT-proBNP, that has an even greater half-life than BNP, are elevated in heart failure and therefore considered to be excellent predictors of disease outcome. Nesiritide, a recombinant human BNP, has been shown to provide symptomatic and haemodynamic improvement in acute decompensated heart failure, although recent reports have suggested an increased short-term risk of death with nesiritide use. This review article describes: the current use of BNP and its inactive precursor NT-proBNP in diagnosis, screening, prognosis and monitoring of therapy for congestive heart failure, the renoprotective actions of natriuretic peptides after renal failure and the controversy around the therapeutic use of the recombinant human BNP nesiritide.
The Journal of biological chemistry, Jan 12, 2010
There is a body of evidence suggesting that Ca 2+ -handling proteins assemble into signaling comp... more There is a body of evidence suggesting that Ca 2+ -handling proteins assemble into signaling complexes required for a fine regulation of Ca 2+ signals, events that regulate a variety of critical cellular processes. Canonical transient receptor potential (TRPC) and Orai proteins have both been proposed to form Ca 2+permeable channels mediating Ca 2+ entry upon agonist stimulation. A number of studies have demonstrated that inositol 1,4,5-trisphosphate receptors (IP 3 Rs) interact with plasma membrane TRPC channels; however at present there is no evidence supporting the interaction between Orai proteins and IP 3 Rs. Here we report that treatment with thapsigargin or cellular agonists results in association of Orai1 with the types I and II IP 3 Rs. In addition, we have found that TRPC3, RACK1 (receptor for activated Ckinase-1) and STIM1 (stromal interaction molecule 1) interact with Orai1 upon stimulation with agonists. TRPC3 expression silencing prevented both the interaction of Orai1 with TRPC3 and, more interestingly, the association of Orai1 with the type I IP 3 R, but not with the type II IP 3 R, thus suggesting that TRPC3 selectively mediates the interaction between Orai1 and the type I IP 3 R. In addition, TRPC3 expression silencing attenuated ATPand CCh-stimulated interaction between RACK1 and the type I IP 3 R, as well as Ca 2+ release and entry. In conclusion, our results indicate that agonist stimulation results in the formation of an Orai1-STIM1-TRPC3-RACK1-type I IP 3 R complex, where TRPC3 plays a central role. This Ca 2+ signaling complex might be important for agonist induced both Ca 2+ release and entry.
Acta Physiologica, 2006
Aim: Natriuretic peptide receptor A (NPR-A) is the main physiological receptor for atrial natriu... more Aim: Natriuretic peptide receptor A (NPR-A) is the main physiological receptor for atrial natriuretic peptide (ANP). Maximal activation of NPR-A guanylyl cyclase (GC) requires ANP binding and ATP interaction with a putative cytoplasmic site. This study investigates the regulatory effect of ATP on GC-coupled NPR-A activity in Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR).Methods: Cyclic GMP production and competitive inhibition of [125I]ANP1−28 binding were performed in solubilized glomerular and papillary renal membranes.Results: Here, we report that incubation of renal glomerular and papillary membranes with ATP induced a concentration-dependent increase in basal and ANP1−28-stimulated GC activity that was significantly greater in SHR than in age-matched WKY. ATPγS was more effective than ATP and induced a greater stimulation of cGMP production in SHR than in WKY. In contrast, in solubilized membranes ATP exerted an inhibitory role on basal and ANP1−28-induced GC activity, suggesting that an accessory protein is required for ATP-induced GC activation. ATP increases NPR-A affinity for ANP1−28 and decreased Bmax in crude and solubilized membranes. Kinetic analysis of GC-coupled NPR-A revealed that ATP reduced the Km and increased the Vmax, an effect that was greater in SHR.Conclusion: Our observations indicate that ATP exerts a greater net effect on NPR-A in SHR than in WKY, which might explain the greater rate of cGMP production observed in SHR compared to WKY.
Peptides, 2002
Dendroaspis natriuretic peptide (DNP) is a recently isolated 38 amino acid peptide that shares st... more Dendroaspis natriuretic peptide (DNP) is a recently isolated 38 amino acid peptide that shares structural and functional properties with the other members of the natriuretic peptide family. The present study demonstrates the presence of DNP-like immunoreactivity in sections of rat aorta, carotid artery and renal vasculature and tubules. DNP-like immunoreactivity was detected in culture aortic vascular smooth muscle cells and medium and is regulated by endothelin-1, angiotensin II and sodium nitroprusside but not by transforming growth factor-. Our observations indicate that DNP elicits a marked inhibitory effect on DNA synthesis in culture rat aortic vascular smooth muscle cells.
Thrombosis and Haemostasis, 2007
Proteins are targets of reactive nitrogen species such as peroxynitrite and nitrogen dioxide. Amo... more Proteins are targets of reactive nitrogen species such as peroxynitrite and nitrogen dioxide. Among the various amino acids in proteins, tyrosine and tryptophan residues are especially susceptible to attack by reactive nitrogen species. On the other hand, protein tyrosine phosphorylation has gained much attention in respect to cellular regulatory events and signal transduction. Peroxynitrite-mediated nitration of peptide YPPPPPW and phosphopeptide pYPPPPPW were studied at pH 7.4. The predominant nitrated products were separated and identified by reverse phase high performance liquid chromatography coupled with electrospray ionization mass spectrometry (LC-MS). The nitration sites were established by tandem electrospray ionization-mass spectrometry (LC-MS/MS). A regulatory effect of tyrosine phosphorylation/dephosphorylation on peptide nitration was observed. YPPPPPW was predominantly nitrated at tyrosine residue while pYPPPPPW was nitrated at tryptophan one. Our results can help in understanding the biochemical significance of the relationship of tyrosine phosphorylation and nitration in proteins.
PloS one, 2014
Here we show that distinct subpopulations of cells exist within traumatic human extremity wounds,... more Here we show that distinct subpopulations of cells exist within traumatic human extremity wounds, each having the ability to differentiate into multiple cells types in vitro. A crude cell suspension derived from traumatized muscle was positively sorted for CD29, CD31, CD34, CD56 or CD91. The cell suspension was also simultaneously negatively sorted for either CD45 or CD117 to exclude hematopoietic stem cells. These subpopulations varied in terms their total numbers and their abilities to grow, migrate, differentiate and secrete cytokines. While all five subpopulations demonstrated equal abilities to undergo osteogenesis, they were distinct in their ability to undergo adipogenesis and vascular endotheliogenesis. The most abundant subpopulations were CD29+ and CD34+, which overlapped significantly. The CD29+ and CD34+ cells had the greatest proliferative and migratory capacity while the CD56+ subpopulation produced the highest amounts of TGFß1 and TGFß2. When cultured under endothelial differentiation conditions the CD29+ and CD34+ cells expressed VE-cadherin, Tie2 and CD31, all markers of endothelial cells. These data indicate that while there are multiple cell types within traumatized muscle that have osteogenic differentiation capacity and may contribute to bone formation in post-traumatic heterotopic ossification (HO), the major contributory cell types are CD29+ and CD34+, which demonstrate endothelial progenitor cell characteristics.
Uploads
Papers by Geoffrey Woodard