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Biomedical subjects

D D Cunningham

Publications and source records attributed to D D Cunningham.

At least 19 recordsLinked to original sources
PubMed2001

Thrombin receptor signaling to cytoskeleton requires Hsp90.

Thrombin is a serine protease that evokes various cellular responses involved in injury and repair of the nervous system through the activation of protease-activated receptor-1 (PAR-1). Signals that modulate cell morphology precede most PAR-1 effects, but the initial signal transduction molecules are not known. Using the yeast two-hybrid system, we identified Hsp90, a chaperone with known signaling properties, as a binding partner of PAR-1. The interaction was confirmed by glutathione S-transferase pull-down, overlay, and co-immunoprecipitation assays. Morphological assays in mouse astrocytes were carried out to evaluate the importance of Hsp90 during cytoskeletal signaling. Reducing Hsp90 levels by antisense treatment or disruption of the Hsp90.PAR-1 complex by the Hsp90-specific drug geldanamycin attenuated thrombin-mediated astrocyte shape changes. Furthermore, overexpression of the PAR-1 cytoplasmic tail abrogated thrombin-induced cytoskeletal changes in neuronal cells. Treatment with geldanamycin specifically inhibited activation of RhoA without affecting thrombin-mediated intracellular calcium release, revealing the regulation of a distinct signaling pathway by Hsp90. Taken together, these studies demonstrate that Hsp90 may be essential for PAR-1-mediated signaling to the cytoskeleton.

Animals↗
PubMed2000

Creatine kinase, an ATP-generating enzyme, is required for thrombin receptor signaling to the cytoskeleton.

Thrombin orchestrates cellular events after injury to the vascular system and extravasation of blood into surrounding tissues. The pathophysiological response to thrombin is mediated by protease-activated receptor-1 (PAR-1), a seven-transmembrane G protein-coupled receptor expressed in the nervous system that is identical to the thrombin receptor in platelets, fibroblasts, and endothelial cells. Once activated by thrombin, PAR-1 induces rapid and dramatic changes in cell morphology, notably the retraction of growth cones, axons, and dendrites in neurons and processes in astrocytes. The signal is conveyed by a series of localized ATP-dependent reactions directed to the actin cytoskeleton. How cells meet the dynamic and localized energy demands during signal transmission is unknown. Using the yeast two-hybrid system, we identified an interaction between PAR-1 cytoplasmic tail and the brain isoform of creatine kinase, a key ATP-generating enzyme that regulates ATP within subcellular compartments. The interaction was confirmed in vitro and in vivo. Reducing creatine kinase levels or its ATP-generating potential inhibited PAR-1-mediated cellular shape changes as well as a PAR-1 signaling pathway involving the activation of RhoA, a small G protein that relays signals to the cytoskeleton. Thrombin-stimulated intracellular calcium release was not affected. Our results suggest that creatine kinase is bound to PAR-1 where it may be poised to provide bursts of site-specific high-energy phosphate necessary for efficient receptor signal transduction during cytoskeletal reorganization.

Adenosine Triphosphate↗
PubMed2000

Vacuum-assisted lancing of the forearm: an effective and less painful approach to blood glucose monitoring.

A vacuum-lancet device was applied to the forearm for the purpose of obtaining capillary blood samples for glucose monitoring with minimal pain. In four clinical trials, a total of 215 individuals aged 12-77 years were tested four times using standard conditions and four times with either a different depth of lancing, different brand of lancet or a larger-sized device. The volume of blood collected using one-half atmosphere of vacuum in 40 sec was measured. The sensation and visual appearance of each lancet puncture on the forearm was recorded. Glucose was measured in forearm and in conventional fingerstick blood samples. The distribution of volumes was skewed to higher values with median values for each trial in the range of 3-10 microL. Ninety-five percent of the lancet sticks were judged as less painful than a fingerstick. Redness and bruising around the lanced sites were noted in some patients but disappeared within a few days. Overall correlation of the forearm versus fingerstick glucose values was 0.96. The vacuum-lancet device was very successful in obtaining capillary blood samples for glucose testing in a relatively painless manner. Incorporation of a glucose measuring system into the device might improve testing compliance among those who fear pain or the sight of blood.

Adolescent↗
PubMed1998

Signaling pathways involved in thrombin-induced cell protection.

This study examined the signal transduction pathways involved in thrombin-induced neuroprotection and compares these results with those of a similar study of thrombin-induced neuronal death. In thrombin-induced protection of astrocytes from hypoglycemia, pretreatment of astrocytes with tyrosine or serine/threonine kinase inhibitors, cytochalasin D, or exoenzyme C3, a potent inhibitor of the small GTPase RhoA, attenuated thrombin-induced protection. These same inhibitors were previously shown to block thrombin-induced cell death, implying a similarity in the cell death and cell-protective pathways. Biochemical assays determined that thrombin increased available RhoA activity, although more slowly and to a lesser extent than occurs in thrombin-induced cell death. A clear difference in these pathways was revealed when a time course study of thrombin-induced cell death indicated that unlike thrombin-induced protection, cells must be exposed to thrombin for >16 h to irreversibly enter the cell death pathway. Addition of lower doses of thrombin every 24 h also induced cell death. These studies indicate that exposure of cells to micromolar concentrations of thrombin alone does not induce cell death, but the continued exposure to thrombin is required. Thus the cell death and protective pathways may share initial signaling proteins, but differences in the amplitude as well as the duration of the signal may result in different final pathways.

ADP Ribose Transferases↗
PubMed1998

Injury-related factors and conditions down-regulate the thrombin receptor (PAR-1) in a human neuronal cell line.

Previous studies have demonstrated that thrombin can induce potent effects on neural cell morphology, biochemistry, and viability. Nearly all of these effects are mediated by proteolytic activation of the thrombin receptor (PAR-1). Mechanisms of PAR-1 regulation in several nonneural cell types have been shown to be novel and cell type specific; however, little is known about PAR-1 regulation in neural cells. In the present study, PAR-1 cell surface expression and regulation were examined in a transformed retinoblast (Ad12 HER 10) cell line using radioiodinated anti-PAR-1 monoclonal antibodies ATAP2, which recognizes intact and cleaved receptors, and SPAN12, which is specific for the intact form of the receptor. Scatchard analysis revealed high-affinity, specific binding to a single affinity class of receptors: K(D) = 3.13 and 5.25 nM, Bmax = 190.1 and 67.8 fmol/mg of protein for 125I-ATAP2 and 125I-SPAN12, respectively. Specificity for PAR-1 was confirmed by demonstrating rapid and near complete decreases for both antibodies following treatment with thrombin or PAR-1 activating peptide (SFLLRN). Differential antibody binding was used to demonstrate rapid and near complete thrombin-induced PAR-1 cleavage and internalization, with protein synthesis-dependent replacement of intact receptors occurring over longer time intervals, but only minimal recycling of cleaved receptors. A variety of factors and conditions were screened for their effects on PAR-1 expression. Significant decreases in PAR-1 expression were induced by the protein kinase C activator phorbol 12-myristate 13-acetate (87% at 3 h), the phospholipid inflammatory mediator lysophosphatidic acid (32% at 3 h), and the injury-related condition hypoglycemia (64 and 100% at 24 h in the absence and presence of dibutyryl cyclic AMP, respectively). The effect of hypoglycemia was shown by RNase protection to be at least partially pretranslational. Finally, thrombin's ability to enhance hypoglycemia-induced cell killing correlated temporally with PAR-1 cell surface expression.

Antibodies, Monoclonal↗
PubMed1997

Thrombin causes a marked delay in skeletal myogenesis that correlates with the delayed expression of myogenin and p21CIP1/WAF1.

Thrombin is a multifunctional serine protease whose activity is regulated in the extravasculature by an extracellular inhibitor, protease nexin-1. Because protease nexin-1 expression has been shown to be regulated during skeletal muscle cell differentiation, we reasoned that thrombin inactivation may be an important requirement for this developmental process. To test this hypothesis, we examined the effects of thrombin on differentiating C2C12 myoblasts. We report here that myogenesis, as scored by myotube formation, is considerably delayed by thrombin. This regulation correlated with delayed expression of myogenin and p21(CIP1/WAF1), both considered critical components of the skeletal muscle cell differentiation program. Regulation occurred at the RNA level, indicating that the effect of thrombin is either transcriptional or post-transcriptional. Furthermore, we present evidence suggesting that this regulation is mediated by the thrombin receptor. Although thrombin is mitogenic for certain cell types, we found that delay of myogenesis in C2C12 cells did not involve a mitogenic signal. Taken together, these results imply that inhibition of the serine protease thrombin may be required for proper progression through the myogenic differentiation program. The data point to potentially important roles that thrombin and protease nexin-1 may play during skeletal muscle development.

Animals↗
PubMed1997

Thrombin induces apoptosis in cultured neurons and astrocytes via a pathway requiring tyrosine kinase and RhoA activities.

Thrombin activity is a factor in acute CNS trauma and may contribute to such chronic neurodegenerative diseases as Alzheimer's disease. Thrombin is a multifunctional serine protease that catalyses the final steps in blood coagulation. However, increasing evidence indicates that thrombin also elicits a variety of cellular and inflammatory responses, including responses from neural cells. Most recently, high concentrations of thrombin were shown to cause cell death in both astrocyte and hippocampal neuron cultures. The purpose of this study was to determine the mechanisms underlying thrombin-induced cell death. Our data show that thrombin appears to cause apoptosis as evidenced by cleavage of DNA into oligonucleosomal-sized fragments, fragmentation of nuclei, and prevention of death by inhibition of protein synthesis. Synthetic peptides that directly activate the thrombin receptor also induced apoptosis, indicating that thrombin-induced cell death occurred via activation of the thrombin receptor. The signal transduction cascade involves tyrosine and serine/threonine kinases and an intact actin cytoskeleton. Additional study revealed the involvement of the small GTP-binding protein RhoA. Thrombin induced RhoA activity in both astrocytes and hippocampal neurons, and inhibition of RhoA activity with exoenzyme C3 attenuated cell death, indicating that thrombin activation of RhoA was necessary for thrombin-induced cell death. Tyrosine kinase inhibitors blocked thrombin induction of RhoA, indicating that tyrosine kinase activity was required upstream of RhoA. These data suggest a sequential linkage of cellular events from which we propose a model for the second messenger cascade induced by thrombin in neural cells that can lead to apoptosis.

Animals↗
PubMed1996

No .NO from NO synthase.

The nitric-oxide synthase (NOS; EC 1.14.13.39) reaction is formulated as a partially tetrahydrobiopterin (H4Bip)-dependent 5-electron oxidation of a terminal guanidino nitrogen of L-arginine (Arg) associated with stoichiometric consumption of dioxygen (O2) and 1.5 mol of NADPH to form L-citrulline (Cit) and nitric oxide (.NO). Analysis of NOS activity has relied largely on indirect methods such as quantification of nitrite/nitrate or the coproduct Cit; we therefore sought to directly quantify .NO formation from purified NOS. However, by two independent methods, NOS did not yield detectable .NO unless superoxide dismutase (SOD; EC 1.15.1.1) was present. In the presence of H4Bip, internal .NO standards were only partially recovered and the dismutation of superoxide (O2-.), which otherwise scavenges. .NO to yield ONOO-, was a plausible mechanism of action of SOD. Under these conditions, a reaction between NADPH and ONOO- resulted in considerable overestimation of enzymatic NADPH consumption. SOD lowered the NADPH:Cit stoichiometry to 0.8-1.1, suggesting either that additional reducing equivalents besides NADPH are required to explain Arg oxidation to .NO or that .NO was not primarily formed. The latter was supported by an additional set of experiments in the absence of H4Bip. Here, recovery of internal .NO standards was unaffected. Thus, a second activity of SOD, the conversion of nitroxyl (NO-) to .NO, was a more likely mechanism of action of SOD. Detection of NOS-derived nitrous oxide (N2O) and hydroxylamine (NH2OH), which cannot arise from .NO decomposition, was consistent with formation of an .NO precursor molecule such as NO-. When, in the presence of SOD, glutathione was added, S-nitrosoglutathione was detected. Our results indicate that .NO is not the primary reaction product of NOS-catalyzed Arg turnover and an alternative reaction mechanism and stoichiometry have to be taken into account.

Electron Transport↗
PubMed1996

Thrombin attenuates neuronal cell death and modulates astrocyte reactivity induced by beta-amyloid in vitro.

beta-Amyloid protein has been implicated as a potential causative agent in the neuropathology associated with Alzheimer's disease. This possibility is supported by observations that beta-amyloid induces neuronal degeneration and astrocyte reactivity in vitro by as yet undefined mechanism(s). In this report, we present data demonstrating that the pathological effects of beta-amyloid on cultured cells are modulated by activation of the thrombin receptor. At concentrations between 50 and 500 nM, thrombin pretreatment significantly attenuates neurotoxicity mediated by fibrillar aggregates of beta 1-42 and beta 25-35 peptides. In cultured astrocytes, the stellate morphology induced by beta 1-42 and beta 25-35 aggregates can be prevented and reversed by thrombin exposures between 10 pM and 1 microM. In contrast, thrombin potentiates rather than attenuates the beta-amyloid-induced increased expression of basic fibroblast growth factor, suggesting that thrombin differentially modulates the effects of beta-amyloid on astrocytes. Thrombin's effects on both neurons and astrocytes are mimicked by thrombin receptor-activating peptide and inhibited by two potent thrombin inhibitors, hirudin and protease nexin-1. These data provide both new insight into the signaling pathways underlying the cellular effects of beta-amyloid and additional support for the role of thrombin as an important mediator of neuropathological events.

Amyloid beta-Peptides↗
PubMed1996

Characterization of the human protease nexin-1 promoter and its regulation by Sp1 through a G/C-rich activation domain.

Protease nexin-1 (PN-1 ) is a potent inhibitor of serine proteases in the extracellular environment. It is abundantly expressed in the nervous system, where it is thought to participate in local injury and repair processes. Although some information has been obtained regarding PN-1 gene structure, relatively little is known about the cis- and trans-acting factors that regulate its expression. Elucidation of these factors should provide a better understanding of PN-1 function during development and wound repair. In this report we describe the characterization of the human PN-1 promoter and identify regulatory domains and a transactivator mediating its transcriptional activity. The promoter is highly G/C rich proximal to the transcriptional start site. It exhibits tissue specificity and is negatively regulated by a silencer element upstream of position -480. A positive regulatory element was mapped between -199 and -45, which contains multiple putative Sp1 consensus binding sites. Electrophoretic mobility shift analysis confirmed that Sp1 specifically binds this region of the PN-1 promoter. DNase I foot-printing revealed six potential Sp1 binding sites between -103 and -56 that were protected by recombinant Sp1. Cotransfection experiments into the Sp1-deficient Drosophila SL2 cell line also showed that Sp1 activates PN-1 promoter activity in a dose-dependent fashion. Thus, our analysis demonstrates that activation of PN-11 transcription is regulated by Sp1 through G/C-rich cis-acting elements in the 5'proximal promoter region.

Amyloid beta-Protein Precursor↗
PubMed1996

Sevoflurane degradation to compound A in anaesthesia breathing systems.

Determination of an effective rate constant and activation energy allowed the application of steady-state theory to predict concentrations of compound A from sevoflurane concentrations, fresh gas flow rate, absorbent temperature and amount of absorbent. Studies by eight research groups were compared. Lower concentrations of compound A than predicted were observed at low flow rates, suggesting that its degradation by the absorbent is important in limiting the maximum observed concentrations in closed and low-flow breathing systems. Trial-to-trial and batch-to-batch variations in compound A concentrations were observed in model system tests of commercial and pilot-plant absorbents. Chemical modification of the absorbent with glycerol lowered concentrations of compound A, possibly by formation of a nucleophilic addition product. An ideal chemical scavenger would remain stable and non-volatile in the absorbent before irreversibly reacting with compound A to form a stable non-volatile product.

Absorption↗
PubMed1995

Analysis of sevoflurane degradation products in vapor phase samples.

Sevoflurane degradation products were measured by GC-flame ionization detection in vapor phase samples using manual and automated injection methods. Sample handling techniques allowed the transfer and storage of samples for up to 72 h. Compound A, fluoromethyl 2,2-difluoro-1-(trifluoromethyl)vinyl ether, was the major vapor phase degradation product formed in simulated clinical conditions. Recoveries of 4-32 ppm (v/v) compound A concentrations using the manual method were in the range of 88-117% (n = 12, mean = 102%, R.S.D. = 9%).

Anesthetics↗
PubMed1995

Thrombin receptor activation stimulates astrocyte proliferation and reversal of stellation by distinct pathways: involvement of tyrosine phosphorylation.

Treatment of cultured type-1 astrocytes with thrombin leads to cell proliferation and reversal of stellation. The half-maximal concentrations of thrombin required for each response are 500 and 2 pM, respectively. To test whether they might be mediated by different receptors, we examined the contribution of the G protein-coupled thrombin receptor to these responses in purified rat astrocytes by using the agonist peptide SFLLRNP. In the absence of added growth factors, SFLLRNP fully mimicked the effects of thrombin at half-maximal concentrations of 30 microM for an increase in cell number and DNA synthesis and 100 nM for the reversal of stellation. The role of protein tyrosine phosphorylation in these events was investigated using antiphosphotyrosine antibodies. Thrombin and SFLLRNP at concentrations at least 10-fold greater than those required for half-maximal reversal of stellation but below those required for mitogenesis induced an identical pattern of tyrosine phosphorylation on several proteins of 55-65, 106, 110-115, and 120-130 kDa. The response was rapid (< 1 min) and transient with a peak response after approximately 2 min. The specific tyrosine kinase inhibitor herbimycin A did not affect thrombin- or SFLLRNP-mediated reversal of stellation at concentrations of up to 1 microM. In contrast, 1 microM herbimycin fully inhibited the ability of thrombin and SFLLRNP to increase cell number and stimulate DNA synthesis. Furthermore, this inhibition by 1 microM herbimycin A corresponded to inhibition of receptor-induced tyrosine phosphorylation. Thus, cell proliferation but not reversal of stellation is dependent on thrombin receptor-activated tyrosine kinase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Thrombin receptor activation protects neurons and astrocytes from cell death produced by environmental insults.

Thrombin is a multifunctional serine protease that is rapidly produced from prothrombin at sites of tissue injury and catalyzes the final steps in blood coagulation. Thrombin also regulates gene expression and process outgrowth in neurons and astrocytes and stimulates proliferation of astrocytes. Since thrombin is produced immediately upon breakdown of the blood-brain barrier we examined its effects on astrocytes and neurons cultured under conditions which resemble those found in vivo following cerebrovascular injury. These studies showed that thrombin markedly protected rat primary astrocytes from cell death induced by hypoglycemia or oxidative stress. Thrombin also protected rat primary hippocampal neurons from cell death produced by hypoglycemia or growth supplement deprivation. Synthetic peptides which directly activate the thrombin receptor also protected astrocytes and neurons from these environmental insults, demonstrating that the thrombin effects were mediated through the thrombin receptor. In contrast to these results with stressed cells, high concentrations of thrombin killed both astrocytes and neurons cultured under normal conditions. All of the effects of thrombin on astrocytes and neurons were blocked by the brain thrombin inhibitor, protease nexin-1 (PN-1). This shows that the effects required the proteolytic activity of thrombin and is consistent with the known proteolytic mechanism by which thrombin activates its receptor. These results indicate that thrombin and PN-1 may regulate the viability of both astrocytes and neurons in early moments following trauma to the CNS or other conditions that alter the blood-brain barrier.

Animals↗
PubMed1995

Cellular localization of thrombin receptor mRNA in rat brain: expression by mesencephalic dopaminergic neurons and codistribution with prothrombin mRNA.

Cell culture studies demonstrating that the serine protease thrombin can induce neuronal and glial process retraction, glial proliferation, and changes in gene expression suggest a role for thrombin in CNS development, plasticity, and response to injury. Most cellular responses to thrombin are mediated by proteolytic activation of the cloned thrombin receptor (TR), a member of the seven transmembrane domain, G-protein-coupled receptor superfamily. As a step toward understanding the role of thrombin and its receptor in the CNS, Northern blot, in situ hybridization, and immunohistochemical techniques were used to analyze the cellular localization of TR mRNA in weanling-age rat brain. TR mRNA was broadly distributed across the neuraxis, although expression was very focal and often anatomically limited within specific neural structures. The greatest hybridization was associated with individual neurons in neocortex, cingulate/retrosplenial cortex, and subiculum, subsets of nuclei in hypothalamus, thalamus, pretectum, and ventral mesencephalon, and discrete cell layers in the hippocampus, cerebellum, and olfactory bulb. Patterns of hybridization included neuronal, glial, and ependymal cells, although white matter was uniformly negative, as were most cerebrovascular endothelial cells. Expression of TR mRNA by astroglia and dopaminergic neurons was confirmed by colocalization with immunoreactivity for glial fibrillary acidic protein (GFAP) in hippocampus and tyrosine hydroxylase in the substantia nigra. Comparison between TR and prothrombin (thrombin's precursor) cRNA hybridization demonstrated distinct but overlapping brain distributions of these transcripts, most clearly evident in postnatally developing, laminated structures. These results suggest widespread utilization of, and multiple physiologic, and possibly pathophysiologic, functions for, the thrombin/TR cell signaling system in the CNS.

Animals↗
PubMed1994

Protease nexin-1, a potent thrombin inhibitor, is reduced around cerebral blood vessels in Alzheimer's disease.

The clotting protease thrombin might contribute to the pathophysiology of central nervous system (CNS) injury and certain diseases by its ability to retract processes on neurons and astrocytes and to stimulate astrocyte proliferation. Protease nexin-1 (PN-1) is a 43 kDa thrombin inhibitor found predominantly in the brain where much of it resides around capillaries and large blood vessels. This location of PN-1 prompted the hypothesis that it may play a protective role against extravasated thrombin released following cerebrovascular injury or under certain pathological conditions. Recent studies indicated that the levels of PN-1 are markedly reduced in the postmortem brains of patients with Alzheimer's disease (AD). It was suggested that this reduction in PN-1 levels was due to the sequestration of PN-1 by extravasated thrombin. In the present study we examined the specific nature of this reduction by immunohistochemical staining of sections from control and AD brains using PN-1 specific antibodies. We show that the levels of PN-1 immunoreactivity around blood vessels and the number of blood vessels exhibiting PN-1 immunoreactivity were markedly reduced in the brains of patients with AD compared to age-matched controls; this reduction was reflected by a decrease in the levels of PN-1 activity and PN-1 protein. Thus an imbalance between PN-1 and thrombin may be a contributing factor in the pathology of AD.

Aged↗
PubMed1994

Thrombin receptor activation induces secretion and nonamyloidogenic processing of amyloid beta-protein precursor.

The amyloid beta-protein (A beta) and protease nexin-2/amyloid beta-protein precursor (PN-2/A beta PP) are major constituents of senile plaques and cerebrovascular deposits in individuals with Alzheimer's disease and related disorders. It has been suggested that the coagulation protease thrombin may process A beta PP in a manner leading to the formation of A beta. Here we investigated the effects of thrombin on the secretion and processing of PN-2/A beta PP and the production of A beta in a cellular system. Incubation of glioblastoma cells with thrombin (1-5 nM) resulted in the accumulation of abnormally processed, carboxyl-terminal-truncated forms of secreted PN-2/A beta PP (approximately 85 kDa) in the culture medium. Higher concentrations of thrombin (> 10 nM) also increased the levels of secreted PN-2/A beta PP in cultured untransfected glioblastoma cells and glioblastoma cells that were stably transfected to overproduce the 695 isoform of A beta PP. Increased secretion of PN-2/A beta PP required the proteolytic activity of thrombin, was induced by activation of the thrombin receptor by agonist peptides, and required activation of protein kinase C. Incubation of the untransfected and transfected glioblastoma cells with thrombin led to decreased levels of soluble A beta in the culture medium consistent with previously suggested mechanisms regarding the secretion of PN-2/A beta PP. Although the present studies suggest that thrombin does not directly contribute to A beta formation, its proteolysis of secreted PN-2/A beta PP may disrupt regions near the carboxyl terminus of the secreted proteins that account for their neuroprotective and cell adhesive properties.

Amyloid beta-Protein Precursor↗