Biomedical subjects

W J Thompson

Publications and source records attributed to W J Thompson.

At least 37 records · Page 2Linked to original sources

PubMed1997

Glial growth factor rescues Schwann cells of mechanoreceptors from denervation-induced apoptosis.

Golgi tendon organs and Pacinian corpuscles are peripheral mechanoreceptors that disappear after denervation during a critical period in early postnatal development. Even if regeneration is allowed to occur, Golgi tendon organs do not reform, and the reformation of Pacinian corpuscles is greatly impaired. The sensory nerve terminals of both types of mechanoreceptors are closely associated with Schwann cells. Here we investigate the changes in the Schwann cells found in Golgi tendon organs and Pacinian corpuscles after nerve resection in the early neonatal period. We report that denervation induces the apoptotic death of these Schwann cells and that this apoptosis can be prevented by administration of a soluble form of neuregulin, glial growth factor 2. Schwann cells associated with these mechanoreceptors are immunoreactive for the neuregulin receptors erbB2, erbB3, and erbB4, and the sensory nerve terminals are immunoreactive for neuregulin. Our results suggest that Schwann cells in developing sensory end organs are trophically dependent on sensory axon terminals and that an axon-derived neuregulin mediates this trophic interaction. The denervation-induced death of mechanoreceptor Schwann cells is correlated with deficiencies in the re-establishment of these sensory end organs by regenerating axons.

Animals↗

PubMed1997

Nerve terminal withdrawal from rat neuromuscular junctions induced by neuregulin and Schwann cells.

Schwann cells (SCs) that cap neuromuscular junctions (nmjs) play roles in guiding nerve terminal growth in paralyzed and partially denervated muscles; however, the role of these cells in the day-to-day maintenance of this synapse is obscure. Neuregulins, alternatively spliced ligands for several erbB receptor tyrosine kinases, are thought to play important roles in cell-cell communication at the nmj, affecting synapse-specific gene expression in muscle fibers and the survival of terminal SCs during development. Here we show that application of a soluble neuregulin isoform, glial growth factor II (GGF2), to developing rat muscles alters terminal SCs, nerve terminals, and muscle fibers. SCs extend processes and migrate from the synapse. Nerve terminals retract from acetylcholine receptor-rich synaptic sites, and their axons grow, in association with SCs, to the ends of the muscle. These axons make effective synapses only after withdrawal of GGF2. These synaptic alterations appear to be induced by the actions of neuregulin on SCs, because SC transplants growing into contact with synaptic sites also caused withdrawal of nerve terminal branches. These results show that SCs can alter synaptic structure at the nmj and implicate these cells in the maintenance of this synapse.

Age Factors↗

PubMed1997

Inhibition of serine-threonine protein phosphatases decreases barrier function of rat pulmonary microvascular endothelial cells.

The flux of multisized fluorescein-isothiocyanate-labeled hydroxy ethyl starch (FITC-HES) macromolecules was used to assess changes in barrier function of rat pulmonary microvascular endothelial cell (RPMVEC) monolayers exposed to protein phosphatase (PP) inhibitors or cGMP analogs and atriopeptin (ANF). Two potent PP inhibitors, calyculin A (CalA) and okadaic acid (OA), increased RPMVEC permeability in a dose- and time-dependent manner, and CalA had a higher intrinsic activity than OA. In contrast, ANF and potent cGMP analogs had no effect on basal RPMVEC permeability. The phosphohistone PP activity contained in RPMVEC sonicates was inhibited by OA with an inhibition profile that suggested at least two components were present, with PP2A accounting for approximately 70% of the OA-inhibitable phosphohistone phosphatase activity. Following separation with heparin-Sepharose chromatography, PP activity exhibited equipotent inhibition by CalA and differential inhibition by OA. Differential inhibition of PP1 and PP2A by OA suggested that PP1 is involved in regulating RPMVEC barrier function. Permeabilized RPMVEC showed increased phosphorylation of several proteins in the presence of phosphatase inhibitors. Treatment with KT 5926, a myosin light chain (MLC) kinase (MLCK) inhibitor, or rolipram, a phosphodiesterase inhibitor, decreased 32P incorporation into immunoprecipitated MLC by CalA and OA. However, this effect did not abolish either the CalA- or OA-induced decrease in the RPMVEC barrier function. Localization of filamentous (F) actin was at the periphery as well as in the cytoplasm and perinuclear region, whereas nonmuscle myosin was seen in the perinuclear region. Neither of these patterns was changed in the presence of CalA. Thus, cGMP does not alter RPMVEC permeability, but inhibition of PP activity results in loss of barrier function by a mechanism independent from MLC phosphorylation.

Animals↗

PubMed1997

Vasoconstriction increases pulmonary nitric oxide synthesis and circulating cyclic GMP.

Vascular shear stress increases when blood flow or blood viscosity increases or when vessel diameter decreases. In the systemic circulation, shear stress is a potent stimulus for endothelial nitric oxide synthesis. We studied isolated rat lungs to determine whether increasing shear stress increases nitric oxide synthesis in the pulmonary circulation. Lungs were given the vasoconstrictor, U46619 (a thromboxane analogue), and perfused at constant flow rates or at constant pressure, since constant pressure perfusion minimizes changes in shear stress with vasoconstriction. The subsequent effect of the NOS inhibitor, N omega-methyl-L-arginine (LMA), or the soluble guanylyl cyclase inhibitor, 6-anilino-5,8-quinolinodione (LY83583) was assessed. Changes in pulmonary vascular resistance (PVR), pulmonary vascular compliance, and perfusate cyclic GMP concentration were measured as indicators of nitric oxide synthesis. The effect of the cyclic GMP-specific (type V) phosphodiesterase inhibitor, zaprinast, on perfusate cyclic GMP concentrations was also examined. An infusion of U46619 consistently increased PVR and decreased compliance. LMA and LY83583 also increased PVR in U46619-treated lungs perfused at constant flow rates, primarily by increasing precapillary resistance. LMA had no effect in U46619-treated lungs perfused at constant pressure. Perfusate cyclic GMP concentrations increased significantly after U46619 in lungs perfused at constant flow rates, but cyclic GMP levels did not change after U46619 in lungs perfused at constant pressure. Zaprinast also increased perfusate cyclic GMP, demonstrating that increases in intracellular cyclic GMP are reflected in circulating cyclic GMP concentrations. We conclude that vasoconstriction with U46619 increases nitric oxide synthesis in isolated rat lungs. Lungs perfused at constant pressure respond differently to NOS inhibitors compared to those perfused at constant flow, suggesting that shear stress may increase nitric oxide synthesis in the lung. Perfusate concentrations of cyclic GMP reflect activation of soluble guanylyl cyclase in this model.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMed1997

Cyclic GMP accumulation in pulmonary microvascular endothelial cells measured by intact cell prelabeling.

Cyclic GMP accumulation in cultured rat pulmonary microvascular endothelial cells (RPMVEC) was studied with a new prelabeling method developed using intact platelets and smooth muscle cells (1). [3H]-hypoxanthine was used to radiolabel the cellular guanine nucleotide pool. Neutral alumina and Dowex-50 double column chromatography was used to purify and quantitate the levels of [3H]-cyclic GMP. Changes in cyclic GMP metabolism in short and long term RPMVEC cultures were studied using rat atrial naturetic factor 8-33 (ANF) and sodium nitroprusside (SNP) in the presence and absence of cyclic nucleotide (CN) phosphodiesterase (PDE) inhibitors. In RPMVEC exogenous hypoxanthine was incorporated into both low (65% uptake) and high (34% uptake) passage cells in a time-dependent manner reaching maximum incorporation near 8 hours. Basal cyclic GMP values in both groups were 0.003% of the total cellular tritium (9 x 10(6) and 4 x 10(6) cpm/10(6) cells, respectively). ANF treatment of prelabeled RPMVEC resulted in a 10- to 12-fold increase in [3H]-cyclic GMP in the absence of CN PDE inhibitors (EC50 = 5.4 nM). However, incubation with SNP showed no changes in cellular cyclic GMP accumulation. Several relatively selective CN PDE inhibitors had no effect on ANF or SNP induced cyclic GMP accumulation in RPMVEC. The ANF induced cGMP accumulation was verified by radioimmunoassay. These studies confirm the utility of the hypoxanthine prelabeling technique to monitor intact microvascular EC cyclic GMP accumulation. Cultured RPMVEC show little or no functional soluble guanylate cyclase or cyclic GMP PDE activity.

Animals↗

PubMed1997

Ca(2+)-inhibitable adenylyl cyclase and pulmonary microvascular permeability.

Intracellular mechanisms responsible for endothelial cell disruption are unknown, although either elevated cytosolic Ca2+ ([Ca2+]i) or decreased adenosine 3',5'-cyclic monophosphate (cAMP) promotes permeability. Recent identification that Ca(2+)-inhibitable adenylyl cyclase establishes an inverse relationship between [Ca2+]i and cAMP in macrovascular endothelial cells provided a possible mechanism of development of permeability. However, these data utilized an in vitro model; lacking was evidence supporting 1) expression of Ca(2+)-inhibitable adenylyl cyclase in pulmonary microvascular endothelium and 2) Ca2+ inhibition of adenylyl cyclase and cAMP content as a paradigm for inflammatory mediator-induced permeability in the intact circulation. We therefore addressed these issues in microvascular endothelial cells derived from rat lung and in an isolated perfused rat lung preparation. Results demonstrate expression of a Ca(2+)-inhibitable adenylyl cyclase in microvascular endothelial cells. Furthermore, data suggest that Ca2+ inhibition of adenylyl cyclase is necessary for development of microvascular permeability in the intact circulation. We conclude Ca2+ inhibition of cAMP represents a critical step in genesis of microvascular permeability in the intact pulmonary circulation.

Adenylyl Cyclase Inhibitors↗

PubMed1996

Nonpeptidal P2 ligands for HIV protease inhibitors: structure-based design, synthesis, and biological evaluation.

Design and synthesis of nonpeptidal bis-tetrahydrofuran ligands based upon the X-ray crystal structure of the HIV-1 protease-inhibitor complex 1 led to replacement of two amide bonds and a 10 pi-aromatic system of Ro 31-8959 class of HIV protease inhibitors. Detailed structure-activity studies have now established that the position of ring oxygens, ring size, and stereochemistry are all crucial to potency. Of particular interest, compound 49 with (3S,3aS,6aS)-bis-Thf is the most potent inhibitor (IC50 value 1.8 +/- 0.2 nM; CIC95 value 46 +/- 4 nM) in this series. The X-ray structure of protein-inhibitor complex 49 has provided insight into the ligand-binding site interactions. As it turned out, both oxygens in the bis-Thf ligands are involved in hydrogen-bonding interactions with Asp 29 and Asp 30 NH present in the S2 subsite of HIV-1 protease. Stereoselective routes have been developed to obtain these novel ligands in optically pure form.

Amino Acid Sequence↗

PubMed1996

Schwann cell apoptosis at developing neuromuscular junctions is regulated by glial growth factor.

Denervated adult mammalian muscle fibres are reinnervated by regenerating axons and, in the case of partially denervated muscles, by sprouts extended from remaining, intact axons. Recent experiments suggest that Schwann cells (SCs) regulate these events, inducing and guiding axonal outgrowth through the processes they extend. In contrast to adults, reinnervation o denervated neonatal muscles is deficient and axonal sprouting is absent. In light of the proposed roles for SCs in these processes, we examined whether SCs in neonatal muscles exhibit altered responses to denervation. We report here that neonatal denervation leads to the rapid, apoptotic death of SCs at rat neuro-muscular junctions. Injection of glial growth factor, a member of the neuregulin family of trophic factors present in developing sensory and motor neurons, prevents this apoptosis in vivo. These results provide further evidence for the importance of SCs in regulating nerve growth and suggest that axon-Schwann cell trophic interactions play a role in the normal development of the neuromuscular system.

Animals↗

PubMed1996

Schwann cells induce and guide sprouting and reinnervation of neuromuscular junctions.

The "terminal' Schwann cells that sit atop the neuromuscular junction sense neuromuscular transmission and respond to perturbations of this transmission by extending long processes. These processes have the ability to induce nerve growth and serve as substrates to guide this growth. These processes thus play major roles in muscle reinnervation and in sprouting. An absence of nerve sprouting is correlated with the apoptotic death of terminal Schwann cells at denervated endplates in neonatal muscles. Thus, Schwann cells appear to participate actively in the maintenance and repair of neuromuscular synapses.

Cell Communication↗

PubMed1996

Our experience in combining mechanics.

The use of more than one orthodontic technique in an orthodontic practice can significantly increase treatment capability. Being able to carry out the efficient and effective use of multiple techniques, however, requires that the appliance and the force systems are designed to do both tipping and translation in an optimal manner, either individually or in a variable combination. The application of this philosophy of combining treatment mechanics has been very favorable in our practice. The case reports and mechanics described were selected from our practice to show the diversity and potential that is possible with combination anchorage treatment mechanics. The key to success in a multiattachment straight wire system is to have the ability to use light tipping movements in combination with rigid translation and to be able to vary the location of either, at any time the need arises during treatment.

Child↗

PubMed1996

Erythro-9-(2-hydroxy-3-nonyl)adenine inhibits cyclic-3',5'-guanosine monophosphate-stimulated phosphodiesterase to reverse hypoxic pulmonary vasoconstriction in the perfused rat lung.

Erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) was shown to reverse the hypoxic pressor response (HPR) in the isolated, blood-perfused rat lung model. EHNA, an adenosine deaminase inhibitor, showed reversal of the HPR in a dose-dependent manner (EC50 = 129 +/- 30 microM). We found that the reversal of HPR by EHNA was not mediated by the adenosine receptors because the EHNA effect was not blocked by the adenosine receptor antagonist, 8-p-sulfophenyl-theophylline (67 microM; n = 6). Pretreatment with a cy-clic-3',5'-adenosine monophosphate (cAMP)-dependent protein kinase inhibitor, Rp-adenosine-3',5'-cyclic monophosphorothioate (0.5 mM; n = 4), blocked EHNA reversal of the HPR. As an alternative mechanism of action, EHNA inhibition of cyclic nucleotide phosphodiesterase(s) isozymes was studied in endothelium intact and denuded pulmonary arteries. Using anion-exchange chromatography the cyclic nucleotide phosphodiesterase (PDE) separated into predominantly PDE families 2 and a mixture of 3 and 4. DEAE fractions showing cAMP hydrolysis activated by 5 microM cyclic-3',5'-guanosine monophosphate (cGMP) had a Km for cAMP of 6.3 microM and an apparent Kact for cGMP of 1.4 microM. EHNA was shown to inhibit PDE2 competitively. In intact vessels, the IC50 for EHNA was 3.3 microM using 0.03 microM [3H]-cAMP substrate assayed in the presence of 2 microM cGMP and in denuded vessels 3.7 microM at 0.03 microM [3H]-cAMP substrate in the presence of 5 microM cGMP. Fractions in which cAMP hydrolysis was inhibited or not affected by 5 microM cGMP (PDE3 and 4, respectively) showed an IC50 of > 200 microM for EHNA. We conclude that reversal of the hypoxic pressor response by EHNA in the isolated, perfused rat lung model occurs with a mechanism involving in part inhibition of smooth muscle PDE2.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1995

A priori prediction of activity for HIV-1 protease inhibitors employing energy minimization in the active site.

We have observed a high correlation between the intermolecular interaction energy (Einter) calculated for HIV-1 protease inhibitor complexes and the observed in vitro enzyme inhibition. A training set of 33 inhibitors containing modifications in the P1' and P2' positions was used to develop a regression equation which relates Einter and pIC50. This correlation was subsequently employed to successfully predict the activity of proposed HIV-1 protease inhibitors in advance of synthesis in a structure-based design program. This included a precursor, 47, to the current phase II clinical candidate, L-735,524 (51). The development of the correlation, its applications, and its limitations are discussed, and the force field (MM2X) and host molecular mechanics program (OPTIMOL) used in this work are described.

Binding Sites↗

PubMed1995

Rolipram and isoproterenol reverse platelet activating factor-induced increases in pulmonary microvascular permeability and vascular resistance.

Platelet activating factor (PAF) is an important mediator of pulmonary microvascular endothelial cell (PMVEC) injury in sepsis. Membrane receptors for PAF have been identified on PMVECs and mediate its actions at least in part by protein kinase C activation. Since rolipram, a family IV cyclic AMP phosphodiesterase inhibitor, and isoproterenol, an adenylate cyclase activator, both reverse ischemia-reperfusion-induced lung permeability, we studied the effects of these agents on PAF-induced pulmonary microvascular permeability. The isolated rat lung model was used in which lungs were ventilated and buffer perfused at constant flow while suspended from a force transducer to monitor lung weight along with arterial (P(a)) and venous (Pv) pressures. Control lungs (n = 6) were infused with PAF (40 nmole/kg) via an arterial port and the capillary permeability coefficient (Kf,c) was determined at 0, 15, and 60 min. The remaining lungs were randomized for infusion with either rolipram (n = 4, 20 mumole/kg) or isoproterenol (n = 4, 5 mumole/kg) via an arterial port 30 min after injury with PAF. In the rolipram- and isoproterenol-treated groups, the Kf,c was determined 15 and 60 min postinfusion with these agents. The control group showed significant elevation in the Kf,c and total pulmonary resistance (Rt). At 15 and 60 min, rolipram and isoproterenol reversed PAF injury as shown by the significant improvement in the Kf,c and Rt. These findings support the concept that increased cyclic AMP is an important mediator in the reversal of PAF-increased PMVEC permeability and pulmonary resistance.

Animals↗

PubMed1995

Schwann cell processes guide regeneration of peripheral axons.

Terminal Schwann cells overlying the neuromuscular junction sprout elaborate processes upon muscle denervation. We show here that motor axons use these processes as guides/substrates during regeneration; in so doing, they escape the confines of endplates and grow between endplates to generate polyneuronal innervation. We also show that Schwann cells in the nerve provide similar guidance. Axons extend from the cut end of a nerve in association with Schwann cell processes and appear to navigate along them. The processes extend from axotomized nerves at the same rate and in the same manner as they do from axon-containing nerves. The rate of process extension limits the rate at which axons regenerate. Thus, Schwann cell processes lead and guide peripheral regeneration.

Animals↗

PubMed1995

Nerve sprouting in muscle is induced and guided by processes extended by Schwann cells.

Partial denervation or paralysis with botulinum toxin, manipulations that induce sprouting of nerve terminals in muscle, also induced terminal Schwann cells to extend processes. These processes were associated with every nerve sprout and in some cases were longer than the sprouts that appeared to be growing along them. Following partial denervation, more than 70% of the nerve sprouts that grew to innervate nearby denervated endplates were associated with Schwann cell processes that had extended from the denervated endplates, i.e., in the direction opposite to nerve growth. Implantation of Schwann cells into an innervated muscle induced sprouting upon contact of an axon or nerve terminal by Schwann cell processes. These observations show that Schwann cells induce and guide axonal sprouting in muscle.

Animals↗

PubMed1995

TNF modulates endothelial properties by decreasing cAMP.

Tumor necrosis factor-alpha (TNF-alpha), a monokine that contributes to vascular dysfunction accompanying the host response to gram-negative sepsis, has been shown to increase vascular permeability in vivo and to diminish the barrier function of cultured endothelial cell (EC) monolayers. The studies reported here indicate that a mechanism through which TNF alters EC barrier function involves a reduction in intracellular adenosine 3',5'-cyclic monophosphate (cAMP) content, due in part to increased cyclic nucleotide phosphodiesterase (CNPDE) activities. TNF increased the diffusional transit of [3H]sorbitol, [3H]inulin, and 125I-labeled albumin across confluent bovine aortic EC monolayers. This effect of TNF was both time and dose dependent and occurred in parallel with a fall in EC cAMP. cAMP analogues, such as dibutyryl cAMP (DBcAMP), prevented TNF-induced perturbation of EC barrier function. TNF also mediated another important alteration in the EC phenotype, in that both mRNA and activity of the anticoagulant cofactor thrombomodulin were reduced after exposure of EC to TNF and were normalized by the addition of DBcAMP. EC monolayers exposed to TNF-alpha showed increased cAMP levels when exposed to 3-isobutyl-1-methylxanthine, a nonspecific CNPDE inhibitor. Ion exchange chromatography of cytosol derived from TNF-treated EC consistently showed an approximately 245% increase in phosphodiesterase (PDE) IV (high-affinity, cAMP-specific PDE) activity as identified by rolipram inhibition. PDE II activity was increased by 150% after TNF-alpha treatment of early passage EC, which was identified by cGMP-activated hydrolysis of cAMP. Western and Northern analyses, as well as activity studies, revealed that TNF treatment did not change the amount of PDE IV protein or mRNA but rather increased the specific activity of the isozyme, suggesting that a posttranslational modification had occurred. These data indicate that activation of EC CNPDE activity and decreased intracellular cAMP may represent a mechanism by which TNF increases EC permeability and promotes a procoagulant EC phenotype.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1995

Adenosine-induced vasodilation: receptor characterization in pulmonary circulation.

Adenosine mediates vascular smooth muscle relaxation in the pulmonary circulation. The A2 receptor has been suggested to mediate adenosine-induced vasodilation (AIV). In this study, the effect(s) of selective adenosine agonist and antagonist on the hypoxic pressor response (HPR) was assessed in the isolated blood-perfused rat lung. Adenosine (0.075-7.5 mM) infusion (0.125 ml/min) into the pulmonary artery dose dependently attenuated the HPR. AIV was mimicked by 10 microM 5'-(N-ethylcarboxamido)adenosine (NECA), a nonselective adenosine agonist. Adenosine- and NECA-induced vasodilation were attenuated by 67 microM 8-(p-sulfophenyl)theophylline. In contrast, NECA-induced vasodilation was not attenuated by the A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine (1 microM). At 10 microM, a minimal vasodilatory effect was seen with the nonselective adenosine agonists CV-1808 and N6-(2-phenylisopropyl)adenosine (R-PIA) compared with NECA. The highly selective A2a agonist 2-[p-(2-carboxyethyl)phenyl amino]-5'-N-ethyl carboxamido adenosine (CGS-21680C, 10 microM) and A1 agonist 2-chloro-N6-cyclopentyladenosine (CCPA, 10 microM) had no vasodilatory effect. Neither the K+ channel blockers tetraethylammonium chloride (10 mM) and glibenclamide (100 microM) nor the NO synthase inhibitor N omega-nitro-L-arginine methyl ester attenuated NECA-induced vasodilation. These findings suggest that AIV is mediated via the A2b receptor and that AIV occurs via an NO-independent mechanism.

Adenosine↗

PubMed1995

Metabolism of L-689,502 by rat liver slices to potent HIV-1 protease inhibitors.

L-689,502, N-[2(R)-hydroxy-1(S)-indanyl]-5(S)-(1,1-dimethylethoxy- carbonyl-amino)-4(S)-hydroxy-6-phenyl-2(R)-(4-[2(R)-(4-morpholinyl) ethoxy]phenyl)methylhexamide, is a potent and specific inhibitor of human immunodeficiency virus-type 1 (HIV-1) protease in vitro. Metabolism of this compound in rat liver slices produced four major and several minor metabolites. The major metabolites were identified as morpholin-2-one, 3'(S)-hydroxyindan and 4'-hydroxyindan analogs, and a 4-O-glucuronic acid conjugate of the parent compound. The metabolites were characterized by Heteronuclear Multiple Quantum Coherence and Nuclear Overhauser Effect techniques in NMR spectroscopy, by MS, and/or comparison with authentic standards. Two of the minor metabolites were similarly characterized as a 2(R)-[4-(2-carboxymethoxy)phenyl]methyl analog and a product with a degraded morpholino ring. The hydroxyindan metabolites were lower in activity than L-689,502, whereas the morpholin-2-one and carboxymethoxyphenyl analogs were approximately 6- and 11-fold more potent as inhibitors of HIV-1 protease, respectively.

Animals↗