Biomedical subjects

T Noll

Publications and source records attributed to T Noll.

At least 19 recordsLinked to original sources

PubMed2006

[The predictive quality of the Psychopathy Checklist-Revised (PCL-R) for violent and sex offenders in Switzerland. A validation study].

In Switzerland, the Hare Psychopathy Checklist-Revised (PCL-R) is administered rather restrictively for risk assessment of recidivism among violent and sexual offenders. The aim of the present study was a first-time evaluation of the predictive validity of the PCL-R for violent and sexual recidivism in Switzerland. The PCL-R scores of 96 violent and sex offenders were evaluated by collecting the data in their psychiatric expert opinions. The scores were then compared to the rates of recidivism as shown in the criminal records. Consistent with previous studies in North America and Europe, the determined predictive accuracy was satisfying. This degree of precision supports the use of the PCL-R for risk assessment of sexual and violent recidivism in Switzerland, as long as the instrument does not constitute the sole criterion to determine future recidivism, but is applied only in combination with a thorough clinical evaluation. The use of precise cut-off scores did not prove to be a valid criterion for the prognosis of recidivism and can therefore not be recommended for Swiss offenders.

Adolescent↗

PubMed2005

Plasma transglutaminase factor XIII induces microvessel ingrowth into biodegradable hydroxyapatite implants in rats.

Coagulation factor XIII is a member of the transglutaminase-family. Transgluaminases cross-link either fibrin monomers in blood coagulation or extracellular proteins in extracellular matrix formation. In early stages of bone healing migration and proliferation of endothelial cells lead to formation of new vessels. The aim of this study was to investigate the angiogenetic activity of plasma factor XIII in bone defects filled with nanoparticulate hydroxyapatite paste. A critical size defect was created in the tibial head of rats which was not filled in group I. In group II the defect was filled with hydroxyapatite paste, and in group III with hydroxyapatite paste enriched with factor XIII. Ten days after surgery angiogenesis in the defects was assessed using immunohistochemistry and confocal laser scanning microscopy. Ac16 antibody was used to detect activation of factor XIII into factor XIIIA. In defects without biomaterial (group I) vessel-rich connective tissue and diffuse distribution of capillaries was observed. In defects filled with pure hydroxyapatite (group II) formation of capillaries was limited to the host bone-hydroxyapatite interface. In contrast, addition of plasma factor XIII to hydroxyapatite (group III) stimulated formation of vessels within the biomaterial. The current study reveals that factor XIII can improve angiogenesis in hydroxyapatite.

Absorbable Implants↗

PubMed2005

[Mechanisms influencing cellular physiology as a concept of treatment for wound healing disturbances].

AIM: Recent knowledge about repair mechanisms in different types of tissue is the basic of actual therapeutic efforts. Center of several experimental and clinical approaches is the influencing of angiogenesis with an also distinct meaning concerning wound healing. Therefore, application of growth factors, gene transfer, and employment of genetically manipulated cells often aim at angiogenesis. Nevertheless, manipulation of angiogenesis also leads to secondary problems such as hyperpermeability followed by impairment of local wound milieu. Our study was done to identify mechanisms to protect from disturbances of endothelial barrier function. METHOD: In a first experimental investigation on cultured endothelial cells, the influence of plasma-transglutaminase (Factor XIII) to endothelial barrier function was studied. In a second step, the influence of Factor XIII on wound healing properties was investigated in patients with a chronic venous ulceration. RESULTS: Activated Factor XIII (FXIIIA*) led to a dose-dependent reduction of endothelial cell permeability of 30 % compared to control with a maximum effect using 1 to 5 U/mL. Clinical investigation revealed a nearly complete reduction of wound secretion. CONCLUSION: Experimental studies revealed that activated Factor XIII stabilizes endothelial barrier under basic conditions as well as under conditions of induced hyperpermeability. Clinical study revealed that Factor XIII also distinctly reduces wound secretion. Therefore, plasma-transglutaminase may offer a new therapeutic option to treat the local or generalized leakage-syndrome.

Administration, Topical↗

PubMed2004

Bioprocess development for the production of a recombinant MUC1 fusion protein expressed by CHO-K1 cells in protein-free medium.

The mucin MUC1 is a candidate for use in specific immunotherapy against breast cancer, but this requires the large-scale production of a MUC1 antigen. In this study, a bioprocess for the expression of a recombinant MUC1 fusion protein with a cancer associated glycosylation in CHO-K1 cells has been developed. Cells permanently expressing parts of the extracellular portion of MUC1 fused to IgG Fc were directly transferred from adherent growth in serum-containing medium to suspension culture in the protein-free ProCHO4-CDM culture medium. Using the Cellferm-pro system, optimal culture parameter as pH and pO(2) were determined in parallel spinner flask batch cultures. A pH of 6.8-7.0 and a pO(2) of 40% of air saturation was found to give best cell growth and productivity of secreted recombinant protein. Specific productivity strongly depended the pO(2) and correlated with the online monitored oxygen uptake rate (OUR) of the cells, which indicates a positive influence of the rate of oxidative phosphorylation on productivity. The optimised conditions were applied to continuous perfusion culture which gave very high cell densities and space time yields of the recombinant MUC1 fusion protein, allowing production at gram scale. The product degradation was much lower in supernatants from continuous perfusion culture compared to batch mode. Antibodies reacting with cancer associated MUC1 glycoforms strongly bound to the fusion protein, indicating that the desired glycoforms were obtained and suggesting that the recombinant MUC1 protein could be tested for use in immunotherapy.

Animals↗

PubMed2004

MEK/MAPK as a signaling element in ATP control of endothelial myosin light chain.

Phosphorylation of endothelial myosin light chains (MLC) is a key mechanism in control of endothelial contractile machinery. Extracellular ATP influences endothelial MLC phosphorylation by either activation of Ca(2+)-dependent MLC kinase or Ca(2+)-independent MLC phosphatase. Here, the role of the MEK/MAPK pathway in this signaling was investigated in porcine aortic endothelial cells. Phosphorylation of ERK2 and phosphorylation of MLC were analyzed in cultured aortic endothelial cells. ATP (10 microM) increased ERK2 phosphorylation from basal 17 +/- 3 to 53 +/- 4%, an effect suppressed in the presence of the MEK inhibitors PD-98059 (20 microM) or U0126 (10 microM). Phosphorylation of ERK2 was not dependent on the ATP-induced cytosolic Ca(2+) rise, because it was unaltered when this was suppressed by the Ca(2+) chelator BAPTA (10 microM) or xestospongin C (3 microM), an inhibitor of the inositol 1,4,5-trisphosphate-sensitive Ca(2+) release mechanism of the endoplasmic reticulum. Phosphorylation of ERK2 was neither induced by the adenosine analog 5'-(N-ethylcarboxamido)adenosine (1 microM) nor inhibited in the presence of the adenosine receptor antagonist 8-phenyltheophylline (10 microM). ATP increased MLC kinase activity, and this was blocked in presence of PD-98059. ATP also increased MLC phosphatase activity, which was not inhibited by PD-98059. The MEK/MAPK pathway is a Ca(2+)-independent part of ATP signaling toward MLC kinase but not of ATP signaling toward MLC phosphatase.

Adenosine Triphosphate↗

PubMed2004

Opposite effect of cAMP signaling in endothelial barriers of different origin.

cAMP-mediated signaling mechanisms may destabilize or stabilize the endothelial barrier, depending on the origin of endothelial cells. Here, microvascular coronary [coronary endothelial cells (CEC)] and macrovascular aortic endothelial cell (AEC) monolayers with opposite responses to cAMP were analyzed. Macromolecule permeability, isometric force, activation state of contractile machinery [indicated by phosphorylation of regulatory myosin light chains (MLC), activity of MLC kinase, and MLC phosphatase], and dynamic changes of adhesion complex proteins (translocation of VE-cadherin and paxillin) were determined. cAMP signaling was stimulated by the adenosine receptor agonist 5'-N-(ethylcarboxamido)-adenosine (NECA), the beta-adrenoceptor agonist isoproterenol (Iso), or by the adenylyl cyclase activator forskolin (FSK). Permeability was increased in CEC and decreased in AEC on stimulation with NECA, Iso, or FSK. The effects could be inhibited by the PKA inhibitor Rp-8-CPT-cAMPS and imitated by the PKA activator Sp-cAMPS. Under cAMP/PKA-dependent stimulation, isometric force and MLC phosphorylation were reduced in monolayers of either cell type, due to an activation of MLC phosphatase. In CEC but not in AEC, FSK induced delocalization of VE-cadherin and paxillin from cellular adhesion complexes as indicated by cell fractionation and immunofluorescence microscopy. In conclusion, decline in contractile activation and isometric force contribute to cAMP/PKA-mediated stabilization of barrier function in AEC. In CEC, this stabilizing effect is overruled by cAMP-induced disintegration of cell adhesion structures.

Animals↗

PubMed2001

Factor XIII prevents development of myocardial edema in children undergoing surgery for congenital heart disease.

In a prospective investigation of perioperative cardiac edema formation requiring a delayed sternal closure, we identified thrombin increase combined with a simultaneous decrease of factor XIII as a probable cause. After experimental studies additionally revealed that factor XIII could protect endothelial barrier function, we did another prospective randomized trial in which factor XIII or placebo was preoperatively substituted. The substitution finally showed distinct effects minimizing the incidence of myocardial swelling. Therefore, the clinical application of factor XIII may have a valuable therapeutic benefit in cases of leakage syndrome during extracorporeal circulation in congenital heart surgery.

Cardiomyopathies↗

PubMed2001

Modulation of early [Ca2+]i rise in metabolically inhibited endothelial cells by xestospongin C.

When energy metabolism is disrupted, endothelial cells lose Ca(2+) from endoplasmic reticulum (ER) and the cytosolic Ca(2+) concentration ([Ca(2+)](i)) increases. The importance of glycolytic energy production and the mechanism of Ca(2+) loss from the ER were analyzed. Endothelial cells from porcine aorta in culture and in situ were used as models. 2-Deoxy-D-glucose (2-DG, 10 mM), an inhibitor of glycolysis, caused an increase in [Ca(2+)](i) (measured with fura 2) within 1 min when total cellular ATP contents were not yet affected. Stimulation of oxidative energy production with pyruvate (5 mM) did not attenuate this 2-DG-induced rise of [Ca(2+)](i), while this maneuver preserved cellular ATP contents. The inhibitor of ER-Ca(2+)-ATPase, thapsigargin (10 nM), augmented the 2-DG-induced rise of [Ca(2+)](i). Xestospongin C (3 microM), an inhibitor of D-myo-inositol 3-phosphate [Ins(3)P]-sensitive ER-Ca(2+) release, abolished the rise. The results demonstrate that the ER of endothelial cells is very sensitive to glycolytic metabolic inhibition. When this occurs, the ER Ca(2+) store is discharged by opening of the Ins(3)P-sensitive release channel. Xestospongin C can effectively suppress the early [Ca(2+)](i) rise in metabolically inhibited endothelial cells.

Adenosine Triphosphate↗

PubMed2000

Mechanism of Ca(2+) overload in endothelial cells exposed to simulated ischemia.

OBJECTIVE: Several studies have shown that myocardial ischemia leads to functional failure of endothelial cells (EC) whereby disturbance of Ca(2+) homeostasis may play an important role. The mechanisms leading to Ca(2+) disbalance in ischemic EC are not fully understood. The aim of this study was to test effects of different components of simulated ischemia (glucose deprivation, anoxia, low extracellular pH (pH(o)) and lactate) on Ca(2+) homeostasis in EC. METHODS: Cytosolic Ca(2+) (Ca(i)), cytosolic pH (pH(i)) and ATP content were measured in cultured rat coronary EC. RESULTS: In normoxic cells 60 min glucose deprivation at pH(o) 7.4 had no effect on pH(i). It only slightly increased Ca(i) and decreased ATP content. Reduction of pH(o) to 6.5 under these conditions led to marked cytosolic acidosis and Ca(i) overload, but had no effect on ATP content. Anoxia at pH(o) 6.5 had no additional effect on Ca(i) overload, but significantly reduced cellular ATP. Addition of 20 mmol/l lactate to anoxia at pH(o) 6.5 accelerated Ca(i) overload due to faster cytosolic acidification. Acidosis-induced Ca(i) overload was prevented by inhibition of Ca(2+) release channels of endoplasmic reticulum (ER) with 3 micromol/l ryanodine or by pre-emptying the ER with thapsigargin. Re-normalisation of pH(o) for 30 min led to recovery of pH(i), but not of Ca(i). CONCLUSION: The ischemic factors leading to cytosolic acidosis (low pH(o) and lactate) cause Ca(i) overload in endothelial cells, while anoxia and glucose deprivation play only a minor role. The ER is the main source for this Ca(i) rise. Ca(i) overload is not readily reversible.

Adenosine Triphosphate↗

PubMed2000

ATP induces dephosphorylation of myosin light chain in endothelial cells.

In cultured porcine aortic endothelial monolayers, the effect of ATP on myosin light chain (MLC) phosphorylation, which controls the endothelial contractile machinery, was studied. ATP (10 microM) reduced MLC phosphorylation but increased cytosolic Ca(2+) concentration ([Ca(2+)](i)). Inhibition of the ATP-evoked [Ca(2+)](i) rise by xestospongin C (10 microM), an inhibitor of the inositol trisphosphate-dependent Ca(2+) release from endoplasmic reticulum, did not affect the ATP-induced dephosphorylation of MLC. MLC dephosphorylation was prevented in the presence of calyculin A (10 nM), an inhibitor of protein phosphatases PP-1 and PP-2A. Thus ATP activates MLC dephosphorylation in a Ca(2+)-independent manner. In the presence of calyculin A, MLC phosphorylation was incremented after addition of ATP, an effect that could be abolished when cells were loaded with the Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N, N,N',N'-tetraacetic acid acetoxymethyl ester (10 microM). Thus ATP also activates a Ca(2+)-dependent kinase acting on MLC. In summary, ATP simultaneously stimulates a functional antagonism toward both phosphorylation and dephosphorylation of MLC in which the dephosphorylation prevails. In endothelial cells, ATP is the first physiological mediator identified to activate MLC dephosphorylation by a Ca(2+)-independent mechanism.

Adenosine Triphosphate↗

PubMed2000

beta-agonistic bronchodilators: comparison of dose/response in working rat hearts.

STUDY OBJECTIVES: Different beta-agonists are compared with regard to their cardiodepressive side effects. DESIGN: The metaphenolic bronchodilators reproterol, salbutamol, fenoterol, and terbutaline were introduced at a dosage of 0.0005 micromol to a maximum of 10 micromol per gram of heart tissue into the isolated working rat heart under hypoxic conditions, and the response was observed during subsequent reoxygenation. As an index of external heart work, aortic flow was measured. Heart rate, coronary flow, and developed pressure were recorded. At the end of heart perfusion, mitochondria were isolated and analyzed for adenosine triphosphatase activity, adenosine triphosphate (ATP) synthesis, and membrane fluidity. Moreover, intact mitochondria and lipid peroxidation were investigated using a model system. MEASUREMENTS AND RESULTS: Compared to controls, reproterol gave the most favorable results, with an increase of 25 to 30% of aortic flow during reoxygenation at a concentration of 10 micromol/g heart tissue. In contrast, both fenoterol and salbutamol at a concentration of 1 micromol/g heart tissue decreased aortic flow during reoxygenation, whereas terbutaline had a negative influence on aortic flow at 0.01 to 0.1 micromol/g heart tissue. Mitochondria of these hearts were isolated at the end of the experiment. Mitochondrial ATP synthesis was increased above controls at nearly all concentrations of reproterol. ATP synthesis was decreased at 1 micromol and 10 micromol fenoterol. As little as 0.0005 micromol terbutaline decreased ATP synthesis by 50%. In intact mitochondria, adenosine diphosphate (ADP) to oxygen ratios were found to be increased with terbutaline and fenoterol, indicating ADP consumption by myokinase activation. Lipid peroxidation was increased in a model system between concentrations of 0.002 micromol/mg and 0.04 micromol/mg phosphatidylcholine by fenoterol and terbutaline, whereas a decrease was noted with reproterol. Membrane fluidity was found increased after addition of reproterol, which supports the evidence of efficient ATP synthesis by this compound. CONCLUSIONS: Cardiodepressive side effects and greater toxicity of fenoterol and terbutaline were found under the conditions of our experiment. Salbutamol and, in particular, reproterol appear much better tolerated. In addition to partial beta-adrenergic agonism, reproterol may exert an inhibitory influence on adenosine receptor sites and phosphodiesterase, which could result in membrane stabilization by saving cyclic adenosine monophosphate or ATP.

Adenosine Triphosphate↗

PubMed2000

A cell-culture reactor for the on-line evaluation of radiopharmaceuticals: evaluation of the lumped constant of FDG in human glioma cells.

UNLABELLED: A fluidized-bed cell-culture reactor with on-line radioactivity detection was developed for the in vitro evaluation of radiopharmaceuticals. The technique was applied to measure the dependency of the lumped constant (LC) of FDG on the glucose concentration in the culture medium in a human glioma cell line. METHODS: Human glioblastoma cells (86HG39) immobilized in open porous microcarriers were cultivated in a continuously operating fluidized-bed bioreactor. At different glucose concentrations in the culture medium, step inputs (0.1 MBq/mL) of FDG were performed and the cellular uptake of FDG was measured on-line and compared with analyzed samples. From these results, the LC of FDG and its dependency on the glucose concentration were calculated. RESULTS: This fluidized-bed technique enabled precise and reproducible adjustment of all relevant experimental parameters, including radiotracer time-concentration course, medium composition, pH, dissolved oxygen and temperature under steady-state conditions, and an on-line determination of the intracellular radiotracer uptake. The immobilized glioma cells formed stable, 3-dimensional, tumor-like spheroids and were continuously proliferating, as proven by an S-phase portion of 25%-40%. For further examination of the cells, an enzymatic method for detachment from the carriers without cellular destruction was introduced. In the FDG experiments, a significant dependency of the LC on the glucose level was found. For normoglycemic glucose concentrations, the LC was determined to be in the range of 0.7+/-0.1, whereas in hypoglycemia LC increased progressively up to a value of 1.22+/-0.01 at a glucose concentration of 3 mmol/L. CONCLUSION: The bioreactor represents an improved in vitro model for the on-line evaluation of radiotracers and combines a wide range of experimental setups and 3-dimensional, tissue-like cell cultivation with a technique for on-line radioactivity detection.

Bioreactors↗

PubMed1999

Calcium antagonists ameliorate ischemia-induced endothelial cell permeability by inhibiting protein kinase C.

BACKGROUND: Dihydropyridines block calcium channels; however, they also influence endothelial cells, which do not express calcium channels. We tested the hypothesis that nifedipine can prevent ischemia-induced endothelial permeability increases by inhibiting protein kinase C (PKC) in cultured porcine endothelial cells. METHODS AND RESULTS: Ischemia was induced by potassium cyanide/deoxyglucose, and permeability was measured by albumin flux. Ion channels were characterized by patch clamp. [Ca2+]i was measured by fura 2. PKC activity was measured by substrate phosphorylation after cell fractionation. PKC isoforms were assessed by Western blot and confocal microscopy. Nifedipine prevented the ischemia-induced increase in permeability in a dose-dependent manner. Ischemia increased [Ca2+]i, which was not affected by nifedipine. Instead, ischemia-induced PKC translocation was prevented by nifedipine. Phorbol ester also increased endothelial cell permeability, which was dose dependently inhibited by nifedipine. The effects of non-calcium-channel-binding dihydropyridine derivatives were similar. Analysis of the PKC isoforms showed that nifedipine prevented ischemia-induced translocation of PKC-alpha and PKC-zeta. Specific inhibition of PKC isoforms with antisense oligodeoxynucleotides demonstrated a major role for PKC-alpha. CONCLUSIONS: Nifedipine exerts a direct effect on endothelial cell permeability that is independent of calcium channels. The inhibition of ischemia-induced permeability by nifedipine seems to be mediated primarily by PKC-alpha inhibition. Anti-ischemic effects of dihydropyridine calcium antagonists could be due in part to their effects on endothelial cell permeability.

Animals↗

PubMed1999

Effect of factor XIII on endothelial barrier function.

The effect of factor XIII on endothelial barrier function was studied in a model of cultured monolayers of porcine aortic endothelial cells and saline-perfused rat hearts. The thrombin-activated plasma factor XIII (1 U/ml) reduced albumin permeability of endothelial monolayers within 20 min by 30 +/- 7% (basal value of 5.9 +/- 0.4 x 10(-6) cm/s), whereas the nonactivated plasma factor XIII had no effect. Reduction of permeability to the same extent, i.e., by 34 +/- 9% could be obtained with the thrombin-activated A subunit of factor XIII (1 U/ml), whereas the iodoacetamide-inactivated A subunit as well as the B subunit had no effect on permeability. Endothelial monolayers exposed to the activated factor XIII A exhibited immunoreactive deposition of itself at interfaces of adjacent cells; however, these were not found on exposure to nonactivated factor XIII A or factor XIII B. Hyperpermeability induced by metabolic inhibition (1 mM potassium cyanide plus 1 mM 2-deoxy-D-glucose) was prevented in the presence of the activated factor XIII A. Likewise, the increase in myocardial water content in ischemic-reperfused rat hearts was prevented in its presence. This study shows that activated factor XIII reduces endothelial permeability. It can prevent the loss of endothelial barrier function under conditions of energy depletion. Its effect seems related to a modification of the paracellular passageways in endothelial monolayers.

Animals↗

PubMed1999

Leishmania major: histone H1 gene expression from the sw3 locus.

Histone H1 in the parasitic protozoan Leishmania is a developmentally regulated protein encoded by the sw3 gene. Here we report that histone H1 variants exist in different Leishmania species and strains of L. major and that they are encoded by polymorphic genes. Amplification of the sw3 gene from the genome of three strains of L. major gave rise to different products in each strain, suggesting the presence of a multicopy gene family. In L. major, these genes were all restricted to a 50-kb Bg/II fragment found on a chromosomal band of 1.3 Mb (chromosome 27). The detection of RFLPs in this locus demonstrated its heterogeneity within several species and strains of Leishmania. Two different copies of sw3 (sw3.0 and sw3.1) were identified after screening a cosmid library containing L. major strain Friedlin genomic DNA. They were identical in their 5' UTRs and open reading frames, but differed in their 3' UTRs. With respect to the originally cloned copy of sw3 from L. major strain LV39, their open reading frames lacked a repeat unit of 9 amino acids. Immunoblots of L. guyanensis parasites transfected with these cosmids revealed that both copies could give rise to the histone H1 protein. The characterization of this locus will now make possible a detailed analysis of the function of histone H1 in Leishmania, as well as permit the dissection of the molecular mechanisms governing the developmental regulation of the sw3 gene.

Amino Acid Sequence↗

PubMed1999

Topical treatment of venous ulcer with fibrin stabilizing factor: experimental investigation of effects on vascular permeability.

BACKGROUND: The clinical observation of distinct reduction of wound secretion tendency during treatment of venous leg ulcer with topically applied factor XIII let us speculate that this enzyme may be involved in modulation of vascular permeability. METHODS: For experimental study porcine aortic endothelial cells were cultured on filter membrane to confluent monolayer. Endothelial covered filters then were used in a two-compartment modell creating an artificial luminal and abluminal compartment. To investigate the influence of factor XIII on endothelial barrier function, we measured the flux of trypan blue-labeled albumin through endothelial monolayers by spectrophotometer. RESULTS: Monolayers that were exposed to factor XIII showed a distinct (n = 10, p < 0.05) decrease of albumin flux in contrast to control. This effect was not dependent on serum substrates. Partially, the permeability reducing effect was due to a certain amount of albumin that is a stabilizing component of Fibrogammin HS. CONCLUSION: Although the mechanism is unclear at the moment, we conclude that reduction of endothelial permeability can be achieved by factor XIII which may play an important role in wound healing of venous leg ulcer.

Administration, Topical↗

PubMed1999

ATP reduces macromolecule permeability of endothelial monolayers despite increasing [Ca2+]i.

We investigated the relationship between the ATP-evoked rise of cytosolic Ca2+ concentration ([Ca2+]i) and barrier function in porcine aortic endothelial monolayers. ATP (0.01-100 microM) induced a transient rise of [Ca2+]i and reduced permeability in a concentration-dependent manner. In contrast, the Ca2+ ionophore ionomycin (1 microM) elicited a rise in [Ca2+]i comparable to that induced by ATP (10 microM), but it increased permeability. For the reduction of permeability, nucleotides were found to be in the following order of potency: ATP = ATPgammaS > ADP = UTP. Blockade of adenosine receptors by 8-phenyltheophylline (10 microM) did not affect ATP (10 microM)-induced reduction of permeability. ATP reduced permeability even in endothelial monolayers that had been loaded with the Ca2+ chelator BAPTA to prevent the rise in [Ca2+]i. U-73122 (1 microM), an inhibitor of phospholipase C (PLC), completely abolished the effect of ATP (10 microM) on permeability. It also abolished the translocation of protein kinase C (PKC) in response to ATP, which could also be achieved by the PKC inhibitors Gö-6976 (100 nM) or bisindolylmaleimide I (1 microM). In the presence of PKC inhibitors, however, the permeability effect of ATP was not affected. The presence of inhibitors of adenylate or guanylate cyclase (50 microM SQ-22536 or 20 microM ODQ) prevented changes in cyclic nucleotides but did not affect the permeability effects of ATP. The study shows that ATP reduces macromolecule permeability via a PLC-mediated mechanism that is independent of the concomitant effects of ATP on cytosolic Ca2+, cyclic nucleotides, or PKC.

Adenosine Triphosphate↗

PubMed1999

The protein phosphatase inhibitor cantharidin alters vascular endothelial cell permeability.

In this study, we characterized the effects of the protein phosphatases type 1 (PP 1) and type 2A (PP 2A) inhibitor cantharidin in endothelial cells. We identified catalytic subunits of PP 1alpha, PP 2Aalpha, and PP 2Abeta immunologically in bovine aortic endothelial cells. Moreover, we detected mRNAs coding for catalytic subunits of PP 1alpha, PP 1beta, and PP 2Aalpha by hybridization with specific DNA probes in total RNA from these cells. Okadaic acid and cantharidin inhibited the activities of catalytic subunits of PP 1 (okadaic acid, 0.01-1 microM; cantharidin, 1-100 microM) and PP 2A (okadaic acid, 0.1 nM to 1 microM; cantharidin, 0.1-100 microM) separated by column chromatography in a concentration-dependent manner. Moreover, cantharidin (1 microM to 1 mM) increased the phosphorylation state of endothelial proteins including the regulatory light chains of myosin without affecting cytosolic calcium concentrations. Cantharidin (5-100 microM) increased the permeability of cultured endothelial cells in a time- and concentration-dependent manner. We suggest that inhibition of PP 1 and PP 2A activities by cantharidin increases endothelial permeability by enhancing the phosphorylation state of endothelial regulatory proteins. Thus, cantharidin might be a useful tool to study the function of protein phosphatases in endothelial barrier function.

Animals↗