The protein phosphatase inhibitor cantharidin induces head and foot formation in buds of Cassiopea andromeda (Rhizostomae, Scyphozoa). (1/89)

The polyps of Cassiopea andromeda produce spindle shaped, freely swimming buds which do not develop a head (a mouth opening surrounded by tentacles) and a foot (a sticky plate at the opposite end) until settlement to a suited substrate. The buds, therewith, look very similar to the planula larvae produced in sexual reproduction. With respect to both, buds and planulae, several peptides and the phorbolester TPA have been found to induce the transformation into a polyp. Here it is shown that cantharidin, a serine/threonine protein phosphatase inhibitor, induces head and foot formation in buds very efficiently in a 30 min treatment, the shortest yet known efficient treatment. Some resultant polyps show malformations which indicate that a bud is ordinary polyp tissue in which preparatory steps of head and foot formation mutually block each other from proceeding. Various compounds related to the transfer of methyl groups have been shown to affect head and foot formation in larvae of the hydrozoon Hydractinia echinata. These compounds including methionine, homocysteine, trigonelline, nicotinic acid and cycloleucine are shown to also interfere with the initiation of the processes which finally lead to head and foot formation in buds of Cassiopea andromeda.  (+info)

The protein phosphatase inhibitor cantharidin alters vascular endothelial cell permeability. (2/89)

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.  (+info)

Penetration of moxifloxacin into peripheral compartments in humans. (3/89)

To characterize the penetration of moxifloxacin (BAY 12-8039) into peripheral target sites, the present study aimed at measuring unbound moxifloxacin concentrations in the interstitial space fluid by means of microdialysis, an innovative clinical sampling technique. In addition, moxifloxacin concentrations were measured in cantharides-induced skin blisters, saliva, and capillary plasma and compared to total- and free-drug concentrations in venous plasma. For this purpose, 12 healthy volunteers received moxifloxacin in an open randomized crossover fashion either as a single oral dose of 400 mg or as a single intravenous infusion of 400 mg over 60 min. An almost-complete equilibration of the free unbound plasma fraction of moxifloxacin with the interstitial space fluid was observed, with mean area under the concentration-time curve (AUC)(interstitial fluid)/AUC(total-plasma) ratios ranging from 0.38 to 0.55 and mean AUC(interstitial fluid)/AUC(free-plasma) ratios ranging from 0.81 to 0.86. The skin blister concentration/plasma concentration ratio reached values above 1.5 after 24 h, indicating a preferential penetration of moxifloxacin into inflamed lesions. The moxifloxacin concentrations in saliva and capillary blood were similar to the corresponding levels in plasma. Our data show that moxifloxacin concentrations attained in the interstitial space fluid in humans and in skin blister fluid following single doses of 400 mg exceed the values for the MIC at which 90% of isolates are inhibited for most clinically relevant bacterial strains, notably including penicillin-resistant Streptococcus pneumoniae. These findings support the use of moxifloxacin for the treatment of soft tissue and respiratory tract infections in humans.  (+info)

A study to determine the pharmacokinetics and inflammatory fluid penetration of gatifloxacin following a single oral dose. (4/89)

A single 400 mg oral dose of gatifloxacin was given to each of nine healthy male volunteers and the concentrations of the drug in plasma, cantharidine-induced inflammatory fluid and urine were measured over the following 24 h. The mean peak concentration in plasma of 4. 1 mg/L was attained at a mean time of 1.8 h post dose. The mean peak concentration in inflammatory fluid was 3.6 mg/L and was attained at a mean time of 4.2 h post dose. The mean plasma elimination half-life of gatifloxacin was 6.8 h and that in inflammatory fluid was 7.2 h. The mean penetration into the inflammatory fluid was 117%. Recovery of drug from urine during the first 24 h post dose was 65% of that administered. Our data suggest that gatifloxacin dosed at 400 mg od should be adequate to treat systemic infections caused by most bacterial species.  (+info)

Phosphatase inhibitor cantharidin blocks adenosine A(1) receptor anti-adrenergic effect in rat cardiac myocytes. (5/89)

Experiments were performed to examine whether the protein phosphatase inhibitor cantharidin blocks the anti-adrenergic effect of adenosine A(1) receptor stimulation. In electrically stimulated adult rat ventricular myocytes loaded with the intracellular calcium concentration ([Ca(2+)](i)) indicator fluo-3, isoproterenol (10 nM) increased systolic [Ca(2+)](i) by 46%, increased twitch amplitude by 56%, and increased total cellular cAMP content by 140%. The adenosine A(1) receptor agonist 2-chloro-N(6)-cyclopentlyadenosine (CCPA) reduced isoproterenol-stimulated [Ca(2+)](i) and contractility by 87 and 80%, respectively, but reduced cAMP content by only 18%. Cantharidin had no effects on myocyte [Ca(2+)](i), contractility, or cAMP in the absence or presence of isoproterenol but blocked the effects of CCPA on [Ca(2+)](i) and contractility by approximately 44%. Cantharidin had no effect on CCPA attenuation of isoproterenol-induced increases in cAMP. Pretreatment with CCPA also reduced the increase in contractile parameters produced by the direct cAMP-dependent protein kinase A (PKA) activator 8-bromocAMP. These results suggest that activation of protein phosphatases mediate, in part, the anti-adrenergic effect of adenosine A(1) receptor activation in ventricular myocardium.  (+info)

Hamster pancreatic beta cell lines with altered sensitivity towards apoptotic signalling by phosphatase inhibitors. (6/89)

Specific inhibitors of serine/threonine phosphatases like okadaic acid can induce apoptotic cell death in the pancreatic beta cell line HIT. Cultivation in stepwise increased concentrations of okadaic acid enabled the isolation of HIT100R cells which proliferate at 100 nM okadaic acid (8 - 10 times the initially lethal concentration). These two cell lines were used to characterize the events triggered by okadaic acid that led to apoptosis. Biochemical markers, e.g. cytochrome c release from mitochondria and increase of caspase-3-like activity, revealed that induction of apoptosis by 100 nM okadaic acid in parental HIT cells started with the release of cytochrome c. In HIT100R cells 500 nM okadaic acid were necessary to induce alterations comparable to those observed with 100 nM okadaic acid in non-resistant HIT cells. In contrast to okadaic acid, the potency of the structurally different phosphatase inhibitor cantharidic acid to induce cytochrome c release, increase of caspase-3-like activity and DNA fragmentation was comparable in HIT and HIT100R cells. Thus, no cross-resistance between these phosphatase inhibitors seemed to exist. Phosphatase activity in extracts from HIT and HIT100R cells did not differ in its total amount or in its sensitivity for okadaic acid. Since higher concentrations of okadaic acid were needed to induce apoptosis in HIT100R cells, a compromised intracellular accumulation of the toxin appeared likely. Functional and structural analysis revealed that this was achieved by the development of the multidrug resistance phenotype in HIT100R cells. The underlying mechanism appeared to be the enhanced expression of the pgp1 but not the pgp2 gene.  (+info)

Protein phosphatase inhibitors arrest cell cycle and reduce branching morphogenesis in fetal rat lung cultures. (7/89)

Protein phosphatase 2A (PP2A) is a key signal transduction intermediate in the regulation of cellular proliferation and differentiation in vitro. However, the role of PP2A in the context of a developing organ is unknown. To explore the role of PP2A in the regulation of lung development, we studied the effect of PP2A inhibition on new airway branching, induction of apoptosis, DNA synthesis, and expression of epithelial marker genes in whole organ explant cultures of embryonic (E14) rat lung. Microdissected lung primordia were cultured in medium containing one of either two PP2A inhibitors, okadaic acid (OA, 0-9 nM) or cantharidin (Can, 0-3,600 nM), or with the PP2B inhibitor deltamethrin (Del, 0-10 microM) as a control for a PP2A-specific effect for 48 h. PP2A inhibition with OA and Can significantly inhibited airway branching and overall lung growth. PP2B inhibition with Del did not affect lung growth or new airway development. Histologically, both PP2A- and PP2B-inhibited explants were similar to controls. Increased apoptosis was not the mechanism of decreased lung growth and new airway branching inasmuch as OA-treated explant sections subjected to the terminal deoxynucleotidyltransferase dUTP nick end labeling reaction demonstrated a decrease in apoptosis. However, PP2A inhibition with OA increased DNA content and 5-bromo-2'-deoxyuridine uptake that correlated with a G(2)/M cell cycle arrest. PP2A inhibition also resulted in altered differentiation of the respiratory epithelium as evidenced by decreased mRNA levels of the early epithelial marker surfactant protein C. These findings suggest that inhibition of protein phosphatases with OA and Can halted mesenchymal cell cycle progression and reduced branching morphogenesis in fetal rat lung explant culture.  (+info)

Cantharidin enhances norepinephrine-induced vasoconstriction in an endothelium-dependent fashion. (8/89)

In this study we characterized the effects of the protein phosphatase (PP) type 1 and type 2A inhibitor cantharidin (Cant) and its structural analogs cantharidic acid and endothall on PP activity, force of contraction, and myosin light chain phosphorylation in rat aorta. All compounds inhibited PP activity in homogenates of rat aorta with a rank order of potency of Cant = cantharidic acid > endothall. However, only Cant increased force of contraction and myosin light chain phosphorylation in intact isolated rat aortic rings. Based on these findings, we investigated the effects of Cant on alpha-adrenoceptor-mediated vasoconstriction. Cant (1 and 3 microM) enhanced norepinephrine-induced contraction in endothelium-intact rat aorta. In contrast, Cant did not affect norepinephrine-induced contraction in endothelium-denuded rat aorta. We suggest that inhibition of PP1 and/or PP2A activities by Cant enhances vascular contractility in endothelium-intact rat aorta by increasing the phosphorylation state of endothelial regulatory proteins.  (+info)