Inhibition of voltage-dependent sodium channels by Ro 31-8220, a 'specific' protein kinase C inhibitor

We find that several protein kinase C (PKC) inhibitors, previously considered to be specific, directly inhibit voltage-dependent Na(+) channels at their useful concentrations. Bisindolylmaleimide I (GF 1092037), IX (Ro 31-8220) and V (an inactive analogue), but not H7 (a non-selective isoquinolinesulfonamide protein kinase inhibitor), inhibited Na(+) channels assessed by several independent criteria: Na(+) channel-dependent glutamate release and [(3)H]batrachotoxinin-A 20-alpha-benzoate binding in rat cortical synaptosomes, veratridine-stimulated 22Na(+) influx in CHO cells expressing rat CNaIIa Na(+) channels and Na(+) currents measured in isolated rat dorsal root ganglion neurons by whole cell patch-clamp recording. These findings limit the usefulness of the bisindolylmaleimide class PKC inhibitors in excitable cells.     

Staurosporine,a non-specific PKC inhibitor,induces keratinocyte differentiation and raises intracellular calcium,but Ro31–8220, a specific inhibitor,does not

The responsiveness of normal human keratinocytes to different modulators of protein kinase C (PKC) was investigated. The PKC agonist TPA, staurosporine (a non-specific inhibitor), and Ro31–8220 (a specific inhibitor) were studied for effect on cell morphology, growth rate, involucrin expression, and intracellular calcium levels. Surprisingly the response to nanomolar concentrations of staurosporine was similar to TPA and induced a fusiform morphology, inhibited growth, increased involucrin levels, and raised intracellular calcium. Staurosporine also increased the number of cornified envelopes, and its action therefore appeared identical to TPA. In contrast, Ro31–8220 had little effect on morphology or growth and blocked both the TPA-induced growth inhibition and calcium rise. Ro31–8220 had no effect on staurosporine-induced growth inhibition but partially reduced its associated calcium rise. These results suggest PKC activation is required for keratinocyte differentiation and that staurosporine acts like a PKC agonist to give a similar effect as TPA. Specific inhibition of PKC by Ro31–8220 inhibits TPA-induced differentiation. © 1994 wiley-Liss, Inc.     

The protein kinase C inhibitors bisindolylmaleimide I (GF 109203x) and IX (Ro 31-8220) are potent inhibitors of glycogen synthase kinase-3 activity

Here we report that the widely used protein kinase C inhibitors, bisindolylmaleimide I and IX, are potent inhibitors of glycogen synthase kinase-3 (GSK-3). Bisindolylmaleimide I and IX inhibited GSK-3 in vitro, when assayed either in cell lysates (IC(50) 360 nM and 6.8 nM, respectively) or in GSK-3beta immunoprecipitates (IC(50) 170 nM and 2.8 nM, respectively) derived from rat epididymal adipocytes. Pretreatment of adipocytes with bisindolylmaleimide I (5 microM) and IX (2 microM) reduced GSK-3 activity in total cell lysates, to 25.1+/-4.3% and 12.9+/-3.0% of control, respectively. By contrast, bisindolylmaleimide V (5 microM), which lacks the functional groups present on bisindolylmaleimide I and IX, had little apparent effect. We propose that bisindolylmaleimide I and IX can directly inhibit GSK-3, and that this may explain some of the previously reported insulin-like effects on glycogen synthase activity.     

Effect of the protein kinase C inhibitor Ro 31-8220 on Ca2+-responses, induced by somatotropin, in swine granulosa cells

Somatotropin effect on Ca2+ responses in pig granulosa cells from antral follicles was investigated using fluorescent dye Fluo-3 AM and chlortetracycline. Ro 31-8220 increased the entry of extracellular calcium and the exit of calcium from intracellular stores. In Ca-free medium Ro 31-8220 exerted no influence on the level of calcium in granulosa cells. The effect of somatotropin on pig granulosa cells is associated with PKC activation. These data suggest the involvement of PKC in the changes of calcium in pig granulosa cells activated by somatotropin.     

Depolarization of the cell membrane causes inhibition of cell locomotion and pinocytosis inAcanthamoeba castellanii andAmoeba proteus

Summary 10 M CCCP protonophore in an acidic medium causes depolarization of the cell membrane and immediate cessation of locomotion inAcanthamoeba castellanii andAmoeba proteus. In the basic media there is no depolarization or inhibition of cell locomotion. Other depolarizing agents (alkali cations, crown molecules) also stop locomotion and induce pinocytosis in amoeba. Pinocytotic uptake of horseradish peroxidase byAcanthamoeba castellanii is increased by 69% in the presence of CCCP in the medium at pH 5.7 but is not influenced at higher pH values. This might indicate that both amoeboid locomotion and pinocytosis are controlled by membrane potential.     

Structure of a cell polarity regulator, a complex between atypical PKC and Par6 PB1 domains

A complex of atypical PKC and Par6 is a common regulator for cell polarity-related processes, which is an essential clue to evolutionary conserved cell polarity regulation. Here, we determined the crystal structure of the complex of PKCiota and Par6alpha PB1 domains to a resolution of 1.5 A. Both PB1 domains adopt a ubiquitin fold. PKCiota PB1 presents an OPR, PC, and AID (OPCA) motif, 28 amino acid residues with acidic and hydrophobic residues, which interacts with the conserved lysine residue of Par6alpha PB1 in a front and back manner. On the interface, several salt bridges are formed including the conserved acidic residues on the OPCA motif of PKCiota PB1 and the conserved lysine residue on the Par6alpha PB1. Structural comparison of the PKCiota and Par6alpha PB1 complex with the p40phox and p67phox PB1 domain complex, subunits of neutrophil NADPH oxidase, reveals that the specific interaction is achieved by tilting the interface so that the insertion or extension in the sequence is engaged in the specificity determinant. The PB1 domain develops the interaction surface on the ubiquitin fold to increase the versatility of molecular interaction.     

Mating effects on the feeding and locomotion of the German cockroach, Blattella germanica

Abstract. Feeding cycles and daily locomotory patterns of the German cockroach, Blattella germanica L. (Dictyoptera: Blattellidae), were correlated with the ovarian development cycle. To meet the nutrient requirement for ovarian development, females increased feeding before forming oothecae. Locomotory activity also increased when females became sexually receptive. All these activities reached a peak just before the formation of oothecae. Ovarian development ceased and locomotion and food consumption decreased during pregnancy. Both mated and virgin females showed similar reproductive cycles, but those of mated females were more precisely timed (intervals between successive oothecae, and pregnancy duration, were 5 ± 0.6 and 17 ± 0.6 days, respectively). However, the intervals between successive oothecae of virgin females were longer and less synchronized. During this longer interval, feeding took place immediately following the discharge of the ootheca, but locomotory activity increased 5 days later when females became sexually receptive. Mated females increased locomotory activities 1 or 2 days before the end of pregnancy, presumably searching for deposition sites for oothecae. Female adults were found to exhibit a daily nocturnal locomotory pattern. However, under the physiological demands of reproduction, the pattern could be changed, for example by increasing activity during photophase when females were sexually receptive. The physiological effects of reproduction override the control of the daily locomotory pattern by its diel clock.     

Loss of GM130 in breast cancer cells and its effects on cell migration,invasion and polarity

Spatially distinct pools of the small GTPase Cdc42 were observed, but the major focus of research so far has been to investigate its signaling at the plasma membrane. We recently showed that the Golgi pool of Cdc42 is relevant for cell polarity and that it is regulated by GM130, a Golgi matrix protein. Loss of GM130 abrogated cell polarity and consistent with the notion that polarity is frequently impaired in cancer, we found that GM130 is downregulated in colorectal cancer. Whether the loss of GM130 solely affects polarity, or whether it affects other processes relevant for tumorigenesis remains unclear. In a panel of breast cancer cells lines, we investigated the consequences of GM130 depletion on traits of relevance for tumor progression, such as survival, proliferation, adhesion, migration and invasion. We show that cellular assays that depend on polarity, such as chemotaxis and wound scratch assays, are only of limited use to investigate the role of polarity modulators in cancer. Depletion of GM130 increases cellular velocity and increases the invasiveness of breast cancer cells, therefore supporting the view that alterations of polarity contribute to tumor progression.     

High-performance liquid chromatographic analysis of the new hypoxic cell radiosensitiser, Ro 03-8799, in biological samples

A high-performance liquid chromatographic method of analysis with UV detection has been developed to measure levels of a new radiosensitiser, Ro 03-8799 and its N-oxide metabolite, in biological fluids and tissues.The accuracy and precision of the method have been determined in both plasma and urine, where the limits of quantitation are 100 and 500 ng/ml, respectively. Typical results are presented from a human volunteer study where samples were analysed by this method.Important aspects of the method, involving both sample handling techniques and chromatographic conditions are discussed.     

Pyrimidine and purine analogues, effects on cell cycle regulation and the role of cell cycle inhibitors to enhance their cytotoxicity

In anti-cancer treatment, deoxynucleoside analogues are widely used in combination chemotherapy. Improvement can be achieved by rational design of novel combinations with cell cycle inhibitors. These compounds inhibit protein kinases, preventing the cell cycle from continuing when affected by deoxynucleoside analogs. The efficacy is dependent on the site of cell cycle inhibition, whether multiple cyclin-dependent kinases are inhibited and whether the inhibitors should be given before or after the deoxynucleoside analogs. The action of cell cycle inhibition in vivo may be limited by unfavorable pharmacokinetics. Preclinical and clinical studies will be discussed, aiming to design improved future strategies.     

Selective effects of calcium chelators on anterograde and retrograde protein transport in the cell

Calcium plays a regulatory role in several aspects of protein trafficking in the cell. Both vesicle fusion and vesicle formation can be inhibited by the addition of calcium chelators. Because the effects of calcium chelators have been studied predominantly in cell-free systems, it is not clear exactly which transport steps in the secretory pathway are sensitive to calcium levels. In this regard, we have studied the effects of calcium chelators on both anterograde and retrograde protein transport in whole cells. Using both cytochemical and biochemical analyses, we find that the anterograde-directed exit of vesicular stomatitis virus G protein and the retrograde-directed exit of Shiga toxin from the Golgi apparatus are both inhibited by calcium chelation. The exit of vesicular stomatitis virus G from a pre-Golgi compartment and the exit of Shiga toxin from an endosomal compartment are sensitive to the membrane-permeant calcium chelator 1,2-bis(2-amino phenoxy)ethane-N,N,N',N'-tetraacetic acid-tetrakis (acetoxymethyl ester) (BAPTA-AM). By contrast, endoplasmic reticulum exit and endocytic internalization from the plasma membrane are not affected by BAPTA. Together, our data show that some, but not all, trafficking steps in the cell may be regulated by calcium. These studies provide a framework for a more detailed analysis of the role of calcium as a regulatory agent during protein transport.     

Water loss, cutaneous resistance, and effects of dehydration on locomotion of Eleutherodactylus frogs

Water loss, cutaneous resistance, and the effects of dehydration on jumping ability were measured in two neotropical frogs, the common coquí (Eleutherodactylus coqui) and the cave coquí (Eleutherodactylus cooki). In both species jumping performance declined with an increase in water loss and a greater duration of exposure to dehydrating conditions. The arboreal species, E. coqui, had a slightly higher rate of water loss and lower cutaneous resistance than the non-arboreal species, E. cooki. However, differences in cutaneous resistance and water loss were too small to explain differences in geographic distributions of these species. In both species, a decline in boundary layer resistance contributed to an increased rate of water loss at higher temperature. Accepted: 4 January 1999     

Differential effects on cell motility, embryonic stem cell self-renewal and senescence by diverse Src kinase family inhibitors

The Src family of non-receptor tyrosine kinases (SFKs) has been shown to play an intricate role in embryonic stem (ES) cell maintenance. In the present study we have focused on the underlying molecular mechanisms responsible for the vastly different effects induced by various commonly used SFK inhibitors. We show that several diverse cell types, including fibroblasts completely lacking SFKs, cannot undergo mitosis in response to SU6656 and that this is caused by an unselective inhibition of Aurora kinases. In contrast, PP2 and PD173952 block motility immediately upon exposure and forces cells to grow in dense colonies. The subsequent halt in proliferation of fibroblast and epithelial cells in the center of the colonies approximately 24 h post-treatment appears to be caused by cell-to-cell contact inhibition rather than a direct effect of SFK kinase inhibition. Interestingly, in addition to generating more homogenous and dense ES cell cultures, without any diverse effect on proliferation, PP2 and PD173652 also promote ES cell self-renewal by reducing the small amount of spontaneous differentiation typically observed under standard ES cell culture conditions. These effects could not be mirrored by the use of Gleevec, a potent inhibitor of c-Abl and PDGFR kinases that are also inhibited by PP2.     

Selective effects of cyclosporin A on functional B cell subsets in the mouse

Cyclosporin A, an immunosuppressive peptide of fungal origin, was believed to selectively affect T lymphocyte functions and to have minimal affects on B lymphocytes. This study shows that, in the mouse, T-dependent B cells and those responding to certain T-independent antigens (so-called TI-1 antigens) are indeed resistant to the drug. However, B cells responsive to other TI antigens (TI-2) are exquisitely sensitive. Thus, doses of the drug that completely abrogate responses to dinitrophenylated (DNP) Ficoll or dextran enhance the response to DNP-lipopolysaccharide and have minimal effects on the response to DNP-Brucella abortus. Virgin T helper cells are sensitive to the drug, whereas primed T cells are not. Cyclosporin A sensitivity therefore represents a novel marker of functional B cell subsets in the mouse and presumably points to fundamental physiologic differences between such subsets.     

Methionine 35 oxidation reduces toxic effects of the amyloid beta-protein fragment (31-35) on human red blood cell

Amyloid beta-peptide, the central constituent of senile plaques in Alzheimer's disease brain, has been shown to be a source of free radical oxidative stress that may lead to neurodegeneration. In particular, it is well known that oxidation of methionine 35, is strongly related to the pathogenesis of Alzheimer's disease, since it represents the residue in the beta-amyloid peptide most susceptible to oxidation "in vivo". In this study, the fragment 31-35 of the beta-amyloid peptide, which has a single methionine at residue 35, was used to investigate the influence of the oxidation state of methionine-35 on the beta-amyloid peptide (31-35) mediated cytotoxic effects. Because no extensive studies have yet addressed whether amyloid beta peptides-mediated toxic effects can occur in the absence of mitochondria, human red blood cells were used as cell model. Exposure of intact red blood cells to beta-amyloid peptide (31-35) induced a marked stimulation (approximately 45%) of the pentose phosphate pathway and a significant inhibition of the red cell enzyme catalase, compared with the results observed in control red blood cells. In contrast, exposure of red blood cells to the beta-amyloid peptide (31-35)-Met35OX i.e. in which the sulfur of methionine is oxidised to sulfoxide, induced a slight activation of PPP (approximately 19%), and an inhibition of catalase activity lower with respect to the results observed in beta-amyloid peptide (31-35)-treated red blood cells. Since the activities of red cell phosphofructokinase, glucose-6-phosphate dehydrogenase, glutathione peroxidase, glutathione reductase and the functionality of hemoglobin were not modified within the red cell following to beta-amyloid peptides exposure, it is likely that beta-amyloid (31-35)-catalase interaction may represent a selective toxic event. Together, these results support the hypothesis that Abeta peptide and the oxidative state of Met-35 may be involved in the mechanisms responsible of neurodegeneration in Alzheimer's disease.     

Mediation of chemoattractant-induced changes in [Ca2+]i and cell shape, polarity, and locomotion by InsP3, DAG, and protein kinase C in newt eosinophils

During chemotaxis large eosinophils from newts exhibit a gradient of [Ca2+]i from rear to front. The direction of the gradient changes on relocation of the chemoattractant source, suggesting that the Ca2+ signal may trigger the cytoskeletal reorganization required for cell reorientation during chemotaxis. The initial stimulatory effect of chemoattractant on [Ca2+]i and the opposite orientations of the intracellular Ca2+ gradient and the external stimulus gradient suggest that more than one chemoattractant-sensitive messenger pathway may be responsible for the generation of spatially graded Ca2+ signals. To identify these messengers, Ca2+ changes were measured in single live cells stimulated with spatially uniform chemoattractant. On stimulation spatially averaged [Ca2+]i increased rapidly from < or = 100 nM to > or = 400 nM and was accompanied by formation of lamellipods. Subsequently cells flattened, polarized and crawled, and [Ca2+]i fluctuated around a mean value of approximately 200 nM. The initial Ca2+ spike was insensitive acutely to removal of extracellular Ca2+ but was abolished by treatments expected to deplete internal Ca2+ stores and by blocking receptors for inositol-trisphosphate, indicating that it is produced by discharge of internal stores, at least some of which are sensitive to InsP3. Activators of protein kinase C (PKC) (diacyl glycerol and phorbol ester) induced flattening and lamellipod activity and suppressed the Ca2+ spike, while cells injected with PKC inhibitors (an inhibitory peptide and low concentrations of heparin-like compounds) produced an enhanced Ca2+ spike on stimulation. Although cell flattening and lamellipod activity were induced by chemoattractant when the normal Ca2+ response was blocked, cells failed to polarize and crawl, indicating that Ca2+ homeostasis is required for these processes. We conclude that InsP3 acting on Ca2+ stores and DAG acting via PKC regulate chemoattractant-induced changes in [Ca2+]i, which in turn control polarization and locomotion. We propose that differences in the spatial distributions of InsP3 and DAG resulting from their respective hydrophilic and lipophilic properties may change Ca2+ distribution in response to stimulus reorientation, enabling the cell to follow the stimulus.