okadaic-acid and methylamine

Macrophage expression of urokinase-type plasminogen activator (uPA) appears to play a role in their release of matrix-bound basic fibroblast growth factor (bFGF) and transforming growth factor-beta (TGF-beta). In experiments reported here, we have examined the potential regulatory effects of bFGF and TGF-beta 1 on macrophage uPA expression. TGF-beta 1 stimulated in a dose- and time-dependent manner the expression of secreted membrane and intracellular uPA activities by a macrophage cell line (RAW264.7). When examined at similar concentrations, bFGF had little effect, and interleukin-1 alpha, tumor necrosis factor-alpha, and monocyte colony stimulating factor had no effect on macrophage uPA expression. Exposure of macrophages to TGF-beta 1 led to a rapid and sustained increase in the steady-state levels of uPA mRNA that was independent of de novo protein synthesis and was completely inhibited by actinomycin D. However, the TGF-beta 1-induced increase in uPA mRNA was largely unaffected by subsequent incubation of cells with actinomycin D. The protein kinase C inhibitor H7 markedly reduced the ability of TGF-beta 1 to stimulate expression of uPA activity. Likewise, okadaic acid and microcystin, inhibitors of serine/threonine phosphatases, potentiated the ability of TGF-beta 1 to upregulate macrophage uPA expression. TGF-beta 1 primed cells converted nearly all added plasminogen to plasmin and expressed sixfold more membrane-bound plasmin than control cells. Preincubation of TGF-beta 1 with either serum or methylamine-modified alpha 2-macroglobulin did not affect its ability to induce macrophage uPA expression. When control and TGF-beta 1-primed macrophages were cultured on matrices containing bound 125I-bFGF, their release of 125I-bFGF was increased five and tenfold, respectively, in the presence of plasminogen. The ability of TGF-beta to induce macrophage uPA expression and the plasmin-dependent release of matrix-bound bFGF may provide an indirect mechanism by which TGF-beta stimulates angiogenesis.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; alpha-Macroglobulins; Animals; Cell Line; Dactinomycin; Drug Synergism; Ethers, Cyclic; Extracellular Matrix; Fibroblast Growth Factor 2; Gene Expression; Humans; Isoquinolines; Macrophages; Methylamines; Mice; Microcystins; Okadaic Acid; Peptides, Cyclic; Piperazines; Plasminogen Activator Inhibitor 1; Protein Kinase C; RNA, Messenger; Transforming Growth Factor beta; Urokinase-Type Plasminogen Activator

1. We studied two-pulse facilitation and delayed release at 0 degree C, because at low temperature facilitation is enhanced and extended whereas delayed release is increased. Our major goal was to test, by a number of approaches, the residual Ca2+ hypothesis of facilitation and delayed release. 2. As we increased the interval between pulses from 30 to 100-200 ms facilitation declined steeply. As we lengthened the interval further facilitation declined more slowly. In our entire series facilitation was still seen at 700 ms, in some preparations facilitation was apparent at 2 s. 3. We measured delayed release in preparations in which excitation-contraction was uncoupled. The decline in the rate of delayed release following the endplate potential (EPP) is similar to the decay of facilitation, both at 0 and 22 degrees C. 4. When we replaced the Ca2+ in the Ringer by Sr2+, facilitation persisted for a longer time, there was significant facilitation 2 s after an EPP. Delayed release also continued longer; the time courses for the decline of facilitation and delayed release were very similar. 5. We measured delayed release after EPPs triggered by electrotonic depolarization in isotonic CaCl2 solution or in Ringer in which the Na+ was replaced by methylamine (these solutions also contained 3,4-diaminopyridine). The time course of delayed release was very similar to that in Ringer. 6. We found that delayed release also facilitated, in the sense that the number of delayed releases, and the rate at which they were released, increased markedly after a second or third closely spaced EPP. The facilitation of delayed release and of EPPs were quantitatively similar. 7. We soaked preparations for 2 h in 200 microM bis-(aminophenoxy) ethane-tetraacetic acid (BAPTA/AM), a cell permeable Ca2+ chelator. In about one-half of these preparations facilitation was clearly diminished, judging from the EPPs evoked by a series of four to five stimuli at 30-ms intervals. The summed results from those preparations in which facilitation was reduced at 30 ms showed that it was also reduced at longer intervals. There was a comparable shortening in delayed release. Facilitation was significantly reduced when we pretreated with ethylene glycol bis-(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (EGTA/AM), another cell-permeable Ca2+ chelator. 8. It has been reported that in BAPTA loaded preparations facilitation during trains of EPPs transiently reappears after e

Topics: Aluminum; Aluminum Chloride; Aluminum Compounds; Animals; Calcium; Calcium Channels; Calcium-Binding Proteins; Chelating Agents; Chlorides; Culture Techniques; Egtazic Acid; Ethers, Cyclic; Hydrogen-Ion Concentration; Ionomycin; Lasalocid; Methylamines; Motor Endplate; Neuromuscular Junction; Okadaic Acid; Ranidae; Reaction Time; Strontium; Synaptic Transmission; Temperature; Terpenes; Thapsigargin