calcimycin and 1-2-didecanoylglycerol

Calcium entry into mature erythrocytes (red blood cells; RBCs) is associated with multiple changes in cell properties. At low intracellular Ca(2+), efflux of potassium and water predominates, leading to changes in erythrocyte rheology. At higher Ca(2+) content, activation of kinases and phosphatases, rupture of membrane-to-skeleton bridges, stimulation of a phospholipid scramblase and phospholipase C, and induction of transglutaminase-mediated protein cross-linking are also observed. Because the physiologic relevance of these latter responses depends partially on whether Ca(2+) entry involves a regulated channel or nonspecific leak, we explored mechanisms that initiate controlled Ca(2+) influx. Protein kinase C (PKC) was considered a prime candidate for the pathway regulator, and phorbol-12 myristate-13 acetate (PMA), a stimulator of PKC, was examined for its influence on erythrocyte Ca(2+). PMA was found to stimulate a rapid, dose-dependent influx of calcium, as demonstrated by the increased fluorescence of an entrapped Ca(2+)-sensitive dye, Fluo-3/AM. The PMA-induced entry was inhibited by staurosporine and the PKC-selective inhibitor chelerythrine chloride, but was activated by the phosphatase inhibitors okadaic acid and calyculin A. The PMA-promoted calcium influx was also inhibited by omega-agatoxin-TK, a calcium channel blocker specific for Ca(v)2.1 channels. To confirm that a Ca(v)2.1-like calcium channel exists in the mature erythrocyte membrane, RBC membrane preparations were immunoblotted with antiserum against the alpha(1A) subunit of the channel. A polypeptide of the expected molecular weight (190 kDa) was visualized. These studies indicate that an omega-agatoxin-TK-sensitive, Ca(v)2.1-like calcium permeability pathway is present in the RBC membrane and that it may function under the control of kinases and phosphatases.

Topics: Adult; Agatoxins; Alkaloids; Benzophenanthridines; Calcimycin; Calcium; Calcium Channel Blockers; Calcium Channels, N-Type; Calcium Signaling; Cell Size; Diglycerides; Dimethyl Sulfoxide; Enzyme Inhibitors; Erythrocytes; Humans; Ion Transport; Ionophores; Marine Toxins; Okadaic Acid; Osmotic Fragility; Oxazoles; Phenanthridines; Phosphoprotein Phosphatases; Protein Kinase C; Spider Venoms; Staurosporine; Tetradecanoylphorbol Acetate

We have examined the effects on X-ray induced malignant transformation in vitro of a number of activators and inhibitors of protein kinase C (PKC). Several of these substances were found to enhance or inhibit transformation, and the extent of the effects on transformation were found to be consistent with the potencies of the substances in activating or inhibiting PKC. Additionally, the observed transformation enhancement was found to be reversed by the presence of the anticarcinogenic protease inhibitors antipain or the Bowman-Birk inhibitor. These results suggest that activation of protein kinase C may be involved in the mechanism of in vitro X-ray induced malignant transformation.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Antipain; Calcimycin; Cell Transformation, Neoplastic; Diglycerides; Enzyme Activation; Isoquinolines; Mice; Mice, Inbred C3H; Neoplasms, Radiation-Induced; Piperazines; Protein Kinase C; Sulfonamides; Trifluoperazine; Trypsin Inhibitor, Bowman-Birk Soybean

The effect of protein kinase C activation and dibutyryl cyclic AMP on oxytocin secretion by ovine luteal tissue slices was investigated. Several putative regulators of luteal oxytocin secretion were also examined. Oxytocin was secreted by luteal tissue slices at a basal rate of 234.4 +/- 32.8 pmol/g per h (n = 24) during 60-min incubations. Activators of protein kinase C: phorbol 12, 13-dibutyrate (n = 8), phorbol 12-myristate, 13-acetate (n = 4) and 1,2-didecanoylglycerol (n = 5), caused a dose-dependent stimulation of oxytocin secretion in the presence of a calcium ionophore (A23187; 0.2 mumol/l). Phospholipase C (PLC; 50-250 units/l) also caused a dose-dependent stimulation of oxytocin secretion by luteal slices. Phospholipase C-stimulated oxytocin secretion was potentiated by the addition of an inhibitor of diacylglycerol kinase (R59 022; n = 4). These data suggest that the activation of protein kinase C has a role in the stimulation of luteal oxytocin secretion. The results are also consistent with the involvement of protein kinase C in PLC-stimulated oxytocin secretion. The cyclic AMP second messenger system does not appear to be involved in the control of oxytocin secretion by the corpus luteum.

Topics: Animals; Bucladesine; Calcimycin; Corpus Luteum; Diglycerides; Dose-Response Relationship, Drug; Enzyme Activation; Female; In Vitro Techniques; Oxytocin; Phorbol 12,13-Dibutyrate; Platelet Activating Factor; Protein Kinase C; Pyrimidinones; Sheep; Tetradecanoylphorbol Acetate; Thiazoles; Type C Phospholipases

The intracellular Ca2+ thresholds for platelet shape change and aggregation by A23187 and palmitoyl lysophosphatidic acid were approximately 350 and 750 nM, respectively, as estimated using quin2. The similar thresholds for these two agonists imply they activate platelets through a similar mechanism. In the absence of cyclooxygenase inhibitors, both agents induce the formation of [3H]inositol phosphates, reflecting the activation of phospholipase C. This activation of phospholipase C is blocked by the cyclooxygenase inhibitor indomethacin. It is suggested that platelet activation by palmitoyl lysophosphatidic acid involves an initial mobilization of intracellular Ca2+ with subsequent activation of phospholipase A2; the arachidonic acid metabolites formed then stimulate phospholipase C.

Topics: Blood Platelets; Calcimycin; Calcium; Cyclooxygenase Inhibitors; Diglycerides; Enzyme Activation; Humans; Indomethacin; Inositol Phosphates; Lysophospholipids; Phosphatidic Acids; Phosphorylation; Platelet Aggregation; Sugar Phosphates; Type C Phospholipases