ucn-1028-c and 1-oleoyl-2-acetylglycerol

We examined the roles of Ca2+ and protein kinase C (PKC) in the cilio-excitatory response to serotonin in pedal ciliary cells from Helisoma trivolvis embryos. Serotonin (5-hydroxytryptamine; 5-HT; 100 micromol/L) induced an increase in ciliary beat frequency (CBF) was abolished by microinjected BAPTA (50 mmol/L), but was only partially inhibited by the phospholipase C inhibitor U-73122 (10 micromol/L). The diacylglycerol analogs 1-oleoyl-2-acetyl-sn-glycerol (100 micromol/L) and 1,2-dioctanoyl-sn-glycerol (100 micromol/L) caused increases in [Ca2+]i that were smaller than those induced by serotonin. In the absence of extracellular Ca2+, 1,2-dioctanoyl-sn-glycerol (100 micromol/L) failed to elicit an increase in both CBF and [Ca2+]i. In contrast, the serotonin-induced increase in CBF persisted in the absence of extracellular Ca2+, although the increase in [Ca2+]i was abolished. PKC inhibitors bisindolylmaleimide (10 and 100 nmol/L) and calphostin C (10 nmol/L) partially inhibited the serotonin-induced increase in CBF, but didn't affect the serotonin-induced change in [Ca2+]i. These findings suggest that an intracellular store-dependent increase in [Ca2+]i mediates the cilio-excitatory response to serotonin. Furthermore, although PKC is able to cause an increase in [Ca2+]i through calcium influx, it contributes to the cilio-excitatory response to 5-HT through a different mechanism.

Topics: Animals; Calcium; Cells, Cultured; Cilia; Diglycerides; Embryo, Nonmammalian; Excitatory Amino Acids; Excitatory Postsynaptic Potentials; Indoles; Ionomycin; Maleimides; Naphthalenes; Protein Kinase C; Protein Kinase Inhibitors; Serotonin; Snails

The aim of the present study was to investigate the implication of protein kinase C (PKC) in the mouse egg activation process. We used OAG (1-oleoyl-2-acetyl-sn-glycerol) as a PKC activator, calphostin C as a specific PKC inhibitor, and the calcium ionophore A23187 as a standard parthenogenetic agent. The exposure of zona-free eggs to 150 microM or 50 microM OAG for 10 min resulted in meiosis II completion in approximately 80% of instances. By contrast, at a lower concentration (25 microM), the PKC stimulator was ineffective as parthenogenetic agent. Shortly after the application of 150 microM OAG, the cytosolic Ca2+ concentration ([Ca2+]i) increased transiently in all the eggs examined, whereas after the addition of 50 microM OAG, [Ca2+]i remained unchanged for at least 20 min. During this period, the activity of M-phase promoting factor (MPF) dramatically decreased and most of the eggs entered anaphase except when the PKC was inhibited by calphostin C. Similarly, MPF inactivation and meiosis resumption were prevented in calphostin C-loaded eggs following treatment with A23187, even though the ionophore-induced Ca2+ signalling was not affected. Taken together, our results indicate that stimulation of PKC is a sufficient and necessary event to induce meiosis resumption in mouse eggs and strongly suggest that, in this species, the mechanism by which a transient calcium burst triggers MPF inactivation involves a PKC-dependent pathway.

Topics: Animals; Calcimycin; Calcium; Diglycerides; Enzyme Activation; Enzyme Inhibitors; Female; Ionophores; Maturation-Promoting Factor; Meiosis; Mesothelin; Mice; Microscopy, Fluorescence; Naphthalenes; Oocytes; Parthenogenesis; Protein Kinase C; Spindle Apparatus

Muscarinic receptor stimulation increases Ca2+ sensitivity, i.e., the amount of force produced at a constant submaximal cytosolic Ca2+ concentration ([Ca2+]i), in permeabilized smooth muscle preparations. It is controversial whether this increase in Ca2+ sensitivity is in part mediated by protein kinase C (PKC). With the use of a beta-escin permeabilized canine tracheal smooth muscle (CTSM) preparation, the effect of four putative PKC inhibitors [calphostin C, chelerythrine chloride, a pseudosubstrate inhibitor for PKC [PKC peptide-(19-31)], and staurosporine] on Ca2+ sensitization induced by acetylcholine (ACh) plus GTP was determined. Preincubation with each of the inhibitors did not affect subsequent Ca2+ sensitization induced by muscarinic receptor stimulation in the presence of a constant submaximal [Ca2+]i, neither did any of these compounds reverse the increase in Ca2+ sensitivity induced by ACh plus GTP. Administration of a 1,2-diacylglycerol analog, 1-oleoyl-2-acetyl-sn-glycerol, did not induce Ca2+ sensitization at a constant submaximal [Ca2+]i. Thus we found no evidence that PKC mediates increases in Ca2+ sensitivity produced by muscarinic receptor stimulation in permeabilized CTSM.

Topics: Acetylcholine; Alkaloids; Animals; Benzophenanthridines; Calcium; Cell Membrane Permeability; Cytosol; Diglycerides; Dogs; Enzyme Inhibitors; Escin; Female; Guanosine Triphosphate; In Vitro Techniques; Kinetics; Male; Muscle Contraction; Muscle, Smooth; Naphthalenes; Peptide Fragments; Phenanthridines; Protein Kinase C; Receptors, Muscarinic; Staurosporine; Trachea

1. The effects of the vasoconstrictor angiotensin II (Ang II) on whole-cell ATP-sensitive K+ currents (IK,ATP) of smooth muscle cells isolated enzymatically from rat mesenteric arteries were investigated using the patch clamp technique. 2. Ang II, at a physiological concentration (100 nM), reduced IK,ATP activated by 0.1 mM internal ATP and 10 microM levcromakalim by 36.4 +/- 2.3%. 3. The protein kinase C (PKC) activator 1-oleoyl-2-acetyl-sn-glycerol (OAG, 1 microM) reduced IK,ATP by 44.1 +/- 2.7%. GDP beta S (1 mM), included in the pipette solution, abolished the inhibition by Ang II, while that by OAG was unaffected. 4. Pretreatment with the PKC inhibitors staurosporine (100 nM) or calphostin C (500 nM) prevented the Ang II-induced inhibition of IK,ATP. 5. Ang II inhibition was unaffected by cell dialysis with PKA inhibitor peptide (5 microM), and the PKA inhibitor Rp-cAMPS (100 microM) did not reduce IK,ATP. 6. Our results suggest that Ang II modulates KATP channels through activation of PKC but not through inhibition of PKA.

Topics: Adenosine Triphosphate; Angiotensin II; Animals; Cromakalim; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Diglycerides; Enzyme Inhibitors; Glyburide; Guanosine Diphosphate; Hypoglycemic Agents; Male; Membrane Potentials; Mesenteric Arteries; Muscle, Smooth, Vascular; Naphthalenes; Patch-Clamp Techniques; Potassium; Potassium Channel Blockers; Potassium Channels; Protein Kinase C; Rats; Rats, Wistar; Staurosporine; Thionucleotides; Vasoconstrictor Agents; Vasodilator Agents

The modulation by protein kinase C (PKC) of the RCK1 K+ channel was investigated in Xenopus oocytes by integration of two-electrode voltage clamp, site-directed mutagenesis and SDS-PAGE analysis techniques. Upon application of beta-phorbol 12-myristate 13-acetate (PMA) the current was inhibited by 50-90%. No changes in the voltage sensitivity of the channel, changes in membrane surface area or selective elimination of RCK1 protein from the plasma membrane could be detected. The inhibition was mimicked by 1-oleoyl-2-acetyl-rac-glycerol (OAG) but not by alphaPMA, and was blocked by staurosporine and calphostin C. Upon deletion of most of the N-terminus a preceding enhancement of about 40% of the current was prominent in response to PKC activation. Its physiological significance is discussed. The N-terminus deletion eliminated 50% of the inhibition. However, phosphorylation of none of the ten classical PKC phosphorylation sites on the channel molecule could account, by itself or in combination with others, for the inhibition. Thus, our results show that PKC activation can modulate the channel conductance in a bimodal fashion. The N-terminus is involved in the inhibition, however, not via its direct phosphorylation.

Topics: Alkaloids; Animals; Base Sequence; Brain; Cell Membrane; Diglycerides; DNA Primers; Enzyme Activation; Enzyme Inhibitors; Female; Kinetics; Membrane Potentials; Models, Structural; Molecular Sequence Data; Mutagenesis, Site-Directed; Naphthalenes; Oocytes; Patch-Clamp Techniques; Potassium Channels; Protein Kinase C; Protein Structure, Secondary; Rats; Recombinant Proteins; Staurosporine; Tetradecanoylphorbol Acetate; Time Factors; Xenopus laevis

The involvement of protein kinase C (PKC) in exocytosis of the mammalian sperm acrosome is still a controversial issue. Work carried out thus far has failed to provide direct evidence for the activation of this enzyme upon stimulation with natural agonists of acrosomal exocytosis. We have therefore used progesterone stimulation of the acrosome reaction in human spermatozoa to clarify this issue. In spermatozoa preincubated under conditions known to support capacitation and fertilization in vitro, treatment with progesterone caused a time-dependent stimulation of phosphorylation of at least eight proteins ranging in size from approximately 20-220 kDa. The inclusion of the PKC inhibitors chelerythrine chloride or calphostin C reduced the observed phosphorylation in a concentration-dependent manner. Exogenously supplied phorbol 12-myristate-13-acetate (PMA) or the permeant diacylglycerol 1-oleoyl-2-acetyl-sn-glycerol (OAG), synthetic activators of PKC, also stimulated phosphorylation in preincubated spermatozoa, but inclusion of calphostin C diminished the response. Furthermore, the prior inclusion of the 1,4-dihydropyridine Ca2+ channel antagonist nifedipine also inhibited phosphorylation, suggesting that PKC is activated downstream of Ca2+ channel opening. Exocytosis triggered by progesterone was significantly inhibited by chelerythrine chloride or calphostin C. Both PMA and OAG triggered exocytosis, but the inclusion of chelerythrine chloride significantly inhibited the response; exocytotic responses were seen only in capacitated cells. These results provide the first direct evidence that PKC activation plays a role in the signal transduction pathway underlying acrosomal exocytosis in progesterone-stimulated capacitated spermatozoa.

Topics: Acrosome; Alkaloids; Benzophenanthridines; Calcium Channel Blockers; Calcium Channels; Diglycerides; Enzyme Activation; Enzyme Inhibitors; Exocytosis; Humans; In Vitro Techniques; Male; Naphthalenes; Nifedipine; Phenanthridines; Phosphorylation; Progesterone; Protein Kinase C; Signal Transduction; Sperm Capacitation; Spermatozoa; Tetradecanoylphorbol Acetate

We evaluated the role of the protein kinase C (PKC) and its isozymes in the activation of human endothelial cells (EC) stimulated by platelet-activating factor (PAF). Exposure of confluent EC to PAF resulted in a rapid and concentration-dependent redistribution of PKC from cytosol to plasma-membrane, rearrangement of cytoskeleton (i.e. decrease in F-actin content and redistribution of vinculin), and finally increase in the transendothelial flux of 125I-albumin. Stimulation of EC with oleylacetylglycerol or phorbol 12-myristate 13-acetate induced the modification of the cytoskeletal structures and the increase of 125I-albumin clearance. Inhibitors of PKC prevented the effects induced by PAF on the cytoskeleton and on the barrier function of the EC monolayer. Confluent EC expressed only alpha, beta, and epsilon PKC isoforms. Biochemical and immunochemical analysis showed that the time course of the PKC isozymes translocation from cytosol to the membrane fraction of EC stimulated by PAF was different: beta isoform was redistributed more quickly than alpha isoform. PAF did not induce translocation of PKC epsilon. These results suggest that activation of PKC alpha and beta is an important signal transduction pathway by which PAF activates endothelial monolayer and modify its function of barrier to macromolecules.

Topics: Actins; Amino Acid Sequence; Antibodies; Cell Membrane; Cells, Cultured; Chromatography, Ion Exchange; Cytoskeletal Proteins; Cytosol; Diglycerides; Diterpenes; Endothelium, Vascular; Enzyme Activation; Ginkgolides; Humans; Isoenzymes; Kinetics; Lactones; Molecular Sequence Data; Naphthalenes; Peptides; Phospholipid Ethers; Platelet Activating Factor; Polycyclic Compounds; Protein Kinase C; Protein Kinase Inhibitors; Tetradecanoylphorbol Acetate; Thiazoles; Vinculin

The ionic mechanism of the effect of extracellularly ejected calcitonin (CT) on the membrane of identified neurons R9 and R10 of Aplysia was investigated with voltage-clamp, micropressure ejection, and ion substitution techniques. Micropressure-ejected CT caused a marked hyperpolarization in the unclamped neuron. Heat-inactivated CT was without effect. Clamping the same neuron at its resting potential level (-60 mV) and re-ejecting CT with the same dose produced a slow outward current (Io(CT), 30-40 sec in duration, 4-6 nA in amplitude) associated with a decrease in input membrane conductance. Io(CT) was decreased by depolarization and increased by hyperpolarization. The extrapolated reversal potential of Io(CT) was approximately +10 mV. Io(CT) was sensitive to changes in the external Na+ concentration but not to changes in K+, Ca2+, and Cl- concentrations. Micropressure-ejected forskolin produced a slow outward current, which, like the current to CT, was associated with a decrease in input membrane conductance, and was sensitive to changes in the external Na+ concentration. Io(CT) was prolonged by bath-applied isobutylmethylxanthine (IBMX) but was not affected by 1-oleoyl-2-acetylglycerol (OAG) and calphostin C. Neither superfusion of the neuron with nordihydroguaiaretic acid (NDGA) nor superfusion with indomethacin caused any changes in Io(CT). These results suggest that extracellular CT can induce a slow outward current associated with a decrease in Na+ conductance, mediated by a receptor-controlled increase in intracellular cyclic adenosine 3',5'-monophosphate.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Aplysia; Calcitonin; Calcium; Colforsin; Cyclic AMP; Diglycerides; Ganglia, Invertebrate; Indomethacin; Masoprocol; Membrane Potentials; Microinjections; Naphthalenes; Neurons; Polycyclic Compounds; Potassium; Protein Kinase C; Sodium; Sodium Channels