calcimycin and phosphatidylinositol-4-phosphate

In isolated synaptosomes from rat brain, 100 microM antimycin A and 10 microM oxamic acid inhibit the 32Pi-labeling of phosphatidylinositol-4,5-bisphosphate (PIP2) and phosphatidylinositol-4-phosphate (PIP) by 90% and 95-99% respectively. 10 mM sodium fluoride inhibits the labeling by 50-60% and 10 mM A23187 inhibits the labeling by 63-70%. Phospholipase A2 inhibits the labeling of PIP2 and PIP by 93-94% and stimulates their degradation by 84-92%. Depolarization of synaptosomes with 75 mM K+ or 100 microM veratrine decreases the labeling of PIP2 and PIP by 66-74%. The decreased labeling results in large part from the Ca(2+)-dependent degradation of 32P-labeled PIP2 and PIP as shown by pulse-chase experiments in which PIP2 and PIP were prelabeled with 32Pi. Depolarization of synaptosomes results in the stimulation of 45Ca2+ uptake with the concomitant hydrolysis of PIP and PIP2. Addition of 1 mM Ca2+ accounts for 25% of the enhanced degradation whereas depolarization with 75 mM K+ accounts for 75% of the enhanced degradation of PIP2 and PIP. Depolarization with 100 mM veratrine results in a 223% increase in inositol trisphosphate as evidenced by stimulation of 45Ca2+ uptake. EGTA (10 mM) and Mg2+ (5-10 mM) inhibit the degradation of PIP and PIP2 and counteract the action of 1 mM Ca2+. Our data demonstrate that 45Ca2+, Mg2+, and membrane depolarization play an important role in the turnover of membrane phosphatidylinositols.

Topics: Animals; Antimycin A; Brain; Calcimycin; Calcium; Egtazic Acid; Magnesium; Male; Oxamic Acid; Phosphates; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositol Phosphates; Phosphatidylinositols; Phospholipases A; Phospholipases A2; Phosphorus Radioisotopes; Rats; Rats, Sprague-Dawley; Sodium Fluoride; Synaptosomes

When ram spermatozoa were treated with Ca2+ and the ionophore A23187 to induce acrosomal exocytosis, a rise in diacylglycerol (DAG) mass was observed, concomitant with a rapid breakdown of [32P]P1-labelled phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate and a rise in [32P]Pi-labelled phosphatidate. Inclusion of the DAG lipase inhibitor RHC 80267 resulted in further but biphasic increases in DAG; there was an increasing accumulation of DAG with concentrations of RHC 80267 up to 10 microM, whereas higher concentrations produced lessening accumulation. Inclusion of RHC 80267 in the ionophore induction system also resulted in significant accelerations of the onset of exocytosis. In spermatozoa stimulated with Ca2+/A23187 and the DAG kinase inhibitor R59022, a similar increase in DAG levels together with stimulation of acrosomal exocytosis were observed. Preincubation of spermatozoa with sn-1-oleoyl-2-acetylglycerol, rac-1-oleoyl-2-acetylglycerol, sn-1,2-dioctanoylglycerol and sn-1,3-dioctanoylglycerol before treatment with Ca2+/A23187 resulted in a dose-dependent stimulation of exocytosis by all these isomers. Neomycin inhibited Ca2+/A23187-induced generation of DAG together with polyphosphoinositide breakdown, as well as acrosomal exocytosis. Inclusion of exogenous DAG, however, overcame the inhibitory effect of neomycin on exocytosis. Our results suggest that DAG has a key role in acrosomal exocytosis and that it acts as a messenger rather than as a substrate from which other active metabolites are generated. The lack of stereospecificity shown by the exogenous DAGs implies that DAG does not act by stimulating protein kinase C, but the metabolite's actual target in the sperm cell is as yet unclear.

Topics: Acrosome; Animals; Calcimycin; Calcium; Cyclohexanones; Diacylglycerol Kinase; Diglycerides; Exocytosis; Lipoprotein Lipase; Male; Neomycin; Phosphatidic Acids; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositol Phosphates; Phosphatidylinositols; Phosphotransferases; Pyrimidinones; Sheep; Spermatozoa; Thiazoles

To clarify the nature of the noradrenaline (NA)-induced contraction, the effects of NA on inositol phospholipid metabolism and the actions of inositol 1,4,5-trisphosphate (InsP3) on skinned muscle of the rabbit mesenteric artery were investigated. NA, in concentrations over 1 nM, reduced the amount of phosphatidylinositol 4,5-bisphosphate (PI-P2) and increased the amount of phosphatidic acid (PA). The maximum reduction in the amount of PI-P2 and the maximum increase in the amount of PA were observed in the presence of 1 microM-NA. With prolonged application of NA, the PI-P2 was gradually restored to near the control level, but with repeated applications of NA separated by rinses with Krebs solution, there was a consistent reduction of PI-P2. The NA-induced PI-P2 breakdown was inhibited by the alpha 1-adrenoceptor blocking agent, prazosin. After incubation of the tissue with radioactive inositol-containing solution, NA transiently increased the amount of radioactive InsP3 which was followed by increases in the amount of inositol 1,4-bisphosphate (InsP2) and inositol monophosphate (InsP). After accumulation of Ca by saponin-treated muscle cells of the dog mesenteric artery dispersed by collagenase, InsP3 released Ca stored in cells but InsP2 did not. A23187 (5 microM) but not InsP3 (up to 10 microM), depleted Ca accumulated in the presence of ATP. In saponin-treated skinned muscle tissues, InsP3 in concentrations over 0.3 microM, produced contraction following accumulation of Ca into the store site. InsP3 released Ca from the same source as caffeine. The release of Ca by InsP3 appeared in a concentration-dependent manner and this release also depended on the amount of Ca stored in cells (the median effective dose (ED50) was 3.0 microM in 0.1 microM-Ca and 1.0 microM in 0.3 microM-Ca). We concluded that NA activates alpha 1-adrenoceptors, thus hydrolysing PI-P2 and synthesizing InsP3. This product can release Ca stored in cells as estimated from the contraction in skinned muscle tissues, and also reduces the residual amount of Ca stored in skinned dispersed muscle cells. Contraction evoked by NA through pharmacomechanical coupling can be explained as a function of InsP3.

Topics: Adenosine Triphosphate; Animals; Caffeine; Calcimycin; Calcium; Dogs; In Vitro Techniques; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Male; Muscle Contraction; Muscle, Smooth, Vascular; Norepinephrine; Phosphatidic Acids; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositol Phosphates; Phosphatidylinositols; Rabbits; Saponins; Sugar Phosphates; Time Factors

The addition of the tumor-promoting phorbol 12-myristate, 13-acetate to rabbit neutrophils greatly potentiates the effect of the calcium ionophore A23187 on [3H]-arachidonic acid release and [32P]-phosphatidic acid generation. At 5 X 10(-8) M A23187, the addition of 20 ng/ml PMA potentiates the action of the ionophore on [3H]-arachidonic acid release by 5-fold. At 5 X 10(-7) M A23187, PMA enhances [32P]-phosphatidic acid production by 1.5-fold. Incubation of the neutrophils with 5 X 10(-7) M ionophore for two minutes causes a significant increase in the [32P] phosphatidic acid production but does not affect the levels of [32P]-phosphatidylinositol or [32P]-phosphatidylinositol 4,5 bis-phosphate. In addition, increasing the sodium chloride concentrations in the suspending medium causes an increase in the level of phosphatidylinositol 4,5 bis-phosphate. These results suggest that the phorbol ester either acting directly or through the activation of protein kinase C modulates significantly the activities of the various forms of phospholipases, particularly A2, and/or increases the availability or amounts of their substrates.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Calcimycin; Drug Synergism; Neutrophils; Phorbols; Phosphatidic Acids; Phosphatidylinositol Phosphates; Phosphatidylinositols; Rabbits; Sodium Chloride; Tetradecanoylphorbol Acetate

Thyrotropin-releasing hormone (TRH) stimulates hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P2) by a phospholipase C (or phosphodiesterase) and elevates cytoplasmic-free Ca2+ concentration ([Ca2+]i) in GH3 pituitary cells. To explore whether hydrolysis of PtdIns-4,5-P2 is secondary to the elevation of [Ca2+]i, we studied the effects of Ca2+ ionophores, A23187 and ionomycin. In cells prelabeled with [3H]myoinositol, A23187 caused a rapid decrease in the levels of [3H]PtdIns-4,5-P2, [3H]PtdIns-4-P, and [3H]PtdIns to 88 +/- 2%, 88 +/- 4%, and 86 +/- 1% of control, respectively, and increased [3H]inositol bisphosphate to 200 +/- 20% at 0.5 min. There was no increase in [3H] Ins-P3; the lack of a measurable increase in [3H]Ins-P3 was not due to its rapid dephosphorylation. In cells prelabeled with [14C]stearic acid, A23187 increased [14C]diacylglycerol and [14C]phosphatidic acid to 166 +/- 20% and 174 +/- 17% of control, respectively. In cells prelabeled with [3H]arachidonic acid, A23187, but not TRH, increased unesterified [3H]arachidonic acid to 166 +/- 8% of control. Similar effects were observed with ionomycin. Hence, Ca2+ ionophores stimulate phosphodiesteratic hydrolysis of PtdIns-4-P but not of PtdIns-4,5-P2 and elevate the level of unesterified arachidonic acid in GH3 cells. These data demonstrate that Ca2+ ionophores affect phosphoinositide metabolism differently than TRH and suggest that TRH stimulation of PtdIns-4,5-P2 hydrolysis is not secondary to the elevation of [Ca2+]i.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Calcimycin; Cell Line; Diglycerides; Ethers; Hydrolysis; Ionomycin; Phosphatidic Acids; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositol Phosphates; Phosphatidylinositols; Pituitary Gland; Rats; Thyrotropin-Releasing Hormone; Time Factors