phosphorus-radioisotopes and 1-2-dioctanoylglycerol

Dysfunction of organs has been reported in diabetic rats, suggesting an association with changes in intracellular signal transduction pathways including phosphatidylinositol (PI) turnover. Diacylglycerol (DG) kinase catalyses the phosphorylation of DG, which is considered to play a major physiological role in the metabolism of the intracellular messenger DG. However, no relation between DG kinase activity and any disease in mammalian tissue has been reported to date. In the present study, we investigated whether the changes in DG kinase activity are related to diabetes. Basal resting level of DG kinase activity changed in tissue isolated from diabetic rats. Decreases in resting activity detected in aorta and kidney and agonist-induced responses differed between these tissues. Submaximal increases in basal activity also were detected in vas deferens and hepatocytes. These changes in DG kinase activity resemble the functional changes associated with complications of diabetes, suggesting that changes in PI turnover followed by DG kinase activity are a key element in the complications. It is the first study about the changes in DG kinase activity in mammalian disease.

Topics: Animals; Aorta; Diabetes Mellitus, Experimental; Diacylglycerol Kinase; Diglycerides; Isotope Labeling; Kidney Cortex; Liver; Male; Phosphates; Phosphatidic Acids; Phosphorus Radioisotopes; Rats; Streptozocin; Vas Deferens

The effects of 4 beta-phorbol-12-myristate-13-acetate (PMA) and 1,2-sn-dioctanoylglycerol (DOCG) on the rate of labeling of phosphatidylcholine (PC) with (32P)phosphate and the rate of formation of (3H)phosphatidylethanol (PET) from PC labeled with (3H)myristic acid were investigated in vitro in minced placentae obtained from first trimester human pregnancies. Maximally effective concentrations of PMA (1 microM) or DOCG (125-250 microM) stimulate PC-labeling with (32P)phosphate along different time courses: responses to DOCG and PMA require 30 and 60 min, respectively. The early response to DOCG is attended by a rapid accumulation of 32P)PCDOCG followed by a decline from the peak value in the second 30 min. The PMA effect is accompanied by increased rate of formation of (32P)phosphatidic acid (PA). Importantly, the effects of PMA and DOCG on PC-labeling are additive and PMA does not have any effect on the labeling of PCDOCG. These findings indicate that PMA stimulates degradation and the attendant turnover of PC, whereas a greater part of the DOCG-effect comes from the stimulation of PC synthesis de novo. Consistent with this notion is the finding that PMA enhances the PC-selective phospholipase D activity (measured by the formation of PET) 2.4-fold, whereas the effect of DOCG is smaller (1.4-1.8-fold) and not additive with that of PMA. The results provide evidence for the presence of functional PC-cycle in the primordial human placenta. The cycle can be triggered by a single addition of PMA and to a lesser extent by DOCG. The smaller effect of DOCG may be related to its short lifetime in the tissue, which is sufficient, however, to stimulate the activity of the regulatory enzyme (CTP: choline cytidylyl transferase) of PC synthesis. Since the effect of PMA on PC-labeling is diminished by protein kinase C inhibitors, this enzyme appears to be involved in the stimulation of PC-cycle by DAG and its analogs.

Topics: Diglycerides; Female; Glycerophospholipids; Humans; In Vitro Techniques; Kinetics; Myristic Acid; Myristic Acids; Phosphates; Phosphatidic Acids; Phosphatidylcholines; Phospholipids; Phosphorus Radioisotopes; Placenta; Pregnancy; Signal Transduction; Tetradecanoylphorbol Acetate; Tritium

The activation of protein kinase C was investigated in digitonin-permeabilized human neuroblastoma SH-SY5Y cells by measuring the phosphorylation of the specific protein kinase C substrate myelin basic protein4-14. The phosphorylation was inhibited by the protein kinase C inhibitory peptide PKC19-36 and was associated to a translocation of the enzyme to the membrane fractions of the SH-SY5Y cells. 1,2-Dioctanoyl-sn-glycerol had no effect on protein kinase C activity unless the calcium concentration was raised to concentrations found in stimulated cells (above 100 nM). Calcium in the absence of other activators did not stimulate protein kinase C. Phorbol 12-myristate 13-acetate was not dependent on calcium for the activation or the translocation of protein kinase C. The induced activation was sustained for 10 min, and thereafter only a small net phosphorylation of the substrate could be detected. Calcium or dioctanoylglycerol, when applied alone, only caused a minor translocation, whereas in combination a marked translocation was observed. Arachidonic acid (10 microM) enhanced protein kinase C activity in the presence of submaximal concentrations of calcium and dioctanoylglycerol. Quinacrine and p-bromophenacyl bromide did not inhibit calcium- and dioctanoylglycerol-induced protein kinase C activity at concentrations which are considered to be sufficient for phospholipase A2 inhibition.

Topics: Acetophenones; Amino Acid Sequence; Arachidonic Acids; Calcium; Cell Membrane; Cell Membrane Permeability; Digitonin; Diglycerides; Dose-Response Relationship, Drug; Enzyme Activation; Humans; Molecular Sequence Data; Myelin Basic Protein; Neuroblastoma; Phospholipases A; Phospholipases A2; Phosphorus; Phosphorus Radioisotopes; Phosphorylation; Protein Kinase C; Quinacrine; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

The incorporation of [32P]inorganic phosphate into membranous, myofibrillar, and cytosolic proteins was studied in Langendorff-perfused guinea pig hearts treated with phorbol 12-myristate 13-acetate (PMA) or 1,2-dioctanoylglycerol (D8G), which are potent activators of protein kinase C. Control hearts were perfused with an inactive phorbol ester (4 alpha-phorbol 12,13-didecanoate), which does not cause activation of protein kinase C. To ensure the blockade of different receptor systems, the perfusions were carried out in the presence of prazosin, propranolol, and atropine. Perfusion of hearts with either PMA (4 microM) or D8G (200 microM) was associated with a negative effect on left ventricular inotropy and relaxation. Examination of the 32P incorporation into various fractions revealed that there were no increases in the degree of phosphorylation of phospholamban in sarcoplasmic reticulum, and troponin I and C protein in the myofibrils, although these proteins were found to be substrates for protein kinase C in vitro. However, in the same hearts, there were significant changes in the 32P incorporation into a 28-kDa cytosolic-protein. Examination of the activity levels of protein kinase C in hearts perfused with PMA indicated a redistribution of this activity from the cytosolic to the membrane fraction, suggesting the activation of the enzyme in vivo. These findings indicate that cardiac regulatory phosphoproteins, which may be phosphorylated by protein kinase C in vitro, are not substrates for protein kinase C in beating hearts perfused with phorbol esters or diacylglycerol analogues.

Topics: Animals; Calcium; Calcium-Binding Proteins; Cyclic AMP; Cytosol; Diglycerides; Guinea Pigs; Heart; Heart Rate; In Vitro Techniques; Inositol 1,4,5-Trisphosphate; Myocardium; Myofibrils; Perfusion; Phosphates; Phosphatidylserines; Phosphoproteins; Phosphorus Radioisotopes; Phosphorylation; Protein Kinase C; Sarcoplasmic Reticulum; Substrate Specificity; Tetradecanoylphorbol Acetate; Troponin; Troponin I; Troponin T