Cardiac and coronary consequences of intracoronary platelet activating factor infusion in the domestic pig

In previous studies we have shown that platelet-activating factor (PAF) is a potent vasoactive substance with deleterious effects on coronary blood flow (CBF) and myocardial performance. The present study further investigates the effects of PAF during its sustained intracoronary infusion in the blood-perfused domestic pig (n = 16). PAF infusion (1-9 nmol/min) produced triphasic changes in CBF (n = 7): an initial brief phase of coronary dilation (14 +/- 2% above baseline), followed by severe reduction in CBF due to increase in coronary vascular resistance and a third phase of escape that was characterized by return of CBF towards baseline in spite of continuing PAF infusion. In 9 remaining pigs PAF infusion had a biphasic response: the first phase of coronary dilation rapidly turned into severe coronary constriction accompanied by severe systemic hypotension and death within a few min. PAF infusion caused a profound rise in systemic arterial and coronary venous thromboxane B2 levels, while 6-keto-PGF1 alpha and leukotriene C4-immunoreactivity levels were not changed. Indomethacin completely blocked the rise in thromboxane level during PAF infusion and abolished the constrictor effect of PAF on the coronary vessels. These data suggest that PAF might play a detrimental role on the coronary circulation and cardiac function, primarily through thromboxane A2 mediated mechanism.     

The final step in the de novo biosynthesis of platelet-activating factor. Properties of a unique CDP-choline:1-alkyl-2-acetyl-sn-glycerol choline-phosphotransferase in microsomes from the renal inner medulla of rats

Final steps in the synthesis of platelet activating factor (PAF) occur via two enzymatic reactions: the acetylation of 1-alkyl-2-lyso-sn-glycero-3-phosphocholine by a specific acetyltransferase or the transfer of the phosphocholine base group from CDP-choline to 1-alkyl-2-acetyl-sn-glycerol by a dithiothreitol (DTT)-insensitive cholinephosphotransferase. Our studies demonstrate that rat kidney inner medulla microsomes synthesize PAF primarily via the DTT-insensitive cholinephosphotransferase since the specific activity of this enzyme is greater than 100-fold higher than the acetyltransferase. The two cholinephosphotransferases that catalyze the biosynthesis of phosphatidylcholine and PAF have similar Mg2+ or Mn2+ requirements and are inhibited by Ca2+. Also topographic experiments indicated that both activities are located on the cytoplasmic face of microsomal vesicles. PAF synthesis was slightly stimulated by 10 mM DTT, whereas the enzymatic synthesis of phosphatidylcholine was inhibited greater than 95% under the same conditions. The concept of two separate enzymes for PAF and phosphatidylcholine synthesis is further substantiated by the differences in the two microsomal cholinephosphotransferase activities with respect to pH optima, substrate specificities, and their sensitivities to temperature, deoxycholate, or ethanol. Study of the substrate specificities of the DTT-insensitive cholinephosphotransferase showed that the enzyme prefers a lipid substrate with 16:0 or 18:1 sn-1-alkyl chains. Short chain esters at the sn-2 position (acetate or propionate) are utilized by the DTT-insensitive cholinephosphotransferase, but analogs with acetamide or methoxy substituents at the sn-2 position are not substrates. Also, CDP-choline is the preferred water-soluble substrate when compared to CDP-ethanolamine. Utilization of endogenous neutral lipids as a substrate by the DTT-insensitive cholinephosphotransferase demonstrated that sufficient levels of alkylacetylglycerols are normally present in rat kidney microsomes to permit the synthesis of physiological quantities of PAF. These data suggest the renal DTT-insensitive cholinephosphotransferase could be a potentially important enzyme in the regulation of systemic blood pressure.     

Cell-free detection and characterization of a novel nerve growth factor-activated protein kinase in PC12 cells

We have developed a cell-free assay to detect and characterize nerve growth factor (NGF)-activated protein kinase activity. Cultured PC12 cells were briefly exposed to NGF, and extracts of these were assayed for phosphorylating activity using exogenously added tyrosine hydroxylase as substrate. Tyrosine hydroxylase was employed since it is an endogenous substrate of NGF-regulated kinase activity and is activated by phosphorylation. In the cell-free assay, extracts prepared from NGF-treated cells yielded a 2-3-fold greater incorporation of phosphate into tyrosine hydroxylase as compared with extracts of control, NGF-untreated cells. Activation did not occur, however, if NGF was added directly to cell extracts. The NGF-stimulated phosphorylating activity appeared to be due to regulation of a protein kinase rather than of a phosphoprotein phosphatase. Characterization of the kinase (designated as kinase N) showed that it is soluble, is detectably activated within 1-3 min after cells are exposed to NGF and maximally activated by 10 min, is half-maximally activated with 0.5 nM NGF and maximally activated with 1 nM NGF, is detectable in the presence of either Mg2+ or Mn2+ but does not require Ca2+, does not require nonmacromolecular cofactors, can use histone H1 as a substrate, and exhibits a 2-fold increase in apparent Vmax in response to NGF but does not undergo a significant change in apparent Km for either ATP or GTP. A number of characteristics of kinase N were assessed including susceptibility to inhibitors, substrate specificity, cofactor requirements, ATP dependence, and lack of down-regulation by prolonged expose to a phorbol ester. These studies indicated that it lacks tyrosine kinase activity and is distinct from a variety of well-characterized protein kinases including cAMP-dependent protein kinase, protein kinase C (Ca2+/phospholipid-dependent enzyme), Ca2+/calmodulin-dependent kinase, and casein kinase II. Preliminary purification data show that the kinase has a basic pI and that it has an apparent Mr of 22,000-25,000. The only amino acid in tyrosine hydroxylase found to be phosphorylated by the semipurified kinase is serine.     

Synthesis and biochemical studies of analogs of platelet-activating factor bearing a methyl group at C2 of the glycerol backbone

Two platelet-activating factor (PAF) analogs containing a methyl group at C2 of the glycerol moiety were synthesized, and some of their biochemical properties were investigated. 1-O-Hexadecyl-2-C,O-dimethyl-rac-glycero-3-phosphocholine (2-methyl-2-methoxy PAF) was prepared in a synthetic scheme beginning with the etherification of 2-methylpropen-1-ol. A reaction sequence involving hydroxylation, tritylation, alkylation, and detritylation afforded 1-O-hexadecyl-2-C,O-dimethyl-rac-glycerol, which was converted into the phosphocholine. A 2-lyso derivative of this PAF analog (2-methyl-lyso PAF) was synthesized from 1-O-hexadecyl-2-C-methyl-3-O-trityl-rac-glycerol. Benzylation followed by detritylation gave 1-O-hexadecyl-2-C-methyl-2-O-benzyl-rac-glycerol, which was converted into the phosphocholine compound. Hydrogenolysis afforded 1-O-hexadecyl-2-C-methyl-rac-glycero-3-phospholine (2-methyl-lyso PAF). The 2-methyl-lyso PAF analog served as a substrate for the acetyl-CoA-dependent acetyltransferase that acetylates 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine. However, 2-methyl-lyso PAF did not have a significant effect on the activities of a CoA-independent transacylase or of the acetylhydrolase that inactivates PAF, and thus does not appear to be a substrate or an inhibitor, respectively, for these enzymes. In addition, this analog exhibited only one-half of the antitumor activity of rac-1-O-alkyl-2-methoxy-rac-glycero-3-phosphocholine in human leukemic (HL-60) cells, and elicited no hypotensive response in rats and no platelet-activating activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis of a thiophosphate analog of dioctanoylphosphatidylcholine: a phospholipase C substrate

Dioctanoylthiophosphatidylcholine, a racemic thiophosphate analog of L-alpha-dioctanoylphosphatidylcholine, has been synthesized and isolated by flash chromatography. In contrast with the didecanoylthiophosphatidylcholine synthesized previously, the analog is easily dispersed on sonication in aqueous media and is rapidly hydrolyzed to produce a free thiol group in the presence of the extracellular phospholipase C from either Bacillus cereus or Clostridium perfringens. When 5,5'-dithiobis (2-nitro-benzoic acid) was included as a thiol reactive chromogenic agent, the resultant measurement of product release, as an increase in absorbance at 412 nm, showed a linear relationship with added enzyme.     

Intramolecular disulfide loop formation in a peptide containing two cysteines

The cyanogen bromide fragment comprising residues 115-181 of Kunitz soybean trypsin inhibitor is a soluble random-coil peptide at pH 7 containing two cysteines separated by eight other amino acids in the primary sequence. Four of the six rate constants have been determined for the three disulfide exchange reactions between this fragment and oxidized and reduced forms of N-acetylcysteine methyl ester. The rate constant for intramolecular loop formation in the fragment containing one thiolate anion and one sulfur connected by a disulfide bond to the small cysteine analogue is 0.36 +/- 0.15 s-1 at 23 degrees C in 3 M guanidine hydrochloride. This measurement provides a frame of reference corresponding to formation of a small but sterically unstrained loop, the fast limit for intramolecular disulfide exchange in a random-coil peptide.     

Crosslinking of isolated cytoskeletal proteins with hemoglobin: a possible damage inflicted to the red cell membrane

Crosslinking of isolated red cell membrane cytoskeletal proteins and hemoglobin mediated by H2O2 was studied. The products of spectrin and hemoglobin interaction were demonstrated electrophoretically to be high-molecular-weight polypeptides crosslinked by nondisulfide covalent bonds. The molecular weight of the protein bands correlated with various combinations of spectrin and hemoglobin chains and the relative amount of the different products was dependent on the molar ratio of the interacting proteins. Free hemin caused spectrin crosslinking as well, but globin in the absence of hemin was inactive. Since the H2O2-mediated reaction resulted in reduction of the spectrin tryptophan fluorescence, the latter was used to monitor the reaction progress under various conditions. Both oxyhemoglobin and methemoglobin were found to be most efficient, whereas cyanmethemoglobin and hemichrome were relatively inactive. Analysis of the data implied that tryptophan oxidation as well as spectrin conformational changes follow an iron-induced crosslinking of the interacting proteins. Actin, the second major protein in the red cell cytoskeleton, behaved similarly to spectrin. The intrinsic fluorescence intensity of both G- and F-actin was decreased upon addition of H2O2 to the mixture of hemoglobin and each of the actin forms. SDS-polyacrylamide gel electrophoresis revealed that G-actin crosslinked one or two hemoglobin chains. F-actin-hemoglobin interaction induced by H2O2 produced very high aggregates that could not penetrate the gel. It is suggested that crosslinking of cytoskeletal proteins in red cells containing membrane-associated hemoglobin provides a rationale for the loss of membrane flexibility.