[en] In order to identify regulatory steps in leukotriene synthesis, the biochemical characteristics of a 5-lipoxygenase activity in a murine mast cell clone (MC-9) were investigated. Supernatants (100,000xg) of sonicated cells metabolized ¹⁴C-arachidonic acid to leukotriene B4 (LTB₄), diastereiomeric 5,12-dihydroxy-eicosatetraenoic acids (5,12-diHETEs), 5-hydroperoxy- and 5-hydroxy-eicosatetraenoic acids (5-HPETE and 5-HETE) and 5-oxo-eicosatetraenoic acid which were identified by high performance liquid chromatography (HPLC) and gas chromatograph-mass spectrometry (GC/MS). Lipoxygenase activity had a pH optimum of 6.9 and was highly dependent upon added calcium. The concentration of calcium for 50% activation (EC₅₀) was 3μM. Activity was also stimulated by ATP (EC₅₀ = 160 μM). Lipoxygenase activity exhibited a biphasic concentration dependence for arachidonic acid with maximum product formation occurring at 35 μM. The activity showed apparent lag phase kinetics which were more pronounced at low protein levels (0.8 mg/ml). The lag phase was also greatly accentuated by glutathione (1 mM) plus glutathione peroxidase (0.4 units/ml). In contrast, addition of any of several hydroperoxides, i.e. 5-, 8-, 9-, or 15-HPETEs (EC₅₀ 1 μM), shortened the lag phase. The results suggest that the cellular levels of hydroperoxides and glutathione peroxidase, as well as calcium and certain nucleotides, may be important factors regulating leukotriene synthesis

[en] MC9 mast cells stimulated by a soluble (calcium ionophore A23187) or by an Fc epsilon-receptor agonist (IgE plus hapten) produce platelet activating factor (PAF). MC9 cells incorporate either exogenous [³H]acetic acid or [³H]lyso-PAF into PAF. PAF was identified by mobility on thin layer chromatography, platelet aggregatory activity inhibitable by known PAF antagonists, and by enzymatic modification. Quantified by aggregation of rabbit platelets, MC9 cells produce 6 pmoles PAF/10(6) cells. MC9 cells express acetyltransferase activity of 0.19 nmole/5 min-mg protein. Analysis of MC9 phospholipids by HPLC showed that MC9 cells contain large amounts of phosphatidylcholine (82 nmoles/10(7) cells) but contain little ether-linked phosphatidylcholine (4 nmoles/10(7) cells)

[en] There exists circumstantial evidence for activation of phospholipase D (PLD) in intact cells. However, because of the complexity of phospholipid remodeling processes, it is essential to distinguish PLD clearly from other phospholipases and phospholipid remodeling enzymes. Therefore, to establish unequivocally PLD activity in dimethyl sulfoxide-differentiated HL-60 granulocytes, to demonstrate the relative contribution of PLD to phospholipid turnover, and to validate the hypothesis that the formation of phosphatidylethanol is an expression of PLD-catalyzed transphosphatidylation, we have developed methodologies to label HL-60 granulocytes in 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (alkyl-PC) with 32P without labeling cellular ATP. These methodologies involve (a) synthesis of alkyl-lysoPC containing 32P by a combination of enzymatic and chemical procedures and (b) incubation of HL-60 granulocytes with this alkyl-[32P] lysoPC which enters the cell and becomes acylated into membrane-associated alkyl-[32P]PC. Upon stimulation of these 32P-labeled cells with the chemotactic peptide, N-formyl-Met-Leu-Phe (fMLP), alkyl-[32P]phosphatidic acid (alkyl-[32P]PA) is formed rapidly. Because, under these conditions, cellular ATP has not been labeled with 32P, alkyl-[32P]PA must be formed via PLD-catalyzed hydrolysis of alkyl-[32P]PC at the terminal phosphodiester bond. This result conclusively demonstrates fMLP-induced activation of PLD in HL-60 granulocytes. These 32P-labeled HL-60 granulocytes have also been stimulated in the presence of ethanol to produce alkyl-[32P]phosphatidylethanol (alkyl-[32P]PEt). Formation of alkyl-[32P]PEt parallels that of alkyl-[32P]PA with respect to time course, fMLP concentration, inhibition by a specific fMLP antagonist (t-butoxycarbonyl-Met-Leu-Phe), and Ca2+ concentration

[en] The H3-histamine receptor provides feedback inhibition of histamine synthesis and release as well as inhibition of other neurotransmitter release. We have characterized this receptor by radioligand binding studies with the H3 agonist N alpha-[3H]methylhistamine ([3H]NAMHA). The results of [3H]NAMHA saturation binding and NAMHA inhibition of [3H]NAMHA binding were consistent with an apparently single class of receptors (KD = 0.37 nM, Bmax = 73 fmol/mg of protein) and competition assays with other agonists and the antagonists impromidine and dimaprit disclosed only a single class of sites. In contrast, inhibition of [3H]NAMHA binding by the specific high affinity H3 antagonist thioperamide revealed two classes of sites (KiA = 5 nM, BmaxA = 30 fmol/mg of protein; KiB = 68 nM, BmaxB = 48 fmol/mg of protein). Burimamide, another antagonist that, like thioperamide, contains a thiourea group, likewise discriminated between two classes of sites. In addition to differences between some antagonist potencies for the two receptors, there is a differential guanine nucleotide sensitivity of the two. The affinity of the H3A receptor for [3H] NAMHA was reduced less than 2-fold, whereas [3H]NAMHA binding to the H3B receptor was undetectable in the presence of guanosine 5'-O-(3-thiotriphosphate). The distinction between H3A and H3B receptor subtypes, the former a high affinity and the latter a low affinity thioperamide site, draws support from published in vitro data