Jefferson R. Surles

Binding and metabolism of platelet-activating factor by human neutrophils.

Human polymorphonuclear neutrophils rapidly incorporated radiolabeled platelet-activating factor, 1-O-[hexadecyl-9, 10-3H2]-2-acetyl-sn-glycero-3-phosphocholine ([3H]PAF), and then metabolized it into its sn-2-fatty acyl derivative. Fractionation of radiolabel-pretreated cells over Percoll gradients revealed that virtually all of the intact [3H]PAF was located in nongranule membranes that were enriched with alkaline phosphatase and cell surface glycoproteins. While still membrane associated, the ligand was rapidly converted to its acyl derivative and then more slowly transferred to specific granules and, to a lesser extent, azurophilic granules. In contrast, neutrophils did not metabolize [3H]PAF at 4 degrees C but rather gradually accumulated it in their alkaline phosphatase-enriched membrane subfractions. These same subfractions contained receptors for the ligand, as determined by their capacity to bind [3H]PAF specifically. Binding was readily saturated, partially reversible, and fit a two receptor model; dissociation constant (Kd) values for high and low affinity sites were 0.2 and 500 nM, respectively. Receptors with similar affinities were detected in whole cells. Furthermore, the potencies of several structural analogues in inhibiting binding of [3H]PAF to membranes correlated closely with their respective potencies in stimulating degranulation responses. Finally, quantitative studies suggested all or most of the cells receptors were membrane associated. We conclude that PAF rapidly enters cellular membranes to bind with specific receptors that trigger function. The intramembranous ligand is also deacetylated, acylated, and then transferred to granules. This metabolism may be sufficiently rapid to limit ligand-receptor binding and distort quantitative analyses of receptors.

Stereospecific activity of 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine and comparison of analogs in the degranulation of platelets and neutrophils

Abstract 1-O-Hexadecyl-2-O-acetyl- sn -glycero-3-phosphocholine (platelet activating factor) stimulated the degranulation of rabbit platelets and human neutrophils, whereas the enantiomer, 3-O-hexadecyl-2-O-acetyl- sn -glycero-1-phosphocholine, was inactive. The analogs compared had the following relative potencies in degranulating platelets and neutrophils: 1-O-hexadecyl-2-O-acetyl- sn -glycero-3-phosphocholine > 1-O-hexadecyl-2-O-ethyl- sn -glycero-3-phosphocholine > rac -1-O-octadecyl-2-O-ethylglycero-3-phosphocholine = 1-O-hexadecyl-2-O-methyl- sn -glycero-3-phosphocholine > rac -1-O-dodecyl-2-O-ethyl-glycero-3-phosphocholine. The deacetylated compound, 1-O-hexadecyl-2-lyso- sn -glycero-3-phosphocholine, and 1-O-hexadecyl-2,2-dimethylpropanediol-3-phosphocholine were inactive. The active analogs selectively desensitized the response to each other in the neutrophils. It is suggested that these compounds may activate cells through interaction with a stereospecific receptor.

Ether lipids inhibit the effects of phorbol diester tumor promoters

Recent studies have shown that the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) stimulates protein kinase C (PKC), whereas the ether-linked phospholipid 1-O-octadecyl-2-O-methyl-rac-glycerol-3-phosphocholine (ET-18-OCH3) inhibits PKC activity in vitro. Therefore, the antitumor effects of ET-18-OCH3 could be due to its inhibition of PKC activity and the effects of tumor promotion. TPA stimulates arachidonic acid release, prostaglandin synthesis, phosphatidylcholine synthesis and the degradation of phosphatidylcholine by phospholipase C in Madin Darby canine kidney (MDCK) cells. Therefore, we have determined the effects of ET-18-OCH3 on these consequences of TPA stimulation. Preliminary experiments determined that ET-18-OCH3 inhibited PKC partially purified from MDCK cells by ion-exchange chromatography on DEAE-cellulose. In addition, ET-18-OCH3, inhibited the TPA-stimulated phosphorylation of a 40,000-dalton protein in intact MDCK cells. These data indicate that ET-18-OCH3 is an effective inhibitor of PKC activity in MDCK cells. In addition, ET-18-OCH3 was found to inhibit arachidonic acid release and prostaglandin synthesis. The inhibition of prostaglandin synthesis appears to be secondary to inhibition of arachidonic acid release, since ET-18-OCH3 does not inhibit TPA-stimulated synthesis of prostaglandin H synthase or the activity of the enzyme directly (Parker, J., Daniel, L. W., and Waite, M. [1987]J. Biol. Chem. 262, 5385–5393). ET-18-OCH3 also inhibits TPA-stimulated phosphatidylcholine synthesis and phosphatidylcholine degradation by phospholipase C. These data provide evidence that the antineoplastic ether lipids inhibit the biochemical effects of the tumor promoter TPA in intact cells and indicate that this inhibition may have a role in their biological activities.

Multigram synthesis of 1-alkylamido phospholipids

Phospholipids containing a 1-alkylamido linkage have shown promisingin vitro neoplastic cell growth inhibitory properties and anti-human immunodeficiency viral activity. We have synthesized a series of alkylamido ether lipid analogues on a milligram scale for initial evaluation, but for furtherin vivo testing of these bioactive phospholipids, synthesis on a larger scale is required. The multigram synthesis of 1-alkylamido ether phospholipids was accomplished by modifying reaction conditions in the amidation step and changing reagents and solvent systems in both the detritylation and phosphorylation steps. This was most crucial in the phosphorylation step, where in the multigram synthesis 2-bromoethyl dichlorophosphate in diethyl ether/tetrahydrofuran (7∶3, vol/vol) gave much improved yields as compared to the 2-chloro-2-oxo-1,3,2-dioxaphospholane reagent. The modifications also resulted in a product that could be more easily purified in sufficient quantities for use inin vivo inhibition studies.