Platelet-activating Factor Receptor Agonist Research Articles

Platelet-Activating Factor Biosynthesis Induced by Various Stimuli in Human HaCaT Keratinocytes

Platelet-activating factor (PAF) is a potent inflammatory mediator that is thought to play a role in cutaneous inflammation. These studies used mass spectrometry to examine the molecular species of PAF precursor glycerophosphocholine lipids (GPC) as well as the biosynthesis of PAF and other sn-2 acetyl-GPC in a human keratinocyte-derived cell line (HaCaT keratinocytes). Approximately 28% of HaCaT keratinocyte GPC consisted of 1-alkyl species, and the relative amounts of the sn-1 alkyl constituents of the PAF precursor 1-alkyl-2-acyl-GPC were as follows: hexadecyl > octadecenyl > octadecyl. Ionophore (A23187)-stimulated HaCaT keratinocytes synthesized both PAF (1-hexadecyl, 1-octadecenyl, and 1-octadecyl species) and less potent 1-acyl analogs (1-palmitoyl, 1-oleoyl, and 1-stearoyl species). PAF production was rapid and maximal by 10 min. The major species of sn-2acetyl-GPC at 2.5 min were 1-hexadecyl-2-acetyl-GPC (2.2 ng/10(6) cells) and 1-palmitoyl-2-acetyl-GPC (2.4 ng/10(6) cells). HaCaT keratinocytes also synthesized PAF and 1-acyl PAF analogs when stimulated with the peptide growth factor endothelin-1 and the nonhydrolyzable PAF receptor agonist carbamyl-PAF. Both 1-hexadecyl-2- acetyl-GPC and 1-palmitoyl-2-acetyl-GPC stimulated intracellular calcium mobilization in HaCaT cells, indicating that these sn-2 acetyl-GPC act in autocrine fashion. These studies revealed that the human keratinocyte-derived cell line HaCaT can synthesize significant amounts of PAF and 1-acyl analogs in vitro from both nonspecific (A23187) and specific (endothelin-1, carbamyl-PAF) stimulation, suggesting a role for this inflammatory lipid mediator in keratinocyte pathophysiology.

Similar coronary vascular effects in the rat perfused heart of platelet-activating factor structural analogues with agonist and antagonist properties.

1. Selective blockade of platelet-activating factor (PAF) receptor subtypes by PAF receptor antagonists has been demonstrated. However, selective activation of PAF receptor subtypes by PAF receptor agonists has not been reported. 2. When structural analogues of PAF that have been shown to possess either agonist or antagonist effects were administered by a bolus injection in the rat perfused heart, they all showed agonist effects. Lower amounts produced vasodilation while higher amounts produced vasodilation followed by vasoconstriction. These coronary vascular effects were typical of that observed with PAF. Lyso-PAF did not show the same typical pattern of coronary vascular effect, confirming that the detergent effect of PAF structural analogues did not play a role in the coronary vascular effects. Other PAF antagonists, CV-6209 and WEB 2170, also did not produce the PAF-like response in the rat perfused heart. 3. The coronary vascular effects of hexanolamine-PAF (H-PAF, putative antagonist) and ethanolamine-PAF (E-PAF, agonist) were further studied. Pretreatment with FR-900452 (a PAF receptor antagonist) or MK-886 (a leukotriene synthesis inhibitor) significantly reduced the vasodilator and vasoconstrictor effects of H-PAF and E-PAF. 4. Pretreatment of rat perfused hearts with low concentrations of H-PAF and E-PAF blocked the response to PAF administration in a dose- and time-dependent manner. However, the pretreatment with either H-PAF or E-PAF did not result in a coronary vascular effect expected of a PAF receptor agonist. These results were compatible with H-PAF and E-PAF behaving as PAF receptor antagonists. 5. In summary, our results demonstrate that several PAF structural analogues possess agonist action in the rat perfused heart. Like the coronary vascular effects of PAF, the effects of H-PAF and E-PAF were blocked by a PAF antagonist (FR-900452) and a leukotriene synthesis inhibitor (MK-886). This suggests that both H-PAF and E-PAF mediate their effect through activation of PAF receptors with a subsequent release of leukotrienes that produced vasodilatation and vasoconstriction. Furthermore, pretreatment of perfused hearts with these compounds blocked the response to PAF in these hearts. Thus these compounds can also behave like a PAF receptor antagonist. This latter action may be due to a gradual receptor inactivation or desensitization by the pretreatment of H-PAF and E-PAF through a PAF receptor agonist effect rather than being a PAF receptor antagonist.

Autocrine stimulation of B lymphocytes by a platelet-activating factor receptor agonist, 1-palmitoyl-2-acetyl-sn-glycero-3-phosphocholine.

Based on previous data which demonstrated the ability of platelet-activating factor (PAF) antagonists to inhibit constitutive immunoglobulin synthesis in B-lymphoblastoid cell lines, we determined the capacity of these cells to synthesize PAF or 1-palmitoyl-2-acetyl-sn-glycero-phosphocholine (PAGPC), an acyl-PAF identified in various cell types. In two B-lymphoblastoid cell lines (LA350 and HSCE-), significant amounts of production of PAGPC were detected, whereas the amount of PAF was below the level of detection in our system. The biologic effects of PAGPC were examined in these cells, both of which have well-characterized PAF receptors. PAGPC induced a concentration-dependent increase in intracellular Ca2+ concentrations and activation of MAP-2 kinase (as detected by immunoblotting and measurements of kinase activity) in these cells. The kinetics and magnitude of these responses were similar to those induced by PAF, and they were inhibited by Web 2086, a PAFR antagonist. Phosphatidylcholine, which differs from PAGPC in that it contains a long fatty acid residue at position 2, did not induce any of these responses. A mutual cross-desensitization of the B lymphoblasts between PAGPC and PAF was observed for Ca2+ mobilization. To induce maximal cell stimulation, approximately 600-fold higher concentrations of PAGPC than of PAF were needed. Because the two B-lymphoblastoid cell lines synthesized significant amounts of PAGPC, this phospholipid may participate in an autocrine stimulation pathway in B cells. Furthermore, the data indicate that PAGPC is an agonist for the PAFR in B lymphoblasts and that the ether linkage in the PAF molecule is not an absolute requirement for activity, although it increases the potency of the ligand.

Binding of carbamyl-platelet-activating factor to the raji lymphoblast platelet-activating factor receptor

Carbamyl-platelet-activating factor (1-hexadecyl-2- N-methylcarbamyl-glycero-3-phosphocholine; CPAF) is an analog of platelet-activating factor (PAF) containing an N-methylcarbamyl moiety at the sn-2 position. CPAF was tested for effects on the Raji lymphoblast PAF receptor. Binding studies conducted at 4°C demonstrated specific binding that reached saturation within 60–80 min. Scatchard analysis of CPAF binding data revealed a single class of CPAF binding sites (14,800/cell) with a K = 2.9 ± 0.9 nM. Competition binding studies with PAF indicated that CPAF has about one-third the potency of native PAF. Unlike PAF, however, CPAF was not significantly metabolized by Raji lymphoblasts at 37°C. CPAF was shown to have PAF-agonistic qualities, since 100 pM to 1 μM CPAF increased free intracellular calcium in a dose-dependent manner. The structurally dissimilar PAF receptor antagonists CV-6209 and alprazolam inhibited the CPAF-induced calcium changes at doses that compared with CPAF binding. Treatment of Raji lymphoblasts with PAF or CPAF (10 pM - 1 μM) did not affect spontaneous proliferation, suggesting that the PAF receptor is not involved in the proliferation process in this cell line. These studies demonstrate that CPAF is a metabolically stable lymphoblast PAF receptor agonist that may provide a useful tool in the further elucidation of the role of PAF in lymphocyte function.