Abstract
Acyl analogs of platelet-activating factor (PAF) (1-acyl-2-acetyl-sn-glycero-3-phosphocholine, acylacetyl -GPC) are the predominant products synthesized during thrombin or ionophore A23187-mediated activation of endothelial cells. However, the biosynthetic pathway responsible for the production of acylacetyl-GPC is not well understood. In the present investigation, we have demonstrated that the acyl analogs of PAF are also the major products from calf pulmonary artery endothelial cells in response to a time-dependent stimulation of ATP (10(-3) M), bradykinin (10(-8) M), or ionophore A23187 (2 microM). In addition, we have found that the CoA-independent PAF:acyllyso-GPC transacetylase recently identified by us is concurrently and transiently induced with maximal 4-fold enhancement at 5 min and returned to near basal level by 10 min treatment of endothelial cells with ATP. Acid phosphatase reduces the increased PAF:acyllyso-GPC transacetylase activity from the homogenates of ATP-activated endothelial cells. Reduced PAF:acyllyso-GPC transacetylase activity can be restored by incubating the acid phosphatase-treated homogenates with ATP (5 mM) and Mg2+ (10 mM). Furthermore, okadaic acid, a protein phosphatase 1 and 2A inhibitor, incubated with endothelial cells in a dose-dependent manner (1-100 nM) for 10-min potentiates and sustained the stimulation of PAF:acyllyso-GPC transacetylase activity by ATP. On the other hand, genistein, tyrphostin-25 (inhibitors of tyrosine-specific protein kinase), and calphostin C (an inhibitor of protein kinase C) block the activation of PAF:acyllyso-GPC transacetylase by ATP. These results are consistent with the notion that ATP regulates the transacetylase activity by reversible activation and inactivation via the phosphorylation and dephosphorylation cycle. ATP also augments the activities of alkyllyso-GPC/acyllyso-GPC:acetyl-CoA acetyltransferase. However, the activation of the acetyltransferases precedes that of the transacetylase with peak activation occurring at 1-2 min of the ATP treatment. In addition, sodium vanadate, also an inhibitor of protein phosphatase, stimulates the increase in the incorporation of [3H]acetate into acyl[3H]acetyl-GPC of the ATP-treated endothelial cells. Collectively, our data show that both acetyltransferases and transacetylase participate in and contribute to the biosynthesis of acyl analogs of PAF in a coordinate fashion in endothelial cells.
Highlights
Pate in and contribute to the biosynthesis of acyl analogs of Platelet-activating factor (PAF) in a coordinate fashion in endothelial cells
We found that acyl analogs of PAF were the major product when calf pulmonary artery endothelial cells were stimulated with ATP (Fig. 1B), bradykinin, or ionophore A23187
It is known that the synthesis of PAF is increased in human umbilical vein endothelial cells in response to interleukin-1 [50], tumor necrosis factor [51], histamine, bradykinin, ATP, peptide leukotrienes LTD4 and LTC4, thrombin [52,53,54], and calcium ionophore A23187 [55] and in bovine blood vessel endothelial cells in response to ATP, bradykinin, angiotensin II, and ionophore A23187 [12, 42]
Summary
17431–17437, 1997 Printed in U.S.A. The Role of Platelet-activating Factor-dependent Transacetylase in the Biosynthesis of 1-Acyl-2-acetyl-sn-glycero-3-phosphocholine by Stimulated Endothelial Cells*. Acyl analogs of platelet-activating factor (PAF) (1-acyl-2-acetyl-sn-glycero-3-phosphocholine, acylacetyl -GPC) are the predominant products synthesized during thrombin or ionophore A23187-mediated activation of endothelial cells. We have demonstrated that the acyl analogs of PAF are the major products from calf pulmonary artery endothelial cells in response to a time-dependent stimulation of ATP (1023 M), bradykinin (1028 M), or ionophore A23187 (2 mM). Acid phosphatase reduces the increased PAF:acyllyso-GPC transacetylase activity from the homogenates of ATP-activated endothelial cells. Okadaic acid, a protein phosphatase 1 and 2A inhibitor, incubated with endothelial cells in a dose-dependent manner (1–100 nM) for 10-min potentiates and sustained the stimulation of PAF:acyllyso-GPC transacetylase activity by ATP. Based on the temporal relationships that exist between the synthesis of acylacetyl-GPC and induction of the transacetylase activity in stimulus-activated cells, our findings suggest that the PAF-dependent transacetylase participates in the production and controls the level of acylacetyl-GPC in endothelial cells
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