Jimmy F. Redman

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.

Protein kinase C regulates leukotriene B4 receptors in human neutrophils

Three protein kinase C (PKC) activators, viz. phorbol myristate acetate, mezerein, and rac‐1‐O‐myristoyl‐2‐acetylglycerol, inhibited human neutrophil binding of [3H] leukotriene B4 (LTB4) by reducing the number of high‐affinity receptors available to the arachidonic acid metabolite. The inhibitory effect occurred in whole cells and cytoplasts but not in isolated membranes; it appeared to involve the activation of PKC rather than direct competition for binding sites. PKC may govern cellular responsiveness by regulating the receptor‐linked bioactions of endogenous mediators like LTB4.

Receptor-independent metabolism of platelet-activating factor by myelogenous cells

Human neutrophils incorporate and metabolize platelet‐activating factor (PAF). We dissociated these events from PAF binding to its receptors. Cells were pretreated with either pronase, a PAF antagonist (L652731), or excess PAF. This reduced PAF receptor numbers by 70 to almost 100% but had no comparable effect upon the neutrophils ability to metabolize PAF. Furthermore, HL‐60 cells efficiently metabolized, but did not specifically bind, PAF. Thus, PAF receptor availability did not correlate with PAF metabolic capacity and we conclude that myelogenous tissues can process this bioactive ligand by a receptor‐independent pathway.