Platelet activating factor (PAF) is a potent mediator in a variety of inflammatory events. Determining whether PAF participates in the bronchial hyperresponsiveness characteristic of asthma is the long term obj ecti ve for which the studies described here represent an initial step. PAF is a potent agonist that causes contraction of guinea pig peripheral lung strips. To determine if specific receptor sites for PAF could be demonstrated in guinea pig lung membranes (GPLM), direct radioligand binding studies were performed with [³H]C₁₆-PAF (l-0-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine) and the PAF antagonists [³H]WEB 2086 and [³H]RP52770. Binding parameters were compared to those from rabbit platelet membranes (RPM). These studies demonstrated specific binding sites for [³H] C₁₆-PAF of high affinity in GPLM with a Kd of 3 nM,• and in RPM with a K(d) of 1 nM. [³H]C₁₆-PAF identified receptor densities in GPLM of 200 fmol/mg protein and in RPM of 1922 fmol/mg protein. In both tissue preparations binding of inhibited to the same maximum degree by C₁₆-PAF, C₁₈-PAF, WEB 2086, and RP52770, all with pseudo-Hill coefficients of unity. The PAF antagonist [³H]WEB 2086 identified a receptor density similar to that of [³H]C₁₆-PAF. The binding of [³H]WEB 2086 was inhibited to the same degree by C₁₆-PAF, C₁₈-PAF, WEB 2086 and RP52770, indicating WEB 2086 and PAF interact at the same receptor sites in both GPLM and RPM. Although inhibition curves for antagonists yielded pseudo-Hill coefficients of unity, inhibition by agonists yielded shallow inhibition curves suggesting two types or states for the PAF receptor. The PAF antagonist [³H]RP52770 was found to be an unsuitable ligand because it labeled a much larger density of binding sites (1200 fmol/mg protein in GPLM, and 10105 fmol/mg protein in RPM) and was inhibited to little or no extent by C₁₆-PAF, C₁₈-PAF, WEB 2086 or lyso-C₁₆-PAF . studies of signal transduction suggest that the binding affinity of the agonists C₁₆-PAF and C₁₈-PAF (but not for the antagonist WEB 2086) is regulated by GTPgamroa- S and Na⁺, providing indirect evidence that the PAF receptor in both tissue preparations is coupled to a guanine nucleotide regulatory protein. However, agonist binding retained shallow inhibition curves indicating heterogeneity of sites with respect to this regulation. Binding affinity for the agonists was not affected by cholera toxin or pertussis toxin. These results indicate PAF receptors in lung tissue could not be distinguished from those in RPM, however, both tissues appear to show heterogeneity of binding indicating the existence of receptor subtypes or states.
| Identifer | oai:union.ndltd.org:arizona.edu/oai:arizona.openrepository.com:10150/185248 |
| Date | January 1990 |
| Creators | Gomez, Jorge. |
| Contributors | Halonen, Marilyn, Olson, George B, Sammons, David W., Yamamura, Henry I., Palmer, John D. |
| Publisher | The University of Arizona. |
| Source Sets | University of Arizona |
| Language | English |
| Detected Language | English |
| Type | text, Dissertation-Reproduction (electronic) |
| Rights | Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. |
Page generated in 0.0162 seconds