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Hepoxilin A3-specific binding in human neutrophils
D Reynaud1, P Demin, C R Pace-Asciak
1Research Institute, Hospital for Sick Children, Toronto, Canada.
Insights
This study identifies specific binding sites for hepoxilin A3 in human neutrophils, revealing a new mechanism for cellular signaling. These findings advance our understanding of hepoxilin A3
Area of Science:
- Biochemistry
- Cell Biology
- Immunology
Background:
- Hepoxilins are known to trigger calcium release from intracellular stores in human neutrophils.
- The precise mechanisms and cellular targets of hepoxilins are not fully elucidated.
Purpose of the Study:
- To investigate the existence and characteristics of specific binding sites for hepoxilin A3 in human neutrophils.
- To characterize the binding kinetics and specificity of hepoxilin A3 to neutrophil membranes.
Main Methods:
- Utilized tritium-labeled hepoxilin A3 (8S) to study binding to broken neutrophil membranes.
- Performed competitive binding assays with unlabeled hepoxilin A3 and related eicosanoids.
- Analyzed binding data using Scatchard analysis to determine binding site parameters.
- Investigated the effect of proteinase K on specific binding to membrane preparations and intact cells.
Main Results:
- Tritium-labeled hepoxilin A3 (8S) demonstrated time-, substrate-, and temperature-dependent binding to broken neutrophil membranes.
- Specific binding was highest at 37°C and was displaced by unlabeled hepoxilin A3 (8S), but not by other eicosanoids like leukotriene B4 or prostaglandins.
- Scatchard analysis indicated a single population of binding sites with an apparent KD of 79.3 ± 9.1 nM and Bmax of 8.86 ± 1.4 pmol/ml.
- Proteinase K treatment inhibited specific binding to broken membranes, but not to intact cells.
Conclusions:
- Neutrophils possess specific binding sites for hepoxilin A3.
- These findings provide the first direct evidence for hepoxilin A3 receptors on neutrophils.
- This discovery opens new avenues for understanding hepoxilin A3-mediated cellular functions.
Abstract:
Hepoxilins have been shown to release calcium from intracellular stores in human neutrophils [Dho, Grinstein, Corey, Su and Pace-Asciak (1990) Biochem. J. 266, 63-68; Laneuville, Reynaud, Grinstein, Nigam and Pace-Asciak (1993) Biochem. J. 295, 393-397]. In this paper we report that tritium-labelled hepoxilin A3 (8S) binds to broken neutrophil membranes in a time-, substrate- and temperature-dependent fashion. Specific binding was displaced with unlabelled hepoxilin A3. Specific binding was greatest at 37 degrees C. Competitive binding was best observed with unlabelled hepoxilin A3 (8S); the glutathione conjugate, HxA3-C (8S or 8R), or 12(S)-hydroxyeicosatetraenoic acid was less active. Similarly inactive in displacing the bound radiolabelled hepoxilin A3 was leukotriene B4 as well as a variety of prostaglandins and thromboxane B2. Formylmethionyl-leucylphenylalanine was similarly inactive in competing for the hepoxilin binding sites. Specific binding was inhibited by pretreatment of the broken membranes during 30 min at 37 degrees C with proteinase K, while specific binding of the intact cells was unaffected. Scatchard analysis of binding data revealed a single population of binding sites with apparent KD and Bmax. of 79.3 +/- 9.1 nM and 8.86 +/- 1.4 pmol/ml per 2 x 10(6) cells (+/- S.E.M.) respectively reflecting approx. 2.67 x 10(6) sites/cell. These results demonstrate for the first time that neutrophils contain specific binding sites to hepoxilin A3.