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Characteristics of receptors for VIP in rat peritoneal macrophage membranes
J R Calvo1, J M Guerrero, M A López-Gonzalez
1Department of Medical Biochemistry and Molecular Biology, School of Medicine, University of Sevilla, Spain.
Abstract:
Vasoactive intestinal peptide (VIP) receptors were investigated in rat peritoneal macrophage membranes (RPMM) using [125I]VIP as ligand. The receptor binding was rapid, reversible, saturable, specific, and dependent on time, temperature, and membrane concentration. The Scatchard analysis of binding data was consistent with the existence of two classes of VIP binding sites with Kd values of 0.60 +/- 0.08 and 275 +/- 39 nM and binding capacities of 580 +/- 71 and 72,500 +/- 810 fmol VIP/mg protein, respectively. The interaction showed a high degree of specificity, as suggested by competitive displacement experiments with several peptides structurally or not structurally related to VIP. These pharmacological studies showed the following order of potency: VIP (IC50 = 1 nM) > rGRF (IC50 = 13 nM) > PHI (IC50 = 421 nM) >> secretin. Glucagon, somatostatin, insulin octapeptide of cholecystokinin [CCK(26-33)], and pancreastatin were ineffective at concentrations up to 1 microM. Binding of [125I]VIP to membranes is markedly reduced by increasing the ionic strength of incubation medium. Treatment of membranes with dithiothreitol, trypsin, and phospholipases A2 and C resulted in a loss of the ability of these membranes to bind VIP. However, treatment with phospholipase D did not affect binding of VIP by membranes. The molecular characterization of VIP receptors in RPMM was performed after [125I]VIP cross-linking to membranes using the cross-linker dithiobis (succinimidyl propionate). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membrane proteins revealed specific [125I]VIP-protein complexes of M(r) 55,000 +/- 1700, 35,000 +/- 900, and 22,000 +/- 500.
Insights
Vasoactive intestinal peptide (VIP) receptors were identified on rat peritoneal macrophage membranes, revealing two distinct binding sites with varying affinities. These VIP receptors play a role in macrophage function.
Area of Science:
- Immunology
- Endocrinology
- Molecular Biology
Background:
- Vasoactive intestinal peptide (VIP) is a neuropeptide with diverse physiological roles.
- Understanding VIP receptor distribution is crucial for elucidating its functions in immune cells.
Purpose of the Study:
- To characterize vasoactive intestinal peptide (VIP) receptors on rat peritoneal macrophage membranes (RPMM).
- To determine the binding kinetics, specificity, and molecular properties of VIP receptors in macrophages.
Main Methods:
- Radioligand binding assays using [125I]VIP.
- Scatchard analysis to quantify receptor binding sites.
- Competitive displacement studies with related peptides.
- Chemical and enzymatic treatments of membranes.
- Photoaffinity labeling and SDS-PAGE for molecular characterization.
Main Results:
- Two classes of VIP binding sites were identified with distinct dissociation constants (Kd) and binding capacities.
- VIP receptor binding demonstrated high specificity, with VIP being the most potent ligand.
- Receptor binding was sensitive to ionic strength and affected by treatments with dithiothreitol, trypsin, and certain phospholipases.
- SDS-PAGE revealed specific [125I]VIP-protein complexes at apparent molecular weights of 55,000, 35,000, and 22,000 Da.
Conclusions:
- Rat peritoneal macrophage membranes possess specific, high-affinity, and low-affinity VIP receptors.
- The characterized VIP receptors are integral membrane proteins with distinct molecular masses.
- These findings contribute to understanding the role of VIP in macrophage biology and immune regulation.