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Functional characterization and mRNA expression of pituitary adenylate cyclase activating polypeptide (PACAP) type I
D Pozo1, M Delgado, C Martinez
1Department of Medical Biochemistry and Molecular Biology, The University of Seville School of Medicine and Virgen Macarena Hospital, Sevilla, Spain.
Abstract:
The present work characterizes the mRNA expression of PACAP type I receptors in rat peritoneal macrophages but not in peritoneal lymphocytes by both retrotranscriptase and polymerase chain reaction (RT-PCR) and homologous Southern hybridization and the stimulation by PACAP27, PACAP38 and vasoactive intestinal peptide (VIP) of sn-1,2-diacylglycerol production in rat peritoneal macrophage membranes. The binding of [125I]PACAP27 was time and cell concentration dependent. Scatchard analysis of displacement of the bound tracer by unlabeled PACAP27 indicates the existence of two classes of binding sites. The dissociation constant (Kd) was 0.64 +/- 0.08 nM and the maximal binding capacity (Bmax) was 8.85 +/- 1.45 fmol/10(6) cells for the high affinity binding site. The low affinity binding site had a Kd of 0.10 +/- 0.06 microM with a Bmax of 300 +/- 21.9 fmol/10(6) cells. Scatchard analysis of VIP displacement data indicated the presence of two classes of binding sites with a Kd and Bmax different to those of PACAP27. These results suggest that PACAP binds to two binding sites, PACAP type I receptors and PACAP type II receptors. The PACAP27-stimulated diacylglycerol production was not affected by treatment with pertussis toxin. However, the presence of GTP partially inhibited this PACAP27 stimulation of 1,2-diacylglycerol in a dose dependent manner, although GTP alone stimulates diacylglycerol accumulation. In conclusion, for the first time we demonstrate by biochemical and molecular biology criteria the existence of PACAP type I receptors on rat peritoneal macrophages and the evidence for coupling with a pertussis toxin-insensitive G regulatory protein.
Insights
This study identifies Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) type I receptors on rat macrophages using RT-PCR and Southern hybridization. These receptors stimulate diacylglycerol production via a pertussis toxin-insensitive G protein.
Area of Science:
- Immunology
- Neuroendocrinology
- Cell Signaling
Background:
- Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) is a neuropeptide with diverse physiological roles.
- The presence and function of PACAP receptors in immune cells, specifically macrophages, remain incompletely understood.
- Understanding PACAP signaling in macrophages is crucial for elucidating immune regulation.
Purpose of the Study:
- To characterize the expression and function of PACAP type I receptors in rat peritoneal macrophages.
- To investigate the signaling pathways coupled to PACAP receptor activation in these cells.
- To determine the binding kinetics and affinity of PACAP and VIP to their receptors on macrophages.
Main Methods:
- Reverse Transcription Polymerase Chain Reaction (RT-PCR) for mRNA expression analysis.
- Southern hybridization for confirmation of receptor gene expression.
- Measurement of sn-1,2-diacylglycerol production in response to PACAP and VIP stimulation.
- Radioligand binding assays using [125I]PACAP27 with Scatchard analysis.
- Investigation of signaling pathway involvement using pertussis toxin and GTP.
Main Results:
- PACAP type I receptor mRNA was detected in rat peritoneal macrophages, but not in lymphocytes.
- PACAP27 and PACAP38 stimulated sn-1,2-diacylglycerol production in macrophage membranes.
- [125I]PACAP27 binding revealed two classes of receptors with distinct affinities (Kd values of 0.64 nM and 0.10 µM).
- Vasoactive Intestinal Peptide (VIP) also bound to two classes of sites with different characteristics.
- PACAP-stimulated diacylglycerol production was insensitive to pertussis toxin but partially inhibited by GTP.
Conclusions:
- This study provides the first biochemical and molecular evidence for PACAP type I receptors on rat peritoneal macrophages.
- PACAP receptors on macrophages appear to couple to a pertussis toxin-insensitive G regulatory protein.
- The findings suggest a novel role for PACAP in regulating macrophage function through diacylglycerol signaling.