Functional characterization and mRNA expression of pituitary adenylate cyclase activating polypeptide (PACAP) type I receptors in rat peritoneal macrophages.
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.
Pozo
D
D
Department of Medical Biochemistry and Molecular Biology, The University of Seville School of Medicine and Virgen Macarena Hospital, Sevilla, Spain.
Delgado
M
M
Martinez
C
C
Gomariz
R P
RP
Guerrero
J M
JM
Calvo
J R
JR
D037361:Adenylate Cyclase Toxin; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism; D004075:Diglycerides / Q000096:biosynthesis; D005786:Gene Expression Regulation / Q000502:physiology*; D006160:Guanosine Triphosphate / Q000494:pharmacology; D007700:Kinetics; D017737:Macrophages, Peritoneal / Q000378:metabolism* / Q000502:physiology; D008297:Male; D009479:Neuropeptides / Q000378:metabolism; D009928:Organ Specificity; D037342:Pertussis Toxin; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis*; D051381:Rats; D017208:Rats, Wistar; D051236:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; D051237:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I; D011979:Receptors, Pituitary Hormone / Q000235:genetics* / Q000378:metabolism*; D051238:Receptors, Vasoactive Intestinal Polypeptide, Type I; D014660:Vasoactive Intestinal Peptide / Q000494:pharmacology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
C011439:1,2-diacylglycerol; C495403:Adcyap1 protein, rat; D037361:Adenylate Cyclase Toxin; D004075:Diglycerides; D009479:Neuropeptides; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D012333:RNA, Messenger; D051236:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; D051237:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I; D011979:Receptors, Pituitary Hormone; D051238:Receptors, Vasoactive Intestinal Polypeptide, Type I; D010566:Virulence Factors, Bordetella; D014660:Vasoactive Intestinal Peptide; D006160:Guanosine Triphosphate; D011065:Poly(ADP-ribose) Polymerases; D037342:Pertussis Toxin