Localization, Characterization, and Second Messenger Coupling of Pituitary Adenylate Cyclase-Activating Polypeptide Receptors in the Fetal Human Adrenal Gland during the Second Trimester of Gestation¹

Abstract

The distribution and pharmacological properties of pituitary adenylate cyclase-activating polypeptide (PACAP) receptors were studied in the fetal human adrenal gland during the second trimester of gestation. Autoradiographic studies, using [125I]PACAP27 as a radioligand, revealed that PACAP-binding sites are exclusively located on chromaffin cells of adrenals from fetuses 14-20 weeks old. Biochemical characterization of binding revealed the occurrence of a single class of PACAP-binding sites with a dissociation constant value of 0.32-0.74 nmol/L and a binding capacity of 0.30-0.81 pmol/mg wet tissue. PACAP27 and PACAP38 were equipotent in competing for [125I]PACAP27 binding (IC50 = 0.28-0.64 nmol/L and 0.15-0.81 nmol/L, respectively), and the Hill coefficients were close to 1. In contrast, vasoactive intestinal polypeptide was much less efficient in displacing the tracer (IC50 = 4-362 nmol/L), and the Hill coefficients were less than 0.6. PACAP38 induced a dose-dependent increase in cAMP production in fetal human adrenal cell suspension (ED50 = 0.07 +/- 0.02 nmol/L), as well as in cells maintained in culture for 5 days (5.4 +/- 1.8 nmol/L). In contrast, PACAP38 induced a modest increase in inositol phosphate formation. These data indicate that type I PACAP receptors are present in the early stages of the human medulla organization during the process of migration of chromaffin cells from the periphery to the central part of the gland. The present results suggest that PACAP could be involved in the regulation of the human adrenochromaffin cells during ontogenesis.

PAC1 Receptor Activation by PACAP-38 Mediates Ca2+ Release from a cAMP-dependent Pool in Human Fetal Adrenal Gland Chromaffin Cells

“…The chemicals used in the present study were obtained from the following sources: RNAqueous TM -4PCR purchased from Ambion (Austin, TX); dithiothreitol, p(dT) [12][13][14][15][16][17][18] , rRNasin Ribonuclease Inhibitor, Moloney murine leukemia virus reverse transcriptase (RT) and DNA ladder from Promega (Madison, WI); deoxy-NTPs and Taq DNA polymerase from Amersham Pharmacia Biotech (Piscataway, NJ); Xestospongin C, U73122, forskolin, thapsigargin, and H-89 from Calbiochem-Novabiochem Corp. (San Diego, CA); methacholine, sodium orthovanadate, nicotine, and DNase from Sigma-Aldrich Canada Ltd (Oakville, Ontario, Canada); collagenase, MEM Eagle's medium and OPTI-MEM from Invitrogen (Burlington, Ontario, Canada); (R p )-cAMPS from Biomol (Plymouth Meeting, PA); Fluo-4 from Molecular Probes (Eugene, OR); PACAP-38 was synthesized by the solid phase methodology as previously described (22); and PACAP (6 -38) was from American Peptide Co. (Sunnyvale, CA). All other chemicals were of A grade purity.…”

“…RNA content and quality were determined photometrically. 5 g of total RNA was denatured (70°C, 10 min) and reverse-transcribed in the presence of 200 M p(dT) [12][13][14][15][16][17][18] at 42°C for 50 min in 20 l of 1ϫ RT buffer (25 mM Tris-HCl, pH 8.3, 37.5 mM KCl, 1.5 mM MgCl 2 ) containing: 15 mM dithiothreitol, 200 M deoxy-NTPs, 25 units of rRNasin ribonuclease inhibitor, and 200 units of Moloney murine leukemia virus RT. Inactivation of the enzyme (70°C, 10 min) was followed by glyceraldehyde-3-phosphate dehydrogenase PCR to assess the quality of the cDNA template (24).…”

“…Cell Culture-Glands were processed as described previously (16). Whole tissues from one or two glands were used for each cell preparation, without separation of fetal zone, neocortex, or chromaffin cells.…”

“…In particular, we have previously demonstrated the occurrence of PACAP-38 (15) and PACAP binding sites (16) in chromaffin cells from 16-to 20-week-old fetal human adrenal glands. Activation of these receptors by PACAP-38 causes stimulation of cAMP production and induces a modest increase in inositol 1,4,5-triphosphate (IP 3 ) formation (16), suggesting a role for the neuropeptide in the developing adrenal gland.…”

“…In particular, we have previously demonstrated the occurrence of PACAP-38 (15) and PACAP binding sites (16) in chromaffin cells from 16-to 20-week-old fetal human adrenal glands. Activation of these receptors by PACAP-38 causes stimulation of cAMP production and induces a modest increase in inositol 1,4,5-triphosphate (IP 3 ) formation (16), suggesting a role for the neuropeptide in the developing adrenal gland. During the process of adrenal gland development, pheochromoblasts originating from the neural crest migrate throughout the fetal cortex, acquiring progressive differentiation through contact with the steroidogenic cells (17,18) (for review see Ref.…”