Abstract
Previous studies have shown that human fetal adrenal gland from 17- to 20-week-old fetuses expressed pituitary adenylate cyclase-activating polypeptide (PACAP) receptors, which were localized on chromaffin cells. The aim of the present study was to identify PACAP receptor isoforms and to determine whether PACAP can affect intracellular calcium concentration ([Ca(2+)](i)) and catecholamine secretion. Using primary cultures and specific stimulation of chromaffin cells, we demonstrate that PACAP-38 induced an increase in [Ca(2+)](i) that was blocked by PACAP (6-38), was independent of external Ca(2+), and originated from thapsigargin-insensitive internal stores. The PACAP-triggered Ca(2+) increase was not affected by inhibition of PLC beta (preincubation with U-73122) or by pretreatment of cells with Xestospongin C, indicating that the inositol 1,4,5-triphosphate-sensitive stores were not mobilized. However, forskolin (FSK), which raises cytosolic cAMP, induced an increase in Ca(2+) similar to that recorded with PACAP-38. Blockage of PKA by H-89 or (R(p))-cAMPS suppressed both PACAP-38 and FSK calcium responses. The effect of PACAP-38 was also abolished by emptying the caffeine/ryanodine-sensitive Ca(2+) stores. Furthermore, treatment of cells with orthovanadate (100 microm) impaired Ca(2+) reloading of PACAP-sensitive stores indicating that PACAP-38 can mobilize Ca(2+) from secretory vesicles. Moreover, PACAP induced catecholamine secretion by chromaffin cells. It is concluded that PACAP-38, through the PAC(1) receptor, acts as a neurotransmitter in human fetal chromaffin cells inducing catecholamine secretion, through nonclassical, recently described, ryanodine/caffeine-sensitive pools, involving a cAMP- and PKA-dependent phosphorylation mechanism.
Highlights
Previous studies have shown that human fetal adrenal gland from 17- to 20-week-old fetuses expressed pituitary adenylate cyclase-activating polypeptide (PACAP) receptors, which were localized on chromaffin cells
Chemicals—The chemicals used in the present study were obtained from the following sources: RNAqueousTM-4PCR purchased from Ambion (Austin, TX); dithiothreitol, p(dT)12–18, 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); (Rp)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)
Molecular Identification of PACAP Receptors—Using autoradiography, we have previously shown that PACAP receptors in the human fetal adrenal gland were localized only on chromaffin cells [16]
Summary
Previous studies have shown that human fetal adrenal gland from 17- to 20-week-old fetuses expressed pituitary adenylate cyclase-activating polypeptide (PACAP) receptors, which were localized on chromaffin cells. It is concluded that PACAP-38, through the PAC1 receptor, acts as a neurotransmitter in human fetal chromaffin cells inducing catecholamine secretion, through nonclassical, recently described, ryanodine/caffeine-sensitive pools, involving a cAMP- and PKA-dependent phosphorylation mechanism. Eight isoforms of PAC1-R, resulting from alternative splicing, have been characterized to date These variants display differential signal transduction properties with regard to adenylyl cyclase and phospholipase C (PLC) stimulation [1, 2]. 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 (IP3) formation [16], suggesting a role for the neuropeptide in the developing. 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]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
