Regulation of neurotrophic peptide expression in sympathetic neurons: quantitative analysis using radioimmunoassay and real-time quantitative polymerase chain reaction.
The regulated expression of the peptide and transcript levels of the neurotrophic peptides, pituitary adenylate cyclase-activating polypeptide (PACAP), galanin and vasoactive intestinal peptide (VIP) were examined in sympathetic neurons of the rat superior cervical ganglion (SCG). Real-time quantitative PCR methods were developed to assess modulation of neuronal peptide precursor protein transcript levels following experimental paradigms of neuropeptidergic plasticity. Oligonucleotide primer, fluorogenic probe and amplification conditions were optimized for maximal assay sensitivity. Depolarization of primary cultured sympathetic neurons stimulated PACAP, galanin, and VIP peptide contents and releases with differing magnitudes and temporal profiles. The rank order of increased neuronal peptide content paralleled the augmented peptide release (VIP>galanin>PACAP). Maximal cellular PACAP and VIP levels were achieved by 72 and 96 h, respectively; galanin levels did not plateau during the treatment period. PACAP transcript elevation was rapid and transient; PACAP mRNA expression diminished at longer depolarization times, which diverged markedly from the sustained high peptide production levels. By contrast, VIP and galanin mRNAs reached maximal levels at later times, and appeared to correlate more closely with peptide production. We previously described multiple proPACAP mRNA variants resulting from alternative 3' untranslated region cleavage and polyadenylation. The shorter depolarization-induced PACAP transcripts exhibit longer half-lives, suggesting that the short proPACAP mRNA variant may function to impart PACAP translational efficiency and sustain PACAP peptide production.
Girard
Béatrice M
BM
Department of Anatomy and Neurobiology, University of Vermont College of Medicine, Burlington, VT 05405, USA.
May
Victor
V
Bora
Susan H
SH
Fina
Frederic
F
Braas
Karen M
KM
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D003199:Computer Systems; D019004:Galanin / Q000235:genetics / Q000378:metabolism*; D020869:Gene Expression Profiling / Q000379:methods*; D005786:Gene Expression Regulation / Q000187:drug effects; D008969:Molecular Sequence Data; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D009479:Neuropeptides / Q000235:genetics / Q000378:metabolism*; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D016133:Polymerase Chain Reaction / Q000379:methods*; D011189:Potassium Chloride / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011863:Radioimmunoassay / Q000379:methods*; D051381:Rats; D013997:Time Factors; D014660:Vasoactive Intestinal Peptide / Q000378:metabolism*
C495403:Adcyap1 protein, rat; D009479:Neuropeptides; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D012333:RNA, Messenger; D014660:Vasoactive Intestinal Peptide; D011189:Potassium Chloride; D019004:Galanin