Involvement of Ca2+ signalling in the vasoactive intestinal peptide and 8-Br-cAMP induction of c-fos mRNA expression.
Vasoactive intestinal peptide (VIP) is known to signal via Gs mediated pathways. VIP stimulated c-fos mRNA expression in a clonal GH3 pituitary tumour cell line, GH3Ca, whereas 8-Br-cAMP only moderately induced c-fos expression. The VIP-induced c-fos expression was inhibited in the presence of EGTA, or the L-type Ca2+ channel blockers verapamil and nifedipine. Measurement of intracellular Ca2+ concentration ([Ca2+]i) by Fura-2 indicates that VIP gradually elevates [Ca2+]i, with the maximum level attained at 4 min following hormone addition. No [Ca2+]i increase could be detected in Ca2+ free buffer or in buffer containing nifedipine or verapamil, which suggests that VIP induced Ca2+ entry from L-type Ca2+ channels. 8-Br-cAMP rapidly increased [Ca2+]i, with a maximum concentration attained within 1 min of its addition and the elevated level maintained for 15 min. In the absence of external Ca2+ or in the presence of verapamil or nifedipine, the sustained Ca2+ increase was abolished whereas the transient Ca2+ peak was unaffected. Depletion of the internal calcium pools by thapsigargin (1 microM, 30 min), on the other hand, blocked the rapid transient [Ca2+], rise, suggesting the biphasic [Ca2+]i elicited by 8-Br-cAMP was due to mobilization from internal Ca2+ pool followed by extracellular flow. Interestingly, pretreatment with thapsigargin greatly potentiated the 8-Br-cAMP-stimulated c-fos expression. Pretreatment of cells with cholera toxin (1 microg/ml, 9 h) to deplete Gs proteins abolished VIP stimulated-[Ca2+] elevation, while it had little effect on the 8-Br-cAMP induced [Ca2+]i rise. Our results show that VIP increased Ca2+ influx from L-type channel through a Gs-mediated mechanism and this Ca2+ entry across the plasma membrane plays a major role in the hormone induced c-fos mRNA expression.
Jang
Y C
YC
Institute of Biochemistry, National Yang-Ming University, Taipei, Taiwan, ROC.
Kao
L S
LS
Wang
F F
FF
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000502:physiology*; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D016760:Proto-Oncogene Proteins c-fos / Q000096:biosynthesis / Q000235:genetics*; D012333:RNA, Messenger; D051381:Rats; D015398:Signal Transduction; D014407:Tumor Cells, Cultured; D014660:Vasoactive Intestinal Peptide / Q000378:metabolism* / Q000494:pharmacology
D016760:Proto-Oncogene Proteins c-fos; D012333:RNA, Messenger; D015124:8-Bromo Cyclic Adenosine Monophosphate; D014660:Vasoactive Intestinal Peptide; D019205:GTP-Binding Protein alpha Subunits, Gs; D002118:Calcium