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Stimulation of catecholamine biosynthesis via the PKC pathway by prolactin-releasing peptide in PC12 rat pheochromocytoma cells

T Nanmoku

T Nanmoku Department of Clinical Pathology, Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan

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K Takekoshi

K Takekoshi Department of Clinical Pathology, Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan

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T Fukuda

T Fukuda Department of Clinical Pathology, Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan

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K Ishii

K Ishii Department of Clinical Pathology, Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan

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K Isobe

K Isobe Department of Clinical Pathology, Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan

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Y Kawakami

Y Kawakami Department of Clinical Pathology, Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan

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We have previously shown that prolactin-releasing peptide (PrRP) stimulates catecholamine release from PC12 cells (rat pheochromocytoma cell line). However, it is not known whether PrRP also affects catecholamine biosynthesis. Thus, we examined the effect of PrRP on catecholamine biosynthesis in PC12 cells. PrRP31 (>10 nM) and PrRP20 (>100 nM) significantly increased the activity and expression level of tyrosine hydroxylase (TH), a rate-limiting enzyme, in catecholamine biosynthesis. However, the PrRP20-stimulated TH activity was markedly weaker than that of PrRP31. PrRP31 (>1 nM) and PrRP20 (>10 nM) significantly induced an increase in the level of PKC activity. Both Ro 32–0432 (a protein kinase C inhibitor) and H89 (a protein kinase A inhibitor) effectively suppressed the PrRP31 (100 nM )-induced TH mRNA level. Next, we examined the effect of PrRP on mitogen-activated protein kinases (MAPKs). PrRP31 (1 μM) significantly induced an increase in the activity of extracellular signal-related kinases (ERKs) and the stress-activated protein kinase/c-jun N terminal kinase (SAPK/JNK). In contrast to ERKs and JNK, PrRP31 did not affect P38 MAPK activity. Consistent with these findings, pretreatment of cells with the MEK-1-inhibitor, PD-98059 (50 μM), significantly inhibited the PrRP31 (100 nM)-induced increase in TH mRNA. These results indicate that PrRP stimulates catecholamine synthesis through both the PKC and PKA pathways in PC12 cells.

Article Type:: Research Article

DOI:: https://doi.org/10.1677/joe.1.05919

Volume/Issue:: Volume 186: Issue 1

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Enzyme-linked immunosorbent assay for human insulin-like growth factor-I using monoclonal and polyclonal antibodies with defined epitope recognition

K. Tamura

K. Tamura
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M. Kobayashi

M. Kobayashi
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S. Suzuki

S. Suzuki
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Y. Ishii

Y. Ishii
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S. Koyama

S. Koyama
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H. Yamada

H. Yamada
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K. Hashimoto

K. Hashimoto
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M. Niwa

M. Niwa
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F. Shibayama

F. Shibayama
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ABSTRACT

Monoclonal antibodies (McAb) and polyclonal antibodies (PcAb) against human insulin-like growth factor-I (somatomedin C; hIGF-I) were produced. Using these two antibodies, an enzyme-linked immunosorbent assay (ELISA) system for hIGF-I was established. The ELISA system was able to detect hIGF-I at a range of 1–25 μg/l, compared with the range of 1–50 μg/l detected by radioimmunoassay (RIA). Human IGF-II and human insulin could not be recognized in this system. The plasma concentrations of IGF-I found using the ELISA agreed well with those found using RIA after conventional Sep-Pak C₁₈ cartridge pretreatment. Epitopes of hIGF-I to McAb and PcAb were investigated by enzymatic digestion of hIGF-I followed by comparing the affinity of the antibodies to the peptides obtained proteolytically. The epitope to McAb was found to be a peptide containing Leu¹⁰-Val¹¹-Asp¹² (epitope 2). Five epitopes to PcAb containing the following key fragments were identified: a conformational structure formed by the disulphide bonds between Cys⁶ and Cys⁴⁸, and between Cys⁴⁷ and Cys⁵² (epitope 1), Leu¹⁰-Val¹¹-Asp¹² (epitope 2), Val¹⁷-Cys¹⁸-Gly¹⁹-Asp²⁰ (epitope 3), Arg²¹-Gly²²-Phe²³-Tyr²⁴ (epitope 4) and Lys⁶⁸-Ser⁶⁹-Ala⁷⁰ (epitope 5). Of these, the peptide containing epitope 5 showed the highest affinity to PcAb. The results indicated that our ELISA system combined recognition by epitope 2 of McAb and recognition by epitope 5 of PcAb to obtain its good specificity.