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European Journal of Pharmacology 403 2000 195–202
www.elsevier.nlrlocaterejphar
Serotonin-induced protein kinase C activation in
cultured rat heart endothelial cells
Hong-Zin Lee
)
, Chun-Hsiung Wu
School of Pharmacy, China Medical College, 91, Hsueh-Shih Road, Taichung 404, Taiwan
Received 6 March 2000; received in revised form 6 July 2000; accepted 11 July 2000
Abstract
This study examined whether serotonin can activate protein kinase C in rat heart endothelial cells. Protein kinase C isozyme
translocation was examined by Western blot analysis with isozyme-specific anti-protein kinase C antibody. In this study, only a protein
kinase C isozyme was found to be translocated from the cytosolic to the particulate fractions after serotonin stimulation. The effect of
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serotonin on the incorporation of P from g- P ATP into peptide substrate was studied as another indicator of protein kinase C
activation. The experiments in this study demonstrated that the Ca
2
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-phospholipid-dependent protein kinase, protein kinase C, was
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activated by serotonin. By investigating H phorbol 12,13-dibutyrate binding to protein kinase C and trypsin-treated protein kinase C
activity, we demonstrated that the site of action of serotonin is probably the regulatory domain of protein kinase C. Finally, we also
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demonstrated that serotonin had no effect on the intracellular concentration of cyclic nucleotides cAMP, cGMP . These findings support
the hypothesis that protein kinase C may be an important participant in serotonin-induced endothelial cell contraction and barrier
dysfunction. q 2000 Elsevier Science B.V. All rights reserved.
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Keywords: 5-HT 5-Hydroxytryptamine, serotonin ; Endothelial cell, rat; Protein kinase C; H Phorbol 12,13-dibutyrate binding; Trypsin-treated protein
kinase C activity; cAMP; cGMP
1. Introduction
Serotonin is a naturally occurring amine with major
effects on a variety of bodily functions. To date, the
studies concerning serotonin have been focused on vascu-
lar and inflammatory responses. Important early studies on
the mechanisms of acute inflammation were performed
with rats and mice and 5-HT receptor antagonists, and
demonstrated the importance of serotonin to the develop-
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ment of inflammation in these models Spector and
.Ž.
Willoughby, 1958a,b, 1959a,b, 1964 . Owen 1977 sug-
gested that administration of serotonin to the plantar sur-
face of the rat paw caused edema with striking extravasa-
tion of albumin. Serotonin was also reported to induce
plasma extravasation as a result of edema formation in
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vivo Pierce et al., 1995; Wang et al., 1996 .
The vascular endothelium functions as a critical and
selective barrier to macromolecular permeability, protect-
ing the underlying tissues from edema. This barrier func-
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Corresponding author. Tel.: q886-4-2058436; fax: q886-4-2039203.
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E-mail address: hong@mail.cmc.edu.tw H.-Z Lee .
tion is modulated by many vasoactive agents in vivo
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Majno and Palade, 1961; Svensjo and Grega, 1986 .
These vasoactive agents, such as thrombin, bradykinin, and
histamine, have been also shown to increase macromolecu-
lar transfer across endothelial monolayers obtained from
human umbilical vein, bovine pulmonary artery and bovine
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aorta in vitro Buchan and Martin, 1992; Schaeffer et al.,
.
1993; Garcia et al., 1986 . Endothelial cell contraction,
with subsequent formation of intercellular gaps, has been
hypothesized as the mechanism by which increased para-
cellular macromolecular transport and increased endothe-
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lial cell permeability occur Majno and Palade, 1961;
.
Boswell et al., 1992; Garcia et al., 1995; Lee, 1997 .
Nowadays, the molecules involved in the regulation of this
contraction are recognized. They include intracellular Ca
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,
protein kinase C, and actin.
Many investigators suggest that protein kinase C activa-
tion is an important signal transduction pathway by which
extracellular mediators increase endothelial macromolecu-
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lar transport Lynch et al., 1990; Stasek et al., 1992;
.
Krizbai et al., 1995; Nagpala et al., 1996 . The activation
of protein kinase C, which can occur as a result of the
0014-2999r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved.
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PII: S0014-29 99 00 00495-7