European Journal of Pharmacology 403 2000 195–202
Serotonin-induced protein kinase C activation in
cultured rat heart endothelial cells
, 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
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
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
-phospholipid-dependent protein kinase, protein kinase C, was
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
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.
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
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-
ment of inflammation in these models Spector and
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
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-
Corresponding author. Tel.: q886-4-2058436; fax: q886-4-2039203.
E-mail address: firstname.lastname@example.org H.-Z Lee .
tion is modulated by many vasoactive agents in vivo
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
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-
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
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-
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.
PII: S0014-29 99 00 00495-7