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pressure In the first 4 weeks the SBP of all rats increased,
accompanied by an increase in weight; however, there was
no significant difference between the ouabain group and the
control group. From the fifth week, the SBP of 45 of the 52
rats in the ouabain group began to increase. Six weeks later
the SBP of the 45 rats were markedly higher than those of the
control group, which received normal saline (142.3±8.2 mmHg
vs 123.4±5.4 mmHg; P<0.01]. These 45 rats were designated
"ouabain-sensitive" (OS) rats. In the remaining 7 rats in the
ouabain group, the SBP did not increase, and these 7 rats
were designated "ouabain-resistant" (OR) rats (Figure 5).
Changes in aortic endothelium in OS and OR
rats In OS rats, the structure of the aortic endothelium was damaged
and the nuclei were depleted. In OR rats, the aortic
endothelium was integrated, the nuclei were clear, and the
endothelial cells were tightly linked to the internal elastic membrane
(Figure 6).
Effect of ouabain on Kv4.2 gene expression in OS and
OR rats Kv4.2 expression was markedly higher in OS rats
than in OR and control rats (P<0.01; Figure 7).
Discussion
Ouabain is one of the most highly hydroxylated, water
soluble and potent cardiac glycosides. It was recognized as
the primary active component of Masai arrow poison more
than 100 years ago by the French anthropologist Arnaud.
Recent work has shown that ouabain (or closely related
isomers) is present in the human
circulation[6].
The effect of ouabain as a sodium pump
(Na+/K+-ATPase) inhibitor is mediated by cell depolarization and
[Na+]i elevation, resulting in activation of voltage-gated
Ca2+ channels and the electrogenic
Ca2+o/3Na+i
exchanger[7]. The effect of ouabain on cell survival is
tissue-specific[8]. Twenty-four hours of treatment with ouabain detaches and kills
Madin-Darby canine kidney (MDCK) epithelial cells;
however, 2 to 4 d treatment with ouabain does not affect the
survival of renal epithelial cells from Rhesus monkeys,
human lymphocytes or vascular smooth muscle cells (VSMC)
from the rat aorta.
Ouabain has been known to act as a hormone and is
possibly involved in the pathogenesis of
hypertension[9]. To investigate the mechanism that is involved in the
vascular remodeling that occurs during hypertension, we studied
the effects of ouabain on HUVEC in culture. We found that
ouabain at physiological concentrations (0.3-0.9 nmol/L)
stimulated the proliferation of HUVEC. However, at
pathological concentrations (0.9-1.8 nmol/L), cell death was
induced, including the swelling phenomenon and
appearance of apoptosis bodies. As Xiao
et al suggested, the neuronal death associated with ouabain treatment consisted
of concurrent apoptotic and necrotic
components[10]. The proteome analysis indicated that ouabain treatment led to
overexpression of numerous soluble and hydrophobic
cellular proteins[11]. The results of mRNA profile analysis
indicated that most of the upregulated genes were related to
signal transduction and metabolism. The candidate genes
were screened from the abovementioned genes and analyzed
further.
We found that the protein kinase gene
(NM_002737) and protein phosphatase genes
(NM_014225 and NM_002719) were upregulated. These 2 genes regulate
signal transduction by catalysis phosphorylation and
dephosphorylation. The protein kinase and protein
phosphatase act on tyrosine, serine, and threonine, related to cell
proliferation and
differentiation[12,13]. DAPK1
(death-associated protein) (NM_004938), a
calcium/calmodulin-dependent serine/threonine kinase, exerts apoptotic effects by
suppressing integrin functions and integrin-mediated
survival signals, and activating a p53-dependent apoptotic
pathway[14]. According to these early responsive gene (ERG),
ouabain mediates the expression of downstream genes, and
makes the endothelial cells in normal metabolism condition.
Also, ouabain upregulates many transcription genes, such
as tumor necrosis factor receptor-associated factor family
member-associated NF-kB activator (TANK)
(NM_004180), E2F4
(NM_001950), GTF21(NM_0999), CREBBP
(NM_004380) and TFIIF (NM_002096).
NF-kB, which is activated by ouabain and TANK, plays a role in immunity
protection, anti-inflammation, vasomotion and cell
proliferation by regulating some cytokine genes and triggering
endothelial cells to secrete
cytokines[15,16]. The results of the present study implied that
NF-kB was activated. The arrestin beta 1 gene
(NM_004041) was upregulated in the mRNA profiles analysis. Members of the arrestin/beta-arrestin
protein family are thought to participate in agonist-mediated
desensitization of G-protein-coupled receptors and cause
specific dampening of cellular responses to stimuli, such as
hormones, neurotransmitters, or sensory signals. Arrestin
beta 1 is a cytosolic protein that acts as a cofactor in the
beta-adrenergic receptor kinase-mediated desensitization of
beta-adrenergic receptors[17]. Physiological ouabain might
be a beta-adrenergic receptor inhibitor according to
upregulation of the arrestin beta 1 gene, which was proved
by the downregulation (Cy5/Cy3=0.374) of the beta
2-adrenergic receptor kinase (ADRBK2) found in the present study.
KCND2, which was found to be upregulated in the present
study, is related to the potassium current, and ouabain is an
inhibitor of sodium pumps. Therefore, in the present paper,
we only focused on KCND2. Voltage-gated potassium (Kv)
channels, which induce outward K+ ion currents, are the most
complex class of voltage-gated ion channels with respect to
both functional and structural
aspects[16]. They consist of 4 sequence-related potassium channel proteins: shaker, shaw,
shab and shal, which are encoded by KCNA, KCNB,
KCNC and KCND, respectively. Their diverse functions include
regulation of neurotransmitter release, heart rate, insulin
secretion, neuronal excitability, epithelial electrolyte
transport, smooth muscle contraction, and cell
volume[18]. Kv4.2/Kv4.3, which are related to the shal subfamily encoded
by KCND, participate in the formation of transient outward
K+ ion current (Ito), are expressed in the epicardium of the
atrium and ventricles, and create the action potential
repolarization I phase.
Kv channels control action potential repolarization,
membrane potential, and action potential frequency in excitable
cells. The gene chip findings of the present study showed
that KCND was upregulated HUVEC after treatment with
ouabain. The SBP of OS rats treated with ouabain increased,
the aortic endothelium became structurally damaged, and
the expression level of Kv4.2 was higher than that of the
control group[19]. The pressure, the structure of the aortic
endothelium, and the Kv4.2 expression level did not change
in OR rats compared with the control group. The findings
indicated that endothelial cells had Kv4.2 channels encoded
by KCND2, which is involved in the cell functions related to
regulating ion transport in the cells. Expression of the
Kv4.2 channel was upregulated after chronic treatment with
ouabain, which might induce changes in the potassium
concentration inside and outside cells, and then indirectly
inhibit the sodium pump of the membrane. The relationship
between Kv4.2 expression, pressure increases and
endothelium damage indicate that Kv channels might be a prominent
factor in the pathological process whereby ouabain induces
hypertension vascular remodeling.
The results of the present study suggest that ouabain
might be involved in controlling the growth and metabolism
of vascular endothelium cells at physiological
concen-trations, but at pathological concentrations it might affect
the structure and function of endothelial cells, and trigger
vascular remodeling in hypertension. Nonetheless, some
questions require further study, for example why ouabain
was able to stimulate proliferation at a concentration of 10
nmol/L, which is higher than that found under pathological
conditions? Is there "autocrine" and "paracrine" ouabain in
endothelial cells, such as occurs for other hormones (eg,
rennin)? We will address these questions in future studies.
Acknowledgement
We would like to thank all members of our ouabain study
group.
References
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