Faculty of Science - Mahidol University

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of key components of the ubiquitin-proteasome pathway (UPP). To
test this postulate, we investigated the effect of prolonged MV on
UPP components and determined the trypsin-like and
peptidylglutamyl peptide hydrolyzing activities of the 20S
proteasome. Adult Sprague-Dawley rats were assigned to either
control or 12-h MV groups (n = 7/group). MV animals were
anesthetized, tracheostomized, and ventilated with room air for 12
h. Animals in the control group were acutely anesthetized but not
exposed to MV. Compared with controls, MV animals demonstrated
increased diaphragmatic mRNA levels of two ubiquitin ligases,
muscle atrophy F-box (+8.3-fold) and muscle ring finger 1 (+19.0fold).
However, MV did not alter mRNA levels of 14-kDa ubiquitinconjugating
enzyme, polyubiquitin, proteasome-activating complex
PA28, or 20S α-subunit 7. Protein levels of 14-kDa ubiquitinconjugating
enzyme and proteasome-activating complex PA28 were
not altered following MV, but 20S α -subunit 7 levels declined (–
17.7%). MV increased diaphragmatic trypsin-like activity (+31%)
but did not alter peptidylglutamyl peptide hydrolyzing activity.
Finally, compared with controls, MV increased ubiquitin-protein
conjugates in both the myofibrillar (+24.9%) and cytosolic (+54.7%)
fractions of the diaphragm. These results are consistent with the
hypothesis that prolonged MV increases diaphragmatic levels of key
components within the UPP and that increases in 20S proteasome
activity contribute to MV-induced diaphragmatic proteolysis and
atrophy.
(Supported by National, Heart, Lung, and Blood Institute Grant)
BARAKOL EXTRACTED FROM CASSIA SIAMEA
STIMULATES CHLORIDE SECRETION IN RAT
COLON (NO. 894)
Chatsri Deachapunya, Sutthasinee Poonyachoti, Watchareewan
Thongsaard, Nateetip Krishnamra
Department of Physiology, Faculty of Science, Mahidol
University E-mail: scnks@mahidol.ac.th
Key words : Barakol, Cassia siamea, chloride secretion
Barakol is a purified extract of Cassia siamea, a plant that
has been used as a laxative in traditional medicine. In this study, the
effect of barakol on anion transport across the rat colon epithelium
was investigated. Colonic epithelium was mounted in Ussing
chambers and bathed with Ringer’s solution. Addition of 1 mM
barakol to the basolateral solution produced a slow increase in shortcircuit
current (Isc) in proximal colon and distal colon by 24.5 +/-
2.2 and 24.2 +/- 1.4 microA/cm(2), respectively. Barakol increased
Isc in a concentration-dependent manner with an EC(50) value of
0.4 mM. The barakol-stimulated increase in Isc was inhibited by
subsequent treatment with 500 microM diphenylamine-2-carboxylic
acid or 400 microM glibenclamide added to the apical solution and
200 microM bumetanide added to the basolateral solution.
Pretreatment of the tissues with 200 microM bumetanide, but not
10 microM amiloride, completely abolished the barakol-increased
Isc. Ion substitution experiments showed an inhibition of barakolstimulated
Isc in chloride-free solution but not in bicarbonate-free
solution. In addition, pretreatment of tissues with 10 microM
tetrodotoxin or 10 microM indomethacin, but not 1 microM atropine
or 10 microM hexamethonium, partially inhibited the Isc response
by barakol. The present results demonstrated the stimulatory effect
of barakol on the bumetanide-sensitive chloride secretion in rat colon.
The effect of barakol was partly mediated by the stimulation of
submucosal nerves and through the release of cyclooxygenase
metabolites. These findings thus provide an explanation for the
underlying mechanism of barakol as a secretagogue in mammalian
colon.
(Supported by The Thailand Research Fund)
Faculty of Science
GDNF FAMILY RECEPTOR ALPHAL PHENOTYPE
OF SPERMATOGONIAL STEM CELLS IN IMMA-
TURE MOUSE TESTIS (NO. 895)
Anyanee Buageaw , Meena Sukhwani , Ahmi Ben-Yehudah ,
Jens Ehmcke , Vanessa Y. Rawe , Chumpol Pholpramool , Kyle
E. Orwig , and Stefan Schlatt
Department of Physiology, Faculty of Science, Mahidol
University. E-mail: sccpp@mahidol.ac.th
Spermatogonial stem cells (SSCs) are essential for
spermatogenesis, and these adult tissue stem cells balance selfrenewal
and differentiation to meet the biological demand of the
testis. The developmental dynamics of SSCs are controlled in part
by factors present in the stem cell niche, which is located on the
basement membrane of seminiferous tubules situated among Sertoli
cells. Sertoli cells produce glial cell line derived neurotrophic factor
(GDNF) and disruption of GDNF expression results in spermatogenic
defects and infertility. GDNF signals through a receptor complex
that includes GDNF family receptor alpha1 (GFRA1), which is
thought to be expressed by SSCs. However, expression of GFRA1
on SSCs has not been confirmed by in vivo functional assay, which
is the only method that allows definitive identification of SSCs.
Therefore, we fractionated mouse pup testis cells based on GFRA1
expression using magnetic activated cell sorting (MACS). GFRA1
sorted and depleted fractions were characterized for germ cell markers
by immunocytochemistry, and stem cell activity by germ cell
transplantation. The GFRA1 positive cell fraction co-eluted with
other markers of SSCs, including ITGA6 and CD9, and was
significantly depleted of KIT positive cells. The transplantation
results confirmed that a subpopulation of SSCs expresses GFRA1,
but that the stem cell pool is heterogeneous with respect to the level
of GFRA1 expression. Interestingly, POU5F1 positive cells were
enriched nearly 15-fold in the GFRA1 selected fraction, possibly
suggesting heterogeneity of developmental potential within the stem
cell pool.
(Supported by Commission on Higher Education Staff Development
Program to A. Baugeaw)
INTERACTIONS OF STEVIOSIDE AND STEVIOL
WITH RENAL ORGANIC ANION TRANSPORTERS
IN S2 CELLS AND MOUSE RENAL CORTICAL
SLICES (NO. 896)
Chutima Srimaroeng 1,2 , Promsuk Jutabha 3 , John B. Pritchard 2 ,
Hitoshi Endou 3 , Varanuj Chatsudthipong 1
1 Department of Physiology, Faculty of Science, Mahidol
University; 2 Laboratory of Pharmacology and Chemistry,
NIEHS, National Institutes of Health, Research Triangle Park,
North Carolina, USA; 3 Department of Pharmacology and
Toxicology, Kyorin University School of Medicine, Tokyo, Japan.
E-mail: scvcs@mahidol.ac.th
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