Faculty of Science - Mahidol University

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334 The mode of carnitine transport in the male reproductive tract is not clearly understood although carnitine plays an important role in male fertility. This study examined the mechanisms of [3H] L-carnitine secretion in a sperm-free isolated segment of the rat distal caput epididymidis perfused in vitro. The secretion varied with the perfusion rate from 0.25 to 1.35 ?l/min in a curvilinear fashion being 4.00 + 0.63 pmol/min.cm at the perfusion rate of 0.78 + 0.03 ?l/ min, and was maintained for 3 hrs. The secretion was not altered when the luminal fluid contained L-carnitine at a concentration 10 folds higher than that in the bath. Replacement of luminal Na + with either N-Methyl-D-glucamine or choline did not suppress the secretion of carnitine. Instead, it was significantly increased. Addition of acetyl-L-carnitine (250 ?M) in the bath failed to inhibit secretion. However, raising the concentration of L-carnitine in the bath from 30 to 500 ?M enhanced the rate of secretion up to 10 folds. The results suggest that secretion of carnitine is carrier mediated. Uptake from the bath into the epididymal cells is achieved by a transporter with low Km and high Vmax, most likely CT1 (OCTN2), which has been localized on the basolateral membrane of the epididymal tubule whilst efflux into the lumen occurs via at least 3 pathways: 1) CT2, probably the main route; 2) a transporter with high Km and Vmax; and 3) a carnitine exchanger. (Supported by Commission on higher Education Staff Development Program to K. Poungpong) INDUCTION OF APOPTOSIS IN THE ABSENCE OF CASPASE-3 BY THE FLAVONE VR-3623 (NO. 888) Kulawee Sujarit 1 , Orawin Prangsaengtong 1 , Samaisukh Sophasan 1 , Vichai Reutrakul 2 1 Department of Physiology, 2 Department of Chemistry, Faculty of Science, Mahidol University, E-mail address: scssj@mahidol.ac.th Key words : VR-3623, MCF-7 cell, caspase-independent The mechanism of the antiproliferative activity of VR-3623, a flavone purified from a Thai tropical plant, Gardenia obtusifolia, on human MCF-7 breast cancer cells, which lack caspase-3 was investigated in vitro in the present study. Treatment of MCF-7 cells with 3 concentrations, 0.8 (GI 50 ), 2, and 8 ?M of VR-3623 for 3-72 hrs. resulted in a dose-and time-dependent increased apoptotic cells as evaluated by 4’,6-diamidino-2-phenylindole (DAPI) staining and poly(ADP-ribose) polymerase (PARP) cleavage. The apoptotic cells characterized by chromatin condensation and DNA fragmentation increased from 8% to 66% as observed at 3 and 72 hrs., respectively, after incubation with 8 ?M of VR-3623. Immuno blot analysis showed the expression of the proteolytic PARP cleavage (85 kDa) at 24 and 72 hrs. Preincubation cells with 10 ?M of Z-VAD-MK, a pan caspase inhibitor, for 1 h. before the addition of 8 ?M of the flavone could alleviate cell death by 13% and completely prevent the cleavage of PARP. Measurement of caspases–9 and –7 activities in the apoptotic cells by using a fluorogenic assay showed that both caspases were activated, but at the low levels throughout the experiments. It is concluded that VR-3623 suppresses the growth of MCF-7 cells by induction of apoptosis and caspase-3 may not be essential in the apoptosis induced by this flavone. (Supported by the Thailand Research Fund and Deptartment of Physiology, Faculty of Science, Mahidol University.) Faculty of Science ANTI-INFLAMMATORY AND IMMUNOMODU- LATORY ACTIVITIES OF STEVIOSIDE AND ITS METABOLITE, STEVIOL ON THP-1 CELLS (NO. 889) Chaiwat Boonkaewwan 1 , Chaivat Toskulkao 1 , Molvibha Vongsakul 2 1 Department of Physiology and 2 Department of Microbiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand. E-mail address: kongchaiwat@hotmail.com; sccts@mahidol.ac.th; scmvs@mahidol.ac.th Key words : anti-inflammation, inflammatory cytokines, stevioside Stevioside (SVS), a natural noncaloric sweetener isolated from Stevia rebaudiana Bertoni, possesses anti-inflammatory and anti-tumor promoting properties; however, no information is available to explain its activity. The aim of this study was to elucidate the anti-inflammatory and immunomodulatory activities of stevioside and its metabolite, steviol (SVO). Stevioside 1 mM significantly suppressed lipopolysaccharide (LPS)-induced release of TNF-α and IL-1β and slightly suppressed nitric oxide release in THP-1 cells without exerting any direct toxic effect, whereas steviol 100 µM did not. Activation of IKKβ and transcription factor NF-κB were suppressed by stevioside, as demonstrated by western blotting. Furthermore, only stevioside induced TNF-α, IL-1β and Nitric oxide release in unstimulated THP-1 cells. Release of TNF-α could be partially neutralized by anti-TLR4 antibody. This study suggested that stevioside attenuates synthesis of inflammatory mediators in LPS-stimulated THP-1 cells by interfering the IKKβ and NF-κB signaling pathway, and stevioside-induced TNF-α secretion is partially mediated through TLR4. (Supported by Mahidol University grants (Government Funds) and Ministry of University Affairs, Thailand) EVALUATION ON THE HEPATOPRO- TECTIVE EFFECTS OF THE STANDARD EXTRACTS FROM Curcuma longa and Andrographis paniculata (NO. 890) Surawat Jariyawat, Kanoknetr Suksen, Aporn Chuncharunee, Samaisukh Sophasan and Pawinee Piyachaturawat Department of Physiology, Graduate Program in Toxicology, Faculty of Science, and Department of Anatomy, Faculty of Medicine, Siriraj Hospital, Mahidol University. E-mail: scsjr@mahidol.ac.th Key words: Curcuma longa, Andrographis paniculata, hepatoprotection This study aims to evaluate the hepatoprotective effect of the standard extracts from Curcuma longa and Andrographis paniculata (Turmeric and AP-extracts). An oral administration of the extract was given to adult male mice 24 h prior to being induced by hepatotoxins and the leakage of hepatic enzymes in plasma was evaluated at 24 h later. The turmeric extract exhibited hepatoprotective activities against CCl 4 but not acetaminophen whereas the AP-extract showed hepatoprotective activities against both CCl 4 - and acetaminophen-induced toxicities. Changes of liver histopathology after treatments were well correlated with the changes PDF created with FinePrint pdfFactory Pro trial version http://www.pdffactory.com
Mahidol University Annual Research Abstracts, Vol. 33 335 of biochemical parameters. By using a human hepatocarcinoma cell line, HepG2, both of the standard extracts showed the protective activities against acetaminophen-induced toxicity. The results suggested that the AP-extract is more effective in providing protection against hepatotoxins. (Supported by a grant from the National Research Council of Thailand-2547) EVALUATION ON THE CHOLERETIC EFFECT OF HYDROXY ACETOPHENONES ON THE SECRETING SPECIES OF BILE ACIDS (NO. 891) Pawinee Piyachaturawat, Suparat Khamdang, Apichart Suksamrarn Department of Physiology and Graduate Program in Toxicology, Fac. of Science, Mahidol University; Department of Chemistry, Fac. of Science, Ramkhamhaeng University. E-mail: scppy@mahidol.ac.th Key words : Bile secretion, Bile acids, Hydroxyacetophenone Choleretic effect of hydroxy acetophenones has been reported to differently increase bile flow rate with varying biliary bile acids output as well as plasma cholesterol (EJP387: 21, 2000). The secretion varied from essential bile acid dependence to essential bile acid independence. The mechanisms by which these compounds exerted different actions remain unclear. This study aims to determine the species of bile acids in the secreted bile after receiving hydroxy acetophenones as bile acids provide the major force for bile formation. The compounds used were 2,4,6-tri, 2,6-di, 2,4-di and 4-mono hydroxy acetophenones (2,4,6-THA, 2,6-DHA, 2,4- DHA and 4-MHA). By using HPLC analysis, four major bile acids species were detected in normal rat bile. They were cholic, chenodeoxycholic, deoxycholic and ursodeoxycholic acids, accounting for about 70, 9, 3, and 18 % of total bile acids, respectively. After administration of the compounds at a dose of 100 mg/kg, id, for 30 min, although the secreted bile acid outputs were markedly increased, their percent distribution of individual species was not different among groups and from the control. 4- MHA did not alter the output. It is suggested that hydroxy acetophenones stimulated biliary secretion of bile acids from the intracellular storage pool in the hepatocyte, not from newly synthesized bile acids. (Supported by Thailand Research Fund and ESTEM, MUA) ENDOGENOUS PROLACTIN MODULATED THE CALCIUM ABSORPTION IN THE JEJUNUM OF SUCKLING RATS (NO. 892) Suwimol Amnattanakul, Narattaphol Charoenphandhu, Liangchai Limlomwongse, and Nateetip Krishnamra Department of Physiology, Faculty of Science, Mahidol University E-mail: scnks@mahidol.ac.th, naratt@narattsys.com Key words : calcium absorption, intestinal segments, prolactin Prolactin has been reported to stimulate intestinal calcium absorption in young and mature, but not aging rats. The present study was performed on suckling rats to elucidate the actions of endogenous prolactin on calcium absorption in various intestinal segments. Before measuring the calcium fluxes, 9-day-old rats were administered for 7 days with 0.9% NaCl, s.c. (control), 3 mg/kg bromocriptine, i.p., twice daily to abolish secretion of endogenous pro lac tin, or bromocriptine plus exogenous 2.5 mg/kg prolactin, s.c. Thereafter, the 16-day-old rats were experimented upon by instilling the 45 Ca-containing solution into the intestinal segments. The results showed that, under a physiological condition, the jejunum had the highest rate of calcium absorption compared with other segments (1.4 ± 0.35 µmol·h –1 ·cm –1 , p < 0.05). The duodenum and ileum also manifested calcium absorption, whereas the colon showed calcium secretion. Lack of endogenous prolactin decreased lumento-plasma and net calcium fluxes in jejunum from 2.07 ± 0.31 to 1.19 ± 0.12 and 1.40 ± 0.35 to 0.88 ± 0.18 µmol·h –1 ·cm –1 (p < 0.05), respectively, and exogenous prolactin restored the jejunal calcium absorption to the control value. Endogenous prolactin also had an effect on the duodenum but, in this case, exogenous prolactin did not reverse the effect of bromocriptine. However, neither ileal nor colonic calcium fluxes were influenced by prolactin. Because luminal sodium concentration has been demonstrated to affect calcium absorption in mature rats, the effect of varying luminal sodium concentrations on calcium fluxes in suckling rats was evaluated. The jejunum was used due to its highest rate of calcium absorption. After filling the jejunal segments with 124 (control), 80, 40 mmol/ L Na + -containing or Na + -free solution, increases in calcium absorption were found to be inversely related to luminal sodium concentrations in both control and bromocriptine-treated rats. The plasma concentration of 45 Ca under luminal sodium free condition was also higher than that of the control condition (2.26% ± 0.07% vs. 2.01% ± 0.09% administered dose, p < 0.05). However, 3 Hmannitol, a marker of the widening of tight junction that was introduced into the lumen, had a stable level in the plasma during an increase in plasma 45 Ca, suggesting that the widening of tight junction was not required for enhanced calcium absorption. In conclusion, calcium absorption in suckling rats was of the highest rate in the jejunum where endogenous prolactin modulated calcium absorption without increasing the paracellular transport of mannitol. (Supported by The Thailand Research Fund and the Graduate Student Grant of the Faculty of Graduate Studies, Mahidol University.) MECHANICAL VENTILATION INDUCES ALTERA- TIONS OF THE UBIQUITIN-PROTEASOME PATHWAY IN THE DIAPHRAGM (NO. 893) Keith C. DeRuisseau, 1 Andreas N. Kavazis, 1 Melissa A. Deering, 1 Darin J. Falk, 1 Darin Van Gammeren, 1 Tossaporn Yimlamai, 1,2 George A. Ordway, 3 and Scott K. Powers 1 1 Department of Applied Physiology and Kinesiology, University of Florida, Gainesville, Florida; 2 Department of Physiology, Faculty of Science, Mahidol University, Bangkok, Thailand; and 3 Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas E-mail: sctyl@mahidol.ac.th Key words: respiratory muscle, proteolysis, proteasome activity Prolonged mechanical ventilation (MV) results in diaphragmatic atrophy due, in part, to an increase in proteolysis. These experiments tested the hypothesis that MV-induced diaphragmatic proteolysis is accompanied by increased expression PDF created with FinePrint pdfFactory Pro trial version http://www.pdffactory.com
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