• Journal of Aquaculture
• /
• v.16 no.1
• /
• pp.8-14
• /
• 2003

To generate expressed sequence tags for genomics research involving genetic linkage analysis, to examine gene expression profiles in muscles of channel catfish in a non-normalized muscle cDNA library, to compare gene expression in young and mature channel catfish muscles using the EST reagents and gene filters to demonstrate the feasibility of functional genomics research in small laboratories. 102 randomly picked cDNA clones were analyzed from the catfish muscle cDNA library. Of the sequences generated, 90.2% of ESTs was identified as known genes by identity comparisons. These 92 clones of known gene products represent transcriptional products of 24 genes. The 10 clones of unknown gene products represent 8 genes. The major transcripts (70.1% of the analyzed ESTs) in the catfish muscle are from many major genes involved in muscle contraction, relaxation, energy metabolism and calcium binding such as alpha actin, creatine kinase, parvalbumin, myosin, troponins, and tropomyosins. Gene expression of the unique ESTs was comparatively studied in the young and adult catfish muscles. Significant differences were observed for aldolase, myostatin, myosin light chain, parvalbumin, and an unknown gene. While myosin light chain and an unknown gene (CM 192) are down-regulated in the mature fish muscle, the aldolase, myostatin, and parvalbumin are significantly up-regulated in the mature fish muscle. Although the physiological significance of the changes in expression levels needs to be further addressed, this research demonstrates the feasibility and power of functional genomics in channel catfish. Channel catfish muscle gene expression profiles provide a valuable molecular muscle physiology blueprint for functional comparative genomics.

• The Korean Journal of Pharmacology
• /
• v.32 no.1
• /
• pp.39-49
• /
• 1996

In the present study, we observed change in intracellular $Ca^{2+}$$([Ca^{2+}]_i)$ as measured with the fluorescent $Ca^{2+}-indicator$ fura-2 in association with force development of the rat basilar arteries during activation by$K^+$ depolarizing solution and U46619, a thromboxane analogue, in the absence and the presence of calcitonin-gent related peptide (CGRP). CGRP (30 and 100 nM) caused a concentration-dependent inhibition of U46619-induced contraction with decrease in $[Ca^{2+}]_i$, whereas it did not exert any effect on the $K^+$ (90 mM)-induced contraction and increase in $[Ca^{2+}]_i$, Further, $[Ca^{2+}]_i-force$ relationships were determined by plotting the ratio of $F_{340}/F_{380}$ $([Ca^{2+}]_i)$ as a function of the force induced by U46619, and the results were compared with those obtained in the presence of CGRP. The curves obtained in the presence of CGRP (30 and 100 nM) were significantly moved to downward without right shift of the curves suggesting that CGRP inhibited the U46619-induced contraction only by mediation of reduction in $[Ca^{2+}]_i$ with out any change in the sensitivity of contractile apparatus to $Ca^{2+}$. The CGRP-induced attenuation of $[Ca^{2+}]_i$ and force development was significantly inhibited under pretreatment with CGRP $(8{\sim}37)$ fragment (100 nM), a CGRP1 receptor antagonist. Both the reduced contraction and reduction in $[Ca^{2+}]_i$ caused by CGRP were fully reversed by pretreatment with charybdotoxin (100 nM) and iberiotoxin (100 nM), large conductance $Ca^{2+}-activated$ $K^+$ channel blockers, but not by apamin (300 nM), a small conductance $Ca^{2+}-activated$ $K^+$ channel blocker, and glibenclamide ( 1 ${\mu}M$), an ATP-sensitive $K^+$ channel blocker. In conclusion, it is suggested that the CGRP1 receptor, upon activation by CGRP, are coupled to opening of $Ca^{2+}-activated$ $K^+$ channel and cause to decrease in $[Ca^{2+}]_i$, thereby leading to vasodilation of the rat basilar artery. However, it is not defined that the mechanism underlying vasodilation whether the $K^+$ channel blockers, charybdotoxin and iberiotoxin directly block the CGRP receptors and that CGRP-evoked relaxation is dependent on the cyclic AMP or $K^+$ channel opening or both actions.

• YAKHAK HOEJI
• /
• v.41 no.6
• /
• pp.797-801
• /
• 1997

Recently we reported that water extracts of Coptidis Rhizomas showed calcium antagonistic action and alpha-adrenoceptor inhibitory action in the vascular smooth muscle. Since ca lcium antagonistic properties are important in the treatment of various diseases including asthma. In the present study, the bronchodilatory effects of crude extract of three kinds of Coptidis Rhizoma (Coptidis chinensis, Coptis japonica and root hair of Coptis japonica) was investigated using rat isolated trachea. The result showed that all extracts relaxed carbachol-contracted tracheal smooth muscle. Concentration-dependently, in which the root hair of Coptis japonica was the least potent. The inhibitory potency expressed in terms of $IC_{50}$ against carbachol contraction was 1.8${\mu}$g/ml and 2.7${\mu}$g/ml for Coptidis chinensis and Coptis japonica, respectively. These extracts also inhibited KCI-contracted tracheal smooth muscle. But the relative potency ($IC_{50}$) was 3.5 and 4.1 folds weaker than carbachol-induced contraction for Coptidics chinenesis and Coptis japonica, respectively. Pretreatment of crude extracts also inhibited carbachol- or KCI-induced contraction, non-competitively. These findings indicate that the extracts have muscarinic blocking as well as $Ca^{2+}$ channel blocking action. When provoked intracellular stored $Ca^{2+}$ release by carbachol in $Ca^{2+}$-free conditions, initial phasic contraction due to $Ca^{2+}$ release was significantly inhibited by the extracts. As taken together, we conclude that water extracts of Coptidis Rhizoma may be beneficial in bronchospasm or other broncheal tube narrowing conditions such as asthma.

• Biomolecules & Therapeutics
• /
• v.27 no.1
• /
• pp.101-106
• /
• 2019

Most diabetic patients experience diabetic mellitus (DM) urinary bladder dysfunction. A number of studies evaluate bladder smooth muscle contraction in DM. In this study, we evaluated the change of bladder smooth muscle contraction between normal rats and DM rats. Furthermore, we used pharmacological inhibitors to determine the differences in the signaling pathways between normal and DM rats. Rats in the DM group received an intraperitoneal injection of 65 mg/kg streptozotocin and measured blood glucose level after 14 days to confirm DM. Bladder smooth muscle contraction was induced using acetylcholine (ACh, $10^{-4}M$). The materials such as, atropine (a muscarinic receptor antagonist), U73122 (a phospholipase C inhibitor), DPCPX (an adenosine $A_1$ receptor antagonist), udenafil (a PDE5 inhibitor), prazosin (an ${\alpha}_1$-receptor antagonist), papaverine (a smooth muscle relaxant), verapamil (a calcium channel blocker), and chelerythrine (a protein kinase C inhibitor) were pre-treated in bladder smooth muscle. We found that the DM rats had lower bladder smooth muscle contractility than normal rats. When prazosin, udenafil, verapamil, and U73122 were pre-treated, there were significant differences between normal and DM rats. Taken together, it was concluded that the change of intracellular $Ca^{2+}$ release mediated by PLC/IP3 and PDE5 activity were responsible for decreased bladder smooth muscle contractility in DM rats.

• The Korean Journal of Physiology and Pharmacology
• /
• v.28 no.4
• /
• pp.335-344
• /
• 2024

Diphenyleneiodonium (DPI) has been widely used as an inhibitor of NADPH oxidase (Nox) to discover its function in cardiac myocytes under various stimuli. However, the effects of DPI itself on Ca²⁺ signaling and contraction in cardiac myocytes under control conditions have not been understood. We investigated the effects of DPI on contraction and Ca²⁺ signaling and their underlying mechanisms using video edge detection, confocal imaging, and whole-cell patch clamp technique in isolated rat cardiac myocytes. Application of DPI suppressed cell shortenings in a concentration-dependent manner (IC₅₀ of ≅0.17 µM) with a maximal inhibition of ~70% at ~100 µM. DPI decreased the magnitude of Ca²⁺ transient and sarcoplasmic reticulum Ca²⁺ content by 20%-30% at 3 µM that is usually used to remove the Nox activity, with no effect on fractional release. There was no significant change in the half-decay time of Ca²⁺ transients by DPI. The L-type Ca²⁺ current (I_Ca) was decreased concentration-dependently by DPI (IC₅₀ of ≅40.3 µM) with ≅13.1%-inhibition at 3 µM. The frequency of Ca²⁺ sparks was reduced by 3 µM DPI (by ~25%), which was resistant to a brief removal of external Ca²⁺ and Na⁺. Mitochondrial superoxide level was reduced by DPI at 3-100 µM. Our data suggest that DPI may suppress L-type Ca²⁺ channel and RyR, thereby attenuating Ca²⁺-induced Ca²⁺ release and contractility in cardiac myocytes, and that such DPI effects may be related to mitochondrial metabolic suppression.

• Korean Journal of Veterinary Research
• /
• v.35 no.2
• /
• pp.245-254
• /
• 1995

The density of ATP-sensitive potassium($K_{APT}$) channels, that open as intracellular ATP concentration falls below a critical level, is very high in skeletal muscle surface membrane and those high density may imply that $K_{ATP}$ channels have very important physiological roles. To elucidate a role of $K_{ATP}$ in relation to fatigue, the modulating effects of potassium channel openers and blockers on the fatigue velocity(FV) of mouse extensor hallucis longus muscle(EHL) were investigated in vitro. Twitch contraction was induced by an electrical field stimulation (EFS: 24-48V, 20ms, 0.2-4Hz) and resulting contraction force was isometrically recorded. The twitch forces were gradually decreased to 25% of initial contraction force(ICF) in $37.52{\pm}1.55sec$($mean{\pm}s.e.m.$, n=135), indicating the fatigue phenomena. The mean velocity for development of the fatigue was measured during the period that twitch force decreased to half($FV_{0/0.5}$) and during the period from half to 25%($FV_{0.5/0.25}$) of ICF. The fatigue was induced once every one hour and the tissue response was stable for up to 4 hours. In control condition, ICF was $5.8{\pm}0.12g$ (n=144) and decreased to 50% of ICF with the mean fatigue velocity of $0.182{\pm}0.006g/sec$($FV_{0/0.5}$, n=135) and from 50% to 25% of ICF with $0.084{\pm}0.004g/sec$($FV_{0.5/0.25}$, n=135). Cromakalim($50{\mu}M$) significantly increased $FV_{0.5/0.25}$(n=4). Glibenclamide($IC_{50}>50{\mu}M$), $Ba^{2+}$($IC_{50}=10{\mu}M$), 4-aminopyridine($FV_{0/0.5}$, $IC_{50}=0.5mM$; $FV_{0.5/0.25}$, $IC_{50}=2mM$) decreased both $FV_{0/0.5}$ and $FV_{0.5/0.25}$ concentration-dependently up to 75%. $TEA^+$(30mM), E-4031($10{\mu}M$), tolbutamide(1mM) decreased $FV_{0.5/0.25}$, but apamin(300nM) and $TEA^+$(10mM) showed no significant effects. Our results suggest that activation of the $K_{ATP}$ channels may be major cause of $K^+$ outflux during development of the fatigue and the isolated EHL muscle could be an useful experimental preparation in studying the fatigue phenomena in skeletal muscle. In addition, the possibility of activation of delayed rectifier during the fatigue development remains to be studied further.

• Journal of Ginseng Research
• /
• v.20 no.3
• /
• pp.274-283
• /
• 1996

In this study, the effects of red ginseng components [ginsenosides (total saponins and $Rg_1$) on the function of ryanodine receptor (RyR) -$Ca^{2+}$ release channel complex protein (named as RyR or $Ca^{2+}$ channel), a membrane protein in sarcoplasmic reticulum (SR) of rabbit skeletal muscle were examined at the SR vesicle's level and the molecular levels with Chaps-solubilized and purified $Ca^{2+}$ channel protein and with reconstituted proteoliposomes by dialysis. The results were as follows. 1. The binding of ryanodine known as inhibitor of muscle contraction to the RyR was decreased at the whole range of concentration ($10^2$~$10^7$%) by these two ginseng components. In heavy SR vesicles, Chaps-solubilized and purified $Ca^{2+}$ channel protein, and reconstituted vesicles, its maximal inhibition by total saponins was shown at the concentration of $10^3$, $10^3$%, and $10^5$% respectively, and by gin- senoside $Rg_1}$) each was $10^3$%, $10^3$%, and $10^4$%. 2. The release of $Ca^{2+}$ ion through $Ca^{2+}$ channel in heavy SR vesicles and reconstituted proteoliposomes was increased as a whole by these two ginseng components, and particularly maximal release by both of them was shown at the range of $10^4$~$10^6$%. These results were seemed to be caused by conformational change of $Ca^{2+}$ release channel protein (RyR) by red ginseng components [ginsenosides (total saponins and $Rg_1}$).

• BMB Reports
• /
• v.28 no.2
• /
• pp.129-137
• /
• 1995

The ryanodine-receptor $Ca^{2+}$ release channel protein in the sarcoplasmic reticulum membrane of rabbit skeletal muscle plays an important role in muscle exitation-contraction (E-C) coupling. Various types of detergents were tested, including Chaps, cholate, octylglucoside, Zwittergents, Mega-9, Lubrol PX, and Triton X-100 for solubilization of this protein. Among these, Chaps and Triton X-100 were found to optionally solubilize the channel complex. Optimum conditions for this solubilization were pH 7.4 with a salt concentration of 1 M. The addition of phospholipid in the solubilization step helped in stabilizing the protein. The purification of the receptor was performed using sucrose density gradient centrifugation. Various methods [dilution, freeze-thaw, adsorption (Biobeads), and dialysis] were investigated to incorporate the Chaps-solubilized and purified $Ca^{2+}$ release channel protein into liposomes made from different types of phospholipids. Of these, a combined method consisting of a dialysis, freeze-thaw and sonication steps yielded the best results. Reconstituted vesicles produced by this method with 95% phosphatidylcholine (from soybean extract) had good function.

• Korean Journal of Veterinary Research
• /
• v.44 no.4
• /
• pp.515-522
• /
• 2004

The vasorelaxant effect of serotonin reuptake inhibitor fluoxetine was investigated in rat isolated thoracic aorta. Fluoxetine induced a concentration-dependent relaxation in aorta precontracted with phenylephrine (PE) and KCl. These relaxations were suppressed by removal of the endothelium (-E) or pretreatment of nitric oxide synthase inhibitors, N(G)-nitro-L-arginine (L-NNA) and N(omega)-nitro-Larginine methyl ester (L-NAME), guanylate cyclase inhibitors, methylene blue (MB) and 1H-[1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one (ODQ), and $Ca^{2+}$ channel blockers, nifedipine and verapamil, in PE-precontracted +E rings. However, fluoxetine-induced relaxations were not suppressed by pretreatment of $K^{+}$ channel blockers, tetrabutylammonium and glibenclamide, in PE-precontracted endothelium intact (+E) rings. The fluoxetine-induced relaxations were not suppressed by removal of the endothelium or pretreatment of LNNA and MB in KCl-precontracted +E rings. Also, fluoxetine inhibited PE-induced sustained contraction in +E rings. These inhibitory effects of fluoxetine on contractions could be reversed by removal of the endothelium or pretreatment of L-NNA, L-NAME, MB, ODQ, nifedipine and verapamil, but not by pretreatment of etrabutylammonium and glibenclamide. These findings suggest that the vasorelaxant effect of fluoxetine is modulated by intracellular $Ca^{2+}$ with an involvement of endothelial NO-cGMP pathway and also may be related to the inhibition of $Ca^{2+}$ entry through voltage-gated channel.

• Journal of Ginseng Research
• /
• v.34 no.4
• /
• pp.314-320
• /
• 2010

The previous reports have showed that ginseng saponins, which are the active ingredients of Panax ginseng, cause the relaxation of artery that are contracted due to a various of hormones or potassium ($K^+$). Recently, we also showed that ginsenosides differentially regulate channel activity. The purpose of this study was to examine whether ginseng saponins affect contraction induced by $K^+$, serotonin (5-HT), or acetylcholine (Ach) in porcine coronary vessel. Treatment with concentrations of ginseng saponins caused a relaxation of 25 mM KCl-induced porcine coronary artery contraction. Also, ginseng saponin induced a significant dose-dependent relaxation of $3\;{\mu}M$ 5-HT-induced porcine coronary artery with the endothelium. In the porcine artery with the endothelium, ginseng saponins induced a relaxation by $3\;{\mu}M$ 5-HT in a concentration-dependent pattern. Ginseng saponins induced relaxation of both 25 mM KCl- and $3\;{\mu}M$ 5-HT-induced coronary artery contraction in the absence and presence of the endothelium. In contrast, treatment with $100\;{\mu}g/mL$ ginseng saponin did not induce relaxation in coronary artery contraction induced by Ach ($0.01\;{\mu}M$ to $30\;{\mu}M$) in the presence of the endothelium, but did cause significant relaxation of coronary artery contractions by Ach ($0.01\;{\mu}M$ to $30\;{\mu}M$) in the absence of the endothelium. These findings indicate that ginseng saponin (> $100\;{\mu}g/mL$) significantly inhibits porcine coronary artery contractions caused by $K^+$, 5-HT, and Ach. Therefore, in this study, we demonstrated that ginseng saponin may show beneficial roles on abnormal coronary contraction.