• Journal of Periodontal and Implant Science
• /
• 제45권2호
• /
• pp.69-75
• /
• 2015

Purpose: Salivary fluid formation is primarily driven by Ca2+-activated, apical efflux of chloride into the lumen of the salivary acinus. The anoctamin1 protein is an anion channel with properties resembling the endogenous calcium-activated chloride channels. In order to better understand the role of anoctamin proteins in salivary exocrine secretion, the expression of the ten members of the anoctamin gene family in the mouse submandibular gland was studied. Methods: Total RNA extracted from mouse submandibular salivary glands was reverse transcribed using primer pairs to amplify the full-length coding regions of each anoctamin gene and was subcloned into plasmid vectors for DNA sequencing. Alternative splice variants were also screened by polymerase chain reaction using primer pairs that amplified six overlapping regions of the complementary DNA of each anoctamin gene, spanning multiple exons. Results: Multiple anoctamin transcripts were found in the mouse submandibular salivary gland, including full-length transcripts of anoctamin1, anoctamin3, anoctamin4, anoctamin5, anoctamin6, anoctamin9, and anoctamin10. Exon-skipping splicing in the N-terminal exons of the anoctamins1, anoctamin5, and anoctamin6 genes resulted in multiple alternative splice variants. No expression of anoctamin2, anoctamin7, or anoctamin8 was found. Conclusions: The predominant anoctamin transcript expressed in the mouse submandibular gland is anoctamin1ac. The chloride channel protein produced by anoctamin1ac is likely responsible for the $Ca^{2+}$-activated chloride efflux, which is the rate-limiting step in salivary exocrine secretion.

• The Korean Journal of Physiology and Pharmacology
• /
• 제10권1호
• /
• pp.19-24
• /
• 2006

The intermediate conductance $Ca^{2+}-activated$ $K^+$ channels (SK4, IKCa1) are present in lymphocytes, and their membrane expression is upregulated by various immunological stimuli. In this study, the activity of SK4 was compared between Bal-17 and WEHI-231 cell lines which represent mature and immature stages of murine B lymphocytes, respectively. The whole-cell patch clamp with high-$Ca^{2+}$ ($0.8{\mu}M$) KCl pipette solution revealed a voltage-independent $K^+$ current that was blocked by clotrimazole (1 mM), an SK4 blocker. The expression of mRNAs for SK4 was confirmed in both Bal-17 and WEHI-231 cells. The density of clotrimazole-sensitive SK4 current was significantly larger in Bal-17 than WEHI-231 cells ($-11.4{\pm}3.1$ Vs. $-5.7{\pm}1.15$ pA/pF). Also, the chronic stimulation of B cell receptors (BCR) by BCR-ligation (anti-IgM Ab, $3{\mu}g$/ml, 8∼12 h) significantly upregulated the amplitude of clotrimazolesensitive current from $-11.4{\pm}3.1$ to $-53.1{\pm}8.6$ pA/pF in Bal-17 cells. In WEHI-231 cells, the effect of BCR-ligation was significantly small ($-5.7{\pm}1.15$ to $-9.0{\pm}1.00$ pA/pF). The differential expression and regulation by BCR-ligation might reflect functional changes in the maturation of B lymphocytes.

• The Korean Journal of Physiology and Pharmacology
• /
• 제13권5호
• /
• pp.393-400
• /
• 2009

NO released by myenteric neurons controls the off contraction induced by electrical field stimulation (EFS) in distal esophageal smooth muscle, but in the presence of nitric oxide synthase (NOS) inhibitor, L-NAME, contraction by EFS occurs at the same time. The authors investigated the intracellular signaling pathways related with G protein and ionic channel EFS-induced contraction using cat esophageal muscles. EFS-induced contractions were significantly suppressed by tetrodotoxin ($1\;{\mu}M$) and atropine ($1\;{\mu}M$). Furthermore, nimodipine inhibited both on and off contractions by EFS in a concentration dependent meaner. The characteristics of 'on' and 'off contraction and the effects of G-proteins, phospholipase, and $K^+$ channel on EFS-induced contraction in smooth muscle were also investigated. Pertussis toxin (PTX, a $G_i$ inactivator) attenuated both EFS-induced contractions. Cholera toxin (CTX, $G_s$ inactivator) also decreased the amplitudes of EFS-induced off and on contractions. However, phospholipase inhibitors did not affect these contractions. Pinacidil (a $K^+$ channel opener) decreased these contractions, and tetraethylammonium (TEA, ${K^+}_{Ca}$ channel blocker) increased them. These results suggest that EFS-induced on and off contractions can be mediated by the activations Gi or Gs proteins, and that L-type $Ca^{2+}$ channel may be activated by G-protein ${\alpha}$ subunits. Furthermore, ${K^+}_{Ca^-}$ channel involve in the depolarization of esophageal smooth muscle. Further studies are required to characterize the physiological regulation of $Ca^{2+}$ channel and to investigate the effects of other $K^+$ channels on EFS-induced on and off contractions.

• The Journal of Korean Physical Therapy
• /
• 제13권1호
• /
• pp.237-243
• /
• 2001

Skeletal muscle cells are activated by ${\alpha}$-motorneurons which release acetylcholine at the neuromuscular junction. This results in a local depolarization of surface membrane which triggers an action potential. The action potential propagates along the surface membrane and also into the T-tubule system. In the triads T-tubules are in close connection with the terminal cisternae of the sarcoplasmic reticulum(SR). The action potential activaies T-tubule voltage sensors(DHP receptors). which activates SR Ca$^{2+}$ release channels(ryanodinc receptors). Ca$^{2+}$ have a key role in skeletal muscle in that an increase of free myoplasmic Ca$^{2+}$ concentration. The process of coupling chemical and electrical signals at the cell surface to the intracellular release of Ca$^{2+}$and ultimate contraction of muscle fibers is termed excitation-contraction coupling(ECC). Coupling of cel1 surface signals to intracellular Ca$^{2+}$ release proceeds by several mechanisms in skeletal muscle cells. This review focus on sarcopiasmic reticulum(SR) Ca$^{2+}$ releasing mechanisms from sarcoplasmic reticulum in the skeletal muscle. The mechanisms include DCCR, CICR, and HCR.

• The Korean Journal of Physiology and Pharmacology
• /
• 제11권5호
• /
• pp.207-213
• /
• 2007

This study was designed to characterize ureteral smooth muscle motility and also to study the effect of forskolin(FSK) and isoproterenol(ISO) on smooth muscle contractility in murine ureter. High $K^+$(50 mM) produced tonic contraction by $0.17{\pm}0.06mN$(n=19). Neuropeptide and neurotransmitters such as serotonin($5{\mu}M$), histamine($20{\mu}M$), and carbarchol(CCh, $10{\sim}50{\mu}M$) did not produce significant contraction. However, CCh($50{\mu}M$) produced slow phasic contraction in the presence of 25 mM $K^+$. Cyclopiazonic acid(CPA, $10{\mu}M$), SR $Ca^{2+}$-ATPase blocker, produced tonic contraction(0.07 mN). Meanwhile, inhibition of mitochondria by protonophore carbnylcyanide m-chlorophenylhydrazone(CCCP) also produced weak tonic contraction(0.01 mN). The possible involvement of $K^+$ channels was also pursued. Tetraethyl ammonium chloride(TEA, 10 mM), glibenclamide($10{\mu}M$) and quinidine($20{\mu}M$) which are known to block $Ca^{2+}$-activated $K^+$ channels($K_{Ca}$ channel), ATP-sensitive $K^+$ channels($K_{ATP}$) and nonselective $K^+$ channel, respectively, did not elicit any significant effect. However, $Ba^{2+}$($1{\sim}2mM$), blocker of inward rectifier $K^+$ channels($K_{IR}$ channel), produced phasic contraction in a reversible manner, which was blocked by $1{\mu}M$ nicardipine, a blocker of dehydropyridine-sensitive voltage-dependent L-type $Ca^{2+}$ channels($VDCC_L$) in smooth muscle membrane. This $Ba^{2+}$-induced phasic contraction was significantly enhanced by $10{\mu}M$ cyclopiazonic acid(CPA) in the frequency and amplitude. Finally, regulation of $Ba^{2+}$-induced contraction was studied by FSK and ISO which are known as adenylyl cyclase activator and $\beta$-adrenergic receptor agonist, respectively. These drugs significantly suppressed the frequency and amplitude of $Ba^{2+}$-induced contraction(p<0.05). These results suggest that $Ba^{2+}$ produces phasic contraction in murine ureteral smooth muscle which can be regulated by FSK and $\beta$-adrenergic stimulation.

• The Korean Journal of Physiology and Pharmacology
• /
• 제25권3호
• /
• pp.227-237
• /
• 2021

Carbon monoxide (CO) is a cardioprotectant and potential cardiovascular therapeutic agent. Human cardiac fibroblasts (HCFs) are important determinants of myocardial structure and function. Large-conductance Ca2+-activated K+ (BK) channel is a potential therapeutic target for cardiovascular disease. We investigated whether CO modulates BK channels and the signaling pathways in HCFs using whole-cell mode patch-clamp recordings. CO-releasing molecules (CORMs; CORM-2 and CORM-3) significantly increased the amplitudes of BK currents (IBK). The CO-induced stimulating effects on IBK were blocked by pre-treatment with specific nitric oxide synthase (NOS) blockers (L-NG-monomethyl arginine citrate and L-NG-nitroarginine methyl ester). 8-bromo-cyclic GMP increased IBK. KT5823 (inhibits PKG) or ODQ (inhibits soluble guanylate cyclase) blocked the CO-stimulating effect on IBK. Moreover, 8-bromo-cyclic AMP also increased IBK, and pre-treatment with KT5720 (inhibits PKA) or SQ22536 (inhibits adenylate cyclase) blocked the CO effect. Pre-treatment with N-ethylmaleimide (a thiol-alkylating reagent) also blocked the CO effect on IBK, and DL-dithiothreitol (a reducing agent) reversed the CO effect. These data suggest that CO activates IBK through NO via the NOS and through the PKG, PKA, and S-nitrosylation pathways.

• The Korean Journal of Physiology and Pharmacology
• /
• 제4권1호
• /
• pp.1-8
• /
• 2000

To investigate the contributions of intrinsic membrane properties to the spontaneous activity of medial vestibular nucleus (MVN) neurons, we assessed the effects of blocking large and small calcium-activated potassium channels by means of patch clamp recordings. Almost all the MVN neurons recorded in neonatal $(P13{\sim}P17)$ rat were shown to have either a single deep after-hyperpolarization (AHP; type A cells), or an early fast and a delayed slow AHP (type B cells). Among the recorded MVN cells, immature action potential shapes were found. Immature type A cell showed single uniform AHP and immature B cell showed a lack of the early fast AHP, and the delayed AHP was separated from the repolarization phase of the spike by a period of isopotentiality. Application of apamin and charybdotoxin (CTX), which selectively block the small and large calcium-activated potassium channels, respectively, resulted in significant changes in spontaneous firings. In both type A and type B cells, CTX (20 nM) resulted in a significant increase in spike frequency but did not induce bursting activity. By contrast, apamin (300 nM) selectively abolished the delayed slow AHP and induced bursting activity in type B cells. Apamin had no effect on the spike frequency of type A cells. These data suggest that there are differential roles of apamin and CTX sensitive potassium conductances in spontaneous firing patterns of MVN neurons, and these conductances are important in regulating the intrinsic rhythmicity and excitability.

• The Korean Journal of Physiology and Pharmacology
• /
• 제7권1호
• /
• pp.47-52
• /
• 2003

The prostate gland contains numerous neuroendocrine cells that are believed to influence the function of the prostate gland. Our recent study demonstrated the expression of both ${\alpha}1$- and ${\alpha}2$-ARs, signaling the release of stored $Ca^{2+}$ and the inhibition of N-type $Ca^{2+}$ channels, respectively, in rat prostate neuroendocrine cells (RPNECs). In this study, the effects of NA on the resting membrane potential (RMP) of RPNECs were investigated using a whole-cell patch clamp method. Fresh RPNECs were dissociated from the ventral lobe of rat prostate and identified from its characteristic shape; round or oval shape with dark cytoplasm. Under zero-current clamp conditions with KCl pipette solution, the resting membrane potential (RMP) of RPNECs was between -35 mV and -85 mV. In those RPNECs with relatively hyperpolarized RMP (＜-60 mV), the application of noradrenaline (NA, $1{\mu}M$) depolarized the membrane to around -40 mV. In contrast, the RPNECs with relatively depolarized RMP (＞-45 mV) showed a transient hyperpolarization and subsequent fluctuation at around -40 mV on application of NA. Under voltage clamp conditions (holding voltage, -40 mV) with CsCl pipette solution, NA evoked a slight inward current (＜-20 pA). NA induced a sharp increase of cytosolic $Ca^{2+}$ concentration ($[Ca^{2+}]_c$), measured by the fura-2 fluorescence, and the voltage clamp study showed the presence of charybdotoxin-sensitive $Ca^{2+}$-activated $K^+$ currents. In summary, adrenergic stimulation induced either depolarization or hyperpolarization of RPNECs, depending on the initial level of RMP. The inward current evoked by NA and the $Ca^{2+}$-activated $K^+$ current might partly explain the depolarization and hyperpolarization, respectively.

• 한국생물물리학회:학술대회논문집
• /
• 한국생물물리학회 1998년도 학술발표회
• /
• pp.33-33
• /
• 1998

Oxidation and reduction of amino acid residues in proteins affect their functional properties. Especially, redox modulation of ion channel activities has been reported in number of ion channel proteins. In this study, we investigated the effects of tertiary-butyl hydrogen peroxide (tBHP) on the large-conductance Ca$\^$2＋/ -activated K$\^$+/(Maxi-K) channel of rat brain using lipid bilayer reconstitution technique.(omitted)

• The Korean Journal of Physiology and Pharmacology
• /
• 제12권6호
• /
• pp.337-342
• /
• 2008

Human bone marrow mesenchymal stem cells (hBM-MSCs) represent a potentially valuable cell type for clinical therapeutic applications. The present study was designed to evaluate the effect of long-term culturing (up to $10^{th}$ passages) of hBM-MSCs from eight individual amyotrophic lateral sclerosis (ALS) patients, focusing on functional ion channels. All hBM-MSCs contain several MSCs markers with no significant differences, whereas the distribution of functional ion channels was shown to be different between cells. Four types of $K^+$ currents, including noise-like $Ca^{+2}$-activated $K^+$ current ($IK_{Ca}$), a transient outward $K^+$ current ($I_{to}$), a delayed rectifier $K^+$ current ($IK_{DR}$), and an inward-rectifier $K^+$ current ($K_{ir}$) were heterogeneously present in these cells, and a TTX-sensitive $Na^+$ current ($I_{Na,TTX}$) was also recorded. In the RT-PCR analysis, Kv1.1,, heag1, Kv4.2, Kir2.1, MaxiK, and hNE-Na were detected. In particular, ($I_{Na,TTX}$) showed a significant passage-dependent increase. This is the first report showing that functional ion channel profiling depend on the cellular passage of hBM-MSCs.