• The Korean Journal of Zoology
• /
• v.38 no.4
• /
• pp.457-464
• /
• 1995

Hela cells, a transformed human epithelial cell line, attach to various substrata but subsequent spreading is specific to collagen or gelatin. The spreading is initiated by the activation of phospholipase $A_2$ (PLA$_2$) which produces arachidonic acid (AA) as a consequence of cell surface collagen receptor clustering. This study examines the mechanism of PLA$_2$activation and which phospholipids are hydrolyzed by PIA$_2$ to release AA in response to Hela cell adhesion to a gelatin substratum. The levels of phosphatidyicholine decreases, among various phospholipids, during attachment and spreading of Hela cells. Lysophosphatidyicholine Is the only lysophospholipids formed during ileLa cell adhesion indicating that clustered collagen receptors activate PLA$_2$to hydrolyze posphatidylcholine to AA and lysophosphatidylcholine. Among various molecular entitles which are known to regulate PLA$_2$ activation, we have previously shown that PLA2 activation is not mediated by either changes in $Ca_2$+ levels, alkalinization of cytoplasmic p11, or activation of protein hinase C. It is also likely that PIA2 activation is not mediated by either pertussis or cholera toxinsensitive G proteins as those toxins do not affect both AA release and cell spreading.

• Progress in Medical Physics
• /
• v.11 no.1
• /
• pp.29-38
• /
• 2000

The signal transduction pathway for most neurotransmitter induced inhibition of $Ca^{2+}$ channels in sympathetic neurons involves a G-protein mediated, membrane-delimited mechanism without the participation of any known protein kinase. However, activation of protein kinase C (PKC) has been proposed as one of the intracellular mechanisms mediating some neurotransmitter induced $Ca^{2+}$ channel inhibition. In the present study, we investigated the effects of phorbol-12, 13-dibutyrate (PDBu) on $Ca^{2+}$ channel currents of acutely dispersed neurons from adult rat superior cervical ganglion (SCG) neurons using whole cell variant of the patch clamp technique. PDBu (500 nM), the activator of PKC, increased $Ca^{2+}$ channel currents and retarded the deactivation of tail currents. The effects of PDBu were voltage dependent and the maximal increase in the current amplitudes was observed between -10 to 10 mV (n=4). PDBu attenuated $Ca^{2+}$ current inhibition induced by norepinephrine (NE), which modulates $Ca^{2+}$ channels via a pertussis toxin (PTX)-sensitive pathway. Inhibition of PDBu by staurosporine (1 $\mu$M) blocked the effects of PDBu on current amplitudes and NE-induced G-protein mediated inhibition of $Ca^{2+}$ currents. Further experiment should be done to know if G-protein or $Ca^{2+}$ channel itself is the target of PKC phosphorvlation.phosphorvlation.

• Molecules and Cells
• /
• v.37 no.9
• /
• pp.656-663
• /
• 2014

Gintonin, a novel, ginseng-derived G protein-coupled lysophosphatidic acid (LPA) receptor ligand, elicits $[Ca^{2+}]_i$ transients in neuronal and non-neuronal cells via pertussis toxin-sensitive and pertussis toxin-insensitive G proteins. The slowly activating delayed rectifier $K^+$ ($I_{Ks}$) channel is a cardiac $K^+$ channel composed of KCNQ1 and KCNE1 subunits. The C terminus of the KCNQ1 channel protein has two calmodulin-binding sites that are involved in regulating $I_{Ks}$ channels. In this study, we investigated the molecular mechanisms of gintonin-mediated activation of human $I_{Ks}$ channel activity by expressing human $I_{Ks}$ channels in Xenopus oocytes. We found that gintonin enhances $I_{Ks}$ channel currents in concentration- and voltage-dependent manners. The $EC_{50}$ for the $I_{Ks}$ channel was $0.05{\pm}0.01{\mu}g/ml$. Gintonin-mediated activation 1 of the $I_{Ks}$ channels was blocked by an LPA1/3 receptor antagonist, an active phospholipase C inhibitor, an $IP_3$ receptor antagonist, and the calcium chelator BAPTA. Gintonin-mediated activation of both the $I_{Ks}$ channel was also blocked by the calmodulin (CaM) blocker calmidazolium. Mutations in the KCNQ1 $[Ca^{2+}]_i$/CaM-binding IQ motif sites (S373P, W392R, or R539W)blocked the action of gintonin on $I_{Ks}$ channel. However, gintonin had no effect on hERG $K^+$ channel activity. These results show that gintonin-mediated enhancement of $I_{Ks}$ channel currents is achieved through binding of the $[Ca^{2+}]_i$/CaM complex to the C terminus of KCNQ1 subunit.

• Journal of Ginseng Research
• /
• v.39 no.2
• /
• pp.169-177
• /
• 2015

Background: Ginsenoside Rd (GSRd), one of the most abundant ingredients of Panax ginseng, protects the heart via multiple mechanisms including the inhibition of $Ca^{2+}$ influx.We intended to explore the effects of GSRd on L-type $Ca^{2+}$ current ($I_{Ca,L}$) and define the mechanism of the suppression of $I_{Ca,L}$ by GSRd. Methods: Perforated-patch recording and whole-cell voltage clamp techniques were applied in isolated rat ventricular myocytes. Results: (1) GSRd reduced $I_{Ca,L}$ peak amplitude in a concentration-dependent manner [half-maximal inhibitory concentration $(IC_{50})=32.4{\pm}7.1{\mu}mol/L$] and up-shifted the current-voltage (I-V) curve. (2) GSRd ($30{\mu}mol/L$) significantly changed the steady-state activation curve of $I_{Ca,L}$ ($V_{0.5}:-19.12{\pm}0.68$ vs. $-6.26{\pm}0.38mV$; n = 5, p < 0.05) and slowed down the recovery of $I_{Ca,L}$ from inactivation [the time content (${\zeta}$) from 91 ms to 136 ms, n = 5, p < 0.01]. (3) A more significant inhibitive effect of GSRd ($100{\mu}mol/L$) was identified in perforated-patch recording when compared with whole-cell recording [$65.7{\pm}3.2%$ (n = 10) vs. $31.4{\pm}5.2%$ (n = 5), p < 0.01]. (4) Pertussis toxin ($G_i$ protein inhibitor) completely abolished the $I_{Ca,L}$ inhibition induced by GSRd. There was a significant difference in inhibition potency between the two cyclic adenosine monophosphate elevating agents (isoprenaline and forskolin) prestimulation [$55{\pm}7.8%$ (n = 5) vs. $17.2{\pm}3.5%$ (n = 5), p < 0.01]. (5) 1H-[1,2,4]Oxadiazolo[4,3-a]-quinoxalin-1-one (a guanylate cyclase inhibitor) and N-acetyl-$\small{L}$-cysteine (a nitric oxide scavenger) partly recovered the $I_{Ca,L}$ inhibition induced by GSRd. (6) Phorbol-12-myristate-13-acetate (a protein kinase C activator) and GF109203X (a protein kinase C inhibitor) did not contribute to the inhibition of GSRd. Conclusion: These findings suggest that GSRd could inhibit $I_{Ca,L}$ through pertussis toxin-sensitive G protein ($G_i$) and a nitric oxide-cyclic guanosine monophosphate-dependent mechanism.

• IMMUNE NETWORK
• /
• v.13 no.6
• /
• pp.295-300
• /
• 2013

Der f 2 is the group 2 major allergen of a house dust mite (Dermatophagoides farinae) and its function has been recently suggested. To determine the optimal condition of sensitization to recombinant Der f 2 (rDer f 2) in murine model of asthma, we compared the effectiveness with different adjuvants in BALB/c and C57BL/6 mice. Mice from both strains sensitized with rDer f 2 by intraperitoneal injection or subcutaneous injection on days 1 and 14. The dosage was $20{\mu}g$. Freund's adjuvants with pertussis toxin (FP) or alum alone were used as adjuvants. On days 28, 29, and 30, mice were challenged intranasally with 0.1% rDer f 2. We evaluated airway hyperresponsivenss, eosinophil proportion in lung lavage, airway inflammation, and serum allergen specific antibody responses. Naive mice were used as controls. Airway hyperresponsiveness was increased in C57BL/6 with FP, and BALB/c with alum (PC200: $13.5{\pm}6.3$, $13.2{\pm}6.7$ vs. >50 mg/ml, p<0.05). The eosinophil proportion was increased in all groups; C57BL/6 with FP, BALB/c with FP, C57BL/6 with alum, BALB/c with alum ($24.8{\pm}3.6$, $20.3{\pm}10.3$, $11.0{\pm}6.9$, $5.7{\pm}2.8$, vs. $0.0{\pm}0.0$%, p<0.05). The serum allergen specific IgE levels were increased in C57BL/6 with FP or alum (OD: $0.8{\pm}1.4$, $1.1{\pm}0.8$, vs. $0.0{\pm}0.0$). C57BL/6 mice were better responders to rDer f 2 and as for adjuvants, Freund's adjuvant with pertussis toxin was better.

• BMB Reports
• /
• v.44 no.3
• /
• pp.205-210
• /
• 2011

Insulin has antiapoptotic activity in various cell types. However, the signaling pathways underlying the antiapoptotic activity of insulin is not yet known. This study was conducted to determine if cAMP affects the antiapoptotic activity of insulin and the activity of PI3K and ERK in CHO cells expressing human insulin receptors (CHO-IR). Insulin-stimulated ERK activity was completely suppressed by cAMP-elevating agents like as pertussis toxin (Ptx) and cholera toxin (Ctx) after 4 h treatment. Insulin-stimulated PKB/Akt activity was not affected at all. Ptx treatment together with insulin increased the number of apoptotic cells and the degree of DNA fragmentation. Ctx or 8-br-cAMP treatment also increased the number of apoptotic cells and stimulated the cleavage of caspase-3 and the hydrolysis of PARP. Taken together, cAMP antagonizes the antiapoptotic activity of insulin and the main target molecule of cAMP in this process is likely ERK, not PI3K-dependent PKB/Akt.

• The Korean Journal of Physiology and Pharmacology
• /
• v.13 no.5
• /
• 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 Korean Journal of Physiology and Pharmacology
• /
• v.2 no.2
• /
• pp.185-192
• /
• 1998

A role of endogenous somatostatin in pancreatic exocrine secretion induced by intrapancreatic cholinergic activation was studied in the isolated rat pancreas perfused with modified Krebs-Henseleit solution. Intrapancreatic neurons were activated by electrical field stimulation (EFS: 15 V, 2 msec and 8 Hz). Pancreatic exocrine secretion, including volume flow and amylase output, and release of somatostatin from the pancreas were respectively determined. Somatostatin cells in the islet were stained with an immunoperoxidase method. EFS significantly increased pancreatic volume flow and amylase output, which were reduced by atropine by 59% and 78%, respectively. Intraarterial infusion of either pertussis toxin or a somatostatin antagonist resulted in a further increase in the EFS-evoked pancreatic secretion. EFS also further elevated exocrine secretion in the pancreas treated with cysteamine, which was completely restored by intraarterial infusion of somatostatin. EFS significantly increased not only the number of immunoreactive somatostatin cells in the islet but also the concentration of immunoreactive somatostatin in portal effluent. It is concluded from the above results that intrapancreatic cholinergic activation elevates pancreatic exocrine secretion as well as release of endogenous somatostatin. Endogenous somatostatin exerts an inhibitory influence on exocrine secretion induced by intrapancreatic cholinergic activation via the islet-acinar portal system in the isolated pancreas of the rat.

• Biomedical Science Letters
• /
• v.14 no.3
• /
• pp.199-201
• /
• 2008

Eosinophil is an improtant leukocyte in the development of various inflammatory diseases. Monocyte chemoattractant protein-1 (MCP-1) acts as a key regulator on monocyte movement, and activation of T cells and NK cells. However, the role of MCP-1 in eosinophils remains to be solved. In the present study, we examined the effect of MCP-1 on eosinophil migration, using human eosinophilic EoL-1 cells as an in vitro model of eosinophils. The surface expression of CCR2 in EoL-1 cells was little detected but MCP-1 strongly induced EoL-1 cell migration in a dose-dependent manner. Increased chemotactic activity due to MCP-1 was blocked by pertussis toxin, a $G_i/G_o$ protein inhibitor and U73122, a phospholipase C (PLC) inhibitor. These results suggest that MCP-1 activates $G_i/G_o$ protein and PLC and this signal pathway is involved in eosinphil movement. This finding supports the elucidation of pathogenic mechanism of eosinophilic inflammation such as asthma and atopic dermatitis.

• Bulletin of the Korean Chemical Society
• /
• v.28 no.6
• /
• pp.1005-1009
• /
• 2007

Leukotriene B4 (LTB4) is a potent chemoattractant for leukocytes and considered to be an inflammatory mediator. Human BLT2 (hBLT2) is a low-affinity G-protein coupled receptor for LTB4 and mediates pertussis toxin-sensitive chemotactic cell movement. Here, we dissected the interaction between hBLT2 and G-protein alpha subunits using GST fusion proteins containing intracellular regions of hBLT2 and various Gα protein including Gα i1, Gα i2, Gα i3, Gα s1, Gα o1, and Gα z. Among the tested Gα subunits, Gα i3 showed the highest binding to the third intracellular loop region of hBLT2 with a dissociation constant (KD) of 5.0 × 10?6 M. These results suggest that Gα i3 has the highest affinity to hBLT2, and the third intracellular loop region of hBLT2 is the major component for the interaction with Gα i3.