The aim of the present paper was to study the role of sodium calcium exchanger (NCX) in the generation of action potentials (APs) in cardiomyocytes during early developmental stage (EDS). The precisely dated embryonic hearts of C57 mice were dissected and enzymatically dissociated to single cells. The changes of APs were recorded by whole-cell patch-clamp technique before and after administration of NCX specific blockers KB-R7943 (5 μmol/L) and SEA0400 (1 μmol/L). The results showed that, both KB-R7943 and SEA0400 had potent negative chronotropic effects on APs of pacemaker-like cells, while such effects were only observed in some ventricular-like cardiomyocytes. The negative chronotropic effect of KB-R7943 on ventricular-like cardiomyocytes was accompanied by shortening of AP duration (APD), whereas such an effect of SEA0400 was paralleled by decrease in velocity of diastolic depolarization (Vdd). From embryonic day 9.5 (E9.5) to E10.5, the negative chronotropic effects of KB-R7943 and SEA0400 on ventricular-like APs of embryonic cardiomyocytes gradually disappeared. These results suggest that, in the short-term development of early embryo, the function of NCX may experience developmental changes as evidenced by different roles of NCX in autorhythmicity and APs generation, indicating that NCX function varies with different conditions of cardiomyocytes.
Action Potentials ; Animals ; Calcium/metabolism* ; Mice ; Myocytes, Cardiac/metabolism* ; Sodium/metabolism* ; Sodium-Calcium Exchanger ; Thiourea/pharmacology*

Supplement effects of ions, sugars, and amino acids on the thermostability of liquefying type alpha-amylase from Bacillus subtilis were examined. The addition of 1 mmol/L Ca2+ or about 50 mmol/L Na+ remarkably stimulated the thermostability of this enzyme among ions examined. The thermostability of the enzyme was enhanced and reduced by the extrinsic addition of 50 mmol/L acidic amino acid such as glutamic acid and alkaline amino acid of the concentrations of sugars from 0 to 1000 mmol/L the thermostability of alpha-amylase increased almost such as arginine, respectively. With the increases linearly. By the co-existence of Na+ or K+ with some amino acids or sugars the thermostability of this enzyme was fairly increased. The changes in the fluorescence intensity of alpha-amylase were examined as a function of the incubation temperature on the enzyme, which showed a good agreement with those of residual activities.
Amino Acids ; pharmacology ; Calcium ; pharmacology ; Carbohydrates ; pharmacology ; Enzyme Stability ; Protein Conformation ; Sodium ; pharmacology ; Temperature ; alpha-Amylases ; chemistry ; metabolism

In order to investigate the effects of Na(2)SeO(3) on expression of VEGF in K562/ADR cells, K562 and K562/ADR cells were treated with Na(2)SeO(3) at dose of 5 and 10 micromol/L. The expressions of VEGF in K562 and K562/ADR cells were detected by ELISA before and at the different time point after treatment. The mutiplie of reversion of resistance was detected by MTT method. The results showed that Na(2)SeO(3) at dose of 10 micromol/L could increase the sensitivity of K562/ADR cell to adriamycin, the multiple of reversion was 3.48. The expression levels of VEGF in K562 and K562/ADR cells increased with prolongation of time cultured, and the VEGF expression levels in K562/ADR cells at the different time points were higher than that in K562 cells (P < 0.05); 5 and 10 micromol/L Na(2)SeO(3) did not suppress expression of VEGF in K562 cells at 72 hours (P > 0.05), and the VEGF level in K562 cells at 96 hours decreased without statistical significance; 5 and 10 micromol/L Na(2)SeO(3) acting for 48 hours did not show suppressive effect on expression of VEGF in K562/ADR cells (P > 0.05), 5 micromol/L Na(2)SeO(3) could decrease the expression of VEGF in K562/ADR cell after treatment for 96 hours, while 10 micromol/L Na(2)SeO(3) could significantly decrease the expression of VEGF in K562/ADR cells treated for 72 hours and 96 hours (P < 0.01). It is concluded that VEGF would be involved in the multidrug resistance of leukemia. Na(2)SeO(3) decreasing expression of VEGF in leukemic cells may be one of the mechanisms reversing multidrug resistance.
Doxorubicin ; pharmacology ; Drug Resistance, Neoplasm ; Humans ; K562 Cells ; Sodium Selenite ; pharmacology ; Vascular Endothelial Growth Factor A ; metabolism

OBJECTIVETo observe the sodium current of acutely isolated alveolar type II cells and its regulation.

METHODSAfter isolation the AT II cells, the whole cell sodium current were recorded in 5 hours with patch-clamp in whole-cell mode, and the effect of amiloride and terbutaline on the current was investigated.

RESULTSThe amiloride-sensitive current of AT II cells were recorded, which could be obviously stimulated by terbutaline.

CONCLUSIONAcutely isolated AT II cells can be valuable for studying electrophysiological characteristics of sodium channels even under pathological conditions.

Amiloride ; pharmacology ; Animals ; Biological Transport ; Cell Separation ; Patch-Clamp Techniques ; Pulmonary Alveoli ; cytology ; metabolism ; Rats ; Rats, Sprague-Dawley ; Sodium ; metabolism ; Sodium Channel Blockers ; pharmacology ; Sodium Channels ; metabolism ; physiology ; Terbutaline ; pharmacology

Cardiac dysfunctions such as myocardial functional failure and ventricular arrhythmia have been largely attributed to intracellular Ca2+ overload. One of the mechanisms of intracellular Ca2+ overload involves a rapid influx of Ca2+ via Na(+)-Ca2+ exchange during the reperfusion which utilizes the accumulation of Na+ in myocytes during ischemic cardiac arrest. Possible sources of the intracellular Na+ accumulation include Na+ channel, Na(+)-H+ exchange, Na(+)-Ca2+ exchange, and Na+ background current. In this study, we studied the role of the Na+ background current in intracellular Na+ accumulation during the cardiac arrest by measuring the Na+ background current in guinea pig ventricular myocytes with whole cell clamp method and evaluating the effects of cardioprotective drugs on the Na+ background current. The results were as follows: (1) The Na+ background inward current at -40 mV membrane potential was larger at Ca2+ free solution than 1.8 mM Ca2+ solution. (2) The Na+ background current was not affected by verapamil. (3) 2 microM O-(N, N-hexamethylene)-amiloride (HMA) decreased the Na+ background current at negative membrane potential. (4) The new cardioprotective drug, R 56865, decreased the Na+ background current. These results suggest that the Na+ background current plays a role in increasing the intracellular Na+ activity during high K+ cardioplegia and the blocking effect of myoprotective drugs, such as R 56865, on the Na+ background current may contribute to myocardial protection after cardioplegia.
Amiloride/pharmacology ; Animal ; Guinea Pigs ; Heart/*drug effects ; Heart Arrest, Induced ; Myocardium/metabolism ; Piperidines/pharmacology ; Potassium/pharmacology ; Sodium/*metabolism ; Support, Non-U.S. Gov't ; Thiazoles/pharmacology ; Verapamil/pharmacology

OBJECTIVETo investigate the protective effects of Cariporide on immature rabbit heart, and to search for the protective mechanism of the Na(+)/H(+) exchange inhibitor on immature rabbit hearts.

METHODSNew Zealand immature rabbits were randomly divided into two groups (n = 12 in each group). The isolated rabbit heart model was involved in this study. The hearts were submitted to 60 minutes of normothermic ischemia with cardioplegia per 20 minutes of reperfusion. Group I received St. Thomas No2 as cardioprotective solution. Group II received St. Thomas No2 with addition of cariporide (10 micro mol/L). The left ventricular function was recorded, including left ventricular systolic pressure (LVSP), left ventricular dystolic pressure (LVDP), coronary artery flow (CAF), mean aortic pressure (MAP), aortic flow (AF) and dp/dt max. The levels of creatine phosphokinase (CK), lactic dehydrogenase (LDH) of coronary sinus venous solution were measured. The ventricular cardiomyocytes isolated from other 6 immature rabbit hearts were subdivided into 3 groups of each heart, which were attained by means of collagenase-perfusion. All cells were incubated with calcium fluoresence indicator Fluo-3/AM, and then the intracellular free calcium was measured under the laser scanning con-focal microscopy. The baseline was measured after isolation without anoxic/re-oxygenation. The control group received anoxic conditions for 60 minutes and re-oxygenation for 30 minutes, then was measured. The experiment group received the same conditions as control group with addition of Cariporide (1 micromol/L).

RESULTSAfter ischaemia/reperfusion, the percentage of recovery of myocardial function in group II was much better than group I; the LVDP, LVSP, MAP, AF, CAF and dp/dt max showed markedly better recovery in group II. The release of CK, LDH was significantly increased in Group I. After anoxic/re-oxygenation, the intracellular free calcium of isolated immature rabbit ventricular myocytes in control group increased significantly than baseline (P < 0.01); there were no significant difference of immature myocardial [Ca(2+)]i between experiment group and baseline (P > 0.05); and the experiment group myocardial [Ca(2+)]i reduced significantly than control (P < 0.01).

CONCLUSIONSCariporide demonstrates significant cardio-protective effects for immature myocardium ischemia/reperfusion, and the protective mechanism may be due to the inhibition of the intracellular free calcium overload.

Animals ; Arrhythmias, Cardiac ; etiology ; Calcium ; metabolism ; Female ; Guanidines ; pharmacology ; Male ; Myocardial Reperfusion Injury ; prevention & control ; Rabbits ; Sodium ; metabolism ; Sodium-Hydrogen Exchangers ; antagonists & inhibitors ; Sulfones ; pharmacology

Different neutralizing agents were used as pH controller to investigate their effects on the growth and succinic acid production of Actinobacillus succinogenes NJ113. The fermentation results showed that Ca(OH)2, CaCO3 and NH4OH were not suitable for succinic acid production by A. succinogenes NJ113 because of their negative effects on cell growth. When Na-base was used, cells would flocculate and lump, and due to the sodium ion concentration reaching to a high level, OD660 dropped sharply after 12 h of fermentation. Mg-base was better because there was no significant inhibition by magnesium ion. Two combined neutralizing agents were used to maintain pH level, one with NaOH and Mg(OH)2 while the other with Na2CO3 and Mg(OH)2. The optimum ratios of the combined neutralizing agents were both 1:1 (g:g) when using 100 g/L glucose. When NaOH and Mg(OH)2 were chosen with the ratio of 1:1(g:g), 69.8 g/L of the succinic acid and 74.5% of the yield was obtained.
Actinobacillus ; genetics ; metabolism ; Fermentation ; Hydrogen-Ion Concentration ; Industrial Microbiology ; Magnesium Hydroxide ; pharmacology ; Sodium Hydroxide ; pharmacology ; Succinic Acid ; metabolism

Kv2.1 channel currents in pancreatic beta-cells are thought to contribute to action potential repolarization and thereby modulate insulin secretion. Because of its central role in this important physiological process, Kv2.1 channel is a promising target for the treatment of type 2 diabetes. Jingzhaotoxin-XI (JZTX-XI) is a novel peptide neurotoxin isolated from the venom of the spider Chilobrachys jingzhao. Two-microelectrode voltage clamp experiments had showed that the toxin inhibited Kv2.1 potassium currents expressed in Xenopus Laevis oocytes. In order to investigate the structure-function relationship of JZTX-XI, the natural toxin and a mutant of JZTX-XI in which Arg3 was replaced by Ala, were synthesized by solid-phase chemistry method with Fmoc-protected amino acids on the PS3 automated peptide synthesizer. Reverse-phase high performance liquid chromatography (RP-HPLC) and matrix assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) were used to monitor the oxidative refolding process of synthetic linear peptides to find the optimal renaturation conditions of these toxins. The experiments also proved that the relative molecular masses of refolded peptides were in accordance with their theoretical molecular masses. RP-HPLC chromatogram of co-injected native and refolded JZTX-XI was a single peak. Under the whole-cell patch-clamp mode, JZTX-XI could completely inhibit hKv2.1 and hNav1.5 channels currents expressed in HEK293T cells with IC50 values of 95.8 nmol/L and 437.1 nmol/L respectively. The mutant R3A-JZTX-XI could also inhibit hKv2.1 and hNav1.5 channel currents expressed in HEK293T cells with IC50 values of 1.22 micromol/L and 1.96 micromol/L respectively. However, the prohibitive levels of R3A-JZTX-XI on hKv2.1 and hNav1.5 channels were reduced by about 12.7 times and 4.5 times respectively, indicating that Arg3 was a key amino acid residue relative to the hKv2.1 channel activity of JZTX-XI, but it is also an amino acid residue correlated with the binding activity of JZTX-XI to hNav1.5 channel. Our findings should be helpful to develop JZTX-XI into a molecular probe and drug candidate targeting to Kv2.1 potassium channel in the pancreas.
Animals ; HEK293 Cells ; Humans ; Insulin-Secreting Cells ; metabolism ; Mutant Proteins ; genetics ; pharmacology ; NAV1.5 Voltage-Gated Sodium Channel ; metabolism ; Neurotoxins ; chemical synthesis ; genetics ; pharmacology ; Protein Refolding ; Shab Potassium Channels ; antagonists & inhibitors ; metabolism ; Sodium Channel Blockers ; pharmacology ; Spider Venoms ; genetics ; pharmacology ; Transfection

OBJECTIVETo explore the effect of terbutaline on sodium transport in rat alveolar type I (ATI) and type II (ATII) cells of rats.

METHODSThe whole cell currents were recorded from ATII cells isolated from rat lungs perfused with or without amiloride (inhibitor of epithelial sodium channel) and ZnCl(2) (inhibitor of cyclic nucleotide-gated cation channel) in the whole cell recording mode using the patch-clamp technique. The effect of terbutaline on the currents was examined.

RESULTSThe main currents recorded from ATII cells were amiloride-sensitive and Zn(2+)-sensitive. The amiloride-sensitive and Zn(2+)-sensitive current shared a similar proportion (P>0.05). Both currents could be significantly increased by terbutaline (P<0.05), and the proportion of amiloride-sensitive current was 1.7 times that of Zn(2+)-sensitive current (P<0.05).

CONCLUSIONThere are functional epithelial sodium channels (ENaC) and cyclic nucleotide-gated cation channels (CNG) on freshly isolated ATII cells, both serving as the main channels for sodium transport. Terbutaline increases the absorption of alveolar fluid primarily by increasing sodium transport of ENaC and CNG on ATI and AT II cells.

Amiloride ; pharmacology ; Animals ; Chlorides ; pharmacology ; Cyclic Nucleotide-Gated Cation Channels ; antagonists & inhibitors ; drug effects ; Male ; Peptides ; pharmacology ; Pulmonary Alveoli ; cytology ; metabolism ; Rats ; Rats, Sprague-Dawley ; Sodium ; metabolism ; Sodium Channels ; drug effects ; Terbutaline ; pharmacology ; Zinc Compounds ; pharmacology

OBJECTIVETo explore the effects of sodium selenite on the expressions of beta-catenin and cyclin D1 in colorectal cancer cells HCT 116 and SW480.

METHODSHCT 116 and SW480 cells were treated by 10 micromol/L sodium selenite at different time points. The expressions and transcription of beta-catenin and cyclin D1 were detected by Western blot analysis and reverse transcriptase polymerase chain reaction (RT-PCR), respectively. Meanwhile, the impact of MG132 (a proteasome inhibitor) pretreatment on the expressions of beta-catenin and cyclin D1 was observed through Western blot analysis. The interaction between beta-catenin and T cell factor 4 (TCF4) after selenite treatment was evaluated using co-immunoprecipitation assay.

RESULTSSodium selenite inhibited the expression of beta-catenin and transcription of its target such as cyclin D1. MG132 pretreatment prevented the inhibition of beta-catenin signaling triggered by selenite in HCT 116 and SW480 cells. Furthermore, selenite treatment disrupted the interaction between beta-catenin and TCF4 in HCT 116 and SW480 cells.

CONCLUSIONSSodium selenite can lower the expression levels of beta-catenin and its target cyclin D1, during which the proteasome-mediated degradative pathway may be involved. The decreased interaction between beta-catenin and TCF4 due to sodium selenite may be also involved in the regulation of beta-catenin signaling.

Cell Line, Tumor ; Colorectal Neoplasms ; metabolism ; Cyclin D1 ; metabolism ; HCT116 Cells ; Humans ; Sodium Selenite ; pharmacology ; beta Catenin ; metabolism