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

OBJECTIVE: To explore the characteristic changes of the peripheral chorda tympanic nerve (CT) electrophysiological responses to salty stimulus and other taste stimuli in rats with the conditioned taste aversion to saltiness. METHODS: Fourteen adult SD male rats were divided into a conditioned taste aversion to salty group (CTA) and a control group (Ctrl) (n=7/group). On the first day of the experiment, rats were given a 0.1 mol/L NaCl intake for 30 min, then, the rats in CTA and Ctrl groups were injected intraperitoneally with 2 ml of 0.15 mol/L LiCl and the same amount of saline respectively. On day 2, 3 and 4, the 30 min consumption of NaCl and distilled water was measured for both groups of rats. On the 4th day after the behavioral test of that day, CT electrophysiological recording experiments were performed on CTA rats and control rats. RESULTS: Compared with the rats in Ctrl group, the electrophysiological characteristics of CT in CTA group rats did not change significantly the responses to the series of NaCl and other four basic taste stimuli (P＞0.05). The amiloride, the epithelial sodium channel blocker, strongly inhibited the response of CT to NaCl in CTA and Ctrl group rats (P＜0.01). CONCLUSION The electrophysiological responses of CT to various gustatory stimuli do not significantly change in rats after the establishment of conditional taste aversion to the saltiness.
Amiloride ; pharmacology ; Animals ; Chorda Tympani Nerve ; physiology ; Conditioning, Classical ; Electrophysiological Phenomena ; Male ; Rats ; Rats, Sprague-Dawley ; Sodium Chloride ; Taste ; physiology

OBJECTIVETo observe the effect of amiloride on the proteinuria of the 5/6 nephrectomy rats.

METHODSTo establish the 5/6 nephrectomy rats model and divide the experiment into 3 groups, sham operated group(Sham), 5/6 nephrectomy model group(NTX) and 5/6 nephrectomy with amiloride-treated group (NTX+amiloride, n=15). The concentration of protein and mRNA of uPAR and the change of podocytes motility were detected by coomassiebluestaining, immunofluorence method and real-time PCR.

RESULTSAt second week, compared with Control group, the 24 h urine protein of NTX group was significantly increased (47.50 ± 28.05 mg vs 14.28 ± 3.8 mg, P = 0.023). There was no statistical significance in 24-hour urine protein between NTX+amiloride group and NTX group (51.56 ± 21.03 mg vs 47.50 ± 28.05 mg, P = 0.748). The same situation was also observed at the time point of 12 week, comparing with NTX group, 24-hour urine protein decreased in Sham group (188.31 ± 29.82 mg vs 21.32 ± 8.59 mg, P = 0.000) and NTX+amiloride group (188.31 ± 29.82 mg vs 121.37 ± 31.14 mg, P=0.000), with statistical significance when comparing with Sham group, the expression of uPAR mRNA in NTX group was significantly increased (9.74 ± 1.44 vs 1.01 ± 0.13, P = 0.000). In contrast, the expression of uPAR mRNA in NTX rats treated with amiloride was significantly lower than in NTX group (9.74 ± 1.44 vs 5.01 ± 1.36, P = 0.000).

CONCLUSIONAmiloride can reduce the proteinuria of the 5/6 nephrectomy rats model of transient proteinuria by inhibiting the induction of uPAR expression.

Amiloride ; pharmacology ; Animals ; Cell Movement ; Disease Models, Animal ; Nephrectomy ; Podocytes ; drug effects ; metabolism ; Proteinuria ; drug therapy ; Rats ; Real-Time Polymerase Chain Reaction ; Receptors, Urokinase Plasminogen Activator ; metabolism

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

We have shown that hyaluronic acid stimulates the proliferation of quiescent NIH 3T3 cells. We have shown that treatment of 1 mg/ml hyaluronic acid results in increase of tyrosine phosphorylation of two proteins, MW 124 kDa and 60 kDa as detected by anti-tyrosine antibodies by Western blot analysis. Maximum phosphorylation occurred within 2 h after addition of 1 mg/ml hyaluronic acid. Stimulation of proliferation was also accompanied by increase in c-Myc protein, which was inhibited by amlloride, an inhibitor of Na+/H+ antiporter and EGTA and increase in the steady state level of pRb, the RB gene product. These results suggest that the intracellular signal transduction pathways that mediate the stimulatory effects of hyaluronic acid on cellular proliferation are similar to those of growth factors.
3T3 Cells ; Amiloride/pharmacology ; Animal ; Cell Division ; Dose-Response Relationship, Drug ; Egtazic Acid/pharmacology ; Hyaluronic Acid/pharmacology* ; Mice ; Mitogens/pharmacology* ; Phosphoproteins/metabolism* ; Phosphorylation ; Proto-Oncogene Proteins c-myc/metabolism* ; Retinoblastoma Protein/metabolism* ; Signal Transduction ; Sodium-Hydrogen Antiporter/antagonists & inhibitors ; Tyrosine

Cell-penetrating peptide (CPP) can be used in pharmaceutics as a highly efficient drug delivery transporter. In this study, four tumor cell lines (MCF-7, MDA-MB-231, C6, and B16F10) were used to observe the uptake of fluorescein isothiocyanate (FITC) labeled CPP and the effects of time and concentration of CPP on cell penetration was studied. The CPP exocytosis on C6 cell line was observed, and its exocytosis kinetics was described by zero order equation. In addition, low-temperature condition (4 degrees C) and endocytosis inhibitors were utilized to investigate the mechanism of CPP uptake by cells. Low-temperature condition did not show significantly inhibition on CPP uptake. Heparin, a membrane glycoprotein receptor inhibitor, showed strong inhibition effect (only 3%-10% of the control) on CPP uptake. Chlorpromazine, chloroquine and 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) showed little effect on CPP uptake. This study indicated that CPP penetration had little selectivity on cell type, but the amount and rate of CPP penetration into cells were related to the type of cell lines. The adsorption of CPP on cell membrane induced by sulfate proteoglycan plays an important role on CPP penetration.
Adsorption ; Amiloride ; analogs & derivatives ; pharmacology ; Cell Line, Tumor ; Cell Membrane ; metabolism ; Cell-Penetrating Peptides ; administration & dosage ; metabolism ; pharmacokinetics ; Chloroquine ; pharmacology ; Chlorpromazine ; pharmacology ; Dose-Response Relationship, Drug ; Exocytosis ; Heparin ; analogs & derivatives ; metabolism ; pharmacology ; Humans ; Proteoglycans ; metabolism ; Temperature ; Time Factors

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

The short-circuit current (I(SC)) technique was used to examine the effects of cAMP-evoking agents, forskolin/IBMX, and a Chinese medicinal formula, Huoxiang-zhengqi liquid (HZL) on HCO(3)(-) secretion by intact porcine distal airway epithelium. The freshly isolated airway epithelial tissue displayed a transepithelial basal current of (94.9±8.2) μA/cm(2), 16.6% and 62.7% of which was inhibited by amiloride (epithelial Na(+) channel blocker, 100 μmol/L) and NPPB (cystic fibrosis transmembrane conductance regulator Cl(-) channel blocker, 100 μmol/L). Substitution of Cl(-) with impermeable gluconate(-) in the K-H bath solution resulted in a basal current of (54.0±6.7) μA/cm(2), which could be abolished by further removal of HCO(3)(-) in the solution, indicating HCO(3)(-) secretion under unstimulated conditions. Application of forskolin/IBMX (10 μmol/L/100 μmol/L) stimulated an increase of (13.8±1.9) μA/cm(2) in I(SC) which could be blocked by Cl(-) channel inhibitor DPC. With Cl(-) and Cl(-)/HCO(3)(-) substitution, forskolin/IBMX evoked an increase of (7.3±0.5) μA/cm(2) in HCO(3)(-)-dependent, DPC-inhibitable I(SC) (I(HCO(3))). Noticeably, basolateral application of HZL (10 μL/mL) in normal K-H solution evoked an I(SC) of (15.9±2.4) μA/cm(2). The EC(50) of this I(SC) was (6.1±1.4) μL/mL. When substituting Cl(-), HZL stimulated an increase of (7.4±1.9) μA/cm(2) in I(HCO(3)), suggesting HZL-induced HCO(3)(-) secretion. After pretreating the epithelial tissues with forskolin/IBMX in Cl(-)-free K-H solution, HZL induced a further increase of (8.4±0.9) μA/cm(2) in I(HCO(3)), and pretreating tissues with HZL did not significantly affect the subsequent forskolin/IBMX-induced I(HCO(3)) response, indicating that HZL- and forskolin/IBMX-induced I(HCO(3)) responses appeared to be independent and be most likely mediated via different cellular mechanisms. Our results suggest that HCO(3)(-) can be secreted by porcine distal airway epithelium under unstimulated and stimulated conditions, and the stimulatory effect of HZL on HCO(3)(-) secretion in the distal airway epithelium shows HZL to be a hopeful new agonist for distal airway HCO(3)(-) secretion that could be of therapeutic significance.
Amiloride ; pharmacology ; Animals ; Bicarbonates ; metabolism ; Biological Transport ; Colforsin ; pharmacology ; Cystic Fibrosis Transmembrane Conductance Regulator ; antagonists & inhibitors ; Drugs, Chinese Herbal ; pharmacology ; Epithelium ; drug effects ; metabolism ; Respiratory System ; drug effects ; metabolism ; Swine

The effects of 5-(N,N-dimethyl)amiloride (DMA) (a blocker of Na(+)/H(+) exchanger or Na(+)/Ca(2+) exchanger) on calcium transient and cell contraction in isolated ventricular myocytes in normal rats and rats with myocardial hypertrophy were examined using ion imaging system with a charge coupled digital camera (CCD camera). Loading myocytes with Fura-2, electrically triggered Ca(2+) transients and cell shortening were measured simultaneously. The results showed that 10 micromol/L DMA increased Ca(2+) transient and cell shortening from 209.60+/-54.96 and 3.07+/-0.97 micrometer to 238.50+/-80.41 and 4.07+/-1.02 micrometer, respectively (P<0.05), which was completely abolished by application of KB-R7943, a specific reverse mode Na(+)/Ca(2+) exchanger blocker. After blocking L-type Ca(2+) channels by nicardipine, DMA also enhanced Ca(2+) transient and cell shortening. In rats with myocardial hypertrophy, DMA showed the common pharmacologic profile as in normal rats but more intense stimulating effects on Ca(2+) transient and cell contraction. The results suggest that DMA increase Ca(2+) transient and cell contraction via stimulating reverse mode Na(+)/ Ca(2+) exchange, and the stimulating effect is more pronounced in rats with myocardial hypertrophy than in normal ones.
Amiloride ; analogs & derivatives ; pharmacology ; Animals ; Calcium ; metabolism ; Cardiomyopathy, Hypertrophic ; drug therapy ; Heart Ventricles ; cytology ; Myocardial Contraction ; drug effects ; Myocytes, Cardiac ; metabolism ; Rats ; Rats, Wistar ; Sodium-Calcium Exchanger ; antagonists & inhibitors ; pharmacology

PURPOSE: The effects of amiloride on cellular toxicity caused by tissue plasminogen activator (tPA) in mouse primary retinal cells were investigated. METHODS: Primary retinal cell cultures were maintained using glial conditioned medium. Commercial tPA and L-arginine were added, and the level of cyclic guanosine monophosphate (cyclic-GMP) in the culture supernatant was assessed using an ELISA assay. We measured the cell viability of cultured retinal cells pretreated with three different concentrations of amiloride (1, 10, and 100 microm) in addition to commercial tPA or L-arginine treatment. RESULTS: After exposing the cultured mouse retinal cells to tPA plus L-arginine or L-arginine alone, cyclic-GMP concentrations were 61.9 +/- 5.1 pmole/mL and 63.1 +/- 6.1 pmole/mL, respectively. However, the control group had a significantly lower concentration of cyclic-GMP (37.2 +/- 3.4 pmole/mL, p < 0.01). The cyclic GMP-dissolved solution did not cause retinal cell death. In the control group and the group treated with 1 microm amiloride and tPA containing L-arginine, the cell viability was 43.7% and 44.5%, respectively. However, cell viability increased to 70.6% with 10 microm amiloride and 78.4% with 100 microm amiloride (p = 0.015). CONCLUSIONS: L-arginine increases intracellular cyclic-GMP and may give rise to retinal cells through this mechanism. In addition, amiloride in concentrations greater than 10 microm protects against L-arginine-induced retinal cell death.
Amiloride/*pharmacology ; Analysis of Variance ; Animals ; Arginine/toxicity ; Cell Death/drug effects ; Cells, Cultured ; Cyclic GMP/pharmacology ; Enzyme-Linked Immunosorbent Assay ; Mice ; Retina/cytology/*drug effects ; Tissue Plasminogen Activator/*toxicity