AIMTo investigate the mechanisms of anti-cancer effect of resveratrol (Res), and the effects of Res in cell apoptosis. The role of Res playing in mitochondrial permeability transition pore (PTP) induction was studied.

METHODSMitochondria was prepared from the liver of Wistar rats. The effects of Res on oxygen consumption of isolated mitochondria from rat liver was measured with Clark-type electrode and resulted in respiration control rate (RCR). Mitochondrial swelling affected by Res was assessed spectrophotometrically, through the changes in absorbance at 540 nm. The PTP opening was learned from the results. Membrane potential of mitochondia was measured through fluorescence spectrophotometry.

RESULTSRes was shown to inhibit the respiration and decrease the RCR of mitochondria. Res can promote the PTP opening mediated by Ca2+. Res was shown to promote the increase of mitochondial membrane potential mediated by Ca2+ and loss of mitochondial membrane potential.

CONCLUSIONRes was shown to inhibit mitochondial respiration and induce PTP opening of mitochondria. These may be one of the pathways that Res showed anti-cancer action and induce cells apoptosis.

Animals ; Antineoplastic Agents, Phytogenic ; pharmacology ; Apoptosis ; drug effects ; Calcium ; metabolism ; Female ; Ion Channels ; drug effects ; metabolism ; Membrane Potentials ; drug effects ; Mitochondria, Liver ; drug effects ; physiology ; Mitochondrial Membrane Transport Proteins ; Mitochondrial Swelling ; drug effects ; Rats ; Rats, Wistar ; Stilbenes ; pharmacology

AIMTo investigate the action of anions and anion channel blockers in the regulation of vascular contraction induced by norepinephrine (NE).

METHODSNE-induced contraction was observed in rat aorta by using routine blood vascular perfusion in vitro.

RESULTSThe anion channel blockers niflumic acid (NFA) and 5-nitro-2-(3-phenoxylpropylamino)-benzoic acid (NPPB) produced inhibitory effects on NE-evoked contractions in the aorta. NE-induced contraction was not significantly changed after the extracellular Na+ was replaced by choline, in contrast, the vascular was relaxed when the extracellular Cl- was replaced by glutamate. Moreover, the vasoconstriction induced by NE was further enhanced with the replacement of the extracellular Cl- by Br-, which was still sensitive to either NFA or NPPB.

CONCLUSIONSAnion channels play an important role in the regulation of blood vascular tone, which may be responsible for the salt-sensitivity hypertension.

Animals ; Anions ; metabolism ; Aorta ; drug effects ; physiology ; In Vitro Techniques ; Ion Channels ; antagonists & inhibitors ; Male ; Muscle Contraction ; drug effects ; physiology ; Muscle, Smooth, Vascular ; drug effects ; physiology ; Norepinephrine ; pharmacology ; Rats ; Rats, Sprague-Dawley

The present study investigated the cellular mechanism underlying the effect of ligustrazine on the ion transport in rat distal colon using the short-circuit current (I(SC)) technique. In freshly isolated colonic strips, basolateral addition of ligustrazine stimulated a rise in I(SC), which was resistant to basolateral application of neuronal sodium channel blocker tetrodotoxin (TTX), but inhibited by 55.2% by basolateral pretreatment with prostaglandin inhibitor indomethacin. The ligustrazine-induced I(SC) increase was inhibited by apical application of Cl(-) channel blockers diphenylamine-2,2'-dicarboxylic acid (DPC) and glibenclamide. Basolaterally administered bumetanide, an inhibitor of Na(+)-K(+)-2 Cl(-) cotransporter, inhibited ligustrazine-evoked current increases by 85.2% and basolateral exposure to Ba(2+), a non-specific potassium channels blocker, and blocked the current by more than 90%, indicating that basolateral Na(+)-K(+)-2 Cl(-) cotransporter and K(+) channels played an important role in the effect of ligustrazine. The results suggested that ligustrazine could stimulate rat distal colon (-) secretion that is mediated by basolateral Na(+)-K(+)-2 Cl(-) cotransporter and K(+) channel.
Animals ; Animals, Newborn ; Calcium Channel Blockers ; pharmacology ; Colon ; metabolism ; physiology ; Epithelial Cells ; metabolism ; Evoked Potentials ; drug effects ; In Vitro Techniques ; Intestinal Mucosa ; cytology ; metabolism ; Ion Transport ; drug effects ; Male ; Potassium Channels ; metabolism ; Pyrazines ; pharmacology ; Rats ; Rats, Sprague-Dawley

For studying the effect of integrin on the [Ca(2+)](i) of mouse eggs and its transmembrane signaling mechanism, zona-free mouse eggs were loaded with calcium probe Fluo-3/AM and the intensity of fluorescence of the eggs treated with different factors was measured through laser confocal microscopy. The results showed that the [Ca(2+)](i) of zona-free mouse eggs was increased when the eggs were treated with RGD peptide, fibronectin (Fn) and anti-mouse integrin subunit alpha(6) and beta(1) monoclonal antibodies, respectively. The [Ca(2+)](i) of the mouse eggs was also increased when the eggs were placed in calcium-free medium and treated with RGD peptide or Fn. The changes in the mouse egg [Ca(2+)](i) caused by RGD and Fn were similar to those caused by sperm. However, the concentration of Ca(2+) of the zona-free mouse eggs pretreated with tyrosine kinase inhibitor was not increased when the eggs were treated in the same way, and, neither was the intracellular calcium increased in those eggs pretreated with PKC inhibitor when the eggs were treated with RGD peptide. It is therefore suggested that the occupancy of integrins on the membrane of mouse eggs by their ligands mediates the release of Ca(2+) and then the increase in the [Ca(2+)](i) of eggs, which is one of the early events of egg activation. The tyrosine kinase signaling pathway and PKC are involved in this process as well.
Animals ; Calcium ; metabolism ; Calcium Channels ; metabolism ; Female ; Integrins ; physiology ; Ion Transport ; drug effects ; Mice ; Oligopeptides ; pharmacology ; Oocytes ; metabolism ; Ovum ; metabolism ; Protein Kinase C ; metabolism ; Protein-Tyrosine Kinases ; metabolism ; Signal Transduction

Swelling-activated chloride currents (ICl.swell) are thought to play a role in several physiologic and pathophysiologic processes and thus represent a target for therapeutic approaches. However, the mechanism of ICl.swell regulation remains unclear. In this study, we used the whole-cell patch-clamp technique to examine the role of protein kinase C (PKC) in the regulation of ICl.swell in human atrial myocytes. Atrial myocytes were isolated from the right atrial appendages of patients undergoing coronary artery bypass and enzymatically dissociated. ICl.swell was evoked in hypotonic solution and recorded using the whole-cell patch-clamp technique. The PKC agonist phorbol dibutyrate (PDBu) enhanced ICl.swell in a concentration-dependent manner, which was reversed in isotonic solution and by a chloride current inhibitor, 9-anthracenecarboxylicacid. Furthermore, the PKC inhibitor bis-indolylmaleimide attenuated the effect and 4α-PDBu, an inactive PDBu analog, had no effect on ICl.swell. These results, obtained using the whole-cell patch-clamp technique, demonstrate the ability of PKC to activate ICl,swell in human atrial myocytes. This observation was consistent with a previous study using a single-channel patch-clamp technique, but differed from some findings in other species.
Anthracenes ; pharmacology ; Chloride Channels ; metabolism ; Chlorides ; agonists ; antagonists & inhibitors ; metabolism ; Culture Media ; metabolism ; pharmacology ; Dose-Response Relationship, Drug ; Evoked Potentials ; drug effects ; physiology ; Heart Atria ; cytology ; drug effects ; metabolism ; Humans ; Hypotonic Solutions ; metabolism ; pharmacology ; Indoles ; pharmacology ; Ion Transport ; drug effects ; Maleimides ; pharmacology ; Myocytes, Cardiac ; cytology ; drug effects ; metabolism ; Patch-Clamp Techniques ; Phorbol 12,13-Dibutyrate ; pharmacology ; Primary Cell Culture ; Protein Kinase C ; metabolism

OBJECTIVETo re-confirm and characterize the biophysical and pharmacological properties of endogenously expressed human acid-sensing ion channel 1a (hASIC1a) current in HEK293 cells with a modified perfusion methods.

METHODSWith cell floating method, which is separating the cultured cell from coverslip and putting the cell in front of perfusion tubing, whole cell patch clamp technique was used to record hASIC1a currents evoked by low pH external solution.

RESULTSUsing cell floating method, the amplitude of hASIC1a currents activated by pH 5.0 in HEK293 cells is twice as large as that by the conventional method where the cells remain attached to coverslip. The time to reach peak at two different recording conditions is (21+/-5) ms and (270+/-25) ms, respectively. Inactivation time constants are (496+/-23) ms and (2284+/-120) ms, respectively. The cell floating method significantly increases the amiloride potency of block on hASIC1a [IC50 is (3.4+/-1.1) micromol/L and (2.4+/- 0.9) micromol/L, respectively]. Both recording methods have similar pH activation EC50 (6.6+/-0.6, 6.6+/-0.7, respectively).

CONCLUSIONASICs channel activation requires fast exchange of extracellular solution with the different pH values. With cell floating method, the presence of hASIC1a current was re-confirmed and the biophysical and pharmacological properties of hASIC1a channel in HEK293 cells were precisely characterized. This method could be used to study all ASICs and other ligand-gated channels that require fast extracellular solution exchange.

Acid Sensing Ion Channels ; Amiloride ; pharmacology ; Biophysics ; instrumentation ; methods ; Cell Culture Techniques ; instrumentation ; methods ; Cell Line ; Cell Membrane ; chemistry ; drug effects ; metabolism ; Culture Media ; chemistry ; pharmacology ; Extracellular Fluid ; chemistry ; metabolism ; Humans ; Hydrogen-Ion Concentration ; drug effects ; Membrane Potentials ; drug effects ; physiology ; Nerve Tissue Proteins ; chemistry ; drug effects ; metabolism ; Neuropharmacology ; instrumentation ; methods ; Patch-Clamp Techniques ; instrumentation ; methods ; Perfusion ; instrumentation ; methods ; Sodium Channel Blockers ; pharmacology ; Sodium Channels ; chemistry ; drug effects ; metabolism ; Time Factors

Muscarinic receptors play key roles in the control of gastrointestinal smooth muscle activity. However, specific physiological functions of each subtype remain to be determined. In this study, the nonselective cation channel activated by carbachol (ICCh) was examined in circular smooth muscle cells of the guinea pig gastric antrum using patch-clamp technique. 4-DAMP inhibited ICCh dose- dependently with IC50 of 1.1 +/- 0.1 nM (n = 6). GTPgS- induced current, however, was not inhibited by 10 nM 4-DAMP. ICCh was not recorded in pertussis- toxin (PTX)-pretreated smooth muscle cells of gastric antrum. ICCh values in response to 10 mM CCh at a holding potential of 60 mV were -330 32 pA (n=4) and -15 +/- 3 pA (n = 6) in the control and PTX-treated cells, respectively (P<0.01). Sensitivities to nanomolar 4-DAMP and PTX suggest the possible involvement of m4 subtype. Using sequence information obtained from cloned guinea pig muscarinic receptor genes, it is possible to amplify the cDNAs encoding m1-m5 from guinea pig brain tissue. Single cell RT-PCR experiments showed that all five subtypes of muscarinic receptor were present in circular smooth muscle cells of the guinea pig gastric antrum. Together with our previous results showing that Go protein is important for activation of ACh-activated NSC channels, our results suggest that ICCh might be activated by acetylcholine through m4 subtype as well as m2 and m3 subtypes in guinea-pig stomach.
Animals ; Base Sequence ; Carbachol/pharmacology ; Cations ; Cholinergic Agonists/pharmacology ; Dose-Response Relationship, Drug ; Drug Interactions ; Guinea Pigs ; Ion Channels/drug effects/metabolism/physiology ; Muscarinic Antagonists/pharmacology ; Muscle Contraction/drug effects ; Muscle, Smooth/drug effects/*metabolism ; Piperidines/pharmacology ; Receptors, Muscarinic/chemistry/classification/*metabolism ; Stomach/drug effects/*metabolism

Diverse subtypes of voltage-gated sodium channels (VGSCs) have been found throughout tissues of the brain, muscles and the heart. Neurotoxins extracted from the venom of the Asian scorpion Buthus martensi Karsch (BmK) act as sodium channel-specific modulators and have therefore been widely used to study VGSCs. α-type neurotoxins, named BmK I, BmK αIV and BmK abT, bind to receptor site-3 on VGSCs and can strongly prolong the inactivation phase of VGSCs. In contrast, β-type neurotoxins, named BmK AS, BmK AS-1, BmK IT and BmK IT2, occupy receptor site-4 on VGSCs and can suppress peak currents and hyperpolarize the activation kinetics of sodium channels. Accumulating evidence from binding assays of scorpion neurotoxins on VGSCs, however, indicate that pharmacological sensitivity of VGSC subtypes to different modulators is much more complex than that suggested by the simple α-type and β-type neurotoxin distinction. Exploring the mechanisms of possible dynamic interactions between site 3-/4-specific modulators and region- and/or species-specific subtypes of VGSCs would therefore greatly expand our understanding of the physiological and pharmacological properties of diverse VGSCs. In this review, we discuss the pharmacological and structural diversity of VGSCs as revealed by studies exploring the binding properties and cross-competitive binding of site 3- or site 4-specific modulators in VGSC subtypes in synaptosomes from distinct tissues of diverse species.
Animals ; Binding Sites ; Binding, Competitive ; Brain ; metabolism ; Heart ; physiology ; Humans ; Insect Proteins ; antagonists & inhibitors ; genetics ; metabolism ; Insecta ; Ion Channel Gating ; drug effects ; physiology ; Kinetics ; Mammals ; Muscles ; metabolism ; Neurotoxins ; chemistry ; classification ; pharmacology ; Protein Binding ; Scorpions ; chemistry ; Sodium ; metabolism ; Sodium Channel Blockers ; pharmacology ; Sodium Channels ; classification ; genetics ; metabolism ; Synaptosomes ; drug effects ; metabolism

OBJECTIVETo detect the expression of voltage-gated Na(+) channel (NaCh) isoforms in rat sinoatrial node and explore their functions.

METHODSExpressions of NaCh isoforms Nav1.1, Nav1.2, Nav1.3, Nav1.5, Nav1.6 and Nav1.7 in the rat sinoatrial node were detected by immunohistochemistry. The functional roles of the NaChs were tested by observing the effect of tetrodotoxin, a specific blocker of NaChs, on the intrinsic heart rate of isolated rat working heart.

RESULTSThe tetrodotoxin- sensitive neuronal isoforms Nav1.1, Nav1.6 and Nav1.7 as well as the tetrodotoxin-resistant cardiac isoform Nav1.5 were present in the rat sinoatrial node, and the neuronal isoforms were more abundant than Nav1.5 (P<0.05). The selective blockade of tetrodotoxin-sensitive isoforms (presumably Nav1.1, Nav1.6 and Nav1.7) by 100 nmol/L tetrodotoxin scarcely affected the intrinsic heart rate (0.5-/+2.9%, P>0.05) while blockade of tetrodotoxin-resistant isoform (presumably Nav1.5) by 2 micromol/L tetrodotoxin resulted in an obvious decline in the intrinsic heart rate (22.1-/+2.1%, P<0.001).

CONCLUSIONSNav1.1, Nav1.5, Nav1.6 and Nav1.7 are all present in rat sinoatrial node. Although neuronal isoforms are more abundant, Nav1.5 seems to contribute more to activity of the sinoatrial node.

Animals ; Heart Rate ; drug effects ; physiology ; Immunohistochemistry ; Ion Channel Gating ; drug effects ; physiology ; Male ; NAV1.1 Voltage-Gated Sodium Channel ; NAV1.5 Voltage-Gated Sodium Channel ; NAV1.6 Voltage-Gated Sodium Channel ; Nerve Tissue Proteins ; biosynthesis ; Protein Isoforms ; biosynthesis ; Rats ; Sinoatrial Node ; drug effects ; metabolism ; physiology ; Sodium Channels ; biosynthesis ; Tetrodotoxin ; pharmacology

Adipose tissue is an important endocrine regulator of glucose metabolism and energy homeostasis. Researches have focused on this tissue not only as a target for pharmacotherapy of obesity and insulin resistance but also as an endocrine tissue with leptin secretion and high insulin sensitivity. Brown adipose tissue (BAT) additionally plays a unique role in thermoregulation through the mitochondrial uncoupling protein 1 (UCP1), which uncouples oxidative phosphorylation. As a genetic tissue ablation model of BAT, we made transgenic mice expressing herpes simplex virus thymidine kinase (HSV-TK) driven by the brown adipocyte- specific UCP1 minimal regulatory element. The HSV-TK transgene was expressed specifically in BAT and more than 35% increase of apoptosis was induced by ganciclovir (GCV) treatment. Nevertheless, the expression level was not high enough to induce BAT ablation in GCV-treated adult mice. Importantly, however, we found that brown adipocytes in the periphery of interscapular BAT were transformed into white adipocyte-like unilocular cells. These cells express white adipocyte-specific leptin protein but are different in the ultrastructure of mitochondria from classical white adipocytes. Our data indicates that atrophy of BAT causes transformation into white adipocyte-like cells in the adult mouse and also suggests that further molecular understanding of adipocyte plasticity using our transgenic mouse model might be beneficial for the development of anti-obesity/anti-diabetic therapies.
Adipose Tissue/*cytology/drug effects/metabolism/ultrastructure ; Aging/physiology ; Animals ; Body Weight ; Carrier Proteins/genetics/metabolism ; *Cell Differentiation/drug effects ; Ganciclovir/pharmacology ; Ion Channels ; Leptin/metabolism ; Membrane Proteins/genetics/metabolism ; Mice ; Mice, Transgenic ; Mitochondrial Proteins ; Obesity/chemically induced ; Organ Specificity ; Thymidine Kinase/genetics/metabolism