Cellular redox state is known to be perturbed during ischemia and that Ca2+ and K+ channels have been shown to have functional thiol groups. In this study, the properties of thiol redox modulation of the ATP-sensitive K+ (KATP) channel were examined in rabbit ventricular myocytes. Rabbit ventricular myocytes were isolated using a Langendorff column for coronary perfusion and collagenase. Single-channel currents were measured in excised membrane patch configuration of patch-clamp technique. The thiol oxidizing agent 5, 5'-dithio-bis- (2-nitro-benzoic acid) (DTNB) inhibited the channel activity, and the inhibitory effect of DTNB was reversed by dithiothreitol (disulfide reducing agent; DTT). DTT itself did not have any effect on the channel activity. However, in the patches excised from the metabolically compromised cells, DTT increased the channel activity. DTT had no effect on the inhibitory action by ATP, showing that thiol oxidation was not involved in the blocking mechanism of ATP. There were no statistical difference in the single channel conductance for the oxidized and reduced states of the channel. Analysis of the open and closed time distributions showed that DTNB had no effect on open and closed time distributions shorter than 4 ms. On the other hand, DTNB decreased the life time of bursts and increased the interburst interval. N-ethylmaleimide (NEM), a substance that reacts with thiol groups of cystein residues in proteins, induced irreversible closure of the channel. The thiol oxidizing agents (DTNB, NEM) inhibited of the KATP channel only, when added to the cytoplasmic side. The results suggested that metabolism-induced changes in the thiol redox can also modulate KATP channel activity and that a modulatory site of thiol redox may be located on the cytoplasmic side of the KATP channel in rabbit ventricular myocytes.
Adenosine Triphosphate ; Collagenases ; Cytoplasm ; Dithionitrobenzoic Acid ; Dithiothreitol ; Ethylmaleimide ; Hand ; Ischemia ; KATP Channels* ; Membranes ; Muscle Cells* ; Oxidants ; Oxidation-Reduction* ; Patch-Clamp Techniques ; Perfusion

Recent studies indicated that cancer cells become resistant to ionizing radiation (IR) and chemotherapy drugs by enhanced DNA repair of the lesions. Therefore, it is expected to increase the killing of cancer cells and reduce drug resistance by inhibiting DNA repair pathways that tumor cells rely on to escape chemotherapy. There are a number of key human DNA repair pathways which depend on multimeric polypeptide activities. For example, Ku heterodimer regulatory DNA binding subunits (Ku70/Ku80) on binding to double strand DNA breaks (DSBs) are able to interact with 470-kDa DNA-dependent protein kinase catalytic subunit (DNA-PKcs), and are essential for DNA-dependent protein kinase (DNA-PK) activity. It has been known that DNA-PK is an important factor for DNA repair and also is a sensor-transmitting damage signal to downstream targets, leading to cell cycles arrest. Our ultimate goal is to develop a treatment of breast tumors by targeting proteins involved in damage-signaling pathway and/or DNA repair. This would greatly facilitate tumor cell cytotoxic activity and programmed cell death through DNA damaging drug treatment. Therefore, we designed a domain of Ku80 mutants that binds to Ku70 but not DNA end binding activity and used the peptide in co-therapy strategy to see whether the targeted inhibition of DNA-PK activity sensitized breast cancer cells to irradiation or chemotherapy drug. We observed that the synthesized peptide (HNI-38) prevented DNA-PKcs from binding to Ku70/Ku80, thus resulting in inactivation of DNA-PK activity. Consequently, the peptide treated cells exhibited poor to no DNA repair, and became highly sensitive to IR or chemotherapy drugs, and the growth of breast cancer cells was inhibited. Additionally, the results obtained in the present study also support the physiological role of resistance of cancer cells to IR or chemotherapy.
Breast Neoplasms ; Catalytic Domain* ; Cell Cycle ; Cell Death ; DNA ; DNA Breaks, Double-Stranded ; DNA Repair ; DNA-Activated Protein Kinase* ; Drug Resistance ; Drug Therapy ; Homicide ; Humans ; Radiation, Ionizing ; United Nations

Alcohol abuse and cigarette smoking have been on the rise worldwide and it has been reported that alcohol and nicotine influence serotonergic neuronal activity in the dorsal raphe. Serotonin (5-hydroxytryptamine, 5-HT) has been implicated in the pathophysiology of various neuropsychiatric disorders. In the present study, the effects of alcohol and nicotine on the synthesis of 5-HT and the expression of tryptophan hydroxylase (TPH), the rate limiting enzyme of 5-HT synthesis, in the dorsal and median raphe of young rats were investigated via immunohistochemistry. The numbers of the 5-HT-positive and TPH-positive cells in raphe nuclei were reduced by alcohol and nicotine treatment, and these numbers were reduced more potently by co-administration of alcohol and nicotine. Based on the results, it can be suggested that the pathogenesis of alcohol- and nicotine-induced neuropsychological disorders involves alcohol- and nicotine-induced suppression of 5-HT synthesis and TPH expression in raphe, and that this may be of particular relevance in the consumption of alcohol and nicotine during adolescence.
Adolescent ; Alcoholism ; Animals ; Humans ; Immunohistochemistry ; Nicotine* ; Raphe Nuclei ; Rats* ; Serotonergic Neurons ; Serotonin* ; Smoking ; Tryptophan Hydroxylase* ; Tryptophan*

To investigate the receptors mediating the regulation of norepinephrine (NE) release in human cerebral cortex slices, we examined the effects of opioid agonists for mu-, delta-, and kappa -receptors on the high potassium (15 mM) -evoked release of [3H]NE. [3H]NE release induced by high potassium was calcium-dependent and tetrodotoxin-sensitive. [D-Pen2, D-Pen5]enkephalin (DPDPE) and deltorphin II (Delt II) inhibited the stimulated release of norepinephrine in a dose-dependent manner. However, Tyr-D-Ala-Gly- (Me) Phe-Gly-ol and U69, 593 did not influence the NE release. Inhibitory effect of DPDPE and Delt-II was antagonized by naloxone, naltrindole, 7-benzylidenaltrexone and naltriben. These results suggest that both delta 1 and delta 2 receptors are involved in regulation of NE release in human cerebral cortex.
Analgesics, Opioid* ; Cerebral Cortex* ; Enkephalin, D-Penicillamine (2,5)- ; Humans* ; Naloxone ; Negotiating ; Norepinephrine ; Potassium ; Receptors, Opioid

To elucidate the mechanism of gentamicin induced renal dysfunction, renal functions and activities of various proximal tubular transport systems were studied in gentamicin-treated rats (Fisher 344). Gentamicin nephrotoxicity was induced by injecting gentamicin sulfate subcutaneously at a dose of 100 mg/kg cntdot day for 7 days. The gentamicin injection resulted in a marked polyuria, hyposthenuria, proteinuria, glycosuria, aminoaciduria, phosphaturia, natriuresis, and kaliuresis, characteristics of aminoglycoside nephropathy. Such renal functional changes occurred in the face of reduced GFR, thus tubular transport functions appeared to be impaired. The polyuria and hyposthenuria were partly associated with a mild osmotic diuresis, but mostly attributed to a reduction in free water reabsorption. In renal cortical brush-border membrane vesicles isolated from gentamicin-treated rats, the Na+ gradient dependent transport of glucose, alanine, phosphate and succinate was significantly attenuated with no changes in Na+/-independent transport and the membrane permeability to Na+. These results indicate that gentamicin treatment induces a defect in free water reabsorption in the distal nephron and impairs various Na+/-cotransport systems in the proximal tubular brush-border membranes, leading to polyuria, hyposthenuria, and increased urinary excretion of Na+ and other solutes.
Alanine ; Animals ; Diuresis ; Gentamicins ; Glucose ; Glycosuria ; Hypophosphatemia, Familial ; Membranes ; Natriuresis ; Nephrons ; Permeability ; Polyuria ; Proteinuria ; Rats* ; Succinic Acid ; Water

In the present study, we characterized the angiotensin II (AII)-induced relaxations in the phenylephrineprecontracted rabbit mesenteric arteries with endothelium. 1) AII-induced relaxation was consistently observed in the rabbit mesenteric arteries with and without endothelium, but not in the aortic segment with endothelium. 2) AII-induced endothelium-dependent relaxation was markedly inhibited by Nw-nitro-L-arginine (L-NNA, 100 micrometer), methylene blue (10 micrometer) and LY83583 (10 micrometer), respectively. 3) Inhibition of cyclooxygenase with indomethacin (10 micrometer) strongly decreased the vasorelaxant response to AII irrespective of the presence of endothelium. 4) 7-Ethoxyresorufin (1 micrometer) and clotrimazole (1 micrometer), inhibitors of cytochrome P-450-dependent arachidonic acid metabolism, greatly attenuated the vasodilator response to All. 5) Carbacyclin, arachidonic acid and prostaglandin F2alpha, (PGF2alpha) caused concentration-dependent relaxations in the mesenteric artery with endothelium, which were inhibited by L-NNA and methylene blue. 6) AII and PGF2alpha, significantly stimulated cyclic GMP formation in the mesenteric arteries with endothelium, which was inhibited by L-NNA and methylene blue, respectively. 7) AII enhanced synthesis of PGF2a and 6-keto PGF1a from the arterial segments with endothelium, which was inhibitable by indomethacin, but not by L-NNA. In conclusion, the vasorelaxant responses to AII of the rabbit mesenteric artery with endothelium are subserved by arachidonic acid and its metabolites produced via activation of cyclooxygenase and cytochrome P-450 enzyme as well as by nitric oxide.
Angiotensin II* ; Angiotensins* ; Arachidonic Acid ; Clotrimazole ; Cyclic GMP ; Cytochrome P-450 Enzyme System ; Cytochromes ; Dinoprost ; Endothelium ; Indomethacin ; Mesenteric Arteries* ; Metabolism ; Methylene Blue ; Nitric Oxide ; Prostaglandin-Endoperoxide Synthases ; Relaxation* ; Vasodilation

The phospholipase C (PLC)-mediated intracellular signal transduction pathway is considered to be involved in the regulation of blood pressure. However, little information is available concerning the distributional and functional significance of PLC in the genetic hypertensive rats. As the first step of knowing the role of PLC on hypertension, we investigated the distribution of 6 PLC isozymes (PLC-beta1, -beta3, -beta4, -gamma1, -gamma2 and -delta1) in the heart and brain, which are concerned with hypertension, in the normotensive Wistar-Kyoto rat (WKY) and spontaneously hypertensive rat (SHR) using the western blotting and immunocytochemistry. The immunoreactivities of PLC isozymes in brain were detected, but there were no distributional and quantitative differences between the WKY and SHR. In the heart, but the immunoreactivities to PLC-beta1 and -gamma2 in the SHR were higher than those in WKY. In immunocytochemistry to confirm these western blotting data, PLC-beta1 and -gamma2 were localized in cardiac myocytes and the intensities of immunoreactivity in SHR were stronger than that in WKY. These results suggest that PLC-beta1 and -gamma2 would have possibility to concern with the establishment of spontaneous hypertension.
Animals ; Blood Pressure ; Blotting, Western ; Brain* ; Heart* ; Hypertension ; Immunohistochemistry ; Isoenzymes* ; Myocytes, Cardiac ; Phospholipases* ; Rats* ; Rats, Inbred SHR ; Signal Transduction ; Type C Phospholipases*

To investigate the properties of ryanodine binding sites of the bird skeletal SR vesicles, SDS PAGE, purification of RyR, and (3H)ryanodine binding study were carried out in the SR vesicles prepared from the chicken pectoral muscle. The chicken SR vesicles have two high molecular weight (HMW) protein bands as in eel SR vesicles on SDS PAGE. The HMW bands on SDS PAGE were found in the (3H) ryanodine peak fraction (Fr3-5) obtained from the purification step of the ryanodine receptor protein. Bmax and KD of the chicken (3H)ryanodine binding sites were 12.52 pmol/mg protein and 14.53 nM, respectively. Specific (3H)ryanodine binding was almost maximal at 50~100 micrometer Ca2+, but was not increased by 5 mM AMP and not inhibited by high Ca2+. Binding was significantly inhibited by 20~100 micrometer ruthenium red and 1 mM tetracaine, but slightly inhibited by Mg2+. From the above results, it is suggested that chicken SR vesicles have the ryanodine binding sites to which the binding of ryanodine is almost maximal at 50~10 micrometer Ca2+, is significantly inhibited by ruthenium red and tetracaine, slightly inhibited by Mg2+, but not affected by AMP and not inhibited by high Ca2+.
Binding Sites* ; Birds ; Chickens* ; Eels ; Electrophoresis, Polyacrylamide Gel ; Molecular Weight ; Ruthenium Red ; Ryanodine ; Ryanodine Receptor Calcium Release Channel ; Sarcoplasmic Reticulum ; Tetracaine

The effect of an organic peroxide, t-butylhydroperoxide (t-BHP), on glutamate uptake was studied in synaptosomes prepared from cerebral cortex. t-BHP inhibited the Na+/-dependent glutamate uptake with no change in the Na+/-independent uptake. This effect of t-BHP was not altered by addition of Ca2+ channel blockers (verapamil, diltiazem and nifedipine) or PLA2 inhibitors (dibucaine, butacaine and quinacrine). However, the effect was prevented by iron chelators (deferoxamine and phenanthroline) and phenolic antioxidants (N,N'-diphenyl-phenylenediamine, butylated hydroxyanisole, and butylated hydroxytoluene). At low concentrations (< 1.0 mM), t-BHP inhibited glutamate uptake without altering lipid peroxidation. Moreover, a large increase in lipid peroxidation by ascorbate/Fe2+ was not accompanied by an inhibition of glutamate uptake. The impairment of glutamate uptake by t-BHP was not intimately related to the change in Na+/-K+/-ATPase activity. These results suggest that inhibition of glutamate uptake by t-BHP is not totally mediated by peroxidation of membrane lipid, but is associated with direct interactions of glutamate transport proteins with t-BHP metabolites. The Ca2+ influx through Ca2+ channel or PLA2 activation may not be involved in the t-BHP inhibition of glutamate transport.
Antioxidants ; Brain* ; Butylated Hydroxyanisole ; Carrier Proteins ; Cerebral Cortex ; Chelating Agents ; Diltiazem ; Glutamic Acid* ; Iron ; Lipid Peroxidation ; Membranes ; Phenol ; Synaptosomes* ; tert-Butylhydroperoxide*

Although cerebellar Purkinje cells display spontaneous electrical activity in vivo and in slice experiments, the mechanism of the spontaneous activity generation has not been clearly understood. The aim of this study was to investigate whether cerebellar Purkinje cells of postnatal rats generate spontaneous electrical activity without synaptic inputs. Dissociated cerebellar Purkinje cells were used for reducing synaptic inputs in the present study. Cerebellar Purkinje cells with dendrites were dissociated from postnatal rats using enzymatic treatment followed by mechanical trituration. Spontaneous electrical activities were recorded from dissociated cells without any stimulus using whole-cell patch clamp configuration. Two types, spontaneously firing or quiescent, of dissociated Purkinje cells were observed in postnatal rats. Both types of cells were identified as Purkinje cells using immunocytochemical staining technique with anti-calbindin after recording. Spontaneously active cells displayed two patterns of firing, repetitive and burst firings. Two thirds of dissociated Purkinje cells displayed repetitive firing and the rest of them did burst firing under same recording condition. Repetitive firing activities were maintained even after further isolation using either physical or pharmacological techniques. Neither high magnesium solution nor excitatory synaptic blockers, AP-5 and DNQX, block the spontaneous activity. These results demonstrate that spontaneous electrical activity of isolated cerebellar Purkinje cells in postnatal rats is generated by intrinsic membrane properties rather than synaptic inputs.
Animals ; Cell Separation ; Dendrites ; Fires ; Magnesium ; Membranes ; Neurons* ; Purkinje Cells ; Rats*