Interstitial cells of Cajal (ICCs) are the pacemaker cells in the gastrointestinal tract, and histamine is known to regulate neuronal activity, control vascular tone, alter endothelial permeability, and modulate gastric acid secretion. However, the action mechanisms of histamine in mouse small intestinal ICCs have not been previously investigated, and thus, in the present study, we investigated the effects of histamine on mouse small intestinal ICCs, and sought to identify the receptors involved. Enzymatic digestions were used to dissociate ICCs from small intestines, and the whole-cell patch-clamp configuration was used to record potentials (in current clamp mode) from cultured ICCs. Histamine was found to depolarize resting membrane potentials concentration dependently, and whereas 2-PEA (a selective H1 receptor agonist) induced membrane depolarizations, Dimaprit (a selective H2-agonist), R-alpha-methylhistamine (R-alpha-MeHa; a selective H3-agonist), and 4-methylhistamine (4-MH; a selective H4-agonist) did not. Pretreatment with Ca(2+)-free solution or thapsigargin (a Ca(2+)-ATPase inhibitor in endoplasmic reticulum) abolished the generation of pacemaker potentials and suppressed histamine-induced membrane depolarization. Furthermore, treatments with U-73122 (a phospholipase C inhibitor) or 5-fluoro-2-indolyl des-chlorohalopemide (FIPI; a phospholipase D inhibitor) blocked histamine-induced membrane depolarizations in ICCs. On the other hand, KT5720 (a protein kinase A inhibitor) did not block histamine-induced membrane depolarization. These results suggest that histamine modulates pacemaker potentials through H1 receptor-mediated pathways via external Ca2+ influx and Ca2+ release from internal stores in a PLC and PLD dependent manner.
Animals ; Carbazoles ; Cyclic AMP-Dependent Protein Kinases ; Dimaprit ; Domperidone ; Estrenes ; Gastric Acid ; Gastrointestinal Tract ; Hand ; Histamine ; Indoles ; Interstitial Cells of Cajal ; Intestine, Small ; Membrane Potentials ; Membranes ; Methylhistamines ; Mice ; Neurons ; Permeability ; Phospholipase D ; Pyridoxal ; Pyrroles ; Pyrrolidinones ; Thapsigargin ; Type C Phospholipases

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

The aim of the present study is to investigate the contribution of Ca2+-activated K+ (KCa) channels and delayed rectifier K+ (KV) channels to the resting membrane potential (RMP) in rabbit middle cerebral arterial smooth muscle cells. The RMP and membrane currents were recorded using the whole-cell patch configuration and single KCa channel was recorded using the outside-out patch configuration. Using the pipette solution containing 0.05 mM EGTA, the RMP was -25.76+/-5.08 mV (n=12) and showed spontaneous transient hyperpolarizations (STHPs). The membrane currents showed time- and voltage-dependent outward currents with spontaneous transient outward currents (STOCs). When we recorded the membrane potential using the pipette solution containing 10 mM EGTA, the RMP was depolarized and did not show STHPs. The membrane currents showed no STOCs but only showed slowly inactivating outward currents. External TEA (1 mM) reversibly inhibited the STHPs, depolarized the RMP, reduced the membrane currents, abolished STOCs, and decreased the open probability of single KCa channel. When KV currents were isolated, the application of 4-AP (5 mM) depolarized the RMP. The important aspect of our results is that KCa channel is responsible for the generation of the STHPs in the membrane potential and plays an important role in the regulation of the RMP and KV channel is also responsible for the regulation of the RMP in rabbit middle cerebral arterial smooth muscle cells.
Egtazic Acid ; Membrane Potentials* ; Membranes ; Muscle, Smooth* ; Myocytes, Smooth Muscle* ; Tea

Adriamycin is a commonly used chemotherapeutic agent for cancer, including acute leukemia, lymphoma, and a number of solid human tumors. However, recent studies have recognized severe cardiotoxicity after an acute dose, which are likely the result of generation of free radicals and lipid peroxidation. Therefore, the clinical uses of adriamycin have been limited. Melatonin, the pineal gland hormone known for its ability to modulate circardian rhythm, has recently been studied in its several functions, including cancer growth inhibition, stimulating the immune system, and acting as an antioxidant and radical scavenging effects. In the present study, we evaluated the effect of melatonin administration on adriamycin-induced cardiotoxicity in rat. Heart slices were prepared using a Stadie-Riggs microtome for the measurement of malondialdehyde (MDA) content used as an index of lipid peroxidation and lactate dehydrogenase (LDH) release as an indicator of lethal cell injury. Serious adriamycin-induced lethality was observed in rat by a single intraperitoneal injection in a dose-dependent manner. A single injection of adriamycin (25 mg/kg, i.p.) induced a lethality rate of 86%, with melatonin (10 mg/kg s.c. for 6 days) treatment reducing the adriamycin-induced lethality rate to 20%. The severe body weight loss caused by adriamycin was also significantly attenuated by melatonin treatment. Treatment of melatonin marked reduced adriamycin-induced the levels of MDA formation and LDH release. A cell damage indicated by the loss of myofibrils, swelling of the mitochondria as well as cytoplasmic vacuolization was seen in adriamycin-treated group. Melatonin attenuated the adriamycin-induced structural alterations. These data provide evidence that melatonin prevents adriamycin-induced cardiotoxicity and might serve as a combination with adriamycin to limit free radical-mediated cardiotoxicity.
Animals ; Body Weight ; Cytoplasm ; Doxorubicin ; Free Radicals ; Heart ; Humans ; Immune System ; Injections, Intraperitoneal ; L-Lactate Dehydrogenase ; Leukemia ; Lipid Peroxidation ; Lymphoma ; Malondialdehyde ; Melatonin* ; Mitochondria ; Myofibrils ; Pineal Gland ; Rats*

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

BACKGROUND: We have previously reported that not only cGMP but also 8-Br-cGMP or 8-pCPT-cGMP, specific and potent stimulators of cGMP-dependent protein kinase (cGMP-PK), increased basal L-type calcium current (ICa) in rabbit ventricular myocytes. Our findings in rabbit ventricular myocytes were entirely different from the earlier findings in different species, suggesting that the activation of cGMP-PK is involved in the facilitation of ICa by cGMP. However, there is no direct evidence that cGMP-PK can stimulate ICa in rabbit ventricular myocytes. In this report, we focused on the direct effect of cGMP-PK an ICa in rabbit ventricular myocytes. METHODS AND RESULTS: We isolated single ventricular myocytes of rabbit hearts by using enzymatic dissociation. Regulation of ICa by cGMP-PK was investigated in rabbit ventricular myocytes using whole-cell voltage clamp method. ICa was elicited by a depolarizing pulse to +10 mV from a holding potential of -40 mV. Extracellular 8-(4-Chlorophenylthio)-guanosine-3',5'-cyclic monophosphate (8-pCPT-cGMP), potent stimulator of cGMP-dependent protein kinase (cGMP-PK), increased basal ICa. cGMP-PK also increased basal ICa. The stimulation of basal ICa by cGMP-PK required both 8-Br-cGMP in low concentration and intracellular ATP to be present. The stimulation of basal ICa by cGMP-PK was blocked by heat inactivation of the cGMP-PK and by bath application of 8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphate, Rp-isomer (Rp-pCPT-cGMP), a phosphodiesterase-resistant cGMP-PK inhibitor. When ICa was increased by internal application of cGMP-PK, IBMX resulted in an additional stimulation of ICa. In the presence of cGMP-PK, already increased ICa was potentiated by bath application of isoprenaline or forskolin or intracellular application of cAMP. CONCLUSIONS: We present evidence that cGMP-PK stimulated basal ICa by a direct phosphorylation of L-type calcium channel or associated regulatory protein in rabbit ventricular myocytes.
1-Methyl-3-isobutylxanthine ; Adenosine Triphosphate ; Baths ; Calcium Channels, L-Type ; Calcium* ; Colforsin ; Heart ; Hot Temperature ; Isoproterenol ; Muscle Cells* ; Phosphorylation ; Protein Kinases*

The purpose of our investigation was to examine the effects of prostaglandin F2alpha (PGF2alpha) on membrane potentials, Ca2+-activated K+ (KCa) channels, and delayed rectifier K+ (KV) channels using the patch-clamp technique in single rabbit middle cerebral arterial smooth muscle cells. PGF2alpha significantly hyperpolarized membrane potentials and increased outward whole-cell K+ currents. PGF2alpha increased open-state probability of KCa channels without the change of the open and closed kinetics. PGF2alpha increased the amplitudes of KV currents with a leftward shift of activation and inactivation curves and a decrease of activation time constant. Our results suggest that the activation of KCa and KV channels, at least in part, may lead to attenuate or counteract vasoconstriction by PGF2alpha in middle cerebral artery.
Dinoprost* ; Kinetics ; Membrane Potentials* ; Membranes* ; Middle Cerebral Artery ; Myocytes, Smooth Muscle ; Patch-Clamp Techniques ; Vasoconstriction

Melatonin, a pineal gland hormone, is believed to act as an antioxidant via the stimulation of radical detoxifying enzymes and scavenging of free radicals. In this study, effects of in vitro and in vivo treatments of melatonin on the cisplatin-induced lipid peroxidation, LDH release and plasma creatinine were determined in rabbit renal cortical cells. The level of malondialdehyde (MDA) was assayed as an index of lipid peroxidation and the level of LDH release as an indicator of cellular damage. In in vitro studies, cisplatin increased the levels of MDA and LDH release in a concentration-and time-dependent manner. Melatonin inhibited the cisplatin-induced lipid peroxidation and LDH release in a concentration-dependent manner. The minimal effective concentration of melatonin that significantly reduced the 300 muM cisplatin-induced lipid peroxidation and LDH release was 1 mM. In in vivo studies, the levels of lipid peroxidation and LDH release in renal cortical cells increased significantly 24 or 48 hours after a single injection of cisplatin (6 mg/kg). When the cisplatin-injected rabbits were pretreated with 10 mg/kg of melatonin, a significant reduction in both lipid peroxidation and LDH release was observed. The plasma creatinine level increased from 0.87+/-0.07 mg/dl in control to 6.33+/-0.54 mg/dl in cisplatin-injected rabbits (P<0.05). Melatonin partially prevented the increase in serum creatinine level (1.98+/-0.11 mg/dl) by cisplatin (P<0.05). In the proximal tubules from cisplatin-treated group, tubular cells had microvilli of variable heights. Necrotic debris was seen in tubular lumens. In most of cells, the mitochondria and lysosomes were increased in frequency. The endocytic vacuoles were not prominent and distribution of the brush border was irregular and shortened. These cisplatin-induced morphological changes were moderate in the melatonin-pretreated group. These results suggest that the toxicity of cisplatin is associated with the generation of reactive oxygen free radicals and that melatonin is a powerful antioxidant, which prevents some of the adverse effects of cisplatin.
Cisplatin ; Creatinine ; Free Radicals ; Lipid Peroxidation ; Lysosomes ; Malondialdehyde ; Melatonin* ; Microvilli ; Mitochondria ; Oxygen ; Pineal Gland ; Plasma ; Rabbits* ; Vacuoles

An impaired smooth muscle cell (SMC) relaxation of coronary artery by alteration of K+ channels would be the most potential explanation for reduced coronary reserve in left ventricular hypertrophy (LVH), however, this possibility has not been investigated. We performed morphometrical analysis of the coronary artery under electron microscopy and measured Ca2+-activated K (KCa) currents and delayed rectifier K (Kdr) currents by whole-cell and inside-out patch-clamp technique in single coronary arterial SMCs from rabbits subjected to isoprenaline-induced cardiac hypertrophy. Coronary arterial SMCs underwent significant changes in ultrastructure. The unitary current amplitude and the open-state probability of KCa channel were significantly reduced in hypertrophy without open-time and closed-time kinetic. The concentration-response curve of KCa channel to Ca2+ is shifted to the right in hypertrophy. The reduction in the mean single channel current and increase in the open channel noise of KCa channel by TEA were more sensitive in hypertrophy. Kdr current density is significantly reduced in hypertrophy without activation and inactivation kinetics. The sensitivity of Kdr current on 4-AP is significantly increased in hypertrophy. This is the first study to report evidence for alterations of KCa channels and Kdr channels in coronary SMCs with LVH. The findings may provide some insight into mechanism of the reduced coronary reserve in LVH.
Cardiomegaly* ; Coronary Vessels* ; Hypertrophy ; Hypertrophy, Left Ventricular ; Kinetics ; Microscopy, Electron ; Myocytes, Smooth Muscle ; Noise ; Patch-Clamp Techniques ; Rabbits ; Relaxation ; Tea

The aim of present study was to define the cellular mechanisms underlying changes in delayed rectifier K+ (KDR) channel function in isoproterenol-induced hypertrophy. It has been proposed that KDR channels play a role in regulation of vascular tone by limiting membrane depolarization in arterial smooth muscle cells. The alterations of the properties of coronary KDR channels have not been studied as a possible mechanism for impaired coronary reserve in cardiac hypertrophy. The present study was carried out to compare the properties of coronary KDR channels in normal and hypertrophied hearts. These channels were measured from rabbit coronary smooth muscle cells using a patch clamp technique. The main findings of the study are as follows: (1) the KDR current density was decreased without changes of the channel kinetics in isoproterenol-induced hypertrophy; (2) the sensitivity of coronary KDR channels to 4-AP was increased in isoproterenol-induced hypertrophy. From the above results, we suggest for the first time that the alteration of KDR channels may limit vasodilating responses to several stimuli and may be involved in impaired coronary reserve in isoproterenol-induced hypertrophy.
Cardiomegaly ; Heart ; Hypertrophy* ; Isoproterenol ; Kinetics ; Membranes ; Muscle Cells* ; Myocytes, Smooth Muscle ; Potassium Channels