Background: Aortic arch (AA) branching patterns vary among different mammalian species.Most previous studies have focused on dogs, whereas those on raccoon dogs remain unexplored. Objectives: The objective of this study was to describe the AA branching pattern in raccoon dogs and compare their morphological features with those of other carnivores. Methods: We prepared silicone cast specimens from a total of 36 raccoon dog carcasses via retrograde injection through the abdominal aorta. The brachiocephalic trunk (BCT) branching patterns were classified based on the relationship between the left and right common carotid arteries. The subclavian artery (SB) branching pattern was examined based on the order of the four major branches: the vertebral artery (VT), costocervical trunk (CCT), superficial cervical artery (SC), and internal thoracic artery (IT). Results: In most cases (88.6%), the BCT branched off from the left common carotid artery and terminated in the right common carotid and right subclavian arteries. In the remaining cases (11.4%), the BCT formed a bicarotid trunk. The SB exhibited various branching patterns, with 26 observed types. Based on the branching order of the four major branches, we identified the main branching pattern, in which the VT branched first (98.6%), the CCT branched second (81.9%), the SC branched third (62.5%), and the IT branched fourth (52.8%). Conclusions The AA branching pattern in raccoon dogs exhibited various branching patterns with both similarities and differences compared to other carnivores.

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

Ischemic preconditioning (IPC) is known to protect the heart against ischemia/reperfusion (IR)-induced injuries, and regional differences in the mitochondrial antioxidant state during IR or IPC may promote the death or survival of viable and infarcted cardiac tissues under oxidative stress. To date, however, the interplay between the mitochondrial antioxidant enzyme system and the level of reactive oxygen species (ROS) in the body has not yet been resolved. In the present study, we examined the effects of IR- and IPC-induced oxidative stresses on mitochondrial function in viable and infarcted cardiac tissues. Our results showed that the mitochondria from viable areas in the IR-induced group were swollen and fused, whereas those in the infarcted area were heavily damaged. IPC protected the mitochondria, thus reducing cardiac injury. We also found that the activity of the mitochondrial antioxidant enzyme system, which includes manganese superoxide dismutase (Mn-SOD), was enhanced in the viable areas compared to the infarcted areas in proportion with decreasing levels of ROS and mitochondrial DNA (mtDNA) damage. These changes were also present between the IPC and IR groups. Regional differences in Mn-SOD expression were shown to be related to a reduction in mtDNA damage as well as to the release of mitochondrial cytochrome c (Cyt c). To the best of our knowledge, this might be the first study to explore the regional mitochondrial changes during IPC. The present findings are expected to help elucidate the molecular mechanism involved in IPC and helpful in the development of new clinical strategies against ischemic heart disease.
Animals ; Cytochromes c ; DNA Damage ; DNA, Mitochondrial ; Heart* ; Ischemic Preconditioning* ; Mitochondria ; Myocardial Ischemia ; Oxidative Stress ; Rats* ; Reactive Oxygen Species ; Superoxide Dismutase ; Superoxides

To characterize cytosolic Ca2+ fluctuations under metabolic inhibition, rat ventricular myocytes were exposed to 200microM 2, 4-dinitrophenol (DNP), and mitochondrial Ca2+, mitochondrial membrane potential (delta psi m), and cytosolic Ca2+ were measured, using Rhod-2 AM, TMRE, and Fluo-4 AM fluorescent dyes, respectively, by Laser Scanning Confocal Microscopy (LSCM). Furthermore, the role of sarcolemmal Na+/Ca2+ exchange (NCX) in cytosolic Ca2+ efflux was studied in KB-R7943 and Na+-free normal Tyrode's solution (143 mM LiCl ). When DNP was applied to cells loaded with Fluo-4 AM, Fluo-4 AM fluorescence intensity initially increased by 70+/-10% within 70+/-10 s, and later by 400+/-200% at 850+/-46 s. Fluorescence intensity of both Rhod-2 AM and TMRE were initially decreased by DNP, coincident with the initial increase of Fluo-4 AM fluorescence intensity. When sarcoplasmic reticulum (SR) Ca2+ was depleted by 1microM thapsigargin plus 10microM ryanodine, the initial increase of Fluo-4 AM fluorescence intensity was unaffected, however, the subsequent progressive increase was abolished. KB-R7943 delayed both the first and the second phases of cytosolic Ca2+ overload, while Na+-free solution accelerated the second. The above results suggest that: 1) the initial rise in cytosolic Ca2+ under DNP results from mitochondrial depolarization; 2) the secondary increase is caused by progressive Ca2+ release from SR; 3) NCX plays an important role in transient cytosolic Ca2+ shifts under metabolic inhibition with DNP.
Animals ; Caffeine ; Cytosol* ; Fluorescence ; Fluorescent Dyes ; Membrane Potential, Mitochondrial ; Microscopy, Confocal ; Mitochondria ; Muscle Cells* ; Rats* ; Ryanodine ; Sarcoplasmic Reticulum ; Thapsigargin

Several signal transduction pathways have been implicated in ischemic preconditioning induced by the activation of ATP-sensitive K+ (KATP) channels. We examined whether protein kinase C (PKC) modulated the activity of KATP channels by recording KATP channel currents in rabbit ventricular myocytes using patch-clamp technique and found that phorbol 12, 13-didecanoate (PDD) enhanced pinacidil-induced KATP channel activity in the cell-attached configuration; and this effect was prevented by bisindolylmaleimide (BIM). KATP channel activity was not increased by 4alpha-PDD. In excised inside-out patches, PKC stimulated KATP channels in the presence of 1 mM ATP, and this effect was abolished in the presence of BIM. Heat-inactivated PKC had no effect on channel activity. PKC-induced activation of KATP channels was reversed by PP2A, and this effect was not detected in the presence of okadaic acid. These results suggest that PKC activates KATP channels in rabbit ventricular myocytes.
Adenosine Triphosphate ; Ischemic Preconditioning ; KATP Channels* ; Muscle Cells* ; Okadaic Acid ; Patch-Clamp Techniques ; Protein Kinase C* ; Protein Kinases* ; Signal Transduction

The purpose of the present study was to evaluate the expression of cardiac marker protein in rabbit cardiac tissue that was exposed to ischemic preconditioning (IPC), or ischemiareperfusion injury (IR) using two-dimensional gel electrophoresis (2DE) and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). We compared 2DE gels of control (uninjured) cardiac tissue with those of IPC and IR cardiac tissue. Expression of one protein was detected in IR heart tissue, however the protein was not detected in the samples of control and IPC tissue. To further characterize the detected protein molecule, the protein in the 2D gel was isolated and subjected to trypsin digestion, followed by MALDI-MS. The protein was identified as myoglobin, which was confirmed also by Western blot analysis. These results are consistent with previous studies of cardiac markers in ischemic hearts, indicating myoglobin as a suitable marker of myocardial injury. In addition, the present use of multiple techniques indicates that proteomic analysis is an appropriate means to identify cardiac markers in studies of IPC and IR.
Blotting, Western ; Digestion ; Electrophoresis, Gel, Two-Dimensional* ; Gels ; Heart ; Ischemia* ; Ischemic Preconditioning ; Mass Spectrometry* ; Myoglobin ; Reperfusion Injury* ; Reperfusion* ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Trypsin

Mitochondrial ATP-sensitive potassium (mitoKATP) channels play a role in early and late ischemic preconditioning. Nevertheless, the subunit composition of mitoKATP channels remains unclear. In this study, we investigated the subunit composition of mitoKATP channels in mitochondria isolated from rat cardiac myocytes. Mitochondria were visualized using the red fluorescence probe, Mitrotracker Red, while mitoKATP channels were visualized using the green fluorescence probe, glibenclamide-BODIPY. The immunofluorescence confocal microscopy revealed the presence of Kir6.1, Kir6.2 and SUR2 present in the cardiac mitochondria. Western blot analysis was carried to further investigate the nature of mitoKATP channels. For SUR proteins, a 140-kDa immunoreactive band that corresponded to SUR2, but no SUR1 was detected. For Kir6.2, three bands (~4, ~6, and ~0 kDa) were detected, and a specific ~6-kDa immunoreactive band corresponding to Kir6.1 was also observed. These observations suggest that the subunits of mitoKATP channels in rat myocytes include Kir6.1, Kir6.2, and a SUR2-related sulfonylurea-binding protein.
Animals ; Blotting, Western ; Fluorescence ; Fluorescent Antibody Technique ; Ischemic Preconditioning ; KATP Channels* ; Microscopy, Confocal ; Mitochondria ; Muscle Cells ; Myocytes, Cardiac* ; Potassium ; Rats*

Ischemic preconditioning (IPC) has been accepted as a heart protection phenomenon against ischemia and reperfusion (I/R) injury. The activation of ATP-sensitive potassium (KATP) channels and the release of myocardial nitric oxide (NO) induced by IPC were demonstrated as the triggers or mediators of IPC. A common action mechanism of NO is a direct or indirect increase in tissue cGMP content. Furthermore, cGMP has also been shown to contribute cardiac protective effect to reduce heart I/R-induced infarction. The present investigation tested the hypothesis that KATP channels attenuate DNA strand breaks and oxidative damage in an in vitro model of I/R utilizing rat ventricular myocytes. We estimated DNA strand breaks and oxidative damage by mean of single cell gel electrophoresis with endonuclease III cutting sites (comet assay). In the I/R model, the level of DNA damage increased massively. Preconditioning with a single 5-min anoxia, diazoxide (100muM), SNAP (300muM) and 8- (4-Chlorophenylthio)-guanosine-3', 5'-cyclic monophosphate (8-pCPT-cGMP) (100muM) followed by 15 min reoxygenation reduced DNA damage level against subsequent 30 min anoxia and 60 min reoxygenation. These protective effects were blocked by the concomitant presence of glibenclamide (50muM), 5-hydroxydecanoate (5-HD) (100muM) and 8- (4-Chlorophenylthio)-guanosine-3', 5'-cyclic monophosphate, Rp-isomer (Rp-8-pCPT-cGMP) (100muM). These results suggest that NO-cGMP-protein kinase G (PKG) pathway contributes to cardioprotective effect of KATP channels in rat ventricular myocytes.
Animals ; Anoxia ; Diazoxide ; DNA ; DNA Damage ; Electrophoresis ; Glyburide ; Heart* ; Infarction ; Ischemia ; Ischemic Preconditioning ; KATP Channels ; Muscle Cells ; Nitric Oxide ; Phosphotransferases* ; Potassium ; Rats* ; Reperfusion

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