Euiyong Kim

ATP-sensitive potassium channels are modulated by intracellular lactate in rabbit ventricular myocytes

During myocardial ischemia, increased anaerobic glycolysis results in the accumulation of large amount of intracellular lactate. Effects of lactate on the ATP-sensitive potassium (KATP) channels were examined in rabbit ventricular myocytes, using the inside-out patch-clamp technique. Millimolar concentrations of lactate, applied to the cytosolic side of the patch membrane, induced openings of the kATP channel. This effect was inhibited by 0.1 mM glybenclamide. Lactate-induced openings of the channel were increased in a dose-dependent fashion. In dose-response relation for lactate, Kd (the lactate concentration producing half-maximal activation) and n (Hill coefficient) were 20 mM and 1.3, respectively (n=5). Activation of KATP channels by lactate occurred even in the presence of 2 mM ATP. Lactate also caused a significant increase in Ki, the ATP concentration causing half-maximal inhibition, from 70 μM in control (n=7) to 232 μM (n=5). From the above results it could be concluded that intracellular lactate modulate kATP channels directly and such modulation may resolve the discrepancy between the low KI in excised membrane patches and high levels of intracellular ATP concentration during myocardial ischemia or hypoxia.

A mathematical model of pacemaker activity recorded from mouse small intestine.

The pacemaker activity of interstitial cells of Cajal (ICCs) has been known to initiate the propagation of slow waves along the whole gastrointestinal tract through spontaneous and repetitive generation of action potentials. We studied the mechanism of the pacemaker activity of ICCs in the mouse small intestine and tested it using a mathematical model. The model includes ion channels, exchanger, pumps and intracellular machinery for Ca2+ regulation. The model also incorporates inositol 1,4,5-triphosphate (IP3) production and IP3-mediated Ca2+ release activities. Most of the parameters were obtained from the literature and were modified to fit the experimental results of ICCs from mouse small intestine. We were then able to compose a mathematical model that simulates the pacemaker activity of ICCs. The model generates pacemaker potentials regularly and repetitively as long as the simulation continues. The frequency was set at 20 min−1 and the duration at 50% repolarization was 639 ms. The resting and overshoot potentials were −78 and +1.2 mV, respectively. The reconstructed pacemaker potentials closely matched those obtained from animal experiments. The model supports the idea that cyclic changes in [Ca2+]i and [IP3] play key roles in the generation of ICC pacemaker activity in the mouse small intestine.

Changes in the Ca2+-activated K+ channels of the coronary artery during left ventricular hypertrophy.

Abstract— It has been suggested that impairment of smooth muscle cell (SMC) function by alterations in the Ca2+-activated K+ (KCa) channels accounts for the reduction in coronary reserve during left ventricular hypertrophy (LVH). However, this hypothesis has not been fully investigated. The main goal of this study was to assess whether the properties of KCa channels in coronary SMCs were altered during LVH. In patch-clamp experiments, the whole-cell currents of the KCa channels were reduced during LVH. The unitary current amplitude and open probability for the KCa channels were significantly reduced in LVH patches compared with control patches. The concentration-response curve of the KCa channel to [Ca2+]i was shifted to the right. Inhibition of the KCa channels by tetraethylammonium (TEA) was more pronounced in LVH cells than in control cells. Western blot analysis indicated no differences in KCa channel expression between the control and LVH coronary SM membranes. In contraction experiments, the effect of high K+ concentration on the resting tension of the LVH coronary artery was greater than on that of the control. The effect of TEA on the resting tension of the LVH coronary artery was reduced compared with the effect on the control. Our findings imply a novel mechanism for reduced coronary reserve during LVH.

Modulation by melatonin of the cardiotoxic and antitumor activities of adriamycin.

In this study, we investigated the effects of melatonin on adriamycin-induced cardiotoxicity both in vivo in rats and in vitro, and on the antitumor activities of adriamycin on MDA-231 and NCI breast cancer cells. Rats that received a single intraperitoneal injection of 25 mg/kg adriamycin showed a mortality rate of 86%, which was reduced to 20% by melatonin treatment (10 mg/kg, SC for 6 days). Melatonin attenuated adriamycin-induced body-weight loss, hemodynamic dysfunction, and the morphologic and biochemical alterations caused by adriamycin. Melatonin also reduced adriamycin-induced nuclear DNA fragmentation, as assessed by the comet assay. In addition, the antitumor activity of adriamycin could be maintained using lower doses of this drug in combination with melatonin. Melatonin treatment in the concentration range of 0.1-2.5 mM inhibited the growth of human breast cancer cells. In terms of oncolytic activity, the combination of adriamycin and melatonin improved the antitumor activity of adriamycin, as indicated by an increase in the number of long-term survivors as well as decreases in body-weight losses resulting from adriamycin treatment. These results indicate that melatonin not only protects against adriamycin-induced cardiotoxicity but also enhances its antitumor activity. This combination of melatonin and adriamycin represents a potentially useful regimen for the treatment of human neoplasms because it allows the use of lower doses of adriamycin, thereby avoiding the toxic side effects associated with this drug.

Effects of histamine on cultured interstitial cells of cajal in murine small intestine.

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 Ca2+-free solution or thapsigargin (a Ca2+-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.

Reciprocal positive regulation between TRPV6 and NUMB in PTEN-deficient prostate cancer cells

Calcium acts as a second messenger and plays a crucial role in signaling pathways involved in cell proliferation. Recently, calcium channels related to calcium influx into the cytosol of epithelial cells have attracted attention as a cancer therapy target. Of these calcium channels, TRPV6 is overexpressed in prostate cancer and is considered an important molecule in the process of metastasis. However, its exact role and mechanism is unclear. NUMB, well-known tumor suppressor gene, is a novel interacting partner of TRPV6. We show that NUMB and TRPV6 have a reciprocal positive regulatory relationship in PC-3 cells. We repeated this experiment in two other prostate cancer cell lines, DU145 and LNCaP. Interestingly, there were no significant changes in TRPV6 expression following NUMB knockdown in DU145. We revealed that the presence or absence of PTEN was the cause of NUMB-TRPV6 function. Loss of PTEN caused a positive correlation of TRPV6-NUMB expression. Collectively, we determined that PTEN is a novel interacting partner of TRPV6 and NUMB. These results demonstrated a novel relationship of NUMB-TRPV6 in prostate cancer cells, and show that PTEN is a novel regulator of this complex.

Study of the association of -667 aquaporin-2 (AQP-2) A/G promoter polymorphism with the incidence and clinical course of chronic kidney disease in Korea

Background. Impaired urinary concentration is uniformly present with advanced disease in chronic renal failure. Aquaporin-2 (AQP-2) is known to be expressed in the renal collecting duct cells and participates in urinary concentration in response to vasopressin. Recently, the study of AQP expression in various forms of chronic kidney disease (CKD) demonstrated a reduction in AQP-2 expression associated with a loss of nephrons and the presence of chronic interstitial fibrosis. No information on aquaporin genetic variations in CKD is available to date. The aim of our study was to evaluate the possible impact of aquaporin-2 genotype on the development and clinical course of CKD. Methods. Blood samples from 259 patients with CKD and 106 ethnicity-, age-, and sex-matched healthy controls were collected, and genomic DNA was extracted. AQP-2 -667 genotype was assessed by PCR, followed by restriction fragment length polymorphism analysis. Results. There were no significant differences in genotype and allele frequencies between the patients and healthy controls (p = 0.3936, p = 0.2941, respectively). In all, 79 (30.5%) patients had the AQP-2 -667 wild-type A/A, 123 (47.5%) were heterozygous for the G allele, and 57 (22.0%) patients showed homozygosity. After subclassification of CKD according to underlying disease, no significant differences were observed between those patients and controls (p = 0.72 for diabetic nephropathy, p = 0.52 for hypertensive nephropathy, p = 0.27 for chronic glomerulonephritis, and p = 0.80 for unknown etiology). Genotype and allele frequencies of the AQP-2 gene polymorphism (rs3759126) of hypertensive patients in pre-ESRD did not show a noticeable difference compared with normal blood pressure patients in pre-ESRD (p = 0.50). No correlation was found to exist between the AQP-2 gene polymorphism (rs3759126) and serum electrolyte levels in pre-ESRD patients (p = 0.38 for serum sodium level and p = 0.44 for serum potassium level). Conclusion. Our data indicate that no association exists between the -667 AQP-2 A/G polymorphism and susceptibility to CKD or its clinical course.

PCHM: A bioinformatic resource for high-throughput human mitochondrial proteome searching and comparison

Mitochondrial proteins associated with a wide spectrum of human diseases and currently large amounts of tissue or organ specific human mitochondrial proteome datasets are generated. However, high-throughput comparative proteomic methods have yet to be applied to extract subtle differences among mitochondria from different tissues or muscle types. The aim of this work was to provide an integrated way to identify and compare huge mitochondrial protein or peptide mass spectral data sets acquired from expert mitochondrial proteome or biomarker discovery community. Proteome comparison of human mitochondria (PCHM) is a web-based analysis environment for manual or automatic analysis of individual peptide mass fingerprints alongside a database of proteins and peptides identified in various organs for human mitochondrial proteins. PCHM provides a suite of graphical tools that allow the virtual plot of peptide mass fingerprinting (PMF) spectra and a fully automatic protein function classification based on gene ontology (GO) annotation system. The new virtual PMF plot is very useful to validate fragmented ion loses of any identical proteins and to remove unwanted foreign ion peaks. Fully automatic protein function classifier provides an easier way to compare the subtle differences of compositionally biased mitochondrial protein functions. PCHM also provides a variety of query algorithms aid in browsing, searching, and accessing complete annotations of data relevant to each mitochondrial protein of interest, which link external databases and users. PCHM will be a useful tool for the systematic and functional characterization of the mitochondrial proteins in relation to human diseases or biological research applications. PCHM can be accessed freely via a web interface http://pchm.inje.ac.kr.

Does strong hypertrophic condition induce fast mitochondrial DNA mutation of rabbit heart

Homo- and heteroplasmic mitochondrial DNA (mtDNA) mutations were observed and identified in an isoproterenol-induced rabbit model of cardiac hypertrophy. Genes encoding proteins essential for catalyzing mitochondrial electron transfer and for generating the proton motive force, such as NADH dehydrogenases (ND2, ND3, ND4, and ND6), cytochrome b, and ATPase 8, showed increased susceptibility for mutation. Specifically, five mutations caused amino acid changes and were located in Complex I and Complex V gene clusters. To our knowledge, this is the first demonstration of a relationship between cardiac hypertrophy induced by a strong sympathetic load and rapid mtDNA mutations.

Pharmacokinetic effect of voriconazole on cyclosporine in the treatment of aspergillosis after renal transplantation.

Azole antifungal agents are essential drugs in the treatment of fungal infections in renal transplant patients. As azoles, these antifungal agents are inhibitors of CYP3A4 and P-glycoprotein (P-gp); and thus therapeutic drug monitoring is important. We evaluated a patient with cutaneous and pulmonary aspergillosis who was successfully treated with voriconazole and a low cyclosporine trough level (3.2 - 27.9 ng/ml) for 3 months. During that period, the patient showed good allograft function with the co-administration of voriconazole and cyclosporine. We measured the patients genotype of MDR1, CYP3A4, CYP3A5 and CYP2C19 enzymes in addition to the intracellular concentration of cyclosporine in peripheral blood mononuclear cells (PBMCs). The intracellular concentration of cyclosporine in PBMC is 3.2 times higher with no functionally defected alleles in MDR1, CYP3A4, CYP3A5 or CYP2C19 enzymes when cyclosporine is co-administered with voriconazole ex vivo. Although other confounding factors causing immunological modulation may exist, it is plausible that low serum and high intracellular cyclosporine concentrations, due to the inhibition of P-gp activity by voriconazole, also contribute to an immunosuppressive state.