Evaluation of hydroxyl radical-scavenging property of purpurogallin using high pressure liquid chromatography

Purpurogallin (PPG) has been used as an additive to edible and non-edible oils or fats to retard oxidation. Its antioxidant mechanism is not known. We investigated the ability of PPG to scavenge exogenously generated hydroxyl radicals (·OH) using a sensitive high pressure liquid chromatographic (HPLC) method. ·OH was generated by photolysis of H₂O₂ (1.25–10 moles) with UV light and was trapped with salicylic acid (500 nmoles). Salicylic acid is hydroxylated to produce ·OH adduct products 2,3-and 2,5-dihydroxybenzoic acid (DHBA). H₂O₂ produced concentration-dependent ·OH as estimated by generation of 2,3- and 2,5-DHBA. PPG (100, 200, 300, 400, 500 and 600 nmoles) produced concentration-dependent decreases in ·OH adduct products (approximately 70% inhibition with 600 nmoles of PPG). It did not affect the peak of standard 2,3- and 2,5-DHBA indicating that the decrease in the adduct product generated by H₂O₂ is due to scavenging of ·OH. These results indicate that photolysis of H₂O₂ by UV light produces ·OH and that PPG scavenges ·OH.     

Antioxidant activity of allicin,an active principle in garlic

Garlic has been claimed to be effective against diseases, in the pathophysiology of which oxygen free radicals (OFRs) have been implicated. Effectiveness of garlic could be due to its ability to scavenge OFRs. However, its antioxidant activity is not known. We investigated the ability of allicin (active ingredient of garlic) contained in the commercial preparation Garlicin to scavenge hydroxyl radicals (·OH) using high pressure liquid chromatographic (HPLC) method. ·OH was generated by photolysis of H₂O₂ (1.25–10 moles/ml) with ultraviolet light and was trapped with salicylic acid which is hydroxylated to produce ·OH adduct products 2,3- and 2,5-dihydroxybenzoic acid (DHBA). H₂O₂ produced a concentration-dependent ·OH as estimated by ·OH adduct products 2,3-DHBA and 2,5-DHBA. Allicin equivalent in Garlicin (1.8, 3.6, 7.2, 14.4, 21.6, 28.8 and 36 g) produced concentration-dependent decreases in the formation of 2,3-DHBA and 2,5-DHBA. The inhibition of formation of 2,3-DHBA and 2,5-DHBA with 1.8 g/ml was 32.36% and 43.2% respectively while with 36.0 g/ml the inhibition was approximately 94.0% and 90.0% respectively. The decrease in ·OH adduct products was due to scavenging of ·OH and not by scavenging of formed ·OH adduct products. Allicin prevented the lipid peroxidation of liver homogenate in a concentration-dependent manner. These results suggest that allicin scavenges ·OH and Garlicin has antioxidant activity.     

Plasma membrane redox system during HL-60 induced differentiation

Summary HL-60 cells were induced to differentiate by exposure to TPA or 1,25-dihydroxyvitamin D₃ (calcitriol). Induction with TPA was in parallel with a modulation of transmembrane redox system. After addition of 2 ng/m1 TPA, transient increases in ferricyanide reductase activity, NAD(H) intracellular levels and short-term response of NAD(H) to 0.4 mM ferricyanide were observed. The role of ascorbate on the differentiation induced by calcitriol also was studied. When HL-60 cells were exposed to 10^–8 M calcitriol in the presence of 0.2 mM ascorbate, specific differentiation markers as NBT reduction or surface antigen CD11b increased significantly with respect to values obtained from treatments with calcitriol alone.     

A dual action of taurine on the delayed rectifier K+ current in embryonic chick cardiomyocytes

Summary Effects of taurine on the delayed rectifier K⁺ channel in isolated 10-day-old embryonic chick ventricular cardiomyocytes were examined at different intracellular Ca²⁺ concentrations ([Ca]_i), using whole-cell voltage and current clamp techniques. Experiments were performed at room temperature (22°C). Test pulses were applied between -20 to +90m V from a holding potential of -40mV. When [Ca]_i was pCa 7, addition of 10 and 20 mM taurine to the bath solution reduced the delayed rectifier K⁺ current (I_K) at +90mV by 17.4 ± 2.8% (n = 5, P < 0.01) and 25.5 ± 2.6% (n = 5, P < 0.001), respectively. In contrast, when [Ca]_i was pCa 10, I_K at +90 mV was enhanced by 19.1 ± 3.1% (n = 7, P < 0.01) at 10mM taurine, and by 29.3 ± 2.4% (n = 7, P < 0.001) at 20mM taurine. The voltage of half-maximum activation (V_1/2) was shifted in a hyperpolarizing direction; at pCa 7, the value was +0.2 ± 2.2mV (n = 5) in control and -10.6 ± 1.8mV (n = 5) in 20mM taurine. At pCa 10, the V_1/2 value was +18.5 ± 4.6mV (n = 5) in control and +6.6 ± 5.2mV (n = 5) in taurine (20mM). Taurine decreased the action potential duration (APD) at pCa 10, but at pCa 7 did not affect it. In addition, taurine enhanced the transient outward current in a concentration-dependent manner. These results indicate that taurine modulates the delayed rectifier K⁺ channel, an effect dependent on [Ca]_i and capable of regulating APD.     

Oxygen free radicals in essential hypertension

Membrane abnormalities in essential hypertensives (EH) are well known. The respiratory burst enzyme, NADPH oxidase is located in the cell membrane of the neutrophil (PMNLs) and its activity is important in generation of oxygen derived free radical (OFR). Recently OFR have been implicated in vascular changes in variety of conditions. An attempt was made to delineate the status of OFR and antioxidants in EH. Ten, age and sex-matched, healthy controls (GpI) and 26 untreated EH (Gp IIA mild-8, Gp IIB Moderate-8, Gp IIC Severe-10) were studied. After clinical examination and basic laboratory evaluation of subjects, neutrophils isolated from their blood were studied. Chemiluminescence (CL) emitted by PMNLs after stimulation was measured (counts/min) in a luminometer and was taken as measure of OFR production and thereby of NADPH oxidase activity. The levels of antioxidants, super oxide dismutase (SOD) and reduced glutathione (GSH), were also estimated. Chemiluminescence was increased significantly (p < 0.01) in Gp IIC (243.04 ± 24.9 × 10³ counts per minute) as compared to Gp IIA (2.80 ± 1.87), Gp IIB (34.54 ± 30.24) and Gp I (0.52 ± 0.15) and SOD was reduced significantly (p < 0.05) in all EH (Gp IIA 3.9 ± 0.3 units per mg protein, Gp IIB 3.5 ± 0.3 and Gp IIC 3.12 ± 0.3) as compared to controls (4.1 ± 0.2). Similarly GSH was reduced (p < 0.05) in EH (Gp IIA 11.2 ± 1.7 mg per gm protein, Gp IIB 8.5 ± 1.1 and Gp IIC 6.6 ± 0.3) as compared to Gp I (13.5 ± 2.5). In essential hypertensives a curvilinear positive correlation was obtained between CL and both systolic (r = 0.7077, p < 0.01) and diastolic (r = 0.7965, p < 0.01) blood pressure. A significant inverse correlation (p < 0.05) was obtained between systolic and diastolic blood pressure on one hand and GSH and SOD on the other. Thus PMNLs of EH have increased emission of CL and depletion of antioxidants. The results indicate that in essential hypertension increased membrane NADPH oxidase activity is present.Abbreviations EH Essential Hypertensives - PMNLs Polymorphonuclear leucocytes - OFR Oxygen derived free radicals - Gp Group - NADPH Reduced Nicotinamide Adenine Dinucleotide Phosphate - CL Chemiluminescence - SOD Superoxide Dismutase - GSH Reduced Glutathione - SBP Systolic blood pressure - DBP Diastolic blood pressure     

Novel effects of propranolol. Release of internal Ca(2+) followed by activation of capacitative Ca(2+) entry in Madin Darby canine kidney cells

The effect of propranolol on Ca(2+) signalling in Madin Darby canine kidney (MDCK) cells was investigated by using fura-2 as a Ca(2+) probe. Propranolol increased cytosolic free Ca(2+) levels ([Ca(2+)](i)) in a concentration-dependent manner between 0.1 and 1 mM. The response was partly inhibited by external Ca(2+) removal. In Ca(2+)-free medium pretreatment with 0.2 mM propranolol partly inhibited the [Ca(2+)](i) rise induced by 1 microM thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+) pump; but pretreatment with thapsigargin abolished propranolol-induced Ca(2+) release. Addition of 3 mM Ca(2+) induced a [Ca(2+)](i) rise after pretreatment with 0.2 mM propranolol in Ca(2+)-free medium. Propranolol (0.2 mM) inhibited 25% of thapsigargin-induced capacitative Ca(2+) entry. Suppression of 1,4,5-trisphosphate (IP(3)) formation by 2 microM U73122, a phospholipase C inhibitor, did not alter 0.2 mM propranolol-induced internal Ca(2+) release. Propranolol (1 mM) also increased [Ca(2+)](i) in human neutrophils. Collectively, we have found that 0.2 mM propranolol increased [Ca(2+)](i) in MDCK cells by releasing Ca(2+) from thapsigargin-sensitive Ca(2+) stores in an IP(3)-independent manner, followed by Ca(2+) influx from external space. Independently, propranolol was able to inhibit thapsigargin-induced capacitative Ca(2+) entry.     

Flouride Promotes Viability and Differentiation of Osteoblast-Like Saos-2 Cells Via BMP/Smads Signaling Pathway

The BMP/Smad signaling pathway plays an important role in the viability and differentiation of osteoblast; however, it is not clear whether this pathway is involved in the fluoride-induced osteoblast differentiation. In this study, we investigated the role of BMP/Smad signaling pathway in fluoride-induced osteoblast-like Saos-2 cells differentiation. Cells were exposed to fluoride of different concentrations (0, 0.1, 0.2, 0.4, 0.8, and 1.6 mM), and cell proliferation was determined using WST assays. The expression of osteoblast marker genes such as osteocalcin (BGP) and bone alkaline phosphatase (BALP) were detected by qRT-PCR. We found that fluoride enhanced the proliferation of Saos-2 cells in a dose-dependent manner and 0.2 mM of fluoride resulted in a higher expression of osteoblast marker genes. In addition, immunofluorescence analysis showed that the promotion effects of 0.2 mM of fluoride on Saos-2 cells differentiation were associated with the activation of the BMP/Smad pathway. Expression of phosphorylated Smad1/5(p-Smad1/5) was higher in cells exposed to 0.2 mM of fluoride. Plasmid expression vectors encoding the short hairpin RNA (shRNA) targeting Smad4 gene were used to block the BMP/Smad pathway, which resulted in a significantly reduced expression of BGP and BALP as well as their corresponding mRNA. The mRNA levels after transfection remained low even in the presence of fluoride. The present results reveal that BMP/Smad signaling pathway was altered during the period of osteogenesis, and that the activities of p-Smad1/5 were required for Saos-2 cells viability and differentiation induced by fluoride.     

Reversal of phosphate induced decreases in force by the benzimidazole pyridazinone,UD-CG 212 CL,in myofilaments from human ventricle

UD-CG 212 Cl, (Fig. 1: 4,5-dihydro-6-[2-(4-hydroxyphenyl)-1H-benzimidazole-5-yl]-5-methyl-3(2H)-pyrid azinone), is the primary metabolite of the positive inotropic agent pimobendan (UDCG 115 BS, Acardi®). Our previous studies [16] showed in detergent extracted preparations of canine ventricular muscle that sub-nanomolar concentrations of UD-CG 212 Cl increased submaximal myofilament force, but only when the activation state had been altered by relatively high (5-10 mM) concentrations of inorganic phosphate (Pi) or relatively low (20 µM) concentrations of MgATP. In the present study, we investigated the effects of UD-CG 212 Cl on the pCa-force relationship of detergent extracted bundles of human cardiac fibers before and after addition of P_i. As expected, treatment with 5 mM P_i depressed maximal force at pCa 4.5 by 27.0 ± 0.4% (mean ± SEM). Force generated at the half-maximally activating Ca²⁺ concentration (pCa₅₀) of control fibers (5.98 ± 0.2) was significantly (p < .05) reduced following the addition of 5 mM P_i (pCa₅₀ = 5.69 ± 0.3). The addition of UD-CG 212 Cl over a range of concentrations (10-^-11>-10-^-6 M) had no effect on Ca²⁺-sensitivity under control conditions, but in the presence of 5 mM P_i, there was a 23.1 ± 0.1% increase in the percent maximal force at pCa5.9. Ca²⁺-sensitivity was also significantly increased in the presence of P_i and 10^-8 M UD-CG 212 Cl (pCa₅₀ = 5.74 ± 0.3, p < .05). We conclude that UD-CG 212 Cl potentially increases sub-maximal force of human ventricular myofilaments with an inotropic action depending on a state of myofilament activation associated with ischemic conditions.     

Novel radio-chromic solution dosimeter for radiotherapy treatment planning

Nitro blue tetrazolium (NBT) solution dosimeters were prepared and investigated based on radiation-induced reduction of NBT²⁺. NBT solution dosimeters containing different concentrations of NBT dye from 1 to 5 mM were prepared in a solution of ethanol. The dosimeters were irradiated with 6 MV X-ray beam at doses up to 30 Gy. The dose sensitivity of NBT solution increases strongly with increase of concentrations of NBT dye. The dose response of NBT dosimeters increases remarkably by addition of various concentrations of sodium formate (0.5, 2.5 and 5 mM). It becomes more remarkable with increasing pH value of NBT-sodium formate dosimeters. The sensitivity of the solution increased fairly with increase of irradiation temperature, therefore, the response of the solutions has to be corrected under actual processing conditions. The stability of solution dosimeters after irradiation was very high up to 30 days.     

All-trans retinoic acid induces apoptosis in acute myeloblastic leukaemia cells

The effect of all-trans retinoic acid (ATRA) on leukaemia cell differentiation, proliferation and induction of apoptosis was studied by using autonomously growing blast cells isolated from eight patients with acute myeloblastic leukaemia (AML) either at diagnosis ( n=4) or at relapse (n=4). No morphological or functional differentiation induced by ATRA was observed in any of the cases studied. In cell cultures, inhibition of leukaemia cell growth by ATRA was obvious, especially in the case of clonogenic cells, and it was both time- and concentration-dependent. Induction of apoptosis was more difficult to achieve. The cells retained over 90% viability in suspension when the ATRA exposure at any of the concentrations studied was 48 h or less. When the time of exposure to ATRA was longer than 48 h, the viability of the cells decreased in a concentration-dependent manner. Apoptosis was observed morphologically in each of the AML cases with 10-5 to 10-8 M ATRA, if the incubation time of cells in ATRA was 72 h. The percentage of apoptotic cells increased with increasing ATRA concentrations from 12± 9% of 10-8 M ATRA to 78±12% of 10-5 M ATRA. The DNA electrophoretic method was able to detect apoptosis in all the AML samples exposed to 10-7 and 10-6 ATRA for 48 h and occasionally in cases where lower concentrations and longer exposure time were used. In conclusion, the present study shows that it is possible to induce apoptotic leukaemia cell death in vitro with ATRA in AML, and this effect is dependent on both concentration and exposure time.     

Role of protein kinase C in alpha(1)-adrenergic regulation of a(Na)(i) in guinea pig ventricular myocytes

We investigated the role of protein kinase C (PKC) in alpha(1)-adrenergic regulation of intracellular Na(+) activity (a(Na)(i)) in single guinea pig ventricular myocytes. a(Na)(i) and membrane potentials were measured with the Na(+)-sensitive indicator sodium-binding benzofuran isophthalate and conventional microelectrodes, respectively, at room temperature (24-26 degrees C) while myocytes were stimulated at a rate of 0.25-0.3 Hz. The PKC activator 4beta-phorbol 12-myristate 13-acetate (PMA) decreased a(Na)(i) in a concentration-dependent manner. PMA (100 nM) produced a maximal decrease in a(Na)(i) of 1.5 mM from 6.5 +/- 0.4 to 5.0 +/- 0.4 mM (means +/- SE, n = 12, P < 0.01). The PMA concentration required for a half-maximal decrease in a(Na)(i) was 0.46 +/- 0.13 nM (n = 3, P < 0.01). An inactive phorbol, 4alpha-phorbol 12-myristate 13-acetate, did not decrease a(Na)(i). The decrease caused by PMA could be blocked by the PKC inhibitors staurosporine and bisindolylmaleimide I (GF-109203X). Stimulation of the alpha(1)-adrenoceptor with 50 microM phenylephrine decreased a(Na)(i) from 6.1 +/- 0.3 to 4.6 +/- 0.3 mM (n = 11, P < 0.01). The decrease in a(Na)(i) produced by phenylephrine was blocked by pretreatment with staurosporine, GF-109203X, or PMA. The decrease in a(Na)(i) produced by PMA was not prevented by pretreatment with tetrodotoxin but was blocked by pretreatment with strophanthidin or high extracellular K(+) concentration. The results suggest that alpha(1)-adrenergic receptor activation results in a decrease in a(Na)(i) via PKC-induced stimulation of the Na(+)-K(+) pump in cardiac myocytes.     

Depressed performance and detoxification enzyme activities of Helicoverpa armigera fed with conventional cotton foliage subjected to methyl jasmonate exposure

Methyl jasmonate (MeJA)‐mediated defense in conventional cotton, Gossypium hirsutum L. (Malvaceae), against cotton bollworm, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae), was investigated with respect to the activities of the detoxification enzymes acetylcholinesterase (AChE), carboxylesterase (CarE), and glutathione S‐transferases (GST) in pupae as well as the performance of larvae. The results suggested that exogenous application of MeJA to cotton leaves depressed the activities of AChE, CarE, and GST of cotton bollworm pupae. Both the absolute and protein‐specific AChE activities of pupae were depressed at all three MeJA concentrations applied as compared with a control, and the effects of 0.4 mM MeJA were significantly higher than those of 0.1 and 0.2 mM. A marked reduction in absolute CarE activity was observed at the 0.4 mM MeJA treatment, whereas the protein‐specific activity was increased by 0.2 and 0.4 mM. Absolute GST activity was significantly depressed only by the 0.4 mM MeJA treatment, whereas protein‐specific GST activity was not markedly affected by MeJA. Protein content of pupae was reduced by 0.4 mM MeJA‐induced defense in cotton leaves. The development time of larvae was protracted and pupal weight was reduced by 0.1 and 0.4 mM MeJA‐treated cotton leaves. Larval weight gain was inhibited significantly on 0.2 and 0.4 mM MeJA‐treated cotton leaves. The results suggested that MeJA‐induced plant defense may have adverse effects on H. armigera. In addition to the inhibition of growth and development, induced defense may also impair the insect's ability to detoxify toxic plant secondary metabolites.     

Hydroxyl radical-scavenging property of indomethacin

The ability of indomethacin to scavenge hydroxyl radical (.OH) using high pressure liquid chromatography (HPLC) was investigated. .OH radical was generated by photolysis of H₂O₂ (1.5–10 mmoles/L) with UV light and was trapped with salicyclic acid (500 nmoles). H₂O₂ produced .OH in a concentration-dependent manner as estimated by .OH adduct products 2,3- and 2,5-dihydroxybenzoic acid (DHBA). Indomethacin in increasing concentrations (5–600 moles/L) produced increasing inhibition of generation of 2,3-DHBA (7–67%) and of 2,5-DHBA (7–77%). The results indicate that indomethacin scavenges .OH in a concentration-dependent manner.     

A combination of FSH and dibutyryl cyclic AMP promote growth and acquisition of meiotic competence of oocytes from early porcine antral follicles

Growing porcine oocytes from early antral follicles (1.2-1.5 mm in diameter) do not mature to metaphase II (MII, 4%) under culture conditions which supported maturation (MII, 95%) of fully grown oocytes from large (4-6 mm) antral follicles. We hypothesized that FSH and dbcAMP supported growth and acquisition of meiotic competence. Growing oocytes (113.0 ± 0.4 μm, mean ± SEM) were cultured for 5 d in medium supplemented with 1 mM dbcAMP, 0.01 IU/mL FSH or both; in these media, oocytes reached, 120.5 ± 0.4, 123.5 ± 0.4 and 125.7 ± 0.2 μm, respectively, after 5 d, and then were matured in vitro for 48 h. Oocytes remained enclosed by cumulus cells when cultured with FSH (82%) or both FSH and dbcAMP (80%), but not with dbcAMP alone (0%). Furthermore, oocytes cultured with FSH maintained trans-zonal projections of cumulus cells. Oocytes remained at the GV stage at higher rates when cultured with dbcAMP and FSH (99%), or dbcAMP (97%), than with FSH (64%), or without either (75%). Following in vitro maturation, oocytes reached MII after in vitro growth with dbcAMP (19%), FSH (11%), or both (68%). When oocytes were cultured with both FSH and dbcAMP, activation of Cdc2 and MAP kinases in growing oocytes was similar to fully grown oocytes. In conclusion, growing porcine oocytes grew and acquired meiotic competence in medium supplemented with dbcAMP and FSH; the former maintained oocytes in meiotic arrest, whereas the latter maintained trans-zonal projections of cumulus cells to oocytes during in vitro growth culture.     

Sperm characteristics in plains (Bison bison bison) versus wood (Bison bison athabascae) bison

The objective was to compare sperm characteristics between the two subspecies of North American bison, plains bison (Bison bison bison) and wood bison (Bison bison athabascae). Frozen-thawed ejaculated sperm from age-matched plains (n = 3) and wood (n = 2) bison were evaluated for morphometry, motility, viability, protein profile, and in vitro fertilization characteristics. Sperm morphometry and motility were assessed with computer-based systems, viability was assessed with SYBR-14 and propidium iodide, and fertilizing ability was determined using a heterologous in vitro fertilization system (using bovine oocytes). For plains versus wood bison, there were significant differences for head width (4.76 ± 0.22 vs 4.71 ± 0.19 μm; mean ± SD), head area (35.64 ± 1.91 vs 34.72 ± 2.64 μm²), head perimeter (23.61 ± 0.68 vs 23.31 ± 0.98 μm), midpiece length (14.58 ± 0.4 vs 14.36 ± 0.51 μm), midpiece width (0.81 ± 0.06 vs 0.79 ± 0.07 μm), and tail length (46.61 ± 2.15 vs 45.98 ± 2.08 μm). However, there was no significant difference in head length (overall, 9.04 ± 0.37 μm), progressive motility (41.16 ± 8.39%), or viability (41.58 ± 5.58%). Based on two-dimensional gel electrophoresis, 93 out of 113 protein spots were similar in their expression patterns. Furthermore, we inferred that differences in sperm biometry between these subspecies did not affect in vitro fertilization percentage (overall, 82.62 ± 12.13%). Based on these findings, we concluded that plains bison were an appropriate research model for developing reproductive technologies for wood bison.     

Sodium and calcium channels in the somatic membrane of artificially differentiated neuroblastoma cells

Using an intracellular dialysis technique a study was made on calcium and sodium inward currents at the neuroblastoma somatic cell membrane in suspension and during the course of artificial morphological differentiation produced by raising the pH of the culture medium to 8.0–8.2. The density of sodium currents in the somata of cells cultured in the suspension averaged 7.3±0.8 µA/µF, while this value varied from 37±5.2 to 54.7±3.6 µA/µF at various stages of culture. These values equalled 1.4±0.2 and 1.1±0.2 to 2.8±0.4 µA/µF in the case of calcium currents. Reciprocal changes were produced in the density of sodium and calcium channels by altering the culture medium.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 18, No. 2, pp. 207–214, March–April, 1986.     

Solid-state fermentation of carob pods for ethanol production

The production of ethanol from carob pods by Saccharomyces cerevisiae in solid-state fermentation was investigated. The maximal ethanol concentration (160±3 g/kg dry pods), ethanol productivity (6.7 ± 0.2 g/kg per hour), ethanol yield (40 ± 1.8%), biomass concentration (7.5 ± 0.4 x 10⁸ cells/g carob pulp) and fermentation efficiency (80 ± 2%) were obtained at an inoculum amount of 3%, a particle size of 0.5 mm, a moisture level of 70%, a pH of 4.5 and a temperature of 30°C. Under the same fermentation conditions both sterilized and non-sterilized carob pods pulp gave the same maximum ethanol concentration.     

Enhancing Effect of Manganese on L-DOPA-Induced Apoptosis in PC12 Cells

L-DOPA and manganese both induce oxidative stress-mediated apoptosis in catecholaminergic PC12 cells. In this study, exposure of PC12 cells to 0.2 mM MnCl2 or 10-20 microM L-DOPA neither affected cell viability, determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, nor induced apoptosis, tested by flow cytometry, fluorescence microscopy, and the TUNEL technique. L-DOPA (50 microM) induced decreases in both cell viability and apoptosis. When 0.2 mM MnCl2 was associated with 10, 20, or 50 microM L-DOPA, a concentration-dependent decrease in cell viability was observed. Apoptotic cell death also occurred. In addition, manganese inhibited L-DOPA effects on dopamine (DA) metabolism (i.e., increases in DA and its acidic metabolite levels in both cell lysate and incubation medium). The antioxidant N-acetyl-L-cysteine significantly inhibited decreases in cell viability, apoptosis, and changes in DA metabolism induced by the manganese association with L-DOPA. An increase in autoxidation of L-DOPA and of newly formed DA is suggested as a mechanism of manganese action. These data show that agents that induce oxidative stress-mediated apoptosis in catecholaminergic cells may act synergistically.     

The role of calcium in the regulation of glucose uptake in isolated working rat heart

Catecholamines or ischemia may increase myocardial glucose uptake by an increase in intracellular calcium. We tested the hypothesis that increasing or decreasing extracellular calcium supply would change glucose uptake. Hearts were perfused for 60 min at a physiological workload with Krebs-Henseleit buffer containing glucose (5 mM) and oleate (0.4 mM; bound to 1% BSA). Calcium concentration was 2.5 mM. In group A (control; n = 12), insulin (1 mU/ml) was added at 30 min. In Group B (n = 7), the calcium concentration was increased to 5.0 and 7.5 mM at 20 min and 40 min, respectively. In Group C (n = 7), verapamil was added at 20 min (0.25 M) and 40 min (1.0 M) to decrease calcium influx. In group D (n = 7), EDTA was added at 20 min (0.5 mM) and at 40 min (1.5 mM) to decrease the free extracellular calcium. Glucose uptake was measured by ³H₂O production from [2-³H]glucose and cardiac work was measured simultaneously. Cardiac power in group B was 8.24 ± 0.60 mW at 2.5 mM calcium, 9.45 ± 0.50 mW at 5 mM calcium and 7.99 ± 0.99 mW at 7.5 mM calcium (n.s.). The addition of verapamil decreased contractile function in a dose-dependent manner (8.50 ± 0.74 vs. 3.11 ± 0.84 vs. 1.48 ± 0.39 mW, p < 0.01) suggesting that verapamil decreased cytosolic calcium concentration. A similar dose-dependent reduction in contractile performance was observed in the EDTA group (8.44 ± 0.81 vs. 7.42 ± 0.96 vs. 4.03 ± 1.32 mW, p < 0.01). Glucose uptake was 1.35 ± 0.11 mol/min/g dry weight under control conditions. Glucose uptake increased threefold with the addition of insulin. Increasing extracellular [Ca²⁺] did not affect glucose uptake. Decreasing Ca²⁺ availability showed a trend towards a decrease in glucose uptake (n.s.), which was minor compared to the decrease in contractile function. We conclude that extracellular calcium does not regulate glucose uptake in the isolated working rat heart in the presence of glucose and fatty acids as substrates. The trend of decreased glucose uptake when calcium supply was limited may be due to dramatically reduced energy demand and not directly due to changes in calcium.     

Characterization of the inhibitory effects of erythromycin and clarithromycin on the HERG potassium channel

Both erythromycin and clarithromycin have been reported to cause QT prolongation and the cardiac arrhythmia torsade de pointes in humans, however direct evidence documenting that these drugs produce this effect by blocking human cardiac ion channels is lacking. The goal of this study was to test the hypothesis that these macrolide antibiotics significantly block the delayed rectifier current (I_Kr) encoded by HERG (the human ether-a-go-go-related gene) at drug concentrations, temperature and ionic conditions mimicking those occurring in human subjects. Potassium currents in HEK 293 cells stably transfected with HERG were recorded using a whole cell voltage clamp method. Exposure of cells to erythromycin reduced the HERG encoded potassium current in a concentration dependent manner with an IC₅₀ of 38.9 ± 1.2 M and Hill Slope factor of 0.4 ± 0.1. Clarithromycin produced a similar concentration-dependent block with an IC₅₀ of 45.7 ± 1.1 M and Hill Slope factor of 1.0 ± 0.1. Erythromycin (25–250 M) and clarithromycin (5 or 25 M) also produced a significant decrease in the integral of the current evoked by an action potential shaped voltage clamp protocol. The results of this study document that both erythromycin and clarithromycin significantly inhibit the HERG potassium current at clinically relevant concentrations.