Dopamine modulates mitochondrial function in viable SH-SY5Y cells possibly via its interaction with complex I: relevance to dopamine pathology in schizophrenia

Deleterious effects of dopamine (DA) involving mitochondrial dysfunction have an important role in DA-associated neuronal disorders, including schizophrenia and Parkinson's disease. DA detrimental effects have been attributed to its ability to be auto-oxidized to toxic reactive oxygen species. Since, unlike Parkinson's disease, schizophrenia does not involve neurodegenerative processes, we suggest a novel mechanism by which DA impairs mitochondrial function without affecting cell viability. DA significantly dissipated mitochondrial membrane potential (delta psi m) in SH-SY5Y cells. Bypassing complex I prevented the DA-induced depolarization. Moreover, DA inhibited complex I but not complex II activity in disrupted mitochondria, suggesting complex I participation in DA-induced mitochondrial dysfunction. We further demonstrated that intact mitochondria can accumulate DA in a saturated manner, with an apparent Km=122.1+/-28.6 nM and Vmax=1.41+/-0.15 pmol/mg protein/min, thereby enabling the interaction between DA and complex I. DA accumulation was an energy and Na+-dependent process. The pharmacological profile of mitochondrial DA uptake differed from that of other characterized DA transporters. Finally, relevance to schizophrenia is demonstrated by an abnormal interaction between DA and complex I in schizophrenic patients. These results suggest a non-lethal interaction between DA and mitochondria possibly via complex I, which can better explain DA-related pathological processes observed in non-degenerative disorders, such as schizophrenia.     

Energetic performance is improved by specific activation of K fluxes through KCa channels in heart mitochondria

Mitochondrial volume regulation depends on K⁺ movement across the inner membrane and a mitochondrial Ca²⁺-dependent K⁺ channel (mitoK_Ca) reportedly contributes to mitochondrial K⁺ uniporter activity. Here we utilize a novel K_Ca channel activator, NS11021, to examine the role of mitoK_Ca in regulating mitochondrial function by measuring K⁺ flux, membrane potential (ΔΨ_m), light scattering, and respiration in guinea pig heart mitochondria. K⁺ uptake and the influence of anions were assessed in mitochondria loaded with the K⁺ sensor PBFI by adding either the chloride (KCl), acetate (KAc), or phosphate (KH₂PO₄) salts of K⁺ to energized mitochondria in a sucrose-based medium. K⁺ fluxes saturated at ∼ 10 mM for each salt, attaining maximal rates of 172 ± 17, 54 ± 2.4, and 33 ± 3.8 nmol K⁺/min/mg in KCl, KAc, or KH₂PO₄, respectively. NS11021 (50 nM) increased the maximal K⁺ uptake rate by 2.5-fold in the presence of KH₂PO₄ or KAc and increased mitochondrial volume, with little effect on ΔΨ_m. In KCl, NS11021 increased K⁺ uptake by only 30% and did not increase volume. The effects of NS11021 on K⁺ uptake were inhibited by the K_Ca toxins charybdotoxin (200 nM) or paxilline (1 μM). Fifty nanomolar of NS11021 increased the mitochondrial respiratory control ratio (RCR) in KH₂PO₄, but not in KCl; however, above 1 μM, NS11021 decreased RCR and depolarized ΔΨ_m. A control compound lacking K_Ca activator properties did not increase K⁺ uptake or volume but had similar nonspecific (toxin-insensitive) effects at high concentrations. The results indicate that activating K⁺ flux through mitoK_Ca mediates a beneficial effect on energetics that depends on mitochondrial swelling with maintained ΔΨ_m.     

Dopamine enhances mtNOS activity: Implications in mitochondrial function

Dopamine and nitric oxide systems can interact in different processes in the central nervous system. Dopamine and oxidation products have been related to mitochondrial dysfunction. In the present study, intact mitochondria and submitochondrial membranes were incubated with different DA concentrations for 5 min. Dopamine (1 mM) increased nitric oxide production in submitochondrial membranes and this effect was partially prevented in the presence of both DA and NOS inhibitor N_ω-nitro-l-arginine (l-NNA). A 46% decrease in state 3 oxygen uptake (active respiration state) was found after 15 mM dopamine incubation. When mitochondria were incubated with 15 mM dopamine in the presence of l-NNA, state 3 respiratory rate was decreased by only 17% showing the involvement of NO. As shown for O₂ consumption, the inhibition of cytochrome oxidase by 1 mM DA was mediated by NO. Hydrogen peroxide production significantly increased after 15 mM DA incubation, being mainly due to its metabolism by MAO. Also, DA-induced depolarization was prevented by the addition of l-NNA showing the involvement of nitric oxide in this process too. This work provides evidence that in the studied conditions, dopamine modifies mitochondrial function by a nitric oxide-dependent pathway.     

Regulation of respiration controlled by mitochondrial creatine kinase in permeabilized cardiac cells in situ: Importance of system level properties

The main focus of this investigation is steady state kinetics of regulation of mitochondrial respiration in permeabilized cardiomyocytes in situ. Complete kinetic analysis of the regulation of respiration by mitochondrial creatine kinase was performed in the presence of pyruvate kinase and phosphoenolpyruvate to simulate interaction of mitochondria with glycolytic enzymes. Such a system analysis revealed striking differences in kinetic behaviour of the MtCK-activated mitochondrial respiration in situ and in vitro. Apparent dissociation constants of MgATP from its binary and ternary complexes with MtCK, K_ia and K_a (1.94 ± 0.86 mM and 2.04 ± 0.14 mM, correspondingly) were increased by several orders of magnitude in situ in comparison with same constants in vitro (0.44 ± 0.08 mM and 0.016 ± 0.01 mM, respectively). Apparent dissociation constants of creatine, K_ib and K_b (2.12 ± 0.21 mM 2.17 ± 0.40 Mm, correspondingly) were significantly decreased in situ in comparison with in vitro mitochondria (28 ± 7 mM and 5 ± 1.2 mM, respectively). Dissociation constant for phosphocreatine was not changed. These data may indicate selective restriction of metabolites' diffusion at the level of mitochondrial outer membrane. It is concluded that mechanisms of the regulation of respiration and energy fluxes in vivo are system level properties which depend on intracellular interactions of mitochondria with cytoskeleton, intracellular MgATPases and cytoplasmic glycolytic system.     

Complex I syndrome in myocardial stunning and the effect of adenosine

Isolated rabbit hearts were exposed to ischemia (I; 15 min) and reperfusion (R; 5-30 min) in a model of stunned myocardium. I/R decreased left-ventricle O₂ consumption (46%) and malate-glutamate-supported mitochondrial state 3 respiration (32%). Activity of complex I was 28% lower after I/R. The pattern observed for the decline in complex I activity was also observed for the reduction in mitochondrial nitric oxide synthase (mtNOS) biochemical (28%) and functional (50%) activities, in accordance with the reported physical and functional interactions between complex I and mtNOS. Malate-glutamate-supported state 4 H₂O₂ production was increased by 78% after I/R. Rabbit heart Mn-SOD concentration in the mitochondrial matrix (7.4 ± 0.7 μM) was not modified by I/R. Mitochondrial phospholipid oxidation products were increased by 42%, whereas protein oxidation was only slightly increased. I/R produced a marked (70%) enhancement in tyrosine nitration of the mitochondrial proteins. Adenosine attenuated postischemic ventricular dysfunction and protected the heart from the declines in O₂ consumption and in complex I and mtNOS activities and from the enhancement of mitochondrial phospholipid oxidation. Rabbit myocardial stunning is associated with a condition of dysfunctional mitochondria named “complex I syndrome.” The beneficial effect of adenosine could be attributed to a better regulation of intracellular cardiomyocyte Ca²⁺ concentration.     

Stimulation of oxidative phosphorylation by calcium in cardiac mitochondria is not influenced by cAMP and PKA activity

Cardiac oxidative ATP generation is finely tuned to match several-fold increases in energy demand. Calcium has been proposed to play a role in the activation of ATP production via PKA phosphorylation in response to intramitochondrial cAMP generation. We evaluated the effect of cAMP, its membrane permeable analogs (dibutyryl-cAMP, 8-bromo-cAMP), and the PKA inhibitor H89 on respiration of isolated pig heart mitochondria. cAMP analogs did not stimulate State 3 respiration of Ca^2 +-depleted mitochondria (82.2 ± 3.6% of control), in contrast to the 2-fold activation induced by 0.95 μM free Ca^2 +, which was unaffected by H89. Using fluorescence and integrating sphere spectroscopy, we determined that Ca^2 + increased the reduction of NADH (8%), and of cytochromes b_H (3%), c₁ (3%), c (4%), and a (2%), together with a doubling of conductances for Complex I + III and Complex IV. None of these changes were induced by cAMP analogs nor abolished by H89. In Ca^2 +-undepleted mitochondria, we observed only slight changes in State 3 respiration rates upon addition of 50 μM cAMP (85 ± 9.9%), dibutyryl-cAMP (80.1 ± 5.2%), 8-bromo-cAMP (88.6 ± 3.3%), or 1 μM H89 (89.7 ± 19.9%) with respect to controls. Similar results were obtained when measuring respiration in heart homogenates. Addition of exogenous PKA with dibutyryl-cAMP or the constitutively active catalytic subunit of PKA to isolated mitochondria decreased State 3 respiration by only 5–15%. These functional studies suggest that alterations in mitochondrial cAMP and PKA activity do not contribute significantly to the acute Ca^2 + stimulation of oxidative phosphorylation.     

The development of mitochondrial membrane affinity chromatography columns for the study of mitochondrial transmembrane proteins

Mitochondrial membrane fragments from U-87 MG (U87MG) and HEK-293 cells were successfully immobilized onto immobilized artificial membrane (IAM) chromatographic support and surface of activated open tubular (OT) silica capillary, resulting in mitochondrial membrane affinity chromatography (MMAC) columns. Translocator protein (TSPO), located in mitochondrial outer membrane as well as sulfonylurea and mitochondrial permeability transition pore (mPTP) receptors, localized to the inner membrane, were characterized. Frontal displacement experiments with multiple concentrations of dipyridamole (DIPY) and PK-11195 were run on MMAC (U87MG) column, and the binding affinities (K_d) determined were 1.08 ± 0.49 and 0.0086 ± 0.0006 μM, respectively, consistent with previously reported values. Furthermore, binding affinities (K_i) for DIPY binding site were determined for TSPO ligands, PK-11195, mesoporphyrin IX, protoporphyrin IX, and rotenone. In addition, the relative ranking of these TSPO ligands based on single displacement studies using DIPY as marker on MMAC (U87MG) was consistent with the obtained K_i values. The immobilization of mitochondrial membrane fragments was also confirmed by confocal microscopy.     

Amyloid beta impairs mitochondrial anterograde transport and degenerates synapses in Alzheimer's disease neurons

Loss of synapses and synaptic damage are the best correlates of cognitive decline identified in patients with Alzheimer's disease (AD), and mitochondrial oxidative damage and synaptic pathology have been identified as early events in the progression of AD. The progressive accumulation of amyloid beta (Aβ) in synapses and synaptic mitochondria are hypothesized to cause synaptic degeneration and cognitive decline in patients with AD. However, the precise mechanistic link between Aβ and mitochondria is not well understood. The purpose of this study was to better understand the effects of Aβ on mitochondrial axonal transport and synaptic alterations in AD. Using mouse hippocampal neurons and Aβ_25-35 peptide, we studied axonal transport of mitochondria, including mitochondrial motility, mitochondrial length and size, mitochondrial index per neurite, and synaptic alterations of the hippocampal neurons. In the PBS-treated neurons, 36.4 ± 4.7% of the observed mitochondria were motile, with 21.0 ± 1.3% moving anterograde and 15.4 ± 3.4% moving retrograde and the average speed of movement was 12.1 ± 1.8 μm/min. In contrast, in the Aβ-treated neurons, the number of motile mitochondria were significantly less, at 20.4 ± 2.6% (P < 0.032), as were those moving anterograde (10.1 ± 2.6%, P < 0.016) relative to PBS-treated neurons, suggesting that the Aβ_25-35 peptide impairs axonal transport of mitochondria in AD neurons. In the Aβ-treated neurons, the average speed of motile mitochondria was also less, at 10.9 ± 1.9 μm/min, and mitochondrial length was significantly decreased. Further, synaptic immunoreactivity was also significantly less in the Aβ-treated neurons relative to the PBS-treated neurons, indicating that Aβ affects synaptic viability. These findings suggest that, in neurons affected by AD, Aβ is toxic, impairs mitochondrial movements, reduces mitochondrial length, and causes synaptic degeneration.     

Blocking c-Jun N-terminal Kinase (JNK) Translocation to the Mitochondria Prevents 6-Hydroxydopamine-induced Toxicity in Vitro and in Vivo

Because oxidative stress and mitochondrial dysfunction are well known contributors to Parkinson disease (PD), we set out to investigate the role mitochondrial JNK plays in the etiology of 6-hydroxydopamine-induced (6-OHDA) oxidative stress, mitochondrial dysfunction, and neurotoxicity in SHSY5Y cells and neuroprotection and motor behavioral protection in vivo. To do this, we utilized a cell-permeable peptide of the outer mitochondrial membrane protein, Sab (SH3BP5), as an inhibitor of JNK mitochondrial translocation. In vitro studies showed that 6-OHDA induced JNK translocation to the mitochondria and that inhibition of mitochondrial JNK signaling by Tat-Sab_KIM1 protected against 6-OHDA-induced oxidative stress, mitochondrial dysfunction, and neurotoxicity. Administration of Tat-Sab_KIM1 via an intracerebral injection into the mid-forebrain bundle increased the number of tyrosine hydroxylase immunoreactive neurons in the substantia nigra pars compacta by 2-fold (p < 0.05) in animals lesioned with 6-OHDA, compared with animals treated only with 6-OHDA into the nigrostriatal pathway. In addition, Tat-Sab_KIM1 decreased the d-amphetamine-induced unilateral rotations associated with the lesion by 30% (p < 0.05). Steady-state brain levels of Tat-Sab_KIM1 at day 7 were 750 nm, which was ∼3.4-fold higher than the IC₅₀ for this peptide versus Sab protein. Collectively, these data suggest that 6-OHDA induced JNK translocation to the mitochondria and that blocking this translocation reduced oxidative stress, mitochondrial dysfunction, and neurotoxicity both in vitro and in vivo. Moreover, the data suggest that inhibitors that block association of JNKs with the mitochondria may be useful neuroprotective agents for the treatment of Parkinson disease.     

Impaired cardiac mitochondrial function and contractile reserve following an acute exposure to environmental particulate matter

Background

It has been suggested that mitochondrial function plays a central role in cardiovascular diseases associated with particulate matter inhalation. The aim of this study was to evaluate this hypothesis, with focus on cardiac O₂ and energetic metabolism, and its impact over cardiac contractility.

Methods

Swiss mice were intranasally instilled with either residual oil fly ash (ROFA) (1.0 mg/kg body weight) or saline solution. After 1, 3 or 5 h of exposure, O₂ consumption was evaluated in heart tissue samples. Mitochondrial respiration, respiratory chain complexes activity, membrane potential and ATP content and production rate were assessed in isolated mitochondria. Cardiac contractile reserve was evaluated according to the Langendorff technique.

Results

Three hours after ROFA exposure, tissue O₂ consumption was significantly decreased by 35% (from 1180 ± 70 to 760 ± 60 ng-at O/min g tissue), as well as mitochondrial rest (state 4) and active (state 3) respiration, by 30 and 24%, respectively (control state 4: 88 ± 5 ng-at O/min mg protein; state 3: 240 ± 20 ng-at O/min mg protein). These findings were associated with decreased complex II activity, mitochondrial depolarization and deficient ATP production. Even though basal contractility was not modified (control: 75 ± 5 mm Hg), isolated perfused hearts failed to properly respond to isoproterenol in ROFA-exposed mice. Tissue O₂ consumption rates positively correlated with cardiac contractile state in controls (r² = 0.8271), but not in treated mice (r² = 0.1396).

General Significance

The present results show an impaired mitochondrial function associated with deficient cardiac contractility, which could represent an early cardiovascular alteration after the exposure to environmental particulate matter.     

Assessment of newly synthesized mitochondrial DNA using BrdU labeling in primary neurons from Alzheimer's disease mice: Implications for impaired mitochondrial biogenesis and synaptic damage

The purpose of our study was to assess mitochondrial biogenesis and distribution in murine primary neurons. Using 5-bromo-2-deoxyuridine (BrdU) incorporation and primary neurons, we studied the mitochondrial biogenesis and mitochondrial distribution in hippocampal neurons from amyloid beta precursor protein (AβPP) transgenic mice and wild-type (WT) neurons treated with oxidative stressors, rotenone and H₂O₂. We found that after 20 h of labeling, BrdU incorporation was specific to porin-positive mitochondria. The proportion of mitochondrial area labeled with BrdU was 40.3 ± 6.3% at 20 h. The number of mitochondria with newly synthesized DNA was higher in AβPP neuronal cell bodies than in the cell bodies of WT neurons (AβPP, 45.23 ± 2.67 BrdU-positive/cell body; WT, 32.92 ± 2.49 BrdU-positive/cell body; p = 0.005). In neurites, the number of BrdU-positive mitochondria decreased in AβPP cultures compared to WT neurons (AβPP, 0.105 ± 0.008 BrdU-positive/μm neurite; WT, 0.220 ± 0.036 BrdU-positive/μm neurite; p = 0.010). Further, BrdU in the cell body increased when neurons were treated with low doses of H₂O₂ (49.6 ± 2.7 BrdU-positive/cell body, p = 0.0002 compared to untreated cells), while the neurites showed decreased BrdU staining (0.122 ± 0.010 BrdU-positive/μm neurite, p = 0.005 compared to the untreated). BrdU labeling was increased in the cell body under rotenone treatment. Additionally, under rotenone treatment, the content of BrdU labeling decreased in neurites. These findings suggest that Aβ and mitochondrial toxins enhance mitochondrial fragmentation in the cell body, and may cause impaired axonal transport of mitochondria leading to synaptic degeneration.     

Unfolding thermodynamics of the Delta-domain in the prohead I subunit of phage HK97: determination by factor analysis of Raman spectra

An early step in the morphogenesis of the double-stranded DNA (dsDNA) bacteriophage HK97 is the assembly of a precursor shell (prohead I) from 420 copies of a 384-residue subunit (gp5). Although formation of prohead I requires direct participation of gp5 residues 2-103 (Δ-domain), this domain is eliminated by viral protease prior to subsequent shell maturation and DNA packaging. The prohead I Δ-domain is thought to resemble a phage scaffolding protein, by virtue of its highly α-helical secondary structure and a tertiary fold that projects inward from the interior surface of the shell. Here, we employ factor analysis of temperature-dependent Raman spectra to characterize the thermostability of the Δ-domain secondary structure and to quantify the thermodynamic parameters of Δ-domain unfolding. The results are compared for the Δ-domain within the prohead I architecture (in situ) and for a recombinantly expressed 111-residue peptide (in vitro). We find that the α-helicity (∼ 70%), median melting temperature (T_m = 58 °C), enthalpy (ΔH_m = 50 ± 5 kcal mol^− 1), entropy (ΔS_m = 150 ± 10 cal mol^− 1 K^− 1), and average cooperative melting unit (〈n_c〉 ∼ 3.5) of the in situ Δ-domain are altered in vitro, indicating specific interdomain interactions within prohead I. Thus, the in vitro Δ-domain, despite an enhanced helical secondary structure (∼ 90% α-helix), exhibits diminished thermostability (T_m = 40 °C; ΔH_m = 27 ± 2 kcal mol^− 1; ΔS_m = 86 ± 6 cal mol^− 1 K^− 1) and noncooperative unfolding (〈n_c〉 ∼ 1) vis-à-vis the in situ Δ-domain. Temperature-dependent Raman markers of subunit side chains, particularly those of Phe and Trp residues, also confirm different local interactions for the in situ and in vitro Δ-domains. The present results clarify the key role of the gp5 Δ-domain in prohead I architecture by providing direct evidence of domain structure stabilization and interdomain interactions within the assembled shell.     

Effect of ethanol on spectral binding, inhibition, and activity of CYP3A4 with an antiretroviral drug nelfinavir

Cytochrome P450 3A4 (CYP3A4) is the most abundant CYP enzyme in the liver and metabolizes approximately 50% of the drugs, including antiretrovirals. Although CYP3A4 induction by ethanol and impact of CYP3A4 on drug metabolism and toxicity is known, CYP3A4-ethanol physical interaction and its impact on drug binding, inhibition, or metabolism is not known. Therefore, we studied the effect of ethanol on binding and inhibition of CYP3A4 with a representative protease inhibitor, nelfinavir, followed by the effect of alcohol on nelfinavir metabolism. Our initial results showed that methanol, ethanol, isopropanol, isobutanol, and isoamyl alcohol bind in the active site of CYP3A4 and exhibit type I spectra. Among these alcohol compounds, ethanol showed the lowest K_D (5.9 ± 0.34 mM), suggesting its strong binding affinity with CYP3A4. Ethanol (20 mM) decreased the K_D of nelfinavir by >5-fold (0.041 ± 0.007 vs. 0.227 ± 0.038 μM). Similarly, 20 mM ethanol decreased the IC₅₀ of nelfinavir by >3-fold (2.6 ± 0.5 vs. 8.3 ± 3.1 μM). These results suggest that ethanol facilitates binding of nelfinavir with CYP3A4. Furthermore, we performed nelfinavir metabolism using LCMS. Although ethanol did not alter k_cat, it decreased the K_m of nelfinavir, suggesting a decrease in catalytic efficiency (k_cat/K_m). This is an important finding because alcoholism is prevalent in HIV-1-infected persons and alcohol is shown to decrease the response to antiretroviral therapy.     

Decolorization of malachite green by cytochrome c in the mitochondria of the fungus Cunninghamella elegans

We studied the decolorization of malachite green (MG) by the fungus Cunninghamella elegans. The mitochondrial activity for MG reduction was increased with a simultaneous increase of a 9-kDa protein, called CeCyt. The presence of cytochrome c in CeCyt protein was determined by optical absorbance spectroscopy with an extinction coefficient (E_550-535) of 19.7 ± 6.3 mM⁻¹ cm⁻¹ and reduction potential of + 261 mV. When purified CeCyt was added into the mitochondria, the specific activity of CeCyt reached 440 ± 122 μmol min⁻¹ mg⁻¹ protein. The inhibition of MG reduction by stigmatellin, but not by antimycin A, indicated a possible linkage of CeCyt activity to the Qo site of the bc1 complex. The RT-PCR results showed tight regulation of the cecyt gene expression by reactive oxygen species. We suggest that CeCyt acts as a protein reductant for MG under oxidative stress in a stationary or secondary growth stage of this fungus.     

Coupling of Mitochondrial Fatty Acid Uptake to Oxidative Flux in the Intact Heart

The coordination of long chain fatty acid (LCFA) transport across the mitochondrial membrane (V_PAL) with subsequent oxidation rate through β-oxidation and the tricarboxylic acid (TCA) cycle (V_tca) has been difficult to characterize in the intact heart. Kinetic analysis of dynamic ¹³C-NMR distinguished these flux rates in isolated rabbit hearts. Hearts were perfused in a 9.4 T magnet with either 0.5 mM [2,4,6,8,10,12,14,16-¹³C₈] palmitate (n = 4), or 0.5 mM ¹³C-labeled palmitate plus 0.08 mM unlabeled butyrate (n = 4). Butyrate is a short chain fatty acid (SCFA) that bypasses the LCFA transporters of mitochondria. In hearts oxidizing palmitate alone, the ratio of V_TCA to V_PAL was 8:1. This is consistent with one molecule of palmitate yielding eight molecules of acetyl-CoA for the subsequent oxidation through the TCA cycle. Addition of butyrate elevated this ratio; V_TCA/V_PAL = 12:1 due to an SCFA-induced increase in V_TCA of 43% (p < 0.05). However, SCFA oxidation did not significantly reduce palmitate transport into the mitochondria: V_PAL = 1.0 ± 0.2 μmol/min/g dw with palmitate alone versus 0.9 ± 0.1 with palmitate plus butyrate. Thus, the products of β-oxidation are preferentially channeled to the TCA cycle, away from mitochondrial efflux via carnitine acetyltransferase.     

Resveratrol Induces a Mitochondrial Complex I-dependent Increase in NADH Oxidation Responsible for Sirtuin Activation in Liver Cells

Resveratrol (RSV) has been shown to be involved in the regulation of energetic metabolism, generating increasing interest in therapeutic use. SIRT1 has been described as the main target of RSV. However, recent reports have challenged the hypothesis of its direct activation by RSV, and the signaling pathways remain elusive. Here, the effects of RSV on mitochondrial metabolism are detailed both in vivo and in vitro using murine and cellular models and isolated enzymes. We demonstrate that low RSV doses (1–5 μm) directly stimulate NADH dehydrogenases and, more specifically, mitochondrial complex I activity (EC₅₀ ∼1 μm). In HepG2 cells, this complex I activation increases the mitochondrial NAD⁺/NADH ratio. This higher NAD⁺ level initiates a SIRT3-dependent increase in the mitochondrial substrate supply pathways (i.e. the tricarboxylic acid cycle and fatty acid oxidation). This effect is also seen in liver mitochondria of RSV-fed animals (50 mg/kg/day). We conclude that the increase in NADH oxidation by complex I is a crucial event for SIRT3 activation by RSV. Our results open up new perspectives in the understanding of the RSV signaling pathway and highlight the critical importance of RSV doses used for future clinical trials.     

N-acetylcysteine, coenzyme Q10 and superoxide dismutase mimetic prevent mitochondrial cell dysfunction and cell death induced by d-galactosamine in primary culture of human hepatocytes

d-Galactosamine (d-GalN) induces reactive oxygen species (ROS) generation and cell death in cultured hepatocytes. The aim of the study was to evaluate the cytoprotective properties of N-acetylcysteine (NAC), coenzyme Q₁₀ (Q₁₀) and the superoxide dismutase (SOD) mimetic against the mitochondrial dysfunction and cell death in d-GalN-treated hepatocytes. Hepatocytes were isolated from liver resections. NAC (0.5 mM), Q₁₀ (30 μM) or MnTBAP (Mn(III)tetrakis(4-benzoic acid) porphyrin chloride (1 mg/mL) were co-administered with d-GalN (40 mM) in hepatocytes. Cell death, oxidative stress, mitochondrial transmembrane potential (MTP), ATP, mitochondrial oxidized/reduced glutathione (GSH) and Q₁₀ ratios, electronic transport chain (ETC) activity, and nuclear- and mitochondria-encoded expression of complex I subunits were determined in hepatocytes. d-GalN induced a transient increase of mitochondrial hyperpolarization and oxidative stress, followed by an increase of oxidized/reduced GSH and Q₁₀ ratios, mitochondrial dysfunction and cell death in hepatocytes. The cytoprotective properties of NAC supplementation were related to a reduction of ROS generation and oxidized/reduced GSH and Q₁₀ ratios, and a recovery of mitochondrial complexes I + III and II + III activities and cellular ATP content. The co-administration of Q₁₀ or MnTBAP recovered oxidized/reduced GSH ratio, and reduced ROS generation, ETC dysfunction and cell death induced by d-GalN. The cytoprotective properties of studied antioxidants were related to an increase of the protein expression of nuclear- and mitochondrial-encoded subunits of complex I. In conclusion, the co-administration of NAC, Q₁₀ and MnTBAP enhanced the expression of complex I subunits, and reduced ROS production, oxidized/reduced GSH ratio, mitochondrial dysfunction and cell death induced by d-GalN in cultured hepatocytes.     

Comparative pharmacokinetics of baicalin and wogonoside by liquid chromatography–mass spectrometry after oral administration of Xiaochaihu Tang and Radix scutellariae extract to rats

The aim of this study was to compare the pharmacokinetics of baicalin and wogonoside in rats following oral administration of Xiaochaihu Tang (Minor Radix Bupleuri Decoction) and Radix scutellariae extract. Thus, a specific LC–MS method was developed and validated for the determination of these flavonoids in the plasma of rats after oral administration Xiaochaihu Tang and Radix scutellariae extract. Chromatographic separation was performed on a Zorbax SB C₁₈ column (150 mm × 4.6 mm, i.d.: 5 μm) with 0.1% formic acid in water and acetonitrile by linear gradient elution. Baicalin, wogonoside and carbamazepine (internal standard, I.S.) were detected in select-ion-monitoring (SIM) mode with a positive electrospray ionization (ESI) interface. The following ions: m/z 447 for baicalin, m/z 461 for wogonoside and m/z 237 for the I.S. were used for quantitative determination. The calibration curves were linear over the concentration ranges from 0.1231 to 6.156 μg mL⁻¹ for baicalin and 0.08832 to 4.416 μg mL⁻¹ for wogonoside. The lower limit of detection (LLOD) based on a signal-to-noise ratio of 2 was 0.06155 μg mL⁻¹ for baicalin and 0.04416 μg mL⁻¹ for wogonoside. Intra-day and inter-day precisions (RSD%) were within 10% and accuracy (RE%) ranged from −6.4 to 4.4%. The extraction recovery at three QC concentrations ranged from 74.7 to 86.0% for baicalin and from 71.3 to 83.7% for wogonoside. The plasma concentrations of baicalin and wogonoside in rats at designated time periods after oral administration were successfully determined using the validated method, pharmacokinetic parameters were estimated by a non-compartment model. Following oral administration of Xiaochaihu Tang and Radix scutellariae extract, the t_1/2 of baicalin was 3.60 ± 0.90 and 5.64 ± 1.67, the C_max1 was 1.64 ± 0.99 and 5.66 ± 2.02, the t_max1 was 0.13 ± 0.05 and 0.20 ± 0.07, the C_max2 was 2.43 ± 0.46 and 3.18 ± 1.66, and the t_max2 were 6.40 ± 1.67 and 5.66 ± 2.02, respectively. Following oral administration of Xiaochaihu Tang and Radix scutellariae extract, the t_1/2 of wogonoside was 4.97 ± 1.68 and 7.71 ± 1.55, the C_max1 was 1.39 ± 0.83 and 1.45 ± 0.37, the t_max1 was 0.21 ± 0.20 and 0.17 ± 0.01, the C_max2 was 1.90 ± 0.55 and 1.42 ± 0.70, and the t_max2 was 5.60 ± 1.67 and 5.20 ± 1.79, respectively. A significant difference (p < 0.05) was observed for t_1/2, and the elimination of baicalin and wogonoside in Xiaochaihu Tang was increased.