In vivo and in vitro conversion of des-1-Asp angiotensin I to angiotensin III

The conversion of exogenous des-Asp angiotensin I to des-Asp angiotensin II (angiotensin III) was studied in vivo in the pulmonary circulation of the intact pentobarbital-anesthetized dog and in vitro in whole blood and heparin-treated plasma by fractionation of ¹²⁵I-labeled peptides and by radioimmunoassay. Conversion and hydrolysis of [¹²⁵I]des-Asp angiotensin I and [¹²⁵I]angiotensin I were compared in vitro and in vivo. The time course of conversion of des-Asp angiotensin I to angiotensin III in vitro was more rapid than that of angiotensin I to II, but the peak concentration of angiotensin III generated was less than angiotensin II due to the more rapid hydrolysis of the des-Asp peptides. Injection of des-Asp angiotensin I into the right atrium was associated with a pressor response and with the appearance of a small amount of angiotensin III in aortic blood. SQ20881 (a nonapeptide converting enzyme inhibitor) abolished both the pressor response and the generation of angiotensin III. After injection of [¹²⁵I]des-Asp angiotensin I, only 10 per cent of the labeled material appearing in arterial blood was angiotensin III; 15 per cent was unchanged des-Asp angiotensin I; 15 per cent was an unidentified metabolite; and 60 per cent was tyrosine. This contrasts with the pattern of peptides recovered after injection of [¹²⁵I]angiotensin I: 70 per cent angiotensin II, 20 per cent intact angiotensin I and only 10 per cent peptide metabolites and tyrosine. Results indicate that conversion of des-Asp angiotensin I to angiotensin III does occur in the pulmonary circulation of the dog but that hydrolysis of the des-Asp peptides by angiotensinases is so rapid that little circulating angiotensin II and only a small pressor response appear. The data suggest that pulmonary conversion of des-Asp angiotensin I is not an important source of circulating angiotensin III in the dog.     

Synthesis and characterization of some new pyrazolines and their inhibitory potencies against carbonic anhydrases

The inhibition of the two human cytosolic carbonic anhydrase (hCA; EC 4.2.1.1) isozymes I and II with some new pyrazoline derivatives was investigated for the first time. The structures of the newly synthesized pyrazoline derivatives were characterized by Fourier transform-infrared spectroscopy, ¹H-/¹³C-nuclear magnetic resonance, and mass spectrometry, and elemental analysis. Compounds 1–6 showed K_i values in the range of 16.4–205.9 nM for hCA I and of 6.08–93.21 nM against hCA II. These hydroxyl and amino group-containing compounds generally were competitive inhibitors. The compounds investigated here showed effective hCA I and II inhibitory effects, in the same range as the clinically used acetazolamide, and might be used as leads for generating enzyme inhibitors, possibly targeting other CA isoforms that have not yet been assayed for their interactions with such agents.     

The effect of drugs and secretagogues on the biosynthesis of gastric mucins.

Gastric mucosal glycoproteins were separated into three well-defined fractions: secreted mucin in vivo, adherent to cells, Fraction I; soluble mucin secreted during incubation in vitro, Fraction II; and a third, Fraction III released solely by pronase digestion (cell bound or intracellular mucin). ¹⁴C-U-D-glucose and ³⁵SO₄ incorporation into glycoprotein fractions II and III was studied during a 4-hr incubation period in vitro, of mucosal scrapings, freed from Fraction I, from rats treated in vivo, with secretin, histamine, aspirin and atropine. The effect of in vitro added puromycin and actinomycin was also studied. Secretin decreased and histamine increased the total amount of protein and glycoprotein in Fraction II. Histamine inhibited incorporation of glucose into this fraction, to a moderate extent. Secretin had no effect on incorporation. Per oral aspirin, administered for 15 min, decreased levels of secreted mucin Fraction I, and strongly inhibited glucose incorporation in Fraction III. Parenterally administered aspirin and atropine decreased mucin secretion in vivo (Fraction I) but activated glucose incorporation slightly in Fraction II and strongly in Fraction III. Puromycin and actinomycin added in vitro to the mucosal scrapings inhibited strongly glucose incorporation in Fraction III, but did not inhibit incorporation in Fraction II. This indicates the existence of two separate phases of mucin biosynthesis and secretion, both necessitating glucose incorporation, but only the first one being sensitive to puromycin and actinomycin. ³⁵SO₄ incorporation was strongly inhibited by all drugs studied. Sulphation of mucins appears to be much more sensitive to drug action than mucin biosynthesis itself. The above results indicate that the described system is suitable for the study in vitro of the time sequence and mechanisms of drug action.     

Involvement of protein kinases in the induction of NO synthase II in human DLD-1 cells

1. Protein phosphorylation is involved in the induction of nitric oxide synthase II (NOS II, iNOS) in several types of animal cells. Here we have investigated the possible involvement of major protein kinases in the induction of NOS II expression in human DLD-1 cells.
2. In DLD-1 cells, interferon-γ alone induced a submaximal NOS II expression; a cytokine mixture consisting of interferon-γ, tumour necrosis factor-α and interleukin-1β produced maximal NOS II induction.
3. Activators of protein kinase A (forskolin, 8-dibutyryl-cyclic AMP), of protein kinase C (tetradecanoylphorbol-13-acetate), and of protein kinase G (8-bromo cyclic GMP) did not induce NOS II mRNA by themselves, nor did they alter NOS II mRNA induction in response to cytokines.
4. Inhibitors of protein kinase A (compound H89), of protein kinase C (bisindolylmaleimide, chelerythrine or staurosporine), of phosphatidylinositol 3-kinase (wortmannin), of p38 mitogen-activated protein kinase (compound SB 203580) and of extracellular signal-regulated kinase (compound PD 98059) also had no influence on basal or cytokine-induced NOS II mRNA expression.
5. Immunoprecipitation kinase assays showed no activation of extracellular signal-regulated kinase or p38 mitogen-activated protein kinase in cytokine-incubated DLD-1 cells. The c-Jun NH₂-terminal kinase was activated by cytokines, but the most efficacious cytokine was tumour necrosis factor-α which did not induce NOS II by itself.
6. In contrast, the protein tyrosine kinase inhibitor tyrphostin B42 (a specific inhibitor of interferon-γ-activated janus kinase 2) and the protein tyrosine kinase inhibitor tyrphostin A25 both reduced CM-induced NOS II mRNA expression in a concentration-dependent manner.
7. These results suggest that activation of NOS II expression in DLD-1 cells is independent of the activities of protein kinases A, C and G, phosphatidylinositol 3-kinase, extracellular signal regulated kinase and p38 mitogen-activated protein kinase, but seems to require protein tyrosine kinase activity, especially the interferon-γ-activated janus kinase 2.

     

Differential contributions of NOS isoforms in the rostral ventrolateral medulla to cardiovascular responses associated with mevinphos intoxication in the rat

The organophosphate poison mevinphos (Mev) elicits cardiovascular responses via nitric oxide (NO) produced on activation of M2 muscarinic receptors (M2R) in the rostral ventrolateral medulla (RVLM), where sympathetic vasomotor tone originates. This study further evaluated the contribution of nitric oxide synthase (NOS) isoforms at the RVLM to this process, using adult Sprague-Dawley rats. Bilateral co-microinjection into the RVLM of the selective NOS I inhibitor (250 pmol), 7-nitroindazole or N(omega)-propyl-L-arginine antagonized the initial sympathoexcitatory cardiovascular responses to Mev (10 nmol). Co-administration of a selective NOS II inhibitor, N6-(1-iminoethyl)-L-lysine (250 or 500 pmol) further enhanced these cardiovascular responses and reversed the secondary sympathoinhibitory actions of Mev. A potent NOS III inhibitor, N5-(1-iminoethyl)-L-ornithine (46 or 92 nmol) was ineffective. We also found that M2R co-localized only with NOS I- or NOS II-immunoreactive RVLM neurons. Furthermore, only NOS I or II in the ventrolateral medulla exhibited an elevation in mRNA or protein levels during the sympathoexcitatory phase, with further up-regulated synthesis of NOS II during the sympathoinhibitory phase of Mev intoxication. We conclude that whereas NOS III is not engaged, NO produced by NOS I and II in the RVLM plays, respectively, a sympathoexcitatory and sympathoinhibitory role in the cardiovascular responses during Mev intoxication.     

2-Amino-4-methylpyridine as a potent inhibitor of inducible NO synthase activity in vitro and in vivo.

1. The ability of 2-amino-4-methylpyridine to inhibit the catalytic activity of the inducible NO synthase (NOS II) enzyme was characterized in vitro and in vivo. 2. In vitro, 2-amino-4-methylpyridine inhibited NOS II activity derived from mouse RAW 264.7 cells with an IC50 of 6 nM. Enzyme kinetic studies indicated that inhibition is competitive with respect to arginine. 2-Amino-4-methylpyridine was less potent on human recombinant NOS II (IC50 = 40 nM) and was still less potent on human recombinant NOS I and NOS III (IC50 = 100 nM). NG-monomethyl-L-arginine (L-NMMA), N6-iminoethyl-L-lysine (L-NIL) and aminoguanidine were much weaker inhibitors of murine NOS II than 2-amino-4-methylpyridine but, unlike 2-amino-4-methylpyridine, retained similar activity on human recombinant NOS II. L-NMMA inhibited all three NOS isoforms with similar potency (IC50S 3-7 microM). In contrast, compared to activity on human recombinant NOS III, L-NIL displayed 10 x selectivity for murine NOS II and 11 x selectivity for human recombinant NOS II while aminoguanidine displayed 7.3 x selectivity for murine NOS II and 3.7 x selectivity for human recombinant NOS II. 3. Mouse RAW 264.7 macrophages produced high levels of nitrite when cultured overnight in the presence of lipopolysaccharide (LPS) and interferon-gamma. Addition of 2-amino-4-methylpyridine at the same time as the LPS and IFN-gamma, dose-dependently reduced the levels of nitrite (IC50 = 1.5 microM) without affecting the induction of NOS II protein. Increasing the extracellular concentration of arginine decreased the potency of 2-amino-4-methylpyridine but at concentrations up to 10 microM, 2-amino-4-methylpyridine did not inhibit the uptake of [3H]-arginine into the cell. Addition of 2-amino-4-methylpyridine after the enzyme was induced also dose-dependently inhibited nitrite production. Together, these data suggest that 2-amino-4-methylpyridine reduces cellular production of NO by competitive inhibition of the catalytic activity of NOS II, in agreement with results obtained from in vitro enzyme kinetic studies. 4. When infused i.v. in conscious unrestrained rats, 2-amino-4-methylpyridine inhibited the rise in plasma nitrate produced in response to intraperitoneal injection of LPS (ID50 = 0.009 mg kg-1 min-1). Larger doses of 2-amino-4-methylpyridine were required to raise mean arterial pressure in untreated conscious rats (ED50 = 0.060 mg kg-1 min-1) indicating 6.9 x selectivity for NOS II over NOS III in vivo. Under the same conditions, L-NMMA was nonselective while L-NIL and aminoguanidine displayed 5.2 x and 8.6 x selectivity respectively. All of these compounds caused significant increases in mean arterial pressure at doses above the ID50 for inhibition of NOS II activity in vivo. 5. 2-Amino-4-methylpyridine also inhibited LPS-induced elevation in plasma nitrate after either subcutaneous (ID50 = 0.3 mg kg-1) or oral (ID50 = 20.8 mg kg-1) administration. 6. These data indicate that 2-amino-4-methylpyridine is a potent inhibitor of NOS II activity in vitro and in vivo with a similar degree of isozyme selectivity to that of L-NIL and aminoguanidine in rodents.     

Treatment with LPS plus INF-γ induces the expression and function of muscarinic acetylcholine receptors,modulating NIH3T3 cell proliferation: participation of NOS and COX

Background and Purpose

LPS and IFN-γ are potent stimuli of inflammation, a process in which fibroblasts are frequently involved. We analysed the effect of treatment with LPS plus IFN-γ on the expression and function of muscarinic acetylcholine receptors in NIH3T3 fibroblasts with regards to proliferation of these cells. We also investigated the participation of NOS and COX, and the role of NF-κB in this process.

Experimental Approach

NIH3T3 cells were treated with LPS (10 ng·mL⁻¹) plus IFN-γ (0.5 ng·mL⁻¹) for 72 h (iNIH3T3 cells). Cell proliferation was evaluated with MTT and protein expression by Western blot analysis. NOS and COX activities were measured by the Griess method and radioimmunoassay respectively.

Key Results

The cholinoceptor agonist carbachol was more effective at stimulating proliferation in iNIH3T3 than in NIH3T3 cells, probably due to the de novo induction of M₃ and M₅ muscarinic receptors independently of NF-κB activation. iNIH3T3 cells produced higher amounts of NO and PGE₂ than NIH3T3 cells, concomitantly with an up-regulation of NOS1 and COX-2, and with the de novo induction of NOS2/3 in inflamed cells. We also found a positive feedback between NOS and COX that could potentiate inflammation.

Conclusions and Implications

Inflammation induced the expression of muscarinic receptors and, therefore,stimulated carbachol-induced proliferation of fibroblasts. Inflammation also up-regulated the expression of NOS and COX-2, thus potentiating the effect of carbachol on NO and PGE₂ production. A positive crosstalk between NOS and COX triggered by carbachol in inflamed cells points to muscarinic receptors as potential therapeutic targets in inflammation.     

Protolytic constants of nizatidine,ranitidine and N,N′-dimethyl-2-nitro-1,1-ethenediamine; spectrophotometric and theoretical investigation

The prototropic exchange equilibria of two drugs, nizatidine (I) and ranitidine (II), and also of structurally related the N,N′-dimethyl-2-nitro-1,1-ethenediamine molecule (III) were investigated. From the changes in electronic spectra in media of various acidity several protonation constants were determined. For nizatidine pK values were −0.82, 1.95, and 6.67; for ranitidine pK values were 1.95 and 8.13; and for III was 2.60. The hydroxylation equilibrium constant in strongly alkaline media was determined too. Corresponding pK_a values were 13.23 for I, 13.26 for II and 13.76 for III. Molecular orbital calculations of electronic spectra confirmed that pK 1.95 for I and II, and pK 2.60 for III, are associated with C-protonation of nitroethenediamine fragment, while all pK_a values correspond to the addition of HO⁻ anion at the same double bond.     

Pharmaceutical Evaluation of Carbamazepine Modifications: Comparative Study for Photostability of Carbamazepine Polymorphs by using Fourier-transformed Reflection-absorption Infrared Spectroscopy and Colorimetric Measurement

Abstract— The tablet surface was evaluated without physical damage by means of Fourier-transform infrared reflection-absorption spectroscopy (FT-IR-RAS) and colorimetric measurement (colour difference, AE) of the carbamazepine polymorphs I, II and III, after photodegradation at two irradiation intensities (30 and 120 J cm^?2 s ^?1) under a near-UV fluorescent lamp. The surface of sample pellets of all crystalline forms turned gradually from white to yellow-orange upon exposure to light, and the discoloration rate of form II was faster than that of forms I and III, indicating that form II was the most unstable of the three. The major photoproducts were identified by HPLC, NMR and MS analyses. The carbamazepine content on the surface of the tablet was determined based on the absorption at 1685 cm^?1 atributable to C = O stretch vibration in the FT-IR-RAS spectra before and after irradiation by a near-UV fluorescent lamp. The semilogarithmic plots of the photodegradation profiles of the various polymorphs were straight lines, including the induction period, indicating that degradation of the drug on the surface followed first-order kinetics. The induction periods of all forms were not significantly different. However, the degradation rate constant of form II at 12.0 Jcm^?2 s ^?1 was 5·1 and 1·5 times larger than those of forms I and III, respectively.     

Isolation of aldose reductase inhibitors from the flowers ofChrysanthemum boreale

The methanol extract from the whole parts of the flowers ofChrysanthemum boreale was found to exhibit a significant inhibition of rat lens aldose reductase (RLAR) activityin vitro. Bioassay guided systematic fractionation of the methanol extract led to the isolation of four flavonoids which were identified as acacetin (I), apigenin (II), luteolin (III) and linarin (IV). Compounds I–III were demonstrated to exhibit a significant inhibition of RLAR. Luteolin (III) was found to be the most potent AR inhibitor with IC₅₀ value of 5×10^?7M.     

Tissue-specific up-regulation of arginase I and II induced by p38 MAPK mediates endothelial dysfunction in type 1 diabetes mellitus

Background and Purpose

Emerging evidence suggests a selective up-regulation of arginase I in diabetes causing coronary artery disease; however, the mechanisms behind this up-regulation are still unknown. Activated p38 MAPK has been reported to increase arginase II in various cardiovascular diseases. We therefore tested the role of p38 MAPK in the regulation of arginase I and II expression and its effect on endothelial dysfunction in diabetes mellitus.

Experimental Approach

Endothelial function was determined in septal coronary (SCA), left anterior descending coronary (LAD) and mesenteric (MA) arteries from healthy and streptozotocin-induced diabetic Wistar rats by wire myographs. Arginase activity and protein levels of arginase I, II, phospho-p38 MAPK and phospho-endothelial NOS (eNOS) (Ser¹¹⁷⁷) were determined in these arteries from diabetic and healthy rats treated with a p38 MAPK inhibitor in vivo.

Key Results

Diabetic SCA and MA displayed impaired endothelium-dependent relaxation, which was prevented by arginase and p38 MAPK inhibition while LAD relaxation was not affected. Arginase I, phospho-p38 MAPK and eNOS protein expression was increased in diabetic coronary arteries. In diabetic MA, however, increased expression of arginase II and phospho-p38 MAPK, increased arginase activity and decreased expression of eNOS were observed. All these effects were reversed by p38 MAPK inhibition.

Conclusions and Implications

Diabetes-induced activation of p38 MAPK causes endothelial dysfunction via selective up-regulation of arginase I expression in coronary arteries and arginase II expression in MA. Therefore, regional differences appear to exist in the arginase isoforms contributing to endothelial dysfunction in type 1 diabetes mellitus.     

Carbonic Anhydrase Inhibitors: Inhibition of Human Erythrocyte Isozymes I and II with a Series of Phenolic Acids

The inhibitory effects of some phenolic acids on the cytosolic human carbonic anhydrase (hCA, EC 4.2.1.1) isozymes hCA I and hCA II were investigated. Ellagic acid, gallic acid, ferulic acid, caffeic acid, quercetin, p-coumaric acid, p-hydroxybenzoic acid, and syringic acid showed K_I values in the range of 99–1061 μm for hCA I and of 105–758 μm against hCA II, respectively. Quercetin (for hCA I), p-coumaric acid (for hCA II), and gallic acid (for hCA II) exhibited competitive inhibitory effects with 4-nitrophenyl acetate as substrate. All of the other phenolic acids were found as non-competitive inhibitors with 4-nitrophenylacetate as substrate for hCA I and hCA II. The phenolic acids investigated here showed thus interesting hCA I and hCA II inhibitory effects and might be used as leads for generating enzyme inhibitors possibly targeting other CA isoforms which have not been yet assayed for their interactions with such agents.     

Purification,characterization and toxicity profile of ricin isoforms from castor beans

The castor seed contains the toxin ricin, one of the most poisonous naturally occurring toxins. The whole of the plant is poisonous, however the seeds are considered the major source of ricin. Ricin exists in different forms in beans of different origin. We investigated the presence of ricin in different isoforms and elucidate some of their structural and biological features isolated from the castor seeds. The isoforms were sub fractionated into ricin I, II and III by chromatography. Their molecular weights lie between 60–65 kDa with difference in their relative electrophoretic mobility. An acidic native PAGE of ricin isoforms at pH 2.9 was performed. Ricin I, II and III are highly cytotoxic against Vero cell line with IC₅₀ values of 60, 30 and 8 ng/ml respectively. Difference in cytotoxicity of isoforms was confirmed through hemagglutination assay, ricin III caused high degree of hemolysis. The preliminary in vivo toxicity studies showed that ricin III is highly toxic. Immunological studies revealed that anti-ricin I and II antibodies are cross reactive with all the ricin variants, whereas the anti-ricin III antibody is highly specific. The present study shows that anti-ricin I and II antibodies can be used for detection of entire ricin isoforms.     

Upregulation of nitric oxide synthase II contributes to apoptotic cell death in the hippocampal CA3 subfield via a cytochrome c/caspase-3 signaling cascade following induction of experimental temporal lobe status epilepticus in the rat

Status epilepticus results in preferential neuronal cell loss in the hippocampus. We evaluated the hypothesis that the repertoire of intracellular events in the vulnerable hippocampal CA3 subfield after induction of experimental temporal lobe status epilepticus entails upregulation of nitric oxide synthase II (NOS II), followed by the release of mitochondrial cytochrome c that triggers the cytosolic caspase-3 cascade, leading to apoptotic cell death. In Sprague-Dawley rats, significant and temporally correlated upregulation of NOS II (3-24h), but not NOS I or II expression, enhanced cytosolic translocation of cytochrome c (days 1 and 3), augmented activated caspase-3 in cytosol (days 1, 3 and 7) and DNA fragmentation (days 1, 3 and 7) was detected bilaterally in the hippocampal CA3 subfield after elicitation of sustained seizure activity by microinjection of kainic acid into the unilateral CA3 subfield. Application bilaterally into the hippocampal CA3 subfield of a selective NOS II inhibitor, S-methylisothiourea, significantly blunted these apoptotic events; a selective NOS I inhibitor, N(omega)-propyl-l-arginine or a potent NOS III inhibitor, N(5)-(1-iminoethyl)-l-ornithine was ineffective. We conclude that upregulation of NOS II contributes to apoptotic cell death in the hippocampal CA3 subfield via a cytochrome c/caspase-3 signaling cascade following the induction of experimental temporal lobe status epilepticus.     

Nitric oxide regulates angiotensin II receptors in vascular smooth muscle cells

The objective of this study was to determine whether an enhanced generation of nitric oxide (NO) causes regulation of angiotensin II receptors in vitro using rat vascular smooth muscle cells in culture. Chronic treatment of cells with a series of NO-generating drugs, sodium nitroprusside, S-nitroso-N-acetylpenicillamine and isosorbide dinitrate for 18h dose and time-dependently decreased [¹²⁵I]-angiotensin II binding to cells without any significant change in affinity. Induction of nitric oxide synthase following lipopolysaccharide (10 and 100 ng/ml) treatment of cells for 18 h increased basal nitric oxide synthase activity with a concomitant increase of nitrite and cyclic cGMP levels in the conditioned media. LPS treatment significantly (P < 0.05) decreased [¹²⁵I]-angiotensin II binding to these cells, an effect that was significantly (P < 0.05) attenuated in the presence of N^G-nitro-L-arginine methyl ester. In contrast, treatment of cells with atrial natriuretic factor, dibutyryl cGMP, 8-bromo-cGMP, NaNO₂ or NaNO₃ failed to significantly alter the affinity or number of [¹²⁵I]-angiotensin II binding sites. These results suggest that NO regulates angiotensin II receptors in vitro through a cGMP-independent mechanism.     

Structure and protein kinase C stimulating activities of lactam analogues of human parathyroid hormone fragment

Five analogues of human parathyroid hormone (hPTH-(20-34)-NH₂, I; cyclo[Lys²⁶-Asp³⁰]-hPTH-(20-34)-NH₂, II; cyclo[Glu²²-Lys²⁶]-hPTH-(20-34)-NH₂, III; cyclo[Lys²⁷-Asp³⁰]-hPTH-(20-34)-NH₂, IV; and [Leu²⁷]-hPTH-(20-34)-NH₂, V) were tested for their ability to promote membrane-bound protein kinase C (PKC) activity in a rat osteosarcoma cell line (ROS 17/2). Analogues I, II and V stimulated PKC activity in the picomolar range, whereas analogues III and IV did not stimulate this activity at any concentration tested. The circular dichroism spectra in neutral, aqueous buffer showed an increase in α-helix in analogues II, III and V as compared to I; this increase appeared to be in the region of the cyclic lactam structure. Analogue IV did not adopt a helical structure, even in the presence of 40% trifluoroethanol, a helix-promoting solvent. The remaining analogues showed a three- to four-fold enhancement of α-helix in this solvent. Analogues II and III had increased retention times in reversed-phase chromatography, as compared to I and IV, This is consistent with a stabilization of amphiphilic helix in analogues II and III compared with I and IV, The data suggest that in the region bounded approximately by residues 24–32, an amphiphilic α-helix is important for correct functional binding to the PTH receptor.     

Nephroprotective effects of nebivolol in 2K1C rats through regulation of the kidney ROS-ADMA-NO pathway

Background

To evaluate the protective effect of nebivolol against kidney damage and elucidate the underlying mechanism in a two-kidney, one-clip (2K1C) rat model.

Rapanone,a naturally occurring benzoquinone,inhibits mitochondrial respiration and induces HepG2 cell death

Rapanone is a natural occurring benzoquinone with several biological effects including unclear cytotoxic mechanisms. Here we addressed if mitochondria are involved in the cytotoxicity of rapanone towards cancer cells by employing hepatic carcinoma (HepG2) cells and isolated rat liver mitochondria. In the HepG2, rapanone (20–40 μM) induced a concentration-dependent mitochondrial membrane potential dissipation, ATP depletion, hydrogen peroxide generation and, phosphatidyl serine externalization; the latter being indicative of apoptosis induction. Rapanone toxicity towards primary rats hepatocytes (IC₅₀ = 35.58 ± 1.50 μM) was lower than that found for HepG2 cells (IC₅₀ = 27.89 ± 0.75 μM). Loading of isolated mitochondria with rapanone (5–20 μM) caused a concentration-dependent inhibition of phosphorylating and uncoupled respirations supported by complex I (glutamate and malate) or the complex II (succinate) substrates, being the latter eliminated by complex IV substrate (TMPD/ascorbate). Rapanone also dissipated mitochondrial membrane potential, depleted ATP content, released Ca²⁺ from Ca²⁺-loaded mitochondria, increased ROS generation, cytochrome c release and membrane fluidity. Further analysis demonstrated that rapanone prevented the cytochrome c reduction in the presence of decylbenzilquinol, identifying complex III as the site of its inhibitory action. Computational docking results of rapanone to cytochrome bc1 (Cyt bc1) complex from the human sources found spontaneous thermodynamic processes for the quinone-Q_o and Q_i binding interactions, supporting the experimental in vitro assays. Collectively, these observations suggest that rapanone impairs mitochondrial respiration by inhibiting electron transport chain at Complex III and promotes mitochondrial dysfunction. This property is potentially involved in rapanone toxicity on cancer cells.     

Experimental osteoporosis induced in female albino rats and its antagonism by Indian black scorpion (Heterometrus bengalensis C.L.Koch) venom

The present study was designed to explore the antiosteoporosis activity of the Indian black scorpion (Heterometrus bengalensis) venom on experimental osteoporosis female albino rats. Sham operated control rats were designated as Gr I, Gr II animals served as osteoporosis control, Gr III osteoporosis rats were treated with SV (1/25th of MLD), Gr IV osteoporosis rats were treated with 1/50th of MLD of SV and Gr V osteoporosis rats were treated with standard (calcium and vit-D3). As compared with the Gr I rats, the Gr II rats showed typical osteoporosis changes in increased of urinary Ca²⁺, PO₄³⁻, CRE, OH-P levels, serum/plasma Ca²⁺, PO₄³⁻, TRAP, IL1, IL6, TNFα and PTH level, bone Ca²⁺, PO₄³⁻, Mg²⁺, Zn²⁺ and decreased level of serum/plasma ALP, EST and PTH, bone Na⁺. In Gr III, Gr IV and Gr V rats, the osteoporosis changes of urine, serum and bone, were significantly restored as compared with the Gr II rats. The bone dimensions, morphology and histological changes observed in Gr II rats were restored in Gr III, Gr IV and Gr V rats. This study confirms that the Indian black scorpion venom may influence bone remodeling process by stimulating bone formation and reducing bone resorption process of osteogenesis.     

Shikonin Directly Inhibits Nitric Oxide Synthases: Possible Targets That Affect Thoracic Aorta Relaxation Response and Nitric Oxide Release From RAW 264.7 Macrophages

Recently, an isomeric mixture of herbal anti-inflammatory naphthoquinones shikonin and alkannin, and their derivatives, have been found to impair cellular responses involving nitric oxide (NO) and NO synthesis, like the acetylcholine-induced relaxation response of rat thoracic aorta and NO release from murine RAW 264.7 macrophages. However, the mechanisms of such effects, including whether NO synthase (NOS) activity is affected, remained unclear. We herein investigate possible targets of shikonin in these NOS-related events. Shikonin by itself dose-dependently inhibited the rat thoracic aorta relaxation in response to acetylcholine (pD′₂ value: 6.29). Its optical enantiomer, alkannin, was equally inhibitory in the aorta relaxation–response assay. In RAW 264.7 cells, shikonin inhibited the lipopolysaccharide-induced NO production by 82% at 1 μM. A cell-free assay to verify direct effects on NOS activity showed that shikonin inhibits all isoforms of NOS (IC₅₀ s, 4–7 μM), suggesting NOS as an inhibition target in both the events. Further possible targets of shikonin that might be involved in the inhibitions of the acetylcholine-induced aorta relaxation response and the NO generation by RAW 264.7 cells are also discussed. It is shown for the first time that shikonin inhibits NOS activity.