Ultraviolet light-induced apoptotic death is impaired by the HMG-CoA reductase inhibitor lovastatin

HMG-CoA reductase inhibitors (i.e., statins) attenuate C-terminal isoprenylation of Rho GTPases, thereby inhibiting UV-C-induced activation of c-Jun-N-terminal kinases/stress-activated protein kinases (JNKs/SAPKs). Inhibition of UV-C-triggered JNK/SAPK activation by lovastatin is due to inhibition of Rac-SEK1/MKK4-mediated phosphorylation of JNKs/SAPKs at Thr183/Tyr185. UV-C-stimulated phosphorylation of p38 kinase (Thr180/Tyr182) is also impaired by lovastatin. Cell killing provoked by UV-C irradiation was significantly inhibited by lovastatin. This was paralleled by a reduced frequency of chromosomal aberrations, accelerated recovery from UV-C-induced transient replication blockage, inhibition of Chk1 kinase activation and impaired cyclinB1 expression. Furthermore, UV-C-induced activation of caspases and apoptotic death was largely reduced by lovastatin. Inhibition of JNK/SAPK by transient overexpression of dominant-negative JNK1/SAPK1 also conferred resistance to UV-C light and attenuated activation of caspase 3. Based on the data, we suggest that lovastatin-provoked resistance to UV-C light is due to the inhibition of UV-C-inducible Rac-SEK1/MKK4-JNK/SAPK-dependent signal mechanisms regulating cell cycle progression and activation of caspases and apoptotic death.     

Anti-lipid deposition effect of HMG-CoA reductase inhibitor, pitavastatin, in a rat model of hypertension and hypercholesterolemia

Since the rat is an atherosclerosis-resistant species, the study of atherosclerosis using rats is limited. The present study was undertaken to develop an atherosclerotic model in rats, to investigate the effect of nitric oxide (NO) inactivation and hyperlipidemia, and to evaluate the effect of pitavastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) inhibitor, on NO inactivation and on hyperlipidemia-induced changes in the cardiovascular system. Four-month-old male spontaneously hypertensive hyperlipidemic rats (SHHR) and Sprague-Dawley (SD) rats were used to study 1) the effect of the period of treatment with N(G)-nitro-L-arginine methyl ester (L-NAME, 100 mg/L) on high fat diet (HFD)-treated SHHR and SD rats, and 2) the effect of pitavastatin (Pit, 0.3 mg/kg/day) on the changes in the aorta of L-NAME- and HFD-treated SHHR and SD rats. L-NAME administration for 1 month then HFD feeding for 2 months markedly increased the deposition of lipids and the thickness of the endothelium in SHHR. Continuous L-NAME treatment with HFD produced severe injury and stripped of endothelium in both strains. The plasma total cholesterol of L-NAME + HFD-treated and L-NAME + HFD + Pit-treated SHHR was significantly higher than that of control SHHR. Lipid deposition, however, was comparatively less in the aorta of L-NAME + HFD + Pit-treated SHHR. The concentration of cholesterol in the aorta of control SHHR was significantly lower than that in the aorta of L-NAME + HFD-treated SHHR, whereas that of L-NAME + HFD + Pit-treated SHHR was the same as that in control SHHR. These data indicated that Pit blocked lipid deposition in the aorta of L-NAME + HFD treated SHHR without changing plasma lipid profiles. In conclusion, NO inactivation and HFD induce lipid deposition in the endothelium, and the HMG-CoA reductase inhibitor blocks the deposition in SHHR.     

Sclerotiorin, from Penicillium frequentans, a potent inhibitor of aldose reductase

Aldose reductase, the first key enzyme in the polyol pathway, is involved in complications of diabetes. Sclerotiorin, isolated and purified from the fermented broth of Penicillium frequentans, inhibited aldose reductase with an IC₅₀ 0.4 μM. The inhibitor also showed antibacterial activity against Bacillus spp.     

HMG-CoA reductase inhibitor fluvastatin prevents angiotensin II-induced cardiac hypertrophy via Rho kinase and inhibition of cyclin D1

HMG-CoA reductase inhibitors, so called statins, decrease cardiac events. Previous studies have shown that HMG-CoA reductase inhibitors inhibit cardiomyocyte hypertrophy in vitro and in vivo by blocking Rho isoprenylation. We have shown that the G1 cell cycle regulatory proteins cyclin D1 and Cdk4 play important roles in cardiomyocyte hypertrophy. However, the relation between Rho and cyclin D1 in cardiomyocyte is unknown. To investigate whether HMG-CoA reductase inhibitors prevent cardiac hypertrophy through attenuation of Rho and cyclin D1, we studied the effect of fluvastatin on angiotensin II-induced cardiomyocyte hypertrophy in vitro and in vivo. Angiotensin II increased the cell surface area and [(3)H]leucine uptake of cultured neonatal rat cardiomyocytes and these changes were suppressed by fluvastatin treatment. Angiotensin II also induced activation of Rho kinase and increased cyclin D1, both of which were also significantly suppressed by fluvastatin. Specific Rho kinase inhibitor, Y-27632 inhibited angiotensin II-induced cardiomyocyte hypertrophy and increased cyclin D1. Overexpression of cyclin D1 by adenoviral gene transfer induced cardiomyocyte hypertrophy, as evidenced by increased cell size and increased protein synthesis; this hypertrophy was not diminished by concomitant treatment with fluvastatin. Infusion of angiotensin II to Wistar rats for 2 weeks induced hypertrophic changes in cardiomyocytes, and this hypertrophy was prevented by oral fluvastatin treatment. These results show that an HMG-CoA reductase inhibitor, fluvastatin, prevents angiotensin II-induced cardiomyocyte hypertrophy in part through inhibition of cyclin D1, which is linked to Rho kinase. This novel mechanism discovered for fluvastatin could be revealed how HMG-CoA reductase inhibitors are preventing cardiac hypertrophy.     

Identification of new potent inhibitor of aldose reductase from Ocimum basilicum

Recent efforts to develop cure for chronic diabetic complications have led to the discovery of potent inhibitors against aldose reductase (AKR1B1, EC 1.1.1.21) whose role in diabetes is well-evident. In the present work, two new natural products were isolated from the ariel part of Ocimum basilicum; 7-(3-hydroxypropyl)-3-methyl-8-β-O-d-glucoside-2H-chromen-2-one (1) and E-4-(6′-hydroxyhex-3′-en-1-yl)phenyl propionate (2) and confirmed their structures with different spectroscopic techniques including NMR spectroscopy etc. The isolated compounds (1, 2) were evaluated for in vitro inhibitory activity against aldose reductase (AKR1B1) and aldehyde reductase (AKR1A1). The natural product (1) showed better inhibitory activity for AKR1B1 with IC₅₀ value of 2.095 ± 0.77 µM compare to standard sorbinil (IC₅₀ = 3.14 ± 0.02 µM). Moreover, the compound (1) also showed multifolds higher activity (IC₅₀ = 0.783 ± 0.07 µM) against AKR1A1 as compared to standard valproic acid (IC₅₀ = 57.4 ± 0.89 µM). However, the natural product (2) showed slightly lower activity for AKR1B1 (IC₅₀ = 4.324 ± 1.25 µM). Moreover, the molecular docking studies of the potent inhibitors were also performed to identify the putative binding modes within the active site of aldose/aldehyde reductases.     

Recognized sequence and conformation in design of linear peptides as a competitive inhibitor for HMG-CoA reductase

This study is an attempt to develop a simple search method for lead peptide candidates, which include constrained structures in a recognized sequence, using the design of a competitive inhibitor for HMG-CoA reductase (HMGR). A structure-functional analysis of previously synthesized peptides proposes that a competitive inhibitory peptide can be designed by maintaining bioactive conformation in a recognized sequence. A conformational aspect of the structure-based approach was applied to the peptide design. By analysis of the projections obtained through a principle component analysis (PCA) for short linear and cyclic peptides, a head-to-tail peptide cycle is considered as a model for its linear analogy. It is proposed that activities of the linear peptides based on an identical amino acid sequence, which are obtained from a less flexible peptide cycle, would be relatively higher than those obtained from more flexible cyclic peptides. The design criterion was formulated in terms of a 'V' parameter, reflecting a relative deviation of an individual peptide cycle from an average statistical peptide cycle based on all optimized structures of the cyclic peptides in set. Twelve peptide cycles were selected for the peptide library. Comparing the calculated 'V' parameters, two cyclic peptides (GLPTGG and GFPTGG) were selected as lead cycles from the library. Based on these sequences, six linear peptides obtained by breaking the cycle at different positions were selected as lead peptide candidates. The linear GFPTGG peptide, showing the highest inhibitory activity against HMGR, increases the inhibitory potency nearly tenfold. Kinetic analysis reveals that the GFPTGG peptide is a competitive inhibitor of HMG-CoA with an equilibrium constant of inhibitor binding (K(i)) of 6.4 +/- 0.3 microM. Conformational data support a conformation of the designed peptides close to the bioactive conformation of the previously synthesized active peptides.     

Design of a highly potent inhibitory peptide acting as a competitive inhibitor of HMG-CoA reductase

This study presents a design of a highly potent and competitive inhibitory peptide for 3-hydroxy-3-methylglutaryl CoA reductase (HMGR). HMGR is the major regulatory enzyme of cholesterol biosynthesis and the target enzyme of many investigations aimed at lowering the rate of cholesterol biosynthesis. In previous studies, the two hypocholesterolemic peptides (LPYP and IAVPGEVA) were isolated and identified from soy protein. Based on these peptide sequences, a number of peptides were designed previously by using the correlation between the conformational flexibility and bioactivity. The design method that was applied in previous studies was slightly modified for the purpose of the current research and 12 new peptides were designed and synthesized. Among all peptides, SFGYVAE showed the highest ability to inhibit HMGR. A kinetic analysis revealed that this peptide is a competitive inhibitor of HMG-CoA with an equilibrium constant of inhibitor binding (K _i) of 12?±?0.4?nM. This is an overall 14,500-fold increase in inhibitory activity compared to the first isolated LPYP peptide from soybeans. Conformational data support a conformation of the designed peptides close to the bioactive conformation of the previously synthesized active peptides.     

Degradation of HMG-CoA reductase in rat liver is cholesterol and ubiquitin independent

In contrast with the accelerated degradation observed in tumor cells in response to sterols, hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase turnover in whole animals was not increased by dietary cholesterol. Furthermore, treating rats with lovastatin to lower hepatic cholesterol levels did not decrease the rate of degradation. The half-life remained in the 6 h range. Co-immunoprecipitation studies revealed that the amount of ubiquitin associated with the reductase was entirely dependent upon the amount of microsomal protein subjected to immunoprecipitation. The results indicate that in liver, neither the rate of reductase protein degradation nor the ubiquitin-proteasome system appear to play roles in mediating changes in HMG-CoA reductase protein levels in response to dietary cholesterol.     

Modeling an active conformation for linear peptides and design of a competitive inhibitor for HMG-CoA reductase

This study presents an approach that can be used to search for lead peptide candidates, including unconstrained structures in a recognized sequence. This approach was performed using the design of a competitive inhibitor for 3-hydroxy-3-methylglutaryl CoA reductase (HMGR). In a previous design for constrained peptides, a head-to-tail cyclic structure of peptide was used as a model of linear analog in searches for lead peptides with a structure close to an active conformation. Analysis of the conformational space occupied by the peptides suggests that an analogical approach can be applied for finding a lead peptide with an unconstrained structure in a recognized sequence via modeling a cycle using fixed residues of the peptide backbone. Using the space obtained by an analysis of the bioactive conformations of statins, eight cyclic peptides were selected for a peptide library based on the YVAE sequence as a recognized motif. For each cycle, the four models were assessed according to the design criterion ("V" parameter) applied for constrained peptides. Three cyclic peptides (FGYVAE, FPYVAE, and FFYVAE) were selected as lead cycles from the library. The linear FGYVAE peptide (IC(50) = 0.4 microM) showed a 1200-fold increase the inhibitory activity compared to the first isolated LPYP peptide (IC(50) = 484 microM) from soybean. Experimental analysis of the modeled peptide structures confirms the appropriateness of the proposed approach for the modeling of active conformations of peptides.     

Ketopantoyl lactone reductase is a conjugated polyketone reductase

Ketopantoyl lactone reductase (EC 1.1.1.168) of Saccharomyces cerevisiae was found to catalyze the reduction of a variety of natural and unnatural conjugated polyketone compounds and quinones, such as isatin, ninhydrin, camphorquinone and beta-naphthoquinone in the presence of NADPH. 5-Bromoisatin is the best substrate for the enzyme (Km = 3.1 mM; Vmax = 650 mumol/min/mg). The enzyme is inhibited by quercetin, and several polyketones. These results suggest that ketopantoyl lactone reductase is a carbonyl reductase which specifically catalyzes the reduction of conjugated polyketones.     

Diminished expression of dihydropteridine reductase is a potent biomarker for hypertensive vessels

To identify the new targets for hypertension, we analyzed the protein expression profiles of aortic smooth muscle in spontaneously hypertensive rats (SHR) of various ages during the development of hypertension, as well as in age‐matched normotensive Wistar–Kyoto (WKY) rats, using a proteomic analysis. The expressions of seven proteins were altered in SHR compared with WKY rats. Of these proteins, NADH dehydrogenase 1α, GSTω1, peroxi‐redoxin I and transgelin were upregulated in SHR compared with WKY rats. On the other hand, the expression of HSP27 and Ran protein decreased in SHR. The diminution of dihydrobiopterin reductase, an enzyme located in the regeneration pathways of tetrahydrobiopterin (BH4), was also prominent in SHR. The results from a PCR analysis revealed that the expression of BH4 biosynthesis enzymes – GTP cyclohydrolase‐1 and sepiapterin reductase – decreased and increased, respectively, in SHR compared with WKY rats. The level of BH4 was less in aortic strips from SHR than from WKY rats. Moreover, treatment with BH4 inhibited aortic smooth muscle contraction induced by serotonin. These results suggest that the deficiency in BH4 regeneration produced by diminished dihydrobiopterin reductase expression is involved in vascular disorders in hypertensive rats.     

The activation state of nitrate reductase is not always correlated with total nitrate reductase activity in leaves

The relation between nitrate reductase (NR; EC 1.6.6.1) activity, activation state and NR protein in leaves of barley (Hordeum vulgare L.) seedlings was investigated. Maximum NR activity (NRA_max) and NR protein content (Western blotting) were modified by growing plants hydroponically at low (0.3 mM) or high (10 mM) nitrate supply. In addition, plants were kept under short-day (8 h light/16 h dark) or long-day (16 h light/8 h dark) conditions in order to manipulate the concentration of nitrate stored in the leaves during the dark phase, and the concentrations of sugars and amino acids accumulated during the light phase, which are potential signalling compounds. Plants were also grown under phosphate deficiency in order to modify their glucose-6-phosphate content. In high-nitrate/long-day conditions, NRA_max and NR protein were almost constant during the whole light period. Low-nitrate/long-day plants had only about 30% of the NRA_max and NR protein of high-nitrate plants. In low-nitrate/long-day plants, NRA_max and NR protein decreased strongly during the second half of the light phase. The decrease was preceded by a strong decrease in the leaf nitrate content. Short daylength generally led to higher nitrate concentrations in leaves. Under short-day/low-nitrate conditions, NRA_max was slightly higher than under long-day conditions and remained almost constant during the day. This correlated with maintenance of higher nitrate concentrations during the short light period. The NR activation state in the light was very similar in high-nitrate and low-nitrate plants, but dark inactivation was twice as high in the high-nitrate plants. Thus, the low NRA_max in low-nitrate/long-day plants was slightly compensated by a higher activation state of NR. Such a partial compensation of a low NR_max by a higher dark activation state was not observed with phosphate-depleted plants. Total leaf concentrations of sugars, of glutamine and glutamate and of glucose-6-phosphate did not correlate with the NR activation state nor with NRA_max. Received: 24 March 1999 / Accepted: 31 May 1999     

Nitrate reductase is not accumulated in chloroplast-ribosome-deficient mutants of higher plants

The activities of nitrite reductase (EC 1.7.7.1) are 60–70% of wild-type activity in pigment-deficient leaves of the chloroplast-ribosomedeficient mutants albostrians (Hordeum vulgare) and iojap (Zea mays). The activity and apoprotein of nitrate reductase (EC 1.6.6.1.) are lacking in the barley mutant. Only very low activities of nitrate reductase can be extracted from leaves of the maize mutant. The molybdenum cofactor of nitrate reductase and xanthine dehydrogenase (EC 1.2.3.2) is present in maize and barley mutant plants. However, it is not inducible by nitrate in pigment-deficient leaves of albostrians. From these results we conclude: (i) Nitrite reductase (a chloroplast enzyme) is synthesized in the cytoplasm and does not need the presence of nitrate reductase for the induction and maintenance if its activity. (ii) The loss or low activity of nitrate reductase is a consequence of the inability of the mutants to accumulate the apoprotein of this enzyme. (iii) The chloroplasts influence the accumulation (i.e. most probably the synthesis) of the nonchloroplast enzyme, nitrate reductase. The accumulation of nitrate reductase needs a chloroplast factor which is not provided by mutant plastids blocked at an early stage of their development.Abbreviations CRM cross-reacting material - Mo-co molybdenum cofactor - NiR nitrite reductase - NR nitrate reductase     

Human carbonyl reductase 4 is a mitochondrial NADPH-dependent quinone reductase

A protein encoded in the gene Cbr4 on human chromosome 4q32.3 belongs to the short-chain dehydrogenase/reductase family. Contrary to the functional annotation as carbonyl reductase 4 (CBR4), we show that the recombinant tetrameric protein, composed of 25-kDa subunits, exhibits NADPH-dependent reductase activity for o- and p-quinones, but not for other aldehydes and ketones. The enzyme was insensitive to dicumarol and quercetin, potent inhibitors of cytosolic quinone reductases. The 25-kDa CBR4 was detected in human liver, kidney and cell lines on Western blotting using anti-CBR4 antibodies. The overexpression of CBR4 in bovine endothelial cells reveals that the enzyme has a non-cleavable mitochondrial targeting signal. We further demonstrate that the in vitro quinone reduction by CBR4 generates superoxide through the redox cycling, and suggest that the enzyme may be involved in the induction of apoptosis by cytotoxic 9,10-phenanthrenequinone.     

MCP-1-induced enhancement of THP-1 adhesion to vascular endothelium was modulated by HMG-CoA reductase inhibitor through RhoA GTPase-, but not ERK1/2-dependent pathway

Monocyte-endothelial interaction plays a pivotal role in atherosclerosis. We previously showed that HMG CoA reductase inhibitor reduces adhesion, however, not the rolling of monocytes to vascular endothelium under flow in vitro. In the present study, we investigated the effect of pitavastatin, a novel HMG CoA reductase inhibitor, on the transition from monocyte rolling on vascular endothelium to stable adhesion induced by MCP-1 under flow (shear stress = 1.0 dyne/cm(2)). Control THP-1 cells rolled on activated (IL-1beta, 4 hours) human umbilical vein endothelial cells (HUVEC) and the number of adhered THP-1 cells were significantly enhanced following the addition of 50 nM of MCP-1 (p < 0.002). In contrast, MCP-1 failed to convert pitavastatin-treated (10 microM, 48 hours) THP-1 rolling to stable adhesion, as compared to baseline adhesion, prior to the addition of MCP-1 (p > 0.4). Pitavastatin-induced changes in THP-1 cells were reversed by treatment with 10 microM of mevalonate, the intermediate of cholesterol biosynthesis. To elucidate the mechanism by which pitavastatin modulates MCP-1-induced THP-1 adhesive interactions, the possible involvement of extracellular signal-regulated kinase 1/2 (ERK1/2) was examined. Western blotting analysis using an anti-ERK1/2 Ab and an antibody against phosphorylated-ERK1/2 (p-ERK) revealed that pitavastatin treatment significantly inhibited the MCP-1-induced phosphorylation of ERK1/2. Further, a RhoA pull-down assay revealed that activation of RhoA GTPase was reduced after pitavastatin treatment. Interestingly, an inhibitor of RhoA GTPase, but not that of the ERK1/2 pathway, attenuated MCP-1-dependent adhesion of THP-1 cells to HUVEC. These findings indicate a role for pitavastatin in modulating the MCP-1-induced phenotypic changes of monocyte-endothelial interactions, which may account for the anti-inflammatory effects of statins.     

Dexamethasone-induced decrease in HMG-CoA reductase and protein-farnesyl transferase activities does not impair ras processing in AR 4-2 J cells

Rat pancreatic acinar cells AR 4-2J respond to dexamethasone by differentiation and a decreased proliferation rate. Protein labelling by [³H]-mevalonolactone, used as a precursor of farnesyl and geranylgeranyl isoprenoid groups, was increased in the presence of dexamethasone. In these same conditions, dexamethasone decreased HMG-CoA reductase activity, leading to a diminished isotopic dilution of the mevalonate precursor. As ras proteins, known to be involved in the regulation of proliferation and differentiation, need to be farnesylated for full biological function, we also measured the level of farnesyl transferase activity and found a dose-dependent decrease in dexamethasone treated cells. Despite these negative effects of dexamethasone on mevalonate pathway, there was no appearance of non-isoprenylated forms of ras, indicating that the level of isoprenoid precursors and farnesyl transferase activity were not limiting in this model.     

Intracerebroventricular and septal injections of arginine vasopressin are not antipyretic in the rabbit

Arginine vasopressin (AVP) has been reported to have an antipyretic effect in the ewe and guinea pig near term. Perfusions with AVP of sites in the septal region also reduced fever in non-pregnant sheep. In the present experiments adult rabbits with third cerebral ventricular or septal cannulas were restrained in a 23°C environment, and rectal temperature was recorded every 10 min. Fever induced by IV administration of leukocytic pyrogen was not reduced by AVP (25–100 ng) given intraventricularly 20 min later. Doses of 1–5 μg AVP injected into the septum likewise were not antipyretic but actually caused an increase in fever. This augmentation of the febrile response is consistent with results of previous studies in this laboratory in which AVP increased hyperthermia in a hot environment and enhanced hyperthermic responses to PGE₂. The data from these experiments provide no evidence that central AVP is an endogenous antipyretic in rabbits; rather, it may be that central AVP augments fever in this species.