The role of protein kinase C in the increased generation in isolated rat hepatocytes of the hydroxyl radical by puromycin aminonucleoside

Puromycin aminonucleoside (PAN) has been known to induce proteinuria. The increased generation of reactive oxygen species (ROS) has been implicated in this toxicity of PAN. We have reported that PAN increases the synthesis of methylguanidine (MG) and creatol which are the products of the reaction of creatinine and the hydroxyl radical in isolated rat hepatocytes. However, the mechanism for the increased ROS induced by PAN is still unclear. In this paper, we investigate the role of protein kinase C (PKC) on the PAN induced reactive oxygen generation in isolated rat hepatocytes. Isolated hepatocytes were incubated in Krebs-Henseleit bicarbonate buffer containing 3% BSA, 16.6 mM creatinine and tested reagents. MG and creatol were determined by high-performance liquid chromatography using 9,10-phenanthrenequinone for the post-labeling. PAN increased MG and creatol synthesis in isolated rat hepatocytes by 60%. 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), a PKC inhibitor, at 10 and 100 μM significantly inhibited MG and creatol synthesis with or without PAN. The inhibition rate is dose dependent from 10 to 100 μM. H1004, a reagent used as control for H-7, did not affect (at 10 μM) or increased little (at 100 μM) the synthesis of MG and creatol. Ro31-8425, a potent PKC inhibitor, significantly inhibited (at 10 μM) MG synthesis in the presence of PAN. PKC in the membrane fraction, a marker of PKC activation, increased over the initial concentration by a factor of 1.65-fold at 60 min incubation and 2.16-fold at 120 min with PAN, while it changed little without PAN. These results indicate that PAN activates PKC resulting in increased hydroxyl radical generation in isolated rat hepatocytes.     

Inhibition by heparin of protein kinase C activation and hydroxyl radical generation in puromycin aminonucleoside treated isolated rat hepatocytes

Heparin has been reported to have many actions similar to calcium-dependent protein kinase (PKC) inhibitors. We have found that puromycin aminonucleoside (PAN) increases hydroxyl radical generation and this was prevented by H-7, a PKC inhibitor in isolated rat hepatocytes. In this study, we investigate the effect of heparin on the increased hydroxyl radical generation as well as PKC activation by PAN in isolated rat hepatocytes. To estimate the amount of hydroxyl radical generation, we measured methylguanidine (MG) and creatol which are the products from the reaction of creatinine and hydroxyl radical. Synthetic rate of MG plus creatol in isolated rat hepatocytes incubated in Krebs-Henseleit bicarbonate buffer containing creatinine and tested reagents were recorded. This rate with or without PAN was 231 ± 11 or 112 ± 5.6 nmol/g wet cells/4 h (mean ± S.E., n = 5), respectively. Heparin concentrations of 3.3, 6.6 and 10 U/ml inhibited MG plus creatol synthesis in the presence of PAN by 30, 38 and 39%, and without PAN by 8.4, 27 and 34%, respectively. Statistical significance was observed except for 3.3 U/ml without PAN. The ratio of PKC in membrane/cytoplasmic fraction, an indicator of PKC activation, increased 2.8- and 3-fold that of the 0 time after 60 and 120 min incubation with PAN while heparin at 10 U/ml almost completely suppressed this increase in the ratio of PKC. The PKC ratio of the membrane/cytoplasmic fraction obtained from hepatocytes with heparin alone or without PAN and heparin was almost unchanged during the tested period. Variation of PKC levels in membrane fraction is similar to that of PKC ratio of the membrane/cytoplasmic fraction. Increased creatol synthesis by PAN and its inhibition by heparin were observed in the same samples as those used for the PKC study.These results indicate that heparin inhibits the increase in hydroxyl radical generation induced by PAN through inhibition of PKC activation in isolated rat hepatocytes.     

Synthesis of creatol, a hydroxyl radical adduct of creatinine and its increase by puromycin aminonucleoside in isolated rat hepatocytes

Creatol is a hydroxyl radical adduct of creatinine and the precursor of methylguanidine (MG), a uremic toxin. We investigate the synthesis of creatol and MG from creatinine and the effect of substances that affect the hydroxyl radical in isolated rat hepatocytes. In the presence of increasing concentrations of creatinine, rising level of creatol were found after 2 h incubation in Krebs-Henseleit bicarbonate buffer. However, further increase of creatol was not observed after 4 and 6 h incubations. On the other hand, MG after 2 h incubation achieved a level of about 50% that of creatol and increased depending on both the creatinine concentration and the incubation period. DMSO, a hydroxyl radical scavenger decreased the generation of creatol and MG by about 50% at 2.5 mM and the inhibition depended on DMSO concentration. Puromycin aminonucleoside (PAN) increased both by about 170%. These findings demonstrated that hepatocytes synthesize creatol prior to MG and are inhibited by a hydroxyl radical scavenger. They also show that PAN increased hydroxyl radical generation in tissue cells.     

Regulation of sterol 27-hydroxylase and an alternative pathway of bile acid biosynthesis in primary cultures of rat hepatocytes

In man, hepatic mitochondrial sterol 27-hydroxylase and microsomal cholesterol 7-hydroxylase initiate distinct pathways of bile acid biosynthesis from cholesterol, the “acidic” and “neutral” pathways, respectively. A similar acidic pathway in the rat has been hypothesized, but its quantitative importance and ability to be regulated at the level of sterol 27-hydroxylase are uncertain. In this study, we explored the molecular regulation of sterol 27-hydroxylase and the acidic pathway of bile acid biosynthesis in primary cultures of adult rat hepatocytes. mRNA and protein turnover rates were approximately 10-fold slower for sterol 27-hydroxylase than for cholesterol 7-hydroxylase. Sterol 27-hydroxylase mRNA was not spontaneously expressed in culture. The sole requirement for preserving sterol 27-hydroxylase mRNA at the level of freshly isolated hepatocytes (0 h) after 72 h was the addition of dexamethasone (0.1 μM; > 7-fold induction). Sterol 27-hydroxylase mRNA, mass and specific activity were not affected by thyroxine (1.0 μM), dibutyryl-cAMP (50 μM), nor squalestatin 1 (150 nM-1.0 μM), an inhibitor of cholesterol biosynthesis. Taurocholate (50 μM), however, repressed sterol 27-hydroxylase mRNA levels by 55%. Sterol 27-hydroxylase specific activity in isolated mitochondria was increased > 10-fold by the addition of 2-hydroxypropyl-β-cyclodextrin. Under culture conditions designed to maximally repress cholesterol 7-hydroxylase and bile acid synthesis from the neutral pathway but maintain sterol 27-hydroxylase mRNA and activity near 0 h levels, bile acid synthesis from [¹⁴C]cholesterol remained relatively high and consisted of β-muricholate, the product of chenodeoxycholate in the rat. We conclude that rat liver harbors a quantitatively important alternative pathway of bile acid biosynthesis and that its initiating enzyme, sterol 27-hydroxylase, may be slowly regulated by glucocorticoids and bile acids.     

The role of cellular oxidases and catalytic iron in the pathogenesis of ethanol-induced liver injury

Free radical generation and catalytic iron have been implicated in the pathogenesis of alcohol-induced liver injury but the source of free radicals is a subject of controversy. The mechanism of ethanol-induced liver injury was investigated in isolated hepatocytes from a rodent model of iron loading in which free radical generation was measured by the determination of alkane production (ethane and pentane). Iron loading (125mg/kg i.p.) increased hepatic non-heme iron 3-fold, increased the prooxidant activity of cytosolic ultrafiltrates 2-fold and doubled ethanol-induced alkane production. The addition of desferrioxamine (20μM), a tight chelator of iron, completely abolished alkane production indicating the importance of catalytic iron. The role of cellular oxidases as a source of ethanol induced free radicals was studied through the use of selective inhibitors. In both the presence and absence of iron loading, selective inhibition of xanthine oxidase with oxipurinol(20μM) diminished ethanol-induced alkane production 0–40%, inhibition of aldehyde oxidase with menadione (20μM) diminished alkane production 36–75%, while the inhibition of aldehyde and xanthine oxidase by feeding tungstate (100mg/kg/day) virtually abolished alkane production. Addition of acetaldehyde(50μM) to hepatocytes generated alkanes at rates comparable to those achieved with ethanol indicating the importance of acetaldehyde metabolism in free radical generation. The cellular oxidases (aldehyde and xanthine oxidase) along with catalytic iron play a fundamental role in the pathogenesis of free radical injury due to ethanol.     

Cyanidin-3-O-beta-glucopyranoside protects myocardium and erythrocytes from oxygen radical-mediated damages

The cyanidin-3- O - β-glucopyranoside (C-3-G) antioxidant capacity towards reactive oxygen species (ROS)-mediated damages was assessed in tissue and cells submitted to increased oxidative stress. In the isolated ischemic and reperfused rat heart, 10 or 30 μM C-3-G protected from both lipid peroxidation (66.7 and 94% inhibition of malondialdehyde (MDA) generation in 10 and 30 μM C-3-G-reperfused hearts, respectively, in comparison with control reperfused hearts) and energy metabolism impairment (higher ATP concentration in 10 and 30 μM C-3-G-reperfused hearts than in control reperfused hearts). These effects were associated to C-3-G permeation within myocardial cells, as indicated by results obtained in the isolated rat heart perfused for 30 min in the recirculating Langendorff mode under normoxia with 10 and 30 μM C-3-G. Protective effects were exerted, in a dose-dependent manner, by C-3-G also in 2 mM hydrogen peroxide-treated human erythrocytes. With respect to MDA formation, an apparent IC 50 of 5.12 μM was calculated for C-3-G (the polyphenol resveratrol used for comparison showed an apparent IC 50 of 38.43 μM). The general indications are that C-3-G (largely diffused in dietary plants and fruits, such as pigmented oranges very common in the Mediterranean diet) represents a powerful natural antioxidant with beneficial effects in case of increased oxidative stress, and at pharmacological concentrations it is able to decrease tissue damages occurring in myocardial ischemia and reperfusion.     

Comparison of hydroxyl radical generation in patients undergoing coronary artery bypass grafting with and without cardiopulmonary bypass

We measured the hydroxyl radical (√OH) generation in fourteen patients undergoing coronary artery bypass grafting (CABG), of whom seven patients underwent on-pump CABG with cardiopulmonary bypass (CPB) and seven patients underwent off-pump CABG without CPB. To detect √OH generation, we measured the urinary excretion of √OH products of creatinine (Cr), creatol (CTL; 5-hydroxycreatinine) and methylguanidine (MG) with HPLC using the one point sampling and collected urine during and after the operation. The urinary CTL value corrected urinary Cr value of on-pump CABG significantly increased about 3-5 times from the beginning of CPB to 4 h after operation compared to the baseline value before CPB in both the collected urine and the one point sampling urine. The urinary MG/Cr value in both groups did not change significantly. Significantly increased √OH generation was found during and soon after on-pump CABG.     

The role of calcium channels in substance P-induced contractile response in the rat iris

This study was undertaken to assess the role of calcium channels in the contractile response induced by substance P in the isolated rat iris. Substance P produced graded and sustained contraction in the rat iris. Pre-incubation of preparations with thapsigargin (1 μM), verapamil (1 μM), isradipine (1 μM) or with ω-conotoxin MCIIA (0.1 μM) did not significantly inhibit substance P-mediated contraction in the isolated rat iris. However, pre-incubation of the preparations with nicardipine (1 μM) or ruthenium red (1 mM) caused parallel displacement to the right of the substance P concentration–response curve without affecting its maximal response. In contrast, amiloride (1 μM), markedly inhibited substance P-mediated contraction (73±5%), while econazole (1 mM) also significantly inhibited (44±11%) substance P-mediated contraction in the isolated rat iris. Collectively, these results suggest that substance P-mediated contractile response in the isolated rat iris depends largely on the influx of external Ca²⁺, by a mechanism which might involve the T-type calcium channels.     

Creatol, an oxidative product of creatinine in hemodialysis patients

Creatol (CTL) is a product resulting from the reaction of creatinine (Cr) with the hydroxyl radical and is identified as a precursor of methylguanidine (MG), a uremic toxin. In this study, we investigated serum CTL levels together with those of Cr and MG in 66 patients who were on maintenance hemodialysis (HD). Prior to dialysis, the mean serum levels of Cr, CTL and MG were 967 (=11.1 mg/dl) ±267 μM, 11.1 ± 4.8μM and 5.8±2.9 μM, respectively. The mean CTL level was about 1.1% that of Cr, and the CTL plus MG level was about 1.4% that of the Cr level. The reduction rates of Cr, CTL and MG by a single HD were 62.6±6.1%, 71.0±10.3% and 51.9±11.6%, respectively. The CTL level at 0.5, 1 and 6h after HD increased rapidly by 20.7±8.7%, 31.7±14.7% and 80.1±27.3%, respectively. There was a significant correlation between CTL or CTL/Cr and parathyroid hormone in patients who had just undergone parathyroidectomy. No significant correlation was found between CTL or CTL/Cr and those factors which seems to be related to the predialysis levels of reactive oxygen. Therefore, because of the good clearance of CTL and its rapid conversion to MG, its usefulness for the estimation of hydroxyl radical generation in HD patients is limited.     

Prolactin-stimulated ornithine decarboxylase induction in rat hepatocytes: coupling to diacylglycerol generation and protein kinase C

The trophic effects of prolactin (PRL) in rat liver have been linked to activation of protein kinase C (PKC). Since alterations in PKC activity imply its activation by 1,2-diacylglycerol (DAG), we tested whether PRL treatment stimulated DAG generation coupled to induction of a growth response in primary hepatocytes. Addition of PRL to hepatocyte cultures significantly increased [3H]-glycerol incorporation into DAG within 5 minutes which was followed by a loss of cytosolic PKC activity by 10 minutes. Prolactin also significantly enhanced radiolabel incorporation into triacylglycerol and phospholipids within 10 minutes and induced ODC activity at 6 hours. Therefore, prolactin-stimulated alterations in PKC activity are preceded by enhanced DAG generation. Moreover, these events appear to be coupled to PRL-stimulated entry of hepatocytes into cell cycle.     

Aldosterone modulates neural vasomotor response in hypertension: role of calcitonin gene-related peptide

Objective: We analyse the effect of aldosterone on vasomotor response induced by electrical field stimulation (EFS) in mesenteric arteries from Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR). Results: Aldosterone (0.001–1 μM) reduced vasoconstrictor response to EFS in a dose- and time-dependent manner only in SHR. Thus, the rest of experiments were performed only in SHR. Aldosterone did not affect either noradrenaline response or release. Effect of aldosterone (1 μM) on EFS response was not affected by N^G-nitro-arginine-methyl esther (100 μM), and was abolished by capsaicin (0.5 μM) and the calcitonin gene-related peptide antagonist (CGRP 8–37, 0.5 μM). Calcitonin gene-related peptide (0.1 nM–0.1 μM) induced a concentration-dependent relaxation, which was enhanced by aldosterone (1 μM). Incubation with either spironolactone (1 μM), glibenclamide (10 μM), RU 486 10 μM, ODQ (10 μM) or cycloheximide (10 μM) significantly reduced the enhancement of CGRP-relaxation produced by aldosterone, while remained unmodified by SQ 22,536. Conclusions: Aldosterone decreases the vasoconstrictor response to EFS in mesenteric arteries from SHR but not from WKY. This effect is mediated by an increased response to the sensory neurotransmitter CGRP, substantially, through glucocorticoid receptors activation. Furthermore, this effect is mediated by an increase of cGMP synthesis and ATP-dependent potassium channel activation.     

Effect of dicumarol,a Nad(P)h: quinone acceptor oxidoreductase 1 (DT-diaphorase) inhibitor on ubiquinone redox cycling in cultured rat hepatocytes

Ubiquinol is considered to serve as an endogenous antioxidant. However, the mechanism by which the redox state of intracellular ubiquinone (UQ) is maintained is not well established. The effect of dicumarol, an inhibitor of NAD(P)H: quinone acceptor oxidoreductase 1 (NQO1=DT-diaphorase, EC 1.6.99.2), on the reduction of UQ in cultured rat hepatocytes was investigated in order to clarify whether or not NQO1 is involved in reducing intracellular UQ. A concentration of 5 μM dicumarol, which does not inhibit cytosolic NADPH-dependent UQ reductase in vitro , was observed to almost completely inhibit NQO1 and thereby to stimulate cytotoxicity of 2-methyl-1,4-naphthoquinone (menadione) in cultured rat hepatocytes. However, 5 μM dicumarol did not inhibit reduction of endogenous UQ-9, as well as exogenous UQ-10 added to the hepatocytes. In addition, it did not stimulate the formation of thiobarbituric acid reactive substances (TBARS) in the hepatocytes. These results suggested that NQO1 is not involved in maintaining UQ in the reduced state in the intact liver cells.     

Genotoxic properties of 4-hydroxyalkenals and analogous aldehydes

4-Hydroxynonenal (HNE), one of the major products of lipid peroxidation, has been demonstrated to induce genotoxic effects in the micromolar range. HNE has too structural domains, a lipophilic tail and a polar head with three functional groups: the aldehyde and hydroxy groups and the trans CC double bond. To evaluate their relative importance, the genotoxic effects of HNE were compared with those of the homologous aldehydes 4-hydroxyhexenal and 4-hydroxyundecenal (different lengths of the lipophilic tail), and the analogous aldehydes 2-trans-nonenal (lacking the OH group) and nonanal (lacking the OH group and the trans CC double bond). This investigation was carried out on primary cultures of adult rat hepatocytes in order to further determine the influence of biotransformation- and/or detoxification reactions.

A 3-h treatment with HNE induces statistically significant levels of SCE at concentrations ≥0.1 μM, micronuclei at concentrations ≥ 1 μM and chromosomal aberrations at a concentration of 10 μM. Compared to HNE the homologous aldehydes induced a significant genotoxic effect at higher concentrations. Statistically significant increases in SCE frequency were obtained at concentrations ≥ 1 μM for 4-hydroxyundecenal and at a concentration of 10 μM for 4-hydroxyhexenal. The induction of chromosomal aberrations was significantly elevated at concentrations of ≥ 10 μM and 10 μM for 4-hydroxyhexenal and 4-hydroxyundecenal, respectively. Except for a 4-hydroxyhexenal concentration of 1 μM, both aldehydes did not induce statistically significant levels of micronucleis.

The HNE analogous aldehydes 2-trans-nonenal and nonanal induced statistically significant frequencies of SCE at concentrations of ≥ 1 μM (nonanal) and ≥ 10 μM (2-trans-nonenal). No significant induction of chromosomal aberrations or micronuclei could be demonstrated.

The structure of the aldehydes investigated appears to influence the cyto- and genotoxic potential in the following ways. (1) The lenght of the lipophilic tail has no influence on chromosomal aberration induction, but appears to determine the yield of SCE and micronuclei, and the cytotoxic potential. (2) The lack of the OH group (2-trans-nonenal) reduces the SCE-inducing potential of the aldehyde shifting the dose-effect curve to higher concentrations. The similar shape compared to SCE induction by HNE indicates that possibly the same active metabolite is formed. (3) The lack of both the OH group and the CC double bond (nonanal) does not result in a complete loss of the SCE-inducing activity. The different shape of the dose-response curve suggests a different metabolism and/or a different mode of interaction with DNA.     

Effects of hemin on rat liver cyclic AMP-dependent protein kinases in cell extracts and intact hepatocytes

Cyclic AMP-dependent protein kinases I and II, partially purified from rat liver cytosol, were inhibited 50% by 40 μM hemin and 100 μM hemin, respectively. With the purified catalytic subunit of cyclic AMP-dependent protein kinase, hemin caused non-competitive inhibition with respect to the peptide substrate and mixed inhibition with respect to ATP. Hemin also inhibited purified phosphorylase b kinase, indicating that hemin concentrations above 10 μM markedly inhibit multiple protein kinases. In isolated intact hepatocytes, hemin inhibited the glucagon-dependent activation of cyclic AMP-dependent protein kinases and the activation of glycogen phosphorylase. For both effects, high heme concentrations (40–60 μM) were required for 50% inhibition. Similar high levels of exogenous hemin inhibited total hepatocyte protein synthesis. By contrast, 5 μM hemin or less was sufficient to raise intracellular heme levels, as indicated by the relative heme-saturation of tryptophan oxygenase in hepatocytes. Hemin, 5 μM, completely repressed induction of 5-aminolevulinate synthase by dexamethasone in hepatocyte primary cultures. Such repression is unlikely to be mediated by inhibition of protein kinases.     

Signal transduction mechanism leading to enhanced proliferation of primary cultured adult rat hepatocytes treated with royal jelly 57-kDa protein

A 57-kDa protein in royal jelly (RJ) was previously shown to stimulate hepatocyte DNA synthesis and prolongs the proliferation of hepatocytes as well as increasing albumin production [Kamakura, M., Suenobu, N., and Fukushima, M. (2001) Biochem. Biophys. Res. Commun. 282, 865-874]. In this study, I investigated the signal transduction mechanisms involved in the induction of hepatocyte DNA synthesis and the promotion of cell survival by this 57-kDa protein in primary cultures of adult rat hepatocytes. Hepatocyte DNA synthesis induced by the 57-kDa protein was not influenced by several alpha- and beta-adrenoceptor antagonists, but was dose-dependently abolished by an inhibitor of a tyrosine-specific protein kinase, genistein. A phospholipase C inhibitor (U-73122) and a protein kinase C (PKC) inhibitor (sphingosine) inhibited 57-kDa protein-stimulated he-patocyte DNA synthesis, whereas a protein kinase A inhibitor (H-89) did not. The 57-kDa protein also activated PKC in rat hepatocytes. Various inhibitors of intracellular signal transduction elements (PD98059, p21 ras farnesyltransferase inhibitor, wortmannin and rapamycin) also blocked hepatocyte DNA synthesis induced by the 57-kDa protein. Furthermore, the 57-kDa protein activated mitogen-activated protein (MAP) kinase in rat hepatocytes. The activation of MAP kinase by the 57-kDa protein was inhibited by PD98059 and sphingosine. The 57-kDa protein also activated protein kinase B, which is a key regulator of cell survival. These results suggest that, like growth factors, the 57-kDa protein activates several important intracellular signaling factors involved in the stimulation of hepatocyte DNA synthesis and the protection of cells from apoptosis.     

Biphasic regulation by N, 2′-O-dibutyryl adenosine 3′, 5′-cyclic monophosphate (dbcAMP) of steroid 21-hydroxylase activity in rat hepatocytes

Steroid 21-hydroxylase activity has been identified in many tissues, including liver. But it is possible that the enzyme found in the liver is different from adrenal 21-hydroxylase. In the adrenal cortex, steroid 21-hydroxylase activity is increased by corticotropin (ACTH); the effect of ACTH is mediated by cyclic AMP (cAMP), and presumably involves a cAMP-dependent protein kinase (PKA). It is not yet clear, however, how extra-adrenal steroid 21-hydroxylase activity is regulated. In the present study, we examined the effect of N⁶, 2′-O-dibutyryl adenosine 3′,5′-cyclic monophosphate (dbcAMP), forskolin, N-[2-(methylamino)ethyl]5-isoquinolinesulfonamide (H-8) and 12-O-tetradecanoylphorbol-13-acetate (TPA) on steroid 21-hydroxylase activity in primary cultures of rat hepatocytes to determine the nature of regulation of extra-adrenal steroid 21-hydroxylase activity. Steroid 21-hydroxylase activity in hepatocytes incubated with 10⁻¹¹M dbcAMP for 24 h was 1.6 times higher than that in control hepatocytes untreated with dbcAMP. On the other hand, steroid 21-hydroxylase activity decreased by 20 and 50% when the cells were incubated with 10⁻⁵ and 10⁻³ M dbcAMP, respectively. The stimulatory effect of 10⁻¹¹ M dbcAMP was not blocked by 10⁻⁵ M H-8 (PKA inhibitor), but the inhibitory effect of 10⁻⁵ or 10⁻³ M cAMP was. TPA did not alter the activity of steroid 21-hydroxylase. These findings indicate that the steroid 21-hydroxylase in rat liver is regulated by mechanisms different from those in the adrenal glands.     

Genistein affects lipogenesis and lipolysis in isolated rat adipocytes

Genistein is a phytoestrogen found in several plants eaten by humans and food-producing animals and exerting a wide spectrum of biological activity. In this experiment, the impact of genistein on lipogenesis and lipolysis was studied in isolated rat adipocytes. Incubation of the cells (10⁶ cells/ml in plastic tubes at 37°C with Krebs-Ringer buffer, 90 min) with genistein (0.01, 0.3, 0.6 and 1 mM) clearly restricted (1 nM) [U-¹⁴C]glucose conversion to total lipids in the absence and presence of insulin. When [¹⁴C]acetate was used as the substrate for lipogenesis, genistein (0.01, 0.1 and 1 mM) exerted a similar effect. Thus, the anti-lipogenetic action of genistein may be an effect not only of alteration in glucose transport and metabolism, but this phytoestrogen can also restrict the fatty acids synthesis and/or their estrification. Incubation of adipocytes with estradiol at the same concentrations also resulted in restriction of lipogenesis, but the effect was less marked. Genistein (0.1 and 1 mM) augmented basal lipolysis in adipocytes. This process was strongly restricted by insulin (1 μM) and H-89 (an inhibitor of protein kinase A; 50 μM) and seems to be primarily due to the inhibitory action of the phytoestrogen on cAMP phosphodiesterase in adipocytes. Genistein at the smallest concentration (0.01 mM) augmented epinephrine-stimulated (1 μM) lipolysis but failed to potentiate lipolysis induced by forskolin (1 μM) or dibutyryl-cAMP (1 mM). These results suggest genistein action on the lipolytic pathways before activation of adenylate cyclase. The restriction of lipolysis stimulated by several lipolytic agents – epinephrine, forskolin and dibutyryl-cAMP were observed when adipocytes were incubated with genistein at highest concentrations (0.1 and 1 mM). These results prove the inhibitory action of this phyestrogen on the final steps of the lipolytic cascade, i.e. on protein kinase A or hormone sensitive lipase. Estradiol, added to the incubation medium, did not affect lipolysis. It can be concluded that genistein significantly affects lipogenesis and lipolysis in isolated rat adipocytes.     

Identification and characterization of a small molecule AMPK activator that treats key components of type 2 diabetes and the metabolic syndrome

AMP-activated protein kinase (AMPK) is a key sensor and regulator of intracellular and whole-body energy metabolism. We have identified a thienopyridone family of AMPK activators. A-769662 directly stimulated partially purified rat liver AMPK (EC₅₀ = 0.8 μM) and inhibited fatty acid synthesis in primary rat hepatocytes (IC₅₀ = 3.2 μM). Short-term treatment of normal Sprague Dawley rats with A-769662 decreased liver malonyl CoA levels and the respiratory exchange ratio, VCO₂/VO_2, indicating an increased rate of whole-body fatty acid oxidation. Treatment of ob/ob mice with 30 mg/kg b.i.d. A-769662 decreased hepatic expression of PEPCK, G6Pase, and FAS, lowered plasma glucose by 40%, reduced body weight gain and significantly decreased both plasma and liver triglyceride levels. These results demonstrate that small molecule-mediated activation of AMPK in vivo is feasible and represents a promising approach for the treatment of type 2 diabetes and the metabolic syndrome.     

Oxidant-sensitive protein phosphorylation in endothelial cells

Reactive oxygen is an important regulator of vascular cell biology; however, the mechanisms involved in transducing signals from oxidants in endothelial cells are poorly defined. Because protein phosphorylation is a major mechanism for signal ransduction, cultured aortic endothelial cells were exposed to nonlethal concentrations of H₂O₂ to examine oxidant-sensitive changes in phosphorylation state. Addition of H₂O₂ increases the phosphorylation of the heat shock protein 27 (HSP27) within 2 min. This response is maximal by 20 min and remains constant for more than 45 min. Levels of intrcellular free Ca²⁺ in endothelial cells did not change following addition of 100 μM H₂O₂, nor did the ability of the cells to respond to bradykinin. H₂O₂-induced phosphorylations were either not affected or were slightly increased in cells pretreated with PKC inhibitors (H-8, staurosporin, or calphostin c). Two-dimensional analysis of phosphoproteins from homogenates of ³²P-labeled cells revealed that phorbol myristate acetate (PMA) did not cause the same degree of HSP27 phosphorylation as H₂O₂. Simultaneous addition of 10 ηM PMA and 50 μM H₂O₂ decreased the oxidant-stimulated phoshorylation of the most acidic HSP27 isoform. These data suggest that signal transduction for H₂O₂-sensitive endothelial cell responses are not only independent of PKC, but may also be suppressed by the action of the kinase.     

Effects of cholecystokinin peptides and neurotensin on dopamine release and metabolism in the rostral and caudal part of the nucleus accumbens using intracerebral dialysis in the anaesthetized rat

By means of intracerebral microdialysis effects of cholecystokinin peptides and neurotensin administered via the microdialysis probe have been studied on dopamine release and metabolism in the nucleus accumbens and neostriatum of the halothane anaesthetized male rat. Levels of extra cellular dopamine (DA) and its metabolites 3,4 dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were assessed in nuc. accumbens (rostral and caudal part) using high performance liquid chromatography in combination with electrochemical detection.

(1) In the rostral part of the nuc. accumbens CCK-8 (10 and 100 μM), CCK-33 (100 μM) but not CCK-4 (10 and 100 μM) increased the levels of DA in the perfusate without increasing the extracellular levels of DOPAC and HVA. (2) In the caudal nuc. accumbens CCK-8 and CCK-4 in concentrations of 10 μM and 100 μ M of CCK-33 had no effect on DA release and metabolism, since the extracellular levels of DA, DOPAC and HVA were not changed. (3) In the rostral nuc. accumbens perfusion with 10 μM of neurotensin but not with any other concentration of neurotensin (0.01, 0.1, 1 and 100 μM) increased the levels of DA in the extracellular fluid. (4) In the caudal nuc. accumbens a 40 min perfusion with neutrotensin produced a concentration dependent increase of the levels of DA in the perfusate (peak action at 10 μ M) which in this case was associated with increases in the extracellular levels of DOPAC and HVA. (5) By means of receptor autoradiography using (3-[¹²⁵I]iodotyrosyl³) neurotensin it was found that a 40 min perfusion with this radioligand in the rostral nuc. accumbens reached a total volume of 0.051 mm³. The diffusion of the radioligand was limited to the rostral or caudal part of the nuc. accumbens depending upon the site of placement of the dialysis probe.

The results indicate the existence of cholecystokinin (CCK) receptors in the rostral nuc. accumbens, which are sensitive to CCK-8 and CCK-33 but not to CCK-4, and which facilitate DA release without producing any detectable increase in DA metabolites. In contrast, such receptors do not appear to play a similar role in the regulation of DA release in the caudal nuc. accumbens, where DA terminals contain CCK-like immunoreactivity. Furthermore, the results indicate that neurotensin receptors exist both in the rostral and caudal nuc. accumbens, where they inter alia enhance the release of DA. In the caudal nuc. accumbens these effects of neurotensin are also associated with an increase of DA metabolites, possibly suggesting that in this region neurotensin receptors may also control DA synthesis.