Inhibition of vascular smooth muscle cell proliferation by chronic hemin treatment

Hemin, an oxidized form of heme, is an essential regulator of gene expression and cell cycle progression. Our laboratory previously reported (34) that chronic hemin treatment of spontaneously hypertensive rats reversed the eutrophic inward remodeling of small peripheral arteries. Whether long-term treatment of cultured vascular smooth muscle cells (VSMCs) with hemin alters the proliferation status of these cells has been unknown. In the present study, hemin treatment at 5 muM for 4, 7, 14, and 21 days significantly inhibited the proliferation of cultured rat aortic VSMCs (A-10 cells) by arresting cells at G0/G1 phases so as to decelerate cell cycle progression. Heme oxygenase (HO) activity and inducible HO-1 protein expression were significantly increased by hemin treatment. HO inhibitor tin protoporphyrin IX (SnPP) abolished the effects of hemin on cell proliferation and HO activity. Interestingly, hemin-induced HO-1 expression was further increased in the presence of SnPP. Hemin treatment had no significant effect on the expression of constitutive HO-2. Expression of p21 protein and the level of reactive oxygen species (ROS) were decreased by hemin treatment, which was reversed by application of SnPP. After removal of hemin from culture medium, inhibited cell proliferation and increased HO-1 expression in VSMCs were returned to control level within 1 wk. Transfection with HO-1 small interfering RNA significantly knocked down HO-1 expression and decreased HO activity, but had no effect on HO-2 expression, in cells treated with or without hemin for 7 days. The inhibitory effect of hemin on cell proliferation was abolished in HO-1 silenced cells. It is concluded that induction of HO-1 and, consequently, increased HO activity are responsible for the chronic inhibitory effect of hemin on VSMC proliferation. Changes in the levels of p21 and ROS might also participate in the cellular effects of hemin.     

Heme oxygenase inhibits human airway smooth muscle proliferation via a bilirubin-dependent modulation of ERK1/2 phosphorylation

The aim of this study was to investigate whether the heme oxygenase (HO) pathway could modulate proliferation of airway smooth muscle (ASM) and the mechanism(s) involved in this phenomenon. In cultured human ASM cells, 10% fetal calf serum or 50 ng/ml platelet-derived growth factor AB induced cell proliferation, extracellular and intracellular reactive oxygen species (ROS) production and ERK1/2 phosphorylation. Pharmacological HO-1 induction (by 10 microm hemin or by 20 microm cobalt-protoporphyrin) and HO inhibition (by 25 microm tin-protoporphyrin or by an antisense oligonucleotide), respectively, reduced and enhanced significantly both cell proliferation and ROS production. Neither the carbon monoxide scavenger myoglobin (5-20 microm) nor the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one could reverse ASM proliferation induced by tin-protoporphyrin, making a role of the CO-cGMP pathway in HO-modulated proliferation unlikely. By contrast, bilirubin (1 microm) and the antioxidant N-acetyl-cysteine (1 mm) significantly reduced mitogen-induced cell proliferation, ROS production, and ERK1/2 phosphorylation. Furthermore, both bilirubin and N-acetyl-cysteine and the ERK1/2 inhibitor PD98059 significantly reversed the effects of HO inhibition on ASM proliferation. These results could be relevant to ASM alterations observed in asthma because activation of the HO pathway prevented the increase in bronchial smooth muscle area induced by repeated ovalbumin challenge in immunized guinea pigs, whereas inhibition of HO had the opposite effect. In conclusion, this study provides evidence for an antiproliferative effect of the HO pathway in ASM in vitro and in vivo through a bilirubin-mediated redox modulation of phosphorylation of ERK1/2.     

Management of Oxidative Stress by Heme Oxygenase-1 in Cisplatin-induced Toxicity in Renal Tubular Cells

Induction of heme oxygenase-1 (HO-1) may serve as an immediate protective response during treatment with the cytostatic drug cisplatin (CDDP). Oxidative pathways participate in the characteristic nephrotoxicity of CDDP. In the present study, cultured tubular cells (LLC-PK1) were used to investigate whether induction of HO provided protection against CDDP by maintaining the cellular redox balance. The antioxidants, &#102 -tocopherol (TOCO) and N -acetylcysteine (NAC), were used to demonstrate that elevation of ROS levels contribute to the development of CDDP-induced cytotoxicity. Chemical modulators of HO activity were used to investigate the role of HO herein. Hemin was used to specifically induce HO-1, while exposure of the cells to tin-protoporphyrin (SnPP) was shown to inhibit HO activity. Hemin treatment prior to CDDP-exposure significantly decreased the generation of ROS to control levels, while inhibition of HO increased the ROS levels beyond the levels measured in cells treated with CDDP alone. Furthermore, HO induction protected significantly against the cytotoxicity of CDDP, although this protection was limited. Similar results were obtained when the cells were preincubated with TOCO, suggesting that mechanisms other than impairment of the redox ratio are important in CDDP-induced loss of cell viability in vitro. In addition, SnPP treatment exacerbated the oxidative response and cytotoxicity of CDDP, especially at low CDDP concentrations. We therefore conclude that HO is able to directly limit the CDDP-induced oxidative stress response and thus serves as safeguard of the cellular redox balance.     

Management of oxidative stress by heme oxygenase-1 in cisplatin-induced toxicity in renal tubular cells

Induction of heme oxygenase-1 (HO-1) may serve as an immediate protective response during treatment with the cytostatic drug cisplatin (CDDP). Oxidative pathways participate in the characteristic nephrotoxicity of CDDP. In the present study, cultured tubular cells (LLC-PK1) were used to investigate whether induction of HO provided protection against CDDP by maintaining the cellular redox balance. The antioxidants, alpha-tocopherol (TOCO) and N-acetylcysteine (NAC), were used to demonstrate that elevation of ROS levels contribute to the development of CDDP-induced cytotoxicity. Chemical modulators of HO activity were used to investigate the role of HO herein. Hemin was used to specifically induce HO-1, while exposure of the cells to tin-protoporphyrin (SnPP) was shown to inhibit HO activity. Hemin treatment prior to CDDP-exposure significantly decreased the generation of ROS to control levels, while inhibition of HO increased the ROS levels beyond the levels measured in cells treated with CDDP alone. Furthermore, HO induction protected significantly against the cytotoxicity of CDDP, although this protection was limited. Similar results were obtained when the cells were preincubated with TOCO, suggesting that mechanisms other than impairment of the redox ratio are important in CDDP-induced loss of cell viability in vitro. In addition, SnPP treatment exacerbated the oxidative response and cytotoxicity of CDDP, especially at low CDDP concentrations. We therefore conclude that HO is able to directly limit the CDDP-induced oxidative stress response and thus serves as safeguard of the cellular redox balance.     

Interaction between endothelial heme oxygenase-2 and endothelin-1 in altered aortic reactivity after hypoxia in rats

The aim of this study was to determine whether increased expression of heme oxygenase (HO) contributes to impairment of aortic contractile responses after hypoxia through effects on reactivity to endothelin-1 (ET-1). Thoracic aortas from normoxic rats and rats exposed to hypoxia (10% O2) for 16 or 48 h were mounted in organ bath myographs for contractile studies, fixed in paraformaldehyde, or frozen in liquid nitrogen for protein extraction. In rings from normoxic rats, the HO inhibitor tin protoporphyrin IX (SnPP IX, 10 microM) did not alter the response to phenylephrine or ET-1. In rings from rats exposed to 16-h hypoxia, maximum tension generated in response to these agonists was higher in endothelium-intact but not -denuded rings in the presence of SnPP IX. In rings from rats exposed to 48-h hypoxia SnPP IX increased contraction in endothelium-intact but not -denuded rings. In endothelium-intact aortic rings from rats exposed to 16-h hypoxia incubated with endothelin A receptor-specific antagonist BQ-123 (10(-7) M), SnPP IX did not alter phenylephrine-induced contraction. Aortic ET-1 protein levels, measured by radioimmunoassay, were increased in rats exposed to hypoxia for 16 and 48 h. Western blotting showed that HO-1 and HO-2 protein were increased after 16 h of hypoxia and returned to near-control levels after 48 h. Increase in HO-1 protein was detected in endothelium-intact and -denuded rings. Removal of endothelium abolished the increase in HO-2 immunoreactivity. Immunohistochemistry localized expression of HO-1 protein to vascular smooth muscle, whereas HO-2 was only detected in endothelium. HO-2 is expressed by aortic endothelial cells early during hypoxic exposure and impairs ET-1-mediated potentiation of contraction to alpha-adrenoceptor stimulation.     

Airway smooth muscle cell tone amplifies contractile function in the presence of chronic cyclic strain

Chronic contractile activation, or tone, in asthma coupled with continuous stretching due to breathing may be involved in altering the contractile function of airway smooth muscle (ASM). Previously, we (11) showed that cytoskeletal remodeling and stiffening responses to acute (2 h) localized stresses were modulated by the level of contractile activation of ASM. Here, we investigated if altered contractility in response to chronic mechanical strain was dependent on repeated modulation of contractile tone. Cultured human ASM cells received 5% cyclic (0.3 Hz), predominantly uniaxial strain for 5 days, with once-daily dosing of either sham, forskolin, carbachol, or histamine to alter tone. Stiffness, contractility (KCl), and "relaxability" (forskolin) were then measured as was cell alignment, myosin light-chain phosphorylation (pMLC), and myosin light-chain kinase (MLCK) content. Cells became aligned and baseline stiffness increased with strain, but repeated lowering of tone inhibited both effects (P < 0.05). Strain also reversed a negative tone-modulation dependence of MLCK, observed in static conditions in agreement with previous reports, with strain and tone together increasing both MLCK and pMLC. Furthermore, contractility increased 176% (SE 59) with repeated tone elevation. These findings indicate that with strain, and not without, repeated tone elevation promoted contractile function through changes in cytoskeletal organization and increased contractile protein. The ability of repeated contractile activation to increase contractility, but only with mechanical stretching, suggests a novel mechanism for increased ASM contractility in asthma and for the role of continuous bronchodilator and corticosteroid therapy in reversing airway hyperresponsiveness.     

TNF-[alpha] modulates murine tracheal rings responsiveness to G-protein-coupled receptor agonists and KCl.

Although the mechanisms that underlie airway hyperresponsiveness in asthma are complex and involve a variety of factors, evidence now suggests that intrinsic abnormalities in airway smooth muscle (ASM) may play an important role. We previously reported that TNF-alpha, a cytokine involved in asthma, augments G-protein-coupled receptor (GPCR) agonist-evoked calcium responses in cultured ASM cells. Here we have extended our previous studies by investigating whether TNF-alpha also modulates the contractile and relaxant responses to GPCR activation using cultured murine tracheal rings. We found that in tracheal rings treated with 50 ng/ml TNF-alpha, carbachol-induced isometric force was significantly increased by 30% compared with those treated with diluent alone (P < 0.05). TNF-alpha also augmented KCl-induced force generation by 70% compared with rings treated with diluent alone (P < 0.01). The enhancing effect of TNF-alpha on carbachol-induced isometric force generation was completely abrogated in the tracheal rings obtained from TNF-alpha receptor (TNFR)1-deficient mice and in control rings treated with a TNF-alpha mutant that solely activates TNFR2. TNF-alpha also attenuated relaxation responsiveness to isoproterenol but not to PGE2 or forskolin. TNF-alpha modulatory effects on GPCR-induced ASM responsiveness were completely abrogated by pertussis toxin, an inhibitor of Gialpha proteins. Taken together, these data suggest that TNF-alpha may participate in the development of airway hyperresponsiveness in asthma via the modulation of ASM responsiveness to both contractile and beta-adrenoceptor GPCR agonists.     

Extracellular matrix proteins differentially regulate airway smooth muscle phenotype and function

Changes in the ECM and increased airway smooth muscle (ASM) mass are major contributors to airway remodeling in asthma and chronic obstructive pulmonary disease. It has recently been demonstrated that ECM proteins may differentially affect proliferation and expression of phenotypic markers of cultured ASM cells. In the present study, we investigated the functional relevance of ECM proteins in the modulation of ASM contractility using bovine tracheal smooth muscle (BTSM) preparations. The results demonstrate that culturing of BSTM strips for 4 days in the presence of fibronectin or collagen I depressed maximal contraction (E(max)) both for methacholine and KCl, which was associated with decreased contractile protein expression. By contrast, both fibronectin and collagen I increased proliferation of cultured BTSM cells. Similar effects were observed for PDGF. Moreover, PDGF augmented fibronectin- and collagen I-induced proliferation in an additive fashion, without an additional effect on contractility or contractile protein expression. The fibronectin-induced depression of contractility was blocked by the integrin antagonist Arg-Gly-Asp-Ser (RGDS) but not by its negative control Gly-Arg-Ala-Asp-Ser-Pro (GRADSP). Laminin, by itself, did not affect contractility or proliferation but reduced the effects of PDGF on these parameters. Strong relationships were found between the ECM-induced changes in E(max) in BTSM strips and their proliferative responses in BSTM cells and for E(max) and contractile protein expression. Our results indicate that ECM proteins differentially regulate both phenotype and function of intact ASM.     

Presoaking with hemin improves salinity tolerance during wheat seed germination

The aim of this study was to investigate whether presoaking with hemin, an inducer of heme oxygenase-1 (HO-1), could alleviate salinity damage during wheat seed germination in comparison with the pretreatment of a well-known nitric oxide (NO) donor sodium nitroprusside (SNP). The results showed that, compared with the samples upon 150 mM NaCl salt stress alone, both 10 ??M hemin and 200 ??M SNP pretreatments could (1) significantly attenuate the inhibition of seed germination and thereafter seedling growth; (2) induce HO expression; (3) enhance amylase activity, thus accelerating the formation of reducing sugar and total soluble sugar; and (4) increase the potassium (K) to sodium (Na) ratio, particularly in the shoot parts. Hemin and SNP could also increase antioxidant enzyme activities, including superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (POD), and ascorbate peroxidase (APX), thus resulting in the alleviation of oxidative damage, as indicated by the decrease of thiobarbituric acid reactive substances (TBARS) content. Moreover, semi-quantitative RT-PCR and isozymatic analysis illustrated that hemin and SNP pretreatment were able to up-regulate the expression of Mn-SOD (especially) and Cu/Zn-SOD gene, and activate SOD isozymatic activities. Since the addition of the NO scavenger methylene blue (MB) differentially reversed the above effects, the protective roles of hemin might be related to the induction of endogenous NO signal. Meanwhile, hemin-driven NO production was confirmed. Together, these results indicated that hemin exerted an advantageous effect on enhancing salinity tolerance during wheat seed germination, which might interact with NO.     

Catalase and superoxide dismutase in the cells of strictly anaerobic microorganisms

Strictly anaerobic microorganisms relating to various physiological groups were screened for catalase and superoxide dismutase (SOD) activity. All of the investigated anaerobes possessed the SOD activity, necessary for protection against toxic products of oxygen reduction. High specific activities of SOD were found in Acetobacterium woodii and Acetobacterium wieringae. Most of the investigated clostridia and acetogens were catalase-negative. A significant activity of catalase was found in Thermohydrogenium kirishiense, in representatives of the genus Desulfotomaculum, and in several methanogens. Methanobrevibacter arboriphilus had an exceptionally high catalase activity after growth in medium supplemented with hemin. Hemin also produced a strong positive effect on the catalase activity in many other anaerobic microorganisms. In methanogens, the activities of the enzymes of antioxidant defense varied in wide ranges depending on the stage of growth and the energy source.     

TAK1 plays a major role in growth factor-induced phenotypic modulation of airway smooth muscle

Increased airway smooth muscle (ASM) mass is a major feature of airway remodeling in asthma and chronic obstructive pulmonary disease. Growth factors induce a proliferative ASM phenotype, characterized by an increased proliferative state and a decreased contractile protein expression, reducing contractility of the muscle. Transforming growth factor-β-activated kinase 1 (TAK1), a mitogen-activated protein kinase kinase kinase, is a key enzyme in proinflammatory signaling in various cell types; however, its function in ASM is unknown. The aim of this study was to investigate the role of TAK1 in growth factor-induced phenotypic modulation of ASM. Using bovine tracheal smooth muscle (BTSM) strips and cells, as well as human tracheal smooth muscle cells, we investigated the role of TAK1 in growth factor-induced proliferation and hypocontractility. Platelet-derived growth factor- (PDGF; 10 ng/ml) and fetal bovine serum (5%)-induced increases in DNA synthesis and cell number in bovine and human cells were significantly inhibited by pretreatment with the specific TAK1 inhibitor LL-Z-1640-2 (5Z-7-oxozeaenol; 100 nM). PDGF-induced DNA synthesis and extracellular signal-regulated kinase-1/2 phosphorylation in BTSM cells were strongly inhibited by both LL-Z-1640-2 pretreatment and transfection of dominant-negative TAK1. In addition, LL-Z-1640-2 inhibited PDGF-induced reduction of BTSM contractility and smooth muscle α-actin expression. The data indicate that TAK1 plays a major role in growth factor-induced phenotypic modulation of ASM.     

Decrease in hepatic cytochrome P-450 and catalase following allylisopropylacetamide: The effect of concomitant hemin administration

It is known that administration of allylisopropylacetamide (AIA) to rats increases δ-aminolevulinic acid synthetase (ALA-S), urine ascorbic acid excretion and decreases hepatic hemoproteins cytochrome P-450 and catalase. Hemin has been shown to inhibit induction of ALA-S by AIA. To investigate further the regulatory role of hemin, AIA was administered to rats over a 5 day period with and without hemin. Hemin did not prevent the decrease in P-450 or catalase caused by AIA but partially inhibited induction of ALA-S and the increase in ascorbic acid excretion. It is unlikely that the hemin is replacing endogenous heme in hemoprotein synthesis and we conclude that the role of hemin in suppressing induction phenomena that follow AIA treatment reflects a regulatory function not related to a role in hemoprotein synthesis.     

The protection by heme oxygenase‐1 induction against thioacetamide‐induced liver toxicity is associated with changes in arginine and asymmetric dimethylarginine

This study was designed to investigate the role of HO‐1 induction in prevention of thioacetamide (TAA)‐induced oxidative stress, inflammation and liver damage. The changes in hepatic dimethylarginine dimethylaminohydrolase (DDAH) activity as well as plasma arginine and asymmetric dimethylarginine (ADMA) levels were also measured to evaluate nitric oxide (NO) bioavailability. Rats were divided into four groups as control, hemin, TAA and hemin + TAA groups. Hemin (50 mg kg^?1, i.p.) was injected to rats 18 h before TAA treatment to induce HO‐1 enzyme expression. Rats were given TAA (300 mg kg^?1, i.p.) and killed 24 h after treatment. Although TAA treatment produced severe hepatic injury, upregulation of HO‐1 ameliorated TAA‐induced liver damage up to some extent as evidence by decreased serum alanine transaminase, aspartate transaminase and arginase activities and histopathological findings. Induction of HO‐1 stimulated antioxidant system and decreased lipid peroxidation in TAA‐treated rats. Myeloperoxidase activity and inducible NO synthase protein expression were decreased, whereas DDAH activity was increased by hemin injection in TAA‐treated rats. Induction of HO‐1 was associated with increased arginine levels and decreased ADMA levels, being the main determinants of NO production, in plasma of TAA‐treated rats. In conclusion, our results indicate that HO‐1 induction alleviated increased oxidative stress and inflammatory reactions together with deterioration in NO production in TAA‐induced liver damage in rats. Copyright © 2012 John Wiley & Sons, Ltd.     

Regulation of asthmatic airway relaxation by estrogen and heat shock protein 90

We tested the hypothesis that asthmatic mouse airways exhibit impaired relaxation to NO donors. Mouse tracheal rings were incubated overnight in serum from asthmatic human subjects or from nonasthmatic controls. The next day, cumulative concentration-response curves (CCRC) to sodium nitroprusside (SNP) and nitroglycerine (NTG) were obtained. Both SNP and NTG relaxed the pre-constricted normal tracheal rings. Tracheal rings exposed to serum from asthmatic patients exhibited a more than a threefold increase in the EC50 of SNP and NTG. Pre-incubation of tracheal rings with heat shock protein 90 inhibitors decreased the relaxation of both normal and asthmatic tracheal rings to SNP and NTG. Pre-incubation with estradiol did not affect normal tracheal ring relaxation but exhibited an increase in asthmatic tracheal ring relaxation, which was abolished by an estrogen receptor (ER) antagonist. ER subtype-selective agonists, but not GPR30 agonists, mimicked the action of estradiol on tracheal ring relaxation. Co-incubation of rings with radicicol and estradiol produced an ER-dependent increase in the relaxation response to SNP of both normal and asthmatic ASM. Estrogen-induced relaxation of ASM was abolished by overnight incubation with radicicol and this was associated with reduced expression of ERβ. These data suggest that asthmatic ASM is considerably less responsive to NO-donors and that both estrogen and hsp90 play important roles in ASM relaxation.     

Catalase and Superoxide Dismutase in the Cells of Strictly Anaerobic Microorganisms

Strictly anaerobic microorganisms relating to various physiological groups were screened for catalase and superoxide dismutase (SOD) activity. All of the investigated anaerobes possessed SOD activity, necessary for protection against toxic products of oxygen reduction. High specific activities of SOD were found in Acetobacterium woodii and Acetobacterium wieringae. Most of the investigated clostridia and acetogens were catalase-negative. A significant activity of catalase was found in Thermohydrogenium kirishiense, in representatives of the genus Desulfotomaculum, and in several methanogens. Methanobrevibacter arboriphilus had an exceptionally high catalase activity after growth in medium supplemented with hemin. Hemin also produced a strong positive effect on the catalase activity in many other anaerobic microorganisms. In methanogens, the activities of the enzymes of antioxidant defense varied in wide ranges depending on the stage of growth and the energy source.     

CAT-2 amplifies the agonist-evoked force of airway smooth muscle by enhancing spermine-mediated phosphatidylinositol-(4)-phosphate-5-kinase-gamma activity

We investigated the effect the loss of the CAT-2 gene (CAT-2-/-) has on lung resistance (R(L)) and tracheal isometric tension. The R(L) of CAT-2-/- mice at a maximal dose of acetylcholine (ACh) was decreased by 33.66% (P = 0.05, n = 8) compared with that of C57BL/6 (B6) mice. The isometric tension of tracheal rings from CAT-2-/- mice showed a significant decrease in carbachol (CCh)-induced force generation (33.01%, P < 0.05, n = 8) compared with controls. The isoproterenol- or the sodium nitroprusside-induced relaxation was not affected in tracheal rings from CAT-2-/- mice. The activity of iNOS and arginase in lung tissue lysates of CAT-2-/- mice was indistinguishable from that of B6 mice. Furthermore, the expression of phospholipase-Cbeta (PLC-beta) and phosphatidylinositol-(4)-phosphate-5-kinase-gamma (PIP-5K-gamma) was examined in the lung tissue of CAT-2-/- and B6 mice. The expression of PIP-5K-gamma but not PLC-beta was significantly reduced in CAT-2-/- compared with B6 mice. The reduced airway smooth muscle (ASM) contractility to CCh seen in the CAT-2-/- tracheal rings was completely reversed by pretreating the rings with 100 muM spermine. This increase in the CAT-2-/- tracheal ring contraction upon spermine pretreatment correlated with a recovery of the expression of PIP-5K-gamma. Our data indicates that CAT-2 exerts control over ASM force development through a spermine-dependent pathway that directly correlates with the expression level of PIP-5K-gamma in the lung.     

Hemin-induced Erk1/2 activation and heme oxygenase-1 expression in human umbilical vein endothelial cells

Hemin has been reported to be protective in the pathological process, but its protective mechanisms have not been precisely defined. Hemin could induce Erk1/2 phosphorylation in astrocyte. Erk1/2 phosphorylation has been proved to be involved in many growth signals cellular transduction. However, little study has been conducted as to the relationship between hemin and Erk1/2 activation in human umbilical vein endothelial cells (HUVECs). The present study aimed to investigate the relationship between hemin and Erk1/2 phosphorylation in HUVECs. The results showed that low concentration of hemin induced and sustained phosphorylation of Erk1/2 for a long time. The HO inhibitor protoporphyrin IX zinc (II) abrogated phosphorylation of Erk1/2 induced by hemin. Biliverdin, one of the metabolites of hemin, obviously induced the Erk1/2 phosphorylation in HUVECs. Both hemin and biliverdin promoted HUVEC cell growth. The results strongly suggested that hemin could induce and sustain Erk1/2 phosphorylation in HUVECs by way of HO-1 induction and biliverdin produced from HO-1 catalysing hemin degradation.     

Hemin-induced Erk1/2 activation and heme oxygenase-1 expression in human umbilical vein endothelial cells

Hemin has been reported to be protective in the pathological process, but its protective mechanisms have not been precisely defined. Hemin could induce Erk1/2 phosphorylation in astrocyte. Erk1/2 phosphorylation has been proved to be involved in many growth signals cellular transduction. However, little study has been conducted as to the relationship between hemin and Erk1/2 activation in human umbilical vein endothelial cells (HUVECs). The present study aimed to investigate the relationship between hemin and Erk1/2 phosphorylation in HUVECs. The results showed that low concentration of hemin induced and sustained phosphorylation of Erk1/2 for a long time. The HO inhibitor protoporphyrin IX zinc (II) abrogated phosphorylation of Erk1/2 induced by hemin. Biliverdin, one of the metabolites of hemin, obviously induced the Erk1/2 phosphorylation in HUVECs. Both hemin and biliverdin promoted HUVEC cell growth. The results strongly suggested that hemin could induce and sustain Erk1/2 phosphorylation in HUVECs by way of HO-1 induction and biliverdin produced from HO-1 catalysing hemin degradation.     

Effects of tin-protoporphyrin IX on blood flow in a rat tumor model

Carbon monoxide (CO), one of the products of heme oxygenase (HO) catalyzed heme degradation, is a vasodilator. The aim of the present study was to clarify the role of HO in blood flow maintenance in tumors. Male BD9 rats bearing subcutaneous transplants of the P22 carcinosarcoma tumor were treated intraperitoneally (i.p.) with either tin-protoporphyrin IX (SnPP; 45 micromol/kg), a selective inhibitor of HO or copper-protoporphyrin IX (CuPP; 45 micromol/kg), used as a negative control. The extent of HO activity inhibition was measured using a spectrophotometric assay of bilirubin production and blood flow rates to the tumor and a range of normal tissues were assessed using the uptake of the radiolabelled tracer, iodo-antipyrine ((125)I-IAP). The animals were cannulated under fentanyl citrate/fluanisone (Hypnorm)/midazolam anesthesia. In the P22 tumor, SnPP, but not CuPP, caused a complete inhibition of HO activity 15 min post-treatment. Administration of SnPP 15 min before blood flow measurements reduced tumor blood flow by 17%, with no effects in any of the normal tissues studied. However, CuPP induced a greater reduction in tumor blood flow than SnPP (45% decrease). Furthermore, CuPP caused a reduction in blood flow to the skin and small intestine but a significant increase to skeletal muscle. The current findings conclusively establish only a minor role played by the HO/CO system in the maintenance of blood flow in this tumor system, despite relatively high levels of HO-1 protein and HO activity. The results also highlight the potential usefulness of CuPP as a tumor blood flow modifier.     

Myosin light chain kinase phosphorylation in tracheal smooth muscle

Purified myosin light chain kinase from smooth muscle is phosphorylated by cyclic AMP-dependent protein kinase, protein kinase C, and the multifunctional calmodulin-dependent protein kinase II. Because phosphorylation in a specific site (site A) by any one of these kinases desensitizes myosin light chain kinase to activation by Ca2+/calmodulin, kinase phosphorylation could play an important role in regulating smooth muscle contractility. This possibility was investigated in 32P-labeled bovine tracheal smooth muscle. Treatment of tissues with carbachol, KCl, isoproterenol, or phorbol 12,13-dibutyrate increased the extent of kinase phosphorylation. Six primary phosphopeptides (A-F) of myosin light chain kinase were identified. Site A was phosphorylated to an appreciable extent only with carbachol or KCl, agents which contract tracheal smooth muscle. The extent of site A phosphorylation correlated to increases in the concentration of Ca2+/calmodulin required for activation. These results show that cyclic AMP-dependent protein kinase and protein kinase C do not affect smooth muscle contractility by phosphorylating site A in myosin light chain kinase. It is proposed that phosphorylation of myosin light chain kinase in site A in contracting tracheal smooth muscle may play a role in the reported desensitization of contractile elements to activation by Ca2+.