Cardiomegaly induced by pressure overload in newborn rats is accompanied by altered expression of the long isoform of Gsα protein and deranged signaling of adenylyl cyclase

G proteins-coupled signaling pathways appear to play a role in the development of cardiac hypertrophy and its progression to heart failure. The present study aimed to investigate trimeric G proteins and adenylyl cyclase signaling in immature as well as in adult rat myocardium during this process caused by pressure overload. Pressure overload was induced in newborn (2-day-old) rats by abdominal aortic banding and myocardial preparations from left ventricular myocardium of immature (10-day-old) and adult (90-day-old) animals were analyzed for the relative content of different G protein subunits and adenylyl cyclase (AC) by immunoblotting with specific antibodies. A functional status of the AC signaling system was also evaluated. Normal maturation of rat heart was accompanied by increased expression of AC type V/VI and VII and of the long isoform (G(s)alphaL) of G(s)alpha protein. In parallel, the amounts of myocardial G(i)alpha/G(o)alpha proteins tended to decrease, and G(q)alpha/G(11)alpha and Gbeta did not change. Interestingly, whereas fluoride-stimulated AC activity increased in the course of maturation, activity of AC measured under other experimental conditions (stimulation by Mn2+, forskolin or isoproterenol) was lower in adult than in young rat myocardium. Pressure overload did not influence distribution of G proteins in immature myocardium, but considerably decreased the content of G(s)alphaL and increased G(o)alpha proteins in hearts of 90-day-old rats. These hearts exhibited worsened functional reserve as compared to age-matched controls and activity of AC was also markedly lower. A considerable reduction in Mn(2+)-stimulated AC activity together with similar decrease in AC activity determined under other stimulation conditions suggests that it is a function of AC catalytic subunit that is primarily impaired in this model of pressure overload.     

Regulation of adenylyl cyclase activity in rat testes by acylated derivatives of peptide 562–572 of a luteinizing hormone receptor

One area of the search for hormonal signaling systems regulators is development of peptides that correspond to the cytoplasmic regions of G protein-coupled receptors (GPCR). Modification of such peptides with hydrophobic radicals increases their efficiency and selectivity. However, at present it has not been studied how the activity of the peptide depends on the localization of hydrophobic radicals, their number, and chemical nature. The aim of this work consisted in synthesis of peptide 562–572 derivatives modified by fatty-acid radicals and corresponding to the C-terminal region of the luteinizing hormone receptor (LHR) and in the study of regulatory effects of the acylated LHR peptides on the basal and hormone-stimulated activity of adenylyl cyclase (AC) in rat tissues. To elucidate the effects of localization of hydrophobic radicals and of their number, modifications of peptide 562–572 were carried out only at the N-or at the C-terminus or at both ends. To study the effect of hydrophobicity, residues of palmitic (Pal) and decanoic (Dec) acids were chosen. Using a solid-phase strategy synthesis was performed of the unmodified peptide NKDTKIAKK-Nle-A^562-572-KA (1) and five of its acylated analogues, N[K(Dec)]DTKIAKK-Nle-A^562-572-KA (2), NKDTKIAKK-Nle-A^562-572-[K(Dec)]A (3), N[K(Dec)]DTKIAKK-Nle-A^562-572-[K(Dec)]A (4), N[K(Pal)]DTKIAKK-Nle-A^562-572-KA (5), and NKDTKIAKK-Nle-A^562-572-[K(Pal)]A (6). Peptide 6 modified with palmitate at the C-terminus to a large extent increased the basal AC activity and reduced the AC stimulating effect of human chorionic gonadotropin (hCG) in testes of rats; peptides 3 and 4 modified with decanoate at the C-terminus were less effective, but exceeded in activity the unmodified peptide 1; and peptides 2 and 5 acylated at the N-terminus were little active. The action of peptides was characterized by tissue and the receptor specificity. Thus, modification of the LHR peptide 562–572 with fatty-acid radicals at the C-terminus enhances its regulatory effect on the functional activity of the adenylyl cyclase system in rat testes, which indicates a promising modification of GPCR peptides with hydrophobic radicals. These data confirm the hypothesis that the hydrophobic radical is to be localized in the locus of GPCR peptide, where a transmembrane domain is located in the receptor.     

Regulation of sialyl-Lewis x epitope expression by TNF-α and EGF in an airway carcinoma cell line

Sialyl-Lewis x epitopes and MUC5AC protein are known to be overexpressed in mucins secreted by patients suffering from various respiratory diseases. To investigate the mechanisms by which airway inflammatory agents mediate the expression of sialyl-Lewis x epitopes and MUC5AC mucin, we examined the effects of tumor necrosis factor (TNF)- and epidermal growth factor (EGF) in the human lung carcinoma cell line, NCI-H292. Basal expression levels of hST3GalIV, FUT3 and C2/4GnT mRNA, involved in the biosynthesis of sialyl-Lewis x, were higher than those of other glycosyltransferases in NCI-H292 cells. TNF- induced expression of hST3GalIV, FUT3, C2/4GnT and MUC5AC mRNAs in NCI-H292 cells. When cells were pretreated with U73122, a phosphatidylinositol-phospholipase C (PI-PLC) inhibitor, the expression of these glycosyltransferase mRNAs was suppressed. Treating cells with EGF induced the down-regulation of these glycosyltransferase mRNAs and sialyl-Lewis x epitopes, while inducing an increase in expression of MUC5AC mRNA. These EGF-mediated effects on the glycosyltransferase and MUC5AC mRNAs were blocked when cells were first exposed to AG1478, an EGF receptor tyrosine kinase inhibitor. These findings suggest that the expression of sialyl-Lewis x epitopes, which is regulated separately from the expression of MUC5AC protein, may be controlled through pathways such as the EGF receptor tyrosine kinase and PI-PLC signaling cascades in NCI-H292 cells. Published in 2005.     

Effect of transmembrane Ca2+ gradient on the coupling of β-adrenergic receptors and adenylyl cyclase

In order to investigate the effect of transmembrane Ca²⁺ gradient on G_s mediated coupling of -AR and adenylyl cyclase, -AR from duck erythrocytes and G_s and adenylyl cyclase from bovine brain cortices were co-reconstituted into asolectin liposomes with different transmembrane Ca²⁺ gradient. These proteoliposomes were proven to be impermeable to water-soluble substances. The results obtained indicate that a physiological transmembrane Ca^2– gradient (1000-fold) is essential for higher stimulation of adenylyl cyclase by hormone-activated -AR via coupling to G_s and can be further enhanced by the decrease of such Ca²⁺ gradient within certain range (100 fold) following Ca²⁺ influx into cells during signal transduction. Fluorescence polarization of DPH revealed that transmembrane Ca²⁺ gradient modulates adenylyl cyclase and its stimulation by hormones through mediating a change in lipid fluidity. Correspondent conformational changes of -AR were also detected from the fluorescence spectra and quenching of Acrylodan-labelled -AR in those proteoliposomes. It is suggested that a proper transmembrane Ca²⁺ gradient is essential for the optimal fluidity of the phospholipid bilayer in the proteoliposomes, which favors the formation of a suitable conformation of the reconstituted -AR and thus promotes the stimulation of adenylyl cyclase activities by hormone-activated -AR via Gs.Abbreviations ATP adenosine triphosphate - -AR -adrenergic receptors - AC adenylyl cyclase - DHA dihydroalprenolol - DPH diphenylhexatriene - [Ca²⁺]_i Ca²⁺ concentration inside proteoliposomes - [Ca²⁺]_o Ca²⁺ concentration outside proteoliposomes - cAMP cyclic adenosine monophosphate - DTT Dithiothreitol - FS fluorescein sulfonate - G_s Stimulatory GTP-binding protein - GTP guanosine triphosphate - GTPS guanosine 5-O-(3-thiotriphosphate) - kDa kilodalton - SDS sodium dodecyl sulfate - Tris N-tris(hydroxymethyl)aminomethane     

Down-regulation of β-adrenoceptors and loss of Gsα subunit levels in ventricular myocardium of rats treated with isoproterenol

We investigated the influence of chronic β-adrenergic stimulation on the β-adrenoceptor-G protein-adenylyl cyclase system in rat ventricular myocardium. The rats received twice-daily injections of 4 mg/kg isoproterenol (ISO) alone or with 8 mg/kg propranolol (PROP) for 4 days. The ISO (10 μM)-induced increase in tissue cAMP production was lower (50%) after chronic ISO treatment than in control myocardium. The β-adrenoceptor density decreased by 43% in ventricular membranes from ISO-treated rats. Immunoblotting techniques using specific antibodies against G proteins revealed that ventricular myocardium contains three Gsα subunit isoforms of 45, 47 and 52kDa. ISO treatment decreased levels of the three Gsα subunits by a total of 40%, while no change in Giα (40/41kDa) and Gcommonβ (35/36kDa) levels were found in the same membrane preparations. The antagonist PROP almost totally blocked the effects of ISO treatment on cAMP, β-adrenoceptors and Gsα subunits. These results suggest that chronic β-adrenergic stimulation causes not only down-regulation of β-adcenoceptors, but also loss of Gsα subunit levels in rat ventricular myocardium.     

Regulation of β-exoglucanase activity production by Saccharomyces cerevisiae in batch and continuous culture

The rate of synthesis and secretion of exo-1–3--glucanase activity closely paralleled the specific rate of growth in exponentially growing Saccharomyces cerevisiae cells in batch culture. When the stationary phase was reached both synthesis and secretion stopped. No activity was synthesized when the cells were maintained in carbon sources that did not allow them to grow. Studies in continuous culture indicate a strong relationship between the synthesis of exoglucanase activity and the specific growth rate. These results are taken as evidence of an essential role of this activity during the yeast budding cycle.Non-standard abbreviations p-NPG p-nitrophenyl--d-glucopyranoside - S_r glucose concentration in the sterile reservoir; , glucose concentration at the steady state - biomass density at the steady state - G glucanase activity - Q _g specific exoglucanase synthesis rate     

Regulation of β-adrenoceptor properties and the lipid milieu in heart muscle membranes during stress

The purpose of this study was to examine changes in fatty acyl chain composition of major cardiac phospholipids in relation to down-regulation of -adrenoceptors during various forms of stress or chronic adrenergic stimulation. Analysis of the fatty acid profile of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in sarcolemma or cardiac muscle membranes showed partial replacement of 18:2n-6 by 20:4n-6 in PC and replacement of both 18:2n-6 and 20:4n-6 by 22:6n-3 in PE during daily administration of epinephrine or norepinephrine for 7 or 15 days, respectively These changes in membrane PC and PE coincided with down-regulation or the decrease in Bmax of -adrenoceptors during adrenergic stimulation. Cardiac membrane response to other forms of stress or chronic adrenergic stimulation such as neonatal stress, restriction stress or restricted food intake was expressed in the same way, that is replacement of 18:2n-6 by 20:4n-6 in PC and replacement of 18:2n-6 and 20:4n-6 by 22:6n-3 in PE.Conclusion: Adaptation to stress includes a decrease in the density of binding sites or down-regulation of -adrenoceptors in sarcolemma synchronized with specific alterations in the fatty acyl chain composition within the membrane bilayer. The changes in the lipid milieu of the membrane may facilitate conformational changes in the transmembrane segment of the receptor forming the ligand binding sites of the -adrenoceptor.     

Does the inhibition of c-myc expression mediate the anti-tumor activity of PPAR's ligands in prostate cancer cell lines?

Peroxisome proliferator-activated receptor gamma (PPARgamma) ligands seem to induce anticancer effects on prostate cancer cells, but the mechanism is not clear. The effect of PPARgamma ligands omega-6 fatty acids and ciglitazone (2-15 microM)--on proliferation, and apoptosis of LNCaP, PC-3, DU145, CA-K and BPH-K cells was studied. PPARgamma ligands led to: (1) reduction of proliferation (20-50%) of all the studied cell lines, (2) stimulation of differentiation of prostate cancer cells through an increased expression (1.5-3-fold: LNCaP, DU145, BPH-K) or reexpression (PC-3, CA-K) of E-cadherin with parallel inhibition of N-cadherin expression (PC-3, CA-K) and (3) down-regulation (1-2-fold) of beta-catenin and c-myc expression. The selective PPARgamma antagonist GW9662 abolished the effect of those ligands on prostate cancer cells. These results suggest that inhibition of beta-catenin and in effect c-myc expression through activation of PPARgamma may help prostate cancer cells to restore several characteristics of normal prostate cells phenotype.     

Regulation of the β-ketoadipate pathway in Rhizobium japonicum and bacteroids by succinate

Rhizobium japonicum 61-A-101 and its bacteroids catabolize phenol and p-hydroxybenzoate. With phenol as a carbon source, utilization started only after a prolonged lag phase while p-hydroxybenzoate was almost instantancously metabolized. Succinate, which supports rapid growth of Rhizobium japonicum, completely repressed respication of phenol; the oxidation of p-hydroxybenzoate was partially inhibited. Pyruvate, supporting slower growth than succinate, retarded the onset of phenol consumption but did not affect its maximum rate.Catabolite repression of phenol utilization by succinate appears to be a characteristic feature of rhizobia. In Pseudomonas putida which also actively metabolizes phenol, succinate had no effect on phenol utilization.     

Regulation of cadherin expression in the chicken neural crest by the Wnt/ β-catenin signaling pathway

In neural crest cell development, the expression of the cell adhesion proteins cadherin-7 and cadherin-11 commences after delamination of the neural crest cells from the neuroepithelium. The canonical Wnt signaling pathway is known to drive this delamination step and is a candidate for inducing expression of these cadherins at this time. This project was initiated to investigate the role of canonical Wnt signaling in the expression of cadherin-7 and cadherin-11 by treating neural crest cells with Wnt3a ligand. Expression of cadherin-11 was first confirmed in the neural crest cells for the chicken embryo. The changes in the expression level of cadherin-7 and -11 following the treatment with Wnt3a ligand were studied using real-time RT-PCR and immunostaining. Statistically significant up-regulation in the mRNA expression of cadherin-7 and cadherin-11 and in the amount of cadherin-7 and cadherin-11 protein found in cell-cell interfaces between neural crest cells was observed in response to Wnt, demonstrating that cadherin-7 and cadherin-11 expressed by the migrating neural crest cells can be regulated by the canonical Wnt pathway.     

Regulation of contractility and metabolic signaling by the β2-adrenergic receptor in rat ventricular muscle

AimsCardiac function is modulated by the sympathetic nervous system through β-adrenergic receptor (β-AR) activity and this represents the main regulatory mechanism for cardiac performance. To date, however, the metabolic and molecular responses to β₂-agonists are not well characterized. Therefore, we studied the inotropic effect and signaling response to selective β₂-AR activation by tulobuterol.Main methodsStrips of rat right ventricle were electrically stimulated (1 Hz) in standard Tyrode solution (95% O₂, 5% CO₂) in the presence of the β₁-antagonist CGP-20712A (1 μM). A cumulative dose–response curve for tulobuterol (0.1–10 μM), in the presence or absence of the phosphodiesterase (PDE) inhibitor IBMX (30 μM), or 10 min incubation (1 μM) with the β₂-agonist tulobuterol was performed.Key findingsβ₂-AR stimulation induced a positive inotropic effect (maximal effect = 33 ± 3.3%) and a decrease in the time required for half relaxation (from 45 ± 0.6 to 31 ± 1.8 ms, ? 30%, p < 0.001) after the inhibition of PDEs. After 10 min of β₂-AR stimulation, p-AMPKα^T172 (54%), p-PKB^T308 (38%), p-AS160^T642 (46%) and p-CREB^S133 (63%) increased, without any change in p-PKA^T197.SignificanceThese results suggest that the regulation of ventricular contractility is not the primary function of the β₂-AR. Rather, β₂-AR could function to activate PKB and AMPK signaling, thereby modulating muscle mass and energetic metabolism of rat ventricular muscle.     

Adaptive basal phosphorylation of eIF2α is responsible for resistance to cellular stress-induced cell death in Pten-null hepatocytes

The α-subunit of eukaryotic initiation factor 2 (eIF2α) is a key translation regulator that plays an important role in cellular stress responses. In the present study, we investigated how eIF2α phosphorylation can be regulated by a tumor suppressor PTEN (phosphatase and tensin homolog deleted on chromosome 10) and how such regulation is used by PTEN-deficient hepatocytes to adapt and cope with oxidative stress. We found that eIF2α was hyperphosphorylated when Pten was deleted, and this process was AKT dependent. Consistent with this finding, we found that the Pten-null cells developed resistance to oxidative glutamate and H(2)O(2)-induced cellular toxicity. We showed that the messenger level of CReP (constitutive repressor of eIF2α phosphorylation), a constitutive phosphatase of eIF2α, was downregulated in Pten-null hepatocytes, providing a possible mechanism through which PTEN/AKT pathway regulates eIF2α phosphorylation. Ectopic expression of CReP restored the sensitivity of the Pten mutant hepatocytes to oxidative stress, confirming the functional significance of the downregulated CReP and upregulated phospho-eIF2α in the resistance of Pten mutant hepatocytes to cellular stress. In summary, our study suggested a novel role of PTEN in regulating stress response through modulating the CReP/eIF2α pathway.     

Paradoxical attenuation of β2-AR function in airway smooth muscle by Gi-mediated counterregulation in transgenic mice overexpressing type 5 adenylyl cyclase

The limiting component within the receptor-G protein-effector complex in airway smooth muscle (ASM) for β(2)-adrenergic receptor (β(2)-AR)-mediated relaxation is unknown. In cardiomyocytes, adenylyl cyclase (AC) is considered the "bottleneck" for β-AR signaling, and gene therapy trials are underway to increase inotropy by increasing cardiac AC expression. We hypothesized that increasing AC in ASM would increase relaxation from β-agonists, thereby providing a strategy for asthma therapy. Transgenic (TG) mice were generated with approximately two- to threefold overexpression of type 5 AC (AC5) in ASM. cAMP and airway relaxation in response to direct activation of AC by forskolin were increased in AC5-TG. Counter to our hypothesis, isoproterenol-mediated airway relaxation was significantly attenuated (～50%) in AC5-TG, as was cAMP production, suggesting compensatory regulatory events limiting β(2)-AR signaling when AC expression is increased. In contrast, acetylcholine-mediated contraction was preserved. G(αi) expression and ERK1/2 activation were markedly increased in AC5-TG (5- and 8-fold, respectively), and β-AR expression was decreased by ～40%. Other G proteins, G protein-coupled receptor kinases, and β-arrestins were unaffected. β-agonist-mediated airway relaxation of AC5-TG was normalized to that of nontransgenic mice by pertussis toxin, implicating β(2)-AR coupling to the increased G(i) as a mechanism of depressed agonist-promoted relaxation in these mice. The decrease in β(2)-AR may account for additional relaxation impairment, given that there is no enhancement over nontransgenic after pertussis toxin, despite AC5 overexpression. ERK1/2 inhibition had no effect on the phenotype. Thus perturbing the ratio of β(2)-AR to AC in ASM by increasing AC fails to improve (and actually decreases) β-agonist efficacy due to counterregulatory events.     

Variation of amino acid concentrations in the medium of HU β-IFN and HU IL-2 producing cell lines

There are different requirements for the nutrient medium of various mammalian cell lines. We have determined the behaviour of the amino acid concentrations in the medium of two growing cell lines used for producing human interleukin 2 and human interferon constitutively. The experiments are based on a fermentation process with a bubble free cell culture aeration system with porous moving membranes, which allows production of high cell densities without foaming. We found interesting alterations in which the actual amino acid ratios are able to trigger consumption and production of a particular component depending on the supply of other possible replacements. Such data indicate the complicated biochemical network of synthesis, conversion and transport phenomena. Finally, we demonstrated the influence of product synthesis upon the amino acid requirements using as an example transformed hu IL-2- and hu -IFN-producing mouse L-cells.Abbreviations CTLL Cytotoxic T Cell Line - DME Dulbeccos Modified Eagle - FCS Foetal Calf Serum - Hepes N(2-Hydroxyethyl)piperazine-N'-2-ethanesulfonic acid - hu -IFN human -Interferon - hu IL-2 human Interleukin 2 - THF Tetrahydrofuran     

Up-regulation of E-cadherin and β-catenin in human hepatocellular carcinoma cell lines by sodium butyrate and interferon-α

Summary Human E-cadherin is a homophilic cell adhesion molecule and its expression is well preserved in normal human hepatocytes; a decrease in its expression has been observed in poorly differentiated hepatocellular carcinoma cells. We examined the alteration of E-cadherin and catenin expressions caused by differentiation inducers in human hepatocellular carcinoma cells. Hepatocellular carcinoma cell lines, HCC-T and HCC-M, were cultured with all-trans retinoic acid (ATRA), dexamethasone (DEX), sodium butyrate, and interferon-α. E-cadherin expression was only up-regulated by butyrate and interferon-α (IFN-α) in both cell lines, studied by means of fluorescence immunostaining and flow cytometry. The localization of E-cadherin staining was shown at their cell membrane. According to the increase in E-cadherin expression, β-catenin expression appeared at the cell membrane of both cell lines when treated with butyrate and IFN-α. Such an appearance was not observed when cells were treated with ATRA and DEX. Western blotting showed that α-and γ-catenin expression was not changed, while only the expression of β-catenin increased. β-Catenin oncogenic activation as a result of amino acid substitutions or interstitial deletions within or including parts of exon 3, which has been demonstrated recently, was not detected in these cell lines by direct deoxyribonucleic acid sequencing. These results suggest that the expression and interaction between E-cadherin and wild-type β-catenin are potentially modulated by butyrate and IFN-α, and that these two agents are potent inhibitors of hepatocellular carcinoma cell invasion and metastasis.