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Biomedical subjects

A Romanelli

Publications and source records attributed to A Romanelli.

At least 19 recordsLinked to original sources
PubMed2006

Clinical and endoscopic presentation of primary gastric lymphoma: a multicentre study.

BACKGROUND: Although the stomach is the most frequent site of intestinal lymphomas, few data are available on both clinical endoscopic presentation of gastric lymphoma and possible differences between low-grade and high-grade lymphomas. METHODS: Clinical, histological and endoscopic records of consecutive patients with primary low-grade or high-grade lymphoma diagnosed were retrieved. Symptoms were categorized as 'alarm' or 'not alarm'. The endoscopic findings were classified as 'normal' or 'abnormal'. RESULTS: Overall, 144 patients with primary gastric lymphoma were detected, including 74 low-grade and 70 high-grade lymphoma. Alarm symptoms, particularly persistent vomiting and weight loss, were more frequently present in patients with high-grade lymphoma than in those with low-grade lymphoma (54% vs. 28%; P = 0.002). Low-grade lymphomas presented as 'normal' appearing mucosa (20% vs. 0%; P = 0.0004) or petechial haemorrhage in the fundus (9% vs. 0%; P = 0.02) more frequently than high-grade lymphomas, being also more often confined to the antrum (47% vs. 27%, P = 0.03) and associated with Helicobacter pylori infection (88% vs. 52%, P < 0.0001). On the contrary, high-grade lymphomas presented more commonly as ulcerative type (70% vs. 52%; P = 0.03), being also more frequently diagnosed in stage >I when compared with low-grade lymphomas (70% vs. 21%, P < 0.0001). CONCLUSIONS: The overall prevalence of alarm symptoms is quite low and may be absent in more than 70% of patients with low-grade lymphoma.

Endoscopy, Gastrointestinal↗
PubMed2006

Combination of vinblastine and bleomycin as first line therapy in advanced classic Kaposi's sarcoma.

BACKGROUND: Classic KS (CKS) mainly affects elderly people, has an irregular course, and is relatively benign for years. However, sometimes the disease may progress rapidly and spread to internal organs, thus necessitating systemic chemotherapy. We therefore decided to carry out a prospective trial using vinblastine and bleomycin, which are active, easy to administer and control, and low cost. METHODS: We treated 29 patients affected by CKS with vinblastine i.v., up to 10 mg in combination with bleomycin i.m., 15 IU every 3 weeks. We administered a median of seven cycles of therapy. RESULTS: All the 29 enrolled patients were evaluated: 21% reached a complete response and 76% had an intermediate response. Toxicity was limited. The maximal response was attained in a median of 5 months, with a mean duration of 4 months. CONCLUSION: The combination of vinblastine and bleomycin achieved a high rate of objective responses in a subgroup of elderly and symptomatic patients, without considerable toxicity. We recommend the combination as first line chemotherapy for advanced CKS.

Aged↗
PubMed2004

Peptide nucleic acids (PNA)-DNA chimeras targeting transcription factors as a tool to modify gene expression.

Peptide nucleic acids (PNAs)-DNA chimeras have been recently described as DNA mimics constituted of a part of PNA and of a part of DNA. We have demonstrated that double stranded molecules based on PNA-DNA chimeras bind to transcription factors in a sequence-dependent manner. Accordingly, these molecules can be used for transcription factor decoy (TFD) pharmacotherapy. Effects of double stranded PNA-DNA chimeras targeting NF-kappaB and Sp1 were determined on in vitro cultured human cells and were found to be comparable to those observed using double-stranded DNA decoys. The TFD molecules based on PNA-DNA chimeras can be further engineered by addition of short peptides facilitating cell penetration and nuclear localization. Therefore, these engineered molecules could be of great interest for in vivo experiments for non-viral gene therapy of a variety of diseases, including neoplastic and viral diseases, for which the TFD approach has been already demonstrated as a very useful strategy.

Apoptosis↗
PubMed2002

Dynamical localization in quasiperiodic driven systems.

We investigate how the time dependence of the Hamiltonian determines the occurrence of dynamical localization (DL) in driven quantum systems with two incommensurate frequencies. If both frequencies are associated to impulsive terms, DL is permanently destroyed. In this case, we show that the evolution is similar to a decoherent case. On the other hand, if both frequencies are associated to smooth driving functions, DL persists although on a time scale longer than in the periodic case. When the driving function consists of a series of pulses of duration sigma, we show that the localization time increases as sigma(-2) as the impulsive limit, sigma-->0, is approached. In the intermediate case, in which only one of the frequencies is associated to an impulsive term in the Hamiltonian, a transition from a localized to a delocalized dynamics takes place at a certain critical value of the strength parameter. We provide an estimate for this critical value, based on analytical considerations. We show how, in all cases, the frequency spectrum of the dynamical response can be used to understand the global features of the motion. All results are numerically checked.

Journal Article↗
PubMed2001

Crystal-state conformation of Calpha,alpha-dialkylated peptides containing chiral beta-homo-residues.

Secondary structure formation and stability are essential features in the knowledge of complex folding topology of biomolecules. To better understand the relationships between preferred conformations and functional properties of beta-homo-amino acids, the synthesis and conformational characterization by X-ray diffraction analysis of peptides containing conformationally constrained Calpha,alpha-dialkylated amino acid residues, such as alpha-aminoisobutyric acid or 1-aminocyclohexane-1-carboxylic acid and a single beta-homoamino acid, differently displaced along the peptide sequence have been carried out. The peptides investigated are: Boc-betaHLeu-(Ac6c)2-OMe, Boc-Ac6c-betaHLeu-(Ac6c)2-OMe and Boc-betaHVal-(Aib)5-OtBu, together with the C-protected beta-homo-residue HCl.H-betaHVal-OMe. The results indicate that the insertion of a betaH-residue at position 1 or 2 of peptides containing strong helix-inducing, bulky Calpha,alpha-disubstituted amino acid residues does not induce any specific conformational preferences. In the crystal state, most of the NH groups of beta-homo residues of tri- and tetrapeptides are not involved in intramolecular hydrogen bonds, thus failing to achieve helical structures similar to those of peptides exclusively constituted of Calpha,alpha-disubstituted amino acid residues. However, by repeating the structural motifs observed in the molecules investigated, a beta-pleated sheet secondary structure, and a new helical structure, named (14/15)-helix, were generated, corresponding to calculated minimum-energy conformations. Our findings, as well as literature data, strongly indicate that conformations of betaH-residues, with the micro torsion angle equal to -60 degrees, are very unlikely.

Alkylation↗
PubMed2001

Molecular interactions with nuclear factor kappaB (NF-kappaB) transcription factors of a PNA-DNA chimera mimicking NF-kappaB binding sites.

The decoy approach against nuclear factor kappaB (NF-kappaB) is a useful tool to alter NF-kappaB dependent gene expression using synthetic oligonucleotides (ODNs) carrying NF-kappaB specific cis-elements. Unfortunately, ODNs are not stable and need to be be extensively modified to be used in vivo or ex vivo. We have previously evaluated the possible use of peptide nucleic acids (PNAs) as decoy molecules. The backbone of PNAs is composed of N-(2-aminoethyl)glycine units, rendering these molecules resistant to both nucleases and proteases. We found that the binding of NF-kappaB transcription factors to PNAs was either very low (binding to PNA-PNA hybrids) or exhibited low stability (binding to PNA-DNA hybrids). The main consideration of the present paper was to determine whether PNA-DNA chimeras mimicking NF-kappaB binding sites are capable of stable interactions with proteins belonging to the NF-kappaB family. Molecular modeling was employed for the design of PNA-DNA chimeras; prediction of molecular interactions between chimeras and NF-kappaB nuclear proteins were investigated by molecular dynamics simulations, and interactions between PNA-DNA chimeras and NF-kappaB proteins were studied by gel shifts. We found significant differences between the structure of duplex NF-kappaB PNA-DNA chimera and duplex NF-kappaB DNA-DNA. However, it was found that these differences do not prevent the duplex PNA-DNA chimera from binding to NF-kappaB transcription factors, being able to suppress the molecular interactions between HIV-1 LTR and p50, p52 and nuclear factors from B-lymphoid cells. Therefore, these results demonstrate that the designed NF-kappaB DNA-PNA chimeras could be used for a decoy approach in gene therapy.

Animals↗
PubMed2001

Complications of tracheal sleeve pneumonectomy: personal experience and overview of the literature.

OBJECTIVES: Tracheal sleeve pneumonectomy, although technically demanding, is considered the choice for tracheobronchial angle cancers. Complications in our 49 tracheal sleeve pneumonectomies are reviewed. Results, complications, and technical aspects are critically discussed. Although series in the literature differ in selection of patients and surgical techniques and extend over long periods, we attempt to compare our experience with results from the literature. METHODS: From 1983 to September 1999, 60 patients eligible for tracheal sleeve pneumonectomy after conventional staging underwent operation. A Sybilla Fome-Cuf ventilation tube (Bivona, Inc, Gary, Ind) was used starting in 1987 to facilitate anastomosis. Since 1993, all patients have undergone video-assisted thoracoscopy immediately before the operation. RESULTS: There were 11 (18.3%) exploratory thoracotomies, 48 right tracheal sleeve pneumonectomies, and 1 left tracheal sleeve pneumonectomy. Among the tracheal sleeve pneumonectomies, we recorded 4 (8.2%) perioperative deaths (myocardial infarction, n = 1; heart failure, n = 1; pulmonary edema, n = 1; gastric ulcer hemorrhage, n = 1; and anastomotic fistula in a patient who received high-dose radiation before the operation, n = 1). We observed 5 (10.2%) complications (lung edema, n = 1; transitory recurrent nerve palsy, n = 2; empyema without fistula cured conservatively, n = 1; and pneumonia, n = 1). Anastomotic stenosis did not occur. Twenty-six (53%) patients are alive 14 to 87 months postoperatively, 12 (24.5%) of these more than 5 years postoperatively. Five (10.2%) died of mediastinal recurrence at 6 and 54 months. Two others (4.1%) died in road accidents. CONCLUSIONS: Tracheal sleeve pneumonectomy is a demanding operation with a high risk of complications. Analysis of literature and personal experience shows that complications can be greatly reduced through accurate selection of patients, precise technique, and optimal postoperative care. Long-term survival equals that obtained after standard pneumonectomy.

Adenocarcinoma↗
PubMed2001

[Blood coagulation monitoring during liver transplantation: Sonoclot analysis and laboratory tests].

BACKGROUND: Aim of this paper is to validate blood coagulation data obtained using the Sonoclot thromboelastographic analyser (Sienco Inc., Morrison, USA) by means of standard laboratory tests during orthotopic liver transplantation (OLT). METHODS DESIGN: comparative study between laboratory data and Sonoclot analysis on simultaneously collected blood samples. SETTING: National Health System Liver Transplantation Center. PATIENTS: fifty-one patients, both males and females, affected by terminal hepatic disease submitted to OLT were enrolled in the study. DATA COLLECTION: simultaneous blood samples were collected during the pre-anhepatic, anhepatic and post-reperfusion phases of OLT; coagulation status was assessed by means of either standard Laboratory tests (INR, aPTT, Fibrinogen, PLT, D-Dimer) and Sonoclot analyser data (SonACT, Rate, Time to Peak, Downward Deflection); a statistical analysis was performed (Pearson s chi(2) test). RESULTS: A statistically significative correlation between the analysed data was found. The Sonoclot analyser was useful in identifying platelets dysfunction and was more sensitive to detect fibrinolysis. CONCLUSIONS: The Sonoclot thromboelastographic analyser is a reliable device for monitoring coagulation during OLT.

Adult↗
PubMed2000

Regulation of ribosomal S6 kinase 2 by effectors of the phosphoinositide 3-kinase pathway.

Ribosomal S6 kinase (S6K1), through phosphorylation of the 40 S ribosomal protein S6 and regulation of 5'-terminal oligopyrimidine tract mRNAs, is an important regulator of cellular translational capacity. S6K1 has also been implicated in regulation of cell size. We have recently identified S6K2, a homolog of S6K1, which phosphorylates S6 in vitro and is regulated by the phosphatidylinositide 3-kinase (PI3-K) and mammalian target of rapamycin pathways in vivo. Here, we characterize S6K2 regulation by PI3-K signaling intermediates and compare its regulation to that of S6K1. We report that S6K2 is activated similarly to S6K1 by the PI3-K effectors phosphoinositide-dependent kinase 1, Cdc42, Rac, and protein kinase Czeta but that S6K2 is more sensitive to basal activation by myristoylated protein kinase Czeta than is S6K1. The C-terminal sequence of S6K2 is divergent from that of S6K1. We find that the S6K2 C terminus plays a greater role in S6K2 regulation than does the S6K1 C terminus by functioning as a potent inhibitor of activation by various agonists. Removal of the S6K2 C terminus results in an enzyme that is hypersensitive to agonist-dependent activation. These data suggest that S6K1 and S6K2 are similarly activated by PI3-K effectors but that sequences unique to S6K2 contribute to stronger inhibition of its kinase activity. Understanding the regulation of the two S6K homologs may provide insight into the physiological roles of these kinases.

Cells, Cultured↗
PubMed2000

Ribosomal S6 kinase 2 inhibition by a potent C-terminal repressor domain is relieved by mitogen-activated protein-extracellular signal-regulated kinase kinase-regulated phosphorylation.

Ribosomal S6 kinase 2 (S6K2) is a recently identified serine/threonine protein kinase that phosphorylates the 40 S ribosomal protein S6 in vitro. S6K2 is highly homologous to S6K1 in the core kinase and linker regulatory domains but differs from S6K1 in the N- and C-terminal regions and is differently localized primarily to the nucleus because of a C-terminal nuclear localization signal unique to S6K2. We have recently demonstrated that S6K2 is regulated similarly to S6K1 by the mammalian target of rapamycin pathway and by multiple PI3-K pathway effectors in vivo. However, deletion of the C-terminal domain of S6K2 enhances kinase activity, whereas analogous deletion of S6K1 is inhibitory. Here, we characterize the S6K2 C-terminal motifs that confer this differential regulation. We demonstrate that the inhibitory effects of the S6K2 C-terminal domain are only partly attributable to the nuclear localization signal but that three C-terminal proline-directed potential mitogen-activated protein kinase phosphorylation sites are critical mediators of this inhibitory effect. Site-specific mutation of these sites to alanine completely desensitizes S6K2 to activating inputs, whereas mutation to aspartic acid to mimic phosphorylation results in an activated enzyme which is hypersensitive to activating inputs. Pretreatment of cells with the mitogen-activated protein-extracellular signal-regulated kinase kinase (MEK) inhibitor U0126 inhibited S6K2 activation to a greater extent than S6K1. Furthermore, S6K2 mutants with C-terminal deletion or acidic phosphorylation site mutations displayed greatly reduced U0126 sensitivity. Thus, MEK-dependent inputs to C-terminal phosphorylation sites appear to be essential for relief of S6K2 inhibition but less critical for activation of S6K1. These data suggest a mechanism by which weak PI3-K agonists can regulate S6 phosphorylation and selective translation in the presence of mitogen-activated protein kinase signaling.

Butadienes↗
PubMed2000

Autografting followed by nonmyeloablative immunosuppressive chemotherapy and allogeneic peripheral-blood hematopoietic stem-cell transplantation as treatment of resistant Hodgkin's disease and non-Hodgkin's lymphoma.

PURPOSE: To investigate the use of a nonmyeloablative fludarabine-based immunosuppressive regimen to allow engraftment of HLA-sibling donors' mobilized stem cells and induction of a graft-versus-lymphoma effect for patients with advanced resistant Hodgkin's disease and non-Hodgkin's lymphoma. PATIENTS AND METHODS: Fifteen patients with Hodgkin's disease (n = 10) and non-Hodgkin's lymphoma (n = 5) were studied. All patients received cyclophosphamide and granulocyte colony-stimulating factor to mobilize autologous hematopoietic stem cells (HSCs). Subsequently, they received high-dose therapy with carmustine, etoposide, cytarabine, and melphalan and reinfusion of HSCs. At a median of 61 days after engraftment, patients were given fludarabine 30 mg/m(2) with cyclophosphamide 300 mg/m(2) daily for 3 days. Donor-mobilized HSC collections were prepared for fresh infusion and were not T-cell depleted. Methotrexate and cyclosporine were used to prevent graft rejection and as graft-versus-host disease (GVHD) prophylaxis. RESULTS: Combined treatment was well tolerated. After mini-allografting, hematologic recovery was prompt. Thirteen patients had 100% donor cell engraftment. Eleven patients achieved complete remission (CR) after the combined procedure. Nine patients, who were in partial remission after autografting, achieved CR after mini-allografting. Seven patients developed >/= grade 2 acute GVHD (aGVHD) and two developed extensive chronic GVHD (cGVHD). Three patients who received the highest number of donor lymphocyte infusions (DLIs) developed grade 3 GVHD (two patients) and extensive cGVHD (one patient). Ten patients are currently alive, and five are in continuous CR. Seven patients received DLI, with five CRs. Five patients died: one of progressive disease, two of progressive disease combined with aGVHD or cGVHD, one of extensive cGVHD, and one of infection. CONCLUSION: Fludarabine/cyclophosphamide was well tolerated and allowed consistent engraftment in lymphoma allografted patients. Response rates were high in this group of refractory and heavily pretreated patients. This dual procedure seems to be most promising in patients with end-stage malignant lymphomas.

Adult↗
PubMed2000

Effect of lengthening of peptide backbone by insertion of chiral beta-homo amino acid residues: conformational behavior of linear peptides containing alternating L-leucine and beta-homo L-leucine residues.

The synthesis and the solution behavior of the linear peptides containing a beta-homo (beta-H) leucine residue-Boc-Leu-beta-HLeu-Leu-OMe, Boc-beta-HLeu-Leu-beta-HLeu-Leu-OMe, and Boc-Leu-beta-HLeu-Leu-beta-HLeu-Leu-OMe-as well as the solid structure of the tripeptide, are reported. The conformational behavior of the peptides was investigated in solution by two-dimensional nmr. Our data support the existence in solution with different families of conformers in rapid interchange. The crystals of the tripeptide are orthorhombic, space group P2(1)2(1)2, with a = 15.829(1) A, b = 29.659(1) A, c = 6.563(1) A, and Z = 4. The structure has been solved by direct methods and refined to final R1 and wR2 indexes of 0.0530 and 0.1436 for 2420 reflections with I > 2sigma(I). In the solid state, the tripeptide does not present intramolecular H bonds, and the peptide backbone of the two leucine residues adopts a quasi-extended conformation. For the beta-HLeu residue, the backbone conformation is specified by the torsion angles straight phi(2) = -120.9(4) degrees, mu(2) = 56.7(4) degrees, psi(3) = -133.2(4) degrees. The side chains of the three residues assume the same conformation (g(-), g(-), trans), and all peptide bonds, except the urethane group at the N-terminus, are in the trans conformation. Preliminary conformational energy calculations carried out on the Ac-NH-beta-HAla-NHMe underline that the conformations with mu angle equal to 180 degrees and 60 degrees assume lower energy with respect to the others. In addition, we found a larger conformational freedom for the psi angle with respect to the straight phi angle.

Crystallography, X-Ray↗
PubMed2000

Computational procedures to explain the different biological activity of DNA/DNA, DNA/PNA and PNA/PNA hybrid molecules mimicking NF-kappaB binding sites.

Peptide nucleic acids (PNA) have recently been proposed as alternative reagents in experiments aimed to the control of gene expression. In PNAs, the pseudopeptide backbone is composed of N-(2-aminoethyl)glycine units and therefore is stable in human serum and cellular extracts. PNAs hybridize with high affinity to complementary sequences of single-stranded RNA and DNA, forming Watson-Crick double helices and giving rise to highly stable (PNA)2-RNA triplexes with RNA targets. Therefore, antisense and antigene PNAs have been synthetized and characterized. The major issue of the present paper is to describe some computational procedures useful to compare the behaviour of PNA double stranded molecules and PNA/DNA hybrids with the behaviour of regular DNA duplexes in generating complexes with DNA-binding proteins. The performed computational analyses clearly allow to predict that the lack of charged phosphate groups and the different shape of helix play a critical role in the binding efficiency of NF-kappaB transcription factors. These computational analyses are in agreement with competitive gel shift and UV-cross linking experiments. These experiments demonstrate that NF-kappaB PNA/PNA hybrids do not interact efficiently with proteins recognizing the NF-kappaB binding sites in genomic sequences. In addition, the data obtained indicate that the same NF-kappaB binding proteins recognize both the NF-kappaB DNA/PNA and DNA/DNA hybrids, but the molecular complexes generated with NF-kappaB DNA/PNA hybrids are less stable than those generated with NF-kappaB target DNA/DNA molecules.

Base Sequence↗
PubMed1999

p70 S6 kinase is regulated by protein kinase Czeta and participates in a phosphoinositide 3-kinase-regulated signalling complex.

p70 S6 kinase (p70S6K) is an important regulator of cell proliferation. Its activation by growth factor requires phosphorylation by various inputs on multiple sites. Data accumulated thus far support a model whereby p70S6K activation requires sequential phosphorylations at proline-directed residues in the putative autoinhibitory pseudosubstrate domain, as well as threonine 389. Threonine 229, a site in the catalytic loop is phosphorylated by phosphoinositide-dependent kinase 1 (PDK-1). Experimental evidence suggests that p70S6K activation requires a phosphoinositide 3-kinase (PI3-K)-dependent signal(s). However, the intermediates between PI3-K and p70S6K remain unclear. Here, we have identified PI3-K-regulated atypical protein kinase C (PKC) isoform PKCzeta as an upstream regulator of p70S6K. In coexpression experiments, we found that a kinase-inactive PKCzeta mutant antagonized activation of p70S6K by epidermal growth factor, PDK-1, and activated Cdc42 and PI3-K. While overexpression of a constitutively active PKCzeta mutant (myristoylated PKCzeta [myr-PKCzeta]) only modestly activated p70S6K, this mutant cooperated with PDK-1 activation of p70S6K. PDK-1-induced activation of a C-terminal truncation mutant of p70S6K was also enhanced by myr-PKCzeta. Moreover, we have found that p70S6K can associate with both PDK-1 and PKCzeta in vivo in a growth factor-independent manner, while PDK-1 and PKCzeta can also associate with each other, suggesting the existence of a multimeric PI3-K signalling complex. This work provides evidence for a link between a phorbol ester-insensitive PKC isoform and p70S6K. The existence of a PI3-K-dependent signalling complex may enable efficient activation of p70S6K in cells.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMed1998

A new collection method for the evaluation of nasal mucus proteins.

BACKGROUND: Different sampling techniques for collecting nasal mucus have been used. The eosinophil and its granule toxin proteins, especially the eosinophil cationic protein (ECP), is an important inflammatory cell. OBJECTIVE: In the healthy subjects (with no acute or chronic disease of the respiratory tract), no detailed quantitative data on mucus nasal proteins have been reported, so the aim of the study is to demonstrate a simple and reliable method for the collection of nasal mucus and for measuring nasal ECP. METHODS: Our method consists in collecting two specimens of mucus from each nasal fossa, using a square of sterile pre-humidified gauze, centrifuged at 2000g for 20 min and stored at -20 degrees C until tested. To evaluate the reproducibility of the technique, 30 healthy subjects were retested after 24 h. RESULTS: The amounts of secretion gathered in the 89 collections did not show any statistical difference between the sexes. In the pooled subjects (n=59), the mean levels of ECP were 108 ng/mL and 325 ng/g and total protein (TP) levels were 701 mg/dL and 2.5 mg/g (expressed in absolute concentration and concentration related to the weight in grams). In the classes, according to age, no statistical differences were found on analysis of the groups by: sex, recovery rate, and ECP and TP concentrations. The Reproducibility Index was very good for the recovery rate (0.78), ECP (ng/mL=0.95, ng/g=0.83) and TP (mg/dL=0.89, mg/g=0.76). CONCLUSION: With our method, the sample collections were taken with minimal stimulation of the mucosa and it was well tolerated by the subjects; the low known dilution factor is also adequate for analysing small quantities of mucus recovered, and the protein concentration can be measured as an absolute value.

Adult↗
PubMed1997

Activation of mitogen-activated protein kinase in freshly isolated rat hepatocytes by both a calcium- and a protein kinase C-dependent pathway.

In the present study, we investigated the role of calcium and protein kinase C (PKC) in the activation of mitogen-activated protein kinase (MAPK) in isolated rat hepatocytes. We found that the glycogenolytic hormone norepinephrine (NE), acting through the alpha1-adrenergic receptor and the G protein Gq, was able to induce a dose- and time-dependent activation of MAPK in hepatocytes. Vasopressin, which acts through a different receptor but also through stimulation of the Gq-dependent pathway, also caused a twofold activation of MAPK. Activation of MAPK by both agonists required the presence of free extracellular calcium and was blocked by the specific PKC inhibitor, Ro 31-8220. MAPK activation was also induced by phorbol myristate acetate (PMA), confirming that a PKC-dependent pathway exists for MAPK activation in liver. Furthermore, calcium-mobilizing agents such as thapsigargin and ionomycin were able to induce an activation of MAPK by a PKC-independent pathway that was totally abolished by preincubation of cells with EGTA. A second pathway for MAPK activation that relies solely on calcium may therefore exist. Ro 31-8220 did not affect phosphorylase activation by NE, vasopressin, thapsigargin, and ionomycin, indicating that PKC inhibition did not interfere with the signaling pathway leading to inositol-1,4,5-triphosphate (IP3)-induced calcium mobilization or with changes in calcium fluxes. The role of MAPK activation by NE and vasopressin in the regulation of hepatic carbohydrate metabolism is discussed.

Animals↗
PubMed1997

Insulin induces Ca2+ influx into isolated rat hepatocyte couplets.

Isolated rat hepatocyte couplets were used to study the direct effect of insulin on intracellular Ca2+ homeostasis. Insulin induced a dose-dependent increase in hepatocellular Ca2+ that was gradual, generally monophasic, and reversible. Chelation of extracellular Ca2+ abolished the insulin-induced Ca2+ response, and this suppression was not related to an effect on insulin binding, as indicated by displacement studies. We thus tested the effect of several Ca2+ channel inhibitors on insulin-induced Ca2+ influx. Verapamil at 20 or 200 microM was without effect, whereas 500 microM nickel and 50 microM gadolinium strongly inhibited insulin-induced Ca2+ entry. Finally, we tested whether insulin-induced Ca2+ movements were implicated in the stimulation of mitogen-activated protein kinase (MAPK) activity, which we measured with the use of an immune-complex assay. Verapamil was without effect on the insulin-dependent stimulation of p44mapk activity, whereas addition of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, nickel, or gadolinium strongly inhibited the effect of the peptide hormone. Our results indicate that insulin triggers Ca2+ influx into hepatocytes, possibly through the opening of channels on the plasma membrane, and that this effect is important for insulin activation of MAPK.

Animals↗