Biomedical subjects

Maria R Mazzoni

Publications and source records attributed to Maria R Mazzoni.

9 recordsLinked to original sources

PubMed2005

Morphiceptin analogues containing a dipeptide mimetic structure: an investigation on the bioactive topology at the mu-receptor.

We describe the design, the conformational behavior, and the biological activity at the mu-opioid receptor of new morphiceptin analogues. In these analogues a recently described dipeptide mimetic structure replaces both the N- and the C-terminal Xaa-Pro dipeptide of morphiceptin. Conformational investigation on the most active analogue, compared to the parent peptide, indicates a high degree of structural tolerance within the mu-opioid receptor binding site. In fact, our results indicate that only the location and the relative orientation of the side chains of the aromatic pharmacophoric residues represent the indispensable structural features for mu-receptor binding. To reach such topological arrangement, opioid peptides can adopt different conformations and configurations. In particular, opioid peptides bearing a proline residue as spacer between the two aromatic residues can adopt, in the active state, both cis and trans configurations at the Tyr(1)-Pro(2) amide bond, each of them with the appropriate backbone and side chains orientations.

Animals↗

PubMed2005

Thrombin modulates the expression of a set of genes including thrombospondin-1 in human microvascular endothelial cells.

Thrombospondin-1 (THBS1) is a large extracellular matrix glycoprotein that affects vasculature systems such as platelet activation, angiogenesis, and wound healing. Increases in THBS1 expression have been liked to disease states including tumor progression, atherosclerosis, and arthritis. The present study focuses on the effects of thrombin activation of the G-protein-coupled, protease-activated receptor-1 (PAR-1) on THBS1 gene expression in the microvascular endothelium. Thrombin-induced changes in gene expression were characterized by microarray analysis of approximately 11,000 different human genes in human microvascular endothelial cells (HMEC-1). Thrombin induced the expression of a set of at least 65 genes including THBS1. Changes in THBS1 mRNA correlated with an increase in the extracellular THBS1 protein concentration. The PAR-1-specific agonist peptide (TFLLRNK-PDK) mimicked thrombin stimulation of THBS1 expression, suggesting that thrombin signaling is through PAR-1. Further studies showed THBS1 expression was sensitive to pertussis toxin and protein kinase C inhibition indicating G(i/o)- and G(q)-mediated pathways. THBS1 up-regulation was also confirmed in human umbilical vein endothelial cells stimulated with thrombin. Analysis of the promoter region of THBS1 and other genes of similar expression profile identified from the microarray predicted an EBOX/EGRF transcription model. Expression of members of each family, MYC and EGR1, respectively, correlated with THBS1 expression. These results suggest thrombin formed at sites of vascular injury increases THBS1 expression into the extracellular matrix via activation of a PAR-1, G(i/o), G(q), EBOX/EGRF-signaling cascade, elucidating regulatory points that may play a role in increased THBS1 expression in disease states.

Adenosine Diphosphate↗

PubMed2004

Modulation of A1 adenosine receptor signaling by peroxynitrite.

Nitric oxide (NO) is a gaseous free radical involved in many pathophysiological processes. During oxidative stress, NO, its derivatives and adenosine are released. Considering adenosine neuroprotective role in the central nervous system (CNS) and toxicity of NO, we investigated the effect of a NO/peroxynitrite (ONOO(-)) donor, 3-morpholinosydnonimine (SIN-1), on A(1) adenosine receptor (A(1)AR) signaling pathway in rat cortical membranes. Membrane treatment with 0.5mM SIN-1 for various periods of time (0-240min) decreased specific binding of the radiolabeled A(1)AR agonist, [3H]N(6)-cyclohexyladenosine ([3H]CHA), in a time-dependent manner, reaching the steady state after 120min. The inhibitory effect of SIN-1 was concentration-dependent, with an EC(50) value of 0.60+/-0.30mM (N=3). Membrane pre-incubation with the superoxide anion (O(2)z.rad;(-)) scavenger superoxide dismutase (SOD) followed by SIN-1 addition, abolished SIN-1 inhibition of [3H]CHA binding. Membrane treatment with 0.5mM SIN-1 for 120min caused a significant 2-fold increase of the K(D) value for [3H]CHA without changing the B(max) value. Moreover, pre-incubation of membranes with A(1)AR agonists, CHA or N(6)-(2-phenylisopropyl)-adenosine (R-PIA) before SIN-1 addition increased the inhibitory effect while the selective A(1)AR antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) had no activity. Membrane treatment with SIN-1 decreased receptor-stimulated guanosine 5'-O-(gamma[35S]thio)triphosphate ([35S]GTPgammaS) binding in a concentration-dependent manner. This treatment influenced [35S]GTPgammaS binding affinity for A(1)AR activated G(i) proteins in cortical membranes. These findings suggest that ONOO(-) modulates A(1)AR signaling pathways by affecting receptor G(i) protein coupling.

Animals↗

PubMed2004

Modulation of endothelin-A receptor, Galpha subunit, and RGS2 expression during H9c2 cardiomyoblast differentiation.

In cardiac myocytes, growth responses depend on activation of G protein-coupled receptors interacting with Gq/11 protein subfamily members. Endothelin receptors of the ETA subtype belong to this receptor group inducing hypertrophic responses. To understand the role of ETA receptors and signal transduction proteins in modulating cell growth, we analyzed the pharmacological profile of this receptor, its level of expression together with those of Galpha subunits and the RGS2 protein in cardiomyoblasts differentiating into the cardiac phenotype. H9c2 rat cardiomyoblasts were grown in the presence of 10% fetal bovine serum (FBS) or 1% FBS plus all-trans-retinoic acid to induce the cardiac phenotype. The pharmacological properties of ETA receptors were investigated by competition-binding experiments, whereas the protein expression profile was analyzed by immunoblot and immunocytochemistry. The pharmacological profile of ETA receptors changed during differentiation of cardiomyoblasts into cardiomyocytes, and the amount of expressed receptor appeared to increase. Immunocytochemistry also showed a marked increase of receptor expression on cell membranes of differentiated cardiomyocytes. Among the other signaling proteins examined, both Galphaq/11 and RGS2 expression decreased in cells with the cardiac phenotype. Our results demonstrate that the expression of key proteins (ETA receptor, Galphaq/11, and RGS2) involved in signal transduction of hypertrophic stimuli is modulated during cell differentiation and correlates with the cardiac phenotype.

Animals↗

PubMed2003

Alkylation of sulfhydryl groups on Galpha(s/olf) subunits by N-ethylmaleimide: regulation by guanine nucleotides.

In rat striatum A(2A) adenosine receptors activate adenylyl cyclase through coupling to G(s)-like proteins, mainly G(olf) that is expressed at high levels in this brain region. In this study we report that the sulfhydryl alkylating reagent, N-ethylmaleimide (NEM), causes a concentration- and time-dependent inhibition of [3H] 2-p-(2-carboxyethyl)phenylethylamino)-5'-N-ethylcarboxamido adenosine ([3H]CGS21680) binding to rat striatal membranes. Membrane treatment with [14C]N-ethylmaleimide ([14C]NEM) labels numerous proteins while addition of 5'-guanylylimidodiphosphate (Gpp(NH)p) reduces labeling of only three protein bands that migrate in SDS-polyacrylamide gel electrophoresis with apparent molecular masses of approximately 52, 45 and 39 kDa, respectively. The 52- and 45-kDa labeled bands show electrophoretic motilities as Galpha(s)-long and Galpha(s)-short/Galpha(olf) subunits. An anti-Galpha(s/olf) antiserum immunoprecipitates two 14C labeled bands of 44 and 39 kDa. The band density decreases by 21-26% when membranes are treated with NEM in the presence of Gpp(NH)p. An anti-A(2A) receptor antibody also immunoprecipitates two 14C labeled bands of 40 and 38 kDa, respectively. However, such protein bands do not show any decrease of their density upon membrane treatment with NEM plus Gpp(NH)p. These results indicate that in rat striatal membranes NEM alkylates sulfhydryl groups of both Galpha(s/olf) subunits and A(2A) adenosine receptors. In addition, cysteine residues of Galpha(s/olf) are easily accessible to modification when the subunit is in the GDP-bound form. The 39- and 38-kDa labeled proteins may represent proteolytic fragments of Galpha(s/olf) and A(2A) adenosine receptor, respectively.

Alkylation↗

PubMed2003

ETA receptor-mediated Ca2+ mobilisation in H9c2 cardiac cells.

Expression and pharmacological properties of endothelin receptors (ETRs) were investigated in H9c2 cardiomyoblasts. The mechanism of receptor-mediated modulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) was examined by measuring fluorescence increase of Fluo-3-loaded cells with flow cytometry. Binding assays showed that [125I]endothelin-1 (ET-1) bound to a single class of high affinity binding sites in cardiomyoblast membranes. Endothelin-3 (ET-3) displaced bound [125I]ET-1 in a biphasic manner, in contrast to an ET(B)-selective agonist, IRL-1620, that was ineffective. The ET(B)-selective antagonist, BQ-788, inhibited [125I]ET-1 binding in a monophasic manner and with low potency. An ET(A)-selective antagonist, BQ-123, competed [125I]ET-1 binding in a monophasic manner. This antagonist was found to be 13-fold more potent than BQ-788. Immunoblotting analysis using anti-ET(A) and -ET(B) antibodies confirmed a predominant expression of the ET(A) receptor. ET-1 induced a concentration-dependent increase of Fluo-3 fluorescence in cardiomyoblasts resuspended in buffer containing 1mM CaCl(2). Treatment of cells with antagonists, PD-145065 and BQ-123, or a phospholipase C-beta inhibitor, U-73122, abolished ET-1-mediated increases in fluorescence. The close structural analogue of U-73122, U-73343, caused a minimal effect on the concentration-response curve of ET-1. ET-3 produced no major increase of Fluo-3 fluorescence. Removal of extracellular Ca(2+) resulted in a shift to the right of the ET-1 concentration-response curve. Both the L-type voltage-operated Ca(2+) channel blocker, nifedipine, and the ryanodine receptor inhibitor, dantrolene, reduced the efficacy of ET-1. Two protein kinase C inhibitors reduced both potency and efficacy of ET-1. Our results demonstrate that ET(A) receptors are expressed and functionally coupled to rise of [Ca(2+)](i) in H9c2 cardiomyoblasts. ET-1-induced [Ca(2+)](i) increase is triggered by Ca(2+) release from intracellular inositol 1,4,5-trisphosphate-gated stores; plasma membrane Ca(2+) channels and ryanodine receptors participate in sustaining the Ca(2+) response. Regulation of channel opening by protein kinase C is also involved in the process of [Ca(2+)](i) increase.

Animals↗

PubMed2003

A structure-activity relationship study on position-2 of the Galpha(s) C-terminal peptide able to inhibit G(s) activation by A2A adenosine receptor.

For some years synthetic peptides corresponding to the C-terminal sequence of Galpha proteins represented an useful tool to study the molecular mechanism of the interaction between these proteins and the G protein coupled receptors. Recently, we have focused our attention on the study of the A(2A) receptor-G(s) protein system. We have synthesised a series of 11-mer peptides from the Galpha(s) C-terminus in which residue at position-2 (Leu(393)) has been alternatively substituted with amino acids having different physico-chemical properties. The aim of our work was to probe the role played by Leu(393) in the receptor/Galpha(s) interaction. All synthetic peptides were tested for their ability to affect the adenylyl cyclase activity stimulated by agonist activation of A(2A) adenosine receptors. Our data point out a relevant role played by the side chain of this residue for a correct G protein/receptor coupling, even though the presence of other residues at position-2 of Galpha(s) C-terminus is tolerated. Furthermore, molecular dynamics calculations on the peptides having greater activity show a correlation between the spatial arrangement of the side chain of residue at position-2 and biological activity of synthetic peptides.

Adenylyl Cyclases↗

PubMed2003

Insights into G protein structure, function, and regulation.

In multicellular organisms from Caenorhabditis elegans to Homo sapiens, the maintenance of homeostasis is dependent on the continual flow and processing of information through a complex network of cells. Moreover, in order for the organism to respond to an ever-changing environment, intercellular signals must be transduced, amplified, and ultimately converted to the appropriate physiological response. The resolution of the molecular events underlying signal response and integration forms the basis of the signal transduction field of research. An evolutionarily highly conserved group of molecules known as heterotrimeric guanine nucleotide-binding proteins (G proteins) are key determinants of the specificity and temporal characteristics of many signaling processes and are the topic of this review. Numerous hormones, neurotransmitters, chemokines, local mediators, and sensory stimuli exert their effects on cells by binding to heptahelical membrane receptors coupled to heterotrimeric G proteins. These highly specialized transducers can modulate the activity of multiple signaling pathways leading to diverse biological responses. In vivo, specific combinations of G alpha- and G beta gamma-subunits are likely required for connecting individual receptors to signaling pathways. The structural determinants of receptor-G protein-effector specificity are not completely understood and, in addition to involving interaction domains of these primary acting proteins, also require the participation of scaffolding and regulatory proteins.

Animals↗

PubMed2002

Conformational analysis of the Galpha(s) protein C-terminal region.

The C-terminal domain of the heterotrimeric G protein a-subunits plays a key role in selective activation of G proteins by their cognate receptors. Several C-terminal fragments of Galpha(s) (from 11 to 21 residues) were recently synthesized. The ability of these peptides to stimulate agonist binding was found to be related to their size. Galpha(s)(380-394) is a 15-mer peptide of intermediate length among those synthesized and tested that displays a biological activity surprisingly weak compared with that of the corresponding 21-mer peptide, shown to be the most active. In the present investigation, Galpha(s)(380-394) was subjected to a conformational NMR analysis in a fluorinated isotropic environment. An NMR structure, calculated on the basis of the data derived from conventional 1D and 2D homonuclear experiments, shows that the C-terminal residues of Galpha(s)(380-394) are involved in a helical arrangement whose length is comparable to that of the most active 21 -mer peptide. A comparative structural refinement of the NMR structures of Galpha(s)(380-394) and Galpha(s)(374-394)C379A was performed using molecular dynamics calculations. The results give structural elements to interpret the role played by both the backbone conformation and the side chain arrangement in determining the activity of the G protein C-terminal fragments. The orientation of the side chains allows the peptides to assume contacts crucial for the G protein/receptor interaction. In the 15-mer peptide the lack as well as the disorder of some N-terminal residues could explain the low biological activity observed.

Amino Acid Sequence↗