Blog Arasys Perfector - Xanya Sofra Weiss

Maddalena Illario, Anna Lina Cavallo, Sara Monaco, Ennio Di Vito, Frank Mueller, Luigi A. Marzano, Giancarlo Troncone, Gianfranco Fenzi, Guido Rossi, and Mario Vitale.

Werecently demonstrated in an immortalized thyroid cell line
that integrin stimulation by fibronectin (FN) simultaneously
activates two signaling pathways: Ras/Raf/MAPK kinase
(Mek)/Erk and calcium (Ca2)/calcium calmodulin-dependent
kinase II (CaMKII). Both signals are necessary to stimulate
Erk phosphorylation because CaMKII modulates Ras-induced
Raf-1 activity. In this study we present evidence that extends
these findings to normal human thyroid cells in primary culture,
demonstrating its biological significance in a more physiological
cell model. In normal thyroid cells, immobilized FNinduced
activation of p21Ras and Erk phosphorylation. This
pathway was responsible for FN-induced cell proliferation.
Concurrent increase of intracellular Ca2 concentration and
CaMKII activation was observed. Both induction of p21Ras
activity and increase of intracellular Ca2 concentration were
mediated by FN binding to
v3 integrin. Inhibition of the
Ca2/CaMKII signal pathway by calmodulin or CaMKII inhibitors
completely abolished the FN-induced Erk phosphorylation.
Binding to FN induced Raf-1 and CaMKII to form a protein
complex, indicating that intersection between Ras/Raf/
Mek/Erk and Ca2/CaMKII signaling pathways occurred at
Raf-1 level. Interruption of the Ca2/CaMKII signal pathway
arrested cell proliferation induced by FN. We also analyzed
thyroid tumor cell lines that displayed concomitant aberrant
integrin expression and signal transduction. These data confirm
that integrin activation by FN in normal thyroid cells
generates Ras/Raf/Mek/Erk and Ca2/CaMKII signaling pathways
and that both are necessary to stimulate cell proliferation,
whereas in thyroid tumors integrin signaling is altered.
(J Clin Endocrinol Metab 90: 2865–2873, 2005)

1. Excitation-contraction coupling is broadly defined as the process linking the action potential to contraction in striated muscle or, more narrowly, as the process coupling surface membrane depolarization to Ca(2+) release from the sarcoplasmic reticulum. 2. We now know that excitation-contraction coupling depends on a macromolecular protein complex or ‘calcium release unit’. The complex extends the extracellular space within the transverse tubule invaginations of the surface membrane, across the transverse tubule membrane into the cytoplasm and then across the sarcoplasmic reticulum membrane and into the lumen of the sarcoplasmic reticulum. 3. The central element of the macromolecular complex is the ryanodine receptor calcium release channel in the sarcoplasmic reticulum membrane. The ryanodine receptor has recruited a surface membrane L-type calcium channel as a ‘voltage sensor’ to detect the action potential and the calcium-binding protein calsequestrin to detect in the environment within the sarcoplasmic reticulum. Consequently, the calcium release channel is able to respond to surface depolarization in a manner that depends on the Ca(2+) load within the calcium store. 4. The molecular components of the ‘calcium release unit’ are the same in skeletal and cardiac muscle. However, the mechanism of excitation-contraction coupling is different. The signal from the voltage sensor to ryanodine receptor is chemical in the heart, depending on an influx of external Ca(2+) through the surface calcium channel. In contrast, conformational coupling links the voltage sensor and the ryanodine receptor in skeletal muscle. 5. Our current understanding of this amazingly efficient molecular signal transduction machine has evolved over the past 50 years. None of the proteins had been identified in the 1950s; indeed, there was debate about whether the molecules involved were, in fact, protein. Nevertheless, a multitude of questions about the molecular interactions and structures of the proteins and their interaction sites remain to be answered and provide a challenge for the next 50 years.

Cruse G, Yang W, Duffy SM, Chachi L, Leyland M, Amrani Y, Bradding P.

BACKGROUND: Mast cells contribute to the pathophysiology of asthma with the sustained release of both preformed and newly generated mediators in response to allergens and other diverse stimuli. Stem cell factor (SCF) is the key human mast cell growth factor, but also primes mast cells for mediator release. SCF expression is markedly increased in asthmatic airways. Short-acting beta(2)-adrenoceptor drugs such as albuterol inhibit human lung mast cell (HLMC) degranulation in vitro in the absence of SCF, but their effect in the presence of SCF is not known. OBJECTIVE: The aim of this study was to elucidate the effects of albuterol on HLMC function in the presence of SCF. METHODS: Mediator release and K(Ca)3.1 ion channel activity were analyzed in purified HLMC. Intracellular signalling and beta(2)-adrenoceptor phosphorylation and internalization were analyzed in the HMC-1 human mast cell line. RESULTS: beta(2)-Adrenoceptor agonist-dependent inhibition of K(Ca)3.1 ion channels and HLMC mediator release was markedly attenuated in the presence of SCF. Remarkably, albuterol actually potentiated IgE-induced histamine release in a dose-dependent manner when both SCF and IgE were present. These effects were related to the SCF-dependent phosphorylation of Tyr350 on the beta(2)-adrenoceptor with immediate uncoupling of the receptor followed by receptor internalization. CONCLUSION: The potentially beneficial effects of beta(2)-adrenoceptor agonists in asthmatic airways may be blunted as a result of the high concentrations of SCF present.

Wingertzahn MA, Ochs RS.

The missing link in our understanding of excitation-contraction coupling (ECC) in skeletal muscle is the mechanism by which Ca2+ increases in the cytosol to trigger contraction. We discuss here a general background of intracellular Ca2+ handling, some characteristics of the major proteins involved in Ca2+ flow during ECC, and mechanisms currently believed to explain the increase in Ca2+ upon stimulation of muscle cells. These mechanisms include the calcium-induced calcium release, the direct coupled mechanism in which a plasma membrane and sarcoplasmic reticulum membrane protein interact, and mechanisms involving Ca2+ secretagogues that are known to elicit increases in calcium in other cells, inositol trisphosphate, and cyclic ADP ribose. We also consider possible roles for proteins associated with the principal calcium release channel of the sarcoplasmic reticulum, the ryanodine receptor. Finally, we discuss malignant hyperthermia, a disease associated directly with aberrant control of muscle cell calcium release. Copyright 1998 Academic Press.

Grit Schaarschmidt, Florian Wegner, Sigrid C. Schwarz, Hartmut Schmidt, Johannes Schwarz

Background: Voltage-gated potassium (Kv) channels are among the earliest ion channels to appear during brain development, suggesting a functional requirement for progenitor cell proliferation and/or differentiation. We tested this hypothesis, using human neural progenitor cells (hNPCs) as a model system.
Methodology/Principal Findings: In proliferating hNPCs a broad spectrum of Kv channel subtypes was identified using quantitative real-time PCR with a predominant expression of the A-type channel Kv4.2. In whole-cell patch-clamp recordings Kv currents were separated into a large transient component characteristic for fast-inactivating A-type potassium channels (IA) and a small, sustained component produced by delayed-rectifying channels (IK). During differentiation the expression of IA as well as A-type channel transcripts dramatically decreased, while IK producing delayed-rectifiers were upregulated. Both Kv currents were differentially inhibited by selective neurotoxins like phrixotoxin-1 and a-dendrotoxin as well as by antagonists like 4-aminopyridine, ammoniumchloride, tetraethylammonium chloride and quinidine. In viability and proliferation assays chronic inhibition of the A-type currents severely disturbed the cell cycle and precluded proper hNPC proliferation, while the blockade of delayed-rectifiers by a-dendrotoxin increased proliferation.
Conclusions/Significance: These findings suggest that A-type potassium currents are essential for proper proliferation of immature multipotent hNPCs.

Pfannkuche K, Liang H, Hannes T, Xi J, Fatima A, Nguemo F, Matzkies M, Wernig M, Jaenisch R, Pillekamp F, Halbach M, Schunkert H, Sarić T, Hescheler J, Reppel M.

AIMS: Induced pluripotent stem (iPS) cells have a developmental potential similar to that of blastocyst-derived embryonic stem (ES) cells and may serve as an autologous source of cells for tissue repair, in vitro disease modelling and toxicity assays. Here we aimed at generating iPS cell-derived cardiomyocytes (CMs) and comparing their molecular and functional characteristics with CMs derived from native murine ES cells. METHODS AND RESULTS: Beating cardiomyocytes were generated using a mass culture system from murine N10 and O9 iPS cells as well as R1 and D3 ES cells. Transcripts of the mesoderm specification factor T-brachyury and non-atrial cardiac specific genes were expressed in differentiating iPS EBs. Using immunocytochemistry to determine the expression and intracellular organisation of cardiac specific structural proteins we demonstrate strong similarity between iPS-CMs and ES-CMs. In line with a previous study electrophysiological analyses showed that hormonal response to beta-adrenergic and muscarinic receptor stimulation was intact. Action potential (AP) recordings suggested that most iPS-CMs measured up to day 23 of differentiation are of ventricular-like type. Application of lidocaine, Cs+, SEA0400 and verapamil+ nifedipine to plated iPS-EBs during multielectrode array (MEA) measurements of extracellular field potentials and intracellular sharp electrode recordings of APs revealed the presence of I(Na), I(f), I(NCX), and I(CaL), respectively, and suggested their involvement in cardiac pacemaking, with I(CaL) being of major importance. Furthermore, iPS-CMs developed and conferred force to avitalized ventricular tissue that was responsive to beta-adrenergic stimulation. CONCLUSIONS: Our data demonstrate that the cardiogenic potential of iPS cells is comparable to that of ES cells and that iPS-CMs possess all fundamental functional elements of a typical cardiac cell, including spontaneous beating, hormonal regulation, cardiac ion channel expression and contractility. Therefore, iPS-CMs can be regarded as a potentially valuable source of cells for in vitro studies and cellular cardiomyoplasty. 2009 S. Karger AG, Basel.

Calderón-Vélez JC, Figueroa-Gordon LC.

The excitation-contraction coupling mechanism was defined as the entire sequence of reactions linking excitation of plasma membrane to activation of contraction in skeletal muscle. By using different techniques, their regulation and interactions have been studied during the last 50 years, defining until now the importance and origin of the calcium ion as a contractile activator and the main proteins involved in the whole mechanism. Furthermore, the study of the ultrastructural basis and pharmacological regulation of the excitation-contraction coupling phenomenon has begun. The excitationcontraction coupling is thought to be altered in situations as ageing, muscle fatigue and some muscle diseases. However, many questions remain to be answered. For example, (1) How excitation-contraction coupling develops and ages? (2) What role does it play in muscle fatigue and other diseases? (3) What is the nature of the interaction between the proteins believed to be involved? The present review describes excitation-contraction coupling in skeletal muscle and techniques used to better understand it as an introduction for discussing unanswered questions regarding excitation-contraction coupling