Measurement of intracellular Ca 2+ mobilization to study GPCR signal transduction

Author(s):  
Anisha Ashokan ◽  
Gopala K. Aradhyam
Author(s):  
Timothy P McMullen ◽  
Evan A Brown ◽  
Nahid Ausrafuggaman ◽  
Alisha Sahu ◽  
Ali D Güler ◽  
...  

Reproduction ◽  
2004 ◽  
Vol 127 (4) ◽  
pp. 455-463 ◽  
Author(s):  
A Talmor-Cohen ◽  
R Tomashov-Matar ◽  
E Eliyahu ◽  
R Shapiro ◽  
R Shalgi

The earliest visible indications for the transition to embryos in mammalian eggs, known as egg activation, are cortical granules exocytosis (CGE) and resumption of meiosis (RM); these events are triggered by the fertilizing spermatozoon through a series of Ca2+transients. The pathways, within the egg, leading to the intracellular Ca2+release and to the downstream cellular events, are currently under intensive investigation. The involvement of Src family kinases (SFKs) in Ca2+release at fertilization is well supported in marine invertebrate eggs but not in mammalian eggs. In a previous study we have shown the expression and localization of Fyn, the first SFK member demonstrated in the mammalian egg. The purpose of the current study was to identify other common SFKs and resolve their function during activation of mammalian eggs. All three kinases examined: Fyn, c-Src and c-Yes are distributed throughout the egg cytoplasm. However, Fyn and c-Yes tend to concentrate at the egg cortex, though only Fyn is localized to the spindle as well. The different localizations of the various SFKs imply the possibility of their different functions within the egg. To examine whether SFKs participate in the signal transduction pathways during egg activation, we employed selective inhibitors of the SFKs activity ((PP2 and SU6656). The results demonstrate that RM, which is triggered by Ca2+elevation, is an SFK-dependent process, while CGE, triggered by either Ca2+elevation or protein kinase C (PKC), is not. The possible involvement of SFKs in the signal transduction pathways that lead from the sperm–egg fusion site downstream of the Ca2+release remains unclear.


2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Leonardo David Herrera-Zúñiga ◽  
Liliana Marisol Moreno-Vargas ◽  
Luck Ballaud ◽  
José Correa-Basurto ◽  
Diego Prada-Gracia ◽  
...  

Abstract In this work, we studied the mechanisms of classical activation and inactivation of signal transduction by the histamine H3 receptor, a 7-helix transmembrane bundle G-Protein Coupled Receptor through long-time-scale atomistic molecular dynamics simulations of the receptor embedded in a hydrated double layer of dipalmitoyl phosphatidyl choline, a zwitterionic polysaturated ordered lipid. Three systems were prepared: the apo receptor, representing the constitutively active receptor; and two holo-receptors—the receptor coupled to the antagonist/inverse agonist ciproxifan, representing the inactive state of the receptor, and the receptor coupled to the endogenous agonist histamine and representing the active state of the receptor. An extensive analysis of the simulation showed that the three states of H3R present significant structural and dynamical differences as well as a complex behavior given that the measured properties interact in multiple and interdependent ways. In addition, the simulations described an unexpected escape of histamine from the orthosteric binding site, in agreement with the experimental modest affinities and rapid off-rates of agonists.


Author(s):  
Victor J. Hruby ◽  
Isabel D. Alves ◽  
Savitha Devanathan ◽  
Zdzislaw Salamon ◽  
Gordon Tollin

2007 ◽  
Vol 102 (3) ◽  
pp. 978-984 ◽  
Author(s):  
Rajat Sethi ◽  
Harjot K. Saini ◽  
Xiaobing Guo ◽  
Xi Wang ◽  
Vijayan Elimban ◽  
...  

To examine whether cardiac hypertrophy is associated with changes in β-adrenoceptor signal transduction mechanisms, pressure overload (PO) was induced by occlusion of the abdominal aorta and volume overload (VO) by creation of an aortocaval shunt for 4 and 24 wk in rats. After hemodynamic assessment of the animals, the left ventricular (LV) particulate fraction was isolated for measurement of β1-adrenoceptors and adenylyl cyclase activity, and cardiomyocytes were isolated for monitoring of the intracellular Ca2+ concentration. Although PO and VO produced cardiac hypertrophy and increased LV end-diastolic pressure at 4 wk, cardiac function was increased in animals subjected to PO but remained unaltered in animals subjected to VO. Cardiac hypertrophy and increased LV end-diastolic pressure were associated with depressed cardiac function at 24 wk of PO or VO, but clinical signs of congestive heart failure were evident only in animals subjected to VO. Isoproterenol-induced increases in cardiac function, activation of adenylyl cyclase activity, and increase in intracellular Ca2+ concentration, as well as β1-adrenoceptor density, were unaltered by PO at 4 wk, augmented by VO at 4 wk, and attenuated by PO and VO at 24 wk. These results suggest that alterations in β1-adrenoceptor signal transduction are dependent on the type and stage of cardiac hypertrophy.


2018 ◽  
Author(s):  
William M. Shaw ◽  
Hitoshi Yamauchi ◽  
Jack Mead ◽  
Glen-Oliver F. Gowers ◽  
David Öling ◽  
...  

AbstractG protein-coupled receptor (GPCR) signaling is the primary method eukaryotes use to respond to specific cues in their environment. However, the relationship between stimulus and response for each GPCR is difficult to predict due to diversity in natural signal transduction architecture and expression. Using genome engineering in yeast, we here constructed an insulated, modular GPCR signal transduction system to study how the response to stimuli can be predictably tuned using synthetic tools. We delineated the contributions of a minimal set of key components via computational and experimental refactoring, identifying simple design principles for rationally tuning the dose-response. Using four different receptors, we demonstrate how this enables cells and consortia to be engineered to respond to desired concentrations of peptides, metabolites and hormones relevant to human health. This work enables rational tuning of cell sensing, while providing a framework to guide reprogramming of GPCR-based signaling in more complex systems.


1999 ◽  
Vol 276 (1) ◽  
pp. E171-E178 ◽  
Author(s):  
N. E. Ajubi ◽  
J. Klein-Nulend ◽  
M. J. Alblas ◽  
E. H. Burger ◽  
P. J. Nijweide

To maintain its structural competence, the skeleton adapts to changes in its mechanical environment. Osteocytes are generally considered the bone mechanosensory cells that translate mechanical signals into biochemical, bone metabolism-regulating stimuli necessary for the adaptive process. Prostaglandins are an important part of this mechanobiochemical signaling. We investigated the signal transduction pathways in osteocytes through which mechanical stress generates an acute release of prostaglandin E2(PGE2). Isolated chicken osteocytes were subjected to 10 min of pulsating fluid flow (PFF; 0.7 ± 0.03 Pa at 5 Hz), and PGE2release was measured. Blockers of Ca2+ entry into the cell or Ca2+ release from internal stores markedly inhibited the PFF-induced PGE2 release, as did disruption of the actin cytoskeleton by cytochalasin B. Specific inhibitors of Ca2+-activated phospholipase C, protein kinase C, and phospholipase A2 also decreased PFF-induced PGE2 release. These results are consistent with the hypothesis that PFF raises intracellular Ca2+ by an enhanced entry through mechanosensitive ion channels in combination with Ca2+- and inositol trisphosphate (the product of phospholipase C)-induced Ca2+ release from intracellular stores. Ca2+ and protein kinase C then stimulate phospholipase A2activity, arachidonic acid production, and ultimately PGE2 release.


Author(s):  
Panagiota Kontou ◽  
Athanasia Pavlopoulou ◽  
Niki Dimou ◽  
Margarita Theodoropoulou ◽  
Georgia Braliou ◽  
...  

2000 ◽  
Vol 78 (3) ◽  
pp. 139-153 ◽  
Author(s):  
John P Chang ◽  
James D Johnson ◽  
Fredrick Van Goor ◽  
Calvin JH Wong ◽  
Warren K Yunker ◽  
...  

The intracellular signal transduction mechanisms mediating maturational gonadotropin and somatotropin secretion in goldfish are reviewed. Several major signaling mechanisms, including changes in intracellular [Ca2+], arachidonic acid cascades, protein kinase C, cyclic AMP/protein kinase A, calmodulin, nitric oxide, and Na+/H+ antiport, are functional in both cell types. However, their relative importance in mediating basal secretion and neuroendocrine-factor-regulated hormone release differs according to cell type. Similarly, agonist- and cell-type-specificity are also present in the transduction pathways leading to neuroendocrine factor-modulated maturational gonadotropin and somatotropin release. Specificity is present not only in the actions of different regulators within the same cell type and with the same ligand in the two cell types, but this also exists between isoforms of the same neuroendocrine factor within a single cell type. Other evidence suggests that function-selectivity of signaling may also result from differential modulation of Ca2+ fluxes from different sources. The interaction of different second messenger systems provide the basis by which regulation of maturational gonadotropin and somatotropin release by multiple neuroendocrine factors can be integrated at the target cell level. Key words: Ca2+ signaling, cAMP, PKC, arachidonic acid, NO.


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