Limits...
Comparative analysis of calcium spikes upon activation of serotonin(1A) and purinergic receptors.

Saxena R, Ganguly S, Chattopadhyay A - PLoS ONE (2012)

Bottom Line: Calcium signaling represents one of the most important signaling cascades in cells and regulates diverse processes such as exocytosis, muscle contraction and relaxation, gene expression and cell growth.In this work, we have described a set of parameters for the analysis of calcium transients that could provide novel insight into mechanisms responsible for maintaining signal specificity by shaping calcium transients.In summary, our analysis offers a novel approach to identify differences in calcium response patterns induced by various stimuli.

View Article: PubMed Central - PubMed

Affiliation: Centre for Cellular and Molecular Biology, Council of Scientific and Industrial Research, Hyderabad, India.

ABSTRACT
Calcium signaling represents one of the most important signaling cascades in cells and regulates diverse processes such as exocytosis, muscle contraction and relaxation, gene expression and cell growth. G protein-coupled receptors (GPCRs) are the most important family of receptors that activate calcium signaling. Since calcium signaling regulates a large number of physiological responses, it is intriguing that how changes in cytosolic calcium levels by a wide range of stimuli lead to signal-specific physiological responses in the cellular interior. In order to address this issue, we have analyzed temporal calcium profiles induced by two GPCRs, the serotonin(1A) and purinergic receptors. In this work, we have described a set of parameters for the analysis of calcium transients that could provide novel insight into mechanisms responsible for maintaining signal specificity by shaping calcium transients. An interesting feature of calcium signaling that has emerged from our analysis is that the profile of individual transients in a calcium response could play an important role in maintaining downstream signal specificity. In summary, our analysis offers a novel approach to identify differences in calcium response patterns induced by various stimuli.

Show MeSH

Related in: MedlinePlus

Dose response plots for calcium signaling induced by ligands in CHO-5-HT1AR cells.The figure shows calcium response in terms of a number of calcium spikes visualized per cell induced with either (A) serotonin or (B) ATP. The curves are nonlinear regression fits to the experimental data using eqn. 1. Data represent means ± SEM of more than 30 cells from at least four independent experiments. See Materials and Methods for more details.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3526489&req=5

pone-0051857-g002: Dose response plots for calcium signaling induced by ligands in CHO-5-HT1AR cells.The figure shows calcium response in terms of a number of calcium spikes visualized per cell induced with either (A) serotonin or (B) ATP. The curves are nonlinear regression fits to the experimental data using eqn. 1. Data represent means ± SEM of more than 30 cells from at least four independent experiments. See Materials and Methods for more details.

Mentions: In order to monitor changes in cytosolic calcium level, we employed a calcium- sensitive fluorophore fluo-3/AM. Fluo-3/AM is a cell permeant variant of a calcium indicator whose fluorescence intensity increases upon binding to calcium [23]. Upon stimulation of fluo-3/AM loaded CHO cells with serotonin or ATP, we observed repetitive calcium transients as opposed to the single broad spike reported earlier [24], [25]. This is possibly due to the fact that we employed higher speed imaging of cells with a temporal resolution of ∼250 ms in contrast to temporal resolution of few sec achieved in earlier reports. The high temporal resolution in our measurements provided us the advantage to visualize ligand-mediated repetitive calcium spikes. Fig. 1A shows a representative series of images scanned after every 7.9 sec showing calcium response in CHO-5-HT1AR cells upon stimulation with serotonin. The temporal intensity profile of calcium response in each cell was obtained by analyzing sequence of images acquired after every ∼250 ms. Such temporal profiles of calcium, induced by serotonin and ATP in single cells are shown in Fig. 1B and C. As mentioned earlier, each calcium spike consists of rise and decay phases as shown in the inset of Fig. 1B. The number of calcium spikes visualized per cell showed a dependence on ligand concentration in both cases (see Fig. 2). The figure shows that the number of calcium spikes increased with an increase in ligand concentration. The dose response curves for serotonin and ATP are shown in Fig. 2A and B, respectively. Dose response curves were fitted with eqn. 1 and EC50 values were calculated for serotonin and ATP. EC50 values obtained were ∼0.28 and ∼0.08 µM in cases of stimulation with serotonin and ATP, respectively. Interestingly, these ligands induced different responses, in terms of number of calcium spikes visualized per cell, corresponding to the same ligand concentration. ATP appeared to be more potent ligand than serotonin in eliciting calcium response. It should be noted that all cells loaded with fluo-3/AM do not exhibit calcium response upon addition of ligand. Their response depends on ligand concentration and type. In addition, the occurrence of oscillations, rather than a single transient, also depends on ligand concentration. For example, probability of occurrence of oscillations increases with increase in ligand concentration.


Comparative analysis of calcium spikes upon activation of serotonin(1A) and purinergic receptors.

Saxena R, Ganguly S, Chattopadhyay A - PLoS ONE (2012)

Dose response plots for calcium signaling induced by ligands in CHO-5-HT1AR cells.The figure shows calcium response in terms of a number of calcium spikes visualized per cell induced with either (A) serotonin or (B) ATP. The curves are nonlinear regression fits to the experimental data using eqn. 1. Data represent means ± SEM of more than 30 cells from at least four independent experiments. See Materials and Methods for more details.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3526489&req=5

pone-0051857-g002: Dose response plots for calcium signaling induced by ligands in CHO-5-HT1AR cells.The figure shows calcium response in terms of a number of calcium spikes visualized per cell induced with either (A) serotonin or (B) ATP. The curves are nonlinear regression fits to the experimental data using eqn. 1. Data represent means ± SEM of more than 30 cells from at least four independent experiments. See Materials and Methods for more details.
Mentions: In order to monitor changes in cytosolic calcium level, we employed a calcium- sensitive fluorophore fluo-3/AM. Fluo-3/AM is a cell permeant variant of a calcium indicator whose fluorescence intensity increases upon binding to calcium [23]. Upon stimulation of fluo-3/AM loaded CHO cells with serotonin or ATP, we observed repetitive calcium transients as opposed to the single broad spike reported earlier [24], [25]. This is possibly due to the fact that we employed higher speed imaging of cells with a temporal resolution of ∼250 ms in contrast to temporal resolution of few sec achieved in earlier reports. The high temporal resolution in our measurements provided us the advantage to visualize ligand-mediated repetitive calcium spikes. Fig. 1A shows a representative series of images scanned after every 7.9 sec showing calcium response in CHO-5-HT1AR cells upon stimulation with serotonin. The temporal intensity profile of calcium response in each cell was obtained by analyzing sequence of images acquired after every ∼250 ms. Such temporal profiles of calcium, induced by serotonin and ATP in single cells are shown in Fig. 1B and C. As mentioned earlier, each calcium spike consists of rise and decay phases as shown in the inset of Fig. 1B. The number of calcium spikes visualized per cell showed a dependence on ligand concentration in both cases (see Fig. 2). The figure shows that the number of calcium spikes increased with an increase in ligand concentration. The dose response curves for serotonin and ATP are shown in Fig. 2A and B, respectively. Dose response curves were fitted with eqn. 1 and EC50 values were calculated for serotonin and ATP. EC50 values obtained were ∼0.28 and ∼0.08 µM in cases of stimulation with serotonin and ATP, respectively. Interestingly, these ligands induced different responses, in terms of number of calcium spikes visualized per cell, corresponding to the same ligand concentration. ATP appeared to be more potent ligand than serotonin in eliciting calcium response. It should be noted that all cells loaded with fluo-3/AM do not exhibit calcium response upon addition of ligand. Their response depends on ligand concentration and type. In addition, the occurrence of oscillations, rather than a single transient, also depends on ligand concentration. For example, probability of occurrence of oscillations increases with increase in ligand concentration.

Bottom Line: Calcium signaling represents one of the most important signaling cascades in cells and regulates diverse processes such as exocytosis, muscle contraction and relaxation, gene expression and cell growth.In this work, we have described a set of parameters for the analysis of calcium transients that could provide novel insight into mechanisms responsible for maintaining signal specificity by shaping calcium transients.In summary, our analysis offers a novel approach to identify differences in calcium response patterns induced by various stimuli.

View Article: PubMed Central - PubMed

Affiliation: Centre for Cellular and Molecular Biology, Council of Scientific and Industrial Research, Hyderabad, India.

ABSTRACT
Calcium signaling represents one of the most important signaling cascades in cells and regulates diverse processes such as exocytosis, muscle contraction and relaxation, gene expression and cell growth. G protein-coupled receptors (GPCRs) are the most important family of receptors that activate calcium signaling. Since calcium signaling regulates a large number of physiological responses, it is intriguing that how changes in cytosolic calcium levels by a wide range of stimuli lead to signal-specific physiological responses in the cellular interior. In order to address this issue, we have analyzed temporal calcium profiles induced by two GPCRs, the serotonin(1A) and purinergic receptors. In this work, we have described a set of parameters for the analysis of calcium transients that could provide novel insight into mechanisms responsible for maintaining signal specificity by shaping calcium transients. An interesting feature of calcium signaling that has emerged from our analysis is that the profile of individual transients in a calcium response could play an important role in maintaining downstream signal specificity. In summary, our analysis offers a novel approach to identify differences in calcium response patterns induced by various stimuli.

Show MeSH
Related in: MedlinePlus