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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.

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Calcium response in CHO-5-HT1AR cells upon stimulation with serotonin or ATP.Panel (A) shows successive images of calcium response in cells upon stimulation with serotonin acquired with a time interval of 7.9 sec. CHO-5-HT1AR cells were loaded with Fluo-3/AM to monitor changes in cytosolic calcium concentration. Time-lapse imaging was performed with a 20×/0.75 NA objective. The scale bar (shown in top left panel) represents 50 µm. Similar response was observed when cells were stimulated with ATP. Representative temporal profiles of calcium response induced by either (B) serotonin or (C) ATP are shown. The concentration of serotonin used was 100 µM and that of ATP was 500 nM, respectively. Both serotonin and ATP induce the occurrence of multiple spikes consisting of rise and decay phases. A typical calcium spike with rise and decay phases is shown in the inset in panel (B). The temporal profile of calcium response was intensity normalized such that the maximum and minimum (basal) intensities corresponded to values 2 and 1, respectively. See Materials and Methods for more details.
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pone-0051857-g001: Calcium response in CHO-5-HT1AR cells upon stimulation with serotonin or ATP.Panel (A) shows successive images of calcium response in cells upon stimulation with serotonin acquired with a time interval of 7.9 sec. CHO-5-HT1AR cells were loaded with Fluo-3/AM to monitor changes in cytosolic calcium concentration. Time-lapse imaging was performed with a 20×/0.75 NA objective. The scale bar (shown in top left panel) represents 50 µm. Similar response was observed when cells were stimulated with ATP. Representative temporal profiles of calcium response induced by either (B) serotonin or (C) ATP are shown. The concentration of serotonin used was 100 µM and that of ATP was 500 nM, respectively. Both serotonin and ATP induce the occurrence of multiple spikes consisting of rise and decay phases. A typical calcium spike with rise and decay phases is shown in the inset in panel (B). The temporal profile of calcium response was intensity normalized such that the maximum and minimum (basal) intensities corresponded to values 2 and 1, respectively. See Materials and Methods for more details.

Mentions: The first category consists of parameters that are described for the dependence of calcium response on ligand concentration. These parameters include maximum-fold increase in the fluorescence of fluo-3/AM upon ligand-mediated increase in cytosolic calcium level (Fmax/Fo) and the apparent time constant (τ) for the release of calcium in the cytoplasm from intracellular stores. Fo and Fmax represent fluorescence intensities of calcium-bound fluo-3/AM corresponding to basal and ligand-induced maximum cytosolic calcium concentrations, respectively. Since increase in the fluorescence of fluo-3/AM is directly proportional to the amount of calcium present, Fmax/Fo would correspond to the maximum-fold increase in calcium concentration upon receptor activation. In order to explore the kinetics of release and uptake of calcium in the cytoplasm, we divided calcium transient into two phases, i.e., rise and decay phases (see inset in Fig. 1B). We chose to analyze the kinetics of only rise phase of the first spike of calcium response (which consists of multiple spikes) since we often observed overlapping spikes, particularly at higher ligand concentrations. The time constant of the rise phase for the first calcium transient was obtained after fitting the normalized data (such that Fo = 1 and Fmax = 2) to a single exponential model as:(2)where F(t) is the fluorescence intensity of calcium-bound fluo-3/AM at time t, Fo is the basal fluorescence intensity of calcium-bound fluo-3/AM, Fmax is the maximum fluorescence intensity of calcium-bound fluo-3/AM upon ligand addition and τ is the apparent time constant for the increase in the fluorescence intensity of calcium bound fluo-3/AM. We evaluated peak characteristics by using the simplest model for spike functions, i.e., Gaussian function (equation 3). It should be noted that these models do not incorporate the molecular mechanism of calcium signaling and give the average parameter for phenomena happening inside cells.


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

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

Calcium response in CHO-5-HT1AR cells upon stimulation with serotonin or ATP.Panel (A) shows successive images of calcium response in cells upon stimulation with serotonin acquired with a time interval of 7.9 sec. CHO-5-HT1AR cells were loaded with Fluo-3/AM to monitor changes in cytosolic calcium concentration. Time-lapse imaging was performed with a 20×/0.75 NA objective. The scale bar (shown in top left panel) represents 50 µm. Similar response was observed when cells were stimulated with ATP. Representative temporal profiles of calcium response induced by either (B) serotonin or (C) ATP are shown. The concentration of serotonin used was 100 µM and that of ATP was 500 nM, respectively. Both serotonin and ATP induce the occurrence of multiple spikes consisting of rise and decay phases. A typical calcium spike with rise and decay phases is shown in the inset in panel (B). The temporal profile of calcium response was intensity normalized such that the maximum and minimum (basal) intensities corresponded to values 2 and 1, respectively. See Materials and Methods for more details.
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Related In: Results  -  Collection

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pone-0051857-g001: Calcium response in CHO-5-HT1AR cells upon stimulation with serotonin or ATP.Panel (A) shows successive images of calcium response in cells upon stimulation with serotonin acquired with a time interval of 7.9 sec. CHO-5-HT1AR cells were loaded with Fluo-3/AM to monitor changes in cytosolic calcium concentration. Time-lapse imaging was performed with a 20×/0.75 NA objective. The scale bar (shown in top left panel) represents 50 µm. Similar response was observed when cells were stimulated with ATP. Representative temporal profiles of calcium response induced by either (B) serotonin or (C) ATP are shown. The concentration of serotonin used was 100 µM and that of ATP was 500 nM, respectively. Both serotonin and ATP induce the occurrence of multiple spikes consisting of rise and decay phases. A typical calcium spike with rise and decay phases is shown in the inset in panel (B). The temporal profile of calcium response was intensity normalized such that the maximum and minimum (basal) intensities corresponded to values 2 and 1, respectively. See Materials and Methods for more details.
Mentions: The first category consists of parameters that are described for the dependence of calcium response on ligand concentration. These parameters include maximum-fold increase in the fluorescence of fluo-3/AM upon ligand-mediated increase in cytosolic calcium level (Fmax/Fo) and the apparent time constant (τ) for the release of calcium in the cytoplasm from intracellular stores. Fo and Fmax represent fluorescence intensities of calcium-bound fluo-3/AM corresponding to basal and ligand-induced maximum cytosolic calcium concentrations, respectively. Since increase in the fluorescence of fluo-3/AM is directly proportional to the amount of calcium present, Fmax/Fo would correspond to the maximum-fold increase in calcium concentration upon receptor activation. In order to explore the kinetics of release and uptake of calcium in the cytoplasm, we divided calcium transient into two phases, i.e., rise and decay phases (see inset in Fig. 1B). We chose to analyze the kinetics of only rise phase of the first spike of calcium response (which consists of multiple spikes) since we often observed overlapping spikes, particularly at higher ligand concentrations. The time constant of the rise phase for the first calcium transient was obtained after fitting the normalized data (such that Fo = 1 and Fmax = 2) to a single exponential model as:(2)where F(t) is the fluorescence intensity of calcium-bound fluo-3/AM at time t, Fo is the basal fluorescence intensity of calcium-bound fluo-3/AM, Fmax is the maximum fluorescence intensity of calcium-bound fluo-3/AM upon ligand addition and τ is the apparent time constant for the increase in the fluorescence intensity of calcium bound fluo-3/AM. We evaluated peak characteristics by using the simplest model for spike functions, i.e., Gaussian function (equation 3). It should be noted that these models do not incorporate the molecular mechanism of calcium signaling and give the average parameter for phenomena happening inside cells.

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