**4. Discussion**

UTP is a full agonist for P2Y<sup>2</sup>

154 Calcium and Signal Transduction

**Figure 8.** Sensitivity of UTP-responsive MSCs to P2Y<sup>2</sup>

**Figure 9.** Contribution of P2Y<sup>1</sup>

in the presence of MRS 2179 or MRS 2211.

to the P2Y<sup>1</sup>

MSCs stimulated by UTP (10 μM), MRS 2768 (10 μM), and the agonists of P2Y4

nonresponsive to 3 μM ADP. (B) When applied alone at 10 μM, antagonists of P2Y<sup>1</sup>

Responsiveness of UTP-sensitive MSCs (n = 95) to MRS 2768 and MRS 4062.

and P2Y4

MSCs to MRS 2768 and MRS 4062, specific agonists of P2Y<sup>2</sup>

[47]. It therefore was unclear whether a particular cell employs either or both of these P2Y receptors for monitoring extracellular UTP. We analyzed the sensitivity of 95 UTP-responsive

Consistently with the analysis of ATP-responsive cells (**Figure 7B**), we found only 9 (9.5%) of 95 UTP-sensitive cells to react to 10 μM MRS 2768 (**Figure 8A**, cell 3 and **Figure 8B**).

and P2Y4

that were identified in MSCs at the population level

agonists. (A) Representative recordings from purinergic

and P2Y13 to ADP responsiveness. (A) Representative MSC responses to 3 μM ADP and

agonist MRS 2365 applied at 300 nM and 10 μM. All cells treated with 10 μM MRS 2179 (n = 65) became

inhibited responses of MSCs to 3 μM ADP (46 cells). (C) Summary of responses of 51 MSCs to 3 μM ADP in control and

receptor MRS 4062 (10 μM), in series. (B)

(MRS 2179) and P2Y13 (MRS 2211)

receptors, respectively.

and P2Y4

Virtually in all cell types, extracellular cues can mobilize intracellular Ca2+ to regulate a variety of diverse cellular functions, such as fertilization, proliferation, secretion, metabolism, gene expression, mobility, and muscle contraction. How can the Ca2+ ion, a chemically simple substance, control so many different physiological processes? The plausible explanation comes from the versatility of Ca2+ signaling mechanisms that can mediate Ca2+ signals with variable kinetics, amplitude, duration, and spatial patterning, depending on cellular context and stimulation [30, 37, 42].

Transduction of multiple agonists involves GPCRs coupled to PLCβ1–4 isoforms that hydrolyze the precursor lipid phosphatidylinositol 4,5-bisphosphate to produce two second messengers, IP<sup>3</sup> and diacylglycerol. The primary mode of action of IP<sup>3</sup> is to bind to IP<sup>3</sup> receptors and release Ca2+ from the endoplasmic reticulum (ER) [30, 51, 52]. Three different isoforms of the IP<sup>3</sup> receptor have been identified (IP<sup>3</sup> R1, IP<sup>3</sup> R2, and IP<sup>3</sup> R3) and shown to serve as a tetrameric IP<sup>3</sup> -gated Ca2+ channel [30, 51–53]. IP<sup>3</sup> R1, IP<sup>3</sup> R2, and IP<sup>3</sup> R3 are distinct by physiological properties, thus allowing cells to generate specific Ca2+ signals with different spatial and temporal characteristics to control diverse cellular functions [30, 52]. In addition to IP<sup>3</sup> , Ca2+ is the primary coregulator of IP<sup>3</sup> receptors [30, 51, 52, 54]. The full activation of the IP<sup>3</sup> receptor occurs when IP<sup>3</sup> has occupied the IP<sup>3</sup> -binding domains on all four subunits [55]. This is associated with a conformational change, which sensitizes the Ca2+-binding site. The binding of cytosolic Ca2+ to this site markedly increases the open probability of the IP<sup>3</sup> receptor channel [54], so that Ca2+ ions released from the ER can additionally stimulate activity of IP<sup>3</sup> receptors. This positive feedback mediates CICR. Meanwhile, the action of cytosolic Ca2+ is bimodal: stimulating IP<sup>3</sup> receptors at low levels, Ca2+ becomes inhibitory above 300 nm [54]. This multimodal control of the IP<sup>3</sup> receptor by IP<sup>3</sup> and Ca2+ is central to various aspects of intracellular Ca2+ signaling [30, 52].

undetectably affecting intracellular Ca2+ below the cut-off concentration, a particular agonist initiated Ca2+ transients that were large and quite similarly shaped at all doses above the threshold (**Figure 2**). In contrast to this step-like dose-response curve, the dependence of response delay on agonist concentration was gradual (**Figure 3**). The inhibitory analysis and Ca2+ uncaging approach showed that agonist transduction universally involved the classical

To reconcile the "all-or-nothing" dose-response curve and gradual dose-delay dependence, we surmised that agonist-evoked Ca2+ signaling in MSCs includes two different but coupled

eration of an initial, presumably local and gradual Ca2+ signal. Next, this local Ca2+ signal stimulates CICR that produces a global Ca2+ signal. Some evidence suggests that the Ca2+ store responsible for the initial Ca2+ signal may be physically separated from the Ca2+ store that provides CICR. Indeed, when cells were overloaded with NP-EGTA due to the twofold excess of NP-EGTA-AM concentration compared to the standard loading protocol (see Methods), a MSC population became poorly sensitive to ATP. However, several UV flashes usually rendered MSC responsive (**Figure 10**). Presumably, overloading with NP-EGTA excessively increased the Ca2+-buffering capacity of the cell cytoplasm, thereby significantly diminishing the initial agonist-induced Ca2+ signal and its speed. The photodistraction of NP-EGTA decreased exogenous Ca2+ buffer to a physiologically more relevant level, thus recovering MSC responsiveness to ATP. Note that in line with multiple reports, relatively slow Ca2+ buffer EGTA is unable to cancel Ca2+-dependent processes mediated by local intracellular Ca2+ signals. For instance, 1 mM EGTA does not prevent activation of Ca2+-gated BK channels by Ca2+ transients originated by both Ca2+ influx via voltage-gated Ca2+ channels and Ca2+ release stimulated by muscarine [58]. By analogy and based on the observation that Ca2+ uncaging was still capable of triggering CICR in MSCs overloaded with NP-EGTA (**Figure 10**), we sug-

production, activation of IP<sup>3</sup>

Calcium Signaling Initiated by Agonists in Mesenchymal Stromal Cells from the Human Adipose…


receptors

157

receptors

receptor

receptors, and gen-

http://dx.doi.org/10.5772/intechopen.79097

phosphoinositide cascade and CICR mechanism (**Figures 4** and **5**) that employed IP<sup>3</sup>

gested that NP-EGTA, slow Ca2+ buffer [59], could hardly repeal stimulation of IP<sup>3</sup>

pool mediating CICR should be spatially separated from agonist-dependent machinery that

**Figure 10.** MSCs overloaded with NP-EGTA became nonresponsive to agonists. In this particular experiment, 76 cells, which have been pre-incubated with 4 μM Fluo-4-AM and 8 μM instead of 4 μM NP-EGTA-AM, were simultaneously assayed. None of these cells generated Ca2+ responses to the first application of 5 μM ATP. As exemplified by the presented fluorescence trace, two sequential Ca2+ uncaging by 4-s UV flashes rendered 7 of 76 cells sensitive to 5 μM

ATP. The same phenomenon was observed in two more similar experiments.

rather than ryanodine receptors (**Figures 4E**, **F** and **5F**, **G**).

stages. Primarily, agonists stimulate IP<sup>3</sup>

by Ca2+ ions released through this IP<sup>3</sup>

In the present work, we studied MSCs from the human adipose tissue and examined intracellular Ca2+ signaling initiated by certain GPCR agonists, including adenosine, ATP, noradrenaline, and some others. Although all first messengers tested here were effective, only a relatively small MSC group responded to a particular agonist. These specifically responsive cell subpopulations overlapped weakly or negligibly, depending on agonists (**Figure 1**). This finding is hardly surprising in light of a widely accepted idea that a MSC population from different sources represents a heterogeneous mixture of diverse cells, including multipotent and more committed progenitor cells [1, 3, 56, 57]. Yet, cultured MSCs are not synchronized and dwell in different phases of the cell cycle. It therefore might be expected that divergent intracellular signaling is inherent in a MSC population containing both proliferating and quiescent cells. The aforementioned factors could underlie intrinsic heterogeneity of a MSC population discussed previously [56, 57]. It also should be mentioned that most of assayed MSCs were found by us nonresponsive to a particular agonist solely in terms of Ca2+ signaling that necessitated coupling of appropriate GPCRs to Ca2+ mobilization. Meanwhile, many GPCR isoforms are in fact promiscuous in that they may be coupled to a variety of downstream signaling pathways, depending on G-proteins involved. For instance, the P2Y1,2,4,6,11 subtypes of purinoreceptors are canonically coupled by Gq /G11 to the phosphoinositide cascade and Ca2+ mobilization, whereas P2Y12,13,14 control cAMP production by inhibiting adenylyl cyclase through G<sup>i</sup> /G<sup>o</sup> . The unique capability of P2Y11 is to stimulate G<sup>s</sup> [18]. In addition, apart from ubiquitous coupling to PLC and adenylyl cyclase, P2Y receptors can also engage effectors such as MAP, PI3, Akt, and PKC kinases; small G-proteins; NO synthase; transactivation of growth factor receptors; and some others [26–29]. Hence, a fraction of MSCs sensitive to a given agonist might be in fact much more abundant than that evaluated by Ca2+ imaging (**Figure 1B**, **C**), because the tested compounds could stimulate not only Ca2+ mobilization but also other signaling events.

The agonist-dependent Ca2+ signaling in MSCs was mostly detailed by us for noradrenaline and certain nucleotides. By using subtype-specific agonists and antagonists, it was shown that mainly a2 -adrenoreceptors mediated Ca2+ mobilization triggered by noradrenaline in adrenergic MSCs (**Figure 6**). In purinergic MSCs, presumably P2Y11 serves as a primary ATP receptor (**Figure 7**), UTP responsiveness is largely mediated by P2Y4 (**Figure 8**), while both P2Y<sup>1</sup> and P2Y13 are involved in detecting ADP (**Figure 9**). The responsivity of MSCs to noradrenaline and ATP and apparently to adenosine, ADP, and UTP exhibited a peculiar dose dependence: undetectably affecting intracellular Ca2+ below the cut-off concentration, a particular agonist initiated Ca2+ transients that were large and quite similarly shaped at all doses above the threshold (**Figure 2**). In contrast to this step-like dose-response curve, the dependence of response delay on agonist concentration was gradual (**Figure 3**). The inhibitory analysis and Ca2+ uncaging approach showed that agonist transduction universally involved the classical phosphoinositide cascade and CICR mechanism (**Figures 4** and **5**) that employed IP<sup>3</sup> receptors rather than ryanodine receptors (**Figures 4E**, **F** and **5F**, **G**).

properties, thus allowing cells to generate specific Ca2+ signals with different spatial and tem-

ated with a conformational change, which sensitizes the Ca2+-binding site. The binding of

This positive feedback mediates CICR. Meanwhile, the action of cytosolic Ca2+ is bimodal:

In the present work, we studied MSCs from the human adipose tissue and examined intracellular Ca2+ signaling initiated by certain GPCR agonists, including adenosine, ATP, noradrenaline, and some others. Although all first messengers tested here were effective, only a relatively small MSC group responded to a particular agonist. These specifically responsive cell subpopulations overlapped weakly or negligibly, depending on agonists (**Figure 1**). This finding is hardly surprising in light of a widely accepted idea that a MSC population from different sources represents a heterogeneous mixture of diverse cells, including multipotent and more committed progenitor cells [1, 3, 56, 57]. Yet, cultured MSCs are not synchronized and dwell in different phases of the cell cycle. It therefore might be expected that divergent intracellular signaling is inherent in a MSC population containing both proliferating and quiescent cells. The aforementioned factors could underlie intrinsic heterogeneity of a MSC population discussed previously [56, 57]. It also should be mentioned that most of assayed MSCs were found by us nonresponsive to a particular agonist solely in terms of Ca2+ signaling that necessitated coupling of appropriate GPCRs to Ca2+ mobilization. Meanwhile, many GPCR isoforms are in fact promiscuous in that they may be coupled to a variety of downstream signaling pathways, depending on G-proteins involved. For instance, the P2Y1,2,4,6,11 subtypes

Ca2+ mobilization, whereas P2Y12,13,14 control cAMP production by inhibiting adenylyl cyclase

ubiquitous coupling to PLC and adenylyl cyclase, P2Y receptors can also engage effectors such as MAP, PI3, Akt, and PKC kinases; small G-proteins; NO synthase; transactivation of growth factor receptors; and some others [26–29]. Hence, a fraction of MSCs sensitive to a given agonist might be in fact much more abundant than that evaluated by Ca2+ imaging (**Figure 1B**, **C**), because the tested compounds could stimulate not only Ca2+ mobilization but

The agonist-dependent Ca2+ signaling in MSCs was mostly detailed by us for noradrenaline and certain nucleotides. By using subtype-specific agonists and antagonists, it was shown that

gic MSCs (**Figure 6**). In purinergic MSCs, presumably P2Y11 serves as a primary ATP receptor

P2Y13 are involved in detecting ADP (**Figure 9**). The responsivity of MSCs to noradrenaline and ATP and apparently to adenosine, ADP, and UTP exhibited a peculiar dose dependence:


. The unique capability of P2Y11 is to stimulate G<sup>s</sup>

receptors [30, 51, 52, 54]. The full activation of the IP<sup>3</sup>

receptors at low levels, Ca2+ becomes inhibitory above 300 nm [54]. This mul-


and Ca2+ is central to various aspects of intracellular

/G11 to the phosphoinositide cascade and

(**Figure 8**), while both P2Y<sup>1</sup>

and

[18]. In addition, apart from

, Ca2+ is

receptor

receptors.

receptor channel

poral characteristics to control diverse cellular functions [30, 52]. In addition to IP<sup>3</sup>

cytosolic Ca2+ to this site markedly increases the open probability of the IP<sup>3</sup>

receptor by IP<sup>3</sup>

[54], so that Ca2+ ions released from the ER can additionally stimulate activity of IP<sup>3</sup>

the primary coregulator of IP<sup>3</sup>

has occupied the IP<sup>3</sup>

of purinoreceptors are canonically coupled by Gq

(**Figure 7**), UTP responsiveness is largely mediated by P2Y4

occurs when IP<sup>3</sup>

156 Calcium and Signal Transduction

stimulating IP<sup>3</sup>

through G<sup>i</sup>

mainly a2

/G<sup>o</sup>

also other signaling events.

timodal control of the IP<sup>3</sup>

Ca2+ signaling [30, 52].

To reconcile the "all-or-nothing" dose-response curve and gradual dose-delay dependence, we surmised that agonist-evoked Ca2+ signaling in MSCs includes two different but coupled stages. Primarily, agonists stimulate IP<sup>3</sup> production, activation of IP<sup>3</sup> receptors, and generation of an initial, presumably local and gradual Ca2+ signal. Next, this local Ca2+ signal stimulates CICR that produces a global Ca2+ signal. Some evidence suggests that the Ca2+ store responsible for the initial Ca2+ signal may be physically separated from the Ca2+ store that provides CICR. Indeed, when cells were overloaded with NP-EGTA due to the twofold excess of NP-EGTA-AM concentration compared to the standard loading protocol (see Methods), a MSC population became poorly sensitive to ATP. However, several UV flashes usually rendered MSC responsive (**Figure 10**). Presumably, overloading with NP-EGTA excessively increased the Ca2+-buffering capacity of the cell cytoplasm, thereby significantly diminishing the initial agonist-induced Ca2+ signal and its speed. The photodistraction of NP-EGTA decreased exogenous Ca2+ buffer to a physiologically more relevant level, thus recovering MSC responsiveness to ATP. Note that in line with multiple reports, relatively slow Ca2+ buffer EGTA is unable to cancel Ca2+-dependent processes mediated by local intracellular Ca2+ signals. For instance, 1 mM EGTA does not prevent activation of Ca2+-gated BK channels by Ca2+ transients originated by both Ca2+ influx via voltage-gated Ca2+ channels and Ca2+ release stimulated by muscarine [58]. By analogy and based on the observation that Ca2+ uncaging was still capable of triggering CICR in MSCs overloaded with NP-EGTA (**Figure 10**), we suggested that NP-EGTA, slow Ca2+ buffer [59], could hardly repeal stimulation of IP<sup>3</sup> receptors by Ca2+ ions released through this IP<sup>3</sup> -gated conduit. If so, the Ca2+ store and IP<sup>3</sup> receptor pool mediating CICR should be spatially separated from agonist-dependent machinery that

**Figure 10.** MSCs overloaded with NP-EGTA became nonresponsive to agonists. In this particular experiment, 76 cells, which have been pre-incubated with 4 μM Fluo-4-AM and 8 μM instead of 4 μM NP-EGTA-AM, were simultaneously assayed. None of these cells generated Ca2+ responses to the first application of 5 μM ATP. As exemplified by the presented fluorescence trace, two sequential Ca2+ uncaging by 4-s UV flashes rendered 7 of 76 cells sensitive to 5 μM ATP. The same phenomenon was observed in two more similar experiments.

generates an initial, local, and gradual Ca2+ signal. Otherwise, it is difficult to explain why in cells overloaded with NP-EGTA, agonist responses disappeared contrary to light responses associated with Ca2+ uncaging (**Figure 10**).

cut-off dose. Of course, the presented model is a simplification of the actual transduction process, and roles for other common contributors to intracellular Ca2+ signaling, including Ca2+ pumps, mitochondria, Ca2+ buffer as well as Ca2+-dependent enzymes and ion channels,

Calcium Signaling Initiated by Agonists in Mesenchymal Stromal Cells from the Human Adipose…

http://dx.doi.org/10.5772/intechopen.79097

159

We thank Dr. V. Yu. Sysoeva for providing MSCs of the first passage. We are thankful to the Russian Science Foundation for support of studies of adrenergic and purinergic transduction

Polina D. Kotova, Olga A. Rogachevskaja, Marina F. Bystrova, Ekaterina N. Kochkina,

Institute of Cell Biophysics of Russian Academy of Sciences, Pushchino, Moscow Region,

[1] Kalinina NI, Sysoeva VY, Rubina KA, Parfenova YV, Tkachuk VA. Mesenchymal stem

[2] Keating A. Mesenchymal stromal cells: New directions. Cell Stem Cell. 2012;**10**:709-716.

[3] Baer PC. Adipose-derived mesenchymal stromal/stem cells: An update on their phenotype in vivo and in vitro. World journal of stem cells. 2014;**6**:256-265. DOI: 10.4252/wjsc.

[4] Nordberg RC, Loboa EG. Our fat future: Translating adipose stem cell therapy. Stem

[5] Casiraghi F, Perico N, Cortinovis M, Remuzzi G. Mesenchymal stromal cells in renal transplantation: Opportunities and challenges. Nature Reviews. Nephrology. 2016;**12**:

[6] Lou G, Chen Z, Zheng M, Liu Y. Mesenchymal stem cell-derived exosomes as a new therapeutic strategy for liver diseases. Experimental & Molecular Medicine. 2017;**49**:e346.

Cells Translational Medicine. 2015;**4**:974-979. DOI: 10.5966/sctm.2015-0071

remain to be elucidated.

**Acknowledgements**

**Author details**

Russia

**References**

v6.i3.256

(grant 18-14-0034) and P2Y receptors (grant 17-75-10127).

\*Address all correspondence to: staskolesnikov@yahoo.com

cells in tissue growth and repair. Acta Naturae. 2011;**3**:30-37

Denis S. Ivashin and Stanislav S. Kolesnikov\*

DOI: 10.1016/j.stem.2012.05.015

241-253. DOI: 10.1038/nrneph.2016.7

DOI: 10.1038/emm.2017.63
