**3.3. Adrenoreceptor subtypes involved in Ca2+ signaling**

Similar to Ca2+ uncaging (**Figure 5A**), uncaging of IP<sup>3</sup>

statistically significant difference (student t-test, p < 0.05).

stimulated by Ca2+ ions liberated from NP-EGTA.

Reportedly, ryanodine and inositol 1,4,5-trisphosphate (IP<sup>3</sup>

which quickly generated a sufficient IP<sup>3</sup>

(5 μM) and uncaging of IP<sup>3</sup>

150 Calcium and Signal Transduction

nergic (n = 14) and adrenergic (n = 6) MSCs (**Figure 5C**). It was therefore possible that Ca2+ uncaging could simulate agonist-like responses by stimulating Ca2+-dependent PLC [42–44],

**Figure 5.** Evidence for Ca2+-induced Ca2+ release in MSCs. (A) Left panel—Ca2+ transients resulted from Ca2+ uncaging in a NP-EGTA loaded cell by UV flashes of varied durations and Ca2+ responses to noradrenaline at the indicated concentrations. Right panel—The superimposition of the responses numbered in (A) as 1 (thick line), 3 (circles), and 4 (thin line). (B) Left panel—Cellular responses to Ca2+ uncaging produced by a 4-s UV flash and to 5 μM ATP. Right panel—The superimposition of the light (thick line) and ATP (thin line) responses shown in the left panel. (C) ATP

E) PLC inhibitor U73122 (2 μM) dumped MSC responsiveness to 0.5 μM noradrenaline (D) and 5 μM ATP (E) but did not prevent agonist response-like Ca2+ transients resulted from Ca2+ uncaging by 4-s UV flashes. (F, G) 2-APB (50 μM) completely abolished biphasic agonist-like responses to Ca2+ uncaging by 4-s UV flashes, while 50 μM ryanodine was ineffective. In the experiments presented in (A–G), emission of a UV laser was weakened by the factor 10, so that Ca2+ uncaging should have lasted for 4 s to liberate as many Ca2+ ions as necessary for stimulating CICR. This gradual release of caged Ca2+ somewhat slowed the rising phase of a biphasic Ca2+ transient produced by CICR, thereby making a lag between a UV flash and a light response clearly visible. (H) Summary of effects of 2 μM U73122, 50 μM ryanodine, or 50 μM 2-APB on Ca2+ transients elicited by 4-s UV flashes. The data are presented as mean ± SD; the asterisk indicates

triggering CICR. To verify this possibility, several adrenergic (n = 12) and purinergic (n = 7) MSCs loaded with NP-EGTA were subjected to Ca2+ uncaging in the presence of U73122. Although this PLC inhibitor expectedly rendered MSCs nonresponsive to the agonists, the cells normally responded to UV flashes (**Figure 5D**, **E**). The ineffectiveness of U73122 (**Figure 5D**, **E**, **H**) provided strong evidence that PLC activation was not obligatory for generating light responses, thereby demonstrating that CICR initiated by UV flashes was directly

channels operating in the endo/sarcoplasmic reticulum, are exclusively responsible for CICR

burst, thereby enhancing activity of IP<sup>3</sup>

by a 2-s UV flash elicited similar responses in a cell loaded with caged-Ins(145)P3/PM. (D,

produced agonist-like responses in puri-

) receptors, Ca2+-gated Ca2+ release

receptors and

Nine human genes encode adrenoreceptors, including α1Α, α1Β, α1D, α2Α, α2Β, α2C, β<sup>1</sup> , β<sup>2</sup> , and β<sup>3</sup> isoforms [45]. Previously, we demonstrated that transcripts for α1Β-, α2Α-, and β<sup>2</sup> adrenoreceptors were invariably present in total MSC preparations [24]. Given that both α<sup>1</sup> and α<sup>2</sup> -adrenoreceptors are routinely coupled to PLC and Ca2+ mobilization in different cells [21, 22], either or both of these isoforms might be responsible for Ca2+ transients generated by MSCs in response to noradrenaline (**Figure 2A**). In contrast, β<sup>2</sup> -adrenoreceptors, which generally involve adenylyl cyclase as a downstream effector [23], could not be an essential contributor to Ca2+ signaling in adrenergic MSCs.

To uncover a role of the particular isoform, we performed recordings using agonists and antagonists specific for α<sup>1</sup> - or α<sup>2</sup> -adrenoreceptors. Overall, 35 noradrenaline-responsive cells were treated with phenylephrine/cirazoline and prazosin (α<sup>1</sup> -agonists and antagonist, respectively) as well as with guanabenz/B-HT 933 and yohimbine (α<sup>2</sup> -agonists and antagonist, respectively). Most of them (29 cells, 83%) were irresponsive to phenylephrine (1–10 μM), and their noradrenaline responses were not inhibited by 10 μM prazosin. In contrast, guanabenz (10–50 μM) and B-HT 933 (10 μM) were quite effective (**Figure 6A**). In particular, 50 μM guanabenz stimulated Ca2+ signaling in all noradrenaline-responsive MSCs (**Figure 6A**–**C**). Consistently, 2 μM yohimbine dumped cellular responses to noradrenaline and guanabenz (**Figure 6A**). Six cells (17%) were sensitive to both 10 μM phenylephrine and 50 μM guanabenz (**Figure 6B**, **C**). These findings indicate that the α<sup>2</sup> -subtype, evidently α2Α, predominantly mediates Ca2+ signaling initiated by noradrenaline in MSCs, although in a minor MSC subpopulation, both α<sup>1</sup> - and α<sup>2</sup> -isoforms could be involved in adrenergic transduction.

#### **3.4. Effects of isoform-specific agonists and antagonists of P2Y receptors**

In mammalians, the P2Y subgroup includes eight GPCRs (P2Y1,2,4,6,11–14) that exhibit certain specificities to nucleotides, depending on species [18, 46]. The expression of purinoreceptors in MSCs was analyzed previously, and transcripts for multiple P2Y receptors were detected, namely, P2Y<sup>1</sup> , P2Y<sup>2</sup> , P2Y4 , P2Y<sup>6</sup> , P2Y11, P2Y13, and P2Y14, while P2Y12 transcripts were not detected in total MSC preparations [47]. Although this P2Y array is sufficient to account for MSC capability to detect ATP, ADP, and UTP, it was impossible to evaluate a contribution of a particular P2Y isoform based on MSC responses to these natural P2Y agonists (**Figure 2B**, **E**, **F**). To address this issue, we used isoform-specific P2Y agonists and antagonists.

agonist MRS 2768 (10 μM) (**Figure 7A**, cell 1 and **Figure 7B**). In a subpopulation of rare MSCs (12 cells) that were capable of generating Ca2+ transients on 3 μM ATP in the presence of NF 340, 11 cells also responded to 10 μM MRS 2768 (**Figure 7A**, cell 2 and **Figure 7B**). Thus, MSCs

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

While the P2Y11 antagonist was highly effective (**Figure 7A**, **B**), most ATP-sensitive MSCs were surprisingly nonresponsive to NF 546 (10 μM), the specific P2Y11 agonist reported to be even more effective than ATP [49]. Among 127 cells that responded to 3 μM ATP, 10 μM NF 546 stimulated Ca2+ signaling solely in 9 cells (7%; **Figure 7C**, **D**). At the moment, we cannot provide any valid explanation for very low efficacy of NF-546 relative to ATP (**Figure 7D**). Perhaps, this synthetic ligand is a biased agonist that enables coupling of P2Y11 to the phosphoinositide cascade by involving only a certain G-protein type, which is absent or relatively

ATP-sensitive MSCs were rendered nonresponsive by 30 μM NF 340, a P2Y11 antagonist, and such cells never responded to 10 μM MRS 2768 (Cell 1). Uncommon cells that remained sensitive to ATP in the presence of 30 μM NF 340 responded to 10 μM MRS 2768 as well (Cell 2). (B) Responsiveness of 181 MSCs to 3 μM ATP and 10 μM MRS 2768 assayed in control and in the presence of NF 340. (C) Representative concurrent recordings from an ordinary cell insensitive to 10 μM NF 546 (Cell 1) and from an occasional cell responsive to this specific P2Y11 agonist (n = 127; Cell 2). (D) Responsiveness of

or both P2Y<sup>2</sup>

and P2Y11 receptors. (A) Representative responses of

agonist MRS 2768 (10 μM). The great majority (93%) of

and P2Y11 to detect

153

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

that were insensitive to NF 340 presumably employed P2Y<sup>2</sup>

less abundant in most of the MSCs.

**Figure 7.** Sensitivity of MSCs to agonists and antagonists of P2Y<sup>2</sup>

two concurrently assayed cells to ATP (3 μM) and to the P2Y<sup>2</sup>

127 MSCs to 3 μM ATP and 10 μM NF 546.

ATP.

The human P2Y family contains two ATP receptors, including specialized P2Y11 and also P2Y<sup>2</sup> that recognizes both UTP and ATP as full equipotent agonists [18]. Although also known as a partial P2Y<sup>1</sup> agonist, ATP was hardly capable of stimulating P2Y<sup>1</sup> -signaling in MSCs at low micromolar concentrations due to much lower efficacy than ADP [48]. We tried to evaluate a contribution of P2Y11 and P2Y<sup>2</sup> to MSC responsiveness to ATP. Among 181 MSCs assayed in this series, 169 cells (93%) became nonresponsive to ATP (3 μM) in the presence of 30 μM NF 340, a specific P2Y11 antagonist. These NF 340-sensitive cells did not respond to the P2Y<sup>2</sup>

**Figure 6.** Sensitivity of MSCs to adrenergic agonists and antagonists. (A) In most (83%) of noradrenaline-sensitive MSCs, α2-receptor agonists B-HT 933 and guanabenz stimulated Ca2+ signaling in contrast to the α1-receptor agonists phenylephrine and cirazoline that were ineffective. Consistently, Ca2+ signaling stimulated in such cells by noradrenaline and guanabenz was canceled in the presence of the α2 antagonist yohimbine, while the α1 antagonist prazosin was ineffective. (B) Small subpopulation (17%) of noradrenaline-sensitive cells responded to both α2 and α1 agonists. (C) Responsiveness of 35 MSCs sequentially stimulated by 0.5 μM noradrenaline, 50 μM guanabenz, and 10 μM phenylephrine.

agonist MRS 2768 (10 μM) (**Figure 7A**, cell 1 and **Figure 7B**). In a subpopulation of rare MSCs (12 cells) that were capable of generating Ca2+ transients on 3 μM ATP in the presence of NF 340, 11 cells also responded to 10 μM MRS 2768 (**Figure 7A**, cell 2 and **Figure 7B**). Thus, MSCs that were insensitive to NF 340 presumably employed P2Y<sup>2</sup> or both P2Y<sup>2</sup> and P2Y11 to detect ATP.

namely, P2Y<sup>1</sup>

152 Calcium and Signal Transduction

a partial P2Y<sup>1</sup>

phenylephrine.

, P2Y<sup>2</sup>

a contribution of P2Y11 and P2Y<sup>2</sup>

, P2Y4

, P2Y<sup>6</sup>

, P2Y11, P2Y13, and P2Y14, while P2Y12 transcripts were not

to MSC responsiveness to ATP. Among 181 MSCs assayed


detected in total MSC preparations [47]. Although this P2Y array is sufficient to account for MSC capability to detect ATP, ADP, and UTP, it was impossible to evaluate a contribution of a particular P2Y isoform based on MSC responses to these natural P2Y agonists (**Figure 2B**, **E**, **F**).

The human P2Y family contains two ATP receptors, including specialized P2Y11 and also P2Y<sup>2</sup> that recognizes both UTP and ATP as full equipotent agonists [18]. Although also known as

micromolar concentrations due to much lower efficacy than ADP [48]. We tried to evaluate

in this series, 169 cells (93%) became nonresponsive to ATP (3 μM) in the presence of 30 μM NF 340, a specific P2Y11 antagonist. These NF 340-sensitive cells did not respond to the P2Y<sup>2</sup>

**Figure 6.** Sensitivity of MSCs to adrenergic agonists and antagonists. (A) In most (83%) of noradrenaline-sensitive MSCs, α2-receptor agonists B-HT 933 and guanabenz stimulated Ca2+ signaling in contrast to the α1-receptor agonists phenylephrine and cirazoline that were ineffective. Consistently, Ca2+ signaling stimulated in such cells by noradrenaline and guanabenz was canceled in the presence of the α2 antagonist yohimbine, while the α1 antagonist prazosin was ineffective. (B) Small subpopulation (17%) of noradrenaline-sensitive cells responded to both α2 and α1 agonists. (C) Responsiveness of 35 MSCs sequentially stimulated by 0.5 μM noradrenaline, 50 μM guanabenz, and 10 μM

To address this issue, we used isoform-specific P2Y agonists and antagonists.

agonist, ATP was hardly capable of stimulating P2Y<sup>1</sup>

While the P2Y11 antagonist was highly effective (**Figure 7A**, **B**), most ATP-sensitive MSCs were surprisingly nonresponsive to NF 546 (10 μM), the specific P2Y11 agonist reported to be even more effective than ATP [49]. Among 127 cells that responded to 3 μM ATP, 10 μM NF 546 stimulated Ca2+ signaling solely in 9 cells (7%; **Figure 7C**, **D**). At the moment, we cannot provide any valid explanation for very low efficacy of NF-546 relative to ATP (**Figure 7D**). Perhaps, this synthetic ligand is a biased agonist that enables coupling of P2Y11 to the phosphoinositide cascade by involving only a certain G-protein type, which is absent or relatively less abundant in most of the MSCs.

**Figure 7.** Sensitivity of MSCs to agonists and antagonists of P2Y<sup>2</sup> and P2Y11 receptors. (A) Representative responses of two concurrently assayed cells to ATP (3 μM) and to the P2Y<sup>2</sup> agonist MRS 2768 (10 μM). The great majority (93%) of ATP-sensitive MSCs were rendered nonresponsive by 30 μM NF 340, a P2Y11 antagonist, and such cells never responded to 10 μM MRS 2768 (Cell 1). Uncommon cells that remained sensitive to ATP in the presence of 30 μM NF 340 responded to 10 μM MRS 2768 as well (Cell 2). (B) Responsiveness of 181 MSCs to 3 μM ATP and 10 μM MRS 2768 assayed in control and in the presence of NF 340. (C) Representative concurrent recordings from an ordinary cell insensitive to 10 μM NF 546 (Cell 1) and from an occasional cell responsive to this specific P2Y11 agonist (n = 127; Cell 2). (D) Responsiveness of 127 MSCs to 3 μM ATP and 10 μM NF 546.

UTP is a full agonist for P2Y<sup>2</sup> and P2Y4 that were identified in MSCs at the population level [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 MSCs to MRS 2768 and MRS 4062, specific agonists of P2Y<sup>2</sup> and P2Y4 receptors, respectively. 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**).

In contrast, 78 cells (82%) responded to 10 μM MRS 4062 (**Figure 7A**, cell 1 and **Figure 7B**).

responsiveness was performed on 102 MSCs sensitive to 3 μM ADP (**Figure 8A**) that might

at submicromolar concentrations [50]. MRS 2365 was ineffective at 100–300 nM but triggered Ca2+ signaling in 16 (25%) of 65 MSCs at 10 μM (**Figure 9A**). Because MRS 2365 specifically

inhibited ADP responses in all MRS 2365-treated MSCs (65 cells; **Figure 9A**). Given that other P2Y receptors were hardly inhibited by 10 μM MRS 2179 [49], the observed effects of the

have been activated by ADP concurrently to mobilize Ca2+ in MSCs. If so, nanomolar MRS

we assayed sensitivity of 51 ADP-responsive MSCs to both MRS 2179 (10 μM) and MRS 2211 (10 μM), a P2Y13 antagonist. It turned out that either of these compounds rendered each of 51 assayed cells nonresponsive to ADP (**Figure 9B**, **C**). Altogether, our findings (**Figure 9A**–**C**)

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

Transduction of multiple agonists involves GPCRs coupled to PLCβ1–4 isoforms that hydrolyze the precursor lipid phosphatidylinositol 4,5-bisphosphate to produce two second mes-

and release Ca2+ from the endoplasmic reticulum (ER) [30, 51, 52]. Three different isoforms of

R1, IP<sup>3</sup>

R2, and IP<sup>3</sup>

R2, and IP<sup>3</sup>

R1, IP<sup>3</sup>

and diacylglycerol. The primary mode of action of IP<sup>3</sup>

and P2Y13 [50], thus triggering Ca2+ signaling in MSCs. This concept predicted that MSCs

negative cells or not coupled to Ca2+ mobilization in a great majority of P2Y4

these contradictory findings, we considered the possibility that both P2Y<sup>1</sup>

indicated that only those MSCs, which functionally expressed both P2Y<sup>1</sup>

was responsible for Ca2+ signaling evoked

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

, P2Y12, and P2Y13. The analysis of ADP

antagonist with IC<sup>50</sup> = 0.15 μM [49],


155


and P2Y13 should

and P2Y13 receptors,

was either expressed in a very small subpopulation of P2Y4

, 65 of 103 ADP-sensitive MSCs were treated with MRS

agonist that displays no activity at P2Y12 and P2Y<sup>13</sup>

receptor were rather inconsistent. To reconcile

, while 10 μM MRS 2365 stimulated activity of both

or P2Y13 was inhibited. In line with this idea,

is to bind to IP<sup>3</sup>

R3) and shown to serve as a tetra-

R3 are distinct by physiological

receptors

and/or P2Y13 receptors, given that P2Y12 transcripts were not found in

with EC50 ~ 1 nM [50], this agonist might bring about a nonspecific action

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

These findings suggested that predominantly P2Y4

Extracellular ADP is detected by cells with P2Y<sup>1</sup>

at 10 μM. On the other hand, MRS 2179 (10 μM), a P2Y<sup>1</sup>

in MSCs by UTP, while P2Y<sup>2</sup>

MSCs. To evaluate a role of the P2Y<sup>1</sup>

2365, a highly potent and selective P2Y<sup>1</sup>

specific agonist and antagonist of the P2Y<sup>1</sup>

2365 was ineffective, activating solely P2Y<sup>1</sup>

would be unable to respond to ADP if either P2Y<sup>1</sup>

receptor have been identified (IP<sup>3</sup>


were capable of generating robust Ca2+ responses to ADP.

be recognized by P2Y<sup>1</sup>

stimulates P2Y<sup>1</sup>

**4. Discussion**

stimulation [30, 37, 42].

sengers, IP<sup>3</sup>

the IP<sup>3</sup>

meric IP<sup>3</sup>

P2Y<sup>1</sup>

**Figure 8.** Sensitivity of UTP-responsive MSCs to P2Y<sup>2</sup> and P2Y4 agonists. (A) Representative recordings from purinergic MSCs stimulated by UTP (10 μM), MRS 2768 (10 μM), and the agonists of P2Y4 receptor MRS 4062 (10 μM), in series. (B) Responsiveness of UTP-sensitive MSCs (n = 95) to MRS 2768 and MRS 4062.

**Figure 9.** Contribution of P2Y<sup>1</sup> and P2Y13 to ADP responsiveness. (A) Representative MSC responses to 3 μM ADP and to the P2Y<sup>1</sup> agonist MRS 2365 applied at 300 nM and 10 μM. All cells treated with 10 μM MRS 2179 (n = 65) became nonresponsive to 3 μM ADP. (B) When applied alone at 10 μM, antagonists of P2Y<sup>1</sup> (MRS 2179) and P2Y13 (MRS 2211) inhibited responses of MSCs to 3 μM ADP (46 cells). (C) Summary of responses of 51 MSCs to 3 μM ADP in control and in the presence of MRS 2179 or MRS 2211.

In contrast, 78 cells (82%) responded to 10 μM MRS 4062 (**Figure 7A**, cell 1 and **Figure 7B**). These findings suggested that predominantly P2Y4 was responsible for Ca2+ signaling evoked in MSCs by UTP, while P2Y<sup>2</sup> was either expressed in a very small subpopulation of P2Y4 negative cells or not coupled to Ca2+ mobilization in a great majority of P2Y4 -positive cells.

Extracellular ADP is detected by cells with P2Y<sup>1</sup> , P2Y12, and P2Y13. The analysis of ADP responsiveness was performed on 102 MSCs sensitive to 3 μM ADP (**Figure 8A**) that might be recognized by P2Y<sup>1</sup> and/or P2Y13 receptors, given that P2Y12 transcripts were not found in MSCs. To evaluate a role of the P2Y<sup>1</sup> , 65 of 103 ADP-sensitive MSCs were treated with MRS 2365, a highly potent and selective P2Y<sup>1</sup> agonist that displays no activity at P2Y12 and P2Y<sup>13</sup> at submicromolar concentrations [50]. MRS 2365 was ineffective at 100–300 nM but triggered Ca2+ signaling in 16 (25%) of 65 MSCs at 10 μM (**Figure 9A**). Because MRS 2365 specifically stimulates P2Y<sup>1</sup> with EC50 ~ 1 nM [50], this agonist might bring about a nonspecific action at 10 μM. On the other hand, MRS 2179 (10 μM), a P2Y<sup>1</sup> antagonist with IC<sup>50</sup> = 0.15 μM [49], inhibited ADP responses in all MRS 2365-treated MSCs (65 cells; **Figure 9A**). Given that other P2Y receptors were hardly inhibited by 10 μM MRS 2179 [49], the observed effects of the specific agonist and antagonist of the P2Y<sup>1</sup> receptor were rather inconsistent. To reconcile these contradictory findings, we considered the possibility that both P2Y<sup>1</sup> and P2Y13 should have been activated by ADP concurrently to mobilize Ca2+ in MSCs. If so, nanomolar MRS 2365 was ineffective, activating solely P2Y<sup>1</sup> , while 10 μM MRS 2365 stimulated activity of both P2Y<sup>1</sup> and P2Y13 [50], thus triggering Ca2+ signaling in MSCs. This concept predicted that MSCs would be unable to respond to ADP if either P2Y<sup>1</sup> or P2Y13 was inhibited. In line with this idea, we assayed sensitivity of 51 ADP-responsive MSCs to both MRS 2179 (10 μM) and MRS 2211 (10 μM), a P2Y13 antagonist. It turned out that either of these compounds rendered each of 51 assayed cells nonresponsive to ADP (**Figure 9B**, **C**). Altogether, our findings (**Figure 9A**–**C**) indicated that only those MSCs, which functionally expressed both P2Y<sup>1</sup> and P2Y13 receptors, were capable of generating robust Ca2+ responses to ADP.
