**3.2. Agonist transduction involves the phosphoinositide cascade and Ca2+-induced Ca2+ release**

In certain experiments, we analyzed coupling of adreno- and purinoreceptors to Ca2+ mobilization in the MSC cytoplasm. When MSCs were pretreated with U73122 (2–5 μM), a poorly reversible inhibitor of PLC, all assayed cells became completely nonresponsive to tested agonists, including noradrenaline (17 cells), ATP (39 cells), adenosine (11 cells), UTP (7 cells), and Calcium Signaling Initiated by Agonists in Mesenchymal Stromal Cells from the Human Adipose… http://dx.doi.org/10.5772/intechopen.79097 147

was intrinsic for the agonist-dependent Ca2+ signaling in general, including purinergic transduction. In particular, submicromolar ATP was ineffective, while the nucleotide elicited Ca2+ transients in the MSC cytoplasm at 1–2 μM and higher (**Figure 2B**). The adenosine responses were characterized by the threshold of 0.2–0.3 μM and were similarly shaped at higher concentrations (9 cells; **Figure 2C**). For ADP- and UTP-responses, the threshold concentrations ranged within 0.5–2 and 3–6 μM, respectively. Although we did not carefully characterize MSC responses to adenosine, ADP, and UTP at widely and gradually varied concentrations, it appeared that dose-response curves for these agonists were also step-like. For example, Ca2+ transients of close magnitudes were usually elicited by adenosine at 0.5 and 5 μM (21 cells),

In the case of noradrenaline and ATP, the dose dependence of MSC responses was carefully evaluated in designated experiments, wherein an agonist dose was gradually varied in a wide range of concentrations (**Figure 2A**, **B**). During this prolonged assay, responsiveness of many cells was liable to rundown, thus impeding the quantitative analysis. Overall, we identified 21 cells that generated sufficiently robust responses to noradrenaline at 30 nM–10 μM with the threshold of 100–200 nM. Among them, 10 cells, which exhibited the same threshold of 150 nM, were taken for the analysis. To compare different experiments, responses of each particular cell recorded at variable agonist concentrations were normalized to a response to 1 μM noradrenaline and superimposed as shown in **Figure 2G**, where different symbols correspond to individual cells. Despite some data scattering, normalized cellular responses were localized in the narrow range of 0.8–1.2 (**Figure 2G**), clearly demonstrating that in all cases, the dose dependence was a step-like rather than gradual. Similar inference came from the analysis of 32 ATP-sensitive cells that showed quite robust responses to the nucleotide gradually applied at 0.5–50 μM. Of them, nine MSCs generated rather similar Ca2+ signals at

One more notable feature of MSC responses was that Ca2+ transients were markedly postponed relative to a moment of agonist application. The characteristic time of response delay (τd, **Figure 3A**) gradually decreased with noradrenaline and ATP concentration (**Figure 3B**, **C**). For instance, Ca2+ transients triggered by noradrenaline were retarded by 38–55 s at the threshold stimulation (**Figure 3A**, left response), whereas the delay was reduced to 17–26 s at the concentration of 1 μM and higher (**Figure 3A**, right response). The detailed assay of the dose-delay dependence was not carried out for the other agonists. Nevertheless, the comparison of MSCs responses obtained at low and saturated concentrations of adenosine, ADP, or UTP revealed a marked decrease in response delay as the agonist dose raised (**Figure 3D**). As discussed below, two distinct mechanisms are presumably responsible for specific dependen-

ADP at 1 and 30 μM (16 cells), and UTP at 3 and 50 μM (11 cells) (**Figure 2C**–**F**).

gradually increasing ATP doses with the threshold of 1 μM (**Figure 2B**, **H**).

cies of the magnitude and delay of MSC responses on agonist concentration.

**Ca2+ release**

146 Calcium and Signal Transduction

**3.2. Agonist transduction involves the phosphoinositide cascade and Ca2+-induced** 

In certain experiments, we analyzed coupling of adreno- and purinoreceptors to Ca2+ mobilization in the MSC cytoplasm. When MSCs were pretreated with U73122 (2–5 μM), a poorly reversible inhibitor of PLC, all assayed cells became completely nonresponsive to tested agonists, including noradrenaline (17 cells), ATP (39 cells), adenosine (11 cells), UTP (7 cells), and

**Figure 3.** Dose dependence of agonist response delay. (A) Representative Ca2+ transients elicited by noradrenaline at 100 nM (threshold concentration) and 500 nM in the same cell. These noradrenaline responses were delayed relative to the moment of agonist application by 55 and 16 s, respectively. The characteristic time of the response delay (τd) was calculated as a time interval necessary for a Ca2+ transient to reach the half-magnitude. (B, C) Response lag versus noradrenaline (B) and ATP (C) concentration. The data were obtained from 10 adrenergic (**Figure 2A**, **G**) and 8 purinergic (**Figure 2B**, **H**) MSCs. (D) Delay of MSC responses to ADP (n = 16), UTP (n = 11), and adenosine (n = 21) at indicated concentrations. In (B–D), the data are presented as mean ± S.D.

ADP (5 cells) (**Figure 4A**–**C**, **G–I**). The inhibitory effect of U73122 on MSC responsiveness was apparently specific as the much less effective analog U73343 (2–5 μM) never canceled MSC responses to the nucleotides (**Figure 4A**–**C**, **G**, **H**). Moreover, the decrease of external Ca2+ from 2 mM to 260 nM weakly or negligibly affected Ca2+ transients elicited by ATP (26 cells), noradrenaline (31 cells), adenosine (7 cells), UTP (14 cells), and ADP (13 cells) (**Figure 4C**, **D**, **G**–**I**). Thus, the agonist-stimulated Ca2+ signaling in MSCs involved GPCRs that were basically coupled by the phosphoinositide cascade to Ca2+ release rather than to Ca2+ entry. Note also that the step-like dose dependence of ATP responses (**Figure 2B**, **H**) and their insignificant sensitivity to external Ca2+ (**Figure 4G**) indicated that P2X receptors could provide only a weak, if any, contribution to Ca2+ signaling triggered by ATP in the MSC cytoplasm.

Given the aforementioned effects of U73122 on MSC responses, there might be little doubt that the IP<sup>3</sup> receptor, a common effector downstream of PLC [30], was involved in transduction of assayed agonist. Expectedly, the IP<sup>3</sup> receptor blocker 2-APB (50 μM) suppressed Ca2+ signaling initiated by ATP (21 cells), noradrenaline (19 cells), adenosine (5 cells), ADP (9 cells), and UTP (10 cells) (**Figure 4D**–**I**)). In contrast, 50 μM ryanodine, a ryanodine receptor

production of an IP<sup>3</sup>

(**Figure 3B**, **C**).

fluorescence acquired at 535 ± 25 nm.

could be determined by the initial gradual Ca2+ signal.

burst and proportional Ca2+ release via IP<sup>3</sup>

problem, we assumed that the agonist transduction occurred in two separated consecutive steps. Initially, an agonist produced a Ca2+ signal most likely being small, local, and gradually dependent on stimulus intensity. When exceeding the threshold, this local and poorly resolved Ca2+ signal pushed massive Ca2+-induced Ca2+ release (CICR) [37–40] to accomplish transduction with a large and global Ca2+ signal. By involving the trigger-like mechanism CICR, a cell generates Ca2+ responses of virtually universal shape and magnitude at different agonist concentrations above the threshold (**Figure 2**). Rising with agonist proportionally, the initial gradual Ca2+ signal reached a CICR threshold for the time that should have shortened with agonist concentration, thus underlying the gradual dose-delay dependence observed

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

To clarify functionality of the CICR mechanism in MSCs and its contribution to agonist responses, we used Ca2+ uncaging that allowed for generating as fast and intensive cytosolic Ca2+ bursts as necessary for initiating the CICR process. In designated experiments, MSCs were loaded with both Fluo-4 and NP-EGTA. The last is photolabile Ca2+ chelator with high affinity to Са2+ (Kd ~ 10−7 М), so that in a resting cell (~100 nm free Са2+), nearly half NP-EGTA molecules are bound to Са2+ ions. The absorption of ultraviolet (UV) light by NP-EGTA disrupts the coordination sphere responsible for Ca2+ binding, thus liberating Ca2+ions and producing a step-like increase in cytosolic Ca2+ [41]. Because a UV laser we employed for uncaging was in fact a biharmonic light source emitting at 351 and 527 nm, a light stimulus caused an optical artifact that was seen as a marked overshoot in a recording trace of cell

In this series, caged Ca2+ was released by moderate UV pulses during several seconds to somehow simulate the suggested Ca2+ signal initially produced by agonists in the MSC cytoplasm. As illustrated in **Figure 5A**, light stimuli triggered in adrenergic MSCs (n = 33) two fundamentally different types of Ca2+ responses. The relatively short, 2-s in the given case, UV pulse produced an optical artifact that was followed by a small Ca2+ jump without evident delay (**Figure 5A**, left panel, response 1 and right panel, thick line). This Ca2+ signal exhibited exponential relaxation presumably mediated by Ca2+ pumps. The sequential 4-s and 6-s UV flashes elicited biphasic Ca2+ transients of nonproportional magnitudes (**Figure 5A**, left panel). Indeed, compared to a 2-s UV pulse, one could expect 4- and 6-s light stimuli to liberate nearly twice and three times more Ca2+ ions, respectively. Meanwhile, 4-, 6-, and 8-s flashes usually triggered the similar Ca2+ transients that exceeded a response to a 2-s pulse by an order of magnitude (**Figure 5A**, left panel). None of the known Ca2+-dependent mechanisms but CICR could amplify and shape an initial Ca2+ signal produced by NP-EGTA photolysis in such a way (**Figure 5A**, right panel, response 1 vs. response 2). In addition, the representative cell (**Figure 5A**, left panel) was insensitive to 50 nM noradrenaline but similarly responded to the agonist at 0.5 and 1 μM concentrations. Similar results were obtained with other eight MSCs that tolerated prolonged serial stimulation with both UV and noradrenaline. Note that biphasic cell responses to light and noradrenaline were quite similar by shape and magnitude (**Figure 5A**, right panel, thin line 2 and circled line 3). Interestingly, light responses exhibited the delay that shortened with UV pulse duration (**Figure 5**, left panel). Similar experiments were performed with purinergic MSCs (n = 23) and basically identical results were obtained (**Figure 5B**). These findings support the idea that the delay of agonist responses (**Figure 3**)

receptors. To address this

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**Figure 4.** Involvement of the phosphoinositide cascade in agonist transduction. (A–C) PLC inhibitor U73122 (2 μM) suppressed MSC responsivity to different agonists, including 0.5 μM noradrenaline (A), 1 μM adenosine (B), and 3 μM ADP (C), while its much less effective analogue U73343 (2 μM) was ineffective in all cases. (C, D) Reduction of external Ca2+ from 2 mM to 260 nM weakly or negligibly affected Ca2+ responses to agonists, including 3 μM ADP and 0.5 μM noradrenaline. Extracellular Ca2+ was not completely removed because MSCs poorly tolerated prolonged exposure to a Ca2+-free solution. (E, F) IP<sup>3</sup> receptor blocker 2-APB (50 μM) reversibly suppressed MSC responses, particularly, to 3 μM ADP, 10 μM UTP, and 5 μM ATP. (F) Caffeine and ryanodine, an agonist and antagonist of ryanodine receptors, respectively, negligibly affected cytosolic Ca2+ and ATP responsivity. (G–I) Summary of effects of indicated compounds and low Ca2+ on MSC responses to the tested agonists; n means the numbers of cells assayed in the particular case. The data are presented as mean ± S.D.; the asterisk indicates statistically significant difference (student t-test, p < 0.05).

antagonist, was ineffective in all cases (**Figure 4F**–**I**). These findings suggested a negligible role for ryanodine receptors in agonist transduction. Consistently, their agonist caffeine (10 mM) insignificantly affected cytosolic Ca2+ in ATP-responsive MSCs (7 cells; **Figure 4F**). It should be noted that 2-APB blocks not only IP<sup>3</sup> receptors but also a variety of Ca2+-entry channels [31–33]. Given however that MSC responsiveness to P2Y agonists insignificantly depended on external Ca2+ and therefore on Ca2+ influx (**Figure 4G**–**I**), we inferred that 2-APB exerted the inhibitory action mainly by targeting IP<sup>3</sup> receptors.

The monotonic and gradual dependence of cellular responses on agonist concentration has been reported for a variety of cellular systems, including those that employ GPCRs coupling to Ca2+ mobilization [34–36]. In contrast, Ca2+ responses were generated by MSCs in an "all-ornothing" manner (**Figure 2**). This step-like dose dependence of response magnitude is poorly explicable and apparently inconsistent with the gradual relation between response delay and agonist concentration (**Figure 3**) if agonist transduction involves solely PLC-dependent production of an IP<sup>3</sup> burst and proportional Ca2+ release via IP<sup>3</sup> receptors. To address this problem, we assumed that the agonist transduction occurred in two separated consecutive steps. Initially, an agonist produced a Ca2+ signal most likely being small, local, and gradually dependent on stimulus intensity. When exceeding the threshold, this local and poorly resolved Ca2+ signal pushed massive Ca2+-induced Ca2+ release (CICR) [37–40] to accomplish transduction with a large and global Ca2+ signal. By involving the trigger-like mechanism CICR, a cell generates Ca2+ responses of virtually universal shape and magnitude at different agonist concentrations above the threshold (**Figure 2**). Rising with agonist proportionally, the initial gradual Ca2+ signal reached a CICR threshold for the time that should have shortened with agonist concentration, thus underlying the gradual dose-delay dependence observed (**Figure 3B**, **C**).

To clarify functionality of the CICR mechanism in MSCs and its contribution to agonist responses, we used Ca2+ uncaging that allowed for generating as fast and intensive cytosolic Ca2+ bursts as necessary for initiating the CICR process. In designated experiments, MSCs were loaded with both Fluo-4 and NP-EGTA. The last is photolabile Ca2+ chelator with high affinity to Са2+ (Kd ~ 10−7 М), so that in a resting cell (~100 nm free Са2+), nearly half NP-EGTA molecules are bound to Са2+ ions. The absorption of ultraviolet (UV) light by NP-EGTA disrupts the coordination sphere responsible for Ca2+ binding, thus liberating Ca2+ions and producing a step-like increase in cytosolic Ca2+ [41]. Because a UV laser we employed for uncaging was in fact a biharmonic light source emitting at 351 and 527 nm, a light stimulus caused an optical artifact that was seen as a marked overshoot in a recording trace of cell fluorescence acquired at 535 ± 25 nm.

In this series, caged Ca2+ was released by moderate UV pulses during several seconds to somehow simulate the suggested Ca2+ signal initially produced by agonists in the MSC cytoplasm. As illustrated in **Figure 5A**, light stimuli triggered in adrenergic MSCs (n = 33) two fundamentally different types of Ca2+ responses. The relatively short, 2-s in the given case, UV pulse produced an optical artifact that was followed by a small Ca2+ jump without evident delay (**Figure 5A**, left panel, response 1 and right panel, thick line). This Ca2+ signal exhibited exponential relaxation presumably mediated by Ca2+ pumps. The sequential 4-s and 6-s UV flashes elicited biphasic Ca2+ transients of nonproportional magnitudes (**Figure 5A**, left panel). Indeed, compared to a 2-s UV pulse, one could expect 4- and 6-s light stimuli to liberate nearly twice and three times more Ca2+ ions, respectively. Meanwhile, 4-, 6-, and 8-s flashes usually triggered the similar Ca2+ transients that exceeded a response to a 2-s pulse by an order of magnitude (**Figure 5A**, left panel). None of the known Ca2+-dependent mechanisms but CICR could amplify and shape an initial Ca2+ signal produced by NP-EGTA photolysis in such a way (**Figure 5A**, right panel, response 1 vs. response 2). In addition, the representative cell (**Figure 5A**, left panel) was insensitive to 50 nM noradrenaline but similarly responded to the agonist at 0.5 and 1 μM concentrations. Similar results were obtained with other eight MSCs that tolerated prolonged serial stimulation with both UV and noradrenaline. Note that biphasic cell responses to light and noradrenaline were quite similar by shape and magnitude (**Figure 5A**, right panel, thin line 2 and circled line 3). Interestingly, light responses exhibited the delay that shortened with UV pulse duration (**Figure 5**, left panel). Similar experiments were performed with purinergic MSCs (n = 23) and basically identical results were obtained (**Figure 5B**). These findings support the idea that the delay of agonist responses (**Figure 3**) could be determined by the initial gradual Ca2+ signal.

antagonist, was ineffective in all cases (**Figure 4F**–**I**). These findings suggested a negligible role for ryanodine receptors in agonist transduction. Consistently, their agonist caffeine (10 mM) insignificantly affected cytosolic Ca2+ in ATP-responsive MSCs (7 cells; **Figure 4F**). It should

**Figure 4.** Involvement of the phosphoinositide cascade in agonist transduction. (A–C) PLC inhibitor U73122 (2 μM) suppressed MSC responsivity to different agonists, including 0.5 μM noradrenaline (A), 1 μM adenosine (B), and 3 μM ADP (C), while its much less effective analogue U73343 (2 μM) was ineffective in all cases. (C, D) Reduction of external Ca2+ from 2 mM to 260 nM weakly or negligibly affected Ca2+ responses to agonists, including 3 μM ADP and 0.5 μM noradrenaline. Extracellular Ca2+ was not completely removed because MSCs poorly tolerated prolonged exposure to

3 μM ADP, 10 μM UTP, and 5 μM ATP. (F) Caffeine and ryanodine, an agonist and antagonist of ryanodine receptors, respectively, negligibly affected cytosolic Ca2+ and ATP responsivity. (G–I) Summary of effects of indicated compounds and low Ca2+ on MSC responses to the tested agonists; n means the numbers of cells assayed in the particular case. The data are presented as mean ± S.D.; the asterisk indicates statistically significant difference (student t-test, p < 0.05).

[31–33]. Given however that MSC responsiveness to P2Y agonists insignificantly depended on external Ca2+ and therefore on Ca2+ influx (**Figure 4G**–**I**), we inferred that 2-APB exerted the

receptors.

The monotonic and gradual dependence of cellular responses on agonist concentration has been reported for a variety of cellular systems, including those that employ GPCRs coupling to Ca2+ mobilization [34–36]. In contrast, Ca2+ responses were generated by MSCs in an "all-ornothing" manner (**Figure 2**). This step-like dose dependence of response magnitude is poorly explicable and apparently inconsistent with the gradual relation between response delay and agonist concentration (**Figure 3**) if agonist transduction involves solely PLC-dependent

receptors but also a variety of Ca2+-entry channels

receptor blocker 2-APB (50 μM) reversibly suppressed MSC responses, particularly, to

be noted that 2-APB blocks not only IP<sup>3</sup>

a Ca2+-free solution. (E, F) IP<sup>3</sup>

148 Calcium and Signal Transduction

inhibitory action mainly by targeting IP<sup>3</sup>

in apparently all cells [39, 42, 44]. To evaluate a relative contribution of IP<sup>3</sup>

cated that Ca2+ uncaging failed to initiate CICR with inhibited IP<sup>3</sup>

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

contributor to Ca2+ signaling in adrenergic MSCs.


benz (**Figure 6B**, **C**). These findings indicate that the α<sup>2</sup>


were treated with phenylephrine/cirazoline and prazosin (α<sup>1</sup>

tively) as well as with guanabenz/B-HT 933 and yohimbine (α<sup>2</sup>

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

antagonists specific for α<sup>1</sup>

population, both α<sup>1</sup>

receptors were responsible for CICR in adrenergic and purinergic MSCs.

by MSCs in response to noradrenaline (**Figure 2A**). In contrast, β<sup>2</sup>

2-APB was removed to restore activity of IP<sup>3</sup>

and β<sup>3</sup>

and α<sup>2</sup>

receptors to CICR in MSCs, we examined effects of their antagonists on Ca2+ signals associated with Ca2+ uncaging. While 50 μM ryanodine was ineffective, 50 μM 2-APB dramatically and reversibly changed a shape and magnitude of UV responses in adrenergic (n = 16) and purinergic (n = 11) MSCs (**Figure 5F**–**H**). In the presence of 50 μM ryanodine, Ca2+ uncaging elicited agonist-like biphasic Ca2+ responses that were delayed relative to stimulatory UV flashes (**Figure 5F**, **G**, 2nd responses). Thus, despite the inhibition of ryanodine receptors, Ca2+ uncaging was still capable of stimulating robust CICR in MSCs responsive to the agonists. With 50 μM 2-APB in the bath, a UV pulse entailed a brief Ca2+ jump that relaxed monotonically and was smaller by the factor 3–4 (**Figure 5F**, **G**, 3rd responses; **Figure 5H**). This indi-

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

transient again (**Figure 5F**, **G**, 4th responses). These observations indicated that basically IP<sup>3</sup>

Nine human genes encode adrenoreceptors, including α1Α, α1Β, α1D, α2Α, α2Β, α2C, β<sup>1</sup>

adrenoreceptors were invariably present in total MSC preparations [24]. Given that both α<sup>1</sup>

generally involve adenylyl cyclase as a downstream effector [23], could not be an essential

To uncover a role of the particular isoform, we performed recordings using agonists and

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 guana-

mediates Ca2+ signaling initiated by noradrenaline in MSCs, although in a minor MSC sub-

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,


isoforms [45]. Previously, we demonstrated that transcripts for α1Β-, α2Α-, and β<sup>2</sup>


and ryanodine

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receptors. Moreover, when





, β<sup>2</sup> ,



receptors, a UV flash triggered a biphasic Ca2+


**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 (5 μM) and uncaging 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, 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 statistically significant difference (student t-test, p < 0.05).

Similar to Ca2+ uncaging (**Figure 5A**), uncaging of IP<sup>3</sup> produced agonist-like responses in purinergic (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], which quickly generated a sufficient IP<sup>3</sup> burst, thereby enhancing activity of IP<sup>3</sup> receptors and 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 stimulated by Ca2+ ions liberated from NP-EGTA.

Reportedly, ryanodine and inositol 1,4,5-trisphosphate (IP<sup>3</sup> ) receptors, Ca2+-gated Ca2+ release channels operating in the endo/sarcoplasmic reticulum, are exclusively responsible for CICR in apparently all cells [39, 42, 44]. To evaluate a relative contribution of IP<sup>3</sup> and ryanodine receptors to CICR in MSCs, we examined effects of their antagonists on Ca2+ signals associated with Ca2+ uncaging. While 50 μM ryanodine was ineffective, 50 μM 2-APB dramatically and reversibly changed a shape and magnitude of UV responses in adrenergic (n = 16) and purinergic (n = 11) MSCs (**Figure 5F**–**H**). In the presence of 50 μM ryanodine, Ca2+ uncaging elicited agonist-like biphasic Ca2+ responses that were delayed relative to stimulatory UV flashes (**Figure 5F**, **G**, 2nd responses). Thus, despite the inhibition of ryanodine receptors, Ca2+ uncaging was still capable of stimulating robust CICR in MSCs responsive to the agonists. With 50 μM 2-APB in the bath, a UV pulse entailed a brief Ca2+ jump that relaxed monotonically and was smaller by the factor 3–4 (**Figure 5F**, **G**, 3rd responses; **Figure 5H**). This indicated that Ca2+ uncaging failed to initiate CICR with inhibited IP<sup>3</sup> receptors. Moreover, when 2-APB was removed to restore activity of IP<sup>3</sup> receptors, a UV flash triggered a biphasic Ca2+ transient again (**Figure 5F**, **G**, 4th responses). These observations indicated that basically IP<sup>3</sup> receptors were responsible for CICR in adrenergic and purinergic MSCs.
