**3.1. Dose dependence of MSC responses to adrenergic and purinergic agonists**

In the present study, we focused on transduction of adrenergic and purinergic agonists capable of stimulating Ca2+ signaling in the MSC cytoplasm. We first aimed at evaluating dose dependencies of cellular responses to tested agonists. The analysis, which initially involved adrenergic transduction, revealed that Ca2+ responses varied with noradrenaline concentration in an "all-or-nothing" fashion. In other words, noradrenaline never caused detectable effects, when applied below 100 nM, but above the threshold of 100–200 nM, it elicited marked Ca2+ transients that were similarly shaped irrespective of agonist concentration (**Figure 2A**).

**Figure 1.** Functional heterogeneity of MSCs from the human adipose tissue. (A) Concurrent monitoring of intracellular Ca2+ in five different cells loaded with Fluo-4. The selected agonists were applied as indicated by the horizontal lines above the upper trace. (B) Fractional distribution of 426 MSCs that responded to at least one from the following serially applied agonists, including 10 μM ATP (adenosine triphosphate), 3 μM ADP (adenosine diphosphate), 10 μM adenosine (Adeno), 20 μM carbachol (carb), 20 μM GABA (gamma-aminobutyric acid), 10 μM glutamic acid (Glut), 0.5 μM noradrenaline (Nor), 10 μM serotonin (Ser), and 10 μM UTP (uridine triphosphate). (C) Distribution of MSC responsiveness to sequentially applied ATP (3 μM), ADP (3 μM), and UTP (10 μM) among a population of 125 cells, each being sensitive to at least one nucleotide.

**Figure 2.** Agonists evoke Ca2+ responses in an "all-or-nothing" manner. (A–E) Monitoring of intracellular Ca2+ in five different MSCs serially stimulated by noradrenaline (A), ATP (B), ADP (C), UTP (D), and adenosine (E) at varied concentrations as indicated. In A, 0.1 mg/ml saponin was applied (arrow) in the end of the recording to demonstrate that Fluo-4 fluorescence was not saturated by Ca2+ bursts elicited by noradrenaline. (F) Summary of MSC responses to adenosine (n = 21; left panel), ADP (n = 16; middle panel) cells, and UTP (n = 11; right panel). For each assayed cell, a response to a particular agonist at low concentration was taken equal 1. The data are presented as mean ± S.D. The difference between responses to adenosine at 0.5 and 5 μM and to ADP at 1 and 30 μM ADP is statistically insignificant (student t-test, p < 0.05). The asterisk indicates significant difference (p < 0.05) of UTP responses at 3 and 50 μM. (G) Superimposed dose dependences of noradrenaline responses recorded from 10 cells that exhibited the threshold of 150 nM. For each cell, serial noradrenaline responses were normalized to a response to 1 μM noradrenaline. (H) Superimposed dose dependences of ATP responses recorded from eight cells that exhibited the threshold of 1 μM. In each case, ATP responses were normalized to a response to 10 μM ATP. In (G) and (H), each particular symbol corresponds

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to an individual cell.

Since we expected to obtain a somewhat gradual dose dependence, we considered the possibility that at concentrations used, noradrenaline might elicit too high Ca2+ transients, which all saturated Fluo-4 fluorescence, thus appearing alike. However, the permeabilizing agent saponin (0.1 mg/mL) evoked marked Ca2+ signals that exceeded noradrenaline responses by the factor of 1.5–2 (17 cells; **Figure 2A**). These observations indicated conclusively that MSC responses to varied noradrenaline could not be equalized due to saturation of the Ca2+ dye. The further analysis of MSC responsivity pointed out that the "all-or-nothing" phenomenon

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**Figure 2.** Agonists evoke Ca2+ responses in an "all-or-nothing" manner. (A–E) Monitoring of intracellular Ca2+ in five different MSCs serially stimulated by noradrenaline (A), ATP (B), ADP (C), UTP (D), and adenosine (E) at varied concentrations as indicated. In A, 0.1 mg/ml saponin was applied (arrow) in the end of the recording to demonstrate that Fluo-4 fluorescence was not saturated by Ca2+ bursts elicited by noradrenaline. (F) Summary of MSC responses to adenosine (n = 21; left panel), ADP (n = 16; middle panel) cells, and UTP (n = 11; right panel). For each assayed cell, a response to a particular agonist at low concentration was taken equal 1. The data are presented as mean ± S.D. The difference between responses to adenosine at 0.5 and 5 μM and to ADP at 1 and 30 μM ADP is statistically insignificant (student t-test, p < 0.05). The asterisk indicates significant difference (p < 0.05) of UTP responses at 3 and 50 μM. (G) Superimposed dose dependences of noradrenaline responses recorded from 10 cells that exhibited the threshold of 150 nM. For each cell, serial noradrenaline responses were normalized to a response to 1 μM noradrenaline. (H) Superimposed dose dependences of ATP responses recorded from eight cells that exhibited the threshold of 1 μM. In each case, ATP responses were normalized to a response to 10 μM ATP. In (G) and (H), each particular symbol corresponds to an individual cell.

Since we expected to obtain a somewhat gradual dose dependence, we considered the possibility that at concentrations used, noradrenaline might elicit too high Ca2+ transients, which all saturated Fluo-4 fluorescence, thus appearing alike. However, the permeabilizing agent saponin (0.1 mg/mL) evoked marked Ca2+ signals that exceeded noradrenaline responses by the factor of 1.5–2 (17 cells; **Figure 2A**). These observations indicated conclusively that MSC responses to varied noradrenaline could not be equalized due to saturation of the Ca2+ dye. The further analysis of MSC responsivity pointed out that the "all-or-nothing" phenomenon

being sensitive to at least one nucleotide.

144 Calcium and Signal Transduction

**Figure 1.** Functional heterogeneity of MSCs from the human adipose tissue. (A) Concurrent monitoring of intracellular Ca2+ in five different cells loaded with Fluo-4. The selected agonists were applied as indicated by the horizontal lines above the upper trace. (B) Fractional distribution of 426 MSCs that responded to at least one from the following serially applied agonists, including 10 μM ATP (adenosine triphosphate), 3 μM ADP (adenosine diphosphate), 10 μM adenosine (Adeno), 20 μM carbachol (carb), 20 μM GABA (gamma-aminobutyric acid), 10 μM glutamic acid (Glut), 0.5 μM noradrenaline (Nor), 10 μM serotonin (Ser), and 10 μM UTP (uridine triphosphate). (C) Distribution of MSC responsiveness to sequentially applied ATP (3 μM), ADP (3 μM), and UTP (10 μM) among a population of 125 cells, each 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), ADP at 1 and 30 μM (16 cells), and UTP at 3 and 50 μM (11 cells) (**Figure 2C**–**F**).

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

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

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

**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

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weak, if any, contribution to Ca2+ signaling triggered by ATP in the MSC cytoplasm.

that the IP<sup>3</sup>

duction of assayed agonist. Expectedly, the IP<sup>3</sup>

concentrations. In (B–D), the data are presented as mean ± S.D.

Given the aforementioned effects of U73122 on MSC responses, there might be little doubt

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

receptor, a common effector downstream of PLC [30], was involved in trans-

receptor blocker 2-APB (50 μM) suppressed
