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

Mesenchymal stromal cells (MSCs) from different sources represent a heterogeneous population of proliferating non-differentiated cells that contain multipotent stem cells capable of originating a variety of mesenchymal cell lineages. By using Ca 2+ imaging and the Ca 2+ dye Fluo -4 , we studied MSCs from the human adipose tissue and examined Ca 2+ signaling initiated by a variety of GPCR ligands, focusing primarily on adrenergic and purinergic agonists. Being characterized by a relative change of Fluo -4 fluorescence, ago-nist-induced Ca 2+ responses were generated in an “all-or-nothing” fashion. Specifically, at relatively low doses, agonists elicited undetectable responses but initiated quite simi- lar Ca 2+ transients at all concentrations above the threshold. The inhibitory analysis and Ca 2+ /IP 3 uncaging pointed at the phosphoinositide cascade as a pivotal pathway responsible for agonist transduction and implicated Ca 2+ -induced Ca 2+ release (CICR) in shaping agonists-dependent Ca 2+ signals. Altogether, our data suggest that agonist transduction in MSCs includes two fundamentally different stages: an agonist initially triggers a local, gradual, and relatively small Ca 2+ signal, which next stimulates CICR to accomplish transduction with a large and global Ca 2+ transient. By involving the trigger-like mechanism CICR, a cell is capable of generating Ca 2+ responses of virtually universal shape and magnitude at different agonist concentrations above the threshold.


Introduction
Mesenchymal stromal cells (MSCs) are described as a heterogeneous cellular pool that includes immature cells responsible for the replenishment of supportive and connective tissues due to their capability of maintaining self-renewal and multipotent differentiation [1][2][3]. By unique biologic properties, cultured MSCs from different sources attract sufficient interest in the fields of regenerative medicine and immunotherapy [4][5][6]. Despite evident progress in MSC biology spurred by the therapeutic potential of these cells, current knowledge on their receptor and signaling systems remains scarce. Evidence exists that MSCs are capable of sensing complex extracellular cues, including hormones, cytokines, and nucleotides [7,8]. This implies that MSCs employ multiple surface receptors and signaling pathways to adjust their physiological functions to specific tissue microenvironment.
Here, we studied MSCs derived from the human adipose tissue and examined Ca 2+ signaling initiated by a variety of agonists of G-protein coupled receptors (GPCRs). We specifically focused on adrenergic and purinergic signaling systems that attracted us for the following reasons. It has been known for a long time that noradrenaline released by sympathetic nerves regulates distinct physiological processes in the adipose tissue such as lipid and glucose metabolism and secretion of distinct signaling molecules, including adipocytokines and cytokines [9]. Hence, MSCs that reside in the adipose tissue can be subjected to the action of noradrenaline and factors released by adipocytes on adrenergic stimulation. Purinergic agonists have been documented as an important factor determining MSC fate [7,8,[10][11][12]. Reportedly, ATP serves both as an adipogenic regulator and an osteogenic factor, while its downstream product adenosine switches off adipogenic differentiation and promotes osteogenesis [13,14]. Damaged tissues are an abundant source of extracellular ATP that may be converted by extracellular nucleotidases to ADP and eventually to adenosine [15]. It therefore might be expected that MSCs are exposed to and regulated by nucleotides and adenosine when these cells migrate in vivo or are transplanted ex vivo into an injured tissue.
The responsiveness to purines and pyrimidines is widespread among eukaryotic cells, which express numerous purinoreceptors from the P1 and P2 families. The P1 subgroup includes four G-protein-coupled receptors (A 1 , A 2A , A 2B , A 3 ) recognizing adenosine as an endogenous agonist [16]. The more diverse P2 family is composed of ionotropic P2X and metabotropic P2Y receptors. P2X receptors are cationic channels specifically gated by ATP, while P2Y receptors are activated by multiple purine and pyrimidine nucleotides or by sugar nucleotides and couple to intracellular second messenger pathways by heteromeric G proteins [17,18]. In mammals, seven genes encode P2X subunits (P2X 1-7 ) that can form homo-and heterotrimeric cation channels with noticeable Ca 2+ permeability [19,20]. The P2Y subfamily includes eight members (P2Y 1,2,4,6,11,12,13,14 ), which are distinct by ligand specificity and coupling to downstream signaling pathways, including the ubiquitous phosphoinositide cascade [17,18].
Nine genes encode human adrenoreceptors, which all belong to the GPCR superfamily and compose three distinctive subgroups, including three α 1 (α 1A , α 1B , α 1D ), three α 2 (α 2A , α 2B , α 2C ), and three β (β 1 , β 2 , β 3 ) receptor subtypes. Canonically, α 1 -adrenoreceptors couple to G q and are ubiquitously involved in Ca 2+ signaling [21]. Although α 2 isoforms widely regulate adenylyl cyclase via G i , their coupling to phospholipase C (PLC) and Ca 2+ mobilization has also been documented [22]. All three β-subtypes are linked to adenylyl cyclase by G s , although β 2 and β 3 also couple to G i , and directly do not control intracellular Ca 2+ [23]. Given that certain isoforms of adrenergic and purinergic receptors are coupled to Ca 2+ mobilization in diverse cell types, we considered Ca 2+ imaging as an adequate approach to detail purinergic and adrenergic transduction in MSCs.

Cell isolation and culturing
MSCs of the first passage were obtained from the Faculty of Basic Medicine at Lomonosov Moscow State University. All procedures that involved human participants were performed in accordance with the ethical standards approved by the Bioethical Committee of the Faculty based on the 1964 Helsinki declaration and its later amendments. The study involved 21 healthy (not suffered from infectious or systemic diseases and malignancies) individuals from 21 to 55 years old, and informed consent was obtained from each participant.
Cells were isolated from subcutaneous fat tissue of healthy donors using enzymatic digestion as previously described [24]. Briefly, the adipose tissue was extensively washed with two volumes of Hank's Balanced Salt Solution (HBSS) containing 5% antibiotic/antimycotic solution (10,000 units of penicillin, 10,000 μg of streptomycin, and 25 μg of Amphotericin B per mL; HyClone), fragmented, and then digested at 37°C for 1 h in the presence of collagenase (200 U/ml, Sigma-Aldrich) and dispase (10 U/ml, BD Biosciences). Enzymatic activity was neutralized by adding an equal volume of culture medium (HyClone™ AdvanceSTEM™ Mesenchymal Stem Cell Basal Medium for human undifferentiated mesenchymal stem cells containing 10% of HyClone™ AdvanceSTEM™ Mesenchymal Stem Cell Growth Supplement (CGS), 1% antibiotic/antimycotic solution (HyClone) and centrifuged at 200 g for 10 min. This led to the sedimentation of diverse cells, including MSCs, macrophages, lymphocytes, and erythrocytes, unlike adipocytes that remained floating. After removal of supernatant, a lysis solution (154 mM NH 4 Cl, 10 mM KHCO 3 , and 0.1 mM EDTA) was added to a cell pellet to lyse erythrocytes, and cell suspension was centrifuged at 200 g for 10 min. Sedimented cells were resuspended in the MSC culture medium and filtered through a 100-μm nylon cell strainer (BD Biosciences). As indicated by flow [24], after isolation and overnight preplating, the obtained cell population contained not only MSC cells that basically represented the most abundant subgroup but also admixed macrophages and lymphocytes. The two last cell subgroups were dramatically depleted by culturing for a week in the MSC culture medium and humidified atmosphere (5% CO 2 ) at 37°C. The obtained MSC population was maintained at a subconfluent level (~80% confluency) and passaged using HyQTase (HyClone). By using the methodology described previously [25], cultured cells were demonstrated to differentiate into the osteogenic, chondrogenic, and adipogenic directions, the finding confirming their multipotency. In experiments, MSCs of the second to fourth passages were usually used.

Preparation of cells for Ca 2+ imaging
Before assaying with Ca 2+ imaging, cells were maintained in a 12-socket plate for 12 h in the medium described above but without antibiotics. For isolation, cells cultured in a 1-ml socket were rinsed twice with the Versene solution (Sigma-Aldrich) that was then substituted for 200 μl HyQTase solution (HyClone) for 3-5 min. The enzymatic treatment was terminated by the addition of a 0.8 ml culture medium to a socket. Next, cells were resuspended, put into a tube, and centrifuged at 50 g for 45 s for moderate sedimentation. Isolated cells were collected by a plastic pipette and plated onto a photometric chamber of nearly 150 μl volume. The last was a disposable coverslip (Menzel-Glaser) with attached ellipsoidal resin wall. The chamber bottom was coated with Cell-Tak (BD Biosciences), enabling strong cell adhesion. Attached cells were then loaded with dyes for 20 min at room temperature (23-25°С) by adding Fluo-4 AM (4 μM) and Pluronic (0.02%; all from Molecular Probes) to a bath solution. Loaded cells were rinsed with the bath solution for several times and kept at 4°C for 1 h prior to recordings. Generally, incubation of MSCs at low temperature stabilized intracellular Ca 2+ and decreased a fraction of spontaneously oscillating cells.

Ca 2+ imaging and uncaging
Experiments were carried out using an inverted fluorescent microscope Axiovert 135 equipped with an objective Plan NeoFluar 20x/0.75 (Zeiss) and a digital EMCCD camera LucaR (Andor Technology). Apart from a transparent light illuminator, the microscope was equipped with a handmade system for epi-illumination via an objective. The epi-illumination was performed using a bifurcational glass fiber. One channel was used for Fluo-4 excitation and transmitted irradiation of a computer-controllable light-emitting diode (LED) LZ1-00B700H (LED Engin). LED emission was filtered with an optical filter ET480/20x (Chroma Technology). Fluo-4 emission was collected at 535 ± 25 nm by using an emission filter ET535/50 m (Chroma Technology). Serial fluorescent images were usually captured every second and analyzed using Imaging Workbench 6 software (INDEC). Within the 1-s acquisition period, the 480 nm LED was switched on for only 200 ms, during which cell fluorescence was collected. This protocol allowed for minimizing photobleaching of Fluo-4 at a sufficiently high signal-to-noise ratio achievable by adjusting LED emission. This enabled us to reliably assay cell responsiveness to different compounds applied serially for up to 60 min. Deviations of cytosolic Ca 2+ from the resting level were quantified by a relative change in the intensity of Fluo-4 fluorescence (ΔF/F 0 ) recorded from an individual cell.
Another channel was connected to a pulsed solid laser TECH-351 Advanced (680 mW) (Laser-Export, Moscow). This unit operated in a two harmonic mode and generated not only 351 nM UV light used for Ca 2+ uncaging but also visible light at 527 nm. The last could penetrate into an emission channel through nonideal optical filters and elicit optical artifacts during uncaging. For Ca 2+ or IP 3 uncaging, cells were loaded with 4 μM Fluo-4-AM (Invitrogen) and 4 μM NP-EGTA-AM (Invitrogen) or 4 μM caged-Ins(145)P3/PM (SiChem) + 0.02% Pluronic (Invitrogen) for 30 min at 23°С. The basic bath solution contained (mM): 110 NaCl, 5.5 KCl, 2 CaCl 2 , 0.8 MgSO 4 , 10 glucose, 10 HEPES-NaOH, and pH 7.4 (≈270 Osm). When necessary, 2 mM CaCl 2 in the bath was replaced with 0.5 mМ EGTA + 0.4 mМ CaCl 2 , thus reducing free Ca 2+ to nearly 260 nM at 23°С as calculated with the Maxchelator program (http://maxchelator. stanford.edu). In this low Ca 2+ bath solution, the glucose concentration was increased to 13 mM to keep osmolarity. All chemicals used in experiments described below were applied by the complete replacement of the bath solution in a 150-μl photometric chamber for nearly 2 s using a perfusion system driven by gravity. The used salts and buffers were from Sigma-Aldrich, and agonists and inhibitors were from Tocris.

Results
In a typical experiment, nearly a hundred of MSCs loaded with Fluo-4 resided in a photometric camera, and their responsiveness to different ligands was assayed with Ca 2+ imaging. Consistently with observations of others [3], functional heterogeneity was characteristic of a MSC population derived from each particular donor. Although a variety of GPCR agonists were found to stimulate Ca 2+ signaling in MSCs, including ATP, ADP, noradrenaline or adrenaline, acetylcholine or its analog carbachol, GABA, glutamate, serotonin, and UTP, only a relatively small group of cells in a given MSC population was specifically responsive to a particular agonist (Figure 1). Overall, nearly 10 3 MSCs were sequentially stimulated by multiple agonists applied at different combinations, and a particular cell was either irresponsive to all stimuli or responded to one, rarely two, particular compound ( Figure 1A-C). ATP-sensitive cells composed the most abundant subgroup of 9-15% (12% on average), depending on MSC preparation ( Figure 1B). The percentage of cells responsive to other agonists was on average: ADP-7.1, adenosine-8.7, carbachol-3.4, GABA-5, glutamate-1.2, noradrenaline-6.7, serotonin-6.6, and UTP-6 ( Figure 1B). The more or less accurate analysis of distribution of MSC responsivity was performed for nucleotides. In designated experiments, wherein cells were sequentially stimulated by ATP, ADP, and UTP, 125 purinergic MSCs were assayed overall, and only 13 cells (10%) were found to respond to all three agonists at the indicated concentrations ( Figure 1C). Both ATP and ADP stimulated Ca 2+ signaling in 40 cells (32%) that did not respond to UTP; 33 cells (26%) preferred the ATP-UTP pair. In addition, 20, 9, and 7 cells (16, 7, and 6%) responded exclusively to ATP, ADP, or UTP, respectively ( Figure 1C).
Thus, the results presented above (Figure 1) clearly demonstrated that responsiveness to a given agonist varied from cell to cell. Note that GPCRs from most subfamilies, e.g. P2Y receptors, can couple to several signaling pathways, depending on cellular context [26][27][28][29]. Hence, in cells nonresponsive in terms of Ca 2+ signaling to a particular agonist, appropriate GPCRs might be either not expressed or not coupled to Ca 2+ mobilization.

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 Ca 2+ 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 Ca 2+ 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 Ca 2+ transients that were similarly shaped irrespective of agonist concentration (Figure 2A).
Since we expected to obtain a somewhat gradual dose dependence, we considered the possibility that at concentrations used, noradrenaline might elicit too high Ca 2+ transients, which all saturated Fluo-4 fluorescence, thus appearing alike. However, the permeabilizing agent saponin (0.1 mg/mL) evoked marked Ca 2+ 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 Ca 2+ dye. The further analysis of MSC responsivity pointed out that the "all-or-nothing" phenomenon  was intrinsic for the agonist-dependent Ca 2+ signaling in general, including purinergic transduction. In particular, submicromolar ATP was ineffective, while the nucleotide elicited Ca 2+ 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, Ca 2+ 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 Ca 2+ signals at gradually increasing ATP doses with the threshold of 1 μM (Figure 2B, H).
One more notable feature of MSC responses was that Ca 2+ transients were markedly postponed relative to a moment of agonist application. The characteristic time of response delay (τ d , Figure 3A) gradually decreased with noradrenaline and ATP concentration ( Figure 3B, C). For instance, Ca 2+ 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.

Agonist transduction involves the phosphoinositide cascade and Ca 2+ -induced Ca 2+ release
In certain experiments, we analyzed coupling of adreno-and purinoreceptors to Ca 2+ 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 Thus, the agonist-stimulated Ca 2+ signaling in MSCs involved GPCRs that were basically coupled by the phosphoinositide cascade to Ca 2+ release rather than to Ca 2+ entry. Note also that the step-like dose dependence of ATP responses (Figure 2B, H) and their insignificant sensitivity to external Ca 2+ ( Figure 4G) indicated that P2X receptors could provide only a weak, if any, contribution to Ca 2+ 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 3 receptor, a common effector downstream of PLC [30], was involved in transduction of assayed agonist. Expectedly, the IP 3 receptor blocker 2-APB (50 μM) suppressed Ca 2+ 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 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 Ca 2+ in ATP-responsive MSCs (7 cells; Figure 4F). It should be noted that 2-APB blocks not only IP 3 receptors but also a variety of Ca 2+ -entry channels [31][32][33]. Given however that MSC responsiveness to P2Y agonists insignificantly depended on external Ca 2+ and therefore on Ca 2+ influx (Figure 4G-I), we inferred that 2-APB exerted the inhibitory action mainly by targeting IP 3 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 Ca 2+ mobilization [34][35][36]. In contrast, Ca 2+ 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 3 burst and proportional Ca 2+ release via IP 3 receptors. To address this problem, we assumed that the agonist transduction occurred in two separated consecutive steps. Initially, an agonist produced a Ca 2+ signal most likely being small, local, and gradually dependent on stimulus intensity. When exceeding the threshold, this local and poorly resolved Ca 2+ signal pushed massive Ca 2+ -induced Ca 2+ release (CICR) [37][38][39][40] to accomplish transduction with a large and global Ca 2+ signal. By involving the trigger-like mechanism CICR, a cell generates Ca 2+ responses of virtually universal shape and magnitude at different agonist concentrations above the threshold (Figure 2). Rising with agonist proportionally, the initial gradual Ca 2+ 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 Ca 2+ uncaging that allowed for generating as fast and intensive cytosolic Ca 2+ 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 Ca 2+ chelator with high affinity to Са 2+ (Kd ~ 10 −7 М), so that in a resting cell (~100 nm free Са 2+ ), nearly half NP-EGTA molecules are bound to Са 2+ ions. The absorption of ultraviolet (UV) light by NP-EGTA disrupts the coordination sphere responsible for Ca 2+ binding, thus liberating Ca 2+ ions and producing a step-like increase in cytosolic Ca 2+ [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 Ca 2+ was released by moderate UV pulses during several seconds to somehow simulate the suggested Ca 2+ 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 Ca 2+ responses. The relatively short, 2-s in the given case, UV pulse produced an optical artifact that was followed by a small Ca 2+ jump without evident delay ( Figure 5A, left panel, response 1 and right panel, thick line). This Ca 2+ signal exhibited exponential relaxation presumably mediated by Ca 2+ pumps. The sequential 4-s and 6-s UV flashes elicited biphasic Ca 2+ 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 Ca 2+ ions, respectively. Meanwhile, 4-, 6-, and 8-s flashes usually triggered the similar Ca 2+ transients that exceeded a response to a 2-s pulse by an order of magnitude ( Figure 5A, left panel). None of the known Ca 2+ -dependent mechanisms but CICR could amplify and shape an initial Ca 2+ 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 Ca 2+ signal.
Similar to Ca 2+ uncaging (Figure 5A), uncaging of IP 3 produced agonist-like responses in purinergic (n = 14) and adrenergic (n = 6) MSCs ( Figure 5C). It was therefore possible that Ca 2+ uncaging could simulate agonist-like responses by stimulating Ca 2+ -dependent PLC [42][43][44], which quickly generated a sufficient IP 3 burst, thereby enhancing activity of IP 3 receptors and triggering CICR. To verify this possibility, several adrenergic (n = 12) and purinergic (n = 7) MSCs loaded with NP-EGTA were subjected to Ca 2+ 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 Ca 2+ ions liberated from NP-EGTA.
Reportedly, ryanodine and inositol 1,4,5-trisphosphate (IP 3 ) receptors, Ca 2+ -gated Ca 2+ 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 3 and ryanodine receptors to CICR in MSCs, we examined effects of their antagonists on Ca 2+ signals associated with Ca 2+ 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, Ca 2+ uncaging elicited agonist-like biphasic Ca 2+ responses that were delayed relative to stimulatory UV flashes (Figure 5F, G, 2nd responses). Thus, despite the inhibition of ryanodine receptors, Ca 2+ 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 Ca 2+ jump that relaxed monotonically and was smaller by the factor 3-4 ( Figure 5F, G, 3rd responses; Figure 5H). This indicated that Ca 2+ uncaging failed to initiate CICR with inhibited IP 3 receptors. Moreover, when 2-APB was removed to restore activity of IP 3 receptors, a UV flash triggered a biphasic Ca 2+ transient again (Figure 5F, G, 4th responses). These observations indicated that basically IP 3 receptors were responsible for CICR in adrenergic and purinergic MSCs.
To address this issue, we used isoform-specific P2Y agonists and antagonists.
The human P2Y family contains two ATP receptors, including specialized P2Y 11 and also P2Y 2 that recognizes both UTP and ATP as full equipotent agonists [18]. Although also known as a partial P2Y 1 agonist, ATP was hardly capable of stimulating P2Y 1 -signaling in MSCs at low micromolar concentrations due to much lower efficacy than ADP [48]. We tried to evaluate a contribution of P2Y 11 and P2Y 2 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 P2Y 11 antagonist. These NF 340-sensitive cells did not respond to the P2Y 2 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 Ca 2+ 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 2 or both P2Y 2 and P2Y 11 to detect ATP.
While the P2Y 11 antagonist was highly effective (Figure 7A, B), most ATP-sensitive MSCs were surprisingly nonresponsive to NF 546 (10 μM), the specific P2Y 11 agonist reported to be even more effective than ATP [49]. Among 127 cells that responded to 3 μM ATP, 10 μM NF 546 stimulated Ca 2+ 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 P2Y 11 to the phosphoinositide cascade by involving only a certain G-protein type, which is absent or relatively less abundant in most of the MSCs. UTP is a full agonist for P2Y 2 and P2Y 4 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 2 and P2Y 4 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). These findings suggested that predominantly P2Y 4 was responsible for Ca 2+ signaling evoked in MSCs by UTP, while P2Y 2 was either expressed in a very small subpopulation of P2Y 4negative cells or not coupled to Ca 2+ mobilization in a great majority of P2Y 4 -positive cells.
Extracellular ADP is detected by cells with P2Y 1 , P2Y 12 , and P2Y 13 . The analysis of ADP responsiveness was performed on 102 MSCs sensitive to 3 μM ADP ( Figure 8A) that might be recognized by P2Y 1 and/or P2Y 13 receptors, given that P2Y 12 transcripts were not found in MSCs. To evaluate a role of the P2Y 1 , 65 of 103 ADP-sensitive MSCs were treated with MRS 2365, a highly potent and selective P2Y 1 agonist that displays no activity at P2Y 12 and P2Y 13 at submicromolar concentrations [50]. MRS 2365 was ineffective at 100-300 nM but triggered Ca 2+ signaling in 16 (25%) of 65 MSCs at 10 μM ( Figure 9A). Because MRS 2365 specifically stimulates P2Y 1 with EC 50 ~ 1 nM [50], this agonist might bring about a nonspecific action at 10 μM. On the other hand, MRS 2179 (10 μM), a P2Y 1 antagonist with IC 50 = 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 1 receptor were rather inconsistent. To reconcile these contradictory findings, we considered the possibility that both P2Y 1 and P2Y 13 should have been activated by ADP concurrently to mobilize Ca 2+ in MSCs. If so, nanomolar MRS 2365 was ineffective, activating solely P2Y 1 , while 10 μM MRS 2365 stimulated activity of both P2Y 1 and P2Y 13 [50], thus triggering Ca 2+ signaling in MSCs. This concept predicted that MSCs would be unable to respond to ADP if either P2Y 1 or P2Y 13 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 P2Y 13 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 1 and P2Y 13 receptors, were capable of generating robust Ca 2+ responses to ADP.

Discussion
Virtually in all cell types, extracellular cues can mobilize intracellular Ca 2+ to regulate a variety of diverse cellular functions, such as fertilization, proliferation, secretion, metabolism, gene expression, mobility, and muscle contraction. How can the Ca 2+ ion, a chemically simple substance, control so many different physiological processes? The plausible explanation comes from the versatility of Ca 2+ signaling mechanisms that can mediate Ca 2+ 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 PLCβ 1-4 isoforms that hydrolyze the precursor lipid phosphatidylinositol 4,5-bisphosphate to produce two second messengers, IP 3 and diacylglycerol. The primary mode of action of IP 3 is to bind to IP 3 receptors and release Ca 2+ from the endoplasmic reticulum (ER) [30,51,52]. Three different isoforms of the IP 3 receptor have been identified (IP 3 R1, IP 3 R2, and IP 3 R3) and shown to serve as a tetrameric IP 3 -gated Ca 2+ channel [30,[51][52][53]. IP 3 R1, IP 3 R2, and IP 3 R3 are distinct by physiological properties, thus allowing cells to generate specific Ca 2+ signals with different spatial and temporal characteristics to control diverse cellular functions [30,52]. In addition to IP 3 , Ca 2+ is the primary coregulator of IP 3 receptors [30,51,52,54]. The full activation of the IP 3 receptor occurs when IP 3 has occupied the IP 3 -binding domains on all four subunits [55]. This is associated with a conformational change, which sensitizes the Ca 2+ -binding site. The binding of cytosolic Ca 2+ to this site markedly increases the open probability of the IP 3 receptor channel [54], so that Ca 2+ ions released from the ER can additionally stimulate activity of IP 3 receptors. This positive feedback mediates CICR. Meanwhile, the action of cytosolic Ca 2+ is bimodal: stimulating IP 3 receptors at low levels, Ca 2+ becomes inhibitory above 300 nm [54]. This multimodal control of the IP 3 receptor by IP 3 and Ca 2+ is central to various aspects of intracellular Ca 2+ signaling [30,52].
In the present work, we studied MSCs from the human adipose tissue and examined intracellular Ca 2+ 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 Ca 2+ signaling that necessitated coupling of appropriate GPCRs to Ca 2+ 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 P2Y 1,2,4,6,11 subtypes of purinoreceptors are canonically coupled by G q /G 11 to the phosphoinositide cascade and Ca 2+ mobilization, whereas P2Y 12,13,14 control cAMP production by inhibiting adenylyl cyclase through G i /G o . The unique capability of P2Y 11 is to stimulate G s [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][27][28][29]. Hence, a fraction of MSCs sensitive to a given agonist might be in fact much more abundant than that evaluated by Ca 2+ imaging ( Figure 1B, C), because the tested compounds could stimulate not only Ca 2+ mobilization but also other signaling events.
The agonist-dependent Ca 2+ 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 a 2 -adrenoreceptors mediated Ca 2+ mobilization triggered by noradrenaline in adrenergic MSCs (Figure 6). In purinergic MSCs, presumably P2Y 11 serves as a primary ATP receptor (Figure 7), UTP responsiveness is largely mediated by P2Y 4 (Figure 8), while both P2Y 1 and P2Y 13 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 Ca 2+ below the cut-off concentration, a particular agonist initiated Ca 2+ 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 Ca 2+ uncaging approach showed that agonist transduction universally involved the classical phosphoinositide cascade and CICR mechanism (Figures 4 and 5) that employed IP 3 receptors rather than ryanodine receptors (Figures 4E, F and 5F, G).
To reconcile the "all-or-nothing" dose-response curve and gradual dose-delay dependence, we surmised that agonist-evoked Ca 2+ signaling in MSCs includes two different but coupled stages. Primarily, agonists stimulate IP 3 production, activation of IP 3 receptors, and generation of an initial, presumably local and gradual Ca 2+ signal. Next, this local Ca 2+ signal stimulates CICR that produces a global Ca 2+ signal. Some evidence suggests that the Ca 2+ store responsible for the initial Ca 2+ signal may be physically separated from the Ca 2+ 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 Ca 2+ -buffering capacity of the cell cytoplasm, thereby significantly diminishing the initial agonist-induced Ca 2+ signal and its speed. The photodistraction of NP-EGTA decreased exogenous Ca 2+ buffer to a physiologically more relevant level, thus recovering MSC responsiveness to ATP. Note that in line with multiple reports, relatively slow Ca 2+ buffer EGTA is unable to cancel Ca 2+ -dependent processes mediated by local intracellular Ca 2+ signals. For instance, 1 mM EGTA does not prevent activation of Ca 2+ -gated BK channels by Ca 2+ transients originated by both Ca 2+ influx via voltage-gated Ca 2+ channels and Ca 2+ release stimulated by muscarine [58]. By analogy and based on the observation that Ca 2+ uncaging was still capable of triggering CICR in MSCs overloaded with NP-EGTA (Figure 10), we suggested that NP-EGTA, slow Ca 2+ buffer [59], could hardly repeal stimulation of IP 3 receptors by Ca 2+ ions released through this IP 3 -gated conduit. If so, the Ca 2+ store and IP 3 receptor pool mediating CICR should be spatially separated from agonist-dependent machinery that generates an initial, local, and gradual Ca 2+ signal. Otherwise, it is difficult to explain why in cells overloaded with NP-EGTA, agonist responses disappeared contrary to light responses associated with Ca 2+ uncaging (Figure 10).

Conclusion
Note in conclusion that the specific features of agonist responses, including kinetics and magnitude, all-or-nothing behavior and gradual dose-response delay were correctly reproduced by Ca 2+ signals elicited by Ca 2+ uncaging ( Figure 5). This supports the idea that agonist-evoked Ca 2+ signaling in MSCs includes two different but coupled stages. Initially, agonists stimulate coupling of suitable GPCRs via appropriate G-proteins to PLC, thus triggering IP 3 production, activation of IP 3 receptors (IP 3 R grad ) followed by the release of Ca 2+ ions from Ca 2+ store. This machinery generates an initial, presumably local and gradual Ca 2+ signal (Figure 11). When exceeding the threshold, this local Ca 2+ signal stimulates CICR that is mediated by IP 3 receptors (IP 3 R CICR ) presumably located in another, spatially separated Ca 2+ store. By involving the trigger-like mechanism CICR, a cell generates Ca 2+ responses of virtually universal shape and magnitude at different agonist concentrations above the Figure 11. Working model of agonist transduction in MSCs. The binding of agonists to GPCRs stimulates PLC-dependent hydrolysis of PIP 2 to DAG and IP 3 . The consequent activation of IP 3 receptors (IP 3 R grad ) mediates Ca 2+ release from related Ca 2+ store, producing an initial Ca 2+ signal that gradually rises with agonist concentration (red curve). As soon as this signal reaches the threshold level (dotted line), the process determining agonist-dependent delay of a cellular response, one stimulates IP 3 receptors of another type (IP 3 R CICR ) in separated Ca 2+ store and triggers CICR. This provides a significant amplification mechanism that finalizes transduction with a large and global Ca 2+ signal (blue curve).
cut-off dose. Of course, the presented model is a simplification of the actual transduction process, and roles for other common contributors to intracellular Ca 2+ signaling, including Ca 2+ pumps, mitochondria, Ca 2+ buffer as well as Ca 2+ -dependent enzymes and ion channels, remain to be elucidated.