**2. Materials and methods**

**1. Introduction**

140 Calcium and Signal Transduction

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 [1–3]. By unique biologic properties, cultured MSCs from different sources attract sufficient interest in the fields of regenerative medicine and immunotherapy [4–6]. 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

Here, we studied MSCs derived from the human adipose tissue and examined Ca2+ 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, 10–12]. 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

The responsiveness to purines and pyrimidines is widespread among eukaryotic cells, which express numerous purinoreceptors from the P1 and P2 families. The P1 subgroup

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 (P2X1–7) that can form homo- and heterotrimeric cation channels with noticeable Ca2+ permeability [19, 20]. The P2Y subfamily includes eight members (P2Y1,2,4,6,11,12,13,14), which are distinct by ligand specificity and coupling to downstream signaling pathways, including the ubiquitous phos-

Nine genes encode human adrenoreceptors, which all belong to the GPCR superfamily and

) receptor subtypes. Canonically, α<sup>1</sup>

, A2A, A2B, A<sup>3</sup>

) recognizing adenosine as an

(α2A, α2B, α2C),

and

(α1A, α1B, α1D), three α<sup>2</sup>


physiological functions to specific tissue microenvironment.

cells migrate *in vivo* or are transplanted *ex vivo* into an injured tissue.

includes four G-protein-coupled receptors (A<sup>1</sup>

compose three distinctive subgroups, including three α<sup>1</sup>

phoinositide cascade [17, 18].

, β<sup>2</sup> , β<sup>3</sup>

and three β (β<sup>1</sup>
