P2Y2 and P2Y6 receptor activation elicits intracellular calcium responses in human adipose-derived mesenchymal stromal cells

Adipose tissue contains self-renewing multipotent cells termed mesenchymal stromal cells. In situ, these cells serve to expand adipose tissue by adipogenesis, but their multipotency has gained interest for use in tissue regeneration. Little is known regarding the repertoire of receptors expressed by adipose-derived mesenchymal stromal cells (AD-MSCs). The purpose of this study was to undertake a comprehensive analysis of purinergic receptor expression. Mesenchymal stromal cells were isolated from human subcutaneous adipose tissue and confirmed by flow cytometry. The expression profile of purinergic receptors was determined by quantitative real-time PCR and immunocytochemistry. The molecular basis for adenine and uracil nucleotide-evoked intracellular calcium responses was determined using Fura-2 measurements. All the known subtypes of P2X and P2Y receptors, excluding P2X2, P2X3 and P2Y12 receptors, were detected at the mRNA and protein level. ATP, ADP and UTP elicited concentration-dependent calcium responses in mesenchymal cells (N = 7–9 donors), with a potency ranking ADP (EC50 1.3 ± 1.0 μM) > ATP (EC50 2.2 ± 1.1 μM) = UTP (3.2 ± 2.8 μM). Cells were unresponsive to UDP (< 30 μM) and UDP-glucose (< 30 μM). ATP responses were attenuated by selective P2Y2 receptor antagonism (AR-C118925XX; IC50 1.1 ± 0.8 μM, 73.0 ± 8.5% max inhibition; N = 7 donors), and UTP responses were abolished. ADP responses were attenuated by the selective P2Y6 receptor antagonist, MRS2587 (IC50 437 ± 133nM, 81.0 ± 8.4% max inhibition; N = 6 donors). These data demonstrate that adenine and uracil nucleotides elicit intracellular calcium responses in human AD-MSCs with a predominant role for P2Y2 and P2Y6 receptor activation. This study furthers understanding about how human adipose-derived mesenchymal stromal cells can respond to external signalling cues

Mechanically Induced Chromatin Condensation Requires Cellular Contractility in Mesenchymal Stem Cells

This work was supported by the National Institutes of Health (R01 AR056624, R01 EB02425, T32 AR007132, and P30 AR050950). Additional support was provided by a Montague Research Award from the Perelman School of Medicine and a University of Pennsylvania University Research Foundation Award

Rapidly and slowly activating components of delayed rectifier K+ current in guinea-pig sino-atrial node pacemaker cells

The components and properties of the delayed rectifier K+ current (IK) in isolated guinea-pig sino-atrial (SA) node pacemaker cells were investigated using the whole-cell configuration of the patch-clamp technique. An envelope of tails test was conducted by applying depolarizing pulses from a holding potential of −50 mV to +30 mV for various durations ranging from 40 to 2000 ms. The ratio of the tail current amplitude elicited upon return to the holding potential to the magnitude of the time-dependent outward current activated during depolarizing steps was dependent on the pulse duration, while after exposure to the selective IKr inhibitor E-4031 (5 μm) this current ratio became practically constant irrespective of the pulse duration. These observations are consistent with the presence of the E-4031-sensitive, rapidly activating and E-4031-resistant, slowly activating components of IK (IKr and IKs, respectively) in guinea-pig SA node cells. The activation range for IKr, defined as the E-4031-sensitive current (half-maximal activation voltage (V1/2) of −26.2 mV) was much more negative than that for IKs, defined as the E-4031-resistant current (V1/2 of +17.2 mV). IKr exhibited a marked inward rectification at potentials positive to −50 mV, whereas IKs showed only a slight rectification. In the current-clamp experiments, bath application of E-4031 (0.5 and 5 μm) initially slowed the repolarization at potentials negative to approximately −30 mV and produced a significant depolarization of the maximum diastolic potential, followed by the arrest of electrical activity, thus indicating that the late phase of the repolarization leading to the maximum diastolic potential at around −60 mV in spontaneous action potentials is primarily produced by IKr in guinea-pig SA node cells. External application of the selective IKs inhibitor 293B (30 μm) also delayed the repolarization process at potentials negative to about −20 mV and induced moderate depolarization of the maximum diastolic potential leading to the arrest of the spontaneous activity. These results provide evidence to suggest that both IKr and IKs are present and play crucial roles in the spontaneous electrical activity of guinea-pig SA node pacemaker cells