Vascular smooth muscle cells (VSMCs) are the predominant cell type in the blood vessel wall. Changes in VSMC actomyosin activity and morphology are prevalent in cardiovascular disease. The actin cytoskeleton actively defines cellular shape and the LInker of Nucleoskeleton and Cytoskeleton (LINC) complex, comprised of nesprin and the Sad1p, UNC-84 (SUN)-domain family members SUN1/2, has emerged as a key regulator of actin cytoskeletal organisation. Although SUN1 and SUN2 function is partially redundant, they possess specific functions and LINC complex composition is tailored for cell-type-specific functions. We investigated the importance of SUN1 and SUN2 in regulating actomyosin activity and cell morphology in VSMCs. We demonstrate that siRNA-mediated depletion of either SUN1 or SUN2 altered VSMC spreading and impaired actomyosin activity and RhoA activity. Importantly, these findings were recapitulated using aortic VSMCs isolated from wild-type and SUN2 knockout (SUN2 KO) mice. Inhibition of actomyosin activity, using the rho-associated, coiled-coil-containing protein kinase1/2 (ROCK1/2) inhibitor Y27632 or blebbistatin, reduced SUN2 mobility in the nuclear envelope and decreased the association between SUN2 and lamin A, confirming that SUN2 dynamics and interactions are influenced by actomyosin activity. We propose that the LINC complex exists in a mechanical feedback circuit with RhoA to regulate VSMC actomyosin activity and morphology.

White adipocytes are key regulators of metabolic homeostasis, which release stored energy as free fatty acids via lipolysis. Adipocytes possess both basal and stimulated lipolytic capacity, but limited information exists regarding the molecular mechanisms that regulate basal lipolysis. Here, we describe a mechanism whereby autocrine purinergic signalling and constitutive P2Y 2 receptor activation suppresses basal lipolysis in primary human in vitro-differentiated adipocytes. We found that human adipocytes possess cytoplasmic Ca 2+ tone due to ATP secretion and constitutive P2Y 2 receptor activation. Pharmacological antagonism or knockdown of P2Y 2 receptors increases intracellular cAMP levels and enhances basal lipolysis. P2Y 2 receptor antagonism works synergistically with phosphodiesterase inhibitors in elevating basal lipolysis, but is dependent upon adenylate cyclase activity. Mechanistically, we suggest that the increased Ca 2+ tone exerts an anti-lipolytic effect by suppression of Ca 2+-sensitive adenylate cyclase isoforms. We also observed that acute enhancement of basal lipolysis following P2Y 2 receptor antagonism alters the profile of secreted adipokines leading to longer-term adaptive decreases in basal lipolysis. Our findings demonstrate that basal lipolysis and adipokine secretion are controlled by autocrine purinergic signalling in human adipocytes.

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 P2Y 12 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 (EC 50 1.3 ± 1.0 μM) > ATP (EC 50 2.2 ± 1.1 μM) = UTP (3.2 ± 2.8 μM). Cells were unresponsive to UDP (< 30 μM) and UDPglucose (< 30 μM). ATP responses were attenuated by selective P2Y 2 receptor antagonism (AR-C118925XX; IC 50 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 P2Y 6 receptor antagonist, MRS2587 (IC 50 437 ± 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 P2Y 2 and P2Y 6 receptor activation. This study furthers understanding about how human adipose-derived mesenchymal stromal cells can respond to external signalling cues.

Background and Purpose: P2X4 is a ligand-gated cation channel activated by extracellular ATP involved in neuropathic pain, inflammation and arterial tone.Experimental Approach: Natural products were screened against human or mouse P2X4 activity using fura-2 loaded 1321N1 cells for measurement of intracellular Ca 2+ responses. Whole-cell currents were measured by patch clamp. Human primary macrophage chemokine release was used to assess effect of taspine on inflammatory cell function. An enzymatic assay was performed to assess the effect of taspine on recombinant PI3-kinase.Key Results: A natural product screen identified taspine as an inhibitor of human P2X4 activity. Taspine inhibits human and mouse P2X4-mediated Ca 2+ influx in 1321N1 cells expressing receptors but lacked activity at human P2X2, P2X3, P2X2/3 and P2X7 receptors. Taspine inhibited the maximal response at human and mouse P2X4 but effective on ATP potency. Taspine has a slow onset rate ($15 min for half-maximal inhibition), irreversible over 30 min of washout. Taspine inhibits P2X4-mediated Ca 2+ signalling in mouse BV-2 microglia cells and human primary macrophage. Taspine inhibited P2X4-mediated CXCL5 secretion in human primary macrophage. Taspine reversed ivermectin-induced potentiation of P2X4 currents in 1321N1 stably expressing cells. The PI3-kinase inhibitor LY294002 mimicked the properties of taspine on P2X4-mediated Ca 2+ influx and whole-cell currents. Taspine directly inhibited the enzymatic activity of recombinant PI3-kinase in a competitive manner. Conclusion and Implications:Taspine is a novel natural product P2X4 receptor inhibitor, mediating its effect through PI3-kinase inhibition rather than receptor antagonism. Taspine can inhibit the pro-inflammatory signalling by P2X4 in human primary macrophage.