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Background and Purpose: Neurons derived from mouse embryonic stem cells (mESCs) are a valuable resource for basic pharmacological research. With the exception of cardiomyocytes, there is relatively little understanding of the pharmacology of stem cell-derived differentiated cells. In this study we investigate P2 receptor agonist effects on GABAergic neurons derived from mESCs.Experimental Approach: mESCs were differentiated into GABAergic neurons in the presence of N2B27 culture medium. At day 24 of differentiation GABAergic neuronal responsiveness to purinergic agonists was investigated using calcium imaging and [3H]-GABA release studies.Key Results: Sub-populations of GABAergic neurons responded to some or all of the adenine and uracil nucleotides ATP, ADP, UTP and UDP (all 100 microM) with elevations of intracellular Ca2+ ([Ca2+]i). The number of neurons responding to ATP was reduced by suramin (100 microM), PPADS (10 microM) and MRS2179 (10 microM), but not by NF023 (10 microM). The response to ATP was modulated by extracellular Zn2+ and pH. Neurons also responded to ATP (100 microM) with the release of [3H]-GABA, an effect completely inhibited by tetrodotoxin (100 nM). Ap4A and 2-methylthioATP both elicited significant [3H]-GABA release. Reverse transcriptase PCR showed the presence of P2X1,2,3,4,5,6 and P2X7, and P2Y1,2 and P2Y6 receptors. mESCs expressed P2X2,5 and P2X7 and P2Y1,2 and P2Y6 receptors.Conclusions and Implications: GABAergic neurons derived from stem cells elevate [Ca2+]i predominantly via the activation of P2X2, P2X4 and P2Y1 receptors. This study shows that mESCs generate good models of neuronal function for in vitro pharmacological investigation. [ABSTRACT FROM AUTHOR]

1: This study investigated the possibility that adenosine receptors modulate the a1-adrenoceptor-mediated contractility of human cultured prostatic stromal cells (HCPSC). 2: The nonselective adenosine receptor agonist, 5'-N-ethylcarboxamido-adenosine (NECA; 10?nM-10?µM), and the A1 adenosine receptor selective agonist, cyclopentyladenosine (CPA; 10?nM-10?µM), elicited significant contractions in HCPSC, with maximum contractile responses of 18±3% and 17±2% reduction in initial cell length, respectively. 3: In the presence of a threshold concentration of phenylephrine (PE) (100?nM), CPA (1?nM-10?µM) caused contractions, with an EC50 of 124±12?nM and maximum contractile response of 37±4%. The A1 adenosine receptor-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX 100?nM) blocked this effect. In the presence of DPCPX (100?nM), NECA (1?nM-10?µM) inhibited contractions elicited by a submaximal concentration of PE (10?µM), with an IC50 of 48±2?nM. The A2A adenosine receptor-selective antagonist 4-(2-[7-amino-2-{furyl}{1,2,4}triazolo{2,3-a}{1,3,5,}triazin-5-yl amino]ethyl)phenol (Zm241385 100?nM) blocked this effect. 4: In BCECF-AM (10?µM)-loaded cells, both CPA (100?pM-1?µM) and NECA (100?pM-10?µM) elicited concentration-dependent decreases in intracellular pH (pHi), with EC50 values of 3.1±0.3 and 6.0±0.3?nM, respectively. The response to NECA was blocked by Zm241385 (100?nM; apparent pKB of 9.4±0.4), but not by DPCPX (100?nM). The maximum response to CPA was blocked by DPCPX (100?nM), and unaffected by Zm241385 (100?nM). 5: NECA (10?nM-10?µM) alone did not increase [3H]-cAMP in HCPSC. In the presence of DPCPX (100?nM), NECA (10?nM-10?µM) caused a concentration dependent increase in [3H]-cAMP, with an EC50 of 1.2±0.1?µM. This response was inhibited by Zm241385 (100?nM). CPA (10?nM-10?µM) had no effect on cAMP, in the presence or absence of forskolin (1?µM). 6: These findings are consistent with a role for adenosine receptors in the modulation of adrenoceptor-mediated contractility in human prostate-derived cells.British Journal of Pharmacology (2004) 141, 302-310. doi:10.1038/sj.bjp.0705535 [ABSTRACT FROM AUTHOR]