The effect of extracellular ATP on adipogenesis was investigated using the mouse 3T3-L1 cell line. Incubation of cells with ATP (1-100 microM) for 5 min induced actin filament reorganization and membrane ruffling mediated through P2Y receptors. Enhancement of preadipocyte migration into fat cell clusters is one of the essential processes of adipose tissue development in vivo and cell migration assays revealed that stimulation of P2Y receptors enhanced chemokinesis (migration) in a concentration dependent manner. In this cell line, growth arrest is required before initiation of differentiation and growth-arrested post-confluent cells can be converted into adipocytes by the presence of the adipogenic hormones dexamethasone, 3-isobutyl-1-methylxanthine and insulin. On the other hand, those hormones alone do not trigger differentiation in proliferating cells. ATP did not induce differentiation when applied alone to either proliferating or postconfluent cells. By contrast, proliferating cells (density <50%) preincubated with ATP for 5 min and subsequently given the adipogenic hormones in the continued presence of ATP, underwent adipocyte differentiation mediated through phospholipase C-coupled P2Y receptors. These adipocytes were found to show very similar characteristics, including morphology and intracellular triacylglycerol accumulation compared with adipocytes differentiated from post-confluent preadipocytes with those adipogenic hormones. When proliferating cells were preincubated with ATP before the addition of the adipogenic hormones, gene expression of aP2 (adipose protein 2) was markedly increased within 6 days, whereas without ATP pretreatment the expression level stayed very low. These results suggest that extracellular ATP renders preadipocytes responsive to adipogenic hormones during the growth phase.

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S U M M A R Y The present study was designed to examine the postnatal developmental changes of atypically shaped cardiomyocytes (ACMs) prepared from the heart of newborn [postnatal day 1 (day-1)] through aged (12-month-old) mice. ACMs were identified as a novel type of self-beating cardiomyocyte with a peculiar morphology in mouse cardiac ventricles. The cell length of ACMs significantly increased during the first three postnatal months and further increased over the following 9 months. In contrast, the population of ACMs was significantly decreased within the first 5 weeks and reached a plateau in the adult stage. ACMs obtained from newborn and adult mice exhibited similar spontaneous action potentials. The expression of the fetal cardiac gene products atrial natriuretic peptide and voltage-gated T-type Ca 21 channel Ca V 3.2 was confirmed by immunostaining in ACMs obtained from both newborn and aged mice. These observations provide evidence that ACMs that exhibit spontaneous beating survive the long-term postnatal development of cardiac ventricles while preserving the expression of fetal cardiac genes. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials. (J Histochem Cytochem 58:543-551, 2010)

Abstract. G protein-coupled receptors (GPCRs) are distributed widely throughout the human body, and nearly 50% of current medicines act on a GPCR. GPCRs are considered to consist of seven transmembrane a -helices that form an a -helical bundle in which agonists and antagonists bind. A 3D structure of the target GPCR is indispensable for designing novel medicines acting on a GPCR. We have previously constructed the 3D structure of human P2Y 1 (hP2Y 1 ) receptor, a GPCR, by homology modeling with the 3D structure of bovine rhodopsin as a template. In the present study, we have employed an in silico screening for compounds that could bind to the hP2Y 1 -receptor model using AutoDock 3.0. We selected 21 of the 30 top-ranked compounds, and by measuring intracellular Ca 2+ concentration, we identified 12 compounds that activated or blocked the hP2Y 1 receptor stably expressed in recombinant CHO cells. 5-Phosphoribosyl-1-pyrophosphate (PRPP) was found to activate the hP2Y 1 receptor with a low ED 50 value of 15 nM. The Ca 2+ assays showed it had no significant effect on P2Y 2 , P2Y 6 , or P2X 2 receptors, but acted as a weak agonist on the P2Y 12 receptor. This is the first study to rationally identify surrogate ligands for the P2Y-receptor family.

This study was designed to examine the autophagy in sino-atrial (SA) nodal cells from the normal adult mouse heart. Autophagy is the cellular process responsible for the degradation and recycling of long-lived and/or damaged cytoplasmic components by lysosomal digestion. In the heart, autophagy is known to occur at a low level under physiological conditions, but to become upregulated when cells are exposed to certain stresses, such as ischemia. We examined whether the basal level of autophagy in SA nodal cells was different from that in ventricular or atrial myocytes. An ultrastructural analysis revealed that the SA nodal cells contained a number of autophagic vacuoles (autophagosomes) with various stages of degradation by lysosomal digestion, whereas the number of those in ventricular or atrial myocytes was either negligible or very small. The immunostaining of autophagosome marker microtubule-associated protein 1 light chain 3 (LC3) and lysosome marker lysosome-associated membrane protein 1 (LAMP1) indicated that the content of both autophagosomes and lysosomes were much greater in SA nodal cells than in ordinary cardiomyocytes. Our results provide evidence that the autophagy is active in normal SA nodal cells, which is not a stress-activated process but a constitutive event in the mouse heart.

OHCA patients fulfilling the criteria unwitnessed by bystanders and asystole in the field and at hospital arrival had universally poor outcomes. Termination of resuscitation after hospital arrival for these patients may decrease unwarranted treatments.

Stimulation of P2 receptors with micromolar concentration of ATP evokes a transient increase in [Ca2+]i (intracellular free Ca2+ concentration), primarily due to release of Ca2+ from intracellular stores; such stimulation also triggers almost complete suppression of thapsigargin-evoked sustained [Ca2+]i increase mediated through a store-operated Ca2+ entry pathway in rat brown adipocytes. We investigated the role of cytoskeletal actin in the inhibitory effect of the extracellular ATP on store-operated Ca2+ entry, using fura 2 fluorescence for continuous measurement of [Ca2+]i, and using Alexa fluor 488-phalloidin staining of actin. Disassembly of actin networks by cytochalasin D (1 microM) or latrunculin A (3 microM) prevented the inhibitory effect of ATP (10 microM) on the thapsigargin (100 nM)-evoked store-operated Ca2+ entry, without changing the effect of ATP in increasing [Ca2+]i. In normal cells, bath application of ATP induced a transient [Ca2+]i increase, consisting of a rapid increase (the rising phase) and the subsequent decrease (the declining phase) to a lower steady level despite the continued presence of the agonist. Disruption of actin assemblies did not significantly affect the rising phase, but prevented the declining phase. Cells incubated with 10 microM ATP for 4 min demonstrated marked accumulations of actin filaments at the cell periphery, showing protrusions at the cell surface; this actin-assembly process is mediated through P2 receptors. In cells treated with cytochalasin D or latrunculin A, extracellular ATP did not induce actin redistribution. These results suggest that the actin reorganization plays a role in ATP-induced inhibition of store-operated Ca2+ entry in rat brown adipocytes.

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