The effect of liver growth stimulation [using the rodent PXR (pregnane X receptor) activator PCN (pregnenolone-16alpha-carbonitrile)] in rats chronically treated with carbon tetrachloride to cause repeated hepatocyte necrosis and liver fibrogenesis was examined. PCN did not inhibit the hepatotoxicity of carbon tetrachloride. However, transdifferentiation of hepatic stellate cells and the extent of fibrosis caused by carbon tetrachloride treatment was significantly inhibited by PCN in vivo. In vitro, PCN directly inhibited hepatic stellate cell transdifferentiation to a profibrogenic phenotype, although the cells did not express the PXR (in contrast with hepatocytes), suggesting that PCN acts independently of the PXR. Mice with a functionally disrupted PXR gene (PXR-/-) did not respond to the antifibrogenic effects of PCN, in contrast with wild-type (PXR+/+) mice, demonstrating an antifibrogenic role for the PXR in vivo. However, PCN inhibited the transdifferentiation of PXR-/--derived mouse hepatic stellate cells in vitro, confirming that there is also a PXR-independent antifibrogenic effect of PCN through a direct interaction with hepatic stellate cells. These data suggest that the PXR is antifibrogenic in rodents in vivo and that a PXR-independent target for PXR activators exists in hepatic stellate cells that also functions to inhibit fibrosis.

Understanding the development and differentiation of the neocortex remains a central focus of neuroscience. While previous studies have examined isolated aspects of cellular and synaptic organization, an integrated functional index of the cortical microcircuit is still lacking. Here we aimed to provide such an index, in the form of spontaneously recurring periods of persistent network activity -or Up states- recorded in mouse cortical slices. These coordinated network dynamics emerge through the orchestrated regulation of multiple cellular and synaptic elements and represent the default activity of the cortical microcircuit. To explore whether spontaneous Up states can capture developmental changes in intracortical networks we obtained local field potential recordings throughout the mouse lifespan. Two independent and complementary methodologies revealed that Up state activity is systematically modified by age, with the largest changes occurring during early development and adolescence. To explore possible regional heterogeneities we also compared the development of Up states in two distinct cortical areas and show that primary somatosensory cortex develops at a faster pace than primary motor cortex. Our findings suggest that in vitro Up states can serve as a functional index of cortical development and differentiation and can provide a baseline for comparing experimental and/or genetic mouse models.

Nicotinic acetylcholine receptors (nAChRs) play an important role in the modulation of many cognitive functions but their role in integrated network activity remains unclear. This is at least partly because of the complexity of the cholinergic circuitry and the difficulty in comparing results from in vivo studies obtained under diverse experimental conditions and types of anesthetics. Hence the role of nAChRs in the synchronization of cortical activity during slow-wave sleep is still controversial, with some studies showing they are involved in ACh-dependent EEG desynchronization, and others suggesting that this effect is mediated exclusively by muscarinic receptors. Here we use an in vitro model of endogenous network activity, in the form of recurring self-maintained depolarized states (Up states), which allows us to examine the role of high-affinity nAChRs on network dynamics in a simpler form of the cortical microcircuit. We find that mice lacking nAChRs containing the ␤2-subunit (␤2-nAChRs) have longer and more frequent Up states, and that this difference is eliminated when ␤2-nAChRs in wild-type mice are blocked. We further show that endogenously released ACh can modulate Up/Down states through the activation of both ␤2-and ␣7-containing nAChRs, but through distinct mechanisms: ␣7-nAChRs affect only the termination of spontaneous Up states, while ␤2-nAChRs also regulate their generation. Finally we provide evidence that the effects of ␤2-subunit-containing, but not ␣7-subunit-containing nAChRs, are mediated through GABA B receptors. To our knowledge this is the first study documenting direct nicotinic modulation of Up/Down state activity.

Ryanodine receptor 2 (RyR2) cDNA has been available for more than 15 years; however, due to the complex nature of ligand gating in this channel, many aspects of recombinant RyR2 function have been unresearched. We established a stable, inducible HEK 293 cell line expressing full-length rabbit RyR2 cDNA and assessed the single-channel properties of the recombinant RyR2, with particular reference to ligand regulation with Ca2+ as the permeant ion. We found that the single-channel conductances of recombinant RyR2 and RyR2 isolated from cardiac muscle are essentially identical, as is irreversible modification by ryanodine. Although it is known that RyR2 expressed in HEK 293 cells is not associated with FKBP12.6, we demonstrate that these channels do not exhibit any discernable disorganized gating characteristics or subconductance states. We also show that the gating of recombinant RyR2 is indistinguishable from that of channels isolated from cardiac muscle when activated by cytosolic Ca2+, caffeine or suramin. The mechanisms underlying ATP activation are also similar; however, the experiments highlighted a novel effect of ATP at physiologically relevant concentrations of 5-10 mM. With Ca2+ as permeant ion, 5-10 mM ATP consistently inactivated recombinant channels (15/16 experiments). Such inactivation was rarely observed with native RyR2 isolated from cardiac muscle (1 in 16 experiments). However, if the channels were purified, inactivation by ATP was then revealed in all experiments. This action of ATP may be relevant for inactivation of sarcoplasmic reticulum Ca2+ release during cardiac excitation-contraction coupling or may represent unnatural behavior that is revealed when RyR2 is purified or expressed in noncardiac systems.

) on the gating of native sheep RyR2, reconstituted into bilayers. Suramin displaces CaM from RyR2 and we have used a gel-shift assay to provide evidence of the mechanism underlying this effect. Finally, using suramin to displace endogenous CaM from RyR2 in permeabilized cardiac cells, we have investigated the effects of 50 nmol·L -1 CaM on sarcoplasmic reticulum (SR) Ca 2+-release. Key results: Ca 2+CaM activated or inhibited single RyR2, but activation was much more likely at low (50-100 nmol·L -1 ) concentrations. Also, suramin displaced CaM from a peptide of the CaM binding domain of RyR2, indicating that, like the skeletal isoform (RyR1), suramin directly competes with CaM for its binding site on the channel. Pre-treatment of rat permeabilized ventricular myocytes with suramin to displace CaM, followed by addition of 50 nmol·L -1 CaM to the mock cytoplasmic solution caused an increase in the frequency of spontaneous Ca 2+ -release events. Application of caffeine demonstrated that 50 nmol·L -1 CaM reduced SR Ca 2+ content. Conclusions and implications: We describe for the first time how Ca 2+CaM is capable, not only of inactivating, but also of activating RyR2 channels in bilayers in a CaM kinase II-independent manner. Similarly, in cardiac cells, CaM stimulates SR Ca 2+-release and the use of caffeine suggests that this is a RyR2-mediated effect. Abbreviations: AIP, autocamtide-2-related inhibitory peptide; CaM, calmodulin; CaMKII, CaM kinase II; EC-coupling, excitation-contraction coupling; RyR2, cardiac ryanodine receptor; RyRF, RyR2 peptide (RSKKAVWHKLL-SKQRKRAVVACFRMAPLYNLP); SR, sarcoplasmic reticulum British Journal of Pharmacology IntroductionThe cardiac ryanodine receptor (RyR2; nomenclature follows Alexander et al., 2008) is the pathway for the release of intracellular Ca 2+ during excitation-contraction (EC) coupling. It also acts as a scaffolding protein localizing numerous other proteins to the dyadic cleft regions. Calmodulin (CaM) binds very tightly to RyR2, but the physiological role of this direct association is unclear (Balshaw et al., 2001;Ai et al., 2005). CaM is a Ca 2+ -sensing protein, which contains four Ca 2+ -binding sites, two on the amino and two on the carboxyl lobe. CaM interacts with numerous proteins, usually in the Ca 2+ -bound form (Ca 2+ CaM), but can also modulate certain proteins in the non-Ca 2+ -bound form (apoCaM). It has been well documented that Ca 2+ CaM can cause partial inhibition of both cardiac and skeletal (RyR1) isoforms of RyR (Fruen et al., 2000;Balshaw et al., 2001). Additionally, apoCaM is known to activate RyR1, but has little effect on RyR2 Tripathy et al., 1995;Fruen et al., 2000).The magnitude of the reported inhibition of RyR2 by Ca 2+ CaM is often exceedingly small (Yamaguchi et al., 2004;Xu and Meissner, 2004) and, interestingly, there are also rare reports suggesting that CaM may activate RyR2 (Fruen et al., 2000;Chugun et al., 2007 Xu and Meissner, 2004) and Ca 2+ -spark generation in cardiac cells (Lukyanenko and Gyorke, 1999;Ai et al., 2005;Guo ...