Muscarinic Receptor-Stimulated Phosphoinositide Turnover in Human SK-N-SH Neuroblastoma Cells: Differential Inhibition by Agents that Elevate Cyclic AMP

The possibility that an increased intracellular concentration of cyclic AMP (cAMP) can regulate the extent of muscarinic receptor-stimulated phosphoinositide (PPI) turnover in the human neuroblastoma cell line SK-N-SH was examined. Addition of either forskolin (or its water-soluble nalog, L-85,8051), theophylline, isobutylmethylxanthine, or Liolera toxin, agents that interact with either the catalytic unit of adenylate cyclase, cAMP phosphodiesterase, or the guanine nucleotide binding protein linked to adenylate cyclase activation, resulted in a 45–181% increase in cAMP concentration and a 27–70% inhibition of carbachol-stimu-lated inositol phosphate release. Through the use of digitonin-permeabilized cells, the site of inhibition was localized to a step at, or distal to, the guanine nucleotide binding protein that regulates phospholipase C activity. In contrast, when intact SK-N-SH cells were exposed to prostaglandin E 1 , the ensuing increases in cAMP were not accompanied by an inhibition of stimulated PPI turnover. These differential effects of increased cAMP concentrations on stimulated PPI turnover may reflect the compartmentation of cAMP within SK-N-SH cells.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66265/1/j.1471-4159.1989.tb08541.x.pd

Identification of Multiple Phosphoinositide-Linked Receptors on Human SK-N-MC Neuroepithelioma Cells

:The biochemical and pharmacological characteristics of receptor-stimulated phosphoinositide (PPI) hydrolysis in human SK-N-MC neuroepithelioma cells have been examined. Of 11 ligands tested, the addition of four, i.e., nor-epinephrine, oxotremorine-M, endothelin-1, and ATP, each resulted in an increased release (three-to eightfold) of inositol phosphates from [ 3 H]inositol-prelabeled cells. Agonist-stimulated PPI turnover was sustained for at least 30 min and required the addition of Ca 2+ for full effect. An increased release of inositol phosphates could also be elicited by the addition of the Ca 2+ ionophore, ionomycin. All four agonists enhanced the release of radiolabeled inositol mono-and bis-phosphates, inositol 1,3,4-trisphosphate, and inositol tetra-kisphosphate. Increases in inositol 1,4,5-trisphosphate were smaller and only consistently observed in the presence of norepinephrine or oxotremorine-M. Norepinephrine-stim-ulated PPI turnover was potently inhibited by prazosin, WB-4101, and 5-methylurapidil ( K 1 , < 2.5 n M ), but was relatively insensitive to chloroethylclonidine pretreatment. This pharmacological profile is consistent with the involvement of an Α 1A -receptor subtype. The presence of an M 1 muscannic cholinergic receptor is also indicated, because pirenzepine blocked oxotremorine-M-stimulated inositol phosphate release ( K 1 = 35 n M ) with a 30-fold greater potency than the M 2 -selective antagonist, AF-DX 116. Of the three endothelins tested, only the addition of endothelin-1 and endothelin-2 promoted PPI hydrolysis, whereas endothelin-3 was essentially inactive. A P 2 nucleotide receptor of broad agonist specificity is also present on these cells and activates PPI turnover in the absence of a generalized increase in plasma membrane permeability. These results indicate that SK-N-MC cells express at least four PPI-linked receptors. Because the functional coupling of three of these receptors, i.e., Α 1A -adrenergic, endothelin, and P 2 nucleotide, has not been extensively characterized previously in neural tissues, the SK-N-MC cell line may provide a useful model system for studies of these receptors and their regulation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66097/1/j.1471-4159.1991.tb06357.x.pd

A Putative M 3 Muscarinic Cholinergic Receptor of High Molecular Weight Couples to Phosphoinositide Hydrolysis in Human SK-N-SH Neuroblastoma Cells

The M 1 -selective (high affinity for pirenzepine) muscarinic acetylcholine receptor (mAChR) antagonist pirenzepine displaced both N -[ 3 H]methylscopolamine ([ 3 H]NMS) and [ 3 H]qui-nuclidinylbenzilate from intact human SK-N-SH neuroblastoma cells with a low affinity ( K i = 869–1,066 nM), a result indicating the predominance of the M 2 or M 3 (low affinity for pirenzepine) receptor subtype in these cells. Whereas a selective M 2 agent, AF-DX 116 {11–2[[2-[(diethylamino)methyl]-1-piperidinyl]-acetyl]-5,11-dihydro-6 H -pyrido[2,3- b ][1,4]benzodiazepin-6-one} bound to the mAChRs with a very low affinity ( K i = 6.0 Μ M ), 4-diphenylacetoxy- N -methylpiperidine methiodide (4-DAMP), an agent that binds with high affinity to the M 3 subtype, potently inhibited [ 3 H]NMS binding ( K i = 7.2 n M ). 4-DAMP was also 1,000-fold more effective than AF-DX 1 16 at blocking stimulated phosphoinositide (PPI) hydrolysis in these cells. Covalent labeling studies (with [ 3 H]propylbenzilylcholine mustard) suggest that the size of the SK-N-SH mAChR (M r = 81.000–98,000) distinguishes it from the predominant mAChR species in rat cerebral cortex (M r =66,000), an M 1 -enriched tissue. These results provide the first demonstration of a neural M 3 mAChR subtype that couples to PPI turnover.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65720/1/j.1471-4159.1988.tb03008.x.pd

Reduction of Muscarinic Receptor Density and of Guanine Nucleotide-Stimulated Phosphoinositide Hydrolysis in Human SH-SY5Y Neuroblastoma Cells Following Long-Term Treatment with 12- O -Tetradecanoylphorbol 13-Acetate or Mezerein

The actions of tumor promoters on the coupling of muscarinic receptors to the hydrolysis of inositol lipids and the generation of Ca 2+ signals were examined in the human neuroblastoma SH-SY5Y cell line. Pretreatment of SH-SY5Y cells with 50 n M 12- O -tetradecanoylphorbol 13-acetate (TPA) for 5 days resulted in neuronal differentiation, a 28% decrease in both N -[ 3 H]methylscopolamine and [ 3 H]-scopolamine binding, and a significantly larger reduction (48%) in agonist-stimulated 3 H-inositol phosphate generation. Whereas mezerein could mimic the effects produced by TPA, the biologically inactive 4 Α -phorbol 12,13-didecanoate was without effect on both antagonist binding and agonist-stimulated phosphoinositide (PPI) turnover. A decline (∼ 50%) in the agonist-mediated rise in cytoplasmic Ca 2+ and a substantial loss of protein kinase C activity also were observed following pretreatment with TPA or mezerein. The ability of fluoride, an agent capable of direct activation of guanine nucleotide binding proteins, to stimulate 3 H-inositol phosphate release was significantly reduced in SH-SY5Y cells treated with these agents. Furthermore, pretreatment of SH-SY5Y neuroblastoma cells with TPA or mezerein impaired 3 H-inositol phosphate formation induced by the addition of either guanosine 5′- O -(3-thiotriphosphate) or carbamylcholine to digitonin-permeabilized cells, but not that elicited by the addition of 2 m M CaCl 2 . Although cells cultured in the presence of serum-free media also exhibited neuronal differentiation, no significant alteration in either muscarinic receptor number or agonist-stimulated PPI hydrolysis was observed. The results suggest that TPA and mezerein decrease agonist-stimulated PPI hydrolysis and Ca 2+ signaling in SH-SY5Y cells not only by a reduction in muscarinic receptor number but also through an inhibition of guanine nucleotide-stimulated PPI turnover.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65548/1/j.1471-4159.1990.tb01227.x.pd

Quench dynamics across quantum critical points

We study the quantum dynamics of a number of model systems as their coupling constants are changed rapidly across a quantum critical point. The primary motivation is provided by the recent experiments of Greiner et al. (Nature 415, 39 (2002)) who studied the response of a Mott insulator of ultracold atoms in an optical lattice to a strong potential gradient. In a previous work (cond-mat/0205169), it had been argued that the resonant response observed at a critical potential gradient could be understood by proximity to an Ising quantum critical point describing the onset of density wave order. Here we obtain numerical results on the evolution of the density wave order as the potential gradient is scanned across the quantum critical point. This is supplemented by studies of the integrable quantum Ising spin chain in a transverse field, where we obtain exact results for the evolution of the Ising order correlations under a time-dependent transverse field. We also study the evolution of transverse superfluid order in the three dimensional case. In all cases, the order parameter is best enhanced in the vicinity of the quantum critical point.Comment: 10 pages, 6 figure

How do healthcare professionals working in accountable care organisations understand patient activation and engagement? Qualitative interviews across two time points.

ObjectiveIf patient engagement is the new 'blockbuster drug' why are we not seeing spectacular effects? Studies have shown that activated patients have improved health outcomes, and patient engagement has become an integral component of value-based payment and delivery models, including accountable care organisations (ACO). Yet the extent to which clinicians and managers at ACOs understand and reliably execute patient engagement in clinical encounters remains unknown. We assessed the use and understanding of patient engagement approaches among frontline clinicians and managers at ACO-affiliated practices.DesignQualitative study; 103 in-depth, semi-structured interviews.ParticipantsSixty clinicians and eight managers were interviewed at two established ACOs.ApproachWe interviewed healthcare professionals about their awareness, attitudes, understanding and experiences of implementing three key approaches to patient engagement and activation: 1) goal-setting, 2) motivational interviewing and 3) shared decision making. Of the 60 clinicians, 33 were interviewed twice leading to 93 clinician interviews. Of the 8 managers, 2 were interviewed twice leading to 10 manager interviews. We used a thematic analysis approach to the data.Key resultsInterviewees recognised the term 'patient activation and engagement' and had favourable attitudes about the utility of the associated skills. However, in-depth probing revealed that although interviewees reported that they used these patient activation and engagement approaches, they have limited understanding of these approaches.ConclusionsWithout understanding the concept of patient activation and the associated approaches of shared decision making and motivational interviewing, effective implementation in routine care seems like a distant goal. Clinical teams in the ACO model would benefit from specificity defining key terms pertaining to the principles of patient activation and engagement. Measuring the degree of understanding with reward that are better-aligned for behaviour change will minimise the notion that these techniques are already being used and help fulfil the potential of patient-centred care

Ergopeptine-Sensitive Calcium-Dependent Protein Phosphorylation System in the Brain

We studied a protein phosphorylation system that is regulated by the dopamine-mimetic ergot bromocriptine. Bromocriptine was found to inhibit selectively the endogenous phosphorylation of a threonine residue(s) in 50,000- and 60,000-dalton proteins in a synaptosome fraction. The bromocriptine-sensitive phosphorylation is stimulated by calcium and by calmodulin, and occurs predominantly in the brain. The inhibitory effect of bromocriptine was not mimicked by 3,4-dihydroxyphenylethylamine or by any of the neurotransmitters and related agents tested, but was mimicked, although less effectively, by other ergots that contain peptide moieties. In the hippocampus, the brain region with the highest content of the 50,000- and 60,000-dalton proteins, the ergopeptine-sensitive protein phosphorylation appears to be localized to interneurons or cell bodies whose axons synapse outside the hippocampus. The results raise the possibility that some of the bromocriptine- and ergopeptine-induced pharmacological effects in the CNS may be mediated by the inhibition of the calcium/calmodulin-dependent phosphorylation of these specific proteins.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65208/1/j.1471-4159.1984.tb02758.x.pd

Inositol Lipids and Signal Transduction in the Nervous System: An Update

The role that inositol lipids play in cellular signaling events in eukaryotic cells remains one of the most intensively investigated areas of cell biology. In this respect, phosphoinositide-mediated signal transduction in the CNS is no exception; major advances have been made since a previous review on this subject (Fisher and Agranoff, 1987). Not only have stimulated phosphoinositide turnover and its physiological sequelae been demonstrated repeatedly in a variety of neural preparations, but, in addition, the detailed molecular mechanisms underlying these events continue to unfold. Here we review the progress that has occurred in selected aspects of this topic since 1987. In the first two sections of this article, emphasis is placed on novel functional roles for the inositol lipids and on recent insights into the molecular characteristics and regulation of three key components of the phosphoinositide signal transduction system, namely, the inositol lipid kinases, phospholipases C (PLCs), and the inositol 1,4,5-trisphosphate[I(1,4,5)P 3 ] receptor. The metabolic fate of I(1,4,5)P 3 in neural tissues, as well as its control, is also detailed. Later we focus on identification of the multiple receptor subtypes that are coupled to inositol lipid turnover and discuss possible strategies for intervention into phosphoinositide-mediated signal transduction. Due to space limitations, an extensive evaluation of the diacylglycerol/protein kinase C (DAG/PKC) limb of the signal transduction pathway is not included (for reviews, see Nishizuka, 1988; Kanoh et al., 1990).Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65626/1/j.1471-4159.1992.tb09273.x.pd

Hydrology of an Evaporite Aquitard Permian Evaporite

Permian evaporite strata of the Palo Duro Basin, Texas Panhandle, are considered to have low permeabilities but are capable of some leakage. Cross-sectional and areal modeling of regional groundwater flow in the Palo Duro Basin indicates leakage from the Upper aquifer through the Evaporite aquitard that contributes approximately 30 percent of the water in the underlying Deep-Basin Brine aquifer. The regional estimate of permeability of the aquitard (˜ 10^-4 md) is in general agreement with laboratory testing of salt permeability. Oxygen and hydrogen isotopic composition and the Cl/Br ratio of the deep-basin brines also suggest leakage through the aquitard. Isotopically nonequilibrated meteoric waters are found to depths of 8,000 ft. Leakage through the aquitard may occur as either matrix or fracture flow. According to petrographic and geochemical studies of the evaporite strata, halite dissolution and minor recrystallization occurred predominantly during Permian time. Post-Permian waters may have altered the top of the Evaporite aquitard. Fracturing is prevalent in the rock strata in the basin; the greatest density of fracturing occurs in areas with tectonic deformation. The potential for leakage may be greatest in these areas.Bureau of Economic Geolog