Feedback Control of Second Messengers Signaling Systems in White Adipose Tissue Adipocytes in Healthy State and Its Loss at Adiposity

Second messengers Ca2+, IP3, cAMP, NO, cGMP, and cADP ribose are incorporated as obligatory elements into multivariable Ca2+-signaling system, which integrates incoming signals of hormones and neurotransmitters in white adipocytes. This cross-controlled system includes two robust generators (RGs) of rhythmic processes, involving phospholipase C- and NO-synthase-dependent signaling networks (PLC-RG and NOS-RG). Multi-loop positive feedback control of both RGs provides their robustness, multistability, signaling interplay, and extreme sensitivity to the alterations of incoming signals of acetylcholine, norepinephrine, insulin, cholecystokinin, atrial natriuretic peptide, bradykinin, and so on. Hypertrophy of cultured adipocytes and of mature cells, isolated from epididymal white adipose tissue (eWAT), results in the loss of rhythmicity and development of general hormonal signaling resistance. Preadipocytes isolated from eWAT of obese mice cannot grow and accumulate lipids in the media devoid of fatty acids. However, even low concentrations of palmitoylcarnitine in the media (1 μM) may result in drastic suppression of mRNA expressions of the proteins of Ca2+-signaling system, especially of NOS-RG. Similar alterations of gene expression are observed in eWAT and liver at adiposity. All this may indicate on universal background pathogenic mechanisms. Treatment modalities, which may help to restore deregulation of Ca2+-signaling system and corresponding tissues dysfunction, are discussed briefly

Deregulation of Ca2+-Signaling Systems in White Adipocytes, Manifested as the Loss of Rhythmic Activity, Underlies the Development of Multiple Hormonal Resistance at Obesity and Type 2 Diabetes

Various types of cells demonstrate ubiquitous rhythmicity registered as simple and complex Ca2+-oscillations, spikes, waves, and triggering phenomena mediated by G-protein and tyrosine kinase coupled receptors. Phospholipase C/IP3-receptors (PLC/IP3R) and endothelial NO-synthase/Ryanodine receptors (NOS/RyR)–dependent Ca2+ signaling systems, organized as multivariate positive feedback generators (PLC-G and NOS-G), underlie this rhythmicity. Loss of rhythmicity at obesity may indicate deregulation of these signaling systems. To issue the impact of cell size, receptors’ interplay, and obesity on the regulation of PLC-G and NOS-G, we applied fluorescent microscopy, immunochemical staining, and inhibitory analysis using cultured adipocytes of epididumal white adipose tissue of mice. Acetylcholine, norepinephrine, atrial natriuretic peptide, bradykinin, cholecystokinin, angiotensin II, and insulin evoked complex [Ca2+]i responses in adipocytes, implicating NOS-G or PLC-G. At low sub-threshold concentrations, acetylcholine and norepinephrine or acetylcholine and peptide hormones (in paired combinations) recruited NOS-G, based on G proteins subunits interplay and signaling amplification. Rhythmicity was cell size- dependent and disappeared in hypertrophied cells filled with lipids. Contrary to control cells, adipocytes of obese hyperglycemic and hypertensive mice, growing on glucose, did not accumulate lipids and demonstrated hormonal resistance being non responsive to any hormone applied. Preincubation of preadipocytes with palmitoyl-L-carnitine (100 nM) provided accumulation of lipids, increased expression and clustering of IP3R and RyR proteins, and partially restored hormonal sensitivity and rhythmicity (5–15% vs. 30–80% in control cells), while adipocytes of diabetic mice were not responsive at all. Here, we presented a detailed kinetic model of NOS-G and discussed its control. Collectively, we may suggest that universal mechanisms underlie loss of rhythmicity, Ca2+-signaling systems deregulation, and development of general hormonal resistance to obesity

To Break or to Brake Neuronal Network Accelerated by Ammonium Ions?

The aim of present study was to investigate the effects of ammonium ions on in vitro neuronal network activity and to search alternative methods of acute ammonia neurotoxicity prevention.Rat hippocampal neuronal and astrocytes co-cultures in vitro, fluorescent microscopy and perforated patch clamp were used to monitor the changes in intracellular Ca2+- and membrane potential produced by ammonium ions and various modulators in the cells implicated in neural networks.Low concentrations of NH4Cl (0.1-4 mM) produce short temporal effects on network activity. Application of 5-8 mM NH4Cl: invariably transforms diverse network firing regimen to identical burst patterns, characterized by substantial neuronal membrane depolarization at plateau phase of potential and high-amplitude Ca2+-oscillations; raises frequency and average for period of oscillations Ca2+-level in all cells implicated in network; results in the appearance of group of «run out» cells with high intracellular Ca2+ and steadily diminished amplitudes of oscillations; increases astrocyte Ca2+-signalling, characterized by the appearance of groups of cells with increased intracellular Ca2+-level and/or chaotic Ca2+-oscillations. Accelerated network activity may be suppressed by the blockade of NMDA or AMPA/kainate-receptors or by overactivation of AMPA/kainite-receptors. Ammonia still activate neuronal firing in the presence of GABA(A) receptors antagonist bicuculline, indicating that «disinhibition phenomenon» is not implicated in the mechanisms of networks acceleration. Network activity may also be slowed down by glycine, agonists of metabotropic inhibitory receptors, betaine, L-carnitine, L-arginine, etc.Obtained results demonstrate that ammonium ions accelerate neuronal networks firing, implicating ionotropic glutamate receptors, having preserved the activities of group of inhibitory ionotropic and metabotropic receptors. This may mean, that ammonia neurotoxicity might be prevented by the activation of various inhibitory receptors (i.e. by the reinforcement of negative feedback control), instead of application of various enzyme inhibitors and receptor antagonists (breaking of neural, metabolic and signaling systems)

Regulation of Papillary Muscle Contractility by NAD and Ammonia Interplay: Contribution of Ion Channels and Exchangers

Various models, including stem cells derived and isolated cardiomyocytes with overexpressed channels, are utilized to analyze the functional interplay of diverse ion currents involved in cardiac automaticity and excitation–contraction coupling control. Here, we used β-NAD and ammonia, known hyperpolarizing and depolarizing agents, respectively, and applied inhibitory analysis to reveal the interplay of several ion channels implicated in rat papillary muscle contractility control. We demonstrated that: 4 mM β-NAD, having no strong impact on resting membrane potential (RMP) and action potential duration (APD90) of ventricular cardiomyocytes, evoked significant suppression of isometric force (F) of paced papillary muscle. Reactive blue 2 restored F to control values, suggesting the involvement of P2Y-receptor-dependent signaling in β-NAD effects. Meantime, 5 mM NH4Cl did not show any effect on F of papillary muscle but resulted in significant RMP depolarization, APD90 shortening, and a rightward shift of I–V relationship for total steady state currents in cardiomyocytes. Paradoxically, NH4Cl, being added after β-NAD and having no effect on RMP, APD, and I–V curve, recovered F to the control values, indicating β-NAD/ammonia antagonism. Blocking of HCN, Kir2.x, and L-type calcium channels, Ca2+-activated K+ channels (SK, IK, and BK), or NCX exchanger reverse mode prevented this effect, indicating consistent cooperation of all currents mediated by these channels and NCX. We suggest that the activation of Kir2.x and HCN channels by extracellular K+, that creates positive and negative feedback, and known ammonia and K+ resemblance, may provide conditions required for the activation of all the chain of channels involved in the interplay. Here, we present a mechanistic model describing an interplay of channels and second messengers, which may explain discovered antagonism of β-NAD and ammonia on rat papillary muscle contractile activity

Acetylcholine Promotes Ca²⁺and NO-Oscillations in Adipocytes Implicating Ca²⁺→NO→cGMP→cADP-ribose→Ca²⁺ Positive Feedback Loop - Modulatory Effects of Norepinephrine and Atrial Natriuretic Peptide

<div><p>Purpose</p><p>This study investigated possible mechanisms of autoregulation of Ca²⁺ signalling pathways in adipocytes responsible for Ca²⁺ and NO oscillations and switching phenomena promoted by acetylcholine (ACh), norepinephrine (NE) and atrial natriuretic peptide (ANP).</p><p>Methods</p><p>Fluorescent microscopy was used to detect changes in Ca²⁺ and NO in cultures of rodent white adipocytes. Agonists and inhibitors were applied to characterize the involvement of various enzymes and Ca²⁺-channels in Ca²⁺ signalling pathways.</p><p>Results</p><p>ACh activating M₃-muscarinic receptors and G_βγ protein dependent phosphatidylinositol 3 kinase induces Ca²⁺ and NO oscillations in adipocytes. At low concentrations of ACh which are insufficient to induce oscillations, NE or α1, α2-adrenergic agonists act by amplifying the effect of ACh to promote Ca²⁺ oscillations or switching phenomena. SNAP, 8-Br-cAMP, NAD and ANP may also produce similar set of dynamic regimes. These regimes arise from activation of the ryanodine receptor (RyR) with the implication of a long positive feedback loop (PFL): Ca²⁺→ NO→cGMP→cADPR→Ca²⁺, which determines periodic or steady operation of a short PFL based on Ca²⁺-induced Ca²⁺ release via RyR by generating cADPR, a coagonist of Ca²⁺ at the RyR. Interplay between these two loops may be responsible for the observed effects. Several other PFLs, based on activation of endothelial nitric oxide synthase or of protein kinase B by Ca²⁺-dependent kinases, may reinforce functioning of main PFL and enhance reliability. All observed regimes are independent of operation of the phospholipase C/Ca²⁺-signalling axis, which may be switched off due to negative feedback arising from phosphorylation of the inositol-3-phosphate receptor by protein kinase G.</p><p>Conclusions</p><p>This study presents a kinetic model of Ca²⁺-signalling system operating in adipocytes and integrating signals from various agonists, which describes it as multivariable multi feedback network with a family of nested positive feedback.</p></div

Suppressive and modulating effects of L-arginine, L-carnitine and acetyl-L-carnitine on neuronal networks activated by ammonium ions.

<p>Cultures 12–18 DIV. The records of representative cells (more than 90% of cells in culture) are presented. All other abbreviations and descriptions as on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134145#pone.0134145.g002" target="_blank">Fig 2</a>. <b>(A, B)</b> Modulating (A) and suppressive (B) effects of L-arginine (10 mM). Typical responses of cells in few (Fig A; 20%) and in most (80%; Fig B) of cultures studied. n = 10. Culture 12 DIV. N = 89 (for Fig A). N = 106 (for Fig B). <b>(C, D)</b> Typical suppressant effect of L-carnitine (10 mM; Fig C) and modulatory effect of acetyl-L-carnitine (10 mM; Fig D) in most of cultures studied (75 and 80%, of cultures. n = 4 and n = 5 correspondingly). Culture 16 DIV. N = 111 (for Fig C). Culture 18 DIV. N = 126 (for Fig D). 200 nM of L-glutamate was added before application of 8 mM NH₄Cl on Fig B, C, D.</p

Simultaneous recordings of membrane potential and of Ca²⁺-oscillations in «run out» cell and representative cell in network activated by 5 mM NH₄Cl.

<p>Neuronal culture 16 DIV. Total number of neuronal cells involved into network is 106. Gaps in the traces represent pauses in data recordings. Here are presented only parts of 3 records. Initial parts were omitted for simplicity. <b>(A)</b> Recording of Ca²⁺-oscillations in «run out» cell. <b>(B)</b> Recording of membrane potential in «run out» cell. <b>(C)</b> Recording of Ca²⁺-oscillations in representative cell (one of 95% cells monitored in network).</p

Transformation of simple and complex intracellular Ca²⁺-oscillations into high-amplitude impulse-shaped Ca²⁺-oscillations by NH₄Cl or bicuculline.

<p>Neuronal cultures 12 DIV. Resting calcium level is outlined by dot-dashed lines. Calcium increment over resting level (VCi = ΔCa (a.u.)/min) is indicated on the Figures as VCi. All other abbreviations as on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134145#pone.0134145.g001" target="_blank">Fig 1</a>. Total number of neuronal cells in networks are: N = 116, 132, 98, 110 for Fig A, B, C and D, correspondingly. <b>(A</b>–<b>C)</b> NH₄Cl induces high-amplitude Ca²⁺-oscillations in representative cells. 200 nM of L-glutamate was added before application of NH₄Cl. <b>(C)</b> The experiment was performed in the presence of 10 μM L-NAME and 200 nM of L-glutamate. <b>(D)</b> 10 μM of bicuculline evokes high-amplitude Ca²⁺-oscillations in spontaneously firing cell. Only parts of total records are presented on Fig B and C. Initial parts were omitted for simplicity.</p