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. 2013 May 16;8(5):e63483.
doi: 10.1371/journal.pone.0063483. Print 2013.

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

Egor A Turovsky  1 , Mariya V Turovskaya, Ludmila P Dolgacheva, Valery P Zinchenko, Vladimir V Dynnik
Affiliations

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

Egor A Turovsky et al. PLoS One. .

Abstract

Purpose: This study investigated possible mechanisms of autoregulation of Ca(2+) signalling pathways in adipocytes responsible for Ca(2+) and NO oscillations and switching phenomena promoted by acetylcholine (ACh), norepinephrine (NE) and atrial natriuretic peptide (ANP).

Methods: Fluorescent microscopy was used to detect changes in Ca(2+) and NO in cultures of rodent white adipocytes. Agonists and inhibitors were applied to characterize the involvement of various enzymes and Ca(2+)-channels in Ca(2+) signalling pathways.

Results: ACh activating M3-muscarinic receptors and Gβγ protein dependent phosphatidylinositol 3 kinase induces Ca(2+) 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 Ca(2+) 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): Ca(2+)→NO→cGMP→cADPR→Ca(2+), which determines periodic or steady operation of a short PFL based on Ca(2+)-induced Ca(2+) release via RyR by generating cADPR, a coagonist of Ca(2+) 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 Ca(2+)-dependent kinases, may reinforce functioning of main PFL and enhance reliability. All observed regimes are independent of operation of the phospholipase C/Ca(2+)-signalling axis, which may be switched off due to negative feedback arising from phosphorylation of the inositol-3-phosphate receptor by protein kinase G.

Conclusions: This study presents a kinetic model of Ca(2+)-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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. The changes of [Ca2+]i in adipocytes upon the effect of 5 µM ACh and inhibitors of mAChR.
A: 5 µM acetylcholine induces oscillations of cytosolic calcium in white adipocytes, as measured by Fura-2 ratio. Here and in later experiments the responses of individual adipocytes are presented. Number of cells in the experiments (n) = 253. B: adipocytes cultured for 24 hours in the presence of 100 ng/ml of pertussis toxin (PTX) are not capable of generating [Ca2+]i oscillations. 92±3,4% of the cells the response to ACh application is absent (grey curve), and the transient signal (black curve) is observed in 8±2,3% of the cells (n = 128). C: in the presence of 10 nM pF-HHSID there is no response to ACh in 90±2,8% of the cells, and the transient signal (black curve) is observed in 10±3,2% of the cells, n = 324. D: in the presence of 10 nM Telenzepine ACh causes [Ca2+]i oscillations in 88±9,4% of adipocytes, while in 13±1,5% of adipocytes the oscillations are absent, n = 8. E: in the presence of 10 nM Methoctramine hydrate the oscillations are observed in 89±3,8% of adipocytes (black curve), while 15±14,8% of the cells give no response, n = 200.
Figure 2
Figure 2. Role of reticular Ca2+ and PLC/IP3R signalling axis in Ca2+oscillations.
A: in the presence of 1 µM inhibitor of SERCA, Thapsigargin, 100% adipocytes do not respond to ACh (n = 160). B: in the presence of 3 µM U73122 the oscillations are observed in 77±10,2% of adipocytes (n = 152). C: in the presence of 1 µM Xestospongin C the oscillations are observed in 80±6,7% of adipocytes (n = 259). D: simultaneous incubation of the cells with 1 µM Xestospongin C and 3 µM U73122 does not suppress [Ca2+]i oscillations in 18±4,1% of adipocytes, which responded with sustained modes of oscillations to ACh (n = 165).
Figure 3
Figure 3. Involvement of PI3Kγ/PKB/eNOS in oscillations induced by ACh.
A: [Ca2+]i response to 5 µM ACh is absent in 84±9,4% of cells in presence 20 nM AS-605290 (PI3K inhibitor) and [Ca2+]i transitory response was recorded in 21±4,9% of cells, (n = 260). B: [Ca2+]i response to 5 µM ACh is absent in 89±7,9% of cells in the presence 10 µM LY-294,002 (PI3K inhibitor), while [Ca2+]i transitory response was recorded in 13±7,4% of cells, (n = 145). C: [Ca2+]i oscillations were inhibited by AKT1/2 (PKB inhibitor) in 68±5,5% cells, (n = 168). D: [Ca2+]i response to 5 µM ACh is absent in 82±8,4% of cells in presence of 1 µM L-NAME (eNOS inhibitor) and [Ca2+]i transitory response was recorded in 18±6,3% of cells, (n = 6).
Figure 4
Figure 4. The potentiation of [Ca2+]i oscillations by α-adrenergic agonists.
A: upon the application of NE (1 µM) in the presence of 10 nM ACh [Ca2+]i the onset of oscillations is observed in 55±5,8% of adipocytes, (n = 144). B: upon the application of 10 nM ACh in the presence of 5 µM NE the oscillations arise in 47±6,8% of the cells, all the rest showing either no response or a transient increase in [Ca2+]i, (n = 110). C: upon the application of NE (5 µM) in the presence of 10 nM M3-cholinergic receptor antagonist (p-F-HHSID) and 10 nM ACh, the transient increase of [Ca2+]i is observed in 73±8,0% of adipocytes, with no periodic modes of Ca2+ detected, (n = 150). The transient increase of calcium upon NE application is a classic demonstration of adrenergic receptor activation. D: the onset of fast Ca2+-oscillations in 37±5,4% of the cells upon application of 3 µM selective agonist of α1-adrenergic receptors, Phenylephrine, (n = 185). E: the generation of impulse shaped oscillations in 24±4,4% of the cells upon application of 3 µM Phenylephrine, (n = 115). F: the onset of relaxation type oscillations with long period in 08±3,4% of the cells, (n = 167). G: upon the application of 3 µM Phenylephrine against 1 nM ACh the Ca2+ signalling system switches to a new steady state with high values of [Ca2+]i n 07±2,1% of adipocytes, (n = 147). H: the transition to a new steady state with high mean values of [Ca2+]i for the oscillation period occurs in single adipocytes, (n = 18). I: the generation of impulse [Ca2+]i oscillations in 24±5,4% of the cells upon the application of 3 µM selective agonist of α2-adrenergic receptor, UK-14,304, against 1 nM Ach, (n = 138). In the absence of ACh (control experiment), upon the application of UK-14,304 adipocytes respond with a single impulse increase of [Ca2+]i, but they never respond with oscillations.
Figure 5
Figure 5. Minimal list of PFLs operating in Ca2+-signalling system (B).
Dasher blue numbered arrows indicate various PFLs appearing in the system and formed by Ca2+ induced Ca2+ release by RyR (1), phosphorylation of RyR by CaMKII (2) and phosphorylation of eNOS by Ca2+(3). For details see the text.
Figure 6
Figure 6. Breakdown of long PFL by eNOS and PKG inhibitors.
A: the changes in [Ca2+]i level (black curve) and NO accumulation in DAF-FM (ΔF/F0, blue curve) upon the application of 5 µM ACh after cell preincubation within 5 minutes with 5 µM inhibitor of PKG, KT5823, in 37±8,6% of cells, (n = 93). B: the effect of of PKG inhibitor, KT5823 (5 µM; curve 1), and KT5823+ inhibitor of NOS, L-NAME (1 mM; curve 2) added 5 minutes before the start of calcium dynamics registration in the cytosol, (n = 137). Slow increase of [Ca2+]i occurs in 51±19,4% of the cells (curve 1) and in 35±8,0% of cells (curve 2). C: the effect of PKG inhibitor, Rp-8-Br-cGMPS, (10 µM) which prevents the onset of periodic modes in all cells and results in a rise of Ca2+ in the cytosol in 30±4,5% of adipocytes (curve 1). The application of 5 µM ACh against a background of Rp-8-Br-cGMPS and NOS inhibitor, 7-NI (1 µM) leads to slow rise of Ca2+ in the cytosol in 11±3,2% of adipocytes (curve 2), n = 214.
Figure 7
Figure 7. The regimes of slow Ca2+ and NO oscillations induced by ACh.
The black curve (1) indicate integral NO level, accumulated by DAF (ΔF/F0), blue curve (2) corresponds to NO (d(ΔF/F0)/dt), the red curve (3) indicate changes in [Ca2+]i.
Figure 8
Figure 8. The induction of various modes of [Ca2+]i oscillations in adipocytes by the intermediates of long PFL(3).
A: upon application of 5 µM SNAP, a donor of NO, the generation of fast Ca2+ oscillations occurs in 34±9,6% of the cells (black curve) and slow high-amplitude Ca2+ oscillations (blue curve) are observed in 11±2,5% of adipocytes, (n = 208). B: the transition of the system to a steady oscillatory mode with a high mean value of Ca2+ for the oscillation period in 15±3,6% of the cells, (n = 97). C: the transition of the system to a new stable stationary state with a high level of calcium in 59±9,8% of the cells, (n = 105). D: [Ca2+]i oscillations with a period of several dozen seconds are induced in 43±6,1% of the cells by the application of 0,1 µM 8Br-cGMP, a permeating analog of cGMP, and are quickly suppressed by an inhibitor of ADPRC – Nicotinamide (NAM), (n = 132). E: [Ca2+]i oscillations with varying period of oscillations are induced in 26±11,0% of adipocytes by the application of 0.1 µM 8Br-cGMP and are quickly suppressed by NAM, (n = 116). F: oscillations caused by the application of 1 µM βNAD are observed in 57±10,3% of adipocytes and are quickly suppressed by nicotinamide (NAM) (n = 163). There are no [Ca2+]i oscillations in 43±8,6% of the cells (grey curve).
Figure 9
Figure 9. The onset of different modes of Ca2+ oscillations in adipocytes under the influence of ANP.
A: the application of 1 µM ANP results in the generation of fast Ca2+ oscillations in 38±10,5% of adipocytes. The addition of 1 mM nicotinamide (NAM) suppresses the periodic modes in all cells, (n = 272). B: the application of 10 µM ANP can, in addition to the typical fast [Ca2+]i oscillations, cause stochastic (chaotic) oscillations in 5±4,2% of adipocytes which are also suppressed with the addition of NAM, (n = 179). C: the effect of potentiation of 1 nM ACh with 1 µM ANP is observed in 43±10,7% of adipocytes, (n = 113). D: the application of 10 µM ANP against the background of oscillations induced by 5 µM ACh results in suppression of Ca2+ oscillations in 7±2,2% of adipocytes, (n = 154). The majority of cells do not demonstrate the suppression of oscillations.
Figure 10
Figure 10. Multivariable multi feedback network with a family of nested PFLs, controlling Ca2+-signalling pathway (B) and integrating the signals from ACh, NE and ANP.
Dashed blue numbered arrows indicate various PFLs operating in the system. Modulating axes, activating PKB and PKG are also presented. For details see the text.
Figure 11
Figure 11. Total kinetic model of Ca2+ signalling in adipopocytes with participation of Ca2+ signalling pathway (A) and (B).
Various types of activation are shown by dashed arrows or dashed lines with the icon (⌢). Various types of inhibition are shown by dashed stroke with the icon (T). Formed PFLs are shown in blue and NFLs in red. Numbered PFLs operating in the pathway (B) correspond to those on Fig. 10. PFLs operating in the pathway are indicated by symbols X and Y in the circles. The description of the schematic is given in the text.
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Publication types

Grants and funding

The study was conducted with the support of the Ministry of Science and Education of Russian Federation, State Contract №16.512.11.2092, Program №7 of Russian Academy of Sciences Presidium (project 01201256033), Program №7 of Russian Academy of Sciences Presidium (project 01201258223), Russian Foundation for Basic Research (project 10-04-01306-a). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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