Experimental data shows that the activation of this system produces inotropy, lusitropy, and chronotropy in the heart, such as increased magnitude and relaxation rates of [Ca2+]i transients and contraction force, and increased heart rhythm. However, excessive stimulation of β1-adrenergic receptors leads to heart dysfunction and heart failure. In this paper, a comprehensive, experimentally based mathematical model of the β1-adrenergic signaling system for mouse ventricular myocytes is developed, which includes major subcellular functional compartments . The model describes biochemical reactions that occur during stimulation of β1-adrenoceptors, changes in ionic currents, and modifications of Ca2+ handling system. Simulations describe the dynamics of major signaling molecules, such as cyclic AMP and protein kinase A, in different subcellular compartments; the effects of inhibition of phosphodiesterases on cAMP production; kinetics and magnitudes of phosphorylation of ion channels, transporters, and Ca2+ handling proteins; modifications of action potential shape and duration; magnitudes and relaxation rates of [Ca2+]i transients; changes in intracellular and transmembrane Ca2+ fluxes; and [Na+]i fluxes and dynamics. The model elucidates complex interactions of ionic currents upon activation of β1-adrenoceptors at different stimulation frequencies, which ultimately lead to a relatively modest increase in action potential duration and significant increase in [Ca2+]i transients. In particular, the model includes two subpopulations of the L-type Ca2+ channels, in caveolae and extracaveolae compartments, and their effects on the action potential and [Ca2+]i transients are investigated. The presented model can be used by researchers for the interpretation of experimental data and for the developments of mathematical models for other species or for pathological conditions.The β While early experimental studies of cardiac cells are predominantly devoted to their electrical activity, and later to Ca2+ dynamics, more recent studies involve investigations of protein signaling systems, which modulate both action potentials and intracellular Ca2+ transients 2+ handling system 1-adrenergic signaling system Cardiac cells belong to a wide class of excitable cells which include electrical activity, Ca1-adrenergic system in ventricular myocytes of different species 1-adrenergic signaling system. The model included biochemical and electrophysiological parts with two major protein kinase A targets, phospholamban and the L-type Ca2+ channel, and consisted of one cytosolic compartment (single-compartment model). Later, a similar model was developed for rabbit ventricular myocytes and included several new PKA targets: ryanodine receptors, troponin I, and slow delayed rectifier K+ current, IKs1-adrenergic signaling system in mouse ventricular myocytes, predominantly on Ca2+ dynamics 1-adrenergic signaling system in guinea pig ventricular myocytes 2+]i transients, and ionic fluxes upon stimulation of β1-adrenoceptors with agonist isoproterenol.Mathematical modeling of protein signaling networks is a supplementary tool for understanding their functions in the heart. Recently, particular attention has been paid to the development of comprehensive models for β1-adrenergic signaling system, were developed 1-adrenergic and M2-muscarinic signaling systems and described the dynamics of cAMP and PKA in different subcellular compartments . The only compartmentalized model of cardiac protein signaling network, which includes both biochemical and electrophysiological parts, β1- and β2-adrenergic and CaMKII-mediated signaling systems, was developed recently for canine ventricular myocytes by Heijman et al. Simultaneously, multi-compartmental models of protein signaling networks, including the β2+ dynamics, and cellular contraction . The model elucidated the mechanism of relatively moderate AP prolongation, significant increase in intracellular [Ca2+]i transients, modification of the intracellular and transmembrane Ca2+ fluxes, and [Na+]i fluxes and dynamics. The model includes two pools of the L-type Ca2+ channels, one in the caveolae and the other in the extracaveolae compartments. The simulations demonstrated their different modulations upon stimulation of β1-ARs and their different effects on the action potentials and [Ca2+]i transients. The model simulated frequency dependences of [Ca2+]i transient decays for control conditions and after stimulation of β1-ARs, and made testable predictions for the frequency dependences of [Ca2+]i transient amplitudes, AP amplitudes and durations. The simulation results are compared to the results obtained from other models of β1-adrenergic signaling system in other species, and the model limitations are discussed.In this paper, we developed a new compartmentalized model for the β1-adrenergic signaling system in mouse ventricular myocytes is a natural extension of the previously published model for action potential and Ca2+ dynamics in mouse ventricular myocytes 1-adrenergic signaling pathway in our model of electrical activity and Ca2+ handling with modifications A mathematical model for the β1-adrenergic signaling system we consider three major functional compartments: caveolae (cav), extracaveolae (ecav), and cytosol (cyt) 2+ concentration does not directly depend on the β1-adrenergic signaling system, we use the same compartmentalization of Ca2+ handling as in Bondarenko et al. ss) of the Ca2+ handling system is completely located in the extracaveolae compartment of the β1-adrenergic signaling system.Our model cell consists of several compartments . For theTotal protein concentrations and their activities are normalized to the cell volume as in 1-adrenergic signaling system is activated by agonist (isoproterenol) , which produce cyclic AMP. cAMP is degraded by phosphodiesterases, three isoforms of which, PDE2, PDE3, and PDE4, are included in our model. Balanced activities of ACs and PDEs establish steady-state levels of cAMP in different compartments. cAMP further activates protein kinase A holoenzyme, which consists of two regulatory and two catalytic subunits. Binding four cAMP molecules to PKA holoenzyme causes dissociation of two catalytic subunits that phosphorylate target proteins, among them are PDE3 and PDE4. β1-ARs are phosphorylated by PKA, as well as by G protein coupled receptor kinase of type 2 (GRK2). PKA is also regulated by heat-stable protein kinase inhibitor (PKI). Intracellular proteins are dephosphorylated by two types of phosphatases, protein phosphatase 1 and 2A. PKA target proteins are located in different compartments. In our model, 20% of the L-type Ca2+ channels , the fast Na+ current, INa, and the phospholemman, which regulates the Na+-K+ pump, INaK, are localized in the caveolae compartment; 80% of the L-type Ca2+ channels, the ryanodine receptors, RyRs, the ultra-rapidly activating delayed rectifier K+ current, IKur, the rapidly inactivating transient outward K+ current, IKto,f, and the time-independent K+ current, IK1, are localized in the extracaveolae compartment; and phospholamban and troponin I are localized in the cytosolic compartment (more details are shown below).In all compartments, the βterenol) . Stimula1-adrenergic receptors are located in non-caveolae fractions. The estimated total concentration of β1-ARs in mouse ventricular myocytes is 0.0103 µM 1-ARs almost evenly between the extracaveolae and cytosolic compartments, with only 1% located in the caveolae compartment  β1-ARs in the caveolae compartment, 1-ARs with bound ligand L (concentration [L]), 1-ARs with bound ligand L and stimulatory G-protein Gs, 1-ARs with bound Gs, 1-ARs, Gs, and Gs.In order to derive algebraic equations which describe ligand-receptor and G-protein-receptor interactions in the caveolae compartment (equations (A.7)-(A.11) in the + and k− are the forward and backward rates, respectively, and K is the dissociation constant (K = k−/k+). The complex formation is described by the differential equation d[AB]/dt = k+[A]·[B] − k−[AB], where [A], [B], and [AB] are the concentrations of the substances A, B, and complex AB, respectively. In steady state, d[AB]/dt = 0, therefore, k+[A]·[B] − k−[AB] = 0. From the last equation, we can determine the concentration of complex AB in the steady-state approximation: [AB] = [A]·[B]/(k−/k+) = [A]·[B]/K.Concentrations of complexes Substitution of the equation for s-proteins, with dissociation of Activation of G1-ARs occurs by both protein kinase A and G-protein-coupled receptor kinase of type 2, GRK2 1-ARs both in ligand- and G-protein-bound states and unbound states, while GRK2 phosphorylation affects only ligand-bound states 1-ARs agonist isoproterenol increased the phosphorylation level of β1-ARs by about 108%  of βout 108% , which i by H-89 . Our mod1-adrenergic pathway, adenylyl cyclases are responsible for synthesis of cAMP from ATP. Our model includes four major types of adenylyl cyclases (AC) found in mouse ventricular myocytes, AC4, AC5, AC6, and AC7 In a βsα, sβγ, according to the experimental data We simulated activation of adenylyl cyclases by the α-subunit of G-protein, Gsαsα is compared to the experimental data for AC4 sβγ. Both figures show good agreement between the experimental and simulated results.1-adrenoceptor agonist isoproterenol on adenylyl cyclase activity in mouse ventricular myocytes , 5th min, and 30th min). It is shown that the AC activity decreases in time, reflecting β1-ARs desensitization (phosphorylation by PKA and GRK2)  found in mouse ventricular myocytes Phosphodiesterases in the βPDE2, PDE3, and PDE4 are activated by cAMP molecules, but with different affinities see . In addiThe effects of the PDE inhibitor IBMX on cAMP production in ventricular myocytes can be estimated by the levels of cAMP at different experimental conditions. 1-adrenergic signaling system which phosphorylates ion channels and proteins of the Ca2+ handling system. Inactive PKA holoenzyme consists of two regulatory subunits R  and two catalytic subunits C. Activation of PKA holoenzyme requires binding four cAMP molecules, in general, with different affinities. PKA holoenzyme with bound four cAMP molecules dissociates two catalytic subunits, which phosphorylates target proteins. In our model, we consider identical affinities for all four binding sites of PKA. Two isoforms of PKA holoenzyme are found in mouse ventricular myocytes, PKAI and PKAII, which differ by their regulatory subunits. PKAI is predominantly located in the cytosolic compartment, and PKAII is the predominant isoform in the caveolae and extracaveolae compartments.cAMP molecules generated in cardiac myocytes by adenylyl cyclases and partially hydrolyzed by phosphodiesterases activate protein kinase A, a major signaling molecule in the βPKAIK and PKAIIK. Some experimental data suggest half-activation constants for PKAIK ∼ 0.1 µM PKAIIK ∼ 0.5 µM PKAIK and PKAIIK in the range ∼ 1.5–3 µM PKAIK and PKAIIK in the nanomolar range were obtained in some experiments because they used free regulatory subunits RI or RII instead of PKA holoenzymes. Experimental data with PKA holoenzymes yielded significantly larger dissociation constants, which are used in our model  on the PKA activity is shown in 2+ current is inhibited by both PP1 and PP2A, and that the portion of the L-type Ca2+ channels and PP2A co-localize with caveolin-3, suggesting caveolae localization for both PP1 and PP2A. PP1 and PP2A are also localized in the cytosolic compartment, as they interact with phospholamban and troponin I There are two major types of protein phosphatases which are important for cardiac myocyte function, protein phosphatase 1 (PP1) and 2A (PP2A). Localization of PP1 and PP2A in three subcellular compartments can be determined by their co-localization with caveolin-3 and by modulation of their targets. Data of Hescheler et al. 1-adrenergic function Inhib1K = 1 nM), we used steady-state approximation to describe I-1-PP1 interaction with corresponding mass conservation relationships:In ventricular myocytes, protein phosphatase 1 is regulated by endogenous inhibitor-1 (I-1) 1-adrenergic signaling system by 1 µM isoproterenol on inhibitor-1 activity. As there is no data for mice, we used the experimental data from guinea pig hearts 1-adrenergic signaling system. It moves freely between cellular compartments and has PKA as a major target. cAMP concentration is determined by the balance between cAMP production by adenylyl cyclases, cAMP degradation by phosphodiesterases, and cAMP diffusion between intracellular compartments  (see (A.131)-(A.133) in cAMP is a one of the major signaling small molecules in the β1-adrenergic signaling system results in an increase of cAMP production by adenylyl cyclases and cAMP degradation by phosphodiesterases in the caveolae, extracaveolae, and cytosolic compartments. 1-ARs desensitization.Stimulation of the βOur model is able to reproduce the time course of the cellular PKA activity, which is determined by the cellular concentration of catalytic subunit  is the sarcolemmal Ca2+ pump, INaCa is the Na+/Ca2+ exchanger, ICab is the Ca2+ background current, INa is the fast Na+ current, INab is the Na+ background current, INaK is the Na+-K+ pump, IKto,f is the rapidly recovering transient outward K+ current, IK1 is the time-independent K+ current, IKur is the ultrarapidly activating delayed rectifier K+ current, IKss is the noninactivating steady-state voltage activated K+ current, IKr is the rapid delayed rectifier K+ current, ICl,Ca is the Ca2+-activated chloride current, and Istim is the stimulus current.Electrical activity of the mouse ventricular myocytes is described by the equation for transmembrane potential CaL, INa, IKto,f, and IKur) as the substrates of the β1-adrenergic signaling system. The Na+/K+ pump is affected by β1-ARs through phosphorylation of phospholemman. One more current, IK1, is also affected by β1-ARs; however, in the voltage range from about −80 to +40 mV, this current does not change significantly. In addition, there are three other phosphorylation substrates, which are the major players in Ca2+ dynamics and are affected by β1-ARs: ryanodine receptors, phospholamban, and troponin I. We consider all these β1-adrenoceptor substrates below as separate modules in the model.In our model, we consider four of these currents  in the new model  and a steady-state inactivation relationship in mouse ventricular myocytes CaLp  compared to the channels in the caveolae compartment (1.42 folds at 1 µM Iso). If the caveolae-located ICaL is responsible for ∼20% of the total current amplitude in control, after exposure to isoproterenol (1 µM) this fraction decreases to ∼15%. The results of simulations are in line with the experimental data of CaL from the population of channels in the caveolae compartment comprises about 15% of the total current.In addition to the time course of Ioterenol . We eval1-adrenergic signaling system, our model is tested in respect to the effects of inhibition of phosphatase activity on the L-type Ca2+ current. CaL traces under heavy buffer conditions (with suppression of Ca2+-induced Ca2+ release) elicited by voltage pulses to 0 mV in control (solid line) and after 1000-s exposure to 1 µM Calyculin A (dashed line) and 600-s exposure to 1 µM isoproterenol (dotted line). In simulations, we consider that Calyculin A inhibits 65% of the activity of both PP1 and PP2A. As seen from simulations in 2+ current. Fitting simulated time courses from CaL with two time constants. Fast (Ca2+-dependent inactivation) and slow (voltage-dependent inactivation) time constants are equal to 7.77 and 22.0 ms, 7.19 and 19.9 ms, and 6.54 and 18.5 ms for control (solid line), after 1000-s exposure to 1 µM Calyculin A (dashed line), and 600-s exposure to 1 µM isoproterenol (dotted line), respectively. These data show significant increase in Ca2+-dependent inactivation of ICaL upon stimulation of the β1-adrenergic signaling system. Similar behavior of ICaL is also observed experimentally with BAPTA in pipette solution 2+-induced Ca2+ release. In the last case, current traces also show bi-exponential decay with the fast and slow time constants 4.83 and 12.4 ms, 4.72 and 12.0 ms, and 4.66 and 12.8 ms for control (solid line), after 1000-s exposure to 1 µM Calyculin A (dashed line), and 600-s exposure to 1 µM isoproterenol (dotted line), respectively. These data show significant increase in the L-type Ca2+ current inactivation with intact Ca2+ dynamics. Simulations also show that activation of β1-ARs increases peak current amplitudes by about two-fold , results in additional small shift in the G/Gmax and steady-state inactivation Na, and the addition of PP2A to pipette solution causes a decrease in INa, which link phosphorylation level of INa to its magnitude. Mechanism of the changes in INa was studied by Zhou et al. + channels in the cell membrane is due to the channel trafficking. More recent analysis of the effects of PKA on INa suggested that phosphorylation and trafficking of INa are related processes: channels’ phosphorylation increases trafficking of fast Na+ channels Activation of the βNa, based on the model CaL, but with some differences. We consider that the amplification of INa occurs through two processes: phosphorylation of the channels and their subsequent trafficking to the cell membrane. As the kinetics of the two processes are indistinguishable in the experiments, we use only one rate constant to characterize the effect of PKA, kphos,tr, which includes both channel’s phosphorylation and trafficking. Two activation pathways are used, one is for non-phosphorylated channels and another is for phosphorylated-trafficked channels  or in close proximity to those couplons ss and the rest are very close to dyads, which suggests their involvement in Ca2+-induced Ca2+ release Experimental studies of ryanodine receptors in rat ventricular myocytes provided evidence of their co-localization in couplons with the L-type CaCaL and INa, we developed a Markov model that consists of two populations of the channels, non-phosphorylated and phosphorylated. For non-phosphorylated channels, we employed our previously developed Markov model for RyRs, which contains two closed (C1 and C2) and two open (O1 and O2) states 1p and C2p) and two open phosphorylated (O1p and O2p) states to the model and the transitions between corresponding non-phosphorylated and phosphorylated states , which increased sensitivity of RyRs by an order of magnitude compared to normal physiological conditions  with the data shown by filled squares  and simulated (solid line) data at relatively large cytosolic Ca2+ concentrations can be due to the method of estimation of the opening probabilities: in the simulations, we used the maximum values of the channel’s opening probabilities, without consideration of the effects of ryanodine receptor inactivation, while in the single-channel experiments, such inactivation is always present and can potentially reduce measured opening probability.In addition, the model simulated dependence of the RyRs open probability as a function of intracellular Cachannels . The exp+-K+ pump shows that ∼30–40% of α1-subunits and 80–90% of β1-subunits are localized in the caveolae-rich membrane fractions +-K+ pump and caveolin-3 co-localization, where ∼70% of those proteins are found in the same fractions +-K+ pump activity was found in caveolae Experimental data on the localization of the Na+-K+ pump, is the third substrate of the β1-adrenergic signaling system located in the caveolae compartment. Activation of β1-ARs increases the function of the Na+-K+ pump by phosphorylation of phospholemman (PLM) through the effective change in dissociation constant Km,Nai+]i concentration +]i in mouse ventricular myocytes after application of 1 µM isoproterenol is shown in + half-saturation constant for the current and effectively increases the pumping rate of Na+ outside the cell. Both time dependences demonstrate similar behavior.In our model, the phospholemman, which regulates the NaNaK as a function of isoproterenol concentration obtained from guinea pig ventricular myocytes , to the extracaveolae compartment.The other substrate of PKA in the extracaveolae compartment in our model is the rapidly recovering transient outward K1-adrenergic signaling system on IKto,f in cardiac cells to1 by isoproterenol (0.1 µM) in human left and right atrial myocytes by about 40–50%. Similar inhibition is obtained in neural cells + current, IA, which is also encoded by Shal-type K+ channels (Kv4.2), after application of 8-br-cAMP (100 µM), and Schwann cells Kto,f at saturating concentrations of isoproterenol  or db-cAMP (10 µM) obtained in Schwann cells Kto,f in control and after application of 10 µM isoproterenol are shown in 1-ARs. A similar level of inhibition of IA is obtained by activation of PKA with saturating concentrations of 8-br-cAMP (100 µM) in neural cells to1 after application of saturating concentrations of isoproterenol in human left and right atrial cells max (13 mV) and steady-state inactivation (6 mV) for isoproterenol-stimulated compared to control cells  is the second substrate that is phosphorylated by PKA in the cytosolic compartment 1-adrenergic signaling system. Upon application of isoproterenol, both experimental data and simulations show saturating TnI phosphorylation levels within 2 minutes of stimulation  with the frequencies from 1 to 5 Hz . Voltage-clamp protocols for ionic currents are described in corresponding figure legends.The resulting model contains 141 ordinary differential equations solved by a fourth-order Runge-Kutta method, with different time steps. A relatively small time step of 0.000002 ms is used during the 10 milliseconds after the initiation of the stimulus current; the rest of the time, the time step is 0.0001 ms. Such small time steps are mainly determined by the very fast activation time constants of ryanodine receptors 2+ dynamics in an apical mouse cardiac cell 1-adrenergic signaling system by using, in considerable part, the methodology from the previously published models 1-adrenergic signaling system. While the model of Hejiman et al. 1-adrenergic signaling system, it is developed for larger species (dog) and is verified with a different set of experimental data. In particular, our model differs from the Hejiman et al. model 1-adrenergic signaling system; 4) the model includes additional modulation of adenylyl cyclases by βγ-subunits of stimulatory G-protein, Gs; 5) the model data is compared in significant part with both absolute and relative magnitudes of the activities of major signaling molecules of the β1-adrenergic signaling system; 6) the model includes two subpopulations of the L-type Ca2+ channels located both in the caveolae and extracaveolae compartments; 7) ryanodine receptors are localized in the extracaveolae compartment. Our model also differs from the recently published Yang and Saucerman model 1-adrenergic signaling system; 2) inclusion of different types of adenylyl cyclases (AC4–7) and phosphodiesterases (PDE2–4), their compartmentalization and specific regulation by drugs ; 3) multicompartmental distribution of protein kinase A targets and the possibility of their separate regulation by drugs; 4) the effects of β1-aderenergic signaling system not only on Ca2+ dynamics, but also on action potential.In this paper, our model for action potential and Ca1-adrenergic signaling system with isoproterenol. In non-stimulated cells, cAMP concentrations in caveolae, extracaveolae, and cytosol are equal to 0.2534 µM, 0.5079 µM, and 0.4078 µM, respectively. Such steady state concentrations are determined by the balance between cAMP production by adenylyl cyclases, cAMP degradation by phosphodiesterases, and cAMP fluxes between the compartments. The steady-state concentrations of cAMP were obtained by running the model equations without electrical stimulations until changes in each variable did not exceed 0.01%. The steady-state distribution of cAMP concentrations are qualitatively similar to the values obtained by Iancu et al. First, we investigated the behavior of cAMP and PKA catalytic subunit concentrations in different cellular compartments in response to the activation of the β1-ARs. The fastest cAMP transient is observed in the caveolae compartment because of the larger relative activities of ACs and PDEs in this compartment . Note that the cellular cAMP concentration transient has a somewhat smaller amplitude due to its normalization to the whole cell volume cellV.ment see . In cavesα) in all subcellular compartments (time-to-peak is ∼10 s). This process is accompanied by activation of phosphodiesterases, with some delay, due to the rising cAMP concentration and phosphorylation of PDEs by catalytic subunits of PKA. The interaction between ACs and PDEs reaches quasi-steady-state by ∼200 s, when their activities become close to each other. The slower part of cAMP transient is determined by the desensitization of β1-ARs when the concentration of Gsα decreases due to its smaller production and faster re-association with Gsβγ to restore Gsαβγ. In addition to the interaction of ACs and PDEs, cAMP fluxes between the subcellular compartments contribute to cAMP transients  and the level of cAMP in each compartment . Concentt = 0 s with simultaneous selective inhibition of PDE3 (90% inhibition) leads to an increase in peak cAMP transient by about 30%. In contrast, simultaneous application of isoproterenol and selective inhibition of PDE4 (90% inhibition) increases peak cAMP value by about ∼150%. This result points to the primary role of PDE4 in cAMP degradation compared to PDE3. Similar results were obtained experimentally in rats In addition to control conditions, we also simulated the effects of PDE3 and PDE4 inhibition on cAMP dynamics . As thert = 200 s for 30 s , and then a pulse of 0.1 µM isoproterenol was applied at 1-adrenergic signaling system affects action potential shape and duration stim = 80 pA/pF, τstim = 1.0 ms) at frequencies 1 and 5 Hz for 300 s without and with application of 1 µM isoproterenol. 50 and APD90 are prolonged by 21.67% and 14.90%, respectively. The simulated data for APD50 prolongation is close to the corresponding experimental data, 13.5±4% 90 prolongation is within the experimental data variation . The L-type Ca2+ current, ICaL, increases almost twice upon stimulation of β1-ARs, and promotes AP prolongation. There is also a decrease in the amplitude of the rapidly recovering transient outward K+ current, IKto,f, which also contributes to AP prolongation. An increase in the amplitude of the ultrarapidly activating K+ current, IKur, is the major opposing effect to AP prolongation, resulting in a relatively small total effect. At 90% of repolarization, four other, relatively small currents contribute to repolarization: the Na+/Ca2+ exchanger current, INaCa, the time-independent K+ current, IK1, the Na+/K+ pump, INaK, and the Na+ background current, INab. . Simulation data shows a correct trend of APD50 prolongation, which increases with stimulation frequency. However, simulated prolongation of APD90 at 5 Hz-stimulation shows somewhat smaller magnitude compared to 4 Hz-data of Tong et al. Simulation data obtained at stimulation frequency 5 Hz , F–J shoectively . These p50 changes from 3.0 to 3.3 ms and APD90 changes from 25.5 to 27.0 ms when stimulation frequency increases from 1 to 5 Hz. Upon exposure to isoproterenol, APD50 increases from 3.65 to 4.2 ms and APD90 ranges from 29.3 to 30.7 ms during the same increase in stimulation frequency. Prolongation of cardiac action potential durations upon application of isoproterenol is consistently observed experimentally in mouse ventricular myocytes We have also simulated the effects of isoproterenol on action potential durations (APDs) at the frequencies ranged from 1 to 5 Hz. Simulation data shows relatively small increase in APDs at different levels of repolarization with stimulation frequency. For example, without isoproterenol, APD2+]i transients obtained from mouse ventricular myocytes  increase in intracellular i transients and intracellular [Na+]i concentration for control conditions and after application of 1 µM isoproterenol upon stimulation with frequency 1 Hz. In control, [Ca2+]i transients approach quasi-steady-state values after a short-time initial decay (negative staircase effect). After application of isoproterenol, [Ca2+]i transients relatively rapidly increase in amplitude during the first ∼300 seconds, and then they slowly decay. Such behavior of [Ca2+]i transients is similar to the experimental data obtained by Despa et al. 2+]i transients is due to the rapid activation of β1-adrenoceptors and cAMP accumulation; relatively slow decay of peak [Ca2+]i is due to β1-adrenoceptors desensitization. In addition, Despa et al. 1-ARs decreases intracellular [Na+]i concentration due to an increased function of the Na+-K+ pump. Our simulation data reproduced this behavior. In control, stimulation of the model cell leads to a slight increase in [Na+]i to saturating values  are shown in + enters the cell through the fast Na+ channels, Na+/Ca2+ exchanger, and background mechanisms , 9.3 µM of Na+ through the Na+/Ca2+ exchanger (16%), and 45.5 µM of Na+ through the background mechanism (77.5%), giving total Na+ influx of 58.6 µM. After application of 1 µM isoproterenol and 1 Hz stimulation, the total Na+ influx into the cell increases to 64.0 µM, resulting in proportional changes of Na+ influx through the fast Na+ channels , increased influx through the Na+/Ca2+ exchanger , and unchanged Na+ influx through the background mechanism . At the larger stimulation frequency (5 Hz), the contribution of the different Na+ entry mechanisms changes. Without isoproterenol, 3.6 µM of Na+ enter the cell during action potential through the fast Na+ channels , 5.8 µM of Na+ through the Na+/Ca2+ exchanger (32%), and 8.9 µM of Na+ through the background mechanism (48%), yielding total Na+ influx of 18.3 µM. Application of 1 µM isoproterenol increases the total Na+ influx into the cell to 24.4 µM, resulting in an increase of Na+ influx through the fast Na+ channels  and the Na+/Ca2+ exchanger , and unchanged Na+ influx through the background mechanism .Experimental data shows the effects of the β as well . In addi+ fluxes and dynamics, and their modifications by the β1-adrenergic signaling system. The model data shows an increased fraction of voltage-dependent Na+ entry into the cell at higher stimulation frequencies and the shift of the balance of the Na+ fluxes upon application of isoproterenol towards outside the cell due to an increased function of the Na+-K+ pump.Thus, our simulations allow for the estimation of Na2+ channels, in caveolin-3-rich and caveolin-3-free membrane fractions, affects differently [Ca2+]i transients and cellular contraction 2+ channels. While the experiments show a 10% decrease in the mean values of the peak of [Ca2+]i transients in the myocytes with blocked caveolae-located L-type Ca2+ channels compared to control, such decrease did not reach statistical significance. Similar small, but not significant, decrease was also observed in myocyte contraction.Experimental data shows that two different populations of the L-type Ca2+ channel block in different cellular compartments on the action potential and [Ca2+]i transients in control and after application of 1 µM isoproterenol  is blocked (APD50 decreased by only 2%), whereas a much larger change in the AP is observed during block of ICaL in the extracaveolae compartment  (APD50 decreased by 14%)  decreases [Ca2+]i transients, similar to the experimental finding by CaL,ecav block such transient is not present (We used the model to investigate the effects of the L-type Caoterenol . The mod by 14%) , A. The CaL,ecav , B. The  present , C.CaL,cav only slightly decreased the total cellular ICaL . More significant effect of ICaL,cav block on [Ca2+]i transients is predicted by simulations with isoproterenol than for control conditions (a decrease by 28%), and [Ca2+]i transients is almost abolished with the ICaL,ecav block , which are different in their cellular localization and function. Experimental data from rat ventricular myocytes by Rybin et al. 1-adrenergic receptors are distributed between caveolae, non-caveolae membrane fractions, and internal membranes, with the majority of the β1-adrenergic receptors being found outside of the caveolae compartment. In contrast, β2-adrenergic receptors (β2-ARs) are localized in the caveolae compartment 1-adrenergic receptors in the caveolae compartment, with the majority of them distributed between the extracaveolae and cytosolic compartments. Such localization suggests different functional consequences: selective activation of β1-adrenergic receptors with isoproterenol in mouse ventricular myocytes leads to a significant increase in [Ca2+]i transient and myocyte contraction, while selective activation of β2-adrenergic receptors with zinterol does not have any effect on myocyte contractility 1-adrenergic receptors also results in phosphorylation of major cytosolic proteins, such as phospholamban and troponin I, while stimulation of β2-adrenergic receptors does not produce this effect The β-adrenergic signaling system plays a significant role in the function of the heart. There are two types of β-adrenergic receptors in cardiomyocytes , five others  are predominantly expressed in non-caveolae membrane domains, and phospholamban and troponin I are located in the cytosol (see Methods for details). It is remarkable that one of the PKA targets, the L-type Ca2+ channel, is expressed in both the caveolae and extracaveolae compartments, with different a physiological role in each domain. In addition to the different distribution of two major protein kinase A isoforms (PKAI and PKAII), there is also a different localization of the major dephosphorylation proteins (protein phosphatase 1 and protein phosphatase 2A). Both PP1 and PP2A are found in the caveolae and cytosolic compartments, however, only PP1 plays an important functional role in the extracaveolae. Such distribution of the targets results in their different phosphorylation kinetics and magnitude, producing a very complex interaction relationship in the effects on action potential and [Ca2+]i transients.Finally, the major protein kinase A phosphorylation targets are also localized in different subcellular compartments. Two of them are found predominantly in the caveolae compartment . Several non-compartmentalized models were developed for β1-adrenergic signaling system in different species, including mice 1-adrenergic receptors on electrical activity and ionic homeostasis. However, as the experimental technique is improving and the new data on subcellular organization of signaling systems is accumulating, more comprehensive models are required for more precise description of the cellular functions. Such important findings include differential localization of the isoforms of the major signaling proteins in the β1-adrenergic signaling system: adenylyl cyclases, phosphodiesterases, and protein kinase A 2+ channels, the major players in cardiac excitation-contraction coupling, and their differential physiological role in cellular function highlights the needs in compartmentalized models of cardiac myocytes 1- and β2-adrenergic, and CaMKII-mediated signaling systems 2+]i transients. Our model of the β1-adrenergic signaling system consists of some elements of the model of Heijman et al. 2+ channels based on the most recent experimental data 1-adrenergic signaling system is clear – separation of the effects of different signaling proteins – the particular role of the signaling molecules in the mechanisms of regulation of the target proteins still needs to be determined.Our model of the β1-adrenergic signaling system moderately prolongs action potentials duration in mouse ventricular myocytes  before and after activation of the β1-adrenergic signaling system is 36 µM and 61 µM, respectively. This estimation correlates with about a two-fold decrease in the time constant of [Ca2+]i relaxation  related to the physiological role of RyR phosphorylation in cardiac function 1-adrenergic signaling system in mouse ventricular myocytes is able to reproduce experimental data on a decrease in [Na+]i concentration upon activation of β1-ARs . With application of isoproterenol, the fraction of Na+ entry decreases for the fast Na+ current to 17%, increases to 46% for the Na+/Ca2+ exchanger, and decreases to 37% for the background mechanism. These fractions are different from those estimated for the larger species, such as rabbits + enter the cell through the fast Na+ current (which is similar to our estimations for mice), 60% through the Na+/Ca2+ exchanger, and 18% through the background mechanisms, suggesting the Na+/Ca2+ exchanger to be the major player in the Na+ transport into the cell. Further experiments are necessary to verify Na+ fluxes structure in mouse ventricular cells without and with application of isoproterenol estimated from the presented model.Our model of the βf β1-ARs . We also1-adrenergic signaling system, it has some limitations. One of these limitations is that some experimental data used for the model verification were obtained from different species, such as rats, canine, and rabbits. In this respect, some of the model parameters can be adjusted to fit the data obtained from mouse ventricular myocytes, when such data becomes available. A second source of the errors in model parameters is the low accuracy of biochemical experiments, which can vary by an order of magnitude (see for example data on PKA activation by cAMP). However, investigations by Soltis and Saucerman 1-adrenergic signaling system is robust, at least in steady-state, even when the model parameters change by 1–2 orders of magnitude. Third, not all model parameters are measured directly in the experiments, and, therefore, are adjusted numerically to fit the experimental data downstream in the signaling pathway. Finally, a fourth limitation comes from the availability of at least two other, β2-adrenergic and CaMKII-mediated, signaling systems that can potentially interact with the β1-adrenergic system. Experimental data with mice shows that the β2-adrenergic signaling system does not affect the β1-adrenergic system in control and upon application of isoproterenol i, compared to Gs in mouse ventricular cells, which inhibits the stimulatory effects of the β2-ARs. The effects of β2-ARs can only be revealed upon additional application of pertussis toxin which suppresses the activity of Gi1-adrenergic signaling system still describes reasonably well most of the available experimental data on mouse ventricular myocytes. The author considers this model as the first step in the development of a more comprehensive compartmentalized model of the adrenergic signaling system in mouse ventricular myocytes.Despite that the presented model was extensively verified by the experimental data and reproduced most effects which result from stimulation of the βAppendix S1Model equations and parameters.(PDF)Click here for additional data file.


Although TiO2 NPs have been shown to induce cellular nanotoxicity and emphysema-like symptoms, whether TiO2 NPs can directly induce mucus secretion from airway cells is currently unknown. Herein, we showed that TiO2 NPs (<75 nm) can directly stimulate mucin secretion from human bronchial ChaGo-K1 epithelial cells via a Ca2+ signaling mediated pathway. The amount of mucin secreted was quantified with enzyme-linked lectin assay (ELLA). The corresponding changes in cytosolic Ca2+ concentration were monitored with Rhod-2, a fluorescent Ca2+ dye. We found that TiO2 NP-evoked mucin secretion was a function of increasing intracellular Ca2+ concentration resulting from an extracellular Ca2+ influx via membrane Ca2+ channels and cytosolic ER Ca2+ release. The calcium-induced calcium release (CICR) mechanism played a major role in further amplifying the intracellular Ca2+ signal and in sustaining a cytosolic Ca2+ increase. This study provides a potential mechanistic link between airborne NPs and the pathoetiology of pulmonary diseases involving mucus hypersecretion.Nanoparticle (NP) exposure has been closely associated with the exacerbation and pathophysiology of many respiratory diseases such as Chronic Obstructive Pulmonary Disease (COPD) and asthma. Mucus hypersecretion and accumulation in the airway are major clinical manifestations commonly found in these diseases. Among a broad spectrum of NPs, titanium dioxide (TiO Mucus is a large, highly glycosylated protein consisting of an array of mucin peptides (apomucin) 2+ channels expressed on the cell membrane allows the influx of extracellular Ca2+, which may serve as the external Ca2+ source 2+]C is usually relayed by triggering a secondary wave of Ca2+ propagation from internal stores, such as the ER 2+ binding sites responsible for triggering Ca2+- induced Ca2+ release (CICR) into the cytosol 2+]C could activate other cytosolic proteins and modulate secretion of mucin, hormones or various neurotransmitters Agonist-induced opening of various Ca2+ levels 2+-dependent secretion through the activation of L-type voltage-gated Ca2+ channels 2 NPs induce mucin secretion via a Ca2+ signaling mediated pathway.NPs have been shown to disturb cellular functions by elevating intracellular Ca2. Cell counts were performed using trypan blue  exclusion and a Bright-Line haemocytometer.The human airway bronchial epithelial cell line ChaGo-K1, obtained from American Type Culture Collection , was used because it expresses MUC proteins and secretes mucin 2 NPs have a surface area of 36 m2/g and the dispersion conductivity is 1040 µS/cm (information from Sigma). All NP samples were sonicated before usage. The concentrations used were 1 mg/ml, 0.75 mg/ml, 0.5 mg/ml, 0.25 mg/ml, 0.1 mg/ml, and 0.05 mg/ml. The range of concentrations used was consistent with the concentrations of TiO2 NPs found in previous reports 2 NPs were reconstituted with Hanks' solution  before being tested. The size of NPs was independently confirmed using homodyne dynamics laser scattering (DLS) as described in previous studies A mixture of anatase and rutile forms of ultrafine titanium (IV) dioxide (<75 nm)  was used in this study because this form has been shown to result in more severe cellular injuries 5 cells per well in a 24-well plate, and incubated for 24 hrs in RPMI 1640 supplemented with 10% FBS. Following 24 hr incubation, the RPMI medium was removed from the cells and the culture was rinsed with Hanks' solution twice before use.Cells were seeded at 2×10d = 570 nM, λEx = 552 nm and λEm = 581)  for 45 minutes. After the dye loading, the cells were rinsed, incubated with either normal Hanks' or Ca2+-free Hanks' solution, and treated with the appropriate TiO2 concentrations. All calcium signaling experiments were carried out on a thermoregulated stage at 37°C mounted on a Nikon microscope . ChaGo-K1 cells were incubated with cadmium chloride  to block the membrane Ca2+ channels 2 NP stimulation. To investigate the interaction between TiO2 and membrane Ca2+ channels, nifedipine , an L-type Ca2+ channel blocker 2. Antioxidant N-acetylcysteine  was also added to ChaGo-K1 cells to study the involvement of reactive oxygen species (ROS) 2 stimulation, and the activation of Ca2+ channels. Thapsigargin  2+ content and to inhibit the CICR mechanism 2+ pool.The cells were then loaded with a Rhod-2 AM dye (1 µM)   in Hanks' solution was incubated with the cells for 5 minutes at 37°C. Calcein is a biological inert green-fluorescent molecule of a molecular mass of 623 Da and an estimated molecular radius of 0.6 nm 2 NP solution containing the calcein dye was then removed and cells were rinsed twice with PBS to remove possible remnants of calcein dye in the extracellular solution. The cells were subsequently stained with a fluorescent nucleus dye, hoechst (10 µM) , for 5 mintues at 37°C and thoroughly rinsed again 2 NP concentrations used in the experiment.ChaGo-K1 cells were seeded at the density of 2×105 cells per well in a 24-well plate and cultured for 24 hrs. ChaGo-K1 cells were then rinsed with PBS and treated with BAPTA-AM , thapsigargin  or ryanodine  for at least 30 minutes. Afterward the cells were stimulated for 15 minutes with the corresponding TiO2 NP concentrations  or ionomycin (1 µM) (positive control) , both prepared in PBS. The supernatant containing secreted mucin was collected and briefly centrifuged at 8,000 rpm to remove the residual TiO2 NPs. The supernatant was then incubated in a 96 well  plate overnight at 4°C. Afterward the 96-well plate was washed with PBST (PBS + 0.05% Tween-20) and then blocked with 1% BSA. The 96 well plate was washed again with PBST and incubated with lectin  , conjugated to horseradish peroxidase  , at 37°C for 1 hr. The substrate, 3,3′,5,5′-Tetramethylbenzidine , was added to each well at room temperature followed by H2SO4  in order to terminate the reaction. The optical density was measured at 450 nm The cells were seeded at 2×102+ signals of more than 200 cells.After staining the treated cells, image analysis was performed with an inverted Nikon Eclipse TE2000-U fluorescent microscope. Each photo was taken at a magnification of 200× and analyzed using SimplePCI . The data shown is a representative of CaThe data was presented as means±SD. Each experiment was performed independently at least three times. Statistical significance was determined using a Student's t-test analysis with p values <0.05 .2 NPs. The particle size distribution ranged from ∼9 to 80 nm due to minor aggregation or agglomeration while the predominant size is ∼50 nm (Dynamic laser scattering (DLS) was used to characterize the TiOs ∼50 nm .2 NPs could generate an increase in [Ca2+]C, ChaGo-K1 cells were loaded with Rhod-2 AM dye and exposed to 0.05–1 mg/ml of TiO2 NPs. The change in [Ca2+]C, as represented by the fluorescence intensity within ChaGo-K1 cells, was monitored for 60 seconds. 2 NPs induced an approximate 150% increase, while lower TiO2 concentrations (<0.1 mg/ml) caused a minor elevation (∼110%) in [Ca2+]C when compared with untreated cells. The effect of TiO2 treatment on the [Ca2+]C of ChaGo-K1 cells followed a concentration-dependent manner  was added in Hanks' solution to chelate possible traces of Ca2+. TiO2 (0.05 mg/ml–1 mg/ml) treatment under Ca2+-free conditions failed to instigate a significant increase in [Ca2+]C (2+ pool is the primary source of the observed cytosolic Ca2+ increase. We then tested whether TiO2 NPs can induce a Ca2+ influx via membrane channels. Blocking the channels with CdCl2 (200 µM) significantly inhibited an increase in [Ca2+]C  2+ level triggered by 1 mg/ml and 0.75 mg/ml TiO2 exposure  was used to elicit mucin secretion as a positive control (Enzyme-linked lectin assay (ELLA) was used to assess the amount of mucin secreted from ChaGo-K1 cells when stimulated with TiOectively . Chelatiecretion . Additioecretion  or ryanoecretion  also res2+ pools . Ionomyc control .2 NPs, can exacerbate airway diseases 2-induced cellular nanotoxicity effects, pulmonary inflammatory responses and emphysema-like pathology 2 NPs can directly trigger mucin secretion has not been resolved. In this study, we demonstrate that TiO2 NPs can stimulate mucin secretion from bronchial epithelial ChaGo-K1 cells via a Ca2+- dependent pathway.Recently, an increasing number of reports have shown that airborne particulate pollution found in both the ambient and working environments, particularly TiO2 NPs can induce mucin secretion that increases as a function of TiO2 NP concentration , the [Ca2+]C increased gradually within the 1st minute , a general Ca2+ channel blocker 2+]C remained low and relatively unchanged with CdCl2. Secondly, nifedipine, a widely used L-type Ca2+ channel blocker, markedly diminished the increase in [Ca2+]C  to test whether the increase in Ca2+ induced by TiO2 NPs could stimulate mucin exocytosis. It is evident that BAPTA significantly inhibited mucin exocytosis , the ER is one of the major internal Ca2+ stores. 2+ had been depleted by pretreatment with thapsigargin, the TiO2 NP-induced [Ca2+]C failed to increase significantly, and the subsequent mucin secretion was abolished. Our data indicates that the ER plays a critical role in relaying TiO2-induced Ca2+ signaling. CICR is a positive feedback mechanism where the ER amplifies a small increase in [Ca2+]C, (e.g. due to voltage-gated Ca2+ influx 2+ from the ER 2+, CICR can generate an overall increase in [Ca2+]C2+]C when applying TiO2 NPs (2-instigated increase in [Ca2+]C was also CICR dependent. The effect of ryanodine was further demonstrated by the lack of mucin secretion under TiO2 NP stimulation (Besides the external CaTiO2 NPs . Therefomulation .2 NPs can activate membrane L-type Ca2+ channels, induce ROS production and possibly disrupt the cellular membrane. Influx of extracellular Ca2+ into the cytoplasm raises [Ca2+]C, which in turn can trigger ryanodine receptors on the ER to release ER resident Ca2+ via the CICR mechanism. A sufficient increase in the cytosolic Ca2+ level results in subsequent mucin secretion. More importantly, our results provide a direct link between airborne particulate matters and the pathogenesis of chronic airway diseases involving mucus hypersecretion and airway obstruction. In addition, we demonstrate that once thought inert and harmless TiO2 NPs can indeed interfere with intracellular Ca2+ signaling, possibly leading to pathological states.In summary, our study indicates that cellular exposure to TiO


As oncogenesis may be associated to the capability of tumor cells to grow independently on Ca2+ influx, it is important to assess whether SOCE regulates EPC-dependent angiogenesis also in tumor patients.Endothelial progenitor cells (EPCs) may be recruited from bone marrow to sustain tumor vascularisation and promote the metastatic switch. Understanding the molecular mechanisms driving EPC proliferation and tubulogenesis could outline novel targets for alternative anti-angiogenic treatments. Store-operated Ca2+ imaging, recombinant sub-membranal and mitochondrial aequorin, real-time polymerase chain reaction, gene silencing techniques and western blot analysis to investigate the expression and the role of SOCE in EPCs isolated from peripheral blood of patients affected by renal cellular carcinoma  as compared to control EPCs (N-EPCs). SOCE, activated by either pharmacological (i.e. cyclopiazonic acid) or physiological (i.e. ATP) stimulation, was significantly higher in RCC-EPCs and was selectively sensitive to BTP-2, and to the trivalent cations, La3+ and Gd3+. Furthermore, 2-APB enhanced thapsigargin-evoked SOCE at low concentrations, whereas higher doses caused SOCE inhibition. Conversely, the anti-angiogenic drug, carboxyamidotriazole (CAI), blocked both SOCE and the intracellular Ca2+ release. SOCE was associated to the over-expression of Orai1, Stim1, and transient receptor potential channel 1 (TRPC1) at both mRNA and protein level The intracellular Ca2+ buffer, BAPTA, BTP-2, and CAI inhibited RCC-EPC proliferation and tubulogenesis. The genetic suppression of Stim1, Orai1, and TRPC1 blocked CPA-evoked SOCE in RCC-EPCs.The present study employed CaSOCE is remodelled in EPCs from RCC patients and stands out as a novel molecular target to interfere with RCC vascularisation due to its ability to control proliferation and tubulogenesis. When ER Ca2+ concentration falls below a threshold level due to InsP3Rs-dependent Ca2+ release, Stim1 proteins rapidly redistribute to peripheral ER sites in close proximity to PM, where they aggregate into multiple puncta 2+ toolkit may be aberrantly expressed in cancer cells 2+ influx in neoplastic cells, which become able to undergo DNA synthesis and proliferation even in absence of extracellular Ca2+ inflow An increase in intracellular Ca2+ imaging, real-time reverse transcriptase polymerase chain reaction (qRT-PCR), gene silencing, western blot analysis, and functional assays to investigate the expression and the role of SOCE in EPCs isolated from RCC patients (RCC-EPCs). We demonstrated that RCC-EPCs display a greater SOCE, which correlated with the over-expression of Orai1, Stim1, and TRPC1 at both mRNA and protein level as compared to control cells (N-EPCs). We further showed that SOCE is selectively affected by the selective blockers, BTP-2, La3+, Gd3+, and 2-aminoethyl diphenylborinate (2-APB). Conversely, carboxyamidotriazole (CAI), an antiangiogenic drug which is in phase II clinical trials of metastatic RCC 2+ influx in RCC-ECFCs. These proteins might, therefore, be conceived as novel molecular targets to adverse tumor neovascularization.Renal cell carcinoma (RCC) accounts for 3.5% and 2.9% of malignant tumors in European men and women, respectively 2+ levels measured in RCC-EPCs and control cells (N-EPCs) were measured upon digital subtraction of the fluorescence background and were not significantly different, the average values of the Fura-2 ratio being 1.44±0.02, n = 137, and 1.41±0.01, n = 245, respectively. Store-operated Ca2+ entry in RCC-EPCs was evaluated by exploiting the classical “Ca2+ add-back” protocol 2+ pool. RCC-EPCs were first exposed to cyclopiazonic acid (CPA) (10 µM), a SERCA inhibitor, which prevents Ca2+ reuptake into ER and promotes Ca2+ efflux into the cytosol through ER leak channels. This manoeuvre results in depletion of ER Ca2+ reservoir and SOCE activation 2+ (0Ca2+), CPA induced a transient elevation in intracellular Ca2+ concentration ([Ca2+]i) in RCC-EPCs due passive ER emptying (2+ (1.5 mM) caused Ca2+ entry through opened store-operated channels and provoked a large increase in [Ca2+]i  lower in the former cells  higher in RCC-EPCs as compared to N-EPCs  upon depletion of the InsP3-sensitive Ca2+ stores and full activation of SOCE with CPA (10 µM)  smaller in RCC-EPCs than in N-EPCs  in RCC-EPCs (The resting Caemptying . Repleni [Ca2+]i . SOCE amer cells . RCC-EPCo N-EPCs . The ago (10 µM) B. TherefA or ATP D. Collecn N-EPCs . This feRCC-EPCs . Ongoing2+ influx in RCC-EPCs, we used aequorin (AEQ) Ca2+ measurements combined with lentiviral gene transfer technology. The N-EPCs and RCC-EPCs were transduced by lentiviral particles expressing AEQ fused with SNAP25 (pm-AEQ) which allows to measuring Ca2+ precisely beneath the plasma membrane. 2+ to the bath after depletion of internal Ca2+ stores by ATP under 0Ca2+ conditions resulted in a significantly higher Ca2+ increase in the sub-plasmalemmal space of RCC-EPCs as compared to N-EPCs. The statistical analysis of these results has been provided in 2+ homeostasis within intracellular organelles, such as mitochondria, the cells were infected with a lentiviral vector expressing AEQ targeted to the mitochondrial lumen by using a cleavable leader sequence of cytochrome peroxidase subunit 8 . In line with Fura-2 experiments, stimulation with 100 µM ATP resulted in significantly lower Ca2+ transient in the mitochondrial lumen of RCC-EPCs as compared with control N-EPCs  as compared to control cells. A more negative VM would enhance the electrochemical gradient driving Ca2+ inrush into the cells and lead to a more robust SOCE M is mainly set by K+ conductances M of about 0 mV, both types of cells were bathed in a solution containing 100 mM KCl (high-K+), as indicated by the Nernst equation for K+, EK  = (−RT/F)*ln([K+]o/[K+]i). 2+ entry.The higher amplitude of store-dependent CaThe expression of Orai1 and Stim1, which mediate SOCE in N-EPCs, was assessed by qRT-PCR analysis of mRNA extracts from RCC-EPCs. The specific primers described in 2+ signalling machinery. In this view, members of the TRPC family of non-selective cation channels, such as TRPC1 and TRPC4, may be involved in store-dependent Ca2+ entry in a number of non-excitable cells Albeit Orai1 and Stim1 have been reported to underlie SOCE in N-EPCs 2+ entry might be successfully exploited to affect proangiogenic Ca2+ signals in solid tumors 2+ influx and proliferation in N-EPCs 2+ entry in response to either CPA or ATP stimulation was operated by store-dependent Ca2+ channels, we utilized the trivalent metal cations, La3+ and Gd3+, which selectively block Orai1-dependent Ca2+ entry at 1–10 µM 2+ entry triggered by CPA (10 µM) was fully abolished by La3+ (10 µM) in both N-EPCs (2+ inflow was induced by ATP (100 µM) (2+]i evoked by CPA and by re-addition of extracellular Ca2+ was suppressed by Gd3+ (10 µM) in both N-EPCs (3+ (10 µM) was probed on ATP-elicited Ca2+ inflow in both cell types (2+ inrush to 2-APB Blockers of Cah N-EPCs  and RCC-h N-EPCs . The sam(100 µM) . Similarh N-EPCs  and RCC-h N-EPCs . The samll types . The pha2+ channels that has entered Phase I and II clinical trials both as single cytostatic agent and in combination with cytotoxic therapies  2+ channels 2+ signals in both N-EPCs and RCC-EPCs. Unlike BTP-2, 10 µM CAI inhibited both CPA-elicited Ca2+ mobilization and CPA-elicited SOCE , however, did not trigger any detectable elevation in [Ca2+]i in both N-EPCs and RCC-EPCs  in EPCs pre-treated with CAI (not shown).CAI is a synthetic small molecule inhibitor of non-voltage-gated Cated SOCE . CAI impn N-EPCs  and RCC-n N-EPCs , whereasn N-EPCs ig. 9F. Mr N-EPCs  or RCC-Er N-EPCs  were stiRCC-EPCs . This re2+ chelator, BAPTA-AM (30 µM), and BTP-2 (20 µM) at doses which have previously been reported to affect cell proliferation in vitro tubulogenesis of both N-EPCs  and RCC-EPCs  the higher amplitude of SOCE in EPCs isolated from patients suffering from RCC correlates to the over-expression of Stim1, Orai1, and TRPC1.As aforementioned, SOCE in N-EPCs is mediated by the physical interaction between Stim1 and Orai1 Material . As a coMaterial B. Simila release . Then, wRCC-EPCs C. The geRCC-EPCs  and RCC-RCC-EPCs . Conversaffected . Collectin vitro tubulogenesis. These data suggest for the first time that the Ca2+ signalling machinery in general, and SOCE in particular, might be exploited to target RCC neovascularisation and improve the outcome of RCC treatments SOCE is emerging as a crucial pathway in the control of EPC proliferation, motility, and tubulogenesis 2+ to the extracellular medium after depletion of endogenous Ca2+ stores with either the specific SERCA inhibitor, CPA, or the InsP3-synthesizing agonist, ATP. By doing so, we found that SOCE is operative and higher in RCC-EPCs in comparison to N-EPCs. These observations were corroborated by the sub-cellular analysis of CPA-elicited Ca2+ inflow in both the sub-membranal space and the mitochondria. The larger amplitude of SOCE in RCC-EPCs was associated to a smaller depletion of the intracellular Ca2+ store, as a consequence of the apparent decrease in ER Ca2+ content (indicated by the lower Ca2+ response to CPA in 0Ca2+) and of InsP3R down-regulation. This finding is not surprising when considering that the fall in luminal Ca2+ content is not linearly correlated to SOCE magnitude 3-sensitive pool which is tightly coupled to SOCE in several cell types 2+ pool that must be emptied for SOCE to be activated is not affected by the global decrease in the ER Ca2+ content observed in RCC-EPCs. On the other hand, a substantial increase in Ca2+ entry is expected to compensate the reduced basal filling of the intracellular Ca2+ reservoir when Ca2+ signals play such a crucial role in controlling cell processes as vital as proliferation and tubulogenesis (see below). As recently reviewed in 2+ influx is more robust in malignant B16B6 melanoma cells than in their non-transformed counterparts 2+ entry in these cells, as demonstrated by the experiments conducted in the presence of high-K+ extracellular solution. An alternative explanation involves the differential expression of the SOCE machinery in RCC-EPCs. SOCE in human EPCs is mediated by the interplay between Stim1, which serves as the ER Ca2+ sensor, and Orai1, which contributes to the channel pore on the plasma membrane 2+ content by Stim1 2+ imaging data, Orai1 and TRPC1 were more abundant in RCC-EPCs, as compared to N-EPCs, at both mRNA and protein level. Furthermore, RCC-EPCs displayed an additional Stim1 variant at 77 kDa, which might contribute to enhance SOCE by increasing the number of Orai1 and TRPC1 proteins activated on the plasma membrane by intracellular store depletion. Interestingly, Stim1 has long been known as a single-pass transmembrane protein of 77 kDa which is subject to translational modifications, such as phosphorylation, S-glutathionylation, and glycosylation, that increase its molecular weight up to 90–100 kDa Drosophila 293T cells display two major bands of Stim1 paralogue, i.e. Stim2, at 105 and 115 kDa as a consequence of their different extent of phosphorylation 2+ inflow. According to the first scenario, Orai1 and TRPC1 form distinct channels on the plasma membrane that are both activated by Stim1 following depletion of the intracellular Ca2+ reservoir. In this case, Orai1 and TRPC1 would mediate Ca2+-selective and non-selective cation currents, respectively. The second hypothesis suggests the assembly of a supramolecular ternary complex involving all of the three proteins upon emptying of the ER Ca2+ pool 2+-selective channel after intracellular Ca2+ release As in previous studies + entry 2+ content and slower decay to the baseline of ATP- and CPA-induced Ca2+ release suggest that other components of the Ca2+ toolkit are aberrantly expressed in RCC-EPCs. Additional experiments are required to assess the intracellular levels of a number of proteins involved in the ER Ca2+ cycle and in Ca2+ extrusion across the plasma membrane 3R transcripts, and the appearance of an additional Stim1 variant suggest that RCC-EPCs display a different gene expression profile as compared to N-EPCs. This feature concurs with the notion that tumor ECs (TECs) exhibit tumor-specific sets of up-regulated genes and chromosomal aberrations 2+ signals when exposed to arachidonic acid (AA), a pro-angiogenic intracellular messenger 2+ toolkit in response to instructive signals emitted from RCC microenvironment An increase in Orai1, but not Stim1, levels has also been reported in several breast cancer cell (BCC) lines 2+ influx and proliferation in N-EPCs is sensitive to BTP-2 2+ entry to drive RCC-EPC proliferation and tubulogenesis could not be given for granted. Indeed, it has long been known that cancer cells may keep on proliferating even in a Ca2+-poor medium, a process that has been termed “habituation” to low extracellular Ca2+2+ entry, but not intracellular Ca2+ release, in RCC-EPCs. The same results were obtained when both types of EPCs were stimulated in the presence of low micromolar concentrations of either La3+ or Gd3+, which selectively target Orai1-mediated Ca2+ inflow. Therefore, BTP-2 and La3+ could be exploited to assess the role played by SOCE in two key processes of EPC-mediated tumor vascularisation, such as cell growth and tubulogenesis. BTP-2 and La3+, as well as the intracellular Ca2+ buffer, BAPTA, blocked RCC-EPC proliferation. The involvement of SOCE in the control of cell growth has also been described in a variety of malignancies 1 and mitosis by regulating the activation of several cyclin-dependent kinases (cdk), such as cdk2 and cdk4, and of calcineurin. BTP-2 and La3+ were also found to impair in vitro tubulogenesis in both N- and RCC-EPCs. This result concurs with the notion that EC adhesion to substrate, spreading, motility and establishment of a patent lumen require extracellular Ca2+ entry 2+-sensitive decoders, such as CaMKII and the neutral protease calpain, that cleaves several focal adhesions proteins at the trailing edge 2+ signalling in EPCs. First, pre-treatment with CAI abolished both phases (i.e. intracellular Ca2+ release and SOCE) of the Ca2+ response to CPA. CAI, however, did not cause any detectable increase in [Ca2+]i when acutely administered to EPCs. Thus CAI did not deplete the intracellular Ca2+ reservoir during the pre-incubation period. Moreover, CAI did not interfere with Fura-2 fluorescence. CPA-evoked Ca2+ efflux from ER is mediated by yet to be identified leakage channels 2+ levels consequent to SERCA inhibition by CPA. Blockage of SOCE in cells pre-treated with CAI might be due to the upstream inhibition of the signal transduction pathway leading to Orai1 activation. Second, CAI suppressed both InsP3-dependent Ca2+ release and SOCE in ATP-stimulated EPCs. Once again, CAI might act by preventing the InsP3-mediated drop in ER Ca2+ levels that must be sensed by Stim1 to stimulate Orai1. The strong impairment of Ca2+ signalling by CAI leads to a significant reduction in the extent of cell growth and tubulogenesis in both N-EPCs and RCC-EPCs. Unlike BTP-2 and La3+, which selectively affects SOCE, we cannot rule out that CAI inhibits EPC by affecting additional signal transduction pathways. It is, however, intriguing that lower doses (i.e. 2 µM) of CAI, which are ineffective on SOCE, do not block but not EPC proliferation and tubulogenesis. Lack of specificity is a hallmark of CAI, which has already been shown to block InsP3 synthesis in mature ECs 3-dependent Ca2+ mobilization in human head and neck squamous cell carcinoma 2+ uniporter 2+ inflow 2+-permeable channels 2+ signals. Albeit our data clearly indicate that SOCE drives proliferation and tubulogenesis in RCC-EPCs, the physiological agonist of this pathway remains to be elucidated. We have previously shown that VEGF recruits SOCE to sustain the repetitive Ca2+ spikes that underlie its pro-angiogenic effect on N-EPCs. Nevertheless, VEGF is ineffective at stimulating detectable Ca2+ signals in RCC-EPCs despite the fact that VEGFR-2 is not down-regulated as compared to control cells. This result might be explained by the reduction in InsP3R levels in these cells. The amount of InsP3 produced by VEGFR-2 might not be sufficient to stimulate Ca2+ release and engage SOCE. The culture medium employed to stimulated cell proliferation and tubulogenesis, i.e. EGM-2, contains a number of additional growth factors and cytokines, such as EGF, bFGF and bovine foetal serum, that might stimulate RCC-EPC by enlisting SOCE We have previously shown that store-dependent Ca2+-permeable channels are regarded as novel molecular targets to exploit in the fight against the cancer 2+ signals in RCC-EPCs. This finding does not rule out the possibility that VEGFR-2 triggers alternative signalling pathways in these cells, such as the phosphoinositide 3-kinase (PI3K)/Akt and the p38 mitogen-activated protein kinase (p38 MAPK) pathways. However, it provides the first molecular explanation for the modest increase in the overall survival and progression-free survival of RCC patients treated with a humanized anti-VEGF neutralizing monoclonal antibody, such as bevacizumab, and the tyrosine kinase inhibitors, sorafenib and sunitinib. These drugs might, indeed, be directed against a target, i.e. VEGFR-2, which is uncoupled from its intracellular signaling cascade in one of the most important cell populations, i.e. EPCs, which contribute to establish the vascular network within the tumor. In this view, the discovery of the role served by Stim1, Orai1, and TRPC1 in driving RCC-EPC proliferation and tubulogenesis might be helpful in devising alternative strategies, such as the local administration of siRNA targeting the molecular components of SOCE or the intratumoral delivery of selective SOCE blockers via the polymer implants that have been recently described In conclusion, this study demonstrated for the first time that SOCE is up-regulated and controls proliferation and tubulogenesis in RCC-EPCs. The higher amplitude of SOCE in these cells is associated to the over-expression of its molecular players, namely Stim1, Orai1, and TRPC1. Targeted therapy based on tyrosine kinase inhibitors does not lead to a significant increase in progression-free survival and survival rates of patients suffering from RCC + CD45− fraction of circulating mononuclear cells, exhibit robust proliferative potential and form capillary-like structures in vitro6 MNCs (range 18–66) were plated on collagen-coated culture dishes (BD Biosciences) in the presence of the endothelial cell growth medium EGM-2 MV Bullet Kit (Lonza) containing endothelial basal medium (EBM-2), 5% foetal bovine serum, recombinant human (rh) EGF, rhVEGF, rhFGF-B, rhIGF-1, ascorbic acid and heparin, and maintained at 37°C in 5% CO2 and humidified atmosphere. Discard of non-adherent cells was performed after 2 days; thereafter medium was changed three times a week. The outgrowth of ECs from adherent MNCs was characterized by the formation of a cluster of cobblestone-appearing cells in vitro Matrigel assay , revealed by 5 mcl of 1∶5 diluted PerCp-streptavidin (Becton Dickinson). An appropriate isotype was used as control. Cells were acquired by a FACS Calibur flow cytometer and analysed by the CEllQuest software (BD Biosciences).Blood samples (40 mL) were obtained from healthy human volunteers aged from 22 to 48 years old (n = 12) and from RCC patients (n = 18). Demographic and clinical characteristics of patients are summarized in ssay see . VEGFR-2For our experiments, we have mainly used endothelial cells obtained from early passage ECFC  with the purpose to avoid  any potential bias due to cell differentiation. However, in order to make sure that the phenotype of the cells did not change throughout the experiments, in preliminary experiments we tested the immunophenotype of both normal ECFCs and ECFCs harvested from RCC patients at different passages and we found no differences, as shown in the Table S1 where the median values of cells expressing the specific antigens are reported. We also tested whether functional differences occurred when early (P2) and late (P6) passage-ECFCs were used by testing the in vitro capacity of capillary network formation in a Matrigel assay and found no differences between early and late passage ECFC-derived cells.2, 1 MgCl2, 10 Glucose, 10 Hepes. In Ca2+-free solution (0Ca2+), Ca2+ was substituted with 2 mM NaCl, and 0.5 mM EGTA was added. Solutions were titrated to pH 7.4 with NaOH. The high-K+ extracellular solution was prepared by replacing 100 mM NaCl with an equimolar amount of KCl. The solution was then titrated to pH 7.4 with KOH. The osmolality of the extracellular solution, as measured with an osmometer , was 300–310 mmol/kg.Physiological salt solution (PSS) had the following composition (in mM): 150 NaCl, 6 KCl, 1.5 CaCl2+]i was monitored by measuring, for each ROI, the ratio of the mean fluorescence emitted at 510 nm when exciting alternatively at 340 and 380 nm (shortly termed “ratio”). An increase in [Ca2+]i causes an increase in the ratio 2+ response to either CPA or ATP was measured as the difference between the ratio at the peak and the mean ratio of 1 min baseline before the peak. SOCE amplitude was measured by averaging 60 sec of signal at the peak and subtracting to this value the mean ratio of 1 min baseline recorded before Ca2+-readdition. Pooled data are given as mean±SE and statistical significance (P<0.05) was evaluated by the Student's t-test for unpaired observations.EPCs were loaded with 4 µM fura-2 acetoxymethyl ester  in PSS for 1 hour min at room temperature. After washing in PSS, the coverslip was fixed to the bottom of a Petri dish and the cells observed by an upright epifluorescence Axiolab microscope , usually equipped with a Zeiss ×40 Achroplan objective . EPCs were excited alternately at 340 and 380 nm, and the emitted light was detected at 510 nm. A first neutral density filter  reduced the overall intensity of the excitation light and a second neutral density filter  was coupled to the 380 nm filter to approach the intensity of the 340 nm light. A round diaphragm was used to increase the contrast. The excitation filters were mounted on a filter wheel . Custom software, working in the LINUX environment, was used to drive the camera  and the filter wheel, and to measure and plot on-line the fluorescence from 10–15 rectangular “regions of interest” (ROI) enclosing 10–15 single cells. Each ROI was identified by a number. Since cell borders were not clearly identifiable, a ROI may not include the whole EPC or may include part of an adjacent EPC. Adjacent ROIs never superimposed. phenyl)-4-methyl-1,2,3-thiadiazole-5-carboxamide (BTP-2) was purchased from Calbiochem . CAI was a gift from the Drug Synthesis and Chemistry Branch, National Cancer Institute . All other chemicals were obtained from Sigma Chemical Co. .Figure S12+ signals induced by CPA and ATP in endothelial progenitor cells.Average of the Ca Endothelial colony forming cells isolated from the peripheral blood of either healthy donors (black tracings) or patients suffering from renal cellular carcinoma (RCC) were exposed to the so-called “Ca2+ add-back” protocol. Briefly, the cells were treated with CPA (10 µM) (A–C) or ATP (100 µM) (D–F) to induce depletion of Ca2+ stores in Ca2+-free medium (0Ca2+) and, subsequently, replaced with Ca2+-containing solution so that store-operated Ca2+ entry (SOCE) could be measured. In each panel, the Ca2+ traces represent the response of cells isolated from three different healthy donors, challenged both with CPA (A–C) and ATP (D–F), and three different patients, again tested both with CPA (A–C) and ATP (D–F). Each tracing is the average of 35–55 cells within one microscopic field. For each panel the two tracings were recorded on the same day.(TIF)Click here for additional data file.Figure S22+ entry pathway in endothelial progenitor cells.Evidence that CPA and ATP activate the same store-operated Ca ATP (100 µM) did not trigger any detectable elevation in [Ca2+]i upon full activation of SOCE with CPA (10 µM) in both N-EPCs (A) and RCC-EPCs (B). No Ca2+ entry occurred in either N-EPCs (C) or RCC-EPCs (D) undergoing the “Ca2+ add-back” protocol in the absence of intracellular Ca2+ release.(TIF)Click here for additional data file.Figure S3Down-regulation of inositol-1,4,5-trisphosphate receptors in endothelial progenitor cells isolated from patients affected by renal cellular carcinoma. Upper panels, mRNA levels were measured by RT-PCR relative to the β-actin internal standard  and the values obtained were reported as ΔCt. Bars represent the mean±SE of at least 4 different experiments each from different RNA extracts. *P<0.05 versus InsP3R1 (1-way ANOVA followed by Newman–Keuls's Q test). Lower panels, gel electrophoresis of the PCR products. The PCR products were of the expected size: InsP3R1, 180 bp; InsP3R2, 158 bp; InsP3R3, 173 bp. The specific primers described in Table S2 have been utilized to examine the expression levels of InsP3R transcripts. MW: molecular weight marker. Blank: reaction without template.(TIF)Click here for additional data file.Figure S4Immunohistochemical localization of the Stim1 protein in endothelial progenitor cells. Stim1 protein was observed in both N-EPCs (A) and RCC-EPCs (B) with a labelling localized within the cytoplasm. Controls in which the primary antibody was substituted by non-immune serum show an absence of labelling (C).(TIF)Click here for additional data file.Figure S5BTP-2 does not affect SOCE at low concentrations. Average±SE of the amplitude of SOCE induced by CPA (10 µM) in both N-EPCs (A) and RCC-EPCs (B) in the presence of different concentrations of BTP-2. These doses were ineffective at inhibiting SOCE (p<0.05). In Panel A, n is equal to 63 (black bar) and 36 (white bar) and p = 0.817 for 200 nM BTP-2, n is equal to 63 (black bar) and 38 (black bar) and p = 0.978 for 500 nM BTP-2, and n is equal to 132 (black bar) and 111 (white bar) and p = 0.103 for 2 µM BTP-2. In Panel B, n is equal to 69 (black bar) and 30 (white bar) and p = 0.597 for 200 nM BTP-2, n is equal to 69 (black bar) and 43 (white bar) and p = 0.811 for 500 nM BTP-2, and n is equal to 175 (black bar) and 109 (white bar) and p = 0.597 for 2 μM BTP-2.(TIF)Click here for additional data file.Figure S62+ levels in endothelial progenitor cells.CAI does not increase intracellular Ca Acute perfusion of CAI at either 2 µM (A) or 10 µM (B) did not elicit any apparent increase in [Ca2+]i in N-EPCs. Each trace is representative of 96 and 138 cells, respectively, isolated from three different donors. Similarly, acute addition of CAI at either 2 µM (C) or 10 µM (D) did not elicit any apparent increase in [Ca2+]i in RCC-EPCs. Each trace is representative of 97 and 118 cells, respectively, isolated from three different patients.(TIF)Click here for additional data file.Figure S72+ entry controls tubulogenesis in endothelial progenitor cells harvested from healthy donors.Store-operated Ca BAPTA (30 μM), BTP-2 (20 μM), and CAI (20 μM) blocked the assembly into tubulary-like structures of N-EPCs.(TIF)Click here for additional data file.Figure S8Low concentrations of BAPTA, BTP-2, and CAI do not affect proliferation and tubulogenesis in endothelial progenitor cells. BAPTA (5 μM), BTP-2 (2 μM), and CAI (2 μM) did not prevent cell growth in both N-EPCs (A) and RCC-EPCs (B). Results are expressed as percentage of growth compared with control (given as 100% growth). C–D, 5 μM BAPTA did not impair tube formation in either N-EPCs (C) or RCC-EPCs (D). E–F, 2 μM BTP-2 had no effect on the assembly of either N-EPCs (E) or RCC-EPCs (F) in a bidimensional tubular network. G and H, 2 μM CAI did not interfere with in vitro tubulogenesis of either N-EPCs (G) or RCC-EPCs (H).(TIF)Click here for additional data file.Figure S9Genistein prevents SOCE in endothelial progenitor cells. Genistein (50 µM) fully inhibited SOCE elicited by CPA (10 µM) in both N-EPCs (A) and RCC-EPCs (B). Genistein was applied to EPCs pre-exposed to CPA under 0Ca2+ conditions 100 sec before restoration of extracellular Ca2+. Each trace is representative of 123 and 142 cells, respectively, isolated from three different donors.(TIF)Click here for additional data file.Figure S10Decreased expression of Sim1, Orai1, and TRPC1 in silenced endothelial progenitor cells harvested from patients affected by renal cellular carcinoma. Western blot and densitometry demonstrating a significant reduction in reduced Stim1 (A), Orai1 (B), and TRPC1 (C) protein expression in silenced RCC-EPCs as compared to control cells . The asterisk indicates s p<0.01 (Student's t-test). Blots representative of three were shown. Lanes were loaded with 10–30 µg of proteins, probed with affinity purified antibodies and processed as described in Materials and Methods. The same blots were stripped and re-probed with anti-β-actin antibody. Bands of the expected molecular weights were shown.(TIF)Click here for additional data file.


Preincubation of granule cells with the Ca2+ ATPase inhibitor, thapsigargin, or the d-myo-inositol 1,4,5-trisphosphate (IP3) receptor antagonist, 2-aminoethoxydiphenyl borate, markedly reduced the stimulatory effect of PACAP on [Ca2+]i. Furthermore, addition of the calcium chelator, EGTA, or exposure of cells to the non-selective Ca2+ channel blocker, NiCl2, significantly attenuated the PACAP-evoked [Ca2+]i increase. Preincubation of granule neurons with the N-type Ca2+ channel blocker, ω-conotoxin GVIA, decreased the PACAP-induced [Ca2+]i response, whereas the L-type Ca2+ channel blocker, nifedipine, and the P- and Q-type Ca2+ channel blocker, ω-conotoxin MVIIC, had no effect. Altogether, these findings indicate that PACAP, acting through PAC1 receptors, provokes an increase in [Ca2+]i in granule neurons, which is mediated by both mobilization of calcium from IP3-sensitive intracellular stores and activation of N-type Ca2+ channel. Some of the activities of PACAP on proliferation, survival, migration, and differentiation of cerebellar granule cells could thus be mediated, at least in part, through these intracellular and/or extracellular calcium fluxes.High concentrations of pituitary adenylate cyclase-activating polypeptide (PACAP) and a high density of PACAP binding sites have been detected in the developing rat cerebellum. In particular, PACAP receptors are actively expressed in immature granule cells, where they activate both adenylyl cyclase and phospholipase C. The aim of the present study was to investigate the ability of PACAP to induce calcium mobilization in cerebellar granule neurons. Administration of PACAP-induced a transient, rapid, and monophasic rise of the cytosolic calcium concentration ([Ca Pituitary adenylate cyclase-activating polypeptide (PACAP) is a 38- or 27-amino acid peptide that was isolated from hypothalamic extracts for its ability to stimulate cAMP formation in anterior pituitary cells  and the cerebella were quickly dissected out.Wistar rats were obtained from a colony raised at the University of Rouen in an accredited animal facility , according to the French recommendations for the care and use of laboratory animals. All experiments were performed in accordance with the French Ministry of Agriculture and the European Communities Council Directive 2010/63/UE of September 22, 2010  under the supervision of authorized investigators . Animals were housed in a temperature-controlled room (22 ± 1°C) under a 12-h light/dark cycle and with The 38-amino acid form of PACAP (PACAP38) was synthesized by solid phase methodology as previously described . Under certain conditions, pharmacological agents were added in the Ringer’s solution 30 min before mounting the glass coverslip on the stage of a Nikon inverted microscope (Eclipse TE200) equipped with a Nikon 60x, 1.4 NA, oil-immersion objective. Fluo-3/AM was excited with the 488-nm laser line, and the emitted fluorescence was recorded with a 500-nm long-pass filter on a Noran OZ confocal laser-scanning microscope , equipped with a standard argon/kripton laser for illumination. Images were acquired as a time series of scan of the same focal plane (512 × 480 pixels at 7.5 images/s) and data processing was carried out using the Intervision software (Noran Instruments). The cells were continuously perifused with Ringer’s solution at constant flow rate (2 mL/min) and temperature (37°C). The perifusion system was also used to deliver test substances at the vicinity of the cultured neuroblasts. Any modification of fluorescence directly reflects a change of intracellular calcium concentration ([Ca2+]i). To determine the increase of [Ca2+]i, the value of the peak amplitude as well as the area under the curve (AUC) of each response profile were calculated by using the PRISM Software . Results are expressed as histograms showing (i) the distribution of cells according to the amplitude of their response to PACAP and (ii) the mean AUCs of their [Ca2+]i increase.Forty-eight hours after plating, granule cells were loaded at 37°C for 45 min with 5 μM fluo-3/AM diluted in culture medium. Then, the calcium-dye-probe was washed off and replaced with a Ringer’s solution containing 10 mM Hepes, 120 mM NaCl, 2 mM KCl, 1.8 mM CaCl18 column , using a gradient of acetonitrile/water containing 0.1% TFA. The specific radioactivity of the tracer was approximately 800 Ci/mmol.PACAP27 was radioiodinated by means of the lactoperoxidase technique as previously described  at 20°C for 1 h in the same buffer, supplemented with 2% BSA. To visualize non-specific binding, slices, or cells were incubated with the radioligand in the presence of 10−6 M PACAP38. At the end of the incubation, sections, or cells were washed with cold Tris buffer and dried under an air stream. Finally, slices were apposed onto Hyperfilm-3H  for 6 days and the radioactivity associated with the cells was counted in a gamma-counter . Tissue slices were photographed by means of a computer-assisted image-analysis station .Twenty-μm-thick sections or granule cells from P8 rat cerebella cultured for 1 day were preincubated at 20°C in 50 mM Tris buffer (pH 7.4) supplemented with 1% bovine serum albumin (BSA), 32 mM saccharose, 5 mM MgClData are expressed as mean ± SEM values from at least three independent experiments. Statistical analyses were conducted with the PRISM software.−6 M PACAP38 provoked a marked increase of [Ca2+]i in more than 90% of the cells  induced a transient, rapid, and monophasic increase in [Ca2+]i followed by gradual return to baseline .Perifusion of cultured cerebellar granule cells with 102+ ATPase inhibitor thapsigargin  induced a substantial reduction of the amplitude of the [Ca2+]i response to 10−6 M PACAP38 in most cells . Preincubation of granule cells with the permeable d-myo-inositol 1,4,5-trisphosphate receptor antagonist 2APB (10−5 M) markedly reduced the [Ca2+]i response to 10−6 M PACAP38. In the presence of 2APB, more than 90% of responding cells exhibited an increase of their fluorescence intensity that was lower than 30% .Preincubation of granule cells with the Ca2+]i response to 10−6 M PACAP38 , significantly reduced the amplitude of the [Ca2+]i peak in response to 10−6 M PACAP38 , significantly attenuated the amplitude of the [Ca125I]PACAP27 as a radioligand. As previously reported [125I]PACAP27 binding was observed in the external granule cell layer of P8 rat cerebella (Figure −6 M PACAP38 to the incubation medium completely displaced [125I]PACAP27 binding (Figure 125I]PACAP27 on cerebellar tissue slices was not impaired (Figure 125I]PACAP27 binding by PACAP38 was not affected (Figure 125I]PACAP27 as a radioligand and it appeared that 6 mM EGTA did not modify the specific binding of [125I]PACAP27 on cultured cells (Figure To verify that EGTA did not influence PACAP binding on its recognition sites, autoradiography experiments were carried out using i response to 10−6 M PACAP38 (p > 0.05; Figures 2+]i rise (p > 0.05; Figures 2+ channel blocker ω-conotoxin GVIA (10−6 M; 30 min; Figures 2+]i increase induced by 10−6 M PACAP38, with 76 and 52% of responding cells exhibiting a ratio of fluorescence intensity greater than 130 in the absence or presence of ω-conotoxin GVIA, respectively (p < 0.001; Figure 2+]i response (p < 0.001; Figure To determine which type of Ca2+ pools in PACAP-induced [Ca2+]i increase in cerebellar neuroblasts has not been previously investigated. Because primary cultures of rat cerebellar granule cells are mainly composed of a single population of cells, the effect of PACAP on the [Ca2+]i response could be monitored on a large number of neurons. Thus, we found that, in 90% of granule cells, infusion of 10−6 M PACAP38 or PACAP27 induced a transient, rapid, and monophasic [Ca2+]i rise, similar to the PACAP-induced Ca2+ response observed in neural NG108-15 cells (Holighaus et al., 2+]i to baseline level. These differences could be explained by the diversity of the PACAP receptor subtype repertoire expressed in each cell type and by the different intracellular signaling systems involved. For instance, in rat gonadotrophs, PACAP stimulates Ca2+ oscillations through activation of VPAC1 coupled to a PTX-insensitive G protein and phospholipase C-β (Hezareh et al., 2+]i, the ability of PACAP to induce [Ca2+]i rise can be ascribed to activation of the PAC1 receptor, as previously reported in primary cultures of rat cortical neurons and astrocytes (Grimaldi and Cavallaro, 2+]i response is mainly mediated by the PAC1 hop1 splice variant (Mustafa et al., 2+]i by stimulating Ca2+ influx via a L-type Ca2+ channel without activating adenylyl cyclase or phospholipase C in response to PACAP (Chatterjee et al., The respective contribution of extracellular and intracellular Ca2+ stores in the stimulatory effect of PACAP on [Ca2+]i. The results showed that depletion of the endoplasmic reticulum Ca2+ store with the Ca2+ ATPase inhibitor thapsigargin reduced the amplitude and the AUC of the PACAP-evoked [Ca2+]i response in cerebellar neurons. Consistent with the notion that intracellular Ca2+ from IP3-sensitive Ca2+ pools could play an important role in the mechanism of action of PACAP, incubation of the cells with 2APB, a cell permeable d-myo-inositol 1,4,5-trisphosphate receptor antagonist, decreased the [Ca2+]i response to PACAP. Such a contribution of intracellular Ca2+ stores to the PACAP-evoked [Ca2+]i response has already been reported in rat gonadotrophs (Rawlings et al., It has previously been demonstrated that, in cerebellar granule neurons, PACAP activates the phospholipase C pathway (Basille et al., 2+ by EGTA or exposure of cells to the non-selective Ca2+ channel blocker NiCl2, also attenuated the stimulatory effect of PACAP38 on [Ca2+]i, indicating that Ca2+ influx is also required for the transient phase of the [Ca2+]i increase. Recruitment of both intracellular and extracellular sources of Ca2+ after activation of PAC1 receptors has already been reported in several models including the rat acinar cell line AR42J (Barnhart et al., 2+ channel was responsible for the stimulatory effect of PACAP on calcium influx, selective blockers of VOCCs were used. Preincubation of granule cells with the N-type Ca2+ channel blocker ω-conotoxin GVIA decreased the PACAP-evoked [Ca2+]i response, whereas the L-type Ca2+ channel blocker nifedipine and the P- and Q-type Ca2+ channel blocker ω-conotoxin MVIIC had no effect. These findings contrast with previous data showing the involvement of L-type Ca2+ channels in the stimulatory effect of PACAP on human neutrophils (Harfi et al., 2+ channels in mouse adrenal chromaffin cells (Hill et al., 2+-channels (Blair and Marshall, Alongside, suppression of extracellular Ca2+ is an essential intracellular messenger required for numerous cellular functions (Carafoli et al., 2+ concentrations have been shown to regulate proliferation (Owens et al., 2+ can exert different effects on immature neurons. For instance, in rat cortical neurons, Ca2+ release from intracellular stores is implicated in neurite elongation, while Ca2+ influx regulates dendritic branching (Ramakers et al., 2+ levels increases the migration rate of granule neuroblasts (Komuro and Rakic, 2+ store plays an essential role in growth and maturation of cerebellar granule cells (Yao et al., 2+ on granule cell development is now well established, the neurotrophic factors able to control Ca2+ levels in cerebellar neuroblasts are not yet clearly identified. Nevertheless, peptides acting on G protein-coupled receptors are thought to be important mediators to control neurite outgrowth and growth cone guidance in cerebellar granule cells through a Ca2+-dependent pathway (Xiang et al., CaIn vivo, PACAP receptors are expressed by precursors of cerebellar granule cells in the external granule cell layer (Basille et al., 2+ increase in maturation of cerebellar neuroblasts is still poorly understood. So far, it has only been shown that calcium is involved in the inhibitory effect of PACAP on granule cell migration (Cameron et al., 2+ entry is essential for granule cell survival (Gallo et al., 2+-dependent manner to indirectly promote granule cell survival (Shintani et al., Granule cells are, by far, the major population of interneurons in the cerebellum and they represent the main source of glutamate, the second most abundant population being GABA-ergic neurons (Voogd and Glickstein, Altogether, these data indicate that some of the activities of PACAP on cerebellar granule cell proliferation, survival, migration, and differentiation involve, at least in part, intracellular and/or extracellular calcium mobilization, but further investigations are needed to decipher the precise role of calcium in each process.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Ganoderma lucidum and Polyporus umbellatus mycelia that inhibits the growth of cancer cells. The results of the present study show that Khz induced apoptosis preferentially in transformed cells and had only minimal effects on non-transformed cells. Furthermore, Khz induced apoptosis by increasing the intracellular Ca2+ concentration ([Ca2+]i) and activating JNK to generate reactive oxygen species (ROS) via NADPH oxidase and the mitochondria. Khz-induced apoptosis was caspase-dependent and occurred via a mitochondrial pathway. ROS generation by NADPH oxidase was critical for Khz-induced apoptosis, and although mitochondrial ROS production was also required, it appeared to occur secondary to ROS generation by NADPH oxidase. Activation of NADPH oxidase was demonstrated by the translocation of regulatory subunits p47phox and p67phox to the cell membrane and was necessary for ROS generation by Khz. Khz triggered a rapid and sustained increase in [Ca2+]i, which activated JNK. JNK plays a key role in the activation of NADPH oxidase because inhibition of its expression or activity abrogated membrane translocation of the p47phox and p67phox subunits and ROS generation. In summary, these data indicate that Khz preferentially induces apoptosis in cancer cells, and the signaling mechanisms involve an increase in [Ca2+]i, JNK activation, and ROS generation via NADPH oxidase and mitochondria.Khz is a compound derived from the fusion of  Ganoderma lucidum have been described in various studies Polyporus umbellatus induces G2/M cell cycle arrest and the induction of apoptosis in HepG2 cells, thereby causing growth suppression Cancer develops from abnormal cellular proliferation or defects in apoptosis that lead to uncontrolled growth G. lucidum , and 2-methoxyestradiol exert their effects by inducing ROS production 2+ concentration ([Ca2+]i) is associated with the induction of apoptosis 2+]i increases, the mitochondria take up Ca2+ and function as a Ca2+-buffer; however, excessive accumulation of mitochondrial Ca2+ triggers apoptosis, at least in part, by inducing ROS generation via the mitochondrial ETC. An increase in cytoplasmic [Ca2+]i can also activate NADPH oxidase, which has been well documented in neutrophils via intracellular Ca2+ leads to the phosphorylation of the p47phox subunit and subsequent enzyme assembly Oxidative stress is widely implicated in apoptotic and non-apoptotic cell death 2+]i that resulted in ROS generation by NADPH oxidase via JNK and, finally, cellular apoptosis.In the present study, we investigated the role of Khz in cellular apoptosis and found that Khz induced a sustained increase in i induced by Khz treatment resulted in ROS generation by NADPH oxidase. The [Ca2+]i increased before the generation of ROS, and Ca2+ chelators such as EGTA and BAPTA-AM abrogated the activation of NADPH oxidase and ROS generation (2+]i induced by Khz treatment (data not shown). Activation of NADPH oxidase by elevated [Ca2+]i is well known in neutrophils and has also been reported for other cell types It is widely accepted that calcium signaling plays an important role in apoptosis neration . However2+-dependent NADPH oxidase activation. JNK was activated within 15 min of Khz treatment in a Ca2+-dependent manner (2+]i but upstream of ROS generation because it was blocked by the Ca2+ chelator BAPTA-AM but not by the ROS scavenger NAC (Finally, JNK was identified as the mediator of Cat manner , and thenger NAC . Furthernger NAC . Most st2+]i, activation of JNK, and ROS generation through NADPH oxidase and mitochondria (In summary, we demonstrated in this study that Khz induces mitochondria-mediated apoptosis preferentially in transformed cells, and the signaling pathway of Khz-induced apoptosis involves an increase in [Cachondria .