The integrative study that included experimentation and mathematical modeling was carried out to analyze dynamic aspects of transient Ca 2 + signaling induced by brief pulses of GPCR agonists in mesenchymal stromal cells from the human adipose tissue (AD-MSCs). The experimental findings argued for IP 3 /Ca 2 + -regulated Ca 2 + release via IP 3 receptors (IP 3 Rs) as a key mechanism mediating agonist-dependent Ca 2 + transients. The consistent signaling circuit was proposed to formalize coupling of agonist binding to Ca 2 + mobilization for mathematical modeling. The model properly simulated the basic phenomenology of agonist transduction in AD-MSCs, which mostly produced single Ca 2 + spikes upon brief stimulation. The spike -like responses were almost invariantly shaped at different agonist doses above a threshold, while response lag markedly decreased with stimulus strength. In AD-MSCs, agonists and IP 3 uncaging elicited similar Ca 2 + transients but IP 3 pulses released Ca 2 + without pronounced delay. This suggested that IP 3 production was rate-limiting in agonist transduction. In a subpopulation of AD-MSCs, brief agonist pulses elicited Ca 2 + bursts crowned by damped oscillations. With properly adjusted parameters of IP 3 R inhibition by cytosolic Ca 2 + , the model reproduced such oscillatory Ca 2 + responses as well. GEM-GECO1 and R-CEPIA1er, the genetically encoded sensors of cytosolic and reticular Ca 2 + , respectively, were co-expressed in HEK-293 cells that also responded to agonists in an "all-or-nothing " manner. The experimentally observed Ca 2 + signals triggered by ACh in both compartments were properly simulated with the suggested signaling circuit. Thus, the performed modeling of the transduction process provides sufficient theoretical basis for deeper interpretation of experimental findings on agonist-induced Ca 2 + signaling in AD-MSCs.

The integrative study that included experimentation and mathematical modeling was carried out to analyze dynamic aspects of transient Ca2+ signaling induced by brief pulses of GPCR agonists in mesenchymal stromal cells from the human adipose tissue (AD-MSCs). The experimental findings argued for IP3/Ca2+-regulated Ca2+ release via IP3 receptors (IP3Rs) as a key mechanism mediating agonist-dependent Ca2+ transients. The consistent signaling circuit was proposed to formalize coupling of agonist binding to Ca2+ mobilization for mathematical modeling. The model properly simulated the basic phenomenology of agonist transduction in AD-MSCs, which mostly produced single Ca2+ spikes upon brief stimulation. The spike-like responses were almost invariantly shaped at different agonist doses above a threshold, while response lag markedly decreased with stimulus strength. In AD-MSCs, agonists and IP3 uncaging elicited similar Ca2+ transients but IP3 pulses released Ca2+ without pronounced delay. This suggested that IP3 production was rate-limiting in agonist transduction. In a subpopulation of AD-MSCs, brief agonist pulses elicited Ca2+ bursts crowned by damped oscillations. With properly adjusted parameters of IP3R inhibition by cytosolic Ca2+, the model reproduced such oscillatory Ca2+ responses as well. GEM-GECO1 and R-CEPIA1er, the genetically encoded sensors of cytosolic and reticular Ca2+, respectively, were co-expressed in HEK-293 cells that also responded to agonists in an "all-or-nothing" manner. The experimentally observed Ca2+ signals triggered by ACh in both compartments were properly simulated with the suggested signaling circuit. Thus, the performed modeling of the transduction process provides sufficient theoretical basis for deeper interpretation of experimental findings on agonist-induced Ca2+ signaling in AD-MSCs.

Intracellular Са2+ controls its own level by regulation of Ca2+ transport across the plasma and organellar membranes, often acting via calmodulin (CaM). Drugs antagonizing CaM action induce an increase in cytosolic Ca2+ concentration in different cells. We have found persistent Са2+ oscillations in cultured white adipocytes in response to calmidazolium (CMZ). They appeared at [CMZ] > 1 μM as repetitive sharp spikes mainly superimposed on a transient or elevated baseline. Similar oscillations were observed when we used trifluoperazine. Oscillations evoked by 5 μM CMZ resulted from the release of stored Ca2+ and were supported by Са2+ entry. Inhibition of store-operated channels by YM-58483 or 2-APB did not change the responses. Phospholipase A2 inhibited by AACOCF3 was responsible for initial Ca2+ mobilization, but not for subsequent oscillations, whereas inhibition of iPLA2 by BEL had no effect. Phospholipase C was partially involved in both stages as revealed with U73122. Intracellular Са2+ stores engaged by CMZ were entirely dependent on thapsigargin. The oscillations existed in the presence of inhibitors of ryanodine or inositol 1,4,5-trisphosphate receptors, or antagonists of Ca2+ transport by lysosome-like acidic stores. Carbenoxolone or octanol, blockers of hemichannels (connexons), when applied for two hours, prevented oscillations but did not affect the initial Са2+ release. Incubation with La3+ for 2 or 24 h inhibited all responses to CMZ, retaining the thapsigargin-induced Ca2+ rise. These results suggest that Ca2+-CaM regulation suppresses La3+-sensitive channels in non-acidic organelles, of which arachidonate-activated channels initiate Ca2+ oscillations, and connexons are intimately implicated in their generation mechanism.

Activation of Ca2+ entry upon cell stimulation by agonists can be accomplished by different mechanisms, including store-operated calcium entry (SOCE) and the action of phospholipase A2 (PLA2) products. In adipocytes there are two relatively independent pathways for Ca2+ mobilization from intracellular stores. In the present work we studied, whether these pathways are coupled with particular mechanisms of Ca2+ entry into the cell. It is shown that acetylcholine (ACh) induces oscillatory responses in cytosolic Ca2+ concentration independently of SOCE inhibition by YM-58483 or 2-APB. These oscillations were abolished by the addition of La3+, which inhibits both store-operated calcium (SOC) and ARC channels (regulated by arachidonic acid, AA). The responses to ACh were suppressed by AACOCF3 inhibiting PLA2 of type IV and VIA (iPLA2). Oscillations evoked by fetal bovine serum (FBS) were distinguished by the baseline spiking and, in contrast, were terminated by YM-58483 and La3+ but were not dependent on AACOCF3. The same cell could respond to ACh and FBS at their sequential addition in any order with the intermediate wash. Oscillatory responses of a similar (base or elevated line) form to phenylephrine decayed only gradually after the inhibition of phospholipase C or inositol 1,4,5-trisphosphate receptor, and were partially attenuated by the inhibitors YM-58483 and La3+ without appreciable influence of AACOCF3. AA at concentrations 1–10 μM caused oscillations when added after spontaneous cessation of ACh-induced oscillations or itself, with a discernible effect produced at lower concentrations after ACh. Calmodulin inhibitor R24571 caused oscillations, which could be suppressed by YM-58483 or AACOCF3 suggesting activation of SOCE and iPLA2, respectively. Taken together, these results indicate that the mechanism of Ca2+ entry activation depends on the signaling pathway involved by an agonist. ACh does not employ SOCE but activates PLA2 with probable participation of the form VIA, which entails the action of its product(s) on ARC channels and likely on lysophospholipid-activated channels. FBS acts through SOCE without participation of PLA2. These two versions can coexist in the case of phenylephrine.

Активация входа Са2+ при стимуляции клеток агонистами может обеспечиваться различными механизмами, включая депо-управляемый вход Ca2+ (store-operated calcium entry, SOCE) и действие продуктов фосфолипазы A2 (PLA2). В связи с существованием в адипоцитах двух относительно независимых путей мобилизации Са2+ из внутриклеточных запасов, в данной работе мы изучили, сопряжены ли эти пути с индивидуальными механизмами входа Са2+ в клетку. Показано, что ацетилхолин (ACh) вызывает колебательные изменения концентрации Са2+ в цитозоле, не зависящие от ингибирования SOCE посредством YM-58483 или 2-APB. Эти колебания устранялись при добавлении La3+, который ингибирует как депо-управляемые Ca2+-каналы, так и ARC-каналы (регулируемые арахидоновой кислотой, АА). Ответы на ACh подавлялись AACOCF3, ингибирующим PLA2 типов IV и VIA (iPLA2). Колебания под действием эмбриональной коровьей сыворотки (FBS), отличавшиеся достижением базального уровня между спайками, напротив, прекращались в присутствии YM-58483 или La3+, но не зависели от AACOCF3. Одна и та же клетка могла отвечать на ACh и FBS при их последовательном добавлении в любом порядке с промежуточной отмывкой. Подобные по форме колебательные ответы (от базального или повышенного уровня) на фенилэфрин постепенно затухали после ингибирования фосфолипазы C или рецептора инозитол-1,4,5-трисфосфата и частично ослаблялись ингибиторами YM-58483 или La3+ без существенного влияния AACOCF3. АА в концентрации 1–10 мкМ вызывала колебания при добавлении как после спонтанного прекращения ACh-индуцированных колебаний, так и самостоятельно, причем после ACh отчетливый эффект достигался при меньших концентрациях АА. Ингибитор кальмодулина R24571 вызывал колебания, которые могли подавляться YM-58483 или AACOCF3, что указывает на активацию, соответственно, SOCE и iPLA2. В целом эти результаты показывают, что механизм активации входа Са2+ зависит от того, какой сигнальный путь затрагивается агонистом. Ацетилхолин не вовлекает SOCE, но активирует PLA2 с вероятным участием формы VIA, что сопровождается действием ее продукта(-ов) на ARC-каналы и, возможно, на лизофосфолипид-активируемые каналы. FBS действует через SOCE без участия PLA2. Эти два варианта могут сосуществовать в случае фенилэфрина.

Experiments on cultured mouse adipocytes (9 days in vitro) using fluorescent microscopy have shown that activation of α 1 - and α 2 -adrenoceptors by norepinephrine (NE) or α 2 -adrenoreceptors by L -arginine evokes transient Ca 2+ signals, while activation of m 3 -cholinoreceptors by acetylcholine (ACh) or betaine causes sustained or damped Ca 2+ oscillations. The presence in the incubation medium of L -arginine at a low concentration (100–200 μM) is necessary for a vigorous manifestation of these effects, apparently due to transition of protein kinase G (PKG) and phosphodiesterase V into an active state. In the presence of 1–10 mM L -arginine, the amplitude of the Ca 2+ transient response to NE increases and signal duration decreases. ACh and NE upon a sequential addition mutually potentiate their effects. Using an inhibitory analysis we show that the observed modes are related to the operation of a signaling pathway with the participation of phosphatidylinositol 3-kinase (PI3K), protein kinase B (PKB), endothelial NO synthase (eNOS), cytoplasmic guanylate cyclase (sGC), protein kinase G (PKG), ADP-ribosyl cyclase (CD38), and the ryanodine receptor (RyR). The formation of several loops of positive feedbacks (PF) and negative feedbacks (NF) in the signaling system is possible: (i) short PF loops due to Ca 2+ -induced Ca 2+ release (CICR) from internal stores through the inositol trisphosphate receptor (IP 3 R) and RyR participating in the transient signal formation; (ii) long PF loop Ca 2+ → eNOS → sGC → PKG → CD38 → RyR → Ca 2+ , which can provide necessary conditions for calcium oscillations arising from short PF loops (CICR); (iii) several NF loops based on PKG-mediated inhibition of IP 3 R and activation of Ca 2+ -ATPases of sarco(endo)plasmic reticulum and of the plasma membrane providing a shutdown of signaling by the pathway phospholipase C → IP 3 R → Ca 2+ and limiting Ca 2+ rise caused by the pathway PI3K → PKB → eNOS → sGC → PKG → CD38 → RyR → Ca 2+ . Convergence of signaling pathways that involve α 1 -, α 2 -, and m 3 -receptors and then Gβγ-subunits of G q and G q proteins acting on PI3Kγ can provide activation of cytoplasmic PKG, which plays a key role in producing transient responses, in activation of Ca 2+ removal and generation of [Ca 2+ ] i oscillations. PKG inhibition (implemented here by KT5823 application) in the presence of any agonist results in rupture of NF loops controlling Ca 2+ transporting systems activity that leads to uncontrolled [Ca 2+ ] i rise and cell death.

In non-excitable cells, several kinds of agonist-induced oscillations of cytosolic Ca 2+ concentration ([Ca 2+ ] i ) are known which differ in their form and generation mechanism. The oscillation source is, as a rule, the regulation of Ca 2+ mobilization from intracellular stores through inositol 1,4,5-trisphosphate (IP 3 ) receptors (IP 3 R) and in some cases through ryanodine receptors (RyR). In the present work, oscillations in single mature adipocytes of mice epididymal fat on the ninth day of cultivation are studied. Cells were stimulated by acetylcholine (ACh) or by fetal bovine serum (FBS). ACh at a concentration of 0.1–5 μM evoked a rise in [Ca 2+ ] i to a peak and subsequent oscillations whose peaks and troughs declined along with increasing amplitude while frequency decreased. In most cells oscillations lasted less than 5 min. The new constant or interspike level exceeded the initial one or was equal to it (at 1 μM ACh). The removal of ACh stopped oscillations immediately. An inhibitor of phospholipase C (U73122) or of IP 3 R (Xestospongin C) did not affect the pattern of responses, which means that the generation of oscillations does not depend on IP 3 . At the same time, suppression of responses by ryanodine, which blocks RyR, was observed. Besides, oscillatory responses were abolished by inhibitors of phosphatidylinositol 3-kinase, NO synthase, and cGMP-dependent protein kinase. FBS (1%) initiated oscillations characterized by return of [Ca 2+ ] i after each peak to the baseline level, occurring prior to stimulation, and by maintenance of roughly constant amplitude and frequency (of the order of 1 min −1 ). Oscillations persisted longer (more than 15 min in 87% of cells) than with ACh. Repeated stimulation of cells by FBS revealed a strongly reduced sensitivity after 1 h of rest, whereas responses to ACh partially restored within 3 min. Investigation of the involvement of IP 3 R and RyR in FBS-induced oscillations gave completely inverse results relative to ACh and demonstrated a leading role of IP 3 R without a considerable contribution of RyR and of its activation pathways. With both stimuli, Ca 2+ entry through the plasma membrane was necessary only as a support of oscillations. The results show that in adipocytes different agonists can engage distinct subsystems of Ca 2+ signaling, each of them generating oscillations with a specific temporal pattern.

Crosstalk mechanisms have not been studied as thoroughly as individual signaling pathways. We exploit experimental and computational approaches to reveal how a concordant interplay between the insulin and epidermal growth factor (EGF) signaling networks can potentiate mitogenic signaling. In HEK293 cells, insulin is a poor activator of the Ras/ERK (extracellular signal‐regulated kinase) cascade, yet it enhances ERK activation by low EGF doses. We find that major crosstalk mechanisms that amplify ERK signaling are localized upstream of Ras and at the Ras/Raf level. Computational modeling unveils how critical network nodes, the adaptor proteins GAB1 and insulin receptor substrate (IRS), Src kinase, and phosphatase SHP2, convert insulin‐induced increase in the phosphatidylinositol‐3,4,5‐triphosphate (PIP3) concentration into enhanced Ras/ERK activity. The model predicts and experiments confirm that insulin‐induced amplification of mitogenic signaling is abolished by disrupting PIP3‐mediated positive feedback via GAB1 and IRS. We demonstrate that GAB1 behaves as a non‐linear amplifier of mitogenic responses and insulin endows EGF signaling with robustness to GAB1 suppression. Our results show the feasibility of using computational models to identify key target combinations and predict complex cellular responses to a mixture of external cues. We present an integrated analysis of crosstalk between the insulin receptor (IR) and epidermal growth factor receptor (EGFR) signaling pathways. Our experimental and computational findings show how systems‐level interactions between the EGFR and IR networks convert the insulin‐induced increase in the phosphatidylinositol‐3,4,5‐triphosphate (PIP3) concentration into enhanced activity of the extracellular signal‐regulated kinase (ERK) pathway. Physiological stimuli never act in isolation, and often cells in the body are simultaneously exposed to EGF and insulin. The EGFR and IR networks share many downstream components, yet their physiological responses to stimuli are different. In cells that express EGFR, including HEK293 cells, EGF acts as a potent activator of mitogenesis through activation of the Ras/ERK pathway. In contrast, mitogenesis and the Ras/ERK pathway are poorly activated by insulin. The main biological function of insulin is metabolic, involving the control of glucose metabolism and stimulation of protein and lipid syntheses. We show that in HEK293 cells, insulin amplifies Ras/ERK activation by low, physiological [EGF], and at saturating [EGF] the insulin effect becomes insignificant. Following 1.5‐ and 15‐min co‐stimulation with EGF plus insulin, the phospho‐ERK level (which is directly related to ERK activity) is significantly larger than the sum of these levels observed for each ligand (Figure 3E, left and right panels), displaying EGF–insulin synergy. The peak ERK activity (at ∼5 min co‐stimulation) does not display synergistic effects (Figure 3E, middle panel). We show that insulin–EGF crosstalk is not a consequence of extra activation of either receptor by co‐stimulation with two ligands, or activation of insulin‐like growth factor receptor‐1 by insulin. Multiple points of crosstalk between EGFR and IR make it difficult to comprehend and predict intricate Ras/ERK signaling dynamics in a cell‐dependent context, using only qualitative arguments. These dynamics depend on a variety of non‐linear interactions and feedback loops. A testable computational model helps us provide insights into the key causative relationships between the input stimuli and Ras/ERK signaling and reveal specific functions of critical network nodes in generating cellular responses (Kholodenko, 2006). Our mechanistic computational model, trained by the data from HEK293 cells, suggests that major crosstalk mechanisms that amplify ERK signaling are localized upstream of Ras and at the Ras/Raf level. Some of the crosstalk interactions affect multiple Ras activation and deactivation routes, which involve the adaptor proteins, Grb2‐associated binder‐1 (GAB1) and insulin receptor substrates (IRS), and the SH2‐domain containing protein tyrosine phosphatase‐2 (SHP2). In the model, EGF and insulin co‐stimulation increases the amount of PIP3 produced by phosphatidylinositol 3‐kinase (PI3K) and further facilitates the GAB1 membrane recruitment and its subsequent tyrosine phosphorylation. An increase in the membrane‐bound phospho‐GAB1 promotes Grb2–SOS binding and increases [SOS] (Ras activator) in close proximity to Ras. At the same time, this gain in phospho‐GAB1 also increases the amounts of RasGAP (Ras deactivator) and SHP2 bound to GAB1. Although SHP2 negatively regulates IR, EGFR, IRS, and GAB1 phosphorylation levels, it has a positive effect on Ras activation, as we showed using a specific SHP2 inhibitor, NSC‐87877 (Chen et al, 2006). This positive effect is related to the formation of the GAB1–SHP2 and IRS–SHP2 complexes and subsequent dephosphorylation of multiple docking sites, involved in RasGAP binding. Simulations predict that the net result of all these interactions is an increase in positive signaling and decrease in negative signaling to Ras, which amplifies the Ras‐GTP level. Additional crosstalk interactions occur at the Ras/Raf level. In the model, at any given Ras‐GTP load, the simultaneous exposure to insulin plus EGF increases Raf activity, relative to insulin alone, owing to EGF‐induced stimulation of tyrosine kinases, which are assumed to belong to the Src family (Wellbrock et al, 2004). We tested the model against the experiment, using kinetic data on responses to multiple perturbations, including different EGF doses, specific inhibitors and small interfering RNA (siRNA). We showed that the PI3K inhibitor wortmannin (WT) suppresses synergistic activation of the Ras/Raf/MEK/ERK pathway by insulin and EGF. The data demonstrate that the total GAB1 phosphorylation level and the concentrations of GAB1‐bound Grb2, SHP2, and PI3K decrease dramatically in WT‐treated cells. We conclude that the loss of insulin–EGF synergy caused by WT arises from the disruption of the GAB1–PI3K positive feedback and the loss of the GAB1‐mediated membrane recruitment of signaling molecules. To get further insight into crosstalk mechanisms, we simulated the dynamics of ERK responses to EGF versus EGF plus insulin in cells with different GAB1 expression levels (Figure 5A). As expected, GAB1 suppression reduces the phospho‐ERK level to a larger degree for EGF than for EGF plus insulin. Model predictions shown in Figure 5B (left panel) illustrate this phospho‐ERK level with decreasing GAB1 at 1.8 min following EGF or EGF plus insulin stimulation. To test the model, HEK293 cells were transfected with targeted siRNA against GAB1 mRNA, resulting in ∼75

Src‐family kinases (SFKs) play a pivotal role in growth factor signaling, mitosis, cell motility and invasiveness. In their basal state, SFKs maintain a closed autoinhibited conformation, where the Src homology 2 domain interacts with an inhibitory phosphotyrosine in the C‐terminus. Activation involves dephosphorylation of this inhibitory phosphotyrosine, followed by intermolecular autophosphorylation of a specific tyrosine residue in the activation loop. The spatiotemporal dynamics of SFK activation controls cell behavior, yet these dynamics remain largely uninvestigated. In the present study, we show that the basic properties of the Src activation/deactivation cycle can bring about complex signaling dynamics, including oscillations, toggle switches and excitable behavior. These intricate dynamics do not require imposed external feedback loops and occur at constant activities of Src inhibitors and activators, such as C‐terminal Src kinase and receptor‐type protein tyrosine phosphatases. We demonstrate that C‐terminal Src kinase and receptor‐type protein tyrosine phosphatase underexpression or their simultaneous overexpression can transform Src response patterns into oscillatory or bistable responses, respectively. Similarly, Src overexpression leads to dysregulation of Src activity, promoting sustained self‐perpetuating oscillations. Distinct types of responses can allow SFKs to trigger different cell‐fate decisions, where cellular outcomes are determined by the stimulation threshold and history. Our mathematical model helps to understand the puzzling experimental observations and suggests conditions where these different kinetic behaviors of SFKs can be tested experimentally.

Calmodulin inhibitor calmidazolium (R24571) in concentrations 1-5 mu M induces a short-term Ca2+ entry in Ehrlich ascite tumor cells (EATC). Ca2+ entry initiates a pulse-like ATP secretion in EATC. Extracellular ATP interacts with P2Y receptors on the cell surface. P2Y-dependent activation of phospholipase C (PLC) generates IP3 and mobilizes Ca2+ from endoplasmic reticulum (ER). Mobilization decreased at the R24571 concentrations higher than 5 mu M. Ca2+-channels induced by R24571 are permeable for Mn2+. Ca2+ entry does not depend on the ER depletion and is suppressed by nordihydroguaretic acid, arachidonic acid (AA), and inhibitors of Ca2+-independent phospholipase A(2) (iPLA2), (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one and palmitoyl trifluoromethyl ketone. The bell-shaped Ca2+ dependence of channel activity is shown. Increasing of Ca2+ concentration to 0.3 mu M activates the channel, but the channel is inhibited completely at 1.3 mu M of Ca2+. The termination of Ca2+ entry is probably due to the inhibition of iPLA2 and/or of the channel at increased intracellular concentrations of AA and Ca2+.

The stimulation of photo- and olfactory receptor cells by light and odorants, respectively, causes a fast change in the cytosolic level of cyclic nucleotides, thereby altering activity of cyclic nucleotide-gated (CNG) channels and producing the generatory current. To study transient gating of CNG channels, we performed computer simulations of macroscopic CNG currents elicited by short agonist pulses using a kinetic model of agonist-induced channel openings. The model includes the consecutive binding of ligand molecules to the multimeric CNG channel and assumes that for a given number of ligands bound, the channel closed-open transitions are fast enough to be in equilibrium. Consistent with experimental findings, the mathematical model demonstrates that the CNG current may decline immediately after abrupt agonist withdrawal, as is the case with retinal channels, or, similar to olfactory channel responses, it may outlast a brief period of agonist application by several hundred milliseconds, exhibiting a plateau. The plateau exists if opening equilibrium constants of the partially liganded channel are close to that of the fully liganded channel. With a larger number of adjacent states open to the similar extent, the more prolonged plateau appears. Thus the values of opening equilibrium constants determine a kinetic phenotype of the macroscopic CNG current. Dose-response curves of CNG currents with a more outlasting plateau showed less apparent cooperativity. For the CNG channel with the tetrameric architecture, these two features could coexist at the realistic Hill coefficient n(H) of about 2. The presented model allows for the interpretation of the effect of calmodulin on the CNG current in terms of the gating constants. Together, our findings account for the kinetic difference between olfactory and retinal CNG channels and point to the former as a potentially inertial element of olfactory transduction machinery.

The properties of the Ca2+ channel induced by a calmodulin inhibitor in Ehrlich ascites tumor cells were investigated using fluorescent indicators Indo-1 and chlortetracycline. The inhibitor of calmodulin calmidazolium (R24571) in concentrations of 1-2 microM induces a short-term Ca2+ entry and a pulse-like ATP secretion. Repeated addition of R24571 also causes a transient Ca2+ signal. Ca2+ channels induced by R24571 are permeable for Mn2+. Ca2+ entry does not depend on endoplasmic reticulum depletion by thapsigargin, ATP, or ionomycin and is suppressed by nordihydroguaretic acid (EC50 = 6.7 microM), quercetin (EC50 = 1.5 microM), dihydroquercetin (EC50 = 17 microM), arachidonic acid (AA) (EC50 = 8.6 microM), and suramin (EC50 = 0.25 +/- 0.05 MM), and weakly depends on temperature in the range of 18 - 37 degrees C. The apparent activation constant for R24571 and the Hill coefficient are 2.5 +/- 0.2 and 4 +/- 0.3 microM, respectively. The products of arachidonic acid oxidation are neither activators nor inhibitors of these channels. The inhibitory effect of nordihydroguaretic acid is indirect and is conceivably caused by the accumulation of arachidonic acid due to suppression of its lipoxygenase-catalyzed oxidation at phospholipase A2 activation. The maximal level of about 1.3 microM in the dependence of Ca2+ signal amplitude on R24571 concentration points to possible inhibition of the channel by increased Ca2+ concentration in the cytosol. The weak dependence on temperature implies that the channel is highly permeable, the chain of enzymic processes is not involved in Ca2+ entry activation, and the mutual compensation of processes with opposite contributions is possible. Using chlortetracycline fluorescence, we have shown in model experiments on calmodulin solution that Ca2+ induces cooperatively a conformational transition of calmodulin with the exposure of a hydrophobic chlortetracycline-Ca(2+)-binding site. The interaction of R24571 with the CaM-Ca2+ complex results in quenching of fluorescence to its level in water, which is interpreted as the elimination of the availability of calmodulin hydrophobic site for chlortetracycline-Ca+. Nordihydroguaretic acid, quercetin, and dihydroquercetin, but not suramin, also interact with calmodulin, but this does not result in the complete closing of its hydrophobic site. It is supposed that the activation of the Ca2+ channel occurs owing to the activation of calmodulin-dependent phospholipase A2 by R24571, which leads to the formation of a low-molecular short-lived secondary messenger, or because of the interaction of R24571 with calmodulin, which directly inhibits the channel. The termination of Ca2+ entry is probably due to the inhibition of phospholipase A2 and/or of the channel at increased concentrations of arachidonic acid and Ca2+.

Signals of cytosolic Ca2+ (Ca2+c) and mitochondrial Ca2+ (Ca2+m) evoked by the activation of purinoreceptors of Ehrlich ascites tumor cells at different extents of inhibition of the mitochondrial Na+/Ca2+ exchanger by tetraphenylphosphonium (TPP+) were investigated. [Ca2+c] was measured by Fura-2 fluorescence, and [Ca2+m] changes were inferred from NAD(P)H fluorescence. The addition of ATP to the cell suspension induced a NAD(P)H response, which replicated Ca2+c signal with some retardation of the peak and a slower decay. In the presence of increasing TPP+ concentrations, NAD(P)H responses evidenced that the rate of [Ca2+m] decay strongly decreases, while the phase of initial rise does not change. The maximal TPP+ dose did not affect [Ca2+c] and NAD(P)H fluorescence in the resting state, as well as ATP-induced [Ca2+c] responses. These data are described in a mathematical model, which accounts for Ca2+ transport through the membranes of endoplasmic reticulum and mitochondria, as well as through the plasma membrane. The model indicates a low rate of the mitochondrial cycle of Ca2+ uptake/efflux at rest and a strong activation of the uptake with increasing [Ca2+c] to which a Hill coefficient of no less than 4 corresponds. Furthermore, the rise of the uptake rate changes in a short time to a decline, and the peak of the rate is markedly ahead of the peak of [Ca2+c].

Effects of Ca2+ ions on the mobilization of Ca2+ from intracellular stores of intact and permeabilized (15 microM digitonin) Ehrlich ascites tumour cells (EATC) have been compared. For permeabilized cells, the dependences of the initial rate and amplitude of Ca2+ mobilization evoked by the addition of 100 nM inositol 1,4,5-trisphosphate (IP3) on preexisting [Ca2+] were bell-shaped within a [Ca2+] range 10(-7)-10(-6) M with the maxima at [Ca2+] = 166 nM. In intact cells, different concentrations of free cytosolic Ca2+ ([Ca2+]i) were produced using low (up to 0.005%) concentrations of digitonin which selectively increased the permeability of the plasma membrane. Stimulation of cells by exogenous ATP at [Ca2+]i = 10(-8)-10(-6) M resulted in Ca2+ mobilization the rate and amplitude of which were maximal at 102-115 nM Ca2+. The experimental Ca2+ dependences were fit by a model which includes channel opening upon Ca2+ binding and transition to the inactive states upon Ca2+ binding to the closed and open channel forms. Three inactivation types (including two particular cases) demonstrate a slight priority of inhibitory binding of Ca2+ only to the open channel, but predict markedly different parameter values. We conclude that an increase in [Ca2+] can stimulate IP3-induced mobilization, but in intact EATC, deviations of [Ca2+]i from the resting level (about 100 nM) attenuate responses to the agonist stimulation.

A model explaining quantal Ca2+ release as an intrinsic property of the inositol 1,4,5-trisphosphate (IP3) receptor has been put forward. The model is based on the hypothesis that the IP3 receptor can catalyze a transformation of the IP, molecule differing from its conventional metabolism. A simple kinetic mechanism is considered, in which IP3-induced Ca2+ channel opening is followed by the step of IP3 conversion and channel closure. Examination of the resulting mathematical model shows that it can reproduce well both partial release of stored Ca2+ and the same responsiveness to subsequent IP3 additions. On incorporation of an additional closed state of the channel, the model describes also a time-dependent channel inactivation at a high IP3 dose. Temperature sensitivity of the catalytic step accounts for the reported elimination of quantal responses and inactivation at low temperature. The transformation product is surmised to be a positional or stereo isomer of IP3.

A general case of the set of two differential equations, describing an open reaction v1 leads to S v reversible E P v2 leads to, has been considered. The requirements to the character of the functions v1([S]), v2([P]) and v([S], [P]) were formulated for the case of existence and absence of alternative steady states and sustained oscillations. The formulae were derived to determine the slope of the unstable portion of the quasi-steady state characteristic. The generalized model of Monod, Wyman and Changeux has been considered as an example of v([S], [P]). It has been shown that with monotonically decreasing v1 and monotonically increasing v2, the alternative steady states and oscillations are possible only in the presence of substrate inhibition or product activation. However, under the joint action of substrate inhibition and product activation, the system will exhibit bistability rather than an oscillatory behavior. In the case of an irreversible two-substrate reaction which can be described by a similar mathematical model, inhibition by the first and second substrate is equivalent to substrate inhibition and product activation.

A simple kinetic model was constructed to study the adaptation of cell energy metabolism to a varying loading. In this model the initiatory step of energy metabolism, in which the initial substrate S is activated at the expense of ATP molecule energy, is catalyzed by an oligomeric enzyme E dissociable at high ATP concentration to monomers E1. It is assumed that the steady state level of monomers E1 in the cell is maintained by constitutive synthesis of E1 molecules, which balances their continuous hydrolysis by proteases. The properties of the kinetic model were studied using a mathematical model which is a system of nonlinear differential equations describing the change with time of the total enzyme E concentration and the concentration of ATP. The main isoclines of this system can intersect in one, two or three points. The mathematical analysis shows that the kinetic model considered exhibits adaptive properties. A sharp increase of the ATPase activity in the model initiates a transient process which leads to a rise in the total enzyme E concentration and in the efficiency of energy metabolism. As a result, the concentration of ATP drops only slightly. The establishment of a new level of the enzyme E concentration may proceed in the oscillatory fashion.