Differential functional properties of Ca2+ stores in pulmonary arterial conduit and resistance myocytes

In smooth muscle cells, oscillations of intracellular Ca2+ concentration ([Ca2+]i) are controlled by inositol 1,4,5-trisphosphate (InsP3) and ryanodine (Ry) receptors on the sarcoplasmic reticulum (SR). Here we show that these Ca2+ oscillations are regulated differentially by InsP3 and Ry receptors in cells dispersed from the main trunk of the pulmonary artery (conduit myocytes) or from tertiary and quaternary arterial branches (resistance myocytes). Ry receptor antagonists inhibit either spontaneous or ATP-induced Ca2+ oscillations in resistance myocytes but they do not affect the oscillations in most conduit myocytes. In contrast, agents that inhibit InsP3 production or activation of InsP3 receptors do not alter the oscillations is resistance myocytes but block them in conduit myocytes. We have also examined the degree of overlap of Ry- and InsP3-sensitive stores in myocytes along the pulmonary arterial tree. In conduit myocytes, depletion of Ry-sensitive stores with repeated application of caffeine in the presence of Ry or in Ca2+ free solutions did not prevent the ATP-induced Ca2+ release from InsP3-dependent stores. However, responsiveness to ATP was completely abolished in resistance myocytes subjected to the same experimental protocol. Thus, InsP3- and Ry-dependent stores appear to be separated in conduit myocytes but joined in resistance myocytes. These data demonstrate for the first time differential properties of intracellular Ca2+ stores and receptors in myocytes distributed along the pulmonary arterial tree and help to explain the distinct functional responses of large and small pulmonary vessels to vasoactive agents.     

Differential segmental activation of Ca-dependent CIand Kchannels in pulmonary arterial myocytes

Differential segmental distribution of electrophysiologically distinct myocytes helps to explain the variability of the pulmonary arteries to vasoactive agents. We have studied whether Ca²⁺-dependent CI⁻(CI_Ca) and K⁺(K_Ca) channels are activated differentially in enzymatically dispersed conduit and resistance myocytes. We measured cytosolic [Ca²⁺] and the changes of membrane current and potential elicited by spontaneous or agonist-induced Ca²⁺oscillations. Conduit arteries contained a heterogeneous cell population with a variable mixture of K_Caand CI_Caconductances. Resistance arteries contained a more homogeneous cell population with predominance of CI_Cachannel activation. The relation between K_Caand CI_Caconductances in a given conduit myocyte determines the size of the V_mchange in response to a rise of cytosolic [Ca²⁺]. Conduit myocytes tend to hyperpolarize towards the K⁺equilibrium potential ( − 90 m V). In resistance myocytes, release of Ca²⁺from stores activates CI_Cachannels and brings V_mto a value close to the chloride equilibrium potential ( − 20 or − 30 m V) thus favouring opening of Ca²⁺channels and Ca²⁺influx. In resistance vessels CI_Cachannels contribute to link agonist-induced Ca²⁺release from stores and membrane depolarization, thus permitting protracted vasoconstriction.     

Stochastic aspects of oscillatory Ca2+ dynamics in hepatocytes

Signal-induced Ca²⁺ oscillations have been observed in many cell types and play a primary role in cell physiology. Although it is the regular character of these oscillations that first catches the attention, a closer look at time series of Ca²⁺ increases reveals that the fluctuations on the period during individual spike trains are far from negligible. Here, we perform a statistical analysis of the regularity of Ca²⁺ oscillations in norepinephrine-stimulated hepatocytes and find that the coefficient of variation lies between 10% and 15%. Stochastic simulations based on Gillespie's algorithm and considering realistic numbers of Ca²⁺ ions and inositol trisphosphate (InsP₃) receptors account for this variability if the receptors are assumed to be grouped in clusters of a few tens of channels. Given the relatively small number of clusters (∼200), the model predicts the existence of repetitive spikes induced by fluctuations (stochastic resonance). Oscillations of this type are found in hepatocytes at subthreshold concentrations of norepinephrine. We next predict with the model that the isoforms of the InsP₃ receptor can affect the variability of the oscillations. In contrast, possible accompanying InsP₃ oscillations have no impact on the robustness of signal-induced repetitive Ca²⁺ spikes.     

ATP and adenosine trigger the interaction of plasma membrane IP3 receptors with protein kinase A in oviductal ciliated cells

We have demonstrated that adenosine did not produce any change of intracellular free Ca²⁺ concentration ([Ca²⁺]i) in oviductal ciliated cells; however, it increased the ATP-induced Ca²⁺ influx through the activation of protein kinase A (PKA). Uncaging of IP₃ and cAMP triggered a larger Ca²⁺ influx than did IP₃ alone. Furthermore, the IP₃ effect was abolished by Xestospongin C, an IP₃ receptor blocker. Whole-cell recordings demonstrated the presence of an ATP-induced Ca²⁺ current, and the addition of adenosine increased the peak of this current. This effect was not observed in the presence of H-89, a PKA inhibitor. Using excised macro-patches of plasma membrane, IP₃ generated a current, which was higher in the presence of the catalytic PKA subunit and this current was blocked by Xestospongin C. We show here that activation of plasma membrane IP₃ receptors directly triggers Ca²⁺ influx in response to ATP and that these receptors are modulated by adenosine-activated PKA.     

Peculiarities of phospholipase C-dependent release of CA2+ from intracellular stores upon activation of choline and purine receptors in myocytes of the guinea-pig small intestine

Using a two-wave fluorescence probe, Fura-2, we studied changes in the intracellular concentration of calcium ions ([Ca²⁺]_i) resulting from activation of muscarinic and purine receptors in single myocytes of the guinea-pig small intestine. Applications of the respective agonists added to the normal Krebs solution (1.0, 10.0, and 100.0 μM carbachol, CCh, as well as 10.0 and 100.0 μM ATP) induced a rise in the [Ca²⁺]_i. Carbachol evoked an increase in the [Ca²⁺]_i, including two components (a rapid and a plateaulike), while ATP under analogous conditions led only to a short-lasting rise in the [Ca²⁺]_i. Transients induced by CCh or ATP applied in different concentrations, which exceeded a certain level, did not significantly differ from each other in their amplitudes, i.e., they were generated according to an all-or-none principle. In the nominally Ca-and Mg-free solution, CCh and ATP induced only rapid increases in the [Ca²⁺]_i in myocytes. The absence of the slow component in the [Ca²⁺]_i elevation upon the action of CCh under such conditions indicates that the effect of ATP, as compared with that of CCh, is not related to activation of the entry of Ca²⁺ ions into cells through voltage-operated calcium channels. After the addition of CCh, repeated application of CCh or ATP induced no effect, while application of CCh after the addition of ATP initiated a rise in the [Ca²⁺]_i. These data show that intracellular calcium stores are depleted completely upon the action of CCh, while they are depleted only partially after the action of ATP. An inhibitor of phospholipase C (PLC), U-73122 (5.0 μM), completely blocked rises in the [Ca²⁺]_i induced by both CCh and ATP; therefore, the release of Ca²⁺ ions from the intracellular calcium stores after application of these agonists is mediated by PLC. We hypothesize that the difference in the release of Ca²⁺ ions from the intracellular stores observed in our experiments upon activation of choline and purine receptors (partial and complete depletion of the stores upon the action of ATP and CCh, respectively) is responsible for the opposite functional effects of the above-mentioned neurotransmitters on smooth muscles. Neirofiziologiya/Neurophysiology, Vol. 38, No. 1, pp. 3–10, January–February, 2006.     

Evaluation of developmental competence,nuclear and ooplasmic maturation of calf oocytes

In this study we evaluated nuclear and ooplasmic maturation of prepuberal calf oocytes to determine a possible cause for their low developmental competency. Calf oocytes resumed meiosis and arrested at the MII stage at rates similar to that of adult animals; however, zygotes derived from calf oocytes cleaved and developed at significantly lower rates. Ooplasmic maturation was assessed during oocyte maturation and fertilization. Transmission electron microscopy revealed that a majority of calf oocytes exhibited some delay in organelle migration and redistribution following maturation. Immunofluorescence microscopy showed that following IVF, a higher percentage of calf oocytes had abnormal chromatin and microtubule configurations than those of adult cattle. These anomalies were characterized by delayed formation of sperm aster and asynchronous pronuclear formation. Microfluorometry was used to characterize the Ca²⁺ responses of calf oocytes to the addition of agonists or after IVF. The addition of thimerosal demonstrated the presence of Ca²⁺ stores in calf oocytes. Injection of near threshold concentrations of inositol 1,4,5-trisphosphate (InsP₃), used to test the sensitivity of the InsP₃R, released significantly less Ca²⁺ in calf than in cow oocytes, whereas higher concentrations of InsP₃ (500 μM) released maximal [Ca²⁺]i in both oocytes. These results suggested that the Ca²⁺ content of intracellular stores was similar, but the sensitivity of the InsP₃R may be different. Following insemination, calf oocytes exhibiting [Ca²⁺]i oscillations displayed comparable amplitude and intervals to cow oocytes; however, a significantly higher number of fertilized calf oocytes failed to show oscillations. Our findings suggest that the low developmental competence of calf oocytes can be attributed, at least in part, to incomplete or delayed ooplasmic maturation. © 1996 Wiley-Liss, Inc.     

Regulation of Ca2+ Release by InsP3 in Single Guinea Pig Hepatocytes and Rat Purkinje Neurons

The repetitive spiking of free cytosolic [Ca²⁺] ([Ca²⁺]_i) during hormonal activation of hepatocytes depends on the activation and subsequent inactivation of InsP₃-evoked Ca²⁺ release. The kinetics of both processes were studied with flash photolytic release of InsP₃ and time resolved measurements of [Ca²⁺]_i in single cells. InsP₃ evoked Ca²⁺ flux into the cytosol was measured as d[Ca²⁺]_i/dt, and the kinetics of Ca²⁺ release compared between hepatocytes and cerebellar Purkinje neurons. In hepatocytes release occurs at InsP₃ concentrations greater than 0.1–0.2 μM. A comparison with photolytic release of metabolically stable 5-thio-InsP₃ suggests that metabolism of InsP₃ is important in determining the minimal concentration needed to produce Ca²⁺ release. A distinct latency or delay of several hundred milliseconds after release of low InsP₃ concentrations decreased to a minimum of 20–30 ms at high concentrations and is reduced to zero by prior increase of [Ca²⁺]_i, suggesting a cooperative action of Ca²⁺ in InsP₃ receptor activation. InsP₃-evoked flux and peak [Ca²⁺]_i increased with InsP₃ concentration up to 5–10 μM, with large variation from cell to cell at each InsP₃ concentration. The duration of InsP₃-evoked flux, measured as 10–90% risetime, showed a good reciprocal correlation with d[Ca²⁺]_i/dt and much less cell to cell variation than the dependence of flux on InsP₃ concentration, suggesting that the rate of termination of the Ca²⁺ flux depends on the free Ca²⁺ flux itself. Comparing this data between hepatocytes and Purkinje neurons shows a similar reciprocal correlation for both, in hepatocytes in the range of low Ca²⁺ flux, up to 50 μM · s⁻¹ and in Purkinje neurons at high flux up to 1,400 μM · s⁻¹. Experiments in which [Ca²⁺]_i was controlled at resting or elevated levels support a mechanism in which InsP₃-evoked Ca²⁺ flux is inhibited by Ca²⁺ inactivation of closed receptor/channels due to Ca²⁺ accumulation local to the release sites. Hepatocytes have a much smaller, more prolonged InsP₃-evoked Ca²⁺ flux than Purkinje neurons. Evidence suggests that these differences in kinetics can be explained by the much lower InsP₃ receptor density in hepatocytes than Purkinje neurons, rather than differences in receptor isoform, and, more generally, that high InsP₃ receptor density promotes fast rising, rapidly inactivating InsP₃-evoked [Ca²⁺]_i transients.     

A re-evaluation of the apparent effects of luminal Ca on inositol 1,4,5-trisphosphate-induced Ca release

It has been suggested that the release of Ca²⁺ from intracellular stores by inositol 1,4,5-trisphosphate (InsP₃) is modulated by the luminal Ca²⁺ content of the stores and that such an effect could underlie the apparent ‘quantal’ nature of InsP3-induced release. Although initial studies failed to find evidence in support of such a modulation, several subsequent reports have indicated luminal Ca²⁺ effects that become apparent only after a greater than 70–75% depletion of Ca²⁺ stores. In these studies, Ca²⁺ release was expressed as a percentage of an A23187-releasable pool which comprised both InsP₃-sensi-tive and InsP₃-insensitive components. In model calculations we have found that the presence of even a minor InsP₃-insensitive component in the total Ca²⁺ pool significantly distorts interpretation of the data. We show that the published results can be accurately duplicated without any requirement for a shift in the true InsP3 sensitivity of Ca²⁺ release if either: (a) the InsP₃-insensitive component does not remain a constant proportion of the total pool during depletion (i.e. depletion disproportionally affects the InsP₃-sensitive component); or (b) during generation of InsP₃-response curves, additional Cal ²⁺ is released from the InsP₃-insensitive component as the InsP₃-sensitive component is progressively emptied. Examination indicates that either, or both, of these conditions apply in the published reports and we conclude that the demonstrated effects of luminal Ca²⁺ may be artifacts.     

Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) and Ca2+ Mobilization

Many physiological processes are controlled by a great diversity of Ca^{2 +} signals that depend on Ca^{2 +} entry into the cell and/or Ca^{2 +} release from internal Ca^{2 +} stores. Ca^{2 +} mobilization from intracellular stores is gated by a family of messengers including inositol-1,4,5-trisphosphate (InsP₃), cyclic ADP-ribose (cADPR), and nicotinic acid adenine dinucleotide phosphate (NAADP). There is increasing evidence for a novel intracellular Ca^{2 +} release channel that may be targeted by NAADP and that displays properties distinctly different from the well-characterized InsP₃ and ryanodine receptors. These channels appear to localize on a wider range of intracellular organelles, including the acidic Ca^{2 +} stores. Activation of the NAADP-sensitive Ca^{2 +} channels evokes complex changes in cytoplasmic Ca^{2 +} levels by means of channel chatter with other intracellular Ca^{2 +} channels. The recent demonstration of changes in intracellular NAADP levels in response to physiologically relevant extracellular stimuli highlights the significance of NAADP as an important regulator of intracellular Ca^{2 +} signaling.     

Investigation of the role of sigma1-receptors in inositol 1,4,5-trisphosphate dependent calcium signaling in hepatocytes

In hepatocytes, as in other cell types, Ca²⁺ signaling is subject to complex regulations, which result largely from the intrinsic characteristics of the different inositol 1,4,5-trisphosphate receptor (InsP₃R) isoforms and from their interactions with other proteins. Although sigma1 receptors (Sig-1Rs) are widely expressed in the liver, their involvement in hepatic Ca²⁺ signaling remains unknown. We here report that in this cell type Sig-1R interact with type 1 isoforms of the InsP₃ receptors (InsP₃R-1). These results obtained by immunoprecipitation experiments are confirmed by the observation that Sig-1R proteins and InsP₃R-1 colocalize in hepatocytes. However, Sig-1R ligands have no effect on InsP₃-induced Ca²⁺ release in hepatocytes. This can be explained by the rather low expression level expression of InsP₃R-1. In contrast, we find that Sig-1R ligands can inhibit agonist-induced Ca²⁺ signaling via an inhibitory effect on InsP₃ synthesis. We show that this inhibition is due to the stimulation of PKC activity by Sig-1R, resulting in the well-known down-regulation of the signaling pathway responsible for the transduction of the extracellular stimulus into InsP₃ synthesis. The PKC sensitive to Sig-1R activity belongs to the family of conventional PKC, but the precise molecular mechanism of this regulation remains to be elucidated.     

Muscarinic Receptor-Mediated Inositol 1,4,5-Trisphosphate Formation in SH-SY5Y Neuroblastoma Cells Is Regulated Acutely by Cytosolic Ca2+ and by Rapid Desensitization

Abstract: Stimulation of muscarinic receptors expressed in SH-SY5Y human neuroblastoma cells resulted in a complex profile of inositol 1,4,5-trisphosphate (InsP₃) accumulation, with a dramatic increase (six- to eightfold) over the first 10 s (the “peak” phase) and subsequently, from ～60 s onward, maintained at a lower but sustained level (the “plateau” phase). Chelation of extracellular Ca²⁺ with EGTA or inhibition of Ca²⁺ channels with Ni²⁺ showed that the plateau phase was dependent upon Ca²⁺ entry. Furthermore, use of thapsigargin and EGTA to discharge and sequester Ca²⁺ from intracellular stores revealed that Ca²⁺ from this source was capable of supporting the peak phase of the InsP₃ response. Carbachol-stimulated phosphoinositidase C activity in permeabilized SH-SY5Y cells was also shown to be highly dependent on free Ca²⁺ concentration (20–100 nM) and suggests that under normal conditions, InsP₃ formation is enhanced by increases in cytosolic free Ca²⁺ concentration that accompany muscarinic receptor activation. Measurement of carbachol-stimulated total inositol phosphate accumulation in the presence of Li⁺ indicated that the initial rate of phosphoinositide hydrolysis (from 0 to 30 s) was about fivefold greater than that from 30 to 300 s. This rapid but partial desensitization of receptor-mediated phosphoinositide hydrolysis provides strong evidence for the mechanism underlying the changes in InsP₃ accumulation over this time. Because very similar data were obtained in Chinese hamster ovary cells transfected with human m3 receptor cDNA, we suggest that although increases in cytosolic free Ca²⁺ concentration amplify InsP₃ formation during stimulation of m3 muscarinic receptors, the primary factor that governs the profile of InsP₃ accumulation is rapid, but partial, desensitization. Such desensitization does not appear to be mediated by changes in cytosolic Ca²⁺ or protein kinase C activity.     

Calcineurin is part of a negative feedback loop in the InsP3/Ca signalling pathway in blowfly salivary glands

The ubiquitous InsP₃/Ca²⁺ signalling pathway is modulated by diverse mechanisms, i.e. feedback of Ca²⁺ and interactions with other signalling pathways. In the salivary glands of the blowfly Calliphora vicina, the hormone serotonin (5-HT) causes a parallel rise in intracellular [Ca²⁺] and [cAMP] via two types of 5-HT receptors. We have shown recently that cAMP/protein kinase A (PKA) sensitizes InsP₃-induced Ca²⁺ release. We have now identified the protein phosphatase that counteracts the effect of PKA on 5-HT-induced InsP₃/Ca²⁺ signalling. We demonstrate that (1) tautomycin and okadaic acid, inhibitors of protein phosphatases PP1 and PP2A, have no effect on 5-HT-induced Ca²⁺ signals; (2) cyclosporin A and FK506, inhibitors of Ca²⁺/calmodulin-activated protein phosphatase calcineurin, cause an increase in the frequency of 5-HT-induced Ca²⁺ oscillations; (3) the sensitizing effect of cyclosporin A on 5-HT-induced Ca²⁺ responses does not involve Ca²⁺ entry into the cells; (4) cyclosporin A increases InsP₃-dependent Ca²⁺ release; (5) inhibition of PKA abolishes the effect of cyclosporin A on the 5-HT-induced Ca²⁺ responses, indicating that PKA and calcineurin act antagonistically on the InsP₃/Ca²⁺ signalling pathway. These findings suggest that calcineurin provides a negative feedback on InsP₃/Ca²⁺ signalling in blowfly salivary glands, counteracting the effect of PKA and desensitizing the signalling cascade at higher 5-HT concentrations.     

Involvement of Ca<Superscript>2+</Superscript> signaling in tachykinin-mediated contractile responses in swine trachea

Summary Neuropeptide tachykinins, present within sensory nerves, have been implicated as neurotransmitters involved in nonadrenergic and noncholinergic airway muscle contraction. The signal transduction pathways of tachykinins on muscle contraction and Ca²⁺ mobilization were investigated in swine trachea. Tachykinins, substance P (SP) and neurokinin A (NKA), concentration (1 nM to 1 μM)-dependently induced contractile responses with removal of epithelium, whereas neurokinin B (NKB) did not alter the muscle tension. The SP- and NKA-evoked muscle contractions were inhibited by NK1-R antagonist L732138, but not by either NK2-R antagonist MDL29913 or NK3-R antagonist SB218795. Consistently, SP-elicited increase in [Ca²⁺]_i was abolished by NK1-R antagonist, neither by NK2-R nor NK3-R antagonists. The SP-induced muscular responses were significantly inhibited by L-type Ca²⁺ channel blocker verapamil and withdrawal of external Ca²⁺. Caffeine (10 mM) or ryanodine (50 μM) also partly suppressed the SP-induced muscle responses. Inhibition of inositol 1,4,5-trisphosphate (InsP₃) receptor with 2-APB (75 μM) potently attenuated SP-evoked Ca²⁺ mobilization and muscle contraction, which was further inhibited by 2-APB under Ca²⁺-free external solution, but not completely. Unexpectedly, simultaneous blockade of InsP₃ receptor and ryanodine receptor (RyR) by 2-APB and ryanodine enhanced SP-evoked muscle contraction and Ca²⁺ mobilization. This potentiation was virtually abolished by removal of external Ca²⁺, suggesting native Ca²⁺ channels may contribute to this phenomenon. These results demonstrate that tachykinins produce a potent muscle contraction associated with Ca²⁺ mobilization via tachykinin NK1- R-dependent activation of multiple signal transduction pathways involving Ca²⁺ influx and release of Ca²⁺ from InsP₃- and ryanodine-sensitive Ca²⁺ stores. Blockade of both InsP₃ receptor and RyR enhances the Ca²⁺ influx through native Ca²⁺ channels in plasma membrane, which is crucial to Ca²⁺ signaling in response to NK1 receptor activation.     

Ca2+ dependence of inositol 1,4,5-trisphosphate-induced Ca2+ release in renal epithelial LLC-PK1 cells

We have studied arginine vasopressin (AVP)-, thapsigargin- and inositol 1,4,5-trisphosphate (InsP₃)-mediated Ca²⁺ release in renal epithelial LLC-PK₁ cells. AVP-induced changes in the intracellular free calcium concentration ([Ca²⁺]_i) were studied in indo-1 loaded single cells by confocal laser cytometry. AVP-mediated Ca²⁺ mobilization was also observed in the absence of extracellular Ca²⁺, but was completely abolished after depletion of the intracellular Ca²⁺ stores by 2 μM thapsigargin. Using ⁴⁵Ca²⁺ fluxes in saponin-permeabilized cell monolayers, we have analysed how InsP₃ affected the Ca²⁺ content of nonmitochondrial Ca²⁺ pools in different loading and release conditions. Less than 10% of the Ca²⁺ was taken up in a thapsigargin-insensitive pool when loading was performed in a medium containing 0.1 μM Ca²⁺. The thapsigargin-insensitive compartment amounted to 35% in the presence of 110 μM Ca²⁺, but Ca²⁺ sequestered in this pool could not be released by InsP₃. The thapsigargin-sensitive Ca²⁺ pool, in contrast, was nearly completely InsP₃ sensitive. A submaximal [InsP₃], however, released only a fraction of the sequestered Ca²⁺. This fraction was dependent on the cytosolic as well as on the luminal [Ca²⁺]. The cytosolic free [Ca²⁺] affected the InsP₃-induced Ca²⁺ release in a biphasic way. Maximal sensitivity toward InsP₃ was found at a free cytosolic [Ca²⁺] between 0.1 and 0.5 μM, whereas higher cytosolic [Ca²⁺] decreased the InsP₃ sensitivity. Other divalent cations or La³⁺ did not provoke similar inhibitory effects on InsP₃-induced Ca²⁺ release. The luminal free [Ca²⁺] was manipulated by varying the time of incubation of Ca²⁺ -loaded cells in an EGTA-containing medium. Reduction of the Ca²⁺ content to one-third of its initial value resulted in a fivefold decrease in the InsP₃ sensitivity of the Ca²⁺ release. © 1993 Wiley-Liss, Inc.     

Elevated InsP3R expression underlies enhanced calcium fluxes and spontaneous extra-systolic calcium release events in hypertrophic cardiac myocytes

Cardiac hypertrophy is associated with profound remodelling of Ca²⁺ signalling pathways. During the early, compensated stages of hypertrophy, Ca²⁺ fluxes may be enhanced to facilitate greater contraction, whereas as the hypertrophic heart decompensates, Ca²⁺ homeostatic mechanisms are dysregulated leading to decreased contractility, arrhythmia and death. Although ryanodine receptor Ca²⁺ release channels (RyR) on the sarcoplasmic reticulum (SR) intracellular Ca²⁺ store are primarily responsible for the Ca²⁺ flux that induces myocyte contraction, a role for Ca²⁺ release via the inositol 1,4,5-trisphosphate receptor (InsP₃R) in cardiac physiology has also emerged. Specifically, InsP₃-induced Ca²⁺ signals generated following myocyte stimulation with an InsP₃-generating agonist (e.g. endothelin, ET-1), lead to modulation of Ca²⁺ signals associated with excitation-contraction coupling (ECC) and the induction of spontaneous Ca²⁺ release events that cause cellular arrhythmia. Using myocytes from spontaneously hypertensive rats (SHR), we recently reported that expression of the type 2 InsP₃R (InsP₃R2) is significantly increased during hypertrophy. Notably, this increased expression was restricted to the junctional SR in close proximity to RyRs. There, enhanced Ca²⁺ release via InsP₃Rs serves to sensitise neighbouring RyRs causing an augmentation of Ca²⁺ fluxes during ECC as well as an increase in non-triggered Ca²⁺ release events. Although the sensitization of RyRs may be a beneficial consequence of elevated InsP₃R expression during hypertrophy, the spontaneous Ca²⁺ release events are potentially of pathological significance giving rise to cardiac arrhythmia. InsP₃R2 expression was also increased in hypertrophic hearts from patients with ischemic dilated cardiomyopathy and aortically-banded mice demonstrating that increased InsP₃R expression may be a general phenomenon that underlies Ca²⁺ changes during hypertrophy.     

Latrunculin A depolarizes starfish oocytes

Depolymerization of the actin cytoskeleton may liberate Ca²⁺ from InsP₃-sensitive stores in some cell types, including starfish oocytes, while inhibiting Ca²⁺ influx in others. However, no information is available on the modulation of membrane potential (V_m) by actin. The present study was aimed to ascertain whether the widely employed actin depolymerizing drug, latrunculin A (Lat A), affects V_m in mature oocytes of the starfish Astropecten aranciacus. Lat A induced a membrane depolarization which was mimicked by cytochalasin D, another popular actin disruptor, and prevented by jasplakinolide, a stabilizer of the actin network. Lat A-elicited depolarization consisted in a positive shift in V_m which reached the threshold of activation of voltage-gated Ca²⁺ channels (VGCC), thus triggering an action potential. Lat A-promoted depolarization lacked the action potential in Ca²⁺-free sea water, while it was abolished upon removal of external Na⁺. Moreover, membrane depolarization was prevented by pre-injection of BAPTA and heparin, but not ryanodine. These data indicate that Lat A induces a membrane depolarization by releasing Ca²⁺ from InsP₃Rs. The Ca²⁺ signal in turn activates a Ca²⁺-dependent Na⁺ entry, which causes the positive shift in V_m and stimulates the VGCC.     

A model for Ca2+ oscillations stimulated by the type 5 metabotropic glutamate receptor: an unusual mechanism based on repetitive, reversible phosphorylation of the receptor

In parallel with experimental investigations, the molecular mechanisms responsible for Ca²⁺ oscillations have been much investigated with computational models. In the vast majority of cell-types, these oscillations rely on the biphasic regulation of the inositol 1,4,5-trisphosphate (InsP₃) receptor by cytosolic Ca²⁺. However, when Ca²⁺ oscillations are initiated by agonist stimulation of the type 5 metabotropic glutamate (mGlu5) receptor, oscillatory behaviour is tightly controlled by repetitive cycles of receptor phosphorylation/dephosphorylation leading to the periodic activation/deactivation of the G protein-activated signalling cascade downstream of this G protein-coupled receptor. We present a minimal model for mGlu5 receptor-induced Ca²⁺ oscillations, taking into account receptor phosphorylation by a protein kinase C isoenzyme sensitive to diacylglycerol but not to Ca²⁺. Depending on the density of receptors and the level of stimulation, the model reproduces Ca²⁺ oscillations based on either a ‘dynamic uncoupling’ mechanism or InsP₃ receptor dynamics. When based on the former mechanism, Ca²⁺ oscillation frequency is insensitive to the level of stimulation, while the level of receptor expression is a determinant of oscillation frequency. When investigating the conditions for the occurrence of oscillations, the model predicts that dynamic uncoupling likely relies on a steep relationship between the activity of PKC and the amount of phosphorylated mGlu5 receptor. Finally, we use the model to simulate the adaptation of the signalling pathway during periods of prolonged stimulation associated with receptor desensitization/internalization. The model suggests that the existence of both oscillatory mechanisms could allow for a significant lengthening of the repetitive Ca²⁺ responses under these conditions.     

InsP3R-associated cGMP Kinase Substrate Determines Inositol 1,4,5-Trisphosphate Receptor Susceptibility to Phosphoregulation by Cyclic Nucleotide-dependent Kinases

Ca²⁺ release through inositol 1,4,5-trisphosphate receptors (InsP₃R) can be modulated by numerous factors, including input from other signal transduction cascades. These events shape the spatio-temporal characteristics of the Ca²⁺ signal and provide fidelity essential for the appropriate activation of effectors. In this study, we investigate the regulation of Ca²⁺ release via InsP₃R following activation of cyclic nucleotide-dependent kinases in the presence and absence of expression of a binding partner InsP₃R-associated cGMP kinase substrate (IRAG). cGMP-dependent kinase (PKG) phosphorylation of only the S2+ InsP₃R-1 subtype resulted in enhanced Ca²⁺ release in the absence of IRAG expression. In contrast, IRAG bound to each InsP₃R subtype, and phosphorylation of IRAG by PKG attenuated Ca²⁺ release through all InsP₃R subtypes. Surprisingly, simply the expression of IRAG attenuated phosphorylation and inhibited the enhanced Ca²⁺ release through InsP₃R-1 following cAMP-dependent protein kinase (PKA) activation. In contrast, IRAG expression did not influence the PKA-enhanced activity of the InsP₃R-2. Phosphorylation of IRAG resulted in reduced Ca²⁺ release through all InsP₃R subtypes during concurrent activation of PKA and PKG, indicating that IRAG modulation is dominant under these conditions. These studies yield mechanistic insight into how cells with various complements of proteins integrate and prioritize signals from ubiquitous signaling pathways.     

Bursting, chaos and birhythmicity originating from self-modulation of the inositol 1,4,5-trisphosphate signal in a model for intracellular Ca2+ oscillations

We investigate the various types of complex Ca²⁺ oscillations which can arise in a model based on the mechanism of Ca²⁺-induced Ca²⁺ release (CICR), that takes into account the Ca²⁺-stimulated degradation of inositol 1,4,5-trisphosphate (InsP₃) by a 3-kinase. This model was previously proposed in the course of an investigation of plausible mechanisms capable of generating complex Ca²⁺ oscillations (Borghans et al., 1997). Besides simple periodic behavior, this model for cytosolic Ca²⁺ oscillations in nonexcitable cells shows complex oscillatory phenomena like bursting or chaos. We show that the model also admits a coexistence between two stable regimes of sustained oscillations (birhythmicity). The occurrence of these various modes of oscillatory behavior is analysed by means of bifurcation diagrams. Complex oscillations are characterized by means of Poincaré sections, power spectra and Lyapounov exponents. The results point to the role of self-modulation of the InsP₃ signal by 3-kinase as a possible source for complex temporal patterns in Ca²⁺ signaling.