Agonist-Evoked Ca2+ Mobilization from Stores Expressing Inositol 1,4,5-Trisphosphate Receptors and Ryanodine Receptors in Cerebellar Granule Neurones

Abstract: The mechanisms involved in Ca²⁺ mobilization evoked by the muscarinic cholinoceptor (mAChR) agonist carbachol (CCh) and N-methyl-d -aspartate (NMDA) in cerebellar granule cells have been investigated. An initial challenge with caffeine greatly reduced the subsequent intracellular Ca²⁺ concentration ([Ca²⁺]_i) response to CCh (to 45 ± 19% of the control), and, similarly, a much reduced caffeine response was detectable after prior stimulation with CCh (to 27 ± 6% of the control). CCh-evoked [Ca²⁺]_i responses were inhibited by preincubation with thapsigargin (10 µM), 2,5-di(tert-butyl)-1,4-benzohydroquinone (BHQ; 25 µM), ryanodine (10 µM), or dantrolene (25 µM). BHQ pretreatment was found to have no effect on the sustained phase of the NMDA-evoked [Ca²⁺]_i response. Both CCh (1 mM) and 1-aminocyclopentane-1S,3R-dicarboxylic acid (ACPD; 200 µM) evoked a much diminished increase in [Ca²⁺]_i in granule cells pretreated with CCh for 24 h compared with vehicle-treated control cells (CCh, 23 ± 14%; ACPD, 27 ± 1% of respective control values). In contrast, a 24-h CCh pretreatment decreased the subsequent inositol 1,4,5-trisphosphate (InsP₃) response to CCh to a much greater extent compared with responses evoked by metabotropic glutamate receptor (mGluR) agonists; this suggests that the former effect on Ca²⁺ mobilization represents a heterologous desensitization of the mGluR-mediated response distal to the pathway second messenger. Furthermore, [Ca²⁺]_i responses to caffeine and NMDA were unaffected by a 24-h pretreatment with CCh. This study indicates that ryanodine receptors, as well as InsP₃ receptors, appear to be crucial to the mAChR-mediated [Ca²⁺]_i response in granule cells. As BHQ apparently differentiates between the CCh- and NMDA-evoked responses, it is possible that the directly InsP₃-sensitive pool is physically different from the ryanodine receptor pool. Also, activation of InsP₃ receptors may not contribute significantly to NMDA-evoked elevation of [Ca²⁺]_i in cerebellar granule cells. A model for the topographic organization of cerebellar granule cell Ca²⁺ stores is proposed.     

Distinct Mechanisms of [Ca2+]i Oscillations in HSY and HSG Cells: Role of Ca2+ Influx and Internal Ca2+ Store Recycling

This study examined [Ca²⁺]_i oscillations in the human salivary gland cell lines, HSY and HSG. Relatively low concentrations of carbachol (CCh) induced oscillatory, and higher [CCh] induced sustained, steady-state increases in [Ca²⁺]_i and K _Ca currents in both cell types. Low IP₃, but not thapsigargin (Tg), induced [Ca²⁺]_i oscillations, whereas Tg blocked CCh-stimulated [Ca²⁺]_i oscillations in both cell types. Unlike in HSG cells, removal of extracellular Ca²⁺ from HSY cells (i) did not affect CCh-stimulated [Ca²⁺]_i oscillations or internal Ca²⁺ store refill, and (ii) converted high [CCh]-induced steady-state increase in [Ca²⁺]_i into oscillations. CCh- or thapsigargin-induced Ca²⁺ influx was higher in HSY, than in HSG, cells. Importantly, HSY cells displayed relatively higher levels of sarcoendoplasmic reticulum Ca²⁺ pump (SERCA) and inositoltrisphosphate receptors (IP₃Rs) than HSG cells. These data demonstrate that [Ca²⁺]_i oscillations in both HSY and HSG cells are primarily determined by the uptake of Ca²⁺ from, and release of Ca²⁺ into, the cytosol by the SERCA and IP₃R activities, respectively. In HSY cells, Ca²⁺ influx does not acutely contribute to this process, although it determines the steady-state increase in [Ca²⁺]_i. In HSG cells, [Ca²⁺]_i oscillations directly depend on Ca²⁺ influx; Ca²⁺ coming into the cell is rapidly taken up into the store and then released into the cytosol. We suggest that the differences in the mechanism of [Ca²⁺]_i oscillations HSY and HSG cells is related to their respective abilities to recycle internal Ca²⁺ stores. Received: 30 October 2000/Revised: 26 February 2001     

Enhanced ER Ca2+ store filling by overexpression of SERCA2b promotes IP3-evoked puffs

Liberation of Ca²⁺ from the endoplasmic reticulum (ER) through inositol trisphosphate receptors (IP₃R) is modulated by the ER Ca²⁺ content, and overexpression of SERCA2b to accelerate Ca²⁺ sequestration into the ER has been shown to potentiate the frequency and amplitude of IP₃-evoked Ca²⁺ waves in Xenopus oocytes. Here, we examined the effects of SERCA overexpression on the elementary IP₃-evoked puffs to elucidate whether ER [Ca²⁺] may modulate IP₃R function via luminal regulatory sites in addition to simply determining the size of the available store and electrochemical driving force for Ca²⁺ release. SERCA2b and Ca²⁺ permeable nicotinic plasmalemmal channels were expressed in oocytes, and hyperpolarizing pulses were delivered to induce Ca²⁺ influx and thereby load ER stores. Puffs evoked by photoreleased IP₃ were significantly potentiated in terms of numbers of responding sites, frequency and amplitude following transient Ca²⁺ influx in SERCA-overexpressing cells, whereas little change was evident with SERCA overexpression alone or following Ca²⁺ influx in control cells not overexpressing SERCA. Intriguingly, we observed the appearance of a new population of puffs that arose after long latencies and had prolonged durations supporting the notion of luminal regulation of IP₃R gating kinetics.     

A novel multi lines analysis tool of Ca2+ dynamics reveals the nonuniformity of Ca2+ propagation

Extracellular stimuli evoke a robust increase in the concentration of intracellular Ca²⁺ ([Ca²⁺]_c) throughout the cell to trigger various cellular responses, such as gene expression and apoptosis. This robust expansion of [Ca²⁺]_c is called Ca²⁺ propagation. To date, it is thought that intracellular second messengers, such as inositol 1,4,5-trisphosphate (IP₃) and intracellular Ca²⁺, and clusters of IP₃ receptors (IP₃Rs) regulate Ca²⁺ propagation. However, little is known about how the elevation in the [Ca²⁺]_c spreads throughout the cell, especially in non-polar cell, including HeLa cell. In this study, we developed a novel multi lines analysis tool. This tool revealed that the velocity of Ca²⁺ propagation was inconstant throughout cell and local concentration of intracellular Ca²⁺ did not contribute to the velocity of Ca²⁺ propagation. Our results suggest that intracellular Ca²⁺ propagation is not merely the result of diffusion of intracellular Ca²⁺, and that, on the contrary, intracellular Ca²⁺ propagation seems to be regulated by more complicated processes.     

Differential Regulation of Multiple Steps in Inositol 1,4,5-Trisphosphate Signaling by Protein Kinase C Shapes Hormone-stimulated Ca2+ Oscillations

How Ca²⁺ oscillations are generated and fine-tuned to yield versatile downstream responses remains to be elucidated. In hepatocytes, G protein-coupled receptor-linked Ca²⁺ oscillations report signal strength via frequency, whereas Ca²⁺ spike amplitude and wave velocity remain constant. IP₃ uncaging also triggers oscillatory Ca²⁺ release, but, in contrast to hormones, Ca²⁺ spike amplitude, width, and wave velocity were dependent on [IP₃] and were not perturbed by phospholipase C (PLC) inhibition. These data indicate that oscillations elicited by IP₃ uncaging are driven by the biphasic regulation of the IP₃ receptor by Ca²⁺, and, unlike hormone-dependent responses, do not require PLC. Removal of extracellular Ca²⁺ did not perturb Ca²⁺ oscillations elicited by IP₃ uncaging, indicating that reloading of endoplasmic reticulum stores via plasma membrane Ca²⁺ influx does not entrain the signal. Activation and inhibition of PKC attenuated hormone-induced Ca²⁺ oscillations but had no effect on Ca²⁺ increases induced by uncaging IP₃. Importantly, PKC activation and inhibition differentially affected Ca²⁺ spike frequencies and kinetics. PKC activation amplifies negative feedback loops at the level of G protein-coupled receptor PLC activity and/or IP₃ metabolism to attenuate IP₃ levels and suppress the generation of Ca²⁺ oscillations. Inhibition of PKC relieves negative feedback regulation of IP₃ accumulation and, thereby, shifts Ca²⁺ oscillations toward sustained responses or dramatically prolonged spikes. PKC down-regulation attenuates phenylephrine-induced Ca²⁺ wave velocity, whereas responses to IP₃ uncaging are enhanced. The ability to assess Ca²⁺ responses in the absence of PLC activity indicates that IP₃ receptor modulation by PKC regulates Ca²⁺ release and wave velocity.     

IP3 receptors and Ca2+ entry

Inositol 1,4,5-trisphosphate receptors (IP₃R) are the most widely expressed intracellular Ca²⁺ release channels. Their activation by IP₃ and Ca²⁺ allows Ca²⁺ to pass rapidly from the ER lumen to the cytosol. The resulting increase in cytosolic [Ca²⁺] may directly regulate cytosolic effectors or fuel Ca²⁺ uptake by other organelles, while the decrease in ER luminal [Ca²⁺] stimulates store-operated Ca²⁺ entry (SOCE). We are close to understanding the structural basis of both IP₃R activation, and the interactions between the ER Ca²⁺-sensor, STIM, and the plasma membrane Ca²⁺ channel, Orai, that lead to SOCE. IP₃Rs are the usual means through which extracellular stimuli, through ER Ca²⁺ release, stimulate SOCE. Here, we review evidence that the IP₃Rs most likely to respond to IP₃ are optimally placed to allow regulation of SOCE. We also consider evidence that IP₃Rs may regulate SOCE downstream of their ability to deplete ER Ca²⁺ stores. Finally, we review evidence that IP₃Rs in the plasma membrane can also directly mediate Ca²⁺ entry in some cells.     

Voltage-Induced Ca2+ Release in Postganglionic Sympathetic Neurons in Adult Mice

Recent studies have provided evidence that depolarization in the absence of extracellular Ca²⁺ can trigger Ca²⁺ release from internal stores in a variety of neuron subtypes. Here we examine whether postganglionic sympathetic neurons are able to mobilize Ca²⁺ from intracellular stores in response to depolarization, independent of Ca²⁺ influx. We measured changes in cytosolic ΔF/F₀ in individual fluo-4 –loaded sympathetic ganglion neurons in response to maintained K⁺ depolarization in the presence (2 mM) and absence of extracellular Ca²⁺ ([Ca²⁺]_e). Progressive elevations in extracellular [K⁺]_e caused increasing membrane depolarizations that were of similar magnitude in 0 and 2 mM [Ca²⁺]_e. Peak amplitude of ΔF/F₀ transients in 2 mM [Ca²⁺]_e increased in a linear fashion as the membrane become more depolarized. Peak elevations of ΔF/F₀ in 0 mM [Ca²⁺]_e were ~5–10% of those evoked at the same membrane potential in 2 mM [Ca²⁺]_e and exhibited an inverse U-shaped dependence on voltage. Both the rise and decay of ΔF/F₀ transients in 0 mM [Ca²⁺]_e were slower than those of ΔF/F₀ transients evoked in 2 mM [Ca²⁺]_e. Rises in ΔF/F₀ evoked by high [K⁺]_e in the absence of extracellular Ca²⁺ were blocked by thapsigargin, an inhibitor of endoplasmic reticulum Ca²⁺ ATPase, or the inositol 1,4,5-triphosphate (IP₃) receptor antagonists 2-aminoethoxydiphenyl borate and xestospongin C, but not by extracellular Cd²⁺, the dihydropyridine antagonist nifedipine, or by ryanodine at concentrations that caused depletion of ryanodine-sensitive Ca²⁺ stores. These results support the notion that postganglionic sympathetic neurons possess the ability to release Ca²⁺ from IP₃-sensitive internal stores in response to membrane depolarization, independent of Ca²⁺ influx.     

Propagation of fast and slow intercellular Ca(2+) waves in primary cultured arterial smooth muscle cells

Smooth muscle contraction is regulated by changes in cytosolic Ca²⁺ concentration ([Ca²⁺]_i). In response to stimulation, Ca²⁺ increase in a single cell can propagate to neighbouring cells through gap junctions, as intercellular Ca²⁺ waves. To investigate the mechanisms underlying Ca²⁺ wave propagation between smooth muscle cells, we used primary cultured rat mesenteric smooth muscle cells (pSMCs). Cells were aligned with the microcontact printing technique and a single pSMC was locally stimulated by mechanical stimulation or by microejection of KCl. Mechanical stimulation evoked two distinct Ca²⁺ waves: (1) a fast wave (2 mm/s) that propagated to all neighbouring cells, and (2) a slow wave (20 μm/s) that was spatially limited in propagation. KCl induced only fast Ca²⁺ waves of the same velocity as the mechanically induced fast waves. Inhibition of gap junctions, voltage-operated calcium channels, inositol 1,4,5-trisphosphate (IP₃) and ryanodine receptors, shows that the fast wave was due to gap junction mediated membrane depolarization and subsequent Ca²⁺ influx through voltage-operated Ca²⁺ channels, whereas, the slow wave was due to Ca²⁺ release primarily through IP₃ receptors. Altogether, these results indicate that temporally and spatially distinct mechanisms allow intercellular communication between SMCs. In intact arteries this may allow fine tuning of vessel tone.     

Modeling Ca2+ feedback on a single inositol 1,4,5-trisphosphate receptor and its modulation by Ca2+ buffers

The inositol 1,4,5-trisphosphate receptor/channel (IP₃R) is a major regulator of intracellular Ca²⁺ signaling, and liberates Ca²⁺ ions from the endoplasmic reticulum in response to binding at cytosolic sites for both IP₃ and Ca²⁺. Although the steady-state gating properties of the IP₃R have been extensively studied and modeled under conditions of fixed [IP₃] and [Ca²⁺], little is known about how Ca²⁺ flux through a channel may modulate the gating of that same channel by feedback onto activating and inhibitory Ca²⁺ binding sites. We thus simulated the dynamics of Ca²⁺ self-feedback on monomeric and tetrameric IP₃R models. A major conclusion is that self-activation depends crucially on stationary cytosolic Ca²⁺ buffers that slow the collapse of the local [Ca²⁺] microdomain after closure. This promotes burst-like reopenings by the rebinding of Ca²⁺ to the activating site; whereas inhibitory actions are substantially independent of stationary buffers but are strongly dependent on the location of the inhibitory Ca²⁺ binding site on the IP₃R in relation to the channel pore.     

Rapid Recycling of Ca2+ between IP3-Sensitive Stores and Lysosomes

Inositol 1,4,5-trisphosphate (IP₃) evokes release of Ca²⁺ from the endoplasmic reticulum (ER), but the resulting Ca²⁺ signals are shaped by interactions with additional intracellular organelles. Bafilomycin A₁, which prevents lysosomal Ca²⁺ uptake by inhibiting H⁺ pumping into lysosomes, increased the amplitude of the initial Ca²⁺ signals evoked by carbachol in human embryonic kidney (HEK) cells. Carbachol alone and carbachol in combination with parathyroid hormone (PTH) evoke Ca²⁺ release from distinct IP₃-sensitive Ca²⁺ stores in HEK cells stably expressing human type 1 PTH receptors. Bafilomycin A₁ similarly exaggerated the Ca²⁺ signals evoked by carbachol or carbachol with PTH, indicating that Ca²⁺ released from distinct IP₃-sensitive Ca²⁺ stores is sequestered by lysosomes. The Ca²⁺ signals resulting from store-operated Ca²⁺ entry, whether evoked by thapsigargin or carbachol, were unaffected by bafilomycin A₁. Using Gd³⁺ (1 mM) to inhibit both Ca²⁺ entry and Ca²⁺ extrusion, HEK cells were repetitively stimulated with carbachol to assess the effectiveness of Ca²⁺ recycling to the ER after IP₃-evoked Ca²⁺ release. Blocking lysosomal Ca²⁺ uptake with bafilomycin A₁ increased the amplitude of each carbachol-evoked Ca²⁺ signal without affecting the rate of Ca²⁺ recycling to the ER. This suggests that Ca²⁺ accumulated by lysosomes is rapidly returned to the ER. We conclude that lysosomes rapidly, reversibly and selectively accumulate the Ca²⁺ released by IP₃ receptors residing within distinct Ca²⁺ stores, but not the Ca²⁺ entering cells via receptor-regulated, store-operated Ca²⁺ entry pathways.     

Inositol 1,4,5‐trisphosphate receptor 1 degradation in mouse eggs and impact on [Ca2+]i oscillations

The initiation of normal embryo development depends on the completion of all events of egg activation. In all species to date, egg activation requires an increase(s) in the intracellular concentration of calcium ([Ca²⁺]_i), which is almost entirely mediated by inositol 1,4,5‐trisphosphate receptor 1 (IP₃R1). In mammalian eggs, fertilization‐induced [Ca²⁺]_i responses exhibit a periodic pattern that are called [Ca²⁺]_i oscillations. These [Ca²⁺]_i oscillations are robust at the beginning of fertilization, which occurs at the second metaphase of meiosis, but wane as zygotes approach the pronuclear stage, time after which in the mouse oscillations cease altogether. Underlying this change in frequency are cellular and biochemical changes associated with egg activation, including degradation of IP₃R1, progression through the cell cycle, and reorganization of intracellular organelles. In this study, we investigated the system requirements for IP₃R1 degradation and examined the impact of the IP₃R1 levels on the pattern of [Ca²⁺]_i oscillations. Using microinjection of IP₃ and of its analogs and conditions that prevent the development of [Ca²⁺]_i oscillations, we show that IP₃R1 degradation requires uniform and persistently elevated levels of IP₃. We also established that progressive degradation of the IP₃R1 results in [Ca²⁺]_i oscillations with diminished periodicity while a near complete depletion of IP₃R1s precludes the initiation of [Ca²⁺]_i oscillations. These results provide insights into the mechanism involved in the generation of [Ca²⁺]_i oscillations in mouse eggs. J. Cell. Physiol. 222:238–247, 2010. © 2009 Wiley‐Liss, Inc.     

Modulation of Endoplasmic Reticulum Ca2+ Store Filling by Cyclic ADP-ribose Promotes Inositol Trisphosphate (IP3)-evoked Ca2+ Signals

In addition to its well established function in activating Ca²⁺ release from the endoplasmic reticulum (ER) through ryanodine receptors (RyR), the second messenger cyclic ADP-ribose (cADPR) also accelerates the activity of SERCA pumps, which sequester Ca²⁺ into the ER. Here, we demonstrate a potential physiological role for cADPR in modulating cellular Ca²⁺ signals via changes in ER Ca²⁺ store content, by imaging Ca²⁺ liberation through inositol trisphosphate receptors (IP₃R) in Xenopus oocytes, which lack RyR. Oocytes were injected with the non-metabolizable analog 3-deaza-cADPR, and cytosolic [Ca²⁺] was transiently elevated by applying voltage-clamp pulses to induce Ca²⁺ influx through expressed plasmalemmal nicotinic channels. We observed a subsequent potentiation of global Ca²⁺ signals evoked by strong photorelease of IP₃, and increased numbers of local Ca²⁺ puffs evoked by weaker photorelease. These effects were not evident with cADPR alone or following cytosolic Ca²⁺ elevation alone, indicating that they did not arise through direct actions of cADPR or Ca²⁺ on the IP₃R, but likely resulted from enhanced ER store filling. Moreover, the appearance of a new population of puffs with longer latencies, prolonged durations, and attenuated amplitudes suggests that luminal ER Ca²⁺ may modulate IP₃R function, in addition to simply determining the size of the available store and the electrochemical driving force for release.     

Imaging of intracellular Ca waves induced by muscarinic receptor stimulation in rat parotid acinar cells

Changes in cytosolic Ca²⁺ concentration ([Ca²⁺]_i) following muscarinic receptor stimulation were studied with digital imaging microscopy in small clusters of Fura-2 loaded rat parotid acinar cells. In the absence of extracellular Ca²⁺, the increase in [Ca²⁺]_i evoked by a high concentration (10 IM) of carbachol (CCh) was initiated in the apical pole of the acinar cells about 0.4 s after stimulation and then rapidly spread as a Ca²⁺ wave toward the basolateral region. The [Ca²⁺]_i reached the maximum high level throughout the cells 1–2 s after stimulation. As Ca²⁺ was eliminated from the extracellular medium, the Ca²⁺ wave was a result of Ca²⁺ release from intracellular stores. The magnitude and velocity of the Ca²⁺ wave decreased with decreasing concentration of CCh, and the increase in [Ca²⁺]_i induced by low CCh concentrations (≤ 0.5 μM) was always larger in the apical region of acinar cells than in the basal region. The Ca²⁺ wave was also observed in isolated single acinar cells, indicating that the maintenance of acinar structure is not essential for the development of the Ca²⁺ wave. Thapsigargin (ThG), an inhibitor of the endoplasmic reticulum Ca²⁺ pump, caused a slow and homogeneous increase in [Ca²⁺]_i throughout the cells. Addition of ThG after CCh, or addition of CCh after ThG, did not stimulate further increases in [Ca²⁺]_i suggesting that the inositol-1,4,5-trisphosphate (InsP₃) and ThG-sensitive Ca²⁺ stores overlap in parotid acinar cells. The present study supports the hypothesis that formation of InsP₃ is essential to trigger the Ca²⁺ wave and that the development of the Ca²⁺ wave may be attributed to regional differences in InsP₃ sensitivity of Ca²⁺ stores. The agonist-induced Ca²⁺ wave is probably a general phenomenon in exocrine acinar cells.     

Oxytocin induced a biphasic increase in the intracellular Ca2+ concentration of porcine myometrial cells: Participation of a pertussis toxin—insensitive G-protein,inositol 1,4,5-trisphosphate—sensitive Ca2+ pool,and Ca2+ channels

This study investigated the underlying mechanisms of oxytocin (OT)-induced increases in intracellular Ca²⁺ concentrations ([Ca²⁺]_i) in acutely dispersed myometrial cells from prepartum sows. A dosedependent increase in [Ca²⁺]_i was induced by OT (0.1 nM to 1 μM) in the presence and absence of extracellular Ca²⁺ ([Ca²⁺]_e). [Ca²⁺]_i was elevated by OT in a biphasic pattern, with a spike followed by a sustained plateau in the presence of [Ca²⁺]_e. However, in the absence of [Ca²⁺]_e, the [Ca²⁺]_i response to OT became monophasic with a lower amplitude and no plateau, and this monophasic increase was abolished by pretreatment with ionomycin, a Ca²⁺ ionophore. Administration of OT (1 μM) for 15 sec increased inositol 1,4,5-trisphosphate (IP₃) formation by 61%. Pretreatment with pertussis toxin (PTX, 1 μg/ml) for 2 hr failed to alter the OT-induced increase in [Ca²⁺]_i and IP₃ formation. U-73122 (30 nM to 3 μM), a phospholipase C (PLC) inhibitor, depressed the rise in [Ca²⁺]_i by OT dose dependently. U-73122 (3 μM) also abolished the OT-induced IP₃ formation. Thapsigargin (2 μM), an inhibitor of Ca²⁺-ATPase in the endoplasmic reticulum, did not increase [Ca²⁺]_i. However, it did time-dependently inhibit the OT-induced increase in [Ca²⁺]_i. Nimodipine (1 μM), a Voltage-dependent Ca²⁺ channel (VDCC) blocker, inhibited the OT-induced plateau by 26%. La³⁺ (1 μM), a nonspecific Ca²⁺ channel blocker, abrogated the OT-induced plateau. In whole-cell patch-clamp studies used to evaluate VDCC activities, OT (0.1 μM) increased Ca²⁺ Current (I_ca) by 40% with no apparent changes in the current-voltage relationship. The OT-induced increase in I_ca reached the maximum in 5 min, and the increase was abolished by nimodipine (1 μM). These results suggested that (1) activation of OT receptors in porcine myometrium evokes a cascade in the PTX-insensitive G-protein–PLC-IP₃ signal transduction, resulting in an increase in [Ca²⁺]_i; (2) the OT-induced increase in [Ca²⁺]_i is characterized by a biphasic pattern, in which the spike is predominately contributed by the intracellular Ca²⁺ release from the IP₃-sensitive pool, and to a lesser extent by Ca²⁺ influx, whereas the plateau is from increased Ca²⁺ influx; and (3) the influx is via VDCC and receptor-operated Ca²⁺ channels. © 1995 Wiley-Liss, Inc.     

Comparison of Ca2+ puffs evoked by extracellular agonists and photoreleased IP3

The inositol trisphosphate (IP₃) signaling pathway evokes local Ca²⁺ signals (Ca²⁺ puffs) that arise from the concerted openings of clustered IP₃ receptor/channels in the ER membrane. Physiological activation is triggered by binding of agonists to G-protein-coupled receptors (GPCRs) on the cell surface, leading to cleavage of phosphatidyl inositol bisphosphate and release of IP₃ into the cytosol. Photorelease of IP₃ from a caged precursor provides a convenient and widely employed means to study the final stage of IP₃-mediated Ca²⁺ liberation, bypassing upstream signaling events to enable more precise control of the timing and relative concentration of cytosolic IP₃. Here, we address whether Ca²⁺ puffs evoked by photoreleased IP₃ fully replicate those arising from physiological agonist stimulation. We imaged puffs in individual SH-SY5Y neuroblastoma cells that were sequentially stimulated by picospritzing extracellular agonist (carbachol, CCH or bradykinin, BK) followed by photorelease of a poorly-metabolized IP₃ analog, i-IP₃. The centroid localizations of fluorescence signals during puffs evoked in the same cells by agonists and photorelease substantially overlapped (within ∼1 μm), suggesting that IP₃ from both sources accesses the same, or closely co-localized clusters of IP₃Rs. Moreover, the time course and spatial spread of puffs evoked by agonists and photorelease matched closely. Because photolysis generates IP₃ uniformly throughout the cytoplasm, our results imply that IP₃ generated in SH-SY5Y cells by activation of receptors to CCH and BK also exerts broadly distributed actions, rather than specifically activating a subpopulation of IP₃Rs that are scaffolded in close proximity to cell surface receptors to form a signaling nanodomain.     

Role of 1,4,5-inositol triphosphate-induced Ca2+ release in pollen tube orientation

 Pollen tube reorientation is a dynamic cellular event crucial for successful fertilization. Previously, it was shown that reorientation is preceded by an asymmetric increase of cytosolic free calcium ([Ca²⁺]_c) in the side of the apex to which the cell will bend. In order to find the targets for this signal transduction pathway, the effects of inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] in the reorientation process were analyzed. Ins(1,4,5)P₃ was artificially increased in different cell domains by localized photoactivation of caged Ins(1,4,5)P₃ and its effects on [Ca²⁺]_c monitored by ion confocal microscopy. It was found that photolysis of caged Ins(1,4,5)P₃ in the nuclear or subapical region resulted in a transient increase in [Ca²⁺]_c and reorientation of the growth axis, while photolysis in the apex frequently resulted in disturbed growth or tip bursting. Perfusion of the cells with the Ins(1,4,5)P₃ receptor blocker heparin prior to photoactivation inhibited the increase in [Ca²⁺]_c and no reorientation was observed. Ca²⁺ release from Ins(1,4,5)P₃-dependent stores localized in the shank of the tube thus seems to be part of the signal transduction pathway that controls tube guidance, although not the primary stimulus leading to reorientation. Received: 5 May 1998 / Accepted: 11 June 1998     

Systems Modeling of Ca2+ Homeostasis and Mobilization in Platelets Mediated by IP3 and Store-Operated Ca2+ Entry

Resting platelets maintain a stable level of low cytoplasmic calcium ([Ca²⁺]_cyt) and high dense tubular system calcium ([Ca²⁺]_dts). During thrombosis, activators cause a transient rise in inositol trisphosphate (IP₃) to trigger calcium mobilization from stores and elevation of [Ca²⁺]_cyt. Another major source of [Ca²⁺]_cyt elevation is store-operated calcium entry (SOCE) through plasmalemmal calcium channels that open in response to store depletion as [Ca²⁺]_dts drops. A 34-species systems model employed kinetics describing IP₃-receptor, DTS-plasmalemma puncta formation, SOCE via assembly of STIM1 and Orai1, and the plasmalemma and sarco/endoplasmic reticulum Ca²⁺-ATPases. Four constraints were imposed: calcium homeostasis before activation; stable in zero extracellular calcium; IP₃-activatable; and functional SOCE. Using a Monte Carlo method to sample three unknown parameters and nine initial concentrations in a 12-dimensional space near measured or expected values, we found that model configurations that were responsive to stimuli and demonstrated significant SOCE required high inner membrane electric potential (>−70 mV) and low resting IP₃ concentrations. The absence of puncta in resting cells was required to prevent spontaneous store depletion in calcium-free media. Ten-fold increases in IP₃ caused saturated calcium mobilization. This systems model represents a critical step in being able to predict platelets’ phenotypes during hemostasis or thrombosis.     

Ca2+-induced Ca2+ Release in Chromaffin Cells Seen from inside the ER with Targeted Aequorin

The presence and physiological role of Ca²⁺-induced Ca²⁺ release (CICR) in nonmuscle excitable cells has been investigated only indirectly through measurements of cytosolic [Ca²⁺] ([Ca²⁺]_c). Using targeted aequorin, we have directly monitored [Ca²⁺] changes inside the ER ([Ca²⁺]_ER) in bovine adrenal chromaffin cells. Ca²⁺ entry induced by cell depolarization triggered a transient Ca²⁺ release from the ER that was highly dependent on [Ca²⁺]_ER and sensitized by low concentrations of caffeine. Caffeine-induced Ca²⁺ release was quantal in nature due to modulation by [Ca²⁺]_ER. Whereas caffeine released essentially all the Ca²⁺ from the ER, inositol 1,4,5-trisphosphate (InsP₃)- producing agonists released only 60–80%. Both InsP₃ and caffeine emptied completely the ER in digitonin-permeabilized cells whereas cyclic ADP-ribose had no effect. Ryanodine induced permanent emptying of the Ca²⁺ stores in a use-dependent manner after activation by caffeine. Fast confocal [Ca²⁺]_c measurements showed that the wave of [Ca²⁺]_c induced by 100-ms depolarizing pulses in voltage-clamped cells was delayed and reduced in intensity in ryanodine-treated cells. Our results indicate that the ER of chromaffin cells behaves mostly as a single homogeneous thapsigargin-sensitive Ca²⁺ pool that can release Ca²⁺ both via InsP₃ receptors or CICR.     

Caspase-3-truncated type 1 inositol 1,4,5-trisphosphate receptor enhances intracellular Ca2+ leak and disturbs Ca2+ signalling

Background information. The IP₃R (inositol 1,4,5‐trisphosphate receptor) is a tetrameric channel that accounts for a large part of the intracellular Ca²⁺ release in virtually all cell types. We have previously demonstrated that caspase‐3‐mediated cleavage of IP₃R1 during cell death generates a C‐terminal fragment of 95 kDa comprising the complete channel domain. Expression of this truncated IP₃R increases the cellular sensitivity to apoptotic stimuli, and it was postulated to be a constitutively active channel. Results. In the present study, we demonstrate that expression of the caspase‐3‐cleaved C‐terminus of IP₃R1 increased the rate of thapsigargin‐mediated Ca²⁺ leak and decreased the rate of Ca²⁺ uptake into the ER (endoplasmic reticulum), although it was not sufficient by itself to deplete intracellular Ca²⁺ stores. We detected the truncated IP₃R1 in different cell types after a challenge with apoptotic stimuli, as well as in aged mouse oocytes. Injection of mRNA corresponding to the truncated IP₃R1 blocked sperm factor‐induced Ca²⁺ oscillations and induced an apoptotic phenotype. Conclusions. In the present study, we show that caspase‐3‐mediated truncation of IP₃R1 enhanced the Ca²⁺ leak from the ER. We suggest a model in which, in normal conditions, the increased Ca²⁺ leak is largely compensated by enhanced Ca²⁺‐uptake activity, whereas in situations where the cellular metabolism is compromised, as occurring in aging oocytes, the Ca²⁺ leak acts as a feed‐forward mechanism to divert the cell into apoptosis.     

Carbamylcholine-induced morphological changes and spatial dynamics of [Ca2+]c in Harderian glands of guinea pigs: calcium-dependent lipid secretion and contraction of myoepithelial cells

To determine whether lipid-secreting cells have cytosolic Ca²⁺ concentration ([Ca²⁺]_c)-related secretory mechanisms, morphological changes and intracellular calcium dynamics of Harderian glands of guinea pigs stimulated by secretagogs were studied by electron microspy and Fura-2/AM digital image analysis. Control glandular cells contained large lipid vacuoles that were bordered by multi-layered membranes. Rough-surfaced endoplasmic reticulum, mitochondria, and smooth-surfaced endoplasmic reticulum may be involved in lipid vacuole formation. Myoepithelial cells surrounded alveoli. After carbamylcholine (CCh, 10^–6, 10^–5, and 10^–3 M) stimulation, lipid materials within the membranous structures were frequently discharged by an exocytotic mechanism. Conspicuous deformation of glandular cells caused by vigorous contraction of myoepithelial cells was observed in isolated alveoli after 10^–6M CCh stimulation, whereas the deformaties of glandular tissues perfused via vessels were small even after 10^–3M CCh stimulation. Connective tissue between glandular alveoli inhibited unbridled myoepithelial-cell contraction. Fura-2/AM digital imaging analysis revealed that CCh stimulation caused an increase in [Ca²⁺]_c in isolated alveoli. The morphological reactions and changes in [Ca²⁺]_c were prevented by atropine. When extracellular calcium ions were absent, enhanced extrusion of lipid vacuoles, myoepithelial-cell contraction, and a rise in [Ca²⁺]_c after CCh stimulation were not observed. Nicotine and catecholamines had no effect on the secretion or on the dynamics of [Ca²⁺]_c. It can be concluded that acetylcholine elicits exocytosis in glandular cells and contraction of the myoepithelial cells of Harderian glands, accompanied by an increase in [Ca²⁺]_c. The dynamics of [Ca²⁺]_c of the gland alveoli are mostly dependent on extracellular Ca²⁺.