The role of inositol 1,4,5-trisphosphate in mobilizing calcium from intracellular stores in the salivary glands of Amblyomma americanum (L.)

Isolated tick salivary glands, permeabilized with digitonin in the presence of the Ca²⁺ uptake inhibitors, sodium azide and vanadate, released Ca²⁺ in response to 20 μM inositol-1,4,5-trisphosphate (IP₃). Inositol-1-phosphate (IP₁) and inositol-1,4-bisphosphate (IP₂) appeared to stimulate an uptake of Ca²⁺ into whole glands. Inositol-1,4,5-trisphosphate caused release of Ca²⁺ from a 100,000 g microsome enriched pellet; however, IP₁ and IP₂ were ineffective in stimulating an uptake or efflux of Ca²⁺. The combined 900 and 11,500 g pellets showed no significant release of Ca²⁺ in response to addition of IP₃. Inositol-1,4,5-trisphosphate concentrations as low as 1 μM are capable of stimulating a significant release of Ca²⁺ from microsomes. Results suggest that intracellular Ca²⁺ is mobilized from microsomal intracellular stores in response to agonists which increase cytosolic IP₃ in tick salivary glands. Results also suggest a possible role for IP₁ and IP₂ or both in stimulating an uptake of Ca²⁺ into vanadate and azide-insensitive intracellular pools.     

Inositol 1,4,5-trisphosphate-induced Ca2+ release from permeabilized mastocytoma cells

The effect of inositol 1,4,5-trisphosphate (IP₃) on Ca²⁺ release in the transformed murine mast cells, mastocytoma P-815 cells permeabilized with digitonin was studied. Ca²⁺ was sequestered by intracellular organelles in the presence of ATP until the medium free Ca²⁺ concentration was lowered to a new steady-state level. The subsequent addition of IP₃ caused a rapid Ca²⁺ release, which was followed by a slow re-uptake of Ca²⁺. Fifty percent of the sequestered Ca²⁺ was released by 10 μM IP₃. Maximal Ca²⁺ release occurred at 10 μM and half maximal activity was at 1.3 μM. These results indicate that IP₃ may function as a messenger of intracellular Ca²⁺ mobilization in mastocytoma 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.     

U-73122 inhibits carbachol-induced increases in [Ca2+]i,IP3, and insulin release in β-TC3 cells

We studied the effects of the aminosteroid U-73122, a putative phospholipase C (PLC) inhibitor, on carbachol-induced increases in insulin release, [Ca²⁺]_i, and IP₃ in β-TC3 cells. Carbachol (0.1–100 μM) increased [Ca²⁺]_i and carbachol (0.1–1000 μM) increased insulin release dose-dependently. Carbachol (100 μM) also increased inositol 1,4,5-trisphosphate (IP₃) production. U-73122 (2–12 νM) inhibited the effects of carbachol on [Ca²⁺]_i and insulin release in a dose-dependent manner, and at the highest dose studied (12 μM) it abolished or greatly attenuated all three effects of carbachol. In contrast, U-73343 (12 μM), the analog of U-73122 that does not inhibit PLC, only inhibited the effect of carbachol on [Ca²⁺]_i by 20% and did not inhibit the effect of carbachol on insulin release. Since carbachol increased IP₃, [Ca²⁺]_i, and insulin release by activating PLC, these results suggested that U-73122 inhibits phospholipase C-depenent processes in β-TC3 cells.     

Intracellular mobilization of Ca by the insect steroid hormone 20-hydroxyecdysone during programmed cell death in silkworm anterior silk glands

20-Hydroxyecdysone (20E) triggers programmed cell death (PCD) and regulates de novo gene expression in the anterior silk glands (ASGs) of the silkworm Bombyx mori. PCD is mediated via a nongenomic pathway that includes Ca²⁺ as a second messenger and the activation of protein kinase C/caspase-3-like protease; however, the steps leading to a concomitant buildup of intracellular Ca²⁺ are unknown. We employed pharmacological tools to identify the components of this pathway. ASGs were cultured in the presence of 1 μM 20E and one of the following inhibitors: a G-protein-coupled receptor (GPCR) inhibitor, a phospholipase C (PLC) inhibitor, an inositol 1,4,5-trisphosphate receptor (IP₃R) antagonist, and an L- or T-type Ca²⁺ channel blocker. The T-type Ca²⁺ channel blocker inhibited 20E-induced nuclear and DNA fragmentation; in contrast, PCD was induced by 20E in Ca²⁺-free medium, indicating that the source of Ca²⁺ is an intracellular reservoir. The IP₃R antagonist inhibited nuclear and DNA fragmentation, suggesting that the endoplasmic reticulum may be the Ca²⁺ source. Finally, the GPCR and PLC inhibitors effectively blocked nuclear and DNA fragmentation. Our results indicate that 20E increases the intracellular level of Ca²⁺ by activating IP₃R, and that this effect may be brought about by the serial activation of GPCR, PLC, and IP₃.     

All-or-none shortening of α-toxin-permeabilized single smooth muscle cells from guinea-pig taenia caecum and acetylcholine-induced Ca2+ sensitization

Isolated single smooth muscle cells from guinea-pig taenia caecum, which responded to acetylcholine (ACh) in an all-or-none manner, were permeabilized by exposure to Staphylococcus aureus α-toxin. Permeabilized cells responded to 0.3 μM or 0.6 μM Ca²⁺ with maximal shortening, showing an all-or-none response. The addition of ACh resulted in a decrease in the concentration of Ca²⁺ required to trigger a threshold response. GDP inhibited, and treatment with GTP mimicked the action of ACh. The addition of inositol 1,4,5-trisphosphate (IP₃) in the permeabilized cells caused a slight cell shortening. These results suggest that α-toxin-permeabilized cells retain their receptor function and show augmentation of Ca²⁺ sensitivity by stimulation of the muscarinic receptor or GTP-binding protein(s). Moreover, it is suggested that the intact cells show an all-or-none response to ACh when the intracellular Ca²⁺ level is greater than the threshold concentration due to signaling being coupled to muscarinic receptor stimulation. This then causes the augmentation of Ca²⁺ sensitivity and reaches the threshold to evoke Ca²⁺ influx and IP₃-induced Ca²⁺ release.     

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.     

Regulation of inositol 1,4,5-trisphosphate-induced Ca2+ release from the endoplasmic reticulum by AMP-activated kinase modulators

The 5' AMP-activated protein kinase (AMPK) is a nutrient-sensitive kinase that plays a key role in the control of cellular energy metabolism. We have explored here the relationship between AMPK and Ca²⁺ signaling by looking at the effect of an AMPK activator (A769662) and an AMPK inhibitor (dorsomorphin) on histamine-induced Ca²⁺-release from the endoplasmic reticulum (ER) in HeLa cells. Our data show that incubation with A769662 (EC₅₀ = 29 μM) inhibited histamine-induced Ca²⁺-release from the ER in intact cells, as well as inositol-1,4,5-trisphosphate (IP₃)-induced Ca²⁺ release in permeabilized cells. On the contrary, dorsomorphin (EC₅₀ = 0.4 μM) activated both histamine and IP₃-induced Ca²⁺-release and reversed the effect of A769662. These results suggest a direct effect of AMPK regulation on IP₃ receptor (IP₃R) function. A phosphoproteomic study did not reveal changes in IP₃R phosphorylation, but showed significant changes in phosphorylation of proteins placed upstream in the IP₃R interactome and in several proteins related with Ca²⁺ metabolism, which could be candidates to mediate the effects observed. In conclusion, our data suggest that AMPK negatively regulates IP₃R. This effect constitutes a novel and very important link between Ca²⁺ signaling and the AMPK pathway.     

Ca2+ Release from Inositol 1,4,5-Trisphosphate-Sensitive Ca2+ Store by Antidepressant Drugs in Cultured Neurons of Rat Frontal Cortex

Abstract: The ability of antidepressant drugs (ADs) to increase the concentration of intracellular Ca²⁺ ([Ca²⁺]_i) was examined in primary cultured neurons from rat frontal cortices using the Ca²⁺-sensitive fluorescent indicator fura-2. Amitriptyline, imipramine, desipramine, and mianserin elicited transient increases in [Ca²⁺]_i in a concentration-dependent manner (100 μM to 1 mM). These four AD-induced [Ca²⁺]_i increases were not altered by the absence of external Ca²⁺ or by the presence of La³⁺ (30 μM), suggesting that these ADs provoked intracellular Ca²⁺ mobilization rather than Ca²⁺ influx. All four ADs increased inositol 1,4,5-trisphosphate (IP₃) contents by 20–60% in the cultured cells. The potency of the IP₃ production by these ADs closely correlated with the AD-induced [Ca²⁺]_i responses. Pretreatment with neomycin, an inhibitor of IP₃ generation, significantly inhibited amitriptyline- and imipramine-induced [Ca²⁺]_i increases. In addition, by initially perfusing with bradykinin (10 μM) or acetylcholine (10 μM), which can stimulate the IP₃ generation and mobilize the intracellular Ca²⁺, the amitriptyline responses were decreased by 76% and 69%, respectively. The amitriptyline-induced [Ca²⁺]_i increases were unaffected by treatment with pertussis toxin. We conclude that high concentrations of amitriptyline and three other ADs mobilize Ca²⁺ from IP₃-sensitive Ca²⁺ stores and that the responses are pertussis toxin-insensitive. However, it seems unlikely that the effects requiring high concentrations of ADs are related to the therapeutic action.     

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.     

Angiotensin II-induced fluid phase endocytosis in human cerebromicrovascular endothelial cells is regulated by the inositol-phosphate signaling pathway

The involvement of the early signaling messengers, inositol tris-phosphate (IP₃), intracellular calcium, [Ca²⁺]_i, and protein kinase C (PKC), in angiotensin II (AII)-induced fluid phase endocytosis was investigated in human brain capillary and microvascular endothelial cells (HCEC). AII (0.01–10 μM) stimulated the uptake of Lucifer yellow CH, an inert dye used as a marker for fluid phase endocytosis, in HCEC by 50–230%. AII also triggered a fast accumulation of IP₃ and a rapid increase in [Ca²⁺]_i in cells loaded with the Ca²⁺-responsive fluorescent dye fura-2. The prompt AII-induced [Ca²⁺]_i spike was not affected by incubating HCEC in Ca²⁺-free medium containing 2 mM EGTA or by pretreating the cultures with the Ca²⁺ channel blockers, methoxyverapamil (D600; 50 μM), nickel (1 mM), or lanthanum (1 mM), suggesting that the activation of AII receptors on HCEC triggers the release of Ca²⁺ from intracellular stores. The AII-triggered increases in IP₃, [Ca²⁺]_i, and Lucifer yellow uptake were inhibited by the nonselective AII receptor antagonist, Sar¹, Val⁵, Ala⁸-AII (SVA-AII), and by the phospholipase C (PLC) inhibitors, neomycin and U-73122. By contrast, the protein kinase C (PKC) inhibitors, staurosporine and calphostin C, failed to affect any of these AII-induced events. This study demonstrates that increased fluid phase endocytotosis induced by AII in human brain capillary endothelium, an event thought to be linked to the observed increases in blood-brain barrier permeability in acute hypertension, is likely dependent on PLC-mediated changes in [Ca²⁺]_i and independent of PKC. © 1996 Wiley-Liss, Inc.     

Low affinity purinergic receptor modulates the response of rat submandibular glands to carbachol and substance P

The effect of extracellular ATP on the intracellular calcium concentration ([Ca²⁺]_i) in rat submandibular glands was tested. The dose-response curve for ATP was biphasic with a first increase in the 1–30 μM concentration range and a further increase at concentrations higher than 100 μM. Among ATP analogs, only benzoyl-ATP stimulated the low affinity component. ATPτS blocked this response. All the other analogs tested reproduced the high-affinity low capacity response. Magnesium and Coomassie blue selectively blocked the low affinity component. High concentrations of ATP blocked the increase of the intracellular calcium concentration [Ca²⁺]_i in response to 100 μM carbachol. By itself, substance P (100 pM-1 μM) increased the [Ca²⁺]_i. One mM ATP potentiated the response to concentrations of substance P higher than 10 nM. This potentiation was reversed by extracellular magnesium. Carbachol 100 μM and substance P (100 pM-1 μM) increased the release of inositol trisphosphate (IP₃) from polyphosphoinositides (polyPI). Activation of the low affinity ATP receptors did not activate the polyPI-specific phospholipase C but inhibited its activation by 100 μM carbachol (−50%) and by 100 nM substance P (−60% at 1 nM substance P and −40% at 100 nM substance P). Substance P induced a strong homologous desensitization: a preincubation with 1 nM substance P nearly completely abolished the response to 1 μM substance P. When the cells were exposed to ATP before the second addition of substance P, the purinergic agonist partially restored the response to the tachykinin without totally reversing the desensitization. It is concluded that two types of purinergic receptors coexist in rat submandibular glands; a high-affinity, low capacity receptor which remains pharmacologically and functionally undefined and a low affinity site, high capacity receptor of the P_2Z type coupled to a non-selective cation channel. The occupancy of these low affinity sites blocks the increase of the [Ca²⁺]_i in response to a muscarinic agonist and the activation of polyPI-specific phospholipase C by carbachol and substance P. It potentiates the effect of high concentrations of substance P on the [Ca²⁺]_i. © 1996 Wiley-Liss, Inc.     

2-Aminoethoxydiphenyl-borate (2-APB) increases excitability in pyramidal neurons

Calcium ions (Ca²⁺) released from inositol trisphosphate (IP₃)-sensitive intracellular stores may participate in both the transient and extended regulation of neuronal excitability in neocortical and hippocampal pyramidal neurons. IP₃ receptor (IP₃R) antagonists represent an important tool for dissociating these consequences of IP₃ generation and IP₃R-dependent internal Ca²⁺ release from the effects of other, concurrently stimulated second messenger signaling cascades and Ca²⁺ sources. In this study, we have described the actions of the IP₃R and store-operated Ca²⁺ channel antagonist, 2-aminoethoxydiphenyl-borate (2-APB), on internal Ca²⁺ release and plasma membrane excitability in neocortical and hippocampal pyramidal neurons. Specifically, we found that a dose of 2-APB (100 μM) sufficient for attenuating or blocking IP₃-mediated internal Ca²⁺ release also raised pyramidal neuron excitability. The 2-APB-dependent increase in excitability reversed upon washout and was characterized by an increase in input resistance, a decrease in the delay to action potential onset, an increase in the width of action potentials, a decrease in the magnitude of afterhyperpolarizations (AHPs), and an increase in the magnitude of post-spike afterdepolarizations (ADPs). From these observations, we conclude that 2-APB potently and reversibly increases neuronal excitability, likely via the inhibition of voltage- and Ca²⁺-dependent potassium (K⁺) conductances.     

Development of a functional assay for Ca2+ release activity of IP3R and expression of an IP3R gene fragment in the baculovirus-insect cell system

Receptor-stimulated phosphoinositide (PI) hydrolysis is an important and ubiquitous mechanism of intracellular signaling. Inositol 1,4,5-trisphosphate (IP₃), generated by phosphoinositide (PI) hydrolysis, binds to and gates an intracellular Ca²⁺ channel, the IP₃ receptor (IP₃R), which is therefore a central component of this signaling cascade. Here we describe the development of a baculovirus (BV)/Sf(S. frugiperda) cell system that can be used to look at IP₃R function. Agonist-evoked changes in intracellular Ca²⁺ levels [Ca²⁺]_i were measured (using Fura2) in Sf cells expressing the gene encoding the muscarinic acetylcholine receptor (vmlAchR). Furthermore, we have constructed a recombinant BV (vlP₃R), with the core of the IP₃R ligand-binding domain from the Drosophila IP₃R, under the polyhedrin promoter. The recombinant protein from such a virus was expected to act as a large ligand sink for IP3, generated by stimulation of vmlAchR. Cells coinfected with recombinant BV carrying the potential dominant-negative vIP₃R construct and vmlAchR have been used to assay the modulation of IP₃R-mediated Ca²⁺ release, by the ligand sink.     

Two mechanisms of calcium oscillations in adipocytes

In non-excitable cells, several kinds of agonist-induced oscillations of cytosolic Ca²⁺ concentration ([Ca²⁺]_i) are known which differ in their form and generation mechanism. The oscillation source is, as a rule, the regulation of Ca²⁺ mobilization from intracellular stores through inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃R) 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 [Ca²⁺]_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 IP₃R (Xestospongin C) did not affect the pattern of responses, which means that the generation of oscillations does not depend on IP₃. 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 [Ca²⁺]_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 IP₃R and RyR in FBS-induced oscillations gave completely inverse results relative to ACh and demonstrated a leading role of IP₃R without a considerable contribution of RyR and of its activation pathways. With both stimuli, Ca²⁺ 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 Ca²⁺ signaling, each of them generating oscillations with a specific temporal pattern.     

Pathophysiological consequences of isoform-specific IP3 receptor mutations

Ca²⁺ signaling governs a diverse range of cellular processes and, as such, is subject to tight regulation. A main component of the complex intracellular Ca²⁺-signaling network is the inositol 1,4,5-trisphosphate (IP₃) receptor (IP₃R), a tetrameric channel that mediates Ca²⁺ release from the endoplasmic reticulum (ER) in response to IP₃. IP₃R function is controlled by a myriad of factors, such as Ca²⁺, ATP, kinases and phosphatases and a plethora of accessory and regulatory proteins. Further complexity in IP₃R-mediated Ca²⁺ signaling is the result of the existence of three main isoforms (IP₃R1, IP₃R2 and IP₃R3) that display distinct functional characteristics and properties. Despite their abundant and overlapping expression profiles, IP₃R1 is highly expressed in neurons, IP₃R2 in cardiomyocytes and hepatocytes and IP₃R3 in rapidly proliferating cells as e.g. epithelial cells. As a consequence, dysfunction and/or dysregulation of IP₃R isoforms will have distinct pathophysiological outcomes, ranging from neurological disorders for IP₃R1 to dysfunctional exocrine tissues and autoimmune diseases for IP₃R2 and -3. Over the past years, several IP₃R mutations have surfaced in the sequence analysis of patient-derived samples. Here, we aimed to provide an integrative overview of the clinically most relevant mutations for each IP₃R isoform and the subsequent molecular mechanisms underlying the etiology of the disease.     

Data-driven modeling of mitochondrial dysfunction in Alzheimer's disease

Intracellular accumulation of oligomeric forms of β amyloid (Aβ) are now believed to play a key role in the earliest phase of Alzheimer's disease (AD) as their rise correlates well with the early symptoms of the disease. Extensive evidence points to impaired neuronal Ca²⁺ homeostasis as a direct consequence of the intracellular Aβ oligomers. However, little is known about the downstream effects of the resulting Ca²⁺ rise on the many intracellular Ca²⁺-dependent pathways. Here we use multiscale modeling in conjunction with patch-clamp electrophysiology of single inositol 1,4,5-trisphosphate (IP₃) receptor (IP₃R) and fluorescence imaging of whole-cell Ca²⁺ response, induced by exogenously applied intracellular Aβ₄₂ oligomers to show that Aβ₄₂ inflicts cytotoxicity by impairing mitochondrial function. Driven by patch-clamp experiments, we first model the kinetics of IP₃R, which is then extended to build a model for the whole-cell Ca²⁺ signals. The whole-cell model is then fitted to fluorescence signals to quantify the overall Ca²⁺ release from the endoplasmic reticulum by intracellular Aβ₄₂ oligomers through G-protein-mediated stimulation of IP₃ production. The estimated IP₃ concentration as a function of intracellular Aβ₄₂ content together with the whole-cell model allows us to show that Aβ₄₂ oligomers impair mitochondrial function through pathological Ca²⁺ uptake and the resulting reduced mitochondrial inner membrane potential, leading to an overall lower ATP and increased production of reactive oxygen species and H₂O₂. We further show that mitochondrial function can be restored by the addition of Ca²⁺ buffer EGTA, in accordance with the observed abrogation of Aβ₄₂ cytotoxicity by EGTA in our live cells experiments.     

Infrared neural stimulation induces intracellular Ca2+ release mediated by phospholipase C

The influence of infrared laser pulses on intracellular Ca²⁺ signaling was investigated in neural cell lines with fluorescent live cell imaging. The probe Fluo‐4 was used to measure Ca²⁺ in HT22 mouse hippocampal neurons and nonelectrically excitable U87 human glioblastoma cells exposed to 50 to 500 ms infrared pulses at 1470 nm. Fluorescence recordings of Fluo‐4 demonstrated that infrared stimulation induced an instantaneous intracellular Ca²⁺ transient with similar dose‐response characteristics in hippocampal neurons and glioblastoma cells (half‐maximal effective energy density EC₅₀ of around 58 J.cm^?2). For both type of cells, the source of the infrared‐induced Ca²⁺ transients was found to originate from intracellular stores and to be mediated by phospholipase C and IP₃‐induced Ca²⁺ release from the endoplasmic reticulum. The activation of phosphoinositide signaling by IR light is a new mechanism of interaction relevant to infrared neural stimulation that will also be widely applicable to nonexcitable cell types. The prospect of infrared optostimulation of the PLC/IP₃ cell signaling cascade has many potential applications including the development of optoceutical therapeutics.      

NaCl-elicited,vacuolar Ca2+ release facilitates prolonged cytosolic Ca2+ signaling in the salt response of Populus euphratica cells

High environmental salt elicits an increase in cytosolic Ca²⁺ ([Ca²⁺]_cyt) in plants, which is generated by extracellular Ca²⁺ influx and Ca²⁺ release from intracellular stores, such as vacuole and endoplasmic reticulum. This study aimed to determine the physiological mechanisms underlying Ca²⁺ release from vacuoles and its role in ionic homeostasis in Populus euphratica. In vivo Ca²⁺ imaging showed that NaCl treatment induced a rapid elevation in [Ca²⁺]_cyt, which was accompanied by a subsequent release of vacuolar Ca²⁺. In cell cultures, NaCl-altered intracellular Ca²⁺ mobilization was abolished by antagonists of inositol (1, 4, 5) trisphosphate (IP₃) and cyclic adenosine diphosphate ribose (cADPR) signaling pathways, but not by slow vacuolar (SV) channel blockers. Furthermore, the NaCl-induced vacuolar Ca²⁺ release was dependent on extracellular ATP, extracellular Ca²⁺ influx, H₂O₂, and NO. In vitro Ca²⁺ flux recordings confirmed that IP₃, cADPR, and Ca²⁺ induced substantial Ca²⁺ efflux from intact vacuoles, but this vacuolar Ca²⁺ flux did not directly respond to ATP, H₂O₂, or NO. Moreover, the IP₃/cADPR-mediated vacuolar Ca²⁺ release enhanced the expression of salt-responsive genes that regulated a wide range of cellular processes required for ion homeostasis, including cytosolic K⁺ maintenance, Na⁺ and Cl⁻ exclusion across the plasma membrane, and Na⁺/H⁺ and Cl⁻/H⁺ exchanges across the vacuolar membrane.     

STIM1 Positively Regulates the Ca2+ Release Activity of the Inositol 1,4,5-Trisphosphate Receptor in Bovine Aortic Endothelial Cells

The endothelium is actively involved in many functions of the cardiovascular system, such as the modulation of arterial pressure and the maintenance of blood flow. These functions require a great versatility of the intracellular Ca²⁺ signaling that resides in the fact that different signals can be encoded by varying the frequency and the amplitude of the Ca²⁺ response. Cells use both extracellular and intracellular Ca²⁺ pools to modulate the intracellular Ca²⁺ concentration. In non-excitable cells, the inositol 1,4,5-trisphosphate receptor (IP₃R), located on the endoplasmic reticulum (ER), is responsible for the release of Ca²⁺ from the intracellular store. The proteins STIM1 and STIM2 are also located on the ER and they are involved in the activation of a store-operated Ca²⁺ entry (SOCE). Due to their Ca²⁺ sensor property and their close proximity with IP₃Rs on the ER, STIMs could modulate the activity of IP₃R. In this study, we showed that STIM1 and STIM2 are expressed in bovine aortic endothelial cells and they both interact with IP₃R. While STIM2 appears to play a minor role, STIM1 plays an important role in the regulation of agonist-induced Ca²⁺ mobilization in BAECs by a positive effect on both the SOCE and the IP₃R-dependent Ca²⁺ release.