Melatonin and IP3-induced Ca2+ release from intracellular stores in the malaria parasite Plasmodium falciparum within infected red blood cells

IP(3)-dependent Ca(2+) signaling controls a myriad of cellular processes in higher eukaryotes and similar signaling pathways are evolutionarily conserved in Plasmodium, the intracellular parasite that causes malaria. We have reported that isolated, permeabilized Plasmodium chabaudi, releases Ca(2+) upon addition of exogenous IP(3). In the present study, we investigated whether the IP(3) signaling pathway operates in intact Plasmodium falciparum, the major disease-causing human malaria parasite. P. falciparum-infected red blood cells (RBCs) in the trophozoite stage were simultaneously loaded with the Ca(2+) indicator Fluo-4/AM and caged-IP(3). Photolytic release of IP(3) elicited a transient Ca(2+) increase in the cytosol of the intact parasite within the RBC. The intracellular Ca(2+) pools of the parasite were selectively discharged, using thapsigargin to deplete endoplasmic reticulum (ER) Ca(2+) and the antimalarial chloroquine to deplete Ca(2+) from acidocalcisomes. These data show that the ER is the major IP(3)-sensitive Ca(2+) store. Previous work has shown that the human host hormone melatonin regulates P. falciparum cell cycle via a Ca(2+)-dependent pathway. In the present study, we demonstrate that melatonin increases inositol-polyphosphate production in intact intraerythrocytic parasite. Moreover, the Ca(2+) responses to melatonin and uncaging of IP(3) were mutually exclusive in infected RBCs. Taken together these data provide evidence that melatonin activates PLC to generate IP(3) and open ER-localized IP(3)-sensitive Ca(2+) channels in P. falciparum. This receptor signaling pathway is likely to be involved in the regulation and synchronization of parasite cell cycle progression.     

Sustained Ca2+ signaling in mouse lacrimal acinar cells due to photolysis of "caged" glycerophosphoryl-myo-inositol 4,5-bisphosphate.

In saponin-permeabilized mouse lacrimal acinar cells, glycerophosphoryl-myo-inositol 4,5-bisphosphate (GPIP2) activated the release of sequestered Ca2+ to the same extent as inositol 1,4,5-trisphosphate ((1,4,5)IP3) but with a potency about 1/10 that of (1,4,5)IP3. In lacrimal gland homogenates, [3H]GPIP2 was metabolized to two compounds which upon anion exchange high performance liquid chromatography eluted at positions indicating that they were [3H]GPIP and [3H]GPIP3. The rate of metabolism of [3H]GPIP2 was much slower than that of [3H](1,4,5)IP3, and its rate of phosphorylation was less than 1% of that of [3H] (1,4,5)IP3. In intact lacrimal cells, photolysis of a microinjected "caged" derivative of GPIP2, 1-(alpha-glycerophosphoryl)-myo-inositol 4,5-bisphosphate P4(5)-1-(2-nitrophenyl)ethyl ester, resulted in sustained activation of Ca2+ signaling; i.e. intracellular Ca2+ release followed by increased entry of Ca2+ across the plasma membrane. These findings indicate that caged GPIP2 should provide a useful tool for producing photolytically initiated, sustained activation of intracellular (1,4,5)IP3 receptors. They also provide strong support for the idea that sustained Ca2+ signaling can be achieved in lacrimal acinar cells by activation of intracellular receptors for (1,4,5)IP3 in the absence of stimulated production of inositol 1,3,4,5-tetrakisphosphate.     

cAMP-dependent protein kinase enhances inositol 1,4,5-trisphosphate-induced Ca2+ release in AR4-2J cells

In non-excitable cells, the inositol 1,4,5-trisphosphate receptor (IP(3)R), a ligand-gated Ca(2+) channel, plays an important role in the control of intracellular Ca(2+). There are three subtypes of IP(3)R that are differentially distributed among cell types. AR4-2J cells express almost exclusively the IP(3)R-2 subtype. The purpose of this study was to investigate the effect of cAMP-dependent protein kinase (PKA) on the activity of IP(3)R-2 in AR4-2J cells. We showed that immunoprecipitated IP(3)R-2 is a good substrate for PKA. Using a back-phosphorylation approach, we showed that endogenous PKA phosphorylates IP(3)R-2 in intact AR4-2J cells. Pretreatment with PKA enhanced IP(3)-induced Ca(2+) release in permeabilized AR4-2J cells. Pretreatment with the cAMP generating agent's forskolin and vasoactive intestinal peptide (VIP) enhanced carbachol (Cch)-induced and epidermal growth factor (EGF)-induced Ca(2+) responses in intact AR4-2J cells. Our results are consistent with an enhancing effect of PKA on IP(3)R-2 activity. This conclusion supports the emerging concept of crosstalk between Ca(2+) signaling and cAMP pathways and thus provides another way by which Ca(2+) signals are finely encoded within non-excitable cells.     

Localization of puff sites adjacent to the plasma membrane: functional and spatial characterization of Ca2+ signaling in SH-SY5Y cells utilizing membrane-permeant caged IP3

The Xenopus oocyte has been a favored model system in which to study spatio-temporal mechanisms of intracellular Ca2+ dynamics, in large part because this giant cell facilitates intracellular injections of Ca2+ indicator dyes, buffers and caged compounds. However, the recent commercial availability of membrane-permeant ester forms of caged IP3 (ci-IP3) and EGTA, now allows for facile loading of these compounds into smaller mammalian cells, permitting control of [IP3]i and cytosolic Ca2+ buffering. Here, we establish the human neuroblastoma SH-SY5Y cell line as an advantageous experimental system for imaging Ca2+ signaling, and characterize IP3-mediated Ca2+ signaling mechanisms in these cells. Flash photo-release of increasing amounts of i-IP3 evokes Ca2+ puffs that transition to waves, but intracellular loading of EGTA decouples release sites, allowing discrete puffs to be studied over a wide range of [IP3]. Puff activity persists for minutes following a single photo-release, pointing to a slow rate of i-IP3 turnover in these cells and suggesting that repetitive Ca2+ spikes with periods of 20-30s are not driven by oscillations in [IP3]. Puff amplitudes are independent of [IP3], whereas their frequencies increase with increasing photo-release. Puff sites in SH-SY5Y cells are not preferentially localized near the nucleus, but instead are concentrated close to the plasma membrane where they can be visualized by total internal reflection microscopy, offering the potential for unprecedented spatio-temporal resolution of Ca2+ puff kinetics.     

Development of calcium releasing activity induced by inositol trisphosphate and cyclic ADP-ribose during in vitro maturation of sea urchin oocytes

During fertilization of sea urchin eggs, the cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) transiently increases (Ca(2+) transient). Increased [Ca(2+)](i) results from a rapid release from intracellular stores, mediated by one or both of two signaling pathways; inositol 1,4,5-trisphosphate (IP(3)) and IP(3) receptor (IP(3)R) or cyclic GMP (cGMP), cyclic ADP-ribose (cADPR) and ryanodine receptor (RyR). During fertilization, cGMP and cADPR increase preceding the Ca(2+) transient, suggesting their contribution to this. If the RyR pathway contributed to the Ca(2+) transient, its Ca(2+) releasing activity would develop in parallel with that of the IP(3) system during maturation of oocytes. Sea urchin oocytes were cultivated in vitro and Ca(2+) transients induced by photolysis of caged IP(3) or caged cADPR were measured during maturation. Oocytes spontaneously began to maturate in seawater. More than 50% of oocytes underwent germinal vesicle breakdown within 25 h and the second meiosis within 35 h, but it took more than 24 h until they became functionally identical to in vivo-matured eggs. Both IP(3) and cADPR induced Ca(2+) transients comparable to those of in vivo-matured eggs later than 24 h from the second meiosis. However, cADPR induced a small Ca(2+) transient even before meiosis, whereas IP(3) and sperm almost did not.     

Down-regulation of inositol 1,4,5-trisphosphate receptor in cells stably expressing the constitutively active angiotensin II N111G-AT(1) receptor

The diverse cellular changes brought about by the expression of a constitutively active receptor are poorly understood. QBI-human embryonic kidney 293A cells stably expressing the constitutively active N111G-AT(1) receptor (N111G cells) showed elevated levels of inositol phosphates and frequent spontaneous intracellular Ca(2+) oscillations. Interestingly, Ca(2+) transients triggered with maximal doses of angiotensin II were much weaker in N111G cells than in wild-type cells. These blunted responses were observed independently of the presence or absence of extracellular Ca(2+) and were also obtained when endogenous muscarinic and purinergic receptors were activated, revealing a heterologous desensitization process. The desensitized component of the Ca(2+) signaling cascade was neither the G protein G(q) nor phospholipase C. The intracellular Ca(2+) store of N111G cells and their mechanism of Ca(2+) entry also appeared to be intact. The most striking adaptive response of N111G cells was a down-regulation of their inositol 1,4,5-trisphosphate receptor (IP(3)R) as revealed by reduced IP(3)-induced Ca(2+) release, lowered [(3)H]IP(3) binding capacity, diminished IP(3)R immunoreactivity, and accelerated IP(3)R degradation involving the lysosomal pathway. Treatment with the inverse agonist EXP3174 reversed the desensitized phenotype of N111G cells. Down-regulation of IP(3)R represents a reversible adaptive response to protect cells against the adverse effects of constitutively active Ca(2+)-mobilizing receptors.     

Highly cooperative dependence of sarco/endoplasmic reticulum calcium ATPase SERCA2a pump activity on cytosolic calcium in living cells

Sarco/endoplasmic reticulum (SR/ER) Ca(2+)-ATPase (SERCA) is an intracellular Ca(2+) pump localized on the SR/ER membrane. The role of SERCA in refilling intracellular Ca(2+) stores is pivotal for maintaining intracellular Ca(2+) homeostasis, and disturbed SERCA activity causes many disease phenotypes, including heart failure, diabetes, cancer, and Alzheimer disease. Although SERCA activity has been described using a simple enzyme activity equation, the dynamics of SERCA activity in living cells is still unknown. To monitor SERCA activity in living cells, we constructed an enhanced CFP (ECFP)- and FlAsH-tagged SERCA2a, designated F-L577, which retains the ATP-dependent Ca(2+) pump activity. The FRET efficiency between ECFP and FlAsH of F-L577 is dependent on the conformational state of the molecule. ER luminal Ca(2+) imaging confirmed that the FRET signal changes directly reflect the Ca(2+) pump activity. Dual imaging of cytosolic Ca(2+) and the FRET signals of F-L577 in intact COS7 cells revealed that SERCA2a activity is coincident with the oscillatory cytosolic Ca(2+) concentration changes evoked by ATP stimulation. The Ca(2+) pump activity of SERCA2a in intact cells can be expressed by the Hill equation with an apparent affinity for Ca(2+) of 0.41 ± 0.0095 μm and a Hill coefficient of 5.7 ± 0.73. These results indicate that in the cellular environment the Ca(2+) dependence of ATPase activation is highly cooperative and that SERCA2a acts as a rapid switch to refill Ca(2+) stores in living cells for shaping the intracellular Ca(2+) dynamics. F-L577 will be useful for future studies on Ca(2+) signaling involving SERCA2a activity.     

Kinetic control of multiple forms of Ca(2+) spikes by inositol trisphosphate in pancreatic acinar cells.

The mechanisms of agonist-induced Ca(2+) spikes have been investigated using a caged inositol 1,4,5-trisphosphate (IP(3)) and a low-affinity Ca(2+) indicator, BTC, in pancreatic acinar cells. Rapid photolysis of caged IP(3) was able to reproduce acetylcholine (ACh)-induced three forms of Ca(2+) spikes: local Ca(2+) spikes and submicromolar (<1 microM) and micromolar (1-15 microM) global Ca(2+) spikes (Ca(2+) waves). These observations indicate that subcellular gradients of IP(3) sensitivity underlie all forms of ACh-induced Ca(2+) spikes, and that the amplitude and extent of Ca(2+) spikes are determined by the concentration of IP(3). IP(3)-induced local Ca(2+) spikes exhibited similar time courses to those generated by ACh, supporting a role for Ca(2+)-induced Ca(2+) release in local Ca(2+) spikes. In contrast, IP(3)- induced global Ca(2+) spikes were consistently faster than those evoked with ACh at all concentrations of IP(3) and ACh, suggesting that production of IP(3) via phospholipase C was slow and limited the spread of the Ca(2+) spikes. Indeed, gradual photolysis of caged IP(3) reproduced ACh-induced slow Ca(2+) spikes. Thus, local and global Ca(2+) spikes involve distinct mechanisms, and the kinetics of global Ca(2+) spikes depends on that of IP(3) production particularly in those cells such as acinar cells where heterogeneity in IP(3) sensitivity plays critical role.     

Calcium signaling in the developing Xenopus myotome.

Embryonic Xenopus myocytes generate spontaneous calcium (Ca(2+)) transients during differentiation in culture. Suppression of these transients disrupts myofibril organization and the formation of sarcomeres through an identified signal transduction cascade. Since transients often occur during myocyte polarization and migration in culture, we hypothesized they might play additional roles in vivo during tissue formation. We have tested this hypothesis by examining Ca(2+) dynamics in the intact Xenopus paraxial mesoderm as it differentiates into the mature myotome. We find that Ca(2+) transients occur in cells of the developing myotome with characteristics remarkably similar to those in cultured myocytes. Transients produced within the myotome are correlated with somitogenesis as well as myocyte maturation. Since transients arise from intracellular stores in cultured myocytes, we examined the functional distribution of both IP(3) and ryanodine receptors in the intact myotome by eliciting Ca(2+) elevations in response to photorelease of caged IP(3) and superfusion of caffeine, respectively. As in culture, transients in vivo depend on Ca(2+) release from ryanodine receptor (RyR) stores, and blocking RyR during development interferes with somite maturation.     

Cell membrane permeable esters of D-myo-inositol 1,4,5-trisphosphate

d-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3, or IP3) is a ubiquitous second messenger that regulates cytosolic Ca2+ activities ([Ca2+]i). To study this signaling branch in intact cells, we have synthesized a caged and cell permeable derivative of IP3, ci-IP3/PM, from myo-inositol in 9 steps. Ci-IP3/PM is a homologue of cm-IP3/PM, a caged and cell permeable IP3 ester developed earlier. In ci-IP3/PM, 2- and 3-hydroxyl groups of myo-inositiol are protected by an isopropylidene group; whereas in cm-IP3/PM, a methoxymethylene is used. Ci-IP3/PM can be loaded into cells non-invasively to high concentrations without activating IP3 receptors (IP3Rs). UV uncaging of loaded ci-IP3 released i-IP3, a potent agonist of IP3Rs, and evoked Ca2+ release from internal stores. Interestingly, elevations of [Ca2+]i by i-IP3 lasted longer than [Ca2+]i transients by m-IP3, the uncaging product of cm-IP3. To understand this difference, we measured the metabolic stability of i-IP3 and m-IP3. Like natural IP3 which is known to be rapidly metabolized in cells, m-IP3 could only be detected within several seconds after uncaging cm-IP3. In contrast, i-IP3 was metabolized at a much slower rate. By exploiting different metabolic rates of m-IP3 and i-IP3, we developed two procedures for activating IP3Rs in cells without UV uncaging. The first method involves photolyzing ci-IP3/PM in vitro to generate i-IP3/PM. Successive additions of low micromolar i-IP3/PM to NIH 3T3 cells caused graded Ca2+ releases, confirming that "quantal Ca2+ release" occurs in fully intact cells with normal ATP supplies and undisrupted endoplasmic reticulum. The second technique utilizes two photon uncaging. After locally illuminating cells loaded with cm-IP3 with femtosecond-pulsed near-infrared light (730 nm), we observed a burst of Ca2+ activity in the uncaging area. This local Ca2+ rise rapidly propagated across cells and could be repeated many times in different sub-cellular locations to produce artificial Ca2+ oscillations of defined amplitudes and frequencies. The complementary advantages of these IP3 prodrugs should provide new approaches for studying IP3-Ca2+ signaling in intact cell populations with high spatiotemporal resolutions.     

DM-nitrophen AM is caged magnesium

DM-nitrophen is a photolabile derivative of EDTA and therefore functions as either caged Ca(2+) or caged Mg(2+). Several papers have been published recently which use the AM ester to load intact cells with DM-nitrophen; under these conditions it is caged Mg(2+). In this commentary, I give a short history of the development and application of caged Ca(2+) probes, explaining why EGTA and BAPTA-based Ca(2+) cages are to be preferred for the photorelease of Ca(2+) under normal intracellular conditions.     

Junctional membrane inositol 1,4,5-trisphosphate receptor complex coordinates sensitization of the silent EGF-induced Ca2+ signaling

Ca(2+) is a highly versatile intracellular signal that regulates many different cellular processes, and cells have developed mechanisms to have exquisite control over Ca(2+) signaling. Epidermal growth factor (EGF), which fails to mobilize intracellular Ca(2+) when administrated alone, becomes capable of evoking [Ca(2+)](i) increase and exocytosis after bradykinin (BK) stimulation in chromaffin cells. Here, we provide evidence that this sensitization process is coordinated by a macromolecular signaling complex comprised of inositol 1,4,5-trisphosphate receptor type I (IP(3)R1), cAMP-dependent protein kinase (PKA), EGF receptor (EGFR), and an A-kinase anchoring protein, yotiao. The IP(3)R complex functions as a focal point to promote Ca(2+) release in two ways: (1) it facilitates PKA-dependent phosphorylation of IP(3)R1 in response to BK-induced elevation of cAMP, and (2) it couples the plasmalemmal EGFR with IP(3)R1 at the Ca(2+) store located juxtaposed to the plasma membrane. Our study illustrates how the junctional membrane IP(3)R complex connects different signaling pathways to define the fidelity and specificity of Ca(2+) signaling.     

Positive regulation of inositol 1,4,5-trisphosphate-induced Ca2+ release by mammalian target of rapamycin (mTOR) in RINm5F cells

The inositol 1,4,5-trisphosphate receptor (IP(3)R), a ligand-gated Ca(2+) channel, is the main regulator of intracellular Ca(2+) mobilization in non-excitable cells. An emerging body of evidence suggests that specific regulatory control of the Ca(2+) signaling pathway is modulated by the activation of additional signaling pathways. In the present study, we investigated the influence of the PI3-kinase/mammalian target of rapamycin (mTOR) pathway on the activity of the IP(3)R/Ca(2+) signaling pathway in RINm5F cells. We used a co-immunoprecipitation approach to show that mTOR physically interacts with IP(3)R-3 in an mTOR activity-dependent manner. We also showed that IP(3)R is phosphorylated by mTOR in cellulo. All the conditions known to modulate mTOR activity (IGF-1, wortmannin, rapamycin, PP242, and nutrient starvation) were shown to modify carbachol-induced Ca(2+) signaling in RINm5F cells. Lastly, we used an assay that directly measures the activity of IP(3)R, to show that mTOR increases the apparent affinity of IP(3)R. Given that mTOR controls cell proliferation and cell homeostasis, and that Ca(2+) plays a key role in these two phenomena, it follows that mTOR facilitates IP(3)R-mediated Ca(2+) release when the nutritional status of cells requires it.     

Spatial microenvironment defines Ca2+ entry and Ca2+ release in salivary gland cells

The difference of Ca(2+) mobilization induced by muscarinic receptor activation between parotid acinar and duct cells was examined. Oxotremorine, a muscarinic-cholinergic agonist, induced intracellular Ca(2+) release and extracellular Ca(2+) entry through store-operated Ca(2+) entry (SOC) and non-SOC channels in acinar cells, but it activated only Ca(2+) entry from non-SOC channels in duct cells. RT-PCR experiments showed that both types of cells expressed the same muscarinic receptor, M3. Given that ATP activated the intracellular Ca(2+) stores, the machinery for intracellular Ca(2+) release was intact in the duct cells. By immunocytochemical experiments, IP(3)R2 colocalized with M3 receptors in the plasma membrane area of acinar cells; in duct cells, IP(3)R2 resided in the region on the opposite side of the M3 receptors. On the other hand, purinergic P2Y2 receptors were found in the apical area of duct cells where they colocalized with IP(3)R2. These results suggest that the expression of the IP(3)Rs near G-protein-coupled receptors is necessary for the activation of intracellular Ca(2+) stores. Therefore, the microenvironment probably affects intracellular Ca(2+) release and Ca(2+) entry.     

Inositol trisphosphate receptors in smooth muscle cells

Inositol 1,4,5-trisphosphate receptors (IP(3)Rs) are a family of tetrameric intracellular calcium (Ca(2+)) release channels that are located on the sarcoplasmic reticulum (SR) membrane of virtually all mammalian cell types, including smooth muscle cells (SMC). Here, we have reviewed literature investigating IP(3)R expression, cellular localization, tissue distribution, activity regulation, communication with ion channels and organelles, generation of Ca(2+) signals, modulation of physiological functions, and alterations in pathologies in SMCs. Three IP(3)R isoforms have been identified, with relative expression and cellular localization of each contributing to signaling differences in diverse SMC types. Several endogenous ligands, kinases, proteins, and other modulators control SMC IP(3)R channel activity. SMC IP(3)Rs communicate with nearby ryanodine-sensitive Ca(2+) channels and mitochondria to influence SR Ca(2+) release and reactive oxygen species generation. IP(3)R-mediated Ca(2+) release can stimulate plasma membrane-localized channels, including transient receptor potential (TRP) channels and store-operated Ca(2+) channels. SMC IP(3)Rs also signal to other proteins via SR Ca(2+) release-independent mechanisms through physical coupling to TRP channels and local communication with large-conductance Ca(2+)-activated potassium channels. IP(3)R-mediated Ca(2+) release generates a wide variety of intracellular Ca(2+) signals, which vary with respect to frequency, amplitude, spatial, and temporal properties. IP(3)R signaling controls multiple SMC functions, including contraction, gene expression, migration, and proliferation. IP(3)R expression and cellular signaling are altered in several SMC diseases, notably asthma, atherosclerosis, diabetes, and hypertension. In summary, IP(3)R-mediated pathways control diverse SMC physiological functions, with pathological alterations in IP(3)R signaling contributing to disease.     

Fluorescent biosensor for quantitative real-time measurements of inositol 1,4,5-trisphosphate in single living cells

The second messenger inositol 1,4,5-trisphosphate (IP(3)) plays a central role in the generation of a variety of spatiotemporally complex intracellular Ca(2+) signals involved in the regulation of many essential physiological processes. Here we describe the development of "LIBRA", a novel ratiometric fluorescent IP(3) biosensor that allows for the quantitative monitoring of intracellular IP(3) concentrations in single living cells in real time. LIBRA consists of the IP(3)-binding domain of the rat type 3 IP(3) receptor fused between the fluorescence resonance energy transfer pair cyan fluorescent protein and yellow fluorescent protein and preceded by a membrane-targeting signal. We show that the LIBRA fluorescent signal is highly selective for IP(3) and unaffected by concentrations of Ca(2+) and ATP in the physiological range. In addition, LIBRA can be calibrated in situ. We demonstrate the utility of LIBRA by monitoring the temporal relationship between the responses intracellular IP(3) and Ca(2+) concentrations in SH-SY5Y cells following acetylcholine stimulation.     

Inositol 1,4,5-trisphosphate directs Ca(2+) flow between mitochondria and the Endoplasmic/Sarcoplasmic reticulum: a role in regulating cardiac autonomic Ca(2+) spiking

The signaling role of the Ca(2+) releaser inositol 1,4, 5-trisphosphate (IP(3)) has been associated with diverse cell functions. Yet, the physiological significance of IP(3) in tissues that feature a ryanodine-sensitive sarcoplasmic reticulum has remained elusive. IP(3) generated by photolysis of caged IP(3) or by purinergic activation of phospholipase Cgamma slowed down or abolished autonomic Ca(2+) spiking in neonatal rat cardiomyocytes. Microinjection of heparin, blocking dominant-negative fusion protein, or anti-phospholipase Cgamma antibody prevented the IP(3)-mediated purinergic effect. IP(3) triggered a ryanodine- and caffeine-insensitive Ca(2+) release restricted to the perinuclear region. In cells loaded with Rhod2 or expressing a mitochondria-targeted cameleon and TMRM to monitor mitochondrial Ca(2+) and potential, IP(3) induced transient Ca(2+) loading and depolarization of the organelles. These mitochondrial changes were associated with Ca(2+) depletion of the sarcoplasmic reticulum and preceded the arrest of cellular Ca(2+) spiking. Thus, IP(3) acting within a restricted cellular region regulates the dynamic of calcium flow between mitochondria and the endoplasmic/sarcoplasmic reticulum. We have thus uncovered a novel role for IP(3) in excitable cells, the regulation of cardiac autonomic activity.     

Photolysis of intracellular caged sphingosine-1-phosphate causes Ca2+ mobilization independently of G-protein-coupled receptors

Sphingosine-1-phosphate (S1P), the product of sphingosine kinase, activates several widely expressed G-protein-coupled receptors (GPCR). S1P might also play a role as second messenger, but this hypothesis has been challenged by recent findings. Here we demonstrate that intracellular S1P can mobilize Ca(2+) in intact cells independently of S1P-GPCR. Within seconds, S1P generated by the photolysis of caged S1P raised the intracellular free Ca(2+) concentration in HEK-293, SKNMC and HepG2 cells, in which the response to extracellularly applied S1P was either blocked or absent. Ca(2+) transients induced by photolysis of caged S1P were caused by Ca(2+) mobilization from thapsigargin-sensitive stores. These results provide direct evidence for a true intracellular action of S1P.     

Effects of a time varying strong magnetic field on release of cytosolic free Ca2+ from intracellular stores in cultured bovine adrenal chromaffin cells

This study was made to explain the mechanisms for the effects of exposure to a time varying 1.51 T magnetic field on the intracellular Ca(2+) signaling pathway. The exposure inhibited an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) in bovine chromaffin cells induced by addition of bradykinin (BK) to a Ca(2+) free medium. The exposure did not change BK induced production of inositol 1,4,5-trisphosphate (IP(3)). [Ca(2+)](i) was markedly increased in IP(3) loaded cells, and this increase was inhibited by the magnetic field exposure. A similar increase in [Ca(2+)](i) by other drugs, which stimulated Ca(2+) release from intracellular Ca(2+) stores, was again inhibited by the same exposure. However, transmembrane Ca(2+) fluxes caused in the presence of thapsigargin were not inhibited by the magnetic field exposure in a Ca(2+) containing medium. Inhibition of the BK induced increase in [Ca(2+)](i) by the exposure for 30 min was mostly recovered 1 h after exposure ended. Our results reveal that the magnetic field exposure inhibits Ca(2+) release from intracellular Ca(2+) stores, but that BK bindings to BK receptors of the cell membrane and intracellular inositol IP(3) production are not influenced.     

No evidence for inositol 1,4,5-trisphosphate-dependent Ca2+ release in isolated fibers of adult mouse skeletal muscle

The presence and role of functional inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)Rs) in adult skeletal muscle are controversial. The current consensus is that, in adult striated muscle, the relative amount of IP(3)Rs is too low and the kinetics of Ca(2+) release from IP(3)R is too slow compared with ryanodine receptors to contribute to the Ca(2+) transient during excitation-contraction coupling. However, it has been suggested that IP(3)-dependent Ca(2+) release may be involved in signaling cascades leading to regulation of muscle gene expression. We have reinvestigated IP(3)-dependent Ca(2+) release in isolated flexor digitorum brevis (FDB) muscle fibers from adult mice. Although Ca(2+) transients were readily induced in cultured C2C12 muscle cells by (a) UTP stimulation, (b) direct injection of IP(3), or (c) photolysis of membrane-permeant caged IP(3), no statistically significant change in calcium signal was detected in adult FDB fibers. We conclude that the IP(3)-IP(3)R system does not appear to affect global calcium levels in adult mouse skeletal muscle.