Subsequent Calcium Release Research Articles

Flupyradifurone activates DUM neuron nicotinic acetylcholine receptors and stimulates an increase in intracellular calcium through the ryanodine receptors

Insect neuronal nicotinic acetylcholine receptors (nAChRs) are transmembrane receptors that play a key role in the development and synaptic plasticity of both vertebrates and invertebrates, and are considered to be major targets of several insecticides. We used dorsal unpaired median (DUM) neurons, which are insect neurosecretory cells, to explore what type of nAChRs are involved in flupyradifurone's (FLU) mode of action, and to study the role of calcium release from intracellular stores in this process. Using whole-cell patch-clamp and fura-2-AM calcium imaging techniques, we found that inhibition of IP3Rs through application of 2-APB reduced FLU inward currents, but did not affect the intracellular calcium release induced by FLU. In contrast, inhibition of RyRs using ryanodine, led to reduction of intracellular calcium increase following FLU pulse application. These results suggested that FLU inward currents are likely due to a combination of the direct effects of FLU on DUM neuron nAChRs and the subsequent calcium release from RyRs.

An Overview on the Hallucinogenic Peyote and Its Alkaloid Mescaline: The Importance of Context, Ceremony and Culture.

Peyote (Lophophora williamsii) is a cactus that contains various biologically active alkaloids-such as pellotine, anhalonidine, hordenine and mescaline. Here, mescaline induces the psychoactive effects of peyote through the activation of the serotonin 5-HT2A receptor and the subsequent release of calcium (Ca2+) from the endoplasmic reticulum (ER). Moreover, an evaluation of the therapeutic benefits of mescaline is also currently the subject of research. It is important to consider that the outcome of taking a psychedelic drug strongly depends on the mindset of the recipient and the context (set and setting principle), including ceremonies and culture. This overview serves to summarise the current state of the knowledge of the metabolism, mechanism of action and clinical application studies of peyote and mescaline. Furthermore, the benefits of the potential of peyote and mescaline are presented in a new light, setting an example for combining a form of treatment embedded in nature and ritually enriched with our current highly innovative Western medicine.

Adrenaline induces calcium signal in astrocytes and vasoconstriction via activation of monoamine oxidase

Adrenaline or epinephrine is a hormone playing an important role in physiology. It is produced de-novo in the brain in very small amounts compared to other catecholamines, including noradrenaline. Although the effects of adrenaline on neurons have been extensively studied, much less is known about the action of this hormone on astrocytes. Here, we studied the effects of adrenaline on astrocytes in primary co-culture of neurons and astrocytes. Application of adrenaline induced calcium signal in both neurons and astrocytes, but only in neurons this effect was dependent on α- and β-receptor antagonists. The effects of adrenaline on astrocytes were less dependent on adrenoreceptors: the antagonist carvedilol had only moderate effect on the calcium signal and the agonist of adrenoreceptors methoxamine induced a signal only in small proportion of the cells. We found that adrenaline in astrocytes activates phospholipase C and subsequent release of calcium from the endoplasmic reticulum. Calcium signal in astrocytes is initiated by the metabolism of adrenaline by the monoamine oxidase (MAO), which activates reactive oxygen species production and induces lipid peroxidation. Inhibitor of MAO selegiline inhibited both adrenaline-induced calcium signal in astrocytes and the vasoconstriction that indicates an important role for monoamine oxidase in adrenaline-induced signalling and function.

Exhaustion of Skeletal Muscle Fibers Within Seconds: Incorporating Phosphate Kinetics Into a Hill-Type Model.

Initiated by neural impulses and subsequent calcium release, skeletal muscle fibers contract (actively generate force) as a result of repetitive power strokes of acto-myosin cross-bridges. The energy required for performing these cross-bridge cycles is provided by the hydrolysis of adenosine triphosphate (ATP). The reaction products, adenosine diphosphate (ADP) and inorganic phosphate (Pi), are then used—among other reactants, such as creatine phosphate—to refuel the ATP energy storage. However, similar to yeasts that perish at the hands of their own waste, the hydrolysis reaction products diminish the chemical potential of ATP and thus inhibit the muscle's force generation as their concentration rises. We suggest to use the term “exhaustion” for force reduction (fatigue) that is caused by combined Pi and ADP accumulation along with a possible reduction in ATP concentration. On the basis of bio-chemical kinetics, we present a model of muscle fiber exhaustion based on hydrolytic ATP-ADP-Pi dynamics, which are assumed to be length- and calcium activity-dependent. Written in terms of differential-algebraic equations, the new sub-model allows to enhance existing Hill-type excitation-contraction models in a straightforward way. Measured time courses of force decay during isometric contractions of rabbit M. gastrocnemius and M. plantaris were employed for model verification, with the finding that our suggested model enhancement proved eminently promising. We discuss implications of our model approach for enhancing muscle models in general, as well as a few aspects regarding the significance of phosphate kinetics as one contributor to muscle fatigue.

L-type calcium channel modulates mechanosensitivity of the cardiomyocyte cell line H9c2

The application of mechanical stimuli to cells often induce increases in intracellular calcium, affecting the regulation of a variety of cell functions. Although the mechanism of mechanotransduction-induced calcium increases has not been fully resolved, the involvement of mechanosensitive ion channels in the plasma membrane and the endoplasmic reticulum has been reported. Here, we demonstrate that voltage-gated L-type calcium channels play a critical role in the mechanosensitive calcium response in H9c2 rat cardiomyocytes. The intracellular calcium level in H9c2 cells increased in a reproducible dose-dependent manner in response to uniaxial stretching. The stretch-activated calcium response (SICR) completely disappeared in calcium-free medium, whereas thapsigargin and cyclopiazonic acid, inhibitors of sarcoendoplasmic reticulum calcium ATPase, partially reduced the SICR. These findings suggest that both calcium influx across the cell membrane and calcium release from the sarcoendoplasmic reticulum are involved in the SICR. Nifedipine, diltiazem, and verapamil, inhibitors of L-type calcium channels, reduced the SICR in a dose-dependent manner. Furthermore, small interfering RNA against the L-type calcium channel α1c subunit diminished the SICR dramatically. Nifedipine also diminished the mechanosensitivity of Langendorff-perfused rat heart. These results suggest that the SICR in H9c2 cardiomyocytes involves the activation of L-type calcium channels and subsequent calcium release from the sarcoendoplasmic reticulum.

Effect of Exposed Surface Area, Volume and Environmental pH on the Calcium Ion Release of Three Commercially Available Tricalcium Silicate Based Dental Cements.

Tricalcium silicate cements (TSC) are used in dental traumatology and endodontics for their bioactivity which is mostly attributed to formation of calcium hydroxide during TSC hydration and its subsequent release of calcium and hydroxide ions. The aim of this study was to determine the effect of volume (Vol), exposed surface area (ESA) and pH of surrounding medium on calcium ion release. Three commercially available hydraulic alkaline dental cements were mixed and condensed into cylindrical tubes of varying length and diameter (n = 6/group). For the effect of ESA and Vol, tubes were immersed in 10 mL of deionized water. To analyze the effect of environmental pH, the tubes were randomly immersed in 10 mL of buffer solutions with varying pH (10.4, 7.4 or 4.4). The solutions were collected and renewed at various time intervals. pH and/or calcium ion release was measured using a pH glass electrode and atomic absorption spectrophotometer respectively. The change of pH, short-term calcium ion release and rate at which calcium ion release reaches maximum were dependent on ESA (p < 0.05) while maximum calcium ion release was dependent on Vol of TSC (p < 0.05). Maximum calcium ion release was significantly higher in acidic solution followed by neutral and alkaline solution (p < 0.05).

Abstract 4611: Novel lysosomotrophic agent inhibits in vivo tumor formation and triggers calcium-dependent cell death in a variety of human cancer cell lines

Abstract In addition to being investigated as probes for diagnostic imaging, lanthanide family of rare earth compounds were shown to be tumor-selective, however the mechanism of anti-cancer activity remains unclear. We synthesized a conjugate of the lanthanide, praseodymium (141Pr), and observed remarkable anti-tumor activity in 2 murine xenograft models (colorectal and prostate cancer), with minimal off target effects. Importantly, Pr-mercaptopyridine oxide (Pr-MPO) elicited strong anti-cancer activity (IC50∼2.5uM) against a host of human cancer cell lines including cisplatin resistant, p53-/- and/or Bax-/- variants. Intrigued by this, we investigated the mechanisms underlying the anti-cancer activity of Pr-MPO. Our earlier findings indicated weak PPARγ agonist-like activity despite any structural similarity to known PPARγ agonists, therefore, we first investigated the effect of Pr-MPO on cell metabolism. An increase in glucose uptake and a significant decrease in pyruvate levels were observed. Indeed, pyruvate delayed cell death while restoring a regulated activity similar to non-treated conditions. Despite the PPARγ agonist-like activity, the anti-cancer effect of Pr-MPO was not dependent on PPARγ signaling. Furthermore, while exposure to Pr-MPO resulted in a drastic reduction in tumor colony forming ability, cell death was caspase independent and associated with a strong autophagic phenotype. Despite the induction of mToR-dependent autophagy, confirmed by western blot analysis of LC3II, p62, and puncta formation upon GFP-LC3 transfection, neither pharmacological inhibition nor the knockdown of Atg7/5 blocked Pr-MPO-induced cell death. Interestingly, Pr-MPO induced a significant increase in intracellular calcium fluorescence, measured by time resolved live imaging, however, unlike thapsigargin, the increase in calcium was not mediated by ER calcium stores. Instead, a significant increase in calcium fluorescence (puncta) was observed within minutes, a pattern distinctly different from the diffuse cytosolic increase upon depleting ER calcium. Notably, chelation of extracellular calcium abrogated the increase in intracellular calcium as well as rescued cells from Pr-MPO-induced cell death, thus suggesting the involvement of store operated calcium entry. Further analysis indicated these puncta being associated with the lysosomes. Therefore, we turned our focus on lysosomes as the “organelle preferé” in Pr-MPO induced cell death. Indeed, Pr-MPO resulted in the induction of lysosomal membrane permeability, which was associated with an overall increase in cathepsin L activity. As the increase in lysosomal calcium fluorescence is rapidly induced by Pr-MPO, a possible target could be a membrane ATPase or the rapid increase in fluorescence could be an indicator for the subsequent release of calcium. These possibilities are the current focus of investigation. Citation Format: Dan Liu, Gregory L. Bellot, Sanjiv K. Yadav, Mark Fivaz, Charnjit Kaur, Gautam Sethi, Shazib Pervaiz. Novel lysosomotrophic agent inhibits in vivo tumor formation and triggers calcium-dependent cell death in a variety of human cancer cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4611. doi:10.1158/1538-7445.AM2014-4611

GABAergic transmission modulates ethanol excitation of ventral tegmental area dopamine neurons

Activation of the dopaminergic (DA) neurons of the ventral tegmental area (VTA) by ethanol has been implicated in its rewarding and reinforcing effects. We previously demonstrated that ethanol enhances GABA release onto VTA-DA neurons via activation of 5-HT 2C receptors and subsequent release of calcium from intracellular stores. Here we demonstrate that excitation of VTA-DA neurons by ethanol is limited by an ethanol-enhancement in GABA release. In this study, we performed whole-cell voltage clamp recordings of miniature inhibitory postsynaptic currents (mIPSCs) and cell-attached recordings of action potential firing from VTA-DA neurons in midbrain slices from young Long Evans rats. Acute exposure to ethanol (75 mM) transiently enhanced the firing rate of VTA-DA neurons as well as the frequency of mIPSCs. Simultaneous blockade of both GABA A and GABA B receptors (Picrotoxin (75 μM) and SCH50911 (20 μM)) disinhibited VTA-DA firing rate whereas a GABA A agonist (muscimol, 1 μM) strongly inhibited firing rate. In the presence of picrotoxin, ethanol enhanced VTA-DA firing rate more than in the absence of picrotoxin. Additionally, a sub-maximal concentration of muscimol together with ethanol inhibited VTA-DA firing rate more than muscimol alone. DAMGO (3 μM) inhibited mIPSC frequency but did not block the ethanol-enhancement in mIPSC frequency. DAMGO (1 and 3 μM) had no effect on VTA-DA firing rate. Naltrexone (60 μM) had no effect on basal or ethanol-enhancement of mIPSC frequency. Additionally, naltrexone (20 and 60 μM) did not block the ethanol-enhancement in VTA-DA firing rate. Overall, the present results indicate that the ethanol enhancement in GABA release onto VTA-DA neurons limits the stimulatory effect of ethanol on VTA-DA neuron activity and may have implications for the rewarding properties of ethanol.

Dopamine Induces Ca2+ Signaling in Astrocytes through Reactive Oxygen Species Generated by Monoamine Oxidase

Dopamine is a neurotransmitter that plays a major role in a variety of brain functions, as well as in disorders such as Parkinson disease and schizophrenia. In cultured astrocytes, we have found that dopamine induces sporadic cytoplasmic calcium ([Ca(2+)](c)) signals. Importantly, we show that the dopamine-induced calcium signaling is receptor-independent in midbrain, cortical, and hippocampal astrocytes. We demonstrate that the calcium signal is initiated by the metabolism of dopamine by monoamine oxidase, which produces reactive oxygen species and induces lipid peroxidation. This stimulates the activation of phospholipase C and subsequent release of calcium from the endoplasmic reticulum via the inositol 1,4,5-trisphosphate receptor mechanism. These findings have major implications on the function of astrocytes that are exposed to dopamine and may contribute to understanding the physiological role of dopamine.

Urotensin II-induced signaling involved in proliferation of vascular smooth muscle cells

The urotensin II receptor, bound by the ligand urotensin II, generates second messengers, ie, inositol triphosphate and diacylglycerol, which stimulate the subsequent release of calcium (Ca(2+)) in vascular smooth muscle cells. Ca(2+) influx leads to the activation of Ca(2+)-dependent kinases (CaMK) via calmodulin binding, resulting in cellular proliferation. We hypothesize that urotensin II signaling in pulmonary arterial vascular smooth muscle cells (Pac1) and primary aortic vascular smooth muscle cells (PAVSMC) results in phosphorylation of Ca(2+)/calmodulin-dependent kinases leading to cellular proliferation. Exposure of Pac1 cultures to urotensin II increased intracellular Ca(2+), subsequently activating Ca(2+)/calmodulin-dependent kinase kinase (CaMKK), and Ca(2+)/calmodulin-dependent kinase Type I (CaMKI), extracellular signal-regulated kinase (ERK 1/2), and protein kinase D. Treatment of Pac1 and PAVSMC with urotensin II increased proliferation as measured by (3)H-thymidine uptake. The urotensin II-induced increase in (3)H-thymidine incorporation was inhibited by a CaMKK inhibitor. Taken together, our results demonstrate that urotensin II stimulation of smooth muscle cells leads to a Ca(2+)/calmodulin-dependent kinase-mediated increase in cellular proliferation.

RANKL Links Arterial Calcification With Osteolysis

See related article, pages 1041–1048 Vascular calcification is a pathological sequence of events that has similarities to the normal physiological process of osteogenesis.1,2 It is thought to result from an imbalance in both local and systemic inhibitors and promoters,3,4 which occurs as a consequence of chronic inflammation, hyperleptinemia, and a deregulation of various bone-regulating proteins. It is common in patients with diabetes and renal failure and in the elderly.4,5 The fact that coronary artery calcification is such a major health problem and the recognition that it is highly correlative with mortality risk6 makes the understanding of vascular calcification a worthy and exciting area of study. In normal skeletal physiology, bone deposition by osteoblasts is in balance with bone resorption by osteoclasts. RANKL (receptor activator of nuclear factor [NF]-κB ligand) and its soluble decoy receptor, osteoprotegerin (OPG), are critical regulators of bone remodeling. Binding of RANKL to its cognate receptor RANK, expressed on osteoclasts and its precursors, induces NF-κB signaling, resulting in NF-κB translocation to the nucleus with a subsequent increase in RelB levels, in turn, stimulating osteoclastic differentiation (Figure, A). OPG, a decoy receptor, expressed by osteoblasts, binds with RANKL, preventing RANK (receptor activator of NF-κB) signaling and thus inhibiting osteoclastogenesis (Figure, B).7 Figure. Bone resorption in the skeleton often coexists with bone formation in the vasculature. A, Binding of RANKL to RANK stimulates osteoclast maturation and destruction of bone through loss of mineral. B, Osteoblasts express OPG the natural decoy receptor for RANKL, preventing RANK activation and inhibiting maturation of osteoclasts thus inhibiting bone resorption. An increased availability of OPG and the block on RANKL signaling supports osteoblast differentiation, maturation, and function allowing the deposition of bone. C, Elevated OPG in the vasculature and increased Ca/P products marks a commitment of …

Urotensin II Induced Signal Involved in Proliferation of Vascular Smooth Muscle Cells

Urotensin II receptor (UIIR), bound by the ligand Urotensin II (UII), generates second messengers IP3 and DAG, which stimulates the subsequent release of calcium (Ca2+) in vascular smooth muscle cells (VSMC). Ca2+ influx leads to the activation of Ca2+ dependent kinases via calmodulin binding, resulting in cellular proliferation. We hypothesize that UII signaling in pulmonary arterial vascular smooth muscle cells (Pac1) and primary aortic vascular smooth muscle cells (PAVSMC) results in phosphorylation of Ca2+/ CaM dependent kinases leading to cellular proliferation. UII exposure to Pac1 cell cultures increased intracellular Ca2+, which subsequently activated Ca2+/CaM dependent kinases (CaMKK &amp; CaMKI), ERK 1/ 2, and PKD (PKC‐µ). Pac1 and PAVSMC cells treated with UII resulted in increased proliferation as measured by 3H thymidine uptake. UII‐induced increase of 3H thymidine incorporation was inhibited by a CaMKK inhibitor. Taken together, our results demonstrate that UII stimulation of smooth muscle cells leads to cellular proliferation, utilizing signaling mechanisms involving Ca2+/CaM dependent kinases.

Spatial segregation of neuronal calcium signals encodes different forms of LTP in rat hippocampus

Calcium regulates numerous processes in the brain. How one signal can coordinate so many diverse actions, even within the same neurone, is the subject of intense investigation. Here we have used two-photon calcium imaging to determine the mechanism that enables calcium to selectively and appropriately induce different forms of long-term potentiation (LTP) in rat hippocampus. Short-lasting LTP (LTP 1) required activation of ryanodine receptors (RyRs), which selectively increased calcium in synaptic spines. LTP of intermediate duration (LTP 2) was dependent on activation of inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) and subsequent calcium release specifically in dendrites. Long-lasting LTP (LTP 3) was selectively dependent on L-type voltage-dependent calcium channels (L-VDCCs), which generated somatic calcium influx. Activation of NMDA receptors was necessary, but not sufficient, for the generation of appropriate calcium signals in spines and dendrites, and the induction of LTP 1 and LTP 2. These results suggest that the selective induction of different forms of LTP is achieved via spatial segregation of functionally distinct calcium signals.

ATP-INDUCED CALCIUM INCREASE AS A POTENTIAL FIRST SIGNAL IN MECHANICAL TISSUE TRAUMA. A LASER SCANNING MICROSCOPIC STUDY ON CULTURED MOUSE SKELETAL MYOCYTES

Although it is known that after major tissue trauma, local incidents in the mechanically destroyed muscle tissue form the basis of subsequently occurring severe inflammatory reactions, the very first events taking place immediately after myocyte destruction have not been studied on the single cell level thus far. Therefore, in this study, the reaction of cultured C2C12 mouse skeletal myocytes to lethal injury was examined using laser scanning microscopy. Mechanical rupture of one single myocyte induced an immediate accumulation of calcium in its cytosol and nuclei, as detected by an increase in the fluorescence intensity of the intracellular calcium-sensitive dye Fluo-3. The intracellular calcium elevation propagated further to the adjacent, noninjured myocytes in a wave-like fashion within seconds. The calcium increase detected in these neighboring cells was higher and up to 1000 times more extended than the physiological calcium spike that induces C2C12 myocyte contraction. Wave propagation did not depend on gap junctional communication but occurred via liberation of nucleotides, mainly ATP, but presumably also UTP and others, from the destroyed cell and subsequent calcium release from the sarcoplasmic reticulum via a purinoceptor-mediated mechanism in the adjacent cells. These findings suggest a decisive role of ATP and related nucleotides in the pathogenesis of tissue trauma because they appear to initiate the signaling mechanism from injured myocytes to the surrounding tissue and potentially to the whole body.

Short-Range Intercellular Calcium Signaling in Bone

The regulation of bone turnover is a complex and finely tuned process. Many factors regulate bone remodeling, including hormones, growth factors, cytokines etc. However, little is known about the signals coupling bone formation to bone resorption, and how mechanical forces are translated into biological effects in bone. Intercellular calcium waves are increases in intracellular calcium concentration in single cells, subsequently propagating to adjacent cells, and can be a possible mechanism for the coupling of bone formation to bone resorption. The aim of the present studies was to investigate whether bone cells are capable of communicating via intercellular calcium signals, and determine by which mechanisms the cells propagate the signals. First, we found that osteoblastic cells can propagate intercellular calcium transients upon mechanical stimulation, and that there are two principally different mechanisms for this propagation. One mechanism involves the secretion of a nucleotide, possibly ATP, acting in an autocrine action to purinergic P2Y2 receptors on the neighboring cells, leading to intracellular IP3 generation and subsequent release of calcium from intracellular stores. The other mechanism involves the passage of a small messenger through gap junctions to the cytoplasm of the neighboring cells, inducing depolarization of the plasma membrane with subsequent opening of membrane bound voltage-operated calcium channels. Next, we found that osteoblasts can propagate these signals to osteoclasts as well. We demonstrated that paracrine action of ATP was responsible for the wave propagation, but now the purinergic P2X7 receptor was involved. Thus, the studies demonstrate that calcium signals can be propagated not only among osteoblasts, but also between osteoblasts and osteoclasts in response to mechanical stimulation. Thus, intercellular calcium signaling can be a mechanism by which mechanical stimuli on bone are translated into biological signals in bone cells, and propagated through the network of cells in bone. Further, the observations offer new pharmacological targets for the modulation of bone turnover, and perhaps even for the treatment of bone metabolic disorders.

The membrane-anchored serine protease, TMPRSS2, activates PAR-2 in prostate cancer cells

TMPRSS2 is a type II transmembrane-bound serine protease that has gained interest owing to its highly localized expression in the prostate and its overexpression in neoplastic prostate epithelium. Once activated, the serine protease domain of TMPRSS2 is released from the cell surface into the extracellular space. PAR (protease-activated receptor)-2 belongs to a family of G-protein-coupled receptors (PAR-1-4) that are activated by specific serine proteases, which are expressed in many normal and malignant cell types. Previous in vitro studies on prostate cancer cells suggest a role for PAR-2 in prostate cancer metastasis. A polyclonal anti-human TMPRSS2 antibody was generated against the TMPRSS2 serine protease domain. The antibody showed specific reactivity with recombinant expressed TMPRSS2, and so was used to extract and purify the cleaved active TMPRSS2 protease from prostate cancer cells. Reverse transcriptase PCR and Western blot analysis were used to show the expression of both TMPRSS2 and PAR-2 in the androgen-dependent LNCaP prostate cancer cell line. Treatment of LNCaP cells with the cellular immunopurified TMPRSS2 protease induced a transient increase in intracellular calcium, which is indicative of G-protein-coupled-receptor activation. This calcium mobilization was inhibited by cellular pre-treatment with a specific PAR-2 antagonist, but not with a PAR-1 antagonist; inhibition of the protease activity also failed to mobilize calcium, suggesting that TMPRSS2 is capable of cleaving and thereby activating the PAR-2 receptor. The calcium mobilization was also inhibited by cellular pre-treatment with suramin or 2-APB (2-aminoethoxydiphenyl borate), indicating that a G-protein pathway is involved and that subsequent calcium release is mainly from intracellular stores. The present study describes how TMPRSS2 may contribute to prostate tumour metastasis via the activation of PAR-2.

Calcium Mobilization and Spontaneous Transient Outward Current Characteristics upon Agonist Activation of P2Y2 Receptors in Smooth Muscle Cells

A quantitative model is provided that links the process of metabotropic receptor activation and sequestration to the generation of inositol 1,4,5-trisphosphate, the subsequent release of calcium from the central sarcoplasmic reticulum, and the consequent release of calcium from subsarcolemma sarcoplasmic reticulum that acts on large-conductance potassium channels to generate spontaneous transient outward currents (STOCs). This model is applied to the case of STOC generation in vascular A7r5 smooth muscle cells that have been transfected with a chimera of the P2Y2 metabotropic receptor and green fluorescent protein (P2Y2-GFP) and exposed to the P2Y2 receptor agonist uridine 5′-triphosphate. The extent of P2Y2-GFP sequestration from the membrane on exposure to uridine 5′-triphosphate, the ensuing changes in cytosolic calcium concentration, as well as the interval between STOCs that are subsequently generated, are used to determine parameter values in the model. With these values, the model gives a good quantitative prediction of the dynamic changes in STOC amplitude observed upon activation of metabotropic P2Y2 receptors in the vascular smooth muscle cell line.

Magnesium sulfate inhibits the oxytocin-induced production of inositol 1,4,5-trisphosphate in cultured human myometrial cells

Objective: The purpose of this study was to determine the effects of magnesium sulfate on inositol trisphosphate production and the mechanism of these effects. Study Design: Myometrium was obtained at the time of cesarean delivery from women before labor at term. Inositol trisphosphate was measured in the primary myometrial cell cultures after stimulation with oxytocin, sodium fluoride, or Bay K 8644 with or without preincubation with magnesium sulfate or nifedipine. Experiments were performed in either calcium-containing or calcium-free medium that contained egtazic acid and after preincubation with the intracellular calcium chelator BAPTA-acetoxymethylester. Inositol trisphosphate production was measured by radioreceptor assay. In separate experiments, changes in intracellular calcium concentrations ([Ca2+]i) were measured with the use of Fura-2 and spectrophotofluorometry. Results: Oxytocin, sodium fluoride, and Bay K 8644 increased inositol trisphosphate production 2- to 4-fold. Preincubation with magnesium sulfate (3 × 10−3 mol/L) for ≥5 minutes decreased oxytocin-, sodium fluoride-, and Bay K 8644-induced inositol trisphosphate production in either calcium-containing or calcium-free media. Preincubation with BAPTA-acetoxymethylester decreased oxytocin-stimulated inositol trisphosphate production by 78% in calcium-containing media and completely prevented the oxytocin response in calcium-free media. Magnesium sulfate decreased inositol trisphosphate production in calcium-containing media but had no additional effect in calcium-free media. Oxytocin and Bay K 8644 increased [Ca2+]i in either calcium-containing or calcium-free media, and magnesium sulfate reduced this in both cases. Conclusion: Magnesium sulfate appears to inhibit phosphatidylinositol-4, 5-bisphosphate-specific phospholipase C activity and subsequent calcium release in cultured myometrial cells by a direct effect on phospholipase C. (Am J Obstet Gynecol 2002;187:419-24.)

Src kinase induces calcium release in Xenopus egg extracts via PLCγ and IP 3-dependent mechanism

Mobilization of intracellular calcium is an indispensable step of fertilization-induced egg activation. Recently, this process has been shown to require the sequential activation of Src family tyrosine kinases, phospholipase Cγ (PLCγ), and inositol-1,4,5-trisphosphate (IP 3)-dependent receptor of endoplasmic reticulum. In the present study, we made an attempt to recapitulate the early events of egg activation by stimulating Src kinase activity in the cell-free extracts of Xenopus eggs. We found that enhanced Src kinase activity can initiate calcium response of low magnitude in cytostatic factor (CSF)-arrested mitotic extracts without releasing them into interphase. The addition of catalytically active recombinant Src kinase, as well as the activation of endogenous Xenopus Src family kinase by hydrogen peroxide (H 2O 2), increased total tyrosine phosphorylation, tyrosine phosphorylation of PLCγ, and IP 3 production in the extracts. The treatment with the Src family kinase-specific inhibitor, PP1, or PLC inhibitor, U73122, or IP 3 receptor antagonist, heparin, prevented calcium release in the extracts. We conclude, therefore, that possible mechanism of Src/H 2O 2 action in the extracts might involve tyrosine phosphorylation and activation of PLCγ, accompanied by the increase in IP 3 content and subsequent calcium release from IP 3-regulated calcium stores. These results also suggest that monitoring calcium signals induced in the Xenopus egg extracts by various components of signaling pathways may provide a particularly useful approach to investigating their role in the signal transduction.

Bi-directional coupling between dihydropyridine receptors and ryanodine receptors

The control of calcium signaling between plasma membrane dihydropyridine receptors (DHPRs or L-type calcium channels) and ryanodine receptors (RyRs or calcium release channels) located in the endoplasmic/sarcoplasmic reticulum (ER/SR) underlies a broad array of functions including skeletal muscle contraction, cardiac performance, arteriole tone, neurosecretion, synaptic plasticity, and gene regulation. It has long been appreciated that DHPR activation of RyRs and subsequent calcium release from intracellular stores represents a key event in the control of these processes. In excitable cells, DHPRs trigger the release of intracellular calcium by promoting the opening of nearby RyRs (termed orthograde coupling). Interestingly, the signaling interaction between DHPRs and RyRs is often bi-directional such that the calcium-conducting activity of DHPR channels is also regulated by its interaction with the RyR (termed retrograde coupling). Recent data indicate that skeletal, cardiac, and neuronal cells utilize fundamentally distinct DHPR/RyR bi-directional coupling mechanisms (chemical and mechanical) in order to control the efficiency, fidelity, and activity of each of these two essential calcium channels. This review will focus on evaluating the nature and molecular determinants of these coupling mechanisms, as well as the extent to which excitable cell function is influenced by bi-directional DHPR/RyR calcium signaling.