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Thirty-six years after the publication of the important article by Busa and Nuccitelli on the variability of intracellular pH (pHi) and the interdependence of pHi and intracellular Ca2+ concentration ([Ca2+]i), little research has been carried out on pHi and calcium signaling. Moreover, the results appear to be contradictory. Some authors claim that the increase in [Ca2+]i is due to a reduction in pHi, others that it is caused by an increase in pHi. The reasons for these conflicting results have not yet been discussed and clarified in an exhaustive manner. The idea that variations in pHi are insignificant, because cellular buffers quickly stabilize the pHi, may be a limiting and fundamentally wrong concept. In fact, it has been shown that protons can move and react in the cell before they are neutralized. Variations in pHi have a remarkable impact on [Ca2+]i and hence on some of the basic biochemical mechanisms of calcium signaling. This paper focuses on the possible triggering role of protons during their short cellular cycle and it suggests a new hypothesis for an IP3 proton dependent mechanism of action., (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

The IP3 receptor binding protein released with inositol 1,4,5-trisphosphate (IRBIT) plays important roles in the regulation of intracellular Ca 2+ signaling and intracellular pH. The mammals express two IRBIT paralogs, i.e., IRBIT1 (encoded by AHCYL1) and IRBIT2 (encoded by AHCYL2). The clawed frog Xenopus laevis oocyte is widely used for biophysical studies on ion channels and transporters. It remains unknown whether endogenous IRBIT is expressed in Xenopus oocytes. Here, we cloned from frog oocyte irbit2.L and irbit2.S, orthologs of mammalian IRBIT2. When over-expressed, the frog IRBITs powerfully stimulate the electrogenic Na + /HCO 3 - cotransporter NBCe1-B as mouse IRBIT2-V2 does. Expression of an isolated Nt fragment of NBCe1-B containing the IRBIT-binding domain greatly decreases NBCe1-B activity in oocytes, suggesting that the basal activity of NBCe1-B contains a large component derived from the stimulation by endogenous frog IRBIT. The frog IRBITs are highly homologous to the mammalian ones in the carboxyl-terminal region, but varies greatly in the amino-terminal (Nt) appendage. Interestingly, truncation study showed that the Nt appendage of IRBIT1 and the long Nt appendage of IRBIT2-V2 modestly enhance, whereas the short Nt appendage of IRBIT2-V4 greatly inhibits the functional interaction between IRBIT and NBCe1-B. Finally, Ala-substitution of Ser68, a key phosphorylation site in the PEST domain of IRBIT, causes distinct functional consequences depending on the structural context of the Nt appendage in different IRBIT isoforms. We conclude that the Nt appendage of IRBITs is not necessary for, but plays an important regulatory role in the functional interaction between IRBIT and NBCe1-B., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

In the body, extracellular stimuli produce inositol 1,4,5-trisphosphate (IP 3 ), an intracellular chemical signal that binds to the IP 3 receptor (IP 3 R) to release calcium ions (Ca 2+ ) from the endoplasmic reticulum. In the past 40 years, the wide-ranging functions mediated by IP 3 R and its genetic defects causing a variety of disorders have been unveiled. Recent cryo-electron microscopy and X-ray crystallography have resolved IP 3 R structures and begun to integrate with concurrent functional studies, which can explicate IP 3 -dependent opening of Ca 2+ -conducting gates placed ∼90 Å away from IP 3 -binding sites and its regulation by Ca 2+ . This review highlights recent research progress on the IP 3 R structure and function. We also propose how protein plasticity within IP 3 R, which involves allosteric gating and assembly transformations accompanied by rapid and chronic structural changes, would enable it to regulate diverse functions at cellular microdomains in pathophysiological states.

Background and Purpose: Metformin, a widely used antidiabetic drug, has been shown to have anti-inflammatory properties; nevertheless, its influence on β -cell meta-inflammation remains unclear. The following study investigated the effects of metformin on meta-inflammatory in β -cells and whether the underlying mechanisms were associated with the G protein-coupled receptor 40-phospholipase C-inositol 1, 4, 5-trisphosphate (GPR40-PLC-IP3) pathway., Materials and Methods: Lipotoxicity-induced β -cells and the high-fat diet-induced obese rat model were used in the study., Results: Metformin-reduced lipotoxicity-induced β -cell meta-inflammatory injury was associated with the expression of GPR40. GPR40 was involved in metformin reversing metabolic inflammation key marker TLR4 activation-mediated β -cell injury. Furthermore, downstream signaling protein PLC-IP3 of GPR40 was involved in the protective effect of metformin on meta-inflammation, and the above process of metformin was partially regulated by AMPK activity. In addition, the anti-inflammatory effects of metformin were observed in obese rats., Conclusion: Metformin can reduce lipotoxicity-induced meta-inflammation in β -cells through the regulation of the GPR40-PLC-IP3 pathway and partially via the regulation of AMPK activity., Competing Interests: The authors have no conflicts to disclose., (Copyright © 2019 Ximei Shen et al.)

Thiamine dependent enzymes are diminished in Alzheimer's disease (AD). Thiamine deficiency in vitro and in rodents is a useful model of this reduction. Thiamine interacts with cellular calcium stores. To directly test the relevance of the thiamine dependent changes to dynamic processes in AD, the interactions must be studied in cells from patients with AD. These studies employed fibroblasts. Mitochondrial dysfunction including reductions in thiamine dependent enzymes and abnormalities in calcium homeostasis and oxidative processes occur in fibroblasts from Alzheimer's Disease (AD) patients. Bombesin-releasable calcium stores (BRCS) from the endoplasmic reticulum (ER) are exaggerated in fibroblasts from patients with AD bearing a presenilin-1 (PS-1) mutation and in control fibroblasts treated with oxidants. ER calcium regulates calcium entry into the cell through capacitative calcium entry (CCE), which is reduced in fibroblasts and neurons from mice bearing PS-1 mutations. Under physiological conditions, mitochondria and ER play important and interactive roles in the regulation of Ca(2+) homeostasis. Thus, the interactions of mitochondria and oxidants with CCE were tested. Inhibition of ER Ca(2+)-ATPase by cyclopiazonic acid (CPA) stimulates CCE. CPA-induced CCE was diminished by inhibition of mitochondrial Ca(2+) export (-60%) or import (-40%). Different aspects of mitochondrial Ca(2+) coupled to CPA-induced-CCE were sensitive to select oxidants. The effects were very different when CCE was examined in the presence of InsP3, a physiological regulator of ER calcium release, and subsequent CCE. CCE under these conditions was only mildly reduced (20-25%) by inhibition of mitochondrial Ca(2+) export, and inhibition of mitochondrial Ca(2+) uptake exaggerated CCE (+53%). However, t-BHP reversed both abnormalities. The results suggest that in the presence of InsP3, mitochondria buffer the local Ca(2+) released from ER following rapid activation of InsP3R and serve as a negative feedback to the CCE. The results suggest that mitochondrial Ca(2+) modifies the depletion and refilling mechanism of ER Ca(2+) stores.

The cardiac hormone atrial natriuretic factor (ANF or ANP) plays significant, well-established roles in a large number of physiological and pathophysiological processes, including water and electrolyte balance, blood pressure regulation, and cardiovascular growth. Understanding the regulation of its production and secretion by atrial cardiomyocytes is incomplete. We have previously established a significant role of G(i/o) protein signaling in modulating ANF secretion as promoted by stretch of the atrial myocardium. In the present study, we investigated the role of G(q) protein signaling and its relationship to G(i/o) protein signaling using pharmacological manipulation of proximal effectors of G(αq) in an ex vivo model of spontaneously beating rat atria. Phospholipase C (PLC) and protein kinase C (PKC) inhibitors dramatically increased basal secretion of ANF. Furthermore, although atrial wall stretch is a potent stimulus for secretion, stretch unexpectedly reduced ANF secretion to basal levels under PLC and PKC inhibitory conditions. Inhibition of the inositol triphosphate receptor did not appear to affect basal secretion but dose-dependently blocked stretch-secretion coupling. The results obtained demonstrate that the PLC and PKC signaling cascades play important albeit unexpected roles in the regulation of basal and stimulated ANF secretion and suggest interplay between the G(q) and G(i/o) protein signaling pathways.

Studies of inositol polyphosphates in the pancreatic β-cell have led to an exciting synergism between new discoveries regarding their cellular roles and new insights into β-cell function. Because the loss or malfunction of the β-cell is central to diabetes, these studies open the possibility of new pharmacological interventions in a disease that has reached epidemic proportions worldwide. Using the β-cell as our prime but not exclusive example, we examine the inositol polyphosphates in three main groups: 1) inositol 1,4,5-trisphosphate and its influence on Ca(2+) signaling, specifically in a cell in which cytoplasmic-free Ca(2+) concentration is principally increased by plasma membrane standing voltage-gated Ca(2+) channels; 2) higher inositol polyphosphates including a novel second messenger inositol 3,4,5,6-tetrakisphosphate and a regulatory role for inositol hexakisphosphate in β-cell Ca(2+) homeostasis and exo- and endocytosis; and 3) inositol pyrophosphates and their role in β-cell exocytosis, together with the exciting possibility of being novel targets for therapy in diabetes. We conclude with some of the new perspectives that are likely to become apparent in the next few years.

Phospholipase C (PLC) converts phosphatidylinositol 4,5-bisphosphate (PIP(2)) to inositol 1,4,5-trisphosphate (IP(3)) and diacylglycerol (DAG). DAG and IP(3) each control diverse cellular processes and are also substrates for synthesis of other important signaling molecules. PLC is thus central to many important interlocking regulatory networks. Mammals express six families of PLCs, each with both unique and overlapping controls over expression and subcellular distribution. Each PLC also responds acutely to its own spectrum of activators that includes heterotrimeric G protein subunits, protein tyrosine kinases, small G proteins, Ca(2+), and phospholipids. Mammalian PLCs are autoinhibited by a region in the catalytic TIM barrel domain that is the target of much of their acute regulation. In combination, the PLCs act as a signaling nexus that integrates numerous signaling inputs, critically governs PIP(2) levels, and regulates production of important second messengers to determine cell behavior over the millisecond to hour timescale.

Autophagy is a process that removes damaged proteins and organelles and is of particular importance in terminally differentiated cells such as cardiomyocytes, where it has primarily a protective role. We investigated the involvement of inositol(1,4,5)trisphosphate (Ins(1,4,5)P(3)) and its receptors in autophagic responses in neonatal rat ventricular myocytes (NRVM). Treatment with the IP(3)-receptor (IP(3)-R) antagonist 2-aminoethoxydiphenyl borate (2-APB) at 5 or 20 μmol/L resulted in an increase in autophagosome content, defined as puncta labeled by antibody to microtubule associated light chain 3 (LC3). 2-APB also increased autophagic flux, indicated by heightened LC3II accumulation, which was further enhanced by bafilomycin (10nmol/L). Expression of Ins(1,4,5)P(3) 5-phosphatase (IP(3)-5-Pase) to deplete Ins(1,4,5)P(3) also increased LC3-labeled puncta and LC3II content, suggesting that Ins(1,4,5)P(3) inhibits autophagy. The IP(3)-R can act as an inhibitory scaffold sequestering the autophagic effector, beclin-1 to its ligand binding domain (LBD). Expression of GFP-IP(3)-R-LBD inhibited autophagic signaling and furthermore, beclin-1 co-immunoprecipitated with the IP(3)-R-LBD. A mutant GFP-IP(3)-R-LBD with reduced ability to bind Ins(1,4,5)P(3) bound beclin-1 and inhibited autophagy similarly to the wild type sequence. These data provide evidence that Ins(1,4,5)P(3) and IP(3)-R act as inhibitors of autophagic responses in cardiomyocytes. By suppressing autophagy, IP(3)-R may contribute to cardiac pathology., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Work over the recent years has greatly expanded our understanding of the specific molecules involved in plant phosphoinositide signaling. Physiological approaches, combined with analytical techniques and genetic mutants have provided tools to understand how individual genes function in this pathway. Several key differences between plants and animals have become apparent. This chapter will highlight the key areas where major differences between plants and animals occur. In particular, phospholipase C and levels of phosphatidylinositol phosphates differ between plants and animals, and may influence how inositol second messengers form and function in plants. Whether inositol 1,4,5-trisphosphate and/or inositol hexakisphosphate (InsP6) function as second messengers in plants is discussed. Recent data on potential, novel roles of InsP6 in plants is considered, along with the existence of a unique InsP6 synthesis pathway. Lastly, the complexity of myo-inositol synthesis in plants is discussed in reference to synthesis of phosphoinositides and impact on plant growth and development.

Electrogenic Na(+)-bicarbonate cotransporter NBCe1 variants contribute to pH(i) regulation, and promote ion reabsorption or secretion by many epithelia. Most Na(+)-coupled bicarbonate transporter (NCBT) families such as NBCe1 contain variants with differences primarily at the cytosolic N and/or C termini that are likely to impart on the transporters different modes of regulation. For example, N-terminal regions of NBCe1 autoregulate activity. Our group previously reported that cytosolic phosphatidylinositol 4,5-bisphosphate (PIP(2)) stimulates heterologously expressed rat NBCe1-A in inside-out macropatches excised from Xenopus laevis oocytes. In the current study on whole oocytes, we used the two-electrode voltage-clamp technique, as well as pH- and voltage-sensitive microelectrodes, to characterize the effect of injecting PIP(2) on the activity of heterologously expressed NBCe1-A, -B, or -C. Injecting PIP(2) (10 μM estimated final) into voltage-clamped oocytes stimulated NBC-mediated, HCO(3)(-)-induced outward currents by >100% for the B and C variants, but not for the A variant. The majority of this stimulation involved PIP(2) hydrolysis and endoplasmic reticulum (ER) Ca(2+) release. Stimulation by PIP(2) injection was mimicked by injecting IP(3), but inhibited by either applying the phospholipase C (PLC) inhibitor U73112 or depleting ER Ca(2+) with prolonged thapsigargin/EGTA treatment. Stimulating the activity of store-operated Ca(2+) channels (SOCCs) to trigger a Ca(2+) influx mimicked the PIP(2)/IP(3) stimulation of the B and C variants. Activating the endogenous G(q) protein-coupled receptor in oocytes with lysophosphatidic acid (LPA) also stimulated the B and C variants in a Ca(2+)-dependent manner, although via an increase in surface expression for the B variant. In simultaneous voltage-clamp and pH(i) studies on NBCe1-C-expressing oocytes, LPA increased the NBC-mediated pH(i)-recovery rate from a CO(2)-induced acid load by ∼80%. Finally, the general kinase inhibitor staurosporine completely inhibited the IP(3)-induced stimulation of NBCe1-C. In summary, injecting PIP(2) stimulates the activity of NBCe1-B and -C expressed in oocytes through an increase in IP(3)/Ca(2+) that involves a staurosporine-sensitive kinase. In conjunction with our previous macropatch findings, PIP(2) regulates NBCe1 through a dual pathway involving both a direct stimulatory effect of PIP(2) on at least NBCe1-A, as well as an indirect stimulatory effect of IP(3)/Ca(2+) on the B and C variants.

Group I metabotropic glutamate receptors (mGluRs) are linked to intracellular Ca(2+) signalling and play important roles related to synaptic plasticity and development. In neurons from the central nucleus of the inferior colliculus (CIC), the activation of these receptors evokes large [Ca(2+) ](i) responses. By using optical imaging of the fluorescent Ca(2+) -sensitive dye Fura-2, we have explored which [Ca(2+) ](i) routes are triggered by group I mGluR activation in young CIC neurons and whether mGluR-induced [Ca(2+) ](i) responses are regulated during postnatal development. In addition, real-time quantitative RT-PCR was used to study the developmental expression of both group I mGluR subtypes, mGluR1 and mGluR5. Application of DHPG, a specific agonist of group I mGluRs, was used on CIC slices from young rats to elicit [Ca(2+) ](i) responses. A majority of responses consisted of an initial thapsigargin-sensitive Ca(2+) peak, related to store depletion, followed by a plateau phase, sensitive to the store-operated Ca(2+) entry blocker 2-APB. During postnatal development, from P6 to P17, DHPG-induced [Ca(2+) ](i) responses changed. The largest Ca(2+) responses were reached at P6, whereas lower peak and plateau responses were found after hearing onset, at P13-P14 and P17. qRT-PCR analysis also revealed important differences in the expression of both mGluR1 and mGluR5 subtypes during development, with the highest levels of both subtypes at P7 and a developmental decrease of both transcripts. Our results suggest both intra- and extracellular routes for [Ca(2+) ](i) increases linked to group I mGluRs in CIC neurons and a regulation of group I mGluR activity and expression during auditory development., (Copyright © 2012 Wiley Periodicals, Inc.)

Background: Inositol 1,4,5-trisphosphate receptors (IP3R) are expressed in almost all animal cells. Three mammalian genes encode closely related IP3R subunits, which assemble into homo- or hetero-tetramers to form intracellular Ca2+ channels., Scope of the Review: In this brief review, we first consider a variety of complementary methods that allow the links between IP3 binding and channel gating to be defined. How does IP3 binding to the IP3-binding core in each IP3R subunit cause opening of a cation-selective pore formed by residues towards the C-terminal? We then describe methods that allow IP3, Ca2+ signals and IP3R mobility to be examined in intact cells. A final section briefly considers genetic analyses of IP3R signalling., Major Conclusions: All IP3R are regulated by both IP3 and Ca2+. This allows them to initiate and regeneratively propagate intracellular Ca2+ signals. The elementary Ca2+ release events evoked by IP3 in intact cells are mediated by very small numbers of active IP3R and the Ca2+-mediated interactions between them. The spatial organization of these Ca2+ signals and their stochastic dependence on so few IP3Rs highlight the need for methods that allow the spatial organization of IP3R signalling to be addressed with single-molecule resolution., General Significance: A variety of complementary methods provide insight into the structural basis of IP3R activation and the contributions of IP3-evoked Ca2+ signals to cellular physiology. This article is part of a Special Issue entitled Biochemical, biophysical and genetic approaches to intracellular calcium signaling., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The role of 3,5,3'-triiodo-l-thyronine (T3) and its metabolite 3,5-diiodo-l-thyronine (T2) in modulating the intracellular Ca(2+) concentration ([Ca(2+)](i)) and endogenous nitric oxide (NO) synthesis was evaluated in pituitary GH(3) cells in the absence or presence of extracellular Ca(2+). When applied in Ca(2+)-free solution, T2 and T3 increased [Ca(2+)](i), in a dose-dependent way, and NO levels. Inhibition of neuronal NO synthase by N(G)-nitro-l-arginine methyl ester and l-n(5)-(1-iminoethyl)ornithine hydrochloride significantly reduced the [Ca(2+)](i) increase induced by T2 and T3. However, while depletion of inositol trisphosphate-dependent Ca(2+) stores did not interfere with the T2- and T3-induced [Ca(2+)](i) increases, the inhibition of phosphatidylinositol 3-kinase by LY-294002 and the dominant negative form of Akt mutated at the ATP binding site prevented these effects. Furthermore, the mitochondrial protonophore carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone prevented the increases in both [Ca(2+)](i) and NO elicited by T2 or T3. Interestingly, rotenone blocked the early [Ca(2+)](i) increases elicited by T2 and T3, while antimycin prevented only that elicited by T3. Inhibition of mitochondrial Na(+)/Ca(2+) exchanger by CGP37157 significantly reduced the [Ca(2+)](i) increases induced by T2 and T3. In the presence of extracellular calcium (1.2 mM), under carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, T2 and T3 increased both [Ca(2+)](i) and intracellular Na(+) concentration; nimodipine reduced the [Ca(2+)](i) increases elicited by T2 and T3, but inhibition of NO synthase and blockade of the Na(+)/H(+) pump by 5-(N-ethyl-N-isopropyl)amiloride prevented only that elicited by T3; and CB-DMB, bisindolylmaleimide, and LY-294002 (inhibitors of the Na(+)/Ca(2+) exchanger, PKC, and phosphatidylinositol 3-kinase, respectively) failed to modify the T2- and T3-induced effects. Collectively, the present results suggest that T2 and T3 exert short-term nongenomic effects on intracellular calcium and NO by modulating plasma membrane and mitochondrial pathways that differ between these iodothyronines.

Oxytocin (OT) is clinically important in gut motility and constitutively reduces duodenum contractility. Intrinsic primary afferent neurons (IPANs), whose physiological classification is as AH cells, are the 1st neurons of the peristaltic reflex pathway. We set out to investigate if this inhibitory effect is mediated by IPANs and to identify the ion channel(s) and intracellular signal transduction pathway that are involved in this effect. Myenteric neurons were isolated from the longitudinal muscle myenteric plexus (LMMP) preparation of rat duodenum and cultured for 16-24 h before electrophysiological recording in whole cell mode and AH cells identified by their electrophysiological characteristics. The cytoplasmic Ca²⁺ concentration ([Ca²⁺](i) ) of isolated neurons was measured using calcium imaging. The concentration of IP(3) in the LMMP and the OT secreted from the LMMP were measured using ELISA. The oxytocin receptor (OTR) and large-conductance calcium-activated potassium (BK(Ca)) channels, as well as the expression of OT and the IPAN marker calbindin 28 K, on the myenteric plexus neurons were localized using double-immunostaining techniques. We found that administration of OT (10⁻⁷ to 10⁻⁵ M) dose dependently hyperpolarized the resting membrane potential and increased the total outward current. The OTR antagonist atosiban or the BK(Ca) channel blocker iberiotoxin (IbTX) blocked the effects of OT suggesting that the increased outward current resulted from BK(Ca) channel opening. OTR and the BK(Ca) α subunit were co-expressed on a subset of myenteric neurons at the LMMP. NS1619 (10⁻⁵ M, a BK(Ca) channel activator) increased the outward current similar to the effect of OT. OT administration also increased [Ca²⁺](i) and the OT-evoked outward current was significantly attenuated by thapsigargin (10⁻⁶ M) or CdCl₂. The effect of OT on the BK(Ca) current was also blocked by pre-treatment with the IP₃ receptor antagonist 2-APB (10⁻⁴ M) or the PLC inhibitor U73122 (10⁻⁵ M). OT (10⁻⁶ M) also increased the IP₃ concentration within the LMMP. Both of the spontaneous and KCl-induced secretion of OT was enhanced by atosiban. Most of OT-immunoreactive cells are also immunoreactive for calbindin 28 K. In summary, we concluded that OT hyperpolarized myenteric IPANs by activating BK(Ca) channels via the OTR-PLC-IP₃-Ca²⁺ signal pathway. OT might modulate IPANs mediated ENS reflex by an autocrine and negative feedback manner., (© 2012 The Authors. Journal of Neurochemistry © 2012 International Society for Neurochemistry.)

Photoactivated probes have gained interest as experimental tools to study intracellular signalling pathways all the way to the molecular level. However technical limitations of the means to activate such compounds have put constraints on their use in spatially highly restricted subcellular areas. The Mosaic digital illumination system uses a high-speed array of individually addressable, tiltable micromirrors to direct continuous-wave laser light onto a specimen with diffraction-limited precision. The system, integrated into a Nikon A1R confocal microscope, was used to uncage Ca²⁺ or IP3 and conduct photobleaching experiments from multiple geometrically complex subcellular regions while simultaneously measuring [Ca²⁺]i with high-speed confocal imaging.

Background/aims: The mechanism underlying transmitter release from astrocytes is not fully understood. The present study examined A(2a) adenosine receptor-mediated glutamate release and intracellular Ca(2+) rise in cultured rat hippocampal astrocytes., Methods: Intracellular amino acids were measured with HPLC. Glutamate release from astrocytes and intracellular Ca(2+) mobilizations were monitored in the NADH imaging, FM1-43 imaging, and fura-2 imaging. The siRNA to silence the A(2a) adenosine receptor-targeted gene was constructed and transfected into cells., Results: Glutamate was condensed in 'synaptic-like vesicle' fractions. In the NADH imaging, CGS21680, an agonist of A2a adenosine receptors, increased NADH fluorescent signals, that reflects glutamate release, and the effect was inhibited by DMPX, an inhibitor of A(2a) adenosine receptors, H-89, a PKA inhibitor, vesicular transport inhibitors, or botulinum toxin-A, an exocytosis inhibitor. In the FM1-43 imaging to see vesicular recycling, CGS21680 decreased FM1-43 fluorescent signals, that was also prevented by DMPX, H-89, vesicular transport inhibitors, or botulinum toxin-A. CGS21680 increased intracellular Ca(2+) concentrations both in Ca(2+)-containing and -free extracellular solution. The Ca(2+) rise was inhibited by DMPX, H-89, or the vesicular transport inhibitor brefeldin A, but it was not affected by inhibitors for phospholipase C, IP(3) receptor, and ryanodine receptor. CGS21680-induced glutamate release and intracellular Ca(2+) rise were prevented by knocking-down A(2a) adenosine receptor., Conclusion: The results of the present study show that A(2a) adenosine receptor/PKA promotes glutamate release from synaptic-like vesicles and stimulates Ca(2+) efflux from an IP(3)- and ryanodine-insensitive intracellular calcium store., (Copyright © 2012 S. Karger AG, Basel.)

Activation of cells by many extracellular agonists leads to the production of inositol 1,4,5-trisphosphate (IP₃). IP₃ is a global messenger that easily diffuses in the cytosol. Its receptor (IP₃R) is a Ca(2+)-release channel located on intracellular membranes, especially the endoplasmic reticulum (ER). The IP₃R has an affinity for IP(3) in the low nanomolar range. A prime regulator of the IP₃R is the Ca(2+) ion itself. Cytosolic Ca(2+) is considered as a co-agonist of the IP₃R, as it strongly increases IP(3)R activity at concentrations up to about 300 nM. In contrast, at higher concentrations, cytosolic Ca(2+) inhibits the IP₃R. Also the luminal Ca(2+) sensitizes the IP₃R. In higher organisms three genes encode for an IP₃R and additional diversity exists as a result of alternative splicing mechanisms and the formation of homo- and heterotetramers. The various IP₃R isoforms have a similar structure and a similar function, but due to differences in their affinity for IP₃, their variable sensitivity to regulatory parameters, their differential interaction with associated proteins, and the variation in their subcellular localization, they participate differently in the formation of intracellular Ca(2+) signals and this affects therefore the physiological consequences of these signals.

Postsynaptic [Ca(2+)](i) changes contribute to several kinds of plasticity in pyramidal neurons. We examined the effects of synaptically activated Ca(2+) waves and NMDA spikes on subsequent Ca(2+) signalling in CA1 pyramidal cell dendrites in hippocampal slices. Tetanic synaptic stimulation evoked a localized Ca(2+) wave in the primary apical dendrites. The [Ca(2+)](i) increase from a backpropagating action potential (bAP) or subthreshold depolarization was reduced if it was generated immediately after the wave. The suppression had a recovery time of 30-60 s. The suppression only occurred where the wave was generated and was not due to a change in bAP amplitude or shape. The suppression also could be generated by Ca(2+) waves evoked by uncaging IP(3), showing that other signalling pathways activated by the synaptic tetanus were not required. The suppression was proportional to the amplitude of the [Ca(2+)](i) change of the Ca(2+) wave and was not blocked by a spectrum of kinase or phosphatase inhibitors, consistent with suppression due to Ca(2+)-dependent inactivation of Ca(2+) channels. The waves also reduced the frequency and amplitude of spontaneous, localized Ca(2+) release events in the dendrites by a different mechanism, probably by depleting the stores at the site of wave generation. The same synaptic tetanus often evoked NMDA spike-mediated [Ca(2+)](i) increases in the oblique dendrites where Ca(2+) waves do not propagate. These NMDA spikes suppressed the [Ca(2+)](i) increase caused by bAPs in those regions. [Ca(2+)](i) increases by Ca(2+) entry through voltage-gated Ca(2+) channels also suppressed the [Ca(2+)](i) increases from subsequent bAPs in regions where the voltage-gated [Ca(2+)](i) increases were largest, showing that all ways of raising [Ca(2+)](i) could cause suppression.

The accumulation of the two branched-chain fatty acids phytanic acid and pristanic acid is known to play an important role in several diseases with peroxisomal impairment, like Refsum disease, Zellweger syndrome and α-methylacyl-CoA racemase deficiency. Recent studies elucidated that the toxic activity of phytanic acid and pristanic acid is mediated by multiple mitochondrial dysfunctions, generation of reactive oxygen species and Ca2+ deregulation via the InsP3-Ca2+ signaling pathway in glial cells. However, the exact signaling mechanism through which both fatty acids mediate toxicity is still under debate. Here, we studied the ability of phytanic acid and pristanic acid to activate the free fatty acid receptor GPR40, a G-protein-coupled receptor, which was described to be involved in the Ca2+ signaling of fatty acids. We treated HEK 293 cells expressing the GPR40 receptor with phytanic acid or pristanic acid. This resulted in a significant increase in the intracellular Ca2+ level, similar to the effect seen after treatment with the synthetic GPR40 agonist GW9508. Furthermore, we demonstrate that the GPR40 activation might be due to an interaction of the carboxylate moiety of fatty acids with the receptor. Our findings indicate that the phytanic acid- and pristanic acid-mediated Ca2+ deregulation can involve the activation of GPR40. Therefore, we suppose that activation of GPR40 might be part of the signaling cascade of the toxicity of phytanic and pristanic acids., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The thyrotropin [thyroid-stimulating hormone (TSH)] receptor (TSHR) is known to acutely and persistently stimulate cAMP signaling and at higher TSH concentrations to acutely stimulate phosphoinositide signaling. We measured persistent signaling by stimulating TSHR-expressing human embryonic kidney-EM293 cells with TSH and measuring cAMP or inositol monophosphate (IP1) production, a measure of phosphoinositide signaling, 60 min or longer after TSH removal. In contrast to persistent cAMP production, persistent IP1 production increased progressively when TSH exposure was increased from 1 to 30 min, whereas the rates of decay of persistent signaling were similar. A small-molecule agonist and a thyroid-stimulating antibody also caused persistent IP1 and cAMP signaling. A small-molecule inverse agonist and a neutral antagonist inhibited TSH-stimulated persistent IP1 production, whereas the inverse agonist but not the neutral antagonist inhibited persistent cAMP production. As with persistent cAMP production, persistent IP1 production was not affected when TSHR internalization was inhibited or enhanced. Moreover, Alexa546-TSH-activated TSHR internalization was not accompanied by Gα(q) coupling protein internalization. Thus, transient exposure to high concentrations of TSH causes persistent phosphoinositide and cAMP signaling that is not dependent on internalization. To our knowledge, this is the first demonstration of persistent activation by any G protein-coupled receptor (GPCR) via the Gα(q) pathway and of two G protein-mediated pathways by any GPCR.

The secretion of fluid and electrolytes by salivary gland acinar cells requires the coordinated regulation of multiple ion channel and transporter proteins, signaling components, and water transport. Importantly, neurotransmitter stimulated increase in the cytosolic free [Ca(2+)] ([Ca(2+)](i)) is critical for the regulation of salivary gland secretion as it regulates several major ion fluxes that together establish the sustained osmotic gradient to drive fluid secretion. The mechanisms that act to modulate these increases in [Ca(2+)](i) are therefore central to the process of salivary fluid secretion. Such modulation involves membrane receptors for neurotransmitters, as well as mechanisms that mediate intracellular Ca(2+) release, and Ca(2+) entry, as well as those that maintain cellular Ca(2+) homeostasis. Together, these mechanisms determine the spatial and temporal aspects of the [Ca(2+)](i) signals that regulate fluid secretion. Molecular cloning of these transporters and channels as well as development of mice lacking these proteins has established the physiological significance of key components that are involved in regulating [Ca(2+)](i) in salivary glands. This review will discuss these important studies and the findings which have led to resolution of the Ca(2+) signaling mechanisms that determine salivary gland fluid secretion.

Liberation of Ca(2+) from the endoplasmic reticulum (ER) through inositol trisphosphate receptors (IP(3)R) is modulated by the ER Ca(2+) content, and overexpression of SERCA2b to accelerate Ca(2+) sequestration into the ER has been shown to potentiate the frequency and amplitude of IP(3)-evoked Ca(2+) waves in Xenopus oocytes. Here, we examined the effects of SERCA overexpression on the elementary IP(3)-evoked puffs to elucidate whether ER [Ca(2+)] may modulate IP(3)R function via luminal regulatory sites in addition to simply determining the size of the available store and electrochemical driving force for Ca(2+) release. SERCA2b and Ca(2+) permeable nicotinic plasmalemmal channels were expressed in oocytes, and hyperpolarizing pulses were delivered to induce Ca(2+) influx and thereby load ER stores. Puffs evoked by photoreleased IP(3) were significantly potentiated in terms of numbers of responding sites, frequency and amplitude following transient Ca(2+) influx in SERCA-overexpressing cells, whereas little change was evident with SERCA overexpression alone or following Ca(2+) influx in control cells not overexpressing SERCA. Intriguingly, we observed the appearance of a new population of puffs that arose after long latencies and had prolonged durations supporting the notion of luminal regulation of IP(3)R gating kinetics., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Galanin (GAL) is a neuropeptide which is up-regulated following neuronal axotomy or inflammation. One subtype of GAL receptor (GalR2) is reported to be expressed in the brain's immune cell population, microglia. In the present study, we investigated the effect of GAL on microglial migration and compared the mechanism with that of bradykinin (BK). GAL significantly increased the migration of rat cultured microglia at 0.1 pM. The GAL-induced signal cascade was partly similar to that induced by BK. It was not dependent on G(i/o) protein but involved activation of protein kinase C, phosphoinositide 3-kinase and Ca(2+)-dependent K(+) channels. However, reverse-mode activation of the Na(+) /Ca(2+) -exchanger 1 was not involved in GAL-induced microglial migration, unlike BK-induced migration. Likewise, nominally-free extracellular Ca(2+) inhibited BK-induced migration but not GAL-induced migration. An inositol-1,4,5-triphosphate receptor antagonist significantly inhibited GAL-induced migration. GAL-induced Ca(2+) signaling did not induce nitric oxide synthase expression, but up-regulated class II major histocompatibility complex expression. These results indicate that activation of inositol-1,4,5-triphosphate receptor and increase in intracellular Ca(2+) are important for GAL-induced migration and immunoreactivity in microglia. The differences in down-stream signal transduction induced by GAL and BK suggest that GAL and BK may control distinct microglial functions under pathological conditions., (© 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.)

CatSpers are calcium (Ca(2+)) channels that are located along the principal piece of mammalian sperm flagella and are directly linked to sperm motility and hyperactivation. It has been observed that Ca(2+) entry through CatSper channels triggers a tail to head Ca(2+) propagation in mouse sperm, as well as a sustained increase of Ca(2+) in the head. Here, we develop a mathematical model to investigate this propagation and sustained increase in the head. A 1-d reaction-diffusion model tracking intracellular Ca(2+) with flux terms for the CatSper channels, a leak flux, and plasma membrane Ca(2+) clearance mechanism is studied. Results of this simple model exhibit tail to head Ca(2+) propagation, but no sustained increase in the head. Therefore, in this model, a simple plasma membrane pump-leak system with diffusion in the cytosol cannot account for these experimentally observed results. It has been proposed that Ca(2+) influx from the CatSper channels induce additional Ca(2+) release from an internal store. We test this hypothesis by examining the possible role of Ca(2+) release from the redundant nuclear envelope (RNE), an inositol 1,4,5-trisphosphate (IP(3)) gated Ca(2+) store in the neck. The simple model is extended to include an equation for IP(3) synthesis, degradation, and diffusion, as well as flux terms for Ca(2+) in the RNE. When IP(3) and the RNE are accounted for, the results of the model exhibit a tail to head Ca(2+) propagation as well as a sustained increase of Ca(2+) in the head.

Intracellular calcium levels ([Ca2+]i) and glucose uptake are central to cardiomyocyte physiology, yet connections between them have not been studied. We investigated whether insulin regulates [Ca2+]i in cultured cardiomyocytes, the participating mechanisms, and their influence on glucose uptake via SLC2 family of facilitative glucose transporter 4 (GLUT4). Primary neonatal rat cardiomyocytes were preloaded with the Ca2+ fluorescent dye fluo3-acetoxymethyl ester compound (AM) and visualized by confocal microscopy. Ca2+ transport pathways were selectively targeted by chemical and molecular inhibition. Glucose uptake was assessed using [3H]2-deoxyglucose, and surface GLUT4 levels were quantified in nonpermeabilized cardiomyocytes transfected with GLUT4-myc-enhanced green fluorescent protein. Insulin elicited a fast, two-component, transient increase in [Ca2+]i. Nifedipine and ryanodine prevented only the first component. The second one was reduced by inositol-1,4,5-trisphosphate (IP3)-receptor-selective inhibitors (xestospongin C, 2 amino-ethoxydiphenylborate), by type 2 IP3 receptor knockdown via small interfering RNA or by transfected Gβγ peptidic inhibitor βARKct. Insulin-stimulated glucose uptake was prevented by bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid-AM, 2-amino-ethoxydiphenylborate, and βARK-ct but not by nifedipine or ryanodine. Similarly, insulin-dependent exofacial exposure of GLUT4-myc-enhanced green fluorescent protein was inhibited by bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid-AM and xestospongin C but not by nifedipine. Phosphatidylinositol 3-kinase and Akt were also required for the second phase of Ca2+ release and GLUT4 translocation. Transfected dominant-negative phosphatidylinositol 3-kinase γ inhibited the latter. In conclusion, in primary neonatal cardiomyocytes, insulin induces an important component of Ca2+ release via IP3 receptor. This component signals to glucose uptake via GLUT4, revealing a so-far unrealized contribution of IP3-sensitive Ca2+ stores to insulin action. This pathway may influence cardiac metabolism in conditions yet to be explored in adult myocardium.

A new paradigm has recently emerged in brain science whereby communications between glial cells and neuron-glia interactions should be considered together with neurons and their networks to understand higher brain functions. In particular, astrocytes, the main type of glial cells in the cortex, have been shown to communicate with neurons and with each other. They are thought to form a gap-junction-coupled syncytium supporting cell-cell communication via propagating Ca(2+) waves. An identified mode of propagation is based on cytoplasm-to-cytoplasm transport of inositol trisphosphate (IP(3)) through gap junctions that locally trigger Ca(2+) pulses via IP(3)-dependent Ca(2+)-induced Ca(2+) release. It is, however, currently unknown whether this intracellular route is able to support the propagation of long-distance regenerative Ca(2+) waves or is restricted to short-distance signaling. Furthermore, the influence of the intracellular signaling dynamics on intercellular propagation remains to be understood. In this work, we propose a model of the gap-junctional route for intercellular Ca(2+) wave propagation in astrocytes. Our model yields two major predictions. First, we show that long-distance regenerative signaling requires nonlinear coupling in the gap junctions. Second, we show that even with nonlinear gap junctions, long-distance regenerative signaling is favored when the internal Ca(2+) dynamics implements frequency modulation-encoding oscillations with pulsating dynamics, while amplitude modulation-encoding dynamics tends to restrict the propagation range. As a result, spatially heterogeneous molecular properties and/or weak couplings are shown to give rise to rich spatiotemporal dynamics that support complex propagation behaviors. These results shed new light on the mechanisms implicated in the propagation of Ca(2+) waves across astrocytes and the precise conditions under which glial cells may participate in information processing in the brain.

Understanding of calcium signaling pathways in cells is essential for elucidating the mechanisms of both normal cell function and cancer development. Calcium messengers play the crucial role for intracellular Ca(2+) release. We propose a new approach to detecting the calcium second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) in cell extracts using surface-enhanced Raman spectroscopy (SERS). Currently available radioreceptor binding and enzymatic assays require extensive sample preparation and take more than 12 h. With a SERS sensor, NAADP can be detected in less than 1 min without any special sample preparation. To the best of our knowledge, this is the first demonstration of using SERS for calcium signaling applications.

Inositol 1,4,5-trisphosphate (IP(3)) is generally viewed as a global messenger that increases cytosolic calcium ion (Ca(2+)) concentration. However, the spatiotemporal dynamics of IP(3) and the functional significance of localized IP(3) production in cell polarity remain largely unknown. Here, we demonstrate the critical role of spatially restricted IP(3) signals in axon guidance. We found that IP(3) and ensuing Ca(2+) signals were produced asymmetrically across growth cones exposed to an extracellular gradient of nerve growth factor (NGF) and mediated growth cone turning responses to NGF. Moreover, photolysis-induced production of IP(3) on one side of a growth cone was sufficient to initiate growth cone turning toward the side with the higher concentration of IP(3). Thus, locally produced IP(3) encodes spatial information that polarizes the growth cone for guided migration.

Inositol 1,4,5-trisphosphate receptors (IP(3)Rs) are intracellular Ca(2+) channels. Their regulation by both IP(3) and Ca(2+) allows interactions between IP(3)Rs to generate a hierarchy of intracellular Ca(2+) release events. These can progress from openings of single IP(3)R, through near-synchronous opening of a few IP(3)Rs within a cluster to much larger signals that give rise to regenerative Ca(2+) waves that can invade the entire cell. We have used patch-clamp recording from excised nuclear membranes of DT40 cells expressing only IP(3)R3 and shown that low concentrations of IP(3) rapidly and reversibly cause IP(3)Rs to assemble into small clusters. In addition to bringing IP(3)Rs close enough to allow Ca(2+) released by one IP(3)R to regulate the activity of its neighbors; clustering also retunes the regulation of IP(3)Rs by IP(3) and Ca(2+). At resting cytosolic [Ca(2+)], lone IP(3)R are more sensitive to IP(3) and the mean channel open time (approximately 10 ms) is twice as long as for clustered IP(3)R. When the cytosolic free [Ca(2+)] is increased to 1 microM, to mimic the conditions that might prevail when an IP(3)R within a cluster opens, clustered IP(3)R are no longer inhibited and their gating becomes coupled. IP(3), by dynamically regulating IP(3)R clustering, both positions IP(3)R for optimal interactions between them and it serves to exaggerate the effects of Ca(2+) within a cluster. During the course of these studies, we have observed that nuclear IP(3)R stably express one of two single channel K(+) conductances (gamma(K) approximately 120 or 200 pS). Here we demonstrate that for both states of the IP(3)R, the effects of IP(3) on clustering are indistinguishable. These observations reinforce our conclusion that IP(3) dynamically regulates assembly of IP(3)Rs into clusters that underlie the hierarchical recruitment of elementary Ca(2+) release events.

Insulin is an hepatic mitogen that promotes liver regeneration. Actions of insulin are mediated by the insulin receptor, which is a receptor tyrosine kinase. It is currently thought that signaling via the insulin receptor occurs at the plasma membrane, where it binds to insulin. Here we report that insulin induces calcium oscillations in isolated rat hepatocytes, and that these calcium signals depend upon activation of phospholipase C and the inositol 1,4,5-trisphosphate receptor, but not upon extracellular calcium. Furthermore, insulin-induced calcium signals occur in the nucleus, and are temporally associated with selective depletion of nuclear phosphatidylinositol bisphosphate and translocation of the insulin receptor to the nucleus. These findings suggest that the insulin receptor translocates to the nucleus to initiate nuclear, inositol 1,4,5-trisphosphate-mediated calcium signals in rat hepatocytes. This novel signaling mechanism may be responsible for insulin's effects on liver growth and regeneration.

In smooth muscle myocytes, agonist-activated release of calcium ions (Ca2+) stored in the sarcoplasmic reticulum (SR) occurs via different but overlapping transduction pathways. Hence, to fully study how SR Ca2+ channels are activated, the simultaneous activation of different Ca2+ signals should be separated. In rat duodenum myocytes, we have previously characterized that acetylcholine (ACh) induces Ca2+ oscillations by binding to its M2 muscarinic receptor and activating the ryanodine receptor subtype 2. Here, we show that ACh simultaneously evokes a Ca2+ signal dependent on activation of inositol 1,4,5-trisphosphate (InsP3) receptor subtype 1. A pharmacologic approach, the use of antisense oligonucleotides directed against InsP3R1, and the expression of a specific biosensor derived from green-fluorescent protein coupled to the pleckstrin homology domain of phospholipase C, suggested that the InsP3R1-dependent Ca2+ signal is transient and due to a transient synthesis of InsP3 via M3 muscarinic receptor. Moreover, we suggest that both M2 and M3 signalling pathways are modulating phosphatidylinositol 4,5-bisphosphate and InsP3 concentration, thus describing closely interacting pathways activated by ACh in duodenum myocytes.

Inositol 1,4,5-trisphosphate (IP(3)) is a ubiquitous intracellular messenger regulating diverse functions in almost all mammalian cell types. It is generated by membrane receptors that couple to phospholipase C (PLC), an enzyme which liberates IP(3) from phosphatidylinositol 4,5-bisphosphate (PIP(2)). The major action of IP(3), which is hydrophilic and thus translocates from the membrane into the cytoplasm, is to induce Ca(2+) release from endogenous stores through IP(3) receptors (IP(3)Rs). Cardiac excitation-contraction coupling relies largely on ryanodine receptor (RyR)-induced Ca(2+) release from the sarcoplasmic reticulum. Myocytes express a significantly larger number of RyRs compared to IP(3)Rs (~100:1), and furthermore they experience substantial fluxes of Ca(2+) with each heartbeat. Therefore, the role of IP(3) and IP(3)-mediated Ca(2+) signaling in cardiac myocytes has long been enigmatic. Recent evidence, however, indicates that despite their paucity cardiac IP(3)Rs may play crucial roles in regulating diverse cardiac functions. Strategic localization of IP(3)Rs in cytoplasmic compartments and the nucleus enables them to participate in subsarcolemmal, bulk cytoplasmic and nuclear Ca(2+) signaling in embryonic stem cell-derived and neonatal cardiomyocytes, and in adult cardiac myocytes from the atria and ventricles. Intriguingly, expression of both IP(3)Rs and membrane receptors that couple to PLC/IP(3) signaling is altered in cardiac disease such as atrial fibrillation or heart failure, suggesting the involvement of IP(3) signaling in the pathology of these diseases. Thus, IP(3) exerts important physiological and pathological functions in the heart, ranging from the regulation of pacemaking, excitation-contraction and excitation-transcription coupling to the initiation and/or progression of arrhythmias, hypertrophy and heart failure.

Calcium (Ca(2+)) release through inositol 1,4,5-trisphosphate receptors (IP(3)Rs) regulates the function of virtually every mammalian cell. Unlike ryanodine receptors, which generate local Ca(2+) events ("sparks") that transmit signals to the juxtaposed cell membrane, a similar functional architecture has not been reported for IP(3)Rs. Here, we have identified spatially fixed, local Ca(2+) release events ("pulsars") in vascular endothelial membrane domains that project through the internal elastic lamina to adjacent smooth muscle membranes. Ca(2+) pulsars are mediated by IP(3)Rs in the endothelial endoplasmic reticulum of these membrane projections. Elevation of IP(3) by the endothelium-dependent vasodilator, acetylcholine, increased the frequency of Ca(2+) pulsars, whereas blunting IP(3) production, blocking IP(3)Rs, or depleting endoplasmic reticulum Ca(2+) inhibited these events. The elementary properties of Ca(2+) pulsars were distinct from ryanodine-receptor-mediated Ca(2+) sparks in smooth muscle and from IP(3)-mediated Ca(2+) puffs in Xenopus oocytes. The intermediate conductance, Ca(2+)-sensitive potassium (K(Ca)3.1) channel also colocalized to the endothelial projections, and blockage of this channel caused an 8-mV depolarization. Inhibition of Ca(2+) pulsars also depolarized to a similar extent, and blocking K(Ca)3.1 channels was without effect in the absence of pulsars. Our results support a mechanism of IP(3) signaling in which Ca(2+) release is spatially restricted to transmit intercellular signals.

Mature oocytes of the annelidan worm Pseudopotamilla occelata have a wide perivitelline space between the oocyte surface and the vitelline envelope and are arrested at the first metaphase (MI). We found a novel two-step Ca2+ increase in normally fertilized oocytes. The first Ca2+ increase originated at a cortex situated underneath a fertilizing sperm on the vitelline envelope, but failed to propagate beyond the center of the oocyte. The first localized Ca2+ increase was then followed by a larger Ca2+ increase starting from the whole oocyte cortex and spreading inwardly to the center. The first localized Ca2+ increase at fertilization was suppressed by the phospholipase C inhibitor U73122, and a similar Ca2+ change was induced by inositol 1,4,5-trisphosphate (IP3). On the other hand, the second global Ca2+ increase in fertilized oocytes was blocked by removal of external Ca2+ or the voltage-gated Ca2+ channel blocker D-600, and a similar Ca2+ change could be mimicked by addition of excess K+ only when external Ca2+ was present. These results suggest that the first localized Ca2+ increase and the second global Ca2+ increase at fertilization are regulated by Ca2+ release from IP3-sensitive stores and Ca2+ influx via voltage-gated Ca2+ channels, respectively. Our data also demonstrated that the localized Ca2+ increase induces the formation of large cytoplasmic protrusion, which helps the fertilizing sperm to enter the oocyte, whereas the following global Ca2+ increase is a prerequisite for the retraction of the cytoplasmic protrusion and the resumption of meiosis from MI.

We have investigated synchronization and propagation of calcium oscillations, mediated by gap junctional excitation transmission. For that purpose we used an experimentally based model of normal rat kidney (NRK) cells, electrically coupled in a one-dimensional configuration (linear strand). Fibroblasts such as NRK cells can form an excitable syncytium and generate spontaneous inositol 1,4,5-trisphosphate (IP(3))-mediated intracellular calcium waves, which may spread over a monolayer culture in a coordinated fashion. An intracellular calcium oscillation in a pacemaker cell causes a membrane depolarization from within that cell via calcium-activated chloride channels, leading to an L-type calcium channel-based action potential (AP) in that cell. This AP is then transmitted to the electrically connected neighbor cell, and the calcium inflow during that transmitted AP triggers a calcium wave in that neighbor cell by opening of IP(3) receptor channels, causing calcium-induced calcium release (CICR). In this way the calcium wave of the pacemaker cell is rapidly propagated by the electrically transmitted AP. Propagation of APs in a strand of cells depends on the number of terminal pacemaker cells, the L-type calcium conductance of the cells, and the electrical coupling between the cells. Our results show that the coupling between IP(3)-mediated calcium oscillations and AP firing provides a robust mechanism for fast propagation of activity across a network of cells, which is representative for many other cell types such as gastrointestinal cells, urethral cells, and pacemaker cells in the heart.

It has been proposed that nuclear and cytosolic Ca(2+) ([Ca(2+)](N) and [Ca(2+)](C)) may be regulated independently. We address here the issue of whether inositol trisphosphate (IP(3)) can, bypassing changes of [Ca(2+)](C), produce direct release of Ca(2+) into the nucleoplasm. We have used targeted aequorins to selectively measure and compare the changes in [Ca(2+)](C) and [Ca(2+)](N) induced by IP(3) in GH(3) pituitary cells. Heparin, an IP(3) inhibitor that does not permeate the nuclear pores, abolished the [Ca(2+)](C) peaks but inhibited only partly the [Ca(2+)](N) peaks. The permeant inhibitor 2-aminoethoxy-diphenyl-borate (2-APB) blocked both responses. Removal of ATP also inhibited more strongly the [Ca(2+)](C) than [Ca(2+)](N) peak. The [Ca(2+)](N) and [Ca(2+)](C) responses differed also in their sensitivity to IP(3), the nuclear response showing higher affinity. Among IP(3) receptors, type 2 (IP(3)R2) has a higher affinity for IP(3) and is not inactivated by ATP removal. We find that IP(3)R2 immunoreactivity is present inside the nucleus whereas the other IP(3)R subtypes are detected only in the cytoplasm. The nuclear envelope (NE) of GH(3) cells showed deep invaginations into the nucleoplasm, with cytosol and cytoplasmic organella inside. These results indicate that GH(3) pituitary cells possess mechanisms able to produce selective increases of [Ca(2+)](N).

Smooth muscle responds to activation of the inositol (1,4,5)-trisphosphate receptor [Ins(1,4,5)P(3)R] with a graded concentration-dependent ("quantal") Ca2+ release from the sarcoplasmic reticulum (SR) store. Graded release seems incompatible both with the finite capacity of the store and the Ca2+-induced Ca2+ release (CICR)-like facility, at Ins(1,4,5)P3Rs, that, once activated, should release the entire content of SR Ca2+. The structural organization of the SR and the regulation of Ins(1,4,5)P3R activity by inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3] and Ca2+ have each been proposed to explain ;quantal' Ca2+ release. Here, we propose that regulation of Ins(1,4,5)P3R activity by lumenal Ca2+ acting at the cytoplasmic aspect of the receptor might explain ;quantal' Ca2+ release in smooth muscle. The entire SR store was found to be lumenally continuous and Ca2+ could diffuse freely throughout: peculiarities of SR structure are unlikely to account for ;quantal' release. While Ca2+ release was regulated by [Ca2+] within the SR, the velocity of release increased (accelerated) during the release process. The extent of acceleration of release determined the peak cytoplasmic [Ca2+] and was attenuated by a reduction in SR [Ca2+] or an increase in cytoplasmic Ca2+ buffering. Positive feedback by released Ca2+ acting at the cytoplasmic aspect of Ins(1,4,5)P3Rs (i.e. CICR-like) might (a) account for the acceleration, (b) provide the regulation of release by SR [Ca2+] and (c) explain the ;quantal' release process itself. During Ca2+ release, SR [Ca2+] and thus unitary Ins(1,4,5)P3R currents decline, CICR reduces and stops. With increasing [Ins(1,4,5)P3], coincidental activation of several neighbouring Ins(1,4,5)P3Rs offsets the reduced Ins(1,4,5)P3R current to renew CICR and Ca2+ release.

Ca2+ uptake by mitochondria might both modulate the cytosolic Ca2+ concentration ([Ca2+]c) and depolarize the mitochondrial membrane potential (delta Psi m) to limit ATP production. To investigate how physiological Ca2+ signaling might affect energy production, delta Psi m was examined during Ca2+ oscillations in smooth muscle cells. In single, voltage-clamped smooth muscle cells, inhibition of mitochondrial Ca2+ accumulation inhibited inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3]-evoked Ca2+ release and prolonged the time required for restoration of [Ca2+]c following activation of plasmalemmal Ca2+ currents (ICa). Ca2+ could be released from mitochondria immediately (within 15 seconds) after a [Ca2+]c rise evoked by Ins(1,4,5)P3 or ICa. Despite this evidence of mitochondrial Ca2+ accumulation, no change in delta Psi m was observed during single or repetitive [Ca2+]c oscillations evoked by these conditions. Occasionally, spontaneous, repetitive, persistent Ca 2+ oscillations were observed. In these cases, mitochondria displayed stochastic delta Psi m depolarizations, which were independent both of events in neighboring mitochondria and of the timing of the [Ca 2+]c oscillations themselves. Such delta Psi m depolarizations could be mimicked by increased exposure to either fluorescence excitation light or the delta Psi m-sensitive dye tetramethylrhodamine ethyl ester (TMRE) and were inhibited by antioxidants (ascorbic acid, catalase, Trolox and TEMPO) or the mitochondrial permeability transition pore (mPTP)-inhibitor cyclosporin A (CsA). Individual mitochondria within smooth muscle cells might depolarize during repetitive Ca2+ oscillations or during oxidative stress but not during the course of single [Ca2+]c transients evoked by Ca2+ influx or store release.

Contractility of airway smooth muscle requires elevation of intracellular calcium concentration. Under resting conditions, airway smooth muscle cells maintain a relatively low intracellular calcium concentration, and activation of the surface receptors by contractile agonists results in an elevation of intracellular calcium, culminating in contraction of the cell. The pattern of elevation of intracellular calcium brought about by agonists is a dynamic process and involves the coordinated activities of ion channels located in the plasma membrane and the sarcoplasmic reticulum. Among the signaling molecules involved in this dynamic calcium regulation in airway smooth muscle cells are inositol 1,4,5-trisphosphate and cyclic ADP-ribose, which mobilize calcium from the sarcoplasmic reticulum by acting via the inositol 1,4,5-trisphosphate and ryanodine receptors, respectively. In addition, calcium influx from the extracellular space is critical for the repletion of the intracellular calcium stores during activation of the cells by agonists. Calcium influx can occur via voltage- and receptor-gated channels in the plasma membrane, as well as by influx that is triggered by depletion of the intracellular stores (i.e., store-operated calcium entry mechanism). Transient receptor potential proteins appear to mediate the calcium influx via receptor- and store-operated channels. Recent studies have shown that proinflammatory cytokines regulate the expression and activity of the pathways involved in intracellular calcium regulation, thereby contributing to airway smooth muscle cell hyperresponsiveness. In this review, we will discuss the specific roles of cyclic ADP-ribose/ryanodine receptor channels and transient receptor potential channels in the regulation of intracellular calcium in airway smooth muscle cells.

We investigated, in monocytic leukemia U937 cells, the effects of docosahexaenoic acid (DHA; 22:6 n-3) on calcium signaling and determined the implication of phospholipase C (PLC) and protein kinase C (PKC) in this pathway. DHA induced dose-dependent increases in [Ca2+]i, which were contributed by intracellular pool, via the production of inositol-1,4,5-triphosphate (IP3) and store-operated Ca2+ (SOC) influx, via opening of Ca2+ release-activated Ca2+ (CRAC) channels. Chemical inhibition of PLC, PKCgamma, and PKCdelta, but not of PKCbeta I/II, PKCalpha, or PKCbetaI, significantly diminished DHA-induced increases in [Ca2+]i. In vitro PKC assays revealed that DHA induced a approximately 2-fold increase in PKCgamma and -delta activities, which were temporally correlated with the DHA-induced increases in [Ca2+]i. In cell-free assays, DHA, but not other structural analogs of fatty acids, activated these PKC isoforms. Competition experiments revealed that DHA-induced activation of both the PKCs was dose-dependently inhibited by phosphatidylserine (PS). Furthermore, DHA induced apoptosis via reactive oxygen species (ROS) production, followed by caspase-3 activation. Chemical inhibition of PKCgamma/delta and of SOC/CRAC channels significantly attenuated both DHA-stimulated ROS production and caspase-3 activity. Our study suggests that DHA-induced activation of PLC/IP3 pathway and activation of PKCgamma/delta, via its action on PS binding site, may be involved in apoptosis in U937 cells.

The purpose of the present study was to explore changes in rat colon motility, and determine the roles of calcium and inositol (1,4,5)-triphosphate (IP3) in colon dysmotility induced by multiple organ dysfunction syndrome (MODS) caused by bacteria peritonitis. The number of stools, the contractility of the muscle strips and the length of smooth muscle cells (SMC) in the colon, the concentration of calcium and IP3 in SMC, and serum nitric oxide were measured. Number of stools, fecal weight, IP3 concentration in SMC and serum nitric oxide concentration were 0.77 +/- 0.52 pellets, 2.51 +/- 0.39 g, 4.14 +/- 2.07 pmol/tube, and 113.95 +/- 37.89 micromol/L, respectively, for the MODS group (N = 11) vs 1.54 +/- 0.64 pellets, 4.32 +/- 0.57 g, 8.19 +/- 3.11 pmol/tube, and 37.42 +/- 19.56 micromol/L for the control group (N = 20; P < 0.05). After treatment with 0.1 mM acetylcholine and 0.1 M potassium chloride, the maximum contraction stress of smooth muscle strips, the length of SMC and the changes of calcium concentration were 593 +/- 81 and 458 +/- 69 g/cm(3), 48.1 +/- 11.8 and 69.2 +/- 15.7 microM, 250 +/- 70 and 167 +/- 48%, respectively, for the control group vs 321 +/- 53 and 284 +/- 56 g/cm(3), 65.1 +/- 18.5 and 87.2 +/- 23.7 microM, 127 +/- 35 and 112 +/- 35% for the MODS group (P < 0.05). Thus, colon contractility was decreased in MODS, a result possibly related to reduced calcium concentration and IP3 in SMC.

The Tec-family protein tyrosine kinase IL-2-inducible T cell kinase (ITK) mediates T cell activation, as does the adaptor protein SLP-76 (SH2-domain-containing leukocyte protein of 76 kD), which forms a complex with ITK and other intracellular signaling enzymes. One of these enzymes is phospholipase C-gamma1 (PLC-gamma1), which mediates T cell receptor (TCR)-stimulated intracellular calcium mobilization leading to the activation of transcription factors such as nuclear factor of activated T cells. The Src-family tyrosine kinase Lck and the Syk-family tyrosine kinase zeta chain-associated protein kinase of 70 kD (ZAP-70), together with ITK, are necessary for the phosphorylation of PLC-gamma1 in response to TCR stimulation. ITK is thought to phosphorylate a specific tyrosine residue of PLC-gamma1 that is required for its activation. The mechanism of activation of ITK appears to involve the interaction between SLP-76 and ITK, which not only initiates ITK activity but is also important to maintain the kinase activity of ITK. This suggests that SLP-76 acts as more than a neutral adaptor in mediating T cell activation; SLP-76 also directly influences the kinase activity of ITK, allowing ITK to phosphorylate PLC-gamma1.

All eukaryotic cells contain the phospholipid phosphatidylinositol 4, 5-bisphosphate (PIP2) that serves multiple roles in signal transduction cascades. Type I phosphatidylinositol-4-phosphate 5-kinase (PIP5KI) catalyzes the synthesis of PIP2 by phosphorylating phosphatidylinositol 4 phosphate. Although the classical isoforms of PIP5KI (designated as alpha, beta, and gamma) all generate the same phospholipid product, they have significantly dissimilar primary structures and expression levels in different tissues, and they appear to localize within different compartments within the cell. Therefore, it appears likely that PIP5KI isoforms have overlapping, but not identical, functions. Here we show that targeted disruption of PIP5KIgamma causes widespread developmental and cellular defects. PIP5KIgamma-null embryos have myocardial developmental defects associated with impaired intracellular junctions that lead to heart failure and extensive prenatal lethality at embryonic day 11.5 of development. Loss of PIP5KIgamma also results in neural tube closure defects that were associated with impaired PIP2 production, adhesion junction formation, and neuronal cell migration. These data, along with those of other PIP5KI isoforms, indicate that individual PIP5KI isoenzymes fulfill specific roles in embryonic development.

Background and Purpose: The thiourea derivative KB-R7943, originally developed as inhibitor of the plasma membrane Na(+)/Ca(2+) exchanger, has been shown to protect against myocardial ischemia-reperfusion injury. We have studied here its effects on mitochondrial Ca(2+) fluxes., Experimental Approach: [Ca(2+)] in cytosol, mitochondria and endoplasmic reticulum (ER), and mitochondrial membrane potential were monitored using both luminescent (targeted aequorins) and fluorescent (fura-2, tetramethylrhodamine ethyl ester) probes in HeLa cells., Key Results: KB-R7943 was also a potent inhibitor of the mitochondrial Ca(2+) uniporter (MCU). In permeabilized HeLa cells, KB-R7943 inhibited mitochondrial Ca(2+) uptake with a Ki of 5.5+/-1.3 microM (mean+/-S.D.). In intact cells, 10 microM KB-R7943 reduced by 80% the mitochondrial [Ca(2+)] peak induced by histamine. KB-R7943 did not modify the mitochondrial membrane potential and had no effect on the mitochondrial Na(+)/Ca(2+) exchanger. KB-R7943 inhibited histamine-induced ER-Ca(2+) release in intact cells, but not in cells loaded with a Ca(2+)-chelator to damp cytosolic [Ca(2+)] changes. Therefore, inhibition of ER-Ca(2+)-release by KB-R7943 was probably due to the increased feedback Ca(2+)-inhibition of inositol 1,4,5-trisphosphate receptors after MCU block. This mechanism also explains why KB-R7943 reversibly blocked histamine-induced cytosolic [Ca(2+)] oscillations in the same range of concentrations required to inhibit MCU., Conclusions and Implications: Inhibition of MCU by KB-R7943 may contribute to its cardioprotective activity by preventing mitochondrial Ca(2+)-overload during ischemia-reperfusion. In addition, the effects of KB-R7943 on Ca(2+) homeostasis provide new evidence for the role of mitochondria modulating Ca(2+)-release and regenerative Ca(2+)-oscillations. Search for permeable and selective MCU inhibitors may yield useful pharmacological tools in the future.

Bradykinin is an important mediator produced during myocardial ischemia and infarction that can activate and/or sensitize cardiac spinal (sympathetic) sensory neurons to trigger chest pain. Because a long-onset latency is associated with the bradykinin effect on cardiac spinal afferents, a cascade of intracellular signaling events is likely involved in the action of bradykinin on cardiac nociceptors. In this study, we determined the signal transduction mechanisms involved in bradykinin stimulation of cardiac nociceptors. Cardiac dorsal root ganglion (DRG) neurons in rats were labeled by intracardiac injection of a fluorescent tracer, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine percholate (DiI). Whole cell current-clamp recordings were performed in acutely isolated DRG neurons. In DiI-labeled DRG neurons, 1 microM bradykinin significantly increased the firing frequency and lowered the membrane potential. Iodoresiniferatoxin, a highly specific transient receptor potential vanilloid type 1 (TRPV1) antagonist, significantly reduced the excitatory effect of bradykinin. Furthermore, the stimulating effect of bradykinin on DiI-labeled DRG neurons was significantly attenuated by baicalein (a selective inhibitor of 12-lipoxygenase) or 2-aminoethyl diphenylborinate [an inositol 1,4,5-trisphosphate (IP(3)) antagonist]. In addition, the effect of bradykinin on cardiac DRG neurons was abolished after the neurons were treated with BAPTA-AM or thapsigargin (to deplete intracellular Ca(2+) stores) but not in the Ca(2+)-free extracellular solution. Collectively, these findings provide new evidence that 12-lipoxygenase products, IP(3), and TRPV1 channels contribute importantly to excitation of cardiac nociceptors by bradykinin. Activation of TRPV1 and the increase in the intracellular Ca(2+) are critically involved in activation/sensitization of cardiac nociceptors by bradykinin.

This article focuses on recent advances in melanocyte biology and physiology. The major function of this neural crest-derived cell is the production of melanins. A "three enzyme theory" in the initiation of pigmentation is put forward and backed up by recent findings. A receptor-independent role for alpha-MSH and the cofactor (6R)-l-erythro-5,6,7,8-terahydrobiopterin (6BH(4)) in the control of tyrosinase is described. The importance of intramelanosomal pH for melanogenesis is covered. Finally, the redundancy of the cAMP and IP3/DAG/calcium signal in melanocytes together with the downstream events are highlighted. The main message of this article is that the intracellular H(2)O(2)- redox-equilibrium controls melanocyte function in a concentration-dependent manner.