IP3 Signaling Research Articles

Activation of IP3R in atrial cardiomyocytes leads to generation of cytosolic cAMP.

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia. Excessive stimulation of the inositol (1,4,5)-trisphosphate (IP3) signaling pathway has been linked to AF through abnormal calcium handling. However, little is known about the mechanisms involved in this process. We expressed the fluorescence resonance energy transfer (FRET)-based cytosolic cyclic adenosine monophosphate (cAMP) sensor EPAC-SH187 in neonatal rat atrial myocytes (NRAMs) and neonatal rat ventricular myocytes (NRVMs). In NRAMs, the addition of the α1-agonist, phenylephrine (PE, 3 µM), resulted in a FRET change of 21.20 ± 7.43%, and the addition of membrane-permeant IP3 derivative 2,3,6-tri-O-butyryl-myo-IP3(1,4,5)-hexakis(acetoxymethyl)ester (IP3-AM, 20 μM) resulted in a peak of 20.31 ± 6.74%. These FRET changes imply an increase in cAMP. Prior application of IP3 receptor (IP3R) inhibitors 2-aminoethyl diphenylborinate (2-APB, 2.5 μM) or Xestospongin-C (0.3 μM) significantly inhibited the change in FRET in NRAMs in response to PE. Xestospongin-C (0.3 μM) significantly inhibited the change in FRET in NRAMs in response to IP3-AM. The FRET change in response to PE in NRVMs was not inhibited by 2-APB or Xestospongin-C. Finally, the localization of cAMP signals was tested by expressing the FRET-based cAMP sensor, AKAP79-CUTie, which targets the intracellular surface of the plasmalemma. We found in NRAMs that PE led to FRET change corresponding to an increase in cAMP that was inhibited by 2-APB and Xestospongin-C. These data support further investigation of the proarrhythmic nature and components of IP3-induced cAMP signaling to identify potential pharmacological targets.NEW & NOTEWORTHY This study shows that indirect activation of the IP3 pathway in atrial myocytes using phenylephrine and direct activation using IP3-AM leads to an increase in cAMP and is in part localized to the cell membrane. These changes can be pharmacologically inhibited using IP3R inhibitors. However, the cAMP rise in ventricular myocytes is independent of IP3R calcium release. Our data support further investigation into the proarrhythmic nature of IP3-induced cAMP signaling.

Bimetallic Nanoplatforms for Prostate Cancer Treatment by Interfering Cellular Communication.

Cellular communication mediated by messenger molecules plays an important role in the progression of cancer. Herein, pH-sensitive zeolitic imidazolate framework-8 (ZIF-8) loaded with PtCl2(OH)2(NH3)2 [i.e., Pt(IV)] bimetallic nanoplatforms were developed for prostate cancer therapy by interfering inositol-1, 4, 5-trisphosphate (IP3)-mediated cellular communication. As an important messenger in cells, the function of IP3 was found to be efficiently interfered with by the Pt(IV)-binding inositol unit. This finding effect of Pt(IV) is totally different from its traditional function as a prodrug of cis-platinum for chemotherapy. The decreased IP3 signal further downregulated the cytoplasmic Ca2+ concentration and downstream signal transduction to inhibit proliferation and invasion of tumor cells. Meanwhile, Zn2+ released from ZIF-8 under an acidic tumor microenvironment decreased adenosine triphosphate biosynthesis, which could further limit the cellular communication. Such a proposed strategy of interfering cellular communication has demonstrated its feasibility in this study, which may provide new perspectives for cancer therapy.

Diacylglycerol kinase is a keystone regulator of signaling relevant to the pathophysiology of asthma.

Signal transduction by G protein-coupled receptors (GPCRs), receptor tyrosine kinases (RTKs) and immunoreceptors converge at the activation of phospholipase C (PLC) for the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). This is a point for second-messenger bifurcation where DAG via protein kinase C (PKC) and IP3 via calcium activate distinct protein targets and regulate cellular functions. IP3 signaling is regulated by multiple calcium influx and efflux proteins involved in calcium homeostasis. A family of lipid kinases belonging to DAG kinases (DGKs) converts DAG to phosphatidic acid (PA), negatively regulating DAG signaling and pathophysiological functions. PA, through a series of biochemical reactions, is recycled to produce new molecules of PIP2. Therefore, DGKs act as a central switch in terminating DAG signaling and resynthesis of membrane phospholipids precursor. Interestingly, calcium and PKC regulate the activation of α and ζ isoforms of DGK that are predominantly expressed in airway and immune cells. Thus, DGK forms a feedback and feedforward control point and plays a crucial role in fine-tuning phospholipid stoichiometry, signaling, and functions. In this review, we discuss the previously underappreciated complex and intriguing DAG/DGK-driven mechanisms in regulating cellular functions associated with asthma, such as contraction and proliferation of airway smooth muscle (ASM) cells and inflammatory activation of immune cells. We highlight the benefits of manipulating DGK activity in mitigating salient features of asthma pathophysiology and shed light on DGK as a molecule of interest for heterogeneous diseases such as asthma.

Cross Talking Calcium, IP3 and Buffer Dynamics Alters ATP and NADH Level in Obese and Normal Hepatocyte Cell.

The cross talk between calcium (Ca2+), IP3 and buffer dynamics regulate various mechanisms in hepatocyte cells. The study of independent systems of calcium, IP3, and buffer signaling provides limited information about cell dynamics. In the current study, coupled reaction-diffusion equations are used to design a cross-talk model for IP3, buffer, and calcium dynamics in a hepatocyte cell. The one-way feedback of calcium, buffer, and IP3 in ATP production, ATP degradation, and NADH production rate is incorporated into the model. Numerical simulation has been done using the Finite Element Method (FEM) along the spatial direction and the Crank-Nicolson (C-N) method along the temporal direction. The numerical results are analysed to determine the effects of alterations in processes of cross-talking dynamics of IP3, buffer, and calcium on ATP and NADH production and degradation rate of ATP in a hepatocyte cell under normal and obesity conditions. The comparative analysis of these findings unveils notable distinctions induced by obesity in calcium dynamics, ATP and NADH synthesis, and ATP degradation kinetics.

Computational modeling of the relationship between morphological heterogeneity and functional responses in mouse hippocampal astrocytes.

Recent studies indicate that astrocytes show heterogeneity in morphology and physiological function. They integrate synaptic signals and release calcium in reaction to active neurons. These calcium signals are not yet fully understood as they are highly dependent on the cell's morphology, which can vary across and within brain regions. We found structural heterogeneity among mouse hippocampal CA1 astrocytes based on geometric features, clustering 741 cells into six classes. Of those, we selected 84 cells and reconstructed their morphology based on confocal microscope images and converted them into multi-compartment models with a high detailedness. We applied a computational biophysical model simulating the intracellular ion and IP3 signaling and diffusion in those 3D cell geometries. The cells were stimulated with three different glutamate stimuli. Calcium mainly oscillated in the stimulated and the neighboring compartment but not in the soma. Significant differences were found in the peak width, mean prominence, and mean peak amplitude of the calcium signal when comparing the signals in the stimulated and neighboring compartments. Overall, this study highlights the influence of the complex morphology of astrocytes on intracellular ionic signaling.

Increased Risk for Atrial Alternans in Rabbit Heart Failure: The Role of Ca2+/Calmodulin-Dependent Kinase II and Inositol-1,4,5-trisphosphate Signaling.

Heart failure (HF) increases the probability of cardiac arrhythmias, including atrial fibrillation (AF), but the mechanisms linking HF to AF are poorly understood. We investigated disturbances in Ca2+ signaling and electrophysiology in rabbit atrial myocytes from normal and failing hearts and identified mechanisms that contribute to the higher risk of atrial arrhythmias in HF. Ca2+ transient (CaT) alternans-beat-to-beat alternations in CaT amplitude-served as indicator of increased arrhythmogenicity. We demonstrate that HF atrial myocytes were more prone to alternans despite no change in action potentials duration and only moderate decrease of L-type Ca2+ current. Ca2+/calmodulin-dependent kinase II (CaMKII) inhibition suppressed CaT alternans. Activation of IP3 signaling by endothelin-1 (ET-1) and angiotensin II (Ang II) resulted in acute, but transient reduction of CaT amplitude and sarcoplasmic reticulum (SR) Ca2+ load, and lowered the alternans risk. However, prolonged exposure to ET-1 and Ang II enhanced SR Ca2+ release and increased the degree of alternans. Inhibition of IP3 receptors prevented the transient ET-1 and Ang II effects and by itself increased the degree of CaT alternans. Our data suggest that activation of CaMKII and IP3 signaling contribute to atrial arrhythmogenesis in HF.

SAT269 Orexin Activates Adrenocortical Steroidogenesis With Upregulation Of BMP Signaling By Human H295R Cells

Abstract Disclosure: Y. Soejima: None. N. Iwata: None. R. Nishioka: None. M. Honda: None. Y. Nakano: None. K. Yamamoto: None. A. Suyama: None. F. Otsuka: None. Orexins are neuropeptides that express primarily in the hypothalamus and are composed of two isoforms, orexin A and orexin B. Orexin A binds to orexin receptor type 1 (OX1R) and type 2 (OX2R), while orexin B binds selectively to OX2R. Orexin has been reported to have important roles in various systems including sleep-wake regulation and feeding behavior in the central nervous system, whereas its receptors are also expressed in peripheral tissues including the endocrine system. The expression of orexin receptors and the effects of orexins on adrenocortical steroidogenesis have been shown in experiments using adrenocortical cells. Bone morphogenetic proteins (BMPs), which are members of the transforming growth factor (TGF)-β superfamily, also play functional roles in various endocrine tissues. In our previous experiments, we have revealed that BMP-6 enhances aldosterone production which is induced by angiotensin II through activation of mitogen-activated protein kinase (MAPK) signaling. We have also shown that peripheral clock genes have functional roles in modulating adrenocortical steroidogenesis by interacting with adrenocortical BMP system. However, the molecular mechanism of physiological effects of orexin and its interaction with BMP signaling remains unclear. In the present study, we investigated the effects of orexin on adrenal steroidogenesis using human adrenocortical H295R cells, by focusing on the interaction with adrenocortical BMP signaling. First, it was confirmed that both OX1R and OX2R were expressed in H295R cells. It was revealed that orexin A (10-300 nM) stimulation for 24 h directly increased mRNA levels of steroidogenic acute regulatory protein (StAR), cytochrome P450 family 11 subfamily B member 2 (CYP11B2) and cytochrome P450 family 17 subfamily A member 1 (CYP17A1) in a concentration-dependent manner. Orexin A further upregulated the expression of these steroidogenic enzymes in H295R cells co-treated with forskolin (FSK). Next, the interrelationship between orexin and BMP signaling was examined. Stimulation with BMP-6 readily activated Smad1/5/9 phosphorylation in H295R cells and the pretreatment with orexin A for 24 h enhanced the Smad1/5/9 phosphorylation induced by BMP-6. On the other hand, BMP-6 significantly suppressed the expression of OX1R but not that of OX2R. In our previous study, we have reported that BMP-6 upregulates the adrenocortical steroidogenesis via the IP3 and MAPK signaling in H295R cells, suggesting that orexin may have a pivotal role as a functional modulator of BMP-6. Collectively, it was revealed that orexin enhances adrenocortical steroidogenesis by regulating BMP signaling with a feedback system in human adrenocortical cells. Here, we will also discuss the relationship with BMP receptors and the effects on endogenous BMP expression. Presentation: Saturday, June 17, 2023

Free fatty acid receptor 1 stimulates cAMP production and gut hormone secretion through Gq-mediated activation of adenylate cyclase 2

ObjectiveFree fatty acid receptor 1 (FFAR1) is highly expressed in enteroendocrine cells of the small intestine and pancreatic beta cells, where FFAR1 agonists function as GLP-1 and insulin secretagogues, respectively. Most efficacious are so-called second-generation synthetic agonists such as AM5262, which, in contrast to endogenous long-chain fatty acids are able to signal through both IP3/Ca2+ and cAMP pathways. Whereas IP3 signaling is to be expected for the mainly Gq-coupled FFAR1, the mechanism behind FFAR1-induced cAMP accumulation remains unclear, although originally proposed to be Gs mediated. Methods and resultsWhen stimulated with AM5262, we observe that FFAR1 can activate the majority of the Gα proteins, except - surprisingly - members of the Gs family. AM5262-induced FFAR1-mediated transcriptional activation through cAMP response element (CREB) was blocked by the specific Gq inhibitor, YM253890. Furthermore, in Gq-deficient cells no CREB signal was observed unless Gq or G11 was reintroduced by transfection. By qPCR we determined that adenylate cyclase 2 (Adcy2) was highly expressed and enriched relative to the nine other Adcys in pro-glucagon expressing enteroendocrine cells. Co-transfection with ADCY2 increased the FFAR1-induced cAMP response 4-5-fold in WT HEK293 cells, an effect fully inhibited by YM253890. Moreover, co-transfection with ADCY2 had no effect in Gq-deficient cells without reintroduction of either Gq or G11. Importantly, although both AM5262/FFAR1 and isoproterenol/β2 adrenergic receptor (β2AR) induced cAMP production was lost in Gs-deficient cells, only the β2AR response was rescued by Gs transfection, whereas co-transfection with ADCY2 was required to rescue the FFAR1 cAMP response. In situ hybridization demonstrated a high degree of co-expression of ADCY2 and FFAR1 in enteroendocrine cells throughout the intestine. Finally, in the enteroendocrine STC-1 and GLUTag cell lines AM5262-induced cAMP accumulation and GLP-1 secretion were both blocked by YM253890. ConclusionsOur results show that Gq signaling is responsible not only for the IP3/Ca2+ but also the cAMP response, which together are required for the highly efficacious hormone secretion induced by second-generation FFAR1 agonists - and that ADCY2 presumably mediates the Gq-driven cAMP response.

Non-inositol 1,4,5-trisphosphate (IP3) receptor IP3-binding proteins

Conventionally, myo-D-inositol 1, 4,5-trisphosphate (IP3) is thought to exert its second messenger effects through the gating of IP3R Ca2+ release channels, located in Ca2+-storage organelles like the endoplasmic reticulum. However, there is considerable indirect evidence to support the concept that IP3 might interact with other, non-IP3R proteins within cells. To explore this possibility further, the Protein Data Bank was searched using the term “IP3”. This resulted in the retrieval of 203 protein structures, the majority of which were members of the IP3R/ryanodine receptor superfamily of channels. Only 49 of these structures were complexed with IP3. These were inspected for their ability to interact with the carbon-1 phosphate of IP3, since this is the least accessible phosphate group of its precursor, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). This reduced the number of structures retrieved to 35, of which 9 were IP3Rs. The remaining 26 structures represent a diverse range of proteins, including inositol-lipid metabolizing enzymes, signal transducers, PH domain containing proteins, cytoskeletal anchor proteins, the TRPV4 ion channel, a retroviral Gag protein and fibroblast growth factor 2. Such proteins may impact on IP3 signalling and its effects on cell-biology. This represents an area open for exploration in the field of IP3 signalling.

Mechanistic insights of neuronal calcium and IP3 signaling system regulating ATP release during ischemia in progression of Alzheimer's disease.

The mechanisms of calcium ([Ca2+]) signaling in various human cells have been widely analyzed by scientists due to its crucial role in human organs like the heartbeat, muscle contractions, bone activity, brain functionality, etc. No study is reported for interdependent [Ca2+] and IP3 mechanics regulating the release of ATP in neuron cells during Ischemia in Alzheimer's disease advancement. In the present investigation, a finite element method (FEM) is framed to explore the interdependence of spatiotemporal [Ca2+] and IP3 signaling mechanics and its role in ATP release during Ischemia as well as in the advancement of Alzheimer's disorder in neuron cells. The results provide us insights of the mutual spatiotemporal impacts of [Ca2+] and IP3 mechanics as well as their contributions to ATP release during Ischemia in neuron cells. The results obtained for the mechanics of interdependent systems differ significantly from the results of simple independent system mechanics and provide new information about the processes of the two systems. From this study, it is concluded that neuronal disorders cannot only be simply attributed to the disturbance caused directly in the processes of calcium signaling mechanics, but also to the disturbances caused in IP3 regulation mechanisms impacting the calcium regulation in the neuron cell and ATP release.

The Pharmacological Potential of Novel Melittin Variants from the Honeybee and Solitary Bees against Inflammation and Cancer.

The venom of honeybees is composed of numerous peptides and proteins and has been used for decades as an anti-inflammatory and anti-cancer agent in traditional medicine. However, the bioactivity of specific biomolecular components has been evaluated for the predominant constituent, melittin. So far, only a few melittin-like peptides from solitary bee species have been investigated, and the molecular mechanisms of bee venoms as therapeutic agents remain largely unknown. Here, the preclinical pharmacological activities of known and proteo-transcriptomically discovered new melittin variants from the honeybee and more ancestral variants from phylogenetically older solitary bees were explored in the context of cancer and inflammation. We studied the effects of melittin peptides on cytotoxicity, second messenger release, and inflammatory markers using primary human cells, non-cancer, and cancerous cell lines. Melittin and some of its variants showed cytotoxic effects, induced Ca2+ signaling and inhibited cAMP production, and prevented LPS-induced NO synthesis but did not affect the IP3 signaling and pro-inflammatory activation of endothelial cells. Compared to the originally-described melittin, some phylogenetically more ancestral variants from solitary bees offer potential therapeutic modalities in modulating the in vitro inflammatory processes, and hindering cancer cell viability/proliferation, including aggressive breast cancers, and are worth further investigation.

Oxidative stress impairs the calcification ability of human dental pulp cells

BackgroundThe relationship between internal root resorption and oxidative stress has not yet been reported. This study aimed to add molecular insight into internal root resorption. The present study was conducted to investigate the effect of hydrogen peroxide (H2O2) as an inducer of oxidative stress on the calcification ability of human dental pulp cells (hDPCs) and the involvement of inositol 1, 4, 5-trisphosphate (IP3).Material and methodshDPCs (Lonza, Basel, Switzerland) were exposed to H2O2. Cell viability and reactive oxygen species (ROS) production were then evaluated. To investigate the effect of H2O2 on the calcification ability of hDPCs, real-time PCR for alkaline phosphatase (ALP) mRNA expression, ALP staining, and Alizarin red staining were performed. Data were compared with those of hDPCs pretreated with 2-aminoethyldiphenylborate (2-APB), which is an IP3 receptor inhibitor.ResultsH2O2 at concentrations above 250 µM significantly reduced cell viability (P < 0.01). More ROS production occurred in 100 µM H2O2-treated hDPCs than in control cells (P < 0.01). 2-APB significantly decreased the production (P < 0.05). H2O2-treated hDPCs showed significant reductions in ALP mRNA expression (P < 0.01), ALP activity (P < 0.01), and mineralized nodule deposition compared with negative control cells (P < 0.01). 2-APB significantly inhibited these reductions (P < 0.01, P < 0.05 and P < 0.01, respectively). Data are representative of three independent experiments with three replicates for each treatment and values are expressed as means ± SD.ConclusionTo the best of our knowledge, this is the first study documenting the involvement of IP3 signaling in the calcification ability of human dental pulp cells impaired by H2O2.

Inhibition of adenylyl cyclase 1 by ST034307 inhibits IP3-evoked changes in sino-atrial node beat rate.

Atrial arrhythmias, such as atrial fibrillation (AF), are a major mortality risk and a leading cause of stroke. The IP3 signalling pathway has been proposed as an atrial-specific target for AF therapy, and atrial IP3 signalling has been linked to the activation of calcium sensitive adenylyl cyclases AC1 and AC8. We investigated the involvement of AC1 in the response of intact mouse atrial tissue and isolated guinea pig atrial and sino-atrial node (SAN) cells to the α-adrenoceptor agonist phenylephrine (PE) using the selective AC1 inhibitor ST034307. The maximum rate change of spontaneously beating mouse right atrial tissue exposed to PE was reduced from 14.5% to 8.2% (p = 0.005) in the presence of 1 μM ST034307, whereas the increase in tension generated in paced left atrial tissue in the presence of PE was not inhibited by ST034307 (Control = 14.2%, ST034307 = 16.3%; p > 0.05). Experiments were performed using isolated guinea pig atrial and SAN cells loaded with Fluo-5F-AM to record changes in calcium transients (CaT) generated by 10 μM PE in the presence and absence of 1 μM ST034307. ST034307 significantly reduced the beating rate of SAN cells (0.34-fold decrease; p = 0.003) but did not inhibit changes in CaT amplitude in response to PE in atrial cells. The results presented here demonstrate pharmacologically the involvement of AC1 in the downstream response of atrial pacemaker activity to α-adrenoreceptor stimulation and IP3R calcium release.

Melatonin Attenuates Vascular Smooth Muscle Contraction Through the γ-Secretase/Notch Intracellular Domain/Myocardin Pathway.

Inositol 1, 4, 5-trisphosphate (IP3) signaling-mediated calcium release drives the contraction of vascular smooth muscles and hence regulates blood vessel volume and blood pressure. Melatonin supplementation has been suggested to be beneficial for hypertension. To determine whether the blood pressure-lowering effect of melatonin was accounted for by IP3 signaling, we evaluated the vasoconstriction response and IP3 signaling in isolated mouse thoracic aortic rings during melatonin incubation. C57BL/6 mice were given intraperitoneal injections daily with melatonin, and the systolic blood pressure and contractility of aortic rings from melatonin-treated mice were decreased, and the contraction suppression effect of melatonin was attributed to the impaired expression of contractile proteins in vascular smooth muscle cells rather than IP3 signaling. Our results further showed that melatonin increased the expression of γ-secretase, which could cleave and release the notch intracellular domain, and the notch intracellular domain prevented the transcription of contractile genes by interfering with the interaction between serum response factor and myocardin, the master regulator of contractile protein. In this article, we report a novel mechanism by which melatonin regulates smooth muscle contractility that does not depend on IP3 signaling.

Three-Dimensional Model of Sub-Plasmalemmal Ca2+ Microdomains Evoked by T Cell Receptor/CD3 Complex Stimulation.

Ca2+ signalling plays an essential role in T cell activation, which is a key step to start an adaptive immune response. During the transition from a quiescent to a fully activated state, Ca2+ microdomains of reduced spatial and temporal extents develop in the junctions between the plasma membrane and the endoplasmic reticulum (ER). These microdomains rely on Ca2+ entry from the extracellular medium, via the ORAI1/STIM1/STIM2 system that mediates store operated Ca2+ entry Store operated calcium entry. The mechanism leading to local store depletion and subsequent Ca2+ entry depends on the activation state of the cells. The initial, smaller microdomains are triggered by D-myo-inositol 1,4,5-trisphosphate (IP3) signalling in response to T cell adhesion. T cell receptor (TCR)/CD3 stimulation then initiates nicotinic acid adenine dinucleotide phosphate signalling, which activates ryanodine receptors (RYR). We have recently developed a mathematical model to elucidate the spatiotemporal Ca2+ dynamics of the microdomains triggered by IP3 signalling in response to T cell adhesion (Gil et al., 2021). This reaction-diffusion model describes the evolution of the cytosolic and endoplasmic reticulum Ca2+ concentrations in a three-dimensional ER-PM junction and was solved using COMSOL Multiphysics. Modelling predicted that adhesion-dependent microdomains result from the concerted activity of IP3 receptors and pre-formed ORAI1-STIM2 complexes. In the present study, we extend this model to include the role of RYRs rapidly after TCR/CD3 stimulation. The involvement of STIM1, which has a lower KD for Ca2+ than STIM2, is also considered. Detailed 3D spatio-temporal simulations show that these Ca2+ microdomains rely on the concerted opening of ∼7 RYRs that are simultaneously active in response to the increase in NAADP induced by T cell stimulation. Opening of these RYRs provoke a local depletion of ER Ca2+ that triggers Ca2+ flux through the ORAI1 channels. Simulations predict that RYRs are most probably located around the junction and that the increase in junctional Ca2+ concentration results from the combination between diffusion of Ca2+ released through the RYRs and Ca2+ entry through ORAI1 in the junction. The computational model moreover provides a tool allowing to investigate how Ca2+ microdomains occur, extend and interact in various states of T cell activation.

HIV infection of astrocytes compromises inter-organelle interactions and inositol phosphate metabolism: A potential mechanism of bystander damage and viral reservoir survival.

HIV-associated neurological dysfunction is observed in more than half of the HIV-infected population, even in the current antiretroviral era. The mechanisms by which HIV mediates CNS dysfunction are not well understood but have been associated with the presence of long-lasting HIV reservoirs. In the CNS, macrophage/microglia and a small population of astrocytes harbor the virus. However, the low number of HIV-infected cells does not correlate with the high degree of damage, suggesting that mechanisms of damage amplification may be involved. Here, we demonstrate that the survival mechanism of HIV-infected cells and the apoptosis of surrounding uninfected cells is regulated by inter-organelle interactions among the mitochondria/Golgi/endoplasmic reticulum system and the associated signaling mediated by IP3 and calcium. We identified that latently HIV-infected astrocytes had elevated intracellular levels of IP3, a master regulator second messenger, which diffuses via gap junctions into neighboring uninfected astrocytes resulting in their apoptosis. In addition, using laser capture microdissection, we confirmed that bystander apoptosis of uninfected astrocytes and the survival of HIV-infected astrocytes were dependent on mitochondrial function, intracellular calcium, and IP3 signaling. Blocking gap junction channels did not prevent an increase in IP3 or inter-organelle dysfunction in HIV-infected cells but reduced the amplification of apoptosis into uninfected neighboring cells. Our data provide a mechanistic explanation for bystander damage induced by surviving infected cells that serve as viral reservoirs and provide potential targets for interventions to reduce the devastating consequences of HIV within the brain.

Signal Transduction of Mineralocorticoid and Angiotensin II Receptors in the Central Control of Sodium Appetite: A Narrative Review.

Sodium appetite is an innate behavior occurring in response to sodium depletion that induces homeostatic responses such as the secretion of the mineralocorticoid hormone aldosterone from the zona glomerulosa of the adrenal cortex and the stimulation of the peptide hormone angiotensin II (ANG II). The synergistic action of these hormones signals to the brain the sodium appetite that represents the increased palatability for salt intake. This narrative review summarizes the main data dealing with the role of mineralocorticoid and ANG II receptors in the central control of sodium appetite. Appropriate keywords and MeSH terms were identified and searched in PubMed. References to original articles and reviews were examined, selected, and discussed. Several brain areas control sodium appetite, including the nucleus of the solitary tract, which contains aldosterone-sensitive HSD2 neurons, and the organum vasculosum lamina terminalis (OVLT) that contains ANG II-sensitive neurons. Furthermore, sodium appetite is under the control of signaling proteins such as mitogen-activated protein kinase (MAPK) and inositol 1,4,5-thriphosphate (IP3). ANG II stimulates salt intake via MAPK, while combined ANG II and aldosterone action induce sodium intake via the IP3 signaling pathway. Finally, aldosterone and ANG II stimulate OVLT neurons and suppress oxytocin secretion inhibiting the neuronal activity of the paraventricular nucleus, thus disinhibiting the OVLT activity to aldosterone and ANG II stimulation.

Receptor-specific Ca2+ oscillation patterns mediated by differential regulation of P2Y purinergic receptors in rat hepatocytes

SummaryExtracellular agonists linked to inositol-1,4,5-trisphosphate (IP3) formation elicit cytosolic Ca2+ oscillations in many cell types, but despite a common signaling pathway, distinct agonist-specific Ca2+ spike patterns are observed. Using qPCR, we show that rat hepatocytes express multiple purinergic P2Y and P2X receptors (R). ADP acting through P2Y1R elicits narrow Ca2+ oscillations, whereas UTP acting through P2Y2R elicits broad Ca2+ oscillations, with composite patterns observed for ATP. P2XRs do not play a role at physiological agonist levels. The discrete Ca2+ signatures reflect differential effects of protein kinase C (PKC), which selectively modifies the falling phase of the Ca2+ spikes. Negative feedback by PKC limits the duration of P2Y1R-induced Ca2+ spikes in a manner that requires extracellular Ca2+. By contrast, P2Y2R is resistant to PKC negative feedback. Thus, the PKC leg of the bifurcated IP3 signaling pathway shapes unique Ca2+ oscillation patterns that allows for distinct cellular responses to different agonists.

Proteolytic activation of Growth-blocking peptides triggers calcium responses through the GPCR Mthl10 during epithelial wound detection.

The presence of a wound triggers surrounding cells to initiate repair mechanisms, but it is not clear how cells initially detect wounds. In epithelial cells, the earliest known wound response, occurring within seconds, is a dramatic increase in cytosolic calcium. Here, we show that wounds in the Drosophila notum trigger cytoplasmic calcium increase by activating extracellular cytokines, Growth-blocking peptides (Gbps), which initiate signaling in surrounding epithelial cells through the G-protein-coupled receptor Methuselah-like 10 (Mthl10). Latent Gbps are present in unwounded tissue and are activated by proteolytic cleavage. Using wing discs, we show that multiple protease families can activate Gbps, suggesting that they act as a generalized protease-detector system. We present experimental and computational evidence that proteases released during wound-induced cell damage and lysis serve as the instructive signal: these proteases liberate Gbp ligands, which bind to Mthl10 receptors on surrounding epithelial cells, and activate downstream release of calcium.

XLα s is a novel activator of PLCβ4

Phospholipase C (PLC) enzymes are essential for normal cellular function. Upon stimulation by GPCRs or RTKs, PLCs hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP2) at cell membranes to generate diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). DAG and IP3 are important second messengers in signal transduction. DAG regulates activity of protein kinase C and IP3 mobilizes intracellular Ca2+, which initiate multiple signaling cascades and cellular processes. Dysregulation of PLC function gives rise to pathological processes. Among six mammalian PLC subfamilies, PLCβ isozymes are activated by Gαq, Gβγ, small G proteins and thus, play significant roles in various cellular activities. Extra-large stimulatory Gα (XLαs) is a large variant of Gαs. Biochemically, XLαs activation by GPCR mediates cAMP generation similarly to Gαs, however, Gαs andXLαs have distinct cellular and physiological functions. He et al. suggests that XLαs is able to stimulate IP3 signaling in vivo. In this study, we aim to identify the PLC that mediates this phenomenon. By co-expressing XLαs with different PLC constructs in cells and measuring total IP production, we identify that XLαs stimulates PLCβ4 to generate IP3. This activity is specific to XLαs because wild-type and constitutively active Gαs do not activate PLCβ4. Using purified proteins, we show that XLαs directly and specifically activates PLCβ4. Interestingly, unlike Gαq, XLαs does not require GTP to activate its effector PLCβ4. We also identify that the helical domain preceding the nucleotide-binding domain in XLαs is required for its ability to stimulate PLCβ4. Further studies are underway to determine the mode of activation of PLCβ4 by XLαs and the role of XLαs – mediated stimulation of PLCβ4 in physiological systems. Overall, we identify XLαs as a novel direct activator of PLCβ4 and this novel signaling axis potentially play important roles physiologically.