Inositol Trisphosphate Production Research Articles

Calcium oscillations in HEK293 cells lacking SOCE suggest the existence of a balanced regulation of IP3 production and degradation

The concentration of free cytosolic Ca2+ is a critical second messenger in almost every cell type, with the signal often being carried by the period of oscillations, or spikes, in the cytosolic Ca2+ concentration. We have previously studied how Ca2+ influx across the plasma membrane affects the period and shape of Ca2+ oscillations in HEK293 cells. However, our theoretical work was unable to explain how the shape of Ca2+ oscillations could change qualitatively, from thin spikes to broad oscillations, during the course of a single time series. Such qualitative changes in oscillation shape are a common feature of HEK293 cells in which STIM1 and 2 have been knocked out. Here, we present an extended version of our earlier model that suggests that such time-dependent qualitative changes in oscillation shape might be the result of balanced positive and negative feedback from Ca2+ to the production and degradation of inositol trisphosphate.

Signal encoding performance of astrocyte-dressed Morris Lecar neurons

There are more support cells called glia in the central nervous system than neurons. Although previous studies have shown that one of these glial cells, astrocytes, can communicate with neurons bidirectionally, giving a silent neuron the ability to fire or changing the neuron’s firing frequency, understanding the role of astrocytes in neuroscience is still in its infancy. With this motivation, in this study, the weak signal encoding performance of different types of astrocyte-dressed Morris Lecar neurons in the presence of noise naturally found in biological systems is systematically investigated. The results showed that astrocyte-dressed Morris Lecar neurons had a subthreshold signal frequency suitable for optimal encoding and that Morris Lecar neurons resonated at this frequency. In addition, the rate of inositol trisphosphate production in the astrocyte has been shown to affect signal detection at different noise intensities and coupling strengths. Finally, simulations performed by including the internal noise of the astrocytes originating from calcium ion channels in the model have shown how the signal transmission changes depending on the calcium channel number, noise intensity, and coupling strength. In this context, the presented study also contains important information in terms of examining stochastic astrocyte dynamics that affect the weak signal detection performance of different types of astrocyte-dressed Morris Lecar neurons.

An olive-derived elenolic acid stimulates hormone release from L-cells and exerts potent beneficial metabolic effects in obese diabetic mice.

Insulin resistance and progressive decline in functional β-cell mass are two key factors for developing type 2 diabetes (T2D), which is largely driven by overweight and obesity, a significant obstacle for effective metabolic control in many patients with T2D. Thus, agents that simultaneously ameliorate obesity and act on multiple pathophysiological components could be more effective for treating T2D. Here, we report that elenolic acid (EA), a phytochemical, is such a dual-action agent. we show that EA dose-dependently stimulates GLP-1 secretion in mouse clonal L-cells and isolated mouse ileum crypts. In addition, EA induces L-cells to secrete peptide YY (PYY). EA induces a rapid increase in intracellular [Ca2+]i and the production of inositol trisphosphate in L-cells, indicating that EA activates phospholipase C (PLC)-mediated signaling. Consistently, inhibition of (PLC) or Gαq ablates EA-stimulated increase of [Ca2+]i and GLP-1 secretion. In vivo, a single dose of EA acutely stimulates GLP-1 and PYY secretion in mice, accompanied with an improved glucose tolerance and insulin levels. Oral administration of EA at a dose of 50 mg/kg/day for 2 weeks normalized the fasting blood glucose and restored glucose tolerance in high-fat diet-induced obese (DIO) mice to levels that were comparable to chow-fed mice. In addition, EA suppresses appetite, reduces food intake, promotes weight loss, and reverses perturbated metabolic variables in obese mice. These results suggest that EA could be a dual-action agent as an alternative or adjuvant treatment for both T2D and obesity.

Calcium Signaling in the Photodamaged Skin: In Vivo Experiments and Mathematical Modeling.

The epidermis forms an essential barrier against a variety of insults. The overall goal of this study was to shed light not only on the effects of accidental epidermal injury, but also on the mechanisms that support laser skin resurfacing with intra-epidermal focal laser-induced photodamage, a widespread medical practice used to treat a range of skin conditions. To this end, we selectively photodamaged a single keratinocyte with intense, focused and pulsed laser radiation, triggering Ca2+ waves in the epidermis of live anesthetized mice with ubiquitous expression of a genetically encoded Ca2+ indicator. Waves expanded radially and rapidly, reaching up to eight orders of bystander cells that remained activated for tens of minutes, without displaying oscillations of the cytosolic free Ca2+ concentration (n}{}{[ {{rm{C}}{{rm{a}}^{2 + }}} ]_c}). By combining in vivo pharmacological dissection with mathematical modeling, we demonstrate that Ca2+ wave propagation depended primarily on the release of ATP, a prime damage-associated molecular patterns (DAMPs), from the hit cell. Increments of the n}{}{[ {{rm{C}}{{rm{a}}^{2 + }}} ]_c} in bystander cells were chiefly due to Ca2+ release from the endoplasmic reticulum (ER), downstream of ATP binding to P2Y purinoceptors. ATP-dependent ATP release though connexin hemichannels (HCs) affected wave propagation at larger distances, where the extracellular ATP concentration was reduced by the combined effect of passive diffusion and hydrolysis due to the action of ectonucleotidases, whereas pannexin channels had no role. Bifurcation analysis suggests basal keratinocytes have too few P2Y receptors (P2YRs) and/or phospholipase C (PLC) to transduce elevated extracellular ATP levels into inositol trisphosphate (IP3) production rates sufficiently large to sustain n}{}{[ {{rm{C}}{{rm{a}}^{2 + }}} ]_c} oscillations.

A mathematical model of intricate calcium dynamics and modulation of calcium signalling by mitochondria in pancreatic acinar cells

• The model is divided into two sub domains: the apical cytosolic sub domain, and the mitochondrial sub domain. However, these two sub domains are connected with the Ca2+. • The proposed model is Class II model. It includes the dynamics of IP3, we take into account the Ca2+ modulation of IP3 generation through PLC, and Ca2+ activated IP3 removal by IP3K. • The biphasic regulation of mitochondrial Ca2+ uptake by apical Ca2+ concentration, and the rapid mode of Ca2+ uptake through mitochondria are considered. • In this model the three types of mechanism by Ca2+ have been well considered. Firstly, the time dependent gating of IPR by Ca2+. Secondly, the Ca2+ enhances the production of IP3. Subsequently, due to the 3- kinase stimulated by Ca2+, degradation of IP3. Thirdly, the gating of uniporter by Ca2+: i.e. Ca2+ activated CU uptake, and subsequently it inactivates the uniporter for further up taking Ca2+. Thus, a [Ca2+] concentration is required for the activation and the inactivation of CU. • How these two sub domain: apical sub domain and the mitochondrial sub domain combine together into a well- mixed model. • The bifurcation analysis is performed to identify the oscillatory structures depending on the different agonist strength to produce complex dynamics in the apical region of the cell. The complex bifurcation points like hopf, period doubling, torus, saddle node bifurcation are studied. The pancreatic acinar cells display wide varieties of Ca 2+ oscillations within the cell. The study of these calcium signals/oscillations are of great interest because they are physiologically important. The Ca 2+ release from the endoplasmic reticulum via inositol trisphosphate receptors (IPR) and the ryanodine receptors (RyR) creates a high cytosolic Ca 2+ signal in the acinar cell. The pancreatic acinar cells are highly polarized cells, and the Ca 2+ response is quite extensive in the apical (peri-granular) region than the basal region of the cell. The Ca 2+ signals which originated in the apical region control the exocytosis process and triggered peri-granular mitochondria (Mito) to uptake Ca 2+ . The peri-granular mitochondria play a critical role in confining cytosolic Ca 2+ elevation by acting as a Ca 2+ buffer barrier. They also participate in shaping the Ca 2+ response by restricting the Ca 2+ signals into the granular region of the acinar cells. They sequestered Ca 2+ very rapidly. Then they release Ca 2+ very slowly to provide Ca 2+ supply to cause Ca 2+ oscillations in the apical region. Thus, herein we proposed a Class II compartmental mathematical model which systematically simulates the complex Ca 2+ oscillations in the apical region, and the peri-granular mitochondria matrix of the pancreatic acinar cells. The model assumes the functional coupling between the IPR and the RyR through the mechanism of calcium induced calcium release (CICR). In the proposed compartmental model of Ca 2+ dynamics, the Ca 2+ dependent production and degradations of (1,4,5) inositol-trisphosphate (IP 3 ) along with the apical parameters, apical endoplasmic reticulum (ER), and the peri-granular mitochondria are considered. The bifurcation analysis of the model is performed which explores the intricate dynamic behaviour of the apical region of the acinar cells, and hence derive the complex Ca 2+ oscillations in both the apical region and the peri-granular mitochondria. The model predicts the different patterns of Ca 2+ oscillation from the regular to sinusoidal to raised-baseline to sustained spikes in the apical region of the acinar cells.

Identification and functional characterization of arginine vasopressin receptor 1A : atypical chemokine receptor 3 heteromers in vascular smooth muscle.

Recent observations suggest that atypical chemokine receptor (ACKR)3 and chemokine (C-X-C motif) receptor (CXCR)4 regulate human vascular smooth muscle function through hetero-oligomerization with α1-adrenoceptors. Here, we show that ACKR3 also regulates arginine vasopressin receptor (AVPR)1A. We observed that ACKR3 agonists inhibit arginine vasopressin (aVP)-induced inositol trisphosphate (IP3) production in human vascular smooth muscle cells (hVSMCs) and antagonize aVP-mediated constriction of isolated arteries. Proximity ligation assays, co-immunoprecipitation and bioluminescence resonance energy transfer experiments suggested that recombinant and endogenous ACKR3 and AVPR1A interact on the cell surface. Interference with ACKR3 : AVPR1A heteromerization using siRNA and peptide analogues of transmembrane domains of ACKR3 abolished aVP-induced IP3 production. aVP stimulation resulted in β-arrestin 2 recruitment to AVPR1A and ACKR3. While ACKR3 activation failed to cross-recruit β-arrestin 2 to AVPR1A, the presence of ACKR3 reduced the efficacy of aVP-induced β-arrestin 2 recruitment to AVPR1A. AVPR1A and ACKR3 co-internalized upon agonist stimulation in hVSMC. These data suggest that AVPR1A : ACKR3 heteromers are constitutively expressed in hVSMC, provide insights into molecular events at the heteromeric receptor complex, and offer a mechanistic basis for interactions between the innate immune and vasoactive neurohormonal systems. Our findings suggest that ACKR3 is a regulator of vascular smooth muscle function and a possible drug target in diseases associated with impaired vascular reactivity.

Low-Intensity Ultrasound Modulates Ca2+ Dynamics in Human Mesenchymal Stem Cells via Connexin 43 Hemichannel.

In recent years, ultrasound has gained attention in new biological applications due to its ability to induce specific biological responses at the cellular level. Although the biophysical mechanisms underlying the interaction between ultrasound and cells are not fully understood, many agree on a pivotal role of Ca2+ signaling through mechanotransduction pathways. Because Ca2+ regulates a vast range of downstream cellular processes, a better understanding of how ultrasound influences Ca2+ signaling could lead to new applications for ultrasound. In this study, we investigated the mechanism of ultrasound-induced Ca2+ mobilization in human mesenchymal stem cells using 47MHz focused ultrasound to stimulate single cells at low intensities (~110mW/cm2). We found that ultrasound exposure triggers opening of connexin 43 hemichannels on the plasma membrane, causing release of ATP into the extracellular space. That ATP then binds to G-protein-coupled P2Y1 purinergic receptors on the membrane, in turn activating phospholipase C, which evokes production of inositol trisphosphate and release of Ca2+ from intracellular stores.

Menthol evokes Ca2+ signals and induces oxidative stress independently of the presence of TRPM8 (menthol) receptor in cancer cells

Menthol is a naturally occurring monoterpene alcohol possessing remarkable biological properties including antipruritic, analgesic, antiseptic, anti-inflammatory and cooling effects. Here, we examined the menthol-evoked Ca2+ signals in breast and prostate cancer cell lines. The effect of menthol (50–500µM) was predicted to be mediated by the transient receptor potential ion channel melastatin subtype 8 (TRPM8). However, the intensity of menthol-evoked Ca2+ signals did not correlate with the expression levels of TRPM8 in breast and prostate cancer cells indicating a TRPM8-independent signaling pathway. Menthol-evoked Ca2+ signals were analyzed in detail in Du 145 prostate cancer cells, as well as in CRISPR/Cas9 TRPM8-knockout Du 145 cells. Menthol (500µM) induced Ca2+ oscillations in both cell lines, thus independent of TRPM8, which were however dependent on the production of inositol trisphosphate. Results based on pharmacological tools point to an involvement of the purinergic pathway in menthol-evoked Ca2+ responses. Finally, menthol (50–500µM) decreased cell viability and induced oxidative stress independently of the presence of TRPM8 channels, despite that temperature-evoked TRPM8-mediated inward currents were significantly decreased in TRPM8-knockout Du 145 cells compared to wild type Du 145 cells.

α1-Adrenergic Receptors Function Within Hetero-Oligomeric Complexes With Atypical Chemokine Receptor 3 and Chemokine (C-X-C motif) Receptor 4 in Vascular Smooth Muscle Cells.

BackgroundRecently, we provided evidence that α1‐adrenergic receptors (ARs) in vascular smooth muscle are regulated by chemokine (C‐X‐C motif) receptor (CXCR) 4 and atypical chemokine receptor 3 (ACKR3). While we showed that CXCR4 controls α1‐ARs through formation of heteromeric receptor complexes in human vascular smooth muscle cells (hVSMCs), the molecular basis underlying cross‐talk between ACKR3 and α1‐ARs is unknown.Methods and ResultsWe show that ACKR3 agonists inhibit inositol trisphosphate production in hVSMCs on stimulation with phenylephrine. In proximity ligation assays and co‐immunoprecipitation experiments, we observed that recombinant and endogenous ACKR3 form heteromeric complexes with α1A/B/D‐AR. While small interfering RNA knockdown of ACKR3 in hVSMCs reduced α1B/D‐AR:ACKR3, CXCR4:ACKR3, and α1B/D‐AR:CXCR4 complexes, small interfering RNA knockdown of CXCR4 reduced α1B/D‐AR:ACKR3 heteromers. Phenylephrine‐induced inositol trisphosphate production from hVSMCs was abolished after ACKR3 and CXCR4 small interfering RNA knockdown. Peptide analogs of transmembrane domains 2/4/7 of ACKR3 showed differential effects on heteromerization between ACKR3, α1A/B/D‐AR, and CXCR4. While the transmembrane domain 2 peptide interfered with α1B/D‐AR:ACKR3 and CXCR4:ACKR3 heteromerization, it increased heteromerization between CXCR4 and α1A/B‐AR. The transmembrane domain 2 peptide inhibited ACKR3 but did not affect α1b‐AR in β‐arrestin recruitment assays. Furthermore, the transmembrane domain 2 peptide inhibited phenylephrine‐induced inositol trisphosphate production in hVSMCs and attenuated phenylephrine‐induced constriction of mesenteric arteries.Conclusionsα1‐ARs form hetero‐oligomeric complexes with the ACKR3:CXCR4 heteromer, which is required for α1B/D‐AR function, and activation of ACKR3 negatively regulates α1‐ARs. G protein–coupled receptor hetero‐oligomerization is a dynamic process, which depends on the relative abundance of available receptor partners. Endogenous α1‐ARs function within a network of hetero‐oligomeric receptor complexes.

Targeted calcium influx boosts cytotoxic T lymphocyte function in the tumour microenvironment

Adoptive cell transfer utilizing tumour-targeting cytotoxic T lymphocytes (CTLs) is one of the most effective immunotherapies against haematological malignancies, but significant clinical success has not yet been achieved in solid tumours due in part to the strong immunosuppressive tumour microenvironment. Here, we show that suppression of CTL killing by CD4+CD25+Foxp3+ regulatory T cell (Treg) is in part mediated by TGFβ-induced inhibition of inositol trisphosphate (IP3) production, leading to a decrease in T cell receptor (TCR)-dependent intracellular Ca2+ response. Highly selective optical control of Ca2+ signalling in adoptively transferred CTLs enhances T cell activation and IFN-γ production in vitro, leading to a significant reduction in tumour growth in mice. Altogether, our findings indicate that the targeted optogenetic stimulation of intracellular Ca2+ signal allows for the remote control of cytotoxic effector functions of adoptively transferred T cells with outstanding spatial resolution by boosting T cell immune responses at the tumour sites.

Targeted calcium influx boosts cytotoxic T lymphocyte function in the tumor microenvironment

Abstract Adoptive cell transfer utilizing tumor-targeting cytotoxic T lymphocytes (CTLs) is one of the most effective immunotherapies against hematological malignancies, but significant clinical success has not yet been achieved in solid tumors due in part to the strong immunosuppressive tumor microenvironment. Systemic or intratumoral delivery of an immune boosting molecule to overcome local suppression has been proposed, but the full potential is limited by non-specific stimulation of tumor growth, metastasis, and angiogenesis. Here, we show that suppression of CTL killing by CD4+CD25+Foxp3+ regulatory T cell (Treg) is mainly mediated by TGFβ-induced inhibition of inositol trisphosphate (IP3) production, leading to a decrease in T cell receptor (TCR)-dependent intracellular Ca2+ response. Both in vitro and in vivo assays revealed that highly selective optical control of Ca2+ signaling in adoptively transferred CTLs was sufficient to overcome immunosuppression at the tumor site by enhancing T cell activation, IFN-γ production and antitumor cytotoxicity, leading to a significant reduction in tumor growth in mice. Together, our findings indicate that the targeted optogenetic stimulation of intracellular Ca2+ signal allows for the remote control of cytotoxic effector functions of adoptively transferred T cells with outstanding spatial resolution by boosting T cell immune responses only at the targeted tumor sites.

Functional and structural consequences of chemokine (C-X-C motif) receptor 4 activation with cognate and non-cognate agonists.

Chemokine (C-X-C motif) receptor 4 (CXCR4) regulates cell trafficking and plays important roles in the immune system. Ubiquitin has recently been identified as an endogenous non-cognate agonist of CXCR4, which activates CXCR4 via interaction sites that are distinct from those of the cognate agonist C-X-C motif chemokine ligand 12 (CXCL12). As compared with CXCL12, chemotactic activities of ubiquitin in primary human cells are poorly characterized. Furthermore, evidence for functional selectivity of CXCR4 agonists is lacking, and structural consequences of ubiquitin binding to CXCR4 are unknown. Here, we show that ubiquitin and CXCL12 have comparable chemotactic activities in normal human peripheral blood mononuclear cells, monocytes, vascular smooth muscle, and endothelial cells. Chemotactic activities of the CXCR4 ligands could be inhibited with the selective CXCR4 antagonist AMD3100 and with a peptide analogue of the second transmembrane domain of CXCR4. In human monocytes, ubiquitin- and CXCL12-induced chemotaxis could be inhibited with pertussis toxin and with inhibitors of phospholipase C, phosphatidylinositol 3 kinase, and extracellular signal-regulated kinase 1/2. Both agonists induced inositol trisphosphate production in vascular smooth muscle cells, which could be inhibited with AMD3100. In β-arrestin recruitment assays, ubiquitin did not sufficiently recruit β-arrestin2 to CXCR4 (EC50>10μM), whereas the EC50 for CXCL12 was 4.6nM (95% confidence interval 3.1-6.1nM). Both agonists induced similar chemical shift changes in the 13C-1H-heteronuclear single quantum correlation (HSQC) spectrum of CXCR4 in membranes, whereas CXCL11 did not significantly alter the 13C-1H-HSQC spectrum of CXCR4. Our findings point towards ubiquitin as a biased agonist of CXCR4.

3D time-varying simulations of Ca2+ dynamics in arterial coupled cells: A massively parallel implementation.

SummaryPreferential locations of atherosclerotic plaque are strongly associated with the areas of low wall shear stress and disturbed haemodynamic characteristics such as flow detachment, flow recirculation and oscillatory flow. The areas of low wall shear stress are also associated with the reduced production of adenosine triphosphate in the endothelial layer, as well as the resulting reduced production of inositol trisphosphate (IP3). The subsequent variation in Ca2+ signalling and nitric oxide synthesis could lead to the impairment of the atheroprotective function played by nitric oxide. In previous studies, it has been suggested that the reduced IP3 and Ca2+ signalling can explain the correlation of atherosclerosis with induced low WSS and disturbed flow characteristics. The massively parallel implementation described in this article provides insight into the dynamics of coupled smooth muscle cells and endothelial cells mapped onto the surface of an idealised arterial bifurcation. We show that variations in coupling parameters, which model normal and pathological conditions, provide vastly different smooth muscle cell Ca2+ dynamics and wave propagation profiles. The extensibility of the coupled cells model and scalability of the implementation provide a solid framework for in silico investigations of the interaction between complex cellular chemistry and the macro‐scale processes determined by fluid dynamics. © 2016 The Authors. International Journal for Numerical Methods in Biomedical Engineering published by John Wiley & Sons Ltd.

Osmoregulatory inositol transporter SMIT1 modulates electrical activity by adjusting PI(4,5)P2 levels

Myo-inositol is an important cellular osmolyte in autoregulation of cell volume and fluid balance, particularly for mammalian brain and kidney cells. We find it also regulates excitability. Myo-inositol is the precursor of phosphoinositides, key signaling lipids including phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. However, whether myo-inositol accumulation during osmoregulation affects signaling and excitability has not been fully explored. We found that overexpression of the Na(+)/myo-inositol cotransporter (SMIT1) and myo-inositol supplementation enlarged intracellular PI(4,5)P2 pools, modulated several PI(4,5)P2-dependent ion channels including KCNQ2/3 channels, and attenuated the action potential firing of superior cervical ganglion neurons. Further experiments using the rapamycin-recruitable phosphatase Sac1 to hydrolyze PI(4)P and the P4M probe to visualize PI(4)P suggested that PI(4)P levels increased after myo-inositol supplementation with SMIT1 expression. Elevated relative levels of PIP and PIP2 were directly confirmed using mass spectrometry. Inositol trisphosphate production and release of calcium from intracellular stores also were augmented after myo-inositol supplementation. Finally, we found that treatment with a hypertonic solution mimicked the effect we observed with SMIT1 overexpression, whereas silencing tonicity-responsive enhancer binding protein prevented these effects. These results show that ion channel function and cellular excitability are under regulation by several "physiological" manipulations that alter the PI(4,5)P2 setpoint. We demonstrate a previously unrecognized linkage between extracellular osmotic changes and the electrical properties of excitable cells.

A Computational Model of the Dendron of the GnRH Neuron.

Gonadotropin-releasing hormone (GnRH) neurons have two major processes that have properties of both dendrites (they receive synaptic input from other neurons) and axons (they actively propagate action potentials to the synaptic terminal). These processes have thus been termed dendrons. We construct a stochastic spatiotemporal model of the dendron of the GnRH neuron, with the goal of studying how stochastic synaptic input along the length of the dendron affects the initiation and propagation of action potentials. We show (1) that synaptic inputs closer to the soma are effective controllers of action potential initiation and electrical bursting and (2) that although the effects on the amplitude and width of propagating action potentials are critically dependent on the timing and location of synaptic input addition, the effects remain small. We conclude that although stochastic synaptic input along the length of the dendron is likely to be a major determinant of action potential initiation, it is an unlikely mechanism for controlling whether or not action potentials reach the synaptic terminal. Thus, the role of synaptic inputs situated along the dendron a long way from the site of action potential initiation remains unclear. We also show that the actions of kisspeptin can result in significant modulation of the amount of calcium released by an action potential at the synaptic terminal. Furthermore, we show that the actions of kisspeptin are greatest when multiple effects operate together and that a kisspeptin-induced increase in firing rate is, by itself, less effective at increasing Ca2+ release than is a combination of an increased firing rate, an increase in Ca2+ influx, and an increase in inositol trisphosphate (IP3) production. We conclude that the inherent synergies in the various actions of kisspeptin make it a likely candidate for the precise control of Ca2+ transients at the synaptic terminal.

Modelling mechanism of calcium oscillations in pancreatic acinar cells.

We present a simple model for calcium oscillations in the pancreatic acinar cells. This model is based on the calcium release from two receptors, inositol trisphosphate receptors (IPR) and ryanodine receptors (RyR) through the process of calcium induced calcium release (CICR). In pancreatic acinar cells, when the Ca²⁺ concentration increases, the mitochondria uptake it very fast to restrict Ca(2+) response in the cell. Afterwards, a much slower release of Ca²⁺ from the mitochondria serves as a calcium supply in the cytosol which causes calcium oscillations. In this paper we discuss a possible mechanism for calcium oscillations based on the interplay among the three calcium stores in the cell: the endoplasmic reticulum (ER), mitochondria and cytosol. Our model predicts that calcium shuttling between ER and mitochondria is a pacemaker role in the generation of Ca²⁺ oscillations. We also consider the calcium dependent production and degradation of (1,4,5) inositol-trisphosphate (IP3), which is a key source of intracellular calcium oscillations in pancreatic acinar cells. In this study we are able to predict the different patterns of calcium oscillations in the cell from sinusoidal to raised-baseline, high frequency and low-frequency baseline spiking.

Divergent signaling mechanisms for venous versus arterial contraction as revealed by endothelin-1

Venous function is underappreciated in its role in blood pressure determination, a physiologic parameter normally ascribed to changes in arterial function. Significant evidence points to the hormone endothelin-1 (ET-1) as being important to venous contributions to blood pressure. We hypothesized that the artery and vein should similarly depend on the signaling pathways stimulated by ET-1, specifically phospholipase C (PLC) activation. This produces two functional arms of signaling: diacylglycerol (DAG; protein kinase C [PKC] activation) and inositol trisphosphate (IP3) production (intracellular calcium release). The model was the male Sprague-Dawley rat. Isolated tissue baths were used to measure isometric contraction. Western blot and immunocytochemical analyses measured the magnitude of expression and site of expression, respectively, of IP3 receptors in smooth muscle/tissue. Pharmacologic methods were used to modify PLC activity and signaling elements downstream of PLC (IP3 receptors, PKC). ET-1-induced contraction was PLC dependent in both tissues as the PLC inhibitor U-73122 significantly reduced contraction in aorta (86% ± 4% of control; P < .05) and vena cava (49% ± 11% of control; P < .05). However, ET-1-induced contraction was not significantly inhibited by the IP3 receptor inhibitor 2-aminoethoxydiphenylborane (100 μM) in vena cava (82% ± 8% of control; P = .23) but was in the aorta (55% ± 4% of control; P < .05). All three IP3 receptor isoforms were located in venous smooth muscle. IP3 receptors were functional in both tissues as the novel membrane-permeable IP3 analogue (Bt-IP3; 10 μM) contracted aorta and vena cava. Similarly, whereas the PKC inhibitor chelerythrine (10 μM) attenuated ET-1-induced contraction in vena cava and aorta (5% ± 2% and 50% ± 5% of control, respectively; P < .05), only the vena cava contracted to the DAG analogue 1-oleoyl-2-acetyl-sn-glycerol. These findings suggest that ET-1 activates PLC in aorta and vena cava, but vena cava contraction to ET-1 may be largely IP3 independent. Rather, DAG—not IP3—may contribute to contraction to ET-1 in vena cava, in part by activation of PKC. These studies outline a fundamental difference between venous and arterial smooth muscle and further reinforce a heterogeneity of vascular smooth muscle function that could be taken advantage of for therapeutic development.

Selective inhibition of angiotensin receptor signaling through Erk1/2 pathway by a novel peptide

A seven-amino acid peptide (PEP7) is encoded within a short open reading frame within exon 2 (E2) in the 5'-leader sequence (5'LS) upstream of the rat ANG 1a-receptor (rAT1aR) mRNA. A chemically synthesized PEP7 markedly inhibited ANG II-induced Erk1/2 activation in cell culture by 62% compared with a scrambled PEP7 (sPEP7) [pErk1/2/Erk1/2 (AU): ANG II, 1.000 ± 0.0, ANG II+PEP7, 0.3812 ± 0.086, ANG II+sPEP7, 1.069 ± 0.18; n = 3]. Under these same conditions, PEP7 had no effect on ANG II-stimulated inositol-trisphosphate production. PEP7 also had no effect on epidermal growth factor- and phorbol methyl ester-induced Erk1/2 activation, suggesting PEP7 selectively inhibits AT1aR-mediated Erk1/2 signaling. PEP7 intracerebroventricularly inhibited ANG II-induced saline intake but had no effect on water intake in male and female rats, indicating PEP7 also selectively inhibits the ANG II-Erk1/2 pathway in vivo since saline drinking is Erk1/2-mediated, while water drinking is not. PEP7 inhibition of ANG II-induced saline ingestion was rapidly reversed by a subsequent intracerebroventricular injection of an oxytocin antagonist, suggesting when PEP7 blocks ANG II-stimulated Erk1/2 activation, animals no longer ingest saline to balance the continued water intake, due to the release of oxytocin and its subsequent inhibitory effects on saline drinking. PEP7 also attenuated ANG II-induced increases in arterial pressure by 35% compared with sPEP7 at the same dose. Thus, we have identified a novel peptide encoded within the rAT1aR E2 that selectively inhibits Erk1/2 activation, resulting in physiological consequences for sodium ingestion and arterial pressure that may have implications for treating sodium-sensitive diseases like hypertension and chronic kidney disease.

Cytotoxicity of Intracellular Aβ42Amyloid Oligomers Involves Ca2+Release from the Endoplasmic Reticulum by Stimulated Production of Inositol Trisphosphate

Oligomeric forms of β-amyloid (Aβ(42)) peptides associated with Alzheimer's disease (AD) disrupt cellular Ca(2+) regulation by liberating Ca(2+) into the cytosol from both extracellular and intracellular sources. We elucidated the actions of intracellular Aβ by imaging Ca(2+) responses to injections of Aβ oligomers into Xenopus oocytes. Two types of signal were observed: (1) local, "channel-like" transients dependent on extracellular Ca(2+) influx, which resembled signals from amlyoid pores formed by extracellular application of oligomers; and (2) local transients and global Ca(2+) waves, resembling Ca(2+) puffs and waves mediated by inositol trisphosphate (IP(3)). The latter responses were suppressed by antagonists of the IP(3) receptor (caffeine and heparin), pretreatment with the G(i)(o)-protein inhibitor pertussis toxin, and pretreatment with lithium to deplete membrane inositol lipids. We show that G-protein-mediated stimulation of IP(3) production and consequent liberation of Ca(2+) from the endoplasmic reticulum by intracellular Aβ oligomers is cytotoxic, potentially representing a novel pathological mechanism in AD which may be further exacerbated by AD-linked mutations in presenilins to promote opening of IP(3) receptor/channels.

Dual Effect of Phosphatidyl (4,5)-Bisphosphate PIP2 on Shaker K+ Channels

Dual Effect of Phosphatidyl (4,5)-Bisphosphate PIP2 on Shaker K+ Channels