IP3 Receptor Expression Research Articles

IP3-receptor expression and co-localization with HCN4 in sinoatrial myocytes

IP3-receptor expression and co-localization with HCN4 in sinoatrial myocytes

A Dominant-Negative Mutant of ANXA7 Impairs Calcium Signaling and Enhances the Proliferation of Prostate Cancer Cells by Downregulating the IP3 Receptor and the PI3K/mTOR Pathway.

Annexin A7/ANXA7 is a calcium-dependent membrane fusion protein with tumor suppressor gene (TSG) properties, which is located on chromosome 10q21 and is thought to function in the regulation of calcium homeostasis and tumorigenesis. However, whether the molecular mechanisms for tumor suppression are also involved in the calcium- and phospholipid-binding properties of ANXA7 remain to be elucidated. We hypothesized that the 4 C-terminal endonexin-fold repeats in ANXA7 (GX(X)GT), which are contained within each of the 4 annexin repeats with 70 amino acids, are responsible for both calcium- and GTP-dependent membrane fusion and the tumor suppressor function. Here, we identified a dominant-negative triple mutant (DNTM/DN-ANXA7J) that dramatically suppressed the ability of ANXA7 to fuse with artificial membranes while also inhibiting tumor cell proliferation and sensitizing cells to cell death. We also found that the [DNTM]ANA7 mutation altered the membrane fusion rate and the ability to bind calcium and phospholipids. In addition, in prostate cancer cells, our data revealed that variations in phosphatidylserine exposure, membrane permeabilization, and cellular apoptosis were associated with differential IP3 receptor expression and PI3K/AKT/mTOR modulation. In conclusion, we discovered a triple mutant of ANXA7, associated with calcium and phospholipid binding, which leads to the loss of several essential functions of ANXA7 pertinent to tumor protection and highlights the importance of the calcium signaling and membrane fusion functions of ANXA7 for preventing tumorigenesis.

Modeling the spatiotemporal properties of crosstalk between RYR and IP3R-mediated Ca2+release in failing cardiomyocytes.

Ryanodine receptors (RyR) and IP3 receptors (IP3R) are Ca2+ channels expressed on the endoplasmic/sarcoplasmic reticulum (ER/SR) membrane in various cell types. Both the spatial localization and the distinct gating properties of these channels contribute to the diverse cellular functions controlled by intracellular Ca2+ signaling. It is known that both RyRs and IP3Rs are expressed on the SR membrane of ventricular cardiomyocytes and that the expression of IP3Rs on the SR at dyadic junctions is increased in cardiac diseases such as hypertrophy and heart failure (HF), and evidence that Ca2+ release through IP3Rs can influence RyR-mediated Ca2+ release has been described. However, despite the suggested functional role for crosstalk between RyRs and IP3Rs, especially under pathological conditions, most previous mathematical models of cardiomyocyte Ca2+ signaling have accounted for only RyRs in isolation. Here, we propose a mathematical model of intracellular Ca2+ signaling that incorporates both RyRs and IP3Rs and can be used to develop quantitative predictions about crosstalk between the two channels. This model considers the spatial arrangement of RyRs and IP3Rs relative to one another based on published immunohistochemistry co-localization studies and simulates the stochastic opening and closing of individual receptors based on previously published models of Ca2+ sparks and puffs. In this model, RyR gating depends on local cytosolic [Ca2+], JSR [Ca2+], and allosteric coupling to neighboring RyRs while IP3R gating depends primarily on local cytosolic [Ca2+] and [IP3]. Based on preliminary results, we hypothesize that the subcellular spatial remodeling and increased expression of IP3Rs that occur in hypertrophy and HF will promote slowed, uncoordinated diastolic Ca2+ sparks, which may contribute to increased risk of arrhythmogenic Ca2+ wave formation and incidence of ventricular arrhythmias observed clinically in patients with hypertrophy and HF.

Intestinal enteroendocrine cells rely on ryanodine and IP3 calcium store receptors for mechanotransduction.

Enteroendocrine cells (EECs) are specialized sensors of luminal forces and chemicals in the gastrointestinal (GI) epithelium that respond to stimulation with a release of signalling molecules such as serotonin (5-HT). For mechanosensitive EECs, force activates Piezo2 channels, which generate a very rapidly activating and inactivating (∼10ms) cationic (Na+ , K+ , Ca2+ ) receptor current. Piezo2 receptor currents lead to a large and persistent increase in intracellular calcium (Ca2+ ) that lasts many seconds to sometimes minutes, suggesting signal amplification. However, intracellular calcium dynamics in EEC mechanotransduction remain poorly understood. The aim of this study was to determine the role of Ca2+ stores in EEC mechanotransduction. Mechanical stimulation of a human EEC cell model (QGP-1) resulted in a rapid increase in cytoplasmic Ca2+ and a slower decrease in ER stores Ca2+ , suggesting the involvement of intracellular Ca2+ stores. Comparing murine primary colonic EECs with colonocytes showed expression of intercellular Ca2+ store receptors, a similar expression of IP3 receptors, but a >30-fold enriched expression of Ryr3 in EECs. In mechanically stimulated primary EECs, Ca2+ responses decreased dramatically by emptying stores and pharmacologically blocking IP3 and RyR1/3 receptors. RyR3 genetic knockdown by siRNA led to a significant decrease in mechanosensitive Ca2+ responses and 5-HT release. In tissue, pressure-induced increase in the Ussing short circuit current was significantly decreased by ryanodine receptor blockade. Our data show that mechanosensitive EECs use intracellular Ca2+ stores to amplify mechanically induced Ca2+ entry, with RyR3 receptors selectively expressed in EECs and involved in Ca2+ signalling, 5-HT release and epithelial secretion. KEY POINTS: A population of enteroendocrine cells (EECs) are specialized mechanosensors of the gastrointestinal (GI) epithelium that respond to mechanical stimulation with the release of important signalling molecules such as serotonin. Mechanical activation of these EECs leads to an increase in intracellular calcium (Ca2+ ) with a longer duration than the stimulus, suggesting intracellular Ca2+ signal amplification. In this study, we profiled the expression of intracellular Ca2+ store receptors and found an enriched expression of the intracellular Ca2+ receptor Ryr3, which contributed to the mechanically evoked increases in intracellular calcium, 5-HT release and epithelial secretion. Our data suggest that mechanosensitive EECsrely on intracellular Ca2+ stores and are selective in their use of Ryr3 for amplification of intracellular Ca2+ . This work advances our understanding of EEC mechanotransduction and may provide novel diagnostic and therapeutic targets for GI motility disorders.

In search of pulmonary hypertension treatments: Effect of 17β-estradiol on PGI2 pathway in human pulmonary artery

IntroductionProstacyclin (PGI2) is synthetized by PGI2 synthase (PGIS) and induces vasorelaxation via activation of cyclic AMP (cAMP) generating IP-receptor. Several components of the PGI2 signaling pathway are reduced in patients with pulmonary hypertension (PH). AimTo study the effect of 17β-estradiol (E2) on the PGI2 signaling pathway in human pulmonary arteries (HPA) and in their smooth muscle cells (hPASMC) derived from Group-3 PH and non-PH patients. MethodsFollowing E2-treatments of isolated HPA and cultured hPASMC, we measured: 6-keto-Prostaglandin F1α (PGI2 stable metabolite) by ELISA, PGIS and IP protein levels by Western blot and HPA vasorelaxations with an organ bath system. ResultsIncubation with E2 (24/48 h, doses ≥ 10 nM) significantly increased the expression of PGIS in hPASMC derived from both PH (65–98%) and non-PH (21–33%) patients, whereas incubation with E2 (2 h, 0.1 and 1 µM) increased 6-keto-PGF1α production in HPA from Group-3 PH patients only, and did not affect 6-keto-PGF1α production in hPASMC from either non-PH or Group-3 PH patients. Increases in IP receptor expression were observed following 10 mM E2-treatment of hPASMC from non-PH (33% after 48 h) and Group-3 PH (23% after 24 h) patient lungs. Finally, preincubation with 100 nM E2 significantly increased arachidonic acid-induced vasorelaxation of HPA from non-PH patient lungs but not of HPA from Group-3 PH patient lungs. ConclusionE2-treatment may help to restore the PGI2-pathway in Group-3 PH.

Modeling the Crosstalk between Ryanodine and IP3 Receptors in Cardiac Myocytes

Ryanodine receptors (RYR) and IP3 receptors (IP3R) are calcium channels expressed on the endoplasmic/sarcoplasmic reticulum (ER/SR) membrane in various cell types. Both the spatial localization and the distinct gating properties of these channels contribute to the diverse cellular functions controlled by intracellular calcium signaling. It is known that both RYRs and IP3Rs are expressed on the SR membrane of cardiac myocytes and that the expression of IP3Rs on the SR at dyadic junctions is increased in cardiac disease, and evidence that calcium release through IP3Rs can influence RyR-mediate calcium release has been described. However, despite the suggested functional role for crosstalk between RYRs and IP3Rs, especially in cardiac disease, most previous mathematical models of myocyte calcium signaling have accounted for only RYRs in isolation. Here, we propose a mathematical model of intracellular calcium signaling that incorporates both RYRs and IP3Rs and can be used to develop quantitative predictions of crosstalk between the two channels. This model considers the spatial arrangement of RYRs and IP3Rs relative to one another based on published co-localization studies using immunohistochemistry and simulates the stochastic opening and closing of individual receptors based on previously published models of calcium sparks and puffs. In these models, gating of RYRs depends on the local cytosolic and JSR calcium concentration as well as the number of neighboring RYRs that are open while IP3R gating depends primarily on local cytosolic calcium and IP3 concentrations. Based on preliminary results, we hypothesize that the subcellular spatial remodeling and increased expression of IP3Rs that occurs in cardiac disease will increase the probability of diastolic calcium spark events, which may contribute to the increased risk of arrhythmia in those with cardiac diseases such as hypertrophy and heart failure.

Changes in IP3 Receptor Expression and Function in Aortic Smooth Muscle of Atherosclerotic Mice

Peroxynitrite is an endothelium-independent vasodilator that induces relaxation via membrane hyperpolarization. The activation of IP3 receptors triggers the opening of potassium channels and hyperpolarization. Previously we found that relaxation to peroxynitrite was maintained during the development of atherosclerosis due to changes in the expression of calcium-regulatory proteins. In this study we investigated: (1) the mechanism of peroxynitrite-induced relaxation in the mouse aorta, (2) the effect of atherosclerosis on relaxation to peroxynitrite and other vasodilators, and (3) the effect of atherosclerosis on the expression and function of the IP3 receptor. Aortic function was studied using wire myography, and atherosclerosis was induced by fat-feeding ApoE^-/- mice. The expression of IP3 receptors was studied using Western blotting and immunohistochemistry. Relaxation to peroxynitrite was attenuated by the IP3 antagonists 2-APB and xestospongin C and also the K_v channel blocker 4-aminopyridine (4-AP). Atherosclerosis attenuated vasodilation to cromakalim and the AMPK activator A769662 but not peroxynitrite. Relaxation was attenuated to a greater extent by 2-APB in atherosclerotic aortae despite the reduced expression of IP3 receptors. 4-AP was less effective in ApoE^-/- mice fat-fed for 4 months. Peroxynitrite relaxation involves an IP3-induced calcium release and K_V channel activation. This mechanism becomes less important as atherosclerosis develops, and relaxation to peroxynitrite may be maintained by increased calcium extrusion.

IP3 Receptor Determines Increased Basal Mitochondrial Calcium And Autophagy Regulation In Mdx Skeletal Muscle

Several reports describe that IP3 receptor (IP3R) is essential for efficient mitochondrial respiration and maintenance of cellular bioenergetics. This is due to the participation of this receptor in calcium transfer from the ER to the mitochondria. Changes in IP3R function can compromise mitochondrial function, changing ATP production and finally altering autophagy. Nevertheless until now there is no detailed study of the IP3R/calcium/autophagy axis in DMD. Our aim was to investigate the participation of IP3R in the regulation of mitochondrial calcium and autophagy in fibers from a DMD animal model, mdx. We analyzed protein levels of LC3, p62, atg5, beclin and bcl‐2 in FDB muscle of mdx (5–7 weeks), electroporated with a short‐hairpin for IP3R. We also evaluated the calcium transfer with mitochondria‐targeted probes that can be electroporated and also mitochondrial potential with TMRE in skeletal muscle fibers. The basal levels of expression of LC3II are diminished in mdx fibers compared with controls. This result correlates with an increased expression of p62 in mdx fibers. We also found differences in other autophagy proteins like atg5, beclin1 and Bcl‐2. When we performed the knockdown for IP3R we observed differences in the expression of almost all of the proteins analyzed. The expression of LC3II was increased in the electroporated mdx fibers with a decrease in p62 expression. We also confirmed that basal mitochondrial calcium level was increased in mdx fibers. Moreover, when we decreased the expression of IP3R, the basal mitochondrial calcium levels returned to normal. Similar result was obtained when we measured the mitochondrial potential in mdx fibers. Collectively, these results suggest that IP3R regulates mitochondrial calcium flux in adult skeletal muscle and is responsible for the increased mitochondrial calcium in mdx fibers. Moreover, knockdown of IP3R can regulate the expression levels of proteins associated with autophagy. Finally, we propose that some inhibitors of the IP3 pathway can be used as a new treatment for the muscle weakness observed in DMD patientsSupport or Funding InformationFONDECYT 3140491(DV), 3140532(FW), 1151293(EJ), ACT‐111 (EJ, SL), FONDAP 15130011(SL)

Computational Analysis of Heart-Failure-Related Remodeling of Cytosolic and Nuclear Calcium Handling in the Canine Atrial Cardiomyocyte

Background: Heart failure (HF) results in extensive remodeling of ventricular cytosolic and nucleoplasmic calcium handling (CaCyt / CaNucl) and produces atrial calcium-handling dysregulation, promoting atrial fibrillation. The molecular mechanisms underlying arrhythmogenic HF-related atrial calcium-handling abnormalities, notably for atrial CaNucl, are largely unknown. Computational modeling has emerged as a tool to study arrhythmogenic mechanisms, but no currently available atrial model can simulate CaNucl, which was addressed here.Methods: CaCyt and CaNucl were measured using confocal microscopy in atrial cardiomyocytes from control or ventricular-tachypacing-induced HF dogs loaded with Fluo4 (1 Hz stimulation). We extended the Ramirez et al. 2000 canine atrial cardiomyocyte model with a nuclear compartment with nuclear-pore channel-mediated calcium diffusion from the cytosol, perinuclear calcium-store (periCs) linked to the sarcoplasmic reticulum (SR), and IP3-receptor-mediated calcium release from periCs to nucleus. IP3-receptor expression in control and HF dogs was evaluated using Western blot.Results: Nucleoplasmic calcium fluorescence ratios exceeded cytosolic levels in experiments (+28% diastolic, +9% systolic) and model (+32% diastolic, +9% systolic). HF significantly increased nuclear and cytosolic fluorescence, and IP3-receptor expression (+20%; P<0.05). We simulated the relative contribution of increased IP3-receptor expression and HF-related atrial CaCyt remodeling (−60% ryanodine-receptor expression, increased SR calcium-uptake; Yeh et al. 2008) to CaNucl remodeling. Increased IP3-receptor expression slightly augmented CaNucl (+5% diastolic, +2% systolic), without affecting CaCyt, whereas CaCyt remodeling strongly increased CaNucl (+28% diastolic, +41% systolic) in the model. The combined effect (IP3-receptor+Cacyt remodeling) was overadditive (+38% diastolic, +46% systolic).Conclusion: We developed the first computational model to simulate atrial CaNucl. HF-related CaCyt and CaNucl remodeling synergistically modulate nuclear calcium transients. The model provides new insights into atrial CaNucl determinants and their regulation in HF, and may help uncover the mechanisms of proarrhythmic atrial gene-expression changes.

Vitamin D3 supplementation increases insulin level by regulating altered IP3 and AMPA receptor expression in the pancreatic islets of streptozotocin-induced diabetic rat

Pancreatic islets, particularly insulin-secreting β cells, share common characteristics with neurons. Glutamate is one of the major excitatory neurotransmitter in the brain and pancreas, and its action is mediated through glutamate receptors. In the present work, we analysed the role of vitamin D3 in the modulation of AMPA receptor subunit and their functional role in insulin release. Radio receptor binding study in diabetic rats showed a significant increase in AMPA receptor density. Insulin AMPA colabelling study showed an altered AMPA GluR2 and GluR4 subunit expression in the pancreatic beta cells. We also found lowered IP3 content and decreased IP3 receptor in pancreas of diabetic rats. The alterations in AMPA and IP3 receptor resulted in reduced cytosolic calcium level concentration, which further blocks Ca2+-mediated insulin release. Vitamin D3 supplementation restored the alteration in vitamin D receptor expression, AMPA receptor density and AMPA and IP3 receptor expression in the pancreatic islets that helps to restore the calcium-mediated insulin secretion. Our study reveals the antidiabetic property of vitamin D3 that is suggested to have therapeutic role through regulating glutamatergic function in diabetic rats.

Antipurinergic therapy corrects the autism-like features in the Fragile X (Fmr1 knockout) mouse model.

BackgroundThis study was designed to test a new approach to drug treatment of autism spectrum disorders (ASDs) in the Fragile X (Fmr1) knockout mouse model.MethodsWe used behavioral analysis, mass spectrometry, metabolomics, electron microscopy, and western analysis to test the hypothesis that the disturbances in social behavior, novelty preference, metabolism, and synapse structure are treatable with antipurinergic therapy (APT).ResultsWeekly treatment with the purinergic antagonist suramin (20 mg/kg intraperitoneally), started at 9 weeks of age, restored normal social behavior, and improved metabolism, and brain synaptosomal structure. Abnormalities in synaptosomal glutamate, endocannabinoid, purinergic, and IP3 receptor expression, complement C1q, TDP43, and amyloid β precursor protein (APP) were corrected. Comprehensive metabolomic analysis identified 20 biochemical pathways associated with symptom improvements. Seventeen pathways were shared with human ASD, and 11 were shared with the maternal immune activation (MIA) model of ASD. These metabolic pathways were previously identified as functionally related mediators of the evolutionarily conserved cell danger response (CDR).ConclusionsThe data show that antipurinergic therapy improves the multisystem, ASD-like features of both the environmental MIA, and the genetic Fragile X models. These abnormalities appeared to be traceable to mitochondria and regulated by purinergic signaling.Electronic supplementary materialThe online version of this article (doi:10.1186/2040-2392-6-1) contains supplementary material, which is available to authorized users.

Functional Inositol 1,4,5-Trisphosphate Receptors Assembled from Concatenated Homo- and Heteromeric Subunits

Vertebrate genomes code for three subtypes of inositol 1,4,5-trisphosphate (IP3) receptors (IP3R1, -2, and -3). Individual IP3R monomers are assembled to form homo- and heterotetrameric channels that mediate Ca(2+) release from intracellular stores. IP3R subtypes are regulated differentially by IP3, Ca(2+), ATP, and various other cellular factors and events. IP3R subtypes are seldom expressed in isolation in individual cell types, and cells often express different complements of IP3R subtypes. When multiple subtypes of IP3R are co-expressed, the subunit composition of channels cannot be specifically defined. Thus, how the subunit composition of heterotetrameric IP3R channels contributes to shaping the spatio-temporal properties of IP3-mediated Ca(2+) signals has been difficult to evaluate. To address this question, we created concatenated IP3R linked by short flexible linkers. Dimeric constructs were expressed in DT40-3KO cells, an IP3R null cell line. The dimeric proteins were localized to membranes, ran as intact dimeric proteins on SDS-PAGE, and migrated as an ∼1100-kDa band on blue native gels exactly as wild type IP3R. Importantly, IP3R channels formed from concatenated dimers were fully functional as indicated by agonist-induced Ca(2+) release. Using single channel "on-nucleus" patch clamp, the channels assembled from homodimers were essentially indistinguishable from those formed by the wild type receptor. However, the activity of channels formed from concatenated IP3R1 and IP3R2 heterodimers was dominated by IP3R2 in terms of the characteristics of regulation by ATP. These studies provide the first insight into the regulation of heterotetrameric IP3R of defined composition. Importantly, the results indicate that the properties of these channels are not simply a blend of those of the constituent IP3R monomers.

Regulation of type 1 IP3 receptor expression by dopamine D2-like receptors via AP-1 and NFATc4 activation

Type 1 inositol 1,4,5-trisphosphate receptors (IP3Rs-1), together with ryanodine receptors, are major calcium channels to regulate intracellular Ca2+ concentration. Although our recent report demonstrates the essential involvement of IP3R-1 up-regulation induced by dopamine D1-like and D2-like receptor (D1 and D2R) stimulation in psychological dependence, exact regulatory mechanisms of IP3R-1 expression by D2Rs have not yet been clarified. Mouse cerebral cortical neurons were treated with inhibitor of Ca2+-related signal transduction pathways coupling to D2Rs and used to analyze the mechanisms of IP3R-1 expression regulated by transcriptional factor. A selective D2R agonist, quinpirole, up-regulated IP3R-1 protein following its mRNA increase, which was significantly inhibited by gallein (a Gβγ modulator), U73122 (a phospholipase C inhibitor), BAPTA-AM (an intracellular calcium chelating reagent), W7 (a calmodulin inhibitor), KN-93 (a calmodulin-dependent protein kinases inhibitor), and FK506 (a calcineurin inhibitor). Immunocytochemical assessment showed that quinpirole increased expression of both cFos and phosphorylated-cJun in nucleus and enhanced translocation of NFATc4 complex to nucleus from cytoplasm. In addition, quinpirole directly recruited bindings between AP-1 and IP3R-1 promoter region and between NFATc4 and IP3R-1 promoter region. These results indicate that D2Rs enhance IP3R-1 gene transcription via increased bindings of AP-1 and NFATc4 to IP3R-1 promoter region after Gβγ activation.

IP3 receptors, stress and apoptosis

Inositol 1,4,5-trisphosphate (IP3) receptors are intracellular calcium channels that are able to release calcium from intracellular stores upon activation by IP3 and modulation by calcium. IP3 receptors are involved in variety of processes during physiological, but also in the pathophysiological states. Unraveling their regulation and function, especially under the pathological situations can result in a development of new therapeutic strategies based on the IP3 receptor´s activation and/or blocking. To the stimuli that can modulate IP3 receptors belong several stress factors (e.g. immobilization stress, oxidative stress and hypoxia) and also apoptosis. Depending on the length and strength of the stress stimulus, expression of IP3 receptors can be increased, or decreased. Therefore, in this minireview modulation of IP3 receptors by some stressors is discussed. Since it was already shown that strong hypoxia might lead to the apoptosis induction, special focus will be given to the hypoxic stress and induction of apoptosis.

Endothelium-Dependent Contractions in Hypertension

Most isolated arteries respond to shear stress and several vasodilator substances, as demonstrated first for acetylcholine,1 by releasing endothelium-derived relaxing factor (or nitric oxide [NO]), and various endothelium-derived hyperpolarizing signals.2,–,5 However, in certain blood vessels, when exposed to stretch, agonists such as thrombin, acetylcholine, and adenosine nucleotides (adenosine diphosphate [ADP] and adenosine triphosphate [ATP]) or calcium ionophores, the endothelium produces diffusible cyclooxygenase (COX)-derived vasoconstrictor prostanoids (endothelium-derived contracting factors [EDCF])6,–,11 Endothelial cells also produce vasoconstrictor peptides, in particular, endothelin-1.12 The attribution of a role to endothelin-1 in instantaneous changes in vascular tone has been made difficult by the almost insurmountable nature of the vasoconstriction caused by the peptide that can only be tempered by NO or calcitonin gene-related peptide.13,14 Likewise, the evidence linking acute release of endothelin-1 to constriction of arteries is still limited.15 Therefore, the present article focuses on the mechanisms leading to the production of endothelial COX-derived vasoconstrictors, in particular, in the rat aorta, which has been the standard preparation used by the author and his collaborators for the study of EDCF-mediated responses However, the occurrence of such EDCF-mediated responses can vary widely, depending on the species and the blood vessel studied. For example, they are prominent in canine veins but not arteries.6 In the mouse, endothelium-dependent contractions are more pronounced in the carotid artery than in the aorta.16,17 Further, in any given blood vessel, the production of endothelium-derived vasoconstrictor prostanoids is exacerbated by aging and disease, in particular, hypertension.10,11,18,19 ### The Trigger: Calcium Acetylcholine (which activates endothelial M3-muscarinic receptors)20 and ADP and ATP (which activate endothelial purinoceptors)21,22 evoke endothelium-dependent contractions and release of calcium from the sarcoplasmic reticulum.23 Lowering the …

Haloperidol increases expression of the inositol 1,4,5-trisphosphate receptors in rat cardiac atria, but not in ventricles

Numerous ligands of sigma receptors are known to prolong the QT interval and therefore cause a variety of arrhythmias. High affinity binding sites for the prototypical sigma ligand haloperidol were found in membranes of cardiac myocytes from adult rats. Activation of sigma 1 receptor leads to a release of calcium from the endoplasmic reticulum that follows increased synthesis of inositol 1,4,5-trisphosphate (IP3). We studied the effect of long-term haloperidol treatment on the expression of sigma 1 receptors, IP3 receptors of type 1 and 2 in the individual parts of the rat heart, in isolated rat cardiomyocytes and in PC12 cells. We have found that prolonged treatment with haloperidol significantly increased mRNA levels of sigma 1 receptors in both atria and ventricles. Sigma 1 receptor's mRNA was increased also in isolated cardiomyocytes. Haloperidol treatment affects the expression of IP3 receptors of type 1 and 2 in cardiac atria, but not in cardiac ventricles. We observed increase in IP3 receptors in differentiated PC12 cells, but not in isolated cardiomyocytes. We propose that this increase might participate in triggering cardiac arrhythmias during haloperidol treatment, which has to be further verified.

Sigma-1 receptor stimulation with fluvoxamine activates Akt–eNOS signaling in the thoracic aorta of ovariectomized rats with abdominal aortic banding

In the present study, we investigated the vasculoprotective effect of sigma-1 receptor stimulation with fluvoxamine on pressure overload hypertrophy-induced vascular injury in the thoracic aorta and defined mechanisms underlying that activity. Wistar rats underwent bilateral ovariectomy, and two weeks later were further treated with abdominal aortic stenosis. To confirm the vasculoprotective role of sigma-1 receptor signaling, we treated rats with the agonist fluvoxamine (at 0.5 and 1.0 mg/kg) and with the antagonist NE-100 (at1.0 mg/kg) for 4 weeks orally once a day after the onset of aortic banding. Interestingly, sigma-1 receptor expression in the thoracic aorta decreased significantly 4 weeks after pressure overload-induced hypertrophy in vehicle treated ovariectomized rats. Fluvoxamine administration significantly attenuated pressure overload-induced vascular injury with concomitant increase in receptor expression and subsequent decrease in IP3 receptor expression. Fluvoxamine treatment also significantly restored pressure overload-induced impaired Akt phosphorylation and stimulated eNOS protein expression as well as Akt-mediated eNOS phosphorylation (Ser1177). Fluvoxamine's vasculoprotective effect was nullified by co-administration of a sigma-1 receptor antagonist. No changes in phosphorylation of ERK1/2 or PKCα in the aorta were observed following pressure overload and after fluvoxamine treatment. Our findings confirm, for the first time, a potential role for sigma-1 receptor expression and signaling in the thoracic aorta in attenuating hypertrophy-induced vascular injury in ovariectomized rats. Thus, we demonstrate, for the first time, a potential role in the thoracic aorta for sigma-1 receptor expression and signaling via Akt–eNOS in attenuating hypertrophy-induced vascular injury in ovariectomized rats.

Smooth Muscle Proliferation and Role of the Prostacyclin (IP) Receptor in Idiopathic Pulmonary Arterial Hypertension

Prostacyclin analogs, used to treat idiopathic pulmonary arterial hypertension (IPAH), are assumed to work through prostacyclin (IP) receptors linked to cyclic AMP (cAMP) generation, although the potential to signal through peroxisome proliferator-activated receptor-γ (PPARγ) exists. IP receptor and PPARγ expression may be depressed in IPAH. We wished to determine if pathways remain functional and if analogs continue to inhibit smooth muscle proliferation. We used Western blotting to determine IP receptor expression in peripheral pulmonary arterial smooth muscle cells (PASMCs) from normal and IPAH lungs and immunohistochemistry to evaluate IP receptor and PPARγ expression in distal arteries. Cell proliferation and cAMP assays assessed analog responses in human and mouse PASMCs and HEK-293 cells. Proliferative rates of IPAH cells were greater than normal human PASMCs. IP receptor protein levels were lower in PASMCs from patients with IPAH, but treprostinil reduced replication and treprostinil-induced cAMP elevation appeared normal. Responses to prostacyclin analogs were largely dependent on the IP receptor and cAMP in normal PASMCs, although in IP(-/-) receptor cells analogs inhibited growth in a cAMP-independent, PPARγ-dependent manner. In IPAH cells, antiproliferative responses to analogs were insensitive to IP receptor or adenylyl cyclase antagonists but were potentiated by a PPARγ agonist and inhibited (∼ 60%) by the PPARγ antagonist GW9662. This coincided with increased PPARγ expression in the medial layer of acinar arteries. The antiproliferative effects of prostacyclin analogs are preserved in IPAH despite IP receptor down-regulation and abnormal coupling. PPARγ may represent a previously unrecognized pathway by which these agents inhibit smooth muscle proliferation.

Role of the Prostanoid EP4 Receptor in Iloprost-mediated Vasodilatation in Pulmonary Hypertension

Iloprost is effective for the treatment of pulmonary hypertension. It acts through elevation of cAMP by binding to the prostacyclin receptor (IP receptor). However, there is evidence that patients with severe pulmonary hypertension have decreased expression of the IP receptor in the remodeled pulmonary arterial smooth muscle. We hypothesized that prostanoid receptors other than the IP receptor are involved in signal transduction by iloprost. Immunoblotting was used to detect the IP and prostanoid EP4 receptor in lung tissue from patients with idiopathic pulmonary arterial hypertension, and immunohistochemistry was used to detect these receptors in lung sections from rats treated with monocrotaline (MCT28d). Protein and mRNA were isolated from pulmonary arterial smooth muscle cells (PASMCs) from control and MCT28d rats treated with AH6809 (an EP2 receptor antagonist) and AH23848 (an EP4 receptor antagonist) in combination with iloprost. Intracellular cAMP was also assessed in these tissues. IP receptor expression was reduced in idiopathic pulmonary arterial hypertension patient lung samples and MCT28d rat lungs compared with the controls. Reverse transcriptase-polymerase chain reaction and immunoblotting of MCT28d rat PASMC extracts revealed scant expression of the IP receptor but stable expression of EP4 receptor, compared with controls. Iloprost-induced elevation in intracellular cAMP in PASMCs was dose-dependently reduced by AH23848, but not by AH6809. Iloprost mediates vasodilatory functions via the EP4 receptor in the case of low IP receptor expression associated with pulmonary arterial hypertension. This is a previously unrecognized mechanism for iloprost, and illustrates that the EP4 receptor may be a novel therapeutic approach for the treatment of pulmonary arterial hypertension.

Hypoxia modulates gene expression of IP3 receptors in rodent cerebellum

Hypoxic brain cell injury is a complex process that results from a series of intracellular events. In this work, we tested whether severe hypoxia for 6 h can affect gene expression and protein levels of intracellular calcium channels, ryanodine receptors, and inositol 1,4,5-trisphosphate receptors in mouse cerebellum. In addition, we tested the effect of hypoxia on cerebellar granular cells of rats. We have found that gene expression of types 1 and 2 IP(3) receptors is significantly increased after the exposure of mice to hypoxic stimulus for 6 h and also in rat cerebellar granular cells. Increased gene expression of IP(3) receptors was reflected in increased protein levels of these channels as well. In this process, reactive oxygen species are most probably involved, as antioxidant quercetin abolished hypoxia-induced increase in both types 1 and 2 IP3 receptor. Ryanodine receptors of types 1 and 2 and sarco(endo)plasmic reticulum Ca(2+)-ATPase were not affected by hypoxia on the level of messenger RNA. To test physiological consequences, we measured levels of intracellular calcium. We observed significantly elevated calcium level in hypoxic compared to normoxic cells. Deeper understanding of mechanisms, through which hypoxia regulates intracellular calcium, could point towards the development of new therapeutic approaches to reduce or suppress the pathological effects of cellular hypoxia, such as those seen in stroke or ischemia.