Systematic Review of Calcium Channels and Intracellular Calcium Signaling: Relevance to Pesticide Neurotoxicity

Pesticides of different chemical classes exert their toxic effects on the nervous system by acting on the different regulatory mechanisms of calcium (Ca²⁺) homeostasis. Pesticides have been shown to alter Ca²⁺ homeostasis, mainly by increasing its intracellular concentration above physiological levels. The pesticide-induced Ca²⁺ overload occurs through two main mechanisms: the entry of Ca²⁺ from the extracellular medium through the different types of Ca²⁺ channels present in the plasma membrane or its release into the cytoplasm from intracellular stocks, mainly from the endoplasmic reticulum. It has also been observed that intracellular increases in the Ca²⁺ concentrations are maintained over time, because pesticides inhibit the enzymes involved in reducing its levels. Thus, the alteration of Ca²⁺ levels can lead to the activation of various signaling pathways that generate oxidative stress, neuroinflammation and, finally, neuronal death. In this review, we also discuss some proposed strategies to counteract the detrimental effects of pesticides on Ca²⁺ homeostasis.     

The LPA3 Receptor: Regulation and Activation of Signaling Pathways

The lysophosphatidic acid 3 receptor (LPA₃) participates in different physiological actions and in the pathogenesis of many diseases through the activation of different signal pathways. Knowledge of the regulation of the function of the LPA₃ receptor is a crucial element for defining its roles in health and disease. This review describes what is known about the signaling pathways activated in terms of its various actions. Next, we review knowledge on the structure of the LPA₃ receptor, the domains found, and the roles that the latter might play in ligand recognition, signaling, and cellular localization. Currently, there is some information on the action of LPA₃ in different cells and whole organisms, but very little is known about the regulation of its function. Areas in which there is a gap in our knowledge are indicated in order to further stimulate experimental work on this receptor and on other members of the LPA receptor family. We are convinced that knowledge on how this receptor is activated, the signaling pathways employed and how the receptor internalization and desensitization are controlled will help design new therapeutic interventions for treating diseases in which the LPA₃ receptor is implicated.     

Permissive Modulation of Sphingosine-1-Phosphate-Enhanced Intracellular Calcium on BKCa Channel of Chromaffin Cells

Sphingosine-1-phosphate (S1P), is a signaling sphingolipid which acts as a bioactive lipid mediator. We assessed whether S1P had multiplex effects in regulating the large-conductance Ca²⁺-activated K⁺ channel (BK_Ca) in catecholamine-secreting chromaffin cells. Using multiple patch-clamp modes, Ca²⁺ imaging, and computational modeling, we evaluated the effects of S1P on the Ca²⁺-activated K⁺ currents (I_K(Ca)) in bovine adrenal chromaffin cells and in a pheochromocytoma cell line (PC12). In outside-out patches, the open probability of BK_Ca channel was reduced with a mean-closed time increment, but without a conductance change in response to a low-concentration S1P (1 µM). The intracellular Ca²⁺ concentration (Ca_i) was elevated in response to a high-dose (10 µM) but not low-dose of S1P. The single-channel activity of BK_Ca was also enhanced by S1P (10 µM) in the cell-attached recording of chromaffin cells. In the whole-cell voltage-clamp, a low-dose S1P (1 µM) suppressed I_K(Ca), whereas a high-dose S1P (10 µM) produced a biphasic response in the amplitude of I_K(Ca), i.e., an initial decrease followed by a sustained increase. The S1P-induced I_K(Ca) enhancement was abolished by BAPTA. Current-clamp studies showed that S1P (1 µM) increased the action potential (AP) firing. Simulation data revealed that the decreased BK_Ca conductance leads to increased AP firings in a modeling chromaffin cell. Over a similar dosage range, S1P (1 µM) inhibited I_K(Ca) and the permissive role of S1P on the BK_Ca activity was also effectively observed in the PC12 cell system. The S1P-mediated I_K(Ca) stimulation may result from the elevated Ca_i, whereas the inhibition of BK_Ca activity by S1P appears to be direct. By the differentiated tailoring BK_Ca channel function, S1P can modulate stimulus-secretion coupling in chromaffin cells.     

The Effect of Sera from Children with Obstructive Sleep Apnea Syndrome (OSAS) on Human Cardiomyocytes Differentiated from Human Embryonic Stem Cells

Obstructive sleep apnea syndrome (OSAS) patients suffer from cardiovascular morbidity, which is the leading cause of death in this disease. Based on our previous work with transformed cell lines and primary rat cardiomyocytes, we determined that upon incubation with sera from pediatric OSAS patients, the cell’s morphology changes, NF-κB pathway is activated, and their beating rate and viability decreases. These results suggest an important link between OSAS, systemic inflammatory signals and end-organ cardiovascular diseases. In this work, we confirmed and expanded these observations on a new in vitro system of beating human cardiomyocytes (CM) differentiated from human embryonic stem cells (hES). Our results show that incubation with pediatric OSAS sera, in contrast to sera from healthy children, induces over-expression of NF-κB p50 and p65 subunits, marked reduction in CMs beating rate, contraction amplitude and a strong reduction in intracellular calcium signal. The use of human CM cells derived from embryonic stem cells has not been previously reported in OSAS research. The results further support the hypothesis that NF-κB dependent inflammatory pathways play an important role in the evolution of cardiovascular morbidity in OSAS. This study uncovers a new model to investigate molecular and functional aspects of cardiovascular pathology in OSAS.     

Insulin Diminishes Superoxide Increase in Cytosol and Mitochondria of Cultured Cortical Neurons Treated with Toxic Glutamate

Glutamate excitotoxicity is involved in the pathogenesis of many disorders, including stroke, traumatic brain injury, and Alzheimer’s disease, for which central insulin resistance is a comorbid condition. Neurotoxicity of glutamate (Glu) is primarily associated with hyperactivation of the ionotropic N-methyl-D-aspartate receptors (NMDARs), causing a sustained increase in intracellular free calcium concentration ([Ca²⁺]_i) and synchronous mitochondrial depolarization and an increase in intracellular superoxide anion radical (O₂^–•) production. Recently, we found that insulin protects neurons against excitotoxicity by decreasing the delayed calcium deregulation (DCD). However, the role of insulin in O₂^–• production in excitotoxicity still needs to be clarified. The present study aims to investigate insulin’s effects on glutamate-evoked O₂^–• generation and DCD using the fluorescent indicators dihydroethidium, MitoSOX Red, and Fura-FF in cortical neurons. We found a linear correlation between [Ca²⁺]_i and [O₂^–•] in primary cultures of the rat neuron exposed to Glu, with insulin significantly reducing the production of intracellular and mitochondrial O₂^–• in the primary cultures of the rat neuron. MK 801, an inhibitor of NMDAR-gated Ca²⁺ influx, completely abrogated the glutamate effects in both the presence and absence of insulin. In experiments in sister cultures, insulin diminished neuronal death and O₂ consumption rate (OCR).     

Mechanisms of Nausea and Vomiting: Current Knowledge and Recent Advances in Intracellular Emetic Signaling Systems

Nausea and vomiting are common gastrointestinal complaints that can be triggered by diverse emetic stimuli through central and/or peripheral nervous systems. Both nausea and vomiting are considered as defense mechanisms when threatening toxins/drugs/bacteria/viruses/fungi enter the body either via the enteral (e.g., the gastrointestinal tract) or parenteral routes, including the blood, skin, and respiratory systems. While vomiting is the act of forceful removal of gastrointestinal contents, nausea is believed to be a subjective sensation that is more difficult to study in nonhuman species. In this review, the authors discuss the anatomical structures, neurotransmitters/mediators, and corresponding receptors, as well as intracellular emetic signaling pathways involved in the processes of nausea and vomiting in diverse animal models as well as humans. While blockade of emetic receptors in the prevention of vomiting is fairly well understood, the potential of new classes of antiemetics altering postreceptor signal transduction mechanisms is currently evolving, which is also reviewed. Finally, future directions within the field will be discussed in terms of important questions that remain to be resolved and advances in technology that may help provide potential answers.     

Antiviral Activity of a Novel Compound CW-33 against Japanese Encephalitis Virus through Inhibiting Intracellular Calcium Overload

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, has five genotypes (I, II, III, IV, and V). JEV genotype I circulates widely in some Asian countries. However, current JEV vaccines based on genotype III strains show low neutralizing capacities against genotype I variants. In addition, JE has no specific treatment, except a few supportive treatments. Compound CW-33, an intermediate synthesized derivative of furoquinolines, was investigated for its antiviral activities against JEV in this study. CW-33 exhibited the less cytotoxicity to Syrian baby hamster kidney (BHK-21) and human medulloblastoma (TE761) cells. CW-33 dose-dependently reduced the cytopathic effect and apoptosis of JEV-infected cells. Supernatant virus yield assay pinpointed CW-33 as having potential anti-JEV activity with IC₅₀ values ranging from 12.7 to 38.5 μM. Time-of-addition assay with CW-33 indicated that simultaneous and post-treatment had no plaque reduction activity, but continuous and simultaneous treatments proved to have highly effective antiviral activity, with IC₅₀ values of 32.7 and 48.5 μM, respectively. CW-33 significantly moderated JEV-triggered Ca²⁺ overload, which correlated with the recovery of mitochondria membrane potential as well as the activation of Akt/mTOR and Jak/STAT1 signals in treated infected cells. Phosphopeptide profiling by LC-MS/MS revealed that CW-33 upregulated proteins from the enzyme modulator category, such as protein phosphatase inhibitor 2 (I-2), Rho GTPase-activating protein 35, ARF GTPase-activating protein GIT2, and putative 3-phosphoinositide-dependent protein kinase 2. These enzyme modulators identified were associated with the activation of Akt/mTOR and Jak/STAT1 signals. Meanwhile, I-2 treatment substantially inhibited the apoptosis of JEV-infected cells. The results demonstrated that CW-33 exhibited a significant potential in the development of anti-JEV agents.     

Calmodulin and Its Binding Proteins in Parkinson’s Disease

Parkinson’s disease (PD) is a neurodegenerative disorder that manifests with rest tremor, muscle rigidity and movement disturbances. At the microscopic level it is characterized by formation of specific intraneuronal inclusions, called Lewy bodies (LBs), and by a progressive loss of dopaminergic neurons in the striatum and substantia nigra. All living cells, among them neurons, rely on Ca²⁺ as a universal carrier of extracellular and intracellular signals that can initiate and control various cellular processes. Disturbances in Ca²⁺ homeostasis and dysfunction of Ca²⁺ signaling pathways may have serious consequences on cells and even result in cell death. Dopaminergic neurons are particularly sensitive to any changes in intracellular Ca²⁺ level. The best known and studied Ca²⁺ sensor in eukaryotic cells is calmodulin. Calmodulin binds Ca²⁺ with high affinity and regulates the activity of a plethora of proteins. In the brain, calmodulin and its binding proteins play a crucial role in regulation of the activity of synaptic proteins and in the maintenance of neuronal plasticity. Thus, any changes in activity of these proteins might be linked to the development and progression of neurodegenerative disorders including PD. This review aims to summarize published results regarding the role of calmodulin and its binding proteins in pathology and pathogenesis of PD.     

The Role of CaMKII and ERK Signaling in Addiction

Nicotine is the predominant addictive compound of tobacco and causes the acquisition of dependence through its interactions with nicotinic acetylcholine receptors and various neurotransmitter releases in the central nervous system. The Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) and extracellular signal-regulated kinase (ERK) play a pivotal role in synaptic plasticity in the hippocampus. CaMKII is involved in long-term potentiation induction, which underlies the consolidation of learning and memory; however, the roles of CaMKII in nicotine and other psychostimulant-induced addiction still require further investigation. This article reviews the molecular mechanisms and crucial roles of CaMKII and ERK in nicotine and other stimulant drug-induced addiction. We also discuss dopamine (DA) receptor signaling involved in nicotine-induced addiction in the brain reward circuitry. In the last section, we introduce the association of polyunsaturated fatty acids and cellular chaperones of fatty acid-binding protein 3 in the context of nicotine-induced addiction in the mouse nucleus accumbens and provide a novel target for the treatment of drug abuse affecting dopaminergic systems.     

Transient Receptor Potential (TRP) Channels in Tumor Vascularization

Tumor diseases are unfortunately quick spreading, even though numerous studies are under way to improve early diagnosis and targeted treatments that take into account both the different characteristics associated with the various tumor types and the conditions of individual patients. In recent years, studies have focused on the role of ion channels in tumor development, as these proteins are involved in several cellular processes relevant to neoplastic transformation. Among all ion channels, many studies have focused on the superfamily of Transient Receptor Potential (TRP) channels, which are non-selective cation channels mediating extracellular Ca²⁺ influx. In this review, we examined the role of different endothelial TRP channel isoforms in tumor vessel formation, a process that is essential in tumor growth and metastasis.     

Rutaecarpine Increases Nitric Oxide Synthesis via eNOS Phosphorylation by TRPV1-Dependent CaMKII and CaMKKβ/AMPK Signaling Pathway in Human Endothelial Cells

Rutaecarpine (RUT) is a bioactive alkaloid isolated from the fruit of Evodia rutaecarpa that exerts a cellular protective effect. However, its protective effects on endothelial cells and its mechanism of action are still unclear. In this study, we demonstrated the effects of RUT on nitric oxide (NO) synthesis via endothelial nitric oxide synthase (eNOS) phosphorylation in endothelial cells and the underlying molecular mechanisms. RUT treatment promoted NO generation by increasing eNOS phosphorylation. Additionally, RUT induced an increase in intracellular Ca²⁺ concentration and phosphorylation of Ca²⁺/calmodulin-dependent protein kinase kinase β (CaMKKβ), AMP-activated protein kinase (AMPK), and Ca²⁺/calmodulin-dependent kinase II (CaMKII). Inhibition of transient receptor potential vanilloid type 1 (TRPV1) attenuated RUT-induced intracellular Ca²⁺ concentration and phosphorylation of CaMKII, CaMKKβ, AMPK, and eNOS. Treatment with KN-62 (a CaMKII inhibitor), Compound C (an AMPK inhibitor), and STO-609 (a CaMKKβ inhibitor) suppressed RUT-induced eNOS phosphorylation and NO generation. Interestingly, RUT attenuated the expression of ICAM-1 and VCAM-1 induced by TNF-α and inhibited the inflammation-related NF-κB signaling pathway. Taken together, these results suggest that RUT promotes NO synthesis and eNOS phosphorylation via the Ca²⁺/CaMKII and CaM/CaMKKβ/AMPK signaling pathways through TRPV1. These findings provide evidence that RUT prevents endothelial dysfunction and benefit cardiovascular health.     

Differential Regulation of Ca2+-Activated Cl− Channel TMEM16A Splice Variants by Membrane PI(4,5)P2

TMEM16A is a Ca²⁺-activated Cl⁻ channel that controls broad cellular processes ranging from mucus secretion to signal transduction and neuronal excitability. Recent studies have reported that membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) is an important cofactor that allosterically regulates TMEM16A channel activity. However, the detailed regulatory actions of PIP₂ in splice variants of TMEM16A remain unclear. Here, we demonstrated that the attenuation of membrane phosphoinositide levels selectively inhibited the current amplitude of the TMEM16A(ac) isoform by decreasing the slow, but not instantaneous, Cl⁻ currents, which are independent of the membrane potential and specific to PI(4,5)P₂ depletion. The attenuation of endogenous PI(4,5)P₂ levels by the activation of Danio rerio voltage-sensitive phosphatase (Dr-VSP) decreased the Cl⁻ currents of TMEM16A(ac) but not the TMEM16A(a) isoform, which was abolished by the co-expression of PIP 5-kinase type-1γ (PIPKIγ). Using the rapamycin-inducible dimerization of exogenous phosphoinositide phosphatases, we further revealed that the stimulatory effects of phosphoinositide on TMEM16A(ac) channels were similar in various membrane potentials and specific to PI(4,5)P₂, not PI4P and PI(3,4,5)P₃. Finally, we also confirmed that PI(4,5)P₂ resynthesis is essential for TMEM16A(ac) recovery from Dr-VSP-induced current inhibition. Our data demonstrate that membrane PI(4,5)P₂ selectively modulates the gating of the TMEM16A(ac) channel in an agonistic manner, which leads to the upregulation of TMEM16A(ac) functions in physiological conditions.     

Acute and Delayed Effects of Mechanical Injury on Calcium Homeostasis and Mitochondrial Potential of Primary Neuroglial Cell Culture: Potential Causal Contributions to Post-Traumatic Syndrome

In vitro models of traumatic brain injury (TBI) help to elucidate the pathological mechanisms responsible for cell dysfunction and death. To simulate in vitro the mechanical brain trauma, primary neuroglial cultures were scratched during different periods of network formation. Fluorescence microscopy was used to measure changes in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and mitochondrial potential (ΔΨm) a few minutes later and on days 3 and 7 after scratching. An increase in [Ca²⁺]_i and a decrease in ΔΨm were observed ~10 s after the injury in cells located no further than 150–200 µm from the scratch border. Ca²⁺ entry into cells during mechanical damage of the primary neuroglial culture occurred predominantly through the NMDA-type glutamate ionotropic channels. MK801, an inhibitor of this type of glutamate receptor, prevented an acute increase in [Ca²⁺]_i in 99% of neurons. Pathological changes in calcium homeostasis persisted in the primary neuroglial culture for one week after injury. Active cell migration in the scratch area occurred on day 11 after neurotrauma and was accompanied by a decrease in the ratio of live to dead cells in the areas adjacent to the injury. Immunohistochemical staining of glial fibrillary acidic protein and β-III tubulin showed that neuronal cells migrated to the injured area earlier than glial cells, but their repair potential was insufficient for survival. Mitochondrial Ca²⁺ overload and a drop in ΔΨm may cause delayed neuronal death and thus play a key role in the development of the post-traumatic syndrome. Preventing prolonged ΔΨm depolarization may be a promising therapeutic approach to improve neuronal survival after traumatic brain injury.     

When Isolated at Full Receptivity,in Vitro Fertilized Wheat (Triticum aestivum,L.) Egg Cells Reveal [Ca2+]cyt Oscillation of Intracellular Origin

During in vitro fertilization of wheat (Triticum aestivum, L.) in egg cells isolated at various developmental stages, changes in cytosolic free calcium ([Ca²⁺]_cyt) were observed. The dynamics of [Ca²⁺]_cyt elevation varied, reflecting the difference in the developmental stage of the eggs used. [Ca²⁺]_cyt oscillation was exclusively observed in fertile, mature egg cells fused with the sperm cell. To determine how [Ca²⁺]_cyt oscillation in mature egg cells is generated, egg cells were incubated in thapsigargin, which proved to be a specific inhibitor of the endoplasmic reticulum (ER) Ca²⁺-ATPase in wheat egg cells. In unfertilized egg cells, the addition of thapsigargin caused an abrupt transient increase in [Ca²⁺]_cyt in the absence of extracellular Ca²⁺, suggesting that an influx pathway for Ca²⁺ is activated by thapsigargin. The [Ca²⁺]_cyt oscillation seemed to require the filling of an intracellular calcium store for the onset of which, calcium influx through the plasma membrane appeared essential. This was demonstrated by omitting extracellular calcium from (or adding GdCl₃ to) the fusion medium, which prevented [Ca²⁺]_cyt oscillation in mature egg cells fused with the sperm. Combined, these data permit the hypothesis that the first sperm-induced transient increase in [Ca²⁺]_cyt depletes an intracellular Ca²⁺ store, triggering an increase in plasma membrane Ca²⁺ permeability, and this enhanced Ca²⁺ influx results in [Ca²⁺]_cyt oscillation.     

Signaling Pathways Used by the Specialized Pro-Resolving Mediator Maresin 2 Regulate Goblet Cell Function: Comparison with Maresin 1

Specialized pro-resolving mediators (SPMs), including Maresins (MaR)-1 and 2, contribute to tear film homeostasis and resolve conjunctival inflammation. We investigated MaR2′s signaling pathways in goblet cells (GC) from rat conjunctiva. Agonist-induced [Ca²⁺]_i and high-molecular weight glycoconjugate secretion were measured. MaR2 increased [Ca²⁺]_i and stimulated secretion. MaR2 and MaR1 stimulate conjunctival goblet cell function, especially secretion, by activating different but overlapping GPCR and signaling pathways, and furthermore counter-regulate histamine stimulated increase in [Ca²⁺]_i. Thus, MaR2 and MaR1 play a role in maintaining the ocular surface and tear film homeostasis in health and disease. As MaR2 and MaR1 modulate conjunctival goblet cell function, they each may have potential as novel, but differing, options for the treatment of ocular surface inflammatory diseases including allergic conjunctivitis and dry eye disease. We conclude that in conjunctival GC MaR2 and MaR1, both increase the [Ca²⁺]_i and stimulate secretion to maintain homeostasis by using one set of different, but overlapping, signaling pathways to increase [Ca²⁺]_i and another set to stimulate secretion. MaR2 also resolves ocular allergy.     

IRAG2 Interacts with IP3-Receptor Types 1, 2, and 3 and Regulates Intracellular Ca2+ in Murine Pancreatic Acinar Cells

The inositol 1,4,5-triphosphate receptor-associated 2 (IRAG2) is also known as Jaw1 or lymphoid-restricted membrane protein (LRMP) and shares homology with the inositol 1,4,5-triphosphate receptor-associated cGMP kinase substrate 1 (IRAG1). IRAG1 interacts with inositol trisphosphate receptors (IP₃ receptors /IP₃R) via its coiled-coil domain and modulates Ca²⁺ release from intracellular stores. Due to the homology of IRAG1 and IRAG2, especially in its coiled-coil domain, it is possible that IRAG2 has similar interaction partners like IRAG1 and that IRAG2 also modulates intracellular Ca²⁺ signaling. In our study, we localized IRAG2 in pancreatic acinar cells of the exocrine pancreas, and we investigated the interaction of IRAG2 with IP₃ receptors and its impact on intracellular Ca²⁺ signaling and exocrine pancreatic function, like amylase secretion. We detected the interaction of IRAG2 with different subtypes of IP₃R and altered Ca²⁺ release in pancreatic acinar cells from mice lacking IRAG2. IRAG2 deficiency decreased basal levels of intracellular Ca²⁺, suggesting that IRAG2 leads to activation of IP₃R under unstimulated basal conditions. Moreover, we observed that loss of IRAG2 impacts the secretion of amylase. Our data, therefore, suggest that IRAG2 modulates intracellular Ca²⁺ signaling, which regulates exocrine pancreatic function.     

Triterpenoid Saponins from Stauntonia chinensis Ameliorate Insulin Resistance via the AMP-Activated Protein Kinase and IR/IRS-1/PI3K/Akt Pathways in Insulin-Resistant HepG2 Cells

Inflammation and oxidative stress play crucial roles in the etiology of type 2 diabetes mellitus. In this study, we examined the anti-diabetic effects of triterpenoid saponins extracted from Stauntonia chinensis on stimulating glucose uptake by insulin-resistant human HepG2 cells. The results showed that saponin 6 significantly increased glucose uptake and glucose catabolism. Saponin 6 also enhanced the phosphorylation of AMP-activated protein kinase (AMPK) and activated the insulin receptor (IR)/insulin receptor substrate-1 (IRS-1)/phosphoinositide 3-kinase (PI3K)/Akt pathway. Therefore, our results suggest that saponins from S. chinensis improve glucose uptake and catabolism in hepatic cells by stimulating the AMPK and the IR/IRS-1/PI3K/Akt signaling pathways. The results also imply that saponins from S. chinensis can enhance glucose uptake and insulin sensitivity, representing a promising treatment for type 2 diabetes mellitus.     

Analysis of Human TAAR8 and Murine Taar8b Mediated Signaling Pathways and Expression Profile

The thyroid hormone derivative 3-iodothyronamine (3-T₁AM) exerts metabolic effects in vivo that contradict known effects of thyroid hormones. 3-T₁AM acts as a trace amine-associated receptor 1 (TAAR1) agonist and activates G_s signaling in vitro. Interestingly, 3-T₁AM-meditated in vivo effects persist in Taar1 knockout-mice indicating that further targets of 3-T₁AM might exist. Here, we investigated another member of the TAAR family, the only scarcely studied mouse and human trace-amine-associated receptor 8 (Taar8b, TAAR8). By RT-qPCR and locked-nucleic-acid (LNA) in situ hybridization, Taar8b expression in different mouse tissues was analyzed. Functionally, we characterized TAAR8 and Taar8b with regard to cell surface expression and signaling via different G-protein-mediated pathways. Cell surface expression was verified by ELISA, and cAMP accumulation was quantified by AlphaScreen for detection of G_s and/or G_i/o signaling. Activation of G-proteins G_q/11 and G_12/13 was analyzed by reporter gene assays. Expression analyses revealed at most marginal Taar8b expression and no gender differences for almost all analyzed tissues. In heart, LNA-in situ hybridization demonstrated the absence of Taar8b expression. We could not identify 3-T₁AM as a ligand for TAAR8 and Taar8b, but both receptors were characterized by a basal G_i/o signaling activity, a so far unknown signaling pathway for TAARs.     

Anti-Thrombotic Effects of Artesunate through Regulation of cAMP and PI3K/MAPK Pathway on Human Platelets

Normal activation of platelets and their aggregation are crucial for proper hemostasis. It appears that excessive or abnormal aggregation of platelets may bring about cardiovascular diseases such as stroke, atherosclerosis, and thrombosis. For this reason, finding a substance that can regulate platelet aggregation or suppress aggregation will aid in the prevention and treatment of cardiovascular diseases. Artesunate is a compound extracted from the plant roots of Artemisia or Scopolia, and its effects have shown to be promising in areas of anticancer and Alzheimer’s disease. However, the role and mechanisms by which artesunate affects the aggregation of platelets and the formation of a thrombus are currently not understood. This study examines the ways artesunate affects the aggregation of platelets and the formation of a thrombus on platelets induced by U46619. As a result, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) production were increased significantly by artesunate relative to the doses, as well as phosphorylated vasodilator-stimulated phosphoprotein (VASP) and inositol 1,4,5-trisphosphate receptor (IP₃R), substrates to cAMP-dependent kinase and cGMP-dependent kinase, in a significant manner. The Ca²⁺, normally mobilized from the dense tubular system, was inhibited due to IP₃R phosphorylation from artesunate, and phosphorylated VASP aided in inhibiting platelet activity via αIIb/β₃ platelet membrane inactivation and inhibiting fibrinogen binding. In addition, MAPK and PI3K/Akt phosphorylation was inhibited via artesunate in a significant manner, causing the production of TXA₂ and intracellular granular secretion (serotonin and ATP release) to be reduced. Therefore, we suggest that artesunate has value as a substance that inhibits platelet aggregation and thrombus formation through an antiplatelet mechanism.