Trypsin-induced elevated contractile responses in a rat model of interstitial cystitis/bladder pain syndrome: Involvement of PAR2 and intracellular Ca2+ release pathways.

AIMS: Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic inflammatory disease with unclear etiology. Different receptors play a role in the pathophysiology including protease activated receptors (PARs). The present study aimed to investigate the subtypes and the effects of PARs on contractility using permeabilized detrusor smooth muscle strips in IC/BPS. MAIN METHODS: IC/BPS was induced by cyclophosphamide injection. Histopathological analysis, PCR for detecting PAR proteins, western blotting for indicating PAR2 protein expression levels and myograph recording for measuring contractile force were used. KEY FINDINGS: The present study reveals that in rat bladder PAR1 and PAR2 but not PAR4 were found to be expressed. The first evidence was revealed where trypsin-induced contractions in rat permeabilized detrusor were potentiated in CYP-induced cystitis. Moreover, the functional inhibition of trypsin-induced contractions by selective PAR2 antagonist (ENMD-1068) and the supporting immunoblotting results emphasized that the main PAR subtype involved in IC/BPS model in rat bladder is PAR2. Our data emphasize the prominent role of IP3 in cystitis pathology besides ryanodine channels. Trypsin-induced Ca2+sensitization contractions were also higher in cystitis. Both Rho kinase and protein kinase C played a role in this increased Ca2+sensitization situation. SIGNIFICANCE: The present paper highlights the intracellular pathways that are involved in trypsin-induced contractions mainly via PAR2 in permeabilized bladder detrusor smooth muscle in a rat model of IC/BPS.

FRET-based assay for intracellular evaluation of α-synuclein aggregation inhibitors.

Aggregation of small neuronal protein α-synuclein (αSyn) in amyloid fibrils is considered to be one of the main causes of Parkinson's disease. Inhibition of this aggregation is a promising approach for disease treatment. Dozens of compounds able to inhibit αSyn fibrillization in solution were developed during the last decade. However, the applicability of most of them in the cellular environment was not established because of the absence of a suitable cell-based assay. In this work, we developed an assay for testing αSyn aggregation inhibitors in cells that is based on fluorescence resonance energy transfer (FRET) between labeled αSyn molecules in fibrils. The assay directly reports the amount of fibrillized αSyn and is more reliable than the assays based on cell viability. Moreover, we showed that cell viability decline does not always correlate with the amount of misfolded αSyn. The developed FRET-based assay does not interfere with the aggregation process and is suitable for high-throughput testing of αSyn aggregation inhibitors. Its application can sort out non-specific inhibitors and thus significantly facilitate the development of drugs for Parkinson`s disease.

Ginsenoside 20(S)-Rh2 promotes cellular pharmacokinetics and intracellular antibacterial activity of levofloxacin against Staphylococcus aureus through drug efflux inhibition and subcellular stabilization.

Intracellular Staphylococcus aureus (S. aureus) often causes clinical failure and relapse after antibiotic treatment. We previously found that 20(S)-ginsenoside Rh2 [20(S)-Rh2] enhanced the therapeutic effect of quinolones in a mouse model of peritonitis, which we attributed to the increased concentrations of quinolones within bacteria. In this study, we investigated the enhancing effect of 20(S)-Rh2 on levofloxacin (LVF) from a perspective of intracellular bacteria. In S. aureus 25923-infected mice, coadministration of LVF (1.5 mg/kg, i.v.) and 20(S)-Rh2 (25, 50 mg/kg, i.g.) markedly increased the survival rate, and decreased intracellular bacteria counts accompanied by increased accumulation of LVF in peritoneal macrophages. In addition, 20(S)-Rh2 (1, 5, 10 µM) dose-dependently increased the uptake and accumulation of LVF in peritoneal macrophages from infected mice without drug treatment. In a model of S. aureus 25923-infected THP-1 macrophages, we showed that 20(S)-Rh2 (1, 5, 10 µM) dose-dependently enhanced the intracellular antibacterial activity of LVF. At the cellular level, 20(S)-Rh2 increased the intracellular accumulation of LVF by inhibiting P-gp and BCRP. PK-PD modeling revealed that 20(S)-Rh2 altered the properties of the cell but not LVF. At the subcellular level, 20(S)-Rh2 did not increase the distribution of LVF in lysosomes but exhibited a stronger sensitizing effect in acidic environments. Molecular dynamics (MD) simulations showed that 20(S)-Rh2 improved the stability of the DNA gyrase-LVF complex in lysosome-like acidic conditions. In conclusion, 20(S)-Rh2 promotes the cellular pharmacokinetics and intracellular antibacterial activities of LVF against S. aureus through efflux transporter inhibition and subcellular stabilization, which is beneficial for infection treatment.

Role of intracellular signaling pathways and their inhibitors in the treatment of inflammation.

Inflammation is not only a defense mechanism of the innate immune system against invaders, but it is also involved in the pathogenesis of many diseases such as atherosclerosis, thrombosis, diabetes, epilepsy, and many neurodegenerative disorders. The World Health Organization (WHO) reports worldwide estimates of people (9.6% in males and 18.0% in females) aged over 60 years, suffering from symptomatic osteoarthritis, and around 339 million suffering from asthma. Other chronic inflammatory diseases, such as ulcerative colitis and Crohn's disease are also highly prevalent. The existing anti-inflammatory agents, both non-steroidal and steroidal, are highly effective; however, their prolonged use is marred by the severity of associated side effects. A holistic approach to ensure patient compliance requires understanding the pathophysiology of inflammation and exploring new targets for drug development. In this regard, various intracellular cell signaling pathways and their signaling molecules have been identified to be associated with inflammation. Therefore, chemical inhibitors of these pathways may be potential candidates for novel anti-inflammatory drug approaches. This review focuses on the anti-inflammatory effect of these inhibitors (for JAK/STAT, MAPK, and mTOR pathways) describing their mechanism of action through literature search, current patents, and molecules under clinical trials.

Detection of intracellular lamellar bodies as a screening marker for fibrotic lesions.

With the rapid increase in application of disinfectants worldwide as a method to block the spread of coronavirus, many new products are being introduced into the market without thorough verification of their impacts on human health and the environment. In the present study, we aimed to propose a screening marker for materials that can induce fibrotic lung disease using disinfectants, which had been demonstrated as causative materials of chronic inflammation and interstitial fibrosis. We first calculated the corresponding LC50 level based on results from cell viability test and exposed the LC50 level of disinfectants to human bronchial epithelial cells for 24 h. Formation of lamellar body-like structures, cleavage of the nuclear matrix, structural damage of mitochondria were found in the cytosol of the treated cells. We also dosed disinfectants by pharyngeal aspiration to mice to determine the LD0 level. The mice were sacrificed on Day 14 after a single dosing, and lamellar body-like structures were observed in the lung tissue of mice. Herein, we hypothesize that DNA damage and metabolic disturbance may play central roles in disinfectant-induced adverse health effects. Additionally, we propose that formation of lamellar bodies can be a screening marker for interstitial fibrosis.

Dehydroeffusol Pprevents Amyloid ß1-42-mediated Hippocampal Neurodegeneration via Reducing Intracellular Zn2+ Toxicity.

Dehydroeffusol, a phenanthrene isolated from Juncus effusus, is a Chinese medicine. To explore an efficacy of dehydroeffusol administration for prevention and cure of Alzheimer's disease, here we examined the effect of dehydroeffusol on amyloid ß1-42 (Aß1-42)-mediated hippocampal neurodegeneration. Dehydroeffusol (15 mg/kg body weight) was orally administered to mice once a day for 6 days and then human Aß1-42 was injected intracerebroventricularly followed by oral administration for 12 days. Neurodegeneration in the dentate granule cell layer, which was determined 2 weeks after Aß1-42 injection, was rescued by dehydroeffusol administration. Aß staining (uptake) was not reduced in the dentate granule cell layer by pre-administration of dehydroeffusol for 6 days, while increase in intracellular Zn2+ induced with Aß1-42 was reduced, suggesting that pre-administration of dehydroeffusol prior to Aß1-42 injection is effective for Aß1-42-mediated neurodegeneration that was linked with intracellular Zn2+ toxicity. As a matter of fact, pre-administration of dehydroeffusol rescued Aß1-42-mediated neurodegeneration. Interestingly, pre-administration of dehydroeffusol increased synthesis of metallothioneins, intracellular Zn2+-binding proteins, in the dentate granule cell layer, which can capture Zn2+ from Zn-Aß1-42 complexes. The present study indicates that pre-administration of dehydroeffusol protects Aß1-42-mediated neurodegeneration in the hippocampus by reducing intracellular Zn2+ toxicity, which is linked with induced synthesis of metallothioneins. Dehydroeffusol, a novel inducer of metallothioneins, may protect Aß1-42-induced pathogenesis in Alzheimer's disease.

Decrease of an intracellular organic osmolyte contributes to the cytotoxicity of organophosphate in neuroblastoma cells in vitro.

Organophosphorus compounds (OP) causes prominent delayed neuropathy in vivo and cytotoxicity to neuronal cells in vitro. The primary target protein of OP's neurotoxicity is neuropathy target esterase (NTE), which can convert phosphatidylcholine (PC) to glycerophosphocholine (GPC). Recent studies reveal that autophagic cell death is important for the initiation and progression of OP-induced neurotoxicity both in vivo and in vitro. However, the mechanism of how OP induces autophagic cell death is unknown. Here it is found that GPC is an important organic osmolyte in the neuroblastoma cells, and treatment with tri-o-cresyl phosphate (TOCP), a representative OP, leads to the decrease of GPC and imbalance of extracellular and intracellular osmolality. Knockdown of GPC metabolizing enzyme glycerophosphodiester phosphodiesterase domain containing 5 (GDPD5) reverses TOCP-induced autophagic cell death, which further supports the notion that the reduced GPC level leads to the autophagic cell death. Furthermore, it is found that autophagic cell death is due to the induction of reactive oxygen species (ROS) and mitochondrial damage by imbalance of osmolality with TOCP treatment. In summary, this study reveals that TOCP treatment decreases GPC level and intracellular osmolality, which induces ROS and mitochondrial damage and leads to the cell death and neurite degradation by autophagy. This study lays the foundation for further investigations on the potential therapeutic approaches for OP neurotoxicity or NTE mutation-related neurological diseases.

ATP-evoked intracellular Ca2+ transients shape the ionic permeability of human microglia from epileptic temporal cortex.

BACKGROUND: Intracellular Ca2+ modulates several microglial activities, such as proliferation, migration, phagocytosis, and inflammatory mediator secretion. Extracellular ATP, the levels of which significantly change during epileptic seizures, activates specific receptors leading to an increase of intracellular free Ca2+ concentration ([Ca2+]i). Here, we aimed to functionally characterize human microglia obtained from cortices of subjects with temporal lobe epilepsy, focusing on the Ca2+-mediated response triggered by purinergic signaling. METHODS: Fura-2 based fluorescence microscopy was used to measure [Ca2+]i in primary cultures of human microglial cells obtained from surgical specimens. The perforated patch-clamp technique, which preserves the cytoplasmic milieu, was used to measure ATP-evoked Ca2+-dependent whole-cell currents. RESULTS: In human microglia extracellular ATP evoked [Ca2+]i increases depend on Ca2+ entry from the extracellular space and on Ca2+ mobilization from intracellular compartments. Extracellular ATP also induced a transient fivefold potentiation of the total transmembrane current, which was completely abolished when [Ca2+]i increases were prevented by removing external Ca2+ and using an intracellular Ca2+ chelator. TRAM-34, a selective KCa3.1 blocker, significantly reduced the ATP-induced current potentiation but did not abolish it. The removal of external Cl- in the presence of TRAM-34 further lowered the ATP-evoked effect. A direct comparison between the ATP-evoked mean current potentiation and mean Ca2+ transient amplitude revealed a linear correlation. Treatment of microglial cells with LPS for 48 h did not prevent the ATP-induced Ca2+ mobilization but completely abolished the ATP-mediated current potentiation. The absence of the Ca2+-evoked K+ current led to a less sustained ATP-evoked Ca2+ entry, as shown by the faster Ca2+ transient kinetics observed in LPS-treated microglia. CONCLUSIONS: Our study confirms a functional role for KCa3.1 channels in human microglia, linking ATP-evoked Ca2+ transients to changes in membrane conductance, with an inflammation-dependent mechanism, and suggests that during brain inflammation the KCa3.1-mediated microglial response to purinergic signaling may be reduced.

Purinergic signalling in host innate immune defence against intracellular pathogens.

Purinergic signalling is an evolutionarily conserved signalling pathway mediated by extracellular nucleotides and nucleosides. Tri- and diphosphonucleotides released from host cells during intracellular pathogen infections activate plasma membrane purinergic type 2 receptors (P2 receptors) that stimulate microbicidal mechanisms in host innate immune cells. P2X ion channels and P2Y G protein-coupled receptors are involved in activating host innate immune defence mechanisms, phagocytosis, phagolysosomal fusion, production of reactive species, acidification of parasitophorous vacuoles, inflammasome activation, and the release of cytokines, chemokines, and other inflammatory mediators. In this review, as part of a special issue in tribute to Geoffrey Burnstock, we discuss advances in understanding the importance of P2 receptors in the host antimicrobial innate mechanisms against intracellular pathogen infections.

Future avenues for Alzheimer's disease detection and therapy: liquid biopsy, intracellular signaling modulation, systems pharmacology drug discovery.

When Alzheimer's disease (AD) disease-modifying therapies will be available, global healthcare systems will be challenged by a large-scale demand for clinical and biological screening. Validation and qualification of globally accessible, minimally-invasive, and time-, cost-saving blood-based biomarkers need to be advanced. Novel pathophysiological mechanisms (and related candidate biomarkers) - including neuroinflammation pathways (TREM2 and YKL-40), axonal degeneration (neurofilament light chain protein), synaptic dysfunction (neurogranin, synaptotagmin, α-synuclein, and SNAP-25) - may be integrated into an expanding pathophysiological and biomarker matrix and, ultimately, integrated into a comprehensive blood-based liquid biopsy, aligned with the evolving ATN + classification system and the precision medicine paradigm. Liquid biopsy-based diagnostic and therapeutic algorithms are increasingly employed in Oncology disease-modifying therapies and medical practice, showing an enormous potential for AD and other brain diseases as well. For AD and other neurodegenerative diseases, newly identified aberrant molecular pathways have been identified as suitable therapeutic targets and are currently investigated by academia/industry-led R&D programs, including the nerve-growth factor pathway in basal forebrain cholinergic neurons, the sigma1 receptor, and the GTPases of the Rho family. Evidence for a clinical long-term effect on cognitive function and brain health span of cholinergic compounds, drug candidates for repositioning programs, and non-pharmacological multidomain interventions (nutrition, cognitive training, and physical activity) is developing as well. Ultimately, novel pharmacological paradigms, such as quantitative systems pharmacology-based integrative/explorative approaches, are gaining momentum to optimize drug discovery and accomplish effective pathway-based strategies for precision medicine. This article is part of the special issue on 'The Quest for Disease-Modifying Therapies for Neurodegenerative Disorders'.

Superior antibacterial activity of gallium based liquid metals due to Ga3+ induced intracellular ROS generation.

Gallium-based liquid metals have increasing applications in a wide variety of emerging areas and they are involved more in frontier studies, the energy industry and additive manufacturing production, and even in daily life. When exposed to open air, large amounts of microorganisms may interact with liquid metals. However, the research of the relationship between pure gallium-based liquid metals and bacterial cells is still limited. In this study, the antibacterial properties of eutectic gallium-indium (EGaIn) alloys were tested against the typical Gram-negative bacteria-Escherichia coli and the Gram-positive bacteria-Staphylococcus aureus and the experimental results displayed that the antibacterial rates reached 100%. We also explored the mechanism of the anti-bacterial properties of EGaIn alloys by measuring the surface composition of the EGaIn film and the concentration of dissolved metal ions. The morphology of the bacterial cells showed that the cell growth and division were influenced by exposure to EGaIn. We also found that the synergistic antibacterial effect came along with the production of reactive oxygen species (ROS). Moreover, the EGaIn film showed enhanced antibacterial activity compared to gallium nitrate at the same initial ion concentration in the solution. This study shows the enormous potential of the anti-bacterial effect of liquid metals.

Regulatory effect of glutathione on treg/Th17 cell balance in allergic rhinitis patients through inhibiting intracellular autophagy.

BACKGROUND: Glutathione is a potential therapy for systemic lupus erythematosus, but its role in allergic rhinitis (AR) has not been determined. This report probed into the actions of glutathione in AR, so as to supplement evidence for a therapeutical countermeasure for AR. METHODS: In this study, peripheral blood mononuclear cells (PBMCs) of patients were extracted and processed with glutathione. PBMCs and nasal mucosa tissues were collected from AR mouse models treated with or without glutathione. The proportions of Th17/Treg cell markers and autophagy-related molecules in the nasal mucosa, PBMCs or Th17/Treg cells were assessed by quantitative real-time polymerase chain reaction (qRT-PCR), Western blot (WB) or flow cytometry analysis, and serum contents of related factors were analyzed by enzyme-linked immunosorbent assay (ELISA). Hematoxylin-eosin (HE) staining was applied to observe the thickness of mouse mucosa. RESULTS: IL-17A, RORγt, Beclin1 and LC3-II/LC3-I levels were increased in AR patients, while Foxp3 and P62 were decreased. The serum contents of IL-17A and eosinophil cationic protein (ECP) in AR patients were elevated, but IL-10 level was reduced. In PBMCs of AR patients, the levels of IL-17A and LC3-II were increased, and the levels of Foxp3 and P62 were decreased, while these changes could be reversed by glutathione. In AR mouse models, glutathione could balance Th17/Treg cells, reduce autophagy, correct the levels of related cytokines in mouse serum, and shrunk mucosa thickness. CONCLUSION: Glutathione could rescue the imbalance of Treg/Th17 cells by suppressing intracellular autophagy, which might be beneficial to the treatment of AR patients.

Intracellular delivery of therapeutic antisense oligonucleotides targeting mRNA coding mitochondrial proteins by cell-penetrating peptides.

Cell-penetrating peptides are a promising therapeutic strategy for a wide variety of degenerative diseases, ageing, and cancer. Among the multitude of cell-penetrating peptides, PepFect14 has been preferentially used in our laboratory for oligonucleotide delivery into cells and in vivo mouse models. However, this activity has mainly been reported towards cytoplasm and nuclei, while the mentioned disorders have been linked to mitochondrial defects. Here, we report a library generated from a combinatorial covalent fusion of a mitochondrial-penetrating peptide, mtCPP1, and PepFect14 in order to deliver therapeutic biomolecules to influence mitochondrial protein expression. The non-covalent complexation of these peptides with oligonucleotides resulted in nano-complexes affecting biological functions in the cytoplasm and on mitochondria. This delivery system proved to efficiently target mitochondrial genes, providing a framework for the development of mitochondrial peptide-based oligonucleotide technologies with the potential to be used as a treatment for patients with mitochondrial disorders.

Cancer Cell Resistance Against the Clinically Investigated Thiosemicarbazone COTI-2 Is Based on Formation of Intracellular Copper Complex Glutathione Adducts and ABCC1-Mediated Efflux.

COTI-2 is a novel anticancer thiosemicarbazone in phase I clinical trial. However, the effects of metal complexation (a main characteristic of thiosemicarbazones) and acquired resistance mechanisms are widely unknown. Therefore, in this study, the copper and iron complexes of COTI-2 were synthesized and evaluated for their anticancer activity and impact on drug resistance in comparison to metal-free thiosemicarbazones. Investigations using Triapine-resistant SW480/Tria and newly established COTI-2-resistant SW480/Coti cells revealed distinct structure-activity relationships. SW480/Coti cells were found to overexpress ABCC1, and COTI-2 being a substrate for this efflux pump. This was unexpected, as ABCC1 has strong selectivity for glutathione adducts. The recognition by ABCC1 could be explained by the reduction kinetics of a ternary Cu-COTI-2 complex with glutathione. Thus, only thiosemicarbazones forming stable, nonreducible copper(II)-glutathione adducts are recognized and, in turn, effluxed by ABCC1. This reveals a crucial connection between copper complex chemistry, glutathione interaction, and the resistance profile of clinically relevant thiosemicarbazones.

Epigallocatechin-3-gallate mobilizes intracellular Ca2+ in prostate cancer cells through combined Ca2+ entry and Ca2+-induced Ca2+ release.

AIMS: To elucidate the mechanism by which (-)-epigallocatechin-3-gallate (EGCG) mediates intracellular Ca2+ increase in androgen-independent prostate cancer (PCa) cells. MAIN METHODS: Following exposure to different doses of EGCG, viability of DU145 and PC3 PCa cells was evaluated by MTT assay and the intracellular Ca2+ dynamics by the fluorescent Ca2+ chelator Fura-2. The expression of different channels was investigated by qPCR analysis and sulfhydryl bonds by Ellman's assay. KEY FINDINGS: EGCG inhibited DU145 and PC3 proliferation with IC50 = 46 and 56 µM, respectively, and induced dose-dependent peaks of internal Ca2+ that were dependent on extracellular Ca2+. The expression of TRPC4 and TRPC6 channels was revealed by qPCR in PC3 cells, but lack of effect by modulators and blockers ruled out an exclusive role for these, as well as for voltage-dependent T-type Ca2+ channels. Application of dithiothreitol and catalase and sulfhydryl (SH) measurements showed that EGCG-induced Ca2+ rise depends on SH oxidation, while the effect of EGTA, dantrolene, and the PLC inhibitor U73122 suggested that EGCG-induced Ca2+ influx acts as a trigger for Ca2+-induced Ca2+ release, involving both ryanodine and IP3 receptors. Different from EGCG, ATP caused a rapid Ca2+ increase, which was independent of external Ca2+, but sensitive to U73122. SIGNIFICANCE: EGCG induces an internal Ca2+ increase in PCa cells by a multi-step mechanism. As dysregulation of cytosolic Ca2+ is directly linked to apoptosis in PCa cells, these data confirm the possibility of using EGCG as a synergistic adjuvant in combined therapies for recalcitrant malignancies like androgen-independent PCa.

Oleuropein promotes hippocampal LTP via intracellular calcium mobilization and Ca2+-permeable AMPA receptor surface recruitment.

Oleuropein (OLE), a major phenolic compound in olive oil, has been demonstrated to possess several pharmacological properties, including neuroprotection. However, the cognitive effects of OLE and its action mechanism have remained unclear. Here, we examined the effect of OLE on long-term potentiation (LTP) using field excitatory postsynaptic potential recorded in the CA1 region of both wild-type and 5XFAD mouse hippocampal slice preparations. In initial experiments with wild-type mice, 100 µM/1 h of OLE produced significant enhancements in the LTPs of Schaffer collateral synapses in the CA1 regions of treated mice, as compared to the vehicle-treated controls. As assessed by surface biotinylation and Western blot analysis, OLE caused a significant increase in protein kinase A (PKA)-mediated phosphorylation and the surface expression of GluA1 containing calcium permeable- amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (CP-AMPARs) in the hippocampus. Furthermore, we found that OLE enhanced LTP induction, while GluA1 phosphorylation occurred in an N-methyl-d-aspartate receptors (NMDARs)-independent manner. The OLE-induced CP-AMPAR trafficking resulted from elevated intracellular Ca2+ levels via regulation of phospholipase C (PLC). Consistently, we also found involvement of NMDAR-independent LTP and GluA1 phosphorylation in 5XFAD transgenic mice hippocampal slices treated with OLE. Together, our findings indicate that OLE may regulate beneficial effects on memory through the facilitation of CP-AMPAR trafficking and synaptic transmission.

Functional modulation of sarcolemmal KATP channels by atrial natriuretic peptide-elicited intracellular signaling in adult rabbit ventricular cardiomyocytes.

ATP-sensitive potassium (KATP) channels couple cell metabolic status to membrane excitability and are crucial for stress adaptation and cytoprotection in the heart. Atrial natriuretic peptide (ANP), a cardiac peptide important for cardiovascular homeostasis, also exhibits cytoprotective features including protection against myocardial ischemia-reperfusion injuries. However, how ANP modulates cardiac KATP channels is largely unknown. In the present study we sought to address this issue by investigating the role of ANP signaling in functional modulation of sarcolemmal KATP (sarcKATP) channels in ventricular myocytes freshly isolated from adult rabbit hearts. Single-channel recordings were performed in combination with pharmacological approaches in the cell-attached patch configuration. Bath application of ANP markedly potentiated sarcKATP channel activities induced by metabolic inhibition with sodium azide, whereas the KATP-stimulating effect of ANP was abrogated by selective inhibition of the natriuretic peptide receptor type A (NPR-A), cGMP-dependent protein kinase (PKG), reactive oxygen species (ROS), extracellular signal-regulated protein kinase (ERK)1/2, Ca2+/calmodulin-dependent protein kinase II (CaMKII), or the ryanodine receptor (RyR). Blockade of RyRs also nullified hydrogen peroxide (H2O2)-induced stimulation of sarcKATP channels in intact cells. Furthermore, single-channel kinetic analyses revealed that ANP enhanced the function of ventricular sarcKATP channels through destabilizing the long closures and facilitating the opening transitions, without affecting the single-channel conductance. In conclusion, here we report that ANP positively modulates the activity of ventricular sarcKATP channels via an intracellular signaling mechanism consisting of NPR-A, PKG, ROS, ERK1/2, CaMKII, and RyR2. This novel mechanism may regulate cardiac excitability and contribute to cytoprotection, in part, by opening myocardial KATP channels.

Intracellular Calcium Responses Encode Action Potential Firing in Spinal Cord Lamina I Neurons.

Maladaptive plasticity of neurons in lamina I of the spinal cord is a lynchpin for the development of chronic pain, and is critically dependent on intracellular calcium signaling. However, the relationship between neuronal activity and intracellular calcium in these neurons is unknown. Here we combined two-photon calcium imaging with whole-cell electrophysiology to determine how action potential firing drives calcium responses within subcellular compartments of male rat spinal cord lamina I neurons. We found that single action potentials generated at the soma increase calcium concentration in the somatic cytosol and nucleus, and these calcium responses invade dendrites and dendritic spines by active backpropagation. Calcium responses in each compartment were dependent on voltage-gated calcium channels, and somatic and nuclear calcium responses were amplified by release of calcium from ryanodine-sensitive intracellular stores. Grouping single action potential-evoked calcium responses by neuron type demonstrated their presence in all defined types, as well as a high degree of similarity in calcium responses between neuron types. With bursts of action potentials, we found that calcium responses have the capacity to encode action potential frequency and number in all compartments, with action potential number being preferentially encoded. Together, these findings indicate that intracellular calcium serves as a readout of neuronal activity within lamina I neurons, providing a unifying mechanism through which activity may regulate plasticity, including that seen in chronic pain.SIGNIFICANCE STATEMENT Despite their critical role in both acute pain sensation and chronic pain, little is known of the fundamental physiology of spinal cord lamina I neurons. This is especially the case with respect to calcium dynamics within these neurons, which could regulate maladaptive plasticity observed in chronic pain. By combining two-photon calcium imaging and patch-clamp electrophysiological recordings from lamina I neurons, we found that action potential firing induces calcium responses within the somatic cytosol, nucleus, dendrites, and dendritic spines of lamina I neurons. Our findings demonstrate the presence of actively backpropagating action potentials, shifting our understanding of how these neurons process information, such that calcium provides a mechanism for lamina I neurons to track their own activity.

Elevated intracellular cAMP concentration mediates growth suppression in glioma cells.

Supressed levels of intracellular cAMP have been associated with malignancy. Thus, elevating cAMP through activation of adenylyl cyclase (AC) or by inhibition of phosphodiesterase (PDE) may be therapeutically beneficial. Here, we demonstrate that elevated cAMP levels suppress growth in C6 cells (a model of glioma) through treatment with forskolin, an AC activator, or a range of small molecule PDE inhibitors with differing selectivity profiles. Forskolin suppressed cell growth in a PKA-dependent manner by inducing a G2/M phase cell cycle arrest. In contrast, trequinsin (a non-selective PDE2/3/7 inhibitor), not only inhibited cell growth via PKA, but also stimulated (independent of PKA) caspase-3/-7 and induced an aneuploidy phenotype. Interestingly, a cocktail of individual PDE 2,3,7 inhibitors suppressed cell growth in a manner analogous to forskolin but not trequinsin. Finally, we demonstrate that concomitant targeting of both AC and PDEs synergistically elevated intracellular cAMP levels thereby potentiating their antiproliferative actions.

In "Vitro" Lps-Stimulated Sertoli Cells Pre-Loaded With Microparticles: Intracellular Activation Pathways.

Sertoli cells (SC) are immune privileged cells with the capacity of modulating the immune response by expressing several immune-regulatory factors. SC have the capacity to respond to external stimuli through innate phagocytic and antibacterial activities. This evidence evoked a potential role of SC as drug carriers and therapeutic agents. Such stimuli drive SC towards a still unknown evolution, the clinical relevance of which as yet remains undisclosed. This study sought to investigate the effects of external stimuli in the form of polymeric microparticles (MP) and bacteria derived endotoxins, such as lipopolysaccharides (LPS), in order to identify the pathways potentially involved in cell phenotype modifications. Compared to single stimulation, when combined, MP and LPS provoked a significant increase in the gene expression of IDO, PD-L1, FAS-L, TLR-3, TLR-4, MHC-II, ICAM-1, TFGß1, BDF123, BDF129, BDF3 and pEP2C. Western Blotting analysis demonstrated up-regulation of the ERK 1-2 and NF-kB p65 phosphorylation ratios. Our study, showing the exponential increase of these mediators upon combined MP and LPS stimulation, suggests a "switch" of SC function from typical cells of the blood-testicular barrier to nonprofessional tolerogenic antigen-presenting cells. Further studies should target the clinical and technological implications of such stimuli-induced SC transformation.