Inhibition Of Phospholipase Research Articles

Induction of Ca2+ signaling and cytotoxic responses of human lung fibroblasts upon an antihistamine drug oxatomide treatment and evaluating the protective effects of Ca2+ chelating.

Oxatomide, an antihistamine drug of the diphenylmethylpiperazine family, has anti-inflammatory effects in airway disease. Because oxatomide was shown to cause diverse physiological responses in several cell models, the impact of oxatomide on Ca2+ signaling and its related physiological effects has not been explored in IMR-90 human fetal lung fibroblasts. This study assessed the effect of oxatomide on cell viability and intracellular free Ca2+ concentrations ([Ca2+]i) and examined whether oxatomide-induced cytotoxicity through Ca2+ signaling in IMR-90 cells. Cell viability was measured by the cell proliferation reagent (WST-1). [Ca2+]i was measured by the Ca2+-sensitive fluorescent dye fura-2. Oxatomide (10-40 μM) concentration dependently reduced cell viability and induced [Ca2+]i rises in IMR-90 cells. This cytotoxic effect was reversed by chelation of cytosolic Ca2+ with BAPTA-AM. In terms of Ca2+ signaling, oxatomide-caused Ca2+ entry was inhibited by modulators of store-operated Ca2+ channels (2-APB and SKF96365) and protein kinase C (PKC) inhibitor (GF109203X). Furthermore, oxatomide-induced Ca2+ influx was confirmed by Mn2+-induced quench of fura-2 fluorescence. In a Ca2+-free medium, preincubation with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin inhibited oxatomide-evoked [Ca2+]i rises. Conversely, treatment with oxatomide abolished thapsigargin-induced [Ca2+]i rises. Inhibition of phospholipase C (PLC) with U73122 also inhibited oxatomide-caused [Ca2+]i rises. In IMR-90 cells, oxatomide-induced cytotoxicity by preceding [Ca2+]i rises involving PKC-sensitive store-operated Ca2+ entry and PLC-dependent Ca2+ release from the endoplasmic reticulum. BAPTA-AM, with its Ca2+ chelating effects, may be a potential compound for preventing oxatomide-induced cytotoxicity.

Exploring the potential therapeutic benefits of 7-methoxy coumarin for neuropathy pain: an in vivo, in vitro, and in silico approach.

7-Methoxycoumarin (7-MC) is well recognized for its anti-inflammatory and anti-nociceptive actions. Its capacity to lessen neuropathic pain hasn't been documented yet. Hence the impact of 7-MC on vincristine-induced peripheral neuropathic pain in rodents was investigated. The investigation also looked at the impact of 7-MC in reducing neuropathic pain via voltage-gated calcium channels and phospholipase enzyme inhibition using pertinent in vitro and in silico methods. Vincristine (0.1mg/kg, i.p., daily) was administered continuously for 7 days to induce peripheral neuropathic pain in mice, with cold allodynia and thermal hyperalgesia and evaluated on the 8th day using the acetone bubble test and hot water tail immersion test. In order to derive the mechanistic approach for ameliorating neuropathic pain, the role of 7-MC in the inhibition of the phospholipase enzyme, gene expression studies on voltage-gated calcium channels using mouse BV2 microglial cells and in silico studies for its calcium channel binding affinity were also performed. The test compounds reduced vincristine-induced cold allodynia and thermal hyperalgesia in mice in a dose-dependent experiments. In vitrostudies on phospholipase inhibition by 7-MC showed an IC50 of 27.08µg/ml and down-regulated the gene expression of calcium channels in the BV2 microglial cell line. In silico docking scores for 7-MCwere higher than the standard drug gabapentin. The compound 7-MC has shown promise in alleviating vincristine-induced peripheral neuropathicin mice. Studies conducted in parallel, both in silico and in vitro have demonstrated that 7-MC effectively reduces neuropathic pain. This pain reduction is achieved through two mechanisms: inhibiting the phospholipase enzyme and blocking voltage-gated calcium channels.

Development and Evaluation of Indole-Based Phospholipase D Inhibitors for Lung Cancer Immunotherapy.

This study explored novel immunomodulatory approaches for cancer treatment, with a specific focus on lung cancer, the leading cause of cancer-related deaths worldwide. We synthesized indole-based phospholipase D (PLD) inhibitors with various substituents to improve anticancer efficacy. Through structure-activity relationship studies, the key compound was identified that significantly inhibiting PLD, suppressing cell growth, viability, and migration in vitro, while inducing apoptosis of lung cancer cells. In silico docking studies confirmed its binding to the PLD1 active site, highlighting the role of specific residues in inhibiting PLD1 activity. The inhibitor modulated oncogenic pathways and immune evasion in lung cancer cells, showing potential for immunotherapy. In vivo experiments in a mouse model showed tumor reduction and immune response alteration. Combining these inhibitors with gemcitabine, an anticancer drug, synergistically enhanced inhibition of lung cancer cell apoptosis and proliferation. This research offers new insights into PLD inhibitor as potential cancer therapeutics.

Phosphatidic acid is involved in regulation of autophagy in neurons in vitro and in vivo.

Major depressive disorder (MDD) is a common and severe psychiatric disease, which does not only lead to variety of neuropsychiatric symptoms, but unfortunately in a relatively large proportion of cases also to suicide. The pathogenesis of MDD still requires definition. We have previously shown that ceramide is increased in the blood plasma of patients with MDD. In mouse models of MDD, which are induced by treatment with corticosterone or application of chronic unpredictable stress, increased blood plasma ceramide also increased and caused an inhibition of phospholipase D in endothelial cells of the hippocampus and reduced phosphatidic acid levels in the hippocampus. Here, we demonstrated that corticosterone treatment of PC12 cells resulted in reduced cellular autophagy, which is corrected by treatment with phosphatidic acid. In vivo, treatment of mice with corticosterone or chronic unpredictable stress also reduced autophagy in hippocampus neurons. Autophagy was normalized upon i.v. injection of phosphatidic acid in these mouse models of MDD. In an attempt to identify targets of phosphatidic acid in neurons, we demonstrated that corticosterone reduced levels of the ganglioside GM1 in PC-12 cells and the hippocampus of mice, which were normalized by treatment of cells or i.v. injection of mice with phosphatidic acid. GM1 application also normalized autophagy in cultured neurons. Phosphatidic acid and GM1 corrected stress-induced alterations in behavior, i.e., mainly anxiety and anhedonia, in experimental MDD in mice. Our data suggest that phosphatidic acid may regulate via GM1 autophagy in neurons.

An acidic polysaccharide promoting GLP-1 secretion from Dendrobium huoshanense protocorm-like bodies: Structure validation and activity exploration

Glucagon-like peptide-1 (GLP-1) as a multifunctional hormone is secreted mainly from enteroendocrine L-cells, and enhancing its endogenous secretion has potential benefits of regulating glucose homeostasis and controlling body weight gain. In the present study, a novel polysaccharide (h-DHP) with high ability to enhance plasma GLP-1 level in mice was isolated from Dendrobium huoshanense protocorm-like bodies under the guidance of activity evaluation. Structural identification showed that h-DHP was an acidic polysaccharide with the molecular weight of 1.38 × 105 Da, and was composed of galactose, glucose, arabinose and glucuronic acid at a molar ratio of 15.7: 11.2: 4.5: 1.0 with a backbone consisting of →5)-α-L-Araf-(1→, →3)-α-D-Galp-(1→, →6)-α-D-Galp-(1→, →3,6)-α-D-Galp-(1→, →6)-β-D-Glcp-(1→ and →4,6)-β-D-Glcp-(1→ along with branches consisting of α-L-Araf-(1→, α-D-Galp-(1→, α-D-GlcAp-(1→, β-D-Glcp-(1→ and →4)-β-D-Glcp-(1→. Animal experiments with different administration routes demonstrated that h-DHP-enhanced plasma GLP-1 level was attributed to h-DHP-promoted GLP-1 secretion in the enteroendocrine L-cells, which was supported by h-DHP-enhanced extracellular GLP-1 level in STC-1 cells. Inhibition of adenylate cyclase and phospholipase C indicated that cAMP and cAMP-triggered intracellular Ca2+ increase participated in h-DHP-promoted GLP-1 secretion. These results suggested that h-DHP has the potential of enhancing endogenous GLP-1 level through h-DHP-promoted and cAMP-mediated GLP-1 secretion from enteroendocrine cells.

Dual therapy with phospholipase and metalloproteinase inhibitors from Sinonatrix annularis alleviated acute kidney and liver injury caused by multiple snake venoms

Snakebite envenomation often induces acute kidney injury (AKI) and acute liver injury (ALI), leading to augmented injuries and poor rehabilitation. Phospholipase A2 (PLA2) and metalloproteinase (SVMP) present in venom are responsible for the envenomation-associated events. In this study, mice envenomed with Deinagkistrodon acutus, Naja atra, or Agkistrodon halys pallas venom exhibited typical AKI and ALI symptoms, including significantly increased plasma levels of myoglobin, free hemoglobin, uric acid, aspartate aminotransferase, and alanine aminotransferase and upregulated expression of kidney NGAL and KIM-1. These effects were significantly inhibited when the mice were pretreated with natural inhibitors of PLA2 and SVMP isolated from Sinonatrix annularis (SaPLIγ and SaMPI). The inhibitors protected the physiological structural integrity of the renal tubules and glomeruli, alleviating inflammatory infiltration and diffuse hemorrhage in the liver. Furthermore, the dual therapy alleviated oxidative stress and apoptosis in the kidneys and liver by mitigating mitochondrial damage, thereby effectively reducing the lethal effect of snake venom in the inhibitor-treated mouse model. This study showed that dual therapy with inhibitors of metalloproteinase and phospholipase can effectively prevent ALI and AKI caused by snake bites. Our findings suggest that intrinsic inhibitors present in snakes are prospective therapeutic agents for multi-organ injuries caused by snake envenoming.

Abstract 138: Platelet RIPK1 Promotes Platelet Activation And Aggregation In Arteriolar Thrombosis And Ischemic Stroke

Receptor-interacting protein Ser/Thr kinase 1 (RIPK1) is a molecular switch for inflammation and cell death in nucleated cells, and its function is controlled by phosphorylation at the Ser166 residue. However, it is unknown whether RIPK1 regulates platelet function in arterial thrombosis. Intravital microscopy using megakaryocyte-specific Ripk1 conditional knockout (CKO) mice ( Ripk1 fl/fl;Pf4-cre ) revealed that platelet Ripk1 promoted platelet thrombus formation at sites of laser-induced cremaster arteriolar thrombosis. Deletion of platelet Ripk1 prolonged the time to occlusion in FeCl 3 -induced carotid arterial thrombosis without affecting tail bleeding times. Further, compared to wild-type (WT) mice, Ripk1 CKO mice exhibited improved neurological deficits and reduced infarct volume in ischemic stroke induced by transient middle cerebral artery occlusion. Flow cytometry and aggregation assays showed that deletion of platelet Ripk1 reduced P-selectin exposure, αIIbβ3 integrin activation, and platelet aggregation induced by thrombin, collagen-related peptides (CRP), and U46619, but not ADP. Pretreatment of Ripk1-null platelets with ADP rescued the aggregation defect. Treatment of platelets with Nec-1s, a RIPK1 inhibitor that blocks RIPK1-Ser166 phosphorylation, did not affect platelet activation and aggregation. Instead, RIPK1-Ser320/321 was rapidly phosphorylated in thrombin- or CRP-stimulated human and mouse platelets. Treatment of platelets with an inhibitor of phospholipase C, p38, or MAPK-activated protein kinase 2 (MK2), but not a protein kinase C inhibitor, decreased RIPK1-Ser320/321 phosphorylation after thrombin or CRP stimulation, implying that the p38-MK2 axis phosphorylates RIPK1-Ser320/321. Using a genetic approach in human megakaryoblastic MEG-01 cells, we found that RIPK1 knockdown reduced granule secretion from stimulated MEG-01 cells, which was rescued by overexpressing WT RIPK1, but not mutant RIPK1-Ser320Ala, in the RIPK1 knockdown MEG-01 cells. These results suggest that platelet RIPK1 enhances granule secretion during platelet activation through p38-MK2-mediated phosphorylation of RIPK1-Ser320/321, thereby contributing to the pathology of thrombotic disease.

Evidence of Active-Forgetting Mechanisms? Blocking Arachidonic Acid Release May Slow Forgetting of Sensitization in Aplysia.

Long-term sensitization in Aplysia is accompanied by a persistent up-regulation of mRNA encoding the peptide neurotransmitter Phe-Met-Arg-Phe-amide (FMRFa), a neuromodulator that opposes the expression of sensitization through activation of the arachidonic acid second-messenger pathway. We completed a preregistered test of the hypothesis that FMRFa plays a critical role in the forgetting of sensitization. Aplysia received long-term sensitization training and were then given whole-body injections of vehicle (N = 27), FMRFa (N = 26), or 4-bromophenacylbromide (4-BPB; N = 31), a phospholipase inhibitor that prevents the release of arachidonic acid. FMRFa produced no changes in forgetting. 4-BPB decreased forgetting measured 6 d after training [d s = 0.55 95% CI(0.01, 1.09)], though the estimated effect size is uncertain. Our results provide preliminary evidence that forgetting of sensitization may be a regulated, active process in Aplysia, but could also indicate a role for arachidonic acid in stabilizing the induction of sensitization.

Abstract 3356: Inhibiting phospholipase D1 reduces pancreatic carcinogenesis in mice

Abstract New therapeutic targets are needed for pancreatic ductal adenocarcinoma (PDAC), a leading cause of cancer-related death worldwide. Phospholipase D (PLD) is a lipid-signaling enzyme that appears to play a critical role in cell proliferation, survival/apoptosis, and downstream oncogenic transformation. Elevated PLD1 or PLD2 (the two classical mammalian isoforms) activity has been shown to promote tumor progression in various cancer types, however, the role and mechanisms of PLD in pancreatic cancer are not yet understood. Thus, the objective is to determine the role of PLD1 regulation in pancreatic carcinogenesis through genetic and pharmacological inhibition. To evaluate the role of PLD1 in tumor growth, we subcutaneously transplanted human MIA PaCa-2 pancreatic cancer cells expressing endogenous PLD1 levels (Ctr KD cells), or MIA PaCa-2 cells in which PLD1 was knocked down (Pld1 KD cells), into immunodeficient nude mice. After 18 days post implantation, tumors that arose from Pld1-KD cells presented a significantly smaller tumor size (p<0.05) and had reduced levels of Bcl-xL and Bcl-2, compared to controls (Ctr KD). Then, to assess the role of PLD1 in the tumor microenvironment, we implanted KPC pancreatic cancer cells into C57BL/6 wild-type (WT) or C57BL/6 PLD1 knockout (Pld1−/−) mice with and without treatment with gemcitabine. Fourteen days after tumor implantation, the growth of KPC tumors was attenuated in Pld1−/− mice by 49% (p<0.05). Treatment with gemcitabine reduced tumor growth by 51.6% (p<0.05) in WT mice and by 79.5% (p<0.03) in Pld1−/− mice. Moreover, we evaluated the effects of PLD1 genetic ablation by crossing LSL-KrasG12D/+; Ptf1Cre/+ (KC) mice into PLD1 knockout (Pld1−/−) mice to generate KC; Pld1−/− mice. At 8 months of age, KC; Pld1−/− mice had significantly smaller pancreases (p<0.05) compared to KC mice. Interestingly, PLD1 ablation increased phosphorylation of ERK and c-Jun in KC; Pld1−/− mice compared to KC mice. Finally, to assess whether pharmacological inhibition of PLD can inhibit pancreatic carcinogenesis, KC mice received daily intraperitoneal injections of a small molecule inhibitor of PLD1 and PLD2 (FIPI) at a dosage of 3 mg/kg of body weight for a duration of 5 weeks. Treatment with FIPI reduced acinar-to-ductal metaplasia (p<0.05) and reduced cell proliferation (p<0.01), when compared to vehicle-treated KC controls. Additionally, FIPI treatment was safe demonstrating no significant differences in body weight between the groups and no significant alteration in the serum levels of liver enzymes, including aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase. In conclusion, these findings reveal PLD1 plays a critical role in pancreatic carcinogenesis and might represent a novel therapeutic target. Citation Format: Hala Amer Addassi, Karen Matsukuma, Gerardo G. Mackenzie. Inhibiting phospholipase D1 reduces pancreatic carcinogenesis in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3356.

Inhibition of phospholipase D promotes neurological function recovery and reduces neuroinflammation after spinal cord injury in mice.

Spinal cord injury (SCI) is a severely disabling disease. Hyperactivation of neuroinflammation is one of the main pathophysiological features of secondary SCI, with phospholipid metabolism playing an important role in regulating inflammation. Phospholipase D (PLD), a critical lipid-signaling molecule, is known to be involved in various physiological processes, including the regulation of inflammation. Despite this knowledge, the specific role of PLD in SCI remains unclear. In this study, we constructed mouse models of SCI and administered PLD inhibitor (FIPI) treatment to investigate the efficacy of PLD. Additionally, transcriptome sequencing and protein microarray analysis of spinal cord tissues were conducted to further elucidate its mechanism of action. The results showed that PLD expression increased after SCI, and inhibition of PLD significantly improved the locomotor ability, reduced glial scarring, and decreased the damage of spinal cord tissues in mice with SCI. Transcriptome sequencing analysis showed that inhibition of PLD altered gene expression in inflammation regulation. Subsequently, the protein microarray analysis of spinal cord tissues revealed variations in numerous inflammatory factors. Biosignature analysis pointed to an association with immunity, thus confirming the results obtained from transcriptome sequencing. Collectively, these observations furnish compelling evidence supporting the anti-inflammatory effect of FIPI in the context of SCI, while also offering important insights into the PLD function which may be a potential therapeutic target for SCI.

Purinergic signaling promotes premature senescence

Extracellular ATP activates P2 purinergic receptors. Whether purinergic signaling is functionally coupled to cellular senescence is largely unknown. We find that oxidative stress induced release of ATP and caused senescence in human lung fibroblasts. Inhibition of P2 receptors limited oxidative stress-induced senescence, while stimulation with exogenous ATP promoted premature senescence. Pharmacological inhibition of P2Y11 receptor (P2Y11R) inhibited premature senescence induced by either oxidative stress or ATP, while stimulation with a P2Y11R agonist was sufficient to induce cellular senescence. Our data show that both extracellular ATP and a P2Y11R agonist induced calcium (Ca++) release from the endoplasmic reticulum (ER) and that either inhibition of phospholipase C or intracellular Ca++ chelation impaired ATP-induced senescence. We also find that Ca++ that was released from the ER, following ATP-mediated activation of phospholipase C, entered mitochondria in a manner dependent on P2Y11R activation. Once in mitochondria, excessive Ca++ promoted the production of reactive oxygen species in a P2Y11R-dependent fashion, which drove development of premature senescence of lung fibroblasts. Finally, we show that conditioned medium derived from senescent lung fibroblasts, which were induced to senesce through the activation of ATP/P2Y11R-mediated signaling, promoted the proliferation of triple-negative breast cancer cells and their tumorigenic potential by secreting amphiregulin. Our study identifies the existence of a novel purinergic signaling pathway that links extracellular ATP to the development of a protumorigenic premature senescent phenotype in lung fibroblasts that is dependent on P2Y11R activation and ER-to-mitochondria calcium signaling.

1-Benzylindoles as inhibitors of cytosolic phospholipase A2α: synthesis, biological activity, aqueous solubility, and cell permeability.

Cytosolic phospholipase A2α (cPLA2α) is considered an interesting target for the development of new anti-inflammatory drugs, as it is significantly involved in the formation of pro-inflammatory lipid mediators. Recently, in a ligand-based virtual screening approach, 2,4-dichlorobenzyl-substituted 4-[2-(indol-3-ylmethylene)hydrazineyl]benzoic acid 7 was found to be an inhibitor of cPLA2α with micromolar activity. This compound has now been systematically varied to increase inhibitory potency. The studies performed led to 5-(1-benzylindol-3-ylmethyl)-2H-tetrazol-2-yl)pentanoic acid derivatives that exhibited submicromolar activity against the enzyme. The most potent compounds were also tested for their water solubility and for permeability in a Caco-2 model. Among other things, it was found that in Caco-2 cells, the pentanoic acid chain of the molecules can be metabolised to a considerable extent to propionic acid by β-oxidation.

Lipid signaling and proline catabolism are activated in barley roots (Hordeum vulgare L.) during recovery from cold stress

Previous findings have shown that phospholipase D (PLD) contributes to the response to long-term chilling stress in barley by regulating the balance of proline (Pro) levels. Although Pro accumulation is one of the most prominent changes in barley roots exposed to this kind of stress, the regulation of its metabolism during recovery from stress remains unclear. Research has mostly focused on the responses to stress per se, and not much is known about the dynamics and mechanisms underlying the subsequent recovery. The present study aimed to evaluate how PLD, its product phosphatidic acid (PA), and diacylglycerol pyrophosphate (DGPP) modulate Pro accumulation in barley during recovery from long-term chilling stress. Pro metabolism involves different pathways and enzymes. The rate-limiting step is mediated by pyrroline-5-carboxylate synthetase (P5CS) in its biosynthesis, and by proline dehydrogenase (ProDH) in its catabolism. We observed that Pro levels decreased in recovering barley roots due to an increase in ProDH activity. The addition of 1-butanol, a PLD inhibitor, reverted this effect and altered the relative gene expression of ProDH. When barley tissues were treated with PA before recovery, the fresh weight of roots increased and ProDH activity was stimulated. These data contribute to our understanding of how acidic membrane phospholipids like PA help to control Pro degradation during recovery from stress.

Inhibition of exosome biogenesis affects cell motility in heterogeneous sub-populations of paediatric-type diffuse high-grade gliomas

BackgroundPaediatric-type diffuse High-Grade Gliomas (PDHGG) are highly heterogeneous tumours which include distinct cell sub-populations co-existing within the same tumour mass. We have previously shown that primary patient-derived and optical barcoded single-cell-derived clones function as interconnected networks. Here, we investigated the role of exosomes as a route for inter-clonal communication mediating PDHGG migration and invasion.ResultsA comprehensive characterisation of seven optical barcoded single-cell-derived clones obtained from two patient-derived cell lines was performed. These analyses highlighted extensive intra-tumour heterogeneity in terms of genetic and transcriptional profiles between clones as well as marked phenotypic differences including distinctive motility patterns. Live single-cell tracking analysis of 3D migration and invasion assays showed that the single-cell-derived clones display a higher speed and longer travelled distance when in co-culture compared to mono-culture conditions. To determine the role of exosomes in PDHGG inter-clonal cross-talks, we isolated exosomes released by different clones and characterised them in terms of marker expression, size and concentration. We demonstrated that exosomes are actively internalized by the cells and that the inhibition of their biogenesis, using the phospholipase inhibitor GW4689, significantly reduced the cell motility in mono-culture and more prominently when the cells from the clones were in co-culture. Analysis of the exosomal miRNAs, performed with a miRNome PCR panel, identified clone-specific miRNAs and a set of miRNA target genes involved in the regulation of cell motility/invasion/migration. These genes were found differentially expressed in co-culture versus mono-culture conditions and their expression levels were significantly modulated upon inhibition of exosome biogenesis.ConclusionsIn conclusion, our study highlights for the first time a key role for exosomes in the inter-clonal communication in PDHGG and suggests that interfering with the exosome biogenesis pathway may be a valuable strategy to inhibit cell motility and dissemination for these specific diseases.

Unravelling the signaling pathway downstream of the neurokinin receptor in atrium: discovering novel anti-arrhythmic targets for treatment of atrial fibrillation

Abstract Background Stimulation of neurokinin receptor (NKR) 3 is anti-fibrillatory by prolonging the action potential duration (APD) of atrial cardiomyocytes via inhibition of a background potassium current (1). The NKR is a Gq/11 protein-coupled receptor, but the downstream intracellular pathway and the end-effector potassium (K+)-channel in atrium are unknown. We aimed at unravelling the components of this pathway in atrial cardiomyocytes. Methods Rabbit (New Zealand White) atrial cardiomyocytes were isolated from Langendorff-perfused hearts by enzymatic dissociation. Human immortalized atrial myocytes (HiAMs) were developed, and kindly provided by our collaborators (2). Adult human atrial cardiomyocytes were obtained by enzymatic dissociation from left atrial appendages (LAA) routinely removed from atrial fibrillation (AF) patients undergoing minimally invasive surgical pulmonary vein isolation in the clinics. Action potentials (APs) and K+ currents were recorded at 36.5°C with the amphotericin-B-perforated patch clamp technique. RNA was isolated from rabbit atrial tissue and sequenced on an Illumina HiSeq4000 platform. Results In rabbit atrial cardiomyocytes, stimulation of NKR3 prolonged APD by ∼50% and inhibited an background outward K+-current by ∼45%. Inhibition of phosphokinase C (PKC) did not prevent APD prolongation by NKR3 stimulation, but application of a diacylglycerol (DAG) analog inhibited an outward K+-current by ∼40% indicating that DAG, but not PKC, is downstream of NKR3. Inhibition of phosphatidylcholine-specific phospholipase C (PC-PLC), but not of phosphoinositide-specific PLC (PI-PLC), reduced APD increase by NKR3 stimulation by five-fold, indicating PC-PLC as the intracellular signaling molecule. In HiAMs, direct PLC activation prolonged APD by 34%. RNA sequencing analysis revealed potential end-targets of the NKR3 pathway in rabbit. Inhibition of small conductance potassium (KCNN2/KCNN3) or TASK-1 (KCNK3) channels (prolonging APD by ∼15%) did not abolish the APD prolongation effect by subsequent NKR3 stimulation (∼50%), indicating that these channels are not the end-effector K+-channels inhibited by NKR3 stimulation. Inhibition of constitutive KACh channels had little effect on APD at baseline. Nevertheless, APD shortening by agonist-induced KACh channel activity could be completely reversed by NKR3 stimulation, indicating that increased outward KACh current can be fully counterbalanced by the reduction in K+ background current. In human atrial cardiomyocytes from AF patients, stimulation of NKR3 prolonged APD by 25%, Conclusions PC-PLC and DAG, but not PI-PLC and PKC, are intracellularly downstream of NKR3 in atrial rabbit cardiomyocytes. Stimulation of the NKR pathway may have an anti-arrhythmic potential in patients with AF.

Inhibition of phospholipase C reduces the capacitation of cryopreserved ovine sperm

This study aimed to evaluate the effect of phospholipase C (PLC) on the capacitation of cryopreserved ovine semen. Sixteen semen samples were cryopreserved with diluent added by 0, 10, or 20 μM of U73122, a PLC inhibitor. The sperm kinetics of the thawed samples were evaluated using the “Computer-assisted Sperm Analysis” system, and the integrity of the plasma and mitochondrial membranes was evaluated using fluorescent probes. Additionally, sperm capacitation and the acrosome reaction with chlortetracycline hydrochloride were evaluated before and after capacitation induction. The results were analysed using analysis of variance and Tukey's test with a 95% probability. Concentrations of 10 or 20 μM of U73122 did not affect the kinetics or number of sperm with intact plasma and mitochondrial membranes. However, after thawing, 10 and 20 μM of the inhibitor reduced the percentage of capacitated and acrosome-reacted sperm. After induction of capacitation, there was a reduction in the number of non-capacitated sperm in all treatment groups, suggesting a reversible effect of U73122. In conclusion, U73122 at concentrations of 10 or 20 μM prevents premature capacitation and acrosome reaction induced by the freezing procedure, without affecting the kinetics and integrity of the sperm membranes.

Quantal size increase induced by the endocannabinoid 2-arachidonoylglycerol requires activation of CGRP receptors in mouse motor synapses.

In mouse motor synapses, the exogenous application of the endocannabinoid (EC) 2-arachidonoylglycerol (2-AG) increases acetylcholine (ACh) quantal size due to the activation of CB1 receptors and the stimulation of ACh vesicular uptake. In the present study, microelectrode recordings of miniature endplate potentials (MEPP) revealed that this effect of 2-AG is independent of brain-derived neurotrophic factor (BDNF) signaling but involves the activation of calcitonin gene-related peptide (CGRP) receptors along with CB1 receptors. Potentiation of MEPP amplitude in the presence of 2-AG was prevented by blockers of CGRP receptors and ryanodine receptors (RyR) and by inhibitors of phospholipase C (PLC) and Ca2+ /calmodulin-dependent protein kinase II (CaMKII). Therefore, we suggest a hypothetical chain of events, which starts from the activation of presynaptic CB1 receptors, involves PLC, RyR, and CaMKII, and results in CGRP release with the subsequent activation of presynaptic CGRP receptors. Activation of CGRP receptors is probably a part of a complex molecular cascade leading to the 2-AG-induced increase in ACh quantal size and MEPP amplitude. We propose that the same chain of events may also take place if 2-AG is endogenously produced in mouse motor synapses, because the increase in MEPP amplitude that follows after prolonged tetanic muscle contractions (30Hz, 2min) was prevented by the blocking of CB1 receptors. This work may help to unveil the previously unknown aspects of the functional interaction between ECs and peptide modulators aimed at the regulation of quantal size and synaptic transmission.

OGD/R-induced ferroptosis and pyroptosis in retinal pigment epithelium cells: Role of PLD1 and PLD2 modulation.

This study investigated the role of phospholipase D (PLD) in retinal ischemia-reperfusion (I/R) injury using an oxygen-glucose deprivation/reperfusion (OGD/R) model commonly used in retinal I/R injury research. To create an in vitro cellular I/R model, pharmacological inhibitors and small interfering RNA (siRNA) were used to target PLD1 and PLD2 in retinal pigment epithelial (RPE) cells. Treatment with PLD inhibitors and siRNA reduced reactive oxygen species (ROS) and malondialdehyde (MDA) induced by OGD/R in RPE cells and increased the levels of superoxide dismutase (SOD) and glutathione (GSH), indicating a reduction in oxidative damage and improvement in the antioxidant system. Next, we showed that inhibiting PLD1 or PLD2 reduced intracellular iron levels and lipid peroxidation, which are critical factors in ferroptosis. Additionally, PLD1 and PLD2 modulated the expression of proteins involved in the regulation of ferroptosis, including GPX4, SLC7A11, FTH1, and ACSL4. We also investigated the roles of PLD1 and PLD2 in preventing pyroptosis, another form of programmed cell death associated with inflammation. Our study found that OGD/R significantly increased the production of pro-inflammatory cytokines and activated caspase-1, NLRP3, ASC, cleaved-caspase 1 (C-caspase-1), and GSDMD-N in RPE cells, indicating pyroptosis induction. However, PLD1 and PLD2 inhibition or knockdown significantly inhibited the production of pro-inflammatory cytokines and activation of the NLRP3 inflammasome, Taken together, our findings support the hypothesis that the PLD signaling pathway plays a key role in OGD/R-induced ferroptosis and pyroptosis induction and may be a potential therapeutic target for preventing or treating retinal dysfunction and degeneration.

A novel in vitro model to study prolonged Pseudomonas aeruginosa infection in the cystic fibrosis bronchial epithelium.

Pseudomonas aeruginosa (PA) is known to chronically infect airways of people with cystic fibrosis (CF) by early adulthood. PA infections can lead to increased airway inflammation and lung tissue damage, ultimately contributing to decreased lung function and quality of life. Existing models of PA infection in vitro commonly utilize 1-6-hour time courses. However, these relatively early time points may not encompass downstream airway cell signaling in response to the chronic PA infections observed in people with cystic fibrosis. To fill this gap in knowledge, the aim of this study was to establish an in vitro model that allows for PA infection of CF bronchial epithelial cells, cultured at the air liquid interface, for 24 hours. Our model shows with an inoculum of 2 x 102 CFUs of PA for 24 hours pro-inflammatory markers such as interleukin 6 and interleukin 8 are upregulated with little decrease in CF bronchial epithelial cell survival or monolayer confluency. Additionally, immunoblotting for phosphorylated phospholipase C gamma, a well-known downstream protein of fibroblast growth factor receptor signaling, showed significantly elevated levels after 24 hours with PA infection that were not seen at earlier timepoints. Finally, inhibition of phospholipase C shows significant downregulation of interleukin 8. Our data suggest that this newly developed in vitro "prolonged PA infection model" recapitulates the elevated inflammatory markers observed in CF, without compromising cell survival. This extended period of PA growth on CF bronchial epithelial cells will have impact on further studies of cell signaling and microbiological studies that were not possible in previous models using shorter PA exposures.

Novel small molecule MRGPRX2 antagonists inhibit a murine model of allergic reaction

Activation of Mas-related G protein-coupled receptor X2 (MRGPRX2) is a crucial non-IgE pathway for mast cell activation associated with allergic reactions and inflammation. Only a few peptides and small compounds targeting MRGPRX2 have been reported, with limited information on their pharmacologic activity. We sought to develop novel small molecule MRGPRX2 antagonists to treat MRGPRX2-mediated allergies and inflammation. A computational approach was used to design novel small molecules as MRGPRX2 antagonists. The short-listed molecules were synthesized and characterized by liquid chromatography and mass spectrometry as well as nuclear magnetic resonance. Inhibitory activity on MRGPRX2 signaling was assessed invitro by using functional bioassays (β-hexosaminidase, calcium flux, and chemokine synthesis) and receptor activation assays (β-arrestin recruitment and Western blot analysis) in human LAD-2 mast cells and HTLA cells. Invivo effects of the novel MRGPRX2 antagonists were assessed using a mouse model of acute allergy and systemic anaphylaxis. The novel small molecules demonstrated higher binding affinity with MRGPRX2 in the docking study. The half-maximal inhibitory concentration is in the low micromolar range (5-21 μM). The small molecules inhibited not only the early phase of mast cell activation but also the late phase, associated with chemokine and prostaglandin release. Further, Western blot analysis revealed inhibition of downstream phospholipase C-γ, extracellular signal-regulated protein kinase 1/2, and Akt signaling pathway. Moreover, in the mouse models of allergies, small molecule administration effectively blocks acute, systemic allergic reactions and inflammation and prevents systemic anaphylaxis. The small molecules might hold a significant therapeutic promise to treat MRGPRX2-mediated allergies and inflammation.