IP3 Receptor Research Articles

KRAP regulates mitochondrial Ca2+ uptake by licensing IP3 receptor activity and stabilizing ER-mitochondrial junctions.

Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) are high-conductance channels that allow the regulated redistribution of Ca2+ from the endoplasmic reticulum (ER) to the cytosol and, at specialized membrane contact sites (MCSs), to other organelles. Only a subset of IP3Rs release Ca2+ to the cytosol in response to IP3. These 'licensed' IP3Rs are associated with Kras-induced actin-interacting protein (KRAP, also known as ITPRID2) beneath the plasma membrane. It is unclear whether KRAP regulates IP3Rs at MCSs. We show, using simultaneous measurements of Ca2+ concentration in the cytosol and mitochondrial matrix, that KRAP also licenses IP3Rs to release Ca2+ to mitochondria. Loss of KRAP abolishes cytosolic and mitochondrial Ca2+ signals evoked by stimulation of IP3Rs via endogenous receptors. KRAP is located at ER-mitochondrial membrane contact sites (ERMCSs) populated by IP3R clusters. Using a proximity ligation assay between IP3R and voltage-dependent anion channel 1 (VDAC1), we show that loss of KRAP reduces the number of ERMCSs. We conclude that KRAP regulates Ca2+ transfer from IP3Rs to mitochondria by both licensing IP3R activity and stabilizing ERMCSs.

Cellular Model for the Analysis of IRBIT-Dependent Regulation of the Type 1 IP<sub>3</sub> Receptor

In vertebrate genomes, three genes encode subunits of IP3 receptors, including IP3R1, IP3R2, and IP3R3. Despite high homology between different subunits, homotetrameric IP3 receptors formed by IP3R1, IP3R2, and IP₃R3 in the endoplasmic reticulum membrane are markedly distinct by their functional features and regulatory mechanisms. It was particularly reported that IP3R1 is specifically regulated by the IP3R binding protein released with IP₃ (IRBIT), which competes with IP3 for binding to IP3R1. In turn, affinity of IRBIT/IP₃R1 binding is regulated by phosphorylation of IRBIT. By using the CRISPR/Cas9 approach to edit the genome of HEK-293 cells, two monoclonal cell lines were generated as a platform for uncovering a role of IRBIT and associated regulatory circuits in control of the IP₃R1 activity. In one line, HEK-IP3R1, IP₃R2, and IP3R3 genes were disrupted, while IP₃R1 was remained functional. Based on this line, the HEK-IP3R1/DIRBIT line was generated, wherein IRBIT (AHCYL1) gene was inactivated. The comparative analysis of ACh-induced Ca2+ signaling in cells of both lines was performed by employing the Ca2+ dye Fluo-4 and Ca2+ imaging. It was particularly shown that ACh mobilized Ca2+ in cells of both lines, which responded to the agonist at widely varied doses in an “all-or-nothing” manner. Yet, HEK-IP₃R1/DIRBIT cells turned out to be less sensitive to ACh compared to HEK-IP₃R1 cells.

Effectiveness of a Prostacyclin IP Receptor Agonist in Patients With Pulmonary Arterial Hypertension in the Real-world Andalusian Setting: The RAMPHA Study

PurposeThe pillar for therapeutic decisions in the evolution of pulmonary arterial hypertension (PAH) is the patients’ prognostic stratification. MethodsA retrospective cohort study was conducted in a Spanish real-world setting to assess the clinical improvement of PAH patients treated with selexipag measured as changes in the risk profile. Secondary objectives were to describe their baseline characteristics, initial risk status, and variables used to assess patient survival and adverse events. FindingsTotal 42 patients (mean age 52.36 [SD: 15.09] years) were included. All had received initial endothelin receptor antagonist treatment and 95.2% dual therapy with phosphodiesterase-5 inhibitor or riociguat. At 6 to 12 months from baseline, patients risk stratification tripled the percentage of patients with low risk, and a trend towards improved risk stratification (P = 0.122). World Health Organization functional class changed, with more patients in milder classes (P = 0.003), and symptom progression slowed down (P < 0.0001). At 3-years, survival was 85.7% and the estimated median survival time was 2.73 years (SD: 1.351; 95% CI: 2.51–2.95). ImplicationsSelexipag did not achieve a significant improvement in risk profile, although it did show an excellent survival rate, effectively improved functional class, and delayed symptom progression in real life. Selexipag was well tolerated and showed a favorable safety profile, supporting a clinical benefit for PAH patients.

Regulatory disturbances in the dynamical signaling systems of and NO in fibroblasts cause fibrotic disorders.

Studying the calcium dynamics within a fibroblast cell individually has provided only a restricted understanding of its functions. However, research efforts focusing on systems biology approaches for such investigations have been largely neglected by researchers until now. Fibroblast cells rely on signaling from calcium and nitric oxide (NO) to maintain their physiological functions and structural stability. Various studies have demonstrated the correlation between NO and the control of dynamics in cells. However, there is currently no existing model to assess the disruptions caused by various factors in regulatory dynamics, potentially resulting in diverse fibrotic disorders. A mathematical model has been developed to investigate the effects of changes in parameters such as buffer, receptor, sarcoplasmic endoplasmic reticulum -ATPase (SERCA) pump, and source influx on the regulation and dysregulation of spatiotemporal calcium and NO dynamics in fibroblast cells. This model is based on a system of reaction-diffusion equations, and numerical simulations are conducted using the finite element method. Disturbances in key processes related to calcium and nitric oxide, including source influx, buffer mechanism, SERCA pump, and inositol trisphosphate receptor, may contribute to deregulation in the calcium and NO dynamics within fibroblasts. The findings also provide new insights into the extent and severity of disorders resulting from alterations in various parameters, potentially leading to deregulation and the development of fibrotic disease.

Nobiletin regulates intracellular Ca2+ levels via IP3R and ameliorates neuroinflammation in Aβ42-induced astrocytes

Astrocytes are the major glial cells in the human brain and provide crucial metabolic and trophic support to neurons. The amyloid-β peptide (Aβ) alter the morphological and functional properties of astrocytes and induce inflammation and calcium dysregulation, contributing to Alzheimer's disease (AD) pathology. Recent studies highlight the role of Toll-like receptor (TLR) 4/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling in inflammation. Reactive oxygen species (ROS) generated due to Aβ, induce apoptosis in the brain cells worsening AD progression. Astrocytic cell surface receptors, such as purinergic receptors (P2Y1 and P2Y2), metabotropic glutamate receptor (mGLUR)5, α7 nicotinic acetylcholine receptor (α7nAChR), and N-methyl-d-aspartate receptors (NMDARs), have been suggested to interact with inositol trisphosphate receptor (IP3R) on the endoplasmic reticulum (ER) to induce Ca2+ movement from ER to cytoplasm, causing Ca2+ dysregulation. We found that the citrus flavonoid nobiletin (NOB) protected primary astrocytes from Aβ42-induced cytotoxicity and inhibited TLR4/NF-κB signaling in Aβ42-induced primary rat astrocytes. NOB was found to regulate Aβ42-induced ROS levels through Keap1-Nrf2 pathway. The receptors P2Y1, P2Y2, mGLUR5, α7nAChR, and NMDARs induced intracellular Ca2+ levels by activating IP3R and NOB regulated them, thereby regulating intracellular Ca2+ levels. Molecular docking analysis revealed a possible interaction between NOB and IP3R in IP3R regulation. Furthermore, RNA sequencing revealed various NOB-mediated biological signaling pathways, such as the AD-presenilin, AD-amyloid secretase, and Wnt signaling pathway, suggesting possible neuroprotective roles of NOB. To conclude, NOB is a promising therapeutic agent for AD and works by modulating AD pathology at various levels in Aβ42-induced primary rat astrocytes.

Lignan 7-hydroxymatairesinol in the context of post-genomic pharmacology

Objective: to determine the molecular mechanisms of action of 7-hydroxymatairesinol (7(OH)MR), the main lignan of spruce extracts, characterized by oncoprotective and anti-inflammatory properties.Material and methods. The analysis of 7(OH)MR was carried out by chemoinformatic approach using the combinatorial theory of solvability and the topological theory of recognition. The postgenomic approach makes it possible to assess the effect of drugs on genome transcription (transcriptome) and on the proteome as a whole.Results. 7(OH)MP has anti-inflammatory (inhibition of 5-lipoxygenase, matrix metalloproteinase 2, mitogen-activated kinase p38-alpha, leukotriene В4 receptor, prostacyclin receptor), and oncoprotective (antioxidant effect due to inhibition of heme oxygenase-2, inhibition of cyclin-dependent kinases 3 and 4, epidermal growth factor, mTOR protein) pharmacological effects.Conclusion. By reducing the expression of proliferative genes and genes involved in chronic inflammation, the 7(OH)MP molecule inhibits the proliferation of tumor cells. Pharmacoinformatic modeling showed that the anti-inflammatory effects of 7(OH)MR may contribute to increased lifespan in animal models.

17-β-estradiol and phytoestrogens elicit NO production and vasodilatation through PI3K, PKA and EGF receptors pathways, evidencing functional selectivity

Endothelial cells express multiple receptors mediating estrogen responses; including the G protein-coupled estrogen receptor (GPER). Past studies on nitric oxide (NO) production elicited by estrogens raised the question whether 17-β-estradiol (E2) and natural phytoestrogens activate equivalent mechanisms. We hypothesized that E2 and phytoestrogens elicit NO production via coupling to distinct intracellular pathways signalling. To this aim, perfusion of E2 and phytoestrogens to the precontracted rat mesentery bed examined vasorelaxation, while fluorescence microscopy on primary endothelial cells cultures quantified single cell NO production determined following 4-amino-5-methylamino-2′,7′-difluoroescein diacetate (DAF) incubation. Daidzein (DAI) and genistein (GEN) induced rapid vasodilatation associated to NO production. Multiple estrogen receptor activity was inferred based on the reduction of DAF-NO signals; G-36 (GPER antagonist) reduced 75 % of all estrogen responses, while fulvestrant (selective nuclear receptor antagonist) reduced significantly more the phytoestrogens responses than E2. The joint application of both antagonists abolished the E2 response but not the phytoestrogen-induced DAF-NO signals. Wortmannin or LY-294002 (PI3K inhibitors), reduced by 90% the E2-evoked signal while altering significantly less the DAI-induced response. In contrast, H-89 (PKA inhibitor), elicited a 23% reduction of the E2-induced signal while blocking 80% of the DAI-induced response. Desmethylxestospongin-B (IP3 receptor antagonist), decreased to equal extent the E2 or the DAI-induced signal. Epidermal growth factor (EGF) induced NO production, cell treatment with AG-1478, an EGF receptor kinase inhibitor reduced 90% DAI-induced response while only 53% the E2-induced signals; highlighting GPER induced EGF receptor trans-modulation. Receptor functional selectivity may explain distinct signalling pathways mediated by E2 and phytoestrogens.

Mitochondria control IP3-evoked Ca2+ release triggered by voltage-dependent Ca2+ entry in intact resistance arteries

The contractility of vascular smooth muscle cells (VSMCs) is a major contributor of vascular tone and blood pressure. In VSMCs, intracellular Ca2+ level determines the contractile activity that generates the force to contract arteries. A major source of Ca2+ is via influx through voltage dependent Ca2+ channels (VDCCs). While mitochondria are now recognised as key regulators of intracellular Ca2+ homeostasis in several cell types, their role in modulating Ca2+ signalling mediated by VDCC in VSMCs is unresolved. Given the potential physiological significance, and unclarified interaction between mitochondria and VDCCs in VSMCs, we hypothesize that mitochondria directly regulate Ca2+ signalling mediated by VDCCs. The interplay between mitochondria and VDCCs was investigated by imaging and analysing intracellular Ca2+ signals in smooth muscle cells in intact arteries from rat mesentery. Depolarization of the plasma membrane potential, by high potassium (20 mM) physiological saline solution, triggered Ca2+ influx through VDCCs and a sustained increase in intracellular Ca2+ on which repetitive Ca2+ oscillations occurred. All Ca2+ signals were abolished by removal of external Ca2+ and by dihydropyridine inhibitors of VDCCs. Significantly, the repetitive Ca2+ oscillations, but not the sustained Ca2+ signals, were blocked by the IP3 receptor inhibitor 2-APB and SERCA inhibitor cyclopiazonic acid. Neither the repetitive Ca2+ oscillations or the sustained Ca2+ signals were altered by the ryanodine receptor inhibitors ryanodine and dantrolene. These results suggest that Ca2+ influx via VDCC triggers Ca2+-induced Ca2+ release at IP3 receptors in intact mesenteric arteries. Depolarization of the mitochondrial membrane potential (ψm), with the uncoupler CCCP or complex I inhibitor rotenone, but not ATP deprivation with the ATP synthesis blocker oligomycin, inhibited VDCC evoked IP3 mediated Ca2+ oscillations but not the sustained Ca2+ signals. Furthermore, in intact arteries, depolarization of ψm directly suppressed inositol triphosphate receptors (IP3Rs) mediated Ca2+ release from the internal Ca2+ store evoked by photolysis of caged IP3. These results suggest that mitochondria regulate Ca2+ release via IP3R triggered by Ca2+ entry via VDCC. In return, Ca2+ entry via VDCCs did not alter ψm, but upregulated ROS production. Together, these results suggest that mitochondria regulate Ca2+-induced Ca2+ release at IP3 receptors triggered by Ca2+ influx via VDCCs but do not directly regulate VDCC activity. British Heart Foundation (RG/F/20/110007; PG/20/9/34859). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Voltage-induced calcium release in Caenorhabditis elegans body muscles

Type 1 voltage-activated calcium channels (CaV1) in the plasma membrane trigger calcium release from the sarcoplasmic reticulum (SR) by two mechanisms. In voltage-induced calcium release (VICR), CaV1 voltage sensing domains are directly coupled to ryanodine receptors (RYRs), an SR calcium channel. In calcium-induced calcium release (CICR), calcium ions flowing through activated CaV1 channels bind and activate RYR channels. VICR is thought to occur exclusively in vertebrate skeletal muscle while CICR occurs in all other muscles (including all invertebrate muscles). Here, we use calcium-activated SLO-2 potassium channels to analyze CaV1-SR coupling in Caenorhabditis elegans body muscles. SLO-2 channels were activated by both VICR and external calcium. VICR-mediated SLO-2 activation requires two SR calcium channels (RYRs and IP3 Receptors), JPH-1/Junctophilin, a PDZ (PSD95, Dlg1, ZO-1 domain) binding domain (PBD) at EGL-19/CaV1's carboxy-terminus, and SHN-1/Shank (a scaffolding protein that binds EGL-19's PBD). Thus, VICR occurs in invertebrate muscles.

Calreticulin regulates hepatic stellate cell activation through modulating TGF-beta-induced Smad signaling

Liver fibrosis is characterized by excessive deposition of extracellular matrix (ECM) as a wound healing process. Activated hepatic stellate cells (HpSCs) are the major producer of the ECM and play a central role in liver fibrogenesis. It has been widely accepted that elimination of activated HpSCs or reversion to a quiescent state can be a feasible strategy for resolving the disease, further highlighting the urgent need for novel therapeutic targets. Calreticulin (CRT) is a molecular chaperone that normally resides in the endoplasmic reticulum (ER), important in protein folding and trafficking through the secretory pathway. CRT also plays a critical role in calcium (Ca2+) homeostasis, with its Ca2+ storage capacity. In the current study, we aimed to demonstrate its function in directing HpSC activation. In a mouse liver injury model, CRT was up-regulated in HpSCs. In cellular experiments, we further showed that this activation was through modulating the canonical TGF-β signaling. As down-regulation of CRT in HpSCs elevated intracellular Ca2+ levels through a form of Ca2+ influx, named store-operated Ca2+ entry (SOCE), we examined whether moderating SOCE affected TGF-β signaling. Interestingly, blocking SOCE had little effect on TGF-β-induced gene expression. In contrast, inhibition of ER Ca2+ release using the inositol trisphosphate receptor inhibitor 2-APB increased TGF-β signaling. Treatment with 2-APB did not alter SOCE but decreased intracellular Ca2+ at the basal level. Indeed, adjusting Ca2+ concentrations by EGTA or BAPTA-AM chelation further enhanced TGF-β-induced signaling. Our results suggest a crucial role of CRT in the liver fibrogenic process through modulating Ca2+ concentrations and TGF-β signaling in HpSCs, which may provide new information and help advance the current discoveries for liver fibrosis.

Transcranial direct current stimulation alters cerebrospinal fluid-interstitial fluid exchange in mouse brain

BackgroundTranscranial direct current stimulation (tDCS) is a non-invasive brain stimulation technique that has gained prominence recently. Clinical studies have explored tDCS as an adjunct to neurologic disease rehabilitation, with evidence suggesting its potential in modulating brain clearance mechanisms. The glymphatic system, a proposed brain waste clearance system, posits that cerebrospinal fluid-interstitial fluid (CSF-ISF) exchange aids in efficient metabolic waste removal. While some studies have linked tDCS to astrocytic inositol trisphosphate (IP3)/Ca2+ signaling, the impact of tDCS on CSF-ISF exchange dynamics remains unclear. HypothesistDCS influences the dynamics of CSF-ISF exchange through astrocytic IP3/Ca2+ signaling. MethodsIn this study, we administered tDCS (0.1 mA for 10 min) to C57BL/6N mice anesthetized with ketamine-xylazine (KX). The anode was positioned on the cranial bone above the cortex, and the cathode was inserted into the neck. Following tDCS, we directly assessed brain fluid dynamics by injecting biotinylated dextran amine (BDA) as a CSF tracer into the cisterna magna (CM). The brain was then extracted after either 30 or 60 min and fixed. After 24 h, the sectioned brain slices were stained with Alexa 594-conjugated streptavidin (SA) to visualize BDA using immunohistochemistry. We conducted Electroencephalography (EEG) recordings and aquaporin 4 (AQP4)/CD31 immunostaining to investigate the underlying mechanisms of tDCS. Additionally, we monitored the efflux of Evans blue, injected into the cisterna magna, using cervical lymph node imaging. Some experiments were subsequently repeated with inositol trisphosphate receptor type 2 (IP3R2) knockout (KO) mice. ResultsPost-tDCS, we observed an increased CSF tracer influx, indicating a modulation of CSF-ISF exchange by tDCS. Additionally, tDCS appeared to enhance the brain's metabolic waste efflux. EEG recordings showed an increase in delta wave post-tDCS. But no significant change in AQP4 expression was detected 30 min post-tDCS. Besides, we found no alteration in CSF-ISF exchange and delta wave activity in IP3R2 KO mice after tDCS. ConclusionOur findings suggest that tDCS augments the glymphatic system's influx and efflux. Through astrocytic IP3/Ca2+ signaling, tDCS was found to modify the delta wave, which correlates positively with brain clearance. This study underscores the potential of tDCS in modulating brain metabolic waste clearance.

Switching from Beraprost to Selexipag in the Treatment of Pulmonary Arterial Hypertension: Insights from a Phase IV Study of the Japanese Registry (The EXCEL Study: EXChange from bEraprost to seLexipag Study).

Pulmonary arterial hypertension (PAH) remains a significant challenge in cardiology, necessitating advancements in treatment strategies. This study explores the safety and efficacy of transitioning patients from beraprost to selexipag, a novel selective prostacyclin receptor agonist, within a Japanese cohort. Employing a multicenter, open-label, prospective design, 25 PAH patients inadequately managed on beraprost were switched to selexipag. Key inclusion criteria included ongoing beraprost therapy for ≥3 months, a diagnosis of PAH confirmed by mean pulmonary artery pressure (mPAP) ≥ 25 mmHg, and current treatment with endothelin receptor antagonists and/or phosphodiesterase type 5 inhibitors. Outcomes assessed were changes in hemodynamic parameters (mPAP, cardiac index, pulmonary vascular resistance) and the 6 min walk distance (6-MWD) over 3-6 months. The study found no statistically significant changes in these parameters post-switch. However, a subset of patients, defined as responders, demonstrated improvements in all measured hemodynamic parameters, suggesting a potential benefit in carefully selected patients. The transition was generally well-tolerated with no serious adverse events reported. This investigation underscores the importance of personalized treatment strategies in PAH, highlighting that certain patients may benefit from switching to selexipag, particularly those previously on higher doses of beraprost. Further research is needed to elucidate the predictors of positive response to selexipag and optimize treatment regimens for this complex condition.

Weighted gene co-expression network analysis reveals key biomarkers and immune infiltration characteristics for bronchial epithelial cells from asthmatic patients.

Asthma ranks among the most prevalent non-communicable diseases worldwide. Previous studies have elucidated the significant role of the immune system in its pathophysiology. Nevertheless, the immune-related mechanisms underlying asthma are complex and still inadequately understood. Thus, our objective was to investigate novel key biomarkers and immune infiltration characteristics associated with asthma by employing integrated bioinformatics tools. In this study, we conducted a weighted gene co-expression network analysis (WGCNA) to identify key modules and genes potentially implicated in asthma. Functional annotation of these key modules and genes was carried out through gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Additionally, we constructed a protein-protein interaction (PPI) network using the STRING database to identify 10 hub genes. Furthermore, we evaluated the relative proportion of immune cells in bronchial epithelial cell samples from 20 healthy individuals and 88 asthmatic patients using CIBERSORT. Finally, we validated the hub genes and explored their correlation with immune infiltration. Furthermore, 20 gene expression modules and 10 hub genes were identified herein. Among them, complement component 3 (C3), prostaglandin I2 receptor (PTGIR), parathyroid hormone-like hormone (PTHLH), and C-X3-C motif chemokine ligand 1 (CX3CL1) were closely correlated with the infiltration of immune cells. They may be novel candidate biomarkers or therapeutic targets for asthma. Furthermore, B cells memory, and plasma cells might play an important role in immune cell infiltration after asthma. C3, PTGIR, CX3CL1, and PTHLH have important clinical diagnostic values and are correlated with infiltration of multiple immune cell types in asthma. These hub genes, B cells memory, and plasma cells may become important biological targets for therapeutic asthma drug screening and drug design.

Role of the 3-mercaptopyruvate sulfurtransferase in colon/colorectal cancers

The 3-mercaptopyruvate sulfurtransferase (MPST) is a protein persulfidase, occurring mainly in mitochondria. Although function of this protein in cancer cells has been already studied, no clear outcome can be postulated up to now. Therefore, we focused on the determination of function of MPST in colon (HCT116 cells)/colorectal (DLD1 cells) cancers. In silico analysis revealed that in gastrointestinal cancers, MPST together with its binding partners can be either of a high risk or might have a protective effect. Silencing of MPST gene resulted in decreased ATP, while acetyl-CoA levels were elevated. Increased apoptosis was detected in cells with silenced MPST gene, which was accompanied by decrease in mitochondrial membrane potential, but no changes in IP3 receptor’s protein. Mitochondria underwent activation of fission and elevated DRP1 expression after MPST silencing. Proliferation and migration of DLD1 and HCT116 cells were markedly affected, showing the importance of MPST protein in colon/colorectal cancer development.

Regulation of BCR-mediated Ca2+ mobilization by MIZ1-TMBIM4 safeguards IgG1+ GC B cell-positive selection.

The transition from immunoglobulin M (IgM) to affinity-matured IgG antibodies is vital for effective humoral immunity. This is facilitated by germinal centers (GCs) through affinity maturation and preferential maintenance of IgG+ B cells over IgM+ B cells. However, it is not known whether the positive selection of the different Ig isotypes within GCs is dependent on specific transcriptional mechanisms. Here, we explored IgG1+ GC B cell transcription factor dependency using a CRISPR-Cas9 screen and conditional mouse genetics. We found that MIZ1 was specifically required for IgG1+ GC B cell survival during positive selection, whereas IgM+ GC B cells were largely independent. Mechanistically, MIZ1 induced TMBIM4, an ancestral anti-apoptotic protein that regulated inositol trisphosphate receptor (IP3R)-mediated calcium (Ca2+) mobilization downstream of B cell receptor (BCR) signaling in IgG1+ B cells. The MIZ1-TMBIM4 axis prevented mitochondrial dysfunction-induced IgG1+ GC cell death caused by excessive Ca2+ accumulation. This study uncovers a unique Ig isotype-specific dependency on a hitherto unidentified mechanism in GC-positive selection.

Dilation of Pregnant Rat Uterine Arteries with Phenols from Extra Virgin Olive Oil Is Endothelium-Dependent and Involves Calcium and Potassium Channels.

During pregnancy, uterine vasculature undergoes significant circumferential growth to increase uterine blood flow, vital for the growing feto-placental unit. However, this process is often compromised in conditions like maternal high blood pressure, particularly in preeclampsia (PE), leading to fetal growth impairment. Currently, there is no cure for PE, partly due to the adverse effects of anti-hypertensive drugs on maternal and fetal health. This study aimed to investigate the vasodilator effect of extra virgin olive oil (EVOO) phenols on the reproductive vasculature, potentially benefiting both mother and fetus. Isolated uterine arteries (UAs) from pregnant rats were tested with EVOO phenols in a pressurized myograph. To elucidate the underlying mechanisms, additional experiments were conducted with specific inhibitors: L-NAME/L-NNA (10-4 M) for nitric oxide synthases, ODQ (10-5 M) for guanylate cyclase, Verapamil (10-5 M) for the L-type calcium channel, Ryanodine (10-5 M) + 2-APB (3 × 10-5 M) for ryanodine and the inositol triphosphate receptors, respectively, and Paxilline (10-5 M) for the large-conductance calcium-activated potassium channel. The results indicated that EVOO-phenols activate Ca2+ signaling pathways, generating nitric oxide, inducing vasodilation via cGMP and BKCa2+ signals in smooth muscle cells. This study suggests the potential use of EVOO phenols to prevent utero-placental blood flow restriction, offering a promising avenue for managing PE.

After wounding, a G-protein coupled receptor promotes the restoration of tension in epithelial cells.

The maintenance of epithelial barrier function involves cellular tension, with cells pulling on their neighbors to maintain epithelial integrity. Wounding interrupts cellular tension, which may serve as an early signal to initiate epithelial repair. To characterize how wounds alter cellular tension we used a laser-recoil assay to map cortical tension around wounds in the epithelial monolayer of the Drosophila pupal notum. Within a minute of wounding, there was widespread loss of cortical tension along both radial and tangential directions. This tension loss was similar to levels observed with Rok inactivation. Tension was subsequently restored around the wound, first in distal cells and then in proximal cells, reaching the wound margin ∼10 min after wounding. Restoring tension required the GPCR Mthl10 and the IP3 receptor, indicating the importance of this calcium signaling pathway known to be activated by cellular damage. Tension restoration correlated with an inward-moving contractile wave that has been previously reported; however, the contractile wave itself was not affected by Mthl10 knockdown. These results indicate that cells may transiently increase tension and contract in the absence of Mthl10 signaling, but that pathway is critical for fully resetting baseline epithelial tension after it is disrupted by wounding.

Agonist-Induced Ca2+ Signaling in HEK-293-Derived Cells Expressing a Single IP3 Receptor Isoform.

In mammals, three genes encode IP3 receptors (IP3Rs), which are involved in agonist-induced Ca2+ signaling in cells of apparently all types. Using the CRISPR/Cas9 approach for disruption of two out of three IP3R genes in HEK-293 cells, we generated three monoclonal cell lines, IP3R1-HEK, IP3R2-HEK, and IP3R3-HEK, with the single functional isoform, IP3R1, IP3R2, and IP3R3, respectively. All engineered cells responded to ACh with Ca2+ transients in an "all-or-nothing" manner, suggesting that each IP3R isotype was capable of mediating CICR. The sensitivity of cells to ACh strongly correlated with the affinity of IP3 binding to an IP3R isoform they expressed. Based on a mathematical model of intracellular Ca2+ signals induced by thapsigargin, a SERCA inhibitor, we developed an approach for estimating relative Ca2+ permeability of Ca2+ store and showed that all three IP3R isoforms contributed to Ca2+ leakage from ER. The relative Ca2+ permeabilities of Ca2+ stores in IP3R1-HEK, IP3R2-HEK, and IP3R3-HEK cells were evaluated as 1:1.75:0.45. Using the genetically encoded sensor R-CEPIA1er for monitoring Ca2+ signals in ER, engineered cells were ranged by resting levels of stored Ca2+ as IP3R3-HEK ≥ IP3R1-HEK > IP3R2-HEK. The developed cell lines could be helpful for further assaying activity, regulation, and pharmacology of individual IP3R isoforms.

Recent advances in canonical versus non-canonical Ca2+-signaling-related anti-apoptotic Bcl-2 functions and prospects for cancer treatment

Cell fate is tightly controlled by a continuous balance between cell survival and cell death inducing mechanisms. B-cell lymphoma 2 (Bcl-2)-family members, composed of effectors and regulators, not only control apoptosis at the level of the mitochondria but also by impacting the intracellular Ca2+ homeostasis and dynamics. On the one hand, anti-apoptotic protein Bcl-2, prevents mitochondrial outer membrane permeabilization (MOMP) by scaffolding and neutralizing proapoptotic Bcl-2-family members via its hydrophobic cleft (region composed of BH-domain 1–3). On the other hand, Bcl-2 suppress pro-apoptotic Ca2+ signals by binding and inhibiting IP3 receptors via its BH4 domain, which is structurally exiled from the hydrophobic cleft by a flexible loop region (FLR). As such, Bcl-2 prevents excessive Ca2+ transfer from ER to mitochondria. Whereas regulation of both pathways requires different functional regions of Bcl-2, both seem to be connected in cancers that overexpress Bcl-2 in a life-promoting dependent manner. Here we discuss the anti-apoptotic canonical and non-canonical role, via calcium signaling, of Bcl-2 in health and cancer and evolving from this the proposed anti-cancer therapies with their shortcomings. We also argue how some cancers, with the major focus on diffuse large B-cell lymphoma (DLBCL) are difficult to treat, although theoretically prime marked for Bcl-2-targeting therapeutics. Further work is needed to understand the non-canonical functions of Bcl-2 also at organelles beyond the mitochondria, the interaction partners outside the Bcl-2 family as well as their ability to target or exploit these functions as therapeutic strategies in diseases.

Spasmolytic Effect of Flopropione Does Not Involve Catechol-O- methyltransferase (COMT) Inhibition.

Flopropione (Flo) has been used for gallstone and urolithiasis as a spasmolytic agent almost exclusively in Japan. According to the package insert, its main mechanism is catechol-O-methyltransferase (COMT) inhibition and anti-serotonergic effect. This is obviously contrary to pharmacological common sense, but it is described that way in pharmacology textbooks and occurs in questions in the National Examination for Pharmacists in Japan. As this is a serious problem in education, we re-examined the action of Flo. The guinea pig ureter was hardly contracted by serotonin, but noradrenaline (NA) elicited repetitive twitch contraction, which was inhibited by Flo. The sphincter of Oddi (SO) exhibited a spontaneous repetitive twitch contraction, which was inhibited by NA and Flo. The inhibitory effect of NA was reversed by α- and β-blockers, whereas that of Flo was not. Entacapone, a representative COMT inhibitor, did not affect the movement of the ureter and the SO. Nifedipine suppressed carbachol-induced contraction of the taenia coli, spontaneous movement of the SO, and NA-induced contraction of the ureter to almost the same extent, whereas Flo did not inhibit the taenia coli, but inhibited the contraction of the SO and the ureter. The inhibitory pattern of Flo resembled that of the ryanodine receptor agonist 4-chloro-m-cresol and the inositol 1,4,5-trisphosphate (IP3) receptor antagonist 2-aminoethoxydiphenyl borate. It is concluded that COMT inhibition or serotonin inhibition is not involved in the spasmolytic action of Flo. Flo might act on ryanodine receptors and/or IP3 receptors, which are responsible for periodic Ca release from Ca stores, to disrupt coordinated Ca dynamics.