Purinergic Stimulation Research Articles

Modulation of purinergic signaling in endothelial cells by tumor microenvironment

Purinergic signaling plays a crucial role in vascular endothelium functions. In particular, ionotropic P2X receptors (P2XRs) are engaged in various intracellular pathways through which endothelial cells (ECs) adapt to external stimuli. However, very little is known about the impact of P2XRs on vascular remodeling during carcinogenesis.We previously demonstrated that high purinergic stimulation impairs the migratory phenotype of tumor-derived endothelial cells (TECs) but not of normal ECs.Since P2XRs are sensitive to different physical and chemical factors, we investigated the impact of tumor microenvironment (TME) on healthy ECs to verify the ability of cancer cells to affect endothelial migratory phenotype through purinergic signaling tuning. More specifically, we focused on P2XR modulation by two different types of TME, mimicking breast and pancreas cancer milieux, which show very different features in terms of vascularization and composition. ECs conditioning with both cancer cell types induced a significant upregulation of some of the most represented P2XR. However, only conditioning with MCF-7 cells and not that with PANC-1 cells was able to alter the migratory phenotype of normal ECs supporting a P2XR-mediated inhibition of cell migration. The differences observed between the two cancer cells could be due to their different proliferative potential and the subsequent different extracellular pH. In addition, in agreement with some of our previous data, the P2XR-induced inhibition of EC migration seems to be independent of calcium signals, as conditioned ECs didn't reveal any changes in the long-lasting responses evoked by purinergic agonists.Collectively, highlighting a significant P2RX modulation by TME, our data strengthen the hypothesis that purinergic signaling may play a central role in vascular remodeling during carcinogenesis. However, the molecular routes upstream and downstream of this modulation remain to be elucidated.

Indole-3-Propionic Acid, a Gut Microbiota-Derived Tryptophan Metabolite, Promotes Endothelial Dysfunction Impairing Purinergic-Induced Nitric Oxide Release in Endothelial Cells.

Different gut microbiota-derived metabolites influence cardiovascular function, and, among all, the role of indole-3-propionic acid (IPA), from tryptophan metabolism, shows controversial effects. The aim of this study was to evaluate its role in endothelial dysfunction. IPA effects were studied on bovine aortic endothelial cells (BAE-1). First, IPA cytotoxicity was evaluated by an MTS assay. Then, the levels of intracellular reactive oxygen species (ROS) were evaluated by a microplate reader or fluorescence microscopy with the CellROX® Green probe, and nitric oxide (NO) production was studied by fluorescence microscopy with the DAR4M-AM probe after acute or chronic treatment. Finally, immunoblotting analysis for endothelial nitric oxide synthase (eNOS) phosphorylation (p-eNOS) was performed. In BAE-1, IPA was not cytotoxic, except for the highest concentration (5 mM) after 48 h of treatment, and it showed neither oxidant nor antioxidant activity. However, the physiological concentration of IPA (1 μM) significantly reduced NO released by adenosine triphosphate (ATP)-stimulated BAE-1. These last data were confirmed by Western blot analysis, where IPA induced a significant reduction in p-eNOS in purinergic-stimulated BAE-1. Given these data, we can speculate that IPA negatively affects the physiological control of vascular tone by impairing the endothelial NO release induced by purinergic stimulation. These results represent a starting point for understanding the mechanisms underlying the relationship between gut microbiota metabolites and cardiometabolic health.

Aerosolized Particulate Matter and Blunting of Ciliary Dynamic Responses: Implications for Veterans and Active Duty Military in Southwest Asia.

Respiratory diseases such as chronic rhinosinusitis and asthma are observed at increased rates in active duty and veteran military members, and they are especially prevalent in individuals who have been deployed in Southwest Asia during Operation Iraqi Freedom and Operation Enduring Freedom. Particulate matter, specifically the fine-grain desert sand found in the Middle East, may be a key source of this pathology because of deleterious effects on mucociliary clearance. With IRB approval, human sinonasal tissue was grown at an air-liquid interface and cultures were exposed to different types and sizes of particulate matter, including sand from Afghanistan and Kuwait. Ciliary dynamic responses to mechanical stimulation and ATP application were assessed following particulate exposure. Particle size of the commercial sand was substantially larger than that of the sand of Afghan or Kuwaiti origin. Following exposure to particulate matter, normal dynamic ciliary responses to mechanical stimulation and ATP application were significantly decreased (P < .01), with corresponding decreases in ATP-induced calcium flux (P < .05). These changes were partially reversible with apical washing after a 16-h period of exposure. After 36 h of exposure to Middle Eastern sand, ciliary responses to purinergic stimulation were completely abolished. There is a neutralization of the dynamic ciliary response following chronic particulate matter exposure, similar to ciliary pathologies observed in patients with chronic rhinosinusitis. Aerosolized particulate matter endured by military personnel in the Southwest Asia may cause dysfunctional mucociliary clearance; these data help to explain the increased prevalence of respiratory pathology in individuals who are or have been deployed in this region.

Pro-inflammatory cytokines stimulate CFTR-dependent anion secretion in pancreatic ductal epithelium.

Loss of CFTR-dependent anion and fluid secretion in the ducts of the exocrine pancreas is thought to contribute to the development of pancreatitis, but little is known about the impact of inflammation on ductal CFTR function. Here we used adult stem cell-derived cell cultures (organoids) obtained from porcine pancreas to evaluate the effects of pro-inflammatory cytokines on CFTR function. Organoids were cultured from porcine pancreas and used to prepare ductal epithelial monolayers. Monolayers were characterized by immunocytochemistry. Epithelial bicarbonate and chloride secretion, and the effect of IL-1β, IL-6, IFN-γ, and TNF-α on CFTR function was assessed by electrophysiology. Immunolocalization of ductal markers, including CFTR, keratin 7, and zonula occludens 1, demonstrated that organoid-derived cells formed a highly polarized epithelium. Stimulation by secretin or VIP triggered CFTR-dependent anion secretion across epithelial monolayers, whereas purinergic receptor stimulation by UTP, elicited CFTR-independent anion secretion. Most of the anion secretory response was attributable to bicarbonate transport. The combination of IL-1β, IL-6, IFN-γ, and TNF-α markedly enhanced CFTR expression and anion secretion across ductal epithelial monolayers, whereas these cytokines had little effect when tested separately. Although TNF-α triggered apoptotic signaling, epithelial barrier function was not significantly affected by cytokine exposure. Pro-inflammatory cytokines enhance CFTR-dependent anion secretion across pancreatic ductal epithelium. We propose that up-regulation of CFTR in the early stages of the inflammatory response, may serve to promote the removal of pathogenic stimuli from the ductal tree, and limit tissue injury.

Fetal bovine serum contains biologically available ATP

ATP is a ubiquitous extracellular messenger released in a wide number of pathophysiological conditions. ATP is known to be present in minute amounts in the extracellular space in healthy tissues and in the blood, and to modulate a multiplicity of cell responses. Cell culture systems are widely used to explore purinergic signaling. We show here that currently used fetal bovine sera contain ATP in the 300–1300 pmol/L range. Serum ATP is associated with albumin as well as with microparticle/microvesicle fraction. Serum microparticles/microvesicles affect in vitro cell responses due to their content of miRNAs, growth factors, and other bioactive molecules. ATP is likely to be one of these bioactive factors found in a variable amount in sera of different commercial sources. ATP in serum supports ATP-dependent biochemical reactions such as the hexokinase-dependent phosphorylation of glucose to glucose 6-phosphate, and affects purinergic signaling. These findings show that cells growing in vitro in serum-supplemented media are exposed to varying levels of extracellular ATP, and thus to varying degrees of purinergic stimulation.

Amplified Ca2+ dynamics and accelerated cell proliferation in breast cancer tissue during purinergic stimulation.

Intracellular Ca2+ dynamics shape malignant behaviors of cancer cells. Whereas previous studies focused on cultured cancer cells, we here used breast organoids and colonic crypts freshly isolated from human and murine surgical biopsies. We performed fluorescence microscopy to evaluate intracellular Ca2+ concentrations in breast and colon cancer tissue with preferential focus on intracellular Ca2+ release in response to purinergic and cholinergic stimuli. Inhibition of the sarco‐/endoplasmic reticulum Ca2+ ATPase with cyclopiazonic acid elicited larger Ca2+ responses in breast cancer tissue, but not in colon cancer tissue, relative to respective normal tissue. The resting intracellular Ca2+ concentration was elevated, and ATP, UTP and acetylcholine induced strongly augmented intracellular Ca2+ responses in breast cancer tissue compared with normal breast tissue. In contrast, resting intracellular Ca2+ levels and acetylcholine‐induced increases in intracellular Ca2+ concentrations were unaffected and ATP‐ and UTP‐induced Ca2+ responses were smaller in colon cancer tissue compared with normal colon tissue. In accordance with the amplified Ca2+ responses, ATP and UTP substantially increased proliferative activity—evaluated by bromodeoxyuridine incorporation—in breast cancer tissue, whereas the effect was minimal in normal breast tissue. ATP caused cell death—identified with ethidium homodimer‐1 staining—in breast cancer tissue only at concentrations above the expected pathophysiological range. We conclude that intracellular Ca2+ responses are amplified in breast cancer tissue, but not in colon cancer tissue, and that nucleotide signaling stimulates breast cancer cell proliferation within the extracellular concentration range typical for solid cancer tissue.

Uncovering Anti‐Arrhythmic Potential of Stellate Ganglion Purinergic Receptors

Cardiovascular disease affects over 30% of people worldwide and is one of the leading causes of death each year. Elevated sympathetic nerve activity is a common feature of cardiovascular disease, contributing to end‐organ damage, morbidity and mortality. Recent findings indicate that short‐circuiting sympathetic nerve overactivity by removal of the stellate ganglion can eradicate arrhythmias, emphasising the need for novel therapeutic targets to correct signalling non‐invasively.We have revealed upregulation of the P2X3 purinergic receptor in stellate ganglia of Spontaneously Hypertensive (SHR) compared to Wistar rats (16 week‐old), via RNASeq transcriptional profiling. We hypothesise that these purinergic receptors within cardiac stellate ganglia play a role in the excessive sympathetic drive to the heart in cardiovascular disease and can initiate cardiac arrhythmias. Thus, we have investigated the functional role of purinergic receptors in the stellate ganglion.Administration of ATP to acutely isolated post‐ganglionic sympathetic neurones from the stellate ganglia of Wistar rats (4‐6 week‐old) evokes a significant increase (Median; 0.21) in [Ca2+]i as measured by Fura‐2AM imaging (n=15, Wilcoxon matched‐pairs test; p&lt;0.0001). This ATP‐induced calcium transient was inhibited by specific P2X3 receptor antagonism with either NF‐10 or AF‐130.Cardiac effects of stellate ganglion purinergic stimulation were investigated in the working heart‐brainstem preparation of Wistar rats (4‐6 week‐old). Increasing doses of ATP (50‐250 µg) delivered via microinjection (50‐250 µL) directly to the right stellate ganglion produced either a tachycardia (18 ± 6 bpm, n=6) or bradycardia (‐24 ± 12 bpm, n=5); these responses were typically found at distinct sites within the ganglion. Preliminary data from SHR suggest that higher doses of ATP (≥250µg) may be arrhythmogenic (n=3).P2X3 purinergic receptors are present in the sympathetic stellate ganglion and contribute to calcium ion flux and cardiac chronotropic regulation. Overexpression of P2X3 receptors is likely to contribute to excessive cardiac sympathetic activity and the development of hypertension and cardiac arrhythmias, making them a promising novel therapeutic target.

Prototypic P2X7 Receptor Agonist, BzATP, Induced the Expression of Unfolded Protein Response Genes in Human M1 Macrophages.

Purinergic receptors stimulation by adenosine triphosphate (ATP) contributes significantly to macrophage activation, and also macrophage cell death. Upon the macrophage activation, the protein load of the endoplasmic reticulum is increased which is resulted in the activation of unfolded protein response (UPR). In the current study, we aimed to evaluate the connection between prototypic P2X7 receptor agonist, extracellular 2'(3')-O-(4-Benzoylbenzoyl)-ATP (BzATP), and the UPR pathway in macrophages. The monocyte-derived macrophages from blood samples of 14 healthy volunteers were skewed toward M1 macrophages after incubation with LPS and IFN-γ. M1 macrophages were treated with 200 µM BzATP. The expression levels of UPR genes, including CHOP, HERP, GADD34, XBP1, and ATF6 in macrophages before and after treatment were measured using real-time polymerase chain reaction. The results demonstrated that the expression of CHOP, HERP, and ATF6 is significantly decreased and the expression level of GADD34 and XBP1 is significantly increased after M1 polarization. BzATP not only significantly increased the expression levels of CHOP, GADD34, ATF6, and HERP but also significantly decreases the XBP1 expression level in M1 macrophages. The present study showed that BzATP induces cellular stress in M1 macrophages by elevating the expression levels of UPR genes including CHOP, GADD34, ATF6, and reducing cell viability.

Paneth Cell Secretion invivo Requires Expression ofTmem16a and Tmem16f.

Paneth cells play a central role in intestinal innate immune response. These cells are localized at the base of small intestinal crypts of Lieberkuhn. The calcium-activated chloride channel TMEM16A and the phospholipid scramblase TMEM16F control intracellular Ca2+ signaling and exocytosis. We analyzed the role of TMEM16A and TMEM16F for Paneth cells secretion. Mice with intestinal epithelial knockout of Tmem16a (Tmem16a-/-) and Tmem16f (Tmem16f-/-) were generated. Tissue structures and Paneth cells were analyzed, and Paneth cell exocytosis was examined in small intestinal organoids invitro. Intracellular Ca2+ signals were measured and were compared between wild-type and Tmem16 knockout mice. Bacterial colonization and intestinal apoptosis were analyzed. Paneth cells in the crypts of Lieberkuhn from Tmem16a-/- and Tmem16f-/- mice demonstrated accumulation of lysozyme. Tmem16a and Tmem16f were localized in wild-type Paneth cells but were absent in cells from knockout animals. Paneth cell number and size were enhanced in the crypt base and mucus accumulated in intestinal goblet cells of knockout animals. Granule fusion and exocytosis on cholinergic and purinergic stimulation were examined online. Both were strongly compromised in the absence of Tmem16a or Tmem16f and were also blocked by inhibition of Tmem16a/f. Purinergic Ca2+ signaling was largely inhibited in Tmem16a knockout mice. Jejunal bacterial content was enhanced in knockout mice, whereas cellular apoptosis was inhibited. The present data demonstrate the role of Tmem16 for exocytosis in Paneth cells. Inhibition or activation of Tmem16a/f is likely to affect microbial content and immune functions present in the small intestine.

Rescue of chloride and bicarbonate transport by elexacaftor-ivacaftor-tezacaftor in organoid-derived CF intestinal and cholangiocyte monolayers

BackgroundIn cystic fibrosis (CF), loss of CF transmembrane conductance regulator (CFTR)-dependent bicarbonate secretion precipitates the accumulation of viscous mucus in the lumen of respiratory and gastrointestinal epithelial tissues. We investigated whether the combination of elexacaftor (ELX), ivacaftor (IVA) and tezacaftor (TEZ), apart from its well-documented effect on chloride transport, also restores Phe508del-CFTR-mediated bicarbonate transport. MethodsEpithelial monolayers were cultured from intestinal and biliary (cholangiocyte) organoids of homozygous Phe508del-CFTR patients and controls. Transcriptome sequencing was performed, and bicarbonate and chloride transport were assessed in the presence or absence of ELX/IVA/TEZ, using the intestinal current measurement technique. ResultsELX/IVA/TEZ markedly enhanced bicarbonate and chloride transport across intestinal epithelium. In biliary epithelium, it failed to enhance CFTR-mediated bicarbonate transport but effectively rescued CFTR-mediated chloride transport, known to be requisite for bicarbonate secretion through the chloride-bicarbonate exchanger AE2 (SLC4A2), which was highly expressed by cholangiocytes. Biliary but not intestinal epithelial cells expressed an alternative anion channel, anoctamin-1/TMEM16A (ANO1), and secreted bicarbonate and chloride upon purinergic receptor stimulation. ConclusionsELX/IVA/TEZ has the potential to restore both chloride and bicarbonate secretion across CF intestinal and biliary epithelia and may counter luminal hyper-acidification in these tissues.

Manipulations of Glutathione Metabolism Modulate IP3-Mediated Store-Operated Ca2+ Entry on Astroglioma Cell Line.

The regulation of the redox status involves the activation of intracellular pathways as Nrf2 which provides hormetic adaptations against oxidative stress in response to environmental stimuli. In the brain, Nrf2 activation upregulates the formation of glutathione (GSH) which is the primary antioxidant system mainly produced by astrocytes. Astrocytes have also been shown to be themselves the target of oxidative stress. However, how changes in the redox status itself could impact the intracellular Ca2+ homeostasis in astrocytes is not known, although this could be of great help to understand the neuronal damage caused by oxidative stress. Indeed, intracellular Ca2+ changes in astrocytes are crucial for their regulatory actions on neuronal networks. We have manipulated GSH concentration in astroglioma cells with selective inhibitors and activators of the enzymes involved in the GSH cycle and analyzed how this could modify Ca2+ homeostasis. IP3-mediated store-operated calcium entry (SOCE), obtained after store depletion elicited by Gq-linked purinergic P2Y receptors activation, are either sensitized or desensitized, following GSH depletion or increase, respectively. The desensitization may involve decreased expression of the proteins STIM2, Orai1, and Orai3 which support SOCE mechanism. The sensitization process revealed by exposing cells to oxidative stress likely involves the increase in the activity of Calcium Release-Activated Channels (CRAC) and/or in their membrane expression. In addition, we observe that GSH depletion drastically impacts P2Y receptor-mediated changes in membrane currents, as evidenced by large increases in Ca2+-dependent K+ currents. We conclude that changes in the redox status of astrocytes could dramatically modify Ca2+ responses to Gq-linked GPCR activation in both directions, by impacting store-dependent Ca2+-channels, and thus modify cellular excitability under purinergic stimulation.

The Tyrosine Phosphatase hPTPRβ Controls the Early Signals and Dopaminergic Cells Viability via the P2X7 Receptor.

ATP, one of the signaling molecules most commonly secreted in the nervous system and capable of stimulating multiple pathways, binds to the ionotropic purinergic receptors, in particular, the P2X7 receptor (P2X7R) and stimulates neuronal cell death. Given this effect of purinergic receptors on the viability of dopaminergic neurons model cells and that Ras GTPases control Erk1/2-regulated mitogen-activated cell proliferation and survival, we have investigated the role of the small GTPases of the Ras superfamily, together with their regulatory and effector molecules as the potential molecular intermediates in the P2X7R-regulated cell death of SN4741 dopaminergic neurons model cells. Here, we demonstrate that the neuronal response to purinergic stimulation involves the Calmodulin/RasGRF1 activation of the small GTPase Ras and Erk1/2. We also demonstrate that tyrosine phosphatase PTPRβ and other tyrosine phosphatases regulate the small GTPase activation pathway and neuronal viability. Our work expands the knowledge on the intracellular responses of dopaminergic cells by identifying new participating molecules and signaling pathways. In this sense, the study of the molecular circuitry of these neurons is key to understanding the functional effects of ATP, as well as considering the importance of these cells in Parkinson’s Disease.

Intracellular calcium and inflammatory markers, mediated by purinergic stimulation, are differentially regulated in monocytes of patients with major depressive disorder

The P2X7 receptor (P2X7R) is a ligand-gated ion channel that is being recognized as a major player in neuropsychiatric disorders such as Major Depressive Disorder (MDD). P2X7R activation is triggered by high extracellular ATP concentrations, leading to channel opening and inducing an increase in cytosolic calcium concentration ([Ca2+]c), that activates the inflammatory pathway. Those receptors are expressed not only in CNS cells but also in peripheral blood cells, where they are activated in response to inflammatory molecules such as bacterial lipopolysaccharide (LPS). LPS induced-tissue damage promotes an elevation of extracellular ATP, triggering the NRLP3-inflammasome assembly and activation that, sequentially, induces caspase-1 cleavage and IL-1β processing and secretion. In this context, we attempt to understand the role of P2X7R in [Ca2+]c homeostasis regulation, inflammasome expression and its pharmacological modulation in MDD. For this purpose, monocytes were isolated from peripheral blood of MDD patients and [Ca2+]c was monitored with the intracellular probe Fura-2. Our results point out to P2X7R as the responsible of the Ca2+ imbalance, as well as TNF-α-dependent activation of caspase-1 in MDD patients. In addition, P2X7R blockade with its specific antagonist, JNJ-47965567, reduces the Ca2+ entry upon Bz-ATP exposure. Altogether, our results point that MDD patients have both, Ca2+ homeostasis alteration and an inflammatory status, which promote an independent-inflammasome activation of caspase-1. Therefore, we propose the pharmacological modulation of P2X7R as a therapeutic approach against MDD symptoms.

Functional expression of TMEM16A in taste bud cells.

Key points Taste transduction occurs in taste buds in the tongue epithelium.The Ca2+‐activated Cl– channels TMEM16A and TMEM16B play relevant physiological roles in several sensory systems.Here, we report that TMEM16A, but not TMEM16B, is expressed in the apical part of taste buds.Large Ca2+‐activated Cl− currents blocked by Ani‐9, a selective inhibitor of TMEM16A, are measured in type I taste cells but not in type II or III taste cells.ATP indirectly activates Ca2+‐activated Cl– currents in type I cells through TMEM16A channels.These results indicate that TMEM16A is functional in type I taste cells and contribute to understanding the largely unknown physiological roles of these cells. The Ca2+‐activated Cl– channels TMEM16A and TMEM16B have relevant roles in many physiological processes including neuronal excitability and regulation of Cl– homeostasis. Here, we examined the presence of Ca2+‐activated Cl– channels in taste cells of mouse vallate papillae by using immunohistochemistry and electrophysiological recordings. By using immunohistochemistry we showed that only TMEM16A, and not TMEM16B, was expressed in taste bud cells where it largely co‐localized with the inwardly rectifying K+ channel KNCJ1 in the apical part of type I cells. By using whole‐cell patch‐clamp recordings in isolated cells from taste buds, we measured an average current of −1083 pA at −100 mV in 1.5 μm Ca2+ and symmetrical Cl– in type I cells. Ion substitution experiments and blockage by Ani‐9, a specific TMEM16A channel blocker, indicated that Ca2+ activated anionic currents through TMEM16A channels. We did not detect any Ca2+‐activated Cl– currents in type II or III taste cells. ATP is released by type II cells in response to various tastants and reaches type I cells where it is hydrolysed by ecto‐ATPases. Type I cells also express P2Y purinergic receptors and stimulation of type I cells with extracellular ATP produced large Ca2+‐activated Cl− currents blocked by Ani‐9, indicating a possible role of TMEM16A in ATP‐mediated signalling. These results provide a definitive demonstration that TMEM16A‐mediated currents are functional in type I taste cells and provide a foundation for future studies investigating physiological roles for these often‐neglected taste cells.

Calmodulin binds and modulates K+-dependent Na+/Ca2+-exchanger isoform 4, NCKX4

The family of K+-dependent Na+/Ca2+-exchangers, NCKX, are important mediators of cellular Ca2+ efflux, particularly in neurons associated with sensory transduction. The NCKX family comprises five proteins, NCKX1–5, each being the product of a different SLC24 gene. NCKX4 (SLC24A4) has been found to have a critical role in termination and adaptation of visual and olfactory signals, melanocortin-dependent satiety signaling, and the maturation of dental enamel. To explore mechanisms that might influence the temporal control of NCKX4 activity, a yeast two-hybrid system was used to search for protein interaction partners. We identified calmodulin as a partner for NCKX4 and confirmed the interaction using glutathione-S-transferase fusion pull-down. Calmodulin binding to NCKX4 was demonstrated in extracts from mouse brain and in transfected HEK293 cells. Calmodulin bound in a Ca2+-dependent manner to a motif present in the central cytosolic loop of NCKX4 and was abolished by the double-mutant I328D/F334D. When cotransfected in HEK293 cells, calmodulin bound to NCKX4 under basal conditions and induced a ∼2.5-fold increase in NCKX4 abundance, but did not influence either cellular location or basal activity. When purinergic stimulation of NCKX4 was examined in these cells, coexpression of wild-type calmodulin, but not a Ca2+ binding-deficient calmodulin mutant, suppressed NCKX4 activation in a time-dependent manner. We propose that Ca2+ binding to calmodulin prepositioned on NCKX4 induces a slow conformational rearrangement that interferes with purinergic stimulation of the exchanger, possibly by obscuring T331, a previously identified potential protein kinase C site.

Abstract P006: Purinergic Receptor Activation Protects Glomerular Microvasculature From Increased Mechanical Stress In Angiotensin Ii-induced Hypertension: A Modeling Study

Angiotensin II (Ang II)-induced hypertension increases afferent and efferent arteriole resistances via the actions of Ang II on the AT1 receptor. In addition to the increased interstitial levels of Ang II, the increased arterial pressure increases interstitial ATP concentrations which act on the purinergic receptors P2X1 and P2X7, to constrict the AA, preventing increases in plasma flow and single nephron GFR (SNGFR). Blockade of the P2 receptors also mitigates the effects of Ang II, thus increasing blood flow and SNGFR, but the resulting increases in mechanical stresses (shear stress and circumferential hoop stress) on the glomerular microvasculature have not been quantified. A mathematical microvascular hemodynamic glomerular model was developed to simulate blood flow and plasma filtration at each of 320 capillary segments in an anatomically-accurate rat glomerular capillary network topology. Afferent and efferent arteriole resistances and network hydraulic conductivity were adjusted to match glomerular hemodynamic data for control, Ang II-induced hypertension and P2X1-blocked conditions (Franco, Martha, et al. Amer. J. Physiology-Renal 313.1 (2017): F9-F19). Ang II infusion increased both afferent and efferent resistances, reducing blood flow while slightly raising glomerular pressure. Blockade of the purinergic receptors reduced both afferent and efferent resistances, maintaining glomerular pressure at hypertensive levels but increasing blood flow significantly, increasing shear stress from 24.9 dynes/cm 2 in hypertensive conditions to 71.3 dynes/cm 2 after purinergic blockade. Because glomerular pressure was maintained, hoop stress barely changed from 69.5 kPa in hypertensive conditions to 70.9 kPa after purinergic blockade. Purinergic blockade also increased hydraulic conductivity and filtering surface area, increasing SNGFR. In hypertension, purinergic stimulation does not prevent the transmission of increased arterial pressure to the glomerular capillaries to reduce capillary hoop stress. However, activation of the purinergic system protects the glomerular microvasculature from increases in shear stress caused by a marked increase in blood flow that would occur in the absence of purinergic stimulation.

Knockout mouse models reveal the contributions of G protein subunits to complement C5a receptor–mediated chemotaxis

G protein-coupled receptor signaling is required for the navigation of immune cells along chemoattractant gradients. However, chemoattractant receptors may couple to more than one type of heterotrimeric G protein, each of which consists of a Gα, Gβ, and Gγ subunit, making it difficult to delineate the critical signaling pathways. Here, we used knockout mouse models and time-lapse microscopy to elucidate Gα and Gβ subunits contributing to complement C5a receptor-mediated chemotaxis. Complement C5a-mediated chemokinesis and chemotaxis were almost completely abolished in macrophages lacking Gnai2 (encoding Gαi2), consistent with a reduced leukocyte recruitment previously observed in Gnai2-/- mice, whereas cells lacking Gnai3 (Gαi3) exhibited only a slight decrease in cell velocity. Surprisingly, C5a-induced Ca2+ transients and lamellipodial membrane spreading were persistent in Gnai2-/- macrophages. Macrophages lacking both Gnaq (Gαq) and Gna11 (Gα11) or both Gna12 (Gα12) and Gna13 (Gα13) had essentially normal chemotaxis, Ca2+ signaling, and cell spreading, except Gna12/Gna13-deficient macrophages had increased cell velocity and elongated trailing ends. Moreover, Gnaq/Gna11-deficient cells did not respond to purinergic receptor P2Y2 stimulation. Genetic deletion of Gna15 (Gα15) virtually abolished C5a-induced Ca2+ transients, but chemotaxis and cell spreading were preserved. Homozygous Gnb1 (Gβ1) deletion was lethal, but mice lacking Gnb2 (Gβ2) were viable. Gnb2-/- macrophages exhibited robust Ca2+ transients and cell spreading, albeit decreased cell velocity and impaired chemotaxis. In summary, complement C5a-mediated chemotaxis requires Gαi2 and Gβ2, but not Ca2+ signaling, and membrane protrusive activity is promoted by G proteins that deplete phosphatidylinositol 4,5-bisphosphate.

TRPV4 and purinergic receptor signalling pathways are separately linked in airway epithelia to CFTR and TMEM16A chloride channels.

Eact is a putative pharmacological activator of TMEM16A. Eact is strongly effective in recombinant Fischer rat thyroid (FRT) cells but not in airway epithelial cells with endogenous TMEM16A expression. Transcriptomic analysis, gene silencing and functional studies in FRT cells reveal that Eact is actually an activator of the Ca2+ -permeable TRPV4 channel. In airway epithelial cells TRPV4 and TMEM16A are expressed in separate cell types. Intracellular Ca2+ elevation by TRPV4stimulation leads to CFTR channel activation. TMEM16A is a Ca2+ -activated Cl- channel expressed in airway epithelial cells, particularly under conditions of mucus hypersecretion. To investigate the role of TMEM16A, we used Eact, a putative TMEM16A pharmacological activator. However, in contrast to purinergic stimulation, we found little effect of Eact on bronchial epithelial cells under conditions of high TMEM16A expression. We hypothesized that Eact is an indirect activator of TMEM16A. By a combination of approaches, including short-circuit current recordings, bulk and single cell RNA sequencing, intracellular Ca2+ imaging and RNA interference, we found that Eact is actually an activator of the Ca2+ -permeable TRPV4 channel and that the modest effect of this compound in bronchial epithelial cells is due to a separate expression of TMEM16A and TRPV4 in different cell types. Importantly, we found that TRPV4stimulation induced activation of the CFTR Cl- channel. Our study reveals the existence of separate Ca2+ signalling pathways linked to different Cl- secretory processes.

Nestin affects fusion pore dynamics in mouse astrocytes

Astrocytes play a homeostatic role in the central nervous system and influence numerous aspects of neurophysiology via intracellular trafficking of vesicles. Intermediate filaments (IFs), also known as nanofilaments, regulate a number of cellular processes including organelle trafficking and adult hippocampal neurogenesis. We have recently demonstrated that the IF protein nestin, a marker of neural stem cells and immature and reactive astrocytes, is also expressed in some astrocytes in the unchallenged hippocampus and regulates neurogenesis through Notch signalling from astrocytes to neural stem cells, possibly via altered trafficking of vesicles containing the Notch ligand Jagged-1. We thus investigated whether nestin affects vesicle dynamics in astrocytes by examining single vesicle interactions with the plasmalemma and vesicle trafficking with high-resolution cell-attached membrane capacitance measurements and confocal microscopy. We used cell cultures of astrocytes from nestin-deficient (Nes-/- ) and wild-type (wt) mice, and fluorescent dextran and Fluo-2 to examine vesicle mobility and intracellular Ca2+ concentration respectively. Nes-/- astrocytes exhibited altered sizes of vesicles undergoing full fission and transient fusion, altered vesicle fusion pore geometry and kinetics, decreased spontaneous vesicle mobility and altered ATP-evoked mobility. Purinergic stimulation evoked Ca2+ signalling that was slightly attenuated in Nes-/- astrocytes, which exhibited more oscillatory Ca2+ responses than wt astrocytes. These results demonstrate at the single vesicle level that nestin regulates vesicle interactions with the plasmalemma and vesicle trafficking, indicating its potential role in astrocyte vesicle-based communication.

Properties of SK3 channel-expressing PDGFRα (+) cells in the rodent urinary bladder

Localisation of platelet-derived growth factor receptor-α (PDGFRα) (+) cells expressing small-conductance Ca2+-activated K+ (SK3) channels in the urinary bladder was investigated, while putative roles of SK3 (+) PDGFRα (+) cells in suppressing detrusor smooth muscle (DSM) spontaneous activity were explored. In guinea-pig bladder, immunohistochemistry for SK3 channels, PDGFRα or vimentin was examined, as were the effects of purinergic agonists on spontaneous phasic contractions (SPCs). In bladder of PDGFRα-GFP mice, the effects of purinergic agonists on intracellular Ca2+ signaling in PDGFRα (+) cells or DSM cells in situ and SPCs were investigated. SK3 (+) cells co-expressing PDGFRα or vimentin were distributed in DSM bundles but not inter-bundle spaces or lamina propria. SK3 (+) cells had a stellate- or spindle-shape cell body extending processes. MRS2365 (100 nM or 1 μM), a P2Y1 agonist, caused a transient contraction without inhibiting SPCs in both DSM and lamina propria. In PDGFRα-GFP mice bladder, MRS2365, (100 nM), ADP (100 μM) or ATP (100 μM) increased the Ca2+ level of PDGFRα (+) cells without suppressing spontaneous Ca2+ transients in neighboring DSM cells, and also failed to suppress SPCs. Preferential localisation of SK3 positive PDGFRα (+) cells in DSM bundles appears to indicate their functional interaction with DSM cells. However, increases in Ca2+ level of PDGFRα (+) cells upon purinergic stimulation are not associated with the inhibition of Ca2+ or contractile activity in DSM cells. Thus, it is unlikely that the SK3-dependent hyperpolarisation generated in SK3 expressing PDGFRα (+) cells is transmitted to DSMs to suppress their excitability.