Retraction notice to "Non-neuronal cholinergic system delays cardiac remodelling in type 1 diabetes" [Heliyon 9 (2023) e17434].

[This retracts the article DOI: 10.1016/j.heliyon.2023.e17434.].

Non-neuronal cholinergic system delays cardiac remodelling in type 1 diabetes.

Aims: Type 1 diabetes mellitus (T1DM) is associated with increased risk of cardiovascular disease (CVD) and mortality. The underlying mechanisms for T1DM-induced heart disease still remains unclear. In this study, we aimed to investigate the effects of cardiac non-neuronal cholinergic system (cNNCS) activation on T1DM-induced cardiac remodelling. Methods: T1DM was induced in C57Bl6 mice using low-dose streptozotocin. Western blot analysis was used to measure the expression of cNNCS components at different time points (4, 8, 12, and 16 weeks after T1DM induction). To assess the potential benefits of cNNCS activation, T1DM was induced in mice with cardiomyocyte-specific overexpression of choline acetyltransferase (ChAT), the enzyme required for acetylcholine (Ac) synthesis. We evaluated the effects of ChAT overexpression on cNNCS components, vascular and cardiac remodelling, and cardiac function. Key findings: Western blot analysis revealed dysregulation of cNNCS components in hearts of T1DM mice. Intracardiac ACh levels were also reduced in T1DM. Activation of ChAT significantly increased intracardiac ACh levels and prevented diabetes-induced dysregulation of cNNCS components. This was associated with preserved microvessel density, reduced apoptosis and fibrosis, and improved cardiac function. Significance: Our study suggests that cNNCS dysregulation may contribute to T1DM-induced cardiac remodelling, and that increasing ACh levels may be a potential therapeutic strategy to prevent or delay T1DM-induced heart disease.

Vasopressin regulation of maternal body fluid balance in pregnancy and lactation: A role for TRPV channels?

Renal water reabsorption increases in pregnancy and lactation to expand maternal blood volume to cope with the cardiovascular demands of the developing fetus and new-born baby. Vasopressin (antidiuretic hormone) promotes renal water reabsorption and its secretion is principally stimulated by body fluid osmolality. Hence, lowered osmolality normally decreases vasopressin secretion. However, despite water retention profoundly reducing osmolality in pregnancy and lactation, vasopressin levels are maintained to drive blood volume expansion. Despite its importance for successful reproduction, the cellular mechanisms that maintain vasopressin secretion in the face of decreased osmolality during pregnancy and lactation are unknown. Vasopressin is secreted by neurons that are intrinsically osmosensitive through expression of N-terminal truncated-transient receptor potential vanilloid-1 channel, ΔN-TRPV1, which is mechanically activated by osmotically-induced cell shrinkage to increase vasopressin neuron activity. Vasopressin neurons also express TRPV4 but the role of TRPV4 in vasopressin neuron function is not well characterised. Here, we summarise our novel evidence showing that TRPV4 forms functional channels with ΔN-TRPV1 that have a greater single-channel conductance compared to channels with ΔN-TRPV1 alone. We propose that upregulation of TRPV4 heteromerisation with ΔN-TRPV1 might maintain vasopressin secretion in pregnancy and lactation to expand blood volume for successful reproduction.

COVID-19 and Liquid Homeostasis in the Lung-A Perspective through the Epithelial Sodium Channel (ENaC) Lens.

Infections with a new corona virus in 2019 lead to the definition of a new disease known as Corona Virus Disease 2019 (COVID-19). The sever cases of COVID-19 and the main cause of death due to virus infection are attributed to respiratory distress. This is associated with the formation of pulmonary oedema that impairs blood oxygenation and hypoxemia as main symptoms of respiratory distress. An important player for the maintenance of a defined liquid environment in lungs needed for normal lung function is the epithelial sodium channel (ENaC). The present article reviews the implications of SARS-CoV-2 infections from the perspective of impaired function of ENaC. The rationale for this perspective is derived from the recognition that viral spike protein and ENaC share a common proteolytic cleavage site. This cleavage site is utilized by the protease furin, that is essential for ENaC activity. Furin cleavage of spike 'activates' the virus protein to enable binding to host cell membrane receptors and initiate cell infection. Based on the importance of proteolytic cleavage for ENaC function and activation of spike, it seems feasible to assume that virus infections are associated with impaired ENaC activity. This is further supported by symptoms of COVID-19 that are reminiscent of impaired ENaC function in the respiratory tract.

Epithelial Sodium Channel δ Subunit Is Expressed in Human Arteries and Has Potential Association With Hypertension.

BACKGROUND: Elevated expression and increased activity of vascular epithelial sodium channel (ENaC) can result in vascular dysfunction in small animal models. However, there is limited or no knowledge on expression and function of ENaC channels in human vasculature. Hence, this study explored the expression and function of ENaC in human arteries and their association with hypertension. METHODS: Human internal mammary artery (IMA) and aorta were obtained from cardiovascular patients undergoing coronary artery bypass graft surgery. Expression of the ENaC subunit was analyzed by polymerase chain reaction, Western blot, and immunohistochemistry. ENaC function was observed by patch-clamp electrophysiology in endothelial cells isolated from IMA. Levels of ENaC subunit expression levels were compared between arteries from normotensive, uncontrolled hypertensive, and controlled hypertensive patients. RESULTS: For the first time, expression of α, ß, γ, and δ was detected at mRNA and protein levels in human IMA and aorta. Single-channel patch-clamp recordings identified both αßγ- and δßγ-like channel conductance in primary endothelial cells isolated and cultured from IMA. Reduced expression of the δ subunit was observed in controlled hypertensive IMA, whereas reduced expression of γ-ENaC was observed in controlled hypertensive aorta. CONCLUSIONS: These data suggest that functional ENaC channels are expressed in human arteries and their expression levels are associated with hypertension.

Epithelial Na+ channel and the glycocalyx: a sweet and salty relationship for arterial shear stress sensing.

PURPOSE OF REVIEW: The ability of endothelial cells to sense mechanical force, and shear stress in particular, is crucial for normal vascular function. This relies on an intact endothelial glycocalyx that facilitates the production of nitric oxide (NO). An emerging arterial shear stress sensor is the epithelial Na+ channel (ENaC). This review highlights existing and new evidence for the interdependent activity of the glycocalyx and ENaC and its implications for vascular function. RECENT FINDINGS: New evidence suggests that the glycocalyx and ENaC are physically connected and that this is important for shear stress sensing. The connection relies on N-glycans attached to glycosylated asparagines of α-ENaC. Removal of specific N-glycans reduced ENaC's shear stress response. Similar effects were observed following degradation of the glycocalyx. Endothelial specific viral transduction of α-ENaC increased blood pressure (∼40 mmHg). This increase was attenuated in animals transduced with an α-ENaC version lacking N-glycans. SUMMARY: These observations indicate that ENaC is connected to the glycocalyx and their activity is interdependent to facilitate arterial shear stress sensation. Future research focusing on how N-glycans mediate this interaction can provide new insights for the understanding of vascular function in health and disease.

Plasticity in Intrinsic Excitability of Hypothalamic Magnocellular Neurosecretory Neurons in Late-Pregnant and Lactating Rats.

Oxytocin and vasopressin secretion from the posterior pituitary gland are required for normal pregnancy and lactation. Oxytocin secretion is relatively low and constant under basal conditions but becomes pulsatile during birth and lactation to stimulate episodic contraction of the uterus for delivery of the fetus and milk ejection during suckling. Vasopressin secretion is maintained in pregnancy and lactation despite reduced osmolality (the principal stimulus for vasopressin secretion) to increase water retention to cope with the cardiovascular demands of pregnancy and lactation. Oxytocin and vasopressin secretion are determined by the action potential (spike) firing of magnocellular neurosecretory neurons of the hypothalamic supraoptic and paraventricular nuclei. In addition to synaptic input activity, spike firing depends on intrinsic excitability conferred by the suite of channels expressed by the neurons. Therefore, we analysed oxytocin and vasopressin neuron activity in anaesthetised non-pregnant, late-pregnant, and lactating rats to test the hypothesis that intrinsic excitability of oxytocin and vasopressin neurons is increased in late pregnancy and lactation to promote oxytocin and vasopressin secretion required for successful pregnancy and lactation. Hazard analysis of spike firing revealed a higher incidence of post-spike hyperexcitability immediately following each spike in oxytocin neurons, but not in vasopressin neurons, in late pregnancy and lactation, which is expected to facilitate high frequency firing during bursts. Despite lower osmolality in late-pregnant and lactating rats, vasopressin neuron activity was not different between non-pregnant, late-pregnant, and lactating rats, and blockade of osmosensitive ΔN-TRPV1 channels inhibited vasopressin neurons to a similar extent in non-pregnant, late-pregnant, and lactating rats. Furthermore, supraoptic nucleus ΔN-TRPV1 mRNA expression was not different between non-pregnant and late-pregnant rats, suggesting that sustained activity of ΔN-TRPV1 channels might maintain vasopressin neuron activity to increase water retention during pregnancy and lactation.

Enhanced Shear Force Responsiveness of Epithelial Na+ Channel's (ENaC) δ Subunit Following the Insertion of N-Glycosylation Motifs Relies on the Extracellular Matrix.

Members of the Degenerin/epithelial Na+ channel (ENaC) protein family and the extracellular cell matrix (ECM) form a mechanosensitive complex. A core feature of this complex are tethers, which connect the channel with the ECM, however, knowledge about the nature of these tethers is scarce. N-glycans of α ENaC were recently identified as potential tethers but whether N-glycans serve as a ubiquitous feature for mechanosensation processes remains unresolved. The purpose of this study was to reveal whether the addition of N-glycans to δ ENaC-which is less responsive to shear force (SF)-increases its SF-responsiveness and whether this relies on a linkage to the ECM. Therefore, N-glycosylation motifs were introduced via site-directed mutagenesis, the resulting proteins expressed with ß and γ ENaC in Xenopus oocytes, and SF-activated currents measured by two-electrode voltage-clamp. The insertion of N-glycosylation motifs increases δ ENaC's SF responsiveness. The inclusion of a glycosylated asparagine (N) at position 487 did increase the molecular mass and provided a channel whose SF response was abolished following ECM degradation via hyaluronidase. This indicates that the addition of N-glycans improves SF-responsiveness and that this effect relies on an intact ECM. These findings further support the role of N-glycans as tethers for mechanotransduction.

Activation of the cardiac non-neuronal cholinergic system prevents the development of diabetes-associated cardiovascular complications.

BACKGROUND: Acetylcholine (ACh) plays a crucial role in the function of the heart. Recent evidence suggests that cardiomyocytes possess a non-neuronal cholinergic system (NNCS) that comprises of choline acetyltransferase (ChAT), choline transporter 1 (CHT1), vesicular acetylcholine transporter (VAChT), acetylcholinesterase (AChE) and type-2 muscarinic ACh receptors (M2AChR) to synthesize, release, degrade ACh as well as for ACh to transduce a signal. NNCS is linked to cardiac cell survival, angiogenesis and glucose metabolism. Impairment of these functions are hallmarks of diabetic heart disease (DHD). The role of the NNCS in DHD is unknown. The aim of this study was to examine the effect of diabetes on cardiac NNCS and determine if activation of cardiac NNCS is beneficial to the diabetic heart. METHODS: Ventricular samples from type-2 diabetic humans and db/db mice were used to measure the expression pattern of NNCS components (ChAT, CHT1, VAChT, AChE and M2AChR) and glucose transporter-4 (GLUT-4) by western blot analysis. To determine the function of the cardiac NNCS in the diabetic heart, a db/db mouse model with cardiac-specific overexpression of ChAT gene was generated (db/db-ChAT-tg). Animals were followed up serially and samples collected at different time points for molecular and histological analysis of cardiac NNCS components and prosurvival and proangiogenic signaling pathways. RESULTS: Immunoblot analysis revealed alterations in the components of cardiac NNCS and GLUT-4 in the type-2 diabetic human and db/db mouse hearts. Interestingly, the dysregulation of cardiac NNCS was followed by the downregulation of GLUT-4 in the db/db mouse heart. Db/db-ChAT-tg mice exhibited preserved cardiac and vascular function in comparison to db/db mice. The improved function was associated with increased cardiac ACh and glucose content, sustained angiogenesis and reduced fibrosis. These beneficial effects were associated with upregulation of the PI3K/Akt/HIF1α signaling pathway, and increased expression of its downstream targets-GLUT-4 and VEGF-A. CONCLUSION: We provide the first evidence for dysregulation of the cardiac NNCS in DHD. Increased cardiac ACh is beneficial and a potential new therapeutic strategy to prevent or delay the development of DHD.

The δ subunit of epithelial sodium channel in humans-a potential player in vascular physiology.

Vascular epithelial sodium channels (ENaCs) made up of canonical α, ß, and γ subunits have attracted more attention recently owing to their physiological role in vascular health and disease. A fourth subunit, δ-ENaC, is expressed in various mammalian species, except mice and rats, which are common animal models for cardiovascular research. Accordingly, δ-ENaC is the least understood subunit. However, the recent discovery of δ subunit in human vascular cells indicates that this subunit may play a significant role in normal/pathological vascular physiology in humans. Channels containing the δ subunit have different biophysical and pharmacological properties compared with channels containing the α subunit, with the potential to alter the vascular function of ENaC in health and disease. Hence, it is important to investigate the expression and function of δ-ENaC in the vasculature to identify whether δ-ENaC is a potential new drug target for the treatment of cardiovascular disease. In this review, we will focus on the existing knowledge of δ-ENaC and implications for vascular physiology and pathophysiology in humans.

Nicotine stimulates ion transport via metabotropic ß4 subunit containing nicotinic ACh receptors.

BACKGROUND AND PURPOSE: Mucociliary clearance is an innate immune process of the airways, essential for removal of respiratory pathogens. It depends on ciliary beat and ion and fluid homeostasis of the epithelium. We have shown that nicotinic ACh receptors (nAChRs) activate ion transport in mouse tracheal epithelium. Yet the receptor subtypes and signalling pathways involved remained unknown. EXPERIMENTAL APPROACH: Transepithelial short circuit currents (ISC ) of freshly isolated mouse tracheae were recorded using the Ussing chamber technique. Changes in [Ca2+ ]i were studied on freshly dissociated mouse tracheal epithelial cells. KEY RESULTS: Apical application of the nAChR agonist nicotine transiently increased ISC . The nicotine effect was abolished by the nAChR antagonist mecamylamine. α-Bungarotoxin (α7 antagonist) had no effect. The agonists epibatidine (α3ß2, α4ß2, α4ß4 and α3ß4) and A-85380 (α4ß2 and α3ß4) increased ISC . The antagonists dihydro-ß-erythroidine (α4ß2, α3ß2, α4ß4 and α3ß4), α-conotoxin MII (α3ß2) and α-conotoxin PnIA (α3ß2) reduced the nicotine effect. Nicotine- and epibatidine-induced currents were unaltered in ß2-/- mice, but in ß4-/- mice no increase was observed. In the presence of thapsigargin (endoplasmatic reticulum Ca2+ -ATPase inhibitor) or the ryanodine receptor antagonists JTV-519 and dantrolene there was a reduction in the nicotine-effect, indicating involvement of Ca2+ release from intracellular stores. Additionally, the PKA inhibitor H-89 and the TMEM16A (Ca2+ -activated chloride channel) inhibitor T16Ainh-A01 significantly reduced the nicotine-effect. CONCLUSION AND IMPLICATIONS: α3ß4 nAChRs are responsible for the nicotine-induced current changes via Ca2+ release from intracellular stores, PKA and ryanodine receptor activation. These nAChRs might be possible targets to stimulate chloride transport via TMEM16A.

Epithelial Na+ Channel (ENaC) Formed by One or Two Subunits Forms Functional Channels That Respond to Shear Force.

Canonical epithelial sodium channels (ENaCs) are heterotrimers formed by α, ß, and γ ENaC subunits in vertebrates and belong to the Degenerin/ENaC family of proteins. Proteins from this family form mechanosensitive channels throughout the animal kingdom. Activity of canonical ENaC is regulated by shear force (SF) mediating Na+ absorption in the kidney and vascular tone of arteries. Expression analysis suggests that non-canonical ENaC, formed by single or only two subunits, exist in certain tissues, but it is unknown if these channels respond to SF. α, ß, γ, and δ ENaC subunits were expressed either alone or in combinations of two subunits in Xenopus oocytes. Amiloride-sensitive currents and the responses to SF were assessed using two-electrode voltage clamp recordings. With the exception of γ ENaC, all homomeric channels provided amiloride-sensitive currents and responded to SF applied via a fluid stream directed onto the oocytes. Channels containing two subunits were also activated by SF. Here, the presence of the γ ENaC subunit when co-expressed with α or δ augmented the SF response in comparison to the αßγ/δßγ ENaC. Overall, we provide evidence that non-canonical ENaC can form channels that respond to SF. This supports a potential function of non-canonical ENaC as mechanosensors in epithelial, vascular, and sensory cells.

Shear force sensing of epithelial Na+ channel (ENaC) relies on N-glycosylated asparagines in the palm and knuckle domains of αENaC.

Mechanosensitive ion channels are crucial for normal cell function and facilitate physiological function, such as blood pressure regulation. So far little is known about the molecular mechanisms of how channels sense mechanical force. Canonical vertebrate epithelial Na+ channel (ENaC) formed by α-, ß-, and γ-subunits is a shear force (SF) sensor and a member of the ENaC/degenerin protein family. ENaC activity in epithelial cells contributes to electrolyte/fluid-homeostasis and blood pressure regulation. Furthermore, ENaC in endothelial cells mediates vascular responsiveness to regulate blood pressure. Here, we provide evidence that ENaC's ability to mediate SF responsiveness relies on the "force-from-filament" principle involving extracellular tethers and the extracellular matrix (ECM). Two glycosylated asparagines, respectively their N-glycans localized in the palm and knuckle domains of αENaC, were identified as potential tethers. Decreased SF-induced ENaC currents were observed following removal of the ECM/glycocalyx, replacement of these glycosylated asparagines, or removal of N-glycans. Endothelial-specific overexpression of αENaC in mice induced hypertension. In contrast, expression of αENaC lacking these glycosylated asparagines blunted this effect. In summary, glycosylated asparagines in the palm and knuckle domains of αENaC are important for SF sensing. In accordance with the force-from-filament principle, they may provide a connection to the ECM that facilitates vascular responsiveness contributing to blood pressure regulation.

Shear force modulates the activity of acid-sensing ion channels at low pH or in the presence of non-proton ligands.

Acid-sensing ion channels (ASICs) belong to the degenerin/epithelial sodium channel protein family that form mechanosensitive ion channels. Evidence as to whether or not ASICs activity is directly modulated by mechanical force is lacking. Human ASICs (hASIC1V3, hASIC2a and hASIC3a) were heterologously expressed as homomeric channels in Xenopus oocytes and two-electrode voltage-clamp recordings were performed. hASIC3a was expressed in HEK-293 cells and currents measured by whole-cell patch-clamp recordings. ASIC currents in response to shear force (SF) were measured at pH 7.4, acidic pH, or in the presence of non-proton ligands at pH 7.4. SF was applied via a fluid stream generated through a pressurized perfusion system. No effect was observed at pH 7.4. Increased transient currents for each homomeric channel were observed when elevated SF was applied in conjunction with acidic pH (6.0-4.0). The sustained current was not (hASIC2a) or only slightly increased (hASIC1V3 and hASIC3a). SF-induced effects were not seen in water injected oocytes and were blocked by amiloride. Non-proton ligands activated a persistent current in hASIC1V3 and cASIC1 (MitTx) and hASIC3a (GMQ) at pH 7.4. Here SF caused a further current increase. Results suggest that ASICs do have an intrinsic ability to respond to mechanical force, supporting their role as mechanosensors in certain local environments.

The non-neuronal cholinergic system in the heart: A comprehensive review.

The autonomic influences on the heart have a ying-yang nature, albeit oversimplified, the interplay between the sympathetic and parasympathetic system (known as the cholinergic system) is often complex and remain poorly understood. Recently, the heart has been recognized to consist of neuronal and non-neuronal cholinergic system (NNCS). The existence of cardiac NNCS has been confirmed by the presence of cholinergic markers in the cardiomyocytes, which are crucial for synthesis (choline acetyltransferase, ChAT), storage (vesicular acetylcholine transporter, VAChT), reuptake of choline for synthesis (high-affinity choline transporter, CHT1) and degradation (acetylcholinesterase, AChE) of acetylcholine (ACh). The non-neuronal ACh released from cardiomyocytes is believed to locally regulate some of the key physiological functions of the heart, such as regulation of heart rate, offsetting hypertrophic signals, maintenance of action potential propagation as well as modulation of cardiac energy metabolism via the muscarinic ACh receptor in an auto/paracrine manner. Apart from this, several studies have also provided evidence for the beneficial role of ACh released from cardiomyocytes against cardiovascular diseases such as sympathetic hyperactivity-induced cardiac remodeling and dysfunction as well as myocardial infarction, confirming the important role of NNCS in disease prevention. In this review, we aim to provide a fundamental overview of cardiac NNCS, and information about its physiological role, regulatory factors as well as its cardioprotective effects. Finally, we propose the different approaches to target cardiac NNCS as an adjunctive treatment to specifically address the withdrawal of neuronal cholinergic system in cardiovascular disease such as heart failure.

Epithelial Na+ channel differentially contributes to shear stress-mediated vascular responsiveness in carotid and mesenteric arteries from mice.

A potential "new player" in arteries for mediating shear stress responses is the epithelial Na+ channel (ENaC). The contribution of ENaC as shear sensor in intact arteries, and particularly different types of arteries (conduit and resistance), is unknown. We investigated the role of ENaC in both conduit (carotid) and resistance (third-order mesenteric) arteries isolated from C57Bl/6J mice. Vessel characteristics were determined at baseline (60 mmHg, no flow) and in response to increased intraluminal pressure and shear stress using a pressure myograph. These protocols were performed in the absence and presence of the ENaC inhibitor amiloride (10 µM) and after inhibition of endothelial nitric oxide synthase (eNOS) by Nω-nitro-l-arginine methyl ester (l-NAME; 100 µM). Under no-flow conditions, amiloride increased internal and external diameters of carotid (13 ± 2%, P < 0.05) but not mesenteric (0.5 ± 0.9%, P > 0.05) arteries. In response to increased intraluminal pressure, amiloride had no effect on the internal diameter of either type of artery. However, amiloride affected the stress-strain curves of mesenteric arteries. With increased shear stress, ENaC-dependent effects were observed in both arteries. In carotid arteries, amiloride augmented flow-mediated dilation (9.2 ± 5.3%) compared with control (no amiloride, 6.2 ± 3.3%, P < 0.05). In mesenteric arteries, amiloride induced a flow-mediated constriction (-11.5 ± 6.6%) compared with control (-2.2 ± 4.5%, P < 0.05). l-NAME mimicked the effect of ENaC inhibition and prevented further amiloride effects in both types of arteries. These observations indicate that ENaC contributes to shear sensing in conduit and resistance arteries. ENaC-mediated effects were associated with NO production but may involve different (artery-dependent) downstream signaling pathways. NEW & NOTEWORTHY The epithelial Na+ channel (ENaC) contributes to shear sensing in conduit and resistance arteries. In conduit arteries ENaC has a role as a vasoconstrictor, whereas in resistance arteries ENaC contributes to vasodilation. Interaction of ENaC with endothelial nitric oxide synthase/nitric oxide signaling to mediate the effects is supported; however, cross talk with other shear stress-dependent signaling pathways cannot be excluded.

Gadolinium released by the linear gadolinium-based contrast-agent Gd-DTPA decreases the activity of human epithelial Na+ channels (ENaCs).

BACKGROUND: Gadolinium-based-contrast-agents (GBCAs) are used for magnetic-resonance-imaging and associated with renal and cardiovascular adverse reactions caused by released Gd3+ ions. Gd3+ is also a modulator of mechano-gated ion channels, including the epithelial Na+ channel (ENaC) that is expressed in kidney epithelium and the vasculature. ENaC is important for salt-/water homeostasis and blood pressure regulation and a likely target of released Gd3+ from GBCAs causing the above-mentioned adverse reactions. Therefore this study examined the effect of Gd3+ and GBCAs on ENaC's activity. METHODS: Human αßγENaC was expressed in Xenopus laevis oocytes and exposed to Gd3+, linear (Gd-DTPA, Magnevist) or cyclic (Dotarem) GBCAs. Transmembrane ion-currents (IM) were recorded by the two-electrode-voltage-clamp technique and Gd3+-release by Gd-DTPA was confirmed by inductively coupled plasma-mass spectrometry. RESULTS: Gd3+ exerts biphasic effects on ENaC's activity: ≤0.3mmol/l decreased IM which was preventable by DEPC (modifies histidines). Strikingly Gd3+≥0.4mmol/l increased IM and this effect was prevented by cysteine-modifying MTSEA. Linear Gd-DTPA and Magnevist mimicked the effect of ≤0.3mmol/l Gd3+, whereas the chelator DTPA showed no effect. Gd3+ and Gd-DTPA increased the IC50 for amiloride, but did not affect ENaC's self-inhibition. Interestingly, cyclic Gd-DOTA (Dotarem) increased IM to a similar extent as its chelator DOTA, suggesting that the chelator rather than released Gd3+ is responsible for this effect. CONCLUSION: These results confirm Gd3+-release from linear Gd-DTPA and indicate that the released Gd3+ amount is sufficient to interfere with ENaC's activity to provide putative explanations for GBCA-related adverse effects.

Mechanosensitive activation of CFTR by increased cell volume and hydrostatic pressure but not shear stress.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl(-) channel that is essential for electrolyte and fluid homeostasis. Preliminary evidence indicates that CFTR is a mechanosensitive channel. In lung epithelia, CFTR is exposed to different mechanical forces such as shear stress (Ss) and membrane distention. The present study questioned whether Ss and/or stretch influence CFTR activity (wild type, ∆F508, G551D). Human CFTR (hCFTR) was heterologously expressed in Xenopus oocytes and the response to the mechanical stimulus and forskolin/IBMX (FI) was measured by two-electrode voltage-clamp experiments. Ss had no influence on hCFTR activity. Injection of an intracellular analogous solution to increase cell volume alone did not affect hCFTR activity. However, hCFTR activity was augmented by injection after pre-stimulation with FI. The response to injection was similar in channels carrying the common mutations ∆F508 and G551D compared to wild type hCFTR. Stretch-induced CFTR activation was further assessed in Ussing chamber measurements using Xenopus lung preparations. Under control conditions increased hydrostatic pressure (HP) decreased the measured ion current including activation of a Cl(-) secretion that was unmasked by the CFTR inhibitor GlyH-101. These data demonstrate activation of CFTR in vitro and in a native pulmonary epithelium in response to mechanical stress. Mechanosensitive regulation of CFTR is highly relevant for pulmonary physiology that relies on ion transport processes facilitated by pulmonary epithelial cells.

Luminal acetylcholine does not affect the activity of the CFTR in tracheal epithelia of pigs.

Fluid homeostasis mediated by the airway epithelium is required for proper lung function, and the CFTR (cystic fibrosis transmembrane conductance regulator) Cl(-) channel is crucial for these processes. Luminal acetylcholine (ACh) acts as an auto-/paracrine mediator to activate Cl(-) channels in airway epithelia and evidence exists showing that nicotinic ACh receptors activate CFTR in murine airway epithelia. The present study investigated whether or not luminal ACh regulates CFTR activity in airway epithelia of pigs, an emerging model for investigations of human airway disease and cystic fibrosis (CF) in particular. Transepithelial ion currents of freshly dissected pig tracheal preparations were measured with Ussing chambers. Application of luminal ACh (100 µM) induced an increase of the short-circuit current (I(SC)). The ACh effect was mimicked by muscarine and pilocarpine (100 µM each) and was sensitive to muscarinic receptor antagonists (atropine, 4-DAMP, pirenzepine). No changes of the I(SC) were observed by nicotine (100 µM) and ACh responses were not affected by nicotine or mecamylamine (25 µM). Luminal application of IBMX (I, 100 µM) and forskolin (F, 10 µM), increase the I(SC) and the I/F-induced current were decreased by the CFTR inhibitor GlyH-101 (GlyH, 50 µM) indicating increased CFTR activity by I/F. In contrast, GlyH did not affect the ACh-induced current, indicating that the ACh response does not involve the activation of the CFTR. Results from this study suggest that luminal ACh does not regulate the activity of the CFTR in tracheal epithelia of pigs which opposes observation from studies using mice airway epithelium.

Phosphocholine-Modified Macromolecules and Canonical Nicotinic Agonists Inhibit ATP-Induced IL-1ß Release.

IL-1ß is a potent proinflammatory cytokine of the innate immune system that is involved in host defense against infection. However, increased production of IL-1ß plays a pathogenic role in various inflammatory diseases, such as rheumatoid arthritis, gout, sepsis, stroke, and transplant rejection. To prevent detrimental collateral damage, IL-1ß release is tightly controlled and typically requires two consecutive danger signals. LPS from Gram-negative bacteria is a prototypical first signal inducing pro-IL-1ß synthesis, whereas extracellular ATP is a typical second signal sensed by the ATP receptor P2X7 that triggers activation of the NLRP3-containing inflammasome, proteolytic cleavage of pro-IL-1ß by caspase-1, and release of mature IL-1ß. Mechanisms controlling IL-1ß release, even in the presence of both danger signals, are needed to protect from collateral damage and are of therapeutic interest. In this article, we show that acetylcholine, choline, phosphocholine, phosphocholine-modified LPS from Haemophilus influenzae, and phosphocholine-modified protein efficiently inhibit ATP-mediated IL-1ß release in human and rat monocytes via nicotinic acetylcholine receptors containing subunits α7, α9, and/or α10. Of note, we identify receptors for phosphocholine-modified macromolecules that are synthesized by microbes and eukaryotic parasites and are well-known modulators of the immune system. Our data suggest that an endogenous anti-inflammatory cholinergic control mechanism effectively controls ATP-mediated release of IL-1ß and that the same mechanism is used by symbionts and misused by parasites to evade innate immune responses of the host.