bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural Heteropolysaccharide Binding to the Visceral Mesothelial Glycocalyx
Authors: Servais Andrew , Kienzle Arne , Valenzuela Cristian , Ysasi Alexandra , Wagner Willi , Ackermann Maximilian , Tsuda Akira , Mentzer Steven
Published: November 2023

Bioadhesives are biopolymers with potential applications in wound healing, drug delivery and tissue engineering. Pectin, a plant‐based heteropolysaccharide, has recently demonstrated potential as a mucoadhesive in the bowel. Since mucoadhesion is a process likely involving the interpenetration of the polymer with mucin chains, we hypothesized that pectin may also be effective at targeting the glycocalyx of visceral mesothelium. To explore the potential role of pectin as a mesothelial bioadhesive, we studied the interaction of various pectin formulations with the mesothelium of lung, liver and bowel. Peel strength and tensile strength of the bioadhesive‐mesothelial interaction was measured by tensilometry. In both high and low methoxyl pectins, bioadhesion was greatest with an equal weight/% formulation with carboxymethylcellulose. Largely independent of the carboxymethylcellulose concentration, the peel strength and tensile strength of the high methoxyl pectin was nearly 3‐fold greater than comparable adhesion of low methoxyl or amidated low methoxyl pectin (p<0.05). Although tissue‐specific variations were observed in lung, liver and bowel mesothelial adhesion, high methoxyl pectin adhesion was consistently greater than low methoxyl formulations. Consistent with a mechanism of polymer‐glycocalyx interpenetration, the high methoxyl pectin adhesion to tissue mesothelium was reversed with hydration and limited by enzyme treatment (pronase). These data indicate that high methoxyl pectins, in an equal weight/% formulation with carboxymethylcellulose, are a promising mesothelial bioadhesive. Further characterization of apparent tissue differences in the mesothelial glycocalyx offers the potential for selective targeting of lung, liver and bowel mesothelium.

Support or Funding Information

NIH T32 HL007734

NIH R01 HL94567
Naturally Occurring Oxidant‐Mediated Protein Amino Acid Conversion
Authors: Suzuki Yuichiro J.
Published: November 2023

Growth factors use reactive oxygen species (ROS) as second messengers. Exact mechanisms of how ROS trigger cell signaling are, however, largely unknown. My laboratory previously demonstrated that protein carbonylation is promoted by ligand/receptor‐mediated cell signaling. Further, ligand/receptor‐mediated protein carbonylation was found to be followed by the reduction of protein carbonyls in a process, which we named ‘decarbonylation’. The discovery of protein decarbonylation suggested that protein carbonylation is reversible. For example, proline residues get oxidized and become glutamyl semialdehyde with a carbonyl group. The present study provides evidence that biological systems contain a mechanism that catalyzes the reduction of glutamyl semialdehyde to proline. Further, since an oxidative product of arginine residues is also glutamyl semialdehyde, protein arginine residues could be converted to proline residues in biological systems. Mass spectrometry analyses supported this hypothesis by showing that two arginine residues (Arg106 and Arg108) of the peroxiredoxin 6‐protein molecule can become proline residues in cultured smooth muscle cells in response to platelet‐derived growth factor. These results reveal that proteins can undergo a naturally occurring oxidant‐mediated protein engineering process, which represents a novel biological mechanism.

Support or Funding Information

NIH
Calcium Imaging Across Large Areas of Intact Vascular Endothelium Reveals Stimulus‐Specific Sensory Cells
Authors: Lee Matthew D. , Wilson Calum , McCarron John G.
Published: November 2023

The endothelium is the innermost layer of blood vessels and it is a complex signal processing centre that controls almost all cardiovascular function. The endothelium uses a multitude of receptors to detect multiple simultaneously arriving extracellular signals that provide cues to physiological state and to determine cellular responses. Ca2+ signals in endothelial cells decode these extracellular signals and encode intracellular information to regulate both single cell activity and physiological behaviour of the entire endothelium [1–3]. Ca2+ regulates the synthesis and release of various vasoactive agents such as nitric oxide and prostacyclin and induces endothelium‐derived hyperpolarising factor. Through various Ca2+‐dependent mediators, the endothelium controls vascular tone, blood‐tissue exchange, recruitment of blood cells, modulates blood clotting and determines the formation of new blood vessels. However, precisely how Ca2+ selectively couples different extracellular signals to various physiological functions is not fully understood. Within single cells, differentiation of extracellular signals may rely on the amplitude, kinetic or spatial features of the Ca2+ signal. Here we report that the endothelium also relies on stimulus‐specific detector cells to resolve various extracellular signals.

To determine how multiple extracellular signals were transduced, we studied Ca2+ signals evoked by two extracellular activators (ATP and carbachol) in large numbers of endothelial cells in intact arteries. The Ca2+ indicator, Cal‐520/AM, was used to image the changes in Ca2+ within ~200 endothelial cells and the data was analysed using a custom analysis package written in Python [3]. Each activator evoked concentration‐dependent increases in the number of cells activated and in the amplitude and frequency of the Ca2+ signal in each cell. The Ca2+ signals for each agonist began in clusters of cells and progressed from there as propagating waves of Ca2+. The number of clusters of cells activated and the extent of propagation of the Ca2+ wave increased with agonist concentration. However, there was no apparent differentiation in the nature of the activator from either the ensemble response or the amplitude, duration or frequency of the Ca2+ signal within cells. Significantly, while, at high concentrations, each agonist evoked a response in every cell in the field, different cells were more sensitive to each agonist. Thus at low concentrations spatially‐distinct cells detected each agonist. Furthermore, the first responding cells for each agonist at all concentrations were spatially‐distinct. Thus we show that, in addition to the spatial temporal properties of the Ca2+ signal within cells, the endothelium uses distinct cells to sensitively detect and specifically decode extracellular activators.
Nongenomic action of cortisol in rainbow trout hepatocytes
Authors: Vijayan Matt , Das Chinmayee , Wildering Willem C
Published: November 2023

Cortisol rapidly activates cell signalling in trout hepatocytes; however, the mechanisms are far from clear. We tested the hypothesis that rapid action of cortisol involves modulation of intracellular Ca2+ levels in trout hepatocytes. Stress level of cortisol reported in fish plasma elicits a rapid increase in intracellular Ca2+ level as determined by Fura‐2AM ratiometric imaging. The increase in intracellular Ca2+ level in trout hepatocytes was also seen with the membrane impermeable form of the steroid (cortisol‐BSA), and this response was not affected by mifepristone (glucocorticoid receptor antagonist). We determined the role of extracellular and intracellular stores of calcium in affecting cortisol‐induced elevation in Ca2+ levels by using EGTA (a chelator) and BAPTA‐AM (inhibitor), respectively. Furthermore, we determined if IP3R pathway was involved in the activation of intracellular stores by using various inhibitors, including PLC inhibitor (U73122), thapsigargin (SERCA blocker) and Rynodine (RyR blocker). Also, L‐type calcium channel blocker Nifedipine and Ca2+release‐activated Ca2+ (CRAC) channel blocker Cpd5J‐4 were utilized to determine the mode of entry of extracellular calcium in response to cortisol stimulation. Overall, our results suggest that cortisol rapidly increases intracellular calcium levels and this may be mediated by the activation of CRAC channel in trout hepatocytes.

Support or Funding Information

This study was supported by the Natural Sciences and Engineering Research Council of Canada Discovery Grant to MMV.
Plasma Exosomes Modulate Signalling in Rainbow Trout Hepatocytes
Authors: Faught Erin , Vijayan Matt
Published: November 2023

Exosomes are a subtype of cell‐secreted extracellular vesicles, approximately 40–100 nm in diameter, that are formed from endosomal compartments. Exosomes carry a payload of proteins and RNA and are thought to play a role in inter‐tissue communication, including regulation of cellular function. Most of these studies are in mammalian models and very little is known about the role of exosomes in lower vertebrates. Recently we showed that the intracellular chaperone heat shock protein 70 (Hsp70) is exported into the plasma of rainbow trout (Oncorhynchus mykiss) in exosomes. Here we tested the hypothesis that plasma exosomes play a role in cell to cell communication and that this is mediated by Hsp70. To determine this, plasma exosomes were tagged with a fluorescent lipophilic dye (DiD) and their uptake monitored in trout hepatocytes in primary culture. Exosomes were taken up by hepatocytes over a 24 h period, but the physiological implications were unclear. We also examined whether exosomes modulate the phosphorylation status of key secondary signalling messengers. Indeed, exosome addition significantly increased phosphorylation of p42/44 MAPK, but not p38 MAPK or cAMP response element‐binding protein (CREB) within 20 min, in trout hepatocytes. Interestingly, when these vesicles were incubated with anti‐Hsp70 antibody, the exosome‐induced increase in p42/p44 MAPK phosphorylation was abolished. This suggests that Hsp70 may be a key player in exosome‐mediated cell to cell signalling in rainbow trout. Overall, this novel finding underscores a key role for exosomes in modulating cellular function during stress in fish.

Support or Funding Information

This study was supported by the Natural Sciences and Engineering Research Council of Canada Discovery Grant to MMV and Post‐Graduate Fellowship to EF.
Expression of angiotensin converting enzyme affects the presentation of MHC class II antigen
Authors: Zhao Tuantuan , Shen Xiao , Shi Peng , Fang Jianmin , Bernstein Kenneth
Published: November 2023

Angiotensin converting enzyme (ACE) in the endoplasmic reticulum has been revealed as the major carboxypeptidase involved in MHC class I antigen processing. ACE is a type I membrane protein and are also present on the surface of cytoplasmic membrane. This study is to understand whether ACE is involved in MHC class II antigen presentation. We find that the macrophages expressing null, normal and excessive ACE have different abilities in presenting model antigens ovalbumin (OVA) and hen egg lysozyme (HEL) in vitro. The differences were observed in the presentation of either the whole antigen proteins or their MHC class II epitopes. The mice with ACE over‐expressed in macrophages are superior over wild‐type (WT) mice in producing CD4+ T cell responses and antibody responses when immunized with OVA in vivo. In addition, ACE can be localized in the endosomic pathway where MHC class II antigen processing and loading take place. The efficiency of macrophages to present MHC class II antigens can be altered by transfecting ACE‐expressing vector or using ACE inhibitors. These data strongly suggest that ACE affects the presentation of MHC class II antigen.
NKCC Regulates Circadian Rhythm Via the Chloride‐Sensing WNK Kinase
Authors: Rodan Aylin , Schellinger Jeffrey , Sun Qifei , Rothenfluh Adrian
Published: November 2023

Objective

Oscillation of intracellular Cl− has been proposed to play a role in circadian rhythms. We examined the role of Cl− transporters and the Cl−‐sensitive WNK (with‐no‐lysine) kinase in the regulation of circadian rhythm in Drosophila melanogaster.

Methods

We measured the circadian period of wild‐type and mutant flies using 24‐hour activity monitoring. We measured chloride concentration in the Drosophila brain pacemaker neurons using the transgenic sensor, ChlopHensor. Results were analyzed in GraphPad Prism using t‐test or one‐way ANOVA with Bonferroni correction for multiple comparisons.

Results

Male flies with null mutations in the Cl− influxing sodium‐potassium‐2‐chloride cotransporter, NKCC (encoded by the gene Ncc69 in flies), have an increased period length compared to controls (mean±SEM: 25.18±0.28 vs. 23.98±0.05 hours, p<0.0001) which is rescued by expression of wild‐type NKCC in the pacemaker neurons (23.78±0.08, p<0.0001 vs NKCC mutant). In NKCC mutant flies, knockdown of the Cl− effluxing potassium‐chloride cotransporter, KCC, in the pacemaker neurons also normalizes period length (23.94±0.27 vs 26.38±0.23 hours, p<0.0001). In control flies, Cl− concentration increased in pacemaker neurons between two hours after lights on (ZT2) and ZT6, from 44±7 to 81±8 mM, whereas in NKCC mutant flies, Cl− did not increase (35±6 to 44±8 mM), resulting in abnormally low intracellular Cl− concentration at ZT6 in the NKCC mutant flies compared to controls (p=0.0027). Cl− inhibits WNK. The NKCC mutant period length phenotype (26.67±0.13 h) was suppressed by knockdown of either wnk (24.70±0.29, p<0.0001) or its downstream kinase fray (homolog of mammalian SPAK/OSR1) (24.34±0.15, p<0.0001) in the pacemaker cells. Conversely, overexpression of a Cl−‐insensitive (activated) wnk (25.34±0.10 h, p=0.0002 vs control) or constitutively active fray (25.56±0.26, p<0.0001 vs control), resulted in increased period length.

Conclusion

Normal activity of the fly NKCC is required to allow an increase in intracellular Cl− in the fly pacemaker neurons between two hours after lights on and six hours. The failure of intracellular Cl− to increase in NKCC mutant flies results in aberrant activation of WNK‐SPAK/OSR1 signaling and prolonged circadian period.

Support or Funding Information

National Institutes of Health
Reduced Expression of Down‐Regulated in Adenoma (DRA) in Murine Enteroid‐Derived Monolayers Following Infection with Salmonella
Authors: Jayaratne Rashini Randika , Lee Beom‐Jae , Lim Eileen , Barrett Kim , Das Soumita
Published: November 2023

Non‐typhoidal Salmonella spp. are the second leading cause of foodborne illness worldwide. Despite its prevalence, the mechanism through which Salmonella infection causes diarrhea is not fully understood. The intestinal epithelium plays a major role in both interacting with bacteria and fluid accumulation. To understand the basic mechanism of Salmonella infection in intestinal epithelial cells, we developed a murine enteroid model that mimics the crypt‐villus axis of the intestines, and contains the four different epithelial subtypes found in the intestine (enterocytes, enteroendocrine, goblet, and Paneth cells). We have previously shown that infection with Salmonella causes decreased expression of the chloride/bicarbonate exchanger SLC26A3 (DRA; Downregulated in Adenoma) in the murine colon. We developed enteroid‐derived monolayers (EDM) from mouse ileum and colon to test whether DRA was similarly down‐regulated following infection with Salmonella. Monolayers were generated from enteroids on either permeable transepithelial supports or cell culture plates. Monolayers were also grown in 8‐well chamber slides to visualize DRA and bacterial internalization. After differentiation, EDM were infected with Salmonella enterica serovar Typhimurium. Expression of DRA and markers for different cell subtypes were studied using RT‐PCR, western blotting, and confocal microscopy. We also measured bacterial internalization using a gentamicin protection assay followed by colony counts. We found bacterial internalization after 1 h followed by decreased expression of DRA after 6 h of Salmonella infection. Interestingly, following infection the proliferation marker Ki67 was upregulated. The intestines contain both secretory (enteroendocrine, goblet, and paneth cells) and absorptive (enterocytes) cell types and their expression was evaluated following infection. The Wnt/NOTCH signaling pathways control cell fate decisions in the intestines. We found that Hes1, a downstream signaling molecule in the NOTCH signaling pathway, was up regulated following infection. Our work shows the potential for EDM to identify molecular mechanisms that may underlie intestinal dysfunction during infection. Ultimately, this model may identify signals that can be targeted for therapeutic development.
Overexpression of SERCA2a alters endoplasmic reticulum Ca2+ handling in WKY but not SHR cardiac sympathetic neurons. A role for altered intracellular Ca2+ handling in hypertensive sympathoexcitation?
Authors: Shanks Julia , Herring Neil , Li Dan , Johnson Errin , Paterson David J
Published: November 2023

Increased sympathetic neurotransmission is well established in both animal and clinical models of hypertension. Altered cardiomyocyte calcium (Ca2+) handling has also been implicated in heart failure, leading to the development of gene therapies to upregulate sarcoendoplasmic reticulum calcium ATPase (SERCA) in cardiomyocytes from heart failure patients. Dysregulation in neuronal Ca2+ handling has previously been reported within isolated neurons of the spontaneously hypertensive rat (SHR). We tested whether overexpressing SERCA2a in isolated cardiac stellate neurons from SHR and Wistar Kyoto rats (WKY) could modulate intracellular Ca2+ transients. Isolated neurons were incubated with either Ad‐MCherry‐SERCA2A or Ad‐MCherry‐Empty (control) for 24 hours immediately post isolation. Ca2+ imaging using fura‐2AM (2.5μM) was carried out three days post transfection. Intracellular Ca2+ changes were measured using Fura‐2AM in response to KCl, caffeine, thapsigargin and FCCP to mobilise intracellular Ca2+ stores. In isolated SHR neurons SERCA2a overexpression facilitated greater depolarisation induced Ca2+ transients than age matched WKY (WKY, Empty: 0.45±0.05 au, n=17. SERCA: 0.66±0.09 au, n=13. SHR, Empty: 0.65±0.06 au, n=18. SERCA: 0.80±0.04 au, n=25. *P<0.05), but no change in ER load (SHR. Empty: 0.16±0.04 au, n=18. SERCA: 0.17±0.02 au, n=25). Whereas ER Ca2+ load was significantly increased post SERCA2a overexpression in the WKY (WKY. Empty: 0.02±0.02 au, n=17. SERCA: 0.11±0.02 au, n=13). Transmission electron microscopy shows a greater abundance of ER in SHR neurons. SERCA2a overexpression resulted in increased mitochondrial Ca2+ load in both SHR and WKY neurons (WKY, Empty: 0.08±0.006 au, n=32. SERCA: 0.11±0.01 au, n=33. SHR, Empty: 0.63±0.009 au, n=25. SERCA: 0.10±0.01 au, n=32) (**P<0.01). Indicating that altered neuronal Ca2+ handling in the SHR may be due to an already present abnormal ER Ca2+ load, with upregulation of SERCA2a further effecting mitochondrial Ca2+ handling and potentially metabolic function in these neurons. The effect of SERCA2a on neurotransmitter release was measured using 3H‐norepinephrine overflow from SD rat atria in response to right stellate ganglia stimulation. Upregulation of atrial SERCA2a resulted in greater neurotransmitter release in compared to control (Empty: 98.7±20.5 cpm, n=7. SERCA: 186.5±28.41 cpm, n=8. *P<0.05). These results demonstrate that SERCA2a upregulation in cardiac sympathetic neurons with broad spectrum promoters results in increased neurotransmission and increased loading of Ca2+ into intracellular stores that trigger greater Ca2+ transients on subsequent neuronal depolarisation.

Support or Funding Information

British Heart Foundation
Shear‐stress sensitive endothelial K channels play major role in flow‐induced vasodilation
Authors: Ahn Sang Joon , Fancher Ibra S , Bian Jing‐tan , Zhang Chong Xu , Gaffin Robert , Phillips Shane A , Levitan Irena
Published: November 2023

Inwardly rectifying K+ (Kir) channels are known to be a putative flow sensor of endothelial cells but their role in the sensitivity of vascular endothelial cells to flow has not been established. In this study, we demonstrate that genetic deficiency of Kir2.1 results in significant loss of flow‐induced activation of the AKT/eNOS cascade, flow‐induced NO release and flow‐induced vasodilation in murine mesenteric arteries. First, we established the primary endothelial cells isolated from murine mesenteric arteries and showed their expression of functional Kir2.1 channels sensitive to shear stress. In order to determine the role of Kir2.1 in flow‐induced endothelial signaling and vasodilaton, we used two approaches: i) a genetic deficiency model, Kir2.1 heterozygous mouse model, a genetically Kir2.1 deficient mouse and ii) i) Ba2+, a well‐known Kir blocker. We observed that, as expected, the Kir2.1 expression level and the flow‐induced whole‐cell currents significantly lower in the endothelial cells from Kir2.1+/− mouse as compared to WT. Furthermore, flow‐induced vasodilation was significantly reduced in Kir2.1+/− mesenteric arteries as compared to WT controls. Exposure of WT arteries to Ba2+ ahad the same effect arteries (92.8±3.2% vs 48±5.6% vs 42.2±2.3%, WT vs WT with Ba2+ vs Kir2.1+/−). Kir2.1 deficiency also resulted in the loss of flow‐induced activation of eNOS and Akt, as well as reduction of NO generation. Notably, EC‐specific over‐expression of Kir2.1 resulted in full rescue of all the flow‐induced functions described above: AKT/eNOS activation, NO release and vasodilation. We also observed that Kir‐independent component of FIV was abrogated by blocking Ca2+‐sensitive K+ channels. Kir2.1 regulation on endothelium‐independent and K+‐induced vasodilation was gone in denuded arteries. Kir2.1+/− mice also showed higher mean blood pressure measured by carotid artery cannulation and increased microvascular resistance measured using a volume‐pressure recording tail cuff. More importantly, blocking of Kir channels also significantly decreased flow‐induced vasodilation in human subcutaneous adipose microvessels. Taken together, our observations suggest that endothelial Kir contribute to flow‐induced vasodilation of mouse mesenteric arteries through NO‐dependent mechanism, whereas Ca2+‐sensitive K+ channels mediate FIV with NO‐independent pathway. Kir2 channels also regulate blood pressure and vascular resistance. Finally, we also show that Kir channels also regulate FIV in human sub‐cutaneous microvessels.

Support or Funding Information

NIH grants HL‐073965 and HL083298 to Irena Levitan NIH grants T32 DK080674 to Ibra Fancher
Glucocorticoid Regulation of Large Conductance, Ca2+‐Activated K Channels in Differentiated Human Bronchial Epithelial Cells
Authors: Zaidman Nathan A , Panoskaltsis‐Mortari Angela , O'Grady Scott M
Published: November 2023

An important component of airway host‐defense involves the process of trapping and removing inspired material through mucociliary clearance (MCC). Regulated transepithelial ion transport supports this process by maintaining the depth of the airway surface liquid (ASL). In the present study we determined the effects of hydrocortisone (HC) on UTP‐stimulated ion transport in differentiated, pseudostratified epithelia derived from normal human bronchial basal cells. UTP stimulated a paxilline‐sensitive, large conductance Ca2+‐activated K (BK) current in control epithelia differentiated in HC containing media, however BK current was not detected in epithelia differentiated in the absence of HC (HC0). The BK opener NS11021 directly activated channels in control epithelia, but in the absence of HC, BK current was only observed when UTP was added after NS11021. The pan‐specific PKC inhibitors GF109203× and Gö6983 blocked UTP‐induced BK activation under control conditions, suggesting that PKC‐mediated phosphorylation was necessary for BK activation. HC0 epithelia expressed significantly more KCNMA1 mRNA transcripts containing the stress‐regulated exon (STREX), a splice‐variant that exhibits altered channel regulation by phosphorylation relative to channels that do not contain the STREX sequence. Furthermore, BK channels and P2Y receptors exhibited distinct cellular localization patterns in overlapping membrane domains at the apical region of differentiated surface cells. These results demonstrate a previously unrecognized role for glucocorticoids in BK channel regulation.

Support or Funding Information

This work was supported by National Institute of Biomedical Imaging and Bioengineering Grant F31 EB‐018707 (N. A. Zaidman) and National Heart, Lung, and Blood Institute Grants R01 HL‐108627 (A. Panoskaltsis‐Mortari) and R01 HL‐110539 (S. M. O'Grady)
A Cyclic‐AMP Dependent Pathway Regulates Intestinal Oxalate Secretion
Authors: Whittamore Jonathan , Hatch Marguerite
Published: November 2023

A primary constituent of kidney stones is calcium oxalate, where elevated urinary oxalate (hyperoxaluria) is a major risk factor. Oxalate is a waste metabolite, and dietary component, with the kidney largely responsible for its excretion. However, the intestine also has a pivotal role, capable of actively secreting oxalate and reducing the endogenous burden. Molecular identification of the membrane‐bound intestinal anion exchangers (AEs) which transport oxalate has raised the possibility of the intestine serving as potential therapeutic target. Despite these advances, the signals and pathways regulating oxalate handling remain largely undefined. Our recent finding that oxalate secretion by the mouse cecum can be dramatically stimulated by the intracellular 2nd messenger cyclic AMP (cAMP) is therefore particularly significant. The objective of this study was therefore focused on identifying the apical transporter and characterizing the corresponding basolateral process involved. At the apical membrane, the Slc26a6 anion exchanger PAT1 (Putative Anion Transporter 1), is a prospective candidate since previous work has demonstrated PAT1 mediates oxalate secretion in the adjacent small intestine. Utilizing in vitro tissue preparations mounted in Ussing chambers, experiments compared ceca from wild‐type and PAT1‐knockout mice. We determined that PAT1 was not directly involved in oxalate secretion, and subsequent experiments revealed evidence for another AE, as well as the cAMP‐stimulated chloride (Cl−) channel, CFTR. At the basolateral membrane, the rate‐limiting step for secretion did not require a Cl−‐dependent AE, as hypothesized, rather our data revealed a novel finding – the involvement of a cation‐Cl− cotransporter – supporting oxalate secretion in response to cAMP.

Support or Funding Information

NIH NIDDK DK‐088892 (M. Hatch)
TRPV4 Expression in Hydrocephalic Rat Choroid Plexus
Authors: Simpson Stefanie , Scherer Andrea , Fulkerson Daniel , Blazer‐Yost Bonnie
Published: November 2023

Hydrocephalus, also known as “water on the brain,” is a condition characterized by an imbalance in cerebral spinal fluid (CSF) production and/or reabsorption or by a blockage in the CSF circulation resulting in enlarged ventricles of the brain. The main production of CSF comes from the choroid plexus (CP), present in the lateral, third, and fourth ventricles. Composed of a high resistance epithelium that surrounds a network of capillaries, the choroid plexus regulates the transport of ions and water between the ventricles and capillaries through channels in the barrier epithelium, thus controlling the production and movement of CSF. When ions are transported across the epithelium, water follows, suggesting aberrant regulation of ion channels may be one cause of hydrocephalus. The mechano‐ and osmo‐sensitive calcium channel, Transient Receptor Potential Vanilloid‐4 (TRPV4), is a potential regulatory channel which is present in CP epithelia. Utilizing the Wpk rat model, which contains a recessive mutation in TMEM67, we are able to model both rapidly progressing (birth to 3 weeks) and slowly progressing (birth to 1 year) hydrocephalic models (homozygous and heterozygous animals, respectively). These genetic models have the advantage of a naturally occurring hydrocephalus as opposed to the commonly used kaolin infusion model where treatment with the caustic compound ablates the epithelial cells that are under investigation. Treatment of Wpk affected rats with a TRPV4 antagonist was found to ameliorate the hydrocephalus in the rapidly progressing model. Immunohistochemistry of brain tissue slices from the hydrocephalic and wild type animals was utilized to visualize and localize TRPV4. At birth, there is an overexpression of TRPV4 in the CP of the hydrocephalic rats compared to wild type animals, and throughout development the expression continues to increase. In both phenotypes, TRPV4 localized to the apical membrane of the CP. In wild type animals and at birth in hydrocephalic animals there is no detectable TRPV4 expression in the ependymal cells lining the ventricle. However, by 15 days of age in hydrocephalic rats, the ependymal cells do express TRPV4. The increase in TRPV4 expression in hydrocephalic rats, combined with the efficacy of TRPV4 antagonist treatment suggests the calcium channel plays a major role in homeostatic CSF production as well as the formation of hydrocephalus.

Support or Funding Information

Hydrocephalus Association Innovator Award; Indiana University Collaborative Research Grant and Indiana Clinical and Translational Sciences Institute CTR Award.
Discrepancy between TRPA1 activation by reversible and irreversible electrophiles suggests involvement of cytosolic cofactors other than polyphosphates.
Authors: Parks Thomas A , Bahia Parmvir K , Taylor‐Clark Tom
Published: November 2023

Transient Receptor Potential Ankyrin 1 (TRPA1) is a non‐selective cation channel that is expressed in a subset of nociceptive afferent sensory nerves. When activated, TRPA1 can evoke nociceptive responses (e.g. pain and defensive reflexes), as well as peripheral neurogenic inflammation. TRPA1 is activated by a host of endogenous (inflammatory mediators) and exogenous (pollutants, food) agonists. Many TRPA1 agonists are electrophilic compounds that covalently modify reactive cysteine residues on TRPA1, leading to activation of the channel. Electrophiles can bind through reversible or irreversible reactions based on the structure of the compound. Here, patch clamp techniques and calcium imaging have been used to characterize human TRPA1 (hTRPA1) channel activity (expressed in HEK293 cells) in the presence of reversible and irreversible electrophiles. Calcium imaging shows that both reversible electrophiles (e.g. allylisothiocyanate [AITC]) and irreversible electrophiles (e.g. iodoacetamide [IA] and N‐ethylmaleimide [NEM]) rapidly induce TRPA1 activation. In contrast, our patch clamp studies show a discrepancy between irreversible and reversible electrophilic TRPA1 activation. Whole cell patch clamp studies exhibit a reduction in the kinetics and magnitude of TRPA1 activation by IA and NEM compared to AITC. Furthermore, single channel patch clamp experiments display reduced open probability with IA and NEM compared to AITC. Both whole cell patch and excised patch clamp recordings were performed in the presence of 5mM polyphosphates. In preliminary gramicidin perforated patch recordings there was no difference in NEM and AITC‐evoked TRPA1 activation. Thus in intact cellular assays (Ca2+ imaging and gramicidin‐perforated patch) there is little difference in TRPA1 activation by reversible and irreversible electrophiles, whereas in dialyzed cellular assays (whole cell patch and excised patch) irreversible electrophiles evoked reduced TRPA1 activation compared to reversible electrophiles. The data suggests that cytosolic factors other than polyphosphates are important for TRPA1 activation by irreversible electrophilic ligands.

Support or Funding Information

NHLBI R01 (HL119802) and Supplement (HL119802‐S1)
The Role of Pore‐Lining Residues of MEC‐4 and MEC‐10 in the Channel's Response to Shear Stress
Authors: Shi Shujie , Kleyman Thomas R.
Published: November 2023

In the nematode Caenorhabditis elegans, gentle body touch is sensed via a multi‐protein mechanotransduction apparatus expressed in the worm's touch receptor neurons. At the core of this mechanotransduction apparatus is a mechanosensitive ion channel formed by two homologous protein, MEC‐4 and MEC‐10. Like other members of the epithelial Na+ channel (ENaC)/degenerin family, the channel's pore is primarily lined by the second transmembrane helixes (TM2) of MEC‐4 and MEC‐10. Strikingly, there are nine and four touch‐insensitive mutations within the MEC‐4 TM2 and MEC‐10 TM2, respectively, highlighting the importance of the pore‐lining residues in transmitting gating signals.

When expressed in Xenopus oocytes, the MEC‐4/MEC‐10 channel is activated by flow‐mediated laminar shear stress (LSS), and both MEC‐4 and MEC‐10 are required for a robust LSS response. To explore the role of the pore‐lining residues of MEC‐4 and MEC‐10 in the channel's regulation by mechanical forces, we introduced point mutations at the specific sites within the MEC‐4 TM2 or MEC‐10 TM2 and examined its response to LSS in Xenopus oocytes. We observed that the LSS response was largely diminished by touch‐insensitive mec‐10 alleles that introduce charged residues into the channel's pore. Both the magnitude and kinetics of channel action by LSS were altered when mutations were introduced at specific sites within MEC‐4 TM2. In addition, we found that channels with high intrinsic Po (MEC‐4 A713D, or MEC‐4 A713C modified with MTSES) showed reduced response to LSS. Together, our results support an essential role of the pore‐forming regions of MEC‐4 and MEC‐10 in channel gating in response to mechanical stimuli.

Support or Funding Information

This work was financially supported, in whole or in part by National Institutes of Health Grants: DK103834 (to S.S.), DK051391 (to T.R.K.), and DK079307 (Pittsburgh Center for Kidney Research)
SCN3A‐encoded Voltage‐gated Sodium Channel NaV1.3 is Specifically Expressed in Human and Mouse Gastrointestinal Enterochromaffin Cells and is Important for Enterochromaffin Cell Excitability
Authors: Strege Peter R. , Knutson Kaitlyn R. , Eggers Samuel J , Wang Fan , Li Joyce , Leiter Andrew , Farrugia Gianrico , Beyder Arthur
Published: November 2023

BACKGROUND

In the gastrointestinal (GI) epithelium, enterochromaffin (EC) cells are enteroendocrine cells that use tryptophan hydroxylase 1 (TPH‐1) to produce serotonin (5‐hydroxytryptamine, 5‐HT). EC cells release 5‐HT in response chemical and mechanical stimuli. EC cell 5‐HT affects GI secretion, motility and sensation. Other neuroendocrine cells are electrically excitable, and several types use SCN3A‐encoded voltage‐gated sodium channel NaV1.3 for electrical excitability. It is unknown whether EC cells are electrically excitable.

AIM

To determine if the EC cells are electrically excitable.

METHODS

Murine TPH1‐CFP small bowel was dissociated, FAC sorted for TPH1‐CFP cells, and ion channel expression was analyzed by RNA‐seq. Murine TPH1‐CFP and human small bowel and colon were analyzed by immunohistochemistry (IHC). After dissociation, primary cultures from TPH1‐CFP mouse jejunum and colon were maintained for 72 hours. Scn3a expression in CFP+, CFP− cultured cells and media was analyzed by single cell polymerase chain reaction (PCR). Voltage‐ and current‐clamp experiments were performed on TPH1‐CFP cells at 24–48 hours.

RESULTS

RNA‐seq of FACS TPH1‐CFP EC cells from murine small bowel showed that Scn3a was the 3rd most densely expressed ion channel gene, and no other ScnXa genes showed significant expression. Scn3a encodes a voltage‐gated sodium channel NaV1.3. By IHC, NaV1.3 is expressed in ~89–93% of the human and mouse small bowel and colon EC cells, which were defined as TPH1‐CFP+ for mouse and 5‐HT+ for human. Single cell PCR of CFP+ cells from primary cultures confirmed Scn3a expression. In voltage‐clamp, we found voltage‐gated fast activating and inactivating voltage‐gated currents in TPH1‐CFP EC cells from murine small bowel (78% of cells, −86±16 pA/pF, n=28), and colon (65% of cells, −66±12 pA/pF, n=14). These currents were abolished by Na+ substitution by NMDG+ and blocked by the NaV1.3 inhibitor ICA‐121431 (IC50 = 131±53 nM). Current‐clamped EC cells showed both elicited and spontaneous action potentials, also abolished by Na+ substitution and NaV1.3 block by ICA‐121431. Spontaneous current‐clamp activity showed action potential bursts of variable amplitude and frequency.

CONCLUSIONS

SCN3A‐encoded voltage‐gated sodium channel NaV1.3 is densely and specifically expressed by the EC cells of the human and mouse small and large bowel. In mouse EC cells, NaV1.3 generates voltage‐gated sodium current which is critical for EC cell electrical excitability.

Support or Funding Information

NIH DK106456, DK84567, DK052766
Involvement of Intracellular Transport in Regulation of TREK‐1 Channel in 293T Cells
Authors: Andharia Naaz , Joseph Ancy , Hayashi Mikio , Okada Masayoshi , Matsuda Hiroko
Published: November 2023

Introduction

Potassium (K+) channels play a crucial roles in the regulation of the resting membrane potential and excitability of various cells. The mammalian two‐pore domain K+ channels are responsible for background or leak K+ currents, one of which is TREK‐1 (TWIK‐related K+ channel). The current of TREK‐1 channel appears to be regulated by intracellular transport, in which the cytosolic C‐terminus has been shown to play a important role in cell surface expression of the channel. An interesting feature of the TREK‐1 channel is the run‐up: i.e. the current through TREK‐1 channels spontaneously increases within several minutes in the whole‐cell patch‐clamp mode. Since C‐terminal domain is required for the surface localization of the TREK‐1 channel, the run‐up might be regulated by transport to the plasma membrane.

Objectives

The present study aimed to investigate the involvement of intracellular transport in the run‐up.

Methods

We stably transduced TREK‐1c or its fusion protein with mCherry to the 293T cells and measured whole‐cell currents with patch‐clamp techniques. Cell surface localization was analyzed with confocal microscopic analysis and protein biotinylation.

Results

Inhibitors of membrane protein transport, N‐methylmaleimide (NEM) and brefeldin‐A, inhibited the run‐up, while an endocytosis inhibitor, pitstop2, facilitated the run‐up. Correspondingly, in cells stably expressing the mCherry‐TREK‐1c fusion protein, NEM decreased the surface localization of mCherry‐TREK‐1c, whereas pitstop2 increased it. The run‐up was inhibited by the intracellular application of a peptide of the C‐terminal fragment, TREK335‐360, corresponding to the interaction site with microtubule associated protein 2 (Mtap2). The extracellular application of an Ezrin inhibitor (NSC668394) also suppressed the surface localization and the run‐up. The co‐application of these inhibitors abolished the TREK‐1c current, suggesting that the additive effects of ezrin and Mtap2 enhance the surface expression of TREK‐1c channels and run‐up.

Conclusion

These results indicate that intracellular protein transport is involved in the run‐up of TREK‐1c currents.

COI: NO

Key words: K+ channel, TREK‐1, intracellular transport, microtubule‐associated protein 2, ezrin

Support or Funding Information

This study was supported by KAKENHI from the JSPS, the SICP from the JST, the Heiwa Nakajima Foundation, the Ryobi Memorial Foundation (to MO), the Mishima Memorial Foundation, the Rotary Yoneyama Memorial Foundation (to NA), and the Fujii Memorial Foundation (to AJ).
Regulation of claudins in renal TJ by Na+,K+‐ATPase alpha 1 subunit
Authors: Larre Maria Isabel , Zhang Jue , Xie Zijian
Published: November 2023

Tight junctions (TJs) are located at the uppermost portion of the lateral membrane of epithelial cells that seal the intracellular interspace between adjacent cells, transforming the epithelial cells into an effective permeable barrier. Tight junctions regulate the movements of substances through the paracellular pathway, and claudins are major determinants of TJ permeability. In the kidneys, the molecular composition of TJ determines the permeability and ion selectivity of different nephron segments along renal tubules. For example, while proximal tubules express claudin‐2, distal nephrons express the claudin‐4 isoform. Little is known about what controls the composition of TJ along the nephron. Previous reports have shown that ouabain, a cardiac glycoside that acts by inhibiting the Na+,K+‐ATPase pump activity, increases TJ sealing degree (10–100 nM), suggesting the participation of Na+,K+‐ATPase in TJ regulation. In this study, with newly developed rescue α1 mutant protocol, we were able to generate unique stable epithelial cell lines that express Na+,K+‐ATPase alpha‐1 with specific mutations in the N domain. The expression of Na+,K+‐ATPase alpha‐1 mutants (A425P and A420P) does not affect the activity of sodium pump but increases the level expression of Src phosphorylation at Y418 and also decrees the expression of caveolin‐1. This study is the first approach to investigated the role of Na+,K+‐ATPase alpha 1 on TJ protein expression and function on kidney epithelial cells.

Results

Using TER measurement, western blot and confocal microscopy we found that A420P and A425P mutation decreases the ion permeability and changes the molecular composition of TJ proteins when compared to wild type Na+,K+‐ATPase alpha‐1. The expression levels and cellular localization of Claudin‐2 and claudin‐4 were altered. Src inhibitor PP2 and pNaKtide were able to decrease the TER values in A425P cell line comparable to wild type. Thus, this work shows that the N domain of Na+/K+‐ATPase alpha‐1 has an important role in tight junction function and this regulation implicates Na+,K+‐ATPase‐Src‐Caveolin‐Claudin pathway. Taken together, these observations have a significant relevance to the altered function of kidneys in physiological and pathological conditions, in which the transport of ion and nutrients are significantly altered.
The membrane trafficking and functionality of the K+‐Cl− co‐transporter KCC2 is regulated by TGF‐β2
Authors: Khakipoor Shokoufeh , Roussa Eleni
Published: November 2023

GABAA response shifts from depolarizing to hyperpolarizing during neuronal maturation and it is well established that K+‐Cl−‐cotransporter KCC2 up‐regulation is responsible for this process. Failure in KCC2 up‐regulation is seen in epilepsy and stroke. Surprisingly, there is not much known about regulatory pathways involved in the KCC2 expression, trafficking, and functional expression. Here, we investigate the effect of transforming growth factor beta 2, TGF‐β2, in the regulation of KCC2 trafficking and functional expression in mouse mature hippocampal neurons and analyze the underlying molecular pathways. Therefore embryonic day 18 primary hippocampal neurons and hindbrain slices were used. KCC2 protein and surface expression is investigated by immunoblotting, surface biotinylation and immunofluorescence. Signaling pathways are studied using siRNA knockdown, co‐immunoprecipitation, and 3D STED microscopy. KCC2 functional expression is measured by somatodendritic Cl− gradient recording via whole cell patch clamp. The results show that KCC2 surface and functional expression is significantly increased in hippocampal neurons treated with TGF‐β2, compared to non‐treated cells. TGF‐β2 treatment leads to cAMP response element‐binding protein (CREB) phosphorylation and Ras‐associated binding protein 11b (Rab11b) gene expression and also increases Rab11b/KCC2 interaction and co‐localization. Knocking down either CREB or Rab11b suppresses KCC2 membrane and functional expression. Pre‐Bötzinger complex neurons in TGF‐β2 deficient mice have a significant decrease in KCC2 membrane expression, compared to wildtype and exogenous TGFβ2 application can rescue this phenotype. We introduce TGF‐β2/CREB/Rab11b as a novel regulatory pathway involved in KCC2 trafficking and functional activity in mouse mature hippocampal neurons.

Support or Funding Information

This work was supported by grants from the Deutsche Forschungsgemeinschaft [grant numbers KR1477/15‐1, SFB 780 TP B02 to K.K. and E.R.].
Dual PDE3/4 Inhibition Attenuates Lithium‐Induced Nephrogenic Diabetes Insipidus
Authors: Borchart Matthew A. , Gumina Diane L. , Kronk Trinity A. , Sim Jae H. , Blount Mitsi A.
Published: November 2023

Acquired nephrogenic diabetes insipidus (NDI) is characterized by the inability of the kidney to concentrate urine and is often an effect of some drug therapies, including a common treatment for bipolar disorder, lithium. Approximately 40% of lithium‐treated patients have polyuria and it is thought that the decreased capacity to concentrate urine in these patients is due to the lithium‐mediated disruption of the cAMP pathway that regulates urea transporter (UT‐A1) and water channel (AQP2) expression and function in the renal medulla. The aim of our study is to identify a mechanism to restore renal cAMP levels in the lithium‐treated kidney and preserve urine concentration capacity. Aged‐matched Sprague‐Dawley (SD) rats were pair‐fed either a standard rodent diet or a diet supplemented with lithium carbonate (40 mmol/kg) for 2 weeks. After treatment, urinary cAMP levels are dampened in lithium‐treated rats and total cAMP‐phosphodiesterase (PDE) activity and mRNA expression of PDE3 and PDE4 isoforms are increased in the inner medulla of lithium‐treated rats. Because PDEs are enzymes that degrade cAMP, inhibition of PDE3 and/or PDE4 could attenuate lithium‐induced NDI. SD rats were treated for two as follows: 1) standard diet, vehicle; 2) standard diet, milrinone (PDE3 inhibitor); 3) standard diet, rolipram (PDE4 inhibitor); 4) standard diet, milrinone, rolipram; 5) lithium diet, vehicle; 6) lithium diet, milrinone (PDE3 inhibitor); 7) lithium diet, rolipram (PDE4 inhibitor); and, 8) lithium diet, milrinone, rolipram. Milrinone treatment of lithium‐fed rats did not prevent polyuria in these animals and did not preserve UT‐A1 and AQP2 expression in the medulla. Similarly, rolipram treatment of lithium‐fed rats yields the same results. When PDE3 and PDE4 are simultaneously inhibited, urine osmolality of lithium‐fed rats is increased compared to vehicle‐treated lithium‐fed rats and UT‐A1 and AQP2 expression is restored to endogenous levels. In summary, lithium lowers renal cAMP and increases cAMP PDE activity and expression. Dual PDE3/PDE4 inhibition attenuates lithium‐induced polyuria in rats – a finding that may allow patients to continue lithium therapy without renal side effects in the future.