bluuebunny/crossref_metadata_2025_split huggingface dataset:
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 InformationThis 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 InformationThis study was supported by the Natural Sciences and Engineering Research Council of Canada Discovery Grant to MMV and Post‐Graduate Fellowship to EF.
NKCC Regulates Circadian Rhythm Via the Chloride‐Sensing WNK Kinase
Authors: Rodan Aylin , Schellinger Jeffrey , Sun Qifei , Rothenfluh Adrian 
Published: November 2023

ObjectiveOscillation 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.MethodsWe 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.ResultsMale 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.ConclusionNormal 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 InformationNational Institutes of Health
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 InformationBritish 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 InformationNIH 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 InformationThis 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 InformationNIH 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 InformationHydrocephalus Association Innovator Award; Indiana University Collaborative Research Grant and Indiana Clinical and Translational Sciences Institute CTR Award.