bluuebunny/crossref_metadata_2025_split huggingface dataset:
Non‐Polarized mpkCCD Cell Model for Aquaporin‐2 Phosphorylation and Membrane Trafficking Study
Authors: CHAN TZU‐HSIEN , YU MING‐JIUN 
Published: June 2021

Aquaporin‐2 (AQP2) is a water channel protein expressed in the kidney collecting duct principal cells responsible for water reabsorption. AQP2's ability to regulate water reabsorption depends on its phosphorylation states and trafficking to and from the apical plasma membrane regulated by the peptide hormone vasopressin. The mpkCCD cells have been an instrumental collecting duct cell model for studying AQP2 phosphorylation and trafficking. However, the cells require a time‐consuming polarization process before they can respond to vasopressin. We sought to circumvent this issue by examining AQP2 phosphorylation and trafficking in non‐polarized AQP2‐overexpressing mpkCCD cells treated with forskolin, which elevates intracellular cyclic AMP (cAMP), a second messenger of vasopressin. We found that forskolin induced AQP2 phosphorylation at serine 269 (S269) by 1.6 folds as observed in polarized mpkCCD and isolated rat inner medullary collecting duct cells treated with vasopressin. Before forskolin stimulation, most AQP2 was intracellular and was not phosphorylated at S269 by confocal immunofluorescence microscopy. In response to forskolin, AQP2 was found in the plasma membrane and was phosphorylated at S269, similar to those observed in the polarized mpkCCD cells and native collecting duct cells stimulated with vasopressin. As a proof of principle, we found that small hairpin RNA‐mediated protein kinase A knockdown reduced AQP2 phosphorylation at S269 and membrane localization in the non‐polarized mpkCCD cells treated with forskolin. Thus, non‐polarized AQP2‐overexpressing mpkCCD cells can be used in lieu of polarized mpkCCD cells when time is a limiting factor including transient small hairpin RNA‐mediated gene knockdown experiments.Support or Funding InformationThis work was supported by the Ministry of Science and Technology, TAIWAN (MOST 107‐2320‐B‐002‐057‐MY3).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Complete NEM Modification of Highly Reactive Cysteines Induces Full TRPA1 Activation
Authors: Parks Thomas , Taylor‐Clark Thomas 
Published: June 2021

Transient Receptor Potential Ankyrin 1 (TRPA1) is a tetrameric, non‐selective, cationic channel that is expressed in the dorsal root ganglia, trigeminal ganglia, and vagal ganglia and is activated by harmful chemicals including pollutants and endogenous irritants produced during inflammation and oxidative stress. Many irritants activate TRPA1 via electrophilic modification of highly reactive cysteine residues located on the cytosolic side of the channel, however the underlying processes that precede activation are poorly understood. Our lab has identified four highly reactive cysteines on TRPA1 (thus 16 highly reactive cysteines per channel) that rapidly bind electrophiles: C273, C621, C665 and C1085. C621 was the most reactive and point mutation studies show that both C621 and C665 are required for activation. In this study, we use cell‐attached single‐channel patch clamp analyses of transiently expressed human TRPA1 (hTRPA1) in HEK293 cells to determine the relationship between irreversible electrophilic binding and channel activity. We recorded stochastic gating events of single channels in response to N‐ethylmaleimide (NEM) treatment. Our open probability (Po) analysis indicates that NEM evoke two distinct hTRPA1 activation profiles: (1) weak activation (low Po) throughout NEM treatment which does not increase with time; (2) weak activation which then suddenly and spontaneously progresses to full activation (near 100% Po). Open time histogram analyses suggest that the weak activation state evoked by NEM has similar open times to spontaneous hTRPA1 activation in control conditions; whereas the full activation state has significantly longer open times. We conclude that the weak and full hTRPA1 activation states by NEM are due to differences in progressive cysteine modification. Furthermore, we suggest that full activation is not the summation of multiple weak states, but is instead a separate activation state that can only be evoked by a particular complement of cysteine modifications.Support or Funding InformationThis research is funded by the National Heart, Lung and Blood Institute (5R01HL119802‐S1).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of Na/K‐ATPase Non‐Enzymatic Function in Proximal Tubule Sodium Handling
Authors: Mukherji Shreya Tapan , Mayes Isabella W , Liu Jiang , McDermott Jeff Paul , Blanco Gustavo , Pierre Sandrine V , Xie Zijian 
Published: June 2021

Systemic levels of endogenous cardiotonic steroids (CTS) increase markedly during salt loading, volume expansion and renal insufficiency, suggesting a physiological role in the regulation of renal Na+ handling. CTS are classically known as specific inhibitors of Na/K‐ATPase (NKA) α1 ion‐pumping function, which is critical for Na+ and water reabsorption in the renal proximal tubule (PT). At much lower concentrations, closer to the range of those reported for endogenous CTS in the blood, they also initiate NKA/Src‐mediated signaling in renal cell cultures. To investigate the respective roles of PT NKA α1/Src signaling and ion‐pumping function in physiological salt handling, we developed a PT‐specific NKA α1 knockout (RPTα1−/−) mouse with PT‐specific rescue by wild‐type NKA α1 (RPT α1WT) or a mutant NKA α1 with intact ion‐pumping function and blunted NKA/Src signaling (RPTα1Y260A).The PT‐specific NKA α1 knockout mouse was generated by crossing SGLT2 (sodium glucose co‐transporter 2)‐Cre mice with Floxed ATP1a1 mice. A SGLT2‐Cre/Rosa 26 system was then used to re‐introduce α1WT or α1Y260A expression at the Rosa26 locus. Mice were born with the expected Mendelian ratio and appeared phenotypically normal, with no detectable change in water and food intake, or body weight. Renal phenotyping was further assessed using metabolic cages in 2–4 month‐old male and female mice.PT‐specific KO and rescue was confirmed by immunohistochemistry in PT cells isolated from RPTα1−/−, RPTα1WT, and RPTα1Y260A mice. Kidney size, morphology and structure appeared overall normal following periodic acid shift (PAS) and trichrome staining. Functionally, in contrast with the predicted outcome based solely on the classical enzymatic function of NKA, a significant decrease in daily urine output (0.55±0.2 vs 1.76±0.3 mL/24h in RPTα1+/+, n=8–10, p<0.01) and absolute Na+ excretion (0.13±0.07 vs 0.35±0.0.5 mmol/24h in RPTα1+/+, n=6, p<0.01) were observed in the absence of PT NKA. The urinary clearance of lithium was significantly decreased in RPTα1−/− (1.42±0.3 vs 4.13±0.5 mL/min in RPTα1+/+, n=3, p<0.05), suggesting that the observed increase in Na+ reabsorption originated in the PT. The urinary output was rescued in the RPTα1WT (2.12±0.3 vs 2.16±0.4 mL/24h in RPTα1WTcontrol, n=7–10, p>0.05) but not in the RPTα1Y260A (0.42±0.1 vs 1.56±0.2 mL/24h in RPTα1Y260Acontrol, n=6–8, p<0.01), which supported the role of NKA α1/Src signaling in the PT Na+ reabsorption.These results support a previously unascertained physiological role of NKA/Src signaling in PT Na+ and water reabsorption, and warrant further investigation to assess the mechanism and possible impact on blood pressure regulation.Support or Funding InformationMarshall Institute of Interdisciplinary Research FundsThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sodium self‐inhibition and proton sensitivity do not correlate with proteolytic processing in epithelial sodium channels containing the δ‐subunit
Authors: Althaus Mike , Gettings Sean Michael , Wichmann Lukas , Krasteva‐Christ Gabriela , Maxeiner Stephan 
Published: June 2021

The epithelial sodium channel (ENaC) is the only constitutively‐active ion channel of the DEG/ENaC protein family and evolved as a mechanism for sodium conservation during tetrapod evolution. We recently characterised a Xenopus laevis ENaC isoform which consists of the δ‐, β‐and γ‐ENaC subunits (Wichmann et al. J Biol Chem 4;293(18):6647–6658). Currents generated by these δβγ‐ENaCs have a slow transient nature due to a pronounced sodium self‐inhibition (SSI). In contrast to canonical ENaCs composed of the α‐, β‐ and γ‐subunits, δβγ‐ENaCs are not stimulated by proteases. The δ‐subunit is not proteolytically processed during intracellular maturation and the presence of the δ‐subunit prevents cleavage of the γ‐subunit at the cell surface. We therefore hypothesised that a lack of proteolytic processing might explain the transient nature of currents generated by Xenopus δβγ‐ENaCs and might indicate an evolutionary prerequisite towards constitutive ENaC activity.We expressed αβγ‐ or δβγ‐ENaC from guinea pigs (Cavia porcellus) as well as Xenopus laevis in Xenopus oocytes and measured currents generated by those isoforms as well as their degree of SSI, response to extracellular chymotrypsin (2 μg/ml) and extracellular acidification (pH 6.0), by two‐electrode voltage‐clamp electrophysiology.In contrast to Xenopus laevis δβγ‐ENaC, currents generated by Cavia porcellus δβγ‐ENaC did not have a transient form and had a profoundly reduced SSI compared to the Cavia porcellus αβγ‐ENaC isoform. Like our previous observations using Xenopus laevis ENaCs, Cavia porcellus αβγ‐ENaC was robustly activated by extracellular chymotrypsin, whereas Cavia porcellus δβγ‐ENaC was insensitive to extracellular protease. Xenopus laevis δβγ‐ENaC was strongly activated by extracellular acidification due to inhibition of SSI, whereas Cavia porcellus δβγ‐ENaC was insensitive to extracellular acidification.We conclude that the absence of proteolytic ENaC activation is not directly linked to a transient ion channel activity. By contrast, a strong SSI and proton sensitivity correlate with a more transient ENaC activity, whereas a lack of SSI and proton insensitivity renders ENaC constitutively‐open. These findings might suggest that changes in the kinetics of SSI and proton sensitivity rather than proteolytic processing determined ENaC evolution towards a constitutively‐active ion channel.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Aging Increases Physiological Function of TRPC3 Channels in Female Arterial Smooth Muscle
Authors: Goldsmith Jacob A. , Evanson Kirk W. 
Published: June 2021

Cardiovascular function declines with age, but patterns of vascular aging differ between the sexes across lifespan. In arterial smooth muscle, transient receptor potential canonical 3 (TRPC3) channels participate in the regulation of intracellular calcium and smooth muscle contractility. The effects of aging on TRPC3 function in female arterial smooth muscle is poorly understood. Here, we investigated physiological function of TRPC3 channels in pressurized, endothelium‐denuded middle cerebral arteries from young (~2 months) and old (~ 20 months) female CD (Sprague Dawley) IGS rats. Myogenic tone (60 mmHg) was similar between young and old cerebral resistance size arteries, however membrane depolarization‐induced vasoconstriction (60 mM K+) was reduced with age. Application of the selective TRPC3 channel inhibitor, Pyr3 (10 μM), elicited vasodilation in both young and old, however Pyr3‐mediated vasodilation was substantially larger in aged females. Application of 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG, 50 μM), a diacylglycerol analog, enhanced myogenic tone to a greater degree in old females as compared to young, while administration of Pyr3+OAG resulted in a greater loss in developed tone in old females. In conclusion, physiological function of TRPC3 channels is elevated with age in female arterial smooth muscle, which could be an important compensatory mechanism to maintain myogenic tone in cerebral resistance size arteries.Support or Funding InformationFlorida State UniversityThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
BIN1 clusters CaV1.2 channels in vascular smooth muscle
Authors: O'Dwyer Samantha , Moreno Claudia , Klug Nicholas , Santana Luis Fernando 
Published: June 2021

Resistance vessel such as mesenteric arteries respond to increases in intravascular pressure by constricting. Ca2+ influx via voltage‐gated CaV1.2 channels in the smooth muscle surrounding these vessels is critical for the development of myogenic tone and thus the regulation of arterial dimeter and blood flow. Our group discovered that CaV1.2 channels have an inherent ability to form clusters and functionally couple in response to local elevations in [Ca2+]i. The consequence of these channels coming together is that they amplify Ca2+ influx during depolarization. However, the mechanisms dictating the arrangement of CaV1.2 channels in smooth muscle cells are unknown. BIN1, a member of the BAR (Bin1‐Amphiphysin‐Rvs) domain superfamily, has been implicated in the trafficking of CaV1.2 channels but has not yet been studied in smooth muscle. In this study, we test the hypothesis that BIN1 promotes the formation of CaV1.2 clusters enhancing overall Ca2+ influx into the vascular smooth muscle and shaping arterial diameter. We report that BIN1 is expressed and translated in arterial smooth muscle while ground state depletion (GSD) super‐resolution imaging suggest that smooth muscle specific down‐regulation of BIN1 diminishes CaV1.2 cluster formation. Using patch clamp electrophysiology and pressurized arteriography, we observed that loss of BIN1 substantially diminishes ICa and attenuates arterial tone. Overall, these results suggest that BIN1 is a critical component in shaping CaV1.2 clustering and plays a physiological role in vascular smooth muscle Ca2+ entry and arterial diameter.Support or Funding InformationThis work is supported by NIH R01 HL085870 (LFS), T32GM099608 (SO), and 18PRE33960249 (SO)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Malignant Hyperthermia: A secondary factor, i.e., intense peripheral vasoconstriction, drives heat retention
Authors: Williams Charles H 
Published: June 2021

MH is a lethal genetic disease at the sodium channel which allows an influx of sodium ions into muscle cells that must be pumped back out by the Na+K+ATPase, thus expending copious amounts of ATP, i.e. a futile cycle, which produces high body temperature. The metabolic rate goes from 5ml/O2/Kg to over 50ml/O2/Kg.A peripheral vasoconstriction develops which shuts down heat loss via radiation thru the skin. This produces a purplish mottled skin color in the skin of a white pig. It also holds the heat internally and raises core temperature up to 118oF. The vasoconstriction is so severe that it increases arterial blood pressure to over 400 mm Hg. Heart rate increases to over 200 bpm and the heart function rapidly degrades resulting in heart failure due to the excessive workload on the heart.Preliminary data suggests that norepinephrine release drives the vasoconstriction and the increased heart rate.All the deleterious effects can be prevented by pretreatment with Organon 9426, a non‐depolarizing muscle relaxant. Sevoflurane, a new general gaseous anesthetic, is a weaker triggering agent than Halothane and can be used for 2–3 hours with minimal MH triggering effect.Succinylcholine is a very potent MH triggering agent and should not be used for human surgery cases.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Interferon alpha/beta receptor 1 inhibition results in delayed onset increased blood pressure and altered vascular remodeling
Authors: Brown Isola A.M. , Good Miranda E. , Peruri Alekhya , Isakson Brant E. 
Published: June 2021

Emerging studies have begun to recognize a role for the immune system in mediating blood pressure, where the depletion or inhibition of immune cells attenuates increased blood pressure in animal models of hypertension. What is less clear, however, is how immunosuppression and the downregulation of specific signaling pathways contributes to the regulation of blood pressure in the absence of a hormonal or other stimulus. The increased incidence of new‐onset hypertension in patients who are prescribed immunosuppressants suggests a relationship between decreased immune activity and hypertension. We investigated this relationship and tested the hypothesis that the suppression of the innate and adaptive immune systems alters vascular structure and blood pressure homeostasis. We induced immunosuppression by treating 11‐week old male C57Bl/6 mice with 4mg of purified anti‐interferon alpha/beta receptor 1 (IFNAR1) antibody via intra‐peritoneal injection. Blood pressure was measured continuously via a radiotelemetry transmitter implanted in the left carotid artery for up to 11 weeks post anti‐IFNAR1 treatment. Following blood pressure recordings, animals were sacrificed and blood vessels including the aorta, renal artery and mesenteric vessel pairs were isolated and analyzed by histology for changes in vascular protein expression and vessel remodeling. Beginning ~10 days post anti‐IFNAR1 treatment, immunosuppressed mice developed increased mean arterial pressure (MAP) during their active period (night), that was attributable to both increased diastolic and systolic blood pressures. Comparatively, only diastolic pressures increased during the rest period (day), resulting in a MAP that was not significantly increased. These changes in pressure were sustained up to 25 days post anti‐IFNAR1 treatment. At 6, 8 and 11 weeks post anti‐IFNAR1, daytime blood pressures in immunosuppressed animals were comparable to control mice while nighttime systolic, diastolic and MAPs remained elevated. Histological analysis of vessels collected 11 weeks after anti‐IFNAR1 treatment showed altered lumen diameter and medial thickness in the aorta and renal arteries. Our findings show a role for immunosuppression in regulating blood pressure homeostasis and vascular structure, and begin to identify mechanisms of remodeling that may be involved. Future work will how investigate key vascular signaling pathways and how they are altered during immunosuppression and the resulting hypertension.Support or Funding InformationFord Foundation Postdoctoral Grant (IAMB), HL 088554 R01 (BEI)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cerebral Vascular Function in a Murine Model of Experimental Cerebral Malaria
Authors: Brooks Steven D , Da Silva Moreira Aline , Carvalho Leonardo , Ackerman Hans 
Published: June 2021

Cerebral Malaria (CM) is among the most severe complications of malarial infection, with a mortality rate of 15–20%. Many survivors of CM develop lasting neurological sequalae, cognitive and behavioral impairments, or epilepsy. However, the etiology of CM is poorly defined, and treatment strategies are limited.Studies in the murine model of experimental cerebral malaria (ECM) have demonstrated cerebral blood flow (CBF) impairments that are associated with decreased nitric oxide (NO) bioavailability, blood brain barrier disruption, endothelial activation, impaired pial vessel reactivity, and cerebral ischemia. While these complications have been observed in vivo, cerebral artery function has not specifically been evaluated in ECM. Therefore, the aim of this study was to interrogate vasoreactivity and NO bioavailability in cerebral arteries (CAs) isolated from ECM mice, with the hypothesis that arterial vasodilatory function would be impaired by ECM.ECM was induced by Plasmodium berghei ANKA (PbA) infection in 10–12 week old C57Bl/6J mice and confirmed by murine coma score. On day 6 post‐infection, CBF was measured by laser speckle imaging. Cerebral arteries (CAs) were dissected from the brain into a modified Krebs buffer. Cerebral vasoreactivity was assessed by ex vivo pressure myography in middle cerebral arteries (MCA) obtained from control (n=4) and ECM (n=7) mice. NO production from dissected vessels was measured in an ex vivo Daf‐2 fluorescence assay (n=6, n=6).As expected, ECM mice had a significant, 25% reduction in CBF. However, ex vivo MCAs from ECM mice maintained robust endothelium‐dependent vasodilation to methacholine (MCh), which was inhibited by L‐NAME. ECM MCAs also exhibited normal dose responses to serotonin‐induced vasoconstriction and nitroprusside‐induced vasodilation, suggesting normal vasoreactivity of ECM cerebral arteries. Similarly, the results of the ex vivo Daf‐2 assay showed that the MCh‐induced NO production from ECM arteries was comparable to controls. To test whether ECM plasma would induce vascular dysfunction in ex vivo CAs, we incubated CAs obtained from ECM (n=2) and control (n=6) mice in plasma collected from ECM or control animals. Surprisingly, we found that plasma from ECM mice increased NO production of both ECM and control CAs.These unexpected results indicate that NO bioavailability and vasoreactivity are preserved in cerebral vessels in ECM, and that circulating factors in the plasma during PbA infection actively stimulate NO production. Our next focus will be on reconciling decreased CBF with preserved vascular function, as well as identifying the NO‐stimulating mediator(s) present within ECM plasma.Support or Funding InformationResearch and funding support from NIAID Division of Intramural Research.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.