bluuebunny/crossref_metadata_2025 huggingface dataset:
PDGF‐induced Vascular Smooth Muscle Cell Migration is Regulated by Coronin 1b
Authors: Williams Holly Colette , Martin Alejandra San , Griendling Kathy K
Published: June 2021

Platelet derived growth factor (PDGF) plays a pivotal role in cardiovascular disease progression, partially by initiating vascular smooth muscle cell (VSMC) migration. Recent studies show that the actin binding protein coronin 1b can regulate actin polymerization and depolymerization via phosphorylation‐regulated binding to Arp2/3 and Slingshot phosphatase (SSH1L). In this study a possible role for coronin 1b in PDGF‐induced VSMC migration was examined. PDGF (10 ng/ml)‐stimulated coronin 1b phosphorylation peaked at 5 minutes (11.8±1.3‐fold above basal) and was maintained for up to 60 minutes. The PKC inhibitors Ro‐32‐0432 (0.5 μM) and GÖ 6976 (0.5 μM) attenuated PDGF‐ induced coronin 1b phosphorylation (88±10% and 72±2%, respectively), implicating PKC‐α or PKC‐β in the phosphorylation response. PDGF stimulation of VSMCs decreased coronin 1b and ArpC2 (a subunit of Arp2/3) interactions at 5–10 min, as determined by co‐immunoprecipitation, but no effect of PDGF on coronin 1b‐SSH1L interaction was observed. Functionally, transfection with siRNA against coronin 1b completely inhibited PDGF‐induced VSMC migration, as measured by modified Boyden chamber assay. These data suggest that PDGF may signal to coronin to coordinate lamellipodia formation and thus migration in VSMCs, and suggest a new therapeutic target for vasculopathies with a significant migratory component.
Modulation of calcium channel subunit expression in vascular smooth muscle cells by transfection with β subunit constructs
Authors: Telemaque Sabine , Flynn Shawn E , Meredith K Brad , Liu Meeiyueh , Davidoff Amy J , Marsh James D
Published: June 2021

Cytosolic Ca2+ is regulated by Ca2+ entry through L‐type calcium channels (LTCC) and release from intracellular Ca2+ stores via the IP3 receptors (IP3R) in vascular smooth muscle cells (VSMC). The aim of this study is to determine if gene transfer of a LTCC dominant‐negative β subunit construct could regulate expression of the other LTCC subunits or IP3R in VSMC. Plasmids encoding the normal β subunit (Full) or the β‐interacting domain (BID, dominant‐negative construct) fused to GFP, were constructed under the control of a SMC‐specific promoter and transfected into A7r5 (VSM) and HL‐1 (cardiac) cells. At 48 hr, cells were visualized under fluorescent microscopy before protein levels were measured by immunoblots in whole cell lysates. Absence of GFP expression in HL‐1 cells confirmed SMC specificity of the promoter. α1C subunit levels tend to be elevated in A7r5 cells expressing Full when compared to GFP (control) whereas levels were not altered in cells expressing BID. IP3R levels were also increased by Full and unchanged by BID overexpression. Neither constructs altered α2 subunit expression. Whether protein levels reflect changes in functional LTCC are being determined by patch clamp. If a dominant‐negative β subunit can be used to reduce Ca2+ influx without affecting expression of the LTCC and other Ca2+ handling proteins, then this may be a viable therapeutic target to attenuate vascular reactivity. (AHA 0755762Z)
Effects of an anoxic insult on hamster hippocampal CA1 pyramidal cells are attenuated with decreasing temperature
Authors: Brown Theodore A , Lewis Carly J , Liepart George H , Ordaz Jeanelle M , Hamilton Jock S , Horowitz John M , Horowitz Barbara A
Published: June 2021

Previous studies showed that CA1 pyramidal cells in the hippocampus of nonhibernating and hibernating Syrian hamsters (Mesocricetus auratus) exhibit greater neuroprotection at 30°C than do those of rats. However, the temperature dependence of the hamster neurons to an anoxic insult has not been studied. We tested the hypothesis that the effect of anoxic exposure on population spike amplitude (PSA) following Shaffer‐collateral/commissural stimulation is attenuated with decreasing temperature. In slices from euthermic hamsters, CA1 pyramidal cell PSA was measured every minute throughout the experiment. Following a 15 minute control period in oxygenated (95%O2/5%CO2) buffer, oxygen was replaced with nitrogen (95%N2/5%CO2) for 15 minutes (the anoxic insult). Oxygenated buffer was then returned to the slices for a 30 minute recovery period. Three minutes after nitrogen exposure at 35°C, PSA had a 75.1 ± 14.9% (mean ± SE) drop from control, compared to a smaller drop (20.3 ± 5.3%) at 30°C. Three minutes after return to oxygenated buffer at 35°C, PSA had recovered by only 5.7 ± 2.9% compared with a 62.4 ± 6.8% recovery at 30°C. Differences are significant (P<0.05), consistent with our hypothesis, and consistent with the proposal that neuroprotection in CA1 pyramidal cells increased as temperature decreased.
Ischemia/Reperfusion Injury Causes an Outward Remodeling of the Middle Cerebral Artery.
Authors: Osmond Jessica M , Dorrance Anne M
Published: June 2021

Currently, the most effective form of treatment for acute ischemic stroke is to recannulate the blocked vessel to reintroduce blood flow to the brain. The long‐term effects of this ischemia/reperfusion injury (I/R) on cerebral vessel structure have not been investigated. To study this, 10‐week‐old Sprague Dawley rats were subjected to middle cerebral artery (MCA) occlusion using the intralumenal suture occlusion technique. After 1 hour of ischemia, the monofilament was withdrawn to allow reperfusion of the brain tissue. Fourteen days later the rats were euthanized and the MCA was studied by pressure myography. I/R caused an increase in the MCA lumen (284.4±5.6 vs 263.7±7.5 μm at 80 mmHg, I/R vs control, p<0.05) and outer diameter (316.6±5.0 vs 295.7±6.1 μm at 80 mmHg, I/R vs control, p<0.05). This change in MCA structure occurred without a change in wall thickness (32.1±1.8 vs 32.0±3.3μm at 80 mmHg, I/R/vs control), wall/lumen ratio (0.11±0.008 vs 0.12±0.014 at 80 mmHg, I/R vs control), or vessel stiffness (β‐coefficient: 5.9±0.4 vs 6.3±0.3, I/R vs control). I/R injury had no effect on the ability of the vessel to generate tone or contract in response to serotonin 2 weeks after the insult. The mechanisms for the outward MCA remodeling remain to be elucidated, as does the effect of the change in structure on the outcome of future ischemic events.
Tetrahydrocurcumin ameliorates mtMMP‐9 mediated mitophagy and mitochondria remodeling in Stroke
Authors: Tyagi Neetu , Qipshidze Natia , Givvimani Srikanth , Kandel Madhavi , Mishra Paras Kumar , Sen Utpal , Johar Alefiya , Tyagi Suresh C
Published: June 2021

Mitophagy is strongly implicated in stroke. Tetrahydrocurcumin(THC) polyphenolic compound, with antioxidant activities. Therefore objective of this study was to determine whether THC ameliorates mitophagy in stroke by reducing matrix metalloproteinase‐9 (MMP‐9) in hyperhomocystemic mice. There weresix groups of mice: WT, WT/Ischemia(IS),WT/IS+ THC,CBS(−/+), CBS(−/+)/IS and CBS(−/+)/IS+THC. Stroke was performed for1 hour. After 24 hours, mice were injected with THC. The infarct area was measured using TTC staining. Permeability was determined by brain edema and Evans Blue extravasation. PCR and Western blot were used todetermine the expression of MMP‐9, TIMPs, mitochondrial redox stress and LC3‐I/II. Mitochondrial respiratory capacity and mitochondrial permeability transition (MPT) were measured fluorescence‐dye. The mitochondrial superoxide and peroxinitrite were detected by using Mito‐SOX and dihydrorhodamine‐123. The result suggested Brain edema and Evans Blue leakage were reduced after stroke in THC treated group as compared to control group along with reduced brain infarct size. Western blot analysis indicated that THC decreased mitochondrial oxidativedamage and ameliorated the MMP‐9 activation and loss of MPT, in part by increasing the protein and mRNA levels of TIMP‐1 and decreasing TIMP‐2 as compared control. This study suggests a therapeutic role of dietary THC in stroke.
Uptake of folates by rat and human brain endothelial cell lines
Authors: Araújo João Ricardo , Gonçalves Pedro , Martel Fátima
Published: June 2021

The blood–brain barrier (BBB) is essential for central nervous system homeostasis. Folates are essential for the biosynthesis of purines, pyrimidines and methionine. Our aim was to characterize 3H‐folic acid (3H‐FA) and 14C‐methyltetrahydrofolic acid (14C‐MTHF) uptake by rat (RBE4) and human (hCMEC/D3) BBB endothelial cell lines. Uptake of 3H‐FA and 14C‐MTHF by RBE4 cells was time‐dependent and linear for the first 2 min of incubation. On the other hand, uptake of these substances by hCMEC/D3 cells showed less time‐dependency. Further experiments were performed in RBE4 cells. Uptake of 3H‐FA was found to be (1) stimulated at an acidic and at an alkaline pH, (2) Na+‐dependent and stimulated when F‐ substituted for Cl−, (3) energy‐independent, (4) inhibited by premetrexed (PTX) and stimulated by cytochalasin D (cyt D) and (5) not affected by MTHF, DIDS, SITS, methotrexate (MTX), monensin (MON) and FA. Uptake of 14C‐MTHF was found to be (1) pH‐independent, (2) Na+, Cl− and energy‐independent, (3) inhibited by PTX and MTX and stimulated by cyt D and (4) not affected by FA, DIDS, SITS, MON and MTHF. RT‐PCR analysis demonstrates mRNA expression of RFC (reduced folate carrier), but neither of FRα (folate receptor α) nor of PCFT (proton‐coupled folate transporter) in RBE4 cells and mRNA expression of both RFC and PCFT, but not of FRα, in hCMEC/D3 cells. Human and rat BBB cells show little capacity for folates uptake.
AT1 receptor‐mediated augmentation of urinary excretion of endogenous Ang II in Val5‐Ang II infused rats
Authors: Shao Weijian , Seth Dale M. , Navar L. Gabriel
Published: June 2021

We previously demonstrated that rats infused chronically with Val5‐Ang II exhibited increased urinary excretion of endogenous Ile5‐Ang II by the 12th day of infusion suggesting the stimulation of endogenous Ang II formation by Val5‐Ang II infusion. To determine the time course of increased urinary Ang II excretion and the effects of AT1 receptor blockade (candesartan, 0.69 ± 0.04 mg/day) on the urinary excretion rates of Ile5‐Ang II in Val5‐Ang II infused rats, Sham (6), Val5‐Ang II infused (5), and Val5‐Ang II infused rats treated with candesartan (6) were evaluated. Val5‐Ang II was infused for 13 days, and Ile5‐Ang II was separated from Val5‐Ang II in urine by HPLC and measured by RIA. SBP increased progressively (206 ± 3.8 mmHg) in Val5‐Ang II infused rats while the candesartan treated group remained normotensive (129.5 ± 6.0 mmHg). Urinary endogenous Ile5‐Ang II excretion rates were elevated by day 9 (2185.7 ± 283.2 fmol/24hr) in Val5‐Ang II infused rats but not in candesartan treated rats (740.6 ± 110.3 fmol/24hr). Thus, AT1 receptor blockade prevented the increase in urinary excretion of endogenous Ang II. These data indicate that the increased urinary endogenous Ang II excretion in Val5‐Ang II infused rats is not due to filtration but is due to secretion into and/or de novo formation of Ang II within the tubular lumen, which is mediated by AT1 receptors.
Immunocytochemical Distribution of the Ang‐(1–7)/Mas Receptor in the Sheep Kidney
Authors: Westwood Brian , Gwathmey TanYa , Shaltout Hossam , Diz Debra , Rose James , Chappell Mark
Published: June 2021

Angiotensin‐(1–7) exhibits functional properties that generally oppose those of angiotensin II (Ang II). In the sheep, low dose infusion of Ang‐(1–7) increases urinary volume and sodium excretion consistent with the natriuretic actions of the peptide in rat and dog. Compelling evidence suggests that the G protein‐coupled receptor mas accounts for the receptor‐mediated actions of Ang‐(1–7). The present studies assessed the distribution of the mas receptor within the adult sheep kidney utilizing an Alomone antibody (Jerusalem, Israel) generated against the human protein. We compared the fluorescent signal of mas to other markers specific for various segments of the nephron. In the cortex, the immunofluorescent signal for mas was predominantly associated with the proximal tubules and the thick ascending limb, but was strikingly absent in the glomerulus and renal vessels. In the medulla, mas was evident in the collecting duct cells that co‐stained for aquaporin‐2, the thick ascending limb and the thin loop of Henle. Interestingly, the mas antibody predominantly stained nuclei in the thin loop. Mas staining was abolished by preabsorption of the primary antibody with the immunogenic peptide. We conclude that the Ang‐(1–7)/mas protein exhibits a unique distribution in the nephron segments that is consistent with natriuretic and diuretic actions of the peptide. Funding: HD17644, HD47584, HL‐56973, HL‐51952.
The role of kidney on sodium balance in AngiotensinII‐induced hypertension.
Authors: Ferreira‐Figueiredo Claudia S.R. , Oliveira‐Souza Maria
Published: June 2021

Aim

Investigate the renal function and modulation of epithelial transporters of rats with angiotensin‐induced hypertension.

Methods

Normal 8‐week‐old male Wistar rats were divided in two groups: control (sham) or treated with 200 ng/kg/min of angiotensin II (ANG II) for 28 days, using osmotic minipumps. Blood pressure (BP) was monitored weekly by tail cuff plethysmography. Renal plasmatic flow (RPF) and glomerular filtration rate (GFR) were determined by para‐aminohippurate sodium and inulin clearance, respectively. The excretion of ions was analyzed and proteic expression of renal transporters or ANG II receptors will be analyzed by Western Blot technique.

Results and Discussion

Our preliminary results animals showed that the dose of ANG II used is sufficient to induce hypertension [110 ± 1,6 (sham) n = 4 versus 158 ± 1,7 (ANG II) mmHg n = 4 *p<0,001]. Urinary Na+ and K+ were reduced in hypertensive rats. RPF and GFR were also reduced [RPF = 13,29 ± 0,8 (sham) n = 14 versus 6,48 ± 0,44 (ANG II) ml/min/kg n = 14 *p<0,001] and [GFR = 8,4 ± 0,52 (sham) n=14 versus 4,73 ± 0,52 (ANG II) ml/min/Kg n = 14 *p<0,001], suggesting changes in renal hemodynamics and tubular function. However, epithelial transporters will be analyzed in the renal cortex and medulla. Financial Support: CNPq and FAPESP
Role of TRPC3 channels in ATP‐induced Ca2+ signaling in principal cells of the inner medullary collecting duct
Authors: Goel Monu , Schilling William
Published: June 2021

TRPC3 channels are expressed in the apical membrane of principal cells of the collecting duct (CD) both in vivo and in cultured mouse IMCD‐3 cells. Studies have shown that ATP‐induced apical‐to‐basolateral transepithelial Ca2+ flux across IMCD‐3 monolayers is increased by over‐expression of TRPC3 and attenuated by a dominant‐negative TRPC3 construct (dn‐TRPC3). To test the hypothesis that Ca2+ entry occurs via TRPC3 channels, Ca2+ permeability of the apical membrane of fura‐2‐loaded IMCD‐3 cells was estimated by the Mn2+ quench technique. Mn2+ influx across the apical membrane was increased 12‐ to 16‐fold by apical ATP, and was blocked by BTP2, an inhibitor of TRPC3 channels with an IC50 of <100 nM. In contrast, Mn2+ influx was increased only ~2‐fold by basolateral ATP. Mn2+ influx was also activated by the diacyl‐glycerol analog, SAG. Apical ATP‐ and SAG‐induced Mn2+ influx was increased by over‐expression of TRPC3 and completely blocked by expression of the dn‐TRPC3 construct. Mn2+ influx was also stimulated ~2‐fold by thapsigargin applied to either the apical or basolateral side, but thapsigargin‐induced flux was unaffected by over‐expression of TRPC3 or dn‐TRPC3. These results demonstrate that stimulation of apical purinergic receptors activates Ca2+ influx across the apical membrane of IMCD‐3 cells via TRPC3 channels by a mechanism that is independent of Ca2+ store depletion. Supported in part by HL097355.
Evidence for ENaC involvement in hypertension produced by NOS1 gene deletion in the collecting duct
Authors: Hyndman Kelly Anne , Elmarakby Ahmed , Pollock David M , Pollock Jennifer S
Published: June 2021

Studies indicate that NOS1 is necessary for proper sodium balance and subsequent blood pressure regulation. We developed collecting duct principle cell‐specific NOS1 knockout (CDNOS1KO) mice using the AQP2‐CRE/LoxP system and found that these mice display a salt‐dependent hypertensive phenotype. We hypothesized that NOS1 in the collecting duct (CD) may regulate the trafficking of the epithelial sodium channel (ENaC) such that CDNOS1KO mice will have greater expression of plasma membrane ENaC in the kidney than flox control mice. Plasma membrane and vesicle fractions from the renal inner medulla of CDNOS1KO and flox control mice were prepared by differential centrifugation and ENaCαβγ expression determined by Western blot analysis. We found a significant increase of ENaCα in the plasma membrane of CDNOS1KO mice (2.0 ± 0.4 RDU/actin, n=4) when compared to flox (1.0 ± 0.2 RDU/actin, n=4, p=0.04). ENaCβγ expression was similar in the plasma membrane from both genotypes as it was for all three subunits in the vesicle fraction. We found that amiloride (3 mg/kg/day ip) significantly lowered the blood pressure in CDNOS1KO on a 4% salt diet (4% salt: 144±2 mmHg; 4% salt + amiloride: 135±0.8 mmHg, n=4, p=0.02). No effects of amiloride on blood pressure were noted in the flox control mice. We conclude that the absence of CD NOS1 leads to an increase of ENaCα in the plasma membrane and may lead to subsequent salt‐sensitive hypertension.
Estrogen induces transient intracellular Ca2+ signals in distal renal tubules
Authors: Praetorius Jeppe , Fenton Robert A , Hofmeister Marlene Vind
Published: June 2021

The renal late distal convoluted tubules (DCT2s) and connecting tubules (CNTs) are sites for fine‐regulating the urinary excretion of various ions including Na+ and Ca2+. Accumulating evidence suggests that estrogen has the ability to induce rapid signaling through activation of plasma membrane‐associated receptors such as GPCR30. We hypothesized that estrogen may evoke acute signaling in the distal renal tubules through such mechanism. DCT2s and CNTs were isolated from transgenic mice expressing enhanced green fluorescent protein (EFGP) driven by the transient receptor potential subfamily V, member 5 (TRPV5) promoter. Intracellular Ca2+ levels ([Ca2+]i) of freshly isolated DCT2s and CNTs increased after 1 min superfusion with 17β‐estradiol (E2, 10−9 M to10−7 M) using live cell Fluo‐4 imaging. The E2‐induced [Ca2+]i signal was dependent on extracellular Ca2+ and was enhanced rather than inhibited by the classical antagonists ICI 182 780. G‐1, an agonist of the membrane associated E2 receptor GPR30, induced a similar response as E2. Preliminary data indicate enhanced TRPV5 immunoreactivity in the apical domain of the cells upon estrogen treatment, while the transepithelial Ca2+ flux in primary cultures of DCT2/CNT did not seem affected. We suggest that E2 can act through non‐genomic signaling pathways in DCT2 and CNT and may acutely alter tubular function.
Glucagon‐like peptide‐1 downregulates Na+/H+ exchanger NHE3 activity in rat renal proximal tubule
Authors: Pacheco Bruna Piccolo Muniz , Lessa Lucilia Maria Abreu , Oricchio Felipe Theocharides , Malnic Gerhard , Girardi Adriana Castello Costa
Published: June 2021

The glucagon‐like peptide‐1 (GLP‐1) receptor has recently been detected in cultured renal proximal tubule cells, where GLP‐1 reduces sodium reabsorption. The present study was undertaken to evaluate the role of GLP‐1 in modulating the in vivo activity of NHE3 which represents the major route for proximal tubular (PT) sodium and bicarbonate reabsorption. Compared to controls, rats treated with 1.0 μg/kg×min GLP (i.v.) for the period of 1 hour exhibited increased urine output (54.3 ± 5.5 vs. 22.4 ± 2.5 mL/min×Kg; P=0.001), fractional sodium excretion (0.41 ± 0.12 vs. 0.14 ± 0.03%; P = 0.02) as well as lithium clearance, an index of end‐proximal tubule delivery (2.2 ± 0.4 vs. 1.3 ± 0.2 mL/min; P < 0.01). Real time RT‐PCR in microdissected rat nephron segments revealed GLP‐1 mRNA expression only in the proximal convoluted tubule. Experiments of stationary microperfusion performed in rat renal PT revealed that GLP‐1 significantly reduced bicarbonate reabsorption (1.16 ± 0.13 vs. 2.11 ± 0.17 nmol/cm2×s; P = 0.002). Reduced PT bicarbonate flux rate was associated with a significant increase of NHE3 phosphorylation at the PKA consensus sites (serines 552 and 605) and decreased expression of the exchanger in microvillus membrane vesicles. We conclude that GLP‐1 has diuretic and natriuretic effects which are mediated at least partially by downregulation of PT NHE3 transport function.
Disruption of sodium and water balance in K+‐adapted BKβ4−/− mice
Authors: Grimm P Richard , Holtzclaw J David , Irsik Debra L , Liu Liping , Sansom Steven C
Published: June 2021

Flow‐induced potassium secretion is a physiological response by which the kidneys increase K+ secretion from the connecting tubule (CNT). Large, calcium‐activated potassium channels (BK), which are responsible for this response, are expressed in principal and intercalated cells (PCs and ICs) of the CNT. BK in the ICs are a complex of α and β4 subunits (BKα/β4), but their physiological function is uncertain. The goal of this study was to determine whether β4 knockout mice (BKβ4−/−) have altered renal salt clearance when fed a K+‐rich diet (5% K+) for 10 days. Under control diet conditions (0.6% K+) no differences were detected between WT and BKβ4−/−. When BKβ4−/− consumed the K+‐rich diet they became volume expanded (Hct − 45.7 ± 0.4% WT vs 35.4 ± 0.4% BKβ4−/−; weight change − 0.42 ± 0.03g WT vs 5.2 ± 0.2g BKβ4−/−). BKβ4−/− had lower urine output (180 ± 7μl/hour vs 206 ± 6μl/hour), UNaV (188 ± 7μmol/day vs 433 ± 13μmol/day), and FENa (0.29 ± 0.03% vs 0.66 ± 0.02%) compared to WT despite having similar food and water consumption. The inability of BKβ4−/− to minimize Na+ absorption led to volume retention, suggesting BKα/β4 of ICs play a role in minimizing Na+ absorption. It is unlikely that the ICs of the CNT are directly responsible for the increased Na+ absorption due to their low Na+/K+‐ATPase expression; but during elevated filtrate delivery, ICs may release paracrine factors that influence Na+ absorption in neighboring PCs.
Reversible glutathionylation of the plasmalemmal Ca2+‐ATPase/pump by the thiol‐oxidizing agent diamide
Authors: Sinkins William G. , Schilling William P.
Published: June 2021

In many cells, oxidative stress (OS) causes a rise in cytosolic free Ca2+ concentration ([Ca2+]i) often leading to necrotic cell death. During OS, proteins can be modified at specific cysteine residues by glutathione (GSH). To determine if the plasmalemmal Ca2+‐ATPase/pump (PMCA) is glutathionylated during OS, membranes from PMCA4‐expressing Sf9 cells or purified PMCA were treated with diamide and biotinylated GSH (biotin‐GSH). Glutathionylated proteins were captured by streptavidin beads and probed for PMCA. Treatment with biotin‐GSH (125 μM) alone resulted in little or no glutathionylation of PMCA. However, robust glutathionylation was observed within 10 minutes in the presence of diamide (100 μM). The reaction was reversed by DTT or glutaredoxin. Native PMCAs in membranes from mouse heart and from cultured bovine aortic endothelial cells (BAEC) were also glutathionylated in vitro. Additionally, PMCA was glutathionylated in response to diamide in intact BAECs loaded with BioGEE, a membrane permeable form of biotin‐GSH. Lastly, glutathionylation of PMCA4 reduced Ca2+‐ATPase activity 29.9 ± 8.9% (n=8). These results demonstrate that PMCA is a target for glutathionylation, and that the modified PMCA has a reduced activity. Inhibition of PMCA by glutathionylation could explain, at least in part, the rise in basal [Ca2+]i that occurs in endothelial cells during periods of OS. Supported in part by HL097355.
Agrin C20 peptide provides neuroprotection by activating a Na,K‐ATPase/Inositol 1,4,5‐Trisphosphate receptor signaling pathway
Authors: Spicarova Zuzana , Liu Xiao Li , Bondar Alexander , Aperia Anita
Published: June 2021

Here we report that the 20kD C‐terminus tail of the heparan sulfate proteoglycan agrin(C20) can activate a calcium signaling pathway that acts to protect neurons from apoptosis. It is now well documented that the ubiquitous integral plasma membrane protein Na,K‐ATPase(NKA) functions both as an ion pump and a signal transducer. Previous studies have shown that ouabain, a specific ligand of the catalytic α subunit of NKA, can trigger signaling pathways that involve activation of Inositol 1,4,5‐trisphosphate receptor (IP3R). In COS7 cells expressing either the ubiquitous endogenous α1 subunit or transfected with the neuron specific α3 subunit, C20 triggers highly regular, slow calcium oscillations with a power spectrum almost identical to that observed for ouabain, and the NKAα binding sites for C20 and for ouabain are shown to partially overlap. Exposure of cells to C20 enhances the interaction between NKA and IP3R, while expression of a peptide that disrupts the interaction between NKA and IP3R significantly reduces the efficacy of agrin signaling. Slow calcium oscillations will typically activate NF‐kB, which in most systems will have an anti‐apoptotic effect. C20, in concentrations that trigger calcium oscillations, also activate NF‐kB and protects from H2O2 apoptosis in neurons. Our results indicate that C20, which in the synapse is cleaved from agrin, may act as an endogenous neuroprotector.
Critical role of the Isoform‐Specific Region in Na,K‐ATPase trafficking and Protein Kinase C‐dependent regulation
Authors: Pierre Sandrine V , Sottejeau Yoann , Belliard Aude , Duran Marie‐Josee , Pressley Thomas A
Published: June 2021

The Isoform Specific Region (ISR) is a region of structural heterogeneity among the 4 isoforms of the Na,K‐ATPase α‐subunit. The ISR participates in PKC regulation of Na,K‐ATPase, a process that involves membrane recycling via clathrin‐coated vesicles. A potential dileucine recognition motif [DE]XXXL[LI] for clathrin adaptors is present in α1 ISR but not in α2. To determine whether a disruption of this motif could explain the change in PKC regulation observed upon ISR exchange, mutants of the rat α1 isoform that replaced the first leucine (L499V) or the glutamate (E495S) of the potential motif were expressed heterologously in opossum kidney cells. In the absence of PKC stimulation, total expression and Na,K‐ATPase mediated 86Rb+uptake were comparable in the mutants‐ and α1‐expressing cells. However, biotinylation experiments revealed an increase of about 80% in the surface expression upon disruption of the motif, as well as a significant reduction of the basal rate of removal from the cell surface. Upon PKC stimulation, activation of Na,K‐ATPase mediated 86Rb+ uptake was doubled in mutant‐expressing cells, comparable to the chimera containing the intact α2 ISR. This suggests that the ISR plays a critical role in the Na,K‐ATPase isoform‐specific response to PKC activation and in basal Na,K‐ATPase expression at the cell surface.
Functional characterization of TMEM16 anion channels
Authors: Owsianik Grzegorz , Prenen Jean , Hermans Diane , Eggermont Jan , Nilius Bernd
Published: June 2021

The family of TMEM16 (or anoctamin) ion channels comprises 10 members that seem to be permeable for anion and activated by intracellular calcium. The best characterized members of this family, TMEM16A and TMEM16B, function as calcium‐activated chloride channels (CaCC) in many physiological processes such as sensory transduction, transepithelial secretion, cardiac and neuronal excitation, fertilization and smooth muscle contraction. Recently, it has been demonstrated that TMEM16A together with TMEM16F/H/J are activated by hypotonic cell swelling (HTS) and might be a part of an epithelial volume‐regulated anion channel (VRAC). To study possible involvement of TMEM16 channels in VRAC function, we examine expression of all TMEM16 genes in cells that display high or low VRAC activity. Quantitative real‐time PCR experiments demonstrated that TMEM16F and TMEM16K mRNA levels were much higher in cells that showed larger VRAC currents. Currents activated by HTS in HEK293 cells were significantly increased when TMEM16F and TMEM16K were overexpressed. However, in contrast to previously published results, TMEM16A did not respond to HTS whereas its CaCC function was not impaired. Further experiments will be performed to identify activation mechanisms and determine permeation properties of TMEM16F/K channels. Supported by Interuniversity Poles of Attraction Program IUAP Nr.3P4/23.
Isoform‐dependent modification of T‐type calcium channels by ProTxII
Authors: Hall Emily Olsen , Edgerton Gabrielle B. , Blumenthal Kenneth M. , Hanck Dorothy A.
Published: June 2021

The 3 isoforms of the T‐type calcium (Ca) channel are differentially expressed in the nervous system and within individual neurons (Perez‐Reyes 2003; McKay et al. 2006). There exist few experimental tools that can differentiate between channel isoforms. Here, we show that ProTxII, a highly charged peptide toxin from the venom of the tarantula Thrixopelma pruriens, differentially modifies the 3 isoforms. CaV3.1 and CaV3.3 channels are sensitive to the toxin when expressed heterologously in HEK cells. In CaV3.1, 2μM toxin produces a positive, dose‐dependent shift in the voltage range of activation of ~20mV and a reduction in macroscopic conductance of ~50%. In CaV3.3 channels 2.5μM ProTxII minimally shifted activation by ~4mV but reduced conductance by ~50%. In CaV3.2, 5μM toxin produces a minimal shift of ~5mV but no reduction in conductance. We examined the sequences of CaV3.1 and CaV3.2 for charge differences in extracellular linkers and p‐loops which may contribute to differential isoform sensitivity. We examined three reciprocal pairs in DIIS3, DII p‐loop, and DIIIS3 of these channels. Reciprocal mutations in the domain II p‐loop profoundly affected toxin sensitivity. In the CaV3.1 background the double mutation E894K/R895T resulted in reduced sensitivity to ProTxII. Conversely the K944E/T945R mutation in the CaV3.2 background conferred sensitivity. Supported by APS Summer Research Fellowship (EOH).
IK1 a crucial component of repolarization reserve is modulated by shifts in [Ca2+]i
Authors: Toth Andras , Nagy Norbert , Virag Laszlo , Varro Andras
Published: June 2021

IK1, a composite current of inward rectifying K+ ion channels (Kir 2.1, 2.2, 2.3 and possibly TWIK‐1, TASK‐1) plays a pivotal role in finalizing the repolarization and adjusting/stabilizing the resting potential. As such, it is an essential component of the repolarization reserve. Its possible modulation by shifts in [Ca2+]i has long been debated. The aim of this study was to investigate the effect of [Ca2+]i modulation on IK1 current in isolated canine ventricular cardiomyocytes. Conventional microelectrode action potential (AP) or “whole cell” patch clamp measurements were combined with optical [Ca2+]i determinations. Elevated [Ca2+]o markedly modulated the shape of the AP, but [Ca2+]i depletion had no substantial effect. Under normal conditions selective IK1 block by 10 μM BaCl2 slightly lengthened the AP (202±5 vs 223±3 ms). When [Ca2+]i was depleted the effect of IK1 block did not significantly differ from that in normal conditions. Elevation of [Ca2+]o to 4 mM, however, nearly doubled the effect of IK1 block (185±8 vs 231±13 ms). IKr inhibition was reduced (205±4 vs 223±4 ms) in the presence of 4 mM [Ca2+]o compared to normal settings (211±3 vs 245±6 ms). Voltage‐clamp measurements demonstrated, that in the presence of elevated [Ca2+]o, IK1 current is also elevated as compared to low Ca2+ settings. We concluded, that shifts in [Ca2+]i may substantially influence the repolarization reserve through a modulatory effect on the IK1 current. Supported by grants LSHM‐CT‐2005‐018833 and CNK 77855.
An extended di‐isoleucine motif mediates Kir2.3 endocytosis via direct binding to the AP‐2 adaptin α/σ2 subunits
Authors: Ortega Bernardo , Mason Amanda K , Welling Paul A
Published: June 2021

A prototypical basolateral membrane potassium channel, Kir2.3, contains a C‐terminal trafficking cassette comprised of a biosynthetic sorting signal, a PDZ binding site, and a novel di‐isoleucine (di‐Ile) endocytic signal that orchestrates polarized trafficking in biosynthetic and endocytotic pathways. Here, we explore how the endocytotic signal is recognized by the AP‐2 clathrin adaptor. Similar to canonical di‐Leu signals, such as in the HIV protein Nef, we found that Kir2.3 signal directly interacts with AP‐2 complex, binding to α/σ2 subunits. Mutation of a key residue in the di‐Leu binding pocket of σ2 abrogated binding to both Kir2.3 and Nef fusion proteins, confirming a common binding site in AP‐2. Extensive mutagenesis analysis of Kir2.3 revealed that additional amino‐acid residues downstream from the di‐Ile motif contribute to the overall binding. Given that Nef motif does not tolerate di‐Leu to di‐Ile substitution, the additional binding sites in Kir2.3 motif may be required to compensate for the sub‐optimal binding provided by the Ile residues. Our data illustrate how diverse endocytic motifs in Kir2.3 and Nef bind to a common hydrophobic pocket in AP‐2, but then require specific amino‐acid residues to confer additional specificity to their bindings. Research supported by NIH & NKF‐MD
MOLECULAR DETERMINANT(S) OF HYPER‐OSMOTICALLY ACTIVATED NKCC1‐MEDIATED K+/K+ EXCHANGE
Authors: Gagnon Kenneth B. , Delpire Eric
Published: June 2021

Na+‐K+‐2Cl− cotransport mediates the movement of two Cl− ions for one Na+ and one K+ ion. Under isosmotic conditions or SPAK/WNK4 activation, the NKCC1‐mediated bumetanide‐sensitive Cl− uptake in Xenopus laevis oocytes, as measured using 36Cl, is twice the value of K+ uptake, as determined using 86Rb. In contrast, under hyperosmotic conditions, there is a very significant activation of the bumetanide‐sensitive K+ uptake with only a minimal increase in Cl− uptake. This indicates that when stimulated by hypertonicity, the cotransporter mediates Na+‐K+‐2Cl− cotransport as well as K+/K+ exchange. Interestingly, this stimulation of K+/K+ exchange was only observed with NKCC1, but not NKCC2. In order to identify the molecular determinant(s) of this NKCC1‐specific activation, we created chimeras of the mouse NKCC1 and the rat NKCC2. Swapping the regulatory amino termini of the cotransporters neither conferred activation to NKCC2, nor prevented activation of NKCC1. Using unique restrictions sites, we created additional chimeric molecules and determined that the second extracellular loop between membrane spanning domains three and four is involved in the hyperosmotic stimulation of K+ uptake (K+/K+ exchange) in NKCC1. However, replacing only this loop in NKCC2 was insufficient for K+ uptake activation, suggesting an interaction between this loop and a yet to be identified region of the cotransporter.
GATMD: γ‐Aminobutyric Acid Transporter Mutagenesis Database
Authors: Kidd Patrick D. , Anderson Cynthia M. , Muster Nemone , Eskandari Sepehr
Published: June 2021

It has been twenty years since the cloning of the first γ‐aminobutyric acid (GABA) transporter (GAT1; SLC6A1) in 1990. During this time, over fifty published studies have provided structure‐function information on investigator‐designed GAT1 mutants. To date, nearly 200 of 599 GAT1 residues have been replaced with different amino acids, and the resulting transporter functional properties have significantly advanced our understanding of this member of the neurotransmitter:sodium symporter family. Given this large body of mutagenesis data, there is merit in having a system which organizes this information and makes it publicly available through a searchable, internet‐based database portal. We first created a relational database which accommodates existing functional and pharmacological data reported on the GABA transporters. Using this database, we manually annotated published data on GAT1 mutants. Finally, we developed a web portal for accessing, searching, and managing the database. We have named this web portal GABA Transporter Mutagenesis Database (GATMD). GATMD is available on the internet at http://physiology.sci.csupomona.edu/GATMD. (Supported by NIH grant SC1GM086344)
Epigenetic regulation of development links adaption and diversification of skeletal phenotypes: a case study in shrews
Authors: Young Rebecca L , Badyaev Alexander V
Published: June 2021

A major goal of evolutionary biology is to understand how diversity is generated. One hypothesis suggests that evolutionary change is facilitated by environmental sensitivity of development and the associated modifications of organismal development that induce selectable phenotypic variation. In vertebrates, growth and development of bones is partially guided by contractions of the attached musculature. During ontogeny, this muscle activity changes from sporadic motility to functional use. Thus, shifts in timing of ossification can either buffer development from environmental signals, or enable environmental signals to influence bone development and generate specialized skeletal adaptations. Here, we outline a conceptual framework for future studies that focuses on mediation of phenotypic plasticity in skeletal development through changes in developmental timing and provide evidence for the importance of developmental timing for adaptation and diversification in shrews.
The Ca2+‐activated Cl‐ channel TMEM16A represents the effector pathway for purinergic receptor‐mediated secretion in liver epithelium.
Authors: Dutta Amal Kumar , Khimji Al‐Karim , Dougherty Michael , Kresge Charles , Woo Kangmee , Esser Victoria , Alpini Gianfranco , Rockey Don
Published: June 2021

In the liver, Cl− channels in the apical membrane of biliary epithelial cells (BECs) provide the driving force for biliary secretion. Extracellular nucleotides are important agonists for Ca2+‐activated Cl‐ channel activation. The molecular identity of the Ca2+‐activated Cl‐ channel is unknown.

Aim

to determine if TMEM16A represents the Ca2+‐activated Cl− channel in BECs.

Methods

Studies were performed in human, mouse, and rat BECs. Membrane Cl− currents were measured by whole‐cell patch clamp and transepithelial secretion (Isc) by Ussing chamber.

Results

TMEM16A mRNA was detected by RT‐PCR in all BEC models. In polarized BEC monolayers, addition of ATP to the apical chamber increased Isc. Pre‐incubation with IL‐4 increased TMEM16A expression and was associated with an increase in Isc. The ATP‐stimulated Isc was completely inhibited by the Cl− channel blockers NPPB and niflumic acid, but insensitive to CFTR‐inh172. In single cells, exposure to ATP rapidly increased [Ca2+]i and activated whole‐cell Cl− currents with a reversal potential of 0 mV and outward rectification. Transfection with TMEM16A siRNA significantly decreased TMEM16A expression and inhibited the ATP‐stimulated whole‐cell currents.

Conclusion

These results demonstrate that TMEM16A is functionally present in BECs and contributes to Ca2+‐activated Cl− secretion in response to extracellular nucleotides. These studies represent the first molecular identification of an alternate, non‐CFTR, Cl− channel in BECs.
Aquaglyceroporins in epithelial and endothelial barriers in the equine hoof
Authors: Asplin Katie , Mobasheri Ali
Published: June 2021

Epidermal keratinocytes in the equine hoof rely on the transport of nutrients across an extracellular matrix (basement membrane), which separates the dermal lamellae from epidermal lamellae. Currently, nothing is known about how water and small solutes are transferred across this barrier. We hypothesize that specific aquaporin (AQP) water channels control fluid flow in equine epidermal hoof keratinocytes, small vessels in the dermis of the hoof, and the digital artery supplying the hoof. Using custom‐designed rabbit polyclonal anti‐sera, we have examined the cellular localization of AQP1, AQP3 and AQP9 within normal equine hoof tissues using immunohistochemistry. AQP3 and AQP9 immunoreactivity was observed in the cytoplasm and plasma membrane of lamellar keratinocytes, although AQP3 staining was stronger. AQP1 and AQP3 immunoreactivity was also demonstrated in the endothelium of small vessels within the dermal hoof tissue and digital artery, while AQP9 only stained smooth muscle of small vessels within the digital artery. Both AQP3 and AQP9 stained the smooth muscle of the digital artery, but only AQP3 demonstrated endothelial staining. These results confirm that aquaporins are present in epithelial and endothelial barriers in the equine hoof and may play a functional role in the transport of water and small solutes, such as glycerol and urea across these barriers. This research was supported by the University of Nottingham.
KCNE1‐induced increase in KCNQ1 currents is not mediated through enhanced membrane expression
Authors: Hammami Sofia , Klaerke Dan A , Willumsen Niels J
Published: June 2021

Association of the voltage‐activated potassium channel KCNQ1 with the accessory protein subunit KCNE1 gives rise to the cardiac IKs delayed rectifier potassium current. Aside from altering the kinetics of the KCNQ1 channel current, KCNE1 also augments the KCNQ1 current. This increase in macroscopic current is due either to an increase in ion channel conductance (γ), the open state probability (Po) or an increase in the number of channels in the plasma membrane (N). The latter can be quantified by measuring the level of KCNQ1 surface expression by using an enzyme‐linked immunoassay. To do this, we employed a HA‐tagged version of the KCNQ1 channel and expressed it in presence or absence of KCNE1 in Xenopus oocytes. The HA‐tag, which is located at the second extracellular loop of the protein, allowed us to “count” the number of KCNQ1 channels expressed in the cell membrane. In parallel, currents were measured with two electrode voltage clamp. The results show that the KCNQ1 surface expression is ~70% lower when KCNE1 is co‐expressed compared to KCNQ1 alone despite a ~20% higher current for the heteromeric KCNQ1/KCNE1. This indicates that the overall increase of the KCNQ1 current, when co‐expressed with KCNE1, is not due to an increase in ion channel surface expression but rather to an increase in single‐channel conductance or in open state probability.
Epithelial Sodium Channel Gating is Modulated by Cysteine‐Palmitoylation
Authors: Mueller Gunhild M , Maarouf Ahmad B. , Kinlough Carol L , Sheng Nan , Kashlan Ossama B. , Luthy Sarah , Okumura Sora , Hughey Rebecca P. , Kleyman Thomas R.
Published: June 2021

The epithelial Na+ channel (ENaC) serves as the final site of renal Na+ absorption in the distal nephron, where the fine tuning of its activity regulates extracellular fluid volume and blood pressure. The three homologous subunits of ENaC (alpha, beta and gamma) have a similar topology with two transmembrane domains, a large extracellular loop, and cytoplasmic N‐ and C‐termini. While various lipids are known regulators of ENaC activity, cytoplasmic Cys‐palmitoylation of ENaC has not been previously described. Palmitoylation of individual epitope‐tagged subunits was assessed with fatty acid‐exchange chemistry, whereby palmitate is replaced by biotin and biotinylated subunits were analyzed by immunoblotting. We observed that only the beta and gamma subunits were modified. The function of palmitoylation adjacent to the membrane was studied by expressing ENaC containing a beta C43,557A mutant in Xenopus oocytes. These mutant channels exhibited significantly enhanced Na+ self inhibition and reduced open probability, when compared with wild type ENaC. In contrast, normal membrane trafficking was observed. Our results indicate that palmitoylation of the beta subunit enhances ENaC gating. (Funding from DK065161, DK065521 and AHA).
Transition metals inhibit epithelial sodium channels via distinct mechanisms
Authors: Sheng Shaohu , Chen Jingxin , Winarski Katie L.
Published: June 2021

The epithelial Na+ channel (ENaC) plays an essential role in body fluid volume homeostasis. We studied the effects of extracellular Mn2+, Co2+, Ni2+ and Hg2+ on human, mouse and rat ENaCs expressed in Xenopus oocytes. External Mn2+ inhibited amiloride‐sensitive currents of human and mouse ENaCs with maximal inhibition of 50% (hENaC) and 30% (mENaC) at 10 mM and did not affect the current of rat ENaC. Extracellular Co2+ inhibited all three ENaCs with estimated inhibitory constants (IC50) of 0.1 mM (hENaC), 2 mM (mENaC) and >10 mM (rENaC). Ni2+ inhibited hENaC, similar to the previously reported effect on mENaC and rENaC, but with 10‐fold higher affinity (IC50 of 50 μM). External Hg2+ blocked all three ENaCs with similar affinity (IC50 of ~10 μM). Mutations of the His residues implicated in Ni2+ block of mENaC greatly reduced the inhibitory effects of Co2+ on human and mouse ENaCs. However, for Ni2+ inhibition of hENaC, αH255A did not change the effect and γH233A significantly increased its maximal inhibition. Mouse ENaC inhibition by Hg2+ was not significantly altered by homologous mutations. Our results indicate that external Mn2+, Co2+ and Ni2+ differentially alter activities of human, mouse and rat ENaCs whereas Hg2+ has a similar inhibition on all three ENaCs. Mutational analyses suggest that these metals inhibit ENaC activity through distinct mechanisms. (Supported by NIH ES014701 and DK079307)
An extracellular subdomain of the epithelial Na+ channel gamma subunit confers Na+ self‐inhibition response
Authors: Winarski Katie L. , Sheng Nan , Chen Jingxin , Kleyman Thomas R. , Sheng Shaohu
Published: June 2021

The epithelial Na+ channel (ENaC) is a key component in regulation of body fluid volume homeostasis. Its activity is controlled in part by Na+ self‐inhibition, a down regulation of channel activity by extracellular Na+. We previously showed that a His residue (γH239) within the extracellular domain (ECD) of γENaC subunit has a critical role in the Na+ self‐inhibition response. We investigated the functional roles of residues in the vicinity of this γH239 by Cys scanning mutagenesis and analyses of Na+ self‐inhibition responses in channel expressing Xenopus oocytes. Significant changes in the speed and magnitude of Na+ self‐inhibition were observed with multiple mutants, which are distributed within a contiguous stretch of 22 residues within the scanned region of 47 residues. Cys substitution at three aromatic residues (γF225C, γW235C and γH239C) resulted in the greatest suppression on Na+ self‐inhibition, suggesting a shared functional role. The distribution pattern of residues whose substitutions significantly altered Na+ self‐inhibition is consistent with secondary structure predictions of one helix. We conclude that the critical γH239 residue functions within a subdomain of γECD that has a specific role in conferring the Na+ self‐inhibition response of ENaC. (Supported by NIH ES014701, DK54354 and DK079307)
Comparison of Na+ Regulation of Exhaled Breath Condensate and Urine in Healthy Humans
Authors: Wheatley Courtney M. , Cassuto Nicholas A. , Foxx‐Lupo William T. , Wong Eric C. , Patanwala Asad E. , Snyder Eric M.
Published: June 2021

In both inner medullary collecting duct (IMCD) cells and alveolar cells stimulation of β2 adrenergic receptors results in renal tubular and alveolar Na+ reabsorption. We sought to explore the relationship between Na+ composition of urine and plasma to that of exhaled breath condensate (EBC, a surrogate for airway surface liquid composition) following β‐agonist administration. We recruited 17 healthy subjects (age=27±9yrs, ht.=170±8cm, wt.=68±12kg, BMI=24±4kg/m2, mean±SD) to complete two visits with administration of either nebulized β‐agonist (albuterol, 2.5mg) or nebulized saline with measures of EBC, plasma, and urine Na+ at baseline and 90 minutes post in a blind crossover design. At baseline there was no relationship between EBC Na+ and urine Na+ nor in urine Na+ and plasma Na+, but there was a relationship between EBC Na+ and plasma Na+ (r=0.35, 1‐tailed p=0.034). Albuterol resulted in a small but insignificant reduction in EBC Na+ (baseline=2.52±1.5, 90min post=2.17±1.4mmol/l), but no change in plasma or urine Na+. Saline resulted in no change in EBC Na+ (baseline=1.96±0.4, 90min post=1.91±0.5) or plasma or urine Na+. These results suggest a relationship between plasma Na+ and EBC Na+, but not between EBC Na+ and urine Na+ likely due to multiple regulatory pathways for renal Na+ handling independent of IMCD cells that constitute the majority of renal Na+ reabsorption and are not also present in the lung.