bluuebunny/crossref_metadata_2025_split huggingface dataset:
Pig Renal Na/K ATPase and Bcl‐2 Proteins: A Co‐Immuno‐Precipitation Study
Authors: Maharjan Chandra Kumar , Heiny Judith , Adragna Norma C , Lauf Peter K 
Published: November 2023

The Na/K ATPase (NKA) has at least two Bcl‐2 like sequences (Cell Physiol. Biochem. Cell Physiol Biochem 31: 257–276, 2013) and, in human lens epithelial cells, co‐immuno‐precipitates with the Bcl‐2 proteins BclXL and Bak (Am J. Physiol. Cell Physiol 2015: 308: C51–C60). Here we searched for immunochemical evidence of the presence of BH3 motif‐containing Bak co‐immuno‐precipitated with the well‐characterized pig renal NKA (PRNKA) (Klodos et al., Kidney Int. 62:2097–2100, 2002).ResultsPRNKA was prepared by the Klodos procedure omitting the final SDS purification step to preserve NKA activity at 0.45 μmol in 5.54 mg/ml. Mouse (ms) anti‐NKA (α6F) antibody (ab) showed the α1 subunit of ~110 kDa in immunoblots (IB) of the PRNKA input, in the protein‐G coated metal bead (PGCMB) ‐ α6F immuno‐pulldown, as well as in PGCMB‐ms and rabbit (rb) IgG pulldowns. A rb polyclonal anti‐human Bak BH3 motif ab (Cell Signaling #3814) together with a secondary (2nd) goat anti‐Rb Fc’ heavy (H) chain ab detected a low level staining of a ~27 kDa peptide in the PRNKA input and a prominent band in the α6F immuno‐pull down suggesting the presence of Bak in the enzyme preparation. IBs with the 2nd anti‐Fc’ H‐chain ab alone, however, not only detected IgG H chains in α6F ab‐, and ms‐ and rb‐IgG immuno‐pull downs, but unexpectedly also in PRNKA input. These findings suggest the presence of 1) Bak, and 2) NKA in PRNKA inputs and in immune and “non‐immune” pulldowns, with an absence of both proteins in ms and rb IgG input only; and 3) an apparent IgG contamination in PRNKA. Because of the latter, our efforts are now directed to discriminate further between Bak and L chains both of very close molecular mass. Whether the triple occurrence of NKA, Bak and IgG in all pull downs is IgG‐molecular specific but not species‐specific or coincidental due to non‐specific adsorption to the protein G beads, or both, is currently under study.Support or Funding InformationSupported by Wright State University Foundation to the Cell Biophysics Laboratory, by the Pharmacology and Toxicology Program to C.K.M., and by NIH to JH.
Oxygen Channels in Red Blood Cells
Authors: Zhao Pan , Geyer R. Ryan , Sim Edward S. , Salameh Ahlam I. , Meyerson Howard , Wass Amanda B. , Moss Fraser J. , Boron Walter F. 
Published: November 2023

It is well known that there are at least three families of membrane proteins—aquaporins (AQPs), rhesus (Rh) proteins, and urea transporters (UTs)—function as channels for CO2 or NH3. In human red blood cells (RBCs), AQP1 and the Rh complex (mainly RhAG) together account for ~90% of membrane CO2 permeability. We hypothesize that most O2 crosses RBC membranes through known polytopic proteins that also act as O2 channels. We quantitated O2 efflux through mouse RBC membranes using stopped‐flow (SF) absorbance spectroscopy to determine the O2 saturation of hemoglobin (Hb). For each mouse, we correct the raw data for modest degrees of hemolysis using our novel SF hemolysis assay, based on the release of carbonic anhydrase (CA) enzymes from RBCs. We find that SF analysis per se increases hemolysis from ~0.5% before a SF experiment to ~5% during a SF experiment on wide type (WT) mouse RBCs. AQP1‐KO, RhAG‐KO and double knockout (dKO) mouse RBCs exhibit similar degrees of hemolysis. After correction for hemolysis, AQP1‐KO decreases the rate constant for O2 efflux (kO2) by ~11%. RhAG‐KO decreases kO2 by ~15%, and dKO decreases kO2 by ~30%. Hematology results shows that mean corpuscular volume (MCV), compared to WT mice, is 8% and 6% higher in RhAG‐KO and dKO mice (p<0.01), respectively, and 2% higher in AQP1‐KO mice (p>0.05). DIC microscopy of living RBCs and microscopic observation of stained blood smears confirms that RBCs from all four groups are normal biconcave disks, and flow cytometry confirms that the major disk diameter is indistinguishable for all four groups. Thus, RBCs from our KO mice probably are a few % thicker than WT RBCs, differences that would not appreciably affect kO2. Mass spectroscopy of RBC ghosts indicates that the KOs do not unexpectedly affect levels of other proteins. Thus, AQP1 and RhAG, the two dominant CO2 channels in human RBCs, together make an important contribution to O2 permeability in mouse RBCs. Our data provide the first evidence that membrane proteins, presumably acting as channels, can enhance O2 permeability, which would be a major paradigm shift.Support or Funding InformationOffice of Naval Research Awards N00014‐14‐1‐0716 and N00014‐15‐1‐2060
Bicaudal‐C in Drosophila as a model of polycystic kidney disease (PKD) and intersection of oxalate nephrolithiasis
Authors: Knier Catherine Grace , Landry Greg M. , Romero Michael F. 
Published: November 2023

BackgroundAutosomal Dominant Polycystic Kidney Disease (ADPKD) is a leading cause of end‐stage renal disease in adults affecting 1 in 800 people in the US. Knocking down bicaudal‐C (BicC) in zebrafish and Xenopus has been reported as a method used to model the cystic changes in ADPKD. Gamberi and colleagues [JASN 25: 538A, 2014] recently presented a similar knockdown in Drosophila melanogaster resulting in cystic Malpighian Tubules (MTs) homologous to the human kidney. Additionally, calcium oxalate (CaOx) kidney stones affect ~1 in 10 people in the US, with the incidence increasing ~2‐fold in PKD patients.MethodsOur group has investigated the PKD phenotype in Drosophila using a Gal4/UAS‐RNAi system to knockdown the expression of BicC in MTs. We initiated CaOx crystal formation by feeding Na‐oxalate or adding to tubule bathing solution [Hirata, AJP 2012].ResultsPenetrance of Uro, CapaR, and c724 drivers in the MTs were compared using UAS‐YFP, resulting in Uro::BicC expression in ~50% of cells with CapaR::BicC approaching 100% and c724 expression specific to MT stellate cells only. MT morphology was analyzed: controls [WT (0%); parental (18%)] occurrence v BicC knockdowns [Uro::BicC (25%); CapaR::BicC (15%); c724::BicC (6.3%)]. Functional tests using Ramsay secretion assays reveal a slower secretion rate in Uro::BicC, CapaR::BicC, and c724::BicC knockdowns compared to WT MTs. In CapaR::BicC knockdowns, slower secretion rates are observed even after stimulation with Drosophila‐kinin. Since there is an increased risk for ADPKD‐related kidney stones, MT calcium oxalate (CaOx) crystallization experiments were performed to quantify crystal formation in BicC knockdown vs WT MTs. While crystals are present in all oxalate exposed MTs, BicC knockdowns using Uro::BicC and c724::BicC form more CaOx crystals than CapaR::BicC and WT. However, crystals formed by Uro::BicC and c724::BicC knockdowns have a significantly smaller total crystal area and average area/individual crystal compared to both CapaR::BicC and WT.ConclusionsThese results suggest that BicC knockdown parallels kidney stone increases associated with human PKD, but does not yet consistently mimic morphologic ADPKD‐induced cystic phenotypes usually observed in PKD models.Support or Funding InformationDK90278, DK101405, DK101405
Store Operated Ca2+ Entry Suppressed TGFβ1‐ Smad3 Signaling Pathway in Glomerular Mesangial Cells
Authors: Chaudhari Sarika , Li Weizu , Ma Rong 
Published: November 2023

Excessive expression of extracellular matrix (ECM) proteins in glomerulus is one of the consistent pathological changes seen in kidney diseases, such as diabetic nephropathy. Transforming growth factor‐ β1 (TGFβ1)‐Smad3 pathway plays a critical role in ECM protein expression and renal fibrosis. Receptor binding of TGFβ1 leads to phosphorylation and subsequent translocation of Smad3 into the nucleus regulating transcription of target genes directly or indirectly.The Orai1‐mediated store operated Ca2+ entry (SOCE) is associated with many physiological and pathological processes in a variety of cells, including glomerular mesangial cells (MCs) which are a major source of ECM proteins. We have previously demonstrated that SOCE in MCs suppressed ECM protein expression in vitro and in vivo. However, the downstream mechanism underlying the SOCE effect is not known. The present study was conducted to test the hypothesis that SOCE suppressed ECM protein expression by inhibiting TGF β1‐Smad3 pathway in MCs. In cultured human MCs, TGFβ1‐induced phosphorylation and translocation of Smad3 was examined in presence and absence of thapsigargin (TG) (1 μM), a classical activator of store‐operated Ca2+ channel. We found that treatment with TGFβ1 (5 ng/ml for 15 hours) significantly increased the expression level of Phospho‐Smad3 (p‐Smad3) evaluated by Western blot. However, this response was markedly inhibited by TG treatment. Consistently, immunocytochemistry and Western blot showed that TGF β1 significantly increased the expression of nuclear Smad3. Again, this TGFβ1‐induced nuclear translocation of Smad3 was prevented by pre‐treatment with TG. Importantly, the TG effect was reversed by La3+ (5 μM) and GSK‐7975A (10 μM), both of which are selective blockers of store‐operated Ca2+ channel. Furthermore, knockdown of Orai1 using siRNA approach significantly augmented TGFβ1‐induced p‐Smad3 expression. Taken together, our results indicate that SOCE in MCs negatively regulates the TGFβ1/Smad3 signaling pathway, and thus could be a potential therapeutic target of kidney disease with glomerular fibrosis.Support or Funding InformationUNTHSC Institutional Seed Grant, UNTHSC Doctoral Student Bridge Grant, Sigma Xi Grants ‐in‐Aid research grant.
Expression of KCNJ1 (ROMK) in the Gastrointestinal Tract
Authors: Han Jaeyong , Lee Seung‐Hun , Ishikawa Yasunobu , Guo Xiaoyun , Xu Shuhua , Wang Tong 
Published: November 2023

It is well understood that the renal outer medullary potassium channel (ROMK; KCNJ1) is expressed in the apical membrane of the renal tubules along the distal nephron and plays an important role in K+ secretion and Na+ and K+ homeostasis. In the gastrointestinal (GI) tract; however, little is known whether ROMK plays a role in K+ secretion. We previously reported that ROMK is functionally expressed in the parietal cells and is involved in gastric acid secretion. Our current study examines ROMK expression and localization in other parts of the GI tract, and also examines whether the expression of ROMK channel is influenced by K+ intake. ROMK expression was examined in the esophagus, ileum, proximal and distal colon of mouse tissue collected from 20‐week old ROMK wild‐type and KO mice. The ROMK mRNA expression was examined by real‐time PCR and immunofluorescence (IF) staining. ROMK specific antibodies were used to study ROMK expression and localization at the protein level. Experimental data show ROMK mRNA expression in esophagus, Ileum, proximal and distal colon in WT mice, but there was no expression detected in these segments of ROMK KO mice. Q‐PCR data showed a slightly increase in ROMK mRNA expression in Ileum, proximal and distal colon in animals treated with high K+ diet (5% K+ diet for 7days) compared with normal K+ dieted animals. IF staining showed very weak ROMK staining in the esophagus, but clearly stained in Ileum, proximal and distal colon. The localization of ROMK is in the epithelial cells; there was no co‐localization of ROMK with the nuclear marker DAPI. In conclusion, ROMK is expressed in the epithelial cells of the Ileum, proximal and distal colon and HK intake might increase ROMK expression in the GI tract.Support or Funding InformationNIH RO1 DK099284 and PO1 DK17433
Effects of TNFα challenge on sensory neuron ion channel expression and temperature sensitivity
Authors: Staunton Caroline Amy , Kumagai Kosuke Amy , Thippeswamy Thimmasettapp , Djourhi Laiche , Barrett‐Jolley Richard Amy 
Published: November 2023

Chronic neuropathic pain is a major worldwide cause of disability with over 116 million adults affected in the US alone. In many cases, direct neuronal effects of cytokines are thought to contribute to the associated temperature and mechanical hypersensitivity. In a previous report we showed that the resting membrane potential of TNFα treated dorsal root ganglion (DRG) neurons was hyperpolarized compared to controls, when measured at 30°C. In this study we extended this work to measure TNFα (24hr, 10ng/ml) induced changes in underlying ion channel biomarker mRNA abundance (with qPCR), absolute resting action potential (AP) frequency and on its temperature sensitivity (using cell‐attached patch, action current recording). DRG neurons were isolated and prepared as described previously [1]. Analysis of whole DRG, glial cell depleted cultures revealed that several ion channels were differentially transcribed following TNFα treatment. Interestingly, there was a statistically significant switch from TRPV4 (transient receptor potential cation channel, subfamily V, member 4) to TRPM2 (transient receptor potential cation channel, subfamily M, member 2) transcript (TRPM2 up >3 fold, TRPV4 down >2 fold; both p<0.001) and significant shifts from KCNA1/2 to KCND2/3). For patch‐clamp experiments, only small DRG neurons (<35pF) were chosen, since they exhibit predominantly temperature and nociceptive phenotypes. Whilst we found that resting action current frequency was lower in TNFα treated neurons than controls, we found that the increase in action current frequency following a +15°C temperature challenge was significantly greater in TNFα treated neurons (1.6 fold increase in control vs 3.5 fold increase after TNFα challenge). These changes are generally smaller than previously reported. Following mathematical simulations, we conclude that this may be due to our use of cell‐attached AP frequency recording. This technique will allow native cellular control of intracellular Ca2+ and may allow endogenous feedback control that may be lost when using conventional whole‐cell patch‐clamp recording.Support or Funding InformationThe authors wish to acknowledge the D‐Board project, which has received funding from the European Union's Seventh Framework Programme for research, technological development and demonstration under grant agreement No. 305815
NMDA Receptors Modulate Brainstem Excitatory Neurotransmission in Spinal Trigeminal Nucleus
Authors: Jennings Ernie A 
Published: November 2023

Nociceptive inputs from craniofacial regions relayed via trigeminal primary afferents terminate primarily in the trigeminal subnucleus caudalis (the medullary dorsal horn, MDH). We have previously reported that increased glutamatergic neurotransmission induced by purinergic receptor activation can be attenuated in vivo and in vitro with N‐methyl‐D‐aspartate receptor (NMDAR) antagonists D‐AP5 and MK‐801, in addition to the P2X2/3 receptor antagonists A317491 and TNP‐ATP. To further characterise the role of NMDAR in trigeminal nociceptive processing, we investigated the effects of NMDA (100μM) and D‐AP5 (40μM) on pharmacologically isolated, evoked excitatory postsynaptic currents (eEPSCs) by using whole‐cell voltage‐clamp recordings from MDH deep laminae in brainstem slices. eEPSCs were evoked by electrical stimulation of trigeminal spinal tract axons. Application of NMDA significantly decreased eEPSC amplitude (n = 9, p < 0.05: Control: 509.8 ± 179.4 pA, NMDA: 413.7 ± 169.6 pA), whereas D‐AP5 did not significantly depress the eEPSC amplitude (n = 7, p > 0.05: Control: 285.9 ± 47.0 pA, AP5: 249.7 ± 48.4 pA). Paired‐stimuli (70ms interstimulus interval) are traditionally used to indicate whether an effect is either pre‐or postsynaptic. This MDH synapse exhibited paired‐pulse depression with a paired‐pulse ratio (PPR) less than 1 under control conditions, indicating a high probability of release. Bath application of D‐AP5 did not alter the PPR (n = 7, p > 0.5, Control: 0.68 ± 0.08, AP5: 0.66 ± 0.05), whereas NMDA resulted in a significant PPR increase (n = 16, p < 0.05, Control: 0.57 ± 0.05, NMDA: 0.70 ± 0.05). With the inclusion of the NMDAR open‐channel blocker MK‐801(1mM) in the pipette internal solution, to exclude potential contributions by postsynaptic NMDARs, NMDA caused a significant increase in PPR (n = 16, p<0.005) and marked depression of the first eEPSC (p < 0.005, Control: 500.5 ± 62.4 pA, NMDA: 445.4 ± 57.3 pA), suggesting that NMDA was not acting on the postsynaptic neuron. Furthermore, the eEPSC decay kinetics were not altered by either D‐AP5 or NMDA when compared to control (D‐AP5: p > 0.1; NMDA: p > 0.1), further suggesting that postsynaptic NMDARs are not predominantly involved in this attenuation. Together, these results suggest NMDARs, located at sites other than on the postsynaptic neuron, modulate glutamatergic neurotransmission between trigeminal primary afferent fibres and neurons in the deep laminae of the medullary dorsal hornSupport or Funding InformationFunding provided by Australian NHMRC project grant 454606
Mechanisms of Passive Inward Transport in Hyperaldosteronism Mutants and Normal Na/K Pumps
Authors: Meyer Dylan , Spontarelli Kerri , Allen Morgan , Olivera J. Fernando , Artigas Pablo 
Published: November 2023

Hyperaldosteronism (HA)‐inducing mutations in the α1 subunit of adrenal Na/K pumps (NKAs) have been found to induce a passive Na influx through the mutated NKAs under near‐physiological conditions (> 100 mM Nao;4.5 mM Ko). Under such conditions, wild type (WT) NKA catalyzes the uphill transport of 3 Na out of the cell in exchange for 2 K using the energy of one ATP molecule, a process that induces an outward current. However, under non‐physiological conditions, WT NKAs passively import H when the [Na]o and [K]o are not enough to saturate externally facing ion bindingsites. Here we use electrophysiology(two electrode voltage clamp and patch clamp) in oocytes expressing mutant NKAs to test the characteristics of the ion pathways through WT and mutant NKAs and whether the inward currents (IIN) induced by HA mutations are large enough to significantly depolarize the adrenal cells, or whether the observed depolarization (~30 mV) is secondary to the distorted ion homeostasis.WT H IIN occurs through a mechanism where the ions traverse the ion binding site that exclusively binds Na (site III). To test the contribution to IIN of ion binding sites I and II (which bind Na or K during the normal cycle), we simultaneously introduced 3 mutations(E327D, N776T, E779D) to disrupt ion binding there by increasing the size of the ion‐binding cavity. Resembling the HA mutations, the triple mutant presented a large IIN carried by Na under physiological conditions. Evaluation of the single mutants attributed this IIN to E327D in trans membrane segment (TM) 4. It has been proposed that the Na ions transported through IIN in two HA mutations, L97R (in TM1) and V325G (in TM4), involves a pathway for Na ions distinct from the H pathway in WT NKAs. To test this proposal, we concomitantly introduced the site III mutation D926N, known to abolish H permeation in WT pumps, with L97R, V325G, and E327D templates. Large IIN was observed in both L97R/D926N and V325G/D926N, while the E327D/D926N lacked IIN. This suggests that the pathway for Na current through these two HA mutants is different than the pathway for H through WT NKA. Additional studies will further clarify whether the effect of D926N on E327D is due to lack of expression, lack of a required partial reaction, or due to a pathway distinct from the HA mutants. We are also using giant inside‐out patches to compare the intracellular NKA ligand (ATP, Na, K, etc.) requirements for Na current through WT and mutant NKAs.To evaluate if the IIN observed in mutant NKAs is enough to significantly depolarize the adrenal cells, we are measuring IIN while counting the total number of NKAs present in the membrane. If the total IIN/NKA molecule is similar to the total IP/NKA molecule in WT pump, it is unlikely that the depolarization is a direct reflection of charge influx through the mutated pumps in the cell (half of the total pumps).Support or Funding InformationFunded by NSF1515434
The Calcium‐Activated Chloride Channel, Anoctamin‐1, is a Critical Regulator of Cell Proliferation Mediated via p44\42 MAPK Phosphorylation and Calcium Signaling
Authors: Franklin Brandon M , Osborn Jeffrey L 
Published: November 2023

Soft tissue repair is a critical function of living organisms which requires restoration of anatomical structure and function following tissue damage. A better understanding of the cellular and molecular mechanisms that control critical cellular processes, such as proliferation, is crucial to the development of clinical therapies in regenerative medicine. We have recently shown that chloride channel inhibition, via 4,4′‐Diisothiocyano‐2,2′‐stilbenedisulfonic acid (DIDS), 4,4′‐Dinitro‐stilbene‐2,2′‐disulfonic acid (DNDS), T16a(inh)‐A01 or Cl− free environment effectively inhibits or delays tail regeneration in larval Ambystoma mexicanum salamanders. Proliferation was assessed via EdU incorporation assay, which revealed that this effect was manifested through regulation of mesenchymal cell proliferation, with all 3 pharmacological treatments reducing proliferation rate in the mesenchyme (Control: 33.5±3.2%; DIDS: 18.13±2.25%; DNDS: 19.64±3.79%; T16a(inh)‐A01: 19.65±1.85% EdU+ cells). There is no effect on proliferation in either neural or epidermal tissues. Western blot analysis demonstrated that phosphorylation of p44/42 MAPK (Erk1/2) is reduced when the calcium activated chloride channel, Tmem16a, is inhibited with T16a(inh)‐A01 in 72 hpa regenerates. These results indicate that the calcium activated chloride channel, anoctamin‐1, regulates proliferation via p44\42 MAPK. A transgenic HEK 293 cell line that overexpresses the anoctamin‐1 channel is being used to further elucidate the channel mechanisms involved in Cl‐channel mediated cell proliferation. Fluorescent calcium indicators will provide semi‐quantitative evidence regarding the influence of anoctamin‐1 on intracellular calcium signaling.Support or Funding InformationSupported by Biomedical Sciences Research Group, LLC.
Cercariae of Proterometra macrostoma may use catecholamines as endogenous neurotransmitters
Authors: Bumpus Erin Nicole , Runyan Keith R , Weitekamp Samuel L , Bartlow Kathleen Marie 
Published: November 2023

Furcocystocercous cercariae of the digenetic trematode parasite Proterametra macrostoma exhibit a characteristic vertical swimming behavior to attract their centrarchid hosts. Previous studies showed that swimming behavior is entirely controlled by the cercarial tail, and may be modulated by factors including light wavelength and mechanical stimulation. We conducted studies using a vertical swim‐up assay to examine the behavioral effects on P. macrostoma cercariae of exogenous application of the catecholamine neurotransmitters dopamine, epinephrine, and norepinephrine. Catecholamine neurotransmitters increased the speed of swimming. Norepinephrine also increased the number of swims per cercaria in a five‐minute period and the time each cercaria spent off the bottom of the water column, implying that behavioral effects of noradrenergic signaling in this species may be ecologically relevant. Studies conducted in whole‐mounted cercariae, using immunofluorescent staining to detect the neuronal marker beta‐III‐tubulin, reveal dense superficial innervation in the posterior cercarial tail in close apposition to striated muscle fibers. Cryosections immunostained for tyrosine hydroxylase (TH) display TH immunoreactivity throughout the distome body and tail of cercariae, particularly in the papillae of the tail, which are thought to be sensory organs. Together, these results suggest that catecholamines are produced and used by P. macrostoma as a neurotransmitter modulating cercarial swimming.Support or Funding InformationThese studies were funded by the Indiana Academy of Science, and by the following funds associated with Earlham College: Gerald Bakker Collaborative Research Endowment Fund, Matthews Student/Faculty Research in Physics/Biological Science, Harry Todd Costello Fund, Stevenson Fund, and Collaborative Research Fund.