bluuebunny/crossref_metadata_2025 huggingface dataset:
Nociceptin/Orphanin FQ (N/OFQ) stimulated diuresis is mediated via inhibition of vasopressin secretion: a role for the hypothalamic paraventricular nucleus (PVN)
Authors: Wainford Richard David , Glatzer Nicholas R , Tasker Jeffrey G , Kapusta Daniel R
Published: June 2021

The role(s) of vasopressin (AVP) and the hypothalamic PVN in mediating the diuretic response to intracerebroventricular (i.c.v.) and PVN microinjection of N/OFQ was examined in rats.

Methods

N/OFQ was injected i.c.v. (5.5 nmol) in conscious instrumented Sprague‐Dawley rats (n=6/group) infused i.v. with isotonic saline (50 μl/min), AVP (170 pg/kg/min) or a V2‐ AVP receptor antagonist (10 nmol/kg/hr) and urine was collected for 80‐min (10‐min periods). In other studies, blood was collected 10‐min post‐i.c.v. or PVN (100 pmol) N/OFQ injection and assayed for AVP. N/OFQ peptide (NOP) receptor immunohistochemistry (IHC) was performed in rat brains expressing GFP in AVP neurons.

Results

I.c.v. and PVN injection of N/OFQ produced a profound diuresis (PVN N/OFQ peak diuresis; 40‐min, Δ164±22 μl/min, P<0.05) in rats. I.v. infusion of a V2‐receptor antagonist increased basal urine output and prevented a further i.c.v. N/OFQ‐induced diuresis. I.v. infusion of AVP reduced basal urine output and prevented the diuretic response to i.c.v. N/OFQ. In naïve rats, 10‐min post‐i.c.v. or PVN N/OFQ, plasma AVP levels were significantly reduced (i.c.v. vehicle, 2.2±0.2 pg/ml; i.c.v. N/OFQ, 1.4±0.1; PVN N/OFQ, 1.3±0.1 P<0.05). IHC studies demonstrated the presence of NOP receptors on AVP expressing PVN magnocellular neurons.

Conclusion

In conscious rats, N/OFQ acts within the PVN, likely at the level of magnocellular neurons, to suppress AVP secretion and thereby produce diuresis. Supported by DK43337, HL 071212, AHA 0855293E, P20 RR018766 to DRK and NS042081 (JGT).
Adrenal hormones are necessary for the hydromineral phenotypes of transgenic mice with central renin‐angiotensin system overactivity
Authors: Grobe Justin L. , Grobe Connie L. , da Rocha Borges Giulianna , Beltz Terry A. , Johnson Alan K. , Sigmund Curt D.
Published: June 2021

To examine the roles of the central renin‐angiotensin system (RAS) in blood pressure and fluid volume homeostasis, our lab previously developed the sRA mouse model, in which human renin is expressed via the neuron‐specific synapsin promoter, and human angiotensinogen via its endogenous promoter. Due to species‐specificity of this reaction, RAS overactivity is restricted to the brain. sRA mice exhibit polydipsia (control, 0.04±0.01 vs sRA, 0.31±0.12 mL/g/d), polyuria (0.08±0.02 vs 0.92±0.04 g/g/d), and a robust sodium appetite (0.94±0.18 vs 3.93±0.07 mEq/d). Here, we examined whether elevated HPA‐axis activity is necessary for these phenotypes. Four weeks after total adrenalectomy, the sodium appetite of sRA mice was completely normalized (0.72±0.21 mEq/d), and the polydipsia (0.11±0.01 mL/g/d) and polyuria (0.15±0.01 g/g/d) were significantly attenuated. In a separate cohort, acute spironolactone treatment partially blocked the sodium intake of sRA mice (3.03±0.31 down to 2.33±0.44 mEq/d), suggesting the involvement of multiple adrenal hormones in this behavior. From these data, we conclude that multiple adrenal hormones are necessary for the dipsogenic and sodium appetite behaviors of mice with chronic overactivity of the brain RAS. Ongoing work is dissecting the contribution of glucocorticoids in hydromineral regulation in sRA mice. (JLG supported by the APS; CLG, AKJ, CDS supported by NIH)
Early sympathetic denervation of splanchnic organs significantly attenuates hypertension and stroke development in stroke‐prone spontaneously hypertensive rats
Authors: Kandlikar Sachin , Dorrance Anne M , Fink Gregory
Published: June 2021

Human and experimental studies have shown that splanchnic sympathetic nervous activity (SNA) is important in the development and maintenance of hypertension. The purpose of this study was to evaluate the importance of splanchnic SNA to the development of hypertension and stroke in stroke‐prone spontaneously hypertensive rats (SHRSP). To accomplish this goal, selective surgical denervation of splanchnic organs was performed by celiac ganglionectomy (CGx) in male SHRSP at 4 weeks of age (70‐80 g body weight). CGx was performed in one group of rats (n=5) while the other group underwent SHAM operation (n=4). Subsequently the rats were fed a regular diet and allowed free access to drinking water. Rats were instrumented with a radiotelemeter for blood pressure (BP) measurement at 12 weeks. After a 7 day recovery, BP was measured for 7 days. BP was significantly lower in CGx rats (138 ± 3.0 mmHg) compared to that of SHAM rats (176 ± 6.9 mmHg). All rats were then fed a Japanese style diet for 4 weeks to study whether CGx protects against stroke. BP increased in both groups but the BP difference between the groups remained. Two strokes occurred in the SHAM group while none occurred in CGx rats. We conclude that splanchnic SNA is critical in the development of hypertension and contributes to the propensity for strokes in SHRSP. (Supported by PO1‐HL070687)
Quantitative proteomic analysis of long‐term vasopressin actions in mpkCCD cells using SILAC and MRM
Authors: Khositseth Sookkasem , Yu M‐J. , Pisitkun T. , Tchapyjnikov D. , Knepper M.A.
Published: June 2021

We employed 'stable isotope labeling with amino acids in cell culture' (SILAC) coupled LC‐MS/MS to identify proteins that change in abundance in response to long‐term vasopressin stimulation in mpkCCD cells. Cells were grown in media containing heavy isotope‐substituted or light non‐substituted amino acids, and either vasopressin analog dDAVP (100pM) or vehicle, respectively. After 5 days, heavy‐ and light‐labeled cell lysates were combined for quantitation by LC‐MS/MS. 65 proteins were increased and 56 were decreased in abundance in response to dDAVP. Increases in 5 proteins from the SILAC results were confirmed by immunoblotting: AQP2, ADD1 (adducin‐1), MAL2, AKAP12, and SLC9A3R1 (NHERF1). For antibody‐independent validation, proteins were selected for targeted quantitative proteomics using multiple‐reaction monitoring (MRM) on a triple quadrupole mass spectrometer. Increases in 4 proteins (AQP2, ASAP2 [ArfGAP with SH3 domain ankyrin repeat and PH domain 2], MON2 [yeast homolog involved in Golgi to endosome transport] and CLMN [calmin]) and a decrease in one protein (NALCN [sodium leak channel non‐selective]) were statistically validated. Overall 1,629 proteins were identified and deposited in a new online database (mpkCCD Proteome Database). These methodologies permit large‐scale targeted quantification, providing an antibody‐independent alternative to multiplexed immunoblotting.
Renal NOS isoforms and splicing in 13 week obese female Zucker rats on antioxidant vs regular diet
Authors: Inman Sharon R , Slyvka Yuriy , Feldman Yelena , Nowak Felicia V
Published: June 2021

NO and NOS activity are important for development of diabetic nephropathy and an antioxidant fortified (AO) diet may protect the kidney in the case of diabetes. Female obese Zucker rats (fa/fa) were studied at 13 weeks of age on regular (REG) and AO diets. AO diet was started at 4 weeks. NOS isoforms (n‐neuronal, e‐endothelial and i‐inducible) and their splice forms were tested with Western blot. The AO diet resulted in lower levels of all NOS isoforms both in kidney cortex and medulla compared to the REG diet. There was no significant difference in NOS expression between cortex and medulla in either group. The following splice forms were present in rat kidneys on AO diet: iNOS (122‐124, 77‐83, 72‐75 kDa), nNOS (110‐113, 76‐77, 68‐70 kDa), eNOS (110‐116, 73‐77, 66‐68 kDa). Animals on the REG diet had the following splice forms: iNOS (114‐118, 76‐79, 70‐72 kDa), nNOS (110‐114, 74‐79, 68‐72 kDa), eNOS (102‐103, 70‐72, 64‐66, 38 kDa). The difference between content of NOS isoforms is the result of an increase in the largest and smallest splice forms of eNOS and iNOS in kidney cortex. In medulla the difference is the result of increase of all splice forms for iNOS and nNOS, but for eNOS only the largest and smallest splice form increased and a new smaller additional 38 kDa form was seen in rats on the REG diet.

Conclusion

AO diet decreased the expression of NOS isoforms in kidneys in obese Zucker rats. Supported by NIH R15 DK073066 to SRI and FVN.
Mast Cells Mediate Stress‐Induced Breakdown in Mucosal Barrier Function in a Porcine Model of Irritable Bowel Syndrome
Authors: Moeser Adam James , Overman Elizabeth Lynne , Blikslager Anthony Thomas
Published: June 2021

Early weaning stress in the pig triggers lifelong stress sensitivity and intestinal barrier dysfunction similar to Irritable Bowel Syndrome (IBS) in humans. We investigated the role of mast cells (MCs) in stress‐induced intestinal barrier dysfunction in early weaned, IBS model pigs. Piglets were removed from their dams at 17 days of age (Early weaning) and housed in a commercial pig nursery facility. At six weeks postweaning, pigs were subjected to social stress for three hours to induce disturbances in colonic barrier function, measured in terms of transepithelial electrical resistance (TER) and mucosal‐to‐serosal 3H‐mannitol fluxes in Ussing chambers. To determine the role of MCs in mucosal barrier disturbances, pigs were pretreated, via intraperitoneal injection, with the MC stabilizer agent Cromolyn (20 mg/kg) or saline vehicle prior to stress. Social stress triggered marked reductions in TER (p<0.05) and elevations 3H‐manntiol flux (p<0.01) compared with unstressed controls indicating impairments in barrier function. Cromolyn inhibited stress‐induced breakdown of mucosal barrier function. In vitro Ussing chamber studies revealed that MC‐mediated colonic barrier disturbances were inhibited by protease inhibitors. These studies show that MCs play a critical role stress‐induced breakdown of mucosal barrier function and that this response is mediated in part by MC proteases.
Anti‐Proliferative Effects of Sera from Rats with Placental Ischemia on Breast Cancer Cells
Authors: Gingery Anne , Gilbert Jeffrey S
Published: June 2021

Although recent epidemiological studies report preeclampsia has long‐term protective effects against breast cancer, the mechanisms underlying these observations remain unknown. Further, it is unclear whether these effects are due to underlying genetic and environmental factors or are sequelae of the altered endocrine milieu and placental ischemia during preeclampsia. To this end, we hypothesized that serum from pregnant rats with placental ischemia‐induced hypertension contains factors that inhibit proliferation of breast cancer cell lines. A variety of breast cancer cell lines and one lung cancer cell line were seeded onto 48 well plates in media with 5% serum from normal pregnant (NP) or reduced uterine perfusion pressure (RUPP) rats. Decreased proliferation, as measured via sulforhodamine B assay, after 24 or 48 hours of treatment with RUPP serum compared to NP serum was found in MBA‐MB‐231 (62.2% ‐ 24h), Hs578T (20.3% ‐ 48h), MCF‐7 (18.5% ‐ 48h) and UMD227 (79.3% ‐ 24h) breast cancer cell lines. Proliferation of the A549 lung carcinoma cell line was not reduced by treatment with RUPP serum. The present data indicate that placental ischemia may play an important and cell specific role in the anticancer effects observed in formerly preeclamptic women. Understanding the mechanisms responsible for these observations may reveal novel therapeutic approaches to cancer prevention and treatment.
Increased intestinal secretory cell differentiation in mice administered total parenteral nutrition
Authors: Kechele Daniel O. , VanDussen Kelli L. , Feng Yongjia , Teitelbaum Daniel H. , Samuelson Linda C.
Published: June 2021

Intestinal atrophy occurs when total parenteral nutrition (TPN) is used to supply nutrients directly into the blood instead of normal ingestion. The cellular basis for the intestinal remodeling has not been fully described. In this study we analyzed the jejunum of mice that received TPN for 7 days compared to freely‐fed sham‐operated and normal C57BL/6 controls. Intestine was characterized by histological staining to assess the four major epithelial cell types: goblet cells, endocrine cells, Paneth cells and enterocytes. PAS/alcian blue staining revealed a 1.5‐fold increase in the number of goblet cells. Moreover the staining characteristics showed a shift from acidic to neutral mucin production in the TPN goblet cells. Increased numbers of endocrine and Paneth cells were also suggested by immunohistochemical staining against the pan‐endocrine cell marker chromogranin A and the Paneth cell marker cryptdin. Thus, TPN administration induced an expansion of all three intestinal secretory cell types. Our findings suggest that TPN disturbed the normal differentiation of multipotential progenitors into secretory vs. absorptive cells, a process known to be regulated by Notch signaling. Future studies will analyze the expression of transcription factors known to be critical effectors of Notch‐mediated lineage determination to understand the mechanism of cellular remodeling in the TPN intestine.

Funded by NIH.
Platelet‐activating factor receptor (PAFR) lysosomal targeting is mediated by p85α subunit of PI3K in enterocytes
Authors: Lu Jing , Caplan Michael , Jilling Tamas
Published: June 2021

PAFR signals via the PI3K pathway to regulate apoptosis in rat small intestinal epithelial cells (IEC‐6). In IEC‐6, over‐expressed p85α regulatory subunit of PI3K and PAFR directly interact with each other and co‐localize to perinuclear vesicles resulting in diminished PAF‐induced apoptosis. We hypothesized that p85α regulates PAFR trafficking and function through lysosomal targeting. Immunostaining revealed that over‐expressed p85α co‐localized with lysosomal associated protein1 (LAMP1) and over‐expressed PAFR. In p85α over‐expressing cells we observed a 2‐fold increase of acid phosphatase (AP) activity in LAMP1‐ and p85α‐positive sucrose gradient fractions. In HT29 colonic epithelial cells, native PAFR was internalized upon 10 to 20 mins of PAF exposure, interacted with native p85α, then recycled to the cell surface following prolonged PAF exposure (40 min). Over‐expressed p85α in HT‐29 reduced the baseline membrane expression of PAFR and upon PAF stimulation, delayed the recycling of PAFR to the surface. Immunoblotting analysis of sucrose gradient fractions from p85α over‐expressing HT‐29 cells revealed that native PAFR was present in p85α‐and LAMP1‐positive fractions that had the highest AP activity. In summary, the inhibition of PAFR signaling by p85α over‐expression is due to removal of receptors from the cell surface, and targeting to lysosomes. Supported by NIH HD37581 and DK062960.
Hexosamine Signaling Mediates Peroxisome Proliferator‐Activated Receptor‐gamma Coactivator (PGC)‐1alpha Suppression During Cardiac Hypertrophy
Authors: Facundo Heberty Fernandes , Brainard Robert Eli , Jones Steven Paul
Published: June 2021

The peroxisome proliferator‐activated receptor‐gamma coactivator‐1α (PGC‐1α) is essential for normal cardiac function. The O‐linked β‐N‐acetylglucosamine post‐translational modification (O‐GlcNAc) of proteins is a glucose‐derived metabolic signal. The relationship between O‐GlcNAc modification and PGC‐1α activity in hypertrophy is unknown. We hypothesized that the metabolic switch from fatty acids to glycolysis during hypertrophy would involve augmented O‐GlcNAc signaling. Transverse aortic constriction in mice in vivo or treatment of neonatal rat cardiac myocytes with phenylephrine significantly enhanced (p<0.05) global O‐GlcNAc signaling and augmented O‐GlcNAc transferase (OGT: adds O‐GlcNAc to proteins) expression. Quantitative real‐time PCR analysis revealed a downregulation of PGC‐1α with concomitant suppression of fatty acid oxidation and mitochondrial biogenesis genes. Reduction of O‐GlcNAc signaling partially rescued the cells from hypertrophic changes (i.e. ANP mRNA elevation), and, reversed the effect of in vitro hypertrophy on PGC‐1α levels and on most of its regulated downstream genes. Interestingly, augmentation of O‐GlcNAc signaling with glucosamine markedly reduced PGC‐1α activity, in the absence of hypertrophy. Similarly, basal expression of PGC‐1α and its downstream genes was elevated in cardiac specific OGT knockout mice. Together, these results reveal the recruitment of O‐GlcNAc signaling as a potentially novel regulator of PGC‐1α activity during cardiac hypertrophy. Furthermore, O‐GlcNAc signaling may mediate constitutive suppression of PGC‐1α activity in the heart. Such findings illuminate new possibilities regarding the inter‐regulation of glycolytic and fatty acid metabolism. Financial support: NIH, American Heart Association
Role of P2XR Channel Purinergic Signaling in Insulin Secretion: Implications in the Treatment of Diabetes
Authors: Williams Clintoria Richards , Contreras Juan L. , Berecek Kathleen H. , Schwiebert Erik M.
Published: June 2021

Secretory defects in β‐cells of islets of Langerhans play a role in diabetes. It is accepted that ATP & zinc are co‐secreted with insulin. However, the physiological roles of these secreted molecules within islets are ill‐defined. We hypothesized that secreted ATP & zinc are autocrine purinergic signaling molecules that activate P2X purinergic receptor (P2XR) channels expressed by β‐cells to enhance glucose‐stimulated insulin secretion (GSIS). To test this postulate, ELISA, bioluminescence and fluorescence assays were used to examine insulin, ATP & zinc secretion, respectively, from β‐cells of islets. RT‐PCR, Western blot and immunofluorescence were used to investigate P2XR channel expression in islets and immortalized β‐cells. Findings confirmed that glucose stimulated release of the endogenous ligands for P2XR channels, ATP and zinc. When these secreted molecules were scavenged or chelated, respectively, GSIS was attenuated. In support, expression of multiple ATP‐gated & zinc‐modulated P2XR channel subtypes was observed in β‐cells. Antagonism of these receptors resulted in blunted GSIS. Furthermore, activation of β‐cell P2XR channels induced insulin secretion in a Ca2+‐dependent manner. We conclude that secreted ATP & zinc have autocrine influence on insulin secretion that is mediated via P2XR channels and propose that the β‐cell P2XR channel signaling pathway is a therapeutic target in diabetes. Supported by an APS Porter Fellowship & a NRSA Predoctoral Fellowship.
A structure‐based approach for detection of thiol oxidoreductases and their catalytic redox‐active cysteine residues
Authors: Marino Stefano Maria , Gladyshev Vadim N
Published: June 2021

Cysteine (Cys) residues often play critical roles in proteins, for example, in the formation of structural disulfide bonds, metal binding, and various catalytic functions. Thiol oxidoreductases, enzymes containing catalytic redox‐active Cys residues (redox Kat Cys), have been extensively studied, but even for these proteins there is little understanding of what distinguishes their redox Cys from other Cys functions. Herein, we characterized thiol oxidoreductases at a structural level and developed an algorithm that can efficiently recognize them by (i) analyzing amino acid and secondary structure composition of the active site and its similarity to known active sites containing redox Kat Cys, and (ii) calculating accessibility, active site location and reactivity of Cys. By applying this procedure to the Saccharomyces cerevisiae proteome, we could identify the majority of known yeast thiol oxidoreductases. This study provides insights into the structural properties of redox Kat Cys and highlights important differences between these residues and other reactive Cys. It should help recognize novel thiol oxidoreductases in structure databases.
Proteinase‐activated receptors in lung surfactant secretion in alveolar type II cells
Authors: Chander Avinash , Chacko Liji B , Killeen Erin , Gerelsaikhan Tudevdagva
Published: June 2021

The epithelial surface of distal lung is continuously exposed to environmental insults that elicit a potentially injurious response involving influx of neutrophils and release of several proteinases to facilitate host‐defense response. These proteinases can activate cellular response through protease‐activated receptors (PARs). The alveolar epithelial type II (T2) cells are important as they synthesize and secrete various components of lung surfactant, which can influence lung compliance and the innate host‐defense in the lung. In this study, we evaluated if one such proteinase, trypsin, influenced the secretion of lung surfactant in T2 cells. The secretion of lung surfactant with trypsin and phorbol myristate acetate (PMA) was measured in 24 h cultured T2 cells that were labeled with [3H]choline. During incubation for two hours, trypsin increased the secretion of lung surfactant in a concentration‐dependent manner. At 1 μg/ml trypsin, the secretion was ~250% of the basal secretion in comparison to ~300% observed with 100ng/ml PMA. Since trypsin can activate PAR2, we evaluated T2 cells for the presence of this receptor. Western blot analysis of freshly isolated and 24 h cultured cells showed that one major band (~45 kDa protein) reacted with the PAR2 antibody suggesting the presence of PAR2 in T2 cells. These results suggest that T2 cells possibly respond to trypsin through PAR2 to increase lung surfactant secretion and contribute to lung host defense following lung injury and neutrophil‐mediated increase in proteinase enzymes.
ENaC trafficking via Rab‐dependent pathways
Authors: Karpushev Alexey , Levchenko Vladislav , Pavlov Tengis S , Lam Vy , Vinnakota Kalyan C , Wakatsuki Tetsuro , Staruschenko Alexander
Published: June 2021

The epithelial Na+ channel (ENaC) is an essential channel responsible for Na+ reabsorption, and it is regulated by different signaling pathways, including small G proteins. Rab proteins are well‐established as a participant in the regulation of trafficking. Co‐expression of Rab11a and Rab3a with ENaC results in a significant increase in channel activity. Biochemical and imaging methods demonstrate that Rab11a colocalized with ENaC and increased ENaC activity by affecting the plasma membrane levels of this channel. Rab11a increases ENaC activity in an additive manner with dominant‐negative dynamin, which is a GTPase responsible for endocytosis. Brefeldin A, an inhibitor of intracellular protein translocation, blocked the stimulatory action of Rab11a on ENaC activity. This is consistent with a mechanism of increased trafficking toward the plasma membrane. We further demonstrate that coexpression of ENaC with Rab11a and SGK1 together had no effect on Rab11a dependent increase of ENaC activity suggesting that SGK1 might be involved into Rab11a dependent vesicle exocytosis. These results demonstrate a novel role for Rab small GTPases in the control of epithelial ion transport. We conclude that ENaC channels, present on the apical plasma membrane, are being exchanged with channels from the intracellular (recycling) endosomes in a Rab11‐dependent manner.

Supported by AHA and ASN.
Src family protein tyrosine kinase (PTK) and MAPK are involved in mediating the effect of low potassium intake (LK) on ROMK channels
Authors: Wang Zhijian , Pan ChuYang , Lin Dao‐Hong , Wang Wen‐Hui
Published: June 2021

We have previously demonstrated that K‐depletion inhibited ROMK‐like small conductance K channels (SK) in the CCD and that the effect is mediated by superoxide anions which stimulate PTK and MAPK. However, K‐depletion caused a severe hypokalemia. Thus, it is not clear whether PTK and MAPK are also involved in regulating SK channels under physiological conditions. The aim of the present study is to examine whether PTK and MAPK play a physiological role in suppressing SK channel activity in response to LK (0.1%). Rats on LK diet for 7 days have a normal plasma K concentration (control, 4.74 and LK, 4.64 mM). However, LK intake significantly stimulates the production of superoxide by 95% in the renal cortex and outer medulla, and increases the phosphorylation of p38 and ERK and the expression of c‐Src. Also, patch‐clamp experiments have demonstrated that LK intake significantly decreased SK channel activity in the CCD. However, Inhibiting p38 and ERK with SB202190 and PD98059 or blocking PTK with herbimycin A significantly stimulates SK channel activity in the CCD in rats on LK. We conclude that MAPK and Src family PTK play a physiological role in inhibiting SK channels in response to LK intake.
Understanding the mechanism responsible for γ6‐dependent modulation of low voltage activated Ca2+ channels by exploring physical interactions
Authors: Garcia Thomas , Best Philip
Published: June 2021

The γ6 subunit of voltage dependent Ca2+ channels is known to decrease Ca2+ current when co‐expressed with the low voltage activated Ca2+ channel subunit α3.1. This effect of γ6 is specific to current density as there is no change in the biophysical properties of the channels when measured at the macroscopic level. Previous studies using chimeric and mutant γ subunits indicate that the first transmembrane domain (TM1) of γ6 and more specifically a GxxxA motif within it are critical for γ6's inhibitory function. In this study, cell surface membrane protein biotinylation, purification, and quantitative western blot demonstrate that the functional effect γ6 confers upon Cav3.1 current is not due to a change in the plasma membrane expression of α3.1. Furthermore, co‐immunoprecipitation assay demonstrates a physical association of γ6 and α3.1 in both HEK cells and atrial myocytes. Subsequently, we engineered chimeric and mutant subunits of γ6 to identify potential interaction site(s). Results from these experiments indicate the functionally critical domain and motif of γ6 are not required for γ6 interaction with α3.1. Further studies are required to determine the region(s) in γ6 involved in interaction with α3.1, as well as the γ6 interaction site within α3.1. Supported by a GIA from the AHA.
Changing S261, including constitutive phosphorylation, enhances AQP2 ubiquitination and internalization
Authors: Deen Peter MT , Tamma Grazia , Stoffels Monique , Hoffert Jason D , Konings Irene BM , Knepper Mark A
Published: June 2021

For renal water reabsorption AVP‐induced phosphorylation of AQP2 at S256 (p256) is needed for its translocation to the apical membrane. Also, phorbol esters (TPA) activation of PKC induces AQP2 ubiquitination at K270, its internalization and lysosomal degradation. But AQP2 can also be phosphorylated at S261 (pS261), which role was studied here. In transfected MDCK cells, forskolin (F) redistributed AQP2 from vesicles to the apical membrane, which was reversed with subsequent F/TPA treatment. As in vivo for AVP, F increased pS256 and decreased pS261 AQP2, which was reversed with F/TPA. For all treatments, AQP2‐S256A and AQP2‐S261A/D always localized to intracellular vesicles, AQP2‐S256D localized in the apical membrane with/without F and internalized with F/TPA, but the changes in pS261 and pS256 were similar to AQP2. AQP2‐S261A/D were more ubiquitinated than wt‐AQP2 and AQP2‐S261D‐K270R, which cannot be ubiquitinated, always localized in the apical membrane. AQP2‐S256D‐S261D, however, localized as AQP2‐S256D. AQP2‐S256D‐Ub always localized to vesicles and was constitutively‐phosphorylated at S261. Thus, as in vivo with AVP, AQP2 is reciprocally changed in S256 and S261 phosphorylation with F and F/TPA, independent of their localization. pS256 is needed and sufficient for AQP2 translocation to the apical membrane, but is overruled by constitutive ubiquitination. Changing S261, including constitutive pS261, enhances AQP2 ubiquitination and internalization, but is overruled by constitutive pS256.
Assay dependent activity of the sodium channel gating modifier protoxin‐I: implications for sodium channel drug discovery
Authors: Bhattacharya Anindya , Wang Qi , Wu Nancy , Wickenden Alan D
Published: June 2021

Sodium channels (NaV1.7) are extremely attractive analgesic drug targets. The Holy Grail in NaV1.7 drug discovery (in‐vitro) is to discover selective NaV1.7 antagonists. Most compounds, if not all bind to a highly conserved, "local anesthetic binding site". Gating modifiers may exert selectivity due to interaction at less conserved allosteric sites. Therefore, we were interested to determine if several of the most commonly used sodium channel screening assays can identify such allosteric hits. Using protoxin‐I (ProTx‐I) we show the ability to detect gating modifiers is assay dependent. Using a voltage‐sensitive dye in FLIPR, ProTX‐I was unable to inhibit veratridine activation of NaV1.7 at concentrations up to 1 μM. Widely used pore blockers (amitriptyline, tetracaine) were effective in this assay format with a pIC50 of 6. Likewise, ProTX‐I was inactive in a radioligand‐binding assay using [3H]‐batrachotoxin as a tracer. In a VIPR FRET assay, ProTX‐1 caused only a small 'delay' in veratridine activation. In contrast , protoxin‐I was a potent blocker (pIC50 of 6.4) of the channel in two automated patch‐clamp assays (IonWorks Quattro and PatchXpress). These findings suggest that gating modulators may be missed in commonly used assays such as FLIPR that use an end‐point read. Carefully optimized VIPR assays or HTEP assays may be the preferred platform to identify sub‐type selective sodium channel blockers.
USP10 regulates post‐endocytic recycling of CFTR in airway epithelial cells
Authors: Bomberger Jennifer , Barnaby Roxanna , Stanton Bruce
Published: June 2021

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) functions as a cyclic AMP‐regulated chloride channel and a regulator of other ion channels and transporters in epithelial tissues. CFTR constitutively cycles between the apical plasma membrane, early endosomes, and recycling endosomes. The aim of the current study was to investigate the role of ubiquitination in regulating the post‐endocytic sorting of CFTR. Employing a deubiquitinating enzyme (DUB) activity assay, we identified the ubiquitin specific protease‐10 (USP10) to be localized and active in early endosomes of polarized airway epithelial cells. Immunoprecipitation studies demonstrated that CFTR interacts with USP10 in early endosomes. siRNA‐mediated knockdown of USP10 increased CFTR ubiquitination and degradation, and reduced apical membrane CFTR abundance. The reduction in cell surface CFTR with USP10 knockdown correlated with a decrease in CFTR‐mediated chloride current. Moreover, overexpression of a dominant negative USP10 mutant (C424A) increased the ubiquitination and degradation of CFTR, while overexpression of WT‐USP10 decreased Ub‐CFTR and increased the expression of CFTR. Finally, siRNA‐mediated knockdown of USP10 resulted in a retargeting of CFTR from recycling endosomes to the lysosomal degradation pathway. These studies demonstrate a role for USP10 in deubiquitinating CFTR, and thus regulating the post‐endocytic recycling of CFTR.
Modulation of CFTR function by reactive oxygen‐nitrogen species
Authors: Denny Sarah , Lazrak Ahmed , Noah James W , Noah Diana L , Matalon Sadis
Published: June 2021

We examined the effects of Reactive Oxygen‐Nitrogen Species (RONS) on chloride (Cl−) currents across Xenopus oocytes heterologously expressing the Cystic Fibrosis Transmembrane conductance Regulator (CFTR), using the two micro‐electrode voltage clamp method. The baseline current value was 310 ± 73 nA (mean ± SE, n = 5). Addition of 10μM Forskolin (an adenylate cyclase activator) activated CFTR and raised the current to 4750 ± 904 nA (mean ± SE, n = 5). Following this activation, the addition of 1mM 3‐morpholinosydnominine (SIN‐1), which generates both superoxide and nitric oxide, along with 10µM Forskolin, resulted in a total inhibition of the Cl− current within 10 minutes to its pre‐activation level. The SIN‐1 mediated decrease in CFTR current was totally absent when SIN‐1 was allowed to decompose to its stable form, SIN‐1c, prior to treatment. Interestingly, the SIN‐1 effect was blocked by intracellular but not extracellular addition of 1mM glutathione (GSH), a RONS scavenger. We conclude that exposure of oocytes expressing CFTR channels to RONS causes a decrease in the Cl− current via an unknown mechanism, potentially involving intracellular pathways that are inhibited by GSH.
Mice presenting skeletal muscle hypertrophic phenotype driven by mIGF‐1 over‐expression have improved carbohydrate metabolism and insulin sensitivity.
Authors: Christoffolete Marcelo Augusto , de Oliveira‐Costa Monique , Okamoto Maristela M. , Turri Antonio , Lohmann Tania Ochi , Ribeiro Miriam Oliveira , Machado Ubiratan Fabres , Musarò Antonio , Moriscot Anselmo Sigari
Published: June 2021

Objective

In this study, we aim to characterize the metabolic profile of mice presenting higher muscle mass driven by mIGF‐1, a muscle specific trophic factor.

Methods

WT and mIGF‐1 TG mice were obtained from our inbreed colony. O2 consumption and CO2 generation was determined in an open circuit respirometer. kITT (%/min) was determined by calculating the slope of the linear regression of neperian logarithm of glycemia values over time in a standard 15 min ivITT test using 0.125U/Kg of insulin. The results were expressed as mean±SEM.

Results

As expected, 3 months old TG mice were heavier than age matched WT mice (33.3 + 0.3 vs 29.7 + 0.4 g, p<0.0001, n=9‐15, respectively). TG mice have a higher non‐protein respiratory quotient then WT (0.9 + 0.03 vs 0.74 + 0.02, p<0.001, n=7, respectively). This data suggested a preference for carbohydrate as major fuel source in TG mice. TG mice have a higher rate of glucose disposal when compared to WT (3.25 + 0.14 vs 2.39 + 0.03 %/min, p<0.01, n=3‐5, respectively). These data altogether reveals a shift in metabolism towards higher carbohydrate utilization and improved insulin sensitivity in mIGF‐1 TG mice.
Natriuretic response to renal medullary endothelin B receptor activation is blunted in chronic angiotensin II‐infused rats
Authors: Kittikulsuth Wararat , Boesen Erika I , Pollock David M
Published: June 2021

Endothelin B (ETB) receptors, highly expressed in the renal medulla, function to promote sodium and water excretion such that the impairment of ETB receptor function results in salt‐dependent hypertension. Since chronic angiotensin (Ang) II also results in salt‐dependent hypertension, we hypothesized that renal medullary ETB receptor function is impaired in Ang II‐dependent hypertension. We compared the diuretic and natriuretic response to intramedullary infusion of ETB receptor agonist sarafotoxin 6c (S6c) in rats treated with Ang II (65 ng/min s.c.) or vehicle for 14 days. Rats were anesthetized and renal perfusion pressure maintained at a normotensive level in Ang II rats via an adjustable clamp placed around aorta. Urine was collected during 2 consecutive 20 min periods for baseline collection followed by 4 additional 20 min periods of intramedullary saline or S6c (0.45 μg/kg/h) infusion. Renal medullary blood flow (MBF) was determined by laser‐doppler flowmetry. In the vehicle group, S6c significantly increased urine flow (Δ8.4+1.7 μL/min; p<0.01) and sodium excretion (Δ0.79+0.24 μmol/min; p<0.01). However, Ang II rats did not display an increase of urine flow (Δ0.7+0.7 μL/min) and sodium excretion (Δ‐0.06+0.04 μmol/min) during S6c infusion. S6c did not affect MBF in either group. These data demonstrate that renal medullary ETB receptor function is impaired in Ang II‐dependent hypertension.
The BK channel in intercalated cells mediates shear stress induced ATP dependent K efflux
Authors: Holtzclaw J. David , Riggle Jakeb D. , Irsik Debra L , Grimm P. Richard , Sansom Steven C
Published: June 2021

Large conductance, Ca2+‐activated K channels (BK) are expressed in both intercalated cells (IC) and principal cells (PC) of the K‐secreting segments of the nephron. However, IC express substantially more BK than PC. We hypothesize that high flow activates BK in IC, reducing IC size, thereby increasing tubule internal diameter and decreasing resistance to flow. To test this hypothesis, K loss was determined in PC and IC subclones of MDCK cells exposed to static or 10 dynes/cm2 shear stress (τ). τ significantly decreased K content in IC, but not PC, by 37% compared to static. This decrease was attenuated by decrease in luminal calcium (Ca), 5 mM TEA, 1 μM Paxilline, or BK‐β4 siRNA, demonstrating that the τ induced K efflux in IC was mediated by BK. Furthermore, exposure of PC and IC to τ led to transient increases in Ca. The addition of 10 μM ATP to IC, but not PC, replicated the τ induced K efflux, which was blocked by 300 μM suramin, 1 μM Paxilline, or BK‐β4 siRNA. Application of ATP significantly increased Ca in PC and IC, by both intracellular and extracellular mechanisms. Finally, wildtype mice on a high K diet demonstrated enlarged distal tubules. This dilation was significantly blunted in BK‐β4−/− mice. These results illustrate that the BK plays a role in flow mediated tubular dilation, presumably by controlling the size and volume of IC lining the distal tubules. Supported by NIH/NIDDK grant R01DK71014 (SCS).
Triolein‐induced renal arterial vasoconstriction and its reversal in a rat model
Authors: Patel Sapna , Adler F. , McIff T. , Poisner A. , Herndon B. , Quinn T. , Molteni A.
Published: June 2021

Fat embolism syndrome (FES) is a respiratory process that may occur after bone fractures and is manifested by severe pulmonary vasoconstriction. It can be modeled in the rat by intravenous (iv) triolein. Our past work has shown the benefits of an ACE‐ inhibitor (captopril) or A2‐type 1 receptor blocker (losartan) on triolein treated pulmonary vessels. To investigate renal vascular effects of FES, we studied the histopathological changes in small arteries with and without treatment by these drugs (n=5/group). Rats received 0.2 ml triolein with necropsy at 24, 48, 96, or 264 hrs. Others received triolein and 50 mg/kg captopril or 10 mg/kg losartan with necropsy at 48 hrs (n=5/group). Saline controls were necropsied at 24 and 264 hrs (4‐5/group). At 400x, 3 vessels per high power field were digitally captured. The diameters of the lumen and medial wall were measured and statistically analyzed. Triolein vs control produced renal vasoconstriction up to 96 hr, p=0.03. Luminal dilation occurred at 48 hr in rats that received treatment with captopril or losartan. Lipid droplets were seen in the glomeruli at all times with the largest at 24 hr, progressively diminishing in size with time. Adventitial edema was evident in triolein‐dosed rats compared to controls (trend, p=0.09). This FES model produces renal vessel constriction that clears independently by 264 hrs. Treatment with captopril or losartan preserves vessel patency. This suggests the reversibility of FES‐related renal vascular changes, and, that the renin‐angiotensin system plays a role in its pathogenesis.

Funding: MA & EJ Asher Orthopedic Research Endowment.
Exercise as an antioxidant decreases age‐associated oxidative stress and protein kinase C activity and restores D1 receptor function in renal proximal tubules of old rats
Authors: George Liza E. , Lokhandwala Mustafa F. , Asghar Mohammad
Published: June 2021

Age‐associated oxidative stress (OS) via protein kinase C (PKC) activation causes D1 receptor G‐protein (D1R/G) uncoupling resulting in inability of dopamine to inhibit Na,K‐ATPase (NKA) in proximal tubules (PT) of old compared to adult Fisher 344 rats. Our hypothesis is that 3‐month exercise in old rats reduces OS, normalizes PKC activity and restores D1R/G coupling in PT. Biochemical, pharmacological and immunological studies were undertaken in PT of exercised and sedentary adult and old rats. We found that OS markers malondialdehyde and protein carbonyls in PT decreased in exercised rats. This was accompanied by an increase in antioxidant enzymes (AEs), superoxide dismutase and hemeoxygenase. Moreover, Nrf‐2 and NFκB in PT nuclei, transcription factors involved in AEs genes transcription, increased in exercised rats. Interestingly, PKC activity decreased and D1R agonist SKF38393‐ mediated 35S‐GTPγS binding and inhibition of NKA increased in PT of exercised rats. Also, D1R numbers and D1R proteins increased in PT of exercised rats. Furthermore, exercise decreased proteinuria and increased phosphaturia in rats. These results suggest beneficial effects of exercise in increasing anti‐oxidant defenses and improving kidney function in general and D1R function in particular in aging. Both Nrf‐2 and NFκB seem to be involved in this phenomenon. [Funding: NIH/NIA AG25056, AG29904].
2‐APB mediated effects on hypoxic calcium influx in rat carotid body glomus cells
Authors: Lahiri Sukhamay , Patel Gopal , Baby Santhosh , Roy Arijit
Published: June 2021

Hypoxia depolarizes the glomus cells of the carotid body and induces calcium rise which was reversed by 2‐APB, an inhibitor of store‐operated calcium channels situated in the endoplasmic reticulum (ER), whereas those by hypercapnia and high extracellular [K+] were not, although both hypoxia and hypercapnia depolarize the glomus cells. Hypoxia‐dependent calcium rise was triggered by a reduction of IP3‐receptors and 2‐APB significantly reversed this response. Further, mitochondrial inhibitions completely overlapped the 2‐APB inhibition, suggesting same mechanism of effects. Also, ATP elevated the [Ca2+]i in normoxia due to calcium influx and the release from the [Ca2+]i stores, both of which were inhibited by 2‐APB. This reaction, as revealed by 2‐APB, is unique and is due to possible involvement of phosphorylation mechanisms in hypoxia, and not operated in hypercapnia. Therefore we hypothesize that store‐operated calcium channels in the ER, where IP3 receptors reside, is reacted with 2‐APB and is critical for the inhibitory response. However, 2‐APB did not block hypercapnic‐ and high extracellular [K+]‐dependent calcium increase.Hypercapnia and high extracellular K+ have no access to this [Ca2+]i stores of ER, and hence have no effects.

Supported by NIH grant 43413.
Blockade of Na+/H+ exchange enhances phrenic burst frequency under control conditions but not in response to hypercapnia in arterially‐perfused adult rat
Authors: Jacobskind Jeremy , Solomon Irene C
Published: June 2021

Increased levels of CO2 (hypercapnic acidosis, HA) produce a marked increase in ventilation. Numerous studies have suggested that this hypercapnia‐induced respiratory excitation results from the fall in intracellular pH (pHi), and that Na+/H+ exchange (NHE) plays an important role in regulating pHi recovery during HA. In CO2 chemosensitive respiratory neurons, however, pHi recovery is not observed during HA. The current study was therefore undertaken to determine the role of NHE in respiratory activity under control conditions and in response to HA. To address this issue, we examined phrenic nerve discharge in response to increasing CO2 from 5% to 10% in an arterially‐perfused adult rat preparation (n=5) under control conditions and during NHE blockade using dimethyl‐amiloride hydrochloride (DMA, 283 µM). Under control conditions, HA increased burst frequency by ~34% (P<0.01) due to a marked reduction in TE (~25%, P<0.01) and a small decrease in TI (~15%, P=0.1). Perfusion with DMA elicited a robust increase in burst frequency of ~65% (P<0.001) due solely to a reduction in TE (P<0.001). Under these conditions, burst frequency was not further altered by HA (P=0.2) although TI was still decreased by ~15% (P<0.05). These results indicate that interference with pHi regulation is capable of enhancing phrenic burst frequency under control conditions but is ineffective in altering burst frequency during HA. Supported by NS045321
Effect of impact exercise on skeletal muscle and bone in OI model mice
Authors: Weber Bettina Anne , Ferreira J. Andries , Carleton Stephanie , McCambridge Amanda , Phillips Charlotte , Brown Marybeth
Published: June 2021

Osteogenesis imperfecta (OI) is a heritable connective tissue disorder in which mutations in type I procollagen genes result in bone deformity and fragility. The OI model mice (oim/+) are heterozygous for a null mutation in the COL1A2 gene of type I collagen and have reduced bone biomechanical integrity compared to wild type (WT) mice. Bone is inherently mechanosensitive, responding and adapting to its mechanical environment. Bone formation occurs in response to high mechanical loads; often changing its geometry to strengthen the skeleton. The largest physiological loads bones typically experience are from muscles. We postulate that even though the OI bone material is biomechanically weaker, the bone will respond to muscle loading, especially during pubertal growth to generate an inherently stronger bone. In a small pilot study, we characterized the muscle mass and contractile generating capacity of non‐exercised and exercised oim/+ mice. We found that therapeutic inclusion of impact exercise can lead to an increase in the contractile generating capacity of the gastrocnemius and soleus muscles as well as bone biomechanical integrity.

This knowledge is important in our ultimate understanding of muscle and bone function in OI model mice and ultimately, humans with this disease.
Epithelial sodium channel (ENaC) and pregnancy
Authors: West Crystal , Zhang Zheng , Ecker Goeffrey , Masilamani Shyama
Published: June 2021

A healthy pregnancy requires avid retention of sodium (Na) and plasma volume expansion (PVE) in order to provide adequate blood supply to the growing fetus. We performed in vivo and in vitro studies to examine the role of the amiloride‐sensitive Na channel (ENaC) in the Na retention and PVE of pregnancy. Late pregnant (LP) rats were in positive Na balance (LP= 2.45±0.11; virgin (V) = 1.15±0.26, mmol/day, p<0.05). A progressive increase in PV was seen in pregnant rats as shown by Evan's Blue dye analysis (mid pregnant (MP) =8.56 ± 0.59; LP=10.06 ± 0.46, ml p<0.05). In vivo ENaC activity was increased in LP vs V rats with a 2 fold greater net natriuretic response to ENaC blockade (benzamil). The protein abundance of a ENaC, the rate‐limiting subunit for channel formation, was increased in MP and LP rats (MP=167 ± 30; LP=168 ± 17, % of virgin rats, p<0.05). There were no differences in mRNA abundance of the ENaC subunits as demonstrated by real‐time RT‐PCR. Immunhistochemistry labeling of ENaC did not show increased cellular trafficking to the apical membrane of collecting duct cells. There was a strong tendency for benzamil to induce a greater PV loss in LP vs MP rats (MP= ‐0.840 ± 0.37; LP= ‐1.70 ± 0.40, ml p=0.06). These findings suggest that the pregnancy‐mediated increase in ENaC activity contributes to the Na retention and PVE of pregnancy. This research was supported by grants from the NHLBI (K22HL66994).
Biology of BRCA2‐DNA recombinase molecular interactions in African trypanosomes
Authors: Hall Mack , Misra Smita , Chaudhuri Gautam
Published: June 2021

BRCA2 is present in many eukaryotes including the protozoan parasite Trypanosoma brucei. One of the major functions of BRCA2 protein is to haul the DNA recombinase protein RAD51 from the cytosol to the nucleus. Binding of BRCA2 to RAD51 is mediated through the consensus BRC repeats of BRCA2. Our hypothesis is that the BRC motif of TbBRCA2 preferentially binds to select RAD51 paralogues to execute its biological function. We amplified the 15 BRC repeat cluster of TbBRCA2 (amino acids 25‐782). We similarly amplified the TbRAD51 (RAD51.1‐RAD51.5) gene ORFs. We cloned these amplicons in appropriate sites in the yeast 2‐hybrid vectors pGBKT7 and pGADT7. Yeast 2‐hybrid analysis revealed that the BRC repeats preferentially bind to TbRAD51.2 and TbRAD51.4. Yeast 2‐hybrid analysis also revealed that a single BRC repeat is enough to bind to these recombinases. Currently, we are investigating the effects of over expressing a single BRC repeat on the homologous recombination/repair process in the parasite.