bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of exercise training on cardiac mitochondrial apoptosis in ovariectomized rats
Authors: Hsu Chih‐Chao , Ou Hsiu‐Chung , Lee Shin‐Da
Published: June 2021

Cardiac apoptosis is found in ovariectomized rats; however, the effects of exercise training on cardiac apoptosis is still unclear in menopausal or bilateral oophorectomied women was available. The purpose of this study was to evaluate the therapeutic effects of exercise training on cardiac mitochondrial apoptotic pathways.

Methods

Eight sham‐operated rats and eight ovariectomized rats at 5–6 months of age were served as negative (sham) and positive (OVX) control. Additional 8 ovariectomized rats underwent treadmill running exercise 1 hour daily for 3 month. After exercise training or sedentary status, the excised hearts were measured by positive TUNEL assays, Hematoxylin‐eosin staining and Western Blotting.

Results

Exercise training significantly prevented the increasing of whole heart weight and OVX‐induced cardiac cell apoptosis. In addition, exercise training decrease the OVX‐induced tBid, Bad, Bax, Bak, cytochrome c overexpression, and caspase‐9 and caspase‐3 activation.

Conclusions

Exercise training suppressed ovariectomy‐induced cardiac apoptosis through inhibiting mitochondrial apoptotic pathways in rat models. The findings may provide one of possible therapeutic approaches for preventing cardiac apoptosis in menopausal or bilateral oophorectomied women.
A kinetic model of endothelial dysfunction from the NOS NO synthesis biochemical pathway
Authors: Kar Saptarshi , Kavdia Mahendra
Published: June 2021

Endothelial nitric oxide synthase (enos) under the influence of shear stress due to blood flow and agonist produces the vasorelaxant nitric oxide (NO). In hyperglycemia, hypertension and hypercholesterolemia, enos uncouples and produces superoxide instead of NO, leading to endothelial dysfunction. One of the key events in the process of endothelial dysfunction involves reduction of enos co‐factor tetrahydrobiopterin (BH4) with respect to dihydrobiopterin (BH2). However, it is not known how this reduction affects NO/superoxide synthesis by enos. In this study, a mathematical kinetic model for biochemical pathways of enos was developed that accounts for both the normal and endothelial dysfunctional state. The effect of the biopterin ratio (BH2:BH4) and kinetic parameters on NO/superoxide synthesis was analyzed. The results predicted that an increase in BH2:BH4 ratio leads to a significant decrease in production of NO and increase in production of superoxide and peroxynitrite. The model also provides a range of kinetic parameters for NOS biochemical pathways over which significant superoxide formation occurs. In addition, superoxide dismutase significantly affects the concentrations of NO, superoxide and peroxynitrite. These results have implications for understanding NOS uncoupling related oxidative stress. Supported by NIH R01 HL084337.
ET‐1‐induced vascular contraction is mediated by RhoA/Rho‐kinase pathway activation via O‐GlcNAcylation
Authors: Lima Victor Vitorino , Giachini Fernanda R , Webb R. Clinton , Tostes Rita C
Published: June 2021

O‐GlcNAc‐proteins and ET‐1‐production are increased in DOCA‐salt rats. We hypothesized that ET‐1 augments O‐GlcNAc levels and this modification contributes to increased vascular contractile responses via activation of RhoA/Rho‐kinase pathway. Incubation of smooth muscle cells with ET‐1 (0.1μM) increased O‐GlcNAc levels and decreased OGA, but not OGT expression, key enzymes in the O‐GlcNAc process. Overnight treatment of rat aortas (no endothelium) with ET‐1 increased contractile response to phenylephrine (PE). However, the effects of ET‐1 were not observed when vessels were previously transfected with an anti‐OGT antibody (chariot OGT) or after incubation with an OGT inhibitor (ST045849 100μM). Aortas from rats chronically infused with low doses of ET‐1 exhibited increased O‐GlcNAc‐proteins and displayed changes in reactivity similar to those induced by PugNAc, an inhibitor of OGA. Augmented aortic contractions to PE, after treatment with PugNAc or ET‐1 were abolished by the Rho kinase inhibitor Y‐27632. ET‐1 or PugNAc increased expression of RhoA and phosphorylated forms of MYPT‐1, a Rho kinase target. The effects of ET‐1 on RhoA and MYPT‐1 expression and activation were not observed after incubation with atrasentan or ST045849. Our data show that ET‐1 augments O‐GlcNAc levels and we speculate that this modification contributes to the vascular changes mediated by RhoA/Rho‐kinase pathway activation.
Protective effect of apigenin on neurons against oxygen‐glucose deprivation/reperfusion induced injury
Authors: Liu Shu‐Qin , Su Fang , Fang Lu‐Mei , Xia Qiang , Zhang Xiong
Published: June 2021

Apigenin, a flavone, has considerable neuroprotective effects by its anti‐oxidative mechanism. However, it is still unclear whether apigenin can protect neurons against oxygen‐glucose deprivation/reperfusion (OGD/R) induced injury. After two hours oxygen‐glucose deprivation and 24 hours reperfusion treatment in primary cultured hippocampal neurons, or ten minutes global cerebral ischemia and followed 24 hours reperfusion induced by Pulsinelli's 4‐vessel occlusion in male Sprague‐Dawley rats, the neuron viability, survival rate and apoptosis rate were evaluated by MTT assay, lactate dehydrogenase (LDH) leakage assay and hoechst staining, respectively. Treatment by OGD/R markedly reduced neuronal viability to 67.60±3.40% of control, increased LDH leakage rate from 17.00±6.25% to 50.49±9.00%, increased apoptosis rate from 7.60±3.56% to 28.90±8.42%. Application of apigenin (10–100 μmol·L−1) during OGD inhibited OGD/R induced neuron injury and apoptosis in a dose‐dependent manner. Besides, the activity of Na+/K+‐ATPase was significantly suppressed in both OGD/R group and global ischemia/reperfusion group. And application of apigenin during ischemia preserved the Na+/K+‐ATPase activity in the both groups. Furthermore, inhibition of Na+/K+‐ATPase with ouabain attenuated the protective effect afforded by apigenin. These data provide the evidence that apigenin has neuroprotective effect against OGD/R induced injury and the protective effect may be associated with its ability to improve Na+/K+‐ATPase activity after OGD/R.
The dietary flavonoid quercetin decreases neuroinflammation in a mouse model of Alzheimer's disease
Authors: JUNG SEUNG HO , Murphy E. Angela , McClellan Jamie L. , Carmichael Martin D. , Davis J. Mark
Published: June 2021

Inflammation plays a fundamental role in the pathogenesis of Alzheimer's disease (AD). The bioactive dietary flavonoid quercetin has received increased interest due to its anti‐inflammatory properties, but its effects on neuroinflammation in AD are not known. The purpose of this study was to determine the effects of dietary flavonoid quercetin on neuroinflammation in AD. Double transgenic female mice (B6C3‐Tg(APPswe, PSEN1dE9) 85Dbo/O) were randomly assigned to either quercetin (QAD) or placebo (PAD) treatments (n=10/group). C57BL/6 mice were used as age matched old (OWT) and young (YWT) controls. Quercetin (1% in mouse chow) (QAD) or placebo (OWT and PAD) was administered from 3–13 months. QAD, OWT and PAD were sacrificed at 13 months of age. YWT were sacrificed at 3 months of age. Brain tissue was collected and analyzed for TNF‐α, IL‐1β, IL‐6 and MCP‐1 mRNA expression using RT‐PCR. YWT showed significantly lower levels of TNF‐α, IL‐1β, IL‐6 and MCP‐1 versus all other groups (P<0.05). Furthermore, PAD showed significantly elevated levels of IL‐1β, IL‐6, and MCP‐1 versus OWT (P<0.05), but quercetin blunted this response for IL‐1β and MCP‐1 (P<0.05). Our data show that quercetin can partly block the increased gene expression of neuroinflammatory factors in AD. These findings may have important implications in AD as well as other neurodegenerative diseases in which inflammation plays a necessary role.
Acid‐sensing ion channel 1 is expressed in cerebral arteries and located near neuronal nitric oxide synthase
Authors: Lin Li‐Hsien , Talman William T.
Published: June 2021

Dramatic tissue acidosis is a common feature of ischemic brain. Responses to acid may be mediated through acid‐sensing ion channels (ASICs), a family of proteins that are activated by acid or modulate acid‐gated subunits. One ASICs subtype, ASIC type 1 (ASIC1), has been implicated in ischemic brain injury. Although ASIC1 is found in many areas of the brain, it is not known if it is expressed in cerebral arteries. Nitric oxide (NO) may modulate ASICs activity and upregulation of neuronal NO synthase (nNOS) in the parasympathetic innervation of cerebral vessels may protect the brain from ischemia. Therefore, we sought to examine the expression of ASIC1 as well as the anatomical relationship between ASIC1 and nNOS in rat cerebral arteries. Confocal images of immunofluorescent stained brain sections showed that ASIC1‐immunoreactivity (IR) was present in smooth muscle layers of the anterior cerebral artery (ACA), middle cerebral artery (MCA), posterior communicating artery (PCA) and basilar artery (BA). This layer was confirmed to be positive for alpha‐smooth muscle actin, a marker for smooth muscle. nNOS‐IR was noted in immediately adjacent endothelial and adventitial layers of ACA, MCA, PCA and BA. The close anatomical location of ASIC1 and nNOS suggests a possible interaction between ASIC1 and nNOS in regulating vasomotor tone and in mediating brain injury induced by ischemia. Support: NIH R01 HL59593 and R01 HL088090.
Chymase‐dependent generation of Angiotensin II from Angiotensin‐(1–12) in human atrial tissue
Authors: Ahmad Sarfaraz , Varagic Jasmina , Westwood Brian , Chappell Mark C. , Strawn William B. , Simmons Tony , Ferrario Carlos M.
Published: June 2021

We showed that angiotensin‐(1–12) [Ang‐(1–12)] is a non‐renin dependent alternate precursor for the generation of cardiac Ang peptides in rat tissue. In this study, we investigated the metabolism of Ang‐(1–12) by plasma membranes (PM) isolated from human atrial appendage tissue from patients undergoing open‐heart surgery. PM were incubated with highly purified 125I‐Ang‐(1–12) at 37° C for 1 h with or without a cocktail of peptidase inhibitors for ACE, ACE2, neprilysin (NEP) and chymase (50 μM each). 125I‐peptide fractions were identified in the supernatant by HPLC coupled to an inline γ‐detector. In the absence of inhibitor, 125I‐Ang‐(1–12) was converted into Ang I (10±7%), Ang II (62±14%), Ang‐(1–7) (7±3%) and other smaller peptides (<2%). In the presence of the all inhibitors, 90±4% of 125I‐Ang‐(1–12) remained intact. The relative contribution of selective inhibition of ACE, ACE2, NEP, and chymase enzyme showed that 125I‐Ang‐(1–12) was primarily converted into Ang II (77±24%) by chymase while its hydrolysis into Ang peptides by ACE, ACE2 and NEP was significantly lower. The activity of individual enzyme was then calculated based on the formation of Ang I, Ang II, Ang‐(1–7) peptides fractions. These results showed a very high chymase activity (54±22 fmol × min−1 × mg protein−1, N=5), followed by ACE2, and NEP (10.5±6 and 7.3±5.6 fmol × min−1 × mg protein−1, respectively) while ACE activity was negligible (2.8±2.1 fmol × min−1 × mg protein−1). In conclusion, we demonstrate for the first time in human cardiac tissue a dominant role of cardiac chymase in the formation of angiotensins peptides from Ang‐(1–12).
EFFECT OF CARVEDILOL AND LOSARTAN ON RENAL RESPONSES TO ANG II AND ADRENERGIC AGONISTS IN FRUCTOSE‐FED RAT
Authors: Abdulla Mohammed H , Sattar Munavvar A , Abdullah Nor A , Khan Md. Abdul Hye , Anand Swarup K. R. L. , Rathore Hassaan A , Ibraheem Zaid O , Johns Edward J
Published: June 2021

Effect of carvedilol and losartan was assessed on insulin sensitivity (IS) and renal responses to angiotensin II (Ang II) and adrenergic agonists in high fructose‐fed model of metabolic syndrome in Sprague‐Dawley rat. Rats were fed with 20% fructose in drinking water (FR) for 8 weeks. FR received either carvedilol (FRC) or losartan (FRL) at (10mg/kg/day po) for 3 weeks starting from week 5 of the experiment. At the end of treatment, an intravenous insulin glucose tolerance test and the renal responses to noradrenaline (NA), phenylephrine (PE), methoxamine (ME) and Ang II were determined. FR exhibited mild hyperglycemia, glucose intolerance and hypertension. In FR, the low IS, as assessed by glucose disappearance rate was restored to that of controls by carvedilol or losartan. The renal responses (%) to NA, PE, ME and Ang II were lower in FR compared to control (control vs FR, 21±5 vs 15±1; 42±6 vs 24±3; 32±7 vs 9±1; 20±5 vs 16±2) respectively. In FR, carvedilol or losartan treatment blunted responses to Ang II to 10±1% and 6±1% respectively. FRC had higher constrictor responses to ME compared to FR (19±3 vs 9±1%). Data demonstrated that Ang II plays an important role in regulating renal haemodynamics in FR and, indicated an interaction between Ang II and adrenergic neurotransmission in these rats. Moreover, carvedilol or losartan treatment improved IS in FR.
Vasopressin Triggers Phosphorylation of Na‐K‐2Cl Cotransporter (NKCC1) in Rat Inner Medullary Collecting Duct (IMCD)
Authors: Chou C‐L , Kassai E.M. , Knepper M.A.
Published: June 2021

Vasopressin‐induced cell swelling in rat IMCD is dependent on the cotransporter NKCC1. NKCC1 is regulated by phosphorylation at Thr203 and Thr208 in other cell types. Here we used R5, a previously characterized antibody recognizing the pThr203/pThr208 double‐phospho‐form of NKCC1, to study vasopressin's effects in rat IMCD suspensions. The V2R‐selective vasopressin analog dDAVP (1 nM) significantly increased the abundance of phosphorylated NKCC1 (168±15%, n=4), an effect mimicked by cAMP analog CPT‐cAMP and inhibited by protein kinase A (PKA) inhibitor H‐89. Immunoblotting using the R5 antibody cross‐preadsorbed with synthetic mono‐phosphopeptides (pThr203 or pThr208) demonstrated that dDAVP increases phosphorylation at Thr203 but not Thr208. Inhibitors of calmodulin (W7) or myosin light chain kinase (ML‐7) did not prevent NKCC1 phosphorylation. Incubating a nonphosphorylated synthetic peptide spanning Thr203 and Thr208 with purified PKA catalytic subunit failed to phosphorylate the peptide, despite phosphorylation of positive‐control peptide. These results show that dDAVP triggers phosphorylation of NKCC1 at Thr203 in native rat IMCD cells and suggest that the effect is indirectly dependent on PKA.
Ion transport homeostasis in Alpha‐1 AMPK KO mice
Authors: Lazo Yoskaly , Baile Goretti , Martínez Laura , Bernal Marisa , Viollet Benoit , Gimenez Ignacio
Published: June 2021

AMP‐activated kinase (AMPK) regulates cell energy balance by modulating ATP synthesis and consumption. In epithelia, AMPK interacts with and regulate ion transport systems, including CFTR, ENaC and NKCC. We studied renal function and measured blood pressure in mice with genetic deletion of the alpha‐1 catalytic subunit of AMPK (a1KO mice; C57Bl6J background). When fed a standard diet (0.3%NaCl) a1KO mice showed mild antidiuresis (WT=0.053 vs. KO=0.038 mL/d/g bw). No significant differences in glomerular filtration rate, sodium or potassium handling were found. When fed low Na+ (0.02%) or high salt (4%NaCl) diets, a1KO mice achieved ion and water balance. Lack of aversion for the high salt diet in a1KO mice suggested increased sodium appetite. Aldosterone urinary excretion was consistently higher in a1KO mice (WT=0.028 vs. KO=0.037 ng/d/g bw). Telemetric 24h blood pressure records showed significantly lower blood pressure in a1KO mice, especially nocturnal diastolic pressure (WT=93.5 vs. KO=86.1 mmHg). We found that a1KO mice lose water and sodium in their feces compared to wild‐type mice. In the kidney, we observed up‐regulation of the alpha‐2 catalytic subunit, which may provide functional compensation for the loss of alpha‐1 activity. Thus, kidneys from a1KO mice apparently have normal function and may be actually compensating for extra renal water and ion losses.

MICIIN Spain BFU2007/62119
Disease‐causing mutation R1185C alters phosphorylation of WNK4 by SGK1 via a calmodulin binding site
Authors: Na Tao , Zhang Wei , Jiang Yi , Peng Ji‐Bin
Published: June 2021

WNK4 is a protein serine/threonine kinase, which is linked to familial hyperkalemic hypertension (FHH). In this study, we evaluated 2 putative calmodulin (CaM) binding sites in WNK4 (amino‐acids 502–525 and 1175–1194) as predicted by a network based CaM target database. The first CaM binding site is close to a cluster of FHH‐causing mutations (E562K, D564A and Q565E) whereas the second contains a FHH‐causing mutation (R1185C) and S1190, a SGK1 phosphorylation site. The two GST‐fusion proteins containing CaM binding sites of WNK4, GST‐WNK4a (492–552) and GST‐WNK4b (1163–1212), exhibited CaM binding ability using pull down assay with CaM‐agarose. Reducing a positive charge in R1185C mutation and introducing a negative charge in phospho‐mimicking S1190D mutation resulted in a reduction of CaM binding ability in GST‐WNK4b. On the other hand, in vitro phosphorylation of GST‐WNK4b by SGK1 was inhibited by CaM in a Ca2+ dependent manner and it was less Ca2+dependent for the R1185C mutant. These results indicate Ca2+‐CaM is involved in the regulation of WNK4 and this regulation may be altered under FHH condition.
SGLT2 mediates glucose reabsorption in the early proximal tubule
Authors: Vallon Volker , Schroth Jana , Platt Kenneth A , Whaley Jean , Thomson Scott , Rieg Timo
Published: June 2021

To elucidate the role of the Na‐glucose transporter SGLT2, we performed in vivo clearance and micropuncture studies under inactin/ketamine anesthesia in gene‐targeted mice lacking Sglt2 (−/−; generated by replacing a 2419 bp region containing exons 1–5 of Slc5a2 by homologous recombination). Blood pressure, glomerular filtration rate and blood glucose levels were not different between Sglt2−/− and wild‐type (WT) mice (101±4 vs. 107±7 mmHg; 6.5±0.7 vs. 6.8±0.6 μl/min/g bw; 8.0±0.8 vs. 7.6±0.6 mM; n=6; NS). Fractional renal reabsorption of glucose was lower in Sglt2−/− mice compared to WT (35±8 vs. 99.7±0.1%; P<0.0001) and varied inversely with the amount of filtered glucose in Sglt2−/− mice (between 60 and 10%). Free‐flow collections of tubular fluid were performed along accessible proximal tubules at the kidney surface to establish a profile for fractional reabsorption of glucose versus fluid (FR‐fluid, FR‐glucose). We found that for early proximal collections (FR‐fluid <40%) 79±7% of the filtered glucose were reabsorbed in WT mice whereas mean net reabsorption of glucose was zero (−0.1±8.0%) in Sglt2−/− mice (P<0.0001). For late proximal collections (FR‐fluid >=40%) FR‐glucose was 93±1% in WT and 22±7% in Sglt2−/− mice (P<0.0001). In conclusion, the results demonstrate that SGLT2 mediates glucose reabsorption in the early proximal tubule and is a major determinant of glucose reabsorption in the kidney.
Activation of a latent nuclear localization signal in the amino‐terminus of γ‐ENaC initiates feedback regulation of channel activity
Authors: Mironova Elena , Stockand James
Published: June 2021

Proteolytic enzymes cleave ENaC at several positions releasing, in part, the NH2 terminus of the γ‐subunit. Cleavage increases ENaC activity by increasing open probability; however, the role of polypeptides cleaved from the channel core remains unclear. We find that the cytosolic NH2‐terminus of γ‐ENaC when expressed as an eGFP‐fusion protein unexpectedly targets to the nucleus being particularly strong in nucleoli. In contrast, the cytosolic COOH‐terminus targets to the cytoplasm and plasma membrane in a manner similar to full‐length subunits. Targeting of the cytosolic NH2‐terminus of γ‐ENaC to the nucleus has functional consequences for co‐expression of eGFP‐fusion proteins containing this segment of the channel, but not the COOH‐terminus, decrease ENaC activity in a dose‐dependant manner. The mechanism of this negative regulation is associated with a decrease in the functional half‐life of ENaC at the plasma membrane. Inspection of the primary amino acid sequence of γ‐ENaC reveals possible nuclear localization signals (NLS) conserved at the extreme NH2‐ terminus and just preceding the first transmembrane domain. Disruption of the putative NLS preceding the first transmembrane domain in γ‐ENaC, but not that at the extreme NH2‐terminus, abolishes both targeting to the nucleus and negative regulation of ENaC activity. These findings are consistent with the release of the NH2‐terminus of γ‐ENaC following cleavage being functionally important for signaling to the nucleus in a manner similar to Notch signaling and release of the cytosolic COOH‐terminal tail of polycystin‐1.
Differential regulation of NaPi2a and NaPi2c by parathyroid hormone
Authors: Okamura Kayo , Giral Hector , Caldas Yupanqui , Sutherland Eileen , Millard Andrew , Levi Moshe , Blaine Judith
Published: June 2021

Parathyroid hormone (PTH) is a critical regulator of renal phosphate (Pi) homeostasis. In the kidney Pi reabsorption is regulated by the sodium phosphate cotransporters NaPi2a and NaPi2c. It has previously been shown that PTH‐induced removal of NaPi2c from the renal proximal tubule brush border membrane (BBM) is approximately 3 times slower than that of NaPi2a. The reason for this is unknown. Using a novel application of total internal reflection fluorescence microscopy (TIR‐FM) to the apical membrane of renal proximal tubule cells we have found that a dynamic actin cytoskeleton and the unconventional myosin motor myosin VI are required for PTH induced removal of NaPi2a from the BBM. Here we demonstrate that a dynamic actin cytoskeleton is required for NaPi2c removal from the BBM in response to PTH. In addition we investigate the role of myosin VI and membrane lipid dynamics in PTH‐induced trafficking of NaPi2c. Differences in the use of motor proteins or clustering of the cotransporters within the BBM may account for the differences in the kinetics of the response of NaPi2a and NaPi2c to PTH.

Supported by NIH K08 DK080989‐01A1.
Role of SPAK binding sites in SLC12 cotransporters basal activity and regulation by WNK3
Authors: Pacheco‐Alvarez Diana , De‐los‐Heros Paola , Ponce‐Coria Jose , Vazquez Norma , Ortal‐Vite Penelope , Rodriguez‐Lobato Erika , Gamba Gerardo
Published: June 2021

With‐no‐lysine kinase 3 (WNK3) is a powerful activator of NKCC1/2 and NCC while is an inhibitor of KCC1 ‐ KCC4. WNKs lie upstream of the STE‐20 kinases SPAK/OSR1. It is known that interaction with SPAK occurs through the SPAK‐binding motif, RFx(V/I). WNK3 contains three such motifs, NCC and NKCC2 one; while KCC4 lacks this motif. In the present study we examined the relevance of SPAK binding sites for NCC, NKCC2, and KCC4 basal activity and regulation by WNK3. The sites of WNK3 (F242A, F745A, or F1337A; one at a time), NCC (F19A), and NKCC2 (F17A) were eliminated. Basal activity and effect of WNK3 was assessed by measuring the tracer 22Na+ or 86Rb+uptake in Xenopus oocytes injected with wild type or mutant cRNAs. Basal activity of NCC‐F19A and NKCC2‐F17A was reduced by ~50%. However, the response to WNK3 and to intracellular chloride depletion was still present. The effect of WNK3 toward NKCC2, but not NCC or KCC4, was eliminated by WNK3‐F1337A. Interestingly, the WNK3 effect towards NCC or KCC4, but not NKCC2, was prevented in WNK3‐F242A. Biochemical analysis revealed that catalytic activity is preserved in both, WNK3‐F242A and WNK3‐F1337A. Thus, we conclude that basal activity of NCC and NKCC2, but not KCC4 requires the presence of an SPAK binding site on the cotransporter. Modulation of NCC, NKCC2, and KCC4 by WNK3 requires SPAK binding sites in the kinase, however, different sites are used for regulation of NKCC2 in contrast to NCC or KCC4.
Isoforms of Na, K‐ATPase in epithelial and endothelial barriers in the equine hoof
Authors: Asplin Katie , Mobasheri Ali
Published: June 2021

Na, K‐ATPase is essential for the maintenance of intracellular sodium and potassium concentrations, thereby maintaining cell volume and electrochemical gradients. Currently, the presence and distribution of sodium pumps has not been characterised in equine hoof tissues. The aim of this study was to characterise the distribution of Na, K‐ATPase pump subunit isoforms within hoof lamellar keratinocytes and small vessels, as well as the digital artery supplying the hoof. Using various isoform specific monoclonal and polyclonal antibodies, we have examined the cellular localization of Na, K‐ATPase α1, α2, α3, β1, and β2, subunits within normal equine hoof tissues using immunohistochemistry. Moderate immunoreactivity for Na, K‐ATPase α1, α2, β1 and weaker staining for Na,K‐ATPase α3 was observed in the cytoplasm and plasma membrane of lamellar keratinocytes. All antibodies except α1 and α3 also demonstrated moderate to strong staining in the smooth muscle and endothelium of small vessels within the dermal hoof tissue. α3, β1, and β2 moderately stained the endothelium, smooth muscle and smaller vessels of the digital artery, while α2 stained only the smooth muscle and small vessels of the digital artery, with no staining observed for α1. These results confirm that Na, K‐ATPase isozymes are present in the equine hoof and may play a functional role in maintaining electrochemical gradients across its epithelial and endothelial barriers. This research was supported by the University of Nottingham.
TMEM16A contributes to Ca2+‐activated Cl− secretion in the renal collecting duct
Authors: Svenningsen Per
Published: June 2021

Extracellular ATP has been shown to activate purinergic signaling in epithelial cells and thereby regulate electrogenic ion transport. In the mouse renal collecting duct cell line M‐1, we found that extracellular ATP (100 nM) elicited Ca2+‐activated Cl− secretion, which was blocked with the purinergic P2‐receptor antagonist suramin and the Cl− inhibitor DPC; however, the channel responsible for the Cl− is unknown. Recently, TMEM16a has been identified as a Ca2+‐activated Cl− channel. We found that TMEM16a was expressed in M‐1 cells at the mRNA and protein level. We generated a M‐1 cell line with stable expression of a Dox‐inducible shRNA against TMEM16a. Induction of shRNA expression led to a knockdown of TMEM16a mRNA and protein. Knockdown of TMEM16a expression had no significant effect on ATP induced Ca2+ in M‐1 cells; however, the Ca2+‐activated Cl− secretion was significantly inhibited. Moreover, we found TMEM16a to be expressed at the apical membrane in the collecting duct of human and mouse kidneys. Taken together, the data suggest that TMEM16a is expressed in the renal collecting duct and is involved in Ca2+‐activated Cl− secretion in human and mouse kidneys.
Angiotensin II – PKC‐mediated translocation of the BK‐β1 subunit to the basolateral membrane of principal cells
Authors: Liu Liping , Grimm Paul R , Holtzclaw John D , Irsik Debra L , Sansom Steven C
Published: June 2021

The pore forming subunit of the large Ca‐activated K channel, BK‐α, with the subunit BKβ1, localizes on the basolateral membrane (BLM) of CCD principal cells (PC) when mice consume a Na‐deficient diet. Because Kcnmb1−/− exhibit profound Na and volume depletion when treated with a Na‐deficient diet, the localization of BK‐α/β1 on the BLM can serve to increase the driving force for Na reabsorption. We investigated the hypothesis that ANGII mediates the BKβ1 incorporation into the BLM of PCs via AT1‐PKC signaling. When cultured on a 3‐dimensional system, MDCK C7‐cells (C7), models of PC, formed cysts with the plasma membrane polarized into distinct apical and BLM membranes, which face the external and internal environments, respectively. Immunohistochemical analysis revealed ANGII (100 nM)‐induced expression of BKβ1 on the BLM; this effect was inhibited by the AT‐1 receptor blocker, Losartan, and the PKC inhibitor, Calphostin C, but not by the AT‐2 blocker, PD123319. C7 were then cultured on T‐75 flasks and membrane and cytosolic proteins were isolated for biotinylation assays and western blots. Total membrane and cytosolic BK‐β1 did not change with ANGII; however, biotinylated BK‐β1 (apical membrane) significantly decreased. We conclude that ANGII, via AT1 and PKC signaling, may mediate the translocation of BKβ1 to the BLM of PC in Na‐deficient conditions.
Effect of AQP1 knock out on mouse exercise tolerance
Authors: Xu Lumei , Zhou Yuehan , Courtney Nicholas A , Radford Thomas S , Boron Walter F
Published: June 2021

The water channel Aquaporin 1 (AQP1) is widely expressed throughout the body. Our lab demonstrated that AQP1 also can act as a CO2 channel when heterologously expressed in oocytes or when studied in the intact proximal tubule. We hypothesized that AQP1 plays a physiologically important role in O2 transport. Because AQP1 is present at high levels in erythrocytes and the pulmonary capillary endothelium, we compared voluntary wheel running over a 24‐h period in AQP1‐null vs wild‐type mice under conditions of hypoxia (ambient [O2] = 16%), normoxia (21%), and hyperoxia (40%). For wild‐type mice, the distances run were 16%: 9.2 ± 0.5 km (n = 28), 21%: 10.7 ± 0.5 km (n = 37), and 40%: 12.1 ± 0.6 km (n = 21). For AQP1‐null mice, the distances were 16%: 4.7 ± 0.5 km (n = 8), 21%: 6.2 ± 0.6 km (n = 13), and 40%: 6.9 ± 0.6 km (n = 12). We performed a linear‐regression analysis of distance run as a function of AQP1 status and [O2], treating [O2] categorically in reference to 21% O2. The AQP1 knockout reduced the distance run by 4.7 ± 0.5 km (p < 0.001), adjusting for [O2]. Compared to 21% O2, reducing [O2] to 16% reduced the distance run by 1.6 ± 0.6 km (p = 0.01), whereas increasing [O2] to 40% increased the distance run by 1.2 ± 0.6 km (p = 0.04), adjusting for AQP1 status. Thus, AQP1‐null mice have a major defect in voluntary exercise tolerance, consistent with the hypothesis that AQP1 plays an important physiological role in O2 transport across plasma membranes.
Placenta copper transport proteins in preeclampsia
Authors: Iseminger Christine V , Anderson Cindy M , Johnson W Thomas
Published: June 2021

Placental insufficiency underlying preeclampsia (PE) is associated with impaired placental angiogenesis. As copper (Cu) is essential to angiogenesis, we investigated differences in the expression of placental Cu transporters Menkes (ATP7A), Wilsons (ATP7B) and the Cu chaperone (CCS) for superoxide dismutase (SOD) in normal pregnancy (NP) and pregnancy complicated by PE. Cu content was determined by atomic absorption spectroscopy. Women pregnant with their first child were enrolled in the first trimester. Serum samples were collected to determine copper status. Dietary and supplemental Cu intakes were determined using a food frequency questionnaire. Placenta samples were collected and frozen for analysis of protein expression by Western blot. In the first trimester, maternal Cu intake (dietary and supplemental) was adequate. There were no significant differences in serum Cu, ceruloplasmin or SOD3 between NP and PE groups. There were no significant differences in placental Cu content between groups. Expression of ATP7A was significantly decreased in PE placentas compared to NP (p<0.05). There was no significant difference in the expression of ATP7B or CCS between groups. Our findings indicate that while maternal Cu status was adequate based on serum markers, impaired placental Cu trafficking may be associated with the development of preeclampsia. Funded by UND New Faculty Scholar Award, APS and USDA/ARS
Going to extremities: is variation in human limb length and proportions a paradox?
Authors: Auerbach Benjamin Miller
Published: June 2021

Group comparisons indicate thermoregulation, nutrition, and locomotor efficiency as factors affecting human limb length and proportional variation. Humans from warmer climates have longer limbs relative to torso length and higher intralimb intermembral indices, likely reflecting thermoregulatory necessity; distal segments have higher surface area‐to‐mass relative than proximal segments, and longer limbs dissipate more heat. Despite this pattern, paradoxically, limb proportions and limb lengths were shown to not correlate in previous research. Subsequent analysis concluded that limb proportions are adaptively constrained and do not closely reflect limb elongation.

Using a global sample of 3680 pre‐industrial skeletons, this study reexamines this incongruity. Coefficients of variation show that variance in proximal relative to distal elements is not universal among all human groups, though distal elements generally have greater variance. Scaling analyses indicate longer limbs have disproportionately longer distal elements and disproportionately shorter proximal elements, following thermoregulatory expectations. Different allometric relationships for proximate and distal elements may obscure these patterns when their lengths are expressed as ratios. Variation in these patterns among groups, and the effects of mechanics and nutrition, are further considered.
β‐adrenergic activation of Cl secretion and K secretion in guinea pig distal colonic mucosa proceeds via separate receptor complexes
Authors: Halm Susan , Zhang Jin , Halm Dan
Published: June 2021

Epinephrine (epi) stimulated ion secretion in isolated mucosa from guinea pig distal colon, measured as short‐circuit current. Epi responses included transient Cl (0~5 min) and sustained K secretion (sec). ICI‐118551 (β2‐selective antagonist) inhibited Cl sec (Kd = 2.7 nM), even though inhibition of K sec required combined β1/β2 antagonism. Co‐immunoprecipitation of β1 and β2 adrenergic receptors (βAdrR) provided support for this combined action. Both β1AdrR and β2AdrR also co‐IPed with arrestin‐2, suggesting an alternate signaling path. Whereas dideoxyadenosine (ddAdo; inhibits transmembrane adenylyl cyclase, tmAC) inhibited only Cl sec, KH7 (inhibits soluble adenylyl cyclase, sAC) inhibited only K sec. Consistent with bicarbonate dependence for sAC, sustained K sec was 42% smaller in HEPES buffered bathing media (CO2/HCO3 free), without altering transient Cl sec. Immunoblot for sAC indicated a presence of 54 and 74 kDa bands, consistent with sAC isoforms found in other tissues. ddAdo suppressed the biphasic epi activation of cAMP levels; KH7 left the early transient cAMP unaltered but lowered sustained levels; early cAMP transients remained in the absence of bicarbonate with sustained levels lowered. Thus, β2AdrR activation produced Cl sec via a transient cAMP elevation from tmAC, while β1AdrR/β2AdrR activation produced K sec via a sustained cAMP from sAC that was bicarbonate dependent. [NIH DK65845]
Renal epithelial Na channel protein expression is reduced in H,K‐ATPase knockout mice
Authors: Greenlee Megan Michelle , Lynch I. Jeanette , Gumz Michelle L. , Cain Brian D. , Wingo Charles S.
Published: June 2021

The importance of the epithelial Na+ channel (ENaC) to Na+ reabsorption is highlighted by genetic gain of function mutations in ENaC that lead to severe hypertension (Liddle's Syndrome). Spicer et al (Am J Phys, 2001) demonstrated that colonic ENaC activity was reduced in HKα2−/− mice, suggesting that H+,K+‐ATPases are required for complete ENaC function. Given the importance of functional ENaC to renal Na+ transport, we investigated whether double knockout of H+,K+‐ATPase α subunits in mice (HKα1,2−/−) affected ENaC expression. Real time quantitative PCR was used to evaluate the mRNA expression of α‐, β‐, and γ‐ENaC subunits in cortex and medulla from wild type (WT) and HKα1,2−/− mice. Cortical α‐ and β‐ENaC mRNA expression was slightly decreased (~20–25%) in HKα1,2−/− mice compared to WT (p=0.10 and 0.08, respectively). Importantly, we observed a 45% reduction in medullary α‐ENaC protein expression (n=3, p<0.05) in HKα1,2−/− mice vs WT, as determined by densitometry of immunoblots. In summary, these data suggest that renal H+,K+‐ATPases are needed for appropriate expression of ENaC subunits and are important for renal Na+ adaptation.

Studies were funded by NIH RO1‐DK‐049750 to C.S.W and 5T32DK007518‐22 to M.M.G.
Identification of epithelial Na+ channel (ENaC) inter‐subunit Cl‐ coordinating residues suggests a trimeric alpha, gamma, beta channel architecture
Authors: Collier Daniel M , Snyder Peter M
Published: June 2021

The extracellular domain of the epithelial Na+ channel (ENaC) is exposed to a wide range of anion concentrations in the kidney. We have previously demonstrated that extracellular Cl− inhibits ENaC activity. To identify residues involved in Cl− inhibition, we mutated residues in the extracellular domain of α‐, β‐ and γENaC that are homologous to the Cl− binding site in ASIC1 and tested the effect of Cl− on the activity of ENaC expressed in Xenopus oocytes. We identified two functionally significant Cl− sites in αβγENaC. One site is formed by residues in the thumb domain of αENaC and palm domain of βENaC. Mutation of residues in this domain decreased Cl− inhibition and decreased Na+ self‐inhibition. The second site is formed by residues at the interface of the thumb domain of βENaC and the palm domain of γENaC. Mutation of these residues also decreased Cl− inhibition, yet had no effect on Na+ self‐inhibition. In contrast, mutations in the thumb domain of γENaC and palm of αENaC had minimal effect on Cl− inhibition or Na+ self‐inhibition. The data demonstrate that Cl− inhibits ENaC activity by two discrete Na+ dependent and Na+ independent mechanisms. Furthermore, our results suggest that ENaC adopts an αγβ rather than αβγ confirmation, providing additional insight into the functional significance of α, β and γENaC subunit interactions.

PMS is supported by NIH HL072256
Role of P97 protein in ENaC recycling
Authors: Malik Bela , Mathur Nithar M , Yue Qiang , Yue Guichun , Duke Billie J , Al‐Khalili Otor , Eaton Douglas C
Published: June 2021

Functional Epithelial Sodium Channels, ENaC, consist of α, β, and γ subunits. A major way to regulate ENaC activity is by controlling the number of ENaC at the apical membrane by either removing ENaC from or inserting ENaC into the cell's apical membrane. Apical ENaC number can be altered by changing the rate of recycling or degradation of internalized ENaC. ENaC, a putative trimer, appears to be internalized as an ubiquitinated multimer. Some internalized ENaC are de‐ubiquitinated by endosomal enzymes and returned to the apical membrane as functional ENaC, but some are finally degraded by the proteasome, an organelle that recognizes and degrades monomeric proteins. Multimeric ENaC must therefore be disassembled before degradation. Disassembly of a multimeric protein requires significant energy that can be provided by a novel protein ATPase, P97. P97 also chaperones individual subunits of ubiquitinated proteins to the proteasome. We elucidated the role of P97 in ENaC recycling by confirming physical interaction between P97 and ENaC subunits by co‐immunoprecipitation. The impact of P97 on ENaC was determined in A6 cells over‐expressing a putative dominant negative P97. ENaC subunit amounts in whole‐cell lysates increased consistent with subunit degradation in these cells.
WNK4 inhibits ENaC activity and reduces γ ENaC subunit expression, but has no effect on β ENaC expression
Authors: Yu Ling , Cai Hui , Duke B J , Otor Al‐Khalili , Eaton Douglas
Published: June 2021

Recently, a serine‐threonine protein kinase WNK4 expressed in the distal nephron has been shown to inhibit ENaC activity; however, the mechanism of this inhibition is unclear. We examined the effect of expressing WNK4 on endogenous ENaC in A6 cells. Single channel recording shows that WNK4 inhibits ENaC activity by reducing apical channel number rather than by changing open probability. Western blots of total as well as apical biotin‐labeled ENaC β and γ subunits show that the γ subunit but not the β subunit is reduced in WNK4 expressing cells. Amiloride‐sensitive trans‐epithelial Na+ current (Isc) of WNK4 expressing cells treated with forskolin increases by the same percentage amount from baseline as forskolin‐treated, sham transfected cells; however, the maximum current amplitude of WNK4 expressing cells is about half of the control likely due to the lower basal ENaC activity. The time course of Isc after forskolin application and removal are the same in control and WNK4 expressing cells, which implies that WNK4 has no effect on PKA stimulated ENaC recycling. Interestingly, even though forskolin increased Isc of control and WNK4 expressing cells, apically expressed β ENaC does not change in either group of cells. Our data imply that WNK4 may regulate the three ENaC subunits in non‐coordinated way, which is consistent with other's reports that the apical residency of ENaC is non‐coordinately regulated.
Mapping a limited inhibitory domain derived from the gamma subunit of ENaC
Authors: Passero Christopher J , Carattino Marcelo D , Myerburg Michael M , Hughey Rebecca P , Kleyman Thomas R
Published: June 2021

Proteases activate the epithelial sodium channel (ENaC) by liberating inhibitory domains from the extracellular regions of its alpha and gamma subunits. Furin and prostasin activate ENaC by cleaving the gamma subunit at sites flanking a 43‐amino acid inhibitory tract. We generated serial deletions within the gamma inhibitory tract of mutant channels lacking protease cleavage sites. Deletion of selected tracts in the gamma subunit of channels lacking protease cleavages sites restored channels activity. Using a library of synthetic peptides corresponding to the area identified by the deletion mutations, the minimal length for inhibition was narrowed down to an 11‐amino acid tract. A synthetic peptide corresponding to this tract inhibits endogenous channels expressed in mouse cortical collecting duct cells and human airway epithelia with an IC50 in the low micromolar range. Further studies using synthetic peptides with single amino acid substitutions determined structural requirements for inhibition. This work was supported by a grant from the National Institutes of Health (DK065161 and F32‐DK080574).
Regulation of ENaC by 1,2‐propanediol, di‐2‐ethylhexyl phthalate and texanol
Authors: Han Dongyun , Molina Raul , Idell Steven , Ji Honglong
Published: June 2021

The volatile organic compounds are potential causes of impaired respiratory system, such as shortness of breath, asthma, wheeze, etc. However, the molecular mechanisms of these symptoms caused by compounds in indoor air remain to be elucidated. In this study, we investigated the effects of 1,2‐propanediol, di‐2‐ethylhexyl phthalate (DEHP) and texanol on the epithelial Na+ channel (ENaC) activity in confluent H441 monolayers, a human bronchoalveolar epithelial cell line and in Xenopus oocytes heterologously expressing human αβγ ENaC. The Ussing chamber studies showed that propanediol (1mM) acutely increased amiloride‐sensitive (AS) short‐circuit current (Isc) levels by 13.7% when it was applied to the apical compartment. However, DEHP (1mM) and texanol (1mM) resulted in a slightly drop of AS Isc levels from 9.2 to 8.7 μA/cm2 and 10.7 to 9.0 μA/cm2, respectively (N=1). Chronically, these three compounds, when applied to the culture medium, increased the equivalent Isc levels in a time‐dependent manner in H441 monolayers. On day 3, Isc levels were increased by propanediol, DEHP, and texanol by 100.3%, 106.7% and 168.0%, respectively. These chronic effects were further corroborated with similar AS Isc in H441 cells using Ussing chamber setup. Taken together, our results suggest that propanediol, DEHP, and texanol lead to elevated ENaC activity in airway, which tightly correlates with airway surface fluid layer. Dehydrated airway may contribute to asthmatic dysfunction followed exposure to these noxious indoor air pollutants. Supported by HL087017 and HL095435.
GABA receptors modulate ventilator‐induced lung injury
Authors: Chintagari Narendranath Reddy , Liu Lin
Published: June 2021

Mechanical ventilators are increasingly used in critical care units. However, they can cause lung injury including pulmonary edema. Our previous studies indicated that γ‐amino butyric acid receptors (GABAR) were involved in alveolar fluid homeostasis. The present study investigated the role of GABAR in ventilator‐induced lung injury. Adult female Sprague‐Dawley rats were subjected to high tidal volume ventilation (HVT) of 40 ml/kg body weight for 1 hour. HVT resulted in lung injury as indicated by increase in total protein in bronchoalveolar fluid, wet‐dry ratio (indication of pulmonary edema) and Evans Blue dye extravasation (indication of vascular damage). Intratracheal administration of GABA (500 μM) prior to ventilation significantly reduced the wet‐dry ratio. GABA‐mediated reduction was effectively blocked by GABA‐A receptor antagonist, bicuculline. The GABA‐mediated effect was not due to the vascular damage, since there were no changes in Evans Blue dye extravasation. However, the decrease in alveolar fluid clearance by HVT was partly prevented by GABA, which was blocked by bicuculline. These results suggest that GABA reduces HVT‐induced pulmonary edema via its effects on alveolar fluid clearance.
Heteropore Characteristics of Type I‐ and Type II‐Like Rat Alveolar Epithelial Cell Monolayers
Authors: KIM YONG HO , BOROK ZEA , CRANDALL EDWARD D , KIM KWANG‐JIN
Published: June 2021

Utilizing primary rat alveolar epithelial cell monolayers (RAECM) grown in the absence (type I cell‐like phenotype, RAECM‐I) or presence (type II cell‐like phenotype, RAECM‐II) of keratinocyte growth factor, we estimated apparent permeability coefficients (Papp) for nine hydrophilic solutes (molecular radii from 0.14 to 2.29 nm) in the apical‐to‐basolateral direction. Under the assumption that passive restricted diffusion of these solutes takes place via equivalent water‐filled pores, pore sizes based on these Papp using Renkin analysis were determined. Results showed that both RAECM‐I and ‐II can best be characterized by two populations of pores. Radii of small and large pores of 0.9 and 4.5 nm in RAECM‐I, and 1.1 and 9.7 nm in RAECM‐II, were estimated. About 98% and 2% of total pore area can be ascribed to small and large pore areas, respectively, in both RAECM‐I and ‐II. However, the number of small pores in RAECM‐II (7.5×109/cm2) was 7‐fold greater than that in RAECM‐I, while the number of large pores (1.4×106/cm2) was about the same in both RAECM‐I and ‐II. These data suggest that passive diffusion of hydrophilic solutes is dependent on the size, distribution and number of equivalent heteropores across alveolar epithelium and the homotypic/heterotypic junctions between alveolar epithelial cells. (Funded by: NIH; Hastings & Whittier Foundations.)
RAGE and ERM proteins in EMT of human alveolar epithelial cells
Authors: Buckley Stephen T , Medina Carlos , Ehrhardt Carsten
Published: June 2021

The receptor for advanced glycation end products (RAGE) purportedly plays an important role in lung homoeostasis, whilst ezrin/radixin/moesin (ERM) proteins serve as important crosslinkers between the plasma membrane and cytoskeleton. Here, we investigated the role of RAGE and ERM proteins in cytokine‐induced epithelial‐mesenchymal transition (EMT) of alveolar epithelial cells (A549).

A549 cells were stimulated with TGF‐β1 and/or Cytomix [IL‐1β + TNF‐α + IFN‐γ]. Changes in expression of EMT markers were measured using immunofluorescence microscopy (IFM) and Western blot (WB). RAGE expression and localisation were evaluated by WB and IFM. Total and phosphorylated ERM levels were assessed by WB. Co‐localisation of F‐actin and ERM proteins was also investigated.

When challenged with TGF‐β1 and/or Cytomix, A549 underwent EMT with a significant change in phenotype. This was coupled with altered expression of RAGE and a marked redistribution of ERM proteins from the cytoplasm to the cell periphery. F‐actin stress fibres formed following induction of EMT, were found to co‐localise with ERM proteins. Moreover, ERM proteins were found to undergo phosphorylation.

Together, these results suggest that both RAGE and ERM proteins may play an important role in EMT of A549 following exposure to TGF‐β1 and/or Cytomix.

STB is funded by an IRSCET postgraduate scholarship.