bluuebunny/crossref_metadata_2025 huggingface dataset:
SLC2A2 gene is upregulated in diabetes and dowregulated in insulin treatment: participation of HNFs (1α, 3β and 4α)
Authors: Silva Aline David , Freitas Helayne Soares , Furuya Daniela Tomie , Poletto Ana Cláudia , Silva Robinson Sabino , Okamoto Maristela Mitiko , Machado Ubiratan Fabres
Published: June 2021

Objective

Investigate the role of HNFs (1á, 3â, 4á) at SLC2A2 (GLUT2 gene) in liver of diabetic and insulin treated diabetic rats.

Methods

3 month old Wistar rats, allocated into non diabetic (C), diabetic (D), diabetic treated with saline (DS) or insulin (DI) for 1, 4 and 6 days (d). The mRNA of GLUT2 and HNFs was analyzed by RT‐PCR. The HNFs binding activity into SLC2A2 promoter was analyzed by EMSA.

Results

In comparison to C, D rats showed: hyperglycemia, polyuria, glycosuria without ketonuria, less gain of weight; increase of: I) GLUT2 (P<0,0001), HNF1á (P<0,05), HNF3â (P<0,01) and HNF4á (P<0,05) mRNA expression, II) bindig activity of HNF1á (P<0,05), HNF3â (P<0,01) and HNF4á (P<0,05) into SLC2A2. In comparison to DS, DI rats showed: reduction of glycemia, increased of body weight; decrease of: I) mRNA expression: GLUT2 after 4 and 6d (P< 0,001), HNF1á after 6d (P<0,05), HNF3â after 4 and 6d (P<0,001) and HNF4á after 6d (P<0,05), II) binding activity into SLC2A2: HNF1á after 4 and 6d (P<0,01), HNF3â after 1, 4 (P<0,05) and 6d (P<0,01) and HNF4á after 6d (P<0,05).

Conclusion

The SLC2A2 overexpression is related to expression and activity of HNFs (1á, 3â, 4á). Furthermore, insulin therapy‐induced regulation of SLC2A2 expression also involves HNFs. Considering the importance of the GLUT2, we hope that the knowledge of the SLC2A2 transcriptional regulation may contribute to control gene expression.

FAPESP 2009/03871‐7
Endothelin‐1‐induced inhibition of NKCC2 activity is blunted in thick ascending limbs from angiotensin II‐hypertensive rats
Authors: Ramseyer Vanesa Daniela , Garvin Jeffrey Lawrence
Published: June 2021

Thick ascending limbs (THALs) absorb 25% of the filtered NaCl. Endothelin‐1 (ET‐1) inhibits NaCl absorption in THALs by increasing NO and reducing Na/K/2Cl cotransporter (NKCC2) activity via ETB receptors. THAL NaCl absorption is enhanced while NO production is reduced in angiotensin II (Ang II)‐hypertensive rats. Thus, we hypothesized that inhibition of NKCC2 by ET‐1 is blunted in Ang II‐induced hypertension. Rats were infused with vehicle or Ang II 200 ng/kg/min. At day 5 we measured: 1) mean arterial blood pressure (MABP) in anesthetized rats; 2) NKCC2 activity in perfused THALs by fluorescence microscopy and; 3) NOS3 and ETB receptor expression in THALs by Western blot. Ang II infusion increased MABP from 84 ± 5 mmHg to 98 ± 1 mmHg (p< 0.01). In vehicle‐treated rats, 1 nM ET‐1 reduced NKCC2 activity from 1.65 ± 0.15 to 0.84 ± 0.14 AU/s (Δ= ‐50 ± 7 %, p<0.01). In contrast, in Ang II‐hypertensive rats, ET‐1 did not decrease NKCC2 activity (from 1.46 ± 0.24 to 1.21 ± 0.22 AU/s). Ang II treatment reduced NOS3 expression by 35 ± 13% (p<0.03) but did not decrease ETB receptor expression (Δ= 10 ± 10%). We conclude that inhibition of NKCC2 activity by ET‐1 is blunted in Ang II‐induced hypertension and this is likely due to a reduction in NOS3 expression rather than ETB receptors. Impaired ET‐1‐induced inhibition of NKCC2 activity could be the cause of the enhanced NaCl absorption seen in this model of hypertension.

Sources of funding: NIH HL 028982 to J.L.G. and AHA 11PRE7510005 to V.D.R
Subunit‐specific regulation of V‐ATPase expression in purified intercalated cells from B1‐deficient mice
Authors: Vedovelli Luca , Rothermel John T. , Finberg Karin E. , Wagner Carsten A. , Brown Dennis , Paunescu Teodor G.
Published: June 2021

Mice deficient in the B1 subunit (B1 KO mice) of the vacuolar proton‐pumping ATPase (V‐ATPase) do not develop metabolic acidosis under baseline conditions. This is due to the insertion of V‐ATPases containing the alternative B2 isoform into the apical membrane of medullary collecting duct intercalated cells (ICs). We previously reported that Western blots (WBs) from whole kidneys showed similar B2 protein levels in B1 KO and wild type (WT) mice. To examine this issue further, we performed WBs from renal medulla membrane and cytosol fractions. Interestingly, both fractions revealed a decrease in the ubiquitous V‐ATPase E1 subunit levels, and WBs of the membrane fraction showed a down‐regulation of the a4 subunit. To compare V‐ATPase expression uniquely in ICs from WT and B1 KO mice, we crossed B1‐EGFP mice, in which EGFP expression is driven by the B1 subunit promoter, with B1 KO mice to generate B1 KO‐EGFP mice. We isolated pure IC populations by FACS from B1‐EGFP and B1 KO‐EGFP mice to compare their V‐ATPase protein levels. We found that V‐ATPase A and E1 subunits are down‐regulated by 59% in B1 KO mice, while the B2 protein level is increased by 57%. This upregulation of B2 compensates for the lack of B1, and is sufficient to maintain acid‐base homeostasis under baseline conditions, even when other V‐ATPase subunits are downregulated. Funding source: NIH DK‐73266, DK‐42956, and DK‐38452.
Insight into the structural differences in transmembrane segment 1 between NBCe1‐A and AE1
Authors: Zhu Quansheng , Newman Debra , Kao Liyo , Liu Weixin , Azimov Rustam , Kurtz Ira
Published: June 2021

The Na+‐HCO3 − cotransporter NBCe1‐A and the Cl−/HCO3 − exchanger AE1 mediate HCO3 − transport across plasma membranes. Although the topology of NBCe1‐A transmembrane segment 1 (TM1) has been modeled based on AE1 because of high sequence homology, our recent studies showed that NBCe1‐A‐TM1 but not AE1‐TM1 is involved in ion translocation. In the present study, we looked into the structural differences between NBCe1‐A‐TM1 and AE1‐TM1. Amino acids Cys389‐Asp452 covering NBCe1‐A‐TM1, and Leu378‐Arg432 covering AE1‐TM1 were individually substituted with cysteines (cys) in a modified NBCe1‐A or AE1 construct (free of reactive cys) and subjected to whole cell labeling with biotin maleimide (BM). The results show a stretch of 48 amino acids (Arg394‐Thr442) in NBCe1‐A, and a stretch of 23 amino acids (Thr398‐Ile421) in AE1 that were not labeled with BM, indicating NBCe1‐A‐TM1 is significantly longer than that of AE1‐TM1. Surprisingly, the deletion in AE1 causing South‐east Asian Ovalocytosis (Ala400‐Ala408) resides in the lipid bilayer forming part of TM1, unlike what has been previously thought. Further analysis with chemical stripping revealed that unlike AE1‐TM1, NBCe1‐A‐TM1 may interact tightly with the N‐terminal cytoplasmic domain. Our study clearly demonstrates that NBCe1‐A‐TM1 and AE1‐TM1 fold significantly differently to perform their respective structure‐functional roles.
Differential responses of proximal tubule Na+/H+ exchanger NHE3 to low pH: comparison between metabolic and respiratory acidosis
Authors: Silva Pedro Henrique Imenez , Girardi Adriana Castello Costa , Rebouças Nancy Amaral
Published: June 2021

The Na+/H+ exchanger isoform 3 (NHE3) is essential for HCO3 − reabsorption in renal proximal tubules. The goal of this work was to evaluate whether there are differences between metabolic and respiratory acidosis with regard to NHE3 modulation and to identify which are the relevant parameters that may trigger these distinct adaptative responses. Metabolic acidosis was achieved by lowering [HCO3 −] in the cell culture medium and respiratory acidosis by increasing pCO2 in the incubator chamber. We found that cell‐surface NHE3 expression increased in response to metabolic and respiratory acidosis. Mild (pH 7.21±0.02) and severe (6.95±0.07) metabolic acidosis increased Nhe3 mRNA levels, while mild (7.11±0.03) and severe (6.86±0.01) respiratory acidosis did not up‐regulate Nhe3 expression. Analyses of the Nhe3 promoter suggested that the regulatory elements sensitive to metabolic acidosis are located in the region spanning −466 to −153 pb. Two SP1 consensus binding sites were localized in this region and metabolic acidosis caused an increment in the SP1 protein levels. We conclude that low pH might be related to NHE3 displacement to the apical membrane, however extracellular pH does not seem to be the parameter that up‐regulates NHE3 expression. Moreover, SP1 was identified as a potential transcription factor that mediates the response to metabolic acidosis in proximal tubule cells. Financial support: CNPq and FAPESP.
Consequences of the laser speckle imaging computation method on analysis of renal autoregulation dynamics
Authors: Scully Christopher , Mitrou Nicholas , Waring Jennifer , Braam Branko , Cupples William A. , Chon Ki H.
Published: June 2021

Laser speckle imaging generates a surface perfusion map by analyzing speckle contrast pattern statistics over either a spatial or temporal set of pixels. Use of a spatial set reduces spatial but increases temporal resolution, while a temporal set increases spatial but decreases temporal resolution. We have found, for renal autoregulation frequencies, signals from single pixels have low signal‐noise ratio (SNR), but averaging pixels in each frame increases the SNR. We compared the moorFLPI (Moor Instruments, UK) imager choice of spatial or temporal statistics for analyzing renal autoregulation in terms of: 1) which provides minimal spatial resolution to achieve adequate SNR and 2) the influence of cardiac pulse aliasing on frequency analysis. Long‐Evans rat kidneys (n=12) were stabilized in vivo, and imaged with the moorFLPI temporal (576×768 pixels, 1Hz) and spatial (113×152 pixels, 25Hz) methods. NxN pixels (N=1…100) in each frame were averaged to generate time‐series, and the power spectrum of each was computed to determine the SNR. We found an adequate SNR was reached at a lower N for the moorFLPI temporal method. Use of the temporal method resulted in cardiac pulse aliasing, but may be used if renal blood flow records are available to identify aliasing. Otherwise, it may be necessary to use the spatial method to prevent aliasing at the expense of spatial resolution. This research was supported by CIHR MOP‐102694.
The Classic Renovascular (Goldblatt) Hypertension (RVHT) is Mediated by Succinate/GPR91 Signaling
Authors: Riquier-Brison Anne DM , Gevorgyan Haykanush A , Peti-Peterdi Janos
Published: June 2021

The novel metabolic receptor GPR91 is key in the activation of the renin‐angiotensin system (RAS). The ligand of GPR91 is the TCA‐cycle intermediate succinate which quickly accumulates in hypoxic tissues. Succinate injection iv in mice causes acute elevations in blood pressure (BP) which is RAS and GPR91 dependent. In the 2‐ kidney, 1‐clip (2K1C) model of RVHT, there is reduced blood flow to the clipped kidney (CK) and hypertension is renin (RAS)‐ dependent. We tested the hypothesis that succinate/GPR91 signaling is involved in the development of RVHT using a novel approach to the 2K1C model as described by Lorenz et al. Wild type (WT) and GPR91 KO mice were either sham operated or a soft cuff was placed on the left renal artery. During 3 weeks, BP was measured daily, and urine and plasma collected weekly. At 3 weeks, systolic BP (SBP) was unchanged in both WT and KO sham groups (WT: 108±2 to 105±14; KO: 104±2 to 91±11 mmHg). SBP increased in cuffed WT from 111±0 to 131±8 mmHg (peaked at week 2 at 148±6 mmHg, P<0.05), but did not change in the cuffed KO (103±13 to 106±9 mmHg). At week 3, kidneys were excised for further analysis. Renin expression increased 3.4±07‐ fold in WT CK, decreased 50% in WT non‐CK (NCK), and was unchanged in the KO CK and NCK. Renal fibrosis in the CK was also improved in GPR91 KO mice. We concluded that GPR91/succinate signaling is a major player in the control of intra‐renal renin (RAS) and BP in RVHT.
Angiotensin II (ANG II), renal perfusion pressure (RPP) and synchronization of cortical blood flow studied using laser speckle perfusion imaging (LSPI)
Authors: Mitrou Nicholas , Scully Chris , Dymond Melissa , Zheng Fei , Waring Jennifer , Braam Branko , Chon Ki , Cupples William
Published: June 2021

Renal autoregulatory mechanisms, myogenic (MR) and tubuloglomerular feedback (TGF), are synchronized among neighboring nephrons. Using LSP I (Moor FLPI) we examined on a physiologically relevant scale the contribution of this synchronization, or coupling, to regulation and autoregulation of renal blood flow. In particular we explore the roles of RPP and ANG II on synchronization at the surface of the kidney cortex. A circular spatial filter (r=562±87 μm) was applied to each frame. Time‐frequency spectra were computed for each pixel and synchronization was assessed by frequency locking within MR and TGF bands. Five male Long Evans rats were studied during spontaneous pressure (SPN, 110±13 mmHg), reduced RPP (LP, 61±2), captopril (1 mg/kg, CAP, 89±12), and LP+CAP, 61±2 mmHg. Surface flux was 1679+227, 1386+172, 2076+126 and 1807+156 units, respectively. Synchronization occurred in both MR and TGF frequency bands at SPN. As expected power in these bands was markedly reduced at LP as was synchronization. Instead, a single dominant frequency developed and was widely synchronized. LP+CAP also showed reduced MR and TGF power, but neither a single dominant frequency nor wide synchronization. The data show that reduced RPP during ACE inhibition reduces synchronization of MR and TGF and suggest that ANG II is a major regulator of the radius of synchronization. Funded by CIHR MOP‐102694
Pravastatin decreases superoxide production by thick ascending limbs
Authors: Soca Rodolfo , Garvin Jeff
Published: June 2021

Hydroxy‐methyl‐glutaryl‐CoA reductase inhibitors (statins) are used in the treatment of hyperlipidemia. Studies suggest that statins have anti‐inflammatory effects. Many of these may be mediated by nitric oxide (NO). Thick ascending limbs (TAL) express NO synthase and we have shown that statins increase NO production in TAL. In separate experiments we have shown that NO can reduce superoxide production. Thus, we hypothesized that pravastatin decreases superoxide production in TAL via NO. We measured superoxide production in 1‐minute periods in medullary TAL suspensions from Sprague‐Dawley rats using the lucigenin assay. The difference in luminescence in the absence and presence of tiron was taken as superoxide production. Tubules were treated with 10 uM pravastatin for 15 minutes. To study the role of NO, tubules were treated with 1 mM L‐NAME. When L‐NAME was used, it was added with the pravastatin. Control TAL produced superoxide at a rate of 1274 +/− 305 AU/min. TAL that had been treated with pravastatin produced superoxide at a rate of 697 +/− 225 AU/min (p < 0.01), 45% less. In the presence of L‐NAME alone, TAL produced superoxide at 1307 +/− 305 AU/min. Tubules treated with pravastatin plus L‐NAME produced superoxide at a rate of 595 ± 277 AU/min, 54% less (p< 0.01 vs. L‐NAME alone). We conclude that: 1) pravastatin inhibits superoxide production by TAL; and 2) NO does not mediate this effect.
COX‐2 inhibition exacerbates SOD1 dowregulation and the progression of renal oxidative stress in response to UUO
Authors: Nilsson Line , Frøkiær Jørgen , Nørregaard Rikke
Published: June 2021

Renal damage and oxidative stress resulting from unilateral ureteral obstruction (UUO) may be aggravated by reactive oxygen species (ROS). Deleterious effects of ROS are attenuated by antioxidant enzymes. We investigated the role of COX‐2 in the progression of renal oxidative stress induced by UUO in mice.

Mice were subjected to UUO and treated with the selective COX‐2 inhibitor parecoxib for 7 days. Using immunoblot analysis, we examined the regulation of antioxidant enzymes, catalase, superoxid dismutase 1 and 2 (SOD1 and −2) in renal cortex and inner medulla. Immunohistochemistry was performed with oxidative stress markers.

COX‐1 and −2 protein were increased in response to UUO. SOD1 and −2 protein were increased in IM in parecoxib treated sham operated mice. Parecoxib treated UUO mice had more reduced SOD1 protein level in IM and cortex than untreated UUO mice, whereas SOD2 and cortical catalase protein levels were unchanged.

COX‐2 inhibition increased the renal SOD1 and −2, consistent with a stress response to increased ROS production. UUO reduced SOD1 and catalase, and inhibited increased SOD1 and −2 seen with COX‐2 inhibition.

In conclusion, suppression of renal antioxidant enzymes by UUO contributes to the progression of renal oxidative stress, a process exacerbated by COX‐2 inhibition.
Antioxidant function of polyamines in human colon cancer cells demonstrated by fluorescence
Authors: Thornton Jonathan M , Osborne David L
Published: June 2021

Polyamines are known to be essential to the growth of eukaryotic cells. Studies have shown that polyamines may prevent oxygen mediated damage. We postulate that a possible function for polyamines in mature enterocytes may relate to an antioxidant function. The focus of this study was to visualize reactive oxygen species under various treatment conditions and determine if alterations in polyamine content may influence ROS mediated fluorescence. Studies used human colon cancer cells (HCT116) grown in culture. Cells were grown using RPMI1640 medium + 10% FCS. Media was supplemented with DFMO, putrescine or DFMO + putrescine. Cells were grown for 24 hours before fluorescent analysis was performed. ROS activity was visualized by using the Image‐iT® fluorescent dye… Cells were loaded with dye and then exposed to 6 mM H2O2. In the cells treated with RPMI1640 + 10% FCS, a baseline fluorescence was observed. Addition of DFMO resulted in a marked increase in the observed fluorescence compared to the control. Addition of putrescine to the DFMO media demonstrated that putrescine could reverse the affects of DFMO treatment. These results indicate that when polyamines are present, human colon cancer cells have a reduced observable load of ROS while the ROS load is increased in the conditions of reduced polyamine levels. This supports the theory that polyamines are functioning as antioxidants in gastrointestinal cells.
Potential role of NADPH oxidase in radiation‐induced pro‐oxidative and pro‐inflammatory pathways in mouse brain
Authors: Cho Hyung Joon , Sonntag William E , Lee Yong Woo
Published: June 2021

The present study was designed to investigate our hypothesis that fractionated whole brain irradiation, a clinically relevant regimen of radiotherapy, produces pro‐oxidative and pro‐inflammatory environments in brain. C57BL/6 mice received either fractionated whole brain irradiation (total cumulative dose of 40 Gy in eight fractions of 5 Gy, twice per week for four weeks) or sham‐irradiation, and were maintained for 4, 8, and 24 h following irradiation. Real‐time RT‐PCR and immunofluorescence staining demonstrated a significant and marked up‐regulation of mRNA and protein expression levels of pro‐inflammatory mediators, such as tumor necrosis factor‐α (TNF‐α) and monocyte chemoattractant protein‐1 (MCP‐1) in irradiated brains compared with the sham‐irradiated controls. Additionally, in situ dihydroethidium (DHE) fluorescence staining revealed that fractionated whole brain irradiation significantly increased production of reactive oxygen species (ROS) in brains. Furthermore, a significant increase in NADPH oxidase‐2 (NOX‐2) immunoreactivity was detected in brains after irradiation. These results suggest that NOX‐2 may play a potential role in whole brain irradiation‐induced pro‐oxidative and pro‐inflammatory pathways in mouse brain. (This work was supported by NINDS R01NS056218)
In vivo modulation of stem cell recruitment and proliferation via bi‐phasic electrical stimulation
Authors: Franklin Brandon Michael , Todd Gregory R , Collett Jason A , Osborn Jeffrey L
Published: June 2021

Tissue regeneration is dependent upon cellular ion channels that may be activated by electrical fields (EF). Modulation of EFs by electrical stimulation (ES) and/or pharmacological ion channel manipulation can either inhibit or induce regeneration in tissues with regenerative capacity. Either bi‐phasic or direct current ES can evoke migration, proliferation and differentiation of stem cells (SC) in vitro. This study hypothesizes that exogenous ES increases the activity of dedifferentiated and resident SC in vivo via activation of ion channel signaling. Bi‐phasic ES (2 volts, 2 Hz) of the skeletal muscle in fresh water crayfish Procambarus clarkii was conducted using implanted electrodes near the distal end of the abdomen. ES was conducted at 24 hours on/off over four cycles (Group I), continuously over 4 days (Group II) and in sham electrode implanted animals (Group III & IV; no stimulation). Relative abundance of SC was quantified by densitometry. Cyclic ES increased SC density (8.69±2.9 units; Group I) over sham experiments (2.03±0.49 units, Group III; P<0.05). Similarly, continual ES increased SC density (3.728±1.1 units, Group II vs. 1.032±0.52 units, Group IV; p<0.05). Thus, in vivo low‐level ES induces SC proliferation similar to previous in vitro studies. Current studies are investigating how pharmacological inhibition of ion channels may alter this effect and how ES changes genomic expression of SC.
Analysis and expression pattern of slc12a genes
Authors: Di Fulvio Mauricio
Published: June 2021

Mammalian cells exhibit a non‐equilibrium distribution of Cl− across their plasma membranes. This distribution plays a key role in cell volume regulation and growth, salt transport, K+ scavenging, hormone secretion and GABA signaling. The [Cl−]i results from the functional balance between Cl− transporters and channels expressed in the cellˈs plasma membrane. The family of solute carriers slc12a is the most relevant one involved in the regulation of the [Cl−]i. This family includes Na+K+2Cl−, Na+Cl− and K+Cl− co‐transporters: NKCCs, NCCs and KCCs, respectively. Two NKCC, one NCC and four KCC genes encode for NKCC1, NKCC2, NCC and KCC1‐4 isoforms. NKCC2 and NCC are considered expressed exclusively in the kidney, whereas NKCC1 is ubiquitously distributed. Similarly, KCC2 is considered a neuron‐specific isoform whilst KCC1, 3 and 4 appear to have less restricted expression patterns. To determine molecular identity, classify known slc12a members and establish patterns of expression we searched and analyzed nucleotide/protein sequences data posted in GenBank, EST and GEO databases. We found that at least two NKCC1 splice variants exist in humans, but different NKCC1s exist in rodents. Some NKCC2 variants were cloned from human kidneys, but were not found in rodents or vice versa. Moreover, at least ten KCC3 splice variants have been cloned from human tissues whilst rodents express a few ones. Further, NKCC2 or NCC mRNAs are detected outside the kidney. Therefore, splice variants of slc12a members may be species‐specific and/or non‐restricted to particular tissues. The molecular classification of slc12a variants and their pattern of expression will aid in the interpretation/extrapolation of functional/pharmacological data.

This work has been supported by the American Diabetes Association (Grant JF‐1001730 to MDiF).
Role of matrix metalloproteinases (MMP) 2 and 9 in TRPV4‐induced lung injury
Authors: Villalta Patricia Caridad , Rocic Petra , Townsley Mary I.
Published: June 2021

TRPV4 (transient receptor potential vanilloid 4) activation increases permeability of the alveolar septal barrier. Since active MMP2 and 9 can also alter septal barrier integrity, in this study we tested whether TRPV4 activation and resultant lung injury are associated with increased MMP 2 and 9 activity in the lung. We developed a high through‐put model to assess lung injury: lungs of C57BL/6 mice were perfused in situ with buffer/4% albumin (non‐recirculating) containing vehicle or the TRPV4 agonist GSK1016790A (3–3000 nM) for 30 min. Terminal measures of permeability and edema were assessed by albumin recovery via ELISA in bronchoalveolar lavage fluid (BALF) and lung wet to dry weight ratio (W/D), respectively. BALF albumin increased at 1000 and 3000 nM GSK1016790A, while lung W/D increased dose‐dependently with an EC50 of 150 nM. Thus, to assess MMP expression and activity, we used Western blotting and zymography of lung lysates respectively, after treatment of lungs with vehicle or GSK1016790A (30, 150, or 1000 nM). While injurious doses of GSK1016790A tended to decrease expression of active forms of MMP2, zymography did not show any change in MMP2 or 9 activity, regardless of GSK1016790A dose. We conclude that TRPV4‐induced lung injury does not increase MMP2 or 9 activity in lung tissue. Nonetheless, vectoral release of MMPs into BALF or into lung effluent cannot be ruled out. Supported by HL066299 and HL076125.
Cigarette Smoke Extract‐Induced Injury in Alveolar Cells in Cell Culture Model Systems
Authors: Downs Charles A , Montgomery David W , Merkle Carrie J
Published: June 2021

We explored the effects of cigarette smoke extract (CSE) on alveolar type I (AT I) cells and microvascular endothelial cells (MVECs) harvested from the lungs of neonatal (7 days), young (3 months) and old (24 months) male Fischer 344 rats. Single and co‐culture models of AT I and MVECs were used. CSE concentration was standardized spectrophotometrically using the smoke from one research grade (1R5F) cigarette dissolved in 10 mL of RPMI 1640 with 10% FBS. Cells were exposed to CSE for 24 hours and then assayed for intracellular ROS, extracellular H2O2, and total antioxidant capacity. Telomere length was determined after exposure to 2% CSE for 3 weeks using qRT‐PCR. ROS production in AT I cells did not change with CSE challenge while H2O2 production by MVECs increased 2 to 2.5 fold. Interestingly, when AT I and MVECs were grown as co‐cultures, H2O2 levels were reduced 1 to 4 fold compared to MVECs cultured alone. The total antioxidant capacity of AT I cells was greater (~8 fold) than MVECs which may explain the reduction of H2O2 levels. Gene expression profiling specific to oxidant and antioxidant pathways revealed 9 genes that were significantly different in MVECs grown in co‐culture compared to single cultures. CSE exposure shortened neonatal telomeres (p<0.05) while CSE exposure did not shorten young and old AT I cells (p<0.05) nor did CSE affect telomere length in young and old MVECs. Gene expression profiling identified 6 genes in neonates and 7 genes in old AT I cells that were affected by CSE exposure and the data suggest that CSE exposure stimulated cell division in neonates and promoted growth arrest in old AT I cells. The results of these studies may provide insight into mechanisms of CSE‐induced injury and possible therapeutic strategies to reduce lung injury secondary to smoking and related environmental exposures.
Effects of Oxytocin on Gastric‐Projecting Neurons in a Rat Model of Functional Dyspepsia
Authors: Babic Tanja , Browning Kirsteen N , Travagli Renato Alberto
Published: June 2021

Emotional stress triggers functional dyspepsia (FD) and gastric dysmotility. Oxytocin (OXY) modulates gastric motility and has a recognized antistressor role. We have shown that although OXY receptors are expressed in the dorsal vagal complex, OXY perfusion does not modulate the critical GABAergic inputs to identified gastric‐projecting preganglionic vagal (DMV) neurons. Here we tested the hypothesis that FD induces an abnormal response to OXY in the gastric‐related vagal circuits.

Following induction of FD, we did whole‐cell patch‐clamp recordings from gastric‐projecting DMV neurons from FD and control rats. In control rats, OXY had no effect on GABA currents in any of the neurons tested unless the neurons were pretreated with the stress hormone corticotrophin releasing factor (CRF). Following CRF, OXY altered GABAcurrents in 6/15 neurons. Conversely, in FD animals, OXY altered GABA currents in 10/25 neurons, independently of CRF. In FD rats, pretreatment of neurons with H89, a protein kinase A (PKA) inhibitor, blocked the effects of OXY on GABA currents in 7/8 neurons tested.

These data show that FD induces an abnormal response to OXY in the gastric‐related vagal circuits similar to that obtained by the stress‐related hormone CRF. We also suggest that that the effects are mediated by activation of cAMP/PKA pathway.

Supported by: NIH DK55330
Trk Moves on Track: retrograde transport of TrkA mediates sensory neuroplasticity in experimental colitis
Authors: QIAO LIYA , Yu Sharon , Gulick Melisa , Xia Chunmei
Published: June 2021

The prototypical neurotrophin nerve growth factor (NGF) has long been known to mediate visceral hypersensitivity caused by inflammation. By employing spinal nerve ligation or by prolonged pre‐treatment with resiniferatoxin (RTX) to desensitize primary afferent neurons, we demonstrate that in the rat TNBS‐induced colonic inflammation causes elevation of NGF in the distal colon where NGF binds to its high affinity receptor, TrkA. The NGF/TrkA complex is then retrogradely transported to the colonic afferent neurons in the dorsal root ganglia (DRG), and induces neuroplasticity by activating extracellular signal‐regulated protein kinase (ERK) 5, the big MAP kinase 1 (BMK1), as well as upregulating brain‐derived neurotrophic factor (BDNF). In these specifically labeled colonic afferent neurons, the phosphorylation level of ERK1/2 is not changed. BDNF up‐regulation in the colonic afferent neurons is significantly attenuated by blockade of ERK5 in vivo by intrathecal injection of a specific MEK inhibitor PD98059. Blockade of ERK5 activity in the ex vivo ganglia‐nerve two compartmented culture also inhibits retrograde NGF‐induced BDNF up‐regulation in the neuronal soma. These findings imply that colonic NGF regulates visceral hypersensitivity through retrograde activation of colonic primary afferent neurons in the DRG involving ERK5‐mediated BDNF up‐regulation. NIDDK DK077917
Multiple factors working in concert regulate blood pressure over a hibernation bout in the Syrian hamster
Authors: Saenz Marissa , Hamilton Jock , Chau Sat , Horowitz John , Chen Chao , Horwitz Barbara
Published: June 2021

Although blood pressure (BP) is regulated as hamsters enter hibernation, the detailed relationship between BP, heart rate (HR), and baroreflex sensitivity (BRS) is not known. We tested the hypothesis that multiple factors regulate BP over a hibernation bout. BP was recorded via a DSI telemetry transmitter in Syrian hamsters (Mesocricetus auratus). Hamsters were then placed into the cold (6±1°C, 8hr Light:16hr Dark) to induce hibernation. Five minute averages of systolic BP, HR and spontaneous BRS were analyzed with Nevrokard. The time for a decrease or increase from 90% to 10% (i.e. “fall or rise time”) of SBP, HR, and BRS were determined. On entry into hibernation (4 animals, 7 bouts total), fall time of HR (194±17 min) and BRS (211±21 min) were significantly (p<0.01) shorter than that of SBP (295±34 min). During the initial phase of entry into hibernation, BP and HR significantly decreased while BRS was maintained, indicating HR may be an important factor in regulating BP. During the later phase of entry, SBP continued to fall and BRS fell rapidly, indicating factors other than simply HR regulate BP. During arousal from hibernation, BRS recovery lagged behind BP and HR, and the rise time of HR, SBP and BRS were not significantly different. Results are consistent with the hypothesis and indicate that multiple factors, arranged in a specific temporal pattern, regulate BP over a hibernation bout. (support: R01 HL091763)
Activated subfornical organ contributes to enhanced sympathoexcitation during chronic heart failure
Authors: Llewellyn Tamra L , Sharma Neeru M , Zheng Hong , Patel Kaushik P
Published: June 2021

A characteristic feature of chronic heart failure (HF) that increases the risk of mortality is elevated sympathetic drive. The subfornical organ (SFO) is involved in neural control of sympathetic drive and may be influenced by circulating peptides such as angiotensin (Ang) II and endothelin (ET)‐1 due to its weak blood‐brain barrier. We hypothesized that an activated SFO, by Ang II/ET‐1, contributes to enhanced sympathetic activity in HF. Sprague‐Dawley rats were subjected to coronary artery ligation to induce HF. HF was confirmed by echocardiography 4 weeks after surgery. The activation of the SFO was assessed by FosB immunohistochemistry. Rats with HF had an increase in FosB‐positive cells in the SFO compared to sham rats (101 vs. 29 FosB‐positive cells). In urethane‐anesthetized rats, microinjection of Ang II (50–200 pmol) into the SFO increased renal sympathetic nerve activity (RSNA), blood pressure, and heart rate to a greater extent in HF than in sham rats (ΔRSNA: 31 vs. 15% of basal value; 100 pmol). HF rats also exhibited an elevated protein expression of AT1 (67%), ETA (69%), ETB (40%) receptors, while they had a reduced level of GABAA receptor (42%) in the SFO. The enhanced activation of the SFO by circulating peptides such as Ang II and ET‐1, that are known to be elevated during HF, may contribute to the sympathoexcitation exhibited in HF. Supported by the UNMC Skala Fellowship and NIH grant HL62222.
Changes in cardiovascular variability in response to stress in rats with two different forms of hypertension
Authors: Hayward Linda F , Hernandez Morgan , Castellanos Mabelin
Published: June 2021

Previously our lab demonstrated an association between an exaggerated response to air jet stress (AJS) in the spontaneously hypertensive rat (SHR) and dysregulation of corticotrophin (CRH) peptide expression in the amygdala (CEA). Changes in the production and release of CEA‐CRH have also been linked to elevated salt‐intake. The present study aimed to evaluate the stress response in normotensive (Sprague Dawley, SD) and hypertensive (SHR vs SD‐DOCA‐salt) rats. Adult 12 wk old male rats (3 to 4/group) were chronically instrumented for blood pressure (BP) and heart rate (HR) measurement and exposed to 20 min. of AJS. At rest, systolic BP variability in the VLF was significantly elevated in the both SHR and SD‐DOCA compared to the SD, and this corresponded to a significantly elevated resting BP (163±6 mmHg). During AJS, the rise in BP was significantly elevated in the SHR compared to both the SD‐DOCA and SD (30±1 vs 20±2 vs 17±2 mmHg, respectively). Alternatively, the rise in HR was lower in the SD‐DOCA. In both the SHR and SD‐DOCA rats HR remained elevated for 10 min post‐AJS compared to the SD. Differences in HR were paralleled by evidence of altered HR HF power during AJS in the SD‐DOCA and altered baroreflex function post stress in the SD‐DOCA and SHR. These data suggest that although high salt intake triggers a sustained hypertension comparable to the SHR, the response to stress is different.
HIF‐1a gene deletion in the nucleus tractus solitarii (NTS) blunts ventilatory acclimatization to hypoxia (VAH)
Authors: Go Ariel D. , Fu Zhenxing , Johnson Randall S. , Powell Frank L.
Published: June 2021

Previously, we found that Hypoxia‐Inducible Factor‐1α (HIF‐1α) expression in the central nervous system (CNS) is necessary for normal VAH in mice. Here we tested the hypothesis that HIF‐1α in the NTS, which receives sensory input from carotid body chemoreceptors, is necessary during hypoxic exposure for normal VAH. We used the Cre‐LoxP strategy and microinjected Adeno‐associated virus expressing Cre‐recombinase (AAV‐Cre) into the NTS of mice carrying conditional HIF‐1α floxed alleles. AAV‐LacZ was microinjected as a control and to determine the extent of AAV expression. One week after microinjection, mice were kept in normoxia (CN) or CH (Pio2 = 70 Torr) for 7 days before measuring the hypoxic ventilatory response with barometric pressure plethysmography. Ventilation (Vi mL/(min kg)) was:

CHRONIC NORMOXIA

Inspired O2 (%)
AAV‐Cre (n=6)
AAV‐LacZ (n=6)

21
2176 ± 158
2059 ± 106

10
2088 ± 181
1905 ± 84

CHRONIC HYPOXIA

Inspired O2 (%)
AAV‐Cre (n=9)
AAV‐LacZ (n=8)

21
2811 ± 121
2949 ± 152

10
3092 ± 245
4566 ± 307

CH significantly increased Vi as expected, but the increases in Vi and breathing frequency in CH were significantly less with AAV‐Cre than with AAV‐LacZ. The results support HIF‐1α in the NTS contributing to the increased CNS gain of the HVR in VAH we reported previously (J. Appl. Physiol. 87: 817–823, 1999). Supported by NIH RO1 HL‐081823 and 1P01 HL 098053.
GAL‐021 –induced respiratory stimulation is associated with increases in carotid sinus nerve and phrenic motoneuron activity in rats
Authors: Baby Santhosh M , Golder Francis J , Peng Sean , Dax Scott L , MacIntyre D Euan , Mannion James C
Published: June 2021

GAL‐021 is a novel respiratory stimulant that increases minute ventilation in rats, mice, and Cynomolgus monkeys. We hypothesized that GAL‐021 increases minute ventilation via mechanisms at the level of the carotid body. Using in vivo electrophysiological techniques, we examined the dose‐dependence of the effects of GAL‐021 (0.01 – 3 mg/kg, IV) on carotid sinus nerve (CSN) activity and phrenic motoneuron activity in mechanically ventilated, vagotomized, and neuromuscularly paralyzed adult rats, and on tidal volume (VT) in anesthetized spontaneously breathing rats. GAL‐021 dose‐dependently increased CSN (ED50 0.13 mg/kg) and phrenic nerve neurogram amplitude (ED50 0.08 mg/kg), values in reasonable accord with effects on VT (ED50 0.04 mg/kg). Thus, GAL‐021 is virtually equipotent in stimulating the afferent, efferent and effector components of the ventilatory response. When administered to conscious rats 7 days after either bilateral CSN transection or sham operation, the ventilatory effects (VT and frequency) of GAL‐021 (0.2 mg/kg, IV) were markedly diminished in the CSN transected rats compared to sham‐operated controls. Collectively, these data suggest that GAL‐021 stimulates breathing, at least in part, by effects on the carotid body resulting in increased carotid sinus nerve activity.
Comparison of the respiratory effects of GAL‐021 in BK α subunit knockout (Slo1−/ −) and wild‐type mice
Authors: Baby Santhosh M , Hoshi Toshinori , Peng Sean , Dax Scott L , Golder Francis J , MacIntyre D Euan , Mannion James C
Published: June 2021

GAL‐021 is a novel respiratory stimulant that increases minute ventilation (Vmin) in rats and Cynomolgus monkeys. Transecting the carotid sinus nerve decreases this effect in rats, suggesting that part of the evoked stimulation is mediated by effects at the level of the carotid body. BK (or Maxi K) channels, which consist of a pore‐forming α‐subunit (slo1) and various â regulatory subunits, are present in the carotid body and have been implicated in oxygen sensing. Hypoxia reportedly decreases BK potassium current and increases carotid sinus nerve activity. In preliminary studies using GH3 cells we demonstrated that GAL‐021 exerts modest inhibition of BK channels (56%I at 10 μM). To test the hypothesis that GAL‐021 may stimulate breathing via inhibition of BK channels, we administered GAL‐021 (0.03 ‐ 3 mg/kg, IV) as bolus injections to isoflurane anesthetized, wild‐type and BK á‐subunit knockout (Slo1−/−) mice. In wild‐type mice, GAL‐021 dose‐dependently increased Vmin (maximum response 143 ± 34 % above baseline; ED50 0.5 mg/kg) consistent with prior studies in rats and monkeys. In Slo1−/− mice the effects of GAL‐021 on Vmin (maximum response 64 ± 24 % above baseline; ED50 2.1 mg/kg) were significantly attenuated (but not abolished) in comparison to the wild‐type. These data suggest that BK channels contribute to the effects of GAL‐021 on ventilation but that non‐BK‐dependent mechanisms are also involved.
Severe sustained hypoxia elicits long‐lasting phrenic motor facilitation (pMF) by an adenosinergic mechanism
Authors: Nichols Nicole L , Devinney Michael J , Mitchell Gordon S
Published: June 2021

Moderate acute intermittent hypoxia (AIH; 3, 5‐min episodes) elicits serotonin‐dependent phrenic long‐term facilitation (pLTF); this form of pLTF is enhanced by spinal adenosine 2A (A2A) receptor inhibition. Severe AIH also elicits pLTF, but via an adenosine A2A receptor dependent (serotonin independent) mechanism. This shift may reflect greater spinal adenosine release/formation during severe AIH. Moderate sustained hypoxia (SH; 9–20‐min) fails to elicit pMF, demonstrating pMF pattern‐sensitivity. However, since severe SH may increase extracellular adenosine levels, we hypothesized that severe SH would elicit pMF via an A2A receptor dependent mechanism. Severe SH (PaO2=25–35; 25 min) was studied in anesthetized, paralyzed and ventilated rats +/− spinal A2A receptor inhibition (MSX‐3, i.t., C4). Severe SH elicits pMF (53%±12% at 60 min post‐SH) vs. time controls (10%±3%; p<0.05, both n=8). MSX‐3 pre‐treatment attenuated severe SH induced pMF (10%±5%; p<0.05, n=7). Thus, severe SH and AIH both elicit spinal A2A‐dependent pMF, unlike moderate SH and AIH (Devinney et al., ibid), indicating that adenosine‐dependent pMF is not pattern sensitive. Understanding mechanisms of pattern sensitivity in respiratory plasticity may enable us to “harness” plasticity as a treatment for respiratory insufficiency.

[Supported by NIH HL080209 and T32 007654 and the Francis Families Foundation]
Spinal A2A receptor inhibition reveals phrenic motor facilitation following moderate sustained hypoxia
Authors: Devinney Michael J , Nichols Nicole L , Mitchell Gordon S
Published: June 2021

Moderate acute intermittent hypoxia (AIH; 3, 5‐min episodes) elicits serotonin‐dependent phrenic long‐term facilitation (pLTF); this form of phrenic motor facilitation (pMF) is enhanced by spinal adenosine 2A (A2A) receptor inhibition. In contrast, moderate sustained hypoxia (SH; 9–20‐min) fails to elicit pMF, demonstrating pLTF pattern‐sensitivity. Since longer moderate hypoxic episodes may increase extracellular adenosine levels and negate pLTF, we hypothesized that A2A receptor blockade reveals pMF after moderate SH. pMF was studied in anesthetized, paralyzed, and ventilated rats ± spinal A2A receptor inhibition (MSX‐3, C4 i.t.). Although, moderate SH (25 min, PaO2 45–55) caused no significant pMF (n=9, 14±5%) vs. time controls (n=8, 10±3%; P >0.05) 60 min post SH, MSX‐3 pre‐treatment revealed significant pMF (n=8, 39±5% %, p<0.05 vs. SH). Thus, A2A receptor activation during moderate SH negates pMF. Since spinal A2A receptor activation and severe SH (25 min, PaO2 25–35) elicit adenosine‐dependent pMF (Nichols et al., ibid), pLTF pattern‐sensitivity may arise from emergent properties of parallel signaling networks giving rise to pMF; inhibitory interactions between these competing mechanisms negates pMF after moderate SH. Understanding mechanisms of pattern‐sensitivity in respiratory plasticity may enable us to better harness plasticity as a therapeutic tool.

Supported by NIH HL080209, T32 007654 and the Francis Families Foundation.
The Involvement of Hypoglycemia in Brain Glycogen Decrease during Exhaustive Exercise
Authors: Matsui Takashi , Soya Hideaki
Published: June 2021

We recently found that prolonged exhaustive exercise with severe hypoglycemia decreases the brain glycogen that is localized in astrocytes as a critical energy source for neurons (J Physiol, 2011), although the mechanism behind this is still uncertain. To examine whether severe hypoglycemia alone would mediate exercise‐induced brain glycogen decrease, we compared the effects of insulin‐ and exhaustive exercise‐induced hypoglycemia on glycogen levels in five brain loci (the cortex, hippocampus, hypothalamus, cerebellum, and brainstem) in adult Wistar rats using a high‐power (10 kW) microwave irradiation method as a gold standard. Both exhaustive exercise and the insulin challenge induced similar severe hypoglycemia (Fig. 1A ). Brain glycogen decreased by approximately 40% with both exhaustive exercise and the insulin challenge, but the glycogen decrease rates in the cortex and hippocampus with exhaustive exercise were greater than in the insulin challenge (Fig. 1B ). Furthermore, decreased blood glucose positively correlated with decreased brain glycogen, but the rate of regression in the cortex, hippocampus, and brainstems of the exercise group were greater than those of the insulin group (Fig. 1C). We found that glycogen decrease, at least in the cortex, hippocampus, and brainstem, would not be due to hypoglycemia alone, suggesting an activity‐dependent brain glycogen decrease. This work was supported in part by KAKENHI (No. 21650166).
Delayed recovery of skeletal muscle mass following hindlimb immobilization in mTOR heterozygous mice
Authors: Lang Susan M. , Kazi Abid A. , Hong-Brown Ly , Lang Charles H.
Published: June 2021

The role of mTOR in regulating muscle protein balance was assessed in WT and mTOR heterozygous (+/−) mice after hindlimb immobilization and recovery, with data compared to the contralateral muscle. A gradual loss of muscle mass, which plateaued by day 7, was seen in WT mice. This response was associated with a sustained reduction in protein synthesis and increased proteasome activity. Atrogin‐1 and MuRF‐1 mRNA was transiently increased but returned to control values by day 7. The decreased muscle mass and protein synthesis and increased proteasome activity did not differ between WT and mTOR+/− mice after immobilization. After 10 days of recovery, WT mice showed no decrement in muscle mass and this accretion resulted from a sustained increase in protein synthesis and normalization of proteasome activity. In contrast, mTOR+/− mice failed to fully replete muscle mass. This defect was caused solely by the lack of a compensatory increase in protein synthesis. Delayed regrowth of the previously immobilized muscle in mTOR+/− mice was associated with a sustained inflammatory response the recovery period as well as a failure to increase IGF‐I. These data suggest mTOR activity is relatively more important in regulating the accretion of muscle mass during recovery than the loss of muscle during the atrophy phase, and that protein synthesis is more sensitive than degradation to the reduction in mTOR during muscle regrowth. (GM38021)
Reduced lung glutamate (GLU) production is the cause of decreased systemic glutamate availability in hyperdynamic sepsis
Authors: Ten Have Gabriella A , Engelen Marielle P , Wolfe Robert R , Deutz Nicolaas E
Published: June 2021

RATIONALE

Previously, we described in patients in the early phase of septic shock that reduced plasma GLU relates to mortality. As the mechanism is unclear, we studied GLU organ net balances (NB) in a Pseudomonas aeroginosa (PM) sepsis induced pig model.

METHODS

Arterial GLU concentrations and transorgan GLU net balances were studied in 12 pigs with sepsis and 8 controls. GLU concentrations were determined with LC‐MS. Data are expressed as (mean (SE) control vs PM) in μM (conc) and nmol/kg bw/min (NB), statistics with Student t‐test.

RESULTS

In PM pigs, arterial GLU concentration (81.3(6.8) vs 38.0(3.7)) and lung GLU net release (7472(1457) vs 1354(1204)) were reduced (resp. 53%, p<0.0001 and 82%, p=0.0088), while Liver net release was unchanged. Gut (195(49) vs 24(34)) and muscle (843(57) vs 376(73)) GLU net uptake were decreased (resp. 88%, p=0.009 and 55%, p=0.0002).

CONCLUSION

The reduced lung GLU production plays a major role in the reduced systemic GLU availability in sepsis.