bluuebunny/crossref_metadata_2025 huggingface dataset:
Interleukin‐6 Contributes to Endothelial Dysfunction in the Cerebral Circulation in Aging
Authors: Didion Sean P. , Chen Xunsheng , Mezzetti Erin M. , Ali M. Irfan
Published: June 2021

The goal of this study was to examine the role of interleukin‐6 (IL‐6), a pro‐inflammatory cytokine, in the endothelial dysfunction associated with aging in the cerebral circulation. Vascular responses were examined in basilar arteries from young (6 mo) and old (24 mo of age) wild‐type (WT) and IL‐6‐deficient mice. Dilatation to acetylcholine (ACh), an endothelium‐dependent agonist, was similar (P>0.05) in basilar arteries from young WT and young IL‐6 deficient mice. For example, 100 μM ACh produced 65±3% and 71±4% dilatation in basilar arteries from young WT and young IL‐6 deficient mice, respectively. With age, responses to ACh were impaired by ~100% in old WT mice as compared to their young counterparts. We have shown previously that this impairment of endothelial function in the basilar artery with age is mediated by reactive oxygen species (ROS). In contrast, the response to 100 μM ACh was partially reduced in old IL‐6 deficient mice, however the degree of impairment was significantly less than that in old WT mice (e.g., 100 μM ACh produced 38±4% and 0±0% dilatation in old IL‐6 deficient and old WT mice, respectively). Responses to nitroprusside (an endothelium‐independent agonist) were not affected (P>0.05) by genotype or aging. These findings suggest that IL‐6, in part and/or in conjunction with ROS, contributes to the endothelial dysfunction observed with aging in the cerebral circulation.
TMEM16A channels generate Ca2+‐activated Cl− currents in cerebral artery smooth muscle cells
Authors: Thomas‐Gatewood Candice , Neeb Zachary P. , Adebiyi Adebowale , Bannister John P. , Jaggar Jonathan H.
Published: June 2021

Transmembrane protein 16A (TMEM16A) channels are recently discovered membrane proteins that display electrophysiological properties similar to Ca2+‐activated chloride (ClCa) channels in native cells. The molecular identity of proteins that generate ClCa currents in smooth muscle cells of resistance‐size arteries is unclear. Here, using molecular biology and patch‐clamp electrophysiology we examined TMEM16A channel expression and characterized Cl− currents in arterial smooth muscle cells of resistance‐size rat cerebral arteries. RT‐PCR amplified transcripts for TMEM16A, but not TMEM16B‐K family members, in isolated pure cerebral artery smooth muscle cells. Western blotting of cerebral artery lysate using an antibody raised against TMEM16A revealed a band consistent with the predicted molecular weight of TMEM16A. Surface biotinylation and immunofluorescence indicated that TMEM16A channels are located predominantly within the arterial smooth muscle cell plasma membrane. ClCa currents recorded in arterial smooth muscle cells displayed properties similar to those of currents generated by recombinant TMEM16A channels expressed in HEK293 cells. A TMEM16A antibody and TMEM16A knockdown using siRNA both attenuated arterial smooth muscle cell ClCa currents. In conclusion, our data indicate that TMEM16A channels generate ClCa currents in cerebral artery smooth muscle cells.
TNFα is central to the augmented myogenic response of skeletal muscle resistance arteries in heart failure
Authors: Kroetsch Jeffrey Thomas , Meissner Anja , Momen Abdul , Husain Mansoor , Bolz Steffen‐Sebastian
Published: June 2021

A hallmark of heart failure (HF) is elevated peripheral resistance, which primarily results from an augmented myogenic response (MR, the intrinsic property of resistance arteries (RA) to adapt diameter to changes in pressure). In cerebral arteries from HF mice, tumor necrosis factor α (TNFα) plays a central role for the enhancement of MRs (through activation of sphingosine kinase‐1). We hypothesized that TNFα is equally important for the augmentation of MRs in skeletal muscle RA and hence, increased peripheral resistance in HF. HF was induced in Male C57/BL6 mice through surgical ligation of the left anterior descending coronary artery. 6–8wks post‐ligation, cremaster muscle RA were isolated and cannulated on a pressure myograph. Elevations in transmural pressure (20–100mmHg in 20mmHg steps) induced MRs that were increased in RA from HF compared to sham‐operated mice (n=7). MRs were not enhanced in RA from HF TNFα−/− mice (n=5). However, treatment with the TNFα scavenger etanercept (1mg/kg 2x/wk for 6wks post‐ligation) did not block the HF‐induced increase in MRs.

Our data suggest that: (i) the augmented MR in skeletal muscle arteries contributes to increased vascular resistance in HF, (ii) TNFα is a mandatory component of this response, and (iii) chronic scavenging of TNFα might be compensated by redundant organization of MR signaling pathways.

Funding: NSERC, HSFO, and CIHR
MECHANISMS INVOLVED IN THE VEIN RELAXATION INDUCED BY THE NEW NITRIC OXIDE (NO) DONOR TERPY IN RENAL HYPERTENSIVE RATS (2K‐1C)
Authors: Paulo Michele , Da Silva Roberto Santana , Bendhack Lusiane M
Published: June 2021

Venous circulation has a potential contribution to the hypertension. This study aimed to investigate the cellular mechanisms of vasodilatation induced by the new NO donor Terpy, in the cava vein from normotensive (2K) and 2K‐1C by the inhibition of soluble guanylyl‐cyclase (sGC) with ODQ, sarcoplasmic reticulum Ca2+‐ATPase (SERCA) with thapsigargin (TG) and K+ channel blockers in cava vein pre‐incubated with L‐NAME to avoid the interference of endogenous NO. The veins were pre‐contracted with endothelin‐1 (ET‐1) after incubation with the inhibitors. It was approved by Ethics Committee. Vasodilatation induced by Terpy was lower in 2K‐1C (pD2:5.42±0.22; ME:56.7±3.1%, n=7) than in 2K veins (pD2:6.10±0.12; ME:70.3±3.2% n=6, p<0.05). It was abolished by ODQ. The non‐selective blockade of K+ channels reduced the ME in 2K (47.4±2.6% n=6, p<0.001) and 2K‐1C veins (45.0±1.1% n=6, p<0.05), blockade of Kv reduced pD2 and ME in 2K (3.56±0.18; 47.5±2.7% n=7, p<0.001) and 2K‐1C veins (3.97±0.21, n=7; 35.4±2.8% n=7, p<0.001), SKca reduced pD2 and ME in 2K (3.87±0.27; 55.4±3.7% n=6, p<0.001) and 2K‐1C veins (4.57±0.19;36.6 ± 1.9% n=6, p<0.001), but KATP had no effect. TG reduced the ME in 2K (31.7±1.5% n=5, p<0.001) and 2K‐1C (25.6±0.7% n=6, p<0.001). In conclusion, the new NO donor induced relaxation in 2K and 2K‐1C rat veins involves the activation of sGC, Kv, SKCa and SERCA. It is impaired in 2K‐1C. Supported by FAPESP and CNPq.
Sub‐cellular localization of bestrophin‐3 in murine blood vessels
Authors: Davis Alison Jane , Orton Kate , Baines Debbie L , Leblanc Normand , Greenwood Iain A
Published: June 2021

We have previously reported that TMEM16A is expressed in smooth muscle, supporting the notion that this newly suggested candidate may contribute to calcium‐activated chloride channel (CACC) conductance. However, the contribution of a previous candidate, Bestrophin, in encoding CACCs remains controversial. Here, we aimed to explore the expression and sub‐cellular localization of Best‐3 in the murine vasculature in order to ascertain its potential contribution to CACC conductance in smooth muscle. Murine blood vessels from female BALB/c mice (6–8 weeks) were isolated for electrophysiological, mRNA and protein (Western blot, sucrose fractionation and immunological) studies. We show for the first time that Best‐3 mRNA (at similar levels to TMEM16A) and protein are present in the mouse vasculature. We also provide new evidence for the presence of Best‐3 smooth muscle splice variant transcripts, and its protein localization to specific sucrose fractions suggesting compartmentalization of this protein within the cell. Consequently, we propose that Best‐3 is expressed in the murine vasculature and remains a viable candidate to contribute to a CACC complex. Supported by the British Heart Foundation.
The effects of short term exercise training on purinergic metabolism in skeletal muscle arterioles
Authors: Stone Audrey J , Evanson Kirk W , Kluess Heidi A
Published: June 2021

The purpose was to determine the effects of short term exercise training on purinergic metabolism in gastrocnemius first‐order arterioles of young female rats. Female rats (2–3mo) were randomly assigned to one of the following four groups: endurance training (ExTr), sedentary for 14 days (SED), no exercise training or sedentary period (BSLN), and exercise trained followed by deconditioning (DECON). The exercise training consisted of running on a treadmill 5 days/week for 2 weeks at 35m/min and on a 7% incline. Adenine nucleotidase activity was measured using whole vessel segments in a spectrophotometric assay. Adenosine (ADO) overflow was measured with a biosensor sensitive to adenosine and inosine. Adenine nucleotidase activity was not different among the groups (BSLN: 0.17±0.08μM phosphate/μg protein, n=12; SED: 0.36±0.11 μM phosphate/μg protein, n=12; ExTr: 0.50±0.14 μM phosphate/μg protein, n=11; DECON: 0.23±0.06 μM phosphate/μg protein, n=13; p>0.01). Average adenosine overflow was highest in the ExTr group (324.9±323 μM ADO, n=4) and lowest in the DECON group (12.23±5.6 μM ADO, n=5; p>0.01 different from all other groups). The pattern of adenosine overflow also differs with exercise training and time. This study suggests that purinergic metabolism does alter with short term exercise training and detraining. This research was supported by the Arkansas Biosciences Institute and the National Institute on Aging.
Small and intermediate conductance Ca2+‐activated K+channels regulate spontaneous contraction of mouse portal vein myocytes
Authors: Blanchette Alexandre , Charbel Chimène , Toussaint Fanny , Greenwood Iain A , Ledoux Jonathan
Published: June 2021

Ca2+‐activated K+ channels (KCa) are key components of vascular myocytes EC coupling through their control of smooth muscle cell membrane potential. Whilst BKCa is expressed ubiquitously in smooth muscle cells, only proliferative VSMCs express KCa3.1 channels. However, mouse portal vein (mPV) is an atypical blood vessel showing spontaneous and rhythmic contractions. Since KCa channels are involved in the regulation of excitability of other cell types like neurons, the contribution of KCa2.x and KCa3.1 channels to the control of mPV excitability was investigated. Expression of KCa2.x and KCa3.1 was detected in mPV by PCR and immunohistochemistry. In single cell patch clamp studies, the RMP of isolated mPV myocytes was hyperpolarized (≈15 mV) by exposure to NS309 (10 μM), a KCa2.x and KCa3.1 channel activator. Inhibition of KCa2.x channels with UCL1689 (3 μM) reversed the effect of NS309 whilst inhibition of KCa3.1 channels (TRAM‐34; 10 μM) had no effect. NS309 significantly decreased the amplitude and increased the frequency of spontaneous contractions of intact mPV. These effects were reversed by application of UCL1689, suggesting that the NS309‐induced changes in spontaneous mPV contractions is carried out by KCa2.x channels. This study suggest that KCa2.x and KCa3.1 channels are expressed in mouse portal vein myocytes and play an important role in regulation of spontaneous contractions.

Supported by FICM
Functional characteristics of alpha adrenoceptors in isolated pressurized rat mesenteric small veins
Authors: Enouri Saad , Johnson Ron , Monteith Gabrielle
Published: June 2021

Vasomotor tone is largely maintained by norepinephrine (NE) released by sympathetic nerves and activating mainly alpha adrenergic receptors (α‐ARs). We investigated the relative contributions of α‐ARs to NE contractile responses in rat mesenteric vessels. Pressure myography was used to study isolated third order mesenteric small veins (6 or 12 mmHg) and arteries (60 or 120 mmHg). NE and phenylephrine (PE) concentration response curves (CRC) were constructed at normal and high transmural pressures in the absence and presence of α‐AR antagonists. Clonidine CRC were obtained at normal transmural pressures. All agonists evoked concentration dependent contractions with NE and PE being more potent in veins compared with arteries. In veins, NE was a more potent agonist than PE. Phentolamine (α‐AR antagonist, 10 μM) and prazosin (α1‐AR antagonist, 100 nM) completely abolished NE responses in all vessels. Yohimbine (α2‐AR antagonist, 100 nM) significantly reduced NE CRC in veins at 6 mmHg. Clonidine responses in veins were significantly reduced by yohimbine. These results indicate the predominant involvement of α1‐ARs in NE‐induced contractions of pressurized mesenteric vessels with no change in the contribution of α1‐ARs as pressure increased. In addition to α1‐ARs, α2‐ARs mediate NE responses in veins at 6 mmHg. This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada.
Transcriptional upregulation of α2δ‐1 subunits elevates CaV1.2 currents in cerebral artery smooth muscle cells of hypertensive rats
Authors: Bannister John P , Thomas‐Gatewood Candice M , Pachuau Judith , adebiyi Adebowale , Luzny Patrik , Jaggar Jonathan H
Published: June 2021

Voltage‐dependent L‐type (CaV1.2) channels contain pore‐forming α1 and auxiliary α2δ subunits. CaV1.2 channels are the principal Ca2+ entry pathway in arterial myocytes and regulate physiological functions, including contractility. CaV1.2 currents are larger in arterial myocytes of hypertensive subjects, leading to elevated arterial contractility, but molecular mechanisms that underlie this pathological alteration are unclear. Here, we investigated the molecular identity and physiological functions of α2δ isoforms in resistance‐size cerebral arteries of normotensive (WKY) and hypertensive (SHR) rats. RT‐PCR indicated that α2δ‐1 was the only α2δ isoform expressed in WKY and SHR arterial myocytes. CaV1.2α1 and α2δ‐1 protein were 2‐ and 2.5‐fold higher in SHR than WKY arteries, respectively. Surface biotinylation indicated that both subunits were present primarily in the plasma membrane of SHR and WKY arteries. Arteries from SHR developed more myogenic tone than WKY. Chronic pregabalin (24h), an α2δ‐1 ligand, decreased surface expression of both subunits, reduced CaV1.2 current density, and dilated pressurized arteries more in SHR than WKY. In summary, our data indicate that transcriptional upregulation of α2δ‐1 elevates surface expression of CaV1.2α1 in myocytes, leading to vasoconstriction. We identify α2δ‐1 as a novel molecular target for inducing vasodilation in hypertensive arteries.
SEX DIFFERENCES IN CONSTRICTOR RESPONSES TO BERAPROST IN RAT MESENTERIC ARTERIOLES: ROLES OF THROMBOXANE VS. PGE2 PROSTANOID RECEPTORS
Authors: Sellers Minga , Stallone John
Published: June 2021

Recently it was reported that prostacyclin (PGI2) and its analogue beraprost (Bpst) cause constriction in rat aorta, likely via thromboxane receptor (TP). We investigated this effect of Bpst in the microvasculature (rat mesenteric arterioles, MA), as well as the role of PGE2 receptors (EP) and whether sex differences exist in the constrictor response to Bpst. Male (M) and female (F) Sprague‐Dawley rats (14–16 wks age) were sacrificed and reactivity to Bpst was measured in MA (100–200 μm dia.) using isometric myography. Concentration‐response curves to Bpst (10−8 – 10−4 M) were obtained in the presence of indomethacin (INDO, 10−5M; COX 1/2 inhibitor) alone or with CAY 10441 (CAY, 10−6M; PGI2 receptor antagonist) and one of the following: SQ 29,548 (SQ, 10−6M; TP antagonist) or AH 6809 (AH, 10−5M; EP antagonist). In F MA, Bpst caused constriction in all groups; the greatest responses were in INDO+CAY and INDO+CAY+AH, suggesting that TP mediates the constrictor responses to Bpst in F MA. In M MA, Bpst also caused constriction in all groups. While INDO+CAY exhibited the greatest response, INDO+CAY+SQ exhibited significantly greater constriction than INDO alone or INDO+CAY+AH, indicating that in M MA, Bpst produces constriction via EP rather than TP. In conclusion, Bpst produces similar constriction in both M and F MA; however, F appear to rely more upon TP, while M relies more on EP for this constrictor effect. (NIH: HL‐080402)
TNFα affects sphingosine‐1‐phosphate signaling through modulating the expression of the cystic fibrosis transmembrane regulator (CFTR) in vascular smooth muscle cells
Authors: Meissner Anja , Bolz Steffen‐Sebastian
Published: June 2021

Cellular effects of the ubiquitous signaling molecule sphingosine‐1‐phosphate (S1P) are tightly controlled by a rheostat between the S1P‐generating enzyme sphingosine kinase 1 (Sphk1) and its functional antagonist S1P phosphohydrolase 1 (SPP1). The intracellular localization of SPP1 requires the import of S1P prior to its degradation. We propose that the import into vascular smooth muscle cells (VSMC) critically depends on the cystic fibrosis transmembrane regulator (CFTR) and that changes in CFTR function regulate S1P signaling. In the vasculature, S1P regulates resistance artery myogenic tone and hence, peripheral resistance. Both parameters are increased in heart failure (HF), a finding that is associated with down‐regulation of vascular CFTR. The pro‐inflammatory cytokine TNFα mechanistically links HF and CFTR because (1) TNFα down‐regulates CFTR mRNA and protein expression, (2) increased TNFα expression and down‐regulation of CFTR in resistance arteries co‐localize in HF and (3) sequestration of TNFα with etanercept normalizes CFTR expression. In addition to these genomic effects, TNFα also acutely controls CFTR function through regulation of its membrane abundance.

Our data suggest that TNFα‐mediated down‐regulation of CFTR function shifts the balance between S1P synthesis and degradation, leading to enhanced bioavailability and vascular/cellular effects of S1P.

Funding: HSFO, CIHR
Role of ZFM1 in smooth muscle cell phenotype regulation
Authors: Cattaruzza Marco , Nogoy Nicole , Hecker Markus
Published: June 2021

Vascular smooth muscle cells (SMC) can rapidly develop a synthetic phenotype in response to growth factors ‐ a phenomenon with major impact on atherosclerosis. Despite its clinical relevance little is known about the mechanisms of this potentially reversible process.

Using primary human cultured SMC and platelet‐derived growth factor (PDGF; 60 U/mL) we here show that the transcriptional repressor ZFM1 is a phenotype‐stabilizing factor in these cells. Exposure of SMC to PDGF leads to proliferation and synthetic gene expression. Interestingly, PDGF causes a rapid repression of ZFM1 gene transcription through replacement of Sp1 from the ZFM1 gene promoter by the transcription factor early growth response‐1. The subsequent drop in ZFM1 abundance disinhibits both proliferation and gene expression and is a prerequisite for PDGF‐induced effects in SMC. Consequently, siRNA‐induced ZFM1 knock down alone is sufficient to increase proliferation and expression of PDGF‐sensitive gene products. Conversely, over‐expression of ZFM1 effectively suppresses all effects of PDGF exposure.

ZFM1 expression levels pivotally determine the SMC phenotype: Whereas its down‐regulation is necessary for PDGF‐induced changes, the maintenance of ZFM1 expression is sufficient to maintain a contractile SMC phenotype.
Birds unadapted to prolonged fasting may not follow the mass loss pattern characteristic of fasting‐adapted species
Authors: Khalilieh Anton , McCue Marshall D , Pinshow Berry
Published: June 2021

Birds, such as penguins and long distance migrants, which are considered adapted to long fasts, sequentially oxidize endogenous carbohydrates, lipids and proteins to meet energy demands. The pattern of nutrient use, metabolic biochemistry, and body mass changes during fasting are generally characterized in three phases; I ‐ post‐absorptive phase; II ‐ protein sparing phase; and III ‐ lethal phase. Little is known about how birds that are not so adapted use endogenous fuels during fasting or food restriction. We hypothesized that those small birds, that do not regularly undergo long fasts have truncated, but otherwise similar fasting phases and sequential changes in fuel oxidation and blood metabolites to fasting‐adapted species. We tested our hypothesis in house sparrows (Passer domesticus) using artificially enriched 13C stable isotope tracers (glucose, palmitic acid, and glycine) and measuring 13CO2 in their breath. We predicted that fasted birds, dosed with 13C enriched tracers, have 13CO2 enrichments in exhaled breath reflecting the sequential changes in fuel oxidation during fasting, each phase being represented by a specific tracer. Our initial data show continuous, parallel oxidation of the tracers during fasting, with no clear division into phases, rather than sequential changes of fuel oxidation similar to fasting‐adapted species and, therefore, do not support our hypotheses.
Potassium ATP channel function is altered in mesenteric arteries of old high salt fed rats
Authors: Whidden Melissa Ann , Basgut Bilgen , Kirichenko Nataliya , Erdos Benedek , Tumer Nihal
Published: June 2021

Hypertension is a risk factor for cardiovascular disease and dietary salt is a contributing factor to high blood pressure. Increased salt intake in the elderly has been shown to impair relaxation of resistance vessels. Therefore, we examined whether a high salt (HS) diet alters the function of KATP channels in mesenteric arteries (MAs) from young and old rats. Young (7 mo) and old (29 mo) F‐344xBN rats were fed a control or HS diet (8% NaCl) for 12 days and 2nd order MAs were used for vascular measurements. Acetylcholine (Ach)‐induced relaxation was significantly reduced with age. Relaxation to Ach was 92 ± 2% in young vs. 81 ± 4% in old arteries. Pretreatment with the KATP channel blocker glibenclamide only reduced relaxation to Ach in young MAs. On a HS diet, dilation to Ach was reduced in old salt MAs (60 ± 3%) when compared with old control arteries (81 ± 4%; P<0.001). Glibenclamide reduced Ach‐induced dilation in young salt MAs but had no effect on old salt MAs. Cromakalim (KATP channel opener) dilation was reduced in old salt MAs when compared with old control arteries. Maximal relaxation to cromakalim was 99 ± 1% in old control vs. 85 ± 5% in old salt arteries (P<0.05). These findings demonstrate that age impairs endothelium‐dependent dilation in MAs. Furthermore, a HS diet alters the function of KATP channels in isolated MAs and affects the mediation of dilator stimuli.

Supported by the Medical Research Service of the Department of Veterans Affairs and NIA T32 AG000196 award.
Regulation of TMEM16A by a novel mechanism involving PKC in biliary epithelium
Authors: Dutta Amal Kumar , Khimji Al‐Karim , Kresge Charles , Rockey Don , Feranchak Drew P.
Published: June 2021

TMEM16A is a Ca2+‐activated Cl− channel in the apical membrane of biliary epithelial cells (BECs), and contributes to biliary secretion and bile formation [Dutta et al. JBC, 2010]. Extracellular nucleotides activate TMEM16A via binding P2 receptors and increasing [Ca2+]i. While PKC contributes to ATP‐mediated secretion in BECs, the role in TMEM16A regulation is unknown.

Aim

To determine if PKC contributes to Cl− transport in BECs through regulation of TMEM16A.

Methods

Studies were performed in human biliary Mz‐cha‐1 cells. Cl− currents were measured by whole‐cell patch clamp.

Results

TMEM16A Cl− currents were activated by exposure to extracellular ATP or increasing Ca2+ in the patch pipette. Currents had a reversal potential of 0 mV, time‐dependent activation and outward rectification. Currents were significantly inhibited by anti‐TMEM16A siRNA. In the presence of the PKC inhibitors Chelerithrine or Gö6976 Cl− currents were significantly inhibited. Conversely, activation by PMA resulted in large Cl− currents. Intracellular dialysis with recombinant PKCα activated Cl− currents with identical biophysical properties to TMEM16A.

Conclusion

PKCα couples ATP‐mediated signaling to TMEM16A activation in BECs. Modulation of kinase signaling pathways may represent a target for increasing biliary secretion and bile formation.

[Studies are supported by AASLD/ALF Liver Scholar Award 2010, Children's Medical Center Foundation grant (Dutta), and NIH NIDDK DK078587]
ELP2, A 4‐PHENYLBUTYRATE INDUCIBLE ELONGATOR COMPONENT, MODULATES HSP70 EXPRESSION
Authors: Suaud Laurence , Panichelli Ashley E , Randell Rachel L , Rubenstein Ronald C
Published: June 2021

Sodium 4‐Phenylbutyrate (4PBA) improves the aberrant trafficking of ΔF508‐CFTR in Cystic Fibrosis (CF) epithelial cells, at least in part by increasing expression of Hsp70 (stress‐induced 70 kDa heat shock protein). To identify other 4PBA‐regulated proteins that may improve ΔF508 trafficking, we performed differential display RT‐PCR on mRNA from IB3‐1 CF bronchiolar epithelial cells treated for 0–24 h with 1 mM 4PBA. StIP‐1, a STAT‐3 (signal transducer and activator of transcription‐3) interacting protein, had transiently increased expression. StIP‐1 is identical to Elongator protein 2 (Elp2). Elongator is a multimeric complex that facilitates mRNA transcription by RNA polymerase II, and is suggested to regulate Hsp70 expression. To investigate regulation of Hsp70 by Elp2, we transfected T84 colonic epithelial cells with small interfering RNAs (siRNA) targeting Elp2. Such depletion of Elp2 reduced Hsp70 protein expression by ~50%. Elp2 is also necessary for activation of STAT‐3 in response to IL‐6, and the Hsp70 promoter contains a functional STAT‐response element. We therefore tested whether 4PBA caused activation of STAT‐3 and altered nuclear protein binding to the STAT response element of the Hsp70 promoter. In IB3‐1 cells, 4PBA caused transient activation of STAT‐3 and increased binding of nuclear proteins to the STAT‐3 element of the Hsp70 promoter. These data suggest a mechanism by which 4PBA, through increased Elp2 expression, would increase Hsp70 expression and promote ΔF508‐CFTR trafficking.
Conserved amino acids in the pre‐TM1 region of the ENaC/Deg ion channel superfamily are important for gating
Authors: Kucher Volodymyr , Boiko Nina , Pochynyuk Oleh , Stockand James D.
Published: June 2021

All members of the ENaC/Deg superfamily contain a conserved His‐Gly sequence in their NH2‐terminus. Substitution of the conserved Gly results in loss of function leading to the salt wasting disorder pseudohypoaldosteronism type I. The exact role of these conserved residues to ENaC function, though, remains largely unknown. Similarly, understanding of structure‐function relations for cytoplasmic domains in ENaC/Deg channels is not as developed as it is for transmembrane and extracellular domains for which there is good crystallographic data. By combining the patch clamp method with heterologous expression of ENaC containing point mutations in these residues, we found that substitution of these critical amino acids dramatically decreases ENaC Po and leads to voltage‐sensing where Po increasing as a function of hyperpolarizing potentials. A functional element, including or near these critical residues, located on the intracellular side of the membrane must interact in an allosteric manner with the channel gate or be part of the gate to produce these results. Voltage‐sensing and loss of function upon substitution of these critical residues are species and subunit independent arguing that this putative functional element is common to all ENaC subunits and possibly all ENaC/Deg subunits. Our results are most consistent with the cytoplasmic domains of ENaC developing important structure that impacts function.
Disruption of membrane trafficking underlies the reduction in Kv1.3 channel surface expression in T lymphocytes during hypoxia
Authors: Chimote Ameet , Vergamini Sue , Filipovich Alexandra , Conforti Laura
Published: June 2021

Hypoxia (H) in solid tumors contributes to decreased immunosurveillance via Kv1.3 channel downregulation and inhibition of T cell function. However, the mechanisms responsible for Kv1.3 downregulation are not understood. We tested the hypothesis that chronic H (CH) reduces Kv1.3 surface expression via alterations in membrane trafficking. Jurkat T cells were maintained either in normoxia (21% O2) or H (1% O2) for 24h and Kv1.3 surface expression was quantified by in‐cell western blot analysis and flow cytometry using a specific Kv1.3 antibody against an extracellular epitope. CH decreased Kv1.3 surface expression in T cells by 25%. To study the involvement of endocytosis, endosome formation was blocked with Bafilomycin A1 (BAF). Comparable inhibition of Kv1.3 surface expression in CH was observed in BAF (27±4%) and control (ctr) (25±3%) cells. On the contrary, inhibition of the Golgi apparatus by Brefeldin A (BFE) prevented Kv1.3 surface expression reduction in CH (ctr 25±2% and BFE −4±3%). Furthermore, blockade of clathrin‐coated vesicle formation in the Golgi by dynasore (DYN), a selective dynamin inhibitor, prevented Kv1.3 surface expression decrease in CH (ctr 23±0.5% and DYN 0.93±1%). Our data suggest that CH disrupts forward trafficking of Kv1.3 protein from the Golgi to the plasma membrane thus contributing to the decrease in Kv1.3 surface expression in T lymphocytes. (NIH 2R01CA095286)
Retinoid‐mediated CYP26a1 expression in primary rat hepatocytes and hepatoma cells
Authors: Li Yang , Chen Guoxun
Published: June 2021

Vitamin A (or retinol) plays a crucial role in health of humans as a micronutrient. Recently we have shown that retinoid synergize with insulin to induce Gck gene expression in primary rat hepatocytes. Based on data from us and others, we believe that VA may be involved in glucose and lipid metabolism in humans.

In cells, retinol (ROL) is reversibly oxidized into retinal (RAL) by retinol dehydrogenase (RDH) and irreversibly into retinoic acid (RA), which is the activator of nuclear receptors‐retinoic acid receptor (RAR) and retinoid X receptor (RXR). We hypothesize that cells may respond to ROL, RAL, and RA depending on the catabolic enzymes of retinoid existing in them. Therefore, we determined and compared the expression levels of CYP26a1, a RA responsive gene, in response to retinoids in both primary rat hepatocytes and HL1C, a rat hepatoma cell line.

We observed that ROL, RAL and RA induced expression of CYP26a1 gene in both primary rat hepatocytes and HL1C cells in a dosage dependent manner. RAL and RA induced the expression of CYP26a1 as early as 3 hours and peaked at 9 hours. In addition, we examined the expression levels of RDH and RALDH in primary hepatocytes and HL1C cells. We found that both types of cells have comparable levels of RDH2 and RALDH1 expression. We are currently trying to confirm their roles in mediating the retinoid catabolism and retinoid‐mediated gene expression in different types of cells.

Sponsor: Guoxun Chen, 1215 W. Cumberland Ave. 229 Jessie Harris Building, Knoxville, TN37996
Role of CPI‐17 phosphorylation in calcium sensitization in smooth muscle of small intrapulmonary airways
Authors: Perez‐Zoghbi Jose F , Mukherjee Seema , Shinde Paurvi
Published: June 2021

Phosphorylation of protein‐kinase C (PKC)‐potentiated myosin phosphatase inhibitor (CPI‐17) plays a role in Ca2+ sensitization in different smooth muscle cell (SMC) types; however its role in small intrapulmonary airways is mostly unknown. We tested the hypothesis that Ca2+ sensitization is accompanied by CPI‐17 phosphorylation. We used phase‐contrast microscopy and Western blots to assess airway contraction, Ca2+ sensitivity and protein phosphorylation in mouse lung slices. We found that stimulation of lung slices with PKC activator phorbol‐12‐myristate‐13‐acetate (PMA, 10 μM) induced strong airway contraction in Ca2+ permeabilized airways due to Ca2+ sensitization. This contraction was reverted by addition of a selective PKC inhibitor (GF109203X, 1 μM) but not by a ROK inhibitor (Y‐27632, 10 μM). Phosphorylation of CPI‐17 increased after stimulation with PMA and CPI‐17 dephosphorylation was observed after subsequent addition of GF109203X. Furthermore, acetylcholine (ACh, 0.5 μM) and serotonin (5‐HT, 0.5 μM) induced strong airway contraction in Ca2+ permeabilized airways which was resistant to GF109203X and blocked by Y‐27632. Phosphorylation of CPI‐17 did not increase significantly after ACh or 5‐HT stimulation. Our results suggest that CPI‐17 phosphorylation is an important mechanism of Ca2+ sensitization but 5‐HT and ACh do not use this pathway in airway SMCs.
TP‐receptor activation potentiates airway stretch‐activated contractions
Authors: Hernandez Jeremy Mark , Janssen Luke Jeffrey
Published: June 2021

A deep inspiration (DI) produces a bronchodilation in healthy individuals. Conversely, in asthmatics, DIs are ineffective in producing bronchodilation, and can even cause bronchoconstriction. Interestingly, the manner by which a DI is able to cause bronchoconstriction via a stretch‐activated contraction (Rstretch) correlates positively with airway inflammation, during which there is increased production of thromboxane A2 (TxA2) and TP‐receptor activation.

In this study we sought to investigate the effect of TP‐receptor activation on airway Rstretch in bovine bronchial segments using the Mayflower organ bath as previously reported by our laboratory. Rstretch was elicited by varying the transmural pressure under isovolumic conditions. Using a pharmacological approach, we showed a reduced Rstretch response in tissues pretreated with indomethacin (Indo), a COX inhibitor; a result mimicked by the TP‐receptor antagonist ICI 192605 and by airway epithelial denudation. U46619, a TP‐receptor agonist elicited enhanced Rstretch responses in a dose‐dependent manner. AH6809, an EP1/DP‐receptor antagonist and AL 8810, an FP‐receptor antagonist had no effect, suggesting PGE2, PGD2, and PGF2 [alpha] are not involved in ASM Rstretch.

These data suggest a role for the TP‐receptor and epithelial release of TxA2 in potentiating the effects of airway Rstretch.

Research Support: CIHR, NSERC
L‐Citrulline Protects Against Hyperoxia‐Induced Increase in Airway Contractile Responses
Authors: Sopi Ramadan B , Mladenov Mitko , Gjorgoski Icko , Jakupaj Muharrem
Published: June 2021

Neonatal hyperoxia increases contraction and decreases relaxation of airways in rat pups due to upregulation of arginase activity and decrease of L‐arginine bioavailability to nitric oxide synthase (NOS). Therefore, we tested the hypothesis that supplementation of L‐citrulline will overcome the increased contractile responses of tracheal smooth muscle (TSM) induced by hyperoxia. Tracheal cylinders were excised from rat pups 12 days old, exposed to hyperoxia or room air for seven days. To induce contraction of TSM, electrical field stimulation (EFS) was applied. Hyperoxia increased EFS‐induced contractile responses as compared to room air animals (1.29 ± 0.1 g vs. 0.83 ± 0.2 g). In vitro supplementation of L‐citrulline decreased the contractile responses in hyperoxic group, but did not show any effect in control group under basal conditions. Inhibition of enzymes involved in L‐citrulline recycling prevented this effect of L‐citrulline. Inhibition of NOS, increased airway contraction in control group but did not affect the responses in hyperoxic group. Incubation with exogenous L‐citrulline reversed the increased contractility.

These data demonstrate that L‐citrulline/L‐arginine cycle is effective in TSM under conditions of reduced L‐arginine bioavailability to NOS. In vitro supplementation of L‐citrulline reverses the increased contractile responses in hyperoxic animals to control levels.
Multi‐cycling of pulmonary surfactant reveals effects of loading history and inhaled environment
Authors: Gouldstone Andrew , Silva Maricris R , Boloorizadeh Parnian
Published: June 2021

The objective of this work was to explore previous published results on lung mechanics and alveolar collapse under different physiologic conditions. Specifically, we had performed indentation experiments on excised lung, and reported immediate, significant and near‐reversible changes in lung stiffness and hysteresis when inhalant gas was changed from room air to 100% oxygen. We hypothesized this was at least in part due to changes in surfactant behavior. To test this, we performed Wilhelmy balance experiments on isolated surfactant (Infasurf) under different loading conditions and gaseous environments. In all the experiments, temperature, humidity and atmospheric composition were controlled, and several compression‐relaxation cycles generated. We found significant effects of several experimental conditions, including oxygen concentration, on the progressive (cycle‐to‐cycle) behavior of the surfactant. The results are relevant to (a) mechanics of breathing, particularly the concept of sigh, and (b) mechanisms of pulmonary oxygen toxicity.
Discovery of the anterior fibulocalcaneus muscle: an anomalous leg muscle
Authors: Lambert H Wayne , Atsas Stavros , Billings Heather J. , Campbell Ferrell R
Published: June 2021

A novel muscle, the anterior fibulocalcaneus, has been discovered bilaterally in the anterior compartment of the leg in three separate cadaveric dissections. This muscle originates from the fibula, anterior crural septum, and the investing fascia of the fibularis tertius. Its tendon passes anterior to the lateral malleolus to insert onto the calcaneus, anterosuperior to the fibular trochlea. Anomalous fibulocalcaneal muscles associated with the lateral malleolus have been noted to cause a variety of ankle pathologies, and supernumerary muscles within the talocrural joint have been utilized for surgical reconstructive procedures. Radiologists and clinicians should be aware of this muscle when embarking in diagnosis and imaging interpretation, especially when chronic ankle pain is localized anterior to the lateral malleolus (Lambert and Atsas, 2010).
Macro vascularization of the talus and relationships for triple arthrodesis
Authors: Pizzimenti Marc A , Haugsdal Jaclyn , Phisitkul Phinit
Published: June 2021

Understanding the macro vascularization of the talus is important for a number of orthopaedic procedures. In the current study, we outlined the general primary arterial supply to the talar and peritalar regions. Two methods were used to construct the extraosseous vascular map of the talus: macro dissection of embalmed specimens; and CT analysis of unembalmed specimens. The general arterial pattern construct, with variations, was determined by comparing the observed vascular trees with those reported in the literature. Unembalmed specimens were injected with latex containing a barium sulfate suspension, from three access points. CT analysis of the specimens provided 3D assessment of the vascular patterning, which in turn permitted reliable measures and locations for major extraosseous branches supplying the talus relative to major anatomical landmarks. Identifying the locations of these vessels can aid in guiding surgical approaches for triple arthrodesis with minimal vascular disruption.

Financial and resource support was provided by the University of Iowa
Analysis of the upper forelimb of Canis dirus to determine physical characteristics that might suggest sociality
Authors: Koper Lindsey , Naples Virginia L
Published: June 2021

Canis dirus guildayi, or the Western Dire wolf, was the most common predator at Rancho La Brea and is believed to be social as are modern wolves. In this study, the upper forelimb, specifically the scapula, humerus, and ulna of C. dirus were examined. Maps determined from examination of ridges, rugosities and other features reflecting alteration of bone surfaces by muscle attachments were created. Interpretation of significance of muscle scars on these bones was made possible by comparison to dissections of the forelimb musculature of Canis latrans (American coyote) and Urocyon cinereagenteus (Red Fox). The dire wolf is considered a social predator, in part from previous studies that characterized specific healed or partially healed skeletal injuries that parallel those in social modern wolves hunting large prey in groups. Further support for sociality in Dire wolves that also specialized in capture of large prey is suggested by anteroposterior scapular lengthening in some larger individuals. This shape, surface area and muscle scar position allowed for a relatively larger origin of the M. serratus ventralis, a muscle that supports the trunk, assists with inspiration and moves the trunk and the scapular region anteroposteriorly. This capability would allow for increased anteroposterior movement of the limb, an advantage for a pursuit predator cursor hunting in cooperation with conspecifics to capture large prey.

Grant Funding Source: Northern Illinois University
Moving Forward with the Mysticete Hyoid: biomechanics of the whale's hyoid in body wave locomotion
Authors: Reidenberg Joy S. , Laitman Jeffrey T.
Published: June 2021

The hyoid apparatus serves as an attachment site for the muscles associated with mastication, deglutition, respiration, and vocalization. In odontocetes (toothed whales, including dolphin and porpoises), the hyoid (basal elements of the hyoid apparatus including the midline basihyal and paired thyrohyals) is large and flattened into a boomerang shape. Inferior movement creates negative pressure in the oral cavity, thus drawing prey into the mouth. Superior movement causes nasopharyngeal pressurization that drives generation of echolocation pulses. In mysticetes (baleen whales), the hyoid is also robust, but rather than being flattened it forms a tubular shape. Hyoid anatomy was studied in 4 mysticete genera (representing 7 species) and compared with hyoids in 14 odontocete genera and 43 terrestrial genera. Results indicate that mysticetes have an unusually thick hyoid periostium that extends caudally in the midline. This thick tissue is sometimes augmented by paired posterior projections of the basihyal, and provides additional surface area for muscular attachments of the infrahyoid muscles. Two prominent rostral projections of the basihyal provide additional surface for attachment of the suprahyoid muscles. These shapes indicate primary movements in the rostro‐caudal axis, e.g., mandibular depression. Contraction of the geniohyoid and sternohyoid muscles while the gape is held closed would cause head depression, thus initiating the body wave of forward swimming. Locomotion is a novel role for the hyoid and may be unique to whales.
Tendon injury and disease: finding a suitable experimental model
Authors: Evans Darrell JR , Agabalyan Natasha A , Stanley Rachael L
Published: June 2021

Cell type and function are poorly defined in tendon in comparison to other cells of the musculoskeletal system. It is not known why tendon cells (tenocytes) fail to fully repair degenerated tendon with a suitable matrix or how and why they are able to ossify their matrix under certain conditions. In some avian species, including the domestic chicken (gallus gallus domesticus), certain tendons of the distal limb undergo apparent spontaneous and ‘normal’ ossification/calcification. Although the appearance of ossified avian tendon has been previously described there have been no studies looking at the developmental processes and mechanisms underlying the ossification or the cell populations involved. We have investigated the nature of the ‘ossified’ tendons tendon taken from the tarsometatarsus region of the hind limb using MRI and histological preparations. We found that the ossified tendons are indistinguishable from bone when examined using MRI and that both flexor and extensor tendons have similar profiles. Histologically, the cells within these tendons have a chondrocytic and osteogenic as well as a tenocytic phenotype, although the cells of each phenotype are regionalised to particular proximal to distal sites along the tendon. Tendon ossification appears to be a endochondral process in terms of the histology but it is unclear whether the cell populations residing in each location are related to each other.
The effects of exercise, methylprednisolone, and a serotonin 5‐HT2br agonist on cardiac remodeling and quadriceps fibrosis in dystrophin deficient mice
Authors: Plochocki Jeffrey H , Costas Jeffrey M , Ho Renita
Published: June 2021

Dilated cardiomyopathy is a leading cause of death in patients with muscular dystrophy (MD). Although there is no cure for MD, exercise and corticosteroids (CS) are commonly prescribed to prolong cardiac function. To test the affects of these treatments on cardiac mass and fibrosis, dystrophin deficient (mdx) mice were separated into control and treatment groups. Additionally, mice were treated with an agonist for the serotonin 5‐HT2b receptor, which is known to play a role in cardiac hypertrophy. The 5‐HT2br agonist was the only treatment that significantly elevated cardiac mass and left ventricular wall thickness above controls, while reducing cardiac fibrosis (P < 0.05). Mice treated with voluntary running exercise exhibited significantly reduced fibrosis compared to controls, but similar cardiac size. CS treatment and CS treatment combined with exercise was not effective at reducing fibrosis and maintaining cardiac mass. Quadriceps fibrosis was significantly diminished only in the CS treated mice relative to controls. Our findings suggest 5‐HT2br agonists may maintain cardiac mass in dystrophin deficient mice and should be further investigated as a treatment modality for MD.
Immunohistochemical localization of pituitary adenylate cyclase‐activating polypeptide (PACAP) and its receptor (PAC‐1) in mouse major salivary glands
Authors: Nonaka Naoko , Nakamachi Tomoya , Shioda Seiji , Nakamura Masanori
Published: June 2021

The control of saliva secretion is mainly under the autonomic nervous control. PACAP is now recognized as the multi‐functional neuropeptide in various organs. We compared the distribution of PACAP and PAC‐1, in major salivary glands; parotid, submandibular and sublingual glands, of young and old C57BL/6 mice. In parotid gland, PACAP was mainly detected in the cells of intercalated ducts. PAC‐1 was detected in the cells of striated duct. In submandibular gland, PACAP was distributed in the nerve fibers adjacent to acini and granular ducts and the strong immunoreaction of PAC‐1 was detected in the tall columnar epithelial cells in granular ducts and some of the cells in the striated ducts. In sublingual gland, while PACAP‐positive neural fibers surrounded the acini, PAC‐1 was expressed mainly in the epithelial cells of striated ducts. These results indicated the PACAP‐positive innervations in three major salivary glands in young and old mice and also suggested the functional differences of PACAP between three salivary glands.

Supported by the Grant‐in‐Aid for Scientific Research (20592148, 21592342) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
Central Vein Fibrosis in Cadaveric Livers of the Elderly
Authors: Mak Ki M. , Hoo Nancy M.
Published: June 2021

Liver fibrosis develops in stages with central vein fibrosis (CVF), perisinusoidal fibrosis (PSF) and portal tract fibrosis (PTF) occurring at early, septal fibrosis at late and cirrhosis at end stage. These lesions were seen in the liver of elderly cadavers with diverse causes of death (Mak et al. FASEB J 2010; 24: 446.11). This study examined 50 cadavers (mean age = 81.3±12.5 yrs) for CVF to evaluate its role in liver fibrosis. Paraffin sections were stained for collagens with Sirius red, a marker for fibrosis. 250 central veins were analyzed by morphometry. The mean vein diameter was 71.1±18.2 μm. CVF was considered present when the thickness of collagens in the rim of the vein exceeded 9 μm with extension of fibrous tissue into the perivenous parenchyma. CVF occurred in 25 livers (50%). Of these, 23 (92%) also had PSF, 12 (48%) PTF, 10 (40%) septa formation, 10 (40%) bridging fibrosis and 1 (4%) cirrhosis. Livers without CVF had a lower incidence of PSF (40%, P = .0002), PTF (28%), septa formation (36%) and bridging fibrosis (20%).

Conclusion/implication

Histogenesis of CVF is associated with PSF in early fibrogenesis. The fibrosis interferes with intrahepatic blood flow, which stimulates advanced fibrogenesis and subsequent portal hypertension. These changes impair hepatic functions and enhance disease susceptibility in older people. Thus fibrosis in the aging liver may have a detrimental health effect in the elderly.