bluuebunny/crossref_metadata_2025 huggingface dataset:
Protective effect of Diosmin on Lipopolysaccharide (LPS) ‐ induced PC12 cell death
Authors: Benzeroual Kenza E , Dholakiya Sanjay L
Published: June 2021

Diosmin is a natural flavones glycoside therapeutically used to improve the symptoms of venous and lymphatic vessel insufficiency and also exhibits anti‐inflammatory properties. In the present study, LPS‐injured PC12 cells were used as an in vitro cell model of Alzheimer's disease (AD) to investigate the neuroprotective effects of diosmin. PC12 cells treated with LPS exhibited an increase in TNF‐alpha, and underwent apoptotic death as determined by western blot and agarose gel electrophoresis assay. The MTT assay results showed that pretreatment with diosmin produced a dose‐dependent increase in cell viability that was maximal at 5 μM of diosmin. Furthermore, western blot analysis showed a dose‐dependent decrease in the expression of TNF‐alpha when PC12 cells were pretreated with diosmin prior to treatment with LPS for 48 hours. Western blot and Caspase‐3 colorimetric assay revealed that diosmin exerts its anti‐apoptotic effects by decreasing the expression of pro‐apoptotic protein Bad, increasing the expression of antiapoptotic protein Bcl2, and decreasing caspase‐3 levels. The present study suggests that diosmin could be used as a neuroprotective agent to slow the progression of neurodegenerative diseases, including AD. Research suported by LIU‐College of Pharmacy
Effect of PRL‐3 phosphatase knockout in cancer and angiogenesis
Authors: Zimmerman Mark W , Isenberg Jeffery S , Homanics Gregg E , Lazo John S
Published: June 2021

Phosphatase of regenerating liver–3 (PRL‐3 or Ptp4a3) is a small, prenylated, protein tyrosine phosphatase that is highly conserved throughout mammalian species. PRL‐3 is considered an attractive therapeutic target for the treatment of metastatic cancer due to high expression levels frequently observed in malignant tumors, distant metastases, and the tumor endothelium. While the mechanistic properties of PRL‐3 are not well understood, the gene has been associated with the progression of multiple human cancer types and increased expression typically correlates with increased tumor invasiveness. Interestingly, our data suggests that increases in PRL‐3 expression can also occur early in a carcinogen‐derived mouse model of colorectal cancer, potentially contributing to primary tumor development. In order to shed light on its function in normal and malignant cells, we have created a novel gene‐targeted mouse model to knockout the PRL‐3 gene product. Importantly, mice that do not express functional PRL‐3 are viable, fertile, and overtly normal. PRL‐3 knockout mice exhibit less angiogenic responses and vascular cell invasion compared to wildtype control mice. These experiments indicate that PRL‐3 likely has unique in vivo functions and may be an effective target for the treatment of malignant disease.
Tmem16a‐induced ClCa currents are regulated via phosphotransferase activity
Authors: Ayon Ramon Jose , Wiwchar Michael , Singer Cherie , Valencik Maria , Greenwood Iain A , Leblanc Normand
Published: June 2021

Ca2+‐activated Cl− currents (ICl(Ca)) represent a major excitatory mechanism in vascular smooth muscle cells (SMCs). In arterial SMCs, ICl(Ca) are down‐regulated by calmodulin‐dependent protein kinase II (CaMKII) and up‐regulated by calcineurin (CaN) and PP1. Recently, Tmem16a has emerged as a gene candidate for the native ICl(Ca) in pulmonary artery SMCs. HEK‐293 cells over‐expressing Tmem16a displayed time‐ and voltage‐dependent Cl− currents when dialyzed with 500 nM free Ca2+ that resembled the properties of ICl(Ca) from vascular SMCs. Similar to ICl(Ca) from VSMCs, Tmem16a‐induced ICl(Ca) also displayed enhanced rundown when cells were dialyzed with 5 mM ATP and this process was attenuated in the absence of ATP. Similar to ICl(Ca) in rabbit VSMCs (Ayon et al., J Biol Chem 284:32507–32521, 2009), dialysis of 30 nM okadaic acid, a nonselective PP1/PP2A inhibitor, impeded the recovery of Tmem16a‐dependent Cl− currents in the absence of ATP. Analysis of mouse Tmem16a protein sequence revealed 5 consensus sites for phosphorylation by CaMKII, which are believed to lie on protein segments facing the cytoplasm. A single point mutation of one of these putative CaMKII phosphorylation sites (Threonine 610 to Alanine) did not alter rundown of the channels. Taken together, these data suggest that much like native ICl(Ca), Tmem16a‐dependent Cl− currents also appear to be regulated by at least one phosphorylation step.
Ah receptor interacts with Nrf2 to mediate induction of NQO1 by 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin and benzo[a]pyrene
Authors: Wang Liping , He Xiaoqing , Bi Yongyi , Szklarz Grazyna , Ma Qiang
Published: June 2021

Ah receptor mediates the induction of NQO1, GSTs, and UGTs by 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) and benzo[a]pyrene (B[a]P); whereas, Nrf2 is critical for the induction of the genes by antioxidants such as tert‐butylhydroquinone (tBHQ). Induction of the genes by AhR agonists or antioxidants requires “dioxin response element” (DRE) or “antioxidant response element” (ARE) respectively. We have used induction of NQO1 as a prototype to elucidate the molecular steps governing the induction of the genes. We previously reported a cross‐interaction between AhR and Nrf2 signal transduction is required for induction of NQO1 by TCDD (Ma et al, Biochem J 377, 205–213, 2004). In this study, we analyzed the interaction between AhR and Nrf2 at the promoter of NQO1. Chromatin immunoprecipitation analyses revealed that treatment with TCDD recruits both AhR and Nrf2 to the promoter region where a DRE and an ARE locate; the finding is in agreement with the result from genetic studies in which induction by TCDD or B[a]P was shown to require both AhR and Nrf2. TCDD‐induced binding of AhR and Nrf2 to DNA is time‐dependent. Consistent with the activation of Nrf2, TCDD treatment inhibits Keap1‐dependent ubiquitination and proteasomal degradation of Nrf2 resulting in the stabilization and nuclear accumulation of Nrf2. Co‐immunoprecipitation experiments revealed that AhR directly interacts with Nrf2 in the presence of TCDD. Our findings demonstrate that AhR interacts with Nrf2 to induce NQO1 and provide a molecular model for studying the induction of NQO1, GSTs, and UGTs by AhR agonists.
Oxygen generating materials for retaining skeletal muscle function
Authors: Ward Catherine Lindsey , Corona Benjamin , Yoo James J , Harrison Benjamin S , Christ George J
Published: June 2021

Particulate oxygen generators (POGs) represent a novel class of biomaterials with numerous potential applications to tissue engineering and regenerative medicine. POGs were evaluated for their ability to preserve muscle function in vitro, using the rat extensor digitorum longus (EDL) muscle as a model system in a simulated hypoxic environment. Whole EDL muscles were isolated from the hindlimb of mature adult rats, assigned to treatment groups (no treatment, needle injection, saline injection, POG injection) and mounted in a 37°C DMT organ bath system and subjected to electric field stimulation (200Hz, 22V, 200ms train, 0.2ms pulse). Muscle function comparisons were made by testing each individual muscle's contractility in 1) oxygenated baths and 2) deoxygenated baths. Statistical analysis was performed between treatment groups using a one‐way ANOVA and between oxygen conditions using a two‐way ANOVA with appropriate post‐hoc testing analysis. POGs provided a sufficient amount of supplemental oxygen during the course of the protocol to support a functioning rat EDL muscle in a hypoxic environment in vitro. This study provides important guidance concerning the amount of oxygen‐generating material needed for engineering skeletal muscle in vitro and for tissue regeneration and repair, as well as prevention of ischemia, in vivo. Funded by NIH 5R01AR57325‐2 & Armed Forces Institute for Regenerative Medicine.
Effects of age and hypertension on α1‐adrenoceptors in the major source arteries of the rat bladder and penis
Authors: Yono Makoto , Tanaka Takanori , Tsuji Shigeki , Irie Shin , Sakata Yukikuni , Otani Masayuki , Latifpour Jamshid
Published: June 2021

As aging and hypertension may impact pelvic arterial blood flow resulting in bladder and penile dysfunction, we investigated effects of age and hypertension on α1‐adrenoceptors in the major source arteries of the rat bladder and penis. Using radioligand receptor binding, real‐time RT‐PCR and fluorescent microsphere infusion techniques, we compared 3 and 22‐month‐old male Fischer rats, and male normotensive Wistar‐Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs). Twenty‐two‐month‐old rats and SHRs had significantly higher total α1‐adrenoceptor density in the internal iliac artery and lower blood flow to the bladder and penis than 3‐month‐old and WKY rats, respectively. RT‐PCR data showed an age and hypertension related increase in the expression of α1B‐adrenoceptor mRNA in the internal iliac, vesical and internal pudendal arteries and a switch from α1A predominance in 3‐month‐old and WKY rats to α1B>α1A in 22‐month‐old rats and SHRs. These findings suggest that pharmacological blockade of the vascular α1B‐adrenoceptor, which could increase pelvic blood flow, may contribute to the improvement of bladder and penile dysfunction in animal models for aging and hypertension. Supported by KAKENHI 21791511, Grant‐in‐Aid for Young Scientists (B) provided by the Ministry of Education, Culture, Sports, Science and Technology of Japan.
Identification of G protein‐coupled receptor (GPCR) targets in pulmonary artery smooth muscle cells
Authors: Aroonsakool Nakon , Kwon Ohmin , Insel Paul A , Murray Fiona
Published: June 2021

The second messenger cAMP is an important regulator of vascular tone and cellular proliferation in the pulmonary artery (PA). Decreased intracellular levels of cAMP in pulmonary artery smooth muscle cells (PASMC) contribute to the abnormal tone and remodeling in the PA that characterizes diseases such as pulmonary arterial hypertension. G‐protein coupled receptors (GPCRs) that couple to Gαs or Gαi, are attractive drug targets to increase cAMP in PASMC. We used a TaqMan® GPCR array to identify novel pulmonary specific GPCRs that could regulate cAMP‐dependent signaling in human PASMC. We found that PASMC express 88 GPCRs, the most abundant of which were orphan receptors. Using real‐time PCR, antibodies and specific agonists and antagonists we validated the most abundant Gαs‐ and Gαi‐coupled GPCRs. Initial efforts focused on the vasoactive intestinal peptide receptor 1 (VIPR1), one of the highest expressed Gαs ‐coupled GPCRs in PASMC. We found that VIPR1 protein is expressed on the plasma membrane of PASMC, activation of which by VIP (1 μM, 10 min) significantly increased cAMP accumulation. These data demonstrate quantitative mRNA expression of GPCRs in PASMC and evidence that such an approach can identify previously unrecognized GPCRs that contribute to physiology and pathophysiology in the pulmonary vasculature. Supported by NIH
S1P‐induced vasoconstriction increased in basilar artery of a subarachnoid hemorrhage model in rats
Authors: Soydan Guray , Xu Jian , Sims John Randall , Waeber Christian
Published: June 2021

Sphingosine‐1‐phosphate (S1P) potently constricts cerebral arteries and plays key functions in the immune, inflammatory, and cardiovascular systems. Inflammation has an important function in the development of cerebral vasospasm after subarachnoid hemorrhage (SAH). S1P is generated and stored in erytrocytes, leukocytes, endothelial cells and platelets and released by platelets during blood clotting. We investigated the functional effect of S1P in basilar artery in a rat subarachnoid hemorrhage model. Using an infusion pump 400μl blood or saline was given with intracysternal injection to young (200–300g) Sprague‐Dawley male rats. After 48 hours brain was removed and basilar artery was isolated and mounted into a wire myograph. Vasoconstricton to KCl, S1P, serotonin (5‐HT), endothelin‐1 (ET‐1), U46619 (a tromboxane A2 agonist) and vasodilatation to acetylcholine (Ach) and sodium nitroprusside (SNP) responses were measured. Compared to saline group, 5‐HT‐, S1P‐ and ET‐1‐induced vasoconstrictions were significantly increased in blood given group whereas KCl and U46619 did not show a difference. There was no difference in Ach‐ and SNP‐induced vasodilations between saline and blood group. In summary, our results suggest that basilar artery constriction to S1P, 5‐HT and ET‐1 are increased in a rat model of SAH and can be a new target for the treatment of vasospasm after SAH.
Measurement of basal cerebral perfusion and metabolic activity in LDLr−/−;hApoB+/+ mice
Authors: Rousseau Jacques , Bolduc Virginie , Thorin Eric , Lecomte Roger
Published: June 2021

The aim of this study was to investigate the feasibility of using positron emission tomography (PET) imaging with readily available radiotracers to assess the effect of atherosclerosis on basal cerebral perfusion and metabolic activity in mice. 13NH3 and 18FDG were selected since the former is widely used for the measurement of myocardial perfusion and the latter is a known marker of cellular glucose metabolism, including in the brain. Adult 12 month‐old male C57Bl/6 mice (n=3) and LDLr−/− ApoB+/+ mice (n=7) with established atherosclerosis were used. The uptake of 13NH3 and 18FDG was measured in both the cortex and the center of the brain. PET images showed consistent higher (30%, p≤0.0001) cerebral 13NH3 uptake that was associated with higher (10%, p≤0.001) cerebral 18FDG uptake in atherosclerotic mice. These data validate the use of PET imaging to measure basal cerebral perfusion and brain metabolic activity in mice. In addition, they suggest that atherosclerosis is associated with higher cerebral perfusion and increased glucose uptake that could reflect hypoxia associated with atherosclerosis, since chronic hypoxia is known to increase brain capillary density and glucose transport across the blood‐brain barrier leading to an enhanced brain 18FDG uptake. Supported by CIHR (MOP 89733).
Improved intraischemic perfusion and pial arteriolar dilation by transfusion of pegylated hemoglobin in the carbon monoxide state
Authors: Cao Suyi , Koehler Raymond C
Published: June 2021

Transfusion of chemically‐modifed, cell‐free hemoglobin (Hb) during middle cerebral artery occlusion (MCAO) can reduce infarct volume, but large amounts are typically required. We tested the concept that transfusion of Hb in the CO state would promote vasodilation during MCAO without requiring large amounts of Hb to be transfused. Transfusion of 10 ml/kg of a 4% solution of pegylated COHb (PEG‐COHb) in anesthetized rats resulted in rapid release of CO and equilibration of CO with red cell‐based Hb (2–3% COHb in whole blood and plasma). Plasma [Hb] was ~0.5 g/dL. Induction of MCAO initially produced 37±8% (±SD) dilation of pial arterioles in the MCA border region that gradually subsided to 7±7% at 2 h in controls. Transfusion of PEG‐COHb at 20 min of MCAO maintained pial arterioles in a dilated state (40±13%) at 2 h, whereas transfusion of albumin (25±13%) or PEG‐Hb without CO (22±8%) had intermediate effects. When transfusion of PEG‐COHb was delayed by 90 min, penumbral laser‐Doppler flow increased from 58% to 80% of pre‐ischemic baseline, whereas transfusion of albumin or PEG‐Hb produced no significant change. We conclude that PEG‐COHb can serve as a CO donor that effectively prevents time‐dependent loss of pial arteriolar dilation during MCAO and improves blood flow in the penumbral region to levels above the threshold typically associated with eventual infarction. (Supported by NIH NS38684).
Effect of plasma on vessel tone and endothelial function
Authors: Palomares Sara Morales , Gardner‐Morse Ira , Cipolla Marilyn J.
Published: June 2021

The majority of isolated vessel experiments are performed with buffer or artificial cerebrospinal fluid in the lumen. Because the presence of plasma could affect vascular function and may be more physiological, we evaluated the influence of plasma on middle cerebral arteries (MCA). Arteries were isolated from male Wistar rats, mounted on glass cannulas in an arteriograph chamber and perfused with physiological saline solution (Ringer's PSS buffer; CTL, n=8) or with plasma (20% in buffer; PL, n=7) pooled from 5 normal rats. After 2 hours of equilibration at 75 mmHg myogenic tone and constriction to the NOS inhibitor L‐NNA (0.1mM) and the cyclooxygenase inhibitor indomethacin (10μM) were compared. The active diameters were significantly smaller at all pressures studied in arteries perfused with plasma than in CTL arteries (at 75mmHg: 243±9μm for CTL vs. 190±9μm for PL; p≤0.001). PL vessels also had greater basal myogenic tone compared with no plasma CTL (at 75mmHg: 10±3% for CTL vs 19±3% for PL; p≤0.05). The perfusion of plasma caused decreased constriction to L‐NNA (41±5% for CTL vs 19±3% for PL; p≤0.01) but did not affect constriction to indomethacin, suggesting the contribution of NO to tone is affected by plasma. In conclusion, there appear to be circulating factors in plasma that increase myogenic tone in the MCA, possibly though decreasing the contribution of NO. Supported by RO1NS043316 and PO1HL95488.
Endothelial TRPC1 and TRPC3 channels contribute to ATP‐mediated relaxation in mouse cerebral arteries
Authors: Kochukov Mikhail Y , Noel Rebecca C , Abramowitz Joel , Birnbaumer Lutz , Marrelli Sean P
Published: June 2021

Canonical transient receptor potential (TRPC) channels in endothelial cells (EC) may play an important role in regulation of vascular tone by mediating Ca2+ dependent relaxation. We tested the role of TRPC1 and C3 in ATP‐mediated Ca2+ signaling and vasorelaxation in mouse posterior cerebral artery (PCA).

Experiments were performed on wild type (WT), TRPC1, and TRPC3 knockout (KO) mice. Isolated PCA were placed in a pressurized artery chamber and monitored for changes in tone by video‐microscopy. ATP was delivered to the endothelium through the artery lumen. Intracellular calcium ([Ca2+]i) was measured by Fura 2 in primary cultures of aorta EC.

PCA from TRPC1 and C3 KO mice developed tone and constricted to endothelin‐1 comparably to WT, but demonstrated impaired vasodilation responses to ATP. In aorta EC, ATP produced an increase in [Ca2+]i insensitive to P2X antagonist NF279 but susceptible to inhibition by TRPC modulator La3+ or PLC blocker U73122. ATP‐induced changes in [Ca2+]i were similar in WT and C3 KO but reduced in C1 KO EC. Pyr3, a TRPC3 blocker, inhibited the [Ca2+]i response significantly in WT but not C3 KO, demonstrating a role of TRPC3 in EC.

Our data support a critical role for TRPC1 and TRPC3 in endothelium‐dependent relaxation to ATP in cerebral arteries. TRPC1 and C3 both appear to be functionally important to ATP‐mediated Ca signaling in aorta EC.

Supported by NIEHS Z01‐ES‐101684 and NIH R01 HL088435
Reduced coronary vasodilatation to diadenosine tetraphosphate after ischemia‐reperfusion in isolated rat heart
Authors: Garcia‐Villalon Angel Luis , Fernandez Nuria , Monge Luis , Dieguez Godofredo
Published: June 2021

Diadenosine polyphosphates increase during coronary ischemia. To analyze their role in coronary ischemia‐reperfusion, the response to diadenosine tetrataphosphate (Ap4A, 10−7 ‐ 10−5 M) was recorded in isolated perfused rat hearts after precontraction of the coronary vasculature with 11‐dideoxy‐1a,9a‐epoxymethanoprostaglandin F2α (U46619, 10−8 ‐3×10−8). In control hearts, Ap4A produced concentration‐dependent vasodilatation both at the basal coronary resting tone or after precontracting coronary vasculature with U46619, and this vasodilatation was reduced by the antagonist of purinergic P2Y receptors reactive blue 2 (2×10−6 M) and the blocker of ATP‐sensitive potassium channels (KATP) glibenclamide (10−5 M). After ischemia‐reperfusion, the vasodilatation to Ap4A diminished, and in this case the relaxation to Ap4A was not modified by reactive blue 2 or glibenclamide. The agonist of P2Y purinergic receptors UTP (10−7 ‐ 10−5 M) produced coronary vasodilatation which also was reduced after ischemia‐reperfusion. These results suggest that the coronary relaxation to Ap4A is reduced after ischemia‐reperfusion, and that this reduction may be due to impaired response of purinergic P2Y receptors and of KATP potassium channels. (Supported by FMMA grant Nº AP57242009 and FIS grant Nº PS09/00394)
Mammalian differences in vagal‐cholinoceptor control of coronary conductance
Authors: Hamut Mutalip , Quail Anthony , Seah Peng , McLeod Damian , Cottee David , White Saxon
Published: June 2021

Although there is no consistency of vagal‐cholinoceptor coronary control mechanisms between species, the anatomical pattern of coronary blood supply in sheep is similar to the dog. The vasodilatation to both acetylcholine (ACh) infusion and electrical stimulation of vagus nerve in the dog, therefore, may also be expected in the sheep. Pulsed Doppler flow probes were implanted on the right (R), circumflex (Cx) and anterior descending (AD) coronary arteries of the sheep. Pressure catheters were placed via the superficial cervical vein and artery in the right atrium and in the aorta respectively. In awake sheep, ACh caused vasodilatation in all three coronary beds. There was a 3‐fold conductance increase in R (p<0.001) compared to 2.5 fold in left‐sided beds (p<0.001). Effective dose 50% (ED50) for Cx was less than that in AD (p<0.05) and in R (p<0.05). In the same sheep under isoflurane anaesthesia and pacing at 150 min−1 direct efferent vagus nerve stimulation caused frequency‐dependent simultaneous vasoconstriction in all three coronary beds. The vasoconstriction was greatest in Cx (p<0.001), intermediate in AD (p<0.001), and least in R (p<0.001). These findings suggest that there are qualitative and quantitative differences between mammalian species. In awake sheep, constrictor cholinoceptors in the media of coronary arteries may play a dominant role. Funding: Hunter Medical Research Institute.
Coronary arterioles of type 2 diabetic patients exhibit diminished dilation to sudden increases in wall shear stress
Authors: Cassuto James , Feher Attila , Labinskyy Nazar , Lansman Steven , Bagi Zsolt
Published: June 2021

Percutaneous coronary intervention (PCI) is commonly associated with coronary no‐reflow (CnRF), a poorly understood phenomenon in which myocardial hypoperfusion persists despite re‐opening the occluded coronary artery. Patients with diabetes (DM) are highly susceptible for developing CnRF. We hypothesized that CnRF is due to the disturbed regulation of coronary arteriolar diameter by wall shear stress (WSS) in DM patients. Coronary arterioles (~100 um) were dissected from atrial appendages and diameter changes were measured to low (10 dyne/cm2) and high (50 dyne/cm2) levels of WSS, as evoked by increases in intraluminal flow ‐ simulating arteriolar WSS changes during PCI. In response to increases in WSS, arterioles from nonDM patients developed significant dilation (19±4% and 8±6%). Arterioles from DM subjects did not respond to increases in WSS (3±4% and −4±1%). Inhibition of NO synthesis with L‐NAME attenuated WSS‐induced dilation in nonDM vessels (5%±3%), while no effect was observed in DM arterioles (−2±2%). The NO donor sodium nitroprusside elicited similar dilation in the two groups. As assessed by quantitative RT‐PCR, there was no difference in eNOS expression in arterioles of DM and nonDM patients. Thus, lack of WSS‐induced NO release in vessels of DM patients leads to a diminished coronary dilation, which may contribute to CnRF. (Supported by: NIH HL104126, PO1HL43023, BHF RE/08/004)
Decreased maternal dietary protein intake during pregnancy alters large conductance, calcium‐activated K channel (BKCa) function in fetal coronary artery smooth muscle cells
Authors: Shukla Praveen , Modgil Amit , Sun Chengwen , Vonnahme Kimberly , Schauer Christopher , Caton Joel , Reynolds Larry , O'Rourke Stephen
Published: June 2021

We have shown that maternal nutrient restriction during pregnancy impairs an EDHF‐like pathway that is mediated via BKCa activation in fetal coronary arteries (CA). We hypothesized that BKCa activity is altered in CA of fetuses from malnourished ewes. Pregnant ewes were fed diets that differed only in metabolizable protein levels (control = 100% vs low protein = 80% of NRC recommendation) from day 100 – 130 of gestation. Fetal CA were then harvested and whole‐cell K currents (−70 to +50mV, 200 ms duration) were obtained in freshly isolated smooth muscle cells (n = 5–6 cells/fetus). In fetal CA smooth muscle cells from ewes fed a low protein diet, the I‐V curve was shifted to the left and peak current density was increased by 31% (low protein = 92.3 ± 7.5 vs. control = 70.5 ± 6.8 pA/pF; n=14). Iberiotoxin (100 nM) almost completely inhibited the K current in cells from the low protein group (72.3 ± 4.8% inhibition), but only partially inhibited this current in controls (31.5 ± 6.2% inhibition). Expression and localization of BKCa α‐ and β1‐subunits was similar in CA from both groups. Restricted maternal protein intake during late pregnancy enhances BKCa activity in fetal CA, without altering BKCa expression or localization, and may represent an adaptation to the dysfunction in the coronary arterial EDHF‐like pathway previously observed in offspring of ewes subjected to decreased nutrient intake during pregnancy (NIH R03HD061532).
The effects of structurally disparate Kv7 activators in the rat vasculature
Authors: Jepps Thomas Andrew , Hansen Rie Schultz , Olesen Søren Peter , Greenwood Iain Andrew
Published: June 2021

Kv7 voltage‐gated potassium channels, encoded by KCNQ1–5 genes, are present functionally in vascular and non‐vascular smooth muscle cells. The aim of the present study was to compare the effect of structurally different activators of Kv7.2–7.5 channels: S‐1, BMS‐204352, ICA27243 and retigabine in the rat mesenteric artery, thoracic aorta and coronary circulation. Isometric tension recording was performed on segments of mesenteric artery and thoracic aorta. In addition the coronary circulation was studied using the Langendorff heart preparation. In mesenteric arteries the Kv7 activators demonstrated EC50 values of: 1.9 μM (BMS204352), 2.5 μM (S‐1), 5.4 μM (ICA27243), and 14.3 μM (retigabine). Application of all Kv7 activators at 10 μM relaxed the thoracic aorta albeit with varying efficacies. In the isolated heart increasing concentrations of Kv7 activators produced a concentration‐dependent increase in coronary flow. This study is the first to fully characterise the effects of various Kv7 channel activators in the rat mesenteric artery, thoracic aorta and coronary artery.

Work was funded by BBSRC‐CASE studentship with NeuroSearch A/S.
Impaired vascular function in female Dentin matrix protein 1 null mice
Authors: Silswal Neerupma , Wacker Michael J , Brotto Marco , Bonewald Lynda , Andresen Jon
Published: June 2021

Dentin matrix protein 1 (DMP1) is highly expressed in mineralized tissues, especially in osteocytes. DMP1 is mainly involved in the control of mineralization and phosphate homeostasis. In humans mutations in DMP1 can lead to autosomal‐recessive hypophosphatemic rickets, which is due to increased production of fibroblast growth factor 23 (FGF23) by osteocytes. Interestingly, we found that aortic vascular smooth muscle expresses FGF receptor type 1 and the co‐receptors α and β‐klotho. DMP1 null mice have low bone mass and high circulating levels of FGF23, yet, cardiovascular function in these mice has not been examined. Therefore, we investigated the aortic vascular function in mature (16–25 week old) Dmp1−/− mice and wild‐type (WT) mice. Responses to ACh (10−4–10−9 M), SNP (10−4–10−9 M), PGF2α (10−4‐10−9 M), 5‐HT (10−5–10−9 M), and caffeine (10 mM) were examined by using isometric tension myography using normo‐phosphate buffers. Endothelium and smooth muscle dependent relaxations were unaltered in Dmp1−/− mice. There were, however, agonist and sex differences in the response to vasoconstrictors such that the contractile response to 5‐HT was selectively impaired (p=0.0013) in female Dmp1−/− mice versus male Dmp1−/− mice and WT mice of either sex. In addition, female Dmp1−/− mice exhibited weaker contractions to caffeine (p=0.02) indicating that SR calcium stores were reduced in these mice. Since the primary defect in Dmp1−/− mice is in the osteocytes it may be that bone releases a factor, possibly FGF23, which alters vascular function. This research was supported by NIH RC2 grant (ARO58962).
Mitochondrial plasticity in the anoxic turtle heart
Authors: Galli Gina LJ , Richards Jeffrey
Published: June 2021

Our objective was to assess mitochondrial respiration and electron transport chain (ETC) activities in permiabilised cardiac fibers from normoxic and anoxic (2 weeks) turtles (Trachemys scripta) acclimated to 5°C. All experiments on animals were conducted in conformance with the FASEB Principles. Mitochondrial maximum ADP stimulated respiration was measured with an Oroboros micro‐respirometer, and respiratory flux through complex I, II and IV of the ETC was assessed by pharmacological intervention. ADP affinity was determined by adding ADP in steps from 0.025 mM to a maximum of 1 mM. All preparations were tested at 5, 13 and 21°C. Anoxic acclimation led to significant reductions in maximum respiration rates through ETC complexes I, II and IV at all temperatures tested, and ADP affinity was significantly lower. State II respiration rates were significantly reduced in anoxic cardiac fibers, leading to a higher respiratory control ratio, which is indicative of a reduction in proton leak. Collectively, these results suggest a reduction in mitochondrial capacity contributes to overall hypometabolism in anoxic turtles. Since complex I of the ETC is a major site of reactive oxygen species generation, a downregulation of this protein may also help to prevent redox damage during anoxic recovery. This research was funded by The Killam Memorial Trust and The Natural Sciences and Educational Research Council of Canada.
Rab5‐dependent endocytosis of KCa2.3
Authors: Gao Yajuan , Devor Daniel C
Published: June 2021

We demonstrated that recycling of KCa2.3 is dependent upon Rab35/EPI64C and RME‐1. Herein, we report our initial studies on the mechanism by which KCa2.3 is retrieved from the plasma membrane. Using a biotin ligase acceptor peptide‐tagged KCa2.3 construct, we demonstrate that expression of a dominant active Rab5 (Q79L) results in the colocalization of endocytosed KCa2.3 with the Rab5‐induced vacuoles. Expression of a dominant negative Rab5 (N133I) resulted in a 2‐fold accumulation of plasma membrane KCa2.3. Also, DN Rab5 slows the endocytosis of KCa2.3, i.e., after 10 min., 19% of KCa2.3 is endocytosed in the presence of WT Rab5, whereas only 5% of KCa2.3 is endocytosed in the presence of DN Rab5. Co‐IP confirmed a close association between Rab5 and KCa2.3. Expression of Rabex‐5 (RabGEF1) GEF inactive mutants or ubiquitin binding mutants resulted in an increased steady‐state expression of KCa2.3 at the plasma membrane. As the Rab5‐dependent endocytic pathway can be either dynamin dependent or independent we determined whether dynamin was required for the endocytosis of KCa2.3. Overexpression of K44A dynamin resulted in an increase in steady‐state plasma membrane KCa2.3, relative to WT dynamin, consistent with an inhibition of endocytosis. In addition, co‐IP confirmed a close association between KCa2.3 and dynamin. In total, our results suggest that KCa2.3 endocytosis is both Rab5‐ and dynamin‐dependent.
A role for MARCKS in phosphoinositide‐dependent regulation of ENaC
Authors: Alli Abdel A , Bao Hui‐Fang , Al‐Khalili Otor , Aldrugh Yasir , Ma He‐Ping , Eaton Douglas C
Published: June 2021

Anionic phosphoinositides (i.e. PIP2 and PIP3) regulate the activity and protein expression of ENaC at the apical membrane. The mechanism by which anionic phosphoinositides are presented to ENaC is unknown. Here, we investigate the association between MARCKS, ENaC, and anionic phosphoinositides in vitro and in situ. Co‐immunoprecipitation experiments revealed a transient association between ENaC gamma subunit and MARCKS in a renal cell line (A6‐2F3). In overlay binding assays, recombinant GST‐MARCKS and GST‐ENaC beta subunit fusion proteins bound various phosphoinositides. Confocal microscopy confirmed the colocalization of MARCKS and PIP2 at the apical membrane of 2F3 cells. After treating 2F3 cells with PMA, immunoblot studies showed an increase in MARCKS phosphorylation, confocal microscopy experiments revealed displacement of MARCKS from the apical membrane, and transepithelial current decreased significantly. The findings presented here suggest a novel mechanism for the regulation of ENaC by phosphoinositides, but will require further investigation.
Leptin promotes KATP channel trafficking through AMPK/GSK3 beta‐mediated inhibition of PTEN in pancreatic beta‐cells
Authors: Park Sun‐Hyun , Lim Ajin , Lee Kyu‐Hee , Lee Suk‐Ho , Jeon Ju‐Hong , Ho Won‐Kyung
Published: June 2021

Leptin has the ability to control glucose homeostasis through activation of ATP‐sensitive potassium (KATP) channel, a key regulator of insulin secretion from pancreatic β‐cells. However, the molecular mechanisms by which leptin controls KATP channel activity are still poorly understood. Here we show that leptin elevates the conductance of KATP channel by promoting trafficking to the plasma membrane in pancreatic β‐cells. Western blot studies using phospho‐specific antibodies showed that leptin increased phosphorylation of AMPK, GSK3β, and PTEN. Since phsophatase activity of PTEN is inactivated by phosphorylation, this result suggests that downstream of leptin signaling to promote KATP channel trafficking involves the activation of AMPK leading to GSK3β‐mediated PTEN inactivation. We demonstrate that AMPK/GSK3β‐mediated inhibition of PTEN is crucial for the leptin‐induced increase in KATP channel trafficking. However, the casein kinase 2 (CK2) and PI3K/Akt pathways is unrelated to the effect of leptin on KATP channel trafficking. Therefore, our results reveal a novel signaling axis that determines leptin regulation of insulin secretion in pancreatic β‐cells. Our findings might help to unravel the peripheral action of leptin on pancreatic β‐cell physiology and glucose homeostasis.
Development of a robust genetic test for hyperkalemic periodic paralysis (HYPP) in quarter horses
Authors: Traver Samantha , Horton Stephen
Published: June 2021

A single nucleotide substitution from cysteine to guanine in a region of the skeletal muscle sodium channel gene (SCN4A) is known to cause an equine genetic disorder known as Hyperkalemic Periodic Paralysis (HYPP). The clinical effects of this disorder range from little or no symptoms to frequent episodes of muscle tremors, weakness, and/or complete collapse. Oligonucleotide primer pairs were designed for both the wild type and mutant alleles of the SCN4A gene for use in Amplification Refractory Mutation System‐PCR (ARMS‐PCR). Genomic DNA from both whole blood and saliva swab samples were isolated from horses with wild type phenotypes for HYPP. As expected for an unaffected individual, our test showed the expected PCR product using the wild type specific primer pair, while the mutant specific primer pair did not generate a product. We then varied annealing temperatures and magnesium concentrations to optimize the PCR and found a temperature of 60°C and 3 mM magnesium to be optimal. We next plan to obtain DNA from both carrier horses and individuals expressing the full‐blown mutant phenotype. DNA isolated from HYPP animals should produce a PCR product with only the mutant specific primer pairs, and carriers for HYPP should produce a product in both wild type and mutant specific PCR reactions.
Protein expression profiling of Caco‐2/HT29‐MTX co‐culture after functionalized carbon nanotube exposure
Authors: Lai Xianyin , Clack James W. , Ntim Susana Ado , Mitra Somenath , Witzmann Frank A.
Published: June 2021

To assess effects of dispersible, functionalized carbon nanotube (CNT) exposure in an in vitro model simulating the digestive tract, Caco‐2/HT29‐MTX cell protein expression was evaluated using liquid chromatography/tandem mass spectrometry (LC‐MS/MS). Three types of CNT, carboxylated single‐wall carbon nanotubes (cSWNT), carboxylated multi‐wall carbon nanotubes (cMWNT), and poly(vinylpyrrolidone) (PVP) polymer functionalized multi‐wall carbon nanotubes (pMWNT), were applied to the co‐cultured cells (75% Caco‐2, 25% HT29‐MTX) at 500 pg/mL and 10 μg/mL for 48 hrs. Proteins were extracted from the cells, reduced, alkylated, and digested. Tryptic peptides were analyzed by LC‐MS/MS. The peptides and proteins were identified using SEQUEST and validated by the Trans‐Proteomic Pipeline. Protein quantification was performed using an in‐house software package, IdentiQuantXLTM. 5007 proteins were identified with >90% confidence and 4199 proteins were quantified. Among these, 803 proteins were differentially expressed (P<0.01). Bioinformatic analysis of the results indicates a significant biological effect of CNTs on Caco‐2/HT29‐MTX protein expression, in the absence of overt toxicity, and merit further study. Supported by NIEHS RC2ES018025.
Evolutionary Medicine: Microevolution of human morphology and its medico‐social impact
Authors: Ruhli Frank J , Henneberg Maciej
Published: June 2021

Human morphology is undergoing evolutionary changes. Hitherto, the socio‐economic and clinical impact of such alterations of human anatomy is mostly underestimated. We present not only current research data on such microevolutionary traits but also address particularly their medico‐social impact. Based on skeletal samples from ancient to modern times as well as based on modern Swiss Armed forces conscription data (N>100,000) we are able to show significant increases in body dimensions (such as stature or weight, BMI), decrease in skeletal robustness as well as an increase in morphological variability. A further particular focus is laid by us on the alterations of the axial skeleton as seen eg. by increased frequencies of spina bifida occulta. Embedded in the research framework of Evolutionary Medicine we will address specific consequences for public health measures (target group for weight reduction programs), medical teaching (variability of anatomical structures) or clinical research (low back pain etiology).
A Tale of Two Compartments: A case study of a bilateral anomalous flexor muscle of the forearm
Authors: Stewart Jillian , Pratt Rebecca
Published: June 2021

During a routine female cadaveric dissection in the prosection lab at Michigan State University, an anomalous flexor muscle of the forearm was uncovered bilaterally. Exploration of the anterior compartment demonstrated that the muscle belly originated from the common flexor attachment and was located proximally in the superficial compartment, deep to the flexor digitorum superficialis. Distally, the tendon of this anomalous muscle belly was discovered in the deep flexor compartment encased by the synovial sheaths of the flexor digitorum profundus (FDP) tendons as it blended via two slips into the tendons of the FDP of the third and fourth digits. The neighboring muscles of the flexor compartments of the forearm displayed normal morphology. Further dissection illustrated that the anomalous bilateral muscle appeared to compress both the proximal ulnar nerve and artery, which ran deep to the muscle belly. This novel muscular variant could have had clinical relevance due to the potential negative effects of vascular compression on the nerve during muscle use. This dissection was supported by a generous donation to the Michigan State University Willed Body Program. Source of Research Support: NA.
A cast‐corrosion technique for assessing bone marrow vascularity for research and teaching using the intraosseous infusion of radiopaque silicone
Authors: Rubal Bernard J. , DeLorenzo Robert A.
Published: June 2021

In this study we examined the medullary vascularity of the tiba, femur, humerus, ulna and sternum of 8 post mortem swine (Sus scrofa). Medullary vascular access was achieved using an introsseous cannulae introduced into cancellous bone. Blood was cleared by infusion of 10 ml of saline, followed by infusion of 15–20 ml of radiopaque silicone casting material adjusted to blood viscosity (MicroFil®, Flow Tech, Carver, MA) under pressures of 35 or 300 mmHg. Casts were then cured for 24–48 hrs at 3°C and specimens imaged using computed tomography(CT) with a slice thickness of 0.5mm. Specimens were deboned and bones demineralized in 17% HCl solution. Decalcified bones were cleared using a water flush and progressive glycerine clearing procedures. Nutrient, emissary and intramedullary conductive vessels were assessed by 3D CT imaging and medullary vascular volume assessed by volume displacement. Microscopic examination done in a neutral density glycerine solution revealed highly dense medullary sinusoids and anastomoses within cancellous regions. Although limited to postmortem applications, the cast‐corrosion method described provides an inexpensive method for demonstrating medullary vascularity within bone. The silicone formulation utilized exhibits minimal shrinkage allowing for assessments of vascular volume and its radiopacity permits correlation with radiography.
Morphometric pelvic muscle map analysis in extant and pleistocene carnivores
Authors: Carlon Burcu , Hubbard Chris
Published: June 2021

Recent studies in our laboratory have developed maps of pelvic musculature attachment sites (muscle maps) for Neofelis nebulosa (clouded leopard), Felis catis (domestic cat) and >Canis latrans (coyote). Correlations were made between actual muscle attachments and bone morphology at the attachment site. Based on this information we can map muscle attachment sites on bones from extinct closely related species. Pleistocene pelvis specimens were examined for bony muscle attachment sites, measured and photographed at the George C. Page Museum in Los Angeles, California. Specimens included Smilodon fatalis (n=9), Panthera atrox (n=4), Canis dirus (n=6), Canis latrans (n=5), and Arctodus simus (n=2). Muscle map surface areas for eight pelvic muscles were determined for each specimen along with pelvic measurements (ilium/ischium: width/length). The measurements were then compared between and within extant and extinct species listed above. Mm. gluteus medius, quadratus femoris, cranial and caudal gemellus surface areas differed significantly in comparisons between the felids, and between felids and non‐felid species in both extinct and extant groups. Pelvic measurements differed primarily between felid and non‐felids. These results suggest that muscle map surface area analysis may be useful for both phylogenetic and morphometric analysis.
Ferula hermonis root extract increases fiber size and myonuclear number within tibialis anterior muscle of exercised rats
Authors: Allouh Mohammed Zohair
Published: June 2021

Ferula hermonis Boiss. is an aphrodisiac plant that grows in the Mediterranean region. It has been reported that acute treatment with the extract from the root of this plant significantly increases serum testosterone in the rat. This study investigates the effects of F. hermonis extract alone or combined with exercise on rat skeletal muscle fibers. Adult male rats were divided into four groups: control sedentary (CS) that had no treatment or exercise; ferula sedentary (FS) that orally treated with ferula extract at a dose of 60 mg/kg/rat every other day over a period of 20 days; control exercised (CE) that trained by swimming for 40 minutes every other day; and ferula exercised (FE) that received combined treatment and exercise. At the end of experiments, the left tibialis anterior muscle was dissected for study from each rat. Fiber diameter, myonuclear number and myonuclear domain size were measured by using immunofluorescent techniques and software analyses. FE group showed significant (P<0.05) increases in muscle weight, fiber size and myonuclear number compared to the remaining groups. However, no significant variations were found among CS, FS and CE groups. This study concludes that exercise combined with administration of F. hermonis extract is more effective in enhancing the growth of skeletal muscle fibers than exercise alone. Funds for this study were provided by the Deanship of Research at JUST (34/2010).

Grant Funding Source: JUST
Calcium signaling regulates gravin‐dependent compartmentalization of PKA
Authors: Schott Micah Bruce , Grove Bryon
Published: June 2021

Gravin (AKAP12) is a multivalent scaffolding protein that anchors PKA to the plasma membrane, but undergoes redistribution to an intracellular compartment in response to PKC activation or increased [Ca2+]i. We postulate that through this mechanism gravin may mediate cross‐talk between Ca2+‐dependent and cAMP‐dependent signaling pathways. To assess this, we examined the effect of Ca2+‐elevating agents on gravin and gravin‐dependent PKA distribution in cells lacking endogenous gravin but expressing EGFP/ EYFP‐gravin constructs. Ionomycin and thapsigargin initiated rapid gravin redistribution into the cytosol, an event which was inhibited by Ca2+ chelation using BAPTA‐AM. Treatment of cells coexpressing gravin‐EYFP and PKA RII‐ECFP with Ca2+‐elevating agents also resulted in PKA redistribution, but only when gravin was present. Consistent with this, treatment with lysophosphatidic acid (LPA) also resulted in rapid gravin redistribution in concert with a rise in [Ca2+]i and resulted in PKA redistribution in cells coexpressing gravin and PKA constructs. However, inhibition studies using BAPTA‐AM and bisindolylmaleimide suggest that in addition to changes in [Ca2+]i, LPA‐mediated gravin dynamics may involve other mechanisms. Studies to further elucidate the mechanism behind LPA‐mediated gravin dynamics are currently underway. This work was supported by funding from UNDSMHS and NIH 5P20RR017699.

Grant Funding Source: Institutional/NIH
Ethanol‐induced oxidative stress in oteoblast‐like cells (UMR 106‐01 BSP)
Authors: Joseph Eugene E , Garcia Beronica
Published: June 2021

Ethanol is known to induce oxidative stress which has been associated with cancer, neurodegenerative and cardiovascular diseases and osteoporosis. Oxidative stress adversely affects bone cells leading to decreased mineralization of bone. Many studies have been done documenting the effect of ethanol on bone. However, the cellular mechanisms leading to the effects of ethanol‐induced oxidative stress and demineralization in bone cell cultures are incompletely understood. In this study, it was hypothesized that ethanol decreases mineralization in UMR cells by inducing oxidative stress. UMR cells were treated with varying concentrations of ethanol and incubated at 37 °C for 60 hours. The cell cultures were treated with 7mM beta‐glycerophosphate for 24 hours to stimulate mineralization. Cell cultures were stained with 40 mM alizarin red to assess mineralization. Mineral content was extracted with 10 mM HCl/70% ethanol and spectrophotometrically quantified using a microplate reader (Spectramax 190). Vector Red staining was also done to test for alkaline phosphatase (ALP). Lactate dehydrogenaes (LDH) concentrations were measured colorimetrically using an LDH bioassay kit. F test ANOVA was used to compare variances and all values in the results were expressed as means ±SD. In all cases, p values of <0.05 were considered statistically significant. The results showed an inverse correlation of ethanol concentrations to both mineral content and alkaline phosphatase concentrations, while a concomitant increase of LDH concentrations was noted with the increasing ethanol concentrations. These results suggest that ethanol causes induced oxidative stress by affecting ALP and LDH activities, leading to an overall demineralization in UMR cell cultures.

(Supported by the Dean's Office of the College of Arts and Sciences of La Sierra University, Riverside, CA).