bluuebunny/crossref_metadata_2025 huggingface dataset:
Histone deacetylases inhibit PIP3‐dependent Rac exchange factor 1 (P‐Rex1) expression in prostate cancer cells via SP1 binding site
Authors: Ha Da , Tu Yaping
Published: June 2021

Previously, we reported that up‐regulation of P‐Rex1, a PIP3 and Gâã ‐activated GEF for Rac, promotes prostate cancer metastasis. Highly‐metastatic prostate cancer PC3 cells display constitutive P‐Rex1 expression whereas the level of P‐Rex1 is almost undetectable in normal prostate epithelial cells and nonmetastatic prostate cancer CWR22 Rv1 cells. Here, we show that P‐Rex1 expression is strongly stimulated by the treatment of TSA, a histone deacetylase inhibitor, in CWR22 Rv1 cells, but not in PC3 cells. Expression level of HDAC1 and HDAC2 are lower in PC3 cells than in CWR22 Rv1 cells. Using CHIP assay, we found that class I histone deacetylase HDAC1 and HDAC2 are bound to the endogenous P‐Rex1 promoter in CWR22 Rv1 cells, but not in PC3 cells. TSA treatment results in the dissociation of HDAC1/HDAC2 and increases of acetylated histone H4 in the promoter region of the P‐Rex1 gene in CWR22 Rv1 cells, subsequently causing the upregulation of P‐Rex1 expression. A putative SP1(Specificity Protein 1) binding site is identified as a TSA‐response element in the P‐Rex1 promoter. Activity of the SP1 transcription factor is indispensable for both TSA‐induced and constitutive P‐Rex1 expression. Further investigation revealed that SP1 may recruit HDAC1 to the P‐Rex1 promoter. Altogether, our results illustrate a SP1‐mediated histone acetylation‐related regulatory mechanism of P‐Rex1 expression in prostate cancer cells.
Effects of extended access and withdrawal on the reinforcing strength of cocaine using a cocaine vs. food concurrent‐choice procedure in rhesus monkeys
Authors: Banks Matthew L , Negus S Stevens
Published: June 2021

Drug withdrawal may increase the reinforcing strength of some classes of abused drugs. However, the effects of cocaine withdrawal on the reinforcing strength of cocaine have produced divergent results. This study examined effects of graded periods of cocaine access and withdrawal on the relative reinforcing strength of cocaine using a cocaine vs. food concurrent‐choice procedure. Cocaine (0 ‐ 0.1 mg/kg/inj) and banana pellets were available under a concurrent‐choice schedule during daily 2‐hr sessions (n=4). Cocaine maintained a dose‐dependent increase in cocaine choice. Subsequently, extended access to cocaine was introduced via availability of 0.1 mg/kg/inj cocaine under a FR 10/ Time Out X schedule during 21hr sessions that followed daily choice sessions. The degree of access was manipulated by varying the time‐out (30, 15, 7.5 min). Extended access with each TO was introduced for 1 week, followed by at least one week of withdrawal. Cocaine intake increased as TO decreased, and peak intake was approximately 11 mg/kg/day. Neither extended cocaine access nor withdrawal altered cocaine choice. Thus, under these conditions, extended cocaine access and withdrawal failed to alter the relative reinforcing strength of cocaine. Research supported by P01‐DA14528 from NIH/NIDA.
Renal sympathetic and systemic vascular responses to cocaine are correlated in conscious rats
Authors: Busauskas Marius , Burris Sarah , Willingham Laura , Knuepfer Mark
Published: June 2021

Cocaine elicits hemodynamic responses that are dependent on activation of central sympathoexcitatory pathways. We reported that greater vascular responses to cocaine are associated with greater increases in sympathetic nerve activity (SNA). We also reported that i.v. or intracerebroventricular propranolol enhances the cocaine induced increase in systemic vascular resistance (SVR). We hypothesized that this increase in SVR was due to a greater increase in SNA. Male Sprague Dawley rats were instrumented for measurement of cardiac output, heart rate, arterial pressure and renal SNA. After a minimum of 3 days for recovery, conscious animals were subjected to cocaine. Ganglionic blockade (trimethaphan, 10 mg/kg i.v.) was used to determine background activity. Cocaine (5 mg/kg i.v.) increased arterial pressure, SVR and renal SNA. Rats, pretreated with propranolol (1 mg/kg i.v.) 10 min. before administration of cocaine, had a greater cocaine‐induced increase in SVR that was associated with a greater increase in renal SNA. Therefore, there was a positive correlation between SVR and renal SNA. We conclude that propranolol increases SVR likely due to an increased central sympathoexcitation. Supported by USPHS NIH R01 DA0017371, DA05180 and training grant 5T32 GM008306.
SmartHomeCage: a platform for automation of rodent behavior monitoring at homecages
Authors: Xie Xinmin Simon , Zhang Jingxi , Xie James , Fang Jidong , Zhou Wei , Khroyan Taline V
Published: June 2021

We have developed a SmartHomeCage system that allow for insertion of normal rodent cages to monitor animal's behaviors in its homecage with bedding, food and water for days. Each apparatus is comprised of multiple sensors and up to 16 apparatuses can be simultaneously operated by one PC. Parameters measured include sleep/wake states, locomotion (traveling distance and speed), rearing, movement patterns and rotations. For validating the system, we showed that cocaine (30 mg/kg, ip) produced an increase in activity including distance traveled and rearing. The nociceptin receptor agonist SR 14150 (10 mg/kg, ip) that was previously shown to induce hypolocomotion produced a decrease in activity. Using a dark box inserted in the normal cage, we were able to assess the anxiolytic effect of diazepam (3mg/kg, i.p.), as indicated by an increased latency of first entry to the dark box and increased the transition number between light and dark compartments. In pilot studies through collaboration with universities, we showed this system is sensitive enough to quantify mouse behavioral changes following experimental spinal injury or middle cerebral artery occlusion. Sleep/wake state automated classification using the SmartHomeCage will be further validated by comparing with simultaneous EEG recordings. This higher throughput behavioral monitoring system for behavioral phenotype of transgenic and gene knockout mice are currently underway.
The effect of cholinergic system in basolateral amygdala (BLA) on the ethanol–induced conditioned place preference (CPP)
Authors: Rostami Parvin , Zarrindast Mohammad Reza , Meshkani Javad , Rezayof Ameneh
Published: June 2021

In this study the effects of bilateral injections of cholinergic agents into the basolateral amygdala (BLA) region on ethanol‐induced conditioned place preference (CPP) were investigated in male Wistar rats. Subcutaneous (s.c.) administration of different doses of ethanol (0.25, 0.5, 1, 1.5 g/kg) produced a partial dose‐dependent CPP as (0.5 g/kg) of ethanol was effective dose. Using a 3‐day schedule of conditioning, it was found that intra‐BLA administration of the nicotinic receptor agonist, nicotine (0.25, 0.5, 0.75,1 and 1.5 ìg/rat) significantly potentiated the ethanol (0.5 g/kg)‐induced CPP. Moreover, intra‐BLA administration of the muscarinic receptor agonist, pilocarine (0.5, 1 and1.5 ìg/rat), potentiated the ethanol (0.5 g/kg)‐induced CPP. Furthermore, intra‐BLA administration of nicotinic receptor antagonist, mecamylamine (0.5, 0.75 and 1 ìg/rat) before an effective dose of nicotine (1 ìg/rat) and Subcutaneous (s.c.) of ethanol (0.5 g/kg) decreased the effect of nicotine‐induced potentiation of the ethanol response. On the other hand, intra‐BLA administration of the muscarinic receptor antagonist, atropine (1, 2, and 4 ìg/rat) inhibited the effect of pilocarpine‐induced potentiation of the ethanol CPP. Intra‐BLA injections of nicotine, mecamylamine, pilocarpine or atropine alone did not induce a significant place preference or place aversion.

Tmu research Grant
Effects of adolescent ethanol exposure on Purkinje cell viability
Authors: Weber John T. , Cooze Jared , Forbes Ashley , French Vanessa
Published: June 2021

Chronic alcoholism can cause damage to the cerebellum, an area of the brain associated with motor learning and coordination. Recently, we found that adolescent exposure to ethanol in rats can cause altered motor function in adulthood. In the current study, we investigated the effects of adolescent ethanol exposure on the viability of cerebellar Purkinje cells (PCs). To simulate binge drinking, male Sprague‐Dawley rats received an intraperitoneal (i.p.) injection of 25% (v/v) ethanol in isotonic saline (3g/kg) for either 7 days (P31‐37) or 4 days (P31,33,35,37). Controls received an i.p. injection of isotonic saline (P31‐37). Animals administered ethanol displayed obvious loss of motor coordination within minutes of injection. Blood alcohol concentration at 30 min was 275± 16 mg/dl, which represents approximately 12‐15 drink equivalents. On P58, rats were sacrificed and coronal slices of the cerebellum were prepared and stained with cresyl violet. Slices from control animals displayed an intact PC layer. However, rats that had received either 4 or 7 injections of ethanol had a significant loss of PCs as compared to controls. Given the importance of PCs in motor control, these findings suggest that PC death caused by adolescent ethanol exposure may lead to long‐lasting adverse effects on motor coordination. Supported by ABMRF/The Foundation for Alcohol Research and Canada Foundation for Innovation.
Functional Interaction between Chronic Nicotinic Acetylcholine Receptor (nAChR) Subtype Agonists and Acute Ethanol‐Induced Cerebellar Ataxia in Mice: Development of Cross‐Tolerance
Authors: Taslim Najla , Dar M. Saeed
Published: June 2021

Well documented functional interaction between nicotine and ethanol may explain their co‐abuse. We have previously reported that attenuation of ethanol ataxia occurs via nAChR subtypes α4 β2 and α7. Now we report possible development of cross‐tolerance between these two psychoactive drugs. CD‐1 male mice weighing 24‐28g were implanted with guide cannulas for chronic intracerebellar (ICB) infusion of drugs. Ataxia was assessed by Rotorod at 15, 30, 45 and 60 min after ethanol (2g/kg;i.p.) challenge. Immunohistochemical study on mice brains was done using α4 and α7 subtype selective antibodies. Chronic ICB infusion of α4 β2‐ and α7‐ selective agonists, RJR‐2403 (31.25, 62.5, 125ng) for 1,3,5,7 days and PNU 282987 (25ng, 250ng, 2.5 μg) for 1, 2, 3, 5 days, respectively, attenuated acute ethanol ataxia dose‐dependently indicating the development of cross‐tolerance between ethanol ataxia and α4 β2 and α7 agonists. Cross‐tolerance was maximal after 5‐day RJR‐2403 treatment and lasted 48 h. However, the cross‐tolerance peaked after 1‐day treatment with PNU 282987 and lasted 72h. Pretreatment with α4 β2‐ and α7‐selective antagonists, DH βE and MLA, abolished the cross‐tolerance indicating nAChR α4 β2 and α7 participation . Immunoreactivity was high for α4 and α7 subtypes in Purkinje cells but sparse in molecular and granular layers. Overall, the results indicate development of cross tolerance between nAChR subtype α4 β2 and α7 selective agonists and ethanol induced ataxia. The observed cross tolerance probably suggests that alcoholics sustain high smoking rate in an attempt to overcome ethnaol's ataxia and other CNS effects thereby contributing to co‐abuse of nicotine and ethanol and subsequent high co‐morbidity associated with these drugs.
Defects in de novo neoangiogenesis in CD34KO mice revealed in a Matrigel chamber model
Authors: Heine Heather L , Baker Jennifer , So Leslie , Ajami Bahareh , Minchinton Andrew , Rossi Fabio , McNagny Kelly , McManus Bruce
Published: June 2021

CD34 is a cell surface sialomucin widely used for hematopoietic stem cell (HSC) and endothelial progenitor cell (EPC) purification and as a marker of most vascular endothelial cells. Despite extensive research, the function of this sialomucin remains elusive. CD34‐deficient (CD34KO) strains of transgenic mice surprisingly have mild phenotypes, with no significant vascular defects. We took advantage of a subcutaneous in vivo Matrigel chamber model to systematically compare angiogenesis, endothelial, pericyte, white blood cell and adipocyte growth and migration in wild type and CD34KO mice. Using immunohistochemical staining for Hoechst 33342, CD31+, NG2+, F4/80, and perilipin, we revealed differences in the frequency of specific cell populations, rate of penetration and relative cell density. Endothelial cells appear to be defective in chambers grown within CD34KO mice. Chamber digestion and analysis of incoming cells by flow cytometry indicates Ter119+ cell numbers are elevated in chambers implanted in CD34KO mice for 6 weeks. Our data provide the first report of an endothelial phenotype in CD34KO mice. As well, the results illustrate how the chamber model of de novo tissue development may be an invaluable method for elucidating endothelial cell subsets that influence neoangiogenesis in vivo. This work is funded by the Canadian Institutes of Health Research.

Grant Funding Source

Canadian Institutes of Health Research
Development of collateral flow pathways in microvascular networks
Authors: Reglin Bettina , Zakrzewicz Andreas , Secomb Timothy W. , Pries Axel R.
Published: June 2021

The formation of functional collaterals from pre‐existing microvessels is an important mechanism to maintain tissue perfusion after vessel obstruction. Here, a previously developed model simulation of inner vessel diameter adaptation in microvascular networks was used to study mechanisms underlying collateralization and arteriogenesis. Changes in hemodynamic and metabolic stimuli after arteriolar occlusion, elicited diameter adaptation and the resulting redistribution of blood flow were analyzed. Results showed that collateral flow pathways were formed including transformation of capillaries and venules to larger arterioles, and partial flow reversal. Initially hypoxic tissue regions (PO2 < 1 mmHg) regained an adequate oxygen level (post‐/pre‐occlusion PO2 = 1.05, Median). Diameter increase in developing collaterals was primarily evoked by: hemodynamic stimuli in the arteriolar portion, hemodynamic and conducted metabolic stimuli in the mid‐section, and local metabolic stimuli close to the capillary beds. Thus, the relative impact of metabolic signals decreases with growing distance from the capillaries downstream of the occlusion site, whereas that of hemodynamic and conducted signals increases.
Effects of an anti‐VEGF administration on whole‐body VEGF distribution assessed by a molecularly‐detailed pharmacokinetic model: a cancer study
Authors: Stefanini Marianne O , Wu Florence TH , Mac Gabhann Feilim , Popel Aleksander S
Published: June 2021

Vascular endothelial growth factor (VEGF), a potent cytokine stimulating angiogenesis, has been reported to be overexpressed in cancer making it a good target for anti‐angiogenic treatment. We constructed a molecularly‐detailed in silico model composed of three compartments (blood plus healthy and diseased tissues). Our model provides insights on the VEGF distribution in each compartment and serves to investigate the possible origins of the plasma VEGF increase observed in cancer patients. We also use this model to predict the effects of anti‐VEGF treatment through different modes of injection (slow infusion, metronomic therapy) on the VEGF profile in each compartment. We find that: 1) an increase of VEGF secretion by the tumor and/or an increase in vascular permeability of VEGF in tumor cannot solely explain the several‐fold plasma VEGF increase observed in cancer patients; 2) the introduction of an anti‐VEGF agent leads to a new pseudo‐steady‐state plasma VEGF level when administered in a metronomic fashion. These findings provide new insights and can be applied to optimizing anti‐angiogenic therapies targeting VEGF.
Spontaneously hypertensive rat mesenteric microvsascular networks undergo angiogenesis in response to a wound healing stimulus
Authors: Aragon Mario , Antoine DeJeune , Murfee Walter Lee
Published: June 2021

Hypertension is associated with increased microvascular resistance due in part to a loss of microvessels, termed microvascular rarefaction. Understanding whether this loss of microvessels is a result of vessel regression or impaired growth requires further investigation of the ability of hypertensive networks to undergo angiogenesis. The objective of this study was to determine the angiogenic response of mesenteric microvascular networks in adult spontaneously hypertensive versus normotensive rats. Mesenteric tissues were harvested from 15‐16 week old spontaneously hypertensive and Wistar rats according to 3 experimental groups: unstimulated, 3 days post and 5 days post wound healing stimulus induced by 20 min exteriorization of the mesentery. Microvascular networks were immunohistochemically identified by PECAM labeling. Consistent with the phenomenon of rarefaction, SHR unstimulated microvascular networks were smaller than normotensive controls. By 3 days post stimulation both SHR and Wistar microvascular networks displayed increased capillary sprouting. By 5 days both SHR and Wistar microvascular networks displayed an increase in vascular area. These observations suggest that hypertensive microvascular networks are able to undergo angiogenesis similar to normotensive networks.
Glutathione levels in ischemia‐induced angiogenesis
Authors: Nijmeh Julie , Moldobaeva Aigul , Wagner Elizabeth
Published: June 2021

We previously have shown, using fluorescent indicators, increased levels of reactive oxygen species (ROS) in a mouse model of lung angiogenesis following left pulmonary artery ligation (LPAL). Furthermore, we demonstrated that ROS play an important role in promoting ischemia‐induced angiogenesis in that treatment with the antioxidant N‐acetylcysteine (NAC) showed reduced neovascularization. In this study, we measured levels of reduced glutathione (GSH), the most abundant antioxidant, and its oxidized form (GSSG) in the lungs as an indicator of ROS levels early after LPAL. Additionally, we assessed the effect of NAC on GSH/GSSG following LPAL. Mice were treated with NAC (ip; 1mg/g body wt), or vehicle before LPAL (‐24hrs, ‐12hrs, 0hr), or sham. After surgery, lungs were harvested (30min, 4hrs, 24hrs) and processed for measuring GSH/GSSG (Oxis kit). Lungs following 4hrs LPAL showed a significant 44% reduction in GSH/GSSG levels compared to sham. NAC treated lungs, however, had an increased baseline GSH/GSSG that did not change with LPAL. These GSH/GSSG measurements confirmed early ROS release after LPAL and further emphasized the pivotal role of ROS ischemia‐induced angiogenesis.
Cannabinoid CB1 receptor inhibition decreases vascular smooth muscle migration and proliferation
Authors: Pacher Pal , Rajesh Mohanraj , Mukhopadhyay Partha , Haskó György
Published: June 2021

Vascular smooth muscle proliferation and migration triggered by inflammatory stimuli and chemoattractants such as platelet‐derived growth factor (PDGF) are key events in the development and progression of atherosclerosis and restenosis. Cannabinoids may modulate cell proliferation and migration in various cell types through cannabinoid receptors. Here we investigated the effects of CB1 receptor antagonist rimonabant (SR141716A), which has recently been shown to have anti‐atherosclerotic effects both in mice and humans on PDGF‐induced proliferation, migration, and signal transduction of human coronary artery smooth muscle cells (HCASMCs). PDGF‐induced Ras and ERK 1/2 activation, while increasing pro‐liferation and migration of HCASMCs, which were dose dependently attenuated by CB1 antagonist, rimonabant. These findings suggest that in addition to improving plasma lipid alterations and decreasing inflammatory cell migration and inflammatory response, CB1 antagonists may exert beneficial effects in atherosclerosis and restenosis by decreasing vascular smooth muscle proliferation and migration.
Role of Adenosine A1 Receptors and P2Y2 Receptors and ERK1/2 Activation in Coronary Atherosclerosis and In‐stent Stenosis
Authors: Long Xin , Elosiebo Raven , Sturek Michael
Published: June 2021

Proliferation of coronary smooth muscle cells (CSMC) plays a pivotal role in atherosclerosis and in‐stent stenosis (ISS). We made the novel discoveries that adenosine A1 receptors (A1R) and P2Y2 receptors via activation of ERK1/2 mediate mitogenesis in CSMC and intact coronary rings. Organ culture (OC) is a useful in vitro model of early coronary artery disease.

Hypothesis

A1R and P2Y2 upregulation and ERK1/2 activation are important for coronary atherosclerosis and ISS. Bare metal stents (BMS) were placed and over‐expanded to 1.3 times normal coronary artery diameter in Ossabaw miniature pigs and ISS assessed after 1, 2, or 4 weeks of recovery. A1R protein and P2Y2 mRNA was upregulated in stented arteries after 1, 2 and 4 weeks of recovery and A1R protein peaked at 1 week, which preceded ISS that peaked at 4 weeks. CCPA, an A1R‐selective agonist, induced robust ERK1/2 activation in BMS vs. non‐stented artery segments. The P2Y2 mRNA was upregulated in coronary segments of 2‐day OC, preceding the development of early coronary atherosclerosis, shown by increased media thickness, collagen content, and nuclei number in coronary segments at 4‐day OC.

Conclusion

In vivo and in vitro data are consistent with A1R and P2Y2 upregulation and activation of ERK1/2 causing coronary atherosclerosis and ISS. Support: NIH RR013223, HL062552, T35 HL007802, AHA Predoctoral Fellowship to X.L.

Grant Funding Source

NIH, AHA
Pro‐inflammatory cytokines mediate the extra‐intestinal thrombus formation associated with inflammatory bowel disease (IBD)
Authors: Yoshida Hideo , Russell Janice , Granger D. Neil
Published: June 2021

IBD is associated with an increased risk for thromboembolism. While the alterations in pro‐ and anti‐coagulant factors that accompany experimental IBD have been recently characterized, the soluble factors released from the inflamed colon that elicit thrombus formation in distant vascular beds remain poorly understood. The objective of this study was to determine whether the pro‐inflammatory cytokines TNF‐a and IL‐1b contribute to the extra‐intestinal thrombosis that is associated with dextran sodium sulfate (DSS)‐induced colitis in mice. Intravital microscopy was used to monitor thrombus formation in arterioles of the cremaster muscle in control and colitic mice. Thrombus formation was induced by the light/dye endothelial injury method and quantified using the time of onset of the thrombus and time to blood flow cessation. Mice were treated with either TNF‐a, IL‐1b, anti‐TNF‐a antibody (mAb) and/or an anti‐IL‐1b mAb. Administration of TNF‐a and/or IL‐1b to control mice accelerated thrombus formation in a manner similar to that noted in DSS‐colitic mice. The accelerated thrombus formation induced by DSS was significantly attenuated by the administration of mAbs directed against TNF‐a or anti‐IL‐1b. These findings indicate that pro‐inflammatory cytokines, presumably derived from the inflamed gut, mediate to the extra‐intestinal thrombosis that accompanies IBD. (P01 DK43785)
Effect of Antioxidant Supplements on Normal and Atherogenic Dieted Rabbit Fibrinolytic Activity
Authors: Beard Elizabeth Letitia , Sokolowski Joseph
Published: June 2021

Ten New Zealand strain rabbits were fed Normal Diet(ND) ( Purina Rabbit Chow‐PRC) vs ten fed Atherogenic Diet(AD) (2% cholesterol, 6% corn oil and 92% PRC). Five animals / diet received the vitamin supplement (VS)=Vitamin C (2gms/day) + Vitamin E (80 IU/day in 0.25 ml. soy lecithin). In 8 weeks, Plasma Fibrinolytic Activity(PFA) was 24.4% in ND+VS vs 14.67% in ND‐VS animals. AD +VS had 16.5% and AD‐VS rabbits 10.4% PFA. In 16 weeks, PFA values were 28.8% in the ND +VS vs 28.8% for ND ‐ VS animals and the AD values were 29.8% +VS and 27.3% ‐VS. FA in homogenized blood platelets was 43.6% in the ND+VS, 35.2 % in ND ‐VS , 43.0% in AD +VS and 42.2% in AD‐VS platelets . Blood Cholesterol values after eight weeks were comparable in the two ND groups. The AD +VS had 915 mg% cholesterol vs 1220mg%for the AD‐VS group. After 16 weeks , cholesterol values in the AD+VS group were slightly above the AD ‐VS group. Plasma and platelet phospholipid levels were not significantly altered by the VS. Addition of the antioxidants to the diet significantly increased FA in the blood plasma and platelets of ND animals. VS slightly increased these values in AD rabbits . At autopsy, the AD+VS group had reduced atherogenic plaque in their aortae compared to AD ‐VS animals.
Septic impairment of capillary blood flow requires activated coagulation pathway and is reversed by ascorbate through eNOS‐dependent dislodging of platelets in capillaries
Authors: Secor Dan Luke , Li Fuyan , Ellis Christopher , Sharpe Michael , Wilson John , Tyml Karel
Published: June 2021

Sepsis impairs capillary blood flow, leading to organ failure. We hypothesized that activation of the coagulation pathway in sepsis contributes to this impairment and that ascorbate reverses impairment by blocking the pathway. Sepsis in mice was induced by feces injection into peritoneum (FIP). Impaired flow in the hindlimb skeletal muscle was assessed with intravital microscopy. At time 0, mice were injected i.v. bolus of saline, platelet‐depleting Ab, P‐selectin blocking Ab or antithrombin, and then assessed at 6 h post‐FIP. Alternatively, eptifibatide (GPIIb/IIIa inhibitor) or ascorbate was injected at 6 h and impairment assessed at 7 h post‐FIP. In some mice, we injected rhodamine 6 G to view platelet and leukocyte adhesion in capillaries at 7 h. Sepsis impaired flow (complete stoppage) in 40 % of capillaries. In wild type mice, platelet depletion, P‐selectin blocking, antithrombin, eptifibatide, and ascorbate all reduced impairment to ~20 %. Sepsis increased the number of adhering platelets ~8 fold (the number of adhering leukocytes was negligible). Blocking of P‐selectin, eptifibatide, and ascorbate reduced this increase down to 3.2 ‐ 4.7 fold. Reduction in impairment and adhesion by ascorbate was not seen in eNOS‐/‐ mice. Thus, impairment of flow requires platelets, is inhibited by anticoagulants, and reversed by ascorbate through eNOS‐dependent dislodging of platelets in capillaries. (Support: HSFO)
Differing responses to TNF in HeLa cells expressing vascular connexins
Authors: Ouellette Yves , Ermilov Leonid G. , Sieck Gary C.
Published: June 2021

We studied the effects of TNF on cell‐to‐cell communication in HeLa cells expressing vascular connexin 37, 40 or 43. HeLa cells were treated with TNF (20 ng/ml) for up to 2 h. In dye‐transfer experiments, carboxyfluroscein (HeLaCx40 and HeLaCx43) or Alexa Fluor‐480 (HeLaCx37) was injected into one cell for 10 s and cell transfer allowed to proceed for 10 m and the number of labeled cells counted. Cell lysates were prepared in Triton X‐100 lysate buffer and detergent‐soluble fractions collected. Cx37, 40 and 43 were detected by Western blot. After 1 hour, TNF treatment resulted in near total loss of dye‐coupling in HeLaCx37 and HeLaCx43 (p<0.02, n=14‐16) and remained constant up to 2 hours. Dye coupling in HeLaCx40 cells remained unchanged after 1 hour and decreased after 2 hours (p<0.05, n=10). Western blots indicated that TNF treatment did not affect detergent solubility of Cx40 and 43. However, TNF caused a significant increase in detergent solubility of Cx37. These results suggest that inflammatory mediators affect connexins differently, and that the loss of Cx37 function may be due to the loss of detergent resistance.
Role of caveolae in regulating large conductance potassium channel activation in coronary arterioles of rats on high fat diet
Authors: Feher Attila , Bagi Zsolt
Published: June 2021

We hypothesized that diet‐induced obesity modulates large conducatance Ca2+ activated K+ (BKCa) channel function in coronary arterioles. Male Wistar rats were fed with normal (control) or high fat diet (HFD 60% of saturated fat, for 10 wk). In isolated coronary arterioles (≈100 µm) acetylcholine (ACh)‐induced, EDHF‐mediated (presence of L‐NAME 2x10‐4 M and indomethacin 10‐5 M) dilations were measured with videomicroscopy. Compared to those of control responses, ACh‐induced dilations were essentially preserved in arterioles of HFD rats. The BKCa inhibitor, iberiotoxin, however elicited a greater reduction in ACh‐evoked dilation in coronary arterioles of HFD rats, than in control vessels. Correspondingly, dilation to BKCa opener NS‐1619 was significantly enhanced in coronary arterioles of HFD rats, when compared to lean controls. Moreover, we found a reduced immunostaining of the caveolin‐1 protein in the smooth muscle layer of coronary arterioles of HFD rats. In control arterioles disrupting caveolae by methyl‐ß ‐cyclodextrin caused similar changes in agonist (ACh and NS‐1619)‐induced dilations, as observed in those of arterioles of HFD rats. We concluded that in coronary arterioles of HFD rats due to the altered function of microvascular caveolae contribution of BKCa channel to EDHF‐mediated dilations is augmented, likely to be responsible for the maintained coronary dilations in obesity.
Peroxide mediates abnormal nitric oxide production in aged rat arteries
Authors: Zhou Xiaosun , Bohlen H Glenn , Unthank Joseph L , Miller Steven J
Published: June 2021

Vascular oxidant stress increases with age and may cause dysregulation of the nitric oxide (NO) system. Our previous results have shown increased basal but suppressed flow‐mediated production of NO in aged resistance arteries that is corrected by antioxidant treatment. This study tested the hypothesis that H2O2 mediates abnormal NO production in resistance arteries of retired breeder (RB) rats. Animal groups consisted of young (Y) and RB Wistar Kyoto rats and RB pre‐treated with apocynin (RB+Apo). Microelectrodes were used to measure in vivo mesenteric artery H2O2 and NO simultaneously at baseline and during flow elevation by temporary occlusion of adjacent arteries. RB had significantly increased basal H2O2 compared with Y (15.6 ± 1.9 vs. 9.4 ± 0.9 uM, p< 0.05). Periarterial H2O2 levels were altered in proportion to flow changes in RB but not Y rats. In RB+Apo, H2O2 baseline was reduced to Y levels (8.2 ± 0.9 uM) and flow‐mediated H2O2 production inhibited. In RB, PEG‐catalase lowered baseline NO (1392 ± 211 to 576 ± 96 nM, p< 0.05) and restored flow‐mediated NO production (1060 ± 253 and 1826 ± 672 nM, 100 and 200% flow increase, respectively). The results suggest that elevated baseline and suppressed flow‐mediated NO production results from near maximal NO activation by H2O2 during aging, and imply that age‐related vascular dysfunction is not the result of reduced NO bioavailability.

Support: HL42898 and HL20605.
Longevity is associated with increased vascular resistance to metabolic stress in P. leucopus
Authors: Ungvari Zoltan , Labinskyy Nazar , Pacher Pal , Mukhopadhyay Partha , Podlutsky Andrej , Austad Steven , Csiszar Anna
Published: June 2021

The oxidative stress hypothesis of aging predicts that cells of long‐lived species exhibit superior resistance to oxidative stress. We tested this hypothesis using two taxonomically related rodents, the white‐footed mouse (Peromyscus leucopus) and the house mouse (Mus musculus) that show a more than two‐fold difference in maximum lifespan potential (8.2 and 3.5 years, respectively). In P.leucopus aortas steady‐state endothelial O2.‐ and H2O2 production and ROS generation by mitochondria were less than in M.musculus vessels. Furthermore, vessels of P.leucopus were more resistant to the pro‐oxidant effects of HG. In M.musculus arteries HG elicited significant up‐regulation of inflammatory markers (TNFá, IL‐6, ICAM‐1, VCAM and MCP‐1). In contrast, the pro‐inflammatory effects of HG were blunted in P.leucopus vessels. Thus, increased lifespan potential in P.leucopus is associated with a decreased cellular ROS generation and increased resistance to the pro‐oxidant and pro‐inflammatory effects of metabolic stress, which accord with the predictions of the oxidative stress hypothesis of aging
Historic contributors to mammalian and bird hibernation research
Authors: Folk G. Edgar , Dickson Eric W. , Thrift Diana L.
Published: June 2021

Sir Julian Huxley inspired students and scientists in 1939 by first reporting the hibernation of hummingbirds. Later, Kayser and Kalabukhov reviewed hibernation experiments done from 1824 to 1960 in their four books published between 1936 and 1960, as well as in a symposium at Harvard University in 1960. At that meeting, 26 papers were presented, and ready to comment on each paper was a distinguished panel consisting of Edward F. Adolph, Ladd Prosser, Donald R. Griffin, and George A. Bartholomew. At this meeting, a speaker, Paavo Suomalainen, invited all 45 participants to a hibernation symposium in Finland to be held a few years later. That symposium was also published. The next 40 years of hibernation research was covered by Charles Lyman, who published about five papers a year relating his experiments to others in the field. His frequent co‐author, Albert R. Dawe, also had a long career publishing his own experiments. The more modern workers, looking for a hormone‐like factor influencing hibernation, include Oeltgen and Dickson. We will discuss the important work of these and other investigators in an oral presentation to the History Group during the 2009 APS meeting.
Peer Review and Oversight of Aero Medical Research during World War II in America: The Committee on Aviation Medicine and its Sub‐Committees
Authors: Dean Jay B.
Published: June 2021

Aero medical research in America was initiated during WWI. Research funding, however, was terminated shortly after hostilities ceased. Aero medical research was restarted in 1934 at Wright Field and later, the Mayo Clinic (1939). Thus, much work needed to be done due to advancements in aeronautics and the looming threat of a global war in 1939. American physiologists learned in 1939 that Germany had been actively researching problems of aviation medicine since the early 1930s. The Committee on Aviation Medicine (CAM) was established in the fall of 1940 and charged with identifying the important physiological problems unique to flight, and locating the few pre‐existing research facilities for hypobaric research. The CAM also had to identify physiologists who could best adapt their research programs to the unexplored problems of aviation medicine. The CAM, chaired by E.F. DuBois, would eventually oversee a total of 37 research projects at 22 academic institutions during WWII, all funded by the Office of Scientific Research and Development. Committee members, recruited primarily from the ranks of the APS, evaluated the scientific merit and progress of each research project monthly/bimonthly. The CAM and its project performers worked closely with the Allied air forces and America's aircraft industry to develop the protective flight gear for waging warfare in the cold, rarefied atmosphere above 20,000 to 40,000 feet.
Respiratory cooling enhances infrared sensing in the South American rattlesnake, Crotalus durissus
Authors: Tattersall Glenn J , Cadena Viviana , Andrade Denis V
Published: June 2021

Snakes possess a profound degree of respiratory evaporation, which can impart significant cooling to the face and head. As pit vipers, rattlesnakes also utilize their forward facing facial pit organs for sensing thermal fluctuations associated with their prey. The greater the difference in temperature between the prey and the pit organ, the greater the thermal flux; cooler pit organs may provide greater infrared detection. We examined the potential for respiratory cooling to enhance the rattlesnake's ability to sense their endothermic prey by exposing two group of snakes to different humidities to manipulate the degree of respiratory cooling, while simultaneously assessing their capacity to track and find murine prey. The latency for snakes to find and eat their prey was significantly longer at high than at low humidity, suggesting cooling increases thermal detection. These differences were accompanied by significant respiratory and facial cooling at low humidity compared to high humidity. Upon initial detection of mice, snakes exhibit a dramatic, slow, respiratory cooling associated, further suggesting that respiratory cooling plays a vital role in thermal detection.
Respiratory accommodations in Rhodnius prolixus in response to increased metabolic rates
Authors: Contreras Heidy Lorena , Bradley Timothy J
Published: June 2021

It has been proposed that discontinuous respiration may have evolved in insects to decrease oxidative damage to tissues when at rest. However if metabolic rate (MR) increases, or the capacity of the respiratory system to deliver O2 to active tissues is altered, respiratory pattern in insects may change to meet aerobic demands. Specifically, we expect that as MR increases insects will transition from discontinuous (DGC) to cyclic and finally to continuous respiration. Preliminary studies have shown that a tenfold increase in MR (via temperature) in Rhodnius changed respiratory patterns from DGC to continuous respiration. However, Rhodnius respiratory responses due to feeding‐induced MR increase are not known. In Rhodnius prolixus metabolic rates (and body volume) may increase up to ten‐fold after a blood meal. They therefore provide an excellent model for studying the effects of specific dynamic action (SDA) on respiratory pattern. We expect that as metabolic rates increase, insects will transition to the different patterns as previously observed. However, due to the large effect of the meal on body volume and MR, differences in the overall MR required to induce the different patterns may be altered.

Funding: APS Porter Physiology Development Award
The Effects of Folic Acid on Endosymbionts, Growth & Metabolites of the Fruit Fly Drosophila melanogaster
Authors: Blatch Sydella , Harrison Jon F.
Published: June 2021

Folic acid is needed for nucleotide synthesis and amino acid metabolism. Its deficiency leads various diseases, but mechanisms are unclear. The fruit fly, Drosophila melanogaster, could be a model for studying folate's biomedical effects but the current literature lacks key information. We examined folic acid in relation to endosymbionts, growth, and metabolites of the fruit fly. First, we show fruit flies consuming diets lacking folic acid received sufficient folates from symbionts, allowing normal development. Second, we show body folate levels were nearly homeostatically‐regulated despite huge variation in dietary folate. When reared for a second generation on these diets, larvae on the very low folate diets were also more viable and retained faster developmental rates, also seen in the first generation. Third, we show folate is needed for DNA synthesis in fruit flies, but not for some single‐carbon cycle reactions for which it is needed in mammals. Thus, folate functions in fruit flies overlap with mammals, but endosymbionts allow fruit flies to fare better when dietary folic acid is low, and flies may have additional metabolic pathways to accomplish functions that in mammals use folate.
Skeletal muscle fatigue is caused by either e‐c coupling, metabolic alterations mechanisms or both
Authors: Santos Bianca Maria Alves , Chang Ruzhang , Garcia Karla P.P. , Gonzalez‐Serratos Hugo
Published: June 2021

Muscle fatigue may be due to a failure in excitation‐contraction coupling or an increase of ATP by products and NO. E‐c coupling alterations occur earlier and metabolic alterations take place later. We investigated these possibilities in frog skeletal muscle by continuous high frequency stimulation and different periods of repetitively stimulations. We judged the extent of e‐c or metabolic possibilities by releasing Ca2+ with 5 mM caffeine prior and immediately after fatigue.

Continuously 60 Hz electric stimulations produced a force dropped to 0.33 ± 0.05 SD the control tetanus force, within a few seconds. The ratio of caffeine force development after and before fatigue was 1.02 ± 0.24 SD. This indicated that the interaction of myosin with actin was not impaired. After repetitive stimulation, the force dropped to 0.85 ± 0.04 SD (6 tetani) and 0.65± 0.11 SD (24 tetani) the control tetanus. Here, the ratio of caffeine force after and before fatigue development was 0.75 ± 0.3 SD. The concentration of caffeine used was the same as before therefore the acto‐myosin interaction was impaired during prolonged repetitive stimulations. The two types of fatigue take place.
Delayed recovery of dysferlin‐null muscle from injurious lengthening contractions is likely due to increased macrophage infiltration
Authors: Roche Joseph A , Vanapalli Renuka , Ru Lisa W , Lovering Richard M , Bloch Robert J
Published: June 2021

The mechanisms underlying dysferlinopathies are unknown. As dysferlin mediates sarcolemmal repair in vitro, we hypothesized that it is necessary for normal recovery from lengthening contractions in vivo.

METHODS

We studied the response to injury from 15 large‐strain lengthening contractions (LSI) or 150 small‐strain lengthening contractions (SSI) in the tibialis anterior muscle in dysferlin‐deficient, A/J (dysf‐) mice, and control, A/WySnJ (dysf+) mice.

RESULTS

Immediately after LSI and SSI, ~40% of torque was lost in both strains of mice. After LSI, dysf+ and dysf‐ muscles took 3 and 14 days, respectively, to recover completely; recovery in dysf‐ but not dysf+ was blunted by irradiation and accompanied by the loss of FDx‐labeled fibers 3‐14 days after injury, and significant myogenesis and inflammation. Recovery from SSI in dysf+ and dysf‐ muscles took 21 and 28 days, respectively; neither strain retained FDx 3 days after injury, and both showed significant myogenesis and inflammation, though inflammatory cell numbers were 5‐fold greater in dysf‐. Most infiltrating cells were CD68+ macrophages. Irradiation limited recovery to ~60% and ~50% of pre‐injury torque in dysf+ and dysf‐ muscles, respectively, but had no effect on inflammation.

CONCLUSIONS

Delayed recovery of dysf‐ muscle from lengthening contractions is associated with decreased myofiber survival and increased macrophage infiltration.

This work was funded by the Jain Foundation.
Diet‐induced obesity alters skeletal muscle satellite cell functional capacity.
Authors: Shortreed Karin Elizabeth , Huang Julianna H , Dhanani Dili , Hawke Thomas J
Published: June 2021

Skeletal muscle satellite cells are integral for normal muscle growth. As skeletal muscle is the largest organ for blood glucose disposal, we were interested to ascertain whether a high fat diet (HFD) would negatively impact muscle satellite cells, and thus have a negative impact on skeletal muscle growth.

Using isolated, intact single fibers, we found that the number of satellite cells in an 'activated state' was significantly enhanced (159 ± 21% of control) while their proliferative capacity was significantly impaired (55 ± 9 % of control).

Preliminary analysis of cells isolated from HFD muscle demonstrate neither a significant impairment in migration capacity, using a Boyd's filter chamber, nor in the ability to fuse or express myosin heavy chain after 48 and 96 hours of exposure to differentiation media.

These preliminary findings suggest that HFD induces a satellite cell state similar to that found in atrophic muscle, where there is an initial enhancement in activation and impairment in proliferative ability. Alternative techniques for assessing satellite cell activation and proliferation are ongoing to confirm these results. Importantly, the potential irreversible effects of diet‐induced obesity on satellite cell function in young muscle requires further investigation to fully define the implications on long‐term muscle growth and regeneration.
EUK‐134, a synthetic superoxide dismutase/catalase mimetic, protects against loss of muscle mass/body mass in diaphragm and gastrocnemius in mdx mice
Authors: Kim Jong‐Hee , Macias BR , Canon C , Courtney S , Lawler John M
Published: June 2021

Duchenne muscular dystrophy (DMD) is characterized by devastating muscle degeneration associated with oxidative stress, loss of contractile tissue, muscle atrophy, muscle weakness and increased fibrosis in respiratory and locomotor muscles. We tested the hypothesis that protection against oxidative stress via the catalase/superoxide dismutase mimetic EUK‐134 will prevent reduction of diaphragm and skeletal muscle mass/body mass during the early inflammatory phase (20‐28 days) in mdx mice. C57BL(wild type) and mdx mice were given EUK‐134 (30mg/kg body weight/day, i.p., injection) for 8 days, beginning at 20 days of age. Body mass was significantly lower in mdx mice (‐37.7%) than wild type but, EUK‐134 increased body mass by 15.5% in mdx mice. Absolute muscle mass was lower in diaphragm (‐44.3%), gastrocnemius (‐53.6%), tibialis anterior (‐58.1%) in mdx mice. Muscle mass/body mass was lower in diaphragm (‐11.4%), gastrocnemius (‐26.3%), tibialis anterior (‐34.4%), but not heart, plantaris, soleus, extensor digitorum longus in mdx mice. EUK‐134 had a significant positive effect in protecting against reduced muscle mass/body mass in diaphragm (+38.7%) and gastrocnemius (+27.1%) in mdx mice. These data indicate that EUK‐134 provide protection against reduced muscle mass/body mass in diaphragm and gastrocnemius in mdx mice during this early phase of muscular dystrophy. Supported by NIH Grant (AR054084).
Skeletal muscle regeneration is attenuated with repression of the cytoskeletal protein, Xin
Authors: Zemanek Bart , Labatia Rita , Atkinson Daniel J , Hawke Thomas J
Published: June 2021

Xin, an actin binding protein, has been implicated in the regulation of satellite cell function and is hypothesized to be essential for skeletal muscle regeneration following injury. To investigate the role of Xin during skeletal muscle regeneration, we inhibited Xin expression using a Xin shRNA adenoviral (XinAd) injection into the right tibialis anterior (TA) of 6 week old male mice, while the left TA served as a control receiving an injection of the virus lacking Xin shRNA. Injury was induced in both TAs 4 days later via cardiotoxin injection. At 5 days post‐injury, muscle fiber area was significantly smaller (Control: 1007 ± 62 μm2 ; Xin Ad: 785± 73 μm2) and the number of centrally located nuclei per muscle fiber was significantly increased (134 ± 9%) in XinAd regenerating muscle compared to control. These deficits in fiber area (Control: 3136±278 μm2;Xin Ad: 2273 ±167 μm2) and centrally located nuclei (137 ± 9% of control) in the XinAd muscles persisted to 14 days regeneration suggesting that the regenerating muscle injected with XinAd is less mature at 5 and 14 days of regeneration. A significant increase in apoptosis (TUNEL) at 5 and 14 days of regeneration (126±6%; 141±12% of control) was observed in the XinAd TA compared to control. These data support the hypothesis that Xin is important for muscle regeneration and future work will investigate the roles of Xin on satellite cell activation and maturation.
Novel Regulation of V‐ATPase by PKA and AMPK in Kidney Intercalated Cells
Authors: Pastor‐Soler Núria M. , Alzamora Rodrigo , Naveed Sajid , Smolak Christy , Gong Fan , Hallows Kenneth R.
Published: June 2021

The metabolic sensor AMP‐activated kinase (AMPK) regulates several transport proteins, potentially coupling ion transport to cellular metabolism. The Vacuolar H+‐ATPase (V‐ATPase) regulates pH homeostasis in intracellular compartments and in the whole organism. Apical membrane V‐ATPase in collecting duct Type‐A intercalated cells (A‐ICs) contributes to net urinary acid excretion and to acid‐base homeostasis. In epididymal proton‐secreting clear cells, AMPK activation prevents alkaline pH and cAMP‐mediated V‐ATPase apical membrane accumulation. To study the effects of AMPK on V‐ATPase subcellular localization in A‐ICs, we exposed kidney slices to two distinct AMPK activators, AICAR and A‐769662. Compared to control kidney slices, each AMPK activator inhibited baseline V‐ATPase apical accumulation in A‐ICs as monitored by immunofluorescence labeling and confocal microscopy. In addition, preincubation of kidney slices with AICAR prevented PKA‐mediated V‐ATPase apical accumulation. Moreover, both PKA and AMPK phosphorylated the V‐ATPase A subunit in vitro and in vivo in HEK‐293 cells. AMPK knockdown enhanced PKA‐dependent A‐subunit phosphorylation in vivo, suggesting an antagonistic effect of AMPK on the regulation of V‐ATPase by PKA. We propose that V‐ATPase activity in kidney is coupled to the sensing of acid‐base status via PKA and to metabolic status via AMPK. (Support by NIH)