bluuebunny/crossref_metadata_2025 huggingface dataset:
Superior antiproliferative activity of histone deacetylase inhibitor apicidin and proteasome inhibitors against human colorectal cancer cells
Authors: Abaza Mohamed Salah , Bahman Abdul‐Majeed , Al‐Attiyah Rajaa
Published: June 2021

The aim of this study was to determine what effect proteasome inhibitors (PSIs) would exert on the response of human colorectal cancer cells to histone deacetylase inhibitor(HDACI) apicidin and also to examine the molecular mechanisms of the combined treatment with apicidin and PSIs as well as its potential to sensitize cancer cells to chemotherapeutic drugs. Growth inhibition of cancer cells by apicidin was potentiated to various degrees in the presence of PSIs in a dose dependent manner. The combinations of apicidin and various PSIs growth arrested colorectal cancer cells in G2‐phase, Go‐G1/S‐phases and S/G2‐M phases in a combination dependent manner. Also, these combinations induced more potent apoptosis compared to single treatment with apicidin and PSIs. Upregulation of p16, p19, p21, p57, caspase‐3, caspase‐3, Bim, Apaf1, Bad and Bak mRNAs and downregulation of Cdk1, Cdk4, Cdk6, Bcl2, cIAP1 and cIAP2 mRNAs were observed. The combinations of apicidin and various PSIs chemosensitized cancer cells to chemotherapeutic drugs. These findings indicated that coadministration of apicidin and PSIs are associated with enhanced antiproliferative activity, apoptosis and chemosensitization of human colorectal cancer cells to conventional chemotherapeutic drugs through selective induction and suppression of genes that play an important role in the cell cycle and apoptosis.
SRJ23, A NEW INVESTIGATIONAL ANTI‐PROSTATE CANCER AGENT
Authors: Wong Hui Chyn , Sagineedu Sreenivasa Rao , Lajis Nordin Haji , Stanslas Johnson
Published: June 2021

In our drug discovery programme, from natural products andrographolide (AGP), the major phytoconstituent of Andrographis paniculata, was identified to have anticancer activity in vitro and in vivo. For improvement of the anti‐cancer potential of AGP, a series of semisynthetic derivatives of this compound was synthesized and tested for in vitro anticancer activity. From the National Cancer Institute (NCI) of USA in vitro anticancer screen, SRJ23 was found to exhibit selective anti‐cancer activity against prostate cancer cell lines (Jada et al., 2008. Br J Pharmacol 155: 641–654). The microculture tetrazolium (MTT) assay was utilized in assessing the in vitro growth inhibition and cytotoxicity of SRJ23 against three different human prostate cancer cell lines, PC‐3, DU‐145 and LNCaP. Subsequently, fluorescence microscopy was performed to determine the morphological cell death. Flow cytometry was used to analyze the cell cycle distribution of treated cells. SRJ23 at low micromolar concentrations exhibited growth inhibition and cytotoxicity in all three prostate cancer cell lines. The activity of the compound was selective towards PC‐3 cells and induced G2/M arrest which led to predominantly apoptotic mode of cell death. Interestingly, SRJ23 induced G1 cell cycle arrest in LNCaP and DU‐145 cell lines. Presently, SRJ23 is being considered a lead compound in the discovery of clinical anti‐prostate cancer agent. This research project is supported by Universiti Putra Malaysia.
Effect of porcine‐serum on viability, proliferation and morphology of hepatic cells
Authors: Osuna‐Martínez Ulises , Gómez‐Solís Antonieta , Alvarez‐Ayala Elizabeth , Reyes‐Esparza Jorge , Rodriguez‐Fragoso Lourdes
Published: June 2021

The aim of this study was to evaluate the effect of porcine‐serum (PS) on viability, proliferation and morphology of hepatic cells. For the study, we used the WRL‐68 cells (ATCC: CL‐48) cultured in MEM supplemented with 10 % fetal bovine serum, 2mol/L glutamine, 1% non essential aminoacid (MEMS). Cells were kept at 37°C, under an atmosphere 95% of air and 5% CO2. We included five groups of treatment: (1) MEMS (Control), (2) MEMS + PS 4 ug/uL, (3) MEMS + PS 8 ug/uL, (4) MEM + PS 4 ug/uL, and (5) MEM + PS 8 ug/uL. Viability (24 hours) and proliferation (24, 48, 72 and 96 hours) were evaluated after treatments by using the MTT assay. We used the Giemsa stain in order to evaluate changes in the cellular morphology. Our results showed that PS (4 and 8 ug/mL) did not affect the viability of hepatic cells, PS did not produce any cytotoxic effect on WRL‐68 cells. We observed that the addition of 4 and 8 ug/mL of PS produce an increase on cell proliferation in approximately 25 % at 24 hours, 27% at 48 hours, 29% at 72 hours and 25% at 96 hours (p < 0.05). We observed that WRL‐68 cells exposed to PS (4 and 8 ug/uL) increased the cellular size and changed the morphology. In conclusion, PS (4 and 8 ug/uL) had not cytotoxic and may act as a growth factor in WRL‐68 cells because is able to increase cell proliferation and cellular size of the hepatic cell.
Lack of Substrate Inhibition in a Monomeric Form of Human Cytosolic SULT2A1
Authors: Cook Ian Thomas , Leyh Thomas , Falany Charles
Published: June 2021

Cytosolic sulfotransferases (SULTs) are important Phase II conjugation enzymes that transfer a SO3 group from 3′‐phosphoadionsine‐5′ phosophosulfate (PAPS) to an acceptor molecule. Substrate inhibition is a common feature of the SULT family, though the mechanism is not well understood. For purification SULT2A1 was expressed as a maltose binding protein (MBP) ‐ fusion protein. The MBP‐SULT2A1 fusion protein eluted as a monomer during gel exclusion chromatography but formed a homodimer after cleavage of the MBP moiety. Kinetic analysis of the MBP‐2A1 monomer and pure SULT2A1 dimer found that both enzymes had similar Km (1.6 and 1.4 μM, respectively) and Vmax (8.6 and 8.4 pmol/min/mol of enzyme, respectively) values; however, MBP‐2A1 did not show substrate inhibition with increasing dehydroepiandrosterone (DHEA) concentrations. Kd values for DHEA binding with SULT2A1 and MBP‐SULT2A1 were 0.6 and 0.7 μM respectively and for PAPS, 0.3 μM for both. These results suggest that the dimerization at the conserved KTVE motif is involved in substrate inhibition.
Homology modeling and molecular dynamics of CYP1A1 and CYP2B1 to explore aryl derivatives metabolism by docking, followed by experimental assays
Authors: Mendieta‐Wejebe Jessica Elena , Correa‐Basurto José , Trujillo‐Ferrara José G. , Rosales‐Hernández Martha Cecilia
Published: June 2021

Since many drugs are metabolized by cytochrome P450 (CYP450), biotransformation studies using these enzymes are valuable in drug development. In this contribution the biotransformation by CYP1A1 and CYP2B1 of two acetylcholinesterase (AChE) inhibitors named 4‐(4′‐hydroxy‐phenylamino)‐4‐oxo propanoic acid (A) and 1H‐pyrrolidine‐1‐(4′‐hydroxy‐phenyl)‐2,5‐dione (B), was investigated theoretically by molecular modeling (docking and molecular dynamics; MD) and experimentally by using liver microsomes from rats pretreated with β‐naphthoflavone and phenobarbital (CYP1A1/CYP2B1 inducers, respectively). First, the target proteins were built under homology modeling and the resulting three dimensional structures were refined by MD, obtaining fifteen snapshots of each isoform. These conformers were used to dock test compounds A and B. Neither A nor B presented interaction of its amide and imide group, respectively, with the heme on any snapshot of CYP1A1. On the other hand when these compounds were docked on CYP2B1, their hydroxyl group was located near the heme iron on the snapshot at 3.5 ns. Therefore, theoretical results suggests that A and B can not generate toxic metabolites. Supporting these results, HPLC analysis showed only one metabolite from A and B, which was identified by GC‐MS as the hydroxylated product. Consequently, experimental results confirmed that neither test compound is toxic.
Administration of ampicillin elevates hepatic primary bile acid synthesis through suppression of ileal FGF15 expression
Authors: Miyata Masaaki , Takamatsu Yuki , Kuribayashi Hideaki , Yamazoe Yasushi
Published: June 2021

Administration of the antibacterial drug ampicillin (ABPC) significantly increased hepatic bile acid concentrations. In the present study, we investigated the mechanisms for the elevation of bile acid levels in ABPC‐treated mice. Hepatic microsomal cholesterol 7α‐hydroxylation was increased 2.0‐fold in ABPC‐treated mice despite higher bile acid levels in the liver and small intestinal lumen. A marked decrease in ileal fibroblast growth factor 15 (FGF15) mRNA level was observed (3% of vehicle‐treated mice) in ABPC‐treated mice. These phenomena were also observed in mice co‐treated with bacitracin/streptomycin/neomycin, which are barely absorbed from the intestine. Secondary bile acid, deoxycholic acid (DCA and tauroDCA) and unconjugated CA contents in the small intestinal lumen were reduced to below detection limits (<0.01 μmol). In ABPC‐treated mice, co‐treatment with tauroDCA reversed reductions in ileal FGF15 mRNA level. ABPC administration to farnesoid X receptor (Fxr)‐null mice also decreased ileal FGF15 mRNA levels. These results suggest that ABPC administration elevates hepatic primary bile acid synthesis, at least in part, through suppression of ileal FGF15 expression.
Aging of the Mesenteric Lymph Pump
Authors: Nagai Takashi , Bridenbaugh Eric , Gashev Anatoliy A
Published: June 2021

All functions of lymphatic system require the effective lymph flow, which cannot exist without effective contractions of muscle cells of lymphatic vessels (LV). However, the mechanisms regulating the lymphatic pumping, including its age‐related alterations, remain greatly under investigated. In this study we tested the hypothesis that contractility of mesenteric LV (MLV) is affected by aging including disturbed NO/flow‐dependent modulation. Parameters of lymphatic pumping of isolated, cannulated & pressurized segments of MLV were measured in F‐344 9‐ and 24‐mo old rats. The influences of incrementally increased transmural pressure and imposed flow were examined before & after treatment with L‐NAME 10‐4 M. We observed that negative inotropic and chronotropic effects of aging in MLV result in a 2–2.5‐fold decrease in their pump productivity. The flow/NO‐dependent regulation of lymphatic pumping is greatly disturbed in aged MLV. At the same time, we observed the evidence of flow‐independent NO release in aged MLV. Data suggest the existence of age‐induced alterations in NO synthase‐dependent modulation of MLV pump activity. We conclude that depletion of contractile reserves in aged MLV diminishes their ability to provide the adequate transport of lymph in mesenteric lymphatic network especially during periods of the increased lymph formation, which would occur after meal consumption. NIH RO1 AG030578.
Effects of environmental temperature and light intensity on blood physiological parameters of broilers
Authors: OLANREWAJU HAMMED AKANDE , BRANTON SCOTT L , PURSWELL JOSEPH L , COLLIER STEPHANIE D , DOZIER WILLIAM A
Published: June 2021

This study examined the effects of atmospheric temperature, light intensity, and their interaction on blood acid‐base balance and electrolytes in broiler chickens under environmentally controlled conditions in 2 trials. The experiment consisted of a factorial arranged in a randomized complete block design with 9 treatments consisted of 3 levels (Low=15.6, Moderate=21.1, High=26.7 °C) of temperatures and 3 levels (0.5, 3.0, 20 lx) of light intensities from d 21 to 56 d of age at 50% RH. Five hundred and forty (30 male/30 female) Ross × Ross 708 chicks were randomly distributed into 9 chambers at 1 d of age. Feed and water were provided ad libitum. Venous blood samples were collected on d 21 (base line), 28, 42, and 56. High temperature significantly (P ≤ 0.05) reduced BW, partial pressure of CO2 (pCO2), bicarbonate, hematocrit, hemoglobin, Ca2+, K+, and Na+ while significantly (P ≤ 0.05) elevated pH level, glucose, osmolality, and anion gap concentrations. Partial pressure of O2 (pO2) was slightly increased when related to ambient temperature. There was no effect of light‐intensity on most of the examined variables. Acid‐base regulation during high ambient temperature and light intensity exposure had not deteriorated despite lower pCO2 that consequently increased blood pH, due to a compensatory decrease in HCO3− concentration. Plasma corticosterone was not affected by temperature, light intensity or their interaction. These results indicate that continuous exposure of broiler chickens to high temperature, high light intensity or their interaction markedly affects some physiological blood variables without inducing stress in broilers.
Inhibition of Rho Kinase by Y27632 Increases Low Oxygen Tension‐Induced ATP Release from Human Red Blood Cells (RBCs)
Authors: Thuet Kelly , Bowles Elizabeth , Sridharan Meera , Adderley Shaquria , Sprague Randy , Stephenson Alan
Published: June 2021

Mechanical deformation and low oxygen (O2) tension were reported to stimulate ATP release from RBCs via a signaling pathway that includes the G protein Gi. Stiffening of the RBC with diamide, which promotes cross linking of cytoskeletal proteins, resulted in decreased low O2 tension‐induced ATP release. In the RBC, spectrin molecules must fold for deformation to occur. Phosphorylation of adducin and moesin by Rho kinase may stabilize cytoskeletal‐membrane interactions. Rho kinase signaling has been linked to increased stiffness of fibroblasts, ventricular myocytes, airway smooth muscle cells and endothelial cells. We hypothesized that Rho kinase inhibits low O2 tension‐induced ATP release from RBCs by stiffening the membrane cytoskeleton. We examined the effect of the Rho kinase inhibitor, Y27632 (1 uM), on low O2 tension‐induced ATP release from RBCs. In human RBCs, ATP release was increased in the presence of Y27632 (p<0.01, n=13). To support the hypothesis that ATP release is not the result of a direct effect of Y27632 to inhibit the activity of Gi, RBCs were incubated with Mastoporan 7, a direct Gi activator. Y27632 had no effect on Mastoporan 7‐ induced ATP release (n=3). These results are consistent with the hypothesis that Rho kinase inhibition increases RBC deformability resulting in augmented ATP release in response to exposure of these cells to low O2 tension. (NIH grant HL089094)
An Experimental and Computational Systems Approach for Studying How Hemodynamics and Bone Marrow‐Derived Cells Coordinate to Regulate Microvascular Remodeling
Authors: Bajikar Sameer S , Meisner Joshua K , Price Richard J
Published: June 2021

Hemodynamics, bone marrow derived cell (BMC) recruitment, and growth factor and chemokine signaling all regulate microvascular remodeling; however, the temporal and spatial coordination of these factors in vivo is poorly understood. Therefore, we are creating experimental and computational systems biology approaches for studying how these factors coordinate in structural remodeling of arteriole networks. We have shown that the dorsal skinfold window chamber (DSFWC) preparation can be used for determining the role of BMCs and chemokine receptors in microvascular remodeling (Nickerson et al., ATVB, 2009). Our new experimental model utilizes a modified mouse DSFWC preparation and a microligation device to occlude single arterioles. Following microligation of a single arcade arteriole, we observed a significant increase in the maximal diameter of “collateral” arterioles circumventing the ligation (65%) compared to “background” arterioles (38%) from day 1 to 7. Measured by microsphere particle imaging velocimetry, collateral arterioles sustained a larger increase in centerline velocity (94%) than background arterioles (36%), showing the experimental model permits observation of collateral vessel remodeling to altered blood flow. Next, a computational network model was developed to characterize network‐wide hemodynamic alterations from microligations. The computer model predicted blood flow increases along the determined “collateral” arteriole pathways and other vessels, which correlated with increases in diameter in vivo from day 1 to 7. These new experimental and computational tools will enable us to test how microvascular remodeling is coordinated through altered hemodynamics and BMC recruitment with high spatio‐temporal resolution. Supported by NIH HL74082.
Transendothelial Transport May Determine Adiponectin Oligomer Functions
Authors: Rutkowski Joseph Michael , Scherer Philipp E
Published: June 2021

Adiponectin has exhibited positive effects in regulation of systemic metabolism and insulin sensitivity through tissues such as liver, skeletal muscle, and centrally in the brain. Circulating as oligomers of 90 to 1080kDa, adiponectin must cross the endothelium to have metabolic effects. Our laboratory has reported differential efficacy of these oligomers on insulin sensitivity. Recent data has also demonstrated oligomer size‐dependence on adiponectin clearance. Only the small trimeric oligomer is found in cerebral spinal fluid. Here, we have measured the Stokes radii of adiponectin to be approximately 5, 10, and 25nm for the trimeric, hexameric, and high molecular weight (HMW) forms, respectively. The HMW size may present transport limitations across endothelial barriers in muscle or in the brain. Using murine 3B‐11, MS1, and bEnd3 endothelial cells in transwell permeability studies, we demonstrate that 3B‐11 and MS1 cells permit equivalent transport of trimeric and HMW adiponectin for their diffusive sizes. The bEnd3 cells, a model of brain endothelium, present an increased barrier to HMW adiponectin. In all cases, however, adiponectin fluxes were less than equivalently‐sized dextrans, suggesting the existence of active adiponectin transport mechanisms. These data demonstrate that the effects of different adiponectin oligomers on tissue metabolism may depend on the endothelium in a given tissue.
Caveolin‐1 limits the contribution of BK(Ca) channel to EDHF‐mediated arteriolar dilation
Authors: Feher Attila , Labinskyy Nazar , Bagi Zsolt
Published: June 2021

Large conductance Ca2+‐activated potassium channels (BKCa) channels are targeted to caveolae microdomains, where Cav‐1 interacts with BK(Ca) channels. The functional consequence of this interaction and its impact on EDHF‐mediated arteriolar dilation is not known. Thus, in this study we set out to examine the role of Cav‐1 in modulating the contribution of BK(Ca) channels to EDHF‐mediated arteriolar dilations. In isolated, pressurized (80 mmHg) gracilis muscle arterioles (≈ 100 μm) of Cav‐1 knockout mice, ACh‐induced, EDHF‐mediated dilations were enhanced (88±5 vs. 79±4 %) and were significantly reduced by selective BK(Ca) channel inhibitor, iberiotoxin (IBTX) (50±6 %), while IBTX had no effect on EDHF‐mediated dilations in wild type mice (75±7 %). Dilations to selective BK(Ca) opener, NS‐1619 were augmented in Cav‐1 knockout mice (58±6 % vs. 26±3 %). Moreover, in control arterioles, disruption of caveolae with methyl‐ß‐cyclodextrin augmented IBTX sensitive, ACh‐induced (29±7 % dilation in the presence of IBTX), and also NS‐1619‐evoked dilations (55±10 %). Immunohistochemistry demonstrated a co‐localization of Cav‐1 and BK(Ca) channels in the smooth muscle layer of control arterioles. Thus, we concluded that under control conditions an interaction between Cav‐1 and BK(Ca) channel limits the contribution of BK(Ca) channel to EDHF‐mediated arteriolar dilation. (AHA 0735540T)
Effects of chronic melatonin treatment in renovascular hypertension
Authors: Almeida Vitor R , Almeida Flavio R , Andersen Monica L , Campos Ruy R , Bergamaschi Cassia T
Published: June 2021

Experimental evidence suggests that reactive oxygen species which occur as a consequence of renal artery stenosis play a significant role in the hypertensive responses to increased Ang II. The aim of this study was to test the hypothesis that melatonin could alleviate renovascular hypertension through its antioxidant effects, as well as by its modulatory role on cardiovascular functions. Male Wistar rats were submitted to Goldblatt surgery (2K‐1C) to induce renovascular hypertension. Melatonin treatment started 5 weeks after surgery (30 mg/Kg, orally, daily) and continuous for 2 weeks. Blood pressure (BP), heart rate (HR) and baroreflex control were evaluated in conscious rats. Melatonin treatment produced a significant reduction in BP when compared to control hypertensive animals (128.1±2,6 x 180.4±16,9 mmHg) with no changes in HR. Hypertension induced profound alterations in the baroreflex control of blood pressure and treatment with melatonin were able to reverse these alterations. In conclusion, melatonin treatment was able to reduce blood pressure and improved baroreflex control in renovascular hypertension, suggesting that melatonin may be of therapeutic use in preventing oxidative stress due to hypertension.
Hemodynamic effects of torcetrapib and cardiotonic steroids in rats
Authors: Ma Xiuying , Zhao Huawei , Forrest Michael J.
Published: June 2021

Torcetrapib (Pfizer), an inhibitor of cholesteryl ester transfer protein (CETP), increases blood pressure and aldosterone in humans and in animal models. We recently reported that torcetrapib‐induced increases in blood pressure are independent of circulating aldosterone but dependent on an intact adrenal‐cortex. In the present study, our goal was to explore whether other adrenal‐derived steroids or mediators, distinct from aldosterone, contribute to torcetrapib‐induced pressor effects in Sprague‐Dawley rats. The studies used conductance catheter technology and pharmacological tools. Initial studies, presented herein, have focused on cardiotonic steroids including ouabain and bufalin. Interestingly, the hemodynamic and pharmacologic profile of these compounds appears distinct from that of torcetrapib. Compared with vehicle, ouabain, bufalin, and torcetrapib increased systemic blood pressure, left ventricular pressure, and total peripheral resistance. However, ouabain and bufalin increased stroke volume and cardiac output whereas torcetrapib did not. In addition, the torcetrapib‐induced pressor effect was inhibited by the calcium channel blocker nifedipine whereas that of ouabain and bufalin was not. In conclusion: 1) these data suggest that the torcetrapib‐induced pressor effect is primarily due to an increase in total peripheral resistance but not cardiac output; 2) the hemodynamic response to torcetrapib is distinct from that of adrenal‐derived cardiotonic steroids.
Polyol pathway contributes to the acute hyperglycemia‐induced contractile dysfunction in perfused heart from rat
Authors: Tang Wai Ho , Cheng Wing Tim , Chung Sookja Kim , Chung Sum Man Stephen
Published: June 2021

The polyol pathway, consisting of aldose reductase (AR) and sorbitol dehydrogenase (SDH), is an alternate metabolic route that converts excess glucose to fructose. Previously, inhibition of AR was found to protect diabetic mice hearts from contractile dysfunction, suggesting that the polyol pathway contributes to hyperglycemia‐induced contractile dysfunction. In this report we show that the polyol pathway also contributes to acute hyperglycemia‐induced cardiac contractile dysfunction. Isolated rat hearts were perfused with either Krebs' buffer containing 10 μM AR inhibitor, Fidarestat, or 1 μM SDH inhibitor, CP‐170,711, and subjected to acute hyperglycemia by perfusing with high glucose medium (33.3 mM) for 2 hours. We find that acute hyperglycemia‐induced contractile dysfunction of the isolated perfused hearts is improved by pharmacological inhibition of the polyol pathway. Under acute hyperglycemia, SERCA is inactivated by the tyrosine nitration, which is contributed by high level of peroxynitrite. However, the activity of NCX is increased, and the activation is probably contributed by reactive oxygen species. All these abnormalities were attenuated by treatment with ARI or SDI. Thus, during acute hyperglycemia, polyol pathway‐induced depletion of GSH and increased level of superoxide probably inactivate SERCA, and stimulate NCX, leading to the abnormalities in contractile function.
Reduced leptin and resistin and increased adiponectin are associated with improved endothelial function during weight loss with low fat compared to low carbohydrate diets
Authors: Phillips Shane A. , Bian Jing‐Tan , Bhutani Surabhi , Varady Krista A.
Published: June 2021

Previously we showed that low fat (LF) diets improved endothelium‐dependent flow mediated dilation (FMD) compared to isocaloric low carbohydrate (LC), high fat diets. The purpose of this study was to determine 1) the relationships between pro‐ (leptin and resistin) and anti‐ (adiponectin) atherogenic adipokines on endothelial function during weight loss with LF and LC diets and 2) if exogenous adiponectin improves microvascular endothelial function in subjects on LC diets. Healthy subjects (BMI: 29–39) were assigned to a LF or an isocaloric LC diet for 6 weeks. Brachial artery FMD was determined with ultrasound. Leptin, resistin, and adiponectin were determined with ELISA (baseline and week 6). In separate studies, endothelium‐dependent acetylcholine (ACh) dilations were determined in isolated resistance arteries from subcutaneous fat biopsies. Body weight, fat mass, and waist circumference was reduced in LF and LC groups (P<0.05). Increased FMD on the LF diet was related to increased adiponectin (r=0.55; P=0.04), decreased leptin (r=−0.78;P=0.03), and decreased resistin (r=−0.47;P=0.01). Leptin (but not resistin) was reduced after LC. Exogenous adiponectin (10μg/ml) reversed impaired ACh dilations in isolated resistance arteries of subjects on LC diets. These results suggest that reduced leptin and resistin and increased adiponectin contribute to improved endothelial function during weight loss on LF but not LC diets. Adiponectin may reverse microvascular endothelial dysfunction induced by high fat, LC diets.
Mitochondria mediated vasodilation by BMS‐191095, a selective mitochondrial KATP channel activator
Authors: Katakam Prasad V.G , Steed Mesia M , Snipes James A , Busija David W
Published: June 2021

Mitochondrial depolarization and subsequent generation of reactive oxygen species (ROS) have been shown to trigger vasodilation via activation of ‘calcium sparks’ and large‐conductance calcium‐activated K+ channels (BKCa). It is unclear if mitochondrial depolarization alone, independent of ROS, is capable of promoting vasodilation. Therefore, we determined the vasodilation induced by BMS‐191095 (BMS), a putative mitochondrial KATP channel opener, in isolated cerebral arteries of Sprague‐Dawley (SD), Zucker obese (ZO) with mitochondrial dysfunction and the lean (ZL) control rats. In SD arteries, 50 μmol/L BMS depolarized mitochondria without producing ROS, and induced vasodilation (% relaxation) in endothelium‐intact arteries (53.1±4.4, n=12) that was diminished by endothelial denudation (36.6±3.9, n=8), nitric oxide (NO) synthase inhibition (27.4±5.3, n=6), and BKCa blockade (21.3±2, n=11) (p<0.05). However, the vasodilation was unaffected by ROS scavenger (45.9±6.7, n=10). In ZO arteries, BMS‐induced vasodilation and mitochondrial depolarization were diminished compared to ZL arteries. Thus, mitochondrial depolarization induces vasodilation via ROS‐independent mechanisms mediated by endothelium, smooth muscle, NO and BKCa. Finally, mitochondrial dysfunction associated with insulin resistance diminishes BMS‐induced vasodilation. Supported by HL‐077731, HL‐030260, HL‐065380.
Canrenoic Acid, a Mineralocorticoid Receptor Antagonist, Attenuates Resistance Artery Remodeling in Stroke‐Prone Spontaneous Hypertensive Rats
Authors: Pires Paulo Wagner , McClain Jonathon Lee , Dorrance Anne McLaren
Published: June 2021

Chronic hypertension causes structural changes in the resistance vasculature through the process of remodeling. Middle cerebral artery (MCA) remodeling increases the risk for ischemic stroke. We have previously shown that the mineralocorticoid receptor (MR) is involved in MCA remodeling. Therefore, we hypothesized that MR antagonism with canrenoic acid (CANR) would attenuate the process. Six‐week old male stroke‐prone spontaneously hypertensive rats (SHRSP) were treated with CANR (20mg/kg/day, n=7) in the drinking water for six weeks. Untreated SHRSP were used as controls (n=12). MCA and mesenteric resistant artery (MRA) passive structure was analyzed by pressure myography. Data are shown as mean ± SEM, CANR vs control respectively. In the MCA, at 80mmHg, CANR treatment increased lumen diameter (277±6 vs 240±5μm, p<0.05) and decreased wall thickness (17.1±1.1 vs 25.6±1.2μm, p<0.05) and wall‐to‐lumen ratio (0.06±0.01 vs 0.11±0.01, p<0.05), without changing vessel stiffness (β‐coefficient: 10.4±1.4 vs 8.16±1.6, p<0.05). In the MRA, at 90 mmHg, CANR treatment reduced wall thickness (17.0±0.7 vs 27±1.7μm, p<0.05) and wall‐to‐lumen ratio (0.16±0.01 vs 0.10±0.01, p<0.05), without changing lumen diameter (274±8 vs 265±9μm) or vessel stiffness (β‐coefficient: 5.5±0.7 vs 5.5±0.5). These data suggest that MR antagonism attenuates cerebral and peripheral remodeling by different mechanisms.
Sphingosine kinase 1 is required for smooth muscle cell phenotypic modulation in response to vascular injury.
Authors: Wamhoff Brian R , Matthews Thomas , Hogan John , Morris Andrew , Karel Yugesh , Macdonald Timothy , Lynch Kevin R
Published: June 2021

Sphingosine‐1‐phosphate (S1P) signaling via S1P receptors plays a critical role in vascular development and disease. We recently showed that the S1P1 receptor promotes vascular smooth muscle cell (SMC) phenotypic modulation in response to injury whereas the S1P2 receptor regulates SMC differentiation. S1P is generated by sphingosine kinase (Sphk), but little is know about Sphk activity in SMCs. Here we show that Sphk1 (not Sphk2) mRNA and activity are increased transiently in response to carotid injury in vivo in both mice and rats. Sphk1 null mice showed reduced neointimal hyperplasia compared to WT mice following carotid injury. Treatment of rat aortic SMCs in vitro with PDGF‐BB resulted in increased Sphk1 (not Sphk2) mRNA, activity and S1P intracellular concentrations. We developed a series of novel pan Sphk inhibitors. Treatment with VPC94075 prevented PDGF‐BB‐induced SMC proliferation and S1P generation; the enantiomer had no effect. We hypothesized that PDGF‐BB‐induced increases of S1P regulate SMC proliferation via autocrine activation of the S1P1 receptor. Treatment of SMCs with VPC44116 (an S1P1 antagonist) attenuated PDGF‐BB‐induced proliferation by ~50%. In conclusion, 1) Sphk1, not Sphk2, is important in regulating endogenous SMC S1P levels in response to vascular injury, and 2) SMC‐generated S1P can feedback via the S1P1 receptor to drive SMC phenotypic modulation. HL081682, GM067958, AHA SDG
Nuclear Factor of Activated T‐cells (NFAT) regulates osteopontin expression in arterial smooth muscle in response to diabetes‐induced hyperglycemia
Authors: Gomez Maria F , Nilsson‐Berglund Lisa M , Zetterqvist Anna V , Nilsson‐Öhman Jenny , Sigvardsson Mikael , Gonzalez‐Bosc Laura V , Smith Maj‐Lis , Salehi Albert , Agardh Elisabet , Nordin‐Fredrikson Gunilla , Agardh Carl‐David , Nilsson Jan , Wamhoff Brian R , Hultgårdh‐Nilsson Anna
Published: June 2021

Hyperglycemia is a recognized risk factor for cardiovascular disease in diabetes. Recently we reported that high glucose activates the transcription factor NFAT in arteries ex vivo. Here, we investigate if hyperglycemia activates NFAT in vivo and whether this leads to vascular complications. Intra peritoneal glucose tolerance test in mice increased NFATc3 nuclear accumulation in vascular smooth muscle. Streptozotocin‐induced diabetes augmented NFATc3 transcriptional activity in arteries of NFAT‐luciferase transgenic mice. Two NFAT responsive sequences were identified in the promoter of the proinflammatory cytokine osteopontin (OPN), known to exacerbate atherosclerosis and restenosis. Activation of NFAT resulted in higher OPN mRNA and protein in native arteries. Glucose‐induced OPN expression was prevented by the calcineurin inhibitor cyclosporin A, the NFAT blocker A‐285222 or the ectonucleotidase apyrase, suggesting a mechanism involving the release of extracellular nucleotides. Further, diabetes resulted in higher OPN expression, which was significantly decreased by in vivo treatment with A‐285222 for 4 weeks or prevented in arteries from NFATc3−/− mice. These results identify a novel molecular mechanism underlying vascular complications of diabetes. Funding: Swedish Heart & Lung Foundations
The nitric oxide (NO) donors [Ru(terpy)(bdq)NO+]3+ (TERPY) and Sodium Nitroprusside (SNP) have different cellular effects in rat basilar arteries relaxation.
Authors: Paulo Michele , Vercesi Juliana Aparecida , Biazzotto Juliana C. , Da Silva Roberto Santana , Bendhack Lusiane Maria
Published: June 2021

This study aimed to verify if the new NO donor (Terpy) induces vasorelaxation in basilar artery from renal hypertensive (2K‐1C) and normotensive (2K) rats and compare this effect with SNP. We also investigated the effects of inhibition of guanylyl‐cyclase with ODQ (1μmol/L) and K+ channels with TEA (1mmol/L). We have analyzed the efficacy (ME) and potency (pD2). The experiments were performed on denuded rings which were pre‐contracted with endothelin‐1 after pre‐incubation with ODQ or TEA. Interestingly, Terpy (1nmol/L to 100mmol/L) did not induce relaxation. On the other hand, SNP induced relaxation in a concentration‐dependent way, which was greater in basilar arteries from normotensive 2K (ME: 91.5±2.5%; pD2: 5.96±0.19, n=6) than in basilar rings from 2K‐1C (ME: 77.8±3.8%; pD2: 4.63±0.15, n=6, P<0.05). TEA had a greater inhibitory effect in 2K arterial rings (ME: 42.0 ± 1.2%, pD2 5.12 ± 0.25, n=6) than in 2K‐1C arteries in which reduced only the maximal relaxation (ME: 19.4 ± 0.8%, n=6) without effect on the potency of the NO donor. ODQ abolished the relaxation induced by SNP in 2K and 2K‐1C arteries. In conclusion, Terpy did not induce relaxation in basilar artery from 2K and 2K‐1C rats and the relaxation induced by SNP involves the activation of soluble guanylyl‐cyclase sensitive to ODQ and K+channels sensitive to TEA. Supported by FAPESP and CNPq.
Effect of Membrane Permeability and Extracellular Diffusion on NO uptake by Erythrocytes
Authors: Deonikar Prabhakar , Kavdia Mahendra
Published: June 2021

Quantification of nitric oxide (NO)‐red blood cells (RBC) reaction metabolites is necessary to evaluate the extent of diffusional resistances for nitric oxide transport in vascular lumen. Delivery of physiologically relevant concentration of nitric oxide and RBC‐hemoglobin are a critical factor in quantification of in vitro NO‐RBC reaction. In this study, we developed a flask bioreactor which delivers gaseous NO to the RBCs and studied the effects of hematocrit (hct) and NO concentrations on NO‐RBC interactions. We developed a computational model of NO transport to single RBCs using experimental data to analyze the effect of RBC membrane permeability on steady state NO concentrations in the bulk of liquid surrounding the RBCs and at the RBC membrane. We observed that the steady state NO concentrations in the bulk of liquid and at the RBC membrane 1) decreased with increase in RBC membrane permeability for a given equilibrium NO concentration and hct; 2) increased with increase available NO concentrations at a given RBC membrane permeability; 3) decreased with increase in hct for a given RBC membrane permeability. Our results indicate that the extracellular diffusion and RBC membrane permeability are significant diffusional barriers for NO transport to the RBCs and the RBC NO membrane permeability should be in the range of 0.0415‐ 0.4 cm/s. Supported by: AHA 0530050N, NIH R01 HL084337 & R15 HL087287.
Heterogeneity in Constrictor Responses Among Selected Inbred Mouse Strains: Influence of Rho‐kinase
Authors: Didion Sean P
Published: June 2021

The goal of this study was to compare and contrast contractile responses among four commonly used inbred mouse strains and to determine the contribution of Rho‐kinase. Contractile responses were examined in carotid arteries (in vitro) from C57BL/6, C3H/He, Balb/c, and SWR mice. We examined constrictor responses to potassium chloride (KCl; 1–100 mM), serotonin (5HT; 1 nM–10 μM), phenylephrine (PE; 1 nM–10 μM), and U46619 (0.03 – 3 mg/ml). 5HT produced a significantly greater degree of contraction in Balb/c mice compared to C57BL/6, SWR and C3H/He mice (eg, 100 μM 5HT produced 0.45±0.06, 0.14±0.03, 0.13±0.02, and 0.08±0.03 mg of contraction in Balb/c, C57Bl/6, SWR, and C3H/He mice, respectively). Contraction to PE and U46619 was also markedly greatest in Balb/c mice. KCl responses were similar in all strains. In order to examine the contribution of Rho‐kinase, carotid arteries were incubated with either vehicle or Y‐27632 (1 μM; Rho‐kinase inhibitor). Y‐27632 reduced contractile function in a constrictor‐ and strain‐dependent manner (eg, contraction to 100 μM 5HT with Y27632 = 0.22±0.04, 0.06±0.01, 0.04±0.01, and 0.03±0.01 mg of force in Balb/c, C57Bl/6, SWR, and C3H/He mice, respectively). These data provide evidence for heterogeneity in constrictor responses among four inbred mouse strains and dependency upon Rho‐kinase.
Clenbuterol increases PGC1a promoter activity via a rapamycin sensitive mechanism
Authors: Philp Andrew , Baar Keith
Published: June 2021

The B2 agonist clenbuterol increases the expression of the peroxisome proliferator activated receptor coactivator‐1a (PGC1a) in skeletal muscle. The aim of this study was to determine the contribution of mTOR towards clenbuterol actions on PGC1a activity and mitochondrial mass in C2C12 myotubes. Transient transfections were carried out on sub‐confluent C2C12 myoblasts using a pGL3‐PGC1a reporter construct. Cells were differentiated for 5 days before treatment for 12h with clenbuterol (0.5–1.5μg/μl), rapamycin (100nm) or combination. Twelve hours of clenbuterol treatment (1.0 ug/ul) increased cell oxygen consumption by 100% (186.5 ± 5.4 to 372.1 ± 37.3 pmole.min‐1 P<0.05). PGC1a promoter activity followed a similar trend increasing by 40% (1.0ug/ul) following clenbuterol treatment (3.25 ± 0.03 to 4.54 ± 0.21 RLU P<0.05). In contrast, 12h Rapamycin treatment decreased PGC1á promoter activity by 22% (2.55 ± 0.3 RLU P<0.05) and cell oxygen consumption from basal conditions (P<0.05). Rapamycin treatment completely blocked the positive effect of clenbuterol on PGC1a promoter activity (−43% 1.87 ± 0.12 RLU P<0.05) and cell oxygen consumption from basal conditions (P<0.05). Given that rapamycin completely blocks the positive effects of clenbuterol, our data suggest that clenbuterol increases PGC1a promoter activity via an mTOR dependent mechanism.
Changing sexes in a complex environment ‐ shifts in skeletal myosin isoforms in coastal populations of California sheephead fish
Authors: Bartlett Jinae Nicole , Loke Kerri M , Young Kelly , Lowe Chris , Rourke Bryan C
Published: June 2021

Myosin heavy chain (MyHC) isoform expression was investigated in California sheephead, Semicossyphus pulcher, a protogynous hermaphroditic fish found in coastal Southern California and Northern Mexico. This species was collected from nine capture sites near the Channel Islands where both overall size, and size of individuals at the transitional sexual stage, varies considerably. We therefore cloned MyHC isoforms and analyzed proteins by SDS‐PAGE to determine if gender, size, water temperature, or geographic location influenced contractile protein expression. The relative percentage of the fast MyHC isoform decreased as sheephead fish transitioned from female to male. The highest expression of slow MyHC isoform was observed in male sheephead. The lowest expression of slow MyHC was seen in Northern and off‐shore female populations, when compared to Southern and near‐shore female populations. Relative percentage of slow MyHC isoform positively correlated with standard length. Thus, in this interesting system where muscle mass undergoes dramatic growth accompanying gender transitions, MyHC isoforms are likely influenced by both sexual development and environment.
What Triggers the Aerobic Dive Limit? Patterns of Muscle Oxygen Depletion during Dives of Emperor Penguins
Authors: Williams Cassondra L. , Ponganis Paul J.
Published: June 2021

The concept that most dives are aerobic and less than the aerobic dive limit (the onset of post‐dive blood lactate accumulation) is a fundamental concept in diving physiology and the interpretation of diving behavior and foraging ecology. The physiological basis of the aerobic dive limit (ADL) is hypothesized to be depletion of the muscle O2 store. A dual‐wavelength near‐infrared spectrophotometer was developed and applied to the pectoral muscle to measure myoglobin O2 saturation levels during dives of emperor penguins (Aptenodytes forsteri) at an isolated dive hole on the sea ice of McMurdo Sound, Antarctica. Data revealed several patterns of O2 depletion. A monotonic decline to near complete O2 depletion during dives at the ADL occurred in some birds. Based on myoglobin concentration in the pectoral muscle and an assumption of no muscle blood flow, this corresponds to an O2 consumption rate of six to twelve ml O2 kg muscle−1 min−1. However, other birds demonstrated different O2 depletion patterns suggesting maintenance of some muscle perfusion during dives. Results support the hypothesis that the onset of post‐dive blood lactate accumulation is secondary to muscle O2 depletion during the dive but that the ADL may vary depending on muscle blood flow patterns. This research was supported by NSF grant OPP 0538594. CLW was supported by a NIH Marine Biotechnology Training Program Fellowship and a UC Regents Fellowship.
Cardiovascular plasticity during hypoxic incubation of alligator (Alligator mississippiensis)
Authors: Eme John , Crossley Dane A , Hicks James W
Published: June 2021

The vertebrate cardiovascular (CV) system becomes operational early in development and must function and mature both anatomically and physiologically. Deviations in developmental environment create challenges that impact CV function and maturation. Reptilian embryos may be particularly impacted given features of development in egg‐laying species, such as hypoxia. Using chronic hypoxic incubation as a stressor (10% O2 beginning at 20% of development), the plasticity of the CV system was assessed in American alligator embryos. Compared to normoxic‐incubated embryos, chronic hypoxic incubation resulted in bradycardia at 90% of incubation, but differing effects on arterial pressure. Assessments of both acute hypoxic and chemically induced chemoreflex indicated maturation of this regulatory mechanism is plastic during embryonic development and blunted by chronic hypoxic incubation. In addition, roles of the sympathetic and parasympathetic nervous systems are altered by hypoxia. These data indicate that maturation of CV function can exhibit a significant degree of phenotypic plasticity in embryonic alligators. NSF Career award IBN IOS‐0845741 to DAC and NSF IOS‐0922756 to JWH.
Regulation of p53 mRNA by AMP Kinase (AMPK) activation in C2C12 myoblasts
Authors: Saleem Ayesha , Shuen Michael , Hood David A.
Published: June 2021

p53 protein is important for basal and exercise‐induced mitochondrial biogenesis in skeletal muscle. As shown previously, pifithrin‐α (15 μM) a specific inhibitor of p53, reduced the AICAR‐induced increase in mitochondrial content in myoblasts. This demonstrates that p53 partly mediates AMPK‐induced mitochondrial adaptations. Here, we investigated whether p53 mRNA content and stability were modulated by AMPK. Two transcript variants (TV) of murine p53 have been discovered. p53 TV2 mRNA was expressed at 60% lower levels than p53 TV1 in C2C12 myoblasts. AICAR treatment (3 days) reduced p53 TV1 and TV2 mRNA levels by 28% and 13%, respectively, compared to control. To investigate whether this reduced mRNA was due to changes in mRNA stability, we generated reporter constructs containing the full length (R2) 3′untranslated region (UTR), or a shorter construct without the AU‐rich region and the poly‐adenylation signal (R1) of p53 TV2 mRNA. Following transfection, luciferase activity of both R1 and R2 was only ~15% of control, indicating that even the shorter 3′UTR could mediate p53 mRNA decay. AICAR treatment further decreased luciferase activity of both constructs by ~35% compared to untreated cells. This suggests that AMPK regulates p53 by reducing mRNA stability and content. Our findings shed light on the complexity of p53 regulation and have significance for p53‐based therapeutic interventions. (Funded by NSERC)
Regulation of Tfam mRNA stability in skeletal muscle fiber types
Authors: D'souza Donna Marie , J Lai Ruanne Y. , Hood David A
Published: June 2021

Changes in mRNA stability may serve as a regulatory mechanism for mitochondrial biogenesis in skeletal muscle. We hypothesized that oxidative capacity would be closely associated with a greater mRNA stability of proteins vital to organelle biogenesis, prompting an enhanced mitochondrial content. Using an in vitro decay assay, the mRNA stability of mitochondrial transcription factor A (Tfam) was assessed, since it regulates the expression of mitochondrial DNA (mtDNA). The decay of Tfam mRNA was slowest in low oxidative fast‐twitch white (FTW) muscle. Decay rates were 1.5‐ and 2.3‐fold greater in fast‐twitch red (FTR) and slow‐twitch red (STR) fibers. Despite this, steady Tfam mRNA levels were not different between fiber types, suggesting increases in transcription in oxidative muscle which parallel mRNA decay. Degradation of mRNA in fiber types matched similar differences in the mRNA destabilizing protein AUF1, particularly the p42 isoform, with a 4.5‐fold difference in protein expression between STR and FTW muscle. A more dramatic difference in the expression of the mRNA stabilizing protein HuR existed between fiber types, which may serve to restrain uncontrolled mRNA decay in highly oxidative muscle. Thus, oxidative muscle types exhibit faster rates of Tfam mRNA turnover than low oxidative muscle, suggesting more precise control of Tfam expression, and thus mtDNA levels, in response to metabolic stress.
The use of SUnSET as a non‐radioactive method for measuring rates of protein synthesis in whole skeletal muscles and in single fibers
Authors: Goodman Craig Andrew , Mabrey Danielle M. , Frey John W. , Lincoln Hannah C. , Miu Man Hing , Schmidt Enrico K. , Pierre Philippe , Hornberger Troy A.
Published: June 2021

SUnSET (SUrface SEnsing of Translation) involves the immunological detection of puromycin in nascent peptides as a means for quantifying rates of protein synthesis (PS). To validate SUnSET against an established technique, plantaris muscles were subjected to synergist ablation (SA) or sham surgery. After 7d, muscles were incubated ex vivo with a flooding dose of 3H‐Phenylalanine (3H‐P) or with puromycin. According to the established 3H‐P method, SA induced a 3.4 fold increase in PS while a western blot (WB) version of SUnSET revealed a 3.6 fold increase, indicating that both techniques produce quantitatively similar results. Furthermore, SUnSET revealed that SA induces a 2.9 fold increase in PS when measurements were performed in vivo. SUnSET was also used to demonstrate that food deprivation induces a ~70% decrease in PS in vivo when measured with WB analysis and similar results were obtained when an immunohistochemical (IHC) version of SUnSET was applied to cross‐sections from control and food deprived samples. Finally, the IHC version of SUnSET was applied to muscles electroporated with constitutively active Akt (CA‐AKT) and it was determined that PS rates were 80% higher in CA‐AKT transfected fibers when compared to non‐transfected fibers in the same section. In conclusion, SUnSET is a simple and inexpensive alternative for measuring PS and allows for measurements to be performed at the single fiber level.