bluuebunny/crossref_metadata_2025 huggingface dataset:
Uric acid does not impact the endothelial‐dependent relaxation of aorta from normal and hypertensive rats
Authors: Szasz Theodora , Linder A Elizabeth , Watts Stephanie W
Published: June 2021

Vascular xanthine oxidase (XO) activity and superoxide, a product of this activity, increase in hypertension. Uric acid (UA), the other XO‐derived product, has a controversial role in modulating vascular reactivity. Hyperuricemia induces hypertension and endothelial dysfunction in rats. Endothelial‐dependent relaxation is decreased in aorta from deoxycorticosterone (DOCA)‐salt hypertensive compared to sham normotensive rats. We hypothesized that UA impairs endothelial‐dependent relaxation in aorta from DOCA rats. Uricemia, ranging from unmeasurable to 547 umol/L in sham and to 506 umol/L in DOCA rats, was not significantly different. UA (1 nM – 100 uM), in the presence of uricase inhibition (oxonic acid 10 uM), did not contract normal aorta. The same combination (100 uM UA, 10 uM oxonic acid) did not modify relaxation to ACh (1 nM – 10 uM) in the normal, sham and DOCA aorta, when compared to vehicle, respectively (response to 10 uM ACh: normal=37±7 vs 48±7, sham=53±15 vs 57±20, DOCA=81±4 vs 85±2 % 20uM PGF2α contraction). Allopurinol (100 uM), the XO inhibitor, did not significantly alter the ACh‐induced relaxation of sham and DOCA aortic rings, when compared to its vehicle (response to 10 uM ACh: sham=61±5 vs 68±9, DOCA=87±6 vs 88±3 % 20 uM PGF2α contraction). These data suggest that XO, at least through its UA arm, does not impact the endothelial function of aorta in normal conditions and in DOCA‐salt hypertension.
AMP Kinase (AMPK) activator 5‐aminoimidazole‐4‐carboxamide‐1‐β‐D‐ribofuranoside (AICAR) alters vascular smooth muscle vasomotor responses in thoracic aorta rings from spontaneously hypertensive (SHR) and Wistar‐Kyoto (WKY) rats
Authors: Ford Rebecca J , Rush James W.E.
Published: June 2021

The metabolic regulatory role of AMPK is well established but potential mechanisms of AMPK in vascular control are not well defined. Aortic rings were isolated from 16–20wk old male SHR and WKY rats and assessed using myography to determine potential vasomotor effects of AICAR. There was a significant drop in resting tension after endothelium‐denuded rings were incubated with AICAR (A; 2mM, 30 min) in SHR (control (C) = −0.14±0.06g, A = −0.97±0.09g, p<0.01) but not in WKY vessels (C = −0.26±0.06g, A = −0.24±0.08g, p=NS, strain*drug p<0.01). Both SHR and WKY denuded vessels relaxed upon exposure to AICAR following phenylephrine pre‐constriction (PE 10−7M; SHR: C = 2.1±2.2%, A = 41.4±11.9%, p<0.01; WKY: C = 3.0±2.2%, A = 23.7±9.7%, p<0.01). In denuded vessels pre‐exposed to AICAR, potassium chloride dose‐response curves were right‐shifted in both SHR and WKY (SHR C vs A p<0.01, WKY C vs A p<0.01) and developed tension to increasing PE concentrations was blunted in both strains at all concentrations (max tension SHR: C = 2.07±0.19g, A = 1.14±0.21g, p<0.01; WKY: C = 2.57±0.19g, A = 1.98±0.21g, p<0.01, strain*drug p<0.01). These results suggest that AMPK can influence vasomotor function in SHR and WKY aortic rings.

Funded by the Heart and Stroke Foundation of Ontario
Age‐dependent changes in P2Y receptors in the hearts of P2Y2 knockout mice
Authors: Tang Chih‐Min , Yokoyama Utako , Sergeeva Okasana , Insel Paul
Published: June 2021

Genetic ablation in mice of the nucleotide receptor P2Y2 leads to salt‐resistant hypertension and changes in several organ systems. In the current study we employed this animal model to investigate age‐dependent changes in cardiac P2Y receptor expression. Using quantitative PCR, we determined the expression level of seven P2Y receptor subtypes, P2Y1, 2, 4, 6, 12, 13 and 14 in hearts of wild‐type (WT) and P2Y2 knockout (KO) mice at age of 4, 12 and 18 months. Hearts of WT mice have both P2Y1 and P2Y2 as the major receptor P2Y subtypes, with lower levels of RNA expression for P2Y6 and P2Y14. All four P2Y subtypes exhibit striking increases in expression in 18 month‐old mice whereas the other three P2Y subtypes have low expression levels and display no significant age‐dependent changes. In contrast, KO mice have elevated expression of all six of the other P2Y subtypes and demonstrate similar age‐dependent patterns of change as do WT mice, with increases in P2Y1, P2Y6 and P2Y14 in the 18‐month old group. These results demonstrate that P2Y2 receptors are an important subtype in the heart, such that their absence results in compensatory expression of other P2Y subtypes. Since P2Y2 KO mice are hypertensive, the observed changes in other cardiac P2Y receptors may represent adaptations to increase in blood pressure as well as to the absence of P2Y2 receptors. (Grant support from NIH, UCSD SIRA and the Ellison Medical Foundation)
Brief History of the Renin‐Angiotensin System
Authors: Igic Rajko
Published: June 2021

We present a short history of the RAS, including biological actions of angiotensin peptides, and the functions of the enzymes that generate and metabolize angiotensins, including the likely effect of manipulating them. Attention is devoted to renin, (pro) renin receptors, angiotensin converting enzyme (ACE), ACE2, chymase, and neprilysin. Also, the rationale for targeting the RAS, based on numerous clinical studies of the ACE inhibitors and AT(1) receptor blockers is included. These two drug classes have protective and beneficial effects preventing functional and structural abnormalities of the cardiovascular system, and reduce the end organ‐damage. Finally, we consider the investigational agents acting on the RAS that have a potential for clinical usage, and give the perspective of pharmacological, immunological and gene targeting of the RAS for treatment of cardiovascular disease.
Incorporating radiology into medical gross anatomy: A study of its efficacy in learning spatial relationships
Authors: Lufler Rebecca Sage , Zumwalt Ann , Hoagland Todd M
Published: June 2021

Radiological imaging is the depiction of anatomical structures and the spatial relationships among them. Visual‐spatial ability (VSA), the ability to mentally manipulate objects in 3D, is an important skill for medical students to apply their anatomical knowledge in the clinical setting. To date, no studies have investigated the effects of cadaver specific CT scans in the gross anatomy lab on students' learning and VSA. We hypothesize that there will be a positive relationship between VSA and practical exam scores, and that use of CT scans will be associated with practical exam scores and increases in students' VSA. 183 first year medical students were provided with cadaver CT scans, and students self selected into groups based on their time spent viewing the scans in lab. VSA was assessed by the Mental Rotations Test, given during orientation and at the end of the course. During the Back and Extremities section, 35% of the students used the CT scans in lab. Preliminary data suggest students who used the CT scans scored significantly higher on the practical exam (p=0.0086). Average VSA of those who used the CT scans was slightly lower than those that did not, but there was a positive correlation between VSA and practical exam scores (p=0.019, r=0.31). This research is supported by the LWW/AAA Education Research Scholarship.

Grant Funding Source

LWW/AAA Education Research Scholarship
Effects of Different Phosphatidylglycerol Species on the Proliferation of Primary Mouse Keratinocytes
Authors: Bollag Wendy B. , Xie Ding , Edwards John
Published: June 2021

Evidence is growing that phosphatidylglycerol (PG) plays a key role in modulating keratinocyte proliferation, as we have previously shown that liposomes derived from egg PG inhibit the proliferation of rapidly dividing cells and stimulate the growth of slowly proliferating keratinocytes. To determine the species of PG most effective at modulating proliferation of primary mouse keratinocytes, primary mouse keratinocytes were treated with liposomes composed of different PG species, and proliferation was measured as [3H]thymidine incorporation into DNA. PG species containing polyunsaturated fatty acids were effective at inhibiting rapidly proliferating keratinocytes, whereas PG species with monounsaturated fatty acids were effective at promoting proliferation in slowly dividing keratinocytes. Thus, palmitoyl, arachidonyl PG (16:0, 20:4, with the first number representing the number of carbon atoms in the fatty acid and the second, the number of double bonds), palmitoyl, linoleoyl PG (16:0,18:2), dilinoleoyl PG (18:2,18:2) and soy PG (a PG mixture with a large percentage of polyunsaturated fatty acids) were particularly effective at inhibiting proliferation in rapidly dividing keratinocytes. Palmitoyl, oleoyl PG (16:0, 18:1) and dioleoyl PG (18:1, 18:1) were especially effective proproliferative PG species. This result suggests that different PG species may signal to diverse effector enzymes to differentially affect keratinocyte proliferation and may be useful for treating skin diseases characterized by excessive or insufficient proliferation. (Supported by NIH grants #AR45212 and AR55022.)
Airway epithelia regulate airway surface liquid glucose concentration
Authors: Pezzulo Alejandro Antonio , Gutierrez Jeydith , Salm Kelly Susan , McConnell Kelly Susan , Karp Philip , Stoltz David Anthony , Zabner Joseph
Published: June 2021

The airway epithelium is lined by a small amount of airway surface liquid (ASL). Studies of exhaled breath condensate suggest that this liquid has a low glucose concentration. We investigated the mechanism that maintains a transepithelial glucose gradient in well‐differentiated human airway epithelia (HAE) grown at the air‐liquid interface. We found that HAE do not have electrogenic glucose transport, suggesting absence of sodium‐glucose cotransporters (SGLT). Polarized HAE cultures maintained a glucose gradient with low apical levels. In bidirectional flux assays, we found that HAE have low paracellular permeability to L‐glucose. Moreover, 2‐deoxyglucose fluxes favored basal to apical transport. Immunofluorescence showed that the facilitated diffusion transporter GLUT‐1 is expressed in the basolateral membrane and GLUT‐10 is expressed in the apical membrane. Finally, we found that increasing ASL glucose results in increased infection with P. aeruginosa in vitro and in vivo. These data suggest that either increased serum glucose concentration or disruption of the paracellular barrier could result in increased glucose in the ASL and increased risk of pulmonary infection.
Proline metabolism and protection against oxidative stress
Authors: Becker Donald F , Zhu Weidong , Natarajan Sathish
Published: June 2021

Proline oxidation by the mitochondrial enzyme proline dehydrogenase is upregulated by the p53 tumor suppressor gene, leading to increases in reactive oxygen species and induction of cellular apoptosis. Our interest in proline, however, is motivated by our recent discoveries indicating that proline protects cells against oxidative stress, a property that has been relatively unexplored in mammalian systems. We have shown that intracellular proline accumulation in mammalian cells correlates with decreased ROS levels and increased protection against oxidative stress. We now show that proline biosynthesis is upregulated under oxidative stress conditions, providing evidence that proline accumulation is a physiological stress response. The mechanism by which proline biosynthesis protects cells against oxidative stress is presently being explored in different cancer cell lines. Support provided by NIH grants P20 RR‐017675‐02 and GM079393.
REDUCED ANEMIA AND IMPROVED IRON AND FOLATE STATUS BEFORE AND AFTER PREGNANCY AMONG FEMALES IN RURAL BANGLADESH IS RELATED TO LENGTH OF AND ADHERENCE TO PERICONCEPTIONAL IRON AND FOLIC ACID SUPPLEMENTATION.
Authors: Khambalia Amina , O'Connor Deborah , Zlotkin Stanley
Published: June 2021

In 2007, Bangladesh's national strategy to reduce anemia included iron and folic acid (IFA) supplementation to adolescent girls and newly married females. We conducted a double‐blinded RCT among married, nulliparous females to determine the impact of daily periconceptional IFA Sprinkles® versus folic acid (FA) Sprinkles® on iron and folate status. Biochemical indices were collected at baseline and after a positive urine pregnancy test (~15 wks gestation), or after 9 mo of supplementation among non‐pregnant females (NPF). Among 88 pregnant females (PF), there was a significant interaction between treatment group and length of supplementation for Hb (p=0.01) and anemia (p=0.02). After 183 study days, IFA improved Hb (127.0 vs 112.5 g/L) and reduced anemia (22.2% vs 66.7%) compared to FA. Group differences for iron status were not significant. Among NPF (n=151), IFA reduced anemia by 81.4% (95% CI: 0.04, 0.90, p=0.04) and ID by 91.8% (95% CI: 0.01, 0.68, p=0.02). Improved ferritin levels were only observed among females in the highest quartile of adherence. Periconceptional FA improved folate status in both groups. Mean ( ±SD) plasma folate increased from 18.0 ( ±7.0) to 33.8 ( ±23.8) nmol/L after 140.3 ( ±65.5) days of supplementation among PF and from 16.6 ( ±6.8) to 31.4 ( ±18.8) nmol/L after 277.4 ( ±5.2) days of supplementation among NPF. Among NPF, adherence was significantly associated with improved plasma folate levels (p<0.0001) and reduced folate deficiency (p=0.002). We conclude that periconceptional IFA supplementation with Sprinkles® significantly improves anemia and iron and folate status before and during pregnancy among females in rural Bangladesh.
The Utility of Zebrafish in Stress Research
Authors: Hart Peter C , Bergner Carisa L , Egan Rupert J , LaPorte Justin L , Smolinsky Amanda N , Amri Hakima , Zukowska Zofia , Glasgow Eric , Kalueff Allan V
Published: June 2021

Zebrafish are becoming widely used in experimental biology and translational modeling of brain disorders, such as epilepsy, addiction and affective disorders. Exploration of novel tank is highly sensitive to various manipulations, and can be used to assess zebrafish stress. Several endpoints can be scored, including latency to initial transition into top half of the tank, number of transitions to the top, erratic movements, and freezing episodes. The use of video tracking techniques introduces additional endpoints, such as velocity and angular velocity. Anxiolytic drugs (e.g., ethanol) tend to reduce anxious behavior (as assessed by shorter latency to initial transition, higher frequency of transitions, and fewer erratic movements or freezing). Chronic treatment with antidepressants (e.g., fluoxetine) also shows significant reduction of anxious behavior. In contrast, alarm pheromone from zebrafish skin provokes a high‐anxiety phenotype, including freezing, erratic movements and markedly reduced exploration. Behaviors observed in these tests can be correlated with physiological (cortisol levels) and genomic (expression of key brain genes) responses. Overall, zebrafish emerge as a promising alternative to other species used in stress research. Supported by SPARC and NARSAD.
Chloride channel ClC‐5 linked to Dent's disease decreases protein level of the intestinal calcium entry channel TRPV6 in Xenopus oocytes
Authors: Na Tao , Zhang Wei , Peng Ji‐Bin
Published: June 2021

TRPV6, a member of the transient receptor potential superfamily of ion channels, mediates transcellular Ca2+ absorption in the intestine. Diet‐dependent hypercalciuria in patients with Dent's disease and in ClC‐5 knock‐down mice promoted us to investigate whether ClC‐5 modulates TRPV6‐mediated Ca2+ transport. When ClC‐5 was expressed in Xenopus laevis oocytes for 2 days, it induced a Gd3+‐sensitive Ca2+ uptake, which was insensitive to L‐type voltage‐gated Ca2+ channel blocker verapamil and nitrendipine. Thus, TRPV6‐mediated Na+ current was used as a measurement of TRPV6 activity instead of radiotracer Ca2+ influx assay. When ClC‐5 was co‐expressed with TRPV6 in oocytes, TRPV6‐induced Na+ current was almost completely suppressed using voltage clamp. Consistent with these results, TRPV6 protein level was greatly diminished by co‐expression with ClC‐5. The decrease in TRPV6 protein level by ClC‐5 was dose‐dependent and correlated with the decrease in TRPV6 Na+ current. To locate the key fragment in ClC‐5 accounting for TRPV6 regulation, amino‐terminal region (amino acids 1‐53), carboxy‐terminal region (amino acids 546‐746) or both of them in ClC‐5 were truncated. Although the three ClC‐5 constructs didn't exhibit outward Cl− current as the wild‐type ClC‐5 did, they retained the ability to inhibit TRPV6‐induced Na+ current by decreasing TRPV6 protein level. Co‐expression of ClC‐5 with TRPV6 in mouse duodenum and in human intestinal cell line Caco‐2 was confirmed by RT‐PCR. Therefore, ClC‐5 may play an important role in regulating TRPV6 protein level, and in turn in modulating intestinal Ca2+ absorption. Further studies are being directed toward the mechanisms by which TRPV6 protein is decreased by ClC‐5.
Cellular methylation regulates hepatocyte expression of selenoprotein P and gluconeogenic enzymes
Authors: Jackson Matthew I , Combs Gerald F
Published: June 2021

S‐adenosylmethionine (SAM) is a methyl donor whose levels are altered in disease, relative to its product, SAH. SAM is also required for metabolism of the nutrient Se. We previously found a positive correlation between Se and Vitamin B12/folic acid but a negative correlation between Se and homocysteine, indicating positive methylation status might facilitate Se metabolism. We also found levels of a selenoprotein, SEPP1, varied by BMI in overweight/obese human subjects. Obesity associates with fatty liver disease accompanied by altered hepatic methylation capacity and decreased levels of plasma indicators of methylation status. To test whether decreased methylation disrupts Se metabolism, we treated human hepatocytes with an inhibitor of transmethylation (adensylhomocysteine hydrolase). After treatment, the SAM/SAH ratio and SEPP1 protein/mRNA were all decreased. The inhibition of SEPP1 expression appears to be transcriptional, not due to decreased mRNA stability or translation. Additionally, glucocorticoid induced upregulation of SEPP1 synthesis was blocked by inhibition of methylation. Showing the generality of our observations, both basal and glucocorticoid‐induced gluconeogenic enzyme expression was also decreased by SEPP1. In summary, we show that hepatocellular SAM‐dependent methylation serves as a regulatory mechanism for both Se homeostasis and gluconeogeneic enzyme expression.
The thioredoxin reductase system is a critical factor in mediating acetaminophen‐induced liver damage
Authors: Carlson Bradley A , Patterson Andrew D , Yoo Min‐Hyuk , Conrad Marcus , Gonzalez Frank J , Hatfield Dolph L
Published: June 2021

Acetaminophen (APAP) overdose is the most common cause of acute liver failure in the US. When taken in excess, APAP is converted to the reactive metabolite NAPQI causing rapid glutathione (GSH) depletion and leading to liver necrosis. These events are initiated by ROS formation in mitochondria which leads to the activation of apoptosis signaling‐regulating kinase 1 (ASK1) and the c‐Jun N‐terminal kinase (JNK) pathway. Since thioredoxin reductase 1 (TR1) is important for attenuating the activation of ASK1 through thioredoxin, the role of TR1 in APAP‐induced liver damage was investigated. Hepatocyte‐specific TR1 knockout (KO) mice were generated and treated with a toxic dose of APAP. Liver toxicity was assessed by measuring the aminotransferase (ALT and AST) activities in serum, and by histological analysis of liver sections. Surprisingly, TR1 KO mice were resistant to APAP toxicity. GSH levels decreased as expected in APAP‐treated control mice, yet were largely unaffected in TR1 KO mice. Unexpectedly, JNK was phosphorylated to a similar extent in TR1 KO mice as APAP‐treated control mice. These results demonstrate an important role for TR1 in modulating APAP‐induced liver damage and provide new insights into APAP‐induced liver toxicity. This research was supported by the Intramural Research Program of the NIH, NCI, CCR.
Distribution of Intravenously Administered Gold Nanoparticles at the Light Microscope Level
Authors: Haines Diana C , Stern Stephen , Hall Jennifer , Patri Anil , McNeil Scott
Published: June 2021

Female mice received an experimental anti‐cancer therapeutic conjugated to a PEGylated gold nanoparticle (GNP) via tail vein either alone, or in combination with ip docetaxel (DTX). Mice were sacrificed at day 10 and 17. Mice receiving the higher doses of GNP had discolored or grey tissues which correlated, microscopically, with a dose‐related “pigment”. At the light microscope level, the greatest concentration was noted in hepatic Kupffer cells, splenic red pulp macrophages and lymph node sinusoidal macrophages. Mild to moderate accumulation was present in the uterine endometrial stoma, renal glomeruli, adrenal cortex and medulla, gastrointestinal lamina propria and submucosa, and bone marrow. Minimal pigment, usually vascular associated, was also noted in other organs. There were no pigment‐associated lesions. The material was interpreted to be aggregated gold nanoparticles and was visible on unstained sections. The recovery group (17‐day) showed decrease in DTX‐associated lesions; however, the GNP‐associated pigment persisted. Increased “severity” of pigment in some organs in the recovery group was due to localized clumping of pigment aggregates. Funded by NCI Contract No. HHSN261200800001E
Prenatal Exposure to Dexamethasone Alters 5HT1A Serotonin Receptor Function and Behavior in Stressed Male Rats
Authors: Shah Darshan Sunil , Vijay Rahul , Page Kathleen Creed
Published: June 2021

Synthetic glucocorticoids (GC) are used to stimulate lung development in fetuses at risk of preterm delivery. Previous studies have shown prenatal exposure to Dexamethasone (Dex) is associated with disturbances serotonergic systems. The current hypothesis is that prenatal exposure to Dex during the third trimester of pregnancy alters 5HT1A receptor function and compromises spatial learning and memory in male rats. Pregnant rat dams were injected daily with 150ug/ml/kg of Dex (sc) from gestation day 14 through 19. Control dams were treated with saline. Adult male offspring were exposed behavior testing an immediately sacrificed. Another group was tested and allowed to return to baseline (basal). Hippocampi were analyzed using a radioligand receptor binding assay and GTPÎ3S incorporation (3H‐MPPF antagonist and 8‐OH‐DPAT agonist respectively). Binding maximum (Bmax) increased for the Dex treated animals under basal and stressed conditions. However, no significant change was seen in the binding affinity (Kd) for either group. Receptor effectiveness was reduced in the Dex animals, however, under basal conditions these animals showed no change in behavior. Dex stressed animals showed a significant increase in anxiety following EPM testing. Elevated GC levels during prenatal development are associated with serotonergic and behavioral dysfunction in adulthood.
Combinatory treatment with mood stabilizers lithium and valproate produces multiple beneficial effects in transgenic mouse models of Huntington's disease
Authors: Chiu Chi‐Tso , Chuang De‐Maw
Published: June 2021

Emerging evidence supports that mood stabilizers lithium and valproate (VPA), via inhibition of glycogen synthase kinase 3 (GSK‐3) and histone deacetylases (HDACs), respectively, have broad neuroprotective and neurotrophic properties. We have shown that co‐treatment with these two drugs produces synergistic neuroprotective effects in cultured neurons against glutamate excitotoxicity, one of the purported mechanisms underlying the pathogenesis of Huntington's disease (HD). This inherited neurodegenerative disease is characterized by movement impairment, cognitive and psychiatric disturbances, as well as premature death. Here we demonstrated that treatment of two mouse models of HD, N171‐82Q and YAC128, with a diet containing both lithium (3 g/kg) and VPA (25 g/kg) exhibited greater effects on suppressing motor deficits and psychiatric abnormalities than monotherapy with each drug alone. This combinatory treatment caused consistent inhibition of GSK‐3β and HDACs, and sustained elevation in brain‐derived neurotrophic factor and heat shock protein 70 in their brains. Most importantly, this co‐treatment markedly prolonged lifespan in N171‐82Q mice. Given that there is no proven treatment for this disease presently, our results suggest a therapeutic potential of combined use of lithium and VPA in HD patients. This work was supported by the Intramural Research Program of the NIMH, NIH.
p23 co‐chaperone is essential for the optimal function of the aryl hydrocarbon receptor in Hepa1c1c7 cells by modulating the AhR content
Authors: Nguyen Phuong Minh , Wang Depeng , Li Yanjie , Chan William K
Published: June 2021

The aryl hydrocarbon receptor (AhR) is a ligand‐activated, basic helix‐loop‐helix‐PAS transcription factor which involves in xenobiotic metabolism and cell growth. Without ligand, the p23‐containing AhR complex is present in the cytoplasm. Although the roles of p23 for the AhR function were suggested, its necessity has been questioned. Our objective is to investigate whether p23 is necessary for the AhR function in Hepa1c1c7 cells. Using shRNA strategy, we generated the p23‐knockdown Hepa1c1c7 stable (p23KD) cells which contained 30% of the wild type p23 content. When we compared p23KD to the wild type (or negative control knockdown stable) cells, we found that in p23KD cells, the total AhR content was reduced to 60% and the nuclear AhR content after 3MC treatment was also reduced to 50%. The 3MC induction of the CYP1A1 content and the DRE‐driven luciferase activity was significantly less pronounced in p23KD cells. The 3MC‐dependent AhR/Arnt heterodimer in the p23KD nuclear extract bound less DRE than the control nuclear extracts in gel shift assays. However, when we normalized the AhR content, all nuclear extracts bound DRE similarly, even though the p23 content was significantly lowered in the p23KD nuclear extract. We concluded that p23 modulates the cellular AhR content which in turn affects the optimal AhR function upon ligand activation. This work is supported by NIH (WKC, R01ES014050).
Extracellular signal‐regulated protein kinases mediate protein kinase C‐ and reactive oxygen species‐dependent stimulation of intracellular cAMP in human eosinophils
Authors: Ezeamuzie Charles I , Philips Elizabeth
Published: June 2021

The role of extracellular signal‐regulated protein kinases 1/2 (ERK1/2) in the signaling pathway of the novel protein kinase C (PKC)‐ and reactive oxygen species (ROS)‐dependent stimulation of intracellular adenosine 3′,5′‐cyclic monophosphate (cAMP) in human eosinophils was studied. Highly purified human eosinophils were stimulated with the PKC activator, phorbol myristate acetate (PMA) and the cAMP response was determined by ELISA. The role of ERK1/2 was studied using specific inhibitors and Western blot analysis. PMA‐stimulated increase in cAMP was confirmed to be dependent on PKC and ROS by using specific inhibitors: Ro 31‐8220 for PKC and diphenyleneiodonium (DPI) for the ROS‐generating NADPH oxidase. Pre‐treating cells with the ERK1/2 inhibitor PD98059, but not p38‐MAPK inhibitor SB203580, nor PI3 kinase inhibitor, wortmannin, abolished the response. PMA treatment induced the phosphorylation of ERK1/2 with a time‐course consistent with a role in the cAMP response. This phosphorylation was abolished by the ERK1/2 inhibitor PD98059 and the PKC inhibitor Ro 31‐8220, but not the NADPH oxidase inhibitor DPI. These results reveal a role for ERK1/2 in the signaling mechanism of PKC‐ and ROS‐dependent stimulation of cAMP production in human eosinophils, and show that ERK1/2 phosphorylation is upstream of ROS production in the signaling pathway. Supported by grant #MR 01/04 from RA, Kuwait University.
The alpha 2A adrenergic receptor evokes activation of p70S6 kinase through G protein and transactivation of EGFR
Authors: Chen Huaping , Lu Roujian , Wang Qin
Published: June 2021

The function of α2A adrenergic receptor (α2AAR) has been implicated in a wide range of physiological activities. The molecular mechanisms behind these activities are topics under intensive investigation. At the cellular level, stimulation of the α2AAR leads to propagation of a number of signaling cascades, such as MAPK, Akt and p70S6 kinase (p70S6K) pathways. In the current study, we characterized α2AAR‐mediated p70S6K activation. Stimulation of the α2AAR evokes activation of p70S6K in both embryonic fibroblasts (MEF) and neurons in a dose‐dependent manner. To delineate the molecular pathway of the α2AAR‐mediated p70S6K activation, pharmacological inhibitors for potential signaling molecules involved were applied on cells. Pertussis toxin treatment of cells blocked α2AAR‐mediated p70S6K activation, indicating the involvement of G proteins in this process. When cells were treated with an EGFR inhibitor or a PI3K inhibitor, α2AAR‐mediated p70S6K activation was also blocked. Additionally, rapamycin treatment diminished α2AAR‐mediated p70S6K activation. Taken together, these data suggests that α2AAR‐mediated activation of p70S6K is a G protein‐dependent process and achieved through transactivation of EGFR, which subsequently activates PI3K and mTOR to stimulate p70S6K. This research was supported by a NIH grant MH081917 (QW) and an AHA SDG grant 0630103N (QW).
Two Distinct Sites on Gβγ are Required for Binding to the N‐Terminus Versus the Activation Site on Adenylyl Cyclase
Authors: Brand Cameron S. , Sadana Rachna , Malik Sundeep , Smrcka Alan V. , Dessauer Carmen W.
Published: June 2021

The heterotrimeric G protein α and βγ subunits are important regulators of adenylyl cyclase (AC), which controls synthesis of the second messenger cyclic AMP. In the inactive state, critical effector sites on Gα and Gβγ are not available. When activated, the subunits either dissociate or rearrange to expose these sites to effectors. However, we have previously shown that AC5 binds to inactive Gs heterotrimer at its N‐terminus (NT), while Gβγ conditional stimulation of AC5 is independent of this binding site. Both FRET and BiFC in live cells support interactions between Gβγ and AC5 that are at least partially dependent on the NT. We hypothesize that Gβγ utilizes two different surfaces to interact with the NT versus the activation sites on AC5 and AC6. In support of this hypothesis, mutations of the Gβγ effector surface were tested for Gβγ conditional stimulation of AC5 and for binding to AC5‐NT. Several Gβγ mutants, including W99A, are unable to support Gβγ activation, but have no effect on Gβγ binding to AC5‐NT. We have also utilized the SIGK peptide as a competition inhibitor of Gβγ “hotspot” interactions. SIGK is a strong inhibitor of Gβγ stimulation of AC6, but has little effect on Gβγ binding to AC6‐NT. Furthermore, Gβγ binds to the NT of many AC isoforms, suggesting that AC anchoring of heterotrimer may be a common theme for ACs to facilitate regulation by both α and βγ subunits.

Supported by NIH grant GM60419 and AHA.
Equilibrium between adenylyl cyclase and phosphodiesterase patterns adrenergic agonist dose‐dependent spatiotemporal cAMP/PKA activities
Authors: Xiang Yang K
Published: June 2021

Beta‐adrenergic receptor induces cAMP/Protein kinase A (PKA) activation to regulate cardiac contraction. Using real‐time detection of cAMP and PKA activities, we show two distinct phases in isoproterenol dose‐dependent responses: a transient and dose‐dependent increase in cAMP and PKA activities at concentrations from 10–12 to 10‐8 M; and a saturated initial increases at concentrations from 10‐8 to 10‐5 M followed by a rapid decrease to different levels that were later sustained in a dose‐dependent manner. The dose‐dependent temporal responses are patterned by equilibrium between receptor‐activated adenylyl cyclase (AC) and phosphodiesterase (PDE). At lower concentrations, cAMP is produced in an agonist dose‐dependent manner with AC as a rate‐limiting factor. However, the cAMP activities are confined within local domains for phosphorylation of local substrates. At higher concentrations, isoproterenol promotes a dose‐dependent selective dissociation of PDE4D but not ACVI from the receptor complex, which shifts the equilibrium between AC and PDE. This shifted balance leads to sustained cAMP accumulation and diffusion for PKA phosphorylation of phospholamban and troponin I, and for myocyte contraction. Our data reveal a new paradigm for adrenergic agonist dose‐dependent cAMP/PKA activities for substrate‐specific phosphorylation dictated by dual regulation of AC and PDE in cardiomyocytes.
Alternative CYP3A4 mRNA Isoforms Are Related to Hepatocyte Differentiation, Liver Development, and Response to Drugs
Authors: Li Dan , Gaedigk Roger , Hart Steven N. , Tregear Hans , Leeder J Steven , Zhong Xiao‐bo
Published: June 2021

Cytochrome P450 3A4 (CYP3A4) metabolizes over 50% of prescribed drugs. CYP3A4 activity changes during liver development and the administration of drugs. Alternative mRNA isoforms occur in over 90% of human genes and are frequently observed in cells responding to developmental and environmental signals. The purpose of this study was to identify CYP3A4 mRNA isoforms in liver and characterize their differential expression during liver development, hepatocyte differentiation, and in response to drugs. cDNA cloning and RNA sequencing (RNA‐Seq) were used to identify CYP3A4 mRNA isoforms. Three isoforms were found in human HepaRG cells and liver tissues, one represented a canonical full length mRNA, one had a shorter 3′‐UTR, and one contained a partial intron 6 retention. The alternative mRNA isoforms were validated by either rapid amplification of cDNA 3′‐end (3′RACE) PCR or end‐point PCR. Quantification of the isoforms by real‐time PCR revealed that expression levels of the shorter 3′‐UTR isoform increased significantly more than the isoform with the canonical 3′‐UTR during liver development, hepatocyte differentiation, and in response to rifampicin, phenobarbital, and dexamethasone.

Conclusion

This study identified two novel CYP3A4 mRNA isoforms. The isoform with the shorter 3′‐UTR might be involved in the regulation of CYP3A4 gene expression during liver development and the administration of some drugs.

This study was supported by 1R01GM087376‐01A1, 1R01ES019487‐01A1, and 5P20‐RR021940 (to X.B.Z), 1R01HD058556‐01A1 (J.S.L), and Lila Self Graduate Fellowship from the University of Kansas (to S.N.H.)
A novel small nucleolar RNA‐derived microRNA‐1291 targets multidrug resistance‐associated protein 1 (MRP1/ABCC1)
Authors: Pan Yuzhuo , Yu Aiming
Published: June 2021

Multidrug resistance‐associated protein (MRP1/ABCC1) is expressed ubiquitously in human tissues including the lung, testis, kidney, pancreas and placenta, and contributes to a variety of physiological functions including the extrusion of endobiotic and xenobiotic toxins. ABCC1 may also confer multidrug resistance (MDR) in cancer cells. As the posttranscriptional regulation of ABCC1 remains elusive, this study aimed to investigate the role of microRNAs in regulation of ABCC1 and the impact on cellular defense against xenobiotic drugs. Bioinformatics analysis showed that miRNA‐1291 might be derived from the small nucleolar RNA SNORA34, and target ABCC1 3′‐untranslated region (3′UTR). Using a novel splinted ligation RNA detection method, SNORA34 was found to be processed to miR‐1291 in PANC‐1 human pancreases cancer cells. Luciferase assay supported the action of miR‐1291 on ABCC1 3′UTR. ABCG2 mRNA and protein expression were dramatically down‐regulated in miR‐1291 stably‐transfected PANC‐1 cells with higher miR‐1291 expression. Intracellular drug accumulation and the sensitivity of cells to doxorubicin or mitoxantrone was significantly increased in PANC‐1 cells with higher miR‐1291 expression. These findings demonstrate the involvement of miR‐1291 in posttranscriptional regulation of ABCC1. Further studies on miRNA signaling may help develop novel strategy to overcome multidrug resistance.
MRP1(ABCC1) function is regulated via RhoA
Authors: Paumi Christian M.
Published: June 2021

Multidrug resistance‐associated protein 1 (MRP1/ABCC1) has been shown to play an important role in the cellular efflux of a broad range of endogenous and exogenous toxins. MRP1 expression is associated with multidrug resistance (MDR) in a variety of cancers. Although much is known about MRP1 function and expression, the post‐translational regulation of MRP1 is poorly characterized. Based on work conducted by Paumi et. al. in 2007 that showed that the function of the yeast MRP1 homologue, Ycf1p, is regulated by the yeast homologue of RhoA, Rho1p, via the GTP‐exchange factor, Tus1p; We have examined the role of RhoA and Tus1p homologue, ARHGEF11 (PDZ‐GEF), in the regulation of MRP1 function. Here we show that MRP1 and RhoA interact by Co‐immunoprecipitation and that inhibition of RhoA activity alters MRP1 cellular function. RhoA overexpression in cancer is strongly associated with a poor chemotherapeutic response in patients. Our preliminary data suggests that one mechanism by which RhoA overexpression results in poor chemotherapeutic response is via increasing MRP1 function. CMP is funded by NIH P20RR020171.
Validation of the histological localization and subcellular polarity of the Oatp4c1 transporter in the rat kidney
Authors: Kuo Kuei‐Ling , McNamara Patrick , Leggas Markos
Published: June 2021

Recently, the organic anion transporting polypeptide 4c1 (4c1) was identified as a novel uptake transporter predominantly expressed on the basolateral membrane of rat kidney proximal tubules. However, in‐vitro studies with MDCKII cells showed that upon transfection 4c1 polarizes to the apical membrane. We used multiple antibodies (N=4) to validate the subcellular localization of 4c1 in rat tissues and in MDCKII and LLCPK1 cells expressing rat 4c1. Antibodies amenable to immunohistochemistry were generated using N‐ and C‐terminal peptide sequences. Apical and basolateral protein markers were used for colocalization studies. Immunofluorescence studies showed that 4c1 polarizes to the apical surface in the in‐vitro cell models. In rat kidney, colocalization of 4c1 with aquaporin 1 but not calbindin in the subcortical region suggest that apical 4c1 expression is localized in proximal straight tubules. 4c1 also colocalizes with P‐glycoprotein and breast cancer resistance protein, but not with multidrug resistance protein 4 in rat kidney. These results were verified by western blot analysis of brush‐border membrane vesicles isolated from the kidney cortex and conclusively demonstrate the apical expression of 4c1 in the rat kidney. To investigate the physiological role of 4c1, future studies will focus on identifying substrates and the driving force for 4c1 mediated transport.
Pregnancy represses induction of hepatobiliary efflux transporters in diabetic mice
Authors: Aleksunes Lauren M , Xu Jialin , Slitt Angela L
Published: June 2021

Efflux transporters are plasma membrane proteins responsible for excreting chemicals and toxicants from the cell. In the liver, efflux transporters including the multidrug resistance‐associated proteins (Mrps) are markedly induced in mice with diabetes. In contrast, other conditions such as pregnancy decrease hepatic Mrp levels. The purpose of the present study was to quantify the mRNA expression of hepatobiliary efflux transporters in pregnant, diabetic mice. Female mice were treated with vehicle or streptozotocin (STZ). Once STZ‐treated mice exhibited elevated glucose levels, they were mated with male mice. mRNA expression was quantified in livers of nonpregnant and pregnant female mice with normal or elevated glucose levels. Livers from nonpregnant, diabetic mice exhibited up‐regulation of Mrp1, 2, 4, 5 and breast cancer resistance protein (Bcrp) mRNA that was attenuated in pregnant, diabetic mice. Mrp3 mRNA was similarly induced in livers of nonpregnant and pregnant diabetic mice. Mrp6 mRNA was decreased by pregnancy, regardless of glucose level. Collectively, these data suggest that pregnancy represses expression of efflux transporters in livers of diabetic mice. Additional studies are necessary to elucidate the transcriptional mechanisms underlying the opposing regulation of hepatobiliary transporters in response to diabetes and pregnancy. Supported by NIH ES016042, ES005022, DK080774.
The ABC transporter Mrp4 (Abcc4) plays a crucial role in normal testosterone production
Authors: Morgan Jessica A , Adachi Masashi , Boyd Kelli , Neale Geoff , Wang Yao , Cheepala Satish , Scheffer George L , Schuetz John D
Published: June 2021

The primary site of testosterone biosynthesis in males occurs in the Leydig cells of the testes. Accordingly, circulating levels of testosterone exhibit a developmental pattern that correlate to the amount of testosterone produced. We show that, in the testes of both mouse and man, Mrp4 is almost exclusively expressed in the Leydig cells. Prior to puberty, despite normal Leydig cell numbers, Mrp4−/− mice have dramatically reduced serum testosterone concentrations when compared to their Mrp4+/+ counterparts. Consistent with this finding, Mrp4−/− mice exhibited both impaired spermatogenesis and prostate development. Nonetheless, adult Mrp4−/− animals are fertile with no obvious reproductive defects. After puberty, circulating testosterone concentrations of the Mrp4−/− mouse rise to levels comparable to those of the Mrp4+/+ mouse. In the Mrp4−/− mouse we have identified a developmental mechanism that accounts for the low levels of testosterone prior to puberty and the unexpected subsequent rise post‐puberty. These findings indicate that Mrp4 function has a crucial role in the appropriate developmental production of systemic testosterone.

Funding provided by the NIH and ALSAC.
Mechanical responses to COREXIT® EC9500A in rat trachea in vitro
Authors: Shimko Michael J. , Zaccone Eric J. , Thompson Janet A. , Kashon Michael L. , Piedimonte Giovanni , Fedan Jeffrey S.
Published: June 2021

Oil dispersant COREXIT® EC9500A (CE) was used to reduce the environmental impact of the Deepwater Horizon oil spill in 2010. Little is known of the pulmonary effects of CE on airway smooth muscle and nerves. We investigated in vitro the effects of CE on rat tracheal preparations. In tracheal strips, CE (0.001 %v/v) elicited relaxation. Contraction occurred at higher concentrations (0.003–0.1 %v/v; EC50 0.0031 %v/v). CE (>0.1 %v/v) elicited relaxation of methacholine (MCh; 3×10−5 M)‐contracted strips (EC50 0.39 %v/v). CE (0.003 %v/v) had no effect on MCh‐induced tone but inhibited neurogenic contractile responses of strips to electrical field stimulation (10 Hz). In the isolated, perfused trachea preparation (IPT), in which agents may be applied separately to the mucosal or serosal surface, CE (0.3–10 %v/v) administered to the mucosal bath resulted in contraction. In MCh‐contracted perfused tracheas, mucosal CE (0.1–10 %v/v) elicited contraction followed by relaxation. Thus, CE was less potent in the IPT compared to strips. CE produced a progressive precipitate at >0.1 %v/v. Our results indicate that CE contracts airway smooth muscle and inhibits excitatory neurotransmission. Relaxation at high CE concentrations cannot be distinguished from precipitate formation or osmotic effects.
Biphasic modulation of airway mesenchymal cell proliferation by hogbarn dust extract
Authors: Dodmane Puttappa R. , Schulte Nancy A. , Wohlgemuth Ian P. , Subramanya Sajana , Romberger Debra J. , Toews Myron L.
Published: June 2021

Workers in concentrated animal feeding operations (CAFOs) often develop chronic inflammatory lung disease. Previous studies have characterized effects of an aqueous hogbarn dust extract (HDE) on inflammation responses of airway epithelial cells. We hypothesized that HDE would also exhibit disease‐relevant effects on airway mesenchymal cells. In both primary isolates of human airway smooth muscle (HASM) cells and the HFL1 human fetal lung fibroblast cell line, HDE at low concentrations stimulated proliferation assessed by [3H]thymidine incorporation, with maximal stimulation at 1%. HDE stimulation was blocked by the EGF receptor tyrosine kinase inhibitor AG1478, implicating EGF receptor involvement, as shown previously for some HDE effects on epithelial cells. At higher concentrations, HDE stimulation decreased, with no stimulation and often significant inhibition induced by HDE at 5% or higher. The stimulatory and inhibitory activities were found in different gel filtration fractions of HDE, though the specific mediators remain unknown. HDE modulation of airway mesenchymal cell proliferation may contribute to airway remodeling in hogbarn workers. Supported by NIH Grant OH008539 to DJR.
Ajoene, a stable garlic byproduct, inhibits high‐fat diet‐induced hepatic steatosis and oxidative injury via LKB1‐mediated AMPK activation
Authors: Han Chang Yeob , Kim Young Woo , Kim Sang Geon
Published: June 2021

Hepatic steatosis is common and may progress to steatohepatitis and cirrhosis. Liver X receptor‐alpha (LXRalpha)‐sterol regulatory element binding protein‐1c (SREBP‐1c) pathway is important for hepatic steatosis. This study investigated the effect of ajoene, a stable garlic byproduct, to inhibit high fat diet (HFD)‐induced hepatic steatosis, and the molecular mechanism. Ajoene treatment attenuated fat accumulation and induction of lipogenic genes in the liver of HFD‐fed mice. Blood biochemical analyses and histopathologic examinations showed that ajoene prevented liver injury with the inhibition of oxidative stress, as evidenced by thiobarbituric acid reactive substances formation and nitrotyrosinylation. Treatment with ajoene inhibited LXRalpha agonist (T0901317)‐mediated SREBP‐1c activation, and transactivation of the lipogenic target genes in hepatocytes. Ajoene was found to activate AMP‐activated protein kinase (AMPK) through LKB1, responsible for the inhibition of p70 ribosomal S6 kinase‐1 (S6K1). The ability of ajoene to repress T0901317‐induced SREBP‐1c expression was antagonized by AMPK inhibition or S6K1 activation, proving the role of the kinases in the anti‐steatotic effect. In conclusion, ajoene has an effect to activate AMPK via LKB1 and inhibit S6K1 activity, contributing to the prevention of SREBP‐1c–mediated hepatic lipogenesis via the inhibition of LXRalpha activity.
Acetaminophen (APAP)‐Induced Hepatotoxicity and Protein Nitration in neuronal Nitric Oxide Synthase (nNOS) Knockout Mice
Authors: Agarwal Rakhee , Hennings Leah , Rafferty Tonya M , Letzig Lynda G , McCullough Sandra , James Laura P , MacMillan‐Crow Lee A , Hinson Jack A
Published: June 2021

In overdose APAP is hepatotoxic. Toxicity occurs by metabolism to N‐acetyl‐p‐benzoquinone imine which covalently binds to proteins followed by protein nitration. Nitration is by peroxynitrite, formed from superoxide and nitric oxide (NO). In freshly isolated hepatocytes we reported that the NO is from nNOS which is believed to be in mitochondria. MnSOD is nitrated by 1 hr in APAP toxicity in mice resulting in MnSOD inactivation. This leads to increased peroxynitrite. To understand the role of nNOS in APAP toxicity and MnSOD nitration, nNOS knockout mice (KO) and wildtype mice (WT) were administered APAP (300 mg/kg). Serum ALT significantly increased at 6 and 8 h in WT and at 8 h in KO. ALT levels in WT were significantly higher than in KO at 6 h but not 8 h. There were no significant differences in hepatic GSH depletion, APAP protein covalent binding, or histopathological evaluation of hepatic necrosis. Hepatic MnSOD activity was significantly lower at 1–4 h in WT and 8 h in KO. MnSOD activity in WT was significantly higher than in KO at 8 h. Western blot analysis for MnSOD nitration indicated a significant increase at 1–8 h in WT and at 6 and 8 h in KO. There was dramatically more nitration of MnSOD in WT than in KO. Nitration in WT was significantly different from KO at 1–6 h. These data indicate that nNOS is important in protein nitration but the role of nNOS and nitration in in vivo toxicity is unclear. Supported by 1 R01 DK079008.