bluuebunny/crossref_metadata_2025 huggingface dataset:
The possible role of serum from septic mice in dystrophin proteolysis in cultured newborn mice cardiomyocytes
Authors: Malvestio Lygia Maria Mouri , Prado Cibele Maria , Celes Mara Rúbia Nunes , Campos Erica Carolina , Ferezin Patricia Mara , Rossi Marcos Antonio
Published: June 2021

Previous results from our laboratory demonstrated reduced dystrophin expression in hearts of septic mice. Dystrophin is responsible to protect the sarcolemma from mechanical stress during cycles of muscle contraction and relaxation. The loss of dystrophin turns the sarcolemma fragile leading to membrane disruption. This study evaluated the dystrophin expression in cultured newborn cardiomyocytes stimulated with serum of mice subjected to experimental sepsis induced by cecal ligation and puncture (CLP) and its relation with calpain and NF‐κB levels. Cardiomyocytes were incubated 5 days after their first spontaneous beating with sera from sham‐operated and septic mice for 24 hs. Serum from septic mice was obtained from male C57Bl/6 6 hs after CLP. Imunofluorescent (IF) staining and western blotting (WB) were performed for evaluation of dystrophin, calpain, and NF‐κB concentration. The cardiomyocytes stimulated with serum from septic mice presented decreased levels of dystrophin and increased levels of calpain and NF‐κB in comparison with cells incubated with sham serum. These results clearly demonstrate that septic serum causes dystrophin proteolysis in cultured newborn cardiomyocytes. This decrease possibly is related to proteolytic action of calpain that could be activate to NF‐κB overexpression contributing to dystrophin damage (supported by FAPESP).
Autophagy in pituitary tumors
Authors: Rotondo Fabio , Horvath Eva , Kovacs Kalman , Syro Luis V , Yamada Shozo
Published: June 2021

Autophagy or self‐cannibalism, is degradation of cytoplasmic constituents providing an alternative energy source and allowing cell survival during hypoxia, nutrient deprivation and metabolic stress. Formation of membranes engulfing part of the cytoplasm, fusion with lysosomes and degradation can be conclusively seen by electron microscopy. To our knowledge, no information is available to date on the role of autophagy in pituitary tumors. We investigated more than 5000 surgically removed human pituitary tumors by electron microscopy. Our results indicate that autophagy is rare and can be demonstrated in every adenohypophysial tumor type. Autophagy is most frequently noted in long acting somatostatin analog treated GH producing and in dopamine agonist treated PRL producing pituitary adenomas. These two treatment modalities which cause tumor shrinkage, clinical and endocrinological improvement, activate autophagy. Many cytotoxic and anticancer drugs stimulate autophagy and counteract the effect of therapy in many tumor types resulting in tumor cell survival. New drugs are needed which inhibit autophagy. It remains to be seen whether anti‐autophagic drugs would suppress the formation of additional nutrients, eliminate the extra energy source and increase the efficiency of drugs used in the treatment of pituitary tumors. This study was supported by the Jarislowsky and Lloyd Carr‐Harris Foundations.
IFN‐gamma specifically targets melanoma stem cells and inhibits in vitro spherogenic growth
Authors: Lee Chung‐Wei , Huang John , Chuang Ya‐Ting , Murphy George F
Published: June 2021

The host immune system can suppress development and progression of cancer. Recent success in cancer immunotherapy validates anti‐cancer strategies by activating innate and adaptive immune responses or suppressing regulatory and inhibitory mechanisms. Among immune modulators, interferon gamma (IFNg) is of special interest because it affects both host immunity and cancer cell kinetics. IFNg has antiproliferative and proapoptotic effects in cancer cells. However, recent data has challenged this paradigm in melanoma cells, suggesting that macrophage‐derived IFNg actually may promote growth of mouse melanomas in vivo. To examine the effect of IFNg in human melanoma cells, we observed that IFNg suppressed growth of susceptible melanoma cell lines A2058 and A375 in a STAT1‐dependent manner, and decreased anchorage‐independent spherogenic growth, an in vitro correlate of cancer stem cell tumorigenicity in animal xenotransplantation models. Interestingly, IFNg did not alter melanoma cell cycle progression or induce apoptosis at the minimal dose that is sufficient to suppress spherogenic growth. IFNg was shown to downregulate mRNA expression of the melanoma stem cell‐associated transcription factor OCT4, and upregulate the tumor suppressor gene XAF1. In conclusion, IFNg specifically suppresses in vitro spherogenic growth that characterizes melanoma stem cells. (NIH NCI R01 CA138231 and T32 HL007627)
Are The Intrarenal Hormones The Culprit in Renal Cell Carcinoma?
Authors: Jackson Cynthia Ann , Canionero Dee Dee
Published: June 2021

Renal Cell Carcinoma (RCC) is a form of kidney cancer that originates in the lining of the renal tubules. Over a decade ago, long‐term use of angiotensin converting enzyme inhibitor to treat hypertension was associated with a decreased incidence of various types of cancer in hypertensive patients. Subsequently, recent studies have demonstrated that angiotensin II, via activation of the angiotensin II type 1 receptor (AT1R), stimulates cellular proliferation and migration and thus may play a role in the pathophysiology of cancer. The present study was designed to evaluate the role of the renin angiotensin system (RAS) in RCC. BrdU proliferation assays were utilized to examine the effects of the various intrarenal components of the RAS in human renal carcinoma cells, ACHN. In addition, immunocytochemistry studies were used to detect the specific signal transduction pathway involved in tumorigenesis. ACHN cells treated with captopril decreased the proliferation rate by 90%. Blockade of angiotensin II receptor subtype 1 (AT1R, losartan‐treated) and subtype 2 (AT2R, PD123319‐treated) significantly decreased proliferation rate by 15% and 85%, respectively. cAMP activity was reduced in the PD123319‐treated cells and augmented in the losartan‐treated cells. The results of these studies demonstrate that angiotensin II via the AT2R rather than the AT1R regulates cellular proliferation in ACHN renal carcinoma cells during tumorigenesis, possibly through the cAMP pathway. These studies will enhance our ability to develop new therapy and/or treatments specifically directed at various biomarkers to reduce the morbidity associated with renal cell carcinoma.
Patterned laminin expression by spheroid‐forming melanoma cells
Authors: Larson Allison R , Lezcano Cecilia , Lee Chung‐Wei , Huang John , Zhan Qian , Murphy George F
Published: June 2021

Vasculogenic mimicry, a collection of laminin networks within tumors is associated with a poor prognosis in melanoma and other cancers. Cultured melanoma cell lines that form spheroids are associated with increased invasiveness in vitro. Accordingly, we hypothesized that spheroid‐forming melanoma cells may also show patterned laminin networks. By immunohistochemistry, the A2058 melanoma cell line showed prominent patterned laminin networks within spheroids that closely mimicked networks that developed in primary melanoma xenografts to NSG mice. Real time PCR analysis of laminin subunits confirmed that anchorage‐independent spheroid‐forming cells expressed a different laminin profile from non‐spheroid‐forming adherent cells. These findings suggest that melanoma spheroids represent a novel approach to studies concerning the nature and significance of vasculogenic mimicry and associated laminin network formation.

Funding provided by NIH Grant SPORE Core: NIH, NCI SPORE in Skin Cancer 5P50CA093683–08, BWH Biomedical Research Institute Microseed Grant, A.L. is supported by BWH Dept of Dermatology NIH Training Grant T32AR007098–38.
The pMAPK/pAMPK ratio modulates the effect of adiponectin on breast cancer cell growth
Authors: Mauro Loredana , Pellegrino Michele , Ricchio Emilia , De Amicis Francesca , Catalano Stefania , Bonofiglio Daniela , Rizza Pietro , Panno Maria Luisa , Ando' Sebastiano
Published: June 2021

Adiponectin, an adipose tissue‐derived hormone, may influence breast cancer progression. Here, we demonstrated in vitro and in vivo that low concentration of adiponectin at (1–5 μg/ml) induced a dicotomic pattern of effects on breast cancer growth, namely a stimulatory action in MCF‐7 cells, mediated by MAPK activation, and an inhibitory effect in MDA‐MB‐231 cells, through the activation of AMPK signaling. The latter signaling appeared markedly potentiated at higher concentration (20–30 μg/ml) in both cell types. In other words the modulatory effect of adiponectin may be driven by pMAPK/pAMPK ratio, which was enhanced at low concentrations in MCF‐7 cells, while it dropped markedly at higher doses in both cell types, supporting the inhibitory effect on cell growth. In addition, low doses of adiponectin induced a marked IGF‐IR phosphorylation. The latter event occurred at the same concentrations generally found in obese women. So, it is reasonable to postulate that in ERalpha‐positive cell line the well‐known IGF‐IR/ERalpha cross talk may activate MAPK, sustaining cell proliferation. Thus, on the basis of the present findings enhancing the circulating levels of adiponectin may represent an important target for novel therapeutic strategies in the treatment of this obesity‐associated malignancy.

The research has been supported by AIRC MFAG 6180.
The Inhibitory Effect of Cibenzolineon Na+/Ca2+ Exchange Current in Cardiac Myocytes
Authors: Watanabe Yasuhide , Kimura Junko
Published: June 2021

Cibenzoline, a class I‐a antiarrhythmic drug, was investigated for the effect on Na+/Ca2+ exchange current (INCX) using the whole‐cell voltage clamp with isolated cardiac ventricular myocytes of guinea pig. INCX was recorded by ramp pulses from the holding potential of −60 mV with the external solution containing 140 mM Na+ and 1 mM Ca2+, and the pipette solution containing 20 mM Na+, 20 mM BAPTA and 10 mM Ca2+ (182 nM free Ca2+). Cibenzoline caused a concentration‐dependent inhibition of INCX. The IC50 values of cibenzoline for the outward and inward components of INCX were 77 microM and 84 microM, respectively, with Hill coefficients of 1. Intracellular application of trypsin via the pipette solution did not change the inhibitory effect of cibenzoline. The inhibitory effect of cibenzoline on INCX at pH 6.5 was smaller than those at pH 7.4 and pH 8.2. Delayed after‐depolarization (DADs) induced by electrical stimulation with ouabain disappeared in the presence of 100 microM cibenzoline. We conclude that cibenzoline inhibits INCX in a supra‐therapeutic range of concentrations possibly from the external or intra‐membrane site of the exchanger molecule.
Angiotensin‐(1–7) stimulates mobilization of bone marrow stem/progenitor cells in diabetes
Authors: Jarajapu Yagna , Vasam Goutham , Pawar Rahul , Bartelmez Stephen
Published: June 2021

Diabetes increases the risk for cardiovascular disease due to impaired function and regeneration of endothelium. Mobilization of bone marrow stem/progenitor cells (BMPCs) that regenerate vascular endothelium, is impaired in diabetes. Angiotensin (Ang)‐(1–7) was shown to increase the vasoreparative potential of diabetic BMPCs. This study evaluates the effect of Ang‐(1–7) on the impaired mobilization of BMPCs in diabetes. Control (Ctrl) or streptozotocin‐induced diabetic (DB) mice were treated with saline or Ang‐(1–7) (100 μg/Kg, IP) for 4 weeks. Circulating BMPCs, Lin−Sca‐1+ cells (C‐LS), were enumerated at 4 weeks. Hind‐limb ischemia (HLI) was induced in Ctrl or DB mice following 4‐week treatment with Ang‐(1–7) and the treatment and C‐LS were enumerated at day 1, 3, 7 and 12. C‐LS were lower in 6‐month DB mice compared to the age‐matched controls (P<0.05) and were partly increased by Ang‐(1–7) treatment. HLI increased C‐LS by 8‐fold in Ctrl and by 3‐fold in DB (P<0.02). Ang‐(1–7) treatment increased C‐LS in DB to 7‐fold in DB. DB was associated with decreased Lin− cells in BM compared to age‐matched Ctrl (P<0.05) that was partly recovered by Ang‐(1–7). Decreased proliferation of BM‐Lin− cells in DB‐mice was enhanced by Ang‐(1–7). These results suggest that Ang‐(1–7) is a promising molecule for accelerating vascular repair by stimulating BMPC mobilization in diabetes.
The effect of nitric oxide on blockade of the norepinephrine transporter, uptake‐1, by atomoxetine
Authors: Simaan Joseph A.
Published: June 2021

We previously showed that fresh synthesis of nitric oxide (NO) mediates transport of substrates across uptake‐1, norepinephrine (NE), tyramine (T), mephentermine, ephedrine and imipramine, and blockade by cocaine. Synthesis of NO was blocked by Nw‐nitro‐L‐arginine (L‐NNA) and the rise in arterial pressure it induces was restored to starting level by an infusion of nitroglycerin (NG), a NO donor. In this study we explored the role of NO synthesis in the blockade of uptake‐1 by atomoxetine, a selective NE uptake blocker used in the treatment of attention deficit disorder, in Sprague‐Dawley rats in which mean arterial pressure was measured. NE (0.05, 0.1, 0.2 ug) that acts directly but is taken up by uptake‐1 to be sequestered in sympathetic nerve terminals, was potentiated after A (0.31 mg/kg) by 45%–89% and not changed further after A‐L‐NNA/NG. The pressor effect of T (0.025, 0.05, 0.1 mg) which acts indirectly by entering across uptake‐1 to release NE, was reduced by 89%–92% after A and restored by 575%–976% after A‐L‐NNA/NG. The pressor effect of methoxamine, not a substrate of uptake‐1, was not affected by pretreatment with A or A‐L‐NNA/NG. It is concluded that fresh synthesis of NO is significantly involved in the blocking activity of A on uptake‐1. Supported by the American University of Beirut.
Electrophysiological Characterization Of Purinergic Receptors In Mouse Enteric Neuron‐Glia Culture
Authors: Gade Aravind R , Akbarali Hamid I
Published: June 2021

Enteric glial cells are known to be associated with neurons in the gut and are responsible for maintaining important functions including motility, mucosal secretion, host defense etc. Purinergic receptors play an important role in glia‐neuronal signaling. In the present study, we examined the effect of purinoceptor agonists on ionic currents in isolated mouse enteric neurons and glia. A mixed population of neuron and glia were prepared from the mouse longitudinal muscle myenteric plexus as recently described (Smith et al., PloS One, 2012;7(9):e45251). Action potentials and robust sodium currents were present in enteric neurons but not in glia. Perfusion with ATP (500uM) resulted in transient inward currents at −60 mV (n=11) characteristic of P2X channel activation in enteric neurons. Enteric glia stained positive for GFAP in culture. Neither action potentials nor sodium currents were observed in glia. ATP (500uM) produced transient outward currents at +120 mV characteristic of P2Y receptor activation (n=5). UTP, a P2Y 2, 4 and 6 specific agonist did not elicit a response in the neurons. Our results suggest that in the enteric neuron‐glia culture, neuronal cells primarily express the P2X receptors and the glial cells primarily express the P2Y receptors. Supported by NIHDK046367.
Pentadecapeptide BPC 157 after 70% liver resection in rats
Authors: Sikiric Predrag , Crvenkovic Dalibor , Sever Marko , Lojo Nermin , Krstonijevic Zoran , Drmic Domagoj , Zenko Anita , Brcic Luka , Zoricic Ivan , Rasic Zarko , Rucman Rudolf , Seiwerth Sven
Published: June 2021

We suggest that stable gastric pentadecapeptide BPC 157 improves function and liver regeneration after 70% liver resection in rats since it can rescue otherwise severe liver lesions after overdose of paracetamol, diclofenac, ibuprofen, insulin, CCl4 or chronic alcohol abuse (for review see, i.e., Curr Pharm Des. 2012). Stable BPC 157 was applied (10 μg, 10 ng) (/kg) either ip once daily or in drinking water (0.16 μg; 0.16 ng/ml/12ml/day) till the sacrifice (at day 3, 7, 14, 21, 28). BPC 157‐rats maintained the weight, exhibited better liver regeneration based on better liver mass/body weight ratio (i.e., 14 days: 0.037 +/−0.004 (BPC 157) vs. 0.023+/− 0.005 (control); 28 days: 0.053+/−0.007 (BPC 157) vs. 0.03 +/− 0.004 (control)) and larger liver volume in pentadecapeptide BPC 157‐rats. Unlike constantly increased AST, ALT, bilirubine levels in controls, BPC 157‐rats after a short post‐surgery increase, mostly presented values comparable to non‐resected animals (conforming functional liver regeneration). Microscopically, controls presented larger areas of liver steatosis and liver fibrosis unlike BPC 157‐rats. Although binuclear cells were sporadically seen in controls, there were less hepatocytes with mitosis compared to all BPC 157 regimens. Supported by Grant 108–1083570‐3635, Croatia
Dihydrotanshinone induced apoptosis in colon cancer cells by p53‐independent but ROS dependent pathway mediated by mitochondria
Authors: WANG Lin , Yeung J. H. K. , Lee W. Y. W. , Zhou X. L. , Hu T. , Cho C. H.
Published: June 2021

Objective

Anti‐cancer action of dihydrotanshinone (DHTS) in colon cancer was evaluated and its underlying mechanisms were studied.

Methods

Cytotoxity of DHTS was measured in colon normal and cancer cells by MTT assay. Apoptotic activity of DHTS was evaluated by FCM. Intracellular Ca2+, ROS generation and MMP were detected by FCM or laser confocal microscopy assay.

Results

DHTS produced a selective cytotoxity against colon cancer cells through induction of apoptosis. The IC50 values for cell viability and apoptosis rates induced by DHTS were similar between HCT116 p53+/+ and HCT116 p53−/−cells. In a separate experiment, pretreatment with N‐Acety‐L‐Cysteine (NAC) or catalase‐PEG, both known to be free radical scavengers, significantly decreased apoptosis induced by DHTS. We also found that DHTS produced a time‐ and dose‐dependent increase in intracellular Ca2+ and ROS generation, which were blocked by pretreatment with BAPTA‐AM (a Ca2+ chelator), catalase‐PEG (a hydrogen peroxide inhibitor) or cotreatment with antimycin A or rotenone. Decrease of MMP was significantly blocked by rotenone or antimycin A while that of mitochondrial ROS was found to be postponed only by antimycin A.

Conclusion

Apoptosis induced by DHTS is p53 independent but ROS dependent. This was initiated from mitochondria dysfunction and promoted by increase of intracellular Ca2+.
Gender Difference in Duodenal HCO3‐ Response to Mucosal Acidification: Importance of Up‐regulation of ASIC3 by Estradiol
Authors: Takeuchi Koji , Ohashi Yumi , Amagase Kikuko , Aihara Eitaro
Published: June 2021

In the present study, we investigated the mechanism responsible for the gender difference in the acid‐induced duodenal HCO3‐ secretion. Male and female SD rats were used after 18 h fasting. Under urethane anesthesia, a proximal duodenal loop was perfused with saline, and HCO3‐ secretion was measured. Mmucosal acidification was performed by exposing the loop to 10 mM HCl for 10 min. Ovariectomy was performed 2 weeks before the experiments, and 17β‐estradiol was given i.m. on day 3, 7 and 10 after the operation. Although no difference was found in the HCO3‐ responses to PGE2 and capsaicin between male and female rats, the response to acid was significantly greater in female rats. However, this different response to acid was dissipated by ovariectomy, and this effect was antagonized by the repeated estradiol treatment. The expression of ASIC3 mRNA was greater in female rats and down‐regulated by ovariectomy, in an estradiol‐dependent manner. These results suggest that the acid induced‐HCO3‐ secretion was greater in female than male rats, and the mechanism for the different response may be accounted for by the up‐regulation of ASIC3 expression in female rats.
Constitutive activity of Gs α R201H is suppressed by disruption of either of two hydrophobic pockets within the protein
Authors: Pals‐Rylaarsdam Robin , Turcic Kyle , Carroll Julie , Tobar‐Rubin Raquel , Janevska Daniela , Din Eraj , Alvarez Rebecca , Choudhry Faiza
Published: June 2021

McCune‐Albright Syndrome (MAS) is a disorder caused by mutations in Gs at R201 that inhibit GTP hydrolysis. We developed a yeast model for MAS using a strain engineered to undergo cell cycle arrest in the presence of constitutively active Gα. We constructed and screened a library of 32,000 additional mutations in a constitutively active Gα gene, and identified 13 potential intragenic suppressor mutations at sites homologous to human Gs. When expressed in HEK cells, three Gs isoforms carrying the MAS (R201H) mutation with one of the 13 substitutions suppressed the MAS mutation's constitutive activity. F142 and L266 participate in hydrophobic packing in the crystal structure. Mutations that reduced the hydrophobicity of either amino acid suppressed the R201H mutation. R231 also suppressed R201H, possibly by altering hydrogen bonds between Switch II and water molecules near the γ phosphate of GTP. The suppressor mutations alone did not alter cellular responses to hormone or constitutively activate the protein. Intragenic suppressor mutations can be used to model potential sites to which small molecule drugs may be targeted to reduce or block the constitutive activity of Gs.

Supported by NIH 1R15ED020190–01
GPR158 and GPR179: a subfamily of orphan GPCRs as a new class of G protein signaling modulators
Authors: Orlandi Cesare , Masuho Ikuo , Posokhova Ekaterina , Cao Yan , Ray Thomas , Hasan Nazarul , Gregg Ronald , Martemyanov Kirill
Published: June 2021

GPR158 and GPR179 are members of a poorly understood subfamily within the class C of GPCRs. Bioinformatics revealed that they share a homology in their 7TM regions, contain several conserved elements at C‐termini, but lack ligand binding domain typical of the class C receptors. GPR158 is broadly distributed across brain regions while GPR179 is specifically expressed in the retina.

We found that GPR158/179 bind to the R7 group of Regulator of G protein Signaling (R7RGS), GTPase accelerating proteins that ensure timely deactivation of GPCR signaling. While GPR179 could bind to all members of R7RGS family, GPR158 forms specific complexes with RGS7. In transfected cells, GPR158/179 recruited R7RGS proteins to the plasma membrane enhancing their ability to deactivate G proteins. GPR158 potentiated RGS‐dependent deactivation of the μ‐opioid receptor signaling, while GPR179 facilitated deactivation of mGluR6. We report that GPR179 forms complexes in native retinas with mGluR6 and TRPM1, the main components of the signaling in ON‐bipolar cells (ON‐BC). Removal of either RGS proteins in these cells results in synaptic transmission deficits and lack of GPR179 prevented their targeting to the dendritic tips of ON‐BC. The ability of GPR158/179 to form heteromers with conventional GPCRs and to enhance RGS activity may be important for the modulation of many physiologically relevant G protein signaling pathways.

Supported by NIH
Regulation of the AGS4–Gαi Interaction by Chemokine Receptors and the Non‐Receptor Guanine Nucleotide Exchange Factor Ric‐8A
Authors: Robichaux William Gene , Oner Sukru Sadik , Lanier Stephen M. , Blumer Joe B.
Published: June 2021

Activator of G‐protein signaling‐4 (AGS4), by virtue of its three G‐protein regulatory (GPR) motifs, is a guanine nucleotide dissociation inhibitor (GDI) for GαiGDP subunits free of Gβγ. Our objective is to define the factors that regulate the GPR‐Gαi interaction. We developed a bioluminescence resonance energy transfer (BRET) system to monitor regulation of the AGS4‐Gαi interaction in live cells. Initially, our BRET system was used to assess the relative BRET signals generated between AGS4 and Gαi subunits Gαi1, Gαi2 and Gαi3. Due to its restricted expression in hematopoietic cell lineages, AGS4 is well positioned to modulate G‐protein signaling via G‐protein coupled chemokine receptors (GPCRs). Indeed, AGS4–Gαi complexes were regulated by CXCR4 activation. Interestingly, we also observed Gαi‐dependent interaction between AGS4 and CXCR4 suggesting that AGS4‐Gαi may interface directly with chemokine receptors, which has broad implications with respect to signal processing via chemokine GPCRs. In addition, we determined that the AGS4–Gαi complex is also regulated by the non‐receptor guanine nucleotide exchange factor (GEF) Ric‐8A, but not by the GEFs AGS1 or GIV/Girdin. These data indicate that the AGS4‐Gαi complex is subject to dynamic regulation by both chemokine receptors and Ric‐8A, and reveal novel insights into the unique role of accessory proteins in G‐protein signal processing.
CB1 Cannabinoid Receptors Signal Through Focal Adhesion Kinase To Activate ERK In Neuronal Cells
Authors: Dalton George D. , Howlett Allyn C.
Published: June 2021

Focal adhesion kinase (FAK) phosphorylated at tyrosine residue 925 (Tyr 925) binds Grb2/Sos which stimulates the tyrosine phosphorylation (Tyr‐P)/activation of the mitogen‐activated protein kinase ERK. We investigated the role that FAK 925 Tyr‐P plays in regulating CB1 cannabinoid receptor (CB1R)‐stimulated ERK Tyr‐P in murine N18TG2 neuroblastoma cells that express endogenous CB1Rs. Immunoblotting experiments revealed the time‐course of CB1R‐stimulated FAK 925 Tyr‐P and ERK Tyr‐P are similar in N18TG2 cells. The synthetic CB1R agonist WIN55212‐2 (10 nM) produced three distinct phases of FAK 925 Tyr‐P and ERK Tyr‐P: Phase I (0–5 min) involved maximal Tyr‐P, Phase II (5–20 min) involved a decline in Tyr‐P, and Phase III (>; 20 min) involved a plateau in Tyr‐P at submaximal levels. Phase I was blocked by the CB1R antagonist SR141716A (1 μM) and pertussis toxin (100 ng/mL) which suggests maximal FAK 925 Tyr‐P/ERK Tyr‐P are mediated by CB1Rs and Gi/o proteins. The Src inhibitor PP2 (2 μM) abolished Phase I FAK 925 Tyr‐P indicating an absolute requirement for Src, while PP2 partially inhibited Phase I ERK Tyr‐P. Phase I FAK 925 Tyr‐P and ERK Tyr‐P also involved transactivation of Flk‐1 vascular endothelial growth factor receptors and epidermal growth factor receptors. The FAK inhibitors Y11 (100 nM) and PF573228 (100 nM) abolished Phase I FAK 925 Tyr‐P, and partially inhibited Phase I ERK Tyr‐ P. These studies demonstrate CB1R‐stimulated maximal ERK activation requires FAK 925 Tyr‐P in neuronal cells. Supported by NIDA grants R01DA003690 (ACH), F32DA026295 (GDD).
Astrocytes produce nitric oxide in response to cholinergic or glutamatergic stimulation
Authors: Wang Xuewei , Lessard Andree , Trudeau Louis‐Eric , Girouard Helene
Published: June 2021

In the brain, it had been demonstrated that nitric oxide (NO) is produced in endothelial cells and neurons by endothelial (eNOS) and neuronal nitric oxide synthase (nNOS), respectively. However, in astrocytes, the source of NO production remains uncertain. The objective of the present study is to determine the sources and activators of NO production in mouse cortical astrocytes. NOS subtypes were identified by immunocytochemistry and electron microscopy. Using cortical astrocytes in culture, NO production was evaluated using 4‐amino‐5‐methylamino‐2′,7′‐difluorofluorescein (DAF‐FM) and confocal microscopy. Elevations of NO were detected in response to a 5 min stimulation with acetylcholine, N‐Methyl‐D‐aspartic acid (NMDA) or trans‐1‐ aminocyclopentane‐1,3‐dicarboxylic acid (t‐ACPD). NO production by acetylcholine seems to be spatially restricted while NO production induced by glutamatergic stimulation is diffuse. These effects were blocked by the NOS competitive inhibitor L‐N‐Nitro‐ Arginine, thus confirming their specificity. Our results suggest that there are two different pathways for NO production in astrocytes, which could have physiological consequences for neuronal and vascular functions in the brain. Supported by NSERC
Evidence that the α1L‐adrenoceptor is the native α1A‐adrenoceptor in rat vas deferens
Authors: Docherty James R
Published: June 2021

A 4th functional subtype of α1‐adrenoceptor, the α1L‐adrenoceptor, present in rat vas deferens, has been proposed at which prazosin has low potency (Ohmura et al., 1992). The subtypes of α1‐adrenoceptor mediating contractions of rat vas deferens to norepinephrine (NE) have been examined employing α1A‐antagonist RS100329 and α1D‐antagonist BMY7378 and the non‐selective antagonist prazosin. In presence of cocaine (3 μM) to block the NE transporter, NE potency (6.14±0.15, n=34) was significantly increased (P<0.05) as compared to in the absence of cocaine (5.30±0.10, n=21). Cocaine amplified contractions to low concentrations of NE. BMY7378 was more potent against contractions to NE in the presence of cocaine, whereas RS100329 was more potent in absence of cocaine. Prazosin, like BMY7378, was more potent in the presence of cocaine, suggesting α1D‐adrenoceptor selectivity. Prazosin pA2 values were 8.34 (slope ‐ 0.78±0.07) in absence of cocaine and 9.01 (slope −0.93±0.12) in presence of cocaine, with the lower value indicative of α1A‐adrenoceptor potency, and the higher value α1D‐adrenoceptor potency. Hence, it is concluded that the α1L‐adrenoceptor reported in rat vas deferens is not a functional isoform of the α1A‐adrenoceptor, it is the native α1A‐adrenoceptor at which prazosin shows lower potency.
Modification of sterol composition in yeast cell membrane from ergosterol to cholesterol and its effect on Ste2 signaling
Authors: Kuroda Kouichi , Morioka Sanae , Shigemori Tomohiro , Hara Keisuke , Morisaska Hironobu , Ueda Mitsuyoshi
Published: June 2021

In human cell membrane, cholesterol is required for functionalization of membrane proteins such as some human G‐protein‐coupled receptors (GPCRs). However, in yeast, ergosterol is the main sterol of cell membrane. Therefore, we investigated the effect of altered sterol composition in yeast cell membrane from ergosterol to cholesterol on GPCR signaling. The cholesterol‐producing yeast instead of ergosterol was constructed by disrupting the ergosterol‐related genes and introducing the genes for cholesterol synthesis. The GC‐MS analysis of the sterol composition in the constructed yeast indicated that cholesterol was successfully produced as one of the major sterols instead of ergosterol. The activity of yeast pheromone GPCR Ste2 in the cholesterol‐producing yeast was evaluated by measuring the activation of the FUS1 promoter. Ste2 signaling was detected even in the cholesterol‐producing yeast, but the maximum signal intensity was weaker than that in wild type yeast. The result suggests that sterol composition is important for the activity of GPCRs. Therefore, the optimization of the sterol composition would extend the range of application of yeast into the analysis of heterogenous GPCRs such as human GPCRs.
Cocaine‐induced locomotor activity in rats selectively‐bred for high and low motivation to voluntarily run
Authors: Brown Jacob Daniel , Green Caroline , Miller Dennis , Booth Frank
Published: June 2021

This study aimed to further characterize a novel colony of Wistar rats selectively‐bred for high (HVR) and low (LVR) motivation to voluntarily run. We aimed to examine any sex differences and line (LVR vs. HVR) differences in locomotor activity (LA) levels in young, sedentary rats.

METHODS

Eighty minutes of LA were measured on 3 successive days to determine novelty‐seeking (Day 1), habituation (Day 2), and cocaine‐induced (Day 3) LA in young (30–38d), male and female HVR/LVR rats.

RESULTS

HVR rats demonstrated greater novelty‐seeking (Day 1) and habituated (Day 2) LA than LVR rats. When analyzed as a difference from saline‐treated rats, cocaine‐induced LA (Day 3) was greater in LVR females than in HVR females. Both LVR and HVR females displayed greater cocaine‐induced LA than LVR and HVR males.

CONCLUSION

Our results suggest that cocaine‐induced LA is dependent on both sex and voluntary running genetic potential. Sex hormone and/or dopaminergic signaling systems are logical targets for future research to elucidate the mechanisms underlying these observations.

Supported by University of Missouri Development Office and the University of Missouri Life Science Fellowship.
Effects of chronic amphetamine treatment on cocaine‐induced facilitation of intracranial self‐stimulation in rats
Authors: Bauer Clayton T. , Banks Matthew L. , Negus S. Stevens
Published: June 2021

Amphetamine is a monoamine releaser and candidate agonist medication for cocaine dependence. Chronic amphetamine treatment decreases cocaine self‐administration by rats, nonhuman primates and humans. This study tested the hypothesis that chronic amphetamine would decrease abuse‐related facilitation of intracranial self‐stimulation (ICSS) by cocaine in rats. Male Sprague‐Dawley rats were equipped with electrodes targeting the medial forebrain bundle and trained to respond under a fixed‐ratio 1 schedule for 0.5 sec trains of electrical stimulation. Stimulation intensity was individually determined in each rat, and frequency varied from 56–158 Hz in 0.05 log units during each session. Effects of 10 mg/kg cocaine on ICSS were determined before, during and after 14‐day continuous infusion with saline (N=5) or amphetamine (0.32 mg/kg/hr, N=5). Saline treatment had no effect on ICSS, and cocaine produced comparable facilitation of ICSS before, during and after saline treatment. Amphetamine facilitated ICSS throughout the 14‐day treatment, and termination of amphetamine decreased ICSS below pre‐treatment baseline. Cocaine facilitated ICSS before and after amphetamine, but during amphetamine treatment, it did not facilitate ICSS above levels produced by amphetamine alone. These findings suggest that chronic amphetamine decreases abuse‐related stimulus effects of cocaine. Funded by R01 DA026946
Uncovering interactions between organic cation transporters and monamine systems: implications for novel antidepressant therapies
Authors: Apple Deana Marie , Horton Rebecca E , Vitela Melissa , Koek Wouter , Daws Lynette C
Published: June 2021

Depression is an important mental health problem. The most commonly prescribed antidepressants are selective serotonin (5‐ HT) reuptake inhibitors (SSRIs), which increase extracellular 5‐HT by blocking the high‐affinity 5‐HT transporter (SERT). Unfortunately, in many patients, SSRIs do not effectively relieve depression symptoms. A potential explanation for their limited effectiveness is the presence of low‐affinity, high‐capacity organic cation transporters (OCTs), which can take up 5‐HT, and may limit the ability of SSRIs to increase extracellular 5‐HT. Using the OCT blocker decynium‐22 (D‐22), we uncovered a significant role for OCTs in 5‐HT uptake when SERT is genetically or pharmacologically compromised. Here we show that in mice lacking the OCT3 subtype, D‐22 no longer enhances SSRI‐induced inhibition of 5‐HT clearance and antidepressant‐like effects. We extend these findings by examining the effects of chronic administration of D‐22, alone and together with monoamine uptake inhibitors, on 5‐HT uptake and antidepressant‐like activity in wild type and OCT3 −/− mice. Our results provide further evidence that OCT3 may contribute to the poor therapeutic outcome of SSRI treatment, and points to D‐22‐sensitive transporters, putatively OCT3, as novel targets for new antidepressant drugs with improved therapeutic potential. Supported by R01 MH093320 (LD & WK), R01 MH064489 (LD), and NARSAD (LD).
Long noncoding RNAs and transcription of cytochrome P450s in mouse liver during maturation
Authors: Peng Lai , Paulson Ariel , Li Hua , He Xi , Lu Hong , Klaassen Curtis D. , Li Linheng , Zhong Xiao‐bo
Published: June 2021

Cytochrome P450 (P450) enzymes, having critical functions in liver to metabolize xeno‐/endo‐biotics, alter their expressions during liver development. The mechanisms regulating the changes are not clear yet. Some long noncoding RNAs (lncRNAs) are known to function in regulation of gene expression during organ maturation. Therefore, we systematically analyzed lncRNAs expressed during liver maturation to determine whether lncRNAs are involved in regulating P450 ontogeny. Male C57BL/6 mouse livers were collected at 12 ages from prenatal to adult (prenatal: GD17.5; neonatal: Day 1, 3, 5, and 10; adolescent: Day 15, 20, 25, and 30; adult: Day 45 and 60, n=3 per age). Poly‐A selected RNAs were sequenced and compared to genes from Ensembl 66 combined with NONCODE v3.0 database. Approximately 15,000 genes were expressed in the developing liver, including ~2,000 lncRNAs. About 70% of these lncRNAs significantly changed their expressions across the ages. Three major ontogenic expression patterns were identified for lncRNAs, which are neonatal‐, adolescent‐, and adult‐enriched groups. The same patterns were also found in P450 genes. A set of lncRNAs highly correlated with expression of each P450 were identified. This is the first attempt to examine the correlation of lncRNAs and P450 ontogeny in liver and will facilitate future study to determine mechanistic roles of lncRNAs in P450 expression during liver maturation.
MiR‐627 mediates the epigenetic mechanism of vitamin D in suppression of colon cancer growth both in vitro and in vivo
Authors: Padi Sathish , Guo Bin
Published: June 2021

Vitamin D has excellent antitumor activities against various types of cancers, but it cannot be used due to its side effect of causing hypercalcemia. To understand its mechanism of action in tumor suppression, we investigated the effects of Calcitriol (Active form of vitamin D) on microRNA expression in colon cancer cells. We identified miR‐627 as the sole miRNA whose expression was stimulated by calcitriol. Furthermore, JMJD1A (jumonji domain containing 1A) was identified as the target of miR‐627. By downregulating JMJD1A, miR‐627 mediated the effects of calcitriol to increase histone H3K9 ethylation and to suppress the expression of growth and differentiation factor 15 (GDF15). Calcitriol also induced miR‐627 and downregulated JMJD1A in tumor xenografts. Calcitriol significantly suppressed the growth of colon tumors in nude mice, whereas colon tumors with stable JMJD1A‐UTR expression showed resistance to the calcitriol. Overexpression of miR‐627 decreased JMJD1A and suppressed colon cancer growth both in vitro and in vivo in nude mice. We also, found that miR‐627 expression was decreased in human colon cancer specimens when compared to its levels in the normal colon tissues. These results suggest that, miR‐627 and JMJD1A may serve as potential targets to exploit the antitumor activity of vitamin D without eliciting its hypercalcemic side effect. This research is supported by NIH Grants: CA130062 & RR015566
Prevention of cyclophosphamide‐induced alopecia by selegiline in a murine model
Authors: Berry Mark D. , Gaiser Alise C. , Chapman Stacy I. , Jackson Patrick
Published: June 2021

One of the most common and psychologically traumatic side effects of cancer therapy is the induction of apoptosis in hair follicle cells, resulting in alopecia. We have tested various compounds for their ability to modify chemotherapy‐induced apoptosis. An initial in vitro screen showed selegiline, clinically used in the control of Parkinson's disease, had a particularly intriguing profile. Selegiline dose‐dependently prevented cisplatin‐and cytosine arabinoside‐induced cell death in phenotypically normal cells, while also increasing cell death in human cancer cells in response to cisplatin. Dose‐response curves for the two effects were comparable, with a maximally effective concentration in both of 10nM. Selegiline was next tested in vivo using a mouse cyclophosphamide‐induced alopecia model. Anaesthetized animals were depilated with a wax/rosin mixture to induce synchronous hair follicle cycling. Alopecia was induced by the administration of 150mg/kg i.p. cyclophosphamide on days 7, 9, and 11 postdepilation. Selegiline was administered i.p. daily, starting on day 7 post‐depilation, at either 0.1 or 1.0mg/kg. Animals were monitored daily and hair re‐growth assessed on days 12, 19 and 26 postdepilation. At 0.1mg/kg selegiline at least partially prevented alopecia in 14/15 mice, with complete hair re‐growth indistinguishable from controls seen in 8/15. At the higher dose of 1mg/kg selegiline was considerably less effective, despite significantly greater monoamine oxidase‐B (MAO‐B) inhibition. The results suggest that selegiline can prevent the toxicity of clinically used cancer chemotherapeutics through an MAO‐B inhibition independent mechanism. This may relate to the p53/GAPDH‐dependent anti‐apoptotic effects of selegiline that have previously been reported. The in vitro studies further suggest that such an effect may occur without a concomitant decrease in chemotherapy efficacy at cancer cells. Financial support provided by NSERC and CFI.
Alterations in the intracellular Ca2+ concentration and vimentin in endothelial cells following tumor cell‐endothelial cell interactions
Authors: Pan Yan , Li Xuejun
Published: June 2021

Intracellular calcium regulation and cytoskeletal proteins in both tumor cells and ECs provide important signaling mechanisms for tumor growth and metastasis. We investigated the influence of tumor cell‐EC interactions on the intracellular calcium ion concentration ([Ca2+]i) and vimentin expression and structural organization in ECs before and after the addition of PC‐3M prostate carcinoma cells. Acrylamide (a known disruptor of intermediate filaments), heparin and nifidipine (calcium channel blocker) was used. PC‐3M cells induced a rapid [Ca2+]i elevation and lead to altered vimentin distribution in ECs at the sites of tumor cell‐EC contact. Tumor cell‐induced [Ca2+]i elevation was significantly inhibited by heparin and nifidipine. Acrylamide‐induced collapse of vimentin filaments shifted the distribution of calcium ions from the cytoplasm to the nucleus and perinuclear field in ECs. Rapid [Ca2+]i elevation and alterations in vimentin distribution occur in ECs at the sites of tumor cell‐EC contact; the structural integrity of vimentin is partly related to the intracellular calcium ion distribution and concentration.
Influence of hepatic P450‐mediated Amiodarone metabolism on Amiodarone‐induced pulmonary toxicity
Authors: Hartog Matthew , Zhu Yi , Lawrence David , Herron Bruce , Ding Xinxin
Published: June 2021

Amiodarone (AMI), a widely used, effective anti‐arrhythmic drug, has severe side effects, including acute lung toxicity and pulmonary fibrosis. Our aim is to test the hypothesis that a decline in liver microsomal P450‐meditated AMI metabolism leads to increased accumulation of AMI in lung and consequently greater pulmonary toxicity. We studied three mouse strains, all on B6 background: wild type (WT), LCN (liver‐specific P450 reductase (CPR) null; has little P450 activity in hepatocytes), and Cpr‐low (has global suppression of CPR expression in all cells). Adult, male mice were treated with AMI (160 mg/kg, i.p.) or vehicle alone, once/day for 3 days. On day 4, tissue AMI levels were determined, and bronchoalveolar lavage (BAL) was analyzed for signs of lung toxicity. Hepatic AMI levels were 1.1‐and 2.7‐fold higher, whereas lung AMI levels were 1.3‐and 4.3‐fold higher, in Cpr‐low and LCN mice, respectively, than in WT mice. As a general marker of lung toxicity, total numbers of cells in BAL were increased by 1.7‐and 2.3‐fold, in Cpr‐low and LCN mice, respectively, compared to treated WT mice. Furthermore, a 2‐fold increase in the number of lymphocytes in BAL was seen in LCN, compared to WT, following AMI treatment. These results support a role of liver P450 in protecting the lung against AMI's acute toxicity. Studies are ongoing to determine if a decrease in hepatic AMI metabolism leads to greater rate of pulmonary fibrosis.
Losartan improves impaired vascular and endothelial responses in mice with allergic asthma
Authors: Ponnoth Dovenia S , Mustafa S. Jamal
Published: June 2021

Asthma is associated with adverse cardiovascular outcomes, though the cause is unclear. We have shown vascular inflammation and altered vascular responses in allergic mice (Ponnoth et al., AJP 2008, 2010). In the present study, we investigated angiotensin II (Ang II) and endothelial aortic responses in a mouse model of allergic asthma. Mice were divided into control (CON) and allergen sensitized‐challenged (SEN) groups. Mice were sensitized i.p. on days 1, 6 with 0.2μg ovalbumin (OVA) followed by 5% OVA aerosol challenges on days 11–13. Organ bath studies were done in both groups on day 14. In the SEN group, Ang II (10μM) produced higher contraction (33.48±3.26% vs 18.06±3.07% in CON; p<0.05) while relaxation to acetylcholine (ACh; 1μM) was lower (30.95±5.73% vs 57.6±4.3% in CON; p<0.05), suggesting endothelium impairment. Ang II type 1receptor antagonist losartan was given i.p. before OVA challenges (10mg/kg daily single dose) on days 11–13 or a single bolus dose (20mg/kg) on day 14 in SEN groups. In both cases, response to ACh (56.1±4.39% and 51.88±5.22%) was similar to CON (55.52±4.86% and losartan treated CON 55.27±5.26%) and was significantly higher than untreated SEN group (29.76±3.2%).Ang II mediated contraction was higher in SEN (33.48±3.26%) vs losartan‐treated SEN (3.15±6.2%). Our data suggest that allergic mice have altered vascular reactivity, which can be improved with losartan. (HL027339, HL094447)
Acetaminophen at low doses depletes airway glutathione and alters respiratory reflex responses
Authors: Smith Gregory J. , Cichocki Joseph A. , Manautou Jose E. , Morris John B.
Published: June 2021

The goal of this study was to determine if acetaminophen (APAP), at non‐hepatotoxic doses, causes physiologically significant depletion of the important antioxidant glutathione (GSH) in the respiratory tract. Airway GSH levels were measured in female C57Bl/6J mice after 30, 60, or 100 mg/kg APAP ip. A dose/time dependent reduction of GSH occurred with maximal depletion (~60% of control) at 1hr (liver) and 2hrs (lung, trachea, and nose). Acrolein is a pro‐oxidant vapor that is detoxified by GSH, but in sufficient concentration stimulates the trigeminal nerve Transient Receptor Potential A1 receptor to elicit the sensory irritation response. We reasoned that if the decrease in GSH induced by APAP was physiologically significant, then APAP would enhance the acrolein‐induced irritation; APAP (100 mg/kg 1 hr prior to 2 ppm acrolein exposure) increased the irritation response 75% over acrolein alone. Nasal GSH averaged to 88, 69, and 59% of control at 30, 60, and 100 mg/kg APAP, respectively. It has been hypothesized that APAP, due to its increased usage and pro‐oxidant properties, has contributed to the 2‐fold increase in asthma prevalence that has occurred since 1980. These results support this hypothesis by indicating that APAP, at doses approximating the therapeutic dose of 15 mg/kg, depletes airway GSH and modulates acute respiratory responses. (Supported by the UConn President's Research Award).
Carotid body denervation (CBD) stunts development of Goldblatt 2 kidney‐1 clip (2K‐1C) hypertension in adult rats
Authors: McBryde Fiona D , Abdala Ana PL , Hendy Emma B , Paton Julian F R
Published: June 2021

The peripheral chemoreflex is sensitised in both the spontaneously hypertensive rat (SHR) and Goldblatt renovascular hypertension (2K1C). We have previously shown CBD to chronically lower arterial pressure (AP) in SHR. Here, we test whether established 2K‐1C hypertension can be ameliorated by CBD. The left renal artery of 6 week old Wistar rats was partially occluded. At 11 weeks of age rats were instrumented to record AP telemetrically, and after a 7 day baseline recording underwent CBD (n=5) or Sham (n=2) surgery. Respiratory rate fell at Week 1 following CBD (75±1 vs. 61±2 breaths/min) but recovered by Week 3 (70±2 breaths/min). AP rose steadily during baseline (+0.9±0.6 mmHg/day CBD; +1.0±0.5 mmHg/day Sham); this continued in Sham (+1.3±1.0 mmHg/day) but not CBD (−0.3±0.9 mmHg/day). Thus, at week 1 AP was unchanged from baseline in CBD rats (183±16 vs. 182±17 mmHg), but increased in Sham (201±18 vs. 210±21mmHg). Spontaneous baroreflex gain increased in CBD (−1.16±0.16 vs. −1.25±0.15 bpm/mmHg) but not Sham (1.1±0.09 vs. 1.0±0.12 bpm/mmHg). Reductions in low (6.3±0.5 vs. 6.0±0.8 mmHg2) and very low frequency (2.8±0.5 vs. 1.8±0.2 mmHg2) spectral power of systolic AP were seen in CBD but not Sham, suggesting sympathoinhibition. In summary, CBD appears to halt the continued AP increase in 2K‐1C rat, possibly via improved baroreflex gain and/or sympathoinhibition. Cibiem & British Heart Foundation funded research.