bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of cell cycle, cell death and EGFR expression in hepatic cells WRL68 treated with ethanol
Authors: Alvarez‐Ayala Elizabeth , Reyes‐Esparza Jorge , Rodriguez‐Fragoso Lourdes
Published: June 2021

Our aim was to study the effect of the ethanol on viability, proliferation and EFGR expression in the hepatic cells (WRL68). For the study, we used the WRL‐68 cells (ATCC: CL‐48) cultured in MEM supplemented with 10 % fetal bovine serum, 2mol/L glutamine, 1% non essential amino acid. Cells were kept at 37°C, under an atmosphere 95% of air and 5% CO2. We treated hepatic cells with ethanol at concentrations of 5, 12, 25, 50, 100, 200, 400 and 800 mg/dL. The viability and cell cycle was analyzed using a MTT assay. Cell cycle and apoptosis were analyzed at different times (cell cycle at 9, 12, 15 and 24 h, and apoptosis at 24 h) by flow cytometry. EGFR expression was analyzed by western‐blot. The ethanol did not modify the viability and cell proliferation. Ethanol induced changes on S phase at 9–15h but did not alter the Go/G1 and G2+M phases in WRL68 cells. Ethanol induces death cell, we observed the presence of apoptotic (40%) and necrotic cells (10%). We observed the EGFR expression in the hepatic cell line WRL68. However, no changes in EGFR expression were observed in cells treated with ethanol. In conclusion, ethanol induces apoptosis, necrosis and changes in cell cycle, but not changes in EGFR expression were observed in hepatic cells WRL‐68.
Proteomic Analysis Identifies Differentially Expressed Proteins in a Breast Primary Tumor and Associated Bone Metastatic Lesion
Authors: Castronovo Vincent , Dumont Bruno , Turtoi Andrei , Greffe Yannick , Wang Ying Hong , Bellahcene Akeila
Published: June 2021

A typical consequence of breast cancer tumor progression is the colonization of the skeleton. Targeted immunotherapy is an option to specifically deliver cytotoxic agents to the malignant lesions. However, the identification of targetable systemically reachable cancer‐associated antigens precludes the development of novel and effective antibody‐based therapies. In this study, we had the unique opportunity to examine a breast cancer primary tumor and the corresponding bone metastatic lesion obtained simultaneously from a fresh autopsy performed on a patient who died from disseminated breast cancer. We were interested in identifying accessible protein biomarkers using a biotinylation procedure on fresh tissue. The biotinylated proteins are captured by streptavidin affinity chromatography and the peptides derived from tryptic digestion are analyzed using the 2D‐HPLC‐MS/MS technique. The comparison of the differential expression of the primary tumor and the bone metastase yielded 234 proteins of which 78 were found to be located in the extracellular matrix and/or in the plasma membrane and represented potential candidates for antibody targeting. Twenty nine of the potential biomarkers were found uniquely expressed in the primary breast cancer while 27 proteins were detected only in the bone metastasis lesion. In particular, several proteins belonging to small leucine rich proteoglycans and integrin families were found up‐regulated in the primary tumor. Our study identifies potential valuable biomarkers for the selective antibody‐based targeted eradication of bone metastases and indicates that bone metastases accessible biomarkers are significantly different from those corresponding to the primary breast tumor.
O‐GlcNAc Causes Increased levels of Tumor Suppressor Protein p53 in Human Breast Cancer MCF‐7 Cells
Authors: Wang Kai , Ho Shiuh‐Rong , Paterson Andrew
Published: June 2021

The tumor suppressor protein p53 may play an anti‐cancer role by activating DNA repair proteins, inducing growth arrest, and initiating apoptosis. The concentration of p53 is controlled by proteasomal degradation. Hence, the proteasome controls important cellular functions such as cell cycle and apoptosis. O‐GlcNAcylation is a dynamic protein post‐translational modification that adds the monosaccharide N‐acetylglucosamine (GlcNAc) to specific serine or threonine residues on target proteins. We have previously found that O‐GlcNAc modification is an endogenous inhibitor of the proteasome. In this study, we tested the hypothesis that O‐GlcNAc modification can cause increased levels of p53 through proteasomal inhibition. Human breast cancer MCF‐7 cells were treated with 5 mM glucosamine (GlcN) for 2, 4, 6, 12, 24 hours. At 24 hours of GlcN treatment, O‐GlcNAc modification was increased 5.3‐fold and p53 levels increased 2.3‐fold compared to vehicle controls. We found that the proteasome had 18.7% decreased activity after 24 hours of GlcN treatment compared to the cells without GlcN treatment. These observations suggest that O‐GlcNAc modification may participate in the apoptosis of cancer cells and be a novel therapeutic target for cancer therapy.
Nicotine upregulates the expression of alpha‐7‐nicotinic receptors on human non small cell lung cancer cells via alpha‐7‐nAChRs and protein kinase C sensitive mechanism
Authors: Lau Jamie K , Brown Kathleen C , Dasgupta Piyali
Published: June 2021

Cigarette smoking is strongly correlated with the onset of non‐small cell lung carcinomas (NSCLC). Nicotine, the active component of cigarettes, induces proliferation and angiogenesis via the α7‐nicotinic acetylcholine receptor (nAChR). Our study examines the effect of long‐term nicotine exposure on α7‐nAChR expression in human NSCLC cells. Chronic exposure to nicotine upregulated the level of α7‐nAChR in a dose dependent manner, with the maximal effect observed between 100nM and 10μM over a period of 12 days. The treatment of human NSCLC cell lines with 100nM nicotine produced a time‐dependent increase of expression of α7‐nAChR starting from 4 to 12 days. Most interestingly, nicotine‐induced upregulation of α7‐nAChR was reversed within 4 days of nicotine exposure; however, the increased expression of α7‐nAChR was irreversible after 12 days of nicotine treatment. Nicotine‐induced α7‐nAChR upregulation was reversed by generalized nAChR‐antagonist mecamylamine and the protein kinase C pathway. Furthermore, α7‐nAChR antagonists α‐bungarotoxin and methyllycaconitine reversed nicotine‐induced increases of α7‐nAChR expression. Our results suggest that nicotine stimulates the expression of α7‐nAChR in human NSCLC cells by an autoregulatory mechanism involving protein kinase C. Funding for our study was supported by the PhRMA Foundation and the Flight Attendant Medical Research Institute YCSA Grant.
A strong inverse correlation between p16INK4a and pRb expression is observed at the level of individual tumor cells in HPV‐negative primary squamous cell lung cancer
Authors: Suh James H , Shroyer Kenneth R
Published: June 2021

Background

A wide range of human papillomavirus detection (<10–>80%) has been reported in pulmonary squamous cell carcinoma (SCC). Our aim is to investigate the potential co‐etiologic role of HPV in a series of North American lung cancers and to evaluate whether p16INK4aoverexpression, a marker of HPV integration in cervical SCC, is seen in pulmonary SCC.

Methods

DNA extracted from formalin‐fixed paraffin‐embedded tissue from 48 consecutive cases of surgically resected pulmonary SCC at Stony Brook University Medical Center (2003–2008) was analyzed for 16 types of HPV by multiplex PCR. Immunohistochemistry was performed to evaluate expression of p16INK4a and pRb.

Results

One case (2%) was positive for HPV DNA by multiplex PCR. Nuclear and cytoplasmic p16INK4a expression in tumor cells was detected by IHC in 22% and focal expression was present in normal bronchial glands in 49%. Nuclear expression of pRb was detected in 84% in a peripheral predominant pattern. An inverse correlation of p16INK4a and pRb expression was observed at the level of individual cells.

Conclusion

HPV is not detected in most pulmonary SCCs from our NE US patient population. p16INK4a overexpression is present in up to 25% of HPV‐negative pulmonary SCC and is inversely correlated with pRb expression at the level of individual tumor cells. Therefore, p16INK4a should not be used to distinguish between primary and HPV‐related metastatic disease.
Autonomous myotube fate specification mediated by Egr3 transcriptional regulation in muscle stretch receptor morphogenesis
Authors: Fernandes Michelle Oliveira , Gruner Katherine , Tourtellotte Warren
Published: June 2021

Muscle spindle stretch receptors mediate myotatic stretch reflexes and limb position information (proprioception) in vertebrates. Sensory (Ia‐afferent) innervation of a small number of myotubes induces their transformation into spindle stretch receptors during skeletal muscle development. Egr3 expression is regulated by Ia‐afferent innervation in developing spindles where it has an essential role in regulating gene expression involved in transducing sensory innervation‐mediated morphogenesis. Whether impaired spindle morphogenesis, which occurs in germline Egr3‐deficient mice, is due to loss of Egr3 expression in skeletal muscle or whether it is due to abnormalities in Ia‐afferent function has not been resolved. Here, we show that loss of Egr3 in skeletal muscles leads to abnormal spindle morphogenesis, ataxia and proprioceptive abnormalities similar to germline Egr3‐deficient mice. By contrast, ablation of Egr3 specifically in Ia‐afferent neurons has no effect on spindle morphogenesis. Thus, Egr3 has a skeletal muscle autonomous role in muscle spindle stretch receptor morphogenesis where it appears to be essential for regulating gene expression required for normal stretch receptor morphogenesis and innervation.
Microglia/macrophages and glioma progression
Authors: Zhai Haiyan , Gravanis Iordanis , Tsirka Stella
Published: June 2021

Gliomas are highly invasive brain tumors with the occurrence of numerous microglia/macrophages in and around the tumor. In this study, we used a glioma‐microglia co‐culture system to observe the details of their interactions. We generated a stable mouse glioma cell line GL261‐EGFP. When primary mouse microglia were treated with GL261‐conditioned medium, the expression of activation markers increased. When microglia were cultured with GL261‐EGFP, the phagocytic activity of microglia was inhibited. To study the contribution of microglia/macrophages to glioma progression in vivo, we used a mouse glioma model with microglia/macrophages ablation. CD11b‐HSVTK transgenic mice were used, which express herpes simplex thymidine kinase (HSVTK) in the cells of monocytic/microglial origin. Ganciclovir (GCV) was administered to the GL261‐EGFP injection site into the striatum. GCV was able to ablate microglia/macrophages in and around the tumor. This resulted in significant decrease in tumor sizes. The mice had much longer survival time as compared to the control group. These results suggest that glioma‐infiltrating microglia/macrophages can promote glioma growth, and that modulating their activity may be beneficial for glioma patients.

Funding: NIH/NINDS R0142168
Scedosporium apiospermum Presenting as an Intracranial Mass in an Immuno‐compromised Patient
Authors: Dabi Alok , Kurukumbi Mohankumar , Khan Raza , Jayam‐Trouth Annapurni
Published: June 2021

Background

Scedosporium apiospermum is ubiquitous filamentous fungi that can cause broad spectrum of opportunistic clinical diseases. These infections can be localized, deep or disseminated. Neurological infections may present as meningitis, meningo‐encephalitis and brain abscess. Here we present a case of S. apiospermum manifesting as intracranial mass in an immunocompromised patient.

Case presentation

A 77 year old male presented with speech slurring for 3 days. He had been on high dose steroids for nephrotic syndrome for last 5 months. Exam revealed left hemiparesis with left facial weakness. Imaging showed ring enhancing right frontoparietal lesion with edema. His condition worsened following a focal seizure with repeat CT head showing rapid increase in size of lesion despite empirical treatment for Toxoplasmosis. He had brain biopsy that revealed Scedosporium apiopsermum infection.

Conclusion

Scedopsorium apiospermum infection is difficult to isolate with conventional investigations unless there is a high index of suspicion. This case is reported to highlight a rising incidence of this rare potentially lethal infection and the prognostic significance of early detection and therapy.
Retinoid receptor‐mediated signaling in motor neurons and its implications for amyotrophic lateral sclerosis
Authors: Kolarcik Christi L. , Bowser Robert
Published: June 2021

Amyotrophic lateral sclerosis (ALS) is defined by progressive degeneration and death of upper and lower motor neurons. Recent work indicates retinoid signaling may contribute to the disease process. Nuclear retinoic acid receptors (RARs) are ligand‐activated transcription factors activated by all‐trans retinoic acid (ATRA); they mediate gene expression by forming heterodimers with retinoid X receptors (RXRs). We investigated the effects of RAR‐mediated signaling on motor neuron survival, morphology and downstream gene expression. Motor neuron‐enriched cultures established from embryonic day 14 rats were treated with pan‐agonists, pan‐antagonists and isotype‐specific agents. Toxicity studies were performed in the presence or absence of either hydrogen peroxide (to model oxidative stress/injury) or glutamate (to model excitotoxicity). In the presence of ATRA, the number of branch points/cell was decreased in motor neurons. RARalpha was localized to proximal and distal processes; RARbeta was localized to the cell body and nucleus. When ATRA was administered in the presence of toxin, motor neuron cell death was delayed. Signaling through RARs appears to maintain long neurites with less branching and delays toxin‐induced cell death. As we have observed increased nuclear RARbeta in sporadic ALS patients, downstream genes modulated by RARbeta are currently being characterized.

Research Support: NIH grant ES013469 (RB); NIH T32 EB001026 (CK); NIH T32 05 TL1 RR024155‐02 (CK)
The effect of a human pancreatic α‐amylase inhibitor, montbretin A, on the chronic treatment of type 2 diabetes
Authors: Yuen Violet G. , Brayer Gary , Withers Stephen , Andersen Raymond , Coleman John , McNeill John
Published: June 2021

Montbretin A, a purified glycosylated acyl flavonol, was isolated from extracts of the plant Crocosmia crocosmiflora. Montbretin A has been shown to be a specific inhibitor of human pancreatic α‐amylase (HPA). The objective of this study was to determine if chronic administration of montbretin A and acarbose, an inhibitor of α‐glucosidases and HPA, could prevent and/or delay the onset of diabetes in the Zucker Diabetic Fatty (ZDF) rat. Montbretin A (7.5 mg/kg/day) and acarbose (10 mg/kg/day) were administered to ZDF and lean littermate controls for 60 days. Plasma was analyzed for glucose, insulin, triglyceride, cholesterol, alanine transaminase (ALT) and 8‐isoprostane. Both montbretin A and acarbose attenuated the increase in plasma glucose levels in the ZDF rat. There was no effect of montbretin A or acarbose on lipid parameters. All ZDF animals had elevated ALT levels; however, ALT levels were significantly higher in the acarbose treated group, which may indicate an exacerbation of liver damage. This study indicates that montbretin A is as effective as acarbose in lowering plasma glucose levels; further studies are ongoing to assess the side effect profile of montbretin A. Project was funded by the Canadian Institutes of Health Research and supported by the Centre of Drug Research and Development.
Obesity decreases the activity of the vascular endocannabinoid system: role of eNOS and AMPK
Authors: Lobato Nubia , Filgueira Fernando , Giachini Fernanda , Carvalho Maria Helena , Webb Clinton , Tostes Rita , Fortes Zuleica
Published: June 2021

AIMS

Since the endocannabinoid system controls the energy balance and the vascular tone, we evaluated the effects of obesity on the activity of this system in the vessel.

METHODS AND RESULTS

Vascular function was assessed in small mesenteric arteries from young obese Zucker rats (OZRs) and their lean counterparts (LZRs). The relaxation to anandamide (AEA), to CB1 and CB2 agonists was decreased in OZRs. CB1 and CB2 blockade almost blunted the AEA response in OZRs. The protein expression of these receptors was decreased in OZRs. Inhibition of AEA transport, NO and prostanoids production decreased the relaxation to AEA only in LZRs, indicating impairment of these mechanisms in OZRs. Signaling pathways mediated by adenylyl cyclase, guanylyl cyclase, PI3K, PKG and PKA are not involved in the decreased relaxation to AEA. ERK inhibition restored the AEA relaxation in OZRs. Only in OZRs, the incubation with AEA increased ERK phosphorylation, which was already increased, indicating activation of contracting pathways by AEA in obesity. AMPK activation restored the relaxation to AEA. Incubation with AEA increased the phosphorylation of eNOS, AMPK and acetyl CoA carboxylase only in LZRs, indicating that these proteins mediate the AEA relaxation in LZRs, and this mechanism is impaired in OZRs.

CONCLUSION

Obesity decreases the activity of the vascular endocannabinoid system by reducing the activity of AMPK and eNOS.

FAPESP
Endothelium‐derived reactive oxygen species impair myogenic tone in type‐2 diabetic rat ophthalmic artery
Authors: Jarajapu Yagna PR , Ito Isamu , Guberski Dennis , Grant Maria
Published: June 2021

We have previously reported that myogenic tone in ophthalmic artery is impaired in obese type‐2 diabetic BBZDR/Wor rat. In this study, we delineated the role of endothelium‐derived reactive oxygen species (ROS) in the attenuated myogenic response. Ophthalmic arteries were isolated from BBZDR/Wor rats (T2D) with 4–5 months duration of diabetes and the age‐matched lean non‐diabetic rats (control). Arterial segments were cannulated in an arteriograph and inner diameter was continuously recorded over a range of intraluminal pressures in the presence or absence of different pharmacological agents. Pressure‐induced constriction in T2D artery was significantly lower compared to that control in the range of 50–150 mmHg. Dilation to carbachol or bradykinin was significantly lower and L‐NAME (non‐specific nitric oxide synthase inhibitor)‐mediated constriction was higher in T2D artery compared to the control when studied at an intraluminal pressure of 70 mmHg. In the presence of 4‐aminoguanidine, myogenic tone T2D artery was comparable to that of control over the pressure range of 50–150 mmHg. Similar results were observed when T3D artery was treated with a scavenger of ROS, N‐acetyl cysteine, NADPH oxidase inhibitor apocynin or peroxynitrite scavenger ebselen. These results suggest that peroxynitrite generated from superoxide and NO acts on arterial smooth muscle and antagonizes myogenic tone.
Potentiation of aspirin induced cardioprotection by minocycline against myocardial ischemia reperfusion injury in streptozotocin diabetic rat
Authors: Bhatt Lokesh Kumar , Addepalli Veeranjaneyulu
Published: June 2021

In the present study we have targeted Matrix Metalloproteinase MMP‐2 and MMP‐9 overactivation in diabetic rats by combination of minocycline and aspirin to attenuate cardiovascular complication of diabetes. Diabetes was induced in male Wistar rats by streptozotocin (55 mg/kg i.p.). Three weeks after diabetes induction, rats were treated with minocycline (50 mg/kg, p.o.), aspirin (50 mg/kg, p.o.), or minocycline (50 mg/kg, p.o.) plus aspirin (50 mg/kg, p.o.) for a period of next three weeks. At the end of sixth week, ischemia/reperfusion injury was induced by ligating left anterior descending coronary artery for 30 min followed by 2hr reperfusion. Percentage infarct volume, arrhythmias, mortality, collagen level and MMP‐2 & MMP‐9 level were significantly increased in vehicle treated diabetic group when compared with normo‐glycemic rats. Treatment with combination of minocycline and aspirin decreased percentage infarct volume, arrhythmias, mortality & collagen level when compared with vehicle‐treated diabetic controls and showed reduced level of MMP‐2 and MMP‐9. Results of the present study suggest that minocycline potentiates cardioprotective effect of aspirin in diabetic rats by enhanced MMP‐2 & MMP‐9 inhibition.

The project was funded by NMIMS University, Mumbai, India.
Isoflurane Preconditioning Modulates the Expression of eNOS and eNOS‐related Genes during Myocardial Ischemia and Reperfusion
Authors: Baotic Ines , Sudhakaran Shaan , Pratt Phil , Kersten Judy
Published: June 2021

Endothelial nitric oxide synthase (eNOS) contributes to isoflurane preconditioning (IsoPC), a form of pharmacological cardioprotection against ischemia‐reperfusion injury. We investigated the effects of IsoPC on the expression of eNOS and eNOS‐related genes: GTP cyclohydrolase‐1 (GTPCH‐1), dimethylaminohydrolase‐2 (DDAH‐2), and protein arginine N‐methyltransferase‐1 (PRMT‐1) during myocardial ischemia and reperfusion. Rats were preconditioned with 1.4% isoflurane administered for 30 min followed by discontinuation for 15 min prior to occlusion of the left anterior descending coronary artery for 30 min. Gene expression in cardiac tissue was quantified by real‐time PCR at five time points: pre‐occlusion, occlusion, 30, 60 and 90 min of reperfusion. Expression of all genes decreased during occlusion and reperfusion in control animals. Isoflurane alone decreased gene expression before coronary artery occlusion, but conversely, attenuated further decreases in gene expression that were observed during occlusion and reperfusion in control rats. Expression of GTPCH‐1, DDAH‐2 and PRMT‐1 recovered to baseline values and eNOS expression was increased after 60 min of reperfusion in the IsoPC compared to the control group. This suggests that IsoPC exerts time‐dependent changes in genes expression pattern that may influence the function of eNOS during and after myocardial ischemia‐reperfusion injury.
“Thrombin sponge”: A potent nanoparticle approach to inhibiting coagulation in acute thrombosis
Authors: Myerson Jacob Wheatley , He Li , Tollefsen Douglas M , Wickline Samuel A
Published: June 2021

An anticoagulant perfluorocarbon (PFC) nanoparticle (NP) was designed as a new antithrombotic with intrinsic magnetic resonance contrast, concentrated therapeutic impact conferred by a thrombin‐absorbing particle surface, and well‐defined pharmacokinetics controlled by the particle itself.

The irreversible thrombin inhibitor, PPACK (Phe(D)‐Pro‐Arg‐Chloromethylketone), was covalently attached to PFC NPs. Optical assay verified that PPACK activity against thrombin and selectivity for thrombin over plasmin was not diminished on the NPs. In vivo activity was assessed for PPACK NPs, PPACK, heparin, non‐functionalized NPs, or saline in C57BL6 mice subjected to laser injury of the carotid artery. Time to thrombotic occlusion of the injured artery was assessed via Doppler flow measurement. Selected arteries were excised to assess NP retention via 19F magnetic resonance (MR).

PPACK NPs exceeded PPACK's activity against thrombin. PPACK NPs outperformed both heparin (p=.001) and PPACK (p=.0006) in delaying occlusion of the carotid artery. PPACK or non‐functionalized NPs failed to delay occlusion of the carotid artery. 19F MR captured PFC NP retention in occluded arteries.

PPACK NPs utilize the long circulating half‐life of the PFC NPs to present thrombin absorbing surfaces that prevent thrombus formation. As potent new antithrombotics that can be traced with 19F MR, PPACK NPs are promising therapeutics.
L‐type Ca2+ (CaL) Channels are Silent During Depolarization of Venous Smooth Muscle
Authors: Thakali Keshari , Kharade Sujay V , Rhee Sung W , Rusch Nancy J
Published: June 2021

Calcium channel blockers (CCBs) reduce cardiac afterload but not preload, suggesting that the venous circulation is insensitive to their dilator action. Indeed we observed that CCBs prevent KCl‐induced constriction in pressurized rat mesenteric arteries (MA) but not veins (MV), suggesting that CaL channels are silent during depolarization of venous smooth muscle cells (SMCs). In patch‐clamp studies, single CaL channel currents in cell‐attached patches were abundant during depolarization from −70 mV to −10 mV in SMCs of MA (NPo= 0.26±0.05) but rarely occurred in MV (NPo=0.02±0.01). Calcium imaging in fluo‐4 loaded vessel segments revealed that KCl (60mM)‐induced depolarization similarly increased intracellular Ca2+ ([Ca]i) in MA and MV by 74% and 66%, respectively. However, whereas the CCB nifedipine (10 μM) blocked KCl‐induced constriction in MA and [Ca]i failed to rise, nifedipine failed to reduce KCl‐induced constrictions in MV and [Ca]i still increased by 34%. Our results suggest that CaL channels fail to open during depolarization of MV and an alternative source of activator Ca2+ mediates KCl‐induced constriction. Work supported by NIH NRSA 1F32 HL095284
Effect of β3 subunit deletion on L‐type calcium channels in mouse small mesenteric arteries
Authors: Kharade Sujay V , Rusch Nancy J
Published: June 2021

The L‐type Ca2+ channels in vascular smooth muscle cells (VSMCs) are presumed to be heteromeric complexes composed of pore‐forming α1C subunits and regulatory β and α2δ subunits. The β subunits emanate from four gene families (β1, β2, β3, β4) and are thought to promote the expression of α1C and uniquely regulate its properties in a subunit–specific manner. Based on studies in aortic VSMCs, it is presumed that the β2 and β3 subunits complex with α1C in VSMCs. The present study used β3 KO mice to define the effect of β3 deletion on the expression and properties of Ca2+ channels in mouse small mesenteric arteries (MA). Western blots showed a 30% loss of α1C expression in MA from β3 KO mice compared to WT mice, presumably reflecting the deletion of α1C‐β3 channel complexes. Single channel recordings of Ba2+ currents in cell‐attached patches of mesenteric VSMCs revealed the persistence of functional Ca2+ channels after β3 deletion. We hypothesize that these currents emanate from α1C‐β2 channel complexes since we failed to detect other known β subunits (β1, β4) on Western blot. In this regard, the β3 KO mouse may provide a unique opportunity to study the properties and regulation of relatively pure populations of vascular α1C‐β2 channels. The β2 subunit is reported to confer novel properties to Ca2+ channel complexes in heterologous expression systems and nonvascular cell types that may affect Ca2+ signaling in the vasculature.
CREB regulation of the CART gene in the rat nucleus accumbens and GH3 cultured cells
Authors: Rogge George Andrew , Jones Douglass C. , Nestler Eric , Kuhar Michael J.
Published: June 2021

Cocaine‐ and amphetamine‐regulated transcript (CART) peptides in the nucleus accumbens (NAc) oppose the rewarding effects of cocaine. Since over expression of the transcription factor (TF) CREB likewise decreases cocaine reward we hypothesized that CREB regulates the CART gene in the rat NAc via interaction at a CART promoter CRE cis‐regulatory element. Electrophoretic mobility shift assays (EMSA) and TF‐antibody super shift assays (SS) with a CRE oligonucleotide identical in sequence to the CART gene CRE site revealed that CREB and ser133 phospho‐CREB from the rat NAc and pituitary‐derived GH3 cells bind to the CRE element of the CART promoter. Furthermore, chromatin immunoprecipitation assays performed with GH3 cells revealed an interaction between the CART promoter region containing the CRE element and both CREB and ser133 phospho‐CREB. To determine if CREB regulates CART expression in the NAc, Herpes simplex virus‐1 vectors (HSV‐CREB) that over expressed CREB were injected into the rat NAc. When compared to the effects of vectors expressing LacZ alone (HSV‐LacZ), HSV‐CREB increased CART mRNA and peptide levels in the NAc as determined by in situ hybridization and Western blotting. In summary, we identified CART as a CREB‐regulated gene in the NAc of rats. Finding CREB‐regulated genes in the NAc offers insight into the molecular mechanisms underlying cocaine's addictive properties.
Differential cocaine‐induced changes in striatal glutamate receptor phosphorylation in low and high cocaine responding rats
Authors: Yamamoto Dorothy , Zahniser Nancy R.
Published: June 2021

Locomotor sensitization induced by repeated exposure to cocaine has been linked to increases in glutamate α‐amino‐3‐hydroxy‐5‐methyl‐isoxazole propionic acid receptor (AMPAR) and N‐methyl D‐aspartate receptor (NMDAR) phosphorylation. Such changes may help to explain why outbred male Sprague‐Dawley rats initially classified as low cocaine responders (LCRs) more readily develop cocaine‐induced locomotor sensitization than those classified as high cocaine responders (HCRs). We used western blots to investigate LCR/HCR changes in AMPAR and NMDAR phosphorylation in the ventral tegmental area (VTA), dorsal striatum (dSTR), and nucleus accumbens (NAc) 40 min after either acute (10 mg/kg, i.p.) or repeated (10 mg/kg, i.p., once daily for 7 days) cocaine. Significant increases in phosphorylation over controls were observed only in LCRS: after acute cocaine at NMDAR NR2B‐Y1472 by 39% and 31% in NAc and dSTR, respectively, and after repeated cocaine at AMPAR GluR1‐Ser845 by 30% in dSTR. No differences were observed in VTA. Our results suggest that increased striatal NMDAR NR2B‐Y1472phosphorylationin LCRs after acute cocaine may contribute to their development of locomotor sensitization and increased AMPAR GluR1‐Ser845 phosphorylation in LCRs may help to maintain their increased locomotor response to a cocaine challenge after repeated dosing. Supported by DA004216, GM007635, DA015050.
Crystallographic comparison of nicotinic ligands in complex with the acetylcholine binding protein
Authors: Talley Todd T. , Park John F. , Wu Joshua , Ho Kwok‐Yiu , Sankaran Banumathi , Taylor Palmer
Published: June 2021

Since the first report of the acetylcholine binding proteins in 2001 a number of X‐ray structures of these proteins in complex with various ligands have been published. Close to 30 structures are publicly available and many still in preparation. The structures consist of peptide toxins, small alkaloid natural products, macrocyclic amines isolated from marine organisms, synthetic and semi‐synthetic pesticides including the neonicotinoids as well as compounds with therapeutic potential. Analyses of these structures highlight many molecular features that contribute to ligand recognition and selectivity. One reoccurring determinant is that tertiary and secondary nitrogen containing ligands show a precise distance relationship with a hydrogen bond from a protonated amine donor to the carbonyl oxygen of Trp 143. In contrast, quaternary ligands are stabilized by an aromatic nest with cation‐π quadrupole interactions being dominant. Many ligands also rely on topographical features removed from this central core forming bonding interactions along the subunit interface and with specific residues present in the complementary subunit. These multiple points of contact allow ligands to select and stabilize specific conformational states of the binding proteins providing a template for the design of new ligands with the potential for greater specificity and efficacy. Supported by R37 GM 108360 and U01 DA 019372.
Pilot Safety Evaluation of Varenicline for the Treatment of Methamphetamine Dependence
Authors: Sevak Rajkumar , Zorick Todd , Miotto Karen , Shoptaw Steven , Swanson Aimee‐Noelle , Clement Clayton , De La Garza Richard , Newton Thomas F , London Edythe
Published: June 2021

Despite the worldwide extent of methamphetamine (MA) dependence, no medication has been shown to effectively treat afflicted individuals. One relatively unexplored approach is modulation of cholinergic system function. Previous human laboratory findings indicate that rivastigmine, a cholinesterase inhibitor, reduces craving for MA. We therefore performed a double‐blind, placebo‐controlled, crossover pilot study of the safety and tolerability of varenicline in eight MA‐dependent subjects. Varenicline is used clinically to aid smoking cessation, and acts as a partial agonist at α4β2 nAChRs with full agonist properties at α7 nAChRs. Oral varenicline dose was titrated over 1 week to reach 1 mg bid, and then was co‐administered with 30 mg MA, delivered in ten intravenous infusions of 3 mg each. Varenicline was found to be safe in combination with MA, producing no cardiac rhythm disturbances or alterations in vital signs. No adverse neuropsychiatric sequelae were detected during varenicline titration and following MA administration. The results suggest that varenicline warrants further investigation as a potential treatment for MA dependence. Supported by P50DA018185 (SS), P20DA022539 (EDL) and MOI RR00865 (UCLA GCRC).
Nicotine and varenicline share discriminative stimulus properties and act through mecamylamine‐sensitive receptors in rhesus monkeys.
Authors: Cunningham Colin Seamus , McMahon Lance R
Published: June 2021

Varenicline is reported to be more effective than nicotine in promoting smoking cessation. To examine pharmacologic factors (i.e., site of action and efficacy at nicotine acetylcholine receptors) that could underlie differences in the clinical effectiveness of these smoking cessation pharmacotherapies, these and other drugs were studied in rhesus monkeys (n=3) discriminating nicotine (1.78 mg/kg, s.c.) from saline. Nicotine and varenicline (ED50 = 0.50 mg/kg and 0.56 mg/kg respectively) produced ≥91% nicotine‐lever responding. Cocaine produced a maximum of 50% nicotine‐lever responding; however, other drugs that act through non‐nicotine receptors (midazolam and ketamine) produced ≤4% nicotine‐lever responding. The nicotine antagonist mecamylamine dose‐dependently antagonized the effects of nicotine and varenicline. These data show that the nicotine discrimination assay is mediated by nicotine receptor agonism and, to some extent, monoamine agonism. The qualitatively similar effects of varenicline and nicotine and the similar antagonism of these agonists suggest that common, mecamylamine‐sensitive receptors mediate the effects of both agonists. Under these conditions in vivo, there is no evidence that nicotine and varenicline differ in their nicotine agonist efficacy. Studies were funded by USPHS grant DA25267.
Nicotinic and muscarinic receptors in the rat dorsal hippocampus mediate the conditioned place preference induced by ethanol
Authors: Rostami Parvin , Zarrindast Mohammad Reza
Published: June 2021

Conditional place preference (CPP) is a behavioral response that can be used to study the neurobiology of reward and addiction behavior. We previously showed that nicotinic receptors in the basolateral amygdale potentiate the ethanol‐induced CPP. This study characterized the role of cholinergic receptors in the dorsal hippocampus in mediating the ethanol‐induced CPP in rats. Daily injection of ethanol (0.25, 0.5, 1 and 1.5 g/kg) s.c. or saline (2 ml/kg) i.p. elicited a dose‐dependent CPP response to ethanol. The maximum response was observed with the 0.5 g/kg ethanol dose, and this dose was used for all further studies. Injections of nicotine (0.5, 0.75 and 1μg/rat) potentiated the ethanol‐induced CPP. Injection of the nicotine receptor antagonist, mecamylamine (1, 2 and 4 μg/rat) inhibited the ethanol induced CPP, as did injection of the muscarinic receptor antagonist atropine (1, 2 and 4 g/rat). Pretreatment with anti‐cholinesterase drug, pilocarpine (0.5, 1 and 1.5 μg/rat) did not alter the ethanol‐induced CPP. This data suggest that both muscarinic and nicotine receptors in the hippocampus may be involved in the acquisition of ethanol induced CPP.
Effect of Chronic in‐vivo Exposure to Opioid versus Cannabinoid Receptor Agonists on CB1 and mu‐Opioid Receptor Protein Level in the Guinea Pig Longitudinal Muscle‐Myenteric Plexus Model
Authors: Maguma Hercules T. , Taylor David A.
Published: June 2021

Similarities in opioid (OP) and cannabinoid (CB) signaling and their effects suggest that tolerance development may utilize similar signaling pathways. Previous studies using the longitudinal muscle‐myenteric plexus (LM/MP) showed selective reduction in potency and efficacy only to cannabinoid1 receptor (CB1) agonists after chronic CB exposure and reduced potency with no effect on efficacy for either OP or CB agonists after chronic OP treatment. The current study investigated the effect of chronic opioid and CB treatment on mu‐opioid (MOR) and CB1 receptor protein level in the LM/MP. We also explored the distribution and potential co‐localization of the receptors in LM/MP neurons. Tolerance was induced by chronic OP or CB receptor agonist injection. Receptor protein levels were assessed using Western blotting and receptor localization by immunofluorescence. Chronic CB treatment significantly reduced (32%) CB1 protein whereas MOR protein levels remained unchanged. Chronic OP treatment altered neither MOR nor CB1 protein levels. Significant co‐localization of CB1 and MOR was observed in the LM/MP with comparable density of immunopositive neurons for each receptor population. The current data reinforce the notion that tolerance observed with chronic CB treatment in this model involves receptor dependent changes, whereas tolerance to OP may involve non‐receptor dependent changes in cell excitability.
The Effects of Positive Allosteric Modulation of the α7 Nicotinic Acetylcholine Receptor in the Five‐Choice Serial Reaction Time Task (5‐CSRTT)
Authors: Horvath Peter , Yoshimura Ryan , Hogenkamp Derk , Gee Kelvin W.
Published: June 2021

Human clinical studies suggest that using nicotine improves attention. Targeting the α7 nicotinic acetylcholine receptor (nAChR) may provide a substantial improvement in cognitive function without side‐effects associated with global activation of other nicotinic receptor subtypes by nicotine. In the present studies, we examine the effect of modulation of the α7 nAChR on Five‐Choice Serial Reaction Time Task (5‐CSRTT) in Sprague‐Dawley rats. The α7 nAChR positive allosteric modulator (PAM), compound 6 (N‐(4‐chlorophenyl)‐α‐[[(chlorophenyl)amino]methylene]‐3‐methyl‐5‐isoxazoleacet‐amide) was tested in the 5‐CSRTT. Compound 6, administered chronically at 0.3 and 1 mg/kg intraperitoneally, significantly increased the rate of acquisition and decreased omissions consistent with improvement in attention. In addition, our data shows that there is a trend for this acquisition effect to be reversed by the nonselective nAChR antagonist mecamylamine. These data are consistent with the hypothesis that activation of α7 nAChRs will enhance cognitive function. This effect on attention also suggests that the α7 nAChRs PAMs may have therapeutic value in the treatment of attention deficit disorders such as attention deficit/hyperactivity disorder (ADHD).
Cognitive impairment and decreased hippocampal neurogenesis after treatment with chemotherapeutic drugs
Authors: Pechnick Robert N , Reyes Kevin C , Das Melanie , Lacayo Liliana M , Farrokhi Catherine , Zonis Svetlana , Chesnokova Vera
Published: June 2021

Post‐chemotherapy cognitive impairment has long been recognized in cancer survivors; however, at the present time the cause(s) is not known. The objective of the present study was to determine the effects of chemotherapeutic drugs on cognitive function and hippocampal neurogenesis in the mouse. Adult female mice were injected once a week for 3 weeks with methotrexate (30 mg/kg/i.p.), cyclophosphamide (100 mg/kg/i.p.) or saline (0.9%/i.p.). One week after the last injection they underwent behavioral testing. Another cohort of mice did not undergo behavioral testing, but were treated with bromodeoxyuridine (BrdU; 50 mg/kg/i.p.; every 2 hr for a total of 4 injections) and sacrificed 24 hr later. There were no significant differences in locomotor activity among the treatment groups; however, spontaneous alternation was impaired in the methotrexate‐treated subjects. BrdU incorporation was not affected in the cyclophosphamide‐treated subjects, but was markedly reduced in the dentate gyrus of the hippocampus after treatment with methotrexate. These results suggest that post‐chemotherapy cognitive impairment might be linked to drug‐induced decreases in hippocampal neurogenesis. Supported by Department of Defense Breast Cancer Research Program grant number BC075629.
The role of Gαo in mu‐opioid signaling and antinociception
Authors: Thomson Jennifer A. , Jutkiewicz Emily M. , Traynor John R.
Published: June 2021

Clinically used opioids (e.g. morphine) elicit their analgesic effects by interacting with the mu‐opioid receptor (MOR), a member of the G protein‐coupled receptor superfamily. Specifically, MOR couples to members of the Gi/o family of inhibitory G proteins. Although MOR has been shown to activate all inhibitory G protein alpha subunits (Gαi/o) in vitro, Gαo is expressed at higher levels in brain than other Gαi/o subtypes, and is especially abundant in regions involved in pain modulation. To evaluate the role of Gαo in MOR signaling and antinociception, we have probed the effect of Gαo knockout on responses to MOR agonists using both in vitro and in vivo techniques. In whole brain homogenates, DAMGO‐stimulated [35S]GTPγS binding was decreased 13% in heterozygous (+/−) Gαo knockout mice and 82% in homozygous (−/−) Gαo knockout mice, as compared with wild‐type controls, although there were no differences in MOR numbers in these mice. In vivo, the antinociceptive effects of morphine and other MOR agonists (i.p.) were decreased significantly in Gαo +/− mice compared with their wild‐type littermates, as measured in the hotplate test. This difference was not observed in Gαi2 or Gαi3 knockout mice. These data implicate Gαo in the modulation of MOR agonist signaling and antinociception. This work was supported by GM077667 (JAT) and DA04087 (JRT).
Competition between Gi/o‐coupled receptors prevents cAMP overshoot following chronic opioid treatment
Authors: Levitt Erica S. , Traynor John R.
Published: June 2021

Use of selective agonists for Gi/o‐coupled receptors endogenously expressed in SH‐SY5Y cells has revealed near complete competition for shared adenylyl cyclase (AC) enzymes, indicating a lack of specific receptor/effector pairs. For example, AC inhibition by the mu opioid agonist DAMGO was not enhanced by addition of agonists to ORL‐1, alpha 2‐adrenergic, cannabinoid or 5‐HT1A receptors. Chronic treatment with an opioid agonist leads to sensitization of AC, so that withdrawal of the agonist and stimulation of AC results in cAMP overshoot. The cAMP overshoot produced by chronic DAMGO treatment was not enhanced when combined chronically with the delta agonist DPDPE, indicating that mu and delta receptors still share AC during chronic treatment. Since opioid and other receptors share AC, cAMP overshoot following chronic opioid treatment should be prevented by heterologous inhibition of cAMP using an agonist to a different Gi/o‐coupled receptor. Indeed, mu opioid overshoot was prevented when agonists to delta opioid, ORL‐1 and alpha 2‐adrenergic receptors were added during withdrawal. Alternatively, delta opioid overshoot was prevented by the mu agonist DAMGO. In contrast, chronic incubation with a mu or delta receptor agonist did not produce heterologous tolerance, confirming that the development of tolerance is a receptor specific effect. Supported by DA23339, GM07767, DA04087.
Functional Selectivity of 9‐OH‐risperidone (paliperidone) versus Risperidone at Dopamine D2 Receptors
Authors: Silva Michelle , Sanchez Teresa A , Berg Kelly A , Clarke William P
Published: June 2021

Functional selectivity (FS) is the term used to describe the ability of drugs to differentially activate signaling cascades coupled to a single receptor subtype. Importantly, differences in the FS profile between drugs acting at the same receptor subtype may underlie differences in therapeutic efficacy and/or adverse effect liability. Paliperidone (PAL) is the 9‐OH metabolite of risperidone (RIS). Both drugs are effective at treating symptoms of schizophrenia and have similar receptor binding profiles. We compared the FS profiles of PAL and RIS for cellular signaling responses at human D2 receptors expressed in CHO or HEK cells. Relative to the reference ligand butaclamol, the relative efficacy of PAL was greater, not different or less than that of RIS, depending upon the response measured. For example, as inverse agonists at the adenylyl cyclase pathway, the relative efficacy of PAL was greater then RIS. However, for reduction of constitutive desensitization, PAL equaled RIS and for increasing receptor density, the relative efficacy of PAL was less than that of RIS. Thus, a small difference in molecular structure can produce pronounced differences in the FS profile of drugs. Moreover, these results suggest that the presence of 9‐OH group can alter the functional response of RIS at human D2 receptors and suggest that the actions of PAL and RIS in vivo may differ as well.
The diacidic ExD motif controls angiotensin II type 2 receptor export from the endoplasmic reticulum
Authors: Zhang Xiaoping , Wu Guangyu
Published: June 2021

The physiological function of angiotensin II (Ang II) is mediated through the Ang II type 1 (AT1R) and type 2 receptor (AT2R), which belong to the superfamily of seven transmembrane cell surface receptors coupled to heterotrimeric G proteins. We have recently demonstrated that AT1R exit from the endoplasmic reticulum (ER) is directed by highly conserved single L residue in the first intracellular loop and F(x) 6LL motif in the C‐terminus. In contrast, the structural determinants for angiotensin II type 2 receptor (AT2R) export trafficking remain undefined. In this study, we have demonstrated that the AT2R mutant lacking the entire C‐terminus was unable to transport to the cell surface. Progressive deletion of the C‐terminus and alanine‐scanning mutagenesis identified two acidic residues, E357 and D359, in the C‐terminus, which are required for AT2R exit out of the ER and transport to the cell surface. Mutation of E357 to A or D, D359 to A or E, ExD to AxA, and ExD to DxE significantly attenuated AT2R transport from the ER to the cell surface in HEK293 and PC12 cells. The mutated AT2R tagged with either GFP or HA was extensively localized within the ER. In contrast, these mutations and deletions did not alter total AT2R expression. These data demonstrate that the diacidic ExD motif plays an important role in modulating export trafficking of newly synthesized AT2R from the ER to the cell surface (R01GM076167).
Attenuation of dynamin‐dependent internalization decreases antinociception during the expression of morphine tolerance
Authors: Macey Tara A. , Fyfe Leon W. , Morgan Michael M. , Ingram Susan L.
Published: June 2021

Mu‐opioid receptor (MOP) internalization has been proposed as a cellular mechanism of morphine tolerance. In order to test this hypothesis in vivo, the fluorescent MOP agonist, DERM‐A594, was microinjected into the periaqueductal gray (vlPAG). Rats were pretreated with either saline (0.4 μl) or morphine (5 μg/0.4 μl) 2X/day for 2 days. On day 3, rats were given either a saline or morphine challenge 30 min prior to DERM‐A594 (300 ng/0.4 μl). Intracellular DERM‐A594 intensity was similar in all groups except for a significant decrease in DERM‐A594 label in morphine tolerant rats given a morphine challenge (F (3, 34) = 3.899, p<0.05). These results suggest that repeated morphine administration increases morphine‐induced internalization of MOPs. To further test this idea, a myr‐dynamin inhibitory peptide (Dyn+) and a scrambled peptide (Dyn−) were microinjected into the vlPAG prior to microinjection of DERM‐A594. Dyn+ reduced DERM‐A594 antinociception compared to Dyn− (15 ± 3 s vs. 33 ± 5 s). Dyn+ also reduced morphine antinociception in both saline (D50 = 8.1 μg vs. Dyn− = 3.8 μg) and morphine pretreated rats (D50 = 10.7 μg vs. Dyn− = 5.8 μg). A comparable shift in D50 values in saline and morphine pretreated rats suggests that Dyn+ attenuates antinociception similarly in both groups. Thus, these studies demonstrate that internalization is important for MOP signaling in the vlPAG. Supported by DA023318(TM), DA015498(MM).