bluuebunny/crossref_metadata_2025_split huggingface dataset:
Anti‐Muscarinic Drug Effects on Murine Cognition: A Pilot Study
Authors: Theobald Robert , Blanchard Ashley , Morgan Cecil James 
Published: November 2023

The importance of cholinergic activity in proper memory function has been well documented with reported memory improvement in mice associated with increased hippocampal cholinergic activity and neurogenesis (Dobryakova, et al, 2014; Jeong, et al, 2014). In the TgCRND8 mouse Alzheimer's Disease (AD) model, Proulx, et al, (2015) demonstrated a disruption in corticothalamic pathways of the prefrontal cortex, suggesting that AD was associated with a loss of intact cholinergic function. Many drugs used as mild sedatives, such as diphenhydramine (DPH), possess significant anti‐muscarinic action, an action that can produce cognitive impairment in mice (Budzynska, et al, 2015). This study tested memory changes by use of a Barnes Maze (BM), a nonstress measure of the ability of mice to remember the location of an escape box. Naïve mice were divided into 5 cohorts (C) and trained using the BM. They received either no drug (Control, C‐1), or oral administration of GDW (Sham, C‐2), atropine (ATR, 0.5 mg/kg, C‐3), DPH, (0.5 mg/kg, C‐4) or DAU 5884 (DAU, 0.25 mg/kg, C‐5). After 3 months treatment was stopped in 50% of mice in each cohort, but testing continued.Results demonstrated a decrease in the ability of mice treated with ATR and DPH to find the escape box measured by increased time and/or distance travelled to escape. Only the ATR treated mice travelled a greater distance to the escape compartment, but both the ATR and the DPH treated mice took more time to find the escape compartment. There was a suggested recovery of memory when treatment was stopped, as seen in time and distance returning towards control. Results are compatible with previous studies with suggested clinical relevance to the choice of sedative agents with anti‐muscarinic effects likely to cause cognitive function decline, especially in the elderly. Additionally, the data supports previous studies indicating that use of M3 selective anti‐muscarinic agents for treatment in patients with overactive bladder to improve quality of life create minimal risk of cognitive decline.
Methamphetamine augments HIV‐1 Tat mediated memory deficits by altering the expression of synaptic proteins and neutrophic factors
Authors: Nookala Anantha Ram , Kumar Anil 
Published: November 2023

Methamphetamine (METH) abuse is common among individuals infected with HIV‐1 and has been shown to affect disease progression towards AIDS. These HIV‐1 infected individuals also exhibit greater neuronal injury and higher cognitive decline. Several HIV‐1 proteins, specifically gp120 and HIV‐1 Tat have been shown to affect neurocognition. HIV‐1 Tat, a viral protein released early during HIV‐1 replication has been implicated in HIV‐associated neurotoxicity mediated through production of pro‐inflammatory cytokines, reactive oxygen species and dysregulation of neuroplasticity, among many others in the central nervous system. However, combined effect of METH and HIV‐1 Tat on neurocognition and its potential effect on neuroplasticity mechanisms is not well documented. The present study was undertaken to determine the combined effect of METH and HIV‐1 Tat on behavior and neuroplasticity markers. Doxycycline (DOX)‐inducible HIV‐1 Tat (1–86) transgenic mice were administered 6mg/kg MA twice a day. Expression of Tat in various brain regions was confirmed by RT‐PCR. Behavior studies were performed in both males and female mice after 3 weeks of MA administration. We employed Y maze and Morris water maze to determine cognition in these mice. Working memory, indicated by number of spontaneous alterations, was decreased significantly in Tat mice that were administered METH. In the water maze task, there was a significant increase in escape latency in all the groups compared to control mice, and the memory deficit was larger in Tat transgenic mice that were administered MA. There is significant decrease in the protein expression of synapsin1, synaptophysin, arg3.1, PSD95 and BDNF in different brain regions. We also looked at the expression levels of a second marker of synaptodendritic integrity, Calmodulin kinase II (CaMKII) by immunoblotting. The results indicate that CaMKII is significantly decreased in HIV‐1 Tat mice that were treated with METH in both sexes. This study therefore provides novel insights into the interaction of HIV‐1 Tat and METH on the dysregulated expression of various plastic proteins in different brain regions.Support or Funding InformationThe work was supported by the grants DA025528, DA025011 and AA020806 from National institute of health (NIH).
Cutting the brakes to prevent Type 2 Diabetes onset: augmenting insulin secretion and conserving pancreatic beta‐cell mass by deleting Gαz
Authors: Wisinski Jaclyn , Reuter Austin , Kimple Michelle 
Published: November 2023

The hyperglycemia of Type 2 Diabetes (T2D) results from a combination of factors, including insulin resistance, impaired and inefficient insulin secretion, and destruction of pancreatic beta‐cells. The second messenger molecule, cyclic AMP (cAMP), amplifies glucose‐stimulated insulin secretion and has been suggested to preserve beta‐cell mass. Pathways that inhibit beta‐cell cAMP production are pathologically upregulated in T2D. One such pathway is mediated by Prostaglandin E2 (PGE2) receptor 3 (EP3), a G protein‐coupled receptor whose expression, as well as that of its natural ligand, PGE2, are both up‐regulated in islets collected from T2D mice and humans. While EP3 is ubiquitously expressed, in the beta‐cell it couples to a unique and non‐ubiquitously expressed G protein, Gz. We hypothesized the genetic elimination of the catalytic a‐subunit of Gz (Gαz) would augment beta‐cell function and mass, preventing or delaying the onset of hyperglycemia in the BTBR ob/ob mouse model of T2D. As expected, Gαz‐null ob/ob mice were as obese and as insulin resistant as wild‐type ob/ob mice, consistent with little to no liver, adipose, or muscle Gaz expression, but displayed near‐normal fasting blood glucose levels. Fasting euglycemia in Gaz‐null ob/ob mice correlated directly with elevated plasma insulin levels, suggesting that Gαz‐null ob/ob beta‐cells were able to better compensate for the increased insulin demand of obesity and insulin resistance. To test this directly, we isolated islets from Gaz‐null and wild‐type ob/ob mice, and found higher insulin content and insulin secretion from Gaz‐null ob/ob islets. To further elucidate the molecular mechanisms augmenting beta cell function in Gaz‐null ob/ob islets, we considered the influence of Gaz on glucagon‐like peptide‐1 (GLP‐1), which is a cAMP‐stimulatory gut/islet hormone. We postulated that, in the beta‐cell, Gaz signaling competes with GLP‐1 receptor signling to block the full effects of GLP‐1 on beta‐cell function and/or mass. To test this hypothesis, we subjected wild‐type and Gaz‐null ob/ob mice to intraperitoneal and oral glucose challenge, and found the Gaz‐null mutation partially restored glucose tolerance after an oral glucose challenge but not an intraperitoneal glucose challenge. Plasma levels of the GLP‐1 were not different between the genotypes, but intra‐islet GLP‐1 produced and secreted by alpha cells was altered in Gaz‐null ob/ob islets. Increased GLP‐1 responsiveness of Gaz‐null ob/ob islets was tested directly ex‐vivo by treatment with the stable GLP‐1 analog, exendin‐4. In sum, we identified a key role for Gαz in the islet pathophysiology of T2D and confirmed that deleting Gaz allowed for a more efficient response of the beta‐cell to a physiologic potentiator of insulin secretion and beta‐cell mass, GLP‐1.Support or Funding InformationMolecular and Applied Nutrition Training Program T32
Targeting and Delivery of Heterotrimeric G‐proteins to Primary Cilia
Authors: Edwards Clare , Lambert Nevin 
Published: November 2023

Primary cilia are fingerlike extensions of the plasma membrane important in cellular signaling and implicated in many disease processes. While much is known about localization and targeting of G‐protein coupled receptors (GPCRs) to cilia, little has been elucidated about the localization and targeting of heterotrimeric G‐proteins that reside downstream. This study aimed to determine if G‐proteins are targeted to the primary ciliary compartment and whether or not they possess the ability to move from the extaciliary membrane into this regulated space. Utilizing confocal microscopy, we imaged Gαi1‐Venus and SSTR3‐mTurq2 expression (a ciliary marker protein) in the primary cilia of NIH3t3 cells and quantified this expression as mean fluorescence in Imagej. Ciliary and membrane fluorescence were compared, and Fluorescence Recovery After Photo bleaching (FRAP) was completed to monitor movement of Gαi1‐V and SSTR3‐mTurq2. A student t‐test revealed that mean fluorescence of Gαi1‐V in the ciliary compartment (n=10, M=77.1, SD=45.8) is not significantly greater than extraciliary membrane fluorescence (n=10, M=75.4, SD=43.2); t(18)=0.08, p<0.05. Additionally, the difference between ciliary Gαi1‐V (n=10, M=77.1, SD=45.8) and membrane Gαi1‐V expression (n=10, M=75.4, SD=43.2) is significantly lower than the difference between expression of SSTR3‐mturq2 in the cilia (n=10, M=189.8, SD=71.5) and on the membrane (n=10, M=48.5, SD=30.1); t(18)=5.31, p<0.05. FRAP analysis of Gαi1‐V (n=6) returned a half recovery time of 12.8 ± 8.24 seconds and a mean mobile fraction of 0.60 ± 0.10, significantly higher than the mobile fraction of SSTR3‐mTurq2 (n=2, MF of 0.04 ± 0.014, t1/2 of 21.6 ± 15.3 seconds); t(6)=4.51, p<0.05. Our data indicate that Gαi1‐V expression is not significantly elevated in the primary cilia, and targeting of heterotrimeric G‐proteins to this space is unlikely. However, there is no evidence of exclusion of Gαi1‐V from the ciliary compartment as expression is visible and able to recover from photo bleaching in a time comparable to diffusion.Support or Funding InformationMedical Scholars Program at Medical College of Georgia.
Quantitative Tissue Distribution and Pharmacokinetics of IW‐1973, a Novel Clinical‐Stage sGC Stimulator, After Oral Administration in Rats
Authors: Banijamali Ali , Wakefield James , Barden Tim , Carvalho Andrew , Zimmer Daniel R , Currie Mark , Hall Katherine , Solon Eric , Masferrer Jaime , Ribadeneira Maria 
Published: November 2023

Soluble guanylate cyclase (sGC) catalyzes the conversion of guanosine‐5′‐triphosphate (GTP) to the secondary messenger cyclic guanosine‐3′,5′‐monophosphate (cGMP) upon binding of nitric oxide (NO). IW‐1973 is a sGC stimulator in clinical development that binds to sGC and enhances NO signaling resulting in an increase in cGMP production. Oral administration of IW‐1973 has been associated with elevations of cGMP‐sGC signaling in many different tissues including liver. Preclinical and clinical studies have shown that sGC stimulators relax smooth muscle, lower blood pressure, and have anti‐fibrotic effects. sGC stimulators with high distribution to tissues may have particular relevance for the treatment of vascular and fibrotic diseases of the lung, heart, liver, and kidney.The tissue distribution and pharmacokinetics (PK) of total radioactivity in male Long‐Evans rats was evaluated by quantitative whole‐body autoradiography (qWBA) following administration of a single oral (PO) dose of 3 mg/kg and 200 μCi/kg [14C]IW‐1973. Tmax was 1 h post‐dose and the t1/2 of total plasma radioactivity was 23.7 h. Analysis of tissues at various timepoints post‐dose revealed rapid and widespread distribution of [14C]IW‐1973‐derived radioactivity. Tissue concentrations in 19 of 44 tissues were observed to be higher than in plasma at all timepoints. Of particular interest were the high tissue to plasma AUCall ratios observed for liver (172), white adipose (28), brown adipose (15), kidney (5–7), heart (5), and lung (3); and the distinctly lower ratios observed in the bone (0.4) and pigmented eye lens (0.1). The preferential tissue distribution of IW‐1973 to organs such as liver and kidney may be a favorable property to treat not only vascular diseases but also those associated with extensive tissue inflammation and fibrosis.Support or Funding InformationIronwood Pharmaceuticals
atRA upregulates SHP expression through RXR activation
Authors: Won Kyoung‐Jae , Jeong Hyunyoung 
Published: November 2023

Retinoids (vitamin A) are used therapeutically as an anticancer agent or to treat dermatological diseases. Common side effects of retinoid include hyperlipidemia. Previous studies have shown that all‐trans retinoic acid (atRA; the bioactive retinoid) upregulates small heterodimer partner (SHP; a transcriptional repressor) and downregulates CYP7A1 (the rate‐limiting enzyme converting cholesterol to a bile acid intermediate), potentially responsible for the side effects. However, the detailed molecular mechanisms remain unclear. In this study, we investigated the role of retinoic acid receptor (RAR; the cognate nuclear receptor for atRA) and its binding partner, retinoid X receptor (RXR), in the transcriptional regulation of SHP and CYP7A1. In primary human hepatocytes and HepaRG cells, atRA increased SHP mRNA level by 12.8‐ and 6.6‐fold, respectively, while dramatically decreasing CYP7A1 mRNA level. In these cells, atRA also increased the levels of SHP pre‐mRNA, suggesting transcriptional activation of SHP by atRA. mRNA half‐life of SHP (determined after actinomycin D treatment in HepaRG cells and human hepatocytes) was not altered by atRA. In HepaRG cells, treatment with an RAR isoform‐specific agonist (i.e., BMS753, CD2314, and BMS961 for RARα, RARβ and RARγ, respectively) caused minimal changes in SHP expression. On the other hand, treatment with an RXR agonist (SR11237) led to increased SHP expression to an extent comparable to that by atRA (3.9‐ and 4.3‐fold, respectively), suggesting that SHP upregulation by atRA is mediated by RXR rather than RAR. mRNA expression of target genes of other permissive nuclear receptors that partner with RXR (i.e., CYP3A4, CYP2B6, and BSEP) was also increased in the cells treated by the RXR agonist to an extent similar to that by atRA. SHP knockdown led to significant de‐repression of CYP7A1 expression in atRA‐treated human hepatocytes (by 80% at 3 h), suggesting a key role of SHP in downregulation of CYP7A1 by atRA. Taken together, results from this study suggest an important role of RXR (rather than RAR) in mediating atRA action on the regulation of hepatic SHP and CYP7A1 expression, as well as expression of other target genes of permissive nuclear receptors that partner with RXR.Support or Funding InformationThis study was supported by NIH (R01 GM112746).
Impact on Drug Responses in Adult Life by Drug Treatment at the Neonatal Age
Authors: Piekos Stephanie , Chen Liming , Zhong Xiao‐bo 
Published: November 2023

The cytochrome P450 enzymes (P450s) are responsible for the metabolism of 75% of drugs currently on the market. However, significant interindividual variations in the enzyme activities of several P450s, including CYP3A4, CYP2B6, CYP2C9, and CYP2C19 exist in the general population. The differences in drug metabolism rates between people alter plasma drug exposure, and may account for the high incidences of adverse drug reactions or low therapeutic efficacy, which are difficult to predict. Identification of factors contributing to these variations is critical to develop strategies to better predict drug response. Several studies in animal models over the past 30 years have proven that adult basal P450 expression is permanently induced following neonatal exposure to the antiepileptic drug phenobarbital. Recently, our own study demonstrated that neonatal treatment with phenobarbital resulted in decreased therapeutic efficacy of omeprazole in proton‐pump inhibition to raise gastric pH in stomachs of adult mice. This decreased efficacy of omeprazole correlated with an upregulation of the P450s that mediate omeprazole metabolism, CYP3A11 and CYP2C29, in the liver of adult mice that received treatment with phenobarbital at the neonatal age. There is now a critical need to investigate whether neonatal treatment with different drugs produces the same permanent variations in P450 expression and metabolism of other drugs administered later in life. We hypothesize that phenytoin, another antiepileptic drug that induces P450s in mice and humans, will also cause permanent overexpression of P450 genes following neonatal exposure, which will affect drug responses in adulthood. Our current ongoing study involves administering phenytoin to neonatal mice, then evaluating expression of CYP3A11, CYP2B10, and CYP2C29, and the responses to omeprazole treatment in adulthood. Gastric pH is measured to evaluate the therapeutic efficacy of omeprazole, livers are harvested for analysis of P450 expression and activity, and blood is collected to measure plasma concentration of omeprazole and its metabolites. If phenytoin produces the same permanent upregulation of several P450s following neonatal exposure with a correlating decrease in omeprazole efficacy, then this phenomenon is not limited to phenobarbital and may be a common consequence of neonatal anti‐epileptic drug treatment. Neonates and infants are commonly administered drugs to treat various indications, such as seizures, and many of these compounds can induce P450s. This work may help prove that induction of P450s due to drug administration in early life can leave lasting alterations in the efficacy of other drugs metabolized by the P450s in later life. To better predict how patients will respond to a drug, a patient's drug history may serve as a factor for predicting variations of P450‐mediated drug metabolism.Support or Funding InformationNIH/NIGMS 1R01GM118367
In vitro metabolite profiling of sildenafil based on metabolomics approach
Authors: Kim Sunjoo , Jo Jun Hyun , Kwon Oh Kwang , Lee Sangkyu 
Published: November 2023

Metabolomics combined with high‐resolution mass spectrometry (HR‐MS) and multivariate data analysis has broad applications in the study of xenobiotic metabolism. Although xenobiotic metabolism is essential to understand toxicity, pharmacokinetic parameters, and excretion pathways, it cannot predict all generated metabolites in biological fluids. In the present study, we used a metabolomics approach to further understand sildenafil phase 1 metabolism in human liver microsomes. Twelve phase 1 metabolites were identified in human liver microsomes, of which, M1–M5 were previously known. The chemical structures of novel metabolites were elucidated by MS2 fragmentation using HR‐MS as follows: M6, reduced sildenafil, M7, N,N‐deethylation and mono‐oxidation; M8, demethanamine, N,N‐deethylation and mono‐hydroxylation; M9, demethanamine and N,N‐deethylation; M10 and M11, mono‐oxidation in the piperazine ring after N‐demethylation; and M12, mono‐oxidation. All metabolites except M1 were formed by CYP3A4 and 3A5. In conclusion, we successfully updated the metabolic pathway of sildenafil in the human liver, including seven novel metabolites, using metabolomics combined with HR‐MS and multivariate data analysis.Support or Funding InformationBK21 Plus KNU Multi‐Omics based Creative Drug Research Team
LXRa Inhibits Autophagy in Hepatocytes through ATG4B Inhibition
Authors: Kim Yun Seok , Nam Hyeon Joo , Kim Sang Geon 
Published: November 2023

Liver steatosis is the most common feature of metabolic syndrome, and may progress to severe disease states. LXRα promotes SREBP‐1c‐dependent lipogenesis and fat accumulation in hepatocytes. However, the effect of LXRα on autophagy in hepatocytes and the consequent changes in energy homeostasis are elusive. This study investigated the role of LXRα modulations in autophagy in association with its impact on the progression of liver steatosis. Treatment of HepG2 cells or primary hepatocytes with T0901317 (an LXRα agonist) decreased the processing of LC3‐I to LC3‐II in a time‐ or concentration‐dependent manner. The effect of LXRα on lysosomal activity from its effect on autophagosome formation was verified using immunofluorescence confocal assays. In primary hepatocytes, LXRα agonist treatment visualized decrease of autophagic flux. Consistently, either ligand activation LXRα or LXRα overexpression decreased Atg4B levels both in vitro and in vivo. Our results provide evidence that LXRα activation inhibits autophagy in hepatocytes as mediated by the inhibition of Atg4B, implying that modulation of LXRα may affect the capacity of autophagy in hepatocytes and thereby contribute to the prevention and treatment of liver steatosis.Support or Funding InformationSupported by the Korean government (NRF‐2015R1A2A1A10052663)
ATP‐Sensitive K+ Channels in Smooth Muscle Cells from Rat Mesenteric Lymph Vessels VESSELS
Authors: Garner Brittney R. , Stolarz Amanda J. , Fletcher Terry W. , Rusch Nancy J. 
Published: November 2023

The ATP‐sensitive potassium (KATP) channel is expressed in plasma membranes of many types of smooth muscle cells (SMCs). It is disinhibited when intracellular ATP concentrations are reduced during conditions of metabolic stress, and the resulting K+ efflux through KATP channels mediates membrane hyperpolarization and SMC relaxation. However, in the lymphatic smooth muscle cells (LSMCs) of rhythmically contracting lymphatic vessels, KATP channels reportedly play an important role in regulating the basal rhythmic contractions that establish lymph flow even in the absence of overt metabolic challenge. The KATP channels in LSMCs also may participate in the dilator responses of lymphatic vessels (LVs) to nitric oxide, inflammatory mediators, and compromise lymph flow in other pathologies associated with oxidative stress1,2,3. Although it is recognized that the properties and the subunit composition of KATP channels show tissue‐specificity, the identity and properties of KATP channels in LSMCs are unknown. The goal of this study was to begin to define the function and properties of KATP channels in rat mesenteric LVs. Isolated rat mesenteric LVs were cannulated on glass pipettes, pressurized at 4.5–6 mmHg, and edge detection was used to continuously monitor changes in vessel diameter. Addition of the KATP channel agonist cromakalim (0.01 μM‐100 μM; half‐log units) progressively increased LV diameter and abolished rhythmic contractions (n=3). In contrast, addition of the KATP channel antagonist, glibenclamide (0.01 μM‐100 μM; half‐log units) resulted in no change in LV diameter but suppressed rhythmic contractions. The latter finding confirmed the important role of KATP channels as regulators of LV contractile function under basal conditions. Subsequently, we have begun to characterize KATP channel currents in SMCs freshly isolated from rat mesenteric LVs. Patch‐clamp studies in the cell‐attached mode reveal unitary K+ currents in symmetrical 145 K+ recording solutions at negative patch potentials near the resting membrane potential of LSMCs. Unitary amplitudes calculated over a range of negative patch potentials from −80 mV to −30 mV indicate a single‐channel conductance of ~ 37 pS. Thus, we have tentatively identified the KATP channel in rat LSMCs as a first step toward defining it's regulation by endogenous substances and pharmacological interventions that may alter lymph flow.Support or Funding InformationSupported by T32 GM106999 (BRG) and R21 CA187325‐01A1 (NJR) from the National Institutes of Health.