bluuebunny/crossref_metadata_2025 huggingface dataset:
EGF Receptor Transactivation by α1‐Adrenoceptors on Prostatic Hyperplastic Cells
Authors: Silva Claudia Lucia M , Nascimento‐Viana Jessica B , Alcántara‐Hernández Rocío , García‐Sáinz J Adolfo , Noël François
Published: November 2023

Benign prostatic hyperplasia (BPH) is a progressive urological disorder related to an imbalance between cell proliferation and apoptosis. BPH is characterized by an increased number of epithelial, smooth muscle and stromal cells. Prostatic enlargement contributes to the lower urinary tract symptoms (LUTS/BPH). The participation of epidermal growth factor (EGF) receptor in G protein‐coupled receptor signaling is a process of transactivation critical for mitogenic activity in some models. Here we focused on the putative role of EGF receptor transactivation mediated by α1D‐adrenoceptors in stromal cell growth. Human BPH cells were treated with phenylephrine (3 μM) or vehicle (control) for either 15 min or 48h, and cell growth and protein expression were examined by Trypan Blue exclusion/MTT and Western blotting assays, respectively. Cell treatment with 3 μM phenylephrine (48h) induced a cell growth effect comparable to 0.1 μM EGF (166.3 ± 13.7 and 157.4 ± 14%, n = 5 experiments performed in quadruplicate, for phenylephrine and EGF, respectively, P < 0.001). The effect of phenylephrine was blocked by the α1D‐adrenoceptor antagonists BMY7378 (50 nM; 92.8 ± 4.7%, n = 5, P < 0.001), LDT3 (50 nM; 94.2 ± 8.2%, n = 5, P < 0.001) and LDT5 (50 nM; 92.6 ± 9.1%, n = 5, P < 0.001) suggesting that these receptors are involved in cell growth. Then we evaluated the downstream α1D‐adrenoceptor signaling. Phenylephrine (3 μM, 15 min) induced p‐ERK expression and the α1D‐adrenoceptor antagonists also blocked this effect (n = 4). Furthermore, the phenylephrine‐induced cell growth was blocked by the metalloproteinase inhibitor GM6001 (10 μM), the selective EGF receptor inhibitor AG1478 (5 μM) and MEK inhibitor PD98059 (1 μM). In conclusion, these results suggest that EGF receptor transactivation by α1D‐adrenoceptor is relevant for human stromal cell proliferation and may contribute to prostatic enlargement and LUTS/BPH.

Support or Funding Information

CNPq, Brazil [455436/2014‐2]
Development of a System for Realtime Assessment of β1‐Adrenergic Receptor Association with Epidermal Growth Factor Receptor
Authors: Guo Shuchi , Carter Rhonda , Tilley Douglas
Published: November 2023

β‐adrenergic receptors (βAR) are G‐protein coupled receptors (GPCR) highly expressed in the heart and essential for coordinating responses to neurohormonal stimulation. βAR stimulation leads to the activation of a number of important proximal signaling pathways. Gs protein‐, β‐arrestin‐ and epidermal growth factor receptor (EGFR)‐dependent signaling pathways each contribute to acute and chronic regulation of cardiac function and remodeling. In heart failure, βAR‐mediated transactivation of EGFR plays a protective role via enhanced cardiomyocyte survival, thus is an attractive pathway to target for potential therapeutic development. We have previously shown that β1AR and EGFR associate in a complex, even at endogenous levels of expression in the heart, and that disruption of this interaction leads to alterations in downstream signaling that may prevent pro‐survival effects. Indirect evidence has suggested that this association is differentially regulated by G protein‐dependent and ‐independent mechanisms, and we hypothesize that GPCR kinases (GRKs) are the proximal regulatory signal for this receptor complex. To begin test the differential roles of G protein‐versus GRK‐dependent regulation of β1AR‐EGFR association, we have developed a series of FRET assays with which to quantify in realtime their impact on cAMP generation, β‐arrestin recruitment, β1AR‐EGFR affinity and EGFR activation. We have applied U2S cells as our initial testing model with adenoviral infection of FRET reporters for each readout, and using isoproterenol (ISO) as a full agonist standard for activation of each pathway, we have successfully established each assay. Using this platform we continue to test the relative impact of G protein‐dependent versus –independent signaling on β1AR‐EGFR association.

Support or Funding Information

NIH HL105414
Ramipril could attenuate thioacetamide‐induced liver fibrosis in rats
Authors: Abdelghany Tarek Mohammed Mamdouh , Abdelsalam Rania Mohsen , Attia Amina Salem , Elsayed Mostafa Elsayed
Published: November 2023

Liver fibrosis results from chronic damage to the liver in conjunction with the accumulation of extracellular matrix proteins including collagen which is a characteristic of most types of chronic liver diseases. Thus, the present study aimed to investigate the possible prophylactic and therapeutic effects of an angiotensin converting enzyme inhibitor, ramipril, on liver fibrosis induced by thioacetamide (TAA) in rats. Rats (n=40) were classified into 4 groups (n=10), normal control, fibrotic control and two fibrotic‐treated in which ramipril 2.5 or 5mg/kg, p.o. was given daily for 6 weeks. Liver fibrosis was induced by TAA (200 mg/kg, ip) twice per week for 6 weeks in all groups except in the normal control group. At the end of the 6th week blood samples were withdrawn from orbital sinus for measurement of plasma AST & ALT and animals were sacrificed and then livers were separated & homogenized for measurement of oxidative stress biomarkers (MDA and reduced GSH), inflammatory markers (TNFα, IL10 and MPO) and a fibrotic marker (hydroxyproline). Furthermore, MMP‐13, TIMP‐1, α‐smooth muscle actin and collagen‐1 gene expressions were estimated using RT‐PCR and TGFβ was estimated using western‐plot method. In the fibrotic group plasma aminotransferase activity, MPO activity MDA, TNFα, Hydroxyproline, Collagen‐1, α smooth muscle actin, TIMP‐1 and TGFβ contents increased coupled by decrease in reduced GSH and MMP‐13 contents. Treatment with ramipril reversed most of the measured parameters. The protective effect of the tested agent could be attributed to its anti‐inflammatory, anti‐fibrotic & anti‐oxidant effects.
Protective effect of ONO‐1301, a long‐acting prostacyclin agonist, on lipopolysaccharide/D‐galactosamine‐induced liver injury
Authors: Misawa Hiroki , Ohashi Wakana , Shimada Yutaka , Hattori Yuichi
Published: November 2023

Prostacyclin serves as a protective, anti‐inflammatory mediator and the long‐acting prostacyclin agonist ONO‐1301 may be useful as an anti‐inflammatory agent. We examined whether two prostacyclin analogs, ONO‐1301 and beraprost, are beneficial in liver injury induced by lipopolysaccharide/D‐galactosamine (LPS/GalN) in mice. Mice were given an intraperitoneal injection of these analogs 1 h before LPS/GalN challenge. Both analogs significantly declined LPS/GalN‐induced increase in serum aminotransferase activity. However, ONO‐1301, but not beraprost, significantly inhibited hepatic gene expression of pro‐inflammatory cytokines, which were sharply elevated by LPS/GalN. The hepatoprotective effect of ONO‐1301, but not beraprost, was also supported by liver histopathological examinations. The mechanisms behind the benefit of ONO‐1301 in reducing LPS/GalN‐induced liver injury did not involve the regulation of liver protective factors, such as hepatocyte growth factor, since they were strikingly changed after LPS/GalN injection regardless of whether ONO‐1301 was treated. Our results indicate ONO‐1301 has the potential as a novel therapeutic modality for the treatment of acute liver injury.
Transgenic Mouse Development for Studying Human Serum Albumin as a Biomarker of Carcinogenic Exposure
Authors: Ding Xinxin , Sheng Jonathan , Wang Yi , Kluetzman Kerri , Zhang Qing‐Yu , Turesky Robert
Published: November 2023

Human albumin is a commonly used protein for studying human exposure to various pharmacologically and toxicologically important chemicals including therapeutic drugs and environmental pollutants. Very often the reactivity of albumin with xenobiotics is studied ex vivo with commercial human albumin or plasma/serum samples. Some studies have characterized the reactivity of albumin with chemicals in rodent models; however differences between the sequences of human and rodent serum albumins can result in formation of different adducts or peptide adducts with different sequences. Our goal is to create a human albumin transgenic mouse model that can be used to establish human protein biomarkers of hazardous xenobiotics for human risk assessment. We have developed a human albumin transgenic mouse model by pronuclear injection and its transgenic genotype and phenotype were characterized by genomic PCR, RT‐PCR, immunochemistry, and UPLC‐MS/MS methods. The transgenic human albumin protein was found to be a full‐length mature protein that coexisted with the endogenous mouse albumin in transgenic mouse serum. The transgenic human albumin protein formed ex vivo adducts with genotoxic metabolites of 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine, a procarcinogenic heterocyclic aromatic amine formed in cooked meat. Studies with human albumin transgenic mouse models can greatly advance our understanding of the reactivity of human albumin with hazardous chemicals in the environment and facilitate the development of analytical methods to measure albumin‐carcinogen adducts in humans.
The Use of Analogs of α‐MSH as Tanning Agents for the Prevention of Melanoma
Authors: Fearce Chelesa T , Swope Viki , Abdel‐Malek Zalfa
Published: November 2023

Melanoma, the deadliest form of skin cancer that originates from melanocytes, continues to increase at an annual rate of 4%. Exposure to solar ultraviolet radiation (UV) is causal for melanoma, especially in those with fair skin and poor tanning ability. α‐Melanocyte stimulating hormone (α‐MSH) plays a key role in regulating pigmentation by binding and activating the melanocortin 1 receptor (MC1R) that is expressed on the cell surface of melanocytes. Additionally, activation of the MC1R increases the DNA repair capacity of melanocytes, thereby reducing the DNA damaging effects of UV and mutations, the underlying cause of melanoma. Certain allelic variants of the MC1R gene are associated with red hair, fair skin, and poor tanning ability. This phenotype increases sensitivity to UV and the risk of melanoma. About 30% of the white population in America is heterozygous for one MC1R variant, which increases melanoma risk. The laboratory is developing a melanoma prevention strategy based on utilizing small peptide analogs of α‐MSH that activate the MC1R. Analogs of α‐MSH will be tested for their ability to protect from the deleterious effects of UV by reducing DNA damage, inflammation and sunburn, and apoptosis, and increasing pigmentation in melanocytes contained in cultured human skin substitutes. The tetrapeptides LK 184 at 10 nM, LK 467 at 100 nM, and the tripeptide LK 514 at 1 μM, were tested for their ability to stimulate pigmentation of engineered human skin substitutes (ESS) containing melanocytes. To determine the effects of LK 467, LK 184, and LK 514 on pigmentation, the ESS were treated for 3, 10 or 15 days daily with either peptide. The effects of the three analogs were quantified using Fontana Masson stain to assess their ability to stimulate pigmentation by synthesizing melanin. The analogs showed promising effects in their ability to induce synthesis of melanin, which induces pigmentation. These experiments helped in the progression of determining the efficacy of these analogs in photoprotection and advance their testing in a clinical trial.
Glucagon‐Like Peptide‐1 (GLP‐1) Receptor Expression on Human Umbilical Vein Endothelial Cells (HUVEC) in Response to Cellular Activation
Authors: Noormohamed Nadia , Kiel Dan
Published: November 2023

Purpose

The Glucagon‐Like Peptide‐1 (GLP‐1) receptor is the target for certain anti‐diabetic medications. The GLP‐1 receptor is a cell‐surface receptor that elicits a number of responses upon activation leading to reduced blood glucose and various other effects. A number of cell‐surface proteins have been shown to change expression level in response to various stimuli, such as nitric oxide and cytokines. Alterations in the level of cell‐surface receptors can increase or decrease responses through that receptor. The purpose of this study is to measure GLP‐1 receptor expression on human umbilical vein endothelial cells (HUVEC) in response to cellular activation by various compounds.

Methods

Nitric oxide and tumor necrosis factor alpha (TNF‐alpha) have been shown to induce changes in levels of cell‐surface receptors. Puerarin is an isoflavone found in a number of plants and herbs that stimulates endothelial nitric oxide synthase, leading to the generation of intracellular nitric oxide. Nitric oxide activates intracellular enzymes, such as guanylate cyclase, leading to the generation of second messengers. TNF‐alpha is a cytokine that increases transcription of various genes including those for cell surface adhesion molecules involved in the inflammatory process. HUVEC monolayers were treated with puerarin, TNF‐alpha, or vehicle. After 24 hours of treatment, cells were harvested. Equivalent amounts of cellular homogenates were separated via electrophoresis, and proteins transferred to a membrane. The membrane was probed with an antibody directed against the GLP‐1 receptor. Visualization of the band corresponding to the GLP‐1 receptor was achieved using chemiluminescence, and quantified using densitometry. Neither of the treatments produced large changes in GLP‐1 receptor expression. GLP‐1 receptor expression on HUVEC may be less responsive to cellular stimulation compared to other proteins on HUVEC.
Integrated Yeast‐mammalian Assay System for Screening of Novel Peptides Activating Glucagon‐like Peptide‐1 Receptor
Authors: Kuroda Kouichi , Shigemori Tomohiro , Ueda Mitsuyoshi
Published: November 2023

Glucagon‐like peptide‐1 (GLP1) is a peptide hormone that is produced in intestinal L‐cells and activates GLP‐1 receptor (GLP1R), a class B G‐protein coupled receptors (GPCRs). GLP1R activation promotes insulin secretion in pancreatic β‐cells. Search of agonists activating GLP1R has been considered to be a promising approach for treating diabetes. To screen peptide agonists acting on GPCRs from peptide library, phage display system has been mainly employed. However, screening is performed based on only the binding ability to receptors, and thus it is necessary to conduct other assays for judging whether the screened peptides are biologically active or not. We have constructed a novel functional screening system that integrates a peptide secretion by yeast cells into a functional detection system by mammalian cells producing GLP1R [1]. This screening system does not require chemical synthesis, purification, and condensation of target peptides. GLP1R‐producing mammalian cells were improved by introducing inducible secretory luciferase as a reporter of signaling activation[2]. Using the constructed yeast‐mammalian assay system for functional screening, novel GLP1R agonists were found from a randomly mutagenized GLP1 library. Our yeast‐mammalian assay system can be applied to direct discovery of novel active peptides against other GPCRs.
LCT Gene Variant ‐13910 C>T, Dairy Intake, and 25‐Hydroxyvitamin D Plasma Levels: A Mendelian Randomization Study
Authors: Alharbi Ohood , El‐Sohemy Ahmed
Published: November 2023

This abstract is from the Experimental Biology 2016 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Background

The LCT ‐13910 C>T gene variant is associated with lactose intolerance (LI) in different ethnic groups. Individuals with LI often limit or avoid dairy consumption, a main dietary source of vitamin D in North America, which may lead to inadequate vitamin D intake. However, the association between LCT genotype and circulating 25(OH)‐vitamin D levels is not clear. The objective of this study was to determine the prevalence of genotypes predictive of LI in different ethnic groups living in Canada and to determine whether LCT genotype is associated with dairy intake and 25(OH)D plasma levels.

Methods

Fasting blood samples were drawn from a total of 1,400 participants aged 20–29 years from the Toronto Nutrigenomics and Health Study for genotyping and plasma 25(OH)D analysis. Dairy intake was assessed using a one‐month, 196‐item Toronto‐modified Willett food frequency questionnaire. The prevalence of the LI gene variant LCT ‐13910C>T was compared using. Using the Mendelian randomization (MR) approach, we used analysis of covariance to determine the association between LCT genotypes and dairy intake, as well as 25(OH)D levels.

Results

Approximately 33% of Caucasians, 99% of East Asians, 74% of South Asians, and 59% of those with other or mixed ethnicities had the CC genotype predictive of LI. The CC genotype was significantly associated with lower total dairy intake (p=0.001), lower skim milk intake (p=0.002), and lower plasma 25(OH)D (p=0.0004). The CC and the CT genotypes were associated with a two‐fold, and 50% greater risk, respectively, of suboptimal plasma 25(OH)D.

Conclusion

The CC genotype predictive of LI is associated with lower plasma 25(OH)D, which is due, at least in part, to lower intake of dairy products, particularly skim milk. Increased risk of suboptimal levels of vitamin D was also observed among those with the CT genotype suggesting an intermediate effect of heterozygotes.

Support or Funding Information: Research support from the Advanced Foods and Materials Network
Association Between LCT ‐13910 C>T Genotype and Plasma 25‐Hydroxyvitamin D with Premenstrual Symptoms
Authors: Jarosz Alicia Caroline , Alharbi Ohood , El‐Sohemy Ahmed
Published: November 2023

Background

Vitamin D has been associated with premenstrual symptoms, but the findings have been inconclusive. Dairy is a good dietary source of vitamin D, but individuals with lactose intolerance consume less dairy and have lower plasma 25(OH)‐vitamin D levels. The LCT ‐ 13910 C>T gene variant is a strong predictor of lactose intolerance where those who have the CC genotype limit their intake of dairy. The objective of this study was to determine the association between 25(OH)‐vitamin D status and LCT genotype on premenstrual symptoms.

Methods

964 women aged 20–29 years participating in the Toronto Nutrigenomics and Health Study provided data on their premenstrual symptoms and fasting blood samples were taken for genotyping and plasma 25(OH) D analysis. Optimal, sufficient, insufficient, and deficient vitamin D status was defined as ≥75, 50–74.9, 30–49.9, and <30 nmol/L, respectively. Logistic regressions were used to calculate the odds ratio (OR) and 95% confidence interval (CI) for the associations between vitamin D status and premenstrual symptoms. A Mendelian Randomization approach was used in the analysis of covariance to determine the association between LCT genotype and premenstrual symptoms.

Results

The odds ratio (95% CI) for experiencing clumsiness was 2.63 (1.32–5.21) for subjects with deficient plasma 25(OH)‐vitamin D status compared to those with optimal status. The corresponding odds ratios (95% CIs) for those experiencing confusion and with deficient or insufficient plasma 25(OH)‐vitamin D were 2.57 (1.42–4.67) and 1.82 (1.09–3.05), respectively, compared to those with optimal status. Plasma 25(OH)‐vitamin D status was not associated with experiencing any of the other premenstrual symptoms (acne, bloating, cramps, mood swings, increased appetite, fatigue, headache, anxiety, sexual desire insomnia, nausea, depression, or desire to be alone). The odds ratios (95% CIs) for experiencing clumsiness and cramps were 2.12 (1.08–4.17) and 0.55 (0.33–0.92), respectively, for individuals with the LCT CC genotype compared to those with the TT genotype. LCT genotype was not significantly associated with any of the other symptoms.

Conclusion

Our findings indicate that plasma 25‐Hydroxyvitamin D status and LCT genotype are associated with certain premenstrual symptoms, suggesting that dairy might be associated with both an increased and decreased risk of certain premenstrual symptoms.

Support or Funding Information

Research support: A.C.J. is a recipient of a Natural Sciences and Engineering Research Council Graduate Scholarship
Reversal of Stem Cell Mobilopathy and Enhanced Vascular Repair by Angiotensin‐(1‐7) in Diabetes
Authors: Vasam Goutham , Jarajapu Yagna
Published: November 2023

Bone marrow stem/progenitor cells (BMSPCs) are mobilized in response to ischemic injury. BMSPCs accelerate vascular repair by re‐endothelialization and revascularization of ischemic areas. Long‐term diabetes causes impairment of mobilization, a.k.a. stem cell mobilopathy, which has now been considered as a major contributing factor for the development of macro and microvascular complications and end‐organ damage. This study evaluated the beneficial effects of Angiotensin (Ang)‐(1‐7), a member of the protective axis of renin angiotensin system, in diabetic stem cell mobilopathy and ischemic vascular repair. Control or streptozotocin (STZ)‐induced type 1 diabetic mice, and type 2 diabetic db/db mice and their lean‐control mice were administered with Ang‐(1‐7) (1 μg/kg/min) or normal saline using subcutaneous osmotic pumps for four weeks. Circulating BMSPCs were enumerated before and after the treatment by flow cytometric enumeration of lineage‐negative (Lin−), Sca‐1 and c‐Kit (LSK) or by Colony Forming Unit (CFU) assay. Hind‐limb ischemia (HLI) was induced by femoral artery ligation; mobilized BMSPCs and blood flow recovery were determined over a period of 10 days post‐HLI by using Laser Doppler blood flow imaging system. Vascular repair was assessed by quantification of capillary density in the ischemic skeletal muscle by immunohistochemistry. The number of circulating BMSPCs are decreased (P<0.01, n=8) in STZ‐ and db/db mice compared to their respective controls. Circulating BMSPCs were increased in control mice in response to ischemic injury, that showed peak response at day‐2 following the injury and returned to normal by day 10 (n=6–8). Blood flow was recovered to 90% of contralateral non‐ischemic limb by day 10. Ischemia‐induced mobilization of BMSPCs was impaired in both models in response to ischemic injury (P<0.001 vs respective controls, n=6–8). This dysfunction was associated with delayed and decreased blood flow recovery (P<0.001, n=6–8), and impaired neovascularization (P<0.001, n=6–8) of ischemic areas. Ang‐(1‐7) treatment increased/normalized the circulating LSK cells, and restored their mobilization following ischemic injury in STZ‐ and db/db mice (n=6–8). Blood flow recovery was accelerated and normalized with enhanced neovascularization of ischemic areas in both models of diabetes by Ang‐(1‐7) compared to the untreated (n=6–8). To our surprise, this protective response was associated with increased Rho‐kinase activity, specifically in the BMSPCs but not in the peripheral cardiovascular tissues (n=5). This study provides compelling evidence for the reversal of diabetic stem cell mobilopathy, and enhanced ischemic vascular repair in diabetes by Ang‐(1‐7) treatment.

Support or Funding Information

This work is partly supported by American Heart Association, and the Core Biology Facility at North Dakota State University that was made possible by NIH‐NIGMS (P30 GM103332‐01).
Toxicological Assessment of Selected Popular Antidiabetic Drugs in Diabetics within Ota, Ogun State, Nigeria
Authors: Ogunlana Olubanke Olujoke , Oyebanji Olufisayo Grace , Ogunlana Oluseyi Ebenezer , Adekeye Bosede Temitope , Adeyemi Alaba Oladipupo , Dokunmu Titilope M. , Iyanda‐Joel Wisdom O , Rotimi Oluwakemi Anuoluwapo , Rotimi Solomon Oladapo , Okwumabua Ogi Emeke
Published: November 2023

The complications associated with diabetes and the new trend of using combination therapy in the management of the disease gave birth to this work, aimed at assessing the hepatotoxic and nephrotoxic effects of selected popularly used antidiabetic medications in type 2 diabetic patients within Ota, Ogun State, Nigeria. The participants, diabetic (n=195) and non‐diabetic (n=30) were divided into the following groups based on their medications: 1 (Non Diabetic control), 2 (Metformin), 3 (Glimepiride), 4 (Glibenclamide), 5 (Metformin and Glimepiride), 6 (Meformin and Glibenclamide), 7 (Metformin, Glimepiride and Glibenclamide) and 8 (Diabetic Dietary control). Serum protein expression profiling, liver and kidney function parameters were assessed in participant's blood. Glyceamic control within the diabetic groups was 29.23%. Urea concentration was significantly increased (p < 0.05) in groups 5 and 7 compared with groups 1 and 8 while the serum creatinine levels in the different groups showed no significant difference. Activities of alkaline phosphatase and aspartate aminotransferase increased significantly (p < 0.05) in group 5 compared with groups 1 and 8. Three low molecular weight proteins likely to be cystatin C, leptin and prophyllin (molecular weights 13, 15 and 18 kDa respectively) were overexpressed in all the diabetic groups. The control of diabetes may best be carried out with dietary control and lifestyle modification as well as good therapeutic drug monitoring for safe assessment of baseline organ function.
Salivary Peptides Regulate Intracellular Calcium in Rat Pancreatic Beta Cells
Authors: Edwards Lincoln P , Fakhouri Walid D , Ardestani Pegah Memar , Zarezadeh Hossein
Published: November 2023

Objectives

Proline Rich Proteins (PRPs), secreted by parotid gland, have a variety of functions including anti‐caries and anti‐bacterial properties, but less is known about their actions outside the oral cavity. One of these proteins, called Parotid Hormone (PH) in Sus scrofa, is believed to play a significant role in the maintenance of healthy dentin and have effects on other organs including pancreas by having regulatory effects on maintaining the sensitivity of beta cells to circulating glucose. Basic Salivary Proline Rich Protein 2 and 3 (PRB2 & PRB3), in human, have the highest sequence homology with porcine Parotid Hormone. Changes in cytosolic calcium ion concentration constitute an important element of signal transduction that may lead to insulin release. We hypothesized that PH, PRB2 and PRB3 are involved in insulin stimulation by regulating the intracellular calcium ion levels in pancreatic beta cells.

Methods

Cultured RIN‐m beta cells from Rattus Norvegicus were used as an in vitro model for measuring intra‐cellular calcium levels using the fluorescent dye Fura2‐AM. Cells in calcium‐rich or calcium‐free medium were treated with PH (34nmoles/200 μL), PRB2 (11nmoles/200μL) or PRB3 (11nmoles/200μL). In some experiments cells were pretreated with the L‐type calcium channel blocker diltiazem (10μM) or with thapsigargin (2μM) to deplete internal calcium stores, before treatment with the peptides.

Results

PH, PRB2 and PRB3 increased intracellular calcium levels in cultured beta cells by 3‐, 10‐ and 3‐fold respectively. In calcium‐free medium, no change was observed in intracellular calcium level after treatment with PH or PRB2; however treatment with PRB3 caused an increase in intracellular calcium level. Interestingly, calcium uptake by cells following PH and PRB2 treatment was partially blocked by diltiazem (10μM), whereas thapsigargin (2μM) treatment blocked the response caused by PRB3.

Conclusion

Salivary peptides PH, PRB2 and PRB3 show differential regulation of intracellular calcium in RIN‐m beta cells. Our data indicates that PH and PRB2 regulate an influx of external calcium partially through L‐type calcium channels, while PRB3 potentially regulates an increase of calcium from internal stores. In summary, our results suggest that PH, PRB2 and PRB3 may have regulatory functions beyond those that have been described in the literature, and may play a role in a parotid‐pancreatic axis.

Support or Funding Information

Departmental funding from the Department of Diagnostic and Biomedical Sciences, University of Texas Health Science Center at Houston, School of Dentistry.
α4‐selective GABAA Receptor Positive Allosteric Modulator Attenuates The “Asthma‐Like Phenotype” In A Murine Asthma Model
Authors: FORKUO GLORIA , Nieman Amanda , Yu Olivia , Guthrie Margaret , Yuan Nina , Kodali Revathi , Jahan Rajwana , Stephen Michael , Poe Michael , Hartzler Benjamin , Emala Charles , Cook James , Stafford Douglas , Arnold Leggy
Published: November 2023

Asthma is a chronic inflammatory disease of the airways characterized by inflammation, mucous metaplasia and airway hyperresponsiveness. The chronic use of current treatment options is associated with adverse side effects, which warrants the development of new asthma therapies. An emerging new target to treat asthma is the GABAA receptor, which is a heteropentameric ligand‐gated chloride channel consisting of combinations of 19 different subunits (α1–6, β1–3, γ1–3, δ, ɛ, π, θ, ρ1–3). Exhaustively studied in the brain, GABAA receptors have lately been linked to inflammation, mucous production and airway hyperresponsiveness. We hypothesize that compounds with specificity towards a particular GABAA receptor assembly act tissue‐specifically and exhibit a well defined pharmacological profile. In this study, we determined the expression of GABAA receptor subunits in mouse lungs and the effects of a novel α4 subtype‐selective GABAAR modulator, XHE‐III‐74EE and its metabolite XHE‐III‐74A (has α4 selectivity and weak α5 selectivity) in an ovalbumin sensitized and challenged (Ova S/C) murine model of asthma. We observed that, chronic treatment with XHE‐III‐74EE but not XHE‐III‐74A significantly reduced airway hyperresponsiveness in Ova S/C mice. In addition, acute treatment with XHE‐III‐74A but not XHE‐III‐74EE decreased airway eosinophilia. Our results support the hypothesis that airway hyper‐responsiveness and inflammation can be targeted with α4/α5 subtype‐selective GABAAR modulators.

Support or Funding Information

National Institute of Neurological Disorders and Stroke [R01NS076517], National Institute of Mental Health [R01MH096463], National Heart, Lung and Blood Institute [R01HL118561], University of Wisconsin Milwaukee, UWM Research Foundation (Catalyst Grant), UWM Department of Chemistry and Biochemistry, and UWM Graduate School (RGI Grant), Milwaukee Institute for Drug Discovery
Therapeutic Potentials of Small‐Molecule Inhibitors Targeting Exchange Protein Activated by cAMP (EPAC)
Authors: Cheng Xiaodong , Zhu Yingmin , Mei Fang
Published: November 2023

The pleotropic second messenger cAMP is an important stress signal that regulates a multitude of physiological functions under normal and diseased conditions. The major cellular effects of cAMP are transduced by two ubiquitously expressed intracellular cAMP receptors, protein kinase A (PKA) and exchange protein directly activated by cAMP (EPAC) in human. Using genetically engineered EPAC knockout mouse models, we have demonstrated that EPAC proteins play important roles in the development of major human diseases, including obesity, cancer and chronic pain etc. Furthermore, we have developed first‐in‐class EPAC specific inhibitors with favorable in vivo pharmacological and toxicological profiles. Applications of EPAC inhibitors in various animal disease models recapitulate the genetic phenotypes of EPAC knockout mice. These Studies validate the therapeutic potentials of small molecule EPAC specific inhibitors.

Support or Funding Information

Research is supported by grants from the National Institute Health (GM061770, GM106218 and AI111464).
Development and therapeutic potential of NOSH‐NSAIDs: A new class of anti‐inflammatory pharmaceuticals
Authors: Kashfi Khosrow
Published: November 2023

Non‐steroidal anti‐inflammatory drugs (NSAIDs) are mainly used to treat pain, reduce fever and inflammation; some also have utility as anti‐platelet agents. There is also a large body of data supporting the notion that NSAIDs are important for cancer prevention. However, their long‐term use is problematic on two accounts: (1) suboptimal efficacy, which is less than 50% and (2) side effects including gastrointestinal, cardiovascular, and renal, which precludes their widespread use. In our search for a “better NSAID”, we developed NOSH‐NSAIDs. These are regular NSAIDs such as aspirin, naproxen, sulindac and others to which nitric oxide (NO) and hydrogen sulfide (H2S) releasing moieties have been covalently attached. Both NO and H2S are gaseous signaling molecules of biological importance. The rational for incorporating these gaseous mediators into NSAID molecules was based on the observations that NO and H2S have some of the same properties as prostaglandins within the gastric mucosa, thus modulating some components of the local mucosal defense systems which should lead to reduced gastrointestinal toxicity and also should embark enhanced cardiovascular and renal safety profiles because NO and H2S have protective roles in the cardiovascular and renal systems. To date we have reported on the synthesis and some general characterizations of three such compounds. NOSH‐aspirin (NBS‐1120), NOSH‐naproxen (AVT‐219), and NOSH‐sulindac (AVT‐18A) have enhanced GI profiles yet they retain all the basic pharmacological activities of their respective native NSAID. Furthermore, we have also reported that these compounds are orders of magnitude more potent than their corresponding NSAID in inhibiting the growth of adenomatous, epithelial, and lymphocytic cancer cell lines, thus demonstrating a generalized property that was tissue‐type independent. In vivo, NBS‐1120 was shown to have chemo‐preventive properties that were superior to aspirin and AVT‐219 was shown to have chemotherapeutic properties. Of interest is also the observation that NBS‐1120 showed synergistic activity with 5‐flurouracil, an established chemotherapeutic agent, in inhibiting colon cancer cell growth. Encouraged by our initial observations, we have formed a company, Avicenna Pharmaceuticals, Inc., around this technology and have forged a path towards an IND application and phase‐1 clinical trials, hopefully to bring these compounds into the clinic.
Antihiperglycemic Effect of 5‐HT2 Receptor Stimulation Induced by indorenate, a serotonin analog
Authors: Hong Enrique , de Lourdes Medina Juana María , Silva‐Arzave Abigail
Published: November 2023

The role of 5‐Hydroxytryptamine in the regulation of glycemia has been a controversial item for some time, however, there are recent findings suggesting that 5‐HT1A receptor stimulation increases the glycemia, while 5‐HT2 receptors do the opposite. Therefore, we decided to investigate the effect of indorenate, a central antihypertensive agent exerting this action by 5‐HT1A central receptor stimulation, but also possessing a 5‐HT2 peripheral receptor stimulation. Since less than 1 % of the oral administered dose penetrates into the CNS, most of the compound could stimulate the 5‐HT2 peripheral receptors leading to an antihyperglycemic effect. The oral dose of glucose (2g/kg) to fasted Wistar rats was followed by a hyperglycemia that was maximal one hr later, this effect was suppressed by the previous administration of an oral dose of 10 mg/kg of indorenate, however, metformin at an oral dose of 300 mg/kg only produce a slight, but significant decrease in hyperglycemia. Pelanserin, a 5‐HT2 antagonist, blocked the effect of indorenate, but WAY 100635, a 5HT‐1A agonist, did not. Indorenate also increase the glucose velocity needed to maintain blood glucose levels constant in an euglycemic insulin clamp in anesthetized Wistar rats. The present data suggest that indorenate may have a potential therapeutic usefulness in both, metabolic syndrome and in type2 diabetes.

Support or Funding Information

Authors are indebt to the SECITIDF for its support.
The Role of O‐GlcNAcylation in Regulating Mitochondrial Function in Cardiomyocytes
Authors: Wright JaLessa Nicole , Kramer Philip , Benavides Gloria , Johnson Michelle S. , Zhang Jianhua , Darley‐Usmar Victor , Chatham John
Published: November 2023

The post‐translational modification of proteins by O‐linked‐N‐acetylglucosamine (O‐GlcNAc) is elevated in diabetes, and has been implicated in the adverse effects of diabetes on the heart including impaired mitochondrial function. Several studies have reported that the adverse effects of hyperglycemia on mitochondrial function are mediated via O‐GlcNAcylation of specific mitochondrial proteins such as NDUFA9 of complex I, complex III subunits core 1 and 2, as well as mitochondrial DNA‐encoded subunit I of complex IV. Moreover, a recent O‐GlcNAc profiling study revealed that numerous mitochondrial proteins were O‐GlcNAcylated, many of which are associated with oxidative phosphorylation i.e., NDUFS1 and ATP5A1. Conversely, it has also been reported that increasing O‐GlcNAc levels with thiamet‐G, a highly selective inhibitor for OGA, which catalyzes the removal of O‐GlcNAc from proteins, had no effect on mitochondrial function. Therefore the goal of this study was to better understand the effects of short‐term increases in O‐GlcNAcylation on mitochondrial function. AC16 cells, an immortalized human cardiac cell line, were treated with thiamet‐G (0.1, 1, 3 and 5 μM) to increase O‐GlcNAc for 1, 2, 4 and 6 h, followed by quantitative assessment of mitochondria bioenergetics using the Seahorse Extracellular Flux Analyzer. In a second series of experiments a 2 h recovery period, following thiamet‐G was introduced prior to measurement of mitochondrial bioenergetics. We found that a 2 h treatment with 3 μM thiamet‐G significantly increased maximal oxygen consumption rates (OCR), from a rate of 14 to 20 pmol/min/μg protein regardless of the duration of treatment. Surprisingly, after a 2 h recovery period there was an approximately 2‐fold decrease in maximal OCR and reserve capacity in untreated cells, while 3μM thiamet‐G doubled OCR to an average of 15 pmol/min/μg protein. In addition to increasing OCR; 3μM thiamet‐G also increased extracellular acidification (ECAR) rates. Thus, augmentation of O‐GlcNAcylation in AC16 cells with thiamet‐G stimulates both OCR and ECAR consistent with increased oxidative and glycolytic metabolism. Interestingly however, at 5μM thiamet‐G both OCR and ECAR were reduced compared to 3μM. In conclusion increasing O‐GlcNAc via thiamet‐G clearly influences both mitochondrial and glycolytic metabolism, suggesting that there is a subtle interaction between O‐GlcNAcylation and cellular metabolism. Future studies will investigate the mechanism(s) underlying the metabolic switch induced by increasing O‐GlcNAc levels as well as determine how this contributes to the response of mitochondria to cellular stress.

I would like to thank the members of the Chatham and Darley‐Usmar labs for their assistance and support with this project.

Support or Funding Information

This work is supported by NIH grant 5T32HL007918‐17, HL101192 and UAB School of Medicine Multi‐Investigator Pilot Grant
CaMKIId As a Nodal Mediator of Insulin Signaling and Adrenergic Signaling in Cardiomyocytes – a Signaling Network Map Profiling by Quantitative Phospho‐proteomics
Authors: Dewan Sukriti , Campos Alex , Wu Shuangding , Stowe Jennifer , Wolf Alex , Bers Donald M , Brill Laurence M , Brown Joan H , McCulloch Andrew
Published: November 2023

Background

Calcium and calmodulin mediated protein kinase II (CaMKIId) plays a key role in regulating cardiac physiology. Upregulated expression and activation of CaMKIId is reported in HF. While cardiac specific over‐expression of CaMKIIdc isoform leads to progressive HF in mice, cardiac specific knockout (KO) of CaMKIId isoform develop hypertrophy but do not progress into end‐stage HF. To elucidate the exact role of CaMKIId as a cardioprotective target, it is pertinent to investigate the signaling mechanisms altered by CaMKIId targets and study the pleiotropic effects of these interactions.

Aim

The purpose of this study is to identify novel CaMKIId signaling targets and interactions thereof, using a sensitive, wide‐range and quantitative, mass spectrometry‐based phosphoproteomics approach in acutely stressed isolated cardiomyocytes.

Methods

To identify putative downstream CaMKIId targets, mice were divided into two groups ‐ CaMKIId KO and control group. Adult cardiomyocytes were freshly isolated by the Langendorff method. A total of 3 biological replicates (pooled from 12 animals) were used per genotype for the present study. Isolated cardiomyocytes were treated with 10uM of isoproterenol for 5 minutes to induce CaMKIId activation via beta‐adrenergic stimulation. Enriched phosphopeptides from CaMKIId KO and control mice samples were differentially quantified high‐resolution liquid chromatography mass spectrometry (LC‐MS/MS). All mass spectra from Phospho‐ and Total samples were analyzed with MaxQuant software. For pathway analysis, proteins with differentially regulated phospho‐sites were keyed in ClueGo (Cytoscape plug‐in) and Ingenuity Pathway Analysis (IPA); and the resulting enriched signaling pathways were functionally grouped by parsing through KEGG, Reactome and Wiki Signaling Pathway databases. These functional signaling groups were then visualized as a comprehensive interconnected network. An interaction map of all enriched signaling pathways with differentially regulated phospho‐proteins was generated. Flourimetric glucose uptake assays and western blots were used to quantify insulin resistance under stress conditions and mechanisms thereof.

Results

Key network hubs and pathways that were predicted to be involved in CaMKIId signaling are cardiac contraction, cAMP signaling, beta‐adrenergic signaling and insulin signaling. Specifically we were able to identify Insulin signaling pathway as a novel pathway being regulated by CaMKIId inhibition.

Conclusion

CaMKIId is a key mediator of contractility, electrophysiology, calcium handling and insulin signaling within cardiomyocytes.

Support or Funding Information

NIH grant 1R01HL105242
Failing Hearts are More Sensitive to Sympathetic, But Less Sensitive to Vagal Induced Atrial Fibrillation in Rats
Authors: Delfiner Matthew S. , Li Ying , Gerdes A. Martin , Zhang Youhua
Published: November 2023

Introduction

Both vagal (VS) and sympathetic (SS) stimulation have been shown to increase atrial fibrillation (AF) inducibility, with VS being more arrhythmogenic in normal hearts. Heart failure (HF) results in autonomic dysfunction, characterized by sympathetic activation and vagal withdrawal, and is associated with an increased incidence of AF. However, it is unknown whether failing hearts respond differently to autonomic stimulation in AF inducibility.

Methods

Myocardial infarction was produced by left anterior coronary artery ligation in 9 rats, with 10 sham‐operated animals serving as controls. Infarcted rats developed HF 2 months after surgery as determined by echocardiography and hemodynamic measurements. AF inducibility was tested under anesthesia at terminal experiments by burst pacing (200 impulses at 50 Hz) in 5 consecutive episodes. The occurrence and duration of spontaneous AF after burst pacing was determined in each episode. AF inducibility was evaluated at baseline, during right cervical vagus nerve stimulation (30 Hz, 0.1 ms duration and 1 mA), and during SS with isoproterenol infusion (0.1ug/kg/min).

Results

See figure. Compared to controls, HF increased AF inducibility. Both the incidence and duration (not shown) were increased at baseline. AF inducibility was augmented in both groups by VS. However, the magnitude of increase was less in the HF group (increased 49% in HF vs 80% in controls). AF inducibility was significantly increased in HF hearts under SS, compared with sham‐controls.

Conclusions

Autonomic stimulation can increase AF inducibility in both HF and control animals. Compared to controls, failing hearts are less sensitive to VS, but more sensitive to SS. The increased sensitivity of failing hearts to SS may explain why “sympathetic AF” is typically seen in patients with structural heart disease and “vagal AF” is usually seen in normal hearts.
Myocardial Electrotonic Coupling Modulates Repolarization Heterogeneities in vivo: Implications for the Assessment of Pro‐Arrhythmic Liabilities in vitro and in silico
Authors: del Rio Carlos L , Hamlin Robert L , Billman George E
Published: November 2023

Passive electrical properties determining electrotonic cell‐to‐cell coupling in the myocardium can modulate repolarization disturbances/heterogeneities; in short, poorly‐coupled cells exert limited electrotonic influence(s) over their neighbors (i.e., lower space constant) facilitating induced/acquired heterogeneities, and therefore, pro‐arrhythmic potential/behaviors. This study investigated, both in vivo and in silico, the effects of changes in myocardial electrotonic coupling on clinically‐relevant indices of arrhythmic liabilities.

Electrotonic coupling in vivo, both at rest and/or during acute ischemia, was modulated by interventions affecting autonomic balance/β‐adrenoceptor (β‐AR) signaling. At rest, acute ischemia induced marked electrotonic uncoupling but β‐AR activation enhanced it; ischemic passive electrical derangements were blunted by vagal‐nerve stimulation and complete/partial β‐AR blockade but were enhanced by exercise. Overall, electrotonic derangements were well correlated with changes in the rate‐corrected QT‐interval (QTc, R2 =0.89, P<0.05) and/or the with duration (terminal‐portion) of the T‐wave (TPEc, R2 = 0.81, P < 0.05); these responses were well‐modeled in silico by introducing (only) aspatially heterogeneous coupling resistance in a one dimensional structure of propagating cardiac action potentials following the Luo‐Rudy formulation. Electrotonic uncoupling also predicted ischemic T‐wave alternans (TWA, R2 = 0.82, P<0.05), a known risk‐marker for malignant arrhythmias in the clinic.

Taken together, these data demonstrate that the degree of ventricular electrotonic uncoupling can explain, at least partially, pro‐arrhythmic electrocardiographic changes reflective of myocardial repolarization abnormalities in vivo, and therefore, it must be considered when examining pro‐arrhythmic liabilities in vitro and/or in silico.