bluuebunny/crossref_metadata_2025 huggingface dataset:
Saikosaponin‐b2 inhibits tumor growth and inflammation of hepatocellular carcinoma by regulating STK4 / IRAK1 / NF‐κB signaling in H22 tumor‐bearing mice
Authors: Gao Zi‐Han , Li Rui‐Fang , Lv Xing‐Zhi , Wang Hong‐Wei , Li San‐Qiang , Wang Jian‐Gang
Published: June 2021

The tumor suppressor serine/threonine‐protein kinase 4 (STK4) may regulate inflammatory signaling in macrophages and thereby protective against inflammation‐associated HCC. Saikosaponin (SS)‐b2, a major bioactive ingredient of saikosaponins from Chinese thorowax root, exhibited anti‐cancer, anti‐inflammatory, anti‐viral, and anti‐oxidant activity. However, the anti‐tumor effect and its signaling mechanism of SS‐b2 on H22 hepatocellular carcinoma remain unknown. This study investigated the anti‐tumor effect of SS‐b2 on H22 hepatocellular carcinoma by regulating STK4/IL‐1 receptor associated kinase 1(IRAK1)/NF‐κB signaling. BALB/c mice were randomly divided into model group (normal saline, 10mL·kg−1·d−1, i.p.), different dose SS‐b2 group (3, 6, 12 mg·kg−1·d−1, i.p.) and doxorubicin group (DOX, 2 mg·kg−1, once every other day, i.p.). The mice were administered for 10 days. The results showed that SS‐b2 had a significant anti‐tumor effect on H22 xenograft mice. The tumor inhibiory rate of SS‐b2 was 16.8%, 32.7% and 55.8% respectively at 3 mg·kg−1, 6 mg·kg−1 and 12 mg·kg−1 dose. SS‐b2 decreased the spleen index in a dose‐independence way but it decreased the liver index only at 12 mg·kg−1 dose. SS‐b2 also significantly reduced the levels of AST and ALT in serum at 6 mg·kg−1 and 12 mg·kg−1. HE Staining revealed that SS‐b2 significantly attenuated the pathological morphology changes. Tumor tissue showed large necrotic, nucleus lysis and vacuole areas in mice with SS‐b2 treatment. Immunohistochemistry results showed that the positive expression of STK4 protein in tumor tissue increased however IRAK1 protein expression decrease significantly in SS‐b2 groups compared with the model group. Moreover, Western blot analysis also showed that the protein expression of STK4 increased whereas the expressions of IRAK1 and NF‐κB protein were decreased in tumor tissue, spleen, and serum in SS‐b2 treatment group. These results suggest that SS‐b2 exerts a significant anti‐tumor effect on H22 xenograft mice by regulating STK4/IRAK1/NF‐κB pathway.

Support or Funding Information

This study was supported by Science and Technology Key Research Fund funded projects of Henan Province, China (No. 142102310137), the Science and Technology Development Project of Luoyang City (No. 1603001A‐3).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
PDE1 Inhibition Attenuates Doxorubicin‐Induced Toxicity in Primary Mouse Cardiomyocytes
Authors: Vigneshwar Navin , Samidurai Arun , Movsesian Matthew , Das Anindita , Kukreja Rakesh C
Published: June 2021

Background

Doxorubicin (DOX) is a widely used and effective chemotherapeutic agent, but is associated with a delayed and progressive cardiomyopathy. Phosphodiesterase 5 (PDE5) inhibitors have been shown to be protective of heart function with DOX co‐treatment in animal models, but PDE5 is expressed at low levels in human heart. In contrast, phosphodiesterase 1 (PDE1) comprises a large fraction of the cGMP‐hydrolytic activity in both human and mouse heart. We hypothesized that PDE1 inhibition would be protect against DOX cardiotoxicity by improving cell survival (cGMP effect) and eliciting inotropic response (a cAMP effect).

Methods and Results

Isolated cardiomyocytes from adult male C57BL/6J mouse were treated with DOX (1 μM) and/or a quinazoline‐based PDE1‐selective inhibitor (5 μM) for 24 hours. Necrosis and apoptosis were assessed by trypan blue staining and TUNEL assay, respectively. Mitochondrial membrane potential (ΔΨm) was evaluated by JC‐1 staining. DOX treatment increased cardiomyocyte necrosis, apoptosis and caused loss of ΔΨm. Treatment with PDE1 inhibitor in combination with DOX significantly (P<0.05) reduced necrosis (37.4±3.9 vs 60.2±4.1% DOX alone, n=6; Figure 1A), apoptosis (17.3±1.22 vs 39.6±2.4% DOX alone, n=8; Figure 1B) and improved ΔΨm (3.1±0.4 vs 0.98±0.01 with DOX alone, n=4; Figure 1C).

Conclusion

PDE1 inhibition has anti‐apoptotic and anti‐necrotic effects against DOX‐induced cardiomyocyte death. PDE1 inhibition in combination with DOX could potentially be an efficient therapeutic strategy to reduce DOX‐induced cardiotoxicity in cancer patients.

Support or Funding Information

R37HL0510454

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Novel Functional Role of TASK‐1 in Hypoxia
Authors: Yu Yang , He Harrison , Hu Keli
Published: June 2021

TASK‐1, a member of the two‐pore domain potassium channel family, is strongly expressed in the heart. However, little is known about the regulation and function of cardiac TASK‐1 channels. The present study was designed to explore whether cardiac TASK‐1 channels play an essential role in the cytoprotection against hypoxia and reoxygenation. A cellular model of hypoxia or hypoxia and oxygenation from rat heart‐derived H9c2 cells and HEK293T cells was employed. The advantage of using HEK293T cells is that they lack endogenous TASK‐1. Hypoxia was induced by incubating cells in an airtight chamber in which O2 was replaced by 95% N2 and 5% CO2 with the glucose‐free Tyrode solution. Trypan blue cell viability assay was utilized in combination with biochemical analysis and immunofluorescence microscopy. The cell viability assay revealed that TASK‐1 expression increased the number of viable cells in HEK293T cells subjected to 2 hours of hypoxia followed by 2 hours of reoxygenation (H/R). To dissect the protective role of TASK‐1, mitochondrial membrane potential (MMP) was assessed by tetramethylrhodamine (TMRE) fluorescence. Our data indicated that MMP was significantly decreased by H/R, but was maintained by TASK‐1 expression. Our work on the subcellular localization of TASK‐1 demonstrated that while TASK‐1 showed a strong plasma membrane localization, a small fraction of TASK‐1 colocalized with the mitochondrial marker MitoTracker in intact cells and/or isolated mitochondria. H/R decreased the expression of TASK‐1 along cell periphery but did not alter colocalization of TASK‐1 with MitoTracker in HEK293T cells and/or H9c2 cells. Western blot analysis further supported the notion that the total cellular TASK‐1 expression from H9c2 cells was significantly reduced by H/R. Moreover, our data showed that Cyclosporin A (CsA), an inhibitor of mitochondrial permeability transition pore (mPTP), prevented reduction in MMP by H/R, the effect was not further enhanced by expressing TASK‐1. In summary, we provided the evidence that TASK‐1 channels are protective against hypoxia and reoxygenation‐induced injury, possibly by their capacity of maintaining the mitochondrial membrane potential via inhibiting mPTP opening.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Renal Effects Induced by Two Phospholipase A2 (Asp‐49 and Lys‐49) Isolated from Bothropoides pauloensis Snake Venom
Authors: Marinho Aline Diogo , Jorge Antonio Rafael Coelho , Filho Adriano Jose Maia Chaves , Junior Francisco Assis Nogueira , Monteiro Helena Serra Azul
Published: June 2021

Acute renal failure (ARF) wich is one of the most serious complications of Bothrops snakebites, is one of the main causes of death and consequences for victims. However, its pathogenesis remains unclear. Considering that isolated toxins are relevant tools for understanding the actions of the whole venom, we studied properties of phospholipases A2 (Asp‐49 and Lys‐49) isolated from Bothrops pauloensis (from State of São Paulo, Southeast region of Brazil), on isolated perfused kidney of rat. The control group perfused with modified Krebs–Henseleit solution alone. The PLA2s (3μg/mL) caused significant effects in isolated rat kidney, Asp‐49 reducing perfusion pressure(PP), the Renal Vascular Resistence (RVR), the Urinary Flow (UF), the Glomerular Filtration Rate (GFR) and the percentage of tubular sodium (%TNa+), chloride (%TCl−) and potassium (%TK+). The Lys‐49 increased PP and UF and decreased the RFG and the %TNa+, %TK+ and %TCl‐. Oxidative stress in kidney tissue was evaluated by TBARS (thiobarbituric acid reactive substances), Nitrite and reduced Glutathione (GSH) level measurement. A significant increase was observed in TBARS (Lys‐49) and Nitrito (Asp‐49 and Lys‐49) and a decrease in GSH levels (Asp‐49) and increase (Lys‐49). The levels of inflammatory cytokines were measured. We analized the inflammatory cytokines, pro‐inflammatory interleukin 1 (IL‐1), tumor necrosis factor‐α (TNF‐α) and anti‐inflammatory interleukin 10 (IL‐10). The results showed high levels of both cytokines (by Lys‐49) and incresed TNF‐α and IL‐10 by Asp‐49. Increased TBARS, Nitrito level and decreased of GSH activity might be implicated in the presence of oxidative stress in patients with snake bite envenomation. These findings demonstrated that PLA2s caused nephrotoxicity in isolated kidney. The characterization of the effects in the isolated kidney gives strong evidences that the acute renal failure induced by these PLA2s is a result of the direct nephrotoxicity which may involve oxidative and inflammatory mechanisms.

Support or Funding Information

CAPES, CNPq, UFC.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Muscadine Grape Extract Treatment Improves Aortic Pulse Wave Velocity and Superior Mesenteric Artery Pulsatility Index in Hypertensive Transgenic (mRen2)27 Female Rats
Authors: Ryalat Fatima , Cruz‐Diaz N , Tallant EA , Gallagher PE , Diz Di , Yamaleyeva LM
Published: June 2021

Hypertension is a major risk factor for heart disease and stroke, the leading causes of death in the United States. Hypertension is characterized by changes in vascular hemodynamics including vascular stiffness of the elastic conduit arteries and increased peripheral vascular resistance of the smaller muscular arteries that are associated with tissue damage. Dietary phenolic compounds are linked to beneficial cardiovascular effects. In this study, we investigated the influence of a muscadine grape extract (MGE; Piedmont Research & Development Corp.) on arterial stiffness and peripheral vascular resistance in hypertensive transgenic (mRen2)27 rats. We also investigated whether sex modulates MGE effects on arterial dysfunction in this rat model. MGE was administered in drinking water (0.2 mg polyphenolics/mL) starting at the age of 14 weeks, control animals were given regular water (n = 5 in each group, control and MGE‐treated female; n = 4 in each group, control and MGE‐treated male). After 6 weeks of treatment, noninvasive cardiovascular hemodynamic measurements were performed using a preclinical ultrasound system, Vevo LAZR (Fujifilm, Visual Sonics). Systolic blood pressure was measured using tail cuff plethysmography (NIBP‐8, Columbus instruments, Ohio). Aortic arch pulse wave velocity (PWV) was used as a measure of arterial stiffness and superior mesenteric artery (SMA) pulsatility index (PI), defined as PI = [(peak systolic velocity‐ end diastolic velocity)/mean flow velocity] was used as an indicator of peripheral vascular resistance. (mRen2)27 rats treated with MGE had lower aortic arch PWV compared with controls in both male (2.31 ± 0.33 vs. 3.79 ± 0.37 m/s, p < 0.05) and female (2.44 ± 0.13 vs. 3.62 ± 0.37 m/s, p < 0.05) rats. In addition, MGE‐treated female rats had lower SMA PI than control females (0.97 ± 0.07 vs. 1.27 ± 0.10, p < 0.05). There were no significant effects of the extract on SMA PI in males (1.19 ± 0.05 vs. 1.34 ± 0.07). These MGE effects were independent of changes in body weight (508 ± 3 vs. 493 ± 9 g in males, 282 ± 5 vs. 280 ± 5 g in females), cardiac index (4.5 ± 0.1 vs. 4.6 ± 0.3 mg/g in males, 4.9 ± 0.3 vs. 4.5 ± 0.2 mg/g in females), systolic blood pressure (188 ± 5 vs. 199 ± 4 mm Hg in males, 191 ± 12 vs. 194 ± 12 mm Hg in females) or cardiac function. In conclusion, six weeks of MGE treatment improved arterial stiffness in both male and female hypertensive rats. More pronounced beneficial vascular effects in females suggest that MGE actions in the arterial vasculature are modulated by sex.

Support or Funding Information

Chronic Disease Fund Research.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Melatonin attenuates bisphenol A‐induced alterations in the cardio‐renal system of rats through the antioxidant defense mechanism
Authors: Ola‐Davies Olufunke Eunice , Olukole Samuel Gbadebo , Oyagbemi Ademola Adetokunbo , Omobowale Temidayo Olutayo , Olaogun Sunday Charles , Adedokun Rahmon Akinyele , Oke Bankole Olusiji
Published: June 2021

Bisphenol A (BPA), an endocrine disrupting chemical (EDC) used in the production of plastics and epoxy resins has been incriminated in a number of biological dysfunctions while melatonin (MLT) is a known antioxidant capable of ameliorating EDC‐induced perturbations. Although limited number of studies exist on the effect of BPA on the cardio‐renal system, there is the dearth of research information on the effect of MLT on rats exposed to environmentally relevant doses of BPA. This study was therefore designed to investigate the effects of MLT on BPA‐induced alterations in the cardio‐renal system of rats. Gravid rats were randomly assigned into five groups (n=5): Group 1 (control) received 0.2 mL 1% dimethyl sulfoxide (DMSO)/99% canola oil as vehicle; Group 2 received BPA at 25 μg/kg/day; Group 3 received BPA at 250 μg/kg/day; Group 4 received BPA at 25 μg/kg/day with concurrent MLT 1 mg/kg/day; and Group 5 received BPA at 250 μg/kg/day with concurrent MLT 1 mg/kg/day. All treatments were administered by gavage from gestational day (GD) 10–21. Thereafter, three male offspring randomly from each litter per group (n=15) were weighed; blood pressure parameters and electrocardiogram taken on post‐natal day (PND) 120. The heart and kidney samples were harvested for markers of oxidative stress and antioxidant defense system, histopathology and histochemistry using standard methods. BPA induced a significant increase in heart rate but significantly decrease systolic, diastolic and mean arterial blood pressure. Exposure to BPA caused significant decrease in P wave interval, PR duration, R wave amplitude and QT interval but significant increase in QRS duration. Also, BPA significantly elevated both cardiac and renal MDA, H2O2, GSH and GPx. Cardiac and renal lesions induced by BPA included erosion of epithelial cells, congestion of blood vessels as well as necrosis. Moreover, BPA induced a significant reduction in cardiac and renal polysaccharide activities. However, the concomitant treatment with MLT attenuated all the observed BPA‐induced alterations of the cardio‐renal system. Hence, MLT attenuates alterations induced by environmentally relevant doses of BPA in the cardio‐renal system.

Support or Funding Information

None

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Effect of Ramadan Fasting on Arginase Activity in Healthy Volunteers
Authors: Shatanawi Alia , Gharaibeh Ahmad M , Gharaibeh Munir N
Published: June 2021

The practice of fasting is performed by many communities for religious or spiritual reasons. Additionally, in the recent years many health benefits were associated with different kinds of fasting. These include: controlling blood glucose levels , reducing insulin resistance, reducing inflammation, improve heart health, lowering blood pressure and boosting brain function while prevent neurodegenerative disorders. Moreover, there is proven evidence on the effects of Ramadan fasting, performed by muslims for one month, on improving vascular health, blood flow and enhancing endothelial function in patients with cardiovascular disease. Fasting was reported to increase nitric oxide (NO) levels and decrease asymmetric dimethylarginine (ADMA); a novel cardiovascular risk factor. Nitric oxide, a potent vasodilator and vascular protective molecule, is produced by endothelial cells by the action of endothelial nitric oxide synthase (eNOS) on L‐arginine. Arginase, an enzyme implicated in many cardiovascular diseases, can compete with eNOS for their common substrate, L‐arginine to produce urea and ornithine. We and others have reported the effects of arginase inhibition on improving endothelial function and NO levels in different cardiovascular diseases and in diabetes.

Our project aims to study the effect of Ramadan fasting on arginase activity in healthy individuals. We measured arginase activity one day before the start of fasting and on the last day of fasting for thirteen young male adults, ages between 18–35 years old. The fasting performed is to withhold from eating, drinking and smoking from sunrise to sunset (about 15 hours) everyday for a period of one month. Plasma arginase activity is measured using a colorimetric reaction of urea production. Our results show a modest though significant decrease in arginase activity of 15.7 ± 4.2%. We also determined basal urea levels and observed no change in these levels. Our results propose one important benefit of fasting through lowering arginase activity thus reserving available L‐arginine for NOS and increasing NO levels. We also noted that basal urea levels did not change. It was expected that not drinking water for long hours could increase chances of dehydration which can increase plasma urea concentrations, but this did not occur. In conclusion, our result show another added health benefit of fasting to the vascular system which is lowering arginase activity. Fasting under doctor supervision can be used in selected patients with vascular diseases to maintain and promote NO levels. We yet have to determine the effects of fasting on other systems of the body such as liver and kidney to preclude any deleterious side effects of this practice.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of Diminazene aceturate treatment on some cardiovascular parameters and antioxidant status of apparently healthy horses
Authors: Omoniwa David , Ogunpolu Blessing , Omoniwa Amy Morayo , Adedokun Rahamon , Olaifa Abayomi , Oyagbemi Ademola , Omobowale Temidayo
Published: June 2021

Introduction

Apart from being a widely used antiprotozoal drug in many parts of Africa, Diminazene aceturate (DIZE), has been shown to have beneficial effects in experimental murine models of hypertension, pulmonary hypertension, myocardial infarction, stroke, atherosclerosis, type 1 diabetes, and eye disease. In this study, we investigated the effect of diminazene aceturate treatment after a single intramuscular administration on some cardiovascular parameters and antioxidant status in apparently healthy Nigerian horses.

Materials and methods

Ten male horses were divided into two groups comprising 5 horses each. Group A was the control group with no drug administered. Group B was treated with DIZE at 3.5 mg/kg, single dose. Baseline data were collected before the commencement of the experiment and thereafter, post‐administration at 0hrs, 24hrs, 48hrs and 96hrs. Base apex electrocardiogram was recorded using a computer ECG machine while oscillometric blood pressure measurement was recorded using a veterinary blood pressure monitor. Markers of oxidative stress were determined in the serum

Result

Single intramuscular treatment caused a significant (p<0.05) increase in the systolic, diastolic and mean arterial blood pressure values when compared with the controls. From the electrocardiogram, sinus rhythm was recorded in both groups however, significant (p<0.05) increase in heart rate, p‐wave duration, QRS complex duration and QTc were observed in the treated group when compared with the control. Oxidative stress markers (malondialdehyde and hydrogen peroxide) significantly (p<0.05) increased while antioxidant enzymes (catalase, glutathione peroxidase and glutathione) were decreased when compared to the control.

Conclusion

This study indicates that the use of DIZE in horses should be with great caution because of the associated cardiovascular physiological alterations, reduction in activities of antioxidant enzymes and increase in markers of oxidative stress.

Support or Funding Information

None

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Angiotensin converting enzyme inhibitors or angiotensin receptors blockers equally enhances skeletal structure via different metabolic influence
Authors: Abuohashish Hatem Mustafa
Published: June 2021

The impact of renin‐angiotensin system (RAS) on bone remodeling and metabolism has gained more interest in recent years, where several RAS metabolites are deemed as potential targets for skeletal diseases. This study compares the effects of angiotensin converting enzyme (ACE) inhibitors and angiotensin‐II (AngII) type‐1 receptor (AT1R) blockers on bone structure and remodeling and investigates the involvement of neutral endopeptidase enzyme (NEP) and angiotensin 1‐9 (Ang1‐9) in ovariectomized (OVX) rats. OVX animals received either captopril or losartan for 6 weeks. Both antihypertensive agents equally attenuated the elevated levels of bone metabolic bio‐markers and restored Ca2+ and phosphorous renal elimination and bone deposition. The altered bone cortical and trabecular morphometry in OVX rats was similarly ameliorated. In femurs heads, the expressions of NEP and Ang1‐9 were enhanced by captopril, while remained unchanged in losartan groups. In addition, both drugs down‐regulated the osteoclastogenesis inducing factor, receptor activator NF‐κB ligand (RANKL), while enhanced osteoprotegerin. Although, both antihypertensive agents had different influence on local skeletal RAS metabolism, losartan and captopril equally restored bone micro‐structure in estrogen deficient animals.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Protective Effects of Ocimum sanctum Leaf Extract against Lead‐induced Toxicity in Rat Brain
Authors: Kilari Eswar A , Putta Swathi , Qureshi Absar Ahmed
Published: June 2021

Lead is a known as multi organ toxicant majorly on neuronal system. The present study evaluates the protective role of Ocimum sanctum leaf extract against lead induced oxidative damage to the different regions of the brain. Albino Wistar rats were divided into three groups each consisting of 10 rats. The Ocimum sanctum extract was administered orally at 200 and 400 mg/kg along with 500ppm of lead acetate dissolved water for four weeks to two groups of rats. The control group was administered with sodium acetate dissolved in water for four weeks. At the end of four weeks study period, the rats were sacrificed and brains were isolated and different regions like cerebellum, hippocampus, frontal cortex and brain stem were separated. The delta ALAD levels were estimated in the serum samples. The antioxidant enzymes (SOD, CAT and GST), non enzymatic antioxidants (GSH), lipid peroxidation (TBRS), metal content, tissue nitrite and protein carbonyl content (PCO) were measured in all separated parts of the brain. Finally the histopathology was conducted for all the selected parts of the brain. The treatment with Ocimum sanctum showed significant elevation of serum delta ALAD levels, antioxidant enzymes (SOD, CAT and GST) and compared to that of lead acetate control group. The lipid peroxidation and PCO were found to be lowered with Ocimum sanctum treatment in lead acetate induced brain toxicity. The histopathological studies indicated that the Ocimum sanctum showed marked amelioration of damaged structure of the parts of the brain caused by the lead acetate in rats. The results indicating that the protective effect was found to be more in cerebellum and hippocampus compared to other parts of brain.

Conclusions

The protective effect of Ocimum sanctum might be due to its antioxidant activity.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Microdialysis Assay for Assessing sGC Stimulator Target Engagement in the Rat Liver
Authors: O'Connell Ryan P. , Hall Katherine C. , Jacobson Sarah , Tobin Jenny V. , Masferrer Jaime L.
Published: June 2021

Dysfunction of the nitric oxide (NO)‐soluble guanylate cyclase (sGC)‐cyclic guanosine monophosphate (cGMP) pathway has been implicated in numerous cardiovascular, metabolic, and fibrotic diseases. sGC stimulators are small molecules that bind sGC and synergize with NO to increase the enzymatic formation of cGMP and amplify downstream signaling. In preclinical models of liver disease, sGC stimulators have been shown to attenuate liver steatosis, inflammation, and fibrosis. To demonstrate their engagement in the sGC‐cGMP pathway in vivo, a unique microdialysis assay was developed to directly measure changes in cGMP levels in the rat liver.

A midline laparotomy was performed on anesthetized male, Sprague Dawley rats (225–250 g) to expose the liver. A 25‐gauge needle was used to place a dialysis probe within the right medial lobe. The probe was perfused with saline at a rate of 2.5 μL/min and dialysate collected in one‐hour periods. Following baseline collection, test compound was administered intravenously, and dialysate collected for the following 2 hours. The NO donor sodium nitroprusside (SNP, 300 nM) was dosed via retrodialysis at study's end as a positive control. cGMP in the dialysate was measured using an enzyme immunoassay.

In a study investigating our clinical‐stage sGC stimulator, praliciguat, an oral 3‐mg/kg dose produced a 3.9 ± 1.7‐fold increase (p<.0001) in cGMP from baseline levels of 0.8 ± 0.1 nM. A 1‐mg/kg dose caused a 1.7 ± 0.1‐fold increase in cGMP (p<.001), while the effect of a 0.3‐mg/kg dose was not significant. On average, SNP induced a 4.7 ± 1‐fold increase in cGMP (p<.0001).

In summary, our microdialysis assay enabled in vivo monitoring of sGC stimulation in the rat liver. Beyond demonstrating the effects of praliciguat, this method can be used to evaluate and characterize other sGC stimulators for their capacity to amplify liver cGMP production. Further, this assay helps to link previously observed hepatic effects of sGC stimulators in disease models with an enhancement of sGC signaling.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ulcerogenic Action of Indomethacin on Gastric and Small Intestinal Mucosa in C57/BL6/J and TRPV1 Knockout Mice
Authors: Filaretova Ludmila , Yarushkina Natalia , Sudalina Maria , Punin Yuriy
Published: June 2021

Capsaicin‐sensitive afferent neurons are involved in maintenance of gastrointestinal mucosa integrity. Capsaicin action is mediated through TRPV1 receptors. The aim of the present study was to elucidate the role of these receptors in providing of protective or/and pathological mechanisms under circumstances of indomethacin (IM)‐induced injury in gastric and small intestinal mucosa. We compared vulnerability of gastric and small intestine mucosa to IM action in mice with genetically deleted TRPV1 receptor and C57/BL6/J mice. IM‐induced injury was assessed by macroscopically as well as histologically; the somatic pain sensitivity was estimated by tail flick latency (tail flick test); plasma corticosterone levels and body weight were also monitored. A single IM administration (35 mg/kg, sc) into fasting (24 h) mice caused the formation of gastric erosions 4 h after injection and, then, after refeeding, formation of the small intestine injury which was visible 24, 48, 72 h after the injection. Although IM‐induced gastrointestinal injury was detectable in both of C57/BL6/J and TRPV1 knockout mice, area of gastric damage was greater in C57/BL6/J than in TRPV1 knockout mice, whereas the small intestine injury (48 and 72 h after IM injection), on contrary, prevailed in TRPV1 knockout mice compared to C57/BL6/J mice. TRPV1 knockout mice also showed an increased tail flick latencies and lower plasma corticosterone levels than C57/BL6/J mice. The data suggests that in TRPV1 knockout mice the gastric mucosa is less vulnerable to ulcerogenic IM action compared to C57/BL6/J mice, whereas their small intestinal mucosa, on contrary, is more susceptible to IM ulcerogenic action than in C57/BL6/J mice.

Support or Funding Information

The study was supported by grant of Russian Science Foundation (RSF) N 14‐15‐00790 and grant of RFBR N 16‐04‐01196a

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Study on the Effect of N453 from Fungi on Inhibiting CFTR Chloride Channels in Intestinal Epithelial Cells
Authors: Chutinun Onjira , Akrimajirachoote Nattaphong , Muanprasat Chatchai
Published: June 2021

Secretory diarrhea remains a global health problem, especially in developing countries. Cyclic AMP‐stimulated intestinal Cl− secretion plays a crucial role in pathogenesis of diarrheas including cholera and traveler's diarrhea. Inhibition of cAMP‐activated Cl− channel is considered as drug target for secretory diarrhea. The main objective of this study was to identify a novel inhibitor of the cystic fibrosis transmembrane conductance regulator (CFTR), cAMP‐activated Cl− channel, from a screen of fungal metabolites. A fungal derivative compound N453 was identified as an active compound that could inhibit cAMP‐activated Cl− secretion in intact T84 cells with a half maximal inhibitory concentration (IC50) of ~ 0.78 μM in Ussing chamber experiments. Importantly, MTT assays revealed that N453 (up to100 μM) did not affect cell viability, suggesting that inhibition of cAMP‐activated Cl− current was not due to cytotoxic effect. In apical Cl− current analysis, N453 suppressed CFTR‐mediated apical Cl− current in a concentration‐dependent manner with IC50 of ~ 2 μM. On the other hand, the Na+‐K+ ATPase activity was not affected by N453. Of interest, N453 concentration‐dependently inhibited basolateral cAMP‐activated K+ channels with an IC50 of ~ 23 μM. In conclusion, this study demonstrated that N453 inhibits Cl− secretion in intestinal epithelial cells, at least in part, by inhibiting CFTR‐mediated apical Cl− current and basolateral cAMP‐activated K+ channels. N453 may be useful as potential therapy of secretory diarrheas.

Support or Funding Information

This work was supported by the NSTDA Chair Professor grant (the Fourth Grant) of the Crown Property Bureau Foundation, Faculty of Science Mahidol University, Faculty of Graduate Studies Mahidol University and Tonkla Ramathibodi.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Pentadecapeptide BPC 157 Counteracts the Adverse Effect of Lithium Overdose in Rats
Authors: Strbe Sanja , Filipcic Igor , Seiwerth Sven , Sikiric Predrag
Published: June 2021

We documented that stable gastric pentadecapeptide BPC 157 mitigates lithium‐induced intoxication in rats. BPC 157, LD1 not achieved, was implemented as an anti‐ulcer peptide in inflammatory bowel disease trials and now in a multiple sclerosis trial (Curr Pharm Des. 2018;24(18):1990–2001). Previously, BPC 157 counteracts consequences of dopamine (D), receptors blockade (neuroleptics‐induced catalepsy, prolonged QT intervals, sphincters dysfunction and gastric lesions), much like over‐stimulation (amphetamine acute and chronic disturbances; much like D‐receptors supersensitivity (amphetamine after haloperidol)), nigrostriatal lesions (MPTP Parkinsogenic neurotoxin), D‐vesicles depletion (reserpine) (see Sikiric et al., Brain‐gut axis and pentadecapeptide BPC 157: Theoretical and practical implications. Curr Neuropharmacol. 2016;14(8):857–865. Review.). Similarly, BPC 157 counteracts immobility more than imipramine in depression‐models (Porsolt's and chronic unpredictable stress‐open field) and induces 5‐HT release in particular brain areas (nigrostriatum) when given peripherally, acute and chronically (see Sikiric et al., Brain‐gut axis and pentadecapeptide BPC 157: Theoretical and practical implications. Curr Neuropharmacol. 2016;14(8):857–865. Review.). In this study, we applied lithium (500 mg/kg/day, intraperitoneally, once daily through 3 subsequent days). Medication (mg/kg) includes BPC 157(0.01; 0,00001), L‐NAME (5.0), L‐arginine (100.0), applied alone and /or together) while control rats received an equivolume of saline (5 mL/kg). Thereafter, at 20 minutes after medication, we assessed muscular weakness (score 1–5) during next 8 minutes. Then, for next 5 minutes, we recorded ECG. At 3 hours thereafter, the brain, heart, quadriceps muscle and diaphragm muscle were used for histopathological analysis. Regularly, lithium immediately produced severe syndrome (severe muscular weakness and prostration, reduced quadriceps muscle fibers and diaphragm, myocardial infarction, and edema of various brain areas, the most prominent in the cerebral cortex). The worsening appeared with subsequent applications. L‐NAME and L‐arginine, given separately, both induced severe aggravation. This aggravation disappeared when L‐NAME and L‐arginine were given together. Contrarily, given alone or together with NO‐agents, BPC 157 reduced muscular weakness and prostration and muscle damage and mitigated lithium induced myocardial damage. BPC 157 reduced nerve damage and brain edema.

Conclusion

BPC 157 can be a therapy of lithium intoxication.

Support or Funding Information

This work was supported by the Ministry of Science, Education and Sports, Republic of Croatia [grant number 108‐1083570‐3635].

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification of BKca channel activation effect of Kushen extract containing Kurarinone to ease urinary incontinence or overactive bladder
Authors: Lee Narasaem , Han Jisun , Jo Heeji , Jang Sung Joo , Ryu Seunghwan , Moh Sang Hyun , Park Chul‐Seung
Published: June 2021

The large‐conductance Ca2+‐activated K+ channel (or BKCa channel) requires membrane depolarization or increased intracellular Ca2+ for its activation. This channel makes one of the most effective ways to generate membrane hyperpolarization and regulates several key physiological processes. In the bladder, BKCa channel evokes the relaxation of urinary bladder smooth muscle and thus represents a therapeutic target for urinary incontinence (UI) or overactive bladder (OAB). In our previous study, we reported that kurarinone, a prenylated flavanone from Sophora flavescens, potently activates BKCa channel and established its therapeutic potential for treating OAB (Lee et al., Mol Pharmacol. 90:140, 2016). Subsequently, we identified five more flavonoids from Kushen, the dry root of S. flavescens. including kuraridinol, a prenylated chalcone, significantly potentiating BKCa channel activity (Lee et al., Biol Pharm Bull. 41:1295, 2018). In this study, we analyzed the components of Kushen extracts isolated with differential ethanol concentrations and tested their efficacy for BKCa channel activation. We found that the potentiation effect of various extracts for BKCa channel correlates well with the concentration of kurainone in the extracts. In addition, the Kushen extracts potently relaxed acetylcholine‐induced contraction of rat bladder smooth muscle and thus decreased the micturition frequency of rats with overactive bladder symptoms. Our results strongly support the idea that the Kushen extract containing kurarinone as the main ingredient can be utilized as nutraceutical or functional food against lower urinary tract symptom such as UI or OAB.

Support or Funding Information

This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry (IPET) through Agri‐Bio industry Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (317070‐4)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The TRPP2 (PC2)‐dependent ion channel of renal primary cilia also requires TRPM3
Authors: Bryant Gillian , Kleene Nancy , Siroky Brian , Landero‐Figueroa Julio , Dixon Brad , Pachciarz Nolan , Lu Lu , Kleene Steve
Published: June 2021

INTRODUCTION

Primary cilia are found in numerous places throughout the body and serve many different functions (e.g., cell migration, intercellular signaling, and sensation). Mutations in primary cilia cause many human diseases. Polycystic kidney disease (PKD) is a ciliopathy that results from mutations in the ciliary proteins polycystin 1, TRPP2/polycystin 2 (PC2), or polyductin/fibrocystin. Although the mechanism is not completely understood, abnormally low intracellular Ca2+ levels are thought to be an early step in cystogenesis.

AIM

We study renal ciliary ion channels in search of a pharmacological means of compensating for the lower cytoplasmic Ca2+. One channel present in renal tissue is TRPM3. When testing cilia with a TRPM3 agonist, we did not detect a channel with traits characteristic of TRPM3, but, to our surprise, the agonist activated the TRPP2‐dependent ciliary channel.

HYPOTHESIS

The TRPP2‐dependent channel also requires TRPM3.

METHODS

We used electrophysiology, qPCR, western blotting, and immunocytochemistry with a murine immortalized renal epithelial cell line.

RESULTS

The TRPP2‐dependent channel was activated by a known agonist of TRPM3 and inhibited by a known antagonist of TRPM3. The TRPP2‐dependent channel current was not detectable when TRPM3 was knocked out with CRISPR/Cas9 genome editing. Knocking out TRPP2 did not reduce TRPM3 mRNA expression, and knocking out TRPM3 did not markedly decrease TRPP2 mRNA expression. Knocking out TRPM3 did not change TRPP2 protein expression. Immunocytochemistry indicated that removal of TRPM3 expression reduced the amount of TRPP2 trafficking to the cilium.

CONCLUSION

The TRPP2‐dependent channel also requires TRPM3. Further studies will be required to determine the nature of the interaction between TRPM3 and TRPP2 and if the interaction can be manipulated to reduce cystogenesis in PKD.

Support or Funding Information

This work was done with financial support from The University of Alabama Hepato/Renal Fibrocystic Disease Core Center, Cincinnati Children's Hospital Medical Center (CCHMC), and the University of Cincinnati.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Rififylin (RFFL), a Novel Regulator of Transient Outward (Ito) Potassium Channels
Authors: Roder Karim , Kabakov Anatoli , Moshal Karni S , Xie An , Turan Nilüfer N , Lu Yichun , Koren Gideon
Published: June 2021

The expression of sarcolemmal ion channels is a net result of the rate of coordinated biosynthesis, proper folding, forward trafficking, recycling, and degradation. The importance of these processes was highlighted by several genome‐wide association studies that reported novel loci that modify the QT interval and are located in or near genes involved in protein trafficking and degradation (Roder et al., 2014; Roder et al. 2018). One of the genes identified, the ring finger and FYVE‐like domain containing E3 ubiquitin protein ligase rififylin (RFFL), has also been shown to regulate cardiac excitation and growth in a congenic rat strain (Gopalakrishnan et al. 2011). As transient outward K+ currents (Ito) are essential during the whole repolarization phase of the action potential in rodents, we hypothesized that RFFL could regulate cardiac excitation through Ito‐encoding channels. Adult rabbit cardiomyocytes with adenovirus‐expressed RFFL exhibited reduced Ito,f. In HEK cells, transient RFFL overexpression caused polyubiquitination and downregulation of total and surface levels of Kv4.2 and Kv4.3 as well as the corresponding current Ito,f. In contrast, in transiently co‐transfected HEK cells, overexpressed RFFL monoubiquitinated and upregulated total and membrane levels of Kv1.4 and the corresponding current Ito,s in a RING domain‐dependent manner. We conclude that RFFL, through its interactions with and ubiquitination of Ito‐encoding channels Kv4.2, Kv4.3 and Kv1.4 regulate their forward trafficking and degradation, which may affect cardiac action potential shape and duration.

Support or Funding Information

NHLBI grant 5R01HL134706‐02 (GK).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
LITAF regulates action potential duration by modulating NEDD4‐1‐mediated degradation of L‐type calcium channels
Authors: Moshal Karni S , Roder Karim , Kabakov Anatoli , Turan Nilüfer N , Xie An , Werdich Andreas A , Kim Tae Yun , Cooper Leroy L , Lu Yichun , Terentyev Dmitry , Choi Bum‐Rak , MacRae Calum A , Koren Gideon
Published: June 2021

Rationale

The QT interval is a predictor of arrhythmias and sudden cardiac death. A genetic variant upstream of the gene encoding LITAF (lipopolysaccharide‐induced tumor necrosis factor) is associated with a prolonged QT interval.

Objective

We sought to determine the role of LITAF in modulating cardiac excitation at the cellular and organ levels.

Methods and Results

Morpholino knockdown of LITAF in zebrafish embryos resulted in a robust increase in calcium transients in optically mapped hearts, which primarily depends on calcium influx. Adenovirally expressed LITAF shortened action potential duration and lowered Cava1c expression in neonatal rabbit cardiomyocytes, whereas a shRNA‐mediated LITAF knockdown had the opposite effect. Using whole‐cell patch clamping and western blotting of 3‐week old rabbit cardiomyocytes, overexpressed LITAF resulted in a decrease in ICa,L and Cava1c expression, whereas a LITAF knockdown increased ICa,L and Cava1c protein levels. Live‐cell confocal calcium imaging detected a decrease in calcium transients in LITAF‐overexpressing adult rabbit cardiomyocytes, which was accompanied by lower Cava1c expression. In tsA201 cells, overexpressed LITAF downregulated the total and surface pools of Cava1c via increased Cava1c ubiquitination and subsequent lysosomal degradation. Co‐immunoprecipitation and immunofluorescence experiments showed complex formation and co‐localization of LITAF and L‐type calcium channels (LTCC) in tsA201 cells and adult rabbit cardiomyocytes, respectively. In tsA201 cells, NEDD4‐1, but not its catalytically inactive form NEDD4‐1‐C867A, increased Cava1c ubiquitination compared to control. Cava1c ubiquitination was further increased by co‐expressed LITAF and NEDD4‐1 but not NEDD4‐1‐C867A. Finally, adenovirally co‐expressed NEDD4‐1 shRNA abolished the negative effect of LITAF on ICa,L and Cava1c expression in 3‐week old rabbit cardiomyocytes.

Conclusions

The data suggest that LITAF promotes NEDD4‐1‐mediated ubiquitination and subsequent degradation of cardiac LTCC, thereby controlling LTCC membrane expression and function and thus APD and the QT interval.

Support or Funding Information

NHLBI grant 5R01HL134706‐02 (GK).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Discovery of Pranlukast in a High‐Throughput Screen for Novel Inhibitors of LRRC8 Volume Regulated Anion Channels
Authors: Figueroa Eric E , Kramer Meghan , Denton Jerod S
Published: June 2021

Cells critically regulate their volume in response to hypotonic cell swelling by transporting chloride and small organic osmolytes out of the cell through volume regulated anion channels (VRACs) encoded by members of the recently defined LRRC8 gene family. VRACs are ubiquitously expressed in mammalian cells and have been implicated in diverse cellular functions in addition to cell volume regulation, including endothelial cell calcium signaling and pancreatic beta cell insulin secretion. The lack of potent and specific pharmacological tool compounds represents a critical barrier to evaluating the integrative physiology and therapeutic potential of VRACs. We therefore carried out a high‐throughput screen (HTS) of 1,184 of FDA approved drugs using a fluorescence‐based assay for novel inhibitors of native VRACs expressed in HEK‐293 cells. The assay measures the quenching of intracellular YFP by iodide influx through VRACs activated by hypotonic‐induced cell swelling. In this screen, the cysteinyl leukotriene receptor 1 (CysLT1R) antagonist, Pranlukast, was identified as a novel inhibitor of VRAC. In YFP quenching assays and patch clamp electrophysiology experiments, Pranlukast inhibits HEK‐293 cell VRAC reversibly and dose‐dependently with an IC50 of ~6 μM and ~239 nM, respectively. Interestingly, the CysLT1R pathway has been implicated in activation of VRAC in other cell types, raising the possibility that Pranlukast inhibits VRAC indirectly via the CysLT1R pathway. Using a series of fluorescence assays and qPCR we show that CysLT1R is not functionally expressed in HEK‐293 cells. Additionally, using a structurally distinct CysLT1R inhibitor, Zafirlukast, did not inhibit VRAC. These studies provide clarifying insights into the putative role of CysLT1 receptor signaling in regulation of VRACs and identify Pranlukast as a structurally novel small‐molecule inhibitor of LRRC8 channels.

Support or Funding Information

Ruth L. Kirschstein National Research Service Award F31 (1F31DK120225‐01)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.