bluuebunny/crossref_metadata_2025 huggingface dataset:
Biomechanical forces differentially regulate signaling in the cortical collecting duct (CCD)
Authors: Carrisoza Rolando , Else Cindy K. , Flores Daniel , Lee Heon G. , Lee Francis Y. , Rohatgi Rajeev , Satlin Lisa M.M
Published: June 2021

Tubular fluid flow (TFF) stimulates Na reabsorption and K secretion in the CCD and subjects CCD cells to biomechanical forces including fluid shear stress (FSS) and circumferential stretch (CS). Intracellular MAPK and extracellular autocrine/paracrine PGE2 signaling regulate cation transport in the CCD and, at least in other systems, are regulated by biomechanical forces. We hypothesized that FSS and CS differentially regulate MAPK signaling and PGE2 release to modulate cation transport in the CCD. Murine CCD cells (mpkCCD) were grown on glass or silicone coated with collagen I, and subjected to 0 or 0.4 dynes/cm2 of FSS or 10% CS. Cells exposed to FSS (3–60 min; n=4–9) expressed greater abundance of phospho(p)‐ERK and p‐p38 than static cells (n=4–9), while CS suppressed p‐p38 expression (n=5) compared to nonstretched cells (n=5). p‐ERK abundance was unaffected by CS. FSS induced PGE2 release; however, CS reduced PGE2 release (0.35±0.07 pg/mL/ug protein [n=8] vs. 0.15±0.02 pg/mL/ug protein [n=9]) into the media bathing mpkCCD cells. In conclusion, FSS and CS differentially regulate p38 activation and PGE2 release in a cell culture model of the CD. We speculate that TFF differentially regulates biomechanical signaling and, in turn, cation transport in the CCD.
Evidence for the existence of calcium signaling domains in a renal cortical collecting duct cell line
Authors: Thai Tiffany L , Bao Hui‐Fang , Liu Bingchen , Ma Heping , Eaton Douglas C
Published: June 2021

The epithelial sodium channel of the kidney (ENaC) is inhibited by activation of the purinergic P2Y2 receptor in the apical membrane but stimulated by basolateral activation of the P2X4 receptor. We hypothesized that regional regulation of [Ca2+]i is responsible for the polarized effects of ATP on ENaC function. To test this, we analyzed [Ca2+]i responses downstream of purinergic agonists in renal A6 cells. Apically, the P2Y2 agonist UTP caused a rapid, transient increase (15–60 sec) in [Ca2+]i observed by loading cells with fluo4‐AM. The P2X4 receptor agonist 2‐meSATP applied basolaterally significantly increased the open probability of ENaC, an effect which was abolished by pretreatment with the Ca2+chelator EDTA (2 mM). In other epithelial cell types, mitochondria form barriers that compartmentalize Ca2+. To test whether such a barrier exists in renal A6 cells, we loaded cells with MitoTracker Red to visualize mitochondria. In Z‐axis sections, we observed strong bands of mitochondria both apically and basolaterally. Our results suggest that the differing effects of apical vs basolateral ATP on ENaC occur through activation of different purinergic receptors, both of which cause increases in [Ca2+]i but that the Ca2+ is confined to two separate compartments, producing differing effects. This work was supported by NIH grants R37‐DK037963 and T32‐DK07656.
Prostaglandin receptor EP2 and EP4 mediated Aquaporin‐2 membrane accumulation does not depend on cAMP
Authors: Olesen Emma Tina Bisgaard , Fenton Robert Andrew
Published: June 2021

We investigated the role of cAMP in aquaporin‐2 (AQP2) apical membrane accumulation (AMA) induced by the prostanoid receptors EP2 and EP4. AQP2 AMA was investigated by cell surface biotinylation and total intracellular cAMP was measured by a TR‐FRET based technique. In MDCK cells stably transfected with AQP2, the EP2 agonist butaprost (BU) (0.1μM) induced time dependent AQP2 AMA which continually increased during 80min stimulation. cAMP increased maximally after 10min stimulation. After agonist removal, although we have previously shown that AQP2 AMA persists for at least 80min, cAMP was reduced to control levels. The EP4 agonist CAY10580 (CA) (3μM) induced maximal AQP2 AMA at 5min. However, cAMP was not increased at any time point examined (5–40min). Similar to MDCK cells, in mpkCCD14 cells CA induced AQP2 AMA which was eliminated 20min after agonist removal, but CA did not increase cAMP. In MDCK cells, the adenylate cyclase inhibitor SQ22536 (500μm) eliminated the effect of BU (50nM) on cAMP levels, but had no effect on BU or CA induced AQP2 AMA at the same concentrations. In conclusion, although AQP2 AMA is usually linked to cAMP, we discovered 1) dissociation between EP2 induced cAMP increase and AQP2 AMA when investigated dynamically and 2) neither EP2 nor EP4 induced AQP2 AMA depends on an increase in cAMP. These studies suggest a novel mechanism for AQP2 trafficking that is non‐cAMP dependent.
The New Mode to Neurohumoral Regulation in Respiratory and Circulatory Systems
Authors: Sun Xing‐Guo
Published: June 2021

We defined the sophisticated organic whole by placing oxygen (along with CO2 and [H+] Trinity) as the core. The regulation through integrative nervous system is still unclear. In order for us to easily comprehend the working mode of respiratory and circulatory regulations, we use an analogy of ¡°sound system regulation mode¡±: 1. The wave signal generated by the circulating blood in the circulatory system due to pulmonary ventilation is compared to the speech acoustic signals produced by person. 2. Multiple peripheral chemoreceptors and baroreceptors are compared to the sonic microphones that sense sound waves; they collect and upload distinct signals. 3. The central nervous system is equivalent to the volume amplifier control system. 4. The central chemoreceptors act as a volume‐adjusting knob; it balances the central incoming/outgoing integration. 5. The respiratory muscle and thorax lung jointly play the role of a speaker.

This concept explains why we can stably maintain our respiration by breath‐by‐breath pattern via respiratory muscles contraction/diastole conversion. Similarly, our circulation can be maintain stable by beat‐by‐beat pattern.
The combined effects of Anastrozole and Testosterone or Trenbolone on Prostate and Levator Ani‐Bulbo Cavernosus Mass
Authors: Beck Darren Thomas , Yarrow Joshua F , McCoy Sean C , Beggs Luke A , Ye Fan , Balaez Alexander , Combs Sarah M , Miller Julie R , Conover Christine F , Borst Stephen E
Published: June 2021

Our purpose was to determine the role of the aromatase enzyme in mediating the myotrophic and prostate enlarging effects of androgens. 10 month old Fisher 344 rats received Sham surgery or orchiectomy (ORX). One week following surgery animals were implanted with motorized micro‐infusion pumps to disperse anastrozole (ANA; aromatase inhibitor) or vehicle at a rate of 2.0 μl/hour. Animals received once weekly IM testosterone enanthate (TE; 7.0mg/week), trenbolone enanthate (TREN; 1.0mg/week), or vehicle. 28 days following pump implantation animals were sacrificed and the prostate and levator ani‐bulbocavernosus (LABC) muscle were excised.

Group (n=3)

Prostate (g)
LABC (g)

SHAM
(a)
0.29±0.064 c
0.88±0.033 b,c,d,e,f,g

ORX
(b)
0.07±0.014 d,e
0.45±0.004 d,e,f,g

ORX+ANA
(c)
0.06±0.003 a,d,e
0.48±0.025 d,e,f,g

ORX+TE
(d)
0.31±0.037 b,c
1.20±0.034 a,b,c

ORX+TE+ANA
(e)
0.31±0.074 b,c
1.22±0.046 a,b,c

ORX+TREN
(f)
0.15±0.064
1.19±0.035 a,b,c

ORX+TREN+ANA
(g)
0.18±0.033
1.19±0.013 a,b,c

Values are mean±SEM. Significance values are reported from one‐way ANOVA and Tukey posthoc group comparison; letters indicate p<0.05 vs. group.

Our findings suggest that estradiol is not required for androgen mediated prostate enlargement. Further, inhibiting the aromatase enzyme did not blunt the myotrophic effects of androgens.

Supported by VA Merit Award to SEB and VA Geriatrics Fellowship to DTB.
Neural and bioenergetic mechanisms of human skeletal muscle fatigue resistance in old age
Authors: Kent‐Braun Jane , Tonson Anne , Deblois Jacob , Fay Jessica , O'Brien Teresa , Foulis Stephen , Kamen Gary , Befroy Douglas , Holste Katherine , Christie Anita
Published: June 2021

There is strong evidence that greater resistance to muscle fatigue in older adults, most often observed during low‐velocity or isometric contractions, has a basis in the bioenergetic response to contractions. However, the role of age‐related changes in motor unit (MU) behavior has not been evaluated in this context. To test the hypothesis that fatigue resistance in old resuls from adaptations in both MU behavior and energetics, we quantified dorsiflexor muscle fatigue (torque‐time integral, %initial); mean MU discharge rate (MUDR) by in‐dwelling electrode and cytosolic pH and inorganic phosphate (Pi) by magnetic resonance spectroscopy during parallel experiments using a protocol of 12 maximal isometric contractions (12s on, 8s off) in 10 young (35±4 yr, mean±SD) and 8 older (74±5) women. Older fatigued less than young (68±7%initial vs 59.7 ±9, p<0.05), had a smaller decrease in MUDR (Δ= −20±10 %initial vs −34±18, p=0.08), and tended to have less acidosis at fatigue (6.88±0.13 vs 6.77±0.16, p=0.18), with no difference in end‐exercise Pi. These data provide the first in vivo evidence of the combined roles of MUDR and energetic responses as mechanisms of fatigue resistance in older adults. NIH AG21094
Age‐associated attenuation of autophagy underlies ryanodine receptor hyperactivity
Authors: Cooper Leroy Leon , Lu Yi Chun , Centracchio Jason , Terentyeva Radmila , Terentyev Dmitry , Koren Gideon
Published: June 2021

Aging is associated with reduced autophagy and altered mitochondrial function; yet, the link between these processes and altered calcium (Ca) homeostasis remains unknown. We used ventricular myocytes from young (5–9 months) and old (4–5 years) rabbit hearts to test the hypothesis that reduced autophagy leads to dysfunctional mitochondria that produces ROS, leading to oxidation of ryanodine receptors (RyRs) and changes in Ca homeostasis. Markers of autophagy were measured via western blotting and β‐galactosidase staining. Age‐associated changes in ROS production, mitochondrial membrane potential, and Ca handling were determined in myocytes using confocal microscopy. Specific markers confirmed reduced autophagy. In aged myocytes, ROS production was increased and mitochondrial membrane was depolarized; both parameters were exacerbated with beta‐adrenergic agonist isoproterenol. Also with isoproterenol, aged myocytes showed decreased Ca transient amplitude, SR Ca load, and latency of spontaneous Ca waves, which were reversed with the mitochondria‐specific antioxidant mito‐TEMPO. These data suggest that age‐related attenuation of autophagy may lead to mitochondrial dysfunction that underlies increased thiol‐oxidation of RyRs. This process could explain the reduced RyR refractoriness leading to spontaneous Ca release in senescent cardiomyocytes making aged hearts more prone to arrhythmia.
Prostaglandin E2 signaling plays an important role in the regulation of the cell cycle progression in C2C12 myoblasts
Authors: Mo Chenglin , Vallejo Julian , Isaacson Janalee , Yang Zhipeng , Wetmore Lori , Allen Tara , Igwe Orisa , Brotto Marco
Published: June 2021

Skeletal muscle myogenesis is a multiple‐stage process, including quiescent satellite cells activation, myoblasts proliferation, withdrawal from the cell cycle, and myogenic differentiation to form multinucleated myotubes. We recently demonstrated that PGE2 signaling is critical in regulation of myogenic differentiation. However, the effect of PGE2 signaling on myoblast proliferation cycle and withdrawal from the cell cycle has not been studied. For this purpose, we employed flow cytometry to analyze the cell cycle of C2C12 myoblasts following treatment with the specific EP1 and EP4 inhibitors, SC 51322 and L161,982, respectively. C2C12 myoblasts were firstly synchronized in G0/G1 phase by serum starvation, followed by treatment with SC51322 and L161,982. Approximate 48% of myoblasts stayed in G0/G1 phase in the control group, while the percentage increased to 65% and 63% after SC51322 and L161,982 treatments for 12h. After 24h, significant inhibition on C2C12 proliferation was observed for both EP1 and EP4 inhibitors. However, the same treatment did not affect C2C12 myoblasts cell cycle withdrawal after 24h of differentiation. Our data demonstrated that PGE2 signaling is important for cell proliferation and cell cycle progression, but not required for cell cycle withdrawal prior to early differentiation.
Hormone Sensitive Lipase knockout mice have higher Post Exercise Insulin Sensitivity despite accumulation of diacylglycerol
Authors: Serup Annette Karen , Alsted Thomas , Wojtaszewski Jørgen , Kiens Bente
Published: June 2021

Aim

To evaluate an accumulation of diacylglycerol (DAG) on post exercise insulin sensitivity.

Method

Insulin‐stimulated 2‐deoxyglucose uptake, using a submaximal insulin concentration, was measured in incubated mouse soleus (SOL) and extensor digitorum longus (EDL) muscle from wildtype (WT) and HSL Knock Out (KO) mice 90 min after completion of 40 min treadmill running.

Results

Post exercise insulin stimulated glucose uptake was 14% and 25% higher in SOL and EDL respectively in HSL KO compared to WT. Intramyocellular triacylglycerol was reduced to the same extent in both WT and HSL KO (p<0.01). Muscle DAG was 40% higher after exercise in HSL KO mice compared to WT (p<0.05), there were no differences in muscle DAG levels at rest. Phosphorylation of AMPK thr172 and ACC ser212 were not increased post exercise in both genotypes. Insulin induced signaling (phosphorylation of AKT ser473 and AKT thr308) was similar in both genotypes in both SOL and EDL. Total GLUT 4 protein expression was 20% and 25% higher in SOL and EDL respectively (p<0.05) in HSL KO compared to WT.

Conclusion

Post exercise insulin sensitivity was higher in HSL KO despite accumulation of DAG. Moreover, the data suggests that reduced fatty acid availability for mitochondrial oxidation plays a role in the observed increased post exercise insulin sensitivity.
The Role of Skeletal Muscle Tribbles 3 (TRB3) on Endoplasmic Reticulum (ER) stress‐ and high fat diet‐induced insulin resistance
Authors: Koh Ho‐Jin , Toyoda Taro , Jung Michelle M , Lee Min‐Young , Hirshman Michael F , Goodyear Laurie J
Published: June 2021

ER stress has been linked to insulin resistance in multiple tissues; however, whether ER stress causes insulin resistance in skeletal muscle has not been explored. ER stress has also been reported to increase TRB3 expression in multiple cell lines. Here, we report that high fat feeding in mice for 6 weeks, and obesity and type 2 diabetes in humans, significantly increased TRB3 expression (1.9‐fold in mice; 1.7‐ and 1.9‐fold in human) and ER stress markers in skeletal muscle. Overexpression of TRB3 in C2C12 muscle cells and mouse tibialis anterior muscles significantly impaired insulin‐stimulated IRS1 Y612 (67% and 22%) and Akt T308 (27% and 23%) phosphorylation. Incubation of C2C12 cells and extensor digitorum longus muscles with ER stressors thapsigargin and tunicamycin increased TRB3 expression and impaired insulin‐stimulated IRS1 Y612 (30–46%) and Akt T308 (35–37%) phosphorylation and glucose uptake (23–36% and 45%), effects reversed in cells with TRB3 RNAi and muscles from TRB3KO mice. Furthermore, TRB3KO mice were protected from high fat diet‐induced insulin resistance in skeletal muscle as TRB3KO mice showed lower body weights (11%) and fasting blood glucose concentration, improved glucose tolerance test (35%), and increased insulin‐stimulated glucose uptake and signaling after 8 weeks on a high fat diet. These data demonstrate that TRB3 mediates ER stress‐induced insulin resistance in skeletal muscle.
Do incisor and molar periodontal ligament mechanoreceptors stimulate antral motility to different degrees?
Authors: Lorber Mortimer
Published: June 2021

Teeth are not ankylosed to their sockets. A periodontal ligament (PDL) lies between each root and its socket. PDL movement during dental occlusion activates its mechanoreceptors (PDLMs)linked to the vagal motor n. to simulate antral motility (Lorber, Can J Physiol Pharmacol 67:29–35, 2000). 10 fasted adult female rats had three 5 min periods: basal, procedure and post‐proc. The procedure was vertical jaw movements at a rate of 1/s. In five rats, only back teeth contacted as in chewing. In the others the edge of a tongue blade between incisors simulated biting. Molar antral motility values were 3.20 ±1.10 (s.d.), 8.40 ± 3.28, 8.60 ± 1.34. Incisor values were 4.80 ± 1.92, 7.40 ± 4.98, 7.40 ± 3.05. Molar occlusion was significant by the Friedman ANOVA, P = 0.032, but incisor occlusion was not, P = 0.074.
Regulation of glucose metabolism by nesfatin‐1
Authors: Li Ziru , Li Yin , Zhang Weizhen
Published: June 2021

Nesfatin‐1 is an 82‐amino acid anorexigenic peptide which also contributes to the regulation of glucose homeostasis. Mechanisms by which nesfatin‐1 modulates glucose metabolism remain largely unknown. Our studies showed that chronic infusion of nesfatin‐1 by a subcutaneous micro‐pump for two weeks significantly improves the glucose tolerance in both lean and high fat diet induced obese mice, while central administration of nesfatin‐1 demonstrated no effect. Plasma insulin increased significantly after nesfatin‐1 infusion in normal chow diet mice, while no further increment was observed in obese mice. Analysis of insulin signaling revealed that both levels of basal and insulin‐stimulated AKT phosphorylation in the skeletal muscle were markedly increased by nesfatin‐1 relative to the control treatment. Consistent with the alteration of pAKT, the expression and membrane translocation of glucose transporter 4 in the skeletal muscle were significantly increased by nesfatin‐1. In conclusion, nesfatin‐1 may improve glucose metabolism by regulating phosphorylation of AKT in skeletal muscle.
Enhanced autophagic flux contributes collagen production in hepatic stellate cells involving TGFbeta1/Smad pathway signaling
Authors: Chen Xing‐Zhou , Yao Wen‐Min , Zhang Guo , Ye Hui‐Lan , Chen Li , Zhang Fa‐Can
Published: June 2021

Autophagy is a conserved lysosomal degradation pathway, which has been shown to be involved in fibrogenesis in multiple organs such as kidney, heart, lung, skin and liver. However, the precise mechanism by which autophagy participates in the hepatic fibrosis remains unclear. Thus this present study was firstly designed to dynamically monitor the specific morphological change of autophagy in primary cultured hepatic stellate cells (HSCs) during the fibrogenesis process. By using fluorescence microscopy and transmission electron microscopy technique, it was found that TGFbeta1 dose‐ and time‐dependently induced increments in autophagosome numbers in HSCs. Secondly, real time RT‐PCR and western blotting was respectively performed to analyze autophagy, apoptosis and TGFbeta1/Smad signaling molecules. In the presence of rapamycin (100nM), an autophagic agonist, Atg5, beclin1, LC3‐II, Smad3 and collagen type I was found to be substantially up‐regulated in HSCs with a caspase3‐independent pattern. On the contrary, either 3‐methyladenine (3MA, 5mM) or hydroxychloroquine (HCQ, 32μg/mL), antagonists for autophagy, was found to dramatically attenuate the impact of rapamycin on the proliferation of HSCs, follow by a corresponding decline in smad3 level and cellular collagen production as well. Furthermore, small interference RNA targeting Atg5, beclin1 was added to pre‐incubate HSCs respectively. It was showed that both Atg5‐ and beclin1‐siRNA amazingly impaired TGFbeta1‐induced expression of collagen type I in these cells. Taken together, our findings indicate a critical working pattern of autophagic flux in HSCs in response to TGFbeta1 and thereby provide a novel intracellular basis for antifibrotic drug development in the future. (Supported by NSFC Grants 30630145, 881172260, 200903 to G.Z. and GWZ200958 to F.Z.)
Mechanisms Underlying Modulation of SLC26A3 Activity by Lactobacillus acidophilus‐secreted Soluble Factors
Authors: Borthakur Alip , Priyamvada Shubha , Kumar Anoop , Gujral Tarunmeet , Natarajan Arivarasu A , Alrefai Waddah A , Dudeja Pradeep K
Published: June 2021

DRA (Down‐Regulated in Adenoma, SLC26A3) mediates apical membrane Cl−/HCO3− exchange activity in mammalian intestinal epithelial cells. Our previous studies showed that soluble factor(s) in the bacteria‐free culture supernatant (CS) of the probiotic Lactobacillus acidophilus (LA) stimulated Cl−/HCO3− exchange activity in Caco‐2 cells via increasing apical membrane DRA levels. Current studies were undertaken to elucidate the mechanisms of LA‐CS enrichment of functional DRA on the apical cell surface. Post‐confluent Caco‐2 monolayers were treated for 3 h with LA‐CS. DRA function was measured as DIDS‐sensitive 125I‐uptake, surface DRA assessed by cell‐surface biotinylation/confocal microscopy. Inhibitors of PI3 kinase and Rac1, as well as methyl‐β‐D‐cyclodextran, a lipid raft‐disrupting agent, abrogated LA‐CS effects on DRA function. LA‐CS enhanced DRA levels in the detergent insoluble (DI) fraction in Caco‐2 cells. Silencing the PDZ protein NHERF2 attenuated LA‐CS enhancement of apical membrane DRA. Molecular size fractionation showed that soluble factors in the LA‐CS that enhance functional DRA on the apical membrane are of low molecular mass (3–10 kD) and are heat‐stable. Our results define novel mechanisms involving lipid rafts, NHERF, and PI3K‐Rac1 pathway in altered trafficking of DRA induced by probiotic‐secreted bioactive factor(s) to modulate intestinal chloride absorption. [Grant support: VA/NIDDK/Bill & Melinda Gates Foundation]
Leucine and methionine share a Na/K‐dependent amino acid transporter in shrimp hepatopancreas
Authors: Duka Ada , Ahearn Gregory A
Published: June 2021

Hepatopancreatic brush border membrane vesicles (BBMV), from Atlantic White shrimp (Litopeneaus setiferus), were used to characterize 3H‐L‐leucine influx and how other essential amino acids and the cations, Na and K, interact with this transport system. L‐leucine uptake occurred against transmembrane concentration gradients in both Na and K media, suggesting that either cation could drive amino acid accumulation. L‐methionine at 20 mM, significantly inhibited 2 min uptakes of 1 mM L‐leucine in both Na‐ and K‐containing incubation media. L‐leucine influxes in both NaCl and KCl media were hyperbolic functions of [L‐leucine], following the Michaelis‐Menten equation. In NaCl, L‐leucine influx displayed a low Km (high affinity) and low Jmax, while in KCl the transport exhibited a high Km (low affinity) and high Jmax. L‐methionine was a competitive inhibitor of L‐leucine influxes in both NaCl and KCl media, (increase in L‐leucine influx Km, but no response in L‐leucine influx Jmax). Potassium was a competitive inhibitor of Na co‐transport with L‐leucine, increasing L‐leucine influx Km in the presence of Na, but having no effect on L‐leucine influx Jmax. Shrimp BBMV transport L‐leucine by a methionine‐shared carrier system stimulated by either Naor Kacting as co‐transport drivers binding to shared activator sites. Agriculture and Food Research Initiative Competitive USDA Grant no. 2010–65206‐20617.
Short duodenal acid exposure elicits protective HCO3− secretary response via Slc26a3, Slc26a9 and CFTR activation and NHE3 inhibition
Authors: Seidler Ursula Elisabeth , Singh Anurag Kumar , Liu Yongjian , Riederer Brigitte , Engelhardt Regina , Thakur Basant Kumar , Soleimani Manoocher
Published: June 2021

The duodenum expresses three apical solute carrier 26 (Slc26) anion transporters. In this study, we assessed the protective HCO3− secretory response to luminal acid in the duodenum of anesthetized Scl26a3, Slc26a6, Slc26a9, and Na+/H+ exchanger isoform 3 (NHE3) knockout (KO) and wildtype (WT) mice, and compared it to that elicited by the proteinkinse A agonist forskolin (FSK). While basal HCO3− secretory rates were reduced in the absence any of the three Slc26 isoforms, the HCO3− secretory response to FSK was not significantly reduced. In contrast, the HCO3− secretory response to a 5 min exposure to luminal acid, pH 2.5, was strongly reduced in the absence of Slc26a3 as well as of Slc26a9 expression, but not of Slc26a6 expression. CFTR inhibitor Inh‐172 reduced the first phase of the acid‐induced HCO3− secretory response, while NHE3 inhibition abolished the sustained phase of the duodenal HCO3− secretory response. Luminal acid exposure resulted in the activation of multiple intracellular signaling pathways, including SPAK, AKT and p38 phosphorylation. It induced a biphasic trafficking of NHE3, first rapidly into the brush border membrane, followed by later endocytosis. We conclude that the long‐lasting duodenal HCO3− secretory response to luminal acid exposure activates multiple duodenocyte signaling pathways and involves changes in trafficking/activity of CFTR, of members of the Slc26 family as well as of NHE3.
Transgenic Mice Containing two Haplotypes of Human Angiotensin Receptor Type 1 Shows Differential Gene Expression and Regulation of Blood Pressure
Authors: Jain Sudhir , Pandey Varunkumar G. , Kumar Ashok
Published: June 2021

Most of the physiological action of renin angiotensin system (RAS) are mediated through the angiotensin receptor type 1 (AT1R). Human AT1R gene promoter has four single nucleotide polymorphisms (SNPs). Our studies have shown that variants ‐ 777T, ‐680T, ‐214A, and ‐119A almost always occur together in Caucasian subjects, creating haplotype I (TTAA or Hap I) and II (AGCG or Hap II) respectively. Our genomic DNA analysis have shown that Hap I of the hAT1R gene is associated with hypertension in Caucasian subjects (p< 0.001). Our transient transfection studies in adrenal cortical (H295R) and vascular smooth muscle (VSMC) cells show that Hap I of hAT1R gene has increased promoter activity as compared to Hap II. In order to understand the physiological effect of these haplotypes in an in vivo situation, we have generated transgenic mice containing 160Kb long hAT1R BAC DNA and differ only in 1.2Kb promoter, containing either Hap I or Hap II. Our studies show that kidney and brain tissues of 8 weeks old transgenic mice containing Hap I have increased hAT1R mRNA levels (6.32 and 8.51 folds respectively) and hAT1R protein (2.2 and 3.2 folds respectively). We also show that transgenic mice containing Hap I of hAT1R gene have a significantly increased systolic blood pressure (10mmHg) as compared to Hap II. Our results suggest that increased expression of hAT1R gene with Hap I may be a contributing factor for hypertension in Caucasian subjects.

Research Support: NIH grants HL081752 and HL66296 to AK
Production of angiotensin within the SFO is sufficient to increase ERK1/2 and CREB activity in the SFO and PVN
Authors: Coble Jeffrey , Grobe Justin , Cassell Martin , Sigmund Curt D
Published: June 2021

Release of angiotensin II (ANG) within the brain increases fluid intake and sodium appetite. We sought to determine the sufficiency of ANG production in the subfornical organ (SFO) to increase fluid intake and sodium appetite by breeding mice expressing human renin under the neuron specific promoter, synapsin (sR), with mice expressing a conditionally inducible human AGT (hAGT) construct (ARed; double transgenic sRARed). Injection of adenovirus encoding Cre‐recombinase (AdCRE) into the lateral ventricle (ICV) of sRARed mice significantly increases hAGT transcript production within the SFO compared to other cardiovascular brain regions (p<0.05, n=12). One month after ICV AdCRE in sRARed mice total fluid intake and sodium appetite (0.436±0.04 mL/g/day; 1.721±0.087 mEq/day) increased compared to littermate controls (0.176±0.04 mL/g/day, P<0.001; 1.257 mEq/day, p<0.05; n=6) in a two‐bottle choice paradigm (water vs. 0.3M NaCl). After ICV AdCRE we observed a relative increase in immunostaining for ERK1/2 and CREB, but not p‐38 MAPK activity in the SFO and paraventricular nucleus (PVN) of sRARedcompared to littermate controls. In conclusion, we demonstrate that activation of ANG production specifically in the SFO is sufficient to increase ERK1/2 and CREB activity in the SFO and PVN, and to increase total fluid intake and sodium appetite.
Anti‐hypertensive effect of chronic ICV angiotensin‐(1–7) in (mRen2)27 transgenic rats is blocked by Mas antagonist.
Authors: Kangussu Lucas Miranda , Santos Robson A.S. , Campagnole‐Santos Maria Jose
Published: June 2021

Overactivity of the renin‐angiotensin system, mainly through Ang II/AT1 axis, is critical in the pathogenesis of cardiovascular diseases. Ang‐(1–7)/Mas axis at the central nervous system may exerts a counter‐regulatory role in hypertension. In this study, Sprague Dawley (SD) and transgenic hypertensive rats (mRen2)27 (TG), instrumented with telemetry probes for arterial pressure (AP) measurements were subjected to 14 days of lateral ventricle (ICV) infusion of Ang‐(1–7) (200 ng/h) or Ang‐(1–7) associated with Mas receptor antagonist (A779, 1mg/h) or saline (0.5 ml/hour) through osmotic mini‐pumps. Ang‐(1–7) ICV attenuated hypertension of TG rats (144±8 mmHg vs 174±3 mmHg, before) similarly during the day and night. The fall in AP was accompanied by a decrease in heart rate only during the day (348±5 beats/min vs 367±6 beats/min). The AP levels were still higher than those of SD rats (102±2 mmHg). Ang‐(1–7) anti‐hypertensive effect was completely blocked by A779 (174±12 mmHg). The increased levels of atrial natriuretic peptide and brain natriuretic peptide in the heart of TG rats were significantly reduced by Ang‐(1–7) infusion and reversed by the concomitant infusion of Ang‐(1–7) and Mas receptor antagonist. These data indicate that the anti‐hypertensive effect induced by chronic increase in Ang‐(1–7) in the brain of (mRen2)27 rats is mediated by Mas receptor.
Acute mineralocorticoid receptor blockade leads to decreased vascular endothelial function in healthy older adults
Authors: Kim Han Kyul , Hwang Moon‐Hyon , Yoo Jeung‐Ki , Luttrell Meredith J. , Meade Thomas H. , English Mark W. , Christou Demetra D.
Published: June 2021

Mineralocorticoid receptors (MR) modulate vascular endothelial function (VEF) in chronic heart failure and in healthy young adults, but their influence in healthy older adults is unknown. To test the hypothesis that MR modulate VEF in healthy aging, we administered acute MR blockade (MRB: 100 mg Eplerenone for 2 days) in a randomized, double‐blind, placebo‐controlled cross‐over study in 22 adults (8 men, 61±1 y; mean±SE, 53–79 y) free from clinical disease. We measured VEF (brachial flow‐mediated dilation [FMD]; ultrasonography), plasma oxidized LDL and vascular endothelial cell protein expression (VECPE) of eNOS, phosphorylated Ser1177 eNOS (PeNOS), NADPH oxidase (p47‐ phox), nitrotyrosine (NT), and TNF‐α (biopsy coupled with quantitative immunofluorescence) after MRB and placebo. In the overall group, FMD decreased (6.9±0.5 vs. 5.6±0.6%, P=0.02; Eplerenone vs. placebo) with MRB. However, MRB had no effect on oxidized LDL and VECPE of eNOS, PeNOS, p47‐phox, NT and TNF‐α (P≥0.4). Greater decreases in FMD were associated with lower baseline white blood cell and neutrophil (r=0.49, P=0.02 and r=0.57, P=0.006, respectively) and greater lymphocyte (r=−0.53, P=0.01) levels. In conclusion, MR modulate VEF in healthy older adults in an immune cell‐specific manner. MR activation appears to be physiologically important for maintaining vascular function.

Supported by NIH Grant AG032067 & AHA Grant 0865117F.
The Antidiabetic Activities of the Aqueous Leaf Extract of Phyllanthus amarus In Some Laboratory Animals
Authors: Adedapo Adeolu Alex , Ofuegbe Sunday Oluwaseun
Published: June 2021

The Antidiabetic Activities of the Aqueous Leaf Extract of Phyllanthus amarus In Some Laboratory Animals

Aims

Phyllanthus amarus has gained popularity in many continents as an herbal remedy hence its aqueous leaf extract is being assessed for its safety potential and antidiabetic properties in some laboratory animals.

Methods

The anti‐diabetic activities of the aqueous extract of the plant were assessed using some standard tests as well as histological changes in liver, kidney and pancreas. Diabetes mellitus was induced in rats using alloxan while glibenclamide at 0.2mg/kg was the reference drug used in this study. Acute toxicity was carried out to determine safe doses for this plant extract.

Results

The extract at 200 and 400mg/kg body weight caused significant reduction of fasting blood glucose, produce significant reduction in the oral glucose tolerance test, marked effect in the hypoglycaemic activity test, and pronounced reduction on the glucose level of diabetic rats. Histopathologically, the architecture of the pancreas appeared intact in the extract‐treated group. The study also showed recovery of the damaged liver cells in the extract‐ treated group.

Conclusion

The plant extract produced antidiabetic activities that are comparable to that of Glibenclamide, and may have validated the basis for the traditional use of Phyllanthus amarus as antidiabetic agent.
Lycopene Inhibits Cyclooxygenase‐2 And Inflammatory Mediator Expression In Microglia
Authors: Lin Hsiao‐Yun , Lin Ho , Lu Dah‐Yuu
Published: June 2021

Microglia is the main immune defense in the central nervous system. Activation of microglia leads to production excessive inflammatory molecules and deleterious consequences for neuronal death. Lycopene, one of the major carotenoids present in tomatoes, shown to exert antioxidant properties and inhibition of cancer cell proliferation. However, the effect of lycopene on neuroinflammatory responses in microglia still remains unknown. In this study, we investigated the signaling pathways involves in lycopene‐inhibited cyclooxygenase‐2 (COX‐2) and inflammatory mediator expression in microglia cells. Here, we demonstrate that lycopene inhibites lipopolysaccharide (LPS)‐induced COX‐2 and inflammatory mediator expression through heme oxygenase‐1 (HO‐1) activation. Our data also demonstrates that stimulation of lycopene increases the phosphorylation of serine–threonine liver kinase B1( LKB1), Calcium/calmodulin‐dependent kinase II( CaMKII) and AMP‐activated protein kinase (AMPK) in microglia. Interestingly, we also found that activation of phosphorylation‐AMPK by lycopene accumulates in the nucleus in microglia. Moreover, pre‐incubated cells with HO‐1 or AMPK pharmacological inhibitors effectively reversed the inhibitory effect of lycopene on LPS‐induced COX‐2 expression. Furthermore, transfection cells with HO‐1 or AMPK siRNA significantly attenuated the inhibitory effect of lycopene as well. These findings suggest that lycopeneinhibites LPS‐induced COX‐2 expression is mediated by HO‐1 activation through the AMPK pathways. Our results also indicate that lycopene is a promising nature product and may be as a useful pharmacological agent for the treatment of neuroinflammation‐associated disorders.
Increased Inducibility of Ventricular Tachycardia in the Type I Diabetic Akita Mouse is Associated with Abnormalities of Ca2+ Homeostasis and Reversed by Pravastatin.
Authors: Jin Hongwei , Rajab Mohammad , Aronovitz Mark , Wang Bo , Park Ho‐Jin , Link Mark , Galper Jonas
Published: June 2021

Diabetes mellitus is associated with increased risk of sudden cardiac death. It's relationship to the development of Ventricular Tachycardia (VT) is not understood. It has recently been suggested that Inhibitors of 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase (statins) might exert anti arrhythmic effects. It is hypothesized that treatment of Type I diabetic Akita mice with Pravastatin decreases the inducibility of VT. Inducibility of VT in Akita mice in response to programmed ventricular stimulation was significantly increased (78.6%) compared to WT (28.6%, p=0.006). Pravastatin treatment for one week (i.p. 20mg/day) decreased the inducibility of VT to 36.84% (p<0.01). Whole cell patch clamp studies of isolated ventricular myocytes from Akita mice demonstrated a high frequency of early and delayed afterdepolarizations ( EADs and DADs) compared to WT type. Pravastatin treatment significantly decreased occurrence of EADs and DADs. Ca2+ reuptake as measured by Ca2+ transient decay time (Tau) was increased in cells from Akita (170.9±10.3 ms) vs WT (138.6±7.3ms, p<0.05) and cytosolic free Ca2+ was increased. Pravastatin significantly improved SR Ca2+ reuptake (Tau=142±5.9ms, p<0.05), decreased cytosolic free Ca2+. Hence Pravastatin might exert and anti‐arrhythmic effect in Type I diabetes by improving Ca2+ homeostasis.
Stromelysin‐1(MMP3) Worsens the Neurovascular Injury in Hyperglycemic Stroke.
Authors: Hafez Sherif , Abdelsaid Mohammed , Coucha Maha , Ergul Adviye
Published: June 2021

Introduction

Acute hyperglycemia (HG) worsens stroke outcomes and increases the risk of cerebral hemorrhage especially with the co‐administration of tissue plasminogen activator (tPA). Matrix metalloproteinase 3 (MMP3) mediates tPA‐induced hemorrhagic transformation (HT) after stroke. However, the role of MMP3 in hyperglycemic stroke is unknown. The goals of the study were to evaluate the impact of the severity of HG 1) on stroke outcomes and 2) on MMP3 activity and expression.

Methods

Mild and severe HG were induced by glucose injections in rats that were subjected to 90 min middle cerebral artery occlusion and 22.5 h reperfusion. Stroke outcome and cerebrovascular MMP3 activity were assessed. Brain microvascular endothelial cells (BVEC) are subjected to oxygen glucose deprivation (OGD) followed by HG and MMP3 expression was measured.

Results

Severe HG significantly increased the infarct size. Mild elevation in blood glucose has significantly increased HT and MMP3 activity in cerebral vasculature. HG increased MMP3 expression in BVEC and hypoxia potentiated this effect.

Conclusion

Mild elevation in blood glucose worsens the vascular injury without increasing the infarct size. Up‐regulation of MMP3 in the cerebrovasculature and endothelial cells suggests that MMP3 highly contributes to the HG induced vascular injury.
Biased signaling by proteinase‐activated receptor 1 (PAR1) via activation with neutrophil and cockroach serine proteinases: tracking of distinct receptor dynamics with dual fluorochrome tagged receptors
Authors: Hollenberg Morley D. , Polley Danny , Ramachandran Rithwik , Mihara Koichiro
Published: June 2021

PAR1 is activated by the unmasking of a ‘tethered’ receptor‐activating sequence by thrombin or by a PAR1‐activating peptide (TFLLR‐NH2). Both agonists cause calcium & MAPKinase (MAPK) signalling. However, we found that neutrophil elastase (NE), proteinase‐3 (PR3) and cockroach allergen trypsin (CAT) disarm PAR1 for thrombin signaling, but selectively stimulate MAPKinase (but not Ca++). We hypothesized that this biased PAR1 signalling by NE, PR3 & CAT would correlate with distinct receptor dynamics (clustering‐internalization). To analyze the dynamics of biased PAR1 activation, we expressed PAR1 with (1) an N‐terminal mCherry red tag that is removed by proteinase, but NOT peptide activation and (2) a C‐terminal EYFP green tag which is retained upon activation by either peptide or enzyme. Dually‐tagged mCherry/EYFP‐PAR1 was visualized as a yellow halo at the cell membrane. PAR1 activation by thrombin caused internalization of ‘green‐labeled’ PAR1, lacking its mCherry signal; whereas TFLLR‐NH2 caused internalization of ‘yellow’ receptor that retains both fluorophores. In contrast, NE, PR3 and CAT activation removed the mCherry tag, leaving the ‘activated’ YFP‐tagged ‘green’ receptor retained at the cell surface. We conclude that differential biased PAR1 signaling correlates with its differential trafficking (internalization: calcium & MAPK vs. membrane retention: MAPK only). Support: CIHR Canada
SKF83959 is a partial agonist, not a functionally selective dopamine D1 receptor ligand as commonly believed
Authors: Lee Sang‐Min , Murthy Vishakantha , Mailman Richard
Published: June 2021

Full dopamine D1 agonists have shown promise in treating several central nervous system disorders. D1‐mediated adenylate cyclase (AC) activation reflects canonical signaling, but some reports have suggested that some D1 ligands also signal via phospholipase C (PLC) activation. It has been proposed and accepted that SKF83959 is a functionally selective D1 agonist that has high intrinsic activity at D1‐mediated PLC activation, but antagonism of D1‐mediated AC activation. Because of anomalies in the pharmacological evidence, we rigorously characterized the pharmacology of SKF83959 in vitro. Contrary to what is commonly assumed, SKF83959 has partial agonist activity at AC activation similar to the prototypical D1 partial agonist, SKF38393. Both SKF83959 and SKF38393 also have partial agonist activity for D1‐mediated β‐arrestin recruitment. Most importantly, no activation of PLC activation was found for either SKF83959 or SKF38393 even at very high pharmacological concentrations (≤300 μM). Thus, these data clearly demonstrate that SKF83959 is a potent low intrinsic activity D1 agonist at AC and β‐arrestin recruitment (consistent with its affinity for the D1 receptor), yet fails to activate PLC. These data show that SKF83959 is not a functionally selective ligand, that its behavioral effects can be explained by known mechanisms, and that its use as a novel probe of D1 receptors is ill‐founded.
The atypical antipsychotic clozapine induces 5‐HT2ARmediated signaling and behavioral events in a beta‐arrestin2‐ independent but Akt‐dependent manner
Authors: Schmid Cullen L , Streicher John M , Bohn Laura M
Published: June 2021

The G protein‐coupled serotonin 2A receptor (5‐HT2AR) is a prominent target for atypical antipsychotic drugs, such as clozapine. Although clozapine is known to inhibit 5‐HT2AR mediated G protein coupling, it differs from classic GPCR antagonists in that it also induces 5‐HT2AR internalization and activates Akt signaling via a 5‐HT2AR‐mediated event. The cognate neurotransmitter at the 5‐HT2AR, serotonin, also induces 5‐HT2AR internalization and Akt phosphorylation, events which require the interaction between the receptor and the intracellular scaffolding and regulatory protein, beta‐arrestin2 (βarr2). Moreover, the 5‐HT2AR/βarr2/Akt interaction is critical for serotonin‐induced, 5‐HT2AR‐mediated behavioral responses in mice. Therefore, we hypothesized that clozapine also utilizes a βarr2‐mediated mechanism to induce 5‐HT2AR signaling and regulatory events, as well as its behavioral effects in mice. Here, we demonstrate that, unlike serotonin, clozapine‐mediates 5‐ HT2AR internalization and Akt phosphorylation independent of receptor interactions with βarr2. Moreover, clozapine‐mediated suppression of MK‐801 and PCP‐induced hyperlocomotion is βarr2 independent, although it is dependent upon Akt. These results demonstrate that serotonin and clozapine utilize differential mechanisms to in the induction of similar events downstream of the 5‐HT2AR. The further elucidation of these mechanisms in vivo may allow for the development of more effective atypical antipsychotic drugs.

Funding from NIDA: DA025158
Visualizing trafficking of the adenosine‐A3 receptor using a fluorescent agonist: a tool for delineating intracellular signalling
Authors: Briddon Steve , Stoddart Leigh , Vernall Andrea , Kellam Barrie , Hill Steve
Published: June 2021

Activation of many G protein‐coupled receptors (GPCRs) leads to β‐arrestin binding and sequestration of the receptor from the cell surface. Whilst internalisation of a GPCR leads to termination of some aspects of receptor‐mediated signalling, recent reports have shown that some GPCRs continue to signal even after internalisation. Here, we show using a mutated version of the adenosine A3 receptor (W243F‐A3AR), that persistent signalling to the cAMP pathway requires receptor internalisation. To investigate this further we developed a fluorescent A3AR agonist, consisting of the agonist NECA conjugated to BODIPY‐630/650‐X via a dipeptide linker. This fluorescent agonist was highly potent at the A3AR ( pEC50 = 9.06 ± 0.17, n=4) and acted as a full agonist in Chinese Hamster Ovary cells expressing this receptor. Confocal microscopy showed a high degree of co‐localisation of the fluorescent agonist with a YFP‐tagged A3AR on the cell surface after a short incubation period (5 min). After longer incubation (30 min), A3AR‐YFP was sequestered from the cell surface to intracellular granules where co‐localisation with the fluorescent agonist was still observed. This fluorescent probe thus allows us to visualise A3AR trafficking and investigate how the receptor continues to signal to second messenger pathways upon internalisation.

We thank the Medical Research Council for financial support.
ADAM17 Regulates Sphingosine 1 phosphate receptor 1 Cell Surface Expression and Downstream Signaling
Authors: Desai Bhushan V , Ludwig Andreas , Malik Asrar B , Mehta Dolly
Published: June 2021

Sphingosine‐1‐phosphate receptor 1 (S1PR1) is a high affinity seven‐membrane G protein coupled receptor for bioactive lipid sphingosine‐1‐phosphate (S1P). S1PR1 plays a critical role in regulating several endothelial cell functions such as proliferation, migration and importantly enhances endothelial barrier function. However, mechanism regulating S1PR1 cell surface retention remains unclear. Members of the ADAM (a disntintegrin and metalloproteases) family of proteases are known for cleaving the ectodomain of a variety of cell surface receptors thereby regulating their activities. Here we addressed the possibility that ADAM family of proteases target S1PR1 and thereby downregulate it's signaling in endothelial cells. We show that treatment of human pulmonary arterial endothelial (HPAE) cells with phorbol‐12‐myristate 13‐acetate (PMA), an activator of ADAM proteases, induced S1PR1 cleavage within 2 hrs, while treatment with a broad range MMP inhibitor (TAPI‐0), stabilized S1PR1 expression. Additionally, PMA‐cleaved S1PR1 failed to localize on the cell surface rendering it refractive to exogenous S1P ligation and in enhancing the endothelial barrier. Interestingly, overexpression of ADAM17, but not ADAM10, in HPAE cells inhibited S1PR1 cell surface expression identifying a novel role of ADAM17 as the protease responsible for targeting S1PR1 and regulating its downstream signaling.
Affinity and Selectivity of Luzindole Analogs in Mouse and Human MT1 and MT2 Melatonin Receptors Transiently Expressed in Mammalian Cells
Authors: Popovska‐Gorevski Marina , McGowan Kathleen A. , Dubocovich Margarita L.
Published: June 2021

The goal of this study was to compare the binding affinity and selectivity of luzindole [(2‐benzyl N‐acetyltryptamine (NAT)] analogs [5‐methoxy‐NAT; 2‐benzyl‐N‐propionyl‐AT (N‐0889); pmethoxy‐benzyl‐NAT (N‐0890); 2‐p‐methyl‐benzyl‐NAT (N‐0891)] in the mouse (m) and human (h) MT1 and MT2 melatonin receptors transiently expressed in COS‐7 cells. Melatonin competed for 2‐[125I]‐iodomelatonin binding for the mouse [Ki (nM) for mMT1: 0.89 and mMT2: 0.52] and the human [Ki (nM) for hMT1: 0.78 and hMT2: 0.37] melatonin receptors with equal affinity. Ki values for luzindole [Ki (nM) for hMT1: 492.3 and mMT1: 504.7] and its analogs to compete for mMT1 and hMT1 were very similar. However, the affinity of luzindole [Ki (nM): 12.1 vs. 740.3], N‐0889, N‐0890 and N‐0891 for hMT2 receptors was significantly higher than for the mMT2 receptor. N‐0889, luzindole, N‐0890, and N‐0891 showed higher selectivity affinity ratios (Ki MT1/MT2) for the hMT2 than the hMT1 melatonin receptors having 31, 41, 56, and 91 fold differences, respectively. In contrast, their selectivity affinity ratios for the mMT1 and mMT2 melatonin receptors were identical. These results show significant differences between the human and the mouse melatonin receptor in terms of the affinities and selectivity ratios of luzindole and its analogs. We conclude that caution is needed when affinities and selectivity ratios are extrapolated to different species.