bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of Ric‐8A activity by phosphorylation (843.4)
Authors: Papasergi Makaia , Gabay Meital , Tall Gregory
Published: June 2021

Ric‐8A is required for the biosynthetic folding of Gαi/q/13. Ric‐8A is also a guanine nucleotide exchange factor (GEF) that stimulates in vitro Gα subunit GTP binding. Although Ric‐8A is crucial for Gα folding, and therefore, proper G protein heterotrimer function, mechanisms by which Ric‐8A itself is regulated have yet to be elucidated. We experimentally identified multiple stoichiometrically phosphorylated sites of recombinant Ric‐8A produced in Sf9 cells and demonstrate that endogenous mammalian Ric‐8A is a phosphorylated protein. Recombinant Ric‐8A was enzymatically dephosphorylated, and the potency of Ric‐8A stimulation of Gα nucleotide exchange was measured. Dephosphorylated Ric‐8A was substantially less potent than phosphorylated Ric‐8A in stimulation of Gαq and Gα13 nucleotide exchange, whereas Gαi activity was largely unaffected by Ric‐8A phosphorylation status. These results were corroborated by comparing the potency of phosphorylated Ric‐8A produced in Sf9 cells to non‐phosphorylated Ric‐8A produced in E. coli. To determine the functional significance of Ric‐8A phosphorylation, identified phospho‐sites and additional sites predicted to be phosphorylated using the Scansite engine were mutated alone or in combination to alanine or phospho‐mimetic aspartic acids. Mutant Ric‐8A proteins were purified and will be tested using GEF and Gα subunit folding assays in cell‐free lysates. The significance of Ric‐8A phosphorylation will be further evaluated in cells. Ric‐8A‐/‐ mouse embryonic stem cells have greatly reduced Gα levels that are rescued by stable Ric‐8A expression. Using this complementation assay we identified Ric‐8A phospho‐mutants that do not rescue the Ric‐8A‐/‐ Gα abundance defects.

Grant Funding Source: NIH RGM0882428
RGS2 protein degradation is mediated by a novel Cullin 4B/F‐box 44 E3 ligase complex (843.8)
Authors: Sjogren Benita , Swaney Steven , Neubig Richard
Published: June 2021

Hypertension and heart failure are major health issues and there is a need to identify more effective treatments and novel drug targets. Regulator of G protein Signaling 2 (RGS2) is highly expressed in both heart and vascular smooth muscle and regulates Gαq signaling. RGS2‐/‐ mice are hypertensive, show enhanced responses to vasoconstrictors and lower tolerability to pressure overload. Over‐expression of RGS2 reduces cardiac hypertrophy and we have shown that pharmacologically enhanced RGS2 protein expression has functional effects on G protein signaling. Therefore, we hypothesize that enhancing RGS2 protein expression is a novel route in treating cardiovascular disease.

RGS2 is rapidly degraded through the proteasome, but the enzymes involved are not known. Thus, the goal of the current study was to identify the molecular machinery responsible for RGS2 protein degradation. Utilizing a β‐galactosidase complementation assay that efficiently quantifies levels of a protein we performed an siRNA screen to identify genes that regulate RGS2 protein levels.

We identified components of a putative cullin‐RING E3 ligase (CRL). siRNA knock‐down of either the substrate recognition component F‐box 44 or cullin 4B (CUL4B), but not cullin 4A, resulted in a significant increase in RGS2 protein levels and stability and overexpression of F‐box 44 CUL4B decreased RGS2 protein levels. We also demonstrated co‐immunoprecipitation of F‐box 44 with both RGS2 and CUL4B.

RGS proteins are difficult drug targets due to their mode of action being through protein‐protein interactions. Inhibiting a more druggable protein within the degradation pathway could be a way to increase RGS2 protein levels and function.

Grant Funding Source: Supported by NIH‐DA023252 and The Swedish Heart‐ and Lung Foundation
The ABC transporter Mrp4/Abcc4 is required for Leydig cell protection from chemotherapeutic drugs (844.1)
Authors: Morgan Jessica , Schuetz John
Published: June 2021

The thiopurines are widely used to treat a variety of diseases such as pediatric acute lymphoblastic leukemia, adult promyelocytic leukemia, and irritable bowel disease. In both human and rodents, one toxic consequence attributed to 6‐mercaptopurine (6MP) is detrimental reproductive effects related to impaired testosterone (T) production. The ABC transporter Mrp4 plays a protective role against 6MP toxicity as it exports 6MP nucleotide metabolites. As Mrp4 is highly expressed in human and rodent Leydig cells (LCs), we hypothesize it protects LCs against 6MP by extruding nucleotides from cells, promoting LC survival to maintain normal T synthesis. Administration of 6MP to Mrp4+/+ and Mrp4‐/‐ mice produced a strong reduction in LCs, testicular T levels, and increased LC death in Mrp4‐/‐ mice. However, as LCs are post‐mitotic, the mechanism of death is unrelated to 6MP nucleotide incorporation into DNA. We show that cultured Mrp4‐/‐ LCs are highly susceptible to 6MP, exhibiting cytotoxicity at doses 6‐fold lower than Mrp4+/+ LCs. Leydig cell death by 6MP appears to involve a mitochondrial pathway as a pan‐caspase inhibitor blocked 6MP‐mediated death. We will report on the mechanisms of this 6MP‐mediated mitochondrial death. An important implication of our study is humans harboring non‐functional Mrp4 alleles may be at risk for LC toxicity and pathophysiological consequences of reduced T concentrations.

Grant Funding Source: This work is supported by grants from the National Institutes of Health (NIH) and ALSAC.
Interaction between riboflavin and BCRP‐mediated drug transport in the mammary gland (844.2)
Authors: Leon‐Cheon John , Dedina Liana , Yang Mingdong , Harper Patricia , Ito Shinya
Published: June 2021

Breastfeeding provides numerous benefits to a child’s growth and development. However, in a lactating mother, expression of Breast Cancer Resistance Protein (BCRP) is significantly increased in the mammary gland. BCRP is an efflux transporter and is responsible for the transport of many different substrates from natural carcinogens (ex. PhIP found in charred meats) to chemotherapeutic drugs. As a result, this poses a risk of exposure to potentially harmful BCRP substrates from a mother’s milk to her child. We hypothesized that riboflavin, a vitamin substrate for BCRP, can act as a potential competitor for BCRP‐mediated excretion of drugs/toxins into milk. We used intravenous topotecan and cimetidine at a dose of 1 and 5 mg/kg as model BCRP substrates in lactating mice. In mice pretreated with 5 mg/kg riboflavin, excretion of topotecan and cimetidine into milk at 30 min postdose was significantly reduced. Mean topotecan levels in the milk were 883 ng/ml (SD: ±465; n=10) in the control and 682 ng/ml (SD: ±316; n=10; p<0.01) in the riboflavin treatment group. For cimetidine, the mean milk levels were 1941 ng/ml (SD: ±387; n=7) in the control, compared to 1555 ng/ml (SD: ±192; n=7; p<0.05) in the riboflavin‐pretreated mice. The milk‐to‐plasma ratios of topotecan and cimetidine in riboflavin‐pretreated mice also showed a significant 35% and 26% decrease respectively. In conclusion, our findings indicate that riboflavin administration does reduce the excretion of other BCRP substrates into a mother’s milk.

Grant Funding Source: Supported by CIHR
Memory deficits and learning skills improved in transgenic mouse model of Alzheimer's disease after date‐rich diet supplementation (845.4)
Authors: Subash Selvaraju , Essa Musthafa , Awlad‐Thani Kathiya , Al‐Adawi Samir , Al‐Asmi Abdullah , Vaishnav Ragini , Guillemin Gilles
Published: June 2021

Although there are no proven ways to delay onset or slow progression of Alzheimer's disease (AD), studies suggest that diet can affect risk. Dates palm fruit are good sources of minerals (Se, Cu, K and Mg), vitamins (C, A, B6, B9, B2, B3), dietary fiber and total phenolics and natural antioxidants (anthocyanins & ferulic acid). Polyphenols have been shown to be neuroprotective in different model systems. We asked whether dietary supplementation with 2% dates or 4% dates would influence behavior and AD like pathology in a transgenic mouse model. The four month old mice with double Swedish APP mutation (APPsw/Tg2576) were purchased from Taconic Farm, NY, USA. The experimental groups of APP‐transgenic mice from the age of 4 months were fed custom‐mix diets (pellets) containing 2% dates or 4% dates. The control groups, i.e. tg mice and wild‐type mice were fed diet without dates. Animals from all groups were undergone behavioral assessments at the age of 4‐5 months and 14‐15 months by Morris water maze test (for spatial memory and learning ability), T maze test (for position discrimination learning ability), rota rod test (for psychomotor coordination), elevated plus maze test (for anxiety‐related behavior) and open field test (anxiety) to analyze the effect of diet rich in dates on learning skills, memory, anxiety, locomotors activity, and motor coordination. Both the diets rich in 2% dates & 4% dates fed tg mice showed a significant improvement in memory, learning ability, anxiety and motor development compared to the AD tg mice on diet without 2% dates & 4% dates. The more effect was observed in 4% dates diet fed animals than 2% fed. Our results suggest that dietary supplementation of date palm fruits may have beneficial effect in reducing the risk, delaying the onset or slowing the progression of Alzheimer's disease.This project was supported by a research grant from The Research Council, Oman (RC/AGR/FOOD/11/01).

Grant Funding Source: Supported by a research grant from The Research Council, Oman (RC/AGR/FOOD/11/01).
Characterizing and treating pulmonary pathology in Marfan syndrome (847.2)
Authors: Sellers Stephanie , Chan Rayleigh , Hirota Jeremy , Mielnik Michael , Hogg James , Bernatchez Pascal
Published: June 2021

Marfan syndrome (MFS) is a connective tissue disorder that leads to life‐threatening complications. While current research focuses on the cardiovascular pathogenesis of MFS, MFS also gives rise to serious pulmonary disease, the study of which is severely lacking. Therefore, we aimed to characterize lung function in a murine model of MFS and investigate the pharmacological effectiveness of Losartan, an angiotensin II receptor type I blocker, and Sildenafil (Viagra), a phosphodiesterase type 5 inhibitor. Our studies demonstrate that MFS mice have significant dilation of the pulmonary artery with highly significant changes lung function measurements as well as significant airspace widening and lung destruction. Notably, Losartan was able to prevent airspace widening and Sildenafil was effective at preventing pulmonary artery dilation but did not improve lung function. This study is the first to report changes in the pulmonary artery and in vivo lung function measures in MFS and investigate the use of Losartan and Sildenafil. As such, it elucidates key aspects of MFS pathogenesis and pharmacological treatment and guides future investigations of pharmacological interventions for MFS pulmonary dysfunction.

Grant Funding Source: Supported by The Canadian Institutes of Health Research
Access to high fat chow, but not sucrose or saccharin, enhances development of locomotor sensitization to cocaine in adolescent female rats (848.8)
Authors: Serafine Katherine , Bentley Todd , France Charles
Published: June 2021

Eating high fat chow enhances cocaine‐induced locomotion and sensitization in adolescent female rats; it is not known whether consumption of other preferred substances (e.g., sucrose or saccharin) also increases sensitivity to cocaine. It is also not known whether daily access to these feeding conditions is necessary to change sensitivity to cocaine, or if weekend access (i.e., 2/7 days per week) is sufficient. Adolescent female Sprague‐Dawley rats had free access to high (34.3%) fat or standard (5.7% fat) chow and free access to water, 10% sucrose solution, or 0.1% saccharin solution for 5 weeks. Rats that had weekend access to high fat chow, sucrose or saccharin had access to water and standard chow on other days. Cumulative doses of cocaine (1‐17.8 mg/kg) enhanced locomotion, and this effect increased in all groups after repeated tests. Although the rate of sensitization was comparable between groups, eating high fat chow daily (but not 2/7 days per week) significantly enhanced cocaine‐induced locomotion compared to controls. Neither daily nor weekend access to sucrose or saccharin enhanced cocaine‐induced sensitization. These results demonstrate that access to high fat chow, but not sucrose or saccharin, enhances development of locomotor sensitization to cocaine in adolescent female rats. As such, vulnerability to abuse cocaine might be increased in adolescent females that eat high fat food.

Grant Funding Source: CPF supported by NIH Grant K05 DA017918 and KMS supported by NIH grant T32 DA031115
Endothelial nlrp3 inflammasome activation associated with lysosomal destabilization during coronary arteritis (851.5)
Authors: Chen Yang , Boini Krishna , Pitzer Ashley , Zhang Yang , Li Pin‐Lan
Published: June 2021

The present study aimed at exploring the mechanisms by which NOD‐like receptor protein 3 (Nlrp3) inflammasomes are activated to result in endothelial dysfunction and coronary arteritis induced by Lactobacillus casei (L. casei) cell wall fragments (LCWE). Endothelial dysfunction and vascular lesion were observed during coronary arteritis in mice treated with LCWE, as shown by media thickening, increases in vascular cell adhesion protein 1 (VCAM‐1) expression, and enhanced adhesion of inflammatory cells to the endothelium including macrophages, neutrophils and T cells. Accompanied with these changes, the inflammasome activation was also shown in the coronary arterial endothelium of these mice, which was characterized by a marked increase in caspase‐1 activity and interleukin‐1β (IL‐1β) production. In isolated cultured endothelial cells (ECs), LCWE induced Nlrp3 inflammasome formation, caspase‐1 activation and IL‐1β production, which were blocked by Nlrp3 gene silencing or lysosome membrane stabilizing agents such as colchicine, dexamethasome, and C2‐ceramide. However, blockade of two activating pathways of Nlrp3 inflammasomes by glibenclamide, a potassium channel blocker or N‐Acetyl‐L‐cysteine (NAC), an oxygen free radical scavenger or antioxidant had no effects on LCWE‐induced inflammsome activation. It was also found that LCWE increased EC lysosomal membrane permeability and triggered a release of lysosomal cathepsin B into the cytosol and that silencing of cathepsin B blocked LCWE‐induced Nlrp3 inflammasome formation and activation in ECs. It is concluded that LCWE activate endothelial Nlrp3 inflammasomes through enhanced lysosomal membrane permeabilization and consequent release of lysosomal cathepsin B in ECs. This activation of endothelial Nlrp3 inflammasomes during LCWE stimulation contributes to the development of coronary arteritis.

Grant Funding Source: Supported by NIH grants HL057244, HL091464 and HL075316
The role of Ca2+ influx pathways in voltage‐dependent STOC production (853.10)
Authors: Hashad Ahmed , Mazumda Neil , Harraz Osama , Welsh Donald
Published: June 2021

Ca2+ sparks are generated in a voltage dependent manner to initiate spontaneous transient outward currents (STOC) that antagonize arterial constriction. In the present study, we defined the mechanisms by which membrane depolarization increases Ca2+ sparks and subsequent STOC production. Using perforated patch clamp electrophysiology and rat cerebral arterial myocytes, we monitored STOC in the presence and absence of agents that modulate Ca2+ entry pathways. Beginning with the CaV3.2 inhibitor Ni2+, STOC frequency decreased in cells held at hyperpolarized voltage (‐40 mV). In contrast, CaV1.2 inhibition with nifedipine suppressed STOC frequency at depolarized potential (‐20 mV). These findings are consistent with the voltage‐dependent profiles of L‐ and T‐type Ca2+ channels; and were replicated in a computational model of Ca2+ spark production. Upon concomitant CaV1.2 and CaV3.2 blockade, we further observed residual voltage‐independent STOC production. This residual component was insensitive to stimulation or inhibition of TRPV4 channels. Further ongoing work aims to investigate the involvement of other Ca2+ entry routes. In summary, distinct Ca2+ channel isoforms govern the voltage‐dependency of STOC production and they do so at defined membrane potentials. These findings have important mechanistic implications to the basis of negative feedback, arterial tone regulation and blood flow control.
Cardiovascular responses to chronic intermittent hypoxia in rats are not affected by previous exposure to hypoxia (856.9)
Authors: Perim Raphael , Bonagamba Leni , Machado Benedito
Published: June 2021

Rats exposed to chronic intermittent hypoxia (CIH) develop hypertension. In the present study we evaluated the effect of a previous protocol of CIH on the cardiovascular responses produced by a new protocol of CIH. Male Wistar rats (45 ‐ 50 g) were divided in 3 groups: Control group (n=24), maintained under normoxia during 35 days; CIH group (n=18), maintained under normoxia during 25 days, followed by 10 days of CIH (6% O2 for 40 s every 9 min, 8 h/day); Pre‐CIH group (n=19), exposed to CIH for 10 days, followed by 15 days under normoxia and then a second exposure to CIH for additional 10 days. At the end of the protocol, rats were instrumented for arterial pressure recording. CIH and Pre‐CIH presented a significant increase in mean arterial pressure (108 ± 2 and 107 ± 2 mmHg, respectively) when compared to the control group (98 ± 1 mmHg), but the increase in both groups exposed to CIH was similar. We conclude that previous exposure to CIH produced no effects on the cardiovascular response to a new protocol of CIH.

Grant Funding Source: FAPESP, CAPES, CNPq
Selective pathways of innate immune responses of heart and kidney during angiotensin II hypertension (857.1)
Authors: Singh Madhu , Cicha Michael , Chapleau Mark , Abboud Francois
Published: June 2021

Angiotensin II (AngII) infusion induces cardiac hypertrophy and hypertension through pro‐inflammatory gene expression in heart and kidney. Because cardiac hypertrophy and hypertension may be uncoupled, we tested the hypothesis that the pathways of activation of innate immune receptors might differ in these phenomena. Specifically, we asked whether (1) MyD88, the ubiquitous adaptor protein of most toll‐like receptors (TLR), and (2) TLR4, that has been implicated in hypertension, are both necessary for the responses to AngII. In an earlier study, we reported that MyD88‐/‐ mice are protected from cardiac hypertrophy and pro‐inflammatory gene expression after myocardial infarction. In contrast, our current results with AngII infusion (733 ng or 3000ng/kg/min) indicate that the pressor response and cardiac hypertrophy seen in WT mice were preserved in MyD88‐/‐ mice. Pro‐inflammatory gene expression was increased in both heart and kidney of WT and MyD88‐/‐, and IL‐6, Mmp9, TGF‐β1 and TGF‐β2 RNA expression increased similarly in MyD88‐/‐ and WT kidney. AngII receptor (AT1R), Nox4, TNF‐α and CTGF were greater in MyD88‐/‐. Thus the pro‐inflammatory effects of AngII are MyD88‐independent. In TLR4‐deficient mice, the AngII induced cardiac hypertrophy was abrogated but the pressor response was enhanced. The systolic pressure after 3 weeks of AngII (1000 ng/kg/min) were 133 mm Hg in WT and 155 mm Hg in TLR4‐deficient mice with corresponding HW/BW ratio of 7.13 and 5.49 (P= 0.03). We conclude that AngII induced cardiac hypertrophy is TLR4 mediated and not dependent on MyD88. The translational relevance is that the molecular targets for the abrogation of end‐organ damage may differ from those for blood pressure reduction in hypertensive disease.

Grant Funding Source: NIH PPG HL14388
A novel role of Meox1 in regulating TGFβ‐induced smooth muscle cell differentiation (866.3)
Authors: Dong Kun , Hsu Amanda , Guo Xia , Chen Shi‐You
Published: June 2021

Mesenchyme homeobox 1 (MEOX1) is expressed in the early developing somite and derivatives during embryogenesis. Somite has been shown to be one of the origins or progenitors for SMC. Therefore, in this study, we studied the role of MEOX1 in SMC differentiation. Smooth muscle cell (SMC) differentiation is an important process during vasculogenesis and angiogenesis. It is well known that alterations of VSMC phenotype play a role in the progression of several cardiovascular disorders including atherosclerosis, hypertension, and restenosis. However, the molecular mechanisms controlling the differentiation and development of VSMC are largely unknown. We found that transforming growth factor‐β (TGF‐β) induces MEOX1 expression in the initial phase of SMC differentiation of pluripotent mesenchymal C3H10T1/2 cells. MEOX1 expression appears to be mediated by both PI3 kinase and Smad3 signaling pathways because blockade of these two pathways by specific inhibitors leads to a decreased expression of MEOX1 and SMC markers. Importantly, knockdown of MEOX1 by specific shRNA suppresses TGF‐β‐induced expression of SMC early markers including SM22α and calponin. MEOX1 overexpression, on the other hand, increases SMC marker expression. These results indicate that MEOX1 is a novel regulator for TGF‐β‐induced SMC differentiation.
Biomechanical regulation of leptin expression in vascular smooth muscle both in vivo and in vitro: a novel vascular‐proteomic and physiological approach (866.9)
Authors: Al Outa Amani , Farhat Zein , Soudani Nadia , Kobaissy Firas , Mechref Yehia , Zhu Rui , Sabra Ramzi , Zeidan Asad
Published: June 2021

Hypertension is considered one of the major risk factors in developing cardiovascular diseases. Hemodynamic abnormality in hypertension is associated with structural and functional alterations which are mainly mediated via leptin. Molecular mechanisms associated with leptin‐mediated blood vessels remodeling both in vivo and in vitro have not been fully elucidated. In this study, we investigated the effect of partial portal vein ligation (PPVL) on leptin expression in vascular smooth muscle (VSMC) and secretion. We used blood vessels organ culture under mechanical stretch in order to investigate molecular mechanisms of leptin‐induced VSMC remodeling. Data showed that leptin expression and ROS production significantly increased in VSMC after 7 and 14 days post PPVL. A significant increase in serum leptin levels was observed in PPVL. Exposing the blood vessels to mechanical stretch (mimicking hypertension) significantly increased nuclear translocation of GATA‐4 and NFAT which were attenuated by the co‐administration of an anti‐leptin receptor antibody and the RhoA inhibitor C3. In addition, vascular‐proteomics platform was performed assessing differential proteome/systems biology profile between stretched and unstretched blood vessels to investigate the underlying mechanisms involved in vascular hypertrophy. A differential display of proteins were identified (7 proteins were upregulated vs. 12 downregulated) using PANTHER (Protein Analysis Through Evolutionary Relationships) system and Pathway Studio 8. Our results indicate that the activation of GATA‐4 & ROS production plays a pivotal role in hypertension signaling, leading to leptin synthesis and VSMC hypertrophy.

Grant Funding Source: Supported by CNRS and MPP at AUB
Mitochondrial dynamics and mitochondrial dysfunction in mesenteric artery: role of homocysteine (867.11)
Authors: Familtseva Anastasia , Kalani Anuradha , Kamat Pradip , Tyagi Neetu , Tyagi Suresh
Published: June 2021

Many studies recently focused on the role of mitochondria in pathogenesis of vascular diseases, but little is known about influence of HHcy on the mitochondrial dynamics, whether it causes mitochondrial dysfunction and vascular alterations. We hypothesize that HHcy alters mitochondrial dynamics which leads to mitochondrial dysfunction following vascular alterations and increased blood flow resistance. To test this hypothesis, we used 12 weeks old C57BL/J6 mice (WT); Cystathionine‐β‐synthase heterozygote knockout (CBS) with genetic HHcy; C3H/HeJ (C3H), which is resistant to atherosclerosis and double‐transgenic CBS/C3H mice. Genotyping was used for the background of CBS and CBS/C3H mice. Western blot (CBS, eNOS, Mfn‐2 and Drp‐1), RT‐ PCR (Bcl‐2, BAX, caspase 3 and caspase 9), ATP/O2 consumption, mitochondrial membrane potential, blood pressure and blood flow measurements in mesenteric artery, Immunohistochemistry (CD31, connexins 40), elastin and collagen staining were also used in this study. Our data showed increase in mitochondrial fission marker (Drp‐1) and collagen in CBS compared to WT and C3H mice. In addition, we observed significant down regulation of Mfn‐2 (mitochondrial fusion marker), CD31, eNOS, connexins 40 (gap junction protein) and elastin in CBS as compared to WT and C3H mice. Interestingly, in CBS/C3H we found that fusion marker, eNOS, CD31 were increased and fission was decreased in mesentery compared to CBS mice. In addition, our data showed the signs of mitochondrial dysfunction (increased caspase 3 and 9) in CBS mice. To conclude, our data suggests that HHcy increased mitochondrial fission causing mitochondrial dysfunction and mesenteric artery alterations with increased blood flow resistance.

Grant Funding Source: National Institutes of Health grants HL‐074185
Carotid body denervation reduces renal sympathetic nerve activity and fibrosis, and increases renal blood flow in congestive heart failure (875.14)
Authors: Marcus Noah , Del Rio Rodrigo , Schultz Harold
Published: June 2021

Background: In congestive heart failure (CHF), carotid body (CB) chemoreceptor activity is enhanced and contributes to increased sympathetic nerve activity (SNA). Decreases in cardiac output and increased sympathetic outflow to the kidneys (RSNA) may reduce renal blood flow (RBF), and contribute to renal fibrosis (RF) which in turn may lead to development of cardio‐renal syndrome. Hypothesis: We hypothesized that denervation of the CB chemoreceptors (CBD) would reduce RSNA and improve RBF and markers of RF in CHF. Methods and Results: In a coronary artery ligation (CAL) model of CHF, Sprague‐Dawley rats display a 40.3±6.3 % reduction in kidney blood flow (measured by echo‐doppler of the renal artery). CHF rats with CBD performed early during the progression of CHF(2 weeks post CAL) showed 17.8±8.5 % reduction in blood flow to the kidney. Accordingly, kidney fibrosis (as quantified by Masson’s trichrome staining) was evident in CHF rats, and CBD reduced the collagen deposition in the kidney by 50%. In a rapid ventricular pacing model of CHF, New Zealand White rabbits have an increased resting RSNA after the development of CHF, and this is decreased after CBD (25%±1max pre‐pace, 43±5%max CHF, 23±2%max CHF‐CBD). Renal blood flow (measured by Transonic flow probe) decreased by 28% with the development of CHF and was restored after CBD. Conclusions: These results indicate that CB chemoreflex contributes to alterations in RSNA and RBF.

Grant Funding Source: Supported by NIH #1F32HL108592‐01A1 and NIH #PO1‐HL62222.
Sleep loss changes neuropeptidase expression and activty in rat brain (876.3)
Authors: Visniauskas Bruna , Dalio Fernanda , Simões Priscila , Mazzacoratti Maria da Graça , Oliveira Vitor , Tufik Sergio , Chagas Jair
Published: June 2021

The effect of sleep deprivation on proteolytic activity in the central nervous system has not been frequently addressed in the literature. We have started to study the changes on peptidase activities in animal models of paradoxical sleep deprivation (PSD), mainly focusing on metallopeptidases like thimet oligopeptidase (EP24.15) known to process neuropeptides. In the present study, we demonstrated that PSD in rats induces changes both in the expression and activity of EP24.15 in the male rat hypothalamus and striatum tissue extracts. In the hypothalamus, the significant decrease in activity and mRNA levels, after PSD, was only totally reversed after 96h of sleep recovery. In the striatum, albeit significative, changes in mRNA and activity do not parallel changes on protein content. Neurotensin, GnRH, dynorphins, enkephalins, orphanin/nociceptin are among the many neuropeptides whose balance can be modified by the modifications on EP24.15 activity and can be, at least partially, related with many of the physiological changes observed in PSD and during the sleep recovery periods.

Grant Funding Source: Supported by FAPESP, CNPq and AFIP
Creating a virtual pharmacology curriculum in a PBL‐intensive, integrated learning environment (88.1)
Authors: Dowhower Karpa Kelly
Published: June 2021

Integrating pharmacology education into a problem‐based learning (PBL) curriculum has proven challenging for many medical schools. In response to pharmacology content deficiencies identified during an assessment of our PBL‐intensive curriculum in 2002‐2003, our institution hired a director of medical pharmacology instruction to oversee efforts to improve the structure of pharmacology instruction in the absence of having a stand‐alone pharmacology course. In this session, the development of a virtual pharmacology curriculum will be explored. This curriculum places emphasis on clearly stated and behaviorally oriented pharmacology learning objectives, pharmacology study guides that correspond to each PBL case, pharmacology review sessions that feature active student involvement in discussions of United States Medical Licensing Examination (USMLE)‐type questions, and increased student accountability through the addition of PBL‐related pharmacology questions on each organ‐based course examination. As a result of these initiatives, we have observed improvements in USMLE Step 1 scores and graduates’ ratings of pharmacology education as determined by the Medical School Graduation Questionnaire. These initiatives are relevant to other medical schools that are also seeking ways to better integrate pharmacology into PBL‐based curricula.
Maternal exercise increases energy efficiency of fetal heart (886.4)
Authors: May Linda , Barrera Daniel , Newcomer Sean , Agbas Abdul
Published: June 2021

Current research suggests exercise during pregnancy benefits the cardiovascular health of offspring. We have studied the effects of exercise during pregnancy on fetal cardiac mitochondrial ability to make energy. We hypothesized that regular maternal exercise during pregnancy increases the mitochondrial (energy) efficiency of fetal heart. Pregnant Duroc X Landrace cross pigs were subjected to exercise or not (controls). Offspring heart tissues were preserved at specific time points. Mitochondrial‐enriched pellets were obtained through differential centrifugation. Protein levels of OXPHOS enzymes, five complexes, were analyzed. Two of the OXPHOS enzyme levels (Complex‐II and ‐III) were decreased in the exercise group compared to controls. Enzyme kinetic analysis of Complex‐II indicated protein levels were low in exercise‐exposed offspring, but the specific activity level was significantly high (p蠄 0.001) compared to controls. There are trends of increased ATP production for exercise exposed fetal hearts. Our findings demonstrate a link between maternal exercise and increased mitochondrial efficiency to produce energy in offspring hearts. Our study begins to elucidate the benefits of maternal exercise on developing fetal hearts. These findings suggest that maternal exercise improves offspring hearts prior to birth and may help protect offspring from heart disease and heart failure later in life.
Kidney oxygenation in the subacute phase of ischemia‐reperfusion injury (890.1)
Authors: Evans Roger , Ow Connie , Abdelkader Amany , Hilliard Lucinda , Malpas Simon , Koeners Maarten
Published: June 2021

We determined the status of kidney tissue oxygenation during the subacute phase of renal ischemia reperfusion injury in 10‐12 week old male Sprague‐Dawley rats. Inner medullary oxygen tension (PO2) was determined using a carbon paste electrode attached to a telemetry transmitter, for 24 hours before and for 5 days after 60 min of bilateral renal ischemia (n=4) or sham (n=4) surgery. In a second protocol, renal tissue PO2 was measured by Clark electrode under thiobutabarbital anesthesia, either 1 or 5 days after recovery from renal ischemia (n = 6 and 3 respectively) or sham surgery (n = 5 and 3 respectively). Inner medullary tissue PO2 measured by telemetry was increased by 45 ± 15% 24 h after reperfusion, but then progressively fell over the next 5 days. Tissue PO2 measured by Clark electrode was well maintained 24 h after ischemia. However, outer medullary PO2 was 69% less 5 days after ischemia than after sham surgery. Our observations suggest that widespread renal hypoxia may not be obligatory in the subacute phase of renal ischemia‐reperfusion injury. However, medullary hypoxia may evolve over a period of days following reperfusion.

Grant Funding Source: Supported by the National Health and Medical Research Council of Australia
Rosiglitazone increases aquaporin 2 transcription and apical expression in renal cells: possible implication for treating nephrogenic diabetes insipidus? (892.10)
Authors: Procino Giuseppe , Milano Serena , Nicoletti Maria , Gerbino Andrea , Mastrofrancesco Lisa , Carmosino Monica , Svelto Maria
Published: June 2021

Aquaporin 2 (AQP2) is the vasopressin‐regulated water channel palying a crucial role in urine concentration in the kidney. Rosiglitazone (RGZ), an agonist of the peroxisome proliferator‐activated receptor gamma (PPARγ), increases AQP2 expression in normal rats. Here we tested whether RGZ could also modulate AQP2 intracellular trafficking in renal cells.

Wild type C57BL‐6 mice were treated for 3 days with 20mg/Kg RGZ to confirm the transcriptional effect on AQP2 in mice. In parallel, we used mouse MCD4 renal collecting duct cells to study the effect of RGZ on AQP2 trafficking to the apical membrane. Apical surface biotinylation and confocal analysis were used to semiquantify the effect. Single cell live microfluorimetry and FRET were used to measure changes in Ca2+ and cAMP intracellular concentrations, respectively.

In mice, RGZ induced a twofold increase of AQP2 compared to control mice.

In MCD4 cells, stimulation with 50µM RGZ for 30 min robustly increased AQP2 trafficking to the apical plasma membrane to an extent comparable to that elicited by forskolin stimulation. Interestingly, RGZ was able to increasely AQP2 phosphorylation at Ser256 concomitant with intracellular Ca++ increase and without apparent intracellular cAMP elevation.

Taken together these results suggest that RGZ might be useful to increase AQP2 transcription and apical trafficking in renal cells bypassing the stimulation of vasopressin receptor which is defective in pathological conditions as in Nephrogenic Diabetes Insipidus.

Grant Funding Source: Telethon GGP12040/ AIFA MRAR08P011
Mathematical model of epithelial Cl‐ secretion determining activity of electro‐neutral ion transporter (892.11)
Authors: Sasamoto Kouhei , Niisato Noami , Marunaka Yoshinori
Published: June 2021

Epithelial Cl‐ secretion plays key roles in prevention of our body from environmental changes by generating driving forces for transepithelial transport of ions and water; for instance, the Cl‐‐secretion‐driven water movement plays an essential role in protection of our body from bacterial and viral infection by producing the liquid covering epithelial apical membrane surface. Transcellular Cl‐ secretion across the epithelium is composed of two steps; 1) the Cl‐ entry step across the basolateral membrane and 2) the Cl‐ releasing step across the apical membrane. The purpose of the present study was to establish a mathematical model expressing characteristics of transcellular Cl‐ secretion via the following three pathways: 1) the Cl‐ entry step across the basolateral membrane into the intracellular space via an electro‐neutral ion transporter, Na+/K+/2Cl‐ cotransporter (NKCC); 2) the Cl‐ secretion step across the apical membrane from the intracellular space via Cl‐ channels; 3) the Cl‐ recycling step across the basolateral membrane via Cl‐ channels. We measured the transcellular Cl‐ secretion by an electrophysiological technique (short‐circuit current) and the apical/basolateral Cl‐ conductance in Ussing chamber by detecting them as NPPB (Cl‐ channel blocker)‐sensitive short‐circuit current and conductance. The mathematical model established in the present study is useful to determine the activity of electro‐neutral ion transporter, NKCC, by combing electrophysiological techniques, providing deep understandings in the research field of epithelial ion transport. Supported by JSPS.
Annexin A2 mediates apical trafficking of renal Na‐K‐2Cl cotransporter (892.22)
Authors: Dathe Christin , Daigeler Anna‐Lena , Jankowski Vera , Jankowski Joachim , Mutig Kerim , Bachmann Sebastian , Paliege Alexander
Published: June 2021

Transport activity of the thick ascending limb (TAL) Na‐K‐2Cl‐cotransporter (NKCC2) critically depends on its association with lipid raft membrane microdomains (LR). The mechanisms involved in the recruitment of NKCC2 into LR have not been elucidated so far. In the present study we hypothesized that the lipid raft scaffolding protein annexin A2 serves as an essential mediator in this process. Detergent‐resistant LR were isolated from rat renal outer medullary tissue samples using discontinuous sucrose density gradient and ultracentrifugation (floating assay). Interaction between NKCC2 and Anxa2 was analyzed by immunoprecipitation, GST pull down, and peptide spot assays. Distribution of the products in the apical TAL membrane was studied by immunofluorescence and immunogold electron microscopy. Trafficking was studied in AVP‐deficient rats and cultured TAL cells using the AVP analogue desmopressin (dDAVP) or low chloride hypotonic stress. Functional relevance of Anxa2 for NKCC2 trafficking was studied using RNAi‐mediated knockdown strategies. Floating assay, immunoprecipitation and colocalization studies confirmed the parallel presence of NKCC2 and Anxa2 in apical membrane LR of the TAL. Binding assays approved physical interaction of Anxa2 and the non‐phosphorylated N‐terminus of NKCC2. Moreover, dDAVP and low chloride hypotonic stress caused parallel trafficking of NKCC2 and Anxa2 in rats and in cultured TAL cells and this response was inhibited by RNAi‐mediated knockdown of Anxa2. In conclusion our data demonstrate that Anxa2 is an essential component of the NKCC2 trafficking protein complex and mediates its recruitment into LR microdomains.
Calcineurin signalling in the thick ascending limb of the kidney (892.30)
Authors: Borschewski Aljona , Dathe Christin , McCormick James A. , Lazelle Rebecca , Willnow Thomas E. , Jankowski Vera , Rosenberger Christian , Mathia Susanne , Ellison David H. , Bachmann Sebastian , Mutig Kerim
Published: June 2021

The calcineurin inhibitors cyclosporine and tacrolimus are widely used for immunosuppressive therapy but hypertension and electrolyte disorders often complicate their application. Hypertension during treatment with tacrolimus has been attributed to inhibition of calcineurin Aα and resultant hyperphosphorylation and activation of the Na+‐Cl‐‐cotransporter (NCC) of the distal convoluted tubule (DCT). Activity of the Na+‐K+‐2Cl‐‐cotransporter (NKCC2) of the thick ascending limb (TAL) is also modulated by the balance between phosphorylation and dephosphorylation reactions. Here we identify NKCC2 as a substrate of calcineurin Aβ and show that cyclosporine activates NKCC2. To further characterize the molecular pathway involved in NKCC2 dephosphorylation we have evaluated SORLA (sorting‐protein‐related receptor with A‐type repeats) knockout mice with nearly complete absence of phosphorylated NKCC2. We establish SORLA as trafficking factor that reduces the apical abundance of calcineurin in TAL thus facilitating NKCC2 phosphorylation. This study elucidates the molecular basis for calcineurin signalling along the TAL. Our results thus have substantial clinical implications for immunosuppressive therapy using calcineurin inhibitors.
MATE1 sequesters organic cations within an intracellular, bafilomycin‐sensitive compartment (892.39)
Authors: Martinez Lucy , Evans Kristen , Dantzler William , Wright Stephen
Published: June 2021

OC secretion across renal proximal tubules (RPTs) involves basolateral OCT2‐mediated uptake from the blood, followed by apical MATE1/2‐mediated efflux into the tubule filtrate. Whereas OCT2 is an electrogenic OC uniporter, MATE is an OC/H exchanger. Epifluorescence microscopy of hMATE1‐expressing CHO cells revealed accumulation of the fluorescent OC, N,N,N‐trimethyl‐2‐[methyl(7‐nitrobenzo[c][l,2,5]oxadiazol‐4‐yl)amino]ethanaminium (NBD‐MTMA) in the cytoplasm and in a smaller, punctate compartment; accumulation in OCT2 expressing cells was restricted to the cytoplasm. A second intracellular compartment was also evident in the multicompartmental kinetics of NBD‐MTMA efflux from MATE1‐CHO cells. Punctate accumulation (20 min) was markedly reduced by coexposure of MATE1‐CHO cells with 1 μM bafilomycin (BAF) and eliminated with 10 μM BAF. BAF had no effect on the initial rate of MATE1‐mediated uptake of NBD‐MTMA (10‐120 sec) suggesting that its effect involved collapse of endosomal pH gradients via inhibition of the V‐type H‐ATPase. The 15 min accumulation of the prototypic OC, [³H]TEA by isolated single non‐perfused rabbit RPTs was also reduced 40% by coexposure to 1 μM BAF. Thus, the native expression in RPTs of MATE protein within endosomes can increase steady‐state OC accumulation. Normal rates of apical endosome turnover may provide a significant parallel pathway for apical OC efflux

Grant Funding Source: NIH grant 5R01DK080801
Deletion of caveolin‐1 decreased the basolateral K channel activity in the early distal convoluted tubule (DCT1) and increased urinary Mg2+ excretion (893.18)
Authors: Zhang Chengbiao , Wang Lijun , Su Xiaotong , Wang Wen‐Hui
Published: June 2021

We used the patch clamp technique to examine the basolateral K channel in the isolated DCT1 of wt and cav‐1 knockout (KO) mice. We identified a 40 pS K channel in the DCT1 of wt (18 patches with K channels from total 35 patches) and of KO mice (25 patches with K channels in total 66 patches). Although K channel Po in DCT1 was similar in wt and KO mice, K channel number per patch was significantly lower in KO than those in wt mice. The whole‐cell K currents in the DCT1 of KO mice were also significantly lower than those of wt and the K reversed potential in DCT1 was less negative in KO mice. This suggests that deletion of cav‐1 decreased the basolateral K channel activity. The expression of c‐Src stimulates KCNJ10 channel, a major basolateral K channel in DCT1, only in cav‐1 cotransfected HEK‐cells but not in un transfected cells. Since the basolateral K channels provide the driving force for apical Mg2+ absorption, we used metabolic cage to examine renal Mg2+ transport in wt and KO mice. Although dietary Mg2+ intake was similar in wt and KO mice, the urinal Mg2+ excretion was significantly higher in KO mice than those of wt mice. Also, plasma Mg2+ concentration was lower (1.2 mg/dl) in KO than that of wt mice (2.2 mg/Dl ). We conclude that deletion of cav‐1 diminished c‐Src‐induced stimulation of KCNJ10 channels and depolarized the cell membrane potential in the DCT1 thereby impairing Mg2+ reabsorption in the DCT and causing hypomagnesaemia .

Grant Funding Source: NIH
Cell biological characterization of the Cohen syndrome‐associated protein COH1 (894.1)
Authors: Lommatzsch Stefanie , Seifert Wenke , Bachmann Sebastian , Kühnisch Jirko , Maritzen Tanja , Horn Denise , Haucke Volker
Published: June 2021

Mutations in COH1 (VPS13B) cause autosomal recessive Cohen syndrome, which is mainly characterized by mental retardation, progressive postnatal microcephaly, facial dysmorphism and progressive retinopathy/myopia, and intermittent neutropenia. Recently, our cell biological analysis identified COH1 as peripheral membrane protein of 3997 amino acids at the cytosolic site of the Golgi apparatus. Initial analysis showed that loss of COH1 upon RNAi impairs the ability of the Golgi ribbon to (re)assemble and thus induces fragmentation into mini‐stacks. Subsequential BFA‐treatment indicates that COH1 regulates the formation of Golgi‐derived membrane tubules consistent with a potential function in intracellular membrane traffic. To further investigate the intracellular function of COH1, we will estimate its impact on endo‐ and exocytosis by live cell imaging analysis of well‐established cargo proteins, such as GFP‐VSVGtsO45, Alexa488‐EGF, and Alexa555‐Tf. Complementary, we will assess a COH1‐protein interaction network with special focus on small GTPases, using different methods, e.g. GST‐pull downs and co‐immunoprecipitations followed by Western blot analysis and mass spectroscopy. The hitherto identified interaction partners become investigated related to their function in transportation, cell polarization, cell migration and ways of signal transduction in neuronal cells. In summary, our study until now identifies COH1 as a Golgi matrix protein required for maintaining Golgi integrity and function. We suggest that our results provide an improved insight into the molecular function of COH1 in Golgi‐associated membrane traffic which in the future may help to unravel its role in brain development, neuronal function, and general pathology in patients with Cohen syndrome.
The role of a UT‐A urea transporter in human small intestine (897.2)
Authors: McGrane Alison , Courtney Danielle , Winter Desmond , Baird Alan , Stewart Gavin
Published: June 2021

Urea transporters (UTs) encoded by the Slc14a1 (UT‐B) and Slc14a2 (UT‐A) genes facilitate the movement of urea across plasma membranes. Urea flux into the gastrointestinal tract enhances the growth of bacterial populations by providing a source of nitrogen, thus maintaining the symbiotic relationship between humans and their intestinal bacteria (Stewart 2011). A previous study identified the UT‐B1 urea transporter within the human colon, localized to the basolateral membrane of colonocytes (Collins et al. 2010). The aim of this present study was to investigate for the expression of UT‐A urea transporters in the human gastrointestinal tract. Using primers for UT‐A1, RT‐PCR analysis detected the expected RNA‐derived 682bp signal primarily in the small intestine, compared to stomach and colon. Using different samples of small intestine cDNA (AMS Biotechnology), UT‐A1 expression was shown to be higher in the jejunum and ileum than the duodenum. Western blotting experiments were then performed using human ileum tissue samples. Using a characterised rat UT‐A1 antibody (L194) (Nielsen et al. 1996), a 120kDa glycosylated UT‐A1 protein could be detected in protein samples from ileostomy patients. In contrast, ileum samples from surgical resection tissue showed UT‐A1 signals of around 170kDa. This study shows that UT‐A1 urea transporters are expressed in the human ileum. Since high levels of bacteria exist in this region, this suggests a physiological function for UT‐A1 in the urea nitrogen salvaging mechanism that is crucial for human gastrointestinal health.

Grant Funding Source: Supported by Science Foundation Ireland (SFI) and University College Dublin (UCD)