bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of high salt diet on age‐related hypertension and central AngII‐induced cardiovascular responses
Authors: Basgut Bilgen , Erdos Benedek , Whidden Melissa , Kirichenko Nataliya , Woods Mary , Tumer Nihal
Published: June 2021

Angiotensin II (AngII) plays an important role in both age‐related and salt‐induced hypertension. The aim of this study was to investigate the effects of age and high‐salt (HS) diet on resting blood pressure (BP) and heart rate (HR), as well as on central AngII‐induced cardiovascular responses. Young (5 mo) and old (27 mo) male F344xBN rats were fed regular chow or HS (8%) for 11 days. BP and HR were measured using radiotelemetry. AngII (10ng/μl/h) or CSF was infused intracerebroventricularly before and during HS for 3 days with osmotic pumps. Old rats had significantly higher BP (108.3±3.6 mmHg) than young (96.1±3.1 mmHg, p<0.05). HS diet caused significant increases in BP in both young and old groups (young: 99.0±3.5, p<0.01; old: 116.3±4.1, p<0.001), but the increase in BP was significantly higher in old rats (p<0.05). In contrast, HR didn't change significantly in either group in response to HS diet. AngII‐induced BP increases were significantly diminished in old rats (4.4±2.0 %) compared with young (12.6±1.3%, p<0.05), while HR responses were similar (15.8±4.0% and 13.5±2.1%). HS diet enhanced AngII‐induced BP and HR elevations in both age groups, but BP increases in old rats (5.9±1.7%) were still significantly lower compared with young (18.8±8.1%, p<0.05). Our findings indicate that sensitivity of BP to HS diet increases with age, and that central AngII‐induced pressor responses are diminished in old rats compared with young both under control conditions and during HS diet treatment.

Supported by the Medical Research Service of the Department of Veterans Affairs, by an award from the American Heart Association and by a TUBITAK scholarship.
The effects of menstrual cycle on cardiovascular response to mental stress and arterial stiffness
Authors: Sone Ryoko , Tan Nobusuke , Yamazaki Fumio
Published: June 2021

The present study examined the effects of menstrual cycle on arterial blood pressure and heart rate (HR) responses to mental stress and cardio‐ankle vascular index (CAVI). The CAVI is a new index of arterial stiffness recently developed by measurement of pulse wave velocity and blood pressure. Eight healthy young women performed the color word test for 2 min followed by CAVI measurement approximately every three days over three menstrual cycles. The differences between the baseline value and the averaged value of systolic (ΔSBP), mean (ΔMBP) and diastolic blood pressures during stress as well as HR during stress (ΔHR) were calculated, respectively. Compared to those during other phases, ΔSBP and ΔMBP showed a tendency to increase during the early follicular and late luteal phases, and these changes correlated with scores on the menstrual distress questionnaire (p<0.05) in a few subjects. The ΔHR and CAVI did not vary with the menstrual cycle. The pressor response to stress did not relate to the CAVI either. Our findings suggest that the effect of the menstrual cycle on pressor response to mental stress differs from person to person and may relate to premenstrual and menstrual symptoms and estrogen action, but arterial stiffness was not affected by the menstrual cycle.
Sympathectomy does not inhibit dexamethasone‐induced hypertension
Authors: Soto‐Pina Alexandra Estela , Kadapakkam Sheela , Mehring Cynthia , Hinojosa‐Laborde Carmen , Strong Randy
Published: June 2021

Dexamethasone (DEX) induces hypertension and increases the expression of tyrosine hydroxylase, the rate limiting step in catecholamine synthesis in the adrenal medulla. We have shown that DEX increases mean arterial pressure (MAP, 119 ± 0.9 mm Hg), and surgical removal of the adrenal medulla does not attenuate this response (120.4 ± 1.3 mm Hg). Our hypothesis is that DEX induces hypertension by systemic sympathetic activation. The purpose of this study was to determine the effect of sympathetic nerve ablation on DEX‐induced hypertension. Fischer 344 rats were implanted with radio‐telemetry transmitters to record MAP and heart rate (HR). After baseline recording, rats underwent chemical sympathectomy with 6‐hydroxydopamine (6‐OHDA, 20 mg/kg, i.p. for 3 days). 6‐OHDA reduced MAP by 7.2 ± 1.0 mmHg, and increased HR by 55±17 bpm. Subsequent treatment with DEX (0.1 mg/day for 7 days) elevated MAP by 12.2 ± 1.6 mmHg and reduced HR by 18±7 bpm. Similar to adrenalmedullectomy, 6‐OHDA did not abolish the hypertensive effects of DEX chronic treatment. This study will provide an insight about the role of the adrenal medulla and peripheral sympathectomy in glucocorticoid‐induced hypertension. Support provided by: Department of Veterans Affairs.
Sex differences in the sympathetic balance of blood pressure: the role of the α‐adrenergic receptors
Authors: Hart Emma C , Charkoudian Nisha , Wallin B Gunnar , Roberts Shelly K , Johnson Christopher P , Joyner Michael J
Published: June 2021

In men, muscle sympathetic nerve activity (MSNA) is positively related to total peripheral resistance (TPR) and inversely related to cardiac output (CO). However, this relationship was not observed in young women. We aimed to investigate whether simultaneous β‐adrenergic stimulation offsets this balance in young women. We measured MSNA (peroneal microneurography), arterial pressure (brachial line), CO (open‐circuit acetylene) and TPR before and after systemic β‐blockade with propranolol in 6 young normotensive women (age; 24 ± 2 yrs, body mass index; 23 ± 1 kg/m2). There was no difference in CO (5.7 ± 0.3 L vs. 5.3 ± 0.4 L), mean arterial pressure (MAP; 95 ± 3 mmHg vs. 95 ± 2 mmHg) before and after beta‐blockade. However, there was significant reduction in heart rate (HR; 61 ± 3 beats/min vs. 55 ± 2 beats/min, P<0.05). β‐blockade caused an increase in TPR (17.4 ± 1.2 mmHg/ml/min vs. 18.5 ± 1.3 mmHg/ml/min; P=0.07) and MSNA (28 ± 3 bursts/100hb vs. 33 ± 3 bursts/100hb, P=0.08). Before β‐blockade there was no relationship of MSNA to TPR, CO or MAP. Following β‐blockade, there was a positive relationship between TPR and MSNA (r=0.60). However, there was no relationship of MSNA to cardiac output (r = −0.12). Interestingly, MAP became related to MSNA (r = 0.56). In summary, the β‐adrenergic receptors appear to play a key role in the sympatho‐hemodynamic balance in young women.

AHA 09POST2170087
The effect of Na+‐Ca2+ exchange on the recovery from Ca2+ dependent inactivation of L‐type Ca2+ channel in cardiomyocytes in pulmonary vein
Authors: Kwon Jeong‐Hoon , Kim Won‐Tae , Kim Hyun‐Ah , Leem Chae Hun
Published: June 2021

Ca2+ dependent inactivation (CDI) of L‐type Ca2+ channel (CaL) is well known phenomena and Ca2+ release from SR greatly affected CaL kinetics, which was known as the release dependent inactivation (RDI). We would like to see whether L‐type Ca2+ kinetics were changed by Na+‐Ca2+ exchange (NCX) which would control the subsarcolemmal Ca2+. We used the isolated cardiomyocytes in main pulmonary vein of rabbit. RDI was induced by applying 2.5 msec prepulse from ‐40 mV to 10 mV and then the same amplitude of 50 msec test pulse was applied with various intervals. In the presence of 0.1 mM EGTA in the pipette solution, the inactivation tau (itau) was increased and the peak current amplitude (PCA) was decreased. As the interval was increased, itau and PCA were progressively returned to control ICaL. In the absence of bath Na+, the initial change was much larger and the recovery process was prolonged. In the presence of BAPTA 10 mM in the pipette solution, these phenomena were disappeared. Ryanodine or thapsigargin also attenuate these phenomena. In conclusion, NCX is an important regulator for the recovery of CaL from CDI by controlling subsarcolemmal Ca2+. (supported by NRF, 2009‐0076234)
Alternans of repolarization and depolarization in ventricular myocytes
Authors: Chourasia Sonam , Agarwal Anuj , Jing Linyuan , Patwardhan Abhijit
Published: June 2021

Several studies suggest that repolarization alternans play an important role in initiation of ventricular arrhythmias. While alternans of duration of repolarization, i.e. of action potential duration (APD), has been extensively investigated, little is known about alternans of amplitude of action potentials (APA). The purpose of our study was to examine the relationship between APD and APA alternans and to determine potential mechanisms of APA alternans. Transmembrane potentials were recorded using glass micro‐electrodes in swine ventricular tissue (n=8). From 51 trials we observed that the incidence of APD alternans was 33% of all action potentials recorded for cycle lengths &*8804; 290 ms. Alternans of APA occurred 50% of times when APD alternans occurred and were concordant (long action potentials were also tall) 98% times. Incidence of APD alternans along with APA alternans was 78% with 96% concordance. These results show that occurrence of APA alternans is not rare. Alternans of APA were accompanied by alternans of |dv/dt|max 77 % of times, suggesting that sodium current may underlie alternans of APA. The potential effect of alternans of depolarization phase of the action potential on conduction suggests that this may also play a role in loss of electrical stability.

Supported by the American Heart Association (Great Rivers Affiliate) and the National Science Foundation (CBET 07350).
Identification of the impact of contention on heart rate variability
Authors: Mongue‐Din Héloïse , Salmon André , Fiszman Marc Y , Fromes Yves
Published: June 2021

Several methods allow monitoring electrocardiogram (ECG) in small animals. Sedation may help to maintain animals; however anaesthetic drugs impair the heart rate variability (HRV). Implantable telemetry devices allow experiments in freely moving animals; but this technique requires surgery and thus a prolonged recovering period is necessary prior to ECG recording. Contention is an alternative to these methods, provided a better understanding of its influence on HRV. This study aimed at investigating the impact of contention on some HRV parameters.

Telemetric and restrained ECG of Syrian hamsters were performed with TA10ETA‐F20 sensors and the EasyCG system, respectively. Time domain analysis based either on statistical or autoregressive approaches showed no significant difference in HRV at baseline. Investigations in the frequency domain revealed that restrains influenced low frequencies, translating a higher sympathetic activity. Pharmacological assays confirmed that restraints affected the sympathovagal balance.

In conclusion, the imprint of contention on HRV was consistent with a shift of the sympathovagal balance towards a higher sympathetic tone.

Funding: Association Française contre les Myopathies
A single exposure to acrolein causes arrhythmogenesis, cardiac electrical dysfunction and decreased heart rate variability in hypertensive rats
Authors: Hazari Mehdi Saeed , Griggs Jennifer , Haykal‐Coates Najwa , Winsett Darrell , Farraj Aimen , Costa Daniel
Published: June 2021

Epidemiological studies demonstrate an association between cardiovascular morbidity, arrhythmias, and exposure to air toxicants such as acrolein. We hypothesized that a single exposure to acrolein would increase arrhythmias and cause changes in the electrocardiogram (ECG) of hypertensive rats. Wistar Kyoto (WKY) and spontaneously hypertensive (SH) rats implanted with radiotelemeters were exposed to either air or 3ppm acrolein for 3 hours, while heart rate (HR) and ECG were continuously monitored. When compared to WKY rats, SH rats had higher baseline HR, prolonged PR interval (PRi), QRS complex duration, and QTc. During acrolein exposure both WKY and SH rats had significantly increased HR, PRi, QTc, and JTc; only PRi returned to baseline levels in both strains after exposure. SH rats had a significant increase in non‐conducted P‐waves and atrial premature beats during exposure to acrolein, which were further augmented after exposure. Although increased, fewer arrhythmias were seen in WKY rats during the same period. Heart rate variability was significantly decreased in SH rats only after exposure, but remained unchanged in WKY rats. Air exposure did not affect any parameters in the WKY or SH rats. These findings suggest that a single exposure to acrolein can increase the sensitivity of the cardiac electrical conduction system and trigger arrhythmias, which may pose a particular health risk to people with cardiovascular disease. Funded by USEPA. (This abstract does not reflect EPA policy.)
Bcl‐2 −/− amd Bim −/− Lung Endothelial Cells Demonstrate Aberrant Adhesion and Migration
Authors: Sorenson Christine M , Sheibani Nader
Published: June 2021

Integration of adhesive, survival and proliferative processes is essential for capillary morphogenesis of endothelial cells (EC) and vascular development. Recent studies indicate that bcl‐2 family members can impact cell adhesion, migration and survival. Unfortunately, the molecular and cellular mechanisms involved are poorly defined. Here we examined how lack of bim and/or bcl‐2 expression impact lung EC function. Lack of bim or bcl‐2 had minimal impact on the steady state rate of apoptosis in lung EC. However, the absence of bcl‐2 or bim had a significant impact on EC adhesion and migration. Lack of bcl‐2 expression decreased lung EC migration, while lack of bim expression increased migration compared to their wild‐type counterparts. Decreased adhesion to fibronectin and vitronectin was observed in both bcl‐2 −/− and bim −/− lung EC, with bcl‐2 −/− EC having very little adhesion to either matrix protein. Capillary morphogenesis was greatly diminished in bcl‐2 −/− EC compared to wild‐type or bim −/− lung EC. We also observed aberrant production of extracellular matrix and eNOS expression in bcl‐2 −/− lung EC, which could contribute to their inability to undergo capillary morphogenesis. Thus, bcl‐2 family members play a central role in modulation of EC proangiogenic properties, which goes beyond their role as simple mediators of mitochondrial homeostasis and apoptosis. Supported by EY018179 and DK067120
Isolation and characterization of lectins from the coronary luminal membrane
Authors: Perez‐Aguilar Sandra , Torres‐Tirado David , Ramiro‐Diaz Juan , Knabb Maureen , Rubio Rafael
Published: June 2021

The luminal membrane of vascular endothelial cells is coated with a variety of proteoglycans, glycosaminoglycans, glycoproteins and glycolipids. These glycosylated structures sense alterations in flow stimuli to mediate myocardial responses. Several lectinic endothelial proteins, such as VCAM‐1, ICAM‐1, PECAM‐1, integrins, have been implicated in the response to flow, and other proteins that respond to flow have yet to be characterized as to their lectinic nature and include B2R, AT1R and the ENaC channels. To characterize the lectinic structures in guinea pig endothelial membranes, columns were produced with bound sugars of mannose, galactose or N‐ acetylglucosamine or a combination of the three sugars. The eluted lectin fraction was tested using dot blots with antibodies against flow‐sensitive proteins. The results show that approximately 10% of the eluted proteins loaded in columns with single sugar are lectins, and 15% of the protein loaded in the three sugar column are lectins. VCAM‐1, ICAM‐1, PECAM‐1 and ENaCs were all found in the eluent of individual sugars or a mix of the three sugars. These results suggest that there are important flow sensitive proteins that are lectinic in nature and present on the luminal surface of the vascular endothelium. Additionally, these results confirm the presence of lectinic ENaC channels in the endothelial glycocalyx.

Funded by CONACyT SEP‐42567, CONACyT‐SALUD 2004‐C01‐156, Fulbright‐Garcia Robles 9583
Neurophysiological mechanisms of recovery of locomotion after spinal injury
Authors: Edgerton V. Reggie , Roy Roland R. , Tillakaratne Niranjala , Gerasimenko Yury
Published: June 2021

The mammalian lumbar spinal cord has the capacity to generate fictive locomotion, i.e. alternating and rhythmic flexion and extension in the absence of oscillating input from either the brain or the periphery, when stimulated pharmacologically or electrically with a tonic stimulation pattern to either the dorsum of the spinal cord or dorsal roots. Although the circuitry within the lumbosacral spinal cord undoubtedly includes those neurons responsible for central pattern generation that can interpret complex proprioceptive input, this feature is not generally recognized as one of its most outstanding. Our experiments show that the spinal circuitry can accurately perceive proprioceptive input to the lumbosacral spinal cord to detect levels of load on the hindlimbs, the speed of a treadmill belt, and the direction of the movement. This input can serve as the source of control of locomotion in the absence of any input from the brain. Further, repetitive use of this spinal circuitry enables it to relearn how to step.
NADPH oxidase (NOX) derived superoxide (O2.‐) upregulates intermediate‐conductance Ca2+ ‐ activated K+ channel (KCa3.1) expression in vascular smooth muscle cells (VSMCs)
Authors: Gole Hope , Bowles Douglas K
Published: June 2021

Reactive oxygen species (ROS), predominantly from NOX, mediate VSMC phenotypic modulation known to play a role in hypertension, atherosclerosis, and hypertrophy. We have previously shown that upregulation of KCa3.1 via increased AP‐1 activity is required for phenotypic modulation of porcine coronary smooth muscle cells (PCSMC). However, the role of ROS in KCa3.1 regulation remains unknown. Our objective was to determine if NOX regulates KCa3.1 expression via AP‐1. Basic fibroblast growth factor (bFGF, 25ng/mL) increased O2.−production in PCSMC, while the NOX inhibitor apocynin (Apo, 1mM) completely abolished O2.− production. bFGF increased KCa3.1 mRNA levels 2.5 fold; while decreasing smooth muscle myosin heavy chain mRNA levels 80% (P< 0.05) in PCSMC. Apo completely inhibited KCa3.1 mRNA upregulation indicating that NOX derived O2.−is required for KCa3.1 upregulation. Acute bFGF significantly increased K+ current, which was inhibited ~50% by the KCa3.1 specific channel blocker TRAM‐34 (100nM) indicating that bFGF causes an upregulation in KCa3.1 channel activity. bFGF increased AP‐1 promoter activity 2.5 fold, while Apo completely inhibited the response to bFGF. These results support a novel mechanism by which NOX derived O2.− regulates KCa3.1 expression through AP‐1.
Expression of KCa2.x and KCa3.1 channels in vascular smooth muscle cells from mouse portal vein
Authors: Blanchette Alexandre , Charbel Chimène , Ledoux Jonathan
Published: June 2021

Calcium‐activated K+ channels are key regulators of smooth muscle membrane potential, and therefore are essential to smooth muscle function. Small and intermediate conductance Ca2+‐activated K+ channels (KCa2.x and KCa3.1) have been reported in vascular myocytes only during proliferation. Portal vein is an atypical blood vessel as it shows action potentials (AP). As KCa2.x channels have been shown to be involved in the control of AP in different cell types, we investigated the presence of KCa2.x and KCa3.1 channels in vascular smooth muscle cells from mouse portal vein (mPV). PCR experiments showed the presence of mRNA for all KCa2.x and KCa3.1 channels in smooth muscle cells from mPV. In Patch clamp experiments, freshly isolated myocytes were dialyzed with a pipette solution containing 1 μM free Ca2+ to stimulate KCa channels. To minimize the contribution of BKCa channels, paxilline (10 μM) was added in the pipette as well as in the superfusion solution. Exposure of myocytes to TRAM‐34 (10 μM), an inhibitor of KCa3.1 channels, induced a decrease of ≈ 65% in membrane currents. However, Apamin (300 nM), a KCa2.x channels blocker, had little if any effect on membrane currents. These data suggest that KCa2.x and KCa3.1 channels are expressed in mPV and may play an important role in the control of membrane potential. This study is financially supported by FRSQ and the Fondation de l'Institut de Cardiologie de Montréal.
4,4‐diisothiocyanatostilbenedisulphonic acid (DIDS) is cytotoxic and does not provide protection against ischemic insults in cultured neurons or astrocytes
Authors: Pamenter Matthew , Gu Xiang , Yoon Michelle , Perkins Guy , Ellisman Mark , Haddad Gabriel
Published: June 2021

DIDS is a putatively cytoprotective compound with numerous cellular targets. Previously our laboratory reported that DIDS provided > 90% cytoprotection in hippocampal slices during ischemic insults (IS); however, recent observations suggest that DIDS‐treated cells may not be viable. To address this inconsistency we examined the effect of DIDS on cellular viability during normoxia and IS in neurons and astrocytes using molecular and biochemical viability assays, electrophysiology, and time‐lapse confocal and transmission electron microscopy (TEM). We found that IS induced membrane depolarization, apoptotic annexin V and necrotic propidium iodide (PI) staining, loss of [ATP], and organelle and DNA damage. DIDS perfusion caused morphological changes in all cells, including rounding off of the cell body and loss of processes. Cellular membrane integrity was better maintained in DIDS‐treated cells than in IS cells; however, DIDS perfusion did not prevent depolarization of cellular membrane potential or [ATP] depletion. Furthermore, in both control and IS cells, annexin V and PI staining were observed within 2 hours of DIDS treatment and were homogenous at 24 hours, while DNA breaks were observed by TUNEL assay. We conclude that DIDS is not cytoprotective against ischemic insults and is cytotoxic to neurons and astrocytes. Previous results implicating DIDS‐mediated cytoprotection should be re‐assessed.
Increased Urinary Renin Excretion Rate in Chronic Ang II‐infused Rats Fed a High Salt Diet leads to augmented urinary Ang II levels.
Authors: McCormack Michael , Liu Liu , Seth Dale M , Kobori Hiroyuki , Navar L Gabriel , Prieto Minolfa C
Published: June 2021

This study examined the urinary excretion of renin (uRen), angiotensinogen (uAGT), and angiotensin II (uAngII) in chronic AngII‐infused Sprague‐Dawley rats fed a high salt (HS) diet. Three groups were studied: 1) AngII‐infused [80 ng/min, 14 d, n=5]; 2) AngII‐infused plus HS [Ang II+HS; 8% NaCl; n=4]; 5) AngII‐infused, HS plus an AngII type 1 receptor blocker (ARB) [Ang II+HS+Candesartan, 25 mg/L, drinking water, 14 d, n=5]. Systolic blood pressures increased in AngII (175±10 mmHg) and were augmented by HS in AngII+HS rats (222±8 mmHg). By day 13, uRen, uAGT and uAngII excretion rates increased in AngII‐infused rats, and in AngII+HS rats: [uRen (AngII: 9±3 vs. AngII+HS: 10±4 ×10‐5 Enzyme Units/day); uAGT (AngII: 1107±106 vs. AngII+HS: 4363±1673 ng/day); uAngII: (HS:1,851±327; AngII: 3,813±431; AngII+HS: 8,570±889 vs. NS: 2080±361 fmol/day; respectively)]. ARB treatment prevented these increases (uRen: 5±3×10‐5 Enzyme Units/day; uAGT: 240±28 ng/day; uAngII: 3,796±1,059 fmol/day). Thus, HS exacerbates the increases in uRen, uAGT, and uAngII as well as the systolic blood pressure in AngII‐dependent hypertensive rats.

NIH [IdeA Program (P20‐RR‐017659‐06) and the Tulane‐BIRCWH Program (K12HD043451)]; and the AHA (09PRE2209079 and 09BGIA2280440).
INFLUENCE OF MATERNAL HIGH SODIUM DIET ON SYSTEMIC AND RENAL RENIN ANGIOTENSIN ALDOSTERONE SYSTEM OF MALES AND FEMALES OFFSPRING WISTAR RATS
Authors: Ramos Débora Rothstein , Costa Nauilo Lima , Jang Karen Lucasechi Lopes , de Oliveira Ivone Braga , Furukawa Luzia Naoko Shinohara
Published: June 2021

Objective

to evaluate the renin angiotensin system (RAS) of male and female offspring, which mothers received high sodium diet during pregnancy.

Methods and Results (mean±SEM p<0.05 n=8/group)

Female Wistar rats received normal salt diet (NSD 1.3%) or high salt diet (HSD 8.0% NaCl) during pregnancy. The offsprings (offNSD, offHSD), males (M) and females (F) were fed NSD until 12 week old. The blood pressure (mmHg) was lower in F (112±2.4) than M offHSD (120±2.4). Aldosterone (pg/mL) was higher in F offNSD (1005±153.3) and offHSD (988±210.2) compared to M offNSD (410±35.8) and offHSD (302±24.9) respectively. The plasma renin activity (ng/mL/h) was lower in M offHSD (2.2±0.6) than M offNSD (4.2±0.4). The renal renin activity (ng/mg tissue/h) was higher in M offHSD (16.4±1.8) than M offNSD (9.0±0.8); and it was lower in F (6.7±1.2) than M offHSD (16.4±1.8). The renin granules (%) was lower in M offHSD (38.4±4.2) than M offNSD (60.7±5.0), and in F offHSD (46.4±4.4) than F offNSD (65.8±4.2). The renin gene expression of renin (IDVrenin/β‐actin) in renal cortex (1.0±0.1) and medulla (0.6±0.06) was lower in M offHSD than M offNSD (1.5±0.2; 1.5±0.2 respectively).

Conclusion

Maternal HSD decreases systemic RAS and gene expression of renal renin in males but not females. Probably there is influence of estrogen in female offspring.

Financial support: FAPESP
EXPRESSION OF COMPONENTS OF THE RENIN‐ANGIOTENSIN SYSTEM IN THE BRAIN OF 2K1C HYPERTENSIVE RATS AFTER EXERCISE TRAINING
Authors: Cangussu Luiza Michelle , Santos Robson AS , Martins Almir , Alzamora Andreia Carvalho , Campagnole‐Santos Maria Jose
Published: June 2021

The renin‐angiotensin system (RAS) modulates the activity of cardiovascular related areas in the brain and may be involved in the improvement of the autonomic function that can be induced by exercise training (ET) in hypertensive rats (HR). In this study, we evaluated mRNA expression of angiotensin receptors, AT1 and MAS, and the enzymes, ACE and ACE2, in the hypothalamus (Hy), ventrolateral medulla (VLM) and cortex (Co) of Goldblatt 2K1C HR submitted to ET (swimming, 60min/day, 5 days/week, for 4 weeks). Systolic arterial pressure (SAP, tail pletismography) was higher in 2K1C (158±6 mmHg; n=6) than in 2K1C‐ET rats (141±4 mmHg; n=6). The SAP of both groups were higher than SHAM (121±4; n=5). 2K1C rats showed increased expression of ACE and ACE2 in the Hy and an increased expression of AT1 in the Hy and VLM in comparison to SHAM. MAS expression was not different in Hy or VLM of 2K1C. ET induced an increase in the expression of ACE2 (3.6±0.4 u.a.; n=4 vs 0.72±0.12 u.a., n=4 in 2K1C) and Mas (3.2±0.4 u.a., n=4 vs 0.6±0.1 u.a., n=4 in 2K1C) and a decrease in AT1 (1.61±0.2 u.a. vs 2.50±0.46 u.a. in 2K1C) in the Hy. However, the expression of ACE, ACE2, Mas and AT1 was not changed in the VLM. No alteration in any of the components was observed in the Co of 2K1C or 2K1C‐ET rats. These data show that ET may induce selective changes in the brain RAS of hypertensive rats favoring the ACE2/MAS axis in the hypothalamus.
Loss of paracrine regulation of ENaC by ATP signaling intrinsic to the distal nephron increases blood pressure
Authors: Pochynyuk Oleh , Bugaj Vladislav , Rieg Timo , Insel Paul , Vallon Volker , Stockand James D
Published: June 2021

ENaC activity is limiting for Na+ reabsorption in the aldosterone‐sensitive distal nephron (ASDN). Improper regulation of ENaC results in sodium imbalance leading to blood pressure disorders. Feedback regulation by the renin‐AngII‐aldosterone system (RAAS) sets ENaC activity. It also is becoming clear that paracrine factors intrinsic to the kidney affect ENaC function. We find that paracrine ATP via P2Y2 receptors and PLC‐dependent signaling acutely decreases ENaC activity in isolated murine ASDN by reducing Po. Genetic deletion of P2Y2 obviates ATP effects on ENaC. Disruption of local ATP signaling in P2Y2 −/− mice contributes to ENaC hyperactivity providing, in part, the mechanism for hypertension in these mice. As expected, ENaC activity in wild type mice has a reciprocal relation with dietary salt feeding. Genetic deletion of P2Y2 overrides regulation of ENaC Po by systemic sodium levels with Po remaining high under all dietary salt conditions. However, the number of active ENaC in the membrane declines with increases in dietary Na+ partially mitigating the effects of increased Po in P2Y2 −/− animals. Clamping systemic mineralocorticoid levels high completely uncouples ENaC in P2Y2 −/− but not wild type mice from normal regulation in response to changes in systemic sodium with ENaC activity being resistant to systemic Na+ levels in DOCA‐treated P2Y2 −/− mice leading to marked salt‐sensitive increases in blood pressure compared to wild‐type mice similarly treated with DOCA.
Evidence for Aldosterone‐mediated regulation of Na‐K ATPase in kidney proximal tubules
Authors: Parks Jason , Barati Michelle T , Salyer Sarah A , Mandal Amritlal , Klein Janet D , Khundmiri Syed J
Published: June 2021

Recent investigations from several laboratories have shown MR expression and increased NHE3, NHE1, and NBCe1 activities in kidney proximal tubules (PT) after aldosterone (Aldo) stimulation. Non‐genomic effects of Aldo have been demonstrated in human kidney PT cells (HK‐2). We hypothesize that Aldo increases Na+/K+ ATPase (Na/K) activity in PT. In the present study we aim to 1) determine if MR is present in human and rat kidney PT 2) determine if Aldo regulates Na/K expression and activity in rat basolateral membranes 3) determine if Aldo increases Na/K subunit in human kidney PT cells. IHC analysis of human and rat kidneys showed presence of MR in PT. Adrenalectomy (ADX) resulted in decreased MR, Na/K, and NHE1 in PT and β and γ ENaC in DCT and CCD. Treatment with Aldo restored MR, Na/K, and NHE1 expression in PT and ENaC expression in DCT and CCD. In human kidney PT cells (HK2, HKC‐5,‐8, and ‐11) Aldo caused translocation of MR into nucleus. Aldo increased Na/K expression and activity in human kidney PT cells. The effects of Aldo on Na/K activity in human kidney PT cells was blocked by specific inhibitors of MR (spironolactone) and by 11β‐HSD inhibitor carbonexolone but not by a GR inhibitor (mifepristone). Treatment with Aldo resulted in decrease in intracellular pH in human kidney PT cells. These results suggest that Aldo may regulate Na/K and NHE1 expression and activity in human and rat kidney PT.

Funded by VAMR and AHA
Pannexin1 is a novel renal ATP release mechanism
Authors: Hanner Fiona , Peti‐Peterdi Janos
Published: June 2021

Purinergic signaling regulates key functions of the renal tubules, including salt and water transport. Due to ectonucleotidase activity along the nephron, it has been suggested that ATP is locally released by tubular cells. Previously, we have shown that the gap junction protein connexin 30 (Cx30) plays a role in ATP release and purinergic mediation of salt reabsorption in the distal nephron/collecting duct. A new candidate for ATP transport is the pannexin1 hemichannel (Px1) which is sensitive to mechanical stress, allows the passage of ATP and unlike Cxs, is insensitive to [Ca2+]e. Therefore we tested the hypothesis that Px1 hemichannels may also mediate ATP release in the distal nephron. Using immunofluorescence, we observed Px1 in the distal convoluted tubules, thin limbs, and select cells of the cortical collecting duct. Px1 function was tested using a luciferase‐based ATP assay and a dye uptake assay in two renal epithelial cell types that endogenously express Px1: MDCK and M1. Px1 was inhibited with either probenecid or Px1 siRNA. Probenecid significantly inhibited ATP release in both M1 (by ~60%) and MDCK (by ~40%) cell types, while Px1 siRNA significantly reduced ATP release in MDCK cells only (by ~30%). Both treatments failed to significantly alter dye uptake in either cell type. Based on these results, we concluded that Px1 is co‐expressed with other Cx isoforms and may regulate ATP release in the distal nephron.
Protein kinase D1 modulates aldosterone‐induced ENaC trafficking and regulation in M1 renal cortical collecting duct cells
Authors: Dooley Ruth , Harvey Brian J , Thomas Warren
Published: June 2021

Aldosterone stimulates the phosphorylation and activation of Protein Kinase D1 in M1 cells through the transactivation of EGFR (1). PKD1 mediates rapid aldosterone‐induced subcellular trafficking of ENaC (2). ENaCα is under transcriptional control of the mineralocorticoid receptor while ENaCβ and ENaCγ are constitutively expressed in the distal nephron. Using siRNA‐mediated stable knockdown of PKD1, transepithelial current measurements (ITE) and confocal microscopy, we examined the role of PKD1 in the regulation of ENaC trafficking and activity in M1 cells. Aldosterone (10nM) increased amiloride‐sensitive ITE from 1 ± 0.21 μA/cm2 to 7 ± 0.86 μA/cm2 (n= 8, p < 0.005) in wild type M1 cells within 24h. This effect was reduced by 71.5% in the PKD1 suppressed cells. ENaCα expression was increased in wild type cells treated with aldosterone for 24h and this response was absent in PKD1 knockdown cells. Aldosterone stimulated the apical membrane insertion of ENaCβ in wild type cells, whereas no trafficking effect was observed in the PKD1 suppressed cells. PKD1 plays a central role in the aldosterone‐mediated regulation of ENaC through both transcriptional control and subcellular trafficking. Funded by Wellcome Trust, Higher Education Authority of Ireland and Science Foundation Ireland.
UCH‐L3 upregulates the epithelial sodium channel in A6 distal nephron cells
Authors: Bao Hui‐Fang , Duke Billie J , Eaton Douglas C
Published: June 2021

Recent studies have shown that the epithelial sodium channel (ENaC) is down‐regulated via its ubiquitination induced by Nedd4‐2 and is upregulated via its deubiquitination induced by Ubiquitin Carboxyl‐Terminal Hydrolase (UCH) L3. In the present study, we used patch‐clamp techniques combined with both confocal microscopy and Western blot methods to further determine how ENaC in A6 distal nephron cells is regulated by UCH‐L3. Patch‐clamp data showed that decreasing the activity of UCH‐L3 with a UCH‐L3 inhibitor significantly reduced the number of active ENaC in the patch. The data from Western blot experiments showed that treatment of A6 cells with the UCH‐L3 inhibitor remarkably reduced the protein levels of α‐, β‐, and γ‐ENaC subunits. Consistent with these data, confocal microscopy experiments showed that the UCH‐L3 inhibitor also reduced the density of α‐ and γ‐ENaC at the apical membrane. These results suggest that UCH‐L3 upregulates ENaC by maintaining its density at the apical membrane.
Angiotensin II‐induced stimulation of NHE3 is dependent on IRBIT and CaMKII
Authors: He Peijian , Yun Chris
Published: June 2021

NHE3 plays an essential role in the reabsorption of NaCl, HCO3−, and fluid in the proximal tubules of the kidney. Accumulating evidence has suggested that angiotensin II (ANG II) at physiological concentrations exerts a stimulatory effect on renal reabsorption of NaCl and filtered water by targeting NHE3. However, the underlying mechanisms remain incompletely elucidated. Herein, we aimed to determine the role of IRBIT in ANG II‐induced stimulation of NHE3 and to elucidate the underlying mechanisms. In OKP cells, 1 nM ANG II stimulated NHE3 activity by ~22%. In comparison, knockdown of IRBIT resulted in ~7% increase, whereas ectopic expression of IRBIT enhanced NHE3 activity by ~33%. Furthermore, ANG II induced over 20% increase in the surface NHE3 expression in OKP cells as determined by surface biotinylation, which was confirmed by immunofluorescence labeling of NHE3 on the apical membrane. CaMKII was shown to phosphorylate IRBIT and, hence, we tested whether CaMKII is required for ANG II‐activation of NHE3. Our results showed that inhibition of CaMKII by KN‐93 or ectopic expression of the natural inhibitor protein of CaMKII resulted in significant suppression of NHE3 activation by ANG II. In conclusion, our current results demonstrate that ANG II regulates NHE3 via CaMKII‐dependent mechanism and identify IRBIT as a physiological regulator of NHE3 in renal proximal epithelial cells.
Defective cAMP‐dependent HCO3‐ Secretion Leads to Abnormal Mucus
Authors: Yang Ning , Garcia Mary Abigail S. , Quinton Paul M.
Published: June 2021

Cystic fibrosis (CF) is a hereditary, fatal disease caused by a unique pathology that appears to begin with thick mucus that obstructs the lumina of hollow organs. In the present study, PAS‐AB Staining, morphometry, pH stat and mucus collection assay methods were used to investigate the formation of abnormal mucus in CF mouse small intestine. We found that in the wild type mouse small intestine, carbachol (100 μM, an intracellular Ca2+ mediated activator) induced goblet cell mucus release, but did not stimulate sustained HCO3− secretion. Whereas, forskolin (10 μM) + IBMX (100 μM) applied to activate intracellular cAMP‐mediated pathways did not have any detectable effect on goblet cell mucus release, but did induce sustained HCO3− secretion. With HCO3− in the basolateral solution, PGE2 (1 μM) + 5‐HT (10 μM), cAMP and Ca2+mediated activators, induced more mucus release from goblet cells in the presence than in the absence of HCO3−. In CF mouse small intestine, PGE2 (1 μM) + 5‐HT (10 μM) also stimulated goblet cell mucus release, but there was no significant difference in mucus releasing goblet cells with or without HCO3− in the basolateral solution. cAMP activated HCO3− secretion was missing in CF intestine. These results suggest that a defect in cAMP‐dependent HCO3− secretion, rather than in mucus release, leads to the abnormal mucus observed in CF mouse.

Supported by Elizabeth Nash Memorial Fellowship
Promotion of Trafficking and Sensitivity to DAG are Involved in TRPC6 Channel Activation by Hydrogen Peroxide
Authors: Graham Sarabeth Marie , Ding Min , Luchowski Rafal , Gryczynski Zygmunt , Ma Rong
Published: June 2021

The present study was performed to examine the effect of H2O2 on TRPC6 channel and the underlying mechanisms in a heterologus expression system. In the TRPC6 expressing HEK293T cells, H2O2 significantly enhanced inward whole cell currents and single channel open probability (NPO) compared with empty vector‐transfected cells. Fura‐2 fluorescence measurements revealed that H2O2 stimulated Ca2+ entry in a dose‐dependent manner in TRPC6‐expressing cells. OAG, a cell membrane permeable homologue of DAG, significantly activated TRPC6 channels in HEK293T cells. The OAG effect was significantly enhanced in the presence of H2O2 (10 μM), suggesting that H2O2 increased the sensitivity of TRPC6 channel to DAG, a physiological stimulus. Moreover, a biotinylation assay showed a clear increase in cell surface TRPC6 protein expression in response to H2O2 (10 μM for 3 min). Consistent with this biochemical evidence, confocal microscopy revealed a significant increase in fluorescence intensity in the cell membrane or sub‐membrane in the first 3 min of H2O2 treatment in TRPC6‐EGFP transfected cells, but not in EGFP‐transfected cells. Taken together, these data indicate that H2O2 is an activator of TRPC6 channel and the activation mechanisms may involve both sensitization of the channel to its physiological stimulus (DAG) as well as promotion of TRPC6 trafficking to the plasma membrane.
Regulation Of Migration and Proliferation of Glioma Cells by Modulating the Ion Channel Activity.
Authors: Rooj Arun Kumar , Fuller Catherine M , Benos Dale J
Published: June 2021

The movement of cations through ion channels is essential for the migration of glioma cells. We have previously reported that malignant glioma cells express an amiloride sensitive cation conductance that is not detected in normal astrocytes. In the present study, we investigate the potential role of this ion channel to regulate the migration and cell cycle processes of glioma cells. We found that the blocking of Acid Sensitive Ion Channel 1 by Psalmotoxin‐1 (40 amino acid synthetic peptide) significantly inhibited both migration and proliferation of D54MG and U251 glioma cells, using in vitro transwell migration and in‐vitro scratch wound assays. We also found that both PcTx‐1 and benzamil caused a significant cell cycle arrest of D54MG cells in G0/G1 (by 18% and 19%, respectively), and reduced accumulation in the S phase (by 61% and 49%, respectively), and G2/M phase (by 51% and 32%, respectively) (n=3–4; P<0.01) at 24h of incubation. PcTX‐1 and benzamil showed the same effect after 48h of incubation time. An unfolded control peptide had no significant effect compared to PcTX‐1. PcTX‐1 also induced upregulation of cyclin dependent kinase inhibitor (CDKI) proteins (p21Cip1 and p27Kip1) in D54MG cells. Thus, our data implicate the potential role of this cation conductance in migration and in progression of the cell cycle in glioma cells. This study was supported by NIH grants CA101952 and DK037206.
An ASIC1 and ENaC hybrid channel is the amiloride sensitive channel in human glioma.
Authors: Kapoor Niren , Lee William , Parpura Vladimir , Fuller Catherine M , Palmer Cheryl Ann , Benos Dale J
Published: June 2021

High grade gliomas express multiple members of the Epithelial Sodium Channel (ENaC)/Degenerin (Deg) family, displaying a basally active amiloride sensitive cation current. We hypothesize that this glioma current is mediated by a hybrid channel composed of a mixture of Epithelial Sodium Channel (ENaC) and Acid Sensing Ion Channel (ASIC) subunits. Using immunohistochemisty we show a higher expression level of ASIC1, αENaC, and γENaC in glioblastoma biopsy tissue compared with non‐neoplastic brain tissue. To test our hypothesis we made dominant negative (DN) cDNAs for ASIC1, αENaC, and γENaC. D54‐MG cells transfected with the DN constructs for ASIC1, αENaC or γENaC showed reduced protein expression and a significant reduction in the amiloride sensitive whole cell current as compared with untransfected D54‐MG cells. Using co‐immunoprecipitation we show interactions between ASIC1, αENaC, and γENaC at the plasma membrane of D54‐MG cells, consistent with these subunits interacting with each other to form an ion channel. Data using Total Internal Resolution Fluorescent Microscopy (TIRFM) also suggest that ASIC1 and ASIC2 interact with γENaC in CHO‐K1 cells. These data suggest that different ENaC/Deg subunits combine to form a hybrid channel that likely underlies the amiloride‐sensitive current in human gliomas. This study was supported by NIH Grants CA101952 and DK037206.
Sphingosine‐1‐Phosphate acutely modulates the CFTR (Cystic Fibrosis Transmembrane Regulator) transporter in an AMPK‐dependent manner
Authors: Malik Firhan Atir , Meissner Anja , Bear Christine , Bolz Steffen Sebastian
Published: June 2021

Introduction

Sphingosine‐1‐Phosphate (S1P) is a primary modulator of resistance artery tone. Its bioavailability is controlled by a rheostat between sphingosine kinase 1 and intracellular S1P phosphohydrolase 1. We have found that the transport by CFTR is the rate‐limiting step for the intracellular hydrolysis of S1P. We hypothesized that S1P limits its own degradation by an AMPK‐dependent modulation of CFTR conductance.

Methods

CFTR conductance was assessed using the iodide efflux technique (conventional and real‐time) in baby hamster kidney (BHK) cells, which stably express human wild type CFTR. AMPK phosphorylation was determined using Western blots

Results

BHK cells were found to endogenously express Sk1, SPP1 and the S1P1 and S1P2 receptors (n=8). S1P (1μM, n=6) significantly reduced iodide conductance by 43% (conventional) and 75% (real‐time). The negative effect of S1P on CFTR iodide conductance was enhanced following S1P2 overexpression but reduced following pretreatment with the S1P2 blocker JTE013 (5μM, n=6). The inhibitory effect of S1P was associated with AMPK phosphorylation and blocked by the AMPK inhibitor Compound C (80μM, n=5). Accordingly, AMPK activation by AICAR mimicked the effect of S1P on iodide conductance.

Conclusions

The S1P2‐ and AMPK‐dependent modulation of CFTR conductance describes a novel pathway through which S1P could potentially regulate its own degradation.
Substrate Specificity of Organic Anion Transporter 2: A Tale of Different Expression Systems?
Authors: Pelis Ryan M
Published: June 2021

The organic anion transporter 2 (OAT2) has been suggested to play an important role in the hepatic elimination of thereapeutic drugs. Available data on OAT2 substrate specificity largely comes from its expression in Xenopus laevis oocytes. The human ortholog of OAT2 (hOAT2) was cloned from human liver and stably expressed in Chinese hamster ovary (CHO) cells and human embryonic kidney (HEK) cells to further define its substrate specificity. hOAT2 mRNA was abundant in the stably selected cells (real‐time PCR), and immunocytochemical localization was consistent with plasma membrane expression. hOAT2‐mediated transport of organic anions that had previously been shown to be transported by hOAT2 in Xenopus oocytes was low to absent, i.e., estrone‐3‐sulfate, taxol and dehydroepiandrosterone. Additionally, several other drugs were tested for their interaction with hOAT2 in these expression systems (e.g., estradiol 17β D‐glucuronide, vinblastine, taurocholate, adefovir, atorvastatin and diclofenac), and the transport protein had little impact on their accumulation in the stable cells. Differences in the substrate specificity of OAT2 expressed in Xenopus oocytes versus mammalian cell lines are apparent. Further investigation is required to determine the substrate specificity and role of OAT2, a mystery transporter, in the hepatic excretion of xenobiotics.
ON THE SUBSTRATE RECOGNITION AND AUTOINHIBITORY DOMAIN OF SPAK
Authors: Gagnon Kenneth B. , Delpire Eric
Published: June 2021

Threonines targeted by SPAK for phosphorylation have been identified in NKCC1, NKCC2, and NCC. However, what constitutes the substrate recognition of the kinase is still unknown. Using site‐directed mutagenesis and functional measurement of NKCC1 activity in Xenopus laevis oocytes, we determined that SPAK recognizes two threonine residues separated by four amino acids. Addition or removal of a single residue abrogated SPAK activation of NKCC1. Although both threonines are followed by hydrophobic residues, only the first residue is essential for SPAK activation of the cotransporter. Negatively charged residues around the sites targeted for phosphorylation by other kinases have been shown to be involved in substrate recognition. Thus, we mutated two aspartate residues flanking the two threonines. Whereas alanine mutation of the upstream residue had no effect, alanine substitution of the downstream residue inhibited cotransporter activity. However, conserved substitution of this residue into glutamate preserved NKCC1 function. SPAK activity requires phosphorylation of two residues by WNK kinases. We found that mutating T243E combined with S383D rendered the kinase constitutively active. Surprisingly, S383A mutation or mutation of residues surrounding this residue also resulted in an active kinase. These data indicate the possibility of an inhibitory domain which is affected by WNK phosphorylation.
PCR Cloning of a Divalent Metal Transporter From Malaria Mosquito Anopheles gambiae tissue cDNA library
Authors: Mandal Anita , Charles Marie , Dines Josica , Merritt Latehia , Singleton Daniell , Brookings Rickell , Meleshkevitch Ella , Boudko Dmitri , Mandal Prabir K
Published: June 2021

Metal ions are vital for many biological processes, such as transcription, respiration and growth (Schaffner et al. 2006)and essential cofactors for oxidative phosphorylation, gene regulation and free‐radical homeostasis. Failure to maintain appropriate levels of metal ions in humans could cause metal ion deficiency disorders, and neurodegenerative diseases (Hediger et al. 1997). The regulation of metal ion transport within neurons is critical for normal brain function (Howitt et al. 2009). Acquisition of metal transition (Zn, Cu, Fe) is mediated by a specific high affinity divalent metal transporter which belongs to a cation efflux protein family with predicted twelve trans‐membrane domains and found to increase tolerance to divalent metal ions like zinc. These proteins are considered to be efflux pumps that remove these ions from cells. Divalent metal transporter 1 (DMT1) is a widely expressed metal ion transporter that is vital for intestinal iron absorption and iron utilization by most cell types throughout the body (Hediger et al. 2007). Cloning and functional characterization of DMT1/NRAMP homolog is reported in mosquito Anopheles albimanus (Martinez‐Barnetche et al. 2008). In this present study, we report the cloning of a divalent metal transporter fragment (~500 bp) from Anopheles gambiae tissue cDNA library by polymerase chain reaction. Supported by Dept of ED HBCU INST AID P031B070074.