bluuebunny/crossref_metadata_2025 huggingface dataset:
TONIC NMDA RECEPTOR‐MEDIATED INWARD CURRENT IN PRE‐SYMPATHETIC PVN NEURONS IS ENHANCED BY HEART FAILURE.
Authors: Chen Qing‐Hui , Dong Ying , Cardoso Leonardo , Shi Peng , Calderon Alfredo S , Andrade Mary A , Toney Glenn M
Published: June 2021

Sympathetic nerve activity (SNA) is increased in heart failure (HF). In rats with HF, activation and blockade of NMDA receptors in the hypothalamic PVN is reported to evoke exaggerated increases and decreases of renal SNA. Such actions could involve enhanced NMDA receptor activation, increased neuronal excitability, or both. To test these possibilities, whole‐cell recordings were made from RVLM‐projecting PVN neurons in brain slices from rats with and without HF. Graded current injections induced graded increases in firing frequency in both groups, but responses to the maximally effective current of 200 pA were significantly greater in the HF group (43 ± 6 Hz, n=5) compared to controls (20 ± 4 Hz, n=6) (P<0.05). In slices perfused with Mg++‐ and TTX‐free aCSF containing picrotoxin (100 μM), blockade of NMDA receptors with AP5 (50 μM) (Vhold: −80 mV) induced a significantly greater (P<0.01) outward shift of holding current in HF (41 ± 5 pA, n=3) compared to control (10 ± 1 pA, n=3) neurons. The functional impact of this was assessed by microinjecting AP5 (3.0 nmol in 50 nl) into the PVN of anesthetized rats. AP5 was confirmed to significantly (P<0.01) reduce RSNA by 25 ± 5 %, LSNA by 27 ± 9 % and MAP by 20 ± 6 mmHg in HF rats (n=3), but not controls (n=6). We conclude that enhanced excitability and tonic NMDA receptor‐mediated inward current among PVN‐RVLM neurons may contribute to enhanced PVN control of SNA in HF. Support: HL071645 (GMT)
Group III muscle afferents are sensitized in rats with heart failure
Authors: Hayes Shawn G.
Published: June 2021

The mechanisms responsible for exaggerated sympathoexcitation of heart failure are unknown, but research suggests that it may be the result of an exaggerated exercise pressor reflex caused by sensitized muscle mechanoreceptors (group III afferents). The afferent arm of this reflex is composed of metabolically sensitive and mechanically sensitive afferents that when stimulated by exercise reflexly increases sympathetic function. To determine the role played by group III afferents in the exaggerated sympathoexcitation of heart failure, I compared the responses of single group III afferents in decerebrate rats with heart failure to those of their healthy counterparts. Group III afferents of heart failure rats had a significantly higher discharge in response to the same stimulus as compared to healthy rats. For example, tendon stretch in healthy rats increased the discharge of group III afferents from a baseline activity 0.17±0.1 to 0.73±0.2 impulses/sec (n=6) whereas tendon stretch in heart failure rats increased the discharge of group III afferents from 0.2 ±0.2 to 2.3±0.7 imp/sec (n=6, p<0.05). Likewise, the responses of group III muscle afferents to static contraction were significantly greater in rats with heart failure rats compared to healthy rats (p<0.05). These sensitized group III afferents may be responsible for the exaggerated sympathetic responses to exercise in heart failure.
Cyclooxygenase products contribute to enhanced muscle reflex in heart failure
Authors: Koba Satoshi , Xing Jihong , Sinoway Lawrence I. , Li Jianhua
Published: June 2021

In heart failure (HF), reflex sympathetic nerve responses to muscle contraction are exaggerated. The present experiment was designed to test the role played by cyclooxygenase products, which are produced within contracting skeletal muscle, in the enhanced muscle reflex of HF. HF was induced by myocardial infarction (MI) after the coronary artery ligation in rats. Echocardiography was performed to determine fractional shortening (FS), an index of the left ventricular function. In decerebrate rats, we examined renal sympathetic nerve activity (RSNA) during 1‐min intermittent (1‐ to 4‐s stimulation‐to‐relaxation) contraction of left triceps surae muscles. RSNA responded synchronously as tension was developed, and the response was significantly (P<0.05) enhanced in MI rats [+28±2 arbitrary unit (a.u.); n=12] with FS <30% compared to control animals (+16±3 a.u.; n=13) with FS > 40% at the same tension development. After intravenous administration of the cyclooxygenase inhibitor indomethacin (5 mg kg−1), the enhanced RSNA response to contraction was significantly reduced in the MI rats by 44% (+15±5 a.u.), but no significant change was found in the control animals (+12±2 a.u.). These data suggest that cyclooxygenase products within active muscle, such as prostaglandins, play a role in the enhanced muscle reflex in HF. Supported by NIH R01 R01 HL075533 & R01 HL078866 (Li) and R01 HL060800 (Sinoway).
Pharmacologic Probes to Differentiate Chronic Autonomic Failure Syndromes
Authors: Goldstein David S , Holmes Courtney , Sharabi Yehonatan
Published: June 2021

Objective: The current clinical classification of chronic autonomic failure (CAF) recognizes four primary forms—pure autonomic failure (PAF), multiple system atrophy (MSA), Parkinson disease (PD), and autoimmune autonomic ganglionopathy (AAG). We provide a pathophysiologic classification scheme, based on neuropharmacologic probes: tyramine (TYR), trimethaphan (TRI) or pentolinium (PEN), yohimbine (YOH), acetylcholine (ACh, quantitative sudomotor axon reflex test, QSART), and 6‐[18F]fluorodopamine (18F‐DA) scanning.

Methods: CAF groups received i.v. TYR, TRI/PEN, YOH, or 18F‐DA or ACh by iontophoresis. Blood pressure (BP) and dihydroxyphenylglycol (DHPG) responses were measured.

Results: PAF involved low cardiac 18F‐DA‐derived radioactivity and attenuated (Atten.) increments (Inc.) to TYR and YOH and decrements (Dec.) to TRI/PEN, in contrast with normal reponses in AAG and MSA. MSA involved augmented (Aug.) responses to YOH and TRI/PEN and normal (Nl.) cardiac 18F‐DA, in contrast with Atten. in PD+NOH.

Conclusions: Pharmacologic probes provide a pathophysiologic classification of chronic autonomic failure that can be utilized in individual patients.
Carbachol microinjections into the dorsal pontine reticular formation repeatedly elicit REM sleep‐like depression of hypoglossal (XII) nerve activity in the rat in situ perfused working heart‐brainstem preparation (WHBP)
Authors: Brandes Ivo Florian , Stettner Georg , Sinner Barbara , Graf Bernhard , Kubin Leszek , Dutschmann Mathias
Published: June 2021

Obstructive sleep apnea (OSA) is characterized by nocturnal episodes of upper airway collapse and hypoxia, with rapid‐eye‐movement sleep (REMS)‐related apneas being longest and most severe. In OSA patients, upper airway muscle tone is reduced during REMS parallel to the postural atonia characteristic of this state. To assess the central neural mechanisms of REMS, in vivo pharmacological models of REM sleep have been used, including those in which REMS‐like state is elicited by microinjections of a cholinergic agonist, carbachol, into the pontine reticular formation (Kubin, 2001). To test whether upper airway motor tone depression similar to that in in vivo models can be elicited in the rat WHBP, injections of carbachol (25–50 nl, 10mM) were made into the dorsomedial pons in n=33 WHBPs while recording XII and phrenic nerve activities (PNA). In 23 preparations, depression of XII nerve activity lasting 16–24 min occurred within 142 s ± 26(SD). A respiratory rate increase from 8 min−1 ± 4 to 18 ± 9 paralleled the depression, whereas PNA was little affected. The depressant episodes could be elicited repeatedly by injections at the same site at >30 min intervals. Thus, the WHBP offers a novel model with which to pharmacologically manipulate cellular environment and study the mechanisms of REMS‐related respiratory depression. Funded by BCCN Göttingen and HL‐47600.
Sex‐specific effect of progesterone on hypoxic ventilatory response (HVR) in newborn rats
Authors: Lefter Raluca , Betry Cecile , Doan Van Diep , Joseph Vincent
Published: June 2021

Progesterone (P) is a respiratory stimulant in adults. Since preterm babies are chronically deprived from placental P and exhibit delayed development of respiratory control and respiratory disorders affecting particularly males, our hypothesis is that there is a sex‐specific effect of P on respiratory control development. Lactating female rats were implanted with osmotic minipump delivering P or vehicle during postnatal days 1–14. Respiratory and metabolic studies were performed using whole body plethysmography in male and female rats at postnatal days 3 (P3) and 12 (P12) under normoxia and hypoxia (10% O2). There was no apparent effect of P in P3 pups, but P decreased ventilation and metabolic rate in P12 females. P increased HVR in P12 rats: slightly in males and dramatically in females. Moreover, in P12 P totally abolished the inhibitory effect of a dopamine D2 receptor antagonist (domperidone injected ip) on HVR in males. In P12 females domperidone injection did not completely abolished the effects of P on HVR. These results suggest that P accelerates the maturation of HVR and induces sex‐specific effects on peripheral chemoreceptors neurochemistry. Founded by FRSQ, Sick Kids Foundation, CIHR.
Chronic intermittent hypoxia abolishes respiratory LTF in rat pups and enhances apnea frequency
Authors: Julien Cecile , Niane Lalah , Bairam Aida , Joseph Vincent
Published: June 2021

We tested the hypothesis that chronic intermittent hypoxia (IH) alters respiratory long‐term facilitation (LTF) induced by acute hypoxic episodes in newborn rats. Rat pups were exposed to normal room air (controls) or chronic IH (IH: O2 levels cycled between 21% and 5% every 10 minutes for 1h; 1h normoxia, 24h/day) during postnatal days 1–10. To assess LTF, we measured minute ventilation (VE ‐ whole body plethysmography) in normoxia and during a recovery period of 2 hours following 10 acute hypoxic episodes. VE gradually increased (+23%) during recovery in controls, but decreased (−15%) in IH rats. In controls, frequency of apneas (≥2 missed breaths, measured before and at the end of recovery) slightly increased following acute hypoxic episodes (from 23±3 to 34±5 apneas/hour), while it dramatically increased in IH rats (from 26±4 to 61±6 apneas/hour). There was no gender‐specific effect. We conclude that in 10‐day old rats: i) LTF is present, ii) chronic IH abolishes LTF and iii) LTF appears to have a protective role against apneas. Neonatal apnea with IH may abolishe endogenous protective mechanisms, and contribute to increase apnea frequency in neonates. (FRSQ, CIHR, Sick kids foundation).
Dynamic cerebral autoregulation during passive heat stress
Authors: Low David A , Wingo Jonathan E. , Keller David M. , Davis Scott L. , Zhang Rong , Crandall Craig G
Published: June 2021

This study tested the hypothesis that passive heating impairs cerebral autoregulation. Transfer function analysis of resting arterial blood pressure (BP) and middle cerebral artery blood velocity (MCAVmean) were conducted on 9 healthy subjects, dressed in a water‐perfused suit, under normothermic (NT) and passive heat stress (HS, Δ core temperature 1.1 ± 0.2 °C, P<0.001) conditions. Passive heating reduced MCAVmean (8 ± 8 cm•s−1, P=0.01), whilst mean arterial BP was maintained (2 ± 5 mm Hg, P=0.36). In the very low frequency range (<0.07 Hz) coherence was lower during HS compared to NT conditions (0.26 ± 0.10 vs 0.57 ± 0.13, respectively, P<0.001). Coherence was >0.5 and similar between NT and HS in the low (0.07–0.20 Hz, 0.64 ± 0.19 vs 0.59 ± 0.10, respectively, P=0.40) and high (0.20–0.35 Hz, 0.63 ± 0.19 vs 0.72 ± 0.14, respectively, P=0.12) frequency ranges. Phase was higher during HS in the low frequency range (NT: 0.55 ± 0.31 vs HS: 0.82 ± 0.17°, P=0.01), whereas it was unchanged by HS in the high frequency range (NT: −0.10 ± 0.20 vs HS: 0.12 ± 0.71°, P=0.14). Transfer gain was higher during HS in the low (NT: 1.53 ± 0.41 vs HS: 1.73 ± 0.38 cm•s −1•mmHg−1, P=0.03) and high (NT: 1.55 ± 0.55 vs HS: 2.06 ± 0.37 cm•s−1•mmHg−1, P=0.03) frequency ranges. Higher transfer function estimates of gain suggest that dynamic cerebral autoregulation is impaired during HS.

Supported by NIH Grant HL‐61388 & HL‐84072
Evidence for Impaired Skeletal Muscle Contraction‐Induced Rapid Vasodilation in Aging Humans
Authors: Carlson Rick E , Kirby Brett S , Voyles Wyatt F , Dinenno Frank A
Published: June 2021

We tested the hypothesis that aging is associated with an impaired contraction‐induced rapid vasodilation in healthy adults. Single contractions of a small muscle mass were employed to isolate the local rapid vasodilatory responses independent of systemic influences on forearm hemodynamics. We measured forearm blood flow (Doppler ultrasound) and arterial blood pressure (finapres) on a beat‐by‐beat basis, and calculated the changes in forearm vascular conductance in response to forearm contractions in 18 young (24 ± 1 yrs) and 13 older (62 ± 2 yrs) healthy subjects. Single, 1‐second dynamic forearm contractions were performed at 5, 10, 20, and 40% of the subjects’ maximal voluntary contraction (MVC). At 5% MVC, there were no significant age‐related differences in contraction‐induced forearm vasodilation. However, the peak vasodilatory responses were impaired ∼40–45% in older adults at 10, 20, and 40% MVC, as were the total vasodilatory responses (∼40–50%; all P<0.05). Additionally, the immediate vasodilation (first cardiac cycle post‐contraction) for the 20% and 40% MVC trials was also impaired ∼50% with age (P<0.05). There were no significant age‐group differences in MVC or forearm fat‐free mass. Under these experimental conditions, the blunted responses with age reflect impaired local contraction‐induced rapid vasodilation.

Supported by AG‐022337, AG‐027150, HL‐087952
Backpack straps decrease upper extremity blood flow
Authors: Neuschwander Timothy , Macias Brandon , Hargens Alan
Published: June 2021

Adults in professions such as the military, firefighting, and mountain rescue routinely carry packs loaded with 60% body weight. Backpack straps are typically situated in a region where they may compress the axillary vein, causing venous hypertension and subsequently decreasing upper extremity blood flow. We hypothesize that upper extremity macro and microvascular blood flow will decrease while wearing a loaded backpack.

Eight healthy volunteers (6 men, 2 women, age range: 18 – 30 years) had the right brachial artery measured with ultrasound Doppler. Index finger pulp microvascular flow was measured using photoplethysmography. Baseline flows were measured immediately before and 10 min after donning a 12kg backpack. Baseline microvascular flow was normalized to 100%; data are presented as means±SE.

After wearing the pack for 10 min, brachial artery blood flow decreased from 2.66±0.36 to 1.52±0.27 mL/s (p<0.05, paired T‐test), and index finger microvascular flow decreased from 100% to 46±6% (p<0.05, paired T‐test).

Even light backpack loads such as 12kg decrease upper extremity macrovascular and microvascular blood flows and may result in a loss of fine motor control and increased fatigue. Redesigning backpack straps to prevent compression of the axillary vein about the clavicle may maintain normal blood flow.

Supported by UCSD Dept. of Orthopaedic Surgery Discretionary Funds.
Cyclooxygenase inhibition attenuates muscle sympathetic nerve responses to passive muscle stretch
Authors: Cui Jian , Moradkhan Raman , Mascarenhas Vernon , Momen Afsana , Sinoway Lawrence I.
Published: June 2021

Previous studies suggest the muscle metabolites may sensitize muscle mechanoreceptors. However, the responsible substance(s) has not been studied thoroughly in humans. This study tested the hypothesis that local cyclooxygenase (COX) inhibition in exercising muscles could attenuate muscle sympathetic nerve activity (MSNA) responses to passive muscle stretch during post exercise muscle ischemia (PEMI). Responses of blood pressure (BP), heart rate, and MSNA to passive stretch of hand and forearm muscles were assessed in 13 healthy subjects during PEMI before and after COX inhibition by local infusion of 6 mg ketorolac tromethamine in saline via Bier block. In the second experiment, the same amount of saline was infused via the Bier block. Ketorolac Bier block significantly decreased the synthesis of COX products. Before ketorolac Bier block, passive stretch evoked significant increase in MSNA (P<0.005) and mean BP (P<0.02). After ketorolac Bier block, passive muscle stretch did not evoke significant responses in MSNA (P=0.11) or in mean BP (P=0.83). Saline Bier block did not attenuate the MSNA responses. These observations indicate that COX inhibition attenuates MSNA responses to stretch during PEMI, and suggest that COX products sensitize muscle mechanoreceptors in humans. Supported by NIH P01 HL077670, M01 RR010732 and AHA 0635245 N.
Impact of metallothionein deficiency on soleus muscle function following acute spinal cord injury
Authors: DeRuisseau Lara R , Recca Daniel M , Bidwell Amy J , Orosz Nadia J , Pompo Kimberly M , DeRuisseau Keith C
Published: June 2021

Metallothioneins (Mts) are small molecular weight proteins that possess metal binding and free radical scavenging properties. Furthermore, Mt is a stress protein that likely participates in the adaptive response of skeletal muscle to stressful stimuli and is up‐regulated following acute spinal cord injury (SCI). We tested the hypothesis that loss of Mt (Mt1 and Mt2; Mt−/ −) would lead to exacerbated atrophy and contractile dysfunction of the soleus muscle following acute SCI (7 days, T9 transection) in Mt−/ − mice compared to control strain mice. Transected control strain (CT; n=5) and transected Mt−/ − (MT; n=6) mice experienced an equivalent % loss of body weight by day 7 of recovery compared to pre‐surgery weight (CT = −8.6 ± 1.6; MT = −11.6 ± 2.4). Soleus muscle mass (mg) of CT (4.3 ± 0.2) and MT (4.6 ± 0.1) groups was lower (p<0.05) than the non‐surgical control strain (NS; n=5; 5.6 ± 0.3). However, preliminary data (n=1–3/group) indicate that a lower maximal specific tension (N/cm2) is generated by the soleus in MT vs. controls (NS=23.7 ± 2.5; CT=22.1; MT 19.0 ± 0.0). In summary, deficiency of Mt1 and Mt2 does not alter the loss of muscle mass but may lead to lower maximal force generating ability of the soleus in response to disuse resulting from spinal cord injury.

Supported by Syracuse University SOE
ISOFORM‐SPECIFIC SUBCELLULAR DISTRIBUTION AND ACTIVATION OF PKC MODULATE ENDOTHELIAL BARRIER INTEGRITY
Authors: Gaudreault Nathalie , Perrin Rachel M , Guo Michael , Clanton Chase , Wu Mack H , Yuan Sarah Y
Published: June 2021

PKC is known to contribute to microvascular injury in diabetes. To determine which PKC isoforms are involved in the regulation of endothelial barrier integrity, we first examined their en face expression in the endothelium of coronary microvessels from diabetic Zucker rats. Immunofluorescence microscopy revealed a significant increase in PKCβII and a decrease in PKCδ expression. To test whether changes in PKCs expression alter microvascular barrier properties, we measured the transendothelial electrical resistance in human coronary microvascular endothelial cell and found that overexpression of PKCβII and inhibition of PKCδ both increased endothelial cell permeability. Using 3D fluorescence microscopy, we observed a distinct translocation of RFP‐tagged PKCβII to the cellular junction upon activation while the δ isoform remained confined to the perinuclear area. Consistently, FRET analysis of PKC activity at distinct subcellular locations demonstrated that PKCβII activity was higher at the cell‐to‐cell junction and plasma membrane surface, whereas PKCδ activity was greater in perinuclear plasma membrane vesicles. Taken together, the data suggest that PKC isoforms target specific subcellular sites leading to differential regulation of endothelial barrier function. Supported by NIH RO1 HL084542 and HL073324.
Decreased Endothelial Cell Resistance Following Knock Down of Claudin 11
Authors: Chitaley Kanchan , Kim Bryan , Thorner Daniel , Engel Karen , Tsubota Yoshi , Wessells Hunter
Published: June 2021

The corpus cavernosum is a specialized vascular organ in which endothelial cells (ECs) are exposed to a wide range of pressures (10–500 mmHg) during flaccidity and rigidity. The tight junction associated gene, claudin 11, is highly expressed in human cavernosal ECs (HCCECs) as compared to ECs from other blood vessels. We tested the hypothesis that claudin 11 plays an important role in barrier function in HCCECs by measuring transendothelial electrical resistance (TEER) following claudin 11 knock down. Ambion siRNA constructs for CLDN11 and a negative control scrambled sequence (SCR) were transfected into HCCECs using DharmaFECT‐1 (Dharmacon). Significant down‐regulation of claudin 11 was verified by Western blot at 2 and 7 days following transfection. Untransfected control (CNT), SCR siRNA, and CLDN11 siRNA transfected ECs were seeded in 6.5‐mm Transwell inserts and integrity of the endothelial monolayer assessed by daily measurements of TEER with an Endohm‐6 electrode chamber and an EVOM voltohmmeter (World Precision Instrument). HCCECs transfected with CLDN11 siRNA had significantly reduced TEER compared to both SCR and CNT groups (% of CNT at 75.5 and 95hr: 73±8.7 and 76±8.9, p<0.05). There was no significant difference in TEER in the SCR siRNA vs. CNT group (% of CNT at 75.5 and 95hr: 97.1±2.2 and 98.5±1.9). These data suggest that claudin 11 is important for cavernosal EC junctions and barrier function.
hTERT overexpression delays replicative senescence but not damage‐induced senescence of vascular endothelial cells isolated from patients with severe CAD
Authors: Voghel Guillaume , Thorin‐Trescases Nathalie , Mamarbachi Maya , Villeneuve Louis , Perrault Louis P , Carrier Michel , Thorin Eric
Published: June 2021

Overexpression of the catalytic subunit of telomerase (hTERT) immortalizes healthy human endothelial cells (EC) but its effect on senescence in pathological conditions is unknown. Our objective was to investigate whether hTERT overexpression would prevent senescence in EC isolated from patients with coronary artery disease (CAD). EC isolated from discarded internal mammary arteries of 11 patients undergoing CABG were infected or not with a lentivirus expressing hTERT and cultured in vitro. We quantified senescence by cytochemical detection of senescence‐associated b‐galactosidase, hTERT activity by real‐time TRAPassay, telomere length by Southern blotting, mRNA levels (p21 and ATM, a DNA damage response protein) by qPCR and oxidative stress by immunostaining of lipid peroxidation (HNE). Compared to control EC, lentiviral infection increased hTERT activity (p<0.05), elongated telomeres (p<0.05), decreased ATM and p21 mRNA levels (p<0.05), increased EC replicative potential (p<0.05) but only delayed the onset of senescence (p<0.05). HNE levels increased in both groups, with time (p<0.05). In conclusion, since no immortalization was observed, we hypothesize that hTERT overexpression delays replicative senescence but cannot bypass stress‐induced senescence associated with CAD.

This study was supported by the Canadian Institutes of Health Research and the Fonds de la Recherche en Santé du Québec.
The immunophilin inhibitor juglone decreases NO bioavailability and vasodilation by inhibiting glutathione S‐transferase
Authors: Mitchell Brett M. , Chiasson Valorie L.
Published: June 2021

Immunophilins are peptidyl‐prolyl isomerases that play a role in immunosuppression and include 3 families: cyclophilins, FKBPs, and the parvulins. We demonstrated previously that depletion of FKBPs by the immunosuppressive drugs rapamycin and FK506 decreases NO production and endothelial function. Recently, the mammalian parvulin inhibitor juglone was reported to be an immunosuppressant, however juglone can also inhibit the intracellular antioxidant glutathione S‐transferase. In this study we determined the effects of juglone on NO bioavailability and endothelial function. Endothelium‐intact mouse aortas treated acutely with juglone (0.1 uM, 30 mins) exhibited significantly decreased maximal NO‐mediated relaxation (relaxation from phenylephrine: juglone = 44 ± 8% vs. control = 80 ± 5%, p<0.05) and a 60% decrease in NO compared to controls. Juglone also elicited a 65% increase in aortic superoxide levels and significantly decreased glutathione S‐transferase activity (juglone = 23 ± 6 nM/min/mL vs. control = 55 ± 10 nM/min/mL, p<0.05). In juglone‐treated aortas, superoxide dismutase restored aortic relaxation to control levels, however the glutathione precursor N‐acetyl‐cysteine had no effect. In conclusion, juglone causes endothelial dysfunction by inhibiting glutathione S‐transferase leading to increased oxidative stress and decreased NO bioavailability. (Supported by HL084299 to BMM)
Effect of intraluminal pressure and tone on smooth muscle Ca2+ oscillations in cremaster muscle arterioles
Authors: Ella Srikanth Reddy , Davis Michael J , Meininger Gerald A , Dora Kim A
Published: June 2021

Arteriolar smooth muscle cells exhibit local variation in intracellular Ca2+ (Ca2+i) levels as shown by phenomena such as sparks, flashes and waves. The exact relationship of such events to levels of intraluminal pressure and myogenic tone, however, remains unclear. Using Fluo 4 (10 μM) loaded and cannulated rat cremaster muscle arterioles, the present studies examined variations in Ca2+i levels. Changes in fluorescence were measured using a spinning disk confocal system coupled to a microscope (x60, 1.2 N.A. objective) and high speed intensified CCD. Measurements of Ca2+i were collected at 30Hz and regions of interest defined within single cells of the vessel wall. Measurements were taken at 10, 40, 70 and 100 mmHg under baseline conditions and following treatment with nifedipine (1 μM) or adenosine (10μM). Under control conditions, increasing intraluminal pressure (10 – 70 mmHg) was associated with a greater number of cells exhibiting oscillations (26 ± 2.9 to 72 ± 3.6 % of cells in a given field) and which occurred with an increasing frequency (0.1 ± 0.02 to 0.27 ± 0.03 Hz). The increase in number of cells exhibiting oscillations and increased frequency of oscillations, with increments in pressure, persisted following either nifedipine or adenosine. In contrast to arterioles exhibiting active myogenic constriction to increases in pressure, vessels exposed to nifedipine or adenosine responded largely passively. Thus, oscillations in Ca2+i are not dependent on active myogenic constriction but are influenced by pressure‐induced distention.
GPR30‐Mediated Vasorelaxation is Enhanced in Female mRen2.Lewis Rats and Attenuated by High Salt
Authors: Lindsey Sarah Hoffmann , Chappell Mark C.
Published: June 2021

Female congenic mRen2.Lewis rats display salt‐dependent increases in blood pressure, but the mechanisms contributing to this phenotype are not known. A newly discovered G‐protein coupled receptor (GPR30) may play a role in the non‐genomic actions of estrogen, and Western analysis of aortic homogenates revealed a single band corresponding to the appropriate molecular weight of GPR30. Therefore, the present studies investigated the vascular responses mediated by GPR30 in isolated aortic rings from Lewis controls or congenics fed a normal (NS, 1%) or high salt diet (HS, 8%). In phenylephrine‐constricted rings, vasorelaxation to ICI 182,780, an agonist to GPR30, was significantly greater in NS (48%) as compared to HS (26%) and Lewis (6%, p<0.05). Relaxation in NS rings was reduced by denuding (22%) or KCl constriction (26%, p<0.05). The selective GPR30 agonist G‐1 also produced greater relaxation in NS (36%) vs. HS (18%) and Lewis (6%, p<0.05) and was attenuated in denuded but not KCl‐constricted rings. Acetylcholine relaxation was not different between groups (p=0.88) suggesting that variations in the GPR30 response are not due to endothelial dysfunction. We conclude that GPR30‐mediated vasorelaxation may offer a protective role in the mRen2.Lewis female rats, and the impaired GPR30 response after exposure to high salt may contribute to the salt‐sensitive phenotype in this strain. Support: HL‐56973 and HL‐51952
Renal Response to Dietary Salt in High Sodium Lithium Countertransport (SLC) Baboons
Authors: Cox Laura A , Glenn Jeremy , Rainwater David L , Haywood Joseph R , Shade Robert E
Published: June 2021

Aim: To identify genes and pathways that regulate SLC activity, a marker for essential hypertension, we challenged normal (N) and high (H) SLC baboons with a high salt (HS) diet and compared renal transcriptome profiles.

Study design: NSLC (256 ± 9) and HSLC (426 ± 17) baboons (n = 5/group) were fed a low salt diet (20 mmol/day NaCl) for 8 weeks then fed a HS diet (150 mmol/day NaCl) for 6 weeks. Kidney biopsy transcriptome data from each diet were used for pairwise, cluster and pathway analyses.

Results: Analysis of HSLC vs NSLC baboons on HS showed differential expression of 8.2% of expressed genes with 148 genes up‐ and 817 genes down‐regulated [including 10 solute carrier family genes]. Eight KEGG pathways were up‐ and 32 KEGG pathways were down‐regulated including oxidative phosphorylation. Cluster analysis of both SLC groups on both diets revealed 5 clusters. One cluster included 396 genes that were salt responsive in NSLC but not HSLC baboons and included protein kinase C, central to calcium‐, PPAR‐, Wnt‐, MAPK‐, and VEGF‐signaling and NADH dehydrogenase, central to oxidative phosphorylation.

Conclusions: Baboon kidney showed salt responsiveness in NSLC baboons for signaling pathways central to sensing stress and nutrients; whereas they were down‐regulated or unchanged in HSLC baboons. This suggests that the HSLC blood pressure phenotype is associated with a lack of response to dietary salt for numerous molecular pathways.
Endothelial cell activation in response to placental ischemia in pregnant rats is mediated by agonistic autoantibodies to the angiotensin type I receptor
Authors: LaMarca Babbette , Roberts Lyndsay , Dukes Matthew P , Murphy Sydney R , Fournier Lillian , Speed Joshua , Cockrell Kathy
Published: June 2021

The release of soluble factors such as endothelin‐1 (ET‐1) and agonistic autoantibodies to the angiotensin II (AngII) type I receptor (AT1‐AA) may be important links between maternal endothelial cell activation and placental ischemia during hypertension in preeclamptic women. We recently demonstrated a role for AT1 receptor activation in mediating ET‐1 production by endothelial cells exposed to serum from pregnant rats with reductions in uterine perfusion pressure (RUPP). The purpose of this study was to determine if activation of the AT1 receptor on endothelial cells in response to placental ischemia is mediated by the AT1‐AA. To achieve this goal, HUVECs were exposed to RUPP serum in the presence and absence of a blocking peptide corresponding to the AT1AA binding site. Arterial pressure was significantly higher in RUPP rats (135+/−2mmHg)) than in normal pregnant rats (106+/−1mmHg). Six hours after exposure to control RUPP serum (n=14), the ET‐1 concentration in cell culture supernatant was 45.8+/− 7.2 pg/ml as compared to 25.0+/− 2.8 pg/ml from cell supernatant exposed to serum from normal pregnant rats (n=17). Interestingly, the increase in ET‐1 production in response to RUPP serum was abolished (20.5+/−3.0 pg/ml; n=17) by pretreatment with the AT1AA blocking peptide. These data indicate that the AT1AA mediates the increase in ET‐1 production induced by serum from pregnant rats exposed to placental ischemia.
Role of calmodulin kinase II in inotropic effect of α1‐adrenergic stimulation in the heart
Authors: Grimm Michael , Huang Katherine , Eschenhagen Thomas , Anderson Mark E. , Brown Joan Heller
Published: June 2021

The multifunctional Ca2+ and calmodulin‐dependent kinase II (CaMKII) may play a role in cardiac function by affecting Ca2+ release or uptake mechanisms through phosphorylation of its downstream targets phospholamban (PLB) and the ryanodine receptor. Here we examined the role of CaMKII in the positive inotropic effect of the α1‐adrenoceptor agonist phenylephrine (PE). In left atria isolated from a genetic mouse model of cardiac CaMKII inhibition (AC3‐I) PLB‐T17 phosphorylation was 20% of wild type (WT) or AC3‐C (scrambled peptide). Respectively, basal force (1 Hz) was 20% lower at 1.8 mM Ca2+, but normal at high Ca2+ (>4.5 mM). The maximal positive inotropic effect of PE, which was more pronounced at low frequencies in WT and AC3‐C, lost its frequency dependence in the AC3‐I atria. Thus, the effect of PE was reduced by 50% at 1 Hz, but was normal at 8 Hz. Post‐rest (30 s, 1 Hz) potentiation of force was also absent. Subsequent studies were carried out examining response to PE in CaMKIIδ knockout mice. Basal and PE (100 μM)‐stimulated left ventricular pressures were not altered in Langendorff perfused hearts from these mice, despite significantly reduced PLB‐T17 phosphorylation after 10 min PE (20% of WT). Our data suggest a role of CaMKII in the positive inotropic effect of atrial α1‐adrenergic stimulation. Further studies are needed to determine the role of CaMKII in cardiac function under pathophysiological conditions.
The senescence‐accelerated mouse: a model for the investigation of age‐related oxidative stress and diastolic dysfunction
Authors: Reed Alana Leigh , Fan Tai‐Hwang Michael , Dudley Samuel C , Sutliff Roy L
Published: June 2021

Diastolic dysfunction (DD) is characterized by impaired active relaxation, increased stiffness, and often progresses to diastolic heart failure. Although DD has been associated with aging and oxidative stress in humans, mechanisms of DD have proven difficult to study due to lack of relevant animal models. We have used the senescence‐accelerated mouse model (SAM) to investigate DD. This model mimics many geriatric disorders observed in humans and consists of two strains, senescence‐prone (SAMP8) and senescence‐resistant controls (SAMR1). Oxidative stress was measured in aortas using electron spin resonance. Pulsed‐wave Doppler echocardiography was used to assess blood flow velocity (E/A), tissue‐Doppler imaging was used to assess mitral annulus velocity (E’/A’), and color M‐mode imaging was used to measure the propagation inflow velocity (Vp). SAMP8 mice had a two‐fold increase in vascular superoxide compared to controls (p<0.05; n=3). The E/A ratio was reduced from 4.2 ± 0.1 cm/s in SAMR1 to 1.9 ± 0.3 cm/s in SAMP8 mice. The E’/A’ ratio was similarly reduced from 1.6 ± 0.1 to 0.6 ± 0.1 in SAMR1 vs. SAMP8 mice, respectively. Finally, Vp was decreased from 42.7 ± 2.4 cm/s in SAMR1 to 24.7 ± 2.0 cm/s in SAMP8 mice (p<0.05; n=3). These data suggest that aging is associated with DD and oxidative stress in this model; therefore, it may be useful in elucidating the mechanisms behind DD.
The role of CD36 and Caveolin‐1 in left ventricular heart function
Authors: Thomas Candice M , Smart Eric J
Published: June 2021

CD36 and caveolin‐3 are known to regulate cardiac myocyte function; in contrast, the role of caveolin‐1 is unknown. To determine if caveolin‐1 effects cardiac function we instrumented CD36 null and caveolin‐1 (cav‐1) null mice to measure changes in intra‐ventricular blood pressure and volume under controlled mechanical ventilation in response to isoproterenol and propranolol. Propranolol caused a decrease in left ventricular end systolic pressure (LVESP) and cardiac output (CO) in CD36 null mice similar to wild‐type mice. At a low concentration, propranolol decreased LVESP and CO in cav‐1 null mice more than two‐fold. CD36 null mice had a higher baseline LVESP than wild‐type mice and isoproterenol caused an additional increase in LVESP and CO parallel to the changes observed in wild‐type mice. In contrast, isoproterenol did not affect LVESP or CO in cav‐1 null mice. CD36/cav‐1 double null mice had an elevated baseline LVESP compared to wild‐type mice, but did not respond to isoproterenol. Propranolol caused a decrease in LVESP and CO parallel to that observed in cav‐1 null mice. These results indicate that the absence of CD36 in mice results in elevated blood pressure that is further elevated in response to sympathetic stimulation and caveolin‐1 plays a regulatory role in the beta adrenergic signaling pathway in the heart.
Dissecting changes in Dpo4 DNA polymerase structure during catalysis by hydrogen‐deuterium exchange mass spectrometry
Authors: Eoff Robert Lawton , Sanchez‐Ponce Raymundo , Guengerich Frederick Peter
Published: June 2021

Sulfolobus solfataricus DNA polymerase IV (Dpo4) has been studied in vitro as a model for Y‐family polymerase catalysis. Some initial kinetic studies with Dpo4 suggested that a non‐covalent step precedes the phosphoryl transfer step in the reaction cycle, which conforms to the “induced‐fit” mechanism for polymerase catalysis. Such a step is often attributed to conformational changes in the enzyme structure, but x‐ray crystal structures of Dpo4 in various catalytic states have failed to reveal the large domain rearrangements that have been observed for polymerases such as bacteriophage T7 DNA polymerase. Our group has used hydrogen‐deuterium exchange in tandem with mass spectrometry in an attempt to resolve structural changes that occur during Dpo4 catalysis. Distinct structural changes were observed upon binding to DNA and upon inclusion of a divalent metal ion. Analysis of these changes may provide insight into non‐covalent steps that define reaction kinetics and contribute to polymerase fidelity.
Locus‐wide Regulation of Mpz Expression by Egr2 and Sox10
Authors: Jang Sung‐Wook , LeBlanc Scott E , Jones Erin A , Svaren John
Published: June 2021

The Egr2/Krox20 transcription factor is a master regulator of the myelination program in peripheral nerve. We have recently demonstrated that Egr2 induces expression of the myelin protein zero (Mpz) gene via its direct binding to a conserved element within the first intron of the Mpz gene. To further elucidate the mechanism of the massive induction of Mpz during myelination, chromatin immunoprecipitation coupled with microarray (ChIP‐chip) analysis was used to identify two additional Egr2 binding sites ‐5 kb upstream of the Mpz transcription start site. Transfection assays revealed that Egr2‐mediated Mpz induction is greatly enhanced in the presence of these sites, which seem to work cooperatively with the intron‐associated enhancer. Interestingly, another important regulator of peripheral nerve myelination, Sox10, also binds to upstream sites in an Egr2‐dependent fashion. Moreover, Sox10 binding at the site is specifically disrupted by a dominant‐negative Egr2 mutant associated with human peripheral neuropathies. Finally, chromosome conformation capture (3C) assays indicated a long‐range interaction between the upstream and the intron enhancer element that is mediated by Egr2. Overall, these results suggest that transactivation of Mpz by Egr2 in myelinating Schwann cells involves interaction between upstream and intron‐associated enhancer elements. This study was supported by the grant (2 R01 HD 41590) from NICHD/NIH.
ISOLATION AND CHARACTERIZATION OF ZEAXANTHIN GLUCOSYLASE (CRTX) FROM PARACOCCUS HAEUNDAENSIS.
Authors: Kim Young Tae , Choi Seong Seok , Seo Yong Bae
Published: June 2021

Zeaxanthin [(3R,3′R)‐β, β ‐carotene‐3,3′diol] is one of the two carotenoids contained within the retina. It is the pigment that gives corn, saffron, and many other plants their characteristic color. Zeaxanthin breaks down to form picrocrocin and safranal, which are responsible for the taste and aroma of saffron. The product of the zeaxanthin glucosylase gene (crtX) mediate the formation of zeaxanthin to zeaxanthin diglucoside. Zeaxanthin glucosylase (CrtX) was isolated from the marine bacterium Paracoccus haeundaensis. The crtX gene consisted of 1248 bp encoding 415 amino acidsresidues. The nucleotide sequence of crtX was analyzed with that of other species and turned out to be well conserved during evolution, which was confirmed from a phylogenetic tress. CrtX recombinant protein was expressed using the E. coli expression vector (pET‐44a(a)+). The plasmid containing crtX gene produced the recombinant protein of approximately 46 kDa. The protein was purified to homogeneity and enzymatically active form. The enzymatic properties of gene product of crtX gene from Paracoccus haeundaensis was analyzed by chromatographic and spectroscopic methods.
Extracellular anion modulation of ASIC3
Authors: Kusama Nobuyoshi , Benson Christopher J.
Published: June 2021

Acid‐sensing ion channels (ASICs) are non‐voltage cation channels gated by extracellular protons. ASICs have chloride‐binding sites in the extracellular domain that are highly conserved between ASIC isoforms. However, the significance of the Cl binding is unknown. We investigated the effect of different anions on heterologously expressed ASIC3 currents recorded by whole‐cell patch‐clamp. Replacement of extracellular Cl with the impermeable and inert anion, methanesulfonate (MS), had no effect upon peak current amplitude, however at the threshold of ASIC3 activation there was a slight potentiation of pH 7‐evoked current in MS. The mechanism involved a shift in steady‐state desensitization whereby Cl caused more desensitization at a given holding pH. In addition, ASIC3 currents desensitized at a faster rate in Cl compared to MS. Other anions, including bromide and thiocyanate, produced the opposite effect of MS; they decreased proton sensitivity and increased kinetic rates. The effects of various anions followed the lytropic series: thiocyanate > Br > Cl > MS. Mutation of the purported Cl binding sites in ASIC3 abolished the modulatory effects of anions. In addition to sensing pH changes, modulation of ASICs by anion fluxes associated with ischemia further positions the channels to sense changes in the extracellular environment.
High NaCl activates c‐Abl kinase, phosphorylating TonEBP/OREBP on Y143, which contributes to its increased nuclear localization
Authors: Gallazzini Morgan , Villers Courtney L , Burg Maurice B , Ferraris Joan D
Published: June 2021

High NaCl activates TonEBP/OREBP (tonicity‐responsive enhancer binding protein/osmotic response element binding protein), resulting in increased transcription of osmoprotective genes. The activation includes nuclear localization of TonEBP/OREBP. An Auxiliary Export Domain (AED) within amino acids 132‐156 of TonEBP/OREBP is involved in its subcellular localization (Tong, JBC 2006). We find that in HEK293 cells: 1) High NaCl increases phosphorylation of Y143. 2) Mutation of Y143 to alanine, which prevents its phosphorylation, reduces nuclear localization of TonEBP/OREBP. 3) Y143 is a consensus c‐Abl phosphorylation site and high NaCl increases c‐Abl kinase activity. 4) c‐Abl phosphorylates Y143 in vitro. 5) c‐Abl coimmunoprecipitates with TonEBP/OREBP, indicating that the proteins are associated. 6) Dominant negative (kinase dead) c‐Abl decreases high NaCl‐induced nuclear localization of TonEBP/OREBP. We conclude that high NaCl increases phosphorylation of TonEBP/OREBP on Y143, catalyzed by c‐Abl kinase, which contributes to increased nuclear localization of TonEBP/OREBP.

This research was supported by the Intramural Research Program of NHLBI.
Hydrogen Peroxide Mediates Post‐Excitatory Depression of Baroreceptor Afferent Activity in Vivo
Authors: Santana‐Filho Valter J , Abboud Francois M , Chapleau Mark W
Published: June 2021

Acute hypertension leads to 'post‐excitatory depression' (PED) of baroreceptor (BR) activity after the period of elevated blood pressure (BP), a phenomenon that contributes to BR resetting. The molecular mechanism of PED is not fully understood. We recently demonstrated that electrical stimulation of isolated BR neurons in vitro generates reactive oxygen species that cause PED of action potential firing after the period of neuronal activation (Hypertension 43(6):1349, 2004). In the present study, we tested the hypothesis that the reactive oxygen species hydrogen peroxide (H2O2) mediates BR PED in vivo. The left aortic depressor nerve (ADN) was draped over a recording/stimulating electrode and crushed centrally to prevent reflex changes in BP in anesthetized C57BL/6 mice. Afferent ADN (BR) activity was recorded before and after a brief period of ADN stimulation (3V, 2ms pulses at 20Hz for 10s). Afferent BR activity was markedly inhibited after the 10s period of electrical stimulation, averaging 50±5% and 80±8% of baseline levels 5 and 60s after the stimulation was terminated (n=7). This PED was abolished after administration of the membrane permeable H2O2 scavenger PEG‐catalase (4000 U, IV) with activity averaging 100±4 and 126±7% of baseline 5 and 60s after ADN stimulation (n=7). PED was unaffected by administration of the saline vehicle in a separate group of mice (n=6). We conclude that H2O2 generated during activation of BR afferents causes PED. This autocrine‐feedback inhibition of BR activity may contribute to rapid BR resetting in acute hypertension. (HL14388, VA)
Alterations in renal sodium and acid‐base transport in H,K‐ATPase null mice
Authors: Greenlee Megan , Lynch Jeanette , Rudin Alicia , Wingo Charles
Published: June 2021

The kidney expresses two H,K‐ATPase isoforms in the collecting duct, HKα1 or HKα2, that function to transport potassium (K) in exchange for protons, particularly in states of K depletion. However, loss of either HKα1 or HKα2 H,K‐ATPase isoforms did not significantly effect either urinary K excretion or systemic acid‐base balance in mice (Spicer et al. J Biol Chem. 2000 275(28); Meneton et al. J Clin Invest. 1998 101(3)). Because another isoform is present in these single knockout animals, we hypothesized that the H,K‐ATPases compensated for each other. To test this hypothesis, the effect of dietary K depletion on renal electrolyte and acid‐base handling was evaluated in double‐knockout mice lacking both HKα1 or HKα2 (HKα1,2−/‐). Surprisingly, HKα1,2−/− mice efficiently conserved urinary K. However, the HKα1,2−/− mice excreted significantly more urinary titratable acid and ammonium, sodium (Na) and chloride (Cl) with no apparent change in blood Na, Cl, pH, or HCO3. With K depletion, HKα1,2−/− mice retained more urinary Na than wild type mice. The evidence suggests that renal H,K‐ATPases may not play an essential role in net renal K conservation. Instead, the pumps may participate directly or indirectly in both Na and acid‐base transport by the kidney.

These studies were supported by an NIH grant (RO1‐DK‐049750).
Suppressed Plasma Angiotensin II and Reduced Antioxidant Enzyme Expression Contribute to Impaired Vascular Relaxation in Dahl Salt‐Sensitive Rats
Authors: Durand Matthew J , Lombard Julian H.
Published: June 2021

Dahl salt‐sensitive (SS) rats exhibit impaired regulation of the renin gene and chronically low plasma angiotensin II (ANG II) levels. Normal ANG II levels and impaired vasodilator responses are restored when chromosome 13 (chr13) from the Brown Norway (BN) rat is introgressed onto the SS genetic background. This study used novel congenic rats [SS.BN‐(D13hmgc41‐D13hmgc23)] containing a 2.0 Mbp portion of chr13 including the BN renin allele to investigate the role of ANG II levels in maintaining vasodilator responses and antioxidant enzyme expression. Male SS and congenic (Ren1‐BN) rats (10‐14 weeks old) were fed a low‐salt diet (0.4% NaCl) after weaning. Responses of isolated middle cerebral arteries (MCA) to acetylcholine (ACh; 10−10 ‐ 10−5 M), reduced PO2 (perfusion and superfusion solution PO2 ≈35‐45 mmHg) and sodium nitroprusside (SNP; 10−12 ‐ 10−4 M) were assessed via video microscopy. MCA from the Ren1‐BN congenic rats dilated in response to ACh and reduced PO2 while MCA from the SS rats did not. Both strains exhibited a dose‐dependent dilation to the NO donor SNP. Cu/Zn SOD expression was significantly reduced in arteries from SS rats vs. Ren1‐BN rats. These results suggest that introduction of a normally functioning renin gene into the SS genetic background restores normal vasodilator responses by increasing antioxidant enzyme expression and reducing vascular oxidative stress. (NIH #HL65289, #HL72920).