bluuebunny/crossref_metadata_2025 huggingface dataset:
AMP‐activated Kinase Inhibits KCNQ1 Channels through Regulation of the Ubiquitin Ligase Nedd4‐2
Authors: Alzamora Rodrigo , Pastor‐Soler Nuria M. , Smolak Christy , Hallows Kenneth R.
Published: June 2021

The KCNQ1 K+ channel ia a regulator of body electrolyte homeostasis. The metabolic sensor AMP‐activated kinase (AMPK) regulates several ion transporters. To determine whether AMPK regulates KCNQ1, we studied the effects of AMPK activation on KCNQ1 currents in Xenopus oocytes. AMPK activation decreased KCNQ1 currents, but AMPK did not phosphorylate KCNQ1 in vitro, suggesting an indirect regulatory mechanism. Recently, it has been shown that Nedd4‐2 inhibits KCNQ1 by decreasing plasma membrane expression. Previous studies from our group have shown that AMPK down‐regulates epithelial Na+ channels via regulation of Nedd4‐2. Therefore, we examined the role of Nedd4‐2 in the AMPK‐dependent regulation of KCNQ1. Channel inhibition by AMPK was blocked in oocytes coexpressing either a dominant‐negative or constitutively active Nedd4‐2 mutant, or a KCNQ1 PY motif mutant, suggesting that AMPK‐dependent modulation of Nedd4‐2 is involved in the regulation. In kidney slices the AMPK activator AICAR induced intracellular redistribution of KCNQ1 from the plasma membrane in the collecting duct. These results suggest that AMPK inhibits KCNQ1 activity by promoting Nedd4‐2‐dependent channel retrieval from the plasma membrane. KCNQ1 inhibition by AMPK may limit cellular K+ recycling under conditions of metabolic stress, thus reducing cellular energy expenditure for ion transport in epithelia. (Funded by NIH and AHA)
Activation of Wnt‐β‐catenin (β‐cat) signaling by cadmium (Cd) in kidney proximal tubule cells
Authors: Thevenod Frank , Chakraborty Prabir K. , Horndasch Manuela , Lee Wing‐Kee , Wolff Natascha A.
Published: June 2021

Cd causes apoptosis of the proximal tubule (PT), but is also a class 1 carcinogen. Using cultured PT cells, we have shown that Cd apoptosis peaks at 24 h, but then decreases over time, which is caused by overexpression of multidrug resistance P‐glycoprotein (Abcb1). One early event associated with Cd nephrotoxicity is disruption of cell‐cell adhesion. β‐cat participates in cell‐cell adhesion by bridging E‐cadherin to β‐cat and as part of the Wnt signaling co‐activates the LEF/TCF DNA‐binding protein. As such, β‐cat transactivates proliferation genes, e. g. c‐myc and cyclin D1, but also Abcb1. The cytoplasmic level of β‐cat is controlled by the proteasome. We showed that Cd decreases transepithelial resistance of PT cells. This was associated with the degradation of E‐cadherin, release of β‐cat into the cytosol and nucleus, and increased expression of c‐myc and Abcb1. Transfection with a β‐cat δN57 mutant, which is not degraded by the proteasome, also increased c‐myc and Abcb1. To investigate whether Cd affects LEF/TCF activity, a luciferase reporter assay (TOPflash/FOPflash) containing LEF/TCF binding sites was used. Cd increased luciferase activity up to 5‐fold in PT cells These results suggest that β‐cat is involved in transcriptional regulation of genes for proliferation and cell survival triggered by Cd, which may induce apoptosis evasion and promote carcinogenesis. Supported by DFG TH 345/10‐1.
EGFR augments cell proliferation in polycystic kidney disease through activation of a novel ion channel
Authors: Zhang Zhi‐Ren , Chu Wen‐Feng , Song Binlin , Gooz Monika , Baldys Aleksander , Kolb Robert J. , Steele Stacy , Houston Amber , Haycraft Courtney J. , Owsianik Grzegorz , Nilius Bernd , Raymond John R. , Komlosi Peter , Bell Phillip D.
Published: June 2021

Previously we have proposed that dysregulation of Ca2+ entry mechanisms due to loss of cilia in collecting duct cells leads to enhanced cell proliferation and cystogenesis. These studies were performed to identify this Ca2+ channel and to determine if it is regulated by the epidermal growth factor receptor (EGFR). Using patch‐clamp, a 23‐pS Ca2+‐permeable channel was found at the apical membrane of orpk cilia(+) and orpk cilia(‐) collecting duct cells. This channel was markedly activated by EGF only from the apical surface in orpk cilia(‐) cells; in orpk cilia(+) cells channel activity was elevated only by basolateral administration of EGF. EGFR was more abundant in orpk cilia(‐) compared to cilia(+) cells and EGF‐induced channel activity was ~3.5‐fold higher in orpk cilia(‐) cells compared to orpk cilia(+) cells. Effects of EGF on this channel were blunted by both AG1478 and PD98059; tyrosine kinase and MAP kinase inhibitors, respectively. Using shRNA, patch‐clamp, and co‐immunoprecipitation we identified the 23‐pS channel as a multimer of TRP channels, TRPP2 and TRPV4. EGR enhanced cell proliferation in orpk cilia(‐) cells, at least in part, through activation of the 23‐pS Ca2+ channel. Our results provide evidence that EGFR activates a TRPP2\TRPV4 channel complex that leads to Ca2+ entry and contributes to hyper‐proliferation of orpk cilia (‐) cells.
Hydrogen sulfide mitigates homocysteine‐induced glomerular injury
Authors: Sen Utpal , Basu Poulami , Metreveli Naira , Kumar Munish , Tyagi Suresh
Published: June 2021

Although recent studies have demonstrated a protective role of H2S in end‐stage organ failure; its role in homocysteine (Hcy)‐induced renal disease is unclear. We hypothesized that H2S protects kidney from Hcy‐induced renal failure. Cystathionine‐β‐synthase heterozygous, (CBS+/‐) and wild type (WT) mice with 2‐kidney (2‐K) and 1‐kidney (1‐K) were used in this study with or without NaHS (H2S donor) supplementation. Plasma Hcy, tissue H2S and urinary protein were measured. Apoptotic cells and O2▸− were detected in the kidney sections. MMP‐2 and ‐9 activities were measured by in vivo imaging and TIMP‐1 expression by immunochemically. Plasma Hcy was elevated in CBS(+/‐) 2‐K, 1‐K and WT 1‐K mice along with increased proteinuria. Interestingly, kidney tissue H2S was attenuated in these groups compared to WT 2‐K. H2S supplement, however, attenuated increased urinary protein secretion. Increased activities of MMP‐2 and ‐9, O2▸− production and apoptotic cells were observed in CBS(+/‐) 2‐K, 1‐K and WT 1‐K mice compared to WT 2‐K. H2S supplement, however, prevented apoptotic cell death, normalized O2▸− production and MMP‐2, ‐9 activities. These results demonstrate that Hcy‐induced renal failure is associated with attenuated endogenous H2S generation and H2S supplement prevent this renal damage, in part, through its antioxidant properties.
DOCA‐induced salt appetite in old Brown‐Norway rats
Authors: Thunhorst Robert L , Beltz Terry G , Johnson Alan Kim
Published: June 2021

Old rats, like the elderly, have diminished drinking responses to most stimuli. In addition, old rats are nearly devoid of renin‐dependent salt appetite. In the present work, we investigated mineralocorticoid‐induced salt appetite in old rats. Young (4 mo), middle‐aged (12 mo) and old (30 mo) male Brown‐Norway rats with ad libitum access to water, 0.3 M NaCl and sodium deficient diet received DOCA (5 mg/kg/day, sc) for six days. In response to DOCA, old rats drank as much saline solution as young rats on an absolute basis (~ 25 ml/day), and about a third as much as middle‐aged rats (~75 ml/day). After adjusting intakes for body weight, the salt appetite responses by age were middle‐aged > young > old rats. In addition, old rats preferred saline less than the other groups as indicated by their significantly reduced saline preference ratios (saline/total fluid intake) compared to young and middle‐aged rats. This is the first demonstration of a significant salt appetite in old animals. We hypothesize that the reduced capacity of old rats to excrete water and sodium loads reduces sodium intake in response to DOCA compared to young and middle‐aged rats. This work was supported by AG‐025465 to RLT and HL‐14388 and DK‐066086 to AKJ.
Antidiuretic Effects of Endothelin A Receptor Antagonism
Authors: Iliescu Radu , Zaugg Christian E. , Reckelhoff Jane F. , Baltatu Ovidiu C.
Published: June 2021

Selective ET‐A receptor antagonism has diuretic and natriuretic effects which may be potentiated by the unopposed activation of ET‐B receptors by endogenous endothelin. A major side effect of selective ET‐A receptor antagonists is fluid retention and edema. We hypothesized that compounds with high selectivity against ET‐A receptors produce fluid retention at concentrations where non‐specific blockade of ET‐B receptors may occur. Incremental doses of the ET‐A receptor antagonist SPP301 (0.003; 0.03; 3 mg/kg; lower dose to target ET‐A receptor, highest dose to target both ET‐A and ET‐B receptors based on a CHO cell assay) were administered i.v. to anesthetized rats (SD, n=6) undergoing saline diuresis (15 mL/kg/h, 1% BSA). Urine was collected from the bladder while blood pressure (BP) and glomerular filtration rate (GFR) were monitored. SPP301 decreased urine output (49; 46; 32 ul/min, p<0.05) and fractional excretion of water (6; 5; 4 %, p<0.05) in a concentration‐dependent manner. Sodium excretion was initially increased but returned to control values after the highest dose of SPP301. GFR was unchanged while BP was reduced by 10 mmHg only by the highest dose of SPP301. Administration of BQ‐788 (ET‐B receptor antagonist, 3mg/kg) after SPP301 3 mg/kg did not further decrease urine output or water excretion and was without effect on GFR. The highest dose of SPP301 caused a decrease of both 24‐hour diuresis (18.3%) and natriuresis (17.6%) with a consequent hematocrit decrease (8.3%) in a hypertensive transgenic model expressing both human renin and angiotensinogen genes. These data indicate that increasing concentrations of SPP301 may also block ET‐B receptors and cause antidiuresis. This effect could explain why fluid retention and edema occur during treatment with selective ET‐A receptor blockers.
Oropharyngeal regulation of urine output and cardiovascular function in rats with osmotic stimulation by sham ingestion
Authors: Knight W. David , Ji Lisa Liping , Campos Pete , Gottlieb Helmut , Cunningham J. Thomas
Published: June 2021

Studies indicate that oropharyngeal afferents participate in the central regulation of hydration status although the role of these afferents in the rat remains controversial. Therefore, we tested the renal and cardiovascular effects of oropharyngeal stimulation with water and isotonic saline. Anesthetized rats were implanted with femoral catheters, esophageal fistulae, and bladder catheters. During the experiment, each rat was infused with isotonic saline (55 µl/min, iv) containing ketamine (1 mg/kg/min) and xylazine (80 mg/kg/min). After acclimation and a 1 h baseline, rats were infused orally (1 ml/min; 2 min.) with either 0.9% isotonic saline (n=4) or water (n=4) and urine was collected in 10 min. aliquots for 1 h. Within minutes, water infusion transiently increased blood pressure and decreased heart rate, and, after thirty minutes, when blood pressure returned to near baseline levels, increased urine output and decreased urinary sodium concentration. Infusions of isotonic saline had no effect. The renal effects of oropharyngeal water infusion are consistent with a hypotonically‐driven inhibition of AVP release that leads to diuresis and decreased sodium concentration. Further, the pressor response to a hypotonic stimulus may serve as an anticipatory reflex to perceived ingestion of a volume that will dilute plasma osmolality. This work supported by R01 HL062576.
Angiotensin II (ANG II) does not utilize the Janus Kinase 2/Signal Transducers of Activated Transcription (JAK/STAT) pathway in the molecular mechanism of blood pressure response to a low salt diet
Authors: Banes‐Berceli Amy K.L. , Sturgis LaShon , Brands Michael W. , Allen Ashlyn J
Published: June 2021

We recently reported that the JAK2 inhibitor AG490 prevented JAK/STAT pathway activation and hypertension during chronic iv. ANG II infusion. It is unknown, however, whether activation of JAK2 is required for ANG II to prevent blood pressure from decreasing in the physiologic response to low salt intake. This was tested by blocking JAK2 activation chronically in rats that were switched from normal‐ to low‐salt intake. Male SD rats with instrumented with artery and vein catheters for 24 hr/day mean arterial pressure (MAP) measurement and iv. infusions. Rats were maintained on a normal‐salt (NS) intake (~ 3 mEq/day), and after baseline measurements chronic iv. infusions of either: vehicle (Veh; saline), the AT1 receptor antagonist Losartan (LOS; 10 mg/kg/day) or AG490 (AG; 10 ng/kg/min) were begun. After a 4‐day control period, rats were switched to low‐salt intake (LS; 0.4 mEq/day), while Veh, LOS and AG infusions continued. There were no differences in MAP between any group under baseline conditions. During the control period, MAP averaged 93+4, 97+3 and 80+2 mm Hg in Veh, AG, and LOS groups, respectively. Switching to LS intake did not change MAP in Veh and AG rats (102+4 and 101+3 mm Hg, respectively); but MAP decreased significantly in LOS rats to 72+4 mm Hg by day 5. These data suggest that JAK2 activation is not involved in ANG II's role in the blood pressure response to LS intake. (HL74167 MWB; HL91177 AB)
Nuclear AT2 receptors mediate angiotensin II‐dependent generation of nitric oxide
Authors: Gwathmey TanYa M. , Pendergrass Karl D. , Pirro Nancy T. , Shaltout Hossam A. , Reid Sean D. , Rose James C. , Chappell Mark C.
Published: June 2021

We recently reported the expression of receptors for the hormone angiotensin II [Ang II] in isolated nuclei of the sheep kidney. The renal cortex contained predominantly AT2 receptors [~70%], while the renal medulla expressed essentially the AT1 subtype [>90%]. The present study examined their functional roles in cortical nuclei of adult sheep. Nuclei were isolated from kidneys of adult (1.5 yr old) sheep by Optiprep density gradient and loaded with the fluorescence dye difluorofluorescein diacetate (DAF) to assess the production of nitric oxide (NO). Ang II (1 nM) significantly increased DAF fluorescence above control (80 +/‐ 11%; P<0.001; N=4); DAF stimulation was abolished by either the AT2 antagonist PD123319 (1µM, P>0.05 vs. control) or the NO synthase inhibitor L‐NAME (1mM, P>0.05 vs. control). Treatment with AT1 receptor antagonist losartan (1µM) did not alter Ang II‐induced NO generation (P>0.05 vs. Ang II). Protein analysis of isolated cortical nuclei revealed a prominent band for both eNOS/NOSIII (135kDa) and the principal NO receptor soluble guanylate cyclase‐β (sGC, 70kDa). We demonstrate that the nuclear AT2 receptor subtype is functionally linked to NO generation and confirm the expression of eNOS and sGC in sheep nuclei. These data suggest that the nucleus contains the necessary signaling components for NO generation and provide further support of a functional intracellular RAS within the kidney.
Modeling the circulation as a supply and demand system
Authors: Johnson Paul C , Yalcin Ozlem , Cabrales Pedro , Intaglietta Marcos
Published: June 2021

Existing concepts of circulatory control focus on the role of the central nervous system in adjusting cardiac output and vascular resistance to maintain a fixed arterial pressure set point. However, arterial pressure level changes in a variety of conditions and it is not clear what mechanism readjusts the postulated set point. Alternatively, we propose that the circulatory system is, at a very fundamental level, a supply and demand system in which demand determines the overall flow of product through the system. In this model the interaction between autoregulatory mechanisms of the peripheral circulation and intrinsic mechanisms of the heart (Starling's Law) determine cardiac output. Arterial pressure is not a regulated variable. This model resembles a modified free‐market economic system in which central control mechanisms modify, rather than set, supply and demand. In principle, this model should be superior to a central command model in optimizing tissue homeostasis. .A model based on this principle predicts the observed effects of decreasing vascular resistance, static exercise and dynamic exercise on cardiac output and arterial pressure. Supported by NIH grant HL 52684
Neuronal nitric oxide synthase (nNOS) and c‐Fos expression in the nucleus tractus solitarius (NTS) of normotensive and hypertensive rats
Authors: Hawkins Megan N , Squiers Jack , Leal Anna K , Mitchell Jere H , Smith Scott A
Published: June 2021

Evidence suggests the skeletal muscle mechanoreflex (MMR) is exaggerated in hypertension and contributes significantly to the heightened pressor response to exercise in this disease. MMR sensory information is processed in the NTS of the medulla oblongata. Under normal conditions, nitric oxide (NO) production via the oxidation of L‐arginine by nNOS within the NTS attenuates MMR activity. However, compared to normotensive Wistar‐Kyoto (WKY) animals, expression of nNOS is decreased in the NTS of spontaneously hypertensive (SHR) rats potentially reducing the buffering capacity of NO. To establish such a link, it must be determined whether nNOS expression (or lack thereof) is present in neurons excited by the MMR. Stimulation of MMR neurons generates expression of the early response gene c‐Fos. Thus, we preferentially stimulated the MMR in WKY and SHR rats via hindlimb muscle stretch (30 sec intervals for one hour) and subsequently examined medullary NTS tissue for both c‐Fos and nNOS expression. Expression of c‐Fos and nNOS was clearly present within the same NTS neurons of WKY rats. Similar results were found in SHR animals though the expression of nNOS was markedly reduced. The findings are consistent with the concept that NO production via nNOS within NTS neurons excited by the MMR may be decreased in hypertension and likely contributes to the development of MMR overactivity in this disease. Supported by NIH HL‐088422
Effect of physical training on hind limb blood flow with femoral artery occlusion in pigs
Authors: Terjung Ronald L , Yang Hsiao Tung , Bowles Douglas K , Laughlin M. Harold
Published: June 2021

Physical training (PT) is an important regimen for intermittent claudicants. To develop collateral circuit supporting the large muscle mass in large mammals, requires development of large caliber conduit vessels. To study whether PT enhances to develop large collateral conduits, male Yucatan pigs (35 kg) received femoral artery occlusion. Flow probes were placed on each external iliac artery and the pigs were given a stress test each wk for eight wks (@1‐5 mile/h). BP in the thoracic aorta was monitored. Pigs were either kept in cage activity (Sed) or ran on treadmill @ 3.5 mi/hr for 35 min/d, for 8 wks (Tr). BP during exercise declined (35%) after PT. Peak BF through the iliac artery observed increased in both Sed and Tr pigs similarly over 8 wks (643±83 to 913±26 ml/min). This increase in BF is similar to the 250±25 ml/min measured in the unobstructed contralateral femoral artery during the same exercise intensity. Thus the increased BF was attributed to collateral flow via the newly formed collateral vessels that were later verified angiographically. BF though the iliac artery remained modestly elevated post‐exercise, compared to the non‐obstructed contralateral iliac artery, characteristic of an ischemic hyperemia response. Our findings demonstrate that a robust collateral vessel development occurs following occlusion of the supply vessel in a large mammal.

Supported by NIH P01HL 52490
Effect of Granulocyte Colony‐Stimulating Factor on Ventricular Vulnerability in Healed Rat Infarcts
Authors: Lee Tsung‐Ming , Chang Nen‐Chung
Published: June 2021

Granulocyte colony‐stimulating factor (G‐CSF) has been used for repair of infarcted myocardium, but concerns have been raised regarding its pro‐arrhythmic potential. Therefore, we analyzed in a rat model of experimental myocardial infarction the influence of G‐CSF treatment on sympathetic remodeling, and expression of nestin. Twenty‐four hours after ligation of the anterior descending artery, male Wistar rats were randomized to either vehicle (MI/C), or G‐CSF (MI/G). At 56 days after infarction, rats in MI/G had significantly higher left ventricular ejection fraction accompanied by a significant decrease in left ventricular end‐diastolic dimension than MI/C. Myocardial norepinephrine levels revealed a significant elevation in MI/C in the border zone compared with sham‐operated rats. Immunohistochemical analysis for tyrosine hydroxylase, growth associated factor 43 and neurofilament also confirmed the change of myocardial norepinephrine. At 5 days, rats in MI/G revealed increased numbers of tissue infiltrated CD34+ cells although similar increase of circulating neutrophils counts between sham/G and MI/G. Compared with MI/C, rats in MI/G showed an increase of nestin structures associated with enhanced sympathetic reinnervation. This was paralleled by a significant upregulation of nestin protein expression and mRNA. Arrhythmic scores during programmed stimulation in MI/G were significantly higher than MI/C, suggesting a pro‐arrhythmic potential. These findings suggest that although G‐CSF administration after infarction improved myocardial function, it resulted in increased sympathetic reinnervation by activation of nestin signal and may modify the arrhythomogenic response to programmed electrical stimulation.
α2b‐Adrenergic receptors modulate baroreflex sensitivity
Authors: Duling Laura , Kanagy Nancy
Published: June 2021

Deoxycorticosterone acetate (DOCA)‐salt hypertension is associated with enhanced sympathetic activity and impaired baroreflex sensitivity (BRS). Blockade of central α2‐adrenoceptors (α2‐AR) attenuates DOCA‐salt hypertension onset and severity and inhibits BRS. Renal α2B‐AR are increased in DOCA‐salt hypertension and regulate sodium retention. We hypothesized mice with decreased α2B‐AR expression would have impaired BRS, attenuated DOCA‐salt hypertension and be more sensitive to uninephrectomy (nx). Blood pressure (BP) and heart rate (HR) were measured using radio‐telemetry in intact and nx heterozygous α2B‐AR null mice (HET) and wild‐type (WT) littermates for 3 control and 14 DOCA‐salt (50 mg DOCA pellet, 1% NaCl, 1% KCl water) treated days. DOCA‐salt increased BP similarly in both intact groups (p<0.05). BP was higher in HET‐nx than HET‐intact mice on all days (p<0.05) but nx did not affect BP in WT groups. HR decreased in a BP‐dependent manner in WT mice but did not change in HET mice. BRS in response to acute changes in BP were evaluated in isoflurane anesthetized HET and WT mice given phenylephrine (IP) to increase BP. HR decreased with increases in BP in WT mice but increased in HET mice. Therefore BRS is impaired in HET mice and α2b‐AR appear to be required for sympathetic withdrawal in response to both acute and chronic increases in BP.
Influence of Diabetes on Baroreflex (BR) Sensitivity of Sympathetic Nerve Activity (SNA)
Authors: Burgess Don , Randall David C , Stocker Sean D
Published: June 2021

We investigated the relationship between blood pressure (BP) and renal SNA in type 1 diabetic rats (n=3; BBDP/Wor; Biomedical Research Models; 472 ± 50 g, mean ± SD) and age‐matched animals (n=2; BBDR/Wor; 529 ± 8 g). Diabetic BBDP/Wor rats were given daily insulin until they had been diabetic for 410 ± 9 days. Then, rats were anesthetized with urethane (1.2 g/kg, iv), and BP and renal SNA were simultaneously recorded at 10 kHz. PE (1‐10 ug/kg) and SNP (2‐20 ug/kg) were administered iv to examine baroreflex sensitivity. Analysis of the BR sensitivity curve indicates that the progression of diabetes leaves the sympathetic neural component of the baroreflex intact (curvature = 0.077 vs. 0.085 mmHg−1, control vs. diabetic.) To simulate SNA mathematically we computed the impulse response function from the transfer function of Kawada et.al. (Conf Proc IEEE Eng Med Biol Soc. 2005;4:3954‐7.) We are able to reproduce the experimentally measured SNA as a function of the experimentally measured BP. In addition, by using a Poisson Spike Generator, we simulated a raw renal nerve recording as a superposition of scaled single‐unit waveforms. This analysis extends the standard BR curve analysis by investigating a linear relationship between BP and SNA throughout the recovery phase and by making a hypothetical connection between SNA and the underlying single unit activity. Supported by RO1 NS39774, NIH HL090826, AHA 0630202N
Mesenteric vascular resistance responses to chronic angiotensin II (AngII) are dependent on dietary salt intake
Authors: Kuroki Marcos T , Ariza Guzman Pilar , Fink Gregory D , Osborn John W
Published: June 2021

We previously reported that total peripheral resistance is increased by AngII in rats consuming a high salt diet, and that this response is attenuated by denervation of the mesenteric vascular bed. Based on these observations, we hypothesize that mesenteric vascular resistance (MVR) is increased in AngII rats consuming a high salt diet. In this study, rats on either a high (2%) or low (0.1%) NaCl diet were chronically instrumented for continuous measurement of MAP and superior mesenteric artery blood flow (MBF). AngII was infused (150 ng/kg/min, sc) for 14 days preceded by 4 days of control. MVR was calculated as MAP/MBF. 0.1% NaCl rats showed minimal responses to AngII. MAP during the control period, and Days 1 and 14 of AngII were 100±2, 103±4 and 104±4 mm Hg respectively. In contrast, in 2% NaCl rats, MAP was 111±3 during control and 126±5 and 140±10, on Days 1 and 14 of AngII. MBF was fairly constant within the two groups. In 0.1% NaCl rats MBF was 12.5±0.9, 12±1, 12.5±0.4 mL/min during control and Days 1 and 14 of AngII. In 2% NaCl rats, MBF averaged 10±1, 8±1, 10±2 mL/min over the same time period. MVR remained stable in 0.1%NaCl rats during control and Days 1 and 14 of AngII (8.0±0.7, 8.6±0.9, 8.3 ±0.6 a.u. respectively). In contrast, MVR increased from a control level of 11±1, to 16±3 on Day 1, and 14±4 a.u. on Day 14 of AngII. We conclude that AngII increases MVR in rats consuming a high salt diet but not those on a low salt diet. HL064178.
Gap junction coupling‐mediated membrane potential oscillations drive activity in cutaneous but not muscle vasoconstrictor sympathetic preganglionic neurones in situ
Authors: Stalbovskiy Alexey O , Paton Julian F , Pickering Anthony E
Published: June 2021

A population of sympathetic preganglionic neurones (SPN) recorded in vitro show gap junction‐mediated oscillations in membrane potential that underlie their spike discharge (Logan et al, 1996, J Physiol), but the role of such coupling in vivo is uncertain. We made whole‐cell recordings from functionally identified SPN in the rat (aged p5‐16) working heart‐brainstem preparation (WHBP, (Paton, 1996, J Neurosci Meths)) to examine the neural mechanisms generating their firing activity.

108 SPNs identified in the T3‐4 segment were functionally categorised by responses to activation of peripheral chemoreflex (NaCN, ia) and diving response (cold to snout). Muscle vasoconstrictor (MVC, 38%) SPN were activated by both tests and converseley cutaneous vasoconstrictor (CVC, 27%) SPN were inhibited by both. MVCs had a higher baseline firing frequency in comparison with CVC (2.6±0.3 vs. 1.4±0.3 Hz; p=0.007) and shorter after‐hyperpolarization (156±10 vs. 296±39 ms, p=0.0001). MVC SPN showed a respiratory‐modulated pattern of firing generated by a torrent of kynurenate‐sensitive, excitatory synaptic inputs. By contrast, the activity of CVC SPN was driven by large, slow, biphasic membrane potential oscillations (n=14 of 15 examined) that resembled filtered action potentials which were relatively insensitive to membrane potential, not blocked by spinal kynurenate and were indistinguishable from those previously reported in vitro. Antidromic activation of CVC SPN evoked short latency depolarization confirming the presence of coupling.

These findings indicate that gap junctional coupling plays an important functional role in the patterning and synchronization of the output from CVC but not MVC SPN.

Funded by BHF.
Analysis of cardiovascular autonomic function from telemetric blood pressure recordings: effects of age, high fat diet and angiotensin II
Authors: Erdos Benedek , Cudykier Idan , Brodsky Peri , Tumer Nihal
Published: June 2021

Assessments of sympathetic/parasympathetic nervous activity and baroreceptor reflex gain (BRG) are important for understanding neural mechanisms of cardiovascular diseases, and evaluation of heart rate variability (HRV) as well as spontaneous BRG from telemetric blood pressure recordings provides a useful tool in experimental rat models. We analyzed changes in these parameters in response to 60% high fat diet feeding and to central infusion of angiotensin II (AngII, 10nmol/h) or losartan (LOS, 15µg/h) in young (5 mo) and old (27mo) F344xBN rats that were equipped with telemetric BP transmitters. Spontaneous BRG was significantly higher in young rats vs. old. High fat feeding, as well as central infusions of both AngII and LOS, reduced BRG in young rats, but were ineffective in old animals. Both low frequency power (LF) of pulse interval data, which reflects sympathetic tone to the heart, and high frequency power (HF), which reflects parasympathetic tone, were significantly lower in old rats compared with young. AngII increased LF in young, but not in old rats, whereas HF remained unchanged in both groups. In contrast, LOS had no effect on either parameter. In response to high fat feeding, LF increased in young, but decreased in old rats, while HF decreased both in young and old animals. These data show that analysis of BRG and HRV can be used to detect age‐, diet‐ and AngII‐induced changes in autonomic function, and that age has a significant effect on how the autonomic nervous system reacts to these stimuli.
Effects of pediatric adiposity on heart rate variability
Authors: Lucas Angela Kim , Drummer Thomas D , Carter Jason R
Published: June 2021

The relationship between obesity and heart rate variability (HRV) has been studied in adults and adolescents, but has not been determined in pediatrics. The purpose of this study was to assess autonomic activity using HRV in a pediatric population. We hypothesized that obese children would have reduced parasympathetic and increased sympathetic activity compared to age‐matched subjects. HRV, arterial pressure, and respiratory rate were recorded in 42 pediatric subjects (age 3‐5, body mass index (BMI) 13‐25 kg/m2). HRV was analyzed in the time domain [heart rate (HR), RR interval (RRI) and RRI standard deviation (RRISD)] and frequency domain [normalized LF (nLF), normalized HF (nHF), and LF/HF ratio]. We used principal components regression analysis to relate BMI to the first three component scores of the six HRV variables. BMI was positively correlated with only the first component (p < 0.001), which was strongly correlated with all original six HRV variables. Specifically, BMI was positively correlated with RRI, RRISD, and nHF (r = 0.8) and negatively correlated with HR, nLF, and LF/HF ratio (r = ‐0.78). Our data demonstrate that higher BMI in the pediatric population is associated with higher parasympathetic and lower sympathetic activity. These findings are contrary to HRV responses observed in adults and adolescents, suggesting complex relationships between age, obesity, and autonomic control of the heart.
Cerebral Autoregulation is affected in Postural Tachycardia Syndrome
Authors: Ocon Anthony James , Clark Debbie , Taneja Indu , Medow Marvin , Stewart Julian M.
Published: June 2021

Orthostatic intolerance (OI) is defined by symptoms and signs while upright, relieved by recumbency. Included are symptoms of CNS malperfusion. Chronic OI, also known as postural tachycardia syndrome (POTS), is associated with excessive upright tachycardia. We hypothesized that CNS symptoms are due to defects of dynamic and static cerebral autoregulation (CA). Nine normocapnic POTS patients and 6 control subjects, ages 15‐27, were continuously monitored for heart rate (HR), mean blood pressure (MAP), end‐tidal CO2, and cerebral blood flow velocity (CBFV) during a supine period and 70° head‐up tilt. Transfer function analysis, with low frequency (LF) between 0.04‐0.15 Hz and high frequency (HF) between 0.15‐0.40 Hz, compared MAP and CBFV. During tilt, POTS had a higher HR than controls (112 ± 14 vs. 83 ± 9 bpm) and lower systolic BP (110 ± 11 vs. 123 ± 4 mmHg). Supine CBFV decreased during tilt in POTS from 66 ± 21 to 54 ± 15.3 cm/s; whereas, in controls it dropped from 67 ± 16.5 to 61 ± 14.9 cm/s. Analysis during tilt showed that POTS exhibited a higher LF and HF coherence than controls (LF: 0.85 ± 0.11 vs. 0.30 ± 0.09; HF: 0.81 ± 0.16 vs. 0.24 ± 0.09). The greater decrease of CBFV during tilt in POTS compared to controls shows decreased static CA affecting CNS perfusion. The high coherence between MAP and CBFV in POTS implies decreased dynamic CA, which permits BP oscillations to further compromise phasic CBF.
Relationship between hippocampal neuron activity and regional cerebral blood flow during sleep‐wake cycle in rats
Authors: Tanji Momoka , Yoshimoto Misa , Miki Kenju
Published: June 2021

The present study was designed to study neurovascular coupling in the hippocampus during sleep‐wake cycle. Wistar male rats were instrumented chronically with multiple electrodes (100 micro‐meter stainless steal wire) for the measurement of the hippocampal CA1 neuron activity and bipolar electrodes for measurement of electroencephalogram (EEG) and electromyogram (EMG). In a separate group, rats were instrumented chronically with a laser‐Doppler flow probe made with a pair of glass fiber (0.25mm outer diameter) in the CA1 region of the hippocampus, and EEG and EMG electrodes. The mean value of the CA1 neuron activity was lowest during REM sleep compared with the other behavioral states including non‐REM sleep, quiet awake, moving, grooming states while the CA1 neuron activity was highest during moving states. However, the mean value of the local cerebral blood flow in the CA1 region of the hippocampus was the highest during REM sleep compared with other behavioral states. These data suggest that neurovascular coupling was modified in behavioral state‐dependent manner. Thus, regional cerebral blood flow in the hippocampus does not always represent changes in neuron activity during sleep‐wake cycle in rats.
Effect of small and intermediate conductance calcium activated potassium channel modulation on cortical cerebral blood flow in vivo
Authors: Dunn Kathryn M. , Nelson Mark T.
Published: June 2021

Pharmacological studies using selective inhibitors and activators of small and intermediate conductance calcium‐activated potassium channels (SKCa and IKCa) indicate that these channels participate in endothelium‐derived hyperpolarization responses and modulate vascular tone in isolated cerebral arteries. However, the relevance of SKCa and IKCa channel activity in the regulation of cerebral blood flow (CBF) in vivo has not been determined. The present study investigated the effect of SKCa and IKCa modulation on cortical CBF in vivo using laser Doppler flowmetry. Superfusion of inhibitors of SKCa and IKCa (apamin, 300 nM, and TRAM‐34, 10 μM, respectively) through a cranial window over the somatosensory cortex in mouse reduced resting cortical CBF by 17 ± 4 % (n=6, p<0.001). This was similar to the reduction in resting CBF with administration of the nonselective nitric oxide synthase inhibitor, N?‐nitro‐L‐arginine (L‐NNA, 1 mM), of 14 ± 6 % (n=6, p<0.01). Superfusion of the SKCa and IKCa activator, NS309, produced a rapid and robust increase in cortical CBF of 37 ± 9 %, which was largely blocked by apamin and TRAM‐34 (n=7, p<0.01). Inhibition of nitric oxide synthesis with L‐NNA had no effect on the increase in CBF in response to NS309. These results indicate that SKCa and IKCa channels are involved in the regulation of cortical CBF in vivo under resting conditions. The results suggest that SKCa and IKCa channels may also mediate in vivo cerebral vascular responses to metabolic and humoral stimuli, and may be potential therapeutic targets for maintenance of cerebral perfusion. This study was supported by National Heart, Lung, and Blood Institute grant HL‐44455.
Effects of insulin resistance on cortical blood flow responses to physiological and pathological stimuli in Zucker obese and lean rats
Authors: Institoris Adam A , Lenti Laura , Gaspar Tamas , Domoki Ferenc , Bari Ferenc , Busija David W.
Published: June 2021

Previous studies have shown that responses of cerebral vascular segments are reduced in insulin resistant rats. However, cortical blood flow (CoBF) responses have not been examined previously. We examined the effect of insulin resistance on CoBF to sensory nerve stimulation (intranasal irrigation with 10−5M capsaicin), to hypercapnia (5 and 10% CO2), to bicuculline‐induced cortical seizure (10−4, 5x 10−4,10−3M), and to systemic hypotension (40mmHg) measured with laser Doppler (LD) flowmetry in a closed cranial window prepared on Zucker lean (ZL) (n=6) and Zucker obese (ZO) (n=4‐6) rats. Capsaicin stimulation caused an increased CoBF for ZL with 6±4% (mean±S.E.M.) vs 0.4±2% for ZO animals. CoBF responses to 5 and 10% CO2 were 19±7% and 33±17% in ZO vs 25±9% and 63±13% in ZL rats, respectively. CoBF changes in response to arterial hypotension were ‐9±4% in ZL and 5±13% in ZO rats. Bicuculline induced a dose‐dependent elevation in CoBF for both ZL (12±9%, 45±13% and 79±19%) and ZO (9±10%, 42±20% and 51±19%) groups. We conclude that insulin resistance has varying effects on CoBF depending upon the type of physiological or pathological challenge.
Carbon monoxide inhibits NADPH oxidase activation during seizures and prevents loss of postictal cerebral vascular function.
Authors: Parfenova Helena , Basuroy Shyamali , Fedinec Alex , Leffler Charles W
Published: June 2021

Prolonged seizures cause sustained neuronal injury and loss of cerebral blood flow regulation that may contribute to adverse neurological outcomes in neonates. Oxidative stress is potentially a key mechanism that initiates brain damage during seizures. In a newborn pig model of neonatal glutamatergic seizures, we investigated oxidative stress in the brain. Seizures lasting over 30 min increased the superoxide level in cerebral vessels and in the brain parenchyma. We hypothesize that NADPH oxidase is a source of superoxide during seizures. Apocynin, the NADPH oxidase inhibitor (20 mg/kg iv), inhibited superoxide elevation in response to seizures in cerebral microvessels and in the brain. Glutamate (0.05‐0.2mM) increased NADPH oxidase activity in cerebral microvascular endothelial cells 1.5‐2 fold. Carbon monoxide (CO, as CORM‐A1), reduced the basal enzyme activity and blocked the glutamate stimulation of NADPH oxidase. CORM‐A1 greatly reduced oxidative stress in ictal piglets and prevented sustained postictal loss of endothelial‐dependent cerebral blood flow regulation. Overall, these data suggest that NADPH oxidase is a major contributor to oxidative stress and cerebrovascular injury caused by seizures and glutamate. CO is a gaseous cytoprotective messenger molecule that preserves cerebrovascular integrity by inhibiting NADPH oxidase and preventing oxidative stress in response to seizures.
CP 55,940 (CP) provides protection from ischemic damage due to middle cerebral artery occlusion (MCAO) via the cannabinoid (CB)1 receptor
Authors: Ellis Jabon L. , Martin John R.
Published: June 2021

Stimulation of CB receptors by WIN 55,212‐2 provides neuroprotection during cerebral ischemia. The present study was undertaken to determine if CP provides similar protection by stimulating the CB1 receptor. Male Sprague‐Dawley rats were divided into 5 groups: 3 groups underwent reversible MCAO using an intraluminal suture; 1 group underwent sham surgery; 1 group served as non‐surgical control. Rats were anesthetized and surgically prepared for MCAO and iv drug administration. The suture was advanced until resistance felt to induce stroke. Rats were given a bolus of AM 251 (AM; 3.0 mg/kg, iv) or vehicle 2.5 h into occlusion. Thirty min later (3 h into occlusion) the rats were given CP (0.3 mg/kg, iv) or vehicle. The suture was withdrawn 3.5 h into occlusion and CP infused (0.9 mg/kg/hr, iv) over the next 5 h. Rats were killed 72 h later, and their brains removed, frozen, and stained for analyzing by image J software. CP reduced the percent infarct volume of the total brain (12.7 ± 2.2 % versus 33.5 ± 6.0 % in the MCAO group; P<0.001). AM 251 prevented the protective effect of CP (39.6 ± 3.5 %; P<0.001 compared to CP and control groups). AM 251 alone increased ischemia (57.6 ± 8.5 %; P<0.001 compared to CP and P<0.05 compared to CP+AM groups). CP provides neuroprotection by stimulation of the CB1 receptor. Supported by a KCOM Graduate Program Grant.
A potential role for the ROS‐HIF‐VEGF pathway in hypoxia‐induced pulmonary vascular leak
Authors: Irwin David , McCord Joe , Majka Susan , Klemm Dwight
Published: June 2021

Acute hypoxia causes pulmonary vascular leak and is involved in the pathogenesis of pulmonary edema associated with inflammation, acute altitude exposure, and other critical illnesses. Reactive oxygen species, HIF‐1 and VEGF have all been implicated in various hypoxic pathologies, yet the ROS‐HIF‐1‐VEGF pathway in pulmonary vascular leak has not been defined. We hypothesized that the ROS‐HIF‐1‐VEGF pathway has an important role in producing hypoxia‐induced pulmonary vascular leak. Human pulmonary artery endothelial cell monolayers (HPAEC) were exposed to either normoxia (21% or acute hypoxia (3% for 24 h and monolayer permeability, H2O2, nuclear HIF‐1α, and cytosolic VEGF were determined. HPAEC were treated with antioxidant cocktail (AO: ascorbate, glutathione, and α‐tocopherol), HIF‐1 siRNA, or VEGF soluble binding protein (sFlt‐1) to delineate the role of the ROS‐HIF‐1‐VEGF pathway in hypoxia‐induced HPAEC leak. Additionally, mice exposed to hypobaric hypoxia (18,000 ft, 10% O2) were treated with the same antioxidant to determine if in‐vitro responses corresponded to in‐vivo hypoxia stress. Hypoxia increased albumin permeability, H2O2 production, nuclear HIF‐1α, and cytosolic VEGF concentration. Treatment with an AO lowered the hypoxia‐induced HPAEC monolayer permeability as well as elevation of HIF‐1α and VEGF. Treatment of hypoxia‐induced HPAEC with either a siRNA designed against HIF‐1α or VEGF antagonist soluble fms‐like tyrosine kinase (sFlt)‐1 decreased monolayer permeability. Mice treated with AO and exposed to hypobaric hypoxia had less pulmonary vascular leak than those that were untreated. Our data suggest that hypoxia‐induced permeability is due, in part, to the ROS‐HIF‐1α ‐VEGF pathway.
Human Peroxiredoxin 6 (Prdx6) gene expression is regulated by keratinocyte growth factor (KGF) through the same antioxidant response element (ARE) that controls its response to oxidative stress
Authors: Chowdhury Ibrul , Feinstein Sheldon I , Fisher Aron B
Published: June 2021

Peroxiredoxin 6 (Prdx6) can reduce lipid hydroperoxides by a unique mechanism employing a single conserved cysteine and also acts as a phospholipase A2. It is highly expressed in lung, both protecting against oxidative stress and regulating pulmonary surfactant phospholipid metabolism. We reported previously that rodent Prdx6 is up‐regulated in development and by oxidative stress. A 1524 base pair fragment of proximal upstream DNA from the human Prdx6 gene was cloned into the pSEAP‐2 reporter vector (BD Bioscience). Transient transfection of this construct into lung cell lines resulted in measurable production of secreted alkaline phosphatase that was increased 3‐4 fold by keratinocyte growth factor (KGF) treatment. β‐galactosidase expressed from a co‐transfected plasmid was used as a control for transfection efficiency. Using promoter deletions and then site‐directed deletion mutagenesis, a consensus Antioxidant Response Element (ARE), located between 357 and 349 nucleotides before the transcriptional start was identified. This element was important in basal and KGF‐induced expression in both A549 cells and primary rat alveolar type II cells. We have previously shown that this ARE controls the Prdx6 response to H2O2 treatment. We conclude that KGF and antioxidants, both activate transcription through the ARE, and may act through Nrf2, a transcription factor known to bind to the ARE. [HL P01‐75587].
Both Peroxidase and Phospholipase A2 Activities of Peroxiredoxin 6 Play a Role in its Protection against Oxidative Stress
Authors: Lien Yu‐Chin , Gao Ling , Gonder Daniel S , Feinstein Sheldon I , Fisher Aron B
Published: June 2021

Peroxiredoxin 6 (Prdx6) is a bifunctional antioxidant enzyme enriched in lung. The goal of our experiments is to determine whether the ability of Prdx6 to protect against oxidative stress is due solely to the active cysteine of the peroxidase or also requires the active center of the phospholipase A2. Our studies are designed to test the ability of Prdx6 containing mutations in these sites to rescue cells from injury due to oxidative stress. We cloned the wild‐type mouse Prdx6 into pIRES2‐ZsGreen1 vector (Promega) that expresses ZsGreen from the same transcript as the cloned gene, using an internal ribosome entry site, and prepared several mutants using site‐directed mutagenesis. We were able to transfect up to 20‐40% of the MLE12 cells with our mouse constructs using Fugene 6 with no visible cytotoxicity. We then tested transfected MLE12 cells for sensitivity to tert‐butyl hydroperoxide, using MTT and neutral red assays. With exposure to 500 uM tert‐butyl hydroperoxide, the survival of wild‐type Prdx6 transfected cells was 3 to 4 fold higher than that of vector transfected cells. The C47S‐Prdx6 (without peroxidase activity) increased survival by less than 2‐fold. Prdx6 constructs with peroxidase but no PLA2 activity showed more of a protective effect. The results suggest that while the peroxidase activity is critical for the antioxidant effect of Prdx6, the PLA2 activity may also contribute. Supported by HL P01‐75587.
Maternal chronic hypoxic stress influences the role of extracellular Ca2+ influx to serotonin‐mediated contractility of pulmonary arteries from fetal sheep
Authors: Goyal Ravi , Vemulakonda Srilakshmi , Mittal Ashwani , Loftin Matthew , Vranken Kurt , Nguyen Dan , Webster Melissa , Blood Arlin B , Pearce William J , Longo Lawrence D , Wilson Sean M
Published: June 2021

Cytosolic Ca2+ is central to pulmonary vascular reactivity and important to pathogenesis due to hypoxic stress, but the role of Ca2+ to disease in the unborn fetus is not well understood. We therefore tested the hypothesis that chronic maternal hypoxia (CH) alters Ca2+‐dependent contractility using wire‐myography and Western immunoblot of pulmonary arteries (PA) isolated from late gestational fetal sheep, where ewes were housed under normoxic conditions or at 12,470 feet for ~ 110 days (CH). Serotonin (5‐HT; 10 μM) dependent contractility was dependent on extracellular Ca2+ in PA from both groups and 10 mM Ni2+ inhibited contractility in CH. However, select Ca2+ entry pathways were altered by CH. The role L‐type Ca2+ channels was lessened by CH as indicated by diminutive action of 10 μM nifedipine (NIF) and reduced α‐subunit protein expression. The role for non‐selective cation channels was also reduced by CH, as determined by serial application of 50 μM SKF 96365 to NIF treated arteries. In comparison, reverse mode‐Na+‐ Ca2+ exchange had little role to the contractility response as 10 μM KB‐R7943 failed to reduce contractility. Given that extracellular Ca2+ removal and Ni2+ effectively reduced contractility while NIF, SKF, and KBR were ineffective, Ni2+ but not SKF sensitive basal Ca2+ influx may have a greater role during 5‐HT‐dependent contractility in fetal PA from CH sheep. Support from NIH, UM, and LLUMC.