bluuebunny/crossref_metadata_2025 huggingface dataset:
Pickpocket1 is a Drosophila Acid‐Sensing Na+ Channel Necessary for Proprioception
Authors: Boiko Nina , Kucher Volodymyr , Eaton Benjamin A. , Stockand James D.
Published: June 2021

The role of acid‐sensing channels during the normal function of the nervous system is poorly defined. Pickpocket1, PPK1, is a DEG/ENaC homolog restrictively expressed in Drosophila class IV multidentritic (md) sensory neurons. These channels are believed to be involved in sensory proprioception, but an exact mechanism is undefined. Using a primary neuronal culture from Drosophila embryos and whole cell patch‐clamping, we identify amiloride‐sensitive sustained (Is) and transient (It) acid‐sensing Na+ currents in md neurons. The robust sustained pH‐sensitive current is Na+ and K+‐selective and sensitive to amiloride but refractory to ruthenium red. A distinct transient sodium current is revealed by relief from inhibition following extracellular acidification. This transient pH‐sensitive current is absent in ppk1 −/− flies and rescued by targeted expression of a PPK1‐transgene identifying it as the PPK1 current. PPK1 function in md sensory neurons is necessary for normal Drosophila larvae proprioception consistent with PPK1 functioning as a molecular mechanosensory transducer. PPK1 activity is not required for intrinsic excitability but rather transduces dynamic sensory input essential to proprioception.
L‐type Ca2+ channel current from on‐cell patch is augmented by H2O2 in rat aortic smooth muscle–derived A7r5 cells
Authors: Ochi Rikuo , Gupte Sachin A
Published: June 2021

L‐type Ca2+ channels are exposed to H2O2 generated within the cells and adjacent cells such as immune cells. Although the effect of H2O2 on ICa,L has long been studied, whether it augments or inhibits ICa,L has not yet been settled. We hypothesized that extracellular and intracellular H2O2 differently modulates ICa,L. Here, we adopted two configurations of patch clamp technique to separately study these modulations in A7r5 cells, a smooth muscle cell line derived from embryonic rat aorta. Whole cell ICa,L recorded with 10 mM Ba2+ as a charge carrier was slightly inhibited with an acceleration of its decay time course by 0.1‐mM H2O2. On‐cell recording enabled us to study the effect of H2O2 without possible complication of direct oxidation of CaV1.2 by external H2O2. Single and multichannel ICa,L were recorded in the presence of 90 mM Ba2+ and Bay K 8644 repetitively applying 500 ms depolarization steps to ‐10 and 0 mV. The mean currents from these currents were significantly increased by 0.1‐mM H2O2 from 0.85 pA to 1.05 pA associated with slow inactivation: ICa,L at 500 ms, 0.56 pA in control and 0.74 pA in the presence of H2O2. Since extracellular H2O2 was not accessible to the patch membrane, these changes reflect intracellular actions of H2O2. In conclusion, ICa,L is augmented by H2O2 via intracellular signaling mechanism in A7r5 cells. Supported by National Heart, Lung, and Blood Institute Grant RO1‐HL‐085352.
Comparison of two types of δENaC channels cloned from human lungs
Authors: JI HONGLONG , Zhao Runzhen , Nie Hongguang , Matalon Sadis
Published: June 2021

δENaC has been detected in human lung epithelial cells. Here we cloned a splicing variant, namely, δ2ENaC in human bronchoalveolar epithelial cells. δ2ENaC possesses 66 extra amino acids attached to the distal amino terminal tail of the δ1ENaC. δ2ENaC was expressed in both alveolar type I and II cells as revealed by in situ hybridization. To compare the biophysical and pharmacological features of the splicing variant with the δ1βγ channels, we examined the whole‐cell and single channel properties comparing with those of δ1βγ and αβγ channels. Divergent from δ1βγ channels, the macroscopic currents of δ2βγ channels were greater; the apparent Na+ affinity was stronger; the apparent dissociation constant for amiloride (Kiamil) but not Evans blue was smaller; the EC50 value of capsazepine activation was less; and the gating kinetics by protons were markedly faster. The single channel activity, unitary conductance, and open probability of δ2βγ channels were significantly greater compared to δ1βγ channels. The potential longevity of δ2βγ at cell surface, as shown by the faster exocytosis and slower endocytosis could contribute to the enhanced channel activity. Our data demonstrate that the channels composed of the splicing variant segregated from both δ1βγ and αβγ channels by strikingly diverse biophysical/pharmacological properties and regulation by physiological signal. Our results may account for the heterogeneities of native amiloride‐sensitive cation channels in human lung and other epithelial tissues. This work was supported by NIH grants HL87017, HL031197, ES015676, ES017218, and National Natural Science Foundation of China (30971181).
1,25‐Dihydroxyvitamin D Enhances Alveolar Fluid Clearance by Up‐Regulating the Expression of Epithelial Sodium Channels
Authors: Nie Hongguang , Yu Tong , Nie Zhuang , Ji Honglong , Li Yan Chun
Published: June 2021

Purpose

Vitamin D is implicated in the pathogenesis of asthma, acute lung injury and other respiratory diseases. 1,25‐ dihydroxyvitamin D (1,25‐VD), the hormonal form of vitamin D, has been shown to reduce vascular permeability and ameliorate lung edema (Chen SF, et al. 1995). Therefore, we speculate that 1,25‐VD may regulate alveolar Na+ transport via targeting epithelial Na+ channels (ENaC), a crucial pathway for alveolar fluid clearance (AFC).

Methods

KM male mice (20~30g) were treated with active vitamin D analogue paricalcitol (daily i.p. injection) for 2 weeks before the in vivo AFC of these mice was measured. Western blotting was used to determine ENaC protein levels in mouse lungs and in human lung H441 epithelial cells treated with 1,25‐VD (20 nM). Whole cell patch‐clamp technique was used to measure ENaC activity in H441 cells.

Results

In vivo total AFC was 31.9 ± 3.8% in paricalcitol‐treated mice, significantly greater than vehicle‐treated controls (19.7 ± 1.9%, n=10, P < 0.05). Amiloride‐sensitive AFC was increased approximately 50% by paricalcitol. Western blot showed that the expression of γ‐ENaC was significantly elevated in 1,25‐VD‐treated H441 cells and in paricalcitol‐treated mouse lungs. Amiloride‐sensitive Na+currents were greater (184%) in 1,25‐VD‐treated H441 cells compared to vehicle‐treated cells.

Conclusions

These observations suggest that vitamin D augments transalveolar fluid clearance, and vitamin D therapy may potentially be used to ameliorate pulmonary edema.

This study was supported by the NSFC (30971181) to HG Nie, and NIH HL87017 to HL Ji.
Modulation of Calu‐3 Anion Channels by H1N1 Infection
Authors: Waugh Taryn A , Ching John C.H. , Carr Anthony P , Townsend Hugh , Loewen Matthew E
Published: June 2021

Influenza virus A infection leads to pulmonary edema through a disruption of the fluid balance in the lungs. Part of this balance is maintained by the regulation of the anion channels in the respiratory epithelium lining the airway. Upon influenza infection this respiratory epithelium produces cytokines that aid in fighting the infection starting around 6 hours. These same cytokines by themselves have been shown to alter anion channel function. In this study, using the Calu‐3 respiratory epithelial cell line, we measure cytokine production by real time quantitative RT‐PCR as well as anion channel function as short‐circuit current produced by a Calu‐3 monolayer in a Ussing Chamber. Viral protein production was shown along with an increase in pro‐inflammatory cytokines at 24 hours post infection. Interestingly, no change in short‐circuit current response was found at 24 hours. A change in short‐circuit current response was first noted at 48 hours post infection. This change in short‐circuit current seems to be a direct effect on anion channel conductance and not a decrease in tissue viability as the resistance of the Calu‐3 monolayer did not significantly change. This study is the first step in understanding the pathophysiology which drives fluid into the lungs during an influenza A infection. This study was supported by SHRF, EHRF, CAHF, CFI and NSERC.
Neonatal inhalation of hyperoxic gas and altered postnatal growth: effects on the pulmonary airways in adulthood
Authors: Harding Richard , O'Reilly Megan , Sozo Foula
Published: June 2021

Our objective was to determine the long‐term effects of neonatal hyperoxia alone on the bronchioles, in the absence of growth restriction. Newborn mice (C57Bl/6J) were raised in hyperoxic gas (65% O2) until postnatal day 7 (P7d), after which they lived in room air until adulthood (P56d). During hyperoxia, dams remained in 65% O2 (H‐E group), or were alternated every 24h between hyperoxia and room air (H‐E/air group) to prevent weight loss and growth restriction of offspring. Controls breathed room air. At P56d, the structure of the bronchioles and lung parenchyma was morphometrically analysed. H‐E dams were lighter 4–13d after delivery whereas weights of H‐E/air dams were not different from controls. During hyperoxia, H‐E pups had reduced growth; H‐E/air pups grew at the same rate as controls. At P56d, lungs of H‐E (n=26) and H‐E/air (n=21) mice had larger alveoli, less parenchymal tissue and thicker airway epithelium than controls (n=27). The airway epithelium of H‐E/air mice had a greater proportion of proliferating and ciliated cells and a lower proportion of Clara cells. H‐E mice had more airway smooth muscle and collagen, whereas collagen was reduced in H‐E/air mice. Neonatal hyperoxia with or without growth restriction induces persistent alterations in the bronchioles and lung parenchyma, which may contribute to lung dysfunction. Research support: National Health and Medical Research Council of Australia.
The Role of the 2‐Pore Domain Potassium Channel Trek‐1 in Regulation of Cytokine Secretion from Alveolar Epithelial Cells
Authors: Schwingshackl Andreas , Teng Bin , Ghosh Manik , Makena Patrudu , Waters Christopher M
Published: June 2021

Release of inflammatory mediators from epithelial cells contributes to the development of Acute Lung Injury, but the underlying mechanisms are poorly understood. We have previously shown that deficiency of the 2‐pore domain potassium (K2P) channel Trek‐1 resulted in decreased IL‐6 and RANTES secretion and increased MCP‐1 secretion from MLE‐12 cells. The objective of this study was to investigate the mechanisms of TNF‐α‐induced cytokine release from Trek‐1‐deficient alveolar epithelial cells.

Methods

We studied expression of protein kinase C (including its isoforms α, β, μ, δ, θ, λ, μ, and ζ), of JNK and p36 kinase, and of the transcription factor p65/NFκB in Trek‐1 deficient (shRNA) MLE‐12 cells after stimulation with TNF‐α.

Results

Trek‐1‐deficiency resulted in decreased expression of total and phosphorylated PKC after stimulation with TNF‐α. In particular, phosphorylation of the PKC isoforms α, β, μ, δ, θ and μ, but not λ and ζ was decreased in Trek‐1‐deficient cells. TNF‐α caused increased phosphorylation of JNK 1/2/3 and decreased phosphorylation of p38 kinase in Trek‐1 deficient cells. Trek‐1‐deficiency did not affect expression of p65/NFκB after stimulation with TNF‐α.

Conclusion

Trek‐1 may regulate cytokine secretion from alveolar epithelial cells via PKC‐, JNK‐ and p38‐dependent pathways.

This study was supported by NIH grant HL 094366 and the Children's Foundation Research Center.
Reduction of Alveolar Type II Cell Volume in Klotho‐deficient Mice With Premature Aging
Authors: Ravikumar Priya , Bellotto Dennis J. , Kuro-o Makoto , Moe Orson W. , Hsia Connie C.W.
Published: June 2021

Klotho mitigates aging in multiple organs. Genetic Klotho deficiency (KL−/−) leads to premature senescence, organ degeneration and death. Age‐related airspace enlargement has been reported in the lungs of KL−/− and haplo‐insufficient (KL+/−) mice (Nature 1997;390:45–51). To examine the relationship between Klotho deficiency and lung structure, we performed morphometry on the right lung of 8–12 wk old KL−/− (n=9) and wild type (KL+/+, n=5) mice. Lungs were fixed by tracheal instillation of 2.5% glutaraldehyde (25cmH2O airway pressure). τhb, harmonic mean thickness of diffusion barrier Mean±SD.

Genotype
KL+/+
KL−/−

Body weight (g)
18.9±3.8
10.1±3.9 *

Right lung vol (mL/kg)
25.55±4.21
28.87±8.03

τhb (μm)
0.549±0.028
0.556±0.049

Volume‐to‐lung volume ratio

Alveolar ducts
0.045±0.007
0.056±0.020 †

Septum
0.113±0.019
0.102±0.004

 Capillary blood
0.040±0.007
0.036±0.002

 Epithelium–type I
0.018±0.003
0.018±0.001

  –type II
0.007±0.001
0.005±0.000 *

 Interstitium–collagen
0.026±0.004
0.023±0.002

  –cell/matrix
0.004±0.001
0.004±0.001

 Endothelium
0.018±0.003
0.018±0.001

p≤0.001,

p<0.05 vs. WT.

KL−/− mice were small and frail. Per kg of body weight, volumes and surface areas of the lung and alveolar components were similar to WT except the volume density of alveolar ducts was 24% higher and that of alveolar type II cells 30% lower. We conclude that Klotho deficiency is associated with diminished reservoir of alveolar progenitor cells, which could contribute to premature aging of the lung.

Supported by NIH F32 HL103043, RO1 AG19712
Impact of Postnatal Hypoxia on MicroRNA Expression in Heart and Muscle of Adult Rats
Authors: Radom-Aizik Shlomit , Kunde Michele , Nance Dwight M. , Zaldivar Frank , Cooper Dan M. , Adams Gregory R.
Published: June 2021

Preterm babies are at risk for lifelong abnormalities in cardiovascular and respiratory systems. The mechanisms and interventions to correct these abnormalities are unknown. Hypoxia is common during the neonatal period in these infants. The acute effects of hypoxia early in life could produce epigenetic programming resulting in long term effects. We studied the acute and long term effects of neonatal hypoxia (Hx, 12% O2) during postnatal days 2 to 20. Acutely, hypoxia severely reduced somatic growth, but produced cardiac right ventricular (RV) hypertrophy that persisted into adulthood (65 days old). The RV of adult Hx rats had higher content of contractile proteins and DNA. At 21 days, the expression of miR‐1 and miR‐206 was significantly downregulated in the RV of Hx rats. The growth of skeletal muscle was disproportionately depressed in Hx rats at 21 days but recovered after 45 days of normoxia. At 21 days the expression of miR‐1 and miR‐206 in skeletal muscle, was upregulated (~2 fold) while miR‐133a was downregulated (~22%). At 65 days of age the expression of miR‐1 remained elevated in the skeletal muscle of male Hx rats. These results indicate that microRNAs participate in reprogramming adaptations to a neonatal hypoxic episode in the heart and skeletal muscles of rats that can persist into adulthood. Supported by NIH Grants P01HD048721 and UL1RR031985.
Upregulation of TMEM16A Underlies Chronic Hypoxia‐induced Increase of Ca2+‐activated Cl− Current in Pulmonary Arterial Smooth Muscle
Authors: Sun Hui , Xia Yang , Paudel Omkar , Yang Xiao-Ru , Sham James SK
Published: June 2021

Ca2+‐activated Cl− channels (CaCCs), encoded by TMEM16A, play an important role in the regulation of vascular tone. We have previously observed an increase in CaCC current (ICl.Ca) and TMEM16A expression in pulmonary arterial smooth muscle cells (PASMCs) and tissue of rats with chronic hypoxic pulmonary hypertension (CPHP, 10 ±0.5% O2 for 3–4 weeks). This study aimed to determine whether chronic hypoxia (CH) alters the Ca2+‐or voltage‐dependence of CaCCs in PASMCs, thereby contributes to the enhanced ICl.Ca. Perforated and conventional whole‐cell patch clamp techniques were used to record the caffeine‐induced ICl.Ca and Ca2+ transients simultaneously on fluo 3/AM‐loaded PASMCs, and ICl.Ca evoked by depolarization at constant high Ca2+, respectively. The activation rates of caffeine‐induced ICl.Ca and Ca2+ release were not different between control and hypoxic PASMCs, and their strong correlation was unaltered by CH at both 0 and −80 mV. At 750 nM of [Ca2+]I, the current‐voltage relation exhibited a strong outward rectification in both control and CH cells with similar rectification index (I+60/I−60=8.7±0.5 vs 8.2±0.8, P>0.05). The steady‐state activation curves from both groups were almost overlapped. The time‐dependent activation of ICl.Ca during depolarization, reflecting the voltage‐dependent Ca2+ binding to the channel, were unchanged in hypoxic PASMCs. These results suggest Ca2+‐ or voltage‐dependent properties of CaCC were unaffected by CH. Thus, the enhanced ICl.Ca is mainly attributable to increased channel expression.

Supported by grants from NIH and AHA.
p22phox in the paraventricular nucleus (PVN) of the brain contributes to diet‐induced obesity (DIO)
Authors: Lob Heinrich Emil , Harrison David G , Mark Allyn L , Davisson Robin L
Published: June 2021

Oxidative stress is implicated in obesity and obesity‐hypertension, but most studies have focused on peripheral adipose and cardiovascular tissues. Recently, a role for brain NADPH oxidase (Nox) has begun to emerge in DIO. We tested the hypothesis that Nox activity in the PVN, a key energy and cardiovascular homeostasis center of the brain, is causally linked to DIO. Mice harboring a floxed allele of p22phox, the required subunit for Nox assembly and activity, underwent bilateral PVN‐targeted injection of an adenovirus encoding Cre recombinase (AdCre) to delete PVN p22phox or control red fluorescent protein (AdRFP). After recovery, mice were fed high fat diet (HFD, 42%) for 8 weeks. Ablation of p22phox in the PVN with AdCre blunted HFD‐induced increases in body weight compared to AdRFP‐injected mice (AdRFP: +11.46±0.49g vs AdCre: +6.9±0.57g, n = 3, p<0.05). Real‐time qPCR confirmed p22phox deletion in PVN. Since Nox is functionally linked to endoplasmic reticulum (ER) stress pathways in some cell types, we examined the effect of deleting p22phox on ER stress biomarkers in PVN of these mice. Preliminary data show that mice with PVN‐targeted ablation of p22phox have lower levels of HFD‐induced GRP78 (−68%, n=2) and GRP94 (−38%, n=2) mRNA in this brain region compared to controls. These data suggest that oxidative and ER stress in the PVN contribute to diet‐induced obesity. HL063887, HL084207
Control of sympathetic and phrenic nerve activity by cholinergic mechanisms in the nucleus of the solitary tract (NTS)
Authors: Furuya Werner Issao , Bassi Mirian , Menani José Vanderlei , Colombari Eduardo , Zoccal Daniel Breseghello , de Almeida Colombari Débora Simões
Published: June 2021

Cholinergic receptors are present in the NTS and acetylcholine (ACH) microinjected into the medial NTS (mNTS) induces hypotension and bradycardia in rats (da Silva et al., 2008; AJP, 295, p R1774). In the present study, we tested the changes in the sympathetic and phrenic nerve activities produced by ACH injected alone or combined with atropine (ATR, muscarinic antagonist) into the mNTS or into the commissural NTS (cNTS). Decorticated arterially‐perfused in situ preparations of male Holtzman rats (60–90 g, n=5–6) were used to record thoracic sympathetic (tSNA) and phrenic nerve (PNA) activities. ACH (10 mM in 60 nL) was injected into the mNTS or cNTS before and 10 min after ATR (17 and 67 mM in 60 nL). Saline was injected as control. ACH into the mNTS reduced the tSNA (−66.1 ± 6.0 % vs. saline: −0.6 ± 3.3 % of baseline) and PNA frequency (−39.7 ± 2.7 % vs. saline: −2.4 ± 2.6 % of baseline), a response attenuated by ATR (67 mM) into the mNTS (tSNA: −29.7 ± 5.2 % and PNA: −12.8 ± 4.6 %, p<0.05). ACH into the cNTS increased PNA frequency (33.1 ± 6.8 %, vs. saline: −6.4 ± 2.7 %), without changing tSNA. ATR (17 mM) into the cNTS reduced the ACH‐induced increase in PNA frequency (8.6 ± 6.4 %, p<0.05). These data suggest that ACH acting on muscarinic receptors in the NTS may play an important role in the control of sympathetic and respiratory activities with distinct responses depending on the NTS level.

Supported by: FAPESP, CNPq.
Central mechanisms activated by leptin to modify hypercapnia‐induced ventilatory responses
Authors: Bassi Mirian , Furuya Werner Issao , Nakamura Natalia Borges , de Almeida Colombari Débora Simões , Menani José Vanderlei , Glass Mogens Lesner , Hall John E , Colombari Eduardo
Published: June 2021

Melanocortin 3/4 receptors (MC3/4R) play a pivotal role in mediated metabolic and cardiovascular actions of leptin. In this study, we evaluated the changes in pulmonary ventilation (VE) to hypercapnia (7% CO2) in rats treated with daily microinjections of leptin and SHU9119 (MC3/4R antagonist) alone or combined into the lateral ventricle (LV) for 7 days. Male Holtzman rats (n=6/group) with stainless steel cannula implanted into the LV were used. Ventilation was recorded by plethysmography. Leptin (5 μg/5μl/day) into the LV reduced body weight by ~17% and increased (P<0.05) the tidal volume (11.0±0.7 vs. saline: 7.8±0.7 ml.kg−1) and the ventilatory response to hypercapnia (1796±140 vs. saline: 1213±126 ml.min−1.kg−1), without changing respiratory frequency. SHU (1 nmol/5μl/day) increased body weight by ~ 13%, without changing the increase in ventilation (1185±53 ml.min−1.kg−1)and in the tidal volume (6.8±0.7 ml.kg−1) produced by hypercapnia. Combined treatment with SHU+leptin also increased body weight (~9%), however, decreased tidal volume (5.2±0.3 ml.kg−1) and ventilatory response to hypercapnia (795±34 ml.min− 1.kg−1, P<0.05). The results suggest that leptin‐induced increase in ventilation depends on the activation of central MC3/4 receptors. In addition, it seems that leptin produces also a parallel secondary inhibitory action on ventilation independent on MC3/4R activation.
Caudal ventrolateral medulla (CVLM) activation by acute hypoxia (AH) is independent of changes in arterial blood pressure (ABP)
Authors: King T Luise , Heesch Cheryl M , Friskey Sarah , Ruyle Brian , Kline David D , Hasser Eileen M
Published: June 2021

Afferent baroreflex and chemoreflex signaling is integrated in the nucleus of the solitary tract and then sent to the CVLM. Hypoxia activates neurons in the CVLM, but also decreases ABP. To evaluate the influence of changes in ABP on CVLM neuronal activation due to AH, we stimulated the arterial chemoreflex while maintaining ABP constant. ABP was monitored in conscious rats (radiotelemetry) during 2 hrs of Normoxia (N: 21% O2, n=4), AH (10% O2, n=5) or AH and i.v. infusion of the vasoconstrictor phenylephrine (0.06–0.3mg/hr) to maintain ABP constant (AH+PE, n=4). Rats were perfused and brainstems processed for examination of Fos‐immunoreactivity (IR) as an index of CVLM activation and tyrosine hydroxylase (TH)‐IR to identify catecholaminergic neurons. AH led to a sustained decrease in ABP (−17±4 mmHg within 15 min). In AH+PE and N rats, ABP remained constant. Changes in breathing frequency and oxygen saturation during hypoxia were similar in AH and AH+PE rats. Compared to N (21±10 cells), hypoxia increased the number of Fos‐IR CVLM neurons whether ABP decreased or was held constant (AH: 117±8; AH+PE: 128±18 cells). Furthermore, the number of activated catecholaminergic CVLM cells was similar in both AH groups (AH: 59+11 vs AH+PE: 59+13). Data suggest that decreased ABP during hypoxia does not contribute substantially to CVLM neuronal activation due to chemoreflex stimulation. NIH Multi‐PI HL 98602
Role of nuclear factor‐kappa B in mitochondria‐derived superoxide‐lowered protein expression of voltage‐gated sodium channels in nodose neurons from heart failure rats
Authors: Tu Huiyin , Liu Jinxu , Li Yu-long
Published: June 2021

Our previous study has shown that chronic heart failure (CHF) reduces protein expression of voltage‐gated sodium (Nav) channels in rat nodose neurons. In the present study, we investigated the involvements of mitochondrial superoxide and nuclear factor‐kappa B (NF‐kB) in CHF‐decreased Nav channel expression in rat nodose neurons. CHF was induced by left coronary ligation. CHF reduced the protein expression of manganese superoxide dismutase (MnSOD) and elevated mitochondrial superoxide level in the nodose neurons. Adenoviral MnSOD (Ad.MnSOD) gene transfection (50 multiplicity of infection) into the nodose neurons normalized the MnSOD expression, reduced the elevation of mitochondrial superoxide, and partially reversed the reduced protein expression of Nav channels in the CHF nodose neurons. Additionally, protein expression of NF‐kB p65 and phosphorylated NF‐kB p65 in the nodose neurons was higher in CHF rats than that in sham rats. Treatment with an NF‐kB inhibitor (caffeic acid phenethyl ester, 10 ìM, 24 h) significantly increased Nav channel density in CHF nodose neurons. Furthermore, Ad.MnSOD inhibited the NF‐kB p65 binding to Nav channel promoter in CHF nodose neurons. These results indicate that mitochondrial superoxide lowered protein expression of Nav channels in CHF nodose neurons via activating NF‐kB.
Regulation of tyrosine hydroxylase by ETA receptors in the olfactory bulb of DOCA‐salt hypertensive rats
Authors: Guil Maria Julia , Morales Vannina Paola , Soria Celeste Victoria , Hope Sandra Ingrid , Bianciotti Liliana Graciela , Vatta Marcelo Sergio
Published: June 2021

We recently showed that olfactory bulb (OB) exposure to endothelins (ETs) differentially modulate tyrosine hydroxylase (TH) activity and expression in normotensive rats. In the present study we evaluated the regulation of TH by ETs in hypertension. BQ610 (selective ETA antagonist) (20uM/ul/min) was intracerebroventriculary applied to normotensive and DOCA‐Salt hypertensive rats. Blood pressure and heart rate were monitored by 60 min and then animals sacrificed to obtain OB where TH activity (radioenzymatic method) and expression of total enzyme and phosphorylated forms (western blot) were assessed. Following BQ610 administration blood pressure and heart rate were reduced in hypertensive rats but no changes were observed in control animals (control, salt or DOCA only). Hypertensive rats exhibited an increase in TH activity as well as in total TH and its phosphorylated forms (pSer19, pSer31 and pSer40) that were diminished to control levels by BQ610 administration. These findings support an interaction between ETs and TH in the OB of DOCA‐salt hypertensive rats likely related with the onset and/or development of hypertension in this animal model (ANPCyT, CONICET, UBA).
Blockade of brain angiotensin II type 1 receptor attenuates angiotensin II‐induced left ventricular end‐diastolic pressure elevation and myocardial hypertrophy via sympathoinhibition
Authors: Shinohara Keisuke , Hirooka Yoshitaka , Kishi Takuya , Sunagawa Kenji
Published: June 2021

Background

Central sympathoexcitation is a cardinal manifestation of heart failure. The aims of the present study were to investigate whether central sympathoexcitation induced by slow presser dose of peripheral angiotensin II (Ang II) infusion deteriorates cardiac function in rats, and if so, to investigate whether blockade of Ang II type 1 receptor (AT1R) in the brain improve the changes of cardiac function via central sympathoinhibition.

Methods and Results

In Sprague‐Dawley rats, the subcutaneous infusion of Ang II at 100 ng·kg−1·min−1 for 14 days increased gradually systolic blood pressure from baseline (+ 15±4 mmHg, n=11). Compared with vehicle infusion, Ang II‐infusion increased plasma Ang II (100±10 vs. 48±8 pg/ml, n=5~9, p<0.05), urinary norepinephrine excretion (1.25±0.03 vs. 0.92±0.05 μg/24 hrs, n=8~12, p<0.001), left ventricular end‐diastolic pressure (LVEDP) (5.0±0.4 vs. 2.7±0.3 mmHg, n=5~8, p<0.001) with myocardial hypertrophy. Chronic intracerebroventricular infusion of AT1R blocker, losartan, attenuated these Ang II‐induced changes. The percent fractional shortening evaluated by echocardiography did not differ between the groups.

Conclusion

Central sympathoexcitation induced by increased plasma Ang II increases LVEDP with myocardial hypertrophy. Furthermore, the blockade of AT1R in the brain reduces the Ang II‐induced changes via sympathoinhibition.
Nonclassical G Protein Coupled Receptor Kinase 5 Regulation of Angiotensin II type 1 Receptor in CATH.a Neurons
Authors: Engler Christopher William , Haack Karla , Zucker Irving
Published: June 2021

Angiotensin II type 1 Receptor (AT1R) plays a pivotal role in the development of heart failure, and as such is upregulated in a number of tissues. AT1R and other G Protein Coupled Receptors are marked for internalization and recycling via G Protein Coupled Receptor Kinase (GRK) phosphorylation. GRK5, a regulator of AT1R, has been shown previously in aorta endothelial cells to stabilize IκBα, an inhibitory element of Nuclear Factor Kappa B (NFκB), a protein also involved in upregulation of AT1R. We hypothesized that in CATH.a neurons, GRK5 may regulate AT1R via interaction with the IκBα/NFκB dimer. Cells stimulated with 100 nM Angiotensin II (Ang II) for 4 hours led to a significant upregulation of AT1R, p65 NFκB and GRK5 proteins (p<0.05). Overexpression of GRK5 led to a normalization of AT1R and p65 NFκB following Ang II stimulation; conversely, silencing of GRK5 heightened Ang II‐mediated AT1R and p65 NFκB upregulation by 100%. Preliminary examination of nuclear and cytosolic fractions showed that GRK5 was present in the nucleus and cytosol, and nuclear localization of GRK5 increased with Ang II stimulation. Co‐immunoprecipitation studies indicated that GRK5 and IκBα are physically associated and dissociate upon Ang II stimulation whereas GRK5 and NFκB do not associate. Together, these data suggest a nontraditional role of GRK5 as a regulator of AT1R ostensibly via its interaction with IκBα. Supported by PO‐1 HL62222.
Arousal in rat pups habituates in response to repeated bouts of hypercapnia: implications for the Sudden Infant Death Syndrome (SIDS)
Authors: Darnall Robert A , Schneider Robert , Webster Caroline , Tobia Chritine
Published: June 2021

In infants, arousal from sleep is an important protective response against hypoxia and hypercapnia. In rat pups, arousal in response to hypoxia habituates with a progressive lengthening of the time to arousal (latency) over repeated bouts. We hypothesized that arousal habituation (AH) would also occur with repeated bouts of hypercapnia and would involve carotid chemoreceptors. We exposed 177 rat pups (P9–P20) to 4 or 8 three minute exposures to either 8% CO2 in room air (CO2/RA), 8% CO2 in 100% O2 (CO2/100%O2), 8% CO2 + 10% O2 (CO2/10%O2), 100% O2 (100%O2), or room air (RA), alternating with 6 minutes of RA. Exposures were begun during sleep and arousal was assessed using behavioral criteria. Arousal latency progressively increased with each exposure to all CO2 containing gas mixtures and mean latencies were shorter than when exposed to either RA or 100%O2 controls. Mean arousal latency was shortest in CO2/10%O2. Arousal latencies with CO2/O2 were longer than those with CO2/RA during trials 5–8. Our results indicate that AH occurs in response to repeated bouts of hypercapnia. The combination of hypercapnia and hypoxia is a more potent arousal stimulus than CO2 alone. Exposure to CO2/O2 resulted in more AH than CO2/RA, suggesting carotid chemoreceptor involvement. AH may prevent adequate protective responses to subsequent exposures to hypoxia and hypercapnia increasing the risk for SIDS. NIHPO1HD36379
Modulation of Tracheobronchial Cough by Sensory Input: In Vivo Experiments and Model Simulations
Authors: Bolser Donald , Poliacek Ivan , Pitts Teresa E , Lindsey Bruce G , Segers Lauren S , Rose Melanie J , Davenport Paul W , Morris Kendall F
Published: June 2021

Cough can be modeled using a network composed of known elements of the respiratory pattern generator; yet the regulation of cough in response to afferent input differs from breathing. Computer simulations of cough based on multineuron recordings in decerebrated cats show similarities and differences with in vivo cough regulation. (i) Integrated cough inspiratory and expiratory motoneuron burst amplitudes are highly correlated in simulations and in decerebrated but not anesthetized cats. (ii) Predictions from simulations of baroreceptor effects on breathing suggested that transiently elevated blood pressure would reduce cough number and cough‐related inspiratory, and expiratory amplitudes as well as prolong cough cycle durations. These effects were seen in both simulations of cough‐baroreceptor stimulation and in vivo. However, preliminary analysis suggests that expulsive phase durations during cough were prolonged in simulations but not in vivo. (iii) Simulations that incorporated linear variations of sensory afferent discharge or conductance of the first central synapse revealed more positive relationships between spatiotemporal features of the cough motor pattern than were seen in vivo. The results support functional “filtering” of sensory afferent input to the cough pattern generator by elements subsequent to the first central synapse. Supported by HL103415, HL89104.
Relationship between total expired air, peak airflow, and cough number in reflex cough
Authors: Hegland Karen Wheeler , Davenport Paul W
Published: June 2021

Reflex cough typically includes multiple cough reaccelerations. The relationship between total expired air volume, peak flow rate and cough volume acceleration has been investigated for multiple reaccelerations during voluntary cough; these data are not available for reflex cough. The goal of this study was to investigate the relationship between reflex cough reaccelerations and cough airflow parameters. Twenty healthy adults volunteered to participate. Participants were seated and outfitted with a facemask and pneumotacograph. Capsaicin (200μM) was delivered via dosimeter attached to the facemask. Cough airflow was recorded across 3 reflex cough trials. Cough epochs (all cough reaccelerations associated with one inhalation) ranged from two (C2) to six (C6) cough reaccelerations. Total expired volume ranged from 1.05 to 2.20 liters of air. The percent of total expired air for a cough was linearly related to the total number of coughs in a cough epoch, peak expiratory flow rate, and cough volume acceleration. Results indicate that greater number of cough reaccelerations in an epoch redistribute the rate and amount of air expired for each cough. Because the rate of airflow during a cough directly contributes to the shearing forces that clear material from the airway, increasing number of cough reaccelerations in an epoch may decrease overall cough effectiveness.
Reversal of opioid –induced respiratory depression by the (+)‐ enantiomer, GAL‐054, but not the (−)‐enantiomer, GAL‐053, of doxapram
Authors: Golder Francis John , Gruber Ryan B , Puskovic Veljko , Peng Sean , Dax Scott L , MacIntyre D Euan , Mannion James C
Published: June 2021

We have demonstrated that the (+) enantiomer of doxapram, GAL‐ 054, is the respiratory stimulant eutomer in drug naïve rats and that its tolerability profile is superior to the (−) enantiomer (or distomer), GAL‐053. Given that doxapram is used clinically to reverse drug‐induced respiratory depression, we hypothesized that GAL‐054 would also be superior to GAL‐053 at reversing opioid‐induced respiratory depression (OIRD). Tidal volume (VT), respiratory frequency (f), minute ventilation (Vmin), and arterial pH and blood gases were measured from conscious rats using whole‐body plethysmography and blood gas analysis before and after bolus morphine injection and then during subsequent doxapram, GAL‐054, and GAL‐053 infusion. Morphine (10 mg/kg, IV) decreased Vmin, by decreasing both VT, and f. GAL‐ 054 (1.0 and 3.0 mg/kg/min) infusion reversed OIRD in a dose‐dependent manner by increasing VT, but not f. In contrast, GAL‐ 053 infusion had minimal effects on breathing in morphine‐treated rats. As expected, morphine increased PaCO2 and decreased pH and PaO2. Doxapram (3 mg/kg/min) and GAL‐054 (3 mg/kg/min) diminished these effects, whereas GAL‐053 infusion (3 mg/kg/min) did not. No adverse events were observed for doxapram or either enantiomer during this study. Collectively, these data support the classification of GAL‐054 as the respiratory stimulant eutomer and GAL‐053 as the distomer of racemic doxapram.
Phrenic motoneuron rate‐coding and recruitment during long‐term facilitation
Authors: Lee Kun-Ze , Fuller David D.
Published: June 2021

Intermittent hypoxia can elicit a progressive and long‐lasting enhancement of phrenic motor output known as long‐term facilitation (LTF). We hypothesized that phrenic LTF occurs due to rate‐coding of previously active phrenic motoneurons (PMNs) and recruitment of previously silent PMNs. A ″single fiber″ phrenic nerve recording technique was used to characterize PMN firing in anesthetized, vagotomized and mechanically ventilated adult rats. PMNs were classified as early‐inspiratory (Early‐I) or late‐inspiratory (Late‐I) based on their discharge onset relative to contralateral phrenic nerve activity during hyperoxic normocapnic baseline conditions. Consistent with prior reports, acute hypoxia increased the discharge frequency (i.e., rate coding) of both Early‐I and Late‐I PMNs and also caused recruitment of previously silent PMNs. During a 45–60 min period following intermittent hypoxia (3–5 brief hypoxic episodes), Late‐I but not Early‐I PMNs showed a progressive enhancement of discharge frequency. In addition, we noted a previously silent PMN that was recruited at ~45 min post‐episodic hypoxia. These preliminary results are consistent with the hypothesis that phrenic LTF is differentially expressed across phenotypically‐defined PMN subtypes. We suggest that the capacity for plasticity may be greater in Late‐I and silent PMNs compared to those cells activated early in the inspiratory effort.
Enhanced activation of sympathetic nerve activity and arterial pressure by the hypothalamic paraventricular nucleus in obese Zucker rats
Authors: Huber Domitila Augusta , Schreihofer Ann Marie
Published: June 2021

Adult obese Zucker rats (OZR) have elevated sympathetic nerve activity (SNA) and mean arterial pressure (MAP) that is driven by the rostral ventrolateral medulla (RVLM), and exaggerated sympatho‐excitatory reflexes associated with an enhanced ability of glutamate to activate the RVLM. Because the PVN contributes to basal SNA and MAP via an excitatory projection to RVLM, we sought to determine whether the PVN may contribute to the elevated SNA and MAP observed in OZR vs. lean Zucker rats (LZR). We also sought to determine whether the ability of the PVN to increase SNA and MAP was exaggerated in OZR vs. LZR. Adult male rats were anesthetized with urethane (1.5 g/kg, iv), ventilated, and paralyzed to record splanchnic SNA and MAP. Compared to LZR, OZR had higher SNA (1.7±0.3 vs. 0.6±0.2 μV, P<0.05) and MAP (128±5 vs. 101±8 mmHg, P<0.05). Inhibition of PVN (muscimol; 100 pmol) evoked larger decreases in AP in OZR (6) vs. LZR (7; −17±3 vs. −8±2 mmHg, P<0.05). This was not due to reduced tonic GABAergic inhibition of PVN because gabazine into PVN (100 pmol) evoked comparable rises in SNA and AP in OZR (5) and LZR (7). Activation of PVN (NMDA, 50 pmol) produced greater increases in SNA (57±3 vs. 42±6 %, P<0.05) and MAP (18±4 vs. 7±2 mmHg, P<0.05) in OZR (6) vs. LZR (7). These data suggest that the PVN contributes to the elevated AP in OZR. Exaggerated responses to activation of the PVN may be due to changes at the PVN or RVLM. Supported by NIH HL086759.
Angiotensin type 1a receptors (AT1a) in the subfornical organ (SFO) are involved in leptin‐induced sympathetically‐mediated brown adipose tissue thermogenesis
Authors: Young Colin N. , Morgan Donald A. , Butler Scott D. , Mark Allyn L. , Davisson Robin L.
Published: June 2021

The adipocyte‐derived hormone leptin acts within the central nervous system to influence body weight regulation. We have shown that AT1a receptors in the SFO are involved in the central weight‐reducing effects of leptin, but the precise mechanisms remain unclear. Here we tested the hypothesis that AT1a receptors in the SFO are critical in central leptin‐induced brown adipose tissue (BAT) sympathetically‐mediated thermogenesis. An adenovirus encoding Cre‐recombinase (Cre) was targeted to the SFO to selectively delete AT1a receptors in AT1a flox mice; viral delivery of LacZ served as control. After 7 days, mice were instrumented for recordings of BAT sympathetic nerve activity (SNA) and leptin was administered intracerebroventricular (ICV; 2ug). ICV leptin caused a robust increase in BAT‐SNA over 4 hours in control mice. This response was completely abolished with SFO‐targeted Cre (%Δ BAT‐SNA 4hr: 290±53 vs. 68±58 spikes/s; LacZ vs. Cre, p<0.05, n=5–6). In line with this, chronic ICV leptin over 4 days resulted in a smaller increase in body temperature in Cre‐treated, compared to LacZ mice, and analysis of BAT revealed reduced thermogenic markers with ablation of AT1a in the SFO (e.g; uncoupling protein 1 mRNA 0.67±0.20; Cre fold LacZ, p=0.056, n=6–7). These findings suggest that AT1a receptors in the SFO play a role in leptin‐induced BAT sympathetically‐mediated thermogenesis.

HL063887, HL084207
Effects of central orexin 1 receptor blockade on locus coeruleus neurons with lateral hypothalamic input
Authors: Verberne Anthony J
Published: June 2021

The locus coeruleus (LC) receives a major orexinergic input and is densely packed with orexin 1 receptors (OX1‐R). Here we examined the effects of central administration of an OX1‐R antagonist on lateral hypothalamic (LH) stimulation‐induced activation of LC neurons. All experiments were conducted using male Sprague Dawley rats under initial isoflurane (1.7–1.9%) anaesthesia followed by urethane (1.3–1.4 g/kg, i.v.). Electrical stimulation of the LH (0.5 Hz, twin 0.5 ms pulses, 3 ms interpulse interval, 300uA) evoked excitatory responses in all LC neurons studied (n = 24) as judged by construction of peri‐stimulus time histograms. LC neurons responded to LH stimulation with an onset latency of 6.5±0.4 ms and a peak latency of 15.3±0.8 ms. Intracerebroventricular administration of the OX1‐R antagonist SB334867A (10 nmol) but not vehicle inhibited LH stimulation‐induced activation of LC neurons by 58±14% (n = 6 neurons). Abolition of LH stimulation‐induced excitatory responses in LC neurons occasionally (n=3/6) revealed constant latency antidromic responses verified by positive collision tests. These findings indicate that the LC receives a robust excitatory input which is mediated, at least in part, by orexin acting at OX1‐Rs. Furthermore, some LC neurons that receive an orexinergic input also project to the LH region. Supported by the Austin Hospital Medical Research Foundation.
Central chemoreflex activation resets the setpoint pressure of baroreflex without compromising its function
Authors: Saku Keita , Hosokawa Kazuya , Sakamoto Takafumi , Onitsuka Ken , Sakamoto Kazuo , Tobushi Tomoyuki , Fujino Takeo , Ide Tomomi , Miyamoto Tadayoshi , Sunagawa Kenji
Published: June 2021

Background

Hypercapnia activates central chemoreflex and induces sympathoexcitation. However, how the chemoreflex modulates baroreflex (BR) function remains unknown. We hypothesized that the chemoreflex resets the BR setpoint pressure without compromising BR function.

Methods and Results

In vagotomized rats (n=5), we vascularily isolated the bilateral carotid sinuses and changed carotid sinus pressure (CSP) from 60 to 170 mmHg. We measured celiac sympathetic nerve activity (SNA) and arterial pressure (AP) with or without central chemoreflex activation. Increases in CSP sigmoidally suppressed SNA, whereas the SNA suppression linearly decreased AP. Hypercapnia (inhalation of 3%CO2, PaCO2 from 39.0±1.9 to 56.9±2.4 mmHg, p<0.001) markedly increased AP ( ▵ AP=31.5±1.4 mmHg, p<0.001) and SNA (▵SNA=57.7±5.5%, p<0.001) irrespective of CSP indicating a parallel upward‐shift of the CSP‐SNA relation. Hypercapnia increased the estimated setpoint pressure from 176.1±10.1 to 194.2±9.0 mmHg (p<0.05). In contrast, hypercapnia did not alter the SNA‐AP relation.

Conclusion

Central chemoreflex modulates hemodynamics by resetting the BR setpoint pressure without compromising its function. The chemoreflex induced pressor response may improve central perfusion and in turn contribute to neural regulation of central metabolic homeostasis. This study was supported by MEXT and MHLW, Japan.
Perivascular blood induces substantial constrictions of isolated basilar artery, which can be reversed by high pCO2
Authors: CSEPLO Peter , Torok Orsolya , Vamos Zoltan , Kosa Dalma , Springo Zsolt , Hamar Janos , Koller Akos
Published: June 2021

The vasoconstrictor mechanisms in acute intracerebral hemorrhage have not been fully clarified. We hypothesized that perivascular hemolysed blood (HB) has substantial effects on the tone and vasomotor responses of isolated cerebral arteries.

Basilar artery (BA) from male rats was isolated and placed in a pressure‐myograph chamber. The diameters of vessels were measured by videomicroscopy. After administration of HB the vasomotor function of vessels was studied in responses to acetylcholine (Ach), sodium‐nitroprusside (SNP), nifedipine (Nife) and CO2, which were obtained in control condition, in the presence of HB, and after washout of HB.

The active diameters of BA were 277±11 μm. HB reduced the basal diameters of BA (to 164±11 μm; 59±4% of AD). After washing out of HB, the active diameters of BA reached the control level: 284±9 μm. In control, Ach, SNP, Nife and CO2 elicited substantial dilations in cerebral arteries (20±2%, 26±2%, 32±3% and 26±2%). In contrast, HB decreased the dilation to ACh, SNP, but not to nifedipin and CO2 (7±1%, 12±2%, 28±3% and 30±2%). After washout of HB NO‐dependent dilations were significantly reduced compared to control (ACh: 6±1%, SNP:14±2%).

Findings of the present study show that extraluminal HB elicits substantial constrictions and inhibits NO‐dependent dilations, but does not affect Ca‐channel blocker and CO2‐induced dilations.
Cold acclimation and central administration of homocysteic acid in rats: a potential model for schizophrenia
Authors: Barney Christopher , Chase Leah , Davis Christopher , Flores Guillermo , Kemink John , Logan Jacqueline , McMurray Mara , Reif Lauren , Sayfie Aaron , Strahle Catherine , Wagner Allyssa
Published: June 2021

Homocysteic acid (HCA) is a neurotoxin and a NMDA receptor agonist that is taken up in GABAergic interneurons by the cystine/glutamate exchanger, System xc‐, and then slowly released. We thus hypothesized that HCA may trigger a slowly developing neurodegeneration of GABAergic interneurons, which might serve as a model for schizophrenia. In addition, we hypothesized that cold acclimation would exacerbate the effects of the HCA. In this study, HCA was injected bilaterally into the lateral cerebral ventricles of rats. Two days later half of the rats were placed in a 5°C environmental chamber. Behavioral testing for motor performance, anxiety, social interaction, hedonistic behavior, and spatial learning and memory began three weeks later. Cold acclimation reduced body weight gain, motor performance and exploratory behavior while HCA decreased exploratory behavior. Neither cold acclimation nor HCA altered spatial learning or memory or social interaction. Both cold acclimation and HCA altered hedonistic behavior as measured with a saccharine preference test. There were no significant interactions between cold acclimation and HCA for any behavioral test. These results indicate that both central administration of HCA and cold acclimation can alter performance in behavioral tests that may relate to schizophrenia‐like behavior in rats but cold acclimation does not potentiate the effects of HCA.