bluuebunny/crossref_metadata_2025 huggingface dataset:
Stop‐action movie of UvrD helicase unwinding DNA
Authors: Yang Wei
Published: June 2021

Molecular mechano‐chemical coupling is a fundamental process in biology. To capture molecular locomotion by X‐ray crystallography, we determined a dozen crystal structures of UvrD‐DNA complexes in the absence or presence of ATP hydrolysis analogs. After combining multiple structures representing each functional state and accounting for crystal lattice effects, we obtained a stop‐action movie of UvrD helicase unwinding DNA one base pair per ATP hydrolyzed. For the first time, we show that each ATP‐hydrolysis cycle delivers a power stroke in two parts. Binding of ATP is coupled with unwinding of one base pair, and release of ADP and Pi is coupled with translocation of the newly unpaired single base. Combining our new mutagenesis, structural and kinetic studies with published data, we have put forward the model of dual active states of UvrD for its dual functions (dsDNA unwinding and RecA removal from ssDNA) in DNA replication and repair. The structural and functional duality is most probably essential for inhibition of unwanted helicase activity.
Post‐transcriptional Regulation of Luteinizing Hormone Receptor Expression by Mevalonate Kinase, a novel mRNA binding Protein
Authors: Nair Anil K , Menon K.M.J.
Published: June 2021

There is increasing evidence that metabolic enzymes act as RNA binding proteins and regulate gene expression post‐transcriptionally. We have identified mevalonate kinase (MVK), an enzyme involved in cholesterol biosynthesis, as a trans‐factor for luteinizing hormone receptor (LHR) mRNA. LHR is one of the crucial G protein‐coupled receptors that plays a key role in mammalian reproduction. MVK binds to the coding region of LHR mRNA and accelerates its degradation in vitro. Translation studies have shown that MVK inhibits translation of LHR mRNA. Endogenous association of LHR mRNA with MVK has also been detected by immunoprecipitation of the ribonucleoprotein complex with MVK antibody. Studies were then performed to examine the structural aspects of MVK required for LHR mRNA recognition. A concentration dependent decrease in LHR mRNA binding activity of MVK by its substrates, ATP and mevalonate, indirectly indicated the involvement of the active site for RNA binding. The role of the active site of MVK for LHR mRNA binding was tested using mutations of the amino acids required for catalysis. The results showed that mutations of Ser146, Glu193, Asp204 and Lys13 reduce LHR mRNA binding activity of MVK. To examine the biological effects of these mutants on LHR mRNA expression, rat LHR mRNA was translated in the presence of the MVK mutant proteins. The results showed that mutations of the active site residues abrogated the inhibitory effect on LHR mRNA translation. From these results we conclude that the intact active site of MVK is required for its binding to LHR mRNA and for its translational suppressor function. (Supported by NIH Grant R37 HD 06656)
Identification of intersubunit bridges essential for ribosome stability in yeast
Authors: Ackell Andrew P. H. , LaRiviere Frederick J.
Published: June 2021

The ribosome is a ribonucleoprotein particle composed of two unequal subunits. Correct association of the two subunits is required for proper ribosome function. X‐ray crystal structures of fully assembled bacterial ribosomes have identified a number of specific interactions, or intersubunit bridges (ISBs), between the large (60S) and small (40S) subunits. Modification interference studies have shown that at least two of these ISBs are important for ribosome stability in bacteria. We are using a similar modification interference approach to identify ISBs that are important for ribosome stability in yeast. To date we have performed sucrose gradient ultracentrifugation under a variety of conditions to dissociate ribosomal subunits from intact 80S ribosomes. Chemical modifications of the purified 40S subunits are currently underway. Subsequently, modified 40S subunits will be allowed to reassociate with native 60S subunits. Presumably, those 40S subunits that are unable to stably reassociate will have been modified at RNA bases involved in important ISBs. We hope these studies will provide insights regarding the function and conservation of ISBs. Supported by the Dreyfus Foundation and the Jeffress Memorial Trust.
A dominant‐lethal screen to identify non‐functional rRNA decay pathway factors in yeast
Authors: Levin Carly S. , LaRiviere Frederick J.
Published: June 2021

Nonfunctional rRNA Decay (NRD), which eliminates nonfunctional ribosomes that have assembled with mutant rRNAs, is a recently discovered quality control mechanism in eukaryotes. To investigate the mechanistic details of NRD, a dominant‐lethal screen was developed to identify trans‐acting factors associated with this pathway. Galactose‐induceable plasmids that encode rRNA genes with mutations in functionally important regions of 18S and 25S rRNA were transformed into Saccharomyces cerevisiae. Transformants were exposed to ultra‐violet (UV) radiation in order to induce genomic DNA mutations. Mutations in genes required for NRD may result in a dominant‐lethal phenotype when mutant rRNAs are expressed in these cells. To determine which cells acquired a dominant lethal phenotype, UV exposed colonies were replica plated onto galactose to allow expression of the plasmid‐encoded mutant rRNAs. To date, we have isolated ten different S. cerevisiae colonies that cannot grow on galactose after UV exposure. Complementation experiments are now underway to determine the identity of the resulting mutant trans‐acting factors and to establish their roles in NRD. Supported by the Dreyfus Foundation and the Jeffress Memorial Trust.
The conductive properties of exponentially dividing LNCaP prostate cancer cells
Authors: Chao Tina , Hamilton Kirk L. , Hessian Paul A. , Butt Grant
Published: June 2021

The intermediate conductance Ca2+‐activated K+ channel (hIK1) is thought to be involved in the cell division of cancer cells. However, direct measurements of the expression and properties of hIK1 during the cell cycle have not been made. Therefore, as an initial step in defining the role of hIK1 in the cell cycle we have measured the conductive properties of exponentially growing LNCaP cells using the whole cell patch clamp technique. Flow cytometry analysis indicated that 24 h after plating 84% of the cells were in the G1 phase, and 7% and 8% of the cells were in the S and G2/M phases, respectively (n = 4). In these cells three distinct conductances were identified in different cells. The majority of the cells (18 out of 29 cells) exhibited an outwardly rectified K+ current. In the presence of 500 μM free Ca2+, this current was inhibited by 10 mM tetraethylammonium (TEA) (n = 8, P < 0.0001) but not the hIK1 inhibitor clotrimazole (10 μM, n = 3, P = 0.11). Furthermore, 1‐EBIO (100 μM), an activator of hIK1, did not stimulate an increase in current in these cells (n = 3, P = 0.27). In a second group of cells (5 of 29) an outwardly rectified current was also seen, but this was relatively insensitive to TEA. In the remaining 5 cells the dominant current was inwardly rectified. 1‐EBIO increased this current from ‐2.7 ± 1.3 to ‐10.8 ± 6.7 pA pF−1 at ERev‐60 mV (n = 3, P = 0.2), but the increase was not significant suggesting the lack of hIK1 activity. Therefore, if hIK1 is involved in proliferation of LNCaP cells, there must be a dramatic change in the expression level or biophysical properties of the channel as the cells proceed through the cell cycle. This work was funded by Grants from the HS & JC Anderson Trust and Lotteries Health.
Role of Phosphorylation in the Inhibition of GDPD5 Activity that Contributes to High NaCl‐ and Urea‐induced Increase of GPC
Authors: Topanurak Supachai , Gallazini Morgan , Ferraris Joan D. , Burg Maurice B.
Published: June 2021

High NaCl increases the abundance of glycerophosphocholine (GPC) in renal medullary and other cells by increasing phospholipase B (NTE) activity and inhibiting glycerophosphocholine phosphodiesterase (GDPD5) activity. Urea also increases GPC, but solely by inhibiting GDPD5. Elevating NaCl or urea bathing HEK293 cells inhibits the activity of GDPD5 immunoprecipitated from the cells and assayed in vitro, suggesting post translational regulation. To investigate the nature of this post translational modification, we transfected recombinant GDPD5‐V5 into HEK293 and increased osmolality to 500 mOsm by adding NaCl or urea. This does not affect migration of GDPD5‐V5 in conventional SDS‐PAGE. However, phosphorylation of GDPD5 that retards its migration is evident in gels containing Phos‐tag, a phosphoprotein chelator. Two retarded bands are evident. High NaCl or urea decreases the upper band, increases the lower one, and increases the mobility of both. We suggest that high NaCl or urea inhibits GDPD5 activity by dephosphorylating it. We are currently attempting to identify the phosphorylation sites by protein mass spectrometry. This research was supported by the Intramural Research Program of NHLBI.
Sequence specific oxidative DNA modifications in the mitochondrial genome of hypoxic human bronchial epithelial and pulmonary artery endothelial cells
Authors: Ruchko Mykhaylo V. , Gorodnya Olenna M. , Pastukh Viktor M. , Gillespie Mark N.
Published: June 2021

Reactive oxygen species used as second messengers in hypoxic signaling oxidatively modify specific bases within hypoxic response elements (HREs) of hypoxia inducible genes (FRBM, 43: 1616, 2007). Because oxidative lesions are restricted to HREs associated with transcriptionally‐active nucleosomes, and since model base modifications influence transcription complex assembly and reporter gene expression, we proposed that controlled oxidative DNA damage and repair in HREs may regulate transcription in hypoxia (AJP‐Lung, 292: L1, 2007). Whether hypoxia causes modifications in transcriptional regulatory sequences of the mitochondrial genome is unknown. Accordingly, we used quantitative Southern blot analyses to detect oxidative base modifications in a 13.8kb mtDNA sequence containing the "fragile region" incriminated in mitochondrial diseases and a 2.7kb sequence containing the D‐loop control region. In human airways epithelial and pulmonary arterial endothelial cells, we found that whereas hypoxia failed to cause lesions in the larger mtDNA sequence, the D‐loop‐containing sequence displayed modifications early after hypoxic exposure that were subsequently removed despite persistent hypoxia. These observations support the intriguing idea that controlled oxidative DNA damage and repair in the D‐loop region may influence mtDNA replication and/or transcription in hypoxia.
Differential Sensitivity of Carotid Body Glomus Cells to Hypoxia and Acidosis
Authors: Lu Yongjun , Whiteis Carol A , Chapleau Mark W , Abboud Francois M
Published: June 2021

We recently demonstrated that TASK channels known to transduce the effect of hypoxia on carotid body glomus cells also contribute to sensing low pH (Circ Res 101, 1009‐1019, 2007). In this study we contrasted the effects of hypoxia (pO2=15 mmHg) and acidosis (pH 6.8) on cytosolic Ca2+ concentration in clusters of glomus cells dissociated from carotid bodies of rats. The cells were loaded with Fluo‐4/AM, a single wavelength fluorescent Ca2+ probe. A microscope (Nikon TE2000‐U) equipped with a CCD camera (Photometrics Cool SNAP‐cf) and imaging analysis software (MetaMorph 6.1) were used to quantitate [Ca2+]i. Baseline [Ca2+]i ranged from 40 to 76 nM (mean 53 ± 2 nM) in 33 glomus cells tested from 7 clusters. Transient increases in [Ca2+]i (?) ranging from 20 to 245 nM were seen in 19 responsive cells. Of these cells, 13 responded to hypoxia (? = 82 ± 17 nM) and 11 cells responded to low pH (? = 111 ± 26 nM). Eight of the 13 hypoxia‐sensitive cells had an increase in [Ca2+]i of 75 ± 24 nM but did not respond to pH 6.8. Conversely, 5 of the 11 pH‐sensitive cells were highly responsive to low pH (? =163 ± 31 nM, n = 5) but showed negligible responses to hypoxia. The results indicate a significant degree of selectivity in responsiveness of individual glomus cells to hypoxia vs. low pH and suggest alternative transduction pathways for hypoxia and acidosis in carotid body chemoreceptors. (HL14388).
TASK and TREK channels in rat carotid body chemoreceptor cells during postnatal development
Authors: Kim Insook , Kim Donghee , Cavanaugh Eric J , Carroll John L
Published: June 2021

Oxygen sensitivity in carotid body (CB) glomus cells are small at birth and fully developed in two weeks, but mechanism for this phenomenon is poorly understood. Two background K+ channels that regulate excitability of CB cells include TASK and TREK channels. Therefore, we hypothesized that changes in oxygen sensitivity during development may be due to modulated gene expression of TASK and TREK channels. To test this idea, expression of TASKs and TREKs mRNA and membrane K+ currents were determined in P0‐1d (postnatal 0‐1 day) and P14‐18d (postnatal 14‐18 days) CB cells. qRT‐PCR was performed using two stable reference genes, PPIA and TBP, and TASK and TREK currents were recorded in cell‐attached patches. TASK‐1 and TASK‐3 channel mRNA showed no significant difference between two age groups (p>0.05). In agreement with this finding, TASK channels of similar open probabilities were recorded from ~90% of patches from both age groups. Unlike TASKs, TREK‐1 and TREK‐2 mRNA in P0‐1d CB were significantly higher than those in P14‐18d CB. Consistent with these results, TREK‐like channels were recorded in ~35% of patches in P0‐1d CB, but none in P14‐18d CB. These results suggest that expression of TREKs undergoes down‐regulation during development, whereas expression of TASKs does not. The changes in TREK channel expression may therefore affect oxygen sensing during development. (Funded by NIH R01 HL54621 and UAMS CUMG).
Perinuclear formation of Reactive Nitrogen Species causes DNA damage in Methicillin‐resistant Staphylococcus Aureus induced sepsis.
Authors: Bansal Kamna , Connelly Rhykka L , Herndon David , Traber Daniel L , Enkhbaatar Perenlei
Published: June 2021

Rationale

MRSA commonly referred to as a 'super bug' because of its resistance to antibiotic therapy, is now a common infection encountered in hospitals and communities. We have previously shown that MRSA causes vascular hyperpermeability, multi‐organ system failure, and death in our ovine model of MRSA‐induced sepsis. We hypothesized that RNS may play a pivotal role in MRSA‐induced DNA damage and consequent cellular death.

Methods

Pulmonary epithelial cell (A549) were challenged with 105 cfu MRSA over a time course of 4 hrs then probed for markers of RNS (2,7‐Dichlorodihydrofluorescein [DCF] and 3‐Nitrotyrosine {3‐NT}), as well as poly ADP‐ribose (PAR), a marker of DNA damage, by confocal imaging and western blot analyses. Neuronal nitric oxide synthase (nNOS) and NADPH oxidase‐4 (NOX4) were localized by confocal microscopy.

Results

MRSA caused nNOS and NOX4 translocation to a perinuclear location, resulting in a 7‐fold increase in RNS (measured by DCF) at the perinuclear compartment compared to normal cells. MRSA also caused PAR (3.4 fold) and 3‐NT (4.8 fold) formation within 1h of MRSA exposure observed by both confocal and western blotting analyses.

Conclusions

MRSA causes DNA damage in alveolar epithelial cells as result of perinuclear formation of RNS .
The M1 muscarinic acetylcholine receptor agonist xanomeline suppresses lethal inflammation
Authors: Pavlov Valentin A. , Ochani Mahendar , Ochani Kanta , Rosas‐Ballina Mauricio , Al‐Abed Yousef , Tracey Kevin J.
Published: June 2021

We have recently indicated a role for brain muscarinic acetylcholine receptors in controlling systemic inflammatory responses (Proc Natl Acad Sci USA, 2006, 5219; Brain Behav Immun, Epub). Here we studied whether xanomeline, a centrally‐acting M1 muscarinic receptor agonist, suppresses the systemic cytokine response and improves survival during lethal endotoxemia. This drug has been studied in the context of Alzheimer's disease, but its anti‐inflammatory effects have not been previously reported. Peripheral drug (5 or 20 mg/kg, i.p.) administration 1h prior to endotoxin (6 mg/kg, i.p.) dose‐dependently suppressed serum TNF levels and significantly improved survival in mice. Pharmacological blockade of brain muscarinic receptors, but not peripheral muscarinic receptors abrogated the survival‐improving drug effect, thus demonstrating that central muscarinic receptors mediated the effect of xanomeline. In addition, administration of xanomeline (20 mg/kg, i.p.) 20h prior to endotoxin significantly reduced serum TNF levels and improved survival. Together these findings demonstrate the long‐lasting anti‐inflammatory efficacy of xanomeline, acting through a brain muscarinic receptor‐dependent mechanism, and indicate the therapeutic potential of centrally‐acting muscarinic M1 agonists in the treatment of inflammatory diseases. This study was funded in part by NIH/NIGMS.
Effects of intermittent hypoxia on regulation of BDNF mRNA in the brainstem and spinal cord of MeCP2 mutants
Authors: Jenkins Victoria K , Balkowiec Agnieszka , Bissonnette John M
Published: June 2021

Brain‐derived neurotrophic factor (BDNF) is necessary and sufficient for the expression of long‐term facilitation, a form of respiratory plasticity that is induced by intermittent hypoxia (IH). BDNF is a target gene for the transcription factor methyl‐CpG‐binding protein 2 (MeCP2), but its role in determining the regulation of BDNF transcripts after IH has not been determined.

MeCP2 null and wild‐type male mice were exposed to three IH intervals (5 minutes of 21% O2, 5 minutes of 8% O2) followed by a 60‐minute post‐hypoxia rest. The ventral half of cervical spinal cord segments 3‐5, pons, and medulla were dissected. RNA was extracted and BDNF levels were evaluated by quantitative RT‐PCR. The IH‐induced increase in expression of transcript 4 in medulla of MeCP2 null mutants exceeded that in wild‐type mice. This transcript trends upward in pons, but stays constant in cervical spinal cord. The results suggest that MeCP2 modulates BDNF expression during IH.
Increased neuropeptide Y innervation in the airway epithelium of mice overexpressing Nerve Growth Factor
Authors: Dey Richard D , Batchelor Thomas P , Hoyle Gary W
Published: June 2021

The neurotrophin, nerve growth factor (NGF), supports nerve growth and axon sprouting of sensory and sympathetic neurons. NGF is elevated in asthmatics and contributes to airway hyperresponsiveness and inflammation. Overexpression of NGF has been reported in lungs of transgenic mice by linking the NGF gene to the Clara cell secretory protein (CCSP) promoter. These mice overexpress catecholamines and the neuropeptide substance P. Since sympathetic nerves respond to NGF, we hypothesized that NPY might be elevated in the NGF‐CCSP mice. NGF‐CCSP and wild‐type mice were prepared for quantitative immunocytochemistry for neuropeptide Y (NPY) and the pan‐neuronal marker PGP 9.5. Nerve fiber density (NFD) of NPY (n=3) in epithelium of the NGF‐CCSP transgenic mice (1.28 + 0.26%) was significantly increased compared to the epithelium of wild‐type mice (0.09 + 0.012%). PGP 9.5 NFD (n=1) in the transgenic mice was also increased (2.86 %) compared to wild‐type (0.07%). These findings support two important conclusions. First, that NGF overexpression substantially enhances NPY innervation of airway epithelium, which is normally very low. Second, the increased PGP 9.5 innervation indicates that NGF‐induced nerve fiber sprouting and growth had occurred within the epithelium. The findings suggest that increased NGF release known to occur during development or environmental exposures may control airway epithelial innervation.

Funded by NIH HL35812 and HL80566.
Effects of smoke exposure on neuropeptide Y‐containing nerves in airway of early postnatal mice
Authors: Wu Zhong.‐Xin , Johnson Jessica , Benders Katherine M. , Dey Richard D.
Published: June 2021

Abundant epidemiological data show that maternal smoking during pregnancy or neonatal and infant periods increases the incidence of respiratory illnesses later in life. However, the underlying mechanism remains unclear. Neuropeptide Y (NPY) produces vasoconstriction of airway vasculature and our previous PCR array data shows that the NPY gene is enhanced by smoke exposure during early prenatal periods. The goal of this proposal is to study the effects of early postnatal smoke exposure on NPY airway innervation. New born mice were directly exposed to either smoke or filtered air for 1, 5 and 10 days (6 hours per day) and the density of NPY‐containing nerves in airway smooth muscle was measured by immunocytochemistry 16 hours after the last smoke exposure. NPY‐containing fibers were mainly found around blood vessels and airway smooth muscle. NPY nerve fiber density in airway smooth muscle were significantly increased from 0.88 0.14 to 4.81 0.68 after 5 days smoke exposure and from 0.69 0.11 to 3.46 0.42 after 10 days smoke exposure. 1 day smoke exposure increased NPY from 0.55 0.07 to 1.18 0.29, but difference was not significant. These data suggest that cigarette smoke exposure enhances NPY expression in airways of newborns.

Funded By: NIH HL 35812 and NIH ES014618
Rat pups from dams fed a partial tryptophan‐deficient diet exhibit a decreased ventilatory response to hypercapnia and slower heart rates
Authors: Penatti Eliana Mori , Barina Alexis E , Harris Alexandra , Raju Sharat , Li Aihua , Kinney Hannah C , Commons Kathryn , Nattie Eugene E
Published: June 2021

Many cases of sudden infant death syndrome (SIDS) are associated with an altered brainstem serotonergic (5‐HT) system, which may affect cardiorespiratory function and contribute to sudden death. High SIDS rates are associated with poverty, raising the possibility that nutritional deficits in tryptophan (TP), a key determinant of 5‐HT synthesis, contribute to the altered 5‐HT system. We hypothesized that exposure of rats during pre‐ and early postnatal life to a diet deficient in TP would result in detrimental physiological effects. Sprague‐Dawley dams were fed either an amino acid based diet (control) or a TP 50% deficient diet (treated) from before mating until the end of the study. We measured ventilation and heart rate (HR) in pups at postnatal days (P) 5, 15 and 25 exposed to air or to 7% CO2. Treated pups weighed 38.4% less than control ones at P25 (P<0.001) although their weight was similar at P5. The CO2 response, represented by the percent increase of minute‐ventilation (V̇E) normalized to oxygen consumption (V̇O2), was reduced (P<0.001) in treated males by 17% at P15 and 18% at P25 compared to controls. HR in treated pups of both genders was lower in air at P15 only (P<0.001). These findings raise the possibility that dietary TP alterations contribute to abnormal development of the brainstem 5‐HT system and thus to SIDS, particularly in males who are affected by SIDS in a 2:1 ratio. (NICHD HD36379)
Respiratory alkalosis is the determinant of the increased production of augmented breaths triggered by hypoxia
Authors: Bell Harold James , Ferguson Carrie , Haouzi Philippe
Published: June 2021

Spontaneous augmented breaths (ABs) are large, transient respiratory disturbances which often lead to apneas or instability in subsequent breath cycles. Hypoxia is known to increase the rate of occurrence of ABs, however the underlying factors mediating this effect are not fully understood. In this study, we examined the role of hypocapnia in the hypoxia‐induced increase in ABs. In studying the ventilatory responses of 5 freely behaving adult rats (489 ± 22 g), we found that an average of 1.0 ± 0.7 ABs occurred in room air (RA) in a standardized 5 minute observation window. Exposure to hypoxia (~10% FiO2) caused a dramatic increase in the number of ABs (13 ± 1.5 in 5 min, P<0.001). When hypoxia was supplemented with 5 % CO2 in the inspired air, the number of ABs returned to a level close to RA conditions (2.4 ± 0.5 p<0.001). CO2 alone had no significant effect. Acetazolamide treatment (100 mg/kg IP 2x/d, for 48 hrs) was found to effectively eliminate the increased prevalence of ABs normally observed during exposure to hypoxia. We therefore conclude that hypoxia promotes the generation of augmented breaths, but only when hypocapnia‐induced alkalosis develops. We further speculate that ABs during hypoxia are expressed as a result of an increased neuronal excitability related to acute respiratory alkalosis. This study received internal funding support from the Heart and Vascular Institute, Penn State College of Medicine.
Gender modulation of respiratory variability during exercise in humans
Authors: BuSha Brett F , Hofmann Cory , Hage Erik
Published: June 2021

The integrative nature of the respiratory control system embeds temporal correlations in the variability of breathing. To test the hypothesis that gender and/or enhanced respiratory drive modifies temporal correlations in respiratory variability during exercise, 6 female and 6 male adult subjects were fitted with a resistive belt to measure chest volume and surface electrodes to measure ECG. Subjects breathed through the mouth while seated motionless on a recumbent bicycle and at two progressive levels of peddling. Breath to breath interval (BBI) was calculated for 144 consecutive breaths and beat to beat interval (RRI) was calculated from 400 consecutive R‐waves in the ECG, time‐indexed with the BBI data. Temporal correlations in the variability of BBI and RRI were quantified with detrended fluctuation analysis (DFA). A significant gender‐exercise interaction in the DFA of the BBI indicated that exercise elicited a greater decrease in the temporal correlation of BBI in female subjects (30%) as compared to male subjects (7%). Exercise resulted in a similar increase in temporal correlations (25%) in the RRI among all subjects. We speculate that the gender‐specific difference in the DFA measurement of the BBI is reflective of subtle differences in the control of breathing between male and female subjects. This work was supported by The College of New Jersey's Mentored Undergraduate Research Experience grant.
Role of the caudal pressor area in cardiorespiratory reflexes
Authors: Takakura Ana C , Moreira Thiago S , Colombari Eduardo
Published: June 2021

Important mechanisms for the cardiovascular regulation are also present in the caudal pressor area (CPA), a site localized within the most caudal part of the ventrolateral medulla. Results from our laboratory had suggested that the pressor mechanisms activated by the stimulation of the CPA depend upon the mechanisms integrated in the nucleus of the solitary tract (NTS). The NTS is the site of the first synapse of the peripheral baroreceptor and chemoreceptor afferent fibers. Here, we test whether the inhibition of CPA could influence the sympathetic baro‐ and chemoreflex. Mean arterial pressure (MAP), splanchnic sympathetic nerve discharge (sSND) and phrenic nerve discharge (PND) were recorded in urethane anesthetized and vagotomized Sprague‐Dawley rats. Bilateral injection of muscimol (GABA‐A agonist, 2 mM ‐ 60 nl) or glycine (1 mM ‐ 60 nl) into the CPA produced a decrease in MAP (104 ± 6, 106 ± 7, respectively vs. saline: 124 ± 3 mmHg) and sSND (‐40 ± 2, ‐39 ± 2%, respectively), but no change on PND. Muscimol or glycine into the CPA reduced the pressor (δ= +8 ± 3, +10 ± 2, respectively vs. saline: +18 ± 6 mmHg) and sympathoexcitatory (+88 ± 8, +74 ± 8, respectively vs. saline: +153 ± 8 %) responses produced by peripheral chemoreceptor activation. However the baroreflex range and gain did not change after muscimol or glycine into the CPA. These data suggest that CPA contributes modestly to resting MAP, sSND and to the sympathoexcitatory effect during stimulation of peripheral chemoreceptors.
Glutamatergic mechanisms on the interaction of sympathetic and respiratory responses to chemoreflex activation in the NTS
Authors: Costa‐Silva João H , Zoccal Daniel B , Machado Benedito H
Published: June 2021

We evaluated the effect of the ionotropic glutamatergic receptor antagonism in different areas of the NTS on baseline respiratory parameters and on chemoreflex responses using an in situ working heart‐brainstem preparation, in which were recorded the activity of the phrenic (PNA) and thoracic sympathetic (tSNA) nerves. Bilateral microinjections of kynurenic acid (KYN 250 mM) were performed into intermediate (iNTS, 0.2‐0.3 mm rostral from calamus scriptorius, CS) and caudal NTS (cNTS, 0.1‐0.3 mm caudal from CS). KYN into cNTS evoked a significant increase in duration and frequency of phrenic burst discharge associated with a slight attenuation of the sympathoexcitation (ΔtSNA: 114±12 vs 67±17 %), but not the tachypnea, evoked by peripheral chemoreflex activation (KCN 0.05%, 50 μL). Besides, KYN microinjected simultaneously into iNTS and cNTS produced higher increases in frequency and duration of phrenic bursts with a significant attenuation of both sympathoexcitatory (ΔtSNA: 103±11 vs 23±5 %) and tachypneic (ΔPNA: 0.4±0.1 vs 0.02±0.06 Hz) responses to chemoreflex activation. The data show that L‐glutamate in iNTS and cNTS plays a tonic inhibitory role on the respiratory activity and the changes in the respiratory pattern due to the glutamate receptor antagonism in the NTS may affect the processing of the ventilatory and sympathetic responses to chemoreflex activation. Supported by FAPESP and CNPq.
Age dependent protein expression of renal acid‐base transporters in rats with congenital ureter obstruction
Authors: Wang Guixian , Topcu Sukru Oguzkan , Djurhuus Jens Christian , Nielsen Soren , Frokiaer Jorgen
Published: June 2021

To clarify the molecular mechanisms of urinary acidification defects in response to congenital partial unilateral ureteral obstruction (PUUO) new born male Wistar rats were subjected to PUUO within the first 24‐48 hours of life to mimic the congenital ureteral obstruction, and were observed for 7 and 14 weeks. PUUO rats were challenged by acid loading with NH4Cl given by gavage for 2 days before termination. Semiquantitative immunoblotting revealed that the abundance of renal acid‐base transporters NHE3, NBC1, NBCn1, pendrin and Na+‐K+‐ATPase were increased in both obstructed and non‐obstructed kidneys 7 weeks after induction of neonatal PUUO. This was confirmed by immunocytochemistry. In contrast, 14 weeks of PUUO caused a reduction in the abundance of NBC1, NBCn1 and Na+‐K+‐ATPase in the obstructed kidney. Consistent with this the plasma pH and HCO3 could maintain normal in PUUO 7 week rats, but dramatically reduced in PUUO 14 week rats under the acid loading. These time/age dependent changes in protein expression were associated with parallel changes in the renal ability to acidify urine in response to acid loading. In conclusion the study demonstrated that 1) the reduction protein abundance of renal acid‐base transporters is time/age dependent in response to PUUO and 2) impairment of urine acidification develops gradually in parallel with the changes in protein expression in response to partial obstruction induced neonatally.
Role of a receptor tyrosine kinase in the HCO3‐induced inhibition of HCO3 reabsorption by rabbit S2 proximal tubule
Authors: Zhou Yuehan
Published: June 2021

Previous work showed that: (1) Proximal tubules (PTs) respond to increases in basolateral (BL) [CO2] or [HCO3] by, respectively, stimulating or inhibiting HCO3 reabsorption (JHCO3), suggesting the presence of CO2/HCO3 sensors near the basolateral membrane. (2) Studies with PD168393 (blocks erbB1 via alkylating a Cys in the ATP‐binding pocket) and BPIQ‐I (competes with ATP) suggest that the CO2 sensitivity of JHCO3 requires an active receptor tyrosine kinase (RTK), probably erbB1. Here, I hypothesize an active RTK also is required for the HCO3 sensitivity of JHCO3. I perfused the lumen of an isolated rabbit S2 PT with 5% CO2/22 mM HCO3 solution. Adding BL 35 nM PD168393 significantly decreased "baseline" JHCO3 (i.e. [HCO3]BL = 22 mM, [CO2]BL = 5%, pHBL = 7.4) from 69 ± 3 (n = 32) to 43 ± 4 pmol mm−1 min−1 ("pmm", n = 6, P < 0.0001). Using an out‐of‐equilibrium (OOE) solution to decrease [HCO3]BL to 0 mM, I found that BL 35 nM PD168393 decreased JHCO3 from 103 ± 6 (n = 7) to 62 ± 7 pmm (n = 6, P < 0.0006) at a fixed [CO2]BL of 5% and pHBL of 7.4. Using OOE solutions to increase [HCO3]BL to 44 mM, I found that BL 35 nM PD168393 had no effect on the already‐low JHCO3 (n = 7, P <0.79). Thus, BL 35 nM PD168393 decreased baseline JHCO3 by 37%, reduced the JHCO3 response at 0 mM HCO3, and eliminated the response at 44 mM. An active RTK, perhaps erbB1, plays a central role in both CO2 and HCO3 signaling regulating H+ secretion by PTs. (Supported by AHA SDG 0735432N)
Telmisartan, an Angiotensin II receptor blocker, abrogates troglitazone‐induced cell signaling coupled to ammoniagenesis in proximal tubule‐like LLC‐PK1 cells
Authors: Oliver Robert E. , Friday Ellen , Welbourne Tomas , Turturro Francesco
Published: June 2021

Troglitazone(TRO) induces a large increase in ammonium production from glutamine via the mitochondrial pathway in proximal tubule‐like LLC‐PK1 cells. To elucidate the upstream site of tro action we measured the acute effects on cell signaling in LLC‐PK1 cells grown in chambers designed for fluorometric ratiometric imagining (BCECF and Fura 2) and in 24 well plates for 18h metabolic studies. TRO (25uM) induces a large fall (p<0.01) in cytosolic pH (7.19±0.09 to 6.85±0.08) and a doubling of cell calcium occurring within 8minutes. Associated with these responses was a similarly rapid and large increase (p<0.05) in phospho‐ERK1/2(p‐ERK to total ERK ratio=1±0.2 for control vs 10.1±3 fold for TRO). Treating the cells with 30uM telmisartan (pre‐incubation for 4minutes and during 8minutes with TRO) markedly reduced both the rise in p‐ERK1/2(10.1±3 to 2.4±0.5) and acidity (6.85±0.08 to 7.11± 0.07) suggesting that telmisartan acutely blocks TRO's upstream site of action. At the downstream site, TRO (25uM) enhanced (p<0.01) mitochondrial ammoniagenesis (NH4+ production 1011±110 to 1678±84nmol/mg/18h) despite a normal extracellular bicarbonate concentration and pH; addition of telmisartan (30uM) to the TRO‐media prevented (p<0.05) the enhanced ammonium production (1193±142 vs 1678±84 nmol/mg/18h for TRO+telmisartan vs TRO). These findings are consonant with angiotensin II‐like receptor involvement in TRO's initial activation of cell signaling pathways underlying the ammoniagenic response in these model porcine proximal tubule‐like cells.
Mitochondrial membrane potential and ammoniagenesis in pig kidney proximal tubule‐like LLC‐PK1 cells
Authors: Oliver Robert E. , Friday Ellen , Welbourne Tomas , Turturro Francesco
Published: June 2021

The hallmarks of metabolic acidosis(MA) in humans are an increase in ammonium production (NH4+PROD) and decrease in sodium reabsorption. Since both proximal tubule activities are dependent upon mitochondrial function, we compared the effect of MA and toglitazone(TRO) on in situ mitochondrial membrane potential (Ψm) and ammoniagenesis in the proximal tubule model LLC‐PK1 cell line. Confluent cells were exposed to DMEM pH 7.4, DMEM pH 6.8 DMEM pH 7.4 plus TRO for 18h after which Ψm and distribution were assessed using TRME and mito‐tracker green staining, respectively. At pH 7.4 mitochondria appear punctate and brightly stained surrounding the nuclei (Hoerscht 33342 staining) whereas with TRO mitochondrial distribution remains unchanged but with Ψm decreasing in a dose‐dependent manner; at 5uM TRO, Ψm staining appears reduced but punctate in contrast to distinctly diffuse cytoplasmic staining despite normal mitochondrial distribution at 50uM TRO. MA reduces Ψm with staining localized to discrete mitochondria similar to 5 uM TRO. MA increased NH4+PROD 59 % (1538±210 vs 2446 ±194 nmol NH4+/mg) while 25uM TRO increased NH4+PROD 136% (1538±210 vs 3624±450 nmol NH4+/mg) consistent with an inverse relationship between Ψm and mitochondrial NH4+PROD. Incubation with MEKK inhibitors UO126 (10uM) and PD98059 (30uM) restored control Ψm at pH 6.8 and with 5, 25 and 50uM TRO while reducing NH4+PROD to a level not different from control at both pH 6.8 (1814±326nmol NH4+/mg) and at 25uM TRO (1820± 419 nmol NH4+/mg). These findings are consonant with p‐ERK‐dependent reduction in proximal tubule mitochondrial Ψm playing a regulatory role in the renal responses to MA.
Contribution of Hypoxia inducible factor‐1α to the profibrotic action of angiotensin II in cultured renal medullary interstitial cells
Authors: Wang Zhengchao , Li Pin‐Lan , Li Ningjun
Published: June 2021

Hypoxia inducible factor (HIF)‐1α has been reported to promote fibrogenesis during the progression of chronic renal diseases. Although angiotensin II (ANG II) was shown to increase HIF‐1α levels in various renal cells, the role of HIF‐1α in ANG II‐induced profibrotic effect has not been determined. The present study was designed to test the hypothesis that HIF‐1α mediates profibrotic effect of ANG II. In cultured renal medullary interstitial cells (RMICs), ANG II (10−6 M) treatment for 20 hours doubled HIF‐1α mRNA levels, which was accompanied by the upregulation of proliferating cell nuclear antigen (PCNA), collagen I/III and tissue inhibitor of metalloproteinases (TIMP)‐1 by 69%, 65% and 26%, respectively. HIF‐1α siRNA decreased HIF‐1α mRNA levels by 60% and completely blocked ANG II‐induced increases in PCNA, collagen and TIMP‐1. Since transdifferentiation is implicated in ANG II‐induced renal tubulointerstitial injury, we also detected the expression of vimentin, a marker of transdifferentiation. It was demonstrated that HIF‐1α siRNA also abolished ANG II‐induced elevation of vimentin mRNA. Our data suggest that HIF‐1α may mediate ANG II‐induced renal fibrotic injury through activation of cell transdifferentiation. (Supported by NIH grant DK54927 and HL89563)
Participation of central G‐alpha i2‐subunit proteins in blood pressure regulation and in countering salt‐sensitive hypertension
Authors: Wainford Richard David , Kapusta Daniel R
Published: June 2021

These studies tested the hypothesis that central Gai/o protein pathways are involved in mediating the cardiovascular depressor responses to central a2‐adrenoceptor activation and in countering the development of hypertension to a high NaCl challenge.

Methods

The a2‐agonist guanabenz (50 μg) was administered by intracerebroventricular (i.c.v.) injection to conscious instrumented Sprague‐Dawley (SD) rats pre‐treated (24‐h) i.c.v. with saline or oligodeoxynucleotides (ODN) against Gai1,i2, i3 and ao subunits (25 μg/each). Mean arterial pressure (MAP) and heart rate were recorded and urine collected for 150‐min (10‐min periods). Separate animals were implanted with miniosmotic pumps delivering i.c.v. Gai2 ODN (25 μg/day) and maintained on normal 0.4% NaCl (NS) or high 8% NaCl (HS) chow for 21‐days prior to study.

Results

In vehicle pretreated SD rats on NS, i.c.v. guanabenz elicited bradycardia, hypotension, diuresis and natriuresis. Selective central down‐regulation of Gai1, i2, i3 or Gao protein did not alter bradycardic or diuretic responses to i.c.v. guanabenz. Gai2 protein down‐regulation prevented the hypotension (peak δMAP [mmHg]; guanabenz, ‐19±2 vs. Gai2 ODN + guanabenz, ‐3±2, P<0.05) and natriuresis to guanabenz (peak δUNaV [ μeq/min]; guanabenz, 7.6±0.8 vs. Gai2 ODN + guanabenz, 2.2±1, P<0.05). When infused (21‐day) with i.c.v. Gai2 ODN, SD rats maintained on HS developed hypertension (MAP [mmHg]; NS + Gai2 ODN, 129±2 vs. HS + Gai2 ODN, 147±3, P<0.05).

Conclusion

Central Gαi2 protein‐gated pathways mediate the hypotensive and natriuretic responses to i.c.v. guanabenz. Further, these data suggest a previously unknown novel role for central Gαi2 protein pathways in preventing the development of salt‐sensitive hypertension. DK43337, HL 071212, AHA 0855293E, P20 RR018766
Cardiovascular and metabolic responses to chronic central infusion of leptin in rats fed a high fat diet
Authors: Dubinion John Harvey , daSilva Alexandre A. , doCarmo Jussara M. , Hall John E
Published: June 2021

Obesity has been reported to be associated with "resistance" to the metabolic effects of leptin. However, previous studies have tested only acute responses to leptin. The purpose of the current study was to test the hypothesis that a high fat (HF) diet causes resistance to the appetite and cardiovascular responses to chronic central leptin infusion. Sprague Dawley (SD) rats were fed a HF diet (40% kcal from fat, n=6) or a normal fat diet (NF, 13% kcal from fat, n=5) for 1 year starting at 3 weeks of age. Radiotelemeters were implanted for continuous monitoring of blood pressure (BP) and heart rate (HR). A 21G cannula was placed into the lateral cerebral ventricle (ICV). After recovery and 5 days of control measurements, leptin was infused ICV at 0.02 μg/kg/min for 10 days. Body weight in HF rats was 14% heavier than NF rats (581±17 vs. 510±17 g) and was associated with significantly higher BP (118±2 vs. 96±1 mmHg). Chronic ICV infusion of leptin decreased caloric intake in HF and NF rats similarly (51 ± 4% vs. 42 ± 1%) by day 5, remaining decreased throughout infusion. Despite decreased food intake and weight loss, BP and HR increased during the last 4 days of leptin infusion in both HF and NF rats (5.4 ± 0.6 vs. 5.0 ± 0.5 mmHg and 17 ± 3 vs. 17 ± 2 bpm). These results suggest this model of dietary‐induced obesity does not develop resistance to the chronic effects of central leptin administration on appetite or BP and HR regulation. (NHLBI PO1 HL51971).
Increased Collecting Duct Renin Protein Expression in Cyp1a1‐Ren2 Transgenic Rats with Inducible Ang II‐dependent Malignant Hypertension.
Authors: Prieto Minolfa C. , Botros Fady T. , Martin Victoria L , Davis Porcha D , Mitchell Kenneth D
Published: June 2021

In angiotensin II (AngII)‐dependent hypertension, renin is upregulated in collecting ducts (CD) despite suppression of plasma renin activity and renin in juxtaglomerular (JG) cells. The transgenic rat model with insertion of the mouse Ren2 gene [Cyp1a1‐Ren2 strain] exhibits renin expression only in the liver, enhancement of PRA, high circulating and intrarenal Ang II levels, and an inducible form of AngII‐dependent malignant hypertension by dietary administration of the aryl hydrocarbon, indole‐3‐carbinol (I3C). Although increased intrarenal AngII content in Cyp1a1‐Ren2 rats can be partially due to internalization via AT1R, de novo intrarenal Ang II formation is suspected in this model. This study determined the renin protein expression in the distal nephron segments as a potential pathway to explain the increased intrarenal AngII content in Cyp1a1‐Ren2 rats. Body weights decreased in hypertensive rats (248±0.2g) compared to controls (349±0.1g). Renin immunostaining was not suppressed in JG cells of Cyp1a1‐Ren2 rats (0.7±0.01 vs. 0.7±0.01DU). In contrast, CD renin immunoreactivity and renal medullary renin protein levels measured by Western blot were 3.8‐fold and 2.6‐fold higher in hypertensive rats than controls. Augmentation of CD renin may contribute to increase intrarenal Ang II content and, thereby, to the increased blood pressure in Cyp1a1‐Ren2 with AngII‐dependent malignant hypertension.
The Effects of Dietary Ascorbic Acid Treatment on Mesenteric Artery Reactivity in Pregnant Rats with Reduced Uterine Perfusion Pressure
Authors: Reho John J , Peck Jennifer , Novak Jackie , Ramirez Rolando J
Published: June 2021

Preeclampsia is a multifaceted disease of pregnancy mediated by poor placental perfusion. The symptoms of the disease include hypertension, hyperuricemia, and proteinuria. Pregnant rats with reduced uteroplacental perfusion (RUPP) have been used as an animal model of preeclampsia. In these animals plasma ascorbate levels are decreased along with increased vascular reactivity similar to what is seen in patients with preeclampsia. Therefore, the aim of this study was to determine the effects of dietary ascorbic acid (AA) treatment to animals with RUPP and to see if detrimental changes in mesenteric vascular reactivity seen in RUPP rats can be ameliorated. Sprague Dawley rats were subjected to RUPP or Sham surgery on Day 14 of gestation. Animals were then given regular water or ascorbic acid water (1 mg/mL) ad libitum post surgery. At gestational Day 21, resistance‐sized mesenteric arteries were harvested and mounted in a pressurized arteriograph. Myogenic reactivity was significantly decreased in AA treated RUPP animals compared to RUPP (p<0.05). AA also reversed RUPP induced decreases in methacholine relaxation seen in RUPP (p<0.05). These results suggest that antioxidant treatment can partially reverse the increased myogenic reactivity and decreased endothelium‐dependent relaxation seen in animals with reductions in uterine perfusion and may provide new insights into the treatment of human preeclampsia.
Autonomic cardiovascular responses to chronic infusions of angiotensin II (ANGII) in wistar kyoto rats (WKY)
Authors: Toward Marie Ann , Kasparov Sergey , Paton Julian F.R.
Published: June 2021

We hypothesized that the hypertension developed during chronic infusion of ANGII results from elevated sympathetic tone and reduced baroreflex sensitivity. Arterial pressure (AP) and heart rate (HR) were recorded via radio telemetry in WKY rats (10‐11 weeks old) chronically infused with ANGII (50, 200 & 800 ng·kg−1·min−1 s.c. for 10 days). Mean AP increased rapidly in the 800 ng group (91 to 151 mmHg by day 8, p<0.05, n=12; peak response), more gradually in the 200 ng group (88 to 117 mmHg by day 10, p<0.001, n=12) and slowly in the 50 ng group (98 to 104 mmHg by day 10, p<0.05, n=12). HR declined initially, apparently due to the baroreflex, in all 3 groups with magnitude and time scale being dose‐dependent. Increases in the very low frequency spectra of AP in both 200 and 800 ng·kg−1·min−1 groups occurred by day 8 suggesting increased sympathetic tone. The spontaneous baroreflex gain (sBRG) showed time and dose dependent changes: the 800 ng group showed an increase peaking on day 2 before declining by day 8 whereas the 200 ng group showed a delayed and blunted elevation in sBRG peaking on day 7 before declining. Thus, peripheral ANGII alters cardiovascular autonomic function which is delayed relative to the onset of hypertension. We propose that ANGII infusion in normotensive rats is a good model for understanding mechanisms of autonomic dysfunction underpinning the development of hypertension. Supported by BBSRC, Pfizer & BHF.
Revealing the role of the autonomic nervous system in the development and maintenance of Goldblatt hypertension in conscious rats
Authors: Oliveira‐Sales Elizabeth B , Toward Marie Ann , Campos Ruy Ribeiro , Paton Julian FR
Published: June 2021

Despite the extensive use of the Goldblatt model of hypertension (two kidney one clip, 2K‐1C) autonomic nervous mechanisms responsible for both the development and maintenance of the hypertension remain unclear. The objective of this study was to analyse cardiovascular autonomic function by power spectral density analysis of arterial pressure and pulse interval measured by radio telemetry for 6 weeks after renal artery clipping. Mean arterial pressure increased from 106±5 to 185±2 mmHg five weeks post clipping (n=6; P<0.05) remaining stable during the 6th week. A tachycardia developed during the 4th week from 406±7 bpm which plateaued by weeks 5 and 6 at 468±10 bpm. The gain of the parasympathetic component of the cardiac baroreflex decreased gradually until the 5th week (from ‐2.4±0.09 to ‐0.8±0.04 bpm/mmHg) when it plateaued, and high frequency power spectral density of the pulse interval decreased from 13.4±0.6 to 8.3±0.01 ms2. In addition, there was an increase in both the low frequency and very low frequency components of the spectra of systolic blood pressure that did not occur until 3 weeks after clipping. The results indicate that 2K‐1C hypertension is associated with: (i) an immediate alteration in cardiac autonomic control characterized by a sympathetic predominance and decreased baroreflex sensitivity; (ii) elevated sympathetic vasomotor drive occurring 3 weeks post clipping that persists into the 6th week. Supported by CAPES.