bluuebunny/crossref_metadata_2025 huggingface dataset:
Epoxyeicosatrienoic Acid‐Induced Pulmonary Vasoconstriction is Mediated by Prostacyclin‐Induced Thromboxane‐Receptor Activation
Authors: Procknow Jesse D , Le Duy , Sprague Randy S , Stephenson Alan H
Published: June 2021

Epoxyeicosatrienoic acids (EETs) were reported to contribute to the increased pulmonary artery (PA) pressure of hypoxia‐induced pulmonary hypertension. EET‐induced PA contraction requires vascular endothelium, cyclooxygenase‐1 (COX‐1) activity and activation of the thromboxane receptor (TP) but not thromboxane synthesis. Here, we show that prostacyclin (PGI2) is a potential mediator of EET‐induced PA contraction. Incubation of rabbit intralobar PA rings with 5,6‐EET at its EC50 for PA contraction (0.7 μM) increased PGI2 synthesis by 2.5 ± 0.2 fold over baseline in an endothelium‐dependent manner. In isolated PA rings, incubation with SC‐560, a selective COX‐1 inhibitor, reduced 5,6‐EET‐induced PA contraction and PGI2 synthesis > 95% (P<0.01). PGI2, but not 6‐keto‐PGF1α, its breakdown product, contracted PA rings in a concentration‐dependent manner, but unlike 5,6‐EET, PGI2‐induced contraction was independent of COX‐1 and endothelium. SQ 29,548 (1 μM), a selective TP receptor antagonist, reduced PGI2‐induced PA contractions > 99% (P<0.01). PGI2‐induced PA contraction was not inhibited by CAY10441 (10 μM), a selective inhibitor of the PGI2 (IP) receptor. These results are consistent with the hypothesis that PGI2 synthesized in the vascular endothelium mediates EET‐induced contraction of intralobar PA via TP‐receptor activation. Support: NIH HL64180, HL089094.
Parasympathetic Nerves Provide Tonic Vasodilator Influences to Cerebral Blood Vessels
Authors: Talman William T. , Dragon Deidre Nitschke , Boysen Nick
Published: June 2021

Stimulation of parasympathetic nerves from the pterygopalatine ganglia (PPG) to cerebral blood vessels leads to vasodilatation. Transection of the nerves reduces vasodilatation with acute hypertension, but the physiological role of the nerves under basal conditions is not known. We hypothesized that they mediate tonic cerebral vasodilatation and tested that hypothesis in freely breathing anesthetized rats instrumented for recording cerebral blood flow (CBF) with laser flowmetry and arterial blood pressure through an arterial cannula. Ipsilateral to the flow probe we exposed the PPG; and, after recording basal arterial pressure, arterial pCO2, and CBF, we quickly resected the PPG. Manipulating the PPG led to increased CBF, but removal of the PPG caused a gradual 28% decrease in cerebral blood flow (decrease of 13.5 ± 6.5 laser units) without changing mean arterial pressure. Cerebrovascular resistance increased after removal of the ganglion from 1.8 ± 0.3 units to 2.9 ± 0.5 units (p<0.01). After removal of the PPG, arterial pCO2 increased from 26.7 ± 3.2 Torr to 40.1 ± 3.7 Torr (p<0.01). Therefore, removal of parasympathetic input to cerebral blood vessels causes cerebral vasoconstriction. The study supports a tonic vasodilatory input to cerebral vessels from parasympathetic nerves. With hypercarbia the tonic effect is underestimated in these studies. Support: VA Merit Review and NIH R01 HL59593.
Receptor activity modifying protein‐1 (RAMP1) overexpression selectively enhances calcitonin gene‐related peptide‐induced vasodilation
Authors: Chrissobolis Sophocles , Zhang Zhongming , Lynch Cynthia M , Kinzenbaw Dale A , Russo Andrew F , Faraci Frank M
Published: June 2021

Calcitonin gene‐related peptide (CGRP), a potent vasodilator, is released during migraine and mediates vasodilation in response to trigeminal nerve activation. CGRP receptors consist of a calcitonin‐like receptor (CLR) and a receptor activity modifying protein‐1 (RAMP1). The aim of this study was to test whether RAMP1 overexpression enhances vasodilation to CGRP. Transgenic mice overexpressing human RAMP1 in most tissues, including blood vessels, were generated using a RAMP1 transgene under control of the widely expressed adenoviral EIIa promoter. In carotid artery in vitro, maximum relaxation to CGRP was increased ∼ 2‐fold in transgenic vs littermate controls, an effect that was inhibited by the CGRP receptor antagonist, CGRP (8–37) (P<0.05). Responses to adrenomedullin (which activates CLR coupled with RAMP2) were similar in both groups (P>0.05). In cerebral circulation, responses to CGRP were selectively increased ∼ 2‐fold in cerebral arterioles in vivo, and ∼ 70% in the basilar artery in vitro in transgenic vs control mice (P<0.05). These data indicate that RAMP1 overexpression selectively enhances vasodilation to CGRP, suggesting responses to CGRP are normally RAMP1‐limited. RAMP1 may represent a therapeutic target to inhibit increased responses to CGRP during migraine, or increase vasodilator responses and CGRP‐mediated signaling in conditions such as ischemia or vasospasm.
Hyaluronidase treatment of the endothelial glycocalyx impairs adenosine mediated recruitment of coronary volume
Authors: Brands Judith , Spaan Jos , Post Mark , Vink Hans , Van Teeffelen Jurgen
Published: June 2021

It is hypothesized that coronary hyperemia by adenosine is accompanied by increased access for blood to the endothelial glycocalyx (EG) (VanTeeffelen, 2005). In the present study, the effect of glycocalyx degradation by hyaluronidase on adenosine mediated volume recruitment was assessed in goats. Labelled red blood cells (RBC) were infused as a bolus in the left main coronary artery in open‐chest anesthetized goats using a perfusion system. Blood samples were collected from the great cardiac vein every 0.5–1 second. Coronary red blood cell volume (Vcor) was calculated by multiplying mean RBC transit time with simultaneously measured coronary RBC flow. This measurement was done in control (con) and during adenosine (ado), before as well as after hyaluronidase treatment. Adenosine‐induced coronary volume recruitment (Vcor,ado/Vcor,con) and flow reserve (Qado/Qcon) were calculated; in control Vcor,ado/Vcor,con was 2.14 ± 0.7 and Qado/Qcon was 2.25 ± 0.8. After hyaluronidase Vcor,ado/Vcor,con reduced to 1.87 ± 0.8 (P<0.05), while Qado/Qcon was not affected (2.22 ± 0.8, N.S. compared to before hyaluronidase). These data demonstrate that enzymatic degradation of the coronary EG impairs the ability of adenosine to recruit coronary vascular volume. It is concluded that recruitment of EG volume contributes to optimal matching of hyperemic coronary flow and volume. Supported by NHF grants 2005T037 and 2003B181.
An Integrated Mathematical and Experimental Model for Nitric Oxide uptake by Red Blood Cells
Authors: Deonikar Prabhakar , Kavdia Mahendra
Published: June 2021

Endothelium‐derived nitric oxide (NO) is a potent vasodilator despite the presence of hemoglobin (Hb) encapsulated in red blood cells (RBC) which has high affinity for scavenging NO (k~ 107 M−1 s−1). NO uptake by RBC‐Hb is reported to be several orders lesser than that of free Hb. Reduced uptake by RBC encapsulated Hb is attributed to several diffusional resistances for NO to react with Hb including extracellular resistance, cell free layer resistance near the vessel wall, resistance in unstirred layer surrounding RBCs and resistance by RBC membrane. The extent of contribution from each diffusional layer towards reduced NO uptake by RBCs is under intense investigation. We developed a novel stirred bioreactor with a headspace to maintain controlled NO concentrations. Using this bioreactor along with an electrochemical NO sensor, we measured the bulk NO concentration in RBC suspensions DI water for various NO concentrations in the bioreactor headspace. We developed a steady state one dimensional mathematical model representing NO biotransport using the data obtained from above experiments to predict NO concentration profile and to determine the role of extracellular diffusional resistance, resistance offered by unstirred layer surrounding RBCs and resistance offered by RBC membrane. This integrated analysis will provide quantitative information about NO_RBC interaction parameters.
Pulsatile laminar flow keeps the vascular endothelium in a quiescent state via the regulation of a Kruppel‐Like Factor 2 pathway
Authors: Young Angela Chien‐hsin , Li Yi‐Shuan Julie , Shyy John J , Chien Shu
Published: June 2021

Vascular endothelial cells (ECs) along the straight part of the arterial tree are generally spared from atherosclerosis by maintaining a quiescent phenotype, thus being protected from EC activation and leukocyte adhesion. The objective of this study is to elucidate the role of Krüppel‐Like Factor 2 (KLF2), a transcription factor that inhibits cell proliferation, in the athero‐protective action of pulsatile laminar flow (PS), which has high shear stress and a large net forward direction that mimics the athero‐protective flow pattern in the straight vessels. We have previously shown that PS inhibits EC proliferation and can induce KLF2. In the current study, using BrdU incorporation and immunostaining, we showed that 24‐hr PS caused a significant reduction of cell proliferation rate to 2% in comparison to 20% in static cells. Knocking down KLF2 (to ∼50%) with siRNA reversed the PS‐reduction of cell proliferation rate up to 11%. These results indicate that the anti‐proliferative action of PS is mediated by KLF2. To elucidate the signaling upstream to KLF2, we found that blocking AMP‐activated protein kinase (AMPK) prevented PS‐induced KLF2 expression. Thus, PS causes an activation of KLF2 through AMPK. These results provide a mechanistic explanation for the athero‐protective action of PS flow via the AMPK‐KLF2 pathway.

(This work is supported by Grant HL064382 and UC San Diego Cardiovascular Scholarship Award)
Angiotensin II increases GABA B receptor expression and activity in the nucleus tractus solitarious of rats
Authors: Yao Fanrong , Zhang Qi , O’Rouke Stephen T. , Sun Chengwen
Published: June 2021

Our previous studies have demonstrated that chronic Ang II treatment increases GABA‐aminobutyric acid (GABA) type B receptor (GBR) expression in neuronal cultures from the nucleus tractus solitarius (NTS). In the present study, we examined the chronic effect of Ang II on GBR expression and GBR activity in the NTS of Sprague Dawley rats. Ang II intracerebroventrical infusion (15 mg/kg/day, 2 weeks), using minipumps, resulted in a significant increase in arterial blood pressure (BP), measured by telemetry (MAP from 104 ± 2 mmHg to 132 ± 4 mmHg). The mRNA levels of GBR in NTS were measured with real‐time PCR demonstrating that Ang II‐infusion induced an 88% increase in the GBR1 mRNA levels. However, GBR2 mRNA levels in the NTS were identical between saline‐infused rats and Ang II‐infused rats. To examine whether GBR activity in NTS is also increased by Ang II‐infusion, we examined the BP response to NTS microinjection of GBR agonist, baclofen, in Ang II‐ or saline‐infusion rats. NTS microinjection of baclofen (50 pmol, 0.1 μl) increased arterial BP in both groups of rats. However, the pressor response to baclofen was enhanced in Ang II‐infusion rat comparing with saline‐infusion rats (31 ± 2 mmHg in Ang II‐infusion rats and 19 ± 2 mmHg in saline‐infusion rats). These experiments indicate that Ang II increases GBR expression and consequently enhances the GABAergic neurotransmission in the NTS. These alterations may lead to dampening of baroreflexes and consequently result in resetting of long‐term blood pressure to a higher level in Ang II‐related hypertension.
The time course of remodeling in rapidly stimulated atrial myocytes: Early electrophysiologic and molecular determinants
Authors: Mace Lisa C , Yermalitskaya Liudmila V , Yang Zhenjiang , Murray Katherine T
Published: June 2021

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia. The arrhythmia is associated with electrical (reduced action potential duration [APD] and ICa) and structural remodeling that increases future AF susceptibility. The time course of the molecular events that initiate this remodeling are not currently known, and is the focus of this investigation using rapidly stimulated atrial myocytes. In rapidly stimulated isolated canine atrial strips, APD90 was reduced within 1.5hr (APD90, ?24.3%, P(0.05) indicative of remodeling. This time course was confirmed in atrial myocytes (HL‐1 cells) subjected to rapid stimulation in culture (APD90, ?36.1%, P(0.01), with an associated reduction in ICa‐L and ICa‐T. Real time quantitative RT‐PCR results revealed no significant change in Ca2+ channel (‐subunit mRNA at this time point, indicating that proteolysis was most likely responsible for reduced ICa. The time course of transcriptional remodeling was variable, with some genes regulated very early (0–3hr: Hspa1a, Aqp4 & Prkci), others gradually over 18hr (Map2k3 & Gucy1a3), and others much later (18hr: Nppa, Nppb & Tgfb1). Thus, the remodeling produced by rapid stimulation of atrial myocytes is complex. Electrical remodeling is achieved within 90 min, while the time course of transcriptional remodeling is highly variable and likely pathway‐specific.
Shp2 Negatively Regulates Growth in Cardiac Myocytes by Controlling Focal Adhesion Kinase and the mTOR/S6 Kinase Pathways
Authors: Marin Talita M. , Clemente Carolina F.M.Z. , Picardi Paty K. , Pascoal Vinicius D.B. , Lopes‐Cendes Iscia T. , Saad Mario J.A. , Franchini Kleber G.
Published: June 2021

Aim

Focal adhesion kinase (FAK) has been assigned a role in the cardiomyocyte hypertrophy. Herein, we investigated whether the Src homology 2 domain‐containing phosphotyrosine phosphatase (Shp2) control FAK activity and its effect on hypertrophy of cardiomyocytes.

Methods

Neonatal rat ventricular myocytes (NRVMs) were stretched in silicone plates (1Hz, 10%). Phosphatase activity was assessed in the anti‐Shp2 immune complex and in samples of Shp2‐PTP domain. FAK/Shp2 association was detected by co‐immunoprecipitation. Shp2 was depleted with siRNA. Hypertrophy of NRVMs was assessed by morphometry and the expression of β‐myosin heavy chain (β‐MHC) transcripts with RT‐PCR.

Results

FAK was most non‐phosphorylated in non‐stretched NRVMS, coinciding with relatively high basal association with Shp2 and Shp2 tyrosine phosphatase activity. Cyclic stretch enhanced FAK phosphorylation at Tyr397 (2.3‐fold) and reduced its association with Shp2 (~85%) and the phosphatase activity of the anti‐Shp2 immune complex (~40%). Shp2‐PTP domain was shown to dephosphorylate FAK immunoprecipitated from NRVMs. Depletion of Shp2 by siRNA enhanced FAK phosphorylation in non‐stretched NRVMs (2.1‐fold). Depletion of Shp2 with siRNA increased ‐MHC transcript and induced hypertrophic morphology (3‐fold) in non‐stretched NRVMs. These effects were accompanied by increased p70S6 kinase phosphorylation at Thr389. Treatment with the FAK/Src pharmacological inhibitor PP2 or rapamycin abolished the hypertrophy induced by Shp2 silencing.

Conclusion

These data indicate that Shp2 controls basal FAK activity in cardiac myocytes, acting as a counter‐regulatory mechanism to antagonize hypertrophy induced by FAK and mTOR/S6K pathways.
Roles of ARF1 and ARF6 in receptor mediated endocytosis of metal‐binding proteins and cadmium‐metallothionein‐1 (CdMT‐1) toxicity in kidney proximal tubule (PT) cells
Authors: Lee Wing‐Kee , Wolff Natascha A. , Abouhamed Marouan , Verroust Pierre J. , Thevenod Frank
Published: June 2021

Filtered metal‐binding proteins, such as transferrin (Tf) or metallothionein‐1 (MT‐1), are apically endocytosed partly via megalin/cubulin in kidney PT where CdMT‐1 internalization causes apoptosis. Small GTPase ARF (ADP‐ribosylation factor) proteins regulate vesicular trafficking. We investigated roles of ARF1 and ARF6, which have been involved in secretory and endocytic pathways, respectively, following Tf, MT‐1 and CdMT‐1 uptake by PT cells. WKPT‐0293 Cl.2 cells derived from rat PT S1‐segment were transfected with hemagglutinin‐tagged wildtype (ARF‐WT) or dominant‐negative (ARF‐DN; ARF1‐T31N, ARF6‐T27N) forms of ARFs. Endogenous ARF1 co‐localized with Rab7 (late endo‐/lysosomal) whereas ARF6 with Rab5a and Rab11 (early and recycling endosomes). Live immunofluorescence staining of megalin showed less surface expression in ARF6‐T27N cells, but not in ARF1‐T31N. Fluorescent Tf mainly accumulated perinuclearly in late endo‐/lysosomes in ARF‐WT but was decreased and/or remained near the plasma membrane in ARF‐DN cells. Intracellular MT‐1 was also reduced in ARF‐DN cells. Moreover, CdMT‐1 toxicity was significantly attenuated in ARF1‐T31N (by 66.1 ± 16.6%, n = 10) or ARF6‐T27N (by 51.0 ± 12.0%, n = 9) cells, compared to respective ARF‐WT. The data support roles for ARF1 and ARF6 in endocytosis and trafficking of Tf/MT‐1 to endo‐/lysosomes and CdMT‐1 toxicity of PT cells.

Funding: DFG TH 345/8‐1
Modulation of Proximal Tubule Sodium‐Proton Exchanger NHE3 by the Glucagon‐Like Peptide 1 Analog Exendin‐4
Authors: Carraro‐Lacroix Luciene Regina , Girardi Adriana Castello Costa
Published: June 2021

The gut incretin hormone glucagon‐like peptide 1 (GLP‐1) is released in response to ingested nutrients and enhances insulin secretion. In addition to its insulinotropic properties, GLP‐1 has been shown to have natriuretic actions paralleled by a diminished proton secretion in humans and animal models. We therefore studied whether exedin‐4, a GLP‐1 analog, affects the activity of the Na/H exchanger NHE3 in the LLC‐PK1 cell line derived from kidney proximal tubule. Expression of GLP‐1 receptor in LLC‐PK1 cells was confirmed at the mRNA level by RT‐PCR. NHE3 transport activity was measured as intracellular pH (pHi) recovery from an acid load using BCECF. The Na‐dependent pHi recovery decreased from 0.43 ± 0.07 (n = 7) to 0.19 ± 0.04 (n = 10) after 30 minutes pretreatment with 500 pM exendin‐4. Reduction of Na/H exchange was accompanied by a significant increase (65 ± 14%) in the phosphorylation of endogenous NHE3. Conversely, inhibition of NHE3 took place without changes in the surface expression of the transporter, as determined by cell surface biotinylation. We conclude that GLP‐1 modulates sodium homeostasis in the kidney, most likely by affecting NHE3 activity.
TRPV5‐promotor driven expression of EGFP produces fluorescence in late DCT, CNT and early CCD
Authors: Praetorius Jeppe , Andrew Fenton Robert , Füechtbauer Ernst‐Martin , Vind Hofmeister Marlene
Published: June 2021

TRPV5 (ECaC) is an apical Ca2+ channel of renal late distal convoluted tubules and connecting tubules (DCT2 and CNT, respectively), and is critically involved in regulated Ca2+ reabsorption. The protein was previously localized to CNT by immunohistochemistry and RT‐PCR analysis. Genetic disruption of TRPV5 leads to hypercalciuria.

Transgenic mice were developed that express EGFP driven by the TRPV5 gene promoter 1) to verify and expand the expression‐profile of TRPV5 and 2) to generate a cell line of the renal tubules expressing this protein as a tool for further studies of TRPV5 biology. TRPV5 promoter‐driven EGFP fluorescence was detected in late DCT2 and CNT in isolated living tubules by confocal microscopy. In addition, EGFP was expressed in some cortical collecting ducts (CCD). Robust EGFP signal was confirmed in sections of paraffin embedded kidney, and the expression of EGFP in both late DCT2/CNT and CCD was verified by double‐fluorescence immunolabeling with various tubule markers. In preliminary analysis, TRPV5 protein expression in DCT2/CNT as well as in CCD using anti‐TRPV5 antibodies coincided with the expression of TRPV5 promoter‐driven EGFP.

Thus, TRPV5 is expressed in DCT2/CNT and CCD, and an EGFP‐expressing transgenic mouse has been generated enabling easy isolation of TRPV5‐expressing Ca2+‐transporting renal tubules. Supported by the Danish National Research Foundation.
Sodium appetite and cocaine sensitization share similar mechanisms
Authors: Acerbo Martin Javier , Johnson Alan Kim
Published: June 2021

Previous studies indicate that the augmented salt appetite induced by repeated sodium depletions may be related to psychostimulant‐induced sensitization. We further investigated this hypothesis by treating the same rats with DOCA to induce salt appetite and with cocaine to induce psychomotor sensitization. In Exp. 1, two groups of rats had either access to sodium (DS) or no access (DnS) and received daily injections of DOCA for 8 days. A third group with sodium access received vehicle (V) injections. All groups had access to distilled water. After a week off, rats were injected with cocaine and placed into cages to monitor activity. The group DnS displayed increased locomotion and showed an increased sensitivity to cocaine compared to V and DS groups. In Exp. 2, rats received daily injections of either cocaine (Coc) or saline (Sal) for 7 days. The Coc and Sal groups were then subdivided into 3 subgroups (DS, DnS, and V) and given DOCA treatment for 7 days. Finally, all rats were given access to sodium. DS rats with Coc history showed increased sodium appetite compared to rats with Sal history, whereas DnS rats with Coc or Sal history did not differ. In summary, DOCA‐induced salt appetite and cocaine‐induced psychomotor responses cross‐sensitized with one another suggesting that sodium appetite induced by DOCA and cocaine‐induced psychomotor sensitization share similar mechanisms.

AHA 0625661Z; NIH DK66086 & HL14388
Pharmacologic Vasopressin Levels May Protect Renal Function in a Porcine (Sus Scrofa) Model of Vasodilatory Septic Shock
Authors: Holt Danielle Boudreau , Delaney Richard R , Hashiro Glenn M , Sato Aileen K , Hernandez Claudia A , Uyehara Catherine F T
Published: June 2021

Acute renal failure commonly occurs with severe sepsis and is associated with high mortality. Pharmacologic administration of vasopressin (VP) increases urine output in septic shock. To elucidate the mechanism by which pharmacologic VP induces diuresis, VP was administered at a low dose of 10 and a high dose of 100 ng/kg/min to piglets with endotoxin‐induced septic shock. VP‐100 produced a 4‐fold increase in urine flow (0.05 ± 0.02 vs. 0.23 ± 0.04 ml/kg/min), creatinine clearance, and filtration fraction compared to VP‐10 while the mean arterial pressure (MAP), delta MAP, and renal blood flow were similar between the groups. High dose VP demonstrated increased osmolar clearance (0.07 ± 0.05 vs. 0.21 ± 0.13 ml/kg/min), Na excretion, and fractional excretion (FE) of sodium (2.04 ± 0.83 vs. 4.51 ± 0.57) despite an increase in aldosterone and no change in renin levels. There was no change in the FE of urea or potassium between groups. The production of dilute urine was evidenced by increased free water clearance (−0.02 ± 0.01 to 0.03 ± 0.02 ml/kg/min), decreased urine osmolality (415 ± 21 vs. 282 ± 22 mOsm/kg H2O), and decreased urine protein (5.3 ± 0.87 vs. 1.9 ± 0.54 mg/ml) observed in the high but not low dose of VP. Thus at high dose VP, the increase in the filtration fraction and the FENa which lead to an increased excretion of dilute urine suggest a tubular osmotic mechanism that may be renal protective in septic shock.
Ion channels profile expression in vestibular endorgans of patients with Meniere's disease and acoustic neuroma.
Authors: Beltran‐Parrazal Luis , Lopez Ivan Axel , Ishiyama Gail , Ishiyama Akira
Published: June 2021

The regulation of ion concentration in the endolymph (K+, Na+, Ca2+ and pH homeostasis) and water permeability are critical for normal function of the vestibular endorgans (VE). Ion channels, transporters, cotransporters and aquaporins are involved in the homeostatic ionic concentration of endolymph. The gene expression profile of these molecules was investigated in utricular macula obtained in surgery for acoustic neuroma (AN) and compared with utricular macula obtained from patients with Meniere's disease (MD). We found in MD patients an up‐regulation of Na(+)/K(+)‐ATPase cotransporter, voltage gated Na+, neuronal Ca++, and K+ neuronal channels. In contrast, we found a down‐regulation of Cl− channels, vesicular monoamine transporters and neurotransmitter transporters to GABA, dopamine, serotonin and glycine. Finally, no significant changes in expression of aquaporins 1, 4 or 6 were detected in utricles form MD patients compared with the AN patients.
Proteomic validation: Searching for a heart of gold
Authors: Parsons Hannah L , Kulpa Jerzy , Brownsey Roger W. , Wambolt Richard W , Allard Michael F.
Published: June 2021

Recent developments in proteomics techniques allow sensitive identification and relative quantitation of proteins in tissues and with increased sensitivity come the ability to detect small changes in protein expression. However, the means by which changes are validated remains incompletely defined. The proteome of mitochondria of hypertrophied hearts from rats with an abdominal aortic constriction (H) was quantitated using amine‐reactive isobaric tagging reagents (iTRAQ®) and tandem mass spectrometry. A small number (15 out of 250) were significantly increased and none decreased significantly. Increases ranged from 10 to 20%, as exemplified by voltage‐dependent anion channel‐1 (VDAC1), with only monoamine oxidase‐A (MAO‐A) showing a substantially greater increase (see table). Traditional immunoblot analysis revealed the significant increase in MAO‐A but not that of VDAC1. The discrepancy in results highlights the relative insensitivity of traditional immunoblot analysis and indicates that more sensitive approaches are essential.

Supported by a grant from the Canadian Institutes for Health Research.
Long term exercise training exacerbates sarcopenia and only modestly attenuates apopotosis
Authors: Rowan Sharon , Hepple Russell T
Published: June 2021

Apoptosis has been implicated in the age‐related loss of muscle known as sarcopenia. In addition, exercise training attenuates some markers of apoptosis. The purpose of this study was to evaluate the impact of a treadmill exercise training program initiated in late middle aged (LMA) rats and continued for 7 mo until senescence (SEN) (a period that spans a dramatic acceleration of muscle mass and functional decline) on gastrocnemius muscle (Gas) mass and markers of apoptosis. 62 male rats were randomly assigned to a training group (T) or sedentary group (S). Contrary to our expectation, following the 7 mo period the Gas mass was lower in T (890 ±60 mg) than C (1143 ± 85 mg; P<0.05). In situ immunolabeling experiments revealed that despite the fact that the loss of fast twitch fibers was markedly lower in T with aging, there was no difference between T and C in the fraction of nuclei that were TUNEL positive (3.0±0.5% vs 2.4±0.4%), or in the number of 8‐hydroxy guanosine positive spots per 100 fibers (1.3±0.3 vs 2.3±0.7). On the other hand, the fraction of fibers positive for activated caspase 3 was less in T (1.0±0.6%) vs C (2.3±0.5%; P<0.05). These results show that exercise training, although protecting fast twitch fibers with aging, had very modest impact on markers of apoptosis activation in Gas muscle of SEN rats.

Supported by CIHR & AHFMR.
Apoptosis does not increase with age in rat extraocular muscle
Authors: McMullen Colleen A , Andrade Francisco H , Dupont‐Versteegden Esther E
Published: June 2021

Sarcopenia decreases quality of life in the elderly. Changes in the structure and function of aging non‐locomotor muscles remain understudied, despite their importance for daily living. The fast and constant activity of the extraocular muscles (EOMs) imposes extreme mechanical and metabolic stresses, and may render EOMs more sensitive to aging. For example, EOMs have a higher incidence of age‐related mitochondrial defects that could result in apoptosis. Apoptosis seems to contribute to sarcopenia in hind limb muscles. Therefore, we hypothesized that apoptosis increases with age in the EOMs. Muscles from 6‐, 18‐, and 30‐mo old male Fisher 344‐Brown Norway rats were used to estimate mean fiber size, to demonstrate apoptosis by TUNEL and ELISA, and to measure caspase‐3, ‐8, ‐9, and ‐12 activity. EOM fiber area decreased 24% from 6 to 30‐mo of age (481±309 to 367±176 sq. μm respectively). TUNEL positive nuclei increased from 6‐mo to 18‐ and 30‐mo (29.41+ 10.95, 40.79+10.68, and 41.28+10.42 percent of fibers to TUNEL positive nuclei respectively). However, mono‐ and oligonucleosomal content was not elevated with age, indicating that TUNEL may have measured DNA damage. Caspase‐3, ‐8, ‐9, and ‐12 activity was not affected by aging. Based on these data, we conclude that in contrast to hind limb muscles, apoptosis does not increase with age in EOM. Other mechanisms must play a role in the age‐related decline in EOM fiber size.
Relationship between aerobic capacity of African American males, serum concentration of insulin‐like growth factor‐1 and growth of prostate cancer cells
Authors: Bond Vernon , Sridhar Rajagoplan , Kassa Alemayehu , Zhang Renshu , Millis Richard
Published: June 2021

Prostate cancer affects blacks disproportionately and its evolution may be affected by a sedentary lifestyle. The growth of prostate tumors may be modulated by androgens and insulin‐like growth factors. This study tested the hypothesis that the serum of males having a low aerobic capacity contains concentrations of substances which promote the growth of prostate cancer cells. Eight low fit (peak oxygen uptake 38< ml · kg−1· min−1) and 9 high fit (peak oxygen uptake >40 ml · kg−1· min−1) males severed as subjects. The serum concentrations of testosterone, insulin‐like growth factor‐1 (IGF‐1) and the relative cell growth of the LNCaP tumor cells were measured. The significance of intergroup differences was evaluated by a t‐test at P<0.05. IGF‐1 concentration was higher in the serum from low fitness group compared to that from the high fitness group. The relative growth of the LNCaP prostate cancer cells was significantly higher in the serum from individuals in the low fitness group than in the high fitness group. There was no significant intergroup difference in serum testosterone level. It is concluded that a lower growth rate of prostate cancer cells may be associated with a lower serum concentration of IGF‐1 in physically active males with a higher aerobic capacity than in sedentary males with a lower aerobic capacity. It is concluded that a sedentary lifestyle may promote the growth of prostate cancers.
Phosphorylation of heat shock factor‐1 by ERK in LPS stimulated RAW cells
Authors: Gupta Aditi , Maity Tapan , Wang San‐Xia , He Ju‐Ren , Hasday Jeffrey D , Singh Ishwar S
Published: June 2021

Heat shock factor‐1 (HSF1) is the predominant stress‐activated transcription factor that undergoes stress‐induced trimerization, phosphorylation and nuclear translocation to regulate transcription. Although characterized as an activator of heat shock protein genes, we found that HSF1 could repress LPS‐induced TNF alpha transcription. LPS also modified HSF1 protein and altered its migration pattern as observed by 2‐D gel electrophoresis. Since LPS is known to activate MAP kinase cascades, we speculated that LPS stimulation causes HSF1 phosphorylation through a MAP kinase dependent mechanism. Using the murine macrophage RAW 264.7 cells we found that LPS stimulation caused a marked decrease in the mobility of HSF‐1 protein as observed on a 7.5% SDS‐gel. This LPS‐induced reduced mobility of HSF1 was abrogated in RAW cells pretreated with MAP kinase inhibitors, or by in vitro phosphatase treatment of cell extracts. Furthermore, co‐immunoprecipitation studies indicated that ERK1/2 physically interacted with HSF1 in RAW cells. However, LPS stimulation did not appear to cause nuclear translocation or trimerization of HSF1 or enhance DNA‐binding activity of HSF1. Our results indicate that LPS induces HSF1 phosphorylation through a MAP kinase dependent pathway and modifies its protein:protein interaction. [Supported by NIH grants GM069431 (ISS), GM066855 and HL69057 (JDH) and VA Merit Review to ISS and JDH].
Amylin excites a subset of proopiomelanocortin (POMC) neurons in the arcuate nucleus (ARC) via enhancing the excitatory glutamatergic synaptic inputs
Authors: Gu Yan , Fan Wei
Published: June 2021

Amylin, released from pancreas in response to nutrient ingestion, exerts anorexic effects. The ARC POMC neurons are essential for energy homeostasis by integrating a variety of peripheral and central signals that regulate energy balance. Amylin binding sites exist in the ARC. We examined the action of amylin on the ARC POMC neurons in hypothalamic slices of POMC‐EGFP transgenic mice. Bath application of amylin (1‐1000 nM) dose‐dependently increased the firing rate in 36 out of 55 POMC cells tested under loose‐patch extracellular recording. In the presence of kynurenic acid and picrotoxin to block synaptic transmission, amylin did not significantly change the firing rate of ARC POMC neurons, suggestive of an underlying presynaptic mechanism. Under voltage clamp recording, bath application of amylin (200 nM) increased the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) in 7 out of 17 POMC cells, and increased the amplitude of sEPSCs in 12 out of 17 POMC cells, while increased the frequency of miniature EPSCs (mEPSCs) but decreased the amplitude of mEPSCs in 6 out of 11 POMC cells tested, respectively. These data suggest that amylin excites a subset of ARC POMC neurons via enhancing the excitatory glutamatergic synaptic inputs. (We thank Dr. Malcolm J Low for providing POMC transgenic mice and Jonathan G Murphy for help with genotyping animals, Supported by NIH grant DK62179 and CSWR foundation.)
The expression of the glutamate transporter EAAC1 is stimulated by all‐trans retinoic acid in C6 rat glioma cells
Authors: Bianchi Massimiliano G. , Gazzola Gian C. , Tognazzi Lucia , Kagechika Hiroyuki , Bussolati Ovidio
Published: June 2021

Several mechanisms modulate the activity of EAAC1, the counterpart of the human EAAT3 carrier encoded by the gene Slc1a1, through changes in transporter trafficking and interaction with regulatory proteins. Much less is known on EAAC1 regulation at the gene level. We investigate here the effects of all‐trans retinoic acid (ATRA) on EAAC1 activity and expression in C6 rat glioma cells. ATRA induced a four‐fold increase of aspartate transport in a dose‐ and time‐dependent manner (EC50 = 120 nM). Kinetic analysis indicated that the stimulation was due to Vmax increase and functional studies pointed to EAAC1 as the target of ATRA. In agreement with these data, Slc1a1 mRNA and EAAC1 protein were much higher in ATRA‐treated cells than in control counterparts, while no expression of GLAST and GLT‐1 transporters was detected under both conditions. ATRA effects on EAAC1 activity were inhibited by the specific RAR inhibitor LE540 and mimicked by AM80, a RAR agonist, but not by the RXR agonists HX630 and PA024. In addition, Slc1a1 mRNA induction was suppressed by cycloheximide, suggesting that a protein intermediate is needed for ATRA effect. In parallel with EAAC1 induction, ATRA treatment caused a huge increase in the expression of the oligodendrocytic marker PLP (proteolipid protein). These results indicate that the expression of Slc1a1 is induced by ATRA through a RAR‐dependent mechanism.
Temperature‐dependent differences in sympathetic neuroeffector transmission in mesenteric arteries and veins in hypertension
Authors: Dong Hua , Galligan James J , Swain Greg
Published: June 2021

Norepinephrine (NE) is released from sympathetic nerves supplying arteries and veins. We studied temperature‐related (28–37 ºC) differences in NE release and clearance using amperometry to measure NE in vitro. Tissues from sham and DOCA‐salt hypertensive rats were studied. Peak NE currents in sham veins declined more with temperature compared to arteries. Peak currents in DOCA‐salt arteries also showed greater temperature sensitivity than those in sham arteries. Currents in sham veins and DOCA‐salt arteries increased faster, decayed more slowly and were more temperature sensitive than those in sham arteries. Idazoxan (blocks α2 adrenergic receptors, α2ARs) and cocaine (blocks the NE transporter, NET) accelerated current rise time in sham arteries only. NE current decay was accelerated by idazoxan in veins but not arteries at 37 ºC and in arteries but not veins at 28 ºC, with a smaller increase in DOCA‐salt arteries at 28 ºC than sham arteries. We conclude that control of NE release from peri‐arterial nerves is more complex and therefore more temperature‐sensitive than in peri‐venous nerves. DOCA‐salt hypertension impairs NE release and clearance in arteries but not veins. α2ARs functional as autoreceptors and possibly as a “clearance receptor” holding NE in the neuroeffector junction. Clearance of NE in sham arteries is NET‐dependent and in sham veins and DOCA‐salt arteries is α2 NE clearance receptor dependent.
Activation of angiotensin II (AngII) type‐2 receptors (AT2R) modulates voltage‐gated Ca2+ currents in dorsomedial NTS (dmNTS) neurons through nitric oxide (NO)
Authors: Wang Gang , Coleman Christal , Glass Michael J , Zhou Ping , Anrather Josef , Pickel Virginia M , Iadecola Costantino
Published: June 2021

Activation of AngII type 1 receptors (AT1R) increases Ca2+ fluxes in dmNTS neurons receiving cardiorespiratory afferents and contributes to neurogenic hypertension. We tested the hypothesis that AT2R‐induced NO production counterbalances AT1R‐mediated Ca2+ fluxes. Dual‐label immuno‐electron microscopy showed that AT2R and nNOS are co‐expressed in rat and mouse dmNTS neurons. In the presence of the AT1R inhibitor losartan (Los; 3μM), AngII (100nM) increased NO production, assessed using DAF‐FM‐DA (48±11%, n=6, p<0.05). The effect was blocked by the AT2R antagonist PD123319 (30 μM), the NO scavenger PTIO (50 μM) or the NOS inhibitor LNNA (1mM). Los + AngII increased NO production in wild type and eNOS‐null mice (p<0.05), but not in nNOS‐null mice (p>0.05). AngII (100nM) increased voltage‐gated L‐type Ca2+ currents in rat dmNTS neurons (+36±3%; n=4; p<0.05). However, in the presence of Los, AngII attenuated Ca2+ currents (−23±5%, n=7, p<0.01), an effect blocked by LNNA and reproduced by the NO donor SNAP (2 μM, −26±5%, n=6, p<0.01) or the AT2R agonist CGP42112A (2 μM, −23±6%, n=4, p<0.05). Thus, AT2R activation increases nNOS‐derived NO, which, in turn, attenuates L‐type Ca2+ currents in dmNTS neurons. The AT2R‐induced inhibition of Ca2+ influx may partially offset the AT1R‐elicited potentiation of Ca2+ influx, mitigating the deleterious effects of AngII on central autonomic neurons (Supported by HL18974).
Angiotensin II (ANG II) Injection into the Rostral Ventrolateral Medulla (RVLM) Elicits Greater Sympathoexcitatory Responses in Salt‐Fed Rats.
Authors: Adams Julye M , Stocker Sean D
Published: June 2021

Increased dietary salt intake enhances sympathetic nerve activity (SNA) and arterial blood pressure (ABP) responses to glutamatergic stimulation of the RVLM. The present study determined whether increased salt intake also enhances SNA and ABP responses to RVLM injection of ANG II. Male Sprague‐Dawley rats were fed chow (0.2% NaCl) and given access to either 0.9% NaCl (SALT) or water (CON) for 2 wks. Baseline mean ABP (MABP) was not different between SALT and CON (117±3 vs 116±3 mmHg, n's > 17). As expected, SALT vs CON rats displayed significantly greater renal SNA (150±5 vs 106±7 %) and MABP (49±2 vs 34±2 mmHg) responses to RVLM injection of L‐glutamate (1 nmol, 60 nL). RVLM injection of ANG II produced greater increases in renal SNA of SALT vs CON rats (n=6) at 0.6 pmol (37±2 vs 16±2%) and 6 pmol (59±4 vs 37±3%; p <0.01). This was associated with a greater increase in MABP of SALT vs CON (n's>8) at 0.6 pmol (11±1 vs 5±1 mmHg) and 6 pmol (20±2 vs 11±1 mmHg; p<0.001). Bilateral injection of the ANG II‐type 1 receptor antagonist, losartan (1nmol), into the RVLM of SALT vs CON rats produced greater decreases in renal SNA (−19±5 vs +7±2 %, p< 0.02) and MABP (−11±1 vs +1±1, p< 0.01). These results indicate that increased dietary salt enhances sympathoexcitatory responses evoked by ANG II in the RVLM and increases the contribution of RVLM ANG II receptor activation to basal renal SNA and ABP.
Localization of 5‐HT1A/B receptors in the rat brainstem, their distribution in IML‐projecting cells and activation during acute hypoxia
Authors: Kline David D. , Dantzler Heather A. , Friskey Sarah A. , Hasser. Eileen M.
Published: June 2021

Serotonin (5‐HT) type‐1 receptors contribute to cardiorespiratory (CR) function. Yet, distribution of these receptors in CR nuclei, and specifically in cells projecting to sympathetic preganglionic neurons in the intermediolateral (IML) cell column, is lacking. Whether 5‐HT1 containing cells are activated during the chemoreflex is also not known. We hypothesized acute hypoxia (10% O2, 3 hr) would stimulate IML‐projecting cells containing 5‐HT1 receptors. Immunohistochemistry in rat brainstem slices retrogradely‐labeled with fluorogold (FG, 1%) in the IML (T2 level) was used to test this notion. Fos‐like reactivity was used as a marker of cell stimulation. Fos, FG, 5‐HT1A and 5‐HT1B distribution was examined. 5‐HT1A and 1B labeling was similarly distributed in the following nuclei: nucleus of the solitary tract (NTS), facial, lateral paragigantocellularis, hypoglossal, ambiguus, rostral and caudal ventral lateral medulla (RVLM, CVLM), and occasionally, the Botzinger and pre‐Botzinger. In the RVLM, ~26% of 5‐HT1A and 18% of 5‐HT1B co‐labeled with FG, and a greater percent of 5‐HT1B than 1A co‐labeled with PNMT. In the NTS, 5‐HT1A/B did not co‐label with FG. Hypoxia increased Fos in the NTS, RVLM and CVLM compared to room air. In the NTS, a small percentage of Fos cells contained 5‐HT1A and in RVLM, some co‐labeled with FG and 5‐HT1B. These data suggest 5‐HT1 receptors may play a role in the CR response to hypoxia.
Role of 5‐hydroxytryptamine (5‐HT2) receptors in the ventrolateral medulla (VLM) in the expression of the 10‐Hz rhythm in sympathetic nerve discharge (SND)
Authors: Barman Susan M , Orer Hakan S , Gebber Gerard L
Published: June 2021

We have shown that chemical inactivation (muscimol or 8‐OHDPAT microinjections) of the medullary raphe selectively reduces the 10‐Hz rhythmic component in SND of baroreceptor‐denervated, urethane‐anesthetized cats. These data suggest that raphe serotonergic neurons play a role in expression of the 10‐Hz rhythm. The current study was designed to identify the brainstem regions and 5‐HT receptor type acted on by these raphe neurons. For this purpose, power in the 10‐Hz band of left and right inferior cardiac SND was measured before and after unilateral microinjections (50 nl/injection) of a 5‐HT2 receptor antagonist (10 mM LY‐53,857) or agonist (10 uM DOI) into the caudal or rostral VLM, 3.8 − 4.2 mm lateral to the midline. LY‐53,857 significantly (p ≫ 0.05) reduced 10‐Hz power (ipsilateral to the injection site) to 32 +/− 8% and 22 +/− 8% of control when injected into the caudal and rostral VLM, respectively. The 10‐Hz power recorded contralateral to the injection site was reduced to a lesser extent. Power at lower frequencies (< 6 Hz) was not significantly changed in either nerve recording. DOI microinjection into either the caudal or rostral VLM produced a change in 10‐Hz power (an increase) opposite to that produced by the 5‐HT2 receptor antagonist. These results support the view that raphe neurons selectively enhance 10‐Hz SND by activating 5‐HT2 receptors on VLM neurons that comprise the rhythm generator. (Supported by HL‐33266.)
Central endocannabinoid modulation of baroreflex‐evoked sympathoinhibition in spontaneously hypertensive rats
Authors: Brozoski Daniel T. , Dean Caron , Hopp Francis A. , Seagard Jeanne L.
Published: June 2021

GABAergic neurotransmission has been reported to be greater in the spontaneously hypertensive rat (SHR) as compared to normotensive Wistar Kyoto (WKY) and Sprague Dawley (SD) rats. Previously, we have shown that endocannabinoids prolong baroreflex inhibition of renal sympathetic nerve activity (RSNA) via modulation of GABAergic neurotransmission in the nucleus tractus solitarius (NTS). Thus we proposed that endocannabinoids could prolong baroreflex‐induced sympathoinhibition more in SHRs than in normotensive rats. This study characterized the physiological role of endogenously released endocannabinoids in the NTS on baroreflex control of RSNA evoked by pressor responses (phenylephrine bolus) or direct stimulation of the baroreflex (NTS microinjection of the excitatory amino acid agonist d,l‐homocysteic acid). Baroreflex responses were compared before and after a microinjection of an indirect cannabinoid 1 receptor agonist, AM404, into the barosensitive region of the NTS of adult male SD rats, WKY rats, and SHRs. AM404 microinjections into the NTS were found to significantly prolong baroreflex‐induced sympathoinhibition in SD and WKY rats, but had an insignificant effect in SHRs. This observation suggests that endocannabinoid modulation of GABAergic transmission in SHRs is reduced and could contribute to elevated sympathetic tone characteristic of this model. VA Medical Research Funds.
Hemoglobin‐based‐oxygen‐carrier treatment exacerbates severity of acute hypoxia induced pulmonary vascular leak
Authors: Jacobs Robert Acton , Hamilton Karyn L. , Tissot van Patot Martha C. , White Molly , Foreman Ben , Gotshall Robert W. , Irwin David C.
Published: June 2021

Acute hypoxic pulmonary vascular leak (HPVL) is associated with increases in pulmonary artery pressure (Ppa) and endothelial permeability. Supplemental oxygen consistently prevents or ameliorates the potentially lethal symptoms related to HPVL. We hypothesized that hemoglobin‐based‐oxygen‐carrier (HBOC) treatment would decrease HPVL by controlling Ppa and/or plasma vascular endothelial growth factor concentration [VEGF] and prevent pulmonary leak despite an acute hypoxic insult. Conscious male Sprague Dawley rats were randomly treated with either Lactated Ringer's solution or HBOC solution and exposed to 4 hours of either normoxia (FiO2 = 21 %) or hypoxia (FiO2 = 10%). Only Ppa increased (p < 0.001) in response to the hypoxia. However, Ppa, pulmonary leak, and [VEGF], all, significantly increased (p = 0.026, 0.030, and 0.047, respectively) in response to HBOC treatment. We conclude that treatment with HBOC not only fails in prevention of HPVL but increases susceptibility by increasing Ppa and/or [VEGF]. Supported by the Defense Advanced Research Projects Agency (DARPA) and the Army Research Office (ARO) contract number W911NF‐06‐1‐0318.
Hypoxia causes perinuclear mitochondrial clustering and nuclear oxidant stress in pulmonary artery endothelial cells (PAECS) via a dynein‐dependent molecular motor
Authors: Swiger Brad , Patel Mita , Pastukh Viktor , Gillespie Mark N. , Al‐Mehdi Abu‐Bakr
Published: June 2021

Accumulating evidence suggests that mitochondria are motile, and may be recruited to subcellular domains where they participate in signal transduction. Mitochondria respond to hypoxia in PAECs by increasing oxygen radical generation, but whether hypoxia alters the disposition of mitochondria in PAECs is unknown, and if so, the molecular mechanism remains to be clarified. Accordingly, we mapped the disposition of Mitotracker‐stained mitochondria in cultured PAECs, and found that hypoxia caused a time‐dependent decrease in mitochondrial density near the plasma membrane and an increase in the perinuclear region. Interruption of the dynein‐dependent molecular motor using a specific siRNA prevented both the perinuclear mitochondrial clustering and, importantly, a hypoxia‐induced nuclear oxidant stress as detected by nuclear DCF fluorescence and by the level of oxidative base modifications in the VEGF promoter. Mitochondria were the source of the DNA‐modifying oxidants as evidenced by the finding that the respiratory chain inhibitor, myxothiazol, prevented the hypoxia‐induced nuclear DCF fluorescence without altering mitochondrial perinuclear clustering. These data suggest that hypoxia causes dynein‐dependent mitochondrial translocation to the perinuclear domain in PAECs that serves to create a nuclear oxidant stress important for hypoxic signaling.

Supported by NIH