bluuebunny/crossref_metadata_2025 huggingface dataset:
Resveratrol Protects Dopaminergic Neurons in Parkinson's Disease Models by Modulating the PKC‐delta Apoptotic Signaling Pathway & Microglial Activation
Authors: Gordon Richard , Hogan Colleen Elizabeth , Kanthasamy Kavin , Vellareddy Anantharam , Kanthasamy Arthi , Kanthasamy Anumantha G
Published: June 2021

Resveratrol is a phytoalexin that has been shown to be neuroprotective in various models of neurodegeneration. Although resveratrol is recognized as a potent antioxidant, emerging evidence suggests this compound can exert its protective function by modulating various signaling pathways including SIRT‐1, NF‐κB, and various kinases. Recent studies from our lab demonstrated that protein kinase Cδ (PKCδ) is an oxidative stress sensitive kinase that is proteolytically activated to induce apoptotic cell death in Parkinson's disease (PD) models. In this study, we examined whether i) resveratrol protects against dopaminergic neurodegeneration in cell culture models of PD ii) resveratrol modulates the PKCδ dependent apoptotic cascade in dopaminergic neuronal cells and iii) resveratrol can suppress the inflammatory response in activated microglia. Resveratrol treatment significantly increased cell viability and attenuated MPP+‐induced DNA fragmentation and caspase‐3 activity in mesencephalic dopaminergic neuronal cells (N27 cells). We also observed that resveratrol effectively blocked the proteolytic cleavage of PKCδ and kinase activity induced by MPP+ treatment. We further confirmed the protective effect of resveratrol in MPP+ treated primary mesencephalic neuron cultures. In BV‐2 microglial cells activated with LPS, pretreatment with resveratrol suppressed pro‐inflammatory cytokine production and nitric oxide release. Collectively, our results demonstrate that resveratrol protects against the degenerative process in dopaminergic neurons by modulating the PKCδ proapoptotic signaling pathway and can also attenuate the inflammatory response in activated microglia
Neuroprotective Factors Secreted by U‐87 Astrocytoma (Astrocytes‐Like) Cells and Their Associated Metabolic Adaptation
Authors: Jaiswal Ashvin R , Wong Yin Yin W. , Dukhande Vikas V. , Lai James C.K.
Published: June 2021

Astrocytes can and do exert neuroprotective effects on their surrounding neurons both in vivo and in vitro. Our previously observation that U‐87 astrocytoma (astrocytes‐like) cells protect neuroblastoma SK‐N‐SH (neurons‐like) cells against neurodegenerative assaults in co‐cultures prompted us to hypothesize U‐87 cells release neuroprotective factors into the medium and protect neurons by scavenging extracellular glutamate and modulating glutamate‐glutamine cycling. To test this hypothesis, we developed a model using U‐87 cells, stage‐wise deprived them of fetal bovine serum, and then treated them with or without pioglitazone for 24 hours. We then collected the medium (“defined conditioned medium”) the U‐87 cells were in. Then we determined the effects of the media on survival of SK‐N‐SH (neurons‐like) cells. Our results indicated that the survival rates of SK‐N‐SH cells when exposed to the conditioned media were higher than those of untreated SK‐N‐SH cells. We noted U‐87 cells appeared to release glia derived neurotrophic factor (GDNF) into the medium. Moreover, in exerting their protective effect, U‐87 cells seemed to alter their protein expression of glutamine synthetase (GS) and glial fibrillary acidic protein (GFAP) when exposed to various stresses. Thus, the results of our studies further elucidate the mechanisms of neuroprotection of astrocytes‐like U‐87 cells and astrocytes in general.
A possible cannabinergic neuroprotective mechanism of action of Lepidium meyenii (Maca)
Authors: Pino‐Figueroa Alejandro J , Maher Timothy J.
Published: June 2021

The pentane extract of the plant Lepidium meyenii (Maca), has been shown to possess neuroprotective activity. This study was designed to examine the involvement of cannabinoid CB1 receptors in the action of some of the lipophilic constituents of Maca, the macamides, in neuronal cell lines. Cells were pretreated with Maca pentane extract (MPX), isolated macamides, anandamide or tetrahydrocannabinol (THC). After 1 hr incubation, cells were subjected to oxidative damage with H2O2. Cell viability was determined 24 hrs later using the trypan blue exclusion cell count method and the spectrophotometric dimethylthiazolyl‐carboxymethoxyphenyl‐sulfophenyl‐tetrazolium (MTS) method. Significant neuroprotective activity was observed with the MPX, macamide‐2, anandamide and THC. O‐2050 (a cannabinoid CB1 receptor silent antagonist) significantly (p<0.05) antagonized the neuroprotective activities. Additionally, the antioxidant activity of the MPX, anandamide and THC was determined using the 2,2′‐azino‐di‐3‐ethylbenzthiazoline sulfonate (ABTS) assay, and found to have no measurable anti‐oxidant capacity. These results demonstrate that some of the lipophilic compounds from Maca may act through the cannabinoid system to protect neurons against oxidative damage. Further studies are required to determine their detailed molecular mechanism.
Mechanism of neurotensin response to methamphetamine self administration
Authors: Hanson Glen R , Hoonakker Amanda J , Alburges Mario E , McDougall Jacob W , Fleckenstein Annette E , Frankel Paul S
Published: June 2021

Owing to the serious consequences of methamphetamine (METH) abuse, the response of neurotensin (NT) systems closely linked with striatal dopamine (DA) pathways was studied using operant conditioning and analyzing the response to self‐administration of METH by contingent lever pressing (SAM) after 4‐h sessions on 5 consecutive d (total of at least 3 mg/kg, iv). Yoked‐METH and saline (YM and YS, respectively) were compared. Results revealed that METH increased dorsal striatal NT levels in the SAM and, to a lesser extent, YM rats, as assessed 6 h after the final session, suggesting that METH self‐administration caused NT changes akin to those reported after high doses of METH administered non‐contingently. We were unable to assess directly if changes in the SAM animals were mediated by DA receptors, as both D1 (SCH23390) and D2 (eticlopride) blockade diminished lever pressing for METH. However, assuming that changes in the striatal NT systems of the YM group were mechanistically similar to those in SAM rats, we pre‐treated YM animals with either SCH23390 or eticlopride prior to each daily session. Results revealed that D1, but not D2, receptor blockade diminished METH‐mediated NT responses in the dorsal striatum, suggesting that the NT response and the associated mechanism in SAM are similar to the effects of a non‐contingent high‐dose of METH treatment. Support: DA09407, 019447, 013367, 00869, 11389, 04222, 00378
The exponential model of demand: A novel, robust, and broadly applicable assay for translational behavioral pharmacology
Authors: Roma Pete , Kaminski Barbara J. , Spiga Ralph , Ator Nancy A. , Hursh Steven R.
Published: June 2021

Methods for quantifying natural and drug reward in clinical and preclinical research often yield mixed results due to procedural limitations or differences between subject populations or treatments. However, the recently introduced “exponential model of demand” may serve as a unifying behavioral methodology for assessing reward and motivation. The model is rooted in behavioral economics and involves a single “price” (fixed‐ratio requirement) per unit of reinforcement per session across several sessions of increasing price. Rather than defining reinforcement as response output or absolute consumption at any one point, the “essential value” of the reinforcer is the rate of decrease in consumption across the range of prices. This change in demand elasticity is calculated in the model as a single value for each subject that is informative yet resilient to dose, potency, concentration, and endogenous biological/genetic confounds. The novel concept of “essential value” is arguably the most sophisticated, comprehensive, and realistic laboratory model of reward and motivation to date. Retrospective and recently collected data support the model's utility. Its impact could be significant for translational behavioral pharmacology, with applications to drug abuse, medications testing, and elucidating the neural and genetic mechanisms mediating reward using a homologous methodology within and across species.
Pharmacological inhibition of dopamine beta‐hydroxylase blocks cocaine‐induced reinstatement in rats
Authors: Cooper Debra Andrea , Schank Jesse , Schroeder Jason , Gaval‐Cruz Meriem , Pozdeyev Nikita , Iuvone Michael , Howell Leonard , Weinshenker David
Published: June 2021

Disulfiram (Antabuse) has recently shown promise as a pharmacotherapy for cocaine dependence, though the mechanisms underlying its efficacy are unclear. One of the many biochemical actions of disulfiram is inhibition of dopamine beta‐hydroxylase (DBH), the enzyme that converts dopamine to norepinephrine (NE); consequently, disulfiram reduces brain NE levels. Noradrenergic signaling is required for reinstatement of drug seeking in rats, a paradigm which is thought to model drug relapse. We therefore hypothesized that DBH inhibition underlies the therapeutic effects of disulfiram. To test this hypothesis, cocaine self‐administration studies were conducted in rats (N=5–8 per group) given pretreatments of vehicle, disulfiram (100 mg/kg i.p.) or the selective DBH inhibitor nepicastat (50 mg/kg i.p.). We found that neither disulfiram nor nepicastat altered lever pressing behavior during the maintenance phase of self‐administration. In contrast, both drugs blocked cocaine‐primed reinstatement of lever pressing following extinction. These results suggest that pharmacological DBH inhibition may be an effective treatment for cocaine dependence, potentially by preventing relapse.
Pain‐related depression of intracranial self‐stimulation in rats: effects of the delta opioid agonist SNC80 and the psychomotor stimulant cocaine
Authors: Rosenberg Marisa Brooke , Folk John E , Rice Kenner C , Negus Sidney Stevens
Published: June 2021

Pain is associated with a stimulation of some behaviors (e.g. withdrawal responses) and a depression of other behaviors (e.g. feeding, locomotion, responding maintained by many types of positive reinforcement). We have argued that analgesic drug development may benefit from complementary evaluation of drug effects on both pain‐stimulated and pain‐depressed behaviors. In this study, intraperitoneal injection of dilute lactic acid (1.8% in a volume of 1ml/kg) was used as a noxious stimulus in rats to stimulate a stretching response and to depress intracranial self‐stimulation (ICSS) of the medial forebrain bundle. The delta opioid agonist SNC80 (1–10 mg/kg, i.p.) dose‐dependently blocked both acid‐stimulated stretching and acid‐induced depression of ICSS without altering control ICSS in the absence of the noxious stimulus. In contrast, cocaine (1–10 mg/kg i.p.) blocked acid‐depressed ICSS only at doses that also facilitated control ICSS, and cocaine had no effect on acid‐stimulated stretching. Thus, the antinociceptive effects of SNC80 in the assay of pain‐depressed ICSS could be dissociated from the non‐selective stimulant effects of cocaine. Supported by NIH grants RO1‐DA11460 and R01‐NS070715.
An Investigation of the Anxiolytic Effects of Aromatherapy in Mice
Authors: Feifel Ariel , Melendez Gilia , Feifel David
Published: June 2021

Certain essential oils, in particular Lavender (Lavandula angustifolia), are claimed by proponents of aromatherapy to reduce anxiety, but there is limited scientific evidence for this. The purpose of this study was to determine whether essential Lavender oil has effects in animal models of anxiety. C57 mice were tested, an elevated plus maze, a common test of anxiety. Each mouse had two trials of five minutes in the maze, one with Lavender oil exposure prior to and during plus maze testing, and the other with exposure to distilled water. Another set of mice were tested in locomotor cages and in startle chambers where their acoustic startle response and prepulse inhibition of startle (PPI) was measured. Overall, mice spent more time and made more entries into closed arms of the elevated maze as expected. There was a tendency for Lavender oil exposure to decrease overall entries compared to water but there was no difference between the Lavender oil versus the water exposure in the percentage of time spent or the percentage of entries into the open arms. Lavender oil did not have any effect on acoustic startle response or PPI, but it did significantly decrease mobility in locomotor cages. These results suggest that Lavender oil does have bioactive, behavioral effects but that these effects are more consistent with non‐specific, sedation rather than anxiolytic‐specific effects.
Oxysterols: Cholesterol Metabolites of Diverse Function in Mice and Men
Authors: Russell David W.
Published: June 2021

Oxysterols are hydroxylated derivatives of cholesterol that play important roles in mammalian cells. The addition of a hydroxyl group to the side chain or ring structures of cholesterol changes the physiochemical properties of the sterol and renders the molecule membrane‐permeable. We have isolated three enzymes that hydroxylate the side chain of cholesterol and have elucidated their biological roles. Cholesterol 24‐hydroxylase is a microsomal cytochrome P450 that is selectively expressed in neurons and plays a major role in the turnover of cholesterol in the brain. Mice that lack this enzyme have defects in learning and synaptic plasticity. Cholesterol 25‐hydroxylase, a microsomal di‐iron‐ containing enzyme, is expressed by macrophages following activation of the innate immune system. Genetic ablation of the enzyme in mice causes hyperimmunoglobulinemia A. Sterol 27‐hydroxylase is a mitochondrial cytochrome P450 expressed in hepatocytes and other cell types, which catalyzes an essential reaction in bile acid synthesis. Inherited mutations in the human gene cause the neurological disorder cerebrotendinous xanthomatosis.
Effects of 2‐APB‐induced synaptic facilitation at developing Xenopus neuromuscular junction
Authors: Liou Jau‐Cheng
Published: June 2021

Here we show that 2‐APB, a compound commonly used as TRP channel inhibitor, dose‐dependently induce a significant facilitation on the frequency of spontaneous neurotransmitter release at developing Xenopus neuromuscular junction through, surprisingly, TRP channel activation. Bath application of universal TRP channel inhibitors either SKF96365 or flufenamic acid cease the 2‐APB‐induced synaptic facilitation. Exclusion of Ca2+ from culture medium or bath application of the pharmacological Ca2+ channel inhibitor cadmium, membrane‐permeable Ca2+ chelator BAPTA‐AM, effectively hampered the facilitation of neurotransmitter release induced by 2‐APB, suggesting Ca2+ influx is requisite for 2‐APB‐induced synaptic facilitation. Blockade of the voltage‐dependent Ca2+ channel with either nifedipine, or verapamil failed to abolish the SSC facilitating effect of 2‐APB. Overall, results from our current studies provide evidences that 2‐APB‐induced the open of TRP channels and Ca2+ influx which result in facilitation of spontaneous neurotransmitter release at developing Xenopus neuromuscular synapse.
Disruption of Cell Shape by Serum Withdrawal is Associated With Rapid Downregulation of Transient Receptor Potential Vanilloid 1 (TRPV1) and β‐Tubulin
Authors: Sheth Sandeep , Jajoo Sarvesh , Mukherjea Debashree , Ramkumar Vickram
Published: June 2021

Serum deprivation is used as a method for studying the effects of various drugs and growth factors on the cell signaling cascades and gene expression in cultured cells. While studying the effects of growth factors on TRPV1 levels in human embryonic kidney (HEK293) cells stably expressing this receptor, we observed a dramatic disruption of cell shape within 3 h of serum withdrawal. The change in cell shape was time‐dependent, was not associated with apoptosis and was fully restored by serum. No change in cell shape to serum withdrawal was observed in normal untransfected HEK cells. The change in cell shape was partly attenuated by ruthenium red, a nonselective inhibitor of TRPV1, but was unaltered by capsaicin, an agonist of this channel, or by knockdown of TRPV1. Interestingly, serum withdrawal was associated with a rapid loss in the expression of TRPV1 and β‐tubulin and decreased activity of p38 mitogen activated protein kinase (MAPK) and AKT‐1. The decreases in TRPV1 levels were mimicked by inhibition of p38 MAPK but not by AKT‐1, while the decrease in β‐tubulin was dependent on both p‐38 MAPK and AKT‐1. Based on these data, we conclude: 1) the regulation of the levels of TRPV1 and β‐tubulin by serum is essential for cell shape and 2) p38 MAPK and/or AKT‐1 pathways mediate the expression of TRPV1 and β‐tubulin in response to serum. This research was supported by a Central Research Committee grant from SIU School of Medicine.
Protein kinase C δ signaling at mitochondria revealed by live cell fluorescence imaging, chemical genetics, and biochemical studies
Authors: Wu‐Zhang Alyssa Xia , Kajimoto Taketoshi , Newton Alexandra C.
Published: June 2021

Mitochondria serve the cell's energy needs and, in response to intracellular signals, execute programmed cell death. Protein kinase C δ (PKCδ), a member of the PKC family of signal transduction isozymes whose dysregulation is implicated in diseases such as cancer and diabetes, has been shown to be essential in mitochondrial‐dependent apoptosis. Here we dissect the molecular mechanisms driving PKCδ interaction with mitochondria. Using FRET‐based imaging, we show that phorbol ester induces acute translocation of PKCδ to mitochondria that is isozyme‐specific, independent of the C2 and C1A domains and of PKC activity, insufficient with the catalytic domain alone, and dependent on the C1B domain. Mitochondrial PKCδ has robust activity at the outer membrane, as revealed by a FRET reporter of PKC activity (CKAR) modified here to report PKCδ activity specifically (δCKAR). Using a PKCδ gate‐keeper mutant with a modified active site, we show that specific inhibition of PKCδ activity neither prevents nor reverses its translocation to mitochondria. Lastly, biochemical studies confirm basal interaction of exogenous PKCδ with and the presence of endogenous PKCδ at mitochondria. These data reveal unique properties of PKCδ that allow isozyme‐specific function at mitochondria. This research was supported by NIH GM43154 (ACN) and NIH Pharmacology Training Grant NIH 2 T32 GM07752 (AXW).
A novel screening system for claudin binder using baculoviral display
Authors: Takahashi Azusa , Kondoh Masuo , Kakutani Hideki , Sakihama Toshiko , Hamakubo Takao , Watari Akihiro , Yagi Kiyohito
Published: June 2021

Recently, it became clear that claudin (CL) plays a pivotal role in tight junction (TJ) barrier function in epithelial cell, and we previously proved that CL can be a target for pharmaceutical therapy. Despite the presence of more than 20 CL family members, few ligands for CL have been identified because it is difficult to prepare CL in an intact form. In this study, we developed a method to screen for CL binders by using the budded baculovirus (BV) display system. CL4‐displaying BV interacted with a CL4 binder, the C‐terminal fragment of Clostridium perfringens enterotoxin (C‐CPE), but it did not interact with mutated C‐CPE that lost CL4‐binding region. C‐CPE did not interact with BV and CL1‐displaying BV. We used CL4‐displaying BV to select CL4‐binding phage in a mixture of a scFv‐phage and C‐CPE‐phage. The percentage of C‐CPE‐phage in the phage mixture increased after the selection, indicating that CL‐displaying BV may be useful for the selection of CL binders. We prepared a C‐CPE phage library by mutating the functional amino acids, and screened the library with CL4‐displaying BV. Then we found that the newly obtained CL4 binders also modulated the TJ barrier. These finding indicate that the CL‐displaying BV system may be a promising method to produce a novel CL binder and modulator.
Angiotensin II‐Induced Vasoconstriction in Skeletal Muscle: Effects of Aging and TNF‐α
Authors: Behnke Bradley Jon , Dominguez James M , Park Yoonjung , Delp Michael D
Published: June 2021

There is increasing evidence that oxidative stress and vascular inflammation contribute to old‐age associated vascular dysfunction. With advancing age there are elevated levels of tumor necrosis factor (TNF)‐α, which, via endothelial vasodilator impairment, may exacerbate the vasoconstrictor responses of angiotensin (ANG)‐II.

Purpose

To determine whether the proinflammatory cytokine TNF‐α contributes to alterations in ANG II‐mediated vasoconstriction with aging.

Methods

Pegylated soluble human TNF type 1 receptor (PEG sTNF‐RI; Amgen Inc) was administered (1 mg/kg, s.c., 3x/wk) for 10 wks in eight young (4–6 mo) and seven old (22–24 mo) male Fischer 344 rats to inhibit soluble TNF‐α binding to receptors. At the end of the TNF‐α inhibition period, gastrocnemius muscle arterioles were isolated and concentration response relations to the cumulative addition of ANG II (3x10−12 to 3x10−5 M) were determined.

Results

Chronic TNF‐α inhibition significantly diminished the vasoconstrictor response of arterioles from ~ 60% vasoconstriction in the old untreated animals to ~ 40% in the old treated animals. Furthermore, age‐associated differences (young vs. old) in ANG II mediated vasoconstriction were eliminated with the chronic TNF‐α inhibition.

Conclusion

With old age there is an enhanced vasoconstriction to ANG II which is due, in part, to diminished endothelial function. Chronic TNF‐α inhibition eliminated age‐related differences in ANG II induced vasoconstriction which suggests that the impaired NOS signaling pathway linked to ANG II receptors with old age may be the result of a proinflammatory state.
Introgression of the Brown Norway Renin Gene onto the Dahl Salt Sensitive Genetic Background Restores Endothelium‐Dependent Vascular Relaxation by Reducing Oxidative Stress in the Cerebral Vasculature
Authors: Durand Matthew J. , Lombard Julian H.
Published: June 2021

Dahl salt sensitive (SS) rats fed low salt (LS) diet are normotensive but have severe endothelial dysfunction, chronically low plasma renin activity and low plasma angiotensin II levels. This study assessed endothelial function in isolated middle cerebral arteries (MCA) from male SS rats and congenic rats receiving a 2.0 Mbp portion of Brown Norway (BN) chromosome 13 containing the BN renin allele (Ren1‐BN). All rats were fed LS diet (0.4% NaCl) since weaning. MCAs from Ren1‐BN congenic rats exhibited dose‐dependent dilation to acetylcholine (10−10 – 10−5 M) whereas MCAs from the SS rats did not. Vascular superoxide (dihydroethidium fluorescence) and hydrogen peroxide levels (Amplex Red) were significantly elevated in cerebral arteries of SS rats compared to Ren1‐BN congenic rats, and chronic antioxidant treatment with tempol (15 mg/kg/day) restored vascular relaxation in SS rats. Western blot analysis of multiple antioxidant enzymes showed significantly greater expression of Cu/Zn superoxide dismutase in cerebral arteries of the Ren1‐BN congenic rats. These results suggest that restoring normal regulation of the renin‐angiotensin system by introgressing the BN renin allele onto the Dahl SS genetic background rescues endothelial function by increasing antioxidant enzyme expression and reducing oxidative stress. (NIH #HL65289, #HL72920, #HL92026).
Modeling Pulmonary Endothelial Serotonin Uptake
Authors: Jardine Bartholomew E , Bassingthwaighte James B
Published: June 2021

A four‐region (capillary plasma, endothelium, ISF, cell) blood tissue exchange model describes data from serotonin uptake experiments in dog lungs. Data from Multiple Indicator Dilution experiments of Rickaby et al. (JAP 51: 405,1981), and Malcorp et al. (JAP 57: 720, 1984) were analyzed to distinguish facilitated transport into the endothelial cells (EC) from permeation via interendothelial clefts. The permeability‐surface area products, PS, for the inter‐EC clefts were low, about 0.2 ml/(g*min). The serotonin endothelial transporter kinetic parameters were Km = ~ 3 uM and Vmax = ~ 4 nmol/(g*min), similar to those found by Rickaby et al. using a simpler two‐region, plasma/endothelium model not accounting for cleft permeation. Because the cleft PS is so small, less than 10% of that in cardiac capillaries, their model fits the data as well as the four‐region one. This validates using the simpler model and supports the methods used by Linehan, Dawson et al (e.g Audi et al JAP 84: 516, 1998) to study lung endothelial substrate handling. (Supported by NIH grants T15HL88516 and EB08407)
Locomotor activity and blood pressure in conscious mice with targeted deletion of the equilibrative nucleoside transporter 1
Authors: Li Lingli , Eisner Christoph , Huang Yuning , Choi Doo‐Sup , Schnermann Jurgen
Published: June 2021

Extracellular adenosine (ado) is regulated by transmembrane transport through both equilibrative and concentrative nucleoside transporters (ENTs and CNTs). Previous studies have reported profound effects of extracellular ado on sleep, anxiety, and locomotor activity. The present experiments were performed in ENT1‐deficient mice to assess the long term effect of impaired ado exchange on arterial blood pressure (MAP) and locomotor activity. Average daily running wheel activity (RWA, revolutions/min) under LD (12h light:12h dark) and DD (24 h dark) was markedly reduced in ENT1 −/− (LD, 3.1 ± 0.1; DD, 2.1 ± 0.1; n=4) vs. WT mice (LD, 9.4 ± 0.7; DD, 11.3 ± 0.3; n=4; p < 0.001). RWA showed comparable circadian periodicity between genotypes, but the periodic strength was greater in WT than ENT1−/−. Spontaneous activity measured with radiotransmitters was also 40% lower in ENT1−/− than WT mice. Telemetric measurements of MAP showed no significant differences between genotypes although pulse pressure was lower in −/− than +/+ mice. Heart rates were lower in ENT1−/− than WT by about 40 bpm during both light and dark periods. Plasma renin concentration (ng Ang I/ml hr) tended to be lower in ENT1 −/− mice without reaching significance. Absence of ENT1 is associated with reduced locomotor activity, perhaps reflecting suppression of neuronal activities by impaired ado transporting capacity.
Changes in aortic augmentation index following systemic β‐adrenergic blockade are dependent on heart rate
Authors: Allen Alexander R , Curry Timothy B. , Hart Emma C. , Charkoudian Nisha , Joyner Michael J. , Casey Darren P.
Published: June 2021

We examined the effects of acute β‐adrenergic blockade on central artery hemodynamics and wave reflection characteristics in young healthy humans. High‐fidelity radial artery waveforms were recorded non‐invasively by applanation tonometry before and after intravenous propranolol (0.25 mg/kg bolus for 5 min followed by a maintenance infusion of 0.004/mg/kg/min for 20–30 min) in fourteen subjects (9 F/5M; 27 ± 2 years). Radial blood pressure (BP) was calibrated from the brachial BP (arterial catheter). Aortic augmentation index (AIa; a measure of amplitude and timing of arterial pressure wave reflection) and roundtrip travel time of the reflected wave to and from the periphery (Δtp; inverse of pulse wave velocity) were calculated from the generated aortic pressure waveform and are indices of central arterial stiffness. Systemic β‐blockade reduced heart rate (HR; 60 ± 2 vs. 53 ± 2 bpm, P < 0.001) and increased AIa (9.9 ± 4.3% vs. 4.3 ± 4.4%, P < 0.05), but did not alter Δtp (156 ± 4 msec vs. 152 ± 2 msec, P = 0.34). The AIa normalized to a HR of 75bpm (AIa@75) was not increased following β‐blockade (P = 0.67). Taken together, our data indicate that the increase in AIa following systemic β‐blockade is a result of a slower HR and not due to changes in β‐ mediated vascular tone.

Supported by NIH K23 DK‐082424 (TBC), AHA 2170087(ECH) and CTSA RR‐024150
Functional interaction between baroreflex and vestibulo‐sympathetic reflex upon head‐up tilt
Authors: Morita Hironobu , Abe Chikara , Kawada Toru , Tanaka Kunihiko , Sugimachi Masaru
Published: June 2021

Head‐up tilt (HUT) elicits gravitational fluid shift and a decrease in arterial pressure (AP), which is thought to be buffered by baroreflex and vestibulo‐sympathetic reflex. However, functional interaction between these reflexes has not been clear. To examine this, renal sympathetic nerve activity (SNA) and AP were measured in anesthetized, vagotomized, and aortic denervated rats with or without vestibular lesion (VL). Isolated carotid sinus pressure (CSP) was increased stepwise from 60 to 180 mmHg with increments of 20 mmHg (30–60 s for each CSP level) while the rat was placed prone flat position and at 60° HUT. HUT shifted the CSP‐SNA relationship (the baroreflex central arc) to a higher SNA, shifted the SNA‐AP relationship (the baroreflex peripheral arc) to a lower AP, and consequently moved the operating point to high SNA while maintaining AP. In VL rats, the HUT‐induced central arc shift was completely abolished, while the peripheral arc shifted to a lower AP; the operating point moved to a lower AP (−26 mmHg). These results indicate that the vestibular system elicits sympathoexcitation upon HUT and shifts the baroreflex central arc to a higher SNA and then maintain baroreflex operating AP.
Activation of Peripheral delta 2 opioid receptors by Deltorphin‐B (Delt‐B) increased cardiac tolerance to arrythmogenic impact of ischemia/reperfusion
Authors: Oeltgen Peter R. , Maslov Leonid N. , Brown Stephen A.
Published: June 2021

We investigated the role of peripheral μ, δ1, δ2 and opioid receptor ligands (ORL1) in the regulation of cardiac tolerance to the arrhythmogenic impact of ischemia reperfusion in rats. The ligands were given prior to 45 min coronary artery occlusion and 10 min reperfusion. The incidence of ventricular arrythmias was determined. Pretreatment with the Delt‐B (150 nmol/kg) reduced incidence of ischemia and reperfusion induced arrhythmias. The δ2 OR agonists Deltorphin‐Dvariant (150 nmol/kg) and Deltorphin‐E (150 nmol/kg) prevented an appearance of arrhythmias only during phase 1b ischemia. The μ OR agonists DAMGO (150 and 1500 nmol/kg) and Dermorphin‐H (150 nmol/kg), the δ1 OR agonist DPDPE (150 and 1500 nmol/kg) and the ORL1 receptor agonist Nociceptin (220 and 1500 nmol/kg) had no effect on the incidence of arrhythmias. Pretreatment with universal opioid antagonist naloxone methiodide and naltrexone and δ2 specific OR antagonist naltriben methane sulfonate (NTB) abolished the antiarrhythmic effect of Delt‐B. The selective δ1 OR antagonist 7‐Benzylidenenaltrexone (BNTX) had no effect. The OR antagonists alone did not exhibit antiarrhythmic properties. δ2 receptor activation by Deltorphins increases tolerance to arrhythmogenic impact of ischemia/reperfusion in rats. Supported by the Office of Navel Research (PRO), a VA Merit Review Grant (SAB) and the Russian Foundation of Basic Research (LNM).
Effects of branched chain amino acids infusion on whole‐body plasma glucose disposal: a pilot study
Authors: Everman Sarah , Mandarino Lawrence J , Puga Guilherme M , Meyer Christian , Katsanos Christos S
Published: June 2021

The physiological importance of the inhibition of muscle glucose uptake simultaneously with the stimulation of muscle protein synthesis, when the plasma concentrations of both glucose and amino acids are elevated, as in the fed state, is not clear. We tested the hypothesis that a branched chain amino acids (BCAA) infusion, known to increase protein accumulation, impairs insulin‐stimulated whole‐body glucose disposal. Two groups of young, healthy males and females (n = 5/group) were studied in the postabsorptive state (3 hours) and during a 3 hour euglycemic, hyperinsulinemic (40 mIU/m‐2/min) clamp. The control group (BAS) was infused with BCAA sufficient to maintain plasma BCAA at the basal concentrations during the clamp, while the other group (HI) received a six hour BCAA infusion (1.66 umol/kg/min) to increase BCAA 4‐fold. Subjects in both groups were insulin sensitive as determined by the HOMA index. Glucose disposal rate during the clamp, as determined by 6,6‐d2 glucose infusion and adjusted to the prevailing plasma insulin concentrations, was lower in the HI group (0.53 umol/kg/min +/− 0.05 vs. 0.93 umol/kg/min +/− 0.11; HI vs. BAS; p=0.005). We conclude that increased concentrations of plasma BCAA decrease whole‐body glucose disposal in healthy humans. This protocol will allow us to study the role of plasma amino acids in modulating glucose uptake in skeletal muscle in humans.
Redox Regulation of Methylation by PRMT1
Authors: Forbes Scott Preston , Cardounel Arturo Juan
Published: June 2021

ADMA and L‐NMMA are endogenous NOS inhibitors derived from the proteolysis of methylated arginine residues on proteins. The methylation is carried out by a group of enzymes referred to as protein‐arginine methyl transferase's (PRMTs). Protein arginine methylation has been demonstrated to regulate key cellular events including transcription, RNA metabolism and protein‐protein interactions and recent evidence suggest that PRMT activity may be under redox control. Therefore, the aim of the current study was to assess the redox‐dependent regulation of PRMT1 activity using recombinant proteins. Results demonstrated a dose dependent increase in PRMT activity in the presence of NO donor DETANONOate (0.1 nM‐1 mM) with a maximal increase of 300% at 1 mM. In contrast, exposure of purified PRMT1 to authentic H2O2 (0.1 nM‐1 mM) and ONOO− (0.1 nM‐1 mM) resulted in decreased PRMT activity with 90% inhibition observed at 1 mM ONOO−. Peroxynitrite inhibition of PRMT1 was accompanied by the formation of nitro‐tyrosine residues on PRMT1 suggesting that tyrosine nitration events are responsible for the inhibition. Increased activity of PRMT1 by exposure to NO would be expected to result in increased formation of the competitive eNOS inhibitors ADMA and L‐NMMA. Thus, NO may form a negative feedback mechanism to regulate activity of eNOS by increasing the activity of PRMT1 and therefore escalating the production of ADMA and L‐NMMA.
A novel technique to study the time course of morphological and functional vascular responses to hypertension in conscious rats
Authors: Stauss Harald Martin , Leick Katie M , Burkle Jason W , Rotella Diane L , Rarick Kevin R , Alterie Jonathan D , Nelson Joshua R , Salter Thomas A , Harvey Michael J , Kim Sung H , Ebnet Thomas J , Anderson Michael G
Published: June 2021

Assessment of morphological vascular responses to exposure to cardiovascular risk factors in experimental animals requires ex vivo experiments that do not allow assessment of the time course of vascular alterations in individual subjects. We used a slit‐lamp biomicroscope (resolution <1 μm) to photograph the long posterior ciliary artery (LPCA) of the iris in conscious normotensive Wistar Kyoto rats (WKY, n=10) and spontaneously hypertensive rats (SHR, n=10) on regular‐salt diet (RS) and in SHR (n=10) on high‐salt diet (HS). The same segment along the LPCA was imaged in consecutive weekly imaging sessions and an imaging software was used to determine the wall to lumen (W/L) ratio. After 4 weeks systolic blood pressure (SBP) did not change in WKY (115±5 vs. 111±5 mmHg) and SHR‐RS (148±7 vs. 155±3 mmHg) but increased significantly in SHR‐HS (139±5 mmHg vs. 166±6 mmHg, p<0.05). The time course of the changes in W/L ratio of the LPCA mirrored the time course of the SBP changes. While W/L ratio did not change in WKY‐RS (+12±10%) and SHR‐RS (+18±17%), there was a 39±14% increase in W/L ratio in SHR‐HS. Since the LPCA was not dilated pharmacologically the W/L ratio assessed in this study may reflect the combination of morphologic alterations and changes in vascular tone. In vivo imaging of the LPCA may allow assessment of the time course of morphological and functional vascular responses to hypertension in conscious rats. (Supported by the American Heart Association)
Hypoxia downregulates peroxisome proliferator‐activated receptor‐gamma in human pulmonary artery smooth muscle cells by NF‐κB and ERK1/2
Authors: Lu Xianghuai , Ramirez Allan , Nanes Mark , Hart C. Michael
Published: June 2021

The ligand‐activated transcription factor, peroxisome proliferator‐activated receptor gamma (PPARγ), regulates metabolism, cell proliferation, and inflammation. We have observed that the PPARγ ligand, rosiglitazone, inhibits hypoxia‐induced proliferation of human pulmonary artery smooth muscle cells (HPASMC) in vitro and hypoxia‐induced pulmonary hypertension in mice in vivo. Furthermore, we have reported that in vitro hypoxia exposure reduced PPARγ protein levels in isolated HPASMC and pulmonary artery endothelial cells (Nisbet, 2009). The current study examined the molecular mechanisms involved in hypoxia regulation of PPARγ. Exposure to 1% oxygen for 72 hours decreased PPARγ1 mRNA and protein levels in HPASMC. Compared to control conditions, hypoxia decreased the activity of a PPARγ luciferase reporter (PPRE‐Luc) by roughly 60%. Inhibiting NF‐κB by applying either p65 siRNA or the NF‐κB specific inhibitor, CAPE, attenuated hypoxia‐induced inhibition of PPRE‐Luc activity. Hypoxia‐induced inhibition of PPRE‐Luc activity was also attenuated by treatment with PD98059, a specific inhibitor of ERK1/2. Mammalian two hybrid assays suggested that PPARγ binds to p65. Deletion of the p65 activation domain (amino acids 308‐551) abrogated p65 binding to PPARγ. These findings suggest that hypoxia activates NF‐κB and MAP kinase pathways to reduce PPAR transcription and PPARγ expression in HPASMC.
Endothelial p120 catenin inhibits LPS‐induced lung inflammatory injury by suppression of MAPK and NF‐κB activation
Authors: Hu Guochang , Wang Yanlei , Sun Yu , Malik Asrar B. , Minshall Richard D.
Published: June 2021

We investigated the role of endothelial p120 catenin in sepsis‐induced lung inflammatory injury. p120 expression in the mouse lung was rapidly downregulated in response to LPS challenge and the level of p120 protein expression is correlated inversely with the severity of sepsis. Depletion of p120 from the mouse pulmonary vasculature using siRNA increased LPS‐induced ICAM‐1 expression, neutrophil recruitment into the lung, production of proinflammatory cytokines TNF‐α and IL‐6, protein permeability, and lung edema formation. LPS‐induced activation of MAPK and the transcription factor NF‐κB in the lung was also significantly enhanced by downregulation of p120. Downregulation of p120 in cultured pulmonary microvascular endothelial cells also enhanced LPS‐induced activation of MAPK and NF‐κB, ICAM‐1 expression, neutrophil adhesion to endothelial cells, and transendothelial neutrophil migration. These effects of LPS were inhibited by overexpression of p120 in endothelial cells. Our results suggest that p120 negatively regulates the lung inflammatory response to LPS by inhibiting the activation of MAPK and NF‐κB and thereby may represent a novel therapeutic strategy for treating sepsis‐associated lung injury. Supported by AHA 0730331N and NIH NHLBI grants 5R01 HL071626 and P01 HL060678.
Commensal bacteria are required for the regeneration of damaged skeletal muscle
Authors: Matsuo Kaori , Fujita Motoo , Nagatomi Ryoichi
Published: June 2021

Regeneration of damaged skeletal muscle requires sufficient supply of nutrients. Fully functional gut assures sufficient supplies of nutrients. Since there are strong lines of evidence that commensal bacteria support gut integrity, we hypothesized that elimination of commensal bacteria may affect muscle recovery from damage. Commensal bacteria of C57BL/6 mice were eliminated by oral administration of antibiotics. Control mice received an equivalent volume of water. Cardiotoxin (CTX) was injected to gatrocnemius muscle to induce muscle damage after the commensal elimination. On different days after CTX, mice were sacrificed and excised gastrocnemius muscle was subjected to immunohistochemical analyses. Although there was no difference in the body weight of antibiotics‐treated and control mice, muscle weight recovery was significantly impaired in antibiotics‐treated even at 20 days after CTX. Histological examination revealed regenerating myogenic cells at day 5 after CTX in both groups. Developmental MHC (dMHC) was positive up to day 7 in the control whereas in the antibody‐treated, dMHC was continuously positive even at day 20. Oral administration of LPS to antibiotics‐treated before CTX effectively restored the delayed recovery of damaged muscle.

Our result suggests that components of commensal bacterial such as LPS favor the recovery of skeletal muscle from muscle damage.
Defining the limits to efficiency in human muscle in vivo
Authors: Nelson Frank Edward , Conley Kevin , Kushmerick Martin
Published: June 2021

How high is too high a value for muscle efficiency? A recent study using an innovative combination of in vivo techniques, 31P NMR with muscle mechanics, found it to be extremely high (68%)(2). To determine if this high efficiency value is within the limits of what we know about muscle efficiency, we explored each of the three factors involved in determining the overall efficiency: efficiency of the cross bridge, loss of mechanical work due to anatomical structure, and energy loss due to activation. The efficiency of the single cross bridge (50%) estimated by Woledge and colleagues(4) is below the current value reported, suggesting it is an over estimate. However, when the efficiency of an array of cross bridges is examined the efficiency increases(3) suggesting 68% is reasonable. Anatomical structure also decreases the maximum efficiency possible, but seems to be a minor factor in the first dorsal interosseous (−5%) based on data from ultrasound images. Recent measurements of the cost of activation in the toadfish (−80%) suggest the value is way too high, but when an estimate of skeletal muscle in mouse (−30%)(1) is used the new estimate seems reasonable. Taken all together the new estimate of contractile efficiency in vivo is a new high, but not too high. Supported by NIH –R01AR041928 and F32AG029064 grants.
Do P2X2/3 and P2X3 receptors play a role in the exercise pressor reflex in cats?
Authors: McCord Jennifer L , Tsuchimochi Hirotsugu , Kaufman Marc P
Published: June 2021

The exercise pressor reflex is due to activation of thin fiber afferents within contracting muscle. These afferents are in part stimulated by ATP activation of purinergic 2X (P2X) receptors during contraction. Although many P2X receptor subtypes exist, the ones that contribute to the exercise pressor reflex are unknown. However, P2X2/3 and P2X3 receptor subtypes are good candidates as they are located on thin fiber afferents and are known to be involved in sensory neurotransmission. To test the hypothesis that P2X2/3 and P2X3 receptors evoke the exercise pressor reflex, we examined the effect of two structurally different P2X2/3 and P2X3 antagonists, A‐317491 (10 μg/kg) and RO‐3 (5μg/kg) on the pressor response to popliteal artery injections of α,β methylene ATP, an agonist for P2X2/3 and P2X3 receptors, and static contraction of the triceps surae muscles in 4 decerebrated cats. We found that both A‐317491 and RO‐3 reduced the pressor response to α,β methylene ATP injection (before blockade Δ 23 ± 4 mmHg; after blockade Δ 14 ± 5 mmHg; P<0.05), suggesting each antagonist was effective in blocking P2X2/3 and P2X3 receptors. In addition, both A‐317491 and RO‐3 reduced the pressor response to contraction (before blockade Δ 30 ± 6 mmHg; after blockade Δ 13 ± 3 mmHg; P<0.05). Our findings, thus far, suggest that P2X2/3 and P2X3 receptors contribute to the exercise pressor reflex in cats. NIH grant AR 051503
P2X receptor blockade attenuates the sympathetic response to exercise in humans
Authors: Cui Jian , Leuenberger Urs A. , Blaha Cheryl , Sinoway Lawrence I.
Published: June 2021

Animal studies suggest that ATP may play a role in evoking the muscle reflex via stimulating P2X receptors on sensory neurons. No data about the role of P2X receptors in the muscle pressor reflex in humans has been reported. We hypothesized that P2X receptor blockade attenuates the exercise pressor response in humans. Blood pressure (Finometer), heart rate, and muscle sympathetic nerve activity (MSNA) from peroneal nerve were assessed during fatiguing isometric handgrip, post exercise muscle ischemia (PEMI), and passive muscle stretch during PEMI in 8 young healthy volunteers. The protocol was performed before and after local infusion of vitamin B6 (Pyridoxine Hydrochloride, a non‐specific blocker of P2 receptors) in saline via Bier block. In the second experiment, the same amount of saline was infused via the Bier block. After Vitamin B6, the MSNA responses to fatiguing handgrip (Δ342±78 vs. Δ548±102), PEMI (Δ280±43 vs. Δ451±85) and passive stretch (Δ338±62 vs. Δ559±107 units/min, all P<0.05) were significantly less than those before B6. The blood pressure responses were also significantly (all P<0.05) less than those before B6. Saline Bier block had no similar effects on the MSNA and blood pressure responses. These data verified our hypothesis that P2X receptor blockade attenuates the exercise pressor response in humans. Supported by AHA 0635245 N, NIH P01 HL077670, and M01 RR010732.
Differential sympathetic activation and blunted cardiovascular diurnal rhythms in a mouse model of hypertrophic cardiomyopathy
Authors: Sabharwal Rasna , Weiss Robert M. , Zimmerman Kathy , Chapleau Mark W.
Published: June 2021

Mutations in the sarcomeric protein α‐tropomyosin (αTM) cause hypertrophic cardiomyopathy (HCM) in humans and mice. We recently reported that mice with HCM, induced by cardiac‐targeted transgenic expression of mutated αTM (Glu180Gly), exhibit myocyte disarray, cardiac fibrosis, left ventricular (LV) dysfunction, and decreased baroreflex sensitivity [Hypertension 2009]. In this study, we tested the hypothesis that sympathetic activity is increased and diurnal variations in heart rate (HR) and blood pressure (BP) are blunted in HCM mice. Cardiac function, BP, HR, locomotor activity and electrocardiograms were measured by echocardiography and telemetry, respectively, in 12–15wk old wild‐type (WT) and HCM mice (n=4, per group). LV ejection fraction and mean BP were significantly reduced in HCM mice. Diurnal changes (night‐day) in BP and HR were markedly attenuated in HCM, despite preserved diurnal changes in locomotor activity (Table). Interestingly, cardiac sympathetic tone (HR response to β‐blocker propranolol) was markedly increased in HCM mice, while vasomotor sympathetic tone (BP response to ganglionic blocker chlorisondamine) was reduced (Table). Furthermore, extensive thrombus in enlarged atria and cardiac arrhythmias were observed in HCM mice.

*P<0.05, HCM vs. WT mice
WT
HCM

Diurnal BP (ΔmmHg)
23±3
10±2 *

Diurnal HR (Δbpm)
76±11
30±14 *

Diurnal Activity (Δcounts/min)
3.9±0.9
6.3±0.8

Cardiac Sympathetic Tone (Δbpm )
−109±12
−200±9 *

Vasomotor Sympathetic Tone (ΔmmHg)
−60±2
−31±4 *

We conclude that cardiovascular diurnal rhythms are blunted and cardiac sympathetic activity is increased in HCM mice. We speculate that altered activity of cardiac afferents innervating the pathologic heart may trigger decreased vasomotor and increased cardiac sympathetic tone and arrhythmias in HCM.