bluuebunny/crossref_metadata_2025 huggingface dataset:
Arsenic binds to redox sensor protein Keap1 directly through the thiol groups of critical cysteines in the linker region of Keap1
Authors: He Xiaoqing , Ma Qiang
Published: June 2021

Arsenic is a well‐documented environmental toxicant and human carcinogen. We have previously reported that arsenic potently induces cytoprotective genes by activating the Nrf2/Keap1/ARE system. Keap1, a Nrf2‐binding partner, is rich in cysteine and may function as a redox sensor of antioxidant inducers. The mechanism by which arsenic and other toxic metals activate Nrf2 is currently unclear. Here we reported that arsenic directly binds with Keap1 through its thiol groups. Fluorescence‐labeled arsenic was shown to bind to purified mouse Keap1 protein in vitro with a high affinity. A thiol reactive agent BAL or excess amount of arsenic competes with the fluorescent arsenic for Keap1 binding. An organoarsenical affinity matrix was developed and was shown to bind with in vitro translated Keap1, purified mouse Keap1, and endogenous Keap1 with a high affinity. Treatment of purified or endogenous Keap1 with arsenic, antioxidant tBHQ, or organoarsenic largely reduced the content of free thiol groups in Keap1. The strongest binding region of Keap1 with PAO matrix was mapped at the linker region of Keap1. Point mutations of cysteine to alanine at the linker region abolished or significantly reduced its binding to the arsenic affinity matrix. Together, these results revealed that arsenic binds with Keap1 directly and the thiol groups of cysteine residues in the linker region of Keap1 play a critical role in arsenic binding.
Gemcitabine in the treatment of relapsed lymphomas
Authors: Boulos Badi M. , Jajeh Ahmad , Tamkus Drimante , Menini Perry
Published: June 2021

Gemcitabine is a diflourinated analyogue of deoxycytidine. It acts as an anti metabilile. It is cell cycle specific and block cell in G1/s interface. It is phosphorylated to its active form and is excreted slowly than other chemotherapeutic drugs. It inhibits DNA synthesis via depletion of cellular deoxynucleotide pools and by way of masked DNA chain termination. A total of 54 patients were treated and have been followed for seven years. Mean age was 54 years. There were 42 males and 12 females. There were 12 Hispanic, 15 White, 24 African American and 3 others. Mycosis fungoides reversal stage (4), Mantle cell NHL (non‐hodgkins lymphoma) (2), Peripheral T‐Cell NHL including NK (natural killer), cell Gamma/Delta sub‐type Lymphoma (7), Anaplastic New cell NHL (2), composite NHL (1), NHL (23), HD (10), other cell sLL/Richter Transformation (5). The response rate was over 75% achieved in patients with NHL with advanced stages III & IV. None of the patients had grade 3 or 4 toxicity. In conclusion gemcitabine is a valid and safe option in the treatment of relapsed and refractory cases of lymphomas.
Herb‐drug interaction involving oseltamivir and Chinese medicine formula
Authors: Chow Moses Sing Sum , Chang Qi , Zuo Joan
Published: June 2021

Aim: The empiric use of certain Chinese medicine formula (CMF) in combination with oseltamivir (O) is advocated for the treatment of deadly avian influenza in Asia. This study aimed to determine the interaction potential of such combination.

Method: Two groups of rats received either O alone or O+CMF (Yin Qiao San/Sang Ju Yin) at equivalent human doses for 5 days. CMF was given 2 h post O dose. Following the last dose, urine as well as plasma O and its active metabolite (M) concentrations were determined, after adding dichlorvos to prevent hydrolysis of O. All drug concentrations were determined by a validated LC/MS/MS method.

Result: The Cmax and area under the concentration time curve of M were significantly reduced in the combination compared to O alone group (1.3±0.2 vs 1.8±0.3 ug/ml and 326±30 vs 452±101 ug.min/ml respectively, P <0.05). The M/ O ratios were 1.96±0.25 vs 2.92± 0.69 respectively (P<0.05). A trend of greater renal clearance of M in the combination group (7.60 vs 6.24 ml/min) was also observed.

Conclusion: Our results indicate that CMF can potentially cause a significant reduction of the active M concentration, most likely due to suppression of O metabolism as well as possibly enhanced M renal clearance. Pharmacokinetic interactions from herb‐drug combinations could potentially involve multiple mechanisms and should be carefully studied before their empiric use. (Funded by Hospital Authority of Hong Kong).
Macrophage‐specific LITAF (lipopolysaccharide induced TNF‐alpha factor) knockout mice (LITAF mac −/−) have a reduced inflammatory response to colonic administration of trinitrobenzene sulfonic acid (TNBS)
Authors: Bushell Kristen Nicole , Leeman Susan E , Amar Salomon , Reed Karen L , Gower Adam C , Stucchi Arthur F , Becker James M
Published: June 2021

Dysregulation of TNF‐α secretion by lamina propria macrophages (LPM) is a hallmark of the inflammatory bowel disease Crohn's Disease (CD). LITAF has been shown to regulate TNF expression in macrophages and macrophage‐specific LITAF mac −/− mice (KO) are resistant to LPS induced sepsis. LITAF protein is increased by 30% in LPM isolated from C57BL/6 mice (WT) after TNBS administration, a well established model of CD (p<0.05). The aim of this study was to compare the colonic inflammatory responses of WT mice to LITAF mac−/− mice after administration of TNBS.

Methods

: WT and KO mice were administered TNBS (3 mg in 50% EtOH) rectally. After 24 hrs, body weights (BW) were determined and levels of the inflammatory marker myeloperoxidase (MPO) and TNF‐α mRNA were measured in colon tissue.

Results

: TNBS administration reduces BW in all animals compared to untreated controls (p<0.05); however, KO mice lost 50% less BW than the WT mice (p<0.05). MPO activity is five fold lower in KO mice compared to WT (p<0.05). Tissue levels of TNF‐α mRNA is also reduced in the KO compared to WT animals (376 ± 156 vs. 843± 253 copies) after TNBS administration.

Conclusions:

LITAF mac −/− have an attenuated inflammatory response to colonic TNBS administration. These results highlight a potential value of anti‐LITAF strategies for therapeutic management of CD. Research Support: BMC departments of Surgery and Pharmacology and NIDCR R01 DE014079.
Inhibition of IFN production by the nrf2 activators, tBHQ and BHA, in activated murine T cells
Authors: Rockwell Cheryl Elizabeth , Klaassen Curtis D
Published: June 2021

Nuclear factor erythroid 2‐related factor 2 (Nrf2) is a transcription factor that is activated by oxidative and other stresses which has been best characterized for its cytoprotective role in the induction of antioxidative and detoxification genes. However, recent studies have demonstrated that Nrf2 also plays an anti‐inflammatory role in macrophages and other myeloid cells. The purpose of the present studies was to determine the effects of Nrf2 activators on T cell function. Accordingly, the current studies show that the Nrf2 activators, tert‐butylhydroquinone (tBHQ) and butylated hydroxyanisole (BHA), suppress IFNγ secretion by anti‐CD3/anti‐CD28‐activated T cells in a concentration‐dependent manner. Likewise, tBHQ and BHA suppress IFNγ mRNA levels in wild‐type T cells but not nrf2‐null T cells, demonstrating that the inhibition of IFNγ occurs at the transcriptional level and is nrf2‐dependent. In addition, Nrf2 activation abrogates the production of other T cell cytokines, including IL‐2 and TNFα. Collectively, these data suggest that Nrf2 activators suppress production of IFNγ, IL‐2, and TNFα by activated T cells which may contribute to the anti‐inflammatory effects of these compounds. Furthermore, the present studies demonstrate that inhibition of IFNγ by Nrf2 activators occurs transcriptionally and is Nrf2‐dependent. (Supported by NIH grants ES09649, ES09716, ES07079, and RR021940.)
AMPK ‐ the fuel gauge of the eukaryotic cell
Authors: Hardie David Grahame
Published: June 2021

The AMP‐activated protein kinase (AMPK) is expressed in essentially all eukaryotic cells and acts as a sensor of cellular energy status. It is a heterotrimer comprised of catalytic alpha subunits and regulatory beta and gamma subunits, and is only active after phosphorylation at Thr‐172 within the kinase domain by upstream kinases. AMPK is activated whenever a metabolic stress causes a decrease in the cellular ATP:ADP ratio: because of the adenylate kinase reaction, this is always accompanied by a much larger increase in the AMP:ATP ratio. Binding of AMP to the gamma subunit of AMPK causes two effects that are antagonized by ATP: (i) it inhibits dephosphorylation of Thr‐172, causing a sensitive switch to the active, phosphorylated form; (ii) it allosterically activates the phosphorylated kinase. The major upstream kinase in most cells is the tumor suppressor LKB1. Since AMPK can cause an arrest of cell growth and the cell cycle, activation of AMPK may explain why LKB1 is a tumor suppressor, although LKB1 also acts upstream of a family of twelve AMPK‐related kinases. In some cell types (e.g. neurones, endothelial cells and T cells), AMPK is also activated by increases in cell calcium, which trigger phosphorylation of Thr‐172 by CaMKK‐beta.

The beta subunit of AMPK in all eukaryotes contains a carbohydrate‐binding module (CBM) that in mammalian cells causes AMPK to associate with glycogen. Recent work from our laboratory suggests that the CBM is a regulatory domain that inhibits AMPK when the reserves of energy in the form of glycogen are high.
RhoA mediates cardiomyocyte survival through FAK‐dependent Akt activation
Authors: Re Dominic Del , Brown Joan Heller
Published: June 2021

The small G‐protein RhoA is a transducer of signals from both GPCRs and the extracellular matrix, influencing cell morphology as well as gene expression. A role for RhoA in cultured cardiomyocyte hypertrophy has been established, however the possibility that RhoA can influence myocyte survival has not been examined. We find that adenoviral expression of activated RhoA protects neonatal rat cardiomyocytes from apoptotic insults induced by peroxide or glucose deprivation. Interestingly, expression of RhoA induces PI3K‐dependent activation of the cardioprotective kinase Akt. It has been reported that RhoA mediates FAK activation and that FAK can directly interact with the p85 regulatory subunit of PI3K. Accordingly, we examined the possible role of FAK in RhoA signaling to Akt. RhoA expression increased FAK phosphorylation and focal adhesion formation in cardiomyocytes in a Rho kinase‐dependent manner. Further, RhoA‐induced Akt activation is prevented by co‐expression of FRNK, a negative regulator of FAK, suggesting that FAK is not only activated but mediates this response. Expression of FRNK also blocked the protective effect afforded by RhoA further implicating FAK in this signaling pathway. The finding that RhoA activation protects cardiomyocytes through FAK‐dependent Akt activation suggests that activation of similar pathways by mechanical stretch could regulate cardiomyocyte survival.

NHLBI R01 HL‐28143
Decreased expression of Gi in erythrocytes from humans with type II diabetes is associated with impaired ATP release in response to decreased oxygen tension
Authors: Hanson Madelyn , Sprague Randy , Bowles Elizabeth , Achilleus David , Stephenson Alan , Ellsworth Mary
Published: June 2021

Erythrocytes (RBCs) release ATP in response to low O2 tension (pO2) via a signaling pathway that requires the G protein, Gi. RBC‐derived ATP is proposed to participate in the distribution of perfusion to meet tissue O2 need. Here, we investigate the hypothesis that reduced Gi levels in RBCs, reported in humans with type II diabetes (DMII), is associated with impaired ATP release in response to reduced pO2. RBCs of rabbits, healthy humans (HH) and humans with DMII were placed in a tonometer and exposed to either 15% or 0% O2 with 6% CO2 (10 min). Reduced pO2 induced ATP release from rabbit and HH RBCs of 93 ± 78% (n= 4, pO2=26 ± 2) and 292 ± 110% (n= 9, pO2= 18 ± 3), respectively. RBCs of humans with DMII failed to release ATP in response to this stimulus (n= 3, pO2= 24 ±1). To determine if RBCs that release ATP in response to reduced pO2 have a role in determining vascular caliber, isolated perfused striated muscle arterioles were exposed to reduced pO2 in the presence and absence of rabbit RBCs (15% hct). When rabbit RBCs were present, vessel diameter increased by 26 ± 11% (n= 7, pO2= 36 ± 5 mm Hg) while vessels perfused with buffer constricted (n= 10, pO2= 28 ± 3 mm Hg). These results demonstrate that decreased Gi levels in RBCs are associated with failure to release ATP in response to reduced pO2 which could contribute to the vascular complications present in patients with DMII. Funded by AHA Fellowship, NIH grants HL‐64180, HL‐89094, ADA grant RA‐133.
Hemorheology in Alzheimer's disease: correlation between real‐time microvascular abnormalities and whole blood viscosity
Authors: Smith Mary Meighan , Chen Peter C. , Cheung Anthony T.
Published: June 2021

We hypothesized that changes in hemorheologic parameters, including vessel diameter, flow velocity, and whole blood viscosity (WBV), would be present in Alzheimer's disease (AD) and would correlate with microvascular abnormalities (vasculopathy). Using the Rheolog™, we obtained WBV profiles, measured at shear rates of 1‐1,000 s−1, for 10 AD patients and age matched non‐AD controls. Vessel diameter, flow velocity, and vasculopathic changes were quantified using computer‐assisted intravital microscopy (CAIM) of the conjunctival microcirculation. A Severity Index (SI), scale 0–15, was computed to reflect degree of vasculopathy. AD patients had significantly higher WBV (3.96±0.29cp vs. 3.34±0.05cp, sheared at 300s−1; P<0.05) and SI (7.00±2.36 vs. 0.61±0.70; P<0.05) than controls. WBV positively correlated (r=0.68; P<0.05) with SI in AD patients. AD patients with severe dementia had significantly higher WBV (4.23±0.19cp vs. 3.68±0.13cp; P<0.05) and SI (8.60±2.30 vs. 5.4±0.89; P<0.05) than less demented patients. Severity of dementia in AD patients did not necessarily correlate with disease duration, but correlated significantly with WBV and severity of vasculopathy. These results strongly suggest the involvement of hemorheologic changes and peripheral vasculopathy in AD.
Sexual and Maturational Difference in Phosphodiesterase mRNA Expression in Rat Skeletal Muscle Microvascular Endothelial Cells
Authors: Wang Jianjie , Huxley Virginia H.
Published: June 2021

The levels of intracellular cAMP and cGMP, important mediators of microvessel permeability (Ps), are regulated predominantly by the rates of synthesis and hydrolysis. Phosphodiesterases (PDE) are central to the stimulation or inhibition of hydrolysis of cAMP and cGMP. Our previous studies demonstrated that basal Ps to albumin was greater in adult males (AM) than adult females (AF), juvenile males (JM) and females (JF), but there were no differences among AF, JM, and JF. Therefore, we hypothesized differential PDE expression in AM relative to AF, JM, and JF, resulting in different levels of cAMP and cGMP and consequent difference in basal Ps. In this study we used endothelial cells (EC) derived from rat skeletal muscle microvessels of AM, AF, JM and JF used for Ps studies to detect PDE 1–5 mRNA expression via real time RT‐PCR analysis. We found that mRNA level of PDE1A was 2.5‐fold higher in JM than JF (n=5). PDE2A expression was dependent on maturity while PDE2A levels were higher in juveniles than adults (n=7) in both sexes, and 2‐fold higher in JF (n=6) than JM (n=7). PDE3B was expressed 2.4‐fold higher in JM (n=7) than AM (n=7). PDE4D expression did not differ among the 4 groups of EC. JM (n=5) expressed higher PDE5A than AM (n=6). The findings indicate differential expression of PDE isoforms among the 4 groups of microvessel EC. However, the relationship between PDE expression and levels of cAMP and cGMP remains to be elucidated.
Understanding the mechanisms of cerebral blood flow with respect to motor learning
Authors: Hart Lucas M. , Jakovcevic Danica , Harder David
Published: June 2021

Neurons in the brain have been shown to undergo long‐term potentiation as a result of motor learning; under similar conditions, astrocytes undergo volume expansion. Neurons and astrocytes make up two of the three components comprising the neurovascular unit (NVU). The NVU matches local cerebral blood flow to neuronal activity resulting in functional hyperemia (FH). This investigation is aimed at developing a technique to study the third component of the NVU, cerebral vasculature, and determine whether FH undergoes plasticity over the course of motor learning. Male rats are pre‐trained in a skilled reaching task for food pellets followed by surgical placement of burr holes bilaterally over the forelimb sensorimotor cortex. A ring is then attached to the skull over each burr hole, which allows a laser Doppler flow probe to be fixed in place during motor training and removed each day following training. Rats are then subjected to a ten‐day training period. We show here that FH responses can be recorded in rats during training in a skilled reaching task. By using this technique to elucidate how FH changes in motor learning, we can provide important information on the plasticity of cerebral vasculature as the brain adapts to a task. This research may assist in future discoveries leading to early diagnosis and rehabilitation of cerebrovascular diseases. This study is funded by the National Institutes of Health grant P01 HL59996.
Elevated arterial wall calcium in small diameter cerebral arteries from rabbit model of subarachnoid hemorrhage
Authors: Nystoriak Matthew A. , Wellman George C.
Published: June 2021

Cytosolic Ca2+ in cerebral artery smooth muscle is a critical determinant of blood vessel diameter and cerebral blood flow. Cerebral vasospasm associated with subarachnoid hemorrhage (SAH) results from complex mechanisms that may involve increased Ca2+ influx via voltage dependent Ca2+ channels (VDCCs). However, intracellular Ca2+ levels in small diameter cerebral arteries following SAH are currently unknown. Here, the objective was to measure arterial wall [Ca2+] of intact pressurized small diameter cerebral arteries from a rabbit model of SAH. Cerebral arteries were mounted in an arteriograph chamber and loaded with Ca2+ indicator dye, Fura‐2. Under basal conditions (6 mM K+ & 20 mmHg), arteries from SAH animals exhibited elevated wall [Ca2+] compared with arteries from control animals. Further, membrane potential depolarization due to elevated extracellular K+ (60 mM) or increased intravascular pressure caused a greater increase in arterial wall Ca2+ in arteries from SAH animals. Elevated arterial wall [Ca2+] corresponded to enhanced constriction at 80 and 110 mmHg and was abolished by VDCC blockers. These results suggest elevated arterial wall [Ca2+] due to increased VDCC activity contributes to enhanced arterial constriction following SAH. This work is supported by the Totman Medical Research Trust Fund, the Peter Martin Brain Aneurysm Endowment, the AHA and the NIH.
Nicotine Impairs eNOS‐Dependent Responses of the Basilar Artery
Authors: Mayhan William G , Arrick Denise M , Sun Hong
Published: June 2021

Our goals were to determine whether acute exposure to nicotine alters eNOS‐dependent responses of the basilar artery and to identify a potential role for activation of NAD(P)H oxidase. We measured diameter of the basilar artery to eNOS‐dependent (acetylcholine) and ‐independent (nitroglycerin) agonists before and during acute infusion of nicotine in the absence or presence of apocynin. In addition, we measure superoxide production by brain tissue to nicotine. We found that eNOS‐dependent vasodilatation was impaired during treatment with nicotine and that apocynin prevented this impairment. Further, the production of superoxide was increased by nicotine and this increase could be inhibited by apocynin. We suggest that nicotine impairs eNOS‐dependent dilatation of the basilar artery by activation of NAD(P)H oxidase.
Caveolin‐1 knockout mice are resistant to the neuroprotective effect of ischemic preconditioning
Authors: Chin‐Lee Blake , Yokoyama Takaakira , Head Brian P , Tsutsumi Yasuo M , Roth David M , Patel Piyush M , Patel Hemal H
Published: June 2021

Caveolin‐1 (Cav‐1) is thought to be a structural component of caveolae in numerous cells. Although no caveolae exist in neurons, Cav‐1 is expressed and may contribute to signaling. We recently showed the involvement of Cav‐1 in protection of isolated neurons. No studies have assessed the role of Cav‐1 in in vivo models of cerebral protection. We hypothesized that ischemic preconditioning, where a sublethal stress protect from a more lethal stress, is absent in Cav‐1 knockout mice. We induced global cerebral ischemia by bilateral carotid artery occlusion (BCAO) with cerebral blood flow (CBF) monitoring. Mice were divided into the following groups: sham, lethal ischemia (LI: BCAO for 12 min), and preconditioning (PC: BCAO for 3 min followed by 24 hr recovery prior to LI; in both wild‐type and Cav‐1 knockout mice). At 3 days following final surgery, the percent of intact CA1 neurons was calculated. Additionally, the plasticity of the posterior communicating arteries on both sides was scored. The percent of intact CA1 neuron in PC group was nearly 3‐fold higher than that in LI group (67.9 ± 7.1 %; PC, 25.8 ± 7.0 %; LI, p<0.001). However, in the PC Cav‐1 knockout mice, similar CA1 neuron loss to the LI group was observed (31.9 ± 9.7 %; PC of Cav‐1 knockout, 25.8 ± 7.0 %; LI of wild‐type, p>0.05). The minimum CBF after BCAO was correlated to the plasticity score. There was a tendency that plasticity score was increased relative to aging in Cav‐1 knockout mice. In conclusion our data suggest that 1) Cav‐1 is important to neuronal protection in vivo and 2) collateral formation appears to be linked to loss of Cav‐1 gene expression and age. Cav‐1 might serve as a unique target for neuronal preconditioning agents.
A beta 1+2 adrenergic receptor antagonist blunts the decrease in the cerebral metabolic ratio during exhaustive exercise in humans
Authors: Seifert Thomas , Overgaard Morten , Rasmussen Peter , Secher Niels , Nielsen Henning Bay
Published: June 2021

Intense exercise decreases the cerebral metabolic ratio of O2 to carbohydrates (glucose + ½ lactate), but whether it is influenced by administration of a beta 1+2 adrenergic receptor antagonist is not known. In eight males incremental cycle ergometry increased the arterial lactate to 15.34 ± 1.46 mM (mean ± SEM) and arterial‐jugular venous (a‐v) difference from −0.02 ± 0.01 mM at rest to 1.01 ± 0.16 mM (P < 0.05). The a‐v difference for glucose increased from 0.66 ± 0.09 to 0.89 ± 0.05 mM (P < 0.05) at exhaustion and the cerebral metabolic ratio decreased from 5.5 ± 0.5 to 3.0 ± 0.1 (P < 0.01). During exercise with beta 1+2 blockade heart rate (69 ± 3 to 58 ± 2 beats/min) and exercise capacity (229 ± 15 W to 195 ± 11 W; P < 0.05) were reduced and arterial lactate reached 9.35 ± 1.34 mM. During exercise with beta 1+2 blockade, the increase in a‐v lactate difference (to 0.48 ± 0.19 mM; P < 0.05) was attenuated and the a‐v glucose difference and the cerebral metabolic ratio remained at levels similar to those at rest. This study demonstrates that beta 1+2 adrenergic blockade blunts the decrease in the cerebral metabolic ratio during exercise by reducing the cerebral uptake of lactate and glucose.
An estrogen‐induced decrease in voltage‐gated calcium channel expression attenuates coronary artery contractility
Authors: Hill Brent J.F. , Rusch Nancy J.
Published: June 2021

Estrogen (E2) can pharmacologically inhibit Ca2+ influx into vascular smooth muscle cells. However, little is known about the ability of E2 to alter vascular Ca2+ channel expression. The goal of our study was to determine if a physiological E2 concentration can decrease the expression of voltage‐gated calcium channels (VGCC) in small coronary arteries. Right coronary arteries were obtained from hearts of reproductively cycling sows. The distal end of the artery was cut into 4 mm rings or longitudinal strips and incubated for 24 hours in E2 (1 nM) or its ethanol solvent. Western blots using protein isolated from the arterial strips demonstrated a decreased expression of the pore‐forming α1C subunit of the VGCC in E2‐treated arteries. In parallel studies, isometric tension recording revealed that E2‐treated arterial rings contracted significantly less to 30 mM KCl. However, E2 and ethanol‐treated rings preconstricted with 60 mM KCl showed similar relaxation responses to E2 (1 nM to 1 μM). These findings suggest that physiological concentrations of E2 down‐regulate VGCC, thereby providing a potentially novel mechanism for limiting coronary vasoconstriction. In the same arteries, E2‐dependent relaxation is intact. By limiting the expression of VGCC, E2 may promote coronary blood flow and potentially lower the incidence of coronary events in pre‐menopausal women. Support: NCRR of the NIH, Grant #P20 RR‐16460
Wave intensity analysis of coronary pressure and velocity for studying coronary‐ventricular interactions
Authors: Siebes Maria , Kolyva Christina , Verhoeff Bart‐Jan , van den Wijngaard Jeroen P.H.M. , Piek Jan J. , Spaan Jos A.E.
Published: June 2021

Coronary wave intensity (WI) represents the energy carried by traveling waves that are generated by cardiac contraction and relaxation.

Distal coronary pressure (Pd) and Doppler velocity (v) were measured in 26 patients at rest and during adenosine‐induced hyperemia before and after revascularization by stent placement. WI was calculated as (dPd/dt) x (dv/dt) and separated into forward (FW) and backward (BW) components during the cardiac cycle.

We found a consistent pattern of BW and FW compression waves during ventricular contraction followed by FW and BW expansion waves in the early ventricular relaxation phase. Individual energies of expansion and compression waves traveling in the same direction were strongly correlated (P< 0.001). The size of BW compression and expansion waves was similar, while FW compression waves were in all cases larger than FW expansion waves (P<0.01). The energy of BW waves at rest was strongly correlated with that during hyperemia (P<0.001). An increase in BW wave energy after stent placement was strongly related to an increase in coronary microvascular conductance (P<0.001).

Our results are consistent with the intramyocardial pump model (BW waves), higher wall stiffness at the end of systole (difference in FW waves) and similar redistribution of vascular resistance at vasodilation (rest vs hyperemia) despite different vascular anatomy.

(Support: NHF grants 2000.090 and 2006B186)
Nitric oxide‐dependent regulation of coronary vascular resistance in hypertension
Authors: Levy Andrew Shawn , Rush James W.E.
Published: June 2021

Hypertension is associated with impaired endothelium dependent relaxations in many vascular beds. Evidence from the coronary vasculature offers conflicting results. The current investigation examined the role of NO in regulating coronary vascular resistance (CVR) in spontaneously hypertensive rats (SHR) and their normotensive counterparts (WKY). Rats were anesthetized, the hearts were removed and the aorta was cannulated to allow for retrograde coronary perfusion using a constant flow Langendorff set‐up. Flow was assigned based on heart weight which was estimated from body weight. SHR had a greater baseline CVR than the WKY (6.72±0.34 vs 4.97±0.62 mmHg/ml/min, p<0.05). Following ET‐1 administration SHR had a greater resistance (9.82±0.75 vs 6.87±0.96 mmHg/ml/min, p<0.05) although the absolute change was not different. There were no differences in maximal reduction in CVR to bradykinin (BK) or sensitivity to BK. Treatment with LNAME caused an increase in resistance in both groups although the change in resistance was not different between the SHR and WKY (3.46±0.41 vs 3.02±0.69 p>0.05). LNAME completely abolished the BK induced reduction in CVR in both the SHR and WKY; the response to sodium nitroprusside was unchanged. Thus, the response to BK is NO dependent in both SHR and WKY. The CVR is elevated in the SHR and inhibition of eNOS increases CVR to a similar extent in both strains.

Funded by NSERC & HSFO
Superoxide balance in aging skeletal muscle
Authors: Furlong Meghan J , Schilling Samantha R , Hruskocy Heather N , Limberg Jacqueline K , Eldridge Marlowe W , Schrage William G
Published: June 2021

The goal of this study was to determine the age‐induced changes in antioxidant and pro‐oxidant status in skeletal muscle. Contracting skeletal muscle is ripe for production of reactive oxygen species, which may contribute to impaired muscle blood flow in older adults by overwhelming NO signaling in arteries. We hypothesized that muscle from older humans would exhibit reduced levels of antioxidant proteins and increased levels of pro‐oxidant proteins. A muscle biopsy was taken from the vastus lateralis muscle in young (18–40 yrs) and older (65–77 yrs) subjects. Muscle fibers were homogenized, adjusted for equal concentrations, and we assessed protein expression via Western blot and ECL. Proteins of interest were superoxide dismutase (SOD‐1, SOD‐2), catalase, and xanthine oxidase (XO). Probing for nitrotyrosine served as a marker for protein oxidative stress. Preliminary results (young n=3, older n = 5) showed that catalase expression was higher in young adults (P=0.04) while SOD‐1 levels were approximately equal. SOD‐2 levels were greater in older adults (P=0.01) as were nitrotyrosine levels. XO levels tended to be higher in the older adult group (P=0.07). These data suggest that aging skeletal muscle shows increased oxidative stress in parallel with lower catalase and higher XO and nitrotyrosine levels. This may ultimately lead to a decrease in NO bioavailability in aging human skeletal muscle during exercise. Funded by the Wisconsin Alumni Research Foundation (WARF).
CHEMICAL MICRO‐SENSORS FOR BIOMEDICAL RESEARCH
Authors: TAHA ZIAD HUSSEIN
Published: June 2021

The design, performance and applications of chemical micro‐sensors for biomedical research are described.

Nitric oxide (NO) sensors have been built to enable the users to measure NO in a wide range of biological and non‐biological samples. The sensors incorporate the sensing elements behind a gas permeable membrane to assure the selectivity of measurements in addition to isolating these elements from any ionic changes in the sample resulting in a stable measurements. Many sensors were built to fit many applications. The sensors are capable of measuring NO at the nanomolar levels with fast response time and wide linearity. The sensors have been used to measure NO in live animals, brain slices, cell culture, tissue samples, and many others. The sensors can indirectly measure nitrite at the nano‐molar level.

A hydrogen sulfide sensor was designed to selectively detect hydrogen sulfide in biological samples. Sensors with tip diamater size of 200–500 micron were prepared. Other sensors include pH, oxygen and carbon monoxide were designed.
Gene expression analysis of circulating leukocytes of rat strains exhibiting long and short survival times following severe hemorrhage
Authors: Bowman Phillip D. , Bynum James A , Klemcke Harold
Published: June 2021

A marked differential survival (0.5 to 6 h) time of multiple rat strains to severe hemorrhage was previously demonstrated (Klemcke et al; Shock, in press 2007). To explore a genetic contribution to differential outcomes, gene expression analysis was performed on long surviving Brown Norway/Mcwi (BN; ∼300 min) and short surviving (DA/OlaHsd (DA;∼40 min)) inbred strains. Anesthetized rats (5 rats per group) were catheterized and, ∼ 24 hours later, 55% of the calculated blood volume was removed during a 26 min period from conscious unrestrained animals. The first ml of blood and the final ml were taken for RNA isolation. RNA was labeled with Cy3 or Cy 5 and hybridized to Agilent rat whole genome microarrays and analyzed for statistical differences in gene expression. No differences in expression were noted between start and finish of hemorrhage. Between strain comparisons of expression patterns indicated large difference between the long surviving BN and short‐surviving DA. 1056 genes were statistically different in their expression with a false discovery rate <5%. Analysis of statistically altered expression patterns with Ingenuity Pathway Analysis™ program indicated differences between long and short surviving strains including pathways for free radical scavenging, organismal survival and mitochondrial dysfunction that may explain differences in survival times.
Patterns of transcriptome expression associated with cardiac injury following E. coli peritonitis
Authors: Goldfarb Roy David , Gandhi Snehal , Cinel Ismail , Wang Qi , Andrew Brooks I , Parrillo Joseph E
Published: June 2021

Cardiac dysfunction is a feature of porcine peritonitis. In order to gain insight into the fundamental mechanisms of this phenotype, gene expression analysis (Affymetrix) was applied to serial cardiac biopsies of sham (n=2) and E. coli infected pigs (n=3). Cardiac samples were taken at basal and hourly after infection. Sham pigs had stable heart rate, cardiac output (CO) and core temperature for the 5 hr period; infected pigs demonstrated an early elevation in CO and ventricular shortening and/or ejection (assessed by echocardiography) followed by development of hypodyanmics. Genes were determined to be differentially regulated at a greater than or less than twofold change and p<0.05. In infected animals, increasing numbers of genes were up‐ or down‐regulated (36, 278, 514, 842 and 1238 at 1, 2, 3, 4 and 5 hr) whereas sham infection altered fewer (247, 67 and 384 genes at 2, 3, 4 hr) Comparing sham vs infected animals at the same time, numbers of significantly altered genes increased with time (32 at basal, to 74, 189 and 601 at 2, 3, and 4 hr post infection.) Gene expression patterns were also examined; 180 genes increased steadily after peritonitis, including many in the cytokine cascade and intracellular signaling; 240 decreased steadily; 9 went up followed by down, including TLR4; and 5 were down to up. These results will provide biomarker and mechanistic insights to pathogenesis of cardiac dysfunction of septic peritonitis and may also help identify some altered novel gene transcription pathways that can serve as new targets for diagnostic tools and therapeutic strategies. All candidate genes will be validated by quantitative PCR.
Ubiquitin Conjugation Regulates Hypotonic Stress‐Induced Trafficking of Short ClC‐3 Channel
Authors: McCloskey Diana T. , Dai Yan‐Ping , Mutafova‐Yambolieva Violeta N. , Hume Joseph R. , Yamboliev Ilia A.
Published: June 2021

Short ClC‐3 channel (sClC‐3) functions include lysosomal acidification and cell volume regulation, requiring lysosomal and surface membrane localization, respectively. As sClC‐3 is primarily a cytosolic protein how ClC‐3‐dependent volume regulation occurs remains undefined. Immunofluorescent labeling of COS‐7 cells revealed both native and expressed ClC‐3 are associated with lysosome‐like vesicles, and less than 10% resides at the cell surface. Hypotonic stress induced export trafficking of sClC‐3 to the cell surface membrane. The surface membrane exposure of sClC‐3 was transient and was followed by spontaneous or hypertonic stress‐induced internalization. Membrane‐associated sClC‐3 underwent ubiquitin conjugation followed by endocytosis of membrane sClC‐3. Inhibition of proteolysis with MG132 and Bafilomycin A potentiated both the surface retention of Flag‐sClC‐3A and the hypotonic stress‐induced Cl− current. Ubiquitin‐specific protease (USP) 7 and much less USP2 or ubiquitin C‐terminal hydrolyze, L1, mediated de‐ubiquitination of sClC‐3 in vitro. These results demonstrate that sClC‐3 can shuttle between the cytosol and cell surface membrane and the latter could contribute to cell volume regulation. Ubiquitination and de‐ubiquitination of sClC‐3 may play a role in the fine regulation of the cell volume during hypotonic stress. This study was supported by NCRR grant P20RR 15581.
OUABAIN RESCUES NEPHROGENESIS IN GROWTH‐FACTOR DEPRIVED EMBRYONIC RAT KIDNEY
Authors: Li Juan , Perini Ilaria , KRUUSMÄGI MARKUS , AIZMAN OLEG , ZELENIN SERGEY , aperia anita
Published: June 2021

Fetal malnutrition and other factors contributing to renal growth retardation result in increased apoptosis and reduction in nephron endowment. Our group has described a novel signaling pathway, where direct interaction between Na,K‐ATPase (NKA) and IP3R triggers slow calcium oscillations and activation of NFkappaB (PNAS 2001, JBC 2003, 2006). This signaling pathway is activated by ouabain, a specific ligand of NKA. Downstream effects include protection from apoptosis and stimulation of cell proliferation (JASN 2006). Here we report activation of this signaling pathway rescues nephrogenesis in growth factor deprived (gfd) embryonic rat kidney. Kidneys from E14 rats were cultured under normal or gfd conditions and studied after 2–3 days in vitro. Exposure to ouabain triggered calcium oscillation and activated NF‐kappaB. Growth factor deprivation retarded ureter branching and formation of new glomeruli and increased apoptotic index. Exposure to nM ouabain prevented these effects. The protective effects of ouabain were abolished by depletion of intracellular calcium stores and by inhibition of NF‐kappaB, underlining the importance of the NKA‐IP3R signaling pathway. The expression of Wt1 and Pax2, which are activated by NF‐kappaB, was increased in ouabain exposed gfd kidneys. Thus we have identified a novel mechanism by which kidney development can be protected under adverse intrauterine circumstances.
Angiotensin II Increases Phosphorylation of the Na,K‐ATPase at Ser‐943 Under the Physiological Conditions Associated with Stimulation of Activity
Authors: Massey Katherine J. , Mattingly Raymond R. , Li Quanwen , Yingst Douglas R.
Published: June 2021

We previously reported that stimulation of activity of rat Na,K‐ATPase expressed in opossum kidney (OK) cells and increases in the amount of α‐1 subunit in the plasma membrane (PM) under physiological conditions using 10−11 M angiotensin II (Ang II) are eliminated by mutating Ser‐943 of the α‐1 subunit to alanine (S943A). Ser‐943 is a putative site for phosphorylation by PKA. Nevertheless, whether it is phosphorylated by PKA under physiological conditions is controversial. To examine if phosphorylation at Ser‐943 is involved in the mechanism by which Ang II alters the activity of the Na,K‐ATPase, we developed lines of OK cells that stably express the AT1a receptor and either the wild‐type rat α‐1 subunit, the S943A mutant or a version with both Ser‐16 and Ser‐23 mutated to alanines. We treated cells +/−10−11 M Ang II for 15 min. at 37° or with IBMX/forskolin. Using a phosphorylation‐sensitive antibody, we showed that Ang II and IBMX/forskolin increased phosphorylation of the α‐1 subunit in cells expressing the wild‐type α‐1 subunit, but not the S943A mutant. Although Ang II does not stimulate the activity of the pump when Ser‐16 or Ser‐23 are mutated to alanines, we also observed increased phosphorylation of the α‐1 subunit in the double mutants S16,23A. From these data we conclude that under physiological conditions associated with stimulation of activity, Ang II increases phosphorylation of the Na,K‐ATPase at Ser‐943. Supported by NIH DK60752.
Aldosterone increases BK channel trafficking in human embryonic kidney cells
Authors: Bari Muhammad R , Grimm Paul R. , Settles Deann C. , Foutz Ruth M. , Holtzclaw David , Sansom Steven C.
Published: June 2021

Large conductance Ca2+‐sensitive K+ channels (BK) reside in the distal nephron where they secrete K+ when the rate of distal flow is increased. A recent study showed that aldosterone (aldo) stimulated BK‐mediated K+ secretion in an isolated mouse distal colon preparation. We used HEK293 cells, expressing BK channels, to determine whether aldo increases the number of BK channels in the plasma membrane by a non‐genomic mechanism. Immunocytochemistry, immunoblotting and RT‐PCR confirmed that HEK293 cells contain mineralocorticoid receptors (MR). Using single channel patch clamp analysis, we determined the 30 min. application of aldo (10 nM) on the NPo (total open time of all channels in a patch), and the percent of observed channels per patch. In cell attached patches (−Vp = −40 mV), aldo increased the NPo of BK from 0.71±0.05 to 1.84±0.41 (p=<0.05, n=3) and the percent of observed BK per patch from 58% to 71% (p<0.05, n=4). Treating cells with aldo plus spironolactone, a MR antagonist, reduced the number of detected BK channels to 50% (p<0.05, n=3). The aldo‐treated cells showed an increased quantity of cell surface biotinylated BK (OD, 1.46±0.19 in control and 3.87±0.76 with aldo, p<0.05, n=3) that was inhibited to OD, 1.32±0.35 by 5 μg/ml brefeldin‐A (p<0.05, n=3). These data suggest that aldosterone, via MR, increases the trafficking of expressed BK channels from the ER to the apical membrane of HEK293 cells. Grant support: NIH RO1‐DK49561 and AHA 0610059Z.
The last residue of the Na, K‐ATPase catalytic subunit isoform specific region (ISR) plays a critical role in Na, K‐ATPase membrane trafficking
Authors: Sottejeau Yoann , Pierre Sandrine V , Duran Marie‐Josee , Carr Deborah L
Published: June 2021

The Isoform Specific Region (ISR) is a region of structural heterogeneity among the 4 isoforms of the Na, K‐ATPase α‐subunit. Replacing α1 ISR (K489NPNASEPKHL499) by α2 ISR sequence (E487REDSPQS‐HV496) results in a doubled response to PKC, suggesting that the ISR participates in isoform‐specific PKC regulation. PKC regulation of α1 involves membrane recycling via clathrin‐coated vesicles. In α1, L499 is immediately followed by another L, making it a potential di‐Leucine (LL) target motif for clathrin adaptor proteins. Replacing α1 ISR by α2 ISR disrupts this LL motif. To test whether this explains the change in PKC regulation observed when tampering with ISR, we compared a L499V mutant of α1 to the full length α1 in a model of heterologous expression of rat constructs in opossum kidney cells. In the absence of PKC stimulation, total expression and Na, K‐ATPase mediated 86Rb+ uptake were comparable in α1L499V and α1 expressing cells. However, biotinylation experiments revealed a higher surface expression of the mutant, suggesting that the mutation affected the steady state level of surface expression of the protein. Upon PKC stimulation, activation of Na, K‐ATPase mediated 86Rb+ uptake was doubled in mutant‐expressing cells. This suggests that the last residue of the ISR plays a critical role in Na,K‐ATPase isoform‐specific response to PKC activation and in steady‐state Na,K‐ATPase expression at the cell surface.
NHE3 becomes phosphorylated during acute hypertension
Authors: Riquier Anne Dominique Marie , Li Eileena , McDonough Alicia Ann
Published: June 2021

When blood pressure (BP) is elevated above baseline, a pressure natriuresis response ensues which is critical to volume and blood pressure homeostasis. The response involves a decrease in Na+ transporter activity along the nephron. We aimed to test the hypothesis that the phosphorylation status of proximal tubule NHE3, medullary NKCC2 and renal AMP kinase is altered during acute hypertension. Background: Dopamine increases NHE3‐P at Ser 552 (Kocinsky, 2005); NKCC2‐P at Thr212/Thr217 is associated with translocation to the plasma membrane (Giménez, 2003); AMPK‐P (Thr172) decreases ENaC activity (Hallows, 2005). BP was increased for 15 min in anesthetized rats by vascular constriction (vs. sham). Kidneys were excised, homogenized with phosphatase and protease inhibitors, and abundance of total and phosphorylated forms of NHE3, NKCC2 and AMPK quantitated with specific antibodies. NHE3‐P at Ser552 doubled in abundance during hypertension. No significant changes in NKCC2‐P or AMPK‐P abundance were evident. Density distribution patterns and lipid domain properties of total and –P forms of NHE3 were identical at baseline BP and after high BP. We conclude that acute hypertension provokes a rapid phosphorylation of NHE3 at ser552 that may participate in the decrease in proximal tubule sodium reabsorption, by provoking changes in NHE3 distribution, activity or protein association. DK34316, NIH Step‐Up (EL).
Aquaporin water channels in the bovine ovary
Authors: Mobasheri Ali , Sawran Ami , Marples David , Luck Martin
Published: June 2021

The follicles of the bovine ovary are large, rapidly growing fluid filled structures. Follicular fluid originates in the ovarian capillary network but nothing is known about how it is transferred over the endothelium, through the ovarian stroma and across the follicle wall into the antrum. We hypothesize that specific aquaporin (AQP) water channels control fluid flow in different regions of the ovary. Using custom‐designed rabbit polyclonal antisera, we have made a preliminary immunohistochemical identification of aquaporin molecules in abattoir‐derived ovarian tissue sections. AQP1 was located in the somatic cells of small, pre‐antral follicles but not in early or large antral follicles. It was also present in the endothelium of stroma and theca interna in most parts of the ovary. AQP1 also stained erythrocytes, and these could be seen penetrating the antrum following the peri‐ovulatory breakdown of the basement membrane. AQP3 and AQP 4 showed clear staining in the oocytes of antral follicles, and AQP4 also exhibited diffuse expression in the stroma. AQP2 was undetected in the ovary, despite strong and specific expression in cortical and medullary collecting ducts of kidneys from the same animals. These results indicate both site‐specific and stage‐specific expression of aquaporins in the ovary. In particular, they suggest that AQP1 and AQP4 influence endothelial and stromal transport and that the transition from pre‐antral to antral development involves a loss of AQP1 expression in the granulosa.
Renal heme oxygenase‐1 upregulation after shock wave lithotripsy
Authors: Clark Daniel Lynn , Handa R. K. , Johnson C. D. , Connors B. A. , Evan A. P.
Published: June 2021

Shock wave lithotripsy (SWL) produces oxidative stress and a rapid inflammatory response in renal tissue. The purpose of this study was to determine the location and level of markers for oxidative stress and inflammation in a porcine model of acute SWL‐induced renal injury. The lower pole calyx of left kidneys of female pigs (30–35 lbs) received 2000 shock waves (SW) at 24 kV, 120 SW/min from a Dornier HM3 lithotripter. Anesthetized animals were subsequently monitored for 4 hours. At 4 hrs post‐treatment, kidneys were perfused with ice‐cold saline, excised, and selected tissue samples were flash‐frozen in liquid N2. Heme oxygenase‐1 protein (HO‐1) was analyzed in renal microsomes by Western blot (Stressgen anti‐HO‐1 antibody SPA‐895). Renal medullary HO‐1/β‐actin ratios were 1.175 ± 0.339 for treated pole (F2), 0.192 ± 0.066 for treated kidney upper pole, and 0.144 ±0.177 for contralateral kidney (n=3). Thus, we observed an eight‐fold induction of HO‐1 in renal medulla at F2 compared to the upper pole medulla of the treated kidney and the contralateral kidney medulla (n=3). In contrast, renal cortex did not show a similar HO‐1 induction. The highly localized HO‐1 induction in SWL‐treated renal medulla parallels our previously reported finding of IL‐6. These data together suggest that an early inflammatory response appears primarily in the treated renal medulla within 4 hours after SWL. Supported by NIH grant DK67133.