bluuebunny/crossref_metadata_2025 huggingface dataset:
Properties of Dissociated Paraventricular Nucleus (PVN) Neurons following Hindlimb Unloading (HU) in Rats
Authors: Kuehl‐Kovarik M Cathleen , Kline David D , Hasser Eileen M
Published: June 2021

Cardiovascular deconditioning (CVD) alters autonomic regulation, including arterial and cardiopulmonary baroreflex function. Cardiopulmonary reflex sympathoinhibition is enhanced in HU rats, an animal model for CVD and the PVN may be involved. We hypothesized that HU alters intrinsic properties of PVN neurons involved in autonomic control. Male rats underwent microinjection of fluorescent, retrograde beads into the spinal cord, allowed to recover and subjected to HU (n = 3) or control (C, n = 3) for 14 days. Rats were euthanized and PVN cells acutely dissociated. Whole cell patch clamp recordings were made on labeled spinally projecting neurons. Input resistance and capacitance were similar in cells from C and HU. Spike frequency adaptation (SFA) was significantly greater in neurons from HU rats compared to C (+35 pA step; 1.8±0.8 vs 5.8±0.9 spikes, p=0.01). Delayed excitation was similar in neurons from both groups. Ionic mechanisms for greater SFA in HU rats were evaluated. In a small number of cells, outward currents were similar between groups. However, in cells (n=5) from HU rats, inward currents appeared to be blunted compared to C (n=6) (@ ‐20 mV: HU = ‐1428±371 pA; C = ‐4918±1142 pA; p = 0.03). Preliminary data are consistent with the concept that, in HU rats, spinally projecting PVN neurons display properties that reduce their excitability. These changes could contribute to altered autonomic regulation in CVD. HL55306
p38 MAPK mediates upregulation of cyclooxygenase‐2 in hypothalamic paraventricular nucleus in rats with heart failure
Authors: Yu Yang , Wei Shun‐Guang , Zhang Zhi‐Hua , Weiss Robert M , Felder Robert B
Published: June 2021

Increased cyclooxygenase‐2 (COX‐2) expression in the paraventricular nucleus of hypothalamus (PVN), with synthesis of prostaglandin E2, contributes to augmented sympathetic nerve activity in heart failure (HF) rats. Increasing evidence has linked the expression of COX‐2 to activation of intracellular mitogen‐activated protein kinase (MAPK) pathways. We hypothesized that activation of central MAPK pathways mediates COX‐2 expression in the PVN in HF. Sprague‐Dawley rats underwent coronary ligation to induce HF or sham surgery (SHAM). HF rats were treated for 4 weeks with intracerebroventricular MAPK inhibitors or vehicle. Compared with SHAM, vehicle treated HF rats had increased (*p<0.05) phosphorylated (normalized to total) p44/42 MAPK (0.22±0.02* vs 0.09±0.01; 0.24±0.02* vs 0.11±0.02), p38 MAPK (0.24±0.03* vs 0.12±0.02) and JNK (0.29±0.03* vs 0.15±0.02), increased COX‐2 mRNA (0.10±0.02* vs 0.04±0.01, normalized to GAPDH) in PVN, and increased PVN neuronal excitation (67±5* vs 25±4). A p38 MAPK inhibitor reduced COX‐2 mRNA in the PVN by 43% (p<0.05, vs vehicle), but p44/42 MAPK and JNK inhibitors had no effect. All three inhibitors significantly decreased excitation of PVN neurons. These data indicate that p38 MAPK plays a critical role in HF‐induced COX‐2 expression in PVN, and so may contribute to augmented sympathetic nerve activity in HF. Supported by a VA Merit Review award.
Infarct rupture and cardiac remodeling after myocardial infarction in 5 different mouse strains
Authors: Janssen Ben , van de Borne Susanne , Brouns‐Strzelecka Agnieszka , Hermans Rob , Blankesteijn Matthijs
Published: June 2021

Background

Transgenic mouse models are used extensively to study the process of infarct healing. However, genetic background may affect the outcome of such studies.

Methods

We compared in 5 different mouse strains (BalbC, C57Bl6, FVB, 129S6 and Swiss) the process of infarct healing after myocardial infarction (MI). Cardiac dimensions were monitored by echocardiography and histology. Cardiac function was determined by intraventricular pressure measurements.

Results

The area at risk was not different between mouse strains. Infarct rupture, which occurred 3‐6 days post‐MI, was most frequent in 129S6 (62%), followed by C57Bl6 (31%), FVB (24%), Swiss (15%) and FVB (3%). This frequency correlated strongly with systolic pressures at 3 days post‐MI, number of inflammatory cells and MMP‐9 activity. Acute heart failure was observed in 25% of FVB mice in the first week after MI, but was rare in other groups. Cardiac dilatation was most marked in Swiss mice and least prominent in surviving 129S6. The dilatation correlated positively with dP/dt values at 14 and 28 days post‐MI. Secondary thinning of the infarct area between 14 and 28 days post‐MI was marked in BalbC (385±55 to 262±26 μm), FVB (480±128 to 300±49 μm) and Swiss (498±135 to 333±72 μm, all P<0.05), but absent in C57Bl6 (409±55 to 402±32 μm) and 129S6 (517±103 to 484±47 µm). This was paralleled by the highest amounts of alpha‐smooth muscle actin positive fibroblasts, so‐called myofibroblasts, in the latter two groups.

Conclusions

Infarct healing in mice strongly depends on genetic background. For studies on the prevention of infarct rupture, 129S6 is the background of choice, whereas BalbC, FVB and Swiss are best suited to study infarct dilatation. The amount of myofibroblasts at 14 days post‐MI predicts the secondary dilatation of the infarct.
Mild hypothermia on reperfusion after warm ischemia improves guinea pig isolated heart function
Authors: Aldakkak Mohammed , Heisner James S , Haumann Johan , Stowe David F , Camara Amadou KS
Published: June 2021

Application of mild hypothermia during the initial critical period of reperfusion after cardiac ischemia may slow mitochondrial and cell metabolism sufficiently to attenuate cardiac damage and better preserve mechanical function. To test this we isolated guinea pig hearts for perfusion at constant pressure with Krebs‐Ringer's solution at pH 7.4. Left ventricular pressure (LVP), coronary flow (CF), and O2 consumption were measured and continuous NADH/FAD and mitochondrial (m) [Ca2+] were assessed in the LV wall by fluorescence spectrophotometry in the absence or presence of indo 1 + MnCl2 to quench cell Ca2+. Hearts were subjected to 30 min ischemia at 37ºC, and then reperfused at either 32ºC or 37ºC for 60 min followed by additional 60 min reperfusion at 37ºC. Values up to reperfusion were similar in both groups. Developed LVP and m[Ca2+] were both lower at 32ºC vs. 37ºC. At the end of reperfusion the 32ºC group showed improved developed LVP (62 % of baseline) when compared to the non‐treatment group (50 %). There were no noticeable improvements in CF, NADH, FAD, and mCa2+ loading during later perfusion. Lower contractility and m[Ca2+] by hypothermia during the critical early reperfusion period may protect the heart during later warm reperfusion.
O‐GlcNAc Transferase Deficiency Exacerbates Metabolic Defects in the Failing Heart
Authors: Watson Lewis Joshua , Facundo Heberty T , Ngoh Gladys , Jones Steven
Published: June 2021

The peroxisome proliferator activated receptor gamma coactivator‐1alpha (PGC‐1α) is a transcriptional activator of many metabolic genes, especially those used in the metabolism of fatty acids. Within the failing heart, a metabolic shift occurs from utilizing mainly fatty acids to relying more on glycolysis. Concurrent with this shift is a reduction in the expression of PGC‐1α and an increase in the glucose‐derived, metabolic post‐translational modification O‐GlcNAc. We hypothesized that O‐GlcNAc signaling would play a role in the control of PGC‐1α during this metabolic change. We subjected wild‐type mice to coronary ligation (HF) or sham. After five days both PGC‐1α and PGC‐1 α levels were significantly reduced (p<0.05) and O‐GlcNAc levels significantly elevated (p<0.05). Using the MerCreMer transgene system coupled with a loxP‐flanked OGT mouse, a cardiomyocyte specific OGT 'KO' was made. After tamoxifen induction, cardiac OGT and O‐GlcNAc levels were significantly reduced (p<0.05) in the transgenic (Tg) group. NTg and Tg mice underwent HF or sham surgery and tissues were harvested after 5 days. Following HF, Tg mice exhibited a greater reduction in PGC levels (figure A, p>0.05) than their NTg counterparts. We conclude that ablation of OGT exacerbates the suppression of PGC‐1α in the failing heart.

Funded by the NIH and American Heart Association.
AMP579 binds to adenosine A2b receptors thus resolving longstanding mystery of its cardioprotective signaling
Authors: Liu Yanping , Yang Xi‐Ming , Walker Sheree , Yang Xiulan , Cohen Michael V , Downey James M
Published: June 2021

The mixed A1/A2a adenosine agonist AMP579 given at reperfusion is very protective in animal models of myocardial infarction; however, neither A1‐ nor A2a‐selective agonists can duplicate its protection despite blocking studies indicating that the protection came from an A2‐type receptor. We recently noted that A2b‐selective agonists are very protective and wondered whether AMP579 might also be an A2b agonist. AMP579 (500nM) for 1h starting at reperfusion protected isolated rabbit hearts exposed to 30 min‐regional ischemia/2h‐reperfusion (12.9±2.2% infarction of risk zone vs 32.0±1.9% in control hearts). A highly selective A2b antagonist PSB1115 (500nM) given for the first 15 min of reperfusion blocked AMP579's protection (32.2±3.1% infarction). We then confirmed A2b binding of AMP579 in human embryonic kidney cells transfected with human A2b receptors in which A2b occupation causes phosphorylation of ERK. AMP579 induced ERK phosphorylation with an EC50 of 250nM. These studies resolve a longstanding mystery related to AMP579's mechanism of protection, and further support the primacy of adenosine A2b receptors in the cardioprotective signaling that can be implemented at reperfusion. Supported by HL‐20648
Fatty acid beta oxidation is the source of malonyl‐CoA for fatty acid chain elongation in rat heart
Authors: Kerner Janos , Minkler Paul , Stoll Maria , Hoppel Charles
Published: June 2021

In rat hearts perfused with [16,16,16‐d3]palmitic acid (M+3), some of the perfused palmitate underwent chain elongation to form M+3 stearoylcarnitine. No M+5 stearoylcarnitine was found indicating that unlabeled malonyl‐CoA served as the chain extender. Based on our data that peroxisomal fatty acid oxidation is the source of carbon units for malonyl‐CoA, in follow‐up experiments we asked whether fatty acid β‐oxidation of palmitate accounted for the chain elongation. Rat hearts were perfused in the working mode with [1,2,3,4‐13C4]palmitic acid (M+4) and the isotopic patterns analyzed using LC/MS/MS and a newly available algorithm. The presence of myristoylcarnitine with M+2 provides evidence for β‐oxidation of perfused M+4 palmitic acid. The relative amount of this mass isotopomer accounts for 82.5%. On the other hand, the 72% relative amount of M+4 species in stearoylcarnitine (corrected by the 21% relative amount of unlabeled, M+0 species) clearly shows that a significant fraction of the perfused palmitate was chain elongated. In addition to M+4 species, there also was a significant amount of M+6 stearoylcarnitine (22% corrected by the M+0 species). The formation of M+6 stearoylcarnitine can be explained only by β‐oxidation of the perfused palmitate and conversion of the M+2 acetyl‐CoA to M+2 malonyl‐CoA which serves as the acceptor for M+4 palmitoyl‐CoA.

Supported by: DK‐066107 and POI AG15885.
Antioxidant status in different genetic strains of rats subjected to severe hemorrhage
Authors: Dubick Michael A , Barr Johnny L , Klemcke Harold G
Published: June 2021

Previous studies in our laboratory demonstrated an 8‐fold difference in survival time after hemorrhage among several rat strains (Klemcke et al., 2008). The present study evaluated whether antioxidant status among strains was associated with these differences. Lung, liver, small intestine and brain were collected 30 min after hemorrhage or handling from 3 groups (control, sham hemorrhage and hemorrhage, n=5/gp) of 3 inbred rats strains (Brown Norway (BN), Fawn Hooded Hypertensive (FHH) and DA) divergent in survival time after hemorrhage (BN>FHH>DA). Tissues were analyzed for antioxidant potential, thiobarbituric acid reactive substances (TBARS), reduced glutathione (GSH), and antioxidant enzyme activities (GSH‐reductase, GSH‐peroxidase, and catalase). For control rats of each strain, these variables were similar when the same tissue was compared. BN rats appeared more responsive to catheterization (sham) and hemorrhage than the other strains, with decreases in brain TBARS, GSH, nitric oxide and lung antioxidant potential compared to controls. For each tissue, as there were no differences between shams and hemorrhage groups, these changes in BN rats did not appear to be related to the hemorrhage. At the time point measured, these data suggest that these rat strains had similar antioxidant status and there were few hemorrhage associated changes in antioxidant levels.

Supported by US Army MRMC.
Regulation of vacuolar H+‐ATPase (V‐ATPase) recycling via a RhoA‐dependent pathway in epididymal clear cells
Authors: Shum Winnie , Da Silva Nicolas , Brown Dennis , Breton Sylvie
Published: June 2021

The V‐ATPase in clear cells is important for luminal acidification in the epididymis, a process that is critical for sperm maturation and storage. We showed that proton secretion in these cells is regulated via V‐ATPase recycling. We now find that RhoA is enriched in clear cells where it is located mainly in the sub‐apical region. In addition, cortical F‐actin was detected beneath the apical membrane of clear cells using a pan‐actin antibody or phalloidin‐TRITC on epididymis sections. In epididymal tubules perfused in vivo, agents known to stimulate V‐ATPase membrane accumulation (1mM cpt‐cAMP, 1mM cpt‐cGMP, 1mM SNP, or 100µM ANGII) significantly reduced the intensity of cortical F‐actin staining. The Rho‐Kinase (ROCK) inhibitor Y27632 (10µM) or the cell permeable Rho inhibitor, Clostridium C3 transferase (3.75 µg/ml) also decreased the intensity of sub‐apical F‐actin staining and induced apical membrane V‐ATPase accumulation. These results provide evidence that depolymerization of the actin cytoskeleton via inhibition of RhoA or its effector ROCK favors apical membrane V‐ATPase accumulation. Thus, cAMP‐ or cGMP‐dependent inhibition of RhoA might be part of the intracellular signaling cascade that is triggered upon agonist‐induced apical membrane V‐ATPase accumulation and subsequent activation of V‐ATPase‐dependent proton secretion in clear cells. This research is supported by NIH grant DK38452.
The adrenaline‐induced colonic K+ secretion is conducted by the ZERO splice variant of KCa1.1 (BK)
Authors: Sorensen Mads Vaarby , Sausbier Mathias , Ruth Peter , Seidler Ursula , Praetorius Helle A , Leipziger Jens
Published: June 2021

Colonic K+ secretion in mammals under go long term hormonal‐ and short term regulation by local agonists. The BK channel has previously been identified as the only luminal K+ channel in resting, Ca2+‐activated and aldosterone‐induced distal K+ secretion. Agonists elevating cAMP are potent activators of colonic K+ secretion. The K+ channel responsible for cAMP‐induced K+ secretion remains to be identified. In this study we used Ussing chambers to identify adrenalin‐induced electrogenic K+ secretion. In mouse colon we found that adrenaline‐induced ion secretion is a compound effect of a dominating anion secretion and a smaller electrically opposing K+ secretion. Using tissue from either BK or CFTR KO and WT mice we were able to isolate the adrenaline‐induced K+ secretion. We found that adrenaline‐induced K+ secretion: 1) is absent in BK−/− tissue, 2) is up‐regulated in mice on a high K+ diet and 3) is present as sustained positive current in colon from CFTR−/− mice. We also identified that colonic enterocytes express STREX and ZERO C‐terminal BK α‐subunit splice variants. Importantly, the ZERO variant known to be activated by cAMP is differentially up‐regulated in animals on high K+ diet. In summary, these results strongly indicate that the adrenalin‐induced distal colonic K+ secretion is mediated by the BK channel and likely involves aldosterone‐induced ZERO splice variant up‐regulation.
Real‐time measurement of transport activity of the human concentrative nucleoside transporter, hCNT3, using a fluorescent reporter
Authors: Johnson Danielle E , Young James D , Campbell Robert E , Casey Joseph R
Published: June 2021

Human concentrative nucleoside transporter, hCNT3, mediates Na+/nucleoside co‐transport, with a low turnover rate (10 nucleoside molecules s−1). hCNT3 also supports H+‐coupled uridine transport, as measured in X. laevis oocytes. We used a new approach to monitor H+/uridine co‐transport in mammalian cells. We fused mNectarine, a new H+‐sensitive red fluorescent protein, to the N‐terminus of hCNT3 to generate mNect‐hCNT3. Fusion of the fluorescent H+ sensor enabled measurement of pH at the intracellular surface of hCNT3. mNectarine fluorescence was monitored in human embryonic kidney 293 cells expressing mNect‐hCNT3 or, as a negative control, cytosolic mNectarine and catalytically dead mutant of the AE1 Cl−/HCO3− exchanger, AE1‐P652C. mNectarine fluorescence values were calibrated for pH using the nigericin/high K+ clamping method. Cells were incubated at the permissive pH for H+‐coupled nucleoside transport, pH 5.5, under Na+‐free conditions. At this pH there was a continuous cytosolic acidification. In mNect‐hCNT3 expressing cells (but not under negative control conditions) the rate of acidification increased upon addition of 0.5 mM uridine, consistent with H+‐coupled uridine transport. We conclude that mNect‐hCNT3 acts as a sensitive self‐referencing sensor of nucleoside transport. Research is supported by the Canadian Institutes of Health Research and the National Cancer Institute of Canada.
Single nucleotide polymorphisms (SNPs) of human SLC26A9
Authors: Chen An‐Ping , Chang Min Hwang , Sindic Aleksandra , Mount David B , Romero Michael F
Published: June 2021

Many diseases are caused by altered epithelial Cl− or HCO3− transport. Mutations in the SLC26A3 transporter are causative for congenital chloride‐losing diarrhea; however, no other SLC26 transporter plays such a direct genetic role in disorders of epithelial Cl− transport. SLC26A9 is expressed in the lung, stomach, intestine and kidney (lesser extent) but its physiological function in these epithelia is not clear. Thus far, no diseases result from SLC26A9 mutations. However, database analysis reveals three single nucleotide polymorphisms (SNPs). We reasoned that the human SLC26A9‐SNPs would provide insight into what a disease phenotype might be functionally manifest. Here we analyzed the ion transport characteristics of human SLC26A9 and its SNPs: V622L, V744M and H748R. Human SLC26A9 and its three SNPs were expressed in Xenopus laevis oocytes. Effects on ion transport were evaluated using ion selective electrodes and voltage clamping. Measuring Cl− and HCO3− flux, our results show that SLC26A9 is a Cl−/HCO3− exchanger. One SNP, V622L, is located in the C‐terminus just before the STAS domain of SLC26A9. V622L has ~50% function (current and Cl− transport). These results suggest that the possession of V622L allele in humans would decrease SLC26A9 function and modulate the risk for lung disease (cystic fibrosis), gut disease (gastric ulcer) or hypertension, due to reduced Cl− transport across epithelial cells.
Bicarbonate exchangers SLC26A3 and SLC26A6 are localized at the apical membrane of intact porcine vas deferens epithelium
Authors: Akoyev Vladimir , Pierucci‐Alves Fernando , Schultz Bruce
Published: June 2021

The goal of this study was to detect bicarbonate exchangers SLC26A3 and SLC26A6 in intact vas deferens epithelia. Bicarbonate uptake by sperm cells while still in the male reproductive tract is required for the development of fertilizing capacity. The expression of SLC26A3 and SLC26A6 at the mRNA level has been reported in the porcine vas deferens (PVD) PVD9902 cell line, but it remains unknown whether SLC26A3 and SLC26A6 are expressed in intact PVD epithelium. Laser scanning confocal microscopy (LSCM) was employed to investigate SLC26A3 and SLC26A6 immunoreactivities in intact PVD and primary PVD cultured (1oPVD) cells. qRT‐PCR was used to determine the mRNA copy number of SLC26A3 and SLC26A6 in total RNA isolated from 1oPVD cells. LSCM demonstrated that SLC26A3 and SLC26A6 immunoreactivities are located at the apical membrane of principal cells in the proximal and distal segments of PVD. In contrast to tissue sections 1oPVD cells (cultured for 14 days) demonstrated diffuse immunolabelling close to apical area. 1oPVD cells expressed 391±70 copies of SLC26A3 and 2324±265 copies of SLC26A6 mRNA per 500 ng of total RNA. The study was supported by the NIH (RO1HD058398) and (RR017686 ‐ Imaging and Molecular Cores).
Base of the thumb domain has a role in ENaC gating
Authors: Ghosh D. Dipon , Sheng Shaohu , Maarouf Ahmad B. , Kleyman Thomas R.
Published: June 2021

Epithelial sodium channels (ENaCs) mediate Na+ transport in the distal nephron and in both airways and alveoli. The regulation of Na+ transport in these areas is crucial for controlling extracellular volume and blood pressure, and airway surface liquid volume. ENaCs are inhibited by external Na+, a process referred to as Na+ self‐inhibition that reflects a reduction in channel open probability. The extracellular domains of ENaC subunits likely have a key role in regulating ENaC gating in response to external Na+. ENaC shares limited homology with acid sensing ion channels (ASICs). Proton gating of ASIC1 has been proposed to involve conformational changes to ultimately affect a Trp residue at the base of the thumb domain. We examined whether a Tyr residue at the base of the thumb domain of all three ENaC subunits, at a location homologous to the Trp residue in ASIC1, affects ENaC gating. Na+ self‐inhibition was measured in Xenopus oocytes expressing wild type channels, as well as channels with an alpha Y418, beta Y356 or gamma Y375 mutation. The alpha Y418 mutation significantly reduced Na+ self‐inhibition, whereas the beta Y356 or gamma Y375 mutants were similar to wild type. Our results suggest that the base of the thumb of the alpha subunit has a role in regulating channel gating in response to external Na+. (Supported by NIH DK54354, ES14701, DK079307 and HHMI)
Evidence for Connexin Hemichannel Activity in Porcine Ocular Nonpigmented Ciliary Epithelium
Authors: Shahidullah Mohammad , Delamere Nicholas A
Published: June 2021

Objective

Recent studies have shown that unpaired connexons form active hemichannels connecting the cytoplasm to the extracellular space. In porcine ciliary body we detected connexin 50 (Cx 50) at the aqueous humor (AH)‐facing basolateral surface and connexin 43 at the apical surface of nonpigmented ciliary epithelium (NPE).

Methods

Connexins were detected by immunological study. To probe for hemichannel activity, cells were cultured on permeable inserts and incubated with the DNA intercalating agent propidium iodide (PI) (Mol. wt. 668.4) or fluorescein dextran (FDex) (Mol. wt 4000), both of which are membrane impermeable. PI or FDex was detected by fluorescence microscopy and fluorimetry.

Results

Under control conditions, only traces of PI entered cultured NPE. PI entry was markedly increased in cells incubated with calcium‐free buffer. FDex did not enter the cultured NPE either in control or calcium‐free medium, indicating size‐limited entry. PI entry also was dependent on [pH]o; significantly higher at pH 7.6 than at pH 7.4 or lower. PI uptake was inhibited by a gap‐junction‐specific blocker 18[alpha] glycyrrhetinic acid (100 [mu]M).

Conclusions

The findings are consistent with the ability of NPE cells to form functional hemichannels. Since Cx 50 is localized to the unapposed basolateral surface of the NPE, we suggest it may act as a hemichannel conduit between the cytoplasm and the AH. EY006915
Functional characterization of L‐type amino acid transport in human choriocarcinoma cell BeWo cell.
Authors: Jin Chun Ji , Anzai Naohiko , He Xin , Fukutomi Toshiyuki , Kimura Toru , Sakurai Hiroyuki
Published: June 2021

System L is a major nutrient transporter system responsible for the Na+‐independent transport of large neutral amino acids including several essential amino acids. In malignant tumors, a system L transporter L‐type amino acid transporter 1(LAT1) is up‐regulated to support tumor cell growth. In this study, we have investigated the properties of [14C]L‐leucine transport in human choriocarcinoma (BeWo) cells. The uptake of [14C]L‐leucine by BeWo cells is Na+‐independent and almost completely inhibited by system L selective inhibitor BCH (IC50 value: 34.4 µM). The profiles of the inhibition of [14C]L‐leucine uptake by amino acids and amino acid‐related compounds in BeWo cells are comparable with those for the LAT1 expressed in Xenopus oocytes. The majority of [14C]L‐leucine uptake is, therefore, mediated by LAT1 in BeWo cells. These results suggest that the transport of neutral amino acids including several essential amino acids into BeWo cell s mediated by LAT and the specific inhibition of LAT in placental cancer cells will be a new rationale for anti‐cancer therapy.
Multi‐drug resistance (MDR1) gene identification and sequence in horses
Authors: Linardi Renata L , Natalini Claudio C , Kousoulas Konstantin G , Huang Li Ju Ti , Stokes Ashley Michelle , Moore Rustin M
Published: June 2021

P‐glycoprotein (P‐gp), a membrane transporter encoded in the multi‐drug resistance (MDR1) gene, has been identified in many species but not in equine. P‐gp interferes with bioavailability and disposition of many drugs and may explain adverse drug‐induced effects after intravenous administration and poor response with oral administration.

The hypothesis is that the MDR1 gene is present in horses. We identified the presence of the equine MDR1 gene, obtained the gene sequence and detected its presence in normal equine tissues.

To identify the equine MDR1 gene, total RNA was extracted from intestinal samples, was reverse transcribed to cDNA and used as a template for PCR. Primers were designed by MacVector 65.3 software using the Gen Bank AY360144 MDR1 cDNA rabbit sequence. Real Time TaqMan RT‐PCR was used to detect the presence of MDR1 gene in the intestine, brain, and spinal cord from six horses.

Our sequence was compared to the MDR1 rabbit sequence and the horse partial MDR1 sequence demonstrated high similarity with this (84%) and other species. Our RT‐PCR data confirmed the presence of the MDR1 gene in the intestine, brain, and spinal cord of all 6 horses.

This data may improve pharmacokinetic and pharmacodynamic studies since the presence of this gene may predispose horses to differences in enteric absorption of some therapeutic agents.

Funding provided by the LSU Equine Health Studies Program.
Luminal adenosine signaling regulates duodenal bicarbonate secretion via A2B receptor and CFTR in rats
Authors: Akiba Yasutada , Ham Maggie , Watanabe Chikako , Nakano Takanari , Kaunitz Jonathan D
Published: June 2021

Duodenal bicarbonate secretion (DBS) is increased by luminal ATP, via activation of enterocyte brush border P2Y1 receptors. Increased DBS augments the activity of intestinal alkaline phosphatase (IAP) activity, which degrades luminal ATP, acting as a negative feedback loop. Since IAP dephosphorylates ATP to ADP, AMP, and adenosine (ADO), and since adenosine deaminase (ADA) is highly expressed in duodenal brush border, we hypothesized that luminal ADO signaling is also involved in DBS regulation.

We measured DBS with flow‐through pH and CO2 electrodes, testing the effect of perfusion of ATP, ADP, AMP or ADO with or without adenosine receptor (A1‐3R) agonists or antagonists, or CFTR inhibitor CFTRinh‐172 on DBS. We also examined the effect of inhibitors of ADA or concentrative nucleoside transporter (CNT) on DBS.

Perfusion of ATP, ADP, AMP or ADO uniformly increased DBS. A2BR agonist NECA increased DBS, whereas ADO‐augmented DBS was inhibited by A2BR antagonist MRS1754 and abolished by CFTRinh‐172. Other AR agonists or antagonists had no effect. A2BR was immunolocalized to the brush border of duodenal villi whereas A2AR to the endothelium. ADA inhibitor EHNA and CNT inhibitor formycin B enhanced ADO‐induced DBS.

Luminal ADO stimulates DBS via A2BR and CFTR. IAP, ADA and CNT may coordinately regulate luminal ADO concentration to modulate ADO‐P1R signaling in rat duodenum.

VA Merit Review, NIH‐NIDDK R01 DK54221
AE1 and carbonic anhydrase II: are they colocalized and do they exhibit direct functional interaction in cell membranes?
Authors: Endeward Volker , Al‐Samir Samer , Sly William S , Gros Gerolf , Papadopoulos Symeon
Published: June 2021

Previous studies have reported that intracellular carbonic anhydrase (CA) II binds to the C‐terminal domain of AE1 and it has been suggested that this interaction directly increases the transport rate of AE1. The goal of our study was a) to detect the intracellular distribution and possible colocalization of CAII and AE 1 in living cells and b) to measure bicarbonate transport rate of AE1 in the absence and presence of CAII in HEK 293 cells und human red blood cells. We expressed fusion proteins of AE1 and yellow fluorescent protein and of CAII and cyan fluorescent protein in HEK 293 cells and observed the distribution of CAII and AE1 with a confocal laser scanning microscope. AE1 was exclusively associated with the plasma membrane, while CAII was homogeneously distributed in the cytoplasm. There was no apparent enrichment of CAII in the plasma membrane. In mass spectrometric measurements of bicarbonate permeability (PHCO3−) we observed a marked increase of PHCO3− over controls upon expression of AE1. Coexpression of AE1 and CAII did not lead to a further increase in PHCO3−. In another study, we measured the bicarbonate permeability of normal and CAII‐ deficient human red blood cells and found no difference in PHCO3−. Both results suggest that there is no direct functional interaction between AE1 and CAII in HEK 293 and human red blood cells.
ACE2 expression in the central nervous system reduces angiotensin‐II‐mediated hypertension and cardiac hypertrophy in transgenic mice.
Authors: Feng Yumei , McIlwain Elizabeth , Varner Kurt , Lazartigues Eric
Published: June 2021

Angiotensin converting enzyme 2 (ACE2) is an important member of the renin angiotensin system, recently identified in the central nervous system. We previously reported that central ACE2 reduces the development of hypertension induced by chronic Ang‐II infusion in transgenic mice (syn‐hACE2) with human ACE2 targeted selectively to neurons. To study the physiological consequences of central ACE2 over‐expression on cardiac function, syn‐hACE2 and control mice (n=8/group) were infused with Ang‐II (600 ng/kg.min sc) for 14 days and cardiac function assessed by echocardiography. Baseline BP (telemetry: 112 ±3 vs. 114 ±4 mmHg), ejection fraction (EF: 45 ±3 vs. 44 ±4%), fractional shortening (FS: 23 ±2 vs. 22 ±2) and left ventricle mass/tibia length (7.9 ±0.4 vs. 7.7 ±0.2) were similar between the two groups (P>0.05). After 2‐week infusion, BP was elevated in controls (135 ±3 mmHg; P<0.01) but not in syn‐hACE2 (101 ±5 mmHg). Although EF and FS were not altered, Ang‐II infusion induced hypertrophy (LV mass/tibia length: 9.1 ±0.3 2PW/LVD: 0.4 ±0.02; P<0.05) in controls. However, in syn‐hACE2 mice, these parameters were significantly reduced (8.2 ±0.2 and 0.3 ±0.01, respectively; P<0.05). These data suggest that ACE2 over‐expression in the brain reduced Ang‐II‐mediated hypertension and protected syn‐hACE2 transgenic mice from sympathetic drive‐mediated cardiac hypertrophy. (AHA 0825459E, NIH RR018766 and HL093178)
Effect of tempol on inward‐rectifier K+ channel regulation of afferent arteriolar tone in diabetic rat kidney
Authors: Troncoso Brindeiro Carmen M. , Carmines Pamela K.
Published: June 2021

We previously reported that inward‐rectifier K+ channels (Kir) contribute to the decreased afferent arteriolar tone that accompanies streptozotocin (STZ)‐induced diabetes in the rat. We hypothesized that the oxidative stress characteristic of diabetes underlies this phenomenon. Sham and STZ rats were chronically‐treated with tempol (SOD mimetic; 9 ± 1 mg/day) or left untreated for 27 days. The in vitro blood‐perfused juxtamedullary nephron technique was used to quantify afferent arteriolar lumen diameter responses to Kir blockade (10, 30, 100 μM Ba2+). In untreated animals, lumen diameter averaged 23.8 ± 1.4 µm in STZ and 18.6 ± 1.9 μm in Sham rats (P=0.05) and the concentration‐dependent response to Ba2+ was exaggerated in the STZ group (100 nM Ba2+: Sham δ = ‐2.0 ± 0.7 μm, STZ δ = ‐7.9 ± 1.4 μm; P<0.001). In tempol‐treated animals, baseline lumen diameter did not differ between Sham and STZ rats, averaging 21.4 ± 1.4 and 21.1 ± 1.6 μm, respectively. Moreover, the afferent arteriolar diameter response to Ba2+ was attenuated in tempol‐treated STZ rats (100 nM Ba2+: δ = ‐2.4 ± 1.3 μm; P<0.05 vs untreated STZ) and did not differ from either untreated or tempol‐treated Sham rats. Thus, tempol normalized baseline diameter and the response to Ba2+ in afferent arterioles from STZ rats. We conclude that oxidative stress plays a role in the Kir‐dependent tonic afferent arteriolar dilation that occurs during STZ‐induced diabetes.
Nuclear Factor kappa B content in internal mammary arteries and saphenous veins in human hypertensive and normotrensive patients
Authors: Watson Ralph Edward , Periera Cristiane Neves , Houghton Dana , Pham Albert Q , Gourineni Nandu , Talbott John D , Fink Gregory D
Published: June 2021

Many cellular genes involved in immunity and inflammation are regulated at the level of transcription by nuclear factor kappa B (NFkB). Products of these genes include TNF‐alpha and interleukin‐6 (IL‐6). Increased plasma levels of these inflammatory cytokines (IL‐6, TNF‐alpha, etc.) are seen in hypertension (HTN), suggesting that vascular inflammation is part of the pathophysiology of HTN. To our knowledge no one has previously measured NFkB in blood vessels of hypertensive patients. Study objective: Measure NFkB tissue levels in internal mammary artery (IMA) and saphenous vein (SV) tissue in hypertensive and normotensive patients undergoing coronary artery bypass graft (CABG) surgery.

Methods

A TransAM NFkB p50 colorimetric ELISA kit was used to measure NFkB content in the vessels in 79 patients undergoing CABG surgery. The patients were interviewed and their hospital charts were abstracted to determine whether the patients had HTN and/or type 2 diabetes.

Results

We found that there was no significant difference in NFkB p50 activity levels in human IMA or SV between hypertensive and normotensive patients undergoing CABG surgery. We also found that the presence of type 2 diabetes did not affect these levels.

Conclusion

These data suggest that vascular inflammation in HTN does not require increased vascular production of NFkB. Grant Support: NIH HL73251.
The Wilms' tumor suppressor WT1 regulates Interleukin‐10 expression in macrophagocytic cells
Authors: Bondke Anja , Kirschner Karin M , Scholz Holger
Published: June 2021

The Wilms' tumor gene, WT1, encodes a zinc finger transcription factor which functions both as a tumor suppressor and a key regulator of embryonic development. WT1 is also expressed in hematopoietic progenitor cells and has been implicated in the pathogenesis of acute myeloid leukemia. However, expression of WT1 by mature monocytes/ macrophages and its potential role in immune function has not been studied previously.

We show that WT1 is upregulated in monocytic cells in response to proinflammatory stimulation and enhances interleukin‐10 (IL‐10) expression.

Silencing of WT1 by siRNA transfection of a mesonephric cell line reduced IL‐10 mRNA levels (cDNA microarray, real‐time PCR) by 80% compared to transfection with a non‐targeting siRNA.

IL‐10 mRNA was increased >15‐fold in cells with inducible expression of the transcriptionally active WT1(‐KTS) protein. In contrast, the WT1(+KTS) isoform did not significantly change IL‐10 mRNA levels.

Moreover, IL‐10 and Wt1 were detected in monocytes from healthy volunteers.

Treatment of murine monocytic J774.1 cells with bacterial lipopolysaccharide (LPS) to activate the innate immune response caused an approx. 5‐fold increase in Wt1 mRNA accompanied by an enhanced IL‐10 expression.

These findings suggest a novel role of WT1 in the regulation of the innate immune response.
Control of muscle sympathetic nerve activity in men and women: calculation of “error signal” and its relationship to resting blood pressure
Authors: Wehrwein Erica , Joyner Michael J , Hart Emma CJ , Vaa Brianna , Strom Nicholas , Wallin B. Gunnar , Charkoudian Nisha
Published: June 2021

Sympathetic neural control has a central role in the regulation of blood pressure. In humans, muscle sympathetic nerve activity (MSNA) is strongly controlled by the arterial baroreflex. Our goal was to evaluate a calculation of blood pressure "error signal" for MSNA burst occurrence and test if there was a gender difference in the relationship between this signal and resting blood pressure. Baseline MSNA was measured in healthy young men (n=49) and women (n=23). T50 (the DBP at which there is a 50% likelihood of an MSNA burst) was calculated. Mean DBP was subtracted from the T50 as an approximate "error signal" for burst activation. We found a positive relationship between this "error signal" and MSNA (bursts/100 heartbeats; BI) in both young men (slope=0.19, r2=0.67, p<0.001) and women (slope=0.32, r2=0.78, p<0.001). Thus, if DBP was lower than T50, there was an increased likelihood of MSNA bursts and vice versa. Women had a greater average "error signal" (i.e., mean T50 ‐ DBP = ‐5 ± 1 in women and ‐2 ± 0.4 in men), a 59 % steeper slope of the error signal:MSNA relationship, and lower mean BI (31 ± 2 vs 42 ± 4, p<0.05), suggesting that mechanisms exist in women to counteract the larger "driving force" for generation of a MSNA burst. These apparent gender differences in control of MSNA may contribute to differences in prevalence of cardiovascular diseases such as hypertension. NIH HL83947, UL1‐RR24150, Swedish Research Council 12170.
Sry3 exogenously delivered to the WKY kidney increases the renal renin‐ angiotensin system
Authors: Milsted Amy , Underwood Adam , Dunphy Gail , Boehme Shannon , Martins Almir , Turner Monte
Published: June 2021

Our laboratory has shown that a locus on the SHR Y chromosome increases blood pressure (BP) in SHR and WKY rats with the SHR Y chromosome (SHR/y rat). A candidate gene is Sry, a transcription factor responsible for testes determination which is also expressed in the kidney. We have found multiple functional Sry loci in our WKY and SHR rats. Sry1 and Sry3 exogenously delivered to the kidney raise BP. Sry1 activates SNS indices whereas Sry3 does not. The objective of this study was to determine if exogenous Sry1 or Sry3 delivered to WKY kidney would elevate indices of the renin‐ angiotensin system (RAS). The expression construct Sry1pEF1 (n=8) or Sry3/pEF1 (n=8), or control vector (n=8) was electroporated to the medulla of the left kidney of WKY rats. After 14 and 21 days the kidneys from half of the groups were analyzed for ACE, angiotensin II, renin and plasma renin activity (PRA) was measured. Renal AngII increased by more than 100% after 21 days in the Sry3 kidneys (p<.01) compared to controls. PRA was increased more than 100% after 14 days in the Sry3 kidneys (p<.05) compared to controls but there was no significant difference in renal renin between groups. Renal ACE content decreased after 21 days in the Sry3 kidneys compared to controls (p<.05). These results are consistent with a role for Sry3 in BP elevation due to elevated RAS, whereas Sry1 has more of a BP influence through SNS activity, as we showed earlier ( RO1HL71579‐01A3).
Progesterone (P4) modulates cytokines expression in temporomandibular joint (TMJ)
Authors: Puri Jyoti , Bellinger Larry , Kramer Phillip
Published: June 2021

OBJECTIVES

Evidence suggests that symptoms of TMJD vary over menstrual cycle indicating that changes in steroid sex hormone modulate TMJ pain. The objective of the study is to determine modulatory role of P4 on cytokine/chemokine expression in TMJ.

METHODS

Sprague Dawley ovariectomized female rats were divided in 2 groups with 1 group receiving progesterone pellets & subcutaneous P4 injections to recapitulate physiological plasma levels of progesterone. Groups were subdivided further & were given bilateral TMJ injections of Complete Freund's adjuvant (CFA) or saline within the superior joint space of TMJ during diestrous‐2 and proestrous phase. So 4 groups include P4/diestrous‐2/saline, P4/diestrous‐2/CFA, P4/proestrous/saline & P4/proestrous/CFA.Rats were sacrificed 24 hrs later. The cytokine/chemokine expression was analyzed in retrodiscal tissue, trigeminal ganglia & subnucleus caudalis using arrays.

RESULTS

The transcript expression of cytokines IL‐6 and IL‐1<â > decreased by 50 & 82 fold &chemokines CCL3, CCL20, CXCL1, CXCL2 and CXCL3 decreased by 103, 91, 86, 118 and 155 fold, comparing the P4/diestrous‐2/saline group to P4/proestrous/saline group suggesting P4 modulates expression of these molecules in TMJ.

CONCLUSION

The data suggest that cyclical plasma levels of P4 in females can modulate cytokines/chemokines expression in TMJ. Supported by NIDCR DE016059‐01 (LLB) & DE15372 (PRK).
A potential role for kisspeptin in puberty onset in sheep
Authors: Seebaugh Amanda M , Nestor Casey C , Hileman Stan
Published: June 2021

In peripubertal sheep, a decrease in inhibition by estradiol leads to an increase in gonadotropin releasing hormone (GnRH), and thus luteinizing hormone (LH), secretion, causing the onset of puberty. The neural mechanisms governing this process remain largely unknown. Kisspeptin is expressed in the arcuate nucleus of the hypothalamus of sheep and stimulates GnRH/LH secretion. We hypothesized that kisspeptin expression would be greater in postpubertal sheep and that its expression would be increased in the absence of steroid negative feedback. Four groups of females were used: prepubertal intact, prepubertal ovariectomized, postpubertal ewes in the early follicular phase, and postpubertal ovariectomized. LH was measured to confirm the endocrine state of each animal. Kisspeptin expression was greater in postpubertal ewes as compared to prepubertal ewes, and was increased by ovariectomy only in the prepubertal group. The increase in kisspeptin cell numbers in postpubertal females is consistent with a role for kisspeptin in puberty onset in the sheep. Further, the increase in kisspeptin expression with ovariectomy in prepubertal females indicates that decreased kisspeptin cell numbers in intact prepubertal females is due to the negative feedback effects of estradiol. NIH Grant 5P20RR016477 to the West Virginia IDeA Network for Biomedical Research Excellence
ERK and p38 MAPK regulate peroxiredoxin 6 targeting to lysosomal‐like organelles in lung epithelium cells
Authors: Sorokina Elena M. , Feinstein Sheldon I. , Fisher Aron B.
Published: June 2021

Peroxiredoxin 6 (Prdx6), a protein with both GSH peroxidase and phospholipase A2 (aiPLA2) activities, participates in lung surfactant phospholipid turnover and protects lung epithelium against oxidative stress. Prdx6 has been localized by immunocytochemistry and sub‐cellular fractionation to cytosol and acidic compartments (lamellar bodies and lysosomes) in lung alveolar epithelium. We propose that Prdx6 subcellular localization determines the balance between the 2 activities since the PLA2 activity has a low pH optimum. We show that ERK and p38 MAPK regulate Prdx6 targeting to lysosomal‐like organellar structures in the MLE12 mouse type II alveolar epithelial cell line. Treatment of MLE12 cells with inhibitors of ERK or p38 MAPK, abolished lysosomal targeting of Prdx6. Inhibition of protein kinase C, an upstream kinase activator of ERK and p38, had a similar effect. Moreover, inhibition of PKC, ERK or p38 resulted in Prdx6 accumulation in endoplasmic reticulum (ER), as detected by co‐localization of Prdx6 with the ER marker, calnexin. These findings suggest that ERK and p38 MAPK regulate a subcellular sorting mechanism which targets Prdx6 to lysosomal organelles, possibly by activation of a chaperone protein that facilitates Prdx6 translocation along the exocytotic pathway, resulting in its sub‐cellular compartmentalization into acidic organelles and consequent activation as a PLA2. [HL019737]
Glucosamine modulates sepsis‐induced changes in cardiac performance
Authors: Kelly Ollie , Zhang Zhenghao , White C. Roger
Published: June 2021

Recent studies suggest an anti‐inflammatory role for glucosamine (GlcN). We tested the hypothesis that GlcN treatment in septic rats prevents defects in cardiac performance. Cecal ligation and puncture (CLP) or sham surgery was performed in male Sprague‐Dawley rats (250‐300g). CLP rats were further randomized to receive GlcN (0.3 mg/g) or saline by ip injection. Echocardiography was performed prior to and 24 hours post surgery. Cardiac dimensions in sham rats were unchanged over this time. In contrast, there was a 6 fold increase in IVSd and PWd and a 5‐fold reduction in LVEDD and LVESD in CLP rats. GlcN treatment attenuated these responses by approximately 50%. Reductions in end‐diastolic volume (EDV), stroke volume (SV) and cardiac output (CO) in CLP rats were also attenuated by GlcN. Fractional shortening, however, was similar in all groups. These data suggest that impaired cardiac performance in CLP rats is due to a reduction in LV filling rather than to a defect in cardiac function per se. CTD110.6 immunoreactivity, a marker of protein O‐GlcNAcylation, was increased in hearts from GlcN‐treated CLP rats. GlcN treatment also increased survival (93%) in CLP rats at 24 hrs compared to saline‐treated CLP rats (70%). It is proposed that GlcN improves cardiac performance and survival in CLP rats by a mechanism involving an increase in protein O‐GlcNAcylation and inhibition of inflammatory pathways.
Can Semilunar Valves Be Quantitated Better with Full Volume Transthoracic 3‐Dimensional Echocardiography than 2‐Dimentional Echocardiography?
Authors: Shizukuda Yukitaka , Harrelson Allan , Shalabh Singhal
Published: June 2021

Full volume 3‐dimensional echocardiography (3DE) may be advantageous for delineation of semilunar valve opening and annulus over 2‐dimensional echocardiography (2DE) because it can visualize the valve's complete structure along with real‐time motion. To evaluate this, we analyzed 3DE data with TomTec 4D Cardioview CAP in 43 consecutive patients (age; 53±14, 21 females, data are mean±SD) who underwent transthoracic 3DE imaging acquisition at standard left parasternal (PS) and apical windows (AP) and whose 2DE acoustic windows were good to excellent. In 3DE, the aortic valve (AV) annulus diameter (AVD) was successfully quantitated in both PS and AP for nearly all patients (100% and 95%, % cases). In PS, the 3DE AV opening area (AVA) was more frequently quantitated (74%) than 2DE AVA (49%, P<0.05 with a chi‐square test). Among cases quantifiable with 3DE, both AVD and AVA in PS showed an excellent correlation with that in AP (R=0.814 and R=0.903, P<0.01 for both with a Pearson correlation test). 3DE AVD also showed significant correlations with 2DE AVD (R=0.769, n=40 in PS, P<0.01). In PS, 3DE AVA correlated significantly with 2DE AVA (R=0.616, n=16, P<0.05). Pulmonic valve annulus diameter and opening area were poorly quantifiable in both PS and AP in 3DE (49% and 9%; 9% and 2%). Our results suggest that 3DE quantification of AV structure is feasible and robust and may provide better assessment of AVA than 2DE.