bluuebunny/crossref_metadata_2025 huggingface dataset:
Phospholipid cellular uptake mediated by cationic lipids affects the inflammatory response
Authors: Lonez Caroline , Vandenbranden Michel , Legat Amandine , Ruysschaert Jean‐Marie
Published: June 2021

We show here that free cationic liposomes inhibit CpG sequences or lipopolysaccharides‐induced TNF‐α secretion by macrophages. Surprisingly, this effect was strictly serum‐dependent. Free cationic liposomes alone did not reveal any anti‐inflammatory activity. By serum fractionation on a density gradient, low‐density lipoproteins and triglyceride‐rich lipoproteins were identified as the serum components that confer to the liposomes an anti‐inflammatory activity1. Phospholipid fractions of these lipoproteins reproduced the effect of the total lipoproteins and could inhibit, in association with diC14‐amidine, the CpG‐induced TNF‐α secretion. Whereas oxidation of LDL phospholipids was a prerequisite to any anti‐inflammatory activity, lipid oxidation is no more required when phospholipids are transported in cationic liposomes. This suggests that cationic mediated‐transport of phospholipids (PC, PS, PE, PG) proceeds through a different route and affects different pathways. Cationic liposomes confer to these phospholipids new properties that can be used to modulate the inflammatory response directed against CpG sequences and lipopolysaccharides.
Contrasting Akt and p38 MAPK phosphorylation profiles in renal proximal tubular cells (RPTCs) exposed to lithium chloride and effects of lithium chloride on RPTC viability
Authors: Johnson Paul Richard , Rajayadhyaksha Medha , Wu Rui , Su Ruifen , Rane Madhavi J.
Published: June 2021

Despite the success of lithium (Li) in the treatment of bipolar disorder, its effects on renal proximal tubular cell (RPTC) survival/death are unclear. Thus, the current study was initiated to determine effects of Li treatment on RPTC viability. RPTCs were treated with or without 10mM lithium chloride at several time points and cell lysates were immunoblotted with various antisera. Li induced Akt phosphorylation at 2 hr, which was down regulated by 24 hr. In contrast, Li induced p38 MAPK phosphorylation as early as 2 hr and was maximally phosphorylated at 24 hr and 48 hr. Akt inhibition failed to block Li‐induced p38 MAPK phosphorylation, suggesting that Akt activation occurs downstream of p38 MAPK. These studies were further confirmed by demonstrating that pharmacological blockade of p38 MAPK inhibited Li‐induced Akt and Hsp27 phosphorylation at 2 hr. However, Hsp27 phosphorylation was independent of Akt activation as demonstrated by Akt inhibition studies. In addition, Akt and p38 MAPK inhibition failed to block Li‐induced GSK‐3β phosphorylation, suggesting an Akt and p38 MAPK independent GSK‐3β inactivation in RPTCs. Furthermore, MTT assay demonstrated Li‐induced RPTC viability at 24 hr and 48 hr. The contributions of p38 MAPK in stimulating Li‐induced RPTC survival is currently under investigation.

This work supported by NIAID [1R56AI059165‐01A2 (MJR)] AHA Scientist Development Grant [G03070 (MJR)]
Imaging PKC activity at MAGUK protein scaffolds
Authors: Gallegos Lisa L , Garcia Erin , Hall Randy , Newton Alexandra C
Published: June 2021

The protein kinase C (PKC) family of Ser/Thr kinases relays extracellular signals to intracellular substrates involved in processes such as cell growth and division, memory and learning, and the immune response. PKC isoforms typically reside in the cytosol in a mature but catalytically‐inactive conformation. Generation of the second messengers diacylglycerol and, for conventional PKCs, calcium causes translocation of PKC to cellular membranes, a hallmark of PKC activation. This activation can be precisely visualized using a genetically‐encoded reporter for PKC activity, CKAR (C kinase activity reporter) (Violin, et al. 2003). We have previously tethered CKAR to various intracellular membranes and analyzed the spatiotemporal dynamics of signaling at the plasma membrane, Golgi, mitochondria, nucleus and cytosol (Gallegos, et al. 2006). In this study, we take our reporters one step further by tethering them to protein scaffolds to visualize agonist‐evoked PKC signaling localized at discrete signaling complexes. Specifically, we used a PDZ array to identify potential scaffolds for the PDZ ligand on PKC and found a strong interaction with members of the membrane‐associated guanylate kinase (MAGUK) family of protein scaffolds. This interaction was confirmed by immunoprecipitation of PKC with two MAGUK family members, SAP97 and PSD95. We show that CKAR can be targeted to MAGUK scaffolds, and that this scaffold‐targeted reporter reads out a different profile of PKC activity compared to untargeted CKAR. The ability to visualize PKC activity at a signaling complex provides insight into the efficiency and specificity of compartmentalized signaling.
Structural modifications of homocysteinylated‐alpha‐2‐macroglobulin
Authors: Catanescu Carmen Otilia , Willard Belinda B. , Kinter Mike T. , Zhloba Alexander A. , Jacobsen Donald W.
Published: June 2021

The effect of homocysteine on alpha‐2‐macroglobulin (a2M) function and structure was investigated by mass spectrometry, circular dichroism, isoelectric focusing, surface plasmon resonance, and UV‐vis spectroscopy. Structural analysis of homocysteinylated‐a2M revealed substantial conformational changes in native a2M compared to the untreated a2M. Proteinase‐activated a2M was more resistant towards homocysteinylation than its native form. Proteinase binding function of a2M was shown to be decreased in the presence of L‐homocysteine but not L‐cysteine, which may indicate that high levels of homocysteine in inflammatory states may decrease the ability of a2M to clear proteinases. Mass spectrometry analysis of homocysteinylated‐a2M identified 8 cysteine‐containing peptides suggesting that at the time of alkylation these cysteine residues were in the reduced form. Moreover, a new disulfide link was observed between C255‐C264, indicating that the free cysteine residues that appear upon homocysteinylation tend to reform disulfide bridges and therefore lead to “scrambled” disulfide bonds that can have a profound effect not just on the three‐dimensional structure of the a2M tetramer but also on its functions. This work was supported by a grant (HL52234, DWJ) from the Heart, Lung, and Blood Institute of the National Institutes of Health.
Development of Interactive Immunoregulatory Gr‐1+ Subsets Induced by Mycobacterial Products: Role of IL‐23 in Mobilization from Bone Marrow.
Authors: van der Veen Roel C
Published: June 2021

Similar to the regulation of vasodilation, the balance between immunosuppressive nitric oxide (NO) and its antagonist superoxide (O2−) regulates expansion of activated T cells in mice. Reduction of NO levels by O2− is essential for T cell expansion and development of autoimmunity. Mice primed with heat‐killed Mycobacterium develop granulocytic Ly‐6G+ and monocytic Ly‐6G− Gr‐1+ subsets in the spleen, which, upon stimulation by activated T cells, produce O2− or NO, respectively, which regulate T‐cell expansion. Here, we report on the development of this ‘Greg system’ in bone marrow (BM). Unlike spleen, naïve BM expressed high levels of Gr‐1+ cells and weak immunoregulatory activity. Upon priming, BM cells expressed strong NO and O2− activity in the Gr‐1 enriched fraction, similar to the spleen, but in addition developed NO activity in the Gr‐1 depleted fraction as well, unlike the spleen. As expected, Ly‐6G+ splenocytes developed poorly in primed IL‐23−/− mice and produced no O2− Unexpectedly, splenocytes expressed reduced NO‐mediated suppression by Gr‐1+ cells as well, indicating a general role for IL‐23 in Greg development. Surprisingly, strong immunoregulatory NO and O2− activities both developed in BM of IL‐23−/− mice. While O2− production was restricted to the Gr‐1+ BM cell fraction, suppressive NO production was strongest in the Gr‐1‐depleted fraction. We conclude that IL‐23 functions at different levels during development of Greg subsets, i.e. mobilization of O2− producing counter‐suppressive granulocytic Greg from BM, and optimal development of suppressive Gr‐1+ monocytic cells within the BM prior to mobilization.
Role of TRAF2 and TRAF3 in CD40 mediated immunoglobulin class switching
Authors: Jabara Haifa H , Sannikova Tatyana , Geha Raif S
Published: June 2021

The isotype switch defect in CD40−/− mice is corrected by wild type (WT) CD40 transgene, but not by a mutant CD40 transgene that does not bind TRAF2 and TRAF3. To define the individual roles of TRAF2 and TRAF3 in CD40 activation of B cells, we introduced mutant CD40 transgenes that selectively lack the ability to bind TRAF2 (ΔTR2), TRAF3 (ΔTR3) or both (ΔTR2,3) into B cells of CD40−/− mice. Serum IgG1 and IgE levels, germinal center formation, CD40‐mediated proliferation, isotype switching, and activation of the non‐canonical NFκB pathway and JNK were partially diminished in ΔTR2 and ΔTR3 mice, and virtually absent in ΔTR2,3 mice. These results suggest that TRAF3 plays a positive role in CD40 driven CSR and that both TRAF2 and TRAF3 are required for optimal CD40 driven isotype switching.
Differential effects of cigarette smoke constituents on pro‐inflammatory dendritic cell functions
Authors: Kroening Paula R , Vassallo Robert
Published: June 2021

Dendritic cells (DC) are key regulators of immunity. Chronic obstructive pulmonary disease [COPD], a destructive lung disease that affects smokers, is associated with chronic airway inflammation. Although increased DC numbers are found in COPD airways, their functional roles in the pathogenesis of COPD are not well‐defined. We postulated that DCs activated by cigarette smoke constituents directly promote chronic airway inflammation. Concordant with this hypothesis, we observed that incubation of DCs with cigarette smoke extract [CSE], and chronic exposure of mice to cigarette smoke, both augmented the generation of neutrophilic chemokines by immature and lipopolysaccharide [LPS] or CD40L‐matured DCs. The generation of IL‐8 by human DCs conditioned with CSE was completely suppressed by the anti‐oxidant n‐acetyl cysteine [NAC]. Cigarette smoke extract and nicotine augmented the production of secreted prostaglandin‐E2 and intracellular cyclo‐oxygenase‐2 in maturing DCs. Whereas the production of IL‐8 by CSE‐conditioned DCs was suppressed by anti‐oxidants, PGE2 production was augmented by anti‐oxidants, and suppressed by inhibitors of nicotinic signaling. These studies indicate that certain pharmacologic strategies such as anti‐oxidant therapy may be only partially effective in mitigating pro‐inflammatory DC‐mediated responses in smokers. Supported by FARMRI research grant to RV.
In vitro conditioned “early” but not “late” effector CD8+ T cells benefit from transient lymphopenia resulting in enhanced anti‐tumor activity
Authors: Diaz‐Montero C. Marcela , AL‐Khami Amir , Naga Osama , Cole David , Salem Mohamed
Published: June 2021

A limitation of adoptive transfer therapy for cancer is the inability of transferred cells to persist. Temporary removal of endogenous lymphocytes increases the survival of transferred cells. The mechanisms behind the beneficial effects of lymphodepletion are starting to emerge; however, less is known about the impact of the phenotypic characteristics of the transferred cells on their ability to benefit from lymphodepletion. We have shown that in vitro priming of CD8+ T cells in the presence of IL‐12 promotes the generation of a subpopulation with an “early activated” phenotype (TEA). Under lymphoreplete conditions, TEAIL‐12 show enhanced survival and anti‐tumor activity when compared to TEAsham. In this study, we show that the anti‐tumor activity of TEAIL‐12 can be further enhanced after transfer into lymphopenic hosts resulting in long term regression of B16 tumors. The significance of these findings relies on the fact that tumor regression in this model is only achieved temporarly. Survival of transferred cells was dependent on their stage of differentiation. In vitro culture of TEAIL‐12 for additional 4 days abrogated their ability to survive under lymphopenic conditions. Our results suggest that long lasting anti‐tumor immunity after adoptive cell transfer could be achieved by utilizing a “fitted” T cell subtype and by promoting its survival and optimal differentiation in vivo through lymphodepletion.
Use of the CRAFTi portable fluorometer to measure serum retinol (vitamin A) concentrations
Authors: Chaimongkol Lakshana , Winichagoon Pattanee , Craft Neal E. , Furr Harold C.
Published: June 2021

Rapid assessment of vitamin A status could be a very useful tool for identifying individuals or groups at risk of vitamin A deficiency. We have evaluated a portable fluorometer modified to measure Retinol that is bound to Retinol Binding Protein in small volumes (25 uL) of serum or plasma without need for solvent extraction. A typical determination requires approximately 30 minutes total time (including collection of blood) but samples can be batch‐processed to increase throughput. Performance of the CRAFTi portable fluorometer was tested by comparing values obtained using it against those obtained by conventional extraction and HPLC analysis of serum retinol on 38 blood samples obtained from women factory workers (lower socioeconomic status) in Bangkok, Thailand. Serum retinol values as determined by HPLC ranged from 0.79 to 2.52 uM (1.60 ± 0.41, mean ± std. dev.); as determined by the fluorometer, from 0.98 to 2.79 uM (1.53 ± 0.43). The slope of the correlation was 0.81 (fluorometer/HPLC). A Bland‐Altman plot showed no systematic bias between the two methods. Furthermore, results by two different analysts using the fluorometer showed good agreement with no evidence of systematic bias. (Supported in part by Sight & Life and Craft Technologies, Inc.)
Dichloroacetate Accelerates Autosomal Recessive Polycystic Kidney Disease in the Pck Rat
Authors: Gattone Vincent H. , Sinders Rachel
Published: June 2021

Dichloroacetate‐DCA is found in chlorinated water and known to be toxic in high doses. DCA is also promoted as a cancer chemotherapeutic agent at low doses, causing an increase in apoptosis of malignant cells and leaving nontransformed cells unaffected. Some chemotherapeutic drugs ameliorate the renal pathology in Polycystic Kidney Disease‐PKD. PKD typically affects both liver and kidney. Renal and biliary epithelia usually exhibits increased proliferation and apoptosis. In the present study we hypothesized that DCA might modify the PKD in Pck rats, an orthologous model of human autosomal recessive PKD. DCA was added to drinking water (75mg/l, a relatively low dose) for 4 week‐old male and female Pck rats, while control rats received tap water. At 8 weeks, rats were evaluated for renal and liver histopathology and serological evidence of disease. Treated male rats had an increased body weight, total kidney weight, volume of renal cystic change and increased proteinuria, but without evidence of increased liver disease. Interestingly, hepatorenal disease in females was unaffected by DCA treatment. In conclusion, data indicated DCA causes an increase in severity of renal disease in male Pck rats.
Gap junctions and the transfer of reactive oxygen species during endothelium‐dependent contractions
Authors: Tang Eva Hoi Ching , Vanhoutte Paul M
Published: June 2021

To assess whether or not reactive oxygen species (ROS) and gap junctions contribute to endothelium‐dependent contractions in the aorta of spontaneously hypertensive rats (SHR), rings with or without endothelium, treated with antioxidants or gap junction inhibitors, were studied by recording of isometric force. Superoxide dismutase and catalase did not affect, while diethyldithiocarbamate or manganese (III) tetrakis(1‐methyl‐4‐pyridyl)porphyrin (cell permeable antioxidants) reduced endothelium‐dependent contractions to acetylcholine and the calcium ionophore A23187. The gap junction inhibitors, 18β‐ glycyrrhetinic acid and carbenoxolone also reduced these contractions. The gap peptides 40Gap27, 37,43Gap27 and 43Gap26 decreased responses to acetylcholine, but only 40Gap27 reduced those to A23187. These findings suggest that ROS contribute to endothelium‐dependent contractions in the SHR aorta. Myoendothelial gap junctions permit the transfer of ROS, explaining why the response is uninterrupted by antioxidants with poor cell permeability but are sensitive to antioxidants with high permeability. The endothelium‐dependent contraction to acetylcholine involves connexins 37, 40 and 43, while only connexin 40 is involved in that to A23187. This study is supported by the RGC of HKU.
Deleterious Cardiovascular Effects of Antenatal Betamethasone Exposure in Young and Adult Sheep
Authors: Shaltout Hossam Abdelnaby , Figueroa Jorge P , Rose James C , Chappell Mark C , Averill David B , Diz Debra I
Published: June 2021

Betamethasone (Beta) administration prior to premature delivery in early third trimester is employed to accelerate lung development and improve survival. Male adult sheep (1.8 years) exposed at 80th day of gestation to Beta (Ad‐Beta) had elevated arterial pressure (MAP), increased left ventricle to body weight ratio (LV/BW), and attenuated baroreflex sensitivity for control of heart rate (BRS) compared to age‐matched controls (Ad‐Veh). We have now determined that young sheep (0.5 years) exposed antenatally to Beta (Y‐Beta) had higher MAP (95 ± 2 vs 77 ± 2 mm Hg; p < 0.05) and lower BRS (3.9 ± 1.2 vs 10.4 ± 1.8 ms/mm Hg; p < 0.05) compared with Y‐Veh. However, at this age there was no difference in LV/BW between Y‐Beta and Y‐Veh groups. The AT1 angiotensin antagonist candesartan (0.3 mg/kg, iv) normalized MAP and improved BRS in Y‐Beta sheep. Heart rate variability (HRV) and blood pressure variability (BPV), measured by time and frequency domain analysis, revealed that Y‐Beta sheep had significantly lower HRV (SDRR, rMSSD, LFRRI, and HFRRI) compared with Y‐Veh and higher BPV (SDMAP, LFSAP). Candesartan partially restored HRV in Y‐Beta sheep and fully corrected BPV. Thus, in utero exposure to Beta increases MAP and impairs baroreflex control of heart rate in young sheep. The deleterious actions of antenatal steroids persisted into adulthood, likely contributing to LV hypertrophy in adult sheep. HD047584
Biphenylalkylacetylhydroquinone ethers suppress the proliferation of murine B16 melanoma cells
Authors: Fernandes Nicolle Valerie , Jung Manfred , Daoud Ali , Mo Huanbiao
Published: June 2021

Hydroquinone, an activator of caspase‐9 activity via reactive oxygen species, and farnesol, a posttranslational down‐regulator of 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase activity suppress the growth of murine B16 melanoma cells. Our previous studies have shown that farnesyl‐O‐acetylhydroquinone has a markedly greater growth‐suppressive activity than that predicted by the responses to the parent compounds. Perillyl alcohol, a modulator of small G‐protein activity, and biphenyl compounds, activators of Fas‐mediated death pathways, suppress B16 growth. A similar synergistic increase in the potency of each compound when ether‐linked to acetylhydroquinone is now reported. Perillyl‐O‐acetylhydroquinone, biphenylethyl‐O‐acetylhydroquinone and biphenylpropyl‐O‐acetylhydroquinone had dose‐dependent impacts on the proliferation of B16 cells with 50% inhibitory concentration (IC50) values of 8.0, 4.2 and 1.4 μmol/L, respectively. The growth‐suppression effected by biphenylpropyl‐O‐acetylhydroquinone was accompanied by a dose‐dependent arrest at the G1/S interface of the cell cycle, an impact greater than that previously reported for farnesyl‐O‐acetylhydroquinone (IC50 = 2.5 μmol/L). These new hydroquinone derivatives may have potential in cancer chemoprevention and/or therapy. TDA, TWU REP & Summer Stipend Award.
Growth inhibition of human cholangiocarcinoma by sorafenib‐based mono‐ and combination treatment
Authors: Höpfner Michael , Baradari Viola , Huether Alexander , Scherübl Hans
Published: June 2021

Background: Treatment options of advanced cholangiocarcinoma (CC) are unsatisfactory and new therapeutic approaches are mandatory. The Ras/Raf/MAPK‐pathway is one of the most significant cellular signaling sequences in the development and maintenance of cancer. Thus inhibition of this key signaling pathway appears to be a promising approach for novel CC‐treatment. Here we investigated the antineoplastic effects of sorafenib (Sor), a novel multi‐kinase inhibitor which potently suppresses wild‐type‐ and mutated Raf‐isoforms. Sor has already shown antineoplastic efficacy in some cancer types but its suitability for treatment of CC has not been investigated so far.

Results: Sor dose‐dependently blocked growth‐factor‐induced MAPK‐activity and inhibited the proliferation CC cells in a time and dose‐dependent manner. At least two mechanisms accounted for the effects observed: G1/G0‐ arrest of the cell cycle and apoptosis. G1/G0‐arrest was associated with upregulation of the cdk‐inhibitor p27 and downregulation of cyclin D1. Combining Sor with doxorubicin, the HDAC inhibitor MS‐275 or IGF‐1R‐inhibition resulted in (over‐) addititve antiproliferative effects. Conclusion: Our study demonstrates that the growth of human CC cells can be potently suppressed by Sor alone or in certain combination therapies and may provide a promising rationale for future in vivo evaluations and clinical trials.
Hyperglycemia activates the Ca2+/calcineurin‐dependent transcription factor NFAT (Nuclear Factor of Activated T‐Cells) in retinal microvessels in vivo
Authors: Zetterqvist Anna V. , Öhman Jenny Nilsson , de Frutos Garcia Sergio , McGuire Paul G. , Bosc Laura Gonzalez , Gomez Maria F.
Published: June 2021

Diabetic retinopathy is a major cause of blindness worldwide. The underlying pathogenesis is not clear but hyperglycemia has been identified as an important risk factor. Endothelial dysfunction, vasoconstriction and changes in Ca2+ signaling have been shown to precede the onset of diabetic retinopathy. We have recently shown that high extracellular glucose activates NFAT in the smooth muscle cells of intact cerebral arteries. This effect involves the local release of extracellular nucleotides, such as UTP, acting on P2Y receptors, leading to increased [Ca2+]i and subsequent activation of calcineurin. Activation of NFAT promotes cell proliferation and the expression of inflammatory markers, such as IL‐6, COX‐2 and osteopontin. We therefore hypothesize whether NFAT may act as a glucose‐sensor in the wall of retinal microvessels. Using confocal immunofluorescence microscopy we show that NFATc3 is expressed in the endothelium of murine retinal microvessels. Acute changes in blood glucose levels induced by IP‐GTT (glucose tolerance test) are readily detected by NFATc3 in vivo. NFAT activation is inhibited by a specific blocker of NFAT as well as by the ecto‐nucleotidase apyrase, suggesting the involvement of extracellular nucleotides also in the endothelial response to high glucose. Supported by: Swedish Heart & Lung Foundations, Swedish Research Medical Council & Royal Physiographic Society in Lund.
Molecular basis of cytokinesis in fission yeast
Authors: Pollard Thomas D. , Vavylonis Dimitrios , Wu Jian‐Qiu , O'shaughnessy Ben
Published: June 2021

Cell division by cytokinesis in animals and fungi depends on a contractile ring of actin filaments and the motor protein myosin‐II. Fluorescence microscopy of live fission yeast cells reveals that they assemble a cytokinetic contractile ring by condensation of a broad band of membrane‐associated nodes. The position of the nucleus determines the initial location of the zone with these nodes by releasing the anillin‐like protein Mid1p, which recruits to nodes myosin‐II followed by other proteins including formin Cdc12p. Actin filaments nucleated by Cdc12p form a network between the nodes and myosin‐II pulls the nodes together into a ring. Our analysis of node motions and numerical simulations show that a simple, unassisted search, capture, pull, and release mechanism can account for contractile ring assembly. A key element in the mechanism is that the connections between actin filaments from one node and myosins in another node are transient, so that myosins produce force on a captured actin filament for only about 20 seconds before being released.
A1 adenosine receptor‐activated protein kinase C signaling in A1 knock‐out mice coronary artery smooth muscle cells
Authors: Ansari Habib R. , Teng Bunyen , Nadeem Ahmed , Schnermann J. , Mustafa S. Jamal
Published: June 2021

A1 adenosine receptor (A1AR) is coupled to Gi/Go proteins, inhibiting adenylate cyclase, but the downstream signaling pathway in smooth muscle cells is unclear. Present study was performed in coronary artery smooth muscle cells (CASMC) from WT and A1AR KO mice to determine the role of protein kinase C (PKC) pathway in A1AR signaling. We used CASMC isolation method from our lab (Teng et al 2006, Am J Physiol Heart Circ Physiol. 290:H1713–20). To detect PKC isoforms, cytosolic and membrane fractions were made from cell homogenates. PKC isoforms were identified by Western blotting. In both WT and A1AR KO, basal levels of PKC α, β, γ, δ, θ, were abundant in cytosolic, and of PKC μ, ε, λ in membrane fractions whereas PKCζ was equal in both fractions. In WT cells treated with A1AR agonist, ENBA (10−5M) increased A1AR expression by 162%, and this was inhibited by 55% compared with control by A1AR antagonist DPCPX (10−6M), whereas there was no effect in A1AR KO. In WT, ENBA activated PKCα expression by 190% over control, and it was inhibited by specific PKCα inhibitor Gö6976 (10−7M), but unchanged in A1AR KO (p<0.05). In organ bath experiments, Gö6976 (10−7M) inhibited ENBA‐induced aortic contraction by 45% in WT, but no effect was noted in A1AR KO. The phospholipase C (PLC) inhibitor U73122 (10−6M) reduced the expression of PLCγ and pERK1/2 by 54% and 51%, respectively (p<0.05). We conclude that A1AR activation by ENBA activates PLC, mainly through PKCα leading to ERK1/2 phosphorylation and contraction in CASMC. Supported by HL‐027339.
Expression of inducible nitric oxide synthase (iNOS) within the ventrolateral medulla in rats following transient middle cerebral artery occlusion and reperfusion
Authors: Ally Ahmmed , Maher Timothy J
Published: June 2021

Majority of stroke cases are due to ischemia in regions supplied by the middle cerebral artery (MCA). We reported that cardiovascular responses during static muscle contractions are attenuated following a transient MCA occlusion (MCAO), and involved differential expression of neuronal nitric oxide synthase (nNOS) protein within the rostral (RVLM) and caudal ventrolateral medulla (CVLM). In this study, we compared cardiovascular responses and inducible NOS (iNOS) protein expression within the left and right sides of both RVLM and CVLM in sham‐operated rats, and in rats with a 90‐minute left‐sided MCAO followed by 24 hour reperfusion. Pressor responses during static muscle contractions were attenuated in MCAO rats when compared to sham rats. Also, iNOS expression within the left RVLM was augmented when compared to the right RVLM in MCAO rats, and to both RVLM quadrants in sham rats. Conversely, compared to sham rats and the right CVLM of MCAO rats, iNOS expression was significantly attenuated in the left CVLM quadrant in left‐sided MCAO rats. Thus, the attenuated pressor response during muscle contraction in MCAO rats is partly due to overexpression and attenuation of iNOS protein within the ipsilateral RVLM and CVLM, respectively. Results demonstrate that differential expression of iNOS protein within the medulla mediates cardiovascular responses during static exercise in pathophysiological conditions.
ENDOTHELIAL NITRIC OXIDE SYNTHASE (eNOS) WITHIN THE PERIAQUEDUCTAL GRAY MATTER DIFFERENTIALLY MODULATES NEUROTRANSMISSION AND CARDIOVASCULAR RESPONSES DURING MECHANICAL, HEAT, AND COLD NOCICEPTION
Authors: Patel Dipan , Chaitoff Kevin A. , Nolan Allisa , Bohlke Mark , Maher Timothy J. , Ally Ahmmed
Published: June 2021

Nitric oxide (NO) is synthesized from L‐arginine via NO synthase (NOS) that exists as 3 isoforms: neuronal, inducible and endothelial (eNOS). We examined the role of eNOS within the periaqueductal gray mater (PAG) on cardiovascular responses and glutamate (GLU) and GABA concentrations during mechanical, thermal, and cold nociception to the hind paw in anesthetized rats. Mechanical stimulus (bilateral hindpaw pinch, 10 sec) increased mean arterial pressure (MAP), heart rate (HR), & GLU in PAG while GABA decreased. Bilateral microdialysis of a selective eNOS antagonist, 7‐nitroindazole (7‐NI; 1μM), into the PAG had no effect on MAP, HR, GLU & GABA during a mechanical stimulus. A heat stimulus (immersing a hindpaw metatarsus in water, 52°C, 10 sec) increased MAP, HR and GLU, while GABA decreased. Administration of 7‐NI into PAG augmented cardiovascular and GLU responses, and attenuated GABA changes. In contrast, a cold water stimulus (10°C, 10 sec) decreased MAP, HR, and GLU, but increased GABA. However, following microdialysis of 7‐NI into the PAG, a cold stimulus evoked pressor and tachycardic responses, augmented GLU and attenuated GABA levels. Results demonstrate that eNOS within the PAG plays a differential role in modulating cardiovascular responses and GLU/GABAergic neurotransmission during mechanical‐, heat‐, and cold‐mediated nociception.
Cell biology of the human proton‐coupled folate transporter (hPCFT) in renal epithelial MDCK cells
Authors: Subramanian Veedamali Subbian , Marchant Jonathan S , Said Hamid M
Published: June 2021

The human proton‐coupled folate transporter (hPCFT) is a recently identified folate transporter. Little is known about the structure‐function relationship of the different domains of this transporter, in particular which regions are important for folate transport as well as targeting of the transporter to the apical cell surface. Our aim in this study was to investigate the role of the COOH‐terminal domain and a well conserved sequence separating transmembrane (TM) domains TM2 and TM3 (DXXGRR; amino acids 109–114) to be important for transport function. Using live cell (MDCK) imaging approaches, we demonstrate that a hPCFT‐YFP construct is functionally expressed at the apical membrane domain and is localized differentially to the human reduced folate carrier (hRFC). In addition, the predicted COOH‐terminal of the hPCFT is not essential for apical targeting or functionality, and that mutations that ablate a consensus beta‐turn sequence separating predicted TM2 and TM3 abolished apical 3H‐folic acid uptake (due to retention in ER). Further, cell surface delivery of hPCFT is disrupted by microtubule depolymerization, and by overexpression of p50. These data presents information regarding structure‐function and membrane targeting of the hPCFT polypeptide, as well as the mechanisms that control its steady state expression in renal epithelial cells. [Supported by grants from the NIH and the DVA].
Gonadal hormone regulation of the renal vasopressin V2 receptor
Authors: Liu Jun , Ji Hong , Zheng Wei , Verbalis Joseph G. , Sandberg Kathryn
Published: June 2021

The renal vasopressin V2 receptor (V2R) plays an important role in the physiological and pathophysiological processes underlying renal escape from arginine vasopressin (AVP)‐induced antidiuresis. The V2R gene is located on the X chromosome and has a high probability of escaping X‐inactivation. Thus, we investigated whether sex differences in expression of the renal V2R exist. We found that kidney V2R mRNA expression was 2.7‐fold higher in female (F) compared to male (M) MF‐1 mice [arbitrary units (AU): F, 8.13 ± 1.64 vs M, 3.06 ± 0.99, n=3; p<0.05]. To determine if these sex differences were a result of gonadal hormone effects, we measured V2R expression 12 weeks after sham‐operation (Sham) or ovariectomy (OVX) in mouse kidneys. Ovariectomy increased V2R mRNA expression by 1.8‐fold in the renal cortex [AU: OVX, 0.58 ± 0.02 vs Sham, 0.32 ± 0.02, n=3; p<0.05] and by 2.2‐fold in the inner medulla [AU: OVX, 4.02 ± 0.85 vs Sham, 1.82 ± 0.13, n=3; p<0.05]. These studies indicate that the renal V2R is regulated by ovarian hormones in female mice. Furthermore, sex differences in renal V2R expression have implications for pathologies associated with aberrant regulation of the V2R, specifically that females may be less able to escape AVP‐induced antidiuresis because of higher renal V2R expression than males (supported by R01 HL57502).
Systemic Sildenafil does not affect maternal hemodynamics or fetal growth in rat pregnancy
Authors: Sasser Jennifer M , Vessell Colleen , Baylis Chris
Published: June 2021

It has been hypothesized that the PDE5 inhibitor sildenafil (SILD) may be useful in the treatment of hypertension during pregnancy. However, we previously showed that increased renal inner medullary PDE5 is involved in the Na retention of pregnancy. The purpose of this study was to determine the effects of oral SILD treatment on maternal plasma volume, Na balance and fetal growth during rat pregnancy. Rats received SILD (10 mg/kg/day (SILD10, n=5) or 50 mg/kg/day (SILD50, n=6)) in food or control diet (CON, n=7) on days 4–20 of pregnancy. On days 14–19, rats were housed in metabolic cages for collection of urine and measurement of Na intake, and hemodynamic and fetal measurement were taken on day 20. Both doses of SILD increased plasma cGMP concentrations (Table, * p<0.05 vs CON); however, sildenafil treatment had no effect on mean arterial pressure (MAP), weight, hematocrit (HCT) or plasma volume (PV). The SILD10 dose slightly reduced sodium retention over the 5 day period, but glomerular filtration rate (creatinine clearance, CCr) was not significantly changed by either SILD treatment. The pup number and size were similar among the groups. Therefore, these studies suggest that oral (systemic) SILD may be safe during pregnancy. The effects of SILD on hypertension during pregnancy should be examined separately.
Disturbed water balance in Ku86−/‐ mice
Authors: Dmitrieva Natalia I , Chen Henry , Nussenzweig André , Burg Maurice B
Published: June 2021

High NaCl induces DNA breaks in cells in culture and in the kidney inner medulla in vivo. Ku86 binds to broken ends of damaged DNA and facilitates DNA repair. Ku86 deficiency compromises adaptation of cells to high NaCl, resulting in cellular hypertrophy, cell cycle arrest and chromosomal breakage (PNAS 102:10730,2005). We hypothesized that the renal function of Ku86−/‐ mice might be impaired because of increased sensitivity of their renal medullary cells to the high NaCl to which they are normally exposed as part of the urinary concentrating mechanism. To analyze the ability of Ku86−/‐ mice to maintain water balance, mice were subjected to different regimes of water consumption. During free access to water, Ku86−/‐ mice drink much more water leading to greater urine volume (2.45*±0.26 vs 1.11±0.09 ml/20g of body weight (BW)), and decreased urine osmolality compared to wild type mice. However, their urinary vasopressin excretion (U‐AVP) is significantly higher (271±76* vs 136±22 pg/24h per 20 g of BW). U‐AVP dramatically increases in Ku86−/‐ mice when water is limited to 3ml per 5g of gel food (599±74* vs 175±30 pg/24h) and they lose weight. Water restriction to 1.7ml/5g causes further weight loss in Ku86−/‐ mice. We conclude that Ku86−/‐ mice do not adapt normally to water restriction, possibly due to decreased responsiveness of renal collecting ducts to vasopressin. Supported by the Intramural Programs of NHLBI and NCI.
Control of hypothalamic malonyl‐CoA by central glucose and leptin
Authors: Lane M. Daniel , Wolfgang Michael , Cha Seung Hun
Published: June 2021

We investigated the responses of intermediates of the hypothalamic malonyl‐CoA signaling pathway to glucose and leptin. Hypothalamic malonyl‐CoA increases in proportion to the carbohydrate content of the diet consumed following food deprivation. After refeeding or peripheral glucose administration, blood glucose and hypothalamic malonyl‐CoA levels peak at 30 and 60 minutes, respectively. The malonyl‐CoA response depends on the amount of glucose administered and is blocked by the icv administration of an inhibitor of glucose metabolism, 2‐deoxyglucose (2‐DG). The change in hypothalamic malonyl‐CoA following glucose administration coincides with the suppression of phosphorylation of AMP kinase (AMPK) and acetyl‐CoA carboxylase. Blockade of glucose utilization in the CNS by icv 2‐DG prevented the effects of glucose on AMPK phosphorylation, malonyl‐CoA and neuropeptide expression in the hypothalamus and on food intake. Icv leptin increased hypothalamic malonyl‐CoA additively with glucose administration. Since leptin‐deficient ob/ob mice showed no defect in the glucose‐ or refeeding‐induced rise in hypothalamic malonyl‐CoA after food deprivation, it is evident that leptin is not required for this effect.
Augmented vasopressin secretion during low sodium diet: Sensitization by volume signals?
Authors: Bie Peter , Molstrom Simon
Published: June 2021

Regulation of vasopressin secretion was studied in conscious dogs and in healthy volunteers. Dogs were fed regular diet or low‐salt diet (0.03 mmol kg−1 d−1) for 7 d. NaCl was infused i.v. at 0.02 mmol kg−1 min−1 in negligible volume for 3 h. Vasopressin V2 receptors were blocked by OPC 31260. Body fluid volume was maintained by servo‐controlled fluid infusion. On regular diet NaCl infusion elevated plasma Na+ and osmolality (pOsm) by 4.5% and elicited a 3‐fold increase in plasma vasopressin (pAVP, 1.58 ±0.24 to 4.39 ±0.40 pg ml−1). At low‐salt diet pAVP increased (1.74 ±0.13 to 4.94 ±0.68 pg ml−1) as a function of pOsm with similar slope (0.31 pg ml−1 (mOsm/kg)−1) but offset to the left by 6.2 mOsm/kg. Similar results were seen during β1‐receptor blockade (metoprolol, 2 mg/kg plus 0.9 mg h−1 kg−1). Human volunteers were studied after 4 d of low‐salt diet. Saline (135 mM) was infused i.v. providing 0.012 mmol Na+ min−1 kg−1 for 4 h (some 1500 ml). Arterial blood pressure (BP) did not change; heart rate (HR) fell by 5.8 ± 1.5 bpm (p<0.01); central venous pressure (CVP) increased by 1.7 ± 0.5 mmHg (p<0.025) concomitant with a fall in pAVP (3.0±0.4 to 1.8±0.1 pg ml−1, p<0.01). Identical results were obtained after metoprolol (0.3 mg/kg plus 0.06 mg h−1 kg−1) except that HR remained constant. The data are compatible with CVP mediated control of pAVP during low‐salt diet.

Supported by the Danish MRC and the Novo Nordisk Foundation.
Essential amino acid supplementation maintains muscle function during bed rest in the elderly
Authors: Ferrando Arny A , Paddon‐Jones Doug , Kortebein Patrick , Ronsen Ola , Lombeida Juan , Symons T. Brock , Wolfe Robert R. , Evans William
Published: June 2021

Older individuals are more likely to experience extended hospitalization. Thus, there is an inherent risk of prolonged inactivity and the accompanying functional decline. We have previously demonstrated that the provision of an essential amino acid (EAA) supplement in younger individuals subjected to 28d of bed rest eliminates the loss of lean body mass (LBM) and ameliorates the loss in muscle function. We hypothesized that EAA administration in older individuals subjected to 10d bed rest would also spare LBM and muscle function. Twenty‐two older subjects were given a placebo (n = 12, 68 ± 5 (SD) yrs, 83 ± 19 kg) or 15g of EAA (n = 10, 71 ± 6, 72 ± 8 kg) 3 times per day throughout 10d of bed rest. Measures of LBM, muscle protein synthesis, and muscle strength and function were determined before and after bed rest. Outcomes were tested statistically by 2X2 ANOVA and Tukey's post‐hoc test. EAA supplementation maintained muscle protein synthetic rate (P=0.025), but did not ameliorate the loss of LBM. EAA supplementation resulted in a preservation of plantar flexion strength (p=0.054), stair ascent power (p=0.029), stair descent power (p=0.026), and floor transfer time (p=0.027). Despite the inability of EAA to preserve LBM, muscle function was maintained. EAA supplementation maintains muscle protein turnover and preserves muscle function in older, inactive individuals.

Project funded by the National Institutes of Health grant PO1 AG023591 (Evans).
Lesions of the Ventral Lamina Terminalis Prevent Salt‐Induced Changes in the Responsiveness of Neurons in the Rostral Ventrolateral Medulla (RVLM)
Authors: Stocker Sean D , Adams Julye M
Published: June 2021

Sympathetic nerve activity (SNA) and arterial blood pressure (ABP) responses to injection of L‐glutamate or GABA into the RVLM are enhanced during elevated dietary salt intake. The present study determined whether sodium‐sensing neurons of the forebrain lamina terminalis mediate these changes. Male Sprague‐dawley rats with or without lesions of the ventral lamina terminalis were given access to normal chow and either water or 0.9% NaCl for 14 days. Injection of L‐glutamate into the RVLM of control rats drinking 0.9% NaCl (57±2 ml/day, n=13) versus water (35±1 ml/day, n=11) produced greater changes in renal SNA (0.1nmol: 81±5 vs 52±5%; 1nmol: 123±8 vs 82±6%) and ABP (0.1nmol: 23±2 vs 10±1; 1nmol: 44±3 vs 24±1 mmHg). In marked contrast, injection of L‐glutamate in lesioned rats drinking 0.9% NaCl (56±7 ml/day, n=5) versus water (42±2 ml/day, n=5) showed similar increases in renal SNA (0.1nmol: 62±9 vs 57±11%; 1nmol: 87±4 vs 85±17%) and ABP (0.1nmol: 15±4 vs 15±3; 1nmol: 29±4 vs 31±5 mmHg). Similarly, injection of GABA produced greater depressor responses in control rats drinking 0.9% NaCl versus water (0.1nmol: −15±1 vs −7±1; 10nmol: −31±3 vs −15±1 mmHg, n>7), and this effect was prevented in lesioned rats (0.1nmol: −6±1 vs −7±1; 10nmol: −12±2 vs −15±1 mmHg, n=3). These findings suggest that increased dietary salt activates neurons in the forebrain lamina terminalis to enhance the responsiveness of RVLM neurons.
Cardiovascular responses to underwater submersion change little after disrupting the RVLM with anti‐DBH‐saporin.
Authors: Panneton W. Michael , Gan Qi , Aguera Jay , Greenwood Vivianne , Harvey Brent
Published: June 2021

Two groups of bulbospinal neurons in the RVLM respond to nasal stimulation with ammonia vapors. One group is slowly‐conducting neurons, probably catecholaminergic, while another involves faster‐conducting neurons, probably noncatecholaminergic. We investigated the C1 catecholaminergic neurons located in the RVLM of rats, and their role in changes of heart rate (HR) and arterial blood pressure (AP) to either diving underwater or nasal stimulation with ammonia vapors. The toxin anti‐DBH‐saporin was injected either directly into the RVLM (20ng/200nl, bilaterally) of one group or into theT2‐T4 spinal cord of another group of rats trained to dive underwater; rats survived for 3–6wks. The dramatic bradycardia (avg. = ~80%) and the increase in BP (avg. =~10%) usually seen in diving rats persisted in both groups. The lesser bradycardia usually seen with nasal stimulation of rats after anesthesia was similar to that after either spinal or medullary injections. However, AP fell −14% in rats with spinal injections, but rose 3% in rats receiving medullary injections. The brains were sectioned and immunostained for tyrosine hydroxylase; positive C1 neurons were counted. The results are discussed in terms of the drop in AP seen after nasal stimulation and spinal injections, the route of injection, and the correlation of physiological data with the loss of C1 neurons. Supported by SLU School of Medicine.
Voluntary exercise modifies gene expression profiles in the nucleus tractus solitarii (NTS) of hypertensive rats
Authors: Potts Jeffrey T , Fong Angelina Y , Mueller Patrick J , McGovern David
Published: June 2021

Regular exercise has beneficial effects on cardiovascular (CV) function. However, its affect on genetic components of brainstem nuceli is poorly understood. Since the NTS is essential for CV regulation, it may be a neural substrate for exercise‐induced CV adaptation. Rats (spontaneously hypertensive, SHR, n=4; Wistar‐Kyoto, WKY, n=4) performed voluntary wheel exercise (EX) for 12 weeks to screen for differentially expressed genes. Additional rats (n=4 per group) served as sedentary (SED) controls. Brainstems were harvested and NTS tissue was obtained by laser capture microscopy. Total RNA was hybridized to cDNA probes (Ratref12, Illumina) containing ~22,000 transcripts. The criterion for differential expression was > 2X difference in signal intensity. There was no significant difference in total distance (722±145 vs 594±91 km) or distance run per day (9±4 vs 7±3 km), SHR vs WKY. In SHR, exercise decreased resting blood pressure (131±3 vs 122±5 mmHg, P<0.05) and heart rate (311±14 vs 288±3 bpm, P<0.05), SHR‐SED vs SHR‐EX. No differences were found in WKY. Nineteen genes were differentially expressed in SHR‐EX (12 genes increased, 7 genes decreased). Of particular interest were genes involved in the inflammatory process (table).

These results form the basis for future studies to elucidate mechanisms and links between voluntary exercise, NTS signaling and CV disease.
Chronic hypoxia (CH)‐induced inflammation down‐regulates soluble guanylate cyclase (sGC) in rat carotid body
Authors: Dinger B , Liu X , He L , Fidone S
Published: June 2021

sGC in oxygen‐sensitive carotid body type I cells is activated by the diffusible gas, nitric oxide (NO). In rats exposed to CH, sGC is down‐regulated, with a corresponding drop in NO‐stimulated production in cGMP. Moreover, CH induces an invasion of activated macrophages and the production of inflammatory cytokines in chemosensory tissue. The present study investigates the relationship between inflammation and the down‐regulation of sGC in type I cells. Quantitative PCR (qPCR) assays of carotid bodies from 7‐day CH (380 Torr) rats demonstrated a 50–500% increase in expression of cytokines interleukin‐1β (IL‐1β), IL‐6, and tumor necrosis factor‐α (TNFα). CH (7 days) elicited a >90% reduction in the expression of sGC. In contrast, animals concurrently treated with anti‐inflammatory drugs, ibuprofen (4mg/kg/day) or bosentan (200mg/kg/day), expressed normal levels of the sGC gene. sGC expression was reduced by 50% in normal type I cells cultured for 48 hrs in the presence of TNFα (50 ng/ml). However, exposure to TNFα did not alter the expression of the gene for tyrosine hydroxylase (TH). Our data indicate that a CHinduced increase in cytokine production directly influences the expression of sGC, a signaling molecule which mediates type I cell inhibition. Inflammation appears to be an important factor in the readjustment of chemoreceptor sensitivity following CH. USPHS Grants NS 12636 and NS 07938.
The stimulation of GIP secretion by lipid absorption – is it dose‐dependent?
Authors: Yoder Stephanie , Kindel Tammy , Li Hui , Xu Maggie , Tso Patrick
Published: June 2021

The incretin hormone glucose‐dependent insulinotropic polypeptide (GIP) enhances glucose‐mediated insulin secretion. Following nutrient ingestion, GIP secretion by enteroendocrine K cells increases rapidly. Previous studies have shown that fat ingestion stimulates GIP release; however, it is unknown if there is a dose‐dependent secretion of incretins to the ingestion of fat. We hypothesize that a direct dose‐dependent relationship exists between the amount of lipid infused and GIP secretion. To test this, lymph fistula rats were used. Under halothane anesthesia, the major mesenteric lymph duct was cannulated, and an infusion tube was placed in the duodenum. Each animal received a single bolus intraduodenally of saline or varying amounts of the fat emulsion Liposyn II. Lymph was continuously collected for 6 hours and analyzed for GIP content. At lipid doses of 27.5 and 55 mg, the secretion of GIP, over the 6 hour period, increased 1 fold above the saline control; at lipid doses of 110, 220, and 440 mg lipid, GIP secretion increased 2 fold above the control. Thus, our data suggest that GIP secretion increases incrementally rather than dose‐dependently in response to a larger lipid dose. Whether this paradigm will be altered in diet induced obese animals is a question of considerable interest to basic and clinical scientists.

Funded by DK076928‐01A(NIH/NIDDK)