bluuebunny/crossref_metadata_2025 huggingface dataset:
Exploring the Role of USP14 in CXCR4‐Mediated Neurodegeneration
Authors: Mines Marjelo Amia , Ding Yun , Goodwin J. Shawn , Fan Guo‐Huang
Published: June 2021

Alzheimer's disease (AD) is one of the most common dementias in today's society. While the cause is unknown, the accumulation of amyloid‐beta protein has been identified as the major factor promoting pathological changes in patients. Chemokine receptor expression, specifically CXCR4, has also been found to be upregulated in various regions in the brains of AD patients. This upregulation may contribute to the pathological changes observed in these patients. Identifying regulatory mechanisms that promote the accumulation of CXCR4 may help us understand the cause for these changes. In the present study, we identify USP14 as a major CXCR4 regulatory protein. This protein is critical in the regulation of CXCR4, as alteration in expression levels of USP14 dramatically affected CXCR4 deubiquitination, degradation, signaling, and chemotaxis. This protein may, therefore, be responsible for the changes in CXCR4 expression and manipulation of USP14 may result in improved patient prognosis. Using the AD mouse model, we explore changes in USP14 expression and assess the effects on AD pathogenesis. USP14 may serve as a possible therapeutic target.
Lysosomal Enhancement Promotes Synaptic Integrity and Functional Recovery in Mouse Models of Alzheimer's Disease
Authors: Butler David , Estick Candice , Batey Lara , Hwang Jeannie , Karim Sanjida , Barnes Sarah N. , Manautou Jose E. , Bahr Ban A.
Published: June 2021

Reducing protein accumulation is essential for treating Alzheimer's disease (AD) by attenuating a pathogenic cascade that leads to synaptic decline. Z‐Phe‐Ala‐diazomethylketone (PADK) increases lysosomal enzymes 2‐ to 9‐fold in vitro and in vivo. This enhancement clears AD‐related proteins and restores synaptic integrity. Here, PADK produced a dose‐dependent increase in cathepsin D without adverse effects. In the first of two transgenic models of AD, APPSwInd mice of 10–11 months exhibited deficits in coordination and spatial memory. PADK at 23 mg/kg × 9 d recovered open‐field intersession habituation (p<0.03) and improved balance beam and rotarod scores in the mice (p<0.001). 6E10 anti‐Aβ staining also revealed a 39% reduction in hippocampal CA1 sp. In a second model, 18–20‐month old APPswe/PS1dE9 mice exhibited a deficit in episodic spontaneous alternation behavior (SAB). PADK at 20 mg/kg × 10 d improved their SAB to control‐level (p=0.01). Synaptic markers GluR1 and NCAM180 were decreased 23–34% in APPswe/PS1dE9 hippocampus compared to non‐tg mice. Corresponding with the SAB improvement, PADK increased GluR1 and NCAM to non‐tg levels (p=0.0001–0.002). PADK also reduced 6E10 staining of CA1 neurons as well as number and size of plaque structures. Lysosomal modulatory drugs that enhance clearance mechanisms thus have the potential to slow the synaptic decline and cognitive deficits associated with AD.
Dendritic pathology and neuronal injury induced by Tat and opiates in a transgenic model of HIV‐1 encephalitis
Authors: Hauser Kurt F , Buch Shreya K , El‐Hage Nazira , Turchen‐Cholewo Jadwiga , Bruce‐Keller Annadora J , Nath Avindra , Knapp Pamela E
Published: June 2021

To assess the mechanisms by which opiate abuse exacerbates HIV‐1 encephalitis (HIVE), morphine (5 mg/day) and/or naltrexone (6 mg/day) were administered via s.c. implant to wild type or transgenic (tg) mice expressing Tat1–86 regulated by a doxycycline‐inducible glial fibrillary acidic protein (GFAP) promoter. Tat induction or morphine exposure significantly increased the proportion of cleaved caspase−3 immunoreactive, but not TUNEL reactive, striatal neurons at 2 d. Despite increases in caspase−3 detection, neuron numbers appeared stable at 2 and 7 d. Instead, Tat induction or morphine exposure caused marked reductions in dendritic length and spine density (Golgi‐impregnated medium spiny neurons) at 7 d. When total spine losses are considered, combined Tat induction and morphine caused additive reductions in dendritic complexity. Neuronal injury was accompanied by significant increases in glial activation (assessed by increased GFAP and F4/80 immunoreactive astrocytes and macrophages/microglia, respectively) and proinflammatory chemokines (RANTES and MCP‐1). The effects of morphine were prevented by naltrexone. Our findings suggest that HIV‐1 Tat and chronic opiates contribute to the neurocognitive defects in neuroAIDS through interactive, non‐lethal reductions in dendritic length and spine density. Support: DA19398, DA18633.
Effects of APS on the Activity of AMPK of STZ‐induced T2DM Rats
Authors: Feng Zou , Jingping Ouyang , Nian Wang , Jie Song
Published: June 2021

To investigate the effects of Astragalus polysaccharide(APS) on the activity of AMPK of STZ‐induced T2DM rats. The T2DM model is duplicated by the high‐fat diet and small dose streptozotocin(STZ, 25 mg/kg,ip). After APS therapy (400 mg/kg/d,ig) for 6 weeks, blood sugar, glycosylated hemoglobin, triglyeride and serum insulin were observed. The insulin sensitivity were evaluated by the comprehensive analysis of OGTT. The activity of AMPK, the activity of ACC, and the expression of total AMPK were measured by Western blotting in skeleton muscle(by using Thr172‐pAMPK antibody, Ser79‐pACC antibody and total AMPK antibody). In this research, fat‐fed STZ‐induced diabetic rats were characterized with hyperglycemia and impaired oral glucose tolerance. After treatment with APS, there was a significant decrease in blood sugar, glycosylated hemoglobin and improved insulin sensitivityin diabetic rats, however, there was no change in blood insulin levels in all rats. The diabetic rats presented that there were obvious degrades in phosphorylation of AMPK and phosphorylation of ACC. After APS therapy, the activity of AMPK and ACC were restored partly. However, total expression of AMPK had no change during diabetes or treatment. The results showed that APS can decrease the content of blood sugar and glycosylated hemoglobin. In this model, the hypoglycemic activity of APS is at least in part by increasing the activity of AMPK and enables insulin‐sensitizing activity. This subject has a financial assistance by NSFC (30370673).
Effect of Progesterone on Hippocampal GABA‐A Receptor Subunit Expression in Mice Lacking PRs
Authors: Reddy Doodipala S. , Zheng Y‐C.
Published: June 2021

Progesterone (P) plays a key role in the pathophysiology of catamenial epilepsy, a menstrual cycle‐related seizure disorder that affects many women with epilepsy. While P's endocrine actions are mediated by the progesterone receptor (PR), P's molecular mechanism of action in seizure activity is not clearly understood. Recently, we proposed an interdependent role for PRs in seizure susceptibility utilizing PR knockout (PRKO) mice based on the emerging evidence of P modulation of GABA‐A receptor plasticity. In this study, we tested the hypothesis that P's actions on hippocampal GABA‐A receptor subunit expression are due in part to PR pathway mediated regulation of subunit expression. We tested this hypothesis utilizing the PRKO transgenic mouse model. Total mRNA isolated from hippocampus in female WT and PRKO mice treated with P for 7 days was utilized for TaqMan PCR. TaqMan reaction conditions were optimized and quantitated by the threshold cycle (Ct) analysis. Chronic P induced a 3‐fold increase in GABA‐A receptor α1‐subunit expression in WT, but not in PRKO mice. However, there was no change in α2‐subunit in both genotypes. These results suggest that progesterone could regulate GABA‐A receptor subunit expression via a PR pathway in the hippocampus, which could be relevant to P mechanisms governing regulation of seizure susceptibility in females. *Supported by NIH grant NS051398*
A simple method for estimation of agonist activity at GPCRs: characterization of responses elicited by M2 and M3 muscarinic receptors
Authors: Tran John A , Chang Alex , Matsui Minoru , Ehlert Frederick J
Published: June 2021

We investigated a parameter termed intrinsic relative activity (RAi), which provides a measure of agonist activity in functional assays. The RAi value is equivalent to the product of observed affinity and intrinsic efficacy of the agonist‐receptor‐G protein complex. To demonstrate that the validity of the RAi analysis, we measured the observed affinity and intrinsic efficacy values of selected agonists using Furchgott's method of partial receptor inactivation and compared the product of these estimates with the corresponding RAi estimate. We found good agreement between RAi and the product of affinity and efficacy in experiments on M3 muscarinic receptor stimulation of contraction of mouse ileum. Since observed affinity and efficacy are parameters inherent to the agonist‐receptor‐G protein complex, the RAi value is expected to be the same for various biological systems utilizing the same receptor‐G protein signaling pathway. We tested this hypothesis by comparing RAi values of muscarinic agonists for inhibiting the electrically paced guinea pig atrium and for inhibiting forskolin stimulated cAMP accumulation in CHO cells expressing the M2 receptor. Our results showed good agreement between the RAi estimates of selected agonists (oxotremorine‐M, oxotremorine, S‐aceclidine, arecoline, bethanechol, pilocarpine and McN‐A‐343) across the two assays.

Supported by NIH grant 69829
Epitope‐tagged knockin mice reveal ligand‐selective internalization that explains differential desensitization of alpha2‐adrenergic responses in native sympathetic neurons
Authors: Lu Roujian , Zhang Youwen , Li Yong , Jiao Kai , Zhou Yi , Limbird Lee , Wang Qin
Published: June 2021

Using a gene targeting approach, we previously generated a knock‐in mouse line, in which N‐terminal hemagglutinin (HA) epitope‐tagged α2Aadrenergic receptor (AR) expression was driven by the endogenous mouse α2AAR locus. This line made it possible, for the first time, to evaluate α2AAR endocytosis and α2AAR‐mediated inhibition of Ca2+ currents in native neurons in response to clonidine and guanfacine, two drugs commonly used in treating attention deficit and hyperactivity disorder (ADHD) and eliciting analgesia through actions on the α2AAR. Exploiting this line, we found that the accelerated desensitization to clonidine compared to suppression of Ca2+ currents by guanfacine paralleled a more marked receptor phosphorylation and endocytosis of α2AAR than guanfacine. Interestingly, however, both guanfacine and clonidine share the same efficacy in coupling receptor occupancy to activation of G proteins, based on findings from receptor binding and α2AAR‐ activation of GTPγS studies. Our data provide strong evidence showing functional relevance of differential regulation of GPCR endocytosis by different ligands on receptor‐mediated signaling in native cells, and may represent an important determinant of therapeutic strategies, i.e. desensitizing or non‐desensitizing signals, depending on the pathophysiology being targeted.
Parathyroid hormone acts as a pharmacological chaperone on the parathyroid hormone receptor
Authors: Klenk Christoph , Lohse Martin J.
Published: June 2021

Parathyroid hormone receptor (PTH1R) belongs to class B G‐protein‐coupled receptors and is the major regulator of extracellular calcium homeostasis. Binding of the native ligand parathyroid hormone (PTH) or biologically active peptide fragments of PTH to the receptor lead to the activation of Gs and Gq/G11 pathways with subsequent stimulation of adenylyl cyclase and phospholipase C. Activation of the PTH1R is followed by the recruitment of beta‐arrestin and subsequently to an efficient internalization of the receptor from the cell surface within several minutes.

Here we show that long‐term stimulation of the PTH1R over several hours with PTH resulted in increased cellular protein levels and enhanced membrane distribution of the receptor. This increase of receptor levels was accompanied by an accumulation of high molecular mass moieties of the PTH1R. Analysis of the maturation steps during biosynthesis of the PTH1R revealed that the increased molecular mass was caused by a differential glycosylation pattern of the receptor. Blocking the export from the endoplasmic reticulum of the newly synthesized receptor by Brefeldin A completely inhibited the PTH induced mass changes. We therefore propose a ligand‐mediated stabilization of immature receptors in the endoplasmic reticulum which could eventually lead to a more efficient maturation of the PTH1R.
Effects of membrane cholesterol modulation on mu opioid receptor signaling
Authors: Levitt Erica , Clark Mary J , Thomson Jennifer A , Traynor John R
Published: June 2021

Methyl‐β‐cyclodextrin (MβCD) is used to deplete membrane cholesterol leading to disruption of lipid rafts. Previously, we have shown that MβCD alters mu opioid‐mediated acute and chronic signaling to adenylyl cyclase in HEK293 cells. The effects of MβCD are due to the removal of membrane cholesterol, since treatment of HEK293 cells expressing the FLAG‐tagged mu receptor (HEK FLAG‐mu) with MβCD that is pre‐complexed to cholesterol restored signaling of the mu agonist, DAMGO. Although, we have confirmed that mu receptors are present in cholesterol‐enriched raft membranes using detergent and non‐detergent based methods, they are also present in non‐raft membranes. Here we use cholesterol modulators to test the hypothesis that the effects observed following cholesterol removal by MβCD are indeed due to disruption of lipid rafts rather than alterations in general membrane environment. Treatment of HEK FLAG‐mu cells with other cholesterol modulators, including cholesterol oxidase (0.5U/ml, 1h) and sphingomyelinase (0.1U/ml, 2h), to disrupt lipid rafts without affecting membrane viscosity, did not alter the ability of DAMGO to interact acutely or chronically with adenylyl cyclase. Therefore, modulation of mu opioid signaling by MβCD appears to be due to the general effects of cholesterol depletion on membrane environment rather than disruption of lipid rafts. Supported by DA04087, DA07267, GM07767, DA22377.
Critical time periods of melatonin exposure enhance osteoblast differentiation from human mesenchymal stem cells
Authors: Witt‐Enderby Paula Ann , Sethi Shalini
Published: June 2021

We have previously shown that melatonin enhances bone stem cell differentiation (hAMSCs) into osteoblasts following 10 days melatonin exposure, that is dependent upon MT2 melatonin receptors (MT2R), internalization and MEK(1/2). We extended the exposure to 21 days as calcium deposition, an essential component of bone differentiation process, takes place between 14 and 21 days in culture. The objective of this study was to determine the critical time periods of melatonin exposure during a 21 day exposure to osteogenic medium. HAMSCs were cultured in osteogenic medium for 21 days and then exposed to melatonin for 2, 5, 10, 14 and 21 days. Melatonin increased alkaline phosphatase (ALP) activity, a differentiation marker, when cells were exposed to melatonin for 2 days or 21 days. Calcium deposition was increased by about 900% in presence of melatonin, but this was seen only when the cells were co‐exposed to melatonin for 21 days. Melatonin's effects on ALP activity at days 2 and 21 were dependent upon MT2R, as these effects were blocked with 4P‐PDOT, an MT2R antagonist. RT‐PCR studies show an increase in early, intermediate and late genes involved in osteoblast differentiation, which is dependent upon the duration of melatonin exposure. These studies indicate that melatonin acting through MT2R can drive the differentiation of hAMSCs into osteoblasts, perhaps by activating different genes at critical time periods.
Activation of Human PLC‐eta2 by Gbetagamma
Authors: Zhou Yixing , Sondek John , Harden T. Kendall
Published: June 2021

The phospholipase C‐η subfamily of PLC isozymes was recently identified, and PLC‐η2 was shown to be activated by Gβ1γ2 after transient expression in COS‐7 cells. We show here that activation of expressed PLC‐η2 also occur in the presence of other Gβγ dimers in addition to Gβ1γ2, and illustrate that their activation occur with a truncation mutant that lacks the long carboxyl terminus. To define the enzymatic properties of PLC‐η2 and its potential direct activation by Gβγ, a construct of PLC‐η2 encompassing PH through C2 domain was purified to homogeneity after expression from a baculovirus in insect cells and assayed after reconstitution with phospholipid vesicles. Purified PLC‐η2 hydrolyzed PI(4,5)P2 with an apparent Km of 9.4 μM and Vmax of 5 μmol/min/mg, similar to activities previously observed with purified PLC‐β or ‐ε. Moreover, Gβ1γ2 stimulated the lipase activity of PLC‐η2 in a concentration dependent manner similar to that observed with purified PLC‐β2. Activation was dependent on the presence of free Gβ1γ2 since its sequestration in the presence of Gαi1 or GRK2‐ct reversed Gβ1γ2‐promoted activation. The PH domain of PLC‐η2 is not required for Gβ1γ2‐mediated regulation since a purified fragment lacking the PH domain nonetheless was activated by Gβ1γ2. Taken together these studies illustrate that PLC‐η2 is a direct downstream effector of Gβγ and therefore, of receptor‐activated heterotrimeric G proteins.
Mutations in the i3N loop calmodulin (CaM)‐binding domain of 5‐HT1A receptor attenuate ERK phosphorylation in CHO‐K1 cells
Authors: Coaxum Sonya Denise , Raymond John R.
Published: June 2021

Our laboratory previously has shown that calmodulin (CaM) binds to two sites in the 5‐HT1A receptor i3 loop; however, the functional significance of the interaction of CaM with the 5‐HT1A receptor is not clear. Because ERK activation is one of the primary signals of the G‐protein coupled 5‐HT1A receptor, we wanted to determine if a receptor deficient in CaM binding has altered ERK activation. We constructed a double point mutation (R223E/K232E) in the 5‐HT1A receptor, and demonstrated that it was well‐expressed after transfection into CHO cells. It binds normally to two agonists (5‐HT and 8‐OH‐DPAT) and maintains intact coupling of the receptor to G‐proteins as demonstrated by GTP‐sensitive high affinity binding. Dot‐blots with biotinylated CaM‐enzyme‐linked avidin system showed that CaM binds to the wild‐type 5‐HT1A peptide in the presence of Ca2+; in contrast, binding of CaM to [R223E/K232E]5‐HT1Ai3N peptide was greatly attenuated, suggesting that the affinity of the mutated 5‐HT1A peptide for CaM was markedly reduced. We stably transfected CHO‐K1 cells with the wild‐type 5‐HT1A receptor or [R223E/K232E]5‐HT1A receptor construct, and measured ERK phosphorylation over time. ERK phosphorylation peaked between 5 and 10 minutes for the wild‐type 5‐HT1A receptor, and peaked at 5 minutes for the mutated [R223E/K232E]5‐HT1A receptor. Interestingly, ERK phosphorylation was decreased dramatically in CHO‐K1 cells transfected with the [R223E/K232E]5‐HT1A receptor construct when compared with the wild‐type 5‐HT1A receptor. These results strongly support a critical role for direct CaM binding to the 5‐HT1A receptor for effective ERK phosphorylation.
Unraveling the molecular mechanism by which the L148S mutation of GPR54 causes idiopathic hypogonadotrophic hypogonadism.
Authors: Wacker Jennifer Lisa , Feller David B. , Tang Xiao‐Bo , DeFino Mia C. , Namkung Yuree , Lyssand John , Mhyre Andrew J. , Tan Xu , Hague Chris
Published: June 2021

The G‐protein coupled receptor GPR54 is an essential component of the molecular signaling cascade that initiates puberty and maintains reproductive competency in mammals. A single point mutation (L148S) in the second intracellular loop of GPR54 causes idiopathic hypogonadotrophic hypogonadism (IHH), a disorder characterized by the failure to initiate puberty, and subsequent infertility. To identify the molecular mechanism by which the L148S mutation causes IHH, we developed a simple, in vitro model to study the localization, ligand‐binding properties, and signaling of GPR54. Confocal microscopy, FACs analysis and radioligand binding experiments demonstrate that the L148S mutation does not inhibit GPR54 expression or kisspeptin binding. On the contrary, the L148S mutation does impair kisspeptin‐GPR54 functional responses (IP3 accumulation and ERK activation). Overexpression of the promiscuous G‐protein G15/16 rescues the functional deficits of the mutant receptor, suggesting that the L148S mutation inhibits G‐protein coupling. Interestingly, this leucine is highly conserved amongst class A GPCRs. In fact, mutation of the corresponding leucine in the α1A‐AR uncouples the receptor from functional responses. Structural homology modeling of GPR54 to the β2‐AR crystal structure suggests that this hydrophobic residue is critical for the docking of Gα subunits to GPCRs.
Positive charged mannose polymer increases myocardial damage induced by coronary ischemia reperfusion injury on in vivo model
Authors: Ita‐Islas Israel , Aviles‐Gama Jorge , Ramirez‐Sanchez Israel , Rubio Rafael , Ceballos Guillermo , Rubio‐Gayosso Ivan
Published: June 2021

Endothelial membrane (EnM) at the coronary vessels is a key element in the pathway involved in the coronary ischemia/reperfusion injury (cIRI). We demonstrated the presence of the mannose receptor (MR) in the coronary EnM. MR mediates a positive (+) inotropic and dromotropic effects in isolated guinea pig hearts. We used mannose polymer (Man‐pol) that has + charge, this polymer was synthesized with a high MW dextran, divinyl sulfone (DVS) as linker for mannose and ethylene diamine to inactivate unbound DVS residues. EnM glycocalyx (Gcx) has been involved in the pathway of the IRI, and its homeostasis is required to down modulate the cIRI. + Charged macromolecules damage the Gcx and exacerbates IRI. Therefore, we evaluated the effect of the +Man‐pol on in vivo cIRI models. We determinate myocardial viability on rat hearts 4 hrs after the cIRI with tetrazolium, and reported as ratio between infarcted area (IA) and total area (TA). +Man‐pol treated hearts 15 min prior reperfusion, significantly increases the damage induced by the cIRI when compared with vehicle treated hearts, IA/TA was 1.7 fold. Detrimental effect of the + charge on the Man‐pol suggests the role of the Gcx on the response against the cIRI. On the other hand, uncharged Man‐pol treated hearts decreased IA/TA by half when compared with vehicle treated hearts; this effect could be induced by the beneficial interaction between Gcx and MR. CONACyT # 46187‐A.
Reduced coronary vasodilatation and increased vasoconstriction to diadenosine pentaphosphate after ischemia‐reperfusion in isolated rat heart
Authors: Garcia‐Villalon Angel Luis , Fernandez Nuria , Monge Luis , Salcedo Adely , Narvaez Raul , Dieguez Godofredo
Published: June 2021

To analyze the role of diadenosine polyphosphates in coronary ischemia‐reperfusion, the coronary response to diadenosine pentaphosphate (AP5A, 10−7 – 10−5 M) was recorded in isolated perfused rat hearts after precontraction of the coronary vasculature with U46619 (10−8 –3×10−8). Under control, AP5A injected intracoronarily produced a small initial increase in perfusion pressure (vasoconstriction, 4±2; 7±2; 14±5 mmHg of increment) followed by a decrease in perfusion pressure (vasodilatation, 21±7; 66±1; 52±18 mmHg of decrease), together with a reduction in both left ventricle contractility (dP/dt, 162±37; 764±182; 1388±392 mmHg/s of reduction) and heart rate. After 30 min of ischemia followed by 15 min of reperfusion, the vasoconstriction produced by AP5A was greater (13±2; 20±3; 47±6 mmHg of increment in perfusion pressure, P<0.01), while the vasodilatation was lower (0±3; 15±5; 1±8 mmHg of reduction in perfusion pressure, P<0.05), than in control. The antagonist of purinergic P2Y receptors reactive blue (2×10−6 M) inhibited the vasodilatation and potentiated the vasoconstriction both in control and after ischemia‐reperfusion. Therefore ischemia‐reperfusion may impair the coronary vasodilatation to AP5A mediated by purinergic P2Y receptors and may enhance vasoconstriction. This later effect might be a consequence of the decreased vasodilatation (Supported, in part, by FMMA, FIS and MEyC)
Local inflammatory response to intraperitoneal (ip) instrumentation
Authors: Flessner Michael F , Credit Kimberly , Henderson Karla , He Zhi , Li Xiaorong , Potter Rebecca , Gomez‐Sanchez Elise
Published: June 2021

After observing cytokine staining in the peritoneum of rats with ip transmitters and aortic catheters, we hypothesized that foreign materials in the cavity cause inflammation. To address this, we aseptically implanted medical grade silicone catheters in the peritoneal cavity of rats. After 1–4 weeks, we cultured the catheter to rule out peritonitis and measured the osmotic volume flux (Josm) and the mannitol mass transfer coefficient (MTC) using plastic chambers affixed to the abdominal wall (AW) peritoneum for 120 min in 12 catheter animals (CATH) and 6 age controls (CON). Peritoneal thickness was measured in trichrome‐stained AW, while angiogenesis was determined with CD31 stain and an optical micrometer. VEGF, FGF, TGFβ showed marked staining in CATH animals with ~none in CON. Large differences (p<.02) were observed in peritoneal thickness (mm±SE: CATH, 267±55 vs CON, 19±3), angiogenesis (#ves/mm±SE: CATH, 51±3 vs CON, 0±0), and MTCs (cm/min × 103 ± SE: CATH, 3.97±.32 vs CON, 2.52±.46), but Josm were not different. We conclude that medical grade silicone in the peritoneal cavity sets up a significant inflammatory response that alters the structure and transport function of the peritoneum.
Metalloproteinases in plasma of the spontaneously hypertensive rats cleave the extracellular domain of beta 2‐adrenergic receptor.
Authors: Rodrigues Stephen Fernandes , Tran Edward , Fortes Zuleica Bruno , Schmid‐Schonbein Geert W
Published: June 2021

We recently observed enhanced metalloproteinase (MMP) activity in spontaneously hypertensive rats (SHR) compared to its normotensive Wistar Kyoto (WKY) rats and lyses of membrane receptors in the SHR by MMPs. The beta2‐adrenergic receptor (B2AR) is susceptible to the action of endogenous MMPs (Biochim Biophys Acta, 802:390, 1984). Thus we hypothesize that MMPs in plasma of SHR are able to lyse B2AR. Aorta of control Wistar rats was exposed for 24h (37°C) to fresh plasma of male Wistar and WKY rats and SHR with and without doxycycline (Doxy 30 μM) or EDTA (10 mM), to reduce MMP activity. The density of extracellular and intracellular domains of B2AR was determined by quantitative immunohistochemistry. The density of the extracellular, but not intracellular, domains of B2AR is reduced in aortic endothelial cells of the SHR compared with WKY or Wistar rats. Treatment of the aorta from Wistars with plasma from SHR, but not from WKY rats, reduced the number of extracellular domain, but not intracellular domain, of B2AR in aortic endothelial cells. Both Doxy and EDTA are able to prevent the cleavage of the extracellular domain. MMPs in plasma of SHR, but not WKY rats, cause cleavage of the extracellular domain of B2AR in endothelial cells of the aorta of Wistars. Thus MMPs may be responsible for the reduced density of the extracellular domain of B2AR in aortic endothelial cells of SHR compared to WKY rats.
“In vivo” and “in vitro” neuroprotectant effect of lipophilic compounds from Lepidium meyenii (Maca)
Authors: Pino Alejandro J , Sarill Miles , Bohlke Mark , Maher Timothy J
Published: June 2021

Neuron death is one of the major pathological factors in stroke and neurodegenerative diseases. However, the drugs targeting cell death remain limited. While the constituents of Maca, a South American plant, are reported to possess various pharmacological properties, no studies have been undertaken to determine their efficacy as neuroprotectants. The aim of this study was to test the Maca's lipophilic constituents in two experimental models: in vivo, in a model of permanent cerebral ischemia, and in vitro in a NMDA‐induced cytotoxicity assay utilizing crayfish neurons. The pentane extract of the root was evaluated in rats prior to and following cerebral ischemia, and 24 hours later TTC staining of viable tissue was used to quantify infarct volumes. Infarct volumes were significantly decreased for the lowest dose (3 mg/kg) and significantly increased for the higher doses (10 & 30 mg/kg) compared to control. Extract fractionation with a silicagel preparative column was guided by HPLC. Crayfish neurons were subjected to NMDA as a neurotoxic agent and treated with vehicle, total pentane extract, or the fractions eluted with a mobile phase (2% methanol in dichloromethane). Neuroprotection by the most lipophilic compounds was demonstrated. These results suggest a potential application of the lipophilic compounds from Maca as neuroprotectants. This research is supported by the MCPHS.
Neuroprotective effects of acetaminophen in a rat model of cerebral ischemia/reperfusion
Authors: Baliga Sunanda S. , Merrill Gary F.
Published: June 2021

Cellular damage experienced during stroke results in disabilities relating to loss of neurologic function. A paucity of effective treatments calls for the use of an inexpensive, readily available, and well‐tolerated drug, such as acetaminophen, in mitigating stroke‐induced damage. We have previously reported the efficacy of acetaminophen against hydroxyl radicals and peroxynitrite anion in myocardial ischemia/reperfusion injury. Similar oxidants cause damage in stroke, thus we hypothesized that acetaminophen would exhibit a comparable level of protection. Male Sprague Dawley rats (350–400g) were subjected to 15 minutes of bilateral carotid artery occlusion (ischemia) combined with systemic hypotension followed by 45 minutes of reperfusion. The cerebrum was assayed for changes in infarct size using TTC, mitochondrial membrane potential (MMP) using flow cytometry (FACS), and mitochondrial area in the hippocampus using electron microscopy. In acetaminophen‐treated rats, infarct size (0.15±0.01 vs 0.13±0.01, P<0.005) and mitochondrial area (0.16±0.006 vs 0.11±0.003, P<0.005) significantly decreased following ischemia/reperfusion and ischemia respectively. FACS analysis using JC‐1 showed an increase in MMP following ischemia/reperfusion. We conclude that acetaminophen has therapeutic potential for treating stroke because it attenuates stroke‐induced damage in rat brains.
Statins Protect Against HIV‐1 and Amyloid beta‐Induced Barrier Dysfunction and Inflammatory Responses in an In Vitro Model of the Blood‐Brain Barrier
Authors: ANDRAS IBOLYA E , Rha GeunBae , Huang Wen , Eum SungYong , Couraud Pierre‐Olivier , Hennig Bernhard , Toborek Michal
Published: June 2021

HIV‐1 infection and prolonged anti‐retroviral therapy were demonstrated to contribute to an increase in amyloid beta deposition in older HIV‐infected individuals. We hypothesize that amyloid beta and HIV‐1 can potentiate their toxic effects on the blood‐brain barrier. To study this hypothesis, we employed an in vitro model of human brain microvascular endothelial cells (HBMEC) co‐cultured with human Jurkat T cells or human monocytes infected with HIV‐1. The presence of HIV‐1 markedly increased fluorescent amyloid beta binding to HBMEC. Amyloid beta significantly amplified E‐selectin promoter activity in HBMEC in the presence of HIV‐1 infected Jurkat T cells. Simvastatin, the HMG‐CoA reductase inhibitor, protected against the increased E‐selectin promoter activity in HBMEC co‐cultured with HIV‐1‐infected Jurkat T cells. In addition, simvastatin reduced the adhesion and transmigration across the BBB of HIV‐1 infected monocytes exposed to amyloid beta. These results indicate that statins, such as simvastatin, may protect the blood‐brain barrier against the injuries and proinflammatory effects induced by HIV‐1 and amyloid beta. Supported by MH63022, MH072567, and NS39254.
The aldosterone synthase inhibitor FAD286 reduces plasma aldosterone concentration (PAC) with a long duration of action in a rat model of subchronic hyperaldosteronism
Authors: Chen Wei , Fu Fumin , Beil Michael , Liu Jing , Hu Chii‐Whei , Liang Guiqing , Maniara Wieslawa , Jeng Arco Y. , Rigel Dean F.
Published: June 2021

FAD286 (FAD) is a prototypical aldosterone synthase (CYP11B2) inhibitor that represents an alternative therapy to mineralocorticoid receptor antagonism. We previously characterized the effects of single oral treatment with FAD in a rat model of acute hyperaldosteronism secondary to angiotensin II (ANG II) infusion. In order to assess the effect of subchronic FAD treatment, we extended these studies using multiple dosing. ANG II was infused (30 ng/kg/min iv) for 11 days in cannulated Sprague‐Dawley rats to elevate PAC to ~2–7 nM. After 1 day (“baseline”), FAD (1, 3, or 10 mg/kg) was administered once daily by oral gavage for 2 or 9 consecutive days. Blood samples were collected at baseline and each subsequent day at 3 (“peak”) or 24 hr (“trough”) after dosing. FAD dose‐dependently lowered peak and trough PAC and elevated peak plasma FAD concentrations (~7200 nM at 10 mg/kg). At the highest dose of FAD, peak‐trough excursions in PAC were negligible despite low‐nM trough FAD concentrations. After discontinuing FAD (10 mg/kg) in the 2‐day‐dosing model, PACs remained blunted for at least 2 more days despite undetectable FAD in plasma. There were no signs of an accumulative drug effect or development of tolerance over the 9 days of FAD treatment. The long duration of action combined with the short PK half‐life suggest that FAD is retained at the target enzyme (CYP11B2) within the adrenal cortical mitochondria.
Insulin Resistance Induced by Low Salt Intake is Linked with RAS Activity and Inflammatory Signaling in Tissues
Authors: Coelho Michella Soares , Negri Heloisa B. , Oliveira Ivone B. , Shimizu Maria HM. , Hirata Aparecida E. , Casarini Dulce E. , Prada Patricia O. , Dolnikoff Miriam S. , Saad Mario JA. , Heimann Joel C.
Published: June 2021

Low salt diet (LS) impairs insulin (INS) signaling and increases angiotensin II (AII) that may enhance JNK and IRS1ser307phosphorylation (PHOS), and inflammatory pathways (IP) with consequent reduction in INS action.

Objective: To evaluate if losartan (LOS) improves INS resistance induced by LS by decreasing JNK and IRS1ser307activity.

Methods: Twelve‐week‐old rats were fed a LS (0.15% NaCl) or normal salt diet (NS: 1.3%) since weaning. Groups were studied: LS+LOS (30 mg/kg/day), LS+vehicle, NS+LOS and NS+vehicle. Blood glucose (GLU), plasma INS and TBARS (oxidative stress marker), HOMA index, and tissue AII (immunohistochemistry and HPLC) were evaluated. The steps of INS signaling and IP were determined by immunoblotting.

Results (mean±SEM, P<0.05, n = 8): INS, HOMA index and TBARS were higher on LS (INS: 712±60) than on NS (INS: 257±40 pmol/l). GLU and TBARS were lower and INS was higher on LS+LOS (TBARS: 4±0.5) than on LS (TBARS: 6±0.6 nmol/ml). Liver and muscle (M) AII were higher on LS (10±1) compared with NS (2±0.9 cell/mm2). Liver IkBα PHOS was lower on LS (20±2) than on NS (100±±4 %) suggesting an activation of IP on LS. Insulin receptor, PI3K and Akt PHOS (90±4) were higher and JNK (120±3) and IRS1ser307 (100±6 %) PHOS were lower in the liver of LS+LOS compared to LS. Similar results were observed in M.

Conclusions: INS resistance induced by LS is associated with dysregulation of INS signaling, activated IP, and higher tissue AII and TBARS. LOS improves INS signaling by decreasing JNK and IRS1ser307 PHOS, with consequent reduction in GLU in LS.

FAPESP
Differential Role of Nitric Oxide in the Modulation of Systemic and Pulmonary Pressure
Authors: Sun Xiaowei , Ku David D.
Published: June 2021

The importance of endogenous NO in the modulation of systemic circulation is well established, but its role in the pulmonary circulation remains unclear. The present study was to investigate the effects of both acute and chronic L‐NAME inhibition of NO production on aortic and pulmonary vasoreactivity. An acute bolus injection of 10 mg/kg L‐NAME resulted in a sustained increase in rat systemic pressure, while the pulmonary pressure remained unaffected. After a bolus injection of 2 μg/kg phenylephrine (PE), a marked transient increase was noted in the systemic, but not pulmonary, arterial pressure. However, in the presence of L‐NAME, similar PE challenge resulted in a marked increase in pulmonary arterial pressure, suggesting that NO release was important counteracting PE‐induced pulmonary constriction. 3 weeks chronic treatment with L‐NAME led to significant systemic hypertension without any effect on pulmonary. But with L‐NAME pretreatment, the development of monocrotaline‐induced pulmonary hypertension was significantly accelerated as compared to control group. These findings demonstrated the differential role of NO in the modulation of systemic and pulmonary circulation. In particular, NO release plays a major role maintaining the basal vascular tone in aortic but not pulmonary arteries. However, its release is obligatory in opposing pulmonary constriction and preventing pulmonary hypertension.
Chronic aortic denervation plus high sodium diet. A model of salt‐sensitive normotension in rat
Authors: Rodríguez‐Martínez Manuel , Rodríguez‐Pérez Adriana S , Brito‐Orta Dolores , López‐Rodríguez Juan F
Published: June 2021

To assess whether progressive arterial baroreceptor denervation results in salt‐sensitive changes in blood pressure, mean arterial pressure (MAP) was measured in six groups of conscious male Wistar rats before and ∼30 days after bilateral: a) sham denervation (SH), b) sino‐aortic denervation (SD) and c) aortic denervation (AD). During the last 21 days of post‐denervation period three groups received high sodium diet (HNa = 8%) and the other three groups low sodium diet (LNa = 0.04%). The remaining baroreflex control of ERSNA was examined in the anesthetized state as %Δ ERSNA vs MAP. The modelling indicated that there were no effects of denervation nor diet on MAP ∼ 30 days after denervation. However, the modelling of Δ MAP (MAP after – MAP before) indicated effect of both denervation and diet but not interaction, the highest and more lasting Δ MAP being observed in AD + HNa group (21± 3 mmHg). The modelling of remaining baroreflex control of ERSNA indicated effect of denervation but not of diet.

Conclusion:

AD + HNa is a novel experimental model of salt‐sensitive normotension, though AD does not impair the remaining baroreflex control of ERSNA.

Supported by: SMRF06‐UASLP

Range %
Slope %/mmHg
X to ½ range mmHg
Lower Plateau %
Gmax

SH
119± 7
−.085±.007
130±5
−85±6
−2.5±0.1

AD
125± 9
−.065±.005*
128±4
−82±5
−2.0±0.2

SD
61± 6*
−.059±.005*
153±7*
−66±6*
−.9±.1*

p<0.05 vs SH.

Conclusion:

AD + HNa is a novel experimental model of salt‐sensitive normotension, though AD does not impair the remaining baroreflex control of ERSNA.

Supported by: SMRF06‐UASLP
Differential long‐term cardiovascular compensations to sustained activation of carotid baroreceptor afferents
Authors: Lohmeier Thomas Edward , Dwyer Terry M , Iliescu Radu , Irwin Eric D , Cates Adam W , Rossing Martin A
Published: June 2021

Prolonged baroreflex activation (PBA) by electrical stimulation of the carotid sinuses produces sustained reductions in mean arterial pressure (MAP) and sympathetic activity. This technique bypasses mechanotransduction at the level of the baroreceptors producing controlled electrical activation of baroreceptor afferents. We used this technique to determine whether there are compensations that diminish the chronic blood pressure lowering effects of the baroreflex during activation with continuous trains of impulses. This was studied in 4 normotensive dogs using 24 hour recordings of MAP and heart rate (HR). Control values for MAP and HR were 93±4 mmHg and 54±2 bpm, respectively. Values for MAP after 1, 2 and 3 weeks of PBA were 64±1, 72±1, and 72±1 mmHg, respectively; the corresponding values for HR were 44±2, 44±1, and 44±1 bpm. Thus, during week 2, there was ~ a 30% reduction in the blood pressure lowering response to PBA, with no effect on PBA induced bradycardia. MAP did return to control levels after cessation of PBA, but only after a modest increase above control during the initial 7 days following discontinuation of PBA. These findings indicate that while PBA produces substantial sustained reductions in MAP, slowly developing central and/or peripheral mechanisms act differentially to partially restore MAP towards control levels without attenuating reflex‐induced decreases in HR. (HL‐51971).
Hypovolemia affects cortical activity patterns associated with the cardiovascular response to moderate lower body negative pressure (LBNP)
Authors: Usselman Charlotte Willemina , Wong Savio W , Kimmerly Derek S , Rothwell Amanda , Shoemaker J Kevin
Published: June 2021

We tested the hypothesis that hypovolemia (HV; reduced blood volume) modifies the cortical autonomic network (CAN) response to moderate (−35 mmHg) LBNP. CAN activity was assessed using functional magnetic resonance imaging (fMRI) with blood oxygen level‐dependent (BOLD) contrast in young healthy subjects (n = 9). Testing was repeated under normovolemia (NV) and HV (spironolactone: 100mg/day for 3 days; randomized). BOLD data were collected at baseline and during 3 repeated 60s bouts of LBNP (separated by 90s rest periods). Random effects analysis revealed BOLD signal changes (p < 0.005, uncorrected) in the CAN during both the onset (baroreceptor unloading) and offset (baroreceptor loading) phases of LBNP. In both NV and HV, LBNP onset induced CAN BOLD signal changes in the thalamus, hippocampus, putamen, mid‐cingulate cortex and posterior insular cortex. In addition, LBNP onset in HV also elicited BOLD signal changes in the caudate nucleus, and anterior and posterior cingulate cortex. Changes in these additional areas were observed again during HV offset, although HV offset did not elicit a change in hippocampal activation. Compared to NV, these results suggest that HV was associated with more widespread CAN activation during the dynamic phases of baroreceptor manipulation.

Supported by the Canadian Space Agency.
Glycerol‐induced fluid shift attenuates the vestibulosympathetic reflex in humans
Authors: Dyckman Damian J , Ray Chester A
Published: June 2021

Classically, the glycerol dehydration test (GDT) has been used to test for the presence of Ménière's disease and can cause acute alterations in vestibular reflexes in both normal and pathological states. The vestibulosympathetic reflex (VSR) elicits increases in muscle sympathetic nerve activity (MSNA) and peripheral vasoconstriction. We hypothesized that the GDT would attenuate the VSR through an acute fluid shift of the inner ear. Nine male subjects (27±1 years) performed head‐down rotation (HDR), which engages the VSR, before and after administration of either the GDT or saline. MSNA (microneurography), arterial blood pressure, and leg blood flow (venous occlusion plethysmography) were measured during HDR. Subjects performed 80‐degree head‐up tilt for up to 30 minutes after the post‐tests. Before drug administration, HDR significantly increased MSNA in burst frequency (Δ5±1 bursts/min, Δ8±1 bursts/min; p<0.01) and total activity (Δ44±13% Δ77±17%; p<0.01), and decreased calf vascular conductance (Δ15±6%, Δ20±3%; p<0.01), in both the saline and glycerol trials, respectively. Post‐saline injection, HDR still significantly increased MSNA (Δ6±2 bursts/min, Δ83±20% total activity; p<0.01) and decreased calf vascular conductance (Δ21±4%, p<0.01), which was not significantly different from pretesting. In contrast, post‐GDT resulted in an attenuation of MSNA (Δ3±1 bursts/min, Δ22±3% total activity) and reduction in calf vascular conductance (Δ7±4%) during HDR. These results suggest that a fluid shift of the inner ear via glycerol dehydration attenuates the VSR. These data provide support that dynamic fluid shifts can have a significant effect on the VSR.

Research supported by NIH DC006459, HL077670, & M01RR10732 and NSBRI CA00404
Integrin‐Fibronectin (FN) Mediated Mechanotransduction of Microvascular Smooth Muscle Cells (VSMC) Involves Activation of RhoA and PKC‐α
Authors: Sun Zhe , Huang Shaoxing , Meininger Gerald A.
Published: June 2021

Integrin‐mediated mechanotransduction plays an important role in the arteriolar myogenic response. Diverse signaling pathways are believed to be involved in this process. To investigate the activation of RhoA and PKC‐α during VSMC mechanotransduction, we utilized atomic force microscopy (AFM) to mechanically stimulate single VSMC cells. Confocal microscopy was used to visualize RhoA and PKC‐α in live VSMCs isolated from rat skeletal muscle arterioles, 70~100 μm Diameter. VSMCs were transfected with either a FRET sensor of RhoA activation or GFP‐tagged PKC‐α. The tips of the AFM probes were fused with a FN‐coated bead (5 μm Dia). The FN‐coated beads were placed in contact (20 minutes) with the VSMC membrane that leads to the formation of an integrin‐mediated cell adhesion complex. A controlled pulling force was applied through the bead adhesion site using the AFM while simultaneously recording the signal from the RhoA‐FRET sensor or the GFP‐PKC‐α. Following pulling, a significant increase in the FRET signal for the RhoA sensor was detected. Also, the pulling was accompanied by translocation of GFP‐PKC‐α to the cell membrane. These changes began to occur within seconds after force application and were observed throughout the cell. Collectively, these data suggested that RhoA and PKC were activated by pulling force applied through integrin‐FN focal adhesion. (Supported by NIH HL‐062863 to GAM and AHA 0765481Z to ZS)
Placenta growth factor gene expression is increased in vascular smooth muscle cells in association with metabolic stress
Authors: Lloyd Pamela G. , Shah Anu , Yin Wei
Published: June 2021

Placenta growth factor (PlGF) is a VEGF‐related protein that is associated with arteriogenesis. We hypothesized that PlGF gene expression in vascular smooth muscle cells (VSMC) would be affected by the presence of endothelial cells (EC) and/or by EC‐derived factors. PlGF mRNA expression was increased in VSMC (N=17) by 24 h of co‐culture with EOMA EC, to 2.67 ± 0.39‐fold of monocultured VSMC level (N=9; p=0.003). Treatment of VSMC with EC‐conditioned culture media (ECM; N=14) for 4 h. also increased PlGF mRNA (to 2.34 ± 0.21 fold of control; N=16, p<0.05). The effect of ECM on PlGF mRNA was transient, since expression was no longer elevated after 24 h. of incubation with ECM (0.85 ± 0.29 fold of control; N=10). Treatment of VSMC with media auto‐conditioned by VSMC also produced a ~2 fold increase in PlGF mRNA. Since the effect was not consistent with the action of a short‐lived endothelial‐specific mediator (such as NO), we next investigated whether metabolic substrates or products might induce PlGF expression. Lactate concentration was 8.17 ± 0.14 mM in EC‐conditioned media (N=14), as opposed to 0.50 ± 0.26 mM in un‐conditioned media (N=8; p<0.001). Lactate correlated with PlGF expression in VSMC after 4 h. of treatment with ECM (R2=0.66; p=0.001). However, exogenous lactate (0–15 mM; N=13) had no effect on PlGF mRNA level in VSMC. These data suggest that PlGF gene expression may be sensitive to glucose deprivation. Support: OSU.