bluuebunny/crossref_metadata_2025 huggingface dataset:
Cytoprotective effect of a synthetic triterpenoid against oxidative stress in human umbilical vein endothelial cells (HUVEC)
Authors: Wang Xinyu Eric , Stavchansky Salomon , Bowman Phillip
Published: June 2021

Induction of phase II enzymes, in particular the 32 kd stress protein heme oxygenase‐1 (HO‐1), is cytoprotective in human endothelial cells. We previously demonstrated that caffeic acid phenethyl ester (CAPE) was cytoprotective against menadione‐induced oxidative stress in HUVEC largely via the induction of HO‐1.* Here, we tested the cytoprotective activity of 1[2‐cyano‐3,12‐dioxooleana‐1,9(11)‐dien‐28‐oyl]imidazole (CDDO‐Im), a new synthetic triterpenoid (Dr. Michael Sporn, Dartmouth University) against oxidative stress. Dose response studies indicated that CDDO‐Im at 100 nM was more cytoprotective against menadione toxicity than an optimal dose of CAPE (20 µM), resulting in endothelial cell survival of 80% compared to 60% for CAPE. Messenger RNA for HO‐1 was increased 90‐fold in the presence of CDDO‐Im, while only 13‐fold by CAPE. Western blot analysis of HO‐1 protein product indicated that by 6 h, CDDO‐Im induced an 8‐fold higher level of HO‐1 while CAPE induced a 2‐fold increase. The results indicate that CDDO‐Im is a much more potent cytoprotectant than CAPE, and this beneficial effect correlates well with the induction of HO‐1.

*Wang X, et al. Eur J Pharmacol. 2008 Sep 4;591(1‐3):28‐35.
Establishing a link between small GTPases and mental health
Authors: Tejada‐Simon Maria Victoria , Gutala Ramana
Published: June 2021

In Fragile X syndrome (the most common type of inherited mental retardation) cognitive impairment is not associated with brain malformations or neuro‐degeneration, but with deficiency in neuronal connectivity due to aberrant spine morphology. Although abnormalities in spines have been associated with impaired cognition in mental retardation, how they generated is not yet well understood. Evidence is pointing to the Rho family of small GTPases (Rac, Rho, Cdc42), which mediate actin reorganization and neuronal morphogenesis. Neuronal plasticity requires actin remodeling and local protein translation. Rac plays a key role in actin remodeling, formation, maturation and maintenance of dendritic spines. By combining biochemistry, electrophysiology and behavioral studies coupled with the use of a Fmr1 knockout mouse as animal model of Fragile X, our investigations suggest that under normal conditions, FMRP (protein missing in Fragile X syndrome) acts as a regulator on the synthesis of Rac. In contrast, in Fmr1 knockout mice, lack of FMRP induces an increase of Rac in brain, leading to abnormal actin remodeling, generating the anomalies reported in the structure of dendritic spines, and resulting in cognitive deficiencies. Additionally, pharmacological manipulations of Rac improve abnormalities observed in Fmr1 knockout mice.
Inhibition of hypothermic preservation‐induced Smac/DIABLO protein expression by diazoxide in donor rat myocardium
Authors: JIANG Jian‐ping , ZHU Li , CHEN Ying‐ying , SHEN Yue‐liang
Published: June 2021

AIM

To investigate the effect of a mitochondrial ATP‐sensitive potassium channel (mitoKATP) opener diazoxide (DE) on Smac/DIABLO protein expressions in rat heart suffered from different duration of hypothermic preservation.

METHODS

The Langendorff model of isolated rat heart was used. After stored in 4ºC Celsior solution with or without DE (30 μmol/L) for different time (0, 3, 6, 9 or 12 h). Cell apoptosis was detected by TUNEL technique. The expression of Smac/ DIABLO protein was also analyzed by Western blotting.

RESULTS

(1) Compared with the hypothermic preservation groups, DE reduced the percentage of apoptotic cells. (2) The peak level of Smac/DIABLO protein expression appeared at 6 h after hypothermic preservation, which was postponed to 9 h by DE. (3) The above effects of DE were attenuated by a mitoKATP channel inhibitor 5‐hydroxydecanoate (5‐HD).

CONCLUSIONS

The findings indicate that in the isolated rat heart DE protects myocardium against different duration of hypothermic preservation injury via opening of mitoKATP channel and inhibition of Smac/DIABLO protein expression.
Anti‐diabetic and anti‐inflammatory effects of an aqueous extract of Ligustrum japonicum in db/db mice with type 2 diabetes
Authors: Lee An Sook , Kang Dae Gill , Lee Ho Sub
Published: June 2021

We investigated the effect of an aqueous extract of Ligustrum japonicum (ALJ) in db/db mice with type 2 diabetes. The mice were treated orally with aqueous extract of Ligustrum japonicum (300 mg/day/kg) for 10 weeks to examine the long‐term effects on hyperglycemia and glomerular histology as well as biochemical and functional abnormalities in kidney. As the results, we found that ALJ treatment markedly reduced hyperglycemia (p<0.01), plasma triglyceride, and plasma creatinine in diabetic db/db mice. Furthermore, treatment of ALJ significantly increased plasma level of high density lipoprotein (HDL)‐cholesterol, urine osmolality, and ameliorated creatinine clearance (Ccr) in diabetic db/db mice. We also found that overexpressions of vascular cellular adhesion molecule (VCAM)‐1 and endothelin (ET)‐1 were observed in aortic tissue of db/db mice, ALJ suppressed both expression of VCAM‐1 and ET‐1 in aorta. In kidney glomerular, the glomeruli enlargement and TGF‐β1 overexpression also found in db/db mice, ALJ significantly decreased those levels. Thus, based on our finding, ALJ could reduce hyperglycemia, vascular inflammation and prevent the development of diabetic nephropathy.
Urodynamic Responses of Juvenile and Adult Cats to Nitric Oxide Donor Administration
Authors: Ignash Sarah J , Theobald Robert J
Published: June 2021

The bladder facilitates collection and voiding of urine. While not clearly understood, it is known that nitric oxide (NO) and estrogen (EST) play a role in bladder relaxation. This study examined the role of EST in conjunction with NO to determine if bladder function is similar in juvenile and adult cats, regardless of gender, and if SIN‐1, a NO donor, enhances relaxation of bladder smooth muscle. SIN‐1 was administered to naïve cats, adults (> 12 months old), male (AM) and female (AF), and juveniles (< 6 months old), male (JM) and female (JF). Urodynamic parameters were measured. Plasma EST and urinary nitrite levels were determined in each group. Our results, compared to previous studies, show that although the EST levels differed within groups, levels were inversely related to urinary nitrite. Also, SIN‐1 increased bladder compliance and decreased frequency of spontaneous contractions and magnitude of MVT. AF had the highest levels of EST, while AM, JM, and JF were similar. The data may explain urodynamic effects of EST depletion in postmenopausal women.
β3‐Adrenergic receptor gene polymorphisms and lower urinary tract function
Authors: Michel Martin C , Teitsma Christine A , Vrydag Wim , Baas Frank , de la Rosette Jean JMCH
Published: June 2021

The β3‐adrenergic receptor (β3AR) plays an important role in urinary bladder relaxation, and the human β3AR gene is polymorphic. Its Trp64Arg single nucleotide polymorphism (SNP) is potentially associated with disease states such as obesity and type 2 diabetes. Therefore, we have explored whether this SNP is associated with an altered bladder function in vivo. DNA was obtained from 1015 men with voiding dysfunction who had undergone full urodynamic phenotyping prior to treatment, and genotyping was performed by MALDI‐TOF. 131 (12.9%) and 6 subjects (0.6%) were heterozygous and homozygous, respectively, for the Arg64 allele, confirming prevalence data in other Caucasian populations. However, there was no association of this allele with any lower urinary tract function parameter including cystometric measurements and symptom scales. Most importantly, bladder compliance, which is under direct control of the β3AR, varied over a wide range within the group but was not associated with genotype. We conclude that the Trp64Arg SNP does not affect baseline urinary bladder function in vivo.

Source of funding

Astellas Europe, EU FP 7 program
Vitamin D regulation of chemerin in human bone marrow adipogenesis
Authors: Roman Alexandra Anna , Sinal Christopher
Published: June 2021

Osteoporosis is a skeletal disorder characterized by low bone mass and deterioration of bone tissue that ultimately leads to an increase in susceptibility to fracture. One cause of bone loss in osteoporosis is a decrease in the number of bone forming osteoblasts. This can occur due to a shift in the differentiation of mesenchymal stem cells (MSCs) within the bone marrow to favour the formation of adipocytes at the expense of forming osteoblasts. Therefore, manipulation of MSC differentiation to favour osteoblast formation over adipocyte formation may offer a novel target for the treatment of osteoporosis. Recently, our laboratory identified chemerin as an adipokine that regulates adipogenesis by activation of chemokine like receptor‐1(CMKLR1). The purpose of this study was to characterize the expression of chemerin and CMKLR1 in human bone marrow MSC adipogenesis and to identify regulators of chemerin within bone. Results indicate that chemerin mRNA and protein expression are altered during adipogenesis. We have also demonstrated that treatment of adipocyte/osteoblast precursors with Vitamin D causes a significant induction in chemerin expression that is mediated through the Vitamin D receptor. These results suggest one mechanism whereby chemerin may be regulated to alter bone formation.
Bradykinin induces COX‐2 expression via MAPKs and NF‐κB in human synovial fibroblast of rheumatoid arthritis
Authors: Chen Yu‐Wen , Yang Chuen‐Mao
Published: June 2021

Bradykinin (BK) is an important inflammatory mediator which has been shown to induce the expression of inflammatory proteins including COX‐2. However, the molecular mechanisms underlying BK‐induced COX‐2 expression in human fibroblast‐like synoviocytes (FLS) which contributes to inflammatory responses in rheumatoid arthritis (RA) were largely unknown. Here, our study aimed to investigate the mechanisms underlying the intracellular signalings involved in COX‐2 expression in BK‐induced inflammatory of human FLS. Western blotting and RT‐PCR analyses showed that BK induced COX‐2 protein and mRNA expression as well as phosphorylation of p42/p44 MAPK, p38 MAPK, and JNK which was attenuated by the inhibitors of MEK1/2 (U0126), p38 MAPK (SB202190), and JNK (SP600125). The involvement of MAPKs in these responses was further confirmed by that transfection with siRNA of p42, p38, or JNK2 attenuated BK‐induced COX‐2 expression. In addition, BK stimulated both IκB‐α degradation and NF‐κB translocation was attenuated by Bay11‐7082, U0126, SB202190, or SP600125. BK‐stimulated NF‐κB translocation into nucleus was further confirmed by immunofluorescence staining. These results suggest that in human FLS, BK‐induced COX‐2 expression was mediated through phosphorylation of MAPKs and NF‐κB pathways.
Serotonin 5‐HT2A receptor activity mediates adipocyte differentiation
Authors: Yu Bangning , Nichols Charles D
Published: June 2021

Serotonin 5‐HT2 receptors are expressed in variety of tissues and play important roles in many processes. The 5‐HT2A receptor is primary known for its role in CNS function. This receptor is, however, expressed in many peripheral tissues where its role remains largely unknown. Here, we present data demonstrating that blockade of 5‐HT2A activity inhibits adipocyte differentiation. This is a process in which pre‐adipocyte fibroblast accumulate lipids and fatty acids and become mature adipocyte. During the initiation of adipocyte differeantiation, transcription factors like CEBP‐b; are increased, which elevates levels of PPAR‐r, facilitating differentiation. Ketanserin, a 5‐HT2A/2C receptor antagonist inhibits both mouse and human adipocyte differeantiation in a dose dependent manner. The IC50 of ketanserin in a primary human preadipocyte model is 78 nM. Using selective 5‐HT2A/2B/2C receptor antagonist, we discovered that blockade of 5‐HT2A or 5‐HT2B receptors but not 5‐HT2C receptors was effective to prevent adipocyte differentiation. Time course analysis indicates that 5‐HT2A receptor activity is necessary very early in the induction phase to facilitate differentiation. Together, these data indicate that 5‐HT2A receptors play an important role in adipocyte differentiation. Studies probing the effects of 5‐HT2A receptor blockade on molecular markers of adipocyte differenetiation are ongoing.
Caspase‐3 Knockdown Maintained norepinephrine‐mediated Contractility of Adult Rat Ventricular Myocyte during Sepsis
Authors: Chopra Mani , Das Padmalaya , Sharma Avadhesh C
Published: June 2021

We hypothesized that norepinephrine (NE)‐induced upregulation of caspase‐3 is responsible for limiting its efficacy as a positive inotrope during sepsis. Therefore, we determined the effect of NE on the contractile function of adult rat ventricular myocytes (ARVM) transfected with caspase‐3 small interfering RNA (caspase‐3 siRNA). Single ARVMs were isolated from septic/sham hearts obtained from male Sprague‐Dawley rats (350‐400g). The effect of NE was determined in caspase‐3 siRNA‐, non‐silencing RNA‐ transfected sham and septic ARVMs. NE produced a significant but less pronounced increase in peak shortening (PS) in septic ARVMs compared to the sham ARVMs. NE exacerbated the sepsis‐induced upregulated expression of caspase‐3 in ARVMs. NE downregulated caspase‐3 fluorescence and increased PS in caspase‐3 siRNA‐transfected septic ARVMs. Caspase‐3 knockdown in the septic ARVMs increased the expression of the contractile proteins, decreased the release of cytochrome c and number of TUNEL‐positive ARVMs. The data suggest that caspase‐3 knockdown ameliorated decreased contractile response of NE during sepsis and demonstrated the regulatory role of caspase‐3 in ARVM contractile dysfunction.
Characterization of sorting nexin‐25, a D1 and D2 dopamine receptor interacting protein that regulates receptor expression and trafficking in HEK293 cells
Authors: Free R Benjamin , Namkung Yoon , Hazelwood Lisa A , Cabrera David M , Sibley David R
Published: June 2021

Dopamine receptors (DARs) do not exist as independent units within the synaptic membrane, but are part of large macromolecular complexes of interacting proteins. Our current studies employ co‐IP assays for DARs coupled with mass spectrometry‐based sequencing to identify interacting partners. One protein identified was sorting nexin‐25. Mammalian sorting nexins (SNXs) have been suggested to regulate intracellular trafficking, internalization, and endosomal recycling or sorting of membrane‐bound cargo. The physiological role of SNX25 is unknown, however, and multiple isoforms of SNX25 have been reported. Using RT‐PCR, we have found that the endogenous isoform in HEK293T cells is a 1004 residue protein containing two putative transmembrane‐spanning regions. When this SNX25 isoform is expressed as an RFP fusion protein, it is localized in distinct clusters near the plasma membrane. When SNX25 is over‐expressed with the DARs, an alteration in the cellular receptor distribution is seen using confocal microscopy. Radioligand binding and functional assays also show that the expression levels of both D1 and D2 DARs are altered. SNX25 over‐expression also appears to limit the internalization of D2 DARs after agonist treatment, but this had only a slight effect on receptor recycling. Overall, these data suggest that SNX25 regulates the trafficking of D1 and D2 DARs and is a novel member of the DAR signalplex.
Membrane cholesterol removal promotes a low agonist affinity state of mu opioid receptors
Authors: Levitt Erica S. , Harris Alexander A. , Traynor John R.
Published: June 2021

Previously, we have shown that removal of plasma membrane cholesterol by the cholesterol‐sequestering agent methyl‐β‐cyclodextrin (MβCD) results in a loss of potency of the mu opioid agonist DAMGO to stimulate GTPγ35S binding or to inhibit adenylyl cyclase. Here, we test the hypothesis that this decrease in potency is due to an uncoupling of the mu opioid receptor from G protein. Approximately half of the mu opioid receptors stably expressed in HEK293 cells were present on the plasma membrane, as determined by whole cell binding. Removal of plasma membrane cholesterol with MβCD significantly decreased the maximum binding of the mu opioid agonist 3H‐DAMGO in a cell membrane preparation. This decrease in agonist binding was partially reversed when cholesterol was added back to the membrane, and could not be explained by changes in total receptor number as measured by binding of the opioid antagonist 3H‐diprenorphine. Rather, MβCD treatment induced the appearance of a low affinity state of the mu opioid receptor as shown by competition of DAMGO with 3H‐diprenorphine. This induced low affinity state was mimicked by the addition of sodium and GTPγS, which uncouples G proteins from receptors. Thus, removal of plasma membrane cholesterol by MβCD decreased the high affinity state of mu opioid receptors which could be explained by an uncoupling of receptor from G protein. Supported by DA07267, DA23339, GM07767, DA04087.
The second intracellular loop of D2R activates Galphai1
Authors: Lyssand John Sverre , Wacker Jennifer L , DeFino Mia C , Tang Xiao‐bo , Tan Xu , Stella Nephi , Zheng Ning , Hague Chris
Published: June 2021

Ligand binding to a G‐protein coupled receptor (GPCR) causes Gα GDP/GTP exchange, Gα dissociation from Gβγ, and ultimately activation/inhibition of down stream effectors. The exact mechanism of G‐protein activation by a GPCR is unknown. We recently published that a single point mutation in the second intracellular loop (ic2) of GPR54 caused decreased G‐protein activation without affecting coupling. This amino acid is a conserved hydrophobic residue amongst GPCRs and may play a role in G‐protein activation by all GPCRs. In support of this, we have shown that a similar mutation in the α1A‐AR also decreases G‐protein activation. Recently Johnston et al (2007) reported the interaction between Gαi1 and the N‐terminal region of the third intracellular loop (ic3) of the D2‐dopamine receptor (D2R). They showed that a peptide derived from the ic3 region of D2R increases Gαi1 activation rate and interacts with critical regions in Gαi1 suggested to be important in G‐protein activation. Using similar techniques, we explored the interaction between a peptide derived from the ic2 of the D2R (D2‐ic2) and purified Gαi1. We have found that D2‐ic2 increases Gai1 activation, but competes with the ic3 peptide. The mechanism of D2‐ic2/Gαi1 activation was assessed by X‐ray crystallography to determine structure. These experiments will elucidate how the second intracellular loop of a GPCR activates heterotrimeric G‐proteins.
Evolution of vertebrate opioid receptors: evidence from cloning and bioinformatics
Authors: Stevens Craig W.
Published: June 2021

The proteins that mediate the analgesic and other effects of opioid drugs and endogenous opioid peptides are known as opioid receptors. Opioid receptors consist of a family of four closely‐related proteins belonging to the large superfamily of G‐protein coupled receptors. The three types of opioid receptors shown unequivocally to mediate analgesia in animal models are the mu (MOR), delta (DOR), and kappa (KOR) opioid receptor proteins. The role of the fourth and newest member of the opioid receptor family, the nociceptin or orphanin FQ receptor (ORL), is not as clear as hyperalgesia, analgesia, and no effect was reported after administration of ORL agonists. There are now cDNA sequences for all four types of opioid receptors that are expressed in the brain of six species from three different classes of vertebrates. This paper presents a comparative analysis of vertebrate opioid receptors using cloning studies from our lab, bioinformatic analyses, and data from recent human genome studies. Results indicate that it is likely that opioid receptors arose by two whole genome duplication events early in vertebrate evolution, that there is a vector of opioid receptor divergence such that receptors in mammals and humans exhibit a higher degree of opioid type‐selectivity, and that the MOR protein shows evidence of the most rapid adaptive evolution. Supported by a National Institutes of Health grant, DA0124482.
Epidermal growth factor induces lysophosphatidic acid type 1 receptor regulation through phosphorylation
Authors: Santana Christian Colin , Vásquez Erendira Avendaño , Sáinz Adolfo García
Published: June 2021

Lysophosphatidic acid (LPA) is a local hormone/autacoid/ growth factor involved in many cellular responses such as: cell migration, proliferation and surviving, platelet aggregation, smooth muscle contraction, neurotransmitter release, establishment of focal adhesions, neurite retraction and cell rounding (among many others). The actions of LPA are mediated through a family of G protein coupled receptors (GPCRs) that includes five subtypes LPA1‐5 receptors which can couple to different G proteins. Phosphorylation of GPCRs is one of the earliest events that regulate their function. Current evidence indicates that homologous desensitization of these receptors mainly involves G protein‐coupled receptor kinases (GRKs), whereas in heterologous desensitization second messenger‐activated kinases play a key role. The primary goal of this work was to study the possibly regulation of LPA1 receptor induced by EGF receptor activation. The LPA1 receptor, tagged with the green fluorescent protein, was transfected into C9 cells. Stimulation with LPA induces an increase in intracellular calcium concentration, LPA1 receptor phosphorylation and internalization. Activation of EGF receptors decreases (50%) LPA‐induced intracellular calcium increase. In addition, EGF receptor activation also induced LPA1 receptor phosphorylation and internalization. This "crosstalk" seems to involve protein kinase C, since pharmacological inhibitors markedly decreased EGF‐induced LPA1 receptor phosphorylation.

This research was partially supported by Grants from DGAPA (IN206302) and CONACyT (079908).
A Cytoplasmic Acidic Amino Acid Motif Mediates PI3‐Kinase‐Sensitive Cell Adhesion and Surface Localization of Class A Scavenger Receptors
Authors: Post Steven , Cholewa Jill M. , Nikolic Dejan M.
Published: June 2021

Class A scavenger receptors (SR‐A) bind a diverse array of ligands and mediate ligand internalization or cell adhesion. The role of specific cytoplasmic domains in coupling SR‐A to ligand internalization or cell adhesion is not clear. The importance of the cytoplasmic tail in regulating SR‐A function was shown by deleting all but six cytoplasmic amino acids (SR‐AΔ1‐49). Although SR‐AΔ1‐49 displayed increased surface localization and mediated cell adhesion, it did not internalize ligand. Moreover, in contrast to the full‐length receptor, cell surface expression and cell adhesion mediated by SR‐AΔ1‐49 was independent of PI3‐kinase (PI3K) activation. To examine the importance of specific amino acids in the cytoplasmic tail of SR‐A, potential regulatory sites were mutated and mutant receptors expressed in HEK cells. Changing the acidic amino acids within a conserved ‐EDAD‐ domain to their amide derivatives (SR‐AQNAN) selectively altered SR‐A localization and regulation. Similar to SR‐AΔ1‐49, cell surface localization and cell adhesion mediated by SR‐AQNAN was independent of PI3K activation. However, ligand internalization mediated by SR‐AQNAN was similar to that of the full‐length receptor. Our results show that acidic amino acids in a cytoplasmic ‐EDAD‐ domain are required for PI3K‐dependent regulation of SR‐A surface localization and are differentially involved in regulating SR‐A function.
Common human alpha2a&c adrenergic receptor polymorphisms do not affect cellular processing nor surface trafficking
Authors: Angelotti Timothy , Sorensen Matt , Hurt Carl
Published: June 2021

Naturally occurring variant genetic forms of α2a and/or α2c adrenergic receptors (α2a&c ARs) have been identified and linked to congestive heart failure. Two well described α2a&c AR variants occur within the 3rd intracellular loop, α2aLys251 and α2cDel322‐325. We investigated if these variant forms exhibited alterations in localization or cellular processing to account for their phenotypic differences.

Human cDNA encoding wild‐type and polymorphic α2a&c ARs were either purchased commercially or created using standard Quick Change mutagenesis techniques, incorporating an amino terminal HA epitope. Each construct was examined with respect to drug binding, biochemical processing (e.g. glycosylation), and surface and total expression (e.g. immunocytochemistry and FACS analysis).

All four cDNA constructs were successfully transfected and expressed in HEK293 cells. RX 821002 binding did not differ greatly between wild‐type and polymorphic forms. Transfected cells were analyzed by immunocytochemistry and FACS; neither polymorphic form demonstrated differences in cell surface or intracellular expression patterns. Lastly, endoglycosidase digestions of transfected cell membranes revealed that the polymorphic forms were processed in a similar manner as their wild‐type counterparts.

It has been shown previously that certain GPCR polymorphisms can affect receptor stability and trafficking. We examined the two most common α2a&c AR polymorphisms and found no discernable difference in receptor expression, drug binding, surface trafficking, or biochemical processing. Therefore, phenotypic differences between wild‐type and polymorphic forms are most likely due to signal transduction, or possibly neuronal specific influences.

This work was supported in part by NINDS K08 NS050654‐01A1 (TA)
The membrane‐proximal C‐terminal Arg/Lys cluster mediates G protein‐coupled receptor interaction with tubulin and is required for receptor transport to the cell surface
Authors: Duvernay Matthew Thomas , Zhang Xiaoping , Dong Chunmin , Wu Guangyu
Published: June 2021

A growing body of evidence supports a pivotal role for microtubules in intracellular protein trafficking. However, the role of microtubule network in biosynthesis of G protein‐coupled receptor (GPCR) remains largely unknown. By using peptide‐conjugated Agarose affinity column and proteomics, we identified α and β‐tubulin interacting with the α2B‐adrenergic receptor (AR) C‐terminus (CT), which was confirmed by GST fusion protein pull down assay using purified tubulin or brain cytosolic extract. Site‐directed mutagenesis of the CT revealed the interaction was mediated by a cluster of basic residues in helix 8, suggestive of an ionic interaction, consistent with the negatively charged EExEEY motif in tubulin as a common docking site for microtubule‐associated proteins. Indeed, NaCl dose‐dependently inhibited α2B‐AR interaction with tubulin. Interestingly, the CT of α2B‐AR and angiotensin II type I receptor (AT1R), but not β2‐AR, physically associated with tubulin and mutation of the Arg/Lys cluster in α2B‐AR and AT1R abolished their export and ERK1/2 activation and the mutated receptors were extensively arrested in the endoplasmic reticulum (ER). Finally, nocodazole treatment blocked α2B‐AR cell surface expression by 60%. These data indicate for the first time that the Arg/Lys cluster‐directed GPCR interaction with tubulin coordinates receptor export from the ER to the plasma membrane (GM076167).
Pharmacological characterization of the α2 adrenergic receptor inhibiting mouse hippocampal CA3 epileptiform activity
Authors: Nelson Brian , Goldenstein Brianna , Xu Ke , Luger Elizabeth , Pribula Jacquline , Wald Jenna , O'Shea Lorraine , Jurgens Chris , Doze Van
Published: June 2021

α2 adrenergic receptor (AR) activation by epinephrine (EPI) inhibits epileptiform bursts in the mouse hippocampus. Which α2AR subtype(s) are involved is unclear. Pharmacological characteristics of α2ARs that inhibit epileptiform activity were investigated in mouse brain slices by examining AR agonists' efficacy and potency, and determining the equilibrium dissociation constants (pKb values) of selective α2AR antagonists. Dexmedetomidine, an imidazoline and guanfacine, a guanidine were the most potent. In contrast, the catecholamines EPI and norepinephrine were the most efficacious. Apparent pKb values calculated for selective α2AR antagonists correlated best with the mouse α2AAR. EPI‐inhibited antiepileptic effects (pKb) were competitively antagonized by atipamezole (8.79), rauwolscine (7.75), WB‐4101 (6.87), prazosin (5.71) and JP‐1302 (5.92). Transgenic α2AAR and α2CAR knockout mice further confirmed that the EPI‐mediated anti‐epileptic effect was through the α2AAR subtype. The α2AAR‐mediated inhibitory actions of EPI on hippocampal CA3 epileptiform burst frequencies were also not sex or age‐dependent. These findings suggest that the activation of α2AAR could provide a new pharmacotherapeutic strategy for treating epilepsy and highlight the need for selective α2AAR agonists. This project was supported by ND EPSCoR, NSF CAREER, NSF REU Site, UND REFUNDU, APS, ASPET and NIH INBRE.
Serum Response Factor involvement in myogenic tone regulation in resistance arteries
Authors: Retailleau Kevin , Toutain Bertrand , Mericskay Mathias , Li Zhenlin , Henrion Daniel , loufrani Laurent
Published: June 2021

Serum Response Factor(SRF) is a transcription factor playing a crucial role in the control of cell cytoskeletal structure. Actin dynamics and RhoA activity are major players in SRF transcriptional activity. In the microcirculation, diameter adjustments in response to change in pressure or flow depend on cytoskeleton integrity. Thus, we hypothesised that SRF deletion will affect mechanotransduction in resistance arteries. Tail caudal arteries from inducible SRF KO mice and control mice were mounted in an arteriograph. Contraction to stepwise increase in pressure (myogenic tone) and relaxation to stepwise increases in flow were determined. Protein expressions were quantificated by western blot. SRF deficiency reduced myogenic tone (CT:16.3±3.2; KO:5.9±2.3) and increased Focal Adhesion Kinase (FAK)(CT:0,23±0,03/KO:0,16±0,02) while P‐FAK/FAK ratio was similar in both groups. Furthermore, no effect was observed in flow‐mediated dilation and receptor dependant contraction (phenylephrine and angiotensine II). This study reported for the first time that SRF inhibition affected specifically myogenic tone. This suggests that SRF is involved in the pressure mechanotransduction in resistance arteries. Due to the central role of myogenic tone in vascular disorders and organs autoregulation this finding opens new perspectives in microcirculation pathophysiology and provides new therapeutic targets.
Insulin Inhibits Low pO2‐Induced ATP Release from Human Erythrocytes (RBCs): Implications for Vascular Control in Pre‐Diabetes
Authors: Hanson Madelyn , Ellsworth Mary , Achilleus David , Bowles Elizabeth , Stephenson Alan , Adderley Shaquria , Sridharan Meera , Sprague Randy
Published: June 2021

RBCs release ATP when exposed to low O2 tension (pO2). This ATP participates in the matching of O2 supply with need in skeletal muscle by stimulating increases in blood flow to areas with increased O2 demand. Here we investigate the hypothesis that hyperinsulinemia, as occurs in pre‐diabetes, inhibits ATP release from RBCs exposed to low pO2, impairing this cell's ability to stimulate vasodilation in response to reduced extraluminal pO2. RBCs were exposed to low O2 (14 ± 1 mm Hg) in the absence and presence of insulin (1 nM). Insulin attenuated ATP release from 16 ± 4 to 8 ± 3 nmoles/4x108 RBCs (n=6, P<0.01). To determine a functional consequence of this action of insulin, we examined the ability of isolated skeletal muscle arterioles perfused with buffer containing RBCs to dilate in response to decreased extraluminal pO2 (21 ± 4 mm Hg, n=10). In vessels perfused with untreated RBCs, low extraluminal pO2 resulted in a 9 ± 3% increase in diameter (P<0.05). In contrast, when RBCs were treated with insulin, the vessels failed to dilate in response to low pO2. These studies show that insulin inhibits ATP release from RBCs in response to reduced pO2 and impairs their ability to stimulate dilation of skeletal muscle arterioles. These results suggest that the hyperinsulinemia of pre‐diabetes could hinder the matching of O2 supply with need in skeletal muscle. Funded by AHA Fellowship (MH), ADA grant RA‐133, NIH grants HL‐64180 and HL‐89094.
Oxygen transport in mesenteric microcirculation with artificially augmented oxygen consumption
Authors: Golub Aleksander Sergeevich , Song Bjorn Kyungsuck , Pittman Roland Nathan
Published: June 2021

The low spatial density of the mesenteric vascular network permits access to relatively isolated microvessels for investigating the mechanisms of O2 transport to tissue. However, the mesenteric tissue has low oxygen consumption rate (VO2) unable to create substantial longitudinal and radial PO2 gradients. An O2 scavenging gel film attached to mesentery preparation was designed as an experimental model of tissue hypoxia with an intact oxygen supply. Phosphorescence quenching microscopy with two (dual‐spot and scanning) PO2 sampling modes was used to obtain longitudinal intravascular PO2 gradients and PO2 profiles in perivascular tissue. The effective oxygen consumption in vivo and in vitro was estimated from analysis of parabolic PO2 profiles typical for combined diffusion/consumption. The VO2 in vitro was found to be within the range 197 ‐ 1527 nl O2 / (cm3 ·s). The effective in vivo VO2 in the mesentery with the gel [1806 nl O2 / (cm3·s)] was 30 times that in the normal tissue, VO2 = 60 nl O2 / (cm3·s). The achieved longitudinal PO2 gradient in arterioles and capillaries was very high, 12.8 mmHg /100 µm. Limitation of the VO2 with a 20 µm thick Parafilm placed between the gel and tissue reduced the effective VO2 from 573 to 232 nl O2 / (cm3 s) and the longitudinal PO2 gradient in microvessels from 14 to 5.3 mmHg /100 µm. Mean PO2 in arterioles increased from 53 to 96 mmHg. Support: AHA‐0655449U and NIH‐HL18292.
A numerical approach for predicting the impact of vascular architecture on blood flow during ischemic repair
Authors: Wood Stephanie Ann , Ghosh Shilpi , Jones Kevin , Gruionu Gabriel , Cardinal Trevor
Published: June 2021

Chronic ischemia results in vascular remodeling characterized by angiogenesis and arteriogenesis as well as reduced resting and hyperemic blood flows. Previous investigations have demonstrated impaired vasoactivity of intramuscular arterioles. However, the impact of vascular growth on blood flow is unknown. The goal of this study was to determine how changes in vascular architecture following arterial occlusion impact blood flow in hindlimb skeletal muscle. Vascular network characteristics were obtained from photomicrographs of India ink casts of the mouse gracilis anterior muscle with and without femoral artery occlusion. Quantitative systems‐level network properties were obtained using Horton's Law of Stream Numbers and the diameter‐defined Strahler method. The Hagen‐Poiseuille equation and conservation of mass were used to solve for flow and pressure throughout the network. The diameter, length, and vessel quantity of control networks were consistent with Horton's Law. The remodeled data yielded a higher diameter ratio, but lower vessel length and quantity ratios compared to normal. Connectivity matrices indicated an overall increased branching in the remodeled vasculature while blood flow appears depressed in the ischemic muscle. Current efforts are aimed at deriving a quantitative expression to represent the effect of varying branch angles on flow resistance.
A model study of vascular microbubble transport in pulsatile blood flow through bifurcating vessels
Authors: Valassis Doug T. , Dodde Robert E. , Eshpuniyani Brijesh , Fowlkes J. Brian , Bull Joseph L.
Published: June 2021

The behavior of long gas bubbles suspended in liquid flowing through bifurcating vessel network was investigated experimentally and theoretically as a model of vascular bubble transport. This work is motivated by a developmental gas embolotherapy in which perflurocarbon microbubbles are selectively formed in vivo and subsequently lodge to occlude blood flow to tumors. The bubbles originate from encapsulated liquid droplets that are small enough to pass through capillaries, allowing intravenous injection. The homogeneity of tumor necrosis depends on the transport and lodging of emboli. A bench top experimental model was used to explore the effects of flow pulsatility, heart rate, gravity, and bifurcation roll angle on bubble splitting and lodging. A corresponding time‐dependent one‐dimensional theoretical model was also developed. At a bifurcation roll angle of 45‐degrees, the most distinct difference in splitting ratios between three physiologic frequencies (1, 1.5, 2 Hz) was observed. As roll angle increased, lodged bubble volume in the first generation channel increased while bubble volume beyond the second bifurcation proportionately decreased. The results elucidate the effects of pulsatile flow and suggest the potential of gas embolotherapy to occlude blood flow to tumors. These finding are also relevant to air embolism.

This work was supported by NIH grant R01EB006476.
The dependence of cell‐free layer thickness in arterioles on systemic hematocrit level
Authors: Yalcin Ozlem , Choi Christine , Chatpun Surapong , Intaglietta Marcos , Johnson Paul
Published: June 2021

Axial migration of red cells in microcirculatory vessels leads to formation of a cell‐free layer (CFL) near the vessel wall. CFL width may be physiologically important in determining hydrodynamic resistance and of NO scavenging by the red cell column, especially in arterioles. It would be expected that the degree of axial migration and width of the CFL would decrease as hematocrit (hct) increases. Our purpose is to determine the hct dependence of CFL width with hemoconcentration and hemodilution. We measured the CFL width in arterioles (20‐55 μm i.d.) in rat cremaster muscle. Dextran 500 was infused to elevate erythrocyte aggregation to normal human levels. Systemic hct was increased stepwise from 40% to 45, 50 and 55% in one group and decreased to 35 and 30% in another group. We measured the CFL thickness and its variations with a high speed video camera and custom built software. To check the performance of this automated method, manual measurements were performed. A 50% increase in hct level (30% to 45%) led to a significant decrease in CFL width (5.15 ± 0.78 μm to 2.69 ± 0.17 μm) (r=‐0.54, p<0.01) while a further 23% increase (45% to 55%) had no effect (2.69 ± 0.17 μm to 2.66 ± 0.16 µm). The variations showed a similar trend. The dependence of CFL width on hct parallels that reported previously for tube hct. Another implication of our results is that NO scavenging may have an inverse relationship with systemic hct.
Sphingosine‐1‐phosphate activates Rac in cultured endothelial cells using conditions that block acute inflammatory response in microvessels
Authors: Adamson R H , Thirkill T L , Curry F E
Published: June 2021

Elevation of endothelial cell intracellular cAMP using combined rolipram (10 µM) and forskolin (5 µM) (RF) blocked microvessel inflammation stimulated by platelet activating factor (PAF) or by bradykinin (Bk) (AJP Heart 294:H1188, 2008). Sphingosine‐1‐phosphate (S1P), believed to act through Rac activation, also attenuated PAF‐ or Bk‐induced acute inflammatory response. Direct activation of Rac using bacterial toxin reduced microvessel PAF response. Therefore we tested whether specific conditions known to attenuate PAF and Bk also induce Rac activation. Using cultured human dermal microvascular endothelial cells (HDMVEC) we compared the S1P dependent activation of Rac, and the Rac activation induced by treatment with RF with vehicle control. S1P (1 min) showed significant concentration (0.1 to 5 µM) dependent Rac activation. Rac activation (1 µM S1P) was maximal at 1 min and diminished over 30 min. In contrast, treatment with RF induced Rac activation greater at 10 to 30 min than at 1 to 5 min. Immunofluorescence demonstrated that both S1P and RF treatment strongly enhanced peripheral cortactin in HDMVEC and induced strong and continuous peripheral VE‐cadherin. The results demonstrate that S1P or RF treatments which we previously demonstrated to attenuate PAF‐ or Bk‐induced permeability in rat mesentery microvessels also significantly stimulate Rac activation and peripheral cortactin in HDMVEC. HL28607
Vascular Endothelial Growth Factor and its Effect on the Fluid Dynamic of the Cervix
Authors: Rhyne Scott Brady , Boucher Shannon Marie , Ohashi Takako , Mowa Chishimba Nathan
Published: June 2021

The role of the microvascular changes in the process of cervical remodeling during protracted labor and preterm births is unclear. VEGF, a potent vascular permeability factor (50,000 > histamine) and angiogenic architect, has been shown to mediate estrogen‐induced uterine edema. We previously have shown that VEGF and its receptors exist in the cervix, and increase as pregnancy progresses. Cervical remodeling is associated with edema, and thus here we hypothesize that VEGF is one of the factors regulating edema during cervical remodeling. Previous data generated from DNA microarray shows that genes involved in edema and vascular permeability are regulated by VEGF, which likely leads to protein extravasation and tissue hydration. In our present study Real Time PCR was used to validate our previous DNA microarray data. This data showed an increase in VCAM‐1 mRNA. Further, we also determined the effects of VEGF on cervical edema in ovariectomized mice. We found a positive correlation between VEGF and cervical edema with increasing concentration. We conclude that VEGF affects cervical edema by regulating genes that modulate vascular leakage such as VCAM‐1.
Reduction in endothelial glycocalyx thickness after hemorrhage
Authors: Torres Filho Ivo P , Vink Hans , Torres Luciana , Pittman Roland
Published: June 2021

Although some evidence suggests that ischemia induces changes in the endothelial glycocalyx (EG), little information is available about the EG following hemorrhagic hypotension (HH). We have employed video analysis to obtain preliminary data on EG changes following hemorrhage. Using intravital videomicroscopy, we studied the spinotrapezius muscle of anesthetized rats subjected to HH (40% blood vol.) for over 1 h. The analog video camera signal was digitized using standard software to DV‐AVI format. About 100 frames of several video clips (3‐5 s each) were processed using specially designed software to derive dimensions of the endothelial glycocalyx from the gap between erythrocytes and the luminal endothelial surface. Frequency distributions of the EG thickness during baseline and during HH showed a shift to lower values following hemorrhage, corresponding to a reduction in EG thickness of nearly 50 % (1.1 µm [range: 0.4 ‐ 4.3 µm] vs 0.6 µm [range: 0.3 ‐ 1.3 µm]). These changes occurred despite similar distributions of anatomic diameters (baseline range: 9 ‐ 24 µm; HH: 10 ‐ 21 µm). The findings suggest that the analysis of EG changes using in vivo video recordings can be used to investigate changes following HH and provide preliminary support for our hypothesis that these low flow states cause reductions in the endothelial surface layer thickness.

Support

NIH, NWO, NHS, (HV: NHF grant 2005T037).
Alignment of angiogenic microvessels and the surrounding collagen matrix under anchored boundary conditions
Authors: Underwood Clayton Jay , Krishnan Laxminarayanan , Fung Allen , Reese Shawn P , James Hoying B , Weiss Jeffery A
Published: June 2021

Previously we have observed an alignment of newly formed microvessels when 3D collagen gel cultures were axially anchored to their culture chamber . In contrast, microvessels in free‐floating gels exhibit a random orientation. The objective of this study was to examine changes in collagen alignment that occur during microvessel outgrowth over time in free‐floating and anchored 3D cultures. Microvessel cultures were fixed after 1 to 6 days in culture and endothelial cells were stained with Isolectin IB4‐Alexa 488. 3D image stacks of microvessels and collagen fibrils were obtained using confocal and confocal reflection microscopy, respectively. Microvessel and collagen fibril organization were quantified using standard image processing algorithms. There was significant alignment of vessels in anchored cultures, starting at 3‐4 days in culture. Similarly, the organization of collagen went from a random organization (anisotropy < 0.1) to a progressively more aligned orientation (anisotropy > 0.25) in a direction consistent with the microvessels. These results suggest that the orientation of microvessel outgrowth and matrix realignment follow similar patterns during angiogenesis. Although these events were highly correlated, it is unclear which event leads in the process of alignment.
Integrating Microvascular, Interstitial, and Lymphatic Function with a Balance Point Characterization of Interstitial Fluid Volume and Protein Regulation
Authors: Dongaonkar Ranjeet M , Laine Glen A , Stewart Randolph H , Quick Christopher M
Published: June 2021

Objective

To develop simple algebraic formulas and an intuitive graphical approach to characterize interstitial fluid volume and protein regulation, and to derive insights that are not available from conventional numerical approaches that are overly complex, conceptually opaque, and lack generality.

Methods

Eight standard equations characterizing fluid and protein flux were solved simultaneously to yield algebraic equations for interstitial fluid pressure and protein concentration as functions of parameters characterizing microvascular, interstitial and lymphatic function. Model results were validated by comparing to reported experimental values.

Results

Equilibrium values of interstitial fluid pressure and protein concentration arise as balance points from the graphical intersection of transmicrovascular flux and lymph flow (analogous to Guyton's classical cardiac output‐venous return curves). Algebraic solutions reveal that interstitial fluid pressure and protein concentration result from a ratio of the microvascular filtration coefficient and the effective lymphatic resistance, and are independent of interstitial compliance. Model results are consistent with reported measurements.

Conclusion

The present work presents a simple, transparent, and general balance point approach to predict lymph flow, interstitial fluid pressure and protein concentration that yields new insights concerning the complex interaction of microvascular, interstitial and lymphatic function. Supported by NIH‐K25HL070608 (CMQ), AHA‐0565116Y (CMQ), AHA‐0365127Y (RHS), CDC‐623086 (GAL).
Metabolic alkalosis vasodilation: role of endothelium and nitric oxide
Authors: Celotto Andrea Carla , Baldo Caroline Floreoto , Capellini Verena Kise , Evora Paulo Roberto
Published: June 2021

For long it has been known that changes in vascular smooth muscle tone plays an important role in blood flow distribution and blood pressure control. pH changes features as one of the factors influencing vascular smooth muscle tone. The aim of the present study was to investigate the effects of mild and severe alkalosis on endothelium‐dependent vasodilatatory responses and the role of nitric oxide (NO) on isolated rat thoracic aorta. We assessed the in vitro (¡̈organ chambers¡̈) effects of increasing pH in vascular function of thoracic aorta rings. pH‐response curves (7.4 to 8.5) were generated using NaOH in phenylephrine pre‐contracted rings. Experiments were conducted in the presence and absence of indomethacin, NG‐nitro‐L‐arginina metil ester (L‐NAME), N5‐(1‐iminoethyl)‐L‐ornithine hydrochloride (L‐NIO), N‐(6‐Aminohexyl)‐5‐chloro‐1‐naphthalenesulfonamide (W‐7), 2,5‐dimethylbenzimidazole (DMB) and methyl‐ƒÒ‐cyclodextrin (CD). Our investigation demonstrated that alkalosis vasodilatory effect on rat thoracic aorta: 1) is endothelium‐dependent and induced by NO; 2) is prostacyclin‐independent; 3) is dependent of the constitutive endothelium NO synthase isoform and of the complex Ca+2/calmodulin; 4) has Na+/Ca+2 exchanger and caveolae as important components in relaxing mechanism. Support: FAPESP; FAEPA‐HC/FMRP.