bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential Ca2+ Coupling of Alpha‐Adrenoreceptors in Murine Mesenteric Arteries and Veins
Authors: Hlavacova Alexandra , Xu Hui , Galligan James J.
Published: June 2021

Mesenteric veins are more sensitive to adrenergic stimulation than arteries and this could be due to different Ca2+ handling mechanisms in arteries and veins. We studied Ca2+ handling in pressurized arteries (60 mmHg) and veins (8 mmHg). Veins were more sensitive to the constrictor effects of norepinephrine (NE) than arteries; EC50 values were 50 and 300 nM respectively. CdCl2 (100 μM), but not nifedipine (1 μM) or GdCl3 (10 μM), blocked NE responses in arteries and veins. Thapsigargin (1 μM) caused a strong contraction in veins that recovered slowly (~30 min). Thapsigargin did not contract arteries. NE responses in arteries and veins were blocked by thapsigargin. In the presence of ouabain (0.1 mM), thapsigargin produced a small arterial contraction. NE responses after in the presence of thapsigargin and ouabain were reduced in arteries but abolished in veins. We conclude that veins are more reactive to NE than arteries. Both vessels use extracellular Ca2+ to support NE‐induced contraction but capacitive Ca2+ entry and L‐type Ca2+ do not contribute to Ca2+ entry in arteries or veins. Facilitation of Ca2+ influx and inhibition of Ca2+ removal by ouabain after intracellular Ca2+ depletion is sufficient for NE to contract arteries, but not veins. Veins have larger Ca2+ stores and more efficient store refilling than arteries; this may account for increased reactivity of veins to NE.
Protective effect of sphingosine 1‐phosphate (S1P) in the cerebral microvasculature
Authors: Edmundson Carmen Lynne , Miller Donald W.
Published: June 2021

Introduction: Sphingosine 1‐phosphate (S1P), an endogenous phospholipid, is involved in pre‐conditioning responses in the heart. The present study evaluated whether exogenous S1P could have a similar protective role in the cerebral microvasculature following hypoxic insult.

Methods: Human brain microvessel endothelial cells (HBMECs) were exposed to oxygen‐glucose deprivation (OGD) for 0–6 hours. Cell viability was assessed using MTT assay immediately after OGD or following a 24‐hr re‐oxygenation period. The effects of S1P on OGD‐mediated cell toxicity was determined by pre‐treating the cells with various concentrations of S1P (0–10 μM) for 0, 1, or 24 hours.

Results: Exposure of HBMECs to OGD resulted in a significant decrease (approximately 20%) in cell viability when assessed immediately after OGD. Pre‐treating HBMEC cells with S1P for 1 hr prior to OGD provided a dose dependent protection, with 1.0 and 10 μM S1P completely reversing the loss in cell viability associated with OGD at all time points examined. However, the protective effects of S1P were not apparent in the 24‐hour pre‐treatment protocol.

Conclusion: Activation of S1P receptors within the brain microvasculature prior to hypoxic events may help prevent toxicity to the cerebral microvasculature.

Funding support provided by Canadian Institutes of Health Research and Manitoba Health Research Council.
MYOGENIC TONE IN JUVENILE RAT MCA IS RESISTANT TO BLOCKADE OF CALCIUM INFLUX AND RELEASE OF INTRACELLULAR CALCIUM
Authors: Charles Shelton M , Zhang Lubo , Longo Lawrence D , Pearce William J , Buchholz John N
Published: June 2021

We measured pressure‐evoked (20–80 mmHg) myogenic tone and wall intracellular calcium ([Ca2+]i) in endothelial denuded, Fura‐2 loaded segments of middle cerebral arteries (MCA) from pup (P14) and adult (6 months) SD rats. Measurements were done in calcium replete HEPES buffer in the absence and presence of, cyclopiazonic acid (CPA, 10 μM) to deplete [Ca2+]i stores or with the calcium channel blocker, lanthanum (La3+, 100 μM), or with La3+ plus CPA. Myogenic tone was developed at each pressure step with corresponding increases in [Ca2+]i, in adult and pup MCA. CPA reduced myogenic tone and [Ca2+]i in the adult MCA, while in the pup CPA decreased myogenic tone with no change in [Ca2+]i and tone remained significantly greater in the pup as compared to the adult. La3+ decreased myogenic tone and [Ca2+]i in the adult MCA and in the pup tone also declined but was greater as compared to adult without any significant decline in [Ca2+]i. In the presence of La3+ and CPA, myogenic tone was completely lost in the adult with a corresponding decline in [Ca2+]i, however, in the pup MCA, myogenic tone while severely depressed, significant tone remained with no significant change in [Ca2+]i. These data suggest that in juvenile cerebral arteries there appears to be a pool of [Ca2+]i that maintains minimal myogenic response when influx and release of calcium are blocked. Supported in part by NIH R01 #HL69078‐01 and NIH P01 # 31226
Inhibiting matrix metalloproteinase‐2 (MMP‐2 reduces endothelial damage in isolated rat hearts during ischemia‐reperfusion
Authors: Fert‐Bober Justyna , Leon Hernando , Sawicka Jolanta , Basran Paul , Schulz Richard , Sawicki Grzegorz
Published: June 2021

Previous studies have shown disruption of the coronary endothelium and increase in its permeability during ischemia‐reperfusion (I/R) which can be linked with MMPs activity. Our group has shown that during I/R, increased activity of MMP‐2 is associated with cardiac dysfunction, which can be prevented by MMP inhibitors. Therefore, we hypothesize that MMPs inhibition reduces the MMP‐2 dependent disruption of the coronary endothelium during I/R.

Isolated rat hearts were perfused in the Langendorff mode at a constant pressure and subjected to 15‐30 min of no‐flow ischemia followed by 30 min of reperfusion. o‐Phenanthroline (Phen), an inhibitor of MMPs, was used to protect the hearts from dysfunction. Endothelial integrity was evaluated by measuring coronary flow, and by measuring the interstitial albumin and serotransferrin in the effluent. MMP‐2 activity was measured in the hearts by zymography.

MMP‐2 activity was increased in heart tissue at the end of ischemia and was correlated with duration of ischemia. The coronary flow was decreased and correlated with the disruption of the endothelial barrier (increased release of albumin and serotransferrin in the coronary effluent). Administration of Phen inhibited both reduction of coronary flow and endothelial barrier disruption.
Store‐Depletion Mediated Agonist‐Activated Contractile Responses in Rat and Human Corporal Smooth Muscle
Authors: Jarajapu Yagna , Christ George , Andersson Karl‐Erik
Published: June 2021

Smooth muscle contraction is dependent on Ca2+‐influx, release of Ca2+ from intracellular stores and Ca2+‐sensitization mechanisms. Depletion of intracellular Ca2+ stores has been shown to cause Ca2+‐influx via transient receptor potential (TRP) channels. In this study we report agonist‐activated contractile responses in rat and human corporal smooth muscle when intracellular Ca2+ stores were depleted. Rat and human corporal smooth muscle strips were mounted in a myograph (DMT‐USA, Inc.) and isometric tension in response to pharmacological treatments were recorded. In the presence of caffeine, thapsigargin and nifedipine, phenylephrine (PE) and endothelin‐1 (ET1) did not evoke contraction in the absence of Ca2+. Reintroduction of extracellular Ca2+ produced concentration‐dependent contractions when increased up to 10 mM, only in the presence of PE or ET1. In rat corporal strips, these responses were completely reversed by SKF96365 and 2APB, that are known to block TRP channels. In human corporal strips, responses evoked by PE were decreased by 2APB and little affected by SKF96365, whereas those evoked by ET1 were decreased by SKF96365 but not by 2APB. This study provides functional evidence for the receptor‐activated Ca2+ influx in the presence of depleted intracellular Ca2+ stores. Characteristics of TRP channels involved in this response need systematic investigation.
In‐vitro metabolism studies with BLX‐1026, a novel Orexin‐2 agonist with anti‐obesity activity
Authors: Mukherjee Aparna , Sen Ananda , Neogi Partha , Dey Deben , Nag Abhijit , Nag Biswajit
Published: June 2021

BLX‐1026 is a water soluble, amino‐acid conjugated small molecule with in vitro Orexin‐2 receptor agonist activity. Orexin receptors have been implicated in non‐exercise activity thermogenesis. Increased levels of Orexin‐2 receptors have been observed in obesity‐resistant rats and decreased Orexin‐A levels observed in obese humans.

Phase I metabolism studies have been conducted with BLX‐1026 in rat, mice, dog, monkey and human liver microsomes and human liver S9 fractions. It is found to be stable in all five species liver microsomes but shows a 61% degradation in the human S9 fractions when incubated for 1 hour. When orally administered to dogs and rats, it is found to have one major metabolite. Upon oral administration to rats and analysis of bile, it converts into the same major metabolite and three minor metabolites of which one is observed to be a glucuronide conjugate indicating Phase II metabolism. Further in vitro studies are underway with BLX‐1026 to further characterize its metabolic pathway.
The in vitro metabolism of Bis‐2‐(bromomethyl)propane‐1,3‐diol (BMP) by hepatic cell fractions and hepatocytes
Authors: Kuester Robert , Hoehle Simone , Sipes I Glenn
Published: June 2021

Bis‐2‐(bromomethyl)propane‐1,3‐diol (BMP) is a brominated flame retardant. Results of a two year bioassay sponsored by the National Toxicology Program have shown it to be multi‐site carcinogen to male and female rats and mice. The studies reported here assessed the in vitro hepatic metabolism of BMP. Hepatic microsomes, cytosol or hepatocytes were obtained from male F‐344 rats. Microsomes (+NADPH) or cytosol (+glutathione) incubated with [14C]‐BMP did not result in formation of metabolites as assessed by HPLC with radiometric detection. Microsomes (+UDPGA) resulted in formation of a BMP‐monoglucuronide (identified by LCMS/MS and by hydrolysis with β‐glucuronidase) at a rate of 1.3 nmol/min/mg protein. Similarly, when cultures of primary hepatocytes from male F‐344 rats were incubated with 50 μM [14C]‐BMP, the BMP‐monoglucuronide was the only BMP derived metabolite detected. It was formed at a rate of 400 pmol/min/106 cells. In all cases, BMP was stoichiometrically converted to its glucuronide. These data as well as in vivo data (not presented) demonstrate that glucuronidation is the predominate, and perhaps only, pathway of BMP metabolism in rat liver. How metabolism of BMP relates to its carcinogenic potential remains to be established. This research was supported in part by the NIEHS NTP Grant No. No1‐ES‐45529 and NIEHS‐sponsored Southwest Environmental Science Center Grant Number P3‐ES‐06694.
In vivo and in vitro metabolism of aromatic amines in transgenic mice with liver‐selective expression of human arylamine N‐acetyltransferase NAT2
Authors: Grant Denis Michael , Sugamori Kim S , Brenneman Debbie
Published: June 2021

To aid studies into the role of liver‐selective expression of human arylamine N‐acetyltransferase NAT2 in tissue patterns of aromatic amine toxicity, we developed a transgenic mouse line that expresses human NAT2 selectively in liver by linking the human NAT2 protein‐coding exon to the mouse albumin promoter, injecting the transgene into mouse oocytes, and testing offspring for transgene incorporation by Southern blotting. We obtained one transgenic founder that possesses a single copy of the human NAT2 coding region. Transgenic animals and offspring of matings with Nat1/Nat2 null mice were assayed for human NAT2 expression using the human NAT2‐selective substrate sulfamethazine (SMZ) and the NAT1‐selective substrate p‐aminosalicylate (PAS) in in vitro enzyme assays and in vivo plasma pharmacokinetic studies. SMZ‐NAT activity was present in liver cytosols from transgenic animals at levels comparable to those observed in livers from human NAT2 rapid acetylators, while activity was absent in Nat1/Nat2 null controls. Activity in colon was low and that in other tissues was undetectable. SMZ was more efficiently cleared in vivo by human NAT2 transgenic mice than non‐transgenics, while the clearance of PAS was similar between the strains. These mice will be a valuable tool to investigate the importance of liver NAT2 expression in the metabolism and toxicity of aromatic amines in humans.
Disparity in expression of CYP1A1 and CYP1B1 and their response to dioxin and benzo[a]pyrene in human mammary epithelial cells from pre‐menopausal African American and Caucasian women
Authors: Goode Gennifer , Humphrey Andria , Prins Petra , Eltom Sakina
Published: June 2021

Although the overall breast cancer (BC) incidence rate for African American (AA) is lower than Caucasian (CA) women, among AA women under the age of 45 the rates are higher. Due to their roles in activation of environmental chemicals to ultimate carcinogens, the products of CYP1A1 and CYP1B1 genes are implicated as risk factors for breast cancer. The objective of this study is to examine the hypothesis that higher incidence of breast cancer in pre‐menopausal AA women is related to a disregulated expression of CYP1A1 & CYP1B1 genes. Our laboratory have isolated and characterized primary cultures of mammary epithelial cells (HMEC) of age‐matched pre‐menopausal AA and CA women, using normal breast tissues from mammoplastic reduction surgery. CYP1A1 & CYP1B1 expression in response to dioxin and benzo[a]pyrene (B[a]P) was measured by real time RT‐PCR in HMEC of ten age matched women donors. RT PCR results showed a marked difference in the expression of CYP1A1/CYP1B1 between AA and CA women in response to dioxin and B[a]P, which correlated to the levels of the Aryl hydrocarbon receptor. Western blot analysis of microsomal CYP1A1 protein showed no correlation to the mRNA expression in AA donors. These findings may contribute to a better understanding of differential role of environmental chemicals on the biology of breast cancer in this ethnic minority group. Supported by RR03032 15, CA91408‐01 and DAMD17‐02‐01‐0483 grants.
Post‐translational Modification and Nuclear Localization of Human Sulfotransferase (SULT) 2B1b
Authors: Falany Charles N , Dumas Nicole A. , He Dongning , Frost Andra R.
Published: June 2021

SULT2B1b is a novel SULT isoform with selectivity for the sulfation of 3β‐hdroxysteroids and cholesterol. SULT2B1b has a unique 53 amino acid 3′‐tail that undergoes Ser phosphorylation and is involved with nuclear translocation in BeWo choriocarcinoma cells. The human SULT2B1 gene encodes two messages utilizing different transcriptional start sites resulting in the incorporation of different first exons. Although both SULT2B1a and 2B1b messages are found in human tissues, only the SULT2B1b protein has been detected. SULT2B1b is present in nuclei of term placental synchiotrophoblast and normal and cancerous breast epithelial cells. The 3′‐tail of SULT2B1b is required for nuclear localization in BeWo cells. Deletion of the unique 3′‐tail prevents translocation to the nuclei. 2D electrophoresis indicates two charge variants of SULT2B1b in BeWo cells; however, the variants are not detected in SULT2B1b lacking the 3′‐tail. Phosphorylation of Ser248 in the 3′‐tail is associated with nuclear localization. Mutation of Ser248 to Ala blocks nuclear translocation of SULT2B1b. Treatment of BeWo cells expressing SULT2B1b with forskolin, a cAMP‐dependent PKA inducer, increases phosphorylation and nuclear localization. SULT2B1b is the first SULT to show post‐translational modification and nuclear localization suggesting a unique function in nuclei. Supported by PHS grant GM38953 to CNF.
Hepatic ABC Transporter Expression in A Hemochromatosis‐Linked Murine Model of Porphyria Cutanea Tarda
Authors: Arch Dorinda D , Phillips John D , Kushner James P , Franklin Michael R
Published: June 2021

Iron overload can lead to hepatic fibrogenesis and high liver iron is frequently associated with porphyria cutanea tarda (PCT), a disease characterized by hepatic accumulation of highly carboxylated porphyrins. To assess whether porphyrin accumulation could arise from impaired anion transport, ABC transporter expression in a porphyria‐susceptible mouse model was investigated by qPCR. Mice with the Urod+/– (uroporphyrinogen decarboxylase), Hfe−/− (hemochromatosis) genotype spontaneously develop PCT, with severely decreased UROD activity and hepatic iron overload. Control animals with only the Urod+/– genotype do not accumulate porphyrins and had no change in expression of twelve ABC transporters examined. Accompanying the high iron accumulation in the Hfe−/− animals, expression of Abca3, Abcb1a, and Abcc4 were increased and expression of Abcc2, Abcc6 and Abcg2 were decreased. In mice with the Urod+/–, Hfe−/− genotype, porphyrin accumulation was accompanied by elevated expression of Abca3 and Abcc4 while Abcc2 and Abcc6 expression was depressed. The elevation in expression of Abcb1a and depression of Abcc2 were no longer present. Novel in these animals was an elevation of Abcc1 expression. The porphyrin accumulation exacerbated the repression of Abcc2 and Abcc6 expression and attenuated the increase in Abca3 seen in Hfe−/− control mice.

Supported USPHS Grants DK20503 and DK07115
Expression of CYP4F2 in human liver and kidney: assessment using specific peptide antibodies
Authors: Lasker Jerome M. , Hirani Vandana , Yarovoy Anton , Kozeska Anita , Dhar Madhurima
Published: June 2021

P450 enzymes comprising the human CYP4F gene subfamily are catalysts of eicosanoid (e.g., 20‐HETE and leukotriene B4) formation and degradation, although the role that individual CYP4F proteins play in these metabolic processes is not well defined. Thus, we developed antibodies to assess tissue‐specific expression and function of CYP4F2, one of four major CYP4F P450s found in human liver and kidney. Polyclonal peptide antibodies elicited in rabbits to CYP4F2 amino acid residues 61–74 (WGHQGMVNPTEEG) and 65–77 (GMVNPTEEGMRVL) recognized only CYP4F2 on immunoblots, and did not cross‐react with CYP4F3b, CYP4F11 or CYP4F12. Immunoquantitation with anti‐CYP4F2 peptide IgG showed highly‐variable CYP4F2 expression in liver (16.4 ± 18.6 pmol/mg; n = 28) and kidney cortex (3.9 ± 3.8 pmol/mg; n = 10), with two dissimilar subjects lacking the hepatic and renal enzyme entirely. Although CYP4F2 efficiently catalyzed ω‐hydroxylation of both arachidonate and oleate, only the former fatty acid‐metabolizing activity correlated strongly (r = 0.926; p < 0.025) with liver CYP4F2 content. Our study provides the first example of peptide antibodies that recognize a single CYP4F P450 found in liver and kidney, namely CYP4F2. Immunoquantitation and correlation analyses performed with these targeted antibodies indicate that CYP4F2 functions as a predominant arachidonate ω‐hydroxylase in human liver (supported by NIH AA07842).
CYP3A4 regulation by DBP and E4BP4 transcription factors
Authors: Maas Kristen E , Le Min , Hines Ronald N
Published: June 2021

The proline and acidic amino acid rich (PAR) family member D‐element binding protein (DBP) induces CYP3A4 promoter activity via a putative element in the proximal promoter. E4 binding protein 4 (E4BP4), also a PAR member, negatively regulates transcription. Given the known DBP and E4BP4 developmental expression pattern, both factors were hypothesized to participate in CYP3A4 developmental regulation. To begin testing this hypothesis, a luciferase reporter construct containing three known PAR binding sites was transiently expressed in HepG2 cells along with DBP and E4BP4 expression vectors. Co‐expression with E4BP4 alone inhibited luciferase expression in a dose‐dependent manner with a maximum 73% response. Co‐expression with DBP alone caused a 270% increase in luciferase activity, but combination with E4BP4 only resulted in a 28% reduction in DPB‐induced promoter activity. In similar experiments using a construct wherein CYP3A4 position ‐771 to +56 directs luciferase expression, E4BP4 was not able to inhibit DPB induced promoter activity. These data suggest that DBP binds PAR responsive elements with higher affinity than E4BP4. Because E4BP4 is expressed in both the fetal and postnatal liver while DBP is only observed postnatally, these data are consistent with an important role for both DPB and E4BP4 in regulating CYP3A4 ontogeny. Supported by Childrenis Research Institute.
Interleukin‐1 in chronic angiotensin II‐high salt diet induced hypertension
Authors: Boesen Erika I , Pollock Jennifer S , Pollock David M
Published: June 2021

IL‐6 and TNF‐α are implicated in hypertension induced by angiotensin II infusion plus high salt diet (AngII‐HS). Since IL‐1β can stimulate production of both cytokines and is implicated in some renal injury models, we investigated the role of IL‐1β in AngII‐HS hypertension. In rats, 14‐day AngII‐HS (AngII at 65 ng/min s.c. + 8% NaCl diet) increased 24‐h average mean arterial pressure (191±3 vs 108±3 mmHg in normal NaCl control rats [NS], P<0.001) and renal cortical immunoreactive IL‐1β content (44±6 vs 21±3 pg/ml, P<0.01). AngII‐HS rats also displayed significantly increased urinary protein (431±33 mg/d) and albumin excretion (2.5±0.3 mg/d) compared to NS rats (22±6 & 0.8±0.3 mg/d, P<0.001). To test whether IL‐1β contributes to renal injury in AngII‐HS hypertension, the effects of 14‐day AngII‐HS (90 ng/min s.c. + 4% NaCl diet) were compared in wild‐type and IL‐1 receptor −/− mice. After a 24‐h baseline urine collection, mice were randomized to AngII‐HS or NS treatment groups. Baseline urine flow and Na+, protein and albumin excretion rates did not significantly differ between the four groups. At day 14, protein excretion was significantly increased by AngII‐HS in wild‐type and IL‐1 receptor −/− mice (3.7±0.9 & 5.2±1.5 mg/d) compared to NS wild‐type and IL‐1 receptor −/− mice (1.3±0.3 & 0.9±0.1 mg/d; P<0.05), however this effect was not significantly different between genotypes. Similarly, urine flow, Na+ and albumin excretion were significantly increased in AngII‐HS mice compared to NS mice (P<0.05), but there were no differences between genotypes. Preliminary data also suggests that the degree of hypertension induced by AngII‐HS is similar in wild‐type and IL‐1 receptor −/− mice. Our results indicate that although IL‐1β production is increased in the renal cortex in AngII‐HS hypertension, IL‐1 receptor signalling does not appear to contribute to the development of proteinuria in this model.
Blood Microvesicles (or Microparticles) Isolation, Identification and Characterization: impact of anticoagulants
Authors: Jayachandran Muthuvel , Litwiller Robert D , Owen Whyte G , Miller Virginia M
Published: June 2021

Heterogeneous, phospholipid‐rich sub‐micron vesicles (“microvesicles”) shed by activated vascular and blood cells may reflect or influence the coagulant propensity of blood. To determine how the anticoagulant used to collect the blood affects microvesicle (Mv) recovery and characteristics, blood was collected from laboratory volunteers and participants of the Kronos Early Estrogen Prevention Study (KEEPS) into tubes containing either sodium citrate, EDTA or hirudin plus tick anticoagulant peptide (H&T). Microvesicles prepared by double centrifugation were analyzed by flow cytometry to define size and surface markers. There were 2–3 times the number of platelet‐derived (CD61 positive) Mv in preparations from citrate and H&T plasma, relative to numbers in EDTA plasmas. Procoagulant activity of Mv was determined by surface phosphatidylserine (PS) expression using annexin‐V‐fluorescein. Annexin‐V (PS) positive Mv did not differ, but total (PS positive and negative) Mv was 2 fold lower from EDTA plasma compared to citrate and H&T. These results suggest that anticoagulation of blood itself alters microvesicle characteristics. Therefore, standardization and specification of the anticoagulant is warranted to identify potential contribution of microvesicles to cardiovascular pathophysiology. Funded by, NIH HL78638, Kronos Research Institute and Mayo Foundation.
Effects of omega‐3 acid ethyl esters and aspirin, alone and in combination, on platelet function in healthy subjects
Authors: Larson Mark K , Ashmore Joseph H , Harris Kristina A , Vogelaar Jessica L , Harris William S
Published: June 2021

Introduction: Omega‐3 fatty acids (n‐3 FA) lower triglycerides and risk of heart attacks. Accordingly, patients at risk are often advised to take both aspirin (ASA) and n‐3 FA. However, both can increase bleeding times, and the effects of the combination on platelet function are unknown. We determined the effects of a prescription n‐3 FA product (P‐OM3) and ASA, alone and in combination, on platelet aggregation assessed by whole blood impedance aggregometry (WBA). Methods: Ten healthy volunteers provided blood samples on four separate occasions: Day 1, baseline; Day 2, 1 day after taking ASA (2 × 325 mg tablets); Day 29, after 28 days of P‐OM3 (4 capsules/d); and Day 30, after 1 day of combined P‐OM3 and ASA. WBA was tested with two concentrations of collagen and with ADP. Results: Aggregation in response to either high‐dose collagen or ADP was not inhibited by ASA alone (vs. baseline), but it was inhibited when combined with P‐OM3 (19.0±2.8 to 13.2±2.9 ohms, and 9.1±3.0 to 6.2±4.2 ohms, respectively, p<0.04 both). Using low‐dose collagen as the agonist, ASA with or without P‐OM3, reduced platelet aggregation vs. baseline (14.7±2.7 vs. ASA+P‐OM3, 2.3±1.1 ohms, p<0.001). Conclusions: P‐OM3 alone did not inhibit platelet aggregation, but it enhanced the inhibitory effect of ASA in response to two common agonists. N‐3 FA may improve ASA resistance.
Changed microvascular adaptation characteristics may explain heterogeneity and hypoxia of tumor perfusion
Authors: Pries Axel R , Cornelissen Annemiek JM , Sloot Anouk A , Dreher Matthew , Hinkeldey Marlene , Höpfner Michael , Dewhirst Mark W , Secomb Timothy W
Published: June 2021

Tumor microcirculation exhibits high structural and functional heterogeneity leading to hypoxia and impairing treatment efficacy. Structural properties of normal vascular beds are controlled by continuous adaptation to local hemodynamic and metabolic stimuli. Here, we tested the hypothesis that tumor microvascular properties are due to changed vascular adaptation characteristics. Control and tumor vascular networks were recorded by intravital microscopy. Hemodynamics and oxygen distribution in these networks were assessed by computer modeling. Vascular diameter adaptation in response to hemodynamic, metabolic and conducted stimuli was simulated. Results were analyzed with respect to diameter heterogeneity at individual bifurcations (AD) and deficit of oxygen delivery relative to demand (Odef). Tumor networks (T) exhibit high AD (0.40) and Odef (0.17) as compared to control networks (C) (AD 0.28; Odef 0.10). Simulated adaptation of T with previously established vascular reaction characteristics of normal tissues led to normalized values for T (AD 0.32; Odef 0.11). In turn, C could be made to mimic T characteristics by assuming increased randomness of vascular growth tendency and by reducing sensitivity to conducted stimuli (AD 0.41; Odef 0.17). Thus, aberrant properties of tumor microvasculature can be explained by changed vascular adaptation characteristics.
Cdc42 increases activation of MMP‐2 in endothelial cells
Authors: Serio Damiano , Ispanovic Eric , Haas Tara L
Published: June 2021

Angiogenesis is stimulated by VEGF and requires basement membrane degradation by the matrix metalloproteinases (MMPs). VEGF induces increased membrane type 1‐MMP on the cell surface (required for MMP‐2 activation) and signals through the RhoGTPase family members. The RhoGTPases and the MAPK p38 are known for their roles upstream of actin cytoskeleton reorganization and are purported to regulate angiogenesis. We hypothesize that Cdc42 mediates the effects of VEGF on activation of MMP‐2 via p38 and actin cytoskeleton reorganization. To investigate this we manipulated Cdc42 signaling with a constitutively active Cdc42 (CA‐Cdc42) TAT‐fusion protein and observed the effects in rat skeletal muscle endothelial cells. Confocal microscopy demonstrated that CA‐Cdc42 increased cortical actin and the proportion of retracted or protruded cells similar to VEGF‐induced actin stress fibre depolymerization. CA‐Cdc42 treatments resulted in increased phospho‐p38 levels as seen with VEGF via western blotting. Furthermore, MMP‐2 activation increased in cells treated with CA‐Cdc42 or VEGF independent of new protein synthesis, as tested by gelatin zymography. This evidence suggests that Cdc42 may mediate the effects of VEGF on actin cytoskeleton reorganization, p38 phosphorylation and the levels of active MMP‐2. Funded by NSERC.
The recovery time course of the endothelial glycocalyx after enzymatic and cytokine degradation.
Authors: Potter Daniel Raymond , Jiang John , Damiano Edward
Published: June 2021

The glycocalyx surface layer on vascular endothelial cells is thought to be a dynamic extracellular matrix, which exhibits changes in thickness and composition in response to local conditions. Previous research has shown that enzymes degrade the glycocalyx, while inflammation causes shedding of the layer. While reconstitution experiments have been performed, the endogenous recovery time of the glycocalyx after degradation has not been addressed. Endothelium with a partially or completely degraded glycocalyx is at risk for increased exposure to blood‐borne free radicals, which, under chronic conditions, could in turn contribute to cardiovascular disease. It is therefore important to ascertain the time course of endogenous recovery of the glycocalyx after degradation. To track glycocalyx recovery, we use micro‐particle image velocimetry in cremaster‐muscle venules to estimate the hydrodynamically relevant thickness of the glycocalyx in mice that have previously been treated with an enzyme (through systemic tail‐vein injection) or an inflammatory cytokine (through scrotal injection). Mice are injected with either hyaluronidase, heparinase III, or TNF‐ α and the glycocalyx thickness is estimated after 1, 3, 5, and 7 days. Preliminary data indicate that it takes over five days for the endothelial glycocalyx to restore itself to its native dimensions of ~ 0.5 μ m. Supported by NIH grant R01 HL76499.
The PPAR Gamma Agonist Rosiglitazone Protects Against Wnt Signaling In Hyperoxia‐Induced Neonatal Rat Lung Injury
Authors: Dasgupta Chiranjib , Wang Ying , Sakurai Reiko , Torday John S , Rehan Virender
Published: June 2021

Bronchopulmonary Dysplasia (BPD) is a consequence of abnormally repaired lung damage. Hyperoxia disrupts critical signaling pathways that support normal lung development. We characterized in vivo hyperoxia induced perturbations in lung morphology and Wnt and TGF‐β signaling in postnatal day (PND) 7 rat lung, and tested if the PPARγ agonist rosiglitazone (RGZ) prevents such perturbations. Newborn rats were grouped as normoxic, hyperoxic (95% O2), and hyperoxic/RGZ (3 mg/kg i.p. daily during 95% O2). PND7 lungs were analyzed by immunoblot, morphometry, and immunohistochemistry (IHC). Hyperoxia caused significant up‐regulation of Wnt signaling markers Lef1 and βcatenin, and TGFβ pathway markers pSMAD3 and SMAD7. The myogenic phenotype markers αSMA and calponin were also up‐regulated. There was significantly reduced alveolar septal thickness, radial alveolar count, but larger alveoli. PPARγ expression, however, was significantly down‐regulated. Hyperoxia‐induced changes were almost completely prevented by RGZ treatment. We demonstrate up‐regulation of Lef1, βcatenin, pSMAD3 and SMAD7 in neonatal rat lungs in hyperoxia. Hyperoxia‐induced simplified alveoli, a hallmark of BPD, along with altered molecular changes, strongly implicating these factors in hyperoxia‐induced lung injury. PPARγ agonists may effectively prevent BPD Grant Support‐NIH HL75405 & HL55268 & TRDRP 15IT‐0250 & 14RT‐0013.
Phosphorylation of caveolin‐1 contributes to the mechanism of oxidant‐induced endothelial hyperpermeability
Authors: Sun Yu , Minshall Richard D. , Visintine David J. , Castellon Maricela , Malik Asrar B. , Hu Guochang
Published: June 2021

Oxidants are thought to increase endothelial permeability by opening interendothelial junctions. Here, we tested whether H2O2‐induced phosphorylation of caveolin‐1 (Cav‐1) stimulates caveolae‐mediated transcellular albumin transport and if this process contributes to protein‐rich edema accumulation. In rat lung microvascular endothelial cells, H2O2 (100 μM ∼ 1.0 mM) induced a concentration‐dependent increase in the uptake (1.5 ∼ 2.7‐fold) and transendothelial transport of 125I‐albumin (3.4 ∼ 7.2‐fold) as well as activation of Src and Src‐dependent phosphorylation of Cav‐1. Furthermore, H2O2 induced stimulation of albumin permeability was blocked by methyl‐β‐cyclodextrin and Cav‐1siRNA. Only with very high concentrations of H2O2 (>5 mM) was there an observable decrease in transendothelial electrical resistance. In the isolated wild type mouse lung, vascular perfusion of H2O2 induced Cav‐1 phosphorylation and lung edema formation, which was abolished in Cav‐1 null lungs. Rescue of Cav‐1 expression resulted in H2O2‐induced lung edema formation, whereas expression of non‐phosphorylatable Cav‐1 mutant (Y14F‐Cav‐1) did not. Thus, H2O2 causes caveolae‐mediated vascular albumin hyperpermeability and induces pulmonary edema formation through enhanced phosphorylation of Cav‐1. Phosphorylation of Cav‐1 may be a therapeutic target in the treatment of oxidant‐induced lung injury.
Fibrocyte, CD13+ Monocyte and CXCR4+/Col+ Macrophage Infiltrates in a Murine Pulmonary Model of Fibrosis Detected Using 8‐color Flow Cytometry
Authors: Achuthanandam Ram , Rafferty Patricia , Cochlin Karyn , Mathur Ashok , Capocasale Renold J , Ort Tatiana , Ekert Jason , Branigan Patrick , Das Anuk , Bugelski Peter
Published: June 2021

A commonly used murine model of pulmonary fibrosis involves administration of bleomycin sulfate. Although this model has been used extensively, the infiltrating cells responsible for the initiation of the fibrotic event is not completely understood. Groups of mice were given an intra‐nasal administration of bleomycin sulfate and euthanized 2, 6 and 14 days post treatment. Phenotypic enumeration was done by 8‐color flow cytometry on day 6 using 16 cell surface markers and 1 intracellular marker for collagen‐I on whole lung digests. Progressive histological changes were observed in paraffin embedded sections stained with Sirius Red for collagen. Soluble collagen protein expression and cytokine levels were determined using the SIRCOL assay and luminex bead immunoassay respectively, from whole lung homogenates. At Day 6, a 3‐fold increase in CD13+ monocytes, a 1.5‐fold increase in CCXR4+/F4\80+/CD11b+/Gr1‐/Col‐1+ macrophages, and a 2‐fold increase in the number of fibrocytes were observed. A statistically significant increase in pro‐inflammatory cytokines including monocyte chemoattractant protein‐1 (MCP‐1) and KC (CXCL‐1) was observed. Our results lead us to conclude that in addition to fibrocytes, CD13+ monocytes and the CXCR4+/Col‐1+ macrophage population may play an important role in the pathogenesis of bleomycin‐induced pulmonary fibrosis.
Regulation of lung epithelial ATP release
Authors: Seminario‐Vidal Lucia , Lazarowski Eduardo
Published: June 2021

Mucociliary clearance, the primary innate defense mechanism of the lung, is controlled by extracellular nucleotides (e.g., ATP) acting on cell surface purinergic receptors. However, the mechanisms by which ATP is released from epithelial cells are poorly understood. To identify signaling elements upstream of ATP release, we examined the effect of GPCR activation on ATP release from lung epithelial A549 cells. The protease activated receptor (PAR) agonist thrombin and the P2Y2‐R agonist UTP induced robust inositol phosphate formation and Ca2+mobilization. RT‐PCR analysis indicated P2Y2‐R and PAR3 mRNA expression. Thrombin elicited a rapid ATP release, which was inhibited by BAPTA‐AM, thapsigargin, or ML‐7 (inhibits Ca2+/CaM‐dependent MLCK). In contrast, UTP caused no ATP release. Thus, signaling elements in addition to Gq/PLCβ/Ca2+may be involved in GPCR‐induced ATP release. PAR‐induced ATP release was decreased by ROCK inhibitors (Y27632 and H1 152), suggesting involvement of Rho/ROCK downstream of G12/13. Mechanical perturbations relevant to the airways (e.g. hypotonic swelling, shear stress) cause robust epithelial ATP release. Pretreatement of cells with ML‐7, Y27632, or H1 152 impaired hypotonicity‐elicited ATP release. Our results suggest that MLCK and Rho activation participate in both receptor‐ and hypotonicity‐induced ATP release from lung epithelial cells.

Supported by NIH P01HL34322
Chronic hypoxia in vivo increases von Willebrand factor expression in lung endothelial cells
Authors: Huertas Alice , Greenberg Steven , Yiming Maimaiti , Bhattacharya Jahar , Bhattacharya Sunita
Published: June 2021

The basis of hypoxia‐induced lung inflammation is not understood. To address this understanding, we considered the role of the endothelial (EC) platelet and leukocyte receptor, von Willebrand factor (vWf). We exposed mice (Swiss Webster) to room air, or 12% O2 for 24 hours. Then, we anesthetized the mice (ketamine: 3ml/kg and xylazine: 1ml/kg, i.p.), opened the chest, injected intra‐cardiac heparin and covered the lungs with chilled buffer (4°C). Through a pulmonary artery cannula we cleared blood in lung vessels by buffer infusion. Then, we infused chilled collagenase (1000 units/ml) and trypsin (3ml), and collected the effluent through a left atrial cannula. After filtering the sample, we treated it with paraformaldehyde (4%) and Triton X‐100 (0.1%). Then, using Alexa 488‐conjugated, specific and non‐specific mAbs (1μg/100 μl) we stained the samples respectively, to detect vWf and to rule out non‐specific ligation. Specific Alexa fluorescence (normalized units) as detected by flow cytometry on 5×104 cells/lung, defined EC. In animals exposed to normoxia, EC fluorescence was 4.2±0.3 units/103 cells (mean±SD; n=3). By contrast, in hypoxia EC fluorescence increased >5 times baseline to 21.2±3.5 units/103cells (n=3, p<0.05). Our findings indicate that hypoxia activated vWf expression in lung EC. In hypoxia, vWf‐induced EC signaling might underlie lung inflammation (Support: HL54157, HL36024).
Role of cyclin dependent kinase 5 (CDK5) and c‐Abl in high NaCl‐induced nuclear localization of the osmoprotective transcription factor, TonEBP/OREBP
Authors: Gallazzini Morgan , Kunin Margarita , Burg Maurice B. , Ferraris Joan D.
Published: June 2021

High NaCl activates TonEBP, resulting in increased transcription of protective genes. Early events include phosphorylation of TonEBP and increase in its nuclear to cytoplasmic (n:c) ratio. The purpose of the present studies was to identify specific amino acids that become phosphorylated in TonEBP and to determine whether they have a role in its nuclear localization. By mass spectrometry we previously identified T135 and Y143 as being phosphorylated in TonEBP. Use of phospho‐specific antibodies confirms phosphorylation at those sites and shows that high NaCl increases it. These amino acids are located in the Auxiliary Export Domain (AED) of TonEBP, which is necessary for its export from the nucleus when NaCl is low. MotifScan identifies T135 as a likely site for phosphorylation by CDK5 and Y143 as a likely c‐Abl site. The CDK5 inhibitors roscovitin and CDK1/5 reduce TonEBP n:c ratio, consistent with the hypothesis that CDK5 contributes to localization of TonEBP by phosphorylating T135. Dominant negative c‐Abl decreases TonEBP n:c ratio in cells exposed to high NaCl, as does prevention of phosphorylation of Y143 by mutating it to alanine. Effects on TonEBP n:c ratio of dominant negative c‐Abl and of mutation of Y143 to alanine are equivalent and non‐additive. We conclude that high NaCl stimulates nuclear localization of TonEBP by phosphorylation on T135 and Y143, most likely catalyzed by CDK5 and c‐Abl, respectively.
LC‐MS/MS identification of amino acids that are phosphorylated in the osmoprotective transcription factor, TonEBP/OREBP
Authors: Kunin Margarita , Simons Brigitte L. , Irarrazabal Carlos , Shen Rong‐Fong , Wang Guanghui , Burg Maurice B. , Ferraris Joan D.
Published: June 2021

High NaCl increases the activity of TonEBP/OREBP, resulting in increased transcription of osmo‐protective genes. High NaCl increases phosphorylation of TonEBP, and there is evidence that the phosphorylation is involved in its activation. The goal of the present studies was aimed to identify which amino acids in TonEBP are phosphorylated and to test their roles. We used HEK293 cells transfected with TonEBP‐1‐547‐V5 or TonEBP‐1‐1531‐V5. The cells were cultured under 200 or 500 mosmol/kg for two hours, nuclear and cytoplasmic proteins were extracted and immunoprecipitated with antibody to TonEBP‐V5. The immunoprecipitate was subjected to in‐solution proteolytic digestion and the phosphopeptides were enriched by IMAC prior to LC‐MS/MS analysis. In order to maximize coverage of possible phosphorylation sites, several different proteolytic enzymes were used. We used Ascore, a probability‐based rating system (Beausoleil at el, 2006), to estimate the probability of correct identification of phosphorylation sites. We find a high probability that S120, S134, T135, S561, S744, S1168, S1364 and S1367 are phosphorylated in at least one of the conditions. An S to A mutation at aa 134, S134A, reduce high NaCl‐dependent nuclear localization of TonEBP, while the phosphorylation at T135 is confirmed by a phosphospecific antibody. Possible functions of the other sites and their confimation by phospho‐specific antibodies are currently in process.

Supported by the Intramural Research Program of NHLBI.
Arachidonic acid (AA) stimulates Ca2+‐activated big conductance K (BK) channel in the cortical collecting duct (CCD) via cytochrome P450 (CYP) epoxygenase‐dependent metabolic pathways.
Authors: Sun Peng , Wang Wen‐hui
Published: June 2021

We used the patch‐clamp technique to study the effect of AA on BK channels in principal cell of the CCD in rat kidney. The dose‐response curve of the AA effect on BK yields that 10 μM AA caused a maximal stimulation of BK and increased NPo from 0.0004 to 0.45. The effect of AA on BK was specific because application of 11,14,17‐eicosatrienoic acid had no effect on BK. To determine the metabolic pathway by which AA‐induced stimulation of BK, we examined the effect of AA in the presence of indomethcin, DDMS and MS‐PPOH. Inhibition of either cyclooxygenase with indomethacin or CYP‐monooxidase with DDMS failed to abolish the stimulatory effect of AA on BK. In contrast, AA‐induced activation of BK was absent in the presence of MS‐PPOH, an inhibitor of CYP epoxygenase. The notion that the stimulative effect of AA on BK is mediated by CYP epoxygenase‐dependent metabolites is also supported by the observation that application of 100 nM 11,12‐epoxyeicosatrienoic acid (EET) mimicked the effect of AA. In contrast, addition of either 8,9‐, or 14,15‐EET failed to stimulate BK. Moreover, application of 11,12‐EET was still able to activate BK in the presence of MS‐PPOH. This suggests that 11,12‐EET is a mediator for the AA‐induced activation of BK. We conclude that AA activates BK activity in the CCD and that the effect of AA is mediated by a CYP epoxygenase‐dependent metabolite, 11,12‐EET.
Synergistic regulation of CFTR‐bicarbonate conductance by protein kinases and glutamate in the human sweat duct
Authors: Reddy M.M. , Quinton Paul M
Published: June 2021

CFTR mutations and the resultant loss of both Cl− and HCO3− conductances of the CF affected epithelial cells seem responsible for many pathological changes in CF. Previous studies have indicated that the Cl− and HCO3− conductance functions of CFTR might be regulated by separate mechanisms (Nature, 423: 756–760, 2003). We further investigated the mechanisms that control CFTR Cl− and HCO3− conductances using electrophysiological techniques on α‐toxin permeabilized human sweat duct as previously described (Nature, 423: 756–760, 2003). Our aim was to test the hypothesis that the Cl−and HCO3− conductance functions of CFTR are regulated differently by protein kinases (PKA and PKG), glutamate and ATP. We found that the kinase agonists (cAMP and cGMP) and cytosolic glutamate maximally stimulated CFTR Cl− conductance with little synergistic action by these agonists. In contrast, these kinase agonists had little effect on CFTR ‐HCO3− conductance (CFTR_gHCO3−) when applied individually. However, simultaneous application of cAMP and cGMP (in the presence of 5 mM ATP) synergistically stimulated CFTR‐gHCO3− to 8.0±2.0 ms/cm2, n=number of ducts=5). Addition of glutamate or its metabolite, α‐ketoglutarate further enhanced CFTR‐gHCO3− to 18.0± 4.0 mS/cm2, n=5. These preliminary data appears to suggest that the Cl− and HCO3− conductance functions of CFTR required distinct conditions for activation and regulation. Supported by NIH‐RO1 DE14352, NIH‐RO1HL08042, USPHS R01 DK 51889 and the Nancy Olmsted Trust
“Diabetes Insipidus” Strain Quail Show Poorly Developed Medullary Cones and Low Aquaporin 2 Water Channel Expression
Authors: YANG YIMU , Minvielle Francis , Kuykindoll Rhonda J , Gasc Jean‐Marie , Yamamoto Tadashi , Nishimura Hiroko
Published: June 2021

A Coturnix japonica strain with clinical signs of diabetes insipidus (DI) was developed after screening for polydipsia and polyuria (homozygous autosomal recessive mutation with unaffected vasotocin system) (Minvielle et al., Poult Sci 86: 249, 2007). To determine the mechanism behind DI manifestation, we examined kidney morphology, molecular expression of AQP2 water channels, and responses to water restriction. DI quail kidneys have poorly developed medullary cones with a significantly (P < 0.01) smaller area per kidney (longitudinal section) (control, CT, 14.7 ± 1.0%; DI, 8.9 ± 1.1%); the number of glomeruli is similar in CT and DI quail. The DI and CT gene and amino acid sequences are nearly identical, including two NPA motifs, a mercury‐sensitive structure, and a putative phosphorylation site. AQP2 mRNA levels measured by in situ riboprobe hybridization and real‐time PCR are low in DI quail. Hematocrit (Hct, %) and plasma osmolality (Posm, mOsmol/l) are also lower (Hct, 42 ± 1; Posm, 297 ± 11) than in CT quail (Hct, 47 ± 1; Posm, 322 ± 3); both increased after 24 h water restriction in DI and CT groups, but DI quail lost more body weight. The results suggest that the renal medulla plays a major role in causing DI manifestation. The mechanism of possible blood volume expansion remains to be elucidated (NSF IOB‐0615359, NIH HL52881).
Role of ATP‐sensitive K+ channels in glucose sensing by immortalized adrenomedullary chromaffin cells
Authors: Piskuric Nikol Anne , Brown Stephen T. , Nurse Colin A.
Published: June 2021

Adrenomedullary chromaffin cells exhibit a critical Ca2+‐dependent catecholamine secretion when directly exposed to perinatal stressors (e.g. hypoxia). Using an immortalized chromaffin cell line (MAH), we investigated their potential sensitivity to low glucose (hypoglycemia) by monitoring changes in intracellular free calcium ([Ca2+]i) with the fluorescent probe Fura‐2. The majority of MAH cells (86%; n=398) were unresponsive on switching from 10 to 0 mM glucose. Interestingly, 0 glucose markedly inhibited (~75%) the rise in [Ca2+]i induced by the depolarizing stimulus high K+ (10 mM). To test the possibility that this was due to KATP channel activation, the KATP channel blocker, glibenclamide (50 μM), was used. In normal K+, glibenclamide increased the proportion of 0 glucose‐responsive cells from 14% to 65%, and the mean Δ[Ca2+]i response increased fourfold. RT‐PCR and Western blot analysis confirmed the presence of both the inward rectifying (Kir6.2) and sulfonylurea (Sur1) subunits of KATP channels. These findings suggest that hypoglycemia‐induced activation of KATP channels may limit [Ca2+]i increases and catecholamine secretion in chromaffin cells. Furthermore, the increased proportion of 0 glucose‐responsive cells in the presence of glibenclamide suggests that hypoglycemia causes a separate excitatory pathway in MAH cells.

Supported by NSERC and the HSFO.