bluuebunny/crossref_metadata_2025 huggingface dataset:
FORMULATION AND INVITRO EVALUATION OF OMEPRAZOLE LIPID MICROSPHERES
Authors: Mohan S Dinesh , Gupta V Rama mohan
Published: June 2021

The current work formulation and invitro evaluation of Omeprazole lipid microspheres for the treatment of zolinger ‐ Ellison syndrome and H.pylori infection was carried out with objective of long‐term treatment for the syndrome. Lipid microspheres have great potential, time tested, safe, and stable at room temperature, easily mass produced yet cheaper. Omeprazole is a benzimidazole derivative, which decreases the gastric secretion, regardless of the primary stimulus with half‐life 1.4 hours in young and 2.9 hours with elderly patient. The treatment period from 6 months to 4 years depending on the condition. The lipid microspheres were prepared by coacervation and phase separation technique. The prepared formulation evaluated for various parameters like particle size, SEM, Drug release study, drug content uniformity, stability studies. The release study shows the formulation follows initially first order and later follows zero order kinetics. The stability studies limits with ICH guidelines. In the present work an attempt was made to study the release pattern of drug Omeprazole from various concentrations of lipid microspheres. The formulation coded D5 shows the maximum release retardant behavior, because of high proportion of lipid incorporation.
Hypoxia, Hyperoxia or pH ? Predominant Factors in Tumor Cell Survival
Authors: Mazzio Elizabeth , Soliman Karam FA
Published: June 2021

Background

The study of hyperoxia vs. hypoxia on tumor cell survival is often studied under experimental conditions that do not control for the shift in pH.

Methods

Here, we examine the molecular genetic effects of (~91% O2 (hyperoxia) vs. ~2.5% O2 (hypoxia)) at a uniform pH ~7.2 using microarrays, HPLC, imaging probes and Western blot.

Results

Hyperoxic and Hypoxic culture conditions for 24 hrs had no adverse effect on viability in malignant N2a cells, provided pH was neutral with ample glucose supply. The hyperoxic transcriptome showed elevation of mRNA of 42 mitochondrial genes, DNA/base excision repair, G protein signaling, protein synthesis, transcription and ubiquitin‐mediated proteolysis. The hypoxic transcriptome corresponded to major elevations in epigenetic (lysine (K)‐specific demethylases, H4k/d/j, Hist4h4, Hist2h3c1, Rara and REST corepressor 2) with greater expression of protein/mRNA for solute carrier family 16, member 3 MCT‐4 (lactate transporter). Blocking MCT‐4 (phloretin) lead to an alkaline shift in both pHex/pHi, corresponding to cell toxicity.

Conclusions

Tumor cells are capable of acclimatizing to extreme variation in O2, but are highly intolerant to factors that invoke alkalinity to the inclusion of rising levels of O2 or impeding lactate release. (Supported by NIH G12RR 03020, 8G12MD007582–28 and 1P20 MD006738–01))
Vasodilators produced during active hyperaemia interact: potassium attenuates the vasodilatory effect of nitric oxide
Authors: Lamb Iain R , Murrant Coral L
Published: June 2021

We previously demonstrated that multiple vasodilators (i.e. nitric oxide (NO), potassium (K+), adenosine (ADO)) are produced in response to skeletal muscle contraction and their ability to produce significant vasodilation differ depending on skeletal muscle stimulation parameters. Given the production of multiple vasodilators resulting from muscle contraction we sought to determine whether the dilators produced altered one another's vasodilatory ability. We tested whether NO's vasodilatory effect was altered in the presence of K+. Using the hamster cremaster muscle preparation in situ we used intravital microscopy to observe arteriolar vasodilation in response to a range of concentrations of an NO donor, S‐nitroso‐N‐acetyl pennicillamine (SNAP, 10−8M‐10−4M) in the absence and the presence of 10mM K+. The vasodilation with 10−8M‐10−4M SNAP (3.1±1.0μm, 7.6±1.4μm, 10.0±1.7μm, 15.7±2.3μm, 17.8±2.1μm respectively) was significantly attenuated in the presence of 10mM K+ (−1.9±1.1μm, −0.2±1.5μm, 1.3±2.5μm, 6.5±3.1μm, 11.5±2.8μm respectively). Our data show that K+ significantly attenuates the vasodilatory effect of NO. Thus, active hyperaemia may not be the sum effects of multiple individual vasodilators but a product of their interactions. This research was funded by NSERC, Canada.
Evaluation of the subacute systemic thrombotic and organ toxicity of nicotine in mice
Authors: Fahim Mohamed Abdelmonem , Nemmar Abderrahim , Al‐Salam Suhail , Dhanasekaran Subramanian , Shafiullah Mohamed , Yasin Javed , Hasan Mohamed Yousif
Published: June 2021

Nicotine is involved in the pathogenesis of hematological and cardiopulmonary diseases. However, the mechanisms underlying these effects are unclear. Presently, we have investigated the effect of nicotine on cerebral microvessel thrombosis, systemic and organs toxicity. Mice were injected with nicotine (1mg/kg, i.p), once a day for 7 days. Briefly, a photo insult of surgically exposed cerebral microvessel, after bolus intravenous injection with fluorescein, were done. The changes in platelet aggregation in microvessels were analysed. In conjunction, superoxide dismutase (SOD), LDH, liver enzymes, creatinine, BUN and histopathology were carried out. Our results revealed a significant reduction in the time required for platelet aggregation following nicotine exposure. Significant decrease in SOD and increase in LDH levels were observed. The liver enzyme showed substantial rise in the AST and ALT levels. The increase in LDH levels emphasizes an induction of systemic inflammation and reduction in SOD levels indicating the oxidative stress involvement in the tissue damages induced by nicotine. Lungs and liver histology showed intra‐vascular hemorrhagic infarction with necrosis and macrophages infiltration with inflammation. We conclude that the sub acute nicotine exposure causes systemic, hepatic and pulmonary toxicity and increases thrombotic event damage in cerebral microvessels.
Variability in the strength of AT1R Ca2+ signaling
Authors: Scott Lena , Bernhem Kristoffer , Brismar Hjalmar , Aperia Anita
Published: June 2021

The renin angiotensin system is a principal physiological regulatory system for blood pressure and the Angiotensin type 1 receptor (AT1R) is an important target for treatment of hypertension.

Using AT1R expressing HEK293a cells we study the Ca2+ response to repeated doses of physiological, 1 nM [Ang II] and pharmacological, 100 nM [Ang II] at 0, 7 and 37 min. AT1R activation results in Ca2+ release from intracellular stores. AT1R signaling has typically been studied at pharmacological 100 nM [Ang II]. At 100 nM [Ang II] a significant desensitization was seen in response to the 2nd dose with partial recovery observed at the 3rd dose. Mean Δ [Ca2+]i was 150, 30 and 70 nM respectively. At 1 nM [Ang II] no desensitization was observed between the first two doses, both at 15 nM, and the response to the 3rd dose, 40 nM, is significantly increased. As recent studies suggest a crosstalk between AT1R and Voltage Gated Calcium Channels, we also studied the effect of blocking Ca2+ channels with Nifedipine, therapeutically used in hypertension treatments, on AT1R response. No change to basal [Ca2+] was seen in presence of Nifedipine, whilst an increase was found in Ca2+ response from repeated doses of 1 nM [Ang II], 130, 110 and 80 nM. The same trend was seen in Podocyte enriched primary cultures.

In summary this study shows novel aspects to the AT1R signaling modulation with implications for the treatment of hypertension.
Relaxin increases renal inner medullary NOS1‐β expression during chronic angiotensin II infusion
Authors: Sasser Jennifer M , Mooney Jennifer N , Baylis Chris
Published: June 2021

Relaxin (RLX) attenuates hypertension and increases nitric oxide (NO) bioavailability. Because NO inhibits sodium reabsorption in the inner medullary collecting duct, this study tested the hypothesis that RLX increases expression of NO synthase (NOS) isoforms in the inner medulla during angiotensin II (ANGII) hypertension. Male Sprague‐Dawley rats were given ANGII (400ng/kg/min) or vehicle (CON) for 6 weeks via minipump. RLX was administered (4 μg/h) to half of the rats for the last 4 weeks (n=6–8/group). ANGII increased blood pressure vs CON (165±5 vs 104±3 mm Hg, p<0.05), and this was reduced by RLX (135±13, p<0.05 vs ANGII and CON). NO bioavailability, assessed by urinary nitrate/nitrite excretion, was reduced during ANGII hypertension (0.8±0.2 vs 2.5±0.2 μmol/day, p<0.05) but restored by RLX treatment (1.8±0.4 μmol/day). NOS3 protein expression and phosphorylation on serine 1177 were comparable in all groups. While no changes were observed in expression of NOS1‐α among groups, NOS1‐β expression was reduced in the renal inner medulla of rats with ANGII hypertension (0.5±0.1 vs 1.0±0.3 RDU, p<0.05) but restored with relaxin treatment (0.8±0.2 RDU, p<0.05 vs ANG). We suggest RLX increases NOS1‐β in the inner medulla to promote NO‐dependent inhibition of sodium reabsorption in the collecting duct, thereby contributing to the lower blood pressure observed following RLX treatment during ANG II infusion.
Inhibition fibroblast growth factor receptor (FGFR) attenuates anti‐aging gene klotho deficiency induced hypertension
Authors: Schuman Mariano , Sun Zhongjie
Published: June 2021

Klotho is a newly‐discovered anti‐aging gene. Klotho (KL) gene delivery prevented progression of spontaneous hypertension and attenuated kidney damage. The purpose of this study is to determine if KL deficiency affects blood pressure and investigate the underlying mechanism. Two groups of wild‐type (WT) and 2 groups of KL heterozygous (+/−) mice were used. Blood pressure was elevated significantly in KL(+/−) mice vs the WT mice (mean BP 126 vs 103 mmHg), suggesting that KL gene deficiency causes hypertension. Plasma level of FGF23 was increased significantly in the KL(+/−) mice. We then inhibited FGF23 by a FGF receptor inhibitor (PD173074). One group of each strain received PD at 1 mg/kg/day (IP) for one week followed by 10 mg/kg for another week, while the other group received vehicle and served as a control. Following the 2‐week treatment, the mice were euthanized and blood, kidney, heart were collected. Distal convoluted tubules (DCT) were isolated from 1 kidney of each animal. PD significantly attenuated hypertension and cardiac hypertrophy. Interestingly, the level of urine norepinephrine was increased in KL(+/−) mice (KL(+/−) 490 ng/ml vs WT 350 ng/ml). PD abolished the KL deficiency‐induced increase in urine norepinephrine. PD also decreased expression of mineralcorticoid receptor (MR) in the DCTs.

Conclusions

Klotho deficiency causes hypertension and cardiac hypertrophy probably via upregulation of FGF23.
Metabolic Dysfunction in Chronic Intermittent Hypoxia‐ Role of Hypothalamic Peptides
Authors: Nedungadi T.Prashant , Mifflin Steve W , Cunningham J.Thomas
Published: June 2021

Obstructive sleep apnea (OSA) is associated with an increase in cardiovascular risk factors that includes metabolic syndrome and hypertension. Our model of Chronic Intermittent Hypoxia (CIH) mimics the hypoxemia and the persistently elevated arterial pressure seen in OSA patients. We examined if metabolic dysfunction associated with CIH is initiated through changes in neuropeptides in the arcuate nucleus (ARH) and paraventricular nucleus (PVN) of the hypothalamus. Adult male rats were exposed to 7 d of CIH or 7 d of normoxia (CON). Increases in body weight were higher in CON compared to CIH (CON: 25 ± 2.5; CIH: 16 ± 2.7). Urinary corticosterone (CORT) was elevated 6 fold in CIH exposed rats. Laser capture microdissection of ARH and PVN followed by RT‐PCR showed significant increase in ÄFosB mRNA in both PVN and ARH (PVN CON: 1± 0.1 vs PVN CIH: 1.96 ± 0.14; ARH CON: 1 ± 0.18 vs ARH CIH: 2.5 ± 0.5), and immunohistochemical observations suggest that CIH elevated FosB in both these regions. Neuropeptide Y (NPY) mRNA levels were elevated after CIH in ARH (CIH: 1.7 ± 0.5 vs CON: 0.3 ± 0.1). In PVN, Insulin receptor (InsR) mRNA was decreased while CORT releasing factor (CRF) mRNA was significantly elevated after CIH (InsR CIH: 1.1 ± 0.3 vs InsR CON: 2.9 ± 0.5; CRF CON: 1.8 ± 0.8 vs CRF CIH: 5.5 ± 1.1). Hence, metabolic dysfunction associated with CIH might be initiated centrally through peptides in the ARH‐PVN hypothalamic circuitry. P01 HL88052
Age‐dependent hypertension and renal sodium handling in the female intrauterine growth restricted rat
Authors: Paixao Ana Durce , Ojeda N B , Intapad S , Fahling J , Alexander B T
Published: June 2021

Hypertension in male offspring exposed to prenatal administration of glucocorticoids involves an increase in sodium transporter abundance. In a rodent model induced by placental insufficiency, female intrauterine growth‐restricted (IUGR) offspring are normotensive after puberty; however, there is an age‐dependent increase in blood pressure that occurs by one year of age, but the mechanism remains unknown. Thus, we hypothesized that an age‐dependent change in renal function and (Na+‐K+) ATPase activity may contribute to the development of hypertension in the female growth‐restricted rat. (Na+‐K+) ATPase activity in the basolateral membrane of the proximal tubule was similar in female growth‐restricted offspring relative to female control at one year of age (Control: 171±44 and IUGR: 76±18 nmol Pi/mg protein/min). Glomerular filtration rate also did not differ upon comparison of female growth‐restricted to female control (Control: 1.89±0.17 and IUGR: 1.57±0.17 ml/min/g kidney weight). In addition, hypertension at one year of age in female growth‐restricted offspring was not associated with changes in 24‐hour sodium or potassium excretion. Thus, these data indicate that alterations in renal sodium transport may not be an underlying mechanism of age‐induced hypertension in the female growth‐restricted rat. HL074927; HL51971; CAPES (Brazil)
REGULATION OF ADRENAL PHENYLETHANOLAMINE N‐METHYLTRANSFERASE GENE EXPRESSION IN A FETAL PROGRAMMING MODEL OF HYPERTENSION
Authors: Khurana Sandhya , Nguyen Phong , Grandbois Julie , Peltsch Heather , Lo Suet , Venkataraman Krishnan , Tai T.C.
Published: June 2021

Prenatal exposure to elevated levels of glucocorticoids (GC) programs for hypertension later in life. The current study examined the impact of prenatal GC exposure on the post‐natal regulation of the gene encoding for phenylethanolamine N‐methyltransferase (PNMT), the enzyme involved in the biosynthesis of the catecholamine, epinephrine. PNMT has been linked to hypertension and is elevated in animal models of hypertension. Results from the current study show that systolic, diastolic and mean arterial blood pressure were elevated in male WKY rat offspring (5 to 17 weeks of age) from dams treated with dexamethasone (DEX, +15%). Adrenal PNMT mRNA was elevated (1.5‐fold) in prenatally DEX‐exposed rats at 17 wks. Analysis of transcriptional regulators of the PNMT gene show that prenatal GC exposure increased mRNA levels of Egr‐1, AP‐2 and GR. Similar increased expression of PNMT protein, along with increased Egr‐1, AP‐2, and GR protein levels were also observed. Furthermore, GMSA showed increased binding of Egr‐1 and GR to the PNMT promoter. These results suggest that increased PNMT gene expression via altered transcriptional activity is a mechanism by which prenatal GC insult may program for hypertension later in life. (Research Funded by CIHR)
Curcumin and analog 2a inhibit â‐catenin pathway and ADPKD cyst growth
Authors: Bello‐Reuss Elsa , Bacallao Robert Louis , Herbert Brittney‐Shea , Lee Moses
Published: June 2021

Inhibition of signaling pathways is actively pursued for ADPKD treatment. Since cyst growth involves many proliferative pathways, multiple‐pathway inhibition could be advantageous. Curcumin inhibits cell‐proliferation pathways activated both in cancer and in ADPKD cyst cells, yet no therapeutic levels are achieved by oral (PO) administration due to poor intestinal absorption and liver inactivation. We studied (a) the effects of Curcumin and Analog 2a (which lacks glucuronidation sites, hence increasing absorption) on proliferation and cyst formation by ADPKD cyst cells in primary cultures and in a telomerase immortalized cell line and on active β‐catenin levels in both primary and cell line cultures; (b) the effects of PO administration of curcumin on cyst development in nude‐mouse implants; (c) the blood and kidney levels of both drugs after PO administration to mice. We found that: 1. Curcumin and Analog 2a were more cytotoxic to cyst cells than to normal kidney cells. 2. Curcumin and Analog 2a inhibited active β‐catenin in cyst cells. 3. Curcumin, undetectable in mouse blood after PO administration, decreased cyst volume in the implants, but not cyst number. 4. Analog 2a was detectable in mouse blood and concentrated in the kidney after PO administration. We conclude that Analog 2a increased effectiveness, decreased toxicity to normal cells and improved bioavailability support the use of new curcumin analogs for ADPKD treatment.
Pendrin gene ablation reduces ENaC surface expression and open probability
Authors: Pech Vladimir , Verlander Jill W , Nanami Masayoshi , Hummler Edith , Rossier Bernard C , Eaton Douglas C , Wall Susan M , Bao Fang H
Published: June 2021

Epithelial sodium channels, ENaC, mediate Na+ absorption and modulate Cl− absorption in the cortical collecting duct (CCD). Since ENaC function is reduced in pendrin KO mice given aldosterone, we determined if pendrin modulates ENaC open probability (PO) and/or surface expression. Pendrin KO and wild type mice were given aldosterone by minipump. ENaC PO and density were measured in split open CCDs. ENaC subcellular distribution was determined by quantitative immunohistochemistry. Transepithelial voltage (VT) and Clabsorption (JCl) were measured in CCDs perfused in vitro. Apical membrane ENaC abundance was lower in pendrin null than in wild type mice in the CCD, although total protein abundance was similar. Single channel recordings showed that both channel number and PO were reduced in pendrin null mice. Benzamil‐sensitive JCl depends on ENaC. We examined the magnitude of benzamil‐sensitive VT and JCl. Both total and benzamil‐sensitive JCl and VT were reduced in pendrin null mice. However, increasing apical ENaC expression by introducing the Liddle's mutation into the pendrin null mouse raised JCl and VT to levels observed in wild type mice. In conclusion, pendrin regulates ENaC activity through regulating both ENaC trafficking and the channel open probability. Constitutive expression of ENaC on the apical membrane by introduction of the Liddle's mutation, restores JCl and VT in the CCD in pendrin null mice.
CFTR and V‐ATPase trafficking and function in the intestinal brush border membrane
Authors: Collaco Anne , Geibel Peter , Kravtsov Dmitri , Ghantaphang Sayali , Lee Beth , Geibel John , Ameen Nadia
Published: June 2021

The multi‐subunit proton pump, vacuolar ATPase (V‐ATPase) and the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel are both regulated by cAMP‐dependent traffic from endosomes to the plasma membrane in ion transporting epithelia. We hypothesized that V‐ATPase traffic and function are linked to CFTR in the intestine. The distribution of V‐ATPase Voa1, Voa2 and V1E subunits and CFTR were examined in wild‐type (WT), CFTR−/− mouse intestine and polarized intestinal CaCo‐2 BBe cells following cAMP stimulation. Functional dependence of V‐ATPase on CFTR was determined in CaCo‐2 BBe cells using the pH sensitive dye BCECF. Co‐immunoprecipitation confirmed Voa1, Voa2 and V1E interaction with CFTR. V‐ATPase Voa1 and Voa2 subunits were localized to the apical and basolateral domains in CaCo‐2 BBe cells. V‐ATPase Voa1 and Voa2 subunits were localized to distinct domains in untreated and treated enterocytes from WT and CFTR−/−intestine. Following cAMP activation, both transporters co‐localized in the apical membrane. Inhibition of V‐ATPase or CFTR significantly decreased proton efflux in CaCo2 BBe cells. These studies suggest functional synergy between both transporters in maintaining intracellular pH; and localization in the intestinal brush border membrane. NIH Grants: R01‐DK‐077065 to N. Ameen and P30‐DK‐34989 to the Yale Liver Center.
Membrane residence time and degradation of KCa3.1 in polarized Fischer rat epithelial (FRT) cells
Authors: Lee Hamilton Kirk , Lee Shih‐Liang (Bob) , Winter Barclay , Balut Corina M. , Devor Daniel C.
Published: June 2021

In this study, we examined the membrane localization, residence time and degradative pathway of KCa3.1, for the first time in a polarized epithelium. To determine the membrane localization of KCa3.1‐BLAP, FRT cells were grown on filters and incubated with either apically‐applied streptavidin or basolaterally‐applied streptavidin. Untransfected cells were used as a negative control. We demonstrated, that KCa3.1‐BLAP was trafficked exclusively to the basolateral membrane of the cells (n=5, both IF and IB). Next, we examined the residence time of KCa3.1 in which FRT cells were incubated with basolaterally‐applied streptavidin and returned back to 37C for 0, 1, 3, 5, 8 and 12 hours. After 2 hours, membrane KCa3.1‐BLAP channels were localized into intracellular vesicles (by IF, n=5) and by 3–4 hours the presence of intracellular KCa3.1‐BLAP was reduced by half (by IB, n=5). Finally, to determine the degradative pathway of KCa3.1‐BLAP, we examined the effects of the lysosomal inhibitors, leupeptin (100 μM) and pepstatin (1μg/ml), on the intracellular fate of KCa3.1‐ BLAP. The presence of leupeptin and pepstatin reduced the degradation of KCa3.1‐BLAP suggesting the channel is degraded via a lysosomal‐dependent pathway (IB, n=5). Our results clearly demonstrate that KCa3.1 is trafficked exclusively to the basolateral membrane of polarized FRT cells, the channels have a half‐life of ~4 hours and that the channels are degraded in the lysosome. This work was supported by the Department of Physiology, Univ. of Otago (KLH) and NIH (DCD).
Detecting Structural Similarity between BK Binding Domains on Iberiotoxin and ASIC1 at Different pH
Authors: Snitsarev Vladislav , Abdulsalam Safanah S , Petroff Elena
Published: June 2021

Previous studies have shown that acid sensing ion channels (ASICs) inhibit calcium‐ and voltage‐activated potassium channels (BK) at physiological pH, and the inhibition is removed at acidic pH (Petroff et al. PNAS 2008; Petroff et al. BBRC 2012). The overlay capabilities of ChemBio3D Ultra 2010 (CambridgeSoft, Cambridge, MA) were used to detect structural similarities between putative BK‐binding domain in ASIC (R191G193K194) and known iberiotoxin (IbTx) domain critical for BK‐binding (R25G26K27).

We compared the structure of (R191G193K194) domain of ASIC1 at low pH (PDP ID: 2QTS) to the structure of IbTx (R25G26K27) domain obtained from Swiss‐Model Repository. We also overlaid the structure of (R191G193K194) domain of ASIC1 at pH of 7.5 (PDB ID: 3S3W) against IbTx binding domain (R25G26K27). Our results show that at pH 7.5, ASIC1's putative BK‐binding domain (R191G193K194) is structurally more similar to the IbTx BK‐binding domain (R25G26K27) than at low pH.

These in silico findings support the previous experimental studies and indicate that the structure of ASIC1 at neutral pH could favor interaction with BK, while at low pH, the conformational changes in ASIC1 make it less likely. This work is supported by the NIH NS070260 grant to EP.

Overlaid R191G193K194 domain of ASIC1 at neutral pH against IbTx's BK‐binding domain (R25G26K27). The distance between the central carbon atoms of each of the residues is shown.

Residues
Distance (Å)

ASIC1 ‐ IbTx

R191 ‐ R25
1.0

G193 ‐ G26
0.9

K194 ‐ K27
0.4

Overlaid R191G193K194 domain of ASIC at low pH against IbTx's BK‐binding domain (R25G26K27). The distance between the central carbon atoms of each of the residues is shown.

Residues
Distance (Å)

ASIC1 ‐ IbTx

R191 ‐ R25
2.5

G193 ‐ G26
2.5

K194 ‐ K27
2.0
Acid evoked coughing in anesthetized guinea pigs depends upon Acid‐Sensing Ion Channel (ASIC)‐dependent activation of capsaicin‐insensitive vagal afferent neurons
Authors: Canning Brendan James , Kollarik Marian , Meeker Sonya , Mori Nanako , Undem Bradley J
Published: June 2021

Citric acid (CA) evoked cough in anesthetized guinea pigs (GPs) depends upon the activation of nodose ganglia neurons that are acid‐sensitive but capsaicin‐insensitive and project to the laryngeal, tracheal and mainstem bronchial mucosa. Using single cell PCR and diminazene (DZ), a potent and selective inhibitor of ASICs, we have addressed the hypothesis that acid evoked cough in anesthetized guinea pigs is ASIC dependent. In vitro, DZ inhibited 1mM CA evoked action potential (AP) discharge in the capsaicin‐insensitive afferent neurons projecting to the trachea (CA evoked 44±7, 36±9, 15±4 and 1±1 APs in control preparations and following treatment with 1, 10 and 100μM DZ, respectively; n=3–5). In vivo, DZ inhibited cough evoked by CA (0.001–2M) applied topically to the tracheal mucosa (CA evoked 8±1, 6±1, 3±2 and 0±0 coughs in control and in GPs pretreated topically with 1, 10 and 100μM DZ, respectively; n=4–8). By contrast, the TRPV1 antagonist SB366791 (10μM) nearly abolished 0.1μM capsaicin‐evoked contractions of the GP trachea in vitro but was without effect on CA evoked cough (8±1 coughs; n=5). Single cell PCR revealed that nodose ganglia neurons retrogradely labeled from the trachea do not express TRPV1 mRNA (0/9) but do (8/9) express either ASIC1 (6/9) and/or ASIC3 (5/9) mRNA. Together, these data implicate ASICs in the acid evoked coughing evoked from the airway mucosa.
Pulmonary lipidosis due to Niemann‐Pick Type C1 (NPC1) deficiency in mice
Authors: Roszell Blair R. , Tao Jian‐Qin , Ning Yue , Yu Kevin J. , Gao Ling , Huang Shaohui , Feinstein Sheldon I. , Bates Sandra R.
Published: June 2021

NPC1, a member of the NPC pathway that facilitates the release of cholesterol from lysosomes, is also found in the limiting membrane of alveolar type II cell (AT2) lamellar bodies (LB). Cholesterol is critical for surface viscosity of pulmonary surfactant but excess cholesterol interferes with surfactant function. Patients with NPC disease and gene‐targeted NPC2 mutant mice exhibit cholesterol accumulation in lysosomes and lung alveolar proteinosis. The goal of this study was to examine the lung disease in the gene‐targeted NPC1‐null mice using histological and biochemical analysis. Compared to wild type (WT) mice, NPC1 mouse lungs were characterized by: a) areas of both severe and mild pathology with thickened septae, excess surfactant in the alveoli, and alveolar macrophages containing surfactant‐like material; b) a 1.5‐fold elevation in phospholipid (PL) and cholesterol in lung tissue; a 4‐ fold increase in PL and a striking increase in cholesterol (40‐fold) in the alveolar surfactant and isolated LBs; and c) a marked increase (160%) in mean area size of the LBs, in contrast to the reduction in LB size in NPC2 mutant mice (50% decrease vs. WT). Thus, an absence of NPC1 protein results in abnormal lung morphology and surfactant composition, indicating that the activity of NPC1 protein is important for the maintenance of normal lung homeostasis. [HL 19737 and NRSA T32–07748]
Role of GADD34 in age‐related differences in hyperoxiainduced stress signaling
Authors: Shenberger Jeffrey , Wu Yuchieh Melissa
Published: June 2021

Hyperoxic exposure activates an adaptive signal transduction pathway in the lung known as the integrated stress response (ISR), which promotes the expression of factors that modulate redox balance, the immune response, and DNA repair. Given that newborns (NB) are more tolerant to the toxic effects of O2 than adults, we conducted the presence study to explore age‐related differences in ISR signaling within the lung. In adult (AD) mice exposed to 95% O2 (Ox), phosphorylated eIF2α, the initiating event of the ISR, increased within 24 hrs and declined by 72 hrs coincident with enhanced GADD34, ATF3, and CHOP expression. In NB, increased p‐2α was delayed until 72 hrs and coincided with lesser increases in GADD34 and ATF3. In MEF and mouse lung epithelial cells (MLE15), Ox increased p‐2α in MLE15 but decreased p‐2α in MEF. Assessment of GADD34/PP1 phosphatase complexes revealed that the amount of PP1 precipitating with GADD34 was 3‐fold greater in MEF. Ox also decreased the association of the PP1 inhibitor I‐1 with GADD34 in both cell types, while the inhibitor BiP was present only in MLE complexes. In animals, 72 hrs of Ox increased GADD34/PP1 association by 70% and decreased I‐1/GADD34 association by 87% in AD relative to NB. These findings show that age‐related and cell specific differences in extent and duration of Ox‐induced ISR activation are likely to be mediated by the composition of GADD34/PP1 phosphatase complexes.
TRPV4 Ca2+ sparklets in myoendothelial projections (MEPs) regulate vascular function
Authors: Sonkusare Swapnil K , Bonev Adrian D. , Dalsgaard Thomas , Santana Luis F , Kotlikoff Michael I , Nelson Mark T
Published: June 2021

Endothelial cells (ECs) lining blood vessels regulate vascular tone. MEPs connect ECs to adjacent smooth muscle cells (SMCs) via gap junctions. We discovered elementary Ca2+ signals (“sparklets”) through single TRPV4 (transient receptor potential vanilloid 4) channels at MEPs (Sonkusare et. al., Science, 2012). TRPV4 sparklets dilate 3rd order mouse mesenteric arteries (MAs) via EC intermediate‐conductance, Ca2+ sensitive K+ (IK) channels. The current study elucidates the mechanism by which muscarinic receptor stimulation dilates MAs via TRPV4 channels. Carbachol (CCh) increased sparklets (5‐fold) ONLY at MEPs, and not elsewhere on the EC membrane. Protein kinase C (PKC) activation similarly increased sparklets only at MEPs. CCh‐activation of sparklets was prevented by PKC inhibitor Go‐6976. Inward rectifier K+ (Kir) currents were present in ECs but not in SMCs. Dilations to CCh, TRPV4 agonist GSK1016790A, and IK/SK opener NS309 were inhibited (70%) by Kir channel blocker Ba2+. The results support a MEP‐localized signaling circuit of PKC‐TRPV4‐IK‐Kir channels which mediates vasodilatory input to the SM.
Serotonin (5‐HT) Receptor Expression and Function in the Kidney and Vasculature is Altered in the Streptozotocin (STZ) rat model of Diabetes Mellitus
Authors: Bhaskaran Subha , Singhal Sara , Banes‐Berceli Amy
Published: June 2021

Diabetes is characterized by vascular and kidney damage. Accumulating evidence suggests that vasoactive substances such as 5‐HT stimulate these pathological processes and plasma levels of 5‐HT are elevated in diabetes. In recent clinical studies 5‐HT2A receptor antagonists substantially decreased albuminuria in type 2 diabetic patients suggesting 5‐HT is mediating pathological processes in vivo. The molecular mechanisms behind the role of 5‐ HT and expression of 5‐HT receptors in vivo in normal and diabetic conditions are currently unknown. Utilizing male Sprague‐ Dawley rats in a type I model of diabetes we used Western blot and immunohistochemical (IHC) analyses to show a significant increase in the expression of 5‐HT2B and 5‐HT2A receptor proteins but not the 5‐HT2C receptor in the diabetic rat kidney. Furthermore, our ex vivo myograph data suggests an increase in the activation of the 5‐HT‐induced intracellular signaling that can stimulate a potentiated contractile response in both thoracic aorta and renal artery in type‐1 diabetic rats which may lead to cardiovascular complications. Investigating both the mechanisms of 5‐HT‐induced intracellular signaling, functional consequences and the expression of 5‐HT receptors, is critical because targeted inhibition may reduce the risk of both kidney damage and vascular abnormality; critical in the treatment for chronic diabetic patients. (NHLBI R00 AKLB)
Role of BKCa channels and PKC in mesenteric artery contractility of diabetic rats
Authors: Suárez Paola Algara , Pérez Cristóbal Joel González , Espinosa‐Tanguma Ricardo
Published: June 2021

The aim of this study was to analyze the effect of diabetes mellitus (DM), acting through protein kinase C (PKC), on vascular tone associated to BKCa channel activity in rat mesenteric artery.

Methods

DM was induced in male Wistar rats by intravenous injection of streptozotocin (STZ, 60mg/kg) and mesenteric artery rings were isolated for isometric force studies. The effects of PKC inhibitor Bisindolymaleimide I (Bis 1) and BKCa channel activator NS 1619 on phenylephrine induced contractions were evaluated and expressed as % force.

Results

NS1619 decreased maximal force in control but not in STZ rats (97.4 ± 16 % vehicle v.s. 81.6 ± 25% NS1619, p=0.011, n=7 for ctrl; 84 ± 13% v.s. 71.8 ± 36%, p=0.54, n = 6 for STZ). Bis1 decreased phenylephrine contraction in both control and STZ rats (61.8 ± 33%, p= 0.003, n =8 ctrl; 34 ± 12%, p=0.0001, n=6 for STZ); although inhibition was more prominent in STZ than in ctrl rats (p= 0.005). Relaxation due to nifedipine was higher for STZ (42±15% for ctrl v.s. 19.5 ±17 for STZ, p=0.031, n=6).

Conclusions

DM leads to elevated PKC and VOCC activity, probably through BKCa channel inhibition which is not reversible by NS1619.

Supported by: C12‐FAI‐03–60.60, UASLP
Inflammation and sexual dimorphism in development of insulin resistance: a dual animal model comparison
Authors: Wintrob Zachary A.P. , Oppong Emmanuel K. , Tse Yu C. , Welt Jeffery M. , Boateng Hans R. , Drumea Ioana , Martorana Michael , Irlam John , LeVea Charles M. , Faitar Silviu L. , Ceacareanu Alice C.
Published: June 2021

Severe combined immunodeficiency (SCID) and immunocompetent BALBc mice strains may constitute resourceful model‐candidates for revealing inflammation‐mediated mechanisms involved in high fat diet (HFD) induced insulin resistance (IR).

This study included 32 animals in groups of 4 for all combinations of male (M), female (F), BALBc, SCID, low fat diet (LFD, 4.1% fat), and HFD (35% fat) for a total of 21 weeks. All groups switched to 10% fat, between week 12 and 15. Biweekly fasting levels of adipokines, inflammatory cytokines and growth factors were assessed. IR was verified by glucose tolerance test at week 13.

By week 13 the HFD‐SCID‐F neither gained weight, nor developed IR; HFD‐BALBc‐F gained weight but did not develop IR, while all HFD‐Ms both gained weight and developed IR. Ultimately the HFD‐SCID‐F gained ~4g and had low insulin levels, while all other HFD groups gained ~10g and had 4–7 fold higher fasting insulin. Adiponectin (ADP) spiked at 3 weeks and remained elevated in HFD‐BALBc‐F. Switching food resulted in upward‐trending insulin reversing only in BALBc‐M and upward‐trending leptin decreased in all but SCID‐F. Liver pathology associated fatty liver disease with IR in HFD.

Concomitant evaluation of these models suggests a combined contribution of inflammation and sexual dimorphism in developing IR. Further research may clarify whether ADP is a mediator of this mechanism.
Green tea (−)‐epigallocatechin gallate inhibits IGF‐I and IGF‐II stimulation of glucose uptake in 3T3‐L1 adipocytes
Authors: Shih Li‐Jane , Ku Hui‐Chen , Tsuei Yi‐Wei , Weng Jueng‐Tsueng , Tsai Shu‐Wei , Lin Ching‐Ling , Kuo Yow‐Chii , Kao Yung‐Hsi
Published: June 2021

Objective

This study investigated the pathways involved in EGCG modulation of IGF‐stimulated glucose uptake in 3T3‐L1 adipocytes.

Methods

Glucose uptake was assayed to measure the uptake of 3H‐2‐deoxyglucose. Western blot analysis was performed to measure levels of Glucose transporters (GLUTs) and IGF signaling molecules.

Results

EGCG inhibited IGF‐I and IGF‐II stimulation of adipocyte glucose uptake in dose‐ and time‐dependent manners. Treatment with 20 μM EGCG for 2 h significantly decreased IGF‐I‐ and IGF‐II‐stimulated glucose uptake by 59% and 64%, respectively. The EGCG receptor [also known as the 67‐kDa laminin receptor (67LR)] was discovered in fat cells. Pretreatment of adipocytes with a 67LR antibody, but not normal rabbit immunoglobulin, prevented the effects of EGCG on IGF‐increased glucose uptake. Moreover, pretreatment with an AMP‐activated protein kinase (AMPK) inhibitor, such as compound C, but not with a glutathione activator, such as N‐acetyl‐L‐cysteine, blocked the anti‐IGF effect of EGCG on adipocyte glucose uptake. Further analysis for subcellular fractions indicated that EGCG decreased the IGF‐induced increases in the GLUT4 translocation from the cytosol to the plasma membrane and that EGCG had no effects on the level of GLUT1 translocation and total amounts of GLUT‐1 and GLUT‐4 proteins. In addition, EGCG suppressed the IGF‐stimulated phosphorylation of PKCζ/λ, but not Akt or ERK, proteins.

Conclusions

These results suggest that EGCG inhibits IGF‐I and IGF‐II stimulation of 3T3‐L1 adipocyte glucose uptake through downregulated GLUT‐4 translocation and IGF signaling and through 67LR‐ and AMPK‐dependent pathways.
Estrogen protects cardiomyocytes against oxidative stress by upregulation of cystathionine γ lyase expression
Authors: Zhu Xiao‐Yan , Wang Long , Cong Binhai , Zhao Wei , Ni Xin
Published: June 2021

The mechanisms of cardioprotective effects of estrogen are not fully understood. Hydrogen sulfide (H2S) is generated predominantly by cystathionine γ lyase (CSE) in myocardium, and has been implicated to have cardioprotective effects. Thus, we hypothesize that endogenous H2S contributes to the cardioprotective effects of estrogen. We found that 17β‐estradiol (E2) increased CSE expression in primary cultured neonatal cardiomyocytes, which was blocked by nonselective estrogen receptor (ER) antagonist ICI 182780 and ERα antagonist MPP, but not by ERβ selective antagonist THC. E2 increased cell survival, decreased caspase‐3 activation and increased Akt phosphorylation in the cells upon hydrogen peroxide treatment. The protective effects of E2 were blocked by either CSE specific inhibitor PAG or knockdown of CSE expression with siRNA. E2‐induced CSE expression was abolished by the protein synthesis inhibitor cycloheximide, suggesting requirement of additional factors. E2 significantly increased Sp1 expression. Importantly, siRNA‐mediated knockdown of Sp1 expression markedly inhibited E2‐mediated upregulation of CSE expression. In conclusion, our data suggest that the cardioprotection of estrogen against oxidative stress is through, at least in part, stimulating CSE expression in cardiomyocytes. CSE induction upon E2 treatment is mainly due to the increased expression of Sp1.
Electrophilic nitro‐fatty acids inhibit vascular inflammation
Authors: Villacorta Luis , Chang Lin , Salvatore Sonia R. , Petrovic‐Djergovic Danica , Zhang Jifeng , Schopfer Francisco J. , Freeman Bruce A. , Chen Y. Eugene
Published: June 2021

Aims

Electrophilic nitroalkene derivatives, products of unsaturated fatty acid nitration, exert long‐term cardiovascular protection in experimental models of metabolic and cardiovascular diseases. The goal of this study is to determine whether low‐dose acute administration of nitro‐fatty acids reduces vascular inflammation in vivo.

Results

Preemptive treatment with nitro‐oleic acid (OA‐NO2), but not oleic acid (OA) inhibits lipopolysaccharide (LPS)‐induced nuclear factor‐κB (NF‐κB) activation both in vivo. An acute treatment with OA‐NO2 in vivo yielding nanomolar concentrations in plasma, is sufficient to inhibit LPS‐induced Toll‐like receptor 4 (TLR4)‐induced cell surface expression in leukocytes, NF‐κB activation and leukocyte recruitment to the vascular endothelium assessed by intravital microscopy.

Conclusions

These studies demonstrate that acute administration of nitro‐fatty acids is effective to reduce vascular inflammation in vivo. These findings reveal a direct role of nitro‐fatty acids in the disruption of the TLR4 signaling and upstream events of the NF‐κB pathway, leading to resolution of pro‐inflammatory activation of NF‐κB in the vasculature.
Functional significance of hsp90‐eNOS interaction in penile tissues
Authors: Yetik‐Anacak Gunay , Ertuna Elif , Ozsarlak‐Sozer Gonen , di Pascoli Saadet Turkseven , Koylu Ersin , Gozen Oguz , Sevin Gulnur , Arun Mehmet , Un Ismail
Published: June 2021

Heat shock protein 90 (hsp90) regulates the NO‐cGMP pathway in several vascular tissues. In the present study, we investigated the existence and functional significance of hsp90‐eNOS interactions in murine penile tissues. Utilizing double‐labeled anti‐eNOS and anti‐hsp90 antibodies we observed co‐localization of hsp90 and eNOS by immunofluorescence in mouse corpus cavernosum (MCC). Additionally, hsp90‐eNOS interaction was demonstrated in co‐immunoprecipitation studies. To study the functional significance of these interactions, we examined the sensitivity of eNOS‐dependent relaxation of MCC to hsp90 inhibitors, radicicol and 17‐AAG. Pretreatment with radicicol (10 μg/ml) or 17AAG (400 nM) for 15 hours disrupted the hsp90‐eNOS complex (as demonstrated by co‐immunoprecitation experiments) and significantly reduced the acetylcholine‐, bradykinin‐ and sphingosine‐induced eNOS‐dependent relaxation of pre‐contracted MCC. Furthermore, 17‐AAG decreased MCC constriction induced by the eNOS inhibitor, LNA. Endothelium‐independent relaxations induced by papaverine or NTG were not affected by hsp90 inhibition, suggesting the absence of sGC regulation by hsp90 in MCC. We conclude that hsp90 forms complexes with eNOS and that it regulates both basal and agonist‐evoked eNOS activation in MCC. (Supported by TUBA GEBIP‐2010, TUBITAK 109S453, EBILTEM 2011/BIL/002 and FABAL).
Role of peroxiredoxin‐1 in regulation of PKG dimerization associated with relaxation to hydrogen peroxide in bovine pulmonary arteries
Authors: Patel Dhara , Kelly Melissa , Kandhi Sharath , Wolin Michael S
Published: June 2021

Previous studies from our laboratory have suggested that thiol oxidation‐elicited dimerization‐activation of the 1α form of protein kinase G (PKG1α) contributes to the relaxation of isolated endothelium removed bovine pulmonary arteries (BPA) to peroxide and responses to hypoxia. We depleted peroxiredoxin‐1 by using siRNA to determine if peroxide metabolism by peroxiredoxin‐1 plays a role in the dimerization‐activation (based on increased VASP phosphorylation) of PKG1α. Hypoxic conditions were used to minimize basal PKG dimerization. Depletion of peroxiredoxin‐1 was observed to attenuate PKG dimerization‐activation (based on decreased VASP phosphorylation). However, peroxiredoxin‐1 depletion did not inhibit PKG dimerization‐activation elicited by inhibiting glucose‐6‐phosphate dehydrogenase (G6PD) with 6‐aminonicotinamide (6‐ AN). 6‐AN promotes NADPH oxidation and a dimerization‐activation of PKG via a mechanism potentially independent of peroxide. Thus, peroxiredoxin‐1 appears to participate in the PKG‐dimerization mediated relaxation to peroxide, but, it does not seem to participate in controlling the NADPH oxidation‐ and PKG‐dimerization associated relaxation to 6‐AN.
P21‐activated kinase inhibits vascular reactivity via inhibition of MLCK
Authors: Chu Ji , Bhattarai Deepa , Cox Charles S , Uray Karen
Published: June 2021

Changes in vascular reactivity contribute to the pathology of a number of different diseases including heart failure, hypertension, severe trauma, and sepsis. P21‐activated kinase (PAK) can alter smooth muscle contractile activity and has been implicated in altering vascular reactivity. The objective of this study was to determine the mechanism by which PAK regulates MLC phosphorylation in vascular smooth muscle to alter vascular reactivity. The effects of PAK activation and inhibition on basal and agonist‐induced aortic contraction in aortic rings and on myosin light chain (MLC) and myosin targeting subunit of MLC phosphatase (MYPT1) phosphorylation and MLC kinase (MLCK) activity in primary aortic smooth muscle cells (ASMC) were determined. Finally, the effects of increased mechanical stimulation on PAK activation were determined in ASMC. PAK activation inhibited both basal tone and agonist induced contraction of rat aortic rings. Conversely, PAK inhibition increased agonist induced contraction and basal tone. In ASMC, PAK activation inhibited MLC phosphorylation and PAK inhibition increased MLC phosphorylation. However, PAK activation increased MYPT1 phosphorylation in ASMC. Increased mechanical stimulation of ASMC increased PAK activation and decreased MLCK activation. These data suggest that PAK inhibits vascular reactivity via decreased inhibition of MLCK.
Non‐muscle (NM) and Smooth Muscle (SM) Myosin II Activation are Regulated by Different Mechanisms During Contractile Stimulation of Airway Smooth Muscle (ASM)
Authors: Zhang Wenwu , Wu Yidi , Gunst Susan J
Published: June 2021

Myosin II is the major motor protein in SM, and is expressed as both SM and NM isoforms. However, the function of non‐muscle myosin II in SM is not understood. We hypothesized that NM and SM myosin II may serve different functions during ASM contraction and be regulated by different mechanisms. We detected both NM myosin IIA and IIB heavy chain isoforms in canine ASM tissues. The 20 kD myosin light chain (MLC) is expressed in both NM and SM isoforms, but these isoforms cannot be distinguished immunologically. We used 2‐D gel electrophoresis to distinguish NM and SM MLC isoforms and analyze their phosphorylation in response to acetylcholine (ACh). NM MLC represented a relatively small proportion of the total cellular MLC (20–30%). Both SM and NM MLCs were phosphorylated in similar proportions in response to ACh stimulation (about 50%). When RhoA was inactivated by expressing the inactive RhoA mutant, RhoA T19N, in muscle tissues, NM MLC phosphorylation was substantially inhibited but there was little effect on SM MLC phosphorylation. The MLC kinase inhibitor, ML‐7, suppressed ACh induced SM myosin II LC phosphorylation, but had little or no effect on NM myosin II MLC phosphorylation. We conclude that MLC kinase primarily regulates the phosphorylation of SM MLC, whereas NM MLC phosphorylation depends on RhoA activation. This suggests SM and NM myosin II play distinct roles in the regulation of SM contraction.

HL29289, HL074099.
Nitric Oxide Stress uncovers pM b2‐adrenergic mediated dilation to isoproterenol mimicked by preventing clathrin endosome formation.
Authors: Frame Mary (Molly) , Calizo Bonnie , Dewar Anthony M , Scarlata Suzanne
Published: June 2021

Nitric oxide exposure induces an oxidative/nitrosative stress (NOX) causing endothelial dysfunction. Our goal was to determine how this stress impacted vasoactive responses to isoproterenol. Intravital microscopy of the hamster cheek pouch was performed (N=20; pentobarbital 70mg/kg i.p.). NOX was induced by applying nitroprusside (10–4M) for 2 minutes via tissue bath. Before NOX, dilation to micropipette isoproterenol was dose dependent (EC50 10–7M). After NOX, dilation was attenuated in the uM range, and a second dilation appeared as a narrow peak from 10–12 to 10–9M, which was inhibited by the b2‐adrenergic receptor (bAR‐2) inverse agonist, carazolol. Preventing clathrin endosome formation with dynasore attenuated dilation to isoproterenol (uM range), and uncovered dilation (pM range), thus mimicking NOX. Dynasore with NOX further increased the low dose potency and efficacy of dilation to isoproterenol (10–14 to 10–9M). To understand the mechanism, we tested whether the fluorescent adrenergic agonist, curcumin, and the bAR‐2 were co‐internalized using FRET in HeLa cells. Before NOX, curcumin is rapidly internalized and co‐localized with the bAR‐2. Moderate NOX delayed co‐localization, while extensive NOX prevented bAR‐2 internalization altogether. Thus, NOX prevented bAR‐2 internalization, and uncovered a low dose dilation to bAR‐2 linked activity. AHA 0655908T