bluuebunny/crossref_metadata_2025 huggingface dataset:
Pro‐osteolytic effects of chronic morphine administration in a model of breast cancer‐induced bone pain.
Authors: Symons‐Liguori Ashley M , Sayers Nicole Marie , Vanderah Todd W
Published: June 2021

Many common cancers have a high propensity for bone metastasis; for breast cancer, bone metastasis affects 70% of advanced patients and is the most common site for secondary tumor relapse. Tumor manifestation disrupts bone homeostasis and results in debilitating chronic skeletal pain that decreases patient functional status, quality of life and survival. Morphine remains the golden standard for managing cancer‐induced bone pain; however, opioids including morphine are associated with a host of side effects that complicate chronic dosing schedules. Furthermore, it has been posited that morphine may accelerate bone wasting in patients; in models of secondary bone metastasis, morphine exacerbates fracture rate, markers of osteolysis, and pain behaviors.

We sought to determine whether morphine exacerbates bone loss in the 66.1‐Balb/C model of breast cancer‐induced bone metastasis (BCBM). Sham and tumor‐inoculated animals received a constant morphine infusion for 7 days (20 mg/kg/day). Morphine produced initial analgesia but failed to manage behaviors indicative of cancer‐induced bone pain over time; additionally, morphine treatment increased TRACP 5b in sera and RANKL in femur extrudate, suggesting that morphine enhances osteolysis in BCBM. In vitro studies evaluated the ability of morphine to induce osteoclastogenesis in the presence of permissive levels of RANKL. Morphine did not appear to affect tumor burden in vivo or in vitro viability of tumor cells. These data suggest that morphine worsens bone loss in the 66.1‐Balb/C model and may act directly on osteoclast cells; however, the exact mechanism of morphine‐induced bone loss is unknown and remains a barrier to improving pain relief in suffering BCBM patients.
Effect of cytochrome P450 isozyme inhibitors on 3,5‐ dichloroaniline nephrotoxicity in vitro
Authors: Rankin Gary O. , Racine Christopher , Ferguson Travis , Preston Deborah , Anestis Dianne
Published: June 2021

Chlorinated anilines are used in making agricultural chemicals, dyes, industrial compounds, and pharmaceuticals, and can induce nephrotoxicity in vivo and in vitro. 3,5‐Dichloroaniline (3,5‐DCA, 1.0 mM) induced nephrotoxicity in isolated rat renal cortical cells (IRCC) following 90 min exposure. IRCC pretreated with non‐selective cytochrome P450 (CYP) inhibitors partially attenuated 3,5‐DCA toxicity, suggesting that CYPs may play a role in 3,5‐ DCA bioactivation. The purpose of the this study was to further explore the role of CYP mediated 3,5‐DCA bioactivation. IRCC were obtained from male Fischer 344 rats and incubated (4 ×106 cells/ml; 3mL) with shaking (90 min) with dimethyl sulfoxide or 3,5‐DCA (1.0mM). IRCC were pretreated with various CYP inhibitors [isoniazid (1.0 mM), ketoconazole (0.1 mM), omeprazole (0.01 mM), diethyldithiocarbamate (DEDTCA; 0.1 mM), oleandomycin triacetate (0.5 mM), or sulfaphenazole (0.1 mM)] and cytotoxity was determined by measuring lactate dehydrogenase release. Pretreatment with DEDTCA, omeprazole, or sulfaphenazole partially attenuated 3,5‐DCA induced nephrotoxicity, while ketoconazole, isoniazid, oleandomycin triacetate had no effect. These results suggest that 3,5‐DCA is bioactivated via multiple pathways, including the CYP2C family. (Supported by NIH Grant 8P20GM103434 to West Virginia IDeA Network for Biomedical Research Excellence)
Tissue‐distribution of claudin‐3/‐4 binder, the C‐terminal fragment of Clostridium perfringens enterotoxin, in mice
Authors: Li Xiangru , Kondoh Masuo , Watari Akihiro , Yagi Kiyohito
Published: June 2021

The C‐terminal fragment of Clostridium perfringens enterotoxin (C‐CPE) is the most well‐characterized claudin‐3 and ‐4 (CL‐3 and ‐4) binder. The mucosal absorption‐enhancing activity of C‐CPE is over 400‐fold more potent than a clinically used enhancer. C‐CPE has potential in cancer therapy and for mucosal vaccination. Since CL‐3 and ‐4 are ubiquitously expressed in heart, lung, liver, kidney, thyroid gland, stomach, and intestine, safety evaluations are important for their future therapeutic use. Here, we investigated the tissue distribution of C‐CPE in mice after intravenous administration. We prepared CF750‐labeled wild‐type C‐CPE and C‐CPE harboring mutant CL‐3/‐4 binding regions. FACS analysis showed that labeled wild‐type C‐CPE bound to CL‐4 but that labeled mutant C‐CPE did not, indicating that labeling did not alter the CL‐binding properties of C‐CPE. Most wild‐type and mutant C‐CPE promptly accumulated in the kidney, and C‐CPE were more distributed in the liver, thyroid gland and intestine than mutant C‐CPE 10 min after administration. The distribution of C‐CPE gradually decreased in the liver, thyroid gland and intestine, but the distribution of C‐CPE increased in the kidney until 48 h after administration. These findings suggest that the influence of CL‐3/‐4 binders on the liver, thyroid gland, intestine and kidney should be considered for future therapeutic applications.
Regulation of flow‐induced intracellular NO levels by endothelial surface glycocalyx
Authors: Fu Bingmei M , Yen Wanyi , Yang Jinlin , Zeng Min , Tarbell John M
Published: June 2021

To test the hypothesis that endothelial surface glycocalyx (ESG) plays a role in mechanosensing and transduction of the microvessel wall, we used fluorescence microscopy to measure the NO levels in the post‐capillary venules of rat mesentery under low and high flow conditions and with/out enzyme treatment for removing heparan sulfate (HS) of the ESG. Rats (SD, 250–300g) were anesthetized with pentobarbital sodium given subcutaneously, the mesentery was gently taken out from the abdominal cavity and arranged on the surface of a glass coverslip for the measurement. After perfusion for 1 hr with 1% BSA Ringer for the control or with 50 mU/mL heparanase III for the treatment, an individual post‐capillary venule (35–50 μm) was loaded for 45 min with 5 μM 4, 5‐Diaminofluorescein diacetate, a membrane permeable fluorescent indictor for NO, then the NO was measured for ~10min under a low flow (< 300 μm/s) as the baseline and for ~60min under a high flow (1500–2000 μm/s mean velocity). In 1 min after switching to the high low, NO increased by 1.14 ± 0.05‐fold (n=3) under control and 1.15 ± 0.04‐fold (n=3) under enzyme treatment. NO continuously increased after 1 min under high flow, reached a plateau of 1.42 ± 0.02‐fold in ~40 min under control and of 1.22 ± 0.03 in ~15min under treatment. The results suggest that ESG participate in EC mechanosensing and transduction through its HS and other components. Supported by NIH R01HL094889–01.
Alcohol intoxication increases microvascular permeability through inactivation of Rac1 and disruption of VE‐cadherin organization
Authors: Doggett Travis , Breslin Jerome
Published: June 2021

We tested the hypothesis that alcohol increases endothelial permeability by inactivating Rac1 and disrupting VE‐cadherin localization. Conscious rats received 2.5 g/kg alcohol or isovolumic water via pre‐implanted gastric catheters. After 30 min, they were anesthetized, prepped for intravital microcopy of the mesenteric microcirculation, and the integrated optical intensity (IOI) of extravasated FITC‐albumin was determined. Transendothelial electrical resistance (TER) of human umbilical vein endothelial cell monolayers also served as an index of barrier function. VE‐cadherin localization was determined by immunofluorescence labeling or in live cells expressing VE‐cadherin‐GFP. Rac1‐GTP was determined by ELISA. The results show that alcohol increases microvascular leak (change in IOI 33.0 ± 16.1 for alcohol vs. 2.86 ± 0.7 for water, p<0.05), and decreases TER when applied at >;20 mM. Alcohol also disrupted VE‐cadherin continuity and significantly decreased Rac1‐GTP (53% ± 5 of control, p<0.01). Addition of 100 μM 8‐CPT‐2′‐O‐Me‐cAMP, an indirect activator of Rac1, at 5 or 10 min post alcohol caused a significantly faster return to baseline TER (respectively 14.9 ± 0.5 and 19.5 ± 0.4 min vs. 59.1 ± 1.5 without activator, p<0.01). The data suggest that alcohol increases endothelial permeability by inactivating Rac1 and disrupting VE‐cadherin at junctions. Supported by NIH R21AA020049 and T32AA007577.
Vasopressin increases blood pressure without inducing pulmonary hypertension in a Sus scrofa model of hemorrhage
Authors: Sarkar Joy , Uyehara Catherine FT , Ichimura Wayne M , Murata Lee‐Ann M , Sato Aileen K , Hernandez Claudia A , Allen Christopher L
Published: June 2021

Distinctive roles of vasoactive hormones in severe hemorrhagic shock are unclear. We recently found a blunted vasopressin (VP) response associated with a higher oxygen debt and an increased pulmonary to systemic vascular resistance (PVR/SVR) ratio in non‐survivors of acute hemorrhage compared to survivors. Hence, we tested the hypothesis that in regulating blood pressure after acute hemorrhage, VP may have distinct effects on pulmonary and systemic hemodynamics, unlike norepinephrine (NE) and phenylephrine (PE). Anesthetized Yorkshire pigs (n=18, 32.3±4.5 kg body wt) underwent rapid hemorrhage (1 to 3 ml/kg/min; 35±5 ml/kg shed volume, 44% mean arterial pressure (MAP) decrease), followed by resuscitation with either normal saline (NS), or NS in combination with VP, NE, or PE titrated to baseline MAPs. All groups had an increase in PVR/SVR during hemorrhage. Administration of NS and PE did not change PVR/SVR. NE further increased PVR/SVR, and induced pulmonary hypertension (PHT). In contrast, VP decreased PVR/SVR by 64%. Results suggest a novel role of VP in differential regulation of pulmonary and systemic arterial pressures, which can prevent PHT and eventual hypoxemia following resuscitation.
Altered protein phosphatase 1 (PP1) activity and expression contribute to the differential regulation of proximal tubule NHE3 before and after onset of hypertension in SHR
Authors: Crajoinas Renato Oliveira , Pessoa Thaissa Dantas , Malnic Gerhard , Girardi Adriana Castello Costa
Published: June 2021

We have previously demonstrated that NHE3 activity is higher in the proximal tubule of young pre‐hypertensive SHR (Y‐SHR) compared to adult hypertensive SHR (A‐SHR). This fall in NHE3 activity with blood pressure increase was associated with a higher ratio of phosphorylated NHE3 at serine 552 to total NHE3 (P‐NHE3/ total). The present study was undertaken to test the hypothesis that the increased levels of phosphorylated NHE3 at the PKA consensus site serine 552 was due to an increase of PKA activity, or a decrease of PP1 activity, or both. To confirm that PKA‐mediated phosphorylation of NHE3 regulation is altered in SHR, we examined the effect of 6MB‐cAMP (an cAMP analog that specifically activates PKA) on both phosphorylation levels and activity of NHE3 in young and adult SHR. We found that the 6MB‐cAMP‐induced increase in the levels of NHE3 phosphorylation at the serine 552 was much higher in the renal cortex from Y‐SHR than from A‐SHR (179 ± 14 vs. 36 ± 4%, P < 0.001). Likewise, the inhibitory effect of 6MB‐cAMP on NHE3‐mediated bicarbonate reabsorption was much more pronounced in proximal tubule from Y‐SHR than from A‐SHR (65 ± 10 vs. 23 ± 9%, P < 0.01). PKA activity remained unchanged between Y‐SHR and A‐SHR. On the other hand, Y‐SHR exhibited higher PP1 activity than A‐SHR (1640 ± 107 vs. 940 ± 119 pM/μg, P < 0.01). Furthermore, PP1α expression was decreased in the renal cortex of A‐SHR compared to Y‐SHR (32 ± 8 %, P < 0.01). We conclude that the differential regulation of proximal tubule NHE3 activity and phosphorylation levels before and after onset of hypertension in SHR is, at least in part, due to changes in the activity and expression of PP1.

Supported by FAPESP
Effect of high‐fat diet (HFD) on blood pressure and placental levels of tumor necrosis factor (TNF)‐α and soluble fms‐like tyrosine kinase (sFlt)‐1 in pregnant rats
Authors: Palei Ana Carolina , Spradley Frank T , Arany Marietta , Cockrell Kathy , Granger Joey P
Published: June 2021

During preeclampsia (PE), placental ischemia increases circulating levels of inflammatory and anti‐angiogenic factors that ultimately leads to hypertension and proteinuria. While obesity is a leading risk factor for developing PE, the mechanisms whereby obesity increases this risk are unclear. Our aims were to examine the effects of HFD on cardiovascular and metabolic systems of normal pregnant rats. Twelve‐week‐old Sprague‐Dawley female rats were fed a normal diet (ND, 13% fat kcal; n=12) or HFD (40% fat kcal). After 9 weeks, rats receiving HFD were grouped in obese prone (OP, weight gain=78.6±2.2 g; n=10) and obese resistant (OR, wg=57.4±2.0 g; n=11) (vs. ND wg=53.6±3.2 g; P<0.05). Rats were then allowed to breed, and mean arterial pressure (MAP) was measured by carotid catheters and tissues were harvested at gestational day 19. Body weight/litter size and visceral fat mass were similar in ND, OR and OP (32.0±2.6 vs. 32.7±2.6 vs. 30.0±1.4 g and 10.9±0.6 vs. 12.2±0.6 vs. 13.2±1.0 g; P>;0.05). MAP was also similar in ND, OR and OP (112±7 vs. 108±3 vs. 110±4 mmHg; P>;0.05). Placental levels of TNFα and sFlt1 were comparable between ND, OR and OP (1.0±0.1 vs. 1.5±0.2 vs. 1.2±0.1 ng/μg and 0.8±0.1 vs.0.9±0.1 vs. 0.9±0.1 pg/μg; P>;0.05). In summary, while HFD increases body weight in OP virgin rats, HFD in OP pregnant rats had no effect on blood pressure regulation or placental levels of TNFα and sFlt1. Funding: HL51971 and HL105324.
HIF‐1alpha activation improves mitochondrial function and morphology in the subtotal nephrectomy model of CKD
Authors: Singh Prabhleen , Pham Hai , Finley Joseph , Perkins Guy , Petrosyan Susanna , Murphy Anne , Patel Hemal
Published: June 2021

HIF‐1α activation in subtotal nephrectomy (STN) lowers elevated renal oxygen consumption, ameliorates hypoxia and improves GFR. HIF‐1α also has significant effects on mitochondrial metabolism. Since the kidney derives most of its energy from oxidative metabolism, we examined the regulation of mitochondrial oxidative metabolism by HIF‐1α in early STN kidney model of CKD.

Mitochondrial oxygen consumption, ATP generation, ROS production and morphology were examined in 1‐week STN rats and DMOG (HIF‐1α inducer) treated STN rats and compared with controls. Mitochondrial oxygen consumption rates were elevated in STN. HIF‐1 α activation lowered the mitochondrial oxygen consumption in both STN and controls. Mitochondrial ATP generation was modestly elevated and ROS (superoxide) production was significantly elevated in STN. HIF‐1 α activation significantly lowered both ATP and ROS production. All results were significant (p<0.05). Mitochondrial morphology was significantly altered in early STN with cortical mitochondria showing an elongated phenotype (fusion) and medullary mitochondrial showing a rounded phenotype (fission). Mitochondrial lengths, volume density and cristae abundance were increased in STN cortical mitochondria compared to control and STN medullary mitochondria. HIF‐1 α activation increased mitochondrial number and volume density in the STN cortical mitochondria and mitochondrial lengths in the control cortical and medullary mitochondria.

In conclusion, there are significant alterations in mitochondrial function and morphology and function in early STN kidney. Several of these are ameliorated by HIF‐1α activation, which may be important in the preservation renal function and morphology and to slow the progression of disease.
Enhanced sensitivity of NKCC2 to β‐adrenergic receptor stimulation in TALs from Dahl salt sensitive (DSS) rats
Authors: Ares Gustavo R , Haque Mohammed Z , Ortiz Pablo A
Published: June 2021

Abnormally enhanced NKCC2 activity and trafficking in the thick ascending limb (TAL) are related to hypertension in DSS rats. In vivo, DSS rats exhibit higher renal sympathetic nerve activity than Dahl salt resistant (DSR) and greater sympathetic‐induced renal Na reabsorption. In Sprague Dawley rats, β‐adrenergic receptor (β‐AR) stimulation enhances surface NKCC2 levels via cAMP and PKA. We hypothesized that the response to β‐AR stimulation is enhanced in DSS rats prior to the development of hypertension resulting in higher surface NKCC2 expression in TALs. We obtained TALs from DSS and DSR rats fed normal salt and measured surface NKCC2 and cAMP. The β‐AR agonist isoproterenol (ISO, 1μM) slightly enhanced surface NKCC2 in DSR (13 ± 9%), but significantly enhanced it in DSS rats (70 ± 24 %, p<0.05). We next studied if this difference was due to higher cAMP production. ISO‐stimulated cAMP production was similar in TALs from DSS and DSR rats (DSR: 106 ± 22, DSS: 143 ± 18 % increase from baseline). To test whether the higher response to ISO in DSS was due to enhanced cAMP‐induced signaling we measured cAMP‐stimulated NKCC2 phosphorylation at Ser126. db‐cAMP (25 μM) stimulated Ser126 phosphorylation in DSS from 1.2 ± 0.3 to 10.5 ± 3.5 a.u, and from 0.6 ± 0.3 to 1.9 ± 0.6 a.u in DSR TALs (p<0.05). We concluded that β‐AR stimulation of NKCC2 trafficking is enhanced in TALs from DSS rats by a mechanism that may involve enhanced cAMP signaling.
The Kv1.3 Blocker PAP‐1 Reduces Infarction and Neurological Deficit in a Rat Model of Reperfusion Stroke
Authors: Chen Yi‐Je , Singh Vikrant , Garing April , Wulff Heike
Published: June 2021

Activated microglia significantly contribute to the secondary inflammatory damage in ischemic stroke and therefore constitute attractive targets for post‐infarct intervention. Microglia express the voltage‐gated Kv1.3 and the calcium‐activated KCa3.1 channel and both channels are involved in microglia mediated neuronal killing, oxidative burst and inflammatory cytokine production. We previously found that KCa3.1 blockade with TRAM‐34 reduces neurological deficit and infarct area in a rat model of ischemic stroke even if initiated 12 hours after reperfusion. We now investigated whether Kv1.3 blockade would have similar effects and whether combined blockade of both microglial K+ channels would offer additional benefits. We subjected male Wistar rats to 90 min of middle cerebral artery occlusion (MCAO) and administered either vehicle or the Kv1.3 blocker PAP‐1 (40 mg/kg i.p. BID) for 7 days starting 12 h after reperfusion. PAP‐1 significantly reduced infarct area and improved neurological deficit in the infarcted hemisphere on day‐7. However, combined blockade of both Kv1.3 and KCa3.1 with PAP‐1 and TRAM‐34 did not further reduce infarct area compared to treatment with either TRAM‐34 or PAP‐1 alone suggesting that blockade of one microglial K+ channel is sufficient to improve outcomes of ischemic stroke.

Supported by RO1 GM076063 from the National Institute of Health.
hERG1 agonist NS1643 inhibits proliferation of ER‐neg breast cancer cells by activating a senescence program
Authors: Gentile Saverio , Lansu Katherine , Ha Geun‐Hyoung , Breuer Eun‐Kyoung
Published: June 2021

Function of the hERG1 potassium channel has been well characterized in the heart where it plays an important role in contraction. However, hERG1 channel is expressed in cancers of different hystogenesis. Although inhibiting hERG1 activity can lead to apoptosis, this approach is not likely to be successful in cancer therapy because of the association between hERG1 blocking agents and cardiac ventricular fibrillation. Nevertheless, the hERG1 agonist NS1643 has been found to protect against cardiac arrhythmia but nothing is known about the effects of this drug in cancer cells.

The most successful breast cancer treatment relies on inhibition of the estrogen (E2) receptor (ER) in mediating the effect of E2 on cell proliferation (Tamoxifen). However, this approach cannot be employed for about 30% of breast tumors as they do not express ERs (ERneg).

Our work demonstrates that application of the hERG1 activator NS1643 to ERneg breast cancer cells strongly inhibits proliferation by causing cell cycle arrest in G1 phase. In addition, cells treated with NS1643 lost invading and migrating capability.

Interestingly, NS1643 treatment did not cause apoptosis but expression level of the tumor suppressor proteins p21 and p16INK4 increased significantly.

In conclusion, these data suggest that hyper stimulation of hERG1 activity in ERneg breast cancer cells can arrest cell proliferation by activating a senescence program.
Ion Channel Phosphorylopathy: a link between genomic variation and human disease.
Authors: Gentile Saverio
Published: June 2021

Many genomic variations that change amino acids in cytoplasmic domains of ion channels have been found to be associated with several diseases, including cardiac arrhythmias and neurodegeneration. However, a mechanism linking these mutations to ion channel malfunction has not been clearly established and in some cases is totally unknown.

Our present work gives an overview on a possible mechanism by which disease‐associated mutations in cytoplasmic domains of several ion channels can affect current activity by creating or disrupting phosphorylation sites for specific kinases. We call these events “Ion Channel Phosphorylopathies”. Understanding Ion Channel Phosphorylopathies offers the opportunity to find a mechanism linking genomic variations to human disease and is crucial to the process of designing an effective pharmacological strategy.
Lack of CFTR in a cystic fibrosis pig model disrupts airway smooth muscle development
Authors: Stoltz David A. , Gansemer Nick D. , Rector Michael V. , Bouzek Drake , Moninger Thomas O. , Meyerholz David K.
Published: June 2021

To better understand the pathogenesis of cystic fibrosis (CF) lung disease, we recently generated CFTR‐targeted pigs. CF pigs develop lung disease typical of human CF. An unexpected finding at birth, prior to the onset of infection and inflammation, was that CF tracheas have a reduced lumen area, irregularly developed airway cartilage, and airway smooth muscle (ASM) with prominent appearing bundles. We hypothesized that these abnormal‐appearing ASM bundles were due to differences in ASM proliferation. To test this hypothesis, tracheas were harvested at birth for histological examination. Despite a reduction in tracheal ASM area in CF, we found that ASM cell density was increased in CF (CF: 0.52±0.06 vs. non‐CF: 0.39±0.02 cells/100 μm2). To investigate if cell proliferation was increased in CF, we stained for the proliferation marker Ki‐67 and found reduced Ki‐67 positive cells in CF (CF: 0.04%±0.01 vs. non‐CF: 0.1%±0.01). This decrease was associated with a reduction in CF ASM cell area (197±19 vs. 259±14 μm2). Finally, electron microscopic studies revealed that at birth CF ASM has a contracted appearance, likely accounting for the reduced cell area. These data suggest that CFTR is important in ASM development and might explain, in part, the increased incidence of airway hyper‐responsiveness in human CF.

This work was supported, in part, by the American Asthma Foundation (Sandler).
Anoctamin‐1 is expressed and functional in the Pulmonary Vascular Endothelium
Authors: Allawzi Ayed Mamdouh , Vang Alexander , Li Anlong , Choudhary Gaurav
Published: June 2021

Background and Objective

The expression and functional role of Anoctamin‐1 (ANO1), a Ca2+ activated Cl channel in the endothelial cells (EC) is unclear. We sought to confirm the presence of ANO1 in the pulmonary vascular EC (PVEC) and the effect of ANO1 activation on EC membrane potential (Vm).

Methods

Whole lung lysates and primary isolated rat lung EC (RLEC) were isolated from 8 week old Sprague‐Dawley rats. ANO1 expression was evaluated in whole lung, commercially available primary rat lung microvascular EC (RLMVEC) and human pulmonary artery EC (HPAEC) lysates by immunoblot, in RLEC and RLMVEC by RT‐PCR, and in RLEC by immunofluorescence (IF). Changes in RLMVEC Vm was assessed using DiBAC4(3) and fluorescent live cell imaging.

Results

Immunoblotting confirmed the presence of ANO1 in whole lung lysates, RLMVEC, and HPAEC. RT‐PCR validated ANO1 expression in RLMVEC and RLEC. IF confirmed the expression of ANO1 on RLEC. Activation of ANO1 with 10μM Eact, a ANO1 opener, resulted in a hyperpolarization of Vm, which was attenuated in presence of a low chloride solution.

Conclusion

ANO1 is expressed in the PVEC and can alter the resting Vm.
The effect of dietary iron restriction against diabetic nephropathy in db/db mice
Authors: Ikeda Yasumasa , Enomoto Hideaki , Tajima Soichiro , Izawa‐Ishizawa Yuki , Kihira Yoshitaka , Ishizawa Keisuke , Tomita Shuhei , Tsuchiya Koichiro , Tamaki Toshiaki
Published: June 2021

Recently body iron reduction has been shown to ameliorate obese and diabetes. In the present study, we tested the protective effects of dietary iron restriction against diabetic nephropathy Male db/db mice at 8 week‐old age, a model of diabetic nephropathy, were divided into 2 groups and fed a normal diet (ND) or a low iron diet (LID) and male db/m mice with ND were employed as control mice. Increasing urinary albumin excretion was observed in db/db mice with ND, but this was suppressed in db/db mice with LID. Histologically, the db/db mice in the ND group had increased glomerular size and mesangial expansion compared to the LID group. Augmented deposition of extracellular matrixes was decreased in db/db mice with LID. Increased superoxide production observed in the kidneys of the ND db/db mice was diminished in the LID group. NADPH oxidase activity and expression of NADPH oxidase components p22phox and NOX4 were augmented in kidney of the ND group, and these were abolished by LID. Renal expression of ferroportin and ferritin heavy chain increased in the kidneys of the db/db mice. Serum iron concentration and urinary iron excretion was significantly higher in ND db/db mice compared to db/m mice and was diminished in the LID group. Taken together, dietary iron restriction exerts a preventive action against the development of diabetic nephropathy through oxidative stress reduction.
Intravascular pressure stimulates functional Kv1.5 surface expression in mesenteric artery smooth muscle cells
Authors: Kidd Michael William , Leo M. Dennis , Narayanan Damodaran , Bannister John P. , Jaggar Jonathan H.
Published: June 2021

Intravascular pressure stimulates arterial depolarization, leading to myocyte voltage‐dependent K+ (KV) channel activation that opposes the resulting vasoconstriction. We tested the hypothesis that membrane potential regulates KV channel surface expression to modulate mesenteric artery contractility. Q‐PCR indicated predominant expression of KV1.5 and 2.1, with lower levels of 1.2 and 2.2 in mesenteric arteries. In isolated arteries at zero pressure, ~70% of total and surface KV1.5 protein was lost in 3 hrs. Total or surface KV2.1 protein did not change. KV1.5 or 2.1 mRNA were also unchanged. Depolarization (30 mM K+) prevented loss of KV1.5 and this was blocked by nimodipine, a voltage‐dependent Ca2+ channel blocker. Bafilomycin, a lysosome inhibitor, prevented KV1.5 loss, and MG‐132, a proteasome inhibitor, inhibited loss by ~50% in non‐pressurized arteries. The functional significance of KV1.5 surface expression was studied in arteries maintained at low (10 mmHg) or physiological (80 mmHg) pressure (3 hrs), with diameter regulation then measured at 80 mmHg. Vasoconstriction to psora‐4, a KV1 inhibitor, or 4‐AP, a broad KV inhibitor, was larger in arteries maintained at physiological pressure than those at low pressure. Data indicate that intravascular pressure inhibits degradation of KV1.5 channels and thus, maintains functional surface expression of these proteins in arterial myocytes. NHLBI/NIH
Smooth muscle microRNAs play a crucial role in regulation of myogenic tone in small mesenteric arteries
Authors: Bhattachariya Anirban , Nordström Ina , Swärd Karl , Hellstrand Per , Albinsson Sebastian
Published: June 2021

Myogenic, pressure‐induced, tone is an intrinsic mechanism for the regulation of blood pressure and blood flow to the organs. Small non‐coding microRNAs (miRNAs) have been implicated in the regulation of smooth muscle differentiation and vascular contractility. However, the role of miRNAs in pressure‐induced contractile responses has not been investigated previously.

The aim of this study was to determine the role of miRNAs in myogenic tone using a pressure myograph and smooth muscle specific, tamoxifen‐inducible knockout of Dicer, an enzyme essential for the biogenesis of miRNAs. The results from these mice were then compared to those of the miR143/145−/− and miR143/145+/− mice. In Dicer KO, myogenic tone was abolished 5 weeks post tamoxifen treatment, even though KCl‐induced contraction was only moderately reduced. A similar phenotype was observed in miR‐143/145−/− mice. Interestingly, myogenic tone was also abolished in miR‐143/145+/− mice, which exhibit normal KCl‐induced contraction. This result indicates that that myogenic constriction is highly sensitive to a reduction of these specific miRNAs. Furthermore, we found that the L‐type calcium channel opener Bay K 8644 partially rescued myogenic tone in Dicer KO and 143/145+/− but not in 143/145−/−. Our results suggest that miRNAs play a crucial role in the regulation of myogenic tone, possibly via regulation of L‐type channel activity
Essential role of calcineurin/NFAT and ROS in mediating mechanical stretch‐induced leptin synthesis and vascular smooth muscle remodeling
Authors: Zeidan Asad , Ghantous Crystal , Farhat Zein , Soudani Nadia , Jaffa Ayad
Published: June 2021

Obesity is associated with hypertension and increased leptin production that contribute to cardiovascular pathology. We used rat portal vein (RPV) organ culture to investigate the effect of mechanical stretch (mimicking hypertension) on secretion of leptin and the effect of exogenous leptin (3.1 nM) on vascular smooth muscle VSMC. Stretching the RPV for 1, 2, 3 6 or 12h significantly up‐regulated leptin gene expressions and leptin synthesis and secretion. MS significantly increased ROS production (10 fold), calcineurin (CN) activity (3 fold) and NFAT nuclear translocation, effects that was significantly attenuated by the coadministration of an anti‐leptin receptor antibody (anti‐OBR; 166 ng/ml), the ROCK inhibitor Y‐27632 (10 μM) as well the RhoA inhibitor C3, (30 μg/ml). Disruption of actin microfilaments with 50nM latrunculin B significantly attenuated MS‐induced ROS production, CN activity and NFAT nuclear translocation. The role of ROS in MS‐induced leptin secretion and expression was further established when pretreatment of NADPH oxidase inhibitor apocynin (1 mM) potently attenuated leptin expression, synthesis and secretion induced by MS. Our results indicate that the activation of CN and ROS production plays a pivotal role in MS signaling, leading to leptin synthesis and VSMC hypertrophy.
The Focal Adhesion: A Regulated Component of Aortic Stiffness
Authors: Saphirstein Robert , Gao Yuan , Jensen Mikkel , Gallant Cynthia , Vetterkind Susanne , Moore Jeffrey , Morgan Kathleen
Published: June 2021

Increased aortic stiffness is a known predictor and cause of cardiovascular disease, but much remains unknown about the sources and mechanisms of aortic stiffness. We tested the hypothesis that the focal adhesions (FAs) connecting vascular smooth muscle cells (VSMCs) to the matrix in the aortic wall are a dynamically regulated component of aortic stiffness. First, we used magnetic tweezers to measure cortical stiffness in A7r5 aortic VSMCs. Lysophosphatidic acid (LPA), an activator of myosin that increases cell contractility, increased cortical stiffness. A small molecule inhibitor of Src‐dependent FA recycling, PP2, significantly inhibited LPA‐induced increases in cortical stiffness, as well as tension‐induced increases in FA size. To extend these findings to the tissue level, we monitored mouse aorta ring stiffness with small sinusoidal length oscillations. The alpha‐agonist phenylephrine, which also increases myosin activation and contractility, increased tissue stress and stiffness in a PP2‐attenuated manner. Phosphotyrosine screening confirmed that the effect of PP2 in vascular tissue involves FA proteins. These data suggest that the transfer of VSMC actomyosin‐generated stress and stiffness to the aortic wall during contractile stimulation depends on FAs. Thus, we identify, for the first time, the FA of the VSMC as a significant subcellular regulator of aortic stiffness. Support: NIH P01 86655
Probing differential subcellular calcium dynamics using targeted fluorescence calcium biosensors in rat pulmonary artery smooth muscle cells
Authors: Subedi Krishna Prasad , Paudel Omkar , Sham James S.K.
Published: June 2021

Intracellular calcium (Ca2+) is involved in distinct cellular functions through global and local signaling in various subcellular compartments. To get better insight if the Ca2+ signals are independently regulated in subcellular compartments, we targeted genetically encoded Ca2+ indicator, D3cpv, into three different subcellular regions and used laser scanning confocal microscopy to study how Ca2+ signals are triggered in the plasma membrane (PM), cytoplasm and nucleus in cultured rat pulmonary artery smooth muscle cells (PASMCs). The in situ Kd for Ca2+ of D3cpv was comparable in the cytoplasm and nucleoplasm, but it was slightly higher in the PM. Application of IP3 or Endothelin‐1 to PASMCs caused rapid increase of fluorescence emission ratio in all three regions. The increase of cytoplasmic/PM Ca2+ preceded the nucleoplasmic Ca2+ and the response was more sustained in the nucleus. Platelet derived growth factor (PDGF) generated variable Ca2+ signals with Ca2+ increase occurred first in the cytoplasm followed by the nucleoplasm in some cells, while an early Ca2+ response in the nucleus was observed in the others. Similarly, the increase in PM Ca2+ was slower than that in the nucleoplasm in some PDGF treated cells. These data suggest a differential regulation of Ca2+ in the cellular compartments that may be essential for the independent regulation of contraction, cell signaling and gene expression in PASMCs.
Effect of GsMTx4 on Mouse Urinary Bladder Smooth Muscle (UBSM) Contractility
Authors: Khan Zulqarnain , Suchyna Thomas M. , Sachs Fred , Meredith Andrea L.
Published: June 2021

Urinary incontinence is a common problem with limited treatment options. Stretch‐activated channels (SACs) enhance UBSM excitability, and have been proposed as a novel target for reducing overactive bladder. The goal of this study was to determine whether an inhibitor of SACs (GsMTx4) decreased UBSM contractions in wild‐type (WT) and BK KO (Kcnma1−/−) bladders, a mouse model for urinary incontinence which harbors a deletion of the BK K+ channel. GsMTx4 was applied to isolated UBSM strips at 0.1–10 μM and spontaneous (phasic) and nerve‐evoked (EFS) contractions were investigated using isometric tension recordings. As expected, baseline BK KO contractile amplitudes were significantly larger than WT. Application of 5 μM GsMTx4 induced a smaller reduction in BK KO phasic amplitude than in WT (KO: −12 ± 0.22%, WT: −31 ± 0.04%), and reliably decreased phasic frequency in BK KO strips (KO: −14 ± 9%, WT: +11 ± 4%). Additionally, 10 μM GsMTx4 suppressed EFS evoked contractions (10 Hz amplitude, BK KO: −19 ± 12%, WT: −4 ± 1%). This data suggests that GsMTx4 may be effective at reducing spontaneous and nerve evoked contractions in overactive UBSM, while having a limited effect on normal UBSM. This work was funded by NIDDK R21–089337 (A.L.M.) and the APS UGSRF (Z.K.).
Increased expression of AT2 receptors in the nucleus of the solitary tract improves baroreflex function in renovascular hypertensive rats.
Authors: Blanch Graziela T , Freiria‐Oliveira Andre H , Rigatto Katya , Li Hongwei , Colombari Eduardo , Sumners Colin , Colombari Debora S A
Published: June 2021

Renovascular hypertensive (2K1C) rats exhibit impaired baroreflexes. Here we studied the effects of increased AT2R expression in the nucleus of the solitary tract (NTS) on baroreflex function of 2K1C rats. The left renal artery was partially obstructed in male Holtzman rats (150–180g) using a silver clip (0.2 mm width). Three weeks later, rats were microinjected in the NTS with either AAV2‐CBA‐AT2R or AAV2‐CBA‐eGFP to elicit chronic increases in AT2R or GFP (control) expression respectively. Thirty days later, femoral artery and vein were cannulated to measure mean arterial pressure (MAP) and heart rate (HR) and perform drug infusions, respectively. The next day, phenylephrine (5 μg/kg, iv) and sodium nitroprusside (30 μg/kg, iv) were used to elicit respective pressor and depressor responses in conscious rats. Reflex HR responses were analyzed to each 10 mmHg change in MAP. Baseline MAP in 2K1C‐AT2R rats (173 ± 7 mmHg) was lower than in 2K1C‐GFP rats (199 ± 9 mmHg; p <0.05). 2K1C‐GFP rats exhibited a significant impairment of the baroreflex (slope: −0.95 ± 0.05, vs. sham‐GFP −1.37 ± 0.06, p<0.05), which was improved in the 2K1C‐AT2R rats (slope: −1.44 ± 0.07). Baroreflex in 2K1C‐AT2R was similar to sham‐AT2R rats (slope: −1.56 ± 0.06) or sham‐GFP. The results suggest that AT2R over expression in the NTS of 2K1C rats improves baroreflex and this may contribute to the reduction in blood pressure observed in these animals.

Supported by:

PNPD/CAPES, NIH and FAPESP/PRONEX.
Opioids modulate cardiac spinal afferent responses to ischemia and other mediators
Authors: Fu Liang‐Wu , Longhurst John
Published: June 2021

Cardiac spinal afferents are activated during myocardial ischemia. Although recent observations show that myocardial ischemia increases the concentrations of opioid peptides and that stimulation of peripheral opioid receptors inhibits chemical‐induced visceral and somatic nociception, the role of opioids in afferent signaling duirng pathophysiological events is unclear. Thus, we hypothesized that peripheral opioids modulate cardiac spinal afferent activity during myocardial ischemia. Nerve activity of single‐unit cardiac afferents in anesthetized cats was recorded from the left sympathetic chain (T2 – T5). Thirty‐three ischemically sensitive afferents (CV=0.34–3.90 m/s) with receptive fields in the left ventricle were identified by their responses to five min of regional ischemia. The responses of these afferents to repeat ischemia or ischemic mediators were further studied. First, epicardial application of naloxone (80 μmol), a non‐selective opioid receptor antagonist, enhanced the responses of 7 cardiac afferents to recurrent myocardial ischemia by 35%, whereas epicardial application of saline did not alter responses of 6 other afferents to repeat ischemia. Second, epicardial naloxone respectively facilitated the responses of other ischemically sensitive cardiac afferents to ATP (n=5) by 54% and bradykinin (BK, n=5) by 52%. In contrast, in the absence of naloxone, the afferents consistently responded to repeated application of ATP (n=5) or BK (n=5). These data suggest that peripheral opioid peptides modulate cardiac spinal afferents responses to ischemia and ischemic mediators like ATP and bradykinin. (Supported by NIH HL66217)
Increased Bilateral External Intercostal EMG Activity during Tracheal Occlusions (TO) in Conscious Rats with Unilateral Cervical Spinal Cord Injury (c‐SCI)
Authors: Jaiswal Poonam B , Sandhu Milap S , Fuller David D , Davenport Paul W
Published: June 2021

Respiratory motor function after c‐SCI is often studied using anesthetized animal preparations with cervical spinal hemisection (C2HS). However, anesthesia can influence respiratory control, and conscious animal studies of chest wall external intercostal (EI) muscle function after C2HS are lacking. We hypothesized that TO in conscious rats would trigger bilateral recruitment of EI muscles as reflected by increased EMG activity 1 week after C2HS. Adult male Sprague Dawley rats were instrumented with wire EMG electrodes implanted bilaterally in the EI muscles and an inflatable vascular cuff around the extra‐thoracic trachea, 1 week prior to a left C2HS injury. TO trials were presented pre and 1 week post‐C2HS in conscious rats. Trachea was obstructed using cuff inflations for 3–5s followed by normal breathing for 10–15s. Data were collected for 2 min before (Pre‐TO), 20 min during (TO) and 2 min after (Post‐TO) TO trials. Pre‐TO integrated EI (intEI) EMG during eupneic breathing was significantly decreased bilaterally 1 week after C2HS. TO significantly increased bilateral intEI EMG, however, post‐TO intEI responses were variable. Conscious rats were able to significantly augment EI muscle EMG activity when presented with a TO 1 week post‐C2HS injury. Therefore, EI muscles play a significant role in maintenance of minute ventilation both during eupneic and loaded breathing 1 week after C2HS in conscious rats.
Respiratory responses to over‐pressurization blast injury rats exposed to hypercapnia
Authors: Adams Sherry , Condrey Jillian A. , Tsai Hsiu‐Wen , Prima Victor , Svetlov Stanislav , Sumners Colin , Davenport Paul
Published: June 2021

Blast traumatic brain injury (TBI) is often the result of an explosion generating an over‐pressurization blast (OB) wave in close proximity to the individual producing closed‐head brain injury. Individuals experiencing blast TBI have respiratory changes directly correlated with the blast‐induced TBI. An OB injury rat model was used in this study to test the hypothesis that exposure to OB would alter respiratory compensation when challenged with hypercapnia. An increase in respiratory drive is expected with an increase in CO2 exposure. The animals were anesthetized and the OB brain injury produced by a compressed air‐driven shock tube directed at the dorsal surface of the head of the rat with an average peak overpressure of 90 psi at the shock‐tube and 52 psi at the dorsal surface of the skull with a total duration of 10 msec of blast exposure. The body was protected from the pressure wave by a plexiglass cover. The diaphragm EMG activity (dEMG) was recorded with electrodes implanted 3–7 days prior to the OB. The dEMG recordings were obtained for 2.5 minutes of baseline recording while on 100% O2, 5 minutes of hypercapnia while receiving 4% CO2, and 2.5 minutes of post‐hypercapnia baseline at 100% O2. There were significant changes seen behavioral functions with increased anxiety and decreased somatosensory reflexes. Dorsal head blast has a dominant effect on cortical function but does not affect hypercapnic sensitivity.
Electromyographic pattern analysis of swallow in both the conscious and anesthetized rat.
Authors: Condrey Jillian Alane , Tsai Hsiu‐Wen , Adams Sherry , Hegland Karen Wheeler , Davenport Paul W
Published: June 2021

Swallow occurs in three phases: oral, pharyngeal and esophageal. The oral phase is voluntary and includes mastication. The vocal folds adduct during the pharyngeal phase causing apnea and the bolus is moved through the pharynx. The esophageal phase moves the bolus to the stomach by relaxing the upper esophageal sphincter and the bolus enters the esophagus with contraction of the pharyngeal constrictor muscles. We hypothesized that the coordination of breathing, swallowing and chewing will result in mastication muscle activity preceding pharyngeal swallow phase that occurs during expiration. EMG electrodes were implanted in rats in the right (RMas) and left masseter (LMas) muscles, right (RCrD) and left cranial digastricus (LCrD) muscles, the right caudal digastricus (RCaD) muscle, the cricothyroid (CT) muscle and the diaphragm (Dia). In conscious rats actively chewing, the swallow occurred with activation of the CT and occurs in phase with the RCrD, LCrD, RCaD and out of phase with the RMas and LMas. Swallows were identified during both Dia EMG inspiration and expiration. In anesthetized rats, swallows occurred after RMas and LMas activity. Swallow CT activity occurred during expiration. These results demonstrate sequential and coordinated EMG activity for mastication, swallow and breathing. Anesthesia affects the respiratory phase coordination for swallow.
Laparotomy disrupts respiratory phase preference for swallow
Authors: Rose Melanie , Nicholas Justine , Pitts Teresa , Davenport Paul , Morris Kendall , Lindsey Bruce , Bolser Donald
Published: June 2021

In humans, swallow occurs predominantly in the expiratory phase (E) of breathing. This phase preference for swallow is thought to contribute to airway protection by limiting the passage of material through the pharyngeal airway to periods in which there is little or no inspiratory (I) airflow. This phase preference is currently attributed to central interactions between the swallow and breathing pattern generators. We speculated that peripheral mechanical factors, such as abdominal pressure, would influence respiratory phase preference for swallow. We induced swallowing in anesthetized spontaneously breathing cats by injection of water into the oropharyngeal airway. In animals with intact abdomens, swallow exhibited respiratory phase preference with 83% during E, 7% during I, 7% during E‐I phase transition and 3% during I‐E transition. In animals with open anterior midline laparotomy, only 38% of swallows occurred during E, 33% during I, 17% during the E‐I transition, and 12% during I‐E. Similar percentage occurrences were observed in these animals during 5 or 10% C02. Electromyogram amplitudes of adductor, cricopharyngeus, and thyrohyoid muscles during swallow did not differ between the two groups. The results support an important role for feedback from somatic and/or visceral thoracoabdominal mechanoreceptors in the coordination of swallow and breathing. Supported by NIH HL89104, HL103415, HL109025, HL89071.
The mechanical advantage of negative intra‐thoracic pressure during swallow
Authors: Pitts Teresa , Gestreau Christian , Rose Melanie J , Davenport Paul W , Morris Kendall F , Bolser Donald C
Published: June 2021

In humans and animals, it is established there is a phrenic nerve burst and contraction of muscles which create negative intrathoracic pressure (costal/crural diaphragm and parasternal) during the pharyngeal phase of swallow. We speculated this swallow activity would change with conditions which create the necessity for more rapid pharyngeal clearance, such as increased respiratory drive. Swallowing was induced in anesthetized spontaneously breathing cats by injection of water into the oropharynx, and respiratory drive was altered by inhalation of 5% and 10% CO2. Electromyograms (EMGs) were recorded from pharyngeal, laryngeal, and chest wall muscles, and intra‐esophageal pressure was recorded from the midthoracic esophagus. The magnitude of negative esophageal pressure during swallow increased by 44% during 5% CO2 and 45% during 10% CO2 compared to baseline. Parasternal and post‐swallow cricopharyngeal (upper esophageal sphincter) EMG activity also increased during swallows that occurred under hypercapnic conditions. Effective pharyngeal swallow may require multiple mechanical events including: positive pressure from oral and pharyngeal muscle contraction propelling the bolus, and negative intra‐thoracic pressure drawing the bolus into the esophagus. Supported by NIH HL89104, HL103415, HL89071, HL109025.
Influence of baclofen on upper airway and spinal motor drive during cough in the anesthetized cat
Authors: Nicholas Justine Nicole , Castillo Daniel , Pitts Teresa E. , Rose Melanie J. , Bolser Donald C.
Published: June 2021

The antitussive effects of (±) baclofen on upper airway muscle activities have not been determined. The hypothesis of this study was that administration of (±) baclofen would suppress upper airway muscle motor activity in a dose dependent manner during cough. Electromyograms (EMGs) of the parasternal, rectus abdominis, thyroarytenoid, posterior cricoarytenoid, and thyrohyoid muscles were measured along with esophageal pressure. Cough was elicited by mechanical stimulation of the lumen of the intrathoracic trachea. Baclofen (±) (3 – 10 μg kg−1 i.a.) induced decreases in EMG amplitude of the rectus abdominis during coughing and cough number per epoch. The durations of the inspiratory and expiratory (E1) phases of cough were prolonged by (±) baclofen. There was no effect of (±) baclofen on the EMG amplitudes of any of the laryngeal muscles, the parasternal, or the duration of the expiratory (E2) phase. Results from the present study indicate differential control mechanisms for laryngeal and spinal muscle motor drive during cough, providing evidence of a control system regulating upper airway activity that is divergent from that controlling drive to spinal motoneurons during cough. Supported by NIH HL89104, HL103415.