bluuebunny/crossref_metadata_2025 huggingface dataset:
The role of hepcidin in anemia of inflammation and anemia of cancer
Authors: Rivera Seth , Pastuszka Martha , Yu Alice , Ganz Tomas
Published: June 2021

Hepcidin is presumed to be the principal mediator of anemia of inflammation (AI) and anemia of cancer (AC). Hepcidin is elevated in these conditions and an inducible hepcidin overexpressing mouse develops a clinical syndrome nearly identical to AI (Roy et al, Blood, 2007). However, no study has determined whether hepcidin is necessary for the development of AI and AC. Using mouse models of AC and AI in hepcidin knockout mice (KO) and wild type controls (WT), we sought to determine whether hepcidin was necessary for the development of anemia in these conditions. In AI, hemoglobin fell to a much lesser extent in KO mice. In AC, hemoglobin fell to a similar degree in WT and KO mice. However, in AC the tumor produced some hepcidin. KO mice have polycythemia at baseline so they are much less anemic than WT mice even in the presence of inflammation and cancer. Hepcidin plays an important role in AI and AC. Future studies will determine whether antagonism of hepcidin after the onset of disease will treat these conditions.
A key role of antizyme in morphological and functional differentiation of pancreatic alpha cells
Authors: Suzuki Jun‐ichiro , Murakami Yasuko , Samejima Keijiro , Ohtani Masahiro , Oka Takami
Published: June 2021

Pancreatic alpha cells produce and secrete glucagon. There is still much to be learned about mechanisms whereby alpha cell differentiation is regulated. We found that AsPC‐1 cells, a human pancreatic cell line, can be induced to differentiate into glucagon‐producing cells by trypsin treatment and subsequent culture in serum‐free medium. These cells expressed glucagon mRNA in response to reduction of glucose concentration in the medium. They also formed cell aggregates resembling the pancreatic islet. We used this system to identify regulatory molecules involved in alpha cell differentiation. Our present study focused on antizyme, a key protein that regulates the biosynthesis and transport of polyamine because antizyme and polyamine levels were high in the pancreas. Antizyme in AsPC‐1 cells was localized in the cytosol during differentiation whereas it was in the nucleus during cell proliferation. Antizyme mRNA and the concentration of spermidine increased when AsPC‐1 cells were induced to differentiate. Transfection of antizyme siRNA into AsPC‐1 cells resulted in specific suppression of antizyme expression and inhibited both morphological and functional differentiation including glucagon gene expression. However, the differentiated state was restored by subsequent transfection of a GFP‐antizyme construct. These results suggested that antizyme is needed for pancreatic alpha cell differentiation.
Regulation of activity dependent transmitter release from chromaffin cells by F‐actin and non‐muscle myosin II
Authors: Doreian Bryan William , Fulop Tiberiu , Smith Corey
Published: June 2021

Adrenal chromaffin cells release catecholamines and neuropeptides upon stimulation. Both transmitter classes are packaged in the same secretory organelle. At basal firing rates, highly soluble catecholamines are released from chromaffin cells by ‘kiss and run’ fusion events. Under acute stress, elevated firing leads to full granule collapse releasing both catecholamine as well as a neuropeptide‐containing proteinacious granule core. Thus, activity dependent transmitter release is regulated by a transition in secretion mode. Here we employ electrochemical, electrophysiological and fluorescence based approaches to investigate the role of actin and myosin in regulating this transition. We show that under basal firing rates, F‐actin networks are maintained stabilizing ‘kiss and run’ fusion events. Under the same stimulation conditions, disruption of F‐actin resulted in conversion to full‐collapse exocytosis. Also, under increased cell stimulation, activation of myosin II and disruption of F‐actin is necessary to drive the full collapse of the granule. Under the same conditions stabilization of F‐actin and inhibition of myosin II resulted in a conversion to ‘kiss and run’ exocytosis. Our results demonstrate that the transition from ‘kiss and run’ to ‘full collapse’ exocytosis depends upon both F‐actin and myosin II, helping to provide essential regulation of the fusion mode and a proper stress response.

This work was supported by a grant from the National Institutes of Health (NIH) 5/T/32/HL07887
Inhibition of prolactin secretion by American ginseng is mediated by ginseng polysaccharide‐induced nitric oxide production
Authors: Murphy Laura L , Arbogast Lydia A , King Mandy L
Published: June 2021

Rats treated with American ginseng exhibit an approximate 50% reduction in serum prolactin levels and a compensatory decrease in hypothalamic dopamine activity. To investigate whether ginseng has direct pituitary effects, anterior pituitaries collected from random cycling female rats were dispersed and cultured for 1 week prior to treatment with water soluble ginseng extract (0 – 2 mg/ml) for 24 hr. Ginseng produced a dose‐dependent decrease in prolactin with maximum suppression (∼50%) within 6 hr at ≥1mg/ml; there was no change in media LH levels. When the ginsenoside and polysaccharide components in ginseng were examined, ginseng polysaccharides (12.5μg/ml) inhibited prolactin whereas ginsenosides had no effect in the pituitary cultures. Ginseng and its polysaccharides both stimulated nitric oxide (NO) production from cultured cells at 24 hr and induced iNOS gene and protein expression at 12 and 24 hr, respectively. The specific iNOS inhibitor 1400W blocked the ginseng/polysaccharide effects on NO production and prolactin release, whereas eNOS and nNOS inhibitors had no effect. Together, these results suggest that the reduction in circulating prolactin levels following ginseng ingestion is due to polysaccharide‐induced pituitary iNOS and NO production and subsequent prolactin suppression.
SK and IK Ca2+‐activated K+ channels as novel pharmacological targets to control urinary bladder smooth muscle excitability and contractility
Authors: Kellett Whitney F. , Cui Xiangli , Hristov Kiril L. , Petkov Georgi V.
Published: June 2021

The role of small conductance Ca2+‐activated K+ (SK) and intermediate conductance Ca2+‐activated K+ (IK) channels in the rat urinary bladder smooth muscle (UBSM) excitability and contractility was studied using the novel SK/IK channel opener – NS309 and the SK and IK channels specific inhibitors, apamin and TRAM‐34, respectively. To identify SK/IK channels in rat UBSM, we employed studies on UBSM contractility and the perforated patch‐clamp technique. NS309 inhibits spontaneous contraction amplitude, frequency, force, and muscle tone of isolated UBSM strips. In current‐clamp mode, NS309 caused ∼2 mV membrane potential hyperpolarization in single UBSM cells. Apamin increased UBSM spontaneous contraction amplitude and frequency, whereas TRAM‐34 had no effect on spontaneous contractility. Pretreatment with NS309 followed by a washout of the drug led to an increase in UBSM contractility indicating for a functional role of the IK channel in this tissue. Both apamin and TRAM‐34 shifted the NS309 concentration‐response curves for spontaneous contractility to the right. Apamin and TRAM‐34 had no significant effect on the resting membrane potential but blocked NS309‐induced hyperpolarization. Collectively, our data reveal a functional role of the IK channel in rat UBSM and suggest that both SK/IK channels are potential therapeutic targets to control overactive urinary bladder. Supported by DK070909 to G.V.P.
Calcineurin A‐alpha is required for the differentiation of basal keratinocyes
Authors: Pena Juan Armando , Losi Jacquelin , Grimwood Ronald , Guler Rebecca , Gooch Jennifer
Published: June 2021

Calcineurin is a serine/threonine phosphatase that is inhibited by the immunosuppressive agents cyclosporine A and FK506. Mice lacking the alpha isoform of the catalytic subunit of calcineurin (CnAα) have been created. On gross inspection, the skin of CnAα KO mice is noted to have decreased elasticity and resilience. Histological analyses reveal changes in the basal keratinocyte layer and an attenuation of cells in the supra‐basal layers, most notably the stratum spinosum. The calcineurin substrate NFATc is highly expressed in the nucleus of basal epidermal cells in WT mice but is cytoplasmic in KO mice, consistent with a loss of calcineurin activity in this layer. PCNA staining identified proliferating cells in the basal layer as expected, but TUNEL assay revealed increased cell death in the supra‐basal layers. Finally, immunohistochemical staining of keratins revealed that there is an increase in cells expressing the basal cell marker keratin 14 in the supra‐basal layers and a marked decrease in expression of the differentiated cell maker keratin 10 in CnAα KO mice. These findings suggest that calcineurin is required for normal differentiation of basal keratinocytes and survival of squamous epidermal cells. Interestingly, this model bears similarities to the inherited disorder Darier's disease and may shed light into the cellular basis of hyperproliferative conditions such as psoriasis.
Effects of pHi change on spontaneous contraction and ionic currents in pregnant rat uterine smooth muscle
Authors: Cho Young‐Eun , Kim Young‐Hwan , Lee Young‐Ho , Ahn Duck Sun
Published: June 2021

It is known that alteration of the intracellular pH(pHi) significantly affects frequency and amplitude of spontaneous contraction in pregnant rat uterine smooth muscle. In this study we examined the effect of pHi change on (i) the spontaneous contraction and intracellular Ca2+ concentration ([Ca2+]i), (ii) the activity and kinetic property of the voltage‐dependent Ca2+ channel and the delayed rectifier K+ channel, and (iii) the activity of Ca2+‐activated K+ channel in pregnant rat myometrium. Intracellular acidosis (20mM Na+‐acetate) decreased in frequency and amplitude of spontaneous contractions. However, intracellular alkalosis (20mM NH4Cl) caused a tonic contraction or marked increase in frequency and amplitude of spontaneous contractions. The force was correlated with the [Ca2+]i transients. Intracellular acidosis decreased the magnitude of the voltage‐dependent Ca2+ current, but intracellular alkalosis increased it. Intracellular acidosis increased the delayed rectifier K+ current but intracellular alkalosis decreased it. Activity of Ca2+‐activated K+ channels (BKCa) was decreased by intracellular acidosis but increased by intracellular alkalosis. These results indicate that pH‐induced change of Ca2+ channels and K+ channels activity may be involved in the pH‐dependent modulation of spontaneous contraction in pregnant rat uterine smooth muscle.
Functional Role of Pacemaking Cells in the Female Reproductive Tract in Health and Disease
Authors: Dixon Rosie , Hwang Sung‐Jin , Ramsey Kyle H. , Ward Sean M
Published: June 2021

Oviducts are lined by smooth muscle which displays rhythmic electrical pacemaker activity that is coupled to mechanical contractions. These contractions, along with cilia beating and epithelial secretions aid the propulsion of the ova to the site of fertilization. Following fertilization, smooth muscle contractions and epithelial secretions are thought to be responsible for the transport of the zygote to the uterus. The contractile activity of oviductal smooth muscle is therefore vital for normal fertility however, it is not understood how these contractions direct ova movement. Aims: (i) examine the cellular mechanisms underlying oviduct smooth muscle excitability; (ii) evaluate whether specialized interstitial cells of Cajal (ICC)‐like cells are responsible for oviduct excitability and (iii) determine the disruption in pacemaker function during inflammation. Results: ICC are responsible for generating pacemaker activity that underlies contractile activity of the oviduct. ICC are disrupted and pacemaker activity damaged by inflammation of the reproductive tract, leading to loss of spontaneous smooth muscle contractions, thus providing an explanation why inflammation can cause tubal infertility. Conclusions: The findings reveal the origin of spontaneous contractions in the oviduct that is necessary for egg and embryonic transport. Grant support was obtained from NIH DK57236 and PHS AI49354.
Alteration of Currents of Large Conductance Calcium‐Activated Potassium Channels by Hyperkalemia in Porcine Arterial Smooth Muscle Cells
Authors: Han Jian‐Guo , Yang Qin , Shen Bing , Yao Xiao‐Qiang , He Guo‐Wei
Published: June 2021

The wide use of hyperkelemic solutions in cardiac surgery including heart transplantation draws our attention on the effect of hyperkalemia on large conductance calcium‐activated potassium channels (BKCa). The BKCa of arterial smooth muscle cells (SMCs) is pivotal in the regulation of vascular tone. SMCs were enzymatically isolated from porcine coronary arteries. Primary cultures of the SMCs (usually 5–7 days) were used for patch‐clamp study. The effect of K+ with different concentrations (5.4, 20, 60, or 120 mM) was investigated on whole‐cell BKCa current with a holding potential of −60 mV (n=5 for each concentration). The rise of extracellular K+ in bath solutions significantly resulted in increase of BKCa currents in a K+‐concentration dependent manner in comparison with the control solution (5.4mM: 31.6±3.4pA/pF; 20mM: 73.6±11.4pA/pF; 60mM: 108.6±20.6pA/pF; 120mM: 135.2±20.5 pA/pF, p<0.05 one‐way ANOVA). Raise of extracellular K+ concentration increases BKCa activity of the porcine coronary arterial SMCs in a dose‐dependent manner. This may be an important part of the effect of hyperkalemic cardioplegia on the coronary circulation and therefore may have clinical implications in the heart preservation.

Acknowledgments: Supported by Hong Kong RGC grant (CUHK4383/03M) and CUHK direct grants (4450171, 4450231, 2041305)
Reactive Oxygen Species Attenuate Endothelium‐Dependent Pulmonary Vasodilation Following Intermittent Hypoxia
Authors: Norton Charles Elbert , Kanagy Nancy , Walker Benjimen , Resta Thomas
Published: June 2021

Exposure of rats to chronic intermittent hypoxia (IH) does not alter endothelium‐derived nitric oxide (EDNO)‐mediated pulmonary vasodilation despite enhanced expression of endothelial nitric oxide synthase (eNOS). Because IH increases vascular oxidative stress in systemic arteries, we hypothesized that increased generation of reactive oxygen species (ROS) following IH reduces EDNO‐dependent pulmonary vasodilation. To test this hypothesis, we examined effects of the ROS scavenger tiron (10 mM) on endothelium‐dependent vasodilatory responses to the calcium ionophore ionomycin (350 nM), in U‐46619‐constricted, salt‐perfused lungs isolated from control and IH rats. IH rats were exposed 20 times/hr to hypoxia (∼5% O2 for 15 secs) for 7 hr/day for 4 weeks. Control animals were subjected to air‐air cycling for a similar duration. Consistent with our hypothesis, we found that ROS scavenging augmented ionomycin‐induced vasodilation in lungs from IH rats, but had no effect in control lungs. In addition, vasodilatory responses were significantly greater in IH compared to control lungs in the presence of tiron. We conclude that the potentially greater capacity for EDNO‐dependent pulmonary vasodilation following IH is inhibited by ROS.
Resveratrol Prevents Monocrotaline‐induced Pulmonary Hypertension in Rats
Authors: Labinskyy Nazar , Csiszar Anna , Wu Joseph M. , Ballabh Praveen , Mathew Rajamma , Podlutsky Andrej , Wolin Michael S , Ungvari Zoltan
Published: June 2021

Progression of pulmonary hypertension (PAH) is associated with increased proliferation of pulmonary arterial smooth muscle cells (PASMCs) and activation of inflammatory processes. We showed previously that resveratrol (RES) in vitro exerts anti‐oxidant anti‐inflammatory effects and attenuates cell proliferation. The present study was undertaken to investigate the efficacy of RES for preventing PAH. Rats injected with monocrotaline (MCT) progressively developed PAH. RES treatment (25 mg/kg/day, p.o., from day 1 post‐MCT) attenuated RV systolic pressure and pulmonary arterial remodeling, decreased expression of inflammatory cytokines (TNFa, IL‐1a, IL‐6, PDGFa/b) and limited the presence of ED‐1 positive cells in the lung. RES in vitro inhibited proliferation of PASMCs. There was an increased expression of eNOS, decreased oxidative stress, down‐regulation of NAD(P)H oxidase subunits and improved endothelial function in small pulmonary arteries of RES‐treated rats. RES treatment also prevented RV hypertrophy and attenuated RV myocardial oxidative stress. In conclusion, RES exerts anti‐inflammatory and anti‐oxidant effects attenuating vascular remodeling and cor pulmonale in experimental PAH.
Magnetic resonance imaging assisted detection of hypoxia induced right ventricular hypertrophy in mice
Authors: Zhou Dan , Sahni‐Fabricant Malika , Szeto Daphne , Mandala Suzanne , Messina Eric , Walker Matthew , Cook Lynn , Hargreaves Richard , Liu Haiying
Published: June 2021

It is well known that chronic hypoxia induces pulmonary hypertension resulting in right ventricular hypertrophy (RVH). Previously, heart histopathology has been the standard assessment tool for assessing right ventricular enlargements in rats and mice. The purpose of our study was to non‐invasively image, with Magnetic Resonance (MR) RVH in mice. Mice were housed in the hypoxia (10% O2) chambers for 2, 3, 5, and 8 wks. Age matched mice housed under the normal room air condition were included as controls. Serial short‐axis MR images of mouse right ventricle (RV) were captured in real time. The volume, wall thickness and ejection fraction (EF) of the RV was assessed using MR. Preliminary results showed that right ventricular volume and wall thickness were not altered after 2 or 3 wks of hypoxia in mice. Following 5 wks of hypoxia, mice showed increased RV wall thickness compared with controls (0.34±0.01 vs. 0.25±0.02 mm, p<0.05) without altering the RV volume and EF. By 8 wks of hypoxia, mice continued to show increased RV wall thickness. In addition, the RV ejection fraction was significantly reduced in hypoxia treated mice (52±1 vs. 69±2%, p<0.01). Our data demonstrated that hypoxia induced RVH in mice can be detected following at least 5 wks of continuous exposure to hypoxia using MR imaging. MR imaging can be applied to monitor heart morphological and functional changes in small animals such as mice.
C‐Fos immunoreactivity (CFI) analysis of sympathetic and non‐sympathetic neural pathways in chronic DOCA‐salt hypertension: effect of benzamil hydrochloride
Authors: Abrams Joanna M , Engeland William C , Osborn John W
Published: June 2021

Intracerebroventricular (ICV) infusion of benzamil attenuates DOCA‐salt hypertension. We examined which brain regions may be involved in this response. Uninephrectomized rats were instrumented with an ICV catheter connected to a minipump delivering either benzamil (16 nmol/day) or vehicle. DOCA (50 mg) or vehicle was implanted subcutaneously, creating four groups: benzamil and DOCA (n=7), benzamil but no DOCA (n=7), DOCA but no benzamil (n=6), or neither benzamil nor DOCA (n=6). Rats drank 0.9% NaCl / 0.2% KCl for 33 days. We analyzed CFI in 5 distinct regions of the hypothalamic paraventricular nucleus (PVN), as well as in supraoptic nucleus (SON), rostroventrolateral medulla (RVLM), and the nucleus of the tractus solitarious (NTS). DOCA increased CFI in all regions of the PVN and in the SON but had no effect in the RVLM or NTS. Benzamil attenuated CFI in two PVN subnuclei: posterior magnocellular neurons and medial parvocellular (mp) neurons, as well as in the SON. These findings support the hypothesis that increasing neural activity plays a role in chronic DOCA‐salt hypertension, and that benzamil attenuates this increase. Benzamil seems to specifically target neurons that play a role in neuroendocrine responses, as it attenuated CFI in the magnocellular vasopressinergic neurons of the PVN and SON, as well as the mp (corticotropin‐releasing hormone positive) neurons of the PVN. (HL064178)
Developing an MRI based method for analyzing differences in blood vessel diameter and brain tissue perfusion in hypertension
Authors: Gillies Robert Milne , Germain Sean , Raizada Mohan , Paton Julian , Walter Glenn
Published: June 2021

We aimed to develop an MRI based protocol to investigate anatomical differences in brainstem blood vessels between spontaneously hypertensive rats (SHR; n=4) and their normotensive progenitor strain Wistar Kyoto (WKY; n=4) rats. These were compared using high‐resolution multislice, 2D time of flight (TOF) Magnetic Resonance Angiography using different gadolinium (Gd) based contrast agents (Gd‐Albumin, Gadodiamide) to image cerebral blood vessels at 4.7T (TR/TE=18ms/4.3ms, matrix=512x320,nslices=80; nex=4, flip=35°, resolution=68x67x500μm3). Tissue perfusion was measured using dynamic contrast enhancement (TR/TE=12ms/2.8ms, matrix=256x128, nslices=1; nex=1, ΔT=1.5s, images=300, flip=15°, resolution=0.137x0.230x1mm3) or by determining changes in tissue transverse relaxation time pre‐ and post‐contrast enhancement. Images were processed and 3D rendered using Osirix (Mac OS10.4 compatible version). We propose that our protocol will allow measurement and comparison of both blood vessel diameters and tissue perfusion in central cardioregulatory regions in the respective rat strains.

Supported by NIH grants HL33610 and 76312.
Roles of eNOS and nNOS in cutaneous vasodilation induced by local warming of the skin and by whole body heat stress in humans
Authors: Kellogg Dean Lundt , Zhao Joan Liu , Wu Yubo
Published: June 2021

We hypothesized that endothelial nitric oxide synthase(eNOS) generation of NO mediates skin vasodilation due to increased local skin temperature(Tloc) and neuronal nitric oxide synthase(nNOS) mediates skin vasodilation due to whole body heat stress(HS). We examined the effects of an eNOS inhibitor, NG‐amino‐L‐arginine(LNAA) and a nNOS inhibitor, N‐omega‐propyl‐L‐arginine(NPLA) on skin blood flow(SkBF) responses to increased Tloc and HS in 2 protocols. In each protocol, 4 microdialysis probes were inserted into skin. SkBF was monitored(LDF). In each protocol, one site was treated with LNAA, another with NPLA, the third with combined LNAA and NPLA(MIX), and the fourth site was untreated. In Protocol 1, LDF/local heating probe holders were used to initially hold Tloc at 34°C and then at 41.5°C. In protocol 2, after a period of normothermia, whole body HS was induced. Results: Protocol‐1: At Tloc=34°C, SkBF did not differ between sites(p>0.05). LNAA and MIX caused similar attenuation of SkBF increases at Tloc=41.5C, but NPLA caused none(p< 0.05 LNAA or MIX vs untreated or NPLA). Protocol‐2: In normothermia, SkBF did not differ between sites(p>0.05). In HS, NPLA and MIX caused similar attenuations of SkBF increases, but LNAA caused none(p< 0.05 NPLA or MIX vs untreated or LNAA). We conclude that eNOS mediates increases in SkBF due to increased Tloc and nNOS mediates increases in SkBF in response to HS.(NIH Grant HL065599)
An indispensable role of TRPC6‐mediated Ca2+ entry in increasing lung microvascular permeability and acute lung injury
Authors: Tauseef Mohammad , Kini Vidisha , Knezevic Nebojsa , Vogel Stephen , Malik Asrar , Birnbaumer Lutz , Mehta Dolly
Published: June 2021

Studies have shown that transient receptor potential cation channels (TRPC) form the principal mechanism to increase intracellular Ca2+ in several cell types including endothelial cells. We showed that thrombin by binding to its receptor PAR‐1 activates transient receptor potential channel 6 (TRPC6), a receptor‐operated Ca2+ channel (ROC), which in turn regulates RhoA‐induced endothelial contraction. In the present study we have investigated microvascular permeability response and lung edema formation in the TRPC6−/− mice. We isolated lungs from TRPC6−/− mice and from strain‐matched wild type (WT) mice and determined the filtration co‐efficient (Kf, c), a measure of hydraulic conductivity across endothelial exchange barrier in response to oleoyl‐2‐acetyl‐sn glycerol (OAG), a cell‐membrane permeable analog of DAG and PAR‐1 peptide. OAG and PAR‐1 increased Kf, c in lungs from WT mice whereas they failed to increase Kf, c in lungs isolated from TRPC6−/− mice. TRPC6 transcript expression was detected in lungs from WT mice but not from TRPC6−/− mouse. Expression of TRPC3, another ROC or TRPC1, a SOC was not altered in TRPC6−/− mice lungs. We also determined endotoxin LPS‐induced acute lung injury (ALI) in WT and TRPC6−/− mice by determining lung wet to dry weight ratio and albumin permeability across the lung microcirculation. We observed that LPS induced lung edema was markedly reduced in TRPC6−/− mice. These intriguing findings suggest that TRPC6 activity is essentially required for increasing lung microvascular permeability and ALI.
Knockdown of Stromal Interaction Molecule 1 (STIM‐1) Decreases Store‐operated Calcium Entry (SOCE) and Attenuates Hypoxic Calcium Response in Pulmonary Artery Smooth Muscle Cells (PASMC)
Authors: Lu Wenju , Wang Jian , Shimoda Larissa A , Sylvester J T
Published: June 2021

The increased intracellular Ca2+ concentration ([Ca2+]i) caused by acute hypoxia in PASMC requires Ca2+ release from sarcoplasmic reticulum (SR) and influx through store‐operated Ca2+ channels (SOCC). Recent evidence in a variety of cell types, including smooth muscle, suggests that STIM‐1 senses Ca2+ concentration within the SR lumen and transduces activation of SOCC upon SR Ca2+ release and depletion. To determine if STIM‐1 plays a role in the [Ca2+]i response to hypoxia in PASMC, we measured [Ca2+]i with the fluorescent Ca2+‐sensitive dye, Fura‐2, in primary cultures of rat distal PASMC treated with nontargeted small interfering RNA (siRNA) or siRNA targeted to STIM‐1. Compared to nontargeted siRNA, STIM‐1 siRNA caused: 1) a 92% decrease in STIM‐1 mRNA, as measured by real‐time quantitative PCR; 2) an 81% decrease in STIM‐1 protein, as measured by Western blotting; 3) no change in mRNA for STIM‐2, a related protein that may regulate STIM‐1; 4) 74% inhibition of the increase in [Ca2+]i caused by restoration of extracellular Ca2+ in cells perfused with Ca2+‐free Krebs Ringer bicarbonate solution (KRBS) containing cyclopiazonic acid to deplete SR Ca2+ stores and nifedipine to block Ca2+ entry through L‐type voltage‐operated Ca2+ channels; 5) 77% inhibition of increase in [Ca2+]i caused by hypoxia (4% O2) in PASMC perfused with normal KRBS; and 6) no inhibition of the increase in [Ca2+]i caused by equimolar (60 mM) substitution of KCl for NaCl in the normal KRBS perfusate. These results indicate that STIM‐1 is expressed in rat distal PASMC and plays an important role in SOCE and the [Ca2+]i response to acute hypoxia in these cells.
Signaling Mechanisms of Protease‐Activate Receptor‐1 Mediated Phosphorylation of Endothelial Nitric Oxide Synthase
Authors: Watts Vabren L. , Eguchi Satoru , Motley Evangeline D.
Published: June 2021

Protease Activated Receptors (PARs) are G protein‐coupled receptors that are known to regulate endothelial nitric oxide synthase (eNOS) activity by phosphorylating the enzyme at various sites. Ser1177 is a positive regulatory site when phosphorylated, whereas, Thr495 phosphorylation negatively regulates eNOS activity. The mechanism for eNOS Thr495 phosphorylation by PAR agonists is unknown. In this study, we used a specific synthetic PAR‐1 activating peptide, TFLLR, and thrombin to assess the role of PAR‐1 involvement in the phosphorylation of eNOS Thr495 in human umbilical vein endothelial cells. Both agonists phosphorylated Thr495 in a time‐ and concentration‐dependent manner. Also, cells pretreated with the PAR‐1 inhibitor, SCH79797, showed a significant decrease in thrombin and TFLLR‐induced phosphorylation of eNOS Thr495. Inhibition of Rho with C3 exoenzyme and Rho‐Kinase with Y‐27632 also caused a significant decrease in thrombin and TFLLR‐induced Thr495 phosphorylation. This suggests that PAR‐1 phosphorylates eNOS Thr495 via activation of G12/13. These findings will contribute to understanding the signaling pathways that regulate eNOS‐induced nitric oxide production. NIH‐SCORE.
AVOIDING A ‘SUPERFICIAL’ UNDERSTANDING OF THE EXCRETION OF WATER; importance of ‘thought’ experiments.
Authors: Halperin Mitchell Lewis , Cheema‐Dhadli Surinder , Shafiee Mohammad , Kamel Kamel S
Published: June 2021

Our objective is to define how the kidney handles a water load by asking two questions. First, “What is the actual signal for the release of vasopressin (VP)?” Our data suggest that the signal is related to the arterial and not the venous plasma Na concentration (PNa). In more detail, when humans consumed identical water loads after an overnight fast slowly (in 2‐hr) or rapidly (0.5‐hr), there was an equal venous PNa, but a 4‐mM lower arterial PNa 30‐min after rapid water ingestion; only these latter subjects had a large water diuresis (12±1 ml/min). Second, “How can we estimate of the volume of filtrate delivered the distal nephron to understand what the urine flow rate should be when VP does not act’? We found a large discrepancy in this estimation using 3 different data sets in rats. First, the (TF/P)Inulin rose 2‐fold between the PCT and the DCT. Second, the medullary interstitial osmolality was ~2500‐mOsm/kg H2O in water‐deprived rats. Third, AQP1 was not found in the descending thin limbs (DtL) of superficial nephrons. We conclude that apparent discrepancies in data can be resolved by recognizing heterogeneity of sampling. First, a lower arterial PNa is needed to induce a water diuresis; hence water that was ingested slowly can be retained for future heat dissipation. Second, washout of the medulla can only influence loops of Henle that have water permeability. Although this seems not to apply to superficial nephrons, examining other data such as the (TF/P)Inulin implies that the DtL is permeable to water even if AQP‐1 were truly absent in superficial nephrons. These points indicate that deeper understanding requires integration of all relevant data.
Three‐dimensional reconstructions of rat renal inner medulla suggest two anatomically separated countercurrent mechanisms for urine concentration
Authors: Pannabecker Thomas Lloyd , Dantzler William H , Layton Anita T , Layton Harold E
Published: June 2021

From 0 to 3.5 mm below inner medullary (IM) border, collecting duct (CD) clusters form an organizing motif with descending thin limbs (DTLs) and descending vasa recta (DVR) lying outside CD clusters. Ascending thin limbs (ATLs) and ascending vasa recta (AVR) are uniformly distributed inside and outside CD clusters. This arrangement suggests two functionally distinct countercurrent systems (CCSs) exist: one inside and one outside CD clusters. CCS inside each cluster delivers NaCl from prebends and corresponding part of ATLs, and urea and water from CDs, to interstitial nodal sites bounded by CDs, AVR, ATLs, and interstitial cells. This CCS (ascending AVR and ATL flows and descending CD flows) produces isolated sites of high osmolality that withdraw water from CDs. This CCS has three subsystems related to distance from IM border: 1) loops with no AQP1 turning within CD clusters in first 1 mm; 2) loops lacking AQP1 for last 60% turning within CD clusters in next 2–2.5 mm; 3) loops lacking AQP1 for last 60% turning next to remaining CDs in final 1.5–2 mm. Last 0.5 mm of this CCS has loops with narrow bends and loops with broad transverse bends. Transverse bends permit NaCl point delivery optimal for generating high osmolality at papilla tip. CCS of loops and vessels outside CD cluster may be shielded from high urea concentrations inside cluster and ensure NaCl and urea absorption from ATLs. (NIH DK‐16294, DK‐42091; NSF DMS‐0715021)
Localization and Regulation of Phosphorylated forms of Aquaporin‐2 in vivo
Authors: Fenton Robert A. , Møller Hanne B. , Nielsen Søren , Hoffert Jason D. , Knepper Mark A.
Published: June 2021

Trafficking of the water channel AQP2 by arginine vasopressin (AVP) is essential for body water homeostasis. Phosphorylation of AQP2 at S256 is essential for membrane targeting. In this study, phosphospecific antibodies were employed to examine the localization and regulation of two novel phospho‐isoforms of AQP2 in vivo. Immunohistochemistry localized p264‐AQP2 and p269‐AQP2 throughout the collecting‐duct and connecting tubule. Confocal laser‐scanning microscopy of normal rat kidneys determined that p264‐AQP2 is expressed both intracellularly and on the apical plasma membrane, whereas p269‐AQP2 is weakly expressed exclusively on the apical plasma membrane. Neither of these phospho‐forms colocalized with markers of the Golgi apparatus, the endoplasmic reticulum or early endosomes. This localization was confirmed by immunogold‐electron microscopy. Immunoblots of Brattleboro rat and Sprague Dawley rat kidneys determined that both phospho‐forms of AQP2 are increased in abundance after short‐term AVP exposure. In Brattleboro rats, p264‐AQP2 was weakly expressed in the absence of AVP, whereas p269 was undetectable. Short‐term AVP exposure increased the abundance of both forms at the apical plasma membrane. The localization of different phospho‐forms of AQP2 to different cellular compartments suggests distinct roles in AQP2 regulation.
Parathyroid hormone (PTH) mediates shear‐induced actin organization and mechanosensitivity via Ras Homologous A (RhoA) GTPase in osteoblasts
Authors: Madden Gregory Russell , Kronbergs Andris , Adams Elizabeth , Golen Kenneth van , Duncan Randall
Published: June 2021

PTH enhances the mechanical loading‐induced increases in bone formation that we postulate results from PTH disruption of the actin cytoskeleton to increase the mechanosensitivity of the osteoblast. Since Ras homologous A (RhoA) GTPase activation promotes actin assembly, we examined the effects of these stimuli on RhoA activation, actin organization, and cellular stiffness. Fluid shear (12 dynes/cm2) increases actin stress fiber formation (ASFF) within 15 min in MC3T3‐E1 osteoblasts that is blocked by pretreatment with 50nM PTH. Using a RhoA G‐LISA assay, we found that shear activated RhoA within 15 min of the onset of shear and that 15 min pretreatment with PTH significantly inhibited this activation. AFM quantification of the stress‐strain relationship of MC3T3‐E1 cells indicated that shear produced a 6­fold increase in cellular stiffness that was blocked by PTH pretreatment. Inhibition of Rho‐dependent kinase (ROCK), an effector protein of RhoA with Y27632 blocked the shear‐induced ASFF as well as the increase in cellular stiffness. These changes in cell stiffness closely correlated with shear‐induced calcium signaling. These studies suggest that PTH inhibits shear‐induced actin organization through the RhoA‐ROCK pathway and that the resultant changes in cell stiffness play a role in the PTH‐mediated enhancement of osteoblast mechanosensitivity. Supported by HHMI and NIH/NIAMS AR043222
Mechanosensation and the primary cilium
Authors: Resnick Andrew
Published: June 2021

The primary cilium has come under increased scrutiny as a site for mechano‐ and chemosensation by cells. We have undertaken a program of study using mouse renal cell lines from the cortical collecting duct and to quantify how mechanical forces arising from fluid shear transduce to a cellular response. Fluid flow through a model nephron has been analysed to determine the in vivo forces. Live cell microscopy is performed to study how the cells respond to both acute and chronic applied mechanical stress. Measured transepithelial ion fluxes are correlated with applied stresses and the cells have been transfected with a GFP chimera shown to localize to the primary cilium.
Loss of contractile function in hyperthermia: roles of oxidant production and arachidonic acid metabolism
Authors: Oliver Scott Ryan , Wright Valerie P , Parinandi Narasimham , Clanton Thomas L
Published: June 2021

The mechanisms for the loss of muscle contractile function in hyperthermia are poorly understood. Previously we demonstrated that oxidant production is elevated in skeletal muscle during hyperthermia, in part through pathways of arachidonic acid (AA) metabolism. Here, we hypothesized that scavenging of oxidants or blocking AA metabolism preserves function in hyperthermia. Rat diaphragm bundles were exposed to 30 min of 40, 41, 42 or 43ºC. Function was unaffected below 42 ºC but at 43ºC a significant loss of peak force (52.8±7.5% of baseline) and an increase in contracture were observed. Treatment with Tiron or Trolox during and after 43ºC exposure or treatment with DTT after exposure had no effect on loss of function. Lipoxygenase (LOX) and cyclooxygenase (COX) inhibitors (baicalein or ETYA; and indomethacin or ibuprofen, respectively) greatly decreased function and increased contracture at 42°C in a dose dependent manner, and even further at 43°C. The inhibitors decreased function to a greater extent when combined at 42°C (−67.9 Δ%max Force ±2.5). Treatment with the PLA2 inhibitor BEL (20μM) also decreased function at 43°C (−84.3 Δ%max Force ±3.8). Overall, results demonstrate that oxidant stress does not appear to contribute significantly to the loss of function in intact muscle, but AA metabolism through LOX and COX pathways are essential in protecting skeletal muscle during hyperthermia. NHLBI 53333
Fasting affects thermoregulation in the cold, depending on time of the day
Authors: Tokizawa Ken , Uchida Yuki , Nagashima Kei
Published: June 2021

BACKGROUND:

We have shown that circadian rhythm of body temperature (Tb) is finely regulated. However, feeding condition is a factor affecting largely the Tb rhythm. During fasting, Tb gradually decreases in the light (inactive) phase, whereas Tb in the dark (active) phase is maintained at the free‐feeding level. The purpose of the present study was to clarify the mechanism involved in the altered Tb rhythm during fasting.

METHODS:

Male ICR mice (2–3 mo old), used for the present study, were housed at an ambient temperature (Ta) of 27ºC in a 12:12‐h light‐dark cycle. After 48‐h fasting, Ta was decreased to 20ºC at ZT1‐4 (early light phase) or ZT13‐16 (early dark phase). Tb (biotelemetry) and oxygen consumption (VO2, indirect calorimetry) were measured.

RESULTS:

During a cold exposure with fasting, Tb decreased by 5.5 ± 2.0ºC at ZT1‐4, and VO2 remained unchanged. However, Tb decreased by only 2.0 ± 0.5ºC at ZT13‐16, which was greater than that in the light phase (p<0.05), and VO2 increased (p<0.05).

CONCLUSION:

These results indicate that fasting attenuates thermoregulatory response to the cold, and the response differs among time of the day. Factors of feeding condition and time would have influences on the mechanism regulating the Tb rhythm. The possible reason will be presented, based on histological study using c‐Fos immunostaining and molecular analysis of UCP1 mRNA expression in the brown adipose tissue.
Role of hypoxia in muscle function and muscular dystrophy
Authors: Mosqueira Matias M , Baby Santosh M , Budak Murat , Bogdanovich Sasha , Willmann Gabriel , Lahiri Sukhhamay , Khurana Tejvir
Published: June 2021

Respiratory insufficiency contributes significantly to the morbidity and, is the commonest cause of mortality, in Duchene's muscular dystrophy (DMD). However, surprisingly little is known mechanistically about 1, basic respiratory physiology in the mdx (DMD‐mouse model) &, 2, effects of hypoxia (high altitude) on muscle function.

To rigorously evaluate mdx respiration, we custom‐built a plethysmograph (with 1 microlitre sensitivity) and exposed awake adult mdx and control mice to graded levels of hypoxia and hypercapnia to determine respiratory rate (RR), tidal volume (VT), and minute ventilation (VE), HVR and HCVR. Additionally, phrenic nerve discharges were recorded from anesthetized mice to understand the integrated ventilatory response of central and peripheral chemoreceptors. Significant abnormalities were found in mdx mice respiratory parameters during normoxia as well as a blunting of the HVR reflex. Finally, we found anatomical, physiological and transcriptome‐level changes in muscle of animals exposed to hypoxia and high altitude (Mt. Marmolejo & Mt. McKinley). These observations support our hypotheses that dystrophic mice have respiratory abnormalities and that hypoxia itself may contribute as a ‘second‐hit’ to muscle patho‐physiology in DMD.
Preservation of endothelial cell/pericyte adhesion by cilostazol in ischemic limb of diabetic mouse
Authors: Suzuki Tomoharu , Matsushita Shonosuke , Sugimori Haruhiko , Sato Fujio , Sakakibara Yuzuru
Published: June 2021

Purpose: Degeneration of pericytes and acellular capillaries are increased in the feet of human diabetic patients. Selective loss of adhesion of pericyte to endothelial cell is known to early event in diabetic microangiopathy. In diabetes, inflammation due to endothelial dysfunction is augmented. Cilostazol, a PDE III inhibitor, is reported to reduce inflammation and oxidative stress in diabetic patients. Based on these findings, we investigated the effect of cilostazol on endothelial/pericyte adhesion in ischemic hindlimb of diabetic mice.

Method: Type I diabetes was induced by intraperitoneal administration of streptozocin (50mg/kg) for five days in both cilostazol‐fed mice and control‐diet‐fed mice. Resection of femoral artery was conducted to develop hindlimb ischemia. Ten days after the procedure, gastrocnemius muscles were resected. Adhesion of pericyte to endothelial cell was assessed by transmission electron microscopy.

Result: The rate of detachment between endothelial cell and pericyte in cilostazol‐fed mice was 1/15, whereas it was 9/8 in non‐cilostazol‐fed mice (p<0.005).

Conclusion: Cilostazol preserved endothelial cell/pericyte adhesion in ischemic limb of diabetic mouse. Cilostazol may provide improvement of capillary morphology in diabetic foot in addition to vasodilation.
Quantitative proteomic analysis of distinct mitochondrial subpopulations in diabetic myocardium
Authors: Dabkowski Erinne R , Williamson Courtney L , Hollander John M
Published: June 2021

Reactive oxygen species resulting from diabetes mellitus contribute to damage and loss of mitochondrial proteins. Two spatially distinct mitochondria populations exist in the cardiac myocyte, interfibrillar mitochondria (IFM), which situate between the myofibrils, and subsarcolemmal mitochondria (SSM), which exist beneath the sarcolemma. The goal of this study was to determine the impact of diabetic insult on proteomic profiles of distinct mitochondrial populations. Swiss Webster mice were made diabetic by streptozotocin injection and sacrificed following diabetes onset. Using 2D‐differential gel electrophoresis and iTRAQTM technology, we determined that proteomic profiles of IFM were affected to a greater extent than SSM, as a result of diabetes. Specific alterations included decreases in ATP synthesizing proteins (ATP synthase, P<0.05) and electron transport chain proteins (cytochrome C oxidase, P<0.05; flavoprotein ubiquinone oxidoreductase, P<0.001). Further, aconitase, a protein prone to oxidation, was decreased in diabetic IFM (P<0.002), while adenine nucleotide translocase, a protein associated with enhanced permeability transition pore opening, was elevated in diabetic IFM (P<0.05). Our results suggest that the IFM are at greater risk for protein loss as a result of diabetes mellitus, which may contribute to the pathogenesis of diabetic cardiomyopathy.

(Supported by AHA 0665237B)
Effects of short‐term exercise combined with either high or low glycemic diets on skeletal muscle lipid content in older, insulin resistant adults
Authors: Haus Jacob M , Covey Kelly K , Jessberger John A , Brooks Latina M , Kashyap Sangeeta R , Dasarathy Sri , Flask Chris A , Kirwan John P
Published: June 2021

We sought to determine whether the reversal of insulin resistance by short‐term exercise combined with different dietary interventions was mediated by changes in IMCL. We hypothesized that 7 consecutive days of exercise plus a low‐glycemic (LG) diet would lead to greater improvements in insulin sensitivity and IMCL than exercise plus a high‐glycemic (HG) diet. Preliminary data are available for 8 men and women (68±1 yr; 32±1 kg/m2) with impaired glucose tolerance. Insulin resistance was determined using the euglycemic‐hyperinsulemic clamp, and IMCL content was measured by localized magnetic resonance spectroscopy of the soleus muscle. Subjects exercised for 60 min/d at an intensity of ~70% VO2max and were provided eucaloric diets (N=4, HG; N=4, LG). The program did not alter VO2max or body weight. Insulin sensitivity (mg/kg/min) increased (P=0.006) in both groups (HG: Pre 3.0±0.5, Post 3.9±0.5; LG: Pre 2.4±0.1, Post 3.9±0.4) and IMCL content (% change) was significantly different between the HG and LG groups (HG: +49%; LG: −40%, P=0.0004). These data indicate that insulin sensitivity improves with short‐term exercise independent of diet or change in IMCL content. The paradox in IMCL content seen with the diet groups may be reflective of alterations in fuel substrate utilization and storage.

Supported by NIH Grants T32 HL007887 (JMH), AG12834 (JPK) and RR018390
Alteration of mitochondria during adipocyte differentiation and mitochondrial dysfunction leads to insulin resistance of adipocytes
Authors: Wang Chih‐Hao , Wei Yau‐Huei
Published: June 2021

Adipocytes play an important role in the regulation of energy metabolism and maintenance of glucose homeostasis in the human body. Recent studies showed that mitochondrial dysfunction is involved in metabolic diseases, such as insulin resistance and type II diabetes. Here, we investigated the importance of mitochondria in the differentiation and function of adipocytes. We used 3T3‐L1 fibroblasts as a model of adipocyte differentiation. First, we examined the changes of mitochondrial biogenesis during adipogenesis. The results showed that mitochondrial biogenesis‐related genes, including PGC‐1α and mtTFA, were up‐regulated during adipogenesis. Besides, we observed that mitochondrial DNA content and the subunits of respiratory enzyme complexes were slightly increased after differentiation. To further investigate the role of mitochondrial dysfunction in insulin resistance and type II diabetes, we treated adipocytes with respiratory inhibitors, oligomycin and antimycin A, respectively. The results showed that mitochondrial dysfunction led to a significant decrease in the sensitivity of the adipocytes to insulin as revealed by a dramatic decrease in Akt phosphorylation. Taken these findings together, we suggest that upregulation of mitochondrial biogenesis plays an important role in the differentiation of adipocytes to execute aerobic metabolism to maintain their normal physiological functions. On the other hand, we demonstrated that mitochondrial dysfunction can lead to insulin resistance of adipocytes in vitro.
AAV Delivery of gp91‐shRNA Stimulates Synthesis and Release of Insulin and Attenuates Blood Glucose Level in Type 2 Diabetes
Authors: Wang Xianfu , Wang Xiuqing , Sun Zhongjie
Published: June 2021

Hypothesis. RNAi inhibition of gp91 improves insulin resistance and decreases blood glucose level.

Methods and Results. Doubled stranded shRNA for gp91phox was synthesized and packaged into the AAV to generate AAV.gp91‐shRNA. Two groups of db/bb mice and 2 groups of lean mice were used. One group of db/db mice and 1 group of lean mice received IV injection of AAV.gp91‐shRNA (1×1011 particles) while the other 2 groups received the same doses of AAV.Control‐shRNA served as controls. Blood glucose levels were elevated in db/db mice treated with AAV.Control‐shRNA. AAV.gp91‐shRNA significantly decreased blood glucose level. Contradictory to our hypothesis, AAV.gp91‐shRNA failed to improve insulin resistance in db/db mice. Interestingly, AAV.gp91‐shRNA increased plasma insulin level significantly (by 2 folds). Immunohistochemical (IHC) data showed that RNAi inhibition of gp91phox significantly increased insulin staining in islet cells, indicating increased insulin synthesis. These data suggest that RNAi inhibition of gp91phox decreased blood glucose level by increasing insulin release.

Conclusions. AAV delivery of gp91‐shRNA may serve as new approach for improving blood glucose level in type 2 diabetes. NADPH oxidase is not involved in insulin resistance in type 2 diabetes.