bluuebunny/crossref_metadata_2025 huggingface dataset:
Medullary “raphé chemosensory amplifier” (RCA) interneurons are mediated by serotonin/substance P and GABA‐synthesizing neurons in situ
Authors: Iceman Kimberly Erin , Harris Michael Bruce
Published: June 2021

Serotonin/substance P (5‐HT/SP) and GABA‐synthesizing neurons in the rat medullary raphé are stimulated and inhibited, respectively, by elevated CO2 (hypercapnia) in vitro and in situ. The raphé nuclei neurons are heterogeneous with respect to neurotransmitter phenotype and responses to hypercapnia. We have identified a type of chemosensitive raphé neuron that is stimulated by hypercapnia but is not serotonergic. We test the hypothesis that these CO2‐stimulated “raphé chemosensory amplifier” (RCA) interneurons receive critical network input from 5‐HT/SP and GABA neurons, mediated by 5HT2A, NK‐1, and GABAA receptors. We assess chemosensitivity of individual raphé RCA interneurons before and after bath pharmacological antagonism of these receptors in the unanesthetized juvenile rat in situ perfused decerebrate brainstem preparation. Coupled with juxtacellular labeling and subsequent immunohistochemistry, we demonstrate RCA interneurons are non‐serotonergic, express NK‐1 receptors, and are dependent on 5HT2A and GABAA receptors for tonic drive and chemosensitivity, suggesting a raphé network sensitive and responsive to hypercapnia. Supported by NIH 2U54NS041069–06A1 and P20GM103395.
The function of cAMP responsive element modulator (CREM) in PDGF induced proliferation of vascular smooth muscle cells
Authors: Seidl Matthias Dodo , Hildebrandt Iris , Klugstedt Christian , Nunes Frank , Endo Shogo , Kojima Nobuhiko , Schmitz Wilhelm , Müller Frank Ulrich
Published: June 2021

The transcription factor CREM plays an important role in cAMP mediated gene expression. Here we want to clarify the role of CREM in vasculature particularly with regard to the regulation of VSMCs proliferation, using CREM (CKO) and CREM isoform ICER (IKO) deficient mice.

CKO mice showed an increase of neointima formation associated with a higher proliferation rate of aortic VSMCs under platelet‐derived growth factor (PDGF) treatment. The PDGF alpha receptor (Pdgfra) promoter was inducible by cAMP and mRNA was upregulated in aortae and PDGF‐treated primary VSMCs of CKO mice. In untreated IKO‐VSMCs the proportion of proliferating cells was higher as compared to WT‐VSMCs, while no differences were observed under PDGF treatment. Induction of cAMP by forskolin reduced the proliferation rate of both IKO and WT‐VSMCs under PDGF treatment.

Accordingly, ICER was linked to the elevated proliferation of VSMCs under non‐stimulated conditions, however, other CREM isoforms than ICER inhibit PDGF‐mediated proliferation of VSMCs possibly by modulation of cAMP‐dependent Pdgfra gene regulation. (supported by DFG)
Molecular Interactions of Serotonin (5‐HT) and Endothelin‐1 (ET‐1) in Vascular Smooth Muscle Cells (VSMC): roles of p42/44 mitogen activated protein kinase (MAPK) and Janus Kinase (JAK) 2 pathways
Authors: Bhaskaran Subha , Zaluski Jeremy , Banes‐Berceli Amy K. L.
Published: June 2021

Diabetes mellitus causes renal and cardiovascular disease complications and evidence suggests elevated levels of 5‐HT and ET‐1 may be involved. Previous data suggests supraphysiological concentrations of 5‐HT can potentiate ET‐1's ability to stimulate DNA synthesis and VSMC proliferation in vitro and may involve activation of p42/44 MAPK and JAK2 signaling pathways. The molecular mechanisms are not yet known. We tested the hypothesis that synergistic interaction between 5‐HT and ET‐1 at physiological concentrations would activate both p42/44 MAPK and JAK/STAT pathways to cause enhanced growth responses. Using cultured rat VSMC and Western Blot analysis our data suggest no effect of acute pre‐incubation with physiological concentrations of ET‐1 and 5‐HT on the activation of p42/44 MAPK and JAK2 proteins in normal or diabetic conditions. However, utilizing myographs to measure contractile responses of rat aorta we found that physiological levels of 5‐HT and ET‐1 each stimulate a potentiation of the contractile response. In contrast to the in vitro data, our ex vivo data suggests that physiological concentrations of 5‐HT and ET‐1 each stimulate enhanced contractile response in aorta. Hence, understanding the mechanisms of 5‐HT/ET‐1 interaction in diabetic disease conditions would provide a route for novel therapies to prevent the development of vascular injury as a complication of diabetes. (NHLBI R00 AKLB)
The G protein‐coupled estrogen receptor 1 (GPER1/GPR30) agonist G‐1 regulates vascular smooth muscle cell Ca2+ handling
Authors: Holm Anders , Olde Björn , Leeb‐Lundberg Fredrik , Nilsson Bengt‐Olof
Published: June 2021

The novel G protein‐coupled estrogen receptor GPER1/GPR30 is implicated in blood pressure regulation but the mechanisms involved have not been identified. Here, we hypothesize that GPER1 controls blood pressure by regulating vascular smooth muscle cell Ca2+ handling. Treatment with the GPER1 agonist G‐1 (1 μM) reduced frequency and amplitude of the spontaneous and synchronous Ca2+ spikes acutely in A7r5 vascular smooth muscle cells expressing mRNA for GPER1. Stimulation with U46619 increased basal Ca2+ level and Ca2+ spike frequency. G‐1 attenuated U46619‐stimulated Ca2+ spike activity but had no effect on U46619‐induced increase in basal level of Ca2+. The GPER1 antagonist G‐15 blocked in part, but not completely, G‐1‐induced down‐regulation of Ca2+ spike activity suggesting that the attenuation of Ca2+ spike activity by G‐1, at least partially, involves GPER1. We established a stable GPER1 knock‐down A7r5 cell clone showing down‐regulation of GPER1 transcript by about 50%. The GPER1 knock‐down cells possessed Ca2+ spike activity and responded to U46619 in a similar manner as the control cells. G‐1 (1 μM) reduced Ca2+ spike activity also in the GPER1 knock‐down cells. In conclusion, we demonstrate that the GPER1 agonist G‐1 regulates vascular smooth muscle cell Ca2+ handling at least partially through GPER1. Supported by the Royal Physiographical Society and Vascular wall program.
Inhibition of Soluble Epoxide Hydrolase Augments Hypoxic Pulmonary Vasoconstriction and Improves Gas Exchange in Mice
Authors: Beloiartsev Arkadi , Ventocilla Christian , Winkler Jeffrey D. , Bloch Kenneth D. , Zapol Warren M.
Published: June 2021

Recent studies report that cytochrome P‐450‐derived epoxyeicosatrienoic acids (EETs) increase pulmonary vascular tone. We hypothesized that inhibiting the metabolism of EETs by inhibition of soluble epoxide hydrolase (sEH) would augment hypoxic pulmonary vasoconstriction (HPV) and improve gas exchange.

Mice were anesthetized, intubated, and ventilated at FIO2=1. Animals received an i.v. infusion of the sEH inhibitor, IK‐950, or vehicle 30 minutes prior to assessing HPV. After thoracotomy, pulmonary artery pressure (PAP) and left lung blood flow (QLPA) were measured before and during transient inferior vena cava occlusion. Left lung pulmonary vascular resistance (PVRL) was calculated from the slope of the PAP‐QLPA relationship. The pulmonary vasoconstrictor response to unilateral left lung hypoxia was estimated by comparing PVRL before and after left main stem bronchial occlusion (LMBO).

Infusion of vehicle or IK‐950 did not change baseline hemodynamic parameters. LMBO increased PVRL, without changing PAP in both vehicle‐and IK‐950‐pretreated mice. Increase in PVRL during LMBO was greater in mice pretreated with IK‐950 than in those pretreated with vehicle (Table). PaO2 after LMBO was greater in mice pretreated with IK‐950 than in those pretreated with vehicle.

Taken together, these results indicate that IK‐950 acts as a selective pulmonary vasoconstrictor of hypoxic lung regions, enhancing the perfusion of ventilated lung regions.
Underdeveloped bradykinin‐dependent vasorelaxation in immature pulmonary arteries from long term hypoxic sheep is not due to loss of cGMP signaling
Authors: Wee Chelsea L , Blum‐Johnston Carla , Blood Quintin , Wilson Rachael H , Blood Arlin B , Longo Lawrence D , Wilson Sean M
Published: June 2021

Endothelial dependent relaxation of the pulmonary vasculature is important during the fetal transition to breathing air. Our studies show bradykinin‐mediated relaxation of the pulmonary vasculature is reduced before birth and that postnatal maturation of nitric oxide (NO) cGMP‐mediated signaling pathways is important to this process. Thus, we tested whether cGMP‐mediated vasorelaxation was blunted before birth by performing wire‐myography on pulmonary arteries (PA) isolated from term‐fetal, newborn, and adult sheep exposed to long term high altitude (3,801 m) hypoxia for >;100 days. The efficacy and potency for serotonin‐induced contraction were evaluated following YC1 activation of soluble guanylate cyclase (sGC) or when cGMP degradation was inhibited with the type 5 phosphodiesterase (PDE5) blocker sildenafil. YC1 and sildenafil decreased the potency for serotonin contraction more prominently in vessels from adults as compared to those from immature sheep. Yet, these drugs reduced the efficacy similarly in nearly all groups, with the exception of adult sheep where YC1 failed to alter the maximum contraction. Overall, sGC and PDE5 dependent cGMP signaling pathways are viable targets for reducing arterial tone and are relevant therapeutic avenues for pulmonary hypertension in newborns and adults. (NSF MRI 0923559, NIH R03HD069746, P01HD031226, R01HD003807, R01HL95973).
Determination of Prdx6 binding sites for its interaction with SP‐A
Authors: Krishnaiah Saikumari Y , Dodia Chandra , Feinstein Sheldon I , Fisher Aron B
Published: June 2021

Our previous studies have shown that the PLA2 activity of Peroxiredoxin 6 (Prdx6) is inhibited by surfactant protein A (SP‐A) through direct protein‐protein interaction. In silico studies using the ZDOCK program (http://zlab.bu.edu/zdock/) suggested that the Prdx6 sequence (195EEEAKKLFPK204) is involved in the interaction with SP‐A. The ability of a peptide comprised of this sequence (Prdx6 peptide) and a scrambled peptide control (PAEKLKAFEKE) to bind to SP‐A were studied by Isothermal Titration Calorimetry at 25°C at pH 4 and pH 7. The resulting data revealed that Prdx6 peptide bound to SP‐A in an exothermic reaction, while the scrambled peptide did not bind. SP‐A reduced PLA2 activity of Prdx6 from 98±3 to 29±3 at pH 4 and from 50±3 to 30±2 nmol/min/mg at pH7. However, in the presence of the Prdx6 peptide, SP‐A inhibition of PLA2 activity was partially reversed (70±2 nmol/min/mg at pH 4 and 43±1 at pH 7). Results for phosphorylated Prdx6 (pPrdx6) were even more striking as SP‐A reduced the PLA2 activity of pPrdx6 from 1659±26 nmol/min/mg to 31±0.4 at pH 4 and from 1712±69 to 34±2 at pH 7 while the presence of the Prdx6 peptide largely reversed the inhibition by approximately 85%. The scrambled peptide had no effect at either pH. Thus, the peptide appears to block the SP‐A binding site on Prdx6 indicating that the identified Prdx6 sequence is involved in the interaction with SP‐A, which results in termination of PLA2 activity.
Caveolin‐1 regulates neuronal regeneration in peripheral nerve crush injury via regulation of Schwann cell function
Authors: Garcia Selma Dee , Dolkas Jennifer , Shubayev Igor , Kellerhals Sarah Elizabeth , Schilling Jan M , Shubayev Veronica I , Patel Hemal H , Kim Youngsoon
Published: June 2021

Peripheral nerve damage occurs in a variety of settings leading to loss of motor function and potential neuropathic pain. Peripheral nerve injury involves complex changes in Schwann cell signaling and metabolism that ultimately lead to regulation of nerve regeneration, though these mechanisms are poorly understood. Caveolins (Cav) are scaffolding proteins that exist in lipid rafts and regulate a myriad of signal transduction. We hypothesized that Cav is critical to Schwann cell response following nerve injury. Knockdown of Cav‐1 slows the speed of axonal regeneration following nerve crush injury. We also observed increased extracellular regulated kinase 1/2 phosphorylation and cell mitosis in Cav‐1 KO nerves compared to wild‐type following injury. Myelin debris clearance and macrophage content declined in Cav‐1 KO nerves. Electron microscopy revealed loss of Schwann cell mitochondrial in cells distal to the crush site. JS1 Schwann cells showed upregulated mitochondrial function via measurement of ATP synthesis and mitochondrial respiration using Seahorse Metabolic Flux Analyzer when Cav‐1 was upregualted. Our studies suggest that loss of Cav‐1 leads to compromised Schwann cell function leading to diminished nerve regeneration following injury and that Cav‐1 targeted to Schwann cells may be a means to positively regulate Schwann cell function in the setting of injury.
Neuroprotective properties of antioxidants in stroke correlate with their effects on ischemic release of glutamate
Authors: Dohare Preeti , Vipani Aarshi , Hyzinski‐Garcia Maria C. , Bowens Nicole H. , Feustel Paul J. , Keller Richard W. , Jourd'heuil David , Mongin Alexander A.
Published: June 2021

Antioxidant agents potently protect against ischemic brain damage in animals, but show no clinical benefits in humans. In the present study we tested the hypothesis that potency of antioxidants in preventing pathological glutamate release can predict their ability to reduce ischemic infarction. Transient focal ischemia in rats was induced by 2‐hr occlusion of the middle cerebral artery. Antioxidants Tempol and Edaravone (500 nmols) were injected into the lateral ventricles prior to ischemia. 72 hrs later, control and antioxidant‐treated animals were evaluated for behavioral deficits and brain damage. Tempol, but not Edaravone, reduced infarction volumes by ~60% and improved neurological outcomes. In parallel microdialysis experiments, Tempol but not Edaravone reduced extracellular levels of the excitatory neurotransmitter glutamate. Paradoxically, when tested in antioxidant assays in vitro, Edaravone was more effective in scavenging the majority of the ischemia related free radicals and oxidants. The only exception was scavenging of superoxide anion, in which Tempol was superior to Edaravone. Overall, these results suggest that neuroprotective properties of Tempol are determined by its ability to scavenge superoxide and prevent pathological glutamate release.
Regulation of cell motility by Na+/H+ exchanger NHE1: implications for cancer development
Authors: Pedersen Stine Falsig
Published: June 2021

Essential roles of ion channels and transporters in regulation of cell motility have been discovered within the last decade. Here, I review recent evidence from our laboratory and others regarding the roles of the Na+/H+ exchanger NHE1 in cell motility, with particular focus on the implications for cancer metastasis. In many cell types, NHE1 localizes to the leading edge of migrating cells and regulates directional cell motility by mechanisms which include the formation of intra‐ and extracellular pH‐gradients and likely also direct protein‐protein interactions and local cell volume changes. In addition, NHE1 localizes strongly to invadopodia: actin‐rich structures assigned essential roles in invading cells. Here, NHE1 appears to be involved in both invadopodia formation and in their extracellular matrix‐degrading function. NHE1 is upregulated in a wide range of human cancers, and inhibition/knockdown of NHE1 attenuates metastasis in several cancer models. However, under some conditions, NHE1 inhibition/knockdown increases adhesiveness and motility, suggesting that the specific role of NHE1 is context‐dependent. In conclusion, NHE1 plays important roles in controlling cell motility. However, to exploit the obvious potential of interfering with NHE1 function to treat cancer metastasis, greater mechanistic understanding of the mechanisms is needed.

Funding: Danish Cancer Society, Danish Research Council
The role of Lysophosphatidic acid in erythropoiesis in K562 human erythroleukemia cell line
Authors: Ho Ya‐Hsuan , Lin Kuan‐Hung , Lee Hsinyu
Published: June 2021

Lysophosphatidic acid (LPA) is an essential factor in the regulation of cellular responses through activating different LPA receptors (LPARs), which belong to a family of G‐protein‐coupled receptors. While LPA has been widely studied for its effects on cardiovascular system development and cancer metastasis, the relationship between LPARs and erythropoiesis remains unclear. The aim of this study is to use human erythroleukemia cell line K562 as a model to investigate the role of LPA and LPARs in erythorocyte development. Our data demonstrated that exogenous addition of LPA had no significant effect on hemin‐induced erythropoiesis by the detection of γ‐globin mRNA expression. However, treatment with Ki16425, a specific antagonist for LPAR1 and LPAR3, significantly decreased γ‐globin mRNA levels. Moreover, treatment with LPAR2 specific agonist, mono‐dodecylphosphate (MDP), also resulted in the reduction of γ‐globin mRNA levels. These results suggested that LPAR1/3 and LPAR2 reciprocally regulate erythropoiesis in K562 cells.
Chronic binge alcohol‐induced skeletal muscle wasting: Implications for satellite cell function
Authors: Decker Laura Elizabeth , LeCapitaine Nicole J , Bagby Greg J , Nelson Steve , Molina Patricia E.
Published: June 2021

Chronic alcohol abuse is associated with skeletal muscle (SKM) myopathy in 40–60% of alcohol abusers. Alcohol‐induced myopathies result from decreased muscle protein synthesis and possibly accelerated muscle proteolysis. The contribution of impaired muscle regenerative capacity is unknown. Muscle stem cells, satellite cells (SC), repair and regenerate muscle tissue. The impact of chronic binge alcohol (CBA) on SC function is unknown. We hypothesized that CBA alters SC phenotype impairing their repair and regeneration capacity. To test this hypothesis, SKM was isolated from macaques after 19 mo daily intragastric sucrose‐or alcohol‐administration. SC regenerative potential was assessed by RT‐qPCR analysis of myogenic transcription factor expression (PAX7, MEF2C, MYOD, MYF5, MYOG). CBA significantly decreased MYF5 expression, a key differentiation transcription factor. Additionally, using picrosirius red staining, we found evidence for SKM fibrosis in CBA animals. Collagen Type I, an extracellular matrix protein secreted during fibrosis, inhibits SC differentiation. These results suggest that CBA impairs SC differentiation potential directly though inhibition of differentiation transcription factor expression and indirectly through SKM fibrosis. We speculate this leads to impaired SKM repair and regeneration and thus, the ability to maintain muscle mass. Support: AA09803, AA11290
Polyamines enhance satellite cell activation and expression of myogenic regulatory factors
Authors: Thornton Kara Jean , Chapalamadugu Kalyan C , Doumit Matthew E , Murdoch Gordon K
Published: June 2021

Satellite cells (SCs) exist amongst other cell types in a unique microenvironment within skeletal muscle. The interactions between SCs and non‐myogenic cells within this niche are poorly characterized. However, most cells behave differently in co‐culture (CC) compared to mono‐culture (MC). Our results indicate that SCs co‐cultured with preadipocytes express less myogenic differentiation factor (MyoD) at 24 (p=0.04) and 48 (p=0.02) hours as well as more sprouty1 (Spry1) at 24 (p=0.04) hours post‐differentiation. Thus, SCs induced to differentiate in a CC with preadipocytes exhibit depressed myogenesis when compared with SCs in MC. Polyamines (putrescine, spermidine and spermine) are potent modulators of cell growth. However, no studies have analyzed effects of these compounds on SCs. Administration of polyamines to SCs in MC and CC systems demonstrates that polyamines increase (p≤0.05) expression of MyoD, myogenin (Myf4), myogenic factor 5 (Myf5) and decrease (p≤0.05) expression of paired box transcripton factor 7 (Pax7) and Spry1. Compared with MC, our CC system better reflects the complexity of the SC niche. Furthermore, our observation of a pro‐myogenic effect of polyamines using CC may indicate a potential natural means of enhancing myogenesis in vivo. We gratefully acknowledge the University of Idaho's Student Grant Program, the USDA NC1184 Hatch Grant and the Idaho Beef Council FY 2013 for funding.
Ion transport across the murine retinal pigment epithelium
Authors: Arnason Sighvatur Saevar , Skarphedinsdottir Sunna Björg , Eysteinsson Thor
Published: June 2021

The retinal pigment epithelium (RPE) is important for the normal transport of ions and water from the subretinal space. Due to the small size of mice, ion transport across fresh murine RPE has not been studied in vitro. Sheets of RPE with the retina attached from normal mice were mounted in EasyMount Ussing chambers with an aperture of 0.031 cm2 (Physiological Instruments Inc) with aireated 38°C Krebs. The short‐circuit current, ISC, and the transepithelial resistance, TER, was recorded with a voltage clamp apparatus. Initial ISC (n=7) was −17,6 ±4,5 μA/cm2, with apical side negative, and TER was 82,8 ±7,6 Ohm*cm2. The following substances were tested (30 min each, both apical and basolateral sides): ATP (100 μM) had no effect on the ISC. Epinephrine (100 μM) increased the ISC by 5%. Bumetanide (100 μM) inhibited the ISC from −16.1 ± 5.2 to −14.5 ± 5.4 μA/cm2. Ouabain (1 mM) induced a biphasic response, a rapid 6 min increase to −17.4 ± 2.6 μA/cm2, and a slower 30 min decrease to 36% of pre‐addition ISC. The Isc was reduced by low Cl− Krebs. Reducing the K+ concentration on the apical side decreased the Isc, which was inhibited by 1 mM BaCl2. The mouse RPE preparation was stable for 2–3 hours. The results indicate that adrenergic but not purinergic receptors affect murine RPE ion transport as measured by the ISC. The ion transport is likely due to Cl− transport with some K+ involvement, with the NaK‐ATPase serving as an important driving force.
Mechanisms of interferon gamma‐induced suppression of aquaporin 1 in murine intestinal epithelium
Authors: Dicay Michael , Ronaghan Natalie , Peplowski Michael , Carati Colin , MacNaughton Wallace K.
Published: June 2021

Chronic diseases like IBD are characterized by high levels of IFNγ and abnormal intestinal transepithelial fluid flux. The water channel protein Aquaporin 1 (AQP1) is widely expressed on intestinal epithelial cells with the primary purpose of regulating transmembrane water flux. To investigate the deficiencies in water transport associated with IBD, we sought to determine the cellular signaling mechanisms by which IFNγ modulates AQP1 expression in the mouse intestinal epithelium.

CMT93 cells treated with IFNγ significantly decreased constitutive AQP1 levels detected by immunofluorescence. Pre‐treatment with either the JAK1 inhibitor or STAT3 siRNA, prior to IFNγ administration resulted in the amelioration of the IFNγ‐induced suppression of AQP1 protein expression. Conversely, IRF‐2 siRNA pre‐treatment further decreased AQP1 protein levels following IFNγ treatment.

Inflammatory cytokines present during IBD are able to influence AQP1 levels and may contribute to the water transport deficiencies associated with these disorders. In this study, the results suggest that decreased epithelial AQP1 expression exerted by IFNγ in CMT93 cells is regulated by JAK1 and STAT3. Of note, IRF‐2 which is normally considered to be a negative regulator of IFNγ signaling, acted in a positive regulatory fashion by further decreasing AQP1 protein signal.

Source of funding: Crohn's & Colitis Foundation of Canada
Sex differences in the effects of β‐estradiol on ENaC current in cell culture
Authors: Mitzelfelt Jeremiah D , Greenlee Megan M , Eaton Douglas C
Published: June 2021

Using cell‐attached patch clamp, we have previously shown that β‐estradiol (E2) activates epithelial sodium channels (ENaC) in alveolar cells. In an attempt to develop a high throughput electrophysiological assay to test the effect of estradiol (E2) and estrogen receptor agonists/antagonists on ENaC, we examined the effect of E2 on amiloride‐sensitive transepithelial current (TEC) in two kidney cell lines (2F3 and mpkccd) with known ENaC activity. E2 had no effect on amiloride sensitive current in either of these cell lines. The lack of an E2 response in the 2F3 and mpkccd cells may result from differences in tissue origin (lung vs. kidney) or sex of the donor animal (male for 2F3 and mpkccd cells, female for L2 cells). To address this question, we measured TEC in MDCK cells, a kidney cell line from a female dog. E2 increased amiloride‐sensitive TEC in MDCK cells. These data suggest that E2 increases ENaC current in females only and may play a role in sex differences in the rate and severity of diseases involving ENaC.

Funding: NIH R37DK037963, K12GM000680 and T32HL076118
The interactive effects of K+ and Cl− on force generation in skeletal muscles: impact at the onset of exercise
Authors: Higgins Amanda Marie , Renaud Jean‐Marc
Published: June 2021

It is now established that interstitial [K+] ([K+]int) increases to 12–14 mM even during moderate exercise. Although it is known that such increases in [K+]int contribute to the decrease in force during fatigue, recent studies suggest that at the onset of exercise small increases in [K+]int actually potentiates force and K+‐induced force depression is prevented by decreases in Cl− conductance (GCl). However, most studies were carried out at temperatures ranging from 25°–30°C or under very specific stimulation frequencies. The objective of this study was to document how the [K+]int – force relationship is modulated at 37°C, at different frequencies and under different GCl in order to better understand how muscle performance can be maximized at the onset of muscle activity.

At a stimulation frequency allowing for maximal tetanic force, soleus muscle started to lose force at 10 and was completely lost by 13. EDL muscles were less sensitive to [K+]int, such that loss of force started at 13 and was completely lost by 15mM. The critical [K+]int required for tetanic force depression to start at 37°C is much higher than the critical [K+]int reported at lower temperatures. Twitch force was potentiated by 80–100% as [K+]int was elevated from 8–11 for soleus and from 8–13.5mM in EDL, while further increases in [K+]int caused a gradual decline in twitch force. Potentiation up to 150% can also occur at frequencies of 1–30Hz in soleus, and 1–100Hz for EDL. The major effect of reducing GCl was an increase in the critical [K+]int that caused a depression of force.
Resistance Training and CLA/n‐3 Administration Improve Myofiber Size and Myogenic Capacity in High Fat Diet‐Fed Mice
Authors: Lee Sang‐Rok , Jo Edward , Khamoui Andy V , Park Bong‐Sup , Zourdos Michael C , Panton Lynn B , Ormsbee Michael J , Kim Jeong‐Su
Published: June 2021

Conjugated linoleic acid (CLA) and omega‐3 fatty acids (n‐3) have received great attention due to their health enhancing benefits. However, the morphogenetic capacity of skeletal muscle during high fat diet (HFD)‐induced obesity and the efficacy of CLA/n‐3 remain to be established. Therefore, we investigated the effects of 20 wks of CLA/n‐3 administration and resistance exercise (RE) on muscle cross sectional area (CSA) and myogenic capacity in HFD‐fed mice. Once sacrificing eight C57BL/6 male mice (age=9 mo), the remaining animals were randomly assigned to five experimental groups (n=10/group): 1) normal diet (C), 2) high fat diet (H), 3) HFD + RE (HE), 4) HFD + CLA/n‐3 (HCN), or 5) HFD + RE + CLA/n‐3 (HECN). Progressive RE was administered on a ladder climbing device 3d/wk for 20 wks. The supplement consisted of 1% CLA and 1% n‐3. Immunohistochemical techniques were employed to evaluate myofiber CSA and the numbers of satellite cell (SC) and myonuclei. Data were analyzed with one‐way ANOVA and significance was set at p<0.05. Muscle CSA significantly reduced in H (−23%) and HCN (−19%) from baseline while maintained in C, HE, and HECN. Only H exhibited a significant decrease in the numbers of SC (−17%) and myonuclei (−19%) from baseline with no change in C, HE, HCN, and HECN. Our findings indicate that 20‐wk HFD induced myofiber atrophy and impaired myogenic capacity. These impairments were improved partially by RE and/or CLA/n‐3 administration.

Study partly supported by Sekwang Inc., Vital Pharmaceuticals, and Ocean Nutrition
Unique roles for 3 specific amino acids in the control of food intake
Authors: Jordi Josua , Lutz Thomas A , Verrey François
Published: June 2021

To maintain nutrient homeostasis the central nervous system integrates signals that promote appetite or satiety. In mammals a protein preload decreases food intake more profoundly than a carbohydrate‐ or a fat preload. We hypothesized that this effect is based on the sensing of specific amino acids (AA) as a signal to control food intake. Orally applied Arg, Lys and Glu decreased subsequent food intake by 50%, 47%, and 35%, respectively, whereas all other proteogenic AA had no effect relative to water control in rats. Decreased food intake correlated with gastric distension as isovolumetric doses of Arg, Lys and Glu increased stomach wet weight by 220%, 260%, and 144%. These behavioral and autonomic responses are controlled by neurons in specific brain areas. Increased neuronal activity, tested by counting c‐Fos positive cells, was detected in the blood‐sensing area postrema and the nucleus of the solitary tract. To test whether circulating AA directly signal to the brain, we administered Arg, Lys and Glu intravenously (2mmol/kg). All three AA induced an anorectic response that was similar to the one induced after oral application. Surgical lesion of the area postrema abolished the anorectic responses of Arg and Glu but not of Lys. We presume that Arg and Glu act in the area postrema to cause the anorectic response, while Lys might stimulate hepatic vagal afferents projecting to the nucleus of the solitary tract.
Transcytosis of insulin across microvascular endothelium
Authors: Azizi Paymon Michael , Wang Changsen , Armstrong Susan M , Klip Amira , Lee Warren L
Published: June 2021

Circulating plasma insulin concentrations are higher than those of the interstitium, suggesting that transport across the microvascular endothelial layer is the rate‐limiting step. It is hypothesized that the metabolic syndrome, characterized by insulin resistance, is influenced by alterations in the trans‐endothelial transport of insulin. However, little is known about how insulin crosses the microvascular endothelium, although in principle it could occur via a paracellular or transcellular route. To date, technical limitations have limited the study of insulin transcytosis. Here we show the development of transwell and total internal reflectance fluorescence microscopy (TIRFM) assays to quantify transcytosis of insulin across endothelial monolayers. The transwell assay measures only the transcellular permeability of insulin and TIRFM measures the fusion of insulin‐containing vesicles at the basal membrane; neither assay is affected by paracellular leak. Using these complementary methods, we show that insulin transcytosis across tissue microvessels is a receptor‐mediated process. Further, that microtubule depolymerization decreases insulin transcytosis. In conclusion, we have developed methods for quantifying the transcytosis of insulin across the microvascular endothelium. We will thus discuss insulin transcytosis under conditions mimicking the metabolic syndrome.
Chronic Carbon Monoxide Treatment Attenuates the Development of Obesity and Remodels Adipocytes in Mice Fed a High Fat Diet
Authors: Hosick Peter Andrew , Al Amoudi Abdulhadi A , Storm Megan V , Gousset Monette U , Pruett Brandon E , Gray Will , Stout Jacob , Stec David E
Published: June 2021

Several studies have demonstrated chronic induction of heme oxygenase‐1 can attenuate the development of obesity; yet, the specific role of its metabolite, carbon monoxide (CO), in mediating this response is not known. The objective of this study was to test the hypothesis that increases in CO mediate the effects of HO‐1 induction in obesity. Mice fed a 60% fat diet were treated with the CO releasing compound, CORM‐A1 (5 mg/kg body weight, i.p.) or the inactive compound iCORM‐A1 (iCO) every 48‐hours for 30 weeks. At 30 weeks of treatment body weights of mice receiving CO injections were significantly reduced compared to iCO treated mice (38 ± 1 vs. 43± 1 grams; p<0.05, n=9/grp). Oxygen consumption measured at 27 weeks of treatment was significantly increased in CO treated mice versus iCO treated mice (96 ± 3 vs. 81 ± 5 ml/LBM/min; p<0.05, n=9/grp). CO treatment decreased the size of epididymal adipocytes compared to iCO treatment (2911 ± 343 vs. 6067 ± 339 μm2; p<0.05, n=3/grp). Chronic CO treatment increased levels of proteins involved in mitochondrial biogenesis such as: NRF‐1, PGC‐1α, Nrf‐2, and UCP‐1 (p<0.05) in adipocytes. In conclusion, our results show chronic CO treatment promotes weight loss and enhances metabolism via up‐regulation of mitochondrial biogenesis, mitochondrial uncoupling, and the remodeling of white adipose tissue thus establishing low level chronic CO treatment as a novel anti‐obesity treatment.
Angiogenesis inhibitor, Sunitinib significantly reduces adipose tissue mass in high fat diet‐induced postmenopausal obese mice
Authors: Gu Jian‐Wei , Makey Kristina L , Chinchar Edmund , Howie Carissa , Miele Lucio
Published: June 2021

We previously reported that postmenopausal obesity is associated with increased angiogenesis in adipose tissue. The Present study determines whether Sunitinib is effective in treating postmenopausal obesity. Ovariectomy (OVX) was performed in 16 eighty week‐old C57BL/6 mice, followed by a high‐fat (60%, HF) diet. After 4 weeks of HF diet, one group received Sunitinib (20 mg/kg/d, IP, n=4) or DMSO (IP, control, n=4), and another group received oral Sunitinib (65±5 mg/kg/d, n=4) or water (control, n=4), the mice continued on HF diet for two weeks. The lean mass (LM) or fat mass (FM) composition was monitored by EchoMRI weekly. Both IP and oral Sunitinib significantly reduced body weight (54.1±2.9 to 39.2±2.3 g and 49.5±2.1 to 32.5±2.5 g, respectively; P<0.01), due to reduced FM (21.6±1.5 to 7.9±0.5 g and 24.9±2.5 to 12.2±0.9 g, respectively; P<0.01), with no changes in LM. Sunitinib caused two‐fold reduction in visceral fat compared to the control group. Oral Sunitinib also reduced food intake compared to the control group. CD31 immunohistochemistry showed a decreased ratio of capillaries to adipocytes. These results support the hypothesis that anti‐angiogenesis therapy could be a good strategy to treat obesity. (NIH/AA013821 and NIH/HL051971)
New experimental model of gastric carcinogenesis in rats with the use of formaldehyde and hydrogen peroxide mixture
Authors: Senchukova Marina , Stadnikov Alexsander A , Bokov Dmitry A
Published: June 2021

This study was designed to develop an animal model of gastric cancer by using a mixture of formaldehyde and hydrogen peroxide. Thirty‐five outbred rats were used. In experimental group, 1.0 ml of the formaldehyde (0.4%) and hydrogen peroxide (0.4%) mixture (1:1) were administered to 30 rats by intragastric gavage on the 1st and 3rd day every 2 weeks for 6 months. Five non‐treated rats served as a control. The mortality rate among treated rats was 13.3%. We found that 6 months after the onset of treatment the 4 of 5 rats had gastric tumor, while after 7.5 months all survived 21 rats showed malignant tumors of the stomach. The tumors were primarily localized within the gastric mucosa, though the invasion of individual tumor cells and their clusters in the muscularis propria and serosa as well as in the lymphatic and blood vessels were also seen. In most cases, the infiltrated gastric mucosa areas (2 to 3) had rigid gastric folds and were 0.7×1.0cm to 1.5×3.0cm in size. We observed signet‐ring cell carcinomas in 80.9% of rats, well‐differentiated tubular adenocarcinoma in 66.6% of rats and moderately differentiated tumors in 23.8% of rats. Metastasis in regional lymph nodes were noted in 3 rats (14.3 %), while 6 animals (28.6%) revealed peritoneal dissemination of the small intestine. In addition, 19.0% of rats showed tumors of the lungs. Therefore, these data demonstrate that our animal model can be used to study gastric carcinogenesis.
Resveratrol relaxes cholecystokinin‐ and KCl‐induced tension in guinea pig gallbladder strips by inhibiting extracellular Ca2+ entry
Authors: Kline Loren W , Karpinski Edward
Published: June 2021

Resveratrol (Res) belongs to a class of polypheloic compounds called stilbenes and is a phytoalexin. Res relaxed cholecystokinin octapeptide (CCK) or KCl‐induced tension. The relaxation was concentration dependent. An in vitro technique was used. Paired t‐tests were used. Differences between mean values with p<0.05 were considered significant. To determine if the PKA/cAMP second messenger system mediated the Res‐induced relaxation, PKA inhibitor 14–22 amide myristolated (PKA‐IM; 180 nM) was used and had no significant effect on Res‐induced relaxation. Bisindolymaleide IV (0.5 μM) and chelerythrine Cl− (5 mM)were used together, a significant (p<0.05) increase in Res‐induced relaxation (61.8±3.5 vs. 73.7±5.6% was observed. 2‐APB (125 μM), significantly (p<0.01) decreased the amount of Res‐induced relaxation (59.7±3.6 vs. 45.9±5.6%). Genistein (10 μM) significantly (p<0.01) increased the Res‐induced relaxation (53.4±2.2 vs. 61.8±3.8%). Neither L‐NMMA (20μM), KT5823 (585 nM), nor fulvestrant (10 μM had a significant effect on the amount of Res‐induced relaxation. Adding Res (75μM) prior to the KCl significantly (p<0.001) decreased the amount of KCl‐induced tension (0.85±0.06 vs. 0.43±0.04 g). Since both CCK and KCl act via L‐type Ca2+ channels and the amount of tension generated is significantly decreased with the prior addition of Res, Res likely exerts its effects via L‐type Ca2+ channels.
Effects of fibrates on duodenal contractions
Authors: Peuler Jacob D. , Belisle Danica , Phelps Laura E.
Published: June 2021

Fibrates (e.g. gemfibrozil) are a class of lipid‐lowering drugs that have been used clinically for over 80 years. But fibrates cause adverse gastrointestinal (GI) side effects, which occur with them all but more frequently with gemfibrozil. Our recent work showed that gemfibrozil could inhibit spontaneous contractility of intact duodenal tissues isolated from female rats. If this occurs in vivo in the form of decreased motility, it would help explain such side effects. In the present work, we found that gemfibrozil can also inhibit spontaneous contractions in duodenum from male rats. Secondly, we found that bezafibrate and fenofibrate can also inhibit spontaneous duodenal contractions but gemfibrozil does so to a greater extent. Thirdly, we found that pretreatment with tetrodotoxin did not affect gemfibrozil's ability to inhibit such contractions. Thus, that ability is not due to an action of gemfibrozil on enteric neuronal activity. Finally, we found that contractions induced by acetylcholine and 5‐hydroxytryptamine can be inhibited by gemfibrozil. Thus, gemfibrozil appears to nonspecifically inhibit duodenal smooth muscle contractions induced by at least two known intestinal contractile substances, an action which if present in vivo may help explain its GI side effects and likely those of other fibrates. Support: MWU Masters Program.
High fat diet decreases glucose‐dependent modulation of 5‐HT responses in gastrointestinal vagal afferent neurons
Authors: Troy Amanda E , Browning Kirsteen N
Published: June 2021

5‐hydroxytryptamine (5‐HT) release from intestinal enteroendocrine cells activates gastrointestinal (GI) vagal afferent neurons inducing a vagally‐mediated gastric relaxation and satiation. Since glucose modulates the response of GI vagal afferent neurons to 5‐HT, we hypothesized that exposure to a high fat diet (HFD) may affect these actions of glucose.

Rats were fed control or HFD (18% or 60% kcal from fat, respectively) from 4 weeks of age for 8 weeks. Electrophysiological recordings were made from acutely dissociated GI vagal afferent neurons and immunocytochemical studies assessed the neuronal distribution of 5‐HT3 receptors.

5‐HT induced an inward current which was sensitive to glucose in the majority (12/13) of control GI vagal afferent neurons. The proportion of 5‐HT‐sensitive GI vagal afferent neurons was unaffected by HFD (5/6 & 5/6 neurons after 1 & 8 weeks respectively, P>;0.05 vs control). HFD attenuated glucose modulation of the 5‐HT response; after 1 week, glucose modulated the 5‐HT response in only 1/5 HFD neurons (151, 183 and 354pA in response to 2.5, 5, and 10mM glucose) while after 8 weeks, 0/5 HFD neurons were affected by glucose. Furthermore, after HFD glucose was unable to alter the proportion of membrane‐associated 5‐HT3 receptors (28±0.8% at 2.5mM to 29±0.7% at 20mM glucose) in contrast to previous control studies (24±1.8% at 2.5mM to 37±2.6% at 20mM glucose).

These data suggest that, while exposure to HFD does not affect the responsiveness of GI vagal afferent neurons to 5‐HT, it diminishes the ability of glucose to regulate 5‐HT responses. This suggests that glucose‐dependent vagal afferent signaling is compromised by relatively short periods of exposure to HFD well in advance of the development of obesity.

Supported by NIH DK078364
Preserved adiposity in the Fisher 344 rat devoid of gut microbiota
Authors: Duca Frank Anthony , Swartz Timothy David , Sakar Yassine , Covasa Mihai
Published: June 2021

The gut microbiota is implicated in affecting host metabolism and energy regulation. Germ‐free (GF) C57BL/6 mice are extremely lean and resistant to diet‐induced obesity; an effect associated with increased intestinal fasting‐induced adipose factor (FIAF) and decreased hepatic lipogenesis. However, whether this altered metabolism observed in the absence of gut microbiota extends to other species commonly used to examine energy metabolism, is unknown. Thus, using the GF Fisher 344 rat, we examined adiposity and associated alterations in intestinal nutrient chemoreceptors and gut peptides, FIAF expression, and markers of hepatic and adipose lipogenesis. We found that, unlike GF mice, GF rats displayed similar body weights and adiposity relative to controls. Along with reductions in intestinal nutrient receptors and gut peptides, the GF state was associated with up‐regulation of intestinal and hepatic FIAF, decreased expression of hepatic FAS, ACC‐1, and SBREP and increased AMPK‐P and ACC‐1‐P. However, GF rats displayed reduced adipocyte FIAF and increased lipogenic enzymes, along with an increase in adipocyte size. These findings show that, despite similarly altered intestinal protein expression and reduced hepatic lipogenesis in the GF state, adiposity is normalized in the Fisher 344 GF rat, unlike the C57Bl/6J GF mouse, through enhancement of adipocyte lipogenesis leading to adipocyte hypertrophy.
Ghrelin and peptide YY increase with weight loss during a 12‐month intervention to reduce dietary energy density in obese women
Authors: Hill Brenna Renee , Rolls Barbara J , Roe Liane S , De Souza Mary Jane , Williams Nancy I
Published: June 2021

The purpose of this study was to determine if low‐energy dense (ED) diets facilitate weight loss through actions on ghrelin (GHR) and PYY, independent of the influence of psychosocial measures of dietary restraint, disinhibition, and tendency toward hunger. Seventy‐one obese women (BMI 30–40 kg/m2) ages 22–60y received dietary counseling to reduce ED. Fasting blood samples were analyzed for total GHR and total PYY by RIA at mo 0, 3, 6, and 12. Restraint, disinhibition, and hunger were assessed by the Eating Inventory. Body weight (−7.8 ± 0.5kg), BMI (−2.9 ± 0.2kg/m2), body fat (−3.0 ± 0.3%) and ED (−1.97 ± 0.21 kJ/g) decreased significantly from mo 0 to 6 (p < 0.05) after which no change occurred from mo 6 to 12. GHR increased in a curvilinear fashion (mo 0: 973 ± 39, mo 3: 1024 ± 37, mo 6: 1109 ± 44, and mo 12: 1063 ± 45pg/ml, p < 0.001) and PYY increased linearly (mo 0: 74.2 ± 3.1, mo 3 76.4 ± 3.2, mo 6: 77.2 ± 3.0, mo 12: 82.8 ± 3.2pg/ml, p < 0.001). ED, body weight, and hunger significantly predicted changes in GHR, with ED being the strongest predictor (GHR = 2674.8 + 291.6*ED ‐ 19.2*BW ‐ 15*H; p < 0.05). There was a trend toward a significant association between ED and PYY (PYY = 115.0 ‐ 43.1*ED; p = 0.05). Results suggest that reductions in ED may promote weight maintenance after a period of weight loss by opposing increases in ghrelin and promoting increases in PYY.
Histidine decarboxylase regulates biliary homeostasis via autocrine histamine signaling
Authors: Francis Heather , Hargrove Laura , Alpini Gianfranco , Meng Fanyin
Published: June 2021

Cholangiocyte homeostasis is regulated by both autocrine/paracrine signaling. In bile duct ligated (BDL) rats large cholangiocyte proliferate, whereas CCl4 induces large cholangiocyte apoptosis. Histidine decarboxylase (HDC) regulates histamine synthesis. We have shown that: (i) cholangiocytes express HDC and secrete histamine; (ii) histamine stimulates biliary growth; and (ii) HDC inhibition decreases biliary growth. No information exists that HDC/histamine regulates cholangiocyte homeostasis. In vivo, normal and 1 wk BDL rats were treated with saline or histamine (0.5 mg/kg) and given CCl4 by gavage (0.5 ml/100 gm). Liver blocks and large cholangiocytes were obtained. HDC, PCNA, TUNEL and CK‐19 biliary expression was measured by immunohistochemistry in liver sections and by qPCR and immunoblots in large cholangiocytes. In vitro, control (MSE‐neg) or stable transfected HDC knockdown large cholangiocytes (MSE‐HDC) were treated with CCl4 (5 mM) in the absence/presence of histamine (10 μM) before evaluating apoptosis and proliferation. After BDL, there was enhanced cholangiocyte proliferation, ductal mass and biliary HDC expression. CCl4 treatment (i) enhanced biliary apoptosis; and (ii) decreased HDC and serum/biliary histamine levels and biliary proliferation and ductal mass, changes that were prevented by histamine. In MSE‐HDC cells there was reduced biliary growth and enhanced apoptosis compared to Mz‐neg. In MSE‐HDC cells treated with CCl4, apoptosis increased compared to MSE‐neg cells treated with CCl4. In Mz‐HDC, CCl4‐induced apoptosis and loss of proliferation was prevented by histamine. HDC is a key regulator of cholangiocyte homeostasis and histamine may be a protective factor after toxic injury. Treatment of cholangiopathies characterized by proliferation or loss may be regulated by the modulation of histamine levels.
Inhibition of RhoA/Rho Kinase Pathway and Contraction by Endogenous and Exogenous Hydrogen Sulfide in Gastrointestinal Smooth Muscle
Authors: Nalli Ancy D , Rajagopal Senthilkumar , Mahavadi Sunila , Grider John R , Murthy Karnam S
Published: June 2021

Inhibitory neurotransmitters, chiefly nitric oxide and vasoactive intestinal peptide, cause MLC20 dephosphorylation and smooth muscle relaxation via inhibition of MLCK and activation of MLCP. H2S produced by commensal bacteria in the colon or synthesized from L‐cysteine via cystathionine‐γ‐lyase (CSE) and cystathionine‐β‐synthase (CBS) regulates smooth muscle tone.

Aim

To examine the expression of CSE and CBS in gastrointestinal smooth muscle and to elucidate the molecular mechanism of H2S‐induced muscle relaxation.

Methods

Expression of CSE and CBS was analyzed by RT‐PCR and western blot. The effect of H2S on carbachol‐induced muscle contraction and Rho kinase activity was examined using an endogenous activator (L‐cysteine) of CSE and CBS and an exogenous H2S donor (NaHS).

Results

Expression of CSE, but not CBS was detected in smooth muscle cells of stomach and colon from mouse, rabbit and human. Carbachol‐induced contraction in muscle strips and muscle cells, and increase in Rho kinase activity were inhibited by L‐cysteine or NaHS in a concentration‐dependent manner (1 to 100 mM). Glibenclamide, an inhibitor of KATP channels and a known target of H2S, had no effect on the inhibition of contraction by H2S.

Conclusion

Both endogenous and exogenous H2S induce muscle relaxation, and the mechanism is via the inhibition of RhoA/Rho kinase activity leading to stimulation of MLCP activity and muscle relaxation.