bluuebunny/crossref_metadata_2025 huggingface dataset:
Thermoregulatory Responses Induced by Central Cholinergic Stimulation During Exercise Are Mediated By Arterial Baroreceptors.
Authors: Pires Washington , Oliveira Bernardo M.S. , Guimarães Juliana B. , Wanner Samuel P. , Lima Milene R.M. , Lima Daniel C. , Haibara Andréa S. , Marubayashi Umeko , Rodrigues Luiz O.C. , Coimbra Cândido C. , Lima Nilo R.V.
Published: June 2021

The aim of this study was to evaluate if the effects of central cholinergic stimulation on thermoregulation during exercise are mediated by arterial baroreceptors. Wistar rats were fitted with cannulae into the lateral cerebral ventricle and subsequently submitted to sinoaortic denervation (Sad) or sham surgery. After two weeks, a catheter was implanted into the ascendant aorta and a thermal sensor into the abdominal cavity. Two days latter they were submitted to exercise on a treadmill at 18 m·min−1 and 5% inclination until fatigue, when tail skin (Ttail) and body temperature (Tb) and pulsatile arterial pressure were measured after the injection of 2 μL of 10 μ 10−3 M physostigmine (Phy) or 0.15 M NaCl solution (Sal) into the cerebral ventricle. Phy‐treated rats had a higher increase in Ttail (30.8 ± 0.6 ºC Phy‐sham vs. 28.0 ± 0.5 ºC Sal‐sham; P<0.05) and lower exercise‐induced increase in Tb (37.9 ± 0.6 ºC Phy‐sham vs. 38.6 ± 1.0 ºC Sal‐sham; P<0.05). In contrast, sinoaortic denervation blunted the Phy injection effects in both Ttail and Tb. Furthermore, Sad rats had a higher increase in Ttail (33.4 ± 0.3 ºC Sad vs. 32.3 ± 0.3 ºC sham; P<0.05) compared with sham group without affecting Tb. We conclude that baroreceptors are involved in thermoregulatory adjustments during exercise. In addition, thermoregulatory responses induced by central cholinergic stimulation during exercise are mediated by arterial baroreceptors.
Phosphorylation of GFAP and lamin B is an early event in reactive astrocytes and may be stimulated by cAMP
Authors: Patel Mayuri P , Ramsey Gregory R , García Dana M , Koke Joseph R
Published: June 2021

Mammalian astrocytes respond to injury by hypertrophy and cell division. Changes in the intermediate filament (IF) cytoskeleton and the lamin nucleo‐skeleton proceeds by phosphorylation of IF subunits, solubilization and reformation after cell division. Monoclonal AB J1‐31 recognizes a phosphoepitope found on GFAP and lamin B in astrocytes and thus is useful for exploring pathways to the reactive state. F98 (a.k.a. 9L) glioblastoma cells have been used to model gliosis in vitro. To test the hypothesis that a cAMP mediated pathway is involved, we added forskolin to confluent cultures of F98 cells and assayed for phosphorylation of lamin B by measuring binding of mAB J1‐31. Forskolin caused a 2X increase in mAB J1‐31 binding to nuclear structures also labeled by anti‐lamin B, and a lesser increase in cytoplasmic labeling of filaments also labeled by anti‐GFAP. Binding of anti‐lamin B and anti‐GFAP to their respective antigens was unaffected by forskolin. Inhibitors of PKA (H89) were not effective in preventing the forskolin induced phosphorylation of lamin B and GFAP, but inhibitors of Ca+2 influx (verapamil) and protein synthesis (cycloheximide) did significantly reduce the effect of forskolin.
Beta‐adrenergic receptor blockers increase ventricular sympathetic innervation by directly enhancing sympathetic axonal sprouting
Authors: Wernli Gwenaelle Laetitia , Bhattacherjee Aritra , Tague Sarah E , Smith Peter G
Published: June 2021

β‐Adrenoceptor antagonists are used to treat a variety of cardiovascular disorders, and discontinuation is associated with severe side effects including palpitations and ventricular dysrhythmias. The objective of this study was to determine if changes in cardiac innervation density might contribute to these adverse effects. Adult rats received saline, the non‐selective β‐blocker propranolol, the selective β1‐blocker metoprolol, or the α‐blocker phentolamine for 7 days by minipump infusion. Ventricular sympathetic innervation increased 48% after propranolol and 44% after metoprolol treatment but was unchanged after phentolamine relative to saline controls. It seems unlikely that sympathetic sprouting is due to altered NGF release as neither α or β receptor agonists influenced NGF release in cultured cardiomyocytes. Propranolol or metoprolol increased neurite outgrowth by 17% and 29% respectively in cultured neonatal superior cervical ganglion neurons, suggesting that β‐blockers exert a direct effect on sympathetic axon outgrowth. The presence of β1 receptors in cultured neurons was confirmed by RT‐PCR. These results indicate that the blockade of β1 adrenergic receptors in vivo or in vitro leads to sympathetic axon sprouting, which may contribute to increased sympathetic tone observed in individuals undergoing withdrawal from β‐adrenergic receptor therapy. Supported by HL079652.
Cellular distribution of heme‐oxygenase (HO), a carbon monoxide (CO)‐producing enzyme, in the hypothalamic supraoptic (SON) and paraventricular (PVN) nuclei
Authors: Reis Wagner Luis , Biancardi Vinicia Campana , Antunes‐Rodrigues Jose , Stern Javier Eduardo
Published: June 2021

In addition to nitric oxide, recent evidence supports carbon monoxide (CO) as an important gaseous molecule that acts as a neurotransmitter in the CNS. Based on recent data supporting CO regulation of neurosecretory hypothalamic function, we aimed to characterize the hypothalamic HO‐CO pathway, by determining the distribution of the HO1 and HO2 isoforms in identified SON and PVN cell types. Robust HO1 and HO2 immunoreactivities (ir) were observed within the SON and PVN. HO1ir was more predominant than HO2ir, both were more densely expressed in the SON than in the PVN, and neuronal and glial‐like staining patterns were observed. Multiple‐labeling studies showed dense HO1ir in magnocellular neurosecretory neurons, and a more predominant expression in oxytocin than in vasopressin cells. HO1ir was also found in nNOS positive neurons. The SON ventral glial lamina and SON/PVN microvasculature also displayed strong HO1ir, which colocalized with the glial markers GFAP and S100. These results support that both neurons and astrocytes constitute cellular sources of CO within the SON and PVN. Ongoing studies evaluate whether HO expression is altered in pathological conditions, including dehydration and heart failure. Supported by NIH HL 090948‐01 to Stern JE and FAPESP 06/50628‐2 to Reis WL.
Maintenance of sympathetic nerve discharge and blood pressure in rats exposed to chronic intermittent hypoxia involves enhanced NMDA receptor function in the hypothalamic PVN
Authors: Dong Ying , Mifflin Steven W , Toney Glenn M
Published: June 2021

Exposure to chronic intermittent hypoxia (CIH) activates the renin‐angiotensin system and raises sympathetic nerve discharge (SND), and mean arterial pressure (MAP). This response profile is consistent with involvement of excitatory forebrain input to the hypothalamic PVN. Here, we investigated control of SND and MAP by PVN NMDA receptors. Control and CIH exposed (3 min 21% O2 ‐ 3 min 10% O2, 8am‐4pm, 7 days) rats were anesthetized and MAP, renal and lumbar SND responses to bilateral PVN injection of the NMDA receptor antagonist AP5 (3.0 nmol in 50 nl/side) were recorded. Baseline MAP was similar across groups. MAP was unchanged by AP5 in control rats (n=4), but decreased by 8 ± 2 mmHg (P<0.05) in the CIH group (n=7). Whereas renal and lumbar SND were not changed by AP5 in control rats, both were reduced (P<0.001) in the CIH group (renal: 83 ± 3, lumbar: 86 ± 1 % baseline). RT‐PCR revealed that NMDA R1 mRNA expression increased by 1.51 ± 0.2 fold in the CIH group compared to controls. We conclude that support of SND and MAP in rats exposed to CIH involves an enhanced role for NMDA receptor activation in the PVN. Similar observations made in other sympathoexcitatory states (e.g., water deprivation, heart failure, AngII‐Salt Hypertension), suggest that similar adaptive processes might underlie PVN‐mediated SND activation in a variety of physiological and disease conditions. Support: P01 HL088052 (SWM & GMT)
Mechanical Tissue Resuscitation (MTR) Reduces Myocardial Reperfusion Injury
Authors: Jordan James E , Morykwas Michael J , Mays Jennifer J , Thompson Edreca A , Argenta Louis C
Published: June 2021

Treating injured tissue with negative pressure re‐establishes physiologic homeostasis and improves tissue survival. Injury sustained following reperfusion of ischemic myocardium involves processes similar to those seen after burns and other wounds. This study explores the possibility of using a mechanical device to achieve local resuscitation of myocardial tissue following acute myocardial infarction.

Anesthetized swine were subjected to 75 min of left coronary artery occlusion and 3 hours of reperfusion. Animals were assigned to 1 of 3 groups: (1) control, treatment with (2) 50 mmHg or (3) 125 mmHg of MTR during reperfusion. All three groups were subjected to equivalent ischemic stress [area of risk (AAR) =12.9± 1.2%, 11.8±1.2% and 11.9±1.4%, respectively]. The application of MTR directly to the injured myocardium for 180 minutes significantly (p<0.001) reduced infarct size in both treatment groups compared to control: 9.3±1.8% (50 mm Hg) vs 26.4±2.1% (control); and 11.9±1.2% (125 mm Hg) vs 26.4±2.1% (control).

Application of a controlled vacuum device directly to the injured myocardium for a defined period of time can achieve MTR and reduce tissue death after ischemia and reperfusion. This treatment strategy offers a simple, effective, clinically relevant, non‐pharmacologic approach to reducing myocardial injury following ischemia and reperfusion.
SPARC Mediates Early Extracellular Matrix Remodeling Following Myocardial Infarction
Authors: McCurdy Sarah M. , Dai Qiuxia , Bradshaw Amy D. , Lindsey Merry L.
Published: June 2021

Secreted protein, acidic, and rich in cysteine (SPARC) is a matricellular protein that helps collagen fibrils assemble. Collagen deposition to form a scar is a key event following a myocardial infarction (MI). Because SPARC roles post‐MI have not been defined, we examined wild type (WT; n=22) and SPARC deficient (SPARC‐null; n=25) mice at day 3 post‐MI. Day 0 unoperated WT (n=28) and SPARC‐null (n=20) mice served as controls. Infarct size was 52±2% for WT and 47±2% for SPARC‐null (p=n.s.), indicating that equal injury stimulus was given. By echocardiography, WT mice increased end diastolic volumes from 45±2 to 83±5 μL (p<0.05 vs day 0 controls). SPARC‐null mice also increased end diastolic volumes, but to a lesser extent (39±3 to 63±5 μL, p<0.05 vs day 0 controls and p<0.05 vs WT day 3 MI), indicating reduced remodeling. Myocyte cross sectional areas increased from 137±5 to 169±4 μm2 in WT and from 132±6 to 163±3 μm2 for SPARC‐null (p<0.05 for both day 0 vs day 3 MI groups), which showed that the hypertrophic response post‐MI was similar. Interestingly, collagen levels in the SPARC‐null mice were 66% of the wild type values, indicating reduced collagen deposition in the absence of SPARC. Our data suggests that SPARC deletion is beneficial in the early time post MI, but whether this benefit persists through longer times needs to be examined. In conclusion, SPARC may play a role in cardiac wound healing and may be a major component of the post‐MI response.
Early phase of ischemic preconditioning in the mouse: effects of endothelial nitric oxide synthase (eNOS), ischemic duration, aging, and gender
Authors: Yang Fuchun , Talukder M.A. Hassan , Zweier Jay L.
Published: June 2021

Ischemic preconditioning (IPC) is a powerful phenomenon that provides effective cardioprotection in mammalian hearts, however, little is known in relation to prolonged ischemia (I), aging and gender. Importantly, the role of eNOS‐mediated nitric oxide (NO) pathway is still controversial. Therefore, we performed an extensive study in wild‐type (WT) and eNOS‐knockout (eNOS‐KO) mice to evaluate whether the infarct limiting effect of IPC depends on eNOS, ischemic periods, ages (12‐16 weeks and 12‐16 months), and gender. Classical IPC was induced by 3‐cycles of 5‐min regional coronary I separated by 5‐min reperfusion (R), and was followed by 30‐min or 60‐min of index I and 24‐hrs R. Control IR protocol had 30‐ or 60‐min I followed by 24‐hrs R. Protection was evaluated by measuring myocardial infarct size as a percentage of area at risk. The major findings are that regardless of age and gender, WT mice exhibited robust IPC effects with significantly smaller myocardial infarction, whereas, eNOS‐KO mice did not. In general, female WT mice had smaller infarction compared to male WT. Importantly, aging and prolonged I caused significantly larger infarction in both genders. Prolonged myocardial ischemia was fatal in eNOS‐KO mice. In conclusion, IPC protects WT mice against in vivo myocardial IR injury regardless of age, gender and/or ischemic duration, but, deletion of eNOS abolishes the cardioprotective effect of early IPC.
Age‐dependent cardiovascular responses to severe haemorrhage in conscious lambs
Authors: Samhan Mohamed A , Qi Wei , Smith Francine G
Published: June 2021

The present study was designed to investigate cardiovascular responses to severe haemorrhage (exceeding the critical point at which compensatory mechanisms fail) in two age groups of conscious lambs (Group I: 1‐2 weeks, N=5; group II: 6‐7 weeks, N=6). Mean arterial pressure (MAP), systolic and diastolic pressures (SAP, DAP), heart rate (HR) and renal blood flow (RBF) were measured for 20 min before (Control, C) and after a fixed haemorrhage of 30% of calculated blood volume. After haemorrhage in group I lambs, MAP decreased from 75±5 [SD] (C) to 55±8 mmHg at 5 min, returning to 61±6 mmHg at 20 min, while in group II lambs, MAP decreased further from 74±4 (C) to 38±7 mmHg at 5 min, returning to 55±6 mmHg at 20 min. The fall in SAP 5 min after haemorrhage was also greater in group II lambs (104±8 (C) to 65±15 mmHg) than in group I lambs (110±14 (C) to 91±8 mmHg), whereas DAP followed changes in MAP in both groups. HR increased in group I lambs from 164±43 (C) to 193±45 at 5 min, decreasing thereafter, whereas in group II lambs, HR was unaltered from 119±20 (C). After haemorrhage, RBF fell to zero at 5 min in both groups, returning towards C at 20 min. These results provide new evidence that the cardiovascular responses to severe haemorrhage are developmentally regulated. Mechanisms underlying these differential responses are not yet known.

This research was supported by the Canadian Institutes of Health Research (CIHR).
Comparison of plasma volume assessments following hemorrhagic shock in pigs
Authors: Colvin Shavaughn Kristin , Dubick Michael A , Martini Wenjun Z
Published: June 2021

Indocyanine green (ICG) dye is frequently used for plasma volume (PV) measurements. However, the accuracy of ICG measurements following severe hemorrhage (H) is unclear due to limited knowledge of its metabolism when liver function is altered by H. This study assessed changes of PV in pigs after H and compared the ICG results with Hct and total protein. Sixteen pigs were divided into controls (n=4) or H with either LR (n=6) or saline (n=6) resuscitation. H was induced by withdrawing 60% of the estimated blood volume from the femoral artery, followed by resuscitation with LR or saline. ICG (25mg/10ml) was injected iv to assess PV at baseline and 6 h after H and resuscitation. Blood samples were taken before each injection for blood gas and chemistry. PV was also calculated based on the changes of Hct and total protein for comparison. There were no changes in PV in controls throughout the study. At 6 h following H and resuscitation, PV by ICG was 121±3% and 120±4% of baseline in the LR and saline groups, respectively. PV calculated by changes of Hct showed 119±1% and 119±2% of baseline in these groups, respectively. Similarly, PV calculated by changes of total protein was 121±6% and 124±3 % of baseline in these groups, respectively. In conclusion, these data suggest that following severe H, ICG remains a reliable method for measurements of PV.

This work was supported by US Army Medical Research and Medical Command.
Acetylcholinesterase inhibitor improves survival from hemorrhage in rodents
Authors: Mathis Keisa W. , Molina Patricia E.
Published: June 2021

Decreased tissue perfusion leading to end organ injury remains a cause of high mortality in hemorrhagic shock (HS) victims. We have demonstrated that intracerebroventricular (ICV) administration of neostigmine, an acetylcholinesterase inhibitor, reverses hypotension following 50% blood loss. We hypothesized that systemic administration of the anti‐cholinesterase physostigmine would act via central cholinergic receptors, enhance sympathetic outflow and improve the pressor response to HS. Intravenous (IV) physostigmine (100µg/kg) increased plasma epinephrine (358%) and norepinephrine (105%), mean arterial pressure (MAP; 12%; p=0.003) and heart rate (21%; p=0.003) within 5 min in conscious male Sprague‐Dawley rats (225‐275g). Mecamylamine (nicotinic antagonist; 50µg, ICV), but not atropine sulfate (muscarinic antagonist; 10µg, ICV), diminished the pressor response to physostigmine. HS (40% blood loss) decreased MAP (50%) resulting in 36% mortality 4d post‐HS. Physostigmine enhanced the HS‐induced rise in plasma epinephrine (159%), reversed hypotension, and improved survival (100% at 4d). These results suggest that physostigmine increases central acetylcholine availability which activates central nicotinic receptors, increases SNS outflow, and in turn improves MAP recovery and survival post‐HS. DOD‐PR‐054196, BoR‐110350057A, NIAAA‐7577, APS Porter Physiology Development Award.
Multiple red cell volumes (RCV) can be determined simultaneously and independently in humans in vivo using red cells (RBCs) labeled at 4 biotin densities
Authors: Mock Donald M , Matthews Nell I , Strauss Ronald G , Burmeister Leon F , Schmidt Robert , Widness John A
Published: June 2021

Anemia is a common and serious problem in critically ill neonates. To investigate the pathophysiology of anemia and to assess responses to red blood cell (RBC) transfusions and erythropoietin therapy, repeated measurement of RCV and blood volume would be useful. We previously reported the in vivo validation of a nonradioactive method for determination of RCV in adults using flow cytometric enumeration of RBCs labeled at one or two biotin densities. Here we report the simultaneous measurement of RCV in 2 adult subjects using 4 populations of RBCs labeled at distinct biotin densities. In one subject, biotinylation reagent concentrations from 9 to 243 μg/mL of RBC produced a mean RCV of 24 ± 0.6 mL/kg; CV= 3%. In the second subject, biotinylation reagent concentrations from 6 to 192 μg/mL of RBC produced a mean RCV of 29 ± 0.8 mL/kg; CV= 3%. RCV values were not different by ANOVA among the 4 densities for either subject. These studies provide evidence that RBCs biotinylated at discrete densities can be used for simultaneous and repeated independent measurements of RCV in vivo in humans. Because only small sample volumes are required and because the subject is not exposed to radiation, we speculate that this RCV method will be useful in investigating the physiology and pathophysiology of anemia in pregnant women, fetuses, and infants. NIH P01 HL046925; Thrasher Research Foundation 02825‐3.
Racial differences in adolescent endothelial function and insulin secretion
Authors: Hoffman Robert Paul
Published: June 2021

African‐American (AA) adults have increased mortality and morbidity from cardiovascular disease. The mechanisms for this likely have their origins in childhood and adolescence. It is therefore crucial that we understand racial differences in this age group. Several studies have indicated that AA adolescents (adol) are insulin resistant compared to Caucasians (Ca). Because insulin resistance has been linked to increased cardiovascular risk and specifically to endothelial dysfunction, we studied endothelial function (EF) differences between AA and Ca adol. Endothelial dysfunction is one of the earliest precursors of cardiovascular disease. We found that post upper arm occlusion forearm blood flow was significantly lower in AA than in Ca adol indicating impaired EF. This impairment was primarily present in obese subjects. Interestingly, insulin sensitivity was not different between groups, but the acute insulin response to glucose (AIRG) in AA was over twice as high as that in Ca. EF was negatively correlated to the AIRG in the whole group and in AA, but not Ca. It, also, negatively correlated with triglycerides in AA. Future research will determine mechanisms for the differences and could potentially focus on intercellular insulin signaling, lipid metabolism, and specific genetic polymorphisms. This will allow the development of treatments to decrease cardiovascular disease in AA.
Sheep red cell survival (RCS) can be followed using 4 different densities of biotin label
Authors: Matthews Nell I , Mock Donald M , Strauss Ronald G , Burmeister Leon F , Schmidt Robert , Widness John A
Published: June 2021

Anemia is a serious problem in critically ill neonates. To investigate anemia, to assess responses to red blood cell (RBC) transfusions and erythropoietin therapy, and to compare sources of RBCs and storage methods, simultaneous measurement of RCS would be useful. Here we report the validation in sheep (a common model for perinatal studies of physiology and erythropoiesis) of a method for independent measurement of RCS using 4 populations of RBCs labeled at distinct biotin densities.

Methods

By varying the biotinylating reagent from 12 to 96 ?g/mL RBC, the density of biotin on sheep RBCs was incrementally increased to produce 4 discrete populations. Biotinylated RBCs (BioRBCs) were reinfused, and blood was sampled over the next 4 months. RCS was determined using flow cytometric enumeration and by scintillation counting.

Results

BioRBCs produced parallel disappearance curves with life spans from the linear phase that were similar to the reference 14C‐cyanate method (mean potential lifespan = 66±2 vs 62 d).

Conclusion

RCS can be measured accurately and simultaneously in sheep using BioRBCs without radiolabeling. NIH P01 HL046925; Thrasher Research Foundation 02825‐3.
Characterization of ATP uptake into isolated pancreatic zymogen granules
Authors: Haanes Kristian Agmund , Novak Ivana
Published: June 2021

Our previous bioimaging studies on pancreatic acinar cells indicated that ATP is stored in granules. Present study aimed to quantify ATP content and characterize ATP uptake into Zymogen Granules (ZG). ZGs were isolated from rat pancreas and stained with Rab3D antibody to confirm identity as described by Chen et al. (Mol. Cell. Proteomics 5(2):306, 2006). ATP and protein were measured using Luciferin/Luciferase and Bradford assays. ATP concentrations in ZG were calculated assuming that ZG protein concentration is 592 mg/ml, as determined by Burnham et al. (Biochem. J. 227(3):743, 1985). Results have shown that the freshly isolated ZGs contained an ATP concentration of 55 ± 7 μM (n=6). Furthermore, ATP uptake into ZGs was characterized using incubation with different ATP concentrations in a buffer (4 mM KCl, sucrose, MES EGTA, pH 6). A Km of 1.4 ± 0.6 mM and a Vmax of 280 ± 60 μM/granule/30 min was calculated using a Eadie‐Hofstee plot (n=5). Loading of 4 mM ATP resulted in ZG ATP concentration of 211 ± 17 μM/30 min. However, when 100 μM DIDS was added, ATP uptake was abolished, i.e. 0 ± 10 µM/granule/30 min (n=7). Bafilomycin, CCCP and Valinomycin did not appear to have significant effects in these assay conditions. In conclusion, this study shows the first quantitative measurements of ATP concentration in zymogen granules and it characterizes a DIDS‐sensitive ATP uptake mechanism.

Support: DNSRC 272‐05‐0420
Lipopolysaccharide (LPS) increases transepithelial sodium absorption in the guinea‐pig trachea by increasing Na+,K+‐ATPase activity
Authors: Dodrill Michael , Fedan Jeffrey S.
Published: June 2021

Earlier, we reported that systemic LPS administration increases transepithelial potential difference (Vt) in the guinea‐pig trachea. The increase in Vt was abolished by the epithelial sodium channel (ENaC) blocker, amiloride, and inhibited by the prostaglandin synthase inhibitor, indomethacin. Here, we hypothesized that LPS increases Na+ absorption by increasing Na+, K+‐ATPase activity. We investigated the effects of LPS on Vt using the isolated, perfused trachea preparation, which allows agents to be added to the luminal or the serosal surface. Eighteen hours after injection with LPS (4 mg/kg, ip), tracheas were removed, mounted on a holder, and perfused with Krebs solution. LPS increased basal Vt (‐34.2 ± 6.1 mV) compared to control (‐5.5 ± 1.8 mV, P < 0.05), consistent with increased cation absorption. Subsequently, apical amiloride (10 μM) reduced Vt to ‐4.5 ± 2.2 in controls and to ‐13.1 ± 2.9 in LPS‐treated animals indicating increased flux of Na+ across the apical membrane. The cation pore‐forming antifungal, amphotericin B (7.5 μM apically) then increased Vt to ‐18.9 ± 2.7 in LPS‐treated tracheas compared to ‐7.0 ± 2.1 in controls. Vt after apical permeabilization is the result of basolateral ion transport and represents the maximum capacity of Na+, K+‐ATPase. We conclude that LPS increases Na+ absorption, at least in part, via ENaC by increasing the activity of the Na+, K+‐ATPase. Funded by NIOSH.
Modulation of folic acid uptake into BeWo cells by cannabinoids
Authors: Araújo João Ricardo , Gonçalves Pedro , Martel Fátima
Published: June 2021

The aim of this study was to investigate the influence of cannabinoids (CB) on the uptake of folic acid (FA) by the choriocarcinoma cell line (BeWo cells). Acutely, anandamide caused a 15% decrease in 3H‐FA uptake (pH 7.5). Moreover, tetrahydrocannabinol (THC) caused a 30% increase, and AM630 a 15% decrease (pH 6.5). Neither the inhibitory effect of anandamide nor the stimulatory effect of THC were changed by the CB receptor type 1 or type 2 antagonists (AM251 and AM630, respectively). Chronically (48 h), THC and AM251 decreased by 20% the uptake of 3H‐FA, and anandamide and AM630 increased it by 30% (pH 7.5). Moreover, CP 55,940 increased uptake of 3H‐FA by 30% (pH 6.5). RT‐PCR analysis showed that the mRNA levels of the Reduced Folate Transporter 1 increased by 9% after chronic treatment with AM630, and the mRNA levels of the Proton‐coupled Folate Transporter decreased by 17% and increased by 18% after chronic treatment with THC and AM251, respectively. In conclusion, uptake of 3H‐FA by BeWo cells is significantly, although not very markedly, changed by several distinct CB receptor agonists and antagonists, either after acute and after chronic exposure of the cells. The acute effects of CB receptor agonists do not seem to be CB receptor‐mediated, and with a few exceptions the chronic effects do not seem to be related to changes in the expression levels of FA transporters.

Supported by FCT (PTDC/SAU‐FCF/67805/2006)
The tight junction protein claudin‐2 forms a paracellular water channel
Authors: Rosenthal Rita , Milatz Susanne , Krug Susanne M. , Günzel Dorothee , Oelrich Beibei , Amasheh Salah , Fromm Michael
Published: June 2021

There is an ongoing dispute concerning the existence of significant water flow across tight junctions (TJ). Instead of distinguishing technically between the possible routes for water, we measured transepithelial water transport in a video‐equipped Ussing chamber before and after TJ perturbation. For this, kidney tubule cells (MDCK‐C7) which lack genuine AQP‐2, claudin‐2, and claudin‐10b were stably transfected with claudin‐2 (C7‐CLDN2) or claudin‐10b (C7‐Cldn10b). Claudin‐2 is typically expressed in leaky epithelia and both form paracellular cation channels. Claudin transfections did not alter the expression of endogenous claudins or of the membranal water channels AQP‐1, ‐3, and ‐4. In the presence of an osmotic gradient (100 mM mannitol) water flow was markedly higher in C7‐CLDN2 cells than in vector controls. Water flow induced by a Na+ gradient (80 mM, high Na+ basolateral) was zero in controls and was present in C7‐CLDN2 cells. The combination of both gradients had additive effects on water flux. Comparison of different clones revealed a correlation between claudin‐2 expression and water flux. In contrast, water fluxes of C7‐Cldn10b cells were not different from that of vector controls. We conclude that the paracellular cation channel claudin‐2, but not claudin‐10b, forms a paracellular water channel and by this mediates paracellular water transport in leaky epithelia. (Supported by DFG FOR 721)
SMALL INTESTINAL GLUCOSE ABSORPTION AFTER GASTRIC BYPASS SURGERY.
Authors: Shah Hiral , Bal Bikram , Frederick Finelli , Koch Timothy R.
Published: June 2021

In small intestine (SI), a Na‐dependent glucose transporter is at apical epithelial cells; a diffusion glucose transporter is at the basolateral membrane. Obese Zucker rats have decreased glucose absorption in SI compared to lean rats (J Nutrition, 1993). After gastric bypass surgery (GBS), small intestinal bacterial overgrowth (SIBO) induces thiamine deficiency (Nutrition Res, 2008). Our hypothesis is that SIBO alters glucose transport in the morbidly obese.

METHODS

We reviewed GBS patients from 1999‐2008 with glucose breath testing (GBT). SIBO is defined by: fasting breath H2 or CH4 at time 0 or rise of >10 ppm </= 30 minutes after 50 grams oral glucose; colonic bacteria defined by: breath H2 or CH4 rise of >10 ppm at >/= 45 minutes after glucose.

RESULTS

Study includes 64 female & 5 male patients, ages 22‐67 years (mean: 48 years). 54 of 69 patients (78%) had a rise in H2 or CH4 at >/= 45 minutes. In these 54, 37 (69%) are H2 producers and 17 (31%) are CH4 producers. Only 31 (57%) of the 54 patients had evidence for SIBO.

CONCLUSIONS

The delayed rise in breath H2 or CH4 in 78% of GBS patients is due to fermentation of glucose by colonic bacteria. This supports our hypothesis that morbidly obese patients have decreased SI glucose transport. We can not differentiate between a defective glucose transporter at an apical or basolateral location. SIBO does not explain this defect, and so it may represent a homeostatic mechanism.
Interaction of a novel urate efflux transporter URATv1 (SLC2A9) with uricosuric agents.
Authors: Anzai Naohiko , Jin Chun Ji , Jutabha Promsuk , Babu Ellappan , Srivastava Sunena , Endou Hitoshi , Sakurai Hiroyuki
Published: June 2021

Hyperuricemia is a significant factor in a variety of diseases, including gout and cardiovascular diseases. And renal excretion largely determines plasma urate concentration. Previously we identified a major urate reabsorptive transporter, URAT1 (SLC22A12), on the apical side of the renal proximal tubular cells. Recently, we identified that URATv1 (SLC2A9) functions as an efflux transporter of urate from the tubular cell. URATv1‐expressed Xenopus oocytes transported urate favoring negative to positive potential direction. Considering its basolateral localization, SLC2A9 is likely to mediate the second step of urate reabsorption. To further characterize the transport properties of URATv1, we performed the urate transport studies in the URATv1 expressed oocytes. URATv1‐mediated urate transport was not trans‐stimulated by intracellularly‐injected glucose, fructose, monocarboxylates such as lactate, nicotinate or pyrazinoate (PZA) for urate. In addition, urate efflux via URATv1 was not enhanced by extracellular glucose or fructose. Urate transport via URATv1 was affected by uricosuric agents probenecid, benzbromarone, phenylbutazone and losartan, but not by monocarboxylates such as lactate, nicotinate, orotate, PZA, or β‐hydroxybutyrate. In summary, different substrate selectivity between URATv1 and URAT1 suggests that targeting URATv1 may lead to a novel therapy for hyperuricemia.
hGLUT9 as a novel urate transporter: its role in liver urate handling and functional study of SLC2A9 SNPs
Authors: Witkowska Kate , Smith Kyla , Karpinski Ed , Young James , O'Neill Debbie , Cheeseman Chris
Published: June 2021

In humans, urate is the final product of purine breakdown in the liver which releases urate into the circulation and the intestine and kidney regulate its excretion. Humans have a high circulating level of urate and small deviations are associated with a variety of metabolic diseases. A genome‐wide association scan correlated SNPs within the SLC2A9 gene, for the hexose transporter GLUT9, with hyperuricemia. This resulted in the identification of this protein as a high capacity urate transporter, which can exchange intracellular urate for extracellular glucose (1). Urate is an organic anion and voltage clamp experiments with GLUT9 expressed in Xenopus oocytes showed that its transport is electrogenic. Immunohistochemistry showed that all of the GLUT9 SNP mutants were expressed in the oocyte plasma membrane and flux studies indicate that all are functional. GLUT9 is expressed in the hepatocyte basolateral membrane and these data support the hypothesis that GLUT9 mediates the efflux of urate down its electrical gradient. Further, in the absence of direct functional effects of the SNPs, it appears that more complex effects of these mutations, perhaps on cytoskeletal interactions or yet to be identified regulatory mechanisms, affect the activity of GLUT9.

1. Mark J. Caulfield, et al (2008) SLC2A9 is a high capacity urate transporter in man. PLoS Medicine 5(10):e197.

Supported by the Canadian Breast Cancer Foundation.
Organic Anion Secretion across the Ciliary Body Epithelium of the Eye
Authors: Pelis Ryan M , Delamere Nicholas A
Published: June 2021

Several organic anions have been shown to accumulate in the ciliary body following intraocular injection. Additionally, clearance of organic anions from the eye is slowed by the organic anion transport inhibitor probenecid. Here, we hypothesized that the ciliary body epithelium would support organic anion secretion in an aqueous humor (AH)‐to‐blood direction. Bovine ciliary body epithelium was mounted in Ussing chambers for determination of unidirectional 3H‐p‐aminohippurate (PAH) fluxes under short‐circuited conditions (at 0.01 mM PAH). The unidirectional AH‐to‐blood flux was 6‐fold greater than the blood‐to‐AH flux (0.6 ± 0.15 vs. 0.1 ± 0.03 nmoles x cm−2 x hr−1), indicating net active secretion. Net active PAH secretion was strongly inhibited by 0.25 mM probenecid. Consistent with the transport data, mRNA (RT‐PCR) and protein (Western blot) analysis revealed several organic anion transporters, including OAT1, MRP2 and MRP4 in the ciliary body. Active organic anion secretion (AH‐to‐blood) by the ciliary body epithelium may influence the effectiveness of thereaputic drugs, such as antivirals, antibiotics and chemotherapeutics, to treat ocular diseases. Supported by NIH (EY006915).
Elevated Hydrostatic Pressure Activates Sodium‐Hydrogen Exchanger‐1 in Rat Optic Nerve Head Astrocytes
Authors: Mandal Amritlal , Shahidullah Mohammad , Delamere Nicholas A , Teran Marcos A
Published: June 2021

Objective

Optic nerve head (ONH) astrocytes are thought to involve in the degeneration of ganglion cells and remodeling of surrounding extracellular matrix in glaucoma. Elevated intraocular pressure (IOP) is the main glaucoma risk factor. Here we report altered sodium hydrogen exchange (NHE) activity in cells exposed to elevated hydrostatic pressure .

Methods

Hydorstatic pressure (HP) of 15 mmHg was applied to the cells in a 20 cm glass cylinder filled with the culture medium equilibrated with 5% CO2/95% air for 2h. Proteins were analyzed by Western blot and cytoplasmic pH (pHi) was measured by BCECF ratiometry.

Results

Elevated HP causes phosphorylation of ERK1/2, ribosomal S6 kinase (p90RSK), and Na‐H exchanger‐1 (NHE1). The MEK/ERK inhibitor UO126 abolished phosphorylation of NHE1 and p90RSK as well as ERK1/2. Although baseline pHi was unaltered in pressure treated cells, the rate of pHi recovery from ammonium chloride acidification was four fold higher. Dimethylamiloride (DMA), an NHE inhibitor, abolished and U0126 partially inhibited the increased rate of pHi recovery in pressure treated astrocytes.

Conclusions

The findings suggest elevated HP caused NHE‐1 activation through phosphorylation following activation of ERK1/2 and p90RSK. These events may lead to the long term changes of protein expression known to occur in pressure‐stressed astrocytes.

Funding

NIH Grant EY014069
Involvement of Na+/HCO3‐ Cotransporter in Intracellular pH Regulation and Anion Secretion in Cultured Rat Epididymal Epithelia
Authors: Zuo Wulin , Li Sheng , Zhang Geng , Ruan Yechun , Zhou Wenliang
Published: June 2021

Intracellular pH(pHi) plays a critical role in many intracellular processes. Several ion transporters, including Na+/H+ exchanger (NHE), Na+/HCO3‐ cotransporter(NBC) and vacuolar H+‐ATPase(H+‐ATPase), contribute to pHi regulation in different types of cells. The present study examined the mechanisms of pHi regulation by NBC in cultured caput epididymal epithelia by using the fluorescent pH sensitive probe, SNARF‐4F. In the HEPES buffered Krebs‐Henseleit(KH) solution, a pHi recovery from NH4Cl induced acidification was completely inhibited by amiloride, the inhibitor of NHE. Then immediate change of the KH solution from HEPES buffered to HCO3‐ buffered would cause another pHi recovery. The pHi recovery in HCO3‐ buffered KH solution was inhibited by 4, 4'diisothiocyanatostilbene‐2, 2′‐disulfonic acid (DIDS) or removal of extracellualr Na+. However, the pHi recovery was not affected by removal of extracellular Cl‐. In addition, the short circuit current measurement showed that adding DIDS to the basolateral side of epididymal epithelial monolayer could inhibit adrenalin induced anion secretion. In summary, these results indicate that not only does NBC play a functional role in pHi regulation of epididymal epithelia, but also regulate the epididymal fluid microenvironment by affecting anion secretion.

This work was supported by the National Natural Science Foundation of China (No.30770817)
Extracellular ATP synthesis regulates ATP release and bicarbonate secretion in rat duodenum
Authors: Watanabe Chikako , Akiba Yasutada , Nakano Takanari , Kaunitz Jonathan D
Published: June 2021

The pH of the alkaline microclimate overlying duodenal enterocyte brush border is regulated by an ecto‐purinergic signaling; intestinal alkaline phosphatase (IAP), extracellular ATP and P2Y receptors regulate duodenal bicarbonate secretion (DBS). IAP inhibition increases DBS and non‐lytic ATP release into the lumen by the mechanism unknown. Ecto‐F1F0‐ATP synthase has been localized to plasma membranes of several cell types. We hypothesized that ecto‐ATP synthase generates extracellular ATP, regulating DBS.

We measured DBS with flow‐through pH and CO2 electrodes with perfusate ATP content measured by luciferin‐luciferase bioassay. We perfused the competitive AP inhibitor glycerol phosphate (GP) with ATP synthase inhibitors oligomycin (Om), piceatannol (Pic) or resveratorol (Res), or the uncoupler CCCP.

GP increased DBS and ATP output. Pic or Res inhibited GP‐induced augmented DBS and ATP output. GP, Pic or Res with GP had no effect on LDH release, confirming no cellular injury. Om or CCCP inhibited GP‐induced ATP output with increased LDH release, consistent with cell injury.

Luminal ATP release unmasked by IAP inhibition is dependent on ecto‐ATP synthase activity, consistent with the presence of ecto‐ATP synthase on the brush border. Ecto‐ATP synthesis and ATP degradation by IAP may coordinately regulate ATP‐P2Y signaling for DBS as part of a novel ecto‐purinergic signaling system.

VA, NIH‐NIDDK
An anatomical resource clinical training center using cadavers as simulated patients: utility, management & best practices
Authors: Johnson James E.
Published: June 2021

Emerging challenges of medical education include the vital task of knowledge application to clinical practice, finding incremental benchmarks of competency in a longitudinal curriculum and the need for a continuum of life‐long learning resources for professionals in practice. We have employed elements of contextual learning theory to guide renovations and to develop new curriculum components meeting these needs. An Anatomical Resource Clinical Training Center (ARCTC) was first opened in 2003 to utilize fresh un‐embalmed human cadavers as simulated patients for the applied learning and competency assessment of professionals in training as well as professionals in practice. ARCTC is used for supervised basic and advanced clinical skill assessment as well as multidisciplinary team training. Each cadaver table includes a display for a ccTV system and wireless resources. In some cases, cadaver CT or X‐Ray images are first acquired for use at the table during procedure training. CSF, venous and arterial volumes can be regulated with catheter lines and carefully prepared skin flaps can be used to unveil deeper structures. ARCTC provides risk free participant praxis. Each year the center provides approximately 4,000 lab encounter hours through clerkship use in the medical curriculum and advanced multi‐disciplinary training to professionals from 16 different clinical services and regional meetings.
Membrane trafficking of NBCe1 but not NBCn1 in rat parotid acinar ParC5 cells
Authors: Grichtchenko irina I , Reyland Mary , Perry Clint J
Published: June 2021

We examined membrane trafficking of "renal" NBCe1‐A and "pancreas" NBCe1‐B, and NBCn1 in ParC5 cells by biotinylation and confocal microscopy using fluorescent tags and endosomal markers. We localized NBCe1 and NBCn1 at the basolateral membrane (BLM) in polarized cells. We blocked constitutive recycling with monensin and detected that both NBCe1‐A/B accumulated in endosomes with a parallel loss from the BLM. We observed that both NBCe1‐A/B undergo a massive carbachol‐stimulated redistribution from the BLM into early and recycling endosomes. We inhibited internalization of NBCe1‐A by transfecting cells with the kinase‐dead (Lys376Arg) PKCd mutant, and inhibited internalization of both NBCe1‐A/B by total PKC inhibitor GF109203X, PKCaβγ inhibitor GÖ6976, and PKCδ inhibitor rottlerin. Our data suggest that "renal" and "pancreas" variants of NBCe1 undergo constitutive and stimulated PKC‐regulated endocytosis in acinar cells. In contrast, NBCn1 remained stably present at the BLM after treatment with monensin, carbachol, or PMA. We speculate that endocytosis of NBCe1, which coincides with the sustained phase of stimulated fluid secretion, could be a part of cell adjustment to a steady state characteristic of a continuous secretory response. At the same time, stable NBCn1 could constantly participate in maintaining acid‐base homeostasis in stimulated saliva‐secreted parotid acinar cells.
Cysteines in Extracellular Loop 3 of NBCe1‐A Form Intra‐ and Inter‐Molecular Disulfide Bonds
Authors: Zhu Quansheng , Azimov Rustam , Kao Liyo , Pushkin Alexander , Kurtz Ira
Published: June 2021

NBCe1‐A cotransports sodium and bicarbonate across the basolateral membrane of the proximal tubule. Four cysteines in the putative NBCe1‐A extracellular loop 3 are conserved among all the SLC4 family sodium coupled bicarbonate transporters. To determine the structural importance of these conserved cysteines, we explored the potential disulfide bonding among the 4 cysteine residues. NBCe1‐A constructs bearing various cysteine mutations were expressed in HEK‐293 cells and examined for the presence of disulfide bonds with the cysteine accessibility method (CAM). Initially we analyzed the aqueous accessibility of the 5 endogenous cytoplasmic cysteines. The results revealed that C120 and C399 are not exposed to the aqueous medium, whereas C389, C992 and C1035 are highly exposed, suggesting that the N‐terminal cytoplasmic region of NBCe1‐A is structured. Mutation of the cytoplasmic cysteines to serines eliminated any free aqueous accessible cysteines indicating that cysteines 583, 585, 630 and 642 in extracellular loop 3 partake in either intra‐ or inter‐monomeric disulfide bonding. Mutation of both cysteine 583 and 585 to serine did not free any cysteines, however, upon treatment with 5mM DTT, free cysteines were detected. Further analysis on mutants where 3 of the 4 cysteines were replaced with serine: 583/585/630 or 583/585/642 showed that the remaining cysteine (642 or 630) in each construct could only be freed after DTT treatment. In conclusion, our data suggests that cysteine 583 and 585 form an intra molecular disulfide bond, whereas cysteine 630 and 642 each form disulfide bonds with the same residue on the opposite monomer, and therefore appears to play an important role in the oligomerization of the cotransporter.

Supported by NIH
Stoichiometry and Inhibitory Pharmacology of Electrogenic Sodium Bicarbonate Cotransporter NBC4c (NBCe2‐C) Expressed in HEK‐293 Cells
Authors: Shao Xuesi , Kao Liyo , Abuladze Natalia , Kurtz Ira
Published: June 2021

NBC4c is an electrogenic sodium bicarbonate cotransporter encoded by SLC4A5. To examine the electrophysiologic properties of NBC4c in a mammalian system, HEK‐293 cells permanently expressing the cotransporter were whole‐cell patch‐clamped. When the patch pipette and bath solutions contained 0 mM HCO3−, increasing [HCO3−]o from 0 to 25 mM induced a mean outward current of 48.4 pA at +45 mV that was blocked by DIDS and tenidap. The inhibitory profiles of DIDS and tenidap were fit to the equation I/I0=1/(1+(C/Ki)n), where Ki is the inhibitory constant, C is the concentration and n is the pseudo Hill coefficient. Tenidap was more potent than DIDS. Tenidap inhibition was voltage dependent with reduced inhibition at more positive voltages. At known intracellular and extracellular Na+ and HCO3− concentrations, HCO3− to Na+ stoichiometry (q) could be calculated with the equation: Erev = RT/(F(q‐1))ln(([Na+]i/[Na+]o)([HCO3−]i/[HCO3−]o)q) where reversal potential Erev (VI=0) was determined from the DIDS‐inhibitable I‐V curves from ‐95 mV to +45 mV. Erev was measured in two independent experiments where [HCO3−]i and [HCO3−]o were equal (25 mM), therefore Erev depended only on [Na+]i/[Na+]o. [Na+]i/[Na+]o values were chosen so that Erev would be significantly different when q = 2 or q = 3 predicted with the above equation. In the first protocol, using [Na+]i/[Na+]o = 40/80 mM, Erev = ‐22.2 ± 3.87 mV. In the second protocol using [Na+]i/[Na+]o = 25/135 mM, Erev = ‐40.5 ± 5.12 mV. These Erev values are close to the calculated values of ‐17.8 and ‐43.3 mV, respectively, assuming q = 2. Our data indicate that NBC4c expressed in HEK‐293 cells transports sodium bicarbonate electrogenically with a stoichiometric ratio of 2 HCO3−: 1 Na+. NBC4c is blocked by DIDS and tenidap with different mechanisms.

Supported by NIH
The other half of the insect SLC4 family: a Cl/HCO3 anion exchanger from mosquito renal tubules
Authors: Piermarini Peter M , Grogan Laura F , Wang Li , Beyenbach Klaus W
Published: June 2021

From the completed genomes of insects it appears that most species possess only two SLC4‐like transporters: (i) a Na‐driven Cl/HCO3 anion exchanger (NDAE) and (ii) a putative Cl/HCO3 anion exchanger (AE). To date, the AE of insects has received little attention compared to the NDAE. The goal of this study was to characterize and localize the SLC4‐like AE expressed in renal (Malpighian) tubules of the yellow‐fever mosquito, Aedes aegypti. Using RT‐PCR on Malpighian tubule cDNA, we identified a full‐length clone (Aedes AE) that encodes 1,243 amino acids with 40% identity to mammalian AEs. Measurements of intracellular pH (pHi) in Xenopus oocytes expressing Aedes‐AE cRNA (AE oocytes) indicate that the rate of pHi recovery after exposure to 5% CO2/33 mM HCO3 is enhanced by lowering extracellular Cl and is blocked by DIDS. No such responses were observed in H2O‐injected oocytes subjected to similar treatments. The above experimental manipulations have minor effects on the membrane potential (Vm) of AE and H2O‐injected oocytes. A polyclonal antibody raised and affinity purified against the Aedes AE labels the stellate cells, but not principal cells, of Malpighian tubules. Taken together, our results indicate that Aedes Malpighian tubules express a DIDS‐sensitive, electroneutral AE and that the role of this AE in stellate cell function deserves further study. Supported by NIH Grant K01‐DK080194‐01 to PMP.