bluuebunny/crossref_metadata_2025_split huggingface dataset:
Temperature Dependence of Delayed Ca2+ Activation of TMEM16F (ANO6) and Its Ca2+ Sensitivity
Authors: Lee Mi‐Ok , Lin Haiyue , Nam Joohyun 
Published: November 2023

TMEM16F, also known as ANO6, belongs to a family of putative Ca2+‐activated Cl− channels (CaCCs). In contrast to other classic CaCCs such as TMEM16A (ANO1), the activation of ANO6 requires very high cytosolic Ca2+ concentration ([Ca2+]c > 3 μM) and strong positive membrane potentials. Moreover, even when [Ca2+]c is high, the ANO6 current appears very slowly, requiring >10 min to reach a steady‐state. These properties were confirmed in all three functioning variants (V1, 2, and 5) of ANO6. The low Ca2+‐sensitivity and slow kinetics of ANO6 suggest that it does not function as an ion channel. In the present study, using whole‐cell patch recordings, we found that the three ANO6 isoforms (V1, 2, and 5) transiently transfected into HEK293T cells were activated by lower [Ca2+]c at physiological temperature (37°C). However, V2 and V5 showed significantly higher sensitivity and were activated by 400 nM [Ca2+]c and V1 by 1 μM [Ca2+]c. Furthermore, V2 and V5 showed significantly greater shortening of the latency time of activation than V1; after preparing the whole‐cell configuration, the currents reached a maximum after 543.3 ± 72.48 s, 56.67 ± 9.55 s, and 62.5 ± 4.53 s for V1, V2, and V5, respectively. The temperature‐dependent effect was reversible. In inside‐out patch, all isoforms of ANO6 exhibited higher calcium sensitivity and channel activity at 37°C than at room temperature. V3 showed no current activity in the inside‐out configuration. These results suggest that nearly all isoforms of ANO6 can be activated at low Ca2+ concentration (≤1 μM) when conditioned at physiological temperature, although they showed different kinetics. Our results suggest that ANO6 functions as ion channel in various tissues and cells depending on the variant forms of ANO6.Support or Funding InformationThis research was supported by Basic Science Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Science, ICT & Future Planning(NRF‐2015R1C1A1A02037738)
MRP4 Influences cAMP Levels in Ciliary Epithelial cells of the Human Eye
Authors: Pelis Ryan M , Ingraham Leslie , Hotchkiss Adam G 
Published: November 2023

The ciliary epithelium is important for aqueous humor inflow and maintenance of intraocular pressure. Treatment of ciliary epithelium with β‐adrenergic agonists leads to increased extracellular cAMP and adenosine and stimulation of aqueous humor inflow. The mechanism by which cAMP gets out ciliary epithelial cells is unknown. The purpose was to determine if the multidrug resistance‐associated protein 4 (MRP4) in ciliary epithelial cells influences intracellular and extracellular cAMP levels. Intracellular and extracellular cAMP levels were monitored in cultured ciliary epithelial cells using a FRET‐based assay. Also examined by RT‐PCR in cultured cells and native human ciliary body was the expression of MRP4, and the expression of receptors and enzymes associated with the cAMP‐adenosine signaling pathway. MRP4 mRNA and protein (immunoblotting) were detected in cultured cells and native human ciliary body. There was a dose‐dependent increase in both intracellular and extracellular cAMP levels following treatment of the cells with isoproterenol (ISO, 0–1 mM). Intracellular cAMP levels were ~10‐fold higher in the presence vs. absence of ISO (1 mM), and extracellular cAMP went from being undetectable to detectable. The β2‐adrenergic receptor was detected in the cultured cells and native ciliary body, but β1‐ and β3‐adrenergic receptors were undetectable. MRP4 mRNA was undetectable by qPCR and RT‐PCR in cultured cells following treatment with siRNA against MRP4. There was no difference in basal intracellular cAMP levels between cells treated with negative control siRNA (control) and MRP4 siRNA. Following treatment with ISO (1 mM) intracellular cAMP levels were 2.7‐fold significantly higher in cells treated with MRP4 siRNA compared to control. There was no difference in extracellular cAMP levels between the two treatments. Also present in both cultured cells and native ciliary body were message for enzymes necessary for conversion of cAMP to adenosine, such as ecto‐5‐nucleotidase (CD73). These data show that MRP4 likely contributes to efflux of cAMP from ciliary epithelial cells where it is then converted to adenosine. The cultured cells should prove useful for further study of the cAMP‐adenosine signaling pathway in the ciliary epithelium of the human eye.Support or Funding InformationNatural Sciences and Engineering Research Council of Canada
Molecular mechanisms mediating the adaptive regulation of human colonic thiamine pyrophosphate (TPP) uptake process
Authors: Srinivasan Padmanabhan , Anandam Kasin Yadunandam , Haidary Habib Ahmad , Said Hamid 
Published: November 2023

Thiamin(vitamin B1) is indispensable for normal cellular metabolism due to itsinvolvement in critical metabolic pathways (e.g., energy metabolism, maintenanceof normal cellular redox state, normal mitochondrial structure and function). Human(mammals) cannot synthesize thiamin, and thus, must obtain the vitamin fromexogenous sources, i. e., diet and gut microbiota. A substantial amount of themicrobiota‐generated thiamin exists in the form of TPP. We have previously shown the existence of a specific and high‐affinity carrier‐mediated uptake process for TPP in human colonocytes (Am J Physiol Gastrointest Liver Physiol. 2012; 303: G389‐95), and have recently determined the molecular identity of the system involved (product of the SLC44A4 gene) (J Biol Chem. 2014; 289: 4405‐16). We have also shown that the colonic TPP uptake process is adaptively regulated by the extracellular substrate level, but nothing is known about the molecular mechanism(s) that mediates this regulation. We addressed this issue in the current investigation using the human colonepithelial NCM460 cells as a model. Maintaining these cells in the presence of high levels of TPP (1 mM) for 2 and 9 days was found to lead to a significant(p ≤ 0.01) reduction in the rate of TPP uptake compared to uptake by cells maintained in the absence of added TPP. While no change in the level of expression of the SLC44A4 mRNA was found in cells maintained for 2 days in the presence of high TPP level [suggesting possible involvement of post translation mechanism(s)], a significant (p ≤ 0.01) decrease in level of SLC44A4 mRNA was found in cells maintained for 9 days. We also observed a decrease in activity of the SLC44A4 promoter in cells maintained for 9 days in high TPP level. The latter was associated with clear histone modifications at the SLC44A4 promoter that involve significant decrease in histone H3K4‐trimethylation and H3K9‐acetylation (euchromatin/activating markers)and a significant increase in histone H3K9‐trimethylation and H3K27‐trimethylation (heterochromatin/repressive markers). While maintaining the cells for 9 days in the presence of high TPP level did not affect the level of expression of the two transcription factors, ELF3 and CREB‐1, that drive the activity of the SLC44A4promoter, their binding to the SLC44A4promoter was decreased as shown by Chip‐qPCR analysis. These studies demonstrate, for the first time, that multiple mechanisms are involved in the adaptive regulation of colonic TPP uptake by extracellular substrate level. At early stages, this regulation involves post‐translational mechanism(s), while long‐term adaptive regulation involves transcriptional and epigenetic mechanisms.Support or Funding InformationSupported by grants from the DVA and the NIH (DK56061 and DK58057).
First identification of mutations in the human SLC5A6 gene associated with brain, immune, bone and intestinal dysfunction
Authors: Subramanian Veedamali , Benke Paul , Constantinescu Alexandru , Said Hamid 
Published: November 2023

Biotin (vitamin B7) is indispensable for normal cellular metabolism due to its involvement in a variety of critical metabolic pathways including fatty acid, amino acid and energy metabolism; it also plays a role in regulating cellular level of reactive oxygen species and gene expression, as well as normal immune function/response. Human (mammalian) cells cannot synthesize biotin endogenously; rather they obtain the vitamin across the plasma membrane via a carrier‐mediated uptake process that involves the human sodium‐dependent multivitamin transporter (hSMVT; product of the SLC5A6 gene); this system also transports pantothenic acid and lipoate. We report here, using whole exome sequencing (GeneDx), the first identification of two mutations in the SLC5A6 gene in a young child: R94X [(CGA>TGA) c280 C>T] and R123L [(CGC>CTC), c368 G>T]. Both of these mutations are located in exon 3 of the SLC5A6 gene. The child exhibited many clinical manifestations including failure to thrive, microcephaly, brain changes, cerebral palsy, developmental delay, immunodeficiency, severe gastro‐esophageal reflux, osteoporosis and pathologic bone fractures. After identification of the hSMVT mutations, the child responded favorably to supplemental administration of pharmacological doses of biotin, pantothenic acid and lipoate. Experimental characterization of the identified mutations utilizing human‐derived intestinal HuTu‐80 and brain U87 cell lines, showed impaired functionality (3H‐biotin uptake) of the two identified hSMVT mutants. In addition, our results (using live‐cell confocal imaging) showed poor expression and cytoplasmic localization of the R94X mutant, while the R123L mutant was predominantly retained in the endoplasmic reticulum. This is the first reporting of mutations in the human SLC5A6 gene that lead to defects in hSMVT function and cell biology, and is associated with a host of clinical abnormalities.Support or Funding InformationSupported by a grant from the DVA, by NIH grants DK58057, DK56057, DK10747, and the Gordon Foundation
Short‐term exposure to chronic intermittent hypoxia (CIH) to model repetitive hypoxemias of sleep apnea is sufficient to confer protection from brain ischemia
Authors: Toney Glenn M. , Wellford Alexander J. , Andrade Mary Ann 
Published: November 2023

Sleep Apnea (SA), a form of sleep disordered breathing, is characterized by repetitive episodes of arterial hypoxemia resulting from interrupted breathing. Often SA results in develop of hypertension, which studies demonstrate results from increased vasoconstriction driven by excessive sympathetic nerve activity. The sympathetic activation response involves recruitment of pre‐sympathetic neurons in the hypothalamic paraventricular nucleus (PVN). Clinical studies report that SA increases the risk of stroke by ~4‐fold. By contrast, pre‐clinical studies in mice suggest that prolonged (~30 days) exposure to CIH is neuroprotective in the middle cerebral artery occlusion (MCAO) model of stroke. Thus available data suggest that neural, glial and/or vascular adaptations to repetitive hypoxia confer protection from brain ischemia. To gain insight into mechanisms of CIH‐induced ischemic protection, effects of short‐term (7 days) exposure to moderate CIH (21% to 10% O2, 10 episodes/h, 8 h/day) on brain tissue infarct volumes were quantified. This was accomplish using a permanent MCAO model as well as a photothrombic lesioning (PTL) model of focal ischemic injury. Results indicate that exposure to CIH for just 7 days reduced the estimated volume of MCAO‐ and hypothalamic PTL‐induced focal ischemic infarcts. In the MCAO model, CIH reduced infarct volume by 31 ± 4 % (SEM, n=5) relative to normoxic controls. CIH more effectively reduced infarct volume in the hypothalamic PTL model by 65 ± 9 % (SEM, n=7). These findings indicate that CIH exposure for only 7 days is sufficient to induce robust protective adaptations. PO2 recordings from multiple brain regions indicate that episodes of CIH cause rapid reductions of tissue O2 availability. However, episodes of brain tissue hypoxia showed little change over consecutive days of CIH exposure, suggesting that CIH does not induce progressive cerebrovascular adaptations that protect brain tissue from experiencing acute hypoxia. Thus findings suggest that adaptations among resident neurons and/or glia promote tissue survival in the face of focal ischemia.Support or Funding InformationSupport: NIH HL088052 & AHA 15GRNT25710176 (GMT)
Dysregulation of Glutathione Synthesis by Enteric Glia During Gastrointestinal Inflammation
Authors: Brown Isola A.M. , Gulbransen Brian D. 
Published: November 2023

Enteric glia, a unique class of peripheral neuroglia, are integral in the maintenance of enteric neuronal circuits and normal gastrointestinal (GI) function. In vitro studies have identified the antioxidant reduced glutathione (GSH) as a vital neuroprotective factor secreted by glia (Abdo et. al 2010). However, little is known about the cellular mechanisms regulating GSH production and the role GSH plays during in vivo inflammation. Here, we identified the cellular components involved in GSH synthesis and investigated the effects of in vivo inhibition of GSH synthesis during GI physiology and pathophysiology.We used immunohistochemistry to localize GSH synthesis proteins and to quantify changes in neuronal density and other cellular markers. We inhibited GSH synthesis in vivo and in vitro with the selective inhibitor Buthionine Sulfoximine (BSO) and induced in vivo inflammation using the 2,4‐dinitro benzene sulfonic acid (DNBS) model of colitis. All experiments used male C57Bl/6 mice, aged 6–8 weeks. Data were analyzed using a Student's t‐test or one/two‐way ANOVA as appropriate.Glutathione is synthesized in a two‐step process. The first and rate‐limiting enzyme glutamate‐cysteine ligase (GCL) is expressed in enteric glia and neuronal varicosities. The second enzyme, glutathione synthetase, is similarly found in both neuronal and glial cell bodies. Immunoreactivity for both enzymes is upregulated in response to in vivo inflammation (p < 0.005). Inhibition of GSH production with the GCL‐inhibitor BSO is sufficient to drive neurodegeneration both in vivo and in vitro (p = 0.053 and p < 0.01 respectively). Mice drinking water supplemented with BSO were protected from colonic shortening (p = 0.054), inflammatory cell infiltration (p < 0.001) and neurodegeneration during colitis.Our works highlights a novel role for the endogenous antioxidant GSH and shows that GSH production by enteric glia is an important neuroprotective mechanism during GI (patho)physiology.Support or Funding InformationF31DK107232 to IBR01DK103723 to BDG
Mechanisms Involved in the Changes of Synaptic Transmission in the Nucleus Tractus Solitarius of Rats Submitted to Sustained Hypoxia
Authors: Lima‐Silveira Ludmila , Accorsi‐Mendonça Daniela , Bonagamba Leni G.H , Machado Benedito H 
Published: November 2023

The sustained hypoxia (SH) is associated with changes in synaptic transmission in the nucleus tractus solitary (NTS) and also with inflammatory processes. In this study, we investigated the effects of anti‐inflammatory treatment on the excitatory synaptic transmission in the NTS neurons and the gene expression of pro‐inflammatory cytokines [interleukin‐1β (IL‐1β), intereleukin‐6 (IL‐6) e tumor necrosis factor‐alfa (TNF‐α)] in this region. Four groups of juvenile wistar rats (100–130g) were evaluated: 1) control (FiO2=20.8%) treated with vehicle (saline + distilled water, i.p), 2) control treated with the anti‐inflammatory Minocycline (30mg/kg, i.p), 3) SH (FiO2=10%) treated with vehicle, 4) SH treated with Minocycline. The experimental protocols were approved by the institutional ethics committee (CEUA 070/2007 and 094/2013). The rats were treated for 3 days and then exposed to SH or normoxia protocol for 24 h. At the end of this period, electrophysiological records of NTS neurons were performed using the whole cell patch‐clamp technique. Genetic analyzes of NTS punches were performed using real‐time qRT‐PCR. The results showed significant changes in the frequency of excitatory postsynaptic spontaneous currents (sEPSCs) in neurons of rats treated with vehicle submitted to SH (2,4±0,3 Hz, n=9) when compared to their control group (0,96±0,2 Hz, n=12; p<0,05). This increase was not observed in neurons of minocycline treated rats submitted to SH (0,7±0,2 Hz, n=9) compared to their control group [(0,7±0,1 Hz, n=10) two‐way ANOVA followed by Bonferroni's post‐test]. A significant increase of the IL‐1β gene expression was observed only in the SH vehicle rats. No changes were observed in relation to cytokines IL‐6 e TNF‐α. These results indicate that the inflammatory process induced by SH, probably due to IL‐1β, participates of the mechanisms related to the increase of excitatory synaptic transmission in NTS neurons.Support or Funding InformationCAPES, CNPq, FAPESP.
Ablation of Astrocytes in the Paraventricular Nucleus Disrupts the Blood‐Brain Barrier and Increases Blood Pressure in Rats
Authors: Setiadi Anthony , May Clive N , Yao Song T 
Published: November 2023

BackgroundPerivascular astrocytes make direct connections to the cerebral vasculature. This close apposition has been shown to be critical for the regulation of blood‐brain barrier (BBB) integrity and function. Recent studies have shown that the BBB becomes disrupted in hypertensive rats, which allows circulating peptides such as angiotensin II, which is normally excluded from the central nervous system, to access brain areas critical for blood pressure control. To date, the association between astrocytes, the BBB and hypertension is still poorly understood.ObjectiveTo determine the importance of astrocytes in maintaining BBB integrity and central control of cardiovascular homeostasis.MethodsTwo groups of male Sprague‐Dawley rats (250–400g), anesthetized (pentobarbital, 60 mg/kg i.p.) and conscious freely‐moving, were bilaterally microinjected with sodium fluoroacetate (50 nL; 10 mM), a specific glial toxin, into the paraventricular nucleus of the hypothalamus (PVN) to ablate perivascular astrocytes. Blood pressure and heart rate were continuously recorded throughout. Evans blue dye (4 mL/kg) was then infused intravenously following microinjections to assess BBB integrity. Changes in baroreceptor reflex function were also studied following fluoroacetate administration.ResultsAblation of PVN astrocytes caused a significant increase in Evans Blue dye extravasation (+16.67 ± 4.9 mean density in arbitrary units, P<0.05) when compared with saline injected controls, indicating a compromised BBB. Furthermore, we found a significant increase in mean arterial pressure in anesthetized (+41.02 ± 5.5 mmHg from baseline, P<0.0001) and conscious rats (+23.82.96 ± 7.8 mmHg from baseline, P<0.05) at 100‐minute and 2‐days following fluoroacetate injection, respectively. However, no changes were seen in the baroreceptor reflex control of heart rate.ConclusionOur results show a functional link between astrocytes, BBB disruption and central regulation of cardiovascular function. These findings suggest that a disrupted BBB, via astrocyte dysfunction, may contribute to the pathophysiology of centrally‐induced cardiovascular diseases such as hypertension.
Hypertension is accompanied with over‐activity of immunity
Authors: Shen Xiao (Gregory) Z. , Zhao Tuantuan , Shi Peng , Bernstein Kenneth E. 
Published: November 2023

Immune system plays an important role in the pathogenesis of hypertension. To understand whether hypertension affects immunity, we studied antigen presenting cells (APCs) isolated from mice with hypertension induced by angiotensin II or L‐NAME. The APCs derived from hypertensive mice were superior in presenting antigens in vitro. To study antigen presentation in vivo, we immunized sham and AngII treated wild type mice with ovalbumin. Consistently, more OVA‐specific CD8+ T cells were induced in the blood and the spleen of hypertensive mice. Moreover, RIP‐mOVA mice were studied because this transgenic mouse line expressing membrane‐bound OVA in pancreatic islet β cells. RIP‐mOVA mice were made hypertensive with either angiotensin II or L‐NAME. When OVA‐specific OT‐I T cells were i.v. infused into RIP‐mOVA mice, the hypertensive mice had more OT‐I cells infiltration into the pancreas and they developed diabetes more rapidly. These data suggest that hypertension itself is associated with over‐activity of the adaptive immune system.Support or Funding InformationNational Nature Science Foudation of China 31270950 and 81670378
Haemodynamic Mechanisms underlying Essential Hypertension Change Across the Adult Age‐Span: The Anglo‐Cardiff Collaborative Trial
Authors: McEniery Carmel , Yasmin Y , McDonnell Barry , Cockcroft John , Wilkinson Ian 
Published: November 2023

Essential hypertension is often viewed and treated as a uniform condition. However, the condition encompasses a number of distinct patterns of blood pressure (BP) elevation such as isolated systolic (ISH), systolic diastolic (SDH) and isolated diastolic hypertension (IDH), which appear to vary in prevalence across the adult age‐span. We hypothesized that different haemodynamic mechanisms account for the age‐related differences in the prevalence of these hypertensive subtypes.Detailed haemodynamic data including seated (clinic) BP, cardiac output (CO), stroke volume (SV), peripheral vascular resistance (PVR) and aortic pulse wave velocity (aPWV) were available in 5337 individuals (2381 males), aged 18–92 years. All individuals were free of cardiovascular disease and medication. Within each gender, subjects were stratified according to age (<30 years, 30–60 years and >60 years) and hypertensive subtype, on the basis of clinic BP readings.ISH was the most common hypertensive subtype in males aged <30 years and was characterized by an elevated CO and SV compared with normotensive individuals. In contrast, SDH and IDH were more common in females aged <30 years and associated with elevated PVR and aPWV compared with normotensives. SDH was the most common subtype in males and females aged 30–60 years and accompanied by increased PVR in both genders. ISH was again the most common subtype in individuals aged >60 years. However, in contrast to younger adults, ISH affected both males and females equally (1:1) and was characterized by a marked elevation in aPWV compared with normotensive individuals.Distinct haemodynamic mechanisms are associated with different hypertensive subtypes in an age‐ and gender‐dependent manner. Detailed physiological profiling and subsequent targeting of therapy towards primary haemodynamic abnormalities could lead to more effective interventions in essential hypertension.