bluuebunny/crossref_metadata_2025_split huggingface dataset:
Role of ATP‐sensitive K+ channels in glucose sensing by immortalized adrenomedullary chromaffin cells
Authors: Piskuric Nikol Anne , Brown Stephen T. , Nurse Colin A. 
Published: June 2021

Adrenomedullary chromaffin cells exhibit a critical Ca2+‐dependent catecholamine secretion when directly exposed to perinatal stressors (e.g. hypoxia). Using an immortalized chromaffin cell line (MAH), we investigated their potential sensitivity to low glucose (hypoglycemia) by monitoring changes in intracellular free calcium ([Ca2+]i) with the fluorescent probe Fura‐2. The majority of MAH cells (86%; n=398) were unresponsive on switching from 10 to 0 mM glucose. Interestingly, 0 glucose markedly inhibited (~75%) the rise in [Ca2+]i induced by the depolarizing stimulus high K+ (10 mM). To test the possibility that this was due to KATP channel activation, the KATP channel blocker, glibenclamide (50 μM), was used. In normal K+, glibenclamide increased the proportion of 0 glucose‐responsive cells from 14% to 65%, and the mean Δ[Ca2+]i response increased fourfold. RT‐PCR and Western blot analysis confirmed the presence of both the inward rectifying (Kir6.2) and sulfonylurea (Sur1) subunits of KATP channels. These findings suggest that hypoglycemia‐induced activation of KATP channels may limit [Ca2+]i increases and catecholamine secretion in chromaffin cells. Furthermore, the increased proportion of 0 glucose‐responsive cells in the presence of glibenclamide suggests that hypoglycemia causes a separate excitatory pathway in MAH cells.Supported by NSERC and the HSFO.
ASIC Channels Inhibit BK Potassium Channels by a Toxin‐Like Extracellular Motif
Authors: Petroff Elena , Price Margaret P , Snitsarev Vladislav , Gong Huiyu , Abboud Francois M , Welsh Michael J 
Published: June 2021

Acid‐sensing ion channels (ASICs) contain a large extracellular domain whose function has remained largely unknown. Within this domain sits a highly conserved sequence which resembles the scorpion toxin sequence that binds and inhibits large conductance calcium‐activated potassium channel (BK). We hypothesized that ASICs might interact with and inhibit BK. In transfected HEK 293 cells, we found that ASIC1a inhibited BK currents. Lowering extracellular pH relieved the inhibition.ASIC2a, −2b and −3 also inhibited BK currents, and the pH‐dependent relief of inhibition varied between ASIC channels. In scorpion toxins, the cationic residues in the RGKC/RFGKC sequence are critical for inhibition. When we mutated the equivalent R and K residues in ASICs to A or E, the inhibition of BK channels was abolished. To test for an interaction of the endogenous proteins, we studied cultured cortical neurons and found an ASIC‐dependent inhibition of BK current and its pH‐dependent relief. BK currents were larger in ASIC1a/2/3 knockout mice, consistent with the lack of inhibition. In addition, the two proteins co‐precipitated from transfected cells and whole brain. These data indicate that a toxin‐like sequence in the extracellular domain of ASICs inhibits BK channels. This novel mechanism for regulating potassium channel activity may influence neuronal excitability. Support: HL014388 to FMA.
Phospholipase A2 and lysophospholipid roles in receptor activation of TRPC5 channel
Authors: AL‐Shawaf Eman , Li Jing , Beech David J. 
Published: June 2021

TRPC5 is a widely expressed calcium‐permeable channel that is stimulated by several agonists acting at various G‐protein coupled receptors. The mechanisms coupling the receptors to TRPC5 are, however, unknown. Because we have shown previously that TRPC5 is activated by intracellular lysophosphatidylcholine (LPC) (Flemming et al 2006, J Biol Chem 281, 4977–82) we investigated the hypothesis that receptor coupling to TRPC5 results, at least in part, from accumulation of LPC as a consequence of receptor‐activation of a calcium‐independent phospholipase A2 (iPLA2). Tetracycline inducible TRPC5‐expressing HEK 293 cells were studied using fluo‐4 to measure calcium‐entry in real‐time 96‐well plate assays. The agonists employed were carbachol (CCh) and sphingosine‐1‐phosphate (S1P), which act at different receptors. Pre‐incubation of cells with LPC, but not arachidonic acid, abolished TRPC5 activation by CCh, reduced stimulation by S1P and left unaffected activation by gadolinium. Bromoenol lactone, an iPLA2 inhibitor, suppressed TRPC5 activation by CCh or S1P but not activation by LPC or gadolinium; a similar effect occurred following knock‐down of iPLA2‐VIA using RNAi. The data suggest receptor‐coupling to TRPC5 involves iPLA2‐VIA and LPC.The research is supported by the Wellcome Trust.
Functional Cysteine Pair in TRPC5 Cation Channel
Authors: Sukumar Piruthivi , Xu Shang‐Zhong , Zeng Fanning , Li Jing , Bahnasi Yahya M , Jiang Lin‐Hua , Sivaprasadarao Asipu , Beech David J 
Published: June 2021

Mammalian homologues of Drosophila TRP proteins form non‐selective cation channels in many cell types. One member of the canonical subfamily of TRP channels, TRPC5, is expressed in a range of tissues including parts of the brain and blood vessels. TRPC5 responds to a multiplicity of activation signals including G protein coupled receptor agonists, lipids and lanthanides (Beech 2007, Handb Exp Pharmacol 179, 109–23). A glutamate residue in the predicted extracellular region between the S5 and S6 transmembrane segments (i.e. the putative turret structure of the channel) is essential for activation by lanthanides (Jung et al 2003 J Biol Chem 278, 3562–71). Amino acid sequence alignment of TRPC1, 4 and 5 revealed the presence of two conserved cysteine residues in close proximity to this glutamate. We hypothesised that these cysteine residues may be functionally important. Experiments were performed on HEK 293 cells over‐expressing TRPC5 channels using patch‐clamp and calcium‐imaging techniques. Reducing agents, acting from the extracellular side of the membrane, were found to have marked stimulatory effects on TRPC5. Mutation of either cysteine residue led to large constitutive currents which were not stimulated by reducing agent or lanthanide, but were inhibited by 2‐aminoethoxydiphenyl borate. The data suggest an accessible inhibitory disulphide bridge in the putative turret of TRPC5.The research is supported by the Wellcome Trust, an Overseas Research Scholarship Award to PS and an Egyptian Ministry of Higher Education Scholarship to YMB.
Overnight food deprivation prevents orexin‐A induced intracellular calcium signaling in duodenal enterocytes
Authors: Flemström Gunnar , Bengtsson Magnus , Mäkelä Kari , Sjöblom Markus , Herzig Karl‐Heinz 
Published: June 2021

Some appetite regulating hormones, including orexin‐A and orexin‐B, are produced in the hypothalamus but also expressed in the intestine. We compared effects of orexin‐A on intracellular calcium [Ca2+]i signaling in enterocytes acutely isolated from duodenum in fed and fasted animals. Method: Lewis × Dark Agouti rats had access to their regular food supply, or were overnight deprived of food (but not water). Animals were sacrificed and duodenal mucosa digested (collagenase/dispase) to yield clusters (10–100 cells) of enterocytes. [Ca2+]i was measured with fluororescence imaging. Results: Orexin‐A (1–100 nM) induced calcium signaling in enterocytes isolated from fed animals. There was a rise in [Ca2+]i to a peak level, and the response was similar to those observed with carbachol or cholecystokinin. In contrast, there were no responses to orexin‐A in enterocytes from overnight food deprived animals. A low concentration of the orexin (OX1) receptor antagonist SB‐33487 (10 nM) abolished the response to 10 nM of orexin‐A and depressed that to 100 nM of the peptide. Conclusion: The orexogenic peptide orexin‐A induces [Ca2+]i signaling in duodenal enterocytes from fed animals and preceeding short fasting abolishes the response. The findings are in line with the reported absence of an electrolyte secretory response to orexin‐A in fasted animals.Supported by the Swedish Research Council and the Finnish Academy
Differential estrogen receptor‐mediated Ca2+ regulation in coronary arterial smooth muscle from intact vs. ovariectomized pigs
Authors: Prakash Y S , Townsend Elizabeth A , Sieck Gary C , Miller Virginia M 
Published: June 2021

Acute administration of estrogen induces vasodilation in several vascular beds including coronary artery smooth muscle (CSM). We tested the hypothesis that the response of CSM to estrogen is mediated via the binding of estrogen to estrogen receptors (ER) and that the response will be influenced by hormonal status. Coronary smooth muscle was prepared from gonadally intact (INT) and. ovariectomized (OVX) female pigs. Immunocytochemistry and receptor binding assays were performed to confirm presence vs. absence of ER. In INT CSM cells, but not OVX CSM, acute administration of 17β‐estradiol resulted in a concentration‐dependent decrease in [Ca2+]i responses to endothelin‐1. [Ca2+]i responses to 17β‐estradiol were comparable between groups. The effect of 17β‐estradiol was inhibited by the ER antagonist ICI 182,780. 17β‐estradiol decreased [Ca2+]i in INT (but not OVX) CSM cells when sarcoplasmic reticulum (SR) Ca2+ reuptake was inhibited, but to a lesser extent than when extracellular Ca2+ was present. This effect was abolished by extracellular lanthanum. These data suggest that 17β‐estradiol modulates [Ca2+]i in CSM via a likely non‐genomic and necessary ER signaling cascade that mediates both plasma membrane Ca2+ influx and efflux, but does not involve SR Ca2+ release.Supported by NIH grants UL1RR024150‐01(Mayo Clinic CTSA awards to YSP & CMP) and HL74309 (GCS)
Regulation of Polycystin‐1 C terminal cleavage by Polycystin‐2
Authors: Bertuccio Claudia Alejandra , Cai Yiqiang , Somlo Stefan , Caplan Michael J 
Published: June 2021

Autosomal dominant polycystic kidney disease is caused by mutations in PKD1 or PKD2, encoding polycystin‐1 (PC1) and polycystin‐2 (PC2), respectively. PC1 undergoes a cleavage that releases its cytoplasmic C‐terminal tail (CTT), which enters the nucleus. We wished to determine whether PC1 cleavage is influenced by PC2 and, if so, whether the PC2 C terminal domain is involved. We generated a fusion protein in which a chimera of Gal4 and VP16 was inserted downstream of the PC1 intracellular CTT domain. PC1‐GalVP was cotransfected with a Gal4‐driven luciferase reporter gene in COS cells. Cotransfection of PC1 and PC2 produced a 2‐fold increase in luciferase activity compared to cells expressing PC1 alone. Western blotting revealed increased PC1 CTT cleavage in the presence of PC2. Luciferase activity did not increase in cells transfected with PC1 and PC2 L703X (lacking a portion of the PC2 C‐terminal). We analyzed a series of truncated PC2 constructs and found that, in contrast to PC2 L703X, PC2 constructs that terminate at residues R742, E787, D847, K859, or R872 stimulated PC1 CTT cleavage to the same extent as full‐length PC‐2. Surprisingly, the A753X and G821X truncated constructs appear to increase PC1 CCT cleavage beyond the levels obtained with full‐length PC2. Thus, the release of the CTT from PC1 is influenced by PC2 and may be regulated by sequences contained within the PC2 C terminal tail.Support by NIH 57328
Dietary omega‐3 fatty acids prevent nitric oxide synthase loss in diabetic kidney disease
Authors: Garman Joe , Xu Qin , Maric Christine 
Published: June 2021

Background: We previously showed that a diet rich in omega‐3 fatty acids attenuates albuminuria, glomerulosclerosis and tubulointerstitial fibrosis associated with diabetes. The aim of the present study was to further examine the mechanisms by which omega‐3 fatty acids exert their renoprotective effect.Methods: The study was performed in 10 week‐old male Sprague‐Dawley non‐diabetic (ND) and streptozotocin‐induced diabetic rats fed normal rat chow (D), or rat chow supplemented with canola oil which is rich in omega‐3 fatty acids (DC) for 30 weeks. Blood pressure was examined by tail cuff sphygmomanometry.Results: D was associated with an increase in systolic blood pressure (BP; ND 153±9; D 198±7; mmHg, p<0.05), decrease in eNOS protein expression in kidney cortex (KC) (eNOS ND 1.29±0.28; D 0.41±0.05, P<0.01) and eNOS and nNOS in red blood cells (RBC) (eNOS ND 1.16±0.12; D 0.27±0.06, P<0.001, nNOS; ND 0.92±0.05; D 0.54±0.10, P<0.05). Diet supplemented with canola oil prevented these changes (BP 162±9; eNOS RBC 1.10±0.07; nNOS RBC 1.48±0.10; eNOS KC 1.50±0.10). No changes were seen in iNOS.Conclusion: Our results show that diet rich in omega‐3 fatty acids prevents the decrease in eNOS and nNOS in the diabetic kidney. These data suggest that a diet rich in omega‐3 fatty acids is beneficial in attenuating hypertension and protects the kidneys by preserving eNOS and nNOS protein expression.
Role of Insulin on The Development of Renal Injury in Streptozotocin‐induced Diabetic Dahl Salt Sensitive Rats
Authors: Hosoya Takuma , Mori Takefumi , Yoneki Yoshimi , Endo Satoshi , Ohsaki Yusuke , Sato Hiroshi , Ito Sadayoshi 
Published: June 2021

Background: Previous studies have suggested that insulin resistance and hyperinsulinemia increase oxidative stress which is implicated in the progression of diabetic nephropathy. The present studies were designed to investigate the role of insulin in the progression of diabetic renal injury.Methods: Dahl salt sensitive rats were administered either with streptozotocin (STZ, 65mg/kg intraperitoneally), control solution (CONT, intraperiotneally), or STZ with insulin (10U/kg/day, subcutaneous infusion) (STZ+INS). Urine was sampled every week in a metabolic cage for urinary protein (UP), TBARS and hydrogen peroxide as a marker of renal injury and oxidative stress. Mean arterial pressure was measured with telemetry method.Results: Urinary protein, TBARS and hydrogen peroxide excretion was significantly increased in STZ group compared to those of CONT group. Mean arterial blood pressure tend to reduce in STZ group but was not different from STZ+INS group. Urinary protein was further increased in STZ+INS group compared to those of STZ group.Conclusion: These results indicate that diabetic induction would induce renal injury in Dahl salt sensitive rats with enhanced oxidative stress. Hyperinsulinemia is involved in the progression of diabetic renal injury independent of its blood pressure.
Intestinal and hepatic cholesterol transporters in Psammomys Obesus, a model of insulin resistance and type 2 diabetes
Authors: Lalonde Genevieve , Delvin Edgard , Elchebly Mounib , Sane Alain Theophile , Lambert Marie , Garofalo Carole , Ziv Ehud , Levy Emile 
Published: June 2021

Poor glycemic control is associated with hyperlipidemia. Our results have recently shown that the increased transport of chylomicrons from the intestine to the circulation in both conditions could potentially result in circulating lipid abnormalities that likely enhance the risk of atherosclerosis. Although protein transporters have been involved in regulating cholesterol movement into and out of enterocytes, little is known about their status in insulin resistance and diabetes. Our objective was to determine mRNA levels of genes such as ABCA‐1, ABCG5/8, NPC1‐L1, Annexin II, CD36, SCP‐2 and SR‐B1, in intestinal and hepatic tissues in Psammomys Obesus. In diabetic animals, analysis of gene expression by Real‐Time PCR revealed a decrease in intestinal and hepatic levels of ABCG5/8 and ABCA1, which act as efflux pumps favoring cholesterol export out of absorptive cells. Furthermore, a decline was observed in intestinal mRNA levels of NPC1‐L1 that plays a major role in cholesterol absorption, whereas no changes were evidenced in the gene expression of intestinal SR‐BI, annexin 2, CD36 and SCP2. In contrast, raised mRNA concentrations characterized annexin‐2 and SR‐BI in the hepatic tissue. Our results suggest that the expression of cholesterol transporters is differently modulated in the intestine and liver in diabetes type 2.Supported by Diabete Canada.
Developmental changes in the pattern of the hypoxic ventilatory response in rabbits
Authors: Stucke Astrid G , Mustapic Sanda , Sanchez Antonio , Stuth Eckehard A , Zuperku Edward J 
Published: June 2021

The ventilatory response to hypoxia in mature mammals consists of an initial increase in ventilatory minute volume with a subsequent decline to a level that remains elevated compared to normoxia, but in neonatal mammals this decline often results in apnea. We investigated the maturation of the hypoxic ventilatory response in rabbits. New Zealand White rabbits between age 14 and 22 days were anesthetized with 1–2 minimum alveolar concentration (MAC) of sevoflurane, tracheotomized, cannulated and paralyzed and mechanically ventilated. Phrenic nerve recordings were performed during normocapnia under 1 MAC sevoflurane anesthesia. Inspiratory O2 concentration (FiO2) was decreased from 21% to 10% for 4–5 minutes or until phrenic nerve activity stopped (apnea). All animals showed an initial increase in peak phrenic nerve activity (PNA) and in phrenic rate. With sustained hypoxia, peak PNA subsequently decreased in all animals. In some animals phrenic rate slowed slightly but a regular rhythm was maintained (adult pattern) while other animals developed apnea or displayed PNA at a slow and irregular rate (immature pattern). The pattern of the hypoxic response was consistent for repeated exposures within each animal. We conclude that in rabbits the change from the immature to adult response pattern to hypoxic exposure occurs during the third week of life.Supported by departmental funds.
Heat acclimation of an adult female with a large surface area of grafted skin: a case report
Authors: Wingo Jonathan E. , Low David A. , Keller David M. , Davis Scott L. , Kowalske Karen J. , Purdue Gary F. , Hunt John L. , Crandall Craig G. 
Published: June 2021

In healthy individuals, heat acclimation improves temperature regulation. It is unknown, however, whether this occurs in individuals with large areas of grafted skin that has impaired blood flow and sweating responses. This study tested the hypothesis that 7 days of heat acclimation would attenuate cardiovascular and thermal strain during exercise‐heat stress of an individual with a large percent total body surface area (TBSA) of grafted skin. A 33‐year‐old female with 75% TBSA grafted skin (14 years post‐injury/grafting) cycled at 45% peak oxygen uptake (50 W) in a climatic chamber set to 40 °C and 30% relative humidity on 7 consecutive days. The goal was to exercise 90 minutes (with a 5‐min break at min 45). Exercise was stopped if core temperature (Tc) reached an upper limit of 39.5 °C. This limit was reached during min 46 of exercise on acclimation day 1, but not until min 65 on day 7. The increases in Tc and heart rate during the first 45 min of exercise (the minimum duration completed for all acclimation bouts) were progressively mitigated with successive days of heat acclimation. Sweat sensitivity (sweat rate increase per 1 °C increase in Tc) in an area of uninjured skin was 30% greater on day 7 than day 1. Heat acclimation improved thermal tolerance of this patient, which could increase safety and comfort during thermal stress and/or exercise.Supported by NIH Grant GM68865
Caloric restriction attenuates developmental myosin heavy chain expression and myostatin signaling in aged muscles
Authors: Hepple Russell , Qin Maggie , Huba Elaine 
Published: June 2021

Aging is associated with an accumulation of muscle fibers expressing developmental myosin heavy chain (dMHC), suggesting they are in the process of regenerating. The cleaved form of myostatin increases in aged muscles and may be involved in impairing regeneration. Since caloric restriction (CR) slows age‐related muscle atrophy, we sought to determine the effect of CR on dMHC expression and to examine how this related to the myostatin signaling pathway. The gastrocnemius muscle from young adult (YA), late middle aged (LMA) and senescent (SEN) ad libitum fed and CR rats was harvested for either dMHC expression in situ or myostatin and ACVR2 receptor expression by Western immunoblot. CR attenuated dMHC expression with aging and although it did not affect the increase in cleaved myostatin, it slowed the increase in one of its receptors, ACVR2. Since the degree of muscle atrophy with aging correlated (r2=0.72) with the proportion of dMHC positive fibers, our results suggest that CR attenuates age‐related muscle atrophy by limiting the accumulation of incompletely regenerated muscle fibers secondary to attenuated myostatin signaling in aged muscles.Supported by AHFMR and CIHR.
Cycling Training effects on resting p38 MAPK activation in previously untrained young subjects
Authors: Schrager Matthew Alan , Fry Andrew C , Bloomer Richard J , Moore Christopher A , Thomason Donald B 
Published: June 2021

p38 is a stress‐activated protein kinase related to apoptosis, but it is not clear what effect an endurance training program has on p38 activation.OBJECTIVE:To quantify p38 activation after an 8‐week endurance training program in sedentary subjects.METHODS:Three men (X±SE; 26.0 ± 1.2 yr) and 6 women (22.0 ± 1.5 yr) participated in an 8‐week cycling training program 3 dwk−1. Pre‐/post‐biopsies were performed, and Western immunoblot analysis with infrared markers was used to identify resting values of total p38 and p‐p38/total p38. In addition, a p38 ‘Activation Index’ (AI) was calculated (p38 AI = total p38 x [0.01 x [p‐p38/total p38]]).RESULTS:VO2 max increased 8.9%, while %body fat decreased 2.1%. Resting total p38 decreased 39.8 ± 15.7% (P < 0.05), but p‐p38/total p38 did not significantly decrease (−8.3 ± 14.9%). However, p38 AI was considerably decreased (pre = 100.0, post = 55.2; P < 0.05).CONCLUSIONS:Our results suggest that this effective endurance‐exercise program does not induce basal stress at the level of skeletal muscle in young previously untrained subjects, as indicated by activation levels of p38. Further study is needed to determine whether these findings apply to more vulnerable populations (e.g. older persons, patients with rehabilitating injuries).Supported by Sigma‐tau HealthSciences, Inc.
Phenotype‐Specific Permeability Transition Pore Regulation in Rat Skeletal Muscle
Authors: Picard Martin , Mattas Jimmy , Ascah Alexis , Csukly Kristina , Godin Richard , Burelle Yan 
Published: June 2021

The aim of this study was to determine whether the susceptibility of mitochondria to opening of the permeability transition pore (PTP) varies according to muscle phenotype represented by the soleus (SOL: 95% type I fibers) and superficial white gastrocnemius (WG: 92% type IIb fibers). Sensitivity to Ca2+‐induced PTP opening was determined with a novel approach using permeabilized myofibers devoid of their contractile apparatus (phantom fibers). Ca2+ uptake in this preparation is specific to mitochondria and calcium retention capacity (CRC) is a good indicator of PTP sensitivity. Mitochondrial volume density was ~ 2 fold greater in SOL vs. WG, as judged by respiratory capacity per mg fiber weight and activity of the marker enzyme citrate synthase (CS). CRC values were 2.2 fold lower in phantom fibers from SOL compared to WG when expressed per unit of CS (CRC: 36.6±9.9 vs. 81.0±26.2 pmol Ca2+/mIU CS; p<0,001; n=4), indicating a pronounced vulnerability to PTP opening in type I vs. IIb fibers. A similar phenomenon was also observed in isolated mitochondria (CRC: <83 and 121 ± 60 nmol Ca2+/mg prot. in SOL and WG). This phenomenon was partly due to a 2‐fold increase in endogenous matrix Ca2+ levels in mitochondria from SOL compared to WG, but not to enhanced ROS production. Expression of the PTP regulatory protein cyclophilin‐D was similar in mitochondria from both muscles. However, expression of ANT, a protein reported to be involved in pore formation and which contributes to Ca2+‐sensitivity of the PTP, was significantly 2.3 fold higher in SOL compared to WG. The present study provides evidence that regulation of the PTP varies according to the phenotype of muscle fibers, which may impact on muscle cell death under conditions of stress.Funded by NSERC.