bluuebunny/crossref_metadata_2025 huggingface dataset:
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.
Deficiency of TRPV4 abolishes shear stress‐induced vasodilation in mice.
Authors: Köhler Ralf , Hartmannsgruber Veronika , Heyken Willm‐Thomas , Kacik Michael , Grgic Ivica , Harteneck Christian , Liedtke Wolfgang , Hoyer Joachim
Published: June 2021

In endothelial sensing of shear stress, the Ca2+‐permeable TRPV4 channel was proposed a candidate mechanosensor. Using TRPV4−/− mice, we investigated whether the absence of endothelial TRPV4 alters shear‐stress‐induced vasodilation. Loss of the TRPV4 protein and function was confirmed by IHC and electrophysiology in endothelium of TRPV4−/− mice. Agonist‐ and shear stress‐induced vasodilation was determined by pressure myography in carotid artery (CA) of TRPV4−/− and TRPV4+/+ mice (WT). In WT CAEC, TRPV4 was activated by the pharmacological TRPV4‐opener 4alphaPDD, arachidonic acid (AA), and by hypotonic cell swelling (HTS). In CAEC of TRPV4−/−, 4alphaPDD did not produce currents and currents in response to AA and HTS were greatly reduced. 4alphaPDD elicited a robust and strictly endothelium‐dependent vasodilation in WT mice, again strikingly absent in TRPV4−/− mice. Shear stress‐induced vasodilation was present in WT, but was eliminated in TRPV4−/− mice. Flow‐induced vasodilation was significantly reduced in TRPV4−/− vs. WT mice. In conclusion, loss of TRPV4 abolishes shear stress‐induced vasodilation. Thus, Ca2+‐influx through endothelial TRPV4 channels is a critical mechanism contributing to endothelial mechanotransduction.
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
Ca2+‐regulated exocytosis modulated by [Cl−]i in ACh‐stimulated antral mucous cells of guinea pig
Authors: Nakahari Takashi
Published: June 2021

The effects of [Cl−]i on ACh‐stimulated exocytosis were studied in guinea pig antral mucous cells. ACh activated Ca2+‐regulated exocytosis (initial phase followed by a sustained phase). Bumetanide or an NO3− solution enhanced it, in contract, NPPB decreased it and eliminated the enhancement. ATP depletion studies demonstrated that exposure of NO3− solution prior to ATP depletion induced an enhanced initial phase followed by a sustained phase, whereas exposure of NO3− solution after ATP depletion induced only a sustained phase. Measurements of [Cl−]i revealed that ACh decreases [Cl−]i. and that bumetanide and NO3− solution decreased [Cl−]i and enhanced the ACh‐evoked [Cl−]i decrease; in contrast, NPPB increased [Cl−]i and inhibited the [Cl−]i decrease induced by ACh, bumetanide or NO3− solution. [Ca2+]i measurements showed that a decrease in [Cl−]i enhanced the ACh‐stimulated [Ca2+]i increase. In conclusion, a decrease in [Cl−]i accelerates ATP‐dependent priming and [Ca2+]i increase, which enhance Ca2+‐regulated exocytosis in ACh‐stimulated antral mucous cells.
Upregulation of interferon‐gamma signaling in Hip1r‐deficient mouse stomach
Authors: Ren Jim H. , Keeley Theresa M , Samuelson Linda C.
Published: June 2021

Gastric inflammation is associated with epithelial cell changes that reflect preneoplastic events. We are using Huntingtin interacting protein 1 related (Hip1r)‐deficient mice to investigate the mechanism for gastric transformation. In the present study, we examined the hypothesis that interferon gamma (IFN‐γ) signaling is involved in the characteristic expansion of mucous neck cells and loss of chief cells observed in these mice. In wild type mice, IFN‐γ receptor 2 was localized to these two cell types by immunohistochemical staining. The Hip1r‐deficient stomach contained inflammatory infiltrates and exhibited increased IFN‐γ receptor 1 and 2 mRNA. Furthermore, targets of IFN‐γ signaling, including the chemokine Cxcl10 and the cell surface mucin Muc1, were up‐regulated in Hip1r‐deficient mice, suggesting increased IFN‐γ signaling. To test whether IFN‐γ is required for the epithelial cell changes, IFN‐γ and Hip1r double knockout mice (IHip DKO) were generated. IFN‐γ receptor mRNAs and the IFN‐γ signaling target Muc1 were normalized in IHip DKO mice, demonstrating that these changes in the Hip1r‐deficient mouse stomach were IFN‐γ dependent. These results suggest that the cytokine IFN‐γ targets the mucus neck/chief cell lineage in the stomach to stimulate cellular transformation, including increased Muc1 and Cxcl10 expression. Supported by NIH RO1 DK056882
Cell Signaling in Space and Time
Authors: Scott John D
Published: June 2021

Multiprotein signaling networks create focal points of enzyme activity that disseminate the intracellular action of many hormones and neurotransmitters. Accordingly, the spatio‐temporal activation of protein kinases and phosphatases is an important factor in controlling where and when phosphorylation events occur. Anchoring proteins provide a molecular framework that orients these enzymes towards selected substrates. A‐kinase anchoring proteins (AKAPs) are signal‐organizing molecules that compartmentalize the cAMP dependent protein kinase, phosphodiesterases and a variety of enzymes that are regulated by second messengers. Using a combination of live cell imaging, biochemical, genetic and electrophysiological techniques I will discuss two emerging principles in AKAP signaling: the combinatorial assembly of different enzymes on the same AKAP backbone; and the dynamic reorganization of AKAP complexes.
Sex differences in the renal handling of protein from high casein and high soy diets in mice
Authors: Rouch Al John , Hanner William , Johnson Derek
Published: June 2021

Soy protein diet appears to reduce the rate of chronic kidney disease (CKD) compared to milk protein diet. The purpose of this study was to compare high soy with high casein consumption on protein excretion in mice. The study contained two exact protocols – one with males and one with females. Each protocol began with 25‐day old mice in metabolic cages. One group consumed 40% casein and the other consumed an isocaloric 40% soy diet for 25 days (n=6 per group). Daily measurements included food intake and protein excretion. In both protocols, the casein group excreted more protein (mg/day) than the soy group as measured on the 25th day (males: 22.9±2.5 vs 12.7±.9, p<.009; females: 10.2±1.7 vs 5.4±8, p<.03). Daily urine protein determined via urinalysis strips supported these differences. Females excreted significantly less protein compared to males despite equal protein consumption. We conclude that high soy intake induces less proteinuria compared to high casein. Female mice excrete less protein compared to males either on high soy or high casein. These data support the benefits of soy protein in CKD patients and indicate sex differences in the renal handling of high protein diets.
Chronic cold stress exacerbates early diabetic kidney damage
Authors: Rogers Jennifer Leigh , Mitchell Adam R , Maric Christine , Myers Adam , Zukowska Zofia , Mulroney Susan E
Published: June 2021

Chronic stress is considered a co‐morbid factor in diabetic complications, but evidence is anecdotal, and without potential mechanisms. Since neuropeptide Y (NPY) is implicated in other stress‐induced pathophysiology, we hypothesize that stress will activate the NPY system and exacerbate early diabetic kidney disease. Diabetes (D) was induced with STZ in adult rats, and after 6 wks rats were placed in 4° water for 2h/day/14 days, then sacrificed. Chronic stress had no effect on plasma NPY levels in female rats. In contrast, stress increased platelet levels of NPY in both non‐diabetic (ND) (23.2±3.9 v 12.2±2.1 ng/ml in control, P<0.01) and D males (18.2±.9 ng/ml, P<0.05 vs cont). Of potential importance is the finding that ND females had 30% greater renal DPPIV/protease activity (which cleaves NPY to NPY 3–36) than ND male kidneys (P<0.05). This activity was decreased to male levels +/− STZ, suggesting that the diabetic female kidney may be exposed to excessive amount of NPY 1–36. Stress significantly increased the tubulointerstitial fibrotic index (in AU) in both sexes in ND rats [Male: 0.72±.06 v 0.54±.02 in cont, P<0.05); Female: 0.68±.04 vs 0.44±.07, P<0.05] and diabetic rats [Male: 1.61±.06 v 1.37±.03 non‐stress, P<0.05; Female: 1.51±.08 v 1.21±.06, P<0.05]. Thus, for the first time we show that chronic stress initiates renal fibrosis in both ND and D kidneys, and sex differences in the stress‐induced NPY system.
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.
Increased neuropeptides in spinal cord with performance of a highly repetitive, forceful task are attenuated with anti‐inflammatory and ergonomic interventions
Authors: Elliott Melanie B , Barr Ann E , Amin Mamta , Harris Michele Y , Bempong Frank , Barbe Mary F
Published: June 2021

In spite of the abundant epidemiological evidence linking exposure to repetitive and/or forceful motion with repetitive strain injuries, effective interventions are still lacking. This study examined the effectiveness of 2 interventions (ergonomic modification and ibuprofen) in reducing nociceptive neuropeptides and inflammation after a highly repetitive, forceful reaching/grasping (HRHF) task. Rats were randomly assigned to 4 groups (ergonomic intervention, medicated, HRHF, or controls; 5–8/group). Controls learned, but did not perform the regimen. HRHF rats reach/grasp every 15 s at 70% of max grip strength (2hr/d, 3d/wk, 6–12 wks). Ergonomic rats performed the HRHF task for 4 wks, then transfer to a low repetition‐negligible force (LRNF) task (reach/grasp every 30 s, 20% of max). Medicated rats receive oral ibuprofen after HRHF task wk 4 and continued the task. Motor function was monitored weekly. Immunochemistry was used to analyze serum cytokines, nerve macrophages, and spinal Substance P (SP) and Neurokinin‐1 (NK‐1). In serum, ibuprofen reduced IL‐1a, interferon‐gamma, MIP2 and MIP3a, while ergonomic intervention reduced IL‐1a and MIP2. Nerve macrophages and spinal SP and NK‐1 expression were reduced by both interventions. These data suggest that a lowered work demand is as effective as ibuprofen in reducing inflammation and neuropeptides induced by repetitive, forceful tasks. NIAMS‐AEBarr
Expressions of phosphorylated cAMP response element‐binding protein and neuronal nitric oxide synthase in the dorsal medulla of Zucker Diabetic Fatty rats
Authors: Ma Shengxing , Li Xiyan , Yi Gloria
Published: June 2021

The purpose of this study was to examine the expressions of phosphorylated cyclic‐AMP response element binding protein (pCREB) and neuronal NO synthase (nNOS) in the brainstem of Zucker Diabetic Fatty (ZDF) rats compared to age‐matched lean rats. Sections of rat medulla were examined by immunolabeling with a polyclonal antibody directed against nNOS and NADPH diaphorase histochemistry, a marker of nNOS activity. The brainstem nuclei were also visualized through double labeling the neurons by immunohistochemistry with anti‐pCREB antibody combined with NAPDH diaphorase reactivity. The number of nNOS immunostaining neurons and NAPDH diaphorase positive neurons were significantly decreased in the gracile nucleus and the medial nucleus tractus solitarius (mNTS), but not altered in other brainstem regions of ZDF rats compared to lean rats. pCREB positive cells containing NAPDH diaphorase reactivity were significantly decreased in the mNTS and gracile nucleus of ZDF rats compared to lean rats. The results show that pCREB neurons endowed with nNOS are decreased in the gracile nucleus and mNTS of ZDF rats. We conclude that reduced pCREB causes decreased nNOS‐NO level in the dorsal medulla, which modifies somatosensory functions in Type II diabetic rats. Supported by ADA 7–07–RA–100 and NIH RO1 AT 002478 to S. Ma.
Maternal Low Protein Diet and Fetal Glucocorticoid Exposure Program Adult Murine Cardiovascular and Endocrine Status
Authors: Roghair Robert D , Segar Jeffrey L , Volk Kenneth A , Aldape Gilbert , Scholz Thomas D , Lamb Fred S
Published: June 2021

Fetal programming of human hypertension (HTN) and the metabolic syndrome is associated with low birth weight. We have modeled this process in mice using iso‐caloric low protein (LP) or normal protein (NP) diets provided to dams from E0 to19. Dams on NP received either saline injections or the 11‐βHSD inhibitor carbenoxolone (CX, 12.5mg/g E10 to 19). Offspring were fed either NP or a high fat (HF) diet. Blood pressure (BP) was recorded by radiotelemetry at baseline, on a high salt diet (9% NaCl), or on high salt plus a nitric oxide synthase (NOS) inhibitor (L‐NAME). Dam caloric intake was not altered by LP or CX, but pup birth weight was reduced (LP 1.7 ± 0.1g, CX 1.9 ± 0.1g, NP 2.2 ± 0.2g). Adult female LP and CX offspring had decreased fat pad weight and blood pressure on either NP or high salt (p<0.05). The decrease in BP was blocked by L‐NAME. In contrast, male LP and CX mice had increased fat pad weight and HTN that was exacerbated by both salt and L‐NAME. HF diet increased blood pressure and fat pad weight in NP mice which became similar to LP and CX. Conclusions: LP or CX impair fetal growth and program HTN in male mice. The HTN is partially compensated by NOS activation and is associated with obesity. Female mice are protected from programmed HTN and display decreased adiposity and enhanced NOS activation.
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.
Cardiovascular and metabolic responses to chronic central MC3/4R antagonism in rats fed a high fat diet
Authors: Dubinion John H , da Silva Alexandre A , Dreher Adrian , Hall John E
Published: June 2021

Our previous results suggest obesity does not cause resistance to the chronic effects of MC3/4R activation in Sprague Dawley (SD) rats fed a high fat (HF) compared to rats fed a normal fat (NF) diet. The purpose of this study was to test the hypothesis that a HF diet alters the appetite and cardiovascular responses to chronic MC3/4R antagonism. SD were fed a HF diet (40% kcal from fat, n=3) or a NF diet (13% kcal from fat, n=8) starting at three weeks of age for at least 20 weeks. Arterial and venous catheters were implanted for continuous monitoring of arterial pressure and heart rate. An intracerebroventricular cannula was implanted for infusion of the MC3/4R antagonist, SHU 9119 (1 nmol/hr) to the lateral ventricle for 10 days. Body weight in the SD fed a HF diet was increased (502±7 vs 477±10 g), and this increase was associated with a significant rise in blood pressure (107.5±0.3 vs 102.1 ±0.4 mmHg). Chronic infusion of SHU increased caloric intake in HF rats almost 30% more than in rats fed a NF diet (209.8±8.6 vs 146.6±4.5 Kcal) Yet, blood pressure and heart rate responses to SHU were comparable in both groups (ΔMAP: −7.2±1.4 vs −4.5±1.3 mmHg and ΔHR: −46.6±1.4 vs −47.6±1.6 bpm) These data suggest that blockade of MC3/4R causes greater increases in caloric intake in rats fed a HF compared to NF diet and that a functional MC3/4R appears to be necessary for obesity to increase blood pressure (PO1 HL51971).
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.
Pharmacologic vasodilation increases muscle perfusion and muscle protein anabolism similarly in elderly and young
Authors: Casperson Shanon , Dillon Edgar Lichar , Angel James A , Sanford Arthur P , Ahmad Masood , Paddon‐Jones Douglas , Volpi Elena , Sheffield‐Moore Melinda
Published: June 2021

We examined muscle protein metabolism in healthy young and elderly during pharmacologic vasodilation of the lower limb vasculature in combination with increased amino acids delivery. We obtained femoral arteriovenous blood samples and v. lateralis muscle biopsies during a primed continuous infusion of L‐[ring‐13C6] Phenylalanine. Muscle protein kinetics were measured in the basal state (BAS) and during a 180 min infusion of sodium nitroprusside (SNP) and amino acids (Premsol 10%) (SNP+AA). Skeletal muscle microvascular recruitment and flow were measured during BAS and SNP+AA using contrast‐enhanced ultrasound (CEU). SNP increased capillary recruitment and microvascular blood flow to a similar extent in both age groups (P ≤ 0.05). Phenylalanine net balance and model derived values for muscle protein synthesis increased significantly during SNP+AA administration, with no differences between age‐groups (P ≤ 0.05). These data demonstrate that an increase in nutritive blood flow in both younger and older individuals facilitates a similar increase in the utilization of amino acids for muscle protein anabolism.
Fructose metabolism to two carbon acids in HepG2 cells
Authors: Knight John , Holmes Ross Philip
Published: June 2021

Fructose intake is a risk factor for calcium oxalate stone disease. It has been hypothesized that fructose metabolism promotes oxalate synthesis leading to the increased stone risk. To assess this relationship we have compared glucose and fructose metabolism in cultured HepG2 cells. Oxalate synthesis was measured by ion chromatography (IC), glyoxylate by HPLC, and other anions including sugar phosphates by IC/MS. The rate of oxalate synthesis with 5mM fructose as the main carbohydrate source, 2.08 ± 0.48 nmoles/mg protein/day was 2.9 times higher than that observed with 5 mM glucose, 0.73 ± 0.14 nmoles/mg protein/day. Dose and time dependent effects were observed. Increased syntheses of glycolate and glyoxylate were not detected, suggesting that these organic acids are not involved in this metabolism. Glyceraldehyde is produced from fructose metabolism but not glucose metabolism. It is possible that the metabolism of this aldehyde results in oxalate synthesis, increasing urinary oxalate excretion and the risk of stone formation.
Maternal Diabetes Increases a Large Conductance Ca2+‐activated K+ (BK) Current of Cardiac Motoneurons in the Nucleus Ambiguus (NA) of Neonatal Mice
Authors: Lin Min , Li Lihua , Epstein Paul N. , Wurster Robert D. , Liu Yeqi , Cheng Zixi (Jack)
Published: June 2021

Maternal diabetes induces baroreflex dysfunction in the offspring. In a companion work (Lin et al. 2008, EB Abstract), we demonstrated that maternal diabetes altered the activity of Ca2+ and Ca2+—activated K+ channels of NA cardiac motoneurons in the neonatal mice of diabetic mothers. In the present study, neonatal mice from FVB control and OVE26 diabetic mothers (mated with FVB fathers) received tracer XRITC injections into the pericardial sac on postnatal days 7–9. Two days later, membrane currents of XRITC‐labeled NA cardiac motoneurons in brainstem slices were recorded using a whole‐cell patch technique. Compared with control, our data indicated that maternal diabetes significantly increased: 1) K+ channel activity of transient outward currents but not persistent currents evoked by voltage steps, 2) Cd2+ (calcium channel blocker cadmium)‐sensitive K+ channel conductance for transient outward currents but not persistent currents (n=8; p<0.05), 3) ChTX (BK channel blocker charybdotoxin)‐sensitive K+ channel conductance for both the transient and persistent outward currents, and the maximum values of ChTX‐sensitive transient and persistent currents at +40 mV (n=13; p<0.05). Thus, we conclude that the increased BK channel activity contributes to the decreased excitability of NA cardiac motoneurons in the neonatal mice of diabetic mothers. NIHRO1 HL‐79636.
L‐arginine Supplementation Reduces Local Cardiac Noradrenergic Neurotransmission in Spontaneously Hypertensive Rats
Authors: Lee Chee Wan , Channon Keith M , Paterson David J
Published: June 2021

Hypertension is associated with noradrenergic hyperactivity. We hypothesised that L‐arginine (L‐arg) supplementation may reduce cardiac noradrenergic hyperactivity in spontaneously hypertensive rats (SHR) via upregulation of nitric oxide synthase (NOS) – cyclic guanosine monophosphate (cGMP) pathway. SHR and Wistar Kyoto rats (WKY) aged 16 weeks were fed with L‐arg (10g/L in drinking water). A control group received no supplementation. A week later, the isolated right atria were used for measurement of electrically evoked [3H]norepinephrine (NE) release. The SHR control group had a significantly lower plasma L‐arg. NE release from the SHR control was higher than the WKY control (340±27% vs 273±25%, p<0.05), but was reduced to 259±13% with L‐arg (p<0.01). Addition of soluble guanylate cyclase (sGC) inhibitor, 1H‐(1,2,4)oxadiazolo(4,3‐a)quinoxaline‐1‐one (10 μM), to organ bath caused a proportionally higher increase of NE release in the L‐arg fed SHR (to 386%) compared with the non‐fed SHR (to 431%) (p<0.01 for paired analysis; p=NS between groups). L‐arg feeding did not reduce NE release in the WKY. Atrial cGMP was lower in the SHR compared with the WKY (2.4±0.4 pmol/mg vs 4.4±0.5 pmol/mg, p<0.05), but increased to 4.1±0.6 pmol/mg protein (p<0.05) with L‐arg. In conclusion, we have shown that L‐arg supplementation corrects the cardiac noradrenergic hyperactivity in the SHR via the NOS‐sGC‐cGMP pathway.
Single cell RT‐PCR indicates lower ASIC2a mRNA expression in aortic baroreceptor neurons of adult SHR vs WKY rats
Authors: Lu Yongjun , Snitsarev Vladislav , Whiteis Carol , Chapleau Mark W , Abboud Francois M
Published: June 2021

We demonstrated that Acid‐Sensing Ion Channel 2 (ASIC2) contributes to mechanoelectrical transduction in baroreceptor neurons. Its expression in nodose sensory ganglia (NG) is decreased in association with development of hypertension in spontaneously hypertensive rats (SHR) (FASEB J, 21:A1407, 2007). Since NG neurons relay sensory signals from various visceral organs we sought to define more specifically the expression of ASIC2a in aortic baroreceptor nodose neurons (ABRNs) of SHR. We hypothesized that ASIC2a expression is reduced in ABRNs of SHR to account for the suppressed depolarizing potential responses to mechanical stimulation we observed earlier in SHR (FASEB J, 19:A607, 2005). ABRNs in NG were labeled with DiI injected into the aortic arch adventitia of SHR and WKY rats at 10 weeks of age. Two weeks later, both NG were removed and neurons were dissociated and cultured. Individual DiI labeled ABRNs and non‐labeled neurons were selected by fluorescence microscopy for quantitative single cell RT‐PCR. The results indicate first that DiI labeled ABRNs (n = 24) had a significant higher level of ASIC2 mRNA (2.99 ± 0.93) than non‐DiI neurons (1.00 ± 0.97) (p < 0.05). Second, the ASIC2a mRNA level in DiI neurons relative to non‐DiI labeled neurons from the same ganglia was significantly lower in SHR (1.97 ± 0.59, n = 12) than in WKY (5.91 ± 0.61, n = 12) (p < 0.05). The single cell RT‐PCR results were confirmed by RT‐PCR of pooled neurons (n = 10/grougp). We propose that the development of hypertension and impaired baroreceptor sensitivity in SHR are associated with significant reduction of ASIC2 expression (HL14388).
A role for spinal vasopressin (V1a) receptors in regulation of arterial pressure (AP) in conscious rats
Authors: Veitenheimer Britta , Osborn John
Published: June 2021

Studies suggest that vasopressinergic PVN neurons increase sympathetic nerve activity (SNA), and therefore AP, via direct projections to the spinal cord and that this pathway is activated during acute osmotic stress. Our laboratory is interested in the role of this pathway in models of chronic osmotic stress such as the DOCA‐salt rat. In this study, we hypothesized that vasopressin can act on spinal V1a receptors to increase AP in conscious rats. Rats were chronically instrumented with telemetry transmitters for measurement of AP and intrathecal catheters for injection of a V1a specific agonist without (N=7) or with (N=6) a V1a specific antagonist pretreatment. Drugs were administered in 10 microliters of saline followed by a 23 microliter saline flush. Injection of the V1a agonist increased AP (33 ± 2 mmHg) and this response was attenuated by pretreatment with the V1a antagonist (22±4). These results are consistent with the hypothesis that spinal V1a receptors may play a role in regulating AP in conscious rats. Initial studies of intrathecal infusion of the V1a antagonist in DOCA‐salt rats suggest these receptors may be active in this chronic model of hypertension. Supported by NIH Grant HL064178.
Doublet and triplet gasps in urethane‐anesthetized neonatal rat
Authors: Poole Oneica Neicola , Yu Hui Jing , Warren Kelly , Solomon Irene C.
Published: June 2021

Previous studies have indicated that unusual gasp patterns (i.e., two or three closely spaced successive gasps termed ‘doublet’ and ‘triplet’ gasps) can be observed in infants with SIDS and non‐SIDS conditions during agonal respiration. Here, we characterize the presence of doublet and triplet gasps recorded in spontaneously breathing urethane‐anesthetized neonatal rats in response to anoxia. Since a marked difference in the number of doublet gasps was recorded in P0‐P6 versus P7‐P12 neonatal rats (P=0.004), the data were divided into two groups. For each group, the total number of gasps was similar (P0–P6, n=133±14 gasps; P7–P12, n=118±16 gasps; P=0.5); however, the incidence of doublet gasps far exceeded that of triplet gasps, with 24±4 vs 2±1 (P<0.001) in P0–P6 rats and 5±1 vs 1±0.4 (P=0.048) in P7–P12 rats. In addition, the latency to the appearance of doublet gasps was longer in P0–P6 vs P7 to P12 rats (431±61 s vs. 161±26 s from the onset of the anoxic gas; P=0.005); the latency to anoxia‐induced gasping was similarly different between the two groups. Our data demonstrate that neonatal rats are capable of producing both doublet and triplet gasps in response to anoxic conditions and that there is a greater likelihood of eliciting doublet rather than triplet gasps. The difference in the number of these unusual gasps between groups suggests that younger neonatal rats are more susceptible to alternate gasping patterns.
Prenatal nicotine and respiratory long term facilitation in neonatal rats
Authors: Fuller David D , Doperalski Nicholas J , Dougherty Brendan J , Sandhu Milap , Reynolds Carie , Hayward Linda F
Published: June 2021

Prenatal nicotine (PN) exposure alters the control of breathing in neonatal rats and the development of serotonergic neurons. Long term facilitation (LTF) is a long‐lasting, serotonin dependent increase in respiratory motor output that occurs in neonates and adults following intermittent hypoxia (IH). In ongoing studies, we are examining the impact of PN on ventilatory LTF in neonatal rats. Dams were implanted with an alzet miniosmotic pump to deliver nicotine (6 mg/kg per day) or saline during pregnancy. Barometric plethysmography was used to record ventilation in unanesthetized rat pups. Rats were exposed to a baseline period (21% O2) and then ten 5‐min bouts of hypoxia (5% O2) followed by a return to baseline conditions. Data recorded following IH were normalized to pre‐hypoxia baseline values. At post‐natal days 15–17, PN rats had a reduced inspiratory tidal volume (p=0.036 vs. saline, 2‐way RM ANOVA) and tended to have increased breathing frequency (p=0.215 vs. saline) over the 30 min period following IH. At 30 min post‐IH, tidal volume was larger than pre‐IH baseline values in saline (p=0.043) but not PN rats (p=0.767). Body weight was not different between PN (44±3 g) and saline rats (42±2 g). These preliminary results (n=4 per group) indicate that expression of LTF in neonatal rats is modulated by exposure to PN. Supported by the Florida Dept. of Health.
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.
Characterizing the breath‐to‐breath hypercapnic ventilatory response in neonatal rats
Authors: Cummings Kevin James , Frappell Peter B.
Published: June 2021

The delay in the appearance of the central component of the ventilatory (VE) response to CO2 depends on the model. A 2–3 s delay has been demonstrated in anesthetised and decerebrate adult cats, versus a 10–20 s delay in awake lambs and dogs. Our aim was to characterise the peripheral and central components of the VE response to CO2 in neonatal rats. A near‐square wave challenge of 8% CO2 in both air and 90% O2 was administered to P4 rats (n=10) for 1 min, with 2 min washout between randomized tests. The air‐CO2 and hyperoxia‐CO2 VE responses were measured with a pneumotach and the slope determined over each breath of the rising phase. Compared to CO2‐hyperoxia, the VE response in CO2‐air occurred about 4 breaths sooner, and the slope of the immediate VE on‐response to CO2 in air was significantly greater than in hyperoxia (P=0.04). Steady‐state responses, determined by comparing the average responses in the last 60 breaths, were not different. These data suggest that for neonatal rats, the carotid bodies contribute significantly to the immediate VE response to CO2, but not to the steady‐state response, and that the central component is delayed only a few breaths relative to the peripheral component. For comparisons between models having different mass‐specific rates of metabolic CO2 production, it may be more appropriate to express the VE delays in terms of breaths rather than absolute time.
Fluid tolerance while running: effect of repeated trials
Authors: Lambert G. Patrick , Lang James , Bull Anthony , Eckerson Joan , Lanspa Stephen , O’Brien John
Published: June 2021

This study assessed tolerance to fluid ingestion with repeated sessions of drinking while running. Seven trained distance runners (age 22 ± 2 yr; VO2max = 54.4 ± 7.1 ml/kg/min) performed six 90 min treadmill runs (~1/week) at 65% VO2max while ingesting a glucose‐electrolyte solution. During run 1, subjects drank ad libitum every 10 min and sweat rate was determined. On runs 2‐6, subjects drank every 10 min at a rate to match the sweat rate determined in run 1. Stomach comfort (1–4 scale) and other gastrointestinal symptoms were also assessed every 10 min. Gastric emptying rate was determined in runs 2 and 6. Subjects consumed significantly (P < 0.05) more fluid during runs 2–6 (mean ± SD; 1247 ± 162 ml), than during run 1 (508 ± 476 ml). Stomach comfort improved (P < 0.05) on runs 5 and 6 (1.7 ± 0.5 units) compared to run 2 (2.3 ± 0.5 units). Gastric emptying rate was not different between runs 2 and 6 (12.0 ± 1.9 ml/min vs. 12.3 ± 2.3 ml/min, respectively). These results indicate repeated sessions of drinking at a rate that matches sweat rate improves stomach comfort, however, gastric emptying rate does not change under such conditions. Supported by the Gatorade Sports Science Institute
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