bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential development of tolerance to μ‐opioid receptor agonists in the mouse colon
Authors: Maguma Hercules Tarambirwa , Dewey William L , Akbarali Hamid I
Published: June 2021

Drawbacks to opioid use include development of analgesic tolerance and persistent constipation. We previously reported that tolerance to morphine develops upon repeated exposure in the ileum but not the colon. The cellular mechanisms of antinociceptive tolerance vary among μ‐opioid receptor agonists. In this study, we assess β‐arr2 deletion on the development of tolerance to different opioids in ileum and colon circular muscle preparations. Each opioid induced contractions in the isolated muscles from C57BL/6 wildtype (WT) and β‐arr2 knockout (KO) mice. Repeated exposure every 30 min with in‐between washes resulted in tolerance to all agonists in ileum of both WT and KO. However, in the colon of WT, tolerance did not develop to either morphine (162 ± 48%; n = 4) or DAMGO (100 ± 10%; n=5) but occurred to fentanyl (62 ± 13%; n=8) and etorphine (38 ± 4%; n=7) when contractions at the 4th exposure were compared to the 1st response. In contrast, all agonists produced tolerance in colon of KO: morphine response at the 4th exposure decreased to 47 ± 4% (n = 5), DAMGO to 52 ± 10% (n=5), fentanyl to 20 ± 5% (n=6) and etorphine 33 ± 7% (n=6). Differences in tolerance among the agonists in colon suggest ligand bias. The deletion of β‐arr2 in colon appears to be necessary for tolerance to morphine and DAMGO but not to fentanyl or etorphine. Supported by NIH DA024009
Some histories of response‐contingent histamine attenuate the punishing effect of histamine
Authors: Truong Yen Nhu Thi , Woods James H
Published: June 2021

Rationale

IV histamine can function as a punisher, but seems less able to serve this function in some choice situations that include lever reversals.

Objective

This study examined whether different histories of response‐contingent IV histamine injection (inj) attenuated the ability of IV histamine to punish sucrose‐maintained responding.

Method

In a two‐lever operant chamber, rats with IV catheters were given a choice between response‐contingent sucrose pellets or response‐contingent IV histamine inj. The lever contingencies were then reversed; the lever that previously delivered only sucrose now delivered sucrose + histamine, and the lever that previously delivered only histamine now delivered only sucrose.

Result

Rats always selected the sucrose‐ alone lever over the histamine‐alone lever in the initial sessions. When the lever that had previously delivered only histamine now delivered only sucrose, and the lever that previously resulted in only sucrose now presented sucrose + histamine, rats very slowly learned to move to the new sucrose‐alone lever. The larger the dose of histamine on the now sucrose + histamine lever, the more slowly the rats learned to avoid this lever.

Conclusion

Drug histories should be considered when evaluating the ability of a drug to punish steady‐state operant choice behavior. NIH 5 T32 GM 7767‐33, NIDA RO1 D0247897.
Characterization of imidazoline I2 receptor agonists‐induced hypothermia in rats
Authors: Thorn David A. , An Xiao-Fei , Zhang Yanan , Pigini Maria , Li Jun-Xu
Published: June 2021

Imidazoline I2 receptors have been implicated in several central nervous system disorders. Although several I2 receptor agonists have been described, no simple and sensitive in vivo bioassay is available for studying I2 receptor ligands. This study examined I2 receptor agonist‐induced hypothermia as a functional in vivo assay of I2 receptor agonism in rats. All the selective I2 receptor agonists examined (2‐BFI, diphenyzoline, phenyzoline, CR4056, tracizoline, BU224 and S22687, 3.2–56 mg/kg, i.p) dose‐dependently produced hypothermia in rats, with varied duration of action. Pharmacological mechanism of the observed hypothermia was studied by combining the I2 receptor agonists (2‐BFI, BU224, tracizoline and diphenyzoline) with imidazoline I2 receptor/α2 adrenoceptor antagonist idazoxan or α2 adrenoceptor antagonist/serotonergic 5‐HT1A receptor agonist yohimbine. Idazoxan but not yohimbine attenuated the hypothermic effects of 2‐BFI, BU224, tracizoline and diphenyzoline, supporting the I2 receptor mechanism. In contrast, both idazoxan and yohimbine attenuated the α2 adrenoceptor agonist clonidine induced hypothermia. Together, these data suggest that imidazoline I2 receptor agonists can produce hypothermic effects, which may be useful as a simple and sensitive in vivo assay for studying I2 receptor ligands.
Immortalized hippocampal cell line H19‐7 express endothelin system genes
Authors: Kanyicska Bela , Liu Huiling
Published: June 2021

Previous studies indicate that endothelins (ETs) may play a neuromodulatory role in the central nervous system. However, there is a paucity of data regarding ETs’ action on specific subsets of neurons. We evaluated the hippocampal cell line H19‐7 (conditionally immortalized by a temperature‐sensitive SV40 large T antigen) as an in vitro model to investigate the role of ETs in neural activity. The relative abundance of endothelin system genes mRNA was assessed by real‐time PCR method using specific primers for endothelins (ET‐1 and ET‐3), endothelin receptors (ETA and ETB), and endothelin converting enzyme‐1 (ECE‐1). Under non‐differentiating conditions (34°C), we found a robust expression of ECE1, ETA and ET‐1, while ETB and ET‐3 mRNA levels were undetectable. The expression patterns of ET system genes were essentially similar when the cells were placed under differentiating condition (39°C) for 24–36 hours. When changes in cytosolic free Ca2+ levels were assessed by the Fura‐2 method, undifferentiated cells were unresponsive to ET1. However, 20 nM ET‐1 elicited a robust bi‐phasic increase of cytosolic free Ca2+ (a hallmark effect of ETA receptor activation) on differentiated cells, and markedly enhanced capacitative Ca2+ entry as well. Thus, differentiated H19‐7 cells seem suitable for mechanistic studies of ETs’ action on neuronal targets.
Morphological changes in raphe nuclei serotonergic fibers of the MPTP treated mouse model of Parkinson's disease
Authors: Nayyar Tultul , Ferguson Marcus C , Ansah Twum A
Published: June 2021

OBJECTIVE

To investigate whether 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) treatment alters the morphology of serotonin (5‐HT) fibers in the in the dorsal (DRN) and median raphe nuclei (MRN) where 5‐HT cell bodies are located.

DESIGN AND METHODS

Male C57BL/6J mice were treated with 4 doses of MPTP 20 mg/kg (i.p.) at every 2 hours and sacrificed after 3 and 16 weeks. Tryptophan hydroxylase (TPH) antibody was used for immunohistochemistry to study the neuronal number and fiber lengths and tail suspension test (TST) to assess depression‐like behavior of the mice.

RESULTS

Increased expression of TPH in the cell bodies and fibers of MPTP treated mice, were noticed without any significant differences in the neuronal number in the DRN and MRN at both 3 and 16 weeks survival times, as compared to control group. However, a significant decrease in fiber lengths in DRN and MRN at 3weeks survival period and in MRN at 16 weeks survival period after MPTP treatment, were observed. The consistent reduction in fiber lengths in the MRN correlated well with our earlier report in MPTP treated mice with preferential decrease in the density of thick beaded 5‐HT axons in the PFC which originate from MRN. Increased immobility as measured by the TST was observed in the MPTP treated mice.

CONCLUSION

MPTP treated mouse may be a useful resource to model depression associated with PD. Supported by NIH SNRP/NINDS grant NS041071.
P450 3A5 is primarily responsible for the formation of the most abundant oxidative metabolite of maraviroc
Authors: Lu Yanhui , Hendrix Craig W. , Bumpus Namandjé N.
Published: June 2021

Maraviroc is an anti‐HIV drug that acts by blocking viral entry into target cells. Although maraviroc is known to be metabolized by the cytochromes P450 (CYP) to several products, a comprehensive analysis of the enzymes involved has yet to be reported; therefore, this work sought to identify the CYPs involved in the oxidative metabolism of maraviroc. Using a panel of cDNA‐expressed CYPs it was determined that the major oxidative metabolite of maraviroc (detected using liquid chromatography‐mass spectrometry) was primarily formed by CYP3A5. Tandem mass spectrometry analysis indicated that the CYP3A5‐mediated oxygen insertion occurred on the difluorocyclohexane ring of maraviroc. For this reaction, the Vmax for CYP3A4 and CYP3A5 was 0.04 and 0.93 pmole/min/pmole P450, respectively. In addition, the Km values were 11.1 μM and 48.9 μM, respectively. Human liver microsomes isolated from donors homozygous for the CYP3A5*3 allele, which results in low expression of CYP3A5, exhibited a 79% decrease in formation of this metabolite as compared to production by human liver microsomes isolated from donors homozygous for the CYP3A5*1 allele. In conclusion, the major oxidative metabolite of maraviroc was found to be formed primarily by CYP3A5. These results indicate that maraviroc may be used as a tool to distinguish the activity of CYP3A5 from that of CYP3A4. Support: PhRMA Foundation Research Starter Grant to N.B.
(−)‐Epicatechin induces calcium and translocation independent eNOS activation in endothelial cells via AKT/HSP90
Authors: Ramirez-Sanchez Israel , Ceballos Guillermo , Villarreal Francisco
Published: June 2021

The consumption of cacao‐derived products provides beneficial cardiovascular effects. Vascular actions of cocoa are related to enhanced nitric oxide (NO) production. These actions can be reproduced by the administration of the flavanol (−)‐epicatechin (EPI). EPI is capable of inducing the synthesis of NO via endothelial NO synthase (eNOS) activation in human coronary artery endothelial cells (HCAEC) through PI3K/AKT/PKA and Ca2+‐CaM/CaMKII pathways. To further understand the mechanisms behind the vascular action of EPI, we investigated the effects of Ca2+ depletion on eNOS translocation, as well as on its activation/phosphorylation. Results demonstrate that under Ca2+‐free conditions, EPI (but not BK) is capable of inducing NO production through eNOS phosphorylation. EPI‐induced translocation of eNOS from the plasmalemma was abolished upon Ca2+ depletion. Thus, under these conditions, EPI can stimulate NO synthesis independent of calmodulin binding to eNOS and of its translocation into the cytoplasm. EPI is able to induce AKT activation as well as the physical association of HSP90‐eNOS. We also examined the effects of EPI on the NO/cGMP/vasodilator‐stimulated phosphoprotein (VASP) pathway in isolated Ca2+‐deprived canine mesenteric arteries. EPI induces the activation of the vasorelaxation‐related pathway and the effects were blocked by nitro‐L‐arginine methyl ester (L‐NAME). Results indicate that EPI can uniquely modulate endothelial responses independently of calcium.
Differential expression of mitoKATP subunits in cardiac mitochondrial subpopulations and the influence of Type I diabetes
Authors: Fancher Ibra Seaphus , Baseler Walter A , Croston Tara L , Thapa Dharendra , Shepherd Danielle , Nichols Cody , Lewis Sara , Jagannathan Rajaganapathi , Asano Shinichi , Dick Gregory M , Hollander John M
Published: June 2021

Mitochondrial dysfunction is an underlying risk for heart failure in Type I diabetes mellitus. MitoKATP channels are likely composed of an inwardly rectifying K+ channel subunit (Kir6.1) and a sulphonylurea receptor (SUR1). MitoKATP regulate mitochondrial volume and membrane potential: two functions disturbed by diabetes mellitus. In the heart, two mitochondrial populations exist: subsarcolemmal (SSM) and interfibrillar (IFM) which differ in their subcellular locations. It is not known whether: a) Kir6.1 or SUR1 expression is similar in IFM and SSM or whether b) diabetes mellitus alters the expression of Kir6.1 or SUR1 subunits in either SSM or IFM. Male FVB mice were made diabetic via multiple low‐dose injections of streptozoticin and examined after 5 weeks. Western blot analyses on cardiac mitochondrial subpopulations revealed Kir6.1 expression in non‐diabetic IFM was greater relative to SSM (p < 0.05), but SUR1 expression was equivalent. Kir6.1 protein was reduced (p < 0.05) in the IFM of diabetic mice. In contrast, diabetes mellitus had no effect on Kir6.1 protein expression in SSM or SUR1 expression in either IFM or SSM. These data show that expression Kir6.1 may be higher in IFM than SSM and that diabetes mellitus reduces Kir6.1 expression in IFM. This suggests that mitoKATP components may be differentially impacted by diabetes mellitus and that these effects may be specific to a subset of mitochondria.
Aging exacerbates microvascular endothelial damage induced by inflammatory factors present in the circulation during sepsis
Authors: Tucsek Zsuzsanna , Gautam Tripti , Sonntag William , Toth Peter , Csiszar Anna , Saito Hiroshi , Szabo Csaba , Ungvari Zoltan
Published: June 2021

The elderly show a significantly elevated mortality rate during sepsis than younger patients, due to their higher propensity to microvascular dysfunction and consequential multi‐organ failure. We tested whether aging renders vascular endothelial cells more susceptible to damage induced by inflammatory factors present in the circulation during sepsis. Primary microvascular endothelial cells derived from young (3 m.o.) and aged (24 m.o.) F344xBN rats were treated with sera obtained from sepsis patients and healthy controls. Apoptotic cell death (caspase 3 and caspase 8 activities) induced by treatment with septic sera was exacerbated in aged endothelial cells as compared to responses obtained in young cells. In aged endothelial cells, compared to young cells, DAF fluorescence, indicating NO production, was decreased both under basal conditions and after treatment with septic sera. Treatment with septic sera elicited greater increases in mitochondrial production of reactive oxygen species (MitoSox fluorescence) and NF‐kappaB activation (reporter gene assay) in aged endothelial cells, as compared to young cells. Collectively, aging increases sensitivity of microvascular endothelial cells to oxidative stress and cellular damage induced by inflammatory factors present in the circulation, which likely contributes to the increased vulnerability of elderly patients to sepsis.
Hsp90 inhibition suppresses LPS‐mediated NFkB target gene expression without affecting NFkB nuclear translocation in human lung microvascular endothelial cells
Authors: Thangjam Gagan S , Joshi Atul D , Snead Connie , Wallace Chris L , Catravas John D
Published: June 2021

Heat shock protein 90 (hsp90) regulates the stability and function of several kinases, including IKB kinases (IKKs). We investigated the mechanisms by which hsp90 inhibitors prevent induction of NFkB‐dependent genes. The hsp90 inhibitor, 17‐AAG, significantly suppressed lipopolysaccharide (LPS)‐mediated IKK‐beta activation and phosphorylation of IKB‐alpha. However, 17‐AAG did not restore IKB‐alpha protein level and was unable to block p65 nuclear translocation and total DNA binding, as reflected by ELISA, in LPS‐stimulated human lung microvascular endothelial cells (HLMVEC). Conversely, prolonged 17‐AAG treatment (>16h) blocked LPS‐mediated induction of NFkB regulated genes, IKB‐alpha, IL‐8 and MMP‐9. Interestingly, binding of p65 to the IKB‐alpha promoter, as reflected by ChIP assay, was reduced by 17‐AAG. Selective pharmacologic inhibition of several key steps in the toll‐like receptor 4 (TLR‐4) signaling pathway revealed that the regulation of NFkB target genes by LPS is dependent on the cAMP‐dependent kinase (PKA) and MAPK pathways. Thus, LPS stimulated p65 (Ser276) phosphorylation in a PKA‐dependent manner; this was significantly inhibited by 17‐AAG and by IKK inhibitors (IKK16 and BAY 11‐7082). We conclude that hsp90 inhibition suppresses NFkB signaling and target gene expression by affecting p65 post‐translational modifications. (Supported by NIH grants: HL093460 and HL101902)
Interference of peroxisome proliferator‐activated receptor‐gamma (PPAG) in vascular muscle enhances myogenic tone in small resistance arteries via protein kinase C (PKC)‐induced inhibition of large conductance Ca2+‐activated K+ channel (BKCa) activity
Authors: Ketsawatsomkron Pimonrat , Lorca Ramon A , England Sarah K , Faraci Frank M , Sigmund Curt D
Published: June 2021

PPARG is a ligand activated transcription factor expressed in vasculature. Targeted expression of dominant negative PPARG to vascular muscle of transgenic mice (S‐P467L) caused hypertension and aortic dysfunction. We hypothesized that smooth muscle PPARG regulates resistance vessel function. We examined myogenic tone and vascular reactivity in small mesenteric arteries (MA) from S‐P467L and non‐transgenic controls (NT) using a pressurized myograph. MA from S‐P467L exhibited significantly increased myogenic tone (at 100 mmHg: S‐P467L 34.5±1.7%, NT 21.0±2.1%, P<0.05). Iberiotoxin (IbTX), an inhibitor of BKCa, induced constriction in NT MA (at 75 mmHg, 10.1±0.6%) but this effect was markedly blunted in S‐P467L (S‐P467L 5.5±0.9%, P<0.05). Whole‐cell K+ current was measured using patch clamp in freshly dissociated smooth muscle cells (SMC) isolated from MA. Total K+current was similar between two groups. However, IbTX only inhibited K+ current in NT (from 40.4±5.6 to 25.4±4.1 pA/pF at 100mV, P<0.01) while having no effect in S‐P467L (from 48.8±7.9 to 50.9±8.5 pA/pF at 100 mV). A PKC inhibitor, chelerythrine chloride restored the sensitivity of S‐P467L SMC to IbTX‐induced blockade of K+ current. Lastly, inhibition of PKC markedly decreased myogenic tone in S‐P467L. We conclude that interference of PPARG function causes increased vascular tone through a reduced BKCa activity by a PKC dependent mechanism.
Role Of MMP9 In Cardiac Stem Cell Differentiation And Autophagy
Authors: Mishra Paras Kumar , Metreveli Naira , Chavali Vishalakshi , Tyagi Neetu , Qipshidze Natia , Sen Utpal , Joshua Irving G. , Tyagi Suresh C.
Published: June 2021

To test the hypothesis that ablation of MMP9 induces cardiac stem cell (CSC) differentiation into cardiomyocytes and inhibits autophagy, we created double knock out (DKO: Ins2+/−/MMP9−/−) mice. The levels of CSC, cardiomyomyocytes and autophagy were determined in the heart of C57BL/6J (WT), Ins2+/− (diabetic) and DKO mice by RT‐PCR, immunocytochemistry, confocal microscopy and Western blotting using c‐kit, troponin and LC3 markers, respectively. The ECM turn over (fibrosis) was measured by Masson Trichrome staining. The cross talk between c‐kit and MMP9 was determined by co‐immunoprecipitation (co‐IP). Interestingly, the degree of fibrosis was correlated with the levels of LC3 and troponin in the WT, Ins2+/− and DKO (mean ± SE: fibrosis −14± 2.1, 34±2.5, 6±1.9; LC3−36±1.5; 39± 0.6, 38±1.0; and troponin−11.2± 0.4, 8.8±0.3, 12.3± 0.28, respectively). Western blot and co‐IP results revealed that induction of MMP9 attenuates c‐kit in the diabetic heart. Based on these findings, it is suggested that robust expression of MMP9 exacerbates autophagy and alleviates cardiomyocytes in diabetes. Our results elicit a novel mechanism, where abrogation of MMP9 promotes differentiation of CSC into cardiomyocytes and improves their survival by inhibiting autophagy.
Structural Basis of Zn2+ Activation of the Epithelial Na+ Channel
Authors: Chen Jingxin , Winarski Katie L , Myerburg Mike M , Pitt Bruce R , Sheng Shaohu
Published: June 2021

Both extracellular and intracellular ions regulate the activity of the epithelial Na+ channel (ENaC). External Zn2+ activates ENaC by reliving Na+ self‐inhibition. We investigated the structural basis for the Zn2+ activation of ENaC by site‐directed and structure‐assisted mutagenesis. The effects of Zn2+ on the mutant channels were examined in Xenopus oocytes using two‐electrode voltage clamp. A scanning of the extracellular domain His residues identified several residues (αH381, γH200, γH202, γH239) where mutations significantly reduced the maximal activation of ENaC by Zn2+. Substitutions at αH445 and βH386 that are homologous to the putative Cl− binding sites of human ENaC prevented Zn2+ from fully activating ENaC. Interestingly, γH88 mutations converted the bell‐shaped Zn2+ dose‐response to a sigmoid one by eliminating the declining phase without affecting the rising phase. Using structural information from the ENaC‐homologous chicken acid sensing ion channel 1, we identified another γENaC residue where mutations resulted in a similar phenotype to that of γH88 mutants. These two residues within the palm subdomain are likely located at a subunit interface. Our results suggest that extracellular Zn2+ bind at least at two distinct sites causing the biphasic dose‐response and one site is located at a subunit interface in the extracellular domain of ENaC. (Supported by NIH ES014701 and DK079307)
Crosstalk between insulin and IGF‐1 receptors in principal cells: implication for ENaC‐mediated sodium reabsorption
Authors: Ilatovskaya Daria V. , Levchenko Vladislav , Pavlov Tengis S. , Staruschenko Alexander
Published: June 2021

Insulin and insulin‐like growth factor 1 (IGF‐1) are recognized as powerful regulators of ENaC in the aldosterone‐sensitive distal nephron (ASDN). Both insulin and IGF‐1 are capable of binding to each other's receptors. Using mice with collecting duct specific knock‐out of insulin receptor (IR) we have recently demonstrated that it is critical for ENaC activity. To study the mechanisms of ENaC regulation by insulin and IGF‐1 we utilized immortalized mpkCCDc14 principal cells. As demonstrated by Western blotting these cells express endogenous ENaC subunits, as well as receptors for insulin and IGF‐1. Insulin and IGF‐1 enhanced transepithelial Na+ transport with EC50 of 12 and 14 nM, respectively. Both insulin and IGF‐1 increased p‐IGF‐1R level. In contrast, insulin only upregulated the level of p‐IR. The effects of both compounds were abolished by IGF‐1R inhibitor, PPP (500 nM; 48 hrs); this inhibitor by itself had no effect on transepithelial current. AG1024 (500 nM), a specific inhibitor of IGF‐1 and insulin receptor tyrosine kinase activity, also diminished insulin and IGF‐1‐stimulated Na+ transport in mpkCCDc14 cells. Thus, we propose that IGF‐1 along with insulin is a key regulator of ENaC activity and both insulin and IGF‐1 receptors are mediating these effects.
Inhibition of αENaC expression and ENaC activity following blockade of the circadian clock‐regulatory kinases CKIδ/ε
Authors: Richards Jacob , Mitzelfelt Megan , Jeffers Lauren , Cheng Kit-Yan , Eaton Douglas , Gumz Michelle L.
Published: June 2021

Increasing evidence suggests that the circadian clock plays an important role in the control of renal function. We previously showed that the circadian clock protein Per1 positively regulates the expression of the alpha subunit of the renal epithelial sodium channel (αENaC). Casein kinase I is a critical regulator of clock proteins. In order to enter the nucleus, Per1 must be phosphorylated by casein kinase Iε (CKIε). Renal cortical collecting duct (mpkCCDc14) cells were treated with the CKIδ/ε inhibitor PF670462. DNA affinity purification assays showed that CKIδ/ε inhibition prevented Per1 from interacting with a target E‐box from the αENaC promoter. Steady state αENaC mRNA levels were reduced more than 60% following CKIδ/ε inhibition. CKIδ/ε inhibition significantly reduced αENaC protein levels. ENaC activity is tightly controlled by aldosterone and αENaC is a well‐known transcriptional target of aldosterone. Inhibition of CKIδ/ε effectively prevented the aldosterone response of αENaC, suggesting an interaction between Per1 and aldosterone‐mediated regulation of this gene. Importantly, single channel analysis in the amphibian renal cell line 2F3 demonstrated that CKIδ/ε inhibition resulted in decreased ENaC NPo. The present study shows for the first time that CKIδ/ε inhibition represses αENaC expression and ENaC activity, providing further evidence for direct control of ENaC by the circadian clock.
Effect of Epithelial Sodium Channel Blockade in Hypertensive Nigerians
Authors: Elias Simiat Olanike , Sofola Soga , Jaja Smith
Published: June 2021

BACKGROUND

Approximately 60% of hypertensive Nigerians are salt‐sensitive. The demonstration of genetic linkage between Liddle syndrome and epithelial sodium channel (ENaC) suggests the physiological involvement of ENaC in blood pressure regulation.

OBJECTIVE

To determine the effect of ENaC blockade on blood pressure in hypertensive Nigerians.

METHODS

Fifty‐three hypertensive subjects were recruited after ethical clearance was given by College of Medicine University of Lagos Research Grants and Experimentation Ethics Committee. Every subject gave an informed written consent. Blood pressure was determined at baseline and after salt‐loading with 200mmol/day of Na+ for 5 days. After one week wash‐out period, the tests were repeated following ingestion of 200mmol/day of Na+ plus amiloride 5mg daily for 5 days.

RESULTS

Blood pressure rose significantly following salt‐loading (SBP, 3.1±2.3%; p < 0.001; DBP 2.9±12.4%; p<0.001). Blockade with amiloride significantly reduced SBP by −7.0±1.4%; p<0.0001 and DBP by −8.5±1.7%; p < 0.0001.

CONCLUSION

There were pressor responses to acute salt‐loading in hypertensive Nigerians. Blockade of epithelial sodium channel led to significant fall in blood pressure.
Renal Medullary Circadian Clock Genes are Altered in Endothelin B Deficient Rats
Authors: Speed Joshua S , Saleh Mohamed A , Pollock David M
Published: June 2021

Blood pressure in humans follows a well‐timed circadian rhythm that is attenuated in a number of hypertensive models. Evidence suggests that these alterations in the circadian pattern of blood pressure may contribute to end stage organ disease associated with hypertension. Our lab has shown that rats lacking the ET‐B receptor have a much larger increase in blood pressure during the active period when placed on a high salt diet (HS) than the control strain. Recent studies indicate that within the kidney, the “clock” genes Bmal, Clock, Per1, and Cry1 also follow a very strict circadian rhythm and may regulate the expression of important sodium channels that influence blood pressure. This study was designed to determine whether the ET‐B receptor within the renal medulla regulates the expression of these “clock” genes. Kidneys from wild type and ET‐B deficient rats were taken during the night (active period) when blood pressure was at its highest point in the circadian cycle (100.1±3.9 mmHg, 109.9±4.2 mmHg, 126.7±4.3mmHg, and 151.5±2.9 mmHg for w/t on normal salt (NS), w/t on HS, ET‐B deficient on NS, and ET‐B deficient on HS respectively). First, we found that, compared to wild type (w/t) rats placed on a NS diet, w/t rat on HS have a significant reduction in Bmal (−8.3±2.9 fold change), Clock (−3.4±0.7 fold change), Per1 (−3.4±0.3 fold change), and Cry1 (−9.5±4.0 fold change) in the inner medulla of the kidney during the active period. In contrast, the reduction of Bmal, Clock, and Cry1 was completely absent in ET‐B deficient rats with no difference in Per1 expression between w/t on HS and ET‐B deficient on HS (−3.4±0.3 vs. −3.0±0.3 fold change respectively). These data suggest that alterations in “clock” gene expression within the renal inner medulla may play an important role in determining the exaggerated circadian blood pressure rhythm in ET‐B deficient rats.
Functions of Ion Transporting Proteins NKA, NKCC and CFTR on the Intestinal Epithelia of Fundulus grandis during Osmotic Challenge
Authors: Meng Yanling , Bodinier Charlotte
Published: June 2021

The Gulf killifish (Fundulus grandis) is a euryhaline species distributed throughout marshes along the Gulf of Mexico, where they may be exposed to a broad range of salinities. This study investigates the cellular distribution and regulation of the ion‐transporting proteins, Na+/K+ ATPase (NKA), Na+/K+/Cl− cotransporter (NKCC), and chloride channel cystic fibrosis transmembrane conductance regulator (CFTR), in the intestine during osmotic challenges. NKA mRNA and protein levels were upregulated in both freshwater (FW) and seawater (SW); NKCC and CFTR mRNA and protein levels also showed variations due to FW or SW acclimation. NKA and CFTR were localized on the basolateral and apical or subapical membrane, respectively in both FW and SW, while NKCC localization was basolateral in FW and apical in SW. To determine the role of these proteins in ion regulation, we used Ussing Chambers, together with pharmacological blockers, and ion substitutions to measure the active Na+ or Cl− transport. NKA, NKCC, and CFTR appear to be implicated in a Cl− and Na+ secretion function in FW acclimated intestinal epithelia. However, in SW, there may be an ion absorption function facilitated by NKA and NKCC, while CFTR may be involved in acid/base regulation rather than Cl− secretion.
Sabertooth cat (Smilodon) Attack Involved Inflicting Pneumothorax
Authors: Wilson Ted , Golbach Lillian P
Published: June 2021

Smilodon fatalis is a large extinct sabertooth cat with a paradoxically fragile mandible. Smilodon crania (n=3) and mandibles (n=4) were compared to Panthera tigris (tiger, n=4) a cat known to use tracheal obstruction during prey attack. Smilodon maxillary canines were longer than those of Tigris (19.2 ± 1.1 vs 6.4 ± 1.1 cm; P<0.001), but had diminutive mandibular canines suggesting the mandible was not used to bite or actively hold prey. Mandibular cross‐sectional area anterior to the first premolar tooth of Smilodon (6.8 ± 3.8 cm2) was significantly less (P<0.04) than Tigris (12.1 ± 1.8 cm2) suggestive of fragility. Distance from mandibular canine tip to condyle distance indexed to cross sectional area for Smilodon and Tigris (4.8 ± 2.1 and 2.4 ± 0.5; P<0.07) also indicates fragility. Smilodon canines were sensitive to fracture with lateral torsion and the narrow mandible would be prone to fracture by prey hooves during sustained strangulation. Smilodon maxillary canine gap was easily sufficient to straddle the ribs of large herbivorous prey (i.e. Bison), which have a partial mediastinum and propensity for bilateral pneumothorax. Therefore, Smilodon probably delivered maximal force at the maxillary canine tip with its mandible fully retracted (closed) and sternum as the point of fulcrum to create deep paired thoracic wall punctures and uni‐ or bilateral pneumothorax.
Altered expression of KATP channel genes after acclimation to hypoxia in goldfish (Carassius auratus)
Authors: DeWitt Judy P. , Ngo Thanh Thu , Yajnik Tanya , Chan Stefanie , Chung Emma , Kang Esther , Cameron John S.
Published: June 2021

Goldfish (Carassius auratus) are highly tolerant of environmental hypoxia, surviving and remaining active for several days in the complete absence of oxygen. Previous work suggests that the hypoxia‐induced activation of ATP‐sensitive potassium (KATP) channels increases tolerance of low oxygen in many species. For goldfish, we have previously characterized a nitric oxide (NO)‐and cGMP‐dependent pathway by which cardiac channel activation occurs in acute hypoxia. The purpose of the present study was to resolve alterations in the expression of genes potentially relevant to KATP channel activity in response to acclimation under hypoxic conditions (2.6 mg O2/l for seven days at 22°C). Quantitative real‐time PCR (qPCR) was used to monitor mRNA transcription of target genes in cardiac muscle. Of the two genes coding for the pore‐forming subunits of cardiac KATP channels (Kir6.1 and Kir6.2), the expression of kcnj8 was unchanged while kcnj11 was downregulated in response to chronic low oxygen. The Kir6.2/Kir6.1 mRNA ratio was decreased by 62%. The expression of hif1a (hypoxia inducible factor) in the heart was enhanced, as was nitric oxide synthase (nos2) and the sulfonylurea receptor regulatory subunit (sur2, abcc9). These data suggest that acclimation to chronic environmental hypoxia enhances KATP channel activity via changes in both structural components and an NO‐dependent signaling cascade. (Supported by an HHMI grant to Wellesley College)
Embryonic temperature produces persistent effects on the capacity for thermal acclimation in adult zebrafish
Authors: Scott Graham R , Schnurr Meghan E , Yin Yi , Johnston Ian A
Published: June 2021

We examined how temperature during embryonic development (22, 27, or 32°C) influences the thermal dependence of swimming performance, muscle phenotype, and gene expression. Temperature treatments were maintained until hatching, after which fish were raised to adulthood at 27°C. Aerobic exercise performance (critical swimming speed) in adult fish was measured 1d after transfer to 22, 27, or 32°C and after 30d acclimation to 16 or 34°C. Developmental temperature had predictable effects on locomotor capacity, with 22°C embryos performing best at 22°C and worst at 32 and 34°C. Surprisingly, performance was ~20% higher in both 32°C and 22°C embryos than in 27°C embryos after 16°C acclimation. These findings were partially explained by differences in the transverse area of red (slow oxidative) and intermediate (fast oxidative) fibers in the axial swimming muscle. RNA‐Seq analysis of the white muscle uncovered large‐scale changes in the transcriptome after acclimation to 16°C. These data suggest that temperature change during a brief window in embryonic development can have a dramatic and persistent effect on thermal acclimation capacity. Supported by the EU‐FP7 project LIFECYCLE and NSERC of Canada.
Increased mitochondrial ROS production is required for ventilator‐induced myonuclear apoptosis in the diaphragm
Authors: Nelson W. Bradley , Smuder Ashley J. , Sollanek Kurt J. , Min Kisuk , Szeto Hazel H. , Powers Scott K.
Published: June 2021

Prolonged mechanical ventilation (MV) results in diaphragm atrophy that is accompanied by myonuclear apoptosis and increased mitochondrial production of reactive oxygen species (ROS). However, it is unknown if this increased mitochondrial ROS production is essential for MV‐induced myonuclear apoptosis in the diaphragm. We tested the hypothesis that during MV, mitochondrial ROS induce the mitochondrial‐mediated apoptotic pathway in the diaphragm.

Methods

Cause‐and‐effect was determined by using a selective mitochondrial targeted antioxidant (SS‐31) that prevents MV‐induced mitochondrial ROS emission. Sprague‐Dawley rats were divided into three groups: 1) control, 2) mechanically ventilated for 12 hrs and 3) mechanically ventilated for 12 hrs with SS‐31.

Results

Compared to control, MV induced a significant increase in diaphragmatic active caspase‐9 and cytosolic cytochrome‐c. Treatment with SS‐31 prevented the MV‐induced increases in these upstream signals of myonuclear apoptosis. Additionally, SS‐31 significantly decreased the MV‐induced presence of diaphragmatic TUNEL positive nuclei, a positive indicator for apoptotic cell death.

Conclusion

These results provide evidence that MV‐induced increases in mitochondrial ROS promote mitochondrial‐mediated myonuclear apoptosis in diaphragm during MV. Supported by the NIH RO1 HL087839 awarded to SKP.
Effects of fatigue on electromechanical delay in human skeletal muscle: new insights from an electromyographic and mechanomyographic combined approach
Authors: Esposito Fabio , Cè Emiliano , Rampichini Susanna , Limonta Eloisa , Veicsteinas Arsenio
Published: June 2021

While tendons are the major contributor to the electromechanical delay (EMD, the latency between the onset of electrical activity and force in a muscle), the relative role of action potential propagation and excitation‐contraction coupling is still undefined. The aim of the study was: (i) to assess the reliability of an electromyographic (EMG) and mechanomyographic (MMG) approach in partitioning EMD; and (ii) to evaluate the effect of fatigue on the muscle and tendon EMD components. Tetanic stimulations were delivered to the medial gastrocnemius of 24 subjects, before and after a fatiguing stimulation. On a different day, the study was repeated for reliability purposes. During contractions, EMG, MMG and force were recorded. EMD and delays between EMG and MMG (Δt EMG‐MMG, muscle component) and MMG and force (Δt MMG‐F, tendon component) were calculated. Intraclass correlation coefficient (ICC) was 0.892, 0.915 and 0.917 for EMD, Δt EMG‐MMG and Δt MMG‐F, respectively. With fatigue, peak force dropped (−26%; P<0.05) and EMD, Δt EMG‐MMG and Δt MMG‐F lengthened from 25.58±2.62 ms, 11.44±0.64 ms and 13.84±2.96 ms to 31.57±4.18 ms (+23%), 12.75±0.66 ms (+12%) and 18.98±4.44 ms (+39%), respectively (P<0.05). EMG‐MMG approach was a reliable tool to assess EMD components. Although fatigue had a stronger effect on EMD tendon component, also muscle component was significantly affected.

(UNIMI PUR grant #12‐1‐5059281‐42)
PCG‐1 alpha over‐expression rescues dystrophic muscle by modifying gene expression
Authors: Hollinger Katrin , Rice Drance , Snella Elizabeth , Selsby Joshua T
Published: June 2021

Duchenne muscular dystrophy is caused by the inability to produce a functional dystrophin protein. Typically, diagnosis is in the preschool years due to locomotor deficits, indicating muscles have already been damaged by the disease. Thus, it is critical that treatments be able to rescue muscle from further deterioration. We have shown that Peroxisome proliferator‐activated receptor γ coactivator‐1α (PGC‐1α) gene transfer rescues dystrophic muscle from disease related decline. To better understand the mechanism underlying the benefits of PGC‐1α over expression, 3 wk old mdx mice were injected in one hind limb with null AAV6 (empty capsid) and in the other with an AAV6 driving expression of PGC‐1α. At six weeks of age solei were collected. Utrophin protein expression was measured by immunohistochemistry and was increased nearly 3‐fold (p<0.05) in PGC‐1α over‐expressing limbs compared to control limbs. PCR arrays were performed to identify genes regulated by PGC‐1α over‐expression. In the PGC‐1α treated soleus expression of genes associated with the dystrophinglycoprotein complex (DGC) were increased by 40–92% (p<0.05), oxidative metabolism by 35–87% (p<0.05), muscle repair by 56–92% (p<0.05), and structural components by 20–300% (p<0.05). These data indicate that PGC‐1α‐mediated rescue of dystrophic muscle is accomplished through numerous contributing mechanisms. Partially supported by CIAG.
Impaired calcium handling in skeletal muscles of mitochondrial‐DNA‐mutator mice
Authors: IVARSSON NIKLAS , Hernández Andrés , Bruton Joseph , Westerblad Håkan
Published: June 2021

Mitochondrial myopathies are diseases of the respiratory chain that lead to impaired muscle function. Mutations in mitochondrial DNA (mtDNA) have been proposed to cause weakness by inducing atrophy of skeletal muscle. Here, we examined muscle function in mice that express a proof‐reading‐deficient version of PolgA and display an increased number of mutations in the mtDNA and a premature aging phenotype, to examine skeletal muscle function. Force and free cytosolic [Ca2+] ([Ca2+]i) were measured in isolated fast‐twitch muscles of 11 month old mice. EDL muscles from mtDNA mutator mice were significantly smaller than controls (7.7 ± 0.3 vs. 11.2 ± 0.6 mg) and produced less force at 100 Hz stimulation (149.2 ± 12.5 vs. 291.1 ± 28.4 mN), indicating reduced force due to muscle atrophy. However, when force was expressed per cross sectional area, mtDNA muscles were weaker than controls (199.8 ± 25.2 vs. 286.7 ± 24.0 kPa). This was accompanied by reduced [Ca2+]i during tetanic contractions in mtDNA muscles (0.9 ± 0.1 vs. 2.1 ± 0.7 μM) and a ~50% reduction in expression of sarcoplasmic reticulum Ca2+‐ATPase‐1 (SERCA1) was observed. In conclusion, skeletal muscle weakness occurs due to both muscle atrophy and impaired Ca2+ handling in muscles from mtDNA mutator mice.
A Novel Method to Measure Glucose Uptake by Single Skeletal Muscle Fibers Reveals a Similar Level of Insulin Resistance for Type IIA, IIB, IIX and IIB/X Fibers from Obese Zucker Rat Epitrochlearis Muscle
Authors: MacKrell James G. , Cartee Gregory D.
Published: June 2021

We have previously developed a method to measure glucose uptake (GU) by single fibers from rat skeletal muscle, showing in normal Wistar rat muscle, insulin‐stimulated GU was significantly greater in fibers expressing IIA vs. all other measured fiber types (IIA> IIB, IIX, IIB/X). Our aim for this study was to use this method to gain insights into insulin resistance. For this purpose we measured glucose uptake by single fibers from Obese Zucker (OZ; a widely used model of insulin resistance) vs. Lean Zucker (LZ) rats. Epitrochlearis muscles were incubated ex vivo with [3H]‐2‐ deoxy‐D‐glucose, with or without insulin, before isolation of ~10–30 single fibers from each muscle. Fiber type (myosin heavy chain, MHC, isoform) and GU were determined for each fiber. Basal GU values by single fibers were not significantly different between OZ and LZ, regardless of MHC isoform expression. For GU by single fibers from insulin‐stimulated muscles, there was a significant main effect of genotype, and post‐hoc analysis indicated that insulin‐stimulated GU values for each fiber type were significantly greater for LZ vs. OZ rats. However, the calculated relative decrement for insulin‐stimulated GU between fibers from OZ vs. LZ did not differ significantly among the fiber types (IIA= 44%, IIB=58%, IIX=52%, IIB/X =52%). These results provide unique information about skeletal muscle GU for a fuller understanding insulin resistance.
30.5±1.5°C Is the Optimal Hypothermia to Protect Hypoxic/Ischemic Heart
Authors: Ning Xue-Han , Sekhar L N , Kupchik N L , Corson M A , Werrbach J H , Tylee T S , Soh YK , Wang E , Villet O M , Ge M , Fan LP , Yao L , Olson A K , Zhu C , Anderson D L , Chen SH , Portman M A
Published: June 2021

Objective

To determine an optimal temperature in hypothermia protection for myocardium.

Methods

Isolated rabbit hearts infused with Langendorff model; Pig hearts in vivo infused with cardiopulmonary bypass; Patient treated with the cooling protocol at Harborview Med Ctr. The animal hearts were treated with a giving range of temperatures prior to and during cardioplegic ischemia. DP, dP/dtmax, cardiac power (CP), etc were monitored.

Results

Following reperfusion the off‐pump function of pig hearts returned to baseline (BL) level in the 29–30°C treatment group. In the 34–35°C group the recovery was significantly lower (P<0.05; DP=44.0± 9.4%, dP/dtmax =28.0±5.9%, CP=21.6±10.3% of BL). The 32.5°C treatment showed some recovery (P<0.05). The isolated rabbit hearts showed similar results to the pig hearts in vivo. One of the authors (XH Ning) suffered from a sudden cardiac arrest for more than 6 minutes in 2009. He was treated with Hypothermic Rescue (32°C central catheter) and had a remarkable recovery.

Conclusion

30.5±1.5°C is an optimal temperature in hypothermia protection. (Partial support from NIH HL‐60666 and CHRMC HR‐5836).
Rate of heat storage does not influence exercise intensity at a fixed rating of perceived exertion
Authors: Cramer Matthew N. , Molgat-Seon Yannick , Carlsen Anthony N. , Jay Ollie
Published: June 2021

This study investigated whether different rates of decline in self‐selected work rate (W) were mediated by early differences in heat storage. Five males cycled at a W to maintain a perceived exertion of 16 (Borg scale) at 35°C and 25°C. Heat production (Hprod) was estimated via indirect calorimetry. Esophageal (Tes) and skin (Tsk) temperatures, as well as local sweat rates on the lower back (LSRback) and thigh (LSRthigh), were measured throughout. The level of muscle activation in vastus lateralis (VL) was assessed via integrated surface electromyography (iEMG). While dry heat loss was lower at 35°C (P<0.01), sweating responses were elevated, indicated by greater LSRthigh at 2 min (P<0.05), a trend toward greater LSRback (P=0.07), and earlier onsets at both sites (P=0.02). Similar Hprod and rates of change in Tes were found in both conditions (P>0.05), yet at 35°C, exercise time was shorter (35°C: 22.6±4.9 min; 25°C: 32.1±10.4 min; P=0.03), and W (% of initial W) declined more rapidly by 15 min (35°C: 89.2±8.2%; 25°C: 82.2±8.0%; P<0.01). Mean Tsk was lower throughout exercise at 25°C (P<0.01). Similar changes in VL iEMG activity were found at 25°C and 35°C (P=0.28). In conclusion, reductions in W occur without early differences in heat storage between air temperatures, implicating factors other than the rate of heat storage, such as Tsk, in the regulation of exercise intensity.

Supported by a NSERC Discovery Grant (O. Jay)
Increased daily‐activity alters thermal perception and behavioral thermoregulation in mice
Authors: Lin Cheng-Hsien , Tokizawa Ken , Uchida Yuki , Nagashima Kei
Published: June 2021

The aim of this study is to know whether physical activity affects animal fs behavioral responses during heat with osmotic stimulus. Male ICR mice (2–4 mo, age) were divided into two groups: one group had free access to running wheel for 8 weeks (WR, n=20) and the other not (NWR, n=20). After subcutaneous injection (1 ml/100 g of body weight) of either isotonic‐ (154 mM, IS) or hypertonic‐ saline (2,500 mM, HS), each mouse was placed in a behavior box with 5 peltier boards at the bottom, where a) thermal mosaic (temperature of each board was randomly chosen among set at 15 Ž, 22 Ž, 28 Ž, 35 Ž, or 39 Ž and was changed each 6 min) or b) operant behavior available (the temperature of each board was set at 39 Ž; right‐end board temperature was changed to 20 Ž for 60 seconds when a mouse moved to left‐first two boards). In the results, WR group had significant operant behavioral responses than NWR group during osmotic stimulus in the heat (2 times higher than NWR) and prefer lower temperature (32.8 Ž vs 34.8 Ž in average). These results indicate that physical activity may improve behavioral thermoregulation in dehydrated mice in the heat. In particular, physical activity may lower the thermal preference.
Thermoregulatory responses are decreased by carbohydrate ingestion through hyperosmotic suppression while the effects were attenuated by insulin in passively heated men
Authors: Suzuki Akina , Okazaki Kazunobu , Imai Daiki , Takeda Ryosuke , Naghavi Nooshin , Matsumura Shinya , Miyagawa Toshiaki
Published: June 2021

Plasma hyperosmolality with thermal sweating or hypertonic saline infusion increases esophageal temperature (Tes) threshold for sweating and cutaneous vasodilation. We assessed whether plasma hyperosmolality with carbohydrate (CHO) ingestion increased Tes threshold. Furthermore, we assessed the effects of insulin secreted with CHO ingestion which was known to modify the sensitivity of thermo‐sensitive neurons on Tes threshold.

Methods

Seven healthy young men performed three trials in a sitting position in an artificial climate chamber (28°C of Ta, 40% of RH). After 10 min baseline, subjects drank water (CNT; water 300 ml), glucose (GLU; water 300 ml + glucose 75 g), or fructose solution (FRU; water 300 ml + fructose 75 g). Twenty min later, they were heated passively (lower legs immersion in 42°C water) for 60 min. Plasma insulin concentration ([Ins]p), plasma osmolality (Posm), percent change in plasma volume (%ΔPV), Tes, sweat rate and cutaneous vascular conductance at forearm and chest were measured.

Results

Posm in GLU and FRU were equally higher than CNT, though there was no difference in %ΔPV among trials. [Ins]p was higher in GLU than CNT and FRU. Tes threshold for cutaneous vasodilation was higher in FRU than CNT and GLU, while similar in CNT and GLU.

Conclusion

Thermoregulatory responses are decreased after CHO ingestion via hyperosmotic suppression, while the effects were attenuated by insulin.