bluuebunny/crossref_metadata_2025 huggingface dataset:
High fat feeding causes insulin resistance and stimulates an increase in mitochondrial content and function in rat skeletal muscle
Authors: Hancock Chad R. , Han Dong‐Ho , Chen May , Holloszy John O.
Published: June 2021

Recent work has suggested that mitochondrial deficiency and dysfunction in skeletal muscle causes the insulin resistance in obesity and type 2 diabetes. It has also been reported that feeding high fat diets to rodents results in a decrease in muscle mitochondria, suggesting that this is the mechanism by which fat feeding causes insulin resistance. The objective of this study was to determine the effects of feeding rats high fat diets on muscle mitochondrial biogenesis and insulin responsiveness. After 4 wk of a high fat diet, maximal insulin stimulated glucose transport activity was decreased in soleus by ~40% (5.9±0.63 and 3.4±0.32 μmol/ml/30min for CH and HFD, p<0.05) and epitrochlearis by about 30% (2.5±0.20 and 1.8±0.14 μ mol/ml/30min for CH and HFD, p<0.05), concomitant with a significant increase in mitochondrial biogenesis evidenced by a ~2 fold increase in a range of mitochondrial marker proteins. An increase in functional mitochondria is evidenced by a ~75% increase in the capacity of triceps muscle to oxidize palmitate (10.3±1.8 and 17.8±2.5 nmol/min/g muscle in CH and HFD, respectively, p<0.05). These findings clearly demonstrate that the insulin resistance induced by high fat feeding is not caused by mitochondrial deficiency in skeletal muscle. This work is supported by NIH 5R01DK018986 and 5F32DK076410.
Diabetes‐related alterations in second messenger regulation of potassium channel activity in corporal smooth muscle cells
Authors: Zhao Weixin , Brink Peter R. , Christ George J
Published: June 2021

Corporal smooth muscle (CSM) relaxation mediates penile erection, and second messenger activation of the KCa and KATP channel subtypes plays a major role in this process. The goal of this study was to evaluate the potential role of altered second messenger activated hyperpolarizing K+ currents in CSM to diabetes (DM)‐related erectile dysfunction (ED). Corporal tissue was excised from the STZ‐DM rats following 2 months of DM. Whole cell currents were recorded from short‐term cultured CSM cells of DM and age‐matched control (AMC) rats; in the absence and presence of either cAMP (prostaglandin E1 (PGE1; 10 uM) or 8‐Br‐cAMP (1 mM)) or cGMP (sodium nitroprusside (SNP; 10 uM) or 8‐Br‐cGMP (1 mM)) activation. Holding potential was −70 mV, with I‐V curves constructed in 10 mV increments from −60 to +100 mV. Resting potentials were lower in DM (−44 ± 1.3 mV; n=40) than AMC (−56 ± 1.4 mV; n=40) (p<0.05). Outward whole cell K+ currents were reduced in DM (206 ± 11 pA; n=40) relative to AMC (321 ± 16 pA; n=40) (p<0.05). K+ currents induced by cGMP, SNP, cAMP and PGE1 were significantly diminished in DM relative to AMC. IBTX (100 nM) decreased cGMP‐activated K+ currents more than glibenclamide (GLIB; 10 uM) in AMC rats, but less than GLIB in DM rats. The data suggest that cGMP and cAMP‐mediated K+ currents are decreased in DM rats, and that altered regulation of the KCa and KATP channel subtypes might contribute to DM‐related ED in rats.
Heat Shock Factor‐1 Knock Out Protects From Doxorubicin‐Induced Heart Failure
Authors: Ilangovan Govindasamy , Vedam Kaushik
Published: June 2021

Doxorubicin (DOX) induces oxidative stress in the heart and congestive heart failure (CHF) among cohort of cancer patients, who are treated with this drug for various malignancies. On the other hand, heat shock factor 1 (HSF‐1) is activated by heat stress and various oxidants. Thus we hypothesized that activation of HSF‐1 by the DOX‐generated oxidants may play a role in the occurrence of CHF and hence knocking HSF‐1 out might protect the heart from DOX. Wild type (HSF‐1+/+) and HSF‐1 knock out (HSF‐1−/−) mice (n=10 in each group) were injected (IP) with DOX at a dose of 6mg/kg for 4 weeks. At the 8th week, CHF was confirmed from the reduced ejection fraction, by echocardiography. There was a significant delay in occurrence of CHF in HSF‐1−/− mice compared to HSF‐1+/+group. In DOX treated HSF‐1+/+ hearts, more than three fold increase in the expression of Hsp25 and phospho (s‐15 & s‐82) Hsp25 (2D western blotting & Ms/Ms analysis), was observed. Further, immunoprecipitation (ip) experiments showed that p53 co‐ip Hsp25 in DOX treated hearts. Consistently, we observed higher Bax (transcripted by p53) in DOX treated HSF‐1+/+ group. However neither increased Hsp25 nor co‐ip of Hsp25 with p53 was observed in HSF‐1−/− group. We conclude from these studies that Hsp25 expression is increased in DOX treated hearts due to oxidative stress, which in turn enhances the p53 dependent pro‐apoptotic proteins such as Bax, resulting in CHF.
Characterization of the Expression and Function of Chemerin and CMKLR1 within the Central Nervous System
Authors: Parlee Sebastian Demian , Salih Wael H , Issa Mark E , Goralski Kerry B
Published: June 2021

Adipose derived signaling molecules (adipokines) play a crucial role in regulating satiety, metabolic homeostasis and body weight via endocrine actions in the CNS. We have identified a novel adipokine, chemerin, which regulates adipogenesis and adipose metabolism through actions on the chemokine‐like receptor 1 (CMKLR1). In the present study the neurological localization of chemerin and CMKLR1 mRNA was determined by dissecting mouse brains into cortex, brain stem, hypothalamus and pituitary. Similar levels of CMKLR1 mRNA expression were detected in each of the brain regions. Conversely, chemerin mRNA expression was enriched in the pituitary. Expression of both genes was significantly reduced in hypothalamus and pituitary of obese (Ob/Ob) mice. In vitro studies confirmed CMKLR1 and chemerin co‐expression in mouse hypothalamus and pituitary cell lines. Immunofluorescence corroborates this data identifying CMKLR1 expression on the hypothalamic cell membrane. These findings suggest that chemerin and CMKLR1 may represent a novel endocrine pathway that is functionally important in the brain centers that regulate feeding and metabolism. Funding: CIHR, NSHRF, DMRF, DPEF
Functional genetics of migraine: analysis of sodium pump and P/Q channel homologs from C elegans
Authors: Tong Xiangyan , Shih Cindy , Ma Shaochun , Gargus J. Jay
Published: June 2021

Familial Hemiplegic Migraine (FHM) provides a unique window into migraine pathophysiology. FHM is caused by missense mutations in 3 genes encoding subunits of a Ca and Na channel and the Na, K‐ATPase. We’ve published that FHM2 mutations produce hypomorphic kinetic alterations in Na/K pump function, but the effects of FHM1 P/Q channel mutations and the mechanism perturbed in common by the 3 loci remain controversial. In C. elegans neurons we were able to define functional interaction between the FHM1 (unc‐2) and FHM2 (eat‐6) homologs, allowing us to define their neuronal signaling pathway. We measured stress‐induced serotonin (5HT) synthesis in the ADF neuron using a tph‐1::GFP promoter reporter construct. Worms were heat‐stressed at 15–25°C and GFP fluorescence quantitated. At all temperatures the unc‐2 mutants have decreased, and the eat‐6 or double mutants have enhanced, tph‐1 synthesis compared to WT (all P<0.001). We also measured aldicarb‐sensitivity and showed unc‐2 hypomorphs have aldicarb‐resistance, whereas eat‐6 hypomorphs are aldicarb‐hypersensitive, with the double mutant, again, the same as eat‐6 alone (all P<0.001). Thus in two different neuronal phenotypes, hypomorphs of the FHM homologs had opposite effects, and in both eat‐6 is epistatic to unc‐2‐‐ meaning upstream in the pathway. Genetic and pharmacological block of calcium signaling proteins reveal novel intermediates in the pathway. Supported by NIH grant to JJG.
The acute effects of close arterial leptin administration on cardiovascular regulation and RVLM presympathetic vasomotor neurons
Authors: Sartor Daniela Maria , Verberne Anthony Maria
Published: June 2021

The gastric source of leptin may exert its effects directly by paracrine activation of vagal afferents, different to the mode of action for adipose leptin. Evidence is emerging for an interactive relationship between leptin and the gastrointestinal hormone cholecystokinin (CCK). The aim of this study was to examine the acute effects of close arterial injection of leptin (10–30 μg/kg) on arterial pressure (AP), heart rate (HR) and firing rate (FR) of presympathetic vasomotor neurons in the rostroventrolateral medulla (RVLM). We recorded AP, HR and RVLM neuronal discharge in anesthetised, paralysed, male Sprague‐Dawley rats. The effects of baroreflex activation, von Bezold‐Jarisch reflex activation (phenylbiguanide, PBG; 5 μg/kg i.v.) and CCK (1 μg/kg, i.v.) were examined. Close arterial leptin administration produced a significant decrease in HR (369 + 12 bpm to 357 + 14 bpm; n=6; P < 0.05; paired t‐test) and AP (107 + 3 mmHg to 85 + 6 mmHg; n=6; P < 0.05) within 5 minutes of administration. All RVLM neurons studied were inhibited by increases in AP and PBG. The FR of RVLM neurons inhibited by CCK (n=3) was significantly attenuated by leptin administration (from 10.6 + 5 to 8.7 + 5 spikes/s) despite the fall in AP, whereas the FR of those not inhibited by CCK (n=3) was either increased or unaffected. These results suggest that leptin may be exerting its cardiovascular effects via a CCK‐dependent mechanism.
Dynamic Visualization of Signaling Activities in Living Cells
Authors: Zhang Jin
Published: June 2021

The complexity and specificity of many forms of signal transduction are widely suspected to require spatial microcompartmentation and dynamic modulation of the activities of protein kinases, phosphatases and second messengers. However, traditional methodologies for detecting signaling events, such as activation of kinases, second messenger production and degradation, are limited in their spatiotemporal resolution and do not allow following these events within the live‐cell context. To achieve dynamic tracking of signaling activities in living cells, genetically encoded fluorescent reporters for protein kinases, second messengers such as cyclic AMP and phosphoinositides have been engineered. Their development and specific examples of their application will be discussed. For instance, this approach has revealed distinct pools of second messengers in specific cellular locations, such as the mitochondria and nucleus. In addition, a novel live‐cell high‐throughput screening method has been developed for identification of new modulators that affect the dynamic activity of kinases and second messengers.
Adenosine A2a receptors in the nucleus of the solitary tract (NTS) do not reset baroreflex functions for renal (RSNA) and adrenal (ASNA) sympathetic nerve activity
Authors: Ichinose Tomoko K , O'Leary Donal S , Scislo Tadeusz J
Published: June 2021

Stimulation of NTS A2a adenosine receptors elicits ‘baroreflex‐like’ decreases in mean arterial pressure (MAP), heart rate and RSNA. In contrast, increases in ASNA occur (AJP 275: H2130‐2139, 1998; AJP 278: H2057‐2068, 2000). Therefore we investigated if NTS A2a receptors affect baroreflex control of RSNA and ASNA. We compared full baroreflex‐response curves (i.v. nitroprusside/phenylephrine) evoked before and after bilateral microinjections into the NTS of selective adenosine A2a receptor agonist (CGS‐21680, 20 pmol/50nL, n=6) or antagonist (ZM‐241385, 40pmol/100nL, n=7) in urethane/α‐chloralose anesthetized male Sprague Dawley rats. CGS‐21680 evoked typical depressor (−23.8 ± 6.2 mmHg) and differential nerve responses (RSNA; −64.7 ± 4.4 Δ%, ASNA; +98.2 ± 11.5 Δ%). In contrast, ZM‐241385 increased RSNA (+10.5 ± 2.8 Δ%) and decreased ASNA (−2.2 ± 0.7 Δ%) without altering MAP. Both the agonist and antagonist did not reset RSNA and ASNA baroreflex curves along the MAP axis. The A2a agonist decreased the range, upper plateau and maximal gain (Gmax) of baroreflex curve for RSNA and increased Gmax and upper plateau for ASNA, the changes consistent with the direct action of the agonist. The A2a antagonist increased the range and upper plateau for RSNA only. We conclude that activation of NTS A2a receptors decreases RSNA and increases ASNA mostly via some non‐baroreflex mechanism(s). NIH HL‐67814
Involvement of nitric oxide (NO) in the nucleus tractus solitarii (NTS) on respiratory but not in cardiovascular responses to chemoreflex activation in awake rats
Authors: Granjeiro Erica M. , Machado Benedito H.
Published: June 2021

There is evidence that NO is a neuromodulator in the NTS. In the present study we evaluated the effect of nitric oxide synthase (NOS) inhibition in the caudal nucleus tractus solitarii (cNTS) on: a) the baseline cardiovascular and respiratory functions, and b) the changes on mean arterial pressure (MAP), heart rate (HR) and respiratory frequency (RF) in response to chemoreflex activation in awake rats. Rats received bilateral guide‐cannulas in the cNTS and the femoral artery and vein were cannulated. RF was evaluated by body‐plethysmography. Chemoreflex was activated with KCN (iv) before and after bilateral microinjections of a non‐selective NOS inhibitor L‐NAME (n=8) or a selective neuronal NOS inhibitor N‐Propyl (n=7) into cNTS. Microinjection of L‐NAME into cNTS produced a significant increase in baseline MAP (100 ± 2 vs 112 ± 3 mmHg) but no changes in HR and RF. Microinjection of N‐Propyl into cNTS did not affect baseline MAP, HR and RF. Microinjection of L‐NAME (138 ± 10 vs 81 ± 11 cpm) or N‐Propyl (159 ± 13 vs 90 ± 17 cpm) into cNTS significantly reduced the tachypneic but had no effect on the magnitude of cardiovascular responses to chemoreflex. The data shows that the activation of the respiratory component of the chemorreflex in the caudal NTS involves nNOS‐derived NO production, while NO does not modulate the cardiovascular responses. Supported by CNPq and FAPESP.
Localization of pancreatic preganglionic neurons within the rat dorsal vagal nucleus
Authors: Mussa Bashair M. , Verberne Anthony J.
Published: June 2021

The dorsal vagal nucleus (DVN) is the main source of the vagal innervation of the pancreas. In vitro studies have demonstrated that the DVN consists of a heterogeneous neuronal population with distinct morphological and electrophysiological characteristics. The aims of this study were (i) to identify DVN pancreatic preganglionic neurons (PPNs) in vivo and (ii) to characterize their responses to cholecystokinin (CCK), a major regulator of pancreatic secretion. Male Sprague Dawley rats anaesthetised with isoflurane (1.5%/100% O2) were used in all experiments. Extracellular single unit recording techniques were used to record the activity of PPNs within the DVN and their locations were marked using Pontamine Sky Blue. PPNs were identified by antidromic activation in response to stimulation of the pancreatic vagus nerve (0.5 Hz, 0.5 ms pulse width, 500μA). They had axonal conduction velocities in the C‐fibre range (< 1 m/s). Twenty‐five PPNs were identified within the rostral, medial and caudal DVN and twenty were tested for responsiveness to CCK. CCK (0.01–10 μg/kg, i.v.) produced three types of responses: (i) PPNs in the medial DVN were completely inhibited by CCK (0.7 ± 0.02 m/s, n = 16), (ii) PPNs in the caudal DVN were slightly activated by CCK (0.6 ± 0.08 m/s, n = 2) and (iii) CCK had no effect on the PPNs in the rostral DVN (0.6 ± 0.01 m/s, n = 2). CCK has complex actions on PPNs that may be related to its effect on exocrine and endocrine secretion. Further investigation of PPNs may allow identification of sub‐groups of DVN neurons that control the pancreas.
Corticosterone (cort) in the dorsal hindbrain (DHB) does not alter the number of neurons in cardiovascular brain regions activated by stress
Authors: Daubert Daisy L. , Looney Benjamin M. , Su Ye , Scheuer Deborah A.
Published: June 2021

Chronic cort in the DHB increases the cardiovascular response to stress, but the mechanism is unknown. We hypothesized that cort in the DHB increases the number of stress‐activated neurons in brain regions involved in cardiovascular control. Pellets containing either 0% (n = 10) or 10% (n = 10) cort were implanted on the DHB. 6 days later, half of the rats in each group were restrained for one hour (stress) and their brains collected an hour later. The remaining animals’ brains were collected without prior restraint. Sectioned brain slices were stained immunohistochemically for cFos. All data is reported as the average number of cFos positive neurons / slice. Stress increased the number of cFos positive neurons (p<0.05) in the nucleus of the solitary tract (1.6±0.7 to 6.6±1.9), caudal ventrolateral medulla (0.6±0.1 to 5.3±1.1), medial amygdala (8.5±2.7 to 18.9±4.2), paraventricular nucleus (8.8±2.0 to 42.2±5.0), dorsomedial hypothalamic nucleus (19.9±4.0 to 40.3±4.8), and arcuate nucleus (7.7±2.2 to 20.8 ±2.1). Stress did not increase cFos expression in the rostral ventrolateral medulla nor the supraoptic nucleus. The cort pellet did not increase the number of cFos positive neurons in any brain region compared to the 0% pellet. We conclude that cort in the DHB does not enhance the cardiovascular stress response by increasing the number of activated neurons involved in cardiovascular control. Supported by NIH HL 076807.
Greater exercise tolerance following VO2max in incremental‐ramp than constant work‐rate exercise
Authors: Murgatroyd Scott R , Ferguson Carrie , Ward Susan A , Whipp Brian J , Longaretti Miriam , Fukuba Yoshiyuki , Miura Akira , Rossiter Harry B
Published: June 2021

The tolerable duration (tlim) of high intensity exercise is well described by a hyperbolic function of work‐rate (WR), where only a fixed amount of work (W′) can be performed above the WR asymptote (CP). We have shown that supra‐CP WRs are not tolerated at the tlim of constant WR (CW) exercise (Coats et al. JAP 95: 483–490, 2003), and so aimed to determine whether this was also the case for incremental‐ramp (IR) exercise. Twelve males performed repeated CW cycle ergometry to estimate CP, W′ and determine VO2max (breath‐by‐breath measurement). Subjects then completed IR and CW exercise to tlim, immediately after which they attempted to sustain a lower WR (110% CP). The amount of supra‐CP work done at tlim was lower (p < 0.01) during IR (15.5 ± 3.6 kJ) than CW and also W′ (18.8 ± 3.4 kJ and 19.8 ± 2.9 kJ, respectively; N.S.), with VO2max (4.2 ± 0.6 l/min) evidence of maximum effort in all tests. tlim at 110% CP was greater (p < 0.01) following IR (range 23–297 s) than CW (6–168 s); the former manifest with no fall in VO2 (4.1 ± 0.7 l/min) despite an 84 ± 15 W reduction in WR and presumably time for it to do so. If the metabolic equivalent of CP is the same during IR and CW, these data suggest that tlim in IR is achieved at VO2max without W′ depletion. We conclude therefore that the ‘profile’ by which intolerance is achieved significantly influences the tlim of subsequent exercise by increasing the rate of W′ depletion and/or accumulation of fatigue‐inducing metabolites.
Cilia‐mediated heat shock protein 27 (hsp27) suppression modulates actin polymerization and organization in wild type mouse and oak ridge polycystic kidney (orpk) cells
Authors: Jones Thomas Joseph , Mell Blair M , Dokas Linda A , Yoder Bradley K , Nauli Surya M
Published: June 2021

Primary cilia play a pivotal role in the development of polycystic kidney disease (PKD), a state with an enhanced propensity for hypertension. Cilia, characterized as mechano/chemo‐sensing organelles, are uniquely situated to communicate extracellular signals to intracellular responses. Because cytoskeletal remodeling plays a significant role in cellular adaptation, we postulated that stress‐induced hsp27, which associates with actin to stabilize the cytoskeleton, could mediate this response. To that end, we evaluated shear and oxidative stress on hsp27 levels and actin polymerization in cilia deficient endothelial cells isolated from the orpk model of PKD. By immunoblotting orpk had a basal suppression of hsp27 compared to wild‐type (wt) cells. Basal suppression could be overcome by oxidative stress but not shear stress. Basal actin levels were consistently elevated in orpk when compared to wt cells and stress had a minimal effect on these levels. Actin organization was inversely correlated with hsp27 levels. Our observations provide evidence that disruption of cilia formation suppresses basal hsp27 levels by an undetermined mechanism and increases the unorganized state of actin. Results suggest primary cilia have a role in regulating actin organization and in providing cytoskeletal adaptation to stress potentially through hsp27. (AHA‐0630257N, NIH‐HL084451)
Resolution of the subunit stoichiometry of the endogenous endothelial ISOC channel
Authors: Cioffi Donna L. , St. Croix Claudette M. , Pitt Bruce R. , Stevens Troy
Published: June 2021

The endothelial ISOC channel is a calcium selective store‐operated calcium (SOC) entry channel whose activation is an important step leading to intercellular gap formation and increased permeability. The transient receptor potential canonical (TRPC) proteins 1 and 4 contribute to ISOC channel structure. SOC entry channels are comprised of four subunits which coalesce to form the channel pore. To date, the number of each type of subunit, i.e. the subunit stoichiometry, remains unknown. We sought to determine the subunit stoichiometry of the endogenous endothelial ISOC channel using Förster Resonance Energy Transfer (FRET). Prior studies demonstrated that TRPC4 interacts with protein 4.1. Thus, protein 4.1 immunoprecipitation was performed to isolate an enriched population of endothelial ISOC channels. TRPC1 and TRPC4 subunits were tagged with fluorophore (Cy3 and Cy5) labeled antibodies. FRET experiments were performed using the sensitized emission approach and confirmed via acceptor photobleaching. Positive FRET transitions occurred between TRPC1‐Cy3 and TRPC4‐Cy5, TRPC1‐Cy3 and TRPC1‐Cy5 as well as between TRPC4‐Cy3 and TRPC4‐Cy5, indicating that the tetrameric ISOC channel is comprised of at least two TRPC1 and at least two TRPC4 subunits. Thus the subunit stoichiometry of the endogenous endothelial ISOC channel is two TRPC1 subunits plus two TRPC4 subunits. Supported by HL60024 and 1F32HL086124.
The temporal relationship of cytokine‐induced expression of genes involved in the metabolism of L‐arginine in bovine pulmonary artery endothelial cells
Authors: Malleske Daniel Thomas , Jin Yi , Nelin Leif D.
Published: June 2021

Inflammatory stimuli have been shown to increase both iNOS and arginase expression and activity. We hypothesized that inflammatory stimuli would result in a transient induction of iNOS and prolonged induction of arginase, thereby representing a mechanism whereby host defense functions predominate early and host repair functions predominate later in the course. To investigate this hypothesis, we studied bovine pulmonary artery endothelial cells (bPAEC) stimulated with lipopolysaccharide (LPS) and tumor necrosis factor–α (TNF‐α) or treated with vehicle. Cells were harvested at 0, 1, 2, 4, 8, 24, 48, 72 and 96 hours after stimulation. We examined expression levels of mRNA for iNOS, arginase II, ornithine decarboxylase (ODC) and ornithine aminotransferase (OAT). Expression levels of iNOS mRNA in LPS/TNF‐α treated cells were increased at 2 hours and peaked by 24 hours with a return to baseline expression between 48 and 72 hours. Conversely, arginase II mRNA expression was increased at 4 hours and remained elevated throughout the experimental period. The mRNA levels of ODC and OAT were relatively high in control cells; although, LPS/TNF‐α stimulation did increase expression. These data are consistent with our hypothesis that inflammatory stimuli result in an early, transient NOS response followed by a sustained arginase response. Taken together, the observed pattern of expression of these enzymes would result in L‐arginine metabolism to NO early in the inflammatory response, followed at 48 to 72 hours by a shift to an arginase‐mediated metabolism of L‐arginine to ornithine and subsequent polyamine and L‐proline production.
A novel role of phosphatase and tensin homolog in regulating transient receptor potential channel 6‐mediated Ca2+‐entry and endothelial permeability
Authors: Kini Vidisha , Mehta Dolly
Published: June 2021

We recently showed that thrombin by binding to its receptor PAR‐1 activates transient receptor potential channel 6 (TRPC6), a receptor‐operated Ca2+ channel (ROC), which in turn controls endothelial permeability. However, mechanisms regulating TRPC6 activity downstream of PAR‐1 remains unclear. Phosphatase and tensin homologue (PTEN) is a tumor suppressor, which dephosphorylates phosphatidylinositol 3,4,5‐triphosphate (PIP3) to maintain phosphatidylinositol 4,5‐biphosphate (PIP2) levels in the membrane. PTEN also contains a C‐terminal PDZ binding motif, known to be involved in mediating protein interaction. We addressed the hypothesis that PAR‐1 activates PTEN which in turn is required for TRPC6 activation and its anchoring to the plasma membrane and for signaling that results in the increased endothelial permeability. We show that PAR‐1 activation within seconds increased PTEN activity. Knock down of endogenous PTEN in endothelial cells markedly reduced Ca2+ entry induced by thrombin as well as by DAG. However, knockdown of PTEN had no effect on thapsigargin induced Ca2+ entry implicating that PTEN specifically regulates ROC activity. We observed that PTEN interacts with TRPC6 which increased after thrombin stimulation. The PTEN tail domain was required for mediating PTEN interaction with TRPC6. Transduction of a PTEN mutant lacking the tail domain and importantly the PDZ binding sequence abolished ROC activity. These findings identify a novel role of PTEN as a potential key regulatory switch responsible for G‐protein coupled receptor induced TRPC6 activation and its role in increased endothelial permeability.
Long‐term effects of low‐dose NOS inhibition in mouse renal medulla
Authors: Stoessel Adelina , Paliege Alexander , Theilig Franziska , Addabbo Francesco , Goligorsky Michael , Bachmann Sebastian
Published: June 2021

Chronic NO deficiency causes endothelial dysfunction of the renal medullary vasculature and increases the expression of the Na,K,2Cl‐cotransporter (NKCC2) of the thick ascending limb. We hypothesized that impaired medullary perfusion and increased NKCC2 activity may promote local hypoxia and fibrosis. To test this, adult male 129J mice were treated with the NOS inhibitor, NG‐methyl‐L‐arginine (L‐NMMA; 0.3 or 0.8 mg/ml in drinking water). Blood pressure was measured by tail plethysmography. Density and structure of medullary blood vessels were evaluated using histochemistry and fine structural analysis. Distribution and quantity of CD31, CD34 and phospho‐NKCC2 were studied. Hypoxic areas were visualized by hypoxyprobe (pimonidazole) staining. L‐NMMA treated groups showed focal expansions of connective tissue deposits in or near vascular bundles. Blood pressure and overall structure and density of medullary microvessels were unaffected. Outer medullary phospho‐NKCC2 immunoreactivity was markedly increased in treated animals, whereas hypoxic signs were unrelated to treatment. We conclude that long term application of sub‐pressor doses of NOS inhibitor in mice increases the activity of outer medullary NKCC2 without major effects on local oxygen tension and vascular integrity. Activation of NKCC2 may be related to fibrotic changes in the renal medulla under reduced availability of NO.
Laser capture microdissection as a tool for deciphering the clinical relevance of molecular signatures of cancer cells
Authors: Wittliff James L
Published: June 2021

Molecular basis of clinical behavior and therapeutic response of human carcinoma is poorly understood. To circumvent cellular heterogeneity and evaluate genomic and proteomic differences of normal and diseased cells, Laser Capture Microdissection (LCM) was employed to isolate distinct cell types non‐disruptively, allowing independent extraction of DNA, RNA or protein analyses under stringent conditions (e.g. RNase‐free & protease‐free). Using an extensive BioRepository, frozen human carcinoma biopsies with clinical follow‐up of 10–18 years were identified and pure populations of 1000–5000 cells were procured by LCM. Total RNA was processed for RT‐qPCR and amplified for microarray analyses (Agilent & Affymetrix). Spectrophotometric and BioAnalyzerTM (Agilent) analyses evaluated aRNA yield, purity and transcript length. Primer sets were created for qPCR (b–actin 200 base, 300 base & 400 base amplicons). FFPE‐extracted transcripts required amplification (ParadiseTM kit, Arcturus) and X3P chips (Affymetrix) requiring ∼200 bases on the 3′ end of aRNA. Gene expression profiles of pure populations of human carcinoma and stromal cells were discovered that correlated with cancer behavior. Significantly, several gene expression signatures were highly associated with clinical outcome suggesting promise for development of novel prognostic tests for cancer management and as aids in drug design.
Sodium butyrate increases di/tripeptide transporter PepT1 expression and function
Authors: Dalmasso Guillaume , Nguyen Hang Thi Thu , Yan Yutao , Charrier‐Hisamuddin Laetita , Sitaraman Shanthi V
Published: June 2021

PepT1 is a di/tripeptide transporter normally expressed in the small intestine but not in the large intestine. This study aimed at investigating PepT1 regulation by sodium butyrate (NaB). We found in vitro, that NaB treatment increased PepT1 promoter activity in a dose‐ and time‐dependent manner, with maximal activity observed in cells treated with 5 mM NaB for 24 hours. Under these conditions, we observed a ~21‐fold increase in hPepT1 promoter activity and a ~2.5‐fold increase in hPepT1 transport activity. These results were confirmed by real‐time RT‐PCR and Western‐blot. Molecular analyses revealed that Cdx2 and CREB transcription factors are essential for NaB‐induced increase of hPepT1 expression and activity. Butyrate‐induced CREB and Cdx2‐promoter binding was confirmed by gel shift and chromatin immunoprecipitation. Moreover, in Cdx2‐overexpressing cells, hPepT1 protein level is higher compared to control cells. Finally, 5 mM butyrate added to drinking water during 24 hours increases the levels of colonic PepT1 expression and activity in mice. Collectively, these data indicate that intracellular accumulation of NaB increases PepT1 expression and activity in colonic epithelial cells.
Pharmacokinetic profiling and behavior characterization of a GalR3 antagonist
Authors: Lundström Linda , Prinssen Eric , Trube Gerhard , Kolczewski Sabine , Wettstein Joseph G , Schuler Franz , Moreau Jean‐Luc , Spooren Will , Gatti Silvia
Published: June 2021

The neuropeptide galanin mediates its effects through three G‐protein coupled receptors (GalR1‐3) and recent studies has suggested a role for the galaninergic system in regulating anxiety and depressive‐like behavior. In order to evaluate the therapeutic potential of GalR3 antagonists as antidepressant and anxiolytic agents we have studied the pharmacokinetic and behavioral characteristics of the only available GalR3 antagonist, SNAP 37889 (Swanson et al. 2005). Chemical stability studies revealed a pH‐dependent stability of the compound which required formulation development to obtain optimal exposure for in vivo studies. Pharmacokinetic studies of the GalR3 antagonist in the rat showed moderate but extended plasma exposure following intraperitoneal administration. Lower exposure was seen after intravenous injection whereas a 4‐fold increased plasma exposure was observed after oral administration. In vitro binding studies in cells stably transfected with human GalR3 confirmed a high affinity binding site for the GalR3 antagonist and functional signaling was carried out in GalR3 and GIRK expressing oocytes. Under the optimal conditions developed, the GalR3 antagonist is being evaluated in relevant behavioral models in rats and mice. Preliminary results indicate a weak anxiolytic effect not associated with sedative properties. A complete profiling of the GalR3 antagonist is ongoing.
Transcriptional Regulation of GI Smooth Muscle in Normal and Diabetic Mice
Authors: Touw Ketrija , Zhou Jiliang , Herring Paul Brian
Published: June 2021

Secondary effects of diabetes can lead to gastroparesis and GI motility dysfunction. Recent studies suggest altered smooth muscle (SM) as well neuronal regulation in these diseases. Consistent with these studies, we found altered expression of smooth muscle contractile proteins in the gut of ob/ob diabetic mice. Utilizing the telokin promoter as a model system, we are elucidating the mechanisms regulating expression of contractile proteins in the gut under normal or diabetic conditions. Telokin is a smooth muscle‐specific protein that is highly expressed in GI SM. Analysis of hprt‐targeted telokin promoter ?‐galactosidase transgenes in vivo, identified a 370bp promoter that is able to mimic expression of endogenous telokin. Within this promoter, a 100bp region is specifically required for telokin expression in GI SM. Analysis of this region revealed evolutionarily conserved binding sites of Sox and SMAD transcription factors. qRT‐PCR analysis demonstrated that Sox4, 5, 6, 7, 12 and 13 are the most abundant Sox proteins in GI SM. Knockdown of Sox7 by siRNA, in GI SM cells, attenuated telokin expression, while knockdown of Sox4, 6 or 12 had no effect. Transduction of GI SMC with dominant negative SMAD4 also attenuated telokin expression. Together these data suggest Sox7 and SMAD proteins are important for regulating expression of contractile proteins in GI SM.

Supported by NIH DK 061130, NIH DK 064466‐05
Growth hormone, prolactin, and sodium acetate have complex and non‐uniform effects on the lipid distribution of a differentiated bovine mammary epithelial cell line
Authors: Fujimoto Brent , Peterson Daniel
Published: June 2021

Non‐polar lipids (NPL) accumulate within the mammary epithelial cell during lactation; however there are no reported systematic studies that utilize NPL composition as a sign of differentiation. Additionally, there are no reported systematic studies on the response of a bovine mammary alveolar cell line (MAC‐T), to a range of lactogenic hormone concentrations, or different lipogenic substrates. In a 3 x 3 factorial arrangement, the effect of growth hormone (GH; 0, 10, 100 ng/ml), and prolactin (PRL; 0, 5, 10 μg/ml) on the lipid composition of MAC‐T was examined using thin layer chromatography. Using the optimal hormone treatment (10 ng/ml GH, 5 μg/ml PRL) for NPL synthesis, inclusion of 10 mM sodium acetate (NaOAc) was assessed to determine the effect of lipogenic substrate on lipid composition. GH had a significant effect on NPL, cholesterol, phosphatidylcholine, phosphatidylinostitol, and sphingomyelin (SM), while PRL only affected SM. NaOAc had a significant effect on NPL, phosphatidylethanolamine and SM. The proportion of NPL peaked with 10 ng/ml GH, but dropped to near‐control levels with 100 ng/ml GH. This study underscores the importance of testing a range of hormone concentrations when assessing lipogenic profile of MAC‐T cells.
Estrogen Augments Shear Stress‐induced Signaling and Gene Expression in Osteoblasts
Authors: Yeh Chiuan‐Ren , Cheng Cheng‐Kung , Chiu Jeng‐Jiann , Chien Shu
Published: June 2021

Estrogen and fluid shear stress play important roles in the pathogenesis of osteoporosis (OP). We investigated the role of interplays between estrogen and shear stress in signal transduction and gene expression in osteoblasts. Both estrogen and shear stress induce transient phosphorylations of extracellular signal‐regulated kinase (ERK) and p38 and expressions of cyclooxygenase‐2 (Cox‐2) and c‐fos in MG63 osteoblast‐like cells. Treatment of MG63 cells with ERK and p38 inhibitors inhibited the estrogen‐induced Cox‐2 and c‐fos expressions. Application of MG63 cells treated with estrogen for 6 h to shear stress (12 dynes/cm2) for 30 min significantly induced increases in ERK and p38 phosphorylations and Cox‐2 and c‐fos expressions, as compared with the cells without estrogen, suggesting estrogen augments the shear stress ‐induced signaling and gene expression in MG63 cells. MG63 cells treated with estrogen for 6 h induced their expression of beta1 integrin, which acts as a mechanoreceptor contributing to estrogen ‐enhancement of signaling and gene expression in MG63 cells in response to shear. Our results suggest that interplays of estrogen and mechanical force exert additive effects on osteoblast responses.
Growth Hormone supplementation up‐regulates collagen expression in human muscle and tendon
Authors: Doessing Simon , Heinemeier Katja , Holm Lars , Reitelseder Soeren , Koskinen Satu , Schjerling Peter , Kjaer Michael
Published: June 2021

Growth Hormone (GH) potentially influences connective tissue in muscle and tendon.

We studied the effect of 14 days of recombinant human GH (rhGH) supplementation and exercise, on the expression of mRNA for Insulin‐Like Growth Factor‐I (IGF‐I), collagen I and collagen III in human skeletal muscle and tendon, in a double blind, randomized, placebo‐controlled crossover design. 10 male subjects received rhGH (0.33–0,50 mg/kg/day) or placebo separated by 4 months. One‐legged exercise (10 × 10 reps at 70% of 1 RM) was performed on the last day of the supplementation period, and 24 hours later tissue was sampled bilaterally from the vastus lateralis muscle and the patella tendon. mRNA expression was measured using real‐time RT‐PCR.

rhGH administration resulted in a 8‐fold rise in circulating GH and a 3‐fold increase in circulating IGF‐I. In addition tendon and muscle connective tissue mRNA expression for IGF‐I increased significantly with GH (p<0.05). Furthermore, muscle and tendon collagen I and III increased significantly in response to rhGH supplementation (p<0.05).

The results indicate a role for rhGH via IGF‐I in increasing collagen content in connective tissue of both tendon and skeletal muscle.
Postnatal Development of Transporter Proteins in Epididymal Clear Cells of Rhesus Monkey (Macaca mulatta)
Authors: Pastor‐Soler Nuria , Smolak Christy , Gong Fan
Published: June 2021

Our understanding of human epididymal physiology is mostly based on rodent studies. However, only half of mouse epididymal genes have orthologs in humans. Moreover, unlike in rodents, androgen exposure in primates is interrupted between infancy and puberty. The epididymal clear cell marker vacuolar H+‐ATPase (V‐ATPase) helps maintain a luminal acidic pH, which is essential for sperm maturation. We hypothesize that developmental expression of clear cell transporter proteins differs between rodents and rhesus. Such differences may be important in understanding human infertility. One‐week‐old rats already have V‐ATPase in clear cells, but we found that this transporter is not expressed until the juvenile stage in rhesus. We also found that adult rhesus epididymis expresses abundant V‐ATPase and carbonic anhydrase, another clear cell marker. In addition, as in human adult epididymis, we detected in rhesus clear cells the cystic fibrosis transmembrane conductance regulator (CFTR), a protein that is much less abundant in rodent clear cells. It is known that mild deficiencies in CFTR function can lead to severe epididymal/vas deferens pathology and infertility in humans. These results highlight the need to systematically investigate the development and physiology of rhesus, rather than rodent, epididymis as a model for understanding human epididymal function.
The pathophysiology of intestinal endometriosis is exacerbated by uncontrollable stress
Authors: Cuevas Marielly , Santiago Olga I , Thompson Kenira J , Flores Idhaliz , Appleyard Caroline B
Published: June 2021

It is not known whether stress affects the severity of endometriosis in women.

Aim:

Assess the effects of chronic stress in an established animal model of intestinal endometriosis.

Methods:

Endometriosis was surgically induced in female rats. One group of rats (endo‐stress) was subjected to uncontrollable swim stress for 10 consecutive days. Endo‐controls had endometriosis with no stress. Sham‐stress had sutures only then swim stress. On day 60 all rats were sacrificed and examined for the presence of endometriotic vesicles. The colons were removed and assessed for macroscopic damage, myeloperoxidase (MPO) activity and histology.

Results:

None of the sham‐stress animals developed vesicles. The endo‐control rats developed vesicles with an average total vesicle length of 6.57 ± 0.96mm per animal. This was increased to 11.26 ± 5.27mm in the endo‐stress group (n=3‐4 ± sem). The endo‐control rats had higher macroscopic and histological colonic damage scores than the sham‐stress animals, which was increased further by stress. The endo‐stress also had the shortest colon length, increased numbers of colonic mast cells, higher MPO levels, and an increase in peritoneal fluid cell infiltration.

Conclusions:

This study presents evidence for the first time of the negative consequences of stress in the progression of endometriosis, most likely through an effect on the immune system. 2G12RR03050, SO6GM00823 & R01HD050559
Induction of arachidonate 12‐lipoxygenase (Alox15) alters intestinal lipid homeostasis during iron‐deficiency
Authors: Collins James F.
Published: June 2021

Gene chip analyses were performed with intestinal mucosa from iron‐deficient and Belgrade (i.e. Dmt1‐deficient) rats. Computational analysis, clustering algorithms and gene ontology analysis revealed enrichment of genes related to lipid metabolism. Most striking was induction of arachidonate 12‐lipoxygenase (Alox15); Alox15 metabolizes polyunsaturated fatty acids (PUFAs) to biologically active lipid mediators. qRT‐PCR studies demonstrated induction of Alox15 throughout the length of the intestine and in the liver of Fe‐deficient (up to 100‐fold) and Belgrade rats. IHC studies showed expression of Alox15 in intestinal and colonic epithelial cells with much stronger expression in the iron‐deficients. We also noted greatly increased PUFA metabolite levels in the iron‐deficient intestine, indicative of lipid peroxidation; the overall pattern of lipid peroxide products was consistent with Alox15 activity. We have thus documented significant regulation of Alox15 by iron in the mammalian small intestine and concomitant alterations in lipid homeostasis. These findings may be of significance as Alox15 may be involved in intestinal epithelial cell differentiation and proliferation. Induction of Alox15 may also have relevance to chronic inflammatory states, such as Inflammatory Bowel Diseases and colon cancer, as these and other pathological conditions are known to result in Anemia of Chronic Disease.
Nicotine causes depolarization, decreased intracellular calcium concentration ([Ca2+]i) and reduced nitric oxide (NO) production in human umbilical artery endothelial cells (HUAECs)
Authors: Pisarcik Sarah , Luke Trevor , Nelin Leif D , Shimoda Larissa A
Published: June 2021

Maternal environmental tobacco smoke (ETS) exposure has been associated with intrauterine growth retardation (IUGR) and low birth weight, both of which increase infant morbidity and mortality. Nicotine, an active component in ETS, is known to decrease birth weight and postnatal weight gain, yet the mechanism remains unclear. We hypothesized that nicotine alters membrane potential (Em)‐dependent regulation of [Ca2+]i and nitric oxide (NO) production in endothelial cells, which could cause umbilical vessel contraction, impairment of blood flow and IUGR. Fluorescent microscopy was used in HUAECs treated with nicotine (10–100ng/ml; 15m–24hr). Fura‐2 was used to measure [Ca2+]i and DiBAC4(3) was used to measure changes in Em. We found that HUAECs treated with 100ng/ml of nicotine showed depolarization and an immediate dramatic decrease in [Ca2+]i. Prolonged nicotine treatment (10 and 100ng/ml; 1–24 hr) also decreased [Ca2+]i . In addition, total nitrite levels in the cell media were measured via Griess reaction following total nitrate reduction. Treatment with nicotine (10ng/ml; 1–4hrs) reduced total nitrite levels in the media in comparison to untreated HUAECs. From these results, we conclude that nicotine, at concentrations comparable to plasma levels in non‐smokers exposed to ETS, caused decreased NO production in HUAECs, perhaps secondary to depolarization and decreased [Ca2+]i. Funded by: HL73859
Ox‐LDL Impairs Bone Marrow Stem Cell Differentiation into Endothelial Cells Possibly through Inhibition of Akt Phosphorylation
Authors: Chu Ling , Hao Hong , Huang Yu , Liu Zehao , Xu Jian , Zweier Jay L , Liu Zhenguo
Published: June 2021

In this study, experiments were designed to investigate the effects of oxidized low density lipoprotein (ox‐LDL) on bone marrow stem cell differentiation into endothelial cells and underlying mechanisms. Adult rat bone marrow multipotent progenitor cells (MAPCs) were used as the source of stem cells, and induced to differentiate into endothelial cells with or without ox‐LDL in the presence of VEGF. The differentiation process was closely monitored with cell morphology and expressions of endothelial markers including vWF, Flk‐1, and CD31 with RT‐PCR and Western blot analysis. As expected, the differentiating stem cells started to express vWF, Flk‐1, and CD31 from day 5 of differentiation, and reached their maximum at day 14. Ox‐LDL (at 9, 18, and 36 ug/ml) significantly inhibited the differentiation of MAPCs into endothelial cells in a dose‐dependent manner (p < 0.05) as reflected by markedly decreased mRNA levels and contents of vWF, Flk‐1, and CD31. Interestedly, the decreased expressions of endothelial markers were associated with significantly reduced Akt phosphorylation during the early phase of differentiation in these cells. These results indicate that ox‐LDL inhibits the differentiation of bone marrow stem cells into endothelial cells possibly through preventing Akt phosphorylation. The data from this study provides a novel mechanism for the development of ox‐LDL‐related cardiovascular diseases.
Involvement of Epithelial Sodium Channel in Pre‐implantation of Mice
Authors: Ruan Ye Chun , Chen Wen Ying , Zhou Wen Liang , Chan Hsiao Chang
Published: June 2021

Our previous study using semi‐quantitative RT‐PCR has demonstrated that the ENaC expression in mouse uteri was enhanced during pre‐implantation with immunohistochemical data showing their location in endometrial epithelium. We hypothesized that ENaC may play a role in fluid absorption to enhance embryo attachment during pre‐implantation. In the present study, inhibitors of potential ion channels/or transporters were injected to the uterine lumen of mice on day 3 of pregnancy and the number of visible implanted embryo of each mouse was counted on day 8. The results showed that amiloride(10μM), the ENaC inhibitor, was observed to significantly (p<0.05, n=9) reduce the implanted embryo number to 6.89±1.55 compared to that of the controls (10.64±0.82), while EIPA(10μM), the inhibitor of Na‐H exchanger (NHE), showed no significant effect, indicating the involvement of ENaC rather than NHE. In addition, La3+(1mM), a non‐selective cation channel inhibitor, showed no significant effect. These in vivo data together with those observed in our previous studies have demonstrated the involvement of ENaC in the pre‐implantation, indicating a significant role of ENaC in the initiation of implantation.