bluuebunny/crossref_metadata_2025 huggingface dataset:
Activated macrophages inhibit enterocyte gap junctions via the release of nitric oxide
Authors: Anand Rahul J. , Dai Shipan , Rippel Christopher , Leaphart Cynthia , Qureshi Faisal , Gribar Steven C. , Kohler Jeff W. , Li Jun , Stolz Donna Beer , Sodhi Chhinder , Hackam David J.
Published: November 2007

Enterocytes exist in close association with tissue macrophages, whose activation during inflammatory processes leads to the release of nitric oxide (NO). Repair from mucosal injury requires the migration of enterocytes into the mucosal defect, a process that requires connexin43 (Cx43)-mediated gap junction communication between adjacent enterocytes. Enterocyte migration is inhibited during inflammatory conditions including necrotizing enterocolitis, in part, through impaired gap junction communication. We now hypothesize that activated macrophages inhibit gap junctions of adjacent enterocytes and seek to determine whether NO release from macrophages was involved. Using a coculture system of enterocytes and macrophages, we now demonstrate that “activation” of macrophages with lipopolysaccharide and interferon reduces the phosphorylation of Cx43 in adjacent enterocytes, an event known to inhibit gap junction communication. The effects of macrophages on enterocyte gap junctions could be reversed by treatment of macrophages with the inducible nitric oxide synthase (iNOS) inhibitor l-Lysine ω-acetamidine hydrochloride (l-NIL) and by incubation with macrophages from iNOS−/−mice, implicating NO in the process. Activated macrophages also caused a NO-dependent redistribution of connexin43 in adjacent enterocytes from the cell surface to an intracellular location, further suggesting NO release may inhibit gap junction function. Treatment of enterocytes with the NO donor S-nitroso- N-acetylpenicillamine (SNAP) markedly inhibited gap junction communication as determined using single cell microinjection of the gap junction tracer Lucifer yellow. Strikingly, activated macrophages inhibited enterocyte migration into a scraped wound, which was reversed by l-NIL pretreatment. These results implicate enterocyte gap junctions as a target of the NO-mediated effects of macrophages during intestinal inflammation, particularly where enterocyte migration is impaired.
Differential effects of ASIC3 and TRPV1 deletion on gastroesophageal sensation in mice
Authors: Bielefeldt Klaus , Davis Brian M.
Published: November 2007

Using a recently developed in vitro preparation of vagal afferent pathways, we examined the role of TRPV1 and ASIC3 on the mechano- and chemosensitive properties of gastroesophageal sensory neurons. Esophagus, stomach, and the intact vagus nerves up to the central terminations were carefully dissected from TRPV1 and ASIC3 knockout mice and wild-type controls. The organ preparation was placed in a superfusion chamber to obtain intracellular recordings from the soma of nodose neurons during luminal stimulation of esophagus and stomach. The proximal esophagus and distal stomach were separately intubated to allow perfusion and graded luminal distension. In wild-type mice, mechanosensitive neurons were activated by low distension pressures and encoded stimulus intensity over the entire range tested. Luminal acidification significantly transiently increased the resting frequency but did not alter responses to subsequent mechanical stimulation. ASIC3 and TRPV1 knockout significantly blunted responses to distension compared with wild-type controls, with deletion of TRPV1 having a more significant effect than ASIC3 deletion. Luminal acidification did not activate mechanosensory neurons in ASIC3 and TRPV1 knockout mice. Our data demonstrate a role of TRPV1 in chemo- and mechanosensation of gastroesophageal afferents. ASIC3 may contribute to acid sensation but plays a more subtle role in responses to distending stimuli. Considering the importance of acid in dyspeptic symptoms and gastroesophageal reflux, TRPV1 or ASIC3 may be an attractive target for treatment strategies in patients who do not respond to acid suppressive therapy.
Endocannabinoids and Liver Disease. I. Endocannabinoids and their receptors in the liver
Authors: Mallat A. , Lotersztajn S.
Published: November 2007

Cannabinoid receptors (CB1 and CB2) and their endogenous ligands (endocannabinoids) have recently emerged as novel mediators of liver diseases. Endogenous activation of CB1 receptors promotes nonalcoholic fatty liver disease (NAFLD) and progression of liver fibrosis associated with chronic liver injury; in addition, CB1 receptors contribute to the pathogenesis of portal hypertension and cirrhotic cardiomyopathy. CB2 receptor-dependent effects are also increasingly characterized, including antifibrogenic effects and regulation of liver inflammation during ischemia-reperfusion and NAFLD. It is likely that the next few years will allow us to delineate whether molecules targeting CB1 and CB2 receptors are useful therapeutic agents for the treatment of chronic liver diseases.
Neural substrate for atrial fibrillation: implications for targeted parasympathetic blockade in the posterior left atrium
Authors: Arora Rishi , Ulphani Joseph S. , Villuendas Roger , Ng Jason , Harvey Laura , Thordson Sarah , Inderyas Firdous , Lu Yi , Gordon David , Denes Pablo , Greene Rodney , Crawford Susan , Decker Robert , Morris Alexander , Goldberger Jeffrey , Kadish Alan H.
Published: November 2007

The parasympathetic (P) nervous system is thought to contribute significantly to focal atrial fibrillation (AF). Thus we hypothesized that P nerve fibers [and related muscarinic (M2) receptors] are preferentially located in the posterior left atrium (PLA) and that selective cholinergic blockade in the PLA can be successfully performed to alter vagal AF substrate. The PLA, pulmonary veins (PVs), and left atrial appendage (LAA) from six dogs were immunostained for sympathetic (S) nerves, P nerves, and M2 receptors. Epicardial electrophysiological mapping was performed in seven additional dogs. The PLA was the most richly innervated, with nerve bundles containing P and S fibers (0.9 ± 1, 3.2 ± 2.5, and 0.17 ± 0.3/cm2 in the PV, PLA, and LAA, respectively, P < 0.001); nerve bundles were located in fibrofatty tissue as well as in surrounding myocardium. P fibers predominated over S fibers within bundles (P-to-S ratio = 4.4, 7.2, and 5.8 in PV, PLA, and LAA, respectively). M2 distribution was also most pronounced in the PLA (17.8 ± 8.3, 14.3 ± 7.3, and 14.5 ± 8 M2-stained cells/cm2 in the PLA, PV, and LAA, respectively, P = 0.012). Left cervical vagal stimulation (VS) caused significant effective refractory period shortening in all regions, with easily inducible AF. Topical application of 1% tropicamide to the PLA significantly attenuated VS-induced effective refractory period shortening in the PLA, PV, and LAA and decreased AF inducibility by 92% ( P < 0.001). We conclude that 1) P fibers and M2 receptors are preferentially located in the PLA, suggesting an important role for this region in creation of vagal AF substrate and 2) targeted P blockade in the PLA is feasible and results in attenuation of vagal responses in the entire left atrium and, consequently, a change in AF substrate.
Estimation of the total peripheral resistance baroreflex impulse response from spontaneous hemodynamic variability
Authors: Chen Xiaoxiao , Kim Jong-Kyung , Sala-Mercado Javier A. , Hammond Robert L. , Elahi Rafat I. , Scislo Tadeusz J. , Swamy Gokul , O'Leary Donal S. , Mukkamala Ramakrishna
Published: November 2007

We previously developed a mathematical analysis technique for estimating the static gain values of the arterial total peripheral resistance (TPR) baroreflex ( GA) and the cardiopulmonary TPR baroreflex ( GC) from small, spontaneous beat-to-beat fluctuations in arterial blood pressure, cardiac output, and stroke volume. Here, we extended the mathematical analysis so as to also estimate the entire arterial TPR baroreflex impulse response [ hA( t)] as well as the lumped arterial compliance (AC). The extended technique may therefore provide a linear dynamic characterization of TPR baroreflex systems during normal physiological conditions from potentially noninvasive measurements. We theoretically evaluated the technique with respect to realistic spontaneous hemodynamic variability generated by a cardiovascular simulator with known system properties. Our results showed that the technique reliably estimated hA( t) [error = 30.2 ± 2.6% for the square root of energy ( EA), 19.7 ± 1.6% for absolute peak amplitude ( PA), 37.3 ± 2.5% for GA, and 33.1 ± 4.9% for the overall time constant] and AC (error = 17.6 ± 4.2%) under various simulator parameter values and reliably tracked changes in GC. We also experimentally evaluated the technique with respect to spontaneous hemodynamic variability measured from seven conscious dogs before and after chronic arterial baroreceptor denervation. Our results showed that the technique correctly predicted the abolishment of hA( t) [ EA = 1.0 ± 0.2 to 0.3 ± 0.1, PA = 0.3 ± 0.1 to 0.1 ± 0.0 s−1, and GA = −2.1 ± 0.6 to 0.3 ± 0.2 ( P < 0.05)] and the enhancement of GC [−0.7 ± 0.44 to −1.8 ± 0.2 ( P < 0.05)] following the chronic intervention. Moreover, the technique yielded estimates whose values were consistent with those reported with more invasive and/or experimentally difficult methods.
Amiloride-sensitive NaCl taste responses are associated with genetic variation of ENaC α-subunit in mice
Authors: Shigemura Noriatsu , Ohkuri Tadahiro , Sadamitsu Chiharu , Yasumatsu Keiko , Yoshida Ryusuke , Beauchamp Gary K. , Bachmanov Alexander A. , Ninomiya Yuzo
Published: November 2007

An epithelial Na+ channel (ENaC) is expressed in taste cells and may be involved in the salt taste transduction. ENaC activity is blocked by amiloride, which in several mammalian species also inhibits taste responses to NaCl. In mice, lingual application of amiloride inhibits NaCl responses in the chorda tympani (CT) gustatory nerve much stronger in the C57BL/6 (B6) strain than in the 129P3/J (129) strain. We examined whether this strain difference is related to gene sequence variation or mRNA expression of three ENaC subunits (α, β, γ). Real-time RT-PCR and in situ hybridization detected no significant strain differences in expression of all three ENaC subunits in fungiform papillae. Sequences of the β- and γENaC subunit genes were also similar in the B6 and 129 strains, but αENaC gene had three single nucleotide polymorphisms (SNPs). One of these SNPs resulted in a substitution of arginine in the B6 strain to tryptophan in the 129 strain (R616W) in the αENaC protein. To examine association of this SNP with amiloride sensitivity of CT responses to NaCl, we produced F2 hybrids between B6 and 129 strains. Amiloride inhibited CT responses to NaCl in F2 hybrids with B6/129 and B6/B6 αENaC R616W genotypes stronger than in F2 hybrids with 129/129 genotype. This suggests that the R616W variation in the αENaC subunit affects amiloride sensitivity of the ENaC channel and provides evidence that ENaC is involved in amiloride-sensitive salt taste responses in mice.
The role of local and systemic renin angiotensin system activation in a genetic model of sympathetic hyperactivity-induced heart failure in mice
Authors: Ferreira J. C. B. , Bacurau A. V. , Evangelista F. S. , Coelho M. A. , Oliveira E. M. , Casarini D. E. , Krieger J. E. , Brum P. C.
Published: November 2007

Sympathetic hyperactivity (SH) and renin angiotensin system (RAS) activation are commonly associated with heart failure (HF), even though the relative contribution of these factors to the cardiac derangement is less understood. The role of SH on RAS components and its consequences for the HF were investigated in mice lacking α2Aand α2Cadrenoceptor knockout (α2A/α2CARKO) that present SH with evidence of HF by 7 mo of age. Cardiac and systemic RAS components and plasma norepinephrine (PN) levels were evaluated in male adult mice at 3 and 7 mo of age. In addition, cardiac morphometric analysis, collagen content, exercise tolerance, and hemodynamic assessments were made. At 3 mo, α2A/α2CARKO mice showed no signs of HF, while displaying elevated PN, activation of local and systemic RAS components, and increased cardiomyocyte width (16%) compared with wild-type mice (WT). In contrast, at 7 mo, α2A/α2CARKO mice presented clear signs of HF accompanied only by cardiac activation of angiotensinogen and ANG II levels and increased collagen content (twofold). Consistent with this local activation of RAS, 8 wk of ANG II AT1receptor blocker treatment restored cardiac structure and function comparable to the WT. Collectively, these data provide direct evidence that cardiac RAS activation plays a major role underlying the structural and functional abnormalities associated with a genetic SH-induced HF in mice.
Relative shortening velocity in locomotor muscles: turkey ankle extensors operate at low V/Vmax
Authors: Gabaldón Annette M. , Nelson Frank E. , Roberts Thomas J.
Published: November 2007

The force-velocity properties of skeletal muscle have an important influence on locomotor performance. All skeletal muscles produce less force the faster they shorten and typically develop maximal power at velocities of ∼30% of maximum shortening velocity (Vmax). We used direct measurements of muscle mechanical function in two ankle extensor muscles of wild turkeys to test the hypothesis that during level running muscles operate at velocities that favor force rather than power. Sonomicrometer measurements of muscle length, tendon strain-gauge measurements of muscle force, and bipolar electromyographs were taken as animals ran over a range of speeds and inclines. These measurements were integrated with previously measured values of muscle Vmaxfor these muscles to calculate relative shortening velocity (V/Vmax). At all speeds for level running the V/Vmaxvalues of the lateral gastrocnemius and the peroneus longus were low (<0.05), corresponding to the region of the force-velocity relationship where the muscles were capable of producing 90% of peak isometric force but only 35% of peak isotonic power. V/Vmaxincreased in response to the demand for mechanical power with increases in running incline and decreased to negative values to absorb energy during downhill running. Measurements of integrated electromyograph activity indicated that the volume of muscle required to produce a given force increased from level to uphill running. This observation is consistent with the idea that V/Vmaxis an important determinant of locomotor cost because it affects the volume of muscle that must be recruited to support body weight.
Deletion of one SERCA2 allele confers protection against bladder wall hypertrophy in a murine model of partial bladder outlet obstruction
Authors: Lassmann Jenny , Sliwoski Joanna , Chang Andy , Canning Douglas A. , Zderic Stephen A.
Published: November 2007

The sarco(endo)plasmic reticulum Ca2+-ATPase2 (SERCA2) is downregulated in cardiac hypertrophy with decompensation. We sought to determine whether mice heterozygous for the SERCA2 allele would develop greater bladder hypertrophy and decompensation than their wild-type littermates following partial bladder outlet obstruction (pBOO). We found that following 4 wk of surgically created pBOO, SERCA2 heterozygous murine bladders showed significantly less hypertrophy, improved in vitro cystometry performance, diminished expression of the slow myosin isoform A analyzed by RT-PCR, a significant drop in nuclear translocation of nuclear factor of activated T cells by EMSA, and decreased cell proliferation within the smooth muscle layer following 5-bromo-2′-deoxyuridine labeling compared with their wild-type littermates. Thus, in contrast to cardiac muscle, deletion of a SERCA2 allele confers protection against bladder hypertrophy in a murine model of pBOO. Compensatory mechanisms in heterozygous mice seem to be related to the calcineurin pathway. Further studies are underway to better define the molecular basis of this observation, which has potential clinical applications.
Role of transporters and ion channels in neuronal injury under hypoxia
Authors: Xue Jin , Zhou Dan , Yao Hang , Haddad Gabriel G.
Published: November 2007

The aims of the current study were to 1) examine the effects of hypoxia and acidosis on cultured cortical neurons and 2) explore the role of transporters and ion channels in hypoxic injury. Cell injury was measured in cultured neurons or hippocampal slices following hypoxia (1% O2) or acidosis (medium pH 6.8) treatment. Inhibitors of transporters and ion channels were employed to investigate their roles in hypoxic injury. Our results showed that 1) neuronal damage was apparent at 5–7 days of hypoxia exposure, i.e., 36–41% of total lactate dehydrogenase was released to medium and 2) acidosis alone did not lead to significant injury compared with nonacidic, normoxic controls. Pharmacological studies revealed 1) no significant difference in neuronal injury between controls (no inhibitor) and inhibition of Na+-K+-ATP pump, voltage-gated Na+channel, ATP-sensitive K+channel, or reverse mode of Na+/Ca2+exchanger under hypoxia; however, 2) inhibition of NBCs with 500 μM DIDS did not cause hypoxic death in either cultured cortical neurons or hippocampal slices; 3) in contrast, inhibition of Na+/H+exchanger isoform 1 (NHE1) with either 10 μM HOE-642 or 2 μM T-162559 resulted in dramatic hypoxic injury (+95% for HOE-642 and +100% for T-162559 relative to normoxic control, P < 0.001) on treatment day 3, when no death occurred for hypoxic controls (no inhibitor). No further damage was observed by NHE1 inhibition on treatment day 5. We conclude that inhibition of NHE1 accelerates hypoxia-induced neuronal damage. In contrast, DIDS rescues neuronal death under hypoxia. Hence, DIDS-sensitive mechanism may be a potential therapeutic target.
Cyclooxygenase mRNA expression in human patellar tendon at rest and after exercise
Authors: Trappe Todd A. , Carroll Chad C. , Jemiolo Bozena , Trappe Scott W. , Døssing Simon , Kjaer Michael , Magnusson S. Peter
Published: November 2007

Exercise has been shown to acutely elevate several metabolic processes in tendon tissue, including collagen turnover and blood flow, and chronically induce changes in tendon properties. Many of these acute metabolic responses to exercise are regulated by the cyclooxygenase (COX) enzymes. We measured the expression levels of COX-1 [variants 1 and 2 (COX-1v1 and COX-1v2)], COX-2, and the recently discovered intron 1-retaining COX-1 variants (COX-1b1, COX-1b2, and COX-1b3) at rest and after resistance exercise (RE). Patellar tendon biopsy samples were taken from six individuals (3 men and 3 women) before and 4 h after a bout of RE (3 sets of 10 repetitions at ∼70% of 1 repetition maximum) and from a separate group of six individuals (3 men and 3 women) before and 24 h after RE and analyzed by real-time RT-PCR. The COX-1 variants were the most abundant COX mRNAs before exercise and remained unchanged ( P > 0.05) after exercise. COX-2 was also expressed in tendon tissue at rest and was unchanged ( P > 0.05) after exercise. The intron 1-retaining COX-1 variants were not detectable in tendon tissue before or after exercise. COX-1 and COX-2 were expressed at much higher levels by the patellar tendon than by quadriceps skeletal muscle, although the overall COX mRNA expression patterns were similar in skeletal muscle and tendon (COX-1v2 > COX-1v1, P < 0.05; ratio of COX-1 to COX-2 ≅ 4:1). These results suggest that COX-1 and COX-2 are constitutively expressed at relatively high levels in human patellar tendon and are likely targets of COX-inhibiting drugs at rest and after physical activity.
Angiotensin II-stimulated Ca2+ entry mechanisms in afferent arterioles: role of transient receptor potential canonical channels and reverse Na+/Ca2+ exchange
Authors: Fellner Susan K. , Arendshorst William J.
Published: November 2007

In afferent arterioles, the signaling events that lead to an increase in cytosolic Ca2+ concentration ([Ca2+]i) and initiation of vascular contraction are increasingly being delineated. We have recently studied angiotensin II (ANG II)-mediated effects on sarcoplasmic reticulum (SR) mobilization of Ca2+ and the role of superoxide and cyclic adenosine diphosphoribose in these processes. In the current study we investigated the participation of transient receptor potential canonical channels (TRPC) and a Na+/Ca2+ exchanger (NCX) in Ca2+ entry mechanisms. Afferent arterioles, isolated with the magnetized polystyrene bead method, were loaded with fura-2 to measure [Ca2+]i ratiometrically. We observed that the Ca2+-dependent chloride channel blocker niflumic acid (10 and 50 μ M) affects neither the peak nor plateau [Ca2+]i response to ANG II. Arterioles were pretreated with ryanodine (100 μM) and TMB-8 to block SR mobilization via the ryanodine receptor and inositol trisphosphate receptor, respectively. The peak [Ca2+]i response to ANG II was reduced by 40%. Addition of 2-aminoethoxydiphenyl borane to block TRPC-mediated Ca2+ entry inhibited the peak [Ca2+]i ANG II response by 80% and the plateau by 74%. Flufenamic acid (FFA; 50 μM), which stimulates TRPC6, caused a sustained increase of [Ca2+]i of 146 nM. This response was unaffected by diltiazem or nifedipine. KB-R7943 (at the low concentration of 10 μM) inhibits reverse (but not forward) mode NCX. KB-R7943 decreased the peak [Ca2+]i response to ANG II by 48% and to FFA by 38%. We conclude that TRPC6 and reverse-mode NCX may be important Ca2+ entry pathways in afferent arterioles.
Homocysteine stimulates monocyte chemoattractant protein-1 expression in the kidney via nuclear factor-κB activation
Authors: Hwang Sun-Young , Woo Connie W. H. , Au-Yeung Kathy K. W. , Siow Yaw L. , Zhu Tong Y. , O Karmin
Published: November 2007

Hyperhomocysteinemia, or an elevation of blood homocysteine (Hcy) levels, is associated with cardiovascular disorders. Although kidney dysfunction is an important risk factor causing hyperhomocysteinemia, the direct effect of Hcy on the kidney is not well documented. There is a positive association between an elevation of blood Hcy levels and the development of chronic kidney disease. Inflammatory response such as increased chemokine expression has been implicated as one of the mechanisms for renal disease. Monocyte chemoattractant protein-1 (MCP-1) is a potent chemokine that is involved in the inflammatory response in renal disease. Nuclear factor-κB (NF-κB) plays an important role in upregulation of MCP-1 expression. We investigated the effect of hyperhomocysteinemia on MCP-1 expression and the molecular mechanism underling such an effect in rat kidneys as well as in proximal tubular cells. Hyperhomocysteinemia was induced in rats fed a high-methionine diet for 12 wk. The MCP-1 mRNA expression and MCP-1 protein levels were significantly increased in kidneys isolated from hyperhomocysteinemic rats. The NF-κB activity was significantly increased in the same kidneys. Pretreatment of hyperhomocysteinemic rats with a NF-κB inhibitor abolished hyperhomocysteinemia-induced MCP-1 expression in the kidney. To confirm the causative role of NF-κB activation in MCP-1 expression, human kidney proximal tubular cells were transfected with decoy NF-κB oligodeoxynucleotide to inhibit NF-κB activation. Such a treatment prevented Hcy-induced MCP-1 mRNA expression in tubular cells. Our results suggest that hyperhomocysteinemia stimulates MCP-1 expression in the kidney via NF-κB activation. Such an inflammatory response may contribute to renal injury associated with hyperhomocysteinemia.
Effect of thyroid hormone on the postnatal renal expression of NHE8
Authors: Gattineni Jyothsna , Sas David , Dagan Amit , Dwarakanath Vangipuram , Baum Michel
Published: November 2007

We previously demonstrated that there are developmental changes in proximal tubule Na+/H+ exchanger (NHE) activity. There is a maturational increase in postnatal brush-border membrane (BBM) vesicle NHE3 protein abundance and decrease in NHE8 protein abundance. The purpose of this study was to determine whether thyroid hormone plays a role in the rat renal maturational isoform switch from NHE8 to NHE3 and whether thyroid hormone regulates NHE8. Administration of thyroid hormone to neonatal rats, before the normal postnatal increase in serum thyroid hormone levels at 3 wk of age, resulted in a premature increase in NHE3/β-actin BBM protein abundance and mRNA abundance. Thyroid hormone also caused a premature decrease in BBM NHE8/β-actin protein abundance, whereas there was no change in mRNA expression (standardized to 28s). Rats made hypothyroid from birth were studied at 28 days, after the normal maturational increase in thyroid hormone. In these hypothyroid adult rats, the maturational increase in BBM NHE3 protein abundance and NHE3 mRNA expression was prevented. In contrast, the developmental decrease in BBM NHE8 protein abundance was prevented in hypothyroid adults, but mRNA expression was unchanged in hypothyroid rats. To determine whether the effect of thyroid hormone was due to a direct epithelial effect, we studied normal rat kidney cells in culture. We recently showed that this cell line expresses NHE8, but does not express NHE3. Thyroid hormone caused a decrease in surface expression of NHE8, determined by biotinylation, but total cellular abundance remained unchanged. NHE8 activity, measured as the sodium-dependent rate of intracellular pH recovery from an acid load, was less with thyroid treatment than control. In conclusion, thyroid hormone plays a potential role in the developmental isoform change from NHE8 to NHE3 and decreases NHE8 activity.
Comparison between voluntary and stimulated contractions of the quadriceps femoris for growth hormone response and muscle damage
Authors: Jubeau Marc , Sartorio Alessandro , Marinone Pier Giulio , Agosti Fiorenza , Hoecke Jacques Van , Nosaka Kazunori , Maffiuletti Nicola A.
Published: November 2007

This study aimed to compare voluntary and stimulated exercise for changes in muscle strength, growth hormone (GH), blood lactate, and markers of muscle damage. Nine healthy men had two leg press exercise bouts separated by 2 wk. In the first bout, the quadriceps muscles were stimulated by biphasic rectangular pulses (75 Hz, duration 400 μs, on-off ratio 6.25–20 s) with current amplitude being consistently increased throughout 40 contractions at maximal tolerable level. In the second bout, 40 voluntary isometric contractions were performed at the same leg press force output as the first bout. Maximal voluntary isometric strength was measured before and after the bouts, and serum GH and blood lactate concentrations were measured before, during, and after exercise. Serum creatine kinase (CK) activity and muscle soreness were assessed before, immediately after, and 24, 48, and 72 h after exercise. Maximal voluntary strength decreased significantly ( P < 0.05) after both bouts, but the magnitude of the decrease was significantly ( P < 0.05) greater for the stimulated contractions (−22%) compared with the voluntary contractions (−9%). Increases in serum GH and lactate concentrations were significantly ( P < 0.05) larger after the stimulation compared with the voluntary exercise. Increases in serum CK activity and muscle soreness were also significantly ( P < 0.05) greater for the stimulation than voluntary exercise. It was concluded that a single bout of electrical stimulation exercise resulted in greater GH response and muscle damage than voluntary exercise.
Exercise training delays cardiac dysfunction and prevents calcium handling abnormalities in sympathetic hyperactivity-induced heart failure mice
Authors: Medeiros Alessandra , Rolim Natale P. L. , Oliveira Rodrigo S. F. , Rosa Kaleizu T. , Mattos Katt C. , Casarini Dulce E. , Irigoyen Maria Claúdia , Krieger Eduardo M. , Krieger José Eduardo , Negrão Carlos Eduardo , Brum Patricia C.
Published: November 2007

Exercise training (ET) is a coadjuvant therapy in preventive cardiology. It delays cardiac dysfunction and exercise intolerance in heart failure (HF); however, the molecular mechanisms underlying its cardioprotection are poorly understood. We tested the hypothesis that ET would prevent Ca2+ handling abnormalities and ventricular dysfunction in sympathetic hyperactivity-induced HF mice. A cohort of male wild-type (WT) and congenic α2A/α2C-adrenoceptor knockout (α2A/α2CARKO) mice with C57BL6/J genetic background (3–5 mo of age) were randomly assigned into untrained and exercise-trained groups. ET consisted of 8-wk swimming session, 60 min, 5 days/wk. Fractional shortening (FS) was assessed by two-dimensional guided M-mode echocardiography. The protein expression of ryanodine receptor (RyR), phospho-Ser2809-RyR, sarcoplasmic reticulum Ca2+ ATPase (SERCA2), Na+/Ca2+ exchanger (NCX), phospholamban (PLN), phospho-Ser16-PLN, and phospho-Thr17-PLN were analyzed by Western blotting. At 3 mo of age, no significant difference in FS and exercise tolerance was observed between WT and α2A/α2CARKO mice. At 5 mo, when cardiac dysfunction is associated with lung edema and increased plasma norepinephrine levels, α2A/α2CARKO mice presented reduced FS paralleled by decreased SERCA2 (26%) and NCX (34%). Conversely, α2A/α2CARKO mice displayed increased phospho-Ser16-PLN (76%) and phospho-Ser2809-RyR (49%). ET in α2A/α2CARKO mice prevented exercise intolerance, ventricular dysfunction, and decreased plasma norepinephrine. ET significantly increased the expression of SERCA2 (58%) and phospho-Ser16-PLN (30%) while it restored the expression of phospho-Ser2809-RyR to WT levels. Collectively, we provide evidence that improved net balance of Ca2+ handling proteins paralleled by a decreased sympathetic activity on ET are, at least in part, compensatory mechanisms against deteriorating ventricular function in HF.
Sex differences in steroidogenesis in skeletal muscle following a single bout of exercise in rats
Authors: Aizawa Katsuji , Iemitsu Motoyuki , Otsuki Takeshi , Maeda Seiji , Miyauchi Takashi , Mesaki Noboru
Published: November 2007

Sex steroid hormones, such as testosterone and estradiol, play important roles in developing both strength and mass of skeletal muscle. Recently, we demonstrated that skeletal muscle can synthesize sex steroid hormones. Whether there are sex differences in basal steroidogenesis or acute exercise-induced alterations of steroidogenesis in the skeletal muscle is unknown. We examined sex differences in the levels of testosterone, estradiol, and steroidogenesis-related enzymes, such as 17β-hydroxysteroid dehydrogenase (HSD), 3β-HSD, and aromatase cytochrome P-450 (P450arom), in the skeletal muscle at rest and after exercise. We studied the gastrocnemius muscles of resting rats (10 wk old) and exercised rats (10 wk old, treadmill running, 30 m/min, 30 min). Basal muscular testosterone levels were higher in males than females, whereas estradiol did not differ between sexes. Additionally, 17β-HSD, 3β-HSD, and P450arom transcript and protein expression were greater in females. After acute exercise, testosterone levels and 17β-HSD expression increased in muscle in both sexes. By comparison, muscular estradiol levels increased in males following exercise but were unchanged in females. Expression of P450arom, which regulates estrogen synthesis, increased after acute exercise in males but decreased after exercise in females. Thus a single bout of exercise can influence the steroidogenic system in skeletal muscle, and these alterations differ between sexes. The acute exercise-induced alteration of steroidogenic enzymes may enhance the local steroidogenesis in the skeletal muscle in both sexes.
Shape Distortion Produced by Isolated Mismatch Between Vision and Proprioception
Authors: Malfait Nicole , Henriques Denise Y. , Gribble Paul L.
Published: November 2007

To investigate the nature of the visuomotor transformation, previous studies have used pointing tasks and examined how adaptation to a spatially localized mismatch between vision and proprioception generalizes across the workspace. Whereas some studies found extensive spatial generalization of single-point remapping, consistent with the hypothesis of a global realignment of visual and proprioceptive spaces, other studies reported limited transfer associated with variations in initial limb posture. Here, we investigated the effects of spatially localized remapping in the context of a visuomanual tracking task. Subjects tracked a visual target tracing a simple two-dimensional geometrical form without visual feedback except at a single point, where the visual display of the hand was shifted relative to its actual position. After adaptation, hand paths exhibited distortions relative to the visual templates that were inconsistent with the idea of a global realignment of visual and proprioceptive spaces. Results of a visuoproprioceptive matching task showed that these distortions were not limited to active movements but also affected perception of passive limb movements.
Rapid Consolidation of Motor Memory in the Vestibuloocular Reflex
Authors: Titley Heather K. , Heskin-Sweezie Raquel , Chung Ji-Yeon J. , Kassardjian Charles D. , Razik Fathima , Broussard Dianne M.
Published: November 2007

Motor memory is relatively labile immediately after learning but can become more stable through consolidation. We investigated consolidation of motor memory in the vestibuloocular reflex (VOR). Cats viewed the world through telescopic lenses during 60 min of passive rotation. Learned decreases (gain-down learning) and increases in the VOR gain (gain-up learning) were measured during sinusoidal rotation at 2 Hz. We found that if rotation in darkness immediately followed learning, the gain of the VOR reverted toward its prelearning value, indicating that expression of the memory was disrupted. If after gain-down learning the cat spent another 60 min stationary without form vision, subsequent disruption did not occur, suggesting that memory had consolidated. Consolidation was less robust for gain-up learning. We conclude that memory in the VOR is initially labile but consolidates rapidly and consistently after gain-down learning.
MUC1 Is a Counter-Receptor for Myelin-Associated Glycoprotein (Siglec-4a) and Their Interaction Contributes to Adhesion in Pancreatic Cancer Perineural Invasion
Authors: Swanson Benjamin J. , McDermott Kimberly M. , Singh Pankaj K. , Eggers John P. , Crocker Paul R. , Hollingsworth Michael A.
Published: November 2007

Perineural invasion in pancreatic adenocarcinoma, a common pathologic phenomenon whereby cancer cells invade and intimately contact the endoneurium of pancreatic nerves, is thought to contribute to both pain and local disease recurrence. MUC1, a type I transmembrane mucin that can affect the adhesive properties of cells, contains a large extracellular tandem repeat domain, which is heavily glycosylated in normal epithelia, but is overexpressed and differentially glycosylated in pancreatic cancer. This altered glycosylation includes the shortened core I O-glycans for monosialyl and disialyl T antigens. Myelin-associated glycoprotein (MAG), a membrane-bound protein expressed on oligodendrocytes and Schwann cells, binds myelin to neurons. MAG's preferred ligands are derivatives of the monosialyl and disialyl T antigen. We investigated whether MUC1 is a counter-receptor for MAG and if their interaction contributed to pancreatic perineural invasion. Results showed that MAG binds pancreatic cells expressing MUC1, that this binding is sialidase-sensitive, and that MAG physically associates with MUC1. Heterotypic adhesion assays between pancreatic cancer cells and Schwann cells revealed that increased expression of MUC1 or MAG enhanced adhesion. Conversely, specific inhibition of MAG or sialyl-T MUC1 partially blocked adhesion. Immunohistochemical analysis of pancreatic perineural invasion showed the expression of both MUC1 and MAG. These results support the hypothesis that the adhesive interactions between MUC1 and MAG are of biological significance in pancreatic cancer perineural invasion. [Cancer Res 2007;67(21):10222–9]
RbAp48 Regulates Cytoskeletal Organization and Morphology by Increasing K-Ras Activity and Signaling through Mitogen-Activated Protein Kinase
Authors: Scuto Anna , Zhang Hongling , Zhao Haiyan , Rivera Maria , Yeatman Timothy J. , Jove Richard , Torres-Roca Javier F.
Published: November 2007

RbAp48 is a WD-40 protein that plays an important role in chromatin metabolism and regulates Ras signaling. Here, we report that RbAp48 is involved in the regulation of cytoskeletal organization, a novel function. First, we show that transfection of RbAp48 into Hs-578T breast cancer cells (Hs-RbAp48-hi) leads to cell size reduction, a rounded cell shape, decreased cellular protrusions, and a higher nuclear/cytoplasmic ratio. Furthermore, we observed cytoskeletal F-actin organization disruption with loss of actin stress fibers and formation of membranous F-actin rings in Hs-RbAp48-hi cells. These morphologic changes were partially reversed by RbAp48 knockdown. Interestingly, mitogen-activated protein kinase (MAPK) was activated in Hs-RbAp48-hi cells, and this activity was also partly reversed by RbAp48 down-regulation. Furthermore, pharmacologic inhibition of MAPK led to the reappearance of organized actin fibers and focal contacts, suggesting MAPK as the effector pathway. Moreover, we show an increase in total Ras activity in Hs-RbAp48-hi cells with K-Ras-GTP becoming the dominant isoform. This reverted to baseline activity levels on RbAp48 small interfering RNA transfection, thus suggesting a direct role for RbAp48 in Ras regulation. Finally, we tested the model in transformed 3T3-K-Ras-G12V fibroblasts. As expected, RbAp48 knockdown in 3T3-K-Ras-hi fibroblasts resulted in reappearance of an organized cytoskeleton and shutdown of K-Ras activity. In conclusion, our data support a model whereby RbAp48 regulates cellular morphology and cytoskeletal organization by increasing K-Ras activity and signaling through MAPK. [Cancer Res 2007;67(20):10317–24]
AS1411 Alters the Localization of a Complex Containing Protein Arginine Methyltransferase 5 and Nucleolin
Authors: Teng Yun , Girvan Allicia C. , Casson Lavona K. , Pierce William M. , Qian Mingwei , Thomas Shelia D. , Bates Paula J.
Published: November 2007

AS1411 is a quadruplex-forming oligonucleotide aptamer that targets nucleolin. It is currently in clinical trials as a treatment for various cancers. We have proposed that AS1411 inhibits cancer cell proliferation by affecting the activities of certain nucleolin-containing complexes. Here, we report that protein arginine methyltransferase 5 (PRMT5), an enzyme that catalyzes the formation of symmetrical dimethylarginine (sDMA), is a nucleolin-associated protein whose localization and activity are altered by AS1411. Levels of PRMT5 were found to be decreased in the nucleus of AS1411-treated DU145 human prostate cancer cells, but increased in the cytoplasm. These changes were dependent on nucleolin and were not observed in cells pretreated with nucleolin-specific small interfering RNA. Treatment with AS1411 altered levels of PRMT5 activity (assessed by sDMA levels) in accord with changes in its localization. In addition, our data indicate that nucleolin itself is a substrate for PRMT5 and that distribution of sDMA-modified nucleolin is altered by AS1411. Because histone arginine methylation by PRMT5 causes transcriptional repression, we also examined expression of selected PRMT5 target genes in AS1411-treated cells. For some genes, including cyclin E2 and tumor suppressor ST7, a significant up-regulation was noted, which corresponded with decreased PRMT5 association with the gene promoter. We conclude that nucleolin is a novel binding partner and substrate for PRMT5, and that AS1411 causes relocalization of the nucleolin-PRMT5 complex from the nucleus to the cytoplasm. Consequently, the nuclear activity of PRMT5 is decreased, leading to derepression of some PRMT5 target genes, which may contribute to the biological effects of AS1411.
PTP1B Contributes to the Oncogenic Properties of Colon Cancer Cells through Src Activation
Authors: Zhu Shudong , Bjorge Jeffrey D. , Fujita Donald J.
Published: November 2007

Src-specific activity has been reported to be elevated in a high percentage of colon cancer cell lines and tumors, but the underlying mechanisms are largely unknown. In this study, we report that, in the seven cancer cell lines tested, Src-specific activity was elevated (5.2- to 18.7-fold) relative to normal colon cells (FHC). This activation of Src correlated with reduced phosphorylation at Y530 of Src, whereas there was no significant change in the level of phosphorylation at Y419. The membrane tyrosine phosphatase activity for a Src family-specific phosphopeptide substrate FCP (Fyn COOH-terminal peptide phosphorylated by Csk) was greatly increased in the cancer cells and was attributed to PTP1B in most of the cell lines. Membrane PTP1B protein levels were also greatly increased. Overexpression of PTP1B increased Src specific activity in colon cancer cells by reducing phosphorylation at Y530 of Src. It also increased anchorage-independent cell growth and this increase was blocked by the Src inhibitor PP2 and Src small interfering RNA (siRNA). Down-regulating PTP1B activity by PTP1B inhibitor CinnGEL 2Me or knocking down PTP1B using siRNA also reduced Src kinase activity and colony formation ability of colon cancer cells. PTP1B siRNA reduced tumor growth in nonobese diabetic/severe combined immunodeficient mice. This study suggests that (a) PTP1B can act as an important activator of Src in colon cancer cells via dephosphorylation at Y530 of Src and (b) elevated levels of PTP1B can increase tumorigenicity of colon cancer cells by activating Src. [Cancer Res 2007;67(21):10129–37]
p53 Determines Multidrug Sensitivity of Childhood Neuroblastoma
Authors: Xue Chengyuan , Haber Michelle , Flemming Claudia , Marshall Glenn M. , Lock Richard B. , MacKenzie Karen L. , Gurova Katerina V. , Norris Murray D. , Gudkov Andrei V.
Published: November 2007

For pediatric cancers like neuroblastoma, the most common extracranial solid tumor of infancy, p53 mutations are rare at diagnosis, but may be acquired after chemotherapy, suggesting a potential role in drug resistance. Heavy metal–selected neuroblastoma cells were found to acquire an unusually broad multidrug resistance (MDR) phenotype but displayed no alterations in genes associated with “classic” MDR. These cells had acquired a mutant p53 gene, linking p53 to drug sensitivity in neuroblastoma. We therefore generated p53-deficient variants in neuroblastoma cell lines with wild-type p53 by transduction of p53-suppressive constructs encoding either short hairpin RNA or a dominant-negative p53 mutant. Analysis of these cells indicated that (a) in contrast to previous reports, wild-type p53 was fully functional in all neuroblastoma lines tested; (b) inactivation of p53 in neuroblastoma cells resulted in establishment of a MDR phenotype; (c) p53-dependent senescence, the primary response of some neuroblastoma cells to DNA damage, is replaced after p53 inactivation by mitotic catastrophe and subsequent apoptosis; (d) knockdown of mutant p53 did not revert the MDR phenotype, suggesting it is determined by p53 inactivation rather than gain of mutant function. These results suggest the importance of p53 status as a prognostic marker of treatment response in neuroblastoma. p53 suppression may have opposite effects on drug sensitivity as determined by analysis of isogenic pairs of tumor cell lines of nonneuroblastoma origin, indicating the importance of tissue context for p53-mediated modulation of tumor cell sensitivity to treatment. [Cancer Res 2007;67(21):10351–60]
Protein Kinase C-α–Mediated Regulation of Low-Density Lipoprotein Receptor–Related Protein and Urokinase Increases Astrocytoma Invasion
Authors: Amos Samson , Mut Melike , diPierro Charles G. , Carpenter Joan E. , Xiao Aizhen , Kohutek Zachary A. , Redpath Gerard T. , Zhao Yunge , Wang Jiahu , Shaffrey Mark E. , Hussaini Isa M.
Published: November 2007

Aggressive and infiltrative invasion is one of the hallmarks of glioblastoma. Low-density lipoprotein receptor–related protein (LRP) is expressed by glioblastoma, but the role of this receptor in astrocytic tumor invasion remains poorly understood. We show that activation of protein kinase C-α (PKC-α) phosphorylated and down-regulated LRP expression. Pretreatment of tumor cells with PKC inhibitors, phosphoinositide 3-kinase (PI3K) inhibitor, PKC-α small interfering RNA (siRNA), and short hairpin RNA abrogated phorbol 12-myristate 13-acetate–induced down-regulation of LRP and inhibited astrocytic tumor invasion in vitro. In xenograft glioblastoma mouse model and in vitro transmembrane invasion assay, LRP-deficient cells, which secreted high levels of urokinase-type plasminogen activator (uPA), invaded extensively the surrounding normal brain tissue, whereas the LRP-overexpressing and uPA-deficient cells did not invade into the surrounding normal brain. siRNA, targeted against uPA in LRP-deficient clones, attenuated their invasive potential. Taken together, our results strongly suggest the involvement of PKC-α/PI3K signaling pathways in the regulation of LRP-mediated astrocytoma invasion. Thus, a strategy of combining small molecule inhibitors of PKC-α and PI3K could provide a new treatment paradigm for glioblastomas. [Cancer Res 2007;67(21):10241–51]
Interaction of the Tumor Metastasis Suppressor Nonmetastatic Protein 23 Homologue H1 and Estrogen Receptor α Alters Estrogen-Responsive Gene Expression
Authors: Curtis Carol D. , Likhite Varsha S. , McLeod Ian X. , Yates John R. , Nardulli Ann M.
Published: November 2007

Metastasis of cancer cells from the primary tumor is associated with poor prognosis and decreased overall survival. One protein implicated in inhibiting metastasis is the tumor metastasis suppressor nonmetastatic protein 23 homologue 1 (NM23-H1). NM23-H1 is a multifunctional protein, which, in addition to limiting metastasis, has DNase and histidine protein kinase activities. We have identified new functions for NM23-H1 in influencing estrogen receptor α (ERα)–mediated gene expression. Using a battery of molecular and biochemical techniques, we show that NM23-H1 interacts with ERα and increases the ERα–estrogen response element (ERE) interaction. When NM23-H1 expression is increased in U2 osteosarcoma and MDA-MB-231 breast cancer cells, transcription of a transiently transfected, estrogen-responsive reporter plasmid is decreased. More importantly, when endogenous NM23-H1 expression is knocked down in MCF-7 human breast cancer cells using small interfering RNA, estrogen responsiveness of the progesterone receptor (PR), Bcl-2, cathepsin D, and cyclin D1 genes, but not the pS2 gene, is enhanced. Furthermore, NM23-H1 associates with the region of the PR gene containing the +90 activator protein 1 site, but not with the ERE-containing region of the pS2 gene, indicating that NM23-H1 mediates gene-specific effects by association with endogenous chromatin. Our studies suggest that the capacity of NM23-H1 to limit the expression of estrogen-responsive genes such as cathepsin D and Bcl-2, which are involved in cell migration, apoptosis, and angiogenesis, may help to explain the metastasis-suppressive effects of this protein. The complementary abilities of ERα and NM23-H1 together to influence gene expression, cell migration, and apoptosis could be key factors in helping to determine tumor cell fate. [Cancer Res 2007;67(21):10600–7]