bluuebunny/crossref_metadata_2025 huggingface dataset:
α4β1-Integrin regulates directionally persistent cell migration in response to shear flow stimulation
Authors: Dikeman Dustin A. , Rivera Rosado Leslie A. , Horn Troy A. , Alves Christina S. , Konstantopoulos Konstantinos , Yang Joy T.
Published: May 2008

α4β1-Integrin plays a pivotal role in cell migration in vivo. This integrin has been shown to regulate the front-back polarity of migrating cells via localized inhibition of α4-integrin/paxillin binding by phosphorylation at the α4-integrin cytoplasmic tail. Here, we demonstrate that α4β1-integrin regulates directionally persistent cell migration via a more complex mechanism in which α4-integrin phosphorylation and paxillin binding act via both cooperative and independent pathways. We show that, in response to shear flow, α4β1-integrin binding to the CS-1 region of fibronectin was necessary and sufficient to promote directionally persistent cell migration when this integrin was ectopically expressed in CHO cells. Under shear flow, the α4β1-integrin-expressing cells formed a fan shape with broad lamellipodia at the front and retracted trailing edges at the back. This “fanning” activity was enhanced by disrupting paxillin binding alone and inhibited by disrupting phosphorylation alone or together with disrupting paxillin binding. Notably, the phosphorylation-disrupting mutation and the double mutation resulted in the formation of long trailing tails, suggesting that α4-integrin phosphorylation is required for trailing edge retraction/detachment independent of paxillin binding. Furthermore, the stable polarity and directional persistence of shear flow-stimulated cells were perturbed by the double mutation but not the single mutations alone, indicating that paxillin binding and α4-integrin phosphorylation can facilitate directionally persistent cell migration in an independent and compensatory manner. These findings provide a new insight into the mechanism by which integrins regulate directionally persistent cell migration.
Rho-family GTPases modulate Ca2+-dependent ATP release from astrocytes
Authors: Blum Andrew E. , Joseph Sheldon M. , Przybylski Ronald J. , Dubyak George R.
Published: May 2008

Previously, we reported that activation of G protein-coupled receptors (GPCR) in 1321N1 human astrocytoma cells elicits a rapid release of ATP that is partially dependent on a Gq/phophospholipase C (PLC)/Ca2+ mobilization signaling cascade. In this study we assessed the role of Rho-family GTPase signaling as an additional pathway for the regulation of ATP release in response to activation of protease-activated receptor-1 (PAR1), lysophosphatidic acid receptor (LPAR), and M3-muscarinic (M3R) GPCRs. Thrombin (or other PAR1 peptide agonists), LPA, and carbachol triggered quantitatively similar Ca2+ mobilization responses, but only thrombin and LPA caused rapid accumulation of active GTP-bound Rho. The ability to elicit Rho activation correlated with the markedly higher efficacy of thrombin and LPA, relative to carbachol, as ATP secretagogues. Clostridium difficile toxin B and Clostridium botulinum C3 exoenzyme, which inhibit Rho-GTPases, attenuated the thrombin- and LPA-stimulated ATP release but did not decrease carbachol-stimulated release. Thus the ability of certain Gq-coupled receptors to additionally stimulate Rho-GTPases acts to strongly potentiate a Ca2+-activated ATP release pathway. However, pharmacological inhibition of Rho kinase I/II or myosin light chain kinase did not attenuate ATP release. PAR1-induced ATP release was also reduced twofold by brefeldin treatment suggesting the possible mobilization of Golgi-derived, ATP-containing secretory vesicles. ATP release was also markedly repressed by the gap junction channel inhibitor carbenoxolone in the absence of any obvious thrombin-induced change in membrane permeability indicative of hemichannel gating.
The inhibitory effect of HKa in endothelial cell tube formation is mediated by disrupting the uPA-uPAR complex and inhibiting its signaling and internalization
Authors: Liu Yuchuan , Cao Dian J. , Sainz Irma M. , Guo Yan-Lin , Colman Robert W.
Published: May 2008

In two-dimensional (2-D) culture systems, we have previously shown that cleaved two-chain high-molecular-weight kininogen (HKa) or its domain 5 induced apoptosis by disrupting urokinase plasminogen activator (uPA) receptor (uPAR)-integrin signal complex formation. In the present study, we used a three-dimensional (3-D) collagen-fibrinogen culture system to monitor the effects of HKa on tube formation. In a 3-D system, HKa significantly inhibited tube and vacuole formation as low as 10 nM, which represents 1.5% of the physiological concentration of high-molecular-weigh kininogen (660 nM), without apparent apoptosis. However, HKa (300 nM) completely inhibited tube formation and increased apoptotic cells about 2-fold by 20–24 h of incubation. uPA-dependent ERK activation and uPAR internalization regulate cell survival and migration. In a 2-D system, we found that exogenous uPA-induced ERK phosphorylation and uPAR internalization were blocked by HKa. In a 3-D system, we found that not only uPA-uPAR association but also the activation of ERK were inhibited by HKa. HKa disrupts the uPA-uPAR complex, inhibiting the signaling pathways, and also inhibits uPAR internalization and regeneration to the cell surface, thereby interfering with uPAR-mediated cell migration, proliferation, and survival. Thus, our data suggest that the suppression of ERK activation and uPAR internalization by HKa contributes to the inhibition of tube formation. We conclude that in this 3-D collagen-fibrinogen gel, HKa modulates the multiple functions of uPAR in endothelial cell tube formation, a process that is closely related to in vivo angiogenesis.
Rho kinases regulate corneal epithelial wound healing
Authors: Yin Jia , Yu Fu-Shin X.
Published: May 2008

We have previously shown that Rho small GTPase is required for modulating both cell migration and proliferation through cytoskeleton reorganization and focal adhesion formation in response to wounding. In the present study, we investigated the role of Rho kinases (ROCKs), major effectors of Rho GTPase, in mediating corneal epithelial wound healing. Both ROCK 1 and 2 were expressed and activated in THCE cells, an SV40-immortalized human corneal epithelial cell (HCEC) line, in response to wounding, lysophosphatidic acid, and heparin-binding EGF-like growth factor (HB-EGF) stimulations. The ROCK inhibitor Y-27632 efficiently antagonized ROCK activities without affecting Rho activation in wounded HCECs. Y-27632 promoted basal and HB-EGF-enhanced scratch wound healing and enhanced cell migration and adhesion to matrices, while retarded HB-EGF induced cell proliferation. E-cadherin- and β-catenin-mediated cell-cell junction and actin cytoskeleton organization were disrupted by Y-27632. Y-27632 impaired the formation and maintenance of tight junction barriers indicated by decreased trans-epithelial resistance and disrupted occludin staining. We conclude that ROCK activities enhance cell proliferation, promote epithelial differentiation, but negatively modulate cell migration and cell adhesion and therefore play a role in regulating corneal epithelial wound healing.
Gastric distension attenuates the hypotensive effect of intraduodenal glucose in healthy older subjects
Authors: Gentilcore Diana , Meyer James H. , Rayner Christopher K. , Horowitz Michael , Jones Karen L.
Published: May 2008

Postprandial hypotension occurs frequently, and current management is suboptimal. Recent studies suggest that the magnitude of the fall in postprandial blood pressure (BP) may be attenuated by gastric distension. The aim of this study was to determine the effect of gastric distension on the hypotensive response to intraduodenal (ID) glucose. Eight healthy subjects (5 males, 3 females, aged 65–76 years) received an ID infusion of either 1) 50 g glucose in 300 ml saline (ID glucose) over 60 min ( t = 0–60 min), 2) 50 g glucose in 300 ml saline over 60 min and intragastric ( 4 ) infusion of 500 ml water between t = 7–10 min (IG water and ID glucose), or 3) ID saline (0.9%) infusion over 60 min and IG infusion of 500 ml water (IG water and ID saline) all followed by ID saline infusion for another 60 min ( t = 60–120 min) on three separate days. BP and heart rate (HR) were measured. Gastric emptying (GE) of the IG water was quantified by two-dimensional ultrasonography. Between t = 0–60 min, systolic and diastolic BP was greater ( P < 0.05 for both) with IG water and ID saline compared with IG water and ID glucose, and less ( P < 0.05 for both) with ID glucose compared with IG water and ID glucose. These effects were evident at relatively low IG volumes (∼300 ml). GE was faster with IG water and ID saline when compared with IG water and ID glucose. We conclude that, in healthy older subjects, IG administration of water markedly attenuates the hypotensive response to ID glucose, presumably as a result of gastric distension.
Impairment of vestibular-mediated cardiovascular response and motor coordination in rats born and reared under hypergravity
Authors: Abe Chikara , Tanaka Kunihiko , Awazu Chihiro , Morita Hironobu
Published: May 2008

It is well known that environmental stimulation is important for the proper development of sensory function. The vestibular system senses gravitational acceleration and then alters cardiovascular and motor functions through reflex pathways. The development of vestibular-mediated cardiovascular and motor functions may depend on the gravitational environment present at birth and during subsequent growth. To examine this hypothesis, arterial pressure (AP) and renal sympathetic nerve activity (RSNA) were monitored during horizontal linear acceleration and performance in a motor coordination task in rats born and reared in 1-G or 2-G environments. Linear acceleration of ±1 G increased AP and RSNA. These responses were attenuated in rats with a vestibular lesion, suggesting that the vestibular system mediated AP and RSNA responses. These responses were also attenuated in rats born in a 2-G environment. AP and RSNA responses were partially restored in these rats when the hypergravity load was removed, and the rats were maintained in a 1-G environment for 1 wk. The AP response to compressed air, which is mediated independently of the vestibular system, did not change in the 2-G environment. Motor coordination was also impaired in the 2-G environment and remained impaired even after 1 wk of unloading. These results indicate that hypergravity impaired both the vestibulo-cardiovascular reflex and motor coordination. The vestibulo-cardiovascular reflex was only impaired temporarily and partially recovered following 1 wk of unloading. In contrast, motor coordination did not return to normal in response to unloading.
Temporal asymmetries of short-term heart period variability are linked to autonomic regulation
Authors: Porta A. , Casali K. R. , Casali A. G. , Gnecchi-Ruscone T. , Tobaldini E. , Montano N. , Lange S. , Geue D. , Cysarz D. , Van Leeuwen P.
Published: May 2008

We exploit time reversibility analysis, checking the invariance of statistical features of a series after time reversal, to detect temporal asymmetries of short-term heart period variability series. Reversibility indexes were extracted from 22 healthy fetuses between 16th to 40th wk of gestation and from 17 healthy humans (aged 21 to 54, median = 28) during graded head-up tilt with table inclination angles randomly selected inside the set {15, 30, 45, 60, 75, 90}. Irreversibility analysis showed that nonlinear dynamics observed in short-term heart period variability are mostly due to asymmetric patterns characterized by bradycardic runs shorter than tachycardic ones. These temporal asymmetries were 1) more likely over short temporal scales than over longer, dominant ones; 2) more frequent during the late period of pregnancy (from 25th to 40th week of gestation); 3) significantly present in healthy humans at rest in supine position; 4) more numerous during 75 and 90° head-up tilt. Results suggest that asymmetric patterns observable in short-term heart period variability might be the result of a fully developed autonomic regulation and that an important shift of the sympathovagal balance toward sympathetic predominance (and vagal withdrawal) can increase their presence.
Adrenomedullin acts in the lateral parabrachial nucleus to increase arterial blood pressure through mechanisms mediated by glutamate and nitric oxide
Authors: Geambasu Adrian , Krukoff Teresa L.
Published: May 2008

Adrenomedullin (ADM) acts in a site-specific manner within autonomic centers of the brain to modulate mean arterial pressure (MAP). To determine the role of ADM in the pontine autonomic center, the lateral parabrachial nucleus (LPBN), we used urethane-anesthetized adult Sprague-Dawley male rats to test the hypothesis that ADM increases MAP at this site through glutamate- and nitric oxide (NO)-dependent mechanisms. ADM microinjected into the LPBN increased MAP in a dose-dependent manner. The pressor effect of ADM (0.01 pmol) had a peak value of 11.9 ± 1.9 mmHg at 2 min and lasted for 7 min. We demonstrated that ADM's effect is receptor mediated by blocking the effect with the ADM receptor antagonist, ADM22-52. We showed that glutamate mediates ADM's pressor response, as this response was blocked using coinjections of ADM with dizolcipine hydrogen maleate or 6-cyano-7-nitroquinoxaline-2,3-dione, N-methyl-d-aspartate (NMDA) and non-NMDA glutamate receptor antagonists, respectively. We tested the roles of NO with coinjections of ADM with either N5-(1-iminoethyl)-l-ornithine or 7-nitroindazole monosodium salt, nonspecific and neuronal NO synthase (NOS) inhibitors, respectively; both inhibitors blocked ADM's pressor effect. Finally, we studied the role of calcium influx in ADM's pressor effect, as intracellular calcium is important in both glutamate and NO neurotransmission. ADM's effect was blocked when nifedipine, an L-type calcium channel blocker, was coinjected with ADM into the LPBN. This study is the first to show that ADM acts in the LPBN to increase MAP through mechanisms dependent on activation of ionotropic glutamate receptors, neuronal and endothelial NOS-mediated NO synthesis, and L-type calcium channel activation.
Role of heme oxygenase-2 in pial arteriolar response to acetylcholine in mice with and without transfusion of cell-free hemoglobin polymers
Authors: Qin Xinyue , Kwansa Herman , Bucci Enrico , Doré Sylvain , Boehning Darren , Shugar David , Koehler Raymond C.
Published: May 2008

Carbon monoxide derived from heme oxygenase (HO) may participate in cerebrovascular regulation under specific circumstances. Previous work has shown that HO contributes to feline pial arteriolar dilation to acetylcholine after transfusion of a cell-free polymeric hemoglobin oxygen carrier. The role of constitutive HO2 in the pial arteriolar dilatory response to acetylcholine was determined by using 1) HO2-null mice (HO2−/−), 2) the HO inhibitor tin protoporphyrin IX (SnPPIX), and 3) 4,5,6,7-tetrabromobenzotriazole (TBB), an inhibitor of casein kinase-2 (CK2)-dependent phosphorylation of HO2. In anesthetized mice, superfusion of a cranial window with SnPPIX decreased arteriolar dilation produced by 10 μM acetylcholine by 51%. After partial polymeric hemoglobin exchange transfusion, the acetylcholine response was normal but was reduced 72% by SnPPIX and 95% by TBB. In HO2−/− mice, the acetylcholine response was modestly reduced by 14% compared with control mice and was unaffected by SnPPIX. After hemoglobin transfusion in HO2−/− mice, acetylcholine responses were also unaffected by SnPPIX and TBB. In contrast, nitric oxide synthase inhibition completely blocked the acetylcholine responses in hemoglobin-transfused HO2−/− mice. We conclude 1) that HO2 activity partially contributes to acetylcholine-induced pial arteriolar dilation in mice, 2) that this contribution is augmented in the presence of a plasma-based hemoglobin polymer and appears to depend on a CK2 kinase mechanism, 3) that nitric oxide synthase activity rather than HO1 activity contributes to the acetylcholine reactivity in HO2−/− mice, and 4) that plasma-based polymeric hemoglobin does not scavenge all of the nitric oxide generated by cerebrovascular acetylcholine stimulation.
Licking and gaping elicited by microstimulation of the nucleus of the solitary tract
Authors: Kinzeler Nicole R. , Travers Susan P.
Published: May 2008

Intraoral infusions of bitter tastants activate expression of the immediate-early gene c-Fos in neurons located in the medial third of the rostral nucleus of the solitary tract (rNST). The distribution of these neurons is distinct from that activated by sour or sweet stimuli. Bitter stimuli are also distinctive because of their potency for eliciting gaping, an oral reflex that functions to actively reject potentially toxic substances. Glossopharyngeal nerve transection profoundly reduces, whereas decerebration spares, the bitter-evoked Fos-like immunoreactivity (FLI) pattern and gaping, implicating the medial rNST as a substrate for the sensory limb of oral rejection. The present experiment tested this hypothesis using microstimulation (100 Hz, 0.2 ms, 5–40 μA) to activate the rNST in awake rats. NST microstimulation elicited licking and gaping, and gaping was evoked from a restricted rNST region. The results indicated some topographic organization in sites effective for evoking gaping, but, in direct conflict with the hypothesis, lateral sites farther from bitter-evoked FLI were more effective than medial sites centered closer to FLI-expressing neurons. The gape-effective sites resemble locations of bitter-responsive neurons recently observed in neurophysiological recordings. These results indicate that bitter-responsive rNST neurons critical for triggering gaping may not express FLI and imply an alternate function for bitter-responsive neurons that do.
Suppression of detrusor-sphincter dysynergia by GABA-receptor activation in the lumbosacral spinal cord in spinal cord-injured rats
Authors: Miyazato Minoru , Sasatomi Kurumi , Hiragata Shiro , Sugaya Kimio , Chancellor Michael B. , de Groat William C. , Yoshimura Naoki
Published: May 2008

We investigated the effects of intrathecal application of GABAA- or GABAB-receptor agonists on detrusor-sphincter dyssynergia (DSD) in spinal cord transection (SCT) rats. Adult female Sprague-Dawley rats were used. At 4 wk after Th9-10 SCT, simultaneous recordings of intravesical pressure and urethral pressure were performed under an awake condition to examine the effect of intrathecal application of GABAA and GABAB agonists (muscimol and baclofen, respectively) or GABAA and GABAB antagonists (bicuculline and saclofen, respectively) at the level of L6-S1 spinal cord. In spinal-intact rats, the effects of bicuculline and saclofen on bladder and urethral activity were also examined. During urethral pressure measurements, DSD characterized by urethral pressure increases during isovolumetric bladder contractions were observed in 95% of SCT rats. However, after intrathecal application of muscimol or baclofen, urethral pressure showed urethral relaxation during isovolumetric bladder contractions. The effective dose to induce inhibition of urethral activity was lower compared with the dose that inhibited bladder contractions. The effect of muscimol and baclofen was antagonized by intrathecal bicuculline and saclofen, respectively. In spinal-intact rats, intrathecal application of bicuculline induced DSD-like changes. These results indicate that GABAA- and GABAB-receptor activation in the spinal cord exerts the inhibitory effects on DSD after SCT. Decreased activation of GABAA receptors due to hypofunction of GABAergic mechanisms in the spinal cord might be responsible, at least in part, for the development of DSD after SCT.
Central infusion of aldosterone synthase inhibitor prevents sympathetic hyperactivity and hypertension by central Na+ in Wistar rats
Authors: Huang Bing S. , White Roselyn A. , Ahmad Monir , Jeng Arco Y. , Leenen Frans H. H.
Published: May 2008

In Wistar rats, increasing cerebrospinal fluid (CSF) Na+ concentration ([Na+]) by intracerebroventricular (ICV) infusion of hypertonic saline causes sympathetic hyperactivity and hypertension that can be prevented by blockade of brain mineralocorticoid receptors (MR). To assess the role of aldosterone produced locally in the brain in the activation of MR in the central nervous system (CNS), Wistar rats were infused ICV with artificial CSF (aCSF), Na+-rich (800 mmol/l) aCSF, aCSF plus the aldosterone synthase inhibitor FAD286 (100 μg·kg−1·day−1), or Na+-rich aCSF plus FAD286. After 2 wk of infusion, rats treated with Na+-rich aCSF exhibited significant increases in aldosterone and corticosterone content in the hypothalamus but not in the hippocampus, as well as increases in resting blood pressure (BP) and sympathoexcitatory responses to air stress, and impairment of arterial baroreflex function. Concomitant ICV infusion of FAD286 prevented the Na+-induced increase in hypothalamic aldosterone but not corticosterone and prevented most of the increases in resting BP and sympathoexcitatory and pressor responses to air stress and the baroreflex impairment. FAD286 had no effects in rats infused with ICV aCSF. In another set of rats, 24-h BP and heart rate were recorded via telemetry before and during a 14-day ICV infusion of Na+-rich aCSF with or without FAD286. Na+-rich aCSF without FAD286 caused sustained increases (∼10 mmHg) in resting mean arterial pressure that were absent in the rats treated with FAD286. These data suggest that in Wistar rats, an increase in CSF [Na+] may increase the biosynthesis of corticosterone and aldosterone in the hypothalamus, and mainly aldosterone activates MR in the CNS leading to sympathetic hyperactivity and hypertension.
Resistance to Ca2+-induced opening of the permeability transition pore differs in mitochondria from glycolytic and oxidative muscles
Authors: Picard Martin , Csukly Kristina , Robillard Marie-Eve , Godin Richard , Ascah Alexis , Bourcier-Lucas Céline , Burelle Yan
Published: May 2008

This study determined whether susceptibility to opening of the permeability transition pore (PTP) varies according to muscle phenotype represented by the slow oxidative soleus (Sol) and superficial white gastrocnemius (WG). Threshold for Ca2+-induced mitochondrial Ca2+ release following PTP opening was determined with a novel approach using permeabilized ghost myofibers. Threshold values for PTP opening were approximately threefold higher in fibers from WG compared with those from Sol (124 ± 47 vs. 30.4 ± 6.8 pmol Ca2+/mU citrate synthase). A similar phenomenon was also observed in isolated mitochondria (threshold: 121 ± 60 vs. 40 ± 10 nmol Ca2+/mg protein in WG and Sol), indicating that this was linked to differences in mitochondrial factors between the two muscles. The resistance of WG fibers to PTP opening was not related to the expression of putative protein modulators (cyclophilin D, adenylate nucleotide translocator-1, and voltage-dependent anion channels) or to difference in respiratory properties and occurred despite the fact that production of reactive oxygen species, which promote pore opening, was higher than in the Sol. However, endogenous matrix Ca2+ measured in mitochondria isolated under resting baseline conditions was approximately twofold lower in the WG than in the Sol (56 ± 4 vs. 111 ± 11 nmol/mg protein), which significantly accounted for the resistance of WG. Together, these results reveal fiber type differences in the sensitivity to Ca2+-induced PTP opening, which may constitute a physiological mechanism to adapt mitochondria to the differences in Ca2+ dynamics between fiber types.
Tamm-Horsfall protein protects the kidney from ischemic injury by decreasing inflammation and altering TLR4 expression
Authors: El-Achkar Tarek M. , Wu Xue-Ru , Rauchman Michael , McCracken Ruth , Kiefer Susan , Dagher Pierre C.
Published: May 2008

Tamm-Horsfall protein (THP) is a glycoprotein with unclear functions expressed exclusively in thick ascending limbs (TAL) of the kidney. Its role in ischemic acute kidney injury is uncertain, with previous data suggesting a possible negative effect by enhancing cast formation and promoting inflammation. Using a recently characterized THP knockout mouse (THP−/−), we investigated the role of THP in renal ischemia-reperfusion injury (IRI). In wild-type mice (THP+/+), THP expression was increased by injury. THP−/− mice developed more functional and histological renal damage after IRI compared with THP+/+. THP−/− kidneys showed more inflammation and tubular necrosis. Cast formation correlated with the severity of injury and was independent of THP presence. THP absence was associated with a more necrotic, rather than apoptotic, phenotype of cell death. The outer medulla was predominantly affected, where significant interstitial neutrophil infiltration was detected in proximity to injured S3 proximal tubular segments and TAL. This coincided with an enhanced expression of the innate immunity receptor Toll-like receptor 4 (TLR4) in S3 segments of THP−/− compared with THP+/+ mice. Specifically, a basolateral S3 expression of TLR4 was more evident in THP−/− kidneys compared with a more apical distribution in THP+/+. Such basolateral location for TLR4 allows a greater interaction with proinflammatory ligands present in the interstitium during ischemia. In conclusion, we are showing a completely novel role for a very old protein in the setting of renal injury. Our data suggest that THP stabilizes the outer medulla in the face of injury by decreasing inflammation, possibly through an effect on TLR4.
Phosphorylation of UT-A1 urea transporter at serines 486 and 499 is important for vasopressin-regulated activity and membrane accumulation
Authors: Blount Mitsi A. , Mistry Abinash C. , Fröhlich Otto , Price S. Russ , Chen Guangping , Sands Jeff M. , Klein Janet D.
Published: May 2008

The UT-A1 urea transporter plays an important role in the urine concentrating mechanism. Vasopressin (or cAMP) increases urea permeability in perfused terminal inner medullary collecting ducts and increases the abundance of phosphorylated UT-A1, suggesting regulation by phosphorylation. We performed a phosphopeptide analysis that strongly suggested that a PKA consensus site(s) in the central loop region of UT-A1 was/were phosphorylated. Serine 486 was most strongly identified, with other potential sites at serine 499 and threonine 524. Phosphomutation constructs of each residue were made and transiently transfected into LLC-PK1 cells to assay for UT-A1 phosphorylation. The basal level of UT-A1 phosphorylation was unaltered by mutation of these sites. We injected oocytes, assayed [14C]urea flux, and determined that mutation of these sites did not alter basal urea transport activity. Next, we tested the effect of stimulating cAMP production with forskolin. Forskolin increased wild-type UT-A1 and T524A phosphorylation in LLC-PK1 cells and increased urea flux in oocytes. In contrast, the S486A and S499A mutants demonstrated loss of forskolin-stimulated UT-A1 phosphorylation and reduced urea flux. In LLC-PK1 cells, we assessed biotinylated UT-A1. Wild-type UT-A1, S486A, and S499A accumulated in the membrane in response to forskolin. However, in the S486A/S499A double mutant, forskolin-stimulated UT-A1 membrane accumulation and urea flux were totally blocked. We conclude that the phosphorylation of UT-A1 on both serines 486 and 499 is important for activity and that this phosphorylation may be involved in UT-A1 membrane accumulation.
Isoforms of renal Na-K-2Cl cotransporter NKCC2: expression and functional significance
Authors: Castrop Hayo , Schnermann Jurgen
Published: May 2008

The renal Na-K-2Cl cotransporter (NKCC2, BSC1) is selectively expressed in the apical membrane of cells of the thick ascending limb of the loop of Henle (TAL) and macula densa. NKCC2-dependent salt transport constitutes the major apical entry pathway for transepithelial salt reabsorption in the TAL. Although NKCC2 is encoded by a single gene ( Slc12a1), differential splicing of the NKCC2 pre-mRNA results in the formation of several alternate transcripts. Thus three full-length splice isoforms of NKCC2 differ in their variable exon 4, resulting in transcripts for NKCC2B, NKCC2A, and NKCC2F. In addition to full-length isoforms, variants with truncated COOH-terminal ends have been described. The various splice isoforms of NKCC2 differ in their localization along the TAL and in their transport characteristics. Data in the literature are reviewed to assess the principles of NKCC2 differential splicing, the localization of NKCC2 splice isoforms along the TAL in various species, and the functional characteristics of the splice isoforms. In addition, we discuss the functional significance of NKCC2 isoforms for TAL salt retrieval and for the specific salt sensor function of macula densa cells based on studies using isoform-specific NKCC2-knockout mice. We suggest that different NKCC2 splice variants cooperate in salt retrieval along the TAL and that the coexpression of two splice variants (NKCC2B and NKCC2A) in the macula densa cells facilitates efficient salt sensing over wide ranges of fluctuating salt concentrations.
Stimulation of lymphocyte responses by angiotensin II promotes kidney injury in hypertension
Authors: Crowley Steven D. , Frey Campbell W. , Gould Samantha K. , Griffiths Robert , Ruiz Phillip , Burchette James L. , Howell David N. , Makhanova Natalia , Yan Ming , Kim Hyung-Suk , Tharaux Pierre-Louis , Coffman Thomas M.
Published: May 2008

Activation of the renin-angiotensin system contributes to the progression of chronic kidney disease. Based on the known cellular effects of ANG II to promote inflammation, we posited that stimulation of lymphocyte responses by ANG II might contribute to the pathogenesis of hypertensive kidney injury. We therefore examined the effects of the immunosuppressive agent mycophenolate mofetil (MMF) on the course of hypertension and kidney disease induced by chronic infusion of ANG II in 129/SvEv mice. Although it had no effect on the severity of hypertension or cardiac hypertrophy, treatment with MMF significantly reduced albuminuria and ameliorated kidney injury, decreasing glomerulosclerosis and reducing lymphocyte infiltration into the renal interstitium. Attenuation of renal pathology with MMF was associated with reduced expression of mRNAs for the proinflammatory cytokines interferon-γ and tumor necrosis factor-α and the profibrotic cytokine transforming growth factor-β. As infiltration of the kidney by T lymphocytes was a prominent feature of ANG II-dependent renal injury, we carried out experiments examining the effects of ANG II on lymphocytes in vitro. We find that exposure of splenic lymphocytes to ANG II causes prominent rearrangements of the actin cytoskeleton. These actions require the activity of Rho kinase. Thus, ANG II exaggerates hypertensive kidney injury by stimulating lymphocyte responses. These proinflammatory actions of ANG II seem to have a proclivity for inducing kidney injury while having negligible actions in the pathogenesis of cardiac hypertrophy.
Protein kinase B/Akt activity is involved in renal TGF-β1-driven epithelial-mesenchymal transition in vitro and in vivo
Authors: Kattla Jayesh J. , Carew Rosemarie M. , Heljić Mediha , Godson Catherine , Brazil Derek P.
Published: May 2008

The molecular pathogenesis of diabetic nephropathy (DN), the leading cause of end-stage renal disease worldwide, is complex and not fully understood. Transforming growth factor-β (TGF-β1) plays a critical role in many fibrotic disorders, including DN. In this study, we report protein kinase B (PKB/Akt) activation as a downstream event contributing to the pathophysiology of DN. We investigated the potential of PKB/Akt to mediate the profibrotic bioactions of TGF-β1 in kidney. Treatment of normal rat kidney epithelial cells (NRK52E) with TGF-β1 resulted in activation of phosphatidylinositol 3-kinase (PI3K) and PKB/Akt as evidenced by increased Ser473phosphorylation and GSK-3β phosphorylation. TGF-β1 also stimulated increased Smad3 phosphorylation in these cells, a response that was insensitive to inhibition of PI3K or PKB/Akt. NRK52E cells displayed a loss of zona occludins 1 and E-cadherin and a gain in vimentin and α-smooth muscle actin expression, consistent with the fibrotic actions of TGF-β1. These effects were blocked with inhibitors of PI3K and PKB/Akt. Furthermore, overexpression of PTEN, the lipid phosphatase regulator of PKB/Akt activation, inhibited TGF-β1-induced PKB/Akt activation. Interestingly, in the Goto-Kakizaki rat model of type 2 diabetes, we also detected increased phosphorylation of PKB/Akt and its downstream target, GSK-3β, in the tubules, relative to that in control Wistar rats. Elevated Smad3 phosphorylation was also detected in kidney extracts from Goto-Kakizaki rats with chronic diabetes. Together, these data suggest that TGF-β1-mediated PKB/Akt activation may be important in renal fibrosis during diabetic nephropathy.
Role of three-dimensional architecture in the urine concentrating mechanism of the rat renal inner medulla
Authors: Pannabecker Thomas L. , Dantzler William H. , Layton Harold E. , Layton Anita T.
Published: May 2008

Recent studies of three-dimensional architecture of rat renal inner medulla (IM) and expression of membrane proteins associated with fluid and solute transport in nephrons and vasculature have revealed structural and transport properties that likely impact the IM urine concentrating mechanism. These studies have shown that 1) IM descending thin limbs (DTLs) have at least two or three functionally distinct subsegments; 2) most ascending thin limbs (ATLs) and about half the ascending vasa recta (AVR) are arranged among clusters of collecting ducts (CDs), which form the organizing motif through the first 3–3.5 mm of the IM, whereas other ATLs and AVR, along with aquaporin-1-positive DTLs and urea transporter B-positive descending vasa recta (DVR), are external to the CD clusters; 3) ATLs, AVR, CDs, and interstitial cells delimit interstitial microdomains within the CD clusters; and 4) many of the longest loops of Henle form bends that include subsegments that run transversely along CDs that lie in the terminal 500 μm of the papilla tip. Based on a more comprehensive understanding of three-dimensional IM architecture, we distinguish two distinct countercurrent systems in the first 3–3.5 mm of the IM (an intra-CD cluster system and an inter-CD cluster system) and a third countercurrent system in the final 1.5–2 mm. Spatial arrangements of loop of Henle subsegments and multiple countercurrent systems throughout four distinct axial IM zones, as well as our initial mathematical model, are consistent with a solute-separation, solute-mixing mechanism for concentrating urine in the IM.
Muscle phosphocreatine kinetics in children and adults at the onset and offset of moderate-intensity exercise
Authors: Barker Alan R. , Welsman Joanne R. , Fulford Jonathan , Welford Deborah , Armstrong Neil
Published: May 2008

The splitting of muscle phosphocreatine (PCr) plays an integral role in the regulation of muscle O2 utilization during a “step” change in metabolic rate. This study tested the hypothesis that the kinetics of muscle PCr would be faster in children compared with adults both at the onset and offset of moderate-intensity exercise, in concert with the previous demonstration of faster phase II pulmonary O2 uptake kinetics in children. Eighteen peri-pubertal children (8 boys, 10 girls) and 16 adults (8 men, 8 women) completed repeated constant work-rate exercise transitions corresponding to 80% of the Pi/PCr intracellular threshold. The changes in quadriceps [PCr], [Pi], [ADP], and pH were determined every 6 s using 31P-magnetic resonance spectroscopy. No significant ( P > 0.05) age- or sex-related differences were found in the PCr kinetic time constant at the onset (boys, 21 ± 4 s; girls, 24 ± 5 s; men, 26 ± 9 s; women, 24 ± 7 s) or offset (boys, 26 ± 5 s; girls, 29 ± 7 s; men, 23 ± 9 s; women 29 ± 7 s) of exercise. Likewise, the estimated theoretical maximal rate of oxidative phosphorylation (Qmax) was independent of age and sex (boys, 1.39 ± 0.20 mM/s; girls, 1.32 ± 0.32 mM/s; men, 2.36 ± 1.18 mM/s; women, 1.51 ± 0.53 mM/s). These results are consistent with the notion that the putative phosphate-linked regulation of muscle O2 utilization is fully mature in peri-pubertal children, which may be attributable to a comparable capacity for mitochondrial oxidative phosphorylation in child and adult muscle.
Shape Selectivity in Primate Frontal Eye Field
Authors: Peng Xinmiao , Sereno Margaret E. , Silva Amanda K. , Lehky Sidney R. , Sereno Anne B.
Published: May 2008

Previous neurophysiological studies of the frontal eye field (FEF) in monkeys have focused on its role in saccade target selection and gaze shift control. It has been argued that FEF neurons indicate the locations of behaviorally significant visual stimuli and are not inherently sensitive to specific features of the visual stimuli per se. Here, for the first time, we directly examined single cell responses to simple, two-dimensional shapes and found that shape selectivity exists in a substantial number of FEF cells during a passive fixation task or during the sample, delay (memory), and eye movement periods in a delayed match to sample (DMTS) task. Our data demonstrate that FEF neurons show sensory and mnemonic selectivity for stimulus shape features whether or not they are behaviorally significant for the task at hand. We also investigated the extent and localization of activation in the FEF using a variety of shape stimuli defined by static or dynamic cues employing functional magentic resonance imaging (fMRI) in anesthetized and paralyzed monkeys. Our fMRI results support the electrophysiological findings by showing significant FEF activation for a variety of shape stimuli and cues in the absence of attentional and motor processing. This shape selectivity in FEF is comparable to previous reports in the ventral pathway, inviting a reconsideration of the functional organization of the visual system.
Dynamic Effects on the Subjective Visual Vertical After Roll Rotation
Authors: Lorincz Erika N. , Hess Bernhard J. M.
Published: May 2008

We investigated in normal human subjects how semicircular canal and otolith signals interact in the estimation of the subjective visual vertical after constant velocity or constant acceleration roll tilt. In the constant velocity paradigm, subjects were rotated in darkness at ±60°/s for five complete cycles before being stopped in one of seven orientations ranging from 0 to ±90° (right/left ear down). In the constant acceleration paradigm, subjects were rotated with an acceleration of +30 or −30°/s2 to the same seven end positions between −90 and +90°, by way of passing once through the upside-down position. The subjective visual vertical was assessed by measuring the setting of a luminous line that appeared at different test delays after stop rotation in otherwise complete darkness. The data suggest that gravitational jerk signals generated by otolith–semicircular canal interactions and/or carried by phasic otolith signals are responsible for the observed transient bias in the estimation of the subjective visual vertical. This transient bias depended on both rotation and tilt direction after constant velocity rotations, but was almost abolished following constant acceleration rotations.
Perceptual Load Modulates Visual Cortex Excitability to Magnetic Stimulation
Authors: Muggleton Neil , Lamb Ruth , Walsh Vincent , Lavie Nilli
Published: May 2008

Much recent research has shown that the level of perceptual load in a task determines the perception of task-irrelevant stimuli and associated neural activity, but the mediating neural mechanisms remain unclear. Here we show that increasing the level of perceptual load in a static letter search task results in an increase in the intensity of transcranial magnetic stimulation over V5/MT required to elicit the perception of a moving phosphene. These findings suggest that the neural mechanisms mediating the effects of perceptual load involve reduced visual cortex excitability in task-unrelated areas.
Identification and Clustering of Event Patterns From In Vivo Multiphoton Optical Recordings of Neuronal Ensembles
Authors: Ozden Ilker , Lee H. Megan , Sullivan Megan R. , Wang Samuel S.-H.
Published: May 2008

In vivo multiphoton fluorescence microscopy allows imaging of cellular structures in brain tissue to depths of hundreds of micrometers and, when combined with the use of activity-dependent indicator dyes, opens the possibility of observing intact, functioning neural circuitry. We have developed tools for analyzing in vivo multiphoton data sets to identify responding structures and events in single cells as well as patterns of activity within the neural ensemble. Data were analyzed from populations of cerebellar Purkinje cell dendrites, which generate calcium-based complex action potentials. For image segmentation, active dendrites were identified using a correlation-based method to group covarying pixels. Firing events were extracted from dendritic fluorescence signals with a 95% detection rate and an 8% false-positive rate. Because an event that begins in one movie frame is sometimes not detected until the next frame, detection delays were compensated using a likelihood-based correction procedure. To identify groups of dendrites that tended to fire synchronously, a k-means-based procedure was developed to analyze pairwise correlations across the population. Because repeated runs of k-means often generated dissimilar clusterings, the runs were combined to determine a consensus cluster number and composition. This procedure, termed meta- k-means, gave clusterings as good as individual runs of k-means, was independent of random initial seeding, and allowed the exclusion of outliers. Our methods should be generally useful for analyzing multicellular activity recordings in a variety of brain structures.
Synaptic Regulation of the Light-Dependent Oscillatory Currents in Starburst Amacrine Cells of the Mouse Retina
Authors: Petit-Jacques Jerome , Bloomfield Stewart A.
Published: May 2008

Responses of on-center starburst amacrine cells to steady light stimuli were recorded in the dark-adapted mouse retina. The response to spots of dim white light appear to show two components, an initial peak that correspond to the onset of the light stimulus and a series of oscillations that ride on top of the initial peak relaxation. The frequency of oscillations during light stimulation was three time higher than the frequency of spontaneous oscillations recorded in the dark. The light-evoked responses in starburst cells were exclusively dependent on the release of glutamate likely from presynaptic bipolar axon terminals and the binding of glutamate to AMPA/kainate receptors because they were blocked by 6-cyano-7-nitroquinoxalene-2,3-dione. The synaptic pathway responsible for the light responses was blocked by AP4, an agonist of metabotropic glutamate receptors that hyperpolarize on-center bipolar cells on activation. Light responses were inhibited by the calcium channel blockers cadmium ions and nifedipine, suggesting that the release of glutamate was calcium dependent. The oscillatory component of the response was specifically inhibited by blocking the glutamate transporter with d-threo-β-benzyloxyaspartic acid, suggesting that glutamate reuptake is necessary for the oscillatory release. GABAergic antagonists bicuculline, SR 95531, and picrotoxin increased the amplitude of the initial peak while they inhibit the frequency of oscillations. TTX had a similar effect. Strychnine, the blocker of glycine receptors did not affect the initial peak but strongly decreased the oscillations frequency. These inhibitory inputs onto the bipolar axon terminals shape and synchronize the oscillatory component.
Recruitment of a Head-Turning Synergy by Low-Frequency Activity in the Primate Superior Colliculus
Authors: Rezvani Sam , Corneil Brian D.
Published: May 2008

Low-frequency activity within the oculomotor system helps bridge sensation and action. Given ocular stability, low-frequency activity sustained by some neurons within the intermediate and deep superior colliculus (dSC) is assumed to be separated from motor output. However, the dSC is an orienting structure and the influence of low-frequency dSC activity at other effectors remains untested. We studied this by simultaneously recording activity from saccade-related dSC neurons and electromyographic (EMG) activity from neck muscles that turn the head. Monkeys performed a gap-saccade paradigm with varying levels of reward expectancy. Despite head restraint and even for relatively small target eccentricities (≤10°), increasing reward expectancy for a given target increased the level of low-frequency activity on dSC neurons encoding saccades to the rewarded target and increased the recruitment of a neck muscle synergy that would turn the head toward the target. The magnitude of neck muscle recruitment correlated positively on a trial-by-trial basis with the level of low-frequency dSC activity, and such correlations were optimized when neck muscle activity was shifted about 20 ms later to account for delays in the tecto-reticulo-spinal pathway. Further, dSC activity discriminated about the side of target presentation approximately 11 ms earlier than neck EMG activity. Considered alongside neck EMG responses evoked causally by SC stimulation, our results are consistent with low-frequency dSC activity recruiting a head-turning synergy. Our results support a brain stem circuit wherein the magnitude of neck muscle recruitment reflects the difference in comparative low-frequency activation across both dSCs, perhaps because of mutually inhibitory interactions within downstream head premotor circuits.
The “Diagonal Effect”: a Systematic Error in Oblique Antisaccades
Authors: Koehn John D. , Roy Elizabeth , Barton Jason J. S.
Published: May 2008

Antisaccades are known to show greater variable error and also a systematic hypometria in their amplitude compared with visually guided prosaccades. In this study, we examined whether their accuracy in direction (as opposed to amplitude) also showed a systematic error. We had human subjects perform prosaccades and antisaccades to goals located at a variety of polar angles. In the first experiment, subjects made prosaccades or antisaccades to one of eight equidistant locations in each block, whereas in the second, they made saccades to one of two equidistant locations per block. In the third, they made antisaccades to one of two locations at different distances but with the same polar angle in each block. Regardless of block design, the results consistently showed a saccadic systematic error, in that oblique antisaccades (but not prosaccades) requiring unequal vertical and horizontal vector components were deviated toward the 45° diagonal meridians. This finding could not be attributed to range effects in either Cartesian or polar coordinates. A perceptual origin of the diagonal effect is suggested by similar systematic errors in other studies of memory-guided manual reaching or perceptual estimation of direction, and may indicate a common spatial bias when there is uncertain information about spatial location.