bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of endogenous TRP1 decreases capacitative Ca2+entry and attenuates pulmonary artery smooth muscle cell proliferation
Authors: Sweeney Michele , Yu Ying , Platoshyn Oleksandr , Zhang Shen , McDaniel Sharon S. , Yuan Jason X.-J.
Published: March 2015

Pulmonary vascular medial hypertrophy due to proliferation of pulmonary artery smooth muscle cells (PASMC) greatly contributes to the increased pulmonary vascular resistance in pulmonary hypertension patients. A rise in cytosolic free Ca2+concentration ([Ca2+]cyt) is an important stimulus for cell growth in PASMC. Resting [Ca2+]cyt, intracellularly stored [Ca2+], capacitative Ca2+entry (CCE), and store-operated Ca2+currents ( ISOC) are greater in proliferating human PASMC than in growth-arrested cells. Expression of TRP1, a transient receptor potential gene proposed to encode the channels responsible for CCE and ISOC, was also upregulated in proliferating PASMC. Our aim was to determine if inhibition of endogenous TRP1 gene expression affects ISOCand CCE and regulates cell proliferation in human PASMC. Cells were treated with an antisense oligonucleotide (AS, for 24 h) specifically designed to cleave TRP1 mRNA and then returned to normal growth medium for 40 h before the experiments. Then, mRNA and protein expression of TRP1 was downregulated, and amplitudes of ISOCand CCE elicited by passive depletion of Ca2+from the sarcoplasmic reticulum using cyclopiazonic acid were significantly reduced in the AS-treated PASMC compared with control. Furthermore, the rate of cell growth was decreased by 50% in AS-treated PASMC. These results indicate that TRP1 may encode a store-operated Ca2+channel that plays a critical role in PASMC proliferation by regulating CCE and intracellular [Ca2+]cyt.
Phosphodiesterase type 4 inhibitor reduces the retention of polymorphonuclear leukocytes in the lung
Authors: Sato Yukio , Sato Shyoko , Yamamoto Tatsuo , Ishikawa Shigemi , Onizuka Masataka , Sakakibara Yuzuru
Published: March 2015

Phosphodiesterase (PDE) type 4 is the predominant PDE isozyme in polymorphonuclear leukocytes (PMN) and plays a key role in the regulation of PMN activation. The aim of this study was to examine the effect of a PDE type 4 inhibitor, rolipram, on the functional changes and the retention of PMN in the lung. In vitro, F-actin content and L-selectin and CD11b expression of PMN stimulated by N-formyl-Met-Leu-Phe were measured by flow cytometry. PMN deformability was evaluated using silicon microchannels. Rolipram reduced the increase of F-actin and CD11b but did not change the decrease of L-selectin. Rolipram inhibited the increase of the transit time of PMN through the microchannel. We evaluated the retention of PMN in the lung in vivo by infusing labeled blood into the vena cava and examining the recovery into aortic root samples in rabbits. Rolipram inhibited the retention of stimulated PMN in the lung. In conclusion, a PDE type 4 inhibitor, rolipram, reduces the retention of PMN in the lung by reducing deformability change and CD11b upregulation of PMN.
Alkalosis stimulates endothelial nitric oxide synthase in cultured human pulmonary arterial endothelial cells
Authors: Mizuno Shiro , Demura Yoshiki , Ameshima Shingo , Okamura Seitaro , Miyamori Isamu , Ishizaki Takeshi
Published: March 2015

To investigate the effect of extracellular pH on endothelial nitric oxide synthase (eNOS) in human pulmonary arteries, we measured eNOS activity and expression as well as some ion channels in human pulmonary arterial endothelial cells (HPAEC) exposed to various pH levels (6.6–8.0). eNOS activity was found to increase with alkalization and decrease with acidification, while Ca2+ uptake into HPAEC increased with alkalization. The addition of 3′,4′-dichlorobenzamil hydrochloride, an inhibitor of the Na+/Ca2+ exchanger (NCX), prevented the increase of eNOS activity with alkalosis. Exposure to alkalosis and acidosis increased eNOS and NCX mRNA levels. These results suggest that an elevation of extracellular pH activates eNOS via the influx of extracellular Ca2+ and that NCX also regulates eNOS activity during alkalosis. Furthermore, NCX may have a tight interaction with eNOS at the level of transcription and might affect pulmonary circulation during alkalosis and acidosis.
Enhanced mtDNA repair capacity protects pulmonary artery endothelial cells from oxidant-mediated death
Authors: Dobson Allison W. , Grishko Valentina , LeDoux Susan P. , Kelley Mark R. , Wilson Glenn L. , Gillespie Mark N.
Published: March 2015

In rat cultured pulmonary arterial (PA), microvascular, and venous endothelial cells (ECs), the rate of mitochondrial (mt) DNA repair is predictive of the severity of xanthine oxidase (XO)-induced mtDNA damage and the sensitivity to XO-mediated cell death. To examine the importance of mtDNA damage and repair more directly, we determined the impact of mitochondrial overexpression of the DNA repair enzyme, Ogg1, on XO-induced mtDNA damage and cell death in PAECs. PAECs were transiently transfected with an Ogg1-mitochondrial targeting sequence construct. Mitochondria-selective overexpression of the transgene product was confirmed microscopically by the observation that immunoreactive Ogg1 colocalized with a mitochondria-specific tracer and, with an oligonucleotide cleavage assay, by a selective enhancement of mitochondrial Ogg1 activity. Overexpression of Ogg1 protected against both XO-induced mtDNA damage, determined by quantitative Southern analysis, and cell death as assessed by trypan blue exclusion and MTS assays. These findings show that mtDNA damage is a direct cause of cell death in XO-treated PAECs.
Hypoxia increases AP-1 binding activity by enhancing capacitative Ca2+entry in human pulmonary artery endothelial cells
Authors: Fantozzi Ivana , Zhang Shen , Platoshyn Oleksandr , Remillard Carmelle V. , Cowling Randy T. , Yuan Jason X.-J.
Published: March 2015

Activating protein (AP)-1 transcription factors modulate expression of genes involved in cell proliferation and migration. Chronic hypoxia increases pulmonary artery smooth muscle cell proliferation by upregulating AP-1-responsive genes encoding for endothelium-derived vasoactive and mitogenic factors implicated in pulmonary hypertension development. The expression of AP-1 transcription factors is sensitive to changes in cytosolic free [Ca2+] ([Ca2+]cyt). Capacitative Ca2+entry (CCE) via store-operated Ca2+channels (SOC) is an important mechanism for raising [Ca2+]cytin pulmonary artery endothelial cells (PAEC). Using combined molecular biological, fluorescence microscopy, and biophysical approaches, we examined the effect of chronic hypoxia (3% O2, 72 h) on AP-1 DNA binding activity, CCE, and transient receptor potential (TRP) gene expression in human (h) PAEC. EMSA showed that AP-1 binding to hPAEC nuclear protein extracts was significantly enhanced by hypoxia, the increase being dependent on store-operated Ca2+influx and sensitive to La3+, an SOC inhibitor. Hypoxia also increased basal [Ca2+]cyt, the amount of CCE produced by store depletion with cyclopiazonic acid, and the amplitude of SOC-mediated currents ( ISOC). The increases of CCE amplitude and ISOCcurrent density by hypoxia were paralleled by enhanced TRPC4 mRNA and protein expression. Hypoxia-enhanced CCE and TRPC4 expression were also attenuated by La3+. These data suggest that hypoxia increases AP-1 binding activity by enhancing Ca2+influx via La3+-sensitive TRP-encoded SOC channels in hPAEC. The Ca2+-mediated increase in AP-1 binding may play an important role in upregulating AP-1-responsive gene expression, in stimulating pulmonary vascular cell proliferation and, ultimately, in pulmonary vascular remodeling in patients with hypoxia-mediated pulmonary hypertension.
Wild-type levels of the mouseForkhead Box f1gene are essential for lung repair
Authors: Kalinichenko Vladimir V. , Zhou Yan , Shin Brian , Stolz Donna Beer , Watkins Simon C. , Whitsett Jeffrey A. , Costa Robert H.
Published: March 2015

The Forkhead Box (Fox) family of transcription factors plays important roles in regulating expression of genes involved in cellular proliferation and differentiation. In a previous study, we showed that newborn foxf1(+/−) mice with diminished Foxf1 levels exhibited abnormal formation of pulmonary alveoli and capillaries and died postnatally. Interestingly, surviving newborn foxf1(+/−) mice exhibited increased pulmonary Foxf1 levels and normal adult lung morphology, suggesting that wild-type Foxf1 levels are required for lung development and function. The present study was conducted to determine whether adult foxf1(+/−) mice were able to undergo lung repair similar to that observed in wild-type mice. We demonstrated that adult foxf1(+/−) mice died from severe lung hemorrhage after butylated hydroxytoluene (BHT) lung injury and that this phenotype was associated with a 10-fold decrease in pulmonary Foxf1 expression and increased alveolar endothelial cell apoptosis that disrupted capillary integrity. Furthermore, BHT-induced lung hemorrhage of adult foxf1(+/−) mice was associated with a drastic reduction in expression of the Flk-1, bone morphogenetic protein-4, surfactant protein B, platelet endothelial cell adhesion molecule, and vascular endothelial cadherin genes, whereas the expression of these genes was either transiently diminished or increased in wild-type lungs after BHT injury. Because these proteins are critical for lung morphogenesis and endothelial homeostasis, their decreased mRNA levels are likely contributing to BHT-induced lung hemorrhage in foxf1(+/−) mice. Collectively, our data suggest that sustained expression of Foxf1 is essential for normal lung repair and endothelial cell survival in response to pulmonary cell injury.
Inhibition of inwardly rectifying K+ channels by cGMP in pulmonary vascular endothelial cells
Authors: Shimoda Larissa A. , Welsh Laura E. , Pearse David B.
Published: March 2015

Endothelial barrier dysfunction is typically triggered by increased intracellular Ca2+concentration. Membrane-permeable analogs of guanosine 3′,5′-cyclic monophosphate (cGMP) prevent disruption of endothelial cell integrity. Because membrane potential ( E m), which influences the electrochemical gradient for Ca2+ influx, is regulated by K+ channels, we investigated the effect of 8-bromo-cGMP on E m and inwardly rectifying K+ (KIR) currents in bovine pulmonary artery and microvascular endothelial cells (BPAEC and BMVEC), using whole cell patch-clamp techniques. Both cell types exhibited inward currents at potentials negative to −50 mV that were abolished by application of 10 μM Ba2+, consistent with KIR current. Ba2+ also depolarized both cell types. 8-Bromo-cGMP (10−3 M) depolarized BPAEC and BMVEC and inhibited KIR current. Pretreatment with Rp-8-cPCT-cGMPS or KT-5823, protein kinase G (PKG) antagonists, did not prevent current inhibition by 8-bromo-cGMP. These data suggest that 8-bromo-cGMP induces depolarization in BPAEC and BMVEC due, in part, to PKG-independent inhibition of KIR current. The depolarization could be a protective mechanism that prevents endothelial cell barrier dysfunction by reducing the driving force for Ca2+ entry.
Bradykinin increases IL-8 generation in airway epithelial cells via COX-2-derived prostanoids
Authors: Rodgers Helen C. , Pang Linhua , Holland Elaine , Corbett Lisa , Range Simon , Knox Alan J.
Published: March 2015

Interleukin (IL)-8, the C-X-C chemokine, is a potent neutrophil chemoattractant that has been implicated in a number of inflammatory airway diseases such as cystic fibrosis. Here we tested the hypothesis that bradykinin, an inflammatory mediator and chloride secretagogue, would increase IL-8 generation in airway epithelial cells through autocrine generation of endogenous prostanoids. Bradykinin increased IL-8 generation in both a non-cystic fibrosis (A549) and cystic fibrosis epithelial cell line (CFTE29[Formula: see text]) that was inhibited by the nonselective cyclooxygenase (COX) inhibitor indomethacin and the COX-2 selective inhibitor NS-398. COX-2 was the only isoform of COX expressed in both cell lines. Furthermore, the COX substrate arachidonic acid and exogenous prostaglandin E2 both increased IL-8 release in A549 cells. These results suggest that bradykinin may contribute to neutrophilic inflammation in the airway by generation of IL-8 from airway epithelial cells. The dependence of this response on endogenous production of prostanoids by COX-2 suggests that selective COX-2 inhibitors may have a role in the treatment of airway diseases characterized by neutrophilic inflammation such as cystic fibrosis or chronic obstructive pulmonary disease.
Metalloproteinase and growth factor interactions: do they play a role in pulmonary fibrosis?
Authors: Winkler Margaret K. , Fowlkes John L.
Published: March 2015

Chronic lung disease due to interstitial fibrosis can be a consequence of acute lung injury and inflammation. The inflammatory response is mediated through the migration of inflammatory cells, actions of proinflammatory cytokines, and the secretion of matrix-degrading proteinases. After the initial inflammatory insult, successful healing of the lung may occur, or alternatively, dysregulated tissue repair can result in scarring and fibrosis. On the basis of recent insights into the mechanisms underlying acute lung injury and its long-term consequences, data suggest that proteinases, such as the matrix metalloproteinases (MMPs), may not only be involved in the breakdown and remodeling that occurs during the injury but may also cause the release of growth factors and cytokines known to influence growth and differentiation of target cells within the lung. Through the release of and activation of fibrosis-promoting cytokines and growth factors such as transforming growth factor-β1, tumor necrosis factor-α, and insulin-like growth factors by MMPs, we propose that these metalloproteinases may be integral to the initiation and progression of pulmonary fibrosis.
Heart rate-arterial blood pressure relationship in conscious rat before vs. after spinal cord transection
Authors: Baldridge Bobby R. , Burgess Don E. , Zimmerman Ethan E. , Carroll Jonathan J. , Sprinkle Aletia G. , Speakman Richard O. , Li Sheng-Gang , Brown David R. , Taylor Robert F. , Dworkin Susan , Randall David C.
Published: March 2015

This experiment quantified the initial disruption and subsequent adaptation of the blood pressure (BP)-heart rate (HR) relationship after spinal cord transection (SCT). BP and HR were recorded for 4 h via an implanted catheter in neurally intact, unanesthetized rats. The animals were then anesthetized, and their spinal cords were severed at T1–T2 ( n = 5) or T4–T5 ( n = 6) or sham lesioned ( n = 4). BP was recorded for 4 h daily over the ensuing 6 days. The neurally intact rat showed a positive cross correlation, with HR leading BP at the peak by 1.8 ± 0.8 (SD) s. The cross correlation in unanesthetized rats ( n= 2) under neuromuscular blockade was also positive, with HR leading. After SCT at T1–T2, the cross correlation became negative, with BP leading HR, and did not change during the next 6 days. The cross correlation also became negative 1–3 days after SCT at T4–T5, but in four rats by day 6 and thereafter the cross correlation progressively reverted to a positive value. We propose that the positive cross correlation with HR leading BP in the intact rat results from an open-loop control that depends on intact supraspinal input to sympathetic preganglionic neurons in the spinal cord. After descending sympathetic pathways were severed at T1–T2, the intact vagal pathway to the sinoatrial node dominated BP regulation via the baroreflex. We suggest that reestablishment of the positive correlation after SCT at T4–T5 was attributable to the surviving sympathetic outflow to the heart and upper vasculature reasserting some effective function, perhaps in association with decreased spinal sympathetic hyperreflexia. The HR-BP cross correlation may index progression of sympathetic dysfunction in pathological processes.
Differential role of PTK and ERK MAPK in superoxide impairment of KATP and KCa channel cerebrovasodilation
Authors: Ross John , Armstead William M.
Published: March 2015

Previously, superoxide (O2 -) has been observed to impair pial artery dilation (PAD) to activators of the ATP-sensitive (KATP) and calcium-sensitive (KCa) K+ channels. This study tested the hypothesis that activation of protein tyrosine kinase (PTK) and the ERK isoform of MAPK by O2 - contribute to impairment of KATP and KCa channel PAD. Exposure of the cerebral cortex to a xanthine oxidase O2 --generating system (OX) blunted PAD to cromakalim, a KATP agonist, but preadministration of genistein, a PTK antagonist, or U-0126, an ERK MAPK inhibitor, almost completely prevented such impairment (11 ± 1 and 22 ± 1 vs. 3 ± 1 and 7 ± 1 vs. 10 ± 1 and 16 ± 2% for cromakalim with 10-8 and 10-6 M PAD during control, OX, and OX + genistein conditions). In contrast, neither genistein nor U-0126 robustly protected PAD to NS-1619, a KCa agonist, after OX exposure (11 ± 1 and 18 ± 2 vs. 1 ± 1 and 2 ± 1 vs. 4 ± 1 and 6 ± 1% for 10-8 and 10-6 M NS-1619 during control, OX, and OX + genistein conditions). These data show that PTK and ERK MAPK activation contribute to O2 --induced KATP and KCa channel PAD impairment and suggest a differential greater role for PTK and ERK MAPK in KATP vs. KCa channel PAD impairment.
Stimulation and blockade of GABAA receptors in the raphe pallidus: effects on body temperature, heart rate, and blood pressure in conscious rats
Authors: Zaretsky Dmitry V. , Zaretskaia Maria V. , DiMicco Joseph A.
Published: March 2015

Studies in anesthetized rats have implicated GABAA receptors in the region of the medullary raphe pallidus (RP) at the level of the facial nucleus in sympathetic nervous regulation of both heart rate and thermoregulatory mechanisms. Therefore, we examined the effect of microinjection of muscimol, a GABAA receptor agonist, and of bicuculline methiodide (BMI), a GABAA receptor antagonist, into the same region of the RP on heart rate, blood pressure, and core body temperature in conscious rats. Microinjection of BMI (40 pmol) into the RP evoked tachycardia that appeared within 1 min and was maximal within 10 min but had little or no effect on blood pressure or body temperature. Microinjection of muscimol (10–80 pmol) at the same sites in the RP evoked marked dose-related decreases in body temperature that developed more slowly (i.e., maximum decreases appearing at 60–75 min after 80 pmol) but had no effect on heart rate or blood pressure. Injection of either agent at sites outside the region had lesser or no effect on the measured parameters. These findings suggest that activity of neurons in the region of the RP plays an important role in the maintenance of body temperature but not heart rate under baseline conditions in conscious rats. Specifically, thermoregulatory neurons in this region appear to be tonically active and contribute to maintenance of body temperature under baseline conditions, while cardiac sympathetic premotor neurons in the RP are not active under these circumstances and thus do not support basal heart rate in conscious rats.
Neurochemical phenotype of hypothalamic neurons showing Fos expression 23 h after intracranial AgRP
Authors: Zheng Huiyuan , Corkern Michele M. , Crousillac Scott M. , Patterson Laurel M. , Phifer Curtis B. , Berthoud Hans-Rudolf
Published: March 2015

Agouti-related protein (AgRP) is coexpressed with neuropeptide Y (NPY) in a population of neurons in the arcuate nucleus (ARC) of the hypothalamus and stimulates food intake for up to 7 days if injected intracerebroventricularly. The prolonged food intake stimulation does not seem to depend on continued competition at the melanocortin-4 receptor (MC4R), because the relatively specific MC4R agonist MTII regains its ability to suppress food intake 24 h after AgRP injection. Intracerebroventricular AgRP also stimulates c-Fos expression 24 h after injection in several brain areas, so the neurons exhibiting delayed Fos expression might be particularly important in feeding behavior. Thus we aimed to identify the neurochemical phenotype of some of these neurons in select hypothalamic areas, using double-label immunohistochemistry. AgRP-injected rats ingested significantly more chow (10.2 ± 0.6 g) vs. saline controls (3.4 ± 0.7 g) in the first 9 h (light phase) after injection. In the lateral hypothalamus (particularly the perifornical area) 23 h after injection, AgRP induced significantly more Fos vs. saline in orexin-A (OXA) neurons (25.6 ± 4.9 vs. 4.8 ± 3.1%), but not in melanin-concentrating hormone (MCH) or cocaine- and amphetamine-regulated transcript (CART) neurons. In the ARC, AgRP induced significantly more Fos in CART (40.6 ± 5.9 vs. 13.4 ± 1.8%) but not NPY neurons. In the paraventricular nucleus, there was no significant difference in Fos expression induced by AgRP vs. saline in oxytocin and CART neurons. We conclude that the long-lasting hyperphagia induced by AgRP is correlated with and possibly partially mediated by hyperactive OXA neurons in the lateral hypothalamus and CART neurons in the ARC, but not by NPY and MCH neurons. The substantial increase in light-phase food intake by AgRP supports a role for the arousing effects of OXA. Activation of CART neurons in the ARC (which likely coexpress proopiomelanocortin) could indicate attempts to activate counterregulatory decreases in food intake.
Muscle interstitial potassium kinetics during intense exhaustive exercise: effect of previous arm exercise
Authors: Nordsborg Nikolai , Mohr Magni , Pedersen Lasse Dannemann , Nielsen Jens Jung , Langberg Henning , Bangsbo Jens
Published: March 2015

Interstitial K+ ([K+]i) was measured in human skeletal muscle by microdialysis during exhaustive leg exercise, with (AL) and without (L) previous intense arm exercise. In addition, the reproducibility of the [K+]i determinations was examined. Possible microdialysis-induced rupture of the sarcolemma was assessed by measurement of carnosine in the dialysate, because carnosine is only expected to be found intracellularly. Changes in [K+]i could be reproduced, when exhaustive leg exercise was performed on two different days, with a between-day difference of ∼0.5 mM at rest and 1.5 mM at exhaustion. The time to exhaustion was shorter in AL than in L (2.7 ± 0.3 vs. 4.0 ± 0.3 min; P < 0.05). Furthermore, [K+]i was higher from 0 to 1.5 min of the intense leg exercise period in AL compared with L (9.2 ± 0.7 vs. 6.4 ± 0.9 mM; P < 0.001) and at exhaustion (11.9 ± 0.5 vs. 10.3 ± 0.6 mM; P < 0.05). The dialysate content of carnosine was elevated by exercise, but low-intensity exercise resulted in higher dialysate carnosine concentrations than subsequent intense exercise. Furthermore, no relationship was found between carnosine concentrations and [K+]i. Thus the present data suggest that microdialysis can be used to determine muscle [K+]i kinetics during intense exercise, when low-intensity exercise is performed before the intense exercise. The high [K+]i levels reached at exhaustion can be expected to cause fatigue, which is supported by the finding that a faster accumulation of interstitial K+, induced by prior arm exercise, was associated with a reduced time to fatigue.
Improved erectile function after Rho-kinase inhibition in a rat castrate model of erectile dysfunction
Authors: Wingard Christopher J. , Johnson John A. , Holmes Andre , Prikosh Anita
Published: March 2015

Androgens are reported to act as strong modulators of erectile function influencing both nitric oxide and vasoconstrictor signaling. Castration results in a depressed erectile response that is associated with a loss of nitric oxide production and increased responsiveness to constrictive agents. The increased vasoconstrictor response may be a result of an active RhoA/Rho-kinase signaling pathway. We report here results of studies designed to test the hypothesis that inhibition of the Rho-kinase pathway restores erectile function in a castrate model by relaxing the smooth muscle. Mean arterial (MAP) and corpus cavernosal (CCP) pressures were monitored during intracavernosal injection of the Rho-kinase inhibitor Y-27632. Castration reduced the maximal erectile response (CCP/MAP) by 33%, and testosterone replacement restored the response (intact, 0.736 ± 0.040; castrate, 0.492 ± 0.022; testosterone, 0.681 ± 0.073). Injection of Y-27632 increased CCP in all experimental groups; it also left shifted the voltage response curve and increased the maximal CCP/MAP response (intact, 0.753 ± 0.091; castrate, 0.782 ± 0.081; testosterone treated, 0.894 ± 0.033). Y-27632 dose dependently relaxed phenylephrine-stimulated cavernosal tissues. Cavernosal tissues showed increased RhoA and Rho-kinase protein levels after castration. Our data support the hypothesis that an active Rho/Rho-kinase pathway contributes to the reduced erectile response after castration due to an upregulation of RhoA/Rho-kinase protein levels and that inhibition of this pathway may serve as an effective treatment for erectile dysfunction.
Cholecystokinin increases cytosolic calcium in a subpopulation of cultured vagal afferent neurons
Authors: Simasko Steven M. , Wiens Jason , Karpiel Adrienne , Covasa Mihai , Ritter Robert C.
Published: March 2015

Imaging fluorescent measurements with fura 2 were used to examine cytosolic calcium signals induced by sulfated CCK octapeptide (CCK-8) in dissociated vagal afferent neurons from adult rat nodose ganglia. We found that 40% (184/465) of the neurons responded to CCK-8 with a transient increase in cytosolic calcium. The threshold concentration of CCK-8 for inducing the response varied from 0.01 to 100 nM. In most neurons (13/16) the response was eliminated by removing extracellular calcium. Depleting intracellular calcium stores with thapsigargin slightly augmented the response. Most neurons were unresponsive to nonsulfated CCK-8. The response was eliminated by the CCK-A receptor antagonist lorglumide. Low concentrations of JMV-180 had no effect; however, high concentrations of JMV-180 reduced responses to CCK-8. These results demonstrate that CCK acts at the low-affinity site of the CCK-A receptor to trigger the entry of extracellular calcium into vagal afferent neurons. Increased cytosolic calcium may participate in acute activation of vagal afferent neurons, or it may initiate long-term changes, which modulate future neuronal responses to sensory stimuli.
NO mediates downregulation of RBF after a prolonged reduction of renal perfusion pressure in SHR
Authors: Sorensen Charlotte Mehlin , Leyssac Paul Peter , Skott Ole , Holstein-Rathlou Niels-Henrik
Published: March 2015

The aim of the study was to investigate mechanisms underlying the downregulation of renal blood flow (RBF) after a prolonged reduction in renal perfusion pressure (RPP) in adult spontaneously hypertensive rats (SHR). We tested the effect on the RBF response of clamping plasma ANG II in sevoflurane-anesthetized SHR. We also tested the effect of general cyclooxygenase (COX) inhibition and inhibition of the inducible COX-2. Furthermore, we assessed the effect of clamping the nitric oxide (NO) system. A prolonged period (15 min) of reduced RPP induced a downregulation of RBF. This was unchanged after clamping of plasma ANG II concentrations, general COX inhibition, and specific inhibition of COX-2. In contrast, clamping the NO system diminished the ability of SHR to downregulate RBF to a lower level. The downregulation of RBF was not associated with a resetting of the lower limit of autoregulation in the control group, in the ANG II-clamped group, or the NO clamped group. However, general COX inhibition and specific COX-2 inhibition enabled downward resetting of the lower limit of autoregulation. In conclusion, in SHR the renin-angiotensin system does not appear to play a major role in the downregulation of RBF after prolonged reduction of RPP. This response appears to be mediated partly by the NO system. We hypothesize that, in SHR, lack of downward resetting of the lower limit of autoregulation in response to a prolonged lowering of RPP could be the result of increased COX-2-mediated production of vasoconstrictory prostaglandins.
Characterization of a new double-filament model of focal cerebral ischemia in heme oxygenase-2-deficient mice
Authors: Goto Shozo , Sampei Kenji , Alkayed Nabil J. , Doré Sylvain , Koehler Raymond C.
Published: March 2015

Variations in vascular anatomy in knockout mouse strains can influence infarct volume after middle cerebral artery (MCA) occlusion (MCAO). In wild-type (WT) and heme oxygenase-2 gene-deleted (HO2-/-) mice, infarcts were not reproducibly achieved with the standard intraluminal filament technique. The present study characterizes a double-filament model of MCAO, which was developed to produce consistent infarcts in both WT and HO2-/- mice. Diameters of most cerebral arteries were similar in WT and HO2-/- mice, although the posterior communicating artery size was variable. In halothane-anesthetized mice, two 6-0 monofilaments with blunted tips were inserted into the left internal carotid artery 6.0 and 4.5 mm past the pterygopalatine artery junction to reside distal and proximal to the origin of the MCA. The tissue “volume at risk” determined by brief dye perfusion in WT (59 ± 2% of hemisphere; ±SE) was similar to HO2-/- (62 ± 4%). The volume of tissue with cerebral blood flow <50 ml·min-1·100 g-1 was similar in WT (35 ± 9%) and HO2-/- (36 ± 11%) during MCAO and at 3 h of reperfusion (<2%). After 1 h MCAO, infarct volume was greater in HO2-/- (44 ± 6%) than WT (25 ± 3%). After increasing MCAO duration to 2 h, the difference between HO2-/- (47 ± 4%) and WT (36 ± 3%) diminished, but infarct volume remained substantially less than the volume at risk. Infusion of tin protoporphyrin IX, an HO inhibitor, during reperfusion after 1 h MCAO increased infarct volume in WT but not significantly in HO2-/- mice, although infarct volume remained less than the volume at risk. Thus greater infarct volume in HO2-/- mice is not attributable to a greater volume at risk, lower intraischemic blood flow, or poor reflow, but rather to a neuroprotective effect of HO2 activity. The double-filament model may be of use as an alternative in other murine knockout strains in which the standard filament model does not yield consistent infarcts.
Sample entropy analysis of neonatal heart rate variability
Authors: Lake Douglas E. , Richman Joshua S. , Griffin M. Pamela , Moorman J. Randall
Published: March 2015

Abnormal heart rate characteristics of reduced variability and transient decelerations are present early in the course of neonatal sepsis. To investigate the dynamics, we calculated sample entropy, a similar but less biased measure than the popular approximate entropy. Both calculate the probability that epochs of window length m that are similar within a tolerance r remain similar at the next point. We studied 89 consecutive admissions to a tertiary care neonatal intensive care unit, among whom there were 21 episodes of sepsis, and we performed numerical simulations. We addressed the fundamental issues of optimal selection of m and r and the impact of missing data. The major findings are that entropy falls before clinical signs of neonatal sepsis and that missing points are well tolerated. The major mechanism, surprisingly, is unrelated to the regularity of the data: entropy estimates inevitably fall in any record with spikes. We propose more informed selection of parameters and reexamination of studies where approximate entropy was interpreted solely as a regularity measure.
Dexamethasone decreases serum and liver IGF-I and maintains liver IGF-I mRNA in parenterally fed rats
Authors: Kritsch K. R. , Murali S. , Adamo M. L. , Ney D. M.
Published: March 2015

Insulin-like growth factor-I (IGF-I) gene expression is regulated by nutritional and hormonal factors. High-dose glucocorticoids decrease food intake, and this confounds studies addressing glucocorticoid effects on IGF-I gene regulation. We investigated alterations in the hepatic IGF-I endocrine system induced by a catabolic dose of dexamethasone (Dex) in rats given adequate nutrition by continuous infusion of total parenteral nutrition (TPN) solution with or without IGF-I administration. The four TPN groups included control, +Dex, +IGF-I, and +IGF-I + Dex ( n = 9–11/group). Dex induced a 12% loss of body weight in association with a 50% decrease in hepatic immunoreactive IGF-I, a 10% decrease in serum IGF-I, and no change in steady-state liver IGF-I mRNA or growth hormone (GH) receptor binding. Exogenous IGF-I increased serum IGF-I, attenuated Dex-induced catabolism, and did not reduce hepatic levels of IGF-I and IGF-I mRNA despite decreased serum GH. These data suggest that Dex-induced catabolism is associated with downregulation of the hepatic IGF-I endocrine system at the translational or posttranslational level when adequate nutrition is provided.
Dynamic interactions between arterial pressure and sympathetic nerve activity: role of arterial baroreceptors
Authors: Julien Claude , Chapuis Bruno , Cheng Yong , Barrès Christian
Published: March 2015

The role of arterial baroreceptors in controlling arterial pressure (AP) variability through changes in sympathetic nerve activity was examined in conscious rats. AP and renal sympathetic nerve activity (RSNA) were measured continuously during 1-h periods in freely behaving rats that had been subjected to sinoaortic baroreceptor denervation (SAD) or a sham operation 2 wk before study ( n = 10 in each group). Fast Fourier transform analysis revealed that chronic SAD did not alter high-frequency (0.75-5 Hz) respiratory-related oscillations of mean AP (MAP) and RSNA, decreased by ∼50% spectral power of both variables in the midfrequency band (MF, 0.27-0.74 Hz) containing the so-called Mayer waves, and induced an eightfold increase in MAP power without altering RSNA power in the low-frequency band (0.005-0.27 Hz). In both groups of rats, coherence between RSNA and MAP was maximal in the MF band and was usually weak at lower frequencies. In SAD rats, the transfer function from RSNA to MAP showed the characteristics of a second-order low-pass filter containing a fixed time delay (∼0.5 s). These results indicate that arterial baroreceptors are not involved in production of respiratory-related oscillations of RSNA but play a major role in the genesis of synchronous oscillations of MAP and RSNA at the frequency of Mayer waves. The weak coupling between slow fluctuations of RSNA and MAP in sham-operated and SAD rats points to the interference of noise sources unrelated to RSNA affecting MAP and of noise sources unrelated to MAP affecting RSNA.
Transecting the gustatory branches of the facial nerve impairs NH4Cl vs. KCl discrimination in rats
Authors: Geran Laura C. , Garcea Mircea , Spector Alan C.
Published: March 2015

Ammonium and potassium chloride share a common taste quality and an amiloride-insensitive route of transduction. An amiloride-sensitive pathway might also be partially activated by these salts, although very few studies have reported effects of amiloride on nonsodium salt perception. This experiment was designed to determine 1) whether rats could discriminate KCl from NH4Cl and, if discrimination was evident, whether performance was impaired with 2) amiloride or 3) gustatory nerve transection. Rats were trained to discriminate KCl from NH4Cl ( n = 8) and NaCl from NH4Cl ( n = 8). Amiloride (100 μM) impaired NaCl vs. NH4Cl but not KCl vs. NH4Cl performance, whereas both groups showed significant impairments after transection of the chorda tympani (CT) and greater superficial petrosal (GSP) branches of the facial nerve. This suggests that rats can discriminate between KCl and NH4Cl and that this discrimination does not rely on an amiloride-sensitive mechanism but does depend on the CT and/or GSP nerves. This experiment supports the hypothesis that the facial nerve is important for salt taste recognition and discrimination.
Metabolic fate of yolk fatty acids in the developing king penguin embryo
Authors: Groscolas René , Fréchard Françoise , Decrock Frédéric , Speake Brian K.
Published: March 2015

This study examines the metabolic fate of total and individual yolk fatty acids (FA) during the embryonic development of the king penguin, a seabird characterized by prolonged incubation (53 days) and hatching (3 days) periods, and a high n-3/n-6 polyunsaturated FA ratio in the egg. Of the ∼15 g of total FA initially present in the egg lipid, 87% was transferred to the embryo by the time of hatching, the remaining 13% being present in the internalized yolk sac of the chick. During the whole incubation, 83% of the transferred FA was oxidized for energy, with only 17% incorporated into embryo lipids. Prehatching ( days 0-49), the fat stores (triacylglycerol) accounted for 58% of the total FA incorporated into embryo lipid. During hatching ( days 49-53), 40% of the FA of the fat stores was mobilized, the mobilization of individual FA being nonselective. At hatch, 53% of the arachidonic acid (20:4n-6) of the initial yolk had been incorporated into embryo lipid compared with only 15% of the total FA and 17-24% of the various n-3 polyunsaturated FA. Similarly, only 32% of the yolk's initial content of 20:4n-6 was oxidized for energy during development compared with 72% of the total FA and 58-66% of the n-3 polyunsaturated FA. The high partitioning of yolk FA toward oxidization and the intense mobilization of fat store FA during hatching most likely reflect the high energy cost of the long incubation and hatching periods of the king penguin. The preferential partitioning of 20:4n-6 into the structural lipid of the embryo in the face of its low content in the yolk may reflect the important roles of this FA in tissue function.
Mitochondrial proliferation in the permanent vs. temporary cold: enzyme activities and mRNA levels in Antarctic and temperate zoarcid fish
Authors: Lucassen M. , Schmidt A. , Eckerle L. G. , Pörtner H.-O.
Published: March 2015

Adjustments in mitochondrial properties and capacities are crucial in acclimatization to seasonal cold and in evolutionary cold adaptation of marine ectotherms. Although long-term compensatory increments in aerobic capacity of fish tissues have frequently been described in response to cold, much less is known about transitional phases and gene expression patterns involved. We investigated the time course of adjustment to acute cold in liver of eurythermal eelpout Zoarces viviparus. Whereas citrate synthase (CS) activity rose progressively in liver, cytochrome c oxidase (COX) activity was not altered during cold acclimation. Species-specific RNA probes were used to determine mRNA levels. CS mRNA (nuclear encoded) displayed a delayed, transient increase in response to cold, such that transcript levels did not parallel the change in enzyme activity. The enzyme activities and mRNA levels in the confamilial Antarctic Pachycara brachycephalum indicate cold compensation of CS activity in this cold-adapted species. The ratio of CS and COX activities was elevated in acclimation and adaptation to cold, indicating enhanced citrate synthesis over respiratory chain capacities in cold-adapted liver mitochondria. This may support enhanced lipid synthesis typically found in cold. The ratio of enzyme activity and transcript levels differed largely between Z. viviparus populations from the Baltic and North Seas, indicating the influence of unidentified parameters other than temperature. Transcript levels may not be tightly correlated with enzyme activities during thermal adaptation and thereafter. The time course of the acclimation process indicates that regulation at the translational and posttranslational levels predominates in adjustment to moderate thermal challenges.
Respiratory cycle timing and fast inspiratory discharge rhythms in the adult decerebrate rat
Authors: Marchenko Vitaliy , Granata Antonio R. , Cohen Morton I.
Published: March 2015

In supracollicular decerebrate paralyzed adult rats, neural respiration was monitored by bilateral phrenic recordings. In the study of respiratory cycle timing, the effects of vagal afferent input (lung inflation) on respiratory phase durations resembled those seen in decerebrate cats. 1) Withholding lung inflation during neural inspiration (I) produced lengthening of I phase duration by 46% (mean, n = 11). 2) Maintaining lung inflation during neural expiration (E) produced lengthening of E phase duration by 112% (mean, n = 4). In the study of fast rhythms in inspiratory discharges, phrenic nerve autospectra and bilateral (left-right) phrenic coherences in 16 rats revealed two types of fast rhythm: 1) high-frequency oscillation (HFO), which had significant coherence peaks ( n = 9, range 106–160 Hz, mean 132 Hz); and 2) medium-frequency oscillation (MFO), which had autospectral peaks but no distinct coherence peaks ( n = 11, range 46–96 Hz, mean 66 Hz). These rhythms resembled MFOs and HFOs in the decerebrate cat, but the modal frequency range was about twice as large. In addition, these frequency values differed markedly from the 20–40 Hz of the rhythms found in earlier studies in neonatal in vitro preparations; the difference may be due to developmental immaturity.
Inhibition of neuronal nitric oxide synthase by 7-nitroindazole attenuates acute lung injury in an ovine model
Authors: Enkhbaatar Perenlei , Murakami Kazunori , Shimoda Katsumi , Mizutani Akio , McGuire Roy , Schmalstieg Frank , Cox Robert , Hawkins Hal , Jodoin Jeffery , Lee Steve , Traber Lillian , Herndon David , Traber Daniel
Published: March 2015

Nitric oxide (NO) has been shown to play a major role in acute lung injury (ALI) after smoke inhalation. In the present study, we developed an ovine sepsis model, created by exposing sheep to smoke inhalation followed by instillation of bacteria into the airway, that mimics human sepsis and pneumonia. We hypothesized that the inhibition of neuronal NO synthase (nNOS) might be beneficial in treating ALI associated with this model. Female sheep ( n = 26) were surgically prepared for the study and given a tracheostomy. This was followed by insufflation of 48 breaths of cotton smoke (40°C) into the airway of each animal and subsequent instillation of live Pseudomonas aeruginosa [5 × 1011colony forming units (CFU)] into each sheep's lung. All sheep were mechanically ventilated using 100% O2. Continuous infusion of 7-nitroindazole (7-NI), an nNOS inhibitor, NG-monomethyl-l-arginine (l-NMMA), a nonspecific NOS inhibitor, or aminoguanidine (AG), an inducible NOS inhibitor, was started 1 h after insult. The administration of 7-NI improved pulmonary gas exchange (PaO2/FiO2; where PaO2is arterial PO2and FiO2is fractional inspired oxygen concentration) and pulmonary shunt fraction and attenuated the increase in lung wet-to-dry weight ratio seen in the nontreated sheep. Histologically, 7-NI prevented airway obstruction. The increase in airway blood flow after injury in the nontreated group was significantly inhibited by 7-NI. The increase in plasma concentration of nitrate and nitrite (NOx) was inhibited by 7-NI as well. Posttreatment with l-NMMA improved the pulmonary gas exchange, but AG did not. The results of the present study show that nNOS may be involved in the pathogenesis of ALI after smoke inhalation injury followed by bacterial instillation in the airway.
Determinants of maximal oxygen uptake in severe acute hypoxia
Authors: Calbet J. A. L. , Boushel R. , Rådegran G. , Søndergaard H. , Wagner P. D. , Saltin B.
Published: March 2015

To unravel the mechanisms by which maximal oxygen uptake (V˙o 2 max) is reduced with severe acute hypoxia in humans, nine Danish lowlanders performed incremental cycle ergometer exercise to exhaustion, while breathing room air (normoxia) or 10.5% O2 in N2(hypoxia, ∼5,300 m above sea level). With hypoxia, exercise PaO2 dropped to 31–34 mmHg and arterial O2 content (CaO2 ) was reduced by 35% ( P < 0.001). Forty-one percent of the reduction in CaO2 was explained by the lower inspired O2 pressure (Pi O2 ) in hypoxia, whereas the rest was due to the impairment of the pulmonary gas exchange, as reflected by the higher alveolar-arterial O2 difference in hypoxia ( P < 0.05). Hypoxia caused a 47% decrease inV˙o 2 max (a greater fall than accountable by reduced CaO2 ). Peak cardiac output decreased by 17% ( P < 0.01), due to equal reductions in both peak heart rate and stroke volume ( P < 0.05). Peak leg blood flow was also lower (by 22%, P < 0.01). Consequently, systemic and leg O2 delivery were reduced by 43 and 47%, respectively, with hypoxia ( P < 0.001) correlating closely with V˙o 2 max( r = 0.98, P < 0.001). Therefore, three main mechanisms account for the reduction ofV˙o 2 max in severe acute hypoxia: 1) reduction of Pi O2 , 2) impairment of pulmonary gas exchange, and 3) reduction of maximal cardiac output and peak leg blood flow, each explaining about one-third of the loss inV˙o 2 max.
Multiple transcription factors regulating the IL-6 gene are activated by cAMP in cultured Caco-2 cells
Authors: Hershko Dan D. , Robb Bruce W. , Luo Guangju , Hasselgren Per-Olof
Published: March 2015

Mucosal and enterocyte IL-6 production is increased during sepsis and endotoxemia. Recent studies suggest that cAMP potentiates IL-6 production in endotoxin- or IL-1β-stimulated enterocytes, but the molecular mechanisms are not known. We examined the role of the transcription factors NF-κB, activator protein (AP)-1, CCAAT/enhancer binding protein (C/EBP), and cAMP response element-binding protein (CREB) in cAMP-induced IL-6 production in cultured Caco-2 cells, a human intestinal epithelial cell line. In addition, the role of the protein kinase A (PKA), protein kinase C (PKC), and mitogen-activated protein (MAP) kinase signaling pathways was examined. Treatment of the cells with IL-1β increased IL-6 production and activated the IL-6 promoter in cells transfected with a luciferase reporter plasmid containing a wild-type IL-6 promoter. These effects of IL-1β were significantly potentiated by cAMP. When the binding sites for the individual transcription factors in the IL-6 promoter were mutated, results indicated that all four transcription factors may be involved in the cAMP-induced activation of the IL-6 gene. Treatment of the Caco-2 cells with cAMP increased the DNA binding activity of CREB, C/EBP, and AP-1, but not NF-κB. By using specific blockers, evidence was found that both PKA and p38 MAP kinase (but not PKC or p42/44 MAP kinase) may be involved in the cAMP-induced potentiation of IL-6 production. The present results suggest that cAMP activates multiple transcription factors involved in the regulation of the IL-6 gene and that the activation of these transcription factors may at least in part explain why cAMP potentiates IL-6 production in stimulated enterocytes.