bluuebunny/crossref_metadata_2025 huggingface dataset:
The rainbow trout skeletal muscle β-adrenergic system: characterization and signaling
Authors: Lortie Michel B. , Moon Thomas W.
Published: March 2015

The presence and functionality of β-adrenoceptors (β-ARs) were examined in red (RM) and white muscle (WM) membranes isolated from the rainbow trout Oncorhynchus mykiss. Specific binding assays revealed the presence of a single class of binding sites with similar affinities in both muscle types ( Kdin nM: 0.14 ± 0.03 and 0.18 ± 0.03 for RM and WM, respectively) but with a significantly higher number of binding sites in RM compared with WM (Bmaxin fmol/mg protein: 3.22 ± 0.11 and 2.60 ± 0.13, respectively). Selective and nonselective β-adrenergic agonists (β-AAs) and antagonists indicated an atypical β-AR pharmacology. This result may represent a nonmammalian β-AR classification or, more likely, the presence of more than one β-AR subtype in trout muscles with similar affinities that could not be kinetically resolved. Adenylyl cyclase (ACase) assays showed a dose-dependent increase in cAMP production as concentrations of β2-AAs increased in both muscle membranes with significantly higher basal cAMP production in RM compared with WM (cAMP production in pmol cAMP · mg protein−1· 10 min−1: 24.67 ± 3.06 and 9.64 ± 3.45, respectively). The agonist-induced increase in cAMP production was blocked by the β-adrenergic antagonist propranolol, while the ACase activator forskolin increased cAMP production by 7- to 14-fold above basal and ∼3-fold above all β-AAs tested. This study demonstrated the presence of atypical β2-ARs on RM and WM membranes of trout, suggesting that β2-AAs may be a tool to enhance protein accretion through this signaling pathway.
Effect of portal hypertension on splenic blood flow, intrasplenic extravasation and systemic blood pressure
Authors: Kaufman Susan , Levasseur Jody
Published: March 2015

We have previously shown that intrasplenic fluid extravasation is important in controlling blood volume. We proposed that, because the splenic vein flows in the portal vein, portal hypertension would increase splenic venous pressure and thus increase intrasplenic microvascular pressure and fluid extravasation. Given that the rat spleen has no capacity to store/release blood, intrasplenic fluid extravasation can be estimated by measuring the difference between splenic arterial inflow and venous outflow. In anesthetized rats, partial ligation of the portal vein rostral to the junction with the splenic vein caused portal venous pressure to rise from 4.5 ± 0.5 to 12.0 ± 0.9 mmHg ( n = 6); there was no change in portal venous pressure downstream of the ligation, although blood flow in the liver fell. Splenic arterial flow did not change, but the arteriovenous flow differential increased from 0.8 ± 0.3 to 1.2 ± 0.1 ml/min ( n = 6), and splenic venous hematocrit rose. Mean arterial pressure fell (101 ± 5.5 to 95 ± 4 mmHg). Splenic afferent nerve activity increased (5.6 ± 0.9 to 16.2 ± 0.7 spikes/s, n = 5). Contrary to our hypothesis, partial ligation of the portal vein caudal to the junction with the splenic vein (same increase in portal venous pressure but no increase in splenic venous pressure) also caused the splenic arteriovenous flow differential to increase (0.6 ± 0.1 to 1.0 ± 0.2 ml/min; n = 8). The increase in intrasplenic fluid efflux and the fall in mean arterial pressure after rostral portal vein ligation were abolished by splenic denervation. We propose there to be an intestinal/hepatic/splenic reflex pathway, through which is mediated the changes in intrasplenic extravasation and systemic blood pressure observed during portal hypertension.
Behavioral dysfunction, brain oxidative stress, and impaired mitochondrial electron transfer in aging mice
Authors: Navarro Ana , Sánchez Del Pino María Jesús , Gómez Carmen , Peralta Juan Luis , Boveris Alberto
Published: March 2015

Behavioral tests, tightrope success, and exploratory activity in a T maze were conducted with male and female mice for 65 wk. Four groups were defined: the lower performance slow males and slow females and the higher performance fast males and fast females. Fast females showed the longest life span and the highest performance, and slow males showed the lowest performance and the shortest life span. Oxidative stress and mitochondrial electron transfer activities were determined in brain of young (28 wk), adult (52 wk), and old (72 wk) mice in a cross-sectional study. Brain thiobarbituric acid reactive substances (TBARS) were increased by 50% in old mice and were ∼15% higher in males than in females and in slow than in fast mice. Brain Cu,Zn-superoxide dismutase (SOD) activity was increased by 52% and Mn-SOD by 108% in old mice. The activities of mitochondrial enzymes NADH-cytochrome c reductase, cytochrome oxidase, and citrate synthase were decreased by 14–58% in old animals. The cumulative toxic effects of oxyradicals are considered the molecular mechanism of the behavioral deficits observed on aging.
Anti-obesity effect of SR141716, a CB1 receptor antagonist, in diet-induced obese mice
Authors: Ravinet Trillou Christine , Arnone Michèle , Delgorge Claire , Gonalons Nadine , Keane Peter , Maffrand Jean-Pierre , Soubrié Philippe
Published: March 2015

Because the CB1 receptor antagonist SR141716 was previously reported to modulate food intake in rodents, we studied its efficacy in reducing obesity in a diet-induced obesity (DIO) model widely used for research on the human obesity syndrome. During a 5-wk treatment, SR141716 (10 mg · kg−1· day−1orally) induced a transient reduction of food intake (−48% on week 1) and a marked but sustained reduction of body weight (−20%) and adiposity (−50%) of DIO mice. Furthermore, SR141716 corrected the insulin resistance and lowered plasma leptin, insulin, and free fatty acid levels. Most of these effects were present, but less pronounced at 3 mg · kg−1· day−1. In addition to its hypophagic action, SR141716 may influence metabolic processes as the body weight loss of SR141716-treated mice was significantly higher during 24-h fasting compared with vehicle-treated animals, and when a 3-day treatment was compared with a pair feeding. SR141716 had no effect in CB1 receptor knockout mice, which confirmed the implication of CB1 receptors in the activity of the compound. These findings suggest that SR141716 has a potential as a novel anti-obesity treatment.
Insulin-induced translocation of facilitative glucose transporters in fetal/neonatal rat skeletal muscle
Authors: He Jing , Thamotharan M. , Devaskar Sherin U.
Published: March 2015

We examined the effect of insulin on fetal/neonatal rat skeletal muscle GLUT-1 and GLUT-4 concentrations and subcellular distribution by employing immunohistochemical analysis and subcellular fractionation followed by Western blot analysis. We observed that insulin did not alter total GLUT-1 or GLUT-4 concentrations or the GLUT-1 subcellular distribution in fetal/neonatal or adult skeletal muscle in 60 min. The basal and insulin-induced changes in subcellular distribution of GLUT-4 were different between the fetal/neonatal and adult skeletal muscle. Under basal conditions, sarcolemma-associated GLUT-4 was higher in the newborn compared with the adult, translating into a higher glucose transport. In contrast, insulin-induced translocation of GLUT-4 to the sarcolemma- and insulin-induced glucose transport was lower in the newborn compared with the adult. This age-related change results in enhanced basal glucose transport to fuel myocytic proliferation and differentiation while relatively curbing the insulin-dependent glucose transport in the newborn.
Postnatal changes in inhibitory effect of C-type natriuretic peptide on secretion of ANP
Authors: Kim Suhn Hee , Han Jeong Hee , Cao Chunhua , Kim Sung Zoo , Cho Kyung Woo
Published: March 2015

To define developmental changes in atrial natriuretic peptide (ANP) secretion and in the cross talk between C-type natriuretic peptide (CNP) and ANP, we performed experiments in isolated perfused nonbeating cardiac atria isolated from rabbits between 1 and 8 wk of age. Changes in atrial pressure resulted in increases in atrial volume that rose with age and reached the peak value at 4 wk. A rise in volume change increased ANP secretion with concomitant translocation of extracellular fluid (ECF) into the atrial lumen, which increased with age and reached the peak value at 4 wk. The positive relationship between stretch-induced ANP secretion and ECF translocation shifted upward and leftward with age. CNP suppressed stretch-induced ANP secretion in the 8-wk-old group but not in the 2- and 4-wk-old groups without differences in ECF translocation and atrial volume. Therefore, the ANP secretion in terms of ECF translocation was markedly suppressed by CNP in the 8-wk-old group but not in the 2- and 4-wk-old groups. The production of cGMP by CNP in atrial tissue membranes was markedly attenuated in young rabbits. However, 8-bromo-cGMP suppressed stretch-induced ANP secretion in 2- and 8-wk-old groups. Natriuretic peptide receptor-B mRNA was similar in both groups. Therefore, we conclude that the inhibitory effect of CNP on atrial ANP secretion is developmentally regulated, being absent during normal cardiac development in young animals and intact in adult animals.
Expression of smooth muscle MyHC B in blood vessels of hypertrophied heart in experimentally hypertensive rats
Authors: Wetzel Katharina , Baltatu Ovidiu , Nafz Benno , Persson Pontus B. , Haase Hannelore , Morano Ingo
Published: March 2015

We demonstrated recently a significantly lower fraction of cardiac precapillary arterioles that expressed smooth muscle myosin heavy chain (MyHC) B (SMB) in spontaneously hypertensive rats. To clarify whether this reduction of SMB expression is of genetic origin, we investigated SMB expression in cardiac precapillary arterioles of normotensive and experimentally hypertensive rats (one clip, one kidney or ANG II minipump). We observed similar SMB expression patterns in precapillary arterioles of experimentally hypertensive rats compared with normotensive controls. These observations suggest that the downregulation of SMB in spontaneously hypertensive rats is of genetic origin rather than an adaptive response to chronically enhanced blood pressure and cardiac hypertrophy.
Hyperleptinemia and reduced TNF-α secretion cause resistance of db/db mice to endotoxin
Authors: Madiehe Abram M. , Mitchell Tiffany D. , Harris Ruth B. S.
Published: March 2015

Leptin deficiency in ob/ob mice increases susceptibility to endotoxic shock, whereas leptin pretreatment protects them against LPS-induced lethality. Lack of the long-form leptin receptor (Ob-Rb) in db/db mice causes resistance. We tested the effects of LPS in C57BL/6J db3J/db3J (BL/3J) mice, which express only the circulating leptin receptors, compared with C57BL/6J db/db (BL/6J) mice, which express all short-form and circulating isoforms of the leptin receptor. Intraperitoneal injections of LPS significantly decreased rectal temperature and increased leptin, corticosterone, and free TNF-α in fed and fasted BL/3J and BL/6J mice. TNF-α was increased three- and fourfold in BL/3J and BL/6J, respectively. LPS (100 μg) caused 50% mortality of fasted BL/6J mice but caused no mortality in fasted BL/3J mice. Pretreatment of fasted BL/3J mice with 30 μg leptin prevented the drop in rectal temperature, blunted the increase in corticosterone, but had no effect on TNF-α induced by 100 μg LPS. Taken together, these data provide evidence that fasted BL/3J mice are more resistant than BL/6J mice to LPS toxicity, presumably due to the absence of leptin receptors in BL/3J mice. This resistance may be due to high levels of free leptin cross-reacting with other cytokine receptors.
Cardiovascular responses to caloric restriction and thermoneutrality in C57BL/6J mice
Authors: Williams T. D. , Chambers J. B. , Henderson R. P. , Rashotte M. E. , Overton J. M.
Published: March 2015

We utilized variations in caloric availability and ambient temperature (Ta) to examine interrelationships between energy expenditure and cardiovascular function in mice. Male C57BL/6J mice ( n = 6) were implanted with telemetry devices and housed in metabolic chambers for measurement of mean arterial pressure (MAP), heart rate (HR), O2consumption (V˙o2), and locomotor activity. Fasting (Ta= 23°C), initiated at the onset of the dark phase, resulted in large and transient depressions in MAP, HR, V˙o2, and locomotor activity that occurred during hours 6–17, which suggests torporlike episodes. Food restriction (14 days, 60% of baseline intake) at Ta= 23°C resulted in progressive reductions in MAP and HR across days that were coupled with an increasing occurrence of episodic torporlike reductions in HR (<300 beats/min) and V˙o2(<1.0 ml/min). Exposure to thermoneutrality (Ta= 30°C, n = 6) reduced baseline light-period MAP (−14 ± 2 mmHg) and HR (−184 ± 12 beats/min). Caloric restriction at thermoneutrality produced further reductions in MAP and HR, but indications of torporlike episodes were absent. The results reveal that mice exhibit robust cardiovascular responses to both acute and chronic negative energy balance. Furthermore, we conclude that Tais a very important consideration when assessing cardiovascular function in mice.
Sympathetic control of the cardiovascular response to acute hypoxemia in the chick embryo
Authors: Mulder A. L. M. , Miedema A. , De Mey J. G. R. , Giussani D. A. , Blanco C. E.
Published: March 2015

In response to an acute hypoxemic insult, the mammalian fetus shows a redistribution of the cardiac output in favor of the heart and brain. Peripheral vasoconstriction contributes to this response and is partly mediated by the release of catecholamines. Two mechanisms of catecholamine release in the fetus are reported: 1) neurogenic sympathetic stimulation and 2) a nonneurogenic mechanism via a direct effect of hypoxemia on chromaffin tissues. In the present study, the effects of sympathetic blockade on plasma catecholamine release and cardiac output distribution in response to acute hypoxemia were studied in the chick embryo at different stages of incubation. Only at the end of the incubation period, sympathetic blockade markedly attenuated the increase in plasma catecholamine concentrations and resulted in a greater fraction of the cardiac output distributed to the carcass. However, these effects did not prevent a significant increase in cardiac output to the brain and heart during acute hypoxemia. These data imply that in the chick embryo the contribution of neurogenic mechanisms to the catecholaminergic response to acute hypoxemia becomes greater by the end of the incubation period.
Effect of AT1 receptor blockade on hepatic redox status in SHR: possible relevance for endothelial function?
Authors: Cediel Eva , Sanz-Rosa David , Oubiña M. Pilar , de las Heras Natalia , Pacheco Francisco R. González , Vegazo Onofre , Jiménez Javier , Cachofeiro Victoria , Lahera Vicente
Published: March 2015

The study investigated whether the amelioration of endothelial dysfunction by candesartan (2 mg·kg-1·day-1; 10 wk) in spontaneously hypertensive rats (SHR) was associated with modification of hepatic redox system. Systolic arterial pressure (SAP) was higher ( P < 0.05) in SHR than in Wistar-Kyoto rats (WKY) and was reduced ( P < 0.05) by candesartan in both strains. Acetylcholine (ACh) relaxations were smaller ( P < 0.05) and contractions induced by ACh + NG-nitro-l-arginine methyl ester (l-NAME) were greater ( P < 0.05) in SHR than in WKY. Treatment with candesartan enhanced ( P < 0.05) ACh relaxations in SHR and reduced ( P < 0.05) ACh + l-NAME contractions in both strains. Expression of aortic endothelial nitric oxide synthase (eNOS) mRNA was similar in WKY and SHR, and candesartan increased ( P < 0.05) it in both strains. Aortic mRNA expression of the subunit p22phox of NAD(P)H oxidase was higher ( P < 0.05) in SHR than in WKY. Treatment with candesartan reduced ( P < 0.05) p22phox expression only in SHR. Malonyl dialdehyde (MDA) levels were higher ( P < 0.05), and the ratio reduced/oxidized glutathione (GSH/GSSG) as well as glutathione peroxidase activity (GPx) were lower ( P < 0.05) in liver homogenates from SHR than from WKY. Candesartan reduced ( P < 0.05) MDA and increased ( P < 0.05) GSH/GSSG ratio without affecting GPx. Vessel, lumen, and media areas were bigger ( P < 0.05) in SHR than in WKY. Candesartan treatment reduced ( P < 0.05) media area in SHR without affecting vessel or lumen area. The results suggest that hypertension is not only associated with elevation of vascular superoxide anions but with alterations of the hepatic redox system, where ANG II is clearly involved. The results further support the key role of ANG II via AT1 receptors for the functional and structural vascular alterations produced by hypertension.
Acute stressor exposure facilitates innate immunity more in physically active than in sedentary rats
Authors: Fleshner Monika , Campisi Jay , Deak Terrence , Greenwood Ben N. , Kintzel Jennifer A. , Leem Ted H. , Smith Taro P. , Sorensen Bristol
Published: March 2015

Most previous stress-immune research focused on the immunosuppressive effects of stress on acquired immunity. More recently, it has become clear that acute stressor exposure can potentiate innate, as well as suppress acquired, immunity. For example, acute stress improves recovery from bacterial inflammation, a classic in vivo measure of innate immunity. The previous work was done in sedentary organisms. Physical activity status can modulate the impact of stress on immune function. The following studies tested the hypothesis that the effect of stress on inflammation after subcutaneous challenge with bacteria ( Escherichia coli) is facilitated by physical activity. The results were that sedentary, stressed rats resolved their inflammation 1–2 days faster and have increased circulating neutrophils compared with their nonstressed, sedentary counterparts. In contrast, physically active, stressed rats resolve their inflammation 3–4 days faster and have increased circulating and inflammatory site neutrophils compared with their nonstressed counterparts. Importantly, the beneficial impact of stress on inflammation recovery and neutrophil migration was greater in the physically active, than sedentary, stressed rats. Thus physical activity status facilitates the positive effect of acute stress on innate immunity.
Volume expansion during NOS substrate donation with l-arginine: regulatory offsetting of renal response?
Authors: Andersen Jens Lundbæk , Sandgaard Niels C. F. , Bie Peter
Published: March 2015

The responses to infusion of nitric oxide synthase substrate (l-arginine 3 mg · kg−1 · min−1) and to slow volume expansion (saline 35 ml/kg for 90 min) alone and in combination were investigated in separate experiments.

l-Arginine left blood pressure and plasma ANG II unaffected but decreased heart rate (6 ± 2 beats/min) and urine osmolality, increased glomerular filtration rate (GFR) transiently, and caused sustained increases in sodium excretion (fourfold) and urine flow (0.2 ± 0.0 to 0.7 ± 0.1 ml/min). Volume expansion increased arterial blood pressure (102 ± 3 to 114 ± 3 mmHg), elevated GFR persistently by 24%, and enhanced sodium excretion to a peak of 251 ± 31 μmol/min, together with marked increases in urine flow, osmolar and free water clearances, whereas plasma ANG II decreased (8.1 ± 1.7 to 1.6 ± 0.3 pg/ml). Combined volume expansion and l-arginine infusion tended to increase arterial blood pressure and increased GFR by 31%, whereas peak sodium excretion was enhanced to 335 ± 23 μmol/min at plasma ANG II levels of 3.0 ± 1.1 pg/ml; urine flow and osmolar clearance were increased at constant free water clearance.

In conclusion, l-arginine 1) increases sodium excretion, 2) decreases basal urine osmolality, 3) exaggerates the natriuretic response to volume expansion by an average of 50% without persistent changes in GFR, and 4) abolishes the increase in free water clearance normally occurring during volume expansion. Thus l-arginine is a natriuretic substance compatible with a role of nitric oxide in sodium homeostasis, possibly by offsetting/shifting the renal response to sodium excess.
Influence of hypoxia and of hypoxemia on the development of cardiac activity in zebrafish larvae
Authors: Jacob E. , Drexel M. , Schwerte T. , Pelster B.
Published: March 2015

Cardiac activity and anaerobic metabolism were analyzed in zebrafish larvae raised under normoxia (Po2= 20 kPa) and under chronic hypoxia (Po2= 10 kPa) at three different temperatures (25, 28, and 31°C). Heart rate increased with development and with temperature. Under normoxia, cardiac output increased significantly at high temperature (31°C), but not at 28 or at 25°C. Under chronic hypoxia, however, heart rate as well as cardiac output increased at all temperatures in larvae at about hatching time or shortly thereafter. Cardiac activity of larvae raised for 2 wk after fertilization with a reduced hemoglobin oxygen-carrying capacity in their blood (hypoxemia; due to the presence of CO or of phenylhydrazine in the incubation water) was not different from control animals. Whole body lactate content of these animals did not increase. Thus there was no indication of a stimulated anaerobic energy metabolism. The increase in cardiac activity observed during hypoxia suggests that at about hatching time receptors are present that sense hypoxic conditions, and this information can be used to induce a stimulation of convective oxygen transport to compensate for a reduction in bulk oxygen diffusion in the face of a reduced oxygen gradient between environmental water and tissues. Under normoxia, however, the Po2gradient between environmental water and tissues and diffusional oxygen transport assure sufficient oxygen supply even if hemoglobin oxygen transport in the blood is severely impaired. Thus, under normoxic conditions and with a normal metabolic rate of the tissues, convective oxygen transport is not required until ∼2 wk after fertilization.
Norepinephrine is not required for reduction of feeding induced by cholecystokinin
Authors: Cannon C. Matson , Palmiter R. D.
Published: March 2015

CCK octapeptide (CCK-8) is released by the gut in response to a meal and acts via CCKA receptors on vagal afferents to induce satiety. However, the central neural pathways by which peripheral CCK-8 affects feeding are poorly understood. In the present study, we tested the hypothesis that norepinephrine (NE) is necessary for satiety induced by peripheral CCK-8 by using mice lacking dopamine β-hydroxylase ( Dbh −/−), the enzyme responsible for synthesizing NE and epinephrine from dopamine. We found that Dbh −/− mice are as responsive to the satiating effects of CCK-8 as their normal littermates.
Ribozyme compromise of adrenomedullin mRNA reveals a physiological role in the regulation of water intake
Authors: Taylor Meghan M. , Samson Willis K.
Published: March 2015

The adrenomedullin (AM) preprohormone is posttranslationally processed to result in two biologically active fragments, AM and proadrenomedullin NH2-terminal 20 peptide (PAMP). AM is thought to play a role in fluid and electrolyte balance by acting in brain to inhibit salt and water appetite and in the kidney to cause diuresis and natriuresis. We previously have shown that AM is necessary for the short-term regulation of salt intake. In this paper, we have designed a ribozyme, a catalytic RNA molecule, which specifically recognizes and cleaves the AM transcript. In cultured vascular smooth muscle cells, ribozyme treatment lowered AM mRNA and reduced peptide content. Intracerebroventricular administration of the ribozyme lowered hypothalamic AM content and led to an exaggerated drinking response in rats, demonstrating that endogenous, brain-derived AM is physiologically relevant and necessary for short-term control of water intake.
Cardiac effects of hypocretin-1 in nucleus ambiguus
Authors: Ciriello John , de Oliveira Cleusa V. R.
Published: March 2015

Although recent studies have reported hypocretin 1 (hcrt-1)-like-immunoreactivity (ir) within the region of the nucleus ambiguus (Amb) in the caudal brain stem, the function of hcrt-1 in the Amb on cardiovascular function is not known. Three series of experiments were done in male Wistar rats to investigate the effects of microinjections of hcrt-1 into Amb on heart rate (HR), mean arterial pressure (MAP), and the arterial baroreceptor reflex. In the first series, a detailed mapping of the distribution of hcrt-1- and hcrt-1 receptor (hcrtR-1)-like-ir was obtained of the Amb region. Although hcrt-1-like- and hcrtR-1-like-ir were found throughout the rostrocaudal extent of the Amb and adjacent ventrolateral medullary reticular formation, most of the hcrtR-1-like-ir was observed in the area just ventral to the compact formation of Amb, in the region of the external formation of the nucleus (Ambe). In the second series, the Amb region that contained hcrt-1 and hcrtR-1-ir was explored for sites that elicited changes in HR and MAP in urethane and α-chloralose-anesthetized rats. Microinjections of hcrt-1 (0.5–2.5 pmol) into the Ambe elicited a dose-related decrease in HR, with little or no direct change in MAP. The small decreases in MAP were found to be secondary to the HR changes. The largest bradycardia responses were elicited from sites in the Ambe. Administration (iv) of the muscarinic receptor antagonist atropine methyl bromide or ipsilateral vagotomy abolished the HR response, indicating that the HR response was due to activation of vagal cardiomotor neurons. In the final series, microinjections of hcrt-1 into the Ambe significantly potentiated the reflex bradycardia elicited by activation of the baroreflex as a result of the increased MAP after the intravenous injection of phenylephrine. These data suggest that hcrt-1 in the Ambe activates neuronal systems that alter the excitability of central circuits that reflexly control the circulation through the activation of vagal preganglionic cardioinhibitory neurons.
Involvement of sympathetic efferents but not capsaicin-sensitive afferents in nociceptin-mediated dual control of rat synovial blood flow
Authors: McDougall Jason J.
Published: March 2015

This study set out to examine the vasomotor effects of the opioid-like peptide nociceptin on knee joint capsular blood flow in urethane-anaesthetized rats. Topical application of nociceptin (10−15–10−8mol) caused a progressive fall in joint perfusion that was significantly inhibited by the specific nociceptin receptor antagonist [Phe1-(CH2-NH)-Gly2] Nociceptin(1–13)-NH2as well as the nonspecific opioid antagonist naloxone. To test whether this constrictor response was sympathetically mediated, we administered nociceptin in animals treated with guanethidine to produce sympathetic blockade or in the presence of the α-adrenoceptor antagonist phentolamine. Both guanethidine treatment and phentolamine coadministration attenuated the constrictor response to nociceptin. Inhibition of nociceptin-mediated vasoconstriction revealed a supplementary hyperemic response that persisted in animals whose knee joints were treated with 1% capsaicin to destroy the articular unmyelinated nerve supply. These results show that, in the rat knee, peripheral administration of nociceptin primarily causes a sympathetically mediated vasoconstriction. In addition, high-dose nociceptin produces a vasodilatatory response that is likely due to the direct action of nociceptin on vascular smooth muscle and not by a neurogenic mechanism.
Role of corticotropin-releasing hormone in stressor-induced alterations of sleep in rat
Authors: Chang Fang-Chia , Opp Mark R.
Published: March 2015

Corticotropin-releasing hormone (CRH) mediates responses to a variety of stressors. We subjected rats to a 1-h period of an acute stressor, physical restraint, and determined the impact on subsequent sleep-wake behavior. Restraint at the beginning of the light period, but not the dark period, increased waking and reduced rapid eye movement sleep without dramatically altering slow-wave sleep (SWS). Electroencephalogram (EEG) slow-wave activity during SWS and brain temperature were increased by this manipulation. Central administration of the CRH receptor antagonist astressin blocked the increase in waking after physical restraint, but not during the period of restraint itself. Blockade of CRH receptors with astressin attenuated the restraint-induced elevation of brain temperature, but not the increase of EEG slow-wave activity during subsequent SWS. Although corticosterone increased after restraint in naive animals, it was not altered by this manipulation in rats well habituated to handling and injection procedures. These results suggest that under these conditions central CRH, but not the hypothalamic-pituitary-adrenal axis, is involved in the alterations in sleep-wake behavior and the modulation of brain temperature of rats exposed to physical restraint.
Iron, lipocalin, and kidney epithelia
Authors: Yang Jun , Mori Kiyoshi , Li Jau Yi , Barasch Jonathan
Published: March 2015

Brilliant new discoveries in the field of iron metabolism have revealed novel transmembrane iron transporters, novel hormones that regulate iron traffic, and iron's control of gene expression. An important role for iron in the embryonic kidney was first identified by Ekblom, who studied transferrin (Landschulz W and Ekblom P. J Biol Chem 260: 15580–15584, 1985; Landschulz W, Thesleff I, and Ekblom P. J Cell Biol 98: 596–601, 1984; Thesleff I, Partanen AM, Landschulz W, Trowbridge IS, and Ekblom P. Differentiation 30: 152– 158, 1985). Nevertheless, how iron traffics to developing organs remains obscure. This review discusses a member of the lipocalin superfamily, 24p3 or neutrophil gelatinase-associated lipocalcin (NGAL), which induces the formation of kidney epithelia. We review the data showing that lipocalins transport low-molecular-weight chemical signals and data indicating that 24p3/NGAL transports iron. We compare 24p3/NGAL to transferrin and a variety of other iron trafficking pathways and suggest specific roles for each in iron transport.
Effect of isolated removal of either basolateral HCO3-or basolateral CO2on HCO3-reabsorption by rabbit S2 proximal tubule
Authors: Zhao Jinhua , Zhou Yuehan , Boron Walter F.
Published: March 2015

The equilibrium [Formula: see text] had made it impossible to determine how isolated changes in basolateral CO2([CO2]) or [Formula: see text] concentration ([[Formula: see text]]), at a fixed basolateral pH, modulate renal [Formula: see text] or reabsorption. In the present study, we have begun to address this issue by measuring [Formula: see text] reabsorption ( JHCO3) and intracellular pH (pHi) in isolated perfused rabbit S2 proximal tubules exposed to three different basolateral (bath) solutions: 1) equilibrated 5% CO2/22 mM [Formula: see text] 7.40, 2) an out-of-equilibrium (OOE) solution containing 5% CO2/pH 7.40 but minimal [Formula: see text] (“pure CO2”), and 3) an OOE solution containing 22 mM [Formula: see text] 7.40 but minimal CO2(“pure [Formula: see text]”). Tubule lumens were constantly perfused with equilibrated 5% CO2/22 mM [Formula: see text]. Compared with the equilibrated bath solution ( JHCO3= 76.5 ± 7.7 pmol·min–1·mm–1, pHi= 7.09 ± 0.04), the pure CO2bath solution increased JHCO3by ∼25% but decreased pHiby 0.19. In contrast, the pure [Formula: see text] bath solution decreased JHCO3by 37% but increased pHiby 0.24. Our data are consistent with two competing hypotheses: 1) the isolated removal of basolateral [Formula: see text] (or CO2) causes a pHidecrease (increase) that in turn raises (lowers) JHCO3; and 2) [Formula: see text] removal raises JHCO3by reducing inhibition of basolateral Na/HCO3cotransport and/or reducing [Formula: see text] backleak, whereas CO2removal lowers JHCO3by reducing stimulation of a CO2sensor.
Extracellular glutathione peroxidase is secreted basolaterally by human renal proximal tubule cells
Authors: Whitin John C. , Bhamre Suvarna , Tham Doris M. , Cohen Harvey J.
Published: March 2015

Extracellular glutathione peroxidase (eGPx) is a secreted selenoenzyme with GPx activity. eGPx protein and activity are found in blood plasma and other extracellular fluids. eGPx in plasma is predominantly derived from the proximal tubules of kidneys in humans. Two types of human proximal tubule cells were cultured on semipermeable polycarbonate membranes to determine whether these cells secrete eGPx in a polarized direction. Immortalized human proximal tubule HK-2 cells and primary human proximal tubule cells formed confluent monolayers when cultured on these membrane inserts in culture dishes, as evidenced by transepithelial resistance. Both cell lines also constituted a barrier to diffusion of a fluoresceinated dextran of 75 kDa, a size similar to eGPx homotetramers. In both cell lines, 6- to 12-fold more35S-methionine-labeled eGPx was immunoprecipitated from the basolateral media than from the apical media, indicating basolateral secretion of eGPx. eGPx was immunolocalized to the extracellular fluid at the basolateral surface of proximal tubules in human kidney. These data support the conclusion that eGPx is secreted through the basolateral membrane of human kidney proximal tubule cells into the extracellular fluid of the kidney, and from there enters blood plasma.
Injury of the renal microvascular endothelium alters barrier function after ischemia
Authors: Sutton Timothy A. , Mang Henry E. , Campos Silvia B. , Sandoval Ruben M. , Yoder Mervin C. , Molitoris Bruce A.
Published: March 2015

The role of renal microvascular endothelial cell injury in the pathophysiology of ischemic acute renal failure (ARF) remains largely unknown. No consistent morphological alterations have been ascribed to the endothelium of the renal microvasculature as a result of ischemia-reperfusion injury. Therefore, the purpose of this study was to examine biochemical markers of endothelial injury and morphological changes in the renal microvascular endothelium in a rodent model of ischemic ARF. Circulating von Willebrand factor (vWF) was measured as a marker of endothelial injury. Twenty-four hours after ischemia, circulating vWF peaked at 124% over baseline values ( P = 0.001). The FVB-TIE2/GFP mouse was utilized to localize morphological changes in the renal microvascular endothelium. Immediately after ischemia, there was a marked increase in F-actin aggregates in the basal and basolateral aspect of renal microvascular endothelial cells in the corticomedullary junction. After 24 h of reperfusion, the pattern of F-actin staining was more similar to that observed under physiological conditions. In addition, alterations in the integrity of the adherens junctions of the renal microvasculature, as demonstrated by loss of localization in vascular endothelial cadherin immunostaining, were observed after 24 h of reperfusion. This observation temporally correlated with the greatest extent of permeability defect in the renal microvasculature as identified using fluorescent dextrans and two-photon intravital imaging. Taken together, these findings indicate that renal vascular endothelial injury occurs in ischemic ARF and may play an important role in the pathophysiology of ischemic ARF.
High NaCl causes Mre11 to leave the nucleus, disrupting DNA damage signaling and repair
Authors: Dmitrieva Natalia I. , Bulavin Dmitry V. , Burg Maurice B.
Published: March 2015

High NaCl causes DNA double-strand breaks and cell cycle arrest, but the mechanism of its genotoxicity has been unclear. In this study, we describe a novel mechanism that contributes to this genotoxicity. The Mre11 exonuclease complex is a central component of DNA damage response. This complex assembles at sites of DNA damage, where it processes DNA ends for subsequent activation of repair and initiates cell cycle checkpoints. However, this does not occur with DNA damage caused by high NaCl. Rather, following high NaCl, Mre11 exits from the nucleus, DNA double-strand breaks accumulate in the S and G2phases of the cell cycle, and DNA repair is inhibited. Furthermore, the exclusion of Mre11 from the nucleus by high NaCl persists following UV or ionizing radiation, also preventing DNA repair in response to those stresses, as evidenced by absence of H2AX phosphorylation at places of DNA damage and by impaired repair of damaged reporter plasmids. Activation of chk1 by phosphorylation on Ser345 generally is required for DNA damage-induced cell cycle arrest. However, chk1 does not become phosphorylated during high NaCl-induced cell cycle arrest. Also, high NaCl prevents ionizing and UV radiation-induced phosphorylation of chk1, but cell cycle arrest still occurs, indicating the existence of alternative mechanisms for the S and G2/M delays. DNA breaks that occur normally during processes such as DNA replication and transcription, as well as damages to DNA induced by genotoxic stresses, ordinarily are rapidly repaired. We propose that inhibition of this repair by high NaCl results in accumulation of DNA damage, accounting for the genotoxicity of high NaCl, and that cell cycle delay induced by high NaCl slows accumulation of DNA damage until the DNA damage-response network can be reactivated.
Mechanisms of secondary hyperparathyroidism
Authors: Silver Justin , Kilav Rachel , Naveh-Many Tally
Published: March 2015

Small decreases in serum Ca2+and more prolonged increases in serum phosphate (Pi) stimulate the parathyroid (PT) to secrete parathyroid hormone (PTH), and 1,25(OH)2D3decreases PTH synthesis and secretion. A prolonged decrease in serum Ca2+and 1,25(OH)2D3, or increase in serum Pi, such as in patients with chronic renal failure, leads to the appropriate secondary increase in serum PTH. This secondary hyperparathyroidism involves increases in PTH gene expression, synthesis, and secretion, and if chronic, to proliferation of the PT cells. Low serum Ca2+leads to an increase in PTH secretion, PTH mRNA stability, and PT cell proliferation. Pialso regulates the PT in a similar manner. The effect of Ca2+on the PT is mediated by a membrane Ca2+receptor. 1,25(OH)2D3decreases PTH gene transcription. Ca2+and Piregulate the PTH gene posttranscriptionally by regulating the binding of PT cytosolic proteins, trans factors, to a defined cissequence in the PTH mRNA 3′-untranslated region, thereby determining the stability of the transcript. PT trans factors and cis elements have been defined.
Cloning of rabbit Kir6.1, SUR2A, and SUR2B: possible candidates for a renal KATP channel
Authors: Brochiero Emmanuelle , Wallendorf Bernadette , Gagnon Dominique , Laprade Raynald , Lapointe Jean-Yves
Published: March 2015

In rabbit proximal tubules, a basolateral ATP- and taurine-sensitive K+ channel (KATP) was shown to be involved in the regulation of the basolateral K+ conductance as a function of the rate of apical Na+ entry. To establish the molecular identity of this channel, we used degenerated primers to look for cDNA transcripts for an inwardly rectifying K+ channel (Kir6.1 and Kir6.2) and sulfonylurea receptors (SUR1, SUR2A, and SUR2B) in a cDNA library obtained from rabbit proximal tubules. PCR products were found only for Kir6.1, SUR2A, and SUR2B. Expression of Kir6.1 in Xenopusoocytes generated an additional K+ current that was found to be sensitive to external barium and intracellular taurine and to changes in intracellular ATP concentrations. To study the specificity of the taurine sensitivity, intracellular taurine was tested on several members of the Kir family expressed in Xenopus oocytes. K+ currents induced by Kir1.1A, Kir2.1, Kir3.2, Kir4.1, or Kir5.1 were insensitive to taurine, but all tested combinations of Kir6.x with or without the SUR subunit were significantly inhibited by taurine. This study suggests that the taurine-sensitive KATP channel of rabbit proximal tubules is formed by a combination of Kir6.1 plus SUR2A and/or SUR2B.
Cd-MT causes endocytosis of brush-border transporters in rat renal proximal tubules
Authors: Sabolic Ivan , Ljubojevic Marija , Herak-Kramberger Carol M. , Brown Dennis
Published: March 2015

Nephrotoxicity in humans and experimental animals due to chronic exposure to cadmium (Cd) is manifested by defects in the reabsorptive and secretory functions of proximal tubules (PT). The main symptoms of Cd nephrotoxicity, including polyuria, phosphaturia, aminoaciduria, glucosuria, and proteinuria, suggest that various brush-border membrane (BBM) transporters are the main targets of Cd. Specific transporters may be either directly inhibited by Cd or lost from the BBM after Cd treatment, or both. We have recently proposed that Cd may impair the vesicle-dependent recycling of BBM transporters by inhibiting vacuolar H+-ATPase (V-ATPase) activity and endocytosis in PT cells (Herak-Kramberger CM, Sabolic I, and Brown D. Kidney Int 53: 1713–1726, 1998). The mechanism underlying the Cd effect was further explored in an in vivo model of experimental Cd nephrotoxicity induced by Cd-metallothionein (Cd-MT; 0.4 mg Cd/kg body mass; a single dose sc) in rats. The time-dependent redistribution of various BBM transporters was examined in this model by fluorescence and gold-labeling immunocytochemistry on tissue sections and by immunoblotting of isolated renal cortical BBM. In PT cells of Cd-MT-treated rats, we observed 1) shortening and loss of microvilli; 2) time-dependent loss of megalin, V-ATPase, aquaporin-1 (AQP1), and type 3 Na+/H+exchanger (NHE3) from the BBM; 3) redistribution of these transporters into vesicles that were randomly scattered throughout the cell cytoplasm; and 4) redistribution of NHE3, but not megalin, into the basolateral plasma membrane. The internalization of BBM transporters was accompanied by fragmentation and loss of microtubules and by an increased abundance of α-tubulin monomers in PT cells. Transporter redistribution was detectable as early as 1 h after Cd-MT treatment and increased in magnitude over the next 12 h. We conclude that the early mechanism of Cd toxicity in PT cells may include a colchicine-like depolymerization of microtubules and impaired vesicle-dependent recycling of various BBM proteins. These processes may lead to a time-dependent loss of cell membrane components, resulting in reabsorptive and secretory defects that occur in Cd-induced nephrotoxicity.
Effect of primary polydipsia on aquaporin and sodium transporter abundance
Authors: Cadnapaphornchai Melissa A. , Summer Sandra N. , Falk Sandor , Thurman Joshua M. , Knepper Mark A. , Schrier Robert W.
Published: March 2015

Chronic primary polydipsia (POLY) in humans is associated with impaired urinary concentrating ability. However, the molecular mechanisms responsible for this finding have not been elucidated. The purpose of this study was to examine the effect of chronic primary POLY on water metabolism and renal aquaporin (AQP) water channels and sodium and urea transporter abundance in rats. Primary POLY was induced in male Sprague-Dawley rats by daily administration of 15 g powdered rat chow mixed in 100 ml water for 10 days. Control rats (CTL) received 15 g powdered rat chow per day and ad libitum drinking water. Rats were studied following this period before further intervention and with a 36-h period of water deprivation to examine maximal urinary concentrating ability. At baseline, POLY rats demonstrated significantly greater water intake (100 ± 1 vs. 22 ± 2 ml/day, P < 0.0001) and urinary output (80 ± 1 vs. 11 ± 1 ml/day, P < 0.0001) and decreased urinary osmolality (159 ± 13 vs. 1,365 ± 188 mosmol/kgH2O, P < 0.001) compared with CTL rats. These findings were accompanied by decreased inner medulla AQP-2 protein abundance in POLY rats compared with CTL rats before water deprivation (76 ± 2 vs. 100 ± 7% CTL mean, P < 0.007). With water deprivation, maximal urinary osmolality was impaired in POLY vs. CTL rats (2,404 ± 148 vs. 3,286 ± 175 mosmol/kgH2O, P < 0.0005). This defect occurred despite higher plasma vasopressin concentrations and similar medullary osmolalities in POLY rats. In response to 36-h water deprivation, inner medulla AQP-2 protein abundance was decreased in POLY rats compared with CTL rats (65 ± 5 vs. 100 ± 5% CTL mean, P < 0.0006). No significant differences were noted in renal protein abundance of either AQP-3 or AQP-4 or sodium and urea transporters. We conclude that the impaired urinary concentrating ability associated with primary POLY in rats is due to impaired osmotic equilibration in the collecting duct that is mediated primarily by decreased AQP-2 protein abundance.
Cycloheximide increases glucocorticoid-stimulated α-ENaC mRNA in collecting duct cells by p38 MAPK-dependent pathway
Authors: Itani Omar A. , Cornish Kristyn L. , Liu Kang Z. , Thomas Christie P.
Published: March 2015

Aldosterone and glucocorticoids (GCs) stimulate Na+reabsorption in the collecting ducts by increasing the activity of the epithelial Na+channel (ENaC). Our laboratory has used Madin-Darby canine kidney-C7 cells to demonstrate that this effect is associated with an increase in α-ENaC gene transcription (Mick VE, Itani OA, Loftus RW, Husted RF, Schmidt TJ, and Thomas CP, Mol Endocrinol 15: 575–588, 2001). Cycloheximide (CHX) superinduced the GC-stimulated α-ENaC expression in a dose-dependent manner, but had no effect on basal or aldosterone-stimulated α-ENaC expression, whereas anisomycin inhibited basal and corticosteroid-stimulated α-ENaC expression. The superinduction of α-ENaC expression was also seen with hypotonicity, was blocked by RU-38486, and was independent of protein synthesis. CHX had no effect on α-ENaC mRNA half-life, confirming that its effect was via an increase in α-ENaC transcription. The effect of CHX and hypotonicity on α-ENaC expression was abolished by SB-202190, indicating an effect mediated via p38 MAPK. Consistent with this scheme, CHX increased pp38 and MKK6, an upstream activator of p38, stimulated α-ENaC promoter activity. These data confirm a model in which CHX activates p38 in Madin-Darby canine kidney-C7 cells to increase α-ENaC gene transcription in a GC-dependent manner.
LPA is a novel lipid regulator of mesangial cell hexokinase activity and HKII isoform expression
Authors: Coy Platina E. , Taneja Navin , Lee Iris , Hecquet Claudie , Bryson Jane M. , Robey R. Brooks
Published: March 2015

The prototypical extracellular phospholipid mediator, lysophosphatidic acid (LPA), exhibits growth factor-like properties and represents an important survival factor in serum. This potent mesangial cell mitogen is increased in conditions associated with glomerular injury. It is also a known activator of the classic mitogen-activated protein kinase (MAPK) pathway, which plays an important role in the regulation of mesangial cell hexokinase (HK) activity. To better understand the mechanisms coupling metabolism to injury, we examined the ability of LPA to regulate HK activity and expression in cultured murine mesangial cells. LPA increased total HK activity in a concentration- and time-dependent manner, with maximal increases of >50% observed within 12 h of exposure to LPA concentrations ≥25 μM (apparent ED502 μM). These effects were associated with increased extracellular signal-regulated kinase (ERK) activity and were prevented by the pharmacological inhibition of either MAPK/ERK kinase or protein kinase C (PKC). Increased HK activity was also associated with increased glucose (Glc) utilization and lactate accumulation, as well as selectively increased HKII isoform abundance. The ability of exogenous LPA to increase HK activity was both Ca2+independent and pertussis toxin insensitive and was mimicked by LPA-generating phospholipase A2. We conclude that LPA constitutes a novel lipid regulator of mesangial cell HK activity and Glc metabolism. This regulation requires sequential activation of both Ca2+-independent PKC and the classic MAPK pathway and culminates in increased HKII abundance. These previously unrecognized metabolic consequences of LPA stimulation have both physiological and pathophysiological implications. They also suggest a novel mechanism whereby metabolism may be coupled to cellular injury via extracellular lipid mediators.