bluuebunny/crossref_metadata_2025 huggingface dataset:
ET-1 stimulates pulmonary arterial smooth muscle cell proliferation via induction of reactive oxygen species
Authors: Wedgwood Stephen , Dettman Robert W. , Black Stephen M.
Published: December 2017

Recent studies implicate reactive oxygen species (ROS) such as superoxide anions and H2O2 in the proliferation of systemic vascular smooth muscle cells (SMCs). However, the role of ROS in SMC proliferation within the pulmonary circulation remains unclear. We investigated the effects of endothelin-1 (ET-1), a potential SMC mitogen, on ROS production and proliferation of fetal pulmonary artery SMCs (FPASMCs). Exposure to ET-1 resulted in increases in superoxide production and viable FPASMCs after 72 h. These increases were prevented by pretreatment with PD-156707. Treatment with pertussis toxin blocked the effects of ET-1, whereas cholera toxin stimulated superoxide production and increased viable cell numbers even in the absence of ET-1. Wortmannin, LY-294002, diphenyleneiodonium (DPI), 4-(2-aminoethyl)benzenesulfonyl fluoride, and apocynin also prevented the ET-1-mediated increases in superoxide production and viable cell numbers. Exposure to H2O2 or diethyldithiocarbamate increased viable cell number by 37% and 50%, respectively. Conversely, ascorbic acid and DPI decreased viable cell number, which appeared to be due to an increase in programmed cell death. Our data suggest that ET-1 exerts a mitogenic effect on FPASMCs via an increase in ROS production and that antioxidants can block this effect via induction of apoptosis. Antioxidant treatment may therefore represent a potential therapy for pulmonary vascular diseases.
Chlorzoxazone or 1-EBIO increases Na+absorption across cystic fibrosis airway epithelial cells
Authors: Gao Lin , Yankaskas James R. , Fuller Catherine M. , Sorscher Eric J. , Matalon Sadis , Forman Henry Jay , Venglarik Charles J.
Published: December 2017

Previous studies demonstrated that chlorzoxazone or 1-ethyl-2-benzimidazolinone (1-EBIO) enhances transepithelial Cl− secretion by increasing basolateral K+ conductance ( G K) (Singh AK, Devor DC, Gerlach AC, Gondor M, Pilewski JM, and Bridges RJ. J Pharmacol Exp Ther 292: 778–787, 2000). Hence these compounds may be useful to treat cystic fibrosis (CF) airway disease. The goal of the present study was to determine whether chlorzoxazone or 1-EBIO altered ion transport across ΔF508-CF transmembrane conductance regulator homozygous CFT1 airway cells. CFT1 monolayers exhibited a basal short-circuit current that was abolished by apical amiloride (inhibition constant 320 nM) as expected for Na+ absorption. The addition of chlorzoxazone (400 μM) or 1-EBIO (2 mM) increased the amiloride-sensitive I sc ∼2.5-fold. This overlapping specificity may preclude use of these compounds as CF therapeutics. Assaying for changes in the basolateral G K with a K+ gradient plus the pore-forming antibiotic amphotericin B revealed that chlorzoxazone or 1-EBIO evoked an ∼10-fold increase in clotrimazole-sensitive G K. In contrast, chlorzoxazone did not alter epithelial Na+ channel-mediated currents across basolateral-permeabilized monolayers or in Xenopus oocytes. These data further suggest that alterations in basolateral G K alone can modulate epithelial Na+ transport.
Amiodarone inhibits lung degradation of SP-A and perturbs the distribution of lysosomal enzymes
Authors: Baritussio Aldo , Marzini Stefano , Agostini Marco , Alberti Antonella , Cimenti Cristina , Bruttomesso Daniela , Manzato Enzo , Quaglino Daniela , Pettenazzo Andrea
Published: December 2017

Amiodarone may induce lung damage by direct toxicity or indirectly through inflammation. To clarify the mechanism of direct toxicity, we briefly exposed rabbit alveolar macrophages to amiodarone and analyzed their morphology, synthesis, and degradation of dipalmitoylphosphatidylcholine (DPPC); distribution of lysosomal enzymes; and uptake of diphtheria toxin and surfactant protein (SP) A used as tracers of the endocytic pathway. Furthermore, in newborn rabbits, we studied the clearance of DPPC and SP-A instilled into the trachea together with increasing amounts of amiodarone. We found that in vitro amiodarone decreases the surface density of mitochondria and lysosomes while increasing the surface density of inclusion bodies, increases the incorporation of choline into DPPC, modifies the distribution of lysosomal enzymes, and does not affect the uptake and processing of diphtheria toxin but inhibits the degradation of SP-A. In vivo amiodarone inhibits the degradation of SP-A but not of DPPC. We conclude that the acute exposure to amiodarone perturbs the endocytic pathway acting after the early endosomes, alters the traffic of lysosomal enzymes, and interferes with the turnover of SP-A.
Cytokine treatment increases arginine metabolism and uptake in bovine pulmonary arterial endothelial cells
Authors: Nelin Leif D. , Nash Heather E. , Chicoine Louis G.
Published: December 2017

l-Arginine (l-Arg) is metabolized to nitric oxide (NO) by NO synthase (NOS) or to urea by arginase (AR). l-Arg is transported into bovine pulmonary arterial endothelial cells (BPAECs) by cationic amino acid transporter-2 (CAT-2). We hypothesized that cytokine treatment would increase l-Arg metabolism and increase CAT-2 mRNA expression. BPAECs were incubated for 24 h in medium (control) or medium with lipopolysaccharide and tumor necrosis factor-α (L-T). L-T increased nitrite production (3.1 ± 0.4 nmol/24 h vs. 1.8 ± 0.1 nmol/24 h for control; P< 0.01) and urea production (83.5 ± 29.5 nmol/24 h vs. 17.8 ± 8.6 nmol/24 h for control; P < 0.05). L-T-treated BPAECs had greater endothelial and inducible NOS mRNA expression compared with control cells. Increasing the medium l-Arg concentration resulted in increased nitrite and urea production in both the control and the L-T-treated BPAECs. L-T treatment resulted in measurable CAT-2 mRNA. L-T increasedl-[3H]Arg uptake (5.78 ± 0.41 pmol vs. 4.45 ± 0.10 pmol for control; P < 0.05). In summary, L-T treatment increased l-Arg metabolism to both NO and urea in BPAECs and resulted in increased levels of CAT-2 mRNA. This suggests that induction of NOS and/or AR is linked to induction of CAT-2 in BPAECs and may represent a mechanism for maintainingl-Arg availability to NOS and/or AR.
Differential expression of KV channel α- and β-subunits in the bovine pulmonary arterial circulation
Authors: Coppock Elizabeth A. , Tamkun Michael M.
Published: December 2017

Resistance pulmonary arteries constrict in response to hypoxia, whereas conduit pulmonary arteries typically do not respond or dilate slightly. One proposed mechanism for this differential response is the variable expression of pulmonary arterial smooth muscle cell voltage-gated K+ (KV) channel subunits (Kv1.2, Kv2.1, Kv1.5, and Kv3.1b) shown to be O2 sensitive in heterologous expression systems. In this study, immunoblotting and immunohistochemistry were used to examine the expression of KV channel α- and β-subunits in the bovine pulmonary arterial circulation to determine whether differential KVchannel subunit distribution is responsible for the distinct sensitivities of pulmonary arteries to hypoxia. Surprisingly, there was little difference in the expression levels of Kv1.2, Kv1.5, and Kv2.1 between conduit and resistance pulmonary arteries. In contrast, expression of the Kv3.1b α-subunit and Kvβ.1, Kvβ1.2, and Kvβ1.3 accessory subunits dramatically increased along the pulmonary arterial tree. The differential expression of all the β-subunits but of only one of the putative O2-sensitive α-subunits suggests that the α-subunits alone are not the O2 sensors but further implicates the auxiliary β-subunits in pulmonary arterial O2 sensing.
Combination of nonspecific PDE inhibitors with inhaled prostacyclin in experimental pulmonary hypertension
Authors: Schermuly Ralph Theo , Roehl Axel , Weissmann Norbert , Ghofrani Hossein Ardeschir , Leuchte Hanno , Grimminger Friedrich , Seeger Werner , Walmrath Dieter
Published: December 2017

Inhalation of aerosolized prostacyclin (PGI2) exerts selective pulmonary vasodilation, but its effect is rapidly lost after termination of nebulization. Amplification of the vasodilatory response to inhaled PGI2 might be achieved by phosphodiesterase (PDE) inhibitors to stabilize its second messenger, cAMP. We established stable pulmonary hypertension in perfused rabbit lungs by continuous infusion of U-46619. Short-term (10-min) aerosolization maneuvers of PGI2 effected a rapid, moderate decrease in pulmonary arterial pressure, with post-PGI2 vasorelaxation being lost within 10–15 min, accompanied by a marginal reduction in shunt flow. Preceding administration of subthreshold doses of the PDE inhibitors theophylline, dipyridamole, and pentoxifylline via the intravascular or inhalational route, which per se did not influence pulmonary hemodynamics, caused more than doubling of the immediate pulmonary arterial pressure drop in response to PGI2 and marked prolongation of the post-PGI2 vasorelaxation to >60 min (all PDE inhibitors via both routes of application). This was accompanied by a reduction in shunt flow in the case of aerosolized theophylline (27.5%), pentoxifylline (30.5%), and dipyridamole (33.4%). Coaerosolization of PGI2 and PDE inhibitors may be considered as a therapeutic strategy in pulmonary hypertension.
Oxygen-induced fetal pulmonary vasodilation is mediated by intracellular calcium activation of KCa channels
Authors: Porter Valerie A. , Rhodes Michael T. , Reeve Helen L. , Cornfield David N.
Published: December 2017

O2 sensing in fetal pulmonary artery smooth muscle is critically important in the successful transition to air breathing at birth. However, the mechanism by which the fetal pulmonary vasculature senses and responds to an acute increase in O2tension is not known. Isolated fetal pulmonary artery smooth muscle cells were kept in primary culture for 5–14 days in a hypoxic environment (20–30 mmHg). These cells showed a 25.1 ± 1.7% decrease in intracellular calcium in response to an acute increase in O2 tension. Low concentrations of caffeine (0.5 mM) and diltiazem also decreased intracellular calcium. The decrease in intracellular calcium concentration in response to increasing O2 was inhibited by iberiotoxin and ryanodine. Freshly isolated fetal pulmonary artery smooth muscle cells exhibited “spontaneous transient outward currents,” indicative of intracellular calcium spark activation of calcium-sensitive potassium channels. The frequency of spontaneous transient outward currents increased when O2 tension was increased to normoxic levels. Increasing fetal pulmonary O2 tension in acutely instrumented fetal sheep increased fetal pulmonary blood flow. Ryanodine attenuated O2-induced pulmonary vasodilation. This study demonstrates that fetal pulmonary vascular smooth muscle cells are capable of responding to an acute increase in O2tension and that this O2 response is mediated by intracellular calcium activation of calcium-sensitive potassium channels.
Acute cigarette smoke exposure induces apoptosis of alveolar macrophages
Authors: Aoshiba Kazutetsu , Tamaoki Jun , Nagai Atsushi
Published: December 2017

Alveolar macrophages (AMs) may play a critical role in cigarette smoke (CS)-related pulmonary diseases. This study was designed to determine whether CS induces apoptosis of AMs. In in vitro studies, mouse, rat, and human AMs and human blood monocyte-derived macrophages cultured with aqueous whole CS extracts underwent apoptosis that was detected by light and electron microscopy and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling. The gas phase of CSE did not cause apoptosis. The CS-induced apoptosis was associated with increased oxidative stress, Bax protein accumulation, mitochondrial dysfunction, and mitochondrial cytochrome c release but was independent of p53, Fas, and caspase activation. This apoptosis was inhibited by antioxidants such as glutathione, ascorbic acid, and α-tocopherol. In in vivo studies where rats were exposed to the smoke from 10 cigarettes over 5 h in an exposure chamber, ∼3% of AMs obtained by bronchoalveolar lavage after 24 h showed apoptosis. These results suggest that acute CS exposure is capable of inducing apoptosis of AMs.
Free radicals in hypoxic rat diaphragm contractility: no role for xanthine oxidase
Authors: Heunks Leo M. A. , Machiels Herwin A. , de Abreu Ronney , Ping Zhu Xiao , van der Heijden Henricus F. M. , Dekhuijzen P. N. Richard
Published: December 2017

Recent evidence indicates that hypoxia enhances the generation of oxidants. Little is known about the role of free radicals in contractility of the rat diaphragm during hypoxia. We hypothesized that antioxidants improve contractility of the hypoxic rat diaphragm and that xanthine oxidase (XO) is an important source of free radicals in the hypoxic diaphragm. The effects of N-acetylcysteine (NAC; 18 mM), Tiron (10 mM), and the XO inhibitor allopurinol (250 μM) were studied on isometric and isotonic force generation during hypoxia (Po2∼7 kPa). NAC and Tiron decreased maximal force generation, slowed the shortening velocity, and decreased the power output. Fatigue rate was decreased in the presence of either NAC or Tiron. Allopurinol did not alter the contractility or fatigability of the diaphragm. During hyperoxia (Po2∼85 kPa), neither NAC nor allopurinol affected the contractility or fatigability of the diaphragm. Thus free radicals play a significant role in diaphragm contractility during hypoxia. Whether antioxidants exert a beneficial or harmful effect on muscle performance depends on the contraction pattern of the muscle. Free radicals generated by XO do not play a role in diaphragm contractility during either hypoxia or hyperoxia.
Regulation of SP-B and SP-C secretion in rat type II cells in primary culture
Authors: Gobran Laurice I. , Rooney Seamus A.
Published: December 2017

Secretion of lung surfactant phospholipids is a highly regulated process. A variety of physiological and pharmacological agents stimulate surfactant phospholipid secretion in isolated type II cells. Although the lipid and hydrophobic protein components of surfactant are believed to be secreted together by exocytosis of lamellar body contents, regulation of surfactant protein (SP) B and SP-C secretion has not previously been examined. To address the question of whether secretion of SP-B and SP-C is stimulated by the same agonists that stimulate phospholipid secretion, we measured secretion of all four SPs under the same conditions used to measure phosphatidylcholine secretion. Freshly isolated rat type II cells were cultured overnight and exposed to known surfactant phospholipid secretagogues for 2.5 h, after which the amounts of SP-A, SP-B, SP-C, and SP-D in the medium were measured with immunoblotting. Secretion of SP-B and SP-C was stimulated three- to fivefold by terbutaline, 5′-( N-ethylcarboxyamido)adenosine, ATP, 12- O-tetradecanoylphorbol 13-acetate, and ionomycin. Similar to their effects on phospholipid secretion, the stimulatory effects of the agonists were abolished by Ro 31-8220. Secretion of SP-A and SP-D was not stimulated by the secretagogues tested. We conclude that secretion of the phospholipid and hydrophobic protein components of surfactant is similarly regulated, whereas secretion of the hydrophilic proteins is regulated differently.
Homogeneous segmental profile of carbon monoxide-mediated pulmonary vasodilation in rats
Authors: Naik Jay S. , Walker Benjimen R.
Published: December 2017

Carbon monoxide (CO) has been proposed to attenuate the vasoconstrictor response to local hypoxia that contributes to pulmonary hypertension. However, the segmental response to CO, as well as its mechanism of action in the pulmonary circulation, has not been fully defined. To investigate the hemodynamic response to exogenous CO, lungs from male Sprague-Dawley rats were perfused with physiological saline solution. Measurements were made of pulmonary arterial, venous, and capillary pressures. Lungs were constricted with the thromboxane mimetic U-46619. To examine the vasodilatory response to CO, 500 μl of CO-equilibrated physiological saline solution or vehicle were injected into the arterial line. Additionally, CO and vehicle responses were examined in the presence of the soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3- a]quinoxalin-1-one (ODQ; 10 μM) or the larger conductance calcium-activated K+(BKCa) channel blockers tetraethylammonium chloride (10 mM) and iberiotoxin (100 nM). CO administration decreased vascular resistance to a similar degree in both vascular segments. This vasodilatory response was completely abolished in lungs pretreated with ODQ. Furthermore, CO administration increased whole lung cGMP content, which was prevented by ODQ. Neither tetraethylammonium chloride nor iberiotoxin affected the CO response. We conclude that exogenous CO administration causes vasodilation in the pulmonary vasculature via a soluble guanylyl cyclase-dependent mechanism that does not likely involve activation of KCachannels.
Mechanisms of sodium fluoride-induced endothelial cell barrier dysfunction: role of MLC phosphorylation
Authors: Wang Peiyi , Verin Alexander D. , Birukova Anna , Gilbert-McClain Lydia I. , Jacobs Keri , Garcia Joe G. N.
Published: December 2017

NaF, a potent G protein activator and Ser/Thr phosphatase inhibitor, significantly increased albumin permeability and decreased transcellular electrical resistance (TER), indicating endothelial cell (EC) barrier impairment. EC barrier dysfunction induced by NaF was accompanied by the development of actin stress fibers, intercellular gap formation, and significant time-dependent increases in myosin light chain (MLC) phosphorylation. However, despite rapid, albeit transient, activation of Ca2+/calmodulin-dependent MLC kinase (MLCK), the specific MLCK inhibitor ML-7 failed to affect NaF-induced MLC phosphorylation, actin cytoskeletal rearrangement, and reductions in TER, suggesting a limited role of MLCK in NaF-induced EC activation. In contrast, strategies to reduce Rho (C3 exoenzyme or toxin B) or to inhibit Rho-associated kinase (Y-27632 or dominant/negative RhoK) dramatically reduced MLC phosphorylation and actin stress fiber formation and significantly attenuated NaF-induced EC barrier dysfunction. Consistent with this role for RhoK activity, NaF selectively inhibited myosin-specific phosphatase activity, whereas the total Ser/Thr phosphatase activity remained unchanged. These data strongly suggest that MLC phosphorylation, mediated primarily by RhoK, and not MLCK, participates in NaF-induced EC actin cytoskeletal changes and barrier dysfunction.
Acute lung injury and lung transplantation influence in vitro subtype conversion of pulmonary surfactant
Authors: Maitra Gayatri , Inchley Kevin , Novick Richard J. , Veldhuizen Ruud A. W. , Lewis James F. , Possmayer Fred
Published: December 2017

The effects of surfactant treatment on surfactant subtype conversion after lung injury were examined. Dogs were subjected to hyperventilation for 8 h with or without surfactant treatment. Lungs were stored for 17 h, and the right lung was transplanted and reperfused for 6 h. Conversion of large aggregate (LA) surfactant to small aggregates was investigated using in vitro surface area cycling. LA from transplanted lungs (Transplant-LA) from the nontreated group converted more rapidly than Transplant-LA from the treated group. Transplant-LA from both groups converted more rapidly than LA from normal lungs. Calculations based on [3H]dipalmitoylphosphatidylcholine in the administered surfactant [bovine lipid extract surfactant (BLES)] showed that the endogenous component of Transplant-LA converted more rapidly than the exogenous component. This indicates exogenous BLES did not equilibrate completely with endogenous surfactant. LA from hyperventilated, stored donor right lungs and from the recipients' native lungs from the nontreated group converted more rapidly than corresponding LA in the BLES-treated group. Similar relative conversions were observed with exogenous components from all lungs. Relative conversion of endogenous component from Transplant-LA was more rapid than that from LA from donor's stored right lung or from the recipient's native right lung. Low levels of phenylmethylsulfonyl fluoride inhibited conversion of Transplant-LA to a greater extent than normal LA. LA from all experimental groups had similar protein levels. These studies show acute lung injury, transplant, ischemia-reperfusion, and surfactant treatment have major effects on surfactant subtype integrity.
DANCE in developing and injured lung
Authors: Jean Jyh-Chang , Eruchalu Ifeanyi , Cao Yu Xia , Joyce-Brady Martin
Published: December 2017

We identified rat developing arteries and neural crest derivatives with multiple epidermal growth factor-like domains ( DANCE) as a developmentally regulated gene using suppression-subtractive hybridization. Northern analysis confirmed a fivefold induction of this mRNA transcript between fetal day 18 and 20 that persisted through postnatal day 17. The level was declining at postnatal day 21 and was similar in adult lung to that at fetal day 18. In adults DANCE mRNA abundance was highest in lung, kidney, and spleen, lower in heart, skeletal muscle, and brain, but absent from liver and thymus. It was abundant in pulmonary artery endothelium and a lung epithelial type 2 cell line, barely detectable in vascular smooth muscle, and absent in fibroblasts. In situ hybridization revealed a regulated pattern of expression in endothelial cells of fetal, postnatal, and adult lung. Because DANCE mRNA was inducible in systemic arteries during recovery from injury, we searched for induction in lung injured by hyperoxia. Mouse DANCE mRNA abundance was unchanged during an acute 3-day exposure period, induced threefold 5 days into the recovery phase, and returned to baseline at days 8, 11, and 14. In situ hybridization at day 5 suggested a diffuse pattern of induction. DANCE may play a role in lung endothelial cell biology during development repair after injury.
Myosin cross-bridge kinetics in airway smooth muscle: a comparative study of humans, rats, and rabbits
Authors: Lecarpentier Y. , Blanc F.-X. , Salmeron S. , Pourny J.-C. , Chemla D. , Coirault C.
Published: December 2017

To analyze the kinetics and unitary force of cross bridges (CBs) in airway smooth muscle (ASM), we proposed a new formalism of Huxley's equations adapted to nonsarcomeric muscles (Huxley AF. Prog Biophys Biophys Chem7: 255–318, 1957). These equations were applied to ASM from rabbits, rats, and humans ( n = 12/group). We tested the hypothesis that species differences in whole ASM mechanics were related to differences in CB mechanics. We calculated the total CB number per square millimeter at peak isometric tension (Ψ ×109), CB unitary force (Π), and the rate constants for CB attachment ( f 1) and detachment ( g 1 and g 2). Total tension, Ψ, and Π were significantly higher in rabbits than in humans and rats. Values of Π were 8.6 ± 0.1 pN in rabbits, 7.6 ± 0.3 pN in humans, and 7.7 ± 0.2 pN in rats. Values of Ψ were 4.0 ± 0.5 in rabbits, 1.2 ± 0.1 in humans, and 1.9 ± 0.2 in rats; f 1 was lower in humans than in rabbits and rats; g 2 was higher in rabbits than in rats and in rats than in humans. In conclusion, ASM mechanical behavior of different species was characterized by specific CB kinetics and CB unitary force.
Ectopic expression of IL-5 identifies an additional CD4+T cell mechanism of airway eosinophil recruitment
Authors: Crosby Jeffrey R. , Shen H. H. , Borchers M. T. , Justice J. P. , Ansay T. , Lee J. J. , Lee N. A.
Published: December 2017

CD4+T cells have a critical role in the development of allergic pulmonary inflammation, including the recruitment of eosinophils to the airway lumen and interstitium. The expression of interleukin (IL)-5 by CD4+cells has, in particular, often been lionized as the central link between allergic inflammation and the concomitant expansion or recruitment of eosinophils. The mechanism(s) by which CD4+T cells mediates eosinophil recruitment was assessed with gene knockout mice deficient for T cells or T cell subtypes and a unique IL-5 transgenic mouse (line NJ.1726) that constitutively overexpresses this cytokine in the lung epithelium. Pulmonary IL-5 expression is significantly attenuated in T cell- and CD4+but not CD8+cell-deficient animals, suggesting an obvious explanation for the lack of eosinophils in the lungs of T cell-deficient and CD4(−/−) mice. However, although the constitutive expression of IL-5 in the lung epithelium of NJ.1726 mice elicited an eosinophilia in the airway lumen of both naive and ovalbumin-treated mice, in the absence of CD4+cells, allergen-mediated eosinophil recruitment to the bronchoalveolar lavage fluid was abolished. Moreover, intranasal instillation of the potent eosinophil-specific chemokine eotaxin-2 was incapable of eliciting eosinophil recruitment in naive and ovalbumin-treated NJ.1726 CD4(−/−) mice, suggesting that eosinophil trafficking during allergic inflammatory responses is a consequence of a CD4+cell-mediated event(s) in addition to IL-5 expression and the establishment of a pulmonary chemokine gradient.
Parathyroid hormone contributes to volume expansion-induced inhibition of proximal reabsorption
Authors: Mercier O. , Bichara M. , Paillard M. , Gardin J. P. , Leviel F.
Published: December 2017

Volume expansion inhibits the proximal reabsorption of water, bicarbonate, and chloride. The present work tested a hypothetical role of parathyroid hormone (PTH) in the expansion effect. We studied 19 Sprague-Dawley rats during a plasma-replete euvolemic state and following 10% body wt colloid-free expansion. In group I, six intact rats, volume expansion decreased plasma ionized calcium concentration ([Ca2+]P) from 2.28 +/- 0.06 to 2.11 +/- 0.04 meq/liter and increased nephrogenous cAMP (NcAMP) from 29 +/- 5 to 66 +/- 10 pmol X min-1 X g kidney wt-1. In group II, six acutely thyroparathyroidectomized (TPTX) rats, [Ca2+]P also fell from 2.18 +/- 0.08 to 1.80 +/- 0.08 meq/liter but NcAMP did not rise significantly (9 +/- 3 vs. 17 +/- 5 pmol X min-1 X g kidney wt-1). These data strongly suggest that stimulation of PTH activity occurred during expansion in intact animals. In group III, seven TPTX rats, volume expansion inhibited proximal reabsorption of total CO2 by 11%, of chloride by 24%, and of water by 19%. Volume expansion-induced reduction in bicarbonate, chloride, and water reabsorption was smaller in TPTX than in intact rats previously studied. We conclude that volume expansion inhibits proximal reabsorption in part by decreasing the active transcellular NaHCO3 and NaCl transport secondary to stimulation of PTH activity.
Characterization of a new model for the study of pressure-natriuresis in the rat
Authors: Roman R. J. , Cowley A. W.
Published: December 2017

The acute relationships between the urinary excretion of sodium and water and renal perfusion pressure were characterized in the rat using a new in vivo model of pressure diuresis. Neural and hormonal influences on the kidney were held constant by denervating the kidney and by maintaining fixed high plasma levels of vasopressin, aldosterone, corticosterone, and norepinephrine levels by intravenous infusion. Renal perfusion pressure (RPP) was varied above and below control using specially designed aortic clamps. Increasing RPP within the autoregulatory range from 90 to 160 mmHg produced 5- to 20-fold increases in urine flow and sodium excretion with no detectable changes in glomerular filtration rate, renal blood flow, or peritubular capillary pressure. The slope of the line relating urine flow and RPP averaged 2 microliter X min-1 X kidney-1 X mmHg-1 rise in RPP. If RPP was first lowered to 90 mmHg, urine flow could be doubled by increasing RPP by as little as 5 mmHg. Uninephrectomy 7-12 days before an experiment did not alter the renal pressure diuresis relationship. The presence of intact renal nerves, however, was found to shift the relationship between urine flow and RPP to the right. The magnitude of the response produced by elevations in pressure using this new model of pressure diuresis was greater than when RPP was increased using carotid occlusion or epinephrine infusion as in previous studies. The results indicate that small changes in arterial pressure may have a greater influence on sodium and water excretion than has been previously recognized.
Parathyroid hormone and dibutyryl cAMP inhibit Na+/H+ exchange in renal brush border vesicles
Authors: Kahn A. M. , Dolson G. M. , Hise M. K. , Bennett S. C. , Weinman E. J.
Published: December 2017

Parathyroid hormone (PTH) and cAMP inhibit sodium, water, and bicarbonate reabsorption in the proximal tubule. We wished to determine whether these agents directly inhibit proximal tubular Na+/H+ exchange. A suspension of rabbit proximal tubules was prepared by enzymatic digestion and Ficoll gradient centrifugation. Oxygen consumption at 37 degrees C was stable over 60 min, averaged 20 nmol X mg protein-1 X min-1, and was inhibited 60% by ouabain. Over 96% of cells excluded trypan blue. From this suspension, brush border membrane vesicles were isolated. The vesicles were enriched 12.7 times in alkaline phosphatase relative to a cortical homogenate and demonstrated pH gradient-stimulated, amiloride-sensitive Na+/H+ countertransport and sodium-phosphate and sodium-D-glucose cotransport. When the tubule suspension was exposed to PTH or dibutyryl cAMP, the activity of Na+/H+ countertransport in the resultant brush border vesicles was inhibited. Neither PTH nor dibutyryl cAMP affected the amiloride-insensitive component of sodium transport or sodium-phosphate or sodium-D-glucose cotransport. The effect of PTH on Na+/H+ counter-transport could not be explained by an alteration in fluidity of the brush border membrane. These experiments demonstrate that PTH and dibutyryl cAMP directly inhibit Na+/H+ countertransport in the brush border membrane of the rabbit proximal tubule.
A model of proximal tubular bicarbonate absorption
Authors: Alpern R. J. , Rector F. C.
Published: December 2017

A model is presented that utilizes determinants of acidification defined from microperfusion studies in the rat to stimulate the effect on absolute bicarbonate absorption along the entire proximal convoluted tubule. Net bicarbonate absorption is considered to consist of active transcellular proton secretion in parallel with passive paracellular bicarbonate diffusion. The rate of proton secretion is calculated as a function of luminal bicarbonate concentration using Michaelis-Menten kinetics. The K1/2 is modified by luminal flow rate and the Vmax by peritubular bicarbonate concentration. Solute-solvent interactions and axial heterogeneity are also included as determinants of proton secretion rate. The model demonstrates that a given percentage stimulation or inhibition of active proton secretion leads to a much smaller effect on absolute proximal bicarbonate absorption along the entire tubular length. This blunting of the stimulation or inhibition is greatest when filtered bicarbonate load is limited by decreases in glomerular filtration rate or plasma bicarbonate concentration. In addition, the model shows that flow dependence is greater at low plasma bicarbonate concentrations, whereas the effect of extracellular fluid volume expansion is greater at high plasma bicarbonate concentrations. Agreement between the model predictions and the results of free-flow micropuncture studies from our laboratory is good with the exception of the effect of raising plasma bicarbonate concentration. This discrepancy is resolvable by allowing the effect of peritubular pH to increase along the length of the tubule, a hypothesis that requires verification.
Nephroimmunopathology and pathophysiology
Authors: Wilson C. B. , Blantz R. C.
Published: December 2017

Immunologic models of renal injury are useful in the study of pathophysiology. Some of these models have already been used in glomerular micropuncture studies and were shown to be approachable with the same techniques that were developed to study normal renal function. The typical decrease in the glomerular permeability coefficient found in such studies is countered by an increase in the hydrostatic pressure gradient, minimizing decreases in single nephron filtration rate. Antibody mechanisms involving either direct glomerular (and tubular) fixation of antibody or accumulation of immune complex materials provide an array of acute and chronic lesions for evaluation with relevance to the bulk of immune glomerular and tubular lesions in humans. The influences of varied and overlapping immune mediator systems are also useful areas for physiologic assessment. The tools of the renal immunopathologist may be useful to the physiologist in identifying and localizing the effects of transport systems central to renal function. The collaborative interaction of investigators skilled in immunology, pathology, and physiology is necessary to achieve optimum scientific value.
Structural adaptation of the distal convoluted tubule to prolonged furosemide treatment
Authors: Kaissling B. , Bachmann S. , Kriz W.
Published: December 2017

Rats were treated with furosemide, continuously applied by implanted minipumps, for 6 days. As compensation for the salt lost with the urine the animals drank large amounts of a salt solution ad libitum. This procedure resulted in an enlargement of kidney cortex. Light and electron microscopic studies revealed a proliferation of the distal convoluted tubule (DCT). The proportion of the DCT in the cortical tissue increased from 5.98 +/- 1.3% in controls to 9.54 +/- 1.7% (P less than 0.01) in treated rats. Basolateral cell membrane amplification in DCT cells increased from 17.33 +/- 2.9 to 38.24 +/- 5.8 (P less than 0.0001) in treated rats, while luminal membrane area per unit tubular length did not change. The structural changes after furosemide treatment in the DCT suggest an increase in active transcellular transport capacity of this segment. It is assumed that the chronically altered Na load of the tubular fluid (due to transport inhibition in the thick ascending limb of Henle's loop) delivered to the DCT may specifically stimulate the transport capacity of this segment by augmentation of basolateral cell membrane area. The changes occurring in the segment situated downstream to the one in which the function is impaired by furosemide emphasize the role of tubular fluid composition in the regulation of transport function.
High-affinity calcium binding sites in luminal and basolateral renal membranes
Authors: Talor Z. , Richison G. , Arruda J. A.
Published: December 2017

We evaluated Ca binding by highly purified luminal (L) and basolateral (BL) tubular membranes prepared from beef kidney. Ca binding was measured by using 45Ca and a rapid-filtration technique. After Ca uptake reached equilibrium, the vesicles were lysed and the amount of 45Ca retained in the membranes was considered the bound Ca. Ca binding in both membranes accounted for approx. 80% of total Ca uptake. Analysis of binding data by Scatchard plot revealed the presence of two distinct types of binding sites in both L and BL membranes. The high-affinity binding sites showed a similar affinity constant of 10(-5)M for both L and BL membranes, but the maximum number of binding sites was 0.75 and 1.6 nmol/mg protein, respectively. In contrast, the low-affinity binding sites were similar regarding affinity constant and maximum number of binding sites in the two membranes. In L and BL membranes, high-affinity binding sites were selective for Ca, as high concentrations of divalent cations were required to inhibit Ca binding. In both membranes Ca binding was inhibited by ruthenium red, LaCl3, and detergents, and it was stimulated by calmodulin inhibitors (trifluoperazine, calmidazolium), ionophore A-23187, and ATP. These results demonstrate that L and BL membranes possess high-affinity binding sites with different capacities but similar characteristics as regards affinity constant and stimulation and inhibition of binding. The data further demonstrate that most of Ca uptake by these membranes represents binding.
Biochemical characterization of renal epithelial cell cultures (LLC-PK1 and MDCK)
Authors: Gstraunthaler G. , Pfaller W. , Kotanko P.
Published: December 2017

The expression of enzymes in LLC-PK1 and MDCK cells was used to study the retention of differentiated properties of the renal epithelial cell lines by a biochemical approach. Activities of marker enzymes, for which intracellular and intranephron localization is known, were determined from crude cell homogenates of LLC-PK1 and MDCK monolayer cultures. The activity patterns of the particular enzymes found were then compared with the in vivo distribution of the enzymes along the rat nephron. LLC-PK1 cells exhibit high activities of apical membrane enzymes when compared with MDCK cells, whereas in the latter high activity of Na-K-ATPase could be detected. The activities of lysosomal enzymes, mitochondrial enzymes, and transaminases were higher in LLC-PK1 than in MDCK cells. Glycolytic enzymes, however, displayed identical activity levels in both the LLC-PK1 and MDCK cells, which may be due to the fact that these are continuous cell lines and to the culture conditions used, since glucose is a major energy source in the culture media.
Cellular heterogeneity of uridine incorporation in collecting tubules: effect of DOCA
Authors: Vandewalle A. , Cluzeaud F. , Chavance M. , Bonvalet J. P.
Published: December 2017

In previous studies we showed that in vitro uridine incorporation along the renal tubule is heterogeneous and that DOCA induces a stimulation of RNA synthesis in distal cortical and medullary structures. The present work examines by autoradiography of isolated tubules and renal tissue sections the cellular heterogeneity of the connecting (CNT) and cortical collecting (CCT) tubules after in vivo injection of [3H]uridine in normal and DOCA-treated rabbits. Data confirmed the profile of uridine incorporation along the tubule, which was found in in vitro experiments, and the DOCA-induced stimulation of RNA synthesis. In microdissected CNT and CCT of control kidneys, statistical analysis of the distribution of labeling revealed the presence of two distinct cell populations: one with low labeling (2-3 silver grains per nucleus) and one with high labeling (10-13), which represent 64 and 36%, respectively (CNT), and 74 and 26%, respectively (CCT), of the whole population. Histological data showed that the respective proportions of intercalated cells (29% in CNT; 21% in CCT) and connecting tubule cells (65%) or principal cells (79%) are close to those of the populations with high or low labeling. In addition, autoradiographs on renal sections directly demonstrated that the labeling of intercalated cells (19.3 silver grains/100 micron2 nuclear surface in CNT; 14.7 in CCT) was three times higher than that of connecting (6.6) or principal (5.8) cells. In isolated CNT and CCT, DOCA induced similar absolute increases in the labeling of the two populations. However, the relative increase was more than two times higher in the population with low labeling (+131% in CNT, +210% in CCT) than in the one with high labeling (+71% and +98%). We conclude that cell population of the collecting cortical tubule (CNT and CCT) is heterogeneous with regard to uridine incorporation, reflecting RNA synthesis.
Maleate-induced stimulation of glutamine metabolism in the intact dog kidney
Authors: Gougoux A. , Vinay P. , Duplain M.
Published: December 2017

Studies were performed in anesthetized normal dogs to evaluate the effects of maleate on renal metabolism. Intravenous administration of maleate (50 mg/kg) markedly increased urinary excretion of glutamine, glutamate, alpha-ketoglutarate, alanine, lactate, pyruvate, and citrate. Despite a fourfold rise in renal cortical concentration of alpha-ketoglutarate, glutamine utilization expressed per 100 ml glomerular filtration rate almost doubled following maleate administration, whereas total ammonia production increased threefold, most of this ammonia being diverted into the renal vein. The renal production of alpha-ketoglutarate rose in a spectacular fashion and was almost equal to the renal utilization of glutamine, indicating a metabolic block at the alpha-ketoglutarate dehydrogenase step. Maleate reduced renal alanine production but did not change lactate utilization. These findings suggest that 1) in the intact dog the mitochondrial entry of glutamine is not regulated only by alpha-ketoglutarate; 2) the deamination of glutamate into alpha-ketoglutarate is accelerated by maleate, probably through an impaired mitochondrial NADH production; 3) the resulting decrement in intramitochondrial glutamate concentration deinhibits the phosphate-dependent glutaminase.
Stimulation of distal potassium secretion by low lumen chloride in the presence of barium
Authors: Ellison D. H. , Velazquez H. , Wright F. S.
Published: December 2017

Potassium secretion into the renal distal tubule is increased when chloride in the tubule fluid is replaced by another anion. The present experiments were done to determine whether this increment in transported potassium traverses a conductive pathway from cell to lumen. Transport rates of potassium, sodium, chloride, and fluid by the renal distal tubule of rats were examined in vivo by continuous microperfusion. The effects of substituting gluconate for chloride in the presence and absence of 5 mM barium in the perfusion fluid were determined. When gluconate replaced chloride in the perfusion solutions, potassium secretion increased (by 44%) without a significant change in transepithelial voltage. Barium in the lumen increased the magnitude of the lumen-negative transepithelial voltage (by 30%) and reduced potassium secretion (by 56%) by inhibiting conductive potassium movement. Barium also decreased both sodium (by 51%) and chloride (by 37%) absorption. Barium did not reduce the stimulation of potassium secretion caused by reducing lumen chloride concentration. Potassium secretion increased (by 77%) when lumen chloride was reduced in the presence of 5 mM barium. We interpret these results by postulating that a cotransport mechanism linking potassium and chloride is present in the luminal membrane of distal tubule cells, that this mechanism operates in parallel with a conductive transport pathway for potassium, and that the K-Cl cotransport mechanism is not inhibited by barium.
In vitro versus in vivo mitochondrial calcium loading in ischemic acute renal failure
Authors: Arnold P. E. , Lumlertgul D. , Burke T. J. , Schrier R. W.
Published: December 2017

Progressive mitochondrial Ca2+ accumulation and respiratory dysfunction have been observed during reperfusion after renal ischemia. The present study demonstrated that normal mitochondria, isolated in the presence of high Ca2+ concentrations, are capable of accumulating large amounts of Ca2+ in vitro and exhibit depressed respiratory rates. Since mitochondria isolated from reperfused ischemic tissue may be exposed to high concentrations of Ca2+ during the isolation procedure, the present study examined the effect of in vitro versus in vivo mitochondrial Ca2+ loading on mitochondrial function during ischemic acute renal failure (ARF) in anesthetized rats. When ruthenium red was added during isolation to prevent mitochondrial Ca2+ exchange with the medium, mitochondrial Ca2+ increased from 10.8 +/- 0.3 to 65.6 +/- 11.6 nmol/mg (P less than 0.001) after 24 h of postischemic reperfusion; this resulted in a 47% reduction in the acceptor-control ratio (ACR) from 4.19 +/- 0.09 to 2.70 +/- 0.13 (P less than 0.001). These data were compared with an increase in mitochondrial Ca2+ from 52.5 +/- 2.9 to 167.6 +/- 25.4 nmol/mg (P less than 0.001) and a 95% fall in ACR (3.84 +/- 0.40 to 1.15 +/- 0.08, P less than 0.001) at 24 h of reperfusion when no ruthenium red was added. However, at each time point examined, in vivo mitochondrial Ca2+ accumulation was shown to account for 50% or more of the mitochondrial respiratory dysfunction observed during ischemic ARF.
Dynamics of glomerular filtration in the river lamprey, Lampetra fluviatilis L
Authors: McVicar A. J. , Rankin J. C.
Published: December 2017

Hydrostatic pressures decreased in the dorsal aorta (from 21.6 +/- 2.0 to 18.7 +/- 0.2 mmHg), proximal (from 19.5 +/- 1.6 to 16.2 +/- 1.7 mmHg) and distal (from 16.7 +/- 0.7 to 13.2 +/- 0.9 mmHg) renal arteries, and glomerular capillaries (from 16.1 +/- 1.1 to 12.3 +/- 0.6 mmHg) of anesthetized lampreys transferred from freshwater to isosmotic 20-30% seawater. Maximal vascular resistance appeared to be in the efferent arteriole; there was a 67% decrease in pressure between glomerular and peritubular capillaries. Plasma oncotic pressure was unchanged. The calculated afferent effective filtration pressure decreased by 87% after transfer and showed a good correlation with single nephron filtration rate. Effective renal plasma flow was high but variable in freshwater lampreys and decreased by 84% after transfer, but glomerular filtration rate did not decrease proportionately and there was a nonsignificant increase in mean filtration fraction from 0.045 +/- 0.022 to 0.080 +/- 0.021. Calculation of glomerular efferent oncotic pressure indicated that filtration equilibrium did not exist in freshwater lampreys but was attained after transfer. The mean coefficient of filtration of freshwater lampreys was 0.028 +/- 0.002 nl X s-1 X mmHg-1.
Tubuloglomerular feedback activity in virgin and 12-day-pregnant rats
Authors: Baylis C. , Blantz R. C.
Published: December 2017

Tubuloglomerular feedback activity was evaluated by micropuncture and microperfusion techniques in virgin and 12-day-pregnant Munich-Wistar rats. Plasma volume increases in pregnancy, which could suppress feedback activity, thus contributing to the rise in glomerular filtration rate observed in normal midterm pregnancy. Late proximal tubules were microperfused at 0, 10, 20, and 40 nl/min and the resulting filtration rate in the same nephron was evaluated. Nephron filtration rate (SNGFR) in proximal and distal tubules of other nephrons was also measured to assess the degree of activation of the tubuloglomerular feedback system and the relation of the spontaneous (normal) late proximal flow rate and SNGFR (distal tubule collections). SNGFR decreased significantly (from the 0 nl/min perfusion value) when late proximal tubules were perfused at 20 and 40 nl/min in both virgin and 12-day-pregnant rats. Tubuloglomerular feedback activity was not suppressed in pregnancy, but the relationship between SNGFR and late proximal tubule perfusion rate was reset for a higher value for SNGFR. The difference between proximal and distal SNGFR suggests that the feedback system was more activated in the virgin than in the pregnant rat. Thus, in spite of the known increases in plasma volume that occur in pregnancy, the kidney does not sense this volume expansion as a stimulus to suppress feedback activity.
Effect of barium on cell volume regulation in rabbit proximal straight tubules
Authors: Welling P. A. , Linshaw M. A. , Sullivan L. P.
Published: December 2017

Rabbit proximal straight tubules swell abruptly when exposed to hypotonic medium but then shrink in a few minutes as they approach their base-line volume following loss of solute and water. Potassium, the major intracellular cation, as well as sodium, is lost during this process. In the present experiments, we studied hypotonic cell volume regulation in the presence of barium, an agent reported to decrease potassium permeability. Exposure to BaCl2 significantly prolonged hypotonic volume recovery in a dose-dependent manner. Tubules depleted of potassium and loaded with sodium chloride by exposure to 10(-4) M ouabain for 1 h swelled osmometrically and subsequently volume regulated in dilute medium. Volume regulation in such tubules is a consequence of transbasement membrane hydrostatic forces. By contrast, tubules similarly loaded with sodium, but also exposed to 10(-3) M BaCl2, volume regulated only minimally in dilute medium, suggesting BaCl2 might also affect sodium movement. However, hypotonic volume regulation was restored in sodium-loaded BaCl2-treated tubules when cells were more effectively depleted of potassium by incubation in 0-mM potassium medium. We conclude that barium retards hypotonic volume regulation primarily because of its effect on potassium movement.