bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of folate uptake in guinea pig placenta
Authors: Sweiry J. H. , Yudilevich D. L.
Published: December 2017

Trophoblast uptake of folate and methotrexate (MTX) was investigated in an in situ or dually perfused (maternal and fetal side) guinea pig placenta by using a single-circulation, paired-tracer technique. For [3H]folate, uptake into trophoblast was rapid (s), high (60–80%) and Na+ independent, and exhibited negligible efflux on both poles of placenta. [3H]folate uptake could be inhibited by folate or 5-methyltetrahydrofolate (CH3THF) but not by equimolar (0.1 microM) MTX, folinic acid, aminopterin, trimoprim, or adenine when these compounds were present in perfusate. Inhibitory effect of folate was time dependent, and its complete reversal by folate-free perfusion required up to 20 min. This suggests the presence of a high-affinity folate carrier that exhibits a slow rate of self exchange. A sudden (bolus) increase of 10 microM folate of CH3THF caused a 70–80% inhibition of [3H]folate uptake, whereas folinic acid, MTX, and trimoprim were two- to threefold less effective. [3H]folate uptake was insensitive to DIDS, SITS, nicotine, ethanol, or phenytoin. For [3H]MTX, uptake was high (60–80%) on both sides of trophoblast, however, as distinct from [3H]folate, rapid and complete efflux followed the initial uptake. [3H]MTX uptake was not inhibited by 0.1 microM MTX, but equimolar folate or CH3THF were highly effective (90%) inhibitors; higher concentration (1 microM) of MTX reduced [3H]MTX uptake by 58%. Transplacental transfer of [3H]folate or [3H]MTX in excess of the leak pathway marker in either direction was not observed. Inhibition obtained by highly concentrated substrate bolus injections indicates saturation (less than 2 microM) of membrane folate carrier.(ABSTRACT TRUNCATED AT 250 WORDS)
Vascular smooth muscle Na+-H+ exchanger kinetics and its activation by angiotensin II
Authors: Vallega G. A. , Canessa M. L. , Berk B. C. , Brock T. A. , Alexander R. W.
Published: December 2017

We have studied the kinetic properties of basal and angiotensin II (ANG II)-stimulated Na+-H+ exchange in cultured rat aortic smooth muscle cells. Initial rates of 22Na+ influx were measured in the presence of ouabain (1 mM) and bumetanide (0.1 mM) with and without amiloride after intracellular acidification by preincubation in Na-free media. The kinetics of amiloride (100 microM)-sensitive Na+ influx were studied under the following conditions: 1) constant intracellular pH (pHi; 6.8) and varying external Na+ (Na+o), which gave a Km of 23.6 +/- 2.0 (SD, n = 3) mM and a maximum velocity (Vmax) of 25 nmol.mg protein-1.min-1 (varying the amiloride concentration gave a Ki of 22 microM for inhibition under these conditions); and 2) constant Na+o (100 mM) and varying pHi (from 7.4 to 6.2), which indicated that amiloride-sensitive Na+ influx was stimulated by cell acidification when an outward H+ gradient was imposed. ANG II-stimulated amiloride-sensitive Na+ influx for up to 30 min with a half-maximal activation 10(-8) M. The pHi dependence from cell pH (pHi 7.2-6.2) of amiloride-sensitive Na+ influx stimulated by ANG II was similar to that of the basal values, a finding indicating that ANG II did not change the affinity of Na+-H+ exchange for intracellular H+. However, at pHi 6.8, ANG II increased the Vmax of amiloride-sensitive Na+ influx from 25 to 33 nmol.mg protein-1.min-1 and markedly decreased the Km for Na+o from 23.6 +/- 7.4 to 3.7 (SD, n = 4; P less than 0.005) mM.(ABSTRACT TRUNCATED AT 250 WORDS)
Sarcomere length and joint kinematics during torque production in frog hindlimb
Authors: Lieber R. L. , Boakes J. L.
Published: December 2017

The relationship between semitendinosus muscle force and knee joint kinematics during isometric torque production was examined in the frog (Rana pipiens) hindlimb. Passive muscle sarcomere length was monitored by laser diffraction during knee rotation, and joint center of rotation was determined later using principles of rigid body kinematics. Contractile force at the distal tibia, resulting from semitendinosus contraction, was also measured, and, using the kinematic data, a torque vs. joint angle curve constructed. Muscle sarcomere length varied from 3.6 micron at full knee extension to 2.0 micron at full knee flexion. Effective lever arm varied almost as a sine function, with optimal lever arm at 90 degrees of flexion. Joint torque increased linearly from 0 to 140 degrees of flexion and then sharply decreased to 160 degrees of flexion. Thus the optimal joint angle occurred at an angle (140 degrees) that was neither the angle at which muscle force was maximum (160 degrees) nor the angle at which the effective lever arm was maximum (90 degrees). These data indicate that knee torque production in the frog results from the interaction between muscular and joint properties and not either property alone.
Osmoregulation by slow changes in aldose reductase and rapid changes in sorbitol flux
Authors: Bagnasco S. M. , Murphy H. R. , Bedford J. J. , Burg M. B.
Published: December 2017

Renal medullary extracellular NaCl concentration is high during antidiuresis. To compensate, the cells accumulate large amounts of nonperturbing, osmotically active solutes (organic “osmolytes”), including sorbitol. GRB-PAP1 is a continuous line of epithelial cells from rabbit inner medulla. These cells accumulate sorbitol when medium NaCl concentration is elevated. The accumulation involves increase in aldose reductase, which catalyzes production of sorbitol from glucose. The purpose of the present study was to investigate control of cell sorbitol once aldose reductase was induced. We measured cell sorbitol, cell-to-medium sorbitol flux, and aldose reductase in cells grown in medium made hyperosmotic (600 mosmol/kg) with added NaCl and at intervals after medium osmolality was reduced to 300 mosmol/kg. In the hyperosmotic medium, cell sorbitol averaged 990 mmol/kg protein (approximately 260 mM), and its flux into the medium was 740 mmol.kg cell protein-1.day-1 (permeability less than 2 X 10(-9) cm/s). Within 5 min after return to isosmotic medium, sorbitol efflux increased greater than 150-fold. By the end of 1 day, cell sorbitol fell 77% but aldose reductase decreased only 10%. Aldose reductase then fell slowly to low levels over 2 wk. Thus renal medullary cells, chronically adapted to high NaCl, reduced their sorbitol level on return to isosmotic conditions by at least two mechanisms: 1) rapid increase in sorbitol flux into the medium, and 2) slow changes in the amount of aldose reductase.
Calcium currents in isolated canine airway smooth muscle cells
Authors: Kotlikoff M. I.
Published: December 2017

Canine tracheal smooth muscle cells were enzymatically dissociated, and individual myocytes were voltage clamped through use of the whole cell, patch-clamp method. Cells dialyzed with solutions high in potassium and bathed in physiological saline demonstrated brief inward currents, followed by large outward currents that inactivated very slowly. When outward currents were blocked, a voltage-activated inward current was observed that activated with depolarizations to voltages positive to -45 mV, with an apparent reversal potential greater than 110 mV, and a peak current at 15 mV. This current was identified as a calcium current on the basis of 1) its presence under conditions in which calcium was the only permeant cation, 2) the lack of a blocking effect of 2 microM tetrodotoxin, and 3) block of the current by Mn2+, Cd2+, and CO2+. Increases in external calcium concentration from 2 to 20 mM resulted in an increase in current amplitude and a shift of voltage activation toward more positive potentials. The current displayed a rapid inactivation phase with a time constant of 16-52 ms, which was well fit by a single exponential. Steady-state inactivation of the calcium current was sigmoidal, with a voltage of half inactivation of -21 mV in 20 mM Ca2+. The principle component of the calcium current was further identified as a transient current on the basis of its rapid inactivation, current-voltage characteristics, and relative insensitivity to dihydropyridine calcium channel blocking agents.
Human alveolar macrophage arachidonic acid metabolism
Authors: Brown G. P. , Monick M. M. , Hunninghake G. W.
Published: December 2017

Metabolites of arachidonic acid are potent modulators of many biological events, and their release from macrophages appears to play an important role in immune and inflammatory processes. In addition, metabolites of the cyclooxygenase or lipoxygenase pathway exhibit distinct biological effects. We used a method to determine if human alveolar macrophages (HAM) could be selectively activated to release products of cyclooxygenase or lipoxygenase pathway of arachidonic acid. HAM obtained by bronchoalveolar lavage from individuals were [3H]arachidonic acid labeled and then stimulated with lipopolysaccharide (LPS) or Ca ionophore A23187. Essentially no arachidonate metabolites were released by unstimulated cells. LPS caused dose- and time-dependent release of arachidonate and only cyclooxygenase products; no lipoxygenase products were detected, even in presence of cyclooxygenase inhibition. Metabolites released in response to LPS included thromboxane B2, prostaglandins D2, F2a, E2, and hydroxyheptadecatrienoic acid. A23187 caused a rapid release of arachidonate and 5-lipoxygenase products, leukotriene B4 and 5-hydroxyeicosatetraenoic acid; no cyclooxygenase inhibition. This demonstrates that HAM are specifically activated to release metabolites derived from cyclooxygenase or lipoxygenase pathway of arachidonic acid. Additionally, shunting down an alternate pathway is not induced by use of inhibitors of either pathway. This suggests alveolar macrophages may enhance or suppress various inflammatory or immune processes in lung, in part, by selective release of various derivatives of arachidonic acid.
Internalization and catabolism of insulin by an established renal cell line
Authors: Yagil C. , Frank B. H. , Rabkin R.
Published: December 2017

Proximal renal tubules are a key site of insulin metabolism. To explore the kinetics and metabolic requirements of insulin internalization and catabolism, we used the opossum kidney cell line, which has proximal tubular-like features and possesses insulin-specific receptors. Internalization was determined by separating membrane-bound insulin from intracellular insulin by exposure to an acidified medium. Internalization of membrane-bound insulin was rapid, and half-maximal internalization occurred within 2.5 min. Degradation products did not accumulate in the cell but appeared in the medium after a delay of 5 min from the onset of internalization. In other experiments, addition of KCN (2 mM) or omission of glucose did not alter degradation, but KCN, combined with the omission of glucose, inhibited degradation by 64%. This was associated with a 240% increase in membrane-bound insulin and an 81% decrease in intracellular insulin. Accordingly, it appears that under these circumstances impaired degradation was a consequence of impaired internalization. In contrast, although 0.1 mM chloroquine, an endosomal-lysosomal inhibitor, also depressed degradation (by 57%), intracellular insulin increased fourfold, indicating failure of intracellular processing. We conclude that these cultured kidney cells rapidly internalize and degrade insulin and that internalization, a prerequisite for degradation, is dependent on energy that can be derived from anaerobic glycolysis or oxidative metabolism. Furthermore, the intracellular degradative processing of insulin by these cells involves a chloroquine-sensitive pathway.
Influence of cytosolic pH on receptor-mediated endocytosis of asialoorosomucoid
Authors: Samuelson A. C. , Stockert R. J. , Novikoff A. B. , Novikoff P. M. , Saez J. C. , Spray D. C. , Wolkoff A. W.
Published: December 2017

Inhibitors of specific steps in the endocytosis of galactose-terminating glycoproteins (asialoglycoproteins) by cultured rat hepatocytes have been described (J. Cell Biol. 98: 375-381, 1984). In particular, substitution of K+ for Na+ in the culture medium results in reduced delivery of ligand to lysosomes; ligand-receptor internalization, dissociation, and segregation remain normal. We have now demonstrated by direct microelectrode measurement that incubation of hepatocytes in K+-substituted medium results in a reduction of intracellular pH by greater than or equal to 0.5 U. In addition, we have shown that reduced intracellular pH in these cells produced by either direct (CO2 diffusion) or indirect (K+ substitution) acidification inhibits ligand delivery to lysosomes. Return of internalized ligand-receptor complex to the cell surface (diacytosis) is also inhibited by these manipulations. These studies suggest that intracellular pH may modulate specific steps involving vesicle translocation and fusion in the receptor-mediated endocytosis of asialoglycoproteins. Similar effects of direct acidification of hepatocytes by CO2 diffusion and indirect acidification by K+ substitution for Na+, on diacytosis and ligand delivery to lysosomes, suggest that K+ substitution may influence these events by altering intracellular pH.
Effect of extracellular acidosis on 45Ca uptake in isolated hypoxic proximal tubules
Authors: Burnier M. , Van Putten V. J. , Schieppati A. , Schrier R. W.
Published: December 2017

Recent in vitro studies have suggested that the presence of extracellular acidosis is protective against the development of oxygen-deprivation injury in several tissues. Because cellular Ca accumulation after renal ischemia may represent a major pathogenic event leading to cellular damage, the purpose of the present study was to examine the effect of extracellular acidosis on 45Ca uptake and desaturation in normal and hypoxic isolated rat proximal tubules. At pH 7.4, an increase in 45Ca uptake was observed in proximal tubules after 30 min of hypoxia. In addition, 45Ca desaturation was increased significantly in hypoxic tubules at pH 7.4. The alterations in 45Ca uptake and desaturation in hypoxic tubules at pH 7.4 were accompanied by significant signs of cellular injury as assessed by the amount of lactate dehydrogenase (LDH) released by the tubules at the end of the hypoxic period (22.5% above control tubules, P less than 0.01) as well as in morphological changes consistent with hypoxic cell injury. In contrast, when maintained at pH 6.9 throughout the study, no difference in 45Ca uptake or desaturation was observed between the control and hypoxic tubules; the hypoxic proximal tubules exhibited a smaller increase in LDH release (12.7% above control tubules) and did not develop the morphological changes observed at pH 7.4. Thus, during hypoxia and reoxygenation at pH 7.4, the increased 45Ca uptake may contribute in part to cellular injury in rat proximal tubules.
Gene transfer of a putative mammalian K+ channel gene from genomic and cosmid DNA
Authors: Mitas M. , Coogan C. , Gargus J. J.
Published: December 2017

We have successfully transfected mouse genomic DNA containing a mutant putative K+ channel gene into murine LMTK- cells. Incorporation of the mutant gene allowed primary transformants to express the mutant cell phenotype of growth at a subthreshold K+ concentration and, furthermore, caused these primary transformants to display the same characteristics of growth and unidirectional K+ flux as the mutant cell line, LTK-1, from which the genomic DNA was derived. A cosmid genomic library was formed from LTK-1 mutant DNA and shown by transfection experiments to contain at least one unique cosmid clone containing a functional copy of the mutant putative K+ channel gene.
Changes in expression of a functional Gi protein in cultured rat heart cells
Authors: Allen I. S. , Gaa S. T. , Rogers T. B.
Published: December 2017

The muscarinic cholinergic agonist, carbachol, and pertussis toxin were used to examine the functional status of the guanine nucleotide-binding protein that inhibits adenylate cyclase (Gi) in cultured neonatal rat heart myocytes. The isoproterenol stimulation of adenylate cyclase activity in myocyte membranes and adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in intact cells (4 days in culture) were insensitive to carbachol (0.1 mM). However, in cells cultured for 11 days, carbachol (0.1 mM) inhibited isoproterenol-stimulated cAMP accumulation by 30%. Angiotensin II (ANG II) was also found to inhibit isoproterenol-stimulated cAMP accumulation in day 11 cells in a dose-dependent manner. Pertussis toxin treatment reversed the inhibitory effects of both ANG II and carbachol, suggesting a role for Gi in the process. Carbachol binding to membranes from day 4 cells was relatively insensitive to guanine nucleotides when compared with binding to membranes from day 11 or adult cells. Furthermore, pertussis toxin-mediated 32P incorporation into a 39- to 41-kDa substrate in day 11 membranes was increased 3.2-fold over that measured in day 4 membranes. These findings support the view that, although Gi is expressed, it is nonfunctional in 4-day-old cultured neonatal rat heart myocytes and acquisition of functional Gi is dependent on culture conditions. Furthermore, the ANG II receptor can couple to Gi in heart.
Separate control of regulatory volume increase and Na+-H+ exchange by cultured renal cells
Authors: Montrose M. H. , Knoblauch C. , Murer H.
Published: December 2017

Suspensions of OK cells (a continuous epithelioid cell line from opossum kidney) are examined by electronic cell sizing, measurements of intracellular pH, and measurements of cellular Na+ and K+. The response of the cells to hypertonic solutions is evaluated in most detail. When shrunken by exposure to hyperosmotic medium (430 mosmol/kg), the cells do not demonstrate a regulatory volume increase (RVI) independent of the solute that is used to increase osmolality [NaCl, N-methyl-D-glucamine-HCl (NMGCl), or sucrose]. In contrast, when cells are preexposed to 190 mosmol/kg medium and then shrunken by exposure to 310 mosmol/kg medium, a volume increase is observed after the addition of 120 mosmol/kg NaCl or NMGCl, but not sucrose. This RVI is sensitive to 1 mM furosemide and removal of Na+ or K+ from the medium, but it is not inhibited by 1 mM amiloride. In the presence of a propionate-induced cellular acidification, a Na+-H+ exchanger in the cells is shown to have a large capacity for net solute uptake and to be inhibited by 1 mM amiloride. Net solute uptake by the Na+-H+ exchanger is sensitive to addition of parathyroid hormone or 8-bromoadenosine 3',5'-cyclic monophosphate but is not stimulated in response to cell shrinkage.
Effect of Na+ on intestinal succinate transport and metabolism in vitro
Authors: Moe A. J. , Mallet R. T. , Jackson M. J. , Hollywood J. A. , Kelleher J. K.
Published: December 2017

The effect of Na+ on 14CO2 production from [14C]succinate was studied in isolated rat enterocytes, and Na+-dependent succinate transport was characterized in pig intestinal brush-border membrane vesicles. The production of 14CO2 from [14C]succinate by enterocytes was decreased 12-fold when Na+ was replaced by N-methyl-D-glucamine in the absence of glutamine and 20-fold in the presence of 0.2 or 0.5 mM glutamine. The ratio of 14CO2 produced from [1,4-14C]succinate to that produced by [2,3-14C]succinate was not affected by Na+ replacement, indicating that the pattern of tricarboxylic acid cycle metabolism was not altered. The uptake of [14C]succinate by brush-border membrane vesicles was stimulated 10-fold in the presence of 100 mM NaCl compared with 100 mM KCl. When succinate uptake was corrected to transport into an osmotically sensitive space, the magnitude of the Na+ stimulation was 20-fold. Succinate transport into brush-border membrane vesicles was Na+ dependent, electroneutral, nonconcentrative, with an apparent Na+-succinate coupling ratio of 2:1. Results of this study indicate that Na+-stimulated CO2 production by enterocytes can be explained by the effect of Na+ on succinate transport across the brush-border membrane.
Reduced Na-K-Cl cotransport in vascular smooth muscle cells from spontaneously hypertensive rats
Authors: O'Donnell M. E. , Owen N. E.
Published: December 2017

We have previously demonstrated the presence of a prominent, cyclic nucleotide-sensitive Na-K-Cl cotransport in vascular smooth muscle cells (VSMC). Others have observed that Na-K-Cl cotransport levels are reduced in erythrocytes of patients with essential hypertension and have proposed that a defect in this Na transport system may play a role in the pathogenesis of the disease. However, such a defect has not been demonstrated in the putative target tissue for essential hypertension, i.e., the VSMC. In the present study, we compared Na-K-Cl cotransport of VSMC from spontaneously hypertensive rats (SHR) with Na-K-Cl cotransport of VSMC from normotensive Wistar-Kyoto rats (WKY). We found that Na-K-Cl cotransport of SHR VSMC is significantly reduced relative to that of WKY VSMC (3.09 vs. 4.39 mumol K.g protein-1.min-1). The apparent ion affinities for Na-K-Cl cotransport of SHR VSMC did not differ from those determined for WKY VSMC. Furthermore, cyclic nucleotide regulation of cotransport also appeared to be the same for the two types of VSMC. In contrast, maximal saturable binding of [3H]bumetanide observed in SHR VSMC was markedly reduced compared with that of WKY VSMC, but the Kd values were similar. Our data suggest that the reduction in cotransport observed in SHR VSMC is the result of a decrease in the number of available cotransport sites.
Assessment of transport capacity of plasmalemmal Ca2+ pump in smooth muscle
Authors: Lucchesi P. A. , Cooney R. A. , Mangsen-Baker C. , Honeyman T. W. , Scheid C. R.
Published: December 2017

In resting smooth muscle, a variety of Ca2+ extrusion processes offset the inward Ca2+ leak. Biochemical studies suggest that the plasmalemmal Ca2+ pump dominates this process; however, this contention could not be proven without a reliable estimate of the inward Ca2+ leak that must be opposed by active transport. Recent studies using dispersed cells from the toad stomach provided such an estimate; thus we examined the capacity of the plasmalemmal Ca2+ pump in this tissue. Membranes were prepared using nitrogen cavitation, high-salt extraction, and flotation on discontinuous sucrose gradients. These membrane vesicles were enriched 16- to 24-fold for plasma membrane markers and exhibited an ATP-dependent uptake of 45Ca that was insensitive to azide or oxalate but sensitive to orthovanadate inhibition and calmodulin stimulation. 45Ca accumulated in the presence of ATP was rapidly released by Ca2+ ionophore but not by caffeine, inositol 1,4,5-trisphosphate, or GTP. Uptake exhibited a high affinity for Ca2+ (Km 0.2 microM) and a high-transport capacity, producing greater than 12,000-fold gradient for Ca2+ and a transmembrane flux rate greater than that observed in resting smooth muscle cells. Thus this enzyme is capable of maintaining steady-state Ca2+ levels in smooth muscle.
Increased abundance of Na+-K+-ATPase mRNAs in response to low external K+
Authors: Pressley T. A. , Ismail-Beigi F. , Gick G. G. , Edelman I. S.
Published: December 2017

Exposure of ARL 15 cells, an established line from adult rat liver, to external K+ concentrations less than 1 mM for 24 h increases Na+-K+ pump abundance (Na+-K+-ATPase) (J. Gen. Physiol. 87:591-606, 1986). We found that treatment of confluent monolayers of ARL 15 cells with low-K+ medium (0.65 mM) caused a 100% increase in total RNA content per plate after 24 h, as well as a 25% increase in DNA and protein content per plate. Concomitant with this growth effect, low-K+ exposure for 6 h elicited 60% increases in mRNA alpha and mRNA beta, the mRNAs that encode the constituent subunits of the Na+-K+-ATPase, in a polyadenylated RNA fraction. At 24 h, however, the abundance of mRNA alpha increased by 290%, whereas mRNA beta increased by only 70%. Moreover, in both control and low-K+-treated cells, mRNA alpha was 30-fold or more greater in abundance than mRNA beta. This discrepancy in abundance was also present in rat liver, but not in cultured MDCK cells. The differences in abundance of mRNA alpha and mRNA beta suggest that the liver may have an unusual subunit composition or biosynthetic mechanism. Nevertheless, the increases in the abundance of mRNA alpha and mRNA beta are sufficient to account for the observed 70-100% increase in Na+-K+-ATPase activity in response to low external K+.
Na+-Ca2+ exchange in sarcolemmal membrane vesicles of dog mesenteric artery
Authors: Matlib M. A.
Published: December 2017

The kinetic characteristics of a Na+-Ca2+ exchange system in the cell membrane of vascular smooth muscle were explored in vitro in isolated sarcolemmal membrane vesicles of dog mesenteric artery. Na+-loaded vesicles rapidly accumulated Ca2+ when an outwardly directed Na+ concentration gradient was created by suspension of the vesicles in a Na+-free medium. This Ca2+ uptake process was reversible depending on the direction and the magnitude of the Na+ concentration gradient across the membrane of the vesicles. Low temperature, monensin, and external Na+ drastically decreased Ca2+ uptake in Na+-loaded vesicles. Monovalent cations K+, Rb+, Li+, and Cs+ did not substitute for Na+ in the exchange process. Divalent cations Ba2+, Cd2+, Mg2+, Mn2+, and Sr2+ inhibited Ca2+ uptake in Na+-loaded vesicles. The order of potency of these divalent cations and concentration which produced 50% inhibition (IC50, microM) were Cd2+(38) greater than Sr2+(110) greater than Ba2+(405) greater than Mn2+(500) greater than Mg2+(greater than 2,500). The trivalent cation La3+ also inhibited Ca2+ uptake (IC50 = 0.175 microM). The apparent Km for free Ca2+ in vesicles loaded with 150 mM NaCl was 2.64 +/- 0.5 microM, and the apparent maximum velocity was 14.8 +/- 1.9 nmol.min-1.mg protein-1. The half of the apparent maximum rate (K0.5) of Ca2+ uptake was observed at 45.5 mM Na+ when loaded internally in the vesicles. Valinomycin in the presence of K+ increased the magnitude of Ca2+ uptake by 16% in Na+-loaded vesicles, indicating that the process may be electrogenic. These data indicate the existence and operation of a specific carrier-mediated Na+-Ca2+ exchange system in sarcolemmal membrane vesicles isolated from a small blood vessel.
Volume and anion dependency of ouabain-resistant K-Rb fluxes in sheep red blood cells
Authors: Lauf P. K.
Published: December 2017

The effect of six different anions on the volume response of ouabain-resistant K transport was systematically studied at extracellular pH (pHo) = 7.4 in sheep red blood cells of both low and high K genotype before and after treatment with the sulfhydryl (SH) reagent N-ethylmaleimide (NEM). In methanesulfonate (CH3SO3), both the apparent Rb permeability (P(app)Rb), calculated from ouabain-resistant Rb influx), and K permeability (PK, calculated from the rate constants of ouabain-resistant zero-trans K efflux, 0k(OR)K) were volume independent and close to 10(-10) cm/s for both cell types, but in Cl, Br, I, SCN, and NO3 they were significantly different in low and high K cells with altered cell volumes. Thus, in 15% osmotically shrunken low K cells, P(app)Rb) and PK were similar regardless of the anions present, but upon 10-15% swelling, they increased to approximately 4-6 X 10(-9) cm/s in Br and 2 X 10(-9) cm/s in Cl and also increased with comparatively small increments in I, SCN, and NO3. Treatment with NEM enhanced both P(app)Rb) and PK, particularly in shrunken low K cells, to approximately 10(-8) cm/s in Br and Cl but not in I, SCN, and NO3. In shrunken or isotonic high K cells, P(app)Rb) and PK were close to 10(-10) cm/s in all anions except for SCN. Swelling and/or NEM increased PK and P(app)Rb) in Cl and Br only two- to threefold.(ABSTRACT TRUNCATED AT 250 WORDS)
Properties of K+ transport in resealed human erythrocyte ghosts
Authors: Brugnara C. , Van Ha T. , Tosteson D. C.
Published: December 2017

We report here our studies on K+ transport in resealed human red cell ghosts (RG) in the presence of 0.1 mM ouabain and 0.01 mM bumetanide, inhibitors of the Na+-K+ pump and Na+-K+-Cl- cotransport, respectively. RG were obtained with the gel-filtration method. K+ efflux from RG was dependent on the pH used in the lysis buffer and increased when the pH used in the lysis buffer and increased when the pH was raised from 5.5 to 8.0. As in intact red cells, RG made from cells of the least dense fraction had a much higher K+ efflux than RG made from cells of the densest fraction. This K+ flux is volume independent and increases when the pH of the flux medium is increased from 6.0 to 8.0. K+ efflux (60-70%) at pH 7.40 from RG made from cells of the least dense fraction is inhibited when Cl- is substituted by nitrate or when the ghosts are resealed in the absence of ATP. This chloride- and ATP-dependent component is markedly reduced in RG made from cells of the densest fraction. An increase in the internal Mg2+ concentration in RG from the least dense fraction induced marked inhibition of K+ efflux. Contrary to intact cells, N-ethylmaleimide (NEM) did not affect K+ efflux from RG. Thus the effects of pH, osmolarity, and NEM on K+ transport in RG are markedly different from those reported in intact erythrocytes.
Bacterial exotoxins and endothelial permeability for water and albumin in vitro
Authors: Suttorp N. , Hessz T. , Seeger W. , Wilke A. , Koob R. , Lutz F. , Drenckhahn D.
Published: December 2017

Effects of Staphylococcus aureus alpha-toxin and Pseudomonas aeruginosa cytotoxin on the permeability of an endothelial monolayer were studied. Porcine pulmonary artery endothelial cells were grown on a polycarbonate membrane, mounted in a chamber, and exposed to a continuous hydrostatic pressure of 10 cmH2O. On application of this trans-endothelial pressure, endothelial monolayer became "sealed," i.e., the filtration rate for water decreased and the reflection coefficient for albumin increased, reaching a plateau after 1-2 h. Sealed monolayer had a hydraulic conductivity of 2.1 X 10(-6) cm.s-1.cmH2O and an albumin reflection coefficient of 0.73. Permeability of the monolayer was increased on addition of an excess of EDTA and reversed on readdition of calcium. Within 60-90 min after addition of 1 microgram/ml alpha-toxin, the filtration rate increased 75-fold, and the albumin reflection coefficient dropped to 0.20. These changes in permeability were accompanied by cell retraction and formation of large intercellular gaps between endothelial cells. Effects of alpha-toxin were abolished by preincubation with neutralizing antibodies and by inhibitors of calmodulin function. Pseudomonas aeruginosa cytotoxin (25 and 50 micrograms/ml) also increased the permeability of the endothelial monolayer, but it was only about one-third as effective as alpha-toxin.
Intracellular sodium flux and high-energy phosphorus metabolites in ischemic skeletal muscle
Authors: Blum H. , Schnall M. D. , Chance B. , Buzby G. P.
Published: December 2017

We have employed concurrent 31P- and 23Na-nuclear magnetic resonance (NMR) spectroscopy in conjunction with the paramagnetic shift reagents dysprosium-chelated tripolyphosphate and triethylenetetramine-hexa-acetic acid to observe the intracellular sodium and phosphorus signals in rat leg muscle. With induced ischemia in the leg, we find slowly falling phosphorylation potential. At a critical value of, associated with energetic failure of the Na+-K+ antiport, the intracellular sodium signal begins to increase. We find the following critical values: log, 3.12 +/- 0.32; pH, 6.86 +/- 0.13; Na+ influx with and without ouabain, 5.1 +/- 4.3 and 4.0 +/- 1.3 mol.l-1.h-1, respectively.
Benzodiazepines inhibit transport-related oxygen consumption in thick ascending limb
Authors: Ziyadeh F. N. , Agus Z. S.
Published: December 2017

Specific binding sites for benzodiazepines (BZD) have been identified in several nonneuronal tissues including the kidney where they are localized predominantly to the tubular epithelium of the thick ascending limb of Henle's loop (TALH). The physiological function of these nonneuronal (peripheral) BZD-binding sites is undefined, but it has been suggested that they may represent receptors for putative endogenous ligands that may modulate cell function. In the current study, we examined the in vitro effects of diazepam and Ro5-4864, a specific peripheral BZD-receptor agonist, on the oxygen consumption of medullary TALH tubule suspensions of rabbit kidney. Maximal inhibition of total oxygen consumption was achieved at a dose of 5 X 10(-4) M of either agent. On average, diazepam and Ro5-4864 reduced total oxygen consumption by 41 and 44%, respectively. The predominant inhibition was in the ouabain-sensitive component of oxygen consumption, which suggests that BZDs inhibit active sodium-chloride transport in the TALH. To assess whether this inhibition depends on sodium uptake, TALH tubules were pretreated with amphotericin B (2 X 10(-6) M) to enhance sodium uptake and stimulate basal oxygen consumption; subsequent addition of Ro5-4864 (5 X 10(-4) M) still reduced oxygen consumption to a residual value that was not different from that in TALH tubules treated with Ro5-4864 but without pretreatment with amphotericin B. This suggests that BZD inhibition of transport-related oxygen consumption is not caused by diminution of sodium uptake into cells and thus appears to be distinct from the effect of furosemide.(ABSTRACT TRUNCATED AT 250 WORDS)
Dependence of force, velocity, and O2 consumption on [Ca2+]o in porcine carotid artery
Authors: Krisanda J. M. , Paul R. J.
Published: December 2017

The relationship between extracellular calcium concentration ([Ca2+]o), isometric force (Fo), unloaded shortening velocity (Vus), and the rate of ATP utilization (JATP) were studied in vascular smooth muscle (VSM) during the steady state of an isometric contraction at 37 degrees C. Experiments were conducted on porcine carotid artery media strip and ring preparations that were stimulated with 109 mM KCl substituted for NaCl. Unloaded shortening velocity was estimated by the "slack" test method. Both Fo and Vus were dependent on [Ca2+]o. Vus at 7.5 mM [Ca2+]o was 1.7 times greater than Vus at 1.6 mM [Ca2+]o. The difference in force at these two Ca2+ concentrations was more variable than Vus, but in general was less than the change in Vus. The rate of ATP utilization was assessed from steady-state measurements of tissue O2 consumption. Increasing [Ca2+]o in the range of 0.15-7.5 mM resulted in an increase in both JATP and Fo. The relation between JATP and Fo was nonlinear with JATP increasing proportionately more than Fo between 1.6 and 7.5 mM Ca2+. The calcium-dependent increase in JATP appears to be primarily related to contractile protein interaction, since the effect of [Ca2+]o on JATP was substantially reduced during stimulation at short muscle lengths (Lmin), where tension development is absent. The economy of tension maintenance was 3.3. times greater when measured in the [Ca2+]o range of 0.15 and 1.6 mM than when measured at 1.6 to 7.5 mM [Ca2+]o. These data indicate that [Ca2+]o may regulate both the number and the rate of cycling of cross bridges in porcine carotid artery.
Epidermal growth factor-mediated effects on equine vascular smooth muscle cells
Authors: Grosenbaugh D. A. , Amoss M. S. , Hood D. M. , Morgan S. J. , Williams J. D.
Published: December 2017

Epidermal growth factor (EGF) receptor binding kinetics and EGF-mediated stimulation of DNA synthesis and cellular proliferation were studied in cultured vascular smooth muscle cells (VSMC) from the equine thoracic aorta. Binding studies, using murine 125I-labeled EGF, indicate the presence of a single class of high-affinity binding sites (apparent KD = 2.8 X 10(-11) M), with an estimated maximal binding capacity of 5,800 sites/cell. EGF stimulated [3H]thymidine uptake in confluent quiescent monolayers in a dose-dependent fashion, half-maximal stimulation occurring at 7.5 X 10(-11) M. Likewise, EGF-mediated cellular proliferation was dose dependent (50% effective dose = 5 X 10(-11) M) under reduced serum concentrations. Equine VSMC contain specific receptors for EGF, and EGF can stimulate DNA synthesis and proliferation in these cultured cells, which suggests that EGF may participate in the proliferative changes observed in equine distal digital peripheral vascular disease.
Ouabain uptake by endocytosis in isolated guinea pig atria
Authors: Nunez-Duran H. , Riboni L. , Ubaldo E. , Kabela E. , Barcenas-Ruiz L.
Published: December 2017

Mammalian cells specifically internalize some molecular species through receptor-mediated endocytosis (RME). We have used four different experimental protocols to investigate whether ouabain enters cardiac cells of guinea pig atrium through this pathway. First, by electron microscope morphometry we found that ouabain increased endocytic vesicles in atrial cells. Second, by scintillation counting we found that [3H]ouabain uptake by the tissue is decreased by three treatments that decrease RME, i.e., NH4Cl, trifluoperazine, and 16 mM [K+]0. Third, by radioautography at the electron microscope level, we checked that in preceding experiments [3H]ouabain was washed out of plasma membrane after 60-min rinse and interiorized into the cardiac cells. Fourth, isometric tension recordings showed that the positive inotropic effect of ouabain was diminished in the presence of inhibitors, whereas that of a hydrophobic analogue, ouabagenin, was not affected. These results suggest that ouabain enters cardiac cells through RME and also that an intracellular site may, at least in part, be responsible for its inotropic effect.
Cytochalasin B inhibition of toad bladder apical membrane responses to ADH
Authors: Wade J. B. , Kachadorian W. A.
Published: December 2017

The possible role of actin microfilaments in antidiuretic hormone (ADH)-induced increases in apical membrane water permeability was investigated in studies that evaluate inhibition by cytochalasin B of both permeability and membrane structural responses in the toad urinary bladder. Experiments were carried out in the absence of a transepithelial osmotic gradient to eliminate possible flow-induced distortions of the response. Measurements of osmotic water permeability after a brief tissue fixation with glutaraldehyde show that cytochalasin B reduces the permeability response to ADH by approximately one-third. Freeze-fracture electron microscopy indicates that the intramembrane particle aggregates, previously found to correlate closely with ADH-induced permeability, are reduced by about the same extent (28%) under these conditions. However, the frequency of apical membrane fusion events was not affected by cytochalasin B treatment. These results suggest that cytochalasin B treatment in the absence of an osmotic gradient alters the ADH-induced permeability through an effect on apical membrane aggregate frequency.
Simultaneous Nomarski and fluorescence imaging during video microscopy of cells
Authors: Foskett J. K.
Published: December 2017

A video microscope designed to allow low light level fluorescence imaging of cells during simultaneous high-resolution differential interference contrast (DIC) imaging, without the fluorescence light losses of 60-90% normally associated with this contrast-enhancement technique, is described. Transmitted light for DIC imaging, filtered at greater than 620 nm, passes through standard DIC optical components, (1/4 lambda-plate, polarizer, and Wollaston prism) before illuminating the cells. Transmitted light and fluorescence emission pass through a second Wollaston prism but not through the analyzer, which is repositioned more distally in the optical path. Prisms designed to reflect light out a side port of the microscope to a video camera have been replaced with a dichroic mirror. This mirror reflects fluorescence emission out the side port to a low light-sensitive video camera. The spectrally distinct transmitted light continues through the dichroic mirror to an overhead camera through a polarizer (analyzer), which completes the DIC optical path. The fluorescence and DIC images can be viewed simultaneously on side-by-side video monitors, examined sequentially by an image-processing computer, or examined simultaneously using a video splitter/inserter. The ability to image cells with high resolution simultaneously with low light level fluorescence imaging should find wide applicability whenever it is necessary or desirable to correlate fluorescence intensity or distribution with specific cell structure or function.
Adenosine receptor-mediated calcium mobilization in cortical collecting tubule cells
Authors: Arend L. J. , Burnatowska-Hledin M. A. , Spielman W. S.
Published: December 2017

To investigate the cellular mechanisms underlying the epithelial actions of adenosine, we studied adenosine receptor-effector coupling in cultured rabbit cortical collecting tubule (RCCT) cells. We previously reported, in RCCT cells isolated by immunodissection, that a potent A2 adenosine analogue [5'-N-ethylcarboxamideadenosine (NECA)] stimulates cAMP production [effective concentration 50% (EC50) = 1 microM], and potent A1 analogues [N6-cyclohexyladenosine (CHA) and R-N6-phenylisopropyladenosine (PIA)] inhibit basal and AVP-stimulated cAMP production (EC50 = 5 nM). The present study was undertaken to determine whether adenosine receptors in RCCT cells are also coupled to a signal transduction system leading to the mobilization of intracellular free calcium. RCCT cells were loaded with the fluorescent calcium indicator, fura-2, and were treated with the adenosine analogues NECA, CHA, and PIA. All three adenosine analogues produced dose-dependent (1 nM-0.1 mM), transient increases in intracellular calcium concentration with equal potency (EC50 = 0.5 microM). Chelation of extracellular calcium with ethyleneglycol-bis(beta-aminoethyl ether)N,N,N',N' tetraacetic acid (EGTA) did not abolish the increase in calcium. The adenosine receptor antagonists, 1,3-diethyl-8-propylxanthine and 8-cyclopentyl-1,3-dipropylxanthine, and pretreatment of RCCT cells with pertussis toxin blocked the increase in calcium. These results demonstrate that RCCT cells have, in addition to adenosine receptors associated with the stimulation and inhibition of cAMP, a pertussis-toxin sensitive receptor system that leads to the mobilization of intracellular calcium.
Binding and internalization of a fluorescent vasopressin analogue by collecting duct cells
Authors: Kirk K. L.
Published: December 2017

The kinetics of binding and internalization of a fluorescent vasopressin analogue [1-desamino-8-rhodamine lysine vasopressin (rhoda LVP)] by principal cells within the microperfused rabbit cortical collecting tubule were quantitatively assessed with computer-assisted video microscopy. At 25 degrees C, binding of rhoda LVP exhibited saturation kinetics with half-maximal binding at 2 nM and maximal binding at concentrations greater than 5 nM. Rhoda LVP binding could be prevented by the simultaneous addition of a 10-fold higher concentration of arginine vasopressin (AVP) or the V2-receptor agonist, 1-desamino-8-D-arginine vasopressin (desmopressin). No obvious internalization or rhoda LVP was detected at 25 degrees C, i.e., the rhoda LVP fluorescence remained localized to the basal pole of each principal cell for at least 100 min after rhoda LVP addition and could be largely reversed by the subsequent addition of AVP. Conversely, warming the cells to 38 degrees C after binding was initiated resulted in a rapid (less than 30 min) migration of the fluorescence into the cell interior and a loss of AVP-displaceable binding from the cell surface. These results document the utility of this noninvasive optical strategy for quantitatively monitoring hormone binding to intact collecting tubule cells and demonstrate that rhoda LVP binds reversibly and with high affinity to V2 receptors on principal cells in the collecting tubule. The internalization (and presumed inactivation) of the hormone-receptor complex at 38 degrees C may contribute to the desensitization of collecting tubule cells to vasopressin at physiological temperature.
Altered junctional permeability between cells transformed by v-ras, v-mos, or v-src
Authors: Atkinson M. M. , Sheridan J. D.
Published: December 2017

Junctional permeability in normal and transformed NRK cells was quantitatively assessed by microinjecting fluorescent dye into one cell of a pair, digitizing the changes of fluorescence intensity using video analysis techniques, and applying the digital values to a solution of Fick's diffusion equation. We show that this approach reliably estimates the junctional permeance of a cell pair. Cells that are temperature sensitive for transformation were shown to also be temperature sensitive vis-a-vis junctional permeance. Thus permeance values were reduced approximately 80-90% on transformation by either a mutant Rous sarcoma virus or a mutant Moloney murine sarcoma virus. Cells transformed by wild-type Kirsten sarcoma virus were also shown to possess levels of junctional permeance significantly lower than nontransformed controls. The transformed junctional phenotype could be observed as early as 15 min after shifting to transformation-permissive conditions. Our results suggest that the oncogenes src, ras, and mos exert their effects on NRK cell junctions via converging pathways, of which one may be phosphorylation of junctional proteins.
Uracil ribonucleotide metabolism in rat and human glomerular epithelial and mesangial cells
Authors: Dumler F. , Cortes P.
Published: December 2017

Uridine diphosphosugars (UDP-sugars: UDP-N-acetylglucosamine, UDP-glucose, and UDP-glucuronic acid) are essential coenzymes for the synthesis of glomerular basement membrane and mesangial matrix (GBM-MM). This study has characterized UDP-sugar metabolism in rat and human glomerular cells in tissue culture. Culture of rat mesangial cells in medium containing dialyzed fetal calf serum resulted in UTP loss (28 +/- 4 nmol.mg DNA-1.h-1); the addition of 2 microM orotate to this medium resulted in net UTP accretion (5.42 +/- 0.06 nmol.mg DNA-1.h-1). Rat mesangial cells demonstrated 16- and 29- to 46-fold greater UTP and UDP-sugar pools, respectively, than whole glomeruli. In human mesangial cells, 6-azauridine (500 microM) decreased UDP-sugar pools by 48% (P less than 0.05), whereas uridine (50 microM) produced a 2.5-fold increase. Human and rat mesangial cells had greater (1.8- to 6.1-fold) UDP-sugar pools than epithelial cells and 1.7-3.4 times greater labeled precursor incorporation into UDP-sugars. In conclusion, glomerular cells utilize both exogenous orotate and uridine for ribonucleotide synthesis, and the extracellular concentration of these precursors markedly influence the formation and cellular content of UDP-sugars. Prominent differences exist between separate glomerular cell populations in their metabolism of UDP-sugars. This may represent diverse activity of glycosylating reactions.
[Ca2+]-dependent myosin phosphorylation in phorbol diester stimulated smooth muscle contraction
Authors: Rembold C. M. , Murphy R. A.
Published: December 2017

Phorbol diesters, potent activators of protein kinase C, can produce a slow contraction in arterial smooth muscle. Such observations have prompted proposals that protein kinase C may have direct regulatory functions in contraction. In this paper, we present evidence that [Ca2+]-dependent myosin light chain phosphorylation is responsible for the contraction induced by low-dose phorbol diester and during force development in response to high-dose phorbol diester stimulation. The relationships between myoplasmic [Ca2+], myosin phosphorylation, and steady-state stress induced by low-dose phorbol dibutyrate were similar to those observed with contractile agonists. However, prolonged exposure to high-dose phorbol dibutyrate induced high stress with elevated phosphorylation that was not associated with elevations in aequorin-estimated [Ca2+]. Our results suggest that phorbol diesters can increase myoplasmic [Ca2+], and the resulting increase in myosin phosphorylation quantitatively explains the contraction.