bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanisms of t-butyl hydroperoxide-induced toxicity to rabbit renal proximal tubules
Authors: Schnellmann R. G.
Published: December 2017

This study examined the mechanisms of t-butyl hydroperoxide (TBHP)-induced oxidative injury to a suspension of rabbit renal proximal tubules. TBHP (0.25-1 mM) produced a specific sequence of intracellular events in the tubules. Initially, TBHP increased tubular glutathione disulfide content and lipid peroxidation. Subsequently, there was an increase in ouabain-sensitive oxygen consumption (indicative of an increase in intracellular sodium concentrations), mitochondrial dysfunction, and a decrease in glutathione content. Finally, cell death, as measured by a decrease in tubular retention of lactate dehydrogenase activity, began between 30 and 60 min. The toxicity was dependent on iron-mediated free radical formation, since the iron chelator, deferoxamine, and the antioxidants, promethazine, butylated hydroxytoluene, and dithiotreitol, prevented the lipid peroxidation, the mitochondrial dysfunction, and cell death. Further studies with the antioxidants provided evidence that lipid peroxidation plays an important role in TBHP toxicity in proximal tubules.
Regulation of glycerinated smooth muscle contraction and relaxation by myosin phosphorylation
Authors: Tanner J. A. , Haeberle J. R. , Meiss R. A.
Published: December 2017

Regulation of isometric force maintenance, isotonic shortening velocity, and muscle stiffness by myosin phosphorylation was examined during both contraction and relaxation of chemically permeabilized (glycerinated) rat uterine smooth muscle. Phosphorylation of the 20,000-Da light chain of myosin (LC20) was manipulated by varying the calcium activity of the bathing solution or by thiophosphorylation of LC20 in the presence of ATP gamma S. With saturating calcium and calmodulin, LC20 phosphorylation was 0.43 mol PO4/mol LC20. This increased to 0.92-0.96 mol PO4/mol LC20 on addition of ATP gamma S. Over the entire range of phosphorylation, there was a significant (P less than 0.001) linear correlation between force and phosphorylation. Stiffness increased monotonically with increasing force; however, the relationship was nonlinear, with stiffness increasing faster at lower levels of activation. Force, stiffness, shortening velocity, and LC20 phosphorylation were compared at identical calcium activities during steady-state conditions of partial contraction and partial relaxation. The ratio of the value of each parameter measured during relaxation to that measured during contraction was 1.11 for force, 1.09 for stiffness, 1.01 for shortening velocity, and 0.83 for LC20 phosphorylation. These results support the hypothesis that contraction and relaxation in glycerinated rat uterine muscle are regulated primarily by phosphorylation and dephosphorylation of LC20.
Metabolic and fiber size properties of cat tibialis anterior motor units
Authors: Martin T. P. , Bodine-Fowler S. , Roy R. R. , Eldred E. , Edgerton V. R.
Published: December 2017

The variability among single muscle fiber enzymatic activities and fiber size within a motor unit was studied in the cat tibialis anterior (TA) muscle. Fourteen units were isolated for physiological testing using standard ventral root filament stimulation techniques, and the muscle fibers of these units were identified by glycogen depletion. The cross-sectional areas, succinate dehydrogenase (SDH) and alpha-glycerolphosphate dehydrogenase (GPD) activities, and the relative alkaline myofibrillar adenosine triphosphate staining densities of a sample of glycogen-depleted and -nondepleted muscle fibers were determined using quantitative histochemical techniques. Each of the unit types previously identified to be present in the TA, based on physiological criteria, were represented by the sample population. The variability among the fibers of a unit was significantly more than the variability among repeated measures on a single fiber for cross-sectional area and SDH and GPD activities. The mean coefficients of variation for SDH and GPD activity within motor unit fibers were 29 and 56%, respectively, whereas the variability between fibers of different units within a muscle was significantly greater (53 and 69%, respectively). Additionally, the mean coefficient of variation for cross-sectional area among motor unit fibers was less than that among fibers not depleted of glycogen (25 vs. 46%). These data suggest that although there is clear evidence for some level of neural control of the properties of a muscle unit (variation within a unit was less than the variation across units), this control is not complete, since the variability among fibers of a single unit was significantly more than the variability found between repeated measurements on a single fiber.
Substance P receptor desensitization requires receptor activation but not phospholipase C
Authors: Sugiya H. , Putney J. W.
Published: December 2017

Previous studies have shown that exposure of parotid acinar cells to substance P at 37 degrees C results in activation of phospholipase C, formation of [3H]inositol 1,4,5-trisphosphate (IP3), and persistent desensitization of the substance P response. In cells treated with antimycin in medium containing glucose, ATP was decreased to approximately 20% of control values, IP3 formation was completely inhibited, but desensitization was unaffected. When cells were treated with antimycin in the absence of glucose, cellular ATP was decreased to approximately 5% of control values, and both IP3 formation and desensitization were blocked. A series of substance P-related peptides increased the formation of [3H]IP3 and induced desensitization of the substance P response with a similar rank order of potencies. The substance P antagonist, [D-Pro, D-Trp]-substance P, inhibited substance P-induced IP3 formation and desensitization but did not induce desensitization. These results suggest that the desensitization of substance P-induced IP3 formation requires agonist activation of a P-type substance P receptor, and that one or more cellular ATP-dependent processes are required for this reaction. However, activation of phospholipase C and the generation of inositol phosphates does not seem to be a prerequisite for desensitization.
Survey of osmolytes in renal cell lines
Authors: Nakanishi T. , Balaban R. S. , Burg M. B.
Published: December 2017

In renal medullas during antidiuresis, the extracellular fluid is hyperosmotic because of high concentrations of NaCl and urea. Under those conditions, the cells contain high concentrations of organic osmolytes, namely sorbitol, myo-inositol, glycerophosphorylcholine (GPC), and betaine to balance the extracellular hyperosmolality. These organic osmolytes increase cell osmolality without perturbing the intracellular milieu in ways that would degrade the function of cellular macromolecules. The present study surveyed a number of cell lines for the ability to survive in media with high concentrations of NaCl and/or urea and for the accumulation of organic osmolytes. Of the renal cell lines tested, MDCK, GRB-MAL1, and A6 cells proliferated in hyperosmotic media, but medullary interstitial cells LLC-PK1 and LLC-PK3 did not proliferate, nor did nonrenal HTC-BH cells, MDCK, LLC-PK1, and LLC-PK3 cells contained higher concentrations of myo-inositol, GPC, and betaine when cultured in media containing high NaCl (with or without high urea) and much lower or undetectable levels of these osmolytes when grown in isosmotic media. Sorbitol, and to a lesser extent myo-inositol, were elevated in GRB-MAL1 cells in media hyperosmotic with NaCl but not in isosmotic media. There was less accumulation of organic osmolytes when only urea was added to increase osmolality. Thus the same osmolytes were accumulated by one or another cell line in vitro as were previously found in renal medullas. These cell lines provide models for studying osmolyte accumulation.
8-BrcAMP does not affect Na-K-2Cl cotransport in winter flounder intestine
Authors: Rao M. C. , Nash N. T.
Published: December 2017

The flounder intestinal epithelium possesses luminal Na-K-2Cl cotransport and K secretory mechanisms that account for the short-circuit current (Isc) across the tissue. This epithelium is also highly cation selective. Bumetanide or 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) completely inhibit Isc, Na-K-2Cl cotransport, and K secretion, whereas 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) inhibits K secretion, partially inhibits Isc, and greatly increases Cl permeability. Although the direct effects of adenosine 3',5'-cyclic monophosphate (cAMP) on other Na-K-2Cl cotransport systems have been examined, the effects of 8-BrcAMP on flounder intestinal Na-K-2Cl cotransport have not been directly measured. In this study, the effects of 8-BrcAMP and bumetanide, either alone or in combination, on the influx (initial rates of uptake) of Cl, Rb, and Na across the luminal surface of the flounder intestine were examined. Bumetanide significantly inhibited Na (50%), Cl, and Rb influx (70% each). In contrast, 8-BrcAMP significantly increased Cl influx in the presence or absence of bumetanide and thereby did not effect the bumetanide-sensitive Cl influx. The nucleotide neither altered bumetanide-sensitive Rb influx nor net transpithelial Na absorption measured under short-circuit conditions but caused a 51-60% decrease in Isc. Measurements of bumetanide-sensitive Na influx exhibited large experimental variability but showed no statistically significant effects of 8-BrcAMP. Prostaglandin E1 (PGE1, 10 microM) and forskolin (10 microM) but not atrial natriuretic factor (1 microM) increased flounder intestinal [cAMP] 200 and 237%, respectively, and, like 8-BrcAMP, increased tissue conductance.(ABSTRACT TRUNCATED AT 250 WORDS)
Growth-dependent regulation of system A in SV40-transformed fetal rat hepatocytes
Authors: Handlogten M. E. , Kilberg M. S.
Published: December 2017

Fetal RLA209-15 hepatocytes, transformed with a temperature-sensitive SV40 mutant, behave like fully differentiated cells at the growth-restrictive temperature of 40 degrees C. Conversely, incubation at the growth-permissive temperature of 33 degrees C results in a transformed phenotype characterized by rapid cell division and decreased production of liver-specific proteins. The results presented here demonstrate that the cells at 33 degrees C exhibited high rates of system A transport, but transfer to 40 degrees C reduced the activity greater than 50% within 24 h. This decline in transport was independent of cell density, although the basal rate of uptake was inversely proportional to cell density in rapidly dividing cells. Transfer of cells from 40 to 33 degrees C resulted in an enhancement of system A activity that was blocked by tunicamycin. Plasma membrane vesicles from cells maintained at either 33 or 40 degrees C retained uptake rates proportional to those in the intact cells; this difference in transport activity could also be demonstrated after detergent solubilization and reconstitution. Collectively, these data indicate that de novo synthesis of the system A carrier is regulated in conjunction with temperature-dependent cell growth in RLA209-15 hepatocytes.
Conversion of esterified fura-2 and indo-1 to Ca2+-sensitive forms by mitochondria
Authors: Gunter T. E. , Restrepo D. , Gunter K. K.
Published: December 2017

Rat liver mitochondria are shown to convert the acetoxymethyl ester forms of fura-2 and indo-1 into Ca2+-dependent forms of these indicators. The excitation spectrum of the Ca2+-dependent conversion product of fura-2 acetoxymethyl ester is shown to be similar to that of the pentacarboxylic acid form of fura-2. A systematic investigation of the ionic strength and pH dependences of the fluorescence of the pentacarboxylic acid forms of these indicators shows small changes within the ranges thought to obtain within the mitochondrial matrix after Ca2+ uptake. Intramitochondrial free Ca2+ levels are studied both before and after Ca2+ sequestration by mitochondria, and a rough estimate is made of the mitochondrial contribution to the Ca2+-dependent fura-2 fluorescence of a hepatocyte suspension.
Blunted cGMP response to ANF in vascular smooth muscle cells of SHR
Authors: Nakamura M. , Nakamura A. , Fine B. , Aviv A.
Published: December 2017

Abnormalities in the coupling of atrial natriuretic factor (ANF) receptors with the guanosine 5'-cyclic monophosphate (cGMP) system in vascular smooth muscle cells (VSMCs) may play a role in the pathophysiology of hypertension in the spontaneously hypertensive rat (SHR). This concept was examined in cultured, aortic VSMCs (passages 6-10) from SHR, Wistar-Kyoto (WKY), and American Wistar (Wis) rats. Quiescent VSMCs of the SHR (serum deprived for 24 h) had higher ANF receptor density (Bmax) and lower affinity [i.e., increased equilibrium dissociation constant (Kd)] than cells from normotensive controls. Maximal binding (Bmax) (specific binding sites/cell) values for these cells were SHR 112,855 +/- 6,951, WKY 48,650 +/- 3,607, and Wis 36,122 +/- 2,607 (means +/- SE; P less than 0.001 for SHR vs. both WKY and Wis). The Kd values were (in nM) SHR 1.20 +/- 0.098, WKY 0.657 +/- 0.065, and Wis 0.37 +/- 0.037 (P less than 0.001 for SHR vs. both WKY and Wis). Despite their higher Bmax, VSMCs of the SHR showed a substantially lower maximal stimulation of cGMP accumulation in response to ANF: 987 +/- 29.3, 1,992 +/- 574.2, and 2,019 +/- 273.8 fmol.4 min-1.10(6) cells-1 for SHR, WKY, and Wis, respectively (P less than 0.01 for SHR vs. Wis and P less than 0.02 for SHR vs. WKY). Further experiments demonstrated that the poor response of SHR VSMCs to the ANF was not due to a population of receptors that did not couple to the particulate guanylate cyclase. Such findings demonstrate a dissociation of the cGMP response to ANF from the binding of the hormone to its receptors in VSMCs of the SHR compared with controls. This appears to represent a primary and innate defect in these cells that may contribute to the hypertensive process in the SHR.
Electrical coupling and pacemaker activity in colonic smooth muscle
Authors: Huizinga J. D. , Shin A. , Chow E.
Published: December 2017

The effect of heptanol on electrical coupling between submucosal circular muscle cells of the dog colon and consequences for slow-wave activity were investigated. Electrotonic potentials showed exponential decay giving a length constant of 2.6 +/- 0.5 mm and a time constant of 157 +/- 48 ms. Heptanol reversibly abolished electrotonic current spread, and subsequently no slow-wave activity was recorded. The length constant decreased to less than 0.2 mm. The input resistance increased from 3 to 36 M omega, suggesting a change from tissue syncytium to electrically isolated cells. D600 (5 X 10(-6) M) also abolished slow wave activity but had opposite effects on electrotonic current spread. The data are consistent with the hypothesis that heptanol reversibly inhibits intercellular coupling, resulting in loss of spread of extracellularly applied current, uncoupling of cells, and loss of pacemaker activity. Regulation of intercellular communication may be important in the control of intestinal motility.
Continuous monitoring of Ca2+ uptake in membrane vesicles with fura-2
Authors: Kargacin M. E. , Scheid C. R. , Honeyman T. W.
Published: December 2017

The Ca2+-selective, fluorescent dye, fura-2, was used to monitor ATP-dependent Ca2+ uptake by membrane vesicles isolated from rabbit skeletal muscle. Micromolar fura-2 concentrations, added outside the vesicles, served as a high-affinity, low-capacity Ca2+ buffer. Changes in fura-2 fluorescence resulted from the decline in free Ca2+ concentration [( Ca2+]free) owing to active Ca2+ accumulation by the vesicles. Ca2+ uptake (delta[Ca2+]total) was calculated from changes in [Ca2+]free and from the Kd value for the fura-2-Ca2+ complex. The velocity of Ca2+ uptake determined in this manner had an apparent [Ca2+]0.5 of approximately 200 nM. The Hill coefficient for dependence of uptake velocity on [Ca2+]free was congruent to 2. Changes in [Ca2+]free and Ca2+ uptake expected for Ca2+ transport by skeletal muscle sarcoplasmic reticulum were determined theoretically from known kinetic parameters and found to be similar to experimental values. This method of directly monitoring Ca2+ uptake can be used to determine the kinetic parameters for Ca2+ transport with small amounts of vesicles and with greater precision than possible with radiometric techniques.
Extensive changes in collagen synthesis and degradation during compensatory lung growth
Authors: McAnulty R. J. , Staple L. H. , Guerreiro D. , Laurent G. J.
Published: December 2017

Unilateral pneumonectomy in rats causes compensatory growth of the remaining lung. This growth involves rapid production of collagen and noncollagen proteins, but the mechanisms for these changes have not been fully investigated. Rates of collagen metabolism were measured using previously validated in vivo methods. Six days after pneumonectomy, a threefold increase in the fractional rate of collagen synthesis was observed (control 11.8 +/- 0.9%/day, pneumonectomy 30.0 +/- 4.6%/day). Collagen degradation rates also increased but returned to normal more rapidly than the synthesis rates. These changes in synthesis and degradation resulted in a 75% increase in collagen content by 28 days. Although degradation of extracellular collagens was apparently increased, the fraction degraded intracellularly decreased by approximately 30%. Noncollagen protein synthesis and degradation rates both increased by approximately 80% (control 44.3 +/- 3.4%/day, pneumonectomy 80.3 +/- 10.2%/day) with a slightly greater increase in synthesis that led to an 85% increase in noncollagen protein content 28 days after pneumonectomy. The data obtained show dramatic changes in protein synthesis and degradation during compensatory lung growth and indicate extensive remodeling of structural elements in lung tissue. The changes for intracellular collagen degradation provide further evidence that this pathway may have an important role in regulating collagen deposition.
MDA content increases in fast- and slow-twitch skeletal muscle with intensity of exercise in a rat
Authors: Alessio H. M. , Goldfarb A. H. , Cutler R. G.
Published: December 2017

Previous work has shown that high-intensity (HI) exercise results in total body increased production of lipid peroxidation. These results suggest that exercise induces a higher level of oxidative stress in muscles leading to the production of various peroxides and aldehydes that are potentially toxic to cells. However, these past studies were carried out only with subjects that were exercised to exhaustion or at a very high intensity. In this paper, we report how a moderate-intensity (MI) exercise, which is more normally experienced, as well as a HI acute bout of exercise influenced oxidative stress-related reactions by measuring malonaldehyde (MDA) and lipid hydroperoxides (LH) in red vastus, white vastus, and soleus muscle. The muscles from untrained male Sprague-Dawley rats were removed immediately after either a HI 1-min run at 45 m/min (n = 8) or a 20-min MI run at 20 m/min (n = 8) and compared with a control group that did not run. MI exercise resulted in a 90% increase in MDA in white vastus and a 62% increase in red vastus muscle (P less than 0.05). HI exercise resulted in a 157, 167, and 83% increase in MDA in white vastus, red vastus, and soleus muscle, respectively. LH values in red and white vastus after HI exercise increased an average of 33%, but this proved not to be statistically significant. These results confirm earlier studies that HI exercise does increase MDA in skeletal muscle, and when intensity of exercise is considerably lowered, elevated MDA is still found but at a relatively lesser amount.
Modulation of angiotensin II binding sites in neuronal cultures by mineralocorticoids
Authors: Sumners C. , Fregly M. J.
Published: December 2017

Previous studies have determined that mineralocorticoid hormones are able to increase the number of angiotensin II (ANG II)-specific binding sites in rat diencephalon and in neuronal cultures and also increase the drinking response elicited by centrally injected ANG II. In the present study, we have examined the specificity and mechanisms of this mineralocorticoid action. In neuronal cultures from the hypothalamus and brain stem (H/BS), both D-aldosterone and deoxycorticosterone acetate (DOCA) caused significant time- and dose-dependent increases in 125I-labeled ANG II-specific binding. This effect was not mimicked by the synthetic glucocorticoid dexamethasone, or by testosterone, beta-estradiol or progesterone. However, the steroid corticosterone induced a moderate increase in [125I] ANG II binding. This may have occurred as a result of its high affinity for the mineralocorticoid type I receptor. DOCA was ineffective in increasing [125I]ANG II specific binding both in neuronal cultures prepared from the cerebellum and in pure astrocytic glial cultures, indicating that this mineralocorticoid effect is specific both for neurons and for certain brain regions. The increase in [125I]ANG II-specific binding elicited by DOCA was abolished by cotreatment with the mineralocorticoid receptor blockers mespirenone or ZK97894 and by cotreatment with cycloheximide. Taken together, these observations suggest that the mineralocorticoid-induced increase in [125I]ANG II-specific binding in H/BS neuronal cultures is a specific event, which is mediated via mineralocorticoid type I receptors and which requires protein synthesis.
Relationship between cytosolic free Ca2+ and Na+-Ca2+ exchange in aortic muscle cells
Authors: Smith J. B. , Zheng T. , Smith L.
Published: December 2017

We examined the relationship between extracellular Na+ ([Na+]o) and cytosolic free Ca2+ ([Ca2+]i) in primary and passaged cultures of aortic muscle cells. Removing [Na+]o increased [Ca2+]i by approximately 10-fold in cells that were Na+ loaded. Decreasing [Na+]o from 140 to 32 mM caused the half-maximal increase in [Ca2+]i. [Ca2+]i exhibited a sigmoidal dependence on [Na+]o. Mg2+, a competitive inhibitor of Na2+-Ca2+ antiport in these cells, antagonized the increase in [Ca2+]i produced by lowering [Na+]o. High K+ decreased the potency of Mg2+ 6.5-fold as previously reported for Na+ gradient-dependent 45Ca2+ influx. In contrast to the Na+-loaded cells, removing [Na+]o caused no detectable change in [Ca2+]i in cells with normal Na+ even though the calculated electrochemical driving force for Na+-Ca2+ exchange was large enough to almost maximally increase [Ca2+]i in the Na+-loaded cells. We conclude that Na+-Ca2+ antiport activity is latent in the unstimulated cell at basal intracellular Na+ and Ca2+.
Inhibitory effect of somatostatin on vagal motoneurons in the rat brain stem in vitro
Authors: Nabekura J. , Mizuno Y. , Oomura Y.
Published: December 2017

Effects of somatostatin-14 (SRIF) on membrane electrical properties were studied in rat brain stem slice preparations maintained in vitro. SRIF hyperpolarized the resting membrane potential and decreased the input resistance of more than two-thirds of the 85 vagal motoneurons tested in the dorsal motor nucleus of the vagus. These effects persisted under synaptic blockade caused by perfusion with a solution containing tetrodotoxin or a Ca2+-free/high-Mg2+ solution and were dependent on the extracellular SRIF concentration (5 X 10(-8) to 1 X 10(-8) M). The Hill coefficient was estimated to be 2. The reversal potential of SRIF-induced hyperpolarization was affected by changing external K+ concentration. The results suggest that, in addition to its well-known peripheral action, SRIF may inhibit secretomotor functions of visceral organs by reducing vagal output in the central nervous system.
Myosin dephosphorylation during rapid relaxation of hog carotid artery smooth muscle
Authors: Driska S. P. , Stein P. G. , Porter R.
Published: December 2017

Changes in myosin light chain phosphorylation were measured during histamine-induced rhythmic contractions of hog carotid artery smooth muscle strips. Histamine made the muscle strips contract spontaneously every 1-5 min, and this allowed measurement of the time course of phosphorylation in relation to force development under conditions where diffusion of the agonist through tissue would not complicate the interpretation of the data. In the absence of histamine, phosphorylation was low [0.12 +/- 0.04 mol P/mol of the 20,000-Da light chain (LC 20)]. Phosphorylation was slightly (but not significantly) higher in the presence of 10 microM histamine in the relaxed state between contractions (0.20 +/- 0.03 mol P/mol LC 20). In muscle strips frozen during force development, when force had reached half of its peak value, phosphorylation was 0.38 +/- 0.06 mol P/mol LC 20. The highest levels of phosphorylation (0.49 +/- 0.04 mol P/mol LC 20) were found in strips frozen at the peak of the rhythmic contractions. Strips frozen when force had declined to half of the peak force showed low levels of phosphorylation (0.17 +/- 0.07 mol P/mol LC 20), indicating that the myosin light chain phosphatase activity was quite high. Mathematical modeling of the kinase and phosphatase reactions suggested that the apparent first-order phosphatase rate constant was at least 0.08 s-1 under these conditions. To obtain a better estimate of this rate constant, a second series of phosphorylation measurements were made early in the relaxation phase of the rhythmic contractions. The highest phosphatase rate constant obtained from these measurements was 0.23 s-1.(ABSTRACT TRUNCATED AT 250 WORDS)
Changes of tracheal smooth muscle stiffness during an isotonic contraction
Authors: Seow C. Y. , Stephens N. L.
Published: December 2017

Stiffness of the series elastic component (SEC) of canine tracheal smooth muscle in isotonic contraction and relaxation was measured by applying small force perturbations to the muscle and measuring the resulting length perturbations. The quick, elastic length transient was taken as the change in length of the SEC (delta L). The force perturbation was a train of 10-Hz rectangular force waves varying from 0 to 10% maximum isometric tension (Po) in magnitude (delta P = 10% Po). Stiffness of the SEC was estimated by the ratio delta P/delta L. The change in SEC stiffness with respect to the change in muscle length was further studied by obtaining the stress-strain curves of the SEC at different muscle lengths using the load-clamping method. The clamps were applied at a fixed time (10 s after stimulation). Length of the muscle 10 s after contraction was controlled by the magnitude of the isotonic afterload. It was found that the apparent SEC stiffness increased as muscle length decreased. This stiffness increase is not likely due to an increase in the number of attached cross bridges, but it is probably due to the gradual diminution of the SEC length itself during muscle shortening.
Na-Ca exchange in ferret red blood cells
Authors: Milanick M. A.
Published: December 2017

Ferrets have high Na (140 mmol/l) red blood cells. To determine whether ferret red cells had a Na-Ca exchange system, Na effluxes via the Na + K + 2Cl cotransrpoter and Ca effluxes via the Ca pump had to be inhibited. This was accomplished by replacing cell chloride with nitrate and by loading the cells with vanadate that inhibits the Ca pump. Under these conditions, extracellular Na (Naout) inhibited Ca influx. Intracellular Na (Nain) was required for the large Ca influx as replacement of Na with Li reduced Ca influx to less than one-tenth of the original rate. Caout stimulated Na efflux by about twofold. The Ca efflux from cells depleted of Na was increased from 0.8 to 3.2 mmol.l packed cells-1.h-1 by the presence of Naout. Cells placed in a Na-free solution accumulated Ca: total intracellular Ca was 20-fold higher than free Caout. Most of this Ca was released on addition of the Ca ionophore, A23187. Because the Na gradient had driven net Ca uphill, the fluxes of Na and Ca are coupled. In a Na-free solution, the K1/2 for Ca influx was usually approximately 10 microM (occasionally approximately 100 microM), and the maximal velocity (Vmax was 1.5-4.4 mmol.l packed cells-1.h-1. At Naout = 150 mM, K1/2 increased 5- to 150-fold. In some cells, Naout decreased Vmax by approximately fourfold, suggesting that Naout does not always compete with Caout.(ABSTRACT TRUNCATED AT 250 WORDS)
A kinetic model of superoxide production from single pulmonary alveolar macrophages
Authors: DiGregorio K. A. , Cilento E. V. , Lantz R. C.
Published: December 2017

A kinetic model was developed to describe the production of superoxide (O2-) by single pulmonary alveolar macrophages (PAM). Model predictions were compared with experimental results obtained from single rat PAM. The O2- was quantified by measuring the reduction of nitro blue tetrazolium (NBT) to a diformazan precipitate (NBTH2) from video-recorded images of individual cells. The kinetic model considered three reactions: 1) the production of extracellular O2- from the reduction of oxygen by NADPH oxidase using intracellular NADPH as the substrate, 2) the subsequent dismutation of O2- to form H2O2, and 3) the reaction of O2- and NBT to form diformazan. NBT specificity for O2- was analyzed by comparing results in the presence and absence of superoxide dismutase (SOD) that catalyzes the dismutation of O2- to H2O2. Measured PAM heterogeneity was accounted for by varying the concentration of intracellular NADPH, its rate of depletion, and the concentration of intracellular NADPH oxidase in the kinetic model. Model predictions compared favorably with experimental results except when SOD was present. This discrepancy may be due to diffusional limitations because NBT is a relatively small molecule (818 mol wt) compared with SOD (34,000 mol wt). In addition, the cell surface is both ruffled and negatively charged, which may introduce steric hindrances and/or electrostatic effects, since SOD is also negatively charged, whereas NBT is positively charged.
Patch clamp of sarcolemmal spheres from stretched skeletal muscle fibers
Authors: Stein P. , Palade P.
Published: December 2017

Stretching frog skeletal muscle fibers to the breaking point results in the rapid formation of numerous large spheres of membrane (5-80 microns diam). The surface of the spheres readily forms gigaohm (G omega) seals against patch pipettes, allowing low-noise single-channel recording. Currents recorded from patches isolated from these spheres indicate that they contain a variety of channels including 1) a small Na+-selective channel seen in the presence of veratridine, 2) a K+-selective channel which is blocked by millimolar Mg-ATP, and 3) a relatively large voltage-dependent Cl- channel which is blocked by Zn2+ and limited in selectivity over other anions [PCl/PMOPS = 3.7; MOPS, 3-(N-morpholino)propanesulfonic acid]. These channels have been described previously and have been identified as markers for sarcolemmal (SL) membrane. Accordingly, this method allows rapid and direct recording of channels in the SL membrane without first having to pretreat fibers with proteolytic enzymes to render the SL accessible to patch pipettes.
Cytosolic magnesium modulates calcium channel activity in mammalian ventricular cells
Authors: Agus Z. S. , Kelepouris E. , Dukes I. , Morad M.
Published: December 2017

The effect of cytosolic free Mg2+ concentration on the regulation of myocardial function was studied by dialyzing isolated guinea pig ventricular myocytes with different internal Mg2+ concentrations [( Mg2+]i). We found that elevation of [Mg2+]i shortened the action potential and suppressed the Ca2+ current. Mean values recorded for action potential duration in cells dialyzed with solutions containing 0, 1.3, and 9.4 mM Mg2+ were 620 +/- 40, 400 +/- 25, and 60 +/- 10, respectively. The suppressive effect of [Mg2+]i on the action potential duration correlated significantly with the suppressive effects of [Mg2+]i on the Ca2+ current. In cells dialyzed with nominally zero Mg2+, calcium current was prominent (3.5 +/- 0.58 nA). At [Mg2+]i of 1.4 mM, calcium current was significantly smaller than in zero [Mg2+]i and was almost completely inhibited by dialysis of the cell with 9.4 mM Mg2+. The Mg2+-induced block of the Ca2+ current was due to steady-state inactivation of the high threshold calcium channel. The block was observed in the presence or absence of adenosine 3',5'-cylic monophosphate and was not reversed by elevation of external Ca2+ concentration, addition of adrenaline, or large negative potentials. These data suggest that cytosolic Mg2+ regulates Ca2+ channel activity by a novel mechanism, unrelated to its effect as a blocking particle of the open channel.
Density-independent isolation of type II pneumocytes after partial pneumonectomy
Authors: Uhal B. D. , Hess G. D. , Rannels D. E.
Published: December 2017

Type II pneumocytes were isolated by immunoglobin G (IgG) panning from the lungs of normal rats and the right lung of rats (180-200 g) subjected to left pneumonectomy. Cells were studied at 7 (pnx-7) and 15 (pnx-15) days postoperative, times during and after, respectively, rapid compensatory growth of the right lung. After 24 h of primary culture, pnx-7 cells contained 32% more protein per DNA, and incorporated thymidine at a rate 224% greater than cells isolated from control rats. Both the protein-to-DNA ratio and thymidine incorporation returned to control values in pnx-15 cells. Uptake of exogenous spermidine also was increased by 50% in pnx-7 cells at 24 h of primary culture and returned to control values in pnx-15 cells. Increased spermidine uptake was due to an increase in the maximal velocity (Vmax) of transport from 30.3 (control) to 45.5 pmol.micrograms DNA-1.h-1 (pnx-7), with no change in the apparent Km of 1.32 microM. No change was observed in the relative rates of phospholipid or neutral lipid biosynthesis. The increases in thymidine incorporation and spermidine uptake were significantly greater than those previously observed [Am. J. Physiol. 254 (Cell Physiol. 23): C684-C690, 1988] in pnx-7 cells isolated by Percoll gradient sedimentation. These results suggest that pnx-7 lungs contain distinct subpopulations of type II pneumocytes, the recovery of which is dependent on the cell isolation protocol employed.
Determinants of atrial natriuretic peptide secretion in cultured atrial myocytes
Authors: Iida H. , Page E.
Published: December 2017

Atrial natriuretic peptide (ANP) secretion by atrial myocytes in 7- to 8-day primary cultures from adult rats was measured by radioimmunoassay under conditions designed to separate primary effects on secretion from effects caused by contractions. Abolishing contraction with 10 microM tetrodotoxin significantly reduced ANP accumulated in 2 h. Raising external Ca2+ concentration from 0.2 to 1.2 mM in the presence of tetrodotoxin did not increase ANP secretion. Substantial ANP secretion persisted in a nominally Ca2+-free medium containing 10 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and was not diminished by 100 microM ryanodine. In the presence of EGTA, 100 nM 12-O-tetradecanoylphorbol 13-acetate (TPA) significantly increased ANP secretion; this increment was unaffected by 100 microM ryanodine. These experiments suggest 1) that in absence of contractions, ANP secretion requires neither transplasmalemmal Ca2+ influx nor ryanodine-inhibitable Ca2+ release by sarcoplasmic reticulum (SR) and 2) that TPA stimulates ANP secretion without requiring Ca influx or ryanodine-inhibitable SR Ca release.
Signal for induction of aldose reductase in renal medullary cells by high external NaCl
Authors: Uchida S. , Garcia-Perez A. , Murphy H. , Burg M.
Published: December 2017

GRB-PAP1 is a continuous line of epithelial cells derived from rabbit renal inner medulla. These cells accumulate large amounts of sorbitol when extracellular NaCl concentration is elevated. The accumulation involves an increase in aldose reductase, an enzyme that catalyzes production of sorbitol from glucose. The purpose of the present studies was to investigate the mechanism by which high NaCl triggers an increase in aldose reductase activity. When NaCl was added to increase medium osmolality from 300 to 500 mosmol/kg, enzyme activity began to increase after 12-24 h, was half-maximal in 2 days, and was maximal by 4 days. To test the effect of other solutes, medium osmolality was increased to 500 mosmol/kg by adding raffinose, urea, or glycerol. Raffinose increased aldose reductase activity as much as NaCl did. In contrast, neither urea nor glycerol affected the enzyme activity. NaCl and raffinose caused a sustained decrease in cell water content and an increase in cell sodium and potassium concentration, but urea did not. In some experiments, ouabain was added either with or without an increase in osmolality. Taking into account all of the experiments, with and without ouabain, we found that aldose reductase activity did not correlate with either cell sodium concentration or cell water content alone. It did correlate, however, with cell potassium concentration and even more strongly with the sum of cell sodium plus potassium concentration. We conclude that the signal by which hyperosmolality triggers an increase in aldose reductase activity most likely involves an increase in intracellular ionic strength.
Tight monolayers of rat alveolar epithelial cells: bioelectric properties and active sodium transport
Authors: Cheek J. M. , Kim K. J. , Crandall E. D.
Published: December 2017

Because the pulmonary alveolar epithelium separates air spaces from a fluid-filled compartment, it is expected that this barrier would be highly resistant to the flow of solutes and water. Investigation of alveolar epithelial resistance has been limited due to the complex anatomy of adult mammalian lung. Previous efforts to study isolated alveolar epithelium cultured on porous substrata yielded leaky monolayers. In this study, alveolar epithelial cells isolated from rat lungs and grown on tissue culture-treated Nucleopore filters resulted in tight monolayers with transepithelial resistance greater than 2,000 omega.cm2. Changes in bioelectric properties of these alveolar epithelial monolayers in response to ouabain, amiloride, and terbutaline are consistent with active sodium transport across a polarized barrier. 22Na flux measurements under short-circuit conditions directly confirm net transepithelial absorption of sodium by alveolar epithelial cells in the apical to basolateral direction, comparable to the observed short-circuit current (4.37 microA/cm2). The transport properties of these tight monolayers may be representative of the characteristics of the mammalian alveolar epithelial barrier in vivo.
Regulation of Na+-H+ exchange in normal NIH-3T3 cells and in NIH-3T3 cells expressing the ras oncogene
Authors: Owen N. E. , Knapik J. , Strebel F. , Tarpley W. G. , Gorman R. R.
Published: December 2017

Our laboratory and others have demonstrated that Na+-H+ exchange can be regulated by two different pathways; one that is mediated by an inositol trisphosphate-stimulated increase in intracellular calcium activity, and one that is mediated by an increase in protein kinase C activity. To determine whether one of these pathways is more important than the other, or whether one pathway is physiologically relevant, we employed normal NIH-3T3 cells (3T3 cells) and NIH-3T3 cells expressing the EJ human bladder ras oncogene (EJ cells). The EJ cells were chosen because they provide a genetic model that does not exhibit serum- or platelet-derived growth factor (PDGF)-stimulated inositol trisphosphate release or Ca2+ mobilization. It was found that serum- or PDGF-stimulated Na+-H+ exchange was more pronounced in EJ cells than in control 3T3 cells. As expected, serum- or PDGF-stimulated Na+-H+ exchange in 3T3 cells was inhibited by chelating intracellular Ca2+ with the intracellular Ca2+ chelator quin2, by the intracellular Ca2+ antagonist 8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate (TMB-8), and by the calmodulin antagonist trifluoperazine. In contrast, these agents did not inhibit serum- or PDGF-stimulated Na+-H+ exchange in EJ cells. Activators of protein kinase C (e.g., 1-oleoyl-2-acetylglycerol or biologically active phorbol esters) were found to stimulate Na+-H+ exchange in EJ cells to the same extent as serum. However, these agents were considerably less effective than serum in control 3T3 cells. Despite these findings, PDGF did not stimulate diacylglycerol levels in EJ cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Effects of muscle length and load on intracellular Ca2+ in tracheal smooth muscle
Authors: Gunst S. J.
Published: December 2017

Canine tracheal smooth muscle strips were loaded with the bioluminescent Ca2+ indicator aequorin, mounted in a tissue bath, and attached to an electromagnetic lever to investigate the effect of changes in muscle length and load on cytosolic free Ca2+. The intracellular Ca2+ concentration ([Ca2+]i) was inversely correlated with muscle length and active force developed during isometric contractions elicited by electrical field stimulation. In addition, quick release to either constant length or constant load at any time point during an active contraction resulted in an increase in [Ca2+]i. These observations are consistent with the possibility that the binding of Ca2+ to contractile or regulatory proteins is decreased when muscle length or active force development is decreased, resulting in the release of bound Ca2+ into the cytoplasm. However, the possibility that changes in muscle length or load affect other mechanisms that regulate [Ca2+]i cannot be excluded.
Cl-dependent K transport in a pure population of volume-regulating human erythrocytes
Authors: O'Neill W. C.
Published: December 2017

Swelling of human red cells activates a putative K-Cl cotransport that is not present at normal cell volume and that disappears after several hours. To determine whether regulatory volume decrease (RVD) is occurring in human erythrocytes and is responsible for the inactivation of K-Cl cotransport, the relationship between cell volume and the inactivation and reactivation of volume-sensitive (VS) K-Cl cotransport was studied. VS K influx into high K cells was transient, whereas influx into low K cells (prepared with nystatin), which are unable to shrink via K efflux, remained fully activated. Likewise, VS K efflux into hypotonic medium disappeared after 100 min in a low K medium but remained activated in a high K medium that prevented cell shrinkage. Cells that had been preincubated in hypotonic medium to inactivate VS K-Cl cotransport showed no significant recovery of VS cotransport after a 6-h incubation in isotonic medium but showed full restoration of VS cotransport after treatment with nystatin in isotonic medium to reequilibrate cell water. A pure fraction of volume-regulating (VR) cells was subsequently isolated by preincubating red cells in hypotonic medium and then subjecting them to further hypotonicity to lyse all non-VR cells. The 2.5% of cells that remained consisted of 16% reticulocytes and exhibited a Cl-dependent RVD in hypotonic medium. VS K-Cl cotransport was enriched 10-fold and Na-K-Cl cotransport was enriched 12-fold in these cells, whereas the enrichment of N-ethylmaleimide (NEM)-activated K-Cl cotransport was only threefold.(ABSTRACT TRUNCATED AT 250 WORDS)
Erythropoietin production in exhypoxic polycythemic mice
Authors: Seferynska I. , Brookins J. , Rice J. C. , Fisher J. W.
Published: December 2017

Our present study was undertaken to determine the serum erythropoietin concentration (radioimmunoassay), hematocrit, red cell mass, and body weight of mice exposed to hypoxia in a hypobaric chamber (0.42 atm, 22 h/day) for 14 days and during the 10 posthypoxic days at ambient pressure to clarify the correlation of the red cell mass and erythropoietin production during hypoxia. The mean serum erythropoietin titer was 326.23 +/- 77.04 mU/ml after 2 days, reached the highest level after 3 days (452.2 +/- 114.5 mU/ml), then gradually declined to a level of 36.5 +/- 11.4 mU/ml after 14 days of hypoxia, and was undetectable during the 10-day posthypoxic period. The hematocrit values were significantly increased from 41.09 +/- 0.50% at day 0 to 51.65 +/- 1.08% after 3 days and to 72.20 +/- 1.53% after 14 days of hypoxia. The red cell mass (calculated from initial body weight) increased from 3.24 +/- 0.1 ml/100 g at day 0 to 7.32 +/- 0.46 ml/100 g after 14 days of hypoxia and declined to 6.66 +/- 0.53 ml/100 g at the end of the 10-day posthypoxic period. The mice lost weight while they were in the hypobaric chamber and showed a significant increase in body weight during the 10-day posthypoxic period. These studies support the concept that chronic intermittent hypoxia causes an early increase, followed by a rapid decline, in erythropoietin production, which is correlated with the gradual increase in red cell mass.
Activation of potassium channels in renal epithelioid cells (MDCK) by extracellular ATP
Authors: Friedrich F. , Weiss H. , Paulmichl M. , Lang F.
Published: December 2017

Extracellular ATP has been shown to stimulate transepithelial chloride transport in confluent Madin-Darby canine kidney (MDCK) cell layers and to enhance potassium conductance in subconfluent MDCK cells. The present study has been performed to test for the effect of extracellular ATP on channel activity in patches from subconfluent MDCK cells. Within 8 s, addition of extracellular ATP (10 mumol/l) leads to a sustained, but fully reversible, appearance of potassium-selective channels in cell-attached patches [increase of open probability from 0.03 +/- 0.02 (n = 10) to 0.50 +/- 0.07 (n = 6)]. With the use of pipettes filled with 145 mmol/l KCl, inwardly rectifying property of the channels is disclosed with a single-channel conductance of 65.7 +/- 3.1 pS (n = 9) at zero potential difference between pipette and bath and with a reversal potential of 75.4 +/- 2.0 mV (n = 5; pipette negative vs. reference in the bath). The open probability of the channels is not significantly modified by altering pipette potential from -50 mV, pipette positive, to 50 mV, pipette negative. At extracellular calcium activities of less than 10 nmol/l, ATP leads to a transient activation of channels. In conclusion, extracellular ATP activates inwardly rectifying potassium channels in the cell membrane of subconfluent MDCK cells. A sustained activation of the channels requires the presence of extracellular calcium and is probably mediated by increases in intracellular calcium.
Rocking promotes differentiated properties in LLC-PK cells by improved oxygenation
Authors: Sahai A. , Cole L. A. , Clarke D. L. , Tannen R. L.
Published: December 2017

Previous studies from our laboratory indicated that in contrast to cells cultured under still conditions, LLC-PK cells cultured under conditions of continuous rocking exhibit pH-modulated ammonia production and also manifest lower levels of lactate dehydrogenase activity. In the present studies, we assessed whether other metabolic features and parameters of differentiation are modified by rocking. Rocked, as contrasted with still, cultures exhibited decreased medium lactate production and increased cellular ATP content consistent with heightened oxidative metabolism. Still cultures did not exhibit either dome formation or sodium-dependent alpha-methylglucoside uptake until the cultures reached full confluency. By contrast, rocked cultures underwent dome formation as well as enhanced alpha-methylglucoside uptake during the growth phase, and these parameters were quantitatively greater than in still culture even after full confluency was achieved. Thus rocking promotes several functions, which can reflect differentiation in LLC-PK cells. To determine whether provision of adequate oxygen accounts for the enhancement of cellular metabolism and differentiated functions by rocking, still cultures grown in a high oxygen concentration of 36% were compared with standard cultures grown in 18% oxygen. The high oxygen environment resulted in the development of pH-modulated ammonia production, a decrease in lactate dehydrogenase activity, lower medium lactate formation, increased ATP levels, more copious dome formation, and increased alpha-methylglucoside uptake. Thus rocking promotes cellular metabolism and differentiated functions by the provision of adequate oxygenation.