bluuebunny/crossref_metadata_2025 huggingface dataset:
Endotoxin alters arachidonate metabolism in pulmonary endothelial cells
Authors: Suttorp N. , Galanos C. , Neuhof H.
Published: December 2017

Endotoxin and lipid A dose dependently (1 ng/ml to 10 micrograms/ml) and time dependently (6-24 h) stimulated the generation of large amounts of prostacyclin in cultured pig pulmonary artery endothelial cells. This effect occurred in the absence of cell detachment and overt cell damage. The presence of at least 1% serum was required but the activation of the complement cascade was not. Endotoxin-treated endothelial cells generated increased amounts of prostacyclin upon stimulation with A23187 and arachidonic acid. Endotoxin-induced activation of arachidonate metabolism could be reduced by 10(-10) M glucocorticoids but not by progesterone. It was further affected by inhibitors of protein and RNA synthesis and calmodulin function. In addition, exposure of endothelial cells to endotoxin resulted in an enhanced synthesis of cyclooxygenase and in a higher enzymatic capacity of prostacyclin synthase. The data indicate that endotoxin in concentrations occurring in the plasma of patients profoundly alters arachidonic acid metabolism in endothelial cells.
Effects of rest duration and ryanodine on changes of extracellular [Ca] in cardiac muscle from rabbits
Authors: MacLeod K. T. , Bers D. M.
Published: December 2017

Cumulative depletions of extracellular Ca were measured using double-barreled Ca-sensitive microelectrodes in the extracellular space of rabbit ventricular muscle. Depletions were produced by 1-Hz stimulation after rest intervals of 10 s to 10 min. With longer rest intervals, depletion size increased while the first postrest contraction decreased in a reciprocal manner. The depletions may represent refilling of sarcoplasmic reticulum (SR) Ca stores that have become partially depleted of Ca during the rest. Within this interpretive framework, the longer the rest interval the lower the SR Ca content, so the SR is then capable of taking up larger amounts of Ca. This may be related to the rest decay of tension of the first postrest beat, since this is thought to be SR dependent. Ryanodine (1 microM) increased the size of the depletions after short rest intervals (less than 2 min) but not after longer (greater than or equal to 2 min) intervals. Ryanodine also increased the rate of Ca loss from the cell on cessation of stimulation. This increased rate of Ca loss with ryanodine may deplete the SR of Ca such that more Ca can be taken up during subsequent stimulation than in untreated muscles. Thus cumulative depletions after short rest intervals are enhanced by ryanodine. When a Ca load was produced during 1) quiescence [by removal of extracellular Na (Nao)] or 2) continuous stimulation (in the presence of 3 microM acetylstrophanthidin), addition of ryanodine (5-10 microM) did not produce any apparent Ca loss. Caffeine (10 mM), added after ryanodine, induced contractures accompanied by Ca efflux, implying there was Ca in the SR after ryanodine exposure. The results of other investigators have suggested that ryanodine may inhibit cardiac SR Ca release. The present study suggests that ryanodine also enhances the loss of cellular (and probably SR) Ca on cessation of stimulation but not when applied during continuous stimulation or quiescence.
Delta alkalinity: a simple method to measure cellular net acid-base fluxes
Authors: Burbea Z. H. , Gullans S. R. , Ben-Yaakov S.
Published: December 2017

Acid-base transport across cell plasma membranes is important for cell homeostasis and growth. Current techniques for quantitatively measuring net acid-base fluxes are generally limited by either assumptions concerning properties of intracellular compartments or use of poorly buffered, nonphysiological solutions. We adapted an approach from marine chemistry to quantitate net acid-base changes in standard physiological media that obviates these problems. This method is based on conservation of charge and involves a simple acid titration of the extracellular medium to an end point, the equivalence point, pHe. For standard physiological solutions containing buffers such as bicarbonate and phosphate, pHe exists in the range of pH 4.0-4.7 and is identified as the pH where dpH/dH+ is maximal in an HCl titration. By determining the quantity of H+ required to reach pHe, one can determine precisely total quantity of proton acceptors (alkalinity) present in physiological pH range. Alkalinity (in meq) is a relative measure of the charge capable of interacting with protons. We show that, unlike pH, changes in alkalinity (delta alkalinity) result only from net acid-base changes in medium. Therefore, by monitoring extracellular delta alkalinity associated with cell function, it is possible to quantitate precisely net acid-base fluxes. Moreover, through a second titration procedure, delta alkalinity can be divided into bicarbonate and nonbicarbonate fractions. As an example, we performed the first direct measurement of net Cl(-)-HCO3- exchange in intact human erythrocytes and observed a Cl(-)-HCO3- exchange ratio of 1.01 +/- 0.03. Overall, delta alkalinity measurements are applicable to numerous cell systems, can be performed with solutions containing a mixture of buffers at normal physiological concentrations (e.g., 25 mM HCO3- and 2 mM HPO4(-2), do not require corrections for CO2 diffusion or loss of CO2 from the solution, and avoid assumptions about intracellular or extracellular buffer properties.
Distribution of meprin in kidneys from mice with high- and low-meprin activity
Authors: Craig S. S. , Reckelhoff J. F. , Bond J. S.
Published: December 2017

An inherited deficiency of a metalloendopeptidase (meprin) activity occurs in kidneys of many inbred mouse strains. To clarify whether meprin protein is present in low-activity strains and determine the distribution of meprin in kidneys of mice with high- and low-meprin activities, kidney slices were stained through the use of the indirect immunoperoxidase technique and examined by light and electron microscopy. Light microscopy at high dilutions of anti-meprin IgG confirmed the brush border localization of meprin in high-meprin activity strains and revealed no detectable cross-reactive material in low-meprin activity strains. However, light and electron microscopy studies that use lower dilutions of anti-meprin immunoglobulin G (IgG) revealed cross-reactivity in low-activity strains, also at the luminal surface of the proximal tubules. Studies at lower magnifications indicated that meprin is primarily associated with the juxtamedullary region of the kidney in both high- and low-activity strains. Western blots of urinary proteins showed significant amounts of meprin-like proteins, but only in the urine of mice with high-meprin activity. The low activity of meprin in some inbred mouse strains is not associated with the presence of the protein in compartments of kidney cells other than the brush border or with secretion of the protein into the urine.
Effects of pH changes on sodium pump fluxes in squid giant axon
Authors: Breitwieser G. E. , Altamirano A. A. , Russell J. M.
Published: December 2017

The effects of independently varying intracellular and extracellular pH on sodium pump fluxes were studied in the squid giant axon. By means of intracellular dialysis, we found that changes of intracellular pH (pHi), but not of extracellular pH, affected ouabain-sensitive Na+ efflux and K+ influx over the pH range of 6.0-8.6. Both fluxes were maximum at a pHi of 7.2-7.4. Variations away from this optimal pHi in either the acidic or alkaline direction resulted in a graded inhibition of both ouabain-sensitive fluxes. The kinetic basis for the inhibitory effect of acidic pHi was examined by comparing the kinetic parameters of activation of ouabain-sensitive sodium efflux by intracellular Na+ (Na+i) and extracellular K+ (K+o) at normal pHi with those at acidic pHi. We found that the inhibitory effect of intracellular acidity results from a reversible decrease in maximum velocity (Vmax), without an effect on the activation parameters for Na+i (K1/2 Na+i) or K+o (K1/2 K+o).
Structure, biochemistry, and assembly of epithelial tight junctions
Authors: Gumbiner B.
Published: December 2017

The zonula occludens (ZO), also referred to as the tight junction, forms the barrier to the diffusion of molecules and ions across the epithelial cell layer through the paracellular space. The level of electrical resistance of the paracellular pathway seems to depend on the number of strands in the ZO observed by freeze-fracture electron microscopy (EM). The ZO also forms the boundary between the compositionally distinct apical and basolateral plasma membrane domains because it is a barrier to the lateral diffusion of lipids and membrane proteins that reside in the extracytoplasmic leaflet of the membrane bilayer. In contrast to its appearance in transmission EM, the tight junction is not a fusion between the outer membrane leaflets of neighboring cells. Rather it consists of protein molecules, including the newly discovered protein ZO-1 and probably others, which bring the plasma membranes into extremely close apposition so as to occlude the extracellular space. Very little is known about the assembly of tight junctions, but several kinds of evidence suggest that they are very dynamic structures. Other elements of the epithelial junctional complex including the zonula adherens (ZA), the Ca2+-dependent cell adhesion molecule uvomorulin, or L-CAM, and actin filaments of the cytoskeleton may participate in the assembly of the ZO.
Culture of type II pneumocytes on a type II cell-derived fibronectin-rich matrix
Authors: Rannels S. R. , Fisher C. S. , Heuser L. J. , Rannels D. E.
Published: December 2017

Recent evidence suggests that during primary culture, type II pneumocytes synthesize and deposit components of an extracellular matrix. The present study investigated the response of freshly isolated type II cells to a preformed, fibronectin-rich matrix synthesized by type II cells over a 6-day interval of primary culture on a plastic surface. Type II cells on 6-day matrix (M6) degraded the preformed matrix and deposited newly synthesized fibronectin more rapidly than cells on plastic, suggesting that M6 itself stimulated type II cell-mediated matrix turnover. In type II cells on plastic, incorporation of radiolabeled thymidine into DNA increased 620 and 1,880% after 2 and 3 days in culture, respectively, as the cells assumed a more flattened phenotype. Although cells on M6 did not divide, both basal rates of thymidine labeling and sensitivity to serum modulators of DNA synthesis were enhanced by the M6 surface, as compared with plastic. Culture of type II cells on surfaces of purified fibronectin enhanced the rate of DNA synthesis in a manner similar to that observed on M6; this effect was blocked by antifibronectin. The data suggest that more rapid fibronectin synthesis and deposition are important components of the response of type II cells to primary culture. Extracellular matrix produced by type II cells appears to be similar to the basement membrane onto which these cells proliferate in vivo after lung injury. A fibronectin-rich surface in itself may thus induce additional extracellular matrix synthesis and further direct cellular differentiation and proliferation.
Fluorescence detection of mitochondrial clusters in mammalian white fat cells in vivo
Authors: DeMartinis F. D. , Ashkin K. T. , Lampe K. T.
Published: December 2017

Rhodamine 123 and epifluorescence microscopy revealed a significant portion of the fat cell's mitochondria existed in the form of clusters or aggregates, whereas the remainder were scattered about the cytoplasm. The aggregates were variable in size and number and apparently bore no fixed relationship to the nucleus or to each other. Mitochondrial clusters were seen in vivo in rat and mouse adipocytes of the mesenteric and epididymal depots, in excised tissue pieces of other depots, and in isolated fat cells. Physiological factors investigated such as species type (rat, mouse, rabbit, dog), sex, age, depot location (superficial vs. deep), fat cell size, hypercholesterolemia, and 24-h fasting had no apparent effect on cluster prevalence or size. Similar aggregates were not visible in several cultured cell lines studied nor in various non-fat cells, capillary endothelial cells, or nerve fibers contained within adipose depots examined. These results indicate that mitochondrial clusters exist naturally in mammalian white fat cells and conclude that they represent a form of cytoplasmic organization whose purposes are not well understood.
Effects of low calcium levels on erythropoietin production by human renal carcinoma cells in culture
Authors: Nagakura K. , Ueno M. , Brookins J. , Beckman B. S. , Fisher J. W.
Published: December 2017

Recent investigations have shown that calcium entry blockers enhance the effects of hypoxia on erythropoietin (Ep) production in vivo. To determine whether deprivation of calcium increases Ep production and/or release, studies were carried out to determine the effects of low levels of extracellular calcium on Ep (radioimmunoassay) secretion in human renal carcinoma cells in culture. Low extracellular calcium levels (0.3 mM) in culture medium significantly (P less than 0.01) enhanced Ep secretion (64-145% increase per day) by renal carcinoma cells in culture when compared with a concentration of 1.9 mM calcium in the control culture medium (23-68% increase per day) incubated for 24 h or more. A 53% increase per day in Ep secretion was also produced by the calmodulin inhibitor trifluoperazine. To determine whether the effects of low calcium levels on Ep production could be due to nonspecific leakage of large intracellular molecules caused by a permeabilization of the cell membrane, the effect of low calcium levels in the cultures of the renal carcinoma cells on lactate dehydrogenase release into the culture medium was studied. Low calcium concentrations failed to significantly enhance lactate dehydrogenase secretion by the renal carcinoma cells. In conclusion, our results indicate a possible involvement of the calcium ion and calmodulin in the biosynthetic pathway for Ep and that calcium may exert a suppressive effect on Ep production.
Isozymes of dog heart Na+-K+-ATPase are immunologically similar to isozymes in brain
Authors: McDonough A. A. , Schmitt C. A.
Published: December 2017

The sodium pump, Na+-K+-ATPase, possesses two populations of cardiac glycoside-binding sites in cardiac tissue. This has been observed in other tissues such as brain where the two sites have been assigned to isozymes of Na+-K+-ATPase termed alpha and alpha +. In a previous study [Am. J. Physiol. 248 (Cell Physiol. 17): C247-C251, 1985], we were unable to demonstrate the presence of an alpha +-form in guinea pig heart sarcolemmal membranes using antibody probes. In the present study, using similar methodology, we show that dog, but not rat or guinea pig, sarcolemmal membranes contain two immunologically distinct alpha-subunits. The antibody-binding characteristics of dog heart alpha and alpha + are similar to the forms found in brain. The relative abundance of the two isozymes, estimated by labeling the sarcolemmal membranes with fluorescein 5'-isothiocyanate, was about equal. We conclude that the two populations of ouabain-binding sites in dog heart may result, at least in part, from the presence of the two isozymes of Na+-K+-ATPase in cardiac tissue of this species.
Intracellular Ca regulation during secretagogue stimulation of the parietal cell
Authors: Negulescu P. A. , Machen T. E.
Published: December 2017

Intracellular [Ca] ([Ca]i) was measured following secretagogue stimulation of rabbit gastric glands loaded with the Ca-sensitive dye fura-2. Glands were mounted on cover slips and placed either in a perfused cuvette (for spectrofluorimetric measurements on whole glands) or in a chamber on the stage of a microscope (for microspectrofluorimetric measurements on single parietal cells within a gland). In parietal cells resting [Ca]i = 91 nM. Either histamine or carbachol caused [Ca]i to increase (spike) rapidly (within 5 s) to greater than 425 nM by releasing Ca from an intracellular store. The two hormones acted on the same store, with carbachol being the more potent releaser. The spike occurred equally well in solutions containing the Ca channel blockers verapamil, nifedipine, Co, or La. After the spike, [Ca]i decreased to a plateau level (130-200 nM) that was dependent on the presence of secretagogue but was independent from the release of the intracellular store. This plateau persisted (up to 40 min) until the addition of antagonist or until the removal of either extracellular Ca or the agonist. Histamine and carbachol were specifically blocked by the H2-antagonist cimetidine and the muscarinic antagonist atropine, respectively. Histamine often induced repeated increases in [Ca]i. A combination of dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) and the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) had no effect on [Ca]i, but if cells were pretreated with histamine, DBcAMP + IBMX would cause [Ca]i to increase. Because exposure to DBcAMP + IBMX stimulates acid secretion yet does not affect [Ca]i, two independent pathways to acid secretion may exist.
Osmotically induced basolateral K+ conductance in turtle colon: lidocaine-induced K+ channel noise
Authors: Dawson D. C. , Van Driessche W. , Helman S. I.
Published: December 2017

The basolateral membrane of amphotericin-treated turtle colon can exhibit two distinct types of K+ conductance, one of which is associated with cell swelling and is blocked by quinidine or lidocaine. Fluctuations in basolateral K+ currents were analyzed under swelling (mucosal KCl) and nonswelling (mucosal K gluconate) conditions. Under nonswelling conditions, it was not possible to detect a spontaneous Lorentzian component in the power density spectrum (PDS) and the addition of lidocaine neither inhibited the macroscopic current nor induced a Lorentzian component in the PDS. Under swelling conditions, however, lidocaine induced a Lorentzian component in the PDS and the corner frequency increased linearly with blocker concentration as expected for reversible blockade of the channel. The gating and conductance properties of osmotically induced channels estimated from a two-state model were similar to those determined recently in single-channel recordings from isolated colonic cells.
Beta-adrenergic stimulation of brown adipocyte proliferation
Authors: Geloen A. , Collet A. J. , Guay G. , Bukowiecki L. J.
Published: December 2017

The mechanisms of brown adipose tissue (BAT) growth were studied by quantitative photonic radioautography using tritiated thymidine to follow mitotic activity. To identify the nature of the adrenergic pathways mediating brown adipocyte proliferation and differentiation, the effects of cold exposure (4 days at 4 degrees C) on BAT growth were compared with those induced by treating rats at 25 degrees C with norepinephrine (a mixed agonist), isoproterenol (a beta-agonist), and phenylephrine (an alpha-agonist). The drugs were continuously administrated via osmotic minipumps (0.375 mumol/h during 4 days) implanted subcutaneously. Cold exposure markedly enhanced the mitotic activity in brown adipocyte precursor cells (interstitial cells and preadipocytes) and endothelial cells forming the numerous capillaries. Norepinephrine mimicked the effects of cold exposure, not only on the mitotic activity, but also on the distribution of the labeling among the various cellular types. Isoproterenol entirely reproduced the effects of norepinephrine both on the labeling index and on the cellular type labeling frequency. In contrast, phenylephrine did not stimulate cell division. These results demonstrate that norepinephrine triggers a coordinated proliferation of brown adipocytes and endothelial cells in warm-exposed rats that is similar to that observed after cold exposure. They also suggest that cold exposure stimulates BAT growth by increasing the release of norepinephrine from sympathetic nerves and that the neurohormone activates mitoses in BAT precursor cells via beta-adrenergic pathways.
Ion transport mediated by copolymers composed of polyoxyethylene and polyoxypropylene
Authors: Atkinson T. P. , Bullock J. O. , Smith T. F. , Mullins R. E. , Hunter R. L.
Published: December 2017

Block copolymers composed of polyoxyethylene and polyoxypropylene were found to increase the influx of Na+ and the efflux of K+ from human erythrocytes. They were, however, ineffective at promoting the transport of Ca2+. The size of the ion fluxes induced by the copolymers correlated with their efficacy in stimulating inflammation. These compounds were also found to induce conductance increases in planar lipid bilayers in a nonvoltage dependent and nonstepwise manner. In both experimental systems, ion transport was facilitated only under temperature and ionic-strength conditions in which the polymers form aggregates in aqueous solution. In neither system did the concentration dependence of transport activity exhibit a pronounced cooperativity. These observations are consistent with the view that aqueous monomers of these surface active agents partition into the membrane, where they facilitate the conductive movement of monovalent cations by means of a carrier type mechanism. As a novel class of ionophores, these substances are of practical interest because they can be water soluble and are potentially reversible.
[ATP]i in Limulus photoreceptors: no correlation with responsiveness or discrete event rate
Authors: Rubin L. J. , Brown J. E.
Published: December 2017

Firefly luciferin-luciferase was microinjected into single, intact photoreceptor cells of Limulus ventral eyes. In addition, total ATP content of whole end organs was measured. An attempt was made to correlate intracellular ATP levels with either the frequency of discrete events or responsiveness to light. Luciferin-luciferase luminescence emitted from a single photoreceptor cell increased after an injection of ATP or P[NH]P and decreased after injection of apyrase. We conclude that the luciferin-luciferase luminescence is monitoring increases as well as decreases in intracellular ATP concentration (ATPi). The ATPi was between 10(-3) and 10(-4) M. Bathing ventral eyes in vanadate or fluoride increased ATP content of whole end organs, increased ATPi in single photoreceptors, and increased the frequency of discrete events. However, apyrase decreased ATPi but did not affect the frequency of discrete events or responsiveness to light until ATPi was dramatically reduced. We conclude that changes in ATP levels are not strictly correlated with changes in responsiveness to light or to changes in the frequency of discrete events.
Characterization of [3H]nifedipine binding to intact vascular smooth muscle cells
Authors: Sumimoto K. , Hirata M. , Kuriyama H.
Published: December 2017

Specific binding of the dihydropyridine Ca2+ antagonist [3H]nifedipine to dispersed smooth muscle cells of the porcine coronary artery was investigated and the findings were compared with the binding to microsomes of smooth muscles. Specific binding to intact cells was saturable and reversible. The dissociation constant was 1.93 +/- 0.42 nM and the maximal binding capacity was 59.6 +/- 12.4 fmol/10(6) cells, as assessed by Scatchard analysis of the equilibrium binding at 25 degrees C. The Kd value with intact cells was slightly higher than that observed with microsomes. Specific binding of [3H]nifedipine to intact cells was completely displaced by unlabeled dihydropyridine derivatives. Among other Ca2+ antagonists, verapamil and d-cis-diltiazem partially and flunarizine completely inhibited the binding. In the case of microsomes, d-cis-diltiazem stimulated the binding of [3H]nifedipine. These results suggest that there may be multiple binding sites for different subclasses of Ca2+ antagonists. Polyvalent cations had no effect on the binding to intact cells. In the case of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA)-treated microsomes, the addition of CaCl2 and BaCl2 increased the Bmax, but the Kd value remained unchanged. MnCl2 and CdCl2 had stimulatory or inhibitory effects, depending on the concentrations, whereas LaCl3 had no effect. The effect of membrane depolarization on the binding was also examined. When the intact cells were incubated in high [K+]o solution for 60 min, the Kd was lowered to 1.4 nM from the control value of 2.0 nM, thereby indicating that [3H]nifedipine binds to Ca2+ channels, with a higher affinity, at depolarized states.
Immune-related intestinal Cl- secretion. I. Effect of histamine on the T84 cell line
Authors: Wasserman S. I. , Barrett K. E. , Huott P. A. , Beuerlein G. , Kagnoff M. F. , Dharmsathaphorn K.
Published: December 2017

The mast cell mediator, histamine, induces a rapid and transient increase in chloride secretion across monolayers of the human colonic epithelial cell line, T84. Threshold stimulation occurred at 3 X 10(-6) M histamine and a maximal effect at 10(-4) M. The effect was reproduced by the H1 agonists 2-methylhistamine and 2-pyridylethylamine, but not by the H2 agonists 4-methylhistamine and dimaprit, suggesting the involvement of an H1 receptor. Additionally, histamine's action was inhibited by an H1 antagonist, diphenhydramine, but not by an H2 antagonist, cimetidine. Histamine treatment increased free cytosolic calcium levels, but not those of adenosine 3',5'-cyclic monophosphate (cAMP) or guanosine 3',5'-cyclic monophosphate (cGMP). The mechanism of chloride secretion induced by histamine resembled that of carbachol, in that both 1) were associated with an increase in free cytosolic calcium, 2) had a site of activation at a basolaterally localized K+ channel, and 3) were potentiated by both cAMP- and cGMP-mediated secretagogues. These results suggest that histamine may act as an intestinal secretagogue via direct interactions with epithelial cells.
Intercellular communication between ciliated cells in culture
Authors: Sanderson M. J. , Chow I. , Dirksen E. R.
Published: December 2017

Cultured mammalian ciliated cells from the respiratory tract respond to mechanical stimulation of their cell surface by displaying a rapid transient increase in beat frequency. Surrounding adjacent and more distal neighboring ciliated cells display a similar frequency response after a short delay that is proportional to their distance from the stimulated cell. To characterize the progression of this communicated response we developed an automated computer-assisted image-analysis system to examine high-speed films of responding cells. Transmission of the frequency response between cells occurs at 0.63 cells/s at 25 degrees C and 1.54 cells/s at 37 degrees C. We have also confirmed that gap junctions exist between cells in both epithelial explants and outgrowths and that adjacent or nonadjacent ciliated, as well as nonciliated, cells are electrically coupled. We postulate that mechanical stimulation and intercellular communication provide a mechanism to regulate beat frequency between ciliated cells in order to facilitate efficient ciliary function and mucus transport.
Ca dependence of Na influx during treatment of rabbit aorta with NE and high K solutions
Authors: Aaronson P. I. , Jones A. W.
Published: December 2017

Cellular influx of 24Na was measured in isolated rabbit aorta during stimulation with 10 microM norepinephrine (NE) or depolarization with 80 mM K solution, using a pulse-labeling, cold-wash technique. NE caused a two- to threefold increase in Na influx; a smaller but significant increase was also observed in depolarized tissues. Basal and NE-induced fluxes at 1 min were significantly increased by a 20-min preincubation in a Ca-free solution containing 2 mM EGTA; elevation of [Mg] in this solution reduced these effects. The high K-induced influx was prevented by a combination of low Ca (30 microns) and elevated Mg (10 mM). The Ca agonist, BAY-K 8644, increased 24Na influx. The Ca antagonist, diltiazem, inhibited the depolarization-stimulated 24Na influx in a concentration-dependent manner, but was less effective in blocking the response to NE. Extension of the preincubation in NE plus Ca-free medium from 30 s to 15 min decreased the influx response and contraction. After exposure to NE in Ca-free solution, 24Na influx remained elevated 10 min after washing out NE in the continued absence of Ca. A second exposure to NE at that time did not increase influx. We propose that a component of 24Na influx during excitation depends directly on a rise in intracellular [Ca]. The role of an indirect effect of [Ca] on metabolic H+ production with subsequent stimulation of the Na+-H+ exchange may also be a factor.
Cultured bovine coronary arterial endothelial cells synthesize HETEs and prostacyclin
Authors: Revtyak G. E. , Johnson A. R. , Campbell W. B.
Published: December 2017

Arachidonic acid metabolism was examined in endothelial cells cultured from bovine coronary arteries. In culture, these cells exhibit specific characteristics of endothelial cells. They form a contact-inhibited monolayer with a cobblestone appearance, contain immunoreactive von Willebrand's factor antigen, and have angiotensin I converting enzyme activity. Prostacyclin was the major prostaglandin synthesized from exogenous and endogenous arachidonic acid in these cells. In addition, exogenous arachidonic acid was metabolized to small amounts of prostaglandin E2 (PGE2) and several relatively nonpolar metabolites including 12-, 15-, and 11-hydroxyeicosatetraenoic acids (12-, 15-, and 11-HETE). Histamine, bradykinin, and thrombin increased PGI2 synthesis in these bovine coronary endothelial cells. Of these agonists, bradykinin was the most potent, increasing basal PGI2 release by fourfold. More vigorous stimulation of the cells with mechanical disruption of the cell monolayer, melittin, or A23187 resulted in release of both PGI2 and PGE2. Pretreatment of cells with exogenous arachidonic acid (10(-5) M) abolished their responsiveness to subsequent stimulation by arachidonic acid or vasoactive agents, but not PGH2. Furthermore, treatment of cells with 15-HPETE (10(-7)-10(-4) M), but not 15-HETE, specifically inhibited basal as well as A23187-stimulated PGI2 release. PGE2 release was increased slightly after 15-HPETE treatment. These studies indicate that bovine coronary endothelial cells can metabolize arachidonic acid to several biologically active products and that PGI2 synthesis by these cells is specifically related to the type of vasoactive agent employed. Both the qualitative pattern and quantity of eicosanoids synthesized by bovine coronary endothelial cells differ substantially from endothelial cells isolated from noncardiac vascular beds.
Aminoglycoside-induced alterations in apical membranes of kidney epithelial cell line (LLC-PK1)
Authors: Inui K. , Saito H. , Iwata T. , Hori R.
Published: December 2017

Aminoglycoside-induced renal cell injury was investigated using the LLC-PK1 pig kidney epithelial cell line. The development of aminopeptidase and gamma-glutamyltransferase, marker enzymes for apical membranes, was inhibited in the LLC-PK1 cells cultured with gentamicin. The inhibitory effect of gentamicin on the enzyme activities was dose dependent and was related to its accumulation within the cells. The development of Na+ -dependent active transport of alpha-methyl-D-glucoside, a nonmetabolizable hexose, was also inhibited by treatment with gentamicin. Inhibitions in apical membrane enzyme activities and Na+ -dependent transport of alpha-methyl-D-glucoside were associated with the elevation of cytosolic free calcium, determined with a fluorescent indicator fura-2. The correlation between the alterations in apical membrane functions and the increase in cytosolic free calcium concentration was also supported by the study using the calcium ionophore A23187. The present data suggest that aminoglycoside-induced alterations in apical membranes of the LLC-PK1 cells are related to the increase in cytosolic free calcium levels.
In vivo 31P-NMR spectroscopy of chronically stimulated canine skeletal muscle
Authors: Clark B. J. , Acker M. A. , McCully K. , Subramanian H. V. , Hammond R. L. , Salmons S. , Chance B. , Stephenson L. W.
Published: December 2017

Chronic stimulation converts skeletal muscle of mixed fiber type to a uniform muscle made up of type I, fatigue-resistant fibers. Here, the bioenergetic correlates of fatigue resistance in conditioned canine latissimus dorsi are assessed with in vivo phosphorus-31 nuclear magnetic resonance (31P-NMR) spectroscopy. After chronic electrical stimulation, five dogs underwent 31P-NMR spectroscopic and isometric tension measurements on conditioned and contralateral control muscle during stimulation for 200, 300, 500, and 800 ms of an 1,100-ms duty cycle. With stimulation, phosphocreatine (PCr) fell proportional to the degree of stimulation in both conditioned and control muscle but fell significantly less in conditioned muscle at all but the least intense stimulation period (200 ms). Isometric tension, expressed as a tension time index per gram muscle, was significantly greater in the conditioned muscle at the two longest stimulation periods. The overall small change in PCr and the lack of a plateau in tension observed in the conditioned muscle are similar to that seen in cardiac muscle during increased energy demand. This study indicates that the conditioned muscle's markedly enhanced resistance to fatigue is in part the result of its increased capacity for oxidative phosphorylation.
Assessment of Na+-H+ exchange activity in phagosomal membranes of human neutrophils
Authors: Grinstein S. , Furuya W.
Published: December 2017

To assess the presence of Na+-H+ exchange in internalized membranes, the phagosomal pH was monitored in suspensions of intact human neutrophils by measuring the fluorescence of ingested bacteria (Micrococcus lysodeikticus) prelabeled with a pH-sensitive dye. Uptake of fluoresceinated bacteria was confirmed by flow cytometry and by phase-contrast and electron microscopy. Manipulation of the cytoplasmic ion content was accomplished by permeabilization of the plasma membrane with nystatin, which did not alter phagosomal permeability. At 37 degrees C, the phagosomal interior acidified at a maximal rate of 0.135 +/- 0.003 pH units/min (n = 10). The endogenous Na+-H+ exchanger does not affect phagosomal acidification, since the rate and extent of the pH change were not altered by 1) omission of intraphagosomal Na+ and 2) addition of the permeant inhibitor methylisobutylamiloride or by trapping amiloride in the phagosome during bacterial ingestion. Moreover, amiloride-sensitive Na+-H+ exchange was not detectable when Na+ or H+ gradients were imposed across the phagosomal membrane. Under comparable conditions, Na+-H+ exchange could be readily detected across the surface membrane. These data imply that the Na+-H+ antiporters are either inactivated in the phagosome or are segregated and not internalized into the phagosomal membrane.
Bicarbonate secretion in rat distal colon in vitro: a measurement technique
Authors: Feldman G. M. , Berman S. F. , Stephenson R. L.
Published: December 2017

To study HCO3- secretion in rat distal colon, we utilized a technique that permits control of electrical and chemical transepithelial gradients. With symmetrical solutions (pH 7.4, [HCO3-] 25 mM, and CO2 tension 40 mmHg) bathing both tissue surfaces and under short-circuit conditions, HCO3- secretion remained stable for greater than 4 h at 1 mueq. h-1.cm-2. As the mucosal solution was alkalinized, the serosal solution was acidified at 3.1 mueq.h-1.cm-2. Ninety-four percent of serosal acidification was accounted for by the rate of metabolic lactic acid generation and transepithelial HCO3- secretion. Clamping transepithelial voltage reversibly affected net HCO3- secretion, and a linear relationship existed between clamped mucosal voltage and net HCO3- flux (r = 0.99); mucosal voltage of -68 mV completely inhibited net secretion. The apparent permeability coefficient of the colon to HCO3- is 2.8 X 10(-6) cm/s. One millimolar ouabain completely inhibited net HCO3- secretion. Acetazolamide (10(-4) M) inhibited secretion by approximately 50%, whereas a 10(-3) M concentration inhibited secretion by 90%. These data demonstrate that net colonic HCO3- secretion can be measured without imposed electrical and chemical gradients and that this flux is voltage sensitive and depends on carbonic anhydrase and Na+-K+-ATPase activities.
Ca2+ inhibits outward current through single K+ channels in canine atrial myocyte sarcolemma
Authors: Bubien J. K. , Van Der Heyde H. , Woods W. T.
Published: December 2017

Single-channel currents in canine atrial cells were recorded by the patch-clamp technique in a bathing solution containing 150 mM [K+] and pipette solutions containing 5 mM [K+]. One kind of current was observed in 56% of 178 cell-free patches and in 3% of 60 patches in the cell-attached configuration. Single-channel amplitude varied in direct proportion to the bath [K+]. Openings of these single channels were prevented when bath [Ca2+] exceeded 1 microM. Below this concentration single-channel percent open time was inversely proportional to log [Ca2+]. Inward current was observed at hyperpolarized membrane potentials in some patches. There was no apparent steady-state voltage sensitivity. These properties suggest that the K+ channel described in this study (gK+LF), a low transition frequency K+ conductor, may be distinct from single K+ channels previously studied in cardiac myocyte sarcolemmae. The single-channel response to "intracellular" free [Ca2+] and the single-channel kinetic characteristics described in this study are similar to the macroscopic "long-lasting transient outward current" (IIO) described by Escande et al. [Am. J. Physiol. 252 (Heart Circ. Physiol. 21): H142-H148, 1987] in human atrial myocytes (tau open = 29.6 ms, tau inactivation = 35.7 ms, respectively). This suggests that gK+LF channels may carry IIO.
Renal tubular vasopressin receptors downregulated by dehydration
Authors: Steiner M. , Phillips M. I.
Published: December 2017

Receptors for arginine vasopressin (AVP) were characterized in tubular epithelial basolateral membranes (BL membranes) prepared from the kidneys of male Sprague-Dawley rats. Association of [3H]AVP was rapid, reversible, and specific. Saturation studies revealed a single class of saturable binding sites with a maximal binding (Bmax) of 184 +/- 15 fmol/mg protein and a KD of 0.61 +/- 0.04 nM. IC50S for AVP, lysine vasopressin, and oxytocin were 0.74 nM, 9.7 nM, and greater than 1 microM, respectively. The V2 receptor antagonist was more than 3,700 times as effective in displacing [3H]AVP than was the V1 antagonist. To investigate the physiological regulation of vasopressin receptors, the effects of elevated levels of circulating AVP on receptor characteristics were studied. Seventy-two-hour water deprivation significantly elevated plasma osmolality and caused an 11.5-fold increase in plasma [AVP]. Scatchard analysis revealed a 38% decrease in the number of AVP receptors on the BL membranes from dehydrated animals. The high-affinity binding sites on the BL membranes fit the pharmacological profile for adenylate cyclase-linked vasopressin receptors (V2), which mediate the antidiuretic action of the hormone. We conclude that physiologically elevated levels of AVP can downregulate vasopressin receptors in the kidney.
Beta-adrenergic actions on membrane electrical properties of dissociated smooth muscle cells
Authors: Yamaguchi H. , Honeyman T. W. , Fay F. S.
Published: December 2017

Studies were carried out to determine the effects of the beta-adrenergic agent, isoproterenol (ISO), on membrane electrical properties in single smooth muscle cells enzymatically dispersed from toad stomach. In cells bathed in buffer of physiological composition, the average resting potential was -56.4 +/- 1.4 mV (mean +/- SE, n = 35). The dominant effect of exposure to ISO was hyperpolarization. The hyperpolarization was apparent in all cells studied and averaged 11.6 +/- 1.2 mV (n = 27). In the majority of the cells, hyperpolarization was accompanied by a decreased input resistance (Rin). Often the change in resistance appeared to lag behind the change in membrane potential. The lack of coincident changes in membrane potential and resistance may reflect a superposition of the outward rectification properties of the membrane on beta-adrenergic-induced increases in ionic conductance. In about half of the cells, an initial small depolarization (3.1 +/- 0.3 mV, n = 14) was accompanied by a small but distinct increase in Rin (12 +/- 2.5%). When membrane potential was made more negative than the estimated equilibrium potential for K+ (EK) by injection of current, ISO also produced biphasic effects, an initial hyperpolarization which reversed to a sustained depolarization to a value (-90 mV) near the estimated EK. The hyperpolarization by ISO could be diminished in a time-dependent manner by previous exposure to ouabain. The inhibition by ouabain, however, appeared to be a fortuitous result of glycoside-induced positive shifts in EK. These observations indicate that the dominant electrophysiological effect of beta-adrenergic stimuli is to hyperpolarize the cell membrane.(ABSTRACT TRUNCATED AT 250 WORDS)
Sodium channels in membrane vesicles from cultured toad bladder cells
Authors: Asher C. , Moran A. , Rossier B. C. , Garty H.
Published: December 2017

Electrical potential-driven 22Na+ fluxes were measured in membrane vesicles prepared from TBM-18(c123) cells (a clone of the established cell line TB-M). Fifty to seventy percent of the tracer uptake in vesicles derived from cells that were cultivated on a porous support were blocked by the diuretic amiloride. The amiloride inhibition constant was less than 0.1 microM, indicating that this flux is mediated by the apical Na+-specific channels. Vesicles prepared from cells that were not grown on a porous support exhibited much smaller amiloride-sensitive fluxes. Two Ca2+-dependent processes that down-regulate the channel conductance and were previously identified in native epithelia were found in the cultured cells as well. Vesicles isolated from cells that were preincubated with 5 X 10(-7) M aldosterone for 16-20 h exhibited higher amiloride-sensitive conductance than vesicles derived from control, steroid-depleted cells. Thus membrane derived from TBM-18(c123) cells can be used to characterize the epithelial Na+ channel and its hormonal regulation.
Lithium movements in resting and chemotactic factor-activated human neutrophils
Authors: Simchowitz L.
Published: December 2017

The ability of the chemotactic factor-activated Na+-H+ exchange system of human neutrophils to bind and transport other cations of the alkali metal series was investigated. After exposure of cells to the tripeptide N-formyl-methionyl-leucyl-phenylalanine (FMLP), the influx of Li+ was measured by flame photometry and correlated with changes in intracellular pH (pHi) derived from the equilibrium distribution of 5,5-dimethyloxazolidine-2,4-dione. In Na+-depleted cells, Li+ (Km approximately equal to 14 mM) could substitute effectively for Na+ (Km approximately equal to 23 mM) at the external translocation site of the carrier, though the maximal transport rate for Li+ (approximately 8 meq.l-1.min-1) was roughly half that for Na+ (approximately 15 meq.l-1.min-1). On the other hand, the carrier lacked appreciable affinity for K+, Rb+, and Cs+. The influx of Li+ from the external solution was accompanied by an equivalent counterefflux of H+ from the internal milieu. The H+ efflux thus induced led to an intracellular alkalinization of approximately 0.7 units, the pHi rising from approximately 7.20 to approximately 7.90. The influx of Li+, as well as the increase in pHi in 140 mM Li+ medium, was competitively inhibited by amiloride (Ki approximately equal to 9 microM). Extracellular H+ also behaved as a competitive inhibitor of Li+ with a Ki of approximately 30 nM (pK'a approximately 7.50). These studies indicate that the FMLP-activated alkali metal cation-H+ exchange mechanism of neutrophils shares a number of features in common with those of Na+-H+ exchangers in a variety of different cells.
Rabbit synoviocyte inositol phospholipid metabolism is stimulated by hydroxyapatite crystals
Authors: Rothenberg R. J. , Cheung H.
Published: December 2017

Inhibition of prostaglandin E2 synthesis partially ameliorates some aspects of synovitis, but joint destruction still progresses. Other aspects of phospholipid metabolism may play a role in synovial tissue pathophysiology. Products of phosphatidylinositol metabolism can activate intracellular processes in response to extracellular stimuli. We asked whether this pathway is activated in synoviocytes in monolayer tissue culture by the addition of hydroxyapatite (HA) crystals in medium. These crystals are found in pathological human synovial fluid. These crystals are associated with the secretion of degradative enzymes and with a destructive arthritis in humans. Rabbit synoviocyte cultures, previously incubated with [3H]inositol to label inositol phospholipids, were stimulated with the addition of hydroxyapatite (180 micrograms/ml) to the cultures. There was enhanced intracellular accumulation of [3H]inositol monophosphate (30-100%) after 4 h. This indicated an increased phospholipase C activity. The radioactivity in [3H]inositol bis- and trisphosphates was too low to reliably measure. The use of [32P]Pi allowed detection of these compounds. In the presence of HA, incorporation of [32P]Pi into phosphatidylinositol, phosphatidylinositol monophosphate, and phosphatidylinositol bisphosphate was increased. In addition, cultures exposed to [32P]Pi during stimulation with HA had an increased content of [32P]inositol monophosphate, bisphosphate, and trisphosphate.
Rat lung type II cell and lamellar body: elemental composition in situ
Authors: Eckenhoff R. G. , Somlyo A. P.
Published: December 2017

We determined the in situ elemental composition of alveolar type II cells (ATII) and lamellar bodies (LB) with electronprobe microanalysis (EPMA) of freeze-dried unstained cryosections (100-200 nm) obtained from lungs frozen in anesthetized rats. Twenty-nine ATII from seven rats were subjected to EPMA. Cytoplasmic (Cyto) composition was the following (in mmol/kg dry wt, mean +/- SE, n = 30): 136 +/- 14.1 Na, 60 +/- 2.8 Mg, 549 +/- 34.8 P, 278 +/- 10.5 S, 158 +/- 7.3 Cl, 525 +/- 26.4 K, and 6.6 +/- 0.9 Ca. LB composition was the following (n = 66): 44 +/- 4.0 Na, 7.9 +/- 0.8 Mg, 1,060 +/- 25.0 P, 79 +/- 4.8 S, 64 +/- 3.6 Cl, 114 +/- 4.1 K, and 30 +/- 0.9 Ca. P and S concentrations were consistent with previous biochemical determinations of phospholipid and protein content of isolated LBs. LBs contain significantly more Ca and less Mg than Cyto. Ca correlated significantly with LB P but not S concentration, and the reported low Ca binding affinity of similar phospholipid mixtures implies a high LB free Ca concentration. Ca was significantly higher in apical and exocytotic LBs compared with those in the perinuclear region. Differences between LB and Cyto monovalent ion concentrations are not entirely due to the difference in hydration revealed by significantly lower K-Cl ratios in LBs. The relative excess of Cl and Ca in LB suggests that these ions may be distributed by active transport systems known to be present in the Golgi apparatus and in Golgi-derived organelles of other cell types.