bluuebunny/crossref_metadata_2025 huggingface dataset:
Uptake of latex particles by pulmonary macrophages: role of calcium
Authors: Parod R. J. , Brain J. D.
Published: December 2017

The uptake of latex particles by both viable and fixed hamster pulmonary macrophages was calcium and trypsin sensitive. Particle uptake did not stimulate the uptake of 45Ca. However, when 45Ca uptake was stimulated with A23187, particle uptake was inhibited. When cobalt was added with A23187, the uptake of 45Ca was inhibited and particle uptake returned to control levels. A23187, cytochalasin B, and A23187 plus cytochalasin B all reduced particle uptake to the same extent. Although both A23187 and ouabain produced similar changes in the intracellular levels of Na+ and K+, only A23187 inhibited particle uptake. We conclude that extracellular Ca2+ promotes particle-cell binding through its interaction with a trypsin-sensitive receptor in the pulmonary macrophage membrane. In contrast, elevated intracellular Ca2+ levels inhibit particle ingestion but not attachment.
Correlation between water flow and intramembrane particle aggregates in toad epidermis
Authors: Brown D. , Grosso A. , DeSousa R. C.
Published: December 2017

In abdominal skins of toads (Bufo marinus) challenged with either vasopressin or isoproterenol, the stimulation of transepithelial water flow was accompanied by the appearance of intramembrane particle (IMP) aggregates. These aggregates were found only in the apical plasma membrane of granular cells of the first-reacting cell layer. The effect of vasopressin varied in magnitude and could be selectively blocked by preincubation of the skins with methohexital. This barbiturate also prevented the appearance of IMP aggregates. Preexposure with propranolol had a similar effect in skins challenged with isoproterenol. Linear regression analysis of net water flow vs. the percentage surface occupied by IMP aggregates showed a significant (P less than 0.001) correlation, which applied to data obtained in six different experimental conditions, i.e., vasopressin or isoproterenol alone and either of these agents in combination with methohexital or propranolol. The results reported here in a nonurinary epithelium (toad epidermis) suggest that the appearance of IMP aggregates is a general response in epithelia undergoing adenosine 3',5'-cyclic monophosphate-mediated changes in water permeability.
Glucose-induced retention of intracellular 45Ca in pancreatic islets
Authors: Andersson T.
Published: December 2017

beta-Cell-rich pancreatic islets from ob/ob mice were loaded with 45Ca in the presence of 20 mM glucose. When the islets were transferred to a nonradioactive incubation medium depleted of Ca2+, most of the 45Ca was lost during the first 10 min of incubation irrespective of whether or not glucose was present. Isolation of subcellular fractions under conditions minimizing 45Ca redistribution disclosed that the presence of 20 mM glucose during the nonradioactive incubation increased the retention of 45Ca in the mitochondrial fraction. This effect was not reproduced by K+ depolarization of the islets. Exposure of islets to 4 mM glucose failed to affect the total content of 45Ca in the islets but increased the incorporation of the isotope into cellular organelles. These results suggest that glucose, even at concentrations below the threshold for stimulation of insulin secretion, enhances the intracellular trapping of calcium.
Nonlinear force response of active smooth muscle subjected to small stretches
Authors: Meiss R. A.
Published: December 2017

Isometrically mounted and electrically stimulated strips of rabbit mesotubarium superius muscles were subjected to sinusoidal length perturbations (delta L) of variable amplitude (0-1% of Lmax) at constant frequency (80 Hz). In both the plateau phase of long-maintained (steady-state) contractions and in brief contraction-relaxation cycles the force perturbation amplitude (delta F) response was directly proportional to delta L (and to the developed force, F) for delta L values less than approximately 0.30% of Lmax. At larger delta L values the magnitude of the delta F response deviated downward from a linear relationship. Statistical analysis of the relationship characterized a "deviation point," the value of delta F and delta L beyond which the delta F response departed from linearity. The value of delta F at the deviation point depended strongly on the developed force, whether it was varied by changing muscle rest length, stimulus strength, or time during a phasic contraction-relaxation cycle. The value of delta L at the deviation point was independent of developed force. Restricting the length of muscle subjected to delta L (while not changing cellular dimensions) showed that the slope of delta F/delta L relationship (the active stiffness) depended inversely on the length of muscle in the system. These findings are consistent with a model in which the active elasticity arises from cross bridges that are borne on myofilaments and that both generate and transmit the muscle force.
EGTA purity and the buffering of calcium ions in physiological solutions
Authors: Miller D. J. , Smith G. L.
Published: December 2017

Results are presented for the purity of three commercial brands of the Ca chelator EGTA [ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid] as determined with pH-metric and oxalate precipitation methods and other techniques. The effect that the observed degree of hydration (2-4%) has on calculated values for [Ca2+] in Ca-EGTA solutions is described with reference to a theoretical dose-response relationship. In addition to the corrections for purity, the adjustments necessary to allow for ionic strength and the correction of pH readings to yield true [H+] activity or concentration values are described. These several corrections result in both a shift and a shape change for the dose-response curve compared with the curve calculated without these corrective procedures. Some authors have already taken some of these aspects into account, but this has not generally been made clear in the literature heretofore. The points raised may help to account for the wide variation of reported [Ca2+] ranges for Ca-sensitive cellular processes.
Aortic perfusion pressure as a determinant of cardiac protein synthesis
Authors: Kira Y. , Kochel P. J. , Gordon E. E. , Morgan H. E.
Published: December 2017

Mechanical parameters and intracellular mediators that may control protein synthesis have been studied in isolated rat hearts subjected to increased aortic pressure or induced to perform cardiac work. Elevation of aortic pressure from 60 to 120 mmHg in Langendorff preparations with glucose, glucose plus insulin, or pyruvate raised the rate of protein synthesis during the 2nd h of perfusion. These effects involved faster rates of both peptide chain initiation and elongation. In working hearts supplied glucose or glucose plus insulin, higher rate of synthesis were observed in both the 1st and 2nd h of perfusion, compared with Langendorff preparations perfused at 60 mmHg. Intracellular levels of glucose 6-phosphate, ATP/ADP ratio, adenylate energy charge, or creatine phosphate/creatine did not correlate with the rate of protein synthesis in beating control hearts. When ventricular pressure development was prevented by ventricular draining and hearts were arrested with tetrodotoxin, protein synthesis still increased as a function of perfusion pressure. Oxygen consumption increased as aortic pressure was raised in beating-drained hearts but was unaffected in arrested-drained hearts. These results indicate that intraventricular pressure development, cardiac contraction, oxygen consumption, glucose 6-phosphate, energy availability, and coronary flow could be dissociated from the stimulatory effect of higher aortic pressures on protein synthesis and suggested that stretch of the ventricular wall, as a consequence of increased aortic pressure, could be the mechanical parameter most closely related to the increase in protein synthesis.
Role of polyamines in differentiation of 3T3-L1 fibroblasts into adipocytes
Authors: Erwin B. G. , Bethell D. R. , Pegg A. E.
Published: December 2017

The role of polyamines in the differentiation of 3T3-L1 fibroblasts into adipose cells was studied. This conversion was blocked by the addition of alpha-difluoromethylornithine, an enzyme-activated irreversible inhibitor of ornithine decarboxylase, which prevented a rise in spermidine content in the differentiating cells. The inhibition of differentiation could be overcome completely by the provision of exogenous putrescine, spermidine, or spermine. Partial reversal could be produced by exposure to nonphysiological homologues of the natural polyamines such as 1,3-diaminopropane, 1,5-diaminopentane, and sym-norspermine. Reversal of the inhibition of differentiation by exogenous polyamines required a period of exposure to the amines, indicating that the lack of differentiation is not due simply to an obligatory role for polyamines in the biosynthesis of lipids. These results indicate that spermidine is required for the differentiation, but spermidine alone was not able to replace insulin and 1-methyl-3-isobutylxanthine in stimulating conversion to adipocytes. Therefore spermidine appears to be necessary but not sufficient for differentiation to occur. Finally, the elevation of spermidine content that occurs during the conversion of fibroblasts to adipocytes did not correlate with an increased activity of the polyamine biosynthetic enzymes. This implies that the increase must be regulated by changes in the rate of degradation or excretion of the polyamines.
Neural coordination of excitation of ferret trachealis muscle
Authors: Coburn R. F.
Published: December 2017

The distribution of junction potentials and synaptic potentials to muscle cells and ganglion cells in the ferret trachealis muscle-nerve plexus preparation was studied with local electrical stimulation of branches of the laryngeal nerve or the interganglionic nerve trunk. Stimulations evoked excitatory junction potentials in muscle cells and fast excitatory postsynaptic potentials in ganglion (AH) cells located throughout the preparation, regardless of the location of the stimulating electrode. Evoked excitatory junction potentials were nearly simultaneous in widely separated muscle cells, suggesting that excitation of different muscle cells is coordinated. The apparent conduction velocity to muscle cells after a local nerve stimulation was 0.1–0.2 m/s. Dissemination of input to ganglion cells and muscle cells was dependent on the integrity of the interganglionic nerve trunk. There was evidence based on analysis of conduction velocities that coupling of electrical activity in different AH ganglion cells and muscle cells was related to the following: 1) an interlacing arrangement of myelinated preganglionic neurons that enter the plexus from multiple branches of the laryngeal nerve, and 2) interlacing neural circuits characterized by synapses between neurons whose cell bodies lie in different ganglia.
Lipolysis and fatty acid transport in rat heart: electron microscopic study
Authors: Wetzel M. G. , Scow R. O.
Published: December 2017

Lamellar structures with a periodicity of 50 A developed in myocytes of glutaraldehyde-fixed heart tissues from young and adult rats when the tissues were incubated with tannic acid (pH 7.4) at 25 degrees C. The increase in lamellar structures (P less than 0.025) was accompanied by a significant decrease in intracellular lipid droplets (P less than 0.025), indicating that tissue lipase was active in fixed tissue and that the lamellar structures were probably composed of fatty acids formed by lipolysis. The lamellar structures in myocytes were located in the lumen of intracellular channels near lipid droplets and mitochondria and in the outer compartment of mitochondria. Lamellar structures were found at the periphery of chylomicrons, in intraendothelial channels, and in extracellular space of incubated fixed tissues from chylomicron-injected young rats. Chylomicron-lipid disappeared from capillaries (P less than 0.025) and lamellar structures with wide interlamellar spacings (80–1,000 A) developed in the extracellular space surrounding capillaries (P less than 0.025) in unfixed heart tissue from chylomicron-injected fasted young rats when the tissue was incubated without tannic acid; lamellar structures did not develop in similarly treated tissue from uninjected rats. Thus the lamellar structures found in extracellular space represent fatty acids derived from lipolyzed chylomicrons. We conclude that fatty acids produced by lipolysis in incubated heart accumulated and spread in an interfacial continuum of external leaflets of cell membranes extending from the capillary lumen to extracellular space and from intracellular lipid droplets to the interior of mitochondria in myocytes. When fatty acids overcrowded the continuum, they formed lamellar extensions of the continuum at different sites along its course through the tissue.
Covalent linkage of [Phe2,Phe(p-N3)3]AVP to vasopressin receptors
Authors: Eggena P. , Fahrenholz F. , Schwartz I. L.
Published: December 2017

We have shown previously ( Eggena et al., Endocrinology 113: 1413-1421, 1983) that [Phe2,Phe(p-N3)3]-AVP induces a prolonged hydrosmotic response in the toad bladder when activated by ultraviolet (UV) light. To determine whether this response is due to covalent binding of the ligand with 8-arginine vasopressin (AVP) receptors, bladders were challenged with the ligand in the presence of AVP or the AVP antagonist, [Phe(p-N3)2]AVP, during photolysis. The permeability of bladders to water was tested subsequently in the absence of hormone or analogue. Bladders with a history of exposure to AVP (or to [Phe-(p-N3)2]AVP) during UV irradiation were considerably less permeable to water than controls, suggesting that [Phe2,Phe(p-N3)3]AVP, AVP, and [Phe(p-N3)2]AVP compete for the same receptor system during photolysis. Other experiments were directed at defining optimal conditions for covalent linkage of [Phe2,Phe(p-N3)3]AVP to receptors. These studies have indicated that two 10-min cycles of UV irradiation are more effective than one and that osmotic water flow at a rate of 1 mg X min-1 X cm-2 during irradiation does not interfere with the ligand-receptor interaction. Acidification of the serosal bath solution to pH 6.5 did not inhibit covalent binding of the ligand to receptors during photolysis. However, the capacity of the ligand-receptor complex to increase bladder permeability to water was markedly inhibited by serosal fluid acidification. These experiments have suggested that [Phe2 ,Phe(p-N3)3]AVP binds covalently to AVP receptors during photolysis and generates a signal that gradually decays as a function of time.(ABSTRACT TRUNCATED AT 250 WORDS)
Evidence that cobalt ion inhibition of prolactin secretion occurs at an intracellular locus
Authors: Thaw C. N. , Raaka E. G. , Gershengorn M. C.
Published: December 2017

Cobalt inhibition of stimulated prolactin secretion has been interpreted as demonstrating an essential role for enhanced calcium influx in the action of thyrotropin-releasing hormone (TRH) in GH3 cells. However, this interpretation is based on the assumption that cobalt ion (Co2+) binds to the external surface of cells to antagonize calcium-mediated processes only by blocking influx of extracellular calcium ion (Ca2+). In this report, we present evidence that Co2+ acts at an intracellular locus (or loci) to inhibit prolactin secretion. When GH3 cells were incubated in medium containing 1.5 mM Ca2+, Co2+ inhibited basal as well as 50 mM K+- and TRH-induced secretion; half-maximal effect occurred between 0.1 and 0.3 mM Co2+. When cells were incubated in medium containing 0.05 and 0.003 mM Ca2+, concentrations that abolish 50 mM K+-induced prolactin secretion, Co2+ still inhibited basal and TRH-stimulated prolactin secretion. Co2+ also inhibited prolactin secretion stimulated by 1-methyl-3-isobutylxanthine, dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP), and vasoactive intestinal peptide, three secretagogues that act to elevate intracellular cAMP, a mechanism which appears not to involve enhanced Ca2+ influx. Last, the presence of Co2+ within the cell was shown by fluorescence quenching of intracellularly trapped Quin 2, a chelator of divalent cations. These data demonstrate that Co2+ enters GH3 cells and that Co2+ inhibition of prolactin secretion does not involve extracellular Ca2+. We suggest that Co2+ not only blocks Ca2+ channels in GH3 cells, but it inhibits prolactin secretion at an intracellular locus (loci). Hence, inhibition by Co2+ should not be interpreted as demonstrating a requirement for Ca2+ influx in stimulated secretion.
Effects of extracellular pH on lactate efflux from frog sartorius muscle
Authors: Seo Y.
Published: December 2017

The isolated sartorius muscle of frog was stimulated under an anaerobic condition, and intracellular lactate concentration and intracellular pH was determined sequentially by 1H-nuclear magnetic resonance, and the efflux of lactate was measured by an enzymatic method on the incubation media with different pH levels. The lactate efflux was shown to increase in proportion to the concentration difference of lactate across the membrane up to 25 mmol X l H2O-1, and the permeability of the membrane to lactate increased with the increment of extracellular pH. H+ efflux was determined from pH change and the buffer capacity of incubation medium. The efflux of H+ rose approximately linearly with the efflux of lactate up to approximately 70 mumol X min-1 X l H2O-1, and the relationship then revealed a clear leveling off. From these results the efflux of lactate was analyzed in terms of a predominantly nonionic or a predominantly ionic efflux mechanism. Analysis showed that the mechanism of efflux was in both the ionic and nonionic form.
Receptor occupancy vs. induction of Na+-K+-ATPase and Na+ transport by aldosterone
Authors: Geering K. , Claire M. , Gaeggeler H. P. , Rossier B. C.
Published: December 2017

In the urinary bladder of the toad Bufo marinus aldosterone (between 0.8 and 100 nM) stimulates Na+ transport [half-maximal induction concentration (K1/2) = 6.5 nM]. At low hormone concentrations (0.8-8 nM), the increase of Na+ transport between 0.75 and 2.5 h is accompanied by a fall in transepithelial resistance (R). Higher hormone concentrations (30-800 nM) induce an additional resistance-independent fraction of Na+ transport within 2.5-8 h. From 6 h on, aldosterone (between 0.2 and 20 nM) stimulates in the same tissue the biosynthesis rate of the alpha- and beta-subunits of Na+-K+-ATPase (K1/2 = 3 and 1.5 nM, respectively). New pump synthesis is thus not a prerequisite for the early mineralocorticoid response but might be linked to the late transport event. The mineralocorticoid response is usually ascribed to interaction with the higher affinity type 1 receptor. In the present study we show, however, that at least 55% of the overall Na+ transport response is linked to nuclear occupation of the lower affinity type 2 receptors [dissociation constant (Kd) = 50 nM, maximum number of binding sites (Nmax) = 315 fmol/mg protein]. Distinct aldosterone effects, such as the fall in R and the increase in Na+-K+-ATPase synthesis, are more closely related to occupation of type 1 receptors (Kd = 0.3 nM, Nmax = 23 fmol/mg protein). At maximal induction of these latter parameters, only about 20% of type 2 receptors are occupied. These results suggest that both types of aldosterone receptors are involved in the mediation of the full mineralocorticoid response: type 1 in the early and late and type 2 particularly in the late tissue response.
Electrophysiological effects of propionate and bicarbonate on gallbladder epithelium
Authors: Petersen K. U. , Reuss L.
Published: December 2017

The effects of propionate and HCO3- on cell membrane potentials in Necturus gallbladder epithelium were investigated using microelectrode techniques in vitro. Bilateral exposure to either propionate or HCO3- (25 mM each) hyperpolarized both cell membranes by about 12 mV. Mucosal addition of either substance produced cyclic changes in voltage of both cell membranes, which attenuated spontaneously, whereas serosal addition caused sustained hyperpolarization. By intraepithelial cable analysis it was shown that both cell membrane conductances rose during the hyperpolarization. Experiments using substitution of mucosal K+ for Na+ revealed that the relative K+ permeability (PK) of the apical membrane was enhanced during the hyperpolarization induced by mucosal (or serosal) propionate (or HCO3-). These effects are mediated by increases in PK at both membranes, with a larger basolateral effect. We suggest that this mechanism accounts for the higher cell membrane potential values measured in epithelia bathed in HCO3--or propionate-containing solutions. Inasmuch as both propionate and HCO3- stimulate fluid absorption in gallbladder epithelium, the increase in cell membrane PK may represent an adaptive response of the cells to regulate their solute content.
Calcium-dependent secretory and redox response to CCK-8 in isolated perfused rat pancreas
Authors: Shibuya I. , Kanno T.
Published: December 2017

Continuous stimulation with 8 pM cholecystokinin octapeptide (CCK-8) induced a gradual increase in pancreatic protein output and little if any change in redox state of cytochromes aa3, b, and c + c1. The protein output was completely abolished when CaCl2 was removed from the perfusing and bathing solution. Continuous stimulation with 200 pM CCK-8 induced the rapid and largest protein output and a distinct reduction of cytochromes and nicotinamide nucleotides. These responses were partially decreased in the Ca2+-deficient environment and enhanced immediately after the replacement with the standard solution. These and other results are compatible with the view that reduction of electron transfer system in the pancreatic acinar cell may be induced by stimulation with the secretagogue in pharmacological concentration and that the reduction may coincide with uptake and retention of cytoplasmic excess Ca2+ by mitochondria.
Ion movements in membrane vesicles: a new fluorescence method and application to smooth muscle
Authors: Grover A. K. , Singh A. P. , Rangachari P. K. , Nicholls P.
Published: December 2017

A method is described for studying ion permeabilities of membrane vesicles based on the principle that when membrane permeability to H+ is very high, the H+ movement is determined by the membrane potential generated by the H+ movement. The rate of H+ movement under these conditions thus gives a measure of the rate of dissipation of this membrane potential by comovement of anions or countermovement of cations present. Thus, by studying the H+ efflux using an impermeant cation and different anions, the membrane permeability to the anions can be assessed. Similarly, the use of an impermeant anion allows the study of the permeation of various cations. H+ movement was followed across the membranes by monitoring a change in the fluorescence intensity of the pH-sensitive dye pyranine trapped inside the membranes. This method when tested using phosphatidylcholine liposomes yielded the expected results, i.e., permeability of the liposomal membrane was: Cl- greater than SO2-4 and K+ greater than Na+. A plasma membrane-enriched fraction loaded with pyranine was isolated from estrogen-dominant rat myometrium. The anion permeability characteristics of this membrane were studied using tetramethylammonium (TMA+) as the poorly permeant cation, and the cation permeability was studied using L-glutamate- as the poorly permeant anion. The anion permeabilities were D-glutamate- less than L-glutamate- less than glutarate2- less than Cl- less than or equal to SO2-4, and the cation permeabilities were TMA+ less than K+ less than Na+. It is hypothesized that the observed anomalously higher Na+ and SO2-4 movements may involve special mechanisms.
Extracellular potassium modifies the structure of kidney epithelial cells in culture
Authors: Waack S. , Walsh-Reitz M. M. , Toback F. G.
Published: December 2017

In animals fed a K-deficient diet, alterations in kidney cell structure and function occur in association with changes in the ionic composition of the extracellular fluid. The hypothesis that the extracellular K concentration mediates these changes in renal tissue was tested in cultures of monkey kidney epithelial cells (BSC-1 line) by reducing the K concentration of the culture medium from the control value of 5.4 to 3.2 mM. Exposure of BSC-1 cells to low-K medium raised the maximal rate of uptake for L-glutamic acid by 39% without a change in apparent Km. To determine whether this alteration in plasma membrane function had a structural correlate, studies of the cell surface were performed using scanning and transmission electron microscopy. Morphometric analysis of scanning electron micrographs revealed that the number of microvilli per cell per unit surface area was 45% greater in cells exposed to low-K medium for 3 min than those exposed to control medium. This observation was confirmed by transmission electron microscopy. The results indicate that an alteration in the extracellular K concentration per se can modify specific structural and functional characteristics of kidney epithelial cells.
Compartmentalization of 11-oxidoreductase within fetal lung alveolus
Authors: Torday J. S. , Post M. , Smith B. T.
Published: December 2017

The role of 11-oxidoreductase in the cellular process of fetal lung surfactant production and its localization within the alveolar domain have been investigated. In organotypic cultures of fetal rat lung, cortisol and cortisone markedly stimulate saturated phosphatidylcholine synthesis by the alveolar type II cell; a 10-fold excess of 11-ketoprogesterone blocks the bioactivity of cortisone. Both cortisol and cortisone also stimulate fibroblast-pneumonocyte factor production, whereas 11-ketoprogesterone blocks the effect of cortisone, but not of cortisol, suggesting that cortisone stimulation of fibroblast-pneumonocyte factor production depends on its conversion to cortisol by 11-oxidoreductase. A survey of the cells that are present in the alveolar domain revealed that 11-oxidoreductase activity is only present in the fibroblast. Localization of both 11-oxidoreductase and fibroblast-pneumonocyte factor production within the same cell emphasizes the significance of 11-oxidoreductase in the regulation of fetal lung surfactant production.
Primary culture of duck salt gland. II. Neurohormonal stimulation of active transport
Authors: Lowy R. J. , Dawson D. C. , Ernst S. A.
Published: December 2017

Primary cultures of structurally polarized sheets of avian salt gland secretory cells were mounted in Lucite chambers for transmural electrophysiological analysis. Transmural resistance values increased during the first 3 days of culture to 293 +/- 35 omega X cm2 and then decreased slowly thereafter. There was little short-circuit current (Isc) in the absence of secretagogues. Serosal addition of either carbachol or epinephrine resulted in a Isc consistent with positive charge flow from mucosa to serosa, thus demonstrating that these cell layers were capable of active ion transport in response to either cholinergic or adrenergic neurohormonal stimulation. Serosal ouabain or furosemide abolished the response to either agonist, while theophylline enhanced the response. Receptor specificity for the electrical responses was shown by selective inhibition of carbachol- and epinephrine-induced Isc by atropine and propranolol, respectively. The results demonstrate that these primary epithelial cell cultures are capable of active ion transport and are sensitive to known inhibitors of secretory transport, and suggest that intracellular coupling mechanisms for hormonal control are retained in culture. These cultures should be useful for studying mechanisms of ion secretory transport and their regulatory control.
NBD-taurine fluorescence as a probe of anion exchange in gallbladder epithelium
Authors: Foskett J. K.
Published: December 2017

Previous work has demonstrated that after osmotic shrinkage of Necturus gallbladder epithelial cells, their volumes are restored to control levels despite the continued presence of the hyperosmotic medium. It has been proposed that activation of parallel neutral Na+-H+ and Cl--HCO-3 exchangers in the apical membrane is necessary for regulatory volume increase. As an independent technique to determine whether and for how long ion flux through the anion exchanger is actually enhanced by exposure to hypertonicity, fluorescence measurements of N-(2-aminoethylsulfonate)-7-nitrobenz-2-oxa-1,3-diazole (NBD-taurine), a substrate of the anion exchanger in red blood cells, have been made in intact Necturus gallbladder. The cells were loaded with the dye by incubation. The tissue was perfused in a miniature chamber placed on the stage of a microscope and viewed with high-magnification optics combined with video. Fluorescence was monitored at frequent intervals with a photomultiplier tube, and transmittance of the tissue to the laser excitation light was monitored with a photodiode. The epithelium was simultaneously observed with transmitted light to control for changes in focus or lateral movement. Exposure of the tissue to a mucosal medium made hypertonic by the addition of mannitol transiently enhanced the efflux of NBD-taurine from the cells in approximately 70% of the tissues examined. In the presence of the anion-exchange inhibitor 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS, 100 microM), hypertonicity enhanced NBD-taurine efflux in only 14% of the preparations.(ABSTRACT TRUNCATED AT 250 WORDS)
Differentiated lines of cells from rabbit renal medullary thick ascending limbs grown on amnion
Authors: Green N. , Algren A. , Hoyer J. , Triche T. , Burg M.
Published: December 2017

Previously we grew differentiated primary epithelial tissue cultures from rabbit renal medullary thick ascending limbs but were unable to subculture them into lines. Now, following the use of amnion as a support during the initial passages, two cell lines have grown from single fragments of medullary thick ascending limbs. Cells have now been in culture past 12 passages over more than 2 yr. On confluence they formed morphologically differentiated epithelial monolayers with polarization of the cells visible on electron microscopy. They had apical zonula occludens and microvilli, lateral cellular interdigitations, and basal membranes flat against the support. “Domes” often were visible when the epithelia formed on dishes, indicative of salt and water transport. Other functional differentiation in some passages of one line or the other included presence of Tamm-Horsfall protein (demonstrated by immunofluorescence) or transepithelial voltage oriented apical surface positive. Both the Tamm-Horsfall protein and the voltage are normally expressed by intact medullary thick ascending limbs and are characteristic of this particular nephron segment.
Salt and acid studies on canine lingual epithelium
Authors: Simon S. A. , Garvin J. L.
Published: December 2017

A section of dog tongue just anterior to the circumvallate papillae was placed in an Ussing chamber and characterized with respect to monovalent cation transport pathways, response to HCl, and the interaction between salt and acid for the purposes of determining whether ion transport through the tongue is related to the transduction process. The primary findings of this work follow. The stimulation of the short-circuit current (Isc) with decreasing pH on the dorsal surface results from a net Cl- efflux through an amiloride- and ouabain-insensitive pathway that changed its selectivity from cationic at neutral pH to anionic at lower pH values. The stimulation is abolished at isotonic NaCl concentrations. This behavior would not be predicted from the proton-binding model to a "sour receptor" as suggested by Beidler (Handbook of Sensory Physiology; Berlin: Springer-Verlag, 1971). In symmetrical solutions of Krebs-Henseleit buffer, Isc is inhibited 30% by 10(-4) M amiloride on the mucosal side and 100% by 10(-3) M ouabain on the serosal side. These results coupled with net Cl- and Na+ flux measurements show that Isc is due to a net Cl- efflux. With 1.0 M NaCl on the dorsal side, 10(-4) M amiloride inhibits Isc 84%. In contrast, when the mucosal surface is bathed in KCl (1.0 M), CsCl (1.0 M), or HCl (10(-3) M), Isc is insensitive to both 10(-4) M amiloride and/or 10(-3) M ouabain. These data suggest that K+, Cs+, and H+ traverse the tongue through different pathways from Na+ and are not energized by a Na+ gradient.
Donnan equilibrium and pH gradient in isolated tracheal apical membrane vesicles
Authors: Langridge-Smith J. E. , Dubinsky W. P.
Published: December 2017

Apical plasma membrane vesicles isolated from bovine tracheal epithelium were found to possess both an electrical potential gradient (delta psi) and a transmembrane pH gradient (delta pH). The delta psi was calculated from the distribution of 86Rb+ in the presence of the ionophore valinomycin, and delta pH was determined from the distribution of [14C]methylamine under conditions of apparent equilibrium. Maximal values for delta psi of -54.3 +/- 2.5 mV and for delta pH of 0.75 +/- 0.07 pH units were obtained under low ionic strength conditions. Increasing the ionic strength by the addition of 50 mM of either permeant or impermeant electrolytes reduced delta psi to near zero values. The delta pH varied in parallel with the delta psi. The results suggest that both 86Rb+ in the presence of valinomycin and H+ are in a Donnan equilibrium with impermeant negative charge in the vesicle interior.
Skeletal muscle protein synthesis and degradation in vitro: effects of temperature
Authors: Essig D. A. , Segal S. S. , White T. P.
Published: December 2017

We compared the structure, function, protein synthesis, and degradation of 70- to 95-mg rat soleus muscles during 120 min of incubation at 20 and 37 degrees C. At 37 degrees C, muscles were characterized by a damaged central core region and a decline of isometric tension development during incubation. Protein synthesis in the core region at 37 degrees C was depressed relative to the peripheral region. At 20 degrees C, developed tension remained constant during incubation, and synthesis rates in the core region were not different from the peripheral region. Compared with fresh muscle, ATP concentration after incubation was not affected by temperature. After equilibration of phenylalanine specific activity between extracellular and intracellular spaces (60 min at 20 degrees C; 30 min at 37 degrees C), rates of protein synthesis at 20 [0.048 nmol tyrosine (Tyr) X mg wet mass-1 X 2 h-1] and 37 degrees C (0.160 nmol Tyr X mg wet mass-1 X 2 h-1) were linear up to 180 and 120 min, respectively. Rates of protein degradation at 20 (0.076 nmol Tyr X mg wet mass-1 X 2 h-1) and 37 degrees C (0.248 nmol Tyr X mg wet mass-1 X 2 h-1) measured after 60 min were linear up to 180 and 120 min, respectively. Incubation at 20 degrees C offers an approach to study 70- to 95-mg muscles in vitro without compromising structure and function.
Polarity of volume-regulatory increase by Necturus gallbladder epithelium
Authors: Marsh D. J. , Spring K. R.
Published: December 2017

Necturus gallbladder epithelial cells respond to the presence of a hypertonic perfusate in either bathing solution by first shrinking due to osmotic water loss and then swelling back to their original volume (volume-regulatory increase). Previous investigations involving increases in the osmolality of the mucosal bath had suggested that volume-regulatory increase was due to the activation of ion exchangers in the apical cell membrane. In the present study the sidedness of the transport processes involved in volume-regulatory increase was investigated. The osmolality of the serosal bath was increased by 18% either in the absence of HCO3- or when an inhibitor of volume-regulatory increase, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), was added to the mucosal or serosal bath. Volume regulation was HCO3- dependent. DIDS was only effective in inhibiting volume regulation when it was added to mucosal bathing solution, suggesting that volume-regulatory increase depended on transport across the apical membrane. Volume-regulatory increase could also be activated by first swelling the cells in hypotonic solution and then returning the tissue to control Ringer solution. The volume-regulatory increase that occurred upon return to control Ringer was also shown to be sensitive to DIDS in the mucosal bath.
Stimulation of net active ion transport across alveolar type II cell monolayers
Authors: Cott G. R. , Sugahara K. , Mason R. J.
Published: December 2017

The active transcellular transport of electrolytes across the alveolar epithelium probably plays an important role in alveolar fluid homeostasis by helping to maintain the alveolus relatively free of fluid. To better understand the factors regulating active ion transport across alveolar epithelial cells, we examined the effect of a number of pharmacologically active agents on the bioelectric properties of alveolar type II cells in primary culture. Alveolar type II cells were isolated from adult male rats and cultured on collagen-coated Millipore filters for 6-14 days. The bioelectric properties of these monolayers were determined in Ussing-type chambers. The addition of 10(-3) M 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) increased the short-circuit current (Isc) from 2.9 +/- 0.75 to 6.9 +/- 0.73 microA/cm2 (means +/- SE; n = 8) and decreased the transepithelial resistance. Cholera toxin, 3-isobutyl-1-methylxanthine, and terbutaline sulfate produced similar increases in Isc and decreases in resistance. The Isc stimulated by 8-BrcAMP was Na but not Cl dependent and could be blocked by amiloride but not by furosemide. Thus 8-BrcAMP and agents that increase intracellular cAMP can stimulate a Na-dependent net active ion transport across alveolar type II cell monolayers. Similar regulatory mechanisms may be involved in controlling solute and fluid movement across the alveolar epithelium in vivo.
Angiotensin II stimulates norepinephrine uptake in hypothalamus-brain stem neuronal cultures
Authors: Sumners C. , Raizada M. K.
Published: December 2017

In this study we have characterized the uptake of [3H]norepinephrine (NE) into neuronal co-cultures of rat hypothalamus and brain stem and have examined the effects of angiotensin II (ANG II) on this uptake. Neuronal co-cultures prepared from the brains of 1-day-old Sprague-Dawley (SD) or Wistar-Kyoto (WKY) rats exhibited sodium-dependent and sodium-independent portions of the total [3H]NE uptake. The sodium-dependent uptake was abolished by blockers such as maprotiline, desmethylimipramine, and xylamine (0.1-100 microM) and is presumably neuronal uptake. The sodium-independent uptake was unaffected by these drugs and is presumably non-neuronal, since nonneuronal co-cultures from SD rats exhibited no significant sodium-dependent or blocker-sensitive uptake. In SD or WKY neuronal co-cultures, ANG II (0.1 nM-10 microM) caused increased [3H]NE uptake during short-term incubations (1-5 min). This stimulatory effect of ANG II was on neuronal NE uptake. Furthermore, it was inhibited by preincubation with saralasin (1-10 microM). Construction of saturation curves and kinetic analyses revealed that ANG II caused an increase in the maximal velocity of uptake of neuronal [3H]NE, but the affinity of the transporter for NE was not altered. With longer-term incubations (15-30 min), ANG II caused a reduction in neuronal [3H]NE uptake. This effect was also blocked by saralasin. However, reliable kinetic analysis was not possible with the longer-term incubations, and it is likely that the inhibitory action of the peptide represents a stimulation of NE release. Therefore, using neuronal co-cultures, we have identified a previously unseen stimulatory action of ANG II on neuronal [3H]NE uptake, which precedes the already documented inhibitory actions.
Clonal variation of cadmium response in human tumor cell lines
Authors: Enger M. D. , Tesmer J. G. , Travis G. L. , Barham S. S.
Published: December 2017

Subpopulations of human tumor-derived cell lines A101D, A204, and A549 were screened for Cd2+ cytotoxic response. Three of six A549, two of seven A101D, and four of seven A204 subpopulations were found to differ significantly from the parental line. A variant subpopulation of A101D (T3) was shown by flow cytometry to be comprised of cells having two distinct DNA histograms. One histogram, type 1, resembles that of normal human fibroblasts. The other, type 2, represents cells with one-third more DNA. Early passage T3 clonal populations were comprised primarily of type 1 cells. With passage, type 1 cells decreased relative to type 2 so that by passage 47 the culture was predominantly type 2. Correspondingly, the A101D T3 subpopulation became more Cd2+ sensitive with time in culture. Subclones having only type 1 DNA histograms were found to be Cd2+ resistant relative to subclones with type 2 histograms, and treatment of A101D T3 cultures having approximately equal amounts of type 1 and 2 cells with 2 microM Cd2+ resulted in the selection of type 1 cells. The enhanced Cd2+ resistance phenotype shown by A101D T3 type 1 cells correlated with reduced Cd2+ uptake and is not attributable to enhanced metallothionein synthesis.
Volume regulation by Amphiuma red blood cells: cytosolic free Ca and alkali metal-H exchange
Authors: Cala P. M. , Mandel L. J. , Murphy E.
Published: December 2017

Osmotic swelling of Amphiuma red blood cells results in activation of electroneutral K-H exchange, whereas cell shrinkage activates an electroneutral Na-H exchange. These K-H and Na-H exchangers function to restore cell volume to normal after cell swelling and shrinkage, respectively. Our previous studies have suggested that Ca plays a role in volume-dependent activation of K-H exchange. In the present studies, intracellular free Ca levels were measured employing the Ca-sensitive extracellular dye arsenazo III and a previously described null-point method. Control values for intracellular free Ca averaged 0.46 +/- 0.15 microM. Cell shrinkage caused this value to decrease to 0.16 +/- 0.11 microM, whereas either cell swelling or addition of 5 microM A23187 resulted in saturation of intracellular Ca buffers, suggesting that both treatments caused an increase in intracellular free Ca. In the presence of 7 microM A23187, the rate of K-H exchange displayed a hyperbolic relationship as a function of extracellular Ca (Cao). The apparent half-maximal concentration for Cao (in the presence of 7 microM A23187) was 0.27 mM for osmotically swollen cells and 1.9 mM for cells in isotonic medium, suggesting that the Ca affinity of a modulating site is increased in swollen cells. Inhibitors of Ca-mediated processes, such as quinidine and the phenothiazines, inhibited K-H exchange. In contrast, the phenothiazines chlorpromazine and trifluoperazine stimulated Na-H exchange by osmotically shrunken cells. These results suggest that increases in intracellular free Ca are involved in stimulating K-H exchange while repressing Na-H exchange in Amphiuma red blood cells.
Downregulation of vasopressin receptors in toad bladder
Authors: Eggena P. , Ma C. L.
Published: December 2017

Binding of tritium-labeled vasopressin [( 3H]AVP) to a broken epithelial cell preparation of the toad's urinary bladder has been related to hormonal action on water and urea transport across the intact tissue. Hormone binding to receptor sites and permeability changes were initiated at the same concentration of hormone (0.4 nM). Half-maximal urea and water permeability responses were observed with 3.1 and 5.6 nM AVP, respectively, although half-maximal receptor saturation required considerably higher concentrations of hormone (less than 500 nM). Because maximal permeability responses were obtained with occupation of approximately 200 fmol/mg protein receptor sites and the total receptor density was in excess of 2,000 fmol/mg protein, there is apparently a receptor reserve in this tissue. The antidiuretic hormone employed by the toad is vasotocin (AVT). This compound was 60-fold more effective than AVP in displacing [3H]AVP from receptor sites. Preincubation of bladders with AVT resulted in downregulation of receptor sites. Although the magnitude of receptor loss was equivalent in the presence or absence of a transmembrane osmotic pressure gradient, the capacity of AVT to induce permeability changes was more markedly reduced in the presence of an osmotic gradient. This observation suggests that the negative-feedback signal initiated by water flow through the hormone target cell diminishes sensitivity to hormone by a mechanism other than by a reduction in the number of surface receptors or by a decrease in their affinity for the hormone.
NaCl transport stimulates prostaglandin release in cultured renal epithelial (MDCK) cells
Authors: Kurtz A. , Pfeilschifter J. , Brown C. D. , Bauer C.
Published: December 2017

Prostaglandins (PGs) can modulate a variety of renal functions, including Na+ and Cl- reabsorption. However, it is not known if a direct interdependence exists between PG synthesis and transport activity. The present study was done to find out whether or not the rate of NaCl transport has an influence on PG synthesis in renal tubular cells. For our studies we used cultures of so-called high-resistance MDCK cells, which were originally derived from canine kidney. This cell type has a loop diuretic- and ouabain-sensitive NaCl transport that can be enhanced by activation of the adenylate cyclase (AC). In MDCK cell cultures we found that each state of increased NaCl transport during stimulation of AC by either epinephrine (10(-6) M), isoprenaline (10(-5) M), or forskolin (10(-5) M) was accompanied by a twofold increase in PG release. During inhibition of NaCl transport by furosemide (10(-4) M) or ouabain (2 X 10(-4) M), stimulation of AC failed to increase PGE2 release, whereas basal PG production was not inhibited by either furosemide or ouabain. Furthermore, PG formation during activation of AC was dependent on the concentration of extracellular Na+, whereas PG formation in the absence of activators of AC was independent of extracellular Na+. These results suggest that increased NaCl transport stimulates PG formation in cultures of high-resistance MDCK cells.
LLC-PK1 cells: cloning of phenotypically stable subpopulations
Authors: Wohlwend A. , Vassalli J. D. , Belin D. , Orci L.
Published: December 2017

The LLC-PK1 pig kidney-derived cell line is morphologically and functionally heterogeneous. We have clonally derived three sublines that differ in their response to calcitonin and in their ability to form domes. The three clones were analyzed for their basal and hormonally induced plasminogen activator production. In contrast to the D + Sc clone, in which calcitonin induced a greater than 100-fold increase in plasminogen activator synthesis, the D + Rc clone did not respond to the hormone; this was related to a deficiency of the cells in calcitonin binding. Transepithelial electrical resistance measurements revealed a direct correlation with the capacity of the cells to form domes; in one of the isolated clones (D-), the lack of dome formation coincided with a low electrical resistance; the D + Sc clone, in which all single cell-derived colonies formed domes, showed a higher electrical resistance than that developed by the original cell line. Thus the LLC-PK1 clones provide a useful in vitro model for the study of epithelial properties.