bluuebunny/crossref_metadata_2025 huggingface dataset:
Network thermodynamic modeling of hormone regulation of active Na+ transport in cultured renal epithelium (A6)
Authors: Fidelman M. L. , Mikulecky D. C.
Published: December 2017

A network thermodynamic model was developed to describe steady-state ion flows (Na+,K+, and Cl-) and related electrical events in a cultured renal epithelium (A6) derived from toad kidney. Three hypotheses for explaining the steady-state increases in short-circuit current (SCC) produced by aldosterone and/or insulin were examined using the model. Changing only the number of basolateral Na+-K+ pumps produced virtually no change in SCC and was ruled out. Changing only the number of apical Na+ channels could produce sufficient increases in SCC but presented problems in the pattern of changes produced in cell ion concentrations and therefore appeared unlikely. Changing both apical and basolateral parameters in a balanced, coordinated manner produced the maximal changes in SCC with the minimal changes in cell ion concentrations and appeared to be the "best" hypothesis. In addition, it was found necessary for tight junction permeability to increase as active Na+ transport increased under open-circuit conditions. Simulations, using these results, compared favorably with experimental data on the stimulatory effects of aldosterone and insulin, both separately and together, on active Na+ transport.
Analysis of structural changes during hypotonic swelling in Ehrlich ascites tumor cells
Authors: Wilkerson E. H. , DiBona D. R. , Schafer J. A.
Published: December 2017

Studies were undertaken to quantify structural changes associated with swelling of Ehrlich ascites tumor cells in hypotonic medium. Cells transferred from isotonic (294 mosmol/kg H2O) to hypotonic (98 mosmol/kg H2O) medium swelled rapidly. Subsequently, approximately 40% of the water initially gained was lost, a phenomenon referred to as volume-regulatory decrease (VRD). During the initial rapid cell swelling, blister-like protrusions or blebs were formed on the cell surface. These blebs were examined by routine light microscopy, differential interference-contrast (DIC) microscopy, and scanning and transmission electron microscopy. Microscopic observations and the distribution of ATPase antibodies indicated that the blebs were formed from plasma membrane. During VRD, the blebs coalesced to form a smooth but expanded membrane surface that appeared to be separated from the original cytoplasm by a layer of less dense ground substance. Computer-assisted morphometry from digitized DIC images of the initial swelling phase indicated that all of the volume gained was sequestered in the blebs. We suggest that bleb formation may allow increases in cell volume without disruption of cytoplasmic organization and may be a protective response to a variety of stressful stimuli. The subsequent VRD is accompanied by reduction of this expanded compartment.
Chemically evoked release of an angiotensin-like peptide from fetal rat brain cells in culture
Authors: Meyer J. M. , Weyhenmeyer J. A.
Published: December 2017

Recent evidence has demonstrated that dissociated fetal rat brain cells in culture synthesize an angiotensin II (ANG II)-like peptide that shares common properties with authentic ANG II, suggesting that brain has a complete renin angiotensin system that is independent of peripheral substrates. Although ANG II has been postulated to function as a neurotransmitter/neuromodulator, the release of ANG II from presynaptic nerve terminals has not been established. To investigate the mechanism of ANG II release, brain cells from 20-day gestational age Sprague-Dawley rats were dissociated by mild trypsinization and grown in culture. The cells were maintained in serum-free medium for 5 days prior to experimental analysis. Cultured brain cells were challenged with 59 mM KCl in the presence or absence of 5 mM CaCl2, and the incubation medium was measured for the release of ANG II by radioimmunoassay and high-performance liquid chromatography. The data demonstrate that K+ stimulation results in a rapid and time-dependent release of ANG II-like peptide that is Ca2+ dependent. We have concluded that these findings are consistent with those for other neurotransmitter/neurohormone systems and therefore provide further support for the role of ANG II as a chemical transmitter in the brain.
Nitrobenzylthioinosine: an in vivo inhibitor of pig erythrocyte energy metabolism
Authors: Young J. D. , Jarvis S. M. , Clanachan A. S. , Henderson J. F. , Paterson A. R.
Published: December 2017

The potential role of plasma nucleosides as metabolic energy substrates for pig erythrocytes, which are impermeable to glucose, was investigated in vivo by infusion of anesthetized pigs with nitrobenzylthioinosine phosphate (NBMPR-P), a soluble prodrug form of the specific nucleoside transport inhibitor, nitrobenzylthioinosine. NBMPR-P administration (1 or 10 mg X kg-1 X h-1) led to complete in vivo blockade of erythrocyte nucleoside transport activity and was associated with a dramatic decrease in the erythrocyte [ATP]-to-[ADP] ratio from 11.4 at time 0 to 2.9 after 4 h (mean results from 3 animals). Plasma inosine concentrations increased progressively from 2-4 microM at time 0 to 20-70 microM after 4 h of drug administration. In contrast, plasma adenosine concentrations remained less than 0.4 microM in all samples. These data suggest that pig erythrocytes utilize plasma inosine as their physiological energy substrate.
Effects of protein reagents on electrotonic coupling in crayfish septate axon
Authors: Campos de Carvalho A. , Ramon F. , Spray D. C.
Published: December 2017

The lateral giant axons of the crayfish nerve cord are composed of segments contributed by each ganglion, which are electrotonically coupled by way of gap junctions. We have investigated the involvement of protein residues in regulating the resistance of crayfish junctional channels by determining effects of group-specific protein reagents. When applied to well-coupled axons, the sulfhydryl group reagents N-ethylmaleimide (NEM) and diamide uncoupled the segments; junctional resistance (Rj) was increased without changing membrane resistance or axoplasmic pH (pHi). The uncoupling produced by NEM could be reversed by alkalinization of the cytoplasm (addition of ammonium chloride to the external medium). Another sulfhydryl reagent (p-chloromercuribenzoic acid) increased Rj to a lesser extent. A disulfide reagent and three amino and three carboxyl group reagents had no effect on the Rj of these axons. The effect of group-specific reagents on partially uncoupled axons was tested by applying the drugs to axons previously exposed to weak acids. N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline recoupled partially uncoupled axons by decreasing Rj and prevented subsequent uncoupling of the junction by low pHi. Another carboxyl group reagent, as well as sulfhydryl and amino group reagents, either had no effect or uncoupled the axons further by increasing Rj. These experimental results suggest that amino acid residues, possibly containing carboxyl and sulfhydryl groups, control the opening and closing of junctional channels and may thus be associated with the channels' active sites.
Glucose alters configuration of gap junctions between pancreatic islet cells
Authors: In't Veld P. A. , Pipeleers D. G. , Gepts W.
Published: December 2017

In rat pancreatic islets, gap junctional subunits (GJS) occur under two different configurations, namely in linear single strands and in polygonal particle aggregates. The present freeze-fracture study demonstrates that GJS can rapidly (dis)assemble into one of these membrane specializations without changes in their total number. Isolation of the pancreatic gland and its perfusion at 2.8 mM glucose is accompanied by a decrease in polygonally packed GJS from 46 to 16%. A rise in medium glucose concentration is followed, within 10 min, by a dose-dependent increase in the percent polygonal particles. This glucose effect on gap junction configuration is calcium dependent and reversible upon glucose removal; it is still entirely detectable when protein synthesis is blocked by cycloheximide. These results indicate that islet gap junctions are dynamic structures that rapidly adjust their configuration to extracellular regulators of beta-cell function. In the light of previous observations, it is suggested that this rapid (dis)assembly of gap junctional structures be considered as a component in the ionic and metabolic coupling between insulin-containing beta-cells of the pancreas.
Fluorescent markers to study membrane retrieval in antidiuretic hormone-treated toad urinary bladder
Authors: Harris H. W. , Wade J. B. , Handler J. S.
Published: December 2017

Antidiuretic hormone (ADH) stimulation of toad urinary bladder causes fusion of intracellular vesicles called aggrephores with the apical plasma membrane of granular cells. Aggrephores contain intramembrane particle aggregates whose appearance in the apical membrane is believed to produce a large increase in its water permeability. ADH removal (ADH washout) is thought to cause the retrieval of aggrephores into granular cell cytoplasm. We studied granular cell uptake of dextran and horseradish peroxidase conjugated with fluorescein, rhodamine, or both during ADH washout. Granular cell uptake of fluorescent dextran was dependent on prior exposure to ADH, a linear function of dextran concentration, and increased by a transepithelial osmotic gradient. Immediately after removal of ADH, granular cell fluorescence was finely dispersed and located near the apical surface. Subsequently, it coalesced into larger bodies. This change was most apparent when a single bladder was subjected to two cycles of ADH stimulation and removal using a dextran containing a different fluorophore for each cycle. The ultrastructural correlate for these fluorescent patterns was identified using rhodamine-labeled horseradish peroxidase. Electron microscopy showed that after detachment from the apical membrane, label was initially in tubular-shaped vesicles near the apical surface. Later, these vesicles clustered near multivesicular bodies and transferred their label to these structures. These tubular vesicles closely resemble the morphology of aggrephores visualized by freeze-fracture electron microscopy. We conclude that these fluorescent compounds can be used as markers for the luminal contents of membrane retrieved during ADH washout and allow detailed study of its intracellular processing.
An established cell line from mouse kidney medullary thick ascending limb. I. Cell culture techniques, morphology, and antigenic expression
Authors: Valentich J. D. , Stokols M. F.
Published: December 2017

The effective use of cultured cells as model systems for investigating the differentiation and regulation of transport processes in renal tubular epithelial cells depends on the availability of functional long-term cell lines derived from specific nephron segments. Conventional culture procedures that treat cells as proliferating microorganisms possess several inherent limitations that could contribute to phenotypic instability and limited proliferative capacity in vitro. In this study, culture techniques were adopted that avoid exposure of cells to proteolytic enzymes, maintain intercellular contacts, and allow cells to remain continually adherent to a collagen gel substratum. This methodology resulted in the development of a continuous epithelial cell line from identified, microdissected segments of the mouse kidney medullary thick ascending limb.
A simple method for incorporating aequorin into mammalian cells
Authors: Borle A. B. , Freudenrich C. C. , Snowdowne K. W.
Published: December 2017

A simple method for incorporating aequorin into mammalian cells to measure cytosolic ionized Ca2+ is described and compared with scrape loading and hypoosmotic treatment (HOST). The procedure consists of incubating the cells for 10 min and centrifuging them at 200 g for 30 s in the presence of aequorin. This method incorporates the same amount of photoprotein as scrape loading but 70% less than HOST. Cytosolic ionized Ca2+ has been measured in hepatocytes, kidney cells and tubules, macrophages, and cardiac myocytes loaded with aequorin by this new procedure.
Skin calcium binding protein: effect of ultraviolet B irradiation
Authors: Rizk M. , Didierjean L. , Pavlovitch J. H.
Published: December 2017

Skin calcium binding protein (SCaBP) has been found in the epidermal basal layer but not in the suprabasal layers, suggesting that its presence is correlated with the position of the cell and/or the cells' low degree of differentiation and high proliferation capacity. Rats were exposed to ultraviolet B (UVB) irradiation (280-320 nm, 0.1 J/cm2) to dissociate these two main characteristics of basal layer cells. Skin biopsy specimens were taken 24 and 48 h after UVB exposure. The [3H]thymidine labeling index, SCaBP localization (indirect immunofluorescence), and SCaBP levels monitored by radioimmunoassay were investigated. The [3H]thymidine labeling index was significantly increased after UVB treatment, and the [3H]thymidine-labeled cells were present not only in the basal layer but also in the suprabasal epidermal layers. Anti-SCaBP immunofluorescence was observed in both suprabasal and basal layers (rather than exclusively in the basal layer, as in control conditions). The radioimmunoassay indicated an overall increase in skin SCaBP content. No evidence was obtained that this increase was due to humoral factors, as no changes in SCaBP concentration on cell proliferation were observed in nonirradiated epidermal areas. Topical application of a single dose of vitamin D3, equivalent to the amount synthesized by UVB exposure, was also without effect. Thus the presence of this marker is correlated with the low degree of cell maturity and the cells' ability to proliferate rather than their basal position.
Studies on final differentiation of rat renal proximal tubular cells in culture
Authors: Larsson S. , Aperia A. , Lechene C.
Published: December 2017

The ontogeny of effective Na and K permeability has been studied in renal epithelial cells isolated from the outermost superficial cortex from adult and young (10-15 days) rats. The cells were cultured for 2-4 days and exhibited phloridzin-inhibitable alpha-methylglucoside uptake, characteristic of renal proximal tubular cells (RPTC). Intracellular concentrations of K, Na, Cl, and P and kinetics of changes in intracellular ionic content after inhibition of Na-K-ATPase with 1 mM ouabain (or by incubation in low-K medium) were measured in individual cells using electron probe analysis. Intracellular concentrations of K, Na, Cl, and P were equivalent in young and adult rat RPTC. Adult rat and young rat cells preincubated in K-free medium rapidly recovered normal intracellular K and Na contents when returned to 5.5 mM K medium. The recovery was almost immediately blocked by ouabain. Effective permeabilities measured as half time of K efflux and Na influx after ouabain inhibition of Na-K-ATPase were higher in adult than in young RPTC cultured for less than 4 days. Effective K and Na permeabilities decreased significantly with increasing time in culture in adult but not in young rat RPTC. Among young rat RPTC, half times of Na and K fluxes were significantly correlated to age. Effective K and Na permeabilities were lower in both young and adult rat RPTC that had been serum deprived for 24 h than in cells that had been continuously cultured in serum. In cells cultured for 3 days and serum deprived for 1 day, the addition of serum significantly increased K and Na permeability both in young and adult RPTC, but the effect was more pronounced in young RPTC where permeability reached the same high values as in adult RPTC continuously cultured in serum. In conclusion, effective Na and K permeabilities and serum activation of "permeability units" change during ontogeny. These ontogenic changes might be blunted after a few days in culture due to dedifferentiation of adult rat RPTC.
Short-term effects of PTH on cultured rat osteoblasts: changes in membrane potential
Authors: Edelman A. , Fritsch J. , Balsan S.
Published: December 2017

The introduction of parathyroid hormone [bPTH (1-34)], 10(-8) M, into the medium of cultured rat osteoblasts results in rapid (less than 1 min) depolarization of the osteoblast membranes. Conventional and pH-sensitive microelectrodes were used to assess the mechanism underlying this change. PTH depolarized cell membrane independently of steady-state membrane potential (Vm). Blocking K+ conductance (Ba2+) and Ca2+-dependent K+ conductance (quinine) depolarized Vm by +13.1 +/- 4.6 (n = 6) and +14.8 +/- 6.7 mV (n = 6), respectively, and both abolished the effect of PTH on Vm. The rate of depolarization was reduced in low-Ca2+ medium. PTH inhibited low Na+-induced cell hyperpolarization, but intracellular pH was not altered by hormone addition. PTH-induced depolarization occurred even when the Na+-K+ pump was blocked with ouabain. A second slower response was seen in cells having a Vm lower than -60 mV, with an increase in negativity 5-15 min after hormone application. The results indicate that PTH rapidly modifies Vm by changes of K+ conductance, which may be the first step in hormonal stimulus-response coupling, and induces delayed, long-term changes in cell status.
Nuclear magnetic resonance measurement of cytosolic free calcium levels in human red blood cells
Authors: Murphy E. , Levy L. , Berkowitz L. R. , Orringer E. P. , Gabel S. A. , London R. E.
Published: December 2017

Red blood cells were loaded with 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid (FBAPTA) by incubation with 50 microM of the acetoxymethyl ester (FBAPTA-AM), and cytosolic free Ca2+ was monitored with 19F-nuclear magnetic resonance (NMR). Loading with 50 microM FBAPTA-AM, which results in a final FBAPTA level of approximately 0.5 mM, caused only a 25-30% fall in cell ATP as measured by 31P-NMR when 5 mM pyruvate was present. Leakage of the NMR active Ca2+ indicator, which results from cell lysis, was corrected for with the addition of extracellular Eu3+ ions, extracellular ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA), or washing. With this method, we have found basal levels of cytosolic free Ca2+ averaging 61 +/- 6 nM (means +/- SE, n = 19). When the intracellular level of FBAPTA was varied from 0.1 to 1.0 mM, there was no correlation between the level of cytosolic free Ca2+ and the level of loading with FBAPTA. Addition of 10 microM of the Ca2+ ionophore A23187 with extracellular Ca2+ set at different levels by Ca2+-EGTA buffers caused an increase in cytosolic free Ca2+ as expected. Furthermore, ATP depletion caused a two- to three-fold increase in cytosolic free Ca2+, consistent with inhibition of Ca2+ efflux via that Ca2+-ATPase.
Vasoactive agents affect mesangial cell adhesion
Authors: Kreisberg J. I. , Venkatachalam M. A.
Published: December 2017

The formation and maintenance of stress fibers in cultured mesangial cells is associated with myosin light chain phosphorylation [Kreisberg et al. Am. J. Physiol. 249 (Renal Fluid Electrolyte Physiol. 18): F227-F235, 1985], a biochemical indicator for activation of actin-myosin interactions. Agents that elevate intracellular levels of adenosine 3',5'-cyclic monophosphate (cAMP) (e.g., isoproterenol) fragment stress fibers and cause myosin light chain dephosphorylation, whereas the addition of contractile agents such as arginine vasopressin (AVP) and prostaglandin E2 (PGE2) reverses these changes. Because stress fiber development in cultured cells is correlated with tight cell to substrate adhesion, we wanted to examine whether vasoactive agents have an effect on mesangial cell adhesion. Both isoproterenol and dibutyryl cAMP (DBcAMP) reduced mesangial cell adherence as measured by a trypsin assay (% detached cells: control 11 +/- 2.4%; isoproterenol plus isobutylmethylxanthine (IBMX) = 48.3 +/- 7.4%; DBcAMP = 29.3 +/- 3.7%; DBcAMP-IBMX = 73 +/- 4.4%). The areas of focal (adhesive) contacts between the cell and substratum as observed by interference-reflexion microscopy were also reduced, being replaced by areas of greater separation (% of the surface in contact with the substratum: control = 7.4 +/- 0.8%; isoproterenol-IBMX = 2.9 +/- 1.1%). Addition of PGE2 or AVP to the incubation medium containing the cAMP-elevating agents prevented the above changes. PGE2 or AVP alone increased mesangial cell adhesion (% detached cells: control 11 +/- 2.4%; PGE2 = 6.8 +/- 0.5%; AVP = 5.1 +/- 1.2%).(ABSTRACT TRUNCATED AT 250 WORDS)
Human platelet osmotic water and nonelectrolyte transport
Authors: Meyer M. M. , Verkman A. S.
Published: December 2017

The osmotic water (Pf) and nonelectrolyte permeability (Ps) properties of human platelets were characterized using the stopped-flow light-scattering technique. At 37 degrees C, Pf = 0.007 +/- 0.001 cm/s, the urea reflection coefficient (sigma urea) = 0.95 +/- 0.04, and Ps for a series of permeant nonelectrolytes was (in cm X s-1 X 10(-6)) 2.1 (urea), 3.5 (glycerol), 3.8 (thiourea), 17 (ethylene glycol), 18 (acetamide), 23 (formamide), and 24 (butyramide). Pf did not depend on the size of the osmotic gradient or on the direction of volume flow. Mercurial sulfhydryl reagents did not inhibit osmotic water transport, and phloretin and phenylurea did not inhibit urea transport. There was a discontinuity in the temperature dependence for both Pf and urea permeability (P urea) at 36 degrees C; enthalpy (delta H) = 25 (greater than 36 degrees C) and 4.4 kcal/mol (less than 36 degrees C) for Pf, and delta H = 26 (greater than 36 degrees C) and 7 kcal/mol (less than 36 degrees C) for P urea. In contrast to the facilitated water and urea transport systems in the red blood cell, these results suggest that the mechanism for water and urea transport in the platelet is primarily by diffusion through membrane phospholipid. A computer-simulated model of platelet circulation through the renal medulla, based on the measured values for Pf, P urea, and sigma urea, indicated that platelets undergo an approximately 40% decrease in volume in the inner medulla and an approximately 20% overshoot in volume as they return to the external isosmotic environment.
Cholinergic regulation of Na absorption by turtle colon: role of basolateral K conductance
Authors: Venglarik C. J. , Dawson D. C.
Published: December 2017

The mechanism underlying the muscarinic inhibition of colonic Na absorption is unknown. In this study the effects of carbachol on active Na transport and basolateral K conductance were compared in the isolated turtle colon. Carbachol produced a biphasic response in both Na transport and basolateral K conductance. The response consisted of a transient activation followed by a sustained inhibition and was blocked by atropine. Submucosal cholinergic neurons were implicated in the regulation of colonic transport by employing depolarizing agents to release endogenous acetylcholine. Depolarizing agents produced a carbachol-like response that was atropine-sensitive. Finally, experiments with the Ca ionophores, A23187 and ionomycin, suggested that the muscarinic response may be mediated, at least in part, by changes in cellular Ca. These experiments provide evidence that cholinergic neurons are present in the turtle colon submucosa, muscarinic agonists cause a change in basolateral K conductance that may be an important event in the regulation of colonic Na absorption, and a Ca second messenger system may be involved in mediating the response.
Mechanical transients initiated by ramp stretch and release to Po in frog muscle fibers
Authors: Cavagna G. A. , Mazzanti M. , Heglund N. C. , Citterio G.
Published: December 2017

Single fibers from the tibialis muscle of Rana temporaria were subjected to ramp stretches during tetanic stimulation at a sarcomere length of approximately 2 microns. Immediately after the stretch, or after different time delays, the active fiber was released against a constant force equal to the isometric force (Po) exerted immediately before the stretch. Four phases were detected after release: an elastic recoil of the fiber's undamped elements, a transient rapid shortening, a marked reduction in the velocity of shortening (often to 0), and an apparently steady shortening (sometimes absent). Increasing the amplitude of the stretch from approximately 2 to 10% of the fiber rest length led to an increase in phase 2 shortening from approximately 5 to 10 nm per half-sarcomere. Phase 2 shortening increased further (up to 14 nm per half-sarcomere) if a time interval of 5-10 ms was left between the end of large ramp stretches and release to Po. After 50- to 100-ms time intervals, shortening occurred in two steps of approximately 5 nm per half-sarcomere each. These findings suggest that phase 2 is due to charging, during and after the stretch, of a damped element, which can then shorten against Po in at least two steps of approximately 5 nm/half sarcomere each.
Voltage-dependent Ba2+ block of K+ channels in apical membrane of frog skin
Authors: De Wolf I. , Van Driessche W.
Published: December 2017

The characteristics of the blockage of apical K+ channels in the frog skin (Rana temporaria) by Ba2+ were investigated with current-voltage measurements and current-fluctuation analysis. Inward K+ currents were recorded with a transepithelial K+ concentration gradient oriented from mucosa to serosa. When Ba2+ (5-10,000 microM) was added to the mucosal solution, the inward K+ current was progressively depressed. This blockage was strongly voltage dependent. The apparent macroscopic Michaelis-Menten constant decreased exponentially with increasing transepithelial voltage (Vt) (mucosa positive). According to Eyring rate theory, the binding site for the Ba2+ ion in the K+ channel was found to be located at a relative electrical distance of 0.72 of the apical membrane potential field, measured from the cytosolic side. From current-voltage (I-V) measurements and Michaelis-Menten kinetics, the Ba2+ dissociation constant was calculated: KBa = 57 microM (Vt = 0 mV). The voltage dependence of the on- and off-rate of the Ba2+-receptor interaction was obtained from the analysis of current fluctuations induced by Ba2+. An exponential relationship between the rates and Vt was obtained, as was found for KBa from the I-V measurements. KBa (at Vt = 0 mV) calculated from the noise experiments was 66 microM.
Solubilization and reconstitution of a chloride transporter from tracheal apical membrane
Authors: Dubinsky W. P. , Monti L. B.
Published: December 2017

Electrogenic Cl- transport in bovine tracheal membrane vesicles was studied by measuring the kinetics of Cl- efflux. Efflux from KCl-loaded vesicles was assayed as the appearance of Cl- in the medium using an anion-specific electrode. Maximal sensitivity was obtained by passage of the vesicles through a Sephadex column immediately before the assay to remove external Cl-. Two components of Cl- transport could be detected. Vesicles exhibited a characteristic spontaneous Cl- efflux that appears to be limited by the cation permeability of the membrane. Addition of the K+-conducting ionophore to increase selectively the permeability to K+ resulted in a stimulation of Cl- efflux. The electrically neutral ionophore nigericin caused only a slight transient increase in Cl- efflux. The valinomycin-dependent flux may be assumed to represent flux through a parallel conductive pathway. Valinomycin-dependent electrogenic Cl- flux was 240 +/- 36 nmol X mg protein-1 X min-1 (n = 4). Soluble extracts obtained with the nonionic detergent octyl glucoside were reconstituted by the freeze-thaw procedure. The reconstituted vesicles exhibited a number of similarities to the native vesicles. Efflux was maximally stimulated with the ionophore valinomycin in the presence of an outwardly directed K+ gradient. Nigericin had no effect on efflux, suggesting the reconstituted transporter is a conductive rather than exchange mechanism. The initial rate of the valinomycin-dependent component was 896 +/- 63 nmol X mg protein-1 X min-1 (n = 5), representing approximately a fourfold increase in specific activity compared with the native membrane vesicle.
Isolation and characterization of a Na-H antiporter-deficient mutant of LLC-PK1 cells
Authors: Agarwal N. , Haggerty J. G. , Adelberg E. A. , Slayman C. W.
Published: December 2017

LLC-PK1 is an established cell line derived from pig kidney epithelium; it differentiates in vitro to form a polarized epithelial sheet, capable of the vectorial transport of solutes and water. We have used a modification of the "proton-suicide" method of Pouyssegur et al. (Proc. Natl. Acad. Sci. USA. 81:4833-4837, 1984) to isolate a mutant of LLC-PK1 cells that is deficient in Na-H antiporter activity. The mutant grows normally at pH 7.0 and above in the presence and absence of bicarbonate; at pH 6.5, however, it requires bicarbonate for growth. Alkalinization of the cytoplasm by the Na-H antiporter thus appears to be essential for growth at acidic pH in the absence of bicarbonate. The antiporter is also essential for the formation of domes (a consequence of vectorial water transport) in the absence of bicarbonate.
Kinase A does not activate phosphatidylinositol phosphorylation in rat lymphocyte membranes
Authors: Coco A. , Macara I. G.
Published: December 2017

The interaction of the dissociated catalytic subunit of adenosine 3',5'-cyclic monophosphate-dependent protein kinase (kinase A) and phosphatidylinositol metabolism has been studied in rat spleen lymphocyte membranes. As reported previously (Sarkadi et al., FEBS Lett. 152: 195-198, 1983) addition of kinase A increased by about twofold the phosphorylation of phosphatidylinositol in lymphocyte membranes at low ATP concentrations. However, we have found that this increase is an artifact of the assay conditions, and that the increase is a consequence of an inhibition of membrane adenosine triphosphatase activity by the protein kinase A. When lipid phosphorylation was measured under initial rate conditions, at high ATP concentrations, the increase was abolished. No effect of kinase A was observed on initial rates of the synthesis or hydrolysis of phosphatidylinositol phosphate. No phosphatidylinositol bisphosphate was produced in the membranes under any of the assay conditions used.
High affinity L-aspartate transport in chick small intestine
Authors: Wingrove T. G. , Kimmich G. A.
Published: December 2017

Epithelial cells isolated from chick small intestine were used to study the mechanism of L-aspartate transport. Two kinetically distinct uptake systems of high (Km' = 16 microM) and low (Km'' = 2.7 mM) affinity are observed. This paper examines the cation dependence and membrane potential sensitivity of the high affinity system. Unidirectional influx studies indicate that extracellular Na+ is an absolute requirement for transport function. Flux is optimal when K+ is present intracellularly, however this cation is not required for Na+-dependent L-aspartate uptake. In the absence of K+, flux enhancement is observed when the intracellular pH is acidic. In contrast, acidic intracellular pH is inhibitory in cells that are preequilibrated with K+. Sodium ([Na+]o greater than [Na+]i gradients, and potassium ([K+]o less than [K+]i) or proton ([H+]o less than [H+]i) gradients can independently energize the Na+-dependent accumulation of L-aspartate above equilibrium levels, suggesting that Na+ and L-aspartate cotransport occurs with concomitant K+ or H+ antiport. L-Aspartate influx is insensitive to membrane potential changes created by inwardly directed anion gradients in the presence or absence of intracellular K+. A model is presented that is consistent with electroneutral Na+-coupled transfer with an ion antiport site of low specificity.
Blocking agents of Ca2+-activated K+ channels in cultured medullary thick ascending limb cells
Authors: Guggino S. E. , Guggino W. B. , Green N. , Sacktor B.
Published: December 2017

Ca2+-activated K+ channels with estimated single channel conductances of 127 +/- 2 pS were identified in the apical cell membrane of clone A3 of cultured medullary thick ascending limb (MTAL) cells. Both Ba2+ and the scorpion toxin, charybdotoxin (CTX), are slow blockers of the channels. An application of 0.1 microM Ba2+ to the intracellular face caused a 50% reduction in fractional open time (fv). Ba2+ block is both concentration and voltage dependent. Concentrations of CTX as low as 2 nM in the extracellular solution caused a significant reduction in fv. Tetraethylammonium (TEA) and quinine are fast blockers of Ca2+-activated K+ channels in MTAL cells. TEA, 400 microM, in the extracellular solution caused a voltage-dependent reduction in channel amplitude, whereas it takes 10 mM in the intracellular solution to reduce channel amplitude by 30%. Micromolar amounts of quinine applied to the intracellular face caused the channels to flicker rapidly between open and blocked states. These results suggest that K+ channels in MTAL cells are homologous to those found in muscle cells, and that these blocking agents may be used to probe the nature of K+ conductances in several nephron segments.
Calcium, cell shrinkage, and prolytic state of human red blood cells
Authors: Crespo L. M. , Novak T. S. , Freedman J. C.
Published: December 2017

The effects of intracellular calcium, or Cac, on the Na permeability of human red blood cells were examined during 3-h incubations with the Ca ionophore A23187 and varied external Ca, Cao. Above 3 microM Cao, Nac increased significantly as ATP decreased. Maintenance of normal ATP with vanadate did not prevent the gain of Nac. Similar amounts of Nac were gained in 3 h by ouabain-treated cells exposed to the K ionophore valinomycin or by cells osmotically shrunken. Cells shrunken with sucrose also exhibited partial loss of Kc. When the cells with increased Nac were subsequently transferred to Na-free, high-K medium, the Nac and Kc that had changed slowly over 3 h returned toward normal within 10 min. The development of irreversible high cation permeability in shrunken cells was not prevented by a variety of transport inhibitors. These observations and cell volume distributions suggest that prolonged shrinkage induces a subpopulation of cells to become highly cation permeable, or "prolytic". The major effect of Cac on Na permeability appears to be an indirect consequence of cell shrinkage due to KCl loss.
Ba2+ induces contraction in swine carotid artery by mobilizing intracellular Ca2+
Authors: Hai C. M. , Murphy R. A.
Published: December 2017

Recent data suggest that Ba2+ activates the smooth muscle contractile apparatus. However, in vitro studies indicate that Ba2+ activates Ca2+ calmodulin-dependent enzymes only at very high concentrations. These observations might suggest that Ba2+ activates the contractile apparatus by a mechanism independent of myosin phosphorylation. We tested this hypothesis using intact and Triton X-100 skinned swine carotid medial strips. In intact tissues, Ba2+ stimulated dose-dependent contractions in the absence of extracellular Ca2+. However, in skinned tissues, Ba2+ induced much lower stress development at concentrations more than three orders of magnitude greater than needed for Ca2+-induced contraction. Ba2+ also failed to maintain stress in skinned tissues previously contracted with 4 microM Ca2+. The Ba2+-induced contraction in intact strips was associated with gradual increases in myosin phosphorylation from a basal level of approximately 5% to a sustained level of 30.0 +/- 0.8%. Partial intracellular calcium depletion with 100 microM histamine, 25 mM caffeine, and 5 mM ethyleneglycol-bis(beta-aminoethyl-ether)-N,N'-tetraacetic acid (EGTA) failed to abolish Ba2+-induced contractions. However, repeated contractions with Ba2+ in the absence of extracellular Ca2+ led to decreases in stress development. These observations suggest that Ba2+ induces stress development by mobilizing intracellular Ca2+ that was not released by histamine and caffeine.
Aldosterone regulation of Na+ transport and Na+-K+-ATPase in A6 cells: role of growth conditions
Authors: Paccolat M. P. , Geering K. , Gaeggeler H. P. , Rossier B. C.
Published: December 2017

The effects of aldosterone on transepithelial sodium transport (measured by the short-circuit current (SCC) and on Na+-K+-adenosine triphosphatase (ATPase) biogenesis have been studied in A6 kidney cells grown on collagen-coated filters in two different media. In medium A, base-line SCCA was close to zero but transmural electrical resistance (RA) was high. Aldosterone (100 nM, t24h) drastically increased SCCA and RA, but only after a 4-h latent period. In medium B, base-line SCCB and RB were significantly higher than in medium A. Aldosterone significantly enhanced SCCB and to a lesser extent RB after a much shorter latent period (approximately 45 min) than in medium A. In medium A, aldosterone elicited a fourfold increase in the relative rate of synthesis of alpha- and beta-subunits of Na+-K+-ATPase. A twofold increase was already observed within the observed latent period. This time course suggests that de novo synthesis of sodium pumps might be one of the critical factors underlying the increase in sodium transport in this growth medium. In medium B, aldosterone elicited a two- to fourfold increase in the relative rate of synthesis of the alpha- and beta-subunits of Na+-K+-ATPase that paralleled SCCB. Thus de novo synthesis of Na+-K+-ATPase is clearly not a prerequisite for the early mineralocorticoid response (t90 min - t180 min), but still could be part of the late mineralocorticoid response (t3 h - t24 h). In both media, the immunochemical cellular pool of Na+-K+-ATPase was apparently not modulated by aldosterone for up to 48 h of incubation.(ABSTRACT TRUNCATED AT 250 WORDS)
Calmodulin antagonists inhibit latch bridges in detergent skinned swine carotid media
Authors: Moreland S. , Little D. K. , Moreland R. S.
Published: December 2017

Ca2+-calmodulin-dependent phosphorylation of the 20,000 apparent molecular weight (Mr) myosin light chain (MLC) is widely believed to be the primary regulator of smooth muscle stress development. Stress maintenance, however, has been hypothesized to be the result of a Ca2+-dependent state called latch, defined as stress maintenance by slowly cycling, dephosphorylated latch bridges. This study was designed to examine the role of calmodulin in the Ca2+ dependence of latch. Swine carotid medial fibers were detergent skinned with Triton X-100 and MLC phosphorylation levels were measured by two-dimensional gel electrophoresis. Experiments were performed in which calmodulin antagonists, W-7 or trifluoperazine (TFP), were used to prevent Ca2+-dependent contractions or to relax previously contracted muscle strips. Both protocols resulted in similar decreases in stress with MLC phosphorylation levels correlating to the level of stress under all conditions. In another series of experiments, the skinned fibers were first contracted with Ca2+ and ATP and then exposed to Ca2+ and cytidine 5'-triphosphate (CTP). This resulted in stress maintenance with basal levels of MLC phosphorylation. W-7 relaxed Ca2+ and CTP maintained stress in a concentration-dependent manner. The potency of W-7 for relaxation of the CTP maintained stress was similar to that for inhibition of the ATP developed stress determined in the first set of experiments. The results demonstrate that calmodulin antagonists inhibit stress associated with MLC phosphorylation and also stress independent of MLC phosphorylation. This suggests that a calmodulin-like, Ca2+-binding protein may be important for latch bridge attachment.
ATP-dependent regulation of cytoplasmic free calcium in nerve terminals
Authors: Rasgado-Flores H. , Blaustein M. P.
Published: December 2017

ATP-dependent Ca uptake was studied in hyperpermeable (saponin treated) rat brain isolated nerve terminals (synaptosomes). The Ca uptake was measured at short incubation times (1-30 s) in the absence and presence of mitochondrial poisons, at various free Ca2+ concentrations (0.03-30 microM). Saponin treatment made the plasma membranes leaky without affecting the ATP-dependent Ca uptake by intracellular organelles. When the free Ca2+ concentration in the incubation medium was varied up to approximately 5 microM free Ca2+, mitochondrial blockers had no effect on the ATP-dependent Ca2+ uptake in the saponin-treated synaptosomes. At higher free Ca2+ concentrations, the blockers inhibited a portion of the ATP-dependent Ca uptake. This indicates that, in the dynamic physiological range of free Ca2+, the nonmitochondrial Ca uptake system (presumably the smooth endoplasmic reticulum, SER) is a more important Ca buffering system than the mitochondrial system. The SER sequesters Ca half maximally at free Ca2+ congruent to 0.4 microM and has a maximal Ca storage capacity of approximately 2 nmol/mg protein. The initial rate of SER Ca uptake is 0.1 nmol X mg protein-1 X s-1. This rate is too slow to account for the very rapid reduction of free Ca2+ that is required to terminate transmitter release immediately after presynaptic depolarization. Nevertheless, Ca sequestration in SER may play an important role in regulating longer term processes such as facilitation and post-tetanic potentiation.
Origin of transport inhibition after omission of serosal sodium
Authors: Nagel W.
Published: December 2017

The omission of sodium from the serosal incubation fluid in isolated frog skins inhibits transcellular Na transport. By the use of intracellular recording with microelectrodes, it has been demonstrated that this inhibition is associated with an increase of the basolateral membrane resistance, resulting in a depolarization of the short-circuited cells. This depolarization in turn accounts for the reduction of Na entry across the apical border. The resistance changes across the outer (apical) border are small in magnitude and unrelated to the inhibition of transcellular transport. The origin of the increase in basolateral membrane resistance, presumably due to decrease of K permeability, is unclear. These data do not support the hypothesis that intracellular Ca regulates the resistance of the apical and basolateral membranes.
Lactate transport by cardiac sarcolemmal vesicles
Authors: Trosper T. L. , Philipson K. D.
Published: December 2017

Page C483: T. L. Trosper and K. D. Philipson. “Lactate transport by cardiac sarcolemmal vesicles.” Page C483: the first sentence of paragraph 2 should read: Recent reports of lactate uptake by isolated cardiac myocytes (14) and perfused hearts (2, 16) presented evidence suggesting that lactate traversal of the cardiac sarcolemma is mediated by a carrier specific for monocarboxylic acids. Page C485: Fig. 1 legend should read: l-Lactate uptake into sarcolemmal vesicles as a function of time at room temperature. 1 mM l-lactate in external medium; vesicles loaded with 280 mM sucrose, pH 7.4. External media: 112 mM NaCl, 56 mM sucrose, pH 7.4, with ( closed circles) or without ( open circles) 5 μM monensin; or 280 mM sucrose, pH 7.4 (X) or 5.9 ( open squares). Data points on upper curves are means ± SD of 3 or more experiments. Lower curves are averages of 2 experiments. Inset: l-lactate uptake at short times from NaCl plus monensin, pH 7.4. Page C485: last line of Fig. 3 legend should read: Slope, which is least-squares fit of the data, gives apparent Km, for l-laxtate of 27 mM. Page C489: References 2, 14, and 16 should read:

2. Dennis, S. C., M. C. Cohn, G. J. Anderson, and D. Garfinkel. Kinetic analysis of monocarboxylate uptake into perfused rat hearts. J. Mol. Cell. Cardiol. 17: 987–995, 1985.

14. Kammermeier, H., B. Wein, and W. Graf. Characteristics of lactate transport in isolated cardiac myocytes. Basic Res. Cardiol. 80,Suppl. 1:57–60, 1985.

16. Mann, G. E., B. V. Zlokovic, AND D. L. Yudilevich. Evidence for a lactate transport system in the sarcolemmal membrane of the perfused rabbit heart. Biochim. Biophys. Acta 819: 241–246, 1985.
Altered Ca fluxes and contractile state during pH changes in cultured heart cells
Authors: Kim D. , Smith T. W.
Published: December 2017

We studied mechanisms underlying changes in myocardial contractile state produced by intracellular (pHi) or extracellular (pHo) changes in pH using cultured chick embryo ventricular cells. A change in pHo of HEPES-buffered medium from 7.4 to 6.0 or to 8.8 changed the amplitude of cell motion by -85 or +60%, and 45Ca uptake at 10 s by -29 or +22%, respectively. The pHo-induced change in Ca uptake was not sensitive to nifedipine (10 microM), but was Na gradient dependent. Changes in pHi produced by NH4Cl or preincubation in media at pH values ranging from 6.0 to 8.8 failed to alter significantly 45Ca uptake or efflux. However, larger changes in pHi were associated with altered Ca uptake. Changes in pHo from 7.4 to 6.0 or to 8.8 were associated with initial changes in 45Ca efflux by +17 or -18%, respectively, and these effects were not Na dependent. Exposure of cells to 20 mM NH4Cl produced intracellular alkalinization and a positive inotropic effect, whereas subsequent removal of NH4Cl caused intracellular acidification and a negative inotropic effect. There was, however, a lack of close temporal relationship between pHi and contractile state. These results indicate that pHo-induced changes in contractile state in cultured heart cells are closely correlated with altered transsarcolemmal Ca movements and presumably are due (at least in part) to these Ca flux changes. In contrast, pHi-induced changes in contractile state appear principally to involve altered Ca handling within the cell and/or altered Ca sensitivity of myofibrils.
Rescue of a wild-type MDCK cell by a ouabain-resistant mutant
Authors: Bolivar J. J. , Lazaro A. , Fernandez S. , Stefani E. , Pena-Cruz V. , Lechene C. , Cereijido M.
Published: December 2017

When wild-type MDCK cells (W-MDCK) were cocultured in mixed monolayers with a ouabain-resistant mutant (R-MDCK), the wild-type cells were protected from the effect of ouabain up to concentrations as high as 100 microM. Rescue depended on the dose of ouabain and on the proportion of each cell type in the coculture. The survival of R-MDCK cells at 1 microM ouabain was not endangered by varying from 1:9 to 9:1 the proportion of W-MDCK cells to be rescued. Ouabain binding revealed two kinds of binding sites in R-MDCK cells, one with high and the other with low affinity. Only the high affinity site was present in W-MDCK cells. Electron probe analysis of individual cells revealed that rescued cells kept a high K and a low Na intracellular contents, similar to control cells. Histograms of intracellular K/Na in cocultured cells treated with ouabain were unimodal. Using microinjection of Lucifer yellow or electrophysiological techniques we estimated that at most 13% of the R-MDCK and W-MDCK cells may be connected at a given time through cell-to-cell junctions. Therefore permanent cell-to-cell communication did not seem to play a central role in the rescue. W-MDCK cells cocultured with R-MDCK cells and subsequently separated, were not rescued. Thus rescue did not seem to depend on the transfer from R-MDCK to W-MDCK cells of either ouabain-resistant Na-K pumps or of information to synthesize them. It is speculated that intercellular communications were sporadic events, so that all cells may become intermittently connected and rescued.