bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of alpha 1- and alpha 2-adrenergic receptors in Cl- transport across frog corneal epithelium
Authors: Chu T. C. , Candia O. A.
Published: December 2017

Norepinephrine, 10(-6) M, reduced Cl- transport by 26% in 75% of isolated frog corneal epithelia. This inhibition was not previously reported. Since beta-adrenergic agonists are known to only stimulate Cl- transport, the action of specific alpha 1- and alpha 2-agonists on Cl- transport and electrical parameters was investigated. Phenylephrine, an alpha 1-agonist always stimulated the Cl(-)-dependent short-circuit current (Isc), but less than the beta-agonists. UK-14,304-18 (UK), a selective alpha 2-agonist, reduced both the Isc (by 31% at 10(-5) M) and the stroma-to-tear unidirectional Cl- flux. UK hyperpolarized the apical membrane potential difference and increased the transepithelial resistance and apical-to-basolateral resistance ratio. UK reduced forskolin-stimulated adenylate cyclase activity by 36%. The electrophysiological effects of UK are consistent with a reduction of the Cl- permeability at the apical membrane. Pretreatment with UK sensitized the tissue for a greater effect by forskolin. Results show that the frog corneal epithelium also possesses alpha 1- and alpha 2-receptors, the latter negatively coupled to the adenylate cyclase system. Cl- transport is thus regulated by an interaction between the positive effects of beta- and alpha 1-stimulation and the negative influence of alpha 2-stimulation.
Low-affinity intestinal L-aspartate transport with 2:1 coupling stoichiometry for Na+/Asp
Authors: Wingrove T. G. , Kimmich G. A.
Published: December 2017

Epithelial cells isolated from chick small intestine were used to define the ionic and electrical characteristics of a low-affinity (Km = 4.1 mM) L-aspartate transport system. L-Glutamate and D-aspartate, but not D-glutamate, were found to inhibit L-aspartate influx, suggesting that this uptake system has a substrate specificity similar to that previously described for a high-affinity (Km = 16 microM) acidic amino acid transporter in the same cells. Low-affinity uptake is Na+ dependent with a Hill coefficient (n) of 1.4. Intracellular K+ moderately enhances but is not required for aspartate influx, and this response is modulated by changes in intracellular pH. The Na+-dependent uptake of aspartate is electroneutral, as evidenced by insensitivity to pronounced changes in delta psi induced by anion gradients or valinomycin in the presence of K+ gradients. Because the above characteristics can be consistent with several transport models, direct measurement of delta Na+-delta Asp coupling stoichiometry were performed. The coupling ratio was determined to be approximately 2.0. A model for intestinal Na+-dependent L-Asp transport is suggested in which each transport cycle involves inward transfer of 2Na+:1Asp+ and outward transfer of K+ or H+ in a net electroneutral set of events.
Oxidants and conductance of cultured epithelial cell monolayers: inositol phospholipid hydrolysis
Authors: Shasby D. M. , Winter M. , Shasby S. S.
Published: December 2017

Reactive oxidants contribute to the alterations in endothelial and epithelial permeability that characterize the inflammatory response. We previously noted that noncytolytic doses of oxidants reversibly decreased the electrical resistance across cultured monolayers of Madin-Darby canine kidney (MDCK) cells (J. Clin. Invest. 76: 1155-1168, 1985). In this investigation, we have found that similar doses of oxidants initiate inositol phospholipid hydrolysis by a phospholipase C in cultured MDCK cells, with resultant increases in inositol polyphosphates, phosphatidic acid, and 1,2 diglycerides. Activation of this pathway is linked to activation of protein kinase C in many cells. The addition of phorbol 12,13-dibutyrate (PDBU) and 1-oleoyl-2-acetyl-sn-glycerol, activators of protein kinase C, decreased the electrical resistance across MDCK monolayers cultured on micropore filters similar to the effects of hydrogen peroxide. In contrast, the addition of 4 alpha-phorbol 12,13-didecanoate, a chemically similar compound that does not activate protein kinase C, did not decrease the electrical resistance. When MDCK monolayers were exposed to PDBU, fixed, and stained with rhodamine phallicidin, the peripheral band of actin in the cells showed a loss of staining density and continuity similar to the changes in phallicidin staining we previously noted in cells exposed to hydrogen peroxide. These data are consistent with the hypothesis that some of the reversible effects of oxidants on epithelial barriers are mediated through phospholipase C hydrolysis of inositol phospholipids with consequent activation of protein kinase C.
Chloride transport across placental microvillous membranes measured by fluorescence
Authors: Illsley N. P. , Glaubensklee C. , Davis B. , Verkman A. S.
Published: December 2017

Chloride transport across human placental microvillous vesicle membrane was investigated using the fluorescent probe SPQ (6-methoxy-N[3-sulfopropyl]quinolinium). Chloride influx (JCl) was calculated from the initial rate of quenching of intravesicular SPQ fluorescence by chloride. JCl measured by SPQ fluorescence was not significantly different from JCl measured by uptake of 36Cl; SPQ did not affect measurements of JCl.JCl was increased 51% by a 58-mV membrane potential (internal positive). Voltage-stimulated JCl showed a saturable dependence on chloride concentration with a dissociation constant (Kd) of 18 +/- 5 mM and was inhibited by diphenylamine-2-carboxylate with an apparent inhibitory constant of 0.13 +/- 0.03 mM. The activation energy calculated for voltage-stimulated JCl was 4.6 +/- 0.6 kcal/mol. JCl was also stimulated by a reduction in the external pH from 7.0 to 5.5 (internal pH = 7.0). pH-stimulated chloride influx was increased by trans-HCO3 (25 mM) and was inhibited by dihydro-4,4'-diisothiocyano-2,2'-disulfonic stilbene. Uptake of 36Cl into microvillous vesicles was stimulated by trans-Cl. pH-stimulated JCl showed a saturable dependence on chloride with a Kd of 38 +/- 6 mM but was not affected by membrane potential. No evidence was found for Na- or K-coupled chloride cotransport. These findings demonstrate the presence of a saturable chloride conductance and an electroneutral chloride-bicarbonate exchanger in the placental microvillous membrane.
Eicosapentaenoic and dihomo gamma linolenic acid metabolism by cultured rat mesangial cells
Authors: Scharschmidt L. A. , Gibbons N. B. , Neuwirth R.
Published: December 2017

To better understand the effects of dietary fatty acid manipulations on glomerular function, we compared mesangial incorporation, release, and metabolism of arachidonic (AA), eicosapentaenoic (EPA), and dihomo gamma linolenic (DHG) acids. We found marked differences in mesangial handling of these fatty acids. AA was incorporated into lipids of mesangial cells much more rapidly than EPA or DHG. Ionophore-induced stimulation of fatty acid release from mesangial cells prelabeled with [14C]AA, [14C]EPA, or [14C]DHG caused a release of labeled AA greater than DHG much less than EPA, respectively. Preloading mesangial cells with DHG or EPA for 24 h reduced subsequent basal, ionophore-, and hormone-stimulated prostaglandin E2 (PGE2) synthesis. Finally, unlike AA, neither EPA nor DHG was converted to a significant extent by mesangial cyclooxygenase or lipoxygenase. Thus the mesangial metabolism of DHG and EPA differs both quantitatively and qualitatively from that of AA. Furthermore, EPA and DHG inhibit metabolism of AA at the level of mesangial cyclooxygenase.
Effect of amiloride on the poorly selective cation channel of larval bullfrog skin
Authors: Hillyard S. D. , Van Driessche W.
Published: December 2017

A small, inward-directed, short-circuit current (SCC) was measured across the isolated skin of larval bullfrogs (Rana catesbeiana) when either NaCl or KCl Ringer solution bathed the mucosal surface. The addition of amiloride, in concentrations of 1-100 microM, produced a stepwise increase in SCC. As SCC values became maximally elevated by amiloride, the plateau value (So) of the Lorentzian component in the power-density spectrum increased, whereas the corner frequency (fc) decreased. This agonist effect of amiloride can be explained by an increase in the open probability and possibly the single-channel current of the larval channel. When the amiloride concentration was increased above 100 microM, the SCC values declined progressively but usually remained above pretreatment values. This suggests an antagonist effect of amiloride that is concurrent with the agonist effect. The removal of Ca2+ from the mucosal Ringers increased SCC in conjunction with an increase in So and a decrease in fc. Under these conditions, the maximal agonist effect of amiloride was observed at concentrations of 10-20 microM. Ca2+ thus exerts an inhibitory effect on the larval cation channel that interferes with the agonist effect of amiloride. The addition of Ba2+ to Ca2+-free preparations lowered SCC and reduced the agonist effect of amiloride.
Temperature-controlled perfusion apparatus for microscope using transparent conducting film heater
Authors: Toyotomi S. , Momose Y.
Published: December 2017

We describe a novel temperature-controlled perfusion apparatus for electrophysiological studies on isolated cells or membrane patches. The apparatus uses a transparent conducting film of indium-tin-oxide as a heating element. The film heater deposited on a glass sheet allows us to construct a structurally simple apparatus that is able not only to heat directly the bath chamber but also to preheat the perfusion solution on the stage of an inverted microscope. The structure ensures a uniform temperature in a bath chamber that has a working area of approximately 6 X 12 mm2 and a capacity of 1 ml. A control unit, designed for operation of the apparatus, regulates the temperature of the bathing solution without introducing electrical noise in a range from ambient temperature to greater than 37 degrees C with an accuracy within +/- 1 degree C. The apparatus is 12 mm in height and designed to fit on the stage of a standard inverted microscope. Since the bath chamber for perfusion experiments is readily interchanged with others, the apparatus can be used widely in microscopic studies of various cells in static solutions.
Initiation of protein synthesis in a cell-free system prepared from rat hepatocytes
Authors: Kimball S. R. , Everson W. V. , Flaim K. E. , Jefferson L. S.
Published: December 2017

A cell-free system, which maintained a linear rate of protein synthesis for up to 20 min of incubation, was prepared from isolated rat hepatocytes. The rate of protein synthesis in the cell-free system was approximately 20% of the rate obtained in isolated hepatocytes or perfused liver. More than 70% of total protein synthesis in the cell-free system was due to reinitiation, as indicated by addition of inhibitors of initiation, i.e., edeine or polyvinyl sulfate. The rate of protein synthesis and formation of 43S initiation complexes in the cell-free system were reduced to 60 and 30% of the control values, respectively, after incubation of hepatocytes in medium deprived of an essential amino acid. Therefore, the cell-free system maintained the defect in initiation induced in the intact cells by amino acid deprivation. The defect in initiation was corrected by addition of either rat liver eukaryotic initiation factor 2 or the guanine nucleotide exchange factor (GEF) to the cell-free system. A role for GEF in the defect in initiation was further implicated by experiments that showed that the activity of the factor was decreased in extracts from livers perfused with medium deficient in amino acids. The cell-free system should provide a valuable tool for investigation of mechanisms involved in the regulation of initiation of protein synthesis.
ADP release from myosin in permeabilized smooth muscle
Authors: Butler T. M. , Pacifico D. S. , Siegman M. J.
Published: December 2017

The purpose of this study was to determine the nucleotide bound to myosin and its rate of release under relaxed and activated conditions in permeabilized rabbit portal veins. Incubation of the muscles in a relaxing solution containing [3H]-ATP resulted in the formation of 60-70 microM radiolabeled ADP in the muscle whether or not the myosin light chains had been thiophosphorylated. This value was similar to the estimate of the concentration of myosin subfragment 1. Upon transfer of the muscles to a chase solution containing no labeled ATP, there was a very slow loss of labeled ADP when the light chains were unphosphorylated, but a much faster release occurred when the light chains were thiophosphorylated. The results suggest that smooth muscle myosin exists primarily in a complex with ADP under both relaxed and phosphorylated conditions and that phosphorylation of all of the light chains results in a large increase in the rate of release of the products of ATP splitting from all of the myosin. Interestingly, the exponential release of ADP in relaxed muscle shows two components, one of which contains about two-thirds of the total ADP and is 5- to 10-fold faster than the other. If the difference in rates of ADP release observed in relaxed muscle persists when the myosin is phosphorylated, then it is possible that there is a 5- to 10-fold difference in rates of cycling for different phosphorylated cross bridges in smooth muscle.
Dependence of stress on cross-bridge phosphorylation in vascular smooth muscle
Authors: Ratz P. H. , Hai C. M. , Murphy R. A.
Published: December 2017

Cross-bridge phosphorylation associated with agonist-stimulated contraction of vascular smooth muscle is often transiently elevated. Such observations led to the concept that phosphorylation of the 20-kDa myosin regulatory light chain (Mp) was required for initial activation and cross-bridge cycling but might not be necessary for steady-state maintenance of stress in the latch state. The possibility that stress maintenance is not regulated by phosphorylation has received some experimental support in contractions induced by phorbol esters and the calcium channel activator BAY K 8644 in which significant increases in Mp were not detected. Our aim was to test the hypothesis that phosphorylation is both necessary and sufficient for activation and for maintenance of steady-state stress. Activation of swine carotid media using agents that bypass receptor activation and elevate Ca2+ influx without mobilizing intracellular Ca2+ stores (BAY K 8644 and ionomycin) produced monotonic increases in both stress and Mp. Transient initial peaks in Mp were absent. Steady-state stress induced by both receptor- and nonreceptor-mediated activation was dependent on small increases in Mp. Increases in Mp greater than 0.3 mol Pi/mol myosin light chain had small effects on stress but produced large increases in the maximum rate of cross-bridge cycling at zero load (Vo). The experimentally determined dependence of stress on Mp was quantitatively predicted by our working hypothesis. This model proposes that Ca2+-stimulated cross-bridge phosphorylation is obligatory for cross-bridge attachment. However, dephosphorylation of attached cross bridges to form noncycling "latch bridges" allows stress maintenance with reduced Mp and cycling.
Glucocorticoid receptor activation in isolated perfused rat hearts
Authors: Czerwinski S. M. , McKee E. E. , Hickson R. C.
Published: December 2017

The formation of unactivated and activated glucocorticoid receptor complexes was studied in intact, isolated, perfused rat hearts in the presence of [3H]triamcinolone acetonide. Receptor activation, as quantified by the DNA-cellulose-binding assay, began to increase within 30 s of perfusion and reached a final steady-state level (t 1/2 = 4.6 min) with 46% of the steroid-receptor complexes bound to DNA-cellulose. With the use of a linear potassium phosphate (KP) gradient (5-400 mM), unactivated receptors eluted from DEAE-cellulose anion exchange columns at approximately 250 mM KP. Two activated receptor forms appeared, which eluted either in the wash fraction (binder IB) or between 50 and 100 mM KP (binder II) and occurred with half times of 1.3 and 2.7 min, respectively. Postperfusion cytosol preparation did not markedly influence the results as receptor binding was reduced by 10% or less when a 100-fold excess of unlabeled triamcinolone acetonide was included in the homogenizing buffer. We conclude from these results that glucocorticoids are able to exert a direct effect on the heart through binding to their own receptor in the absence of endogenous hormones. The time dependency of receptor activation supports a physiological role for this process. However, activation rates, determined from conformational changes associated with altered DEAE-cellulose elution profiles and appearance of activated receptor forms, occur earlier and may not be coordinated with the rate of activation as quantified by DNA-cellulose binding.
Intracellular Ca requirements for stimulus-secretion coupling in parietal cell
Authors: Negulescu P. A. , Reenstra W. W. , Machen T. E.
Published: December 2017

The role of cytosolic free Ca (Cai) in stimulating acid production by the parietal cell in response to the secretagogues carbachol (carb), histamine (hist), and dibutyryl adenosine 3',5'-cyclic monophosphate plus 3-isobutyl-1-methylxanthine (DBcAMP + IBMX) was evaluated. Microfluorimetry with fura-2 was used to measure Cai in single parietal cells within intact rabbit gastric glands. Acid production was determined in parallel experiments by monitoring the accumulation of [14C]aminopyrine (AP) in suspensions of glands. Carb increased peak Cai levels to 1 microM in a dose-dependent manner [concentration for half-maximal response (K0.5) = 8 microM] that correlated well with the dose dependence of carb-stimulated AP accumulation (K0.5 = 18 microM). The Ca chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) was used to attenuate secretagogue-induced Cai increases. Incubating glands for 10 min with 1 and 10 microM BAPTA/AM caused resting Cai to decrease from 119 to 93 and 80 nM, respectively. BAPTA/AM, 1 and 10 microM, blocked carb-stimulated increases in Cai by 60 and 90% and AP accumulation by 50 and 90%. Hist, which increases cytosolic cAMP, caused small and relatively infrequent increases in Cai. Even so, hist-stimulated AP accumulation was inhibited 8 and 40% by 1 and 10 microM BAPTA. DBcAMP had no effect on Cai, and AP accumulation caused by DBcAMP was unaffected by the concentrations of BAPTA tested. These data suggest that carb requires an increase in Cai as a secretory signal. Hist also exhibited some Cai dependence, which may be attributable to either a small increase in Cai or the necessity of having a specific basal level of Cai. A Cai requirement for DBcAMP-stimulated acid secretion was not detected. Thus the parietal cell possesses both Ca-dependent and Ca-independent stimulatory pathways, and at least one secretagogue (hist) may utilize both pathways.
Effects of charge on membrane processing in the proximal nephron
Authors: Peterson D. R. , Kubillus S. , Binstock W. , Zikos D.
Published: December 2017

To examine the effects of molecular charge on membrane processing in renal tubular cells, the distribution of cationic and anionic ferritin was characterized in microperfused proximal nephron segments. During the first 7 min of proximal tubule perfusion, cationic ferritin was observed 1) bound to the brush-border membrane, 2) in apically positioned vesicles and vacuoles, 3) in lysosomes, 4) in vesicles adjacent to the basolateral plasmalemma, and 5) bound to the basolateral plasmalemma. Compared with anionic ferritin, the distribution of cationic ferritin was characterized by 1) a smaller relative grain density for lysosomes, 2) an accumulation of granules in an enlarged pool of apical cytoplasmic vesicles and vacuoles, and 3) a greater number of granules reaching the basolateral plasmalemma. During incubation directly in the presence of isolated renal cortical microvilli, binding of cationic ferritin increased significantly as pH was lowered from 8.0 to 4.5 and was greater than that of anionic ferritin, which varied little with pH. The data indicate that the molecular charge of endocytosed substances affects routing and membrane processing in proximal tubular cells, suggesting that their membrane-binding characteristics may influence transport patterns.
Immune mediators increase adherence of T-lymphocytes to human lung fibroblasts
Authors: Hampson F. , Monick M. , Peterson M. W. , Hunninghake G. W.
Published: December 2017

The purpose of the present study was to determine whether immunological mediators that are known to be released in the lungs of patients with active sarcoidosis might increase the adherence of human T-lymphocytes to human lung fibroblasts. The studies demonstrate that interleukin 1 (IL-1), tumor necrosis factor (TNF), gamma-interferon (IFN), and interleukin 2 (IL-2) increase the adherence of T-lymphocytes to fibroblasts in a dose- and time-dependent manner. An effect of IL-1, TNF, and IFN was observed at concentrations less than 1 ng/ml; an effect of IL-2 was not observed unless greater than or equal to 60 ng/ml of the mediator was used. The effect of the mediators was primarily on the fibroblasts; however, a significant increase in adherence was also observed when the T-lymphocytes were preincubated with the mediators. These observations suggest that there may be an intimate relationship between the immune response and the fibrotic response in the lungs of patients with pulmonary sarcoidosis or in other disorders where tissue fibrosis is mediated by immunological processes.
Detection of La3+ influx in ventricular cells by indo-1 fluorescence
Authors: Peeters G. A. , Kohmoto O. , Barry W. H.
Published: December 2017

We exposed indo-1-loaded cultured embryonic chick ventricular cells to 0.03-1.0 mM extracellular lanthanum concentration ([La3+]o) and simultaneously measured cell contractile motion and the 410/480 nm fluorescence intensity ratio. After exposure to La3+, ventricular cells stopped contracting and relaxed within seconds, and the 410/480 fluorescence ratio increased. The increase in the 410/480 signal was related to [La3+]o but was not affected by short exposures to zero extracellular calcium concentration ([Ca2+]o) or caffeine, suggesting that the fluorescence was not caused by a La3+-induced increase in intracellular calcium concentration ([Ca2+]i) but rather to increased intracellular lanthanum concentration ([La3+]i). In vitro studies confirmed that indo-1 fluorescence was sensitive to La3+. The increase in [La3+]i in 0.1 mM [La3+]o was directly related to intracellular sodium concentration ([Na+]i), suggesting that La3+ entered cells via Na+-La3+ exchange. In contrast to ventricular cells, which have a functionally distinct Na+-Ca2+ exchange system, exposure of indo-1-loaded cultured bovine endothelial cells to La3+ failed to produce an increase in [La3+]i. These results indicate that exposure of ventricular cells to 0.1-1.0 mM [La3+]o results in a [La3+]i greater than 250 nM within 1 min. Therefore, changes in myocardial 45Ca2+ fluxes and contents induced by La3+ cannot be ascribed solely to extracellular La3+ effects.
Molecular characterization of rat skeletal muscle myosin light chain kinase
Authors: Herring B. P. , Nunnally M. H. , Gallagher P. J. , Stull J. T.
Published: December 2017

A 1.85-kilobase (kb) cDNA has been isolated that encodes the catalytic and calmodulin binding domains of rat skeletal muscle myosin light chain kinase. The cDNA hybridized to a 3.3-kb RNA present in fast- and slow-twitch skeletal muscles. The reported enzymatic activity (3-fold greater in fast- than slow-twitch skeletal muscles) reflects the relative abundance of this RNA in the two types of skeletal muscle. No hybridization of the cDNA was detected to RNA isolated from smooth or nonmuscle tissues. The clone cross hybridized to a 2.2-kb RNA present in cardiac tissue. Ribonuclease protection analysis of skeletal and cardiac muscle RNA revealed major differences in the two hybridizing RNAs. Thus rat skeletal muscle contains a single myosin light chain kinase isoform, which is distinct from the cardiac, smooth, and nonmuscle forms.
Homologous rat hepatic protease inhibitor genes show divergent functional responses to inflammation
Authors: Schwarzenberg S. J. , Yoon J. B. , Sharp H. L. , Seelig S.
Published: December 2017

The genes encoding three distinct serine protease inhibitors (Spi) have been cloned from rat liver. These inhibitors are highly homologous with each other and are similar to alpha 1-antitrypsin at the nucleic and amino acid sequence level. Although previous investigators have examined the regulation of the Spi 2 locus by inflammation, the use of various techniques and the complexity of this genetic locus have led to incomplete and somewhat confusing results. Oligonucleotide probes specific for Spi 2.1, Spi 2.2, Spi 2.3, and a 3' mouse cDNA probe for alpha 1-antitrypsin mRNA were used to measure these mRNA after induction of inflammation with subcutaneous turpentine in Fischer rats. alpha 1-Antitrypsin mRNA increased 1.8-fold, and Spi 2.2 increased 7-fold. In contrast, Spi 2.1 and 2.3 mRNA sequences decreased fourfold. The maximal changes occurred between 24 and 48 h after inflammation, with a gradual return toward normal over the next 4 days. Since Spi 2.1, Spi 2.3, and alpha 1-antitrypsin mRNA sequences are responsive to growth hormone, two other growth hormone-responsive mRNA sequences, alpha 2u-globulin and insulin-like growth factor I, were measured, and they also decreased after induction of inflammation. The results of this study show that, despite a marked similarity of nucleotide sequence, Spi 2.1 and 2.3 genes respond very differently from Spi 2.2 and alpha 1-antitrypsin to both growth hormone and inflammation. We speculate that the functions of Spi 2.1 and 2.3 products are different from those of Spi 2.2 and alpha 1-antitrypsin and may involve the regulation of growth.
Influence of fiber type and muscle source on Ca2+ sensitivity of rat fibers
Authors: Laszewski-Williams B. , Ruff R. L. , Gordon A. M.
Published: December 2017

This study investigated the influence of muscle source and fiber type on the calcium sensitivity of skinned rat skeletal muscle fibers from predominantly slow muscles [soleus (SOL) and adductor longus (AL)], mixed muscle [posterior gracilis (PG)], and predominantly fast-twitch muscle [extensor digitorum longus (EDL)]. Fibers were characterized histochemically and by one-dimensional protein gel electrophoresis, and calcium-tension relationships were determined. Fiber type and muscle source had significant effects on the negative log of the calcium concentration associated with half-maximal tension (pCa1/2). Slow-twitch fibers had larger values of pCa1/2 than did fast-twitch fibers. Slow-twitch fibers from the predominantly slow muscles, SOL and AL, had similar values of pCa1/2 but slightly smaller values than from the mixed muscle, PG. Fast-glycolytic (FG) fibers from the predominantly fast muscle, EDL, had a higher pCa1/2 than fibers from the mixed fiber type muscle, PG. There were no differences between the pCa1/2 associated with FG and fast-oxidative-glycolytic fibers.
Interactions of TPA and insulin on Na+ transport across frog skin
Authors: Civan M. M. , Peterson-Yantorno K. , George K. , O'Brien T. G.
Published: December 2017

The phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) activates protein kinase C (PKC) and produces an early stimulation of Na+ transport across frog skin. The ionic basis for this stimulation was studied with combined transepithelial and intracellular electrical measurements. In an initial series of experiments, TPA approximately doubled the amiloride-sensitive short-circuit current (ISC), apical Na+ permeability (PapNa), and apical membrane conductance without affecting the basolateral membrane conductance. The apical effects led to a marked depolarization of the short-circuited skin and a small increase in intracellular Na+ concentration. TPAs increase of PapNa was sufficient to explain the stimulation of basolateral Na+ transport when both the voltage and substrate dependence of the pump were taken into account. After the early stimulation, TPA later depressed ISC. Added at this point (congruent to 1-2 h after TPA administration), insulin had no effect on ISC, whereas a partial response to vasopressin was still observed. Measured either early or late after TPA addition, the phorbol ester reduced insulin binding by congruent to 40%. Insofar as 60% of the specific binding is retained, the abolishment of insulin's natriferic response is unlikely to result from the TPA-induced reduction in hormonal binding. The data provide further support for the concept that activation of PKC produces an early stimulation of Na+ transport by increasing apical Na+ permeability, and that part of insulin's natriferic effect may be mediated by PKC activation.
Matrix-derived soluble components influence type II pneumocytes in primary culture
Authors: Rannels S. R. , Grove R. N. , Rannels D. E.
Published: December 2017

Type II pulmonary epithelial cells cultured on a plastic surface fail to retain differentiated form and function. During the first 3 days in primary culture, the cells flatten and lose characteristic lamellar inclusions; they increase in size and exhibit accelerated rates of protein synthesis and thymidine incorporation. These transitions are inhibited markedly if the cells are plated on matrigel (MG), a laminin-rich surface derived from the Englebreth-Holm-Swarm (EHS) sarcoma. Soluble components released from matrigel (MGS) mimic some of the effects of the solid gel. As on a plastic surface, the cells flatten when exposed to MGS during culture. In contrast, MGS inhibits thymidine incorporation and protein synthesis; it is most effective when added early in the culture interval. Direct contact of the cells with the matrigel surface itself is always more effective than maximal MGS activity. The effects of MGS are not reproduced by purified laminin or by transforming growth factor-beta, both of which are present in matrigel. These results indicate that the effects of the solid matrigel surface on cell morphology are caused in part by direct cell-matrix contact but that additional effects, such as decreased DNA synthesis, can be mediated by activity of solubilized gel components. They further provide a model wherein changes in type II cell morphology and function, which typically occur in parallel during primary culture, can be separated experimentally.
Na+-Ca2+ exchange in cardiac sarcolemma: modulation of Ca2+ affinity by exercise
Authors: Tibbits G. F. , Kashihara H. , O'Reilly K.
Published: December 2017

The high activity of the cardiac Na+-Ca2+ exchanger has led to the suggestion that it plays an important role in the regulation of myocardial contractility. We have proposed that exercise training increases stroke volume as a consequence of an enhanced contractility caused by an adaptation in Ca2+ transport across the cardiac plasma membrane (sarcolemma). The present study examined the possibility that the Na+-Ca2+ exchanger in heart muscle is modified in response to training. Sprague-Dawley rats (female, n = 72) were randomly divided into exercise-trained (T) and sedentary control (C) groups. As a result of the 11-wk treadmill-training paradigm, group T had a 7.6% higher (P less than 0.005) heart-to-body weight ratio and a 36% increase (P less than 0.01) in gastrocnemius mitochondrial enzyme activity. Na+-Ca2+ exchange was studied in highly purified sarcolemmal vesicles using rapid-quenching techniques. The absolute initial rate of uptake was significantly higher in T vs. C at calcium concentrations [( Ca2+]) ranging from 10 to 80 microM. This increased uptake appears to be due solely to the fact that the apparent Km of the myocardial Na+-Ca2+ exchanger for Ca2+ was significantly lower in T vs. C (15.7 +/- 1.1 vs. 36.1 +/- 2.6 microM), since the maximum velocity was unchanged. The observed increase in the affinity of the exchanger for Ca2+ is not attributable to group differences in vesicular purity, cross-contamination, or passive Ca2+ efflux. This observation is consistent with observed alterations in sarcolemmal composition in response to exercise training. We propose that the modification of the Na+-Ca2+ exchanger may play an important role in the adaptation of the heart to exercise.
K-independent Na-Cl cotransport in bovine tracheal epithelial cells
Authors: Musch M. W. , Field M.
Published: December 2017

Uptakes of 22Na, 36Cl, and 86Rb into isolated bovine tracheal epithelial cells were measured in the presence and absence of 10 microM bumetanide. Preincubation with ouabain (0.5 mM) did not alter initial rates of Na and Cl uptakes but prolonged from 0.5 or less to 2 min the period in which Na uptake is linear with time. Initial rates of bumetanide-inhibitable Na and Cl uptakes (influxes), measured for 2 min in identical solutions, were similar in magnitude (bumetanide-sensitive Cl influx/bumetanide-sensitive Na influx = 1.2). Omission of K did not affect bumetanide-sensitive Na or Cl influx. Cl influx was not affected by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid. Amiloride (0.1 mM) partially inhibited the bumetanide-insensitive Na influx but had no effect on the bumetanide-sensitive Na influx. Rb influx was not affected by bumetanide but was markedly reduced by ouabain and slightly reduced by Ba, the combination being additive. Half-maximally inhibitory concentration values for inhibition of Cl influx by 4 sulfamoylbenzoic acid derivatives were as follows (in mumol/l): 0.125 benzmetanide; 0.64 bumetanide; 16 piretanide; and 26 furosemide. Affinities of Na and Cl for the bumetanide-inhibitable cotransport process were determined by measuring bumetanide-sensitive Cl influx at varying [Na] and bumetanide-sensitive Na influx at varying [Cl]. Both plots were hyperbolic, and the K0.5 values for Na and Cl were 4.1 and 53.9 mM, respectively. Bumetanide-inhibitable Cl influx was not altered by secretory stimuli (epinephrine, A23187) but was more than doubled by osmotic shrinkage (200 mM mannitol or sucrose).(ABSTRACT TRUNCATED AT 250 WORDS)
Effects of substrate and hypoxia on smooth muscle metabolism and contraction
Authors: Wendt I. R.
Published: December 2017

Suprabasal heat production, oxygen consumption, and lactate production were measured, together with force, in 30-s isometric contractions of longitudinal smooth muscle from rabbit urinary bladder at 27 degrees C. Either glucose or pyruvate was provided as exogenous substrate. Under aerobic conditions with glucose as substrate, force averaged 95 mN/mm2 and heat production 121 mJ/g. Oxygen consumption (0.18 mumol/g) could account for only two-thirds of the total energy expenditure represented as heat production. The remaining one-third was accounted for by aerobic lactate production (0.36 mumol/g). When pyruvate replaced glucose as substrate, both the force developed and the total heat liberated were unchanged. Oxygen consumption, however, increased by approximately 40% (to 0.25 mumol/g) and was able to fully account for the measured heat production. The frequency of spontaneous contractions under aerobic conditions was always reduced in the presence of pyruvate. Under anaerobic conditions force was essentially unaltered, and heat production was only slightly reduced (101 mJ/g) with glucose present. Lactate production increased threefold over that under aerobic conditions. With pyruvate as substrate both force and heat production declined markedly (to less than 5% of the aerobic values). The results indicate that under aerobic conditions and with glucose as substrate, smooth muscle of rabbit urinary bladder generates about one-third of its suprabasal energy requirements through glycolysis and that glycolysis can be further accelerated under anaerobic conditions to provide sufficient energy to sustain contraction. If pyruvate replaces glucose as substrate, the metabolism shifts to being virtually all oxidative, and contraction can no longer be sustained in the absence of oxygen.
Effect of loop diuretics on bullfrog cornea epithelium
Authors: Nagel W. , Carrasquer G.
Published: December 2017

The effect of loop diuretics on Cl transport was studied on an in vitro preparation of the bullfrog cornea. Bumetanide (10(-4) M) or furosemide (10(-3) M) added to the stromal solution decreased Cl transport measured as the short-circuit current (Isc) to values near zero. Concomitantly, transepithelial conductance (gt) decreased, whereas the intracellular potential (Vo) hyperpolarized and the fractional resistance of the apical membrane (fRo) increased. Substitution of SO4 for Cl in the tear-side solution led to prompt changes in Isc, gt, Vo, and fRo, characteristic of appreciable passive Cl movement across the apical membrane before and after inhibition. Epinephrine (10(-4) M) was similarly effective on apical membrane conductance in inhibited tissues as under control conditions, but the effective electromotive force for transepithelial Cl transport was reduced to approximately 25%. Intracellular Cl activity, measured with ion-selective microelectrodes, decreased so much that the difference in electrochemical Cl potential divided by the Faraday constant (delta mu Cl/F) was close to zero after inhibition of Isc by bumetanide. Apical Cl permeability remained essentially unchanged. Accordingly, loop diuretics inhibit Cl transport in the Cl-secreting cornea epithelium by blocking the Na-Cl symport without secondary apical effects, as believed for other Cl-reabsorbing epithelia.
Rat vs. rabbit ventricle: Ca flux and intracellular Na assessed by ion-selective microelectrodes
Authors: Shattock M. J. , Bers D. M.
Published: December 2017

Trans sarcolemmal Ca movements in rabbit and rat ventricular muscle were compared using extracellular double-barreled Ca-selective microelectrodes. In rabbit ventricle, steady-state twitches were associated with transient extracellular Ca (Cao) depletions, indicative of Ca uptake during the twitch. In contrast, steady-state twitches in rat ventricle were associated with net cellular Ca extrusion. Rest periods in rabbit ventricle lead to a net loss of cell Ca and resumption of stimulation induces a net uptake of Ca by the cells. Conversely, in rat ventricle rest periods lead to cellular Ca gain and resumption of stimulation induces a net Ca loss from the cells. Thus stimulation is associated with net Ca gain in rabbit ventricle and net Ca loss in rat ventricle. These observations provide an explanation for some of the functional differences between rat and rabbit ventricle (e.g., negative force-frequency staircase and rest potentiation in rat vs. positive staircase and rest decay in rabbit). Resting intracellular Na activity (alpha iNa) was 12.7 +/- 0.6 mM in rat and 7.2 +/- 0.5 mM in rabbit ventricle. This alpha iNa in rat ventricle is sufficiently high that Ca entry via Na-Ca exchange is thermodynamically favored at the resting membrane potential. This may explain why rest potentiation is observed in rat ventricle. In contrast, the lower alpha iNa in rabbit ventricle would favor Ca extrusion via Na-Ca exchange at rest (and consequent rest decay). In rat ventricle, the increase of intracellular [Ca] ([Ca]i) associated with contraction, coupled with the short action potential duration, strongly favor Ca extrusion via Na-Ca exchange and explain the observed Cao accumulation observed during twitches in rat. The high plateau of the rabbit ventricular action potential tends to prevent Ca extrusion via Na-Ca exchange during the contraction and explains the Cao depletions observed in rabbit. It is concluded that the higher alpha iNa and shorter action potential duration in rat vs. rabbit ventricle can explain many of the functional differences observed in these tissues.
Spectrophotometry of b-type cytochromes in rat brain in vivo and in vitro
Authors: Piantadosi C. A.
Published: December 2017

Terminal oxidase inhibitors such as cyanide (CN) and carbon monoxide (CO) produce different absorption changes in the intact brain, suggesting different mitochondrial responses to the inhibitors. In the present study, the nature of the cytochromes involved in CO and CN responses in vivo was investigated by low-temperature spectroscopy of rat brain, frozen in situ, and of preparations of brain homogenate and isolated mitochondria. Comparison of the spectra from different preparations at the high resolution afforded by low-temperature spectroscopy indicated that absorption responses to CO in vivo originated from mitochondrial b cytochromes. Further detailed spectral analysis of mitochondrial preparations revealed three CN-insensitive b cytochromes in nonsynaptic brain mitochondria; one cytochrome could be reduced by succinate in the presence of CN, the second could be reduced by succinate plus ATP, and the third could be reduced only by anaerobiosis. The spectral characteristics of the mitochondrial b cytochromes, when compared with spectra from CO-exposed brain tissue frozen in situ, strongly implicated the energy-dependent cytochrome b in the oxidation-reduction (redox) responses caused by CO in vivo.
Mitogenesis by serum and PDGF is independent of PI degradation and PKC in VSMC
Authors: Kihara M. , Robinson P. J. , Buck S. H. , Dage R. C.
Published: December 2017

Capacities of serum, platelet-derived growth factor (PDGF), and fibroblast growth factor (FGF) on phosphatidylinositol (PI) degradation and cell growth were compared in cultured vascular smooth muscle cells (VSMC) from rat aorta. The role of protein kinase C (PKC) in growth control was also evaluated using polymixin B, a selective inhibitor of PKC. Both dialyzed and nondialyzed fetal bovine serum (FBS) in concentrations from 2 to 20% stimulated [3H]thymidine incorporation into DNA and cell growth without producing corresponding increases in PI turnover. Moreover, both PDGF (40-160 ng/ml) and FGF (6.25-150 ng/ml) also stimulated mitogenesis, but PDGF was more effective although less potent. Mitogenic amounts of PDGF did not stimulate PI turnover, whereas a maximally mitogenic amount of FGF (50 ng/ml) did produce a slight increase. Polymixin B inhibited PKC activity (IC50, 32 microM) from these cells but failed to suppress DNA synthesis produced by 10% FBS or PDGF (50 ng/ml). However, it did suppress that by FGF (50 ng/ml). Angiotensin II (10(-11)-10(-7) M) and phorbol 12,13-dibutyrate (PDB, 1-20 nM) were not mitogenic in the presence or absence of insulin (10 micrograms/ml) or the calcium ionophore A23187 (0.25-4 microM), under serum-free conditions. Instead, PDB inhibited mitogenesis of cells maintained under 0.2% FBS or stimulated with insulin (10 micrograms/ml).(ABSTRACT TRUNCATED AT 250 WORDS)
Stilbene disulfonate blockade of colonic secretory Cl- channels in planar lipid bilayers
Authors: Bridges R. J. , Worrell R. T. , Frizzell R. A. , Benos D. J.
Published: December 2017

We studied blockade by 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS) of a secretory Cl- channel from colonic enterocyte plasma membrane vesicles incorporated into planar lipid bilayer membranes. Except for intermittent long-lived closed periods (100 ms to several min), the control channel open probability (Po) was greater than 90%. DNDS, added to the cis or vesicle-containing side, which corresponds to the outer membrane side of the channel, caused a dramatic increase in the number of current transitions from the open-to-closed state. DNDS caused a concentration-dependent decrease in Po with a maximum inhibition of 95 +/- 2.0% and a half-maximal inhibitory concentration of 3.3 +/- 1.4 microM. DNDS added to the trans side of the channel had no effect on either the single-channel conductance or kinetic behavior of the channel. Kinetic analysis revealed that DNDS blockade from the cis side could be explained by a linear, closed-open-blocked, kinetic scheme. The estimated DNDS block rate constants were kon = 3.2 X 10(7) M-1.s-1 and koff = 52 s-1, yielding an equilibrium dissociation constant (KD) of 2.1 +/- 0.38 microM, similar to the Ki for inhibition of Po. The effects of DNDS were fully reversible after perfusion of the cis compartment with DNDS-free solution. In contrast, the covalently reactive 4,4'-diisothiocyano-substituted stilbene disulfonate caused an irreversible blockade of the Cl- channel.
Isolation and culture of HCO3- -secreting intercalated cells
Authors: Van Adelsberg J. , Edwards J. C. , Herzlinger D. , Cannon C. , Rater M. , al-Awqati Q.
Published: December 2017

Intercalated cells of the distal nephron secrete either H+ or HCO3-. We have succeeded in isolating HCO3- -secreting intercalated cells from the rabbit kidney. When seeded onto collagen-coated permeable supports, these cells form monolayers with a resistance of 595 +/- 75 omega.cm2. The monolayers maintain the characteristics of an epithelium, with apical microvillae and tight junctions. They display the same polarity as do HCO3- -secreting intercalated cells in vivo, namely apical peanut lectin binding and apical Cl- -HCO3- exchange. The monolayers are capable of transepithelial HCO3- transport via this exchanger. The rate of Cl- -dependent transepithelial HCO3- transport is 4 +/- 0.4 nmol.min-1.cm-2. Transepithelial HCO3- transport is completely abolished by 50 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid applied to the apical side of the monolayer. These cultured HCO3- -secreting intercalated cells should prove useful for defining the cellular regulation of HCO3- secretion.
Extracellular Na+ electrode for monitoring net Na+ flux in cell suspensions
Authors: Brady H. R. , Kone B. C. , Gullans S. R.
Published: December 2017

A computer-linked extracellular sodium-sensitive electrode system is described that is suitable for the routine measurement of net Na+ transport in cell suspensions. The commercially available Na+ electrode exhibited high selectivity for Na+ over other cations and a rapid response time (less than 3 s). This system resolved changes of 0.4 mM in the presence of 147 mM extracellular Na+. Measurements of Na+ transport in suspensions of rabbit proximal tubules showed that ouabain caused a dose-dependent net Na+ influx with an inhibitor constant (Ki) of 2.5 +/- 0.2 microM and a maximal velocity (Vmax) of 229 +/- 7 nmol Na+.min-1.mg protein-1. This compared favorably with the ouabain-induced K+ efflux (Ki = 2.4 microM; Vmax = 160 +/- 3.3 nmol K+.min-1.mg protein-1) and the ouabain-induced inhibition of respiration (Ki = 3.3 microM; Vmax = 11.8 nmol O2.min-1.mg protein-1). In addition, Ba2+, a K+ channel blocker known to depolarize the cell, caused a net Na+ efflux, whereas glucose, a Na+-cotransported solute, promoted a net Na+ influx. This system should be a powerful tool for continuous monitoring of net Na+ fluxes in cell suspensions.
The search for the endogenous digitalis: an alternative hypothesis
Authors: Kelly R. A. , Smith T. W.
Published: December 2017

The universal presence of a binding site for cardiac glycosides on Na+-K+-ATPase has engendered speculation as to whether it also serves as a receptor for an endogenous digitalis-like hormone or autacoid. If such a hormone were to exist, it could play a role in sodium homeostasis and in the pathophysiology of primary hypertension and uremia. However, we believe that this hypothesis rests on unproven assumptions. Although typical of many toxins and drugs, binding to a single protein that acts as both its receptor and effector mechanism at the cell membrane, thereby directly affecting transmembrane ion flux, would be unusual for a hormone or autacoid. As an alternative hypothesis for the evolutionary conservation of the cardiac glycoside binding site, we suggest that its endogenous ligand may exist within the cell. After cotranslational insertion of the alpha- and beta-subunits into the membrane of the rough endoplasmic reticulum, Na+-K+-ATPase, like most integral membrane proteins, 1) must be targeted through a complex network of intracellular organelles to the correct plasmalemmal domain, 2) must be monitored for appropriate protein conformation and subunit assembly, and perhaps 3) could have its catalytic function regulated before insertion in the cell membrane. Because the lumina of the endoplasmic reticulum, Golgi, and other organelles and vesicles are topologically equivalent to the outside of the cell, all three functions could be subserved by an intraorganellar ligand for the cardiac glycoside binding site.