bluuebunny/crossref_metadata_2025 huggingface dataset:
alpha-Ketoacids scavenge H2O2 in vitro and in vivo and reduce menadione-induced DNA injury and cytotoxicity
Authors: Nath K. A. , Ngo E. O. , Hebbel R. P. , Croatt A. J. , Zhou B. , Nutter L. M.
Published: December 2017

We demonstrate that alpha-ketoacids reduce and, in some instances, abrogate menadione-induced DNA damage and cytotoxicity in the human breast cancer cell line, MCF7. We confirm that alpha-ketoacids quench the copious amounts of H2O2 generated by menadione while these alpha-ketoacids undergo nonenzymatic oxidative decarboxylation; our data thus support enhanced H2O2 production as an important pathway for menadione-induced DNA damage and cytotoxicity. We also demonstrate that alpha-ketoacids scavenge H2O2 generated by mitochondria and microsomes when these organelles are exposed to menadione; additionally, alpha-ketoacids protect oxidant-vulnerable enzymes against functional impairment induced by H2O2. Finally, we provide the first in vivo demonstration that acute elevations in concentrations of alpha-ketoacids in rat tissues and urine scavenge H2O2. We conclude that enhanced H2O2 production is a major pathway for menadione-induced DNA damage and cytotoxicity and that the diverse alpha-ketoacids present within the cell must be considered, along with glutathione peroxidase and catalase, as part of the intracellular antioxidant defense mechanisms that regulate the ambient levels of H2O2.
Kinetics of [Ca]i decline in cardiac myocytes depend on peak [Ca]i
Authors: Bers D. M. , Berlin J. R.
Published: December 2017

The rate of decline of free intracellular Ca concentration ([Ca]i) is a potentially useful index of the function of Ca transport systems. However, interpretations of these results may depend on multiple Ca transport systems and interaction with intracellular Ca binding sites. We measured [Ca]i in voltage-clamped ventricular myocytes isolated from rat hearts using indo 1 fluorescence. Conditions were chosen where [Ca]i decline was expected to depend almost exclusively on the sarcoplasmic reticulum Ca pump. The half time of [Ca]i decline (t1/2) decreased as the amplitude of the intracellular Ca (Cai) transient increased. This is not the result that would be expected from a transport system where the transport rate is a linear function of free [Ca]i. In this case the time constant of [Ca]i decline (tau) should be independent of the peak value of [Ca]i. This is also true if linear buffering of Cai is included. We develop a simple but more realistic theoretical framework where Ca transport rate and Ca binding both depend on free [Ca]i with Michaelis-Menten type functions. We demonstrate that the observed decline in apparent tau with increasing peak [Ca]i is entirely expected on theoretical grounds and over a wide range of characteristics for Ca transport and binding. We conclude that one cannot draw inferences about the intrinsic Ca transport function based on tau values unless the Cai transient has a comparable size.
Modulation of the delayed rectifier K+ current in neurons by an angiotensin II type 2 receptor fragment
Authors: Kang J. , Richards E. M. , Posner P. , Sumners C.
Published: December 2017

Angiotensin II (ANG II) stimulates the delayed rectifier K+ current (IK) in neurons cultured from rat hypothalamus and brain stem via AT2 receptors, and this effect involves activation of a Gi protein and protein phosphatase 2A (PP2A). However, there was no evidence that the AT2 receptor involved in this response was the same as the recently cloned AT2 receptor. In the present study, intracellular injection of a 22-amino acid peptide (PEP-22) corresponding to the putative third intracellular loop of the cloned AT2 receptor elicited an increase in IK in cultured neurons that was similar to the effect produced by ANG II. Furthermore, this effect of PEP-22 was abolished by pertussis toxin (200 ng/ml, 24 h) pretreatment and also by superfusion of the PP2A inhibitor okadaic acid (10 nM), suggesting the involvement of Gi protein and PP2A, respectively. Intracellular injection of a random peptide or normal pipette solution did not affect neuronal IK. This is direct evidence to link the cloned AT2 receptor to a defined response elicited by ANG II.
Kinetics of 2-deoxyglucose transport in skeletal muscle: effects of insulin and contractions
Authors: Hansen P. , Gulve E. , Gao J. , Schluter J. , Mueckler M. , Holloszy J.
Published: December 2017

There is some controversy regarding whether insulin or contractile activity alters the affinity of skeletal muscle glucose transporters for glucose and its analogues. The effects of insulin and contractions on the kinetics of glucose transport were therefore reexamined in isolated rat skeletal muscles. Concentration-dependent rates of 2-deoxyglucose (2-DG) transport were measured in the absence or presence of insulin (2 mU/ml) in the epitrochlearis and split soleus muscles. The apparent half-maximal saturating substrate concentration (Km) for basal 2-DG transport (approximately 12 mM) was similar for the split soleus and epitrochlearis, and the apparent Km was not changed by insulin in either muscle type. The presence of 2 mU/ml insulin increased the maximal transport velocity (Vmax) approximately fourfold in the epitrochlearis and approximately eightfold in the split soleus. In the epitrochlearis, in vitro muscle contractions also resulted in an approximately fourfold increases in Vmax with no change in apparent Km. The combined effects of insulin and contractions on Vmax were completely additive, but the apparent Km was not different from insulin alone. The apparent Km values for basal and insulin-stimulated glucose transport were further characterized in the epitrochlearis isolated from transgenic mice overexpressing the GLUT-1 isoform in the sarcolemma and their nontransgenic littermates. The apparent Km for basal 2-DG transport in the transgenic muscle (9 mM) was not significantly different from the apparent Km for insulin-stimulated transport in the control muscle (10 mM). The present study provides evidence that insulin and contractions, either alone or in combination, increase glucose transport activity in skeletal muscle by increasing Vmax, with no significant change in Km. Our results also suggest that, in intact skeletal muscle, the Km for basal glucose transport (a process mediated primarily by GLUT-1) is similar to the Km values for stimulated transport, mediated predominantly by GLUT-4.
Thrombin receptor-activating peptide sensitizes the human endothelial thrombin receptor
Authors: Kruse H. J. , Mayerhofer C. , Siess W. , Weber P. C.
Published: December 2017

Receptor-operated effects of alpha-thrombin and of the thrombin receptor-activating peptide TRAP14 on cytoplasmic Ca2+ concentration ([Ca2+]i) were examined in fura 2-loaded endothelial cells. Experiments with hirudin showed that alpha-thrombin-induced Ca2+ influx requires the continuous presence of active alpha-thrombin. YFLLRNP, known to antagonize alpha-thrombin- and TRAP7-induced [Ca2+]i transients in platelets, did not antagonize [Ca2+]i transients in response to alpha-thrombin and TRAP14 in human umbilical vein endothelial cells (HUVEC). Repetitive short-term stimulations with alpha-thrombin desensitized [Ca2+]i transients to subsequent stimulations with either alpha-thrombin or TRAP14. In contrast, repeated short-term stimulations with TRAP14 sensitized [Ca2+]i transients to subsequent stimulations with either agonist. Blockade of Ca2+ influx by SKF-96365 abolished the sensitizing effect of TRAP14. The results indicate distinct characteristics of platelet and endothelial thrombin receptors and suggest that alpha-thrombin and TRAP14 activate the receptor differently. It appears that receptor desensitization occurs independently of TRAP14 binding and, hence, tethered ligand binding to and activation of the receptor. Persistent receptor desensitization after alpha-thrombin seems to depend on both alpha-thrombin binding to the hirudin-like receptor domain and the irreversible proteolytic cleavage of the receptor. It does not involve the TRAP14/tethered ligand binding site of the receptor. TRAP14 primes the receptor by a mechanism mediated by Ca2+ influx.
Nitric oxide donor SIN-1 inhibits mammalian cardiac calcium current through cGMP-dependent protein kinase
Authors: Wahler G. M. , Dollinger S. J.
Published: December 2017

The effect of the nitric oxide (NO) donor SIN-1 (3-morpholino-sydnonimine) on the calcium current (ICa) was examined in guinea pig ventricular myocytes. SIN-1 had little effect on basal ICa. After moderate stimulation of ICa with 10 nM isoproterenol (ISO), 10 microM SIN-1 caused either stimulation or inhibition of ICa; 100 microM SIN-1 consistently caused inhibition. SIN-1 (1-100 microM) inhibited ICa equally following considerable enhancement of ICa by either 1 microM ISO or 100 microM 3-isobutyl-1-methylxanthine, a nonspecific phosphodiesterase (PDE) inhibitor. SIN-1 (100 microM) also inhibited ICa equally following enhancement by either 10 microM pipette adenosine 3',5'-cyclic monophosphate (cAMP) or hydrolysis-resistant 8-bromo-cAMP. Thus the inhibitory effect of SIN-1 appears independent of PDEs. Addition of LY-83583 (a blocker of guanylate cyclase) to the pipette or superfusion with KT-5823 [a blocker of the guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase] suppressed the inhibitory effect of SIN-1. We conclude that NO is an important modulator of beta-adrenergic effects on ICa and that the mechanism of NO inhibition of ICa in mammalian cardiac cells involves the cGMP-dependent protein kinase.
Hormonal regulation, pharmacology, and membrane sorting of vertebrate Na+/H+ exchanger isoforms
Authors: Noel J. , Pouyssegur J.
Published: December 2017

Since the cloning of the first member of the Na+/H+ exchanger (NHE) family, termed NHE1, four NHE isoforms have been cloned (NHE2, NHE3, NHE4, and the trout beta-NHE) and expressed in exchanger-deficient cell lines. All these isoforms exhibit significant identity to NHE1 and possess a similar hydropathy profile with two highly conserved transmembrane segments presumably involved in ion transport. These isoforms are allosterically activated by intracellular H+, regulate intracellular pH in a Na(+)-dependent manner, and are inhibited by amiloride and 5-amino derivatives with distinct Ki values. NHE1 is the amiloride-sensitive, growth factor-activatable, and ubiquitously expressed NHE known to regulate intracellular pH and cellular volume. NHE2, NHE3, and NHE4 are, however, restricted in their tissue distribution, suggesting roles in specialized functions of these epithelial tissues. In this review we present and discuss the most recent advances in the molecular and biochemical features, hormonal and growth factor activation, specific expression, and membrane sorting of the members of this NHE family.
Troponin I isoforms and differential effects of acidic pH on soleus and cardiac myofilaments
Authors: Wattanapermpool J. , Reiser P. J. , Solaro R. J.
Published: December 2017

Differences in pH sensitivity of tension generation between developing and adult cardiac myofilaments, which contain the same isoform of troponin C (TnC), have been proposed to be due to troponin I (TnI) isoform switching from the slow skeletal (ss) to cardiac (c) TnI isoforms (21). We investigated the effects of acidic pH on Ca(2+)-activation of force in chemically skinned preparations of adult rat trabeculae and single soleus fibers that also share the same TnC isoform. Compared with the soleus fibers, trabeculae demonstrated a greater suppression of tension and a rightward shift in pCa50 (-log half-maximally activating molar Ca2+ concentration) when pH was decreased from 7.0 to 6.2. The pH-induced shift in pCa50 in soleus fibers did not change with sarcomere length. Troponin subunit interactions were also investigated, using cardiac troponin C (cTnCIA) labeled with a fluorescent probe, 2-(4'-iodoacetamidoanilino)-naphthalene-6-sulfonic acid. Under acidic conditions, cTnCIA demonstrated a decrease in Ca(2+)-affinity. This decrease was amplified both in the binary complex cTnCIA-cTnI and in the complex cTnCIA-cTnI-cTnT-tropomyosin to the same extent. In contrast, substitution of ssTnI for cTnI in these complexes produced the same decrease in Ca2+ affinity in response to acidic pH as cTnCIA alone. These results support our hypothesis that differential effects of pH on tension generation and Ca2+ sensitivity between soleus fibers and trabeculae are due to the presence of different isoforms of TnI.
Downregulation of the oxytocin receptor on cultured astroglial cells
Authors: Di Scala-Guenot D. , Strosser M. T.
Published: December 2017

Cultured astroglial cells obtained from rat fetal hypothalamus express oxytocin (OT) receptors, which have been previously characterized (Di Scala-Guenot and Strosser. Biochem. J. 284: 491-497, 1992), with a radioiodinated OT antagonist. In these cells, at steady-state binding at 37 degrees C, ice-cold acidic treatment released 10% of the bound ligand; with pronase treatment, 52% of the tracer was released. Because the binding was performed with an antagonist, one could assume that the radiolabeled ligand remains locked into the membrane in a state insensitive to the stripping agents rather than being internalized. Receptor downregulation induced by OT was concentration- and time-dependent, leading to a 72% loss of maximal binding capacity without changing the affinity of the receptor. On removal of OT the binding capacity recovered partially and the restoration process was blocked by monensin (20 microM) but not by cycloheximide (20 micrograms/ml), suggesting involvement of receptor recycling. Concerning the early mechanisms involved in the downregulation processes, uncoupling of the receptor from the G protein and the receptor phosphorylation by protein kinase C could be demonstrated. Treatment of the cells with the OT antagonist d(CH2)5OVT was shown to facilitate radioligand binding and to protect the receptor against OT-induced downregulation.
Smooth muscle stretch-activated phospholipase C activity
Authors: Matsumoto H. , Baron C. B. , Coburn R. F.
Published: December 2017

Rabbit aortic muscles were stretched from a holding length of 0.6 maximum length (Lmax) to lengths as great as 1.0 Lmax and the new length maintained. When muscles were stretched to 1.0 Lmax, inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and inositol 1,4-bisphosphate [Ins(1,4)P2] contents were increased at 375 ms (uncorrected for freezing time) poststretch to 209 +/- 27 and 139.8 +/- 12% (SE), respectively, of control values. Increases in Ins(1,4,5)P3 and Ins(1,4)P2 contents reached an apparent maximum at approximately 500 ms, i.e., to 243.7 +/- 15.8 and 180.9 +/- 16.2% of control, and were decreased to near control levels at 1,700 ms poststretch. The stretch threshold for phospholipase C (PLC) activation was 0.85 Lmax. The latency to onset of PLC activation, correcting for the time for freezing, was 275 to 375 ms. Maximal PLC activity was 91 pmol.s-1.100 nmol total lipid P(i)-1, which corresponded to 10% of total phosphatidylinositol bisphosphate being hydrolyzed per second. The mechanism of stretch-activated PLC activity involved influx of Ca2+ via gadolinium-sensitive ion channels, but not via nifedipine-sensitive ion channels.
Impaired EDRF production by endothelial cells exposed to fibrin monomer and FDP
Authors: Freedman J. E. , Fabian A. , Loscalzo J.
Published: December 2017

Fibrin and fibrinogen and their proteolytic degradation products, found within the atheroma, may contribute to vascular dysfunction. We monitored the production of endothelium-derived relaxing factor (EDRF) by bovine aortic endothelial cells (BAEC) after exposure to fibrinogen, fibrin monomer (FM), and fibrin and fibrinogen degradation products (FDP). Cells incubated with FM and FDP, compared with cells incubated with fibrinogen, were less able to inhibit platelet aggregation, and this effect correlated with a concentration-dependent decrease in EDRF production: BAEC incubated with FM or FDP, but not fibrinogen, produced significantly less nitric oxide (NO), as determined using a photolysis-chemiluminescence system. Northern analysis of BAEC incubated with fibrinogen, FM, or FDP and probed for constitutive bovine endothelial NO synthase mRNA demonstrated decreased expression in cells exposed to FDP or FM. These data show that FM and FDP reduce EDRF produced by BAEC and attenuate constitutive NO synthase expression. These effects may represent a mechanism by which thrombotic determinants adversely affect endothelial function and thereby potentially impair vasomotor responses and contribute to atherothrombogenesis.
Aldosterone stimulation of GTP hydrolysis in membranes from renal epithelia
Authors: Sariban-Sohraby S. , Mies F. , Abramow M. , Fisher R. S.
Published: December 2017

Specific hydrolysis of GTP catalyzed by membranes prepared from A6 epithelial cells grown on porous supports was measured. Aldosterone treatment of the cells for 4 h increased Na+ transport and stimulated GTP hydrolysis by apical membranes in vitro more than twofold over basal levels. This stimulation was attributed to an increase in maximum velocity with little change in Michaelis-Menten constant values. Na+ transport rate and GTP hydrolysis were linearly correlated after aldosterone. This relationship was maintained when aldosterone's response was blunted by various inhibitors. Spironolactone decreased both the hormone-stimulated guanosinetriphosphatase (GTPase) and the Na+ transport rate. Pertussis toxin, which exerted minimal effects on basal rates, reduced the increase of Na+ current normally observed after aldosterone and the hormone stimulation of GTPase activity. The expression of classical Gi/Go-type G proteins was not increased after hormone treatment. When A6 cells were grown on nonporous plastic dishes, aldosterone neither stimulated GTPase activity nor increased amiloride-blockable 22Na+ fluxes. We propose that activation of one or more G proteins in the apical membrane of A6 cells is directly involved in the natriferic action of aldosterone.
Myosin phosphorylation enhances rate of force development in fast-twitch skeletal muscle
Authors: Vandenboom R. , Grange R. W. , Houston M. E.
Published: December 2017

Skeletal muscle force output is regulated through Ca(2+)-mediated alterations of the rate at which cross bridges make the transition from non-force-generating to force-generating states, defined by the rate constant fapp. In skinned-fiber models, phosphate incorporation by the regulatory light chain (R-LC) subunits of myosin increases fapp independent of Ca2+, thus increasing the Ca2+ sensitivity for the rate and extent of steady-state force development. The goal of this study was to determine whether phosphate incorporation by the R-LC subunits of skeletal muscle is related to the maximal rate of isometric force development (+dF/dtmax) in intact muscle. Changes in myosin phosphate content and contractile performance were analyzed at selected times after the application of a 5-Hz 20-s conditioning stimulus (CS) employed specifically to elevate R-LC phosphate content in mouse extensor digitorum longus at 25 degrees C. R-LC phosphate content (in mol phosphate/mol R-LC) increased from 0.13 +/- 0.04 at rest to 0.68 +/- 0.02 20 s after the CS and by 360 s after the CS R-LC phosphate content had decayed to 0.37 +/- 0.06. Values obtained for twitch and tetanic +dF/dtmax after the CS were strongly correlated to R-LC phosphate content (r = 0.97 and 0.96, respectively), suggesting that phosphate incorporation by skeletal myosin R-LC contributes to an enhanced rate of isometric force development in fast-twitch skeletal muscle.
Ammonia permeability of erythrocyte membrane studied by 14N and 15N saturation transfer NMR spectroscopy
Authors: Labotka R. J. , Lundberg P. , Kuchel P. W.
Published: December 2017

The permeability of biological membranes to the rapidly penetrating compound ammonia is extremely difficult to study due to the lack of readily available radionuclides. 14N and 15N saturation transfer nuclear magnetic resonance (NMR) experiments were used to measure the erythrocyte membrane permeability of ammonia under equilibrium exchange conditions. When 14N spectra from erythrocytes suspended in NH4Cl solution were obtained in the presence of the extracellular shift reagent dysprosium tripolyphosphate, intracellular and extracellular ammonia signals were readily resolved. Comparison with 15N spectra from erythrocyte suspensions containing 15N4Cl revealed that the intracellular [14N]ammonia signals were 100% NMR visible. 14N and 15N saturation transfer NMR experiments showed similar influx rates and permeabilities, indicating no loss of saturation transfer due to quadrupolar relaxation of 14N nuclei upon membrane passage. Ammonia influx was directly proportional to concentration (0.39 +/- 0.012 fmol.cell-1.s-1.mM-1 at pH 7.0) and not saturable, which is consistent with passive diffusion. Apparent ammonia permeability increased with pH over the range of pH 6-8 as the fraction of free NH3 increased. However, diffusion through unstirred layers became increasingly rate limiting. The permeability of the unstirred layers (1.1 +/- 0.45 x 10(-3) cm/s) was considerably lower than that of NH3 (0.21 +/- 0.014 cm/s). The Arrhenius activation energy for NH3 permeability was 49.5 +/- 11.8 kJ/mol. No evidence for NH+4 influx over the time domain of these experiments was found.
Metabolism of cyclic ADP-ribose in opossum kidney renal epithelial cells
Authors: Beers K. W. , Chini E. N. , Lee H. C. , Dousa T. P.
Published: December 2017

We have previously shown that NAD+ inhibits renal Na(+)-Pi symport; however, the biochemical mechanism of NAD+ in this action is not clarified. We now propose that NAD+ acts indirectly by first being converted to cyclic ADP-ribose (cADPR), a potent stimulator of intracellular Ca2+ mobilization. In permeabilized opossum kidney (OK) cells, a cell line often employed as a model for study of proximal tubular epithelial transport, cADPR is synthesized from beta-NAD+ in a substrate concentration (0.01-1 mM) and time-dependent manner. That cADPR was generated from beta-NAD+ by OK cells was verified by coelution with authentic cADPR on anion exchange high-performance liquid chromatography and by homologous desensitization of the Ca2+ release bioassay to authentic cADPR. cADPR synthesized by permeabilized OK cells was not influenced by the addition of parathyroid hormone. The OK cell also contains the enzyme activity necessary to catalyze catabolism of cADPR. Identification of these two key enzyme activities of cADPR metabolism in OK cells is consistent with a possible role of cADPR in regulation of the Na(+)-Pi symporter by NAD+ in response to metabolic stimuli. d
Lysine uptake by human placental microvillous membrane: comparison of system y+ with basal membrane
Authors: Furesz T. C. , Moe A. J. , Smith C. H.
Published: December 2017

Transport of lysine by microvillous membranes was investigated by characterization of L-[3H]lysine uptake in membrane vesicles isolated from human placentas. At least one Na(+)-independent system was observed at 22 degrees C and two systems at 37 degrees C. Lysine concentration dependence data were fit by a one- or two-system model with a Michaelis-Menten constant (Km) of 124 +/- 28 microM and a maximum velocity (Vmax) of 33.1 +/- 7.7 pmol.mg protein-1.min-1 at 22 degrees C and with Km values of 1 +/- 0.6 and 245 +/- 51 microM and Vmax values of 0.14 +/- 0.07 and 45.8 +/- 8.7 pmol.mg protein-1.30 s-1 at 37 degrees C. In the presence of N-ethylmaleimide, the uptake (37 degrees C) data were fit by a one-system model with kinetic parameters similar to the lower Km system. Uptake of L-lysine in the absence of Na+ was inhibited completely by L-arginine, L-histidine, and L-homoarginine. In the presence of Na+, uptake was inhibited completely by these same three amino acids and L-leucine but only partially by other neutral amino acids. To compare directly microvillous and basal membrane from the same placenta, we examined the inhibition of 20 microM lysine uptake in the presence of Na+. Inhibition by L-leucine was similar in the two membranes. However, L-homoserine, L-alanine, and L-phenylalanine over a wide concentration range inhibited substantially less in microvillous (at both temperatures) than in basal membrane.(ABSTRACT TRUNCATED AT 250 WORDS)
Analysis of possible mechanisms for in vitro oscillations of insulin secretion
Authors: Maki L. W. , Keizer J.
Published: December 2017

We explore possible kinetic mechanisms responsible for the oscillatory (pulsatile) secretion of insulin observed in vitro when pancreatic islets or islet-derived cells are perifused with glucose. Three primary processes are included: 1) glucose stimulation of insulin secretion, controlled by glucokinase; 2) uptake of glucose through GLUT transporters; and 3) glucose metabolism. Perifusion is approximated as a limiting case of a chemical flow reactor. Using experimentally determined rate laws for the three processes, we examine the effects of exogenous insulin as a phenomenological activator and inhibitor of secretion. The resulting differential equations support oscillations using either direct or indirect inhibition by insulin. The oscillations have many of the characteristics observed in vitro, although the indirect model is in better overall agreement with experiment. We conclude that the mechanisms explored here may help explain insulin oscillations for HIT cells, but not for islets, and predict that oscillations can be modulated by including insulin in the perifusion medium.
Urea activation of K-Cl transport in human erythrocytes
Authors: Kaji D. M. , Gasson C.
Published: December 2017

This report prompted us to examine the effect of urea on K-Cl cotransport in human erythrocytes. In human erythrocytes, urea activated K-Cl cotransport reversibly and in a concentration-dependent manner. Pretreatment with okadaic acid abolished the urea activation of transport, suggesting that exposure to urea resulted in net dephosphorylation of the transporter or a key regulator and that the action of urea was exerted proximal to the phosphorylation-dephosphorylation step. At a concentration of 200 mM, urea activated K-Cl cotransport without any delay, even in the absence of cell swelling. However, with increasing urea concentrations, an appreciable increase in lag time was observed before the final steady-state flux was reached, suggesting that urea inhibits a regulatory kinase. The latter conclusion was also supported by the finding that, at any given urea concentration, the lag time for activation was greater than the lag time for deactivation. Mg depletion activated cotransport, and urea had no additional stimulatory effect in Mg-depleted cells. In urea-pretreated cells, swelling further activated cotransport, but without any measurable delay, in contrast to a time lag of 8 min when control cells (not exposed to urea) were swollen. The latter finding suggests that urea promotes the conversion of transporters from the resting to the partially activated state. These findings raise the possibility that high concentrations of urea in the renal medulla may play a role in the decrease in cell volume that occurs during the maturation of reticulocytes and young erythrocytes, both in normal subjects and in subjects with hemoglobinopathies.
MRF4, Myf-5, and myogenin mRNAs in the adaptive responses of mature rat muscle
Authors: Jacobs-El J. , Zhou M. Y. , Russell B.
Published: December 2017

We studied the possible role of specific muscle regulatory factors (MRF) in the adaptive response to changes in contractile activity in mature skeletal muscle. The tibialis anterior muscle of anesthetized female rats was subjected to low-frequency stimulation, static stretch, or a combination of both. Message levels of MRF were observed after 2 h of activity, and the subsequent 20-h recovery period by slot blot and in situ hybridizations for MRF4, Myf-5, and myogenin. A combination of stimulation and stretch for 2 h increased MRF4 (11.6 +/- 5.3-fold) and Myf-5 (6.6 +/- 1.4-fold). In situ hybridization showed abundance in some regions of the muscle with positive staining near peripheral nuclei of both large and small fibers. Message levels remained high for 30 min and declined to near control levels by 20 h of recovery. Myogenin mRNA levels were unaffected by any manipulations. Neither stretch alone nor 10 Hz of electrical stimulation alone induced a significant increase in MRF. We conclude that myonuclei, and possibly activated myoblasts, increase expression of Myf-5 and MRF4 after a combination of both stimulation and stretch for 2 h.
Urokinase receptor mediates mechanical force transfer across the cell surface
Authors: Wang N. , Planus E. , Pouchelet M. , Fredberg J. J. , Barlovatz-Meimon G.
Published: December 2017

The tripartite complex formed by the urokinase receptor, urokinase, and its inhibitor is an enzymatic system that controls plasmin formation involved in degradation of extracellular matrix proteins. With the use of magnetic twisting cytometry with urokinase-coated ferromagnetic beads, we applied mechanical stress directly to the urokinase receptor on the surface of human myogenic cells in culture. The stiffness and the stiffening response measured through the urokinase receptor resembled those of integrins, which are linked mechanically to the cytoskeleton. Furthermore, stiffness decreased with disruption of actin microfilaments. These results demonstrate that the urokinase receptor is coupled mechanically to the cytoskeleton. Inhibition of the tripartite complex formation with antibodies led to a twofold increase in cytoskeletal stiffness. A stiffened cytoskeleton might impede cytoskeletal remodeling and reorganization and thus impede cell motility. Our results demonstrate that the urokinase receptor mediates mechanical force transfer across the cell surface. As such, it is a novel pathway to regulate cytoskeletal stiffness and, thereby, possibly to modulate motility of normal and abnormal adherent cells.
Adenosine attenuates calcium paradox injury: role of adenosine A1 receptor
Authors: Suleiman J. , Ashraf M.
Published: December 2017

The present study was conducted to test the hypothesis that adenosine attenuates the Ca2+ paradox (PD) injury via stimulation of adenosine A1 receptors linked to Gi proteins in the isolated rat heart. Treatment of adenosine reduced maximum lactate dehydrogenase release and ATP loss compared with regular Ca2+ PD. Recovery of mechanical activity after Ca2+ repletion was observed only in heart treated with adenosine before and during the Ca2+ PD. Significant preservation of myocytes was observed in adenosine-treated hearts compared with the regular Ca2+ PD. Adenosine exerted its effects in a dose-dependent manner, being maximum at 100 microM. The protective effects were mediated by adenosine A1 receptor activation since the adenosine A1 receptor agonist N6-phenylisopropyladenosine provided protection similar to adenosine-treated heart and was blocked by A1 receptor antagonist and pertussis toxin. This study suggests that protection by adenosine against the lethal injury of the Ca2+ PD is mediated by adenosine A1 receptor and a pertussis toxin-sensitive inhibiting G protein.
UVB radiation-activated genes induced by transcriptional and posttranscriptional mechanisms in rat keratinocytes
Authors: Rosen C. F. , Poon R. , Drucker D. J.
Published: December 2017

The mechanisms utilized by ultraviolet B (UVB) radiation in the regulation of gene expression, as well as the genetic targets for transmission of the UVB signal, remain incompletely understood. To elucidate the mechanisms and targets for UVB activation in mammalian cells, we screened a keratinocyte cDNA library with differentially subtracted UVB-enriched cDNA probes. Twenty-three UVB-induced cDNA clones were isolated, including cDNAs for keratin, elongation factor-1 alpha, ferritin heavy chain, thioltransferase, cyclin G, cornifin, cellubrevin, poly(A) binding protein, and the surfeit locus. The temporal kinetics of maximal RNA induction following UVB exposure were heterogeneous, varying from 1 to 24 h post-UVB radiation. Analysis of the regulation of gene expression demonstrated that the levels of most UVB-induced mRNAs were also independently induced by serum and cycloheximide, features previously described for genes induced by DNA damage and members of the immediate early gene family. In contrast to results from studies of immediate early genes, treatment of keratinocytes with both serum and cycloheximide resulted in superinduction of only one mRNA transcript. Nuclear run-on assays demonstrated that UVB radiation increased the transcription rate in 8 of 23 genes, suggesting that UVB radiation utilizes both transcriptional and posttranscriptional mechanisms for the modulation of keratinocyte gene expression. The identification of a group of UVB-inducible keratinocyte genes should prove useful for the characterization of the genomic response to UVB radiation and the analysis of the molecular mechanisms underlying the UVB regulation of gene expression.
Muscarinic suppression of ATP-sensitive K+ channel in rabbit esophageal smooth muscle
Authors: Hatakeyama N. , Wang Q. , Goyal R. K. , Akbarali H. I.
Published: December 2017

Smooth muscle cells from the rabbit esophageal muscularis mucosae were studied for the presence of ATP-sensitive K+ channel (KATP) and its inhibition by carbachol. Lemakalim (10 microM), a synthetic K+ channel opener, increased whole cell currents by -174 +/- 15 pA with 0.1 mM intracellular ATP concentration ([ATP]i) and -70 +/- 11 pA with 5 mM [ATP]i. Glibenclamide (10 microM) completely abolished the lemakalim-induced currents. These currents were therefore denoted as KATP. Carbachol (10 microM) suppressed KATP by 74 +/- 4% with 10 mM intracellular ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) concentration and 100% when EGTA was omitted from the pipette solution. Carbachol suppression was attenuated to 23 +/- 16% by the M3 receptor antagonist, p-flurohexahydrosiladifenidol (0.1 microM). KATP was also suppressed by phorbol 12-myristate 13-acetate (PMA; 100 nM) by 63 +/- 9%. The effects of both PMA and carbachol were significantly reduced by inhibitors of protein kinase C and tyrosine kinase. These results suggest that carbachol suppression of KATP is via M3 receptor subtype and the signaling pathway involves Ca2+, protein kinase C, and tyrosine kinase.
Norepinephrine and ANG II stimulate secretion of TGF-beta by neonatal rat cardiac fibroblasts in vitro
Authors: Fisher S. A. , Absher M.
Published: December 2017

Transforming growth factor-beta (TGF-beta) is a ubiquitous growth-regulating protein that is capable of influencing the growth and function of heart cells in vitro. To better understand the role TGF-beta might play as a paracrine mediator of cardiac hypertrophy, the expression, secretion, and growth effects of TGF-beta were examined. Neonatal cardiac fibroblasts in vitro secreted latent TGF-beta 1 and TGF-beta 2 as high as 15 ng/10(6) cells. Angiotensin II (ANG II) and norepinephrine (NE) each augmented up to threefold the expression and secretion of latent TGF-beta 1 and TGF-beta 2 and also induced a shift in isoform predominance from beta 1 to beta 2. Each agent individually produced hypertrophic growth of neonatal cardiocytes and hyperplastic growth of cardiac fibroblasts. Paradoxically, the combination of NE and ANG II at intermediate and high concentrations resulted in less TGF-beta secretion (compared with either agent alone) and in hypertrophic growth of fibroblasts. These results suggest that the growth-promoting effects of ANG II and NE may in part be mediated via a paracrine stimulation of TGF-beta secretion.
Cell-matrix interaction in bone: type I collagen modulates signal transduction in osteoblast-like cells
Authors: Green J. , Schotland S. , Stauber D. J. , Kleeman C. R. , Clemens T. L.
Published: December 2017

Cell interaction with extracellular matrix (ECM) modulates cell growth and differentiation. By using in vitro culture systems, we tested the effect of type I collagen (Coll-I) on signal transduction mechanisms in the osteosarcoma cell line UMR-106 and in primary cultures from neonatal rat calvariae. Cells were cultured for 72 h on Coll-I gel matrix and compared with control cells plated on plastic surfaces. Agonist-dependent and voltage-dependent rises in cytosolic Ca2+ concentration ([Ca2+]i; measured by fura 2 fluorometry) were significantly blunted in cells cultured on Coll-I compared with cells grown on plastic. In UMR-106 cells, the collagen matrix effect was mimicked by 24-h incubation with soluble Coll-I or short peptides containing the arginine-glycine-aspartate motif. Accumulation of cellular adenosine 3',5'-cyclic monophosphate (cAMP) stimulated by parathyroid hormone, cholera toxin, and forskolin was augmented (50-150%) in cells plated on Coll-I vs. control. The collagen effect on both [Ca2+]i- and adenylate cyclase-signaling pathways in UMR-106 cells was abrogated in the presence of protein kinase C (PKC) depletion or inhibition. Also, Coll-I induced a twofold increase in membrane-bound PKC without changing cytosolic PKC activity. Thus, by altering PKC activity, Coll-I modulates the [Ca2+]i- and cAMP-signaling pathways in osteoblasts. This, in turn, may influence bone remodeling processes.
Caffeine alters cardiac myofilament activity and regulation independently of Ca2+ binding to troponin C
Authors: Powers F. M. , Solaro R. J.
Published: December 2017

We investigated the mechanism by which caffeine influences myofilament responsiveness to Ca2+ by measuring isometric force, Ca2+ binding, and ATPase activity of dog cardiac myofilament proteins. Caffeine (20 mM) increased submaximal and depressed maximal force in skinned fiber bundles. Although the Ca2+ sensitivity of myofilament activity was increased by caffeine, there was no effect on Ca2+ binding to troponin C (TnC) in skinned fiber bundles. To determine if caffeine altered actin-myosin interaction or affected myosin directly, myofibrillar, actomyosin, and myosin ATPase activities were measured. Maximal Ca(2+)-activated myofibrillar Mg(2+)-ATPase activity was depressed by 20 mM caffeine, whereas submaximal Mg(2+)-ATPase activities were not changed. Actomyosin Mg(2+)-ATPase activity was significantly depressed by caffeine concentrations > or = 15 mM. Myosin Ca(2+)-ATPase activity was depressed by caffeine, whereas Mg(2+)-ATPase and K(EDTA)-ATPase activities were not affected. These data suggest that caffeine affects myofilament function via a mechanism that is independent of TnC-Ca2+ binding but that may involve direct effects on actin-cross-bridge interaction.
Tetramethylammonium is a muscarinic agonist in rat heart
Authors: Kennedy R. H. , Wyeth R. P. , Gerner P. , Liu S. , Fontenot H. J. , Seifen E.
Published: December 2017

Studies were designed to determine if tetramethylammonium (TMA), a quaternary amine that structurally resembles the cationic portion of acetylcholine, can affect cardiac function by acting on muscarinic receptors. Experiments examined effects of this cation on 1) the spontaneous beating rate of right atrial preparations isolated from rats, 2) force of contraction in isoproterenol-treated (0.1 microM) rat papillary muscle, and 3) quinuclidinyl benzilate ([3H]QNB) binding to rat ventricular membranes. TMA elicited concentration-dependent (0.5-50 mM) negative chronotropic and negative inotropic actions that were antagonized by the muscarinic receptor antagonist atropine. Radioligand studies showed that TMA acts as both a competitive and noncompetitive antagonist of [3H]QNB binding; the apparent dissociation constant for [3H]QNB was increased (0.092 +/- 0.025 nM in the absence of TMA; 1.14 +/- 0.204 nM in the presence of 50 mM TMA), whereas binding site density was decreased (148 +/- 26 and 65 +/- 4 fmol/mg in the absence and presence of 50 mM TMA, respectively). These results suggest that extracellular TMA can alter the function of rat heart by stimulating muscarinic receptors. This action should be considered when using this quaternary amine as a cation substitute.
HIV-gp120 activates large-conductance apamin-sensitive potassium channels in rat astrocytes
Authors: Bubien J. K. , Benveniste E. N. , Benos D. J.
Published: December 2017

Central nervous system (CNS) involvement usually occurs in individuals infected with human immunodeficiency virus type 1 (HIV-1). Evidence is now accumulating that neurons and astrocytes may be functionally compromised by exposure to viral components or cellular factors released from HIV-1-infected macrophages and/or microglia. We have previously reported that the HIV coat protein gp120 stimulates Na+/H+ exchange in primary cultured rat astrocytes, which, ultimately, results in the activation of a K+ conductance. In this report we characterize the electrophysiological and biophysical properties of the channels responsible for the gp120-induced increase in K+ conductance. These K+ channels had a relatively large unitary conductance (147 pS), were not gated by voltage, were sensitive to changes in H+ concentration at their cytosolic face, were specifically inhibited by apamin, and were insensitive to charybdotoxin and tetraethylammonium. The activation of these channels by gp120 is referable to cellular alkalinization subsequent to Na+/H+ exchange stimulation; gp120 failed to activate these K+ channels in the absence of external Na+ or in the presence of amiloride, an inhibitor of Na+/H+ exchange. Subsequent K+ loss from the astrocyte into the restricted extracellular space surrounding neurons can then lead to neuronal depolarization, activation of voltage-sensitive Ca2+ channels, and, eventually, cell death. Thus abnormal activation of astrocyte K+ channels by gp120 may contribute to the CNS pathophysiology associated with HIV-1 infection.
A cloned renal epithelial Na+ channel protein displays stretch activation in planar lipid bilayers
Authors: Awayda M. S. , Ismailov I. I. , Berdiev B. K. , Benos D. J.
Published: December 2017

We have previously cloned a bovine renal epithelial channel homologue (alpha-bENaC) belonging to the epithelial Na+ channel (ENaC) family. With the use of a rabbit nuclease-treated in vitro translation system, mRNA coding for alpha-bENaC was translated and the polypeptide products were reconstituted into liposomes. On incorporation into planar lipid bilayers, in vitro-translated alpha-bENaC protein 1) displayed voltage-independent Na+ channel activity with a single-channel conductance of 40 pS, 2) was mechanosensitive in that the single-channel open probability was maximally activated with a hydrostatic pressure gradient of 0.26 mmHg across the bilayer, 3) was blocked by low concentrations of amiloride [apparent inhibitory constant of amiloride (K(i)amil approximately 150 nM], and 4) was cation selective with a Li+:Na+:K+ permselectivity of 2:1:0.14 under nonstretched conditions. These pharmacological and selectivity characteristics were altered to a lower amiloride affinity (K(i)amil > 25 microM) and a lack of monovalent cation selectivity in the presence of a hydrostatic pressure gradient. This observation of stretch activation (SA) of alpha-bENaC was confirmed in dual electrode recordings of heterologously expressed alpha-bENaC whole cell currents in Xenopus oocytes swelled by the injection of 15 nl of a 100 mM KCl solution. We conclude that alpha-bENaC, and by analogy other ENaCs, represent a novel family of cloned SA channels.
Inositol phosphate structural requisites for Ca2+ influx
Authors: DeLisle S. , Mayr G. W. , Welsh M. J.
Published: December 2017

To understand how inositol phosphates (InsP) cause Ca2+ influx, we injected 37 highly purified compounds containing a total of 49 InsP positional isomers into Xenopus oocytes. The eight InsP that stimulated Ca2+ influx were those that had the highest potency at releasing intracellular Ca2+, indicating that their common target was the inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] receptor. To cause Ca2+ influx, these InsP had to be injected in a much higher concentration than the minimal concentration required to release intracellular Ca2+. Such high InsP concentrations could inhibit ongoing oscillatory intracellular Ca2+ release. In addition, we found that InsPs could not elicit further intracellular Ca2+ release during the course of Ca2+ influx. Our data are consistent with the “capacitative Ca2+ entry” hypothesis, which states that InsP stimulate Ca2+ influx by depleting the InsP-sensitive intracellular Ca2+ store. In this context, we would suggest that to deplete the InsP-sensitive intracellular Ca2+ store, InsP may have to be present in a sufficiently high concentration to override the oscillatory Ca(2+)-refilling mechanisms of the stores.
cAMP-independent regulation of CFTR by the actin cytoskeleton
Authors: Prat A. G. , Xiao Y. F. , Ausiello D. A. , Cantiello H. F.
Published: December 2017

Protein kinase A (PKA)-activation of epithelial Na+ channels requires actin filaments. Mouse mammary adenocarcinoma cells expressing the human cystic fibrosis transmembrane conductance regulator (CFTR) or mock transfectants were used to determine whether CFTR is also modulated by the actin cytoskeleton. The actin filament disrupter cytochalasin D (CD; approximately 5 micrograms/ml) readily activated whole cell currents in CFTR but not in mock-transfected (MOCK) cells. Addition of actin to the cytosolic side of quiescent excised inside-out patches of CFTR but not MOCK cells also activated CFTR. The actin-activated Cl- channels (symmetrical Cl-) had a linear conductance of 9.3 pS and were inhibited by diphenylamine-2-carboxylate and monoclonal antibodies raised against CFTR. Channel activity was also blocked by addition of the actin-binding proteins deoxyribonuclease I and filamin. Incubation of CFTR cells with CD (approximately 15 micrograms/ml) for > 6 h prevented CFTR activation by the addition of either 8-bromoadenosine 3',5'-cyclic monophosphate plus forskolin under whole cell conditions or PKA under excised inside-out conditions. However, CFTR activation was restored by subsequent addition of actin. The data indicate that CFTR is regulated by actin filaments whose effect may, in turn, be associated with the PKA-dependent pathway.
MRF4, Myf-5, and myogenin mRNAs in the adaptive responses of mature rat muscle
Authors: Jacobs-El J. , Zhou M. -Y. , Russell B.
Published: December 2017

Pages C1045-C1052: J. Jacobs-El, M.-Y. Zhou, and B. Russell. “ MRF4, Myf-5, and myogenin mRNAs in the adaptive responses of mature rat muscle.” Pages C1049 and C1051, Figs. 4 and 6 were inadvertently switched, although the legends as placed are correct. The correct Figs. 4 and 6 with their legends are as follows.

(See PDF)
Imaging real-time neurite outgrowth and cytoskeletal reorganization with an atomic force microscope
Authors: Lal R. , Drake B. , Blumberg D. , Saner D. R. , Hansma P. K. , Feinstein S. C.
Published: December 2017

An atomic force microscope was used to image the morphology and structural reorganization of rat NIH/3T3 fibroblasts and PC-12 cells growing in petri dishes. NIH/3T3 fibroblasts had a uniform morphology and an extensive cytoskeletal network. Cell thickness varied from approximately 2-3 microns above the nucleus to approximately 20-30 nm over the distal processes, and cytoskeletal fibers as small as 30 nm wide were observed. Imaging over an extended period of time showed a limited degree of cytoskeletal reorganization. Localized force dissection did not induce significant retraction of cellular processes and immediate cell death. Differentiating PC-12 cells with a neuronal phenotype had a nonuniform morphology, abundant cytoskeletal elements, neuritic processes, and growth cones. The cell thickness varied from approximately 5-8 microns over the nucleus to approximately 100-500 nm over the neuritic processes; growth cones approximately 50-700 nm wide and end structures approximately 30-150 nm wide were visible. Repeated imaging showed reorganization of the growth cone, especially the appearance and disappearance of beadlike features and fibrous organization. Thus an atomic force microscope can be used for high-resolution real-time studies of the dynamic subcellular mechanisms that drive cell behavior.