PubMed Health⌕ Search

Biomedical subjects

J N Loeb

Publications and source records attributed to J N Loeb.

At least 19 recordsLinked to original sources
PubMed1992

Enhancement of glucose transport in response to inhibition of oxidative metabolism: pre- and posttranslational mechanisms.

Addition of 5 mM sodium azide to Clone 9 cells, a rat liver cell line characterized by intracellular glucose concentrations of less than 10% that of the external medium and limited glycogen stores, results in a 50-80% reduction in cell ATP content within 20 min which then recovers to near-basal levels within 1 h and is subsequently maintained at normal levels for 24 h despite continuing the presence of the inhibitor. Associated with this adaptive response is a striking stimulation of facilitated glucose transport, mediated by the GLUT-1 transporter, that exhibits "early" and "late" phases that appear to be mechanistically different. During the early phase of the response (0-2 h), glucose transport rate is enhanced 12-fold in the absence of any change in cell GLUT-1 or GLUT-1 mRNA content. In contrast, the late phase of the response (8-24 h) is characterized by a further large stimulation of glucose transport (to 1.6 times the 2-h value) that is associated with 2- to 3- and 6- to 10-fold increments in cell GLUT-1 and GLUT-1 mRNA content, respectively. In time course studies an increase in GLUT-1 mRNA content was observed at 4 h and preceded the increment in GLUT-1 which became detectable after 8 h of exposure to azide. A marked induction of GLUT-1 mRNA by azide was also demonstrable in cells incubated in medium containing higher concentrations of glucose (10.6 mM), although the increment was approximately 20% less than when cells were incubated in standard medium (containing 5.6 mM glucose).(ABSTRACT TRUNCATED AT 250 WORDS)

3-O-Methylglucose↗
PubMed1991

Regulation of glucose transport in Clone 9 cells by thyroid hormone.

Triiodothyronine (T3) is found to stimulate cytochalasin B-inhibitable glucose transport in Clone 9 cells, a 'non-transformed' rat liver cell line. After an initial lag period of more than 3 h, glucose transport rate is significantly increased at 6 h and reaches more than 3-times the control rate at 24 h. The enhancement of glucose transport by T3 is due to an increase in transport Vmax and occurs in the absence of a change in either the Km for glucose transport (approximately 3 mM) or the Ki for inhibition of transport by cytochalasin B ((1-2).10(-7) M). Consistent with the observed Ki for cytochalasin B, Northern blot analysis of RNA from control and T3-treated cells employing cDNA probes encoding GTs of the human erythrocyte/rat brain/HepG2 cell transporter (GLUT-1), rat muscle/fat cell transporter (GLUT-4), and rat liver transporter (GLUT-2) types indicates expression of only the GLUT-1 mRNA isoform in these cells. The abundance of GLUT-1 mRNA increases approx. 1.9-fold after 24 h of T3 treatment and is accompanied by an approx. 1.3-fold increase in the abundance of GLUT-1 in whole-cell extracts as demonstrated by Western blot analysis employing a polyclonal antibody directed against the 13 amino acid C-terminal peptide of GLUT-1. The more than 3-fold stimulation of glucose transport at 24 h substantially exceeds the fractional increment in transporter abundance suggesting that, in addition to increasing total GLUT-1 abundance, exposure to T3 may result in a translocation of transporters to the plasma membrane or an activation of pre-existing membrane transporter sites.

3-O-Methylglucose↗
PubMed1990

Stimulation of glucose transport in Clone 9 cells by exposure to alkaline pH.

Incubation of a rat liver cell line (Clone 9) for 2 h at pH 8.5 was found to result in a profound (5- to 8-fold) stimulation of cytochalasin B-inhibitable glucose transport. The enhancement of glucose transport after exposure to elevated external pH (achieved by lowering the CO2 tension in a bicarbonate-containing medium) was demonstrable within 15 min, was half-maximal at pH 8.0, and was near-maximal at pH 8.6. Intracellular pH rose linearly with incremental changes in external pH, from pH 7.45 to 8.6 with a slope of 0.6. The increase in transport activity in response to incubation at alkaline pH was accompanied by a parallel increase in lactate production and persisted for more than an hour after external pH was restored to normal. During the latter period, intracellular glucose concentration (less than 10% of that of the external medium under control conditions) increased greater than 10-fold to approximate that in the extracellular medium. Incubation of these cells at pH 8.5 for 2 h resulted in a complete resistance of cell ATP levels to challenge with 5 mM cyanide, suggesting that the adaptive facilitation of glucose transport was of sufficient magnitude to permit a marked stimulation of glycolytic ATP synthesis on inhibition of oxidative phosphorylation. The enhancement of glucose transport was attributable to an increase in the maximum velocity (Vmax) rather than to any change in the Michaelis constant (Km) for transport and was not prevented by cycloheximide. It is concluded that the marked stimulation of glucose transport resulting from exposure of these "low-glucose" cells to alkaline pH reflects either an increase in the abundance of functional glucose transporters in the plasma membrane or an increase in their catalytic turnover rate.

3-O-Methylglucose↗
PubMed1989

Enhanced glucose transport in response to inhibition of respiration in Clone 9 cells.

An acceleration of ATP synthesis by anaerobic glycolysis provides important compensation for interference with respiration in a variety of cells. Effective compensation for an inhibition of respiration, however, can occur in cells in which glucose entry is rate limiting only if sufficient glucose becomes available through an enhancement of transport. We present here a detailed study of the effects of inhibition of respiration in Clone 9 cells, a continuous cell line characterized by low internal glucose concentrations (less than 10% that of the external medium) and minimal stores of glycogen. Exposure of these cells to 5 mM cyanide results in a 90% fall in cell ATP and a twofold rise in cell Na+ within 20 min. By the end of 1 h, however, there is a 4.5- to 7-fold increase in cytochalasin B-inhibitable glucose transport that is accompanied by a parallel increase in the rate of lactate production, a partial recovery of cell ATP, and no further rise in cell Na+. The acute fall in ATP resulting from a submaximally effective concentration of cyanide (0.5 mM) is moreover followed by a time-dependent recovery of cell ATP to near-normal levels and subsequent resistance to challenge with even 5 mM cyanide. The stimulation of facilitative glucose transport resulting from exposure to cyanide is attributable to an increase in maximal velocity rather than to a change in Km and persists for more than 2 h after removal of the inhibitor. These results demonstrate that, in these cells characterized by low internal glucose concentrations, regulation of glucose entry is of central importance in ATP homeostasis and that a major component of the adaptive response to an inhibition of respiration is a time-dependent increase in glucose transport.

3-O-Methylglucose↗
PubMed1988

Kinetic analysis of Na,K-activated adenosine triphosphatase induced by low external K+ in a rat liver cell line.

Exposure of ARL 15 cells to medium containing reduced concentrations of K+ (0.65 mM) elicited a 50-100% increase in Na,K-ATPase activity. The inhibition by ouabain of both the basal and the induced enzyme conformed to a single-site model (KI = 1 x 10(-4) M). The low K+-induced increment in Na,K-ATPase activity was accompanied by an equivalent increase in the abundance of Na,K-pump sites estimated by ouabain-stabilized ("back-door") phosphorylation, such that the calculated catalytic turnover number of approximately 8000/min was minimally changed. Comparison of the dependence of ouabain-inhibitable K+ uptake on intracellular Na+ and on extracellular K+ concentrations in control and low K+-treated cells revealed no change in the respective half-maximal stimulatory concentrations for these cations, whereas the maximal rate of active K+ uptake in cells exposed to low external K+ increased by nearly 100%. The derived Hill coefficients for active K+ transport rate were also unchanged by the low K+ treatment (i.e. approximately 1.4 for extracellular K+ and 2.6 for intracellular Na+). Na,K-ATPase activity of basal and low K+-induced cells calculated from the measured maximal Na,K transport rate closely approximated the Na,K-ATPase activity measured enzymatically in unfractionated cell lysates under Vmax conditions, suggesting that all or most of the Na,K-ATPase enzymatic units present in both basal and stimulated states are functionally active. Northern blot analysis of RNA isolated from control cells indicated the presence of the Na,K-ATPase alpha-I isoform of the enzyme which increased by nearly 200% following incubation of the cells in low-K+ medium. By contrast, the alpha-II and alpha-III mRNAs were undetectable in either the basal or low K+-stimulated state. These results indicate that the Na,K-ATPase induced by incubation of ARL 15 cells in low-K+ medium is kinetically and functionally indistinguishable from the basal enzyme, and that only the alpha-I isoform is expressed under control and low-K+ conditions.

Animals↗
PubMed1988

Selective induction of high-ouabain-affinity isoform of Na+-K+-ATPase by thyroid hormone.

The administration of thyroid hormone is known to result in an induction of the Na+-K+-adenosinetriphosphatase (Na+-K+-ATPase) in rat skeletal muscle and other thyroid hormone-responsive tissues. Since the Na+-K+-ATPase in a variety of mammalian tissues has recently been reported to exist in at least two forms distinguishable by differing affinities for the inhibitory cardiac glycoside ouabain, we have studied the effects of 3,3',5-triiodo-L-thyronine (T3) treatment on these two forms of the enzyme in rat diaphragm. The inhibition of Na+-K+-ATPase activity in a crude membrane fraction by varying concentrations of ouabain conformed to a biphasic pattern consistent with the presence of two distinct isoforms with inhibition constants (KIs) for ouabain of approximately 10(-7) and 10(-4) M, respectively. Treatment of hypothyroid rats with T3 (50 micrograms/100 g body wt on 3 alternate days) nearly tripled that portion of the Na+-K+-ATPase activity corresponding to the high-ouabain-affinity form (increased by 178 +/- 24%), whereas the enzyme activity corresponding to the low-ouabain-affinity form was only slightly changed (increased by 20 +/- 5%). Measurement of the specific binding of [3H]ouabain to these membranes confirmed the presence of a class of high-affinity ouabain binding sites with a dissociation constant (Kd) of slightly less than 10(-7) M, whose maximal binding capacity was increased by T3 treatment by 185%. The calculated catalytic turnover associated with the high-affinity site was 70-80 molecules ATP hydrolyzed.site-1.s-1 and was unchanged by T3 treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Time course of Na,K transport and other metabolic responses to thyroid hormone in clone 9 cells.

To elucidate the relationship between the stimulation of Na+ and K+ fluxes by thyroid hormone and the induction of the Na,K-ATPase, we performed a detailed comparison of the time courses of these hormonal effects in a rat liver cell line, clone 9. Stimulations of passive K+ efflux, passive Na+ influx, and ouabain-inhibitable K+ uptake were all evident within 6-12 h of exposure of cells to T3 (10(-7) M). The time course of the induction of Na,K-ATPase activity closely paralleled that of the increase in the rate of Na+ and K+ fluxes. The maximal stimulatory effects of T3 on ouabain-inhibitable K+ uptake and Na,K-ATPase activity at 72 h were +49% and +36%, respectively. Intracellular Na+ and K+ contents were virtually unchanged during these increases in ion fluxes and Na,K-ATPase activity, suggesting an efficient homeostatic adaptation to the augmented passive "leak" of Na+ and K+ down their transmembrane concentration gradients. T3 treatment for 72 h was also shown to stimulate both lactate production (+62%) and [3H]2-deoxyglucose uptake (+82%) in these cells. The onset of these effects appeared to precede that of the stimulation of Na+ and K+ fluxes, being detectable at 4 h. Neither these latter effects of T3 nor the stimulation of ouabain-inhibitable K+ uptake could be demonstrated when RNA or protein synthesis was inhibited by actinomycin D or cycloheximide, respectively. It is concluded that in clone 9 cells thyroid hormone causes increases in passive Na+ influx, passive K+ efflux, active Na,K transport, and Na,K-ATPase activity whose time courses are closely parallel.

Animals↗
PubMed1987

Ionic dependence of active Na-K transport: "clamping" of cellular Na+ with monensin.

The Na+ ionophore monensin was used to study the Na+- and K+-dependence of ouabain-inhibitable 86Rb+ uptake in ARL 15 cells, a rat liver cell line. Graded concentrations of monensin rapidly induced incremental elevations of cellular Na+ that were stable for up to 2 h. In experiments in which cellular Na+ was thus "clamped" at various levels, the activation curve for ouabain-inhibitable 86Rb+ uptake as a function of intracellular Na+ was found to be steepest near basal Na+ levels (Hill coefficient approximately equal to 2.4), indicating that these cells can respond to relatively large changes in passive Na+ entry by increasing the race of Na-K pump function with only minimal increases in cellular Na+. Exposure of cells to monensin also permitted examination of the extracellular-K+ dependence of ouabain-inhibitable 86Rb+ uptake in the presence of saturating intracellular Na+ and yielded a Hill coefficient of approximately 1.5. The rate of ATP hydrolysis calculated from measurements of the maximal rate of ouabain-inhibitable 86Rb+ uptake in intact cells was similar to the enzymatic Vmax of the Na+-K+-ATPase in cell lysates, suggesting that the Na+-K+-ATPase activity in these broken-cell preparations closely reflects the functional transport capacity of the Na-K pump.

Animals↗
PubMed1987

Hormone binding and coupled response relationships in systems dependent on the generation of secondary mediators.

The relationship between hormone binding and biological response curves is discussed for instances in which the response is dependent upon the generation of a secondary mediator of hormone action. In the simplest model, where the rate of mediator generation is directly proportional to the level of hormone receptor occupancy, and degradation of the secondary mediator follows first-order kinetics, the form of the biological response curve is identical to that for hormone receptor occupancy, but the curve is displaced to the left such that a half-maximal biologic response occurs at less than 50% hormone receptor occupancy (spare-receptor phenomenon). The same is shown to be the case when the model is iterated to include a sequence of coupled intermediate binding reactions intervening between initial hormone binding and the final biological response. When, in contrast, the degradation of one or more of these coupled intermediates is not strictly first order, but instead shows standard Michaelis-Menten kinetics, the response curve, while again remaining parallel to the curve for hormone binding, can now move to the right of the binding curve such that a high threshold is observed for the biological effect, and a half-maximal response may not occur until the level of hormone receptor occupancy is well over 50%. Some consequences of this model are discussed with special reference to the sensitivity and speed of reversibility of the biological response, implications for responses at pharmacological as opposed to physiological concentrations of hormone, and parallels which can be extended to coupled enzymatic reactions of the Michaelis-Menten type.

Calcium↗
PubMed1986

Stimulation of Na,K-activated adenosine triphosphatase and active transport by low external K+ in a rat liver cell line.

Exposure of ARL 15 cells, an established line from adult rat liver, to concentrations of external K+ below 1 mM caused a rapid fall in intracellular K+ and a corresponding rise in intracellular Na+ that became maximal within 12 h. Upon continued exposure to low external K+, these initial changes were followed by a striking recovery such that, by 24 h, intracellular Na+ and K+ concentrations approached their control values. Concomitant with this recovery, there was a substantial increase in Na,K-ATPase specific activity that was detectable at 12 h and maximal at 24 h. After restoration of the external K+ concentration, the elevated level of enzyme activity showed little change for at least 24 h. In contrast, restoration of external K+ resulted in a rapid rise in intracellular K+ and a fall in Na+ such that within 30 min the Na+/K+ ratio was lower than in control cells. This overshoot, together with a demonstrated increase in active 86Rb+ uptake under "Vmax" conditions, confirms that the enhancement in Na,K-ATPase specific activity in response to low external K+ represents an increase in functional Na,K pumping capacity.

Animals↗
PubMed1986

Stimulation of potassium efflux in rat liver by a low dose of thyroid hormone: evidence for enhanced cation permeability in the absence of Na,K-ATPase induction.

The effects of a low dose of T3 on passive cellular K+ efflux and Na,K-ATPase activity were studied in hypothyroid rat liver. Male Sprague-Dawley rats were rendered hypothyroid by 4 weeks of a low iodine diet with 0.5% NaClO4 added to the drinking water, and for the last 2 weeks of this period received daily sc injections of either T3 (1 microgram/100 g BW) or diluent alone. At the end of this time, both the passive efflux of 86Rb+ (a K+ analog) from liver slices isotopically prelabeled in vitro and Na,K-ATPase activity in liver homogenates were determined. The T3 treatment regimen resulted in a 55% increase in the 86Rb+ efflux rate constant (P less than 0.003), while, in contrast, Na,K-ATPase activity remained unchanged. These results show that T3, even at a low dose, enhances passive K+ efflux from liver slices and that, consistent with previous observations, this enhancement can occur in the absence of any detectable change in the number of Na,K pumps. Since the rate of Na,K pump function appears in general to be limited by the rate of passive cation permeation, rather than by Na,K pump number per se, these observations provide additional evidence that increased cation permeability may play a role in the stimulation of active cation transport by thyroid hormone.

Animals↗
PubMed1986

Stimulation of active Na+ and K+ transport by thyroid hormone in a rat liver cell line: role of enhanced Na+ entry.

A continuous cell line derived from rat liver (ARL 15) has been identified that responds to thyroid hormone with a stimulation of active Na,K transport. Stimulation of ouabain-inhibitable K+ uptake, which is half-maximal at a T3 concentration of 1.4 X 10(-10) M, is accompanied by corresponding increases in passive Na+ influx and in passive K+ efflux. The enhancement of Na+ and K+ fluxes by T3 is shown to be accompanied by smaller and equivalent increases both in enzymatically measured Na,K-ATPase activity and in maximal ouabain-sensitive Na,K transport in the presence of the Na+ ionophore monensin. The demonstration that both passive Na+ influx and passive fractional K+ efflux are simultaneously increased by T3 supports the earlier suggestion that the stimulation of active Na,K transport by thyroid hormone is attributable, at least in part, to an enhancement of rate-limiting passive Na+ and K+ fluxes by an increase in membrane permeability.

Animals↗
PubMed1984

Early enhancement of passive potassium efflux from rat liver by thyroid hormone: relation to induction of Na,K-ATPase.

The effect of thyroid hormone treatment on the passive efflux of 42K+ from rat liver slices was studied to determine whether an increase in permeability might play a role in the known enhancement of active monovalent cation transport and Na,K-ATPase activity induced by thyroid hormone. Initial studies showed that the sc injection of 50 micrograms T3/100 g BW on 3 alternate days resulted in an 82% increase in the 42K+ passive efflux rate constant in liver slices and a 23% increase in the Na,K-ATPase activity of liver homogenates. Subsequent investigation of the relative time courses of these two hormonal effects showed that after a single injection of T3 the rate constant for 42K+ efflux increased within 6 h and reached a plateau between 24 and 48 h. In contrast, Na,K-ATPase activity was not augmented until 24 h and continued to rise between 24 and 48 h. These results suggest that an early increase in cation permeability (with resultant cation leak) may account for the known stimulatory effect of thyroid hormone on active monovalent cation transport in rat liver. The relatively late increase in Na,K-ATPase activity might then be regarded as an adaptive cellular response to increased passive cation fluxes rather than a direct effect of thyroid hormone. The magnitude and early onset of the effect of thyroid hormone treatment on cellular 42K+ efflux raise the possibility that an increase in passive cation permeability may be a proximal event in the mediation of thyroid hormone action.

Animals↗
PubMed1983

The concentration dependence of active K+ transport in the turkey erythrocyte. Hill analysis and evidence for positive cooperativity between ion binding sites.

A mathematical model is presented which describes the theoretical relationship between ligand concentration and physiological response for systems in which the response is dependent upon simultaneous occupancy of two receptor ligand-binding sites. The treatment considers both the possibility of intrinsic differences between the binding sites with regard to ligand affinity, as well as the possibility of mutually induced changes in affinity resulting from allosteric interactions. Unlike the Monod-Wyman-Changeux formulation for allosteric enzymes, the general model put forward here makes double occupancy an absolute requirement for enzymatic function. It is shown that such a model leads to the prediction of a curvilinear Hill plot from which one can obtain an explicit estimate of the degree of allosteric interaction between the two ligand binding sites as well as the Gibbs standard free energy change for the overall binding reaction. It is then shown that, in the specific instance of Na, K-ATPase-mediated K+ transport by the turkey erythrocyte, the configuration of the Hill curve describing the rate of ouabain-sensitive K+ transport as a function of external K+ concentration conforms closely to that predicted by the model described above. The results are of particular interest because they indicate a strongly cooperative interaction between the two K+ binding sites on the transport protein such that occupancy of one site results in an enhancement of the affinity of the other site for K+ by a minimum of 15- to 20-fold. Finally, we consider in detail a model of the Monod-Wyman-Changeux type in which, by contrast, both singly and doubly occupied forms of the enzyme are assumed to be catalytically active, and which we analogously extend to allow for the possibility of asymmetry between the two ligand binding sites. Although it is shown that the two models can not be differentiated from each other in the present experimental system, they yield virtually identical estimates for the degree of positive cooperativity between the two K+ binding sites.

Animals↗
PubMed1983

The influence of hyperthyroidism and hypothyroidism on alpha- and beta-adrenergic receptor systems and adrenergic responsiveness.

A detailed review has been conducted of studies addressing dressing the subject of the influence of thyroid hormone on alpha- and beta-adrenergic receptors and adrenergic responsiveness in a wide range of experimental animals and tissues. The studies summarized in the present article have been restricted to those in which explicit measurements of receptor number were made by the use of appropriate radioligands. Particular emphasis is given to an examination of the relationship between thyroid hormone-induced changes in alpha- and beta-adrenergic receptor number and accompanying changes in adenylate cyclase activity and more distal adrenergic responses. Although in many instances thyroid hormone-induced changes in receptor number are reflected in coordinate changes in adrenergic sensitivity, this is shown to be by no means uniformly the case. In contrasting instances, modifications at other more distal sites in the sequence of events mediating catecholamine hormone action are responsible for biochemical and physiological changes in catecholamine responsiveness induced by thyroid hormone.

Adenylyl Cyclases↗