Effects of methylprednisolone on the energy metabolism of quiescent and conA-stimulated thymocytes of the rat

1993 ◽  
Vol 13 (1) ◽  
pp. 41-52 ◽  
Author(s):  
Frank Buttgereit ◽  
Martin D. Brand ◽  
Marianne Müller
Keyword(s):  
Plasma Membrane ◽  
Oxygen Consumption ◽  
Energy Metabolism ◽  
Concanavalin A ◽  
Glucocorticoid Receptors ◽  
High Concentrations ◽  
Subsequent Activation ◽  
Respiratory Stimulation ◽  
Stimulation Of

The short-term effects of high concentrations of Methylprednisolone (MP) on the energy metabolism of quiescent and Concanavalin A-stimulated rat thymocytes were investigated in vitro. Concanavalin A (ConA) stimulated the respiration rate of quiescent thymocytes by 35%. Addition of more than 0.15 mg MP/107 cells to ConA-stimulated cells reversed this respiratory stimulation; in addition, higher concentrations of MP caused a similar progressive decrease in the rate of respiration of both quiescent and ConA-stimulated cells. Similarly, the stimulation of respiration by ConA was greatly reduced in MP-treated cells. MP addition lowered cytoplasmic [Ca2+] and, at high concentrations, abolished the ability of ConA to increase [Ca2+]. Thus MP both reverses and prevents the immediate stimulation of thymocytes by ConA. In quiescent thymocytes, MP strongly inhibited that part of the oxygen consumption used to drive the cycle of Na+ influx across the plasma membrane and Na+ efflux on the Na+K+-ATPase, but did not inhibit oxygen consumption used to drive protein synthesis. In ConA-stimulated thymocytes MP had the same effects and also strongly inhibited oxygen consumption dependent on the cycle of Ca2+ influx across the plasma membrane and Ca2+ efflux on the Ca2+-ATPase, but had little effect on oxygen consumption used to drive RNA and DNA synthesis. These results show that MP prevents cation cycling in thymocytes (either by preventing cation influx or by inhibiting cation pumps) and prevents mitogenic stimulation of the cells. The high MP concentration required and the speed of onset of the effect (lless than 30s) provide strong evidence that these effects of MP are not mediated by glucocorticoid receptors and subsequent activation of gene expression. They may be caused by direct effects of MP on the properties of the plasma membrane. These effects are considered to be, at least partially, responsible for the beneficial results that currently have been obtained using MP megadoses in various clinical situations.

scholarly journals Regulation of interleukin 6 receptor expression in human monocytes and monocyte-derived macrophages. Comparison with the expression in human hepatocytes.

1989 ◽  
Vol 170 (5) ◽  
pp. 1537-1549 ◽  
Author(s):  
J Bauer ◽  
T M Bauer ◽  
T Kalb ◽  
T Taga ◽  
G Lengyel ◽  
...  
Keyword(s):  
Plasma Cells ◽  
Human Peripheral Blood ◽  
Receptor Expression ◽  
Mrna Levels ◽  
Blood Monocytes ◽  
Inflammatory Conditions ◽  
High Concentrations ◽  
Human Primary Hepatocytes ◽  
Stimulation Of

IL-6 is a cytokine with pleiotropic biological functions, including induction of the hepatic acute phase response and differentiation of activated B cells into Ig-secreting plasma cells. We found that human peripheral blood monocytes express the IL-6-R, which is undetectable on the large majority of lymphocytes of healthy individuals. Stimulation of monocytes by endotoxin or IL-1 causes a rapid downregulation of IL-6-R mRNA levels and a concomitant enhancement of IL-6 mRNA expression. IL-6 itself was found to suppress the IL-6-R at high concentrations. A gradual decrease of IL-6-R mRNA levels was observed along in vitro maturation of monocytes into macrophages. We show that downregulation of IL-6-R mRNA levels by IL-1 and IL-6 is monocyte specific, since IL-6-R expression is stimulated by both IL-1 and IL-6 in cultured human primary hepatocytes. Our data indicate that under noninflammatory conditions, monocytes may play a role in binding of trace amounts of circulating IL-6. Repression of monocytic IL-6-R and stimulation of hepatocytic IL-6-R synthesis may represent a shift of the IL-6 tissue targets under inflammatory conditions.

scholarly journals In Vitro Stimulation of Hamster Lymphocytes with Concanavalin A

1972 ◽  
Vol 5 (3) ◽  
pp. 339-345 ◽  
Author(s):  
S. B. Singh ◽  
S. S. Tevethia
Keyword(s):  
Concanavalin A ◽  
Stimulation Of

Effects of endogenous calcium transport inhibitor from heart muscle on the active calcium uptake and passive calcium release properties of sarcoplasmic reticulum

1989 ◽  
Vol 67 (9) ◽  
pp. 999-1006 ◽  
Author(s):  
Njanoor Narayanan ◽  
Philip Bedard ◽  
Trilochan S. Waraich
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Heart Muscle ◽  
Calcium Release ◽  
Membrane Vesicles ◽  
Transport Inhibitor ◽  
Low Concentrations ◽  
Active Calcium ◽  
High Concentrations ◽  
Stimulation Of

In the present study, the effects of the cytosolic Ca2+ transport inhibitor on ATP-dependent Ca2+ uptake by, and unidirectional passive Ca2+ release from, sarcoplassmic reticulum enriched membrane vesicles were examined in parallel experiments to determine whether inhibitor-mediated enhancement in Ca2+ efflux contributes to inhibition of net Ca2+ uptake. When assays were performed at pH 6.8 in the presence of oxalate, low concentrations (<100 μg/mL) of the inhibitor caused substantial inhibition of Ca2+ uptake by SR (28–50%). At this pH, low concentrations of the inhibitor did not cause enhancement of passive Ca2+ release from actively Ca2+-loaded sarcoplasmic reticulum. Under these conditions, high concentrations (>100 μg/mL) of the inhibitor caused stimulation of passive Ca2+ release but to a much lesser extent when compared with the extent of inhibition of active Ca2+ uptake (i.e., twofold greater inhibition of Ca2+ uptake than stimulation of Ca2+ release). When Ca2+ uptake and release assays were carried out at pH 7.4, the Ca2+ release promoting action of the inhibitor became more pronounced, such that the magnitude of enhancement in Ca2+ release at varying concentrations of the inhibitor (20–200 μg/mL) was not markedly different from the magnitude of inhibition of Ca2+ uptake. In the absence of oxalate in the assay medium, inhibition of Ca2+ uptake was observed at alkaline but not acidic pH. These findings imply that the inhibition of Ca2+ uptake observed at pH 6.8 is mainly due to decrease in the rate of active Ca2+ transport into the membrane vesicles rather than stimulation of passive Ca2+ efflux; at alkaline pH (pH 7.4), enhanced Ca2+ efflux contributes substantially, if not exclusively, to the decrease in Ca2+ uptake observed in the presence of the inhibitor. It is suggested that if the cytosolic inhibitor has actions similar to those observed in vitro in intact cardiac muscle, acid–base status of the intracellular fluid would be a major factor influencing the nature of its effects (inhibition of Ca2+ uptake or stimulation of Ca2+ release) on transmembrane Ca2+ fluxes across the sarcoplasmic reticulum.Key words: sarcoplasmic reticulum, Ca2+ uptake, Ca2+ release, endogenous inhibitor, heart muscle.

scholarly journals Antigen-stimulated Activation of Phospholipase D1b by Rac1, ARF6, and PKCα in RBL-2H3 Cells

2002 ◽  
Vol 13 (4) ◽  
pp. 1252-1262 ◽  
Author(s):  
Dale J. Powner ◽  
Matthew N. Hodgkin ◽  
Michael J.O. Wakelam
Keyword(s):  
Plasma Membrane ◽  
Cell Types ◽  
Enzyme Activation ◽  
Dominant Negative ◽  
Ph Domain ◽  
Phosphatidylinositol 3 Kinase ◽  
Stimulation Of ◽  
Phosphatidylinositol 3

Phospholipase D (PLD) activity can be detected in response to many agonists in most cell types; however, the pathway from receptor occupation to enzyme activation remains unclear. In vitro PLD1b activity is phosphatidylinositol 4,5-bisphosphate dependent via an N-terminal PH domain and is stimulated by Rho, ARF, and PKC family proteins, combinations of which cooperatively increase this activity. Here we provide the first evidence for the in vivo regulation of PLD1b at the molecular level. Antigen stimulation of RBL-2H3 cells induces the colocalization of PLD1b with Rac1, ARF6, and PKCα at the plasma membrane in actin-rich structures, simultaneously with cooperatively increasing PLD activity. Activation is both specific and direct because dominant negative mutants of Rac1 and ARF6 inhibit stimulated PLD activity, and surface plasmon resonance reveals that the regulatory proteins bind directly and independently to PLD1b. This also indicates that PLD1b can concurrently interact with a member from each regulator family. Our results show that in contrast to PLD1b's translocation to the plasma membrane, PLD activation is phosphatidylinositol 3-kinase dependent. Therefore, because inactive, dominant negative GTPases do not activate PLD1b, we propose that activation results from phosphatidylinositol 3-kinase–dependent stimulation of Rac1, ARF6, and PKCα.

scholarly journals Differential cytotoxic effects of graphene and graphene oxide on skin keratinocytes

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Marco Pelin ◽  
Laura Fusco ◽  
Verónica León ◽  
Cristina Martín ◽  
Alejandro Criado ◽  
...  
Keyword(s):  
Graphene Oxide ◽  
Plasma Membrane ◽  
Membrane Damage ◽  
Skin Toxicity ◽  
Mitochondrial Activity ◽  
Iodide Uptake ◽  
Cytotoxic Effects ◽  
Plasma Membrane Damage ◽  
High Concentrations

Abstract Impressive properties make graphene-based materials (GBMs) promising tools for nanoelectronics and biomedicine. However, safety concerns need to be cleared before mass production of GBMs starts. As skin, together with lungs, displays the highest exposure to GBMs, it is of fundamental importance to understand what happens when GBMs get in contact with skin cells. The present study was carried out on HaCaT keratinocytes, an in vitro model of skin toxicity, on which the effects of four GBMs were evaluated: a few layer graphene, prepared by ball-milling treatment (FLG), and three samples of graphene oxide (GOs, a research-grade GO1, and two commercial GOs, GO2 and GO3). Even though no significant effects were observed after 24 h, after 72 h the less oxidized compound (FLG) was the less cytotoxic, inducing mitochondrial and plasma-membrane damages with EC50s of 62.8 μg/mL (WST-8 assay) and 45.5 μg/mL (propidium iodide uptake), respectively. By contrast, the largest and most oxidized compound, GO3, was the most cytotoxic, inducing mitochondrial and plasma-membrane damages with EC50s of 5.4 and 2.9 μg/mL, respectively. These results suggest that only high concentrations and long exposure times to FLG and GOs could impair mitochondrial activity associated with plasma membrane damage, suggesting low cytotoxic effects at the skin level.

ALDOSTERONE STIMULATION IN VITRO

1967 ◽  
Vol 54 (1) ◽  
pp. 63-72 ◽  
Author(s):  
Jürg Müller ◽  
W. Joe Weick
Keyword(s):  
Response Curve ◽  
Gel Filtration ◽  
Monovalent Cations ◽  
Deficient Diet ◽  
Rat Serum ◽  
Assay Procedure ◽  
Serum Fractions ◽  
High Concentrations ◽  
Stimulation Of

ABSTRACT The effect of rat serum and serum fractions on biosynthesis of aldosterone, corticosterone and deoxycorticosterone was investigated by a previously described in vitro assay procedure, using adrenal sections from rats which had been kept on a sodium-deficient diet. Addition of small amounts of serum to the incubation medium significantly stimulated aldosterone and deoxycorticosterone production. An almost linear log-dose/response curve was obtained over a 1:100 concentration range. Stimulation of corticosterone biosynthesis was observed only at high concentrations of serum. Whereas most of the aldosterone- and deoxycorticosterone-stimulating activities were dialysable, most of the corticosterone-stimulating activity remained in the non-dialysable fraction. Ion-exchange and gel filtration chromatography indicated that the unknown dialysable aldosterone-stimulating substance was different from the known aldosterone-stimulating agents, i. e. angiotensin II and monovalent cations.

Extreme hyperinsulinemia unmasks insulin’s effect to stimulate protein synthesis in the human forearm

1998 ◽  
Vol 274 (6) ◽  
pp. E1067-E1074 ◽  
Author(s):  
Teresa A. Hillier ◽  
David A. Fryburg ◽  
Linda A. Jahn ◽  
Eugene J. Barrett
Keyword(s):  
Protein Synthesis ◽  
Muscle Protein ◽  
Muscle Protein Synthesis ◽  
In Vivo Studies ◽  
Skeletal Muscle Protein ◽  
High Concentrations ◽  
Arterial Plasma ◽  
Stimulation Of

Insulin clearly stimulates skeletal muscle protein synthesis in vitro. Surprisingly, this effect has been difficult to reproduce in vivo. As in vitro studies have typically used much higher insulin concentrations than in vivo studies, we examined whether these concentration differences could explain the discrepancy between in vitro and in vivo observations. In 14 healthy volunteers, we raised forearm insulin concentrations 1,000-fold above basal levels while maintaining euglycemia for 4 h. Amino acids (AA) were given to either maintain basal arterial ( n = 4) or venous plasma ( n = 6) AA or increment arterial plasma AA by 100% ( n = 4) in the forearm. We measured forearm muscle glucose, lactate, oxygen, phenylalanine balance, and [3H]phenylalanine kinetics at baseline and at 4 h of insulin infusion. Extreme hyperinsulinemia strongly reversed postabsorptive muscle’s phenylalanine balance from a net release to an uptake ( P < 0.001). This marked anabolic effect resulted from a dramatic stimulation of protein synthesis ( P < 0.01) and a modest decline in protein degradation. Furthermore, this effect was seen even when basal arterial or venous aminoacidemia was maintained. With marked hyperinsulinemia, protein synthesis increased further when plasma AA concentrations were also increased ( P< 0.05). Forearm blood flow rose at least twofold with the combined insulin and AA infusion ( P< 0.01), and this was consistent in all groups. These results demonstrate an effect of high concentrations of insulin to markedly stimulate muscle protein synthesis in vivo in adults, even when AA concentrations are not increased. This is similar to prior in vitro reports but distinct from physiological hyperinsulinemia in vivo where stimulation of protein synthesis does not occur. Therefore, the current findings suggest that the differences in insulin concentrations used in prior studies may largely explain the previously reported discrepancy between insulin action on protein synthesis in adult muscle in vivo vs. in vitro.

scholarly journals Ca2+-dependent and Ca2+-independent degradation of phosphatidylinositol in rabbit vas deferens

1981 ◽  
Vol 194 (1) ◽  
pp. 129-136 ◽  
Author(s):  
K Egawa ◽  
B Sacktor ◽  
T Takenawa
Keyword(s):  
Smooth Muscle ◽  
Plasma Membrane ◽  
Vas Deferens ◽  
The Other ◽  
Ionophore A23187 ◽  
K Concentration ◽  
Rabbit Vas Deferens ◽  
Dependent Mechanism ◽  
Stimulation Of

The effects of Ca2+ and acetylcholine on the degradation and synthesis of phosphatidylinositol in rabbit vas deferens was studied in vitro by a pulse–chase technique and by measuring the content of the phospholipid in the tissue. Ca2+-dependent degradation of phosphatidylinositol was found in slices and homogenates prelabelled with myo-[2-3H]inositol. The phosphatidylinositol content of the slices also decreased by a Ca2+-dependent mechanism. On the other hand, removal of intracellular Ca2+ with the ionophore A23187 and EGTA increased the amount of phosphatidylinositol. These results indicate that the intracellular Ca2+ concentration has an important role in regulating the phosphatidylinositol content of the tissue. Increasing the extracellular K+ concentration, which causes an increase in plasma-membrane Ca2+ permeability, did not enhance phosphatidylinositol breakdown nor decrease its tissue content. However, phosphatidylinositol synthesis was clearly inhibited. After stimulation of the smooth muscle with acetylcholine, degradation of phosphatidylinositol was enhanced. Furthermore, the content of phosphatidylinositol in the tissue also decreased. These phenomena were evident even in the absence of Ca2+. The acetylcholine-induced degradation of phosphatidylinositol was blocked by the muscarinic antagonist atropine, but not by the nicotinic antagonist (+)-tubocurarine. The acetylcholine-induced decrease in the phosphatidylinositol content of the tissue led to the compensatory synthesis of phosphatidylinositol. Synthesis was separated from degradation in the same tissue. Compensatory synthesis was inhibited by acetylcholine. The degradation of phosphatidylinositol induced by acetylcholine was not inhibited by 8-bromoguanosine 3′:5′-cyclic monophosphate, indicating that the degradative process was not mediated by an increase in the cyclic nucleotide.

scholarly journals Early plasma-membrane-potential changes during stimulation of lymphocytes by concanavalin A.

1983 ◽  
Vol 210 (3) ◽  
pp. 885-891 ◽  
Author(s):  
S M Felber ◽  
M D Brand
Keyword(s):  
Plasma Membrane ◽  
Membrane Potential ◽  
Concanavalin A ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
K Channel ◽  
Ionophore A23187 ◽  
Resting Cells ◽  
Plasma Membrane Potential ◽  
Stimulation Of

1. We have monitored the plasma-membrane potential of lymphocytes by measuring the accumulation of the lipophilic cation methyltriphenylphosphonium (TPMP+) in the presence of the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). 2. The mitogen concanavalin A causes a decrease in TPMP+ accumulation by pig lymphocytes corresponding to a 3 mV depolarization with 2 1/2 min. Concanavalin A does not alter 86Rb+ uptake in the first 30 min. 3. In contrast concanavalin A increased TPMP+ accumulation and the rate of Rb+ uptake in mouse thymocytes. This is consistent with a previous proposal that the mitogen induces a hyperpolarization of mouse thymocytes as a result of stimulation of a Ca2+-dependent K+ channel. 4. Studies with the calcium ionophore A23187 and quinine (an inhibitor of the Ca2+-dependent K+ channel) suggest that the channel is partially closed in mouse resting thymocytes but is almost fully active in pig resting cells. Thus concanavalin A hyperpolarizes mouse thymocytes by activating the Ca2+-dependent K+ channel but cannot do so in pig lymphocytes because the channel is already maximally activated. 5. The 3mV depolarization of pig cells cannot be explained by a decrease in electrogenic K+ permeability.

The influence of Methylprednisolone on the energy metabolism of Ehrlich ascites tumour cells

1994 ◽  
Vol 14 (6) ◽  
pp. 283-290 ◽  
Author(s):  
Frank Buttgereit ◽  
Marianne Müller ◽  
Karsten Wolbart ◽  
Bernhard Thiele ◽  
Falk Hiepe
Keyword(s):  
Plasma Membrane ◽  
Oxygen Consumption ◽  
Energy Metabolism ◽  
Membrane Permeability ◽  
Plasma Membrane Permeability ◽  
Ehrlich Ascites ◽  
Ehrlich Ascites Tumour ◽  
Cellular Oxygen ◽  
Ascites Tumour Cells ◽  
Ehrlich Ascites Tumour Cells

Using Ehrlich ascites tumour cells, the short-term effects of the therapeutic glucocorticoid Methylprednisolone (MP) on the cellular energy metabolism were studied. ATP-consuming processes involved in the rapid MP effects were identified indirectly from the effects of MP on cellular oxygen consumption related to the inhibition of respiration by selective inhibitors of Ca2+-ATPase and protein synthesis. The effects of MP on plasma membrane permeability for Ca2+ ions and phospholipid turnover were studied directly by using confocal laser scanning microscopy and tracerkinetic measurements, respectively. MP inhibited cellular oxygen consumption, suppressed the inhibitory effect of lanthanum but not that of cycloheximide on oxygen consumption, blocked the [Ca2+]i rise in response to calcium ionophore A 23187, and decreased phospholipid turnover. MP acted instantly in a dose-dependent manner. The observed effects of MP are discussed in relation to the hypothesis that the drug has direct membrane effect affecting plasma membrane permeability and function.

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