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PubMed1992

Methotrexate transport in the human intestine. Evidence for heterogeneity.

The transport of methotrexate (MTX) was investigated in organ-cultured endoscopic biopsy specimens of intestinal mucosa from normal subjects. In biopsy specimens from the proximal small intestine incubated with [3H]MTX (0.1 microM) for 2 hr at pH 5.5, [3H]MTX accumulated in the intracellular fluid to a concentration 3.5-fold higher than that of the medium, but at pH 6.5 and 7.5, the concentration was the same as that of the medium. In biopsy specimens from the cecum incubated under similar conditions, no accumulation against a concentration gradient was found. However, the accumulation of MTX was significantly higher at pH 5.5 than at 7.5. At [MTX] = 0.1 microM, the initial rate of MTX transport in the small intestine was significantly affected by medium pH and was optimal at pH 5.5. The relationship between the initial rate of uptake and medium [H+] was hyperbolic, suggestive of saturability with respect to [H+] with a Km of 132.2 nm [H+], corresponding to a medium pH of 6.88. At medium [MTX] = 10 microM, this effect was abolished. At pH 5.5, the relationship between the initial rate of uptake and medium [MTX] was sigmoidal, suggestive of a positive cooperativity, with napp of 1.8. [MTX] at 0.5 Vmax was 20.37 microM. Folic acid inhibited 37% of MTX flux. At pH 7.5, the relationship between the initial rate of uptake of MTX and medium [MTX] was linear. These data indicate the presence of a proton-dependent active transport of MTX in the human proximal small intestine, which is partially shared with folic acid.

Biological Transport↗
PubMed1988

Calcium: a program in BASIC for calculating the composition of solutions with specified free concentrations of calcium, magnesium and other divalent cations.

A BASIC program is presented which facilitates the formulation of biologically relevant chemical solutions containing specified free concentrations of as many as three divalent metal cations (Ca2+, Mg2+ and the choice of a third divalent cation) at any pH in the presence of as many as three ligands (EGTA, ATP and GTP). The program uses the law of mass action and the absolute stability (association) constants found in the literature to calculate the total concentration of divalent metal cation needed to achieve a desired free concentration. The user enters the pH, the concentrations of the ligands used and the desired free concentrations of the divalent cations. This program was developed for use in a wide range of biological applications, particularly the rapid design of solutions which mimic certain aspects of intracellular fluid.

Adenosine Triphosphate↗
PubMed1994

Indirect fluorescence determination of lactate and pyruvate in single erythrocytes by capillary electrophoresis.

A scheme of using fluorescein as the fluorophore for indirect detection of anions was demonstrated. This system is quite stable at a fluorescein concentration of 100 microM even without any other buffer components. Different injection modes affect the limit of detection (LOD). A LOD of about 20 amol was obtained for lactate under optimal conditions. Lactate and pyruvate in the intracellular fluid of erythrocytes were measured in this manner. The average amounts in a single erythrocyte for lactate and pyruvate are 1.3 and 2.1 fmol, respectively, or a ratio of 1.6 for pyruvate to lactate. Variations of the absolute amounts and the ratios are fairly large among a group of 27 cells examined. This is consistent with the difference of cells in size and composition. Although the migration times changed by up to 20% during a series of runs from the influence of concomitants in the cells, the migration time ratio was maintained around 1.072 with 3% relative standard deviation.

Adult↗
PubMed1975

A pressure vessel model for nerve compression.

This paper suggests that compression block in nerve can be better interpreted in terms of a pressure vessel model for nerve fiber distension that the more common models based upon tubes with more or less rigid walls. In this model resistance to compression is due to the elasticity of the cell membrane at locations where displaced intracellular fluid tends to distend it. Because the stresses in pressure vessels increase with the size of the vessel, the theory predicts that the percentage deformation should be larger in larger diameter fibers.

Mathematics↗
PubMed1992

Competition for transport of amino acids into rat heart: effect of competitors on protein synthesis and degradation.

Transport of the neutral amino acids, 2-(methylamino)isobutyrate (MeAIB) and Phe, was examined in isolated rat hearts perfused by the Langendorff method. Hearts were perfused by recirculating for various time periods buffer containing [14C]-MeAIB or [14C]-Phe plus desired additions. Uptake of MeAIB was linear for approximately 30 minutes; Phe uptake was linear for a maximum of 5 minutes, and reached a steady state after 15 minutes. Km and Vmax for MeAIB were 1.1 +/- 0.03 mmol/L and 37.7 +/- 0.4 pmol/microL intracellular fluid (ICF)/min; values for Phe were 1.8 +/- 0.02 mmol/L and 364 +/- 5 pmol/microL ICF/minute. Uptake of MeAIB (0.2 mmol/L) was reduced 95% in the presence of Ser (10 mmol/L), and less severely by large neutral amino acids ([LNAA], 10 mmol/L) such as Phe and Leu (by 46% and 54%, respectively). Uptake of Phe (0.2 mmol/L) was reduced by LNAA such as Val, Leu, and Ile (by 51%, 78%, and 81%, respectively), or by commercial preparations used in parenteral nutrition, eg, Travasol or Travasol plus extra branched-chain amino acids (BCAA) (Branchamin); Ser had little effect (8% reduction). Insulin in the perfusion medium increased the fractional rate of protein synthesis. Individual BCAA at physiological concentrations (0.2 mmol/L) did not alter the rate of protein synthesis. Branchamin or Travasol plus Branchamin also had no effect on the rate of protein synthesis in heart, but did depress the rate of degradation. These studies suggest that amino acid transport into heart may be affected by normal levels of plasma amino acids, whereas protein synthesis is not.

Amino Acids↗
PubMed1995

Effect of albuterol treatment on subsequent dialytic potassium removal.

End-stage renal disease patients presenting with severe hyperkalemia are frequently treated with albuterol to lower their plasma potassium acutely, until emergent hemodialysis can be initiated. Such treatment stimulates potassium shifts from the extracellular to the intracellular fluid compartments. The resulting reduction of potassium concentration gradient between the blood and dialysate may potentially attenuate the efficacy of potassium removal during the ensuing hemodialysis treatment. To evaluate the effect of prior albuterol treatment on dialytic potassium removal, seven chronic hemodialysis patients were studied prospectively on two separate occasions. In one study the patients received 20 mg nebulized albuterol 30 minutes before dialysis; in the control study, albuterol treatment was omitted. Plasma potassium decreased 30 minutes after albuterol treatment (-0.84 +/- 0.06 mmol/L; P < 0.001) and remained unchanged in the corresponding period of the control experiment. Plasma potassium decreased during dialysis in both experimental protocols, but was significantly lower throughout dialysis in the albuterol study, as compared with the control study. Cumulative dialytic potassium removal was significantly lower following albuterol pretreatment compared with the control experiment (29.0 +/- 5.7 mmol v 49.6 +/- 7.0 mmol; P < 0.001). These observations suggest that acute albuterol therapy in patients with end-stage renal disease may substantially decrease potassium removal in the ensuing hemodialysis session. This may lead to rebound hyperkalemia several hours after the dialysis treatment.

Adult↗
PubMed1986

Carcass minerals in chickens (Gallus domesticus) during growth.

Moisture, sodium, potassium, calcium, phosphate and magnesium were determined in the carcass of male chickens (Gallus domesticus) between the ages of 1 and 70 days. Moisture decreased rapidly during the first 3 days of life, followed by a more moderate decline. Sodium and potassium concentrations decreased with age, but not in a parallel fashion. Some of the changes with age could be explained on the basis of an extra- and intracellular fluid shifts. Calcium and phosphate concentration increased with age, reflecting skeletal calcification. Carcass magnesium concentration increased during the first 3 days of life and did not change significantly thereafter.

Aging↗
PubMed1984

Glucose transport and metabolism in the perfused hindquarters of lean and obese-hyperglycemic (db/db) mice. Effects of insulin and electrical stimulation.

Changes in glucose transport and metabolism in skeletal muscles of the obese-diabetic mice (db/db) was characterized using the perfused mouse hindquarter preparation. Metabolism of [5-3H]glucose, uptake of 3-O-[methyl-3H]glucose (methylglucose) and [2-14C]deoxyglucose (deoxyglucose) was studied under resting, electrically stimulated contracting, and insulin-stimulated conditions. Basal rate of methylglucose uptake was 255 +/- 18 and 180 +/- 9 microliter/15 min per ml intracellular fluid space for lean and db/db mice, respectively. The V- of methylglucose transport was decreased with no change in Km in the db/db mice. Both electrical stimulation and insulin (1/mU/ml) increased methylglucose uptake rate 2-fold in both lean and obese mice. We observed no significant change in insulin sensitivity in the db/db mice in stimulating methylglucose uptake which was subnormal under all conditions. Similar results were obtained using deoxyglucose. Likewise, uptake of glucose and 3H2O production from [5-3H]glucose were significantly reduced, both at rest and during electrically stimulated contraction in the db/db mouse. However, lactate production in the electrically stimulated db/db mouse preparations was not significantly different from that in the lean mice. These data suggest a major contribution from an impaired glucose transport activity to the reduction in glucose metabolism in the db/db mouse skeletal muscle.

3-O-Methylglucose↗
PubMed1984

Electrolytes and plasma volume regulation in hypovolemic shock.

The concept of hypovolemic shock and its effects on plasma volume were described in the 1700s. It has been left for more recent investigators to elucidate the role of plasma volume and its mechanisms of regulation and interaction with the interstitial and intracellular fluid spaces. These interactions are discussed, and a therapeutic regimen of fluid administration based on a clinical classification of hypovolemic hemorrhagic states is presented. The merits and characteristics of the current fluids available for resuscitation are reviewed. The choices of fluid for resuscitation should be made with the overall goal of an efficient and effective restoration of circulating volume without producing excessive, avoidable, potentially harmful distortion of other organ systems and body fluid compartments.

Electrolytes↗
PubMed2006

Formal agent-based modelling of intracellular chemical interactions.

Individual-based or agent-based models have proved useful in a variety of different biological contexts. This paper presents an agent-based model using a formal computational modelling approach to model a crucial biological system--the intracellular NF-kappaB signalling pathway. The pathway is vital to immune response regulation, and is fundamental to basic survival in a range of species. Alterations in pathway regulation underlie many diseases, including atherosclerosis and arthritis. Our modelling of individual molecules, receptors and genes provides a more comprehensive outline of regulatory network mechanisms than previously possible with equation-based approaches. The model has been validated with data obtained from single cell experimental analysis.

Intracellular Fluid↗
PubMed2004

Intracellular pH-dependent peroxynitrite-evoked synergistic death of glucose-deprived astrocytes.

Previously, we reported that glucose-deprived astrocytes were highly vulnerable to peroxynitrite (ONOO-). Here we demonstrate that the increased vulnerability caused by glucose deprivation and ONOO- depends on intracellular pH. The ONOO- releasing reagent 3-morpholinosydnonimine (SIN-1) markedly induced the release of lactate dehydrogenase (LDH, the marker of cytotoxicity) in glucose-deprived astrocytes. Morphological studies and caspase activity assay showed that astrocytes treated together with glucose deprivation and ONOO- died mostly in a necrotic mode. Alkalinization of pH from 7.4 to 7.8 increased LDH release, whereas acidification from pH 7.4 to 7.0 decreased it. However, intracellular pH (pHi), not extracellular pH (pHe), appeared to play a critical role in the synergistic death. Thus, without a change in pHe (7.4) cytosolic acidification by a weak acid salt, sodium acetate, and a Na+/H+ antiporter inhibitor, amiloride, reduced LDH release. In contrast, a weak base, NH4Cl, and a Na+/H+ antiporter stimulator, monensin, increased pHi and greatly enhanced LDH release. The augmented death was found to be due, in part, to the preceding decrease in the level of reduced glutathione, the ONOO- scavenger, and collapse of the mitochondrial transmembrane potential at alkaline pH.

Amiloride↗
PubMed2004

Intracellular trafficking of hormone receptors.

Agonist binding stimulates endocytosis of hormone receptors via vesicular uptake mechanisms. Interactions of the intracellular domains of receptors with specific targeting proteins are crucial for sorting of internalized receptor in endosomes. Some receptors are targeted for very rapid (e.g. beta2-adrenergic receptor) or slower (e.g. AT1 angiotensin receptor) recycling pathways, whereas others are targeted to lysosomes for degradation (e.g. EGF receptor or PAR1 protease-activated receptor). This review discusses the mechanisms involved in these processes, which regulate surface receptor expression and set the stage for intracellular signaling of G protein-coupled and growth factor receptors.

Animals↗
PubMed1997

Calcium waves in a model with a random spatially discrete distribution of Ca2+ release sites.

We study the propagation of intracellular calcium waves in a model that features Ca2+ release from discrete sites in the endoplasmic reticulum membrane and random spatial distribution of these sites. The results of our simulations qualitatively reproduce the experimentally observed behavior of the waves. When the level of the channel activator inositol trisphosphate is low, the wave undergoes fragmentation and eventually vanishes at a finite distance from the region of initiation, a phenomenon we refer to as an abortive wave. With increasing activator concentration, the mean distance of propagation increases. Above a critical level of activator, the wave becomes stable. We show that the heterogeneous distribution of Ca2+ channels is the cause of this phenomenon.

Animals↗
PubMed2000

Involvement of protein kinase C in superoxide anion-induced activation of nuclear factor-kappa B in human endothelial cells.

OBJECTIVE: Nuclear factor-kappa B (NF-kappa B) plays an important role in the regulation of redox-sensitive genes which are related to the pathogenesis of various vascular diseases. Although oxygen free-radicals are known to activate NF-kappa B, the signaling pathway of oxygen free radical-induced NF-kappa B activation remains largely unclear. Thus, this study was performed to examine the possible involvement of protein kinase C (PKC) in the oxygen free radical-induced NF-kappa B activation in human umbilical vein endothelial cells (HUVECs). METHODS: Superoxide anion was generated by xanthine and xanthine oxidase. An electrophoretic mobility shift assay (EMSA) was performed using a kappa B-motif oligonucleotide and nuclear extracts from HUVECs. Immunoblot analysis using an antibody against I kappa B alpha, phosphorylated by I kappa B alpha kinase, or myristoylated alanine-rich C kinase substrate (MARCKS) phosphorylated by protein kinase C was carried out. An NF-kappa B luciferase reporter gene assay was also performed. RESULTS: The treatment of the cells with superoxide anion for 60 min increased the NF-kappa B/DNA binding activity. Immunoblot analysis showed that superoxide anion induced phosphorylation of I kappa B alpha within 10 min. Furthermore, phosphorylation of MARCKS occurred more rapidly than phosphorylation of I kappa B alpha. Pretreatment of the cells with calphostin C (100-400 nmol/l) and chelerythrine chloride (5-10 mumol/l), inhibitors of PKC, abolished the superoxide anion-induced NF-kappa B activation. Down-regulation of endogenous PKC by long-term exposure to phorbol 12-myristate 13-acetate decreased the superoxide anion-induced NF-kappa B activation to a basal level. Superoxide anion induced the luciferase reporter gene and this induction was completely inhibited by calphostin C (200 nmol/l) and 4,5-dihydroxy-1,3-benzene disulfonic acid (tiron). CONCLUSION: These results suggest that PKC is involved in the activation of NF-kappa B by superoxide anion in human endothelial cells.

1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium ↗
PubMed1999

The RAMP2/CRLR complex is a functional adrenomedullin receptor in human endothelial and vascular smooth muscle cells.

Adrenomedullin, a potently hypotensive peptide isolated from human pheochromocytoma, is known to elicit a rise in cAMP levels within mammalian endothelial and smooth muscle cells. Until now, however, little has been known about the adrenomedullin receptor. Recently, a group called receptor activity-modifying proteins that complex with the calcitonin receptor-like receptor, and thereby regulate its transport and ligand specificity, were identified. Here we show that mRNA for both the calcitonin receptor-like receptor and the receptor activity-modifying protein 2, but not the receptor activity-modifying protein 1 or receptor activity-modifying protein 3, are expressed in human endothelial and vascular smooth muscle cells. We also found that adrenomedullin increased cAMP levels in HeLa EBNA and 293 EBNA cells, expressing both the receptor activity-modifying protein 2 and calcitonin receptor-like receptor proteins. Thus, the receptor activity-modifying protein 2/calcitonin receptor-like receptor complex apparently serves as a functional adrenomedullin receptor in human endothelial and vascular smooth muscle cells.

Adrenomedullin↗
PubMed1976

"Anabolic" effects of methandienone in men undergoing athletic training.

After failure to confirm an anabolic action of testosterone and its derivatives in rats, methandienone ('Dianabol', an "anabolic steroid" used by athletes) has been given to 11 athletic men during a course of weight-training, in a double-blind, crossover experiment. The dose of methandienone was 100 mg/day for 6 wk. Body weight and composition, muscular strength and performance, and indices of endocrine function were studied. Compared with the placebo period, on methandienone the subjects gained weight (mean 3-3 kg +/- 0-6 kg) and accumulated a disproportionately large amount of potassium (420+/-68 mmol); the increase in weight was confined to the lean part of the body, and the muscles increased in size. Strength and performance improved over each training period, but not significantly differently on drug and placebo. On the drug, plasma-cortisol concentration and urinary cortisol excretion increased, and plasma-testosterone decreased. Although the weight and body-composition changes may demonstrate an anabolic action of methandienone in man, they may alternatively have been caused by an increase in intracellular fluid, and the question of anabolic action therefore remains open.

Adult↗
PubMed2003

Refilling of cortical calcium stores in Paramecium cells: in situ analysis in correlation with store-operated calcium influx.

This is the first thorough study of refilling of a cortical calcium store in a secretory cell after stimulation in which we combined widely different methodologies. Stimulation of dense-core vesicle ("trichocysts") exocytosis in Paramecium involves a Ca(2+) -influx" superimposed to Ca(2+) -release from cortical stores ("alveolar sacs" (ASs)). In quenched-flow experiments, membrane fusion frequency rose with increasing [Ca(2+)](o) in the medium, from approximately 20-25% at [Ca(2+)](o) < or = 0.25 microM to 100% at [Ca(2+)](o) between 2 and 10 microM, i.e. close to the range of estimated local intracellular [Ca(2+)] during membrane fusion. Next, we analyzed Ca(2+)-specific fluorochrome signals during stimulation under different conditions. Treatment with actin-reactive drugs had no effect on Ca(2+) -signaling. In double trigger experiments, with BAPTA in the second secretagogue application (BAPTA only for stimulation and analysis), the cortical Ca(2+) -signal (due solely to Ca(2+) released from cortical stores) recovered with t(1/2) approximately 65 min. When ASs were analyzed in situ by X-ray microanalysis after different trigger times (+Ca(2+)(o)), t(1/2) for store refilling was similar, approximately 60 min. These values are similar to previously measured 45Ca(2+) -uptake by isolated ASs. In sum we find, (i) exogenous Ca(2+) increases exocytosis/membrane fusion performance with EC(50)=0.7 microM, (ii) Ca(2+) -signaling in this system is not sensitive to actin-reactive drugs, and (iii) refilling of these cortical calcium stores goes on over hours and thus is much slower than expected.

Actins↗
PubMed2002

Peripheral lymphocytes and intracellular cytokines in C282Y homozygous hemochromatosis patients.

BACKGROUND/AIMS: Several abnormalities in the immune status of hereditary hemochromatosis patients have been reported. We evaluated the peripheral blood lymphocytes phenotype and cytokine profile of CD8(+) and CD4(+) T cells in C282Y homozygous hereditary hemochromatosis patients compared to control subjects. METHODS: Peripheral blood lymphocytes from 17 asymptomatic patients and 14 control subjects were analyzed. We determined the distribution of lymphocyte subsets and investigated at single-cell level by flow-cytometry the potential of cytokines production. The frequency of cytokine (interferon gamma, tumor necrosis factor alpha, interleukin 2 (IL-2), IL-4, IL-5, IL-10 and IL-13) producing cells was assessed in total T-lymphocytes, CD3(+)CD8(+) and CD3(+)CD4(+) subsets. RESULTS: The patients studied showed a significant decrease of total lymphocyte count, T CD4(+)CD3(+), CD28(+), CD8(+)CD28(+) lymphocytes and natural killer (NK) CD56(+)CD16(+)CD3(-) cells. The reduction of CD28(+) and CD8(+)CD28(+) lymphocyte count was inversely related to transferrin saturation index. An increase in the ability of T-cells to produce all the cytokines studied and a major increase in IL-4 and IL-10 production in the CD3(+)CD8(+) subset was found. Our results demonstrate that activated Th1 and Th2 lymphocytes coexist in the peripheral blood of hereditary hemochromatosis patients and that T-cytotoxic (Tc) 2 subset is more expanded than in control population. CONCLUSIONS: The association of a decreased number of T CD8(+) cytotoxic lymphocytes and NK cells, and the development of Tc2 cells in asymptomatic C282Y homozygous patients represents an imbalance in their immune function that might contribute to the high incidence of hepatocarcinoma.

Adult↗