Glucose transport in isolated fat cells. Procedures for measurement of the intracellular water space.
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Adipose Tissue↗
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Kinetic analysis of the mechanism of action of the multidrug transporter.
To clarify the mechanistic role of PGP (P-glycoprotein) in multidrug transport, we constructed a kinetic model composed of four compartments: (1) the extracellular space; (2) the space in the membrane; (3) the intracellular space; and (4) the pore-like space in the PGP molecule. The kinetics of the concentration of ADM (adriamycin) in each compartment were formulated based on the assumptions that (a) the movement of ADM between two compartments by diffusion is dependent on a dynamic distribution coefficient introduced here, (b) the uptake of ADM into the pore-like structure by the pump mechanism activated by ATP is described by enzyme kinetics, (c) the movement of ADM out of the pore-like structure to the extracellular medium through a valve-like mechanism is also expressed by enzyme kinetics. The mathematical analysis of the exact solution can explain the distinct effects of verapamil and vanadate on the accumulation and release of ADM, where verapamil inhibits the efflux by the valve-like mechanism and vanadate blocks the influx by the pump mechanism. We also performed a numerical calculation with this model for a quantitative explanation and found the valid parameter values to fit the experimental data. These results support the modified hydrophobic vacuum cleaner model.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
23Na and 39K nuclear magnetic resonance studies of perfused rat hearts. Discrimination of intra- and extracellular ions using a shift reagent.
High-resolution 23Na and 39K nuclear magnetic resonance (NMR) spectra of perfused, beating rat hearts have been obtained in the absence and presence of the downfield shift reagent Dy(TTHA)3- in the perfusing medium. Evidence indicates that Dy(TTHA)3- enters essentially all extracellular spaces but does not enter intracellular spaces. It can thus be used to discriminate the resonances of the ions in these spaces. Experiments supporting this conclusion include interventions that inhibit the Na+/K+ pump such as the inclusion of ouabain in and the exclusion of K+ from the perfusing medium. In each of these experiments, a peak corresponding to intracellular sodium increased in intensity. In the latter experiment, the increase was reversed when the concentration of K+ in the perfusing medium was returned to normal. When the concentration of Ca2+ in the perfusing medium was also returned to normal, the previously quiescent heart resumed beating. In the beating heart where the Na+/K+ pump was not inhibited, the intensity of the intracellular Na+ resonance was less than 20% of that expected. Although the data are more sparse, the NMR visibility of the intracellular K+ signal appears to be no more than 20%.
Animals↗
Evidence for an organized lattice in the intercellular space of vestibular sensory cat epithelia.
The intracellular space between sensory hair cells and nerve endings and between supporting cells of the vestibular epithelia in the cat contains a fine network of microstructures arranged obliquely between the cell membranes of adjacent cells. In situ immersion-perfusion fixation, electron microscopy complemented by lanthanum negative-contrast and freeze-fracture methods suggest that these intercellular structures are tubular with an external diameter between 3 and 6 nm and an internal diameter of 1.5 nm. They could serve as intercytoplasmic tunnels. We discuss the evidence concerning the analogy between the present microchannels and the passageways regularly arranged in the intercellular space of gap-junctions which would permit the transport of low molecular weight substances from one cell to other.
Animals↗
Demonstration of fatty acid domains in membranes produced by lipolysis in mouse adipose tissue. A freeze-fracture study.
Fatty acids produced by isoproterenol-stimulated lipolysis in mouse adipose tissue incubated at pH 7.4 formed myelin figures when the tissue was processed at pH 9.0. Myelin figures, visualized with freeze-fracture electron microscopy, were found in intracellular channels of adipocytes, extracellular space, intracellular channels of endothelial cells, and capillary lumen. The E-fracture face of plasma membranes of adipocytes and endothelial cells and intracellular membranes of adipocytes contained areas that were free of particles. These areas, which were continuous with particle-studded areas of the E-fracture faces, were irregular in shape, sometimes circular or oblong, other times long and narrow. The surfaces of particle-free areas were flat, concave, convex, and often corrugated, with multiple folds that sometimes abutted on myelin figures. We conclude that the particle-free areas are composed of partially ionized fatty acids located in the external leaflets of plasma and intracellular membranes of adipocytes and endothelium. They were formed by fatty acids that entered leaflets at pH 9.0, probably from lipolyzed lipid droplets in adipocytes, moved in a continuum of membrane leaflets between and within cells, overcrowded the leaflets, and subsequently produced corrugations and lamellar extensions (myelin figures) of leaflets.
Adipose Tissue↗
Validation of transport measurements in skeletal muscle with N-13 amino acids using a rabbit isolated hindlimb model.
We are studying the transport of C-11 and N-13 labeled amino acids in tumor-bearing rabbits to determine the role of amino acid transport in the pathogenesis of muscle wasting in cancer. To validate a new, in vivo, method for measuring transport in skeletal muscle with these compounds, an isolated hindlimb model was developed in rabbits. The limb was perfused with a non-recirculating, normothermic, constant pressure system and a cell-free perfusate. Hemodynamic and metabolic parameters were measured during the first 75 min. of perfusion and found to remain normal and stable. Flow varied directly with perfusion pressure over the normal range of resting flows in the intact rabbit hindlimb. Time-activity curves (TAC's) were recorded from the medial thigh following bolus co-injection of L-[amide N-13] glutamine or N-13 L-glutamate with Tc-99m human serum albumin (HSA) into the femoral artery. Regional plasma flow was determined from the Tc-99m data. The N-13 TAC's consistently manifested a three-phased washout with half times of approximately 30 sec., 5 min. and 2 hr. Capillary and cellular transport parameters were computed from the N-13 data using a double barrier, single capillary model of capillary and cellular transport and assuming that the three washout components result, respectively, from tracer throughput, extraction into the interstitial space and extraction into the intracellular space. This interpretation was validated and the sensitivity of the technique to transport processes demonstrated by examining the effects on the N-13 TAC's and computed transport parameters of several factors known to influence cellular transport of amino acids, viz., the insulin concentration, amino acid concentration and pH of the perfusate. Time-activity curves and transport parameters for N-13 L-glutamine in the isolated limb were very similar to those observed in the intact rabbit hindlimb, suggesting that studies in the perfused model are indicative of amino acid transport in vivo. The methodology described here is especially well suited for studying the specific effects on transport of factors which influence amino acid metabolism in skeletal muscle (e.g., hormones and monokines).
Amino Acids↗
The electrochemical potential across mycoplasmal membranes.
The electrochemical proton gradient across mycoplasmal membranes was studied. The transmembrane proton-motive potential, delta p, is composed of two parameters, a transmembrane electric potential difference, delta psi, and a transmembrane proton gradient, delta pH, according to the formula delta p = delta psi -(A x delta pH). Membrane potentials were determined with use of potential-sensitive cyanine dyes. The delta psi for both Mycoplasma mycoides subspecies capri and Mycoplasma gallisepticum was -48 mV +/- 10%, with the inside negative; the delta psi of Acholeplasma laidlawii was -28mV +/- 20%. The delta pH was determined by measuring the distribution of [14C]5,5-dimethyl oxazolidine-2,4-dione between the intracellular space and the medium. The intracellular pH of glycolyzing mycoplasmas was generally more alkaline than the extracellular medium: at an external pH of 7.0, the internal pH was 7.4 and hence delta pH = 0.4, a value corresponding to -24 mV. Thus, the delta p of both M. mycoides subspecies capri and M. gallisepticum was calculated to be -72 mV and that of A. laidlawii, to be -52 mV. The data further indicate that the delta p is generated by a membrane-bound electrogenic, proton-translocating adenosine triphosphatase that operates in the direction of hydrolysis of adenosine triphosphatase, which formed by glycolysis, and leads to proton extrusion.
Acholeplasma laidlawii↗
Pharmacokinetic optimisation of the treatment of bacterial central nervous system infections.
Central nervous system (CNS) infections caused by bacteria with reduced sensitivity to antibacterials are an increasing worldwide challenge. In successfully treating these infections the following conditions should be considered: (i) Antibacterials do not distribute homogeneously in the central nervous compartments [cerebrospinal fluid (CSF), extracellular space of the nervous tissue, intracellular space of the neurons, glial cells and leucocytes]. Even within the CSF, after intravenous administration, a ventriculo-lumbar concentration gradient is often observed. (ii) Valid parameters of drug entry into the CSF are the CSF: serum concentration ratio in steady state and the CSF: serum ratio of the area under the concentration-time curves (AUCCSF/AUCS). Frequently, the elimination half-life (t1/2 beta) in CSF is longer than t1/2 beta in serum. (iii) For most antibacterials, lipophilicity, molecular weight and serum protein binding determine the drug entry into the CSF and brain tissue. With an intact blood-CSF and blood-brain barrier, the entry of hydrophilic antibacterials (beta-lactam antibacterials, glycopeptides) into the CNS compartments is poor and increases during meningeal inflammation. More lipophilic compounds [metronidazole, quinolones, rifampicin (rifampin) and chloramphenicol] are less dependent on the function of the blood-CSF and blood-brain barrier. (iv) Determination of the minimal inhibitory concentrations (MIC) of the causative organism is necessary for optimisation of treatment. (v) For rapid sterilisation of CSF, drug concentrations of at least 10 times MIC are required. The minimum CSF concentration: MIC ratio ensuring successful therapy is unknown. Strategies to achieve optimum antibacterial concentrations in the presence of minor disturbances of the blood-CSF and blood-brain barrier include, the increased use of low toxicity antibacterials (e.g., beta-lactam antibiotics), the use of moderately lipophilic compounds, and the combination of intravenous and intraventricular administration. Antibacterials which do not interfere with bacterial cell wall synthesis delay and/or decrease the liberation of proinflammatory bacterial products, delay or inhibit tumour necrosis factor release, and may reduce brain oedema in experimental meningitis. Conclusive evidence of the reduction of neuronal damage by this approach, however, is lacking.
Anti-Bacterial Agents↗
Guidelines for the treatment of acidaemia with THAM.
THAM (trometamol; tris-hydroxymethyl aminomethane) is a biologically inert amino alcohol of low toxicity, which buffers carbon dioxide and acids in vitro and in vivo. At 37 degrees C, the pK (the pH at which the weak conjugate acid or base in the solution is 50% ionised) of THAM is 7.8, making it a more effective buffer than bicarbonate in the physiological range of blood pH. THAM is a proton acceptor with a stoichiometric equivalence of titrating 1 proton per molecule. In vivo, THAM supplements the buffering capacity of the blood bicarbonate system, accepting a proton, generating bicarbonate and decreasing the partial pressure of carbon dioxide in arterial blood (paCO2). It rapidly distributes through the extracellular space and slowly penetrates the intracellular space, except for erythrocytes and hepatocytes, and it is excreted by the kidney in its protonated form at a rate that slightly exceeds creatinine clearance. Unlike bicarbonate, which requires an open system for carbon dioxide elimination in order to exert its buffering effect, THAM is effective in a closed or semiclosed system, and maintains its buffering power in the presence of hypothermia. THAM rapidly restores pH and acid-base regulation in acidaemia caused by carbon dioxide retention or metabolic acid accumulation, which have the potential to impair organ function. Tissue irritation and venous thrombosis at the site of administration occurs with THAM base (pH 10.4) administered through a peripheral or umbilical vein: THAM acetate 0.3 mol/L (pH 8.6) is well tolerated, does not cause tissue or venous irritation and is the only formulation available in the US. In large doses, THAM may induce respiratory depression and hypoglycaemia, which will require ventilatory assistance and glucose administration. The initial loading dose of THAM acetate 0.3 mol/L in the treatment of acidaemia may be estimated as follows: THAM (ml of 0.3 mol/L solution) = lean body-weight (kg) x base deficit (mmol/L). The maximum daily dose is 15 mmol/kg for an adult (3.5L of a 0.3 mol/L solution in a 70kg patient). When disturbances result in severe hypercapnic or metabolic acidaemia, which overwhelms the capacity of normal pH homeostatic mechanisms (pH < or = 7.20), the use of THAM within a 'therapeutic window' is an effective therapy. It may restore the pH of the internal milieu, thus permitting the homeostatic mechanisms of acid-base regulation to assume their normal function. In the treatment of respiratory failure, THAM has been used in conjunction with hypothermia and controlled hypercapnia. Other indications are diabetic or renal acidosis, salicylate or barbiturate intoxication, and increased intracranial pressure associated with cerebral trauma. THAM is also used in cardioplegic solutions, during liver transplantation and for chemolysis of renal calculi. THAM administration must follow established guidelines, along with concurrent monitoring of acid-base status (blood gas analysis), ventilation, and plasma electrolytes and glucose.
Acidosis↗
Role of Na(+)-Ca2+ exchange system in the pathogenesis of myocardial ischemia-reperfusion damage.
Results from a systematic experiment on isolated perfused rat heart and isolated myocytes of adult rat showed that the mechanism of calcium influx during myocardial ischemia-reperfusion is due to the development of intracellular sodium overload during ischemic period; on reperfusion, the high intracellular Na+ content activated the reverse direction of Na(+)-Ca2+ exchange over myocardial sarcolemma (SL), thus a large quantity of extracellular Ca2+ fluxed over the SL to the intracellular space, forming a condition of intracellular Ca2+ overload, which leads to irreversible damage of the myocardium.
Animals↗
Effect of intrahepatic arterial infusion of 131I-labelled lipiodol on hepatocellular carcinoma of rat.
Intrahepatic arterial infusion of 131I-labelled lipiodol was performed to study the intrahepatic distribution of lipiodol and to determine the radiation effect on 3'-Methyl-4-Dimethylaminobenzene (DAB) induced hepatocellular carcinoma (HCC) in rats. From the findings of the softex films and scintigrams of the liver, according to the time course, lipiodol was found to accumulate in the tumour tissues parallel with the degree of perfusion, and it remained in the tumour tissues for an extended period probably due to the delay of the degradation of lipiodol compared to that in non tumour tissues. It was noticed that the lipiodol, accumulated and deposited selectively in the tumour tissues, existed mostly in the extracellular space but not in the intracellular space. In histological studies of the resected specimens of the tumours, complete necrosis of hepatocellular carcinoma was recognized 2 to 8 weeks after the infusion of 131I-labelled lipiodol, while there was none in the control group where cold lipiodol was employed. These results suggest that the most effect on hepatocellular carcinoma in this study is caused by the radiation effect of 131I-labelled lipiodol.
Animals↗
Importance of the plasma refilling rate in the genesis of hypovolaemic hypotension during regular dialysis and controlled sequential ultrafiltration-haemodialysis.
The effects of ultrafiltration (UF) on plasma volume (PV) have been studied in eight patients using regular dialysis (RD) and controlled sequential ultrafiltration-haemodialysis (CSU) performed with a Rhodial 75 dialysis system. For a given value of UF the reduction of PV is determined by the plasma refilling rate. During CSU ultrafiltration induces a rapid increase in oncotic pressure without decreasing plasma osmolality. The high plasma refilling rate which can reach 1500 ml/hr allows moderate hypovolaemia despite high rates of UF and contributes to the usual good clinical tolerance of CSU. During RD a rapid decrease in plasma osmolality contributes to a water shift from the vascular space towards the interstitial and intracellular spaces and severe hypovolaemia can occur despite moderate ultrafiltration. CSU offers an adequate treatment for sodium overloaded patients with hypervolaemia, but is of no benefit in routine conditions.
Humans↗
Extracellular apparent diffusion in rat brain.
The apparent diffusion coefficients (ADCs) of a series of markers concentrated in the extracellular space of normal rat brain were measured to evaluate, by inference, the ADC of water in the extracellular space. The markers (mannitol, phenylphosphonate, and polyethylene glycols) are defined as "compartment selective" because tissue culture experiments demonstrate some leakage into the intracellular space, making them less "compartment specific" than commonly believed. These primarily extracellular markers have ADCs similar to those of intracellular metabolites of comparable hydrodynamic radius, suggesting that water ADC values in the intra- and extracellular spaces are similar. If this is the case, then it is unlikely that a net shift of water from the extra- to the intracellular space contributes significantly to the reduction in water ADC detected following brain injury. Rather, this reduction is more likely due primarily to a reduction of the ADC of intracellular water associated with injury.
Analysis of Variance↗
Rapid water and slow sodium excretion of acetated Ringer's solution dehydrates cells.
UNLABELLED: Acetated Ringer's solution is a slightly hypotonic infusion fluid (osmolality 270 mosmol/kg) that has inspired the belief that the fluid causes a shift of water volume to the intracellular space. We assessed the role of the kidney in modifying this volume shift by infusing 25 mL/kg of Ringer's acetate solution (mean, 1565 mL) over a time period of 15, 30, 45, and 80 min on different occasions in 5 healthy female volunteers. Regardless of the rate of administration, the excreted urine contained only half as much sodium (mean, 67 mmol/L) as the infused fluid. As there was only a slight increase of 0.9 mmol/L in the serum sodium level, mass balance calculations indicated that 274 mL of water had shifted from the intracellular to the extracellular space 30 min after the infusions ended (P < 0.001). This fluid shift was also maintained over the subsequent 90 min. In conclusion, infusion of Ringer's acetate solution does not promote cellular swelling as a result of the excretion of urine that is low in sodium. A slight dehydration of fluid from the intracellular space still persisted when our measurements ended 2 h after completing the infusion. IMPLICATIONS: The kidney promotes slight cellular dehydration after infusion of Ringer's acetate solution by rapid excretion of water, whereas natriuresis occurs more slowly. In volunteers, this translocation amounted to 18% of the infused fluid volume and persisted for at least 2 h.
Adult↗
Regulation of cell volume in the perfused rat liver by hormones.
The effect of hormones on cell volume was studied in isolated perfused rat liver by assessing the intracellular water space as the difference between a [3H]inulin- and a [14C]urea-accessible space. The intracellular water space (control value 559 +/- 7 microliters/g of liver; n = 88) increased on addition of insulin (35 nM) or phenylephrine (5 microM) by 12 or 8% respectively, whereas it decreased with cyclic AMP (cAMP; 50 microM), glucagon (100 nM) or adenosine (50 microM) by 9, 13 or 6% respectively. Both insulin and glucagon exerted half-maximal effects on cell volume and cellular K+ balance at hormone concentrations found physiologically in the portal vein. Adenosine-induced cell shrinkage was explained by a net K+ release from the liver. Phenylephrine (5 microM) led to cell swelling by about 8%, which was additive to insulin-induced swelling. Extracellular ATP (20 microM) induced cell shrinkage by about 6%; this was additive to adenosine-induced shrinkage. Vasopressin (15 nM) did not appreciably change cell volume, but induced marked cell shrinkage when glucagon or cAMP was present. Insulin- and phenylephrine-induced cell swelling was counteracted by cAMP. Hormone-induced changes of intracellular water space could sufficiently explain accompanying liver mass changes induced by glucagon, cAMP, adenosine or vasopressin, but not those by phenylephrine and extracellular ATP. The data show that liver cell volume is subject to hormonal regulation, in part owing to modification of cellular K+ balance. Glucagon- and insulin-induced cell volume changes occur already in the presence of physiological hormone concentrations. The effects of Ca2(+)-mobilizing hormones on cell volume are not uniform. In view of the recently established role of cell volume changes in modulating liver cell function, the present findings open a new perspective on the mechanisms of hormone action in liver, underlining our previous hypothesis that cell volume changes may represent a 'second messenger' of hormone action.
Adenosine↗
Uptake of labelled noradrenaline by isolated atria.
The uptake of labelled noradrenaline by isolated rabbit atria has been studied, and the rate of outflow of radioactivity after a period of loading has been recorded. There is first a rapid outflow presumably from extracellular space, followed by a slow outflow presumably from intracellular space. Cocaine greatly diminished the intracellular uptake. Tyramine caused an increased outflow of radioactivity from intracellular sites which was not due to the increase in atrial rate, since noradrenaline which increased the rate more than tyramine had less effect on the outflow.
Animals↗
Early disturbance of calcium translocation across the plasma membrane in toxic liver injury.
An increased influx and/or a decreased extrusion of calcium across the plasma membrane resulting in an increase in cytosolic-free calcium could play an important role in the initiation of irreversible cell injury. Therefore, the translocation of calcium across the plasma membrane was probed in the perfused rat liver using multiple indicator dilution methodology. The sucrose space corresponding to the extracellular space amounted to 0.35 +/- 0.13 ml per gm liver, and the water space corresponding to the extra- and intracellular spaces was 0.97 +/- 0.08 ml per gm. The calcium space was always slightly larger (0.42 +/- 0.10 ml per gm) than the sucrose space. The calcium space further increased during perfusion with the calcium ionophore A 23187, whereas the sucrose space remained unchanged. Two hours after administration to intact rats of acetaminophen (2 gm per kg) and carbon tetrachloride (2 ml per kg), respectively, the calcium space had increased markedly relative to the sucrose space and relative to the water space, indicating an increased accessibility of the cells to extracellular calcium. Similarly, reperfusion of livers after 90 min of ischemia was associated with an increase in calcium space relative to the sucrose and water spaces. These studies indicate that, in three models of acute liver injury, the net influx of calcium across the plasma membrane is increased early in the evolution of the injury before irreversible damage occurs.
Acetaminophen↗
Rheogenic sodium transport in a tight epithelium, the amphibian skin.
1. Intracellular potentials from frog and toad skins were measured to identify rheogenic components of active Na transport across the basolateral membrane. Transcellular current flow and associated R . I-drops were blocked with amiloride or Na-free mucosal solution. 2. The potential difference across the basolateral membrane was found to be hyperpolarized by 18 . 5 +/- 1 . 6 mV above the steady-state value immediately after blockage of apical membrane Na conductance. The hyperpolarization disappeared within 15--25 min. 3. The final steady-state value of 93 . 1 +/- 2 . 5 mV was slightly less than reasonable estimates of the K equilibrium potential. 4. The hyperpolarization could not be observed 3--5 min after addition of ouabain (10(-4) M). 5. Both the magnitude and duration of the hyperpolarization correlate directly with the amount of Na accumulated in the intracellular space. 6. A fraction of the intracellular potential was missing when Na transport was re-established after long term blockage of apical membrane Na entry. It reappeared within 10--20 min. 7. It is suggested that the hyperpolarization is due to rheogenic Na transport across the basolateral membranes. This transport mechanism may contribute some 30--50% of the electrical gradient for passive Na entry across the mucosal membrane. 8. A coupling ratio between pumped fluxes of Na and K of about 2:1 is calculated from the data.
Animals↗